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various metabolic pathways are defi- the enzyme should be delayed until 2 months after the cient or fail to function properly. These abnormalities hemolytic episode when reticulocytes are at a steady state are almost always inherited defects. Erythrocytes with (normal) and the erythrocytes produced after the hemo- intrinsic defects are susceptible to early destruction, and lytic episode have aged. Screening tests for the enzyme when this destruction exceeds the marrow capacity to include the fluorescent dye test, cytochemical staining, replace cells, hemolytic anemia results. These hemolytic dye reduction test, and ascorbate cyanide test. Definitive anemias are known as chronic or hereditary nonspherocytic testing requires quantitation of the enzyme or genetic hemolytic anemias. testing for mutations. Erythrocyte enzyme deficiencies can compromise the Deficiencies of enzymes in the glycolytic pathway integrity of the cell membrane or hemoglobin and lead decrease ATP production and lead to hemolysis. Cation to hemolysis. The HMP shunt provides the cell-reducing pump activity may be impaired and osmotic fragility may power, protecting it from oxidant damage. Defects in be increased. Pyruvate kinase (PK) is the most common this shunt allow hemoglobin to be oxidized and dena- enzyme abnormality in this pathway. Many PK enzyme tured to Heinz bodies. The Heinz bodies damage the mutants exist, resulting in a diverse array of clinical and cell membrane. The finding of bite cells (degmacytes) laboratory findings. Reticulocytosis may be significant. on the blood smear is evidence that Heinz bodies have The blood film is remarkable for the presence of irregu- been pitted from the cells. The most common deficiency larly contracted cells and echinocytes. Heinz bodies and in this pathway is G6PD deficiency, a sex-linked disor- bite cells are not found. Screening and definitive tests der. This enzyme has many different variants, some of for the enzyme are based on fluorescence. Several other which cause severe hemolysis and others mild hemolysis. rare enzyme defects that also lead to hemolysis have been In most cases, hemolysis is sporadic, occurring during identified. 396 Chapter 18 Review Questions Level I 7. Following a hemolytic episode, which of the following is a common finding in G6PD deficiency? (Objective 5) 1. What are the two main metabolic pathways that erythrocytes use for glucose metabolism? (Objective 1) a. Reticulocytosis a. Krebs cycle and glycolytic pathway b. Appearance of burr cells on the blood smear b. Hexokinase and Krebs cycle c. Increased haptoglobin c. Oxidative phosphorylation and glycolytic pathway d. Decreased unconjugated bilirubin d. Hexosemonophosphate shunt and glycolytic 8. An abnormal erythrocyte resulting from splenic removal pathway of Heinz bodies in erythrocytes is called: (Objective 7) 2. The main protective functions of erythrocyte enzymes a. macrocyte result from which of the following? (Objective 2) b. target cell a. Electron transport and cation pumping using ATP c. bite cell b. Cation pumping using ATP and protection of d. dacryocyte hemoglobin by reduced glutathione c. Protection of hemoglobin by reduced glutathione 9. Following a hemolytic episode in a G6PD-deficient and electron transport individual, a characteristic finding on a blood smear d. Cation pumping and bilirubin production is: (Objective 7) a. increased polychromasia 3. In G6PD deficiency, anemia ultimately results from: b. echinocytes (Objective 2) c. Howell-jolly bodies a. buildup of 2,3-BPG and poor iron binding d. Pappenheimer bodies b. inability to maintain enough ATP to pump cations c. oxidative damage to hemoglobin and splenic 10. What compound can induce anemia in G6PD defi- removal of erythrocytes ciency? (Objective 8) d. membrane protein defects and loss of erythrocyte a. Aspirin flexibility b. Vitamin C 4. Which is the most common erythrocyte enzyme defi- c. Iron ciency? (Objective 3) d. Primaquine a. Pyruvate kinase Level II b. Hexokinase 1. Why should G6PD testing be delayed for an individ- c. Glucose phosphate isomerase ual following a hemolytic episode? (Objective 1) d. Glucose-6-phosphate dehydrogenase a. The level of glucose must have time to replenish. 5. Which is true for the inheritance pattern for G6PD? b. Heinz bodies can interfere with the test method. (Objective 4) c. Deficient cells have been selectively destroyed. a. It is X-linked and only found in males. d. The patient needs time to build up iron stores. b. It is autosomal dominant and affects all offspring. c. It is X-linked; however, females can be affected. 2. An 18-year-old black male was suspected of having G6PD deficiency when he experienced hemolytic ane- d. It is autosomal, and males are affected; females are mia after administration of primaquine. An erythro- carriers. cyte G6PD analysis performed on blood taken 2 days after symptoms appeared was normal. A reticulocyte 6. Which of the following is a quantitative test for count revealed 12% reticulocytes at this time. These G6PD? (Objective 6) results suggest that: (Objective 7) a. Rate reduction test a. the patient definitely does not have G6PD defi- b. Fluorescent spot test ciency but could have pyruvate kinase deficiency c. Dye reduction test b. another G6PD test should be done in several months d. Ascorbate cyanide test when the reticulocyte count returns to normal Hemolytic Anemia: Enzyme Deficiencies 397 c. leukocytes could be contaminating the sample, giv- c. Heinz bodies are not formed in pyruvate kinase ing a false result deficiency. d. the patient probably has the G6PD-Mediterranean d. Pyruvate kinase deficiency does not cause abnor- variant mal erythrocyte morphology. 3. The main consequence of enzyme defects in the glyco- 7. Hereditary nonspherocytic hemolytic anemia syn- lytic pathway is: (Objective 3) drome is associated with which G6PD variants? (Objective 4) a. decreased ATP production b. Heinz body formation a. Class I c. decreased formation of reduced glutathione b. Class II d. decreased formation of 2,3-BPG c. Class III d. Class IV 4. Which of the following G6PD isoenzyme variants will not result in hemolysis? (Objective 4) 8. Harmful peroxides in the erythrocyte are neutralized by which of the following? (Objective 2) a. G6PD-Mediterranean b. G6PD-B a. ATP production c. G6PD-Canton b. Heinz bodies d. G6PD@A- c. Bilirubin d. Glutathione 5. The blood smear of a patient with chronic nonsphero- cytic hemolytic anemia reveals echinocytes, acantho- 9. A patient is suspected of having a form of G6PD defi- cytes, target cells, and irregularly contracted cells. ciency but is not affected by ingestion of fava beans. Which follow-up test could help define the cause of How can this be explained? (Objective 4) this anemia? (Objective 6) a. The patient has the G6PD-Mediterranean type. a. PEP fluorescent test b. The G6PD-B type of G6PD is present. b. Ascorbate cyanide test c. The patient does not have G6PD deficiency. c. Fluorescent spot test d. A mild form of G6PD is present. d. Quantitation of G6PD 10. Which lab test is most useful in screening for G6PD 6. Explain why bite cells are not characteristic of pyru- deficiency? (Objective 1) vate kinase deficiency. (Objectives 3, 5, 6) a. Fluorescent spot test a. The spleen removes them as they are formed. b. CBC b. The erythrocyte forms a spherocyte as inclusions c. Reticulocyte count are removed. d. PEP fluorescent test References 1. van Solinge, W. W., & van Wijk, R. (2016) Erythocyte enzyme 6. Luzzatto, L., Usanga, E. A., & Reddy, S. (1969). Glucose-6- disorders. In: K. Kaushansky, M. A. Lichtman, J. T. Prchal, M. phosphate dehydrogenase deficient red cells: Resistance to M. Levi, O. W. Press, L. J. Burns & M. A. Caligiuri, eds. Williams infection by malarial parasites. Science, 164(3881), 839–842. Hematology (9th ed., pp. 689–724). New York: McGraw-Hill. 7. Cappadoro, M., Giribaldi, G., O’Brien, E., Turrini, F., Mannu, F., 2. Herz, F., Kaplan, E., & Scheye, E. S. (1970). Diagnosis of erythro- Ulliers, D, . . . & Arese, P. (1998). Early phagocytosis of glucose- cyte glucose-6-phosphate dehydrogenase deficiency in the Negro 6-phosphate dehydrogenase (G6PD)-deficient erythrocytes para- male despite hemolytic crisis. Blood, 35(1), 90–93. sitized by plasmodium falciparum can explain malaria protection 3. Ringelhahn, B. (1972). A simple laboratory procedure for the in G6PD deficiency. Blood, 92(7), 2527–2534. recognition of A- (African type) G6PD deficiency in acute haemo- 8. Carter, N., Pamba, A., Duparc, S., & Waitumbi, J.N. (2011). Fre- lytic crisis. Clinica Chimica Acta, 36(1), 272–274. quency of glucose-6-phosphate dehydrogenase deficiency in 4. Department of Defense Instruction, Number 6465.01 (2015, July malaria patients from six African countries enrolled in two ran- 17). Erythrocyte glucose-6-phosphate dehydrogenase deficiency (G6PD) domized anti-malarial clinical trials. Malaria Journal, 10(241). doi: and sickle cell trait screening programs. Retrieved from www.dtic 10.1186/1475-2875-10-241 .mil/whs/directives/corres/pdf/646501p.pdf. 9. Guindo, A., Fairhurst, R. M., Doumbo, O. K., Wellems, T. E., & 5. Price, E. A., Otis, S., & Schrier, S. L. (2013). Red blood cell enzy- Diallo, D. A. (2007). X-linked G6PD deficiency protects hemizy- mopathies. In: R. Hoffman, E. J. Benz Jr, L. E. Silberstein, H. gous males but not heterozygous females against severe malaria. Heslop, J. Weitz, & J. Anaztasi, eds. Hematology Basic Principles PLoS Medicine, 4(3), e66. and Practice (6th ed., pp. 581-591). Philadelphia: Elsevier. 398 Chapter 18 10. Bienzle, U., Lucas, A., Ayeni, O., & Luzzatto, L. (1972). Glucose- dehydrogenase deficiency?: A systematic review of clinical studies 6-phosphate dehydrogenase and malaria: Greater resistance of focusing on patients transfused with glucose-6-phosphate dehydro- females heterozygous for enzyme deficiency and of males with genase-deficient red cells. Transfusion Medicine Reviews, 28(10), 7–17. non-deficient variant. Lancet, 1(7742), 107–110. 28. Ward, P. C., Schwartz, B. S., & White, J. G. (1983). Heinz-body 11. Luzzatto, L., & Seneca, E. (2014). G6PD deficiency: A classic anemia: “bite cell” variant—a light and electron microscopic example of pharmacogenetic with on-going clinical implications. study. American Journal of Hematology, 15(2), 135–146. British Journal of Haematology, 164(4), 469–480. 29. Glassman, A. B., & Harmening, D. M. (2009). Intracorpuscular 12. Arese, P., & DeFlora, A. (1990), Pathophysiology of hemolysis defects II: Hereditary enzyme deficiencies. In: D. Harmening, ed. in glucose-6-phosphate dehydrogenase deficiency. Seminars in Clinical hematology and fundamentals of hemostasis (pp. 164–172). Hematology, 27(1), 1–40. Philadelphia: F.A. Davis. 13. WHO Working Group. (1989). Glucose-6-phosphate dehydrogenase 30. Shannon, K., & Buchanan, G.R. (1982). Severe hemolytic anemia deficiency. Bulletin of the World Health Organization, 67(6), 601–611. in black children with glucose-6-phosphate dehydrogenase defi- 14. Costa, E., Cabeda, J. M., Vieira, E., Pinto, R., Pereira, S. A., ciency. Pediatrics, 70(3), 364–369. Ferraz, L., . . . Barbot, J. (2000). Glucose-6-phosphate 31. Nadarajan, V., Shanmugam, H., Sthaneshwar, P., Jayaranee, S., dehydrogenase Aveiro: a de novo mutation associated with Sultan, K. S., Ang, C., & Arumugam, S. (2011). Modification to chronic nonspherocytic hemolytic anemia. Blood, 95(4), 1499–1501. reporting of qualitative fluorescent spot test results improves 15. Darr, S., Vulliamy, T. J., Kaeda, J., Mason, P. J., & Luzzatto, L. detection of glucose-6-phosphate dehydrogenase (G6PD)- (1996). Molecular characterization of G6PD deficiency in Oman. deficient heterozygote female newborns. International Journal of Human Heredity, 46(3), 172–176. Laboratory Hematology, 33(5), 463–470. 16. Sarkar, S., Prakash, D., Marwaha, R. K., Garewal, G., Kumar, L., 32. Wong, F. L., Boo, N. Y., Ainoon, O., & Wong, M. K. (2009). Com- Singhi, S., & Walia, B. N. (1993). Acute intravascular haemolysis parison of detection of glucose-6-phosphate dehydrogenase defi- in glucose-6-phosphate dehydrogenase deficiency. Annals of ciency using fluorescent spot test, enzyme assay and molecular Tropical Paediatrics, 13(4), 391–394. method for prediction of severe neonatal hyperbilirubinaemia. 17. Arese, P., Turrini, F., & Schwarzer, E. (2005). Band 3/complement- Singapore Medical Journal, 50(1), 62–67. mediated recognition and removal of normally senescent and 33. Kim, S., Nguon, C., Guillard, B., Duong, S., Chy, S., Sum, pathological human erythrocytes. Cell Physiology and Biochemistry, S., . . . Menard, D. (2011). Performance of the CareStartTM G6PD 16(4-6), 133–146. deficiency screening test, a point-of-care diagnostic for prima- 18. Nafa, L., Reghis, A., Osmani, N., Baghli, L., Benabadji, M., quine therapy screening. PLoS One, 6(12), e28357. Kaplan, J. C., . . . Luzzatto, L. (1993). G6PD Aures: A new muta- 34. Tinley, K. E., Loughlin, A. M., Jepson, A., & Barnett, E. D. (2010). tion 148 Ile S Thr2 causing mild G6PD deficiency is associated Evaluation of a rapid qualitative enzyme chromatographic test with favism. Human Molecular Genetics, 2(1), 81–82. for glucose-6-phosphate dehydrogenase deficiency. American 19. Vives Corrons, J. S., Feliu, E., Pujades, M. A., Cardellach, F., Journal of Tropical Medicine and Hygiene, 82(2), 210–214. Rozman, C., Carreras, A., . . . Zuazu, F. J. (1982). Severe glucose- 35. Van Noorden, C. J., Dolbeare, F., & Aten, J. (1989). Flow cytofluo- 6-phosphate dehydrogenase (G6PD) deficiency associated rometric analysis of enzyme reactions based on quenching of with chronic |
hemolytic anemia, granulocyte dysfunction, and fluorescence by the final reaction product: detection of glucose- increased susceptibility to infections: description of a new molec- 6-phosphate dehydrogenase deficiency in human erythrocytes. ular variant (G6PD Barcelona). Blood, 59(2), 428–434. Journal of Histochemistry and Cytochemistry, 37(9), 1313–1318. 20. Roos, D., van Zwieten, R., Wijnen, J. T., Gómez-Gallego, F., de Boer, 36. Miao, J. K., Chen, Q. X., Bao, L. M., Huang, Y., Zhang, J., Wan, K. M., Stevens, D., . . . Beutler, E. (1999). Molecular basis and enzy- X., . . . Li, T. Y. (2013). Determination of optimal cutoff value to accu- matic properties of glucose-6-phosphate dehydrogenase volendam, rately identify glucose-6-phosphate dehydrogenase-deficient het- leading to chronic nonspherocytic anemia, granulocyte dysfunction, erozygous female neonates. Clinica Chimica Acta, 23(424), 131–135. and increased susceptibility to infections. Blood, 94(9), 2955–2962. 37. Loos, H., Roos, D., Weening, R., & Houwerzijl, J. (1976). Familial 21. Gray, G. R., Klebanoff, S. J., Stamatoyannopoulus, G., Naiman, deficiency of glutathione reductase in human blood cells. Blood, S. C., Kilman, M. R., Austin, T., . . . Robinson, G. C. (1973). Neu- 48(1), 53–72. trophil dysfunction, chronic granulomatous disease and non- 38. Kamerbeek, N. M., van Zwieten, R., de Boer, M., Morren, G., Vuil, spherocytic haemolytic anemia caused by complete deficiency of H., Bannink, N., . . . Roos, D. (2007). Molecular basis of glutathione glucose-6-phosphate dehydrogenase. Lancet, 2(7828), 530–534. reductase deficiency in human blood cells. Blood, 109(8), 3560–3566. 22. Wolach, B., Ashkenazi, M., Grossmann, R., Gavrieli, R., Fried- 39. Zanella, A., Fermo, E., Bianchi, P., Chiarelli, L. R., & Valentini, man, Z., Bashan, N., & Roos, D. (2004). Diurnal fluctuation of G. (2007). Pyruvate kinase deficiency: The genotype–phenotype leukocyte G6PD activity: a possible explanation for the normal association. Blood Review, 21(4), 217–231. neutrophil bactericidal activity and the low incidence of pyogenic 40. Institute of Medical Genetics in Cardiff. (2017). The Human Gene infections in patients with severe G6PD deficiency in Israel. Pedi- Mutation Database. Available from www.hgmd.cf.ac.uk. atric Research, 55(5), 807–813. 41. Pissard, S., de Montalembert, M., Bachir, D., Max-Audit, I., 23. Theodorsson, E., Birgens, H., & Hagve, T.A. (2007). Haemoglo- Goossens, M., Wajcman, H., & Bader-Meunier, B. (2007). Pyruvate binopathies and glucose-6-phosphate dehydrogenase deficiency kinase (PK) deficiency in newborns: The pitfalls of diagnosis. in a Scandinavian perspective. Scandinavian Journal of Clinical Journal of Pediatrics, 150(4), 443–445. Laboratory Investigation, 67(1), 3–10. 42. Hennekam, L., Beemer, F., Cats, B., Jansen, G., & Staal, G. (1990). 24. Johnson, L. H., Bhutani, V. K., & Brown, A. K. (2002). System- Hydrops fetalis associated with red cell pyruvate kinase defi- based approach to management of neonatal jaundice and preven- ciency. Genetic Counseling, 1(1), 75–79. tion of kernicterus. Journal of Pediatrics, 140(4), 396–403. 43. Tsang, S. S., & Feng, C. S. (1993). A modified screening procedure 25. Kaplan, M., Hammerman, C., & Bhutani, V. K. (2015). Parental to detect pyruvate kinase deficiency. American Journal of Clinical education and the WHO neonatal screening program: A quarter Pathology, 99(2), 128–131. century later. Journal of Perinatology, 35(10), 779–784. 44. Whitney, K. M., & Lothrop Jr., C. D. (1995). Genetic test for pyru- 26. Mesner, O., Hammerman, C., Goldschmidt, D., Rudensky, B. vate kinase deficiency of Basenjis. Journal of the American Veteri- Bader, D., & Kaplan, M. (2004). Glucose-6-phosphate dehydro- nary Medicine Association, 207(7), 918–921. genase activity in male premature and term neonates. Archives of 45. Vora, S., Corash, L., Engel, W. K., Durham, S., Seaman, C., & Disease in Childhood. Fetal and Neonatal Edition, 89(6), F555–557. Piomelli, S. (1980). The molecular mechanism of the inherited 27. Renzaho, A. M., Husser, E., & Polonsky, M. (2014). Should phosphofructokinase deficiency associated with hemolysis and blood donors be routinely screened for glucose-6-phosphate myopathy. Blood, 55(4), 629–635. Chapter 19 Hemolytic Anemia: Immune Anemias Linda A. Smith, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. List the antibody systems or specificities a. Cold agglutinin syndrome usually involved in: b. Warm autoimmune hemolytic anemia a. Cold agglutinin syndrome (WAIHA) b. Paroxysmal cold hemoglobinuria (PCH) 4. Describe and recognize the typical hema- c. Warm autoimmune hemolytic anemias tologic laboratory findings in ABO and Rh (WAIHA) hemolytic disease of the fetus and newborn 2. Describe the purpose and general procedure (HDFN). for the direct antiglobulin test (DAT) and 5. Contrast alloimmune and autoimmune identify the typical DAT profile (polyspecific hemolytic anemias including stimulus for AHG, anti-IgG, anti-C3) found in patients antibody production, site of hemolysis, type with cold agglutinin syndrome and warm of antibody involved, and direct antiglobu- autoimmune hemolytic anemia. lin test (DAT) and indirect antiglobulin test 3. Describe and recognize the characteristic (IAT) reactions. hematologic findings associated with the following conditions: Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare the pathophysiology of extra- 2. Given a set of laboratory data, determine vascular and intravascular hemolysis in the underlying mechanism of hemolysis and immune hemolytic anemia (IHA) including suggest confirmatory tests. site of destruction, immunoglobulin class of antibody, and underlying mechanism. 399 400 Chapter 19 3. Contrast the different mechanisms of drug- 6. Interpret the results of laboratory tests for induced immune hemolytic anemia and HDFN, and determine whether evidence of tests used for confirmation, and identify the hemolysis is present. drugs commonly involved. 7. Compare and contrast the pathophysiol- 4. Describe the mechanism of hemolysis, select ogy and clinical findings of an immediate the confirmatory tests for paroxysmal cold transfusion reaction with those of a delayed hemoglobinuria (PCH), and evaluate the reaction. results. 8. List the causes of the secondary types of 5. Compare the prenatal and postnatal patho- cold agglutinin syndrome and warm auto- physiology of hemolytic disease of the fetus immune hemolytic anemia and identify key and newborn (HDFN). laboratory results linking the cause and the autoimmune condition. Chapter Outline Objectives—Level I and Level II 399 Mechanisms of Hemolysis 404 Key Terms 400 Laboratory Identification of Sensitized Red Background Basics 400 Cells 405 Case Study 401 Autoimmune Hemolytic Anemias (AIHA) 407 Overview 401 Alloimmune Hemolytic Anemia 417 Introduction 401 Summary 423 Classification of Immune Hemolytic Anemias 401 Review Questions 423 Sites and Factors that Affect Hemolysis 403 References 425 Key Terms Alloimmune hemolytic anemia Donath-Landsteiner (D-L) antibody Immune hemolytic anemia (IHA) Antihuman globulin (AHG) Drug-induced hemolysis Immune tolerance Autoimmune hemolytic anemia Erythroblastosis fetalis Indirect antiglobulin test (IAT) (AIHA) Hemolytic disease of the fetus and Kernicterus Cold agglutinin syndrome (CAS) newborn (HDFN) Paroxysmal cold hemoglobinuria Direct antiglobulin test (DAT) Hemolytic transfusion reaction (PCH) Background Basics The information in this chapter builds on the concepts Level I learned in previous chapters. Additionally, a basic under- • Summarize the normal production, life span, and standing of immunology principles and an introduction destruction of erythrocytes. (Chapter 5) to immunohematology will be helpful. To maximize your • List the reference interval for the hematology param- learning experience, you should review these concepts eters: hemoglobin, hematocrit, erythrocyte count, before starting this unit of study: and reticulocytes. (Chapters 10, 11) Hemolytic Anemia: Immune Anemias 401 • List the different intrinsic and extrinsic factors that destruction to laboratory parameters in the d iagnosis can result in hemolytic anemia. (Chapter 11) of abnormal hemolysis. (Chapters 5, 11) • Describe the basic structure of immunoglobulin and • Choose laboratory tests to assist in diagnosing its normal physiologic role. (Chapter 8) anemia in a cost-efficient and effective manner. (Chapter 11) Level II • Identify clinical signs of hemolytic anemia and • Describe the role of erythropoietin and regulation of changes that occur in laboratory tests that signal pos- its production. (Chapter 5) sible hemolytic anemia. (Chapter 11) • Differentiate intravascular and extravascular destruction of erythrocytes and relate these types of CASE STUDY Introduction We refer to this case study throughout the chapter. When an immune-mediated process (antibody and/or com- plement) destroys erythrocytes prematurely, the disorder Nancy, a 28-year-old female, made an appointment is referred to as an immune hemolytic anemia (IHA). The with her physician because she was feeling tired all individual, however, might or might not become anemic, the time and was short of breath with minor exer- depending on the severity of hemolysis and the ability of tion. She indicated that the symptoms had been the bone marrow to compensate for erythrocyte loss. Diag- ongoing for about 3 weeks. She has no known his- nosis of anemia (and underlying hemolysis) is determined tory of chronic diseases. by laboratory findings such as a decrease in hemoglobin Consider the initial laboratory tests that should and hematocrit, an increase in reticulocytes and/or uncon- be performed to evaluate this patient’s condition jugated bilirubin, and a decrease in serum haptoglobin. based on clinical history and symptoms. Initial confirmation of the underlying immune mechanism is accomplished by demonstrating attachment of antibody or complement to the patient’s erythrocytes. Table 11-9 (Chapter 11) summarizes some common laboratory values Overview characteristically seen in hemolytic anemia. This chapter compares the different types of immune- mediated hemolytic anemias: autoimmune, alloimmune, Classification Of Immune and drug induced. The underlying mechanism for each of these anemias involves the reaction of an antibody Hemolytic Anemias with erythrocyte antigens and subsequent cell destruc- tion by either intravascular or extravascular processes. Determining the underlying process of immune hemolysis The chapter also compares mechanisms of intravascular is important because each type requires a specific treatment and extravascular hemolysis and describes the tests nec- regimen. Initially, IHA can be classified into three broad cat- essary to detect erythrocyte sensitization and identify the egories based on the stimulus for antibody production1,2 causative antibody. (Table 19-1). These are: The chapter discusses pathophysiology, clinical pre- • Autoimmune hemolytic anemia sentation and laboratory evaluation, differential diagnosis, • Drug-induced hemolytic anemia and therapy for each of the major types of autoimmune • Alloimmune hemolytic anemia hemolytic anemia. Hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN) are Autoimmune hemolytic anemia (AIHA) is a com- included as examples of alloimmune hemolytic anemia. plex and incompletely understood process characterized The causative antibody and clinical presentation for acute by an immune reaction against self-antigens and increased and delayed transfusion reactions are compared and the destruction of erythrocytes. Destruction may be mediated laboratory tests required to confirm hemolytic transfusion by anti-erythrocyte antibodies. Antibody alone, antibody reactions are described. The section on HDFN compares with complement, or complement alone. Individuals pro- ABO-HDFN and Rh-HDFN. The treatment of a fetus and a duce antibodies against their own erythrocyte antigens newborn with this condition as well as preventative mea- (autoantibodies), which are usually directed against high- sures are included. incidence antigens (antigens present on the erythrocytes of 402 Chapter 19 react best at body temperature (37°C); the anemia they pro- Table 19.1 Classification of Immune Hemolytic Anemias duce is termed warm autoimmune hemolytic anemia (WAIHA). Classification Causes About 70% of the AIHAs are of the warm type. Most warm Autoimmune • Warm-reactive antibodies (37°C) autoantibodies are of the IgG class (most frequently IgG1) Primary or idiopathic and cause extravascular hemolysis of the erythrocyte. A few Secondary warm-reacting autoantibodies of either the IgM or IgA class • Autoimmune disorders (systemic lupus erythe- have been identified. Cold hemolytic anemias, on the other matosus, rheumatoid arthritis, and others) hand, are usually due to the presence of an IgM antibody • Chronic lymphocytic leukemia and other immu- noproliferative diseases with an optimal thermal reactivity below 30°C. Hemolysis • Viral infections with cold-reacting antibodies results from IgM binding to • Neoplastic disorders and activating complement. The IgM antibody attaches to • Chronic inflammatory diseases erythrocytes in the cold and fixes complement. After warm- • Cold-reactive antibodies (less than 30°C) ing, the antibody dissociates from the cell, but the comple- Primary or idiopathic (cold hemagglutinin disease) ment remains, either causing direct cell lysis or initiating Secondary extravascular destruction. Included in the cold hemolytic • Infectious diseases (Mycoplasma pneumoniae, anemia classification is a special condition, paroxysmal cold Epstein-Barr virus, other organisms) hemoglobinuria (PCH), which is characterized by a cold- • Lymphoproliferative disorders Paroxysmal cold hemoglobinuria reacting IgG antibody capable of fixing complement. • Idiopathic A third category, mixed-type autoimmune hemolytic ane- • Secondary mia, demonstrates both warm-reacting (IgG) autoantibodies Viral syndromes and cold-reacting (IgM) autoantibodies. • Syphilis (tertiary) Drugs that attach to the erythrocyte membrane or alter • Mixed type it in some way can cause drug-induced hemolysis. Histori- • DAT negative cally, several |
different mechanisms of hemolysis have been Drug induced • Drug dependent hypothesized based on whether the drug binds directly to • Drug independent the cell, reacts with an antibody in the circulation to form an • Nonimmunologic protein adsorption (NIPA) immune complex that binds to the cell, or alters the eryth- Alloimmune • Hemolytic transfusion reaction rocyte antigens to stimulate formation of autoantibodies. • Hemolytic disease of the fetus and newborn Now, however, these antibodies are broadly classified as either drug dependent or drug independent based on reac- tions of patient’s erythrocytes and the drug in the in vitro most people). The autoantibodies characteristically react not test systems. These mechanisms are discussed in detail later only with the individual’s own erythrocytes but also with in this chapter. the erythrocytes of other individuals carrying that antigen. AIHA occurs because of antibody development to an Autoimmune hemolytic anemias are further classified erythrocyte antigen that the individual lacks. When an indi- as warm or cold hemolytic anemia based on clinical symp- vidual is exposed to erythrocytes from another person, there toms and on the optimal temperature at which the antibody could be antigens on the transfused cells that are not pres- reacts in vivo and in vitro (Table 19-2). Some antibodies ent on the recipient’s erythrocytes. Therefore, the recipient’s Table 19.2 Characteristics of Agglutinins in Hemolytic Anemia Warm-Reacting Antibodies Cold-Reacting Antibodies Immunoglobulin (Ig) class IgG IgM IgM (rare) IgG (PCH only) IgA (usually with IgG) Optimal reactivity 37°C Less than 30°C, usually less than 10°C Mechanism of hemolysis Extravascular Intravascular: complement-mediated lysis Attachment of membrane-bound IgG or C3b to macro- Extravascular: attachment of membrane-bound C3b to phage receptors macrophage receptors Specificity Usually broad specificity anti-Rh Usually autoanti-I Occasionally autoanti-i PCH: autoanti-P PCH, paroxysmal cold hemoglobinuria. Hemolytic Anemia: Immune Anemias 403 lymphocytes recognize antigens on the transfused cells as foreign and stimulate the production of antibodies (alloan- Sites And Factors That tibodies). In contrast to autoantibodies, these alloantibodies Affect Hemolysis react only with the antigens on the transfused cells or cells from individuals who possess the antigen. The alloanti- Regardless of whether it is caused by alloantibodies or bodies do not react with the individual’s own erythrocytes. autoantibodies, hemolysis can be intravascular or extra- Examples of alloimmune hemolytic anemia are: vascular, depending on the class of antibody involved and whether the complement cascade has been completely • HDFN in which the mother makes antibodies against activated. Most immune-mediated hemolysis is extravas- antigens on the fetal erythrocytes cular. Erythrocytes sensitized (coated) with antibody • Transfusion reactions in which the recipient makes anti- (IgG) or complement components (e.g., C3b) attach to bodies to antigens on the transfused (donor) cells macrophages in the spleen or liver via macrophage recep- The presence of alloantibodies can be detected in vitro tors for the Fc portion of IgG or the C3b component of by performing an antibody screen in which the patient’s complement. These cells are then phagocytized serum reacts with commercial erythrocytes containing most (Figure 19-1). Intravascular hemolysis occurs if the com- of the clinically significant antigens. An autocontrol consist- plement cascade is activated through C9 (the membrane ing of the patient’s serum and erythrocytes can also be set attack complex), resulting in lysis of the cell. The rate at up. When only alloantibodies are present, the autocontrol which hemolysis occurs in hemolytic anemia is related to shows no hemolysis or agglutination, whereas the mixture several factors.1 Table 19-3 summarizes the major factors of patient’s serum and the commercial cells produce agglu- and their effects. tination and, in rare cases, hemolysis. Checkpoint 19.2 Checkpoint 19.1 Explain how the class of immunoglobulin, amount of antibody What are the three major categories of immune hemolytic ane- bound, and thermal reactivity of the antibody affect hemolysis. mia, and how is antibody production stimulated in each type? CASE STUDY (continued from page 401) Macrophage Nancy’s initial complete blood count (CBC) shows Fc receptor C3b receptor a hemoglobin value of 7.0 g/dL and a hematocrit of 21%. Her white blood cell (WBC) count and RBC RBC C3b platelet count are within the reference interval. Antibody 1. What are some reasons for her to have a low hemoglobin value? Figure 19.1 Immune-mediated extravascular hemolysis. Erythrocytes sensitized with antibody or complement (C3b) attach to macrophages via specific cell receptors for these immune proteins. Table 19.3 Factors Affecting the Rate of Hemolysis in Immune Hemolytic Anemias Factor Effect Class of immunoglobulin coating the erythrocytes For IgG subclasses, the affinity of macrophage receptors and rate of hemolysis is greatest for IgG3 and IgG1 Quantity of Ig molecules per erythrocyte Immunoglobulins coat a cell with a high density (large number) of corresponding antigens more heavily than a cell with low density (fewer) antigens Titer of the antibody Higher titer is more likely to cause increased hemolysis Ability of the Ig to activate complement IgM and IgG can activate complement; for IgG, the ability to activate complement is IgG3 7 IgG1 7 IgG2 Thermal amplitude of the antibody Warm-reacti ng (37°C) antibodies can cause hemolysis but cold-reacting (0–4°C) antibodies usually do not The activity level of macrophages Suppression of Fc receptors decreases rate of hemolysis Complement components on the membrane Macrophages have receptors for C3b but no effective receptor for C3d 404 Chapter 19 Mechanisms Of Hemolysis and destruction of cells and microorganisms. The major roles of complement in immune hemolytic anemias are The mechanism for hemolysis is based on whether IgM, sensitization and lysis of erythrocytes. Sensitization occurs IgG, and/or complement are/is present on the erythro- when only a portion of the cascade is activated and depos- cyte. Specific phagocytic cells in the spleen or liver initiate ited on the erythrocyte membrane; lysis results when the extravascular hemolysis of cells coated with IgG or comple- entire system is activated. ment. Complete activation (through C9) of the complement The complement proteins are designated numerically cascade results in intravascular hemolysis. The type of lysis (e.g., C1, C2, C3) or by letters or historical names. Comple- (intravascular or extravascular) can affect rate of hemolysis ment proteins normally circulate in an inactive state but and number of erythrocytes destroyed. Intravascular hemo- under certain circumstances become activated in a cascade- lysis may affect up to 200 mL erythrocytes/hour versus only like fashion. An activated component is identified by plac- 20 cells/hour with extravascular hemolysis.2 ing a bar over the component (C5). The complement cascade can be initiated by at least IgG-Mediated Hemolysis three separate mechanisms: classic, alternate, and lec- IgG mediates erythrocyte destruction by first attaching to tin. The usual and most important activation pathway in the erythrocyte membrane antigens through the Fab por- IHA is the classic, although on some occasions the alter- tion of the Ig molecule. The Fc portion of the bound IgG is nate pathway can be activated (Figure 19-2). The classic exposed and binds to Fc receptors (FcgR@I, -II, -III) on mac- complement pathway is initiated by an antigen–antibody rophages in the red pulp of the spleen. After binding, the reaction involving IgM as well as IgG1 and IgG3 antibod- macrophage pits the antigen–antibody (Ag/Ab) complex, ies (IgG2 antibodies activate but less efficiently). The first fragmenting the cell membrane, which then reseals itself. complement component (C1q) must attach to two antibody With repeated splenic passage, the erythrocyte continues to Fc regions, requiring that two antibody-binding sites be lose membrane and gradually assumes a spherocytic shape. near. Thus, the attachment of complement depends on the As the cell becomes more spherocytic, it becomes rigid and density or concentration of antibody molecules and their less deformable and is eventually phagocytized by splenic spatial arrangement when attached to antigens on the cell macrophages. Alternatively, the antibody-sensitized cell can surface. Only one IgM molecule (a pentameric structure) be entirely engulfed by the macrophages (phagocytosis). is required to activate the complement pathway, whereas Natural killer (NK) cells and neutrophils also have Fc two IgG molecules (monomeric) are required; as a result, gR. Neutrophils are capable of phagocytosis (FcgR@I,@III). Inter- IgM is much more likely to activate complement than IgG. action of antibody-coated cells with NK cells usually results Therefore, the nature of the antibody involved is an impor- in the recognition and lysis of cells by NK cells, which is tant determinant of the extent of erythrocyte destruction by referred to as antibody-dependent cell-mediated cytotox- complement. icity (ADCC). This occurs through binding of IgG by the Activation of the complement cascade is initiated FcgRIII (CD16) on NK cells. when the first complement component, C1, binds to two As the spleen becomes saturated with sensitized red Fc regions of IgG or IgM antibody molecules. This attach- blood cells (RBCs) (RBCs coated with antibody and/or ment initiates activation of the other complement compo- complement), the liver assists in removing the cells. Lightly nents (C4, C2, C3). The complex containing C3b activates coated cells are more efficiently removed in the spleen due the terminal components, C5 to C9. Activation of these to the sluggish splenic blood flow. The liver can be of some terminal complement components, known as the membrane importance in removing heavily sensitized cells. The splenic attack complex (MAC), is responsible for the lytic attack on tissue proliferates in response to an increase in erythrocyte the erythrocyte membrane. Membrane leakage begins at the sequestration and can be responsible for splenic enlarge- C8-activation stage when a transmembrane pore is formed. ment (splenomegaly) in chronic warm-immune hemolytic C9 prevents the pore from resealing, resulting in osmotic anemias (Chapter 3). In immune hemolytic anemias in lysis. The alternate pathway of complement activation, which complement is activated, it, as well as IgG, is present which bypasses C1, C2, and C4 and activates C3 directly, on the erythrocyte membrane, which enhances phagocyto- can be initiated by aggregated IgG, IgA, and IgE as well as sis by increasing the likelihood of the cell binding to either by several polysaccharides and liposaccharides. the Fc and/or C3b receptors of macrophages. If complement activation on the erythrocyte membrane is complete (C1 S C9), intravascular hemolysis occurs. However, activation of complement does not always go Complement-Mediated Hemolysis through C9 and thus does not always lead to direct cell The complement system consists of more than 20 serum lysis. More commonly, activation proceeds only through proteins responsible for several diverse biological activities C3 on the erythrocyte membrane (in which case the cell is including the mediation of acute inflammatory responses said to be sensitized). As C3 is activated, it is broken down Hemolytic Anemia: Immune Anemias 405 into two fragments: C3a, which is released into the plasma, encounters macrophages. C3c dissociates from the mem- and C3b, which remains attached to the cell membrane. brane, but C3d remains attached. Erythrocytes coated with The sensitized cell with attached C3b is totally or partially C3d have a normal survival because macrophages have no engulfed by binding to the C3b receptors (CR1, CR3) of receptors for this complement component. Thus, the bal- macrophages in the liver (most complement-coated cells are ance between C3b deposition on the membrane and C3b removed in this organ). Because of the enzymatic action of inactivation determines the susceptibility of erythrocytes to the protein C3b inactivator in plasma, C3b on erythrocytes phagocytosis by macrophages via the C3b receptors. can be further cleaved to form C3c and C3d before the cell In addition to C3b inactivator, other inhibitors of com- plement activation exist. Classic Pathway Alternate Pathway IgM-Mediated Hemolysis C1q,r,s Ag/Ab, polysaccharides In cold agglutinin syndrome, IgM molecules attach to the Ag/Ab Ca++ erythrocyte membrane, but these sensitized cells are not C1q,r,s removed from circulation in the same manner as those sensi- attaches to Fc of Ag/Ab Factor D tized with IgG because macrophages do not have receptors for the Fc portion of IgM. However, IgM is an efficient acti- C1q,r,s,-Ag/Ab C3b+Factor B C3b,Bb Mg++ vator of complement, and cells can be lysed intravascularly if complement activation through C9 is complete. If acti- Properdin (P) C1q,r,s,-Ag/Ab vation is incomplete and only C3b coats the erythrocytes, C4 they can be destroyed extravascularly via adherence to CR1 C4a C3b,P,Bb and CR3 receptors on macrophages. Adherence of the cell C1q,r,s,-Ag/Ab-C4b to macrophages via complement receptors and subsequent C2 C3 C2b phagocytosis, however, is less efficient than immune adher- C1q,r,s,-Ag/Ab-C4b,2a ence (adherence mediated via immunoglobulin) and phago- C3 C3b inactivator cytosis via macrophage Fcg receptors. It has been estimated |
C3a Ag/Ab-4b,2a,3b that more than 100,000 molecules of the complement com- ponent C3b are required on the cell surface to induce effec- tive macrophage phagocytosis via complement alone. C3b C5 Inhibition C5a is also inefficient in promoting adherence to macrophages Activation C5b because much of the C3b is inactivated to C3d. Thus, extra- C6 vascular hemolysis of cells sensitized with complement is C5b,6 not as severe as hemolysis of cells sensitized with IgG. C7 In addition to activating complement, IgM antibodies C5b,6,7 agglutinate cells. In vitro, agglutination is a useful phenom- C8 enon for detecting the presence of cold agglutinins. C5b,6,7,8 C9 C5b,6,7,8,9 Checkpoint 19.3 Compare the mechanisms of IgG-mediated hemolysis with Figure 19.2 The complement cascade. The central event in those of IgM-mediated hemolysis. complement activation is the activation of C3 by C3 convertases. This can occur by two separate but interrelated mechanisms, the classic and alternate pathways. The classic complement pathway is initiated by an antigen–antibody reaction. The antigen–antibody Laboratory Identification of complex activates the C1q, r, s complex, which in turn activates C4 by proteolytic cleavage to C4a and C4b. C2 binds to C4b and Sensitized Red Cells is proteolytically cleaved by C1s to form C2a and C2b. The C4b2a complex serves as C3 convertase. In the alternate pathway, C3b Suspicion of immune hemolytic anemia indicates specific serves as the cofactor of the C3-cleaving enzyme complex (C3b, tests to detect and identify the causative antibody. In gen- P, Bb), also known as C3 convertase. Thus, C3b serves to prime its eral, two distinct techniques are used: own activation. The C3b formed through the classic pathway can directly initiate the assembly of the alternate pathway C3 convertase. • Agglutination in saline, which will detect IgM C3 can also be activated by spontaneous hydrolysis. The C3b antibodies complexes formed by the classic and alternate pathways activate C5 to C5a and C5b. Membrane damage is initiated by the assembly of • Antihuman globulin (AHG) test, which will detect IgG C5b with C6, 7, 8, 9. antibodies and/or complement Spontaneous ©UTHSCSA 1993 406 Chapter 19 IgM antibodies can be detected by agglutination reac- are used to specifically identify what component is on the tions using test sera (antibody) and appropriate erythro- erythrocyte. cytes suspended in saline, but IgG antibodies cannot. The The two types of tests using AHG are: difference in the ability of IgG and IgM to cause aggluti- • Direct antiglobulin test detects erythrocytes coated nation in saline can be explained by the difference in size with antibody or complement in vivo of the two antibodies in relation to the zeta potential. The erythrocyte zeta potential is an electrostatic potential cre- • Indirect antiglobulin test detects antibodies in the ated by the ionic cloud surrounding erythrocytes when plasma or serum they are suspended in saline. This force tends to keep the erythrocytes about 25 nm apart in solution. Thus, an anti- Direct Antiglobulin Test body must be large enough to span the 25 nm gap between The direct antiglobulin test (DAT) detects erythrocytes that cells to agglutinate saline- or plasma-suspended cells. The have been sensitized with antibody and/or complement in IgM pentamer has a possible span of 35 nm; therefore, it vivo. This test should always be performed in suspected can overcome the electrostatic forces separating the cells cases of AIHA because it differentiates AIHA from all other and cause agglutination (Figure 19-3). However, the maxi- types of hemolytic anemia, confirming the immunologic mum span of an IgG molecule is about 14 nm, and it cannot basis of erythrocyte destruction. Results must be evaluated bridge antigens on two separate cells to cause agglutina- in conjunction with clinical symptoms and other laboratory tion. Thus, detection of IgG antibodies requires a different data.3 Specimens collected in tubes with ethylenediamine- technique using AHG, which is an antibody to human IgG. tetraacetate (EDTA) are preferred to clotted specimens for AHG can connect the antibody molecules on separate cells the DAT procedure. EDTA chelates Ca++ and Mg++, pre- and cause agglutination. venting the in vitro binding of complement to red cells The AHG test (historically referred to as the Coombs that can be mediated by naturally occurring cold-reactive test) is a laboratory procedure designed to detect erythro- antibodies (e.g., autoanti-I). However, complement that has cytes sensitized with IgG and/or complement. Polyspecific been bound in vivo will be detected. Polyspecific AHG is AHG is composed of antibodies to both IgG and C3, capa- added to saline-washed patient cells, and agglutination is ble of attaching to the Fc region of IgG immunoglobulins considered positive evidence for the presence of IgG and/or or complement components on two separate cells, bridg- complement components on the cells due to in vivo coating. ing the distance between them and leading to the lattice A positive test with polyspecific AHG should be fol- formation known as agglutination. Monoclonal antiserum lowed by a DAT with monospecific AHG antiserum that against IgG only (anti-IgG AHG) or C3 only (anti-C3 AHG) reacts specifically with either IgG or complement to deter- mine the type of proteins bound to the erythrocyte. Either or both monospecific IgG-AHG and C3b-AHG can be positive. If the anti-IgG test is positive, the antibody can be removed from the cell by an elution process, and the resulting elu- IgG ate (solution containing the antibody) tested to identify the specificity of the antibody. 14nm If an autoantibody is IgM, only complement usually is detected on the erythrocytes as the IgM tends to dissociate RBC RBC from the cells in the warmer part of the circulation. The poly- 25 nm specific and the anti-C3 monospecific DAT test will be positive; the anti-IgG will be negative. An elution procedure is not used if only complement is detected on the erythrocyte because no antibody would be recovered for identification. In general, if the antibody coating the cells is IgA, the DAT will be negative because the AHG reagent cannot detect that immunoglobulin class. IgA-specific or IgM-specific AHG is not routinely avail- 35 nm able in most clinical laboratories but may be used in reference IgM pentamer laboratories for workup of difficult-to-diagnose cases. Figure 19.3 The zeta potential of erythrocytes keeps the cells about 25 nm apart when suspended in saline. IgG antibodies have Indirect Antiglobulin Test a span of about 14 nm, not enough to bridge the gap between cells The indirect antiglobulin test (IAT), sometimes referred to and cause agglutination. IgM antibodies, however, are pentamers with a span of about 35 nm, a distance sufficient to bridge the space as an antibody screen, is used to detect antibodies in the between cells and cause agglutination. patient’s serum. A positive IAT indicates alloimmunization Hemolytic Anemia: Immune Anemias 407 (immunization to antigens from another individual) and/ Positive DAT in Normal Individuals or the presence of autoantibody in the patient’s serum. In the IAT, free antibody is detected by incubating the patient’s Some healthy blood donors and hospitalized patients can serum with reagent erythrocytes of known antigenic have a positive DAT but not shortened erythrocyte survival. makeup (commonly referred to as screening cells). An auto- The reason for this observation is not clear,1 but factors that control consisting of the patient’s serum and erythrocytes could be responsible include.21,22 may also be included. After a specified incubation period at 1. The individual’s macrophages may not be as active in 37°C, the cells are washed free of excess serum, and AHG removing sensitized cells as the macrophages in indi- antiserum is added. If the patient’s antibody has attached viduals with hemolytic disease. to the corresponding erythrocyte antigen during the incuba- 2. The amount of antibody bound to cells might not be tion period, the reagent cells will agglutinate with AHG. sufficient to cause decreased erythrocyte life span. Further testing to identify the specificity of the antibody is 3. Macrophages might not recognize the subclass of anti- then carried out. body sensitizing the cell. Macrophage Fc receptors have low affinity for the IgG2 and IgG4 subclasses. Eryth- rocytes coated with these immunoglobulins will give Checkpoint 19.4 a positive direct DAT, but in vivo, survival of the cells Compare the purpose of the DAT and the IAT, and state the type will be normal. of specimen used for each test. 4. The thermal amplitude of the antibody may be less than 37°C 5. The positive DAT can be due to the presence of cer- Negative DAT in AIHA tain complement fragments on erythrocytes. Increased amounts of C3d can be found on the erythrocytes, but In some cases of AIHA, antibody cannot be detected because this component is not detected by macrophage on the patient’s cells or in the serum.4,5,6,7,8,9,10 This can receptors, erythrocytes will not have decreased survival. result from an insufficient number of IgG molecules on 6. Patients with hypergammaglobulinemia or receiving the erythrocyte for detection, autoantibodies of the IgA high-dose intravenous immunoglobulin (IVIG) or anti- or IgM class, or the presence of autoantibodies with a low lymphocyte globulin could have a positive DAT affinity for the erythrocyte.1,7,11 The DAT can detect as because of nonspecific binding of immunoglobulins to few as 100–500 molecules of IgG per cell or 400–1100 mol- the erythrocytes. ecules of complement per cell. However, in vivo removal of sensitized cells by macrophages can occur when cells are coated with fewer IgG molecules. Thus, the in vivo CASE STUDY (continued from page 403) life span of the sensitized cell can be significantly short- ened, as evidenced by the clinical findings of a typical When examining Nancy’s peripheral blood smear, DAT-positive hemolytic anemia, but the concentration the laboratory professional noted that spherocytes of antibodies on the cell could be insufficient to give a were present. The reticulocyte count was elevated. positive DAT.4,8 Newer, more sensitive techniques, such The laboratory professional called the blood bank as enzyme-linked DAT, gel tests, dual DAT procedure, and found that the DAT on Nancy was positive Polybrene tests, immunoblotting, and flow cytometry, can with polyspecific AHG, anti-IgG, but negative with detect lower concentrations of antibodies than the conven- anticomplement. tional DAT technique.9,12,13,14,15,16,17 The dual DAT test is 2. What is the significance of the spherocytes? able to identify IgM bound to erythrocytes in WAIHA.18 IgA or IgM autoantibodies are not detected using polyspe- 3. Based on these results, what do you suspect is cific AHG. However, if complement has been activated, going on in Nancy’s blood? Explain. it can be detected if sufficient molecules are present.1,11,19 In other cases the polyclonal activation in WAIHA may result in increased levels of reactive IgA.20 Specialized antihuman globulin reagents with anti-IgA or anti-IgM Autoimmune Hemolytic specificity is used in some reference laboratories to detect erythrocyte sensitization by immunoglobulins of these Anemias (AIHA) classes. In general, these cases of DAT-negative hemolysis Usually a person’s immune system recognizes self-antigens respond to steroid therapy in a similar manner to DAT- (those that appear on the individual’s own cells) and does positive cases.4 not mount an immune response to these antigens. These 408 Chapter 19 antigens can, however, stimulate an immune response if rate of hemolysis and hemoglobin levels (LAI). The type of injected into another individual because the recipient’s lysis may affect hemoglobin levels, since intravascular lysis immune system recognizes them as being “foreign.” The may destroy more cells per hour than extravascular lysis.2 immune regulatory mechanisms that govern response to Although WAIHA can occur at any age, the incidence self-antigens are collectively known as immune tolerance. increases after age 40. Childhood incidence peaks in the first It is generally accepted that autoimmune diseases occur 4 years of life, often after acute infections, and may pres- as the result of several factors including genetic predisposi- ent with more severe symptoms than adult onset.11,20,30,31 tion, exposure to infectious agents that can induce antibody About 60% of the cases of WAIHA are idiopathic and can be production due to molecular mimicry, and defects in the acute or chronic. In acute idiopathic WAIHA, severe anemia mechanism regulating immune tolerance including altered develops over 2 to 3 days, but the hemolysis is self-limited levels of CR1, and lack of effective T-regulatory cells (TReg). with a duration of several weeks to several years. In other T-regulatory cells are a subpopulation of T-cells that have instances, the hemolysis is chronic and does not abate. a role in immune tolerance and maintaining self-tolerance. |
The underlying disorders most frequently associated The cells are known to inhibit T-cell proliferation and cyto- with secondary WAIHA are: kine production and help prevent autoimmunity.23 They • Lymphoproliferative disease, including chronic lym- also inhibit self-reactive T-cells. Decreased numbers of TRegs phocytic leukemia (CLL) and Hodgkin’s disease have been found in patients with autoimmune hemolytic (Chapter 28). CLL, found most frequently in older anemia.23,24,25 In addition, there is evidence for the role of adults, is the disease most frequently associated with Th17 cells in autoimmunity, including diseases such as rheu- the development of secondary WAIHA. WAIHA often matoid arthritis and systemic lupus erythematosus.26,27,28 develops late in the disease when the immune dysregu- The Th17 cells belong to a novel class of CD4+ T-cells that lation is greatest. Many children with idiopathic WAIHA secrete several cytokines including IL-17, IL-21, and IL-22, eventually develop a lymphoproliferative disease. which may play a role in development of autoimmune dis- ease. The level of IL-17, which is pro-inflammatory cytokine • Neoplastic diseases that amplifies inflammation, is increased in patients with • Other autoimmune disorders, including systemic lupus autoimmune hemolytic anemia. Significantly elevated lev- erythematosus and rheumatoid arthritis, ulcerative els of Th 17 cells correlate with disease activity.26,27 Another colitis, and Crohn’s disease. Those patients with SLE factor that may influence development of autoimmune dis- and WAIHA often also have increased risk for immune eases, including warm autoimmune hemolytic anemia, is an thrombocytopenia, which may or may not reflect levels imbalance of specific cytokines. IL-10 helps to promote anti- necessary for diagnosis of Evan’s syndrome.32 body production by B-cells while IL-12 suppresses humoral • Certain viral and bacterial infections, especially in immune response and promotes cell-mediated responses.24 infants and young children In one study patients with warm autoimmune hemolytic • Vaccinations anemia showed an imbalance in the levels of these cyto- kines.23 Warm or cold AIHA is also categorized based on • Solid organ transplant whether there is an underlying disease associated with it. Presence of the underlying disorder can complicate The two categories are: initial diagnosis and treatment of hemolytic anemia. In • Primary or idiopathic: no underlying disease identified many cases, the AIHA is resolved by treating the underly- ing disease. • Secondary: underlying disease present PATHOPHYSIOLOGY Warm Autoimmune Hemolytic The autoantibody in WAIHA is reactive with antigens on Anemia the patient’s erythrocytes. Most often the specificity of the antibody is directed against polypeptide antigens of the Rh Warm autoimmune hemolytic anemia (WAIHA) is the most system, although other antigen systems can be involved. common form of AIHA (up to 70% of cases).29 It is most When directed against the Rh system, the antibody can be often mediated by IgG antibodies (usually IgG1 or IgG3) specific for a single antigen (such as autoanti-e) or more whose optimal in vivo reactivity is at 37°C. In a majority commonly, the antibody reacts with a complex Rh antigen (90%) of WAIHA cases, erythrocytes are sensitized with IgG found on all erythrocytes except Rh null or Rh-deleted and complement or IgG alone. About 13% of cases are sensi- cells. The epitope against which these latter antibodies are tized with complement alone.29 WAIHA is rarely associated directed has not been defined. Recent research suggests that with IgM-only or IgA-only sensitization. In cases where the antibody can be against a component of the erythrocyte IgM is involved, the presence of complement alone on the membrane such as glycophorins, band 3 protein, or protein erythrocyte may be a clue.10 The subclass of IgG may affect band 4.1. Hemolytic Anemia: Immune Anemias 409 Most hemolysis in WAIHA is extravascular via splenic macrophages. Subclass of IgG present may affect the degree Table 19.4 Laboratory Findings Associated with WAIHA of hemolysis, with IgG3 associated with lower hemoglo- Common findings • Positive DAT bin levels.22 Although complement is not needed for cell • Normocytic, normochromic anemia destruction, if both antibody and complement are on the • Increased reticulocytes cell membrane, phagocytosis is enhanced. If erythrocyte • Spherocytes and other erythrocyte abnormalities destruction exceeds the compensatory capacity of the bone • Presence of autoantibody in the serum marrow to produce new cells, anemia develops. Direct • Increased serum bilirubin (total and complement-mediated intravascular hemolysis associated unconjugated) with IgM antibodies in WAIHA is rare. • Decreased serum haptoglobin • Elevated lactate dehydrogenase (LD) CLINICAL PRESENTATION • Positive antibody screen with all cells The most common presenting symptoms in idiopathic including autocontrol WAIHA are related to anemia. Progressive weakness, dizzi- • Incompatible crossmatches with all donors ness, dyspnea on exertion, back or abdominal pain, and jaundice are common. In secondary WAIHA, signs and Other laboratory findings • Increased osmotic fragility that can be associated with • Increased urine and fecal urobilinogen symptoms of the underlying disorder can obscure the fea- hemolysis in WAIHA • Hemoglobinemia,a hemoglobinuria, tures of the hemolytic anemia. The patient can present with methemoglobinemia, hemosiderinuria signs and symptoms of both the underlying disease and a Seen only in acute hemolytic episode. hemolysis or just the disease. The signs of WAIHA in a few patients can precede development of the underlying dis- ease. Although mild to moderate splenomegaly is present other poikilocytes are characteristic (Figure 19-4). Sphero- in more than 50% of patients with WAIHA, massive splenic cytes found in AIHA are usually more heterogeneous than enlargement suggests an underlying lymphoproliferative are the spherocytes associated with hereditary spherocytosis. disorder. Hepatomegaly is found in about one-third of This anisocytosis is readily noted upon examining the blood patients with primary WAIHA. smear and is indicated by an increase in red cell distribution width (RDW) on automated hematology analyzers. Eryth- rophagocytosis by monocytes is rarely seen. The mono- Checkpoint 19.5 cyte-engulfed erythrocyte is readily detected if the cell still What are the clinical findings and the immune stimuli for contains its pink-staining hemoglobin. If the hemoglobin has WAIHA? leaked out of the cell, however, only colorless vacuoles are seen. Leukocyte counts are normal or increased with neutro- philia in idiopathic WAIHA but can vary in the secondary Laboratory Evaluation form based on the underlying disease. Platelet counts are Refer to Table 19-4 for the most common laboratory findings associated with WAIHA. Findings such as a positive DAT, autoantibody in the serum, and presence of spherocytes on the peripheral blood smear reflect the immune-mediated destruction of the erythrocyte. Peripheral Blood Moderate to severe normocytic, normo- chromic anemia is typical and decreased hemoglobin is the most direct indicator of clinical severity.33 In well-compen- sated hemolytic disease, anemia can be mild or absent, and the only abnormal parameters may be a positive DAT and an increase in reticulocytes. Reticulocytes are invariably increased in uncomplicated hemolytic disease. The reticu- locyte production index can be as high as 6 or 7. Depending on the degree of reticulocytosis, macrocytosis can be present. Presence of reticulocytopenia may represent an autoimmune reaction against erythrocyte precursors and presents a more severe presentation with clinical necessity for transfusion.25,34 Figure 19.4 A blood smear from a patient with warm autoimmune hemolytic anemia (WAIHA). The marked anisocytosis The blood smear frequently shows erythrocyte abnor- is due to the presence of spherocytes and large polychromatophilic malities that suggest a hemolytic process. Polychromasia, erythrocytes. The nucleated cells are orthochromic normoblasts nucleated red blood cells, spherocytes, schistocytes, and (PB; Wright stain, 1000* original magnification). 410 Chapter 19 usually normal or slightly increased. Evan’s syndrome results the cross-match. In addition, approximately 30% of patients when severe thrombocytopenia accompanies WAIHA. with WAIHA have underlying alloantibodies, which can Bone Marrow Bone marrow examination is not necessary present an additional challenge to finding compatible blood because the autoantibody can mask their presence.35 for the diagnosis of WAIHA. The bone marrow can show erythroid hyperplasia, depending on the degree of hemoly- If the patient has an alloantibody, blood negative for the sis. Erythrophagocytosis by macrophages can be seen. Com- specific antigen must be administered. If serologically com- pensatory bone marrow response can be less than expected patible blood cannot be found because of the presence of the with concomitant folic acid deficiency. In chronic hemolysis, autoantibody, donor cells demonstrating the least incom- the folic acid requirement increases two to three times nor- patibility are usually chosen. These transfusions appear to mal; without folic acid supplements, the stores are quickly be safe and have little risk of increased hemolysis.36 In some depleted. If the patient contracts viral infections associated cases, the blood transfused is matched only for ABO and Rh with bone marrow suppression, life-threatening anemia type. The clinical problems with this approach, however, (aplastic crisis) can occur. are two-fold: donor cells (1) are often destroyed as rapidly as the patient’s own erythrocytes and (2) can also stimulate Other Laboratory Tests The DAT is a useful test to distin- the production of alloantibodies. The precautions of delay- guish the immune nature of this hemolytic anemia from ing or denying transfusion to patients who require it due nonimmune-mediated hemolytic anemias. The test is usu- to lack of serologically compatible blood may jeopardize ally positive with polyspecific AHG and anti-IgG mono- their survival.37 In some cases, the use of flow cytometry specific AHG antiserum. Only about 30% of the cases of can help evaluate the survival of donor cells in patients WAIHA have a reaction with anti-C3, either with or without with WAIHA. In other cases, such as individuals who must the concurrent presence of IgG. Complement only, IgM- or receive serial transfusions, finding phenotypically matched IgA-mediated lysis in WAIHA is rare.11,19 donor blood can decrease the risk of stimulating alloanti- Reaction of patients’ serum with commercial screening bodies. In patients who require serial transfusion, genotyp- cells typically shows agglutination with all cells when AHG ing of the patient may be an option that permits selection of is added. The autocontrol (patient serum and patient eryth- antigen-matched units without interference from autoanti- rocytes) shows similar reactions. Other laboratory findings body-coated cells and may provide cells with better in vivo are nonspecific but reflect the hemolytic component of the survival.38,39 If the autoantibody has an identifiable single condition (Table 19-4). specificity (such as autoanti-e), the donor blood chosen for DIFFERENTIAL DIAGNOSIS the compatibility test should be negative for the antigen. The diagnosis of WAIHA requires clinical features of hemo- Therapies for WAIHA focus on decreasing the produc- lysis as well as serologic evidence. WAIHA with the pres- tion of autoantibodies and slowing down the destruction of ence of spherocytes can be differentiated from hereditary erythrocytes. These therapies include:35,37,40 spherocytosis (HS) by the DAT. Antibodies are not respon- • Corticosteroids The standard initial or first-line ther- sible for forming spherocytes in HS; therefore, the DAT is apy for patients with WAIHA is often a course of negative. The autohemolysis test is abnormal in both HS immunosuppressive drugs such as corticosteroids. and in WAIHA with spherocytes. The addition of glucose Corticosteroids are used to produce immunosuppres- significantly corrects autohemolysis in HS, but not in sion by decreasing lymphocyte proliferation and sup- WAIHA. The peripheral blood smear also gives clues to pressing macrophage sequestration of sensitized cells diagnosis. The spherocytes in HS appear as a rather homo- by affecting the Fc receptors. Although many patients geneous population, but WAIHA has a mixture of sphero- show a good initial response with a decrease in eryth- cytes, microspherocytes, and normocytes. rocyte destruction, few patients experience complete remission with this therapy. A decrease in production of Checkpoint 19.6 autoantibodies is often demonstrated by a decrease in What is the DAT pattern in WAIHA? Explain why spherocytes the strength of the DAT reaction and indirect antiglob- are commonly seen in WAIHA. ulin test. Response usually begins within the second week of treatments, and many patients require long- term low-dose maintenance to prevent relapse. Patients THERAPY who do not respond are often placed on second-line Transfusion therapy is not necessary in self-limiting hemo- therapy. Patients with underlying lymphoproliferative lytic disorders without life-threatening hypoxemic anemia. diseases or SLE often show a reduced response to cor- When transfusion is indicated (more often associated with ticosteroids, necessitating second-line therapy.35,40,41,42,43 acute onset), finding a suitable donor is difficult because the Standard second-line treatments with the most efficacy patient’s autoantibody usually reacts with all donor cells in for individuals who do not respond to corticosteroids Hemolytic Anemia: Immune Anemias 411 includes splenectomy or the use of monoclonal anti- some refractory patients as adjunct therapy.44 |
There is body treatment (rituximab). no recommended therapy for secondary WAIHA.35,40 • Splenectomy Splenectomy has been a standard second- Treatment of the underlying disease in patients with line therapy for years and provides long-term remission secondary WAIHA is important. Often resolution for some patients.35,40 Removal of the spleen decreases of the disease leads to decreased production of the the destruction of IgG-coated erythrocytes that would autoantibody. normally be removed by splenic macrophages. If, however, the antibody concentration remains high, the destruction of sensitized erythrocytes can continue in Cold Autoimmune Hemolytic the liver. Some evidence suggests that splenectomy can Anemia be more beneficial in patients with idiopathic WAIHA Cold AIHA, also termed cold agglutinin disease (CAD) or than in those with the secondary type. The primary cold agglutinin syndrome (CAS), is associated with an IgM complication is sepsis related to infections, especially antibody that fixes complement and is reactive below 37°C. with encapsulated bacteria. This disorder, which comprises 16–30% of the AIHAs, is less • Rituximab (monoclonal anti-CD20) Rituximab has common in children than adults. IgA and IgG antibodies been used in the treatment of clonal B-cell malignan- rarely have been implicated in hemolysis in CAS. cies, including chronic lymphocytic leukemia, in CAS, like WAIHA, is either idiopathic or second- cases of autoimmune collagen vascular disease and ary (Table 19-5). Unlike the autoantibody associated with in patients with cold agglutinin syndrome. It is rec- WAIHA, however, this autoantibody is routinely present ommended as a second-line therapy in patients with in the plasma and is often not of clinical significance. Idio- idiopathic and secondary WAIHA that are refractory to pathic CAS is usually chronic, occurring after age 50 with other treatments and is being used in place of splenec- a peak onset after age 70. The antibody involved is usually tomy in many patients.18 Children appear to respond a monoclonal IgM/k light chain with autoanti-I specificity. better than adults.40 Rituximab depletes the B cells that The secondary type can occur after an infectious disease produce autoantibodies but does not apparently affect or be associated with lymphoproliferative disease. The sec- plasma cells or hematopoietic stem cells. ondary type associated with infectious disease is usually • Danazol This drug is a synthetic anabolic steroid that has an acute, self-limiting form that has an onset 1–3 weeks been used as a first-line treatment with corticosteroids. after infection and resolves within 4–6 weeks.45 Most of • Cytotoxic drugs Various alternative therapies are avail- these autoantibodies are polyclonal and have a specificity able for patients who do not respond to first- or second- for antigens of the Ii system. Anti-I is usually associated line therapy or who have recurrent disease. Cytotoxic with Mycoplasma pneumoniae infections but may also be drugs such as cyclophosphamide, cyclosporine A, vin- associated with viral infections including HIV. Anti-i can cristine-loaded platelets, mycophenolate mofetil, and be seen in infectious mononucleosis. Up to 75% of chronic azathioprine cause general suppression of the immune CAS have an underlying bone marrow lymphoprolifera- system, which decreases synthesis of autoantibody. tive B cell disorder.46,47,48,49,50 The clonality can be demon- strated via flow cytometry of immunohistochemistry. The • High-dose intravenous immunoglobulin (IVIG) This secondary chronic form of CAS associated with lymphop- treatment blocks Fc receptors on macrophages and roliferative disorders, such as lymphoma or Waldenstrom’s affects T- and B-cell function by increasing TReg cells or macroglobulinemia, is typically found in older individuals. reducing B-cell function. It has a variable success rate The antibody in these cases is usually a monoclonal IgMk and is most often used as an adjunct therapy with cor- ticosteroids or in selected patients with severe anemia who are refractory to drugs. Table 19.5 Autoimmune Hemolytic Anemia Caused by • Therapeutic plasma exchange and plasmapheresis Cold-Reacting Antibodies This procedure can dilute or temporarily remove the Cold agglutinin syndrome Primary (idiopathic) autoantibody from the patient’s circulation. It has been (CAS) Secondary successful in reducing antibody load for a short time in • Infection associated some cases but is not a satisfactory long-term therapy. It • Mycoplasma pneumonia infections, infectious mononucleosis is now recommended as a “last ditch” effort for patients • Lymphoproliferative disease associated with severe hemolysis.35,40 Paroxysmal cold Primary (idiopathic) • Hematopoietic stem cell transplants are not recom- hemoglobinuria Secondary mended as their effectiveness has not been proven. Use • Viral infections of erythropoiesis-stimulating agents has been used in • Tertiary syphilis 412 Chapter 19 protein. A more severe type of cold AIHA, paroxysmal cold cell volume (MCV) is falsely elevated (Table 19-6). These hemoglobinuria (PCH), is associated with a biphasic cold- erroneous values occur when erythrocyte agglutinates are reacting IgG antibody and is discussed in the next section. sized and counted as single cells. The hematocrit, calcu- lated from the erroneous erythrocyte count and MCV, is PATHOPHYSIOLOGY falsely low. The mean cell hemoglobin (MCH) and mean The severity of CAS is related to the thermal range of the cell hemoglobin concentration (MCHC) calculated from the antibody. Cold-reacting antibodies with a wide range of erythrocyte count and hematocrit are falsely elevated. The thermal activity (up to 32°C) can cause problems when the hemoglobin assay is accurate because the cells are lysed peripheral circulation cools to this temperature. Comple- to determine this parameter. Accurate cell counts can be ment-mediated lysis accounts for most of the erythrocyte obtained by warming the blood and diluting reagents to destruction. 37°C before performing the test. Visible autoagglutination The cold-reacting antibody is usually directed against in tubes of anticoagulated blood can be observed as the the I antigen, which is expressed on erythrocytes of almost blood cools to room temperature. all adults. I antigen specificity of the antibody can be defined When blood counts are performed at 37°C, the results by reactivity of the patient’s serum with all adult erythro- indicate a mild to moderate normocytic, normochromic cytes but minimal or no reactivity with cord cells (which anemia. The blood film shows polychromasia, some sphe- lack the I antigen). In CAS associated with infectious mono- rocytes, rouleaux, or clumps of erythrocytes, and some- nucleosis however, the antibody may have anti-i specific- times nucleated red cells (Figure 19-5). Erythrophagocytosis ity. These antibodies react strongly with cord cells because can be seen but is more typical on smears made from buffy the i antigen is generally expressed strongly on cord cells coats after the blood has incubated at room temperature. and erythrocytes of children younger than 2 years old and Leukocyte and platelet counts are usually normal. Leuko- weakly or not at all with adult erythrocytes. The second cytosis can occur during acute hemolysis as the result of a most common specificity for cold auto agglutinins is anti-Pr, bone marrow stress response. The bone marrow exhibits which can be stimulated by infections with rubella virus or normoblastic hyperplasia often with an increase in lym- the varicella virus. The Pr antigens are expressed on both phoid cells. Patients with chronic CAS may show decreased adult and infant erythrocytes. C3 and/or C4 levels. Patients also can have a decreased CLINICAL PRESENTATION value for the CH50 assay, a functional hemolytic assay In some instances, CAS is associated with a chronic hemo- that measures the integrity of the entire complement cas- lytic anemia with or without jaundice. In others, hemoly- cade. Bilirubin and LD may be increased depending on the sis is episodic and associated only with chilling. degree of hemolysis. Erythrocyte agglutination occurs in areas of the body that cool to the thermal range of the antibody and cause sludg- ing of the blood flow within capillaries. Vascular changes including acrocyanosis in which the hands and/or feet Table 19.6 Criteria for Clinical Diagnosis of Cold turn blue and cold can occur. In Raynaud’s phenomenon, Agglutinin Syndrome (CAS) pain can be accompanied by a characteristic pattern of Clinical history • Acrocyanosis color changes in the skin from white (due to spasm of the • Hemoglobinuria on exposure to cold vessels) to blue (caused by cyanosis) to red (which indi- Laboratory evaluation • Serological cates a return of blood flow to the area). These conditions DAT: Positive with polyspecific AHG primarily affect the extremities, especially the tip of the Negative with anti-IgG; Positive with nose, fingers, toes, and ears. Hemoglobinuria accompa- anti-C3 IAT: Antibody showing characteristic reac- nies the acute hemolytic attacks. Splenomegaly can be tions at less than 25°C present (Table 19-6). Cold agglutinin titer more than 1000 at 4°C • False increase in MCV, MCH, and MCHC • False decrease in erythrocyte count Checkpoint 19.7 • Normocytic, normochromic anemia Describe the mechanism of cell destruction in CAS. • Reticulocytosis • Spherocytes, agglutinated RBCs, rouleaux, nucleated RBCs on blood smear • Increased bilirubin (total and unconjugated) LABORATORY EVALUATION • Increased LD The first indication of the presence of unsuspected cold • Decreased haptoglobin agglutinins often is from blood counts performed on elec- • Hemoglobinemia, hemoglobinuria in acute tronic cell counters. The erythrocyte count is inappropri- hemolysis ately decreased for the hemoglobin content, and the mean • Hemosiderinuria in chronic hemolysis Hemolytic Anemia: Immune Anemias 413 or only weakly positive with benign cold agglutinins. The DAT, utilizing monospecific anti-IgG, is negative both in CAS and benign cold agglutinins. Historically, the cold agglutinin test was performed when CAS was suspected. This test demonstrates the ability of the pathologic antibody to agglutinate the patient’s cells at temperatures from 0°C to 20°C in saline. The reaction is reversible with agglutinates dispersing at 37°C. With benign cold antibodies, agglutination occurs at 0–4°C and can occur up to 20°C. Titers of benign cold agglutinins reach 1:64 in normal individuals; in cold agglutinin disease, the titer is usually more than 1:1000. Titers of 1:256 or more with a positive DAT using monospecific anti-C3 antisera and a negative DAT using monospecific anti-IgG antisera are Figure 19.5 Cold autoimmune hemolytic anemia from a highly suggestive of cold agglutinin disease. patient with chronic lymphocytic leukemia. Some of the erythrocytes are in small clumps. Spherocytes are also present (peripheral blood, Wright stain, 1000* original magnification). Checkpoint 19.9 Compare the DAT findings and the antibody specificity in WAIHA and CAS. Checkpoint 19.8 Explain why the MCV, MCH, and MCHC can be falsely increased when blood from someone with CAS is tested using an auto- THERAPY mated cell counter. The most effective therapy in primary CAS is usually achieved by keeping the individual’s extremities warm. Corticosteroids are not usually effective. Chemotherapy DIFFERENTIATION OF CAS AGGLUTININS FROM using rituximab has been somewhat effective in primary BENIGN COLD AGGLUTININS CAS and CAS associated with an underlying lympho- The serum of most normal individuals exhibits the pres- proliferative disease and is recommended as a first-line ence of cold autoantibodies with anti-I specificity when the therapy.35,40 When used with fludarabine, the response rate serum and cells are incubated at 4°C. These antibodies are for patients increases. Recently, Eculizumab, a monoclonal termed benign cold autoagglutinins because their thermal antibody that binds to C5, has been used to treat patients amplitude and concentration (titer) are not high enough refractory to rituximab. This complement inhibitor binds to to cause clinical problems. When pathologic cold aggluti- C5 and inhibits formation of the membrane attack complex nins (increased titer and increased thermal amplitude) are (MAC), thus inhibiting hemolysis.51 Another new drug tar- suspected as the cause of anemia, laboratory tests should gets the serine protease C1s, thus preventing attachment be performed to differentiate pathologic cold agglutinins of the C1 molecule to antibody and preventing intravas- from the benign ones (Table 19-7). The DAT with polyspe- cular hemolysis.46,52,53 Plasma exchange can be used in cific AHG and monospecific anticomplement antiserum is acute hemolytic episodes because most IgM is distributed positive in pathologic cold agglutinin disease but negative in the intravascular spaces. However, plasma exchange is Table 19.7 Comparison of Characteristics of Pathologic Cold Agglutinins Found in CAS with Those of Benign Cold Agglutinins Found in Normal Individuals Pathologic Agglutinins Benign Agglutinins Antibody class IgM IgM Antibody specificity Usually anti-I but in secondary CAS can be anti-i anti-I Antibody clonality Monoclonal in idiopathic type and secondary type due to lympho- Polyclonal proliferative disease; polyclonal in secondary type due to infec- tious disease Thermal amplitude 0–30°C 0–4°C Agglutination at room temperature Significant Not present Titer Usually more than 1:1000 Less than 1:64 DAT Positive with polyspecific AHG and monospecific anticomplement Negative 414 Chapter 19 effective for only a short time because |
protein regeneration CLINICAL PRESENTATION half-life is ∼5 days. Splenectomy, which can be effective in Hemoglobinuria is the most common clinical symptom. WAIHA, is not effective in CAS because the C3b-coated cells The patient can also experience jaundice, pallor, and are destroyed primarily by Kupffer cells in the liver. Theo- hepatosplenomegaly. Raynaud’s phenomenon can occur retically, patients with strongly reacting cold agglutinins during acute episodes followed by jaundice. Rare cases may have complications if surgical procedures requiring of acute renal failure have been associated with severe hypothermia are undertaken. There is little evidence this hemolysis.61 is a great risk.51,54,55 Like secondary WAIHA, resolution of LABORATORY EVALUATION secondary CAS usually occurs when the underlying condi- The degree of anemia depends on the frequency and sever- tion has been treated. ity of hemolytic attacks. During the attack, hemoglobin con- centration drops sharply accompanied by hemoglobinemia, Paroxysmal Cold Hemoglobinuria methemalbuminemia, and hemoglobinuria. Hemoglobin Paroxysmal cold hemoglobinuria (PCH) is a rare but acute values can decrease to as low as 5 g/dL. Neutropenia, a and rapidly progressive autoimmune hemolytic disorder neutrophilic shift to the left, reticulocytopenia, and sphe- that is characterized by massive intermittent acute hemoly- rocytes can accompany erythrocyte lysis. Other abnormal sis and hemoglobinuria. Although it is the cause of about 2% erythrocyte morphology including anisocytosis and poi- of autoimmune hemolytic anemias overall, it is the cause of kilocytosis as well as erythrocyte fragments may be seen.61 30–40% of AIHA in children and is most frequent in children Serum bilirubin, blood urea nitrogen, and lactic dehydro- under the age of 5 years. Historically, PCH was associated genase (LD) are elevated, whereas serum complement and with congenital or tertiary syphilis in adults. It is now most haptoglobin are decreased. Erythrophagocytosis occurs often seen in children with viral and bacterial infections, more commonly in PCH than in other types of AIHA, and although it has been rarely reported in adults due to various the phagocytic cells usually involved are segmented neu- clinical presentations.56 The infections linked to PCH include trophils, not monocytes. Epstein-Barr virus, cytomegalovirus, measles, mumps, Hae- Antibodies on the cells are not usually detected by the mophilus influenzae, and Klebsiella pneumoniae. Infections have DAT because the D-L antibody elutes at warm tempera- been linked with Parvovirus 19 as the P antigen serves as a tures. A weakly positive DAT with anticomplement AHG receptor for this virus. Post-vaccination PCH has also been can appear and persist for several days after the hemolytic reported.57,58 PCH is usually a transient disorder that appears episode. The IAT can be positive if performed in the cold. 5 days to 3 weeks after infection onset but can persist up to 3 Normal erythrocytes incubated with patient serum react months. It generally resolves spontaneously, but transfusion more positively in the IAT than will patient cells. can be required in cases of severe, life-threatening anemia. D-L antibodies are usually present in low titers (less Treatment with plasma exchange or immunosuppressive than 1:32) and express a low thermal amplitude, but their drugs may be used in cases of severe hemolysis. presence can be verified by the D-L test, which employs a biphasic reaction (Table 19-8). In this test, the patient’s PATHOPHYSIOLOGY blood is collected in two clot tubes (serum, not plasma, is PCH was the first hemolytic anemia for which a mechanism required); one is incubated at 4°C for 30 minutes and the of hemolysis was established. This hemolytic anemia is dis- other at 37°C for 30 minutes. Both tubes are then incubated tinct from the other cold AIHAs because of the nature of the at 37°C. If the D-L antibody is present, it causes hemolysis in antibody involved. It is caused by a biphasic complement- the tube initially incubated at 4°C and then warmed to 37°C. fixing IgG antibody, the Donath-Landsteiner (D-L) anti- No hemolysis is present in the tube kept at 37°C. Hemoly- body, which has an auto-anti-P specificity. Although IgG sis in this test can also occur in cold agglutinin syndrome is the most common antibody class involved in PCH, there (CAS), but the hemolysis occurs very slowly. See Table 19-9 have been reports of IgM and IgA.58,59,60 In rare cases of IgM for a comparison of PCH and CAS. class antibody, the specificity was biphasic anti-I, while oth- ers were anti-P specificity.58,60 Biphasic refers to the two tem- THERAPY peratures necessary for optimal lysis of the erythrocytes. The PCH associated with acute infections terminates upon antibody reacts with erythrocytes in the capillaries at temper- recovery from the infection. Steroids are not usually atures less than 20°C and avidly binds the early acting com- helpful. Transfusion can be required if the hemolysis is plement components. Upon warming to 37°C, the antibody severe. In rare cases when the hemolysis persists, plas- molecule detaches from the cell, but the membrane attack mapheresis can be used. Rituximab has also been used complement components are activated on the cell membrane as therapy in rare adult cases. The chronic form of the causing cell lysis. Because the antibody repeatedly attaches disease is best treated by avoiding exposure to the cold. and detaches from erythrocytes with subsequent comple- Since hemolysis is primarily intravascular, splenectomy ment activation, it can cause significant hemolysis. is not indicated.59 Hemolytic Anemia: Immune Anemias 415 Both C3 and IgG can be detected on the erythrocyte in Table 19.8 Donath-Landsteiner (D-L) Test for Detecting the Presence of D-L Antibodiesa the DAT. The cold-reacting antibody often has specificity for the Ii system antigens. The warm-reacting autoantibody is Patient’s Whole Blooda Control Test like those found in classic WAIHA and often has a complex Incubate for 30 min at 37°C 4°C Rh specificity. Incubate for 30 min at 37°C 37°C Patients usually respond well to treatment with cortico- Centrifuge: observe plasma steroids and do not require transfusion. Rituximab has been for presence of hemolysis used in cases with underlying lymphoproliferative disease.5 Interpretation D-L antibodies present No hemolysis Hemolysis Drug-Induced Hemolytic Anemias No D-L antibodies present No hemolysis No hemolysis Although the occurrence is rare, certain drugs can cause a Two tubes of patient’s whole blood are used; one tube serves as the control and the immune cytopenias that involve one or more cell lineages other as the test. including neutrophils, platelets, and erythrocytes. Anemia, thrombocytopenia, and agranulocytosis can occur together or separately. It has been proposed that a drug’s ability to Checkpoint 19.10 induce production of antibodies against different cell lin- Compare the antibody specificity and the confirmatory test for eages is related to its affinity for the cells. The greater the PCH and CAS. affinity, the more likely sensitization against the drug–cell complex is to occur. Drug-induced immune hemolytic anemia is a relatively uncommon acquired condition pre- Mixed-Type AIHA cipitated by certain drugs. The drug itself does not cause erythrocyte injury, and not all individuals taking the drug Mixed-type AIHA is characterized by the presence of a develop this immune reaction. warm-reacting IgG autoantibody and a cold-reacting IgM More than 135 drugs have been found to induce a autoantibody that has both high titer and increased ther- positive DAT or an immune-mediated hemolytic anemia.65 mal amplitude (reacts at more than 30°C). It is less common The classes of drugs implicated include antimicrobials, non- in children than adults.62 About 50% of the cases are idio- steroidal anti-inflammatory drugs, antineoplastic drugs, pathic; most of the remainder are associated with diseases diuretics, and antidiabetic drugs. Note that second- and such as systemic lupus erythematosus or lymphoprolifera- third-generation cephalosporins such as cefotetan and tive diseases.63,64 Patients may have symptoms that are con- ceftriaxone constitute the majority of cases of drug- sistent with each type and frequently present with an acute, induced immune hemolytic anemia and are responsible for more severe anemia and can have a more chronic course most fatalities with ceftriaxone causing more than 50% of the with intermittent exacerbations.34,62 Reticulocytopenia has fatalities. Piperacillin is now the most common drug asso- also been noted in several cases of mixed WAIHA and DAT- ciated with DIHA.66 Chemotherapeutic drugs, especially negative hemolysis.34 The IgG antibody mediates extravas- purine analogs such as fludarabine that are used to treat cular hemolysis, and the IgM is responsible for complement patients with leukemia and some types of lymphoma, are fixation and intravascular hemolysis. In some cases, the another class of drugs associated with DIHA. Drug-induced cold-reacting antibody triggers increased hemolysis and immune hemolysis must be distinguished from both drug- hemoglobinuria when the patient is exposed to the cold. induced nonimmune hemolysis that occurs secondarily to Table 19.9 Comparison of Cold Agglutinin Syndrome (CAS) and Paroxysmal Cold Hemoglobinuria (PCH) CAS PCH Patient Usually adults more than 50 years of age Usually children after viral infection Clinical findings Acrocyanosis Chills, fever, hemoglobinuria DAT Positive with polyspecific AHG and monospecific C3 Positive with polyspecific AHG and monospecific C3 Donath-Landsteiner test Negative Positive Antibody class IgM Biphasic IgG (D-L) Antibody specificity Anti-I Anti-P Thermal amplitude of antibody Up to 30°C Under 20°C Hemolysis Chronic extravascular/intravascular Acute intravascular Therapy Avoid the cold Supportive; treatment of underlying illness 416 Chapter 19 erythrocyte metabolic defects such as G6PD deficiency and MECHANISM OF IN VIVO ACTION FOR DRUG-INDE- from spontaneous autoimmune disorders. This is important PENDENT ANTIBODIES because drug-induced, immune hemolytic anemias are the In the drug-independent mechanism, the drug binds to the result of an immune response to drug-induced alteration of erythrocyte; however, the antibody is produced against the erythrocyte. The resolution for this immune hemolysis primarily erythrocyte epitopes. This can be due to altera- involves withdrawal of the offending drug and support- tion of the erythrocyte membrane by the drug or molecu- ive treatment, not the use of immunosuppressives or other lar mimicry. This mechanism mimics warm autoimmune therapy. hemolytic anemia in that the patient’s serum reacts with all Although historically there were four mechanisms used erythrocytes against which it is tested. It is important that to describe the mechanisms of drug-induced hemolysis these be distinguished so that appropriate treatment can be (drug adsorption, immune complex, membrane modifica- initiated.69,70 The mechanism by which antibody produc- tion, and autoantibody-like), with our current understand- tion is induced is unknown; however, evidence indicates ing of the process a “unifying hypothesis” has been that the drug alters normal erythrocyte antigens so they proposed to explain antibody formation in drug-induced are no longer recognized as self. Historically this mecha- hemolytic anemias. The hypothesis proposes that once a nism was linked to the antihypertensive drug Aldomet drug binds to the erythrocyte membrane, antibodies can be (a@methyldopa). In approximately 20% of patients on the produced to react with epitopes specific to the drug, with drug, a positive DAT develops after about 3–6 months. epitopes that represent a combination of drug and erythro- However, hemolytic anemia develops in only 1% of these cyte proteins, or with epitopes primarily on the erythrocyte patients. Fludarabine now is the drug most commonly asso- membrane. This helps to explain how patients can develop ciated with this type of drug-independent mechanism for more than one type of drug-induced antibody. antibody formation, although other drugs have been impli- Currently, two major types of drug-induced antibodies cated when there is an underlying condition.5,65,71 Eryth- have been described: drug dependent and drug indepen- rocyte destruction is extravascular, and anemia develops dent. They are named based on the type of in vitro testing gradually. If the drug is withdrawn, the antibody produc- required for identification. Drug-dependent antibodies are tion gradually stops, but the DAT can remain positive for those that require the presence of the drug during in vitro years. The DAT is dose dependent. The larger the dose testing. Drug-independent antibodies are those that react of the drug, the more likely the patient is to have a posi- in vitro without the presence of the drug. A third category, tive DAT. The DAT using anti-IgG is positive but because nonimmunological protein adsorption (NIPA), causes posi- complement is rarely activated, the DAT using anti-C3 is tive DAT but is rarely linked with hemolysis.66 usually negative. Workups for investigating drug-induced hemolytic In nonimmunological protein adsorption (NIPA) some anemia may be quite extensive. They can require DAT, drugs, b@lactamase inhibitors, cefotetan, and platinum- reaction of patient’s serum with drug-coated cells, and rou- based chemotherapeutic agents such as oxaliplatin can tine antibody investigations. This is important to be able to alter the erythrocyte membrane so that |
immunoglobu- distinguish these reactions from transfusion reactions, true lins and other serum proteins bind nonspecifically to the autoantibody formation, or other clinical conditions.65,67 membrane.66,70 No drug antibody is involved. DAT using polyspecific AHG is positive; the anti-IgG or anti-C3 can MECHANISM OF IN VIVO ACTION FOR be positive or negative. This mechanism is known to cause DRUG-DEPENDENT ANTIBODIES hemolysis in some patients. The coated cells are removed The mechanism of action for the drug-dependent type anti- via macrophage receptors.65,70 bodies can occur in two ways. In one, the drug binds cova- Table 19-10 contains a summary of the characteris- lently to the erythrocyte and antibodies are produced only tics of each mechanism. Regardless of the underlying against the drug epitopes. Antibody forms and attaches mechanism, erythrocytes sensitized with either anti- to the drug-coated erythrocyte, which is then removed body and/or complement have a shortened life span. by splenic macrophages. Drugs often associated with this Each of these mechanisms causes a positive DAT with mechanism are intravenous penicillin, piperacillin, and polyspecific AHG and with either anti-IgG and/or anti- cephotetan. The second mechanism for drug-dependent C3 AHG. antibody occurs when the drug binds only weakly to the erythrocyte and antibody is formed to epitopes that consist of both drug and erythrocyte membrane. The antibody can Checkpoint 19.11 fix complement and cause intravascular lysis. Ceftriaxone is Compare the different types of drug-induced hemolysis includ- one of the drugs commonly associated with this mechanism ing the type of hemolysis, the drug usually associated with the and is linked to severe intravascular hemolysis often within mechanism, and the DAT profile. hours of administration.66,68 Hemolytic Anemia: Immune Anemias 417 Table 19.10 Summary of Classic Mechanisms of Drug-Induced Immune Hemolytic Anemia Type (Prototypic Drug) Action Direct Antiglobulin Test Mechanism of Cell Destruction Drug dependent Drug bound to cell S antibody forms primarily Polyspecific AHG positive, anti-IgG Extravascular adhesion to to drug epitopes and binds to drug positive, anti-C3 can be positive macrophages via FcgR and phagocytosis Drug binds loosely to erythrocyte S antibody Polyspecific AHG positive, anti-IgG Intravascular complement- forms to epitopes of drug and cell S activates negative, anti-C3 positive mediated lysis complement cascade S antibody leaves cell Drug independent Drug adheres to cell membrane S antibody Polyspecific AHG positive, anti-IgG Extravascular adhesion to Autoantibody-like forms primarily against epitopes on erythrocyte positive, anti-C3 positive or negative macrophages via FcgR and membrane S antibody reacts with erythrocyte phagocytosis Nonimmune protein Modification of cell membrane that results in Polyspecific AHG positive, mono- Hemolysis can result adsorption nonimmunologically absorbed IgG, IgA, IgM, C3 specific can be positive or negative autoimmunity, or passenger lymphocyte syndrome. Pas- CASE STUDY (continued from page 407) senger lymphocyte syndrome is an immune hemolytic process Two days later, Nancy’s hemoglobin dropped to that develops often within the first week post-transplant. 50 g/L. The physician ordered several more tests. It may occur following solid organ (heart, lung, kidney), She had a positive IAT, and the antibody reacted bone marrow, or stem cell transplant.75 The donor B lym- with all cells including her own. Other test results phocytes and/or plasma cells transplanted with the organ indicated that this patient had systemic lupus or bone marrow produce antibodies against the recipient’s erythematosus. blood group antigens. Although ABO incompatibility is most frequently implicated, other blood group system anti- 4. What type of antibody appears to be present in bodies (anti-D, anti-K, or other Rh system antibodies) may Nancy? Explain. be involved.75,76 Development of autoantibodies after solid 5. What is the relationship of Nancy’s primary organ transplant is rare but is more common after bone mar- disease, systemic lupus erythematosus, and her row or stem cell transplants.34,35 Hemostatic expansion post- anemia? HSCT may trigger loss of self-tolerance, leading to increases in autoreactive cells.73,77 Post-HSCT infections may also trigger development of antibodies that have the ability to cross react with human erythrocyte antigens, stimulating Alloimmune Hemolytic an autoimmune process.77 Blood transfusions can, in some patients, lead to the development of autoantibodies. In most Anemia cases, the hemolysis from passenger lymphocyte syndrome is usually self-limiting as the donor B lymphocytes gener- Hemolytic anemia induced by an individual’s immuni- ally do not survive.74 zation with erythrocyte antigens on the infused cells of Another type of immune-mediated hemolysis is recog- another individual is known as alloimmune hemolytic nized in patients receiving high-dose intravenous immu- anemia. The patient’s erythrocytes lack the antigen(s) pres- noglobulin (IVIG) therapy (more than 2 g/kg body weight ent on infused cells. These transfused antigens are recog- or 100 g IVIG in 2–4 days). IVIG, made from the plasma nized as foreign and induce the recipient to form antibodies of thousands of donors, is used in treating a variety of that, in turn, react with the transfused cells. This type of autoimmune and immunodeficiency syndromes including immunologic destruction of erythrocytes is characteristic Guillian-Barre syndrome, Kawasaki disease, immune-medi- of transfusion reactions and hemolytic disease of the fetus ated thrombocytopenia, and antibody-mediated transplant and newborn (HDFN). Factors such as the immunogenicity rejection. of the antigen, number of transfusions, and function of the A two-hit theory is proposed to explain the hemolysis recipient’s immune system can influence the development seen with IVIG. The first factor is that the IVIG recipient is a of alloantibodies. non–Group O individual (Group A, Group B, or Group AB). There is evidence of the production not only of alloan- Most cases are seen with individuals with Group A blood. tibodies in patients undergoing solid organ and allogeneic The second factor is the presence of an underlying inflam- bone marrow/stem cell transplants but also of autoanti- matory condition.78,79 The non-immune-stimulated anti-A bodies.72,73,74 Post-transplant immune-mediated hemolysis and anti-B in IVIG may attach to erythrocytes. The under- can be linked to major blood group incompatibility, pas- lying condition activates splenic macrophages to increase sive transfer of antibody from the donor, development of lysis of antibody or complement coated cells. Despite the 418 Chapter 19 relatively common positive DAT and hemolysis caused by complications of the reaction (e.g., shock, renal failure, dis- anti-A or anti-B, there is no evidence that anti-D in IVIG seminated intravascular coagulation [DIC]). Laboratory causes lysis. investigation of the reaction must be performed. Lysis develops in about one-third of cases as early as The delayed hemolytic transfusion reaction is usually 24 hours after treatment. In other cases, it may take as the result of an anamnestic response in which the donor long as 2 weeks post-treatment.79 The lysis is usually erythrocytes contain an antigen to which the patient has self-limiting and primarily extravascular and may result been previously sensitized. In these cases, the antibody was in a slight decrease in hemoglobin, a positive DAT.78,80,81 not detectable prior to transfusion, but the infused eryth- Patients receiving high dose IVIG should have hemoglobin rocytes restimulate antibody production, destroying the levels followed at 36 and 96 hours post-infusion.81 donor cells containing the antigen. In a delayed transfu- sion reaction, some patients show no clinical signs, and the Hemolytic Transfusion Reactions reaction is detected only when laboratory tests demonstrate that the expected increase in hemoglobin has not occurred Transfusion of blood can cause a hemolytic transfusion or when, upon subsequent testing, demonstrate a positive reaction as the result of the interaction of foreign (nonself) DAT or presence of a previously unidentified alloantibody. antigens on transfused erythrocytes and alloantibodies Other patients can present with dark urine or vague symp- in the patient’s plasma. In contrast to AIHA, the antibod- toms such as fever and fatigue post-transfusion, which will ies produced in transfusion reactions cause immunologic trigger an investigation. Patients at highest risk for delayed destruction of donor cells but do not react with the eryth- reactions are those who receive multiple transfusions over rocytes of the person making the antibody. However, many their lifetime (e.g., patients with aplastic anemia, sickle cell of the same factors involved in autoimmune hemolytic ane- anemia). Patients with sickle cell anemia or other hemo- mia (e.g., antibody class/subclass, complement activation, globinopathies may undergo hyperhemolysis in which not antigen density, antibody titer, efficiency of Fc receptors) only the donor cells but also the patient’s own erythrocytes are also involved in this immune response.82 The two types are lysed.82,84 of transfusion reactions involving alloantibodies to eryth- rocyte antigens are acute (occurring within 24 hours) and PATHOPHYSIOLOGY delayed (occurring 2–14 days after transfusion) (Table 19-11). An acute hemolytic transfusion reaction is characterized by An acute hemolytic transfusion reaction results when the intravascular hemolysis with hemoglobinuria because of infused erythrocytes react with antibodies that already exist complete activation of the complement cascade. IgM anti- in the recipient, usually ABO system antibodies, although bodies usually mediate this type of hemolysis of donor cells, other system antibodies have been implicated.83 This type although IgG antibodies are rarely involved. Acute hemo- of reaction usually is caused by clerical or other human lytic transfusion reaction is typical of an ABO incompatibil- error that results in an ABO mismatch between donor and ity and begins very shortly after the infusion of the donor recipient. For example, the patient is given the wrong unit unit has begun. As little as 10 mL of ABO-incompatible of blood or is misidentified by the phlebotomist, nurse, or blood can trigger symptoms of an acute hemolytic laboratory personnel. Most errors occur in patient care areas, reaction.29 As cells are lysed, the release of erythrocyte mem- not in the transfusion service area of the laboratory. Patients brane phospholipids can activate the intrinsic and extrinsic given the wrong blood exhibit classic clinical signs includ- coagulation cascade (Chapter 32). The resulting consump- ing increased pulse rate, hypotension, chills and fever, pain tive coagulopathy (disseminated intravascular coagulation) (chest, flank, or at the infusion site), or difficulty breathing. can damage the kidney by deposition of fibrin in the micro- When an acute transfusion reaction is suspected, the transfu- vasculature (Chapters 32, 34). The intravascular hemolysis sion must be stopped immediately because of the potential may induce a “cytokine storm” with increased production of Table 19.11 Comparison of Acute and Delayed Hemolytic Transfusion Reactions Acute Delayed Timing Immediate (within 24 hours) 2–14 days Underlying cause Usually ABO antibodies Other antibodies: often Kidd system (anamnestic response) Hemolysis Intravascular Extravascular; rare, slower intravascular if antibody capable of fixing complement Symptoms Fever, chills, back pain, hypotension, pain at site of infusion Uncommon (fever, hemoglobinuria) Laboratory findings Hemoglobinemia Positive DAT May see hemoglobinuria Positive DAT (possible) Antibody in eluate Hemolytic Anemia: Immune Anemias 419 pro-inflammatory cytokines such as increased tumor necro- LABORATORY EVALUATION sis factor@a (TNF@a) and other interleukins that can mediate The laboratory findings vary, depending on whether the some of the clinical symptoms.29,82 Mortality rates from ABO transfusion reaction is acute or delayed. Intravascular hemo- acute hemolytic transfusion reactions range from 10–50%. lysis usually accompanies the acute reaction and typically Extravascular hemolysis is typical of a delayed hemo- can be associated with hemoglobinemia, hemoglobinuria, lytic transfusion reaction occurring when erythrocytes methemoglobinemia, and decreased haptoglobin and hemo- are coated with IgG antibodies and removed via macro- pexin. Extravascular hemolysis usually accompanies the phage Fc receptors in the spleen. The speed of the removal delayed reaction. The DAT might or might not be positive depends on the amount of antibody on the cell. Comple- in acute hemolytic transfusion reactions, depending on the ment is not usually involved but when present can enhance extent of erythrocyte destruction. The DAT is usually posi- phagocytosis. tive in a delayed hemolytic transfusion reaction but might Delayed transfusion reactions occur 2–14 days after a not be detected until days after transfusion. A patient who is transfusion. Although the antibody cannot be detected in suspected of experiencing either an acute or delayed trans- pretransfusion testing because its concentration is lower fusion reaction should have a transfusion reaction workup. than the sensitivity level of the test, antigens on infused donor cells induce a secondary (anamnestic) antigenic response. The antibody produced is usually IgG, and Checkpoint 19.13 hemolysis is extravascular. The first indication of a delayed Compare the characteristic laboratory findings in acute hemo- lytic transfusion reactions and in delayed transfusion reactions. reaction is a drop in the hemoglobin concentration several days after the transfusion with no signs of bleeding. Intra- vascular hemolysis can also occur but is less pronounced THERAPY than in acute reactions. Laboratory investigation reveals a The most important immediate action to take when an acute |
positive DAT because of antibody-coated donor cells in the transfusion reaction occurs is to terminate the transfusion. patient’s circulation. Antibodies characteristically associ- A major effort should be made to maintain urine flow to ated with a delayed transfusion reaction are in the Kidd prevent renal damage. Shock and bleeding require immedi- system (anti@Jka, anti@Jkb), although antibodies to other anti- ate attention. In a delayed reaction, treatment is generally gens have also caused delayed reactions. not required, although in potentially severe cases red blood CLINICAL PRESENTATION exchange may be required.86 Future units of blood given to Symptoms of an immediate transfusion reaction begin within the patient, however, must lack the antigen to which the minutes to hours after beginning the transfusion. A variety of patient has made an antibody. Cases of hyperhemolysis nonspecific symptoms, including fever, chills, low back pain, treatment with monoclonal antibodies that affect the sensations of chest compression or burning at the site of infu- immune response (Rituximab or Eculizumab) has shown sion, hypotension, and nausea and vomiting, can occur. Unless variable effectiveness.84,87 the transfusion is immediately stopped, shock can occur. Anuria due to tubular necrosis secondary to inadequate renal blood flow and bleeding due to DIC are common complica- CASE STUDY (continued from page 417) tions. The severity of the reaction and extent of organ damage The clinician wants to start Nancy’s therapy and are directly proportional to the amount of blood infused. give her a transfusion. Although some types of non-erythrocyte-related trans- 6. How would knowing that Nancy had not been fusion reaction (e.g., allergic, febrile, nonhemolytic) are transfused in the last several months help you common in the pediatric population, delayed hemolytic decide on the underlying cause of the antibody? transfusion reactions are uncommon in the pediatric popu- lation compared with the adult population.85 Most delayed 7. What would you tell the clinician about giving transfusion reactions cause few signs or symptoms. The a transfusion? most common signs are malaise and unexplained fever sev- 8. What kind of therapy could be used? eral days after the transfusion. Some patients notice the presence of hemoglobin in the urine. Hemolytic Disease of the Fetus and Checkpoint 19.12 Newborn (HDFN) Compare the underlying mechanisms, pathophysiology, and clinical symptoms of an acute hemolytic transfusion reaction Hemolytic disease of the fetus and newborn (HDFN) is an with a delayed one. alloimmune disease associated with increased erythrocyte destruction during fetal and neonatal life and is caused by 420 Chapter 19 fetomaternal blood group incompatibility. Maternal anti- or transfusion. Normally, the placenta does not allow free bodies are transported across the placenta and react with passage of erythrocytes from fetal to maternal circulation, antigens of paternal origin on the fetal erythrocytes.29 The but small numbers of erythrocytes can enter the maternal three categories of HDFN are: ABO caused by anti-A, anti- circulation during gestation. Additionally, small amounts B, and/or anti-A, B; Rh(D) caused by anti-D; and “other” of fetal blood also can enter the mother’s circulation during caused by alloantibodies to other Rh system antigens (C, delivery. The risk of sensitization increases as the volume of c, E, e) or antibodies to other blood group system antigens the fetal bleed increases. If the fetal-maternal bleed is suf- (e.g., Kell, Kidd, Duffy). More than 95% of HDFN cases are ficient to stimulate the production of maternal antibodies, due to either anti-D or ABO system antibodies. Although subsequent pregnancies could be at risk for HDFN. HDFN caused by ABO antibodies is more common than Although three classes of immunoglobulins can be HDFN caused by anti-D, Rh(D) incompatibility causes more produced during the mother’s immunization—IgG, IgM, severe disease (Table 19-12). The antibodies most commonly IgA—only IgG can cross the placenta and cause HDFN. The associated with the remaining HDFN cases include anti-K, IgG antibody is actively transported across the placenta and anti-c, anti-C, and anti-E, although any IgG antibody can causes destruction of fetal erythrocytes. The fetus/newborn be implicated. HDFN caused by anti-D and anti-K are the has anemia and bilirubinemia of varying degrees of severity most severe. HDFN caused by anti-K is unique in that the based on the strength of the immune response and degree anti-K suppresses erythropoiesis in the fetus and is respon- of hemolysis. sible for the early destruction of erythroid progenitor cells, In ABO-HDFN, the mother already has the naturally leading to a more severe anemia and increased numbers of occurring ABO system antibodies, generally a mixture of IgM intrauterine transfusions.88,89,90 and IgG anti-A, anti-B, and/or anti-A,B. The pre-existing IgG antibodies can cross the placenta to destroy fetal cells, PATHOPHYSIOLOGY potentially affecting all pregnancies including the first. The pathophysiology of HDFN involves initial sensitiza- Approximately 15–20% of the group A or group B babies tion and antibody production, in utero effects, and postnatal born to group O mothers develop serologic evidence of effects. Four conditions must be met for HDFN to occur: ABO-HDFN (positive DAT).3 In HDFN caused by all other • The mother must be exposed (sensitized) to an erythro- antibodies including anti-D, the mother generally lacks cyte antigen that she lacks. preformed antibodies capable of recognizing fetal antigens. • The fetus must possess the antigen to which the mother The firstborn is not usually affected because the first preg- has been sensitized. nancy serves as the sensitizing event. In each subsequent • The mother must produce antibodies to the foreign pregnancy with an antigen-positive fetus, the risk for sever- antigens. ity of HDFN increases as the antibody response increases. • The mother’s antibody must be able to cross the pla- Prenatal Period If destruction of fetal erythrocytes is severe centa and enter the fetal circulation enough in utero, the fetus becomes severely anemic and can develop complications as a result. Extramedullary hemato- Sensitization The mother may have been exposed to for- poiesis occurs in the liver and spleen, causing their enlarge- eign (nonself) erythrocyte antigens by previous pregnancy ment. Because of hemolysis, the unconjugated (indirect) Table 19.12 Comparison of Hemolytic Disease of the Fetus and Newborn Caused by ABO and Rh(D) Feature Rh ABO Other Antibody Immune IgG Nonimmune IgG Immune IgG Blood group Mother Rh negative Mother, group O; newborn, group A or B Mother lacks antigen that is on fetal cell Baby Rh positive Obstetric history Only pregnancies after the first are usually First pregnancy and subsequent preg- Pregnancy can be first if mother previ- affected nancies can be affected ously sensitized by transfusion. If preg- nancy is sensitizing event, usually second and subsequent pregnancies affected Clinical findings Moderate to severe anemia and Mild anemia if present; mild to moderate Mild to severe anemia and bilirubinemia bilirubinemia bilirubinemia with a peak 24–48 hours after birth Laboratory findings DAT positive DAT weakly positive or negative DAT positive No spherocytes Spherocytes present Therapy Exchange transfusion if severe Phototherapy Phototherapy and/or exchange transfu- sion, if severe Hemolytic Anemia: Immune Anemias 421 bilirubin concentration increases. In the fetus, this biliru- the greatest risk thereafter. In Rh(D) incompatibility, the bin crosses the placenta and is conjugated and excreted by cord blood hemoglobin can be low normal at birth (nor- the mother. With procedures such as amniocentesis, the mal hemoglobin at birth is 14–20 g/dL), and the newborn amount of bilirubin in amniotic fluid can be measured to may not appear jaundiced. However, significant hemoly- help determine the relative severity of hemolysis. Cordo- sis occurring in the first 24 hours of life outside the womb centesis in which a sample of the fetal blood is taken via a results in anemia with pallor and jaundice. In severe cases, needle inserted into the umbilical vein allows testing of the hepatosplenomegaly can be present. Severe anemia can be fetal blood for blood type, DAT, hemoglobin, and bilirubin. accompanied by heart failure and edema. As the level of Noninvasive procedures such as Doppler ultrasound can unconjugated bilirubin rises, kernicterus can occur and can help predict anemia based on blood flow in the fetus. In be fatal in severe cases. The risk of hyperbilirubinemia in recent years, methods to obtain fetal cells or cell-free DNA premature infants is even greater because of the inability of from the maternal circulation have been introduced. These the premature liver to excrete the excess bilirubin. methods help in predicting Rh-HDFN and eliminating the ABO incompatibility is not as severe as Rh incompati- need for invasive procedures such as amniocentesis. Pre- bility. The clinical course is usually benign, and hemolysis natal fetal genotyping diagnostic procedures have been is minimal. Within 24–48 hours after birth, the infant developed. Although they were initially designed to detect appears jaundiced, but kernicterus is extremely rare. Ane- the D antigen, they have been expanded to detect other Rh mia is mild and pallor is uncommon. Hepatosplenomegaly, system antigens (C, c, E, e) and K, Fy, and JK genotypes as if present, is mild. well and to predict paternal zygosity.91,92 HDFN is signifi- cantly more severe when women have multiple antibodies that include anti-D than those with anti-D alone or when Checkpoint 19.14 specificities of the antibodies do not include anti-D.93,94 This Compare the pathophysiology and clinical findings of ABO- may be because more antigen sites are coated with antibody, HDFN and Rh-HDFN. leading to a more aggressive immune response. The most serious complication of HDFN is cardiac failure and hydrops fetalis, which occurs when the fetus is LABORATORY EVALUATION unable to produce sufficient erythrocytes. Hydrops fetalis Laboratory tests are essential to identify the etiology is characterized by edema and accumulation of fluid in the of HDFN, determine prognosis, and select appropriate peritoneal, pericardial, or pleural cavities. treatment. Classic prenatal testing on a pregnant woman Erythroblastosis fetalis, a term also used to describe includes ABO and Rh typing of the erythrocytes as well as characteristics of HDFN, reflects the presence of large an antibody screen (IAT) on her serum. If the IAT is positive, numbers of nucleated erythrocytes found in the newborn’s the antibody is identified so that an assessment of HDFN peripheral blood in very severe cases. risk can be performed. The postnatal HDFN workup will Postnatal Period Erythrocyte destruction persists after involve laboratory tests on both the mother and the infant. birth because of maternal antibodies in the newborn’s Rh Incompatibility The DAT in Rh incompatibility is circulation. After birth, the newborn must conjugate and usually positive, reflecting antibody coating of the new- excrete the bilirubin on its own. In the neonate, albumin born’s erythrocytes. About 50% of affected infants have a levels for bilirubin transport are limited, and liver glucuro- cord blood hemoglobin concentration of less than 14 g/dL. nyl transferase for bilirubin conjugation is low; therefore, Because the capillary blood hemoglobin can be up to 4 g/dL considerable amounts of toxic unconjugated bilirubin can higher due to placental transfer of blood at birth, the cord accumulate in the newborn after delivery. In the unconju- blood hemoglobin concentration is most useful as an indica- gated state, bilirubin is toxic because it is lipid soluble and tor of anemia at birth and a baseline to follow destruction of can easily cross cell membranes. This form of bilirubin has erythrocytes after birth. A direct relationship exists between a high affinity for basal ganglia of the central nervous sys- the initial cord blood hemoglobin level and the severity of tem. Thus, the excess unconjugated bilirubin can lead to the disease. Lower cord hemoglobin levels at birth are asso- kernicterus, an irreversible form of brain damage. The con- ciated with a more severe clinical course. After birth, hemo- jugated form of bilirubin cannot cause this problem because globin levels can fall at the rate of 3 g/dL/day. The lowest it is water soluble and lipid insoluble and cannot cross cell hemoglobin values are present at 3–4 days. The erythro- membranes. cytes are macrocytic and normochromic. Reticulocytes are CLINICAL PRESENTATION markedly increased, sometimes reaching 60%. Nucleated Anemia resulting from increased cell destruction is the red cells are markedly increased in the peripheral blood greatest risk to both the fetus in utero and to the newborn (10@100 * 103/mcL) reflecting the rapid production of cells with HDFN in the first 24 hours of life. Bilirubinemia is in response to erythrocyte destruction. Normal infants also 422 Chapter 19 have nucleated red cells in the peripheral blood, but their successful delivery. In severe cases of HDFN the mother values are |
much lower (0.2@2.0 * 103/mcL). may also undergo routine plasmapheresis to remove circu- A blood smear shows marked polychromasia, mild or lating antibody until the fetus can be delivered. IVIG may absent poikilocytosis, and few, if any, spherocytes. In some be used in conjunction with plasmapheresis to suppress an cases, the leukocyte count is increased to 30 * 103/mcL immune response.95,96 or more due to a rise in neutrophils reflecting the marrow In mild cases postnatally, the newborn is treated with response to stress (the normal leukocyte count at birth is phototherapy, which slowly lowers the toxic bilirubin 15920 * 103/mcL). However, recent studies have shown level. Although toxic levels of bilirubin are 19–20 mg/dL, that a neutropenia is often present, regardless of the type exchange transfusion is usually performed before that level of HDFN.4 A significant neutrophilic shift to the left often is reached. Exchange transfusions can also be indicated if occurs. The platelet count is usually normal, but thrombo- the bilirubin is rising more than 1 mg/dL/hour or if there cytopenia can develop in severe cases. is significant anemia. The transfusion has several beneficial Cord blood bilirubin is elevated in Rh-HDFN but is effects: usually less than 5.5 mg/dL. However, the elevated bili- • Removes plasma containing maternal antibodies and rubin does not accurately reflect the severity of hemolysis dilutes the concentration of remaining antibodies because bilirubin produced before birth readily crosses the placenta and is metabolized by the mother. The newborn’s • Removes some of the antibody-coated erythrocytes serum bilirubin peaks on the third or fourth day and can • Lowers the level of bilirubin reach 40–50 mg/dL if not treated. Most bilirubin is in the • Treats the anemia toxic unconjugated form. Full-term infants with bilirubin concentrations more than 10 mg/dL are at increased risk RH IMMUNE GLOBULIN (RHIG) for kernicterus, and premature infants can develop it with The passive injection of Rh immunoglobulin (RhIG) levels as low as 8–10 mg/dL. that contains anti-D prevents maternal immunization. ABO Incompatibility In the case of ABO incompatibility, a About 7–8% of Rh-negative women develop antibodies weakly positive DAT is found in the cord blood, but it often to Rh-positive cells after the birth of an Rh-positive ABO- becomes negative within 12 hours. The weak reaction is due compatible infant. The routine use of prophylactic RhIG in to the small number of anti-A or anti-B antibodies attached Rh-negative women during gestation (at 28 weeks) and fol- to the erythrocyte. Bilirubin is not usually significantly ele- lowing the birth of an Rh-positive child has decreased the vated, but the bilirubin level 6 hours after birth can be used incidence of HDFN considerably, although other changes to predict development of hyperbilirubinemia in severe in prenatal care and number of pregnancies also may have cases.5 The peripheral blood smear in severe cases can show affected the incidence. Most women (92%) who develop increased numbers of nucleated erythrocytes and the pres- anti-D during pregnancy do so at 28 weeks or later. There- ence of schistocytes, spherocytes, and polychromasia. fore, antepartum administration of RhIG is given between weeks 28 and 30 of gestation to any Rh-negative woman who has not developed anti-D. The RhIG acts as an immu- Checkpoint 19.15 nosuppressant, depressing the production of immune IgG. Compare the laboratory findings including the peripheral blood However, this prenatal anti-D binds to fetal cells and can smear and the DAT for newborns with ABO-HDFN and those result in a positive DAT and must be evaluated for clinical with Rh-HDFN. and serological significance. Postnatally, a dose of RhIG given within 72 hours of birth protects against the consequences of a fetal-maternal THERAPY bleed. Rh-positive erythrocytes of a fetus that enter the The major efforts of therapy are to prevent hyperbilirubi- mother’s circulation at birth can stimulate the mother’s nemia and anemia. If the destruction of erythrocytes and immune system to make antibodies. As in the antepar- degree of anemia appear to affect the viability of the fetus, tum administration of RhIG, postnatal RhIG can bind fetal an intrauterine transfusion can be given. Non-invasive cells and mediate their removal in the spleen. The dose methods such as Doppler velocity have, in many cases, of RhIG should be determined based on the number of replaced amniocentesis and cordocentesis to determine fetal cells in the maternal circulation. Several tests can be the degree of anemia. These techniques, in conjunction used to estimate the volume of fetal maternal hemorrhage with molecular methods that identify fetal DNA for detect- (FMH), including the qualitative rosette and the quantita- ing in utero hemolysis have allowed decisions to be made tive K leihauer-Betke tests, and, more recently, immuno- on whether an intrauterine transfusion should be given. fluorescent flow cytometry using a monoclonal antibody Intrauterine transfusions often must be given on a routine against fetal hemoglobin to determine the number of fetal basis until the fetus reaches a gestational age that allows cells present. Hemolytic Anemia: Immune Anemias 423 Summary Immune hemolytic anemia (IHA) is mediated by antibodies appears to cause a change in the erythrocyte membrane that and/or complement and can be classified as autoimmune, causes the body to recognize it as foreign and produce an alloimmune, or drug induced, depending on the underly- antibody against the cell. ing process. Patients with IHA have a positive DAT due Alloimmune hemolytic anemia has two presentations: to immunoglobulin and/or complement on the cell. The transfusion reaction and hemolytic disease of the newborn. autoimmune hemolytic anemias (AIHA) occur when the In both conditions, infusion of erythrocytes containing for- offending antibody reacts with an antigen on the patient’s eign (nonself) antigens stimulates the antibody. This sensiti- erythrocytes (self-antigen). These antibodies can also react zation causes the production of alloantibodies to the foreign with erythrocytes of most other persons. AIHA are either antigens, resulting in the cell’s destruction. The antibody idiopathic or secondary to an underlying disease and are can be detected in the serum and cause a positive DAT. further classified as warm or cold, depending on the ther- Transfusion reactions can be acute (immediate) or mal reactivity of the causative antibody. WAIHA is most delayed (2–14 days after transfusion). Acute reactions most often caused by IgG antibodies that react at body tempera- often involve an ABO mismatch between donor and recipient ture (37°C) and cause extravascular hemolysis. Cold hem- and result in intravascular hemolysis because of complement agglutinins are IgM antibodies that generally react at less activation. Delayed transfusion reactions are due to non-ABO than 20°C but are efficient complement activators and can antibodies. These reactions result in extravascular lysis and cause intravascular hemolysis. In both cases, the DAT is are characterized by a positive DAT. The causative antibody positive, and the IAT procedure can detect the antibody. An must be identified so that future transfused erythrocytes lack IgG biphasic antibody that binds complement in the cool the antigen corresponding to the patient’s antibody. peripheral circulation mediates PCH, another type of cold ABO, Rh(D) or other blood group system antibod- autoimmune hemolytic condition. As the coated cells reach ies that react with fetal erythrocytes can cause hemolytic warmer portions of the circulation, the complement cascade disease of the fetus and newborn (HDFN). ABO-HDFN is is activated and intravascular hemolysis occurs. generally milder than Rh-HDFN and can occur in any preg- In drug-induced immune hemolytic anemia, the drug nancy because prior antigenic sensitization is not required. binds to the erythrocyte membrane and induces antibody Rh and other types of HDFN require prior sensitization. formation, which occurs by several mechanisms, depending Maternal antibodies that coat fetal cells can cause anemia on the causative drug. The antibodies can be drug depen- and/or elevated bilirubin in the newborn. Maternal and dent, and the drug must be present for the antibody to react. newborn ABO and Rh group, maternal antibody, and new- In autoantibody formation (drug independent), the drug born DAT are used to evaluate HDFN. Review Questions Level I 3. Which of the following parameters on an automated hematology instrument could be seen in cases of cold 1. The characteristic erythrocyte seen in a peripheral agglutinin syndrome (CAS)? (Objective 3) blood smear in WAIHA is a(n): (Objective 3) a. Falsely elevated MCV a. macrocyte b. Falsely elevated RBC count b. spherocyte c. Falsely decreased MCHC c. dacrocyte d. Falsely decreased hemoglobin d. elliptocyte 4. One purpose of the DAT is to: (Objective 2) 2. Which of the following might be observed on a peripheral blood smear in cases of cold autoimmune a. detect erythrocytes coated with immunoglobulin hemolytic anemia? (Objective 3) in vivo a. Helmet cells b. detect antibodies in the serum b. Macrocytes c. neutralize serum complement c. Agglutination d. prevent agglutination by IgM antibodies d. Spherocytes 424 Chapter 19 5. Intravascular hemolysis is characteristic of which of c. penicillin the following alloantibody situations? (Objective 5) d. rituximab a. Delayed hemolytic transfusion reaction 2. Because a newborn shows evidence of jaundice, a b. ABO-HDFN workup for HDFN is started. The newborn has a c. Rh-HDFN weakly positive DAT with anti-IgG. The mother is d. Acute hemolytic transfusion reaction group O, Rh negative, and the newborn is group A, Rh negative. The blood smear shows evidence 6. A patient with WAIHA would most likely have serum of spherocytes. What is the most likely cause? that reacts in which of these patterns? (Objective 5) (Objective 6) Serum + Own Serum + Erythrocytes a. Rh-HDFN Erythrocytes of Others b. ABO-HDFN a. Negative Negative c. Combined ABO- and Rh-HDFN b. Negative Positive d. Cold agglutinins c. Positive Positive 3. Which autoimmune syndrome is characterized by the d. Positive Negative presence of a biphasic complement-fixing IgG antibody? (Objective 4) 7. Which of the following is not considered to be a con- dition caused by autoantibodies? (Objective 5) a. PCH b. WAIHA a. PCH c. Cold agglutinin syndrome b. CAS d. Immune complex drug induced c. Delayed transfusion reaction d. Drug-induced hemolytic anemia 4. Intravenous administration of what drug is char- acteristically associated with covalent attachment 8. The Donath-Landsteiner antibody is found in which of the drug to the erythrocyte membrane and type of hemolytic anemia? (Objective 1) drug-dependent antibody formation? (Objective 3) a. Warm autoimmune hemolytic anemia a. Penicillin b. Paroxysmal cold hemoglobinuria b. Aldomet c. Cold autoimmune hemolytic anemia c. Quinidine d. Drug-induced hemolytic anemia d. Third-generation cephalosporins 9. Which of the following is not part of the typical 5. Based on maternal and fetal testing, a newborn is sus- peripheral blood picture in Rh-HDFN? (Objective 4) pected of having Rh-HDFN. Which of the following a. Macrocytes peripheral blood smear morphologies might be seen? (Objective 6) b. Polychromasia c. Spherocytes a. Microcytic, hypochromic erythrocytes, decreased reticulocytes d. Increased nucleated erythrocytes b. Microcytic, normochromic erythrocytes, increased 10. The antibody found in PCH is: (Objective 1) nucleated red blood cells c. Macrocytic, hypochromic erythrocytes, decreased a. IgM reticulocytes b. IgG d. Macrocytic, normochromic erythrocytes, increased c. directed against the Rh antigens nucleated red blood cells d. directed against the ABO antigens 6. What is the most likely mechanism of hemolysis in Level II WAIHA? (Objective 1) 1. The drug(s) most often associated with drug-induced a. Increased sensitivity to complement hemolytic anemia is/are: (Objective 3) b. Fixing of complement by IgM antibody a. aldomet c. Biphasic reactions by IgG antibodies b. second- and third-generation cephalosporins d. Phagocytosis of IgG coated erythrocytes Hemolytic Anemia: Immune Anemias 425 7. A patient who received a transfusion 6 days ago is Spherocytes are present. The physician suspects suspected of having a delayed transfusion reaction. hemolytic anemia, and laboratory tests suggest a Which of the following would not be a characteristic hemolytic process with an increase in reticulocytes finding? (Objective 7) and bilirubin. Based on this information, what is the most likely problem? (Objective 8) a. Positive DAT b. Positive IAT a. Secondary CAD c. Hemoglobinuria b. Secondary PCH d. Decreased hemoglobin c. Secondary WAIHA d. Secondary cold agglutinins 8. How would you interpret the following results of a Donath-Landsteiner test? (Objective 4) 10. The following DAT results were seen in a 35-year- old female who was being investigated for a drug- Patient incubated at 4°C and then 37°C Hemolysis induced hemolytic anemia. She had a hemoglobin of Control incubated only at 37°C No hemolysis 70 g/L, and her serum reacted with all cells against a. Positive which it was |
tested. What drug is the most likely cause? (Objective 3) b. Negative c. Invalid because of control reaction DAT (polyspecific) Positive d. Equivocal—repeat in 2 weeks DAT anti-IgG Positive DAT anti-C3 Negative 9. A 75-year-old man presents to the physician with a. Penicillin complaints of weakness and fatigue. His leukocyte b. Quinidine count is elevated, and his hemoglobin is 60 g/L. The peripheral blood smear shows that most the cells are c. Cephalosporin small, mature lymphocytes. The diagnosis is CLL. d. Fludarabine References 1. Meulenbroek, E. M., Wouters, D., & Zeerleder, S. S. (2015). Lyse in autoimmune hemolytic anemia is a footprint for difficult-to- or not to lyse: Clinical significance of red blood cell autoantibod- detect IgM autoantibodies. Hematologica, 100, 1407–1414. ies. Blood Reviews, 29, 369–376. doi: 10.3324/haematol.2015.128991 2. Barcellini, W. (2015). Pitfalls in the diagnosis of autoimmune 11. Branstetter, C. N., Hankins, J. S., Moreau, D., & Nottage, K. A. haemolytic anaemia. Blood Transfusion, 13, 3–5. (2015). Severe autoimmune hemolytic anemia in an infant caused 3. Zantek, N. D., Koepsell, S. A., Tharp, D. R., & Cohn, C. S. (2012). by warm-reactive IgM and IgA autoantibodies: A case report and The direct antiglobulin test: A critical step in the evaluation of review of the literature. Journal Pediatric Hematology and Oncology, hemolysis. American Journal of Hematology, 87, 707–709. 37, 468–471. 4. Kamesaki, R., Toyotsuji, T., & Kajii, E. (2013). Characterization of 12. Losos, M., Hamad, D., Joshi, S., Scrape, S., & Chen, J. (2016). direct antiglobulin test-negative autoimmune hemolytic anemia: Warm autoimmune hemolytic anemia and direct antiglobulin A study of 154 cases. American Journal of Hematology, 88, 93–96. testing with a false-negative result in a 53-year-old man: The 5. Salama, A., & Mayer, B. (2014). Diagnostic pitfalls of drug- DAT will set you free. Lab Medicine, 47, 227–32. doi: 10.1093/ induced immune hemolytic anemia. Immunohematology, 30, 80–84. labmed/lmw018 6. Fetzko, S., Ahmed, A., & Cooing, L. (2015). Finding the elusive 13. Bloch, E. M., Sakac, D., Branch, H. A., Cserti-Gazdewich, C., and causative autoantibody: An atypical case of autoimmune Pendergast, J., Pavenski, K., . . . Branch, D. R. (2015). Western hemolytic anemia. Clinical Case Reports, 3(4), 227–230. immunoblotting as a new tool for investigating direct antiglobu- doi: 10.1002/ccr3.203 lin test-negative autoimmune hemolytic anemia. Transfusion, 55, 7. Bajpayee, A., Dubey, A., Verman. A., & Chaudhary, R. K. (2014). 1529–1537. A report of a rare case of autoimmune haemolytic anaemia in a 14. Fayek, M. H., Saad, A. A., Eissa, D. G., Tawfik, L. M., & Kamal, patient with Hodgkin’s disease in whom routine serology was G. (2012). Role of gel test and flow cytometry in diagnosis of negative. Blood Transfusion, 12(Suppl.), s299–s301. Coombs’ negative autoimmune haemolytic anaemia. International 8. Park, B. S., Park, S., Jin, K., Kim, Y., Park, K. M., Lee, J. Journal of Laboratory Hematology, 34, 311–319. N., . . . Kim, Y. W. (2014). Coombs-negative autoimmune hemo- 15. Alzate, M. A., Manrique, L. G., Bolanos, N. I., Duarte, M., lytic anemia in Crohn’s disease. American Journal of Case Reports, Coral-Alvarado, P., & Gonzalez, J. M. (2014). Simultaneous 15, 550–553. doi: 10.126959/AJCR.892136 detection of IgG, IgM, IgA complexes and C3d attached to 9. Segel, G. B., & Lichtman, M. A. (2014). Direct antiglobulin erythrocytes by flow cytometry. International Journal of Laboratory (“Coombs”) test-negative autoimmune hemolytic anemia: A Hematology, 37, 382–389. review. Blood Cells, Molecules and Diseases, 52, 152–160. 16. Bartolmas, T., & Salama, A. (2010) A dual antiglobulin test for the 10. Meulenbroek, E. M., deHaas, H., Brouwer, C., Folman, detection of weak or nonagglutinating immunoglobulin warm Zeerleder, S. S., & Wouters, D. (2015). Complement deposition autoantibodies. Transfusion, 50, 1131–1134. 426 Chapter 19 17. Thedswad, A., Taka, O., & Wanachiwanawin, W. (2016). Signifi- 37. Yurek, S. Mayer, B., Almahallawi, M., Pruss, A., & Salama, A. cances of red cell bound immunoglobulin G as detected by flow (2015). Precautions surrounding blood transfusion in autoim- cytometry in patients with Coombs-negative immune hemolysis. mune haemolytic anaemias are overestimated. Blood Transfusion, Transfusion Medicine, 26, 130–137. doi: 10.1111/tme.12282 13, 616–621. doi: 10.2450/2015.0326.14 18. Barcellini, W. (2015). Immune hemolysis: Diagnosis and treat- 38. El Kenz, H., Efira, A., Le, P. Q., Thiry, C., Valsamis, J., ment recommendations. Seminars in Hematology, 52, 304–312. Azerad, M. A., . . . Corazza, F. (2014). Transfusion support of 19. Palla, A. R., Khimani, F., & Craig, M. D. (2013). Warm autoim- autoimmune hemolytic anemia: How could the blood group mune hemolytic anemia with a direct antiglobulin test positive genotyping help? Translational Research, 163, 36–42. for C3 and negative for IgG: A case study and analytical literature 39. Castilho, L. (2016). Solving cases in autoimmune hemolytic ane- review of incidence and severity. Clinical Medicine Insights, 6, mia. ISBT Science Series, 12, 25–31. 57–60. doi: 10.4137/CCRep.S11469 40. Salama, A. (2015). Treatment options for primary autoimmune 20. McGann, P. T., McDade, J., Mortier, N. A., Combs, M. R., & hemolytic anemia: A short comprehensive review. Transfusion Ware, R. E. (2010). IgA-mediated autoimmune hemolytic anemia Medicine Hemotherapy, 42, 294–301. in an infant. Pediatric Blood Cancer, 56, 837–839. 41. Guidice, V., Rosamilio, R., Ferrara, I., Seneca, E., Serio, B., & 21. Bicakci, Z., Ozturkmen, S., Akyay, A., & Olcay, L. (2012). False Sellari, C. (2016). Efficacy and safety of splenectomy in adult positive result of the direct antiglobulin test (DAT): The role of autoimmune hemolytic anemia. Open Medicine, 11, 374–380. the elevated level of Immunoglobulin G. Pediatric Hematology and doi: 10.1515/med-2016-0068 Oncology, 29, 611–619. 42. Roumier, M., Loustau, V., Guillaid, C., Langille, L., Mahevas, M., 22. Lai, M., DeStefano, V., & Landoli, R. (2014). Haemoglobin levels Khellar, M., . . . Michel, M. (2014). Characteristics and outcome in autoimmune haemolytic anaemias at diagnosis: Relationship of warm autoimmune hemolytic anemia in adults: New insights with immunoprotein on red blood cells. Immunology Research, 60, based on a single-center experience with 60 patients. American 127–131. Journal of Hematology, 89, e150–e155. doi: 10.1002/ajh.23767 23. Ahmed, E., Elgohary, T., & Ibrahim, H. (2011). Naturally occurring 43. Newman, K., Owlia, M. B., El-Hemaidi, I., & Akhtri, M. (2013). regulatory T cells and interleukins 10 and 12 in the pathogenesis Management of immune cytopenias in patients with systemic of idiopathic warm autoimmune hemolytic anemia. Journal of lupus erythematosus—old and new. Autoimmunity Reviews, 12, Investigational Allergology & Clinical Immunology, 21, 297–304. 784–791. 24. Arce-Sillas, A., Alvarez-Luquin, D. D., Tamaya-Dominguez, B., 44. Salama, A., Hartnack, D., Lindemann, H. W., Lange, H. J., Gomez-Fuentes, S., Trejo-Garcia, A., Melo-Sals, M, . . . Adalid- Rummel, M., & Loew, A. (2014). The effect of erythropoiesis- Peralta, L. (2016). Regulatory T cells: Molecular actions on effec- stimulating agents in patients with therapy-refractory autoim- tor cells in immune regulation. Journal of Immunology Research, mune hemolytic anemia. Transfusion Medicine and Hemotherapy, 2016, 1–12. doi: http://dx.doi.org/0.1155/2016/1720827 41, 462–468. 25. Barcellini, W. (2015). New insights in the pathogenesis of autoim- 45. Swiecicki, P. L., Hegerova, L. T., & Gertz, M. A. (2013). Cold mune hemolytic anemia. Transfusion Medicine and Hemotherapy, agglutinin disease. Blood, 122, 1114–1121. 42, 287–293. 46. Shi, J., Rose, E. L., Singh, A., Hussain, S., Stagliano, N. E., 26. Han, L., Yang, J., Wang, X., Li, D., Lv, L., & Li, B. (2015). Th17 cells Perry, G. C., . . . Panicker, S. (2014). TNT003, an inhibitor of the in autoimmune disease. Frontiers in Medicine, 9, 10–19. serine protease C1s, prevents complement activation induced by 27. Xu, L., Zhang, T., Liu, Z., Li, Q., Xu, Z., & Ren, T. (2012). Critical cold agglutinins. Blood, 123, 4015–4022. role of Th17 cells in development of autoimmune hemolytic ane- 47. Berentsen, S., & Tjonnfjord, G. E. (2012). Diagnosis and treatment mia. Experimental Hematology, 40, 994–1004. of cold agglutinin mediated autoimmune hemolytic anemia. 28. Waite, J. C, & Skokos, D. (2012). The Th17 response and inflam- Blood Reviews, 26, 107–115. matory autoimmune disease. International Journal of Inflammation. 48. Randen, U., Troen, G., Tierens, A., Steen, C., Warsame, A., doi: 10.1155/2012/819467. Belske, K., . . . Delale, J. (2014). Primary cold agglutinin-associ- 29. Fung, M. K., Grossman, B. J., Hillyer, C. D., & Westoff, C. M., eds. ated lymphoproliferative disease: A B-cell lymphoma of the bone (2014). Technical manual (18th ed.). Bethesda, MD: AABB. marrow distinct from lymphoplasmacytic lymphoma. Haemato- 30. Sankaran, J., Rodriguez, V., Jacob, E. K, Kreuter J. D., & Go, R. S. logica, 99, 497–504. doi: 10.3324/haematol.2013.091702 (2016). Autoimmune hemolytic anemia in children: Mayo clinical 49. Berentsen, S. (2015). Role of complement in autoimmune hemo- experience. Journal Pediatric Hematology Oncology, 38, e120–e124. lytic anemia. Transfusion Medicine and Hemotherapy, 42, 303–310. 31. Vagace, J. M., Bajo, R., & Gervasini, G. (2014). Diagnostic and 50. Berentsen, S., Randen, U., & Tjonnfjord, G. E. (2015). Cold therapeutic challenges of primary autoimmune haemolytic agglutinin-mediated autoimmune hemolytic anemia. Hematology anaemia in children. Archives of Diseases in Childhood, 99, 668–673. Oncology Clinics of North America, 29, 455–471. doi:10.1136/archdischild-2013-305748 51. Wouters, D., & Zeerleder, S. (2015). Complement inhibitors to 32. Domiciano, D. S. & Shinjo, S. K. (2010). Autoimmune hemolytic treat IgM-mediated autoimmune hemolysis. Haematologica, 100, anemia in systemic lupus erythematosus: Association with 1388–1395. thrombocytopenia. Clinical Rheumatology, 29, 1427–1431. 52. Tesfaye, A., & Broome, C. (2016). A novel approach for treatment 33. Barcellini, W., & Fattizzo, B. (2015). Clinical applications of of cold agglutinin syndrome-related severe hemolysis. Journal of hemolytic markers in the differential diagnosis and manage- Hematology, 5, 30-33. ment of hemolytic anemias. Disease Markers. doi: http://dx.doi. 53. Berentsen, S. S. (2014). Complement, Cold agglutinins, and ther- org/10.1155/2015635670 apy. Blood, 123, 4010–4012. 34. Barcellini, W., Fattizzo, B., Zaninoni, A., Radice, T., Nichele, I., 54. Jain, M. D., Cabrerizo-Sanchez, R., Karkouti, K., Yau, T., DiBona, E., . . . Zanella, A. (2014). Clinical heterogeneity and pre- Pendergast, J. M., & Cserti-Gazdewich, C. M. (2013). Seek and dictors of outcome in primary autoimmune hemolytic anemia: A you shall find - but then what do you do?: Cold agglutinins in GIMEMA study of 308 patients. Blood, 124, 2930–2936. cardiopulmonary bypass and a single-center experience with 35. Zanella, A., & Barcellini, W. (2014). Treatment of autoimmune cold agglutinin screening before cardiac surgery. Transfusion hemolytic anemias. Haematologica, 99, 1547–1554. Medicine Reviews, 27, 65–73. 36. Park, S. H., Choe, W. H., & Kwon, S. W. (2015). Red blood cell 55. Barbara, D. W., Mauermann, W. J., Neal, J. R., Abel, M. D., Schaff, transfusion in patients with autoantibodies: Is it effective and safe H. V., & Winters, J. L. (2013). Cold agglutinins in patients under- without increasing hemolysis risk? Annals of Laboratory Medicine, going cardiac surgery requiring cardiopulmonary bypass. The 35, 436–444. doi: http://dx.doi.org/10.3343/alm.2015.35.4.436 Journal of Thoracic and Cardiovascular Surgery, 146, 668–680. Hemolytic Anemia: Immune Anemias 427 56. Zeller, M. P., Arnold, D. M., Al Habsi, K., Cserti-Gazdewich, C., Aguera-Morales, M. L, . . . Aljama-Garcia, P. (2015). Passenger Delage, G., Lebrun, A, . . . . Heddle, N. M. (2016). Paroxysmal lymphocyte syndrome after simultaneous pancreas-kidney cold hemoglobinuria: A difficult diagnosis in adult patients. transplantation: A case report of an unusual cause of alloimmune Transfusion, 57, 137–143. hemolytic anemia. Transplantation Proceedings, 47, 2667–2668. 57. Shanbhag, S., & Spivak, J. (2015). Paroxysmal cold hemoglobin- 76. Karanth, P., Birchall, J., Day, S., Unsworth, D. J., & Avanan, R. uria. Heamtology Oncology Clinics of North America, 29, 473–478. (2014). Immune hemolysis resulting from passenger lymphocyte 58. Hayashi, H., Yasutomi, M., Hayashi, T., Yuasa, M., Kawakita, A., syndrome derived anti-Rh (D) reactivity after kidney transplanta- Hata, I., . . . Yusei, O. (2013). Paroxysmal cold hemoglobinuria tion: A case report and literature review. Transplantation, 97(9), caused by IgM-class Donath-Landsteiner antibody. Pediatrics e54–e55. doi: 10.1097/TP00000000000000100 International, 55(5), 664–666. 77. Holbro, A., Abinum, M., & Daikeler, T. (2012). Management of 59. Whipple, N. S., Moreau, D. A. B., Moulds, J. M., Hankins, J. S., autoimmune diseases after haematopoietic stem cell transplanta- Wang, W. C., & Notage, K. A. (2015). Paroxysmal cold hemoglo- tion. British Journal of Haematology, 157, 281–290. binuria due to an IgA Donath-Landsteiner antibody. Pediatric 78. Pendergast, J., Willie-Ramharack, K., Sampson, L., LAroche, V., Blood Cancer, 62, 2044–2046. & Tranch, D. R. (2105). The role of inflammation in intravenous 60. Karafin, M. S., Shirey, R. S., Ness, P. M., King, K. E., & Keefer, J. immune globulin-mediated hemolysis. Transfusion, 55, S65–S73. (2012). A case study of a child with chronic hemolytic anemia 79. Berg, R., Shebl, A., Kimber, M. C., Abraham, M., & Schreiber, G. due to a Donath-Landsteiner positive, IgM anti-I autoantibody. |
B. (2015). Hemolytic events associated with intravenous immune Pediatric Blood Cancer, 59, 953–955. globulin therapy: A qualitative analysis of 263 cases reported 61. Slemp, S. N., Davisson S. M., Layten, J., Cipkala, D. A. & to four manufacturers between 2003 and 2012. Transfusion, 55, Waxman, D. A. (2014). Two case studies and a review of paroxys- S36–S46. mal cold hemoglobinuria. Lab Medicine, 46(3), 253–258. 80. Padmore, R. (2015). Possible mechanisms for intravenous 62. Bass, G. F., Tuscano, E. T., & Tuscanom, J. M. (2014). Diagnosis immunoglobulin-associate hemolysis: Clues obtained from and classification of autoimmune hemolytic anemia. Autoimmu- review of clinical case reports. Transfusion, 55, S59–S64. nity Reviews, 13, 560–564. 81. Scott, D. E., & Epstein, J. S. (2015). Safeguarding immune globulin 63. Hirano, Y., Itonaga, T., Yasudo, H., Isojima, T., Miura, K., recipients against hemolysis: What do we know and where do we Harita, Y., . . . Oka, A. (2016). Systemic lupus erythematosus pre- go? Transfusion, 55, S122–S126. senting with mixed-type fulminant autoimmune hemolytic 82. Zimring, J. C., & Spitalnik, S. L. (2015). Pathobiology of transfu- anemia. Pediatrics International, 527–530. doi: 10.111/ped.12849 sion reactions. Annual Review of Pathologic Mechanisms of Disease, 64. Ogura, M. M. (2014). A mixed-type autoimmune hemolytic 10, 83–110. anemia with immune thrombocytopenia related with myositis 83. Irani, M. S., Figueroa, D., & Savage, G. (2015) Acute hemolytic and post-transplantation lymphoproliferative disorder. Annals of transfusion reaction due to anti-Leb. Transfusion, 55, 2486–2488. Hematology, 93, 869–871. 84. Boonyasampant, M., Weitz, I. C., Kay, B., Boonchalermvichian, C., 65. Garratty, G., & Arndt, P. A. (2014). Drugs that have been shown to Liebman, H. A., & Shulman, I. A. (2015). Life-threatening delayed cause drug-induced immune hemolytic anemia or positive direct hyperhemolytic transfusion reaction in a patient with sickle cell antiglobulin tests: Some interesting findings since 2007. Immuno- disease: Effective treatment with eculizumab followed by ritux- hematology, 30, 66–79. imab. Transfusion, 55, 2398–2403. 66. Arndt, P. A. (2014). Drug-induced immune hemolytic anemia: 85. Oakley, F. D., Woods, M., Arnold, S., & Young, P. P. (2015). Trans- The last 30 years of changes. Immunohematology, 30, 44–54. fusion reactions in pediatric compared with adult patients: A 67. Leger, R. M., Arndt, P. A., & Garratty, G. (2014). How we investi- look at rate, reaction type, and associated products. Transfusion, gate drug-induced immune hemolytic anemia. Immunohematol- 55, 563–570. ogy, 30, 85–94. 86. Tormey, C. A., & Stack, G. (2013). Limiting the extent of a delayed 68. Arndt, P. A., Leger, R., & Garratty, G. (2012). Serologic character- hemolytic transfusion reaction with automated red blood cell istics of ceftriaxone antibodies in 25 patients with drug-induced exchange. Archives of Pathology and Laboratory Medicine, 137, immune hemolytic anemia. Transfusion, 52, 602–612. 861–864. 69. Mayer, B., Bartolmas, T., Yurek, S., & Salama, A. (2015). Variabil- 87. Gupta, S., Fenves, A., Nance, S. T., Sykes, D. B., & Dzik, W. (2015). ity of findings in drug-induced immune haemolytic anaemia: Hyperhemolysis syndrome in a patient without a hemoglobin- Experience over 20 years in a single centre. Transfusion Medicine opathy, unresponsive to treatment with eculizumab. Transfusion, and Hemotherapy, 42, 333–339. 55, 623–628. 70. Garratty, G. (2010). Immune hemolytic anemia associated with 88. de Haas, M., Thurik, F. F., Koelewijn, J. M., & Van der Schoot, C. drug therapy. Blood Reviews, 24, 143–150. E. (2015). Haemolytic disease of the fetus and newborn. 71. Satori, A., Staley, B., & Skipper, A. (2014). Drug-induced auto- Vox Sanguinis, 109, 99–113. immune hemolytic anemia in a 78-year-old African-American 89. Rath, M. E., Smits-Wintjens, V. E., Lindenburg, I. T., Brand, A., man with chronic lymphocytic leukemia. Lab Medicine, 45(3), van Kamp, I. L., Oepkes, D., . . . Lopriore, E. (2011). Exchange e105–e108. transfusions and top-up transfusions in neonates with Kell hae- 72. Holbro, A., & Passweg, J. R. (2015). Management of hemolytic molytic disease compared to Rh D haemolytic disease. anemia following allogeneic stem cell transplantation. Vox Sanguinis, 100, 312–316. Hematology, 2015, 376–384. 90. Denomme, G. A. (2015). Kell and Kx blood systems. Immunohema- 73. Rovira, J., Cid, J., Gutierrez-Garcia, G., Pereira, A., Fernandez- tology, 31, 14–19. Aviles, F., Rosinol, L, . . . Lozano, M. (2013). Fatal immune 91. Fasano, R. M. (2016). Hemolytic disease of the fetus and newborn in hemolytic anemia following allogeneic stem cell transplantation: the molecular era. Seminars in Fetal & Neonatal Medicine, 21, 28–34. Report of 2 cases and review of literature. Transfusion Medicine 92. Pirelli, K. L., Pietz, B. C., Johnson, S. T., Pinder, H. I., & Bellissimo, Reviews, 27, 168–170. D. B. (2010). Molecular determination of RHD zygosity: Predict- 74. Nadarajah, L., Ashman, N., Thuraisingham, R., Barber, C., ing risk of hemolytic disease of the fetus and newborn related to Allard, S., & Green, L. (2013). Literature review of passenger anti-D. Prenatal Diagnosis, 30, 1207–1212. lymphocyte syndrome following renal transplantation and two 93. Smith, H. M., Shirey, R. S., Thoman, S. K., & Jackson, J. B. (2013). case reports. American Journal of Transplantation, 13, 1594–1600. Prevalence of clinically significant red blood cell alloantibodies in 75. Hurtarte-Sandoval, A. R., Navarro-Cabello, M. D., Alvarez-Rivas, pregnant women at a large tertiary-care facility. Immuohematology, M. A., Robles-Lopez, A. I., Salmeron-Rodrguez, M. D., 29, 127–130. 428 Chapter 19 94. Markham, K. B., Rossi, K. Q., Nagaraja, H. N., & O’Shaughnessy, 96. Houston, B. L., Govia, R., Abou-Setta, A. M., Reid, G. J., Hadfield, R. W. (2015). Hemolytic disease of the fetus and newborn due M., Menard, C, . . . Zarychanski, R. (2015). Severe Rh alloim- to multiple maternal antibodies. American Journal of Obstetrics & munization and hemolytic disease of the fetus managed with Gynecology, 213, e1–e5. plasmapheresis, intravenous immunoglobulin and intrauterine 95. Fernandez-Alba, J. J., Leon, R., Gonzalez-Macias, C., Paz, A., transfusion: A case report. Transfusion and Apheresis Science, 53, Prado, F., Moreno, L. J., . . . Torrejon, R. (2014). Treatment of D 399–402. alloimmunization in pregnancy with plasmapheresis and intra- venous immune globulin: Case report. Transfusion and Apheresis Science, 51, 70–72. Chapter 20 Hemolytic Anemia: Nonimmune Defects Linda A. Smith, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define microangiopathic hemolytic anemia thrombocytopenic purpura (TTP), and (MAHA) and list several associated disorders hemolytic uremic syndrome (HUS). and the age group most commonly affected. 3. Recognize the characteristic erythrocyte 2. Describe the general morphology and morphology of MAHA on a stained blood hematologic values associated with MAHA film. and criteria that distinguish disseminated 4. List organisms that can cause erythrocyte intravascular coagulation (DIC), thrombotic hemolysis. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Summarize the general pathophysiology for 4. Given a set of data and clinical history, MAHA. determine whether MAHA is a probable 2. Compare and contrast the clinical findings, diagnosis, identify the possible etiology, underlying cause, treatment, and character- and propose follow-up tests that should be istic findings for erythrocytes, platelet count, performed. and coagulation tests for each of the follow- 5. Compare the underlying mechanism of ing types of MAHA: hemolysis by the following infectious a. Hemolytic uremic syndrome (HUS) agents: b. Thrombotic thrombocytopenic purpura a. Plasmodium parasites (malaria) (TTP) b. Babesia parasites c. Disseminated intravascular coagulation c. Bartonella bacteria (DIC) d. Clostridium bacteria 3. Define exercise-induced hemoglobinuria. 429 430 Chapter 20 Chapter Outline Objectives—Level I and Level II 429 Hemolytic Anemia Caused by Physical Key Terms 430 Injury to the Erythrocyte 431 Background Basics 430 Hemolytic Anemias Caused by Antagonists in the Blood 440 Case Study 430 Summary 443 Overview 431 Review Questions 443 Introduction 431 References 445 Key Terms Cryosupernatant Hemolytic uremic syndrome (HUS) Thrombotic thrombocytopenic Disseminated intravascular Microangiopathic hemolytic purpura (TTP) coagulation (DIC) anemia (MAHA) von Willebrand factor (VWF) Fresh frozen plasma (FFP) Plasma exchange Hemolysis, elevated liver enzymes and low platelet (HELLP) syndrome Background Basics The information in this chapter builds on concepts • Identify the intrinsic hemolytic anemias, and describe learned in previous chapters. To maximize your learn- how they differ from extrinsic hemolytic anemias. ing experience, you should review the following (Chapter 11) concepts: Level II Level I • Describe the structure and function of the major • Describe the normal production, life span, and proteins of the erythrocyte membrane. (Chapter 5, 17) destruction of the erythrocyte. (Chapter 5) • Identify the key tests that can be used in diagnosis • List the reference intervals for basic adult hematology of anemia, and identify clinical signs of anemia. parameters. (Chapters 1, 10, inside book covers) (Chapter 11) • Identify sources of defects that lead to hemolytic • Describe the different nonimmune mechanisms of anemia. (Chapter 11) hemolysis and how they are detected. (Chapters 5, • Review the immune hemolytic anemias, and describe 11, 17, 18) how they differ from other hemolytic anemias. • Identify the laboratory tests that differentiate immune (Chapter 19) from nonimmune anemia. (Chapters 11, 19) CASE STUDY contraceptives but was not taking any other drugs. We refer to this case study throughout the chapter. Her initial laboratory tests showed: Mai, a 35-year-old woman, saw her physician. Hb 6 g/dL (60g/L) She complained of weakness, low-grade fever, Hct 18% (0.18 L/L) and periods of forgetfulness, and memory loss for WBC 8.9 * 103>mcL the last week or so. She denied any viral illness before the onset of symptoms. She was on oral Consider reflex testing that could be helpful in identifying the cause of the anemia. Hemolytic Anemia: Nonimmune Defects 431 Overview or with foreign surfaces such as artificial heart valves com- monly induce such damage. There are other causes of injury, This chapter deals with the mechanisms of hemolysis not including Shiga toxin from organisms such as Esherichia coli included in the chapters on membrane defects (Chap- (E. coli) 0157:H7. Infectious agents such as Plasmodium sp. ter 17), metabolic deficiencies (Chapter 18), or immune and Babesia sp. can cause injury to the erythrocytes during mechanisms (Chapter 19) but includes microangiopathic their intracellular life cycle. Some drugs and chemicals can hemolytic anemias (MAHAs). Hemolytic uremic syndrome cause membrane oxidant injury, leading to intravascular (HUS), an MAHA, is discussed in detail including disease hemolysis or removal of the damaged cell by the spleen association, pathophysiology, and clinical presentation and (Table 20-1). laboratory evaluation. It also provides a brief overview of thrombotic thrombocytopenic purpura (TTP), which is dis- cussed in detail in Chapter 35. Uncommon causes of hemo- lysis such as hypertension, mechanical heart devices, burns, Hemolytic Anemia Caused exercise, and infectious agents are also briefly discussed. by Physical Injury to the Erythrocyte Introduction Intravascular and/or extravascular hemolysis and striking Erythrocytes that have normal hemoglobin structure, abnormal shapes of the circulating peripheral blood eryth- enzymes, and membranes can be prematurely destroyed by rocytes, including fragments (schistocytes) and helmet cells factors extrinsic to the cell. This destruction can be immune characterize hemolytic anemia caused by traumatic physi- mediated via antibodies and/or complement (Chapter 19). cal injury to the erythrocytes in the vascular circulation. However nonimmune factors also can cause either extravas- cular or intravascular hemolysis, depending on the type and Thrombotic Microangiopathic extent of injury to the erythrocyte. This chapter discusses nonimmune causes that lead to premature erythrocyte Anemia (TMA) destruction. Erythrocytes can undergo traumatic physical Thrombotic microangiopathic anemia (TMA) is an inclusive injury in the peripheral circulation, resulting in the pres- term referring to several conditions in which a hemolytic ence of schistocytes in the peripheral blood. Contact with process is caused by microcirculatory lesions. The condi- fibrin strands or platelet aggregates in the microcirculation tions described by this term are sometimes referred to as Table 20.1 Hemolytic Anemias Caused by Nonimmune Antagonists in the Erythrocyte Environment Category Antagonist Mode of Hemolysis Thrombotic microangiopathic anemia Thrombi in microcirculation Physical damage to erythrocytes by microthrombi Malignant hypertension Unknown Physical damage to erythrocytes Other physical trauma Exercise-induced hemoglobinuria External force Fragmentation of erythrocytes from exces- Oxidative stress sive external force as they pass through microcapillaries Thermal injury Heat Thermal damage to erythrocyte membrane proteins Traumatic cardiac Physical stress Erythrocyte fragmentation Infectious agents Plasmodium sp. Direct parasitization of erythrocyte; hypersplen- ism; acute intravascular hemolysis (P. falciparum infection) Babesia sp. Invasion of the erythrocyte and cell lysis Bartonella sp. Invasion of the erythrocyte and cell lysis Clostridium sp. Hemolytic toxins Animal venoms Snake bites Mechanical cell damage due to DIC Spider bites Venom Chemicals and drugs Water Osmotic lysis Oxidants Hemoglobin denaturation Lead Erythrocyte membrane |
damage 432 Chapter 20 the microangiopathic hemolytic anemias (MAHA) and are characterized by the presence of schistocytes, thrombocyto- CASE STUDY (continued from page 430) penia, and ischemic damage to organs.1,2,3,4,5 In these condi- Mai’s peripheral blood smear showed moderate tions, damage to the endothelial lining of the small vessels schistocytes and polychromasia. results in deposits of fibrin (which sometimes includes 1. What are some conditions that result in the pres- platelets) within the vessel. In addition, neutrophils’ role ence of schistocytes? in inflammation and thrombosis through neutrophil extra- cellular traps (NETs) has been explored6 (Chapter 7). As the erythrocytes are forced through the fibrin strands, the membrane can be sliced open. Sometimes the erythrocyte HEMOLYTIC UREMIC SYNDROME membrane reseals itself, leading to abnormal erythrocyte Hemolytic uremic syndrome (HUS) is a multisystem dis- shapes that are noted as schistocytes and keratocytes on order first described in the mid-1950s. It is characterized by the peripheral blood smear (Chapter 10). These damaged a triad of clinical findings: cells are often prematurely removed in the spleen (extra- • Hemolytic anemia with erythrocyte fragmentation vascular hemolysis). Severely damaged erythrocytes can be destroyed intravascularly. Depending on the underly- • Thrombocytopenia ing pathology, leukocytes can be increased and platelets • Acute nephropathy, which can include acute renal can be decreased. Evidence of intravascular coagulation failure (formation of fibrin within the blood vessels) and fibrino- Most individuals recover renal function.1,4,7 In some cases, lysis (breakdown of fibrin) could be present. The plasma there can also be evidence of mild neurologic problems, concentration of markers of hemolysis (bilirubin and hap- although only a small percentage develop severe symptoms. toglobin) varies depending on the type and extent of hemo- HUS can be subdivided into several categories. In this lysis (Chapter 11). text, classic STEC-HUS, secondary HUS (due to other infec- The underlying diseases and conditions responsible tious and noninfectious causes), and atypical HUS are dis- for TMA include classic Shiga toxin–producing E. coli hemo- cussed (Table 20-3). A previous categorization of two groups lytic uremic syndrome (STEC-HUS), atypical hemolytic uremic (D+ HUS and D- HUS) based on the presence or absence syndrome (aHUS), and thrombotic thrombocytopenic purpura of a bloody diarrheal prodrome is no longer recommended. (TTP). Other conditions that are characterized by microan- giopathic hemolytic anemia include malignant hyperten- Shiga Toxin–Producing E. Coli Hemolytic Uremic Syndrome sion, autoimmune disorders, sepsis, disseminated cancer, (Stec-Hus) Classic STEC-HUS is related to the presence and pregnancy (eclampsia, preeclampsia). A severe form of an infectious organism that produces Shiga-like toxin of preeclampsia characterized by hemolysis, elevated (other organisms that do not produce Shiga-like toxin may liver enzymes and low platelet counts (HELLP) syndrome also cause HUS). STEC-HUS is the most common form and can also cause MAHA. HUS and TTP can present with represents about 90% of cases. Onset tends to occur in chil- similar initial clinical symptoms, but the underlying etiol- dren between the ages of 6 months and 10 years; however, ogy, the age group affected, and the target organ(s) differ most cases are seen in children up to the age of 5 years. It (Table 20-2). is also considered the most common cause of acute renal failure in children and can lead to chronic renal insuffi- ciency (Table 20-2). Adult onset of this type is infrequent Checkpoint 20.1 and occurs in those over 16 years of age. The elderly are at What abnormal erythrocytes characterize microangiopathic highest risk for severe disease. hemolytic anemia? How are these cells formed? Classic STEC-HUS is characterized by bloody diarrhea (or hemorrhagic colitis) with more than 90% of the cases Table 20.2 Comparison of Characteristics Associated with STEC-HUS and TTP TTP HUS Adults ages 20–50 Children less than 5 years old Hemolytic anemia with red cell fragmentation Hemolytic anemia with red cell fragmentation Renal dysfunction (mild to moderate) Acute renal failure Thrombocytopenia Thrombocytopenia Severe CNS symptoms Mild CNS symptoms Fever Hemolytic Anemia: Nonimmune Defects 433 associated with gastrointestinal infections by specific sero- the individual had an invasive infection such as pneumo- types of Escherichia coli, which produce Shiga toxin (Stx). nia or bacteremia and the causative organism belonged to Most other cases are associated with infection by Shigella a serotype not included in pneumococcal vaccines. Occa- dysenteriae Type 1.7 The incubation period from infection sional cases have been linked to viral infections such as to onset of diarrhea is usually 5–6 days but can range from Epstein-Barr virus, human immunodeficiency virus, and 1–14 days. However, only about 15% of individuals who are cytomegalovirus. infected with Shiga toxin-producing E. coli develop HUS. Secondary HUS that is due to noninfectious include In the United States, the most common serotype of connective tissue diseases such as systemic lupus erythe- Shiga toxin–producing E. coli (STEC), or enterohemorrhagic matosus and lupus anticoagulant, as well as some types of E. coli (EHEC), is E. coli O157:H7. Other enterohemorrhagic cancer (especially stomach, colon, and breast) and diabe- serotypes including 026, 011, 0104, and 0145 have been iden- tes.2,3 The disease has been reported in young women with tified.8,9,10 The causative organism is not part of the normal complications of pregnancy, after normal childbirth, or with flora of humans but is found in the gastrointestinal tract of the use of oral contraceptives. Many drugs including anti- a small percentage of cattle. Most human infections have platelet drugs and antineoplastic agents have been reported been traced to the ingestion of incompletely cooked beef to cause rare cases of HUS. HUS secondary to other diseases contaminated with the organism, but the organism can also has a higher risk of recurrence and a lower survival rate be transmitted by the fecal-oral route (inadequate hand than cases that are associated with colitis or have no identi- washing). fiable trigger (primary). Other factors such as elevated neutrophil counts, Atypical HUS The term atypical HUS (aHUS) is used to increased C-reactive protein (CRP), and fever are associated describe cases of HUS caused by mutations in complement with an increased risk for developing HUS. Those under regulatory proteins or autoantibodies to these proteins.13,14,15 In 5 years of age and over 75 years of age are also at greater many cases these are inherited or familial. Episodes of aHUS risk.11 The use of antibiotics or antimotility drugs for treat- often recur.16 Almost 50% of cases are mutations related to three ing individuals with diarrhea from E. coli O157:H7 has also regulatory proteins associated with the alternate complement been associated with increased risk for developing HUS.11,12 pathway. These proteins, which function to control ampli- Secondary HUS The term, secondary HUS is often used fication of the pathway by C3bBb, are factor H, membrane for post-infectious HUS not caused by STEC or caused cofactor protein (MCP, CD46), and factor I. Other mutations in complement factor B and C3 have been identified.14,17,18,19,20,21 by other noninfectious causes (Table 20-3). Streptococcus pneumoniae (S. pneumoniae) is the organism most com- In many cases, these mutations do not manifest until a trigger- ing event occurs.18,19 monly linked to the post-infectious type. In most cases, An autoantibody against factor H has also been implicated in up to 15% of cases.14 The impaired regula- tion of the complement cascade leads to increased endothelial Table 20.3 deposition of C3, increased C5a, activation of the membrane Types of HUS and Associated Conditions attack complex (MAC), glomerular endothelial cell destruc- That Can Precipitate HUS tion, and microvascular thrombosis.20 Type of HUS Associated Condition CLASSIC STEC-HUS Diarrhea related (classic) Post-infection with: Checkpoint 20.2 • Escherichia coli O157:H7 What are the two types of HUS, and what organisms or dis- • other serotypes of E. coli eases are most commonly associated with each type? • Shigella dysenteriae serotype SECONDARY Post-infection with: • S. pneumoniae Pathophysiology More than 70% of the cases of HUS • Viral infections have been associated with damage to the renal glomeru- Immunosuppression related lar capillary endothelium by the Shiga toxin produced Chemotherapy/cytotoxic drugs by E. coli O157:H7 and S. dysenteriae Type 1.8,9 Once the ATYPICAL Mutations in the genes for comple- organism enters the human gastrointestinal tract, it colo- ment regulatory proteins nizes and forms an attachment lesion and begins to secrete Autoantibodies to complement regulatory proteins virulence factors including Stx. The organism’s toxin is Pregnancy or oral contraceptive absorbed into the circulation through damaged gastroin- related testinal tissue and onto the surface of neutrophils (Figure OTHER Renal and bone marrow transplantation 20-1). Neutrophils then deliver the Stx to the kidney. This Pregnancy or oral contraceptive toxin has a predilection for endothelial cells of the micro- related vasculature of the glomerulus and exerts a direct toxic 434 Chapter 20 Shiga toxin produced by organism Catabolic enzymes (especially leukocyte elastase) and oxi- dative products released from the granules of activated neutrophils have been implicated in causing additional Local damage to colon mucosa endothelial damage. Toxin enters circulation Checkpoint 20.3 Explain how infection with E. coli O157:H7 results in intravas- cular hemolysis. Toxin damages endothelial cells of capillaries in glomeruli Clinical Presentation STEC-HUS occurs in previously Platelet activation/fibrin microthrombi form Renal failure healthy children with the highest incidence in the first year of life. The onset is acute with sudden pallor, abdominal pain, vomiting, foul-smelling and bloody diarrhea, and mac- Erythrocytes damaged as trapped in thrombi roscopic hematuria. Other symptoms include a low-grade fever, hypertension, petechiae, bruising, and jaundice. The most important and/or serious complication of HUS is acute Schistocytes renal failure, which can lead to chronic renal insufficiency in some children. The duration of oliguria and anuria is vari- able. Organs other than the kidney can be affected as well. Intravasular hemolysis Splenic sequestration Regardless the organ affected, the pathology is the same (i.e., thrombosis of the microcirculation). Central ner- Figure 20.1 A possible mechanism for damage by Shiga-like vous system symptoms can result directly from microan- toxin of Escherichia coli O157:H7. giopathy of the central nervous system or from resulting hypertension. Lethargy and minor seizures are the most effect when bound to the endothelial cells. The toxin’s B common symptoms. Hepatomegaly can be present; sple- subunits bind to plasma membrane Gb3 receptors while nomegaly is less common. Hyperglycemia is common in the A subunit inhibits ribosomal protein synthesis, leading children because of pancreatic damage secondary to HUS. to cell death. Infiltrates of inflammatory cells and lipo- aHUS may occur at any age and clinical findings are like polysaccharides and the production of pro-inflammatory those of STEC-HUS.19 Most cases have an abrupt onset. cytokines such as IL-8 and tumor necrosis factor@a and chemokines contribute to the cytotoxic damage in glo- Laboratory Evaluation A moderate to severe normocytic, merular and renal tubular cells.7,9,10,11 Endothelial damage normochromic anemia is typical with hemoglobin levels as leads to the release of prothrombotic-, vasoactive-, and low as 3–4 g/dL (median values 7–9 g/dL) (Table 20-4). The platelet-aggregating substances that cause platelet activa- peripheral blood smear shows fragmented and deformed tion with the subsequent formation of thrombi. Although damage primarily occurs in the renal microvasculature, other organ systems (central nervous system, heart, liver) Table 20.4 Laboratory Findings in HUS and TTP can be affected. The resulting thrombotic microangiopa- Evidence of hemolysis Decreased hemoglobin/hematocrit thy that traps erythrocytes and causes fragmentation is Increased reticulocytes/ polychromasia responsible for the schistocytes commonly seen in HUS. Thrombocytopenia In addition, Shiga toxin has been shown to transiently Leukocytosis with shift to the left activate the alternative complement pathway and inhibit Presence of schistocytes regulatory proteins.11,21,22,23 Evidence of intravascular Hemoglobinemia hemolysis Hemoglobinuria In S. pneumoniae infections, capillary damage is medi- Decreased haptoglobin ated by the bacterial enzyme neuraminidase.12,24 Neuramin- Increased total and unconjugated serum bilirubin idase cleaves cell membrane glycoproteins and glycolipids, Increased lactic dehydrogenase facilitating tissue invasion by the bacteria and exposing the (LD) normally hidden T-antigen (Thomsen-Friedenreich antigen) Evidence of thrombotic Thrombocytopenia microangiopathy Fibrin degradation products (nor- on capillary walls, platelets, and erythrocytes. Naturally mal to slightly increased); D-dimer occurring anti-T antibodies cause agglutination of cells and increased platelets leading to thrombosis in the small vessels. Some PT and APTT (normal to slightly abnormal) evidence suggests that increased C3b deposition on cells Factors I, V, VIII (normal to may lead to cell destruction via the complement pathway. increased) Hemolytic Anemia: Nonimmune Defects 435 cells (schistocytes, burr cells, helmet cells, spherocytes) with later section “Disseminated Intravascular Coagulation”), the degree of anemia correlated directly with the degree coagulation screening tests are abnormal. The |
direct and of morphologic change (Figure 20-2). Polychromasia and indirect antiglobulin tests are usually negative, reflecting a an occasional nucleated erythrocyte can be seen. A leuko- nonimmune pathology. cytosis with a shift to the left is common. Platelet counts Urinalysis results show moderate to massive amounts vary from low normal to markedly decreased with a median of protein (1–2 g/24 hours to 10 g/24 hours), gross or value of 50 * 103>mcL. The duration of thrombocytopenia microscopic hematuria, increased numbers of neutrophils is 1–2 weeks. (pyuria), and casts (hyaline, granular, renal epithelial), Hemoglobinemia with an increase in total serum bili- reflecting the damage to the glomerulus. The presence of rubin (2–3 mg/dL) and a decrease in serum haptoglobin hemosiderin in the urine sediment reflects chronic intravas- reflects chronic intravascular hemolysis. Serum lactate cular hemolysis (Chapter 11). dehydrogenase (LD) is markedly elevated, and cardiac enzymes can be elevated because of myocardial dam- age. Blood urea nitrogen (BUN), and creatinine levels are Checkpoint 20.4 What are the typical erythrocyte morphology and coagulation increased, and the glomerular filtration rate is decreased, test results in children with HUS? reflecting renal damage. Metabolic acidosis, hyponatremia, and hypokalemia are common. Screening for complement abnormalities (CH50 assay, C3 and C4 levels, complement Therapy Mild to moderately severe cases of STEC-HUS factor H [CFH] and complement factor I [CFI] levels) can have the best prognosis for recovery (more than 80%). be indicated in patients with symptoms of aHUS.22,23 The With improvement in early diagnosis and supportive care, CH50 is a test of total complement activity and is reported especially during oliguric or anuric phases, the mortal- as the reciprocal of the serum dilution that lyses 50% of a ity of the disease has been reduced to 5–15%. Supportive red cell suspension. In addition, tests for antibodies to these care includes close observation, blood transfusion if neces- factors, flow cytometry for MCP on white blood cells, and sary, control of electrolyte and water imbalances, control of molecular studies to identify mutations in specific genes hypertension, and peritoneal dialysis in anuria.7,15,25 Plate- may be indicated.15 In HUS caused by S. pneumoniae, the let transfusions are not recommended because they can direct antiglobulin test (DAT) may be positive.7 exacerbate the thrombotic process but may be required in Screening tests for coagulation abnormalities include some patients with excessive bleeding. Antibiotic therapy the prothrombin time (PT) and activated partial throm- in STEC-HUS is not recommended.12 The beneficial use of boplastin time (APTT) (Chapter 32). Many cases of HUS fresh frozen plasma exchange (removal of patient plasma exhibit no detectable consumption of coagulation factors. and replacement with donor plasma) has not been shown The PT can be normal or slightly prolonged, but the APTT to be efficacious in patients with STEC-HUS. In contrast, is usually normal. Although fibrin-degradation products patients with aHUS, especially that caused by complement (fragments of fibrin produced by plasmin degradation of dysregulation, can benefit from plasma exchange or plasma fibrin) and D-dimer are elevated, disseminated intravascu- infusions. Plasma infusions are contraindicated in patients lar coagulation (DIC) is rare (Chapter 34). In DIC (see the with HUS who have a positive direct antiglobulin test or who are infected with S. pneumoniae because of the presence of naturally occurring anti-T in the infused plasma. Potentially promising preventative measures in indi- viduals infected with Shiga toxin-producing E. coli is the use of monoclonal antibodies against Shiga toxin to provide passive immunity or the use of Shiga toxin receptor ana- logs.12,15,25 There is only a small window of time (3–5 days) from onset of diarrhea in which treatment can be effective, so detection of the toxin in stool or blood by flow cytometry must be performed. Although plasma exchange has been the primary therapy for aHUS, the understanding of the etiology has changed treatment recommendations. The drug eculizamab is a recombinant, monoclonal anti-C5 antibody. Originally Figure 20.2 used to treat paroxysmal nocturnal hemoglobinuria, it has A peripheral blood smear from a patient with hemolytic uremic syndrome. The platelets are markedly decreased. proved to be effective in aHUS.14,15,19 The drug suppresses Schistocytes and spherocytes are present (Wright-Giemsa stain, complement activation by binding to C5 and preventing 1000* magnification). cleavage into C5a and C5b, thus preventing progression to 436 Chapter 20 the membrane attack complex (MAC). This drug is now rec- capillaries and arterioles in several organs including the ommended as a first-line therapy in aHUS.14,25,26 Treatment kidneys, heart, brain, and pancreas (VWF is a plasma pro- should be initiated as soon as STEC-HUS and ADAMST13 tein needed for platelets to adhere to collagen). Although deficiency have been ruled out and may be required for these thrombi contain platelets and sometimes immunoglo- life.14 Kidney transplant in patients with aHUS has poor bin and complement, there is little fibrin, inflammation, or outcome because of the recurring nature of the condition.19 subendothelial exposure as in disseminated intravascular coagulation (DIC). 27 THROMBOTIC THROMBOCYTOPENIC PURPURA Thrombotic thrombocytopenic purpura (TTP) is another Ultra large multimers of VWF normally are cleaved relatively uncommon disorder in which platelet aggrega- into smaller forms by the protease ADAMTS13 (a dis- tion on the microvascular endothelium results in serious integrin and metalloprotease with thrombospondin complications. In most cases, it is an acute disorder that type 1 motif) (Chapter 35). It is now known that a defi- affects young adults (ages 20–50 with a peak incidence in ciency in ADAMTS13 is the cause of TTP. Because of an the third decade). TTP occurs more frequently in females ADAMTS13 deficiency, the ultra large VWF multimers than males. TTP can be congenital or acquired (nonidio- remain attached to the endothelial cells and adhere to pathic or idiopathic). Genetic mutations in ADAMTS13 platelets, inducing platelet aggregation and formation of Chapter 32 may be associated with a single gene or combi- platelet thrombi. As erythrocytes are forced through the nations of mutations in multiple genes.17,27 thrombi, fragmentation occurs. There is some evidence Various clinical events have been identified as possible that these ULVWF may also activate the alternative com- precipitating factors in acquired TTP (Table 20-5). Infections plement pathway.5,6,27 The familial form of TTP occurs are the most common precipitating factor (40%) followed because of a mutation in the ADAMTS13 gene, resulting by pregnancy (10–25%). Without treatment, TTP has a mor- tality rate more than 90% because of multiorgan failure. in a deficient/dysfunctional enzyme. The acquired type The disorder is discussed in detail in Chapter 35, but an is caused by autoantibodies against ADAMTS13, which overview comparison with HUS in this chapter highlights block its activity. several key findings. Clinical Presentation The clinical aspects of TTP are like Pathophysiology TTP is characterized by microthrombi those of HUS except that TTP occurs most often in young composed of platelets and unusually large forms of von adults and involves more organ systems. Neurologic symp- Willebrand factor (VWF) (Chapters 31, 32) that occlude toms are more prominent, renal dysfunction is less severe, and the mortality rate is higher than in HUS (Table 20-2). Symptoms can be eliminated with early treatment, although some patients recovering from TTP can have permanent Table 20.5 Some Reported Clinical Conditions That Can manifestations of renal damage and require dialysis. Be Precipitating Factors in TTP Laboratory Evaluation Typical laboratory results are Conditions Examples shown in Table 20-4. The hemoglobin is usually less than Infections Bacterial—enteric organisms (E. 10.5 g/dL (average 8.0–9.0 g/dL). The mean cell volume coli, Shigella, Salmonella, Campy- lobacter, or Yersinia species) (MCV) is variable, either normal or decreased if there is marked erythrocyte fragmentation or increased in the Bacterial—other (S. pneumoniae, Legionella, Mycoplasma species) presence of reticulocytosis. The mean corpuscular hemo- Viral (HIV, EBV, influenza, herpes globin (MCH) and mean cell hemoglobin concentration simplex) (MCHC) are normal. Reticulocytes and nucleated eryth- Drugs Antimicrobials—penicillin rocytes can be found in the peripheral blood, reflecting Ticlopidine, Chemotherapeutic the bone marrow response to hemolysis. The most striking agents blood finding is the abundance of schistocytes (generally Connective tissue diseases Systemic lupus erythematosus more than 1%). Leukocytosis with counts of more than Rheumatoid arthritis 20 * 103>mcL occurs in 50% of patients and is usually Ankylosing spondylitis accompanied by a shift to the left. Thrombocytopenia is Sjögren’s syndrome often severe (8@44 * 103>mcL) because of consumption of Miscellaneous Bee sting platelets in the formation of microthrombi. Megakaryo- Dog bite cytes are abundant in the bone marrow, reflecting an Carbon monoxide poisoning increased hematopoietic response to the consumption of Pregnancy or oral-contraceptive platelets.28 related Coagulation tests are usually normal or only slightly Lymphomas and carcinomas abnormal in TTP; this helps to differentiate TTP from DIC Hemolytic Anemia: Nonimmune Defects 437 in which an increase in D-dimer as well as a prolonged PT, APTT, and thrombin time occur (Chapter 34). ADAMTS13 Table 20.6 Causes of Disseminated Intravascular Coagulation (DIC) activity level is the primary test to distinguish TTP from other TMas; the activity level is usually 610, and may be Bacterial sepsis Endotoxins as low as 5%.27,28 Exotoxins Hemoglobinemia, hemoglobinuria, decreased hapto- Neoplasm Solid tumors globin levels, and increased total and unconjugated serum Myeloproliferative disorders bilirubin are direct evidence of intravascular hemolysis. The Serious trauma —— direct antiglobulin test (DAT) is negative. Obstetrical complications Therapy Studies have shown that plasma exchange with Immunologic disorders Hemolytic transfusion reactions fresh frozen plasma (FFP) can be effective in providing the Transplant rejection needed ADAMTS13 protease.29,30 It is also effective in Miscellaneous Venom—snake or insect removing autoantibody (in acquired TTP) and is usually Drugs continued until the platelet count has recovered.31 Cryosu- pernatant, which lacks the large VWF multimers present in FFP, yet still contains the needed VWF cleaving protease, activation of plasma procoagulant proteins lead to deposi- can be used.32 Treatment options for those with autoanti- tion of fibrin and formation of widespread microthrombi in bodies include corticosteroids, the monoclonal antibody the microvasculature. As erythrocytes become entangled in rituximab (anti-CD20), antiplatelet or platelet-inhibiting the fibrin meshwork in the capillaries (clothesline effect), agents, intravenous administration of steroids, or combina- they fragment to form schistocytes. Complications that result tions of corticosteroids and plasma. Targeted therapies include thrombotic occlusion of vessels, bleeding because of including use of recombinant ADAMTS13 may be a future consumption of coagulation proteins, and ultimately organ option.31,33 failure. Hemolysis is not usually severe, but the effects of the consumptive coagulopathy can cause severe thrombocyto- penia and serious bleeding complications. Checkpoint 20.5 The typical findings on the blood smear include How does the clinical presentation of TTP differ from that of the presence of schistocytes and thrombocytopenia HUS? How is it similar? (Figure 20-3). The presence of schistocytes is not specific for DIC and they may only be present in low numbers.37 However, the abnormal coagulation tests help distinguish CASE STUDY this condition from others (TTP and HUS) that give a similar (continued from page 432) picture on a peripheral blood smear and increase diagnostic As Mai was questioned further, she indicated that accuracy.28,37 Abnormal coagulation tests include: she had noticed many bruises on her extremities. Her platelet count was 31 * 103>mcL. She had a • Prolonged prothrombin time (PT), activated partial 2.5% reticulocyte count. thromboplastin time (APTT), and thrombin time (TT) 2. What is the significance of these results? • Elevated D-dimer test 3. Why might the clinician order coagulation tests? DISSEMINATED INTRAVASCULAR COAGULATION Disseminated intravascular coagulation (DIC) is a complex thrombohemorrhagic condition in which the normal coagu- lation process (coagulation and fibrinolysis) is altered by an underlying condition. The more common conditions that precipitate DIC include bacterial sepsis, neoplasms, immu- nologic disorders, or trauma.34,35 It may also be observed as an obstetrical complication associated with severe pre- eclampsia, intrauterine fetal death, or post-partum hemor- rhage36 (Table 20-6). DIC can be initiated by damage to the endothelial lining of vessels, which causes release of throm- Figure 20.3 Peripheral blood from patient with disseminated boplastic substances that activate the coagulation mecha- intravascular coagulation. Notice the schistocytes, spherocytes, and nism. As a result, platelet activation and aggregation and thrombocytopenia (Wright stain, 1000* magnification). 438 Chapter 20 • Decreased platelet count of pregnancies with eclampsia develop HELLP syndrome • Increased fibrin degradation products (FDP) with a mortality rate of about 1%. The peripheral blood findings are like those found in • Decreased fibrinogen (often less than 150 mg/dL) TTP, HUS, and other microangiopathic conditions. Over- • Decreased antithrombin (AT) all, however, the hemolysis and thrombocytopenia are |
less In some cases, specific tests such as procalcitonin, an inflam- severe than those associated with TTP or HUS. Liver dam- matory biomarker, can be useful if septic DIC is suspected. age is due primarily to obstruction of hepatic sinusoids and In these cases, there may also be decreased protein C and can lead to subsequent hepatic hemorrhage or necrosis. decreased antithrombin.34,38 Microvascular fibrin-like deposits that resemble those in Treatment can include erythrocyte and platelet trans- TTP/HUS are responsible for the presence of schistocytes. fusions as well as infusion of fresh frozen plasma or factor Laboratory markers are used to determine the presence concentrates to replace coagulation factors. Some patients of HELLP. The liver enzyme most frequently measured may require specific fibrinogen replacement therapy.36 Most is aspartate aminotransferase (AST), and concentrations important, however, are the treatment and resolution of the more than 70 IU/L are common. Increased total (more than underlying disorder responsible for the DIC. The etiology, 1.2 mg/dL) and/or unconjugated bilirubin, increased LD diagnosis, and treatment of DIC is discussed further in (generally more than 600 IU/L), and decreased haptoglo- Chapter 34. bin can also be seen in HELLP. Coagulation tests such as the PT and APTT are usually normal until late in the dis- ease course. The platelet count decreases (usually less than Checkpoint 20.6 100 * 103>mcL) as a result of platelet aggregation and con- Explain how DIC can be differentiated from TTP and HUS based sumption at the site of endothelial damage.43 Although DIC on coagulation tests. infrequently occurs as a complication of HELLP, patients with lower platelet counts (less than 50 * 103>uL) can be at increased risk. There is evidence that some patients HELLP SYNDROME have a compensated form of DIC based on changes in The HELLP syndrome is an obstetric complication char- D-dimer values. Acute tubular necrosis with renal failure, acterized by hemolysis, elevated liver enzymes, and a low hepatic rupture, and pulmonary edema can also occur as platelet count that usually develops prior to childbirth, often complications.40 between the 26th and 28th week of gestation.39,40 Up to 30% Corticosteroid therapy can be useful in controlling cell of cases develop within 48 hours post-delivery.40 A partial destruction and decreasing liver enzymes if the fetus can- or incomplete form in which only 1 or 2 of the characteristic not be delivered immediately. Plasma exchange is rarely findings is present has also been described. HELLP must used as a treatment except in those whose values do not be distinguished from preeclampsia, TTP, and complement- normalize within 48 hours post-delivery or who progress induced thrombotic microangiopathy.41,42 The etiology and to post-partum thrombotic syndrome. The use of plasma pathogenesis are not well understood, but an association exchange will remove procoagulant factors.40,45 The use of with pregnancy-induced hypertension and DIC is sug- cortisone dexamethasone has eliminated the need for plate- gested.43 As with TTP and HUS, the precipitating factor is let transfusions in most patients with platelet counts less often unknown, but the clinical aspects are characterized than 50 * 103>mcL. by capillary endothelial damage and intravascular platelet activation as well as microangiopathic hemolytic anemia.39 Several gene variations including ones in the FAS and coag- CASE STUDY (continued from page 437) ulation Factor V Leiden have shown to increase risk. Placen- Mai’s PT and APTT were slightly prolonged. The tal factors may induce an inflammatory response that leads fibrinogen levels were slightly decreased. to activation of the complement and/or coagulation cas- cade.43 Although a significant prevalence of HELLP occurs 4. What do these findings indicate about the under- in patients with antiphospholipid syndrome, there seems lying problem? to be no direct association with the presence of IgM or IgG anticardiolipin antibodies or anti-b2-glycoprotein-I antibod- ies.44 Some experts consider HELLP to be a severe form of preeclampsia or eclampsia, which shares some of their char- MALIGNANT HYPERTENSION acteristics such as hypertension, proteinuria, and thrombo- Malignant hypertension can present with a low platelet cytopenia, but it is distinguished from them by the presence count, erythrocyte fragmentation, and renal failure. In addi- of hemolysis and elevated liver enzymes. Severe cases can tion, the presence of schistocytes, low platelet count, and compromise fetal growth and survival. Approximately 10% increased LD has been used to predict renal insufficiency as Hemolytic Anemia: Nonimmune Defects 439 well as recovery.The mechanism of hemolysis is unknown. depends on the percentage of body surface area burned. It may be caused by endothelial injury, fibrinoid necrosis Hemolysis probably results from the direct effect of heat- of arterioles, or deposition of fibrin fed by thromboplas- causing protein denaturation of spectrin in the eryth- tic substances released from membranes of lysed erythro- rocyte membrane. If erythrocytes are heated to 48 °C in cytes.15,46,47 Some individuals with malignant hypertension vitro, spectrin degradation causes a loss of elasticity and have decreased levels of ADAMTS13 activity; this may be deformability. In addition, the fatty acid and lipoprotein caused by an increased release of VWF that occurs because metabolism in both plasma and erythrocytes are altered of endothelial cell stimulation. after burn injuries, which can contribute to abnormal erythrocyte morphology. Peripheral blood smears show OTHER CONDITIONS ASSOCIATED WITH MAHA erythrocyte budding, schistocytes, and spherocytes. After Hematopoietic stem cell transplant recipients may show 48 hours, signs of hemolysis such as hemoglobinuria and transplant-associated thrombotic microangiopathy (TA- hemoglobinemia decrease. Thermal injury to erythrocytes TMA). TA-TMA may represent a form of graft-versus- also has occurred during hemodialysis when the dialysate host disease (GVHD) and result from endothelial damage is overheated. induced by donor cytotoxic T-cells or by treatment-related endothelial cell injury.48,49 It is characterized by many of EXERCISE-INDUCED HEMOGLOBINURIA the same diagnostic criteria as other TMAs including schis- Exercise-induced hemoglobinuria (sometimes described tocytes, decreased platelet counts, decreased hemoglobin, as march or runner’s hemoglobinuria) describes a transient and increased LD. The alternative and classical complement hemolysis occurring after strenuous exercise and often pathways are involved.49 Although the condition can resem- involves contact with a hard surface (e.g., running, tennis, ble TTP, ADAMTS13 activity is more than 5%. The kidney marching). The hemolysis is probably because of physical is most commonly affected but lungs, gastrointestinal tract, injury to erythrocytes as they pass through the microvas- and brain may also be affected. Treatment may include use of plasma exchange and eculizamab. 48,49 culature. Plasma hemoglobin and haptoglobin levels indi- cate that the primary cause of the transient hemolysis is Cancer-related MAHA (CR-MAHA) is uncommon but intravascular lysis.57,58 However, it is not seen in all indi- may be related to destruction of erythrocytes and plate- viduals participating in these activities and is occasion- lets in small vessels in cancerous tissue. Patients have a ally seen in other physical activities such as swimming, DAT-negative hemolytic anemia with schistocytes and thrombocytopenia.50 cycling, and rowing in which contact with a hard surface is limited. Hand drumming or percussion hemolysis has Drugs may also be linked to TMA in a few circum- a similar pathogenesis, only the damage is initiated in the stances. The process must be distinguished from immune- palms of the hands, not the soles of the feet.59 In recent mediated lysis (drug dependent antibodies or drug independent antibodies).51,52 years, the role of exercise-induced oxidative stress and red cell age have been recognized as potential additional causes of lysis, especially in normally sedentary indi- Other Erythrocyte Physical Trauma viduals who participate in strenuous exercise. Increased Resulting in Hemolytic Anemia osmotic fragility and decreased deformability leading to TRAUMATIC CARDIAC HEMOLYTIC ANEMIA intravascular hemolysis were noted in these individuals. Hemolytic anemia is a recognized complication following In addition, changes in erythrocyte membrane proteins surgical insertion of prosthetic heart valves or with aortic such as spectrin, especially in older cells, can increase sus- valve stenosis.53,54,55 However, massive hemolysis is rarely ceptibility to extravascular hemolysis during strenuous a complication.53 Unlike the microangiopathic anemia seen exercise. with TTP or DIC, the platelet count usually does not signifi- In contrast to the other hemolytic conditions dis- cantly decrease.53,56 Excessive acceleration or turbulence of cussed so far in this chapter, no erythrocyte fragments blood flow around the valve can fragment the erythrocytes appear on the peripheral blood smear, but the hallmarks as a result of “shear stress.” Many erythrocyte fragments of intravascular hemolysis—hemoglobinemia and hemo- are apparent on the blood smear. The spleen removes some globinuria—can be present. The passage of reddish urine of the severely traumatized cells, but many cells undergo immediately after exercise and for several hours thereaf- intravascular hemolysis. Newly designed prosthetic valves ter is usually the only complaint from affected individu- have helped decrease the shear stress and resulting eryth- als. Anemia and decrease in total erythrocyte volume are rocyte fragmentation. uncommon because less than 1% of the erythrocytes are hemolyzed during an episode.60 Individuals can present THERMAL INJURY with a slightly increased MCV because of increased reticu- Hemolytic anemia occurs within the first 24–46 hours locytes. Iron deficiency can occur if exercise and hemolysis after extensive thermal burns, and the degree of hemolysis are frequent.60 440 Chapter 20 methods, including invasion of the erythrocyte by parasites, CASE STUDY (continued from page 438) damage to the cell membrane, production of hemolysins Mai’s symptoms continued to worsen with that lyse the erythrocyte, or stimulation of antibodies or f requent seizures, headaches, and dizziness. immune complex deposits that result in increased phago- Her urinalysis results showed a 2+ protein and cytosis of the cell.61 moderate blood. However, she had normal urinary volume. Infectious Agents 5. Based on these results, what is the most likely Parasites and bacteria can infect erythrocytes and lead condition associated with these clinical and directly to their destruction. Alternatively, toxins produced laboratory results? Explain. by infectious agents can cause hemolysis. 6. What therapy might be used? MALARIAL PARASITES The anemia accompanying malaria is due directly and indirectly to the intracellular malarial parasites that spend part of their life cycle in the erythrocyte (Figure 20-4). The Hemolytic Anemias anemia resulting from this infection is usually a mild, nor- Caused by Antagonists in mocytic normochromic anemia but can be severe in infec- tion with Plasmodium falciparum because of the high levels of The Blood parasitemia. Thrombocytopenia can also be present. There are some kits available to detect parasite-specific antigens Antagonists such as drugs or venoms and infectious in the blood, but definitive diagnosis involves finding the organisms can cause premature erythrocyte destruction life cycle stage within the erythrocyte on a peripheral blood (Table 20-1). This hemolysis is precipitated by multiple smear. Infection with P. falciparum, a cause of severe anemia a b c Figure 20.4 Peripheral blood smears from patients with malaria. (a) A ring form of malaria in the erythrocyte. (b) An immature schizont form of malaria in the erythrocyte. (c) A gametocyte of Plasmodium falciparum in the erythrocyte (Wright stain, 1000* magnification). Hemolytic Anemia: Nonimmune Defects 441 in children, can be accompanied by ineffective erythropoi- esis and decreased reticulocytes. The hemoglobin in these cases can reach levels as low as 5 g/dL. In addition, poor diet, malnutrition, and decreased iron and folate stores contribute to the severity of the anemia. Exchange transfu- sions can be used in the severest cases to remove infected erythrocytes. The release of the intraerythrocytic parasite from the cell results in the cell destruction. Also, the spleen can remove the entire parasitized cell, or splenic macrophages can pit the parasite from the erythrocyte, damaging the cell membrane. The decreased deformability of the erythrocyte is due to changes in the cell membrane by splenic pitting, remodeling of the erythrocyte cytoskeleton by parasite proteins, as well as the presence of the large intracellular organism itself.62,63,64 The resulting decreased deformability can lead to removal of the cell by the spleen. Anemia can also result from an immune-mediated process. Antimalarial Figure 20.5 Peripheral blood smear of a patient with antibodies react with malarial antigens on the erythrocyte babesiosis. Several infected erythrocytes are in the field (Wright membrane, resulting in removal of the sensitized cell by the stain, 1000* magnification). splenic macrophages. In some cases, the concentration of complement regulatory proteins decrease, which can facili- doubles or tetrads in the form of a Maltese cross. Travel tate complement-mediated hemolysis. history, the absence of the characteristic banana-shaped Blackwater fever, an uncommon complication of infec- gametocytes, and the lack of pigment help distinguish it tion with P. falciparum, is characterized by massive acute from P. |
falciparum. Most infections are asymptomatic, but intravascular hemolysis with hemoglobinemia, hemoglo- some cases present with a flulike syndrome. Generally, binuria, methemalbuminemia, and hyperbilirubinemia. there is 1–10% parasitemia. Extravascular hemolysis can However, the parasitemia level is often low. The mechanism occur in a manner like that seen with malaria. A mild to that precipitates this is unclear. One possible mechanism is moderate normocytic anemia as well as thrombocytopenia the development of an autoantibody to the infected eryth- can be present. Other possible laboratory findings include rocyte. Another is a direct reaction to the drug quinine or to increased reticulocyte count, liver enzymes, and bilirubin. repeated incomplete treatment with the drug. Quinine can In a rare fulminating infection, severe anemia, intravascular act as a hapten to stimulate formation of a drug-dependent hemolysis, and hemoglobinuria are seen.67 Complications antibody that has complement-fixing ability. In some cases, associated with intravascular hemolysis include renal fail- the direct antiglobulin test (DAT) can be positive with either ure and DIC. Individuals at greatest risk of complications monospecific anticomplement or anti-IgG. Use of synthetic are the elderly and those who are splenectomized or immu- quinine drugs has considerably decreased the frequency of nosuppressed.68 Patients who are splenectomized generally this complication. It is possible that other antimalarial drugs have a more severe clinical presentation and higher levels of including mefloquine could trigger this response. parasitemia. In severe cases of hemolysis or renal complica- tions, exchange transfusion may be indicated. BABESIOSIS Babesiosis, a protozoan infection of rodents and cattle, is most commonly transmitted to humans by the bite of a hard Checkpoint 20.7 tick. However, the organism can be acquired transplacen- Why do malaria and babesiosis result in anemia? tally and through packed red blood cell transfusions. In the United States, it occurs most frequently in the New England area but cases can be found in Virginia and the Midwest. BARTONELLOSIS The most common organism for insect and transfusion Gram-negative bacterial organisms in the genus Barton- transmission is Babesia microti; however, other species such ella are zoonotic pathogens transmitted by blood-sucking as B. divergensa and B. duncani have been linked to trans- arthropods or by direct inoculation by the scratch or bite of a mission.65,66,67 On the peripheral blood smear, the parasites mammal. The organisms infect erythrocytes and endothelial appear as intracellular, pleomorphic, 1 - 5 mcM 1mm2 cells. Infection by Bartonella bacilliformis is associated with ring-like structures resembling ring-form trophozoites of Carrion’s disease, which is currently restricted to Columbia, Plasmodium falciparum (Figure 20-5). Some can appear as Peru, and Ecuador. Travelers to these areas are at risk for 442 Chapter 20 acquiring infections, which may manifest months to years Thomsen-Friedenrich (T-antigen), which can react with non- after leaving the endemic area.69 Other Bartonella species immune autoantibody. The peripheral blood smear shows infect erythrocytes but are not associated with hemolytic many microspherocytes and few erythrocyte fragments. manifestations. The disease is transmitted by the bite of a female sand-fly and is biphasic with the acute phase char- acterized by an often fatal syndrome consisting of myalgia, Animal Venoms high fever, and an acute, severe hemolytic anemia (Oroya Venoms injected by bees, wasps, spiders, and scorpions may fever) with onset in approximately 21 days after inocula- cause hemolysis in some susceptible individuals. One of the tion. Hepatosplenomegaly and jaundice can also be present. more common bites that can result in hemolysis is that of the Changes in T-lymphocyte counts and levels of cytokines brown recluse spider (Loxosceles reclusa). The characteristic such as IL-10 can also be associated with acute infection. symptom of envenomation is a localized lesion that shows Some patients may suffer temporary immunosuppression.70 signs of inflammation and a central thrombosis surrounded The disease can progress to coma and death within weeks by ischemic areas. However, up to 15% of individuals, and has a high mortality in untreated cases, especially if especially children, the elderly, and immunocompromised other comorbidities exist.71 If the patient survives, a chronic patients, develop systemic symptoms including nausea and phase characterized by an inflammatory reaction presents vomiting, fever, jaundice, and intravascular hemolysis that weeks to months later. In this phase of the disease the can be severe.77,78 Patients have leukocytosis, anemia, hema- organisms invade endothelial cells. The disease is charac- turia, thrombocytopenia, and increased levels of creatine terized by a granulomatous reaction (verruga peruana) with phosphokinase (CK). There are some reports of a positive the appearance of cutaneous hemangioma-like lesions that DAT with complement on the erythrocyte after bites.79 The can contain bacteria, neutrophils, macrophages, endothelial mechanism of venom damage appears to involve sphingo- cells, and deposits of immunoglobulins.72 The pleomorphic myelinase D2, which cleaves glycophorin from the erythro- coccobacillary, organisms are readily visualized as single, cyte membrane.80 Hyaluronidase and other esterases may paired, or chained organisms on or within erythrocytes on also be involved in cell damage.81 This decreases the struc- Wright- or Giemsa-stained peripheral blood smears during tural integrity of the membrane and increases its sensitivity the course of the disease. The organism releases several pro- to complement-mediated lysis. Systemic loxoscelism may teins including deformin that are responsible for inducing be treated with plasma exchange.82 the pitting or invagination of the erythrocyte membrane. Although snake bites rarely cause hemolysis directly, These structures, as well as other proteins such as hemo- hemolysis can be the result of DIC or a venom-induced lysins, can serve as entry portals for the bacteria and help consumption coagulopathy (VICC) resulting from activa- explain the mechanism of cell destruction.72,73 tion of the coagulation pathway. Snake venom may contain a mixture of biologically active proteins (including proco- CLOSTRIDIUM PERFRINGENS agulant toxins, anticoagulant toxins, protein C activators, C. perfringens is part of the normal flora of the gastrointes- and platelet aggregation inducers and inhibitors that affect tinal tract. Infection may present as a transient bacteremia hemostasis.83 Phospholipid A2 can degrade lecithin in the or a life-threatening condition. It is one of the few organ- cell membrane.84,85 VICC has no systemic microthrombi, but isms (along with C. septicum and C. novyi) that can cause the D-dimer levels and activated PTT are elevated.85 a rapid, massive intravascular hemolysis.73,74 Although this complication is very rare, it carries a high mortality risk unless aggressively treated with antimicrobials and Chemicals and Drugs surgical drainage.75 Patients with neoplasms such as colon Various chemicals and drugs have been identified as possi- cancer or invasive tumors of the genitourinary tract are at ble causes of erythrocyte hemolysis; many of these are dose highest risk. The bacteria produce several potent exotoxins dependent. In addition to erythrocyte hemolysis, chemicals that affect host cell membranes. The major hemolytic toxin and drugs can also produce methemoglobinemia and cya- 1a@toxin2 is a phospholipase C that hydrolyzes sphingomy- nosis, or in some instances bone marrow aplasia. elin, phospholipids, and lecithin present in the erythrocyte Hemoglobinemia and hemoglobinuria can occur membrane and leads to changes in membrane integrity and because of osmotic lysis of erythrocytes when water enters the presence of spherocytes. Streptolysin O and perfringo- the vascular system during transurethral resection or when lysin have been implicated in development of DIC in these inappropriate solutions are used during a blood transfusion. patients.76 Fever, thrombocytopenia, neutrophilia, hemoglo- Some drugs known to cause hemolysis in G6PD-defi- binemia, and hemoglobinuria are present. Anuria or acute cient persons can also cause hemolysis in normal persons renal failure can develop as a result of the hemoglobinuria. if the dose is sufficiently high. The hemolysis mechanism is Lysis of the erythrocyte or other cells can cause DIC. Bac- like that in G6PD deficiency with hemoglobin denaturation terial neuraminidase may expose cryptic antigens such as and Heinz body formation because of strong oxidants. Hemolytic Anemia: Nonimmune Defects 443 Anemia associated with lead poisoning is usually iron within mitochondria (Chapter 12). However, lead classified with sideroblastic anemias because the patho- also damages the erythrocyte membrane, which is mani- physiologic and hematologic findings are similar. Lead fested by an increase in osmotic fragility and mechanical inhibits heme synthesis, causing an accumulation of fragility. Summary Mechanisms of nonimmune damage to the erythrocyte disorder in which platelet aggregation and unusually large are varied. Hemolytic anemia caused by traumatic physi- forms of von Willebrand factor (VWF) on the microvascu- cal injury to the erythrocytes in the vascular circulation lar endothelium leads to formation of platelet thrombi that can be characterized by extravascular or intravascular occlude capillaries and arterioles in multiple organs. The hemolysis. Microangiopathic hemolytic anemia refers to underlying cause of TTP is a deficiency in the ADAMTS13 a group of anemias caused by microcirculatory lesions that protease that cleaves the ultralarge multimers of VWF injure the erythrocytes, producing schistocytes. Of those into smaller forms. HELLP syndrome, another MAHA, is discussed in this chapter, HUS and TTP are the more com- thought to be a severe form of preeclampsia. HELLP is monly encountered. Classic HUS is the most common and characterized by hemolysis, elevated liver enzymes, and is mediated by the Shiga-like toxin of E. coli O157:H7. The a low platelet count. Fibrin-like deposits are responsible toxin enters the gastrointestinal tract and damages the for the presence of schistocytes. Antagonists in the blood mucosa. Once it enters the circulation, it has a predilection such as venoms and infectious organisms can also cause for the endothelial cells of the glomerular microvascula- hemolytic anemia. Intraerythrocytic parasitic infections ture and exerts a toxic effect. Endothelial damage leads with organisms such as Plasmodium sp. or Babesia sp. can to release of prothrombotic substances that cause plate- cause hemolysis and anemia without the presence of schis- let activation and formation of thrombi. The thrombi trap tocytes. In susceptible individuals, drugs or chemicals can erythrocytes and cause fragmentation of the cells. TTP is a also lead to hemolysis. Review Questions Level I c. spur cell anemia d. immune hemolytic anemia 1. Which of the following results is associated with HUS and TTP? (Objective 2) 4. Which of the following organisms does not cause damage of the erythrocyte because of an intraerythro- a. Increased haptoglobin cytic life cycle? (Objective 4) b. Thrombocytopenia a. Plasmodium falciparum c. Reticulocytopenia b. Babesia sp. d. Decreased LD c. Bartonella sp. 2. One of the major criteria that distinguishes DIC from d. Clostridium perfringens other causes of microangiopathic hemolytic anemia is: (Objective 2) 5. A characteristic finding on a blood smear in MAHA is the presence of: (Objective 3) a. the presence of schistocytes b. thrombocytopenia a. target cells c. decreased hemoglobin b. spur cells d. an abnormal coagulation test c. schistocytes d. echinocytes 3. A patient who has anemia with an increased reticulo- cyte count, increased bilirubin, and many schistocytes 6. All the following are characterized as causes of on the blood smear could have: (Objective 2) MAHA except: (Objective 1) a. MAHA a. TTP b. high cholesterol in the blood b. DIC 444 Chapter 20 c. HUS 2. A 34-year-old woman is brought into the ER after fall- d. March hemoglobinuria ing off a ladder while painting her house. Selected lab results include: 7. All the following are associated with HUS except: (Objective 2) Hb 8.0 g/dL PT 36 seconds (80 g/L) a. thrombocytosis Hct 25% APTT 775 seconds b. nucleated RBCs in peripheral blood (0.25 L/L) c. schistocytes Platelet 20 * 103>mcL Fibrinogen 100 mg/Dl d. reticulocytosis count 8. MAHA is most frequently caused by: (Objective 1) Peripheral blood smear shows schistocytes. Given these results, what is the most likely diagnosis? a. microcirculatory lesions (Objectives 2, 4) b. immune destruction a. HELLP syndrome c. antagonists in the blood b. TTP d. plasma lipid abnormalities c. DIC 9. Intravascular hemolysis in MAHA would be d. Traumatic hemolytic anemia associated with which of the following parameters? (Objective 2) 3. Laboratory results for a child who had easy bruis- ing, tiredness, difficulty breathing, and decreased Bilirubin Haptoglobin urinary output were hemoglobin: 6.5 g/dL; platelets: A. decreased decreased 41 * 103>mcL; PT and APTT within normal reference B. decreased increased intervals. His mother indicated he had an episode of bloody diarrhea about 2 weeks earlier but it had not C. increased decreased recurred. Based on the clinical and limited laboratory D. increased increased findings, what is the most likely condition? (Objectives 2, 4) 10. MAHA caused by HUS is usually seen in which age group? (Objective 1) a. TTP a. Children younger than 5 years of age b. Bartonellosis b. |
Females between 20 and 50 years of age c. Clostridium sp. infection c. Either sex younger than 50 years of age d. HUS d. Males older than 16 years of age 4. The most likely age group for developing TTP is: (Objective 2) Level II a. female children younger than 1 year 1. A 43-year-old woman presents to her physician with a 3-week history of fatigue, constant headache, and b. women between 20 and 50 years low-grade fever. Selected laboratory results include: c. either gender younger than 5 years d. men older than 16 years Hb 7.5 g/dL Platelet count 16 * 103>mcL (75 g/L) 5. Which of the following disorders is not characterized Hct 23% Reticulocytes 11% by the presence of schistocytes? (Objective 3) (0.23 L/L) a. March hemoglobinuria RDW 15 b. Insertion of a prosthetic valve Peripheral blood smear showed schistocytes. Which c. Third-degree burns of the following drugs that the patient was taking d. Malignant hypertension could cause these symptoms and lab values? (Objectives 2, 4) 6. A patient with a deficiency in the VWF protease a. Ticlopidine ADAMTS13 would be at risk to develop which condi- tion? (Objective 2) b. Aspirin c. Estrogens a. HUS d. Quinine b. Spur cell anemia Hemolytic Anemia: Nonimmune Defects 445 c. TTP Her platelet count was less than 60 * 103>mcL and d. Hereditary acanthocytosis her hemoglobin was 7.5 g/dL. She had no history of chronic disease. What laboratory test(s) might give a 7. The formation of schistocytes in MAHA is primarily clue to the underlying cause? (Objective 4) due to: (Objective 1) a. Haptoglobin a. pitting by splenic macrophages b. Reticulocyte count b. defective cell membranes c. Liver enzymes c. increased membrane phospholipids d. APTT and PT d. shearing of erythrocytes by fibrin threads 10. Hemolytic toxins are the major cause of intravascular 8. Plasma exchange is used as a primary treatment for hemolysis in diseases or conditions caused by which which of the following? (Objective 2) of the following organisms? (Objective 5) a. HUS a. Plasmodium falciparum b. TTP b. Babesia sp. c. Abetalipoproteinemia c. Bartonella bacilliformis d. DIC d. Clostridium perfringens 9. The peripheral blood smear from a 6-months preg- nant woman showed the presence of schistocytes. References 1. Shenkman, B., & Einav, Y. (2014). Thrombotic thrombocytope- and atypical hemolytic uremic syndrome. Pathologic Biologic, 63, nic purpura and other thrombotic microangiopathic hemolytic 136–143. anemias: Diagnosis and classification. Autoimmunity Reviews, 13, 13. Geerdink, L. M., Westra, D., vanWijk, J. A. E., Dorresteja, E. 584–586. K., Lilien, M. R., Davin, J. C, . . . van de Kar, N. C. A. J. (2012). 2. George, J. N., & Charania R. S. (2013). Evaluation of patients Atypical hemolytic uremic syndrome in children: Complement with microangiopathic hemolytic anemia and thrombocytopenia. mutations and clinical characteristics. Pediatric Nephrology, 27, Seminars in Thrombosis and Hemostasis, 39, 153–160. 1283–1291. 3. Mariotte, E., & Veyradier, A. (2015). Thrombotic thrombocytope- 14. Kavanagh, D., Goodship, T. H., & Richards, A. (2013). Atypi- nic purpura: From diagnosis to therapy. Current Opinion in Criti- cal hemolytic uremic syndrome. Seminars in Nephrology, 33(5), cal Care, 21(6), 593–601. 508–530. 4. Tsai, H. (2013). Thrombotic thrombocytopenia purpura and the 15. Tsai, H. (2014). A mechanistic approach to the diagnosis and atypical hemolytic uremic syndrome. Hematology Oncology Clinics management of atypical hemolytic uremic syndrome. Transfusion of North America, 27, 565–584. Medicine Reviews, 28, 187–197. 5. George, J. N., & Nester, C. M. (2014). Syndromes of thrombotic 16. Bu, F., Borsa, N., Gianluigi, A., & Smith, R. J. H. (2012). Familial microangiopathy. New England Journal of Medicine, 371, 654–666. atypical hemolytic uremic syndrome: A review of its genetic and 6. Matevoysan, K., & Sarode, R. (2015). Thrombosis, microangiopa- clinical aspects. Clinical and Developmental Immunology, Article ID thies, and inflammation. Seminars in Thrombosis and Hemostasis, No. 370626. doi: 10.1155/2012/370426 41, 556–562. 17. Vieira-Martins, P., El Sissy, C., Bordereau, P., Gruber, A., Rosain, 7. Mele, C., Remuzzi, G., & Noris, M. (2014). Hemolytic uremic syn- J., & Fremeaux-Bacchi, V. (2016). Defining the genetics of throm- drome. Seminars in Immunopathology, 36, 399–420. botic microangiopathies. Transfusion and Apheresis Science, 54, 8. Gould, L. H., Mody, R. K., Ong, K. L., Clogher, P., Cronquist, A. 212–219. B., Garman, K. N, . . . Griffin, P. M. (2013). Increased recognition 18. Conway, E. M. (2015). HUS and the case for complement. Blood, of non-O157 Shiga-toxin-producing Escherichia coli infections in 126(18), 2085–2090. the United States during 2000–2010: Epidemiologic features and 19. Nayer, A., & Asif, A. (2016). Atypical hemolytic-uremic syn- comparison with E. coli O157 infections. Foodborne Pathogens and drome: A clinical review. American Journal of Therapeutics, 23, Disease, 10, 453–462. doi: 10.1089/pd.2012.1401 e151–158. 9. Pennington, H. (2011). Escherichia coli O157. The Lancet, 376, 20. Teoh, C. W., Riedl, M., & Licht, C. (2016). The alterative pathway 1428–1435. of complement and the thrombotic microangiopathies. Transfu- 10. Frank, C., Werber, D., Cramer, J. P., Askar, M., Faber, M., an der sion and Apheresis Science, 54, 220–231. Heiden, A., . . . Krause, G. (2011). Epidemic profile of Shiga-toxin 21. Turner, N., Sartain, S., & Moake, J. (2015). Ultralarge Von Wil- producing Escherichia coli O104:H4 outbreak in Germany. New lebrand factor-induced platelet clumping and activation of the England Journal of Medicine, 365, 1771–1780. alternative complement pathway in thrombotic thrombocytope- 11. Keir, L. S. (2015). Shiga toxin associated hemolytic uremic syn- nic pupura and hemolytic-uremic syndrome. Hematology Oncol- drome. Hematology Oncology Clinics of North America, 29, 525–539. ogy Clinics of North America, 29, 509–524. 12. Picard, C., Burtey, S., Bornet, C., Curti, C., Montana, M., & Vanelle, P. (2015). Pathophysiology and treatment of typical 446 Chapter 20 22. Kerr, H., & Richards, A. (2012). Complement-mediated injury and microangiopathy, thrombotic thrombocytopenia and other imita- protection of endothelium: Lessons from atypical uraemic syn- tors. European Journal of Obstetrics and Gynecology and Reproductive drome. Immunobiology, 217, 195–203. Biology, 189, 68–72. 23. Johnson, S., & Waters, A. (2012). Is complement a culprit in infec- 43. Abildgaard, U., & Heimdal, K. (2013). Pathogenesis of the syn- tion-induced forms of haemoytic uraemic syndrome? Immunobiol- drome of hemolysis, elevated liver enzymes, and low platelet ogy, 217, 235–243. count (HELLP): A review. European Journal of Obstetrics and Gyne- 24. Banarjee, R., Hersh, A. L., Newland, J., Beckmann, S. E., Polgreen, cology and Reproductive Biology, 166, 117–123. P. M., Bender, J., . . . Shah, S. S. (2011). Streptococcus pneumoniae- 44. Appenzeller, S., Souza, F. H. C., deSouza, A. W. S., Shoenfeld, Y., associated hemolytic uremic syndrome among children in North & de Carvalho, J. F. (2011). HELLP syndrome and its relationship America. Pediatric Infectious Disease Journal, 30, 736–739. with antiphospholipid syndrome and antiphospholipid antibod- 25. Goldwater, P. N., & Bettelheim, K. A. (2012). Treatment of entero- ies. Seminars in Arthritis and Rheumatism, 41, 517–523. hemorrhagic Escherichia coli (EHEC) infection and hemolytic ure- 45. Simetka, O., Klat, J., Gumulec J., Dolezalkova E., Salounova D., mic syndrome (HUS). BMC Medicine, 10, 12. & Kacerovsky, M. (2015). Early identification of women with 26. Legendre, C. M., Licht, C., Muus, P., Greenbaum, L. A., Babu, S., HELLP syndrome who need plasma exchange after delivery. Bedrosian, C., . . . Loirat, C. (2013). Terminal complement inhibi- Transfusion and Apheresis Science, 52, 54–59. tor Eculizumab in atypical hemolytic-uremic syndrome. New 46. Akimoto, T., Muto, S., Ito, C., Takahashi, H., Takeda, S., Ando, England Journal of Medicine, 23, 2169–2181. Y., & Kusano, E. (2011) Clinical features of malignant hyperten- 27. Sadler, J. E. (2015). What’s new in the diagnosis and pathophysi- sion with thrombotic microangiopathy. Clinical and Experimental ology of thrombotic thrombocytopenic purpura? Hematology, Hypertension, 33, 77–83. 2015, 631–636. 47. Khanal, N., Dahal, S., Upadhyay, S., Bhatt, V. R., & Bierman, P. J. 28. Park, Y. A., Waldrum, M. R., & Marques, M. B. (2010). Platelet (2015). Differentiating malignant hypertension-induced throm- count and prothrombin time help distinguish thrombotic throm- botic microangiopathy from thrombotic thrombocytopenic pur- bocytopenic purpura–hemolytic uremic syndrome from dis- pura. Therapeutic Advances in Hematology, 6, 97–102. seminated intravascular coagulation. American Journal of Clinical 48. Kim, S. S., Patel, M., Yum, K., & Keyzner A. (2014). Hematopoetic Pathology, 133, 460–465. stem cell transplant-associated thrombotic microangiopathy: 29. Clar, W. F. (2012). Thrombotic microangiopathy: Current knowl- Review of pharmacological treatment options. Transfusion, 55, edge and outcomes with plasma exchange. Seminars in Dialysis, 452–458. 25, 214–219. 49. Jodele, S., Laskin, B. L., Dandoy, C. E., Myers, K. C., El-Bietar, J., 30. George, J. N. (2010). How I treat patients with thrombotic throm- Davies, S. M., . . . Dixon, B. P. (2015). A new paradigm: Diagnosis bocytopenic purpura. Blood, 116, 4060–4069. and management of HSCT-associated thrombotic microangi- 31. George, J. N. (2012). Corticosteroids and rituximab as adjunctive opathy as multi-system endothelial injury. Blood Reviews, 29, treatments for thrombotic thrombocytopenic purpura. American 191–204. Journal of Hematology, 87, S88–S91. 50. Lechner, K., & Obermeier, H. L. (2012). Cancer-related microan- 32. Coppo, P., & Froissart, A. (2015). Treatment of thrombotic throm- giopathic hemolytic anemia. Medicine, 91(4), 195–205. bocytopenic purpura beyond therapeutic plasma exchange. 51. Al-Nouri, Z. L., Reese, J. A., Terrell, D. R., Vesely, S. K., & George, Hematology, 2015, 637–643. J. N. (2015). Drug-induced thrombotic microangiopathy: A sys- 33. Hori, Y., Hayakawa, M., Isonishi, A., Soejima, K., Matsumoto, M., tematic review of published reports. Blood, 125(4), 616–618. & Fujimura, Y. (2013). ADAMTS13 unbound to larger vonWil- 52. Reese, J. A., Bougie, D. W., Curtis, B. R., Terrell, D. R., Vesely, S. K., lebrand factor multimers in cryosupernatant: Implications for Aster, R. H., . . . George, J. N. (2015). Drug-induced thrombotic selection of plasma preparations for thrombotic thrombocytope- microangiopathy: Experience of the Oklahoma Registry and the nia purpura treatment. Transfusion, 53, 3192–3202. Blood Center of Wisconsin. American Journal of Hematology, 90(5), 34. Wada, H., Matsumoto, T., Yamashita, Y., & Hatada, T. (2014). 406–410. Disseminated intravascular coagulation: Testing and diagnosis. 53. Naik, A. V., Bhalgat, P. S., Bhadane N. S., & Joshi S. V. (2016). Very Clinica Chimica Acta, 436, 130–134. early onset traumatic hemolysis following mitral valve repair in a 35. Levi, M. (2013). Diagnosis and treatment of disseminated pediatric patient. Indian Heart Journal, 68, 5237–5240. intravascular coagulation. International Journal of Laboratory 54. Vahidkhah, K., Cordasco, D., Abbasi, M., Ge, L., Tseng, E., Bagchi, Hematology, 36, 228–236. doi: 10.1111/ijlh.12221 P., & Azadani, A. N. (2016). Flow-induced damage to blood cells 36. Hossain, N., & Paidas M. J. (2013) Disseminated intravascular in aortic valve stenosis. Annals of Biomedical Engineering, 44(9), coagulation. Seminars in Perinatology, 37, 257–266. 2724–2736. 37. Lesesve, J. F., Martin, M., Banasiak, C., Ander-Kerneis, E., Barde, 55. Choi, J. H., Park, Y. H., Yun, K. W., Lee, S. H., Kim, J. S., Kim, V., Lusina, D., . . . Lecompte, T. (2013). Schistocytes in dissemi- J., . . . Chun, K. J. (2013). Intractable hemolytic anemia after mitral nated intravascular coagulation. International Journal of Laboratory valve repair: A report of three cases. Echocardiography, 30, Hematology, 36, 439–441. E281–E284. doi: 10.1111/echo.12293 38. Favaloro, E. J. (2010). Laboratory testing in disseminated intra- 56. Najib, M. Q., Vinales, K. L., Paripati, H. R., Kundranda, M. N., vascular coagulation. Seminars in Thrombosis and Hemostasis, 36, Valdwz, R., Rihal, C. S., . . . Chaliki, H. P. (2011). An unusual pre- 458–467. sentation of hemolytic anemia in a patient with prosthetic mitral 39. Dusse, L. M., Alpoim, P. N., Silva, J. T., Rios, D. R. A., Brandao, A. valve. Echocardiography, 28, E112–E114. H., & Cabral, A. C. V. (2015). Revisiting HELLP syndrome. Clinica 57. Lippi, G., Schena, F., Salvagno, G. L., Aloe, R., Banfi, G., & Guidi, Chimica Acta, 451, 117–120. G. C. (2012). Foot-strike haemolysis after a 60-km ultramarathon. 40. Erkurt, M. A., Berber, I., Berktas, H. B., Kuku, I., Kaya, E., Koro- Blood Transfusion, 10, 377–383. glu, M., . . . Ozgul, M. (2015). A life-saving therapy in Class I 58. Sureira, T. M., Amancia, O. S., & Braga, J. A. P. (2012). Influence HELLP syndrome: Therapeutic plasma exchange. Transfusion and of artistic gymnastics on iron nutritional status and exercise- Apheresis Science, 52, 194–198. induced hemolysis in female athletes. International Journal of Pro- 41. George, J. N., Nester C. M., & McIntosh J. J. (2015). Syndromes of tein Nutrition and Exercise Metabolism, 22, 243–250. thrombotic microangiopathy associated with pregnancy. Hematol- 59. Vasudev, M., Bresnahan, B. A., Cohen, E. P., Hari, P. N., Hari- ogy, 2015, 644–648. haran, S., & Vasudev, B. S. (2011). Percussion |
hemoglobinuria – a 42. Pourrat, O., Coudroy, R., & Pierre, F. (2015). Differen- novel term for hand-trauma-induced mechanical hemolysis: A tiation between severe HELLP syndrome and thrombotic care report. Journal of Medical Case Reports, 5, 508–511. Hemolytic Anemia: Nonimmune Defects 447 60. Robach, P., Boisson R. C., Vincent, L., Lundby, C., Moutereau, 73. Simon, T. G., Bradley, J., Jones, A., & Carino, G. (2014). Massive S., Gergele, L., . . . Millet, G. Y. (2014). Hemolysis induced by intravascular hemolysis from Clostridium perfringens septicemia: an extreme mountain ultra-marathon is not associated with a A review. Journal of Intensive Care Medicine, 29(6), 327–333. decrease in total red blood cell volume. Scandinavian Journal of 74. Cochrane, J., Bland, L., & Noble, M. (2015). Intravascular hemoly- Medicine and Science in Sports, 24, 18–27. sis and septicemia due to Clostridium perfringens emphysematous 61. McCullough, J. (2014). RBCs as targets of infection. Hematology, cholecystitis and hepatic abscesses. Case Reports in Medicine, 2015. 2014(1), 404–409. doi: http:/dx.doi.org/10.1155/2015/523402 62. Dasari, R., & Bhakdi, S. (2012). Pathogenesis of malaria revisited. 75. Watt, J., Amini, A., Mosier, J., Gustafson, M., Wynne, J., Friese, Medical Microbiology and Immunology, 201(4), 599–604. R., . . . O’Keeffe, T. (2012). Treatment of severe hemolytic anemia 63. Hosseini, S. M., & Feng, J. J. (2012). How malaria parasites reduce caused by Clostridium perfringens sepsis in liver transplant recipi- the deformability of infected red blood cells. Biophysical Journal, ent. Surgical Infections, 13, 60–62. 103, 1–10. 76. Hashiba, M., Tomino, A., Takenaka, N., Hattori, T., Kano, H., 64. Mohandas, N., & An, X. (2012). Malaria and human red cells. Tsuda, M., & Takeyama, N. (2016). Clostridium perfringens infec- Medical Microbiology and Immunology, 201(4), 593–598. tion in a febrile patient with severe hemolytic anemia. American 65. Leiby, D. A. (2011). Transfusion-transmitted Babesia spp: Bulls-eye Journal of Case Reports, 17, 219–223. doi: 10:12659/AJCR.895721 on Babesia microti. Clinical Microbiology Reviews, 24(1), 14–28. 77. Rosen, J. L., Dumitru, J. K., Langley, E. W., & Olivier, C. A. M. 66. Herwaldt, B. L., Linden, J. V., Bosserman, E., Young, C., (2012). Emergency department death from systemic loxoscelism. Olkowska, D., & Wilson, M. (2011). Transfusion-associated babe- Annals of Emergency Medicine, 60(4), 439–441. siosis in the United States: A description of cases. Annals of Inter- 78. Hubbard, J. J., & James, L. P. (2011). Complications and outcomes nal Medicine, 155, 509–520. of brown recluse spider bites in children. Clinical Pediatrics, 50, 67. Cursino-Santos, J. R., Halverson, G., Rodriguez, M., Narla, M., 252–258. & Lobo, C. A. (2014). Identification of binding domains on red 79. McDade, J., Aygun, B., & Ware, R. E. (2010). Brown recluse spider blood cell glycophorins for Babesia divergens. Transfusion, 54, (Loxosceles reclusa) envenomation leading to acute hemolytic ane- 982–989. mia in six adolescents. Journal of Pediatrics, 156, 155–157. 68. Shatzel, J. J., Donohoe, K., Chu, N. Q., Garraty, G., Mody, K., 80. Malaque, C. M. S., Santoro, M. L., Cardoso, J. L. C., Conde, M. R., Bengston, E. M., . . . Dunbar, N. M. (2014). Profound autoimmune Novaes, C. T. G., Risk, J. Y., Fan, H. W. (2011). Clinical picture and hemolysis and Evans syndrome in two asplenic patients with laboratorial valuation in human loxoscelism. Toxicon, 58, 664–671. babesiosis. Transfusion, 55, 661–665. 81. Levin, C., Bonstein, L., Lauterbach, R., Mader, R., Rozemman, D., 69. Breitschwerdt, E. B., (2014). Bartonellosis: One health perspec- & Koren, A. (2014). Immune-mediated mechanism for thrombo- tives for an emerging infectious disease. ILAR Journal, 55(1), cytopenia after Loxosceles spider bite. Pediatric Blood and Cancer, 46–58. doi: 10.1093/ilar/ilu015 61, 1466–1468. 70. Huarcaya, E., Best, I., Rodriguez-Tafur, J., Maguina, C., S olorzano, 82. Said, A., Hmiel, P., Goldsmith, M., Dietzen, D., & Hartman, M. E. N., Menacho, J., . . . Ventosilla, P. (2011). Cytokines and (2014). Successful use of plasma exchange for profound hemoly- T-lymphocyte count in patients in the acute and chronic phases sis in a child with loxoscelism. Pediatrics, 134, e1464–e1467. of Bartonella bacilliformis infection in an endemic area in Peru: A doi: 10.1542/peds.2013-3338 pilot study. Revista do Instituto de Medicina Tropical de Sao Paulo, 53, 83. McCleary, R. J. R., & Kini, R. M. (2013). Snake bites and hemosta- 149–154. doi: http://dx.doi.org/10.1590/S0036-46652011000300006 sis/thrombosis. Thrombosis Research, 132, 642–646. 71. Angelakis E., & Raoult, D. (2014). Pathogenicity and treatment of 84. Bhagwat, K., & Amar, L. (2013). Blood hemoglobin, lactate dehy- Bartonella infections. International Journal of Antimicrobial Agents, drogenase and total creatine kinase combinely as markers of 44, 16–25. hemolysis and rhabdomyolysis associated with snakebite. Inter- 72. Pulliainen, A. T., & Dehio, C. (2012). Persistence of Bartonella sp. national Journal of Toxicology and Pharmacological Research, 5(1), 5–8. Stealth pathogens: From subclinical infections to vasoprolifera- 85. Berling, I., & Isbister, G. K. (2015). Hematologic effects and com- tive tumor formation. FEMS Microbiology Reviews, 36, 563–599. plications of snake envenomation. Transfusion Medicine Reviews, doi: 10.1111/j.1574-6976.2012.00324.x 29, 82–89. This page intentionally left blank Section Four Nonmalignant Disorders of Leukocytes 449 Chapter 21 Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes Kristin Landis-Piwowar, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Recognize neutrophilia from hematologic 6. Identify neutropenia from hematologic data data and name the common disorders and list the common disorders associated associated with neutrophilia. with neutropenia. 2. Explain the quantitative and qualitative 7. Recognize the conditions associated with neutrophil response to acute bacterial false or pseudo-neutropenia. infections. 8. Identify neutrophil nuclear alterations 3. Identify immature granulocytes and including Pelger-Huët, hypersegmentation, morphologic changes (toxic granulation, and pyknotic forms on stained blood films Döhle bodies, intracellular organisms, and and microscopic pictures. vacuoles) often seen in reactive neutrophilia. 9. Recognize morulae, Alder-Reilly granules, 4. Define and recognize leukemoid reaction, and Chédiak-Higashi inclusions on stained leukoerythroblastosis, and pyknotic nuclei blood films and microscopic pictures. on stained blood films and microscopic 10. State the common conditions associated pictures. with abnormal eosinophil, basophil, and 5. Distinguish leukemoid reaction from monocyte counts. chronic myeloid leukemia based on 11. Define Gaucher and Niemann-Pick diseases. laboratory data. 450 Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 451 Objectives—Level II At the end of this unit of study, the student should be able to: 1. Assess the etiology, associated conditions, functional abnormalities (Chédiak-Higashi, and peripheral blood findings for immediate, Alder-Reilly, May-Hegglin, and chronic acute, chronic, and reactive neutrophilia. granulomatous diseases) and differentiate 2. Compare and contrast the hematologic and their cellular abnormalities. clinical features for leukemoid reaction and 8. Evaluate alterations in the relative and/or chronic myeloid leukemia (CML). absolute numbers of eosinophils, basophils, 3. Organize neutropenia to include etiology and monocytes and associate them with the and associated conditions as well as blood clinical condition of the patient. and bone marrow findings. 9. Evaluate the etiology, laboratory findings, 4. Recognize, evaluate, and select appropriate and clinical features of the lysosomal storage corrective action for false neutropenia. disorders. 5. Appraise the nuclear abnormalities of 10. Identify and differentiate the abnormal neutrophils including Pelger-Huët, pseudo- macrophages seen in Gaucher disease, Pelger-Huët, hypersegmentation, and Niemann-Pick disease, and sea-blue pyknotic nuclei, and reconcile them with the histiocytosis syndrome. appropriate clinical conditions of the patient. 11. Construct an efficient and cost-effective 6. Appraise the cytoplasmic abnormalities of reflex testing pathway for following-up neutrophils including toxic granulation, neutrophilia, neutropenia, and qualitative Döhle bodies, vacuoles, intracellular organ- granulocyte abnormalities. isms, and morulae and reconcile them with 12. Evaluate a case study from a patient with a the patient’s appropriate clinical condition. nonmalignant granulocyte disorder. 7. Recognize and summarize the clinical features of the inherited granulocyte Chapter Outline Objectives—Level I and Level II 450, 451 Eosinophil Disorders 464 Key Terms 451 Basophil and Mast Cell Disorders 466 Background Basics 452 Monocyte/Macrophage Disorders 466 Case Study 452 Summary 468 Overview 452 Review Questions 469 Introduction 452 References 471 Neutrophil Disorders 453 Key Terms Agranulocytosis Döhle body Hypereosinophilic syndrome Basophilia Egress (HES) Demargination Hypereosinophilia Leukemoid reaction 452 Chapter 21 Leukocytosis Monocytosis Pseudo-neutrophilia Leukoerythroblastic reaction Morulae Reactive neutrophilia Leukopenia Myelophthisis Sea-blue histiocytosis syndrome Lysosomal storage disorders Neutropenia Shift to the left/left shift Mastocytosis Neutrophilia Toxic granules Monocytopenia Pelger-Huët anomaly (PHA) Background Basics The information in this chapter builds on concepts Level II learned in previous chapters. To maximize your learning • Describe the role of specific neutrophil granules and experience, you should review before starting this unit enzymes in antimicrobial systems. (Chapter 7) of study: • Identify normal macrophages and discuss their role in the bone marrow and the rest of the monocyte- Level I macrophage system. (Chapter 7) • Summarize the production, kinetics, distribution, life • Describe leukocyte maturation and proliferation pools span, and basic function of neutrophils and mono- in the bone marrow; describe the role of cytokines in cytes. (Chapter 7) bone marrow release of leukocytes; describe the pro- • Describe how leukocytes are counted and differ- cess of leukocyte egress to tissue. (Chapters 3, 4, 7) entiated; recognize normal and immature granulo- • Correlate the function of the hematopoietic organs to cytes. (Chapters 7, 10, 37) leukocyte distribution and demise. (Chapter 3) acquired and inherited physiologic states. The laboratory CASE STUDY professional must recognize these abnormalities and cor- We refer to this case throughout the chapter. relate them with the patient’s clinical condition. Dennis, a 24-year-old man, was taken to emer- gency surgery to repair several bone fractures sustained in an automobile accident. His previ- Introduction ous medical history was unremarkable. He was in Changes in leukocyte concentration and morphology excellent health prior to the accident. are often the body’s normal responses to various disease Consider why Dennis’s condition could result processes and toxic challenges. Most often, one class of in abnormal hematologic test results and the possi- leukocyte is affected more than the others, providing ble complications that could occur during Dennis’s an important clue to diagnosis. The type of cell affected treatment and recovery. depends in a large part on its function (e.g., bacterial infec- tion commonly results in an absolute neutrophilia, viral infections are characterized by an absolute lymphocytosis, Overview and certain parasitic infections cause an eosinophilia). Thus, the absolute concentrations of each leukocyte class aids in This chapter discusses benign changes in granulocytes and the differential diagnosis of a patient, especially when the monocytes as a response to various nonmalignant disease total leukocyte concentration is abnormal. states and toxic challenges. These changes include both Leukocytosis refers to a condition in which the total quantitative and qualitative variations that can be detected leukocyte count is more than 11.0 * 103/mcL in an adult. by laboratory professionals. The chapter is organized by See Appendix D, Table B for leukocyte and differential class of leukocyte (neutrophils, eosinophils, basophils, reference intervals. Although leukocytosis usually occurs and monocytes) and by the changes in quantity and qual- because of an increase in neutrophils, it can also be related to ity (morphology) of those leukocytes. These variations an increase in lymphocytes or (rarely) in monocytes, eosino- in leukocyte number and appearance are correlated with phils, or basophils.1 Quantitative variations of leukocytes Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 453 are evaluated by performing a total leukocyte count and a Automated hematology analyzers detect neutrophil counts differential count. The absolute concentration of each type outside the reference interval, but they do not detect quali- of leukocyte can be calculated from these two values as tative changes in the neutrophils. Detection of these changes follows (Chapter 7): requires careful microscopic examination of stained blood films and provides information for differential diagnosis, Differential count (in decimal form) * WBC count especially in patients with recurring bacterial infections. (* 103/mcL) = Absolute cell count (* 103/mcL) Quantitative Disorders Checkpoint 21.1 Quantitative neutrophil abnormalities can result from An adult patient’s total leukocyte count is 5.0 * 103/mcL. There malignant or benign disorders. Malignant disorders are are 60% segmented neutrophils, 35% lymphocytes, and 5% caused by neoplastic transformation of hematopoietic stem monocytes on the differential. Calculate the absolute number of cells and are discussed in Chapters 23–28. Benign disorders each cell type. Is each of these relative and absolute cell counts normal or abnormal? are usually acquired and most often occur as a consequence of a physiologic insult (stressful stimulus that affects nor- mal function and can result in morbidity). Three interre- Leukopenia refers to a condition in which the total leu- lated mechanisms affecting neutrophil concentration in the kocyte count falls to less than 4.5 * 103/mcL. This condi- peripheral |
blood include: (1) bone marrow production and tion is usually the result of a decrease in neutrophils, but release of neutrophils, (2) rate of neutrophil egress to tissue lymphocytes and other leukocytes may also contribute. or survival time in blood, and (3) ratio of marginating to Morphologic or qualitative variations in leukocytes circulating neutrophils in peripheral blood. are noted by examination of the stained blood smear NEUTROPHILIA (Chapters 10, 37). Some qualitative changes affect cell func- The reference interval for neutrophil concentration varies tion, whereas others do not. Variations in the appearance of with age and race, which emphasizes the importance of eval- the cell together with its concentration can provide specific uating the count for each demographic group. Neutrophilia clues to the pathologic process. refers to an increase in the total circulating absolute neu- trophil concentration (ANC). In adults, neutrophilia occurs when the ANC exceeds 7.0 * 103/mcL. See Appendix D, Neutrophil Disorders Table B for age- and race-specific reference intervals. Because neutrophils are the most numerous type of leuko- Neutrophilia that is not caused by malignancy most cyte that circulates in the peripheral blood in adults, quan- often occurs as a response to a physiologic or pathologic titative disorders of neutrophils are often accompanied by process and is termed reactive neutrophilia; it can be imme- changes in the total leukocyte count. Although neutrophilia diate, acute, or chronic and can involve any or all of the (increase in neutrophils) is more common than neutropenia three mechanisms listed in Table 21-1. (decrease in neutrophils), the consequences to the health of Immediate Neutrophilia Of the neutrophils inside a a patient are more severe when the neutrophil count is low. blood vessel, approximately 50% freely circulate, and the Table 21.1 Conditions Associated with Neutrophilia Immediate Neutrophilia Acute Neutrophilia Chronic Neutrophilia Strenuous exercise Acute bacterial, fungal, and some viral infections Persistence of infections that cause acute neutrophilia Stress Inflammatory processes Persistence of inflammation and chronic inflammatory • Tissue damage from burns, trauma, or surgery disorders • Collagen, vascular, and autoimmune disorders • Hypersensitivity reactions Pain Drugs, hormones, and toxins Drugs, hormones, and toxins Extreme temperatures Colony-stimulating factors, vaccines, venoms Metabolic and endocrine disorders • Eclampsia • Adrenocorticotropic hormone Childbirth Acute hemorrhage or hemolysis Hematologic neoplasms Epinephrine Hereditary and congenital disorders Anesthesia Various cancers Emotional stimuli (panic, severe stress) 454 Chapter 21 remaining 50% are loosely attached to the endothelial cells neutrophil count may be normal, but toxic morphologi- of the blood vessel (marginated; Chapter 7). Immediate neu- cal changes may be observed including toxic granulation, trophilia can occur without pathologic stimulus and is prob- Döhle bodies, and cytoplasmic vacuoles (discussed later in ably a simple redistribution of the marginating pool to the this chapter and illustrated in Figure 21-1). circulating pool. Routine laboratory analysis of a peripheral Bacterial Infection Bacterial infection is the most com- blood sample detects only those neutrophils in the circulat- mon cause of neutrophilia, especially infection caused by ing pool. pyogenic organisms such as staphylococci and streptococci Although the increase in circulating neutrophils occurs (Table 21-1). Depending on the virulence of the microorgan- rapidly in immediate neutrophilia, it is transient (lasting ism, extent of infection, and response of the host, the neu- only 20–30 minutes). Furthermore, immediate neutrophila trophil count can range from 7.0- 70 * 103/mcL, although is independent of bone marrow output and tissue egress the count is usually in the range of 10- 25 * 103/mcL. As (movement of neutrophils out of the circulation and into the the demand for neutrophils at the site of infection increases, tissues). Therefore, immediate neutrophilia is also referred the early response of the bone marrow is to increase out- to as pseudo-neutrophilia or demargination because no put of storage neutrophils to the peripheral blood, causing actual change in the number of neutrophils within the vas- a left shift. The inflow of neutrophils from the bone mar- culature occurs. The increased circulating neutrophils are row to the blood continues until it exceeds the neutrophil typically mature, normal cells. This redistribution of neutro- outflow to the tissues, causing an absolute neutrophilia. phils causes the physiologic neutrophilia that accompanies In very severe infections, the storage pool of neutrophils active exercise, epinephrine administration, anesthesia, and can become exhausted, the mitotic pool may be unable anxiety and can increase the neutrophil count by as much to keep up with the demand, and neutropenia develops.4 as twofold.2 Neutropenia in overwhelming infection is a poor prognos- Acute Neutrophilia Acute neutrophilia occurs when neu- tic indicator. Chronic bacterial infection can lead to chronic trophils egress from the bone marrow storage pool into stimulation of the marrow, whereby the production of neu- the peripheral blood. Within hours following a pathologic trophils remains high and a new steady state of production stimulus (e.g., bacterial infection, toxin) the circulating pool develops. of neutrophils can increase by as much as 10-fold.3 Neutrophilia is neither a unique nor an absolute finding The neutrophilia is far more pronounced than in in bacterial infections. Infections with other organisms such pseudo-neutrophilia, and the proportion of immature neu- as fungi, rickettsia, spirochetes, and parasites also can cause trophils can be increased. More bands appear if the tissue a neutrophilia. Certain bacterial infections are characterized demand for neutrophils creates an acute shortage of seg- by neutropenia rather than neutrophilia.4 Rarely, bacterial mented neutrophils in the bone marrow storage pool. Con- infections lead to lymphocytosis rather than neutrophilia, tinued demand in extreme circumstances can result in the release of metamyelocytes.2 As bone marrow production increases and the storage pool is replenished, the leukocyte differential returns to normal. Chronic Neutrophilia Chronic neutrophilia generally follows acute neutrophilia and occurs if the stimulus for neutrophils continues beyond a few days. This results in a depleted storage pool and increased production of the mitotic pool in an attempt to meet the demand for neutro- phils. In this state, the marrow shows increased numbers of early neutrophil precursors including myeloblasts, pro- myelocytes, and myelocytes. The blood contains increased numbers of bands, metamyelocytes, myelocytes, and (rarely) promyelocytes. An increase in the concentration of immature forms of leukocytes in the circulation is termed a shift to the left or left shift. Conditions Associated with Neutrophilia Chronic neutro- philia caused by benign or toxic conditions is usually charac- Figure 21.1 A leukemoid reaction. There is an increased leukocyte count and a left shift. The cells have heavy toxic terized by a total leukocyte count of less than 50 * 103/mcL granulation and Döhle bodies (arrows), suggesting an infectious or and a left shift. If immature cells are present they are usu- toxic reactive leukocytosis (peripheral blood, Wright-Giemsa stain, ally bands and metamyelocytes. In some instances, the 1000* magnification). Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 455 as is the case with whooping cough caused by Bordetella release toxic intracellular enzymes (granules) and oxygen pertussis infection. Although typically associated with a metabolites. These toxic substances mediate the inflamma- lymphocytosis, viruses can also cause neutrophilia in the tory process by injuring other body cells and propagating earliest stages of infection. the formation of additional chemotactic factors that attract more leukocytes. CASE STUDY (Continued from page 452) Leukemoid Reaction Extreme neutrophilic reactions to severe infections or necrotizing tissue can produce a Laboratory results on Dennis, the trauma patient, 2 leukemoid reaction (Figure 21-1). A leukemoid reaction is days after surgery are as follows: a benign proliferation of leukocytes characterized by (1) a WBC 14.5 * 103/mcL WBC total leukocyte count usually less than 50 * 103/mcL and Differential (2) numerous circulating leukocyte precursors.5 In a neu- trophilic-leukemoid reaction, the number of circulating Hb 12.9 g/dL Segmented 5% neutrophil precursors is increased including bands, meta- neutrophils myelocytes, myelocytes and promyelocytes, and rarely, PLT 180 * 103/mcL Band 50% blasts. neutrophils Leukemoid reactions can produce a blood picture Lymphocytes 40% indistinguishable from that of chronic myeloid leukemia Monocytes 5% (CML; Chapter 24). If a differential diagnosis cannot be determined by routine hematologic parameters, then cyto- Urine, blood, and wound cultures were ordered. genetic studies, molecular analysis, and leukocyte alkaline 1. What results, if any, are abnormal? phosphatase (LAP) stain scores can be helpful (Table 21-2). 2. Contrary to leukemia, a leukemoid reaction is transient, What is the most likely reason for these results? disappearing when the inciting stimulus is removed. A leukemoid reaction can occur in chronic infections (such as tuberculosis) and may present with toxic granules, Döhle Tissue Destruction/Injury, Inflammation, and Metabolic bodies, and vacuoles.5 In addition, leukemoid reactions can Alterations Conditions other than infection that may result accompany carcinoma of the lung, stomach, breast, or liver in a neutrophilia include tissue necrosis, inflammation, cer- and other inflammatory processes.6 tain metabolic conditions, and drug intoxication (Table 21-1). All of these conditions produce neutrophilia by increasing neutrophil egress from the bone marrow into the circulation Checkpoint 21.2 in response to increased neutrophil diapedesis to the tissue. How can CML be distinguished from a leukemoid reaction? Examples of these conditions include rheumatoid arthritis, tis- sue infarctions, burns, neoplasms, trauma, uremia, and gout. Leukoerythroblastic Reaction A leukoerythroblastic Although leukocytes defend the body against foreign reaction (Figure 21-2) is characterized by the presence substances, they also contribute to the inflammatory pro- of nucleated erythrocytes and a neutrophilic left shift in cess. Damaged tissue releases cytokines that act as chemo- the peripheral blood. The total neutrophil count can be taxins, causing neutrophils to leave the vessels and move increased, decreased, or normal. Erythrocytes in this condi- toward the injury site. In gout, for example, deposits of tion often exhibit poikilocytosis with teardrop shapes and uric acid crystals in joints attract neutrophils to the area. anisocytosis. Leukoerythroblastosis is most often associ- In the process of phagocytosis and death, the leukocytes ated with chronic neoplastic myeloproliferative conditions, Table 21.2 Comparison of Laboratory Results in Leukemoid Reactions and Chronic Myeloid Leukemia (CML) Leukemoid Reaction CML Leukocyte count Less than 50 * 103/mcL Usually greater than 50 * 103/mcL Leukocyte differential Shift to the left with bands, metamyelocytes, Shift to the left with immature cells including promyelocytes and myelocytes; neutrophil toxic changes and blasts; increased eosinophils and basophils Erythrocyte count Normal Often decreased Platelets Usually normal Increased or decreased Philadelphia chromosome Absent Usually present BCR/ABL1 gene mutation Absent Present Clinical Related to primary condition Systemic (splenomegaly, enlarged nodes, bone pain) 456 Chapter 21 Checkpoint 21.3 What is the difference between a leukemoid reaction and a leu- koerythroblastic reaction? NEUTROPENIA Neutropenia occurs when the neutrophil count is less than 1.592.0 * 103/mcL (varies depending on ethnicity). Agranulocytosis, a term that refers to a neutrophil count less than 0.5 * 103/mcL is associated with a high prob- ability of infection. Basophils and eosinophils are also com- monly depleted in severe neutropenia. True neutropenia can occur because of (1) decreased bone marrow production, (2) increased cell loss (from immune destruction or increased neutrophil egress to the tissue), or (3) pseudo-neutropenia (increased neutrophilic margination). An artificial neutropenia can result from neutrophil agglutination, disintegration, and laboratory Figure 21.2 A leukoerythroblastic reaction. There are instrument problems. Table 21-3 lists the most common nucleated erythrocytes and a left shift with band neutrophils causes of neutropenia (note that some causes of neutrope- and a myelocyte (peripheral blood, Wright-Giemsa stain, 1000* nia are also seen in neutrophilia). magnification). Decreased Bone Marrow Production Neutropenia can especially myelofibrosis, myelophthisis (replacement of develop as a result of decreased bone marrow production. normal hematopoietic tissue in the bone marrow by fibrosis, In this case, the bone marrow shows myeloid hypopla- leukemia, or metastatic cancer cells), and severe hemolytic sia, and the myeloid-to-erythroid (M:E) ratio is decreased anemias such as Rh hemolytic disease of the fetus and new- (Chapter 38). Defective neutrophil production depletes the born (HDFN; Chapter 19). bone marrow storage pool, decreases neutrophil egress to Stimulated Bone Marrow States Patients whose bone tissues, and reduces both the peripheral blood circulating marrow has been stimulated by hematopoietic growth and marginating pools. Immature cells may enter the blood factors or cytokines such as granulocyte monocyte-colony– in an attempt to alleviate the neutrophil shortage; however, stimulating factor (GM-CSF; CSF2) can produce a rapid cells younger than bands are less efficient in phagocytosis. increase of total white cell counts and release of leukocyte The end result is a lack of an adequate number of neutro- precursors including blasts. Cytokines are used to replenish phils |
at inflammatory sites, resulting in increased risk for leukocytes after bone marrow transplant, high-dose chemo- overwhelming infections. therapy, bone marrow failure, or prior to autologous blood Stem Cell Disorders Decreased bone marrow produc- donation or stem cell apheresis7 (Chapter 29). tion can occur when hematopoietic stem cells fail to pro- Corticosteroid therapy produces a neutrophilia that liferate as in aplastic anemia, following radiotherapy or occurs as a result of increased bone marrow output accom- panied by decreased migration of neutrophils to the tis- sues (by inhibition of the ability of neutrophils to adhere Table 21.3 Causes of Leukopenia and/or Neutropenia to vessel walls).8 This inhibition of neutrophil diapedesis can in part explain the increased incidence of bacterial Causes Examples infections in patients on steroid therapy even though the Infections Viral and overwhelming bacterial such as blood neutrophil count is increased. Steroids also decrease sepsis the number and inhibit the function of monocytes/ Physical agents and Radiation, benzene chemicals macrophages.9 Drugs Chemotherapy, certain drugs in the classes Physiologic Leukocytosis Physiologic leukocytosis and of sedatives, anti-inflammatory, antibacterial, antithyroid, and antihistamines neutrophilia are present at birth and for the first few days Hematologic disorders Acute leukemia, megaloblastic and aplastic of life. The leukocytosis can be accompanied by a slight left anemia, splenomegaly shift. Physiologic stressors including exposure to extreme Alloantibodies or Systemic lupus erythematosus temperatures, emotional stimuli, exercise, and labor dur- autoantibodies ing childbirth can cause neutrophilia, generally without a Hereditary or congenital Familial neutropenia, cyclic neutropenia left shift. disorders Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 457 chemotherapy, or with infiltration of hematopoietic tis- SBDS genes.12 Most disorders display neutrophil precursor sue by malignant cells (myelophthisis). Of new leukemia responsiveness to large doses of G-CSF.11 Familial neutrope- cases, 50% present with a total white count that is less than nia is a rare benign anomaly characterized by an absolute 5.0 * 103/mcL and an absolute neutrophil count that is decrease in neutrophils but usually a normal total leuko- less than 1.0 * 103/mcL.10 This leukopenia and neutrope- cyte count, and no infectious complications. It is transmitted nia occurs when normal precursor cells in the bone marrow as an autosomal dominant trait and is usually detected by are replaced by malignant cells, but the malignant cells have chance. Table 21-4 provides a more complete list of congeni- not egressed to the peripheral blood in significant numbers. tal neutropenic disorders.2,12 Megaloblastic Anemia Neutropenia is a characteristic find- Increased Cell Loss Neutropenia can occur as the result of ing in megaloblastic anemia (Chapter 16) and myelodys- increased neutrophil diapedesis. In severe or early infection, plastic syndromes (Chapter 25). In these cases, however, the the bone marrow may not produce cells as rapidly as they marrow is usually hyperplastic. Neutropenia results not from are being utilized, resulting in neutropenia. Various viral, marrow failure but from destruction of abnormal myeloid bacterial, rickettsial, and protozoan infections induce tissue precursors in the bone marrow (ineffective granulopoiesis). damage that increases the demand for and destruction of Chemicals/Drugs A wide variety of drugs and chemicals neutrophils. Marked toxic changes to the granulocytes often are associated with leukopenia and neutropenia if given in accompany neutropenia resulting from severe infections. sufficient dosage (Table 21-3). Chemotherapy and radia- Prognosis in these cases is very poor because the infecting tion treatments for cancer are nonselective and are com- organisms are able to prevail over the body’s immune system. mon causes of not only neutropenia but also pancytopenia. Immune Neutropenia Antibodies directed against neutrophil- The decreased bone marrow production of granulocytes specific antigens can cause a decrease in the number of predisposes patients receiving treatment for malignan- neutrophils. Leukocytes are destroyed in a manner similar cies to frequent and serious infections. Chemotherapeutic to erythrocytes in immune hemolytic anemia (Chapter 17). drugs induce apoptosis in mitotically active cells by a vari- In some cases, drugs precipitate an immunologic response ety of mechanisms including direct DNA damage, altering leading to a sudden disappearance of neutrophils from the folate receptors, or inhibiting enzymes needed for mitosis. circulation. The immunologic mechanism can include direct Although the action of chemotherapeutic drugs reduces cancer cell proliferation, normal hematopoietic precursor cells in the bone marrow also are actively dividing and Table 21.4 Congenital Neutropenic Disorders their proliferation is inhibited as well. Prophylactic use of antibiotics and GM-CSF has reduced mortality in patients Disorder Inheritance receiving chemotherapy, but infections due to neutropenia Disorders of production remain a serious complication.9 Drug-induced neutropenia Cyclic neutropenia AD also can result from allergic (immunologic) reactions to Familial neutropenia AD drugs. Women, older patients, and patients with a history Fanconi pancytopenia AR of allergies are more commonly affected. Reticular dysgenesis AR Congenital Neutropenia Several rare inherited disorders Severe congenital neutrophilia AD, AR cause neutropenia related to decreased bone marrow pro- Wiskott-Aldrich syndrome XLR duction. Periodic or cyclic neutropenia is a curious form of Disorders of RNA synthesis and processing neutropenia that begins in infancy or childhood and occurs Cartilage-hair hypoplasia AR in regular 21- to 30-day cycles. Cyclic neutropenia is inher- Dyskeratosis congenital XLR, AD, AR ited as an autosomal dominant trait and is due to mutations Shwachman-Diamond syndrome AR in the gene for neutrophil elastase (ELANE).10 The severely Disorders of metabolism neutropenic period (less than 0.5 * 103/mcL) lasts for sev- Barth syndrome AR, XLR eral days and is marked by frequent infections. Between Glycogen Storage disease, type 1b AR the neutropenic attacks, the patient is asymptomatic. Severe Pearson’s syndrome Mitochondrial congenital neutropenias include a heterogenous group of Disorders of vesicular transport rare, often fatal disorders marked by extreme neutropenia Chédiak-Higashi syndrome AR (less than 0.2 * 103/mcL). The total leukocyte count is often Cohen syndrome AR within the reference interval. One form of SCN is inherited Griscelli syndrome, type II AR in an autosomal dominant manner and involves mutations in the neutrophil elastase gene. Others, such as Kostmann Hermansky-Pudlak syndrome, type II AR syndrome, display autosomal recessive inheritance, and p14 Deficiency Probable AR involve a number of other genes, including the HAX1 and AD, autosomal dominant; AR, autosomal recessive; XLR, X-linked recessive. 458 Chapter 21 cell lysis or sensitization and subsequent sequestration of False Neutropenia It is important for the laboratory profes- neutrophils in the spleen.2 The two types of immune neu- sional to recognize when neutropenia is a result of labora- tropenia are alloimmune and autoimmune. tory in vitro manipulations of blood. Four in vitro causes of Alloimmune neonatal neutropenia occurs when mater- a low neutrophil count include: nal antibodies are directed against paternal-origin antigens • EDTA-induced neutrophil adherence to erythrocytes on fetal neutrophils and are transferred across the placenta. Affected infants are susceptible to infections for up to 4 • Disintegration of neutrophils over time prior to testing months or until the neutropenia is resolved.2 This immune • Disruption of abnormally fragile leukocytes during process is similar to that found in hemolytic disease of the preparation of the blood for testing fetus and newborn (HDFN) except that the firstborn child • Neutrophil aggregation can be affected. Alloimmune neutropenia can also be the result of a transfusion reaction. Neutrophils can adhere to erythrocytes (rarely) when the Autoimmune neutropenia (AIN) is categorized as blood is drawn in EDTA, causing an erroneously low auto- either primary or secondary. Primary AIN is not associated mated white count. If observed on the stained smears, blood with other diseases and occurs predominantly in young can be recollected by finger stick to make manual dilutions children. This condition of unknown etiology develops as and blood smears without utilizing EDTA (Chapter 37). antibody-coated neutrophils are sequestered and destroyed Neutrophils disintegrate in blood collection tubes faster by the spleen. Patients develop fever and recurrent infec- than other leukocytes. If there is a delay in testing the blood, tions. Spontaneous remission usually occurs after a period the neutrophil count can be erroneously decreased. In some of 13–20 months. Secondary autoimmune neutropenia gen- pathologic conditions, the leukocytes are more fragile than erally occurs in older patients, many of whom have been normal and can rupture with the manipulations of prepar- diagnosed with another autoimmune disorder such as sys- ing blood for testing in the laboratory. Finally, the neutro- temic lupus erythematosus (SLE) or rheumatoid arthritis phil count can be falsely decreased if the neutrophils clump (AR). In secondary AIN, antineutrophil antibodies are not together because of the presence of paraproteins (proteins the only cause of the neutropenia, and the actual target of appearing in large quantity because of other pathological the antibodies is not always known.13 conditions). Infections associated with AIN are not usually life threatening and are treated with routine antibiotic therapy. Checkpoint 21.4 Intravenous doses of immunoglobulin can be used in severe How can the correct white cell count be determined when neu- cases. The total leukocyte count is usually normal or near trophils clump in the presence of EDTA? normal, but the neutrophil count is decreased. G-CSF ther- apy can be indicated for patients with severe infections. Immune neutropenia is difficult to diagnose, but is Qualitative or Morphologic aided by testing for antineutrophil antibodies or neutrophil Abnormalities surface antigens by various methods including agglutina- Automated hematology analyzers do not detect or flag tion tests, immunofluorescence, and enzyme-linked immu- neutrophil morphologic abnormalities. Laboratory profes- nosorbent assay (ELISA). The availability of these complex sionals must microscopically evaluate stained blood smears tests varies widely among laboratories. to identify cytoplasmic and/or nuclear morphologic abnor- Hypersplenism Hypersplenism can result in a selective malities in neutrophils. Cytoplasmic abnormalities are the splenic culling of neutrophils producing mild neutropenia. most common, and most cytoplasmic changes (Döhle bod- The bone marrow in this case exhibits neutrophilic hyper- ies, toxic granulation, and vacuoles) are reactive, transient plasia. Thrombocytopenia and (occasionally) anemia can changes accompanying infectious states. The correct identi- also accompany hypersplenism. fication of alterations such as intracellular microorganisms Pseudo-Neutropenia Pseudo-neutropenia is similar to can lead to the prompt diagnosis and treatment of life- pseudo-neutrophilia in that it is produced by alterations in threatening infections, whereas recognition of Pelger-Huët the circulating and marginated pools. Pseudo-neutropenia or hypersegmented neutrophils can point to the diagnosis results from the transfer of circulating neutrophils to the of specific conditions that can prove elusive without the marginated neutrophil pool with no change in the total morphologic information. peripheral blood neutrophil pool. This temporary shift is NUCLEAR ABNORMALITIES characteristic of some infections with endotoxin production Pelger-Huët Anomaly Pelger-Huët anomaly (Figure 21-3) and of hypersensitivity reactions. Because of the selective is a benign anomaly inherited in an autosomal dominant margination of neutrophils, the total leukocyte count drops fashion and occurs in about 1 in 5000 individuals. The neu- and a relative lymphocytosis develops. trophil nucleus does not segment beyond the two-lobed Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 459 a b Figure 21.3 Pelger-Huët anomaly. (a) Note pince-nez, or eyeglass-shaped, nuclei. These mature cells could easily be confused for bands if not for the highly clumped chromatin. (b) The nucleated neutrophil is the nonsegmented or round nucleus form that can be found (peripheral blood, Wright-Giemsa stain, 1000* magnification). stage and can appear as a single, round nucleus with no likely appear in a blood picture with far more than 10% segmentation. The presence of excessive coarse chroma- (likely more than 50%) of the neutrophils showing three or tin clumping in the nucleus aids in the differentiation of more nuclear lobes and possibly immature myeloid cells.18 bilobed cells from band neutrophils. The bilobed nucleus Hypersegmentation Larger-than-normal neutrophils with has a characteristic dumbbell shape with the two lobes six or more nuclear segments (hypersegmented neutro- connected by a thin strand of chromatin. Cells with this phils) or five or more neutrophils with five lobes are com- appearance are often called pince-nez cells (French name mon and early indicators of megaloblastic anemia. These for the style of eyeglasses without earpieces). Rod-shaped cells are found together with pancytopenia and macro and peanut-shaped nuclei can also be found. Individuals ovalocytes that typically accompany deficiencies of folate heterozygous for Pelger-Huët anomaly have neutrophil or vitamin B12 (Chapter 15). Reported cases of hereditary nuclei that are primarily bilobed, whereas the majority of hypersegmentation of neutrophils, a benign condition, are neutrophils in homozygous individuals have a round or rarely significant but need to be distinguished from multi- oval nucleus with only a few cells having the classic bilobed lobed nuclei that are associated with disease.19 shape.16 |
The cell is functionally normal, and individu- als with hereditary Pelger-Huët anomaly do not display Pyknotic Nucleus Pyknotic, or apoptotic, nuclei (Figure increased susceptibility to bacterial infections.16 The signifi- 21-4) are found in dying neutrophils in blood or body fluid cance of recognizing this anomaly lies in differentiating the benign hereditary condition from a left shift that can occur during infections. Acquired- or pseudo-Pelger-Huët anomaly can pres- ent in myeloproliferative disorders and myelodysplastic states. The neutrophils in pseudo-Pelger-Huët anomaly (sometimes described as pelgeroid) are frequently hypo- granular because of a lack of secondary granules, and the nuclei acquire a round rather than a dumbbell shape. The chromatin appears with intense clumping, aiding in differ- entiation of these mononuclear cells from myelocytes. It is particularly important that laboratory pro- fessionals distinguish between the Pelger-Huët and pseudo-Pelger-Huët anomalies because the presence of the acquired form can be used to aid in the diagnosis of myelodysplasia and malignancy.17 To distinguish the two, the inherited Pelger-Huët anomaly usually presents with Figure 21.4 The nucleated cell at the top is a dying neutrophil less than 10% of the neutrophils displaying three or more with a pykonotic nucleus. Note the smooth nuclear material that nuclear lobes without the presence of immature myeloid cells is breaking up (peripheral blood, Wright-Giemsa stain, 1000* (meta- and myelocytes). Pseudo-Pelger-Huët neutrophils magnification). 460 Chapter 21 preparations. The nuclear chromatin condenses and the seg- ments disappear, becoming smooth, dark-staining spheres. If the nucleus is round, these apoptotic cells can be confused with nucleated erythrocytes. Checkpoint 21.5 Describe the difference between hypersegmented and hypo- segmented neutrophils and pyknotic nuclei. In what conditions are each seen? CYTOPLASMIC ABNORMALITIES Cytoplasmic inclusions are often found in infectious states and when present, give the health care provider important diagnostic information (Table 21-5). These inclusions are Döhle bodies, toxic granules, vacuoles, and intracellular organisms. Figure 21.5 Arrow points to neutrophil with three bluish inclusions (Döhle bodies) (peripheral blood; Wright-Giemsa stain, Döhle Bodies Döhle bodies are light gray-blue oval 1000* magnification). inclusions in the cytoplasm of neutrophils and eosinophils (Figure 21-5). Found near the periphery of the cell, Döhle bodies are composed of aggregates of rough endoplasmic are discharged to fight bacteria. Toxic granulation is seen reticulum. They can be seen in severe infections, burns, in the same conditions as Döhle bodies. It is important to and cancer and as a result of toxic drugs. Döhle bodies distinguish toxic granules and inclusions from toxic gran- should be looked for whenever toxic granulation or other ules or inclusions can appear as artifacts that can occur reactive morphologic changes are present because they fre- with increased staining time or decreased pH of the buffer quently occur together. Döhle bodies are similar in appear- used in the staining process. True toxic granulation will not ance to the cytoplasmic inclusions found in May-Hegglin appear equally in all neutrophils. anomaly (described later in the “May-Hegglin Anomaly” Cytoplasmic Vacuoles Cytoplasmic vacuoles appear as section). clear, unstained circular areas. Vacuoles probably repre- Toxic Granules Toxic granules are large, deeply stained, sent the end stage of phagocytosis (Figure 21-7). They are blue-black primary granules in the cytoplasm of segmented usually seen in the same conditions as toxic granulation neutrophils and sometimes in bands and metamyelocytes and Döhle bodies. Cytoplasmic vacuoles in neutrophils (Figure 21-6). Primary (nonspecific) granules normally from a fresh specimen correlate highly with the presence lose their basophilia as the cell matures, so even though of septicemia. about one-third of the granules in the mature neutrophil Vacuoles can also appear as an artifact in smears made are primary granules, their presence is not observable by from blood that has been collected and stored in EDTA. light microscopy. In contrast, toxic primary granules retain Vacuoles related to storage are more likely to be smaller their basophilia in the mature neutrophil, perhaps because and more uniformly dispersed than those in toxic states. of a lack of maturation. Additionally, toxic primary gran- Making smears from fresh blood without anticoagulant can ules can become more apparent as the secondary granules eliminate the vacuole artifact. Table 21.5 Cytoplasmic Inclusions Found in Neutrophils in Infectious Conditions Inclusion Morphologic Characteristics Composition Associated Conditions Döhle body Light gray-blue oval near cell periphery Rough endoplasmic reticulum (RNA) Infections, burns, cancer, or inflammatory states Toxic granules Large blue-black granules Primary granules Same as Döhle body Cytoplasmic vacuole Clear, unstained circular area Open spaces from phagocytosis Same as Döhle body Bacteria Small basophilic rods or cocci Phagocytized organisms Bacteremia or sepsis Fungi Round or oval basophilic inclusions Phagocytized fungal organisms Systemic fungal infections often in slightly larger than bacteria immunosuppressed patients Morulae Basophilic, granular; irregularly shaped Clusters of Ehrlichia rickettsial Ehrlichiosis organisms Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 461 Figure 21.6 A segmented neutrophil and a band neutrophil Figure 21.7 Intracellular microorganisms. Note the vacuoles with toxic granulation (peripheral blood, Wright-Giemsa stain, and toxic granulation in the cells (peripheral blood, Wright-Giemsa 1000* magnification). stain, 1000* magnification). agent of human granulocytic ehrlichiosis. Ehrlichia sp. are CASE STUDY (Continued from page 455) tick-borne, small, obligate intracellular, coccobacilli bac- All cultures were negative for bacteria and fungi. teria. They infect leukocytes where they multiply within On further examination of the blood smear, it was phagosomes. The intracellular organisms are pleomorphic, noted that no abnormal cytoplasmic features were appearing as basophilic, condensed, or loose aggregates of observed in the neutrophils. However, the nuclei of organisms, which tend to appear spherical. most of the bands appeared more condensed than Ehrlichiosis is characterized by high fever, leukopenia, normal, and many had two lobes in a dumbbell thrombocytopenia, and elevated liver enzymes.20,21 Most shape. cases of ehrlichiosis occur in April through September when nymphal ticks are most active. The intracellular microcol- 3. Given the leukocyte morphology and cul- onies of Ehrlichia, called morulae, are observed in leuko- tures, what additional condition must now be cytes on stained blood films (Figure 21-8). The presence of considered? morulae in leukocytes can be the first diagnostic finding of 4. Explain the clinical significance of the nuclear Ehrlichia infection. The leukocyte eventually ruptures and anomaly described in Dennis. releases the organisms that then infect other leukocytes. The pathogenesis of ehrlichiosis can be related to direct cellu- 5. Why is the white cell count elevated? lar injury by the bacteria or a cascade of inflammatory or immune events. Intracellular Organisms In most infections, including sep- Confirmation of infection is made through serologic ticemia, the causative agents are not demonstrable in the determination of antibody titers using indirect fluorescent peripheral blood. However, microorganisms seen inside neutrophils should always be considered a significant finding, and the physician should be notified immediately (Figure 21-7). Intracellular Histoplasma, or Candida, is sometimes found on blood smears from patients with HIV or other severe immunosuppression. Organisms found outside cells must be interpreted with care. On stained blood smears, it is cru- cial to distinguish whether the microorganisms came from contaminated equipment or stain or are actually present in the patient’s blood. Organisms must also be distinguished from other cytoplasmic material and precipitated stain. All bacteria and yeasts stain basophilic with Wright’s stain. In the United States, two known species of Ehrlichia Figure 21.8 Morulae in ehrlichiosis. This segmented infect humans. E. chaffeensis infects monocytes and is the neutrophil from a patient with human granulocytic ehrlichiosis causative agent of human monocytic ehrlichiosis (HME), contains two dense, basophilic inclusions called morulae (peripheral whereas E. ewingii infects granulocytes and is the causative blood, Wright-Giemsa stain, 1000* magnification). 462 Chapter 21 antibody (IFA) assays or by identification of DNA sequences by polymerase chain reaction (PCR). Peripheral blood cyto- penia is probably the result of sequestration of infected cells in the spleen, liver, and lymph nodes. The bone marrow is usually hypercellular.22,23 Anaplasma phagocytophilum also infects neutrophils and is the causative agent of human granulocytic anaplasmosis (HGA). The clinical manifestations and laboratory findings are similar to that of Ehrlichia infection, and morulae are observed in the neutrophils.20 INHERITED FUNCTIONAL ABNORMALITIES Functional neutrophil abnormalities are almost always inherited and can be accompanied by morphologic abnor- Figure 21.9 Lymphocyte from Hurler’s disease malities. It is suggested that granulocyte functional abnor- (mucopolysaccharidoses). Note the halo around the granules malities be suspected in patients with recurrent, severe (peripheral blood, Wright-Giemsa stain, 1000* magnification). infections, abscesses, and delayed wound healing and in mutations of the CHS1 gene, which codes for a lysosomal antibiotic resistant sepsis. See Table 21-6 for a summary of trafficking regulatory protein. CHS is characterized by the functional defects and their clinical features. defects in granule morphogenesis, and death usually occurs Alder-Reilly Anomaly Alder-Reilly anomaly is an inherited during the first or second decade of life because of recurrent condition characterized by the presence of large purplish bacterial infections (Figure 21-10). Giant gray-green perox- granules in the cytoplasm of all leukocytes. Similar mor- idase-positive bodies and giant lysosomes are found in the phology is seen in disorders such as Hurler’s syndrome and cytoplasm of leukocytes as well as most granule-containing Hunter’s syndrome, both of which are lysosomal storage cells of other tissues. These bodies are formed by fusion of disorders (discussed later in the “Monocyte/Macrophage primary, nonspecific azurophilic granules. This abnormal Disorders” section; see also Figure 21-9). These disorders are fusion of cytoplasmic membranes prevents the granules characterized by incompletely degraded mucopolysaccha- from being delivered into the phagosomes to participate in rides that accumulate in the lysosomes and appear as large killing of ingested bacteria. Neutropenia and thrombocyto- granules.24 The granules can resemble toxic granulation in penia are frequent complications as the disease progresses. neutrophils and can also appear in lymphocytes. They tend The patients have skin hypopigmentation, silvery hair, and to occur in clusters in the shape of dots or commas and are photophobia from an abnormality of melanosomes. Lymph- surrounded by vacuoles. The inclusions frequently are seen adenopathy and hepatosplenomegaly are characteristic.25 only in cells of the bone marrow, not in the peripheral blood, May-Hegglin Anomaly May-Hegglin anomaly is a rare, but in either case, the cells function normally. inherited, autosomal dominant trait in which granulocytes Chédiak-Higashi Syndrome Chédiak-Higashi syndrome contain inclusions consisting mainly of RNA from rough (CHS) is a rare autosomal recessive disorder associated with endoplasmic reticulum, which are similar in appearance to Table 21.6 Inherited Qualitative Neutrophil Abnormalities Condition Morphologic or Functional Defect Clinical Features Alder-Reilly anomaly Large, purplish cytoplasmic granules in all leukocytes Associated with mucopolysaccharidosis such as Cells function normally Hurler’s syndrome Chédiak-Higashi syndrome Giant fused granules in neutrophils and lymphs Serious, often fatal condition with repeated pyrogenic Cells engulf but do not kill microorganisms infections May-Hegglin anomaly Blue, Döhle-like cytoplasmic inclusions in all Bleeding tendency from associated thrombocytopenia granulocytes Cells function normally Chronic granulomatous disease (CGD) Defective respiratory burst Recurrent infections, especially in childhood Cells engulf but don’t kill microorganisms Myeloperoxidase deficiency Low or absent myeloperoxidase enzyme Usually benign; other bactericidal systems prevent Cell morphology normal most infections Leukocyte adhesion deficiency (LAD) Absence of cell-surface adhesion proteins affecting Serious condition with recurrent infections and high multiple cell functions mortality Cell morphology normal Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 463 from mutations in any of the five genes that encode the sub- units of the NADPH oxidase.25 Patients suffer from recur- rent, often life-threatening, infections with opportunistic pathogens (bacterial and fungal) that result in the forma- tion of granulomas. CGD can be diagnosed in childhood but in some cases, the onset of symptoms may not occur until early adulthood. The affected cells in CGD are morphologically normal but cannot generate antimicrobial oxygen metabolites, such as H2O2. Therefore, the neutrophils can phagocytize micro- organisms but cannot kill them. Ingested microorganisms produce hydrogen peroxide as a by-product of oxidative a b metabolism; those that are catalase-positive are not killed because they destroy the H2O2 of their own metabolism. Figure 21.10 (a) Neutrophil from Chédiak-Higashi syndrome. Catalase-positive organisms continue to grow intracellu- (b) Lymphocyte from the same patient as in a. Note the bluish-gray inclusion bodies (peripheral blood, Wright-Giemsa stain; 1000* larly where they are protected from antibiotics, whereas magnification). catalase-negative organisms kill themselves by generat- ing H2O2 that can be used in the phagocytic cell’s myelo- Döhle bodies (Figure 21-11). The inclusions can be distin- peroxidase-dependent antimicrobial defense mechanisms guished from true Döhle bodies because they are usually (Chapter 7). larger and rounder in shape.26 The Döhle body-like inclu- The peripheral blood |
neutrophil count is normal but sions result because of a defect in the myosin heavy-chain increases in the presence of infection. Immunoglobulin lev- type IIA gene (MYHA gene) that leads to the precipitation of els are often increased because of chronic infection.28 Treat- non-muscle myosin heavy chains. The neutrophils function ment involves the use of prophylactic antibiotics and early normally. Variable thrombocytopenia with giant platelets treatment of infections. The nitroblue tetrazolium slide test is characteristic. The only apparent clinical symptom that (NBT) is useful in detecting the abnormal oxygen metabo- patients may exhibit is abnormal bleeding related to the low lism of neutrophils in CGD. Neutrophils are mixed with platelet count. nitroblue tetrazolium and microorganisms. In normal indi- Chronic Granulomatous Disease Chronic granulomatous viduals, the leukocytes phagocytize the microorganisms, disease (CGD) is an inherited disorder (65% X-linked, 35% initiating an increase in oxygen uptake. This process leads autosomal recessive)27 characterized by defects in the respi- to an accumulation of oxygen metabolites that reduce the ratory burst oxidase (NADPH oxidase) system. CGD results NBT to a blue-black compound, which shows in the cell as dark crystals. Neutrophils from individuals with CGD cannot mobilize a respiratory burst, so no dark crystals appear.29 The dihydrorhodamine 123 (DHR123) assay using flow cytometry is replacing the NBT test in some laboratories. The DHR123 assay (also referred to as the neutrophil oxida- tive burst assay), incubates granulocytes with bacteria and the dye DHR123.30 After the neutrophils phagocytize the bacteria, they activate nicotinamide adenine dinucleotide phosphate oxidase and produce reactive oxygen metabo- lites (the respiratory burst). These metabolites oxidize DHR123 to fluorescent rhodamine 123, which is detected by flow cytometry. In healthy adults, the reference interval for granulocytes with phagocytic activity is 80–100%. Myeloperoxidase Deficiency Myeloperoxidase (MPO) deficiency is, for the most part, a benign autosomal reces- sive disorder characterized by an absence of myeloperoxi- dase in neutrophils and monocytes. Although neutrophils Figure 21.11 May-Hegglin anomaly. There is a neutrophil use MPO in the bactericidal process, an increase in bacterial with a Döhle-like structure in the cytoplasm and a large platelet infections is not usually seen in MPO deficiency, even in (arrow; peripheral blood, Wright-Giemsa stain, 1000* magnification). homozygous individuals. The neutrophils are able to utilize 464 Chapter 21 alternative antimicrobial systems to kill the microorganisms thrombasthenia (Chapter 33). LAD III is associated with (although somewhat more slowly; Chapter 7). However, mutations in KINDLIN-3 a protein involved in integrin patients with MPO deficiency and diabetes mellitus can activation,25 experience disseminated fungal infections (usually with Candida albicans).31 Checkpoint 21.6 Certain hematology analyzers can detect the presence Explain how you can determine whether toxic granulation and or absence of MPO in a two-stage cytochemical reaction that vacuoles in the neutrophils are due to the patient’s condition differentiates cells based on stain and size characteristics. or to artifact. Regarding MPO, neutrophils, eosinophils, and monocytes are usually positive and basophils and lymphocytes are negative. The neutrophils in patients with MPO deficiency are depicted as large, unstained cells. On peripheral blood Eosinophil Disorders smears, the neutrophils appear morphologically normal. Disorders involving exclusively eosinophils are rare. An Leukocyte Adhesion Deficiency There are three forms of increase in the circulating number of these cells can indicate leukocyte adhesion deficiency, LAD I, LAD II and LAD III. a potentially serious condition. Because the lower limit of the Features of all three include recurrent soft tissue bacterial reference interval is very low, a decrease is difficult to deter- and fungal infections with persistent leukocytosis and gran- mine and is probably not significant. Eosinopenia can be seen ulocytosis due to increased stimulation of the bone marrow, in acute infections and inflammatory reactions and with the and decreased neutrophil emigration into the tissues. Treat- administration of glucocorticosteroids. Glucocorticosteroids ment includes prophylactic antibiotics and early, aggressive and epinephrine inhibit eosinophil release from the bone treatment of infections. Mortality rate in childhood can be marrow and increase their margination.32 Increased eosin- high for severe cases of Type I and Type III LAD, and bone ophils, hypereosinophilia (HE), can be classified as clonal marrow transplantation is recommended.25 (neoplastic, also called primary) or reactive (non-clonal, also LAD I is a rare, autosomal recessive disorder char- called secondary).33 In either instance, the HE may or may acterized by decreased or absent leukocyte cell-surface not be associated with specific HE-mediated organ damage, adhesion proteins (b2@integrins, also termed CD11/CD18 referred to as hypereosinophilic syndrome (HES).34 In addi- complex; Chapter 7). Neutrophils from patients with LAD tion there are rare cases of familial or hereditary HE. Finally, I have multiple functional defects, including impaired occasionally HE may be found in the absence of evidence of adhesion to endothelial cells, chemotaxis, phagocytosis, the previous three conditions, and is referred to as idiopathic respiratory burst activation, and degranulation. Because of HE, or HE of undetermined/unknown significance.34 defective adhesion proteins, the neutrophils cannot adhere to endothelial cells of the blood vessel walls and exit the Nonclonal (Reactive) circulation. In addition, LAD I neutrophils are not able to recognize the presence of the complement C3bi fragment on Hypereosinophilia microorganisms, so phagocytosis is not stimulated. The fre- Hypereosinophilia refers to an increase in eosinophils more quency and severity of the infections in LAD I depends on than 0.4 * 103/L. Reactive HE appears to be induced by the amount of CD11/CD18 the cells express. Diagnosis can cytokines secreted from T lymphocytes. This type of eosin- be made by flow cytometric analysis of neutrophil CD11b ophilia is polyclonal and the common myeloid progenitor levels using a monoclonal antibody.29 (CMP) cell is normal. Various conditions associated with LAD II also is a rare autosomal recessive disorder the cellular immune response (mediated by T lymphocytes) characterized by failure of endothelial cells to synthesize are characterized by HE including tissue-invasive parasites, the ligand for leukocyte L-selectin (Sialy@Lex). Leukocyte allergic conditions, respiratory tract disorders, gastrointes- function is normal, but the cells fail to adhere to the vessel tinal diseases, and skin and connective tissue disorders and wall endothelium and cannot exit the circulation to enter diseases.34 Parasites are the most common cause of secondary the tissues in host defense responses. LAD II red cells are HE worldwide. When the eosinophil concentration is high Lewis antigen negative, and have the rare Bombay (hh) and immature forms are present, the blood picture can resem- phenotype. These cells are unable to form certain fucose ble that seen in chronic eosinophilic leukemia (Chapter 24). carbohydrate linkages. The mutation in LAD II is in a mem- The conditions associated with HE are listed in Table 21-7. brane fucose transporter, and results in a generalized loss Tissue invasion by parasites produces an eosinophilia of expression of fucosylated molecules on the cell surface.25 more pronounced than parasitic infestation of the gut or LAD III has only been described in a small number of blood. Eosinophils are especially effective in fighting tis- patients. In addition to defects in leukocyte adhesion, the sue larvae of parasites; they readily adhere to larvae coated patients have a bleeding disorder similar to Glanzmann with IgG, IgE, and/or complement. Larvae are too large Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 465 Table 21.7 Conditions Associated with Quantitative Changes of Eosinophils, Basophils, and Mast Cells Eosinopenia Acute infections, inflammatory reactions, administration of glucocorticosteroids Hypereosinophilia (HE) Nonclonal (reactive) Parasitic infection Allergic conditions, especially asthma, dermatitis, and drug reactions Clonal (neoplastic) CEL, NOS; eosinophilia with abnormalities of PDGFRA, PDGFRB, or FGFR1, and eosinophilia that occurs in other MPNs Idiopathic No known cause Familial Unknown Basopenia Inflammatory states following immunologic reactions Basophilia Immediate hypersensitivity reactions Endocrinopathies Infectious diseases Figure 21.12 Hypereosinophilic syndrome. Note that the cells Chronic myeloproliferative disorders, especially are all mature eosinophils (peripheral blood, Wright-Giemsa stain, CML; chronic basophilic leukemia 1000* magnification). Mastocytosis Immediate hypersensitivity reactions Connective tissue disorders Infectious diseases neoplastic disorder with mutations in genes that code for Neoplastic disorders such as lymphoprolifera- platelet derived growth factor receptor (PDGFR) or fibro- tive diseases and hematopoietic stem cell diseases blast growth factor receptor 1 (FGFR1). These disorders are considered myeloproliferative neoplasms and are discussed CEL, NOS, chronic eosinophilic leukemia, not otherwise specified; CML, chronic myeloid leukemia; FGFR1, fibroblast growth factor receptor 1; MPN, myeloproliferative neoplasm; in Chapter 24. PDGFR, platelet-derived growth factor receptors (PDGFRA, PDGFRB) Idiopathic Hypereosinophilia for phagocytosis, so the eosinophil molds itself around the In other cases, the cause for the HE is unknown and clon- larva to destroy it. Intracellular eosinophilic granules fuse ality cannot be proven. These disorders are collectively with the eosinophil membrane and expel their contents into known as idiopathic HE. The identification of genetic muta- the space between the cell and the larva. The granular sub- tions has decreased the number of cases that are considered stances attack the larva wall, partially digesting it.35 idiopathic.34 A moderate increase in eosinophils frequently char- Chronic HE can cause extensive tissue damage as acterizes allergic disorders (asthma, dermatitis, and drug the granules are released from disintegrating eosinophils reactions). Large numbers can also be found in nasal dis- hypereosinophilic syndrome (HES). In many HES cases, charges and sputum of allergic individuals as well as in the large numbers of circulating eosinophils damage the heart. peripheral blood. Charcot-Leyden crystals formed from either eosinophil HE also accompanies a disorder termed pulmonary cytoplasm or granules can be found in exudates and tissues infiltrate with eosinophilia (PIE) syndrome characterized where large numbers of eosinophils migrate and disinte- by asthma, pulmonary infiltrating eosinophils, central ner- grate. Treatment of HES with corticosteroids, hydroxyurea, vous system anomalies, peripheral neuropathy, polyateritis and/or a@interferon is sometimes effective in reducing the nodosa, and local or systemic eosinophilia. This syndrome eosinophil count. HES is most commonly seen in males can be produced by parasitic or bacterial infections, allergic (more than 90%) and is rarely seen in children.37 reactions, or collagen disorders. In some cases, no cause can Idiopathic HES must be differentiated from reactive be found.36 eosinophilias and clonal eosinophilias (such as eosinophilic leukemia). Eosinophilic leukemia usually presents with Clonal (Neoplastic) myeloblasts and eosinophilic myelocytes, whereas idio- Hypereosinophilia pathic HES and reactive eosinophilia present with mature eosinophils. In addition, an abnormal clonal chromosome Primary HE is characterized by a persistent blood eosino- karyotype or molecular mutation suggests eosinophilic leu- philia of more than 1.5 * 103/mcL with tissue infiltration kemia or another clonal variant rather than idiopathic HES (Figure 21-12). It may be a clonal myeloid or lymphoid or reactive eosinophilia (Chapter 24). 466 Chapter 21 Basophil and Mast Cell gene rearrangement suggests the diagnosis of acute baso- philic leukemia, an extremely rare condition. Disorders A decrease in basophils is even more difficult to establish than eosinopenia. Scanning a blood smear with The cells’ cytoplasmic granule-associated preformed media- 100* magnification will reveal a rare basophil in normal tors, including histamine and certain proteases, their lipid individuals. Decreases in basophils are seen in inflamma- mediators (such as prostaglandin D2 and leukotriene C4), tory states and following immunologic reactions. which are generated upon activation of the cells, and their Mastocytosis can be found in a number of disorders cytokines, growth factors, and chemokines contribute to in which the number of mast cells is increased as much as many of the characteristic signs and symptoms of these dis- fourfold in affected tissues (most commonly the skin) and eases. However, mast cells and basophils also contribute to in conjunction with certain neoplastic disorders (Table 21-7). protective host responses associated with IgE production, No clinical disorder that involves a decrease in mast cells especially those directed against parasites. has been identified; however, long-term treatment with glu- Both basophils and mast cells are important in inflamma- cocorticoids can lower the number of mast cells.38 tory and immediate allergic reactions. They both express the receptor for immunoglobulin (Ig) E (FceRI). Their immune Checkpoint 21.7 function can be IgE-dependent (IgE binds to high-affinity Why are the basophil and eosinophil counts important when receptors on these cells) or IgE-independent (triggered by assessing the benign or neoplastic nature of a disorder? complement fragment C5a binding to C5a receptors on these cells). Basophils and mast cells mediate an immune response through granules that contain histamine and some proteases, lipid molecules (i.e., prostaglandin), and cytokines, growth Monocyte/Macrophage factors, and chemokines. Extracellular degranulation can be induced by physical destruction of the cell, chemical sub- Disorders stances (toxins, venoms, proteases), endogenous mechanisms Quantitative disorders are associated with monocytes, (tissue proteases, |
cationic proteins), and immune mechanisms. whereas qualitative disorders are associated with both The number of basophils and mast cells increases at monocytes and macrophages. The qualitative disorders are sites of inflammation. Basophils migrate from the blood by inherited lysosomal storage disorders. adhering to the endothelium (mediated by several families of adhesion molecules and receptors).38 Basophilia refers to an increase in basophils greater Quantitative Disorders than 0.2 * 103/mcL and is associated with immediate Monocytosis occurs when the absolute monocyte count hypersensitivity reactions and chronic myeloproliferative is more than 0.8 * 103/mcL (Figure 21-14). It is seen most disorders (Table 21-7, Figure 21-13). An absolute basophilia often in inflammatory conditions and certain malignancies is often helpful in distinguishing CML from a leukemoid (Table 21-8). Monocytosis occurring in the recovery stage reaction or other benign leukocytosis. A basophil count of acute infections and in agranulocytosis is considered a exceeding 80% of the total leukocyte population and the favorable sign. Monocytes also play an important role in the absence of the Philadelphia chromosome and/or BCR/ABL1 cellular response against mycobacterium in tuberculosis. Figure 21.13 Peripheral blood from a patient with CML and Figure 21.14 Peripheral blood from a patient with reactive 30% basophils (peripheral blood, Wright-Giemsa stain, 1000* monocytosis showing two reactive monocytes (peripheral blood, magnification). Wright-Giemsa stain, 1000* magnification). Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 467 In MPS, the accumulating macromolecules are found pri- Table 21.8 Conditions Associated with Quantitative marily in connective tissue. Changes of Monocytes Neoplastic Myelodysplastic/myeloproliferative neoplasms GAUCHER DISEASE Chronic myelomonocytic leukemia Gaucher (pronounced “go-shay”) disease is an inherited Juvenile myelomonocytic leukemia autosomal recessive disorder characterized by a deficiency Chronic myeloid leukemia of b@glucosidase (an enzyme needed to break down the lipid Acute monocytic, myelomonocytic, and myelocytic glucocerebroside). In this disease, the macrophage is unable leukemias to digest the stroma of ingested cells, and the lipid glucocer- Reactive Inflammatory conditions ebroside accumulates. The clinical findings (splenomegaly Collagen diseases Immune disorders and bone pain) of the disease are related to the accumula- Certain infections (e.g., TB, syphilis) tion of this lipid in macrophages mainly in the spleen, liver, Monocytopenia Stem cell disorders such as aplastic anemia and bone marrow. The macrophages (Gaucher cells) are large (209100 mcM[mM]) with small eccentric nuclei, and the cytoplasm appears wrinkled or striated40 (Figure 21-15). Unexplained monocytosis has been reported to be asso- The spleen and liver can become greatly enlarged. Leukope- ciated with as many as 62% of all malignancies. Monocy- nia, thrombocytopenia, and anemia can occur from seques- tosis can be seen in myelodysplastic states, acute myeloid tration by an enlarged spleen. Diagnosis of Gaucher disease leukemias, and chronic myeloid leukemia and in approxi- is determined by the detection of insufficient b@glucosidase mately 25% of Hodgkin lymphomas. In these conditions, enzyme activity in peripheral blood leukocytes.40 the monocyte is probably a part of a reactive process to Cells similar to Gaucher cells can be found in the mar- the neoplasm rather than a part of the clonal neoplasm row of individuals with a rapid granulocyte turnover, itself. Neoplastic proliferation of monocytes occurs in acute especially in chronic myeloid leukemia. The accumulation monocytic leukemia and acute and chronic myelomonocytic of lipid in these disorders does not result from enzyme leukemia (Chapters 24, 26). deficiency but from the inability of the macrophage to Monocytopenia refers to a concentration of monocytes keep up with the flow of fat into the cell from the increased less than 0.2 * 103/mcL and is found in stem cell disorders cell turnover. Differential diagnosis of Gaucher disease such as aplastic anemia. Monocytopenia is difficult to estab- can be confirmed by demonstrating decreased leukocyte lish because of the low normal levels of these cells. b@glucosidase activity, whereas the enzyme level is normal or increased in myeloproliferative disorders. Qualitative Disorders NIEMANN-PICK DISEASE Lysosomal storage disorders include a large group of inher- Niemann-Pick disease includes a group of rare disorders ited disorders. All nucleated cells contain lysosomes used that are related autosomal recessive diseases. Signs of the as a part of the cell’s recycling system. Lysosomes contain disease begin in infancy with poor physical development. various enzymes (including glucosidases, lipases, proteases, The spleen and liver are greatly enlarged. The disease is and nucleases) that are involved in degradative processes. often fatal by 3 years of age. In Niemann-Pick type A and Defects in any of these enzymes can lead to the accumula- tion of either nondegraded substrates or catabolic products that are unable to be transported out of the lysosome. This accumulation can lead to cell dysfunction and pathologi- cal phenotypes. Most of these disorders are inherited in an autosomal recessive pattern.39 The type of storage material that accumulates (glyco- proteins, glycosphingolipids, mucolipids, mucopolysac- charides, etc.) can be used to classify lysosomal storage disorders. Based on this accumulated storage material, there are two main categories: mucopolysaccharidoses (MPS) and lipidoses. Many of these disorders cause detect- able morphology in granulocytes (see the section “Alder- Reilly Anomaly” earlier in this chapter) and monocytes/ macrophages. Disorders based on the presence of abnormal macrophages in hematologic tissue, a common presenting feature, include Gaucher disease, Niemann-Pick disease, Figure 21.15 Gaucher macrophages in bone marrow and sea-blue histiocytosis (see the “Histiocytoses” section). (peripheral blood, Wright-Giemsa stain, 1000* magnification). 468 Chapter 21 type B disease, the defect is a deficiency of sphingomy- (LCH); class II—non-Langerhans histiocytoses (non-LCH), elinase (an enzyme needed to break down lipids), result- which encompasses the histiocytoses of mononuclear ing in excessive sphingomyelin storage. Macrophages with phagocytes; and class III—malignant histiocytoses. a foamy appearance are found in lymphoid tissue and The Langerhans cell is an immature dendritic cell usu- the bone marrow (Figure 21-16). The foam cells are large ally found in the epidermis, oral and vaginal mucosa, and (20–100 mcM) with an eccentric nucleus and globular cyto- lungs. They differ from other tissue cells by characteris- plasmic inclusions. Leukopenia and thrombocytopenia can tic racquet-shaped structural inclusions known as Birbeck occur from increased sequestration by the enlarged spleen, granules. Monocyte/macrophage histiocytes contribute to and blood lymphocytes can contain several vacuoles that reactive (in response to an inflammatory stimulus) and are lipid-filled lysosomes.41 malignant disorders. Reactive macrophage histiocytes can be seen in benign proliferative diseases (xanthoma dissemi- MISCELLANEOUS LYSOSOMAL STORAGE DISORDERS nata, juvenile xanthogranuloma), in nonmalignant hemo- Tay-Sachs disease, Sandhoff disease, and Wolman’s disease phagocytic diseases (fulminant hemophagocytic syndrome, are inherited lipid storage diseases and Hurler’s and Hunter’s histiocytosis with massive lymphadenopathy), and in sev- syndromes are inherited MPS. These disorders are character- eral of the storage disorders (Gaucher, Niemann-Pick, and ized by a deficiency of one or more enzymes that metabolize sea-blue histiocytosis). lipids or mucopolysaccharides. As a result of these enzyme Sea-blue histiocytosis syndrome is a rare inherited deficiencies, abnormal concentrations of these macromole- disorder characterized by splenomegaly and thrombocyto- cules accumulate in the lysosomes of tissue cells. These condi- penia. Sea-blue staining macrophages laden with lipid are tions affect mostly nonhematologic tissue and are often fatal. found in the liver, spleen, and bone marrow (Figure 21-17). There are no specific findings in the peripheral blood. Lipid- laden macrophages can be present in the bone marrow.42 The cell is large (in diameter) with a dense eccentric nucleus and cytoplasm that contains blue or blue-green granules. HISTIOCYTOSES Considerable variation in clinical manifestations is present, Two different types of cells are known as histiocytes: Lang- but in most patients, the course of the disease is benign. Sea- erhans cells/dendritic cells and monocytes/macrophages. blue histiocytes can also be seen in a variety of disorders of Both cell types are antigen processing and antigen present- lipid metabolism and in association with various other dis- ing cells and share a common progenitor. The three classes orders including Niemann-Pick disease, Gaucher disease, of histiocytoses are class I—Langerhans cell histiocytoses some hematopoietic diseases, and in certain infections. Figure 21.16 Macrophage in the bone marrow of a patient with Niemann-Pick disease. Note the foamy cytoplasm with inclusions Figure 21.17 Sea-blue histiocyte from bone marrow (peripheral blood, Wright-Giemsa stain, 1000* magnification). (peripheral blood, Wright-Giemsa stain, 1000* magnification. Summary Leukocytes respond to toxic, infectious, and inflammatory leukocyte types (depending on the cell’s function). Thus, a dif- processes to defend the tissues and limit and/or eliminate the ferential count and the total leukocyte count aid in diagnosis. disease process or toxic challenge. This can involve a change Neutrophilia, an increase in neutrophils, most often in leukocyte concentration, most often increasing one or more occurs as a result of a reaction to a physiologic or pathologic Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 469 process (most commonly, bacterial infection). Tissue injury disease and myeloperoxidase deficiency. Leukocyte adhe- or inflammation can also cause a neutrophilia. Neutropenia, sion deficiency I (LAD I) is identified by the absence of a decrease in neutrophils, is less commonly encountered. It leukocyte cell-surface adhesion proteins that inhibit the can be caused by drugs, immune mechanisms, or decreased adhesion of neutrophils to endothelial cells so the neutro- bone marrow production. Several inherited conditions are phils cannot exit the blood. Other rare leukocyte functional characterized by neutropenia and recurrent infections. Mor- abnormalities include Alder-Reilly, Chédiak-Higashi, and May- phologic abnormalities of neutrophils can be found in infec- Hegglin anomalies. tious states and are important to identify on stained blood Eosinophils increase in infections with parasites, smears. These include Döhle bodies, toxic granulation, and allergic conditions, hypersensitivity reactions, cancer, and cytoplasmic vacuoles. Other morphologic abnormalities chronic inflammatory states. Basophilia is seen in hypersen- include pince-nez cells found in Pelger-Huët anomaly and sitivity reactions, infections, and chronic myeloproliferative morulae found in ehrlichiosis. disorders, especially chronic myeloid leukemia. Monocyto- Functional and morphologic abnormalities of the sis is found in a wide variety of conditions, especially malig- leukocyte characterize a number of inherited conditions. nancies. Macrophage disorders are commonly associated Defects in the generation of oxidizing radicals after phago- with a group of lysosomal storage disorders in which these cytosing bacteria characterize chronic granulomatous cells are unable to completely digest phagocytosed material. Review Questions Level I c. vacuoles 1. Which of the following hematologic values would d. primary granules you expect if the peripheral blood smear revealed 5. Which of the following is a common reason for toxic granulation, Döhle bodies, and vacuoles in neutropenia in hospitalized patients? (Objective 6) neutrophils? (Objectives 1, 2) a. Chronic myeloproliferative disorders such as CML a. WBC: 4.0 * 103/mcL b. Chemotherapy and/or radiation treatment for b. The differential shows 15% bands cancer c. The differential shows 20% eosinophils c. Childbirth d. Hb: 10 g/dL; platelets 20 * 103/mcL d. Lack of exercise 2. The white count in an adult is 2.0 * 103/mcL The 6. Which of the following causes a false neutropenia? differential shows 60% segmented neutrophils. Which (Objective 7) of the following correctly describes these results? a. EDTA-induced agglutination (Objective 6) b. Bone marrow aplasia a. Normal c. Splenomegaly b. Leukocytosis and neutrophilia d. Immune neutropenia c. Leukopenia with normal number of neutrophils 7. Which of the following can be used to distinguish a d. Leukopenia and neutropenia leukemoid reaction from CML? (Objective 5) 3. Which of the following is the most common cause of a. A total WBC count more than 25 * 103/mcL neutrophilia? (Objective 1) b. The presence/absence of immature cells of the a. Bacterial infection myeloid lineage b. Acute leukemia c. Presence/absence of BCR/ABL1 gene mutation c. Chemotherapy d. Presence/absence of autoantibodies d. Aplastic anemia 8. Which of the following is a common cause of 4. Blue-gray oval inclusions composed of RNA near the eosinophilia? (Objective 10) periphery of neutrophils is a description of: a. Parasitic infection (Objective 3) b. Eosinophilic leukemia a. toxic granules c. CML b. Döhle bodies d. Acute hemorrhage 470 Chapter 21 9. What substances build up and are ingested by 3. Which of the following correctly describes the macrophages in qualitative macrophage disorders neutrophil concentration if the patient is an infant? such as Gaucher disease? (Objective 11) (Objective 1) a. Proteins a. Pseudo-neutrophilia b. Carbohydrates b. Leukemoid reaction c. Lipids c. Leukoerythroblastic reaction d. Rough endoplasmic reticulum d. Physiologic neutrophilia 10. Distinct, large, unidentified inclusions are found in 4. Which of the following correctly describes the the cytoplasm of many granulocytes. Select the best differential if the patient is an adult? (Objective 1) course of action. (Objective 9) a. Pseudo-neutrophilia a. Ignore them because they probably are not significant. b. Leukoerythroblastic |
reaction b. Report them as intracellular yeast. c. Agranulocytosis c. Suspect rare or unusual conditions such as d. Physiologic neutrophilia Chédiak-Higashi. 5. A leukoerythroblastic reaction can be associated with: d. Report them as toxic granulation. (Objectives 1, 2, 12) Level II a. chronic granulomatous disease b. Chédiak-Higashi syndrome c. severe hemolytic anemia Use the following case study to answer review questions 1–5. d. leukocyte adhesion deficiency A patient’s white count is 30.0 * 103/mcL. The 6. Alder-Reilly anomaly can be differentiated from toxic differential is as follows: granulation by: (Objectives 7, 11) Segmented neutrophils 54% a. presence/absence of other toxic features Band neutrophils 10% (leukocytosis, toxic granulation, Döhle bodies, Metamyelocytes 2% vacuoles) Lymphocytes 26% b. the presence of hypersegmentation in neutrophils Monocytes 5% c. other CBC parameters abnormal (RBC, HCT, or Eosinophils 3% PLT) 6 Nucleated RBCs/100 WBCs d. patient symptoms related to an allergic reaction 7. May-Hegglin can be differentiated from conditions with toxic Döhle bodies by: (Objectives 7, 11) 1. Select the additional information most important to a. presence of thrombocytosis assess whether these results are normal or indicate a b. presence or absence of other toxic features such as disease process. (Objective 1) toxic granulation a. Platelet count c. foam cells in the bone marrow b. RBC count d. patient history of recent trauma c. Patient history 8. Pelger-Huët anomaly can be differentiated from an d. Patient age increase in band neutrophils by: (Objectives 5, 11) 2. Which of the following correctly describes the absolute a. flow cytometry for the presence of CD11b neutrophil count for the patient? (Objectives 1, 12) b. abnormal inclusions found in cells other than a. Normal for both an infant and an adult neutrophils b. Neutrophilia for both an infant and an adult c. other CBC parameters abnormal (RBC, HCT, or c. Normal for an infant and neutrophilia for an adult PLT) d. Normal for an adult and neutrophilia for an infant d. finding all bilobed segmented neutrophils Nonmalignant Disorders of Leukocytes: Granulocytes and Monocytes 471 9. Chédiak-Higashi can be differentiated from c. The neutrophil count was too high for an adult. intracellular yeasts or morulae by: (Objectives 6, 7, 11) d. The patient suffered from congenital neutropenia. a. presence of giant platelets 11. Leukemia can be differentiated from infection by: b. abnormal inclusions in neutrophils and (Objectives 2, 11) lymphocytes a. lack of glucosidase enzyme activity in monocytes c. other CBC parameters abnormal (RBC, HCT, or PLT) b. a shift to the left in granulocytic cells d. lack of glucosidase enzyme activity c. BCR/ABL1 gene mutation d. flow cytometry for the presence of CD11b 10. An adult patient’s white blood cell count was 10.1 * 103/mcL 12. A sample with toxic vacuoles can be differentiated and the absolute neutrophil count from a sample with prolonged storage by: was 1.3 * 103/mcL. The medical laboratory scientist (Objectives 4, 6, 11) who analyzed the data suspected a false neutropenia and requested a redraw for the patient. Which of the a. presence of other toxic features (leukocytosis, toxic following could have caused the medical laboratory granulation, Döhle bodies, vacuoles) scientist to question the CBC results? (Objective 4) b. abnormal inclusions found in lymphocytes a. The blood specimen was drawn 6 days before c. other abnormal CBC parameters (RBC, HCT, or testing. PLT) b. The patient’s history indicated hypersplenism. d. family history References 1. Kumar, V., Abbas, A. K., Mitchell, R. N., & Aster, J. C. (2005). 10. Lichtman, M. A., & Liesveld, J. L. (2006). Acute myelogenous leu- Diseases of white blood cells, lymph nodes, spleen, and thymus. kemia. In: T. J. Kipps & U. Seligsohn, eds. Williams hematology (6th In: V. Kumar, A. K. Abbas, & J. C. Aster, eds. Robbins and Cotran ed., pp. 1047–1084). New York: McGraw-Hill. pathologic basis of disease (9th ed., pp. 593–594). Philadelphia: 11. Freifeld, A. G., Bow, E. J., Sepkowitz, K. A., Boeckh, M. J., Ito, J. I., Elsevier Saunders. Mullen, C. A., . . . Wingard, J. R. (2011). Infectious Diseases 2. Lichtman, M. A., Kipps, T. J., Kaushansky, K., Prchal, J. T., & Levi, Society of America. Clinical practice guideline for the use of M. M. (2011). Neutropenia and neutrophilia. In: M. S. Lichtman, antimicrobial agents in neutropenic patients with cancer: 2010 K. Kaushansky, T. J. Kipps, J. F. Prchal, & M. M. Levi, eds. update by the Infectious Diseases Society of America. Clinical Williams hematology (8th ed., pp. 168–179). New York: McGraw-Hill. Infectious Diseases, 52(4), 427–431. doi: 10.1093/cid/ciq147 3. Furze, R. C., & Rankin, S. M. (2008). Neutrophil mobilization 12. Rice L. & Jung M. (2018). Neutrophilic Leukocytosis, neutrope- and clearance in the bone marrow. Immunology, 125(3), 281–288. nia, monocytosis and monocytopenia. In: R. Hoffman, E. J. Benz, doi: 10.1111/j.1365-2567.2008.02950.x L. E. Silberstein, H. E. Heslop, J. I. Weitz, J. Anastasi, . . . . S. A. 4. Alves-Filho, J. C., Freitas, A. D., Spiller, F., Souto, F. O., & Cunha, Abutalib, eds. Hematology, basic principles and practice (7th ed., pp. F. Q. (2008). The role of neutrophils in severe sepsis. Shock, 675–681). Philadelphia: Elsevier. 30(Suppl. 1), 3–9. doi: 10.1097/shk.0b013e3181818466 13. Hestdal, K., Welte, K., Lie, S., Keller, J., Ruscetti, F., & 5. Dinauer, M. C., & Coates, T. D. (2018). Disorders of p hagocyte Abrahamsen, T. (1993). Severe congenital neutropenia: abnormal function. In: R. Hoffman, E. J. Benz, L. E. Silberstein, H. E. Heslop, growth and differentiation of myeloid progenitors to granulocyte J. I. Weitz, J. Anastasi . . . S. A. Abutalib, eds. Hematology: Basic colony-stimulating factor (G-CSF) but normal response to G-CSF principles and practice (7th ed., pp. 691–709). Philadelphia: Elsevier. plus stem cell factor. Blood, 82, 2991–2997. 6. Mukhopadhyay, S., Mukhopadhyay, S., Banki, K., & Mahajan S. 14. Boxer, L. A., & Newburger, P. E. (2007). A molecular classification (2004). Leukemoid reaction: A diagnostic clue in metastatic of congenital neutropenia syndromes. Pediatric Blood and Cancer, carcinoma mimicking classic Hodgkin lymphoma. Archives of 49(5), 609–614. doi: 10.1002/pbc.21282 Pathology and Laboratory Medicine, 128(12), 1445–1447. 15. Capsoni, F., Sarzi-Puttini, P., & Zanella, A. (2005). Primary and 7. Moore, A. J., Vu, M. A., & Strickland, S. A. (2014). Supportive secondary autoimmune neutropenia. Arthritis Research and care in hematologic malignancies. In: J. P. Greer, D. A. Arber, B. Therapy, 7, 208–214. doi: 10.1186/ar1803 Glader, A. F. List, R. T. Means Jr., & F. Paraskevas, eds. Wintrobe’s 16. Speeckaert, M. M., Verhelst, C., Koch, A., Speeckaert, R., & Lacquet, clinical hematology (13th ed., pp. 1426–1466). Philadelphia: F. (2009). Pelger-Huët anomaly: A critical review of the literature. Lippincott, Willams & Wilkins. Acta Haematologica, 121(4), 202–206. doi: 10.1159/000220333 8. Lichtman, M. A., Kaushansky, K., Kipps, T. J., Prchal, J. T., & Levi, 17. Cunningham, J. M., Patnaik, M. M., Hammerschmidt, D. E., & M. M. (2006). Neutropenia and neutrophilia. In: M. S. Lichtman, Vercellotti, G. M. (2009). Historical perspective and c linical K. Kaushansky, T. J. Kipps, J. F. Prchal, & M. M. Levi, eds. Williams implications of the Pelger-Huet cell. American Journal of hematology (8th ed., pp. 168–179). New York: McGraw-Hill. Hematology, 84(2), 116–119. doi: 10.1002/ajh.21320 9. Coutinho, A. E., & Chapman, K. E. (2011). The anti-inflammatory 18. Constantino, B. T. (2005). Pelger-Huët anomaly—Morphology, and immunosuppressive effects of glucocorticoids, recent mechanism, and significance in the peripheral blood film. developments and mechanistic insights. Molecular and Cellular Laboratory Medicine, 36(2), 103–107. doi: 10.1309/ Endocrinology, 335(1), 2–13. doi: 10.1016/j.mce.2010.04.005 hw3w-cyxn-dyyk-eq38 472 Chapter 21 19. Bohinjec, J. (1981). Myelokathexis: Chronic neutropenia with abscess. American Journal of Medicine, 66(1), 149–153. doi: hyperplastic bone marrow and hypersegmented neutrophils in 10.1016/0002-9343(79)90519-9 two siblings. Blut, 42(3), 191–196. doi: 10.1007/bf01026389 32. Wardlaw, A. J. (2017). Eosinophils and related disorders. In: 20. Thomas, R. J., Dumler, J. S., & Carlyon, J. A. (2014). Urrent K. Kaushansky, J. T. Prchal, O. W. Press, M. A. Lichtman, management of human granulocytic anaplasmosis, human mono- M. Levi, & L. J. Burns, eds. Williams hematology (9th ed., cytic ehrlichiosis and Ehrlichia ewingii ehrlichiosis. Expert Review pp. 947–964). New York: McGraw-Hill Professional. of Anti-Infective Therapy, 7(6), 709–722. doi: 10.1586/eri.09.44 33. Tefferi, A., Patnaik, M. M., & Pardanani, A. (2006). Eosinophilia: 21. Wormser, G. P., Dattwyler, R. J., Shapiro, E. D., Halperin, J. J., Secondary, clonal and idiopathic. British Journal of Haematology, Steere, A. C., Klempner, M. S., . . . Nadelman, R. B. (2006). The 133(5), 468–492. doi: 10.1111/j.1365-2141.2006.06038.x clinical a ssessment, treatment, and prevention of lyme disease, 34. Valent P., Reiter A., & Gotlib J. (2018). Eosinophilia, human granulocytic anaplasmosis, and babesiosis: Clinical prac- eosinophil-associated diseases, eosinophilic leukemias, and the tice guidelines by the Infectious Diseases Society of America. hypereosinophilic syndromes. In: R. Hoffman, E. J. Benz Jr., L. E. Clinical Infectious Diseases, 43(9), 1089–1134. doi: 10.1086/508667 Silberstein, H. Heslop, J. Weitz, J. Anastasi . . . S. A. Abutalib, eds. 22. Bakken, J. S., & Dumler, J. S. (2000). Human granulocytic ehrlichi- Hematology: Basic principles and practice (7th ed., pp. 1151–1169). osis. Clinical Infectious Diseases, 31(2), 554–560. doi: 10.1086/313948 New York: Churchill-Livingstone. 23. Hamilton, K. S., Standaert, S. M., & Kinney, M. C. (2004). 35. Powell, N., Walker, M. M., & Talley, N. J. (2010). Gastrointestinal Characteristic peripheral blood findings in human ehrlichiosis. eosinophils in health, disease and functional disorders. Modern Pathology, 17(5), 512–517. doi: 10.1038/modpathol.3800075 Nature Reviews Gastroenterology and Hepatology, 7(3), 146–156. 24. Van Hoof, F.V. (1974). Mucopolysaccaridosis and mucolipidoses. doi: 10.1038/nrgastro.2010.5 Journal of Clinical Pathology, Supplement (Royal College of Pathol- 36. Linch, S. N., & Gold, J. A. (2011). The role of eosinophils in ogists). 8, 64–93. non-parasitic infections. Endocrine, Metabolic and Immune Disorders - 25. Dinauer, M. C., & Coates, T. D. (2018). Disorders of phagocyte Drug Targets, 11(2), 165–172. doi: 10.2174/187153011795564188 function. In: R. Hoffman, E. J. Benz, L. E. Silberstein, H. E. Heslop, 37. Oermann, C. M., Panesar, K. S., Langston, C., Larsen, G. L., J. I. Weitz, J. Anastasi . . . S. A. Abutalib, eds. Hematology: Basic Menendez, A. A., Schofield, D. E., . . . Fan L. L. (2000). Pulmonary principles and practice (7th ed., pp. 691–709). Philadelphia: Elsevier. infiltrates with eosinophilia syndromes in children. Journal of 26. Bennet, J. S. (2009). Hereditary disorders of platelet function. In: Pediatrics, 136(3), 351–358. doi: 10.1067/mpd.2000.103350 R. Hoffman, B. Furie, P. McGlave, L. E. Silberstein, S. J. Shattil, & 38. Galli, S. J., Metcalfe, D. D., & Dvorak, A. M. (2017). Basophils and E. J. Benz, eds. Hematology: Basic principles and practice (5th ed., pp. mast cells and their disorders. In: K. Kaushansky, J. T. Prchal, O. W. 2134–2135). Philadelphia: Elsevier Churchill Livingstone. Press, M. A. Lichtman, M. Levi, & L. J. Burns, eds. Williams hematol- 27. Assari, T. (2006). Chronic granulomatous disease; fundamental ogy (6th ed., pp. 801–816). New York: McGraw-Hill Education. stages in our understanding of CGD. Medical Immunology. 39. Grabowski, G. A. (2008). Phenotype, diagnosis, and treatment doi: 10.1186/1476-9433-5-4 of Gaucher's disease. Lancet, 372(9645), 1263–1271. doi: 10.1016/ 28. Seger, R. A. (2008). Modern management of chronic granulomatous S0140-6736(08)61522-6 disease. British Journal of Haematology, 140q(3), 255–266. 40. Jmoudiak, M., & Futerman, A. H. (2005). G aucher disease: doi: 10.1111/j.1365-2141.2007.06880.x Pathological mechanisms and modern m anagement. British 29. Borregaard, N. (2017). Disorders of neutrophil function. In: M. Journal of Haematology, 129(2), 178–188. doi: 10.1111/j.1365-2141. A. Lichtman, K. Kaushansky, J. T. Prchal, M. M. Levi, L. J. Burns, 2004.05351.x & J. O. Armitage, eds. Williams manual of hematology (9th ed., pp. 41. Zimran, A., Elstein, D. (2017). Lipid storage diseases. In: K. 165–174). New York: McGraw-Hill Education. Kaushansky, J. T. Prchal, O. W. Press, M. A. Lichtman, M. Levi, & 30. Crockard, A., Thompson, J., Boyd, N., Haughton, D., Mccluskey, L. J. Burns, eds. Williams hematology (6th ed., pp. 801–816). D., & Turner, C. (1997). Diagnosis and carrier detection of chronic New York: McGraw-Hill Education. granulomatous disease in five families by flow cytometry. 42. McGovern, M. M., & Desnick, R. J. (2014). Lysosoal abnormalities International Archives of Allergy and Immunology, 114(2), 144–152. of the monocyte-macrophage system: Gaucher and Niemann- doi: 10.1159/000237660 Pick diseases. In: J. P. Greer, D. A. Arber, B. Glader, A. F. List, 31. Cech, P., Stalder, H., Widmann, J. J., Rohner, A., & Miescher, R. T. Means Jr., & F. Paraskevas, eds. Wintrobe’s clinical hematology P. A. (1979). Leukocyte myeloperoxidase deficiency and (13th ed., pp. 1302–1306). Philadelphia: Lippincott, Willams & diabetes mellitus associated with Candida albicans liver Wilkins. Chapter 22 Nonmalignant Lymphocyte Disorders Roslyn Mcqueen, PhD Author’s Note: I |
acknowledge Sue S. Beglinger, MS, who wrote this chapter in previous editions. This chapter retains many of her sections. Objectives—Level I At the end of this unit of study, the student should be able to: 1. Identify the infectious agent and describe 6. Describe clinical symptoms of disorders the clinical symptoms associated with in which a leukocytosis is caused by infectious mononucleosis. lymphocytosis. 2. Describe and recognize the reactive 7. State the complications associated with morphology of lymphocytes found in cytomegalovirus (CMV) infections. infectious mononucleosis. 8. Identify absolute and relative 3. Relate the heterophile antibody test to lymphocytopenia and lymphocytosis, infectious mononucleosis. and list conditions associated with these 4. Given a differential and leukocyte count, cal- abnormal counts. culate an absolute lymphocyte count and dif- 9. Explain the pathophysiology of HIV ferentiate it from a relative lymphocyte count. infections, and describe how it affects 5. Identify reactive cell morphology associated lymphocytes. with viral infections, and compare it to 10. Describe the abnormal hematological normal lymphocyte morphology. findings associated with AIDS. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Assess and resolve/explain conflicting and serologic tests in suspected Epstein-Barr results from peripheral blood morphology virus (EBV) infection. 473 474 Chapter 22 2. Describe the pathophysiology of infectious 8. Assess a patient case using the AIDS case mononucleosis. surveillance criteria of the Centers for 3. Assess and correlate antibody titers found Disease Control and Prevention (CDC) in infectious mononucleosis with respect to and recommend appropriate laboratory the various EBV viral antigens. testing. 4. State the pathophysiology of toxoplasmo- 9. Explain the cytopenia, identify the defect, sis infections, and explain the resulting and recognize the laboratory features lymphocytosis. in congenital qualitative disorders of lymphocytes. 5. Differentiate benign lymphocytic leukemoid reactions from neoplastic lymphoprolifera- 10. Evaluate a case study from a patient with a tive disorders by laboratory results and lymphoproliferative disorder and conclude characteristics of cell morphology. from the medical history and laboratory 6. Define the pathophysiology of CMV results the most likely diagnosis for the infection, and give clinical findings disorder. associated with it. 11. Identify the cytokine associated with 7. In Bordetella pertussis infection, propose hyper IgE syndrome (HIES) and explain the cause of lymphocytosis and recognize its role in the pathophysiology of the laboratory features associated with it. disorder. Chapter Outline Objectives—Level I and Level II 473 Lymphocytosis 475 Key Terms 474 Lymphocytopenia 482 Background Basics 474 Summary 489 Case Study 475 Review Questions 490 Overview 475 References 492 Introduction 475 Key Terms Acquired immune deficiency Hyper IgE syndrome (HIES) Reactive lymphocytosis syndrome (AIDS) Immunosuppressed Severe combined immunodefi- Advanced HIV disease (AHD) Infectious mononucleosis ciency (SCID) syndrome Bordetella pertussis Lymphocytic leukemoid reaction Toxoplasmosis Cytomegalovirus (CMV) Opportunistic organisms Viral load Epstein-Barr virus (EBV) Persistent polyclonal B-cell Heterophile antibody lymphocytosis (PPBL) Background Basics The information in this chapter builds on the concepts Level I learned in previous chapters. To maximize your learning • Describe normal and reactive lymphocyte experience, you should review the following concepts morphology and identify the distinguishing before starting this unit of study: characteristics of T and B lymphocytes. (Chapter 8) Nonmalignant Lymphocyte Disorders 475 • Calculate absolute cell counts and summarize summarize the structure and function of each of the relationship between the hematocrit and the immunoglobulins, and describe the immune hemoglobin. (Chapters 7, 10) response. (Chapter 8) • Define antigen and antibody, describe their roles in • Describe the structure and function of the hemato- infectious and noninfectious diseases, and summa- poietic organs and tissue. (Chapter 3) rize the immune response. (Chapter 8) • Explain the principle and the application of direct Level II antiglobulin tests in diagnosis of immune-mediated • Describe the process of T and B lymphocyte dif- anemia. (Chapter 19) ferentiation and the function of each subtype, lymphocytosis. Some acquired disorders result in a lym- CASE STUDY phocytopenia that can compromise the function of the We refer to this case study throughout the chapter. immune system. In acquired disorders affecting lympho- Heather, a 54-day-old female, was admitted to the cytes, the change in lymphocyte concentration and mor- hospital because of recurrent respiratory distress phology is a reactive process. In contrast, in congenital and failure to gain weight. She was born prema- disorders involving lymphocytes, the primary defect is turely at 35 weeks gestation by urgent Cesarean within the lymphocytic system. section. Her mother was immune to rubella and had negative serologic tests for syphilis. Consider why the lymphocytic system should Lymphocytosis be evaluated in this child and the possible etiology Lymphocytosis, an increase in lymphocytes, can result of repeated respiratory problems. from an absolute or relative increase in lymphocytes. Lymphocytosis can occur with or without a leukocyto- sis. The classification of lymphocytosis varies with age. Overview The lymphocyte concentration in children is normally higher than in adults and varies with the child’s age. The This chapter discusses benign conditions associ- reader is directed to Table B, inside cover, for the reference ated with quantitative and qualitative alterations in interval for such counts for children of various ages and lymphocytes. The first part of the chapter describes adults. Lymphocytosis in young children is defined as an conditions associated with lymphocytosis, includ- absolute lymphocyte count greater than 8.8 * 103/mcL, ing infectious mononucleosis and other acquired while a relative lymphocytosis exists when the differen- disorders c haracterized by l ymphocytosis. The later tial exceeds 65%. Absolute lymphocytosis occurs in adults part of the chapter discusses lymphocytopenia and when the lymphocyte count exceeds 4.8 * 103/mcL, while immune d eficiency states, both acquired and congenital. relative lymphocytosis is present when the lymphocyte Additionally, it discusses laboratory evaluation for the differential exceeds 35% (differs with race). An absolute diagnosis and differentiation of nonmalignant lympho- lymphocytosis can occur without relative lymphocytosis, cytic disorders from neoplastic lymphoproliferative and a relative lymphocytosis can occur without absolute disease. lymphocytosis. Lymphocytosis is usually a self-limiting, reactive pro- cess that occurs in response to an infection or inflamma- Introduction tory condition. Both T and B lymphocytes are commonly affected, but their function remains normal. Occasionally, Evidence of disease, especially infectious disease, can viral infections can cause functional impairment of the lym- be observed by finding abnormal concentrations of lym- phocytes, yielding both a qualitative disorder and quantita- phocytes and/or reactive lymphocytes on a peripheral tive changes. blood smear. This finding helps direct the physician’s Once lymphocytes have been stimulated by an subsequent workup of the patient and aids in the initia- infection or inflammatory condition, they enter vari- tion of appropriate therapy. Most disorders affecting lym- ous states of activation, resulting in a morphologically phocytes are acquired and are characterized by a reactive heterogeneous population of cells on stained blood 476 Chapter 22 smears (Figure 22-1). These activated cells can appear large with irregular shapes, cytoplasmic basophilia, Table 22.1 Conditions Associated with Lymphocytosis granules and vacuoles observed. The nuclear chroma- Benign (Nonmalignant) tin usually becomes more dispersed (Chapter 8). Cells Conditions Neoplastic Conditions with these activation features are commonly referred Viral Infections Acute lymphoblastic leukemia to as reactive or atypical lymphocytes. The preferred • Adenovirus Mature B cell neoplasms Mature T and NK cell term is reactive lymphocytes because these cells are not, • Cytomegalovirus (CMV) neoplasms in fact, “ atypical.” Occasionally, intense proliferation • Coxsackie virus Posttransplant • Epstein-Barr virus (infectious lymphoproliferative disorder of lymphoid elements in the lymph nodes and spleen mononucleosis) occurs, causing lymphadenopathy and splenomegaly, • Human herpesvirus type 6 respectively. • Human herpesvirus type 8 T lymphocytes normally compose about 60–80% of • Human T lymphotrophic virus type 1 peripheral blood lymphocytes. Thus, increases in the • Primary human immunodeficiency concentration of T lymphocytes are more likely to cause virus (HIV) infection changes in the relative lymphocyte count than are increases Nonviral Infections in B lymphocytes. Absolute lymphocytosis is not usu- • Bordetella pertussis ally accompanied by leukocytosis except in infectious • Brucellosis mononucleosis, Bordetella pertussis infection, cytomega- • Rickettsia lovirus infection, and lymphocytic leukemia. A relative • Shigellosis • Toxoplasma gondii lymphocytosis secondary to neutropenia that occurs in a • Tuberculosis variety of viral infections is more commonly found. The Miscellaneous absolute lymphocyte count is calculated as: • Persistent polyclonal B cell lymphocytosis %Lymphocytes (decimal form) * WBC count (* 109/L) • Physiologic stress = Absolute cell count (* 109/L) • Drug reactions It is important to differentiate benign conditions asso- ciated with lymphocytosis from neoplastic or malignant lymphoproliferative disorders (Table 22-1). The presence of heterogeneous reactive lymphocytes, positive sero- CASE STUDY (continued from page 475) logic tests for the presence of specific antibodies against Admission CBC on Heidi was WBC: 7.6 * 109/L; infectious organisms, and absence of anemia and thrombo- Hct: 55%; Plt: 242 * 109/L; and differential: 84% cytopenia favor a benign diagnosis. This chapter includes segs, 2% bands, 4% lymphocytes, 8% monocytes, a discussion of the more common disorders associated and 2% eosinophils. with a reactive lymphocytosis. 1. Does this patient have a leukocytosis, leukope- nia, or neither? Explain. 2. Does this patient have an abnormal lymphocyte count? Explain. Infectious Mononucleosis Infectious mononucleosis is a self-limiting lymphoprolif- erative disease caused by infection with Epstein-Barr virus (EBV). It is one of the most common human viruses and a member of the herpes virus family. EBV infection occurs worldwide, with 90% of people EBV infected at some point in their lives.1 Not considered highly contagious, the disease Figure 22.1 is transmitted through direct contact through body fluids, Forms of stimulated lymphocytes from a case of infectious mononucleosis. There are two immunoblasts (one o’clock primarily saliva. and eight o’clock) and a reactive lymph (three o’clock) (peripheral Infectious mononucleosis was initially described in blood, Wright-Giemsa stain, 1000* magnification). 1920 by Sprunt and Evans as an acute infectious disease Nonmalignant Lymphocyte Disorders 477 accompanied by atypical large peripheral blood lympho- that resemble activated natural killer cells are present early cytes.2 Later, Downey described the morphologic presen- in the disease. Cytotoxic T lymphocytes inhibit the activa- tations of lymphocytes commonly observed in infectious tion and proliferation of EBV-infected B lymphocytes and mononucleosis. The morphology ranged from a small participate in the cell-mediated immune response. The lymphocyte with a lobed nucleus and scant cytoplasm, to majority of the reactive lymphocytes seen in the periph- a large lymphocyte with abundant cytoplasm (“ballerina eral blood are these CD8+ cytotoxic T lymphocytes. Others skirt” cells) that reaches out and abuts adjacent cells, and include, g/d T cells and CD16+ CD56+ natural killer (NK) finally, to a large lymphocyte with finely reticular chromatin cells stimulated in response to EBV-infected B cells. NK cells and abundant cytoplasm.3 These cells represent morpho- are also emerging as important players during infectious logic changes observed in CD8 T lymphocyte activated in mononucleosis.7 response to EBV-infected cells.4 CLINICAL PRESENTATION Infectious mononucleosis is common worldwide and Early symptoms include lethargy, headache, fever, chills, usually affects young adults; the peak age for infection sore throat, nausea, and anoxia. The classic triad presen- is 14–24 years of age. About 90% of adults that have had tation of symptoms are fever, pharyngitis, and lymphade- exposure possess lifelong immunity.5 Infection in children nopathy (Table 22-2).7 Children younger than 10 years of from lower-income groups usually occurs before 4 years of age are often asymptomatic but have reactive lymphocytes age; in more affluent populations, peak infection incidence and elevated C-reactive protein (CRP). Increases in liver occurs during adolescence.6 aminotransferases are generally found in children more A major function of T lymphocytes is to provide cel- than 10 years of age.7 The cervical, axillary, and inguinal lular immunity, thereby limiting the viral growth of EBV- lymph nodes are commonly enlarged. Splenomegaly occurs infected B lymphocytes. Immune-compromised individuals in 50–75% of these patients, and hepatomegaly occurs in are at increased risk of serious infection. EBV-associated B about 25%. Occasionally, jaundice develops. Hematologic cell tumors and lymphoproliferative syndromes can occur complications that can occur during or immediately after in transplant patients and patients with acquired immune the disease include autoimmune hemolytic anemia, throm- deficiency syndrome (AIDS). These patients have severe T bocytopenia, agranulocytosis, and (very rarely) aplas- lymphocyte immunodeficiency. Lacking a T lymphocyte tic anemia. The disease is usually self-limiting, resolving response, male children with the rare X-linked lymphopro- within a few weeks. liferative disorder (XLP) are unable to limit EBV infection of B lymphocytes. As a result, |
a fatal polyclonal B lymphocyte LABORATORY EVALUATION proliferation occurs.7 Hematologic findings provide important clues for diagnosis in infectious mononucleosis. Serologic tests can confirm the EBV PATHOPHYSIOLOGY diagnosis. EBV attaches to a receptor on the B lymphocyte mem- Peripheral Blood During active viral infection, an intense brane designated CD21, which is the receptor for the C3d proliferation of lymphocytes occurs within affected complement component.7 The virus infects resting B lym- lymph nodes. The leukocyte count is usually increased phocytes as well as epithelial cells of the oropharynx and (12925 * 103/mcL) primarily because of an absolute lym- cervix. Binding of the virus to the B lymphocyte activates phocytosis. Lymphocytosis begins about 1 week after symp- the cell and induces the expression of the activation marker toms appear, peaks at 2 to 3 weeks, and remains elevated for CD23, which is the receptor for a B lymphocyte growth fac- tor.7 Once internalized, the virus is incorporated into the B lymphocyte genome, instructing the host cell to begin production of EBV proteins. These viral proteins are then Table 22.2 Summary of Typical Clinical Presentation and expressed on the cell membrane. Thus, EBV-infected cells Laboratory Evaluation in Infectious Mononucleosis express markers of activated B-lymphocytes as well as viral Clinical Presentation Laboratory Evaluation markers. The viral genome is maintained in the lymphocyte nucleus and passed on to the cell’s progeny. This results • Lymphadenopathy • Leukocytosis in EBV-immortalized B lymphocytes and possible latent • Fever • Lymphocytosis infection. • Lethargy • Elevated C-reactive protein A complex, multifaceted cellular immune response • Sore throat • More than 20% reactive (atypical) lymphocytes controls acute EBV infection. In the first week of illness, • Splenomegaly • Immunoblasts present a polyclonal increase in immunoglobulins occurs. During the second week, however, the number of immunoglobulin- • Headache • Heterophile antibodies present secreting B lymphocytes decreases because of the action of • Positive antigen tests for EBV CD8+ cytotoxic T lymphocytes. Activated cytotoxic T cells • Elevated aminotransferases (ALT, AST) 478 Chapter 22 2 to 8 weeks. Lymphocytes usually constitute about 50% of Bone Marrow Bone marrow aspirations in EBV infection the leukocyte differential with about 20% reactive lympho- are not indicated, but when performed show hyperplasia of cytes. The platelet count is often mildly decreased; concen- all cellular elements except neutrophils. trations of less than 100 * 103/mcL are rare. Serologic Tests Serologic tests are used to differentiate this Various forms of reactive lymphocytes can be found in disease from similar more serious diseases (e.g., diphtheria, the peripheral blood (Figure 22-2). Typical cells are irregular hepatitis). The blood of patients with infectious mononucle- in shape and have large amounts of spreading cytoplasm osis contains greatly increased concentrations of transient with irregular basophilia. Other reactive cells can have deep heterophile antibodies that agglutinate sheep or horse blue cytoplasm and vacuoles. Immunoblasts are usually erythrocytes but are not specific for EBV8 (a heterophile present early in the disease. Plasmacytoid lymphocytes and antibody reacts with antigens common to multiple species). an occasional plasma cell also can be found. When present, Antibodies specific for EBV can be identified by first absorb- immunoblasts should be distinguished from leukemic lym- ing the nonspecific heterophile antibodies from patient phoblasts to prevent a misdiagnosis. The chromatin pattern serum with guinea pig antigen and testing the absorbed of leukemic lymphoblasts is usually finer than the reticular serum with horse erythrocytes. The infectious mononu- chromatin of immunoblasts. In addition, immunoblasts gen- cleosis IgM antibodies react with horse erythrocytes. Posi- erally have a lower N:C ratio with more abundant, some- tive agglutination of horse erythrocytes by treated serum times vacuolated, cytoplasm. Another important criterion indicates EBV infection. A negative result usually indicates that helps differentiate infectious mononucleosis from leu- infection by some other virus. On occasion, the level of anti- kemia is the morphologic heterogeneity of the lymphocyte body is not yet high enough to be detected; therefore, the population, characteristic of viral infections, whereas leuke- test should be repeated a week later if patient symptoms mia usually has a relatively homogeneous cell population. continue. Other diseases associated with a reactive lymphocyto- Rapid, specific, and sensitive slide agglutination or sis can mimic the blood picture of infectious mononucleo- solid phase immunoassay tests are available to determine sis. These include cytomegalovirus infection, viral hepatitis, the presence of infectious mononucleosis heterophile anti- and toxoplasmosis. bodies. The infected individual also produces antibodies specific for the viral capsid antigen (VCA) of EBV at vari- ous stages of infection, which can be detected earlier than the heterophile antibodies. VCA-IgM rises first, followed by VCA-IgG, which when present, signals the development of immunity.6 Antibodies to EBV nuclear antigens (EBNA) rise during early convalescence and persist together with VCA-IgG (Table 22-3). Thus, the presence of EBNA excludes an acute infection. A patient with all clinical manifestations and peripheral blood findings of infectious mononucleosis occasionally does not have a positive heterophile test (heterophile- negative syndrome). In 10–20% of adult cases and 50% of children younger than 10 years of age, the test is nega- tive in the presence of EBV infection. In other cases, the heterophile-negative syndrome is caused by a non-EBV Figure 22.2 viral infection. The most likely causative agent is cyto- Two reactive forms of lymphocytes from a case of infectious mononucleosis. Note the cytoplasmic vacuoles and megalovirus. Antibody responses might not be detected cytoplasmic basophilia and irregular shapes (peripheral blood, in immunosuppressed individuals (those in whom the Wright-Giemsa stain, 1000* magnification). immune response is suppressed either naturally, artificially, Table 22.3 Antibodies to EBV Found in Infectious Mononucleosis Stage of Infection Heterophile Antibodies VCA-IgM (titer) VCA-IgG (titer) EBNA (titer) Acute (0–3 months) Present 71:160 71:60 Not detected Recent (3–12 months) Present Not detected 71:160 71:10 Past (712 months) Present Not detected 71:40 71:40 VCA, viral capsid antigen; EBNA, EBV nuclear antigen. Nonmalignant Lymphocyte Disorders 479 or pathologically).8 Viral load testing can be important in test is negative. Biopsy of lymph nodes shows a reactive immune-compromised patients who develop fulminant follicular hyperplasia and can play an important role in liver disease when the disease process is not self-limiting.9 diagnosis. Diagnosis of an active infection is confirmed by Other laboratory tests can be abnormal, depending on seroconversion and a rising titer of antibodies to T. gondii. the presence or absence of complications. Hepatitis of some Immunologically compromised hosts have a more severe degree is common and can be a severe complication. An infection.13 The most common hematologic complication is increase in both direct and indirect bilirubin fractions and hemolytic anemia, which can be severe. Molecular tests for an increase in serum liver enzymes are common findings in T. gondii DNA are available. the presence of hepatitis. A rare complication of infectious mononucleosis is hemolytic anemia. The anemia appears Cytomegalovirus to be caused by cold agglutinins directed against the eryth- Cytomegalovirus (CMV) is a worldwide pathogen causing rocyte I-antigen. a spectrum of infection that varies depending on whether THERAPY the host is immunocompetent or immunocompromised. Because the disease is normally self-limited, therapy is sup- CMV is a member of the Herpesviridae family which portive. Bed rest is recommended if fever and myalgia are includes the Epstein-Barr virus, herpes simplex virus, present. Strenuous exercise should be avoided for several varicella-zoster virus, and herpesvirus 6, 7, and 8. These weeks, especially if splenomegaly is present. Antibiotics are viruses have the ability to remain latent within the host, not useful except in the presence of secondary infections. reactivating and shedding when the host’s immune system Antiviral drugs may be helpful for immune-compromised is compromised.14 Serologic tests show that 45 to100% of the individuals.9 U.S. population has prior exposure to CMV. The virus may be shed in body fluids, such as urine, saliva, blood, tears, Checkpoint 22.1 semen, and breast milk. It is also the virus most frequently A patient with lymphocytosis showing reactive lymphocyte transmitted from mother to the developing fetus before or morphology with large, basophilic cells, fine chromatin, and a during birth.15 visible nucleolus has a negative infectious mononucleosis sero- CLINICAL PRESENTATION logic test. What is a possible cause for this altered lymphocyte morphology? In the immunocompetent individual, CMV infection is asymptomatic or produces mild flu-like symptoms. Symptoms, when apparent, develop 9 to 60 days after Toxoplasmosis primary infection. It is characterized by prolonged fever, lymphadenopathy, and splenomegaly. In contrast to infec- Toxoplasmosis is a parasitic disease caused by Toxoplasma tious mononucleosis, pharyngitis is uncommon.16 After gondii (T. gondii). This obligate intracellular parasite can infection, the virus remains alive and is usually dormant multiply in all body cells except erythrocytes, although the within the person's body for life. Recurrent disease rarely preferred target organs are the lymph nodes, brain, heart, occurs unless the person's immune system is suppressed and lungs. Contact with this intracellular protozoan may due to therapeutic drugs or disease. occur through direct ingestion of food or water contami- Infection with CMV can be the result of congenital or nated with cat feces containing oocysts, ingestion of tis- acquired infection. Congenital infection in neonates occurs sue cysts in uncooked meat, transplacental infection of the when the virus from the infected pregnant woman crosses fetus, WBC transfusion, or organ transplantation. Disease the placenta and infects the fetus. The newborn can dem- may occur through acute infection after recent contact with onstrate jaundice, microcephaly, and hepatosplenomegaly. T. gondii cysts or oocysts or through endogenous reactivation.10 However, only about 10% of infected infants exhibit clini- Infections are often asymptomatic, but pose a signifi- cal evidence of the disease. The most common hematologic cant risk to the fetus of a pregnant woman. Transplacental findings in neonates are thrombocytopenia and hemolytic infection can cause abortion, jaundice, hepatosplenomegaly, anemia. chorioretinitis, hydrocephalus, microcephaly, cerebral calci- Acquired infection is spread by close contact, blood fication, and mental retardation.11 Acquired infections can transfusions, and sexual contact. CMV infection in the sometimes cause symptoms resembling infectious mono- immunosuppressed can be life-threatening. It is the most nucleosis. Toxoplasmosis seropositivity is more common in common viral infection complicating tissue transplants and rural than urban children.12 Laboratory evaluations reveal is a significant cause of morbidity and mortality in immu- leukocytosis with a relative lymphocytosis or (more rarely) nocompromised patients.17 However, treatment with anti- an absolute lymphocytosis, and an increase in reactive lym- viral drugs and immunosuppressive therapy dramatically phocytes. Most reactive cells are morphologically similar to decreases the incidence of CMV complications in organ lymphoblasts or lymphoma cells. The heterophile antibody transplantation.18 480 Chapter 22 LABORATORY EVALUATION Most viral infections have the potential to elicit an immune response resulting in lymphocytosis. CMV is the most common cause of heterophile-negative infectious mononu- cleosis. Morphologically, the lymphocytes appear reactive, exhibiting a similar morphology to infectious mononucle- osis.19 The diagnosis is made by serologic testing, cultur- ing the virus, or by detecting the virus's genetic material (CMV DNA). There are two types of CMV antibodies that are pro- duced in response to a CMV infection, IgM and IgG (Chap- ter 8). IgM antibodies are the first to be produced by the body in response to a CMV infection and last 3 to 4 months. CMV IgG is produced early in primary infection and per- sists lifelong. Rising titers of IgG can be used as markers of Figure 22.3 Lymphoproliferation resulting from B. pertussis acute infection. A positive test for CMV IgG indicates that a (whooping cough). Note the numerous small lymphocytes with person was infected with CMV at some time during his or condensed chromatin. This self-limiting peripheral blood picture her life, but does not indicate when a person was infected.20 must be distinguished from that of CLL (peripheral blood, Wright- False-positive reactions have resulted from the presence of Giemsa stain, 1000* magnification). rheumatoid factors. The lymphocytes are small cells with condensed chromatin Laboratory evaluation of the CBC indicates a leukocy- and indistinct nucleoli.21 tosis with an absolute lymphocytosis. Many lymphocytes A pertussis toxin secreted by the bacteria causes an show a reactive morphology, but the heterophile antibody accumulation of lymphocytes in the blood by recruiting test is negative. CMV is thought to infect neutrophils, which lymphocytes into the peripheral circulation and blocking serve as a means of transporting the virus to other body their migration back into lymphoid tissue. The toxin inter- sites.20 Aspartate aminotransferases liver function tests may feres with expression of L-selectin (CD62L) on all leuko- be mildly or moderately |
elevated along with evidence of cytes, but lymphocytes are particularly sensitive. Decreased subclinical hemolysis. Diagnosis is confirmed by demon- cellularity of the lymph nodes accompanies rapid periph- strating the virus in the urine or blood using a viral DNA eral lymphocytosis. The ratio of CD4 to CD8 lymphocytes (molecular) assay or by a rise in the cytomegalovirus anti- remains normal.22 Laboratory diagnostic methods include body titer (except in immunocompromised patients). culture, serology, immunophenotyping, and PCR.23 Culture is the gold standard and is specific but not sensitive. The Bordetella Pertussis advantage of culture is that it allows antibiotic sensitivity testing and epidemiological typing. Serologic diagnosis is Pertussis, also known as whooping cough, was one of the based on demonstrating antipertussis toxin antibodies. It is most common childhood diseases in the 20th century and preferred for older children and adults who present after a major cause of childhood mortality. Bordetella pertussis a prolonged coughing illness. PCR for B. pertussis DNA is (B. pertussis), a fastidious, aerobic, Gram-negative cocco- most sensitive in detecting infection when used during the bacillus produces an acute respiratory infection marked first 3 weeks of cough24 and is most useful in acutely ill by severe, spasmodic (whooping) coughing episodes. infants. B. pertussis produces a number of virulence factors, includ- The incidence of whooping cough infections continues ing pertussis toxin, adenylate cyclase toxin, filamentous to rise, suggesting that pertussis immunizations given in hemagglutinin, and hemolysin, which contribute to its childhood do not yield lasting immunity. Studies show that pathologic manifestations.21 Transmission is by droplets 20 to 30% of adults with a cough that lingers 6 to 8 weeks directly from person to person, where the bacteria will are positive for B. pertussis. The increased incidence of infec- colonize the ciliated cells of the respiratory mucosa, and tions also may be due to increased sensitivity of PCR detec- undergo rapid multiplication. tions.24 The infections cause a lingering cough in adults, and Infection with B. pertussis causes a blood picture very passing it on to infants can be fatal.25 similar to that of reactive viral lymphocytosis (Figure 22-3). The leukocyte count typically rises to 15925 * 103/mcL but can reach 50 * 103/mcL. The rise in leukocytes is caused Reactive Lymphocytosis by an absolute lymphocytosis of T, B, and natural killer A reactive lymphocytosis process, previously called infec- (NK) lymphocytes along with increases in neutrophils tious lymphocytosis, is a reactive immune response asso- and monocytes. Granulocytes may show toxic changes. ciated with several common viruses that infect children, Nonmalignant Lymphocyte Disorders 481 usually between the ages of 2 and 10 years (often coxsackie Plasmacytosis and adenovirus). It is characterized by an increase in blood lymphocytes, often up to 30 * 103/mcL and occasionally as Plasma cells are not normally found in the peripheral blood high as 100 * 103/mcL, which might be mistaken for acute and constitute 4% of the cells in the bone marrow. Most leukemia. Eosinophilia may be present. Patients are usually plasma cells are found in the medullary cores of lymph asymptomatic but may have fever, abdominal pain, or diar- nodes, although they can occasionally be found in the rhea. There is no lymph node enlargement or splenomegaly. peripheral blood with intense stimulation of the immune Symptoms only last a few days, but the lymphocytosis may system. This can occur in some viral and bacterial infections persist for several weeks. such as rubeola, infectious mononucleosis, toxoplasmosis, An increased relative lymphocyte count with the syphilis, and lung infections (pneumonia, tuberculosis, presence of reactive or immature-appearing lymphocytes abscesses). Circulating plasma cells can also be found in (lymphocytic leukemoid reaction) characterizes the disorders associated with elevated gamma globulin such lymphocytic reaction. Many of the lymphocytes are large, as multiple myeloma, skin diseases, cirrhosis of the liver, reactive cells with deep blue cytoplasm and fine chroma- collagen disorders, and sarcoidosis. tin and may show cytoplasmic vacuoles (Figure 22-4). Normal plasma cell morphology is included with the Reactive cells are usually nonclonal T lymphocytes and discussion of lymphocyte morphology in Chapter 8. A mor- large granular lymphocytes. In some viral infections, lym- phologic variation of the reactive plasma cell is called the phocytopenia and neutropenia precede lymphocytosis. flame cell, named for its reddish-purple cytoplasm. A glyco- As the infection subsides, plasmacytoid lymphocytes can protein produced in the rough endoplasmic reticulum (RER) be found (Figure 22-5). The heterophile antibodies test is causes the red tinge, and the presence of ribosomes causes negative. the purple tinge. These cells contain more immunoglobulin In some cases, a lymphocytic leukemoid reaction than normal plasma cells. Flame cells have been associated resembles chronic lymphocytic leukemia (CLL; Chapter with IgA plasma cell myeloma but are now recognized in a 29). Bone marrow aspiration, however, shows minimal (if variety of immune pathologies and are occasionally found any) increase in lymphocytes in a lymphocytic leukemoid in normal bone marrow. reaction. In contrast to CLL, lymphadenopathy and spleno- megaly are usually absent. In addition, patients with a lym- Persistent Polyclonal B-Cell phocytic leukemoid reaction are usually young, whereas CLL patients are usually older adults. Lymphocytosis The reactive lymphocytosis can be distinguished from a Persistent polyclonal B-cell lymphocytosis (PPBL) is a lymphoproliferative disorder based on a pleomorphic ver- rare disorder found primarily in the female adult smoker. sus monomorphic presentation, respectively. Most reactive It is characterized by chronic, stable, persistent, polyclonal lymphocytosis shows a spectrum of morphologic changes lymphocytosis with atypical binucleated lymphocytes with including variations in sizes and shapes. abundant cytoplasm in the peripheral blood. There is often Figure 22.4 An immunoblast found in a patient with a Figure 22.5 A plasmacytoid lymphocyte. Note the deep viral infection. This cell must be distinguished from a leukemic basophilic cytoplasm and eccentric nucleus. These cells are associated lymphoblast (peripheral blood, Wright-Giemsa stain, 1000* with infectious states. Note also the neutrophil with toxic granulation magnification). (peripheral blood, Wright-Giemsa stain, 1000* magnification). 482 Chapter 22 evidence of an HLA-DR7 haplotype, partial insertion of to mount an immune response is possible, resulting in chromosome 3, i(3q), and multiple IgH/Bcl-2 rearrange- immunodeficiency. Most individuals with lymphocyto- ments that suggest PPBL could lead to a malignant pro- penia have a reduced absolute number of T cells, particu- cess.26 The disorder is often asymptomatic and found by larly the number of CD4+ T cells. chance on a routine blood analysis. Hematologic evaluation reveals a moderate increase Lymphocyte Sequestration and in the absolute lymphocyte counts (more than 4 * 109/L) without evidence for infection or other conditions that can Destruction increase the lymphocyte count. Specific morphologic fea- Lymphocytopenia results from decreased production or tures predictive of the diagnosis include basophilic vacu- increased destruction of lymphocytes, changes in lympho- olated cytoplasm and monocytoid changes.26 There is an cyte circulation patterns, and other unknown causes (Table expansion of CD27+ immunoglobulin (Ig) M+ IgD+ B@cells. 22-4). Various acquired or inherited diseases, conditions and There is a polyclonal increase in serum IgM but low IgG and factors are associated with lymphocytopenia. Corticosteroid IgA levels. Bone marrow examination reveals lymphocytic therapy causes a sharp drop in circulating lymphocytes infiltrates. within 4 hours. The decrease is caused by sequestration of Symptoms can include fever, fatigue, weight loss, recur- lymphocytes in the bone marrow. Values return to normal rent chest infections, or generalized lymphadenopathy. within 12–24 hours after cessation of therapy. Acute inflam- PPBL may evolve into a variety of malignant lymphop- matory conditions, including viral and bacterial infections, roliferative disorders including non-Hodgkin lymphoma, also can be associated with a transient lymphocytopenia. diffuse large cell lymphoma, splenic marginal zone lym- Malignancies with an associated lymphocytopenia is a phoma, and other clonal solid tumors.26 poor prognostic sign. Systemic lupus erythematosus is fre- Additional plasma cell variants include Mott cells, Rus- quently associated with a lymphocytopenia presumably sell bodies, and Dutcher bodies. These variations are associ- caused by autoantibodies produced against these cells. ated with the malignant disorders plasma cell leukemia and Chemotherapeutic alkylating drugs for malignancy, such as multiple myeloma (Chapter 28). cyclophosphamide, cause the death of T and B-lymphocytes in both interphase and mitosis. Malnutrition is the most common cause of lymphocytopenia. Starvation causes Lymphocytopenia thymic involution and depletion of T lymphocytes. Both congenital and acquired immune deficiency disorders are The lymphocytes are essential for providing immunity and associated with lymphocytopenia.27 protecting the body from infections. Lymphocytopenia is Irradiation causes a prolonged suppression of lym- defined as an abnormally low level of lymphocytes in the phocyte production. CD4+ helper lymphocytes are more blood. The blood value is age dependent and defined as sensitive to radiation than are CD8+ lymphocytes. It an absolute lymphocyte count of less than 1.0 * 103/mcL appears that small daily fractions of radiation are more for adults or less than 3.0 * 103/mcL in children younger damaging to lymphocytes than periodic large doses. With than 5 years of age. In general, lymphocyte counts less periodic radiation, the lymphocytes can renew during than 2 * 103/mcL are abnormal in this population. When periods of nonradiation. Aggressive treatment of hema- the lymphocyte count is decreased, an impaired ability tologic malignancies with chemotherapeutics or ionizing Table 22.4 Conditions Associated with Lymphocytopenia Infectious Diseases Immune Disorders Malignancy Drugs Other Human immunodeficiency virus Systemic lupus Hodgkin’s disease Corticosteroid therapy Acute inflammatory (HIV) erythematosus conditions Severe acute respiratory syn- Ataxia-telangiectasia Diffuse large B cell Radiation and Aplastic anemia drome (SARS) lymphoma chemotherapy Respiratory syncytial virus (RSV) Wiskott-Aldrich syndrome Peripheral T cell lymphoma Steroid therapy Malnutrition West Nile virus encephalitis Severe combined immuno- Stress deficiency disease Measles Sjögren syndrome Acute and chronic renal disease Ehrlichia Salmonella typhi Nonmalignant Lymphocyte Disorders 483 radiation can lead to immunodeficiency by depleting and recombinant viruses. Subtype B predominates in the short-lived, antigen-activated B lymphocytes.27 Americas, Caribbean, and Western Europe.29 Clinical Presentation Patients experience weight loss, Checkpoint 22.2 fever, lymphadenopathy, thrush, chronic rash, and intermit- Why is lymphocytopenia a concern if there is no accompanying tent diarrhea. In the pre-antiretroviral era, patients infected leukopenia? with HIV had a grave prognosis. Current highly active anti- retroviral treatment (HAART) protocols provide effective therapy to reduce viral replication and delay the onset of AIDS for HIV-infected people.29 CASE STUDY (continued from page 476) Not all individuals infected with the HIV virus Heidi weighed 2 pounds 1 ounce at birth. Tests for (HIV-positive individuals) have the clinical condition CMV and toxoplasma infections were negative. known as AIDS. AIDS is defined by the occurrence of At 4 days of age, she was transferred to a special repeated infections with multiple opportunistic organisms facility for feeding and growth m onitoring. She and an increase in malignancies, especially Kaposi’s sar- developed a diaper rash that failed to respond coma, in individuals infected with HIV. to many measures. No thrush was found. Transmission rates between individuals is dictated by At 44 days, she developed pneumonia from the viral load in the HIV-infected source. Transmission of coagulase-negative staphylococci that responded to the virus occurs through sexual intercourse, blood/blood antibiotics. Her WBC count was 12.3 * 103/mcL product transfusion (93%), mother-to-child transmission, with a differential of segs 42%, bands 5%, and needle-sharing. Infants born to HIV-infected mothers lymphocytes 1%, monocytes 28%, eosinophils 23%, are at increased risk for perinatal HIV infection. Estimated and basophils 1%. rates for mother-to-baby transmission in the absence of anti- retroviral therapy is 23%.30 3. What is the absolute lymphocyte count? Surveillance Hiv Case Definition In 1982, the U.S. Centers 4. What possible causes exist for these opportunis- for Disease Control and Prevention (CDC) developed a case tic infections? definition of AIDS for surveillance purposes. It is revised periodically as more data are collected about the disease.31 The 2014 revision of Surveillance Case Definition for HIV infection describes the clinical criteria and “opportunis- Immune Deficiency Disorders tic i llnesses” for presumptive evidence of HIV infection31 (Table 22-5) and standardizes data collection for improved Immune deficiency disorders are characterized by impaired therapeutic treatment. Symptoms of recurrent infections with function of one or more of the components of the immune opportunistic infections warrant suspicion of HIV infection. system, T, B, or NK lymphocytes. In some disorders, the numbers of these cells are also reduced. This results in an inability to mount a normal adaptive immune response. These disorders are characterized clinically by an increase Table 22.5 Opportunistic Illnesses in HIV Infection in infections and neoplasms and can be sub-grouped as Microbial Infections Malignancies Other |
acquired or congenital. Recurrent bacterial Cervical cancer AIDS dementia Acquired immunodeficiency syndrome (AIDS) is an infections syndrome infectious disorder characterized by lymphocytopenia. Candidiasis Kaposi’s sarcoma Encephalopathy Because of the frequency with which it is encountered in Coccidioidomycosis Lymphoma Progressive multifocal the clinical hematology laboratory, it is discussed in more leukoencephalopathy detail next and is followed by a discussion of the congenital Cryptococcosis Wasting syndrome immune deficiency disorders. Cryptosporidiosis Cytomegalovirus ACQUIRED IMMUNODEFICIENCY SYNDROME Herpes simplex Acquired Immunodeficiency Syndrome (AIDS) is a Histoplasmosis highly lethal immune deficiency disease first described in 1981.28 The disease is caused by infection with a retro- Isosporiasis virus, human immunodeficiency virus type I (HIV-1) or Mycobacterium type 2 (HIV-2). HIV-1 is comprised of four distinct viruses, Pneumocystis carinii types M, N, O, and P. Group M is the globally predomi- Salmonella nant viral strain and is further divided into nine subtypes Toxoplasmosis 484 Chapter 22 The revised criteria apply to adults and children over by binding to both the CD4 protein that is part of the T-cell 18 months of age and combined the HIV classification sys- receptor (TCR; Chapter 8), and chemokine receptor CCR5.32 tem and AIDS case definition into a single case definition Once in the cell, HIV sheds its viral coat and uses reverse for HIV infection. This revised case definition requires lab- transcriptase to make a DNA copy of the viral RNA. Viral oratory-confirmed evidence of HIV infection. The Council DNA is then integrated into the host cell DNA where the of State and Territorial Epidemiologists (CSTE), in coop- virus replicates. Viral infection of these CD4 lymphocytes eration with pediatricians, also described the criteria for causes cytolysis, rapid selective depletion of this lympho- a negative diagnosis for children …18 months old born to cyte subset, and eventually lymphocytopenia. Monocytes HIV-infected mothers. The child does not meet the criteria and macrophages also have the CD4 and CCR5 proteins and for HIV/AIDS diagnosis if antibody tests or virologic tests are infected but not destroyed by the virus. Rare individuals are negative after 4–6 months of age and the child does not are homozygous for a 32-base pair deletion in the gene for have an AIDS-defining condition.31 Almost all children born CCR5 (CCR5 delta 32), resulting in lack of protein expres- to HIV-infected mothers have anti-HIV IgG antibodies at sion on the cell surface. These individuals are resistant to birth because of placental transmission of maternal antibod- HIV infections.33 ies. However, only 15–30% of the children are infected with Cell-mediated immunity and humoral immunity are HIV. Thus, anti-HIV IgG tests in this population are not reli- abnormal. Cell-mediated immunity declines as CD4+ T able until after 18 months of age. In these cases, tests for lymphocyte-helper function for monocytes, macrophages, the detection of HIV nucleic acid performed after the infant and other T lymphocytes declines. Humoral responses are is at least one month of age are almost always positive if exaggerated with polyclonal B lymphocyte proliferation, the virus is present.30 In addition, the 2014 CDC surveil- increased immunoglobulin production, and hypergam- lance HIV data allow for staging of HIV infections based maglobulinemia. Although the B lymphocytes have a poor on immunophenotyping of CD4 lymphocytes and clinical response to mitogens in vitro, they secrete immunoglobulin conditions (Table 22-6). For surveillance purposes, the stag- spontaneously. This suggests the B lymphocytes are already ing of disease illness only progresses; it is never reclassified activated and unable to react to further in vitro stimulation. as a less severe stage. The CD8+ cytotoxic T lymphocytes attempt to respond to The World Health Organization (WHO) uses similar HIV infection to control viral replication during primary criteria for diagnosing HIV infection as the CDC, but WHO infection and reduce the viral plasma level in chronic infec- splits the CDC stage 2 into two separate stages: stage 2 and tion. Because the immune response to HIV is defective and stage 3. Those patients with decreased lymph counts but does not clear the virus, the immune system remains con- not requiring treatment are classified as stage 2, and those tinually activated with high rates of T cell turnover that patients with decreased CD4+ T lymphs and conditions that eventually leads to T cell exhaustion and depletion.32 require antiretroviral therapy for advanced HIV disease Laboratory Evaluation Multiple hematologic abnormali- (AHD) as stage 3. WHO classifies patients with both clini- ties, including leukopenia, lymphocytopenia, anemia, and cal conditions and the diagnostic lymphopenia as stage 4 thrombocytopenia, are found in HIV infections (Table 22-7). AIDS.31 Leukopenia is usually related to lymphocytopenia, although Pathophysiology The main etiologic agent of AIDS in the neutropenia also can be present. Lymphocytes can include United States is the retrovirus, HIV-1. A related but immuno- reactive forms. Mild to moderate normocytic, normochromic logically distinct virus, HIV-2, is endemic to regions of West anemia is present in the majority of HIV-infected individu- Africa. The virus selectively infects helper T lymphocytes als and worsens as the disease progresses.32 Inflammatory Table 22.6 CDC and WHO Staging of HIV Infection for Adolescents and Adults CD4+ T Lymph CD4+ T Lymph Counta (percentagea of total CDC Staging WHO Staging AIDS-Defining Condition(s) (cells/mcL) lymphocytes) Stage 1 HIV infection Stage 1 HIV infection None CD4+ lymphs Ú 500 CD4+ lymphs Ú 26 Stage 2 HIV infection Stage 2 HIV infection None CD4+ lymphs 2009499 CD4+ lymphs 14925 Stage 3 HIV infection (AHD) Clinical conditions requiring CD4+ lymphs 2009499 CD4+ lymphs 14925 retroviral treatment Stage 3 AIDS Stage 4 AIDS Clinical condition present CD4+ lymphs 6 200 CD4+ lymphs 6 14 a Staging requires either CD4+ T lymph count or percent CD4+ T lymphs of total lymphocytes. Adapted from Appendix, MMWR Recommendations and Reports, 63(RR-03), 1–10, April 2014. Nonmalignant Lymphocyte Disorders 485 Immediate evaluation and treatment for HIV exposure Table 22.7 Laboratory Evaluation in HIV Infections has proven to be very effective in eliminating or significantly • Leukopenia • Thrombocytopenia minimizing the risk of HIV infection and AIDS. Health • Anemia • Decreased CD4 counts care workers with occupational exposure (e.g., needlestick • Macrocytosis (in patients treated • Positive molecular tests for injury) should receive immediate antiretroviral therapy.37 with zidovudine) HIV-1 RNA The CDC recommends initiation of antiretroviral therapy within 36 hours of a needlestick injury from a potential HIV source. Studies show that as time elapses after exposure, cytokines may play a role in suppressing erythropoiesis in viral replications exceed the control of the antiretroviral a manner similar to that found in anemia of chronic disease therapy. After 72 hours, therapy to prevent infection is no (ACD; Chapter 12). Macrocytosis (MCV more than 100fL) longer effective.37 Combination therapy should consist of occurs in up to 70% of patients 2 weeks after receiving zid- two or more antiretroviral drugs and continue for 4 weeks. ovudine.33 Antierythrocyte antibodies can be found in up to Laboratory evaluation for adverse effects should be consid- 20% of patients with hypergammaglobulinemia. These anti- ered after 2 weeks. bodies react like polyagglutinins and cause a positive direct Recent HIV research studies have demonstrated a antiglobulin test (DAT, Coombs’ test; Chapter 19). Immune cross-reacting antibody that develops late in HIV infection thrombocytopenia, indistinguishable from i diopathic of patients with decreased CD4+ T cells and high viral loads, thrombocytopenic purpura (ITP), is common (Chapter 33). which has a neutralizing effect on HIV proteins. The anti- Iron studies are similar to those found in ACD with low body is specifically targeted at the envelope protein, gp120. serum iron and TIBC and increased serum ferritin. This antibody response, although too late to eliminate HIV infection once established, may prove to be effective in find- Disease Monitoring The severity of CD4 lymphocytopenia ing a means for HIV immunization.32,38 and concentration of plasma HIV-1 RNA copies correlate with the severity of disease. The normal CD4:CD8 ratio in periph- eral blood is about 2:1. In AIDS, this ratio reverses progres- Checkpoint 22.3 sively and permanently because of destruction of the CD4+ T Why does infection with HIV result in an increased chance for lymphocytes. The CD4+ T lymphocyte count is performed at opportunistic infections? initial diagnosis and measured periodically to monitor dis- ease progression. AIDS (CDC stage 3 HIV infection) is defined OTHER ACQUIRED IMMUNE DEFICIENCY DISORDERS by a CD4+ T lymphocyte count of less than 200/mcL (mL) or Other acquired immune deficiency disorders are diagnosed a CD4+ T@lymphocyte concentration less than 14% of total based on functional abnormalities of lymphocytes. The lymphocytes.33 In addition, the viral load is monitored by lymphocyte count can be normal but in many cases, it is measuring the number of copies of HIV-1 RNA.34 decreased. Acquired defects of either T or B lymphocytes can result in serious clinical manifestations. Some inflam- Therapy No cure for AIDS currently exists. Treatment with matory states transiently impede the response of T lympho- zidovudine (azodothymidine, AZT) and protease inhibi- cytes to antigen. These include idiopathic granulomatous tors lengthen the time between HIV seropositivity and the disorders and malignancy. Severe infection by one micro- onset of AIDS. Improved antiretroviral treatments have organism sometimes impedes the ability of T lymphocytes postponed the onset of AIDS indefinitely in some patients.34 to react to other infectious organisms. Starvation or severe In 2008, a medical group in Germany reported an protein deficiency also can severely affect the functional apparent cure for an HIV-1 positive patient who had ability of the T lymphocyte. received a bone marrow transplant (BMT) for acute myeloid leukemia.35 The donor was homozygous for the CCR5 delta CONGENITAL IMMUNE DEFICIENCY DISORDERS 32 mutation, and the transplanted hematologic cells were Congenital disorders are usually characterized by a resistant to viral infection by HIV-1. Although widely cov- decrease in lymphocytes and impairment in either cell- ered in both the scientific and popular (lay) literature, this mediated immunity (T lymphocytes), humoral immunity information has not made a significant impact to date on (B lymphocytes), or both (Table 22-8). In contrast to the reac- the treatment strategies for most AIDS patients. BMT is not tive morphologic heterogeneity of lymphocytes associated an option for most AIDS patients, due to both the logistic with viral disorders, lymphocytes in congenital disorders improbability of finding an HLA-compatible/CCR5 delta 32 are usually normal in appearance. The functional impair- homozygous donor and the cost and medical risks involved ment of the immune response is often apparent from birth with the procedure. However, it has sparked interest in the or at a very young age when children present with recurrent possibility of a type of gene therapy in which the CCR5 gene infections. Due to extensive research using molecular tech- is modified in an effort to prevent the HIV virus from being niques, the genetic mutations causing immunodeficiency able to infect hematologic cells.36 are being identified. 486 Chapter 22 Severe Combined Immunodeficiency Syndrome Severe is impaired (Chapter 4). IL-7/IL-7R signaling is required combined immunodeficiency (SCID) syndrome includes for T cell development, and in its absence, T cell lym- a heterogeneous group of rare genetic disorders result- phopoiesis fails. Thus, X-linked SCID is c haracterized by ing in major qualitative immune defects involving both absent T and NK lymphocytes and a hypoplastic thymus.41 humoral and cellular immune functions. These disorders B lymphocytes are normal in number but are nonfunctional include numerous and diverse genetic mutations with dif- due to lack of T-helper function, and immunoglobulin lev- ferent inheritance patterns and varied severity in clinical els are severely depressed. Family history is important in manifestation. Most are inherited as sex-linked or autoso- determining the mode of inheritance of SCID, although a mal-recessive traits, but some autosomal dominant forms negative family history of the disease does not rule out an have been reported. About 75% of individuals with SCID X-linked disease. Up to one-third of the cases present as a are males because the most common form of SCID is an spontaneous mutation.41 X-linked disorder. Females who carry the abnormal X-linked SCID gene Both the T and B lymphoid lineages are functionally have normal immunity. These carriers can be detected deficient. Subgroups of SCID are now defined on the basis by molecular assays for IL-2R mutations in the g@chain of which lymphocytes are absent. If only the T cells are locus using cells other than lymphocytes. The normal absent, it is termed T-B+NK+ SCID; if both T and B cells mature female cell population is a mosaic |
with one or are absent, it is T-B-NK+ SCID; and in those patients (usu- the other X chromosome inactivated (X-inactivation [also ally with the most severe lymphopenia) in whom all three called lyonization] is the normal process by which one of lymphocyte subsets are absent, it is T-B-NK- SCID.39 In all the copies of the X chromosome present in the female is cases both the T and B lymphoid lineages are functionally inactivated and transcriptionally silent). In female SCID deficient. Because T cell help is required in generating an carriers, h owever, only lymphocytes carrying normal, antibody response against protein antigens, disorders in non-inactivated X chromosomes are found, rather than the which T lymphocytes are absent result in peripheral blood expected mixture of cells with normal and abnormal X chro- B lymphocytes that are unresponsive to most mitogens, mosome inactivation. Random X chromosome inactivation and immunoglobulin production is decreased. However, occurs in these carriers, but the gene product of the mutant these B lymphocytes respond normally when incubated X chromosome does not support lymphocyte maturation. with normal T lymphocytes in vitro.40 The absolute lym- Thus, lymphocytes with the mutant X chromosome fail to phocyte count is variable but often is decreased to less than develop, and the only lymphocytes found in carriers have 1.0 * 103/mcL. Lymphocyte counts in SCID patients may the active normal X chromosome. be normal in the neonate, especially if they are able to gen- erate B cells and NK cells, but eventually they develop a Checkpoint 22.4 profound lymphopenia. Would you expect female carriers of X-linked SCIDS to be more Lymph node examination reveals a lack of plasma cells, susceptible to infection than the normal population? Why or B lymphocytes, and T lymphocytes. No lymphoid cells are why not? found in the spleen, tonsils, or intestinal tract. The bone marrow also is deficient in plasma cells and lymphocyte Autosomal SCID The autosomal forms of SCID exhibit precursors. severe deficiencies of both T and B lymphocytes. The Frequent recurrent infections, skin rashes, diarrhea, most common form, found in about 30–40% of autosomal- and failure to thrive are characteristic findings in infants recessive SCID (15% of all SCID patients), is due to an with SCID. Death related to overwhelming sepsis usu- adenosine deaminase (ADA) deficiency that results in a ally occurs within the first 2 years of life if untreated. depletion of T, B, and NK lymphocytes, T-B-NK-.41 The Bone marrow transplantation, immunoglobulin therapy, adenosine deaminase gene is located at chromosome and gene therapy are the only options for successful treatment.40 20q13.11 (Table 22-8). Both point mutations and gene dele- tions have been associated with ADA deficiency. Sex-Linked SCID Classic sex-linked (X-linked) SCID is the Another enzyme deficiency, purine nucleoside most common form of inherited severe combined immuno- phosphorylase (PNP), is the cause of ∼2, of SCID cases. deficiency, accounting for about 45% of cases. This form of The gene for PNP is located at chromosome 14q13.1. Both SCID (T-B+NK- SCID) has been mapped to the long arm of these enzymes degrade purines. Without PNP, accumu- of the X chromosome (Xq13.1–13.3) and is associated with lation of toxic DNA metabolites (deoxyadenosine triphos- a loss-of-function mutation in the gene for the g@chain of phate/dATP and deoxyguanosine triphosphate/dGTP) the IL-2 receptor (Table 22-8). Because the g@chain also is an occurs, inhibiting normal T and B cell development. The essential subunit of the receptors for IL-4, IL-7, IL-9, IL-15, two disorders can be differentiated by the presence of NK and IL-21, hematopoietic regulation by all of these cytokines cells and the ADA enzyme in PNP.41 Nonmalignant Lymphocyte Disorders 487 Table 22.8 Laboratory Evaluation in Selected Immunodeficiency Disorders Disorder Immunoglobulins B-Lymphs T-/NK-Lymphs Genetic Loci SCID X-linked T- B+ NK- T IgG, IgE, IgA; N IgM N; abnormal function Absent IL2RG (Xq13.1) Autosomal recessive T- B+ NK+ T IgG, IgE, IgA; N IgM N; abnormal function T-lymphs absent ZAP-70 (2q12); IL7R (5p13) T- B- NK+ T IgG, IgM, IgA, IgE Absent T-lymphs absent RAG-1 and RAG-2 (11p13); PNP (14q13.1) T- B+ NK- T IgG, IgE, IgA; N IgM N; abnormal function Absent JAK3 (19p13.1) T- B- NK- T IgG, IgM, IgA, IgE Absent Absent ADA (20q13.11, ADA deficiency); AK2 (1p34, reticular dysgenesis) MHC class II deficiency T IgG, IgE, IgA; N IgM N N or T T-lymphs CIITA (16p13); RFXANK (19p12); T+/- B+ NK+ RFX5 (1q21); RFXAP (13q14) Other Immunodeficiency Syndromes X-linked X-linked agammaglobulinemia T IgG, IgM, IgA Mature cells absent N BTK mutations (Xq21.3–q22) (T+ B- NK+) Wiskott-Aldrich syndrome T IgM; N/T IgA; N; abnormal function N or T T-lymphs WAS mutations (Xp11.22–11.3) (T+ B- NK+) c IgE; N/c IgG Autosomal diGeorge syndrome N or c IgG; IgM; IgA N number; delayed T-lymphs TBX1 (del 22q11.2) (T- B+ NK+); AD maturation Absent Ataxia-telangiectasia; AR N/c IgM; N/c IgG; IgE; IgA N; abnormal function T T-lymphs ATM (11q22–23) Hyper IgE syndrome (HIES); N IgM, IgG, IgA; c IgE N T T-lymphs DOCK8 (9p24.3); STAT3 AR and AD (17q21.31) T , decreased; c , increased; N, normal; ADA, deaminase; AK2, adenylate kinase 2; AD, autosomal dominant; AR, autosomal recessive; ATM, ataxia telangiectasia mutated; BTK, Bruton’s tyrosine kinase; CIITA, Class II MHC transactivator; DOCK8, dedicator of cytokinesis 8; IL2RG, interleukin 2 receptor, gamma chain; IL7R, interleukin 7 receptor 7 alpha chain; JAK3, Janus kinase 3; PNP, purine nucleoside phosphorylase; RAG, recombination activating gene; RfX5, Regulatory factor X-5; RfXAP, RfX-associated protein; RfXANK, RfX-associated ankyrin containing protein; SCID, severe combined immunodeficiency syndrome; STAT3, signal transducer activator of transcription 3; TBX1, T-box-containing transcription factor; WAS, Wiskott-Aldrich syndrome; ZAP70, z@chain associated protein kinase-70. Other defects include a deficiency of MHC class II gene expression, interleukin-2 receptor a@chain (IL@2Ra) defi- 7. Are the lymphocytes more likely to be morpho- ciency, mutations in the RAG-1 and RAG-2 genes (which logically heterogeneous or homogeneous? Why? catalyze VDJ recombination), and defective assembly of the 8. What confirmatory test is indicated? T cell receptor–CD3 complex (Chapter 8).40 Wiskott-Aldrich Syndrome Wiskott-Aldrich syndrome CASE STUDY (continued from page 483) (WAS) is a sex-linked recessive disease characterized by the All of Heidi’s immunoglobulin levels were triad of eczema, thrombocytopenia, and immunodeficiency, decreased. T and B lymphocyte counts were severely resulting in recurrent infections. The first signs of WAS are decreased. The peripheral blood smear showed usually petechiae and bruising, resulting from a low plate- anisocytosis, poikilocytosis (schistocytes), polychro- let count. Spontaneous nosebleeds and bloody diarrhea are matophilia, and two nucleated RBCs/100 WBCs. common. Eczema develops within the first month of life Her thymus was not detectable on chest films. while recurrent bacterial infections develop by 3 months.42 5. Is this child more likely to have a congenital or About two-thirds of affected children have a family history acquired immune deficiency? of the disease; in one-third there is an apparent spontane- ous mutation. Untreated, most children die before 10 years 6. If she has a congenital immune deficiency, is it of age as a result of infection or bleeding. Those who sur- more likely that she has X-linked or autosomal vive longer can develop neoplasms of the histiocytic, lym- SCIDS? phocytic, or myelocytic lineages. EBV infections in WAS patients can lead to lymphoma.43 488 Chapter 22 Laboratory evaluation plays an important role in the or maternal X chromosomes and are asymptomatic. This diagnosis of WAS (Table 22-9). There is a progressive decrease nonrandom inactivation pattern is found in the carrier’s in thymic-dependent immunity and depletion of paracorti- T and B lymphocytes, granulocytes, monocytes, and mega- cal areas in the lymph nodes, leading to abnormal lympho- karyocytes, indicating that all hematopoietic cells in WAS cyte function. The absolute numbers of CD4+ and CD8+ are affected by the presence of the WAS gene. lymphocytes and their ratio is normal to variable. Typically, Therapy includes treatment for bleeding and infection. CD8+ lymphocytes are decreased, but numbers vary over Splenectomy usually results in correction of the platelet time for individuals. Circulating B-lymphocyte numbers are count and normalized platelet volume and significantly normal, but antibody production is abnormal.43 Serum IgM reduces the risk of bleeding complications. Bone marrow levels are decreased, but IgE and IgA levels are increased. transplant before 5 years of age has an 85% cure rate.43 IgG concentrations are usually normal (Table 22-8). One of the most consistent findings is the low or absent Checkpoint 22.5 level of circulating antibodies to the blood group antigens.43 What laboratory findings suggest WAS in a child, and how is the These children are unable to produce antibodies to polysaccha- diagnosis confirmed? ride antigens, a T lymphocyte-independent phenomenon. This suggests that there is an intrinsic B lymphocyte abnormality. DiGeorge Syndrome DiGeorge syndrome is a congenital A low platelet count with abnormal bleeding in the neo- immunodeficiency marked by the absence or hypoplasia of natal period is one of the first clinical signs of WAS. Patients the thymus, hypoparathyroidism, heart defects, and dys- have a severe thrombocytopenia (less than 70 * 103/mcL) morphic facies. Hypocalcemia is typical, and the presenting with small-size platelets.43 Platelets are intrinsically abnor- symptom can be seizure resulting from hypocalcemia. Usu- mal with decreased expression of surface glycoproteins ally a decrease in peripheral blood T lymphocytes occurs IIb, IIIa, and IV and the defective expression of CD62P and as well as a decrease in the cellularity of the T lymphocyte CD63. The prothrombin time and activated partial throm- regions of peripheral lymphoid tissue. The low lymphocyte boplastin time are normal, indicating the coagulation factor count is related to a decreased number of CD4+ lympho- proteins are adequate (Chapters 31 and 32). Megakaryocytes cytes (Table 22-8). T lymphocyte function varies. Children in the bone marrow are normal or increased in number and with a hypoplastic thymus may be able to produce enough morphologically normal. lymphocytes with normal function to maintain immuno- Genetic mutations involve the Wiskott-Aldrich competence. B lymphocytes are normal in number and syndrome (WAS) gene on the short arm of the X chromo- function, and immunoglobulin levels are normal. Infants some between Xp11.22 and Xp11.3. The WAS gene codes exhibit increased susceptibility to viral, fungal, and bacterial for the WAS protein (WASp), which is mainly expressed infections that are frequently overwhelming. Death occurs in hematopoietic cells. Females are carriers; affected males in the first year unless thymic grafts are performed. do not pass the deficiency to their male children. PCR tech- Cytogenetic studies on these children show a chromo- niques detect 98% of affected males and are the primary some 22q11.2 deletion; this deletion is thought to affect an diagnostic tests when WAS is suspected.43 Molecular analy- estimated 1 in 4,000 people, and is considered autosomal sis using restriction fragment length polymorphisms reveals dominant because a deletion in one copy of chromosome 22 that female carriers have selective inactivation of the WAS in each cell is sufficient to cause the condition.44 This defect X chromosome rather than random inactivation of paternal also is found in a parent of a child with DiGeorge syndrome in 25% of the cases. Table 22.9 Sex-Linked Agammaglobulinemia Sex-linked (X-linked) Laboratory Features in Wiskott-Aldrich Syndrome (WAS) agammaglobulinemia (Bruton’s disease) is inherited as a sex-linked disease characterized by frequent respiratory Feature Description and skin infections with extracellular, catalase-negative, Platelets Decreased concentration, small size, pyogenic bacteria. Molecular analysis has revealed that abnormal platelet function the genetic defect is on the long arm of the X chromo- Lymphocytes Decreased or normal concentration, some, (Xq21.3–22; Table 22-8).39 More than 90% of patients T lymphocytes variable, B lymphocytes usually normal have a loss-of-function mutation of a tyrosine kinase gene, Immunoglobulin IgM decreased, IgE and IgA increased, IgG Bruton’s tyrosine kinase (BTK). The genetic mutation results normal/increased in a block in B lymphocyte maturation at the pre-B lympho- Antibodies to blood group Absent or decreased cyte stage. The variable and constant regions of the IgM antigens immunoglobulin chain fail to connect. PCR Detects WAS gene mutation Peripheral blood lymphocyte counts are normal as are T Ig, immunoglobulin; PCR, polymerase chain reaction. lymphocytes; there is, however, a decrease in B lymphocytes Nonmalignant Lymphocyte Disorders 489 and an absence of plasma cells in lymph nodes. The serum skin abscesses, recurrent lung infections, and eosinophilia. concentrations of IgG, IgM, and IgA are decreased or absent. The disorder has both autosomal dominant and recessive Cell-mediated immune function is normal. Monthly injec- forms. These HIESs are associated with several mutations tions of gamma globulin |
are effective in preventing severe (Table 22-8). There are two forms of HIES: a dominant form infections. caused by mutations in STAT3 and a recessive form, for Female carriers of this disease have normal immunity. which a genetic cause is unclear. All of their B lymphocytes carry the paternal, normal X Autosomal-dominant HIES, the most common disease chromosome, suggesting that the normal X chromosome in this group, results from STAT3 gene mutations and has confers a survival advantage. a variety of connective tissue and skeletal abnormalities.46 JAK-STAT (Chapter 4) mutations alter cytoplasmic signal- Ataxia-Telangiectasia Ataxia-telangiectasia (AT) is inher- ing pathways, which cause deficiencies in multiple cell lin- ited as an autosomal-recessive disease that results from eages. CD4+ lymphocytes, B lymphocytes, myeloid cells, mutations in the ataxia-telangiectasia mutated (ATM) gene keratinocytes, osteoclasts, and monocytes are all decreased. at chromosome 11q22–23. The ATM protein is involved Autosomal-dominant HIES additionally results in connec- in signaling pathways involved in cellular responses to tive tissue, skeletal, vascular, and dental abnormalities. It is DNA damage. The disease is characterized by progressive the most frequent cause of immune deficiency with HIES.46 neurologic disease, immune dysfunction, and predisposi- STAT3 is necessary for normal IL-17 cytokine signaling and tion to malignancy. Affected individuals are ataxic and in regulation. Deficiencies in IL-17 due to STAT3 mutations childhood or adolescence develop telangiectasias. Ataxia lead to decreased differentiation of T helper 17 (Th17) lym- refers to uncoordinated movements, such as when walking. phocytes and result in a primary immunodeficiency. Both A telangiectasia is a vascular lesion formed by a dilation of a T and NK lymphocytes are decreased. IL-17 also controls group of blood vessels that appears as a red line or r adiating eosinophilotropic chemokines that drive eosinophil pro- limbs (spider). Telangiectasias appear as tiny, red, spider- duction. Patients have an eosinophilia, numerous tissue like veins. Chronic respiratory infection and lymphoid abnormalities including facial, dental, skeletal, and soft malignancy are the most common causes of death. tissue abnormalities, short limbs, and osteoporosis and are Patients with AT have a defect in cell-mediated immu- susceptible to repeated infections. Recurrent infections are nity with hypoplasia or dysplasia of the thymus gland and characterized by increased levels of serum IgM but deficien- depletion of T lymphocyte areas in the lymph nodes. B lym- cies in the other immunoglobulins, especially IgG. phocyte function also is abnormal with impaired isotype Autosomal-recessive HIES is caused by homozygous switching (Chapter 8). Lymphocytopenia with a reversed null mutations in TYK2 (tyrosine kinase 2) or DOCK8 CD4:CD8 ratio exists along with deficiencies in IgA, IgG, (dedicator of cytokinesis 8) genes.47 This mutation impairs and IgE. IgM levels are increased. Cytogenetic analysis T lymphocyte function, which results in a lack of specific reveals excessive chromosome breakage and rearrange- antibody formation, recurrent viral infections, candidiasis, ments in cultured cells and clonal abnormalities of chro- mosome 7 or 14.45 dermatitis, and the increased serum IgE. Both X-linked and autosomal recessive mutations have Hyper IgE Syndrome The hyper IgE syndromes (HIES) been identified that affect T lymphocyte activation because (originally named Job's syndrome) are rare, primary of abnormalities in cytokine signaling. Patients have quali- immunodeficiency diseases that involve both humoral tative defects in both T and B lymphocytes with recur- and cellular immunity deficiencies. They are characterized rent infections and extreme susceptibility to opportunistic by elevated serum IgE, eczema, recurrent staphylococcal infections.46 Summary Lymphocytes mount an immune response in inflammatory response to eliminate foreign antigens, the cell itself also or infectious states. In these states, the lymphocyte morphol- can serve as the site of infection for some viruses that use ogy often includes various reactive forms, immunoblasts, lymphocyte membrane receptors to attach to and invade and possibly plasmacytoid cells. Lymphocytosis is defined the cell. as an absolute lymphocyte count exceeding 4 * 103/mcL, Infectious mononucleosis is a common self-limiting whereas lymphocytopenia is defined as a total lymphocyte lymphoproliferative disorder caused by infection with EBV. count less than 1.0 * 103/mcL. Quantitative changes (either Laboratory diagnosis of this disorder includes serologic increased or decreased) in the total lymphocyte concentra- testing for heterophile antibodies and identification of reac- tion occur. Although the lymphocyte induces an immune tive lymphocytes on Romanowsky-stained blood smears. 490 Chapter 22 Lymphocytopenia, on the other hand, typically reflects currently is no cure, but extensive research is investigating depletion of T cells, the most abundant lymphocyte sub- a neutralizing immune response that may lead to vaccine type in the blood. The most common cause of such T cell development. depletion is a viral infection, such as infection with the Congenital qualitative disorders of lymphocytes human immunodeficiency virus, although other causes include a wide variety of immunodeficiency disorders. exist. AIDS is a disease caused by infection of the CD4+ T Either the T or B lymphocyte or both can be affected. These lymphocyte with the retrovirus HIV-1. The virus suppresses are usually very serious disorders in which most affected the immune response by replicating within and destroying individuals succumb to the disease in childhood. As molec- CD4+ lymphocytes. CD4+ lymphocyte levels and viral loads ular testing improves detection, more genetic mutations will are used to monitor disease progression in HIV-infected elucidate the origins of these primary immunodeficiencies patients. Antiretroviral treatments in combination with pro- so that treatment options can be explored. Bone marrow/ tease inhibitors slow the progression of the disease. There stem cell transplant is the only treatment in many cases. Review Questions Level I 5. A 29-year-old female patient was seen for abdomi- nal fullness, fever, and lethargy. Her total leuko- 1. Advanced HIV disease (AHD) patients have numer- cyte count was 11.8 * 103/mcL. The differential ous infections because the HIV virus has infected: showed 32% neutrophils, 65% lymphocytes, and (Objective 9) 3% monocytes. A few of her lymphocytes were a. neutrophils small round lymphocytes with a high N:C ratio and b. CD8+ lymphocytes overall size similar to the red cells, but many of her lymphocytes appeared large with basophilic cyto- c. CD4+ lymphocytes plasm and increased N:C ratio. This patient should d. B-lymphocytes be considered for: (Objective 5) 2. According to the CDC definition, a patient is consid- a. a urinary tract infection ered to have progressed from AHD to AIDS when the b. hepatitis following is detected: (Objective 10) c. bacterial meningitis a. a leukopenia d. Streptococcus pneumonia b. a CD4:CD8 ratio of 2:1 6. The morphology of lymphocytes found in infectious c. the absolute CD4+ count of less than 200/mcL mononucleosis is described as: (Objective 2) d. the absolute CD4+ count of less than 500/mcL a. segmented nucleus with pinkish-tan cytoplasm 3. Which of the following infectious agents is associated b. horseshoe-shaped nucleus with blue/gray with a newborn found to have jaundice and micro- cytoplasm cephaly with an enlarged spleen and decreased plate- let and red cell counts? (Objective 7) c. irregular shaped nucleus with spreading, deep blue cytoplasm a. Cytomegalovirus d. small cells with condensed chromatin and deep b. Epstein-Barr virus blue cytoplasm c. Toxoplasmosis gondii 7. A 2-year-old child has a total leukocyte count of d. Bordetella pertussis 10 * 103/mcL and 60% lymphocytes. Which of the 4. A teenager is seen at the clinic for a sore throat and following best describes the child’s blood count? enlarged cervical lymph nodes. He has reactive, atypi- (Objectives 4, 8) cal lymphocytes along with an elevated CRP and a. Absolute lymphocytosis elevated aminotransferases. He should also be tested b. Relative lymphocytosis for: (Objectives 1, 3) c. Normal lymphocyte count for the age given a. pertussis antitoxin antibodies d. Absolute lymphocytopenia b. HIV antibodies c. Toxoplasmosis antibodies d. heterophile antibodies Nonmalignant Lymphocyte Disorders 491 c. decreased reactive B lymphocytes with decreased Use this case study for questions 8–10. cytotoxic T lymphocytes A 19-year-old female college student went to student d. increased reactive B lymphocytes with increased health complaining of lethargy and a sore throat for cytotoxic T lymphocytes the past two weeks. Physical exam shows pharyn- 3. Epstein-Barr virus infects lymphocytes by attaching to gitis, lymphadenopathy, and splenomegaly with a which receptor? (Objective 2) total leukocyte count of 11 * 103/mcL and 70% lym- phocytes (50% of lymphs are reactive). a. CD4 b. CD8 c. CD21 d. TCR 8. She probably has: (Objective 1) a. HIV b. hepatitis Use this case study to answer questions 4 and 5. c. X-linked SCIDS A young male sees his physician for an ongoing d. infectious mononucleosis cough. He is found to have a total leukocyte count of 9. Her absolute lymphocyte count is: (Objective 4) 23 * 103/mcL with a differential of 22% neutrophils, a. 10 * 103/mcL 72% lymphocytes, 5% monocytes, and 1% eosino- b. 11 * 103/mL phils. The majority of the lymphocytes appear small with dense chromatin. c. 5.5 * 103/MCL d. 7.7 * 103/MCL 10. The best description of this patient’s leukocyte count is a(n): (Objectives 4, 8) 4. Which of the following describes his blood count? (Objective 7) a. relative lymphocytopenia a. Absolute neutrophilia b. relative neutrophilia b. Relative neutrophilia c. absolute lymphocytosis c. Absolute lymphocytosis d. absolute neutrophilia d. Relative eosinophilia Level II 5. The most likely cause of illness is: (Objectives 5, 7, 10) 1. A 17-year-old female patient is seen at the clinic because she has a lingering cough. She had a total leukocytosis a. Epstein-Barr virus of 23 * 103/mcL with 79% normal- looking lympho- b. Bordetella pertussis cytes. She should be screened for: (Objective 6) c. Cytomegalovirus a. pertussis antitoxin antibodies d. hepatitis b. VCA IgM 6. After taking care of a pair of cats, a 25-year-old male c. EBNA saw his physician for fever of unknown origin. He d. CMV antibodies had a total leukocyte count of 16 * 103/mcL with 78% lymphocytes, many showing reactivity, baso- 2. A patient with lethargy, pharyngitis, and lymphade- philia, and increased cytoplasm. His monospot test nopathy is found to have many lymphocytes that are was negative. What other testing should be consid- large with spreading, irregular cytoplasm and some ered? (Objective 4) with deep basophilia. A study of the lymphyocyte subsets would be expected to show: (Objective 2) a. Screening for Toxoplasmosis gondii a. decreased reactive B lymphocytes with increased b. Screening for HIV cytotoxic T lymphocytes c. Detection of pertussis antitoxin antibodies b. increased reactive B lymphocytes with decreased d. Detection of hepatitis B antigen cytotoxic T lymphocytes 492 Chapter 22 c. AHD Use this case study for questions 7–9. d. AIDS A 39-year-old male went to the clinic with com- 8. Which lymphocytes are periodically counted to moni- plaints of nagging cough, weight loss, diarrhea, and tor the disease? (Objective 8) low-grade temperature. Results of physical exami- nation showed lymphadenopathy, congested lungs, a. Infected B lymphocytes and increased heart rate. Slight splenomegaly and b. CD4+T lymphocytes hepatomegaly were noted. A CBC and flow cytome- c. CD8+T lymphocytes try studies were ordered. Histologic examination of d. Natural killer lymphocytes sputum with Gomori’s methenamine silver nitrate stain revealed Pneumocystis carinii. 9. Which laboratory test will be used to follow this patient’s disease? (Objective 8) Laboratory data Differential WBC 2.8 * 103/mcL Segmented 68% a. HIV-1 viral load count neutrophils b. Throat swab RBC 3.86 * 106/mcL Lymphocytes 21% c. Serologic test for heterophile antibody count d. PCR for genetic mutations Hb 13.6 g/dL Monocytes 10% 10. Immune deficiency associated with HIES is attributed (136 g/L) to the abnormal signaling or loss of cytokine: Hct 0.41 L/L Eosinophils 1% (Objective 11) Platelet 104 * 103/mcL Positive for HIV-1 a. IL-2 count antibodies b. IL-7 c. IL-15 d. IL-17 7. What clinical condition does this patient have? (Objectives 8, 10) a. Congenital immune deficiency b. Infectious mononucleosis References 1. Zhang, X., Dawson, C. W., He, Z., & Huang, P. (2012). Immune 7. Balfour, H. H. Jr., Dunmire, S. K., & Hogquist, K. A. (2015). evasion strategies of the human gamma-herpesviruses: Infectious mononucleosis. Clinical Translational Immunology, 4(2), e33. implications for viral tumorigenesis. Journal of Medical Virology, 8. Ebell, M. (2004). Epstein-Barr virus infectious mononucleosis. 84(2), 272–281. American Family Physician, 70, 1279–1287. 2. Sprunt, T. P. E. (1920). FA mononuclear leucocytosis in reaction 9. Vouloumanou, E. K., Rafailidis, P. I., & Falagas, M. E. (2012). to acute infections (“infectious mononucleosis”). Johns Hopkins Current diagnosis and management of infectious mononucleosis. Hospital Bulletin, 31, 410–417. Current Opinions in Hematology, 19, 14–20. 3. Downey, H. M. (1923). CA Acute |
lymphadenosis compared 10. Montoya, J. G., & Liesenfeld, O. (2004). Toxoplasmosis. Lancet, with acute lymphatic leukemia. Archives of Internal Medicine, 32, 363, 1965–1976 82–112. 11. Berger, F., Goulet, V., Le Strat, Y., & Desenclos, J. C. (2009). 4. Foerster, J. (1999). Infectious mononucleosis. In: G. R. Lee, J. Toxoplasmosis among pregnant women in France: Risk factors Foerster, J. N. Lukens, F. Paraskevas, J. P. Greer, G. M. Rodgers, and change of prevalence between 1995 and 2003. Review of & J. M. Baron, eds. Wintrobe’s clinical hematology (10th ed., Epidemiology Sante Publique, 57, 241–248. pp. 1926–1955). Baltimore: Williams & Wilkins. 12. Taylor, M., Lennon, B., Holland, C., & Cafferkey, M. (1997). 5. Dunmire, S. K., Odumade, O. A., Porter, J. L., Reyes-Genere, J., & Community study of toxoplasma antibodies in urban and rural Schmeling, D.O., (2014). Primary EBV infection induces an school children age 4 to 8 years. Archives of Disease in Childhood, expression profile distinct from other viruses but similar to 77, 406–409. hemophagocytic syndromes. PLoS One, 9, e85422. 13. Leal, F. E., Cavazzana, C. L., Andrade, H. F., Galisteo, A. J., 6. Balfour, H. H. Jr., Sifakis, F., Sliman, J. A., Knight, J. A., Schmeling, Mendonça, J. S., & Kallas, E. G. (2007). Toxoplasma gondii D. O., & Thomas, W. (2013). Age-specific prevalence of Epstein- pneumonia in immunocompetent subjects: Case report and Barr virus infection among individuals aged 6–19 years in the review. Clinical Infectious Diseases, 44, e62–e66. United States and factors affecting its acquisition. Journal of 14. Taylor, G. (2003). Cytomegalovirus. American Family Physician, Infectious Disease, 208, 1286–1293. 67(3), 519–524. Nonmalignant Lymphocyte Disorders 493 15. Stagno, S., Remington, J. S., & Klein, J. O. (2001). Cytomegalovirus. 31. Centers for Disease Control and Prevention. (2014). Revised In: J. S. Remington, & J. O. Klein, eds. Infectious diseases of the surveillance case definition for HIV infection–United States, 2014. fetus and newborn infant, 5th (pp. 389–424). Philadelphia: W.B. MMWR Recommendation and Reports, 63(RR-03), 1–10. Saunders. 32. Liebman, H. A., & Tulpule, A. (2013). Hematologic manifestations 16. Cannon, M. J., Schmid, D. S., & Hyde, T. B. (2010). Review of of AIDS. In: R. Hoffman, E. J. Benz Jr., L.E. Silberstein, H. E. cytomegalovirus seroprevalence and demographic characteristics Heslop, J. I. Weitz, & J. Anastasi, eds. Hematology: Basic principles associated with infection. Reviews in Medical Virology, 20(4), 202–213. and practice 6th ed., (pp. 2191–2207). Philadelphia: Churchill 17. Dropului, L. (2006). The impact of cytomegalovirus infections Livingstone. on solid organ transplantation. Advanced Studies in Medicine, 6, 33. Toren, K. G., Buskin, S. E., Dombrowski, J. C., Cassels, S. L., & 303–304, 319–328. Golden, M. R. (2016). Time from HIV diagnosis to viral load 18. Steininger, C. (2007). Clinical relevance of cytomegalovirus suppression: 2007–2013. Sexually Transmitted Diseases, 43(1), 34–40. infection in patients with disorders of the immune system. 34. Günthard, H. F., Saag, M. S., Benson, C. A., del Rio, C., Eron, J. J., & Clinical Microbiology and Infection, 13(10), 953–963. Gallant, J. E., (2016). Antiretroviral drugs for treatment and 19. Bravender, T. (2010). Epstein-Barr virus, cytomegalovirus, and prevention of HIV infection in adults: 2016 Recommendations infectious mononucleosis. Adolescent Medicine State of the Art of the International Antiviral Society-USA Panel. Journal of the Review, 21(2), 251–264. American Medical Association, 316(2), 191–210. 20. De Paschale, M., & Clerici, P. (2012) Serological diagnosis of 35. Hütter, G., Nowak, D., Mossner, M., Ganepola, S., Mübig, A., & Epstein-Barr virus infection: Problems and solutions. World Allers, K., (2009). Long-term control of HIV by CCR5 Delta Journal of Virology, 1(1), 31–43. 32/Delta 32 stem cell transplantation. New England Journal of 21. Crowcroft, N., & Pebody, R. (2006). Recent developments in Medicine, 360, 692–698. pertussis. Lancet, 367, 926–936. 36. Allers, K., Hütter, G., Hofmann, J., Loddenkemper, C., Rieger, 22. Hodge, G., Hodge, S., Markus, C., Lawrence, A., & Han, P. (2003). K., & Thiel, E., (2011). Evidence for the cure of HIV infection by A marked decreased in L-selectin expression by leucocytes in CCR5∆32/∆32 stem cell transplantation. Blood, 117, 2791–2799. infants with Bordetella pertussis infection: Leucocytosis explained? 37. New York State Department of Health AIDS Institute. (2010). HIV Respirology, 8, 157–162. prophylaxis following occupational exposure. NYSDoH, pp. 2–10. 23. Mattoo, S., & Cherry, J. D. (2005). Molecular pathogenesis, Retrieved from www.hivguidelines.org. epidemiology, and clinical manifestations of respiratory 38. Grays, E. S., Madiga, M. C., Hemanus, T., Moore, P. L., infections due to Bordetella pertussis and other Bordetella Wibmer, C. K., & Tumba, N. L., (2011). The neutralization subspecies. Clinical Microbiology Review, 18(2), 326–382. breadth of HIV-1 develops incrementally over four years and is 24. Pawloski, L. C., Plikaytis, B. D., Martin, M. D., Martin, S. associated with cd4+ T cell decline and high viral load during W., Prince, H. E., & Lapé-Nixon, M., (2016). Evaluation of acute infection. Journal of Virology, 85, 4828–4840. commercial assays for single-point diagnosis of pertussis in the 39. Lewis, D. B., Nadeau, K. C., & Cohen, A. C. (2009). Disorders US. Journal of the Pediatric Infectious Disease Society, 6(3), e15-e21. of lymphocyte function. In: R. Hoffman, E. J. Benz, S. J. Shattil, B. 25. Dworkin, M. S. (2005). Adults are whooping, but are internists Furie, L. E. Silberstein, P. McGlave, & H. Heslop, eds. Hematology: listening? Annals of Internal Medicine, 142, 832–835. Basic principles and practice, 5th ed., (pp. 721–745). Philadelphia: 26. Troussard, S., Cornet, E., Lesesve, J. F., Kourel, C., & Mossafa, Churchill Livingstone. H. (2008). Polycolonal B-cell lymphocytosis with binucleated 40. Cirillo, E., Giardino, G., Gallo, V., D’Assante, R., Grasso, F., & lymphocytes (PPBL). OncoTargets and Therapy, 1, 59–66. Romano, R., (2015). Severe combined immunodeficiency–an 27. Broudy, V. C., & Harrington, R. D. (2016). Hematologic update. Annals of the New York Academy of Science, 1356, 90–106. Manifestations of Acquired Immunodeficency Syndrome. In: K. Kaushansky, M. A. Lichtman, J. Prchal, M. M. Levi, O. W. Press, 41. Hersfield, M. (2011). Adenosine deaminase deficiency. L. J. Burns, & C. Michael, eds. Williams hematology, 9th ed. GeneReviews. Retrieved from www.ncbi.nlm.nih.gov/ (pp. 1239-1274). New York: McGraw-Hill. Pubmed/20301656. 28. Sharp, P. M., & Hahn, B. H. (2011). Origins of HIV and the AIDS 42. Chandra, S., Bronicki, L., Nagaraj, C. B., & Zhang, K. (n. d.). pandemic. Cold Spring Harbor Perspectives in Medicine, 1:a006841. WAS-related disorders. GeneReviews. Retrieved from www.ncbi. nlm.nih.gov/Pubmed/20301357. 29. Mofenson, L. M., Brady, M. T., Danner, S. P., Dominguez, K. L., Hazra, & R., Handelsman, E., (2009). Guidelines for the 43. Filipovich, A. H., Johnson, J., & Zhang, K. (2011). WAS-related prevention and treatment of opportunistic infections among HIV- disorders. GeneReviews. Retrieved from www.ncbi.nlm.nih.gov/ exposed and HIV-infected children: Recommendations from CDC, Pubmed/20301357. the National Institutes of Health, the HIV Medicine Association of 44. Fomin, A. B., Pastorino, A. C., Kim, C. A., Pereira, C. A., Carneiro- the Infectious Diseases Society of America, the Pediatric Infectious Sampaio, M., & Abe-Jacob, C. M. (2010). DiGeorge syndrome: Diseases Society, and the American Academy of Pediatrics. A not so rare disease. Clinics (Sao Paulo), 65, 865–869. MMWR Recommendations and Reports, 58(RR-11), 1–166. 45. Forte, W. C., Menezes, M. C., Dionigi, P. C., & Bastos, C. L. 30. Siberry, G. K., Abzug, M. J., Nachman, S., Brady, M. T., (2005). Different clinical and laboratory evolutions in ataxia- Dominguez, & K. L., Handelsman, E., (2013). Guidelines for the telangiectasia syndrome: report of four cases. Allergology and prevention and treatment of opportunistic infections in HIV- Immunopathology, 33, 199–203. exposed and HIV-infected children: Recommendations from 46. Tangye, S. G., Cook, M. C., & Fulcher, D. A. (2009). Brief the National Institutes of Health, Centers for Disease Control reviews: insights into the role of STAT3 in human lymphocyte and Prevention, the HIV Medicine Association of the Infectious differentiation as revealed by the hyper-IgE syndrome. Diseases Society of America, the Pediatric Infectious Diseases Journal of Immunology, 182, 21–28. Society, and the American Academy of Pediatrics. Pediatric 47. Su, H. D., Jing, H., & Zhang, Q. (2011). Dock8 deficiency. Infectious Disease Journal, 32(Suppl. 2), i-KK4. Annals of the New York Academy of Science, 1246, 26–33. This page intentionally left blank Section Five Neoplastic Hematologic Disorders 495 Chapter 23 Introduction to Hematopoietic Neoplasms Gideon H. Labiner, MS Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define and differentiate the terms neoplasm 5. Compare and contrast the laboratory and malignant and identify hematopoi- findings of the acute and chronic leukemias etic disorders that can be included in each and myeloid and lymphoid leukemias. category. 6. Define and differentiate proto-oncogenes 2. Compare and contrast the general and oncogenes and summarize their characteristics of the myelodysplastic relationship to neoplastic processes. syndromes (MDSs), myeloproliferative 7. Correlate patient age to the overall incidence neoplasms (MPNs), and acute and chronic of the hematopoietic neoplasms. leukemias. 8. Explain the usefulness of immunological 3. Describe the World Health Organization (WHO) techniques, chromosome analysis, and classification system used for MDSs, MPNs, molecular genetic analysis, in the diagnosis the leukemias, and lymphoid neoplasms. and prognosis of hematopoietic neoplasms. 4. List the various laboratory methods used 9. State the prognosis and survival rates of the to classify the hematopoietic neoplasms. hematopoietic neoplasms. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Explain how proto-oncogenes are activated and 2. Describe the effects of radiation on the inci- the role that oncogenes and tumor suppressor dence of leukemia. genes and their protein products play in the 3. Differentiate between the acute and etiology of hematopoietic neoplasms. chronic myeloid and lymphoid leukemias 496 Introduction to Hematopoietic Neoplasms 497 based on their clinical and hematologic leukemia (AML, ALL), and mature lymphoid findings. neoplasms, and justify a patient diagnosis 4. Reconcile the use of chemotherapy for based on these features. treatment of leukemia. 8. Select laboratory procedures appropriate 5. Compare and contrast treatment options for confirming cell lineage and diagnosis for the hematopoietic neoplasms including in hematopoietic neoplasms. possible complications. 9. Define epigenetics and explain its role 6. Name the leukemogenic factors of leukemia in cancer. and propose how each contributes to the 10. Define cancer stem cell and explain its development of leukemia. similarities to the hematopoietic stem 7. Compare and contrast laboratory features cell (HSC). of MDS, MPN, acute myeloid and lymphoid Chapter Outline Objectives—Level I and Level II 496 Laboratory Evaluation 506 Key Terms 497 Hematopoietic Neoplasm Classification 506 Background Basics 498 Laboratory Procedures for Diagnosing and Case Study 498 Classifying Neoplasms 507 Overview 498 Prognosis and Treatment of Neoplastic Disorders 509 Introduction 498 Summary 512 Pathophysiology 499 Review Questions 513 Epidemiology 505 References 515 Clinical Presentation 505 Key Terms Acute lymphocytic leukemia (ALL) Cytogenetic remission Mature neoplasm Acute myeloid leukemia (AML) Epigenetics Minimum residual disease (MRD) Auer rod French-American-British (FAB) Molecular remission Benign Hematologic remission Myelodysplastic syndrome (MDS) Cancer-initiating cell Induction therapy Myeloproliferative neoplasm Cancer stem cell Leukemia (MPN) Chronic lymphocytic leukemia Leukemic stem cell (LSC) Neoplasm (CLL) Leukemogenesis Oncogenes Chronic myelogenous leukemia Lymphoma Precursor neoplasm (CML) Maintenance chemotherapy Proto-oncogene Consolidation therapy Malignant Tumor suppressor gene 498 Chapter 23 Background Basics The information in this chapter serves as a general introduc- Level II tion to the hematopoietic neoplasms (Chapters 24–28). To • Describe the actions of cytokines, cytokine recep- maximize your learning experience, you should review the tors, signaling pathways, and transcription factors. following concepts before starting this unit of study: (Chapters 2, 4) • Summarize the cell cycle and identify factors that Level I affect it. (Chapter 2) • Summarize the origin and differentiation of the • Describe the normal structure and function of the hematopoietic cells. (Chapters 2, 4) bone marrow, spleen, and lymph nodes. (Chapter 3) • Describe the morphologic characteristics of the hematopoietic cells. (Chapters 5, 7, 8) syndromes. The acute myeloid neoplasms are described in CASE STUDY Chapter 26, the acute lymphoid neoplasms are described We refer to this case study throughout the chapter. in Chapter 27, and Chapter 28 includes a discussion of the Agnes, a 72-year-old female, saw her physician mature lymphoid neoplasms. The chapters on flow cytom- for a persistent cough and fatigue. She had always etry (Chapter 40), chromosome analysis (Chapter 41), and been in good health and played golf regularly. molecular analysis (Chapter 42), are referenced frequently Upon examination, she was noted to be pale and and provide additional information on laboratory proce- had slight splenomegaly. A CBC revealed the WBC dures used in diagnosis, prognosis, and treatment. count was |
83.9 * 109/L. Consider possible explanations for this test result and which reflex tests should be performed. Introduction Neoplasm (tumor) means “new growth.” Neoplasms arise as a consequence of dysregulated proliferation of a single Overview transformed cell. Genetic mutations in the transformed cell reduce or eliminate the cell’s dependence on external cyto- This chapter is the first in a section that discusses the hema- kines (growth factors) to regulate proliferation. topoietic neoplasms. It provides a general introduction to Neoplasms are either benign or malignant. Benign neoplastic hematologic disorders that are discussed in neoplasms are formed from highly organized, differenti- detail in Chapters 24–28. It describes oncogenesis includ- ated cells and do not spread or invade surrounding tis- ing how oncogenes are activated and their association with sue. Malignancy means “deadly” or “having the potential hematopoietic disease. This is followed by a description of for producing death.” A malignant neoplasm is a clone how neoplasms are classified and characterized according of abnormal, anaplastic, proliferating cells, which often to cell lineage, degree of cell differentiation, morphology, have the potential to metastasize (spread). Only malignant cytochemistry, immunophenotype, and genetic abnormali- tumors are correctly referred to as cancer. Although cancer ties. The etiology and pathophysiology of the leukemias are is actually a malignancy of epithelial tissue, common use examined using general clinical and laboratory findings. of the term includes all malignant neoplasms. A benign Finally, prognosis and treatment modalities for the disor- neoplasm can be premalignant and progress with further ders are considered. genetic mutations to a malignant neoplasm (Figure 23-1). A comprehensive table that includes the diagnosis, Malignant neoplasms of the bone marrow are collec- immunophenotype, chromosome translocation, and geno- tively known as leukemia. These neoplasms are grouped typic finding is included in Appendix B. This table should according to cell lineage as lymphoid, myeloid, and histio- be referred to when reading the chapters in this section. cytic/dendritic cell.1 The myeloid and lymphoid neoplasms This introductory chapter should be read before progress- are further subgrouped as precursor neoplasms (acute) and ing to the other chapters in this section. The other chapters mature neoplasms (usually chronic). A comparison of clini- are organized according to the World Health Organization cal and laboratory findings associated with these precursor (WHO) classification of hematopoietic and lymphopoi- and mature subgroups is listed in Table 23-1. etic neoplasms. Chapter 24 includes the myeloprolifera- Precursor neoplasms are characterized by genetic tive neoplasms. Chapter 25 includes the myelodysplastic mutations that promote proliferation and survival and/or Introduction to Hematopoietic Neoplasms 499 MPN Reactive leukocytosis MDS Acute Leukemoid reaction CLL Leukemia Plasma cell neoplasms Figure 23.1 The spectrum of hematopoietic proliferation ranges from benign to malignant outcomes. Benign myeloid and lymphoid proliferation is usually a reactive process (reactive leukocytosis; leukemoid reaction). Mature myeloid and lymphoid neoplasms include myeloproliferative neoplasms (MPNs), and myelodysplastic syndromes (MDSs), chronic lymphocytic leukemia (CLL), and plasma cell neoplasms. These neoplasms are derived from a mutated precursor cell that divides incessantly but has Figure 23.2 Acute myeloid leukemia. Note the large number some capacity to mature. Acute leukemia is a malignant precursor of myeloblasts with no mature granulocytes present (Wright stain, neoplasm characterized by unregulated cell proliferation and a 1000* magnification, peripheral blood). block in maturation. Mature neoplasms can progress with additional genetic mutations and terminate in acute leukemia. Abnormal proliferation of lymphoid cells sometimes occurs within the lymphatic tissue or lymph nodes. These that block differentiation into mature hematopoietic cells. solid tumors are referred to as lymphoma. If the lymphoma Thus, in acute leukemia (AL), a precursor neoplasm, there affects the bone marrow and lymphoma cells are found in is a gap in the normal maturation pyramid of cells and the peripheral circulation, the leukemic phase of lymphoma presents with many blasts (Figure 23-2) and some mature is present. forms of the affected lineage, but a decrease in intermedi- Failure of normal hematopoiesis is the most serious ate maturational stages. The mature cells seen in the bone consequence of malignant neoplasms. As the neoplastic marrow and peripheral blood arise from proliferation of cell population increases, the concentration of normal cells the residual normal hematopoietic stem cells (HSCs) in decreases, resulting in the inevitable cytopenias of normal the bone marrow. The excess of blasts primarily reflects blood cells (Figure 23-4). If the neoplasm is not treated, the proliferation of the abnormal malignant clone that fails to patient usually succumbs to infections secondary to granu- undergo maturation. locytopenia or bleeding secondary to thrombocytopenia. Mature neoplasms are also characterized by an increase in proliferation and survival of neoplastic cells, but in con- trast to precursor neoplasms, maturation of progenitor cells is nearly normal. The result is leukocytosis with the pre- Pathophysiology dominant leukemic cells being amitotic, mature or partially Cancer is a disease of gene mutations. Hematopoietic mature cells, with normal function. Thus, the bone marrow neoplasms are believed to occur as the result of a somatic and peripheral blood usually show an increase in concen- mutation(s) of a single hematopoietic stem or progenitor tration of cells at all maturation stages (Figure 23-3). The cell.2 Evidence for the clonal evolution of neoplastic cells mature myeloid neoplasms include subgroups in which the comes from cytogenetic studies. More than 50% of individu- cells exhibit dysplastic features (myelodysplasia). als with leukemia show an acquired abnormal karyotype in Table 23.1 Comparison of Precursor and Mature Hematopoietic Neoplasms Precursor (acute neoplasm) Mature (chronic neoplasm) Age All ages Adults Clinical onset Sudden Insidious Course of disease (untreated) Weeks–months Months–years Predominant cell Blasts, some mature forms Mature forms Anemia Mild–severe Mild Thrombocytopenia Mild–severe Mild WBC Variable Increased 500 Chapter 23 normal HSCs (Chapter 3). While more restricted progeni- tor cells, such as committed lymphoid progenitor (CLP) or committed myeloid progenitor (CMP) cells, do not normally have self-renewal capacity, they may acquire mutation(s) that reactivate the self-renewal program(s) and that allow them to become cancer-initiating cells.2 Many of the signal- ing pathways that have been shown to regulate normal stem cell development also play a role in cancer cell proliferation.2,3 The cancer-initiating cell gives rise to the cancer stem cell that divides incessantly to generate a tumor of “identi- cal” sibling cells or clones and sustains malignant growth. Most cancers are not truly clonal but consist of hetero- geneous cell populations. Only small subsets of cells are capable of extensive proliferation (cancer stem cells) within Figure 23.3 Chronic myelogenous leukemia. Note the a tumor. Identification of the hematopoietic form of a can- large number of granulocytic cells in various stages of maturation cer stem cell, or leukemic stem cell (LSC), for each type of including blasts, metamyelocyte, bands, and segmented neutrophils leukemia has become a major focus of research because the (Wright stain, 1000* magnification, peripheral blood). LSC is responsible for propagating the leukemia. hematopoietic cells whereas other somatic cells are normal. Using cytogenetic markers, normal and malignant cells can Checkpoint 23.1 be demonstrated to populate the marrow simultaneously. In A patient has 50% monoblasts in the bone marrow. Which untreated leukemias and during relapse, the leukemic cells precursor cell could be the cancer-initiating cell? dominate, whereas during remission, usually only normal cells can be detected. The mutations leading to malignant transformation of the cancer stem cell or LSC often are associated with a chro- Cancer Stem Cells mosome alteration that is observed as an abnormal karyotype Hematopoietic neoplasms arise when a normal hematopoi- when studying cells in mitosis (Chapter 41). When chromo- etic precursor cell acquires a cancer-initiating mutation. The some studies are normal, aberrations in DNA at the molecu- cell in which this genetic mutation(s) occurs is termed the lar level may be found (Chapter 42). In either case, genomic cell of origin or cancer-initiating cell (Figure 23-5). The can- changes in the cancer cells lead to a survival and/or prolifera- cer-initiating cell can be a hematopoietic stem cell (HSC) or tion advantage over normal cells and to the neoplastic expan- more differentiated progenitor cells.2,3 A hallmark of hemato- sion of the affected cancer stem cell and its progeny. In acute logic malignancies is the capacity for unlimited self-renewal leukemia, this unregulated proliferation is accompanied by of the cancer-initiating cell, which is also a characteristic of an arrest in maturation at the blast cell stage (Figure 23-5). Checkpoint 23.2 Neoplastic cells in peripheral blood A 62-year-old male presents with an elevated leukocyte count, mild anemia, and a slightly decreased platelet count. His phy- sician suspects leukemia. Explain why the erythrocytes and platelets are affected. Molecular Basis of Cancer The cancer cell genotype is generally maintained (stably inherited) during cell division. This implies that the tumor Normal cells in peripheral blood cell DNA determines the disease phenotype. Time ONCOGENES Figure 23.4 When inoculated into animals, certain viruses are capable of Clonal expansion of neoplastic cells in the bone marrow over a period of time leads to a decrease in the causing tumors. These tumor viruses carry discrete genetic concentration of normal cells in both the bone marrow and elements, oncogenes, which are responsible for inducing peripheral blood. malignant cell transformation. The proteins encoded by Bone marrow stem cell pool Introduction to Hematopoietic Neoplasms 501 Development of a Development of a normal hematopoietic neoplasm hematopoietic cell HSCs Leukemic Progenitors mutation(s) Blasts AL Figure 23.5 A hematopoietic neoplasm (left) is derived from a single cancer-initiating cell. The leukemic mutation(s) (black arrows) that transform a normal hematopoietic precursor cell to a cancer-initiating cell can occur at the hematopoietic stem cell (HSC) or more committed progenitor. If the cancer-initiating cell is a committed progenitor, then the mutations must include the capacity for self-renewal (blue arrows). If the mutation also can result in a block to terminal differentiation—the resulting malignancy will be an acute leukemia (AL). Residual “normal” HSCs and committed progenitors in the marrow will still be capable of producing mature cells (right). the oncogenes play important roles in the cell cycle, such • Growth factor receptors When activated to an onco- as initiation of DNA replication and transcriptional control gene, the mutated receptors are capable of trigger- of genes. Importantly, many viral oncogenes have normal ing growth-promoting signals, even in the absence of counterparts in the human genome, called proto-oncogenes. ligand (cytokine) binding. Thus, the human genome carries genes with the potential • Signal transducers The normal function of these pro- to dramatically alter cell growth and to cause malignancy teins (the largest class of proto-oncogenes) is to pass when altered or activated to an oncogene.4 receptor signals to downstream targets. Many of these One of the defining features of cancer cells is their abil- proto-oncogenes encode protein-tyrosine kinases found ity to proliferate under conditions in which normal cells on the inner surface of the membrane. Often the onco- do not.5 The proteins encoded by proto-oncogenes func- genic form of these genes produces signaling molecules tion in the signaling pathways by which cells receive and that exist in a constantly activated state in the absence execute growth instructions. The mutations that convert of growth factor/receptor interaction and signaling. proto-oncogenes to oncogenes (referred to as oncogene acti- • Transcription factors These proteins bind DNA and vation) are often either structural mutations resulting in the function to control the expression of cellular genes continuous (constitutive) activity of a protein without an required for proliferation. incoming signal or mutations in gene regulation that lead to the production of a protein at the wrong place or time. The Thus, proto-oncogenes are genes that regulate the ini- result in either case is a persistent internal growth signal tiation of DNA replication, cell division, the commitment to that is uncoupled from environmental controls. It is pos- cellular differentiation, and/or apoptosis (Chapter 2). Their sible that any gene playing a key role in cellular growth activation to an oncogene disrupts the growth-control appa- can become an oncogene if mutated in an appropriate way. ratus of the cell. Proto-oncogene activation occurs by one of In general, the proto-oncogenes that have been identi- three genetic mechanisms: mutation, gene rearrangement, fied serve one of the following functions in normal growth or gene amplification. The result is either (1) a qualitative control4 (Chapters 2, 4): change in function of the gene’s protein product, resulting in enhanced activity, (2) a protein that is no longer subject to • Growth factors These molecules provide the signals to the control of regulatory factors, or (3) |
a quantitative change grow and when activated to an “oncogene” result in an (increased production) of an otherwise normal protein. autocrine growth stimulation. 502 Chapter 23 TUMOR SUPPRESSOR GENES in most cells. Acquired mutations of RB (i.e., nonfamilial) Cancer is widely accepted to be a multi-hit phenomenon, are found in about 25% of sporadic cancers and have been resulting from several independent genetic alterations observed in various hematopoietic neoplasms. occurring sequentially within a single cell. Specific tumor Inactivation of the p53 gene, also a tumor suppressor suppressor genes function to inhibit cell growth in normal gene, is seen in more than half of all human cancers, mak- cells. Thus, in addition to oncogene activation that results ing it the most common genetic defect detectable in human in growth-promoting activity, tumor cells often have inac- tumors.8,9 Interestingly, a damaged p53 gene can be inherited tivating mutations of growth-suppressing genes that can (like familial retinoblastoma) resulting in Li-Fraumeni syn- also contribute to tumor development. Mutations in tumor drome and an inherited susceptibility to a variety of cancers suppressor genes behave differently from oncogene muta- including hematopoietic neoplasms.10,11 In affected individ- tions (Table 23-2). Oncogene mutations tend to be activating uals, 50% develop cancer by age 30 and 90% by age 70. The mutations, which functionally are dominant to wild-type function of p53 in cell cycle regulation (Chapter 2) is to block (nonmutated) gene products; they produce proliferation cell cycle progression in the event of damaged DNA or to signals even when a single copy of the oncogene is pres- trigger apoptosis if the damaged DNA cannot be repaired. ent. Tumor suppressor mutations, on the other hand, are The p53 protein is a major component of the body’s anti- recessive, loss-of-function mutations. Mutation in one gene tumor army, serving as a “molecular policeman” monitor- copy usually has no effect as long as a sufficient amount of ing the integrity of the genome. Loss of function of the p53 normally functioning wild-type protein remains. However, gene facilitates tumor formation by allowing damaged cells mutations in both copies of a tumor suppressor gene lead to to proceed through the cell cycle and continue to replicate. complete loss of its normal, growth-inhibitory effect. EPIGENETICS Understanding the function of tumor suppressor genes In addition to the mutations observed in various oncogenes has been greatly aided by studies of rare cancers that run and tumor suppressor genes, epigenetic alterations may also in families in which affected family members appear to be observed in a malignancy.12,13 Epigenetics is defined as inherit susceptibility to and develop certain kinds of tumors heritable changes in gene expression not caused by changes at rates much higher than the normal population. The first in DNA sequence, but rather to the ability of a gene to be of these familial cancers to be explained at the molecular expressed. Epigenetic changes are stable from one cell gen- level was the inherited susceptibility to retinoblastoma (a eration to the next after each mitotic event. These changes tumor of the eye) in certain families.6,7 Although retinoblas- play an important role in normal development and differ- toma can occur sporadically, about one-third of the cases entiation and are associated with “silencing” genes and occur in related siblings, suggesting an inherited suscep- chromatin condensation into heterochromatin (Chapter 2). tibility to the disease. The development of retinoblastoma Major epigenetic changes include DNA methylation of CpG requires two mutations that inactivate both of the RB loci dinucleotides, histone acetylation/deacetylation reactions, on each of the chromosomes 13. In the familial form of the and microRNAs. disease, the affected children inherit one mutant RB allele and one normal allele. Retinoblastoma (or another malig- DNA METHYLATION nancy) develops when acquired mutations eliminate the The methylation pattern of a gene determines its expression. function of the remaining normal (wild-type) allele. Thus, In a malignancy, there can be regions of the genome that are the RB gene acts as a tumor suppressor gene that normally inappropriately demethylated whereas other regions may functions to arrest excessive growth of cells. As is typically be aberrantly methylated. The list of genes that acquire true of tumor suppressor genes, one RB copy is sufficient hypermethylation of CpGs in their promoter regions and to keep growth in check, but loss of both copies eliminates contribute to tumorigenesis is growing.14 This hypermeth- the tumor suppressor function, and a tumor develops. The ylation is associated with transcriptional silencing of these protein product of the RB gene (Rb protein) is not specific genes and is the explanation for one of the most common to retinal tissue but serves as a universal cell cycle brake causes of loss of function of key tumor suppressor genes. Table 23.2 Properties of Oncogenes and Tumor Suppressor Genes Property Oncogenes Tumor Suppressor Gene Nature of mutation Dominant (one mutated allele displays the phenotype) Recessive (both alleles must be mutated to display the phenotype) Gain of function Loss of function Inherited mutant allele Never observed Rare—basis for inherited predisposition in cancers Somatic mutations in cancers Yes Yes Introduction to Hematopoietic Neoplasms 503 HISTONE DEACETYLATION Amplification Modifications to histone proteins are also seen in malignan- Translocation Retroviral insertion cies. Histones that are acetylated allow for chromatin to be loosely compacted and therefore allows for gene expres- + sion. Hypo-acetylated histones bind tightly to the phos- G1 phate backbone of DNA and help maintain chromatin in Cyclin D1 p16 Rb an inactive state, thus silencing gene expression. Various Cdk4 types of malignant cells use enzymes called histone deacety- S – – lases (HDACs) to hypo-acetylate key histone proteins. This + results in the silencing of tumor suppressor genes and, Mutation Mutation Deletion Amplification Deletion likewise, cell proliferation rather than differentiation. One Methylation Mutation Tumor viruses approach to treating cancer patients involves demethylat- ing agents or HDAC inhibitors to reverse epigenetic changes Figure 23.6 Alterations of the G1 checkpoint that can lead associated with certain types of cancer.14 to malignancy. Loss-of-function alterations in cell cycle negative regulators (i.e., the tumor suppressor gene products p16 or Rb) can MICRORNAs contribute to uncontrolled proliferation. Similarly, gain-of-function MicroRNAs (miRNAs) are a class of non-protein coding, mutations of positive regulators of proliferation can contribute to single-stranded RNA molecules (about 22 nucleotides in uncontrolled proliferation (i.e., the proto-oncogene gene products length) that help to control cell differentiation through post- Cyclin D, Cdk4). = inhibition of the pathway; + = an alteration that transcriptional degradation of their target mRNAs (Chap- increases activity of the indicated proteins; - = an alteration that decreases activity of the indicated proteins. ter 2). Aberrant expression of miRNAs leads to malignant transformation in a variety of cancers including hematologic malignancies by two general mechanisms: (1) increased in cyclin-dependent kinases can also lead to a loss of cell miRNA expression can lead to the degradation of tumor cycle regulation. For instance, mutations that lead to overex- suppressor gene transcripts, and (2) decreased miRNA expres- pression of Cdk4 (a kinase regulated by cyclin D) have been sion can lead to the over-expression of proto- oncogenes. reported in a number of human tumors and contributes MiRNAs are increasingly being used as biomarkers of diag- to the excessive growth characteristics of those diseases. nosis, prognosis, and prediction of response to therapy.15 Therefore, the p16-cyclin D-Rb pathway, which controls the G1 checkpoint in cell cycle regulation, is believed to play a pivotal role in tumorigenesis (Figure 23-6). Some investiga- Checkpoint 23.3 tors have proposed that a mutation involving at least one Mutations in proto-oncogenes predisposing to malignancy are member of this checkpoint must occur in order for a malig- nant phenotype to be established.16 said to be dominant mutations, whereas mutations in tumor suppressor genes are said to behave as recessive mutations, requiring loss of both alleles. Explain this difference in behavior of the gene products. Checkpoint 23.4 A cell contains a mutation that blocks expression of p16. What is the effect (if any) on the daughter cells produced? CELL CYCLE CHECKPOINTS AND CANCER A common feature of many cancer cells is the loss of regu- lation of cell cycle checkpoints (Chapter 2), either by over- APOPTOSIS AND CANCER expression of positive regulators (for example, cyclins and The accumulation of an excess number of cells, a characteris- Cdks) or the loss of function of negative regulators (the Cdk tic of malignancies, can result from increased cell proliferation inhibitors, p53, or Rb).15 Cyclin D, cyclin E, and cyclin A are (see previous discussion of cell cycle checkpoints and can- overexpressed in a variety of human cancers and function cer) and/or to decreased cell death (apoptosis).5 Thus, muta- as oncogenes in their mutated configuration. Often, spe- tions of genes important in regulating apoptosis have also cific chromosomal translocations activate the expression been identified as oncogenes and tumor suppressor genes. of a cyclin gene by placing it under the influence of other These include loss-of-function mutations that initiate apop- transcriptional control elements. For example, the t(11;14) tosis such as p53, Bax, and other pro-apoptotic Bcl-2 family translocation seen in some B-lymphocyte malignancies members as well as overexpression of antiapoptotic proteins places the cyclin D gene under regulatory control of the such as Bcl-2 and other Bcl-2 family members that function immunoglobulin heavy chain locus, resulting in activation to inhibit apoptosis17 (Chapter 2). Bcl-2 is overexpressed of cyclin D expression at inappropriate times during a cell’s in most cases of B-cell follicular lymphoma, many cases life. Thus, the result of the t(11;14) translocation is an onco- of B-cell chronic lymphocytic leukemia (CLL), and some genic form of cyclin D (Chapter 41). Furthermore, mutations cases of acute myeloid l eukemia (AML)18 (Chapters 26–28). # 504 Chapter 23 Mutations in Bax (resulting in loss of pro-apoptotic func- Radiation, some chemicals, and drugs can cause chro- tion) have been reported in about 20% of leukemic cell lines. mosome mutations. Ionizing radiation has long been rec- The result is production of cells with an extended life span, ognized as capable of inducing leukemia, which is evident increased proliferation capacity, and diminished cell death. from observations of human exposure to radiation from nuclear reactions, therapeutic radiation, and occupational Leukemogenesis exposure to radiation. An increase in leukemia has been observed after treatment with alkylating agents and other From studies on laboratory animals, several factors have chemotherapeutic drugs used in treatment of many kinds been suggested as playing roles in leukemogenesis: (1) of malignancy. The only chemical that is specifically impli- genetic susceptibility, (2) somatic mutation, (3) viral infec- cated in causing leukemia other than those used as medica- tion, and (4) immunologic dysfunction (Table 23-3). tions is benzene. GENETIC SUSCEPTIBILITY The outcome of chromosome breaks and translocations Strong evidence suggests that hereditary factors and abnormal can lead to the activation of oncogenes and ultimately result genetic material have important leukemogenic effects. A num- in the aberrant expression of the protein product (Appen- ber of individuals who have congenital abnormalities associ- dix B). Furthermore, breaks and translocations within gene ated with karyotypic abnormalities have a markedly increased sequences can produce hybrid (fusion) genes and a new pro- risk of developing acute leukemia. Each of these genetic events tein product. For example, the balanced translocation between has the potential to activate proto-oncogenes or eliminate the chromosomes 9 and 22 in chronic myelogenous leukemia function of a tumor suppressor gene. The best known of the (CML) results in the formation of a BCR-ABL fusion gene that genetic abnormalities associated with leukemia is Down syn- encodes an abnormal tyrosine kinase (TK) protein. The TK is drome in which the extra chromosome 21 may participate in constitutively activated to transduce signals of cell survival, translocations.19,20 Various other congenital disorders also are proliferation, and resistance to apoptosis. Consequently, the associated with an increased risk for leukemia.21,22 cell containing the translocation is able to persist under condi- tions that would not normally support its survival. SOMATIC MUTATION A somatic cell mutation is an acquired change in the genetic VIRAL INFECTION material of cells other than those involved in reproduction. Retroviruses have been shown to cause leukemia in labo- Mutations in the chromosome near proto-oncogenes likely ratory animals, and a few malignancies can be traced to a play a role in neoplasm development. More than 50% of viral infection of cells in humans.23 Retroviruses contain a patients with leukemia have acquired abnormal karyotypes reverse transcriptase that |
allows them to produce a DNA and cytogenetic studies have revealed specific, consistent copy of the viral RNA core. The DNA can then be copied mutations in certain subgroups of hematopoietic neoplasms.23 to produce more viral cores or can be incorporated into the host cell’s nuclear DNA. The strongest support for the exis- tence of a leukemogenic virus in humans comes from the Table 23.3 Factors that May Play a Role in isolation of several human retroviruses known as human Leukemogenesis T-cell leukemia/lymphoma virus (HTLV-I, II, V) and human Factor Example immunodeficiency virus (HIV-1) from cell lines of patients with mature T-cell malignancies.24 Exactly how viruses Genetic susceptibility Down syndrome induce leukemia is unclear, but the incorporation of the Fanconi anemia viral genome into host DNA is suspected to lead to activa- Kleinfelter syndrome tion of proto-oncogenes. Bloom syndrome Wiskott-Aldrich syndrome IMMUNOLOGIC DYSFUNCTION Diamond-Blackfan syndrome An increased incidence of lymphocytic leukemia has been observed in both congenital and acquired immunologic Xeroderma pigmentosum disorders. These disorders include the hereditary immu- Li-Fraumeni syndrome nologic diseases Wiskott-Aldrich syndrome, Bruton type Somatic mutation Radiation X-linked agammaglobulinemia, and ataxia telangiectasia. Chemicals An association between long-term treatment of patients Drugs with immunosuppressive drugs (e.g., renal transplant) Viral infection Retroviruses—HTLV-I, II, V, HIV-1 and leukemia also has been observed. Possibly, a break- Immunologic disorders Wiskott-Aldrich syndrome down in the cell-mediated immunologic self-surveillance Bruton type X-linked agammaglobulinemia system and/or deficient production of antibodies against Ataxia telangiectasia foreign antigens leads to the emergence and survival of Immunosuppressive therapy neoplastic cells. Introduction to Hematopoietic Neoplasms 505 MISCELLANEOUS FACTORS Although acute leukemia occurs at all ages, peak incidence Certain hematologic diseases appear to pose a leukemo- occurs in the first decade, particularly from the ages of 2 to genic risk. Leukemia development in some patients appears 5 followed by a decreasing incidence in the second and third to be related to the treatment used for the primary disease, decade. Thereafter, the incidence begins to increase, rising but in others, no such relationship can be found. The high- steeply after age 50. The cellular type of leukemia occur- est incidence of acute leukemia is found in individuals with ring at these peak periods differs significantly. Most child- other neoplastic bone marrow disorders, such as myelo- hood acute leukemias are of the lymphoid type, whereas proliferative neoplasms (MPNs) and myelodysplastic those occurring in adults are typically myeloid. Chronic syndrome (MDS),25,26,27 prompting some hematologists to leukemias are rare in children. Chronic myelogenous leu- use the words preleukemia or premalignant for these disor- kemia occurs most often in young to middle-aged adults, ders. Additional genetic mutations (or epigenetic altera- and chronic lymphocytic leukemia is diagnosed primarily tions) occur as the preleukemic disease progresses to the in older adults. MPN and MDS occur most often in middle- malignancy, leukemia. Other hematopoietic diseases with age to older adults. an increased incidence of leukemia include paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia, and multiple myeloma. Interestingly, all of these hematologic disorders are considered stem cell disorders in which the Clinical Presentation primary hematologic defect lies in the myeloid or lymphoid Failure of the normal triad of hematopoiesis is the most progenitor cells or in the pluripotential stem cells. serious consequence of hematopoietic neoplasms and the No single factor is responsible for the genetic altera- most frequent symptoms are related to erythrocytopenia, tions that result in hematopoietic neoplasms; rather, a vari- thrombocytopenia, and/or neutropenia. The major clini- ety of etiologic factors produce the malignancy.28,29,30 The cal problems are anemia, infection, and bleeding episodes. cause varies from patient to patient, and some individuals Weight loss and bone pain that results from marrow expan- are more susceptible than others to malignancies. sion are also common complaints. Physical examination can show hepatosplenomegaly and, occasionally, lymph- adenopathy. Organomegaly is more common in mature Checkpoint 23.5 cell leukemias (chronic leukemias) than in the precursor Does a 3-year-old child with Down syndrome have an increased (acute) forms. risk of developing leukemia? Why or why not? Although the disease originates in the bone marrow, neoplastic cells can infiltrate any tissue of the body, especially the spleen, liver, lymph nodes, central nervous Epidemiology system, and skin. The lesions produced vary from rashes to tumors. Skin infiltration is most commonly found in The Leukemia and Lymphoma Society predicted that in acute myeloid leukemia (AML), particularly those with 2016, about 171,000 new cases of leukemia, lymphoma, and a monocytic component. Central nervous system (CNS) myeloma would have been reported, accounting for 10% of involvement is common in acute lymphoblastic leukemia new cases of cancer in the United States.31 About 31% more (ALL) of childhood.32 Chloromas, which are green tumor males are living with leukemia than females. masses of immature leukocytes, are associated with AML Approximately 50% of all leukemias are diagnosed and CML and are usually found in bone but can be found as acute. Although some difference in the incidence of the throughout the body. The green color, which fades to a dirty acute leukemias exists between countries and regions of yellow after exposure to air, is responsible for the descrip- countries, the differences are not great. tive name given to this unique clinical finding. Presumably, Of particular interest are the incidence and morpho- the green color results from the myeloperoxidase content of logic variation of leukemia among age groups (Table 23-4). the malignant cells. Table 23.4 Age Groups Typically Found in Acute (Precursor) and Chronic (Mature) Leukemias Neoplasm Age Acute lymphocytic leukemia (ALL) Children 2–5 years old Chronic lymphocyte leukemia (CLL) Adults more than 50 years old Acute myeloid leukemia (AML) Adults Chronic myeloid leukemia (CML) and other myelopro-liferative neoplasms Adults 506 Chapter 23 Laboratory Evaluation Maturation abnormalities are commonly present in all three cell lines. Megaloblastoid erythropoiesis can be Cell counts and morphology are variable in hematopoietic prominent but is unresponsive to vitamin B12 or folic acid neoplasms (Table 23-5). A normocytic (occasionally macro- treatment. cytic) normochromic anemia is often present at diagnosis. Because of the intense increase in cell turnover, other If not present initially, anemia invariably develops during laboratory tests reflecting cell destruction could be abnor- progression of the disease. mal. An increase in uric acid, which is a normal product of The platelet count varies. Thrombocytopenia is usu- nucleic acid metabolism, is a consistent finding in all types ally present at diagnosis in precursor neoplasms (acute of leukemia. The rate of excretion can increase to 50 times leukemia). Thrombocytosis is a common initial finding in normal. Serum lactic dehydrogenase (LD) levels appear to some of the mature neoplasms but can decrease with dis- correlate closely with the concentration of leukemic cells. ease progression. Platelet morphology and function may Isoenzyme studies reveal that the LD is derived from imma- be abnormal. Large hypogranular platelet forms are com- ture leukocyte precursors. Muramidase (lysozyme) is a mon, and circulating micromegakaryocytes occasionally lysosomal enzyme present in monocytes and granulocytes. are present. The serum and urine muramidase concentration in leuke- The leukocyte count can be normal, increased, or mia is highly variable and is related to the cellular type. decreased. More than 50% of patients with AML do not The highest concentrations are found in neoplasms with a have a significant leukocytosis at diagnosis. However, if monocytic component. left untreated, leukocytosis eventually develops. On the other hand, in the mature myeloproliferative and lym- phoproliferative neoplasms, leukocytosis at diagnosis is a prominent finding. Normal or decreased leukocyte Checkpoint 23.6 counts are typical in myelodysplastic syndrome (MDS). Why is finding Auer rods an important factor in the diagnosis Regardless of the leukocyte count, an increase in imma- of leukemia? ture precursors is found in most cases. Blasts are especially prominent in the precursor leukemias. Unique pink-stain- ing granular inclusions called Auer rods can be found in the blast cells and promyelocytes of some acute myeloid Hematopoietic Neoplasm leukemias. These Auer rods are believed to be formed from fused primary granules. When AML is suspected, finding Classification Auer rods can help to establish the diagnosis because they Classifications of hematopoietic neoplasms are considered are not found in ALL. to be important for three reasons: The bone marrow is hypercellular but occasionally is 1. They provide clinicians and researchers a way to study, normocellular or hypocellular. Reticulin is increased, often select, and compare various therapeutic regimens. worsening with disease progression. Blasts are usually increased. A minimum of 20% blasts is recommended for a 2. They provide a system for diagnosis using clearly defined diagnosis of AML. The cutoff of less than 20% blasts is used clinical features and laboratory findings. to differentiate the mature myeloid neoplasms (MPN and 3. They permit meaningful associations of genetic abnor- MDS) from precusor myeloid neoplasms. malities with pathogenesis of neoplastic disease. Table 23.5 Characteristic Hematologic Findings in Hematopoietic Neoplasms Neoplasm Leukocytes Platelets Bone Marrow Other Acute leukemia Normal or increased; blasts Decreased Hypercellular, more than 20% Auer rods in AML present blasts MDS Normal or decreased; blasts can Variable Hypercellular, occasionally hypo- Dysplastic be present cellular, less than 20% blasts MPN (includes chronic Increased; shift to left but predomi- Usually increased Hypercellular, occasionally hypo- myelogenous leukemia (CML) nance of mature forms cellular, less than 20% blasts MDS/MPN Increased blasts and other imma- Usually Hypercellular, less than 20% blasts Dysplastic ture forms decreased CLL Increased mature lymphocytes; Variable Hypercellular, less than 20% blasts Smudge cells; less than neutropenia 10% prolymphocytes MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; CLL, chronic lymphocytic leukemia Introduction to Hematopoietic Neoplasms 507 Before 2001, hematopoietic neoplasm classification was primarily based on cell lineage as determined by the CASE STUDY (continued from page 498) morphology and cytochemistry of the neoplastic cells. The CBC results on Agnes were: The system was known as the FAB (French-American- WBC 83.9 * 103/mcL Differential British) classification system.33 Later, immunopheno- RBC 3.15 * 106/mcL Segs 12% typing was added to the schema. More recently, genetic Hb 9.5 g/dL (95 g/L) Lymphs 88% features, prior therapy, and a history of myelodysplasia Hct 29% (0.29 L/L) are recognized to have a significant impact on the clini- Platelets 130 * 103 cal behavior of the hematopoietic neoplasms. Thus, in /mcL 2001, the Society for Hematopathology and the Euro- 1. Given Agnes’s laboratory results, would this pean Association of Hematopathologists developed the most likely be considered an acute or chronic World Health Organization (WHO) classification to bet- leukemia? Explain. ter define the hematopoietic neoplasms; this classifica- 2. What group of leukemia (cell lineage) is sug- tion was updated in 2008 and again in 2016.34,35,36,37 This gested by Agnes’s blood cell differential results? classification uses morphologic, immunophenotype, and molecular analyses to determine cell lineage and degree 3. What would you expect the blast count in the of maturation (Table 23-6). These analyses also help to bone marrow to be? determine whether cell proliferation is effective or ineffec- tive and to detect whether cells are cytologically normal or dysplastic. Clinical features such as prior therapy, age, and history of MDS are correlated with genetic findings, morphology, and immunophenotype to define subgroups Laboratory Procedures for of myeloid and lymphoid neoplasms.35,36,37,38 Cytogenetic and molecular genetic studies are necessary to categorize Diagnosing and Classifying those neoplasms that are genetically defined, to establish a baseline, to assess genetic evolution and progression, Neoplasms and to monitor response of the disease to therapy. In some Initial evaluation of patients with a hematopoietic neoplasm cases, molecular analysis can detect gene mutations when should include collection of peripheral blood and bone mar- the cytogenetic profile is normal. These studies that define row specimens prior to therapy. Bone marrow should include cell lineage, degree of differentiation, and dysplasia and aspirate as well as biopsy specimens. Morphology and blast detect genetic abnormalities should be performed at ini- count are performed on Romanowsky-stained smears. In tial evaluation.36 addition to obtaining a blast count, these cells must be differ- The classification according to lineage of the neoplastic entiated. Differentiation of leukemic blasts allows classifica- cells includes three groups: myeloid, lymphoid, and his- tion of acute leukemia into cell lineage (lymphoid or myeloid) tiocytic/dendritic (Table 23-6). Mast cells are derived from and various subtypes (Table 23-7). In the acute leukemias, hematopoietic progenitor cells and possess myeloid cell identification of the cell lineage of leukemic blasts is often characteristics. Thus, mast cell disease can also be consid- difficult when performed by morphology alone unless Auer ered a myeloid disorder. |
rods are present. However, the distinction of cell lineage is Table 23.6 WHO Classification of Hematopoietic, Lymphopoietic, and Histiocytic/Dendritic Neoplasms Classification Diseases Included Hematopoietic Myeloproliferative neoplasms (MPNs) Myeloid and lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, FGFR1, or PCM1-JAK2 Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) Myelodysplastic syndromes (MDSs) Acute myeloid leukemia (AML) and related neoplasms Acute leukemia of ambiguous lineage Lymphopoietic Precursor lymphoid neoplasms (T and B lymphoblastic leukemia/lymphoma) Mature B-cell neoplasms (including chronic lymphocytic leukemia, CLL) Mature T-cell and NK-cell neoplasms Hodgkin lymphoma Posttransplant lymphoproliferative disorders (PTLD) Histocytic and Dendritic Histiocytic and dendritic cell neoplasms Subgroups not included. See Appendix C for classification that includes subgroups. 508 Chapter 23 Table 23.7 Comparison of Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML)a ALL AML Age Common in children Common in adults Hematologic presentation Anemia, neutropenia, thrombocytopenia, lympho- Anemia, neutropenia, thrombocytopenia, myeloblasts, blasts, prolymphocytes promyelocytes Prominent cell morphology Small-to-medium lymphoblasts, fine chromatin with Medium-to-large myeloblasts with distinct nucleoli, fine nuclear scanty to abundant cytoplasm, indistinct nucleoli chromatin, and abundant basophilic cytoplasm, possible Auer rods Cytochemistry PAS positive, peroxidase negative, SBB negative, TdT PAS negative, peroxidase positive, SBB positive, TdT negative or positive, chloroacetate esterase (specific) negative positive, chloroacetate esterase positive Immunophenotype B Cell: CD19, CD20, cytoplasmic CD79a and CD22, CD34, HLA-DR, CD117, CD13, CD33, CD15 CD10, CD19 (can also have CD13 and CD33); T cell: CD1a, CD2, CD3, CD4, CD5, CD7, CD8 PAS = periodic acid Schiff; SBB = Sudan black B; TdT = terminal deoxynucleotidyl transferase a Genetic abnormalities are also used to differentiate ALL and AML. important for selecting the appropriate therapy. Morphology, that of megaloblastic cells. As a result, although the nucleus immunologic marker analysis (immunophenotype), and/or appears very immature, the cytoplasm of leukemic blasts may genetic studies can differentiate blasts. Cytogenetic analysis contain constituents normally present only in more mature on a bone marrow specimen should be performed, and addi- cells or can lack one or more constituents expressed by their tional molecular genetic studies should be guided by clinical, normal counterparts. In addition, abnormal accumulation laboratory, and morphologic information. and distribution of cellular metabolites is possible. Distinction of the mature neoplasms is not as difficult as in acute leukemias because the maturation of the neo- Immunologic Analysis plastic cell in mature neoplasms is not blocked, and the cell matures into recognizable blood cells. Immunophenotyping Immunologic analysis is based on identifying specific is not usually necessary in classification of MDS and MPN, membrane antigens (surface markers) characteristically but genetic studies are helpful in subgrouping. All data are found on a particular cell lineage. Immunologic techniques correlated into a report that gives the WHO diagnosis.35,36 with monoclonal antibodies are widely used to identify cell membrane antigens on a variety of cells (Chapter 40). The Cytochemical Analysis development of a large number of monoclonal antibodies that react with surface antigens on normal and neoplastic In hematology, cytochemistry refers to in vitro staining of cells cells and the technical advances in flow cytometry have that allows microscopic examination of the cells’ chemical greatly enhanced the ability to define leukemic cell lineage, composition. Cell morphology is not significantly altered in the the stage of cell development, and clonality. By utilizing a staining process. Most cellular cytochemical markers represent panel of monoclonal antibodies, a more complete picture of organelle-associated enzymes and other proteins. The cells are the cells’ lineage can be determined. incubated with substrates that react with specific cellular con- Immunophenotyping plays a central role in diagnosing stituents. If the specific constituent is present in the cell, the hematopoietic and lymphoid neoplasms. It includes identify- constituent’s reaction with the substrate is confirmed by the ing the lineage of blasts in the blast phase of CML, subtyping formation of a colored product. The stained cells are examined the myeloid leukemias, and identifying the lineage of those and evaluated on smears with a light microscope, although leukemias that lack specific morphologic characteristics such electron microscopy occasionally is necessary to identify very as minimally differentiated AML from ALL and subtyping weak reactions at the subcellular level. The results of these the lymphoid neoplasms as T or B cell. Immunologic patterns cellular reactions in normal and disease states are well estab- can help plan molecular analysis because some immunophe- lished. Cytochemistry is particularly helpful in differentiating notypes are associated with particular genetic mutations. The the lymphoid or myeloid lineage of the blasts in precursor identification of cell antigens can also help direct treatment leukemias when morphologic identification on Romanowsky- because some antigens are targeted for specific therapy (e.g., stained smears is difficult (Table 23-7). Cytochemistry is also rituximab, an anti-CD20 drug). If marrow cell suspensions helpful in subgrouping AML and related precursor neoplasms. are not available, immunophenotyping can be done using Staining procedures are described in Chapter 37. immunohistochemical analysis on bone marrow biopsy It is important to remember that the blast cells seen in specimens. See Chapter 40 for a more thorough discussion leukemias are neoplastic and can therefore differ from normal of monoclonal antibodies and their use in the identification blasts in both morphology and metabolic activity. Leukemic of neoplastic hematopoietic disorders. Appendix B includes cells often display nuclear/cytoplasmic asynchrony similar to the antigenic patterns in hematologic neoplasms. Introduction to Hematopoietic Neoplasms 509 Genetic Analysis Molecular analysis also is helpful in providing clues to the pathogenesis of hematopoietic neoplasms. For Genetic analysis includes cytogenetics to determine the cell example, the specific t(15;17) (q22;q12) mutation found in karyotype and molecular methods to identify specific gene acute promyelocytic leukemia produces an abnormal form mutations—both of which can be key to differentiate and of the nuclear hormone receptor, retinoic acid receptor@a define some neoplasms. In some cases, the karyotype will (RAR@a). The abnormal receptor is involved in the matu- appear normal but mutations can be found using molecu- ration blockade seen in the neoplastic promyelocytic cells. lar techniques such as polymerase chain reaction (PCR), When patients who possess this mutation are treated with DNA sequencing, and fluorescent in situ hybridization retinoic acid derivatives, the cells are induced to differenti- (FISH) (Chapter 42). ate into mature granulocytes. CYTOGENETICS Advances in cytogenetics have enabled cytogeneticists to Checkpoint 23.7 identify characteristic nonrandom abnormal karyotypes in A patient has 35% blasts in the bone marrow. They do not the majority of the acute leukemias and in some MPNs and show any specific morphologic characteristics that will allow MDSs.39 Some specific chromosome changes are consistently them to be classified according to cell lineage. What are the associated with a particular neoplastic subgroup and thus next steps that the clinical laboratory scientist should take with are helpful in diagnosis. For example, the t(15;17)(q22;q12) is this specimen? diagnostic of acute promyelocytic leukemia, and the Philadel- phia chromosome characterized by t(9;22)(q34;q11.2) confirms a clinical diagnosis of CML. In the lymphoid leukemias, non- Prognosis and Treatment random chromosome changes are often associated with either the B or T lymphocyte lineage and provide important prog- of Neoplastic Disorders nostic as well as diagnostic information. Chromosomal rear- rangements and their accompanying molecular abnormalities Studies of the hematopoietic neoplasms that have eluci- can identify distinct clinical groups with a predictable clinical dated the pathogenesis of these disorders have led to new course and response to specific therapy. In addition to helping treatment modalities that significantly changed the prog- physicians evaluate their patients, cytogenetic studies provide nosis for patients. new insights into the pathogenesis of neoplastic diseases. When cytogenetic abnormalities are present before therapy, their presence or absence can be used to identify Prognosis remission, relapse, and minimal residual disease after ther- Before the 1960s, a patient diagnosed with acute leukemia apy. If the cytogenetic abnormality identified before therapy could expect to die within a few months. With new treat- is still present after therapy, it is evidence that neoplastic ment modalities, remission rates for both ALL and AML cells remain in the bone marrow. In some cases, additional have improved dramatically. Remission was defined origi- chromosome aberrations can be identified during the course nally as a period of time in which there were no clinical or of the disease or after a period of remission. The finding of hematologic signs of the disease. More recently with the var- additional chromosome aberrations is not usually a signal ious diagnostic and monitoring modalities available, several of disease progression. Thus, physicians may order multiple levels of remission can be defined. For treatment purposes, cytogenetic analyses (Chapter 41). complete remission (response) is defined as the total absence of disease according to the test used (e.g., hematologic ver- MOLECULAR ANALYSIS sus cytogenetic versus molecular). Hematologic remission Molecular genetic analysis, the process of using DNA tech- refers to the absence of neoplastic cells in the peripheral nology to identify genetic defects at the molecular level, blood and bone marrow and the return to normal levels of is being used increasingly as a diagnostic tool in studying hematologic parameters. Cytogenetic remission refers to the neoplasms. In some cases, the chromosome karyotype is absence of recognized cytogenetic abnormalities associated normal, but a genetic mutation can be identified. About 5% with a given neoplastic disease. Molecular remission refers of patients with CML do not show the typical Philadelphia to the absence of detectable molecular abnormalities using chromosome on karyotyping, but the BCR/ABL1 mutation PCR or related molecular technologies. can be identified by molecular techniques (Chapters 24 Sensitive molecular testing such as PCR can detect the and 42). When present, this helps establish or confirm the presence of less than 1 in 106 tumor cells (Chapter 42), whereas diagnosis. The JAK2 mutation, JAK2(V617F), plays a piv- the sensitivity of detection of malignant cells using cytoge- otal role in the pathogenesis of many BCR/ABL1–negative netics is much lower. Thus, a complete molecular response MPNs. Thus, diagnostic algorithms for MPN now consider is highly desirable and the most promising evidence that the the mutational status of JAK2. neoplasm has been eliminated. A combination of negative 510 Chapter 23 “traditional” tests (peripheral blood and bone marrow blast also kills many normal cells. Complications of traditional count and cytogenetics) and positive molecular tests (PCR/ therapy include bleeding because of decreased platelet FISH) is sometimes referred to as a state of minimum resid- counts, infections from suppression of granulocytes, and ual disease (MRD). The designation “partial response” is anemia because of erythrocyte suppression in the marrow. used when the relevant laboratory values have a significant Supplemental support with recombinant growth factors can decrease without achieving a total absence of disease. A “major sometimes be used to mitigate the cytopenias. response” can include either a complete or partial response. Most drugs used to treat leukemia are included in three Survival in acute leukemias varies with age and group— groups: antimetabolites, alkylating agents, and antibiotics. ALL or AML. Approximately 90% of children treated for ALL The antimetabolites are purine or pyrimidine antagonists, can be expected to enter a prolonged remission with an indef- which inhibit the synthesis of DNA. These drugs kill cells inite period of survival.39 The prognosis for ALL in adults is in cycle, affecting any rapidly dividing cell. In addition to not as good as that for children. Only 10–15% of patients typi- leukemic cells, the antimetabolites also kill cells lining the cally achieve long-term survival. Patients who receive bone gut, germinal epithelium of the hair follicles, and normal marrow or stem cell transplants, especially younger patients, hematopoietic cells. This leads to complications of gastro- have a better prognosis. Patients who had a previous MDS intestinal disturbances, loss of hair, and life-threatening or chronic MPN respond poorly to standard chemotherapy. cytopenias. The alkylating agents (chemical compounds Survival in the chronic neoplasms is longer. For CML containing alkyl groups) are not specific for cells in cycle patients receiving imatinib as initial therapy, overall survival but kill both resting and proliferating cells. These drugs at 60 months is 89%.40 After onset of blast crisis, survival is gen- attach to DNA molecules, interfering with DNA synthesis. erally only 1–2 months. Survival in CLL depends on its sever- As a class, they are mutagenic and carcinogenic; they frag- ity at diagnosis and ranges from 30 to more than 120 months. ment and clump chromosomes, inactivate DNA viruses, Patients with other MDS and MPN diseases usually survive and inhibit mitosis but not protein function. The side without treatment for a year or more and even |
longer for some effects of these compounds include myelosuppression, subtypes. Prognosis for the lymphomas depends on the cell stomatitis, and nausea and vomiting. Antibiotics bind to type and can range from 6 months to 10 years or longer. both DNA and RNA molecules, interfering with cell repli- cation. Toxic effects of this therapy are similar to those of alkylating agents. Since the 1970s, various drug combinations have been CASE STUDY (continued from page 507) found to be more effective than single drug administration. 4. Would you expect Agnes to survive more than The drugs commonly used, and their modes of action are 3 years or succumb fairly quickly after treatment? included in Table 23-8. Therapy for most leukemias is divided into several phases. The induction therapy phase is designed to induce the disease into complete remission (i.e., eradicating the Treatment leukemic blast population). Once a complete remission has been achieved, it is often followed by a continuation Therapeutic success rates differ by disease and the patient’s of treatment, referred to as maintenance chemotherapy or condition at diagnosis. Often a complete hematologic or consolidation therapy. The purpose of maintenance ther- cytogenetic remission is achieved initially, only to be fol- apy is to eradicate any remaining leukemic cells. lowed by a return of the disease (relapse) after a period of The treatment regimen for AML and ALL is similar, time. Most currently used treatment regimens target the although the combination of antileukemic agents differs. In actively proliferating cancer cells and might not, in fact, be both diagnoses, the purpose of chemotherapy is the same: effective against the leukemic stem cells (LSCs) (also referred to eradicate the leukemic blasts. Central nervous system to as the cancer stem cells). Thus, the relapse seen in some (CNS) involvement is a common feature of ALL but not patients following a complete remission is likely because of of AML. Therefore, CNS prophylactic treatment (cranial the reemergence of the disease from a quiescent LSC, which irradiation and/or intrathecal chemotherapy) is part of the was not eliminated by the treatment regimen utilized. therapy regimen for ALL. CHEMOTHERAPY Permanent remission in CLL is rare. Treatment is Chemotherapy remains the treatment of choice for many conservative and usually reserved for patients with more leukemias. The goal of this type of therapy is to eradicate all aggressive forms of the disease. Treatment for MPN and malignant cells within the bone marrow, allowing repopu- MDS also is primarily supportive and designed to improve lation by residual normal hematopoietic precursors. The the quality of the patient’s life. Drugs designed to reverse problem with this type of therapy is that the drugs used epigenetic alterations in MDS recently have been intro- in treatment are not specific for leukemic cells. Treatment duced (see “Epigenetic Therapy”). Introduction to Hematopoietic Neoplasms 511 Table 23.8 Chemotherapeutic Agents Usually Used in Acute Leukemia (AL) Treatment Drug Class Action Doxorubicin Anthracycline antibiotic Inhibits DNA and RNA synthesis Daunorubicin Anthracycline antibiotic Inhibits DNA and RNA synthesis Idarubicin Anthracycline antibiotic Inhibits DNA and RNA synthesis 5-Azacytidine Pyrimidine antimetabolite Inhibits DNA and RNA synthesis 6-Thioguanine Purine antimetabolite Inhibits purine synthesis Methotrexate Folic acid antimetabolite Inhibits pyrimidine synthesis 6-Mercaptopurine Purine antimetabolite Inhibits pyrimidine synthesis Cytosine arabinoside Pyrimidine antimetabolite Inhibits DNA synthesis Prednisone Synthetic glucocorticoid Lyses lymphoblasts Vincristine Plant alkaloid Inhibits RNA synthesis and assembly of mitotic spindles Asparaginase Escherichia coli enzyme Depletes endogenous asparagines Cyclophosphamide Synthetic alkylating agent Cross-links DNA strands MOLECULAR-TARGETED THERAPY removing some of the patient’s marrow while the patient is As the genetic mysteries of hematologic neoplasms are in complete remission. The marrow specimen is then treated being resolved, novel therapies that target genetic muta- to remove any residual leukemic cells and cryopreserved. tions are used to silence the gene’s expression or that of Chemotherapy and/or radiotherapy are administered to the mutated protein or to reactivate silenced genes. The the patient to remove all traces of leukemia, and the treated therapies appear to be better tolerated than the traditional marrow is given back to the patient. chemotherapy regimens. Two targeted therapies are in cur- Autologous bone marrow transplantation has been rent use as first-line therapy: imatinib for CML and all-trans applied to patients in remission and to those in early relapse. retinoic acid (ATRA) for acute promyelocytic leukemia.41 Overall survival appears to be better in those transplanted Although hematopoietic stem cell transplantation had been during the first complete remission. Bone marrow transplan- recommended as first-line therapy for CML because it was tation appears to be successful in many cases; the number of the only treatment with the potential for cure, recent stud- patients who are undergoing this type of therapy is increasing. ies reveal that survival is superior in patients receiving drug treatment (interferon and/or imatinib).42 Rituximab, STEM CELL TRANSPLANTS cytolytic monoclonal anti-CD20 antibody, is a treatment Peripheral blood as well as bone marrow stem cells can directed against cells that have the CD20 protein (primarily be used to re-establish hematopoiesis in the marrow after B cells). These therapies are discussed in Chapters 24 and 26. intensive chemotherapy or radiotherapy in a process called stem cell transplantation. In this procedure, apheresis is used EPIGENETIC THERAPY to collect stem cells from the peripheral circulation, usu- With the recognition of the contribution of epigenetic altera- ally after they have been mobilized (induced to exit the tions to the neoplastic process, a number of drugs have been marrow) by cytokines such as G-CSF. These stem cells can developed and are in various stages of clinical trials. Both come from either the patient (autologous) or from a suit- demethylating agents (e.g., azacitidine) and histone deacet- able donor (allogeneic). People who receive allogeneic stem ylase inhibitors (HDAC-I) (e.g., valproic acid, phenylbutyr- cells are given drugs to prevent rejection. Production of ate) are being evaluated with promising early results.43,44,45 new blood cells usually becomes established in 10–21 days BONE MARROW TRANSPLANT following infusion of the stem cells. Stem cell transplanta- Bone marrow transplants have provided hope as a possible tion is still a fairly new and complex treatment for leuke- cure for hematopoietic disorders; the highest rate of success mia. A more thorough discussion of hematopoietic stem in transplant patients has occurred with those younger than cell transplantation can be found in Chapter 29. 40 years of age in a first remission with a closely matched donor. In this procedure, drugs and irradiation are used to induce remission and eradicate any evidence of leukemic CASE STUDY (continued from page 510) cells. Bone marrow from a suitable donor is then transplanted into the patient to supply a source of normal stem cells. 5. Is Agnes a suitable candidate for a bone marrow Autologous transplants have been used when a com- transplant? Why or why not? patible donor cannot be found. This procedure involves 512 Chapter 23 HEMATOPOIETIC GROWTH FACTORS In excessive amounts, the uric acid precipitates in renal Recombinant hematopoietic growth factors are used in sup- tubules, leading to renal failure (uric acid nephropathy). portive care of acute leukemia patients. Erythropoietin is Lysed cells also can release procoagulants into the vas- used in the treatment of chemotherapy-related anemia.46 cular system, leading to disseminated intravascular coagu- Granulocyte colony-stimulating factor (G-CSF) and gran- lation. In this case, the resulting decrease in platelets and ulocyte-macrophage colony-stimulating factor (GM-CSF) coagulation factors can lead to hemorrhage. This compli- aid in decreasing the incidence of severe neutropenia and cation is especially prevalent in acute promyelocytic leu- infection in patients receiving myelosuppressive chemo- kemia. The granules of the promyelocytes contain potent therapy.44 Interleukin-11 promotes the maturation of mega- activators of the coagulation factors (Chapter 33). karyocytes by stimulating stem cells and megakaryocyte Since chemotherapeutics destroy normal as well as leu- progenitor cells.47 Research in the area of identifying addi- kemic cells. The cytopenias that develop during aggressive tional hematopoietic growth-stimulating factors continues chemotherapy can lead to death from infection, bleeding, in the hope of accelerating hematopoietic recovery in che- or complications of anemia. To prevent these life-threaten- motherapy patients. ing episodes, the patient may need supportive treatment including transfusions with blood components and/or COMPLICATIONS OF TREATMENT Treatment for leukemia actually can aggravate the patient’s cytokines as well as antimicrobial therapy. clinical situation. Although uric acid levels are commonly elevated in leukemia because of the increase in cell turnover, the concentration of uric acid can drastically increase dur- CASE STUDY (continued from page 511) ing effective therapy because of the release of nucleic acids by lysed cells. Uric acid is a normal end product of nucleic 6. What types of treatment are available for Agnes? acid degradation and is excreted mainly by the kidney. Summary A neoplasm is an unregulated production of cells that can in the regulatory mechanisms of cell proliferation and dif- be either benign or malignant. The WHO classification of ferentiation. Epigenetic alterations also play an important neoplastic disorders of the bone marrow hematopoietic role in silencing tumor suppressor genes in neoplastic cells. cells groups the disorders according to lineage of the neo- Differentiation and classification of acute leukemias plastic cells: myeloid, lymphoid, and histiocytic/dendritic. depend on accurately identifying the blast cell popula- The myeloid and lymphoid neoplasms are subgrouped tion. Because the lineage of blast cells is sometimes diffi- as precursor and mature neoplasms. Morphology, genetic cult to differentiate using only morphologic characteristics, abnormalities, immunophenotype, and clinical features immunologic phenotyping using monoclonal antibodies define these major groups. The mature myeloid subgroups are employed routinely to help identify blast phenotypes include myeloproliferative neoplasms, myeloproliferative/ and stage of cell differentiation. Chromosome and molecu- myelodysplastic neoplasms, and myelodysplastic syn- lar genetic analyses are helpful because specific mutations dromes. The precursor myeloid group includes the acute often are associated with specific types of leukemias. myeloid leukemias. The lymphoid group includes precur- Hematologic findings of hematopoietic neoplasms sor B-cell, mature B-cell, precursor T-cell, and mature T-cell include anemia, thrombocytopenia (in acute leukemia and neoplasms. The mature lymphoid group includes CLL. The MDS), and often leukocytosis. A leukocytic shift to the left is precursor lymphoid group includes ALL. consistently found with a combination of blasts and mature Oncogenes are mutated forms of proto-oncogenes and cells in acute leukemia. In the chronic leukemias and other are known to contribute to tumorigenesis. Many proto- chronic neoplastic stem cell disorders, cells appear more on oncogene protein products are involved in regulating cell a continuum from immature to mature. Morphologic abnor- growth and include hematopoietic growth factors as well malities of neoplastic cells are not unusual. Auer rods can as their cellular receptors, signaling proteins, and tran- be found in blasts of AML. scription factors. Proto-oncogenes can be mutated to onco- Historically, hematopoietic neoplasms have been genes by mutagens, viruses, or chromosome breaks and treated using a combination of cytotoxic drugs (che- translocations. Oncogenes can cause production of abnor- motherapy). The goal is to induce remission by eradi- mal growth factors, abnormal amounts of growth factors, cating the leukemic cells. Hematopoietic stem cell abnormal growth factor receptors, or other abnormalities transplants can be used to restore the marrow after Introduction to Hematopoietic Neoplasms 513 intense chemotherapy or radiotherapy. New approaches factors is used in some cases to stimulate leukemic cells include drugs to reverse epigenetic modifications char- to proliferate, making them more susceptible to cytotoxic acteristic of neoplastic cells and other drugs targeted drugs. This therapy also has been used to decrease the at the exact molecular abnormality associated with the neutropenic, anemic, and thrombocytopenic period after neoplastic cell. Treatment with hematopoietic growth chemotherapy or radiotherapy. Review Questions Level I c. ALL 1. Auer rods are inclusions found in: (Objective 5) d. CLL a. myeloblasts 7. Acute lymphoblastic leukemia occurs with greatest b. lymphoblasts frequency in which age group? (Objective 7) c. erythrocytes a. 2–5 years d. prolymphocytes b. 10–15 years c. 20–30 years 2. Chromosome changes in hematologic neoplasms are: (Objective 8) d. More than 50 years a. used to aid in diagnosis 8. Chronic leukemias primarily affect: (Objectives 2, 7) b. present in AL but not MPN or MDS a. adults, progress slowly, and have mature cells c. associated with a poor outcome in circulation d. not usually present b. children, progress rapidly, and have mature cells in peripheral circulation 3. Genes that can cause tumors if activated are: c. young adults, progress slowly, and have immature (Objective 6) cells in peripheral circulation a. cancer genes d. all ages, |
progress rapidly, and have immature cells b. proto-oncogenes in peripheral circulation c. preleukemia genes 9. The WHO system classifies a 19-year-old patient’s d. tumor suppressor genes bone marrow as a precursor B-cell leukemia. Which of the following best describes this leukemia? 4. A common characteristic of acute lymphoblastic (Objectives 2, 3, 5) leukemia is: (Objective 2) a. CLL a. BCR/ABL1 gene mutation b. CML b. bone pain c. AML c. many blast cells with Auer rods d. ALL d. leukocytopenia 10. Immunophenotyping of blast cells is important to: 5. Which of the following does the WHO c lassification (Objective 8) use to subgroup the ALL into T- and B-cell neoplasms? (Objective 3) a. identify the leukemia’s etiology a. Clinical presentation b. help determine cell lineage b. Immunophenotype c. determine whether cytogenetic analysis is necessary c. Cytogenetics d. replace the need for molecular analysis d. Morphology 11. The minimum percentage of blast cells required for a 6. A leukemia that shows a profusion of granulocytes at diagnosis of acute myeloid leukemia using the WHO all stages of development from blasts to segmented classification is: (Objective 3) neutrophils is: (Objectives 2, 5) a. 20% a. CML b. 30% b. AML 514 Chapter 23 c. 40% 4. A patient with a hypercellular, dysplastic bone mar- d. 50% row; anemia; and neutropenia in peripheral blood most likely has which of the following neoplasms? 12. Proteins encoded by proto-oncogenes serve to: (Objective 7) (Objective 6) a. MPN a. demethylate oncogenes b. AML b. inactivate tumor suppressor genes c. MDS c. cause unregulated cell proliferation d. ALL d. provide signaling pathways for normal cell growth control 5. A 52-year-old female was admitted to the hospital for minor elective surgery. Her pre-operative CBC was: 13. Which of these genes cause unregulated cell growth? WBC 49.4 * 103/mcL WBC Differential (Objective 6) RBC 4.50 * 106/mcL 3% segs a. Oncogenes Hb 12.7 g/dL 97% lymphs b. Antioncogenes Hct 38% PLT 213 * 103/mcL c. Proto-oncogenes d. Tumor suppressor genes What is the best explanation for the cause of this patient’s leukocytosis and lymphocytosis? Level II (Objectives 3, 7) 1. If the cell of origin for a neoplastic tumor undergoes a. CLL genetic mutations that gives the cell the ability to b. AML self-renew and blocks terminal differentiation, the c. ALL resulting malignancy will be: (Objective 11) d. CML a. acute leukemia b. chronic lymphocytic leukemia c. chronic myelogenous leukemia Use the following information to answer questions 6–8: d. myeloproliferative neoplasm A 43-year-old male had been working with the 2. Which of the following factors has not been proposed Peace Corps in Mexico for the past 10 years. His as playing a role in causing leukemia? (Objective 6) primary responsibilities were taking radiographs a. Benzene and doing laboratory work at the various clin- ics. He had been complaining of weakness and b. Radiation fatigue for about a month and had had several c. Living at high altitudes severe nosebleeds. His CBC upon admission to d. Chromosome translocations the hospital was: 3. A 3-year-old child with Down syndrome pres- WBC 25.6 * 103/mcL Differential ents with pallor, fatigue, lymphadenopathy, and RBC 3.11 * 106/mcL 75% blasts with Auer rods hepatosplenomegaly. The initial CBC results were: Hb 8.9 g/dL 20% lymphs (Objectives 3, 7) Hct 26.7% 3% monos WBC 18.7 * 103/mcL WBC Differential PLT 13 * 103/mcL 2% segs RBC 2.34 * 106/mcL 27% segs Hb 5.8 g/dL 10% lymphs Hct 17.4% 63% blasts PLT 130 * 103/mcL 6. Which leukemia is this patient most likely to have? These findings are suggestive of: (Objectives 3, 7) a. MDS a. ALL b. CLL b. AML c. AML c. CLL d. ALL d. CML Introduction to Hematopoietic Neoplasms 515 7. What would be the most likely causative agent of the 9. Heritable changes in gene expression not from leukemia? (Objective 2) changes in DNA are known as: (Objective 10) a. Virus a. chromosomal mutations b. Age b. pseudo-mutations c. Ionizing radiation c. cytogenetic mutations d. Hepatitis d. epigenetic changes 8. What treatment would result in the best prognosis 10. Mutations in tumor suppressor genes can result in: for this patient if there were no complicating factors? (Objective 1) (Objective 5) a. inhibition of normal cell growth a. Hematopoietic growth factors b. tumor development b. Stem cell transplantation c. activation of oncogenes c. Radiation therapy d. triggering growth-promoting signals in the absence d. Chemotherapy of ligand binding References 1. Arber, D. A., Orazi, A., Hasserjian, R. P., Brunning, R. D., Le Beau, 12. Esteller, M. (2008). Epigenetics in cancer. New England Journal of M. M., Porwit, A., . . . Thiele, J. (2017). Introduction and Overview Medicien, 35, 1148–1192. of the classification of the Myeloid Neoplasms. In: S. H. Swerd- 13. Sharma, S., Kelly, T.K., & Jones, P. A. (2010). Epigenetics in cancer. low, E. Campo, N. L. Harris, E. S. Jaffe, S. A. Pileri, H. Stein, & Carcinogenesis, 31, 27–36. J. Thiele, eds. WHO Classification of Tumours of Haematopoietic and 14. Dawson, M.A., & Kouzarides, T. (2012). Cancer epigenetics: Lymphoid Tissues (Revised 4 ed., pp. 15–28). Lyon, France: Interna- from mechanism to therapy. Cell, 150(1), 12–27. doi: 10.1016/ tional Agency for Research on Cancer. j.cell.2012.06.013 2. Magee, J. A., Piskounova, E., & Morrison, S. J. (2012). Cancer stem 15. Weiss, C. N., & Ito, K. (2017). A macro view of microRNAs: The cells: Impact, heterogeneity, and uncertainty. Cancer Cell, 21(3), discovery of microRNAs and their role in hematopoiesis and 283–296. doi: 10.1016/j.ccr.2012.03.003 hematologic disease. International Review of Cell and Molecular 3. Passegue, E., Jamieson, C. H. M., Ailles, L. E., & Weissman, I. L. Biology, 334, 99–175. doi: 10.1016/bs.ircmb.2017.03.007 (2003). Normal and leukemic hematopoiesis: Are leukemias a 16. Blain, S. W. (2008). Switching cyclin D-Cdk4 kinase activity on stem cell disorder or a reacquisition of stem cell characteristics? and off. Cell Cycle, 7(7), 892–898. doi: 10.4161/cc.7.7.5637 Proceedings of the National Academy of Sciences USA, 100, 11842– 17. Nobori, T., Miura, K., Wu, D. J., Lois, A., Takabayashi, K., & 11849. doi: 10.1073/pnas.2034201100 Carson, D. A. (1994). Deletions of the cyclin-dependent kinase-4 4. Croce, C. E. (2008). Oncogenes and cancer. New England Journal of inhibitor gene in multiple human cancers. Nature, 368(6473), Medicine, 358, 502–511. 753–756. doi: 10.1038/368753a0 5. Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: 18. Adams, J. M., & Cory, S. (2007). The Bcl-2 apoptotic switch in the next generation. Cell, 144(5), 646–674. doi: 10.1016/ cancer development and therapy. Oncogene, 26(9), 1324–1337. doi: j.cell.2011.02.013 10.1038/sj.onc.1210220 6. Di Flore, R., D’Anneo, A., Tesoriere, G., & Vento, R. (2013). RB1 19. Wojcik, I., Szybka, M., Golanska, M., Rieske, P., Blonski, J. Z., in cancer: Different mechanisms of RB1 inactivation and altera- Robak, T., & Bartkowiak, J. (2005). Abnormalities of the P53, tions of pRb pathway in tumorigenesis. Journal of Cell Physiology, MDM2, BCL2 and BAX genes in acute leukemias. Neoplasma, 228(8), 1676–1687. doi:10.1002/jcp.24329. 52(4), 318–324. 7. Burkart, D. L., & Sage, J. (2008). Cellular mechanisms of tumour 20. Xavier, A. C., & Taub, J. W. (2010). Acute leukemia in children suppression by the retinoblastoma gene. Nature Reviews Cancer, with Down syndrome. Heamatologica, 95(7), 1043–1045. doi: 8(9), 671–682. doi: 10.1038/nrc2399 10.3324/haematol.2010.024968 8. Bargonetti, J., & Manfredi, J. J. (2002). Multiple roles of the tumor 21. Ross, J. A., Spector, L. G., Robison, L. L., & Olshan, A. F. (2005). suppressor p53. Current Opinion in Oncology, 14(1), 86–91. Epidemiology of leukemia in children with Down syndrome. 9. Pietsch, E. C., Sykes, S. M., McMahon, S. B., & Murphy, M. E. Pediatric Blood & Cancer, 44(1), 8–12. doi: 10.1002/pbc.20165 (2008). The p53 family and programmed cell death. Oncogene, 22. Gao, Q., Horowitz, M., Roulston, D., Hagos, F., Zhao, N., Feireich, 27(50), 6507–6521. doi: 10.1038/onc.2008.315 E.J., . . . Olopade, O. I. (2000). Susceptibility gene for familial 10. Malkin, D. (2011). Li-Fraumeni syndrome. Genes Cancer, 2, acute myeloid leukemia associated with loss of 5q and/or 7q is 475–484. not localized on the commonly deleted portion of 5q. Genes Chro- 11. Malkin, D., Li, F. P., Strong, L. C., Fraumeni Jr., J. F., Nelson, C. E., mosomes Cancer, 28(2), 164–172. Kim, D. H., . . . , Tainsky, M. A. (1990). Germline p53 muta- 23. Darbary, H., Stoler, D. L., & Anderson, G. A. (2009). Family cancer tions in a familial syndrome of breast cancer, sarcomas and syndromes: inherited deficiencies in systems for the maintenance other neoplasms. Science, 250(4985), 1233–1238. doi: 10.1126/ of genomic integrity. Surgical and Oncological Clinics of North science.1978757 America, 18(1), 1–17. doi: 10.1016/j.soc.2008.08.001 516 Chapter 23 24. Dayaram, T., & Marriott, S. J. (2008). Effect of transforming The 2008 revision of the World Health Organization (WHO) clas- viruses on molecular mechanisms associated with cancer. Journal sification of myeloid neoplasms and acute leukemia: Rationale of Cell Physiology, 216(2), 309–314. doi: 10.1002/jcp.21439 and important changes. Blood, 114(5), 937–951. doi: 10.1182/ 25. Chan, J. K., & Warner, C. G. (2012). Dynamic roles for NF-kB in blood-2009-03-209262 HTLV-I and HIV-1 retroviral pathogenesis. Immunological Review, 36. Campo, E., Swerdlow, S. H., Harris, N. L., Pileri, S., Stein, H., & 246(1), 286–310. doi: 10.1111/j.1600-065X.2012.01094.x Jaffe, E. S. (2011). The 2008 WHO classification of lymphoid neo- 26. Jaffe, E. S., Harris, N. L., Stein, H., & Vardiman, J.W. (2001). plasms and beyond: evolving concepts and practical applications. Pathology and genetics. Tumours of haematopoietic and lymphoid Blood, 117(19), 5019–5032. doi: 10.1182/blood-2011-01-293050 tissues. World Health Organization Classification of Tumours. 37. Arber, D. A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M. J., Washington, DC: IARC Press. Le Beau, M. M. . . . Vardiman, J.W. (2016). The 2016 revision of the 27. Kumar, T., Mandla, S. G., & Greer, W. L. (2000). Familial myelo- World Health Organization classification of myeloid neoplasms dysplastic syndrome with early age of onset. American Journal of and acute leukemia. Blood, 127(20), 2391–2405. doi: 10.1182/ Hematology, 64(1), 53–58. blood-2016-03-643544 28. Occhipinti, E., Correa, H., Yu, I., & Craver, R. (2005). Comparison 38. Swerdlow, S. H., Campo, E., Pileri, S. A., Harris, N. L., Stein, H., of two classifications for pediatric myelodysplastic and myelo- Siebert, R., . . . Jaffe, E. S. (2016). The 2016 revision of the World proliferative disorders. Pediatric Blood Cancer, 44 (3), 240–244. Health Organization classification of lymphoid neoplasms. Blood, doi: 10.1002/pbc.20174 127(20), 2375–2390. doi: 10.1182/blood-2016-01-643569 29. Baiocchi, R. A. (2016). Classification of Malignant Lymphoid 39. Terwilleger T., & Abdul-Hay, M. (2017). Acute lymphoblastic Disorders. In: K. Kaushansky, M. A. Lichtman, J. T. Prchal, M. M. leukemia: A comprehensive review and 2017 update. Blood Cancer Levi, O. W. Press, L. J. Burns, & M. A. Caligiuri, eds. Williams Journal, 7(6), e577. doi: 10.1038/bcj.2017.53 Hematology (9th ed., pp. 1493–1504). New York: McGraw-Hill 40. Hughes, T. (2006). ABL kinase inhibitor therapy for CML: Base- Education. line assessments and response monitoring. Hematology: American 30. Brunning, R. D., Orazi, A., Germing, U., Le Beau, M.M., Society of Hematology Education Program, 2006(1), 211–218. doi: Porwit, A., Baumann, I., . . . Hellstrom-Lindberg, E. (2008). 10.1182/asheducation-2006.1.211 Myelodysplastic syndromes/neoplasms, overview. In: 41. Ikeda, A., Shankar, D. B., Watanabe, M., Tamanoi, F., Moore, T. B., S. H. Swerdlow, E. Campo, N. L. Harris, E.S. Jaffe, S.A. Pileri, & Sakamoto, K. M. (2006). Molecular targets and the treatment of H. Stein, . . . J.W. Vardimaneds, eds. WHO Classification of myeloid leukemia. Molecular Genetic Metabolism, 88(3), 216–224. Tumours of Haematopoietic and Lymphoid Tissues (pp. 87–107). doi: 10.1016/j.ymgme.2006.03.011 Lyon, France: IARC. 42. Hehlmann, R., Berger, U., Pfirrmann, M., Heimpel, H., Hochhaus, 31. Paltiel, O., Friedlander, Y., Deutsch, L., Yanetz, R., Calderon- A., Hasford, J., . . . Gratwohl, A. (2007). Drug treatment is supe- Margalit, R., Tiram, E., . . . , Manor, O. (2007). The interval rior to allografting as first-line therapy in chronic myeloid leuke- between cancer diagnosis among mothers and offspring in mia. Blood, 109(11), 4686–4692. doi: 10.1182/blood-2006-11-055186 a population-based cohort. Familial Cancer, 6(1), 121–129. 43. Jones, P. A., & Baylin, S. B. (2007). The epigenomics of cancer. Cell, doi: 10.1007/s10689-006-9113-9 128, 683–692. 32. Leukemia and Lymphoma Society. (2017, January). Leuke- 44. Garcia-Manero, G., & Issa, J. P. (2005). Histone deacetylase mia facts and statistics. Retrieved from http://www.lls.org/ inhibitors: A review of their clinical status as a ntineoplastic facts-and-statistics/facts-and-statistics-overview. agents. Cancer Investigation, 23(7), 635–642. doi: 33. Larson, R. A. (2016). Acute Lymphoblastic Leukemia. In: K. 10.1080/07357900500283119 Kaushansky, M. A. Lichtman, J. T. Prchal, M. M. Levi, O. W. |
Press, 45. Garcia-Manero, G. (2007). Modifying the epigenome as a thera- L. J. Burns, & M. A. Caligiuri, eds. Williams Hematology (9th ed., peutic strategy in myelodysplasia. Hematology: Amercian Society pp. 1505–1526). New York, NY: McGraw-Hill Education. of Hematology Education Program, 2007(1), 405–411. doi: 10.1182/ 34. Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, asheducation-2007.1.405 D. A., Gralnick, H. R., & Sultan, C. (1976). Proposals for the 46. Parsons, S. K. (2000). Oncology practice patterns in the use of classification of the acute leukaemias. French-American-British hematopoietic growth factors. Current Opinion in Pediatrics, 12(1), (FAB) Co-operative Group. British Journal of Haematology, 33(4), 10–17. 451–458. 47. Kaushansky, K. (2000). Use of thrombopoietic growth factors in 35. Vardiman, J. W., Thiele, J., Arber, D. A., Brunning, R. D., acute leukemia. Leukemia, 14(3), 505–508. Borowitz, M. J., Porwit, A., . . . Bloomfield, C. D. (2009). Chapter 24 Myeloproliferative Neoplasms Tim R. Randolph, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Identify the major lineages involved with 5. Describe and recognize the peripheral blood the various myeloproliferative neoplasms findings in CML patients and in those with (MPNs): chronic myeloid leukemia blast crisis. (CML), polycythemia vera (PV), essential 6. List and recognize laboratory findings thrombocythemia (ET), and primary typically associated with PMF. myelofibrosis (PMF). 7. Define criteria that indicate a 2. Recognize abnormal complete blood transformation of an MPN into a blast crisis. count (CBC) results that suggest an MPN. 8. Compare the lab findings in primary PV to 3. Explain the diagnostic chromosome those in secondary polycythemia. abnormality associated with CML and 9. Describe and recognize the characteristic its significance in acute lymphoblastic peripheral blood picture found in ET. leukemia (ALL). 10. Describe and recognize the peripheral 4. Identify the peak incidence of CML blood findings in patients with clonal according to age and sex distribution. hypereosinophila and mastocytosis. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Differentiate the subgroups of 2. Contrast laboratory findings in MPNs and myeloproliferative neoplasms (MPNs) from myelodysplastic syndromes (MDSs) as well other reactive and neoplastic diseases based as acute leukemia (AL). on laboratory findings in the peripheral 3. Describe molecular gene mutations in blood, bone marrow, and other diagnostic MPNs and hypothesize how these changes laboratory tests. in the pluripotential stem cell lead to MPNs 517 518 Chapter 24 and blast crisis (Philadelphia c hromosome, 8. Use laboratory results and clinical findings BCR/ABL1 rearrangement p53, p16, JAK2 to differentiate essential thrombocythemia (V617F), MPL (W515), CALR, FIP1L1/ from nonmalignant conditions that result in PDGFRA, ETV6-PDGFRB, and FGFR1 thrombocytosis. mutations, and expression of growth factor 9. Use the criteria suggested for diagnosing receptors). CML, chronic eosinophilic leukemia, not 4. Differentiate CML from a benign leukemoid otherwise specified (CEL-NOS), and chronic reaction using laboratory tests. neutrophilic leukemia (CNL) to distinguish 5. Assess the role of platelet-derived growth these disorders. factor (PDGF) in the fibrosis associated with 10. Differentiate unclassifiable myeloprolifera- primary myelofibrosis (PMF). tive neoplasms from other subgroups of 6. Assess laboratory results including the MPNs based on laboratory features. evaluation of peripheral blood and bone 11. Differentiate eosinophilic disorders using marrow smears using the diagnostic criteria laboratory results and genetic mutations. associated with PMF. 12. Describe systemic mastocytosis by giving 7. Compare clinical and laboratory findings, the criteria for its diagnosis. and interpret laboratory findings in 13. Using peripheral blood, bone marrow find- relative polycythemia and absolute ings, and patient medical history, determine polycythemia. the classification of MPNs. Chapter Outline Objectives—Level I and Level II 517 Chronic Myeloid Leukemia (CML) 522 Key Terms 518 Chronic Neutrophilic Leukemia (CNL) 530 Background Basics 519 Essential Thrombocythemia (ET) 531 Case Study 519 Polycythemia Vera (PV) 535 Overview 519 Primary Myelofibrosis (PMF) 540 Introduction 519 Myeloproliferative Neoplasm, PART I: Overview of Myeloproliferative Unclassifiable (MPN, U) 545 Neoplasms (MPNs) 520 Clonal Hypereosinophilia 545 Classification 520 Mast Cell Disease (Mastocytosis) 548 Pathophysiology 521 Summary 549 General Features 521 Review Questions 550 PART II: Subgroups of MPNs 522 References 552 Key Terms Blast crisis Janus kinase 2 (JAK2) gene Chronic eosinophilic leukemia, not otherwise Leukemic hiatus specified (CEL-NOS) Panmyelosis Chronic myeloid leukemia (CML) Plethora Chronic neutrophilic leukemia (CNL) Polycythemia vera (PV) Clonal hypereosinophilia Primary myelofibrosis (PMF) Essential thrombocythemia (ET) Spent phase Hypereosinophilic syndrome (HES) Systemic mastocytosis Myeloproliferative Neoplasms 519 Background Basics The information in this chapter builds on concepts Level II learned in previous chapters. To maximize your learn- • Describe the influence of growth factors on hemato- ing experience, you should review these related concepts poietic cell proliferation. (Chapter 4) before starting this unit of study: • Explain the evaluation of red cell mass based on changes in fluid volume. (Chapter 11) Level I • Explain the hemoglobin–oxygen dissociation curve. • Outline the cell cycle; describe stem cell differen- (Chapter 6) tiation and maturation for the various myeloid lin- eages. (Chapters 2, 3) • Explain how proto-oncogenes are activated and their role in the etiology of hematopoietic neoplasms. • Describe and recognize morphology for the various (Chapters 2, 23) stages of myeloid maturation. (Chapter 7) • Describe how cell marker panels can be used to dif- • Calculate red cell indices. (Chapters 10, 36) ferentiate hematopoietic neoplasms. (Chapters 23, 40) • Use appropriate morphologic terms to describe size • Discuss the value of cytogenetic studies in suspected and chromia of red cells in anemic states. (Chapters hematopoietic neoplasms. (Chapters 23, 41) 10, 11) • Explain how the use of molecular tests can assist in • Outline and explain the classification of hematopoi- diagnosing hematopoietic neoplasms. (Chapters 23, 42) etic neoplasms. (Chapter 23) • Summarize the relationship of oncogenes to the neo- plastic process (Chapter 23) laboratory evaluation, and therapy are included for each CASE STUDY subgroup. These are followed by an explanation of how to We refer to this case throughout the chapter. differentiate MPNs from diseases with similar laboratory Roger, a 52-year-old man with hyperuricemia, findings. This is a lengthy chapter, so it is divided into two went to the clinic for a follow-up evaluation for sections to help the reader organize the study of this group splenomegaly. His palpable spleen, noted 18 of important disorders. months earlier, had been gradually enlarging. He originally denied fatigue, fever, and discomfort. He was examined, and a CBC was ordered. The Introduction results revealed leukocytosis, thrombocytosis, and Hematopoiesis is a highly regulated process whereby a anemia. normal steady-state production of hematopoietic cells in Consider how reflex laboratory testing can the bone marrow and destruction of senescent cells in the assist in diagnosing this patient. tissues maintain a constant peripheral blood cell concentra- tion (Chapters 2, 3). Hematopoietic neoplasms result from acquired mutations in hematopoietic stem cells, allowing them to escape the regulatory controls for proliferation, Overview natural cell death (apoptosis), and/or differentiation in the bone marrow (Chapters 2, 23). This chapter presents the group of neoplastic but not truly In the former French-American-British (FAB) classification malignant disorders called myeloproliferative neoplasms system, neoplastic disorders of hematopoietic cells typically (MPNs). MPNs must be distinguished from other neoplas- were grouped into three main categories: m yeloproliferative tic and benign hematologic disorders for the physician disorders (MPDs), myelodysplastic states or syndromes to select appropriate therapy for the patient. The chapter (MDSs), and acute leukemias (ALs), including both myeloid begins with the classification, pathophysiology, and general and lymphoid subtypes. This classification system used the characteristics of MPNs, which provide the groundwork for blast count, lineage commitment, cell morphology, level of a more detailed explanation and description of each sub- differentiation of the neoplastic cells, cytochemistry, and group. More specific pathophysiology, clinical presentation, immunophenotyping to classify the diseases. 520 Chapter 24 The World Health Organization (WHO) developed a Hematologic classification is based on the most affected cell newer classification system for hematopoietic neoplasms line (Table 24-1). In the WHO classification,2 the spectrum that integrates cytogenetics, DNA analysis, and clinical of myeloproliferative neoplasms includes: features with cytochemistry and immunophenotyping. In • Chronic myeloid leukemia (CML), Philadelphia (Ph) 2016, the WHO published an update to this classification chromosome positive with BCR-ABL1 fusion gene pres- system. The WHO classification of myeloid disorders ent, t(9;22) (q34;q12) includes the same three categories as the original FAB system and the previous WHO system, AML, MPN, and • Chronic neutrophilic leukemia (CNL), BCR-ABL1- MDS, but adds additional categories to include: myeloid/ negative lymphoid neoplasms with eosinophilia and rearrange- • Essential thrombocythemia (ET) ment of PDGFRA, PDGFRB, or FGFR1; the myelodys- • Polycythemia vera (PV) plastic/myeloproliferative neoplasms (MDS/MPN); • Primary myelofibrosis (PMF, formerly known as myelo- blastic plasmacytoid dendritic cell neoplasm; and acute fibrosis with myeloid metaplasia [MMM] and chronic idio- leukemias of ambiguous lineage. In addition, each group pathic myelofibrosis [CIMF]) has s ubcategories based on clinical history, genetic and laboratory f indings. The WHO proposed MDS/MPN cat- • PMF, prefibrotic/early stage egory includes neoplasms that are proliferative like MPNs • PMF, overt fibrotic stage but have dysplastic features like MDS. MDS/MPNs are • Myeloproliferative neoplasm, unclassifiable (MPN, U) discussed in Chapter 25 with MDS. • Myeloid and lymphoid neoplasms associated with In the WHO classification, the percentage of blasts, eosinophilia and PDGFRA, PDGFRB, or FGFR1 degree of cell maturation, and dysplasia are critical assess- mutations ments initially used to classify the hematopoietic neo- • Chronic eosinophilic leukemia, not otherwise specified plasms. A blast count of more than 20% is necessary for (CEL-NOS) an AL diagnosis, whereas the nonacute leukemia disorders are diagnosed with less than 20% blasts. MPNs and MDS • Mastocytosis are characterized by an autonomous, neoplastic clonal Important criteria in the WHO classification system of proliferation of hematopoietic precursors. Increased num- the MPNs are (1) Ph chromosome must be present or if not bers of erythrocytes, leukocytes, and/or platelets in both the identified by routine cytogenetic analysis, the BCR/ABL1 bone marrow and peripheral blood generally distinguish fusion gene must be detected for a diagnosis of CML and (2) MPNs from MDS. MDS, on the other hand, is most com- the minimum bone marrow blast count to distinguish MPNs monly characterized by a hyperproliferative bone marrow, from AL is 20% (MPNs have less than 20% blasts). Because dysplastic maturation, and increased apoptosis that result identification of Ph is required for a diagnosis of CML, Ph- in peripheral blood cytopenias. Both MPNs and MDS have negative cases with myelodysplastic and myeloproliferative the potential of evolving into acute leukemia. features are included in the WHO MDS/MPN group and are called atypical CML (aCML). The classification of these PART I disorders is not always clear because of overlapping clinical OVERVIEW OF MYELOPROLIFERATIVE NEOPLASMS (MPNs) Table 24.1 Classification of Myeloproliferative Neoplasms (MPNs) by Predominance of Cell Types Classification Involved Cell Line Myeloproliferative Neoplasm Myeloid Chronic myeloid leukemia (CML) and chronic The term myeloproliferative syndrome, coined by William neutrophilic leukemia (CNL), and sometimes primary myelofibrosis (PMF) Dameshek in 1951, describes a group of disorders that Megakaryocytic Essential thrombocythemia (ET) result from an unchecked, autonomous clonal proliferation of cellular elements in the bone marrow.1 Under the WHO Erythroid Polycythemia vera (PV) classification system, myeloproliferative neoplasms are gen- Fibroblasta Primary myelofibrosis (PMF) erally characterized by panhypercellularity (panmyelosis) Eosinophil Chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) of the bone marrow accompanied by erythrocytosis, granu- Mast cell Mastocytosis locytosis, and/or thrombocytosis in the peripheral blood. Variable Myeloproliferative neoplasm, unclassifiable Although trilineage cell involvement (erythrocytic, granu- (MPN, U) locytic, thrombocytic) is characteristic of MPNs, one cell a The fibroblast in PMF is not a part of the neoplastic process but is increased because of a line is usually more prominently affected than the others. reactive process. Myeloproliferative Neoplasms 521 Table 24.2 Differential Features of Myeloproliferative Neoplasms Parameter CML CNL ET PV PMF CEL-NOS Peripheral blood Hematocrit N or c N or c N or T c c c T T Leukocyte c c c c N or c N or c c or c c c Platelets c or T N or c c c c c N,c , T N Immature c c c Slight c (610%) Rare Absent or c c c N or c granulocytes LAP T c N or c N or c N,c , T - Philadelphia Present Absent Absent Absent Absent Absent chromosome Spleen size N or c c N or c c c c c N Bone marrow |
fibrosis Absent or c Absent Absent or c Absent or c c c c Absent or c N, normal; T , decreased; c , c c , c c c , slight, moderate, marked increase, respectively; LAP, leukocyte alkaline phosphatase; CML, chronic myeloid leukemia; CNL, chronic neutrophilic leukemia; ET, essential thrombocythemia; PV, polycythemia vera; PMF, primary myelofibrosis; CEL-NOS, chronic eosinophilic leukemia, not otherwise specified. and laboratory features between subgroups at different times during the disease course (Table 24-2). General Features Myeloproliferative neoplasms usually occur in middle-aged and older adults (peak frequency in the fifth to seventh Pathophysiology decades of life). They are rare in children. The onset of the disease is gradual, evolving over months or even years. The The primary defect in MPNs appears to be in the pluripoten- clinical, laboratory, and morphologic findings frequently tial hematopoietic stem cell (HSC)3 (Figure 24-1). A clone of overlap among the specific MPN disorders. Clinical find- abnormal hematopoietic stem cells and their progeny pref- ings can include hemorrhage, thrombosis, infection, pallor, erentially expand until normal hematopoietic cell growth and weakness. Anemia or polycythemia, leukoerythroblas- is inhibited and the majority of functioning bone marrow tosis, leukocytosis, thrombocytosis with bizarre platelet is derived from the abnormal clone. Excessive prolifera- morphology, and bone marrow fibrosis are common labo- tion of hematopoietic cells occurs through various mecha- ratory findings that can occur in almost any of the MPN nisms that require multiple genetic mutations3 (Chapter 23). subtypes. Commitment, differentiation, and maturation of the abnormal clone are generally preserved, leading to increased numbers of mature cells in the peripheral blood, usually with one lin- eage (erythroid, myeloid, or megakaryocytic) predominating. Erythrocyte Finding uniform biochemical, cytogenetic, or molecu- lar genetic abnormalities in hematopoietic cells from the HSC Platelets bone marrow or peripheral blood of patients with MPNs suggests that these cells were derived from a single mutant MPN stem cell (i.e., clonal origin) (Chapter 23). The abnormali- Granulocytes ties are not present in other somatic cells, indicating that the mutations are acquired rather than inherited. The most consistent chromosome abnormality, the Ph chromosome, is found in all hematopoietic blood cells in patients with CML. The Janus kinase 2 (JAK2) gene, which codes for Monocyte Macrophage a tyrosine kinase involved in cell signaling, is mutated in almost all cases of PV and some cases of ET and PMF. These Figure 24.1 This schematic of hematopoietic cell mutations are discussed in more detail later in the chap- development shows that a mutation in the pluripotential ter. The application of molecular biology in the study of hematopoietic stem cell, HSC (CD34+ ), results in clonal proliferation hematopoietic neoplasms has been useful in identifying the of the progeny of that cell, all containing the mutation. Thus, all cell lines will be affected. The HSC is thought to be the original specific genetic abnormality at the DNA, mRNA, and pro- neoplastic cell in CML and other MPNs. Arrows indicate intermediate tein levels (Chapter 42). These studies support the clonality steps to mature cells that are not shown. MPN, myeloproliferative hypothesis of MPNs. neoplasm. 522 Chapter 24 When present, anemia is caused by ineffective eryth- Hepatosplenomegaly is a common clinical finding. Extra- ropoiesis, marrow fibrosis, and/or a shortened red cell medullary hematopoiesis can also occur in benign diseases survival because of splenic sequestration resulting in such as chronic hemolytic anemias, but the hematopoiesis splenomegaly. While decreased bone marrow iron can be in these disorders is confined to the erythrocyte lineage. In observed, it does not reflect true iron deficiency.4 For this contrast, all cell lineages are present in the extramedullary reason, serum ferritin and serum iron are more reliable esti- masses that accompany an MPN. Marrow fibrosis likely mates of iron deficiency in the presence of MPNs than is the causes distortion of marrow sinusoids, which permit HSCs evaluation of bone marrow iron. to escape into the sinusoids and gain entry to the peripheral Thrombocytosis may be present, and if it is classi- blood.7 The HSCs then lodge in extramedullary sites, such fied as the primary disorder, it is believed to result from as the spleen, to proliferate and differentiate. an autonomous, unregulated proliferation of megakaryo- MPNs carry a significant risk of terminating in acute cytes. The level of interleukin-6 (IL-6) and thrombopoietin leukemia. This transition may result from chromosome (TPO), cytokines that promote megakaryopoiesis, is normal instability in the original mutant stem cell8 or as the result in MPNs, whereas the level is usually increased in second- of leukemogenic chemotherapy and radiotherapy for the ary or reactive thrombocytosis.5 Platelet membrane pro- original myeloproliferative neoplasm (Chapter 23). teins (glycoprotein IIb/IIIa, von Willebrand factor [VWF], fibrinogen, fibronectin, and vitronectin) are significantly Checkpoint 24.1 decreased in many patients with MPNs.5 Genetic changes In essential thrombocythemia, all hematopoietic lines have altering growth factor receptors, including the thrombopoi- increased cell proliferation. Which lineage has the greatest etin receptor (myeloproliferative leukemia virus oncogene increase? [MPL]), and mutations in the Janus kinase (JAK) signal- ing pathways result in increased megakaryopoiesis and enhanced activation and aggregation of platelets.6 PART II The bone marrow is usually hypercellular at onset SUBGROUPS OF MPNs of the MPN but often becomes fibrotic during the course of the disease. Fibroblasts are a part of the bone marrow stroma, which provides a suitable microenvironment for developing hematopoietic cells (Chapters 3, 38). Fibroblasts Chronic Myeloid Leukemia produce reticular fibers that form a three-dimensional sup- porting network for vascular sinuses and hematopoietic (CML) elements. Fibrosis refers to an increase in fibroblasts and Chronic myeloid leukemia (CML), also known as chronic reticular fibers. Fibrosis in MPNs is thought to be a reac- granulocytic leukemia (CGL), chronic myeloid leukemia, and chronic tive process that is secondary to the increased production myelocytic leukemia, is the best-defined MPN. It is characterized of cytokines from the abnormal hematopoietic cells (primar- by a neoplastic growth of primarily myeloid cells in the bone ily megakaryocytes). As evidence of their benign prolifera- marrow with an extreme elevation of these cells in the periph- tion, fibroblasts exhibit normal karyotypes. Although often eral blood. Erythrocytic and megakaryocytic lineages can also considered the hallmark of PMF, fibrosis can be seen in the expand and extramedullary granulocytic proliferation in the other MPNs as well. spleen and liver reflects progression of the disease. Reactive fibrosis can result from the intramedullary The natural course of the disease occurs in three phases: release of cytokines from platelets, megakaryocytes, and chronic, accelerated, and blast crisis. The initial chronic phase malignant cells that are mitogenic for fibroblasts. Human responds well to therapy. Use of traditional chemotherapy platelet-derived growth factor (PDGF) stimulates growth can usually restore and maintain normal health for months and cell division of fibroblasts as well as other cells. The or years. CML can eventually t ransform into an accelerated platelet concentration of PDGF in patients with MPNs is phase and then into blast phase or acute leukemia. The acute significantly decreased, likely because of excessive release. leukemic phase can be either acute myeloid leukemia (AML) The serum PDGF level is significantly higher in patients or lymphoblastic leukemia (LL, also called acute LL [ALL]). with PMF and ET than in other MPNs or in normal con- After progression to the blast crisis phase, the prognosis is trols. Not surprisingly, PMF and ET are the two MPNs with poor with a survival of less than 6 months using traditional the most significant degree of fibrosis.5 Increased fibroblast chemotherapy. H owever, 70–90% of patients who are treated proliferation and function leads to an increase in collagen, in the chronic phase with tyrosine kinase inhibitors (TKIs; laminin, and fibronectin in the medullary cavity. e.g., imatinib mesylate) experience a 5-year progression- When marrow fibrosis supervenes over the course of free survival. It is anticipated that long-term survival will the disease, the major sites of hematopoiesis become the improve with the use of second- and third-generation TKIs extramedullary tissues, particularly the liver and spleen. used as rescue therapy and as first-line treatment. Myeloproliferative Neoplasms 523 Etiology and Pathophysiology Philadelphia chromosome translocation The Ph chromosome (Ph) is an acquired chromosomal Chrs 9 Chrs 22 translocation that results in a fusion gene called BCR/ ABL1. This fusion gene is present in all neoplastic hemato- poietic cells in CML except T lymphocytes and sometimes q11.2 BCR B lymphocytes and is necessary for the diagnosis of CML (Figure 24-2) (Chapters 41, 42). The Ph chromosome was the first chromosome abnormality found consistently asso- Chrs 22q2 ciated with a malignant disease. It is not found in other BCR/ABL1 (Ph+chromosome) somatic cells or fibroblasts. In some cases, the Ph chromosome can be detected months before the diagnosis of CML. Once the chromo- some is identified, it will rise and fall as a reflection of tumor burden and can be used as a measure of disease pro- gression, remission, and relapse. Because all hematopoietic q34 ABL1 cells are involved in the neoplastic process (evidenced by the presence of Ph in those cells), the original neoplastic Chrs 9q1 cell (cell of origin) is most likely the HSC (Figure 24-1). ABL1/BCR Molecular biology has dramatically increased under- Normal CML standing of the role of the Ph in CML. The Ph results from a balanced reciprocal translocation between the Figure 24.3 The Philadelphia translocation in CML. long arms of chromosomes 9 and 22, t(9;22)(q34;q11.2) Arrows indicate the chromosome breakpoints at 9q34 (ABL1 (Figure 24-3). On chromosome 9, the breakpoint is spread gene) and 22q11.2 (BCR gene) in the genes directly involved in across a 90 kb region within the first exon of ABL1, trans- the translocation. The translocation results in a lengthened 9q+ locating the 3′ end of exon 1 and exons 2–11 (in which chromosome and a shortened 22q- chromosome (Ph chromosome). the tyrosine kinase domains reside) to chromosome 22. The breakpoint in chromosome 22 occurs within the BCR exon 13 or 14 of BCR. The resulting Ph has a 5′ BCR head gene, a large 70-kb gene with 25 exons. The breakpoint (exons 1-13 or 1-14) from chromosome 22 and a 3′ ABL1 usually occurs in an area known as the major breakpoint tail (exons 2–11) from chromosome 9 (Figure 24-4). This cluster region (M-BCR). When the breakpoint occurs in hybrid gene is then transcribed into an 8.5-kb fusion the M-BCR, two different fusion genes are formed (BCR/ ABL1 and ABL1/BCR). Both fusion genes join the translo- cated portion of the ABL1 gene (exons 2–11) with either ABL1 gene (Chromosome 9) 1b 1a a2 a11 5' 3' BCR gene (Chromosome 22) e1 e1e2 e6 e13 e14 e19 5' 3' m-BCR M-BCR micro-BCR e1a2 BCR ABL1 p190 e13a2 BCR ABL1 p210 e14a2 BCR ABL1 Figure 24.4 Exon maps of the normal BCR and ABL1 genes, and the BCR/ABL1 fusion gene formed by the Ph chromosome translocation found in CML and de novo acute leukemias. Breakpoints in the ABL1 gene occur upstream of exon 2 (a2, downward arrow). Breakpoints in the major breakpoint cluster region (M-BCR) of the BCR gene occur in exon 13 (e13) and exon 14 (e14) in CML; breakpoints can Figure 24.2 Karyotype from a patient with CML showing also occur in the minor breakpoint cluster region (m-BCR, 3′ of exon1 the Ph chromosome translocation, t(9:22)(q34;q11.2). The Ph [e1]) in acute leukemias. Following BCR/ABL1 fusion, the resulting chromosome is chromosome 22. protein of 210 (p210; e13a2 or e14a2) or 190 (p190; e1a2) kD is formed. 524 Chapter 24 mRNA, in contrast to the normal ABL1, which is tran- scribed to a 6- or 7-kb mRNA.9 Table 24.4 Clinical Uses of Molecular Analysis for the BCR/ABL1 Gene Rearrangement10 Translation of the 8.5-kb fusion mRNA creates a new, abnormal fusion protein, with a molecular mass of 210 • Differential diagnosis of CML kD (p210, normal ABL1 is a 145-kD protein; F igure 24-4). • Diagnosis of CML when Ph is absent This fusion protein produces constitutive tyrosine kinase • Differentiation of CML in blast crisis from de novo ALL when Ph is present activity, increasing autophosphorylation within the cell • Confirmation of a diagnosis of CML when the patient presents in the that activates intracellular signal transduction pathways, blast crisis phase of CML and results in abnormal gene expression (Chapters 2, • Monitoring of CML patients on tyrosine kinase inhibitor (TKI) therapy to determine response, resistance, or relapse 23). Tyrosine kinase enzymes regulate metabolic path- • |
Detection of minimal residual disease in post-bone marrow transplant ways by transferring phosphate groups from phosphate patients donors (ATP and GTP) to target proteins at tyrosine residues, activating the protein. Some serve as recep- tors for growth factors (RTK [receptor tyrosine kinase]). Table 24.5 Genetic Rearrangements and Related The oncogenic role of p210 is found in association with Proteins Found in Philadelphia Chromosome Positive CML, increased granulocyte colony–stimulating factor (G-CSF) ALL, and Philadelphia Chromosome Negative MPN, or ALL and PDGF and suppression of apoptosis in hematopoi- etic cells.9 Over a period of several years, the t(9;22) cell Clinical Condition Involvement of Ph/BCR Size of TK line replaces the normal marrow cells, and the clinical presentation of CML is observed. The reciprocal fusion Normal Ph- , BCR- 145 kD gene, ABL1/BCR1, is transcribed in approximately two- CML Ph+ , BCR+ 210 kD thirds of CML patients, although its significance is not Ph- , BCR+ 210 kD clear. Characteristics of the ABL1 and BCR proto-onco- Other MPNs Ph- , BCR- 145 kD genes, as well as the BCR/ABL1 fusion oncogene, are ALL Ph+ , BCR+ 210 kD (CML blast described in Table 24-3. crisis) Detection of the BCR gene rearrangement has sev- Ph+ , BCR- (minor BCR+ ) 190 kD (? de novo ALL) eral clinical uses in the diagnosis and prognosis of CML Ph- , BCR- 145 kD (? de novo ALL) (Table 24-4).9,10 About 5–10% of patients with the CML Ph+ , BCR- (micro BCR+ ) 230 kD (variant CML) phenotype lack Ph. In these cases, the translocation is not TK, tyrosine kinase; CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; detected at the karyotypic level by cytogenetic studies but Ph+ , Philadelphia chromosome positive; Ph- , Philadelphia chromosome negative; BCR+ , rearrangement within the M-BCR region; BCR- , no rearrangement within the M-BCR is detected at the molecular level by reverse transcriptase region. polymerase chain reaction (RT-PCR) or fluorescent in situ hybridization (FISH), verifying that the molecular BCR/ ABL1 fusion has occurred (Chapters 41, 42). The malig- DISEASE PROGRESSION AND ADDITIONAL CHROMO- nant cells in these cases also express the 8.5-kb chimeric SOME/MOLECULAR MUTATIONS mRNA and the p210 protein (Table 24-5). Historically, or in untreated cases, progression of CML Cases of phenotypic CML that are both Ph chromo- is marked by an accelerated phase followed by an acute some negative and BCR/ABL1-negative are usually CNL, phase (blast crisis). In about 80% of patients, this progres- aCML, or possibly cases of chronic myelomonocytic leu- sion is preceded or accompanied by the development of kemia (CMML) if absolute monocytosis and leukocytosis additional chromosomal abnormalities (Table 24-6).9 Thus, are present. The last two disorders are now considered repeated chromosome analysis in patients with CML can MDS/MPN but all have high proliferative rates.11 be helpful in predicting disease progression. Table 24.3 Characteristics of the Proto-Oncogenes and Oncogenes Involved in the BCR/ABL1 Gene Rearrangements in CML Gene Size No. Exons Breakpoint Transcript(s) Protein Location Activity ABL1 More than 230 kb 11 5′ of exon II 6 kb; 7 kb p145 Nucleus Tyrosine kinase (normal) (100–200 kb) BCR More than 100 kb 25 BCR (5.8 kb) 4.5 kb; 6.7 kb p160 Cytoplasm Serine/Threonine kinase BCR/ABL1 Varies Varies Fusion hybrid 8.5 kb p210 Plasma Tyrosine kinase fusion membrane (increased) Kb, kilobases. Myeloproliferative Neoplasms 525 and older. It is most prevalent in the seventh, eighth, and Table 24.6 Additional Chromosomal Changes in Blast ninth decades of life and is almost equally distributed Crisis of CML between sexes. CML can occur in young adults more so Rate of Occurrence Chromosomal Change than other MPNs. Although rare, CML can occur in child- Frequent Duplication of Ph chromosome hood. The incidence of CML is highest in countries that Trisomy 8 are more economically advanced. The question arises, Isochromosome 17 however, as to whether the higher incidences reflect the Loss of Y chromosome ability to detect and diagnose the disease because of Rare Translocation (15;17) increased availability of advanced medicine and diag- Translocation (3;21)(q26;q22) nostic tools in these countries versus a higher mutagen exposure. Translocation (3;3)/inversion (3) The disease has an insidious onset with the most com- Very rare Deletion of chromosome 5(-5) or the long arm (5q- ) mon symptoms being increased weakness, loss of stamina, Deletion of chromosome 7(-7) or the long unexplained fever, night sweats, weight loss, and feelings arm (7q- ) of fullness in the abdomen (hepatosplenomegaly). Gastro- intestinal tract bleeding or retinal hemorrhages are occa- sionally the first signs of the disease. Physical examination At the molecular level, mutations in the p53 gene, a reveals pallor, tenderness over the lower sternum, spleno- tumor suppressor gene, are found in at least 25% of patients megaly, and occasionally hepatomegaly. Lymphadenopa- in blast crisis, especially in those with myeloid blast crisis,9 thy is not typical but when present, it suggests an onset while mutations/alterations of p53 in the chronic state of of the acute phase of the disease. Petechiae and ecchymo- CML are rare. Other tumor suppressor genes such as p16 ses reflect the presence of quantitative and/or qualitative and RB and oncogenes such as BCL-2 and AML1 can also platelet abnormalities. Some individuals are asymptom- acquire mutations that contribute to the development of atic, and CML is found incidentally during examination blast crisis9 (Chapter 23). for other medical problems or during a routine physical PHILADELPHIA (PH) CHROMOSOME IN ACUTE examination. LEUKEMIAS Any organ eventually can be infiltrated with myeloid About 2–5% of childhood ALL, 25% of adult ALL, and elements, but extramedullary masses in areas other than some cases of AML carry the ph chromosome at diagnosis the spleen and liver are uncommon findings in the chronic (Chapters 25, 26). Ph-positive AML cases may actually be phase. On fresh incision, extramedullary masses appear CML in blast crisis that were not diagnosed in the chronic green, presumably because of the presence of the myeloid (CML) stage. In about 50% of the Ph-positive ALLs, the enzyme myeloperoxidase. These greenish tumors have BCR/ABL1 protein, p210, is present and these cases prob- been called chloromas. The green color fades to a dirty yel- ably represent the blast crisis phase of CML. In the remain- low when the tissue is exposed to air. ing 50% of Ph-positive all, the breakpoints on chromosome Without intervention, symptoms worsen over the 22 fall 5′ to the M-BCR within the first intron of the BCR next 3–5 years, and increased debilitation heralds the gene (minor breakpoint cluster region, M-BRC). These onset of the blast phase. With the onset of blast crisis, leukemias express a distinct translation product from the response to therapy is poor and survival is less than 6 mRNA hybrid termed the p190 kD protein9 (Figure 24-4). months using traditional chemotherapy. Treatment with These Ph-positive ALLs may actually be de novo acute leu- tyrosine kinase inhibitors has dramatically improved kemia cases (Table 24-5). Like p210, the p190 protein also patient outcomes. shows an increased tyrosine kinase activity, but its role in the development of the ALL phenotype is unclear. Laboratory Evaluation PERIPHERAL BLOOD Checkpoint 24.2 The most striking abnormality in the peripheral blood A patient has the CML phenotype, but the genetic karyotype does not show the Ph chromosome. If this is truly a CML, what is the extreme leukocytosis. The white count is usually should molecular analysis show? greater than 100 * 103/mcL, with a median of about 170 * 103/mcL. Patients diagnosed early may have a Clinical Presentation leukocyte count of 25-75 * 103/mcL. Thrombocytosis, which can exceed 1000 * 103/mcL, and variation in plate- CML is the most common MPN, accounting for 15–20% let shape are found in more than half of the patients. If of all leukemia cases. It can occur at any age, but the inci- thrombocytosis is a new observation, blast crisis is prob- dence increases dramatically among those 55 years of age ably imminent; during blast crisis, thrombocytopenia is 526 Chapter 24 a common finding. Platelet function also is frequently The marrow differential count of leukocyte precursors often abnormal. Megakaryocyte fragments and micromega- is within the reference interval. The typical leukemic hiatus karyocytes may be found (Figure 24-5). (lack of developing cells between immature cells [blasts] and At the time of diagnosis, a mild to moderate normo- mature cells [segmented neutrophils]) that is characteristic cytic, normochromic anemia is typical with a hemoglobin of AL is not present in CML. Auer rods may be found in the concentration in the range of 9–13 g/dL. The severity of myeloblasts during blast crisis, but this is an unusual find- anemia is proportional to the increase in leukocytes. Eryth- ing. Erythropoiesis is normoblastic because the increased rocyte morphology is generally normal, but nucleated tyrosine kinase activity does not directly affect erythropoi- erythrocytes can be found. Reticulocytes are normal or esis, but normoblasts can be decreased. Megakaryocytes slightly increased. are usually increased with frequent immature and atypi- Blood smears exhibit a shift to the left with all stages cal forms. In contrast to the large megakaryocytes found of granulocyte maturation present (Figure 24-6). The pre- in other subgroups of MPNs, CML typically reveals small dominant cells are the segmented neutrophils and myelo- megakaryocytes. cytes. Promyelocytes and blasts do not usually exceed 20% Gaucher-like cells (histiocytes exhibiting a wrinkled of the leukocytes in the peripheral blood. Eosinophils and tissue paper appearance of the cytoplasm) have been basophils are often increased in both relative and abso- observed in the bone marrow (Chapter 21). However, these lute terms. Increasing blast or basophil numbers herald Gaucher-like cells do not occur because of the lack of the blast crisis. Monocytes are moderately increased. Signs of b@glucocerebrosidase enzyme as in Gaucher’s d isease myeloid dysplasia including pseudo–Pelger-Huët anomaly but because of the overload of cerebrosides caused by (hyposegmentation of the neutrophil nucleus [Chapter 21]) increased cell turnover. The histiocytes in CML have nor- and decreased leukocyte alkaline phosphatase (LAP) are mal to increased amounts of b@glucocerebrosidase, but the frequent (Chapters 23, 37). Low or absent LAP is charac- cells cannot process the excess cerebrosides fast enough to teristic but not specific for CML. Monocytosis, myeloid dys- prevent them from accumulating. The marrow can become plasia, and micromegakaryocytes are overlapping features fibrotic late in the course of the disease. If the patient does found in both CML and chronic myelomonocytic leukemia not see a physician until the fibrosis is prominent, an inap- (CMML), but the presence of the Ph will differentiate the propriate diagnosis of PMF could be considered. At this two disorders. point, chromosomal (Ph) or molecular (BCR/ABL1) analysis can establish the correct diagnosis. BONE MARROW Other nonspecific findings related to the increased The bone marrow is 90–100% cellular with a striking increase proliferation of cells can be present. Total serum in the myeloid-to-erythroid ratio (10:1 to 50:1) reflective of cobalamin and the unsaturated cobalamin binding capac- the myelopoiesis. The active red marrow can extend into ity are increased. Serum haptocorrins are often elevated the long bones. Cortical thinning and erosion of the tra- (Chapter 15). These findings are probably related to the beculae can be present. The hematopoietic marrow cells are increased number of granulocytes that are thought to syn- primarily immature granulocytes with less than 20% blasts thesize these proteins. Uric acid and lactate dehydrogenase by WHO classification, an important characteristic that (LD) are elevated, secondarily to increased cell turnover. distinguishes CML from all forms of acute leukemia (AL).2 Muramidase is normal or only slightly increased. Figure 24.5 Arrow points to a micromegakaryocyte in the Figure 24.6 CML with leukocytosis and a shift to the left peripheral blood of a patient with CML (peripheral blood, Wright- (peripheral blood, Wright-Giemsa stain, 1000* magnification). Giemsa stain, 1000* magnification). Myeloproliferative Neoplasms 527 Checkpoint 24.3 Describe the peripheral blood differential of a CML patient. CASE STUDY (continued from page 519) Physical examination revealed a slightly enlarged liver and palpable spleen. Roger had hyperurice- mia. Blood counts showed: Hb 11.6 g/dL Hct 35% RBC 3.6 * 106/mcL WBC 26.2 * 103/mcL Figure 24.7 Peripheral blood film from a patient with CML in Platelets 853 * 103 accelerated phase. There is an increase in blasts. Arrows point to /mcL blasts (Wright-Giemsa stain, 1000* magnification). The blood cell differential showed marked aniso- cytosis, poikilocytosis with many teardrops, and at any time after the initial diagnosis, and often precedes numerous nucleated red blood |
cells. Immature the final stage of blast transformation (blast crisis).2 Clinical myeloid cells were found along with basophilia features reflect an increase in debilitation including pyrexia and large platelets. (fever), night sweats, weight loss, increased weakness, mal- 1. What are Roger’s MCV and MCHC? aise, bone pain, and lymphadenopathy. About 30% of those patients in the accelerated phase die before developing 2. How would you classify his anemia blast crisis. morphologically? Although blast crisis typically develops after a short, 3. Based on Roger’s history and current laboratory accelerated phase, about one-third of cases abruptly data, what other tests should be performed? develop a blast transformation. After onset of blast crisis, survival is about 1–2 months using classical chemotherapy and is dramatically improved for most patients receiving Terminal Phase TKI therapy. The clinical features in blast crisis are similar to those of acute leukemia (Chapters 26, 27). If untreated, the typical CML course progresses to the The hematologic criteria for identifying blast crisis is accelerated stage approximately 3–5 years after diagnosis made by finding moderate to marked diffuse bone mar- (Figure 24-7). Transition to the accelerated phase is h eralded row fibrosis and greater than or equal to 20% blasts2 in the by one or more of the changes listed in Table 24-7, can occur peripheral blood or bone marrow of a patient previously Table 24.7 Accelerated Phase of CML Characterized by the presence of one or more of the following: Peripheral Blood/Bone Marrow Findings Molecular Findings Therapy-Specific Findings Other Persistent or increasing WBC count (more than Additional clonal chromosomal Hematologic resistance to the Persistent or increasing 10 * 103/mcL), unresponsive to therapy abnormalities in Ph+ cells at diagnosis first TKI splenomegaly (second Ph, trisomy 8, isochromosome (unresponsive to therapy) 17q, trisomy 19), complex karyotype, or abnormalities of 3q26.2 Ten to 19% myeloblasts in peripheral blood or Any new clonal chromosomal Any hematological, cytogenetic, bone marrow abnormality in Ph+ cells that occurs or molecular indications of during therapy resistance to two sequential TKIs Basophils in peripheral blood Occurrence of two or more mutations in BCR/ABL1 during TKI therapy Persistent thrombocytopenia (less than 100 * 103/mcL), unresponsive to therapy Persistent thrombocytosis (greater than 1000 * 103/mcL), unresponsive to therapy 528 Chapter 24 Table 24.8 Blast Crisis Phase of CML Characterized by the presence of one or more of the following: • Greater than or equal to 20% blasts in the bone marrow or nucleated cells in the peripheral blood • Extramedullary blast proliferation • Presence of large clusters of blasts in the bone marrow biopsy specimen diagnosed as having CML (Table 24-8 and Figure 24-8). Any type of blast involvement is possible including myeloid, lymphoid, erythroid, or megakaryocytic cells. Because blast morphology alone is often not sufficient to identify the type of blast involved, cytochemical, enzymatic, ultra- structural, and immunophenotyping and molecular studies Figure 24.8 Peripheral blood from a patient with CML in blast are necessary (Chapters 23, 37, 40). crisis. Note the cluster of blasts with vacuoles (Wright-Giemsa stain, About 65–75% of blast crises are myeloblastic, and 25–35% 1000* magnification). of blast crises are lymphoblastic. The lymphoblasts in blast cri- sis are immunologically typed as common acute lymphocytic leukemia antigen positive (CALLA+/CD10+ ) (Chapters 8, quinone 2 (NQ02), and c-raf. Imatinib b inding inhibits 27, 40) and demonstrate an elevated terminal deoxynucleoti- kinase activity and prevents cell signal transduction.14,15,16 dyl transferase (TdT) suggesting that these cells belong to the This range of binding explains the potential use of imatinib B lymphocyte lineage (Chapter 8). Erythroblastic and mega- in other conditions but raises questions about clinical effi- karyoblastic crises are uncommon. cacy and toxicity.14 Imatinib is generally well tolerated with mild to moder- Therapy ate side effects, primarily involving the gastrointestinal tract and skin. However, it is teratogenic in laboratory animals. The purpose of CML therapy is to reduce the leukocyte mass, Imatinib is also effective in delaying disease progression in restore bone marrow function, reduce splenomegaly, and patients in blast crisis.12,13 abolish symptoms. Leukapheresis is sometimes used initially Patients initially responsive to imatinib therapy can to reduce the leukocyte mass when excessive numbers of cells develop resistance (secondary resistance or loss of response), result in a significant increase in blood viscosity. Supportive whereas other patients might not respond to initial therapy measures during therapeutic regimens include transfusion to (primary resistance; Table 24-9).13 The majority of patients in treat severe anemia and antibiotics to treat infections. the advanced stages of CML show a measurable response to Before the discovery of molecularly targeted therapy imatinib although fewer of them reach remission compared for malignant diseases, the use of interferon@a, plus cyta- with those treated in the chronic phase. rabine was considered standard therapy for patients with In the majority of cases, loss of response (second- CML who were not candidates for an allogeneic HSC trans- ary resistance; Table 24-9) is related to development plant. Interferon@a, a glycoprotein, has a myelosuppressive of additional mutations in the kinase domain of BCR/ effect directly inhibiting myeloid progenitor cells. It induces a remission in 55–75% of CML patients and, in some cases, eliminates the Ph clone. A cytogenetic response is more Table 24.9 Resistance to Imatinib in Patients with CML favorable in patients who are treated early in the chronic Resistance Definition phase of the disease.9 Primary resistance • Complete hematologic response is not IMATINIB achieved by 3–6 months Currently, the tyrosine kinase inhibitor (TKI) imatinib • Cytogenetic response is not achieved by 6 months (more than 95% Ph+ metaphases mesylate (Gleevec®), is considered the first treatment option persist) except in pregnant patients.12,13 It is a molecular-targeted • Partial cytogenetic response is not achieved therapy. Imatinib competitively binds to the ATP binding by 12 months (more than 35% Ph+ metaphases persist) site of the tyrosine kinase of ABL1 (BCR/ABL1), stem cell fac- Secondary resistance • Loss of a complete hematologic response tor receptor (c-Kit), platelet-derived growth factor receptor • Loss of a partial or complete cytogenetic (PDGFR), lymphocyte-specific kinase (Lck), vascular endo- response thelial growth factor receptor (VEGFR), colony s timulating • Increasing levels of BCR/ABL1 as assessed factor receptor-1 (CSFR-1), NAD(P)H dehydrogenase, by real-time PCR Myeloproliferative Neoplasms 529 ABL1.9 A particular ABL1 protein mutant, T315I, in which Ponatinib is considered a third-generation TKI and was threonine is mutated to isoleucine at the gatekeeper posi- also FDA approved in 2012 as a rescue therapy for patients tion of the kinase domain does not respond to any of the who are resistant to imatinib, dasatinib, and nilotinib. TKIs currently used in CML therapy and represents about Most notable is that Ponatinib is effective against the T315I 15% of i matinib resistant patients.14 Mutation screening is mutation. It can inhibit a wide range of tyrosine kinases to suggested for those who do not have an adequate initial include PDGFR, c-Kit, FGFR, VEGFR, and Flt-3.14 response or who experience a loss of response to imatinib Baseline assessment for CML patients prior to start- because the position of the m utation within the kinase ing imatinib therapy should include bone marrow mor- domain can be clinically r elevant. Some mutations have phologic analysis, cytogenetics, and RT-PCR to determine inferior survival (P-loop of BCR/ABL1 protein kinase muta- baseline level of BCR/ABL1 transcripts. Measuring BCR/ tions) compared with mutations at other sites.15 ABL1 mRNA levels in plasma has been found to correspond Several therapeutic approaches for patients who with patient tumor burden and is suggested as a monitor- experience imatinib resistance exist. Dose escalation is ing standard for patients.16 The RT-PCR assay of blood often the first course of action. Remission can be recovered should be performed at least every 3 months after therapy by imatinib dose escalation when the loss of remission is begins, or a single assay at 6 months can be performed if the caused by the development of a second Ph in the HSC or patient is responding.17 Bone marrow cytogenetics should BCR/ABL1 gene duplication. Occasionally, relapse because be performed every 6 months until a major c ytogenetic of certain mutations in the ATP binding site can be over- response (MCyR; less than 35% Ph+ ), is achieved. A com- come through imatinib dose escalation.16 plete cytogenetic response (0% Ph+ ) and BCR/ABL1- When dose escalation fails, switching to an alterna- negative results (complete molecular response) in treated tive tyrosine kinase inhibitor is the treatment of choice. patients predict a favorable outcome. Incomplete response Dasatinib (BMS354825) is a tyrosine kinase inhibitor that, (primary resistance) indicates the presence of minimal like imatinib, attaches to the ATP binding site of the ABL1 residual disease, and increasing BCR/ABL1 results indicate portion of the BCR/ABL1 protein. It is effective against all the potential for disease relapse (secondary resistance). imatinib resistant mutants currently identified except the HSC TRANSPLANTATION T315I mutation. Allogenic HSC transplantation can be an option for patients Nilotinib (AMN107) is a structural derivative of ima- who meet the bone marrow transplant criteria. Limitations tinib that binds to and stabilizes the ABL protein ATP bind- are based on patient age (less than 65 years) and availability ing site with 30-times greater potency than imatinib. Like of a compatible donor.9 High-dose chemoradiotherapy or dasatinib, nilotinib is effective against all imatinib resistant imatinib therapy is followed by HSC transplantation from mutants currently identified except the T315I mutation. syngeneic or allogenenic donors. Stem cell transplantation is In September 2012, the U.S. Food and Drug Administra- most successful when administered during the first chronic tion approved bosutinib for the treatment of chronic, accel- phase of CML. Of patients receiving HLA-matched sibling erated, or blast phase Philadelphia chromosome positive stem cells, 86% have survival rates that exceed 3 years.9 (Ph+ ) chronic myeloid leukemia (CML) in adult patients with resistance or intolerance to prior therapy. Like dasat- inib and nilotinib, it is effective against most imatinib-resis- Differential Diagnosis tant mutants except the T315I mutation. Preliminary results Many infectious, inflammatory, or malignant disorders and suggest that second-generation TKIs could be superior first- severe hemorrhage or hemolysis can cause a leukemoid line therapies over imatinib for new CML patients.16 reaction (Chapter 21) that resembles CML (Table 24-10). Table 24.10 Comparison of Peripheral Blood Features of CML and Leukemoid Reactions Laboratory Parameter CML Leukemoid Reaction Leukocytes Moderate to marked leukocytosis, blasts and promyelocytes in Mild to moderate leukocytosis, toxic granulation, peripheral blood (deep left shift), toxic changes usually absent, Döhle bodies and vacuoles present, blasts absent, eosinophilia and basophilia, neutrophils with single-lobed nuclei promyelocytes rare, no absolute basophilia or and hypogranular forms can be present eosinophilia Platelets Often increased with abnormal morphological forms present, occa- Usually normal sional micromegakaryocytes Erythrocytes Anemia usually present, variable anisocytosis, poikilocytosis, NRBC Anemia rarely, NRBC not typical present LAP Low Increased Chromosome karyotype Ph or BCR/ABL1 translocation present Normal 530 Chapter 24 At times, the clinical findings of a leukemoid reaction per- mit an accurate diagnosis, but in some cases, differential Table 24.11 Diagnostic Criteria for Chronic Neutrophilic Leukemia2 diagnosis requires further investigation. In a leukemoid reaction, leukocytosis is generally accompanied by a pre- Peripheral blood leukocytosis greater than or equal to 25 * 103/mcL dominance of segmented neutrophils and bands on the • Bands and segmented neutrophils more than 80% white blood count (WBC) blood smear (myelocytes, metamyelocytes, promyelo- • Immature granulocytes less than 10% WBC cytes, and blasts are few in number compared with CML). • Myeloblasts less than 1% WBC Toxic granulation, cytoplasmic vacuoles, and Döhle bod- • No dysgranulopoiesis ies in granulocytes often accompany benign toxic leukocy- Hypercellular BM tosis in leukemoid reactions and are not common in CML. • Increased percent of neutrophilic granulocytes In contrast to CML, monocytes, eosinophils, and baso- • Less than 5% myeloblasts phils are generally not elevated in a leukemoid reaction. • Normal neutrophil maturation pattern Other diagnostic tests (elevated LAP score and • Normal or left shift of megakaryocytes absence of Ph) are helpful in differentiating a leukemoid Absence of Ph chromosome or BCR/ABL1 mutation Absence of mutations of PDGFRA, PDGFRB, or FGFR1 reaction from CML. Splenomegaly is uncommon in a leu- No evidence of other MPNs kemoid reaction. Although a bone marrow examination No evidence of an MDS or MDS/MPN disorder; monocytes less than is rarely necessary to make a differential diagnosis, the 1 * 103/mcL; no dysplastic changes in granulocytes or other myeloid cells marrow in a leukemoid reaction can be |
hypercellular, but Presence of CSF3R T618I or other activating CSF3R mutation in contrast to CML, the maturation of granulocytic cells If CSF3R T618I is absent, persistent neutrophilia for at least 3 months, splenomegaly, no identifiable cause of reactive neutrophilia including is orderly. the absence of a plasma cell neoplasm OR demonstration of clonality CML occasionally resembles PMF. Distinguishing of myeloid cells by cytogenetic or molecular studies. features of PMF include markedly abnormal erythrocyte morphology with nucleated erythrocytes and immature leukocytes (leukoerythroblastic reaction), an increased Etiology and Pathophysiology LAP score, bone marrow fibrosis, and the absence of the Ph. The cause of CNL has not been identified. About 20% of CNL is another MPN that may resemble CML but does cases are associated with an underlying neoplasm, par- not have the BCR/ABL1 mutation. It must be distinguished ticularly multiple myeloma. However, clonality of the from CML because in the absence of the BCR/ABL1 muta- neutrophils when neutrophilia is associated with multiple tion, imatinib is not an effective treatment. Because of the myeloma has not been demonstrated. Thus, neutrophil pro- similarity of CNL to CML, it is discussed next. liferation may be related to abnormal cytokine production in this setting. The CNL cell of origin is probably the HSC. Normal apoptotic signaling is likely disrupted. Checkpoint 24.4 Cytogenetics are normal in most patients, but up to 25% What clinical, peripheral blood, and genetic features differentiate have mutations including 20 q- , 21+ , 11q- , and +8, +9. Ph CML from an infectious process? and BCR/ABL1 fusion gene are absent. If plasma cell prolifera- tion is present, clonality of the neutrophils should be established by cytogenetic or molecular studies for a diagnosis of CNL. Chronic Neutrophilic Clinical Presentation Leukemia (CNL) CNL is a rare MPN, with approximately 150 reported cases. The median age at diagnosis is 65 years. The male-to-female Chronic neutrophilic leukemia (CNL) is an MPN ratio is about 1:1. Most patients are asymptomatic at the time characterized by a sustained increase in neutrophils in of diagnosis, but fatigue, weight loss, easy bruising, bone the peripheral blood with a slight shift to the left and pain, and night sweats can occur. Hepatosplenomegaly is no Ph or BCR/ABL1 mutation. Monocytosis and baso- usually present. philia are absent, which helps to distinguish CNL from myelodysplasia and CML, respectively. The bone mar- row is hypercellular because of increased neutrophilic Laboratory Evaluation granulocyte proliferation, but dysplasia is absent. All The most notable feature is neutrophilia (more than causes of a reactive neutrophilia and other MPNs must 25 * 103/mcL) (Figure 24-9). Mature segmented forms be ruled out for a diagnosis of CNL, so it is a diagnosis of and bands predominate, and more immature cells account exclusion. The diagnostic criteria for CNL18 are defined for less than 10% of the leukocytes. Neutrophils can appear in Table 24-11. toxic but not dysplastic. Both the RBC count and platelet Myeloproliferative Neoplasms 531 Essential Thrombocythemia (ET) Essential thrombocythemia (ET) is a myeloprolifera- tive neoplasm affecting primarily the megakaryocytic lineage. Sustained proliferation of megakaryocytes in the marrow and extreme thrombocytosis in the peripheral blood with thrombocytopathy (a qualitative disorder of platelets) occurs. Previously, considerable contro- versy existed concerning the inclusion of ET as a spe- cific entity in the myeloproliferative disorders because thrombocytosis is often a component of CML, PMF, and PV. However, ET is now firmly established as a hemato- Figure 24.9 logic neoplasm with d istinct clinical manifestations and Peripheral blood from a patient with CNL. Note complications.19 the increased numbers of mature neutrophils (Wright-Giemsa stain, 1000* magnification). Synonyms of ET include primary thrombocythemia, hemorrhagic thrombocythemia, primary thrombocytosis, and idiopathic thrombocytosis. morphology are normal. Platelets are usually present in normal concentration, but thrombocytopenia can develop Etiology and Pathophysiology as the disease progresses and the spleen enlarges. Bone mar- row is hypercellular with an M:E ratio that can reach 20:1 or ET is a neoplastic disorder of the HSCs usually resulting higher. Granulocytic hyperplasia is present, but dysplasia, in clonal hematopoiesis affecting all three lineages, but Auer rods in blasts, and increased blasts are absent. in some cases, involving only the megakaryocytes.20 The Excessive erythroid and megakaroycytic p roliferation may clonal population of cells appears hypersensitive to some be present. The LAP score is usually increased. A muta- cytokines, including IL-3 and IL-6, but the clones are not tion in the colony stimulating factor 3 receptor (CSF3R) is hypersensitive to GM-CSF. Sensitivity to the inhibitory strongly associated with CNL. The Ph chromosome and effects of TGF@b is decreased, minimizing inhibition of BCR/ABL1 fusion gene are absent. thrombopoiesis. Thus, a combination of increased sensi- tivity to some cytokines that promote platelet production coupled with a decreased sensitivity to negative regulators Therapy could account for the increased megakaryocyte prolifera- Hydroxyurea is a first-line therapy for CNL. Response lasts tion characteristic of ET. for about 12 months. Second-line therapy is interferon@a. Expression of MPL, the TPO receptor, and its mRNA Allogeneic stem cell transplantation is a potentially curative are generally decreased in ET, serum levels of thrombopoi- treatment for those patients who are eligible. etin are normal or slightly elevated in most patients, yet Median survival is 2.5 years. CNL also has an accel- proliferation of progenitor cells ensues.20 erated phase that is marked by progressive neutrophilia unresponsive to treatment, anemia, thrombocytopenia, and JAK2(V617F) splenomegaly.9 Blasts and other immature cells can be pres- The normal JAK2 protein is a cytoplasmic protein kinase ent in the peripheral blood. closely associated with cytokine receptors and thus is dis- tributed almost exclusively near the cell membrane. When Differential Diagnosis a receptor binds cytokines, the JAK2 protein is transphos- phorylated and activated. In turn, JAK2 phosphorylates CNL must be differentiated from CML and physiologic signal transducers and activators of transcription (STAT) causes of neutrophilia such as infection or inflammation. proteins. The JAK–receptor complex also activates other Differential diagnosis from other myeloid neoplasms signaling pathways. Several inhibitory control mecha- requires an absence of circulating blasts, absolute monocy- nisms constrain the normal JAK2/STAT activation path- tosis, eosinophilia, and basophilia. way. The JAK2 protein has two homologous domains: JH1, which has functional (kinase domain) activity, and JH2, Checkpoint 24.5 which lacks kinase activity (pseudo-kinase domain). The What is the most important feature that separates all other JH2 domain normally interacts with the JH1 domain to forms of MPNs from CML? inhibit kinase activity and to modulate or regulate recep- tor signaling.20 532 Chapter 24 In 2005, a gain-of-function mutation in the JAK2 Clinical Presentation tyrosine kinase gene, JAK2(V617F), was identified in patients with PV and later in ET and PMF.20 JAK2(V617F) is Although a relatively rare disorder, the incidence of ET found on chromosome band 9p24 and encodes a mutated peaks primarily from 50–60 years of age and secondarily tyrosine kinase activator of cell signal transduction. The from 20–30 years of age. The older group of patients has single nucleotide somatic mutation in exon 14 (substitution no gender predilection, but the younger age group pre- of thymine for guanine) results in the substitution of the dominantly involves women. The overall incidence is about 1.592.4/100,000 people annually.20 amino acid valine for phenylalanine at position 617. The amino acid substitution occurs in the pseudo-kinase JH2 The presenting symptoms of patients with ET are vari- domain of the JAK2 protein. This reduces the JH2 inhibitory able. Extreme thrombocytosis is frequently detected. Many function and results in constitutive activation. Furthermore, of these patients are asymptomatic, and their diagnosis is the mutation allows for downstream phosphorylation of made incidentally.20 Symptomatic patients most commonly STAT molecules independent of cytokine interaction with present with thrombosis (primarily involving the microvas- the receptor (i.e., autonomous signaling; Chapter 3). This culature) that can result in pulmonary embolisms or minor JAK2(V617F) gain-of-function mutation gives the cells a bleeding. Neurologic complications are common (e.g., proliferation advantage. headache, paresthesias of the extremities) and are associ- Presence of the JAK2(V617F) mutation appears to ated with platelet-mediated ischemia and thrombosis. Cir- increase the risk of thrombosis proportional to the degree culatory insufficiency involving the microvasculature of the of leukocytosis with no correlation to platelet count.20 The toes and fingers is frequent and associated with pain and connection between leukocytosis and thrombosis may occasionally gangrene. Hemorrhagic episodes can occur, be the result of tissue factor expression by neutrophils, primarily involving the gastrointestinal tract, skin, urinary platelet P-selectin expression, and aggregate formation tract, and oral mucosal membranes. These problems appear between platelets and leukocytes that lead to interac- to be more frequent in patients older than 59 years of age tion with the endothelium in areas of inflammation and with thrombosis occurring more frequently than bleeding at the lower platelet concentrations.20,29 injury.21,22 A single JAK2(V617F) mutation is more commonly About half of the patients have a palpable spleen, but encountered in ET, whereas two JAK2(V617F) mutations splenomegaly is usually slight. Occasionally, splenic atro- are associated with PV.23,24,25,26 This dosage effect is asso- phy resulting from repeated splenic thrombosis and silent ciated with disease transformation from ET to PV and infarctions is seen. When this occurs, it is associated with with disease progression in patients with PV. Therefore, typical morphologic alterations on the peripheral blood JAK2(V617F) burden is implicated as having a dominant smear as discussed in the next section. role in the pathophysiology of Ph negative MPNs.27 The mutation also results in increased responsiveness to eryth- Laboratory Evaluation ropoietin (EPO) and IL-3. JAK2(V617F) is found in about PERIPHERAL BLOOD 50% of patients with PMF and most patients (greater than The most striking finding in the peripheral blood is extreme 95%) with polycythemia vera.20,29,28 and consistent thrombocytosis (Figure 24-10). Platelet counts Mutations in cytokine receptors for EPO, TPO, are greater than 450 * 103/mcL and often range from 1000 and G-CSF may activate JAK2 in those cases that are to 5000 * 103/mcL. The peripheral blood smear can show JAK2(V617F) negative. giant bizarre platelets, and platelets can appear in aggre- gates. Megakaryocytes and megakaryocyte fragments may MPL MUTATIONS be present. However, in many cases, platelet morphology Two mutations in the thrombopoietin receptor MPL appears normal. Abnormalities in platelet aggregation and (MPLW515L and MPLW515K) occur in approximately 4% of adhesion suggest defects in platelet function (Chapter 36). patients with JAK2(V617F)-negative ET and 10% with PMF. Anemia, if present, is generally proportional to the This mutation results in cytokine-independent growth and constitutive downstream signaling pathways.20 severity of bleeding and is usually normocytic; however, long-standing or recurrent hemorrhagic episodes can lead CALRETICULIN (CALR) MUTATIONS to iron deficiency and a microcytic, hypochromic anemia. Calreticulin (CALR) mutations also affect the JAK-STAT In about one-third of the patients, slight erythrocytosis is pathway. At least four mutations have been described in present and can cause confusion with polycythemia vera. CALR; the mutations are associated with a higher platelet Aggregated platelets can lead to an erroneous increase in count and lower WBC and hemoglobin levels compared the erythrocyte count on automated cell counters. Therefore, to ET patients with a JAK2 mutation.22 Thrombosis occurs hemoglobin determinations are better to assess the patient’s at half the rate of ET patients with a JAK2 mutation and anemic status. The reticulocyte count can be increased if patients do not progress to PV.22,31 bleeding is present, in which case mild polychromatophilia Myeloproliferative Neoplasms 533 Table 24.12 Platelet Abnormalities Found in Essential Thrombocythemia • Decreased or mutated MPL receptors for thrombopoietin (TPO) • Shortened platelet survival • Increased plasma b@thromboglobulin (b@TG) • Increased urinary thromboxane B2 (TXB2) • Acquired Von Willebrand disease • Defective epinephrine, collagen, ADP-induced platelet aggregation • Decreased ATP secretion • Acquired storage pool deficiency because of abnormal in vivo platelet activation common findings including defective platelet aggregation Figure 24.10 Essential thrombocythemia. Platelets are with epinephrine, ADP, and collagen. A loss of platelet markedly increased, and a giant form (arrow) is present (peripheral a@adrenergic receptors associated with reduced epineph- blood, Wright-Giemsa stain, 1000* magnification). rine-induced aggregation is characteristic of an MPN and is useful in differentiating ET from secondary thrombocytosis. Spontaneous in vitro platelet aggregation, or hyperaggrega- may be noted. Peripheral blood abnormalities second- bility, is a common finding. In vivo platelet aggregation is ary to autosplenectomy can occur if the spleen has been likely if increased plasma b@thromboglobulin and platelet infarcted (Chapter 3). These abnormalities include Howell- factor 4 (released from platelet a@granules) levels are found. Jolly bodies, nucleated erythrocytes, and poikilocytosis. Other platelet |
abnormalities that have been described in A leukocytosis ranging from 22 to 40 * 103/mcL is association with ET are included in Table 24-12.32 almost always present. Occasional metamyelocytes and A form of acquired von Willebrand disease (VWD) has myelocytes can be found with ET. Mild eosinophilia and been described in association with excessively high platelet basophilia also are observed. The LAP score may be nor- counts and ET. The increase in number of circulating plate- mal or increased; it is rarely low. Nucleated erythrocytes are lets is associated with adsorption of larger VWF multim- found in 25% of patients. ers and their removal from the circulation. The laboratory ET patients must not meet any criteria for features are characteristic of Type 2 VWD with a decrease BCR/ABL1 + CML, PV, PMF, MDS, or other myeloid or absence of large VWF multimers and reduced levels of neoplasms and must demonstrate JAK2(V617F), MPL, or ristocetin cofactor activity (Chapters 34, 36). CALR mutations. Minor criteria include presence of a clonal MOLECULAR GENETICS marker or absence of evidence of reactive thrombocytosis.2 A low incidence of clonal chromosomal cytogenetic abnor- BONE MARROW malities (about 5%) is found in ET. However, mutations in The bone marrow exhibits marked hyperplasia with a JAK2, MPL, and CALR genes function to drive the disor- striking increase in megakaryocytes often with clustering der and are found in both sporatic and familial cases.22 The of megakaryocytes along the sinusoidal borders. The back- JAK2(V617F) and MPL(W515L) mutations are the most com- ground of stained slides shows many platelets. The mega- mon.22,32 Most triple-negative patients express other somatic karyocytes are large with abundant mature cytoplasm and mutations in JAK2 and MPL.32,33 frequently increased nuclear lobulation. Mitotic forms are OTHER LABORATORY EVALUATION increased. Erythroid and myeloid hyperplasia also are evi- Other laboratory tests may be abnormal. Serum cobala- dent. Stains for iron reveal normal or decreased stores in min and the unsaturated cobalamin binding capacity are the context of normal serum ferritin levels. In about 25% of increased. An increase in cell turnover can cause serum uric cases, reticulin is increased, but significant fibrosis is gener- ally not seen.20 acid, lactate dehydrogenase (LD), and acid phosphatase to be elevated. Serum potassium can be elevated as a result TESTS OF HEMOSTASIS of in vitro release of potassium from platelets (pseudohy- Laboratory tests alone are unreliable in predicting bleeding perkalemia). The spurious nature of this hyperkalemia can or thrombotic complications in ET. The prothrombin time be verified by performing a simultaneous potassium assay (PT) and activated partial thromboplastin time (APTT) are on plasma, which should be normal. Arterial blood gases usually normal, but evidence of low-grade disseminated may reveal a pseudohypoxia if the sample is not tested intravascular coagulation (DIC) may be present. Platelet promptly because of the in vitro consumption of oxygen by aggregation studies are frequently abnormal with the most the increased numbers of platelets. 534 Chapter 24 Prognosis and Therapy Table 24.13 Conditions Associated with Thrombocytosis Median survival for patients with ET is 19.8 years.22 Essential thrombocythemia (ET) Transformation into primary myelofibrosis or acute leu- Polycythemia vera (PV) kemia occurs at a rate of 10% and 5%, respectively, and a Chronic myeloid leukemia (CML) Primary myelofibrosis (PMF) JAK2 mutation increases the risk of transformation into Secondary thrombocytosis polycythemia vera.20 The prognosis appears to be better in • Chronic inflammatory disorders younger patients. The most common causes of death are • Acute hemorrhage thrombosis and bleeding. • Hemolytic anemia Controversy exists as to which patients with ET require • Hodgkin’s disease therapy. It is generally agreed that patients with a history • Metastatic carcinoma of thrombosis or cardiovascular risk factors require therapy • Lymphoma • Postsplenectomy to reduce the platelet count. Plateletpheresis can quickly • Postoperative reduce the platelet count below 1000 * 103/mcL for con- • Iron deficiency trol of vascular accidents. Anticoagulants and drugs to inhibit platelet function are used to control thrombosis, and aspirin therapy is recommended for all ET patients unless contraindicated.20 is normal as are leukocytes and erythrocytes, and spleno- CHEMOTHERAPY megaly is absent. Cytoreductive therapy frequently includes use of Differentiating ET from PV can be difficult. However, hydroxyurea and anagrelide. Hydroxyurea is an effec- marked erythrocytosis with clinical findings suggestive tive form of cytoreductive therapy in patients to lower of hypervolemia is more typical of PV. The Polycythemia both the leukocyte and platelet counts and lower the risk Vera Study Group (PVSG) proposed a set of diagnostic of thrombosis. Although anagrelide (inhibitor of mega- criteria for ET, which were adopted as diagnostic criteria karyocyte differentiation) can be used to reduce only the in the WHO classification (Table 24-14). The first criterion, platelet count, its side effects are not well tolerated.20 The a platelet count greater than 600 * 103/mcL later reduced leukemogenic potential of these therapeutic agents is of by the WHO group to 450 * 103/mcL excludes many concern. The benefit of specific therapy in asymptom- cases of secondary thrombocytosis. The second criterion, atic patients has not been established. Therapeutic trials increased megakaryocytes in the bone marrow, is necessary with b@interferon show improvement in both hematologic to confirm the diagnosis of ET and rule out other causes of clonal thrombocythemia.36 parameters and clinical symptoms on nearly all patients.32 Withdrawal of interferon, however, leads to recurrence of thrombocytosis. MOLECULAR TARGETS Table 24.14 WHO Diagnostic Criteria for Essential Thrombocythemia (ET)2Use of JAK inhibitors is being investigated primarily in PMF but is beginning to be studied in patients with advanced Major Criteria* ET and PV. These include ICNB018424 (ruxolitinib) and • Platelet count greater than 450 * 103/mcL CEP-701 (lestaurtinib).30,31 Patient response to lestaurtinib • Bone marrow biopsy shows megakaryocytic hyperplasia (enlarged, mature megakaryocytes). No significant increase or left shift in neu- is not as promising as that for ruxolitinib,35 a JAK i nhibitor trophil granulopoiesis or erythropoiesis and very rarely minor (grade 1) that binds the ATP-binding site of JAK (wild-type and increase in reticulin fibers. JAK2[V617F]).9,14 • Not meeting WHO criteria for BCR/ABL1 + CML, PV, PMF, MDS, and other myeloid neoplasms • Presence of JAK2, CALR, or MPL mutations Differential Diagnosis Minor Criteria Although the other MPNs have certain diagnostic markers, • Presence of a clonal marker or absence of evidence for reactive thrombocytosis ET is largely a diagnosis of exclusion. Essential thrombo- cytosis must be differentiated from a secondary, reactive * Diagnosis of ET requires meeting all four major criteria or the first three major criteria and one minor criteria. thrombocytosis (Table 24-13) associated with many acute and chronic infections, inflammatory diseases, carcino- mas, and Hodgkin’s disease. The platelet count in ET often Checkpoint 24.6 exceeds 1000 * 103/mcL and is persistent over a period Is a patient who has a platelet count of 846 * 103/mcL, spleno- of months or years. Secondary or reactive thrombocyto- megaly, and abnormal platelet function tests of hyperaggrega- sis rarely reaches 1000 * 103/mcL and is transitory. In tion likely to have reactive or essential thrombocytosis? Explain. addition, platelet function in secondary thrombocytosis Myeloproliferative Neoplasms 535 Polycythemia Vera (PV) Table 24.15 Classification of Polycythemia The term polycythemia literally means an increase in the cel- Classification Associated Conditions lular elements of the blood. However, it is most commonly Polycythemia vera (primary) Normal or decreased erythropoietin used to describe an increase in erythrocytes exclusive of leu- levels; autonomous cell proliferation kocytes and platelets. Polycythemia vera (PV) is a myelo- Secondary polycythemia High altitude proliferative neoplasm characterized by an unregulated (erythropoietin driven) Chronic obstructive pulmonary disease proliferation of primarily the erythroid elements in the bone Obesity (Pickwickian syndrome) marrow and an increase in erythrocyte concentration in the Inappropriate erythropoietin production peripheral blood. In addition to autonomous proliferation Tumors (e.g., hepatoma, uterine of erythroid cells, leukocytes and platelets can also prolifer- fibroma, renal carcinoma) ate uncontrolled, resulting in a pancytosis (an increase in all Renal ischemia hematopoietic cells in the blood). PV has several synonyms, Familial erythrocytosis including polycythemia rubra vera, primary polycythemia, ery- Hemoglobins with high oxygen affinity thremia, and Osler’s disease. Congenital decrease in erythrocyte 2,3-BPG Relative polycythemia Gaisböck’s syndrome (stress poly- Classification cythemia, spurious polycythemia, pseudopolycythemia) Polycythemia is a general term used to describe erythrocyto- Dehydration sis resulting in an increase in both hemoglobin concentra- tion and red cell mass (RCM) or hematocrit (Chapter 10). When evaluating a patient for polycythemia, it is impor- stimulus of erythrocytosis—hence, the name secondary tant to determine whether these parameters are elevated polycythemia—and is associated with elevated plasma because of an absolute increase in total erythrocyte mass EPO levels. PV results from a primary, unregulated, or (absolute erythrocytosis) or from a decrease in plasma vol- dysregulated increase in erythrocyte production. Rela- ume (relative erythrocytosis) (Figure 24-11). Although an tive polycythemia is characterized by a normal or even absolute erythrocytosis suggests a PV diagnosis, polycy- decreased RCM and occurs as a result of a decreased themia that is secondary to tissue hypoxia, cardiac or pul- plasma volume. It is generally a mild polycythemia result- monary disease, and abnormal hemoglobins should also ing from dehydration, hemoconcentration, or a condition be considered. known as Gaisböck’s syndrome. In an attempt to clarify the pathogenesis of the disor- der, polycythemia is classified into three different groups: Etiology and Pathophysiology PV, secondary polycythemia, and relative polycythe- mia (Table 24-15). Both PV and secondary polycythemia The panhyperplasia often associated with PV suggests a result from an absolute increase in the total body RCM. clonal stem cell defect, and cytogenetic studies have con- Secondary polycythemia can be distinguished from PV firmed its clonal nature.37 Evidence of clonality persists in by a distinct, although not always apparent, physiologic cells even during complete remission. Although all lineages in the peripheral blood can be increased in PV, an increase in erythropoiesis is the out- standing feature. Possible mechanisms for this increase are suggested in Table 24-16.38 In vitro studies using cell culture systems show that PV bone marrow cells can form erythroid colonies without the addition of erythropoietin, suggesting 60% Hct that increased proliferation results from an unregulated neoplastic proliferation of stem cells.37 Other patients’ bone 45% Hct 55% Hct Table 24.16 Possible Mechanisms for Increased Normal Absolute Relative Erythropoiesis in Polycythemia Vera polycythemia polycythemia • Erythropoietin-independent proliferation of neoplastic progenitor cells Figure 24.11 The hematocrit can be increased because of • Hypersensitivity of erythroid progenitor cells to erythropoietin an absolute increase in erythrocyte mass, a condition known as • Hypersensitivity of erythroid progenitor cells to growth factors other than erythropoietin absolute polycythemia (center) or a decrease in plasma volume, a condition known as relative polycythemia (right). • Inhibition of apoptosis in progenitor cells 536 Chapter 24 marrow cells show increased sensitivity to EPO, insulin-like These include MPL,48 the lymphocyte-specific adapter growth factor 1, and IL-3, forming in vitro colonies at sig- protein LNK,49 and an anti-apoptotic protein member of nificantly reduced cytokine concentrations and likely giving the Bcl-2 family called Bcl - xL.43 PV progenitor cells a growth advantage.39,40 The erythroid MPL mutations promote thrombopoietin independent maturation is morphologically normal and the erythrocytes activation of the MPL protein and the resulting throm- function normally and have a normal lifespan. bocytosis associated with ET and PV. Furthermore, the protein LNK functions to downregulate JAK/STAT signal- JAK2 ing following normal erythropoietin and thrombopoietin Several mutations, most affecting the Janus kinase–STAT sig- stimulation to return cell production to the steady state. nal transduction pathway, have been identified in patients Loss of LNK function results in continued erythroid and with PV. As is the case in ET, three mutations in particular thrombocytic production.49 Finally, increased expression drive the disease (JAK2, MPL, and CALR) and can occur of Bcl - xL leads to inhibition of apoptosis in progenitor in both sporatic and familial forms.22 Some patients pres- cells43 (Chapter 2). The defect in programmed cell death ent with none of these three mutations and are called triple creates an accumulation of altered, hypersensitive stem negative. The majority of triple-negative patients possess a and progenitor cells. Thrombopoietin receptor hyperre- somatic mutation in JAK2 and/or MPL.37 The JAK2(V617F) sponsiveness and resistance to apoptosis are also found in mutation is seen in almost all patients (more than 95%) with the megakaryocytic lineage, resulting in the common find- PV.40 In addition, about 5% of PV cases carry other muta- ing of thrombocytosis associated with PV.6,43 tions in exon 12 of JAK2 |
that disrupt normal JAK2 function. Since the JAK2 (V617F) is not found in all PV patients, it is EPIGENETICS hypothesized that JAK2(V617F) is not the sole transform- Epigenetic changes contribute to the malignant transfor- ing mutation, and many suspect it is not even the initiat- mation of PV. In addition to the TET2 mutation described ing event.41,42 This theory is supported by evidence that in earlier, somatic mutations in the gene for the isocitrate some patients with JAK2 mutated myeloproliferative neo- dehydrogenase (IDH) 1 enzyme that functions in the citric plasms that evolved into AML, the resulting myeloblasts acid cycle manifest epigenetic modifications. IDH1 nor- do not bear the JAK2 mutation.43 Therefore, one or more mally functions to convert isocitrate to a@ketoglutarate with mutations are likely to precede the JAK2(V617F) mutation the associated production of NADPH. Mutations have also and are necessary to predispose patients to developing the been identified in IDH2 in patients with MPNs. Epigenetic JAK2(V617F) mutation and the resulting MPN. analysis of IDH1/2 mutations in AML patients has demon- Two mutations that may precede the JAK2(V617F) strated hypermethylation. Mutations in these two enzymes mutation include TET2 (ten-eleven translocation) and a occur in PV, ET, and PMF at a rate of 1–5% and are much germline single nucleotide polymorphism (SNP) in the more frequent (26%) in the later stages of an MPN and after JAK2 locus (rs10974944).44 The TET2 protein functions as transformation to acute leukemia.33 It has been speculated a catalyst in the hydroxylation of 5-methylcytosine (5-mC), that mutational analysis of IDH1 and IDH2 might be useful converting 5-mC to 5-hydroxylmethylcytosine (5-hmC).45,46 as a marker for disease progression in patients with MPNs. It is thought that 5-hmC is an intermediate base in the demethylation of DNA and therefore serves as an epigen- GENES AND PRESENTATION OF PHENOTYPE etic regulator that participates in driving myeloid prolif- Because the JAK2(V617F) mutation is found in nearly all eration (Chapter 23). Mutations that delete or lead to loss patients with PV and in about half of the patients with ET of function in TET2 are thought to occur in the HSC and and PMF, it is unclear as to how one mutation can result contribute to myeloid rather than lymphoid neoplasms.47 in three different conditions. It is possible that a dosage Clonal analysis of immature progenitor cells in patients effect of the JAK2 mutation may play an important role in with JAK2 and TET2 mutations suggest that TET2 precedes determining disease phenotype since most patients with ET JAK2 with the exception of one report of familial MPN for are heterozygous for JAK2, whereas PV patients tend to be which the opposite is suspected.48 homozygous. Given the suspicion of a pre-JAK2 mutation in A germline polymorphism in the JAK2 gene (rs10974944) the MPNs and the discovery of mutated MPL, the following is found between exons 12 and 13. This polymorphism mutational sequence has been proposed50: increases the likelihood of developing the JAK2(V617F) • A pre-JAK2 mutation produces a hyperproliferative mutation and the resulting MPN by three- to four-fold.44 clone with increased susceptibility to additional muta- tions. Either the MPL or JAK2 mutation occurs, trigger- OTHER MUTATIONS ing the ET phenotype. The JAK2 mutation may produce In the absence of the JAK2(V617F) mutation, other mutations preferential triggering of the MPL receptor over the have been identified that disrupt the normal function of EPO receptor because MPL receptors have a higher the JAK/STAT pathway resulting in myeloid proliferation. density on the HSC surface. Myeloproliferative Neoplasms 537 • When a second JAK2 mutation occurs, sufficient JAK2 survival exceeds 10–20 years.37 Factors that adversely affect proteins exist to trigger the EPO receptors converting survival are age, abnormal karyotype, leukocytosis, and a the disease phenotype to PV. history of thrombosis.20 • Additional mutations evolve the phenotype to that of PMF, but thrombocytosis remains because of chemo- Laboratory Evaluation kines released in response to the fibrosis. PERIPHERAL BLOOD Clinical Presentation The most striking peripheral blood finding in PV is an abso- lute erythrocytosis in the range of 6910 * 106/mcL, with a Polycythemia vera has an estimated prevalence of hemoglobin concentration more than 18.5 g/dL in males 44957/100,000 people.20 Familial cases of ET have been and 16.5 g/dL in females. The hematocrit in females is usu- identified.20,31 It occurs most often between the ages of ally greater than 48% and in males is greater than 52%. The 40–60 years with a peak incidence in the sixth decade of total RCM is increased, greater than 25% of mean normal; life. The disease is rare in children. It occurs more frequently the plasma volume can be normal, elevated, or decreased. in males than females and is more common in whites than Early in the disease, the erythrocytes are normocytic, nor- blacks, particularly in those of Jewish descent.40 PV has been mochromic; however, after repeated therapeutic phlebot- reported to occur in several members of the same family, omy, iron-deficient erythropoiesis can result in microcytic suggesting a familial predisposition may exist. hypochromic cells. Patients with PV occasionally present The disease onset is usually gradual with a history of with iron deficiency secondary to occult blood loss resulting mild symptoms for several years. In some cases, PV is found from abnormal platelet function. This can create a confusing in asymptomatic individuals. When symptoms are present, peripheral blood picture because the concentration of eryth- they are typically related to the increased erythrocyte mass rocytes is normal to increased with significant microcytosis, and the associated cardiovascular disease because of the simulating a thalassemia (Chapter 14). Nucleated erythro- hyperviscosity of the blood. Headache, weakness, pruritus, cytes can be found. On the blood smear, the erythrocytes weight loss, and fatigue are the most common symptoms. typically appear crowded even at the feathered edge. The Pruritus is attributed to hyperhistaminemia that can be reticulocyte count is normal or slightly elevated. spontaneous or induced by hot showers or baths. Itching is Leukocytosis in the range of 129 20 * 103/mcL occurs generalized with absence of a rash. in about two-thirds of the cases because of an increase in About one-third of the patients experience throm- granulocyte production. Early in the disease, there can be botic or hemorrhagic episodes; however, the CALR muta- a relative granulocytosis and a relative lymphopenia with tion imparts a reduced thrombotic risk especially in ET a normal total leukocyte count. A shift to the left can be patients.20,37 Myocardial infarctions, retinal vein thrombo- found with the presence of myelocytes and metamyelo- sis, thrombophlebitis, and cerebral ischemia can occur at cytes, but finding promyelocytes, blasts, or excessive num- any stage of the disease and occasionally may be the first bers of immature myeloid cells is unusual. Relative and indication of the disease. absolute basophilia is common. The LAP score is usually When the hematocrit exceeds 60%, the blood viscos- higher than 100. ity increases steeply, decreasing blood flow and increasing Megakaryocytic hyperplasia in the bone marrow peripheral vascular resistance. These interactions produce accompanied by an increase in platelet production is a hypertension in about 50% of the patients with PV. Plethora consistent finding in PV. In some patients, the megakaryo- (a florid complexion resulting from an excessive amount of cytes proliferate without overexpression of the receptor blood), especially on the face but also on the hands, feet, and for thrombopoietin (those patients with a mutation in the ears, is a common finding on physical examination. MPL gene) and have decreased apoptosis as do the ery- After 2–10 years, bone marrow failure may develop, throid progenitor cells.40,43 The platelet count is greater accompanied by clinical findings that include an increase in than 400 * 103/mcL in 20% of PV patients and occasion- splenomegaly, anemia, and bleeding. Laboratory results are ally exceeds 1000 * 103/mcL. Giant platelets can be found likely to reveal a decreased platelet count and decreasing on the blood smear. Qualitative platelet abnormalities are hematocrit. Together, these findings are known as the spent reflected by abnormal aggregation to one or more aggregat- phase and often herald the transition to AML. Postpolycy- ing agents—epinephrine, collagen, adenosine diphosphate themic myelofibrosis develops in about 30% of PV cases (ADP), or thrombin (Chapters 33, 36). Lack of aggregation and splenomegaly is a characteristic finding at the spent with epinephrine is the most common abnormality. The PT phase. Acute leukemia develops as an abrupt transition in and APTT are usually normal (Chapters 34, 36). Abnormal 5–10% of patients. Leukemia appears to develop at a higher multimeric forms of von Willebrand factor (VWF) are found rate in patients treated with myelosuppressive drugs than in about half of PV patients and may lead to a diagnosis of in those treated with phlebotomy alone. Overall median acquired VWD40 (Chapter 34). 538 Chapter 24 Advanced disease is accompanied by striking morpho- resulting from tissue hypoxia, EPO levels are elevated, and logic changes in erythrocytes (Figure 24-12). The peripheral arterial oxygen saturation levels are decreased. When sec- blood picture may resemble that of myelofibrosis with a leu- ondary polycythemia occurs from an inappropriate increase koerythroblastic anemia, poikilocytosis with dacryocytes, in EPO, oxygen saturation levels are usually normal. and thrombocytopenia. In cases that advance to acute leuke- Other laboratory tests also can be abnormal. Serum mia, the blood picture exhibits anemia with marked eryth- uric acid is greater than 7 mg/dL in two-thirds of the rocyte abnormalities, thrombocytopenia, and blast cells. patients and can cause symptoms of gout. The increase probably reflects an increase in the turnover of nucleic BONE MARROW acids from the increased number of blood cells. Serum Most patients with PV have a moderate to marked increase cobalamin-binding capacity in most of the untreated PV in bone marrow cellularity. The hypercellularity is greater patients is increased, primarily because of the increase in than is seen in secondary polycythemia, and hematopoietic haptocorrins derived from granulocytes. Serum cobalamin marrow can extend into the long bones. Granulopoiesis as also is increased but not in proportion to the unsaturated well as erythropoiesis is often increased; consequently, the binding capacity. M:E ratio is usually normal. Although the relative number of myeloblasts is not increased, one of the most significant MOLECULAR GENETICS findings is an increase in megakaryocytes. Eosinophils Because of the high frequency of the JAK2(V617F) muta- are also often increased. Sometimes bone marrow biop- tion in PV, peripheral blood screening for JAK2(V617F) in sies reveal a slight to marked increase in fibrotic material the initial evaluation of patients suspected of having PV or reticulin, but it is generally directly proportional to the has been recommended. Cytogenetic abnormalities includ- degree of cellularity (e.g., more cellular marrows demon- ing chromosomal aneuploidy and partial deletions may strating more reticulin). Iron stores are usually absent, pre- be found. The most consistent abnormality is a trisomy 8 sumably because of a diversion of iron from storage sites to or 9, an abnormally long chromosome 1, and partial dele- the large numbers of developing erythroblasts. In the post- tions of chromosome 13 and 20. The frequency of multiple polycythemic stage, the bone marrow reveals reticulin and karyotypic abnormalities increases after years of treatment collagen fibrosis. Cellularity varies but is often hypocellular and occurs in more than 80% of patients who develop acute with prominent clusters of megakaryocytes. Erythropoiesis leukemia. Thus, progression from a normal to an abnormal and granulopoiesis decrease. A shift to the left can be pres- karyotype is an adverse prognostic indicator. ent, but blasts are usually less than 10% and dysplasia is unusual. Prognosis and Therapy OTHER LABORATORY EVALUATION There is no known cure for PV, but treatment usually pro- EPO levels are normal or low in PV, and arterial oxygen longs survival. Without treatment, 50% of the patients saturation levels are normal. In secondary polycythemia survive about 18 months. With only phlebotomy as the palliative treatment, survival extends to about 14 years. Thrombosis is the most frequent complication, and often patients are given antiplatelet therapy.37 PHLEBOTOMY Two types of therapy, phlebotomy and myelosuppressive therapy, have been used historically. Therapeutic phle- botomy is performed to keep the hematocrit below 45% in men and 42% in women and to intentionally reduce iron supplies. Lack of iron is expected to slow the production of erythrocytes, reducing erythrocytosis and the accompany- ing symptoms. CHEMOTHERAPY Myelosuppressive or cytoreductive therapy utilizing hydroxyurea, alkylators, interferon, radioactive phosphorus (32P), and anagrelide have been used to |
reduce the amount of proliferating hematopoietic cells and for patients at risk Figure 24.12 Peripheral blood from a patient with PV for vascular events.36,37 Hydroxyurea inhibits ribonucleo- showing thrombocytosis and large platelets. A reactive lymphocyte and segmented neutrophil are in the center. Poikilocytosis with target tide reductase and carries less risk of secondary leukemia cells, pencil cells, and acanthocytes is present (Wright-Giemsa stain, than does busulphan, an alkylating agent. Younger patients 600* original magnification). who are intolerant or resistant to hydroxyurea can be treated Myeloproliferative Neoplasms 539 with a@interferon; busulfan is used for older patients.37 blood oxygen saturation can also occur in severe obstructive Low-dose pegylated interferon a@2a has been shown to lung disease and in obesity. The hematocrit is generally not normalize blood counts in patients with PV and ET, and higher than 57% in these cases. on sustained therapy, a 30–40% chance that patients could Inappropriate Increase in Erythropoietin A nonphysio- develop a significant molecular response and a significant logic increase in EPO (inappropriate) has been described decrease in the JAK2(V617F) allele burden. Pegylated inter- in association with certain tumors that appear to secrete feron a@2a can retard the progression of ET and PV toward EPO or an EPO-like substance. About 50% of patients who PMF.51 Patients receiving 32P therapy have a mean survival experience this have renal tumors. Other tumors that have of 12 years, yet these patients show a progressive incidence been associated with erythrocytosis include those of the of malignant complications. liver, cerebellum, uterus, adrenals, ovaries, lung, and MOLECULAR TARGETS thymus. In almost all cases, EPO levels return to normal Research to find a molecularly targeted therapy specific and the erythrocytosis disappears after resection of the for the abnormal JAK2 kinase similar to the use of ima- tumor. Renal cysts are also associated with polycythe- tinib in CML is ongoing. The JAK2 inhibitor, ruxolitinib, mia, possibly because of localized pressure and hypoxia showed promising results in clinical trials. In PV patients, to the juxtaglomerular apparatus, resulting in increased 97% achieved phlebotomy independence and many EPO secretion. In some patients with hypertension, renal showed complete remission.52 CEP-701 (lestauritinib, a artery disease, and renal transplants, renal ischemia can JAK inhibitor) therapy also revealed a decreased need occur, resulting in erythrocytosis secondary to increased for phlebotomy and a reduction in JAK2(V617F) allele EPO production. burden.53 Familial Polycythemia Inherited hemoglobin variants with increased oxygen affinity cause tissue hypoxia and are Differential Diagnosis associated with a secondary erythrocytosis. Because of the It is essential that PV be differentiated from the more benign increased oxygen affinity, less oxygen is released to the tis- causes of secondary erythrocytosis and relative polycythe- sues, stimulating erythropoietin production. Inherited defi- mia so that effective therapy can be initiated. ciency of 2,3-BPG also results in decreased oxygen release to tissues. These inherited conditions are usually found in SECONDARY POLYCYTHEMIA young children and in other family members as well. Secondary polycythemia can be classified into the follow- ing groups: Neonatal Polycythemia In neonates, hematocrits of 48% are common (Table A in Appendix). The etiology is attributed 1. Polycythemia caused by an increase in EPO as a normal to placental transfusion that occurs as a result of late cord physiologic response to tissue hypoxia clamping (7–10 seconds after delivery) and/or increased 2. Polycythemia caused by an inappropriate, nonphysi- erythropoiesis stimulated by intrauterine hypoxia.40 ologic increase in erythropoietin production 3. Familial polycythemia associated with high oxygen- Checkpoint 24.7 affinity hemoglobin variants Renal tumors can produce an inappropriate amount of EPO, 4. Neonatal polycythemia associated with intrauterine resulting in what type of polycythemia? hypoxia or late cord clamping Tissue Hypoxia A decreased arterial oxygen saturation Relative Polycythemia and subsequent tissue hypoxia are the most common cause Relative polycythemia is a mild polycythemia resulting from of secondary polycythemia. The polycythemia disappears dehydration, hemoconcentration, or a condition known as when the underlying cause is identified and effectively Gaisböck’s syndrome, which has several synonyms, includ- treated. Residents of high-altitude areas demonstrate a ing spurious polycythemia, pseudopolycythemia, and stress significant increase in hemoglobin and hematocrit that is erythrocytosis. Patients with these conditions have a rela- progressively elevated at high altitudes. The decrease in tive polycythemia and hypertension with nephropathy or barometric pressure at high altitudes decreases the inspired relative polycythemia associated with emotional stress.40 oxygen tension. As a result, less oxygen enters the eryth- RCM is essentially normal. High hematocrit and hemo- rocytes in the pulmonary alveoli, and the arterial blood globin concentrations appear to result from a combination oxygen saturation decreases (Chapters 6, 11). Chronic of high-normal erythrocyte concentrations with a low- hyperventilation partially compensates for the reduced pO2 normal plasma volume. The most common symptoms in the lungs. Compensation at the cellular level involves an are light-headedness, headaches, and dizziness. Plethora increase in 2,3-BPG, facilitating the transfer of oxygen to the is common but splenomegaly is rare. These patients tissues. Tissue hypoxia secondary to a decrease in arterial have a high incidence of thromboembolic complications 540 Chapter 24 Table 24.17 Polycythemia Vera Diagnostic Criteria as Table 24.18 Differential Features of Polycythemia Defined by WHO2 Feature PV Secondary Relative Major criteria 1. Hb greater than 16.5 g/dL in men or 16.0 g/dL in women OR Spleen size c N N Hct greater than 49% in men and 48% in RCM Greater than or c N women OR equal to 36 mL/kg Increased RBC mass greater than 25% (males) mean normal Greater than or 2. Bone marrow showing hypercellularity for age equal to 32 mL/kg with trilineage growth (panmyelosis) i ncluding (females) prominent erythroid, granulocytic, and Leukocyte count c N N megakaryocytic proliferation with pleomorphic, mature megakaryocytes (difference is size) Platelet count c N N 3. Presence of JAK2(V617F) or JAK2 exon Serum cobalamin c N N 12 mutation Arterial O2 saturation N T N Minor criterion 1. Subnormal serum erythropoietin level Bone marrow Panhyperplasia, Erythroid N reticulin deposits hyperplasia LAP N to c N N and cardiovascular disease. Although the hemoglobin, Iron stores T N N hematocrit, and e rythrocyte counts are increased, leuko- cytes and platelets are normal. Bone marrow cellularity is EPO N, T N, c N normal with no increase in megakaryocytes or reticulin. Chromosome Abnormal, greater N N Bone marrow iron stores are absent in 50% of the patients, studies than 90% JAK2 (V617F)+ but serum iron studies are normal. Chromosome karyo- types are almost always normal. N, normal; c , increased; T , decreased; LAP, leukocyte alkaline phosphatase; EPO, erythropoietin. LABORATORY DIFFERENTIATION OF POLYCYTHEMIA The WHO-defined diagnostic criteria for PV include ini- tial determination of total RCM or hemoglobin to establish Genetic Studies Studies should include molecular analysis the presence of an absolute polycythemia37 (Table 24-17). for the JAK2(V617F) mutation found in exon 14. In addi- However, in some cases with hemoglobin levels below the tion, 10 different mutations have been identified in exon 12 value used for a diagnosis of PV and thrombocytosis, the of JAK2 that are associated with erythrocytosis. Karyotype RCM may be higher than required, thus revealing an occult screening for trisomies of chromosome 8 and 9 as well as erythrocytosis.54 Without the RCM, these cases would have deletions of 13 or 20 may also be performed.40 been diagnosed as essential thrombocythemia. This sug- gests that RCM is an important test to perform when PV Bone Marrow Changes A bone marrow assessment is suspected. Determination of serum EPO levels is impor- can be helpful in patients in whom EPO is not low and tant because it helps to distinguish between primary and JAK2(V617F) is not detected, but an elevated RCM and secondary polycythemia. If EPO levels are low, screening clinical symptoms suggest PV. Histologic changes for PV for the JAK2(V617F) mutation and bone marrow histology include hypercellularity with increased erythroid precur- should be performed.40 An elevated EPO indicates second- sors, increased granulocytic and megakaryocytic cells with ary polycythemia. megakaryocyte clusters, and reticulin fibrosis.40 In any classification scheme, recognizing the possibility of two coexisting disease states is imperative. For instance, Checkpoint 24.8 a patient can have both PV and a secondary polycythemia Which of these conditions—iron deficiency, smoking, emphy- as occurs in chronic obstructive pulmonary disease. Refer sema, pregnancy, dehydration—are associated with an abso- to Table 24-18 for differentiating features of polycythemia lute increase in RCM? vera from secondary and relative polycythemia. Erythropoietin Measurement EPO is critical in differ- entiating PV from secondary polycythemias. With the JAK2(V617F) mutation, erythropoiesis occurs without the Primary Myelofibrosis (PMF) need for EPO stimulation. Serum EPO levels are usually Primary myelofibrosis (PMF) is a clonal hematopoietic very low or not detectable in PV. Secondary causes of poly- stem cell disorder with splenomegaly, leukoerythroblastosis, cythemia are related to elevated EPO levels either because extramedullary hematopoiesis (myeloid metaplasia), and of hypoxia or an inappropriate release of EPO from the kid- progressive bone marrow fibrosis.55,56 It is the least common neys (tumors and renal carcinomas). but most aggressive of the myeloproliferative neoplasms. Myeloproliferative Neoplasms 541 The proliferation of hematopoietic cells in the early stages Most PMF patients have a somatic mutation in one of of the disease is neoplastic and presents either as a de novo three driver mutations that affect the JAK-STAT pathway condition or as an evolutionary consequence of PV or ET. (JAK2, MPL, CALR).51 A small subset of PMF patients do not The fibroblast (collagen-producing cell) is an important express any of the three driver mutations, termed triple nega- component of normal bone marrow in which the fibro- tive, and are associated with a worse prognosis. Mutations blasts provide a support structure for hematopoietic cells. in ten-eleven-translocation-2 (TET2), additional sex comb Fibroblast proliferation in PMF is reactive and secondary to like 1 (ASXL1), DNA methyltransferase 3A (DNMT3A), and the underlying disorder. Fibrotic tissue eventually disrupts enhancer of Zeste homolog 2 (EZH2) have also been identified the normal architecture and replaces hematopoietic tissue in patients with PMF, but these mutations affect epigenetic reg- in the bone marrow. Excessive marrow fibrosis inhibits ulation rather than the JAK-STAT pathway.33 These mutations normal hematopoiesis, producing marrow hypoplasia and constitutively activate cell signaling and DNA replication. stimulates myeloid metaplasia. Myeloid metaplasia usually CYTOKINE INVOLVEMENT occurs in both the spleen and the liver. These organs can Megakaryocytes play an important role in the pathogenic become massive in size as the result of islands of proliferat- development of the abnormal PMF marrow. In areas of ing erythroid, myeloid, and megakaryocytic elements. The megakaryocyte necrosis, fibroblast proliferation and colla- extramedullary hematopoiesis is similar to that occurring gen deposition often are prominent. This stromal reaction during embryonic hematopoiesis. is a cytokine-mediated process.57 PDGF, epidermal growth PMF has been known by many synonyms. Most of the factor (EGF), and transforming growth factor beta (TGF@b) different names were attempts to describe the typical blood, are contained in the a@granules of megakaryocytes and bone marrow, and spleen abnormalities. Some of the terms platelets, and all stimulate the growth and proliferation of that have been used include agnogenic myeloid metaplasia, fibroblasts. Reduced platelet concentrations of PDGF and myelofibrosis with myeloid metaplasia, chronic idiopathic myelo- increased levels of serum PDGF are characteristic of PMF. fibrosis, aleukemic myelosis, myelosclerosis, splenomegalic myelo- This condition is thought to represent the abnormal release phthisis, and leukoerythroblastic anemia. or leakage of the growth factor from the platelet. PDGF does not stimulate synthesis of collagen, laminin, or fibro- Etiology and Pathophysiology nectin, but TGF@b stimulates increased expression of genes for fibronectin and collagen while it decreases synthesis of PMF is associated with a profound hyperplasia of mor- collagenase-like enzymes. Thus, the net effect is the accu- phologically abnormal megakaryocytes (dysplastic and mulation of bone marrow stromal elements.57 necrotic). In most cases, only megakaryocytes and granulo- cytes are involved, but all three lineages, including erythro- GENE MUTATIONS cytes, can be involved in the disease process. PMF is often About 50% of patients can have a somatic mutation in preceded by a hypercellular phase of variable duration. The either the JAK2 or MPL genes in a hematopoietic stem cell. disease evolves from this prefibrotic stage with minimal JAK2(V617F) allele homozygosity is identified more often reticulin fibrosis to a fibrotic stage with marked reticulin in PV and PMF than in ET. The JAK2(V617F) allele burden or collagen fibrosis. Thus, at diagnosis, the bone marrow has been associated with disease progression.58,59,60,61 On the can exhibit varying degrees of fibrosis. The fibrosis is |
not other hand, PMF patients negative for JAK2(V617F) and with considered part of the primary abnormal clonal prolifera- lower JAK2(V617F) burden experience shorter survival. This tion but is a secondary reactive event occurring in response may indicate that there is another clone as yet not identified to the progeny of the clonal hematopoietic cells and, in fact, that overrides the V617F-negative clone and results in a more fibroblasts do not contain the chromosome abnormalities aggressive disease phenotype.59 The proto-oncogene CBL is found in the hematopoietic cells. mutated primarily in juvenile myelomomocytic leukemia Understanding of this disease has increased consid- (JMML) or chronic myelomonocytic leukemia (CMML) and erably with a better understanding of normal bone mar- has been identified in 6% of patients with PMF.62 Chromosome row structure and the changes that occur in myelofibrotic aberrations, when present, are restricted to cells derived from marrow associated with megakaryocyte growth factors the mutated HSCs. Some of these cells are highly sensitive to that mediate fibrogenesis.57 The bone marrow extracellular or independent from regulation by their respective stimula- matrix or microenvironment (stroma) supports hematopoi- tory factors. The disorder has been reported to terminate in etic cell proliferation (Chapter 3). Myelofibrotic stroma is ALL as well as AML in some patients. characterized by an increase in total collagen, fibroblasts, vitronectin (a cytoadhesion molecule), fibronectin (a cytoad- Checkpoint 24.9 hesion molecule normally limited to megakaryocytes and What growth factors are primarily responsible for stimulating walls of blood vessels), and laminin (a glycoprotein that fibrogenesis in the bone marrow? supports adhesion and growth of cells). 542 Chapter 24 Clinical Presentation and the effectiveness of extramedullary hematopoiesis. The anemia becomes more severe with the progression PMF generally affects individuals older than 50 years of the disease and is aggravated in some patients by the of age and seems to occur equally between sexes. It combination of splenomegaly, which causes sequestration rarely occurs in childhood. Its onset is gradual, and of erythrocytes, and expanded plasma volume (dilutional the disease is chronic. Early in the disease process, the anemia). patient might not have symptoms, making the time of The presence of abnormal erythrocyte morphol- onset difficult to determine. If symptoms are present, ogy is an important feature of PMF. The most typical they are usually related to anemia or pressure from an poikilocyte is the dacryocyte, although elliptocytes and enlarged spleen. Bleeding occasionally is a present- ovalocytes are also present (Figure 24-13). A few nucle- ing symptom. Patients complain of weakness, weight ated erythrocytes are usually found and sometimes can loss, loss of appetite, night sweats, pruritus, pain in be numerous. B asophilic stippling is a common finding. the extremities, bone pain, discomfort in the upper left Reticulocytosis is typical, ranging from 2 to 15%. The quadrant, and fever. The major physical findings are majority of patients have an absolute reticulocyte more splenomegaly (in 90%), h epatomegaly (in 50%), pallor, than 60 * 103/mcL. and petechiae.63,64 Myeloid metaplasia is found in the The leukocyte count is usually elevated but can be spleen and frequently in the liver and can be found in normal or, less often, decreased upon initial presentation. the kidney, adrenal glands, p eritoneal and extraperi- The count generally ranges from 15 to 30 * 103/mcL. toneal s urfaces, skin, lymph nodes, and spinal cord. A leukocyte count above 60 * 103/mcL prior to splenec- Osteosclerosis is a frequent finding and, when found in tomy is rare. As the disease progresses, the leukocyte association with splenomegaly, suggests a diagnosis of concentration does not decrease as quickly as the erythro- myelofibrosis. cytes and platelets. An orderly progression of immature An atypical acute form of the disease has been described granulocytes is characteristically found. Blasts generally with a rapid and progressive course of a few months to 1 compose less than 5% of circulating leukocytes. Other year. Anemia develops rapidly, and the leukocyte count is common findings include basophilia, eosinophilia, and decreased. The bone marrow in these cases exhibits a pro- pseudo–Pelger-Huët anomaly. The LAP is elevated or liferation of reticular and collagen fibers. normal but occasionally is decreased. Low LAP scores Patients with systemic lupus erythrematosus (SLE) correlate with leukopenia. When elevated, the LAP score can present with myelofibrosis that is morphologically helps to differentiate this disease from CML. The Ph chro- indistinguishable from the myelofibrosis of PMF. These mosome is not present. patients also have various peripheral blood cytopenias Platelets can be decreased, normal, or increased. similar to those found in PMF but not splenomegaly. The Higher counts are associated with early disease stages; myelofibrosis in SLE has been referred to as autoimmune thrombocytopenia is usually found in the later stages. myelofibrosis. Patients with myelofibrosis and an absence of splenomegaly should have an antinuclear antibody (ANA) test to rule out SLE.65 Laboratory Evaluation PERIPHERAL BLOOD The typical peripheral blood findings for PMF reflect both qualitative and quantitative cellular abnormali- ties. At diagnosis, there may be anemia, leukocytosis with a left shift, and thrombocytosis. Pancytopenia and leukoerythroblastosis with striking anisocytosis and poikilocytosis is typical at later stages of the disease (Figure 24-13). The anemia is usually normocytic, normo- chromic, but hypochromia may be found if the individual has a history of hemorrhage or hemolysis. Between 35 and 54% of patients with PMF have a hemoglobin level of less than 10 g/dL.56 Folic acid deficiency can develop as a result of increased utilization by the neoplastic clone and Figure 24.13 Peripheral blood from a patient with PMF. Leukoerythroblastic picture with an erythroblast and a myeloblast is associated with a macrocytic anemia. Anemia uncom- below it. The arrow points to a large platelet. Other abnormal plicated by iron deficiency or folic acid deficiency cor- platelets are present. Note the poikilocytosis with dacryocytes relates directly with the extent of bone marrow fibrosis (Wright-Giemsa stain, 1000* magnification). Myeloproliferative Neoplasms 543 Thrombocytopenia is often attributed to excessive splenic pooling. The platelets can appear dysplastic: typically CASE STUDY (continued from page 527) giant, bizarre, and frequently hypogranular. Circulating During the office visit, Roger stated his symptoms of megakaryocyte fragments, mononuclear micromega- fatigue, weakness, dyspnea, bone pain, and abdomi- karyocytes, and naked megakaryocyte nuclei can exist. nal discomfort. A bone marrow biopsy was ordered. The micromegakaryocytes can present an identification The aspiration was unsuccessful, but the marrow problem because they frequently resemble lympho- biopsy showed moderate to marked hyperplasia, cytes. However, important differentiating features of clusters of platelets, abnormal megakaryocyte mor- micromegakaryocytes are the presence of demarcation phology, and fibrotic marrow spaces. membranes with bull’s-eye granules in the cytoplasm and 4. What diagnoses do these results suggest? cytoplasmic blebbing. Qualitative platelet abnormalities including abnormal aggregation and adhesiveness, and 5. Give a reason for the unsuccessful, dry-tap bone defective platelet procoagulant activity on exposure to marrow aspiration. collagen are consistent findings. 6. What characteristic peripheral blood morpholo- Among patients with PMF, 15% have major hemolytic gies correlate with the bone marrow picture and episodes during the course of their disease.56 Hemosiderin- physical exam? uria and decreased haptoglobin are found in about 10% of patients, suggesting intravascular hemolysis. The cause of hemolysis can be hypersplenism, PNH-like defective eryth- rocytes, and antierythrocyte antibodies. A bleeding diathesis ranging from petechiae and ecchy- moses to life-threatening hemorrhage can be found in some patients, likely resulting from a combination of throm- bocytopenia and/or abnormal platelet function. Hemo- static abnormalities suggestive of chronic DIC, including decreased platelet count, decreased concentration of factors V and VIII, and increased fibrin degradation products, can be present.56 Other laboratory tests for PMF are frequently abnor- mal. Serum uric acid and LD are elevated in most patients. Serum cobalamin can be slightly increased but is usually normal. Checkpoint 24.10 What erythrocyte morphologic feature is a hallmark for Figure 24.14 Bone marrow stained with reticulin stain shows myelofibrosis? increased collagen (black-staining fibers) in a patient with PMF (BM biopsy, reticulin stain, 100* magnification). BONE MARROW The bone marrow is difficult to penetrate and frequently MOLECULAR GENETICS yields a dry tap. If aspiration is successful, smears may No specific cytogenetic abnormality is diagnostic for PMF. show no abnormalities; biopsy specimens are needed However, cytogenetic analysis is important to differentiate to reveal the extent of fibrosis. In most PMF cases, myelofibrosis from other myeloproliferative disorders, par- the marrow is hypercellular with varying degrees of ticularly CML, which also can have some degree of fibrosis. diffuse fibrosis and focal aggregates of megakaryocytes The Ph chromosome is not present in PMF. Trisomy or dele- (Figure 24-14). tion of group C chromosomes (chromosomes 6–12) is also Three bone marrow histologic patterns have associated with myelofibrosis. Complete or partial loss of been described: (1) panhyperplasia with absence of chromosomes 5, 7, and 20 is associated with PMF patients myelofibrosis but a slight increase in connective tissue treated with chemotherapy. reticulin, (2) myeloid atrophy with fibrosis, prominent Most patients with PMF have mutations in one of the collagen and reticulin fibers, and cellularity less than three driver genes, (JAK2, MPL, and CALR).51 JAK2(V617F) 30% and (3) myelofibrosis and myelosclerosis with bony is the most common of the three driver mutations fol- trabeculae occupying 30% of the biopsy and extensive lowed by CALR, then MPL. In addition to the driver muta- fibrosis. tions, most PMF patients bear mutations in genes affecting 544 Chapter 24 Table 24.19 WHO Diagnostic Criteria for Overt PMF2 Major criteria* 1. Megakaryocytic proliferation and atypia, accompanied by either reticulin and/or collagen fibrosis grades 2 or 3 2. Not meeting the WHO criteria for BCR/ABL1 + CML, PV, ET, MDS, or other myeloid neoplasms 3. Presence of JAK2, CALR, or MPL mutation or in the absence of these mutations, presence of another clonal marker, or absence of minor reactive BM myelofibrosis Minor criteria 1. Presence of at least one of the following, confirmed in two consecutive determinations a. Anemia not attributed to a comorbid condition b. Leukocytosis greater than 11 * 103/mcL c. Palpable splenomegaly d. LDH increased to above upper limit of institution’s reference range e. Leukoerythroblastosis * Diagnosis of prePMF requires meeting all three major criteria and at least one minor criteria. epigenetic modifications like TET2, ASXL1, DNMT3A, and Differential Diagnosis EZH2.51 The diagnostic algorithm for PMF was revised by WHO in 2016 (Table 24-19). Differentiating PMF from other conditions associated with fibrosis is essential to ensure appropriate therapeutic Prognosis and Therapy regimens (Table 24-20). Splenomegaly, anemia, and a leu- koerythroblastic blood picture are significant findings in The average survival time after diagnosis is 4–5 years. The both myelofibrosis and CML. In myelofibrosis, the leuko- main causes of death are infection, hemorrhage, thrombosis, cyte count is generally less than 50 * 103/mcL, whereas in and cardiac failure. About 10–15% of patients progress to an CML, the count is expected to be higher. In myelofibrosis, acute myeloid leukemia and some to acute lymphoblastic the granulocyte left shift is less pronounced and poikilo- leukemia. cytosis is striking. The bone marrow in myelofibrosis is Therapy is selected to achieve two particular outcomes: fibrous with large numbers of megakaryocytes. In CML, the improve cytopenia and reduce massive splenomegaly. Cor- bone marrow can also exhibit some fibrosis, but the most ticosteroids, androgens, and recombinant erythropoietin abnormal finding is the myeloid hyperplasia. The serum are used to stimulate erythropoiesis to reduce anemia, but cobalamin level is not as elevated in myelofibrosis as it is in patients may still require periodic transfusions.56 When ane- CML. The LAP score in myelofibrosis is variable, but when mia cannot be controlled, splenectomy can be considered. elevated, it is strong evidence against CML. The most reli- Irradiation has been suggested to decrease spleen size able test to differentiate CML and PMF is cytogenetic analy- in an attempt to relieve symptoms or to decrease excessive sis for the Ph chromosome. erythrocyte destruction. Hydroxyurea has been used, but it Differentiating PMF from polycythemia vera, espe- can cause severe pancytopenia. Thalidomide and lenalido- cially in the later stages, is more difficult. The later stages mide also have been used to treat splenomegaly as well as of PV can be accompanied by increased marrow fibrosis chemotherapeutic agents such as 2-chlorodeoxyadenosine and actual transformation to PMF can occur. When throm- (2-CdA or Leustatin) when the splenomegaly is advanced.66 bocytosis is the principal initial hematologic finding, PMF Neuropathy has been reported with thalidomide, and can be confused with ET. A bone marrow biopsy aids in myelosuppression |
can occur with lenalidomide. A similar the differentiation, revealing fibrosis in PMF. agent, pomalidomide, has produced anemia and symptom relief without neuropathy and myelosuppression.67 Alloge- neic stem cell transplantation is the only curative therapy for PMF, but the advanced age of many patients is a factor Table 24.20 Conditions Associated with Marrow Fibrosis in the high mortality rate. Neoplasms Other JAK inhibitors are being investigated with varying Primary myelofibrosis Miliary tuberculosis success. Ruxolitinib, a JAK1/2 inhibitor that when admin- Chronic myeloid leukemia Fungal infection istered to patients with advanced PMF at maximum toler- Polycythemia vera Granulomas ated doses daily, resulted in clinical improvement for some Essential thrombocythemia Marrow damage by radiation or patients.68 However, ruxolitinib did not impact JAK2(V617F) Megakaryocytic leukemia chemicals allele burden. CYT387, another JAK1/2 inhibitor, is associ- Metastatic carcinoma ated with splenic reduction and some degree of symptom Hairy cell leukemia Lymphoma relief.36 Clinical trials with drugs that are JAK2/FLT3 inhibi- Hodgkin’s disease tors have variable clinical effects.69 Acute megakaryocytic leukemia Myeloproliferative Neoplasms 545 the action of eosinophilic cytokines, GM-CSF, IL-3, and CASE STUDY (continued from page 543) IL-5. IL-5 is relatively lineage specific for eosinophils Over the next several months, Roger experienced (Chapters 4, 7). The cells are released into the peripheral increasing splenomegaly and abdominal discom- blood and rapidly migrate to tissues where they per- fort. Cytogenetic studies revealed a trisomy 8. form their p hysiologic function. Diagnosis of disorders associated with eosinophilia may be urgent because of 7. What is the most likely explanation for the potential damage to organs infiltrated with eosinophils increased splenomegaly? and subsequent release of cytokines, enzymes, and other 8. What are possible outcomes of this disorder? proteins. The term hypereosinophilic syndrome (HES) describes a group of disorders that demonstrate an abso- lute eosinophil count of greater than 1.5 * 103/mcL that persists for more than 4 weeks.70 The eosinophilia can result Myeloproliferative from a clonal (neoplastic) disorder or a nonclonal (benign or reactive) disorder or can be of unknown origin (idiopathic- Neoplasm, Unclassifiable hypereosinophilic syndrome, I-HES) (Table 24-21). Reactive, nonclonal (benign) hypereosinophilic dis- (MPN, U) orders result from increased production of eosinophil cytokines secondary to or associated with another diag- Myeloproliferative neoplasm, unclassified (MPN, U) is the nosis (Chapters 7, 21). Diseases typically associated with diagnosis for cases that have the characteristic clinical, lab- benign eosinophilia must be ruled out before considering oratory, and morphologic features of an MPN but do not meet the specific criteria for one of the other MPN catego- ries or have features that overlap two or more categories. The Ph chromosome and BCR/ABL1 fusion gene are absent, Table 24.21 Conditions of Hypereosinophilia and the cell of origin is most likely the HSC. Conditions Examples Most cases of MPN, U are either very early stages of Neoplastic Myeloproliferative neoplasms PV, PMF, or ET or are at a late stage of advanced MPN in conditions (clonal) • Chronic eosinophilic leukemia, not otherwise which extensive myelofibrosis, osteosclerosis, or transfor- specified (CEL-NOS) mation to an aggressive stage obscures the true disorder. • Chronic myeloid leukemia (CML) Follow-up at intervals can permit an accurate diagnosis. • Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) A specific diagnosis should be made as soon as possible • Chronic myelomonocytic leukemia (CMML) because of the therapeutic implications for each of the neo- • Myelodysplastic syndromes plastic disorders. • Systemic mastocytosis (SM) The incidence of MPN, U is unknown but can be • Myeloid and lymphoid neoplasms associated as high as 20% of MPNs. Clinical features are similar to with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 those found in other MPNs, including splenomegaly and • Myeloid and lymphoid neoplasms associated hepatomegaly. with PDGFRA rearrangement Leukocytes and platelets can be increased. Hemo- • Myeloid neoplasms associated with PDGFRB globin is variable. The bone marrow is hypercellular and rearrangement • Myeloid and lymphoid neoplasms associated often shows megakaryocytic hyperplasia and variable with FGFR1 abnormalities granulocytic and erythroid proliferation. In advanced Acute leukemias (AML or ALL) stages, the bone marrow is fibrotic or osteomyeloscle- Benign, reactive Parasitic infection rotic. If there are 10–19% blasts in the peripheral blood, conditions the disease is diagnosed as an accelerated stage. No spe- (nonclonal) cific cytogenetic or molecular abnormalities are identified Asthma with this disorder. Allergies Skin diseases Loeffler’s syndrome Clonal Hypereosinophilia Vasculitis Drug hypersensitivity Eosinophils are derived from the colony-forming units Idiopathic- generating granulocytes, erythroblasts, macrophages, hypereosinophilic and megakaryocytes (CFU-GEMM and CMP) through syndrome 546 Chapter 24 factor receptor 1 (FGFR1). These gene mutations result in Table 24.22 Suggested Information and Testing to Exclude constitutive activation of intrinsic tyrosine kinase activ- Diseases Associated with Reactive (secondary) Eosinophilia ity (Chapter 4). All three disorders can present as a chronic Clinical Patient history myeloproliferative disorder. Less frequently, they can Physical examination present as AML or precursor T lymphoblastic leukemia/ Laboratory tests Complete blood count and differential lymphoma. Recognition of these disorders is important Bone marrow aspiration and biopsy because the mutations result in abnormal tyrosine kinases Routine chemistries that may respond to treatment with imatinib or other TKIs. Serum IgE Eosinophilia is variable but present in most cases. Cobalamin MYELOID AND LYMPHOID NEOPLASMS WITH PDGFRA HIV serology REARRANGEMENT Serology testing and stool analysis for parasites A gain of function tyrosine kinase fusion gene was discov- Other tests Pulmonary function tests ered when some HES patients who were unresponsive to Chest and abdominal CT scan corticosteroid therapy were found to respond to imatinib therapy. The gene fusion was found to occur between the FIP1L1 gene and the PDGFRA gene. The mutation results a neoplastic diagnosis (Table 24-21). See Table 24-22 from a small interstitial deletion in chromosome 4, del(4) for tests to exclude diseases associated with reactive (q12q12), resulting in the FIP1L1/PDGFRA (F/P) gene70 and (secondary) eosinophilia.71 It is especially important an abnormal, constitutively activated tyrosine kinase pro- to perform serological testing to rule out infection with tein. The cell of origin is the HSC. The detection of a fusion Strongyloides sp. because patients with this infection who gene in a particular lineage does not always correlate with are given corticosteroids (common treatment for I-HES) the morphological presentation. can experience dissemination of the disease, which can be fatal.72 Once it is found that the hypereosinophilic condi- Clinical Presentation Most of the cases involving the F/P tion is not clonal, testing to identify reactive conditions mutation are found in males (17:1 male-to-female ratio), and such as allergies, parasitic infections, and asthma, must age at onset is 25–55 years.70 There is evidence of eosinophil- be performed. In some cases, no underlying cause for the related tissue damage and tissue fibrosis. Splenomegaly is eosinophilia can be found and clonality cannot be proven. present. This disorder is known as I-HES (Table 24-21). Many dis- Laboratory Evaluation The initial laboratory finding is orders that were previously considered as I-HES are now usually hypereosinophilia but may be AML or T lympho- included in subgroups with specific genetic abnormalities. blastic leukemia/lymphoma. Eosinophils are mature with Clonal hypereosinophilia is considered a progenitor few myelocytes or promyelocytes. Eosinophil granulation cell disorder (primary) that includes a new myeloid/lym- may be sparse with vacuoles present. Hyper- or hyposeg- phoid neoplasm group added by the WHO in 2008 termed mentation of the nucleus can be seen. A few patients have myeloid and lymphoid neoplasms with eosinophilia and an increase in blasts, but the peripheral blood and/or bone abnormalities of platelet-derived growth factor receptor a marrow contain less than 20% blasts. Anemia and throm- (PDGFRA), PDGFR b (PDGFRB), or fibroblast growth fac- bocytopenia may be present. Increased serum tryptase tor receptor 1 (FGFR1)70 and chronic eosinophilic leukemia, and cobalamin are typical. The bone marrow is hypercel- not otherwise specified (CEL-NOS), a subgroup of MPNs lular with increased eosinophil precursors. Charcot-Ley- (Table 24-21). The eosinophil may also be a part of the neo- den crystals may be seen. Many cases show an increase in plastic clone of cells found in other neoplasms such as MPNs, spindle-shaped atypical mast cells with a CD25+ immuno- MDS, AML, and ALL. Cytogenetics or molecular analysis phenotype. These findings also are present in a significant can help clarify the origin of eosinophils in these neoplasms. number of patients who have systemic mastocytosis with increased atypical (spindle-shaped) mast cells in the bone Myeloid and Lymphoid Neoplasms marrow and a mutation in the stem cell factor receptor c-Kit. Associated with Eosinophilia and The F/P gene can be demonstrated by RT-PCR or FISH tech- niques (Chapters 41, 42). PDGFRA, PDGFRB, or FGFR1 Mutations Therapy Although most patients who harbor the F/P mutation respond to imatinib therapy, variations within These are a group of neoplastic eosinophil disorders caused the F/P mutation have recently been identified as resistant by mutations in genes that encode the a@ or b@chains of the to imatinib. The second generation nilotinib and the third protein tyrosine kinases platelet-derived growth factor generation ponatinib TKIs have shown promising results in receptors (PDGFRA, PDGFRB) or in the fibroblast growth imatinib-resistant patients.73 Myeloproliferative Neoplasms 547 MYELOID NEOPLASMS WITH PDGFRB that incorporate part of the FGFR1 gene. All fusion genes REARRANGEMENT encode a tyrosine kinase that has constitutive activity. Other The myeloid neoplasms with PDGFRB rearrangement are cytogenetic abnormalities can occur. The postulated cell of characterized by a t(5;12) (q33;p13) rearrangement (most origin is the HSC. commonly) to form the ETV6-PDGFRB fusion gene. Other Clinical Presentation There is a male predominance with translocations with a 5q31–33 breakpoint can occur and lead the myeloid and lymphoid neoplasms in the FGFR1 abnor- to other fusion genes, but they are not included in this sub- malities subgroup (1.5:1), and a wide age range at onset group and are not likely to respond to imatinib therapy. The (3–84 years) is observed. postulated cell of origin is a multipotential HSC that is able to differentiate to neutrophils, monocytes, eosinophils, and Laboratory Evaluation This neoplasm presents as a probably mast cells. hypereosinophilia, or if transformed to an acute neo- plasm, as an AML or precursor T or B lymphoblastic Clinical Presentation Myeloid neoplasms with PDGFRB leukemia/lymphoma or as a mixed phenotype acute rearrangement can present as CMML with eosinophilia, leukemia. In patients who are diagnosed in acute trans- aCML, or as MPN with eosinophilia. Acute (blast) transfor- formation, the myeloblasts, lymphoblasts, and eosino- mation can occur. There is a 2:1 male predominance and a phils belong to the neoplastic clone. The chronic phase wide variability in age (8–72 years) at onset. usually has eosinophilia, neutrophilia, and occasionally Laboratory Evaluation The leukocytes are increased with monocytosis. a variable increase in eosinophils, neutrophils, monocytes, Therapy Prognosis is poor. Tyrosine kinase inhibitors are and precursor cells. Anemia and thrombocytopenia may be not currently effective although second- and third-line TKIs present. The bone marrow is hypercellular with an increase are showing promising results.73 Bone marrow transplant in mast cells that can be spindle shaped. Reticulin can be can be indicated in the chronic phase. increased. The peripheral blood and bone marrow have less than 20% blasts. Molecular analysis using primers for all known breakpoints to confirm ETV6-PDGFRB is recom- Chronic Eosinophilic Leukemia, Not mended. Cytogenetic analysis usually reveals the t(5;12). Otherwise Specified (CEL-NOS) Therapy Before imatinib therapy, survival with myeloid CEL-NOS is a clonal myeloproliferative neoplasm that neoplasms with PDGFRB rearrangement was less than 2 presents with eosinophilia not classified as another years. Patients usually respond to imatinib treatment.73 neoplastic condition and does not have the PDGFRA, Survival is expected to improve with early diagnosis and PDGFRB, or FGFR1 mutations. In addition, reactive treatment before organ damage occurs. eosinophilia, diseases associated with abnormal release MYELOID AND LYMPHOID NEOPLASMS WITH FGFR1 of cytokines (IL-2, IL-3, IL-5, or GM-CSF), or presence of ABNORMALITIES aberrant T cells must be ruled out. The diagnostic crite- The myeloid and lymphoid neoplasms with FGFR1 abnor- ria for CEL-NOS70 are listed in Table 24-23. The cell of malities subgroup of eosinophilic neoplasms are charac- origin for this leukemia is probably the HSC. In some terized by an 8p11 breakpoint, which, depending on the cases, cytogenetic abnormalities such as trisomy 8 can chromosome partner, results in a variety of fusion genes be identified.70 Table 24.23 Diagnostic Criteria for Chronic Eosinophilic Leukemia, Not Otherwise Specified (CEL-NOS)—Persistent Eosinophilia Greater Than or Equal to 1.5 * 103/mcL in Blood • Less than 20% blasts in blood and bone marrow; no inv(16)(p13.1q22) or t(16;16)(p13.1:q22) or other diagnostic feature of |
AML • Exclude eosinophilic disorders that bear the PDGFRA, PDGFRB, or FGFR1 mutations • No BCR/ABL1 fusion gene; absence of other MPNs, MDS/MPN • Exclude all secondary causes of eosinophilia • Exclude neoplastic disorders with secondary eosinophilia • Exclude neoplastic disorders when eosinophils are a part of the neoplastic clone (e.g., AML) • Exclude presence of an aberrant phenotypic T-cell population • With all of the preceding conditions and evidence of clonality or greater than 2% blasts in the peripheral blood and greater than 5% blasts in the bone marrow, diagnosis is CEL If a patient has persistent eosinophilia but does not meet these criteria, the diagnosis can be reactive eosinophilia, idiopathic hypereosinophilia, or idiopathic hypereosinophilic syndrome. Gotlib, J. (2012). World Health Organization-defined eosinophilic disorders: 2012 update on diagnosis, risk stratification and management. American Journal of Hematology, 87(9), 903–914; Vardiman, J. W., Thiele, J., Arber, D. A., et al. (2009). The 2008 revision of the World Health Organization (WHO) classification of the myeloid neoplasms and acute leukemia: Rationale and important changes. Blood, 114, 937–951. Bain, B. J., Gilliland, D. G., Vardiman, J. W., et al. (2008). Chronic eosinophilic leukemia, not otherwise specified. In S. H. Swerdlow, E. Campo, N. L. Harris, et al. (Eds.), WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (4th ed.). Lyon, France: IARC Press. 548 Chapter 24 CLINICAL PRESENTATION Idiopathic Hypereosinophilic CEL-NOS is most often diagnosed in middle-aged men (male-to-female ratio is 9:1). Presenting symptoms include Syndrome (I-HES) fever and significant weight loss. Clinical features include I-HES is a diagnosis of exclusion. If the patient has central nervous system (CNS) irregularities, hepatospleno- persistent eosinophilia (eosinophil count greater than megaly, congestive heart failure, pulmonary fibrosis, and or equal to 1.5 * 103/mcL for at least 6 months) and occasionally lymphadenopathy. Release of excessive eosin- does not meet the criteria for CEL (Table 24-23) or a ophilic granules in the blood causes fibrosis of the endo- familial eosinophilia (autosomal dominant disorder), thelial cells resulting in peripheral vasculitis, gangrene of the d iagnosis may be I-HES. I-HES usually has organ- digits, and organ damage, particularly of the heart and associated i nvolvement and no evidence of clonality lungs.70 or T-cell abnormality (T cells secrete eosinophil cyto- kines). Pathogenesis is unknown. As more molecular LABORATORY EVALUATION genetic mutations have been found and more diagnos- CEL-NOS is characterized by a peripheral blood tic techniques have become available, this diagnosis eosinophilia of 1.5 * 103/mcL or more. There is evidence has been made less frequently. First-line therapy for of clonality and/or more than 2% blasts in the peripheral most patients with I-HES is the use of c orticosteroids.74 blood or 5–19% blasts in the bone marrow. The leukocyte Patients with I-HES should be m onitored regularly count is usually greater than 30 * 103/mcL with 30–70% because follow-up evidence may show that the condi- eosinophils. Anemia and thrombocytopenia can be present. tion is leukemic. The LAP score is normal, but the serum cobalamin, uric acid, and muramidase are frequently elevated. The bone marrow shows a left shift with many eosinophilic myelo- cytes. Marrow fibrosis is a common finding, and Charcot- Mast Cell Disease Leyden crystals are often seen. (Mastocytosis) THERAPY When therapy is ineffective, the prognosis for patients with Mast cell disorders are heterogeneous and are c haracterized CEL-NOS is poor. Few live beyond 1 year after diagnosis. by the abnormal proliferation of mast cells in one or more The major cause of death is congestive heart failure from organ systems. Mast cells share a c ommon progenitor cell tissue injury. with myelomonocytic cells. Based on the 2016 revised WHO diagnostic criteria for MPNs, m astocytosis is no longer DIFFERENTIAL DIAGNOSIS considered an MPN; however, the WHO classification is Ruling out conditions associated with reactive eosinophilia included in Table 24-24. is important. CEL-NOS also should be differentiated from The two major groups of mast cell disorders are clonal eosinophilia found in other hematologic disorders cutaneous and systemic. Cutaneous disease is based (Table 24-21). If there is no evidence of a reactive eosino- on the presence of histological skin lesions and typical philia, clonality, or increase in blasts, the diagnosis should clinical signs but no systemic involvement. It is typically be I-HES. found in children. Table 24.24 Criteria for Diagnosis of Systemic Mastocytosis (SM) and Mast Cell Leukemia (MCL) SM requires the major criteria and one minor criteria or three minor criteria. Major criteria • Bone marrow or other organs that show multifocal, dense infiltrates of tryptase positive mast cells (more than 15 cells in aggregates) Minor criteria • Biopsy of bone marrow or other organs shows spindle-shaped or atypical morphology in more than 25% of mast cells or more than 25% of all mast cells in the bone marrow are immature or atypical • Presence of c-Kit point mutation (codon 816) in mast cells from bone marrow or other organ • Mast cells in peripheral blood, bone marrow, or other tissue coexpress CD2 and/or CD25 in addition to normal mast cell markers • In absence of associated clonal myeloid disorder, total serum tryptase is greater than 20 ng/mL Mast cell leukemia criteria • Criteria for SM fulfilled • Diffuse infiltration by atypical immature mast cells shown in bone marrow biopsy Myeloproliferative Neoplasms 549 Systemic disease or systemic mastocytosis (SM) cells in bone marrow, and multiorgan failure (Table 24-24). involves multifocal histological lesions in the bone marrow The aleukemic mast cell leukemia variant has less than and other organs. There may be anemia and an increase or 10% mast cells in the peripheral blood. The bone marrow decrease in leukocytes and platelets, and eosinophilia can mast cells can be immature and blast-like. Mast cells with be marked. bilobed or polylobed nuclei can be found. Signs of myelo- Genetic studies reveal that SM is a clonal disorder proliferation and dysplasia can be p resent, but the criteria characterized by a somatic mutation of the c-Kit proto- for another hematologic disorder are not. The mast cells are oncogene. Stem cell factor receptor is encoded for by c-Kit tryptase positive and express c-Kit. Serum tryptase levels (tyrosine kinase receptor) and is a major regulator of mast are elevated. Pancytopenia characterizes later stages of the cell development, and hematopoietic stem cells. When disease when bone marrow failure occurs. mutated, the receptor is constitutively activated, giving Most patients are adults with symptoms related to the cell a proliferative advantage.75 proteins or mediators released from mast cells, which One subgroup of systemic mast cell disease is mast include hypotension, flushing, and diarrhea. Weight loss, cell leukemia (MCL), an aggressive disease with a short bone pain, and organomegaly occur later in the disease. No survival. The criteria for its diagnosis are the fulfillment standard therapy or long-term cure exists. Antimediator of the criteria for systemic mastocytosis, circulating mast drugs such as aspirin and antihistamines are used to relieve cells in the peripheral blood (greater than or equal to 10%) symptoms. Experience with chemotherapy and bone mar- mast cells comprising greater than 20% of the nucleated row transplant is lacking.75 Summary The myeloproliferative neoplasms (MPNs) are character- in gene expression and abnormal control of cell growth. ized as a group of clonal stem cell disorders differentiated Abnormal karyotypes in hematopoietic cells can be found by neoplastic production of one or more of the hemato- in any of the subgroups. The best-characterized abnormal- poietic lineages in bone marrow and peripheral blood. ity is the Philadelphia (Ph) chromosome found in all indi- The WHO classification of MPN includes the subgroups viduals with CML. The Ph chromosome is the result of a chronic myeloid leukemia (CML); chronic neutrophilic translocation of genetic material between chromosomes leukemia (CNL); essential thrombocythemia (ET); poly- 9 and 22 [t(9;22) (q34;q11.2)]. Results of molecular testing cythemia vera (PV); primary myelofibrosis (PMF); myelo- of CML patients, whether positive or negative for the Ph proliferative neoplasm, unclassifiable (MPN, U); myeloid chromosome, reveals a BCR/ABL1 fusion gene encoding an and lymphoid neoplasms associated with eosinophilia abnormal tyrosine kinase protein, p210. The function of the and PDGFRA, PDGFRB, or FGFR1 mutations; chronic abnormal tyrosine kinase results in the pathogenesis of CML. eosinophilic leukemia, not otherwise specified (CEL- JAK2(V617F), MPL(W515), and CALR mutations are com- NOS). The 2016 revised WHO criteria excluded mastocy- mon in other MPNs. Each driver mutation modulates the tosis from the MPNs but it is still discussed in this section. JAK-STAT pathway affecting cellular signal transduction. Although all hematopoietic cell lineages can be The survival of patients with MPNs varies according involved in the unregulated proliferation in MPNs, one lin- to the subgroup and complications of thrombosis. Patients eage is usually involved more than the others. The myeloid with PV and ET appear to survive longer than patients cells are primarily affected in CML, CNL, and eosinophil with CML or PMF. Any of the subgroups can evolve into disorders; the erythrocytes are affected in PV; the plate- acute leukemia with or without specific therapy. Currently, lets/megakaryocytes are affected in ET; and mast cells are stem cell transplantation is the only cure, but few patients elevated in mastocytosis. The most characteristic finding of qualify for the procedure. However, molecular-targeted PMF is a nonmalignant proliferation of fibroblasts in the therapy and antiplatelet drugs give favorable outcomes and bone marrow. Splenomegaly, bone marrow fibrosis, and improve prognosis. Imatinib mesylate revolutionized CML megakaryocytic hyperplasia are findings common to all therapy and a cadre of second- and third-generation TKIs subgroups. are being tested for potential use as first-line therapy, rescue The underlying pathophysiology appears to be chro- therapy, or combination therapy. Several JAK2 inhibitors mosomal rearrangements that occur in the regions of proto- are being tested for effectiveness against the JAK2(V617F)- oncogenes leading to qualitative or quantitative alterations positive MPNs. 550 Chapter 24 Review Questions Level I 8. A 50-year-old man was admitted to the e mergency room for chest pain and a blood count was ordered. 1. The most prominent cell line in CML is the: (Objective 1) The results showed erythrocyte count 6.5 * 106/mcL; a. erythroid hematocrit 0.60 L/L (60%); leukocyte count b. myeloid 15 * 103/mcL; and platelet count 500 * 103/mcL. These results indicate: (Objective 2) c. megakaryocyte d. fibroblast a. the need for further investigation of a possible diagnosis of MPN 2. The most prominent cell line found in polycythemia b. normal findings for an adult male vera (PV) is: (Objective 1) c. the patient has experienced a thrombotic episode a. erythroid d. a malfunction of the cell-counting instrument b. myeloid 9. A patient previously diagnosed with CML now has a c. megakaryocyte platelet count of 540 * 103/mcL and a leukocyte count d. fibroblast of 350 * 103/mcL with a peripheral blood differential showing 15% segmented neutrophils, 23% bands, 2% 3. The Philadelphia chromosome (Ph) results from a metamyelocytes, 35% blasts, 6% lymphocytes, 4% translocation of chromosomes: (Objective 3) monocytes, 6% eosinophils, and 8% basophils. These a. 8 and 14 results are most consistent with: (Objective 5) b. 9 and 22 a. CML c. 12 and 17 b. ET d. 15 and 17 c. CML in blast crisis d. PMF 4. The peak age for CML is: (Objective 4) 10. A patient presenting in the ER with a platelet count of a. <5 years greater than 1000 * 103/mcL and a leukocyte count of b. 15–30 years 25 * 103/mcL with a normochromic, normocytic ane- c. 40–59 years mia should be evaluated for: (Objective 9) d. 60 years or more a. Ph chromosome 5. The Philadelphia chromosome (Ph) can be found in b. essential thrombocythemia patients with: (Objective 3) c. PMF d. primary polycythemia a. CML b. ALL Level II c. ET d. PV Use this case study to answer questions 1–5. A 45-year-old Caucasian female was admitted to the 6. Which of the following represent a characteristic hospital from the emergency room. She had experi- peripheral blood finding in patients with PMF? enced pain in the upper quadrant and bloating for the (Objective 6) past several weeks. She had multiple bruises on her a. Elliptocytes legs and arms. She also stated that her gums bled easily b. Dacryocytes when she brushed her teeth. She had been unusually c. Target cells tired and lost about 10 lb in the last 2 months. Results of physical examination showed a massive spleen. The d. Schistocytes following laboratory results |
were noted on admission. 7. PV can be distinguished from secondary polycythe- Hb 7.4 g/dL WBC Differential mia by measuring: (Objective 8) Erythrocyt 2.9 * 106/mcL 31% segmented a. hematocrit ount neutrophils b. plasma volume Hct 22% 26% bands c. hemoglobin concentration RDW 18.0 8% metamyelocytes d. erythropoietin Myeloproliferative Neoplasms 551 c. Leukopenia Leukocyte 520 * 103/mcL 11% myelocytes d. Myelodysplastic count 5. What is the best description of the bone marrow? Platelet 960 * 103/mcL 4% promyelocytes (Objective 1) count a. Decreased M:E ratio and increased cellularity 2% blasts b. Increased M:E ratio and decreased cellularity 4% lymphocytes c. Increased M:E ratio and increased cellularity 3% monocytes d. Decreased M:E ratio and decreased cellularity 5% eosinophils 6% basophils 6. Extensive bone marrow fibrosis, leukoerythroblastic peripheral blood, and the presence of anisocytosis 4 nucleated with dacryocytes are most characteristic of which erythrocytes/100 MPN? (Objective 6) leukocytes a. CML Occasional micro- megakaryocytes b. PV c. ET Anisocytosis and poikilocytosis were moderate. d. PMF A bone marrow aspiration was performed. The marrow was 90% cellular with a myeloid-to-ery- 7. A 68-year-old man was seen in the clinic for lethargy, throid ratio of 10:1. The majority of the cells were dyspnea, and light-headedness. Results of his blood neutrophilic precursors. Eosinophils and basophils counts were erythrocyte count 5.0 * 106/mcL; hema- were increased. Myeloblasts accounted for 10% of tocrit 55%; leukocyte count 60 * 103/mcL; platelet the nucleated cells. Megakaryocytes were increased. count 70 * 103/mcL. His differential showed a shift to the left in myeloid elements with 40% eosinophils. The bone marrow revealed 10% blasts. He does not 1. What findings suggest that this patient has a defect in have the PDGFRA, PDGFRB, or FGFR1 mutations or the pluripotential stem cell rather than a benign pro- the Ph chromosome. Secondary causes of eosinophilia liferation of hematopoietic cells? (Objective 4) have been ruled out. He most likely has: (Objective 10) a. The presence of a leukoerythroblastic blood picture a. PV b. The involvement of several cell lineages in the prolifer- b. CML in blast crisis ative process including neutrophilic cells and platelets c. essential thrombocythemia c. The shift to the left in the neutrophilic cell line d. chronic eosinophilic leukemia d. An increase in the RDW 8. A molecular test should be performed on the patient 2. Molecular analysis (RT-PCR) revealed the presence of in question 7 for which mutation? (Objective 3) a BCR/ABL1 fusion product. Based on this informa- tion, what myeloproliferative disorder is present? a. JAK2(V617F) (Objectives 1, 3) b. BCR/ABL1 a. CML c. FIP1L1/PDGFRA b. PV d. MPL(W515) c. ET 9. Which of the following does not cause secondary d. PMF polycythemia? (Objective 7) 3. What cytochemical stain is used to help differentiate a a. Chronic obstructive pulmonary disease leukemoid reaction from CML? (Objectives 1, 4) b. Smoking a. Myeloperoxidase c. Emphysema b. New methylene blue d. Dehydration c. Leukocyte alkaline phosphatase d. Perl’s Prussian blue 10. Which of the following gene mutations are uniquely associated with clonal hypereosinophilia? (Objective 11) 4. Which of the following terms most accurately describes this patient’s peripheral blood picture? a. JAK2(V617F) (Objectives 10, 13) b. F1P1L1/PDGFRA a. Leukemoid reaction c. MPL(W515) b. Leukoerythroblastic d. BCR/ABL1 552 Chapter 24 References 1. Dameshek, W. (1951). Some speculations on the 17. Guilhot, F., Roy, L., & Tomowiak, C. (2012). Current treatment myeloproliferative syndromes. Blood, 6(4), 372–375. strategies in chronic myeloid leukemia. Current Opinion in 2. Arber, D. A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M. Hematology, 19(2), 102–109. doi: 10.1097/MOH.0b013e32834ff610 J., Le Beau, M. M., . . . Vardiman, J. W. (2016). The 2016 revision 18. Ma, W., Tseng, R., Gorre, M., Jilani, I., Keating, M., Kantarjian, to the World Health organization classification of myeloid H., . . . Albitar, M. (2007). Plasma RNA as an alternative to cells neoplasms and acute leukemia. Blood, 127(20), 2391–2405. doi: for monitoring molecular response in patients with chronic 10.1182/blood-2016-03-643544 myeloid leukemia. Haematologica, 92(2), 170–175. doi: 10.3324/ 3. Them, N. C., & Kralovics, R. (2013). Genetic basis of MPN: haematol.10360 Beyond JAK2-V617F. Current Hematologic Malignancy Reports, 8(4), 19. Brain, B. J., Brunning, R. D., Orazi, A., & Thiele, J. (2017). 299–306. doi: 10.1007/s11899-013-0184-z Chronic neutrophilic leukemia. In: S. H. Swerdlow, E. Campo, 4. Cervantes, F., Salgado, C., & Rozman, C. (1991). Assessment N. L. Harris, E. S. Jaffe, S. A. Pileri, & H. Stein, eds. World Health of iron stores in hospitalized patients. American Journal of Organization Classification of Tumours (4th ed., pp. 37–38). Lyon, Clinical Pathology, 95(1),105-106. doi: https://doi.org/10.1093/ France: International Agency for Research on Cancer. ajcp/95.1.105a 20. Aruch, D., & Mascarenhas, J. (2016). Contemporary approach 5. Bleeker, J. S., & Hogan, W. J. (2011). Thrombocytosis: to essential thrombocythemia and polycythemia vera. Diagnostic evaluation, thrombotic risk stratification, and risk- Current Opinion in Hematology, 23(2), 150–160. doi: 10.1097/ based management strategies. Thrombosis, 2011, 1–16. doi: moh.0000000000000216 10.1155/2011/536062 21. Beer, P. A., & Green, A.R. (2016). Essential thrombocythemia. In: K. 6. Kaushansky, K. (2006). Hematopoietic growth factors, signaling Kaushansky, J. T. Prchal, O. W. Press, M. A. Lichtman, M. Levi, & and the chronic myeloproliferative disorders. Cytokine and Growth L. J. Burns, eds. Williams hematology (9th ed., pp. 1307–1318). New Factor Reviews, 17(6), 423–430. doi: 10.1016/j.cytogfr.2006.09.005 York: McGraw-Hill Education 7. Dickstein, J. I., & Vardiman, J. W. (1993). Issues in the pathology 22. Passamonti, F., Mora, B., & Maffioli, M. (2016). New molecular and diagnosis of the chronic myeloproliferative disorders and the genetics in the diagnosis and treatment of myeloproliferative myelodysplastic syndromes. American Journal of Clinical Pathology, neoplasms. Current Opinion in Hematology, 23(2), 137–143. doi: 99(4), 513-525. 10.1093/ajcp/99.4.513 10.1097/moh.0000000000000218 8. Brazma, D., Grace, C., Howard, J., Melo, J. V., Holyoke, T., 23. Arellano-Rodrigo, E., Alvarez-Larran, A., Reverter, J. C., Villamor, Apperley, J. F., & Nacheva, E. P. (2007). Genomic profile of N., Colomer, D., & Cervantes, F. (2006). Increased platelet and chronic myelogenous leukemia: Imbalances associated with leukocyte activation as contributing mechanisms for thrombosis disease progression. Genes, Chromosomes and Cancer, 46(11), in essential thrombocythemia and correlation with JAK2 1039–1050. doi: 10.1002/gcc.20487 mutational status. Haematologica, 91(2), 169–175. 9. Liesveld, J. L., & Lichtman, M. A. (2016). Chronic myelogenous 24. Darbousset, R., Thomas, G. M., Mezouar, S., Frere, C., Bonier, leukemia and related disorders. In: K. Kaushansky, J. T. Prchal, R., Mackman, N., . . . Panicot-Dubois, L. (2012). Tissue factor- O. W. Press, M. A. Lichtman, M. Levi, L. J. Burns, & M. Caligiuri, positive neutrophils bind to injured endothelial wall and initiate eds. Williams hematology (9th ed., pp. 1437–1492). New York: thrombus formation. Blood, 120(10), 2133–2143. doi: 10.1182/ McGraw-Hill Education. blood-2012-06-437772 10. Foroni, L., Gerrard, G., Nna, E., Khorashad, J. S., Stevens, D., 25. Tefferi, A., & Elliot, M. (2007). Thrombosis in myeloproliferative Swale, B., . . . Marin, D. (2009). Technical aspects and clinical disorders: Prevalence, prognostic factors, and the role of applications in measuring BCR-ABL1 transcripts number in leukocytes and JAK2V617F. Seminars in Thrombosis and chronic myelogenous leukemia. American Journal of Hematology, Hemostasis, 33(4), 313–320. doi: 10.1055/s-2007-976165 84(8), 517–522. doi: 10.1002/ajh.21457 26. Campbell, P. J., Scott, L. M., Buck, G., Wheatley, K., East, C. L., 11. Vakil, E., & Tefferi, A. (2011). BCR-ABL1—Negative Marsden, J. T., . . . A. (2005). Definition of subtypes of essential myeloproliferative neoplasms: A review of molecular biology, thrombocythaemia and relation to polycythemia vera based diagnosis, and treatment. Clinical Lymphoma Myeloma and on JAK2 V617F mutation status: A prospective study. Lancet, Leukemia, 11(Suppl. 1), 37–45. doi: 10.1016/j.clml.2011.04.002 366(9501), 1945–1953. doi: 10.1016/S0140-6736(05)67785-9 12. Yilmaz, M., Kantarjian, H., Jabbour, E., O'Brien, S., Borthakur, G., 27. Vainchenker, W., Delhommeau, F., Constantinescu, S. N., & Verstovsek, S., . . . Cortes, J. (2013). Similar outcome of patients Bernard, O. A. (2011). New mutations and pathogenesis of with chronic myeloid leukemia treated with imatinib in or out myeloproliferative neoplasms. Blood, 118(7), 1723–1735. doi: of clinical trials. Clinical Lymphoma, Myeloma and Leukemia, 13(6), 10.1182/blood-2011-02-292102 693–696. doi: 10.1016/j.clml.2013.05.011 28. Dahabreh, I. J., Zoi, K., Giannouli, S., Zoi, C., Loukopoulos, D., 13. Nadal, E., & Olavarria, E. (2004). Imatinib mesylate (Gleevec/ & Voulgarelis, M. (2009). Is JAK2 V617F mutation more than a Glivec) a molecular-targeted therapy for chronic myeloid diagnostic index? Leukemia Research, 33(1), 67–73. doi: 10.1016/j. leukaemia and other malignancies. International Journal of Clinical leukres.2008.06.006 Practice, 58(5), 511–516. doi: 10.1111/j.1368-5031.2004.00173.x 29. Vannucchi, A. M., Antonioli, E., Guglielmelli, P., Rambaldi, 14. Green, M. R., Newton, M. D., & Fancher, K. M. (2016). Off-target A., Barosi, G., Marchioli, R., . . . Barbui, T. (2007). Clinical effects of BCR-ABL and JAK2 inhibitors. American Journal of profile of homozygous JAK2 617V>F mutation in patients with Clinical Oncology, 39(1), 76–84. doi: 10.1097/coc.0000000000000023 polycythemia vera or essential thrombocythemia. Blood, 110(3), 15. Milojkovic, D., & Apperley, J. (2009). Mechanisms of resistance 840–846. doi: 10.1182/blood-2006-12-064287 to imatinib and second-generation tyrosine inhibitors in chronic 30. Vannucchi, A. M., Antonioli, E., Guglielmelli, P., Longo, G., myeloid leukemia. Clinical Cancer Research, 15(24), 7519–7527. doi: Pancrazzi, A., Ponziani, V., . . . Barbui, T. (2007). Prospective 10.1158/1078-0432.ccr-09-1068 identification of high-risk polycythemia vera patients based 16. Gibbons, D. L, Pricl, S., Kantarjian, H., Cortes, J., & Quitas- on JAK2V617F allele burden. Leukemia, 21(9), 1952–1959. doi: Cardama, A. (2011). The rise and fall of gatekeeper mutations?: 10.1038/sj.leu.2404854 The BCR-ABL1 T315I paradigm. Cancer, 118(2), 293–299. doi: 31. Nussenzveig, R. H., Swierczek, S. I., Jelinek, J., Gaikwad, A., Liu, 10.1002/cncr.26225 E., Verstovsek, S., . . . Prchal, T. J. (2007). Polycythemia vera is not Myeloproliferative Neoplasms 553 initiated by JAK2V617F mutation. Experimental Hematology, 35(1), 46. Ko, M., Huang, Y., Jankowska, A. M., Pape, U. J., Tahiliani, M., 32–38. doi: 10.1016/j.exphem.2006.11.012 Bandukwala, H. S., . . . Rao, A. (2010). Impaired hydroxylation of 32. Thiele, J., Kvasnicka, H. M., Orazi, A., Gianelli, U., Tefferi, A., 5-methylcytosine in myeloid cancers with mutant TET2. Nature, Gisslinger, H., & Barbui, T. (2017). Essential thrombocythaemia. In: 468(7325), 839–843. doi: 10.1038/nature09586 S. H. Swerdlow, E. Campo, N. L. Harris, E. S. Jaffe, S. A. Pileri, & 47. Saint-Martin, C., Leroy, G., Delhommeau, F., Panelatti, G., H. Stein, eds. World Health Organization Classification of Tumours Dupont, S., James, C., . . . Bellanne-Chantelot, C. (2009). Analysis (4th ed., pp. 50–53). Lyon, France: International Agency for of the ten-eleven translocation 2 (TET2) gene in familial Research on Cancer. myeloproliferative neoplasms. Blood, 114(8), 1628–1632. doi: 33. Tefferi, A. (2010). Novel mutations and their functional and 10.1182/blood-2009-01-197525 clinical relevance in myeloproliferative neoplasms: JAK2, MPL, 48. Abdel-Wahab, O., & Levine, R. L. (2010). Genetics of the TET2, ASXL1, CBL, IDH and IKZF1. Leukemia, 24(6), 1128–1138. myeloproliferative neoplasms. Myeloproliferative Neoplasms, 39–68. doi: 10.1038/leu.2010.69 doi: 10.1007/978-1-60761-266-7_2 34. Michiels, J. J., Berneman, Z., Schroyens, W., Koudstaal, P. J., 49. Lasho, T. L., Pardanani, A., & Tefferi, A. (2010). LNK mutations Lindemans, J., Neumann, H. A., & Van Vliet, H. H. (2006). in JAK2 mutation-negative erythrocytosis. New England Journal of Platelet-mediated erythromelalgic, cerebral, ocular and coronary Medicine, 363(12), 1189–1190. doi: 10.1056/NEJMc1006966 microvascular ischemic and thrombotic manifestations in 50. Dupont, S., Masse, A., James, C., Teyssandier, I., Lecluse, Y., patients with essential thrombocythemia and polycythemia Larbret, F., . . . Delhommeau, F. (2007). The JAK2 617V>F vera: A distinct aspirin-responsive and coumadin-resistant mutation triggers erythropoietin hypersensitivity and terminal arterial thrombophilia. Platelets, 17(8), 528–544. doi: erythroid amplification in primary cells from patients with 10.1080/09537100600758677 polycythemia vera. Blood, 110(3), 1013–1021. doi: 10.1182/ 35. Barosi, G., Birgegard, G., Finazzi, G., Griesshammer, M., blood-2006-10-054940 Harrison, C., Hasselbalch, H. C., . . . Barbui, T. (2009). Response 51. Hobbs, G. S., & Rampal, R. K. (2015). Clinical and molecular criteria for essential thrombocythemia and polycythemia vera: genetic characterization of myelofibrosis. Current Opinion in Result of a European LeukemiaNet consensus conference. Blood, Hematology, 22(2), 177–183. doi: 10.1097/moh.0000000000000122 113(20), 4829–4833. doi: 10.1182/blood-2008-09-176818 52. Moliterno, A. R., Hexner, E., Roboz, G. J., Carroll, M., Luger, S., 36. Verstovsek, S., Passamonti, F., Rambaldi, A., Barosi, G., Rosen, P., Mascarenhas, J . . . .& Bensen-Kennedy, D. M. (2009). An open- Levy, R . . . .& Vannucchi, A. (2009). A phase 2 study of INCB018424, label study of CEP-701 in patients with JAK2V617F-positive PV an oral, selective JAK1/JAK2 inhibitor, in patients with advanced and ET: Update of 39 enrolled patients. Blood, 114, 753. polycythemia vera (PV) and essential thrombocythemia (ET) 53. Tefferi, A. (2012). JAK inhibitors for myeloproliferative refractory to hydroxyuria. Blood, 113, 4829–4833. neoplasms: Clarifying facts from myths. Blood, 119(12), 2721– 37. Prchal, J. F., & Prchal, J. T. |
(2016). Polycythemia vera. In: K. 2730. doi: 10.1182/blood-2011-11-395228 Kaushansky, J. T. Prchal, O. W. Press, M. A. Lichtman, M. Levi, L. 54. Cassinat, B., Laguillier, C., Gardin, C., Beco, V. D., Burcheri, J. Burns, & M. Caligiuri, eds. Williams hematology (9th ed., S., Fenaux, P., . . . Kiladjian, J. (2007). Classification of pp. 1291–1306). New York: McGraw-Hill Education myeloproliferative disorders in the JAK2 era: Is there a role 38. Tefferi, A., & Spivak, J. L. (2005). Polycythemia vera: Scientific for red cell mass? Leukemia, 22(2), 452–453. doi: 10.1038/ advances and current practice. Seminars in Hematology, 42(4), sj.leu.2404908 206–220. doi: 10.1053/j.seminhematol.2005.08.003 55. Mascarenhas, J., Najfeld, V., Kremyanskaya, M., Keyzner, A., 39. Tefferi, A. (2003). Polycythemia vera: A comprehensive review Salama, M. E., & Hoffman, R. (2018). Primary myelofibrosis. and clinical recommendations. Mayo Clinic Proceedings, 78(2), In: R. Hoffman, E. J. Benz Jr., L. E. Silberstein, H. E. Heslop, J. I. 174–194. doi: 10.4065/78.2.174 Weitz, & J. Anastasi, eds. Hematology: Basic principles and practice 40. Cao, M., Olsen, R. J., & Zu, Y. (2006). Polycythemia (7th ed., pp. 1125–1150). Philadelphia: Elsevier. vera: New clincopathologic perspectives. Archives of 56. Lichtman, M. A., & Prchal, J. T. (2016). Primary myelofibrosis. In: Pathology and Laboratory Medicine, 130(8), 1126–1132. doi: K. Kaushansky, J. T. Prchal, O. W. Press, M. A. Lichtman, M. Levi, 10.1043/1543-2165(2006)130[1126:PV]2.0.CO;2 & L. J. Burns, eds. Williams hematology (9th ed., pp. 1319–1340). 41. Scott, L. M., Tong, W., Levine, R. L., Scott, M. A., Beer, P. A., New York: McGraw-Hill Education. Stratton, M. R., . . . Green, A. R. (2007). JAK2 exon 12 mutations 57. Arana-Yi, C., Quintas-Cardama, A., Giles, F., Thomas, D., in polycythemia vera and idiopathic erythrocytosis. New England Carrasco-Yalan, A., Cortes, J., . . . Verstovsek, S. (2006). Advances Journal of Medicine, 356(5), 459–468. doi: 10.1056/NEJMoa065202 in the therapy of chronic idiopathic myelofibrosis. The Oncologist, 42. Kralovics, R., Teo, S. S., Theocharides, A., Buser, A. S., Tichelli, 11(8), 929–943. doi: 10.1634/theoncologist.11-8-929 A., & Skoda, R. C. (2006). Acquisition of the V617F mutation 58. Vannucchi, A. M., Antonioli, E., Guglielmelli, P., Pardanani, A., of JAK2 is a late genetic event in a subset of patients with & Tefferi, A. (2008). Clinical correlates of JAK2V617F presence myeloproliferative disorders. Blood, 108(4), 1377–1380. doi: or allele burden in myeloproliferative neoplasms: A critical 10.1182/blood-2005-11-009605 reappraisal. Leukemia, 22(7), 1299–1307. doi: 10.1038/leu.2008.113 43. Theocharides, A., Boissinot, M., Girodon, F., Garand, R., Teo, 59. Tefferi, A., Lasho, T. L., Huang, J., Finke, C., Mesa, R. A., Li, C. S., Lippert, E., . . . Skoda, R. C. (2007). Leukemic blasts in Y., . . . Pardanani, A. (2008). Low JAK2V617F allele burden in transformed JAK2-V617F-positive myeloproliferative disorders primary myelofibrosis, compared to either a higher allele burden or are frequently negative for the JAK2-V617F mutation. Blood, unmutated status, is associated with inferior overall and leukemia- 110(1), 375–379. doi: 10.1182/blood-2006-12-062125 free survival. Leukemia, 22(4), 756–761. doi: 10.1038/sj.leu.2405097 44. Kilpivaaro, O., Mukherjee, S., Schram, A. M., Wadleigh, M., 60. Scott, L. M., Scott, M. A., Campbell, P. J., & Green, A. R. Mullally, A., Ebert, B. L., . . . Levine, R. L. (2009). A germline (2006). Progenitors homozygous for the V617F mutation occur JAK2 SNP is associated with predisposition to the development in most patients with polycythemia vera but not essential of JAK2(V617F)-positive myeloproliferative neoplasms. Nature thrombocythemia. Blood, 108(7), 2435–2437. doi: 10.1182/ Genetics, 41(4), 455–459. doi: 10.1038/ng.342 blood-2006-04-018259 45. Ito, S., D'Alessio, A. C., Taranova, O. V., Hong, K., Sowers, 61. Guglielmelli, P., Barosi, G., Specchia, G., Rambaldi, A., Coco, F. L. C., & Zhang, Y. (2010). Role of Tet proteins in 5mC to L., Antonioli, E., . . . Vannucchi, A. M. (2009). Identification of 5hmC conversion, ES-cell self-renewal and inner cell mass patients with poorer survival in primary myelofibrosis based on specifications. Nature, 466(7310), 1129–1133. doi: 10.1038/ the burden of JAK2V617F mutated allele. Blood, 114(8), 1477–1483. nature09303 doi: 10.1182/blood-2009-04-216044 554 Chapter 24 62. Grand, F. H., Hidalgo-Curtis, C. E., Ernst, T., Zoi, K., Zoi, 69. Pardanani, A., Gotlib, J. R., Jamieson, C., Cortes, J. E., Talpaz, C., Mcguire, C., . . . Cross, N. C. (2009). Frequent CBL M., Stone, R. M., . . . Tefferi, A. (2011). Safety and efficacy of mutations associated with 11q acquired uniparental disomy in TG101348, a selective JAK2 inhibitor in myelofibrosis. Journal of myeloproliferative neoplasms. Blood, 113(24), 6182–6192. doi: Clinical Oncology: Official Journal of the American Society of Clinical 10.1182/blood-2008-12-194548 Oncology, 29(7), 789–796. doi: 10.1200/JCO.2010.32.8021 63. Mesa, R. A., Schwager, S., Radia, D., Cheville, A., Hussein, K., 70. Bain, B. J., Horny, H. P., Hasserjain, R. P., & Orazi, A. (2017). Chronic Niblack, J., . . . Tefferi, A. (2009). The Myelofibrosis Symptom eosinophilic leukemia, not otherwise specified. In: S. H. Swerdlow, Assessment Form (MFSAF): An evidence-based brief inventory to E. Campo, N. L. Harris, E. S. Jaffe, S. A. Pileri, & H. Stein, eds. World measure quality of life and symptomatic response to treatment in Health Organization Classification of Tumours (4th ed., pp. 54–56). myelofibrosis. Leukemia Research, 33(9), 1199–1203. doi: 10.1016/j. Lyon, France: International Agency for Research on Cancer. leukres.2009.01.03 71. Klion, A. D. (2005). Recent advances in the diagnosis and 64. Mesa, R. A., Niblack, J., Wadleigh, M., Verstovsek, S., Camoriano, treatment of hypereosinophilic syndromes. ASH Education Book, J., Barnes, S., . . . Tefferi, A. (2007). The burden of fatigue and 2005(1), 209–214. doi: 10.1182/asheducation-2005.1.209 quality of life in myeloproliferative disorders (MPDs). Cancer, 72. Montes, M., Sawhney, C., & Barros, N. (2010). Strongyloides 109(1), 68–76. doi: 10.1002/cncr.22365 stercoralis: There but not seen. Current Opinion in Infectious 65. Pullarkat, V., Bass, R. D., Gong, J. Z., Feinstein, D. I., & Brynes, Diseases, 23(5), 500–504. doi: 10.1097/qco.0b013e32833df718 R. K. (2002). Primary autoimmune myelofibrosis: Definition 73. Havelange, V., & Demoulin, J. B. (2013). Review of current of a distinct clinicopathologic syndrome. American Journal of classification, molecular alterations, and tyrosine kinase inhibitor Hematology, 72(1), 8–12. doi: 10.1002/ajh.10258 therapies in myeloproliferative disorders with hypereosinophilia. 66. Tefferi, A., Cortes, J., Verstovsek, S., Mesa, R. A., Thomas, D., Journal of Blood Medicine, 4, 111–121. doi: 10.2147/JBM.S33142 Lasho, T. L., . . . Kantarjian, H. M. (2006). Lenalidomide therapy 74. Vardiman, J. W., Thiele, J., Arber, D. A., Brunning, R. D., in myelofibrosis with myeloid metaplasia. Blood, 108(4), Borowitz, M. J., Porwit, A., . . . Bloomfield, C. D. (2009). The 2008 1158–1164. doi: 10.1182/blood-2006-02-004572 revision of the World Health Organization (WHO) classification 67. Faoro, L. N., Tefferi, A., & Mesa, R. A. (2005). Long-term of myeloid neoplasms and acute leukemia: Rationale and analysis of the palliative benefit of 2-chlorodeoxyadenosine important changes. Blood, 114(5), 937–951. doi: 10.1182/ for myelofibrosis with myeloid metaplasia. European Journal of blood-2009-03-209262 Haematology, 74(2), 117–120. doi: 10.1111/j.1600-0609.2004.00370.x 75. Horny, H. P., Akin, C., Aber, D. A., Peterson, L. C., Tefferi, A., 68. Verstovsek, S., Kantarjian, H., Mesa, R. A., Pardanani, A. D., Metcalfe, D. D., . . . Valent, P. (2017). Mastocytosis. In: S. H. Cortes-Franco, J., Thomas, D. A., . . . Tefferi, A. (2010). Safety Swerdlow, E. Campo, N. L. Harris, E. S. Jaffe, S. A. Pileri, & H. and efficacy of INC018424, a JAK1 and JAK2 inhibitor, in Stein, eds. World Health Organization Classification of Tumours (4th myelofibrosis. New England Journal of Medicine, 363(12), ed., pp. 61–70). Lyon, France: International Agency for Research 1117–1127. doi: 10.1056/NEJMoa1002028 on Cancer. Chapter 25 Myelodysplastic Syndromes Sara A. Taylor, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define myelodysplastic syndrome (MDS) and 4. Define the WHO category of myelodysplastic/ list general characteristics of these diseases. myeloproliferative neoplasms (MDS/MPNs), 2. List the six subgroups of MDS recognized and list its general characteristics. by the 2016 World Health Organization 5. List the five subgroups of MDS/MPNs and (WHO) Classification System and identify identify key morphological and clinical key morphological and clinical criteria that criteria as well as laboratory findings that distinguish each group. distinguish each group. 3. Describe laboratory findings and recognize changes in morphology that are characteristic of this group of disorders. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Describe the pathophysiology of MDS. 5. Assess the results of cytogenetic and 2. Distinguish among agranular blasts, molecular tests and correlate them with a granular blasts, and promyelocytes. diagnosis of MDS. 3. Summarize the treatment and prognosis of 6. List and briefly describe the MDS variants MDS. not listed in the six WHO subgroups. 4. Explain the relationship between MDS and 7. Differentiate MDS from MPN, acute acute leukemia. leukemia, and other hematologic 555 556 Chapter 25 abnormalities using peripheral blood, bone 9. Select laboratory tests that are helpful in marrow, and cytogenetic characteristics. diagnosing and differentiating MDS and 8. Differentiate MDS/MPNs from MDS, MDS/MPNs. MPN, acute leukemia, and other 10. Evaluate a patient’s laboratory and c linical hematologic abnormalities using peripheral findings, and propose a diagnostic MDS blood, bone marrow, and cytogenetic or MDS/MPNs subgroup based on those characteristics. findings. Chapter Outline Objectives—Level I and Level II 555 Classification 567 Key Terms 556 Description of MDS Subgroups 568 Background Basics 556 Variables of MDS Subgroups 571 Case Study 557 Differential Diagnosis 571 Overview 557 Prognosis 572 Introduction 557 Therapy 573 Pathophysiology 557 Myelodysplastic/Myeloproliferative Neoplasms Incidence 560 (MDS/MPNs) 574 Clinical Presentation 560 Summary 576 Laboratory Evaluation 560 Review Questions 577 Blast and Precursor Cell Classification 565 References 580 Key Terms Atypical chronic myeloid leukemia Juvenile myelomonocytic leukemia Myelodysplastic syndrome with (aCML, BCR/ABL1-) (JMML) ring sideroblasts (MDS-RS) CHIP Micromegakaryocyte Myelodysplastic syndrome Chronic myelomonocytic leukemia Myelodysplastic syndromes (MDS) with multilineage dysplasia (CMML) Myelodysplastic/ (MDS-MLD) Dysplasia myeloproliferative neoplasms Myelodysplastic syndrome Dyspoiesis (MDS/MPNs) with single lineage dysplasia Endomitosis Myelodysplastic syndrome with (MDS-SLD) excess blasts (MDS-EB) Ring sideroblasts ICUS Background Basics The information in this chapter builds on the concepts • Describe and recognize the cell morphology described learned in previous chapters. To maximize your learning by these terms: ring sideroblasts (Chapter 12), experience, you should review the following concepts Pelger-Huët anomaly (Chapter 21), and megaloblas- before starting this unit of study: toid erythropoiesis (Chapter 15). • Explain what ineffective hematopoiesis means. Level I (Chapters 11, 24) • Create a schematic depicting derivation of different • Summarize the history and basis of the classification types of blood cells from the pluripotent hematopoi- system to classify malignant leukocyte disorders. etic stem cell. (Chapter 2) (Chapter 23) Myelodysplastic Syndromes 557 • Describe normal leukocyte development, differ- • Explain how oncogenes and hematopoietic growth entiation, and concentrations in the peripheral factors affect cellular maturation and prolifera- blood. (Chapter 8) tion. (Chapters 3, 23) • Explain the role of epigenetics in cellular develop- Level II ment. (Chapter 2) • Describe the use of immunological features that dis- • Explain the use of cytogenetics and flow cytometry in tinguish the different types of blasts. (Chapters 23, 40) the diagnosis of malignant disorders. (Chapters 40, 41) and, in some cases, transformation into a condition indis- CASE STUDY tinguishable from acute leukemia. MDS occurs most com- We refer to this case study throughout the chapter. monly in the elderly, although it is diagnosed occasionally in children.1,2 Hancock, a 65-year-old white male, was seen in tri- age with complaints of fatigue, malaise, anorexia, Before the 1980s, much confusion and disagreement and hemoptysis of recent onset. A complete blood existed in the literature concerning the criteria for defining, count (CBC) was ordered and revealed anemia and subgrouping, and naming MDS. Because of the predisposi- a shift to the left in granulocytes. Hematopoietic tion of MDS to terminate in leukemia, the term preleukemia cells showed dysplastic features. commonly was used to describe these disorders. However, Consider diagnostic probabilities and reflex the evolution of MDS to acute leukemia is not obligatory, testing that could provide differential diagnostic and, in fact, many patients die of intercurrent disease or information. complications of the cytopenia before evolving to leukemia. Most hematologists currently consider the terms dysmyelo- poietic syndrome and myelodysplastic syndrome to be more acceptable than preleukemia or other synonyms in describing these hematologic disorders. In this book, the term myelo- Overview dysplastic syndrome is used. This chapter describes the neoplastic hematopoietic dis- orders known as the myelodysplastic syndromes (MDS). It Pathophysiology begins with a description of the pathophysiology, inci- dence, and general clinical and laboratory features, includ- Myeolodysplastic syndromes arise from proliferation of ing peripheral blood and bone marrow findings. |
Genetic stem cells that have acquired a defect (somatic mutation) findings common to MDS are discussed. This is followed that prevents them from differentiating, maturing, and pro- by a discussion of the classification of the disorders into liferating normally. The stem cell abnormality most often morphologic subgroups according to the 2016 classification involves the myeloid progenitor cell; the lymphocytic cell of the World Health Organization. Each subgroup is then lines are rarely affected. Many of the afflicted stem cells do described with specific features that allow it to be classi- differentiate into mature cells but exhibit flawed and ineffec- fied. Variants are also described. Prognosis and therapy are tive progression so that their numbers decrease in the periph- discussed and the chapter concludes with a discussion of eral blood (cytopenia), a phenomenon known as ineffective myelodysplastic/myeloproliferative overlap syndromes. hematopoiesis. In addition to decreased numbers, the myelo- dysplastic cells develop abnormal morphology and exhibit compromised function. Gradually, the stem cells lose their Introduction ability to differentiate, and in some cases, they transform into cells that are consistent with acute leukemia.2,3 The myelodysplastic syndromes (MDS) are considered pri- Somatic mutations in hematopoietic cells that lead to mary, neoplastic, pluripotential stem cell disorders. They are clonal expansion of the abnormal cells can be detected in characterized by one or more peripheral blood cytopenias people with normal blood counts, without dysplasia, and with prominent maturation abnormalities (dyspoiesis or with no apparent disease. These mutations are commonly dysplasia) in the bone marrow. The peripheral blood cyto- acquired during the aging process. The presence of these penias result from ineffective hematopoiesis and increased mutations confers an increased risk of a hematologic malig- apoptosis as evidenced by an accompanying bone mar- nancy diagnosis. This condition is called clonal hematopoiesis row hyperplasia. These relatively common entities evolve of indeterminate potential (CHIP), analogous to monoclonal progressively, leading to the aggravation of the cytopenias gammopathy of undetermined significance4 (Chapter 28). 558 Chapter 25 On the other hand, there are patients with unexplainable, ABNORMAL KARYOTYPES persistent cytopenia, but no abnormal marrow morphology Nearly every chromosome has been shown to exhibit and no known MDS-associated somatic mutation or abnor- genomic aberration in MDS. Moreover, abnormal cytogenetic mal karyotype. This is known as idiopathic cytopenia of unde- patterns in MDS are heterogeneous; chromosome abnor- termined significance (ICUS). These patients are monitored; malities can present either as the sole aberration in MDS or some will subsequently be diagnosed with MDS or AML.4 can occur as part of a complex karyotype. The most com- Myelodysplastic syndromes are described as either monly encountered abnormal karyotypes in MDS are the primary (de novo origin) or secondary (developing after -5/del(5q), -7/del(7q), +8, del(20q), and -Y, suggesting treatment for another malignancy). The cause of either is that genes located in these regions contribute to the patho- uncertain, but myelodysplastic syndromes are acquired physiology of this syndrome.7,8 Table 25-1 describes the most neoplasms. When the MDS is the primary type, environ- frequently mutated genes in MDS. Many mutations and mental insults such as exposure to genotoxic chemicals karyotypic abnormalities implicated in the development of (benzene), radiation, and viral infection are all etiologic MDS are epigenetic regulators, splicing factors, signaling pro- agents that could contribute to MDS development. teins, and tumor suppressors.9,10 MDS patients often display If MDS develops after chemotherapy with alkylat- more than one functional type of mutation, and only 10% of ing agents or topoisomerase II inhibitors either in conjunc- MDS patients lack any of the commonly aberrant genes. tion with radiation treatment or alone, the MDS is known The cytogenetic abnormalities associated with MDS can as the secondary type.1,3 Regardless of whether the MDS be MDS-defining in a cytopenic patient even in the absence of develops after environmental insult or previous therapy, a diagnostic morphologic dysplasia. However, the abnormality multistep pathophysiology is believed to be necessary for must be demonstrated by conventional karyotyping and not MDS to develop. MDS appears to develop as a result of FISH or gene sequencing. On the other hand, the presence complex interactions between compromised hematopoietic of +8, -Y, or del(20q) is not considered as MDS-defining if stem cells and the microenvironment of the bone marrow.5,6 there are no diagnostic morphologic findings associated with Although nearly 50% of MDS patients present with normal MDS.11 The only cytogenetic or molecular genetic abnormal- chromosomes, genetic instability of the myeloid stem cell ity that defines a specific MDS subtype is del(5q). almost certainly contributes to the development of MDS. −5 and del(5q) -5 and del(5q) are among the most fre- That MDS is primarily a disease present in elderly patients quently noted genetic abnormalities in MDS, found in lends credence to the implication of genomic instability as 10–40% of myelodysplastic syndromes.7,8,9 The WHO clas- a contributing factor toward disease progression. sification of myeloid neoplasms includes an MDS subtype named for this cytogenetic abnormality when it exists as Checkpoint 25.1 the sole aberration: MDS with isolated del(5q). Based on How does the pathophysiology of primary MDS differ from sec- data that shows no adverse effect if there is one additional ondary MDS? chromosomal abnormality in addition to the del(5q), MDS with del(5q) may be diagnosed if there is one additional cytogenetic abnormality except if that abnormality is mono- Cytogenetics, Epigenetics, and Single somy 7 or del(7q).11 Gene Mutations Three commonly deleted regions (CDRs) are found on the long arm of chromosome 5, located among bands The abnormal MDS clone is characterized by altered func- 5q31–33.2,9 The loss of many of the genes in these CDRs tion of genes that result from chromosomal abnormalities, seems to have a role in the pathophysiology of hematopoi- gene silencing (epigenetic changes), or single gene muta- etic malignancies; their mechanisms of action include the tions. “Unbalanced” genetic abnormalities (which result control of cell signaling, transcription, cell cycle, antioxi- in a larger or smaller amount of DNA in the cell, such as dant levels, apoptosis, cell differentiation/proliferation, and trisomies or whole or partial chromosomal deletions) are tumor suppression. Whole or partial losses of chromosome characteristically seen in the MDS and are thought to be 5 are mostly somatically acquired; often they are associated responsible for the ineffective hematopoiesis and cytope- with earlier treatment with radiotherapy, alkylating agents, nias.2 These chromosomal abnormalities are in contrast to or DNA topoisomerase II antagonists.11 the balanced abnormalities that maintain the normal quan- tity of DNA (e.g., reciprocal translocations) seen primarily −7 and del(7q) Karyotypic abnormalities on chromosome in myeloproliferative neoplasms (MPNs) and acute myeloid 7 appear with up to 50% incidence in MDS patients.7,8,9 The leukemia (AML) (Chapters 24 and 26, respectively). More- -7 karyotype presents with severe refractory cytopenias over, many chromosomal alterations involve gene deletions, and increased incidence of severe infections.6 Three distinct suggesting that tumor suppressor genes or DNA repair CDRs have been identified on chromosome 7, one at 7q22, genes are altered (Chapter 23). and the other two at 7q312–33 and 7q35–36.8 It is very likely Myelodysplastic Syndromes 559 that the long arm of chromosome 7 is well populated with deletions of chromosome 5, chromosome 7, or both are fre- tumor suppressor genes.8,9 quently components of a complex karyotype.9 +8 Trisomy 8 occurs with about 5% frequency in MDS. EPIGENETIC CHANGES Individuals with MDS who present with trisomy 8 in their Epigenetic changes to the genome refer to changes in genetic progenitor cells robustly express apoptosis genes as well as information that is not encoded in the DNA sequence itself, compromised immune response genes, which could under- but influenced by molecules “around” the nucleotides lie their MDS phenotype.9 of DNA (Chapter 23). Epigenetic modifications to DNA del(20q) A deletion of the long arm of chromosome 20 is changes the way that the genes are expressed. In the past seen in about 5% of MDS cases. Genes located in this area several years, studies of epigenetic changes to DNA have of the genome that are crucial to normal hematopoietic pro- concentrated mostly on DNA methylation and histone gression include ASXL1 (Table 25-1).7,8,12 modification. DNA methylation is the addition of methyl groups to cytosine-rich regions (CpG regions) commonly −Y MDS patients can present with a loss of the Y chromo- found within gene-promoter regions. CpG hypermethyl- some, but the frequency is variable, and the significance of ation silences the transcript; the contribution of hypermeth- the missing chromosome remains uncertain. -Y is found in ylation of tumor suppressor gene promoters in MDS has many malignant diseases but also in healthy, aging men.13 been established for many years. Complex Karyotype Approximately 20% of patients Histone acetylation adds acetyl groups to DNA, relax- exhibiting primary MDS and nearly 90% of MDS patients ing it and making it more available to transcriptional diagnosed with treatment-related MDS exhibit a complex machinery, which allows genes to be expressed. Histone karyotype. One of the significant differences between pri- methylation can be either repressive or activating, depend- mary and secondary MDS is the degree of genomic abnor- ing on the gene involved. mality between the two. Primary MDS is associated with More recently, the concept of epigenetic changes to single chromosomal abnormalities, whereas complex chro- DNA has been expanded to include demethylation, ubiq- mosomal abnormalities are often seen in secondary MDS.9 uitinylation, microRNAs, and long non-coding RNAs.14,15,16 Complex karyotypes generally involve the presence of more The putative mechanisms of epigenetic influence on the than three chromosomal abnormalities. Whole or partial development of MDS include increased self-renewal and Table 25.1 Significant Mutated Genes in MDS Gene Location Frequency Significance ASXL1 20q11.1 ∼11% Epigenetic control (histone modification) BCOR Xp11.4 ∼5% Transcription repressor CBL 11q23 2% Negative regulator of signal transduction; frequent in MPN/MDS DNMT3A 2p23 ∼8% Epigenetic control (CpG methylation) ETV6 12p13 Less than 5% Transcription factor EZH2 7q36 ∼5% Tumor suppressor signaling and prevention of apoptosis IDH1/2 2q33/15q26 Less than 5 % Cell metabolism; epigenetic control (DNA methylation) K-RAS 12p12.1 Less than 1% Activating mutations stimulate MAPK signaling JAK2 9p24 ∼5% Kinase signaling NPM1 5(q35) ∼5% Putative proto-oncogene; product inactivates p53 N-RAS 1p13.2 Less than 5 % Activating mutations stimulate MAPK signaling PRPF8 17p13.3 Less than 5 % Splicing factor RUNX1 21q22.12 ~ 10 % Transcription factor important for hematopoietic differentiation SETBP1 18q21.1 2–5% Proto-oncogene; mutations associated with proliferation SF3B1 2q33 ∼30% Splicing factor SRSF2 17q25 Less than 15% Splicing factor TET2 4q24 ∼20% DNA methylation; tumor suppressor TP53 17p13.1 Less than 10% Transcription factor; tumor suppressor U2AF1 21q22 ~ 7 % Splicing factor ASXL1, additional sex combs like 1; BCOR, BCL6 corepressor; CBL, Casitas B-lineage lymphoma; ETV6, ets variant 6; EZH2, enhancer of zeste 2 polycomb repressive complex 2 s ubunit; IDH1/2, isocitrate dehydrogenase 1/2; JAK2, janus kinase 2; NPM1, nuclephosmin 1; N-RAS, neuroblastoma rat sarcoma; K-RAS, Kirsten rat sarcoma; PRPF8, pre-mRNA processing factor 8; RUNX1, runt-related transcription factor 1; SF3B1, splicing factor 3b; SRSF2, serine/arginine-rich splicing factor 2; TET2, tet methylcytosine dioxygenase 2; TP53, tumor protein p53; U2AF1, U2 small nuclear RNA auxiliary factor 1. 560 Chapter 25 inhibited differentiation of the hematopoietic stem cell (HSC). Multiple genes, including those for cell cycle regu- Incidence lators, tumor suppressors, DNA repair, and apoptosis, have MDS occurs primarily in individuals older than 50 years of been identified as targets for methylation. Silencing any of age and has a slight male predominance, except in the form these critical genes could contribute to the development of with isolated 5q deletion, which predominates in women. MDS; it is believed that epigenetic changes constitute a sig- The incidence rate of MDS in the United States is 4.4 per nificant factor in the progression of MDS into AML.14,15,16 100,000, but the incidence increases with age. The median The influence of epigenetic changes in the development age at diagnosis is 65–70 years.2 The frequency of MDS diag- of MDS will certainly be more fully elucidated in the future. noses seems to be rising. This rise could be the result of an Treatment with drugs that modify epigenetic transforma- increase in the awareness of MDS on the part of physicians tion, such as hypomethylating agents, are examined in the and clinical laboratory professionals, in the application of section on MDS treatment. diagnostic procedures in elderly individuals, or in the num- ber of elderly in the population. ONCOGENES AND TUMOR SUPPRESSOR GENES Control of cellular differentiation, maturation, and prolifer- ation occurs through properly functioning proto-oncogenes and tumor suppressor genes |
(Chapter 23) and when these Clinical Presentation genes are mutated, neoplasia can result.3 Although muta- The most frequent presenting symptoms, fatigue and tions in the proto-oncogenes JAK2 (Chapter 24), RAS, and weakness, are related to an anemia that is nonresponsive RUNX1 and the tumor suppressor gene TP53 are most com- to treatment. Less commonly, hemorrhagic symptoms and monly observed, the overall incidence of single gene muta- infection precede diagnosis. These symptoms are related to tions is low7,11,16,17 (Table 25-1). thrombocytopenia and neutropenia. Many individuals can N- and K-RAS mutations have been observed in 10–15% be asymptomatic, and their disease is discovered on rou- of patients with MDS. The most common mutation is a tine laboratory screening.18 Infection is a common complica- single base change in codon 12 of the N-RAS (neuroblas- tion in patients diagnosed with MDS, particularly for those toma-ras) family. RAS proteins normally function in growth patients diagnosed with an aggressive MDS subgroup or factor-mediated signaling, and mutation is associated with who present with neutropenia (less than 1 * 109/L). Infec- a higher risk of AML transformation and a worse progno- tion and bleeding are the most common causes of death.18 sis. RUNX1 is a regulatory transcription factor for hema- Splenomegaly and/or hepatomegaly are uncommon. topoietic differentiation, and point mutations have been observed in 20% of MDS patients. Mutations in the tumor suppressor gene TP53 (Chapter 23) appear in approximately 20% of MDS patients Laboratory Evaluation and are most commonly associated with secondary MDS. MDS presents with cytopenia(s) and a range of abnormal Normal p53 protein functions to maintain genetic stability morphologic features (dysplasia) that can be demonstrated in cells and TP53 mutations in MDS patients are linked to a on stained peripheral blood and bone marrow smears. poor prognosis.7,11,16,17 The quality of the peripheral blood smear and bone mar- row specimen is very important as poor quality smears can Proliferation Abnormalities result in misinterpretation of dysplasia. Smears should be made from blood that has been exposed to anticoagulants In vitro studies suggest that excessive premature intramed- for less than 2 hours. Criteria for classification of MDS into ullary cell death of hematologic precursors via apoptosis subtypes based on morphology and cell counts are pre- contributes to ineffective hematopoiesis and peripheral sented in a later section of this chapter. Included here are blood cytopenias seen in early MDS. The various gene the general hematologic features used to initially define the alterations of the MDS clone result in an intrinsic increase presence of an MDS (Table 25-2). in susceptibility of the clone to apoptosis.2,5 Increased levels of tumor necrosis factor (TNF)@a in MDS patients has been linked to increased apoptosis of the MDS clone and normal Peripheral Blood hematopoietic cells.2,5 Progression to leukemia is associated The peripheral blood characteristics of MDS are cytope- with a reduction in apoptosis, thereby allowing the expan- nias and dysplasia. Cytopenias are defined as hemoglo- sion of the neoplastic clone. bin less than 10 g/dL, absolute neutrophil count less than 1.8 * 109/L, and platelets less than 100 * 109/L. Anemia Checkpoint 25.2 is the most consistent finding, occurring in more than 80% How do epigenetics contribute to the pathophysiology of MDS? of the cases. Bicytopenia occurs in 30% of the cases and pan- cytopenia in 15%.19 Less commonly, an isolated neutropenia Myelodysplastic Syndromes 561 Table 25.2 Hematologic Abnormalities in Myelodysplastic Syndromes Erythroid Series Myeloid Series Thrombocyte Series Peripheral blood Anemia Neutropenia Thrombocytopenia or thrombocytosis Anisocytosis, dimorphism Hypogranulation, abnormal granulation Giant forms Poikilocytosis Shift to the left Hypogranulation Macrocytes, oval macrocytes Nuclear abnormalities Micromegakaryocytes Basophilic stippling Pseudo-Pelger-Huët Functional abnormalities Nucleated RBCs Ring nuclei Howell-Jolly bodies Monocytosis Sideroblasts Reticulocytopenia Bone marrow Megaloblastoid erythropoiesis Abnormal granules in promyelocytes Micromegakaryocytes Nuclear fragmentation and budding Increase in granular and agranular blasts Megakaryocytes with multiple separated Karyorrhexis Absence of secondary granules nuclei Multiple nuclei Nuclear abnormalities Large mononuclear megakaryocytes Defective hemoglobinization Decreased myeloperoxidase Hypogranulation or large abnormal gran- ules in megakaryocytes Vacuolization Auer rods in blasts Ring sideroblasts or thrombocytopenia is found. Dysplastic features of one or a common initial finding in the refractory anemia with more cell lines are typical. Because of the critical importance ring sideroblasts (RARS) subgroup. Reticulocytes show of recognizing dysplasia in the diagnosis of MDS, smears an absolute decrease in number but can appear normal should be made from samples that have been anticoagu- if only the uncorrected or relative number (percent) is lated for 2 hours or less. Morphologic changes from pro- reported. longed exposure to anticoagulant can easily be confused In addition to anemia, qualitative abnormalities indica- with dysplastic changes in cells. Functional abnormalities tive of dyserythropoiesis are present. These include aniso- of hematologic cells are also common. Studies show that cytosis, poikilocytosis, basophilic stippling, Howell-Jolly the higher the degree and number of cytopenias, the worse bodies, and nucleated erythrocytes. Often hemoglobin F is the prognosis.19 increased (5–6%) and distributed in a heterogeneous pattern. Acquired hemoglobin H has also been found in MDS.19 Other ERYTHROCYTES erythrocyte changes include altered A, B, and I antigens, The degree of anemia is variable, but the hemoglobin is enzyme changes, and an acquired erythrocyte membrane less than 10 g/dL. The erythrocytes are usually macrocytic change similar but not identical to that found in paroxysmal (Figure 25-1) and less often normocytic. Oval macrocytes nocturnal hemoglobinuria (PNH)19 (Chapter 17). similar to those in megaloblastic anemia are frequently present, but patients have normal vitamin B12 and folate LEUKOCYTES levels. A dimorphic anemia with both oval macrocytes Neutropenia is the second most common cytopenia or normocytes and microcytic hypochromic cells is also observed in MDS and is present in approximately 50% of patients at the time of diagnosis.20 Neutropenia can be accompanied by a shift to the left with the finding of meta- myelocytes and myelocytes on the peripheral blood smear. Blasts and promyelocytes can also be present. Morphologic abnormalities in granulocytes indicative of dysgranulopoiesis are considered a hallmark finding in MDS. Dysgranulopoiesis is characterized by agranular or hypogranular neutrophils, persistent basophilia of the cyto- plasm, abnormal appearing granules, hyposegmentation (pseudo-Pelger-Huët) (Figure 25-2), or hypersegmentation of the nucleus and donut- or ring-shaped nuclei. Care should be taken to distinguish neutrophils with the pseudo-Pelger- Huët anomaly and hypogranulation from lymphocytes and Figure 25.1 A peripheral blood film of a patient with MDS. to distinguish neutrophilic-band forms with hypogranula- Note macrocytic cells and anisocytosis (Peripheral blood, Wright- tion from monocytes. Neutrophils can also demonstrate Giemsa stain, 1000* magnification). enzyme defects, such as decreased myeloperoxidase (MPO) 562 Chapter 25 Figure 25.4 Arrow points to a large agranular platelet in a Figure 25.2 Peripheral blood film from a patient with MDS peripheral blood film from a patient with MDS (Wright-Giemsa stain, showing neutrophils with the pseudo-Pelger-Huët nucleus. One 1000* magnification). cell’s nucleus is eye glass shaped (pince-nez) and the other is single lobed. The nuclear chromatin is condensed, and the cells contain MICROMEGAKARYOCYTES granules, making identification possible. In many cases, these types of neutrophils are agranular, making differentiation of them Sometimes circulating micromegakaryocytes (small abnor- from lymphocytes difficult (Peripheral blood, Wright-Giemsa stain, mal megakaryocytes), also called dwarf megakaryocytes, 1000* magnification). can be found in MDS and MPNs. Micromegakaryocytes can be difficult to identify and are frequently overlooked and decreased leukocyte alkaline phosphatase (LAP). In unless cytoplasmic tags or blebs are present (Figure 25-5). some cases, neutrophils exhibit severe functional impair- Micromegakaryocytes are believed to represent abnor- ment, including defective bactericidal, phagocytic, or che- mal megakaryocytes that have reduced ability to undergo motactic properties. Absolute monocytosis is a common endomitosis (megakaryocytes undergo a unique matura- finding even in leukopenic conditions. tion process whereby the DNA content duplicates without cell division, resulting in a polyploid nucleus (endomitosis; PLATELETS Chapter 31). Most micromegakaryocytes have a single-lobed Qualitative and quantitative platelet abnormalities are nucleus and are the size of a lymphocyte. Morphologi- often present. The platelet count can be normal, increased, cally, they can be confused with lymphocytes but can be or decreased. Approximately 25–50% of patients have distinguished by cytoplasmic tags of one or more platelets mild to moderate thrombocytopenia when diagnosed.21 attached to a nucleus. Some can have pale blue, foamy, or Giant platelets, hypogranular platelets, and platelets with vacuolated cytoplasm that resembles a nongranular plate- large fused granules can be seen in the peripheral blood let. The nuclear structure is variable, but many cells have (Figures 25-3 and 25-4). Functional platelet abnormalities include abnormal adhesion and aggregation. As a result, platelet function tests can be abnormal. Figure 25.5 Arrow points to a micromegakaryocyte in a peripheral blood film from a patient with MDS. Note the dense chromatin structure and the irregular rim of cytoplasm with Figure 25.3 Arrows point to large platelets in a peripheral cytoplasmic tags. An agranular platelet in the field matches the blood film from a patient with MDS (Wright-Giemsa stain, 1000* appearance of the micromegakaryocyte cytoplasm (Wright-Giemsa magnification). stain, 1000* magnification). Myelodysplastic Syndromes 563 very densely clumped chromatin and stain dark blue-black complex and variable, but one of their functions is to secrete with Wright-Giemsa stain. Others can have a finer, looser growth factors and cytokines that are necessary for hema- chromatin. These cells also are found in the bone marrow. topoietic cell development and survival. It is presumed that atypical synergy of the hematopoietic cells and the ancil- Checkpoint 25.3 lary bone marrow cells contribute to the ineffective hema- How does the typical peripheral blood picture in MDS differ from topoiesis observed in MDS. An unfavorable bone marrow that in aplastic anemia (Chapter 16)? microenvironment may be hypoxic, inflamed, lacking in appropriate cytokines and/or growth factors, and gener- ally contrary to normal hematopoietic development.5,6 Collection of bone marrow samples are necessary for CASE STUDY (continued from page 557) morphological examination since identification of the char- The results of the CBC on Hancock were: acteristic dyshematopoietic element of MDS is important in RBC 1.60 * 1012/L WBC Differential establishing a diagnosis. Moreover, bone marrow aspirate provides material for molecular diagnostic testing, which Hb 5.8 g/dL (58 44% segmented may be essential for diagnosis. In most cases, the bone g/L) neutrophils marrow is hypercellular; the hypercellular bone marrow in Hct 0.17 L/L (17%) 7% band conjunction with peripheral blood pancytopenia is a nota- neutrophils ble feature of myelodysplastic syndromes. Asynchrony in WBC 10.5 * 109/L 6% lymphocytes nuclear and cytoplasmic development, known as dyshema- Platelets 39 * 109/L 28% eosinophils topoiesis, is evident in all the hematopoietic cell lines.2,21 In MDS, the number of myeloblasts is less than 20%. Reticulocyte 0.8% 1% metamyelocytes count 1% myelocytes DYSERYTHROPOIESIS The most common bone marrow finding in MDS is nuclear- 9% promyelocytes cytoplasmic asynchrony similar to that seen in megaloblas- 4% blasts tic anemia (Chapter 15). However, the chromatin is usually The neutrophilic cells show marked hypo- hypercondensed. This nuclear chromatin pattern is often segmentation and hypogranulation. Red blood described as megaloblastoid (Figure 25-6). The abnormal cell (RBC) morphology includes anisocytosis and erythrocytic maturation is not responsive to vitamin B12 or poikilocytosis, teardrop cells, ovalocytes, and folic acid therapy. Giant, multinucleated erythroid precur- schistocytes. sors can be found (Figure 25-7). Other nuclear abnormalities include fragmentation, abnormal shape, budding, karryo- 1. In what cell lines is cytopenia present? hexis, and irregular staining properties. The cytoplasm of 2. What abnormalities are present in the differential? erythroid precursors can show defective hemoglobiniza- tion, vacuoles, and basophilic stippling. The presence 3. What evidence of dyspoiesis is seen in the leuko- of ring sideroblasts, reflecting the abnormal erythrocyte cyte morphology? metabolism, is a common finding. The International Work- 4. Calculate the mean cell volume (MCV). What ing Group on Morphology of MDS (IWGM-MDS) defines peripheral blood findings are helpful to rule out ring sideroblasts as erythroblasts in which at least five megaloblastic anemia? 5. What features of the differential resemble chronic myeloid leukemia (CML)? What helps to distin- guish this case from CML? Bone Marrow The bone marrow microenvironment contains pluripotent hematopoietic stem cells, capable of differentiating into all the peripheral blood cells. There are many other types of cells in the bone marrow that are ancillary to the devel- opment of hematopoietic progenitor cells; these include Figure 25.6 Megaloblastoid erythroblasts in the bone marrow osteoblasts, vascular endothelial cells, mesenchymal stem of a patient with MDS. Note the condensed chromatin and Howell- cells, and macrophages. The roles of these ancillary cells are Jolly bodies (Wright-Giemsa stain, 1000* magnification). |
564 Chapter 25 Figure 25.8 Bone marrow from a patient with MDS. Note the metamyelocytes (arrows) and bands with a lack of secondary granules. The bands have light bluish-pink cytoplasm, and the Figure 25.7 Arrow points to a multinucleated metamyelocytes have a darker bluish cytoplasm (Wright-Giemsa polychromatophilic erythroblast in the bone marrow of a patient stain, 1000* magnification). with MDS. It is also megaloblastoid (Wright-Giemsa stain, 1000* magnification). DYSMEGAKARYOPOIESIS Megakaryocytes can be decreased, normal, or increased. mitochondrial iron deposits encircle at least one-third or The presence of micromegakaryocytes, large mononuclear more of the circumference of the nucleus.22 megakaryocytes, and megakaryocytes with other abnormal nuclear configurations (Figure 25-9) reflect abnormalities in DYSGRANULOPOIESIS maturation. Megakaryocyte nuclei can be hyperlobulated Granulopoiesis is usually normal to increased in patients or hypolobulated and can show multiple widely separated with MDS unless the overall marrow is hypocellular. Abnor- nuclei. The lack of granules or presence of giant abnormal mal granulocyte maturation (dysgranulopoiesis), however, granules is also characteristic. Although dysmegakaryo- is almost always present (Figure 25-8). One of the major poietic morphology is potentially useful for diagnosis and findings of dysgranulopoiesis in the bone marrow is abnor- prognostication, identification of dysplasia is subject to mal staining of the primary granules in promyelocytes and individual interpretation.23 myelocytes. Sometimes the granules are larger than normal, and at other times are absent. Secondary granules can be absent in myelocytes and other more mature neutrophils, Molecular Diagnostics giving rise to the hypogranular peripheral blood neutro- Morphology contributes greatly to the diagnosis of MDS, phils. Irregular cytoplasmic basophilia with a dense rim of but genomic instability figures importantly in the develop- peripheral basophilia is also characteristic. Nuclear abnor- ment of myelodysplastic syndromes. Although it is becom- malities similar to those found in the peripheral blood ing increasingly essential to obtain information about a granulocytes can be present in bone marrow granulocytes. patient’s genetic profile for prognosis of MDS and overall a b Figure 25.9 (a) Abnormal megakaryocyte (arrow) from a patient with MDS. (b) Arrow points to a micromegakaryocyte in the bone marrow of a patient with MDS. Note the megaloblastoid features of the surrounding cells (Both are bone marrow; Wright-Giemsa stain, 1000* magnification). Myelodysplastic Syndromes 565 survival, at the present time, gene mutations are insufficient for a diagnosis of MDS and cannot be used as presumptive 6. Which of the hematopoietic cell lines exhibit evidence for a diagnosis. Information about chromosome dyshematopoiesis in the bone marrow? aberrations provides useful information to clinicians for diagnosing, prognosticating, tracking, and treating MDS. 7. How would you classify the bone marrow To date, it hasn’t been possible to assign specific genetic cellularity? abnormalities to the development of MDS, but certain aber- 8. What does the M:E ratio indicate? rations repeatedly have been identified in MDS patients. In broad terms, mutations associated with MDS affect 9. Identify at least two features of the bone marrow DNA methylation, DNA repair, chromatin/histone modi- that are compatible with a diagnosis of MDS. fication, transcription regulation, tumor suppression, and 10. What chemistry tests would be helpful to rule signal transduction.11,14–17 It is interesting to note that the out megaloblastic anemia? genetic profiles of patients with primary versus secondary MDS differ; those with primary MDS tend to have single chromosomal abnormalities, whereas patients with second- ary MDS are more likely to exhibit complex chromosomal abnormalities.9 DNA amplification, hybridization tech- Blast and Precursor Cell niques, and gene sequencing are molecular techniques that are employed to help identify genetic irregularities. Cyto- Classification genetic studies (karyotyping; Chapter 41) are also useful in For patients with MDS, the blast count appears to be the the diagnosis of MDS. most important prognostic indicator of survival and pro- gression to acute leukemia. The maximum number of blasts Additional Laboratory Evaluation compatible with a diagnosis of MDS is 19%, whereas the Serum iron and ferritin levels are normal or increased, minimum criterion for a diagnosis of acute leukemia is at and the total iron-binding capacity (TIBC) is normal or least 20% blasts. Correctly identifying blasts and type and decreased, distinguishing MDS from iron-deficiency ane- degree of dysplasia is important in MDS because these char- mia (IDA). Cobalamin (vitamin B acteristics are the basis of classifying MDS subtypes and dif- 12) and folic acid levels are normal to increased, a feature that helps to differentiate ferentiating them from AML with myelodysplasia-related MDS with megaloblastoid features from megaloblastic ane- changes (AML-MRC; Chapter 26).24 It is essential that suffi- mias with the typical megaloblastic features. Lactic dehy- cient cells be evaluated in order to accurately identify blasts drogenase (LD) and uric acid levels can be increased as a and precursor cells in suspected cases of MDS.25 The blast result of ineffective hematopoiesis. count in myeloid neoplasms is expressed as a percentage of all nucleated cells in the bone marrow, including eryth- roblasts. A total of 500 nucleated cells should be counted Checkpoint 25.4 in the bone marrow and 200 nucleated cells counted in the Why is serum cobalamin serum folate level or bone marrow iron peripheral blood. If cytopenia in the peripheral blood is stain important in diagnosing MDS? severe, a buffy coat should be used to perform the count. In 2005 and 2006, IWGM-MDS developed new diagnostic cri- teria for MDS and redefined MDS myeloblast morphology. CASE STUDY (continued from page 563) In addition to redefining myeloblast morphology, it pro- posed criteria necessary to define normal and dysplastic A bone marrow was performed on Hancock. The promyelocytes and ring sideroblasts. The morphological marrow showed a cellularity of about 75%. There categories of early cells in MDS established by the IWGM- was myeloid hyperplasia with 9% blasts, 26% MDS are granular myeloblasts, agranular myeloblasts, promyelocytes, 18% myelocytes, 6% metamyelo- normal promyelocytes, dysplastic promyelocytes, and ring cytes, 4% bands, and 37% eosinophils. The ratio of sideroblasts22 (Figure 25-10). myeloid-to-erythroid precursors (M:E) was 12:1. The myelocytes were hypogranular, and some had two nuclei. The erythroid precursors showed Myeloblasts megaloblastoid changes. Megakaryocytes were Myeloblasts are defined as either granular or agranu- adequate in number but showed abnormal forms lar. Granular blasts possess azurophilic granules and can with nuclear separation and single nucleated exhibit Auer rods, although this is an unusual finding. Gran- forms. ular blasts include the former type II and type III blasts, but today there is no differentiation of blasts based on the 566 Chapter 25 Dysplastic Agranular blast Granular blast Promyelocyte promyelocyte • High • Same morphologic • Central or eccentric • Irregular cytoplasmic nuclear/cytoplasmic characteristics as nucleus with nucleoli basophilia ratio agranular blast • Clearly visible Golgi • Poorly developed • Nucleoli and fine • Cytoplasmic granules zone Golgi zone nuclear chromatin • Azurophilic granules • Hyper- or • Basophilic cytoplasm • Basophilic cytoplasm hypogranularity or irregular distribution (clumps) of granules Figure 25.10 Blasts and promyelocytes as defined by the IWGM-MDS. number of granules observed in the cytoplasm. Agranular which can complicate the morphologic differentiation of blasts correspond to the earlier type I classification of MDS granular blasts and promyelocytes. Dysplastic promy- blasts. Whether granular or agranular, the blasts of MDS elocytes differ from normal promyelocytes in that their should exhibit visible nucleoli, fine nuclear chromatin, scant chromatin can vary from fine and lacy to coarse, their azu- basophilic cytoplasm, and, very importantly, should lack a rophilic granules can be of variable quantity and distribu- Golgi zone22,23 (Figure 25-11). tion, their cytoplasm can stain irregularly, and their Golgi zone may be only faintly visible.22,23 Promyelocytes Differentiating between granular blasts and promyelocytes Ring Sideroblasts is essential. Promyelocytes present with most of the features The IWGM-MDS has defined three types of sideroblasts, of granular blasts, including fine azurophilic cytoplasmic types 1, 2, and 3. granules. The IWGM-MDS has determined that the distin- Type 1 sideroblasts are defined as possessing fewer guishing feature between the two cells is the presence of a than five siderotic cytoplasmic granules. distinct nonstaining, perinuclear Golgi zone seen in pro- Type 2 sideroblasts have five or more siderotic granules, myelocytes. Promyelocytes of MDS can exhibit dysplasia, but they lack perinuclear distribution. Type 3 sideroblasts a b Figure 25.11 (a) Agranular myeloblast. (b) Granular myeloblasts (arrows) (Both: peripheral blood, Wright-Giemsa stain, 1000* magnification). Myelodysplastic Syndromes 567 exhibit five or more siderotic granules arranged around the nucleus of the erythroblast so that they encircle at least one- Classification third of the nuclear perimeter. A system to describe, define, and name diseases is impor- The IWGM-MDS recommends that a minimum of tant so that they can be diagnosed, treated, and studied. In 100 nucleated erythrocytes at all stages of maturation be 1982, the French-American-British (FAB) group proposed a included in the count. Note in the section “Classification” classification scheme for the myelodysplastic syndromes. that the WHO still defines the required number of ring sid- This classification defined five subgroups based on morpho- eroblasts as 15%.22,23 logical characteristics, such as the blast count and degree of dyspoiesis in the peripheral blood and bone marrow.2,24 Although the FAB classification system was the bench- Checkpoint 25.5 mark for diagnosing MDS for the past two decades, it did not Why is it important to correctly identify the number of blasts when evaluating the peripheral blood or bone marrow smear of incorporate the newer diagnostic technologies such as cytoge- a patient suspected of having MDS? netics and immunophenotyping.2 In 2001, the WHO published a new classification system developed by a group of American and European pathologists, hematologists, and oncologists. Immunological Identification This new system incorporated morphology, immunopheno- of Blasts type, and genetics with clinical and prognostic features into a classification of all neoplastic diseases of the hematopoietic In 2008, a new classification system devised by the WHO and lymphoid tissues (Chapter 23). This resulted in signifi- included immunophenotype abnormalities in the bone cant changes in the classification of MDS. The WHO system marrow cells of suspected MDS as an additional diagnostic of classification is updated and revised periodically. Major and prognostic tool (this classification system was updated changes in the 2008 revision eliminated previous categories in 2016). Immunophenotyping with flow cytometry detects of MDS and added new groups; the current subclasses of MDS the expression of antigen abnormalities in all cell lines. It are listed in Table 25-4. The most recent 2016 update mostly is most useful when marrow morphology and cytogenet- changes the terminology used to identify the various sub- ics are inconclusive, and to distinguish MDS cases with groups included in MDS. Terms such as “refractory anemia” a hypoplastic bone marrow from other bone marrow fail- and “refractory cytopenia” have been replaced with the term ure disorders, such as aplastic anemia.24,25 Aberrant anti- “myelodysplastic syndrome” amended with the modifiers: genicity observed in MDS includes lymphoid antigens single/multilineage dysplasia, ring sideroblasts (and single/ on myeloid cells, over- or under-expression of expected multilineage dysplasia), excess blasts, unclassifiable, and antigens, the presence of immature antigens on mature del(5q).11 A provisional category, refractory cytopenia of child- cells, and expression of mature antigens on immature cells hood (RCC), was added in the 2008 revision and remains in (Table 25-3).24,25 place in the 2016 update. The 2016 WHO Classification System Table 25.3 Characteristic Flow Cytometric Analysis of Myelodysplastic Syndromes Abnormal Expression Decreased Expression CD5, CD7, CD13, CD33, CD56 Progenitors, neutrophils, monocytes CD10 Neutrophils CD36, CD71 Erythrocyte precursors Side Scatter Neutrophils, monocytes CD14/CD36, CD16, CD36 Monocytes CD45 Progenitors, neutrophils, monocytes CD19 Neutrophils, Monocytes CD11b, CD14, HLA-DR Monocytes Increased Expression Loss of Synchronized Expression CD11b, CD15 Progenitors CD11b/CD13, CD11b/CD16, CD13/ Neutrophils CD36 Neutrophils CD16, CD15/CD10 Erythrocyte precursors CD117 Neutrophils, monocytes CD71/CD235 HLA-DR Neutrophils Side Scatter Progenitors CD11b/HLA-DR, CD34 Neutrophils, monocytes Decreased Populations Increased Populations CD19+/CD10+ B@cells CD34+/CD19@ Progenitors CD19+/CD34+ B@cells CD34+/CD38@/dim Progenitors CD117+ Progenitors CD117+ Erythrocyte progenitors CD34+/HLA@DR Progenitors 568 Chapter 25 Table 25.4 2016 World Health Organization Classification Criteria for Myelodysplastic Syndromes Classification Peripheral Blood Bone Marrow MDS with single lineage Unicytopenia or bicytopenia Unilineage dysplasia dysplasia (MDS-SLD) Rare or no blasts (less than 1%) 10% or more of cells in affected lineage are dysplastic No Auer rods Less than 5% blasts, less than 15% ring sideroblast No Auer rods MDS with ring sideroblasts (MDS-RS) • MDS-RS and single lineage dys- Anemia Erythroid dysplasia plasia (MDS-RS-SLD) Rare or no blasts (less than 1%) Less than 5% other blasts No Auer rods 15% or more ring sideroblasts • MDS-RS and multilineage Anemia No Auer rods dysplasia (MDS-RS-MLD) Rare or no |
blasts (less than 1%) Dysplasia in 10% or more of cells of two or more lineages No Auer rods less than 5% other blasts 15% or more ring sideroblasts No Auer rods MDS with multilineage Uni-, bi-, or pancytopenia Dysplasia in 10% or more of cells of two or more lineages dysplasia (MDS-MLD) Rare or no blasts (less than 1%) Less than 5% blasts in marrow No Auer rods Variable percentage of ring sideroblasts Less than 1 * 109/L monocytes No Auer rods Refractory anemia with excess blasts (MDS-EB) • MDS-EB-1 Uni-, bi-, or pancytopenias Unilineage or multilineage dysplasia Less than 5% blasts 5–9% blasts • MDS-EB-2 No Auer rods No Auer rods Uni-, bi-, or pancytopenia Unilineage or multilineage dysplasia 5–19% blasts 10–19% blasts { Auer rods { Auer rods MDS with isolated del(5q) Anemia and/or neutropenia Normal to increased megakaryocytes with hypolobulated nuclei Rare or no blasts Unilineage or multilineage dysplasia less than 5% blasts No Auer rods Isolated del(5q) cytogenetic abnormality No Auer rods MDS, unclassifiable (MDS-U) Uni-, bi-, or pancytopenia Unequivocal dysplasia in less than 10% of cells lines less than 5% blasts Uni-, bi-, pancytopenia No ring sideroblasts Rare or no blasts (less than 1%) Cytogenetic abnormalities characteristic of MDS Childhood MDS Less than 2% blasts; dysplasia in Less than 5% blasts; hypocellular Clusters of 20 or more erythroid precursors • Refractory cytopenia of childhood neutrophils More than 10% cells with uni-dysplasia, bi-, or multilineage dysplasia criteria for each subgroup of MDS are shown in Table 25-4. • MDS-RS and single lineage dysplasia (MDS-RS-SLD) Studies to evaluate the clinical usefulness of the WHO clas- • MDS-RS and multilineage dysplasia (MDS-RS-MLD) sification conclude that it clearly identifies more homogenous 3. MDS with multilineage dysplasia (MDS-MLD) subgroups and enables clinicians to select the best treatments and better predict prognosis and clinical responses.11 At this 4. MDS excess blasts (MDS-EB) time, most clinicians have adopted the 2016 WHO Classifica- • MDS-EB-1 tion System, and it is followed in this chapter. • MDS-EB-2 5. MDS with isolated del(5q) 6. MDS, unclassifiable Description of MDS 7. Childhood MDS Subgroups • Refractory cytopenia of childhood (provisional) The 2016 WHO Classification System identifies the follow- MDS with Single Lineage Dysplasia ing myeloplastic syndromes: (MDS-SLD) 1. MDS with single lineage dysplasia (MDS-SLD) The category MDS with single lineage dysplasia is for 2. MDS with ring sideroblasts (MDS-RS) cases with isolated cytopenia or bicytopenia accompanied Myelodysplastic Syndromes 569 by unilineage dysplasia. It includes refractory anemia MDS with Multilineage Dysplasia (RA) and less common cases of refractory neutropenia and refractory thrombocytopenia. These cases together make up (MDS-MLD) approximately 10–20% of all types of MDS; refractory neu- MDS with multilineage dysplasia (MDS-MLD) is char- tropenia and refractory thrombocytopenia represent only acterized by dysplastic features in at least 10% of the cells 1–2%. In general, these cases have an extended course with in two or more cell lineages, less than 5% blasts in the bone the rate of progression to AML of less than 5% at 5 years. marrow, and less than 1% blasts in the peripheral blood. Most patients present with a normocytic, normochomic, or This is one of the most common subgroups, representing sometimes macrocytic anemia. Variation in size and shape 30–40% of all MDS cases. Studies have shown that individ- of the red blood cells is common. Less than 1% blasts are uals with MDS-MLD have a worse prognosis than those present in the peripheral blood and less than 5% blasts with dysplasia in only one lineage; thus, a new subgroup are in the bone marrow. The bone marrow is hypercellular was added in 2001.25 Patients usually display cytopenias in because of increased erythropoiesis or sometimes normo- two or more lineages and have less than 1 * 109/L mono- cellular. Dyserythropoiesis is present without prominent cytes present in the peripheral blood. Ring sideroblasts can ring sideroblasts. In MDS with single lineage dysplasia, exceed 15% of the nucleated red blood cells in the marrow. dysplasia is seen in less than 1–2% in the granulocytic and Chromosomal abnormalities can be present in up to 50% of thrombocytic cells. cases in this category, some being complex. The frequency Chromosomal analysis is frequently helpful in diagnos- of evolution to AML is approximately 10% within 2 years ing MDS with single lineage dysplasia because of the mini- of onset. mal morphologic changes in some cases. Excluding other reactive causes of cytopenias and associated dysplasia is also important. Patients with pancytopenia and morpho- MDS with Excess Blasts (MDS-EB) logic dysplasia of only one cell line should be classified as In MDS with excess blasts (MDS-EB), cytopenia occurs MDS, unclassified rather than this category. in at least two lineages and conspicuous qualitative abnor- malities are in all three lineages. The anemia is normocytic or slightly macrocytic with reticulocytopenia. Evidence MDS with Ring Sideroblasts of dysgranulopoiesis is prominent. Monocytosis without (MDS-RS) leukocytosis can be present, but the absolute monocyte count does not exceed 1 * 109/L, and serum and urinary The 2016 revision of the WHO classification of MDS changes lysozyme levels are normal. Platelet abnormalities include the name of refractory anemia with ring sideroblasts to giant forms, abnormal granularity, and functional aberra- MDS with ring sideroblasts (MDS-RS) and divides this tions. Sometimes circulating micromegakaryocytes can be subgroup further, into MDS-RS with single lineage dyspla- found. sia and MDS-RS with multilineage dysplasia.22 Mutations to The bone marrow is hypercellular but less often is a spliceosome gene SB3B1 is often seen in MDS, it appears normocellular with varying degrees of granulocytic and to be an early mutation event in the development of MDS. erythrocytic hyperplasia. All three lineages show signs of This mutation correlates well with the appearance of ring dyshematopoiesis. Blast numbers below 20% define MDS; sideroblasts. The presence or absence of a mutated SF3B1 blasts in excess of this number changes the neoplasm to gene and the number of ring sideroblasts both contribute to AML. Abnormal promyelocytes can be present. These the inclusion of an MDS syndrome into the MDS-RS class; abnormal cells have blast-like nuclei with nucleoli and no if there are between 5 and 15% ring sideroblasts among the chromatin condensation, and the cytoplasm contains large erythroid precursors, the SF3B1 mutation must be present bizarre granules. Ring sideroblasts may be increased, but for inclusion in MDS-RS. If there are at least 15% ring sid- the elevated blast count differentiates MDS-EB from MDS- eroblasts, the presence of mutated SF3B1 becomes irrelevant RS. In some cases, differentiating MDS-EB from acute leuke- since the presence of at least 15% ring sideroblasts qualifies mia is difficult. Serial examinations are sometimes necessary the syndrome as MDS-RS.2,22 The anemia is usually macro- to make an accurate diagnosis. cytic and is less often normocytic. Sometimes evidence of a There are two subgroups of MDS-EB based on the blast dual population of normochromic and hypochromic cells count, emphasizing the importance of determining blast exists. The peripheral blood shows reticulocytopenia and quantity for purposes of therapy.26 In MDS-EB-1 there are often leukopenia. less than 5% blasts in the peripheral blood and 5–9% blasts The bone marrow is hypercellular with megaloblastoid in the bone marrow. Auer rods are not present in the blood dyserythropoiesis if the subclassification is determined to be or bone marrow. In MDS-EB-2 there are 5–19% blasts in the a single lineage dysplasia. If dysgranulopoiesis and/or dys- peripheral blood and 10–19% blasts in the bone marrow. megakaryopoiesis are also present, the classification becomes MDS-EB-2 may have Auer rods present. Generally, the num- MDS with ring sideroblasts and multilineage dysplasia. ber of bone marrow blasts serves as a prognostic indicator, 570 Chapter 25 with patients having 5–9% bone marrow blasts experienc- third type of MDS-U, one that is based on defining cytoge- ing a longer median survival time than those patients with netic abnormality. In this unclassifiable MDS type, there are 10–19% blasts.27 Up to 50% of patients with the increased rare or no blasts in the peripheral blood, dysplastic changes blast count progress on to AML, and even patients with to single or multiple cell lineages, and Auer rods are not the lower blast count have a 25% likelihood of progress- observed. Up to 15% ringed sideroblasts may be present, ing to acute leukemia. Current WHO classification of MDS but other blasts in the bone marrow are less than 5%. To be is based on the percentage of blasts in total bone marrow placed in this category the cells must exhibit an MDS defin- nucleated cells, but there is a movement toward calculating ing abnormality.26 blast percentage from nonerythroid nucleated cells only.17,27 Recent studies indicate that enumeration of blasts from Refractory Cytopenia of Childhood nonerythroid nucleated cells rather than from total nucle- ated cells in the bone marrow allows a superior prognostic Childhood MDS is rare and accounts for less than 5% of assessment of MDS.27 cases of hematopoietic neoplasms in children. The mani- festation of MDS in adults and in children has major dif- MDS with Isolated del(5q) ferences, and pediatric hematologists deemed previous classification systems not useful.1,2 MDS patients with an isolated deletion of the long arm of The 2008 WHO classification of MDS introduced chromosome 5 (del[5q] or 5q- ) and no other chromosomal a provisional entity, refractory cytopenia of childhood abnormalities appear to have a unique disease course char- (RCC), specifically for childhood cases of MDS with less acterized by a favorable prognosis and low risk of transfor- than 2% blasts in the peripheral blood and less than 5% mation into AML. Since there is no adverse effect associated in the marrow and persistent cytopenias associated with with this subgroup having one additional chromosomal dysplasia in any or all cell lines (this is retained in the 2016 abnormality, MDS with isolated del(5q) can be diagnosed WHO classification). Other childhood cases of MDS with if there is an additional cytogenetic abnormality except if 2–19% blasts in the peripheral blood and 5–19% blasts that abnormality is monosomy 7 or del(7q). Women have a in the bone marrow should be categorized the same as marked predominance of cases (70%), and the mean age at for adults. Dysplasia in children with MDS is less pro- presentation is 66 years. The main features are macrocytic nounced, and the more aggressive subtype (MDS-EB) pre- anemia, moderate leukopenia, normal to increased plate- dominates; progression to acute leukemia is faster than let count, hypolobulated megakaryocytes, and less than in adults. Age of 2 years or less and a hemoglobin F level 1% blasts in the peripheral blood and less than 5% in the of 10% or higher are associated with a poor prognosis.30 bone marrow. The bone marrow is usually hypercellular or Cytogenetic abnormalities are seen in approximately 70% normocellular with normal to increased megakaryocytes, of cases, and monosomy 7 is the most common cytoge- some with hyperlobulated nuclei.28 The JAK2 V617F muta- netic change. Unlike adults, abnormalities of chromosome tion has recently been found in a small subset of patients 7 do not seem to be associated with a poor prognosis in with isolated del(5q). These patients have slightly higher children. One-third of children with MDS have genetic WBC and platelet counts but no separate classification has predisposition syndromes, such as Down syndrome.30 been established to date.29 These cases have been moved to a new category, myeloid leukemia in Down syndrome, and are now excluded from MDS, Unclassifiable (MDS-U) the MDS.31 MDS-U has variable presentation, and cases assigned to this category do not fit into any of the defined WHO subgroups. Checkpoint 25.6 There are several types of unclassifiable myelodysplastic When a diagnosis of MDS is considered for a patient, why must syndromes, and they present with variable findings. MDS-U other causes of reactive cytopenias be ruled out? What type of with 1% peripheral blood blasts also presents with uni-, bi-, testing helps determine the diagnosis of MDS? or pancytopenia and dysplastic changes in one or more cell lines. Auer rods are not seen in either the peripheral blood or in bone marrow cells. There are no ringed sideroblasts and the percentage of other blasts in the bone marrow is CASE STUDY (continued from page 565) less than 5%. MDS-U with single lineage dysplasia has rare Review Hancock’s peripheral blood and bone mar- or no blasts in the peripheral blood, but pancytopenia, and row features previously identified. |
dysplastic changes in a single cell line. Auer rods are not seen in either the peripheral blood or in bone marrow cells. 11. What is the most likely MDS subgroup, and on There are no ringed sideroblasts and the percentage of other what criteria is the answer based? blasts in the bone marrow are less than 5%. Finally, there is a Myelodysplastic Syndromes 571 Variables of MDS typically have pancytopenia, excess blasts, hypocellular bone marrow with fibrosis, trilineage dysplasia, small Subgroups megakaryocytes with hypolobulated nuclei, and no hep- atomegaly or prominent splenomegaly.34 The increased A number of patients have blood and/or marrow find- fibrosis is thought to be produced by the liberation of cyto- ings that cause diagnosis and/or classification problems. kines such as transforming growth factor@b (TGF@b) and Some of these occur often enough to consider them as platelet-derived growth factor (PDGF) from dysplastic variables within MDS subgroups, but the 2008 WHO megakaryocytes.33, 34 classification does not formally recognize them. The recommendation is that these cases be classified accord- ing to one of the subgroups and note the unusual mor- Therapy-Related Myelodysplasia phologic features, such as “MDS-EB-2 with hypoplastic In 2008, the WHO Classification of Hematopoietic Neo- marrow.”3,18 plasms and the International Agency for Research on Cancer placed neoplasms that develop after a patient has received Hypoplastic MDS chemotherapy or radiation therapy for malignancies into a special category, therapy-related myeloid neoplasm (t-MN). Although most MDS cases are associated with hypercellular The category includes therapy-related myelodysplastic syn- or normocellular bone marrows, about 5–10% have hypocel- dromes (t-MDS) and therapy-related acute myeloid leuke- lular marrows. The hypocellular marrow does not appear mia (t-AML).35 to affect survival rate, nor does it influence progression to Development of MDS or acute leukemia (AL) appears AML.29 In these cases, bone marrow biopsy is necessary to to be related to the therapy’s duration, amount, and repeti- exclude a diagnosis of aplastic anemia or hypoplastic AML. tion as well as the patient’s age. The highest incidence of This distinction is important because the diagnosis influ- t-MDS occurs 3–8 years after treatment, and long-term use ences treatment and prognosis. of DNA alkylating agents appears to present the highest Hypoplastic MDS should be considered when the risk.2,7 On a molecular level there is a high frequency of bone marrow cellularity is less than 30% or less than 20% TP53 gene mutations, suggesting that aberrant DNA repair in patients older than 60 years of age. The criteria for MDS and dysregulated cell apoptosis underlie development of must be met in hypoplastic cases as well as in the hyper- myeloid neoplasms.36 Mutational analysis in patients who cellular or normocellular cases. Dysplasia can be difficult have developed t-MDS reveal aberrant gene expression to identify, and dyserythropoiesis has been described in related to mitochondrial function, protein synthesis, DNA aplastic anemia. Dysmegakaryopoiesis and dysgranulo- repair, and regulation of hematopoiesis. poiesis, however, are most characteristic of MDS and can be helpful findings. In addition, atypical localization of immature precursors (ALIP), indicating abnormal bone marrow architecture, is typical of MDS. If present, chromo- Differential Diagnosis somal abnormalities help distinguish MDS from aplastic anemia as does immunophenotyping by flow cytometry MDS must be differentiated from other causes of cytope- for CD34 and the presence of megakaryocytes.28 The dis- nia and dysplasia that mimic MDS.4,37 Possible reactive tinction of MDS from AML can be made based on the etiologies of dysplasia should be considered, especially blast count. A count greater than 19% indicates AML. when the dysplasia is limited to a single cell line. Dys- Although the pathophysiology of hypoplastic MDS is plastic cells in excess of 10% (the MDS threshold) may unknown, secretion of inhibitory cytokines by autoreactive occur in normal individuals and in non-neoplastic causes or clonal-involved T cells is believed to suppress normal of cytopenia. A smaller percentage of dysplastic nor- hematopoiesis.32 moblasts and megakaryocytes may be seen in the bone marrow of all age groups37 and disorders with erythroid MDS with Fibrosis hyperplasia can be associated with significant erythroid dysplasia. The differential diagnosis of MDS from other Mild to moderate fibrosis has been described in up to causes of dysplasia is particularly important in, but not 50% of patients with MDS; marked fibrosis can be seen limited to, deficiencies of vitamin B12, folate, and copper, in 10–15% of cases.27,33 The incidence of fibrosis appears abusive alcohol consumption, chemotherapeutic drugs, to be even higher in therapy-related MDS. If fibrosis is and certain infections. present, other diagnoses including primary myelofibrosis Nutritional deficiency of vitamin B12 and folate are (Chapter 24), chronic myelogenous leukemia (Chapter 24), associated with cytopenias and dysplasia of all three cell and acute megakaryoblastic leukemia (Chapter 26) should lineages; dysplastic changes include macro-ovalocytes, be considered and excluded. MDS patients with fibrosis giant metamyelocytes and bands, nuclear cytoplasmic 572 Chapter 25 asynchrony, and hypersegmented neutrophils. Vitamin The WHO Classification-Based Prognostic Scoring B12 and serum methylmalonic acid (MMA) should be mea- System (WPSS) was published in 2011 (Table 25-5).This sured to evaluate vitamin B12 if the classic morphologic prognostic scoring system utilizes the WHO category of abnormalities of hypersgmented neutrophils and macro- MDS classification, karyotype, and transfusion require- ovalocytes are found. ments and assigns patients to five risk groups of survival Copper deficiency may masquerade as MDS with ane- and probability of leukemic progression.1,44 mia, neutropenia, and thrombocytopenia in the peripheral In 2012 a revised International Prognostic Scoring blood.38,39 The bone marrow reveals vacuoles in erythroid System (IPSS-R) was published that integrated additional and myeloid precursors, iron containing plasma cells, and clinical features to improve prognostic assessment1,44 decrease in granulocyte precursors and ring sideroblasts; (Table 25-6). The IPSS-R incorporated additional new these changes are often found in copper-deficient individu- cytogenetics and clinical features including the degree of als who have increased zinc supplementation. cytopenia of three lineages (erythroid, platelets, and neu- Alcohol abuse can cause direct suppression of the bone trophils). The karyotypes were split into five cytogenetic marrow, resulting in cytopenias. Associated nutritional prognostic subgroups, rather than the three subgroups deficiencies of folate and copper can lead to morphologic used in the original IPSS, and the marrow blast percent- changes, as described above. Vacuolated erythroblasts in age values were adjusted. It was further refined by cat- the bone marrow is a transient feature of acute alcohol use. egorizing patients into five rather than four prognostic Many medications including chemotherapeutic agents, categories. The IPSS-R is based on a much larger combined antibiotics, and some immunosuppressive agents can cause database of patients provided by the International Working hematopoietic dysplasia. G-CSF can cause an increase in Group for Prognosis in MDS (IWG-PM) and is expected to peripheral blood blasts as well as hypergranular and hypol- provide an improved prognostic categorization model.1,44 obular neutrophils. Infection with Parvovirus B19 can cause decreased ery- throid precursors and giant pronormoblasts.40 HIV infection CASE STUDY (continued from page 570) is associated with trilineage dysplastic changes and abnor- malities of bone marrow cellularity.41 Hepatitis C infection Hancock’s karyotope showed multiple complex may cause dyserythropoiesis.42 abnormalities. In some diagnostically ambiguous cases the patient is 12. Using the IPSS-R, what is the prognosis for diagnosed with CHIP or ICUS and followed until further Hancock? information on their condition is available.4 Prognosis Table 25.5 WHO Classification-Based Prognostic Scoring System (WPSS) of Myelodydysplastic Syndromes The median survival for all types of MDS is less than 2 years; however, some patients can survive many years Points WHO Category Karyotypea Transfusion requirementbwith continuous transfusion therapy. The mortality rate varies from 58–72%. Leukemic transformation ranges in 0 MDS-SLD, MDS-RS- Good None incidence from 10–40%. The likelihood of transformation to SLD, MDS-5q AML increases with the presence of severe cytopenias, more 1 MDS-MLD, Intermediate Regular MDS-RS-MLD overt dysplastic features of cells, and complex chromosome 2 MDS-EB-1 Poor – abnormalities.43,44 3 MDS-EB-2 – – The International Prognostic Scoring System (IPSS) Use summed points to determine risk category was developed in 1997 to assist physicians in predicting WPSS Risk Group Score prognosis and selection of optimal therapy for individuals diagnosed with MDS. This system was developed using Very low 0 data from seven large studies that had previously gen- Low 1 erated prognostic systems and was accepted worldwide. Intermediate 2 The prognostic score was based on the percentage of blasts High 3–4 in the bone marrow, cytogenetic abnormalities, and num- Very high 5–6 ber of cytopenias, and patients were divided into four risk a Karyotypes: Good = normal, -Y, del(5q- ) only, del(20q- ) only. Poor = complex (3 or groups with distinct risks of death and leukemic trans- more abnormalities), or chromosome 7 abnormalities. Intermediate = all others. b Transfusion dependency is defined as at least one red blood cell transfusion every 8 formation. Low scores indicate prolonged survival.1,17,43,44 weeks over a period of 4 months. Myelodysplastic Syndromes 573 Table 25.6 International Prognostic Scoring System-Revised (IPSS-R) for the Myelodysplastic Syndromes Prognostic Variable 0 0.5 1 1.5 2 3 4 Marrow blasts % …2 72965 5–10 710 Karyotypea Very Good Good Intermediate Poor Very Poor Hemoglobin (g/dL) Ú10 8–10 68 Platelets (*109/L) Ú100 5096100 650 Absolute neutrophil Ú0.8 60.8 count (*109/L) IPSS Risk Group Median Survival (years); Score no treatment Very low 8.8 …1.5 Low 5.3 71.593 Intermediate 3.0 7394.5 High 1.6 74.596 Very high 0.8 76 a Cytogenetic prognostic subgroups include: Very Good [-Y, del(11q)]; Good [normal, del(5q), del(12p), del(20q), double including del(5q)]; Intermediate [del(7q), +8, +19, i(17q), any other single or double independent clones]; Poor [-7, inv(3)/t(3q)/del(3q), double including -7/del(7q), complex: three abnormalities]; Very Poor (complex: more than three abnormalities). Therapy myelocytic cell lines may contribute to the development of MDS.2 Myelodysplastic syndromes are so heterogeneous that The U.S. Food and Drug Administration (FDA) has therapy must be based on the patient’s risk stratifica- approved three drugs for the treatment of MDS: azacitidine, tion, transfusion needs, the number of blasts seen, and decitabine, and lenalidomide. Azacitidine and its deoxy more recently, the patient’s cytogenetic profile. Patients derivative decitabine are pyrimidine nucleoside analogues should be stratified into lower-risk patients and higher- of cytidine. As such, they incorporate into DNA to act as risk patients. Lower-risk patients include those whose risk false substrates and in this way reversibly inhibit DNA score is determined to be Low or Intermediate-1 (IPSS); methyltransferase to block DNA methylation. The result- Very Low, Low, or Intermediate (IPSS-R); or Very Low, ing hypomethylation can activate tumor suppressor genes Low, Intermediate (WPSS). Higher-risk patients are those silenced by hypermethylation, producing an antitumor whose risk score is Intermediate-2 or High (IPSS); Inter- effect. Although the mechanism of action of lenalidomide mediate, High, or Very High (IPSS-R); or High, Very High remains incompletely understood, it appears to have multi- (WPSS).2 ple mechanisms of action including direct antitumor effect, The therapeutic strategy for patients evaluated as inhibition of the microenvironment that supports tumor lower risk is to improve their relevant cytopenias. Lineage cells, and an immunosuppressive function. Interestingly in appropriate growth factors may be effective in counter- MDS with del(5q), lenalidomide inhibits the malignant cell ing the cytopenias of MDS. Antibiotics are necessary for line, but in MDS without 5q deletions, lenalidomide exerts infections that are likely to follow neutropenia but are not its effect by promoting erythropoiesis.44,45 recommended as a prophylactic treatment. Since transfu- The treatment options for patients with higher-risk sions are often indicated to relieve anemia in MDS, iron MDS could include the same treatment options for lower overload is a concern. The National Comprehensive Can- risk patients but traditionally have also utilized classi- cer Network (NCCN) recommends the use of iron chela- cal combination therapy with anthrocycline and cytara- tion therapy in patients with ferritin levels 2500 mg/L or bine. The most important prognostic factor governing more.2,25,43 the response of patients to such AML-like therapy is the Immunosuppressive therapy such as that with cyclo- patient’s karyotype. Patients with -7, del (7q), or com- sporine or antithymocyte globulin (ATG) is controver- plex karyotype do not respond favorably to this regimen.45 sial but may be considered in some lower-risk patients. Allogeneic stem cell transplantation is usually not recom- ATG consists of pooled antibodies against human mended for patients with lower-risk MDS but remains a T-l ymphocytes. This treatment can be effective because viable treatment option for children and higher risk patients abnormal autoimmune activity of activated T-cells against and is potentially curative.2,25,44 574 Chapter 25 Myelodysplastic/ Clinical symptoms can |
result from cytopenia(s) when present, dysplastic cells that do not function properly, and Myeloproliferative leukemic infiltration of various organs. Splenomegaly and hepatomegaly are commonly found, but the clinical pre- Neoplasms (MDS/MPNS) sentation is highly variable. The incidence varies widely, depending on the specific disease, as do the prognosis and The category of myelodysplastic/myeloproliferative neo- tendency for clonal evolution and disease progression. The plasms (MDS/MPNs) includes clonal hematopoietic neo- disorders included in MDS/MPN are: plasms that, at the time of initial presentation, have some clinical, laboratory, or morphologic findings of both an MDS • Chronic myelomonocytic leukemia (CMML) and an MPN.24,46 Typically, patients with MDS/MPN have • Atypical chronic myeloid leukemia, BCR/ABL1- a hypercellular bone marrow associated with proliferation • Juvenile myelomonocytic leukemia (JMML) in one or more of the myeloid lineages. Frequently, the pro- liferation is effective and results in increased numbers of • MDS/MPN with ring sideroblasts and thrombocytosis circulating cells. However, these cells commonly are mor- (MDS/MPN-RS-T) phologically and functionally dysplastic. While one or more • MDS/MPN, unclassifiable (Table 25-7) lineages can show hyperproliferation, one or more other lineages can exhibit ineffective proliferation and resulting Chronic Myelomonocytic Leukemia cytopenia(s). The percentage of blasts in the bone marrow and peripheral blood is always less than 20% At present, (CMML) there are no identified genetic defects specific for any of the Chronic myelomonocytic leukemia (CMML) is a clonal entities included in this category. However, recurring chro- hematopoietic neoplasm associated with a persistent mosomal and molecular abnormalities have been described monocytosis (greater than 1 * 109/L) in the peripheral (Table 25-7). blood (Figure 25-12). Although the finding of absolute Table 25.7 The Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPNs) Classification Peripheral Blood Bone Marrow Genetics Immunophenotype Chronic myelomonocytic Monocytosis 610% blasts (including BCR/ABL1- CD33, CD13+ , leukemia (CMML-1) More than 1 * 109/L; less promonocytes); dysplasia CD14, CD68, than 5% blasts (including of one or more myeloid CD64 variable; lysozyme+ promonocytes) lineages CMML-2 5–19% blasts (including 10–19% blasts (including (same as CMML-1) (same as CMML-1) promonocytes) promonocytes or when Auer rods present, regardless of blast/promonocyte count) Atypical chronic Leukocytosis (less than Hypercellular; less than 20% BCR/ABL1- CD33, CD13+ , MPO+ myeloid leukemia 10% monocytes); less than blasts; dysgranulopoiesis; No evidence of PDGFA, (aCML, BCR/ABL1-) 20% blasts; more than 10% without dyserythropoiesis PDGFRB, or FGFR1 promyelocytes, myelocytes, or megakaryopoiesis rearrangement metamyelocytes No PCM1-JAK2 No/minimal basophilia Juvenile myelomonocytic Monocytosis Hypercellular; less than 20% BCR/ABL1- CD33, CD13+ , leukemia (JMML) More than 1 * 109/L; less blasts; dysgranulopoiesis; Somatic mutation in CD14, CD68, than 20% blasts occasionally dyserythropoiesis PTPN11, KRAS, NRAS, CD64 variable; lysozyme+ or NF1 mutation, germ line CBL mutation, loss of heterozygosity of CBL, monosomy 7 Myelodysplasic/ Anemia with or without More than 15% ring SF3B1 mutation; myeloproliferative multilineage dysplasia; less siderblasts BCR@ABL1-; neoplasm with ring than 1% blasts; persistent Less than 5% blasts no rearrangement of sideroblasts and thrombocytosis PDGFRA, PDGFRB, thrombocytosis FGFR1; no PCM1-JAK2, (MDS/MPN-RS-T) no 3;3(q21;q26), inv(3)) (q21;q26), del(5q) Myelodysplastic/ Anemia, leukocytosis and/ Hypercellular; proliferation in BCR/ABL1- Nondiagnostic myeloproliferative or thrombocytosis; less than any or all myeloid lineages neoplasm, unclassifiable 20% blasts (MDS/MPN, U) Myelodysplastic Syndromes 575 characterized by primary involvement of the neutrophil series with leukocytosis involving dysplastic immature and mature neutrophils. Multilineage dysplasia is common. The annual incidence of aCML, BCR/ABL1- is about 3 in 100,000. Most patients are older with the median age at diagnosis in the seventh to eighth decade. Median survival time is less than 20 months, and progression to acute leuke- mia is seen in 25–40% of cases. The cell of origin is believed to be the common myeloid progenitor cell (CMP). Reported cytogenetic abnormalities are similar to CMML. The BCR/ABL1 fusion gene is absent, and the specific genes SETBP1 and ETNK1 are mutated in about 30% of the cases.47 For a diagnosis of aCML, BCR/ABL1-, the peripheral blood WBC must be in excess of 13 * 109/L with increased Figure 25.12 dysplastic neutrophils and their precursors. Promyelocytes, Increased monocytes in the peripheral blood myelocytes, and metamyelocytes comprise more than 10% from a patient with CMML. (Wright stain, 1000* magnification) and blasts constitute less than 20% of the peripheral blood leukocytes. Absolute monocytosis and basophilia are usu- monocytosis is required for diagnosis, other hematologic ally absent, but their presence doesn’t preclude a diagnosis findings are remarkably variable. Leukocytosis, leukopenia, of aCML. Dysplasia and ineffective hematopoiesis in other neutrophilia, and neutropenia are all possibilities in CMML. cell lines frequently result in anemia and thrombocytopenia. Dyshematopoiesis can range from minimal expression in a aCML, BCR/ABL1- typically presents with a hypercel- single lineage to marked dysplasia in all the lineages. lular bone marrow. Dysplastic granulocytes account for most The cell of origin is believed to be the HSC. Clonal cyto- of the hypercellularity, dysplasia of the erythrocytic and genetic abnormalities are found in 20–40% of patients, but megakaryocytic lineages may or may not be present. Immu- none are specific. The most frequently reported abnormali- nophenotype results are typical for the neutrophil lineage. ties include SRSF2, TET2, and ASXL1. Somewhat less fre- quently seen gene mutations include SETBP1, NRAS/KRAS, RUNX1, CBL, and EZH2 (Table 25-1). Checkpoint 25.7 The WHO recommendations of diagnostic criteria for How do the laboratory findings of aCML, BCR/ABL1- differ from CMML are as follows: peripheral blood monocytosis greater those observed in CML (Chapter 24)? than 1 * 109/L, less than 20% blasts (myeloblasts, monoblasts, and promonocytes) in the peripheral blood and bone marrow, dysplasia in one or more myeloid lineages, absence of the BCR/ Juvenile Myelomonocytic Leukemia ABL1 fusion gene, and no rearrangement of PDGFRA, PDG- Juvenile myelomonocytic leukemia (JMML) is an aggres- FRB, or FGFR1 genes.23,46 The WHO recommends classifying sive childhood myeloproliferative disorder characterized CMML into two subgroups based on blasts or blast equiva- by proliferation and immaturity of the granulocytic and lents (promyelocytes) seen in the bone marrow (Table 25-7), monocytic lineages in the peripheral blood and bone mar- but recently stratification of CMML into three blast-based row. Dysmyelopoiesis and dyserythropoiesis but rarely groups has been suggested for better prognostication. The dysmegakaryopoiesis are evident in the bone marrow. Con- three proposed groups are CMML-0 when there are less than comitantly, as a result of the myelomonocytic expansion in 2% blasts in the PB and less than 5% BM blasts; CMML-1 the bone marrow, JMML patients often present with anemia when PB/BM blasts are 2–4%/5–9%; and CMML-2 for cases and thrombocytopenia. with blasts of 5–19% in the PB and 10–19% in the BM.46 The cell of origin is believed to be the HSC. Gain of function mutations of N-RAS and K-RAS and disruption Atypical Chronic Myeloid Leukemia of tumor suppressor genes are essential initiating events in (aCML, BCR/ABL1−) the development of JMML. Moreover, somatic mutations in PTPN11 (that encodes a protein tyrosine phosphatase) have The 2008 revision of the WHO classification of myeloid been found in 35% of JMML cases and have been recom- neoplasms renamed atypical chronic myeloid leukemia mended for inclusion in the diagnostic criteria.48 Monosomy (aCML) to aCML BCR/ABL1- to emphasize that the disor- 7 can also be included for diagnostic purposes. der is not merely an atypical CML. Atypical chronic myeloid The annual incidence of JMML is 0.13/100,000 children leukemia (aCML, BCR/ABL1-) is a variant of MDS/MPN (age 0–14 years of age). It accounts for less than 2–3% of all 576 Chapter 25 leukemia in children. The age at diagnosis is variable (from in this subtype even if the SF3B1 mutation is detected, 1 month to early adolescence), but 75% of cases are diag- whereas a myelodysplastic syndrome may be classified as nosed in children less than 3 years old. A significant associa- MDS-RS with less than 15% ring sideroblasts as long as the tion of JMML with neurofibromatosis type 1 (NF-1) exists, SF3B1 mutation is present. MDS/MPN-RS-T often presents with about 10% of cases of JMML occurring in children with with the JAK2V617F mutation as well. The JAK2 mutation is this diagnosis. The overall prognosis is poor, although sur- seen in several myeloproliferative diseases, and putatively vival times can vary based on the type of therapy chosen. underlies the thrombocytosis seen in these neoplasms.32 Without effective treatment, most children die from organ failure as the result of leukemic infiltration. About 10–20% Myelodysplastic/Myeloproliferative progress to acute leukemia. The following criteria have been established for the Neoplasm, Unclassifiable (MDS/ diagnosis of JMML: absolute monocytosis (more than MPN, U) 1 * 109/L), less than 20% blasts in the peripheral blood This subcategory, MDS/MPN, U, is used for cases that have and bone marrow, absence of the BCR/ABL1 fusion gene clinical, laboratory, and morphologic features that support a in addition to at least two of the following: myeloid pre- diagnosis of both MDS and MPN but do not meet the crite- cursors in the peripheral blood, leukocytosis of more than ria of the other entities included in the MDS/MPN category. 10 * 109/L, increased fetal hemoglobin for the patient’s No cytogenetic or molecular genetic findings are specific for demographic, or hypersensitivity of the hematopoietic pro- this group. The presence of the BCR/ABL1 fusion gene must genitor to GM-CSF. always be excluded. The MDS/MPN, U disorders are characterized by the MDS/MPN with Ring Sideroblasts proliferation of one or more of the myeloid lineages that and Thrombocytosis (MDS/ are ineffective and/or dysplastic. Proliferation of the other MPN-RS-T) myeloid lineages is generally effective with or without dysplasia. Anemia (with or without macrocytosis) usually In earlier classifications, this category was known as refrac- occurs, occasionally with a dimorphic population of RBCs. tory anemia with ring sideroblasts and thrombocytotosis The bone marrow is hypercellular and can show prolifera- (RARS-T).23 MDS/MPN-RS-T is very similar to MDS-RS tion in any or all of the myeloid lineages. Dysplastic features with respect to the ring sideroblasts present in the erythroid are present in at least one lineage. Typically, the patient has precursors of the bone marrow. In MDS-RS, the presence of a less than 20% blasts in the blood and bone marrow and mutated SF3B1 gene is well correlated with the appearance prominent myeloproliferative features associated with of ring sideroblasts and this is true for MDS/MPN-RS-T as either thrombocytosis or leukocytosis. The bone marrow well. With MDS/MPN with ring sideroblasts and thrombocy- and peripheral blood are always involved; extramedullary tosis, at least 15% ring sideroblasts are required for inclusion tissues (liver, spleen) are sometimes also involved. Summary The myelodysplastic syndromes are pluripotential hema- bone marrow is abnormal with megaloblastoid features com- topoietic stem cell disorders characterized by one or more monly present. Neutrophils can show hyposegmentation of peripheral blood cytopenias and prominent cellular matura- the nucleus and hypogranulation. Megakaryocytes also show tion abnormalities. The bone marrow is usually normocellular megaloblastoid features. Platelets can be large and agranular. or hypercellular, indicating a high degree of ineffective hema- The WHO group has classified the MDSs into six sub- topoiesis. The most commonly encountered abnormal karyo- groups depending on the blast count, degree of dyspoie- types in MDS are the -5/del(5q), -7/del (7q), +8, del(20q), sis and cytopenias, and presence of abnormal cytogenetic -Y, and complex karyotype. Mutated genes of significance findings. These include MDS-SLD, MDS-RS (subclasses involve RNA splicing machinery, signaling pathways, tran- MDS-RS-SLD and MDS-RS-MLD), MDS-MLD, MDS-EB scription regulation, DNA repair, and epigenetic alterations (subclasses MDS-EB-1 and MDS-EB-2), MDS with isolated to the genome. Some of the most frequently mutated genes del(5q), and MDS, unclassifiable. Subgroups with higher include TET2, IDH1/2, ASXL1, EZH2, ETV6, FLT3, JAK2, blast counts and involvement of multiple lineages in dys- NPM1, N-RAS, K-RAS, RUNX1, TP53, and SETBP1. poiesis and complex cytogenetic abnormalities are more Although anemia is the most common cytopenia, neu- aggressive disorders. MDS frequently terminates in acute tropenia and thrombocytopenia also occur. Erythrocytes are leukemia; treatment is variable because of the heteroge- macrocytic or less frequently normocytic. Erythropoiesis in the neous presentation of the disease. Treatment options are Myelodysplastic Syndromes 577 made based on multiple clinical parameters including the myelodysplastic syndrome simultaneously. This category patient’s cytogenetic profile. includes CMML, aCML BCR/ABL1-, JMML, MDS/MPN- The myelodysplastic/myeloproliferative neoplasm RS-T, and MDS/MPN, U. An important diagnostic consid- category (MDS/MPN) includes disorders that have eration is absence of the BCR/ABL1 fusion gene in these features of both a myeloproliferative neoplasm and a patients. Review Questions Level I c. hypercellular 1. Which subgroup of MDS has refractory cytopenia d. fibrotic with unilineage dysplasia? (Objective 2) 7. The most common dyserythropoietic |
finding in the a. MDS-MLD bone marrow in MDS is: (Objective 3) b. MDS-EB-1 a. megaloblastoid development c. MDS associated with isolated del(5q) b. impaired hemoglobinization d. MDS-SLD c. pseudo-Pelger-Huet cells d. agranular cytoplasm 2. Dysplasia in 10% or more of cells of two or more lineages, less than 5% other blasts, 15% or more ring 8. Which of the following help define myelodysplastic sideroblasts, and no Auer rods in the bone marrow syndrome? (Objective 1) suggests a diagnosis of: (Objective 5) 1. Characterized by one or more peripheral blood a. MDS-RS-MLD cytopenias b. MDS-RS 2. Maturation abnormalities (dyspoiesis) in the bone c. MDS-MLD marrow blood cells d. MDS-EB 3. Microcytic, hypochromic RBCs in the peripheral blood 3. The WHO classification system of MDS is based on: a. 1 and 2 only (Objective 2) b. 2 and 3 only a. morphology c. 1 and 3 only b. immunophenotype d. 1, 2, and 3 c. genetics 9. Which test(s) is(are) most helpful to differentiate a sus- d. all of the above pected MDS from megaloblastic anemia? (Objective 3) 4. The type of anemia usually seen in MDS is: a. MCV and MCHC (Objective 1) b. Serum ferritin a. macrocytic, normochromic c. Serum folate and cobalamin b. normocytic, normochromic d. bone marrow cellularity c. microcytic, hypochromic 10. Which MDS subgroup occasionally may have Auer d. normocytic, hypochromic rods? (Objective 2) 5. The most common “cytopenia(s)” seen in MDS is/are: a. MDS-MLD (Objectives 3 and 5) b. MDS with isolated del(5q)- syndrome a. leukopenia c. MDS-EB-1 b. thrombocytopenia d. MDS-EB-2 c. anemia 11. A dimorphic red blood cell population is most d. a combination of two of the above characteristic of which MDS subgroup? (Objective 2) 6. The typical bone marrow cellularity in MDS is: a. MDS-SLD (Objective 3) b. MDS-RS a. hypocellular c. MDS-EB b. normocellular d. CMML 578 Chapter 25 12. A patient presents with the following symptoms and a. 1 and 3 laboratory data: Weak, e asily fatigued, night sweats, and b. 2 and 4 dyspnea on exertion. The spleen was slightly enlarged. c. 1, 2, and 3 WBC = 6.0 * 103/mcL, RBC = 2.80 * 106/mcL, Hb = 85g/L, MCV = 103 fL, Plt = 220 * 103/mcL. d. 1, 2, 3, and 4 The peripheral blood film showed macro-ovalocytes 17. Dysgranulopoiesis may be seen on a peripheral blood and hypersegmented neutrophils. Less than 5% blasts film of an MDS patient as: (Objective 3) and a few ringed sideroblasts were seen in the bone marrow. Serum folate and cobalamin levels were 1. agranular or hypogranular neutrophils normal. What is the most likely diagnosis? 2. hypersegmented neutrophils (Objective 2) 3. pseudo-Pelger-Huet cells a. MDS, subgroup MDS-SLD 4. lingering cytoplasmic basophilia in mature b. MDS, subgroup MDS-RS neutrophils c. Megaloblastic anemia a. 1 and 3 d. Sideroblastic anemia b. 2 and 4 c. 1, 2, and 3 13. Which MDS subgroup is found predominantly in women? (Objective 2) d. 1, 2, 3, and 4 a. MDS with isolated del (5q) 18. Evidence of dysmegakaryocytopoiesis may be seen on a peripheral blood film as: (Objective 3) b. MDS-SLD c. MDS-MLD 1. Micromegakaryocytes d. MDS-RS 2. Increased platelets 3. Hypogranular platelets 14. Which of the following terms are currently acceptable a. 1 and 3 terminologies for MDS? (Objective 1) b. 2 1. Preleukemia c. 1, 2, and 3 2. Myelodysplastic syndrome d. 1 and 2 3. Smouldering leukemia 4. Dysmyelopoietic syndrome Level II a. 1 and 3 1. A cell resembling a blast that contains primary b. 2 and 4 granules and lacks a Golgi zone would be classified c. 1, 2, and 3 as a(n): (Objective 2) d. 1, 2, 3, and 4 a. granular blast b. agranular blast 15. Which of the following, when present in the bone marrow, indicate a typical MDS? (Objective 1) c. promyelocyte d. dysplastic promyelocyte 1. Hypercellularity 2. Abnormal maturation (dyspoiesis) 2. The contrast between a hypercellular bone marrow 3. Less than 20% blasts and a cytopenic peripheral blood film seen in MDS is attributed to: (Objective 1) 4. Increased amounts of fibrotic tissue a. 1 and 3 a. premature destruction of abnormal cells in the bone marrow (ineffective hematopoiesis) b. 2 and 4 b. production of blood cells outside the bone marrow c. 1, 2, and 3 (extramedullary hematopoiesis) d. 1, 2, 3, and 4 c. splenic sequestration 16. Which of the following are peripheral blood signs of d. immune destruction of cells in the peripheral an MDS? (Objective 1) blood 1. Macro-ovalocytes 3. Which of the following would be most helpful to dif- 2. Pseudo-Pelger-Huet cells ferentiate CMML from CML? (Objective 8) 3. Giant platelets with decreased granulation a. Karyotype 4. Cytopenia, alone or in combination b. Presence of nucleated RBCs Myelodysplastic Syndromes 579 c. Elevated leukocyte count 8. The most common chromosomal abnormality found d. Percentage of bone marrow blasts in the MDS is: (Objective 5) 4. The most effective treatment for MDS is currently a. 5q- considered to be: (Objective 3) b. 7q- a. hematopoietic growth factors c. trisomy 8 (+8) b. chemotherapy d. t(9;22) c. bone marrow transplant 9. 5q- syndrome is frequently associated with which d. immunotherapy morphological abnormality? (Objective 5) a. pseudo-Pelger-Huet cells b. hyperlobulated megakaryocytes Use the following information to answer questions 5–7, c. ring sideroblasts A patient presents with the following laboratory data: d. megaloblastoid erythropoiesis RBC 2.30 * 106/mcL Case Study Hb 78 g/L Hct 24% Use this information to answer Questions 10–12. MCV 104 fL RDW 20 A patient’s primary presenting symptoms were fatigue and shortness of breath. WBC 8.5 * 103/mcL Plt 140 * 103/mcL RBC decreased Hb 8.5 g/dL The differential was normal except for 2% metamyelocytes. MCV 104 fL Oval macrocytes, a few abnormal NRBC, and a few siderocytes WBC were seen. The bone marrow contained 3% blasts and exhibited 3.4 * 103/mcL hypercellularity with megaloblastoid development in erythroid Plt 180 * 103/mcL cells. Previous treatment with cobalamin and folate was not effective. The peripheral blood film showed macrocytes, atypical platelets, 5. What is the most probable MDS subgroup? (Objective hypogranulation of neutrophils, and pseudo-Pelger-Huet cells. 10) The differential revealed a few myelocytes, metamyelocytes, and 2% blasts. The bone marrow was hypercellular with erythroid a. MDS-EB-1 and granulocytic hyperplasia. Dysplasia was evident in all b. MDS-RS three cell lines. There were 8% myeloblasts present and 20% c. MDS-SLD ring sideroblasts. d. MDS-EB-2 6. What other hematologic disorder does this peripheral 10. Ringed sideroblasts are defined as nucleated red blood picture resemble? (Objective 7) blood cells in which iron deposits encircle at least how much of the nucleus? (Objectives 1 [Level I] a. Iron-deficiency anemia and 5) b. Aplastic anemia a. One-third c. Megaloblastic anemia b. One-half d. Anemia of chronic disease c. One-fourth 7. In reference to Question 6, what laboratory test(s) d. The whole nucleus would be helpful to distinguish the two disorders? (Objective 9) 11. Which MDS subgroup is most likely? (Objective 8) a. Lactate dehydrogenase a. MDS-RS b. Uric acid b. MDS-EB-2 c. Serum folate and cobalamin c. MDS-EB-1 d. Serum ferritin d. MDS-MLD 580 Chapter 25 12. What morphological finding distinguishes MDS-EB-1 17. According to the IPSS, which factors have a major from MDS-EB-2? (Objective 8) impact on disease prognosis? (Objective 3) a. MDS-EB-2 presents with monocytosis 1. Degree of cytogenetic abnormalities b. MDS-EB-2 presents with an increased platelet 2. Number of blasts in the bone marrow count 3. Number of cytopenias in the peripheral blood c. MDS-EB-2 has a greater number of blasts in the 4. Number of blasts in the peripheral blood bone marrow a. 2 and 4 d. MDS-EB-2 presents with erythroid hyperplasia in b. 1, 2, 3, and 4 bone marrow c. 1, 2, and 3 13. Complex karyotype abnormalities are present in what d. 1 and 3 percentage of therapy-related MDS? (Objective 5) 18. Atypical CML can best be distinguished from CML by a. 50% which of the following? (Objective 8) b. 90% a. Dysplastic cells in the bone marrow c. 20% b. Leukocytosis d. 10% c. Absence of BCR/ABL1 fusion gene 14. Presence of micromegakaryocytes may be distin- d. Abnormal karyotype guished from lymphocytes by: (Objective 9) 19. MDS is usually diagnosed in elderly patients. This is a. cytogenetics evidence that which of the following is a contributing b. presence of cytoplasmic tags on the nucleus factor to disease development? (Objective 1) c. Wright’s stain a. Genetic instability of myeloid stem cell d. epigenetics b. Epigenetic changes 15. The origin of the MDS is considered to be: (Objective 1) c. Changes in bone marrow microenvironment a. enzyme deficiency d. Inherited chromosomal disorders b. abnormal stem cell 20. Which of the following would distinguish an c. abnormal DNA synthesis MDS/MPN from MDS? (Objective 8) d. congenital genetic defect a. Less than 20% blasts in the bone marrow 16. Which would help to distinguish MDS with a b. Hypercellular bone marrow hypoplastic marrow from aplastic anemia? c. Leukocytosis (Objective 7) d. Presence of dysplastic cells 1. Chromosomal abnormalities 21. A 70-year-old patient is suspected to have an MDS. 2. Pancytopenia His peripheral blood shows dysplasia, cytopenias in 3. Dysgranulocytosis and dysmegakaryocytosis two cell lines, and an increased number of monocytes. 4. Absolute decrease in reticulocytes This would best be classified as: (Objectives 6 and 8) a. 2 and 4 a. JMML b. 1, 2, and 3 b. MDS-MLD c. 1 and 3 c. aCML d. 1, 2, 3, and 4 d. CMML References 1. National Comprehensive Cancer network. NCCN clinical 4. Steensma, D. P., Bejar, R., Jaiswal, S., Lindsley, R. C., Sekeres, M. A., practice guidelines in oncology. Myelodysplastic Syndromes. Hasserjian, R. P., & Ebert, B.L. (2015). Clonal hematopoiesis of V. 1. 2016. Accessed on 8/8/2016 from www.nccn.org indeterminate potential and its distinction from myelodysplastic 2. Adès, L., Itzykson, R., & Fenaux, P. (2014). Myelodysplastic syndromes. Blood, 126, 9–16. syndromes. Lancet, 383(9936), 2239–2252. 5. Rankin, E. B., Narla, A., Park, J. K., Lin, S., & Sakamoto, K. M. 3. Bejar, R., & Steensma, D. P. (2014). Recent developments in (2015). Biology of the bone marrow microenvironment and myelodysplastic syndromes. Blood, 124(18), 2793–2803. myelodysplastic syndromes. Mol Genet Metab, 116(1), 24–28. Myelodysplastic Syndromes 581 6. Cogle, C. R., Saki, N., Khodadi, E., Li, J., Shahjahani, M., & by flow cytometry in myeloid neoplasms. British Journal of Azizidoost, S. (2015). Bone marrow niche in the myelodysplastic Haematology. (153), 421–436. syndromes. Leuk Res, 39(10), 1020–1027. 25. Porwit, A. (2015). Is there a role for flow cytometry in the 7. Visconte, V., Tiu, R. V., & Rogers, H. J. (2014). Pathogenesis of evaluation of patients with myelodysplastic syndromes? myelodysplastic syndromes: An overview of molecular and Current Hematological Malignancy Reports, 10(3), 309–317. non-molecular aspects of the disease. Blood Res, 49(4), 216–227. 26. Bennett, J. M. (2016). Changes in the updated 2016: WHO 8. Davids, M. S., & Steensma, D. P. (2010). The molecular classification of myelodysplastic syndromes and related myeloid pathogenesis of myelodysplastic syndromes. Cancer Biol Ther, neoplasms. Clinical Lymphoma Myeloma and Leukemia, 16(11), 10(4), 309–319. 607–609. 9. Kawankar, N., & Rao Vundinti, B. (2011). Cytogenetic 27. Gangat, N., Patnaik, M. M., & Tefferi, A. (2016). Myelodysplastic abnormalities in myelodysplastic syndrome: An overview. syndromes: Contemporary review and how we treat. American Hematology, 16(3), 131–138. Journal of Hematology, 91(1), 76–89. 10. Zahid, M. F., Malik, U. A., Sohail, M., Hassan, I. N., Ali, S., & 28. Feng, G., Gale, R. P., Cui, W., Cai, W., Huang, G., Xu, Z., . . . Shaukat, M. H. S. (2017). Cytogenetic abnormalities in Zhang, H. (2016). A systematic classification of megakaryocytic myelodysplastic syndromes: An overview. International Journal dysplasia and its impact on prognosis for patients with myelo- of Hematology-Oncology and Stem Cell Research, 11(3), 231–239. dysplastic syndromes. Experimental hematology. Oncology, 11. Arber, D. A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M. J., 5(1), 1. Le Beau, M. M., . . . Vardiman, J. W. (2016). The 2016 revision to 29. Zhou, J., Orazi, A., & Czader, M. B. (2011). Myelodysplastic the World Health Organization (WHO) classification of myeloid syndromes. Seminars in diagnostic pathology, 28(4), 258–272. neoplasms and acute leukemia. Blood, 127, 2391–2405. 30. Arenillas, L., Calvo, X., Luño, E., Senent, L., Alonso, E., Ramos, F., 12. Zhang, L., Padron, E., & Lancet, J. (2015). The molecular basis . . . Montoro, J. (2016). Considering bone marrow blasts from and clinical |
significance of genetic mutations identified in nonerythroid cellularity improves the prognostic evaluation of myelodysplastic syndromes. Leukemia Research, 39(1), 6–17. myelodysplastic syndromes. Journal of Clinical Oncology, 34(27), 13. Ganster, C., Kämpfe, D., Jung, K., Braulke, F., Shirneshan, K., 3284–3292. Machherndl-Spandl, S., . . . Haase, D. (2015). New data shed light 31. Mateos, M. K., Barbaric, D., Byatt, S. A., Sutton, R., & on Y-loss-related pathogenesis in myelodysplastic syndromes. Marshall, G. M. (2015). Down syndrome and leukemia: Insights Genes, Chromosomes Cancer, 54(12), 717–724. into leukemogenesis and translational targets. Translational 14. Itzykson, R., & Fenaux, P. (2014) Epigenetics of myelodysplastic Pediatrics, 4(2), 76. syndromes. Leukemia. 28(3), 497–506. 32. Hamdi, W., Ogawara, H., Handa, H., Tsukamoto, N., Nojima, Y., & 15. Jhanwar, S. C. (2015). Genetic and epigenetic pathways in Murakami, H. (2009). Clinical significance of regulatory T cells myelodysplastic syndromes: A brief overview. Advances in in patients with myelodysplastic syndrome. European Journal of Biological Regulation, 58, 28–37. Haematology, 82(3), 201–207. 16. Itzykson, R., Kosmider, O., & Fenaux, P. (2013). Somatic 33. Scott, B. L., & Deeg, H. J. (2010). Myelodysplastic syndromes. mutations and epigenetic abnormalities in myelodysplastic Annual Review of Medicine, 61, 345–358. syndromes. Best Pract Res Clin Haematol, 26(4), 355–364. 34. Verburgh, E., Achten, R., Maes, B., Hagemeijer, A., Boogaerts, M., 17. Gill, H., Leung, A. Y., & Kwong, Y. L. (2016). Molecular and De Wolf-Peeters, C., & Verhoef, G. (2003). Additional prognostic cellular mechanisms of myelodysplastic syndrome: Implications value of bone marrow histology in patients subclassified on targeted therapy. International Journal of Molecular Sciences, according to the International Prognostic Scoring System for 17(4), 440. myelodysplastic syndromes. Journal of Clinical Oncology, 21(2), 18. Garcia-Manero, G. (2015). Myelodysplastic syndromes: 2015 273–282. update on diagnosis, risk-stratification and management. 35. Zhang, L., & Wang, S. A. (2014). A focused review of hema- Americam Journal of Hematology 90(9), 831–841. topoietic neoplasms occurring in the therapy-related setting. 19. Mughal, T. I., Cross, N. C., Padron, E., Tiu, R. V., Savona, M., International Journal of Clinical and Experimental Pathology, 7(7), Malcovati, L., . . . Orazi, A. (2015). An international MDS/MPN 3512–3523. working group’s perspective and recommendations on molecular 36. Greenberg, P. L., Tuechler, H., Schanz, J., Sanz, G., pathogenesis, diagnosis and clinical characterization of Garcia-Manero, G., Solé, F., . . . Kantarjian, H. (2012). Revised myelodysplastic/myeloproliferative neoplasms. Haematologica, international p rognostic scoring system for myelodysplastic 100(9), 1117–1130. syndromes. Blood, 120(12), 2454–2465. 20. Steensma, D. P., & Bennett, J. M. (2006). The myelodysplastic 37. Steensma, D.P. (2012). Dysplasia has a differential diagnosis: syndromes: Diagnosis and treatment. Mayo Clinic Proceedings, Distinguishing genuine myelodysplastic syndroms (MDS) from 81(1), 104–130. mimics, imitators, copycats and impostors. Current Hematologic 21. Bejar, R., & Steensma, D. P. (2010). Myelodysplastic syndromes. Malignancy Reports, 7, 310–320. In Kaushansky, K., Lichtman, M., Prchal, J., Levi, M., Press, O., 38. Halfdanarson, T. R., Kumar, N., Li, C-Y., & Phyliky, R. L. (2008). Burns, L., Caligiuri, M., eds. Williams Hematology 9th ed. Haematological manifestations of copper deficiency: A (pp. 1341–1272). New York: McGraw-Hill. retrospective review. European Journal of Haematology, 80, 22. Mufti, G. J., Bennett, J. M., Goasguen, J., Bain, B. J., Baumann, I., 523–531. Brunning, R., . . . Jinnai, I. (2008). Diagnosis and classification 39. Gabreyes, A. A., Abbasi, H. N., Forbes, K. P., McQuaker, G., of myelodysplastic syndrome: International working group Duncan, A., & Morrison, I. (2013). Hypocupremia associated on morphology of myelodysplastic syndrome (IWGM-MDS) cytopenia and myelopathy: A national retrospective review. consensus proposals for the definition and enumeration of European Journal of Haematology, 90, 1–9. myeloblasts and ring sideroblasts. Haematologica, 93(11), 40. Young, N. S., & Brown, K. E., (2004). Parvovirus B19. NEJM, 350, 1712–1717. 586–597. 23. Giagounidis, A., & Haase, D. (2013). Morphology, cytogenetics 41. Bain, B. J. (1997). The haematological features of HIV infection. and classification of MDS. Best Practice & Research Clinical British Journal of Haematology, 99, 1–8. Haematology, 26(4), 337–353. 42. Klco, J. M., Geng, B., Brunt, E. M., Hassan, A., Nguyen, R-D., 24. Ossenkoppele, G. J., van de Loosdrecht, A. A., & Schuurhuis. G. J. Kreisel, F. H., & Frater, J. L. (2010). Bone marrow biopsy in (2011) Review of the relevance of aberrant antigen expression patients with hepatitis C virus infection: Spectrum of findings 582 Chapter 25 and diagnostic utility. American Journal of Hematology, 85, 46. Foucar, K. (2009). Myelodysplastic/myeloproliferative n eoplasms. 106–110. American Journal of Clinical Pathology, 32(2), 281–289. 43. Jonas, B. A., & Greenberg, P. L. (2015). MDS prognostic scoring 47. Sochacki, A. L., Fischer, M. A., & Savona, M. R. (2016). systems-Past, present, and future. Best Practice & Research Clinical Therapeutic approaches in myelofibrosis and myelodysplastic/ Haematology 28(1), 3–13. myeloproliferative overlap syndromes. Oncological Targets and 44. Meers, S. (2015). The myelodysplastic syndromes: The era of Therapy, 9, 2273. understanding. European Journal of Haematology, 94(5), 379–390. 48. Niemeyer, C. M., & Kratz, C. P. (2008). Paediatric myelodysplastic 45. Bhatia, R., & Deeg, H. J. (2011). Treatment-related myelodysplastic syndromes and juvenile myelomonocytic leukaemia: Molecular syndrome-Molecular characteristics and therapy. Current Opinion classification and treatment options. British Journal of H aematology, in Hematology, 18(2), 77. 140(6), 610–624. Chapter 26 Acute Myeloid Leukemias Kyle Riding, PhD Objectives—Level I By the end of this unit of study, the student should be able to: 1. Define acute leukemia (AL) and acute myeloid leukocytes, thrombocytes, and blasts) seen leukemia (AML). in AML. 2. State the differences between acute myeloid 5. Describe the typical bone marrow picture leukemia (AML) and acute lymphoblastic (cellularity, M:E ratio, blasts) seen in AML. leukemia (ALL). 6. Give the typical cytochemistry and immu- 3. List and define the common variants of AML nophenotype results that help differentiate as defined by the World Health Organiza- AML from other ALs. tion (WHO) classification. 7. Describe Auer rods and their significance. 4. Describe and recognize the typical peripheral blood picture (erythrocytes, Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare and contrast the various presenta- 4. Correlate Wright stain morphology of the tions of AML. AML subgroups with cytochemical stains, 2. Predict the most likely leukemia subtype flow cytometry and genetic testing. based on patient history, physical assess- 5. Evaluate peripheral blood results in relation ment, and laboratory findings. to oncological therapy. 3. Correlate cellular presentation of AML with 6. Evaluate patient data from the medical his- prognosis and common complications. tory and the laboratory results to determine 583 584 Chapter 26 whether a disorder can be classified and, 7. Explain the criteria used for the 2016 WHO if not, specify the additional testing to be classification of AML. performed. Chapter Outline Objectives—Level I and Level II 583 Laboratory Evaluation 585 Key Terms 584 Classification 587 Background Basics 584 Therapy 601 Case Study 584 Summary 602 Overview 585 Review Questions 602 Introduction 585 References 604 Etiology and Pathophysiology 585 Key Terms Acute leukemia (AL) B-lymphoid cell antigens Recurrent genetic abnormalities Acute myeloid leukemia (AML) Dysplasia T-lymphoid cell antigens Auer rods Leukemias of ambiguous lineage WHO classification Background Basics The information in this chapter builds on concepts learned Level II in previous chapters. To maximize your learning experi- • Summarize the role of oncogenes and growth fac- ence, you should review and have an understanding of the tors in cell proliferation, differentiation, and matura- following concepts before starting this unit of study: tion. (Chapters 2, 4, and 23) • Describe the role of molecular analysis in diagnosing Level I and treating acute leukemia. (Chapters 23, 42) • Describe the origin and differentiation of hematopoi- • Describe the use of immunophenotyping in acute etic cells. (Chapter 4) leukemia. (Chapters 23, 40) • Summarize the maturation, differentiation, and • Describe the role of cytogenetics in diagnosis, function of leukocytes. (Chapter 7) treatment, and prognosis of acute leukemia. • Outline the classification and general laboratory (Chapters 23, 41) findings of acute leukemias. (Chapter 23) • List and describe the criteria used to d ifferentiate acute leukemias from other hematologic neoplasms. (Chapter 23) CASE STUDY a chemistry panel. He was sent to the local emer- We refer to this case study throughout the chapter. gency room when his CBC revealed a WBC count of 120 * 106/mcL and potential blasts being noted Jonathan, a 12-year-old Caucasian male, had been by the physician office analyzer. He was admitted in excellent health until he was seen at his primary to the hospital for further evaluation. care physician for a sore throat, lack of energy, and Consider what additional laboratory testing a purple rash. The physician noted splenomegaly could assist in diagnosing Jonathan. and ordered a CBC with differential along with Acute Myeloid Leukemias 585 Overview older FAB system to fall out of favor with healthcare practi- tioners now using the WHO classification in its place. This chapter describes the acute myeloid (also referred to as myelogenous) leukemias (AMLs). It begins with the general laboratory findings in AML followed by a specific descrip- tion of each subgroup in the World Health Organization Etiology And (WHO) classification. Clinical and laboratory findings are Pathophysiology described with an emphasis on defining characteristics. The chapter concludes with the current types of therapy used AML is a disease characterized by two fundamental cellular to treat AML. features: the ability to proliferate continuously and aberrant or arrested development1 (Figure 26-1). Excessive prolifera- tion can be the result of mutations affecting growth factors, growth factor receptors, signaling pathway components, and Introduction transcription factors that regulate genes involved in cell sur- All acute leukemias (ALs) are stem cell disorders character- vival and proliferation (Chapter 23). More than half of the ized by malignant neoplastic proliferation and accumula- cases of AML display cytogenetic abnormalities. Most are tion of immature and nonfunctional hematopoietic cells in balanced, reciprocal chromosomal translocations with many the bone marrow. The disease arises due to a succession of of the translocation break points located at the loci for genes somatic mutations in a hematopoietic stem or progenitor encoding transcription factors and other signaling mole- cell. The neoplastic cells show increased proliferation and/ cules. The most common consequence of the translocation is or decreased programmed cell death (apoptosis). The net the generation of a fusion gene that codes for a novel fusion effect is expansion of the leukemic clone and a decrease in protein. The fusion proteins that are formed usually alter normal cells.1 the normal function of one or both of the rearranged genes Acute leukemia is a clonal expansion of a single trans- and modify the normal programs of cell proliferation, dif- formed cell; therefore, all ALs begin long before any clinical ferentiation, and survival. In addition, other types of genetic signs and symptoms appear. As the leukemic cells expand abnormalities (e.g., epigenetic alterations) likely interact in number, the classic triad of anemia, infection, and bleed- with the cytogenetic mutations (called the multistep origin of ing seen in acute leukemia occur as a result of “normal” malignancy), resulting in the full leukemic transformation.1 hematopoietic cell cytopenias. Death often occurs from either infection or hemorrhage in weeks to months unless therapeutic intervention occurs.2 Laboratory Evaluation The two major categories of acute leukemias are clas- sified according to the origin of the cell with the primary Peripheral Blood defect: acute myeloid leukemia (AML) and acute lympho- The peripheral blood picture is variable in AML. Although blastic leukemia (ALL). If the defect primarily affects the it is traditional to describe leukemias as having elevated maturation and differentiation of the common myeloid pro- leukocyte counts, 50% of the cases have decreased counts or genitor (CMP) cell, the leukemia is classified as AML. If the counts within the reference interval at the time of diagnosis. defect primarily affects the common lymphoid progenitor The leukocyte count ranges from less than 1 * 103/mcL to (CLP) cell, the leukemia is classified as ALL (Chapter 27). greater than 100 * 103/mcL. Regardless of the leukocyte The acute leukemias are classified into subtypes. The concentration, the presence of blasts on the blood smear most reliable parameters for defining and classifying neo- suggests the AL diagnosis. The current WHO definition of plastic cells in AML into subtypes are cell markers (cell sur- AL requires 20% or more blasts in the peripheral blood or face or internal antigens) defined by immunologic probes bone marrow.5 Upon preliminary diagnosis, cases must be and genetic abnormalities identified with cytogenetic and classified based on a combination of immunologic, cyto- |
molecular studies (Chapters 40–42). In the late 1970s, the genetic and molecular genetic methods and cytochemical first internationally accepted classification for the acute leu- tests (Chapter 23). Typically, the myeloblast seen in AML kemias, the French-American-British (FAB) classification, is approximately 20 mcM in diameter with variably promi- was based on a combination of neoplastic cell morphol- nent nucleoli in a nucleus composed primarily of dispersed ogy and cytochemical cellular reactions.3 This classification chromatin (euchromatin or transcriptionally active DNA). remained essentially unchanged until 1999 when the WHO A Golgi apparatus is present but is not easily visualized. and the International Society of Hematology proposed a Although blasts normally do not have granules visible by new classification.4 The incorporation of cytogenetic and bright-field microscopy, neoplastic blasts do not always molecular information as well as myelodysplasia and clini- develop normally and can have granules in the RNA-rich cal findings into the characterization of AL has caused the cytoplasm (Chapter 25). 586 Chapter 26 AML AML CML MDS AML AML NP Granulocyte GMP MP Monocyte HSC CMP MkP Platelets MkEP EP Erythrocyte Figure 26.1 AML may originate from any of the cells that fall within the paths of the downward arrows. Importantly, the AML cell of origin acquires the capacity for self-renewal and maturation arrest. AML, acute myeloid leukemia; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; CMP, common myeloid progenitor cell; NP, neutrophil progenitor; MP, monocyte progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor; MkEP, megakaryocyte erythrocyte progenitor; GMP, granulocyte monocyte progenitor; CMP, common myeloid progenitor; HSC, hematopoietic stem cell. Erythrocytes are typically decreased, and a hemoglobin differentiated monocytic or myelocytic cells (promyelo- value less than 10 g/dL is common. Erythrocytes can be cytes). They are not found in lymphoid blasts. slightly macrocytic because of their inability to successfully Other abnormal findings on the blood smear can compete with neoplastic cells for folate or cobalamin and/ include monocytosis and neutropenia. Monocytosis fre- or early release of marrow reticulocytes. The red cell distri- quently precedes overt leukemia. Neutrophils can dem- bution width (RDW) is often elevated. Erythrocyte inclu- onstrate signs of dysplasia including hyposegmentation, sions, such as Howell-Jolly bodies, Pappenheimer bodies, hypogranulation, and small nuclei with hypercondensed and basophilic stippling, reflective of erythrocyte matura- chromatin. Signs of myelodysplasia are especially common tion defects, may be present. Nucleated erythrocytes may in promyelocytic leukemia.6 Eosinophils and basophils can also be present. be mildly to markedly increased. When present, basophilia Platelets are typically decreased. Hypogranular plate- can help to differentiate leukemia from a leukemoid reac- lets and occasional enlarged forms (giant platelets) may be tion. Absolute basophilia is not present in a leukemoid present. As the disease progresses, more immature platelet reaction. A blast phase of chronic myeloid leukemia (CML) forms, such as megakaryocytic fragments, may be seen. The should be excluded by evaluating BCR/ABL1. platelet count might not correlate with the potential compli- cation of bleeding because qualitative platelet defects may also be present. Bone Marrow If the physician suspects AL but no blast cells are When a diagnosis of AML, MDS, or MPN is suspected, detected on the peripheral blood smear or if the leukocyte the first step is to evaluate the bone marrow. Bone mar- count is low (less than 2 * 103/mcL) a buffy-coat smear row (BM) testing should include both aspirate and biopsy may be prepared and often reveals the presence of blast specimens. The quality of marrow specimen is critical for cells when they are present in very low concentrations. all subsequent analyses. Typically, the BM presentation is Finding blasts with azurophilic granules is helpful in iden- hypercellular with decreased fat content (relative to age- tifying the myeloid nature of the leukemia. The presence of related normals), a predominance of blasts, and sometimes Auer rods (fused or coalesced primary granules) in blasts an increase in fibrosis. excludes a diagnosis of ALL. Auer rods are primarily found According to the WHO criteria for AL, blasts must in myeloblasts and on rare occasions in monoblasts or more compose 20% or more of the nonerythroid nucleated cells Acute Myeloid Leukemias 587 to distinguish AL from myelodysplastic syndromes. Fre- quently, the blast count is close to 100%. Auer rods are Classification present in BM blasts in about half of AML cases. Normal The 2016 revision to the WHO classification is the result- erythropoiesis, megakaryocytopoiesis, and granulopoiesis ing work of a worldwide clinical advisory committee that are decreased or absent in the marrow aspirate. Additional updated the 2008 classification of the hematopoietic neo- assessment of the subtypes of AML evaluates only the plasms. As in the past, the 2016 WHO classification expands myeloid cells, not lymphocytes, plasma cells, mast cells, the parameters used to classify the neoplastic disorders.7 The macrophages, or nucleated erythrocytes. parameters include microscopic morphology, cytochemistry Cells may be clumped together, occasionally forming flow cytometry, cytogenetics, molecular genetic abnormali- sheets of infiltrate that disturb the usual marrow architec- ties, and clinical findings. The WHO major AML subgroups, ture. A larger infiltrate that disrupts marrow architecture each with variants or subtypes, are listed in Table 26-1. may make it difficult to obtain appropriately spiculated bone marrow aspirates. In addition to light microscopic Identification of Cell Lineage morphologic evaluation, BM samples should be sent for Because blast cells are immature cells, they are often difficult flow cytometry, cytogenetics, and molecular genetics. AML to identify by morphology alone using light microscopy. is the diagnosis if blasts possess a t(8;21, inv(16), or t(15;17), Cytochemistry, molecular testing, and immunophenotyping regardless of the blast percentage, when certain clinical give additional information that can help define cell lineage parameters are met.6 Table 26.1 2016 WHO Classification of Acute Myeloid Checkpoint 26.1 Leukemias (AML) What results would you expect to find on the CBC and differen- tial in a suspected case of AL? 1. AML with recurrent genetic abnormalities • AML with t(8;21)(q22;q22.1), RUNX1-RUNX1T1 • AML with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); CBFB-MYH11 Other Laboratory Evaluation • AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A • APL (promyelocytic) with PML-RARA Other laboratory findings can reflect the increased prolifera- • AML with t(6;9) (p23;q34.1); DEK-NUP214 tion and turnover of cells. Hyperuricemia and increased • AML with inv(3)(q21.3;q26.2) or t(3;3)(q21.3;q26.2); GATA2, lactate dehydrogenase (LD) are common findings resulting MECOM • AML (megakaryoblastic) with t(1;22)(p13.3;q13.3); RBM15-MKL1 from the increased cell turnover. When present, hypercalce- • AML with mutated NPM1 mia is thought to be caused by increased bone resorption • AML with biallelic mutation of CEBPA associated with leukemic proliferation in the bone marrow. • AML with BCR-ABL1 (new provisional entry) Increased serum and urine muramidase are typical findings • AML with mutated RUNX1 (new provisional entry) in leukemias with a monocytic component. 2. AML with myelodysplasia-related changes AML arising from a previous myelodysplastic or myelodysplastic/ myeloproliferative neoplasm CASE STUDY (continued from page 584) 3. Therapy-related myeloid neoplasms Includes (t-AML), (t-MDS), and (t-MDS/MPN) in patients who receive Admission laboratory data on Jonathan are as cytotoxic therapies (alkylating agents, ionizing radiation, topoisomer- follows: ase, and others) 4. AML not otherwise specified RBC 3.2 * 106/mcL WBC Differential • AML minimally differentiated Hb 9.7 g/dL Blasts 75% • AML without maturation Hct 30 % Neutrophils 6% • AML with maturation PLT 31 * 103 / mcL Promyelocytes 2% • Acute myelomonocytic leukemia • Acute monoblastic and monocytic leukemia WBC 120 * 103/mcL Myelocytes 8% • Pure erythroid leukemia Metamyelocytes 9% • Acute megakaryoblastic leukemia Note: Auer Rods seen in some blasts • Acute basophilic leukemia Erythrocyte morphology: Rare schistocytes and dacryocytes seen. • Acute panmyelosis with myelofibrosis 1. What clues do you have that this patient could 5. Myeloid sarcoma have an acute leukemia? Tumor mass consisting of myeloid blasts with or without maturation occurring in an extramedullary site 2. Based on the presenting data, what additional 6. Myeloid proliferations related to Down syndrome testing might be of value? • Transient abnormal myelopoiesis • Myeloid leukemia associated with Down syndrome 588 Chapter 26 (Chapters 40, 41, 42). When both cytochemistry and immu- a limited, representative panel of monoclonal antibodies. nophenotyping are used, most AL cases can be classified as Common cell markers used in identification include CD2, being of lymphoid or myeloid origin. Rarely, a population CD3, CD4, CD5, CD7, CD10, CD13, CD14, CD15, CD16, of malignant blasts is cytochemically negative by conven- CD19, CD22, CD33, MPO, HLA-DR, CD34, CD45, CD56, tional methods and nonreactive with both lymphoid and CD64, and CD117.9 myeloid monoclonal antibodies. Such leukemias are classi- The first panel of monoclonal antibodies should differ- fied as undifferentiated.5 entiate AML from ALL and T-cell ALL (T-ALL) from B-cell The acute leukemias of ambiguous lineage encompass ALL (B-ALL). Use of a panel of antibodies such as those leukemias that show no clear evidence of differentiation to listed in Table 26-2 can usually discriminate AML from ALL. a single lineage. This group includes leukemias without Individual facilities have their own preferred panels of anti- lineage-specific antigens (acute undifferentiated leukemia) bodies. Panels should include typing for the myeloid anti- and leukemias that express antigens of more than one lin- gens, the B-lymphoid cell antigens (CD19, CD20, CD22, eage, making it impossible to determine any one lineage and CD79a), and the T-lymphoid cell antigens (CD2, CD3, (mixed phenotype acute leukemias, MPAL). These leuke- CD5, and CD7). Several aberrant antigens are sometimes mias are discussed in Chapter 27. found on neoplastic cells (e.g., the CD7 [T-lymphoid] anti- gen can be found on neoplastic myeloid cells in AML). CYTOCHEMISTRY The CD34 marker is also present on the least differentiated The common cytochemical stains include the myeloperoxi- myeloid cells and early lymphoid cells. The monoclonal dase (MPO), Sudan black B (SBB), naphthol AS-D chloroac- antibodies that react with most cases of AML include CD13, etate esterase (specific esterase), and a@naphthyl esterases CD15, CD33, CD64, and CD117.8 (nonspecific esterase) (Chapter 34). Granulocytic cells stain The CMP cell is capable of differentiation into granulo- positive with MPO and with SBB; lymphoblasts are nega- cytes, erythrocytes, monocytes, and megakaryocytes. Thus, tive. Thus, these stains help differentiate the acute myeloid if the neoplastic clone has “early” myeloid antigens, a sec- leukemias from the acute lymphoblastic leukemias. The ond panel of monoclonal antibodies should include anti- esterase stain helps differentiate precursor granulocytic bodies to subtype the AML into granulocytic, monocytic, cells from precursor monocytic cells. Granulocytic cells erythrocytic, and megakaryocytic lineages (Table 26-3). stain positive with naphthol AS-D chloroacetate esterase, and cells of monocytic lineage stain positive with nonspe- cific esterase. Most institutions use immunophenotyping and cytogenetics as first-line tests to define cell lineage. CASE STUDY (continued from page 588) Flow cytometry or immunohistochemistry can be used 4. If the cells from Jonathan’s BM were immu- to demonstrate terminal deoxynucleotidyl transferase (TdT) nophenotyped, which of these—CD13, CD33, in individual cells. Although originally thought to be a lym- CD20, CD2, CD7, CD10, CD19—would you phoid specific marker, TdT is found on more immature expect to be positive? hematopoietic cells, sometimes including those of myeloid lineage. Therefore, TdT cannot be the sole determinant of lymphoid lineage. CYTOGENETIC ANALYSES Two-thirds of patients with AML have detectable cytoge- netic abnormalities that include aneuploidies (variation in CASE STUDY (continued from page 587) total chromosome number) and translocations.1 Commonly 3. Based on the peripheral blood examination, what observed aneuploidies include trisomy 8, monosomy 7, cytochemical stain results would you expect to monosomy 21, trisomy 21, and loss of an X or Y chromo- find on Jonathan’s neoplastic cells? some. Translocations result in fusion genes that are either Table 26.2 Differentiation of ALL from AML Using IMMUNOPHENOTYPING Immunophenotyping with Selected Monoclonal Antibodies Immunophenotyping by flow cytometry has become a nec- Cell Marker essary component of AL classification, particularly when the presence of more than one neoplastic cell population Leukemia CD13, CD19, CD20, CD2, CD3, HLA-DR CD10 Type CD33 CD22, CD79a CD5, CD7 is suspected.8 Immunophenotyping by flow cytometry follows a specific sequence of testing with monoclonal AML + + - - - antibodies. The use of extensive panels is costly and time B-lymphocyte + - + + - consuming. In most cases, lineage can be determined using T-lymphocyte - - - { + Acute Myeloid Leukemias 589 Table 26.3 Morphologic, Immunophenotypic, and Cytochemical Results Used to Classify AML Subcategories Cell Markers with Monoclonal Antibodies CD71 CD41, CD42, Other Markers That AML Subgroup Morphology Cytochemistry HLA-DR CD117 CD34 CD13 CD33 CD11b CD14 Glycophorin A CD61 May Be Present AML not otherwise |
specified AML minimal Minimal evidence of Myeloperoxidase - + + + + + + + + - - - - CD7, CD38 TdTa differentiated myeloid differentiation (less than 3% of blasts) No maturation seen Sudan black B- Agranular cytoplasm (less than 3% of blasts) Specific esterase - AML without Evidence of myeloid dif- Myeloperoxidase + + + + + + + + + +/- + - - CD7, Lysozyme maturation ferentiation, no matura- (greater than 3% of blasts) tion seen, Auer rods or Sudan black B+ some granulation (greater than 3% of blasts) Specific esterase + AML with All stages of neutrophil Myeloperoxidase + +/- + + +/- + + + + + - - - - CD4, CD15, CD19, maturation maturation Sudan black B+ Lysozyme Pseudo–Pelger-Huët Specific esterase + Hypogranulation Esoinophilic precursors possible Acute myelomono- Neutrophil and mono- Myeloperoxidase + - + - + + + + + + +/- - - CD4, CD11c CD36, cytic leukemia cytic precursors present Nonspecific esterase +/- CD64, CD68, (AMML) Vacuolization Specific esterase + Lysozyme Acute monoblastic/ Monoblast/monocyte Myeloperoxidase - + + - + + + + + + - +/- CD11c, CD15 monocytic leukemia dominance Nonspecific esterase +/- CD65,CD4 (AMoL) Hemophagocytosis Specific esterase + CD64, Lysozyme present Nuclear lobulation Pure erythroid 80% or more immature Myeloperoxidase + +/- + - + + + + - - + - - leukemia erythroid precursors; Sudan black B+ 30% or more are pro- Nonspecific esterase +/- erythroblasts; less than PAS + (erythroblasts) 20% myeloblasts pres- ent amongall cells in the bone marrow Acute megakaryo- Cytopenia with or with- Myeloperoxidase - + + + - + - - - +/- - blastic leukemia out thrombocytopenia Sudan black B- (AMkL) Basophilic agranular Nonspecific esterase +/- blasts with pseudopods Specific esterase - Micro-megakaryocytes PAS+ Platelet peroxidase + Acute basophilic Basophilic precursors Metachromatic positivity + + + - + - - - + + CD123, CD203, leukemia (ABL) Vacuolization with toluidine blue CD9 Acute panmyelosis Pancytopenia Myeloperoxidase + + + + + + - - +/- +/- Lysozyme with myelofibrosis Dysplastic changes in neutrophils and platelets (Continued) 590 Chapter 26 Table 26.3 Continued Cell Markers with Monoclonal Antibodies CD71 CD41, CD42, Other Markers That AML Subgroup Morphology Cytochemistry HLA-DR CD117 CD34 CD13 CD33 CD11b CD14 Glycophorin A CD61 May Be Present Therapy-related myeloid neoplasms (t-MDS, t-AML, t-MDS/MPN) Most have multilineage Myeloperoxidase + No con- CD13, CD33, CD34, dysplasia Sudan black B+ sistent CD7, Basophilia is often Increased iron in ring pheno- CD56 present formation type Pancytopenia or isolated PAS+ cytopenia Specific esterase + Associated with Nonspecific esterease + unbalanced loss of genetic material often involving chromosomes 5 and or 7 AML with myelodysplasia-related changes Following MDS or Dysplasia required in Myeloperoxidase + No con- CD13, CD33, CD34, MDS/MPNs 50% or more of two cell Sudan black B+ sistent CD7, CD56, Multidrug With a cytogenetic lines Increased iron in ring pheno- resistance glycoprotein abnormality charac- Hypogranular neutro- formation type receptor (MDR-1) teristic of MDS phils, pseudo-Pelger- PAS+ With multilineage Huët nuclei dysplasia without Dyserythropoiesis with NPM1 or biallelic megaloblastoid changes, CEBPA mutations ringed sideroblasts, nuclear fragments or vacuoles Dysmegakaryopoiesis with micromegakaryo- cytes, hypolobulated nuclei, and other dysplastic signs a TdT is usually used to identify early lymphoid precursors but can also be found in 10–20% of AML Specific esterase = Napthol AS-d chloroacetate esterase; nonspecific esterase = Alpha - naphthyl esterase Acute Myeloid Leukemias 591 beneficial for proliferation and survival or disrupt differ- sensitivity than cytogenetics for diagnosing and following entiation and maturation. Additional genetic abnormalities the progression of the disease. The AMLs in this category can develop in subclones as the disease progresses. generally have a high rate of complete remission and a more Characteristic, nonrandom cytogenetic abnormalities favorable prognosis. Table 26-4 outlines the morphologies are observed in the WHO classifications of AML with recur- frequently encountered for each recurrent abnormality. rent genetic abnormalities and frequently in therapy- AML with T(8;21)(Q22;Q22.1); RUNX1-RUNX1T1 This sub- related myeloid neoplasms. These nonrandom chromosome category is found in 5–10% of AML cases (previously clas- abnormalities are discussed in subsequent sections. If the sified as AML with maturation). It occurs predominantly in expected abnormal karyotype is not found, fluorescence in children and adults younger than 60 years of age.10 Myelo- situ hybridization (FISH) or molecular analysis is some- blasts are usually large with abundant basophilic cytoplasm times helpful. containing numerous azurophilic granules, perinuclear clearing, and Auer rods. Some blasts may show very large granules (pseudo-Chédiak-Higashi granules). Promyelo- Checkpoint 26.2 cytes, myelocytes, and mature neutrophils with variable Explain why molecular analysis is not performed on all sus- dysplasia are usually present in the bone marrow. Dyspla- pected cases of acute leukemia. sia in other cell lines is uncommon. Eosinophilia can be present. Cytochemical and immunophenotypic results are typical for myeloblasts (Appendix B). The leukemic “cell of WHO Classification of AML origin” is believed to be the hematopoietic stem cell (HSC; Figure 26-1). AML WITH RECURRENT GENETIC ABNORMALITIES The t(8;21)(q22;q22) translocation repositions the 5′ The AML with recurrent genetic abnormalities classi- region of the RUNX1 gene (chromosome 21, previously fication is used when chromosomal or molecular-level termed AML1) with the 3′ region of the RUNX1T1 gene abnormalities, are found. In most cases, the chromosomal (chromosome 8; also called ETO). The result is a fusion rearrangements create a fusion gene, encoding a novel protein called RUNX1-RUNX1T1 (also known as RUNX1- fusion (chimeric) protein while the molecular-level muta- ETO). RUNX1 is a member of a family of genes called tions involve genes that impair cellular differentiation and core@binding factor@a (CBFa). CBFa proteins such as RUNX1 apoptosis. Molecular techniques (RT-PCR) have a higher normally form a heterodimeric complex with CBFb proteins Table 26.4 Cytogenetic and Morphologic Results Used to Classify AML with Recurrent Cytogenetic Abnormalities AML with recurrent genetic abnormalities Morphology AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 Maturation in the neutrophil lineage Auer rods AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) or t(16;16) Acute myelomonocytic leukemia (AMML Eo) (p13.1;q22); CBFB-MYH11 Maturation in both the neutrophil and monocytic lineage, abnormal eosinophils Eosinophils 5% or more Abnormal eosinophilic granules present in promyelocyte or myelocyte stage AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A Monoblasts and promonocytes predominate, can see vacuoles and granules Acute promyelocytic leukemia with PML-RARA Hypergranular variant Promyelocytes dominate Multiple Auer rods in one cell Microgranular variant Bi-lobed nuclei No visible granularity AML with t(6;9)(p23;q34.1); DEK-NUP214 With or without monocytic features and often associated with basophilia and multilineage dysplasia AML with inv(3)(q21.3;q26.2) or t(3;3) (q21.3;q26.2); GATA2, Associated with elevated platelets and atypical megakaryocytes (mono or MECOM bilobed) and multilineage dysplasia AML with t(1;22)(p13.3;q13.3), RBM15-MLK1 Megakaryocytic lineage with maturation AML with mutated NPM1 Myelomonocytic or monocytic features presents in older people with a normal karyotype AML with biallelic mutated CEBPA Myeloid leukemia with or without maturation AML with mutated RUNX1 (provisional entity) Can show myelomonocytic or monoblastic features AML with BCR-ABL1(provisional entity) 592 Chapter 26 in order to bind to DNA as functional transcription factors. detectable by conventional cytogenetics, so FISH or molecu- The RUNX1-RUNX1T1 fusion protein acts as a transcrip- lar studies must be performed.7 tional repressor by blocking the normal DNA binding func- tion of RUNX1, resulting in its inability to regulate cellular differentiation. Furthermore, the RUNX1-RUNX1T1 fusion Checkpoint 26.3 What does recurrent genetic abnormality mean in the context of leads to the activation of other genes involved in cellular AML, and what is the general outcome of these abnormalities? proliferation. The outcome is blocked differentiation and increased proliferation of the leukemic cells.11 AML with INV(16)(P13.1;Q22) OR T(16;16)(P13.1;Q22); CBFB- AML with PML-RARA Acute promyelocytic leukemia MYH11 This subcategory is identified in 7% of patients (APL) with PML-RARA is an AML in which abnormal pro- with AML12 and generally presents with monocytic and myelocytes predominate. Both hypergranular (“typical” granulocytic maturation and the presence of abnormal APL) and hypogranular or microgranular presentations eosinophils in the bone marrow. The most striking abnor- can be seen. APL can occur at any age, but most patients mality of the eosinophils is the presence of immature (baso- are adults in middle life. APL constitutes 5–8% of AML.7 philic) granules that would normally predominate at the The presenting signs for both hypergranular and hypo- promyelocyte and myelocyte stages (Chapter 7). The immu- granular APL often include acute disseminated intravas- nophenotypic and cytochemical results reflect the presence cular coagulation (DIC; Chapter 34).15 The most common of both neutrophilic and monocytic lineages (Appendix B). clinical finding at initial diagnosis is bleeding. The release of The leukemic cell of origin is believed to be an HSC. procoagulant material from promyelocytic granules likely The inv(16)(p13.1;q22) and t(16;16)(p13.1;q22) both initiates DIC, a serious complicating factor of the disease. result in the fusion of the core-binding factor gene CBFB Cytotoxic therapy for APL can potentiate or aggravate this (16q22) to the smooth muscle myosin heavy chain gene complication. Not only do the lysed promyelocytes release SMMHC (MYH11) at 16p13.1. Because of difficulties in large amounts of procoagulant material but also tissue correctly identifying these mutations with traditional cyto- factor–containing particles are released from dying cells genetics, FISH or RT-PCR may be necessary to document during cytotoxic therapy. Evidence of secondary fibrinoly- the mutation. The CBFb - MYH11 fusion protein binds to sis as a component of the DIC syndrome is also present. RUNX1 and represses its transcription factor function.13 Heparin therapy can be administered with initiation of che- motherapy to prevent or modulate the DIC. Other abnor- AML with T(9;11)(P21.3;Q23.3); MLLT3-KMT2A These malities of coagulation can be present. leukemias are usually associated with monocytic features (monoblasts and promonocytes). They can occur at any Hypergranular APL. Most patients with the hypergranular age but are more common in children (up to 25% of child- subtype are leukopenic or exhibit only slightly increased hood AML cases and 2–5% of all AML cases).14 Identical leukocyte counts. Most cells in the BM are abnormal pro- cytogenetic abnormalities can also be found in therapy- myelocytes with heavy azurophilic granulation. The gran- related AML (see the section “Therapy-Related Myeloid ules can be so densely packed that they obscure the nucleus Neoplasms”). Patients can have extramedullary myeloid (Figure 26-2a). Some cells filled with fine dustlike granules (monocytic) sarcomas (extramedullary leukemia) and tissue also can be present. Cells with multiple Auer rods, some- infiltration (gingiva, skin) and may present with dissemi- times occurring in bundles, are characteristic with cyto- nated intravascular coagulation. Monoblasts and promono- plasm that is frequently clear and pale blue, but cells can cytes can have scattered azurophilic granules and vacuoles contain only a few azurophilic granules or lakes of clear and give phenotypic results typical for the monocytic lin- pink material16 (Figure 26-3). In some cases, the typical eage (Table 26-4). The leukemic cell of origin is believed to hypergranularity of promyelocytes is less evident in the be the HSC. peripheral blood than in the bone marrow.17 The nucleus The KMT2A (formerly MLL) gene (11q23) is involved in varies in shape but is often folded or indented or sometimes a number of leukemia-associated translocations with differ- bilobed. Often a large number of promyelocytes appear to ent partner chromosomes. The KMT2A protein is a DNA- be disrupted on the blood smear with free azurophilic gran- binding protein that interacts with other nuclear proteins ules and Auer rods intermingled with intact cells. Anemia and permits the association of transcription factors that help and thrombocytopenia are typical findings. regulate transcription. The most common translocations Microgranular APL Variant. In contrast to typical APL, the involving 11q23 seen in childhood AML are t(9;11)(p21;q23), leukocyte count in microgranular APL is usually markedly t(11;19) (q23;p13.1), and t(11;19)(q23;p13.3). More than 80 increased.16 The predominant cell in the peripheral blood different translocations involving KMT2A are described is a promyelocyte with a bilobular, reniform, or multilobed with more than 50 different translocation partner genes. Up nucleus (resembling that of a monocyte) and cytoplasm with to one-third of KMT2A translocations in AML are not an apparent paucity of granules on Wright-stained smears Acute Myeloid Leukemias 593 a b Figure 26.2 (a) Peripheral blood film from a patient with acute promyelocytic leukemia (APL), hypergranular variant. These promyelocytes have an irregularly shaped nucleus and numerous azurophilic granules. (b) Peripheral blood film from a patient with APL, microgranular variant. Note the bilobed nuclei and absent or fine granules in these abnormal promyelocytes. (Wright-Giemsa stain, 1000* magnification) (Figure 26-2b). The apparent absence of granules is because A |
form of APL in which some promyelocytes contain of their submicroscopic size, but they are readily visible metachromatic granules (when stained with toluidine blue) with cytochemistry (myeloperoxidase, MPO positive). has been described.18 Distinctive features include folded The nuclear chromatin is fine with nucleoli often visible. nuclei, hypergranular cytoplasm, and coarse metachromatic A small abnormal promyelocyte with a bilobed nucleus, granules. deeply basophilic cytoplasm, and sometimes cytoplasmic Diagnostic Gene Rearrangement. The PML-RARA fusion projections is present as a minor population of cells in is consistently associated with APL. The break point most cases and occasionally is the predominant cell. When involves the retinoic acid receptor@a (RARa) gene on chro- cytoplasmic projections are present, the cells can resemble mosome 17 and the promyelocytic leukemia (PML) gene megakaryoblasts. Cells with multiple Auer rods are scarce on chromosome 15 and is limited to the neoplastic cells. or absent, but single Auer rods can be found. In contrast Some APL patients have a cytogenetically normal karyo- to the hypogranular appearance of peripheral blood pro- type, but molecular analysis has shown submicroscopic myelocytes, the BM promyelocytes may be more typical insertion of RARa into PML, leading to the expression of the cells found in the hypergranular form of APL. See of the PML/RARa transcript. These findings suggest Appendix B for cytochemical and immunophenotypic that the t(15;17) translocation/fusion protein is involved results for APL. in the pathogenesis of the disease. However, other vari- ant translocations have been reported in APL, all involv- ing the RARa gene translocated with different target genes, including promyelocytic zinc finger (PLZF) on chromosome 11(t[11;17][9q23;q21]), nuclear mitotic apparatus (NuMA) on chromosome 15 (t[11;17][q13;q21]), nucleophosmin (NPM1) on chromosome 5 (t[5;17][q23;q12]), and signal transducer and activator of transcription 5b (STAT5b) on chromosome 17 (t[17;17]).19 The complex routes by which the PML-RARA fusion protein can be found led the 2016 WHO classification to be more inclusive and not focus on the t(15;17) muta- tion in the diagnosis and instead rely on the presence of the fusion protein.7 The typical t(15;17) gene rearrangement results in a fusion PML/RARa gene and a reciprocal RARa/PML gene. The PML/RARa mRNA has been identified in all APL Figure 26.3 A cell from promyelocytic leukemia. Notice patients and the reverse translocation RARa/PML mRNA the bundle of Auer rods (arrow). (Wright-Giemsa stain, 1000* is found in about 75% of APL patients.20 Molecular analysis magnification) of the PML/RARa gene is important in monitoring therapy 594 Chapter 26 and relapse because the gene disappears with successful destroys the dividing stem cells that bear the t(15;17) trans- treatment but returns as an early marker of relapse. Also, location and induces a durable remission.1 some patients have molecular evidence of the fusion protein About 25% of patients given RA therapy become acutely product (fusion gene) in the absence of a detectable cytoge- ill associated with the death and lysis of the large number netic abnormality. of cells. The mortality rate in these individuals can be high. The RARa protein is a nuclear hormone receptor that The illness is similar to capillary leak syndrome with fever, binds to specific DNA sequences (RA-responsive elements) respiratory disease, renal impairment, and hemorrhage.23 and controls transcription of specific target genes under the control of retinoid hormones. RARa forms a complex with a second protein, retinoid-X receptor (RXRa). In the Checkpoint 26.4 absence of RA, the RARa/RXRa complex represses tran- Why is it important to perform molecular studies on patients scription by recruiting corepressor proteins and inducing with AML with t(15;17)? histone deacetylation.20 Physiologic concentrations of RA normally induce a conformational change in the retinoic acid receptors, causing release of the corepressor molecules AML with T(6;9)(P23;Q34.1); DEK-NUP214 This AML pres- and recruitment of coactivator molecules, resulting in gene transcription and granulocytic differentiation.21 ents with or without monocytic features often associated with basophilia and multilineage dysplasia. It is detected in PML is a growth suppressor nuclear protein normally 0.7–1.8% of AML and occurs in both children and adults.7 found in complex macromolecular structures containing AML with t(6;9)(p23;q34) usually presents with anemia and numerous other nuclear proteins. The PML/RARa fusion thrombocytopenia, and often with pancytopenia. In adults, protein binds to the RA-responsive elements of the target the white blood cell count is generally lower than other genes. Like the native protein, the fusion protein recruits AML categories.24 Morphologically, they most commonly corepressor molecules and in the absence of RA inhibits present with AML with maturation or acute myelomono- transcription of RA-responsive genes. Although the fusion cytic leukemia. Auer rods are present in one-third of cases. protein can bind RA, physiologic concentrations of RA are Marrow and peripheral blood basophilia is seen in 44–62% not sufficient to induce the release of the corepressor pro- of cases. Most cases show evidence of granulocytic and ery- teins from PML/RARa and repression of gene transcrip- throid dysplasia. tion is maintained.21 Thus, the cells do not differentiate into The t(6;9)(p23;q34) results in a fusion of the DEK gene granulocytes. on chromosome 6 with the NUP214 (formerly known as Pharmacological concentrations of RA (all-transretinoic CAN) gene on chromosome 9. This results in a nucleoporin acid [ATRA]) have proven to be an effective treatment for fusion protein that acts as an aberrant transcription factor inducing complete hematologic remission in APL (but the and alters nuclear transport by binding to soluble transport remission is generally short lived). ATRA induces the neo- factors.25 The t(6;9) is the sole abnormality in the majority of plastic promyelocytes to differentiate to mature granulo- cytes, thus overcoming the maturation arrest.22 cases, but some patients have the t(6;9) in association with It is thought a complex karyotype. that a high concentration of RA, as is given in induction therapy for APL, overcomes the interference with receptor AML with INV(3)(Q21.3;Q26.2) OR T(3;3)(Q21.3;Q26.2); activation (it promotes release of the corepressor molecules GATA2, MECOM This AML can present de novo or arise and assembly of the coactivator molecules on the target from a prior MDS. It is often associated with normal or genes, allowing gene transcription and cellular differentia- elevated peripheral blood platelet count and has increased tion). Cells previously unable to mature and initiate apop- atypical megakaryocytes with mono- or bi-lobated nuclei tosis are then able to do so, causing a transitory situation and associated multilineage dysplasia. AML with inv(3) known as hyperleukocytosis. accounts for 1–2% of all AML.26 It occurs most commonly Unfortunately, the stem cells bearing the t(15;17) trans- in adults with no sex predilection. Patients present with location are unaffected by ATRA and continue to proliferate, anemia and a normal or elevated platelet count. Some pres- resulting in relapse of the disease. The current approach ent with hepatosplenomegaly. A subset of cases may have to therapy is to induce hematologic remission with ATRA less than 20% blasts at the time of diagnosis with features and then administer traditional chemotherapy (to try to of chronic myelomonocytic leukemia. Marrow eosinophils, eradicate the leukemic stem cells). When the promyelocytes basophils, and mast cells may be increased. The BM shows are first induced to differentiate and are no longer pres- small hypo-lobated megakaryocytes. ent in large numbers, the subsequent chemotherapy does A variety of abnormalities in the long arm of chromo- not result in the massive death of promyelocytes (and the some 3 occur in myeloid malignancies; inv(3)(q21;q26.2) release of procoagulants), thus avoiding or minimizing the and t(3;3)(q21;q26.2) are the most common. The t(3;3) life-threatening effects of DIC. Instead, the chemotherapy (q21;q26.2) involves the repositioning of a GATA2 enhancer Acute Myeloid Leukemias 595 that simultaneously causes activation of the EVI1 gene or show extramedullary involvement with the most frequently the MECOM complex locus (MDS1-EVI1 genes) and leads affected sites being the gingiva, lymph nodes, and skin. to leukemogenesis, and ihematopoietic cell transforma- NPM1 mutation is found in approximately 35% of AML tion.27–29 Secondary karyotypic abnormalities are common patients.34 NPM1 mutations can also be seen in AML with with inv(3)(q21;q26.2) and t(3;3)(q21;q26.2) with monosomy and without maturation and in acute erythroid leukemia. 7 most common, occurring in approximately 50% of cases, The diagnosis of AML with mutated NPM1 is made followed by 5q deletions and complex karyotypes. based on the identification of the mutation by molecular studies and is usually associated with a normal karyotype; AML with T(1;22)(P13.3;Q13.32); RBM15-MKL1 This AML 5–15% show chromosomal abnormalities including trisomy generally shows maturation in the megakaryocytic lineage. 8 and del(9q). It is uncommon, representing less than 1% of all cases. It occurs de novo and is restricted to infants and young AML with Biallelic Mutations of CEBPA Acute myeloid children with most cases occurring in the first 6 months leukemia with mutated CEBPA is usually seen in AML of life. The clinical presentation is marked organomegaly, with or without maturation. Some cases can show myelo- especially hepatosplenomegaly. Patients present with ane- monocytic or monoblastic features. The CEBPA protein is mia and thrombocytopenia with a moderately elevated normally expressed at low levels in HSCs, increases in con- white blood cell count. The blasts are similar to those of centration as cells mature to the CMP stage, and decreases acute megakaryoblastic leukemia. Small and large mega- again as cells mature to terminally differentiated neutro- karyoblasts may be present along with morphologically phils or monocytes. Loss of CEBPA gene expression leads undifferentiated blasts resembling lymphoblasts. The to maturation arrest at the CMP.35 megakaryoblasts are usually medium to large in size with CEBPA mutations usually present de novo, and are seen an indented nucleus, fine reticular chromatin, and one to in 5–15% of AML.36 There is no age or sex difference. Patients three nucleoli. The cytoplasm is basophilic and often agran- present with a higher hemoglobin level and lower platelet ular and may show distinct blebs or pseudopod formation. counts, lower LD levels, and higher peripheral blood blast Micromegakaryocytes are common. Dysplasia in the ery- count when compared with CEBPA non-mutated AML. throid and granulocytic cell lines is usually not present. AML with a normal karyotype and biallelic CEBPA muta- The t(1;22)(p13;q13) translocation results in the fusion tion is associated with a favorable prognosis while those of the RNA-binding motif protein-15 (RBM15) gene (1p13) with only a single CEBPA mutation do not have the same with the megakaryoblastic leukemia-1 (MKL1) gene (22q13) outcome.7 This favorable outcome is only associated with and ultimately, expression of the RBM15–MKL1 fusion pro- biallelic mutations, thus the 2016 WHO classification distin- tein. In most cases, this is the sole abnormality. guishes between single and double mutation disease. The function of RBM15 is not well defined. It appears to act as a transcriptional repressor that negatively regu- Provisional Subclasses and Miscellaneous Mutations lates megakaryocyte development. When RBM15 is lost or As in prior editions, the 2016 WHO classification uses the nonfunctional, megakaryocytes are increased in number in provisional entity designation to introduce new subclasses the BM and spleen.30 MKL1 encodes a transcriptional coacti- to the recurrent genetic abnormalities subtypes. These enti- vator of genes involved in megakaryocyte differentiation ties are included based on new data that highlights the role and maturation.31 The precise role of the fusion protein these mutations play in disease progression and response in AML is unclear; however, it may modulate chromatin to therapy. The FLT3 mutation is also discussed below due organization (epigenetic control) in regions important for to its importance in prognosis. megakaryocytic development.32 AML With BCR-ABL1 BCR-ABL1 mutations are found in a AML with Mutated NPM1 This form of AML usually number of neoplastic hematopathologies including an esti- involves mutations in exon 12 of the NPM1 gene. The NPM mated 1% of de novo AML. As is the case in other disease protein is a chaperone of proteins that shuttles between the processes, this mutation is more prevalent among males in nucleus and cytoplasm. NPM1 gene mutations appear to the sixth decade of life.37 Without appropriate clinical his- cause loss-of-function (Chapter 23) of the NPM protein to tory, differentiating Philadelphia chromosome positive de the extent that NPM remains in the cytoplasm and can no novo AML from blastic transformations of CML may be dif- longer translocate to the nucleus.33 ficult, but its presence in AML is important as it indicates This AML frequently has myelomonocytic or mono- that tyrosine kinase therapy may be beneficial.7 cytic features and presents de novo in older adults with Examination of peripheral blood often shows variation a normal karyotype. Usually there is no |
history of a prior in the white blood count, but median counts are approxi- MDS or MPN. Patients often have anemia and thrombocy- mately 19.0 * 103/mcL. Typically the cells present are topenia and a higher white blood cell count than other AML immature myeloid cells, although occasional cases involve types. There is a slight female predominance. Patients can a predominance of monocytic or megakaryocytic elements. 596 Chapter 26 Statistically significant differences in the presence of In these cases, AML occurs as a late complication of cyto- splenomegaly and absolute basophilia have been noted toxic chemotherapy and/or radiation therapy administered between CML and AML patients with this mutation. for a prior malignancy. This group of neoplasms is best con- However a more definitive approach for separating de sidered together as a unique clinical syndrome. novo AML from blast transformation in CML is being Therapy-related neoplasms are thought to be the sought for those patients with limited clinical information consequence of mutations induced by cytotoxic therapy. present at diagnosis.7,37 Cytotoxic agents commonly implicated include alkylating agents, topoisomerase II inhibitors, and ionizing radia- AML with Mutated RUNX1 This new provisional subtype tion therapy. Two subsets are recognized. The most com- is defined by RUNX1 gene mutation and is found pre- mon occurs 5–10 years after receiving the alkylating drug dominantly in patients who develop AML with minimal or radiation. The patient often presents with t-MDS, and a differentiation. Megakaryoblastic subtypes of AML may minority will present with t-MDS/MPN or t-AML. Patients also demonstrate the mutation. Myelodysplastic-related commonly have an unbalanced loss of genetic material alterations in hematopoietic lineages are often noted, even involving chromosome 5 and/or 7. In the second subset, the in cases of de novo AML. The RUNX1 gene is located on latency period is shorter, about 1–5 years, and follows treat- the long arm of chromosome 21 and plays an important ment with topoisomerase II inhibitors. Most of the patients role in the development of hematopoietic stem cells during embryonic and fetal development.38 in this category present with acute leukemia. Approximately 70% of patients have unbalanced chro- While the gene mutation itself is not known to be leu- mosomal losses, mainly with chromosome 5 and/or 7, and kemogenic, it is often found with other genetic mutations or are often associated with additional abnormalities including aneuploidy states.39 It is included in the 2016 WHO classi- del(13q), del(20q), del(11q), del(3p), -17, -18, -21, +8. This fication because its presence is associated with unfavorable outcomes and resistance to therapy.7,39 type of AML generally has a poor prognosis.1 AML WITH MYELODYSPLASIA-RELATED CHANGES FLT3 and AML Accurate assessment of prognosis is central This category is an acute leukemia with morphological to the management of AML. The FLT3 gene mutations are features of a myelodysplastic neoplasm, a prior history of associated with a worse prognosis. The FLT3 gene is located myelodysplastic neoplasm, a myelodysplastic/myelopro- at 13q12 and encodes a tyrosine kinase receptor involved liferative neoplasm or an MDS-related cytogenetic abnor- in hematopoietic stem cell differentiation and proliferation. mality. To classify as AML with myelodysplasia-related The FLT3 protein is expressed on progenitor cells and blasts changes by morphology, the dysplasia must be present in most cases of AML. FLT3 mutations may occur with any in 50% of cells in at least two cell lineages. Dyserythro- AML type and in MDS and can occur with or without addi- poiesis is characterized by megaloblastosis, karyorrhexis, tional cytogenetic abnormalities.40 Mutations lead to con- nuclear irregularity, multinucleation, ring sideroblasts, or stitutively activated FLT3 that transduces signals for cell cytoplasmic vacuoles. Dysgranulopoiesis is characterized growth and inhibition of apoptosis. FLT3 gene mutations by neutrophils with hypogranular cytoplasm, hyposeg- occur in approximately 30% of adult and 12% of pediatric patients with de novo AML.40 mentation (pseudo–Pelger-Huët anomaly), or abnormal segmentation. Dysmegakaryopoiesis is characterized by The two primary types of FLT3 mutations are internal micromegakaryocytes, megakaryocytes with non-lobated tandem duplication (FLT3-ITD) seen in 30–40% of cases, and nuclei, or multiple nuclei. The chromosomal abnormalities mutations affecting the second tyrosine kinase domain found in this category are similar to those found in MDS (TKD).5 FLT3-ITD mutations are associated with an adverse and often involve complex karyotypes with -7/del(7q) and outcome, but the significance of FLT3-TKD mutations -5/del(5q) being the most common (Chapter 25). Trisomy remains controversial.7 Although FLT3 mutations are not 8 and del(20q) are common in MDS but are not considered included as a defining criterion for WHO classification, they are important for prognosis and should be evaluated.5 to be disease-specific abnormalities and would not be suf- ficient by themselves to be considered a case of AML with myelodysplasia-related changes. The 2016 WHO classifica- Checkpoint 26.5 tion has determined that multilineage dysplasia with either Would the FLT3, NPM1, and CEBPA genes be classified as NPM1 or bialleic mutations of CEBPA cannot be classified tumor suppressor or proto-oncogenes? under AML with myelodysplastic-related changes.7 AMLS NOT OTHERWISE SPECIFIED THERAPY-RELATED MYELOID NEOPLASMS This AML category includes cases of AML that do not fulfill This category includes therapy-related acute myeloid leuke- criteria for any of the other described groups.5 The subcat- mia (t-AML), myelodysplastic syndrome (t-MDS), and myelo- egories in this group are primarily differentiated on mor- dysplastic/ myeloproliferative neoplasms (t-MDS/MPNs). phology and cytochemical features. The defining criterion Acute Myeloid Leukemias 597 is 20% or more blasts (the blast percentage includes promy- elocytes in APL and promonocytes in AML with monocytic Table 26.5 Cytochemical Reactions for Auer Rods differentiation). For pure erythroid leukemias, the blast SBB + percentage is based on the percentage of abnormal erythro- Myeloperoxidase (MPO) + blasts. Immunophenotypic and cytochemical results for this Napthol AS-D chloroacetate esterase { category of AML are included in Table 26-3. PAS { AML WITH MINIMAL DIFFERENTIATION AML with Romanowsky + or - (occasionally seen only with MPO or SBB) minimal differentiation is a rare leukemia (less than 5% of all AML cases) and is characterized by lack of evidence of myeloid differentiation by morphology (absence of gran- The defining feature is its lack of cellular maturation; less ules) or cytochemistry (63, of the blasts are positive for than 10% of all granulocytic cells show evidence of matura- SBB or MPO).41 This subtype is generally associated with a tion beyond the myeloblast stage (Figure 26-4). poor prognosis. The blasts, however, demonstrate myeloid The predominant cell in the peripheral blood is usually differentiation by flow cytometry for the myeloid lineage a poorly differentiated myeloblast with fine lacy chromatin (CD13 and CD33). Most cases are also CD34, CD38, and and nucleoli. Myeloblasts are usually 90% or more of non- HLA@DR+ . No unique chromosomal abnormalities are erythroid cells in the BM. with more than 3% of blasts posi- associated with this subtype. The most common previously tive for MPO and/or SBB. The blasts may have azurophilic reported abnormalities are complex karyotypes and unbal- granules, Auer rods, and vacuoles. If no evidence of gran- anced abnormalities such as -5/del(5q), -7/del(7q), +8, ules or Auer rods is present, the blasts can resemble lym- and del(11q), but the presence of these abnormalities now phoblasts and must be differentiated by immunophenotype. places them in a different category (Table 26-1).10 Because Cytochemical staining reactions for Auer rods are similar to the blasts in the minimally differentiated AML group have reactions for myeloblasts (Table 26-5). There is no demon- no morphologic differentiating features, immunophenotyp- strated association between AML without maturation and ing should be used to exclude the lymphoid lineage. The specific chromosomal abnormalities. cell of origin is thought to be the HSC.42 AML WITHOUT MATURATION Characterized by a high percentage of BM blasts without significant evidence of maturation to more mature neutrophils, this variant AML Checkpoint 26.6 without maturation can occur at any age but most com- What hematologic features help distinguish AML minimally differentiated from AML without maturation? monly in adulthood. It accounts for 5–10% of the cases of AML. Patients usually present with BM failure; however, there can be a leukocytosis with markedly increased blasts. AML WITH MATURATION AML with maturation is characterized by 20% or more blasts with evidence of mat- uration to more mature neutrophils (greater than 10% of cells at differentiated stages of maturation: promyelocytes, myelocytes, metamyelocytes). Monocytes and their pre- cursors constitute less than 20% of the marrow cells. This type of AML occurs in all age groups (20% of patients are younger than 25 years of age, and 45% are older than 60 years of age) and accounts for about 10% of AML cases.40 Patients often present with anemia, thrombocytopenia, and neutropenia. Blasts with and without azurophilic granulation can be present, and blasts frequently contain Auer rods. Vari- able dysplasia, including myeloid hypogranulation, nuclear hypo- and hypersegmentation, and occasionally binucle- ated myeloid cells, can be seen (Figure 26-5). The BM is hypercellular, and myeloblasts make up 20–89% of the Figure 26.4 Peripheral blood film from a patient with AML. nonerythroid nucleated cells. Eosinophils and sometimes The large mononuclear cells are myeloblasts. The cell at the left (arrow) is a myeloblast with an Auer rod. Note the high nuclear- basophils can be increased. No demonstrated association to-cytoplasmic ratio, the fine lacy chromatin, and the prominent between AML with maturation and specific recurrent chro- nucleoli. (Wright-Giemsa stain, 1000* magnification) mosomal abnormalities exists. 598 Chapter 26 Figure 26.5 Figure 26.6 Peripheral blood film from a case of acute Peripheral blood film from a patient with AML myelomonocytic leukemia (AMML). Monoblasts and promonocytes with maturation. Myeloblasts are at the bottom and hypogranulated are present. (Wright-Giemsa stain, 1000* magnification) segmented neutrophils are at the top. (Wright-Giemsa stain; 1000* magnification) Additional laboratory testing is required when (1) the Checkpoint 26.7 BM findings are as previously described, but the peripheral A patient with AML has a peripheral blood differential that blood monocyte count is less than 5 * 103/mcL or (2) the includes 91% myeloblasts, 4% promyelocytes, 3% granulo- peripheral blood monocyte count is 5 * 103/mcL or more, cytes, and 2% monocytes, and 30% of the blasts are positive but the BM has less than 20% monocytic cells. In these cases, with MPO. Which category of AML is the most likely diagnosis? Explain. ancillary laboratory tests such as lysozyme levels or cyto- chemical methods can be utilized to confirm the presence of a significant monocytic component and establish a diag- Acute Myelomonocytic Leukemia (AMML) Characterized nosis of AMML (Table 26-6). by proliferation of both myelocytic and monocytic precur- sors, the BM in acute myelomonocytic leukemia (AMML) is hypercellular with 20% or more blasts; neutrophils and their precursors as well as monocytes and their precursors each Table 26.6 Nonimmunophenotypic Criteria for Diagnosis compose 20% or more of marrow cells. AMML accounts for of AMML 5–10% of AML cases. Some patients have a history of chronic 1. Bone marrow Blasts 20% or more of nonerythroid cells myelomonocytic leukemia. Infiltrations of leukemic cells in Monocytic cells 20% or more of nonerythroid cells extramedullary sites are more frequent than in the pure Granulocytic cells 20% or more of nonerythroid cells granulocytic variants. Serum and urinary levels of murami- and peripheral blood 5 * 103/mcL or more monocytic cells dase (lysozyme) are usually elevated because of the mono- or if peripheral blood cytic proliferation. Less than 5 * 103/mcL monocytic cells The peripheral blood leukocyte count in AMML is Requires ancillary Serum or urinary lysozyme 3* normal tests usually increased. Monocytic cells (monoblasts, promono- or naphthol AS-D chloroacetate esterase and cytes, monocytes) are increased to 5 * 103/mcL or more a@naphthyl acetate esterase (+ ) in blasts (Figure 26-6). Anemia and thrombocytopenia are present or naphthol AS-D acetate esterase with and in almost all cases. The myeloblasts appear similar to blasts without NaFl reveal more than 20% monocytic cells in bone marrow in AML with differentiation. Monoblasts are usually large with abundant bluish-gray cytoplasm and can show pseu- 2. Peripheral blood 5 * 103/mcL or more monocytic cells dopods. Scattered fine azurophilic granules and vacuoles and bone marrow Blasts 20% or more of nonerythroid cells may be present. The nucleus is round or convoluted with Monocytic cells less than 20% of nonerythroid cells delicate chromatin and one or more prominent nucleoli. Requires ancillary Serum or urinary lysozyme 3* normal tests The monoblasts and myeloblasts demonstrate the expected cytochemical and immunophenotypic results for their or naphthol AS-D chloroacetate esterase and a@naphthyl acetate esterase (+ ) in blasts respective lineages |
(Table 26-5). The BM can reveal eryth- or naphthol AS-D acetate esterase with and rophagocytosis by monocytes. The cell of origin is thought without NaFl reveal more than 20% monocytic to be the hematopoietic stem cell.42 cells in bone marrow Acute Myeloid Leukemias 599 Acute Monoblastic Leukemia and Acute Monocytic Leuke- aspect of this disease is the degree of extramedullary leuke- mia (AMOL) These subtypes are myeloid leukemias in mic proliferation (gingival and cutaneous infiltration, cen- which 80% or more of the leukemic cells are monocytic tral nervous system involvement). As with AMML, serum (including monoblasts, promonocytes, monocytes). The and urine muramidase are moderately elevated. Monocytic neutrophilic component, if present, is less than 20% of the cells account for 80% or more of cells in the bone marrow. cells. The majority of the monocytic cells (usually 80% or Monocytes in the peripheral blood are increased, and mono- more) found in acute monoblastic leukemia are monoblasts blasts are often present. The monoblasts are large (up to 40 (Figure 26-7). The majority of the monocytic cells in acute mcM) with abundant, variably basophilic cytoplasm. Pseu- monocytic leukemia are promonocytes (Figure 26-8). Acute dopods with translucent cytoplasm are common, and fine monoblastic leukemia accounts for 5–8% of AML cases, and azurophilic granules can be present. The nucleus is round acute monocytic leukemia accounts for 3–6% of them.42 This or oval with delicate chromatin and one or more prominent disease is usually seen in children or young adults. nucleoli, but Auer rods are usually not found. Dyshemato- The most common clinical findings are weakness, poiesis is not conspicuous. Promonocytes have a more irreg- bleeding, and a diffuse erythematous skin rash. One notable ular and convoluted nucleus, and nucleoli can be present. a b Figure 26.7 (a) A smear from a bone marrow aspirate of a patient with acute -monoblastic leukemia. The cells are predominately monoblasts and promonocytes. (b) Monoblasts in peripheral blood from a case of acute monoblastic leukemia. (Wright-Giemsa stain, 1000* magnification) a b Figure 26.8 (a) A smear from a bone marrow aspirate of a patient with acute monocytic leukemia. There is a predominance of promonocytes and monoblasts (Wright-Giemsa stain, 1000* magnification). (b) Monocytic cells in peripheral blood, including promonocytes and monoblasts, from acute monocytic leukemia (Wright-Giemsa stain; 1000* magnification). 600 Chapter 26 The cytoplasm is less basophilic than that of the monoblast with a ground glass appearance. Fine azurophilic granules Checkpoint 26.8 are often present. What are the WHO2016 criteria for a diagnosis of pure erythroid Abnormalities of the long arm of chromosome 11 with leukemia? translocations or deletions are often found in monocytic leu- kemias. The t(8;16) abnormality is also found and is asso- Acute Megakaryoblastic Leukemia (AMKL) The mega- ciated with hemophagocytosis. The expression of c-FOS karyoblastic subgroup of AML is an acute leukemia in proto-oncogene on chromosome 14 appears to be enhanced which 50% or more of the leukemic blasts are of the mega- in acute leukemias with monocytic lineage involvement.1 karyocytic lineage. It occurs in both adults and children and This gene has been linked to normal monocyte–macrophage constitutes less than 5% of all AML cases.42 This category differentiation. The cell of origin for this AML subtype is excludes AML with myelodysplasia-related changes, AML believed to be an HSC.7 with t(1;22)(p13;q13), inv(3)(q21;q26), t(3;3)(q21;q26.2), and Down syndrome-related cases. Pure Erythroid Leukemia (PEL) The only change to the Patients usually present with cytopenias; although category of AML not otherwise specified in the 2016 WHO most have thrombocytopenia, some can have thrombocy- classification involves AL involving the erythrocytic lin- tosis. Dysplastic features in the neutrophils and platelets eage. Classification of erthrocytic leukemias has always may be present. There is no significant organomegaly. The been challenging due to overlapping features with myelo- BM megakaryoblasts are usually medium to large (12–18 dysplastic syndromes and other subtypes of AML. The 2016 mcM) with basophilic, often agranular, cytoplasm. The cells classification has simplified the criteria for this subtype, frequently show distinct pseudopod formation. Small blasts now known as pure erythroid leukemia.7 resembling lymphoblasts can also be present. On careful Bone marrow analysis shows more than 80% of bone examination of the peripheral blood smear, circulating marrow cells are erythroid precursors with 30% or more micromegakaryocytes and undifferentiated blasts can be being proerythroblasts. Cases with fewer erythroid pre- found. However, the finding of cytoplasmic blebs suggests cursors or an increase in myeloblasts may be classified as that these cells are megakaryocytic. Megakaryocytic frag- AML or myelodysplastic syndrome, based on other criteria ments also can be present. according to the 2016 WHO classification system. The BM BM aspiration often results in a dry tap because of erythroblasts are distinctly abnormal with bizarre morpho- extensive marrow fibrosis associated with an expanded logical features. Giant multilobular or multinucleated forms megakaryocyte lineage. In these cases, marrow biopsy are common. Other features include nuclear budding and may be required and usually reveals increased fibro- fragmentation, cytoplasmic vacuoles, Howell-Jolly bodies, blasts and/or increased reticulin as well as more than 20% ringed sideroblasts, and megaloblastoid changes. Erythro- blasts. It has been suggested that megakaryocytes secrete phagocytosis of the abnormal erythroblasts is a common a number of mitogenic factors that stimulate fibroblast finding. Dysmegakaryopoiesis is common with mononu- proliferation. clear forms or micromegakaryoblasts present. Neutrophils Blasts can be identified as megakaryocytic by immu- can exhibit hypogranularity and pseudo–Pelger-Huët nophenotyping flow cytometry for CD41, CD61, and anomaly. The leukocyte alkaline phosphatase score is nor- CD42b, and cytochemistry. The blasts are highly variable, mal or increased. Leukocytes and platelets are usually ranging from small round cells with scant cytoplasm and decreased. dense heavy chromatin to cells with moderately abundant The most dominant changes in the peripheral blood cytoplasm with or without granules and nuclei with lacy are anemia with striking poikilocytosis and anisocytosis. chromatin and prominent nucleoli. Some blasts can have There are a large number of nucleated erythrocytes that are cytoplasmic blebs. Megakaryocytes with shedding platelets dysplastic with megaloblastoid nuclei and/or bi- or multi- occasionally are present. Dysplasia of all cell lines is a com- nucleated cells in the more immature stages. The cytoplasm mon finding. frequently contains vacuoles. No unique chromosomal abnormality is associated Normoblasts are typically periodic acid-Schiff (PAS) with acute megakaryoblastic leukemia in adults. negative; however, in erythroleukemia, erythroblasts can demonstrate coarse positivity in either a diffuse or granu- Acute Basophilic Leukemia (ABL) The primary differen- lar pattern. PAS-positive erythroblasts are occasionally also tiation of this acute leukemia is along the basophil lineage. found in MDS, other subgroups of AML, iron-deficiency Some cases develop as a blast transformation phase of CML. anemia, thalassemia, severe hemolytic anemia, and some- This is a very rare form of AML with few reported cases times in megaloblastic anemia. Erythroblasts usually react (less than 1% of all AML cases). The most characteristic positively with antibodies to glycophorin A (CD71) or feature by cytochemistry is metachromatic positivity with hemoglobin A. toluidine blue.7 The blasts can show diffuse staining with Acute Myeloid Leukemias 601 acid phosphatase and occasionally PAS positivity. They are in reticulin fibrosis resulting in a dry tap. The blasts are usually negative for typical cytochemical stains for myeloid positive for the same markers as in TAM except that 50% cells but express myeloid markers (e.g., CD13, CD33). No of cases are negative for CD34, and 30% of cases are nega- consistent chromosomal abnormality has been identified tive for CD56. In addition to trisomy 21, somatic mutations with this subtype of AML. The cell of origin is believed to of the gene encoding the transcription factor GATA1 are be a myeloid precursor committed to the basophil lineage. considered pathognomonic of TAM or MDS/AML of The differential diagnosis includes a blast phase of a myelo- Down syndrome. proliferative neoplasm and other AML with basophilia such as AML with t(6;9)(p23;q34). ACUTE PANMYELOSIS WITH MYELOFIBROSIS CASE STUDY (continued from page 588) This disorder is associated with an acute panmyeloid pro- liferation and fibrosis of the bone marrow. A rare form of A BM aspirate and biopsy were performed on Jon- AML, it occurs primarily in adults. The major differential athan to aid in diagnosis. The BM biopsy revealed diagnosis is with acute megakaryoblastic leukemia (with a hypercellular marrow and an M:E ratio of 9.1:1. fibrosis) or other types of AML with associated marrow The predominant cells were blasts with Auer rods fibrosis and/or with chronic idiopathic myelofibrosis. frequently present. This diagnosis is indicated when the proliferative process 5. Based on the morphology, what is the most likely involves all major myeloid lineages (granulocytes, erythroid AML classification? cells, and megakaryocytes [i.e., panmyelosis]). Dysplastic, 6. What are at least two recurrent genetic mutations small megakaryocytes are characteristic. The number of seen in this type of presentation? blasts varies but is usually between 20 and 25%. Diagnosis requires BM biopsy and immunohistology. MYELOID SARCOMA This designation refers to a disease process in which a tumor mass of myeloblasts or immature myeloid cells occurs in an Therapy extramedullary site or in bone. It can occur concurrently Traditional chemotherapy for AML is designed to reduce with acute or chronic myeloid leukemias or other myelo- tumor load as rapidly as possible. Newer treatment modal- proliferative or myelodysplastic disorders. The immuno- ities include molecularly targeted therapies (e.g., ATRA), phenotype and cytochemistry reflect the specific myeloid epigenetic-targeted therapies (demethylating agents, his- cell(s) involved in the malignant process (i.e., myeloblasts, tone deacetylase inhibitors), and autologous or allogeneic promyelocytes, occasionally monocytes). BM transplants and infusions of donor lymphocyte cells with total body irradiation to increase leukemic cell destruc- MYELOID PROLIFERATIONS RELATED TO DOWN tion. Current research is focusing on novel therapies includ- SYNDROME ing the use of monoclonal antibodies and gene therapy and Individuals with Down syndrome have been found to the destruction of the cellular matrix that supports the neo- have an increased risk of leukemia, estimated at 10- to 100- plastic tissue. fold. They also are at risk for developing transient abnor- Evaluation of peripheral blood counts is essential to mal myelopoiesis (TAM), which is a unique disorder of support patients during chemotherapy. Development of at- Down syndrome. The newborn with TAM presents with risk stages such as pancytopenia, severe granulocytopenia, clinical and morphologic findings indistinguishable from and/or thrombocytopenia must be monitored so that early AML. The blasts are of the megakaryocytic lineage. At pre- intervention can occur (growth factors to stimulate hema- sentation, thrombocytopenia is the most common feature. topoietic cell recovery and/or transfusions). There may be a marked leukocytosis with an increase in BM transplantation (Chapter 29), whether allogeneic or blasts. Hepatosplenomegaly may be present. In the major- autologous, remains the only therapeutic choice that cur- ity of cases, spontaneous remission occurs within the first rently provides the potential for a prolonged (10+ years) three months of life. In most cases, the blasts are positive disease-free survival for most patients with AML. for CD34, CD56, CD117, CD13, CD33, CD7, CD4, CD41, CD42, and CD61. In addition to trisomy 21, acquired GATA1 mutations are present in blast cells of TAM. Chil- dren with Down syndrome have a 50-fold increase in inci- Checkpoint 26.9 dence of acute leukemia during the first 5 years of life. Predict the peripheral blood picture of a patient on antifolate About 1–2% of children with Down syndrome will develop chemotherapy. AML during this time. The BM core may show an increase 602 Chapter 26 Summary The ALs compose a heterogeneous group of neoplastic ALL (MPO- ). Subgrouping the AMLs requires additional stem cell disorders characterized by unregulated prolif- cytochemical stains, immunophenotyping, and cytoge- eration and blocked maturation. The two major groups of netic analysis. The AML cases known as AML not other- AL are AML and ALL, which the 2016 WHO Classifica- wise specified are primarily classified by morphology and tion System further categorizes into subtypes based on cytochemistry. morphologic criteria, cytochemical stains, immunologic The onset of AML is usually abrupt; without treatment, analysis, and cytogenetic and molecular abnormalities. it progresses rapidly. Symptoms are related to anemia, The 2016 WHO classification system recognizes AML thrombocytopenia, and/or neutropenia. Splenomegaly, subtypes with recurrent genetic abnormalities, with mul- hepatomegaly, and lymphadenopathy are common find- tilineage dysplasia, and following cytotoxic therapy. It ings. Hematologic findings include a macrocytic or nor- replaces the FAB classification system that was based on mocytic, normochromic anemia, thrombocytopenia, and a morphology and cytochemistry and updates the prior decreased, normal, |
or increased leukocyte count. Blasts are WHO classifications based on updated knowledge related almost always found in the peripheral blood. A BM exami- to AML diagnosis and treatment. MPO and/or SBB cyto- nation is always indicated if leukemia is suspected. The BM chemical staining help differentiate AML (MPO+ ) from reveals 20% or more blasts. Review Questions Level I 4. An Auer rod is best described as (Objective 7): 1. A differential report notes the presence of more than a. threads of chromosomal DNA found within the 20% blasts. This number supports the diagnosis of: cytoplasm (Objective 1) b. inappropriately fused primary granules a. CML c. excessive accumulations of ribosomal RNA b. AML d. Golgi apparatus that have not been enzymatically c. CLL degraded d. MDS 5. The leukemia that belongs to the WHO classifica- tion of AML with recurrent genetic abnormalities is: 2. The presence of blasts with no evidence of myeloid (Objective 3) differentiation by morphology and less than 3% posi- tive for SBB or MPO is commonly seen in: a. APL with PML-RARA (Objective 2) b. megakaryoblastic leukemia a. AML—minimally differentiated c. pure erythroid leukemia b. AML—without maturation d. AML minimally differentiated c. acute basophilic leukemia 6. Which of the following subcategories of AML give a d. myeloid sarcoma positive result with a@naphthyl esterases? (Objective 6) 3. Which of the following best describes the bone marrow of a patient with AML? (Objective 5) a. AML without maturation a. M:E ratio of 1:4, normocellular, and 15% b. Acute monocytic leukemia lymphoblasts c. Pure erythroid leukemia b. M:E ratio of 10:1, hypercellular, and 35% d. APL with PML-RARA myeloblasts c. M:E ratio of 1:5, hypocellular, and 11% myeloblasts d. M:E ratio of 4:1, hyperceullar, and 40% lymphoblasts Acute Myeloid Leukemias 603 7. A patient who had been treated previously for myelo- 2. The WHO classification of AML is based on: dysplasia developed AML. What subgroup is most (Objectives 2, 7) appropriate for a diagnosis in this case? (Objective 3) a. genetic abnormalities a. AML with recurrent genetic abnormalities b. morphology and cytochemistry of blasts b. AML with MDS-related changes c. immunophenotyping of blasts c. t-AML d. all of the above d. AML with differentiation 3. Which of the following leukemias is associated with 8. The minimum number of blasts in the peripheral cells containing an abundance of Auer rods occurring blood or bone marrow needed for a diagnosis of AML in bundles? (Objective 2) is: (Objectives 1, 4) a. APL a. 30% b. AML with multilineage dysplasia b. 10% c. AML t(8;21) c. 50% d. AML therapy related d. 20% 4. Which of these leukemic blasts demonstrate t(1;22) 9. Pure erythroid leukemia must demonstrate which (p13;q13) positivity? (Objective 4) of the following features within the bone marrow? (Objectives, 3, 5) a. Erythroblasts a. More than 20% myeloblasts b. Myeloblasts b. More than 30% orthrochromic erythroblasts c. Megakaryoblasts c. Less than 20% megakaryocytes d. Monoblasts d. Less than 80% erythroid precursors 5. A bone marrow from a 20-year-old male revealed 10. The presence of de novo AML with BCR-ABL1 as a 40% agranular blasts. Cytochemical stains were provisional entity was added to the WHO classifica- negative for MPO, SBB, and specific and nonspecific tion of AML due to: (Objective 3) esterases. What immunophenotype panel should be used to help identify the blast cell lineage? a. the presence of the mutation definitively differenti- (Objective 6) ates de novo AML from CML a. CD13, CD33, CD34, CD10, CD19, CD2, CD3, CD5 b. therapy with tyrosine kinase inhibitors may improve outcomes b. CD11a, CD14, CD10, CD2, CD3, CD71, CD61 c. data suggests that BCR-ABL1 is more appropri- c. CD13, CD33, CD34, CD11b, CD71, CD41 ately linked to AML rather than CML d. CD41, CD61, CD13, CD15, CD38, CD117 d. the prognosis for this mutation is better using standard therapies 6. A patient has a leukocyte count of 75 * 103/mcL. A large number of blast cells are present in the Level II peripheral blood smear. Cytochemical stain reveals positive staining with SBB. Immunophenotyping is 1. In attempting to subtype a case of acute l eukemia, CD13+ , CD33+ , CD19- , CD22- , CD3- , CD5- . a laboratory professional noted that the blasts were Karyotyping reveals t(8;21)(q22;q22). Based on the negative with myeloperoxidase, SBB, specific and results, these cells would be considered: nonspecific esterase but positive when stained (Objective 4) with PAS and platelet peroxidase. These blasts should show positivity with which of the following a. Myeloblasts monoclonal antibody(ies)? (Objectives 2, 4) b. Stem cells a. CD41, CD42, CD61 c. Monoblasts b. CD24 d. Promyelocytes c. CD7 d. Glycophorin A 604 Chapter 26 7. A patient with leukemia is receiving chemotherapy 9. The presence of CD34, CD13, and CD14 is typically including a drug that is an antagonist to folic acid associated with a positive: (Objective 4) metabolism. Which of the following types of erythro- a. PAS stain cytes will be produced? (Objective 5) b. TdT a. Macrocytes c. CD71 b. Microcytes d. myeloperoxidase c. Spherocytes d. Codocytes 10. All transretinoic acid therapy is used in leukemia patients with this genetic abnormality: 8. Hypergranular promyelocytic leukemia is character- (Objectives 3, 5) ized by which cytogenetic abnormality? (Objective 2,4) a. t(9;22) a. t(15;17) b. t(8;21) b. 11q23 abnormality c. trisomy 8 c. t(8;21) d. t(15;17) d. inv(16)(p13;q22) References 1. Lichtman, M. A., & Liesveld, J. L. (2015). Acute myelogenous leu- translocation: Clinical consequences and biological implications. kemia. In: O. W. Press, ed. Williams hematology (9th ed., Journal of Biomedicine and Biotechnology, 2011, 104631. pp. 1373–1436). Philadelphia: McGraw-Hill Education. 11. Khan, M., Cortes, J., Kadia, T., Naqvi, K., Brandt, M., Pierce, 2. Lechner, K., Geissler, K., Jäger, U., Greinix, H., & Kalhs, P. (1999). S., . . . & Kornblau, S. (2017). Clinical outcomes and co-occurring Treatment of acute leukemia. Annals of Oncology, 10(Suppl. 6), mutations in patients with RUNX1-mutated acute myeloid 45–51. leukemia. International Journal of Molecular Sciences, 18(8), 1618. 3. Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, D. doi: 10.3390/ijms18081618 A., Gralnick, H. R., & Sultan, C. (1976). Proposals for the Classi- 12. Reilly, J. T. (2005). Pathogenesis of acute myeloid leukaemia fication of the Acute Leukaemias French-American-British (FAB) and inv(16)(p13;q22): A paradigm for understanding Co-operative Group. British Journal of Haematology, 33(4), 451–458. leukaemogenesis? British Journal of Haematology, 128(1), 18–34. doi: 10.1111/j.1365-2141.1976.tb03563.x doi: 10.1111/j.1365-2141.2004.05236.x 4. Harris, N. L., Jaffe, E. S., Diebold, J., Flandrin, G., Muller- Hermelink, 13. Goyama, S., & Mulloy, J. C. (2011). Molecular pathogenesis of H. K., Vardiman, J., . . . & Bloomfield, C. D. (1999). The World core binding factor leukemia: Current knowledge and future Health Organization Classification of Neoplastic Diseases of the prospects. International Journal of Hematology, 94(2), 126–133. Hematopoietic and Lymphoid Tissues. Annals of Oncology, 10(12), doi: 10.1007/s12185-011-0858-z 1419–1432. doi: 10.1023/a:1008375931236 14. Deschler, B., & Lübbert, M. (2006). Acute myeloid leukemia: 5. Vardiman, J. W., Thiele, J., Arber, D. A., Brunning, R. D., epidemiology and etiology. Cancer, 107(9), 2099-2107. Borowitz, M. J., Porwit, A., . . . & Bloomfield, C. D. (2009). The 15. Meijers, J., Oudijk, E. J. D., Mosnier, L. O., Bos, R., Bouma, B. N., 2008 revision of the World Health Organization (WHO) clas- Nieuwenhuis, H. K., & Fijnheer, R. (2000). Reduced a ctivity sification of myeloid neoplasms and acute leukemia: rationale of TAFI (thrombin-activatable fibrinolysis inhibitor) in acute and important changes. Blood, 114(5), 937–951. doi: 10.1182/ promyelocytic leukaemia. British Journal of Haematology, 108, blood-2009-03-209262 518–523. 6. Swerdlow, S. H., Campo, E., Pileri, S. A., Harris, N. L., Stein, H., 16. Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, Siebert, R., . . . Jaffe, E., eds. (2008). World Health Organization Clas- D. A. G., Gralnick, H. R., & Sultan, C. (1980). A variant form of sification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, hypergranular promyelocytic leukaemia (M3). British Journal of France: IARC Press. Haematology, 44, 169–170. 7. Arber, D. A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M. J., 17. Castoldi, G. L., Liso, V., Specchia, G., & Tomasi, P. (1994). Acute Le Beau, M. M., . . . & Vardiman, J. W. (2016). The 2016 revision promyelocytic leukemia: Morphological aspects. Leukemia, 8, to the World Health Organization Classification of Myeloid Neo- 1441–1446. plasms and Acute Leukemia. Blood, 128(3), 462–463. doi: 10.1182/ 18. Gianni, M., Ponzanelli, I., Mologni, L., Reichert, U., Rambaldi, blood-2016-06-721662 A., Terao, M., & Garattini, E. (2000). Retinoid-dependent growth 8. Craig, F. E., & Foon, K. A. (2008). Flow cytometric immunophe- inhibition, differentiation and apoptosis in acute promyelocytic notyping for hematologic neoplasms. Blood,111(8), 3941–3967. leukemia cells. Expression and activation of caspases. Cell Death doi: 10.1182/blood-2007-11-120535 and Differentiation, 7(5), 447–460. doi: 10.1038/sj.cdd.4400673 9. Döhner, H., Estey, E. H., Amadori, S., Appelbaum, F. R., Büchner, 19. Redner, R. L. (2002). Variations on a theme: The alternate trans- T., Burnett, A. K., . . . & Lo-Coco, F. (2010). Diagnosis and man- locations in APL. Leukemia, 16(10), 1927–1932. doi: 10.1038/ agement of acute myeloid leukemia in adults: Recommendations sj.leu.2402720 from an international expert panel, on behalf of the European 20. Walz, C., Grimwade, D., Saussele, S., Lengfelder, E., Haferlach, LeukemiaNet. Blood, 115, 453–474. C., Schnittger, S., . . . & Reiter, A. (2010). Atypical mRNA fusions 10. Reikvam, H., Hatfield, K. J., Kittang, A. O. Hovland, R., & in PML-RARA positive, RARA-PML negative acute promyelo- Bruserud, Ø. (2011). Acute myeloid leukemia with the t(8;21) cytic leukemia. Genes, Chromosomes and Cancer, 49(5), 471–479. Acute Myeloid Leukemias 605 21. Grignani, F., De Matteis, S., Nervi, C., Tomassoni, L., Gelmetti, V., that is required for cytokine-independent proliferation. Cioce, M., . . . & Seiser, C. (1998). Fusion proteins of the retinoic PLoS ONE, 7(8). doi: 10.1371/journal.pone.0042965 acid receptor@a recruit histone deacetylase in promyelocytic leu- 33. Falini, B., Mecucci, C., Tiacci, E., Alcalay, M., Rosati, R., kaemia. Nature, 391(6669), 815. Pasqualucci, L., . . . & Bigerna, B. (2005). Cytoplasmic nucleo- 22. Tomita, A., Kiyoi, H., & Naoe, T. (2013). Mechanisms of action phosmin in acute myelogenous leukemia with a normal and resistance to all-trans retinoic acid (ATRA) and arsenic triox- karyotype. New England Journal of Medicine, 352(3), 254–266. ide (As2O3) in acute promyelocytic leukemia. International Journal 34. Verhaak, R. G. (2005). Mutations in nucleophosmin (NPM1) in of Hematology, 97(6), 717–725. doi: 10.1007/s12185-013-1354-4 acute myeloid leukemia (AML): Association with other gene 23. Tallman, M. S., Andersen, J. W., Schiffer, C. A., Appelbaum, F. R., abnormalities and previously established gene expression signa- Feusner, J. H., Ogden, A., . . . & Wiernik, P. H. (2000). Clinical tures and their favorable prognostic significance. Blood, 106(12), description of 44 patients with acute promyelocytic leukemia 3747–3754. doi: 10.1182/blood-2005-05-2168 who developed the retinoic acid syndrome: Presented in part at 35. Paz-Priel, I., & Friedman, A. (2011). C/EBPa dysregulation in the meeting of the American Society of Hematology, San Diego, AML and ALL. Critical Reviews in Oncogenesis, 16(1–2), 93–102. CA, December 1997. Blood, 95(1), 90–95. doi: 10.1615/critrevoncog.v16.i1-2.90 24. Slovak, M. L., Gundacker, H., Bloomfield, C. D., Dewald, G., 36. Wouters, B. J., Lowenberg, B., Erpelinck-Verschueren, C. A., Appelbaum, F. R., Larson, R. A., . . . & Willman, C. L. (2006). A Putten, W. L., Valk, P. J., & Delwel, R. (2009). Double CEBPA retrospective study of 69 patients with t (6; 9)(p23; q34) AML mutations, but not single CEBPA mutations, define a subgroup of emphasizes the need for a prospective, multicenter initiative acute myeloid leukemia with a distinctive gene expression pro- for rare ‘poor prognosis’ myeloid malignancies. Leukemia, 20(7), file that is uniquely associated with a favorable outcome. Blood, 1295-1297. doi: 10.1038/sj.leu.2404233 113(13), 3088–3091. doi: 10.1182/blood-2008-09-179895 25. Scandura, J. M., Boccuni, P., Cammenga, J., & Nimer, S. D. (2002). 37. Soupir, C. P., Vergilio, J. A., Cin, P. D., Muzikansky, A., Kantarjian, Transcription factor fusions in acute leukemia: variations on a H., Jones, D., & Hasserjian, R. P. (2007). Philadelphia chromo- theme. Oncogene, 21(21), 3422–3444. doi: 10.1038/sj.onc.1205315 some–positive acute myeloid leukemia: a rare aggressive leuke- 26. Byrd, J. C. (2002). Pretreatment cytogenetic abnormalities are mia with clinicopathologic features distinct from chronic myeloid predictive of induction success, cumulative incidence of relapse, leukemia in myeloid blast crisis. American journal of clinical and overall survival in adult patients with de novo acute myeloid pathology, 127(4), 642–650. leukemia: Results from Cancer and Leukemia Group B (CALGB 38. Schnittger, S., Dicker, F., Kern, W., Wendland, |
N., Sundermann, J., 8461). Blood, 100(13), 4325–4336. doi: 10.1182/blood-2002-03-0772 Alpermann, T., . . . & Haferlach, T. (2011). RUNX1 mutations are 27. Groschel, S., Sanders, M. A., Hoogenboezem, R., de Wit, E., Britta, frequent in de novo AML with noncomplex karyotype and confer B. A., Erpelinck, C., . . . & Rombouts, E. J. (2014, June). A single an unfavorable prognosis. Blood, 117(8), 2348–2357. doi: 10.1182/ oncogenic enhancer rearrangement causes concomitant EVI1 and blood-2009-11-255976 GATA2 deregulation in leukemia. Cell, 157, 369–381. 39. Gaidzik, V. I., Bullinger, L., Schlenk, R. F., Zimmermann, A. S., 28. Yamazaki, H., Suzuki, M., Otsuki, A., Shimizu, R., Bresnick, E. H., Röck, J., Paschka, P., . . . & Späth, D. (2011). RUNX1 mutations in Engel, J. D., & Yamamoto, M. (2014). A remote GATA2 hemato- acute myeloid leukemia: results from a comprehensive genetic poietic enhancer drives leukemogenesis in inv (3)(q21; q26) by and clinical analysis from the AML study group. Journal of Clini- activating EVI1 expression. Cancer Cell, 25(4), 415–427. cal Oncology, 29(10), 1364–1372. 29. Lahortiga, I., Vázquez, I., Agirre, X., Larrayoz, M. J., Vizmanos, J. L., 40. Kadia, T. M., Ravandi, F., Cortes, J., & Kantarjian, H. (2015). Gozzetti, A., . . . & Odero, M. D. (2004). Molecular heterogeneity in Toward individualized therapy in acute myeloid leukemia. JAMA AML/MDS patients with 3q21q26 rearrangements. Genes, Chro- Oncology, 1(6), 820–828. doi: 10.1001/jamaoncol.2015.0617 mosomes and Cancer, 40(3), 179–189. doi: 10.1002/gcc.20033 41. Patel, K. P., Khokhar, F. A., Muzzafar, T., You, M. J., Bueso-Ramos, 30. Raffel, G. D., Mercher, T., Shigematsu, H., Williams, I. R., Cullen, C. E., Ravandi, F., . . . & Medeiros, L. J. (2013). TdT expression in D. E., Akashi, K., . . . & Gilliland, D. G. (2007). Ott1 (Rbm15) has acute myeloid leukemia with minimal differentiation is associ- pleiotropic roles in hematopoietic development. Proceedings of the ated with distinctive clinicopathological features and better over- National Academy of Sciences, 104(14), 6001–6006. all survival following stem cell transplantation. Modern Pathology, 31. Cheng, E. C., Luo, Q., Bruscia, E. M., Renda, M. J., Troy, J. A., 26(2), 195–203. doi: 10.1038/modpathol.2012.142 Massaro, S. A., . . . & Sun, Y. (2009). Role for MKL1 in megakaryo- 42. Jaffe, E. S., Harris, N.L., & Vardiman. J.W., eds. (2001). World cytic maturation. Blood, 113(12), 2826–2834. Health Organization Classification of Tumours: Pathology and genet- 32. Lee, J., & Skalnik, D. G. (2012). Rbm15-Mkl1 interacts with the ics of tumours of haematopoietic and lymphoid tissues. Lyon, France: Setd1b histone H3-Lys4 methyltransferase via a SPOC domain IARC Press. Chapter 27 Precursor Lymphoid Neoplasms Kyle Riding, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define acute lymphoblastic leukemia (ALL) 4. Give the typical results of flow cytometric and lymphoblastic lymphoma (LBL), and analysis and genetic findings in ALL. differentiate them from acute myeloid 5. Summarize the clinical signs and symptoms leukemia (AML). and the most frequent age groups associated 2. List and define the variants of ALL/LBL with ALL. according to the WHO classification. 6. Define the rare acute leukemias that are not 3. Describe and recognize the typical p eripheral included in the AML and ALL groups. blood picture (erythrocytes, leukocytes, blasts, thrombocytes) seen in ALL. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare and contrast the various 4. Correlate Wright stained blast morphology presentations of ALL/LBL. in the ALL subgroups with flow cytometry 2. Predict the most likely WHO or and genetic testing results. immunophenotypic subgroup based on 5. Evaluate peripheral blood results in relation patient history, physical assessment, and to response to oncological therapy (e.g., laboratory findings. complete or partial remission, relapse). 3. Correlate cellular presentation with 6. Identify acute leukemia (AL) from a prognosis and common complications in peripheral blood smear and recommend ALL/LBL. laboratory tests that may be useful in 606 Precursor Lymphoid Neoplasms 607 differentiation of acute leukemia of 8. Correlate clinical and laboratory findings ambiguous lineage, AML, ALL, and the with prognosis in ALL/LBL. subgroups of ALL. 9. Contrast the clinical and laboratory findings 7. Define the phases and purposes of of ALL to LBL. chemotherapy for ALL. Chapter Outline Objectives—Level I and Level II 606 Laboratory Evaluation 609 Key Terms 607 Identification of Cell Lineage 611 Background Basics 607 WHO Classification 612 Case Study 608 Therapy 617 Overview 608 Summary 618 Introduction 608 Review Questions 618 Etiology and Pathophysiology 608 References 620 Clinical Presentation 608 Key Terms Acute undifferentiated leukemia Biphenotypic acute leukemia Minimal residual disease (MRD) (AUL) Consolidation therapy T cell acute lymphoblastic B cell acute lymphoblastic Induction therapy leukemia leukemia Maintenance chemotherapy Bilineage acute leukemia Background Basics The information in this chapter will build upon concepts Level II learned in previous chapters. To maximize your learning • Summarize the role of oncogenes and growth factors experience, you should review and have an understand- in cell proliferation, differentiation, and maturation. ing of these concepts before starting this unit of study: (Chapters 4, 23) • Diagram the maturation pathway for T and B lym- Level I phocytes. (Chapter 8) • Summarize the origin and differentiation of hemato- • Describe the role of molecular analysis in diagnosis poietic cells. (Chapter 4) and treatment of acute leukemia. (Chapter 42) • Describe the maturation, differentiation, and func- • Describe the use of immunophenotyping in acute tion of the lymphocytes. (Chapter 8) leukemia. (Chapters 23, 40) • Outline the classification and general laboratory find- • Describe the role of cytogenetics and diagnosis and ings of the acute leukemias. (Chapter 23) treatment of acute leukemia. (Chapters 23, 41) • Summarize the typical laboratory findings that define acute myeloid leukemia. (Chapter 26) 608 Chapter 27 two entities are not always distinct. Tissue involvement CASE STUDY can occur in ALL, and peripheral blood and bone marrow We refer to this case study throughout the chapter. involvement can occur in LBL. It is thought that ALL and Dan, a 4-year-old white male, was seen by his LBL are different clinical expressions of the same disease physician for symptoms of easy fatigue and bruis- and therefore are grouped together in the WHO classifica- ing. His mother stated that until one month ago, tion. They arise from a malignant change in the hematopoi- he was a “typical kid.” Since then she has noticed etic stem cell, common lymphoid progenitor (CLP), or more increased lassitude, a regression to more babylike differentiated progenitors of the T or B cell lineage. behavior, and loss of appetite. For the past two days, his temperature has been 100°F. Upon physi- cal examination, the child presented as pale, quiet, Etiology and and of appropriate size for his age. Most systems were unremarkable with the exception of several Pathophysiology small firm lymph nodes felt in the cervical and Similar to AML (Chapter 26), the ALLs are hematologic dis- auxiliary regions. His CBC revealed a WBC count orders characterized by malignant neoplastic proliferation of 40.2 * 109/L with 90% blasts. and accumulation of immature and dysfunctional hema- Consider the laboratory testing that could help topoietic cells in the bone marrow. Multiple somatically in diagnosing Dan’s illness. acquired mutations within a stem cell prior to its differen- tiation into a lymphoid progenitor or within a lymphoid precursor cell gives rise to a clone of malignant lympho- cytes. Thus, ALL/LBL can be either T or B cell phenotypes. Overview How the cells acquire these mutations is unknown, but a combination of leukemogenic factors could be responsible This chapter discusses the acute lymphoblastic leukemias (Chapter 23). The types of genetic alterations include chro- and lymphomas. A summary of clinical signs and symp- mosomal rearrangements and abnormalities of cell ploidy as toms is juxtaposed with the hematologic findings. The 2016 well as point mutations in oncogenes or tumor suppressor World Health Organization (WHO) Classification System genes that alter cellular functions (Chapter 23). These geneti- is used. The emphasis is on the laboratory techniques and cally abnormal cells proliferate in an unregulated manner results used to diagnose and classify the neoplasms. The with altered responses to growth and antigrowth signals rare acute leukemias that are not grouped into either acute and exhibit maturation arrest at any one of several early myeloid leukemia (AML) or acute lymphoblastic leukemia stages of differentiation. They divide and synthesize DNA (ALL) are termed acute leukemias of ambiguous lineage and are more slowly than normal hematopoietic cells, but they have also reviewed. ALL may also be referred to as lymphoblastic enhanced self-renewal and increased resistance to apoptotic leukemia (LL) since the term acute is considered by some to signals.1 Thus, the leukemic clone expands and the neoplas- be redundant with the term lymphoblastic. tic cells accumulate. Impairment of normal hematopoiesis as the leukemic clone expands is the primary cause of concern. Introduction Lymphoid malignancies include a wide spectrum of syn- Clinical Presentation dromes from disorders that primarily involve the bone Although ALL has been diagnosed in all age groups, the inci- marrow and peripheral blood (leukemias) to those that ini- dence of the disease demonstrates two clear frequency clus- tially present as solid tissue masses (lymphomas) primarily ters, one between the ages of 2 and 5 years and another in the involving lymphoid organs (lymph nodes, tonsils, spleen, sixth decade. In elderly people, the signs and s ymptoms— thymus, and the lymphoid tissue of the gastrointestinal complaints of fatigue, infections, and b ruising—are typically tract). These lymphoid neoplasms may be composed primar- more acute than in children. The onset of the disease in chil- ily of mature or precursor lymphoid cells, a distinction that dren can be insidious or abrupt with nonspecific signs and serves as a means for classification. The mature lymphoid symptoms. leukemias and lymphomas are discussed in Chapter 28. This The clinical picture of anemia, increased infections, and chapter includes a description of the precursor or immature thrombocytopenia results from bone marrow failure because lymphoid leukemias (ALL) and lymphomas (lymphoblastic of the replacement of normal marrow elements by leukemic lymphoma [LBL]). Although ALL is primarily a neoplasm lymphoblasts. Pallor, fatigue, and shortness of breath reflect of the bone marrow and peripheral blood and LBL is a neo- the anemia, which tends to become symptomatic below plasm that presents as a solid mass in lymphoid tissue, the 10 g/dL of hemoglobin. Fever can result from an infection Precursor Lymphoid Neoplasms 609 or remain classified as a fever of unknown origin, perhaps because of the release of endogenous pyrogens and other Table 27.1 Initial Laboratory Findings Characteristic of ALL inflammatory compounds. Both petechiae and ecchymoses are present in more than 50% of the patients. Bone pain, especially Peripheral blood • Leukocyte count usually increased but can be normal or decreased tenderness of the long bones, presents in about 80% of chil- • Neutropenia dren with ALL, and is often excused as “growing pains” but • Lymphoblasts present is actually from the expansion of marrow space by leukemic • Normocytic, normochromic anemia cells. Weight loss with or without anorexia is common because • Thrombocytopenia of the negative nitrogen balance of metabolically abnormal Bone marrow • Hypercellular cells. Leukemia infiltrates are responsible for splenomegaly, • Increased lymphoblasts, usually more than hepatomegaly, and lymphadenopathy when present. 25%, not less than 20% B cell ALL is characterized by inappropriate secretion of monoclonal immunoglobulins that can increase blood most commonly in patients with T cell ALL. The absolute viscosity and impair blood flow through the microcircula- neutrophil count is decreased in 20–40% of patients, even tion (hyperviscosity syndrome). These immunoglobulins when there is leukocytosis, and correlates strongly with also can impair granulocyte and platelet function, induce the incidence of infection.1 Although no established mini- pathologic rouleaux, interact with coagulation proteins, and mum percentage of blasts defines ALL (as there is for AML), interfere with coagulation. Production of autoantibodies can WHO recommends that the diagnosis of ALL not be made lead to autoimmune hemolytic anemia, autoimmune throm- if there are less than 20% blasts within the bone marrow.3 bocytopenia, and autoimmune neutropenia. Extramedul- Many treatment protocols use the criterion of a minimum of lary involvement is common, affecting the central nervous 25% blasts within the bone marrow for a diagnosis of ALL. system, lymph nodes, spleen, liver, and testis. T and B lymphoblasts are not distinguishable by mor- The neoplasm can present with primary involvement |
of phology. Circulating lymphoblasts are found in the periph- nodal and extranodal sites with minimal or no involvement eral blood in 90% of cases.1 They vary from small cells with of bone marrow and peripheral blood. The disease is then scant to moderate light basophilic or gray-blue cytoplasm, referred to as LBL. Sites of involvement in B cell LBL include a high N:C ratio, slightly condensed chromatin, and indis- skin, soft tissue, central nervous system, and lymph nodes. tinct nucleoli to larger cells with a moderate amount of In T cell LBL, mediastinal (thymic) involvement is com- cytoplasm, finely dispersed chromatin, and distinct nucle- mon. When the patient presents with a neoplastic lymphoid oli1 (Figures 27-1 and 27-2). The nucleus can be irregularly mass and lymphoblasts in the bone marrow, the distinction shaped. between lymphoma and leukemia is arbitrary. However, The larger blasts may be intermixed with the smaller when peripheral blood and bone marrow are extensively blasts (Figure 27-2). The cytoplasm can contain vacuoles involved, the disease is known as lymphoblastic leukemia. and amphophilic granules (that stain a pinkish to purple Without leukemia, LBL is usually asymptomatic. color) that are probably lysosomal in origin.4 These granules T cell ALL and LBL tend to have more aggressive clini- can make it difficult to distinguish these lymphoblasts from cal behavior than B cell malignancies. T cell malignancies myeloblasts. often involve extranodal and extramedullary sites.1 Lymph- Unless accompanied by bleeding, a normochromic adenopathy and hepatospenomegaly are usually present. normocytic anemia is common. Anisocytosis, poikilocyto- sis, and nucleated RBCs are usually not present.5 There is Checkpoint 27.1 typically thrombocytopenia (48952 * 109/L). Compare the typical age groups in which AML and ALL are found. CASE STUDY (continued from page 608) The physician ordered a CBC on Dan. The results Laboratory Evaluation were as follows: Evaluation of the peripheral blood and bone marrow is criti- WBC 40.2 * 109/L WBC Differential cal for making a diagnosis of ALL (Table 27-1). RBC 3.45 * 1012/L Neutrophils 8% Peripheral Blood Hb 9.7 g/dL Monocytes 1% Hct 32% Eosinophils 1% The white blood cell count can be increased, decreased, or MCV 92.7 fL Blasts 90% within the reference interval.2,3 Hyperleukocytosis (greater than 100.0 * 109/L) occurs in 11–23% of all patients and 610 Chapter 27 a b Figure 27.1 (a) Lymphoblasts in peripheral blood from acute lymphoblastic leukemia. Notice the nuclear cleavage, condensed chromatin, and high N:C ratio. (b) Lymphoblasts in the bone marrow from the patient with ALL in (a). Note cells with a high N:C ratio, fine chromatin, and nucleoli (both Wright-Giemsa stain, 1000* magnification). The bone marrow in most patients reveals a marrow densely MCH 28.1 pg populated with lymphoblasts (greater than 65%) and aspi- ration may not be possible. In this case, biopsy is indicated. MCHC 30.3 The morphology of the cells in the marrow mirrors that RDW 17.3 seen in the peripheral blood. Auer rods are not present in PLT 63 * 109/L lymphoblasts; if Auer rods are seen, the blasts are distin- On a scan of the peripheral blood smear, rare guished as myeloblasts. nucleated erythrocytes were seen, and the platelets appeared decreased in number. Tissue Involvement 1. Based on these data, what would be the initial Lymphoma is the term used to describe lymphoid neoplasms interpretation of Dan’s presentation? when there is mass lesion of lymphoid (Chapter 3) or other tissue with no or little involvement of the bone marrow and peripheral blood. In B-LBL, lymph nodes and other Bone Marrow involved tissue show diffuse or paracortical patterns of infiltration by malignant lymphoid cells. There are usually The bone marrow is preferred for diagnosis because there numerous mitotic figures. There may be a focal “starry sky” are no circulating blasts at diagnosis in up to 10% of patients pattern due to macrophage phagocytosis of apoptotic tumor with ALL. Marrow cells also are better for genetic studies. cells. In T-LBL, lymph nodes can show total architectural a b Figure 27.2 (a) Lymphoblasts in peripheral blood from acute lymphoblastic leukemia. Note the fine chromatin and nucleoli (Wright- Giemsa stain, 1000* magnification). (b) Lymphoblasts in the bone marrow from patient in (a). Note the blasts with vacuoles (Wright-Giemsa stain, 500* magnification). Precursor Lymphoid Neoplasms 611 effacement that involves the capsule. Sometimes the morphologic examination of Romanowsky-stained smears involvement in the paracortical area is only partial, spar- alone. When morphologic examination cannot differenti- ing the germinal center. A multinodular pattern and “starry ate lymphoblasts from other blasts, cytochemistry may be sky” effect can be present. helpful (Chapter 37). In lymphoblasts, the myeloperoxi- dase (MPO) and Sudan black B (SBB) stains are negative, Other Laboratory Evaluation which are usually sufficient to differentiate lymphoblasts from myeloblasts. The periodic acid-Schiff (PAS) reaction As with other leukemias, other laboratory findings are con- usually demonstrates a coarse granular positivity in lym- sistent with increased cellular metabolism and, in general, phoblasts, especially those with a T cell immunophenotype. the extent of abnormality of the various laboratory param- The PAS reactivity may be present in AML, but the granular eters is proportional to the tumor burden. Hyperurice- pattern is superimposed on a diffusely positive background, mia and an increased lactate dehydrogenase are common whereas there is no background positivity in lymphoblasts. expressions of cell turnover. Hypercalcemia may occur, which is thought to be from bone resorption associated with leukemic infiltration in the bone marrow. The typical con- Terminal Deoxynucleotidyl sequence of leukemic infiltration of the kidney is impaired Transferase (TdT) renal function with increased serum creatinine, phospho- Certain intracellular enzymes are helpful in identifying rus, blood urea nitrogen, and uric acid. Because the central cellular subtypes. The most important of these is terminal nervous system (CNS) is a frequent site for extramedullary deoxynucleotidyl transferase (TdT), a DNA polymerase metastasis of ALL, cerebrospinal fluid analysis may be used found in cell nuclei. Its presence may be determined by throughout treatment to investigate the possibility of leu- direct enzyme assay, by indirect immunofluorescence, or kemic infiltration. with monoclonal antibodies. This enzyme is not present in normal mature lymphocytes but can be found in 65% of the Checkpoint 27.2 total thymic population of lymphocytes with the TdT posi- Explain the difference between ALL and LBL. tive cells localized in the cortex.6 It can also be found in very early B cells and occasionally very early myeloblasts and therefore, about 1–3% of normal bone marrow cells are TdT positive. Its value in ALL is to differentiate early precursor Identification of Cell lymphoblasts from more mature cells. Lineage Immunophenotyping The first step in classifying acute leukemia (AL) is to differ- entiate ALL from AML, usually by identifying the lineage The immunologic phenotype of neoplastic cells is helpful of the blasts. Although cell morphology on Romanowsky- in determining lineage of the neoplastic cells, differentiat- stained smears and cytochemistry can help distinguish ing a benign from a neoplastic process, classification and ALL from AML, classification of ALL/LBL into subgroups prognosis of the disease, and detection of minimal residual relies on immunologic, cytogenetic, and molecular genetic disease (MRD) after treatment. The appearance of specific methods. cell markers (antigenic determinants) is developmentally Immunophenotyping is used to determine the cells’ regulated in normal lymphoid cells (Chapter 8). Some anti- lineage (myeloid or lymphoid), subtype (T or B cell), and genic determinants appear at a very early stage of devel- maturation stage. Cytogenetics and molecular analysis opment and disappear with age, while others appear on can provide evidence of clonality, reveal distinct genetic mature cells. Studies of surface and intracellular markers abnormalities associated with subgroups of ALL/LBL, reveal that lymphoblasts in ALL share markers with nor- mal lymphoid counterparts in various stages of maturation7 and provide important prognostic information. Identifica- tion of these cellular characteristics can reveal the biologic (Chapter 8). However, immunophenotyping also has dem- subtypes, which is important for optimizing treatment onstrated that while some leukemic cells have phenotypes outcomes. of normal cells, others can show asynchronous gene expres- sion, resulting in an inappropriate combination of antigens Morphology and Cytochemistry or lineage plasticity8 (Figure 27-3). Because no one marker is specific for any neoplasm, a panel of antibodies must be Morphology is important since some neoplasms have used for accurate diagnosis. characteristic or diagnostic cell features. However, differ- Immunophenotyping is important in differentiat- entiation of ALL from AML (AML minimally differenti- ing ALL from minimally differentiated AML and in iden- ated or AML without maturation) may not be possible by tifying subtypes of the neoplastic lymphoblast (T or B). 612 Chapter 27 Progenitor Pro-B ALL Intermediate Pre-B ALL HCL Multiple cell pre-B ALL CLL myeloma PLL (plasma cell) TdT TdT TdT (mature) CD34 cCD22 CD19 CD19 CD19 CD38 CD19 CD34 CD10 CD20 CD20 cIg CD19 CD201/2 sCD22 CD22 cCD22 cIg sIg Figure 27.3 B lymphocyte maturation pathway with leukemic and other lymphoproliferative counterparts. ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; PLL, prolymphocytic leukemia; HCL, hairy cell leukemia; clg, cytoplasmic immunoglobulin; slg, surface immunoglobulin. Immunophenotyping also is valuable in recognizing leuke- signaling pathways that govern cell proliferation, such as mias of mixed lineage. After therapy, immunophenotyping RAS (Chapter 2).10,11 is used in combination with molecular studies (e.g., poly- merase chain reaction [PCR]/fluorescence in situ hybridiza- CASE STUDY (continued from page 609) tion [FISH]; Chapter 42) to determine MRD. 1. What tests should be used as the initial follow Checkpoint 27.3 up in this case? Why is it necessary to immunophenotype the lympho- blasts in ALL if they have been identified as lymphoblasts morphologically? Who Classification Cytogenetic Analysis The WHO Classification defines two subgroups of pre- Chromosomal abnormalities are present in 75% of ALL. cursor ALL/LBL: Precursor B cell and precursor T cell This information has diagnostic and prognostic implica- neoplasms (leukemia/lymphoma; Table 27-2). These tions. Some specific abnormalities are recurrent and used precursor neoplasms with bone marrow and peripheral to subgroup the B cell ALL/LBL. blood involvement are acute lymphoblastic leukemias, whereas precursor T cell and precursor B cell neoplasms Molecular Analysis presenting as solid tumors are lymphoblastic lymphomas (Chapter 28). Molecular analysis can determine the presence of rear- B cell ALL/LBLs that are characterized by recurrent rangements of immunoglobin heavy (IGH) chain genes genetic abnormalities are subgrouped as ALL with recurrent and/or light chain genes and T cell receptor (TCR) genes cytogenetic abnormalities (see Appendix B for these subgroups (Chapter 8). Rearrangement of IGH chain genes occurs and cellular characteristics). The remainder are subgrouped before cellular expression of immunoglobulin (Ig) and is as ALL not otherwise specified (NOS). Although Burkitt an early genetic marker of B cell ontogeny.9 Rearrangement lymphoma was classified as ALL in the FAB classification of the TCR polypeptide genes is an early sign of T cell lin- (L3), it is suggested that the B cell ALL designation not be eage. Molecular analysis of these gene rearrangements can used to designate Burkitt leukemia/lymphoma.1 Burkitt be used to establish clonality of the T or B cell populations lymphoma is included in the WHO classification of mature as well as cell lineage. Clonal gene rearrangement helps to B cell neoplasms and is discussed in Chapter 28. There are differentiate neoplastic lymphoid cells from normal lym- no recurrent cytogenetic changes observed that are specific phoid cells. However, IGH gene rearrangement is seen in to LBL.9 The subgrouping of T cell ALL has been a topic of some instances of T cell ALL, and TCR gene rearrangement considerable debate but recently had one subset created in occurs in some cases of B cell ALL.9 Thus, identification of the 2016 WHO update classification (Appendix B).12 additional markers of B and T cell lineage commitment is required for diagnosis. Point mutations are less frequently B Lymphoblastic Leukemia/ observed but may include genes of major cell-cycle reg- ulator proteins such as p53, various cyclin-dependent Lymphoma kinase inhibitors (CDKi), including p16, p15, and p14, Precursor B cell leukemia (B cell ALL) is a neoplasm of lym- and activating mutations of proto-oncogenes involved in phoblasts committed to the B cell lineage, which involves Precursor Lymphoid Neoplasms 613 Table 27.2 Classification of Lymphoid Leukemia and Lymphoma by the World Health Organization Subgroup Subgroup Subgroups B cell neoplasms Precursor B cell neoplasms Lymphoblastic leukemia B cell ALL /LBL with recurrent genetic abnormalities Lymphoblastic lymphoma (see Appendix B) B cell ALL /LBL not otherwise specified Mature B cell neoplasms (See Chapter 28) (See Chapter 28) T |
cell neoplasms Precursor T cell neoplasms Lymphoblastic leukemia Lymphoblastic lymphoma Mature T cell neoplasms (See Chapter 28) (See Chapter 28) the bone marrow and peripheral blood. Occasionally, the IMMUNOPHENOTYPING disease can present with primary involvement of the lymph Immunophenotyping can identify markers on neoplastic nodes or extranodal sites, in which case it is called B lym- B cells that correlate to normal stages of maturation within phoblastic lymphoma (B-LBL). Although arbitrary and with the B cell lineage (pro-B, intermediate pre-B, and pre-B; exceptions, if a patient has a mass lesion and 25% or fewer Table 27-3). However, for therapeutic purposes, it is neces- lymphoblasts in the bone marrow, the term lymphoma is sary to distinguish only between precursor B cell, mature preferred. B cell, and T cell immunophenotypes.13 It is recommended B cell ALL accounts for 80–85% of the cases of ALL that panels for cell identification include at least one highly in children and about 70% of the cases of ALL in adults. sensitive marker, CD19, and a highly specific marker, cyto- In general, children between the ages of 2 and 10 years plasmic CD79a and CD22, for B cell lineage.12 of age diagnosed with precursor B cell ALL have a good The earliest stage of differentiation is the pro-B cell prognosis (long-term event-free survival more than 80%). and is characterized by an immunophenoptype pattern Adult event-free survival is lower, 30–50%, depending of TdT+ , HLA@DR+ , and almost always CD19+ and on the disease subtype and the patient’s age at diag- CD79a+ . The intermediate pre-B cell (also known as the nosis (there is an inverse relationship between age and common ALL cell) expresses CD10 (the common ALL anti- prognosis). gen, or CALLA).14,15 The most mature precursor B cell Table 27.3 Summary of Laboratory Features Helpful in Classification of ALL Common ALL Characteristic Pro-B Pre-B Precursor T (intermediate pre-B) Gene rearrangement Immunoglobulin (Ig) +/- + + -a T cell receptor (TCR) - - - +b Immunologic features Cytoplasmic Ig - + + - Surface Ig - - +/- - Immunophenotype CD34 + +/- - + CD19 + + + - CD22 +1c2 +1c2 +1s2 - CD10 - + +/- - CD20 - +/- + - CD2, CD3, CD5, CD7 - - - + Cytochemistry TdT + + + + PAS - - - + a About 20% of T ALL exhibits rearrangement of Ig gene b A small number of B ALL exhibits rearrangement of TCR gene + , positive; - , negative; (c), cytoplasmic; (s), surface. 614 Chapter 27 differentiation stage is the pre-B cell, which can be CD10 The rearrangements are more heterogeneous than originally negative but positive for CD20, surface CD22 (sCD22), thought which has caused a shift in the nomenclature used and cytoplasmic immunoglobulin (clg). However, surface by the 2016 WHO classification system. The presence of a immunoglobulin is absent. Malignancies involving more rearrangement in this gene is known to carry prognostic mature stages of B cell maturation are discussed in the clas- implications depending on the age of the patient and may sification of mature lymphoid neoplasms (Chapter 28). warrant hematopoietic stem cell transplantation in younger The majority of ALL/LBL presentations in both chil- patients to improve prognosis.20 dren and adults arise from precursor or immature B cells The t(9;22)(q34.1;q11.2)/BCR-ABL1 translocation is that typically have the same immunophenotype as normal more common in adults (10–15%) than children with B cell immature B cells (CD10, CD19, CD34, CD22, and TdT). This ALL.21 In most childhood cases, the BCR/ABL1 translocation makes the distinction between normal and neoplastic cells results in a p190 kD fusion protein, whereas in adult cases, challenging, especially when assessing minimal residual about one-half of the translocations produce the p210 kD disease unless the abnormal cells have an additional or protein present in CML. The remainder produce the p190 distinct marker.16 Up to 50% of B cell ALL cells coexpress protein (Chapter 24). a myeloid associated antigen—CD13, CD15, or C33—that The t(5;14)(q31.1;q32.3) results in a translocation can help in the differentiation between normal and neoplas- between the IL3 gene and the IGH2 gene, which results in a tic. Chapter 40 and Appendix B describe immunopheno- variable eosinophilia that is reactive, not clonal. Blasts are types associated with specific leukemias. CD19+ and CD10+ . The diagnosis can be made on immun- ophenotypic and genetic information regardless of the blast CYTOGENETICS AND PATHOGENESIS count in the bone marrow. Cytogenetic abnormalities associated with B cell ALL include translocations, hypodiploidy, and hyperdiploidy.10 Hyperdiploid B cell ALL is common in children and is seen in 20–26% of B ALL cases.23 Almost all cases The most significant translocations that have distinctive of hyperdiploid B cell ALL have mutations in the recep- clinical or phenotypic properties and important prognostic information are:17 tor tyrosine kinase FLT-3, resulting in constitutive activa- tion of the receptor.24 Hypodiploid clones may also have • t(12;21)(p13.2;q22.1)/ETV6-RUNX1 structural abnormalities but none are specific for diag- • t(v;11q23.3); KMT2A rearrangement nosis. Cytogenetic abnormalities considered to be poor • t(9;22)(q34;q11)/BCR-ABL1 prognostic factors are t(4;11)(q21;q23)/MLL-AFF1 (9% of cases), t(1;19)(q23.3;p13.3)/PBX1-E2A (5% of cases), t(9;22) • t(5;14)(q31;q32.3)/IL3-IGH2 (q34;q11.2)/BCR-ABL1 (4% of cases), and hypodiploidy (5% • t(1;19)(q23.3;p13.3)/E2A-PBX1(TCF3-PBX1) of cases).1,25 • Translocations involving tyrosine kinases or cytokine B ALL with intrachromosomal amplification of chromo- receptors (BCR-ABL1-like mutations) some 21 (iAMP21) and BCR-ABL1-like mutations are the • Intrachromosomal amplification of chromosome 21 newest provisional entities within the 2016 WHO classifi- (iAMP21) cation. iAMP21 involves chromosome 21 containing five or more copies of the RUNX1 gene being present that requires The most common translocation (present in about 25% aggressive therapy and is found predominantly in children. of cases) in childhood B cell ALL is t(12;21)(p13;q22.3), pro- Similarly, the BCR-ABL1-like mutations are found mostly in ducing the ETV6(TEL)-RUNX1(AML1) fusion gene.18,19 Both children and again carry a poor prognosis but may respond the ETV6 and RUNX1 genes are translocated in other types well to tyrosine kinase inhibitors.12 of leukemia—including t(5;12), t(8;21), and t(3;21)—that are found in various subtypes of AML. The ETV6-RUNX1 PROGNOSIS fusion protein is believed to initiate and drive leukemo- Precursor B cell ALL generally has a good prognosis. In genesis in cells carrying the translocation by disrupting the pediatric population, the complete remission rate the normal processes of cell differentiation, apoptosis, cell approaches 95%; in adults, it is 60–85%.26 Long-term, event- adhesion, and response to DNA damage.17 Expression of free survival is lower for both patient groups (∼80% and the ETV6-RUNX1 fusion protein in B cell ALL is associ- 30–50%, respectively). Generally, hyperdiploidy (more ated with an excellent prognosis with event-free survival than 50 chromosomes) is a better prognostic finding than approaching 90%. hypodiploidy (less than 45 chromosomes) with event-free Infants who present with ALL commonly carry the survival rates nearly 90%. Patients with hyperdiploid chro- t(v;11q23.3) translocation that rearranges the KMT2A gene mosome counts less than 50 have a worse prognosis.7,8 (formerly MLL gene). The protein encoded by the KMT2A Positive predictive factors in children include diagnosis gene is involved in chromatin remodeling and epigenetic at ages 1–9, hyperdiploid chromosomes or t(12;21), and a transcriptional control of cell-cycle regulatory proteins.18 low or normal white blood cell (WBC) count at diagnosis.1 Precursor Lymphoid Neoplasms 615 Adverse factors include very young age (less than 1 year), high WBC counts at diagnosis, and other cytogenetic Checkpoint 27.4 abnormalities listed in the section “Cytogenetics and A patient has 50% blasts in his bone marrow. Immunophe- Pathogenesis” (Table 27-4).27 notyping is CD19 positive, but CD20, CD2, CD10, and CD7 negative. What additional testing can be helpful to distinguish the immunologic subgroup of this leukemia? T Lymphoblastic Leukemia/ Lymphoma CYTOGENETICS AND PATHOGENESIS Accounting for approximately 15% of the cases of childhood T cell lineage can be determined by detecting rearrange- ALL and approximately 25% of the cases of adult ALL, pre- ment of the TCR genes using molecular methods. There are cursor T cell leukemia (T cell ALL) involves lymphoblasts four TCR genes that are capable of rearranging and encode committed to T cell lineage in the bone marrow and periph- the a@, b@, g@, and d@chains of the TCR (Chapter 8). Detection eral blood. If the primary site of involvement is a lymph of a monoclonal TCR gene rearrangement suggests a neo- node or extranodal site, the disease is termed T lymphoblastic plasm of the T cell lineage, but about 20% of T cell ALL cases lymphoma (T-LBL). exhibit simultaneous rearrangement of one of the immuno- Precursor T cell ALL usually presents with a high WBC globulin genes. In addition, a few cases of B cell ALL have count and often a mediastinal mass (because of leukemic shown TCR gene rearrangement. infiltration of the thymus) or other tissue masses. The lym- About one-third of the cases of T cell ALL have translo- phoblasts are similar to those seen in B cell ALL, although cations involving the a and d T cell receptor loci (14q11.2), they are more likely to be variable in size, and cytoplasmic the b locus (7q35), or the g locus (7p14–15). The translo- vacuoles can be present. The cytochemistry is also similar cations involve a variety of partner genes, including the to that seen in B cell ALL, but acid phosphatase can show transcription factors MYC (8q24.1), TAL1 (1p32), RBTN1 focal intense positivity in T cell ALL. (11p15), RBTN2 (11p13), HOX11 (10q24), and HOX11L2 (5q35).31 The result is usually a dysregulation of the part- IMMUNOPHENOTYPING ner gene, resulting in growth enhancement. In addition, The lymphoblasts in T cell ALL are usually TdT+ , CD7+ , molecular mutations of the NOTCH1 gene are found in and CD3+ (Table 27-5). However, only CD3 is considered more than 50% of cases of T cell ALL.32 NOTCH1 is a trans- lineage specific. There is variable expression of CD1, CD2, membrane receptor that is involved in the regulation of CD4, CD5, CD8, and CD10.28 Lymphoblasts frequently normal early T cell development. MYC, the downstream coexpress CD4 and CD8, indicative of the cortical stage of target of NOTCH1, is involved in the growth of neoplastic thymocyte differentiation (“double-positive cells”).25 At the cells. However, the NOTCH1 mutation is rare in ETP ALL; medullary stage of differentiation, the cells are either CD4+ mutations more frequently associated with myeloid malig- or CD8+ . CD79a, generally considered to be a B lineage nancies (e.g., FLT3, IDH1, and GATA2) are seen in this form marker, has been observed in some cases. One or more of of T cell ALL.12,29 the myeloid-associated markers (CD13, CD15, CD33) can occasionally be seen. As in B cell ALL, T cell ALL also can PROGNOSIS be stratified into differentiation stages, with cytoplasmic Although acute precursor T cell leukemia was considered a CD3, CD2, and CD7 appearing in the earliest stage followed high-risk disease with a poorer prognosis than B cell ALL, by CD5, CD1a, and subsequently, the appearance of mem- current therapeutic protocols are improving the prognosis brane CD3.25 for this disease. T cell ALL in children increases the risk for The 2016 WHO classification differentiates early T induction failure and relapse. In adults, the prognosis of precursor (ETP) ALL as a unique group. The characteristic T cell ALL may be better than of B cell ALL. This may be immunophenotype of ETP ALL is expression of CD7 while related to a lower incidence of adverse cytogenetic muta- lacking expression of CD1a and CD8 and expression of at tions in T cell ALL. least one myeloid/stem cell marker such as CD34, CD117, or CD65.12 The prognosis for these patients is exceptionally poor.29,22 CASE STUDY (continued from page 612) 3. If the flow cytometry pattern showed a positive Table 27.4 Poor Prognostic Indicators in ALL CD10, what would be the classification of this acute leukemia? Clinical findings Laboratory findings • Infants (less than 1 year old) • High blast counts 4. In this situation, would the therapeutic outcome • Patients beyond puberty • Presence of BCR-ABL1 or be considered as favorable or bleak? Why? • CNS or mediastinal involvement BCR-ABL1-like mutations 616 Chapter 27 Table 27.5 Cellular Markers Useful in Diagnosis and Classification of T Cell ALL T Cell CD19, Developmental CD20, Ig genes TCR genes Stage HLA-DR TdT CD34 CD2 CDla CD3 CD7 CD8 CD4 CD10 CD22 rearrangement rearrangement Pro-T +/- +/- +/- - - + (c) + - - +/- - - +/- Pre-T +/- +/- +/- + |
- + (c) + - - +/- - - +/- Cortical T +/- +/- - + + + (c) + + + +/- - - +/- Medullary T +/- +/- - + - + (c) + +a or - +a or - +/- - - +/- Early-T +/- +/- +/- +/- - +/- + - +/- - - - +/- a Either CD4 or CD8+ . (c), cytoplasmic; - , absent; + , present; +/- , may be present; Ig, immunoglobulin; TCR, T cell receptor. morphologic and/or immunophenotypic characteristics of Checkpoint 27.5 both myeloid and lymphoid cells (or of both T and B lym- A 3-year-old patient has 45% lymphoblasts in the bone marrow. phocytes). Terminology regarding these disorders has been If the cells tested positive for CD19, CD10, and CD34, what is confusing. If two distinct populations of blasts are identifi- the most likely immunologic subgroup? Why should cytogenet- able, each expressing markers of a distinct lineage (i.e., one ics be performed on this patient? population myeloid and one population lymphoid or one population T lymphoid and one population B lymphoid), Acute Leukemias of Ambiguous the disease has been called bilineage acute leukemia. If the blasts coexpress myeloid and T or B lineage-specific Lineage markers or concurrent T and B lineage markers, the disease This WHO group of leukemias include malignant neo- has been called biphenotypic acute leukemia.29 Also rec- plasms in which the morphology, cytochemistry, undefined ognized is a leukemia with myeloid/natural killer (NK) cell genetic alterations, and immunophenotype lack sufficient acute leukemia with markers of both NK cells (CD56) and information to classify them within a given lineage or in myeloid lineage (CD13/CD33/MPO).29 WHO classification which the blasts have morphologic and/or immunopheno- attempts to clarify the terminology confusion and refers to typic characteristics of more than one lineage. these biphenotypic and bilineage leukemias by the gen- eral term mixed phenotype acute leukemia (MPAL). The terms ACUTE UNDIFFERENTIATED LEUKEMIA B/myeloid leukemia and T/myeloid leukemia are used to des- Although determination of the myeloid or lymphoid origin of ignate leukemias containing the two lineages specified blasts is important for treatment decisions, doing so is some- regardless of whether one or two populations of blasts are times not possible with current methods. The acute undiffer- seen. Specific mutations in the BCR-ABL1 gene and rear- entiated leukemia (AUL) category includes acute leukemias rangement of the KMT2A gene are distinct subtypes of in which the morphology, cytochemistry, and immunophe- MPAL according to the WHO classification mainly due to notype of the proliferating blasts lack sufficient information prognosis (i.e., poor prognosis of KMT2A rearrangement) to classify them as either myeloid or lymphoid origin. In this and therapeutic (i.e., TKI therapy for BCR-ABL1 muta- case, the leukemia is classified as acute undifferentiated leu- tions) reasons.12 However, they carry the same ambiguity kemia.28,33 Electron microscopic studies can sometimes detect in regard to cellular lineage of the MPAL leukemias not oth- ultrastructural evidence of primary granules and/or peroxi- erwise specified. dase, a finding that would indicate acute myeloid leukemia. The MPAL designation is reserved for cases in which Immunophenotyping should be done with a comprehensive lineage assignment is ambiguous. It should not be used panel of monoclonal antibodies to exclude neoplastic cells when an ALL aberrantly expresses one or two myeloid of unusual lineages such as basophils and NK-cell precur- antigenic markers or an AML aberrantly expresses one or sors. Absence of primary granules and/or MPO with elec- two lymphoid antigenic markers. These are considered tron microscopy with other negative or ambiguous findings myeloid antigen–positive ALL and lymphoid antigen–positive indicates a diagnosis of AUL. It is predominantly diagnosed AML, respectively. Because many markers are only lineage in adults, and only about one-third of patients respond to the associated, not lineage specific, coexpression of only one or chemotherapy regimens for AML and ALL. two cross-lineage antigens is insufficient for a diagnosis of ACUTE LEUKEMIAS WITH LINEAGE HETEROGENEITY biphenotypic leukemia. The category acute leukemias with lineage heterogene- Lack of lineage specificity (lineage infidelity) could be ity includes those leukemias in which the blasts have the result of genetic misprogramming, or the leukemic clone Precursor Lymphoid Neoplasms 617 could represent a bipotential cell that has retained both lym- express inappropriate combinations of antigens, making phoid and myeloid markers during development. Although diagnosis challenging. Treatment protocols and prognosis mixed lineage leukemias are uncommon, their identifica- are proving to be more effective and accurate when the leu- tion is important to determine their actual occurrence, to kemic cell lineage is accurately classified immunologically. identify appropriate therapy, and to correlate karyotypic Therapy response and detection of residual leukemic cells abnormalities.34,35 A large percentage of the acute leukemias (MRD) is possible using immunophenotyping and genetic of ambiguous lineage has associated cytogenetic abnormali- testing. ties. The cell of origin is thought to be the multipotential Chemotherapy for ALL is divided into several phases. hematopoietic stem cell. The induction therapy phase is designed to reduce the dis- Current methodology using two or more immuno- ease to complete remission (i.e., eradicating the leukemic logic markers in a double-labeling technique distinguishes blast population). This is followed by a CNS prophylactic whether the lymphoid and myeloid antigens are on the phase. CNS leukemia is the most common form of relapse in same cell or separate cells.28 This can be done with flow young children who have not undergone specific treatment cytometry or immunohistochemistry on tissue sections. to the brain and spinal column early in remission. The two Another procedure is to stain smears with MPO to identify potential modes of treatment in the CNS prophylactic phase myeloid cells coupled with flow cytometry to detect the T or are cranium irradiation and/or intrathecal chemotherapy. B lymphoid population. Cranial irradiation is seldom a component of most current treatment protocols because of the risk of neurocognitive deficits and endocrinopathy and of inducing a second can- Checkpoint 27.6 cer.1 The third phase is maintenance chemotherapy, also A patient with acute leukemia has two morphologically different types of blasts. One population is positive for CD7 and CD2. called cytoreductive therapy or remission consolidation therapy.1The other is positive for CD33 and CD13. What is the most The need for this type of therapy is controver- appropriate classification of this leukemia? sial. Some studies have shown a slight increase in survival with its use, whereas others reveal no improvement. Before the institution of CNS prophylactic treatment, there was a Natural Killer Cell Lymphoblastic high incidence of relapse. The maintenance therapy was designed to prevent this relapse and prolong remission. The Leukemia/Lymphoma purpose of maintenance therapy is to eradicate any remain- Natural killer (NK) cell lymphoblastic leukemia is a neo- ing leukemic cells. Drug treatment usually continues for 2–3 plasm that is difficult to define because (1) no specific mark- years. The relapse rate after cessation of all therapy is about ers for human NK-cell progenitors have been identified and 25% in the pediatric patient population. (2) NK-cell progenitors can express markers that are seen Allogeneic hematopoietic stem cell transplantation in T cell ALL (CD7, CD2, CD5, and cytoplasmic CD3). The (HSCT) remains controversial. Currently, most clinicians CD56 marker was previously thought to identify NK leu- consider it to be of benefit to some high-risk adult patients kemia but many of these cases are now known to be plas- and has shown promise in infants with KMTA2 gene rear- macytoid dendritic cell leukemia. More specific markers for rangements.1,20 Patients who relapse while on therapy or precursor NK cells are CD94 and CD161, but they are not after only a short remission are often considered candidates usually tested. Until more specific panels for NK cells are for HSCT. The use of umbilical cord blood as a source of developed, the 2016 WHO recommends that NK-cell lym- hematopoietic stem cells is being considered more fre- phoblastic leukemia remain a provisional entity but distinct quently, especially in the pediatric patient population and separate from other lymphoblastic or ambiguous lin- because it does not require the same degree of histocompat- eage leukemia subtypes.12,36 ibility as do transplants using peripheral blood or marrow stem cells. Relapse is defined as the reappearance of leukemic cells Therapy anywhere in the body, although the bone marrow is the most common site. Leukemic relapse occasionally occurs New treatment protocols have raised the complete at extramedullary sites. Most relapses occur during treat- remission rate for B cell ALL to more than 95% in chil- ment or within the first two years after completion. Rarely, dren and 60–85% in adults. The prognosis of T cell ALL/ relapses have been observed up to ten years after the induc- LBL is not as favorable as B cell ALL/LBL. This may be tion of remission. Relapse indicates a poor outcome for most in part because of the presence of high-risk clinical fea- patients, especially if it occurs during therapy or after only tures (Table 27-4). A significant number of acute leukemias a brief initial remission. 618 Chapter 27 Summary Acute leukemias represent a heterogeneous group of pre- Regardless of subtype, the onset of ALL is usually cursor hematopoietic neoplasms characterized by unregu- abrupt and without treatment, progresses. Symptoms are lated proliferation, arrested maturation, and/or ineffective related to anemia, thrombocytopenia, and/or neutropenia. apoptosis. The World Health Organization classification Splenomegaly, hepatomegaly, and lymphadenopathy are categorizes two major groups: precursor (acute) myeloid common findings. Hematologic findings of ALL include a and precursor (acute) lymphoid. The precursor lymphoid normocytic, normochromic anemia, and thrombocytope- group includes lymphoblastic lymphomas (LBL) and acute nia and a decreased, normal, or increased leukocyte count. lymphoblastic leukemia (ALL). LBL presents with a mass of Blasts are almost always found in the peripheral blood. malignant lymphoid cells in nodal or extranodal sites with Although the WHO classification does not require a mini- little or no bone marrow and peripheral blood involvement. mum number of blasts for a diagnosis of ALL/LBL as it ALL presents with bone marrow and peripheral blood does for AML, it is recommended that a diagnosis of ALL involvement. The two groups are considered to be differ- not be made for blast counts less than 20%. ent clinical presentations of the same disease. Acute leukemia with lineage heterogeneity includes ALL/LBL is subdivided into precursor B cell and pre- two situations in which ALs have characteristics of T and cursor T cell based on the lineage of the neoplastic blast B cells or myeloid and lymphoid cells. Mixed phenotype population. Further subgrouping is based on morphology, acute leukemia (MPAL) describes ALs with blasts that pos- genetic or karyotypic mutational status, and immunophe- sess markers of multiple lineages or two different popu- notype of blasts. Various cytogenetic and molecular changes lations of blasts. While natural killer cell lymphoblastic are described in ALL, including t(12;21)(p13;q22)/ETV6- leukemia is considered a unique group of lymphoblastic RUNX1 most commonly and t(9;22)(q34;q11.2)/BCR-ABL1 leukemia, difficulties in creating specific panels that detect that is associated with a poor prognosis. their presence is an ongoing diagnostic challenge. Review Questions Level I c. acute lymphoblastic leukemia 1. Acute lymphoblastic leukemia is characterized by the d. acute leukemia of ambiguous lineage presence of: (Objective 1) 4. The presence of CD19 and CD22(c) and absence of a. less than 20% small resting lymphoblasts in the CD10 (CALLA) on neoplastic lymphoblasts is most bone marrow often a sign of: (Objective 4) b. hypercellular bone marrow with increased a. biphenotypic leukemia lymphoblasts b. pro-B ALL c. 30% or more lymphoblasts with erythroid c. pre-B ALL dysplasia in the bone marrow d. T cell ALL d. more than 30% myeloblasts in the peripheral blood 5. The anemia most often seen in patients with ALL is: 2. Acute lymphoblastic leukemia is most often seen in (Objective 3) patients: (Objective 5) a. microcytic hypochromic a. older than 60 years b. normocytic normochromic b. between the ages of 35 and 60 years c. macrocytic normochromic c. between the ages of 10 and 35 years d. macrocytic hypochromic d. younger than 5 years 6. Leukemia of ambiguous lineage is most often described as the presence of: (Objectives 4, 6) 3. ALL characterized by the presence of small basophilic lymphoblasts with a round nucleus, finely g ranular a. large, basophilic blasts with slight to moderate chromatin, and inconspicuous nucleoli can be granulation classified as: (Objective 3) b. blasts demonstrating both CD10 and C33 a. ALL—Burkitt’s type c. |
CD3 and CD4 markers b. acute biphenotypic leukemia d. no demonstrable markers using immunophenotyping Precursor Lymphoid Neoplasms 619 7. The oversecretion of monoclonal immunoglobulins is a. precursor B ALL most often seen in: (Objectives 3, 5) b. AUL a. B cell ALL c. T cell ALL b. T cell ALL d. bilineage ALL c. B cell LBL 4. An adult patient with splenomegaly has an increase in d. T cell LBL mononuclear cells in the peripheral blood. The bone 8. CD2, CD3, and CD4 are present on the cells found in marrow was filled with a heterogeneous collection a patient with a previously undiagnosed leukemia. of blasts with no granulation. Flow c ytometry shows These markers are characteristic of: (Objective 4) a positive CD20 and CD10 and negative surface immunoglobulin. Which of the following conditions a. pre-B cell ALL is most likely? (Objective 4) b. pro-B cell ALL a. Intermediate pre-B ALL (CALLA) c. T cell ALL b. Pro-B ALL d. ALL—Burkitt’s type c. Pre-T cell ALL 9. Acute biphenotypic leukemia is characterized by the d. Cortical T cell ALL presence of: (Objective 6) 5. Which of the following represents the most common a. ultrastructual evidence of primary granules chromosomal translocation found in ALL? b. CD19, CD20, and CD23 (Objective 4) c. CD2, CD4, CD8, and presence of myeloperoxidase a. t(1;19)(q23;p13.3)/PBX1-E2A d. negative immunophenotyping b. t(12;21)(p13;q22)/ETV6-RUNX1 10. Which of the following laboratory results are most c. t(4;11)(q21;q23)/MLL-AFF1 commonly found in ALL? (Objective 3) d. t(9;22)(q34;q11)/BCR-ABL1 a. Eosinophilia and basophilia 6. The CD10 antigen found in ALL is also known as the: b. Neutropenia and thrombocytopenia (Objective 4) c. Neutrophilia and thrombocytopenia a. B cell ALL antigen d. Lymphocytosis and thrombocytosis b. T cell ALL antigen Level II c. CALLA antigen 1. Cells that are positive for t(9;22) are characteristic of d. NK-cell ALL antigen which of the following? (Objective 4) 7. The most likely patient presentation for ALL in an a. T cell ALL adult includes: (Objective 2) b. Reactive lymphocytosis a. male aged 18–30 with mild anemia and no c. Acute undifferentiated leukemia thrombocytopenia d. B cell ALL b. male older than 60 years with significant anemia and thrombocytopenia 2. Monoclonal rearrangement of the TCR genes is associated with blasts that have the following c. female 10–25 years old with significant bone pain immunophenotype: (Objective 2) and hemolytic anemia d. female 30–50 years old with hemorrhage and bulky a. CD2+ , CD4+ , CD19+ nodes b. CD19+ , CD20+ , CD10+ c. CD3+ , CD13- , CD34+ 8. The objective of induction therapy includes: (Objective 7) d. CD2+ CD3+ , CD4+ a. reduction in the tumor burden 3. A peripheral blood smear is noted to have 80% blasts. b. elimination of tumor cells in the central nervous There are two different types of blast cells present: system one that is small, with a round nucleus, indistinct nucleoli, and scanty cytoplasm while the second is a c. replacement with peripheral blood stem cells from large blast with basophilic cytoplasm. One possible another person interpretation for this is: (Objectives 1, 6) d. prevention of relapse 620 Chapter 27 9. Poor prognostic findings for patients with ALL 10. A large mediastinal mass is found in a 60-year-old include: (Objective 8) male. The bone marrow has 15% lymphoblasts. This is most characteristic of: (Objective 9) a. young age at time of diagnosis b. presence of chromosomal mutations such as a. precursor T cell LBL BCR/ABL1 b. ALL—Burkitt’s type c. hyperploidy c. precursor T cell ALL d. presence of TEL-AML1 gene d. precursor B cell ALL References 1. Larson, R. A. (2015). Acute lymphoblastic leukemia. In K. Neoplasms and Acute Leukemia. Blood, 128(3), 462–463. Kaushansky, M. S. Lichtman, J. T. Prchal, M. M. Levi, O. W. Press, doi: 10.1182/blood-2016-06-721662 L. J. Burns, & M. A. Caligiuri (Eds.), Williams Hematology (9th ed., 13. Pui, C. H., & Evans, W. E. (2006). Treatment of acute lymphoblas- pp. 1505–1526). New York: McGraw-Hill Education. tic leukemia. New England Journal of Medicine, 354(2), 166–178. 2. Borowitz, M. J., Chan, J. K., Downing, J. R., Le Beau, M. M., & 14. Basso, G., Case, C., & Dellorto, M. (2007). Diagnosis and genetic Arber, D. A. (2017). Precursor lymphoid neoplasms. In subtypes of leukemia combining gene expression and flow S. Swerdlow, E. Campo, N. Harris, E. Jaffe, S. Pileri, H. Stein, & cytometry. Blood Cells, Molecules, and Diseases, 39(2), 164–168. H., Thiele, J. (Eds.), WHO Classification of Tumours of Haemato- doi: 10.1016/j.bcmd.2007.05.004 poietic and Lymphoid Tissues (4th ed., pp. 199–214). Lyon, France: 15. Schultz, K. R., Pullen, D. J., Sather, H. N., Shuster, J. J., Devidas, M., International Agency for Research on Cancer. Borowitz, M. J., . . . Camitta, B. M. (2006). Risk- and 3. Borowitz, M. J., & Chan, J. K. C. (2008). Precursor lymphoid neo- response-based classification of childhood B-precursor acute plasms. In: S. Swerdlow, E. Campo, N. Harris, E. Jaffe, S. Pileri, & lymphoblastic leukemia: a combined analysis of p rognostic H. Stein, eds. WHO Classification of Tumours of Haematopoietic and markers from the Pediatric Oncology Group (POG) and Lymphoid Tissues (3rd ed., pp 167–178). Lyon, France: Interna- Children’s Cancer Group (CCG). Blood, 109(3), 926–935. tional Agency for Research on Cancer. doi: 10.1182/blood-2006-01-024729 4. Pitman, S. D., & Huang, Q. (2007). Granular acute lymphoblastic 16. Harrison, C. J., Haas, O., Harbott, J., Biondi, A., Stanulla, M., leukemia: A case report and literature review. American Journal of Trka, J., & Izraeli, S. (2010). Detection of prognostically relevant Hematology, 82(9), 834–837. doi: 10.1002/ajh.20922 genetic abnormalities in childhood B-cell precursor acute lym- 5. Majumder, D., Banerjee, D., Chandra, S., Banerjee, S., & phoblastic leukaemia: Recommendations from the Biology and Chakrabarti, A. (2006). Red cell morphology in leukemia, hypo- Diagnosis Committee of the International Berlin-Frankfürt- plastic anemia and myelodysplastic syndrome. Pathophysiology, Münster study group. British Journal of Haematology, 151(2), 13(4), 217–225. doi: 10.1016/j.pathophys.2006.06.002 132–142. doi: 10.1111/j.1365-2141.2010.08314.x 6. Kaleem, Z., Crawford, E., Pathan, M. H., Jasper, L., Covinsky, M. 17. Ney-Garcia, D. R., Liehr, T., Bhatt, S., Souza, M. T., Matos, R. R., A., Johnson, L. R., & White, G. (2003). Flow cytometric analysis of Pimenta, G., . . . Silva, M. L. (2012). Childhood B-cell progenitor acute leukemias: Diagnostic utility and critical analysis of data. acute lymphoblastic leukemia presenting a three-way t(11;12;21) Archives of Pathology and Laboratory Medicine, 127(1), 42–48. (q14;p13;q22) with a RUNX1 gene signal on chromosome 11. 7. Kersey, J., Nesbit, M., Hallgren, H., Sabad, A., Yunis, E., & International Journal of Hematology, 95(1), 112–114. doi: 10.1007/ Gajl-Peczalska, K. (1975). Evidence for origin of c ertain s12185-011-0981-x childhood acute lymphoblastic leukemias and lymphomas 18. Fuka, G., Kauer, M., Kofler, R., Haas, O. A., & Panzer-Grümayer, in thymus-derived lymphocytes. Cancer, 36(4), 1348–1352. R. (2011). The leukemia-specific fusion gene ETV6/RUNX1 doi: 10.1002/1097-0142(197510)36:4<1348::aid-cncr2820360424> perturbs distinct key biological functions primarily by gene 3.0.co;2-v repression. PLoS ONE, 6(10). doi: 10.1371/journal.pone.0026348 8. Hurwitz, C. A., Loken, M. R., Graham, M. L., Karp, J. E., 19. Bernt, K. M., & Armstrong, S. A. (2011). Targeting epigenetic Borowitz, M. J., Pullen, D. J., & Civin, C. I. (1988). Asynchronous programs in MLL-rearranged leukemias. Hematology, 2011(1), antigen expression in B-lineage acute lymphoblastic leukemia. 354–360. doi: 10.1182/asheducation-2011.1.354 Blood, 72(1), 299–307. 20. Kato, M., Hasegawa, D., Koh, K., Kato, K., Takita, J., Inagaki, J., 9. Burns, M., Armstrong, S. A., & Gutierrez, A. (2018). Pathobiology . . . Kato, K. (2014). Allogeneic haematopoietic stem cell trans- of acute lymphoblastic leukemia. In R. Hoffman, E. J. Benz Jr., plantation for infant acute lymphoblastic leukaemia with KMT2A L. E. Silberstein, H. Heslop, J. Weitz, & J. Anastasi, Hematology: (MLL) rearrangements: A retrospective study from the paediatric Basic principles and practice (7th ed., pp. 1005–1019). Philadelphia: acute lymphoblastic leukaemia working group of the Japan Soci- Elsevier. ety for Haematopoietic Cel. British Journal of Haematology, 168(4), 10. Tyner, J. W., Jemal, A. M., Thayer, M., Druker, B. J., & Chang, B. H. 564–570. doi: 10.1111/bjh.13174 (2011). Targeting survivin and p53 in pediatric acute lymphoblas- 21. Xing, H., Yang, X., Liu, T., Lin, J., Chen, X., & Gong, Y. (2012). The tic leukemia. Leukemia, 26(4), 623–632. doi: 10.1038/leu.2011.249 study of resistant mechanisms and reversal in an imatinib resis- 11. Kelly, K. M., Burkhardt, B., & Bollard, C. M. (2018). Malignant tant Ph acute lymphoblastic leukemia cell line. Leukemia Research, lymphomas in childhood. In: R. Hoffman, E. J. Benz Jr., 36(4), 509–513. doi: 10.1016/j.leukres.2011.12.018 L. E. Silberstein, H. Heslop, J. Weitz, & J. Anastasi. Hematology: 22. Arico, M., Valsecchi, M. G., Camitta, B., Schrappe, M., Chessells, Basic principles and practice (7th ed., pp. 1330–1342). Philadelphia: J., Baruchel, A., . . . Masera, G. (2000). Outcome of treatment in Elsevier. children with Philadelphia chromosome-positive acute lym- 12. Arber, D. A., Orazi, A., & Hasserjian, R. (2016). The 2016 revi- phoblastic leukemia. New England Journal of Medicine, 342(14), sion to the World Health Organization Classification of Myeloid 998–1006. doi: 10.1056/NEJM200004063421402 Precursor Lymphoid Neoplasms 621 23. Najfeld, V. (2013). Conventional and molecular cytogenetic basis significance of early T-cell precursor acute lymphoblastic leu- of hematologic malignancies. In R. Hoffman, E. J. Benz Jr., kaemia: Results of the Tokyo Children’s Cancer Study Group L. E. Silberstein, H. Heslop, J. Weitz, & J. Anastasi (Eds.), Study L99-15. British Journal of Haematology, 156(3), 358–365. Hematology: Basic Principles and Practice (6th ed., pp. 728–780). doi: 10.1111/j.1365-2141.2011.08955.x Philadelphia: Elsevier. 31. Baiocchi, R. A. (2016). Classification of Malignant Lymphoid 24. Markovic, A., Mackenzie, K. L., & Lock, R. B. (2005). FLT-3: A Disorders. In: K. Kaushansky, M. A. Lichtman, J. T. Prchal, M. new focus in the understanding of acute leukemia. Interna- M. Levi, O. W. Press, L. J. Burns, & M. A. Caligiuri, eds. Williams tional Journal of Biochemistry and Cell Biology, 37(6), 1168–1172. Hematology (9th ed., pp.1493–1504). New York: McGraw-Hill doi: 10.1016/j.biocel.2004.12.005 Education. 25. Armstrong, S. A., & Look, A. T. (2005). Molecular genetics of 32. Sharma, V. M., Draheim, K. M., & Kelliher, M. A. (2007). The acute lymphoblastic leukemia. Journal of Clinical Oncology, 23(26), Notch1/c-Myc pathway in T cell leukemia. Cell Cycle, 6(8), 6306–6315. doi: 10.1200/JCO.2005.05.047 927–930. doi: 10.4161/cc.6.8.4134 26. Schwab, C., & Harrison, C. J. (2011). Acute lymphoblastic 33. Béné, M. C., & Porwit, A. (2012). Acute leukemias of ambiguous leukaemia. Methods in Molecular Biology, 730, 99–117. lineage. Seminars in Diagnostic Pathology, 29(1), 12–18. doi: 10.1007/978-1-61779-074-4_8 doi: 10.1053/j.semdp.2011.08.004 27. Van der Veer, A., Waanders, E., Pieters, R., Willemse, M. E., Van Reijmersdal, S. V., Russell, L. J., . . . M. L. Den Boer (2013). 34. Scott, A. A., Head, D. R., Kopecky, K. J., Appelbaum, F. R., Theil, Independent prognostic value of BCR-ABL1-like signature and K. S., Grever, M. R., . . . Licht, J. D. (1994). HLA-DR-, CD33, CD56, IKZF1 deletion, but not high CRLF2 expression, in children with CD16- myeloid/natural killer cell acute leukemia: A previously B-cell precursor ALL. Blood, 122(15), 2622–2629. doi: 10.1182/ unrecognized form of acute leukemia potentially misdiagnosed blood-2012-10-462358 as French-American-British acute myeloid leukemia-M3. Blood, 84(1), 244–255. 28. Lewis, R. E., Cruse, J. M., Sanders, C. M., Webb, R. N., Tillman, B. F., Beason, K. L., . . . Koehler, J. (2006). The immunophenotype 35. Vela, J. A., Monteserin, M. C., Delgado, I., Benito, L., & Oña, F. of pre-TALL/LBL revisited. Experimental and Molecular Pathology, (2000). Aberrant Immunophenotypes Detected by Flow Cytom- 81(2), 162–165. doi: 10.1016/j.yexmp.2006.06.006 etry in Acute Lymphoblastic Leukemia. Leukemia and Lymphoma, 29. Coustan-Smith, E., Mullighan, C. G., Onciu, M., Behm, F. G., 36(3–4), 275–284. doi: 10.3109/10428190009148848 Raimondi, S. C., Pei, D., . . . Campana, D. (2009). Early T-cell 36. Borowitz, M. J., Bene, M. C., Harris, N. L., Porwit, A., Matutes, E., & precursor leukaemia: A subtype of very high-risk acute Arber, D. A. (2017). Acute leukemias of ambiguous lineage. In S. lymphoblastic leukaemia. The Lancet Oncology, 10(2), 147–156. Swerdlow, E. Campo, N. Harris, E. Jaffe, S. Pileri, H. Stein, & H., doi: 10.1016/s1470-2045(08)70314-0 Thiele, J. (Eds.), WHO Classification of Tumours of Haematopoietic and 30. Inukai, T., Kiyokawa, N., Campana, D., Coustan-Smith, E., Lymphoid Tissues (4th ed., pp. 179–187). Lyon, France: International Kikuchi, A., Kobayashi, M., . . . Ohara, A. (2011). Clinical Agency for Research on Cancer. Chapter 28 Mature Lymphoid Neoplasms Katalin Kelemen, MD, PhD Fiona E. Craig, MD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Describe the clinical presentation of patients 7. List and describe the |
chronic leukemic with mature lymphoid neoplasms. lymphoproliferative disorders. 2. Describe how the diagnosis of a lymphoid 8. Recognize and differentiate abnormal and neoplasm is made. normal lymphocytes on a stained peripheral 3. Differentiate among chronic lymphocytic blood smear and associate their presence leukemia (CLL), lymphoma, and multiple with a clinical diagnosis. myeloma based on peripheral blood 9. Describe and apply a multidisciplinary findings and ancillary studies. approach to the classification and staging 4. Describe the histology of a normal lymph of lymphoid neoplasms. node. 10. Compare the laboratory and clinical 5. Summarize the causes of lymphadenopathy. findings of multiple myeloma and lymphoplasmacytic lymphoma. 6. Contrast the morphology of Hodgkin and non-Hodgkin lymphoma. 11. Define monoclonal gammopathy. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Describe the use of immunophenotyping 3. Compare and contrast the laboratory and genotyping in detecting clonality and features characteristic of the following list the characteristic results of the more non-Hodgkin lymphomas: common mature lymphoid neoplasms. a. Small lymphocytic lymphoma 2. Contrast the features of low-grade and b. Follicular lymphoma high-grade lymphoma. c. Mantle cell lymphoma 622 Mature Lymphoid Neoplasms 623 d. MALT lymphoma 6. Compare and contrast the laboratory e. Lymphoplasmacytic lymphoma features characteristic of the following f. Diffuse large B cell lymphoma plasma cell disorders: g. Burkitt lymphoma a. Plasmacytoma h. Anaplastic large cell lymphoma b. Symptomatic plasma cell myeloma i. Peripheral T cell lymphoma c. Monoclonal gammopathy of undetermined 4. Compare and contrast the laboratory significance features characteristic of Hodgkin 7. Recognize and identify the peripheral blood lymphoma (HL) subtypes. abnormalities associated with CLL, B-PLL, 5. Compare and contrast the clinical HCL, T-LGL, Sézary syndrome, plasma cell and laboratory features characteristic myeloma, and non-Hodgkin lymphoma. of the following chronic leukemic 8. Describe the etiology and pathophysiology lymphoproliferative disorders: of lymphoid neoplasms. a. Chronic lymphocytic leukemia (CLL) 9. Differentiate reactive from malignant b. B-prolymphocytic leukemia (B-PLL) proliferations of lymphoid cells using c. Hairy cell leukemia (HCL) clinical and laboratory data. d. T-prolymphocytic leukemia (T-PLL) e. Large granular lymphocytic leukemia (T-LGL) f. Sézary syndrome Chapter Outline Objectives—Level I and Level II 622 Diagnosis and WHO Classification 625 Key Terms 623 Mature B Cell Neoplasms 626 Background Basics 624 Mature T and NK Cell Neoplasms 636 Case Study 624 Hodgkin Lymphoma (HL) 640 Overview 624 Summary 642 Introduction 624 Review Questions 642 Etiology and Pathophysiology 624 References 644 Key Terms Anaplastic large cell lymphoma Leukemia Richter’s transformation (ALCL) Lymphoepithelial lesion Rouleaux BCL-2 gene Lymphoma Sézary cell Bence-Jones proteinuria Monoclonal gammopathy of unde- Smudge cell Butt cell termined significance (MGUS) Stage Dutcher body Plasma cell neoplasm Starry-sky appearance Flame cell Plasmacytoma Tingible body macrophages Hairy cell Popcorn cell (L&H cell) Waldenström macroglobulinemia Hallmark cell Prolymphocyte Lacunar cell Reed-Sternberg (R-S) cell 624 Chapter 28 Background Basics The information in this chapter builds on concepts • Outline the classification and summarize the general learned in previous chapters. To maximize your learn- characteristics of acute leukemia. (Chapter 23) ing experience, you should review these concepts before starting this unit of study: Level II • Explain the pathophysiology of neoplasia. Level I (Chapters 2, 23) • Describe the structure and function of lymph • Identify the etiology of acute leukemia. (Chapter 23) nodes. (Chapter 3) to the lymphoid lineage (Chapters 23, 27). Although, in gen- CASE STUDY eral, these broad categories are useful, distinction between We refer to this case study throughout the chapter. leukemia and lymphoma is not always clear-cut. Leukemia Julia, a 56-year-old female, presented with a cells can infiltrate tissue, and lymphoma can involve the 4-month history of generalized painless lymphade- bone marrow and peripheral blood. nopathy and fatigue. A CBC revealed leukocytosis resulting from lymphocytosis. Consider laboratory features that can help dif- Etiology and ferentiate a reactive lymphocytosis from a neoplas- tic lymphocytosis. Pathophysiology The genesis of lymphoid neoplasms is thought to be a mul- tistep process involving acquired genetic, inherited genetic, and environmental factors. The lymphoma cells are thought Overview to derive from a single precursor cell (they are clonal). Lymphoid neoplasms can be divided into mature and pre- cursor neoplasms, based on the stage of maturation of the Acquired Genetic Factors neoplastic cells. The precursor lymphoid neoplasms are dis- As described for the acute leukemias, acquired alterations cussed in Chapter 27. This chapter focuses on the mature of proto-oncogenes and tumor suppressor genes have been lymphoid neoplasms, and discusses their etiology and associated with the development of mature lymphoid pathophysiology, how a diagnosis is established, the clas- neoplasms. Additional targets for genetic damage are the sification scheme, and unique features of selected subtypes. genes involved in programmed cell death (apoptosis [e.g., BCL-2]; Chapter 2). The BCL-2 gene on chromosome 18 is involved in the pathophysiology of follicular lymphoma. Introduction Translocation of the BCL-2 gene to the region of the immu- noglobulin heavy chain gene, t(14;18), causes overexpres- Mature lymphoid neoplasms represent a heterogeneous sion of the BCL-2 gene. The resulting increase in BCL-2 group of disorders that can be divided into two broad cat- protein leads to an inhibition of apoptosis. Decreased cell egories based on the distribution of disease: lymphoma death results in an accumulation of lymphocytes within and leukemia. Lymphomas are malignant neoplasms that the lymph node. Therefore, low-grade follicular lymphoma present as tumor masses primarily involving the lymphoid appears to arise from cell persistence rather than uncon- organs, including lymph nodes, tonsils, spleen, thymus, trolled cell proliferation. and lymphoid tissue of other organs. Although most lym- phomas are composed of mature lymphoid cells, blastic malignancies do occur (lymphoblastic lymphoma). Leukemia Inherited Genetic Factors is a malignant neoplasm that primarily involves the bone Some inherited immunodeficiency syndromes are associ- marrow and peripheral blood. The chronic leukemic lym- ated with an increased incidence of malignant lymphoma, phoid neoplasms are composed of mature lymphocytes and including ataxia telangiectasia, Wiskott-Aldrich syndrome, usually have an insidious onset and an indolent course. In severe combined immunodeficiency (SCID), X-linked lym- contrast, lymphoblastic leukemia (also referred to as acute phoproliferative disorder, and autoimmune lymphoprolif- lymphoblastic leukemia [ALL]) is composed of blasts belonging erative syndrome (ALPS). Mature Lymphoid Neoplasms 625 Environmental Factors cell type. Less frequently, malignant lymphoid cells can be recognized under the microscope because of an abnormal Viral and bacterial infections may play a role in lym- or bizarre cell appearance. Mature lymphoid neoplasms phoma genesis. Epstein-Barr virus (EBV) is associated have widely different clinical symptoms and aggressive- with the development of several forms of lymphoid neo- ness, making the implementation of a generic grading sys- plasm including African Burkitt lymphoma, some cases of tem difficult. endemic Burkitt lymphoma, Hodgkin lymphoma (HL), and lymphoma associated with inherited or acquired immuno- deficiency. EBV infection is acquired orally and is often Laboratory Evaluation manifest clinically as infectious mononucleosis. The virus Clonal rearrangement of immunoglobulin or T cell recep- infects B lymphocytes where it remains latent under the tor genes or the presence of an abnormal translocation can immune system’s control. The EBV-infected cells can pro- be used to identify neoplastic lymphocytes (Chapter 42). liferate if the host becomes immunocompromised and/or The presence of a specific translocation can also assist in the B lymphocytes acquire additional genetic abnormalities determining the lymphoma subtype, as in the BCL-2 gene such as the C-MYC translocation. rearrangement in follicular lymphoma (Chapters 41 and 42). Another infectious agent associated with the devel- Flow cytometric studies can be used to demonstrate clonal- opment of non-Hodgkin lymphoma (NHL) is Helicobacter ity by demonstrating expression of only one of the immu- pylori. Patients with H. pylori–induced inflammation of noglobulin light chains (k or l) on B cells. The presence of the stomach have a high incidence of gastric lymphoma an abnormal phenotype such as CD5- T lymphocytes can of mucosa-associated lymphoid tissue (MALT) lymphoma support the diagnosis of a T cell lymphoma (Chapter 40). type. Chronic Helicobacter infection leads to antigen-driven Immunophenotyping of the lymphoma cells assists in sub- T lymphocyte stimulation and subsequent B lymphocyte classification, determining prognosis, and detecting resid- activation. The B lymphocytes initially are polyclonal and ual disease following treatment. depend entirely on T lymphocyte stimulation. With time, the B cell population can proliferate autonomously. If the Prognosis B lymphocyte proliferation still depends on T lymphocyte stimulation, the lymphoma can regress following removal The 2008 World Health Organization (WHO) scheme uses of the antigenic stimulus with antimicrobial therapy. Lym- morphologic, phenotypic, and genotypic features to clas- phoma that is proliferating independent of antigenic stim- sify mature lymphoid neoplasms into distinct disease ulation requires more drastic therapy including excision entities that have a predictable prognosis and response to and/or chemotherapy. therapy (Table 28-1). The latest revision of the WHO clas- sification published in 2016 incorporates new molecular advances derived from whole genome sequencing while Checkpoint 28.1 retaining the principles of the 2008 classification.1 Some of How does the BCL-2 gene rearrangement promote lymphoma these disease entities have a long indolent course and are genesis? often referred to as being low-grade (e.g., small lympho- cytic lymphoma). In contrast, some subtypes are clinically aggressive and, without treatment, kill the patient rapidly. Diagnosis and Who These are referred to as aggressive or high grade (e.g., Burkitt lymphoma). Classification The diagnosis and classification of mature lymphoid neo- Therapy plasms involve integration of clinical information with Current therapeutic regimens are actually more effective at the morphologic appearance and the results of ancillary treating high-grade than low-grade lymphoid neoplasms, studies, such as flow cytometry, chromosome analysis, and many patients treated for high-grade lymphoma are and molecular analysis. For most cases of leukemia, mor- cured of their disease. Often patients with low-grade lym- phologic evaluation is performed on peripheral blood and phoid neoplasms are treated for symptomatic relief rather bone marrow (aspirate and biopsy) samples. A diagnosis than the intent to cure. In general, histologic sections from of lymphoma requires a tissue biopsy or a fine-needle aspi- lower-grade lymphoma more often demonstrate a nodular rate of a mass. In general, normal or reactive proliferations growth pattern, smaller cells, lower mitotic activity, and of lymphocytes contain a mixture of cells varying in size, an absence of apoptosis. Higher-grade lymphoma usually shape, and staining characteristics. The cells present in has a diffuse growth pattern, is often composed of larger lymphoid neoplasms are usually more homogeneous than cells, and displays more numerous mitoses and apoptotic reactive lymphocytes because of the expansion of a single bodies. 626 Chapter 28 Table 28.1 WHO 2016 Classification of Selected Mature Table 28.2 Ann Arbor Staging System for Malignant Lymphoid Neoplasms Lymphoma Mature B Cell Chronic lymphocytic leukemia/small lymphocytic I Single lymph node region or single extralymphatic site 1IE2 Neoplasms lymphoma II Two or more lymph node regions on same side of diaphragm or B cell prolymphocytic leukemia with involvement of limited contiguous extralymphatic site 1IIE2 Hairy cell leukemia III Lymph node regions on both sides of diaphragm, which can Follicular lymphoma include spleen 1IIIS2 and/or limited contiguous extralymphatic Mantle cell lymphoma site 1IIIE2 Extranodal marginal zone lymphoma of mucosa- IV Multiple or disseminated foci of involvement of one or more associated lymphoid tissue (MALT lymphoma) extralymphatic organs or tissues with or without lymphatic Splenic marginal zone lymphoma involvement Nodal marginal zone lymphoma Lymphoplasmacytic lymphoma Diffuse large B cell lymphoma (DLBCL), not other- wise specified Mature B Cell Neoplasms Primary mediastinal (thymic) B cell lymphoma Chronic Lymphocytic Leukemia/ Burkitt lymphoma Plasma cell myeloma Small Lymphocytic Lymphoma Monoclonal gammopathy of undetermined Chronic lymphocytic leukemia (CLL) and small lymphocytic significance lymphoma (SLL) represent different clinical manifestations of Monoclonal immunoglobulin deposition diseases one disease entity (CLL/SLL). Patients with CLL present with Mature T and NK T cell prolymphocytic leukemia cell Neoplasms peripheral blood lymphocytosis but often develop lymph T cell large granular lymphocytic leukemia Sézary syndrome node involvement. In contrast, patients with SLL present Adult T cell leukemia/lymphoma with lymphadenopathy but often develop peripheral blood Extranodal NK/T cell lymphoma, nasal type and bone marrow disease. At first diagnosis of CLL/SLL, Enteropathy-associated T cell lymphoma patients are often asymptomatic. As the disease progresses, Hepatosplenic T cell lymphoma anemia, thrombocytopenia, and neutropenia may develop Subcutaneous panniculitis-like T cell lymphoma due to replacement of the bone marrow hematopoietic cells Angioimmunoblastic T cell lymphoma by neoplastic lymphocytes, hypersplenism, and immune- Anaplastic large cell lymphoma (ALCL), ALK positive mediated cell destruction, among other causes (Chapter 19). Anaplastic large cell lymphoma |
(ALCL), ALK negative Peripheral T cell lymphoma, not otherwise specified PERIPHERAL BLOOD Hodgkin Nodular lymphocyte-predominant Hodgkin A diagnosis of CLL requires a sustained absolute lymphocyto- lymphoma lymphoma sis more than 5 * 103/mcL with characteristic morphologic Classical Hodgkin lymphoma: nodular sclerosis, and phenotypic findings. The lymphocytes are small and mixed cellularity, lymphocyte rich, and lymphocyte depleted appear mature with scant cytoplasm. The nuclei are usually round, and the chromatin is regularly clumped. Nucleoli are inconspicuous (Figure 28-1a). A few large p rolymphocytes Staging are usually present but represent 610, of all lymphocytes (Figure 28-1a). Prolymphocytes have abundant pale-staining The prognosis of a patient with a lymphoid neoplasm is cytoplasm and a large central prominent nucleolus. Smudge not only related to the pathologic findings but also clinical cells represent CLL cells that burst open during smear prepa- parameters, such as the extent and distribution of disease ration (Figure 28-1a). Though common in CLL, smudge cells (stage). Patients with widespread lymphoma usually have are nonspecific and they should not be used to diagnose CLL. a worse prognosis. Determining the stage of disease usu- The number of smudge cells can be reduced by mixing a drop ally involves radiologic studies, peripheral blood exami- of albumin with a drop of blood prior to making the smear. nation, and bone marrow aspiration and biopsy. The Ann Arbor scheme is often used to stage malignant lymphoma IMMUNOPHENOTYPING (Table 28-2). Bone marrow involvement indicates dissemi- Although a provisional diagnosis usually can be made fol- nated disease, Stage IV. lowing smear examination, immunophenotyping is often used to establish a definitive diagnosis. CLL is characterized by aberrant expression of the T lymphocyte antigen CD5. Checkpoint 28.2 The CLL cells usually have weak surface expression of CD20 How does staging differ from grading in characterizing the lym- and monoclonal immunoglobulin. Expression of CD23 and phoid neoplasms? CD200, and lack of FMC-7 positivity distinguishes CLL from leukemic mantle cell lymphoma (MCL). A recently Mature Lymphoid Neoplasms 627 a b Figure 28.1 (a) Chronic lymphocytic leukemia. Small round lymphocytes with clumped chromatin, a larger prolymphocyte with a prominent nucleolus, and numerous smudge cells (arrows) (peripheral blood, Wright stain, 1000* magnification). (b) Small lymphocytic lymphoma. The periphery of the image displays many small lymphocytes with round nuclei and clumped chromatin. The center of the image contains a vague nodule containing pale-staining larger cells (proliferation center; lymph node biopsy, H&E stain, 50* magnification). recognized useful marker is the Lymphoid enhancer bind- status of the immunoglobulin (Ig) heavy chain gene variable ing factor 1 (LEF-1), which is expressed in CLL/SLL but not region by a molecular sequencing method1VH2. CLL with a in mantle cell lymphoma, marginal zone lymphoma and more aggressive behavior is associated with higher levels follicular lymphoma.1 of expression of CD38 and ZAP-70 as determined by flow Flow cytometric studies occasionally detect a small cytometry and an unmutated Ig VH gene. population of cells with a phenotype characteristic of CLL but representing less than the required absolute count of CYTOGENETICS AND PROGNOSIS 5 * 103/mcL. If there is no clinical evidence of an overt The use of routine cytogenetic studies is limited in CLL/SLL lymphoid neoplasm, the diagnosis of monoclonal B lympho- because the leukemic cells do not proliferate well in culture. cytosis (MBL) is rendered. MBL precedes virtually all cases Fluorescence in situ hybridization (FISH), a more suitable of CLL. Based on recent recommendations of the revised cytogenetic technique, is able to detect chromosomal abnor- WHO classification, “low-count” MBL (defined as less than malities in approximately half of all CLL/SLL patients. The 0.5 * 103/mcL) does not require a clinical follow-up other most common chromosomal abnormality, del 13q14-23.1, is than routine medical care, while a “high-count” MBL (more associated with a relatively good prognosis. The following than 0.5 * 103/mcL) requires a yearly follow-up.1 chromosomal abnormalities, listed in order of decreasing frequency, are associated with a worse prognosis: trisomy LYMPH NODE BIOPSY 12, deletion 11q22.3-23, deletion 6q21-23, and deletions at A lymph node biopsy performed in CLL/SLL reveals an 17p13.1 (p53 aberrations), 14q abnormalities and complex essentially diffuse infiltrate of small mature lymphocytes. chromosomal abnormalities. The small lymphocytes have dense, regularly clumped chromatin and lack nucleoli (Figure 28-1b). At low power, TRANSFORMATION vaguely nodular structures, the so-called proliferation cen- Less than 10% of patients with CLL/SLL develop trans- ters (Figure 28-1b) containing larger cells with pale-staining formation to a B cell lymphoid neoplasm with a worse cytoplasm and prominent eosinophilic nucleoli can be seen.1 prognosis. Over time, the number of prolymphocytes can increase, often in association with acquired genetic abnor- MOLECULAR PROGNOSTICS malities, although progression to prolymphocytic leukemia The majority of patients with CLL/SLL experience a slowly is unusual. Richter’s transformation is the transition to an progressing indolent course and require treatment only for aggressive large B cell lymphoma, which occurs in approxi- symptom relief. It has been recently recognized that CLL/SLL mately 2–8% of patients with CLL/SLL.2 is a rather heterogeneous disorder and a subset of patients requires earlier treatment due to a more aggressive course THERAPY and a shorter overall survival. Prognostic markers are labo- The Rai and Binet prognostic staging systems, which are ratory markers that correlate with a certain clinical behavior based on physical examination and blood counts, can be and are often evaluated at diagnosis in an attempt to predict used to assess the need for therapy and the specific type outcome. Commonly used prognostic markers include the of therapy.3 Typically, patients with early stage CLL do not expression of proteins CD38 and ZAP-70 and the mutational receive therapy but are monitored for disease progression. 628 Chapter 28 Standard therapy for those who are symptomatic or have advanced disease is chemotherapy with fludarabine, cyclo- phosphamide, and rituximab (anti-CD20). New small- molecule inhibitors (e.g., ibrutinib) that work inside the cell to block activity of enzymes responsible for cell signaling may change the standard treatment of B cell malignancies. B Cell Prolymphocytic Leukemia B cell prolymphocytic leukemia (B-PLL) is an aggressive, rare, leukemic disorder that often does not respond to treat- ment. B-PLL can arise de novo, or as a progression of CLL or another low-grade lymphoma, such as splenic marginal zone lymphoma. Patients with B-PLL usually have promi- nent splenomegaly but minimal lymphadenopathy. PERIPHERAL BLOOD The CBC in patients with B-PLL reveals marked absolute Figure 28.2 Prolymphocytic leukemia. Numerous large lymphocytosis, often more than 300 * 103/mcL, anemia, lymphoid cells with prominent nucleoli (prolymphocytes) (peripheral blood, Wright stain, 1000* magnification). and thrombocytopenia. The neoplastic cells have a charac- teristic appearance of “prolymphocytes”: large cells with moderate amounts of pale basophilic cytoplasm, moder- ately condensed chromatin, and a single prominent nucleo- Hairy Cell Leukemia lus (Figure 28-2). Prolymphocytes represent less than 10% Hairy cell leukemia (HCL; Figure 28-3) is an uncommon of the lymphocytes seen in CLL and more than 55% of the B cell neoplasm presenting in middle age. Males have a lymphocytes present in PLL. Cases with 11–55% prolym- significantly higher incidence (male-to-female ratio = 7:1). phocytes have an unpredictable course. At presentation, patients usually have massive spleno- megaly but lack lymphadenopathy. Extensive bone mar- IMMUNOPHENOTYPING row involvement often exists; therefore, patients with HCL The phenotype of B-PLL differs from that of CLL in demon- usually present with pancytopenia. strating more variable expression of CD5, stronger intensity expression of surface immunoglobulin and CD20, positivity PERIPHERAL BLOOD with FMC-7, and absence of CD23. B-PLL is not associated The white blood cell (WBC) count is low because of both with a characteristic chromosome abnormality, but the short neutropenia and monocytopenia; patients thus have an arm of chromosome 17 is often deleted, del(17p), resulting increased susceptibility to infection, especially with myco- in inactivation of the p53 gene (a tumor suppressor gene). bacterial organisms. Although neoplastic cells (hairy cells) B-PLL does not respond well to chemotherapy and has a are usually present on a peripheral smear, they are usually poor prognosis. too few to elevate the WBC count. The hairy cells have a a b Figure 28.3 Hairy cell leukemia. (a) Two abnormal lymphocytes with abundant pale-staining cytoplasm with hair-like projections and relatively finely distributed chromatin (peripheral blood, Wright stain, 1000* magnification). (b) Replacement of hematopoietic precursors by abnormal small lymphocytes with abundant clear cytoplasm (“fried-egg” appearance) (bone marrow, H&E stain, 1000* magnification). Mature Lymphoid Neoplasms 629 characteristic abnormal appearance with abundant pale- Follicular Lymphoma staining cytoplasm, circumferential cytoplasmic projections (“hairs”), usually oval or reniform nuclei, and relatively Follicular lymphoma (FL; grade 1; Figure 28-4) is one of fine chromatin (Figure 28-3a). Flow cytometric immuno- the most common types of mature B cell lymphoid neo- phenotyping is used to establish a diagnosis of HCL. HCL plasm within the United States, second only to diffuse cells are mature B lymphocytes that are positive for CD19, large B cell lymphoma. Patients usually present with gen- CD20 (strong intensity), CD22, CD25, CD103, and CD11c. eralized painless lymphadenopathy, most have advanced There is usually strong-intensity monoclonal surface stage disease (Figure 28-4a) with bone marrow involve- immunoglobulin present. Molecular diagnostics reveal the ment (Figure 28-4a), and a few have peripheral blood BRAF V600E mutation in the vast majority of cases (nearly involvement (Figure 28-4b). Involvement of other extra- 100%) and is generally recognized as the causal genetic nodal sites is unusual. event of HCL.4 LYMPH NODE BIOPSY BONE MARROW Lymph node biopsy of FL reveals an infiltrate of lymphoid Bone marrow involvement is often diffuse, and the neoplas- cells forming poorly circumscribed nodules that resemble tic cells are usually surrounded by fibrosis, preventing their follicular germinal centers (Figure 28-4c). In addition, the aspiration from the bone marrow (resulting in a “dry tap”). lymphoma cells grow in a diffuse pattern in some areas. Bone marrow biopsy sections reveal a monotonous infil- Neoplastic follicles differ from reactive follicles in that trate of abnormal lymphocytes with small nuclei and abun- neoplastic follicles lack evidence of lymphocyte apoptosis. dant pale-staining cytoplasm (“fried egg” appearance)5 This is manifest on histologic sections as a lack of macro- (Figure 28-3b). phages engulfing fragments of dead cells (tingible body macrophages), which are usually found in areas of exten- TISSUE INVOLVEMENT sive apoptosis (reactive germinal centers and high-grade Splenectomy specimens reveal marked expansion of the red lymphoma). The neoplastic infiltrate always contains a pulp because of an infiltrate of abnormal cells with the fried mixed population of small cleaved (centrocytes) and large egg appearance. Lakes of erythrocytes are often formed cells (centroblasts) but is often more homogeneous than a between the tumor cells (pseudosinuses). Immunohisto- normal germinal center (Figure 28-4d). The proportion of chemical stains for the B cell antigen CD20 can be used to large cells varies and is used to separate FL into three grades highlight the infiltrate. Other features characteristic of HCL (1, 2, or 3). include Annexin A1 and CD123 staining and BRAF V600E In situ follicular neoplasia (ISFN) is a term used for non- mutation.4,5 HCL is an indolent disease. Long-lasting com- neoplastic follicles colonized by follicular lymphoma cells. plete remissions are often obtained with the chemotherapy ISFN exhibits a low-rate progression, but it may be associ- agent 2-chlorodeoxyadenosine (2-CDA/cladribine), while ated with lymphoma elsewhere and therefore, it requires more recently the use of BRAF inhibitors and immunother- additional clinical assessment.1 FL cells in the peripheral apy (anti-CD20 monoclonal antibody) are used for long- blood and bone marrow usually have very irregular nuclear lasting clinical responses. outlines and a deep indentation (cleft) of the nuclear mem- brane (“buttock” or “butt” cells; Figure 28-4b). Checkpoint 28.3 IMMUNOPHENOTYPING AND CYTOGENETICS Chronic lymphoid malignancies compose a heterogeneous Conventional histologic evaluation is usually insufficient group. What characteristics allow these malignancies to be to establish a diagnosis of FL. Immunophenotyping can grouped together? be used to confirm the presence of lymphoma (monoclo- nal lymphocytes) and confirm the subtype of lymphoma 1CD10+2. Most FL cases arise because of a chromosome CASE STUDY (continued from page 624) translocation t(14;18) involving the BCL-2 gene that leads Julia’s CBC revealed a WBC count of 20 * 103/mcL to overexpression of BCL-2 protein in lymphocytes. with 60% lymphocytes. Examination of the periph- BCL-2 protein inhibits individual cell death (apoptosis), eral blood revealed mature lymphocytes with allowing follicle center cells to accumulate and produce scant cytoplasm, clumped chromatin, and irregular lymphadenopathy. nuclear outlines. PROGNOSIS AND TREATMENT 1. What is the differential diagnosis? Most patients with FL have an indolent |
disease with a median survival of 7–9 years. Patients with grade 3 FL 2. What studies could be performed to establish the are sometimes cured. However, the lower-grade FLs diagnosis? (grades 1 and 2) are incurable with current therapies and are therefore treated for relief of symptoms. Duodenal 630 Chapter 28 a b c d Figure 28.4 Follicular lymphoma, grade 1. (a) Bone marrow involvement by low grade follicular lymphoma displaying a characteristic paratrabecular growth pattern (bone marrow biopsy, H&E stain, 20* magnification). (b) Circulating lymphoma cells with irregular nuclear outlines (peripheral blood, Wright stain, 1000* magnification). (c) Loss of the normal lymph node architecture. The abnormal infiltrate forms numerous poorly defined nodules (follicles) (lymph node biopsy, H&E stain, 20* magnification). (d) Neoplastic follicle composed of a relatively homogeneous population of small lymphoid cells containing angulated, twisted nuclei (lymph node biopsy, H&E stain, 200* magnification). follicular lymphoma and pediatric-type follicular lym- CASE STUDY (continued from page 629) phoma are two recently recognized subtypes that are Julia’s cervical lymph node biopsy reveals efface- often localized and may not require systemic treatment ment of the normal architecture by a lymphoid other than local excision.1 Low-grade FL can progress to infiltrate with a nodular growth pattern. The a diffuse large cell lymphoma with a median survival of nodules contain a relatively homogeneous popu- less than 1 year. lation of small lymphocytes and a few admixed large cells. The infiltrating lymphoid cells have Mantle Cell Lymphoma (MCL) irregular nuclear outlines. Tingible body macro- phages are lacking, and very few mitotic figures Mantle cell lymphoma usually presents with disseminated are present. disease involving multiple lymph node groups, bone mar- row, peripheral blood, spleen, liver, and gastrointestinal 3. What is the cause of the lymphadenopathy? Is tract. Gastrointestinal tract involvement can present as this process low grade or high grade? multiple polyps involving the small bowel (lymphomatous polyposis; Figure 28-5a). Mature Lymphoid Neoplasms 631 a b c d Figure 28.5 Mantle cell lymphoma. (a), (b) Abnormal lymphoid infiltrate in a lymph node with a uniform infiltrate of small lymphoid cells with slightly irregular nuclear outlines (lymph node biopsy, H&E stain, (a) 200* , (b) 500* magnification). (c) Cyclin D1 nuclear overexpression (lymph node biopsy, immunohistochemistry stain, 500* magnification). (d) SOX11 nuclear overexpression (lymph node biopsy, immunohistochemical stain, 500* magnification). PERIPHERAL BLOOD IMMUNOPHENOTYPING AND CYTOGENETICS MCL is a B cell lymphoma composed of small- to medium- Establishing an MCL diagnosis requires the use of ancillary sized lymphoid cells with irregular nuclear contours studies. The following immunophenotype is characteris- (Figure 28-5). MCL is associated with a t(11;14)(q13;q32) tic of MCL: CD19+ , CD5+ , CD23- , FMC-7+ , and sIg+ IgH/CCND1 translocation resulting in overexpression of (strong intensity), and almost all are positive for cyclin D1 Cyclin D1, a regulatory protein participating in progression and SOX11 by immunohistochemistry6 (Figure 28-5d). The of cells from the G1 to S phase of the cell cycle (Chapter 2). IgH/CCND1 translocation can be demonstrated by FISH An indolent clinical variant is the leukemic, non-nodal studies in the vast majority of cases of MCL. MCL, which involves peripheral blood, bone marrow, PROGNOSIS and spleen without lymph node involvement. Blastoid MCL is a relatively aggressive lymphoma with a poor and pleomorphic MCL (composed of blast-like or large response to current therapies. However, recent findings cells, respectively) represent more aggressive lymphomas regarding the pathophysiology of the disease may help and usually carry additional molecular and cytogenetic in the development of new therapeutic approaches.1 Cur- abnormalities. rently, the overall median survival is approximately 3–4 LYMPH NODE BIOPSY years. Therefore, distinction of MCL from the other small On histologic sections, MCL can demonstrate either a dif- lymphoid B cell neoplasms (follicular lymphoma, SLL, and fuse or vaguely nodular growth pattern (Figure 28-5b). The MALT lymphoma [see “Extranodal Marginal Zone Lym- mantle zone pattern is characterized by a neoplastic infil- phoma of Mucosa-Associated Lymphoid Tissue” below]) trate surrounding reactive germinal centers (Figure 28-5c). is important. 632 Chapter 28 Extranodal Marginal Zone immunoglobulin on lymphocytes) or paraffin section Lymphoma of Mucosa-Associated immunohistochemistry (cytoplasmic immunoglobulin in cells demonstrating plasma cell differentiation). In contrast Lymphoid Tissue to FL and small lymphocytic lymphoma, MALT lymphoma Patients with extranodal marginal zone lymphoma of is usually negative for CD10 and CD5. mucosa-associated lymphoid tissue (MALT) usually pres- TREATMENT ent with localized extranodal disease (e.g., involving the MALT lymphoma is an indolent disease, though it has the stomach, salivary gland, lacrimal gland, thyroid, lung). A potential to transform to higher-grade, large B cell lym- preceding chronic inflammatory disorder such as chronic phoma in a subset of patients. Although, patients with gastritis from Helicobacter pylori infection or autoimmune H. pylori–associated gastric MALT lymphoma are often disease (Sjögren’s syndrome or Hashimoto’s thyroiditis) cured with antimicrobial therapy, the polymerase chain often occurs. reaction (PCR) assay for a monoclonal B cell population can LYMPH NODE BIOPSY remain positive after complete remission. MALT lymphoma A biopsy of MALT lymphoma (Figure 28-6a) reveals an infil- that is unresponsive to antimicrobial therapy or that occurs trate of small to intermediate size lymphocytes intimately at other sites is often treated with local excision or radiation associated with epithelial cells (e.g., gastric mucosa, salivary therapy. gland ducts). Epithelial structures infiltrated by neoplastic lymphocytes are referred to as lymphoepithelial lesions Lymphoplasmacytic Lymphoma (Figure 28-6a). MALT lymphoma is composed predomi- Lymphoplasmacytic lymphoma (LPL) is a neoplasm that nantly of small lymphocytes with round or slightly cleaved primarily involves the bone marrow and sometimes the nuclei that often have abundant pale-staining cytoplasm spleen and lymph nodes and is composed of a mixture of and are referred to as monocytoid B cells because of their neoplastic small lymphocytes and plasma cells. LPL must resemblance to monocytes (Figure 28-6b). MALT lymphoma be distinguished from small B cell lymphoid neoplasms, can contain admixed neoplastic plasma cells. In addition other types of lymphoma that can have plasmacytic dif- to the neoplastic cells, the infiltrate often contains benign ferentiation, such as MALT lymphoma, and neoplasms germinal centers. Infiltration of benign germinal centers by composed entirely of plasma cells (plasma cell neoplasms). neoplastic cells is referred to as follicular colonization. Unlike those with plasma cell neoplasm multiple myeloma, IMMUNOPHENOTYPING patients with LPL lack lytic bone lesions. The neoplas- The differential diagnosis includes other lymphomas tic plasma cells in LPL often secrete monotypic immuno- composed of small lymphocytes, including SLL, MCL, globulin, leading to a paraprotein, often of IgM type. High and FL. Ancillary studies can assist in obtaining the cor- plasma levels of the large pentamer IgM result in hypervis- rect diagnosis. Restriction of immunoglobulin light chains cosity that can lead to poor circulation in small blood ves- can be demonstrated by immunophenotyping (surface sels, visual impairment, headache, dizziness, and deafness. a b Figure 28.6 Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). (a) Parotid salivary gland involved by MALT lymphoma. Two lymphoepithelial lesions are present demonstrating infiltration of ducts by neoplastic lymphoid cells (parotid gland biopsy, H&E stain, 50* magnification). (b) Lymphoid infiltrate of MALT lymphoma. Many of the neoplastic cells have abundant pale- staining cytoplasm (i.e., a “monocytoid” appearance) (parotid gland biopsy, H&E stain, 100* magnification). Mature Lymphoid Neoplasms 633 Waldenström macroglobulinemia is the combination of transformation from a lower-grade lymphoma (e.g., follicu- LPL with bone marrow involvement and an IgM monoclo- lar lymphoma or small lymphocytic lymphoma), whereas nal paraprotein. others arise de novo. Though several histologic variants are recognized, morphologic and immunophenotypic features LYMPH NODE BIOPSY have limited value in predicting prognosis in DLCL. Histologic sections usually demonstrate a diffuse infiltrate composed of lymphocytes, plasma cells, and lymphoid cells GENOMIC PROFILING AND IMMUNOHISTOCHEMISTRY that demonstrate some evidence of differentiation toward Gene expression profiling has divided DLBCL into germi- plasma cells (plasmacytoid lymphocytes). Immunohisto- nal center B cell–like (GCB) and activated B cell–like (ABC) chemistry can be performed to demonstrate the presence subgroups. These subgroups differ in their chromosomal of cytoplasmic monoclonal immunoglobulin light chain (k alterations, molecular pathways and clinical outcome, with or l) and IgM heavy chain. the ABC type DLBCL associated with a worse outcome then the GCB type. Since genomic profiling is difficult to MOLECULAR EVALUATION perform, a simple immunohistochemical approach using While no specific chromosomal abnormalities are recog- antibodies to CD10, BCL6, and IRF4/MUM1 (so-called nized in LPL, about 90% of LPL or Waldenstrom macroglob- Haas algorithm) provide a useful surrogate to differentiate ulinemia have MYD88 L265P mutations. This mutation is between ABC and GCB.8 also found in a significant proportion of IgM but not IgG or IgA monoclonal gammopathy of undetermined significance MOLECULAR PROGNOSTICS (MGUS), and in certain diffuse large B cell lymphomas, but MYC gene alterations play an important role in the prog- not in plasma cell myeloma.7 nosis of DLBCL. The MYC gene is rearranged in 5-15% of DLBCL and is frequently associated with BCL2 or, less fre- TREATMENT quently, with BCL6 (so-called “double-hit” or “triple-hit” Treatment of symptomatic patients includes rituximab, lymphomas).9 These lymphomas have been recently recog- which can be used in combination with chemotherapy. nized as a separate entity designated as “High-grade B cell Autologous bone marrow transplant is a recent treatment lymphoma with rearrangement of MYC and BCL2 and/or option. Plasmapheresis to treat hyperviscosity is used to BCL6.”1 Additional important prognostic markers are the reduce paraprotein levels. The median survival for patients expression of MYC and BCL2 proteins by the lymphoma, with LPL is typically 5–10 years. which are commonly expressed without the presence of a translocation. “Double expressor” lymphomas (MYC Diffuse Large B Cell Lymphoma and BCL2 positive by immunohistochemistry) are also Diffuse large B cell lymphoma (DLBCL) is a heteroge- associated with a worse outcome, though not as aggres- neous group of tumors composed of large B lymphoid sive as the “double-hit” or “triple-hit” DLBCL.10 Finally, cells (Figure 28-7). Some DLBCLs develop as a result of the importance of Epstein-Barr virus in the pathogenesis and prognosis is acknowledged by recent introduction of the category “EBV-positive DLBCL.”11 With multi-agent chemotherapy and anti-CD20 monoclonal antibody ther- apy (rituximab), the long-term remission rate of DLBCL is 50–60%. CASE STUDY (continued from page 630) The previous biopsy had established a diagnosis of low-grade NHL, follicular type. Julia received multi-agent chemotherapy for symptomatic relief. Two years following the diagnosis, she returned with rapidly expanding lymph nodes in her neck. Repeat biopsy revealed effacement of the lymph node architecture by a diffuse infiltrate of large B cells. Figure 28.7 Diffuse large B cell lymphoma. Abnormal infiltrate 5. What is the diagnosis? composed of large lymphoid cells with pale-staining, vesicular 6. What is the relationship of this disease to the pre- chromatin, irregular nuclear outlines, and basophilic nucleoli. Several vious diagnosis? mitotic figures are present (lymph node biopsy, H&E stain, 200* magnification). 634 Chapter 28 Burkitt Lymphoma lymphoblastic leukemia/lymphoma and DLBCL. Most cases of Burkitt lymphoma have an isolated chromosome Burkitt lymphoma is a high-grade NHL with a high inci- translocation leading to rearrangement of the MYC and IGH dence in Africa (endemic subtype). It represents approxi- genes, typically t(8;14) or less frequently t(2;8) or t(8;22). mately one-third of all pediatric lymphomas occurring MYC gene rearrangement occurs in only 5% of DLBCL. This outside Africa (sporadic Burkitt lymphoma). Many adult distinction is important because the therapeutic regimen cases occur in immunocompromised individuals such as used for Burkitt lymphoma usually differs from that used those infected with the HIV virus. Burkitt lymphoma often for DLBCL. With the appropriate aggressive multiagent involves extranodal sites. Endemic Burkitt lymphoma has chemotherapy, Burkitt lymphoma is potentially curable. a predilection for involvement of the facial bones and jaw; sporadic Burkitt lymphoma often presents with disease involving the intestine, ovaries, or kidney. EBV is thought Plasma Cell Neoplasms to play a role in the pathophysiology of Burkitt lymphoma. The plasma cell neoplasms are considered to be a group of DNA of EBV is present in most cases of endemic Burkitt lym- diseases composed of immunoglobulin-secreting cells in the phoma and approximately one-third of the HIV-associated absence of neoplastic B lymphocytes. It is important to dis- tumors. EBV is found less frequently in the sporadic form. tinguish this group of neoplasms from the mature lymphoid neoplasms that can demonstrate plasmacytic differentiation LYMPH NODE BIOPSY (e.g., LPL). A biopsy of Burkitt lymphoma usually reveals a diffuse infil- Serum or urine protein electrophoresis (SPEP or UPEP) trate of neoplastic |
cells demonstrating a “starry sky” appear- reveal increased protein with a narrow range of electropho- ance (Figure 28-8). The “sky” represents the blue nuclei of the retic mobility (M spike) (Figure 28-9a, b). The M spike can neoplastic lymphocytes; the “stars” are formed by scattered be further characterized by immunofixation electrophoresis pale-staining tingible body macrophages. The infiltrating (IFE) to confirm the presence of a monoclonal protein and lymphoid cells are intermediate in size with nuclei approxi- determine the immunoglobulin class (Figure 28-9c). The mately the same size as those of the tingible body macro- monoclonal protein usually contains one immunoglobulin phages. Multiple small nucleoli are usually present, and light chain (k or l) and one heavy chain with the follow- mitotic figures and apoptotic bodies are frequent. The latter ing incidence: IgG 7 IgA 7 IgM 7 IgD 7 IgE. Normal two features are characteristic of high-grade lymphoma. immunoglobulin production is usually decreased, leading IMMUNOPHENOTYPING AND MOLECULAR to functional hypogammaglobulinemia. Plasma cell neo- EVALUATION plasms can produce an excess of immunoglobulin light Burkitt lymphoma exhibits a germinal center B cell-type chains, light chains only, heavy chains only, or no immu- immunophenotype: CD19+ , sIg+ , CD10+ , CD5- . The noglobulin (nonsecretory). Patients with light chain only differential diagnosis for Burkitt lymphoma includes B disease can have a normal SPEP because the protein passes into the urine. The presence of free immunoglobulin light chains in the urine is referred to as Bence-Jones proteinuria. The plasma cell neoplasms can be divided into disease entities based on the distribution and extent of disease (soli- tary versus multiple lesions and bone versus extraosseous) and the characteristics of the immunosecretory protein produced (class of heavy chain and the presence of amy- loid production, immunoglobulin heavy, or light chain; Table 28-3). PLASMA CELL MYELOMA Plasma cell myeloma is a bone marrow based, multifocal plasma cell neoplasm associated with an M protein in serum and/or urine. Several clinical variants range from asymp- tomatic to an aggressive disease that is identified on the presentation, radiologic findings, and laboratory informa- Figure 28.8 Burkitt lymphoma. Intermediate size lymphocytes tion. Symptomatic plasma cell myeloma is characterized by an with multiple nucleoli and scant cytoplasm. Numerous mitotic figures M protein in the urine or serum, clonal plasma cells in the are present. The presence of apoptotic bodies indicates individual bone marrow whose symptoms are hypercalcemia, renal cell necrosis. There is a “starry-sky” appearance because of pale- staining tingible body macrophages scattered in an infiltrate that insufficiency, anemia, lytic bone lesions (CRAB), and evi- appears basophilic because of staining of the tumor cell nuclei dence of related organ or tissue impairment. Patients often (lymph node biopsy, H&E stain, 100* magnification). present with bone pain and/or pathologic fractures because Mature Lymphoid Neoplasms 635 9/10/99 ABN 1392 Monoclonal spike 1681 Albumin 1685 Monoclonal serum protein Normal control 723 a1 a2 b g a b Free k light chains IgG k SPEP IgG IgA IgM K l c Figure 28.9 Monoclonal gammopathy. (a) Serum protein electrophoresis. Sample 1681 displays a band in the early gamma region. (b) Densitometry scan reveals a “spike” in the gamma region. A similar spike was found in the urine. (c) Immunofixation electrophoresis performed on a urine sample reveals two monoclonal bands composed of IgG kappa and free kappa light chains (Bence-Jones proteins). of tumor infiltration. Asymptomatic (smoldering) myeloma is replacing the normal hematopoietic cells (Figure 28-10a, b). characterized by M protein in serum at a level more than Neoplastic plasma cells vary from normal to abnormal forms 30 g/L and/or 10% or more of clonal plasma cells in the bone with more finely distributed chromatin or nucleoli. Intra- marrow but no organ or tissue impairment or symptoms. nuclear inclusions composed of immunoglobulin (Dutcher bodies; Figure 28-11a) or red-tinged cytoplasm (flame cells; Cytogenetics Cytogenetic evaluation by FISH distin- Figure 28-11b). Intracytoplasmic inclusions composed of guishes two genetic subtypes based on chromosome immunoglobulin can occur as single inclusions (Russell content: hyperdiploid and nonhyperdiploid.12 The hyper- body) or multiple inclusions (Mott cell; F igure 28-11c), they diploid group includes trisomies of chromosomes 3, 5, 7, 9, can also be seen in reactive plasma cells. Neoplastic plasma 11, 15, 19, and 21. The nonhyperdiploid group is character- cells rarely circulate in the blood, but the peripheral smear ized by reciprocal translocations, deletions, and complex is often abnormal because of stacking of the erythrocytes karyotypes. The most frequent translocation involves the (rouleaux formation; Figure 28-11d; Chapter 10). immunoglobulin heavy chain variable region on chromo- some 14q32 and involves cyclin D1. MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS) Peripheral Blood and Bone Marrow Examination of the Monoclonal gammopathy of undetermined significance bone marrow reveals an abnormal infiltrate of plasma cells (MGUS) is used to describe a low level of serum monoclonal g b a2 a1 Albumin 636 Chapter 28 for symptomatic plasma cell myeloma. In contrast, plasmacy- Table 28.3 Key Features of Selected Plasma Cell toma of extraosseous locations, such as the upper respiratory Neoplasms tract, have a good prognosis with only rare recurrence after Neoplasm Features excision and a low incidence of dissemination. Sometimes, Symptomatic plasma M spike in serum or urine distinguishing extraosseous plasmacytoma from MALT lym- cell myeloma Clonal plasma cells in bone marrow phoma with plasmacytic differentiation is difficult. Related organ or tissue damage (CRAB) Patients under 65 years of age can be treated with high- Asymptomatic plasma M spike in serum or urine (more than 30 g/L) dose chemotherapy followed by autologous or allogeneic cell myeloma Clonal plasma cells in bone marrow greater stem cell transplant. Those over the age of 65 can be treated than 10% with chemotherapy (melphalan) and prednisone. The prog- No related organ or tissue damage nosis of symptomatic plasma cell myeloma is poor with a Monoclonal gammop- M spike in serum or urine (less than 30 g/L) athy of undetermined median survival of 3–4 years. Patients with asymptomatic Clonal plasma cells in bone marrow 610, significance (MGUS) plasma cell myeloma can have stable disease for many years No related organ or tissue damage but often progress to symptomatic myeloma. Plasmacytoma Solitary collection of clonal plasma cells Can have M spike No clonal plasma cells elsewhere in bone marrow No related organ or tissue damage Mature T and NK Cell CRAB, hypercalcemia renal insufficiency, anemia, bone lesions. Neoplasms T cell and NK cell lymphomas are less common than B cell protein without evidence of an overt neoplasm. Therefore, lymphomas, as they comprise only 10–15% of NHLs in MGUS is a diagnosis that requires exclusion of other plasma the United States and Western Europe. Despite their low cell and lymphoid malignancies. The prevalence of a mono- prevalence, they represent a highly heterogeneous group clonal serum spike increases with age and is present in 3% of diseases. T cell lymphoma and systemic anaplastic large of asymptomatic patients over 70 years of age. There is no cell lymphoma arise in peripheral lymphoid organs (mainly treatment for MGUS. Although most patients have stable lymph nodes) and resemble CD4+ effector T cells. Other, disease and die of other causes, MGUS can progress to an less common T cell lymphomas (hepatosplenic T cell lym- overt plasma cell neoplasm. phoma, subcutaneous panniculitis-like T cell lymphoma PLASMACYTOMA and enteropathy-associated T cell lymphoma) are related A plasmacytoma is a localized, tumorous collection of clonal to the innate immune system and have a tendency to arise plasma cells. The prognosis of a plasmacytoma is related to its in mucosal or cutaneous sites, more commonly express location. Many patients with plasmacytoma of bone develop gamma/delta T cell receptors, and cytotoxic granule- additional plasmacytomas and ultimately meet the criteria associated proteins. Cytogenetic translocations, specific a b Figure 28.10 Symptomatic plasma cell myeloma. (a) Skull x-ray demonstrating multiple lytic bone lesions giving a “moth-eaten” appearance. (b) Replacement of bone marrow hematopoietic precursors by an infiltrate of plasma cells (bone marrow aspirate, Wright stain, 1000* magnification). Mature Lymphoid Neoplasms 637 a b c d Figure 28.11 Symptomatic plasma cell myeloma. (a) Plasma cell with intranuclear inclusion indicated with arrow (Dutcher body) (H&E stain, 1000* magnification). (b) Many plasma cells with a red tinge to the cytoplasm imparting a flame-cell appearance (bone marrow, Wright stain, 1000* magnification). (c) Plasma cell with multiple cytoplasmic inclusions (Mott cell) (bone marrow, Wright stain, 1000* magnification). (d) Stacking of peripheral blood erythrocytes resulting from the presence of increased immunoglobulin (Rouleaux) (peripheral blood, Wright stain; 1000* magnification). cytogenetic abnormalities, and disease-defining mutations classified as peripheral T cell lymphoma, not otherwise specified are uncommon in T and NK cell lymphomas. The WHO (PTCL NOS). The morphologic appearance of PTCL, NOS classification divides the mature T and NK cell neoplasms is heterogeneous with the neoplastic cells often exhibiting based on their primary clinical presentation into nodal, abundant clear or pale-staining cytoplasm and irregular extranodal and leukemic categories. Association with nuclear outlines. Histiocytes, plasma cells, and eosinophils Epstein-Barr virus, immunophenotype (CD4+ versus are admixed with the neoplastic cells. Immunophenotyping CD8+ ) and the presence of cytotoxic or NK-associated demonstrates expression of pan–T cell antigens. The pres- markers also play a role in the classification. ence of an abnormal immunophenotype (CD5- or CD7- ) or clonal rearrangement of the T cell receptor can assist in Nodal T and NK Cell Lymphomas the distinction of T cell lymphoma from a reactive process. Nodal cases usually show a CD4+/CD8- phenotype. Cyto- PERIPHERAL T CELL LYMPHOMA, NOT OTHERWISE genetics usually reveals a complex karyotype. PTCL, NOS SPECIFIED (NOS) usually presents with peripheral lymph node involvement A large category of peripheral T cell lymphoma (30% in though bone marrow, spleen, and liver can be also involved. Western countries) cannot be placed into one of the other In general, peripheral T cell lymphoma is an aggressive dis- subtypes in the current classification. This category is ease with a poor response to therapy and frequent relapses. 638 Chapter 28 Combination chemotherapy and stem cell transplantation systemic multiagent chemotherapy, but the rate of relapse is offer cure in a subset of patients. high. Brentuximab-vedotin is a recently emerged anti-CD30 antibody-drug conjugate that has shown great success in ANGIOIMMUNOBLASTIC T CELL LYMPHOMA (AITL) the treatment of ALCL and other CD30+ lymphomas.13 Angioimmunoblastic T cell lymphoma (AITL) presents with Systemic ALCL, ALK negative is a lymphoma morphologi- generalized lymphadenopathy, hepatosplenomegaly, ane- cally and immunophenotyically similar to ALCL, ALK+ , but mia and hypergammaglobulinemia. Lymph node biopsy lacks ALK translocations and ALK protein expression. The shows effacement of the paracortex by a polymorphic patients are typically older and clinically have a worse prog- inflammatory infiltrate, frequent large immunoblasts and nosis than ALCL, though still better prognosis than PTCL, a marked vascular proliferation. Residual B cell follicles are NOS. Other subtypes of ALK negative ALCL include primary often present and irregular proliferation of follicular den- cutaneous ALCL and a recently introduced category of ALCL dritic cells is typical. It has been recently recognized that the associated with breast implant.1 Both of these entities resemble neoplastic cells in AITL may be derived from intrafollicular systemic ALCL morphologically and immunophenotypically, T cells; they express a number of T follicular cell markers, but they are usually localized to their respective extranodal including CD10, CXCL13, and CD270 (PD-1). Virtually all location and show an indolent course. cases of AITL contain increased numbers of EBV-infected B cells. A clonal T cell population is usually present and in a subset of cases, a B cell clone also may be detected. Extranodal T and NK Cell ANAPLASTIC LARGE CELL LYMPHOMA (ALCL) Lymphomas One of the best-characterized subtypes of T/NK cell lym- HEPATOSPLENIC T CELL LYMPHOMA (HSTCL) phoma is anaplastic large cell lymphoma (ALCL). ALCL is HSTCL a rare extranodal lymphoma that arises from the composed of large bizarre anaplastic cells with horseshoe or cytotoxic g/d or a/b T cells of the splenic red pulp. Most wreath-like appearance (“hallmark” cells; Figure 28-12a). cases are young adult males who present with massive ALCL is positive for the CD30 antigen, may express pan-T hepatosplenomegaly, anemia, thrombocytopenia but no cell antigens such as CD2 and CD3 and frequently, cytotoxic lymphadenopathy. A subset of cases arise after solid organ granule proteins. Although ALCL is derived from T cells, transplantation or in the setting of treatment of inflamma- frequent loss of T cell antigen expression can result in a tory bowel disease. This lymphoma |
infiltrates cords and null phenotype (lack of expression of either T or B cell anti- sinuses of the splenic red pulp, hepatic sinusoids and bone gens). Most ALCL cases involving lymph nodes contain the marrow sinuses. The characteristic immunophenotype is translocation t(2;5) that joins the nucleophosmin (NPM1) CD2+ , CD3+ , CD4- , CD5- , CD7+ and CD8+ . Most cases and anaplastic large-cell kinase (ALK) genes. This leads to exhibit g/d T cell receptors (TCR), though a minor subset is abnormal expression of ALK protein that can be detected a/b TCR positive. Karyotypic studies often show isochro- by immunohistochemistry (Figure 28-12b). Approximately mosome 7q. The disease is aggressive, and most patients 70% of patients with systemic ALCL go into remission with die within two years. a b Figure 28.12 Anaplastic large cell lymphoma. (a) Abnormal infiltrate of large cells with eosinophilic cytoplasm, including cells with multilobated nuclei (lymph node biopsy, H&E stain, 100* magnification). (b) ALK expression (lymph node biopsy, immunohistochemical stain, 200* magnification). Mature Lymphoid Neoplasms 639 EXTRANODAL NK/T CELL LYMPHOMA, NASAL TYPE T CELL LARGE GRANULAR LYMPHOCYTIC (T-LGL) Nasal NK/T cell lymphomas are rare in the United States and LEUKEMIA they are most frequent in East Asia and among the Native T-LGL leukemia often presents with a modest lymphocytosis American populations of Mexico and Central and South composed of cells with abundant pale-staining cytoplasm, America. This lymphoma is aggressive with an angiocentric azurophilic cytoplasmic granules, and nuclei with mature and angiodestructive growth producing areas of geographic clumped chromatin. Large granular lymphocytes are normal necrosis. Most of these lymphomas are EBV-associated and components of the peripheral blood and represent NK-like represent either true NK cell lymphomas or cytotoxic T cell T cells (CD3+ ) or NK cells (CD3- ) (Chapter 8). T-LGL leu- lymphomas. Expression of the NK cell associated antigen kemia cells show the following phenotype: CD2+ , CD3+ , CD56 is usually present. Non-nasal presentation can occur CD4- , CD5- , CD7+ , CD8+ , CD16+ , CD56+/- , and in skin, soft tissue, gastrointestinal tract and testis. CD57+/- . Loss of pan T cell antigens and demonstration of clonal T cell receptor rearrangement supports the diagnosis of SUBCUTANEOUS PANNICULITIS-LIKE T CELL a lymphoma as opposed to a reactive proliferation. In addition LYMPHOMA to lymphocytosis, anemia, neutropenia, and thrombocytope- This rare subtype of T cell lymphoma presents as multiple nia are common features. Although splenomegaly is com- erythematous subcutaneous nodules on the legs, trunk or mon, lymphadenopathy and hepatomegaly are uncommon. both. This lymphoma tends to remain localized to the sub- Approximately 25% of patients with T-LGL leukemia have cutaneous tissue, but may be complicated by a severe and symptomatic rheumatoid arthritis. The triad of rheumatoid often fatal hemophagocytic syndrome. Histologically the arthritis, splenomegaly, and neutropenia defines Felty’s syn- lymphoma exhibits a growth pattern similar to the inflam- drome. Patients with T-LGL leukemia usually have an indo- matory condition panniculitis. Lymphoma cells are usually lent course with more than 80% overall survival after 10 years. CD8 positive, a/b TCR positive and express cytotoxic gran- ule proteins. ADULT T CELL LEUKEMIA/LYMPHOMA (ATLL) ATLL is a T cell neoplasm caused by Human T cell leukemia ENTEROPATHY-ASSOCIATED T CELL virus-1 (HTLV-1) and it is more prevalent where the ret- LYMPHOMA (EATL) rovirus is endemic, in Southwestern Japan, Central Africa, EATL is a rare aggressive lymphoma that arises from the and the Caribbean basin. The disease involves peripheral intraepithelial lymphocytes of the small intestine as a blood, bone marrow, lymph nodes, and skin. Circulating long-term complication of celiac disease. Patients present lymphoma cells have hyperlobated, cloverleaf-like nuclei. with abdominal pain and weight loss. Acute obstruction The neoplastic cells are CD4+ and express CD25 and FoxP3, or perforation is less common. Histologically the tumors a phenotype characteristic of regulatory T cells. ATLL exhib- vary from proliferation of abnormal intraepithelial lym- its clonal T cell gene rearrangements and clonal integration phocytes to single or multiple tumor masses along the of the HTLV-1 genome. small intestine. The tumor cells are pleomorphic and show brisk mitotic activity. The phenotype is often CD2+ , CD3+ , SÉZARY SYNDROME CD4- , CD5- , CD7+ , CD8 and a/b TCR positive. Many Sézary syndrome, the leukemic presentation of cutane- cases demonstrate a clonal T cell gene rearrangement. ous T cell lymphoma, is defined by the combination of erythroderma (red skin), generalized lymphadenopathy, Leukemic T and NK Cell and neoplastic T cells (Sézary cells) in the skin, lymph Lymphomas nodes, and peripheral blood. Sézary cells, the neoplastic T lymphocytes, have irregular, convoluted (cerebriform) T CELL PROLYMPHOCYTIC LEUKEMIA (T-PLL) nuclear outlines (Figure 28-13). Abnormal cells can be T-PLL is a rare leukemic disorder of adults that presents counted in a blood smear leukocyte differential to deter- with marked lymphocytosis and splenomegaly. Lymph- mine a Sézary count; an absolute count of greater than adenopathy, hepatomegaly, and skin infiltration may be 1000 cells/mm3 (1 * 103/mcL) is required for diagnosis. present. The lymphoma cells are small or medium in size Sézary cells are mature helper T cells (CD3+ , CD4+ ) but and can have convoluted nuclear outlines and a prominent usually lack expression of CD7. The presence of a CD4:CD8 nucleolus. T-PLL has a mature phenotype with expression ratio of greater than 10, aberrant antigen expression by flow of CD3, CD2, CD5, and CD7. Most cases are CD4+ , though cytometry, and a clonal T cell receptor gene rearrangement some cases express both CD4 and CD8. The most frequent support the diagnosis. Sézary syndrome is closely related to cytogenetic abnormalities include inv(14)q11q32, del(11q), the primary cutaneous T cell lymphoma mycosis fungoides i(8q), and trisomy 8q. T-PLL is an aggressive disorder with (MF). MF is usually characterized by cutaneous patches and a median survival time of only 7.5 months. A subset of cases plaques, rather than diffuse erythroderma, and has less fre- shows a good clinical response to anti-CD52 monoclonal quent blood involvement. Treatment is usually palliative to antibody therapy. relieve symptoms and improve the quality of life. Clinical 640 Chapter 28 Table 28.4 Classification of Hodgkin Lymphoma Subtype Sclerosis Lymphocytes Tumor Cells Variants NLPHL - + + + + + L&H CHL NS Present + + + + Lacunar MC - + + + + - LR - + + + + + - LD -/+ + + + + + - NS, nodular sclerosis classical HL; MC, mixed cellularity classical HL; LD, lymphocyte depletion classical HL; LR, lymphocyte-rich classic HL; LP, lymphocyte-predominant HL; L&H, malignant cells characteristic of LP HL; + , few; + + + + , many. The reactive small lymphocytes that surround the neoplas- tic cells of NLPHL often include an unusual subset of CD57 positive T cells. Figure 28.13 Sézary syndrome. Abnormal large lymphocytes with relatively finely distributed chromatin and numerous nuclear folds (Sézary cell) (peripheral blood, H&E stain, 1000* Classical Hodgkin Lymphoma (CHL) magnification). The tumor cells in classical Hodgkin lymphoma (CHL) have large prominent eosinophilic nucleoli and coarse nuclear trials with ultraviolet B radiation and high-dose chemo- membranes. Reed-Sternberg (R-S) cells have two or more therapy and radiation with stem cell transplant are ongoing. nuclear lobes containing inclusion-like nucleoli and an area of perinucleolar clearing imparting an owl’s-eye appear- ance (Figure 28-15a). The reactive cells accompanying the Hodgkin Lymphoma (HL) neoplastic cells of classical HL include a heterogeneous Hodgkin lymphoma is a lymph node based neoplasm com- mixture of small lymphocytes, histiocytes, eosinophils, and posed of a small number of large mononucleated or mul- plasma cells. The tumor cells of classical HL represent acti- tinucleated neoplastic cells residing in a heterogeneous vated post-germinal center B lymphocytes. They express non-neoplastic cell infiltrate. HL is classified into two main CD30 and PAX5 and express CD15 in 75–80% of cases. They diseases, namely, nodular lymphocyte predominant HL may variably express other B cell markers CD20 or CD79a, (NLPHL) and Classical HL (CHL). Separation of these two but strong uniform positivity for these markers is not char- broad categories of lymphoma is important because they acteristic and should suggest an alternative diagnosis. CHL are treated with different combinations of chemotherapeutic cells carry mutated IgV genes, indicating that they represent agents. CHL is further classified into nodular sclerosis (NS), B cell lineage-derived cells, though they do not express the mixed cellularity (MC), lymphocyte-rich (LR), and lympho- transcription factors OCT2 and BOB1 together. They can cyte depletion (LD) subtypes (Table 28-4) based on the cel- also be positive for EBV. lular background and the extent of fibrosis. Nodular Lymphocyte-Predominant Hodgkin Lymphoma (NLPHL) Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is composed of nodular or nodular and diffuse prolifera- tion of large neoplastic cells known as popcorn cells (L&H cells) residing in a meshwork of follicular dendritic cells and many small B cells. The popcorn cells characteris- tically have a large multilobated nucleus with delicate nuclear membranes, finely granular chromatin, and small indistinct nucleoli (Figure 28-14). Unlike the neoplastic cells of classical HL, those of NLPHL have a B cell phe- Figure 28.14 Nodular lymphocyte predominant Hodgkin lymphoma. Large L&H (“popcorn”) cell with multilobated nucleus, notype—CD45+ , CD19+ , CD20+ , CD79a+ , and PAX5+— delicate nuclear membranes, and small basophilic nucleoli. The and they express the B cell transcription factors OCT2 and background contains many histiocytes with pale-staining eosinophilic BOB1. By definition, they lack staining for CD30 and CD15. cytoplasm and small lymphocytes (H&E stain, 200* magnification). Mature Lymphoid Neoplasms 641 A diagnosis of mixed cellularity HL is made after exclud- Checkpoint 28.4 ing the other classic subtypes: NS, LR, and LD. However, Name and describe the cell that is characteristic of Hodgkin the histologic subtypes of classical HL do not have inde- lymphoma. pendent prognostic significance. The stage of HL is one of the most important prognostic markers. The 5-year survival for patients with Stage I or IIA Hodgkin lym- The nodular sclerosis subtype of CHL is character- phoma currently is approximately 90% and for Stage ized by C-shaped bands of birefringent fibrous tissue IV is 60–70%. Treatment options depend on the stage of (Figure 28-15b), nodular aggregates of cells, and tumor disease and can include chemotherapy with or without cell variants with cytoplasmic clearing and delicate, radiation.1 multilobated nuclei (lacunar cells; Figure 28-15c). The lymphocyte-rich form of CHL has a background of reactive cells including many small lymphocytes, but in contrast to Checkpoint 28.5 NLPHL, the neoplastic cells have the phenotype of classi- What clinical finding differentiates multiple myeloma from other cal HL. Lymphocyte-depleted HL is a rare subtype that has plasma cell neoplasms? many tumor cells and only rare reactive lymphocytes. a b c Figure 28.15 Classical Hodgkin lymphoma, nodular sclerosis. (a) Reed-Sternberg cell with two nuclear lobes and prominent, eosinophilic nucleoli giving an owl’s eye appearance. The background contains many small lymphocytes and a few pale eosinophilic histiocytes (lymph node biopsy, H&E stain, 200* magnification). (b) Bands of fibrous tissue isolate nodules containing an abnormal cellular infiltrate (nodular sclerosing pattern) (lymph node biopsy, H&E stain, 20* magnification). (c) Lacunar cells with abundant clear-staining cytoplasm, delicate nuclear membranes, and small basophilic nucleoli (lymph node biopsy, H&E stain, 100* magnification). 642 Chapter 28 Summary The mature lymphoid neoplasms represent a diverse group usually requires a combination of conventional morphol- of neoplasms that vary in clinical presentation, morpho- ogy, immunophenotyping, molecular analysis, and cyto- logic appearance, immunophenotype, and genotype. The genetics. HL differs from other lymphomas clinically and WHO classifies these neoplasms into mature B cell and histologically; it is composed of large tumor cells that do mature T/NK cell neoplasms. As in the precursor B cell not resemble a normal cell counterpart and are accom- and T cell neoplasms, the leukemic entities are character- panied by many reactive cells. HL has several histologic ized by peripheral blood and bone marrow involvement subtypes that differ in phenotype, appearance of the large whereas the lymphomas have tumorous masses involving neoplastic cells, and nature of the reactive component. the lymph nodes and other lymphoid tissue. The malignant Although the classification of lymphoid malignancies is cells often resemble one or more stages of normal lympho- complicated, it is important to separate distinct disease cyte development and, therefore, must be distinguished entities that have a predictable outcome and response to from reactive proliferations of these cells. The diagnosis specific therapy. Review Questions Level I 4. A patient with absolute lymphocytosis of 4.0 |
* 103/mcL was found to have a monotypic B cell 1. Which of the following is the most likely distribution population by flow cytometry. The i munophenotype of disease in a patient with leukemia? was consistent with CLL/SLL. This indicates: (Objective 3) (Objective 9) a. Lytic bone lesion a. CLL/SLL b. Tumorous mass involving lymph nodes b. monoclonal B lymphocytosis (MBL) c. Tumorous mass involving the tonsil c. Richter’s transformation d. Widespread involvement of the bone marrow d. prolymphocytic transformation of CLL 2. A 60-year-old male undergoing a routine CBC 5. You are asked to perform a peripheral blood smear was found to have lymphocytosis. The peripheral review on a 56-year-old male with a history of lytic smear revealed a uniform population of small bone lesions, hypercalcemia, and a recent diagnosis mature l ymphocytes. Which of the following is of plasmacytoma. Which of the following is the most helpful in differentiating a benign from a malignant likely peripheral blood finding? (Objective 10) lymphocytosis? (Objectives 8, 9) a. Plasmacytosis a. Cytochemistry b. Lymphocytosis b. Immunophenotyping c. Agglutination c. Morphology d. Rouleaux d. Absolute lymphocyte count 6. A bone marrow biopsy was performed on a patient 3. Diagnosis of a lymphoid neoplasm is supported by with bone pain. The bone marrow differential which of the following findings? (Objective 2) revealed 40% plasma cells. Serum electrophoresis a. Clonal immunoglobulin gene rearrangement showed an increased protein with an M spike. What b. Population of lymphocytes expressing only one would you expect to find on the peripheral blood immunoglobulin light chain smear in this patient? (Objectives 3, 10) c. Population of lymphocytes expressing an a bnormal a. Rouleaux phenotype b. Spherocytes d. All of the above c. Plasma cells d. More than 20% blasts Mature Lymphoid Neoplasms 643 7. The WHO classification of lymphoid neoplasms rec- 3. A lymph node biopsy is performed on a patient with ommends which of the following studies to identify lymphadenopathy. Which of the following findings neoplastic cells and type of lymphoid malignancy? is(are) characteristic of a reactive proliferation rather (Objective 9) than malignant lymphoma? (Objective 9) a. Genotype a. A mixed population of cells varying in size, shape, b. Morphology and color c. Phenotype b. Clonality of cells d. All of the above c. Presence of large, bizarre cells d. Many cells with mitotic activity 8. The Reed-Sternberg cell is found in what type of lym- phoid malignancy? (Objective 6) 4. A CBC performed on a 65-year-old female reveals a. Chronic lymphocytic leukemia lymphocytosis. A bone marrow reveals replacement of the hematopoietic precursors by an infiltrate b. Multiple myeloma of mature lymphocytes. Which of the following c. HL is the most likely course of the disease? d. NHL (Objective 5) 9. A peripheral smear performed on a 35-year-old male a. Cure following therapy with lymphocytosis reveals numerous smudge cells. b. Rapid progression Which of the following does this finding indicate? c. Slow progression (Objectives 3, 8) d. Spontaneous remission a. Chronic lymphocytic leukemia b. Infectious mononucleosis 5. Which of the following phenotypes is characteristic of chronic lymphocytic leukemia? (Objective 1) c. Lymphoblastic leukemia d. None because the finding is not diagnostic a. CD19+ , CD5+ , CD23+ b. CD19+ , CD5- , CD23+ 10. Peripheral smear examination of a patient with c. CD19+ , CD5+ , CD23- chronic lymphocytic leukemia reveals 8% prolympho- cytes. This finding is consistent with which of the fol- d. CD19+ , CD5- , CD23- lowing diagnoses? (Objective 8) 6. A CBC performed on an 80-year-old female reveals a. Chronic lymphocytic leukemia an absolute lymphocyte count of 8 * 103/mcL. b. Prolymphocytic leukemia Examination of the peripheral blood smear reveals a uniform population of small lymphocytes. Which of c. Prolymphocytoid transformation of CLL the following procedures is most likely to establish a d. Richter’s transformation diagnosis? (Objectives 1, 5) Level II a. Cytogenetics 1. Which of the following is(are) involved in the patho- b. Flow cytometry immunophenotyping physiology of low-grade follicular lymphoma? c. Immunoglobulin gene rearrangement (Objective 8) d. Lymph node biopsy a. Genes involved in apoptosis 7. A patient presents with pancytopenia and massive b. Inherited immunodeficiency splenomegaly but no lymphadenopathy. Unusual c. Proto-oncogenes cells noted on the blood smear had abundant d. Tumor suppressor genes pale-staining cytoplasm and cytoplasmic projections. The nuclei were oval with a fine chromatin pattern. 2. Which type of lymphoma can antimicrobial therapy Which of the following test results would be helpful cure? (Objective 8) to establish the diagnosis? (Objective 5) a. Burkitt lymphoma a. Clonal T cell gene rearrangement b. Hodgkin lymphoma b. t(11;14) IGH/CCND1 by FISH c. MALT lymphoma c. BRAF V600E mutation d. HIV-associated non-Hodgkin lymphoma d. POSITIVE myeloperoxidase cytochemical staining 644 Chapter 28 8. A CBC performed on a 60-year-old male with a his- b. The patient has a new lymphoid neoplasm: chronic tory of rheumatoid arthritis revealed neutropenia lymphocytic leukemia. and an absolute lymphocytosis. Examination of the c. The lymphoma has transformed. peripheral smear revealed many lymphocytes with d. The patient has developed a therapy-related abundant pale-staining cytoplasm and cytoplasmic neoplasm. granules. Which of the following is the most likely phenotype? (Objectives 1, 5) 10. A lymph node biopsy revealed an infiltrate of small a. CD3+ , CD2+ , CD57+ lymphoid cells with a vaguely nodular growth pat- tern. Flow cytometry revealed a monoclonal popula- b. CD19+ , CD5+ , CD57- tion of B cells expressing CD5. Immunohistochemistry c. CD2+ , CD3- , CD56+ revealed nuclear staining for cyclin-D1 protein. Which d. CD19+ , CD10+ , CD34+ of the following translocations is associated with these neoplastic cells? 9. A patient previously diagnosed with SLL is found to (Objective 3) have peripheral blood lymphocytosis. The lympho- cytes are small and have round nuclei with clumped a. t(11;14) chromatin. Which of the following is the most appro- b. t(15;17) priate interpretation? (Objective 3) c. t(1;14) a. This is a different presentation (CLL) of the same d. t(8;14) follicular lymphoma disease process. References 1. Swerdlow, S. H., Campo, E., Pileri S. A., Harris, N. L., Stein, H., Waldenstrom’s macroglobulinemia. New England Journal of Siebert, R., . . . , Zelenetz, A.D. (2016). The 2016 revision of the Medicine, 367, 826–833. World Health Organization classification of lymphoid neoplasms. 8. Hans, C. P., Weisenburger, D. D., Greiner, T. C., Gascoyne, R. D., Blood, 127, 2375–2390. Delabie, J., Ott, G., . . . Chan, W.C. (2004). Confirmation of the 2. Tandon, B., Peterson, L., Gao, J., Nelson, B., Ma, S., Rosen, S., & molecular classification of diffuse large B-cell lymphoma by Chen, Y.H. (2011). Nuclear overexpression of lymphoid-enhancer- immunohistochemistry using a tissue microarray. Blood, 103, binding factor1 identifies chronic lymphocytic leukemia/small 275–282. lymphocytic lymphoma in small B-cell lymphomas. Modern 9. Swerdlow, S. H. (2014). Diagnosis of “double-hit” diffuse large Pathology, 24, 1433–1443. B-cell lymphoma and B-cell lymphoma, unclassifiable, with 3. Hallek, M. (2013). Chronic lymphocytic leukemia: 2013 update features intermediate between DLBCL and Burkitt lymphoma: on diagnosis, risk stratification and treatment. American Journal of When and how, FISH versus IHC. Hematology American Society of Hematology, 88, 803–816. Hematology Educational Program, 2014, 90–99. 4. Falini, B., Martelli, M. P., & Tiacci, E. (2016). BRAF-V600E 10. Kabube, K., & Campo, E. (2015). MYC alterations in diffuse large mutation in hairy cell leukemia: from bench to bedside. Blood. B-cell lymphomas. Seminars of Hematology, 52, 97–106. doi: 10.1182/blood-2016-07-418434 11. Said, J. (2015). The expanding spectrum of EBV+ lymphomas. 5. Blombery, P. A., Wong, S. Q., & Hewitt, C. A. (2012). Detection of Blood, 126, 827–828. BRAF mutations in patients with hairy cell leukemia and related 12. Chesi, M., & Bergsagel, P. L. (2013). Molecular pathogenesis lymphoproliferative disorders. Haematologica, 97, 780–783. of multiple myeloma: Basic and clinical updates. International 6. Mozos, A., Royo, C., Harmann, E., De Jong, D., Batro, C., Valera, Journal of Hematology, 97, 313–323. A., . . . Chuang, S. (2009). SOX11 expression is highly specific 13. Younes, A., Bartlett, N. L., Leonard, J. P., Kennedy, D. A., for mantle cell lymphoma and identifies the cyclin D1-negative Lynch, C. M., Sievers, E. L., & Forero-Torres, A. (2010). subtype. Haematologica, 94, 1555–1562. Brentuximab vedotin (SGN-35) for relapsed CD30-positive 7. Treon, S. P., Xu, L., Yang, G., Zhou, Y., Liu, X., Cao, Y., . . . lymphomas. New England Journal of Medicine, 363, 1812–1821. Tripsas, C. (2012). MYD88 L265P somatic mutation in Chapter 29 Hematopoietic Stem Cell Transplantation Aamir Ehsan, MD Andrea Yunes, MD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Describe the sources of hematopoietic stem 5. Identify the infections that are serious cells and characteristics of each source. complications during the peritransplant 2. Identify the diseases that can be treated with period. different sources of hematopoietic stem cells. 6. Describe the significance and effects of graft- 3. List characteristics of stem cells. versus-host disease (GVHD), and compare them to those of the graft-versus-leukemia 4. Describe the significance of ABO and HLA (GVL) process. antigens for stem cell transplant. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Summarize the collection and processing of 5. Formulate the sequence of events for hematopoietic stem cells, and assess the suc- a patient who will receive a stem cell cess of these procedures. transplant. 2. Explain the role of the clinical laboratory 6. Select laboratory tests used to determine professional in stem cell transplantation. engraftment and assess engraftment given 3. Select and outline methods to enumerate the results obtained by these tests. hematopoietic stem cells. 7. Differentiate between the types of stem cell 4. Explain the complications of stem cell transplantation (SCT), and select the most transplant, and assess the patient’s risk of appropriate type for the patient’s clinical developing them. condition. 645 646 Chapter 29 Chapter Outline Objectives—Level I and Level II 645 Collection Target for Stem Cells 653 Key Terms 646 Hematopoietic Engraftment 654 Background Basics 646 Role of the Clinical Laboratory Professional in Stem Case Study 647 Cell Transplantation 654 Overview 647 Graft-versus-Host Disease and Graft-versus-Leukemia Introduction 647 Effect 655 Origin and Differentiation of Hematopoietic Stem Complications Associated with Stem Cell Cells 647 Transplantation 656 Sources of Hematopoietic Stem Cells and Types of Increased Availability and Success of Stem Cell Stem Cell Transplants 648 Transplantation 657 Collection and Processing of Hematopoietic Stem Summary 658 Cells 650 Review Questions 658 Quantitation of Hematopoietic Stem Cells 652 References 660 Key Terms Allogeneic Cryopreserved Polymorphic Apheresis Engraftment Progenitor cells Autologous Graft-versus-host disease (GVHD) Purging Chimerism Graft-versus-leukemia (GVL) Syngeneic Clonogenic Passenger lymphocyte syndrome Conditioning regimen (PLS) Background Basics This chapter builds on concepts learned in previous Level II chapters. To maximize your learning experience, you • Describe the role of cytokines and bone marrow should review the following material before starting this microenvironment in maturation and differentiation unit of study: of hematopoietic stem cells; explain the role of onco- genes in cancer development. (Chapters 2, 4, 23) Level I • Explain the use of molecular genetic technology and • Describe the origin and differentiation of hematopoi- cytogenetics in diagnosis and prognosis of neoplastic etic cells. (Chapters 2, 3, 4) hematopoietic disorders. (Chapters 41, 42) • Outline the classification and explain the etiology • Correlate subgroups of neoplastic hematologic and pathophysiology of neoplastic hematologic disorders with laboratory findings and prognosis. disorders. (Chapter 23) (Chapter 23) • Explain the role of chemotherapy and radiotherapy in treatment of neoplastic hematologic disorders. (Chapters 23–28) Hematopoietic Stem Cell Transplantation 647 CASE STUDY Introduction We refer to this case study throughout the chapter. Hematopoietic stem cell transplantation (SCT) is a recog- Brandon, a 35-year-old male (weight 80 kg), was nized therapeutic modality for leukemias, lymphomas, recently diagnosed with acute myeloid leukemia solid organ tumors, and a variety of metabolic and immu- (AML) and received induction chemotherapy. Day nologic disorders (Table 29-1). The concept of SCT came 21 bone marrow revealed no evidence of residual from experiments done over six decades ago when it was leukemia. Two weeks later, circulating blasts observed that mice given intravenous marrow infusions could overcome lethal doses of radiation.1,2 were seen in the peripheral blood. Brandon was Later, in the evaluated for a hematopoietic stem cell transplant 1960s, the first successful bone marrow transplant was per- (SCT). formed on a leukemia patient using marrow donated by the Consider the laboratory’s role in evaluating the leukemic patient’s brother. transplant, collecting |
and processing the stem cells, Stem cells can be obtained from the patient (autolo- and determining engraftment. gous), from a genetically dissimilar but human leuko- cyte antigen (HLA)-matched donor (allogeneic), an HLA-matched sibling donor (allogeneic sibling), or an iden- tical twin (syngeneic). HLA genes control the body’s ability to mount an immune response. These genes code for histo- Overview compatibility antigens found on the surface of essentially all nucleated cells (Chapters 7, 8). After the early successful The use of stem cells in the therapy of neoplastic hemato- allogeneic bone marrow SCTs using fresh anticoagulated poietic disorders has become commonplace. The labora- bone marrow from HLA-matched siblings, clinical practice tory’s role in this therapy is critical and requires that the has expanded to include the use of autologous bone mar- clinical laboratory professional knows not only the associ- row stem cells, autologous and allogeneic peripheral blood ated laboratory tests but also why the tests are necessary stem cells (PBSCs), and umbilical cord stem cells. and how they relate to the overall process of stem cell transplantation (SCT). The stem cell transplant procedure is also referred to as bone marrow transplant, peripheral blood stem cell transplant, and umbilical cord stem cell transplant Origin and Differentiation depending on the source of the stem cells. This chapter reviews the sources of hematopoietic stem cells (HSCs); of Hematopoietic Stem the use of allogeneic, autologous, and umbilical cord stem cells in treating a variety of malignant and nonmalignant Cells disorders; the mobilization, collection, and enumeration Multipotential hematopoietic stem cells (HSCs) are rare, of mononuclear cells (MNCs) and CD34+ cells; the pro- undifferentiated cells that can be found in the bone marrow cedures used to assess transplant success (engraftment); and occasionally in the peripheral blood of healthy indi- and the role of the clinical laboratory professional during viduals. Self-renewal and multilineage differentiation are this process. the two distinct biologic properties that make HSCs unique. Table 29.1 Diseases Treatable with Hematopoietic Stem Cell Transplantation Hematologic Nonhematologic Neoplastic Non-neoplastic Acute lymphoblastic leukemia Aplastic anemia Solid organ malignancies (breast and ovarian cancer, neuro- Acute myeloid leukemia Paroxysmal nocturnal hemoglobinuria blastoma, Wilms’ tumor, germ cell tumor) Chronic myeloid leukemia Thalassemia Inborn errors of metabolism (e.g., Hurler’s syndrome) Primary myelofibrosis Sickle cell disease Severe combined immunodeficiency disease Chronic lymphocytic leukemia Fanconi’s anemia Wiskott-Aldrich syndrome Myelodysplastic syndrome Congenital pure red cell aplasia Congenital leukocyte dysfunction syndromes Non-Hodgkin lymphoma Malignant osteopetrosis Hodgkin lymphoma Multiple myeloma 648 Chapter 29 Multipotential HSCs differentiate into myeloid and lymphoid cells harvested from peripheral blood (PBSCs) are more progenitor cells (see Figure 4-3 in Chapter 4). With additional common. Allogeneic transplants are often indicated when divisions, their progeny differentiate into committed single the disease process involves the patient’s own stem cells lineage cells and become progressively restricted to prolif- (Table 29-2). Allogeneic transplant can be performed for eration. The multipotential progenitor cells and committed treatment of acute leukemias, chronic myeloid leukemia cells lack the property of self-renewal. The common myeloid (CML), aplastic anemia, hemoglobinopathies, immune defi- progenitor cells (CMPs) produce three types of committed ciencies, and metabolic genetic disorders.4,5 progenitor cells that differentiate into erythroid (EP), gran- Several important factors must be considered in order ulocyte-macrophage (GMP), and megakaryocytic (MkP) cell to achieve successful allogeneic SCT. First, adequate num- pathways. The common lymphoid progenitor cells (CLPs) bers of HSCs should be present in the graft so that when differentiate into T cells, B cells, natural killer cells, and lym- infused into the recipient’s circulation, engraftment in the phoid dendritic cells. The committed progenitor cells have marrow microenvironment will occur. Second, the recipi- been called colony-forming units (CFUs) because they give ent’s immune system should tolerate donor stem cells rise to colonies of differentiated progeny in vitro (Chapter 4). so that graft rejection does not occur. Third, the donor’s From these committed progenitors arise morphologically rec- immune cells should tolerate host tissue to avoid severe ognizable precursors of differentiated cells (pronormoblast, graft-versus-host disease (GVHD), which occurs when myeloblast, monoblast, megakaryoblast, lymphoblast) that immunocompetent donor T lymphocytes recognize HLA further differentiate into mature cells. The terms progenitor antigens on the host cells as foreign and initiate cell injury. cell and stem cell are used interchangeably when discussing Fourth, passenger lymphocyte syndrome may occur in cells used for transplantation purposes. cases of ABO mismatched donors and recipients. Lastly, the donor’s immune cells should destroy recipient resid- ual tumor or leukemic cells called graft-versus-leukemia Sources of Hematopoietic (GVL). PLS, GVHD, and GVL are discussed later. Highly incompatible tissues are almost certain to be Stem Cells and Types of rejected. Compatibility is based on the immune system’s recognition of certain cell markers as “self.” The most critical Stem Cell Transplants of these cell markers appear to be the HLA molecules, also known as the major histocompatibility complex (MHC). The HSCs for clinical use can be harvested from the bone mar- row, peripheral blood, and umbilical cord blood (UCB).3 HLA system is highly polymorphic (genetically variable). Genes present on the short arm of chromosome 6 Fetal bone marrow and liver are also rich in stem cells. encode HLA antigens (Chapter 8). Class I HLA antigens are However, fetal stem cells are primordial and therefore encoded by three loci: HLA-A, HLA-B, and HLA-C. Class II unusable for research or transplant, and stem cells from the HLA antigens are encoded by another three loci: HLA-DR, liver are not capable of transdifferentiation to hematopoi- HLA-DP, and HLA-DQ. Serological and molecular testing etic stem cells. Unlike other tissues and organs destined for methods are available to determine an individual’s HLA transplant, HSCs are not routinely harvested from cadav- type.6,7 Although the candidate donors and recipients are ers because in most situations, the stem cell donor receives tested for HLA-A, -B, -C, -DR, and -DQ antigens, an HLA hematopoietic cytokines for a few days before the stem cells match requires only that the donor and recipient have com- are harvested to maximize stem cell yield. patible HLA-A, HLA-B, and usually HLA-DR antigens. An optimum clinical result occurs when the donor and recipi- Allogeneic Stem Cell Transplantation ent are matched at the HLA-A, HLA-B, and HLA-DR loci In the past, the usual source of stem cells has been the (6 loci in all). For UCB stem cells, the six-point match is used bone marrow, but now allogeneic transplants using stem since UCB cells lack thymic education and are less likely Table 29.2 Type of Transplant, Sources of Stem Cells, and Diseases for Which the Cells Are Used Type of Transplant Source of Stem Cells Diseases Allogeneic Bone marrow Acute leukemias, chronic myeloid leukemia, non-neoplastic hemato- Peripheral blood logic conditions (Table 29-1), immunologic and inherited diseases Cord blood Autologous Peripheral blood Hodgkin lymphoma, non-Hodgkin lymphoma, solid organ tumors, Bone marrow plasma cell myeloma Syngeneic Peripheral blood Any disorder treated by allogeneic/autologous transplant except a Bone marrow genetic disease Hematopoietic Stem Cell Transplantation 649 to cause GVHD. Even with a complete match, graft rejec- pure red cell aplasia (recipient’s antibodies directed against tion and GVHD can occur, suggesting that other antigens donor ABO blood type) or delayed hemolysis (from donor can play a role in donor/graft reactions. On the other hand, B lymphocytes producing ABO antibodies against residual successful transplant can occur even with some degree of recipient RBCs). This adverse effect is known as passenger HLA mismatch.8,9 lymphocyte syndrome (PLS) and occurs due to a major or Approximately 30% of patients requiring allogeneic minor ABO incompatibility between donor and recipient. transplant have a matched sibling donor (10 of 10 loci; Stem cell donors are tested for infectious diseases HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ), and another required by the Food and Drug Administration (FDA) for 2–5% have a partially matched related donor (acceptable volunteer blood donors. Most transplant centers absolutely for use: 7–8 of 10).10 If family members do not match or exclude donors only if they are found to have serologic evi- are not available, the search for an unrelated HLA-matched dence of HIV infection. donor is made through the National Marrow Donor Pro- Before receiving the allogeneic HSC infusion, the recip- gram (NMDP) and World Marrow Donor Association ient is treated with conditioning chemotherapy and/or total (WMDA).11,12 This type of transplant is called a matched body irradiation.13 Depending on the underlying disease, unrelated donor (MUD) transplant. The NMDP and WMDA this regimen can serve two purposes. First, it provides an promote volunteer donation of bone marrow and periph- antitumor effect if the transplant is being done as part of eral blood stem cells and establish and promote the practice a treatment regimen for a malignant disease. Second, it is of sharing stem cell sources by need rather than the donor’s immunosuppressive to enable the recipient to better tolerate geographic location.12 Although millions of donors are in the donor cells. The goal of giving high-dose c hemotherapy/ the pool, the MUD transplant has certain limitations. First, radiotherapy is to destroy all malignant cells, but the because of HLA polymorphisms, locating HLA-matched therapy is also toxic to normal bone marrow cells. The donors is difficult. Second, the length of time to find a com- patient’s marrow is then rescued with infused donor stem patible allograft is often about 4–6 months. Third, the cost cells. As in solid organ transplantation, recipients must of donor search and procurement is high. Fourth, the racial also be given additional immunosuppressive therapy such distribution in the NMDP is unbalanced, limiting its useful- as cyclosporine or tacrolimus to minimize graft rejection ness for patients of minority origins, particularly those of and GVHD. African, Mexican, and Asian heritage. However, consider- Syngeneic transplantation is the use of bone marrow able efforts are being made to recruit minority donors and or PBSCs from an identical twin. This type of transplant is in 2010, 40% of new donors in the NMDP registry were from rare but could be used for any disorder treated by alloge- diverse ethnic and racial backgrounds. Finally, the chance neic or autologous transplant except genetic diseases that of graft failure, GVHD, and opportunistic infections with affect both twins. Syngeneic transplants should have no risk MUD transplant increases. of rejection or GVHD, and no special immunosuppressive When options for a donor are few, bone marrow trans- drugs need be given. plants have been performed with haplo-identical stem cells. These stem cells are matched for only one haplotype or one Autologous Stem Cell set of HLA antigens (3 of 6 loci). The other haplotype is mismatched. With this type of stem cell transplant, the Transplantation donor can be a sibling, a child, or, in some cases, a first- Autologous stem cell transplantation infuses the patient’s own degree relative. Parents are not used as donors. The risk bone marrow or PBSCs. These stem cells are collected prior of life- threatening GVHD, however, is significant. Research to intensive or myeloablative chemotherapy and/or radio- is ongoing to decrease the risk of GVHD by reducing the therapy, which seek to eliminate the malignant cells. After number of transplanted T cells. The haplotype match is therapy, the collected stem cells are infused into the patient rarely needed currently because of advances in the ability to re-establish hematopoiesis. Autologous transplantation to expand the numbers of cord blood stem cells. is usually indicated when the patient’s stem cells are not The initial donor selection for an allogeneic transplant affected by the disease and the underlying disease is poten- is based on HLA compatibility with the recipient. ABO tially responsive to high-dose chemotherapy and/or radio- blood group antigens of the donor and recipient do not therapy. This type of transplant is commonly utilized for need to be matched to accomplish successful transplanta- Hodgkin lymphoma; non-Hodgkin lymphoma; plasma cell tion because these antigens are not expressed on HSCs, and myeloma; solid organ tumors including breast, ovarian, and recipients will convert to the blood type of the donor or testicular cancers; and pediatric neoplasms such as neuro- become a mixed chimera of donor and recipient blood types. blastoma and Wilms’ tumor (Table 29-2). Although more than 30% of SCT recipients receive HSCs Autologous PBSC transplant can be performed even if from ABO-incompatible donors, there are potential adverse the disease involves the patient’s own marrow. However, in consequences, including delayed red blood cell recovery or this case, a number of other factors should be evaluated: age, 650 Chapter 29 |
underlying disease, degree of marrow involvement, response donors, pilot programs for banking unrelated donor cord to previous chemotherapy, and donor availability. The use blood were initiated in various countries around the world. of autologous stem cells for transplant in a subset of acute Cord blood contains sufficient numbers of HSCs for leukemias, myeloproliferative disorders, and myelodysplas- engraftment in most recipients weighing less than 40 kg. tic syndrome (MDS) has been attempted but without great However, cord blood transplants have been attempted with success.14,15,16 some success in patients weighing more than 40 kg. To Autologous stem cell transplantation has both advan- obtain an adequate HSC dose, the transplantation of two tages and disadvantages. One important advantage is that partially HLA-matched cord blood units has been used it is not necessary to identify HLA-matched donors. Graft with success in some adults.22 See Table 29-3 for the advan- rejection and GVHD do not occur, so immunosuppression tages and disadvantages of using cord blood over marrow is not used. Peritransplant mortality is low, and older or PBSCs. Clinical studies are currently underway to patients tolerate the procedure relatively well. Some disad- increase the number of UCB stem cells in vitro to achieve a vantages of autologous transplant include the possibility of usable number of SCTs for adult transplants. neoplastic cells in the stem cell product that could cause disease recurrence, difficulty in obtaining an adequate num- ber of stem cells if the patient has received extensive prior CASE STUDY (continued from page 647) therapy, and the absence of the GVL effect that is sometimes Bone marrow from Brandon was examined, and seen with allogeneic transplant. GVL is a favorable effect 6% blasts were present. He has four siblings. seen when immunocompetent donor T cells present in the allograft destroy the recipient’s leukemic cells. The signifi- 1. Is Brandon a candidate for SCT? cance of GVL is described later in the chapter. 2. If yes, what form of transplant is required? 3. What testing should Brandon undergo to pro- Checkpoint 29.1 ceed with the transplant? A physician is evaluating a 28-year-old patient with a history of acute lymphoblastic leukemia for PBSC transplantation. The laboratory professional found circulating leukemic blasts in the peripheral blood. Is this patient a candidate for an autologous PBSC transplant? Checkpoint 29.2 What would be the best form of transplant for a patient with CML who needs an SCT, and what antigen type needs to be matched? Umbilical Cord Blood Stem Cell Transplantation Umbilical cord blood (UCB) contains sufficient numbers of HSCs to provide short-term and long-term engraftment in Collection and Processing related and unrelated recipients.17,18,19 The first UCB- derived SCT was performed in a 5-year-old boy with of Hematopoietic Stem Fanconi’s anemia in 1988.20 Clinical data indicate that UCB from siblings and unrelated donors can be used to Cells reconstitute hematopoiesis in patients with malignant and Collecting and processing HSCs requires a team of trained nonmalignant disorders.17,18,19,20,21 As a result of the early laboratory professionals to process the cells, perform successes with UCB SCT transplantation from sibling testing, and assess engraftment, failure, and complications. Table 29.3 Advantages and Disadvantages of Using Cord Blood Stem Cells versus Marrow Stem Cells from Donors Advantages Disadvantages Cord blood can be abundantly available if parents donate after birth. Total number of stem cells is generally inadequate for most adult transplant Cord blood stem cells can be collected without risk to mother or infant. patients. Cord blood can be frozen and made readily available as needed. GVL effect can be reduced. Ethnic balance of a given population of donors can be maintained. Only one unit is available from one donor. The risk of latent viral contamination (CMV, EBV) is low. Cord blood does not contain mature T cells and therefore is likely to be less immunogenic and will reduce the incidence of GVHD. Requires less stringent HLA matching. CMV, cytomegalovirus; EBV, Epstein-Barr virus; GVHD, graft-versus-host disease; GVL, graft-versus-leukemia. Hematopoietic Stem Cell Transplantation 651 Bone Marrow CASE STUDY (continued from page 650) Collection of bone marrow stem cells (allogeneic or autolo- An HLA-matched sibling has agreed to be a donor gous) is a surgical procedure using general or local anes- for Brandon. thesia. The marrow is harvested from the posterior iliac crest with specific harvest needles and syringes. Approxi- 4. Should the source of stem cells be peripheral mately 10–15 mL of marrow per kilogram of recipient blood or bone marrow? Why? body weight is harvested to provide an adequate num- ber of mononuclear cells (MNCs). When the marrow is harvested, it contains a mixture of red blood cells, white Umbilical Cord Blood blood cells, MNCs, platelets, plasma, and fat particles. If a marrow recipient is ABO compatible with the donor, the If UCB is to be collected as a source of stem cells, the mother marrow can be infused immediately after filtering the fat must sign an informed consent, and a complete medical and small bone particles. If the donor and recipient are history of the parents is obtained. Cord blood is usually ABO incompatible, erythrocytes and/or plasma must collected from a delivered placenta or sometimes from an be removed to avoid adverse reactions (e.g., PLS) in the undelivered placenta (after the baby is delivered and the recipient upon administration. In some cases, the marrow cord is being clamped). A large-bore needle is inserted into can be further processed to remove a mononuclear cell the umbilical vein to collect blood in a sterile collection bag layer that can be frozen and stored. Stem cell collection containing anticoagulant. Several methods are used to sep- by this method is rarely used now and is largely replaced arate MNCs from the whole cord blood: Ficoll-Hypaque, by peripheral blood stem cell collection (see “Peripheral modified Ficoll-Hypaque gradient method, and addition of Blood”). hydroxyethyl starch (HES).25,26 The cord blood is not usu- ally processed if certain conditions are present (Table 29-4). Peripheral Blood Because stem cells are rare in the peripheral blood (less Purging than 0.01%), various regimens are used to mobilize stem Purging is a technique to reduce possible tumor cell con- cells in the marrow, force these cells to enter the periph- tamination or in the setting of allogeneic transplant, to eral blood, and increase the number of circulating MNCs. reduce the number of T lymphocytes present in the product. Transplanting more stem cells means more rapid hemato- Purging is performed on stem cell products that have been poietic recovery.23 For autologous PBSC collection, patients collected by apheresis. Depending on the source of stem can receive cytotoxic chemotherapy (e.g., cyclophospha- cells and clinical conditions in which the cells are used, vari- mide), hematopoietic cytokines (e.g., granulocyte colony- ous purging techniques (mechanical, immunological, and stimulating factor [G-CSF], granulocyte-macrophage pharmacological) are available.27 colony-stimulating factor [GM-CSF]), or a combination In cases of an allogeneic SCT, GVHD, an immunologic of chemotherapy and cytokines to mobilize stem cells. response that results from the presence of the donor’s T For allogeneic PBSC collection, hematopoietic cytokines lymphocytes that can interact with the host’s cells, is a (G-CSF or GM-CSF) are given to normal, healthy donors potential complication. Purging the donor T lymphocytes to mobilize stem cells. from the allograft can prevent or reduce its severity. How- After cytotoxic and/or cytokine therapy, the stem ever, some of these donor T cells seem to be necessary for cells in the marrow are mobilized to the peripheral blood marrow engraftment and for achievement of the GVL effect. where they can be collected by apheresis, an automated GVL, which is a favorable response, is usually seen because procedure that uses a blood cell separator. Whole blood the T cells present in the graft can have an active role in is withdrawn from the donor and anticoagulated. The components are separated using centrifugation or fil- tration, and the desired component is collected (in this Table 29.4 Conditions in Which Stem Cells Are Not case, mononuclear cells that contain HSCs). The remain- Usually Collected and/or Processed from UCB ing components are combined and infused back into the Inadequate volume collected from individual placenta (less than 40 mL) donor. Preterm delivery (less than 36 weeks) The optimum timing for collection of PBSCs after cyto- Fever higher than 100°F (38°C) in mother toxic and cyotokine therapy is sometimes unpredictable, Premature rupture of membranes and the time of harvest varies depending on the mobiliza- tion protocol. Some centers determine the timing of PBSC Meconium staining of fluid collection based on a preapheresis peripheral blood CD34 Family history of inherited diseases count.24 Congenital anomalies in infant 652 Chapter 29 destroying the residual leukemic cells in the recipient. In rescue in cases of disease relapse. Because the engraftment support of this possibility, patients receiving T cell–depleted outcome depends on the number of stem cells present grafts are known to be at higher risk for disease relapse in the product, the HSCs (from bone marrow, PBSCs, or than patients receiving unmanipulated grafts for the same cord blood) are quantitated by one of the three methods disease. described next. Cryopreservation and Storage of Determination of Mononuclear Hematopoietic Stem Cells Cell Count HSCs from bone marrow, PBSCs, or cord blood can be effec- Stem cells are mononuclear cells (as are immature myeloid tively cryopreserved (stored at very low temperatures) to cells, lymphocytes, and monocytes); thus, counting the maintain cell viability for months or years. When the stem MNCs provides a very indirect estimate of the number of cells are frozen or thawed, cell injury or loss of stem cell stem cells present in the sample (Figure 29-1). The volume viability results from intracellular crystal formation and cel- of cells necessary to achieve a satisfactory dose for success- lular dehydration. ful engraftment can be determined from the MNC count. To achieve optimum cryopreservation without compro- The dose is usually specified as the number of MNCs per mising the cell viability, a cryoprotectant agent is used.28 kilogram of the recipient’s body weight. Laboratories use The most common cryoprotectants are 10% dimethyl- automated cell counters or hemacytometers to count the sulfoxide (DMSO) or 5% DMSO with 6% hydroxyethyl MNCs. starch (HES). The DMSO diffuses rapidly into the cells and increases intracellular solute concentration, thus decreasing CD34 Enumeration by Flow intracellular ice crystal formation and preventing cell lysis. The DMSO provides a protective barrier during the freezing Cytometry and thawing processes. After optimizing the concentration Enumerating cells that express CD34 antigen (stem cells and of the stem cells in the presence of cryoprotectant, a sample progenitor cells) by immunophenotyping has become a rou- is obtained from the SCT product and inoculated on tissue tine practice. With use of a flow cytometer and fluorescent culture media for colony assays. The SCT product is tested dyes, anti-CD34 antibodies offer a rapid method of enumer- for bacterial contamination using a blood culture bottle. ating these cells. Flow cytometric enumeration of CD34+ cells has been shown to be the most useful indicator of the Infusion of Hematopoietic Stem Cells hematopoietic reconstitutive capacity of SCT. The major difficulty with the analysis of CD34+ cells The patient to be infused (recipient) is premedicated with is the low number of cells present in the specimen. The antihistamine and antiemetic (sometimes diuretics and/or method’s sensitivity is 1 in 10,000. Procedures to measure steroids). The frozen stem cell bags are thawed in a 37°C CD34+ cells vary widely. Optimally, 100 CD34+ cells and water bath immediately before the infusion. The infusion 75,000 CD45+ (leukocytes) events should be acquired to is carried out as quickly as possible to dilute the cell sus- pension with the patient’s blood volume because DMSO in liquid phase can be toxic to stem cells. The most common adverse reactions associated with cryopreserved stem cells are chills, nausea and vomiting, fever, allergic reactions, transient cough, and shortness of breath. Adverse events from HSC infusion can be related to DMSO-induced his- tamine release; therefore, some centers thaw and dilute or wash the cell suspension to remove the DMSO, but cell loss and clumping can be a problem with this approach. Quantitation of Hematopoietic Stem Cells The number of stem cells or CD34+ cells that can be obtained Figure 29.1 Wright’s stain slide prepared for the manual varies greatly from donor to donor. For PBSC, more than differential count from the stem cell product showing myeloid one apheresis procedure is |
needed to acquire the appro- precursors and mononuclear cells (peripheral blood, Wright’s stain, priate dose for engraftment, hematopoietic recovery, and 1000* magnification). Hematopoietic Stem Cell Transplantation 653 optimize the accuracy of analysis.28,29 The CD34+ count is Commercial kits are now available to aid in the standardiza- calculated in conjunction with the total leukocyte counts.29,30 tion of clonogenic assays, although this method is rarely CD34+ counts can be performed on peripheral blood and used in clinical practice This assay is performed by stem cell the stem cell product. Results should be reported as soon laboratory processing personnel only. If there is no growth as possible because the decision to collect more stem cells of colony-forming units (CFUs) or burst-forming units depends on CD34+ yield. If the CD34+ cells are lower than (BFUs) after the incubation, the graft may be inadequate required, the decision can be made to perform (or postpone) and a new donor or collection must be initiated. more apheresis or to mobilize the stem cells in a different manner. Some centers determine the timing of PBSC collec- Checkpoint 29.3 tion based on a peripheral blood CD34+ count.24,31 Common You receive a peripheral blood specimen in the hematology lab- practice is to collect an additional unit of cells for cryopreser- oratory with a request for a mononuclear cell count and analysis vation, regardless of the CD34+ count, unless the first col- of CD34+ cells. Without any other information, why should you lection has an extremely elevated CD34+ count and the unit consider this a STAT request? can be split for infusion of an adequate amount of cells. Cell Culture for Colony Forming Units Collection Target for Stem Flow cytometry analysis identifies cells only by their anti- Cells genic markers and thus cannot identify cells that are clo- The recipient’s body weight determines the required dose nogenic (capable of proliferating and forming colonies of of stem cells. The exact number of HSCs needed to ensure cells when grown in vitro); only culture techniques can engraftment is not known, but cell doses of demonstrate clonogenicity. An in vitro clonogenic assay is 2.595.0 * 106 CD34+ cells/kg of recipient weight are desir- a culture system containing stem cells and various growth able; earlier engraftment is observed with even higher factors. The culture is incubated for 14–15 days on a semi- doses.34,35 For a transplant using UCB, greater than 3.0 * 107 solid medium (agar or methylcellulose). The colonies gener- of total nucleated cells per kg of recipient weight is ated from committed myeloid and erythroid hematopoietic desirable. progenitors can be differentiated and counted (Figure 29-2). The advantage of the clonogenic assay is that this sys- tem tests the capacity of progenitor cells to divide and can CASE STUDY (continued from page 651) predict the engraftment potential of the stem cell prod- PBSCs were collected by apheresis from the sibling. uct.32,33 The 2-week incubation period needed in this method Enumeration of CD34 count indicates that the total is a distinct disadvantage when data are needed for imme- CD34 + cells collected are 6 * 106/kg. diate clinical decisions. The results of clonogenic assay sys- tems from different institutions vary greatly because of 5. Is this an adequate dose for SCT? variability in reagent lots and the level of staff expertise. a b Figure 29.2 Cell culture colonies. (a) Erythroid colonies. (b) Myeloid colonies (original magnification *100, no stain on semisolid medium). 654 Chapter 29 Hematopoietic (Chapter 42). STRs are short sequences of DNA repeated numerous times. Individuals can have unique numbers of Engraftment copies of the repeat element, and this individual copy num- ber is referred to as a polymorphism. SCT generally involves The conditioning regimen given to patients for SCT causes donation between closely related individuals, so multiple severe myelosuppression and can affect T and B cell immu- STR loci must be analyzed to differentiate donor from recip- nologic responses (usually in an allogeneic setting). After ient cells. Loci that discriminate between individuals are the conditioning regimen, the HSCs are infused to restore said to be informative; loci that do not discriminate are said marrow function. A period of pancytopenia typically fol- to be non-informative. lows during which the patient is susceptible to develop- ing infections and bleeding complications. To reduce the period of pancytopenia, which can last from a few days to Checkpoint 29.4 2–4 weeks, growth factors such as G-CSF or GM-CSF can be A physician wants to evaluate the engraftment in a male patient given.35,36 Recovery of myeloid cells usually occurs first and who received SCT from his brother 4 months ago. What labora- is followed by platelet and red cell recovery. tory tests should be performed to make this assessment? Evidence of Initial Engraftment Evidence of hematopoietic engraftment exists when the CASE STUDY (continued from page 653) absolute neutrophil count is greater than 500/mcL (mL). The Stem cells were collected and frozen for Brandon. platelet count must be greater than 20,000/mcL, with no need for platelet transfusions for at least 7 days. Compared 6. Does Brandon need to undergo any form of ther- with bone marrow transplants, the amount of time for cel- apy before the transplant? lular recovery in a recipient following a PBSC transplant is somewhat shorter. After UCB stem cell transplants, neutro- phil recovery can take 5–6 weeks, and the platelets usually engraft late (median time 80 days).27 Role of the Clinical Evidence of Long-Term Engraftment Laboratory Professional in When all hematopoietic cells in an SCT recipient are derived from an allogeneic donor, the condition is referred to as full Stem Cell Transplantation or complete chimerism (the presence of only donor hema- When the decision to perform an SCT has been made, col- topoietic cells in a recipient). When a recipient’s hemato- lecting and processing stem cells involves dedicated clinical poietic cells persist together with donor cells after SCT, the laboratory staff who work as a team (Figure 29-3). The SCT condition is referred to as partial or mixed chimerism. process involves the clinical laboratory professionals work- To evaluate the long-term engraftment of HSCs, vari- ing in the departments of hematology, apheresis, blood bank, ous laboratory methods have evolved.37 Detection of red microbiology, flow cytometry, molecular/HLA, cytogenetics, cell antigens was among the first tests used to evaluate and bone marrow transplant laboratories (Figure 29-4). donor cell engraftment. For example, if the recipient’s blood Consider a patient for whom the clinical decision for group is O and the donor’s blood group is A, the recipi- autologous SCT has been made. This patient will receive ent can type as group A after hematopoietic engraftment cytotoxic chemotherapy, G-CSF, or both. For PBSC collec- with complete chimerism. However, because of the long life tion, the patient will undergo apheresis. In an allogeneic span of red cells and multiple blood transfusions during transplant setting, both the donor and recipient cells are transplantation, the red cell antigens are no longer used for typed for HLA antigens in the molecular/HLA laboratory chimerism studies. before collecting stem cells. The apheresis staff will spend Several molecular techniques can be used to evaluate 4 to 6 hours with the patient to get a good yield of MNCs. chimerism in transplant patients. If the donor and recipient After collecting the product, the sample will be sent to the are of opposite sex, fluorescent in situ hybridization (FISH) hematology and flow cytometry labs where WBCs/MNCs analysis of X and Y chromosomes can be used to monitor and CD34+ counts will be performed on a sample of the engraftment (Chapter 42).38,39 stem cell product collected by apheresis. Currently, the most widely used methodology for mon- The bone marrow transplant laboratory staff will process itoring chimerism is by polymerase chain reaction amplifi- and cryopreserve the stem cells for future use. The cell via- cation of short tandem repeat (STR) loci followed by bility will be determined and samples for bacterial cultures capillary electrophoresis to distinguish the amplicons38,39,40 obtained. On the day of transplant, the clinical laboratory Hematopoietic Stem Cell Transplantation 655 Factors to be considered for the SCT Steps involved in stem cell processing 1. Age of patient 2. Underlying disease 3. Involvement of patient’s own bone marrow or stem cells 4. Degree of marrow involvement Autologous Allogeneic Cord blood 5. Response to previous chemotherapy (a) PBSC—apheresis (a) PBSC—apheresis MNCs are separated 6. Donor availability (b) Marrow—harvested (b) Marrow—harvested and collected. in the operating in the operating room room Autologous stem cell transplant Allogeneic stem cell —PBSC (usually) transplant Mononuclear cells are collected in a bag. —Bone marrow Purging can be performed; technique varies according to the intended use of Search for HLA-matched siblings the product. or family members Samples taken for WBC/MNC counts, CD34 enumeration by flow cytometry, CFU analysis, routine microbiological Not available Available cultures, and cell viability. MNCs are frozen with cryoprotectant. MUD transplant Cord blood —Bone marrow transplant —PBSC Patient receives preparative regimen for SCT (chemotherapy/total body irradiation.) Figure 29.3 Decision-making process in stem cell transplantation. The patient is usually evaluated for SCT at the time the primary diagnosis is made. Numerous factors must be MNC are thawed and infused. (After infusion, G-CSF/GM-CSF can be given to considered before deciding on autologous or allogeneic SCT. For potentially reduce the period of pancytopenia.) allogeneic SCT, a donor is selected based on HLA compatibility. If family members do not match or are not available, the search for an unrelated HLA-matched donor is made through the NMDP. The UCB Patient evaluated for engraftment. registry/banks is/are also options. In general, if the disease involves the patient’s own stem cells, allogeneic transplant is a valid option. PBSC, peripheral blood stem cell; MUD, matched unrelated donor. Short-term engraftment Long-term engraftment staff (usually from the bone marrow laboratory) will thaw Absolute neutrophil count >500 mcL Chimerism studies Platelet count >20,000/mcL with no the stem cell units at the patient bedside for the infusion. transfusion After transplant, the hematology department will eval- uate initial (short-term) engraftment with a complete blood count. For evaluation of long-term engraftment, the sample Figure 29.4 Steps involved in stem cell transplantation will be sent to the cytogenetic and molecular/HLA labora- from the time the patient is diagnosed with the disease to the final tories to perform chimerism studies. Throughout the course infusion of stem cell product. of the transplant, the blood bank personnel will provide special blood components required during the transplanta- (interleukin-1, tumor necrosis factor [TNF]). This leads to tion process. tissue injury.41,42 Three factors necessary for GVHD to occur are the pres- ence of immunocompetent donor T lymphocytes, HLA allo- Graft-Versus-Host Disease antigens, and an immunosuppressed host. GVHD is more and Graft-Versus-Leukemia frequent and severe in recipients of partially HLA-matched sibling allografts or in grafts from unrelated donors.8 However, Effect GVHD also occurs after HLA-identical sibling donor allografts, indicating the role of other HLA minor antigens or factors that The exact mechanism for GVHD is not clearly known. are not usually investigated at the time of transplant. However, its suggested pathogenesis is that immunocom- Overall, the risk of acute GVHD (for the degree of HLA petent donor T lymphocytes recognize HLA antigens on mismatch) is less in recipients of UCB-derived SCT than in the host (recipient) cells as foreign and initiate secondary recipients of a marrow or PBSC transplant, perhaps because inflammatory injury mediated by inflammatory cytokines of the immature nature of cord blood immune cells. GVHD 656 Chapter 29 can be prevented or its severity reduced by decreasing the number of donor T lymphocytes from the graft (purging) Table 29.5 Causes of Graft Failure and/or administering medications such as cyclosporine A, Inadequate number of stem cells methotrexate, steroids, cyclophosphamide, antithymocyte HLA disparity between donor and recipient globulin, and tacrolimus.43 Significant T cell depletion of the allograft Recipients of T cell–depleted marrow experience Decreased degree of immunosuppression GVHD less frequently but have a higher incidence of dis- ease relapse and higher graft failure than patients treated with unmanipulated marrow for the same disease. T lym- GRAFT-VERSUS-HOST DISEASE (GVHD) phocytes are known to be essential for marrow engraftment Classification of GVHD can be either acute or chronic. Acute and for achieving desirable GVL effect, which is seen when GVHD generally occurs in the first 3 months after allogeneic immunocompetent donor T cells present in the allograft SCT and usually involves the recipient’s skin (maculopapu- destroy the |
recipient’s residual leukemic cells.44 For exam- lar dermatitis), liver (elevation of bilirubin, abnormal liver ple, in allograft recipients with CML who relapse after function tests), and gastrointestinal tract (nausea, vomiting, allogeneic SCT, infusion of small numbers of lymphocytes diarrhea). Chronic GVHD is defined as symptoms appearing collected by apheresis from the original graft donor (donor 80–100 days after transplantation. lymphocyte infusion or DLI)45 can induce a potent GVL PASSENGER LYMPHOCYTE SYNDROME (PLS) effect and re-establish complete remission in most patients. PLS is a complication that occurs when there is an ABO The critical issue of how many T lymphocytes or which incompatibility between the donor and the recipient. When subpopulations of T lymphocytes need to be removed from marrow is collected that is ABO incompatible (e.g., type A the allograft to avoid GVHD but allow marrow engraftment donor and type O recipient), it is necessary to perform RBC and the GVL effect is unknown. The T-cell concentration depletion of the unit before infusion (3 to 5% residual RBCs). and subsets required to achieve long-term engraftment, The process is performed in the stem cell laboratory in which control of GVHD, and beneficial GVL effect also depend on red cells are sedimented and centrifuged from the unit. The the disease and the degree of HLA disparity. plasma with the stem cells is then extracted from the unit and tested for viability and cell count; this manipulated product is then infused into the recipient. Clinical staff monitor the Complications Associated patients for hemolysis and treat if necessary. It is important to note that some hemolysis occurs in all cases, as it is not with Stem Cell possible to deplete all RBCs from the marrow product. Transplantation PERITRANSPLANT INFECTIONS During the peritransplant period when the patient has been Complications with SCT can be categorized as early and immunosuppressed and has a low neutrophil count, the risk late. Complications that occur during the first 100 days after of opportunistic infection is very high. These infections dur- transplantation are considered early (acute) complications; ing the first 3 months post-transplant can be serious but those that occur from after 100 days (more than 3 months) rarely life threatening. Infections can be bacterial, protozoal, to several years after transplantation are considered late fungal, and/or viral. Reactivated cytomegalovirus (CMV), a (chronic) complications. member of the herpes virus family, and herpes simplex are the most common causes of infection. In the United States, Early Complications the range of CMV prevalence in the population is 50–85%. SCT patients are particularly vulnerable to CMV infection, Early complications include graft failure, GVHD, peritrans- which can be primary, but superinfection with a second plant infections, and recurrence of malignant disease. strain or a reactivation of latent disease also can occur. The GRAFT FAILURE complications include pneumonitis, gastroenteritis, and ret- Primary graft failure is the failure to establish hematologic initis. Fatal CMV pneumonia occurs in 10–15% of patients. engraftment and can be defined as failure to attain an abso- The relative risk for CMV infection among SCT patients lute neutrophil count more than 500/mcL by 28 days after depends on the serological status of both the donor and the transplant. Secondary graft failure is the loss of an established recipient.46 A seronegative recipient of a seropositive trans- graft and is defined as the redevelopment of pancytopenia plant is at significant risk of primary transfusion-transmitted at any time after primary engraftment. These complications CMV infection. Seropositive recipients of seropositive trans- can be seen in the allogeneic transplant setting but rarely plants can develop reactivation or superinfection of CMV. in the autologous setting. See Table 29-5 for causes of graft A donor for a CMV-negative patient should be an HLA- failure. compatible person who is seronegative for CMV. A positive Hematopoietic Stem Cell Transplantation 657 test for CMV IgG antibodies is not a criterion for rejection to delayed infections. These complications include hypothy- of the donor. SCT patients who are at significant risk for roidism, hypogonadism, cataracts, growth retardation in acquiring transfusion-transmitted CMV also should receive pediatric patients, neuropathies, and sometimes develop- cellular components that carry a reduced risk of CMV. CMV ment of post-transplant lymphoproliferative disorders and resides in leukocytes, and leukocyte-reduced cellular blood second malignancies (MDS and leukemia). components can prevent CMV infection.47,48,49 However, the equivalent efficacy of leukocyte-reduced and CMV-sero- negative cellular components in preventing transfusion- Checkpoint 29.5 associated (TA) CMV has not yet been established. Most A CMV-seronegative patient requires SCT. Two HLA-matched transplant physicians believe that if a CMV-seronegative donors are available. Is it important to know the stem cell donor’s CMV status? If the stem cell donor is CMV seronegative recipient receives stem cells from a CMV-seronegative and the patient requires red cell transfusion during the peritrans- donor, all subsequent blood components received should plant period, what blood components (in terms of CMV status) be CMV-seronegative. If the CMV-negative blood compo- would you select for this patient? nent is not available, transfusing a leukocyte-reduced fil- tered component can effectively prevent the transmission of CMV disease. CASE STUDY (continued from page 654) RECURRENCE OF MALIGNANT DISEASE Patients who undergo autologous transplantation are Brandon received stem cells from his HLA-matched more at risk of dying from disease recurrence than from sibling that successfully engrafted. Three months transplant-related complications. After an autologous trans- later, he developed diarrhea, skin rash, and plant, recurrence of the original disease can occur because jaundice. of incomplete eradication of malignant cells in the patient 7. What could be the possible cause for this? or the presence of residual neoplastic cells in the autograft that have not been adequately eliminated by purging tech- niques. Autologous transplant recipients also do not have the benefit of a graft-versus-leukemia effect. Patients who receive an allogeneic rather than an autologous transplant Increased Availability for hematologic malignancies have a lower relapse rate; however, the risk of relapse varies widely from 5 to 70%. and Success of Stem Cell This variability depends on the diagnosis and stage of the Transplantation disease, the degree of immunosuppression, the degree of match or mismatch of the allograft, and the presence or About 28,000 autologous and 21,000 allogeneic hematopoi- absence of GVHD. etic stem cell transplants are performed every year world- wide,51 and the number increases by about 10–20% annually. OTHER COMPLICATIONS Outcomes are improving, with reduced mortality that can Symptoms and clinical signs of toxicity related to chemo- be related to reductions in organ damage, infection, and therapy and radiotherapy including mucositis, myocarditis, severe GVHD.52 According to one study of patients who pericarditis, pneumonitis, hemorrhagic cystitis, and adverse survived at least 2 years after autologous SCT for a hema- drug reactions can occur after SCT. A serious liver disorder tologic neoplasm, about 69% were alive 10 years after trans- that complicates up to 50% of marrow transplants is called plantation.53 These statistics indicate that it is imperative veno-occlusive disease (or sinusoidal obstruction syndrome). for clinical laboratory professionals to work closely with It is characterized by right upper quadrant pain, weight physicians who perform these procedures and stay abreast gain, and jaundice and is diagnosed clinically. Other fea- of developments in this area. tures include ascites, hepatomegaly, hyperbilirubinemia, encephalopathy, and renal or multiorgan failure. Risk fac- Gene Therapy tors include chronic liver disease, previous SCT, and the use of busulfan and cyclophosphamide as the cytotoxic In addition to SCT, gene therapy is a technology that offers preparative treatment. 50 the possibility for a complete cure for genetic disease.54 A complete discussion of this technology is beyond the scope Late Complications of this book. The goal of gene therapy is to introduce a func- tional copy of the patient’s defective gene into a sufficient Late complications can be secondary to pretransplantation number of the appropriate cell types (such as stem cells) and chemotherapy/radiotherapy, continued effects of acute to have sufficient expression of that gene so that its prod- complications, and/or immunosuppressive states leading uct functions to correct the deficiency. Attempts have been 658 Chapter 29 made to use genetically engineered viruses to carry the DNA immune deficiency, and hemophilia B. For stem cell gene of interest into host cells. Viral genes required for propaga- therapy to find more widespread clinical use, several impor- tion are replaced with a working copy of the human gene. tant related issues—such as the isolation of the appropriate This form of therapy has been used (with variable success) cell population for transduction, efficiency of the transduc- to treat conditions such as adenosine deaminase deficiency, tion process, and safe insertion of the functionally correct chronic granulomatous disease, X-linked combined severe gene into the recipient’s genome—must be overcome.54 Summary SCT is a recognized therapeutic modality for leukemias, cells. For allogeneic PBSC collection, hematopoietic cyto- lymphomas, solid organ tumors, and a variety of meta- kines can be given to the donor. The number of stem cells bolic/immunologic disorders. Sources of HSCs are bone collected varies from donor to donor, and engraftment par- marrow, peripheral blood, and umbilical cord blood. For tially depends on the number of stem cells in the product. an autologous transplant, the patient’s own stem cells are The number of stem cells in the transplant collection can be collected, frozen, and used later for hematopoietic reconsti- determined by quantitating the MNCs, cells, and CFUs/ tution. For an allogeneic transplant, the donor is selected BFUs. Before transplant, the patient undergoes conditioning based on the best HLA match from either family members chemotherapy/radiotherapy, after which the stem cells are or an unrelated donor. A team of health care professionals, infused. Routine blood counts and chimerism studies (in including clinical laboratory professionals, is needed to col- allogeneic SCT only) can be performed to assess engraft- lect, process, and quantitate stem cells before administering ment. Complications of SCT include graft failure, GVHD, them to the patient. For autologous PBSC collection, patients PSL, opportunistic infections, and recurrence of malignant can receive chemotherapy and cytokines to mobilize stem disease. Review Questions Level I 4. What is a syngeneic transplant? (Objective 1) 1. Which of the following properties can be attributable a. Infusion of stem cells from any donor into the to stem cells? (Objective 3) recipient a. Self-renewal b. Infusion of patient’s own stem cells b. Multilineage differentiation c. Infusion of stem cells from an identical twin c. Mononuclear morphology d. Infusion of stem cells from different species d. All the above 5. What cells are usually CD34+? (Objective 3) 2. What is (are) the source(s) of hematopoietic stem a. Maturing myeloid cells cells? (Objective 1) b. Stem cells a. Bone marrow c. B lymphocytes b. Peripheral blood d. T lymphocytes c. Umbilical cord blood 6. Which of the following is correct regarding allogeneic d. All of the above stem cell transplant and ABO antigens? (Objective 4) 3. What form of transplant can be used for inherited a. ABO antigens do not need to be matched. genetic diseases? (Objective 2) b. ABO antigens are strongly expressed on stem cells. a. Autologous stem cell transplant c. The O group patient should not receive stem cells b. Allogeneic stem cell transplant from A or B blood group donors. c. Syngeneic stem cell transplant d. ABO antigens should always be matched. d. Stored autologous cord blood Hematopoietic Stem Cell Transplantation 659 7. What is the single most important factor to consider c. received both allogeneic and autologous SCT in donor selection for an allogeneic transplant? d. received autologous SCT for only solid organ (Objective 4) tumors a. HLA compatibility with the recipient 4. What are the three factors necessary to set the stage b. ABO compatibility with the recipient for GVHD? (Objective 4) c. CMV status of the donor a. Immunocompetent donor T lymphocytes, ABO d. HLA and ABO compatibility with the recipient antigens, and immunosuppressed host 8. Which of the following infections can be serious in the b. Immunosuppressed donor T lymphocytes, HLA peritransplant period? (Objective 5) alloantigens, and immunosuppressed host c. Immunocompetent donor B lymphocytes, HLA a. Bacterial alloantigens, and immunosuppressed host b. Fungal d. Immunocompetent donor T lymphocytes, HLA c. Cytomegalovirus alloantigens, and immunosuppressed host d. All of the above 5. What procedure usually is used to quantify HSCs? 9. Severe GVHD usually is seen in which group of (Objective 3) patients? (Objective 6) a. Lymphocyte count a. Syngeneic SCT b. CD4+ count by flow cytometry b. Autologous SCT c. |
Monocyte count by automated cell counter c. Unmatched allogeneic SCT d. CD34+ count by flow cytometry d. Umbilical cord stem transplant 6. Patients who receive what type of stem cell transplant 10. What organ(s)/tissue(s) is(are) usually involved in are more at risk for disease recurrence than of GVHD? (Objective 6) transplant-related complications? (Objective 4) a. Skin only a. Allogenic b. Skin, liver, and/or the gastrointestinal tract b. Syngeneic c. Liver only c. Cord blood d. Gastrointestinal tract only d. Autologous Level II 7. What test is appropriate to determine long-term engraftment of HSCs only when donor and recipient 1. What does the term chimerism mean? (Objective 6) are of the opposite sex? (Objective 6) a. Recovery of recipient’s red cells a. Detection of RBC antigens b. Absence of malignant cells b. Fluorescent in situ hybridization with sex c. Presence of donor hematopoietic stem cells in a chromosomes recipient c. CD34+ cell enumeration by flow cytometry d. Recovery of patient’s white cells and platelets only d. Platelet and neutrophil counts 2. What is the most appropriate form of transplant for an 8. GVL effect is seen after SCT in patients who have adult patient with CML weighing 80 kg? (Objective 7) received: (Objective 4) a. Autologous SCT a. autologous transplant b. Umbilical cord SCT b. allogeneic transplant c. Allogeneic SCT c. either autologous or allogeneic transplant d. None of the above; transplant is not indicated in d. umbilical cord transplant patients with CML 3. A risk for GVHD is carried by a patient who has: (Objective 4) a. received allogeneic SCT b. received autologous SCT 660 Chapter 29 9. For autologous peripheral SCT, the patient usually 10. What is the sequence in which successful engraftment undergoes therapy in which sequence? (Objective 5) occurs after SCT? (Objective 6) a. Myeloablative therapy, apheresis, and then G-CSF a. Neutrophils are increased first followed by an b. G-CSF followed by myeloablative therapy and increase in platelets. then apheresis b. Platelets are increased first followed by an increase c. Apheresis to collect CD34+ cells, myeloabla- in neutrophils. tive therapy, and then infusion of collected c. Red cells are increased first followed by an increase CD34+ cells. in platelets. d. Myeloablative therapy and then apheresis to col- d. Red cells and platelets are increased first followed lect CD34+ cells by an increase in neutrophils. References 1. Jacobson, L. O., Marks, E. K., Robson, M. J., & Zirkle, R. E. (1949). 16. van der Lans, M. C. M., Witkamp, F. E., Oldenmenger, W. H., & Effect of spleen protection on mortality following x-irradiation. Broers, A. E. C. (2017). Five phases of recovery and rehabilitation Journal of Laboratory and Clinical Medicine, 34, 1538–1543. after allogeneic stem cell transplantation: A qualitative study. 2. Lorenz, E., Uphoff, D., Reid, T. R., & Shelton, E. (1951). Modifica- Cancer Nursing, 00, 1–8. tion of irradiation injury in mice and guinea pigs by bone mar- 17. Ballen, K. K. (2005). New trends in umbilical cord blood trans- row injections. Journal of the National Cancer Institute, 12, 197–201. plantation. Blood, 105(10), 3786–3792. 3. Wilson, A., & Trumpp, A. (2006). Bone marrow hematopoietic 18. Wagner, J. E., Rosenthal, J., Sweetman, R., Shu, X. O., Davies, S. M., stem cell niches. Nature Reviews Immunology, 6(2), 93–106. Ramsay, N. K., . . . Cairo, M. S. (1996). Successful transplantation 4. Speck, B., Bortin, M. M., Champlin, R., Goldman, J. M., of HLA-matched and HLA-mismatched umbilical cord blood Herzig, R. H., Mcglave, P. B., . . . Rimm, A. A. (1984). Allogeneic from unrelated donors: Analysis of engraftment and acute graft- bone-marrow transplantation for chronic myelogenous leukae- versus-host disease. Blood, 88, 795–802. mia. Lancet, 1, 665–668. 19. Gluckman, E., Rocha, V., Boyer-Chammard, A., Locatelli, F., 5. Copeland, E. A. (2006). Hematopoietic stem cell transplantation. Arcese, W., Pasquini, R., . . . Chastang, C. (1997). Outcome of New England Journal of Medicine, 354(17), 1813–1826. cord-blood transplantation from related and unrelated donors. 6. Gam, R., Shah, P., Crossland, R. E., Norden, J., Dickinson, A. M., & Eurocord Transplant Group and the European Blood and Mar- Dressel, R. (2017). Genetic association of hematopoietic stem cell row Transplantation Group. New England Journal of Medicine, 337, transplantation outcome beyond histocompatibility genes. Fron- 373–381. tiers in Immunology, 8, 380. 20. Gluckman, E., Broxmeyer, H. E., Auerbach, A. D., Friedman, H. S., 7. Beatty, P. G. (1994). The immunogenetics of bone marrow trans- Douglas, G. W., Devergie, A., . . . Lehn, P. (1989). Hematopoietic plantation. Transfusion Medicine Reviews, 8, 45–58. reconstitution in a patient with Fanconi’s anemia by means of 8. Beatty, P. G., Clift, R. A., Mickelson, E. M., Nisperos, B. B., umbilical-cord blood from an HLA-identical sibling. New England Flournoy, N., Martin, P. J., . . . Hansen, J. A. (1985). Marrow trans- Journal of Medicine, 321, 1174–1178. plantation from related donors other than HLA-identical siblings. 21. Rubinstein, P., Carrier, C., Scaradavou, A., Kurtzberg, J., New England Journal of Medicine, 313, 765–771. Adamson, J., Migliaccio, A. R., . . . Stevens, C. E. (1998). Outcomes 9. Vogelsang, G. B., Hess, A. D., & Santos, G. W. (1988). Acute graft- among 562 recipients of placental-blood transplants from unre- versus-host disease: Clinical characteristics in the cyclosporine lated donors. New England Journal of Medicine, 339, 1565–1577. era. Medicine, 67, 163–174. 22. Scaradavou, A., Brunstein, C. G., Eapen, M., Le-Rademacher, J., 10. Kernan, N. A., Bartsch, G., Ash, R. C., Beatty, P. G., Champlin, R., Barker, J. N., Chao, N., . . . Shpall, E. J. (2013). Double unit grafts Filipovich, A., . . . Blume, K. G. (1993). Analysis of 462 transplan- successfully extend the application of umbilical cord blood trans- tations from unrelated donors facilitated by the National Marrow plantation in adults with acute leukemia. Blood, 121, 752–758. Donor Program. New England Journal of Medicine, 328, 593–602. 23. Storch, E., Mark, T., Avecilla, S., Pagan, C., Rhodes, J., Shore, T., . . . 11. Be the Match. (n.d.). National Marrow Donor Program Survey. Cushing, M. (2015). A novel hematopoietic progenitor cell mobi- Retrieved from www.marrow.org. lization and collection algorithm based on preemptive CD34 enu- 12. Perumbeti, A., Sacher, R. A., & Besa, E. C. (2012). Hematopoietic meration. Transfusion, 55(8), 2010–2016. doi: 10.1111/trf.13076 stem cell transplantation. Medscape Reference. Retrieved from emed- 24. Schots, R., Van Riet, I., Damiaens, S., Flament, J., Lacor, P., icine.medscape.com/article/208954-overview. Staelens, Y., . . . De Waele, M. (1996). The absolute number of 13. Heslop, H. E. (2012). Overview of hematopoietic stem cell circulating CD34+ cells predicts the number of hematopoietic transplantation. In R. Hoffman, E. J. Benz Jr, L. E. Silberstein, stem cells that can be collected by apheresis. Bone Marrow H. E. Heslop, J. I. Weitz & J. Anastasi (Eds.), Hematology: Basic Transplant, 17, 509–515. principles and practice, 6th ed., (pp. 1542–1545). Philadelphia: 25. Rubinstein, P., Dobrila, L., Rosenfield, R. E., Adamson, J. W., Saunders. Migliaccio, G., Migliaccio, A. R., . . . Stevens, C. E. (1995). Process- 14. Stein, A. S., & Forman, S. J. (1999). Autologous hematopoietic ing and cryopreservation of placental/umbilical cord blood for stem cell transplantation for acute myeloid leukemia. In E. D. unrelated bone marrow reconstitution. Proceedings of the National Thomas, K. G. Blume, & S. J. Forman (Eds.), Hematopoietic stem Academy of Science USA, 92(10), 119–122. cell transplantation, 2nd ed., (pp. 963–977). Boston: Blackwell 26. Wissel, M. E., & Lasky, L. C. (1995). Progenitor processing and Science. cryopreservation. In: M. E. Brecher, L. C. Lasky, R. A. Sacher, & 15. Armitage, J. O. (1994). Bone marrow transplantation. New England L.A. Issitt, eds. Hematopoietic progenitor cells: Processing, standards, Journal of Medicine, 330, 827–838. and practice (pp. 109–124). Bethesda, MD: AABB. Hematopoietic Stem Cell Transplantation 661 27. Harris, D. T., Schumacher, M. J., Rychlik, S., Booth, A., Acevedo, 41. Jadus, M. R., & Wepsic, H. T. (1992). The role of cytokines in A., Rubinstein, P., . . . Boyse, E. A. (1994). Collection, separation, graft-versus-host reactions and disease. Bone Marrow Transplanta- and cryopreservation of umbilical cord blood for use in trans- tion, 10, 1–14. plantation. Bone Marrow Transplant, 13, 135–143. 42. Krenger, W., & Ferrara, J. L. (1996). Dysregulation of cytokines 28. Sims, L. C., Brecher, M. E., Gertis, K., Jenkins, A., Nickischer, D., during graft-versus-host disease. Journal of Hematotherapy, 5, 3–14. Schmitz, J. L., . . . Bentley, S. A. (1997). Enumeration of CD34- 43. Chao, N. J., Schmidt, G. M., Niland, J. C., Amylon, M. D., Dagis, positive stem cells: Evaluation and comparison of three methods. A. C., Long, G. D., . . . Forman, S. J. (1993). Cyclosporine, metho- Journal of Hematotherapy, 6, 213–226. trexate, and prednisone compared with cyclosporine and pred- 29. Sutherland, D. R., Anderson, L., Keeney, M., Nayar, R., & nisone for prophylaxis of acute graft-versus-host disease. New Chin-Yee, I. (1996). The ISHAGE guidelines for CD34+ cell deter- England Journal of Medicine, 329, 1225–1230. mination by flow cytometry. International Society of Hematother- 44. Porter, D., & Antin, J. (1996). Graft-versus-leukemia effect of apy and Graft Engineering. Journal of Hematotherapy, 5, 213–226. allogeneic bone marrow transplantation and donor mononuclear 30. Keeney, M., Chin-Yee, I., Weir, K., Popma, J., Nayar, R., & cell infusions. In: J. Winter, ed. Blood stem cell transplantation Sutherland, D. R. (1998). Single platform flow cytometric absolute (pp. 57–85). Norwell, MA: Kluwer Academic. CD34+ cell counts based on the ISHAGE guidelines. Interna- 45. Mackinnon, S., Papadopoulos, E. B., Carabasi, M. H., Reich, L., tional Society of Hematotherapy and Graft Engineering. Cytom- Collins, N. H., Boulad, F., . . . Kernan, N. A. (1995). Adoptive etry, 34, 61–70. immunotherapy evaluating escalating doses of donor leukocytes 31. Gambell, P., Herbert, K., Dickinson, M., Stokes, K., Bressel, M., for relapse of chronic myeloid leukemia after bone marrow trans- Wall, D., . . . Prince, H. M. (2012). Peripheral blood CD34+ cell plantation: Separation of graft-versus-leukemia responses from enumeration as a predictor of apheresis yield: An analysis of graft-versus-host disease. Blood, 86, 1261–1268. more than 1,000 collections. Biology of Blood and Marrow Transplan- 46. Bowden, R. A., Slichter, S. J., Sayers, M. H., Mori, M., Cays, M. J., tation, 18(5), 763–772. & Meyers, J. D. (1991). Use of leukocyte-depleted platelets and 32. AABB. (2015). Standards for cellular therapy services (7th ed.). cytomegalovirus-seronegative red blood cells for prevention Bethesda, MD: Author. of primary cytomegalovirus infection after marrow transplant. 33. Douay, L., Gorin, N. C., Mary, J. Y., Lemarie, E., Lopez, M., Blood, 78, 246–250. Najman, A., . . . Salmon, C. (1986). Recovery of CFU-GM from 47. AABB. (1997). Leukocyte reduction for the prevention of transfusion- cryopreserved marrow and in vivo evaluation after autologous transmitted cytomegalovirus (Bulletin No. 97-2). Bethesda, MD: bone marrow transplantation are predictive of engraftment. Author. Experimental Hematology, 14, 358–365. 48. Bowden, R. A., Slichter, S. J., Sayers, M., Weisdorf, D., Cays, M., 34. Bensinger, W., Appelbaum, F., Rowley, S., Storb, R., Sanders., J., Schoch, G., . . . Miller, W. (1995). A comparison of filtered Lilleby, K., . . . Weaver, C. (1995). Factors that influence collection leukocyte-reduced and cytomegalovirus (CMV) seronegative and engraftment of autologous peripheral-blood stem cells. Jour- blood products for the prevention of transfusion-associated CMV nal of Clinical Oncology, 13, 2547–2555. infection after marrow transplant. Blood, 86, 3598–3603. 35. Haas, R., Mohle, R., Fruhauf, S., Goldschmidt, H., Witt, B., 49. Preiksaitis, J. K. (2000). The cytomegalovirus-“safe” blood prod- Flentje., M., . . . Hunstein, W. (1994). Patient characteristics asso- uct: Is leukoreduction equivalent to antibody screening? Transfu- ciated with successful mobilizing and autografting of periph- sion Medical Reviews, 14, 112–136. eral blood progenitor cells in malignant lymphoma. Blood, 83, 50. Dignan, F. L., Wynn, R. F., Hadzic, N., Karani, J., Quaglia, A., 3787–3794. Pagliuca, A., . . . Potter, M. N. (2013). BCSH/BSBMT guideline: 36. Siena, S., Schiavo, R., Pedrazzoli, P., & Carlo-Stella, C. (2000). Diagnosis and management of veno-occlusive disease (sinusoidal Therapeutic relevance of CD34 cell dose in blood cell trans- obstruction syndrome) following haematopoietic stem cell plantation for cancer therapy. Journal of Clinical Oncology, 18(6), transplantation. British Journal of Haematology, 163(4), 444–457. 1360–1377. 51. Gratwohl, A., Baldomero, H., Aljurf, M., Pasquini, M. C., 37. Bryant, E., & Martin, P. J. (1999). Documentation of engraftment Bouzas, L. F., Yoshimi, A., . . . Kodera, Y. (2010). Hematopoietic and characterization of chimerism following hematopoietic cell stem cell transplantation: A global perspective. Journal of the transplantation. In E. D. Thomas, K. G. Blume, & S. |
J. Forman American Medical Association, 303, 1617–1624. (Eds.), Hematopoietic stem cell transplantation (2nd ed., 52. Gooley, T. A., Chien, J. W., Pergam, S. A., Hingorani, S., Sorror, M. L., pp. 197–206). Boston: Blackwell Science. Boeckh, M., . . . Martin, P. J. (2010). Reduced mortality after allo- 38. Murphey, K. (2013). Chimerism analysis following hematopoi- geneic hematopoietic-cell transplantation. New England Journal of etic stem cell transplantation. Methods in Molecular Biology, 999, Medicine, 363, 2091–2101. 137–149. 53. Bhatia, S., Robison, L. L., Francisco, L., Carter, A., Liu, Y., Grant, M., 39. Manasatienkij, C., & Chatchawin, R. (2012). Clinical application . . . Forman, S. J. (2005). Late mortality in survivors of autologous of forensic DNA analysis: A literature review. Journal of the Medi- hematopoietic-cell transplantation: Report from the Bone Marrow cal Association Thailand, 95(10), 1357–1363. Transplant Survivor Study. Blood, 105, 4215–4222. 40. Seattle Cancer Care Alliance. (2013). Chimerism-testing: Chimerism 54. Chinen, J., & Puck, J. M. (2004). Perspectives of gene therapy for testing/engraftment analysis. Retrieved from www.seattlecca.org/ primary immunodeficiencies. Current Opinion in Allergy and Clini- Chimerism-Testing.cfm. cal Immunology, 4(6), 523–527. This page intentionally left blank Section Six Body Fluids 663 Chapter 30 Morphologic Analysis of Body Fluids in the Hematology Laboratory Jerelyn Walters, BS Objectives—Level I At the end of this unit of study, the student should be able to: 1. List the types of body fluids studied in the 6. Describe the appearance of bacterial clinical laboratory, and describe the body and fungal organisms in Wright-stained cavities in which they are found. preparations. 2. List physical characteristics of various 7. List the differentiating morphologic fluids. features of benign and malignant cells 3. Describe cell-counting and slide preparation on Wright-stained preparations. techniques 8. List the types of crystals that can be found 4. List and describe the normal cells seen in in joint fluids. each body fluid type. 9. Describe routine hematologic methods for 5. Describe various artifact types that can be semen analysis. seen in body fluid preparations including 10. Interpret automated lamellar body counts in those in cytocentrifuged specimens. amniotic fluid. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Identify the procedure used to obtain each 3. Define and differentiate exudates and type of body fluid. transudates, chylous and pseudochylous fluids. 2. Describe the production of serous, synovial, 4. Identify common cell types seen in CSF, and cerebrospinal fluid (CSF). serous, synovial, and bronchoalveolar lavage fluids. 664 Morphologic Analysis of Body Fluids in the Hematology Laboratory 665 5. Evaluate the significance of microorganisms 8. Define birefringence, and explain its use in found in Wright-stained body fluid prepara- the identification of crystals in body fluids. tions and suggest methods to confirm them. 9. Identify and differentiate the various 6. Compare and contrast the morphologic types of crystals that can be found in joint distinction between benign tissue cells and fluids, and associate them with particular malignant cells in cytocentrifuged Wright- disorders. stained preparations of body fluids, and 10. Given CSF parameters, differentiate a true identify these cells when seen. hemorrhage from a traumatic tap. 7. Recognize erythrophagocytosis, and explain its significance in CSF. Chapter Outline Objectives—Level I and Level II 664 Types of Body Fluids 666 Key Terms 665 Hematologic Analysis of Body Fluids 670 Background Basics 665 Analysis of Other Fluids 693 Case Study 666 Summary 698 Overview 666 Review Questions 698 Introduction 666 References 700 Key Terms Arachnoid mater Effusion fluid Pleocytosis Ascites Exudate Pleural cavity Ascitic fluid Lamellar body counts (LBCs) Pseudochylous Birefringence Meninges Semen Bronchoalveolar lavage (BAL) Pericardial cavity Sperm Central nervous system (CNS) Pericardium Synovium Cerebrospinal fluid (CSF) Peritoneal cavity Transudate Chylous Peritoneum Dura mater Pia mater Background Basics Although the study of body fluids is not generally included Level I in textbooks of hematology, most clinical hematology • Recognize the common types of normal cells found laboratories have the responsibility to perform total cell in the peripheral blood. (Chapters 1, 5, 7, 8, 10) counts and differential cell counts on body fluid samples. • List the major types of neoplastic hematopoietic dis- Although much of this chapter can be read independently orders. (Chapter 23) of the other chapters, the chapter builds on the general knowledge of hematologic morphology, cell counting, and • Describe and understand the use of the hemocytom- hematologic neoplasms. You should review the following eter to perform manual cell counts. (Chapter 37) concepts before beginning this chapter: • Describe the major types of lymphoma. (Chapter 28) 666 Chapter 30 Level II • Describe and identify the cells associated with • Recognize reactive hematopoietic cells found in the the various types of acute (precursor) leukemia. peripheral blood. (Chapters 7, 8, 10, 21, 22) (Chapters 26, 27) • Describe and recognize the hematopoietic precur- • Compare and contrast the various types of lympho- sors found in the normal bone marrow. (Chapters 4, proliferative disorders. (Chapter 28) 5, 7, 8, 38) CASE STUDY Introduction We refer to this case study throughout the chapter. The hematology laboratory plays an increasingly important Carolyn, a 51-year-old woman who is otherwise in role in the morphologic evaluation of body fluids. This is her usual state of good health, has flu-like symp- primarily because of the use of the cytocentrifuge, which toms. After two weeks, her cough and fever persist. markedly improves morphology over the previously used Antibiotics were started, and 2 days later she has direct smear technique. The cytocentrifuge is an instrument severe right-side chest pain and worsening short- used to prepare slides from body fluid specimens other than ness of breath. Radiologic studies show a large peripheral blood. The sample is centrifuged directly onto effusion in the right pleural cavity. A thoracentesis a glass slide and yields a concentrate of cells with excel- is performed, and 1 liter of thick, yellow fluid is lent morphology. Properly prepared, a cytocentrifuge slide aspirated. yields an approximate 20-fold concentration of cells. As you read and study this chapter, think about The Wright-stained cytocentrifuge-prepared slide is the type of fluid this could be and the laboratory made in the hematology laboratory to perform a differen- studies that should be done on the fluid to assist in tial of the nucleated cells present in the fluid. In addition, diagnosis. this slide is valuable in making many important diagnoses, both benign and malignant. Most malignant cells, including hematopoietic malignancies, carcinomas, and sarcomas, can be recognized microscopically. The hematopoietic malig- nancies are generally easier to diagnose from the Wright- Overview stained slide than from the routine cytology preparation, which is prepared by alcohol fixation of cells and Papani- The hematology laboratory usually receives body fluids for colaou staining. Another advantage of the slides made in cell counts and morphologic evaluation. The types of cells the hematology laboratory is that they are prepared within found, and their concentration are helpful information for an hour of specimen receipt, whereas cytology prepara- the clinician in arriving at a diagnosis. The cells that can be tions take longer. Additionally, important nonmalignant found in these fluids include white blood cells (WBCs) and findings, such as intracellular bacteria and fungi, which red blood cells (RBCs) as well as tissue and tumor cells. The can be detected on a Wright-stained slide, frequently are cytocentrifuge is used to prepare slide preparations for mor- not seen on cytology slides. Hematologic slides, however, phologic review, and Wright stain commonly is used to stain should not replace cytology preparations, which have better the cells on the slide. Identifying and differentiating the var- retention of nuclear detail and are superior to hematologic ious types of cells seen in body fluids requires experience. slides when attempting to determine the specific type of car- However, all laboratory professionals who perform body cinoma or sarcoma present. Both techniques are necessary fluid analysis should be able to differentiate the types of and aid in arriving at the most accurate diagnosis. WBCs, normal tissue lining cells, and most malignant cells from benign cells. This chapter describes the sites where body fluids are found, the methods by which the fluids are obtained, and an explanation of why fluids accumulate at Types of Body Fluids these sites. The focus of the chapter is the hematologic anal- The body fluids discussed in this chapter are those com- ysis of these fluids and identification of the types of cells monly analyzed in the hematology laboratory, including and inclusions that can be found in each fluid. The artifacts pleural, peritoneal, and less commonly pericardial fluids that can be found and the way to differentiate them from that, as a group, are called serous fluids. Also included are significant inclusions are also described and depicted. This cerebral spinal fluid from the central nervous system (CNS) chapter is presented in atlas format because this is the most and synovial fluid from joint spaces. The medical tech- helpful way to discuss the morphology of body fluid cells. niques for obtaining these fluids (Table 30-1) are, at times, Morphologic Analysis of Body Fluids in the Hematology Laboratory 667 The pleural membrane’s purpose is to provide a moist sur- Table 30.1 Sites of Origin of Body Fluids, Procedure to face to minimize friction between the lung and chest wall Obtain Fluid, and Type of Fluid as respiration occurs (Figure 30-1). Anatomic Site Procedure Fluid Obtained The peritoneal cavity consists of the space between the Pleural cavity Thoracentesis Pleural fluid inside of the abdominal wall and the outside of the stom- Pericardial cavity Pericardial aspiration Pericardial fluid ach, small and large intestine, liver, and superior aspect of Peritoneal cavity Paracentesis Peritoneal fluid the urinary bladder and uterus. The kidneys are positioned (ascitic fluid) posterior to the peritoneal membrane (the peritoneum) and Joint space Joint aspiration Synovial fluid are referred to as retroperitoneal (Figure 30-2). (arthrocentesis) Spinal cord Spinal tap, lumbar puncture Cerebrospinal fluid Pericardial cavity Left lung inappropriately used to identify the type of fluid. For exam- Parietal ple, the laboratory can receive a fluid labeled thoracentesis pleura fluid, but for accuracy of reporting results, the fluid should Heart Visceral be identified as a pleural fluid. This chapter also reviews pleura hematologic testing of less common fluids, including bron- Pleural Pleural chial lavage fluid, semen for routine analysis, and amniotic cavity effusion fluid used to obtain lamellar body counts. Serous Fluids Right lung The pleural, pericardial, and peritoneal cavities are similar in structure and function. These cavities are often referred to as spaces (i.e., pleural space or peritoneal space) but are only Mesothelial potential spaces and do not normally contain any apprecia- cells ble amount of fluid. These potential spaces are the minimal gap between the serous membrane structures that comprise the pleural, peritoneal, or pericardial membrane. The serous Figure 30.1 Diagram of right lung illustrating the pleural cavity membranes are identified as the parietal membrane, which is the and inset showing mesothelial cells of the parietal and visceral outer membrane, and the visceral membrane, which is the inner pleura. Illustration of heart shows the pericardial cavity. Diagram of membrane closest to the internal organs. These membranes the lung shows the abnormal accumulation of fluid (effusion) in the are composed of a continuous single layer of mesothelial cells pleural cavity compressing the lower left lung. and submesothelial connective tissue. Normally, there is just enough fluid between these two serous membrane surfaces to keep them moist. For example, the adult pleural space nor- Visceral peritoneum Liver mally contains less than 15 mL of fluid. The fluid that forms in these spaces is an ultrafiltrate of plasma, which means large molecules such as protein cholesterol, and cellular elements Stomach normally do not pass through the membrane in appreciable Pancreas amounts. The parietal membrane produces the serous fluid Intestine Duodenum that the visceral membrane absorbs at a dynamic rate. Fluid Retroperitoneal transfer across the pleural membranes is thought to be as space high as 5,000–10,000 mL per 24 hours in normal adults. Fluid Peritoneal production depends on four factors known as Starling forces: space capillary hydrostatic pressure, plasma oncotic pressure, lym- Kidney phatic resorption, and capillary permeability.1,2 Any patho- Parietal peritoneum logic state affecting one or several of these four factors can result in an abnormal collection of fluid. As a group, excess Bladder Rectum fluids that form in the pleural, peritoneal, or pericardial space are referred to as effusion fluids, although peritoneal |
effu- sion fluid is often referred to as ascitic fluid or ascites. Figure 30.2 Abdominal cavity illustrating the peritoneal space The pleural cavities (left and right) consist of the space between the inside of the abdominal wall and the outside of the liver, between the lung and the inside portion of the chest wall. stomach, intestine, and dome of the bladder. 668 Chapter 30 The pericardial cavity is the space between the out- Differentiation of transudate and exudate effusion ermost aspect of the heart and the innermost aspect of the fluids can assist the physician in determining whether the pericardial sac. The pericardium is the membrane that cause of the effusion is a systemic disease (transudate) or a provides a continuous covering of the pericardial cavity primary pathologic local disease (exudate). Although hema- (Figure 30-1). With advances in imaging, pericardial fluids tologic results such as cell counts, and cell types are not are seen infrequently in the laboratory. included in the transudate/exudate differential equation, Effusion fluids are traditionally classified as transu- they do provide significant information in discerning the dates or exudates to aid in clinical diagnosis. An effusion cause of many exudates. can accumulate from a systemic disease state (transudate) or a primary pathologic state of the area (exudate). Transu- Cerebrospinal Fluid dates are frequently a result of increased capillary hydro- The central nervous system (CNS), consisting of the brain static pressure as seen with congestive heart failure or of and spinal cord, is normally lined by special membranes decreased plasma oncotic pressure as seen with hypopro- referred to as meningeal membranes, or meninges, that protect teinemia because of nephrotic syndrome or liver failure. it. The meninges consist of a relatively thick dura mater, The capillary hydrostatic pressure and plasma oncotic the outermost membrane; a thinner arachnoid mater, the pressure control the rate of filtration and reabsorption middle membrane; and an innermost pia mater that lies of the serous fluid. Changes in these pressures can cause directly on the surface of the brain and spinal cord. fluid to accumulate, but the fluid is still an ultrafiltrate of The cerebrospinal fluid (CSF) occupies the subarach- plasma, resulting in an effusion fluid that is fairly clear and noid space between the arachnoid mater and pia mater and uncomplicated. protects the brain and spinal cord from sudden changes An exudate is formed by increased capillary perme- in pressure, provides a stable chemical environment, and ability and/or decreased lymphatic resorption. Many dif- allows for the exchange of nutrients and waste products ferent pathologic processes, such as bacterial infections, (Figure 30-3). The choroid plexus cells and ependymal lin- viral infections, neoplasms, and collagen vascular diseases, ing cells found in the ventricles produce the CSF. This CSF can cause an exudative effusion. These diseases can damage circulates through the ventricular system in the cerebrum, the serous membrane, rendering it incapable of providing cerebellum, and brain stem and completely covers the an ultrafiltrate of plasma. As a result, plasma constituents surface of the brain and spinal cord. The CSF, a product normally filtered such as protein, cholesterol, and cellular of ultrafiltration and active secretion, is made at a rate of elements can seep into the fluid resulting in a more com- approximately 21 mL/hour5 and is reabsorbed by the arach- plicated fluid. noid cells. The total CSF volume in adults is 90–150 mL, but Although traditional criteria for differentiating tran- the constant production and reabsorption of nearly 500 mL sudates from exudates included specific gravity measure- per day results in a dynamic fluid that is replaced every ments and leukocyte counts, the current criteria for pleural 5–6 hours. Neonates have a CSF volume of 10–60 mL. The effusions, known as Light’s criteria, use serum and fluid mea- four primary reasons to test CSF in the laboratory are2: surements for protein and lactate dehydrogenase (LDH).3 According to Light’s criteria, if a pleural effusion meets one • To detect meningitis of the following criteria, it is classified as an exudate. • To detect a subarachnoid hemorrhage • Pleural fluid/serum protein ratio more than 0.5 • To detect a malignancy • Pleural fluid/serum LDH ratio more than 0.6 • To detect demyelinating disease • Pleural fluid LDH more than 2 3 the normal upper limit Meningitis is an inflammation of the meningeal mem- for serum LDH branes most often caused by viral, bacterial, or fungal infec- If the patient has congestive heart failure or cirrho- tion in the CSF. Regardless of the cause, the universal finding sis and the pleural fluid meets the criteria of an exudate in meningitis is an elevated leukocyte count. The presence by a small amount, it is recommended that the difference of an elevated leukocyte count is called p leocytosis, a term between the serum protein and pleural fluid protein be cal- unique to CSF. Determining the cause of meningitis is of culated. If the difference is greater than 3.1 g/dL, the fluid great importance because bacterial meningitis can be life- is probably a transudate.3 threatening. Although serious, viral meningitis is rarely Current criteria for differentiating peritoneal effusions fatal. Fungal meningitis is more commonly seen in indi- use a calculation known as the serum–ascites albumin gradi- viduals with compromised immune systems. ent (SAGG).4 If the serum albumin minus the fluid albumin Subarachnoid hemorrhage (SAH) is bleeding into the is equal to or greater than 1.1 g/dL the fluid is classified space between the two innermost meningeal membranes— as a transudate. A result of less than 1.1 g/dL indicates an the arachnoid membrane and the pia mater. SAH is a life- exudate. threatening event with a mortality rate as high as 25–50%. Morphologic Analysis of Body Fluids in the Hematology Laboratory 669 Skull Brain Subarachnoid space with CSF Spinal cord Skull Vertibral bone Dura mater Subdural space Arachnoid mater Subarachnoid space Pia mater Figure 30.3 Central nervous system showing the subarachnoid space containing cerebral spinal fluid (CSF) covering the brain and spinal cord. Inset illustrates the meninges: dura mater, arachnoid mater, and pia mater. Thus, prompt diagnosis is critically important. The current Currently there is no D-dimer test that has been gold standard for detection of an SAH is computed tomog- approved for CSF testing by the FDA, although a fair amount raphy (CT), which has a reported sensitivity and specificity of published literature describing its use does exist.6,9 approaching 100% if performed within 6 hours of the onset The detection of malignant cells in CSF, including leu- of symptoms (e.g., severe or “thunderclap” headache).6 kemic cells, can be critically important in both treatment and Unfortunately, the sensitivity and specificity decrease if test- prognosis. Many current chemotherapeutic agents do not ing extends beyond 6 hours. In cases when patients delay cross the blood brain barrier; thus, malignant cells harbored seeking treatment or CT might not be readily available, the in the CNS can be a source of disease relapse. Intrathecal physician likely performs a lumbar puncture and submits chemotherapy (injection directly into the spinal canal) is a CSF to the hematology laboratory for testing to assess the therapeutic option in these cases. Malignant cells found in probability of an SAH. A patient with an SAH has significant RBCs in the spinal fluid. However, the diagnostic accuracy of detecting an SAH by examination of CSF is an imperfect Table 30.2 Criteria for Differentiation of Subarachnoid Hemorrhage (SAH) and Traumatic Tap science at best. A lumbar puncture can result in a “traumatic tap,” meaning peripheral blood is introduced into the CSF Subarachnoid Hemorrhage Traumatic Tap sample during the puncture. It is estimated that 20% of lum- Equal amount of blood in all tubes Decreasing amount of blood in bar punctures are traumatic, making it difficult to determine sequential tubes the actual source of the RBCs. See Table 30-2 for traditional Blood does not clot Blood clots detected in 1 or more tubes criteria for differentiating a traumatic tap from a true SAH. It should be noted, however, that the differentiating char- Xanthochromia Colorless supernatant Negative D-dimer testa Positive D-dimer testaacteristics are not infallible because it is possible to have a traumatic tap overlying an SAH.7,8 a Currently, no FDA-approved D-dimer test is used for CSF testing. 670 Chapter 30 the CSF are often from metastatic disease, most commonly from breast and lung tumors. Primary brain tumors seldom involve the meninges, and the malignant cells from these tumors are not often seen in the CSF. Examination of the CSF for the detection of demyelin- Bone Synovial membrane ating disease includes immunoglobulin and electrophore- sis testing, which is typically not performed in the general Cartilage Synovial hematology laboratory. fluid Checkpoint 30.1 To obtain a sample of CSF for analysis, the needle must be inserted into what area of the central nervous system? Figure 30.4 Knee joint illustrating synovial membrane lining and synovial fluid. Synovial Fluid Some bony joints of the body are lined with special mem- tool in diagnosing interstitial lung diseases, infections, branes called the synovium that normally consists of a and malignancies. BAL is especially helpful in diagnosing single layer of synovial cells (Figure 30-4). The joint space infections caused by difficult-to-culture organisms such as contains synovial fluid that acts as a lubricant and a trans- Pneumocystis jirovecii (formerly P. carinii). Other conditions port medium for nutrients to get to the joint’s bone surfaces. that have specific findings in BAL include alveolar proteino- Minimal fluid normally is present with the largest synovial sis, pulmonary hemorrhage syndrome, eosinophilic pneu- joint, the knee, containing less than 4 mL. The synovial fluid monia, hypersensitivity pneumonitis, and sarcoidosis.10 is produced in part by the synovial cells and is an ultrafil- The hematologic analysis of BAL centers on the enu- trate of plasma. Synovial fluid also contains a mucopolysac- meration and morphologic differentiation of the nucle- charide called hyaluronic acid, which sometimes makes the ated cells present. The concentration of cells varies with fluid so thick that it hampers laboratory studies. A small the quality of the sample (amount of saline infused and amount of hyaluronidase powder can be added to an aliquot retrieved) but as with other body fluids, the type of cell of synovial fluid to decrease the viscosity and allow accu- present typically has more importance than the cell count rate cell counting and proper slide preparation. Synovial itself. Most cells normally present are macrophages (more fluids are not identified as transudates or exudates for diag- than 80%) and a few hematopoietic cells, primarily lympho- nostic purposes but are classified based on laboratory find- cytes (considered a contaminant caused by the procedure).11 ings (Table 30-3). The two conditions in which laboratory Both fungal and bacterial microorganisms can be seen in the results are essentially diagnostic of specific joint disease are hematology-stained smear and should be noted if present, a positive gram stain and culture indicating infection, and but proper identification of these elements should be left to the identification of monosodium urate or calcium pyro- the microbiology laboratory. phosphate dehydrate crystals indicating a crystal-induced arthritis.1 Bronchoalveolar Lavage (BAL) Hematologic Analysis A bronchoalveolar lavage (BAL) is not a normally occur- of Body Fluids ring fluid but is obtained by a diagnostic procedure of infus- ing and removing a sterile saline solution into the alveolar Specimen Collection and Handling and bronchial airspaces of the lung via a bronchoscope. The Normal serous and synovial fluid do not clot. However, lavage procedure allows harvesting the cellular and noncel- body fluids are submitted to the laboratory because of a sus- lular elements of the lower respiratory tract; it is a valuable pected abnormal condition. That, along with the possibility Table 30.3 Diagnostic Joint Fluid Groups Group IV Crystal Group I Noninflammatory Group II Inflammatory Group III Septic Group V Hemorrhagic Induced Osteoarthritis, traumatic Rheumatoid arthritis, lupus Infection: bacterial, Gout, pseudo gout Traumatic injury, arthritis, other degenerative erythematosus, other mycobacterial, or fungal hemophilia joint disorders immunologic disorders Morphologic Analysis of Body Fluids in the Hematology Laboratory 671 of peripheral blood contamination introduced in the process print through a sample to differentiate hazy from cloudy. of obtaining the sample, increases the possibility of clotting Color can vary widely from colorless to yellow, pink, red, occurring in the sample. To ensure accurate cell counts, the or brown. Samples can also be described as grossly bloody. preferred sample for hematology analysis is one submitted CSF is |
normally clear and colorless. As few as 200 in an anticoagulant tube containing EDTA or sodium hepa- WBCs/mcL or 400 RBCs/mcL can cause a hazy appear- rin. There are anecdotal reports of lithium heparin forming ance. CSF that is bloody requires examination of all tubes rhomboid crystals that can be confused with calcium pyro- received to aid in differentiating a traumatic tap from a sub- phosphate dehydrate crystals, so it should be avoided as an arachnoid hemorrhage. The presence of blood because of a anticoagulant for synovial fluids.12 traumatic tap decreases as the fluid is collected in sequen- Viscous synovial fluids should be treated with hyal- tial tubes; thus, the last tube collected will be less bloody uronidase before performing cell counts or preparing than the first tube collected. An equal amount of blood in cytocentrifuge slides. The volume of fluid received by the all tubes is suggestive of a subarachnoid hemorrhage. Some laboratory should be recorded. laboratories perform RBC counts on the first and last tube to Normal CSF also does not clot, but it is never submitted aid in identifying the cause of the bloody CSF. A traumatic in an anticoagulant tube because the presence of a clot is tap is suggested when the total RBC count in the first tube an indicator of a traumatic tap rather than a subarachnoid collected is significantly higher than that in the last tube hemorrhage.1 CSF is typically collected in a sequential series collected. of three or four sterile tubes without anticoagulant. The CSF should always be examined for xanthochromia, a last tube collected should be used for hematologic analysis yellow, orange, or pink coloration of the CSF, often caused because it is the least likely to be affected by any periph- by the presence of breakdown products of red blood cells; eral blood contamination because of performing the lumbar it is usually thought to indicate true CNS hemorrhage puncture. Knowing the sequence in which the tubes were (Figure 30-5). If the CSF is cloudy or bloody, an aliquot of collected to differentiate a subarachnoid bleed from a trau- fluid should be centrifuged, and the supernatant examined matic tap is essential. The total volume of CSF and number for xanthochromia. Xanthochromia appears within in sev- of tubes received in the laboratory should be recorded. eral hours of the bleed and can persist for a week or more. BAL samples do not require anticoagulants, and a por- Xanthochromia also can occur if a grossly bloody fluid from tion of the lavage is usually submitted to the hematology a traumatic tap sits for some time before it is centrifuged or laboratory in a sterile container. Whereas there is presently the amount of plasma accompanying the blood introduced no published consensus for the laboratory handling or pro- during collection is sufficient to color the supernatant. cessing of BAL, it is generally agreed the sample should not Additionally, patients with very high serum bilirubin levels be filtered to remove any interfering mucus because this can have spillover into the CSF, causing the fluid to appear will result in loss of cells.1 Samples can be treated with a xanthochromic. More than 90% of patients with a serum bil- commercially available mucolytic agent such as Mucolexx™ irubin of 10–15 mg/dL have xanthochromic CSF. Infection to digest the mucus and allow accurate cell counting and or inflammatory conditions with high CSF protein levels cytocentrifuge slide preparation.13 (greater than 150 mg/dL) can also exhibit xanthochromia.1 Clotted samples of any type of body fluid negatively affect the accuracy of both cell count and differential results. However, because body fluids are considered “irretriev- able” samples, clotted samples should not be summarily rejected for analysis. Small fibrin clots can be removed from the sample and the cell count reported as “approximate, small clots present.” The approximate count can be helpful if it indicates an increased cell count and, in truth, the type of cell present often has more importance than the actual cell count. Cell counts should not be performed on samples that are solidly clotted, but a valid attempt should be made to get some cells on a slide either by cytocentrifuging any free liquid or by a touch-prep technique. Physical Characteristics The clarity and color of all fluids should be noted and reported. Clarity can be graded simply as clear, hazy, or Figure 30.5 CSF samples showing xanthochromic (left) and cloudy. Some laboratory professionals use the ability to read colorless (right) supernatant. 672 Chapter 30 Spectrophotometric methods for a more accurate detection of xanthochromia have been proposed, but recent literature CASE STUDY (continued from page 666) supports visual examination as a more clinically appropri- Radiologic studies show a large effusion in the ate method.14 Long considered one of the better indicators right pleural cavity. A thoracentesis is performed, of a subarachnoid hemorrhage, xanthochromia does have and 1 liter of thick, yellow fluid is aspirated. limitations in its predictive accuracy and can indicate a pre- Laboratory studies show a total protein of vious rather than current bleed. 4.5 g/dL (serum = 6 g/dL), lactate dehydrogenase Normal serous fluids (pleural, peritoneal, pericardial) 40 U/L (serum = 50 U/L), and total leukocyte are clear and pale yellow. Increased cellular elements, infec- count of 20,000/mcL with 90% segmented tion, or malignancy can cause hazy or cloudy fluids. Bloody neutrophils, 10% histiocytes, and many fluids can be caused by a traumatic tap, pulmonary or degenerating cells. intestinal infarction, malignancy, or trauma. It is generally 1. Is this a transudate or exudate? not necessary to examine the supernatant of serous fluids unless a chylous fluid is suspected. 2. Is this a chylous fluid? A chylous effusion has a characteristic milky, opaque appearance that remains in the supernatant after centrifuga- tion. Chylous effusions result from lymphatic vessel leakage and are not commonly seen. In the pleural cavity (chylotho- Cell Counting rax), this results from leakage of the major thoracic duct. In Cell counts of body fluids should be performed as soon as the peritoneal cavity, chylous effusions result from blockage possible because of the risk of WBC lysis. WBCs, especially of the lymphatic vessels. In both the pleural and peritoneal neutrophils, are less stable in body fluids because they are cavities, chylous effusions most often result from a malig- not in the usual nutritive plasma environment where they nancy such as lymphoma or carcinoma, or from trauma. normally exist. This type of fluid is rich in chylomicrons, has elevated tri- A common rule is to perform the cell count within glycerides, normal cholesterol, and its predominant cells 1 hour of collection. This 1-hour limit is critical for accu- are lymphocytes.2,15 A pseudochylous effusion is also milky rate CSF analysis because studies have shown significant and results from a chronic, long-standing effusion because WBC loss, particularly neutrophils, at 1 hour post collec- of conditions such as tuberculosis and rheumatoid pleuri- tion.17 Serous and synovial fluids have been shown to have tis.2 Pseudochylous effusions do not contain chylomicrons improved cellular stability when refrigerated for 24 hours, and have elevated cholesterol and low triglycerides. Cho- but these guidelines may not apply to fluids that form lesterol crystals are commonly seen, as is a mixed reactive because of sepsis or inflammation. cell population with many inflammatory and necrotic cells. Cell counts can be performed by manual or automated Synovial fluid is normally pale yellow, clear, and vis- methods. Automated methods provide more efficiency and cous. Hazy or cloudy synovial fluid can be the result of precision compared with manual methods. Regardless of infection, inflammation, or crystal formation. Bloody syno- the method used, the WBC count performed on body fluids vial fluid is often the result of trauma or traumatic tap but is more correctly reported as a total nucleated cell count can be seen in hemophilic hemarthrosis and poorly con- (TNCC). Monocytes, macrophages, normal tissue lining trolled anticoagulation therapy. cells, and some malignant cells often have similar cellular Viscosity is an important component of synovial fluid. size and nuclear content, making it impossible to count The high viscosity results from the glycoproteins, hyal- just the WBCs; thus, all nucleated cells should be included uronic acid, and lubricin produced by the synovial lining in the count. Cell counts performed on CSF traditionally cells. As seen in rheumatoid arthritis and other inflamma- include both the TNCC and the RBC count because the tory conditions, inflammation can cause a decrease in the RBCs can be helpful in detecting a subarachnoid hemor- viscosity often to the point that the synovial fluid has the rhage. As mentioned, the RBC count can be performed on consistency of a serous fluid. Viscosity can be assessed at both the first and last tube collected when there is suspicion the bedside or in the laboratory by performing the simple of a traumatic tap as an aid in differentiating the two condi- string test. Synovial fluid with normal viscosity will form a tions. RBC counts are of limited use in serous and synovial string more than 5–10 cm in length or longer when dripped fluids and are not performed routinely.2 When necessary, from a syringe or disposable transfer pipet. Inflammatory RBC counts in grossly bloody or hemorrhagic fluids should synovial fluid forms small drops like water.16 Viscosity is be performed by automated methods. A hematocrit can be typically reported as normal or decreased. more helpful in these cases. The color and clarity of BAL samples can have limited Synovial fluid should be treated with hyaluronidase clinical correlation because of variances in volume retrieved before cell counting or slide preparation. A small amount and concentration, but should be reported none the less. of dry hyaluronidase powder pinched between two clean Morphologic Analysis of Body Fluids in the Hematology Laboratory 673 applicator sticks should be added to a 0.5–1 mL aliquot of the fluid.18 The sample is then mixed gently for 20–30 sec- Table 30.4 Example of Suggested Dilutions for Manual Cell Counts in Hazy, Cloudy, and Bloody Body Fluids onds until it liquefies. If the sample aliquot is still viscous, an additional small amount of hyaluronidase powder can Hazy (1:2) Cloudy (1:5)a Bloody (1:10) be added. The dry powder will not cause a dilutional effect. 1-part fluid 1-part fluid 1-part fluid Cell counting should be performed immediately following 1-part diluent 4 parts diluent 9 parts diluent hyaluronidase treatment. a Mucoid BAL samples should be treated with a muco- For example, for a 1:5 dilution, use 100 mcL of fluid and 400 mcL of diluent. lytic agent before performing the cell count and preparing the slide. Mucolytic agents are usually liquid and can add a error. The cell counts on each side should match within significant dilution to the fluid. This dilution must be taken 10 cells on cell counts less than 100/mcL and 10% on cell into consideration when calculating the final cell count counts greater than 100/mcL.19 result. The average number of cells counted on both sides is used to calculate the final result. The area of the hemocy- MANUAL CELL COUNTS tometer counted is determined by the cellularity of the sam- Manual cell counts are performed using a hemocytom- ple. Low cell counts require counting all nine squares on eter. Disposable hemocytometers, such as the C-chip™, each side of the hemocytometer. Higher cell counts can be are preferred over the standard glass Neubauer chamber. performed by counting the four large corner squares (WBC The disposable hemocytometer has an integral coverslip, area on each side). If numerous RBCs are present, as in a is for single use, and avoids the hazards of cleaning glass bloody CSF, the usual RBC area (five small squares within hemocytometers. The Neubauer hemocytometer consists of the center large square) can be used to count the red blood a glass counting chamber and a separate glass coverslip that cells (1/5, or 0.2 of 1 mm2). The laboratory should provide must be properly placed for accurate counting. The hemo- guidelines indicating acceptable dilutions and the area to cytometer has two counting chambers, one on each side. be counted based on cellularity (online student resource Each counting chamber is divided into nine large squares Table 30-1). that are further subdivided into smaller squares. Each of the The cell count is calculated using the standard hemo- nine large squares measures 1 mm2. The coverslip gives the cytometer formula |
by considering the dilution made, if any, counting chamber a depth of 0.1 mm; thus, the total volume and the area counted. The formula for calculating hemocy- of each counting chamber is 0.9 mm3. (Chapter 37 describes tometer cell counts is detailed use of a hemocytometer.) Total cell count (cells/mm3) = Clear fluids can be counted undiluted. The pipet used Mean number of cells on both sides * Reciprocal of dilution to insert the fluid into the counting chamber can be rinsed with a stain such as new methylene blue before drawing Number of large squares on one side * Area of square * Depth up the fluid to assist in differentiating RBCs from WBCs when performing the cell count. To prevent any dilutional effect, the stain should be thoroughly expelled from the EXAMPLE pipet before drawing the sample. Sufficient stain clings to The number of cells counted in four large corner squares on the wall of the pipet to stain the WBCs, allowing more accu- each side of the hemocytometer: rate identification. Side 1: 115 Dilutions should be made for hazy, cloudy, or bloody Side 2: 125 fluids based on an estimate of cellularity that can be per- formed by observing a cover-slipped drop of fluid on a Mean of cells counted: 120 slide. Dilutions can be made with sterile saline or isotonic Dilution = 1 : 5 instrument diluent for counting both RBCs and WBCs. Sam- Area counted: four large squares (4 mm2) ples with increased RBCs can make it difficult to identify the Depth: 0.1 mm WBCs. These samples can be diluted with a commercially 120 * 5 available diluent containing acetic acid (to lyse the RBCs) = 1,500 cells/mm3 (mcL or 1.5 * 109/L) 4 * 1 mm2 * 0.1 mm or a combined acetic acid/dye solution (to stain the WBCs) such as Turk’s solution. Dilutions must be made with accu- rate pipetting devices (Table 30-4). A second person should verify manual calculations per- Both chambers (sides) of the hemocytometer should be formed in the laboratory whenever possible. plated with the fluid so that the count is performed in dupli- Quality control (QC) should be performed for manual cate. If a dilution is necessary, duplicate dilutions should body fluid cell counts. Acceptable QC methods include be used to fill each side of the chamber to reduce potential commercially available controls or a procedural control. 674 Chapter 30 An acceptable procedural control is to correlate the nucle- neonates decreases with age, so the reference interval for ated cell count with the number of nucleated cells seen per children up to 1 year old is 0–20 cells/mcL and 0–10 cells/ field on the properly made cytocentrifuge-prepared slide.20 mcL from age 1 year to adolescence.3 In both adults and As a rule, a properly prepared cytocentrifuge slide concen- neonates, the predominant WBCs normally present are trates the cells 20-fold. Thus, a fluid sample with a TNCC of monocytes and lymphocytes. 3/mcL should have less than 100 nucleated cells on the The most important considerations for the interpreta- cytocentrifuge slide. If a 100-cell differential can easily be tion of results are the types of WBCs present and the cor- performed on the slide, the TNCC result of 3 cells/mcL relation with clinical findings. An increased WBC count in should be suspect. Conversely, a TNCC of 30/mcL should CSF indicates meningitis. Increased counts with predomi- provide sufficient cells on the cytocentrifuge slide to easily nantly neutrophils are associated with bacterial meningitis, perform a 100-cell manual differential. whereas those with predominantly lymphocytes are associ- AUTOMATED CELL COUNTS ated with viral meningitis. Normally, RBCs are not present. Automated body fluid cell counts can be performed on The presence of RBCs indicates a subarachnoid hemorrhage a variety of current generation hematology analyzers21,22 or blood introduced by a traumatic tap. (Chapter 39). It is imperative to know and understand the The total WBC count cannot be interpreted without the acceptable fluid types, analytic measurement range, clini- total RBCs count. When a specimen is obtained as a trau- cal reportable range, and result interpretation for the ana- matic tap, the WBCs and RBCs reflect the same WBC/RBC lyzer in use. As with manual cell counts, synovial fluid ratio as the patient’s peripheral blood. A general rule is to should be treated with hyaluronidase before automated expect 1–2 WBCs for every 1000 RBCs in the CSF in this situ- cell counting, and BAL samples should be treated with a ation. For example, if the total WBC count in a CSF specimen mucolytic agent. is 10/mcL and the RBC count is 10,000/mcL, no significant Restrictive low linearity limits on some analyzers can increase of WBCs (pleocytosis) is evident. If, however, the preclude analysis of clear, colorless fluids such as CSF, but total RBC count is 100/mcL with a WBC count of 10/mcL, more cellular fluids can usually be analyzed. For example, a significant increase of WBCs indicates a pathologic state. if the lower limit of an instrument’s analytic measurement Serous Fluids: Pleural, Peritoneal, Pericardial Normal range for nucleated cells is 100/mcL, the clinical report- serous fluids have a total nucleated cell count less than able range for serous and synovial fluids could be less 1000/mcL. The cells are predominantly mononuclear cells than 100/mcL because less than 100 nucleated cells is well and can include a few mesothelial lining cells. Increased cell within the normal range for these fluids. Newer hematol- counts with a predominance of neutrophils suggest infec- ogy analyzers have dedicated body fluid channels that use tion. An increased cell count with a predominance of lym- fluorescent dyes and extended count times to achieve an phocytes is associated with a lymphoproliferative disorder, analytical measurement range accurate to one nucleated tuberculosis, or a chylous effusion. RBCs are not normally cell per m icroliter.23 Bloody fluids can be analyzed on most seen in serous fluids. Bloody serous fluid can result from a hematology analyzers. Regardless of the cause of the bloody malignancy, trauma, or a traumatic tap. sample—traumatic tap or hemorrhagic condition—the blood present is peripheral blood, which these instruments Synovial Fluids Normal synovial fluid has a total nucle- were designed to analyze. ated cell count of less than 200/mcL with predominantly Interpreting acceptability of results is critically impor- mononuclear cells. Increased cell counts are associated with tant. Body fluids often contain debris and noncellular ele- inflammation and infection. Certain diagnoses can be sus- ments that can interfere with accuracy. The scattergram or pected when comparing the total WBC count with percent histogram accompanying the cell count must be examined of segmented neutrophils present; however, there is signifi- for any interference. Because scattergrams and histograms cant overlap, and morphologic examination by cytocentri- associated with cell counts vary by analyzer, it is essential fuge preparations can be extremely helpful.24,25 to understand and appropriately interpret results generated Joint fluids that have a total WBC count of 50,000– by the analyzer in use. 200,000/mcL suggest an infectious or crystal-induced etiol- ogy. If the differential count shows 90% or more segmented CELL COUNT REFERENCE INTERVALS AND neutrophils, an infectious agent is most likely, and cultures CORRELATION WITH DISEASE must be obtained. Microorganisms can be seen in joint fluid The number and types of cells found in body fluids is if present in sufficient numbers and have the same morphol- important information that is used with clinical findings to ogy as previously described. Bacterial organisms are more help make a diagnosis. common, and pathogenic yeasts are seen only rarely. Cerebrospinal Fluid The reference interval for WBCs in When the total WBC counts is in the range of 2,000 to CSF is 0–5 cells/mcL for adults and 0–30 cells/mcL for 200,000/mcL with more than 50% neutrophils in the dif- neonates.2,7 The higher upper reference interval seen in ferential count, entities such as rheumatoid arthritis (RA), Morphologic Analysis of Body Fluids in the Hematology Laboratory 675 systemic lupus erythematosus (SLE), and Reiter’s syndrome should be considered. Bronchoalveolar Lavage Reference intervals for nucle- ated cell counts in BAL samples are difficult to determine because the concentration depends on the volume of fluid infused and the return yield. Studies performed on normal subjects show a range of nucleated cell counts of 100–400/mcL.26 Microscopic findings in BAL samples have more importance than the cell count itself. Nucleated Cell Differential Body fluid analysis requires not only enumeration of cells but also microscopic examination. The types of cells present as well as artifacts and crystals can be identified microscopi- cally.27 In many cases, the types of cells present can be more important than the cell count. Morphologic examination Figure 30.7 Cytocentrifuge slide assembly with disposable and differentiation of cells require concentration techniques dual chamber cytofunnel. that preserve cellular integrity and morphology. morphologic examination, the amount of fluid used should CYTOCENTRIFUGE SLIDE PREPARATION be based on the cell count. Specific manufacturer instruc- Cytocentrifugation is a method that concentrates cells in a tions should be followed. Fluid should be added to the monolayer on a defined area of a slide with minimal mor- chamber with a disposable pipet. A drop of sterile 10–22% phologic distortion. A cytocentrifuge is a centrifuge with albumin should be added to the chamber when preparing a bowl or rotor, usually removable, that can hold multiple slides on serous fluids and CSF. The albumin helps preserve slide assemblies (Figure 30-6). The cytocentrifuge slide cellular morphology in low protein samples and improves assembly consists of a funnel-shaped chamber, an absor- the adhesion of cells to the slide. Albumin should not be bent filter material, and a slide. A clip holds the components added to synovial samples because they have a high protein together. Disposable chambers with attached filter mate- content. To obtain suitable slides for bloody samples, dilu- rial are available as are commercially available slides with tion of the RBCs to less than 5000/mcL is recommended.1 In pre-marked zones to identify the area where the cells are samples with low nucleated cell counts, this can be counter- deposited. Slides should be made in duplicate, or commer- productive because doing so can result in too few nucleated cially available dual chambers and slides should be used cells available. An alternate option for bloody serous fluid (Figure 30-7). As with cell counts, slides should be made and CSF is to make a small dilution using 10% acetic acid to as soon as possible to avoid cellular degeneration and vis- lyse the RBCs. Acetic acid should never be added to syno- cous synovial samples should be treated with hyaluroni- vial fluids because it can cause precipitation of the proteins. dase before slide preparation. To produce the best slides for Cytocentrifuges with adjustable speed, time, and accel- eration can be adjusted for optimum slide preparation on various fluids. As a rule, serous fluids and CSF samples should be cytocentrifuged at a low speed (600 rpm) for 10 minutes with low acceleration. Synovial fluids can be cyto- centrifuged at the same speed and time settings, but high acceleration, if available, can be helpful in harvesting cells in these samples. Care must be taken when disassembling the slide assembly following cytocentrifugation. After removing the clip, the sample chamber must be pulled directly away from the slide. Allowing the chamber to slide up or down the slide will disrupt and remove the cells. Allow the slide to dry before staining with a Wright or Wright-Giemsa stain. MANUAL SLIDE PREPARATION Cytocentrifugation is the gold standard for body fluid slide preparation. However, there may be rare instances when a Figure 30.6 Cytocentrifuge with removable head. cytocentrifuge is not available or functional, in which case a 676 Chapter 30 smear must be made manually. Samples with low nucleated cell counts (less than 1,000 or 2,000/mcL) should be concen- trated before making the smear. An aliquot (1–2 mL) of the fluid can be centrifuged to concentrate the cells in a but- ton on the bottom of the tube. Centrifuging at a low speed (1000–1500 rpm) for 10 minutes causes less disruption of the cells than centrifuging at a higher speed. The supernatant is removed, and the button of cells is gently resuspended. One drop of 10–22% albumin should be added to the resus- pended cells for serous and CSF samples. A drop of the con- centrated cells can then be used to prepare a wedge smear similar to |
a peripheral blood smear; the wedge smear can be made directly from the sample on highly cellular fluids. CELL TYPES AND MICROSCOPIC FINDINGS COMMON TO ALL FLUIDS Figure 30.8 Artifactual change in neutrophils showing nuclear Segmented neutrophils (segs) are frequently seen in the lobes thrown to the periphery of the cytoplasm (Slides in this chapter are all cytocentrifuge-prepared body fluids, Wright stained, pleural, pericardial, and peritoneal fluids in varying num- magnification 1000* unless otherwise noted). bers. The neutrophils have the same appearance as in peripheral blood smears. However, cytocentrifuge artefac- tual changes sometimes can be seen with nuclear segments being thrown to the periphery of the cytoplasm, creating a “windmill” and hypersegmented appearance (Figure 30-8; all slides in this chapter were cytocentrifuge-prepared body fluids, Wright stained, magnification 1000* , unless otherwise noted). Degeneration of neutrophils is seen more frequently in body fluid samples than in peripheral blood smears. Peripheral blood cells, especially neutrophils, are less stable in body fluids because they are not in their nor- mal plasma environment. Studies have shown that in CSF, up to 32% of the neutrophils can be lost in 1 hour because of degeneration.17 The dying cells show cytoplasmic vacu- olization and separation of nuclear segments with dense- staining chromatin (Figure 30-9). These cells can be mistaken for nucleated RBCs and even yeast organisms. Neutrophilic Figure 30.9 In the center (arrow) is a degenerating neutrophil precursors, such as promyelocytes, myelocytes, and meta- with separation of nuclear lobes and dense staining of the myelocytes, are not commonly seen, but if present they can chromatin. represent a chronic inflammatory process or true marrow disorder, such as myeloproliferative disorders, myelodys- plastic states, and leukemia. Myeloblasts are usually seen only in the latter three. Lymphocytes are frequently present in all types of fluids in variable numbers. The lymphocytes vary in mor- phology from small to large and transformed (reactive). In cytocentrifuge preparations, the lymphocyte nucleoli can be artifactually more prominent than in peripheral blood smears, the nuclear shape can be irregular, and the cyto- plasm can have artifactual projections3 (Figures 30-10 and 30-11). If neoplastic lymphocytes (leukemias, lymphomas) are present, the morphology depends on the type of neo- plasm, and the cells are usually homogeneous in appear- ance. Flow cytometry or immunoperoxidase techniques can be helpful in distinguishing benign versus malignant Figure 30.10 Artifactual change in lymphocytes with overly lymphocytes. prominent nucleoli. Morphologic Analysis of Body Fluids in the Hematology Laboratory 677 Figure 30.11 Artifactual change in lymphocytes with Figure 30.13 Histiocyte (arrow) in pleural fluid. cytoplasmic projections. Monocytes in body fluids can appear like that seen Eosinophils, basophils, and mast cells can be present in peripheral blood smears or can be larger with abun- in small numbers in pleural, pericardial, peritoneal, or joint dant, vacuolated cytoplasm (histiocyte), or they can have fluids. Increased numbers of these cell types are seen in actual phagocytosed material (macrophage, phagocyte) various disorders and can correlate with peripheral blood (Figures 30-12 through 30-14). The distinction among the eosinophilia or basophilia.4 Eosinophils are frequently seen three morphologic types (monocytes, histiocytes, and mac- in nonspecific or idiopathic effusions and can be present in rophages) is not clinically important, and these cells can effusions caused by various infectious agents, malignancies, be reported as a combined population of mono/macro- and connective tissue disorders. Mast cells can be distin- phages. In some cases, however, the phagocytosed mate- guished from basophils because mast cells have a round rial or organisms can be diagnostically important. Vacuoles (not segmented) nucleus and a higher number of cytoplas- that develop in phagocytic cells can fuse into a single large mic granules than basophils. The granules in mast cells are vacuole resulting in a “signet ring” appearance with the smaller than those seen in basophils (Figures 30-17 and nucleus flattened against the cell membrane (Figure 30-15). 30-18). An increase in the number of basophils can correlate These signet ring cells can be seen in both benign and malig- with myeloproliferative disorders involving body fluids. nant conditions. Only a few monocytes are present in CSF, Benign tissue cells can be seen in any of the body fluids and histiocytes/macrophages usually are seen in the CSF and must be differentiated from malignant cells (Table 30-5). only in pathologic states. Plasma cells are not seen in normal The morphology of these tissue cells varies according to the fluids and usually are present only in chronic inflammatory fluid space and is discussed in the “Microscopic Findings in disorders (Figure 30-16). Specific Fluids” section. Figure 30.12 Monocytes in pleural fluid. Figure 30.14 Macrophage in pleural fluid. 678 Chapter 30 Figure 30.15 Macrophage in pleural fluid with single large Figure 30.17 Basophil (arrow) in pleural fluid. vacuole giving a “signet ring” appearance. When performing the nucleated cell differential on like yeast organisms, even budding yeasts if two particles body fluids, all cell types should be differentiated if possible. are closely associated. Starch particles usually have a refrac- Abnormal or reactive cells that cannot clearly be assigned to tile center that is not a characteristic feature of yeast. They a specific category should be counted as unclassified cells are also birefringent (discussed in the section “Joint Fluid”), and described. Clumps of cells (benign or malignant) should showing as bright Maltese cross-like figures with polarized be noted because they can adversely affect the count’s accu- light (Figure 30-19). Precipitated stain can look like bacte- racy. A comment such as “cells present in clumps, count rial organisms. If the precipitate appears to be intracellular, could be falsely decreased” should be reported. changing the fine focus usually reveals the precipitate to be in a different plane of focus than the cell. True intracel- MORPHOLOGIC FINDINGS RESULTING lular bacteria are in the same plane of focus as the cell. Stain FROM ARTIFACT precipitate is darker than bacteria and variable in size and Some artifactual findings such as peripheral displacement can be seen extracellularly, sometimes in distant areas of the of the nucleus in neutrophils, lymphocyte cytoplasmic slide (Figure 30-20). In difficult cases, an extra slide should extensions, and overly prominent nucleoli of lymphocytes be prepared for a gram stain. The cytocentrifuge exagger- have already been mentioned (Table 30-6). Other artifactual ates early cellular degeneration, and cellular nuclei show changes can resemble actual pathologic findings, so inter- irregular nuclear margins and separating chromatin. This pretation must be made cautiously. Starch particles can be can be mistaken for malignant cells (Figure 30-21). an in vitro contaminant in any type of body fluid. Although most surgical gloves in use are now powder free, starch NONSPECIFIC REACTIVE CHANGES can be on sterilized surgical gloves used by the physician The term nonspecific reactive changes refers to effusions that obtaining fluid from the patient. Starch particles can look have an inflammatory cell response that is not diagnostic Figure 30.16 Plasma cell in pleural fluid. Figure 30.18 Mast cell (arrow) in pleural fluid. Morphologic Analysis of Body Fluids in the Hematology Laboratory 679 Table 30.5 Normal Existing Tissue Cells Found in Various Table 30.6 Artifacts That Can Be Seen with Wright- Body Fluids Stained Cytocentrifuge Prepared Slides and Potential Mistaken Interpretation Fluid Type Normal Tissue Cells Cerebrospinal Ependymal, choroid plexus cells, Artifact Mistaken Interpretation arachnoid Peripheral localization of nuclear Hypersegmented neutrophils Pleural, pericardial, peritoneal Mesothelial lobes in neutrophils Joint Synovial Degenerating neutrophil Yeast organisms, nucleated RBCs Bronchoalveolar lavage Bronchial Overprominence of nucleoli in Blast cells lymphocytes Cytoplasmic projections of Hairy cell leukemia for any specific disorder. In various pathologic states, cer- lymphocytes tain types of WBCs can be present in increased numbers. Single large vacuole in histiocyte Signet ring cell carcinoma Bacterial infections have a predominance of segmented neu- Starch particles Yeast organisms, crystals trophils, whereas viral, fungal, and mycobacterial infections Stain precipitate Bacterial organisms can have a predominance of lymphocytes or show a mixed Degenerating tissue cells Malignant cells inflammatory response. As in peripheral blood, neutrophils can have toxic most helpful feature in distinguishing reactive lympho- granulation, Döhle bodies, and cytoplasmic vacuoles cytes from lymphoma cells is that the former consists of (Chapter 21). a heterogeneous population of cells with varying nuclear Lymphocytes are frequently reactive and trans- shape, amount of cytoplasm, and degree of cytoplasmic formed, simulating lymphoma cells (Chapters 22, 28). The basophilia (Figures 30-22 and 30-23). Lymphoma cells are a b c Figure 30.19 (a) Starch particles with plain light resembling yeast. (b) Starch particles as seen with polarized light appearing as Maltese cross shape. (c) Starch particles with polarized light and quartz compensator. 680 Chapter 30 Figure 30.20 Stain precipitate on top of the cell with Figure 30.22 Reactive lymphocytes in pleural fluid. precipitate in focus and cells slightly out of focus. homogeneous with the same nuclear and cytoplasmic fea- provide additional protection and cell stability when mak- tures. The morphology of the lymphoma cells depends on ing cytocentrifuge slides of serous and cerebral spinal fluids the particular type of lymphoma. can be a source of bacterial contamination. Most pathogenic yeasts are found in CSF rather than in MICROORGANISMS pleural, pericardial, or peritoneal fluids and can be found Most types of pathogenic bacterial and fungal organisms intracellularly. The different types of pathogenic yeast show stain with Wright stain and are detectable on a routine cyto- some distinguishing features on Wright-stained slides. This centrifuge preparation. Bacteria stain blue regardless of the morphologic variance can be used as a clue to an initial Gram stain features. Recognizing the presence of intracellu- impression of the specific type of yeast, but cultures must be lar organisms is important because it indicates true pathoge- obtained for definitive identification. The most frequently nicity rather than in vitro contamination (Figure 30-24). Once seen fungal organisms in fluids are Cryptococcus, Histo- bacteria have been recognized with Wright stain, preparing plasma, Candida albicans, and Candida tropicalis. a second cytocentrifuge slide for a Gram stain is helpful to confirm the presence of bacteria and to give additional MALIGNANT CELLS IN BODY FLUIDS information to the physician while cultures are pending. Malignant cells can be seen in any fluid but are found more Caution must be used when suspected bacteria are seen in often in serous fluids. They are rare in synovial fluid. Malig- synovial fluids treated with hyaluronidase. Hyaluronidase nant cells in serous fluids pose the greatest difficulty for the products can be labeled as sterile, but on rare occasions can laboratory because they are often difficult to differentiate contain nonviable bacteria. Thus, the cytocentrifuge slide from mesothelial cells. Differentiating characteristics are made for a confirmation Gram stain should be prepared discussed in the section “Microscopic Findings in Specific using the untreated fluid. Likewise, the albumin used to Fluids.” Figure 30.21 Early cell degeneration. Figure 30.23 Reactive lymphocyte (arrow) in pleural fluid. Morphologic Analysis of Body Fluids in the Hematology Laboratory 681 Figure 30.24 Figure 30.25 Pleural fluid. Intracellular and extracellular bacterial cocci in joint fluid. In some patients, a diagnosis of malignancy may than on peripheral blood smears, and the nuclear membrane already have been established by other tissue sampling can be surprisingly irregular. Auer rods can be seen, and, if (biopsy or excision), and finding the malignant cells in necessary, unstained slides can be prepared for cytochemi- fluid establishes a condition of tumor metastasis. For other cal stains and terminal deoxynucleotidyl transferase (TdT) patients, finding malignant cells in fluid can be the initial to differentiate the blasts (Chapters 23, 37). Lymphoblasts diagnosis of a malignancy, and if a sample has not been sent have a very high nuclear-cytoplasmic ratio, and the nucleus to cytology, the recognition of malignancy by the hematol- can be folded or convoluted. ogy laboratory is critical in establishing an early diagnosis. The morphology of non-Hodgkin lymphoma in the Malignant cells in fluids can usually be distinguished as body fluids depends on the particular type of lymphoma. hematopoietic in origin (leukemia, lymphoma) versus non- Again, the nuclear membrane can be surprisingly irregular. hematopoietic (carcinoma, sarcoma), and in some cases, Large-cell lymphoma has cells that are moderate to large additional specification of cell type is also possible. It is with irregular nuclei, partially clumped chromatin, and important to look at the entire cellular area of the slide with sometimes prominent nucleoli (Figure 30-26). The cyto- a low power objective (10* ) to detect suspicious clusters plasm is low to moderate in amount and basophilic. The of cells. cells are discohesive |
(a loosely joined cluster), but if the fluid is very cellular, the cells can be thrown together and have the appearance of carcinoma cell clusters; nuclear molding CASE STUDY (continued from page 672) is not seen. Burkitt lymphoma has intermediate size cells A cytocentrifuged, Wright-stained slide is prepared, with more than one nucleoli and an immature blast-like and a photomicrograph is taken (Figure 30-25). chromatin (Figure 30-27). Prominent cytoplasmic vacuoles frequently are apparent. Small-cell lymphoma is the most 3. What is an appropriate next step to determine difficult to diagnose and can look like a benign lymphocytic whether the material seen is debris or true organisms? 4. Some of the large tissue cells showed features of degeneration and suspicious for malignancy. How should this be interpreted? Almost any type of hematopoietic malignancy, includ- ing lymphocytic and nonlymphocytic leukemias, lympho- mas, Hodgkin disease, and plasma cell neoplasms,28 can be found in body fluids. Generally, the abnormal cells found in the body fluids in these disorders have the same morpho- logic features as peripheral blood and bone marrow cells. The acute leukemias only occasionally involve the pleural, pericardial, or peritoneal cavities but more often are seen in the CSF. Blasts appear larger on cytocentrifuge preparations Figure 30.26 Large-cell lymphoma in pleural fluid. 682 Chapter 30 Figure 30.27 Burkitt lymphoma (small noncleaved cell) in Figure 30.29 Small lymphocytic lymphoma. pleural fluid. infiltrate. In these cases, flow cytometry is valuable in dem- onstrating a clonal population of cells (Chapter 40). T-cell Checkpoint 30.2 lymphoma can show markedly irregular, convoluted nuclei; A 32-year-old woman has right-side chest pain and shortness however, marker studies are necessary to confirm the T- of breath that has worsened over a two-week period. Chest radiologic studies reveal a right pleural effusion, and a thoracen- or B-cell origin of the neoplastic cells (Figures 30-28 and tesis is performed. The pleural fluid specimen on a cytocentri- 30-29). Cell origin identification can be accomplished by fuged, Wright-stained slide reveals cells like that seen in Figure flow cytometry or immunoperoxidase techniques on cyto- 30-10. What is the best interpretation of this finding? If there is a centrifuge-prepared slides. strong concern that this can represent a low-grade lymphoma, Primary effusion (body cavity) lymphoma is a high- what would be the best way to determine whether these are grade disease found only in a body cavity without an asso- benign or malignant lymphocytes? ciated solid tumor mass. This unique malignancy has been reported in patients who are immunocompromised, usu- ally HIV positive, and is associated with human Herpes Hodgkin lymphoma can occasionally be seen to involve virus 8 (HHV-8; also known as Kaposi sarcoma–associated pleural fluid (Figures 30-30 and 30-31). The malignant herpes virus, KSHV).29–31 The morphology of the cells in Hodgkin cell is large with a moderate to abundant amount the effusion is like the morphology of cells found in ana- of cytoplasm, large nuclei, and prominent nucleoli. If the plastic large-cell lymphoma, diffuse large B-cell lymphoma, nucleus is bilobed or if the cell has two nuclei, it can be or small lymphocytic lymphoma. These malignant cells are a Reed-Sternberg cell. The other cells present consist of usually B-cell derived but lack surface-associated antigens varying numbers of small lymphocytes, eosinophils, his- for T- or B-cell lineage. tiocytes, and plasma cells. If other tissue biopsy has already Figure 30.28 Lymphoblastic lymphoma, T-cell type, in pleural fluid. Figure 30.30 Hodgkin disease, pleural fluid. Morphologic Analysis of Body Fluids in the Hematology Laboratory 683 Figure 30.32 Pleural fluid. Figure 30.31 Hodgkin disease with Reed-Sternberg cell (arrow), pleural fluid. Mesothelial cells can show nonspecific reactive changes, which include multinuclearity, presence of nucleoli, mitotic established a patient’s Hodgkin disease diagnosis, the activity, basophilic staining, and sometimes an increase in malignant cells (either Hodgkin, Reed-Sternberg, or mul- cell size (Figures 30-34 and 30-35). Occasionally there also is tinucleated variants) must still be identified in the effusion an increased nuclear-cytoplasmic ratio and nuclear folding sample to diagnose the fluid’s involvement. simulating carcinoma. Reactive mesothelial and malignant cells must be dif- CASE STUDY ferentiated. Reactive mesothelial cells can tend to cluster (continued from page 681) and appear cohesive, but the clusters appear flat rather than After 3 weeks, Carolyn improved significantly, the ball-like clusters of malignant cells. Because reactive but the chest pain and effusion did not resolve. A mesothelial cells are often seen in clusters, it can be chal- repeat thoracentesis was performed. Laboratory lenging to differentiate the benign and malignant cells in studies show protein 4.7 g/dL (serum = 60 g/dL), pleural and peritoneal fluids. A useful feature of mesothe- lactate dehydrogenase 50 U/L (serum = 60 U/L), lial cells is their overall uniformity when viewed under low and total nucleated cell count 3,000/mcL. A cyto- power. The round-to-oval nuclei are uniform in appearance centrifuged, Wright-stained slide is examined, and as is the generally low nuclear-to-cytoplasmic (N:C) ratio. the differential count shows 30% segmented neu- Clusters of mesothelial cells are flat, and the individual cells trophils, 20% lymphocytes, 10% histiocytes, and can be distinguished often showing a clear separation or 40% tissue cells. See Figure 30-32 for a photomicro- “window” separating the cells in the cluster (Figure 30-36). graph of the tissue cells. In cases when distinguishing reactive mesothelial cells from 5. Is this an exudate or transudate? malignant cells is difficult, cytology preparations usually are definitive because alcohol fixation and Papanicolaou 6. What is the most appropriate interpretation of these findings? MICROSCOPIC FINDINGS IN SPECIFIC FLUIDS Serous: Pleural, Peritoneal, Pericardial Benign mesothelial cells can be seen in the pleural, pericardial, and peritoneal fluids. These are large cells that have a moderate to abun- dant amount of cytoplasm. The cytoplasm can be light or dark blue and occasionally contain granules or phagocy- tosed debris. The nucleus is eccentric with a homogeneous chromatin pattern. Nucleoli can be seen, and if present, are blue with a smooth membrane (Figure 30-33). The pleural, pericardial, and peritoneal fluids also can contain malignant cells, and their identification is critical for making an accurate diagnosis.32–34 Any one sample can have only a few malignant cells that are difficult to find. Figure 30.33 Benign mesothelial cells in pleural fluid. 684 Chapter 30 Figure 30.34 Reactive mesothelial cells in pleural fluid. Figure 30.36 Reactive mesothelial cells in pleural fluid. stain yield better nuclear detail. As a rule, malignant cells do together. For example, one type of malignancy can show not have this uniform appearance, and the nuclear shapes smooth nuclear membranes but with unevenly distributed and sizes can vary dramatically. The N:C ratio is often high, chromatin and irregular nucleoli (Table 30-8). and clusters of cells can be ball-like, making distinguishing The most common nonhematopoietic malignancies the individual cells difficult. seen in body fluids are small-cell carcinoma and adenocar- General features that can be seen in almost any type cinoma. Small-cell carcinoma cells have the same general of malignant cell include an irregular nuclear membrane, morphologic findings of malignant cells but can be distin- unevenly distributed chromatin, and nucleoli that also have guished from other types of carcinoma cells because of the an irregular membrane (Table 30-7).33 The N:C ratio varies characteristic high N:C ratio, blast-like chromatin, absence with small carcinoma cells having minimal cytoplasm and of nucleoli or nonprominent nucleoli, and frequent nuclear adenocarcinoma cells having as much or more cytoplasm molding (the process of the nucleus of one cell molding as a benign mesothelial cell. The nuclear membrane irregu- around the shape of an adjacent cell) (Figure 30-38). Nuclear larity can be jagged or have multiple folds. When nucleoli molding occurs with cohesive growth of cells requiring the are present, they are frequently prominent and irregular presence of tight junctions between the cytoplasmic mem- in shape (Figure 30-37). Mitotic activity alone is not a reli- branes of the cells. Therefore, nuclear molding can be seen able indication of malignancy because reactive mesothelial in any type of carcinoma but is most often seen with small- cells can undergo mitosis. Nor are cytoplasmic vacuoles a cell carcinoma. Some cells also can have a paranuclear “blue reliable finding for malignancy because their presence can body,” which is an inclusion that has not yet been character- be part of early degeneration in many cells. None of the ized and can represent early cell degeneration or phagocy- features described can be used alone to diagnose malig- tosed material. Depending on the cell’s orientation, the blue nancy. All the features must be looked for and evaluated body can appear to be intranuclear and has been described in small-cell carcinoma but rarely in sarcoma.35 The blue body is seen only with air-dried, Wright-stained prepara- tions (Figure 30-39). If the malignant cells are noncohesive, Table 30.7 Comparison of Morphologic Features of Reactive Mesothelial Cells versus Malignant Cells Reactive Mesothelial Cell Features Malignant Cells Cells Nuclear membrane Smooth Irregular, jagged Chromatin Evenly distributed Unevenly distributed Nucleoli Absent or present with Prominent, fre- smooth membrane quently multiple, irregular membrane Nuclear molding None Present in non- hematopoietic Figure 30.35 Multinucleated mesothelial cell in pleural fluid. malignancies Morphologic Analysis of Body Fluids in the Hematology Laboratory 685 a b c Figure 30.37 (a) Malignant cells (adenocarcinoma), pleural fluid. (b) Benign mesothelial cell to contrast with features of malignant cell. (c) Single malignant cell (adenocarcinoma), pleural fluid. small-cell carcinoma could be mistaken for a hematopoietic a small-cell carcinoma cell with a moderate to abundant malignancy, and finding a blue body would be a good clue amount of cytoplasm (Figure 30-40). The nuclear chromatin for the diagnosis of small cell carcinoma. is partially clumped and heterogeneous and has prominent Adenocarcinoma differs from small cell carcinoma in nucleoli. The presence of cytoplasmic vacuoles is not spe- that the overall size of an adenocarcinoma cell is larger than cific and can represent early cell degeneration. Table 30.8 General Morphologic Findings of Benign Mesothelial Cells and Malignant Cellsa Small-Cell Large-Cell Mesothelial Cell Adenocarcinoma Leukemic Blasts Carcinoma Lymphoma Cell size Large, 15–30 mcM Large to giant Moderate to large Moderate to large Small to moderate Chromatin Loose, homogeneous Partially clumped, Slightly coarse, Partially clumped, Smooth, lacelike, heterogeneous homogeneous heterogeneous homogeneous Nucleoli None to small and Prominent, multiple, None to small, Small to prominent, Variable regular irregular not prominent irregular Nuclear membrane Smooth Irregular, jagged Irregular, jagged, folded Irregular, jagged, folded Smooth or irregular, folded N:C ratio Low, 1:3–5 Low, 1:3 or less High, 1:1.25 High to moderate, High to moderate, 1:1.25–1:2 1:1.25–1:1.75 Intercellular Individual or clumped, Usually clumped, Clumped with nuclear Individual, no clumping, Individual, no clumping, relationship no nuclear molding { nuclear molding molding, occasionally no nuclear molding no nuclear molding individual a Any given cell can show variable features, so all must be evaluated before deciding whether a body fluid sample is benign or malignant. No single feature can be used to diagnose malignancy. 686 Chapter 30 a b Figure 30.38 (a) Small-cell carcinoma, pleural fluid showing tight cell clusters (original magnification, 25* ). (b) Small-cell carcinoma. Other types of carcinoma and sarcoma can be found in In rare cases, spontaneous formation of lupus ery- body fluids. The features seen with Wright stain are not as thematosus (LE) cells is seen in serous fluids. An LE cell specific as a cytology preparation; the latter is necessary to is a macrophage, either neutrophil or monocyte, that has specifically identify the type of malignancy. For example, phagocytosed a nucleus showing a homogeneous, smooth squamous cell carcinoma can look like adenocarcinoma chromatin pattern. Finding these cells is suspicious but not with Wright stain; however, in most cases, the two are read- diagnostic for SLE. Other autoimmune disorders can also ily distinguishable on cytology preparations. Certain types show the LE cell phenomenon. Nevertheless, the identifi- of malignant cells can contain clues to their cellular origin. cation of these cells can be extremely helpful in arriving Melanoma cells can have melanin pigment that is demon- at a diagnosis difficult to make. The LE cell should not be strable with Wright stain, and hepatocellular carcinoma can mistaken for simple phagocytosis of cells by macrophages, have bile pigment. The presence of these pigments can be which is frequently seen. The chromatin of the usual phago- suspected with Wright stain but must be confirmed with cytosed cell is neither smooth nor homogeneous. more specific staining techniques. Cerebrospinal Fluid The tissue cells (choroid plexus and ependymal) that can be seen |
in CSF tend to cluster and can Checkpoint 30.3 have cytoplasmic granules and slightly irregular nuclei What are the best features to use in determining whether tissue (Figure 30-41). The arachnoid cells are frequently seen as cells are benign or malignant when examining a cytocentrifuged, a syncytium with a mass of cytoplasm containing several Wright-stained slide of a body fluid specimen? nuclei (Figure 30-42). These benign tissue cells are usually seen in CSF only from infants and adults who have had Figure 30.39 Small-cell carcinoma, paranuclear “blue body” (arrow) in malignant cell. Figure 30.40 Adenocarcinoma in pleural fluid. Morphologic Analysis of Body Fluids in the Hematology Laboratory 687 Figure 30.41 Choroid plexus cells in CSF. Figure 30.43 Reactive lymphocytes in CSF. some type of manipulation such as recent neurosurgery or Erythrophagocytosis and hemosiderin can be seen on other shunt placement. The appearance of these cells from a spi- body fluids and should be reported but has more clinical nal tap procedure in an adult is very unusual. CSF tissue significance in CSF. cells can resemble mesothelial cells, but they are found only in the serous spaces, not in CSF. Checkpoint 30.4 As with the pleural, pericardial, and peritoneal fluids, A wife finds her 47-year-old husband comatose at home. During reactive lymphocytes must be distinguished from lym- examination in the emergency room, a spinal tap is performed, phoma cells (Figure 30-43). Hematopoietic precursors, as and grossly bloody spinal fluid is obtained. The total RBC count well as megakaryocytes, can be present if the spinal tap in the first tube is the same as that in the third tube. A cytocen- needle penetrated the vertebral bone, drawing back a por- trifuged, Wright-stained slide shows findings like that seen in tion of bone marrow. This is most often seen in infants but Figure 30-44. What is the most appropriate interpretation of can occur in adults with osteoarthritis. these findings? A definitive sign of CNS hemorrhage is phagocyto- sis of erythrocytes by histiocytes (erythrophagocytosis) Because mesothelial cells are not found in the CSF, the (Figure 30-44). It takes approximately 18 hours for his- presence of any large tissue cells should be considered sus- tiocytes to mobilize and phagocytose erythrocytes after a picious for malignancy (Figures 30-46 and 30-47). Malig- hemorrhage; thus, erythrophagocytosis is not an immediate nant cells, however, must be differentiated from the benign indicator of a current CNS hemorrhage. In older hemor- choroid plexus cells, ependymal cells, and arachnoid cells rhages, hemosiderin and hematoidin crystals can be seen by evaluating them for standard features of malignancy intracellularly or extracellularly (Figure 30-45). Hemosid- as described earlier. Cytology preparations are usually erin and hematoidin are products of hemoglobin catabolism. definitive. Figure 30.42 Arachnoid cell syncytium in CSF. Figure 30.44 Erythrophagocytosis in CSF. 688 Chapter 30 Figure 30.45 Hematoidin crystals in macrophage in CSF. Figure 30.47 Large-cell undifferentiated carcinoma with intense chemical acute meningitis, CSF. Cells from primary CNS neoplasms rarely are found HIV-positive. The lymphomas in these patients are high in the CSF. Medulloblastoma is a malignant tumor usu- grade and frequently correspond to Burkitt lymphoma or ally occurring in the cerebellum of pediatric patients and diffuse large B-cell lymphoma (Figure 30-53). has a cellular morphology like that of small-cell carcinoma The most common yeast organism seen in CSF is cryp- (Figures 30-48 and 30-49). The patient history from the phy- tococcus (online student resource Figure 30-7). When crypto- sician is necessary to distinguish the tumor’s origin. coccus is suspected from the cytocentrifuge-prepared slide, Acute lymphoblastic leukemia more often involves the the microbiology laboratory should perform an India ink CNS than acute nonlymphocytic leukemia (Figures 30-50 preparation to confirm the presence of the characteristic through 30-52). When erythrocytes are present, care must large capsule of cryptococcus. If very few organisms are be taken not to overinterpret the presence of blasts that sim- present, however, the India ink preparation can be nega- ply represent peripheral blood contamination. If no eryth- tive because unconcentrated CSF is used. Cultures must be rocytes are present, even a low number of blasts (1–2%) obtained to confirm the type of organism present. can indicate CNS involvement.36 Special studies such as cytochemistry stains, TdT, and surface markers by immu- CASE STUDY (continued from page 683) noperoxidase stains or flow cytometry can be helpful21 After an extensive work up, Carolyn is found to (Chapters 23 and 40). have a primary adenocarcinoma of the right upper Any type of lymphoma can involve the CNS, but the lung lobe. A surgical resection is performed. Six high-grade lymphomas such as lymphoblastic, diffuse large months later, the patient has severe headaches. A B-cell, and Burkitt lymphoma are more often seen. Primary spinal tap is performed, and a photomicrograph of CNS lymphoma is seen more often in patients who are the cells is as seen in Figure 30-54. 7. What is the most appropriate interpretation of these cells? Joint Fluid The tissue cells present in joint fluids are syno- vial lining cells and have a similar appearance to mesothe- lial cells with a somewhat denser cytoplasm (Figure 30-55). While they can resemble mesothelial cells, mesothelial cells only occur in the serous spaces and are not present in the synovial spaces. Synovial cells can become proliferative in a reactive setting like mesothelial cells. Reactive synovial cells also can be multinucleated and occur in clusters, and their nuclei can have small, regular nucleoli. Malignant cells can be seen in synovial fluid; however, this is extremely rare.37 The so-called rheumatoid arthritis (RA) cell seen in Figure 30.46 Adenocarcinoma, CSF. synovial fluid is a neutrophil containing granules of immune Morphologic Analysis of Body Fluids in the Hematology Laboratory 689 Figure 30.48 Small-cell carcinoma, CSF. Figure 30.51 Acute myelomonocytic leukemia, CSF. Figure 30.49 Medulloblastoma, CSF. Figure 30.52 Blast crisis of chronic myelocytic leukemia with myeloblasts in CSF. Figure 30.53 High-grade lymphoma, small noncleaved cell Figure 30.50 Acute lymphoblastic leukemia, CSF. type, CSF, pleural fluid. 690 Chapter 30 Figure 30.55 Synovial cells in joint fluid. sufficient numbers of crystals are present, they can be seen with plain light microscopy. However, polarized light must Figure 30.54 Cerebral Spinal Fluid (CSF). be used to confirm birefringence.39 If fewer crystals are pres- ent, polarization is necessary to see them initially. Every complexes. These cells are not specific for a diagnosis of RA. joint fluid sent to the hematology laboratory for cell counts The Reiter’s cell is a macrophage with vacuoles containing should have a crystal examination.40 Although Wright-stain debris of phagocytosed neutrophils. The debris also can be techniques can result in the dissolution of the monosodium unrecognizable blue material. These cells are also nonspe- urate crystals, calcium pyrophosphate crystals are not mois- cific and not diagnostic for Reiter’s disease and are usually ture sensitive and can readily be observed in stained prepa- classified as neutrophages or monomacrophages. rations (Table 30-9). Although crystals can be seen on occasion in other body Birefringence refers to a material’s ability to refract fluids (most commonly cholesterol in serous fluids), they light rays. It is determined by using a polarizing micro- have far greater diagnostic importance in joint fluid. The scope. A fixed light filter (analyzer) is placed above the three most common types of crystals present in joint fluid specimen and a rotating filter (polarizer) is placed below are monosodium urate crystals seen in gout, calcium pyro- the specimen. Both filters allow light to pass in only one phosphate crystals seen in pseudo gout, and cholesterol direction. When the polarizer is rotated 90 degrees to the crystals present in different types of chronic arthritis such as analyzer, no light can pass, yielding a “dark field.” If the RA.38 Examination for crystals on a cytocentrifuge-prepared specimen contains birefringent material, it changes the slide is superior to a standard wet preparation because the direction (refracts) of the light rays, allowing them to pass cytocentrifuge concentrates the specimen. Samples that are through the analyzer, and the birefringent material appears negative with wet preparation can show crystals on the as a bright particle or crystal.39 A quartz compensator, often concentrated cytocentrifuge slide. Using cytocentrifuge-pre- referred to as 1 red compensator filter, is used to further iden- pared slides also decreases the biologic hazard when han- tify a crystal by determining the velocity of the light rays dling wet preparations. Cytocentrifuge slides prepared for passing through the crystal’s grain (axis). crystal examination should not be stained. Uric acid crys- Monosodium urate (MSU) crystals should be reported tals are very hygroscopic and dissolve easily if any water as intracellular and/or extracellular. MSU crystals are or moisture is present in either the fixative or the stain. If typically long, thin, and needlelike with pointed ends Table 30.9 Morphologic Comparisons of Commonly Seen Birefringent Crystals and Particles Color Parallel to Quartz Crystal Birefringence Morphology Compensator Monosodium urate Strong Yellow Long, thin, needlelike, intra- and extracellular Calcium pyrophosphate Weak Blue Short, rectangular, intra- and extracellular Cholesterol Strong Variable Large plate-like, notched, extracellular Steroids Strong Variable Amorphous, intra- and extracellular Talc particles Strong Yellow and blue Maltese cross shape, extracellular Morphologic Analysis of Body Fluids in the Hematology Laboratory 691 (Figure 30-56a). They can be seen singly or in bundles. when turned perpendicular to the axis of the compensator, MSU crystals are strongly birefringent and are brilliant with the color changes to blue 2,7,39 (Figure 30-56c). polarized light (Figure 30-56b). A quartz compensator must Calcium pyrophosphate (CPP) crystals also can be seen be used to determine positive or negative birefringence. intracellularly and/or extracellularly. CPP crystals are typi- MSU crystals are negatively birefringent and when aligned cally short, rectangular, and weakly birefringent, so they parallel to the axis of the compensator, show a yellow color; can be difficult to see with polarized light (Figures 30-57a a a b b c c Figure 30.56 (a) MSU crystals with plain light, (b) polarized Figure 30.57 CPP crystals with (a) plain light, (b) polarized light, (c) quartz compensator with yellow crystal parallel to quartz line light, (c) quartz compensator with yellow crystal perpendicular to and blue crystal perpendicular to quartz line. quartz line and blue crystal parallel to quartz line. 692 Chapter 30 and 30-57b). When aligned parallel to the axis of a quartz compensator, the CPP crystals are blue but change to yellow when they are perpendicular to the axis of the compensator2,7,39 (Figure 30-57c). A joint fluid occasionally has both MSU and CPP crystals; the presence of one does not exclude the other. Cholesterol crystals have a characteristic notched-plate shape and are birefringent (Figure 30-58). These crystals are present in chronically inflamed joints as are seen in RA. Starch particles are distinguished from pathogenic crys- tals by a characteristic Maltese cross shape with polarized light (Figure 30-19). If a joint has been injected with steroids, the steroid par- ticles can be seen intracellularly and extracellulary. Steroid particles do not have a crystal shape and are amorphous but a birefringent (Figure 30-59). A microscopic finding that can be important in joint fluids is the presence of polyethylene fragments. These frag- ments, essentially pieces of plastic, stain bright blue with most Wright stains and appear as artifact or a contaminant, which they can be because plastic devices are used to obtain and process most fluids (syringes, cytocentrifuge cham- bers, etc.). However, plastic is also a component in artificial joint replacements, and increased numbers of polyethylene pieces seen in the fluid in these cases can indicate failure of the replacement joint (Figure 30-60). Checkpoint 30.5 A 57-year-old man has an acutely swollen, painful, reddened b joint in his left great toe. Joint fluid is aspirated, and a photo- micrograph is taken (Figure 30-61). This picture is taken with Figure 30.59 Steroid particles in joint fluid: (a) polarized light, polarized light using a quartz compensator. What is the most (b) quartz compensator. appropriate interpretation of this finding? Figure 30.58 Cholesterol crystals in joint fluid. Figure 30.60 Polyethylene pieces (arrow) in joint fluid. Morphologic Analysis of Body Fluids in the Hematology Laboratory 693 lining cells are referred to as creola bodies and can be con- fused with malignant cells. The presence of the cilia should help correctly identify these cells as bronchial lining cells. BAL samples from smokers show increased monomacro- phages, many with irregular, black inclusions. This is often called “smoker’s lung,” and the inclusions are carbon par- ticles. They should not be mistaken |
for hemosiderin or a microorganism (Figure 30-63). Epithelial cells can be seen but also are considered a contaminant of the procedure pri- marily from the upper airway. The laboratory professional typically includes these contaminant cells in an “other” cat- egory of the differential. Figure 30.61 Synovial fluid. Amniotic Fluid Lamellar Body Analysis of Other Fluids Counts Lamellar body counts (LBCs) in amniotic fluid are an The hematology laboratory usually is responsible for assessment of fetal lung maturity (FLM) and the associated performing analysis on other fluids including BAL fluid risk of developing respiratory distress syndrome (RDS) for determining conditions in the lower respiratory tract, in the premature newborn. RDS is caused by insufficient amniotic fluid lamellar body counts to determine fetal surfactant in the newborn’s lungs. Lamellar bodies are lung maturity, and semen to assess the number and mor- densely packed layers of phospholipid secreted by the type phology of sperm as well as other characteristics of the II alveolar cells and are the storage packages for surfactant fluid. in amniotic fluid. The number of lamellar bodies present in BAL Fluid the amniotic fluid is proportional to the amount of surfac- tant available. Lamellar bodies are similar in size and den- Tissue cells seen in BAL samples are bronchial lining cells. sity to platelets and can be counted in the platelet channel They are considered a contaminant in lavage samples of most hematology analyzers.41 A meta-analysis of studies because they indicate sampling from the bronchial tree showed that the LBCs performed equally as well, and in rather than the lower respiratory tract.1 Bronchial lining some cases, better than the lecithin-to-sphingomyelin (L:S) cells are unique in that they have a row of cilia at one end ratio in predicting RDS.42 (Figure 30-62). The cilia can become detached because of Lamellar bodies can be counted by both impedance normal physiological processes and appear as unattached and optical platelet-counting methodologies. However, it tufts called ciliocytophthoria. Ball-like clusters of bronchial is critical that the laboratory establish a maturity cutoff for Figure 30.62 Ciliated bronchial lining cell (arrow), neutrophils, Figure 30.63 Bronchoalveolar lavage: monomacrophages and monomacrophages in bronchoalveolar lavage sample. with carbon inclusions (arrows) in smoker’s lung. 694 Chapter 30 the methodology used.43 As with previous FLM methods, acceptability of results as with all samples analyzed on an two different cutoffs need to be determined: automated hematology instrument. An example of an amniotic fluid LBC with acceptable • One cutoff to indicate maturity when the LBC is higher platelet impedance histogram is shown in Figure 30-64. An than the cutoff limit example of an unacceptable platelet impedance histogram • A second cutoff to indicate immaturity when the LBC indicating probable interference with the accuracy of the is less than the cutoff limit result is seen in Figure 30-65. In these instances, the LBC LBCs that fall between the two cutoffs are considered cannot be performed because of interfering substances. indeterminate. The platelet channel result, if acceptable, is reported as According to CLSI document C58-A,44 three approaches the LBC. to establishing the cutoffs are acceptable. The first is to com- Vaginal pool samples can be tested for lamellar bodies pare LBCs to newborn outcomes. This approach requires but are not the recommended sample. Vaginal pool samples access to newborn records and cooperation with neonatolo- can be contaminated with blood, mucus, bacteria, or urine, gists in accurately defining RDS. Because of the low inci- which can have unpredictable effects on the LBC. If it is dence of RDS, this method can take considerable time. necessary to test a vaginal pool sample, the specimen type/ A second approach to establishing the cutoffs is to source should be clearly indicated on the report.44 Commer- compare the LBC to an existing FLM method such as the cial QC and proficiency testing are available for LBCs. L/S ratio. The lamellar body cutoff that consistently clas- sifies samples as mature when compared with the exist- ing method can appropriately be used for the mature PLT cutoff. This approach can require access to newborn medi- cal records to resolve any discrepancies between the two methods. The third approach is split sample analysis with a laboratory that is already performing and reporting a validated lamellar body count. Various published cutoffs list a mature LBC cutoff of 40,000–50,000/mcL and an immature cutoff of 15,000–19,000/mcL. Values between these cutoffs are reported as indeterminate. It should be noted that most of published references used an imped- ance platelet-counting methodology, and optical counts 40fL can vary significantly. Known interfering substances are meconium and blood. Samples contaminated with meconium are not Figure 30.64 Amniotic fluid LBC with acceptable platelet (PLT) impedance channel histogram. acceptable for LBC analysis because meconium contamina- tion causes a falsely elevated LBC. Blood, in sufficient quan- tity, causes a paradoxical biphasic interference resulting in PLT an initial increase in LBCs (presumably from the introduc- tion of peripheral platelets) followed by a decrease in LBCs most likely because of the lamellar bodies trapped in fibrin strands. Samples contaminated with blood can be tested for LBCs if the RBC count is less than 31,000/mcL.45 An aliquot of the well-mixed amniotic fluid sample should be placed in a clear tube, if necessary, to observe the sample for blood or meconium contamination. LBCs in amniotic fluid are stable for 33 days if refrigerated (4°C). The sample should not be frozen or centrifuged because either of these actions falsely decrease the LBC.44,45 To perform the LBC, the sample is mixed by inver- 40fL sion for a minimum of 10 times and analyzed on an auto- mated hematology instrument that provides a histogram Figure 30.65 An unacceptable platelet (PLT) impedance or scattergram display of the platelet channel result. The histogram indicating probable interference on the right slope histogram or scattergram display should be examined for affecting the accuracy of the result. Morphologic Analysis of Body Fluids in the Hematology Laboratory 695 Semen Analysis should be thin, runny, and homogeneous. Gelatinous par- ticles can be seen in normal liquefied semen sample. Semen analysis is an important test in assessing male fertil- Samples that do not liquefy impede the accurate mea- ity. Semen is the fluid discharged during ejaculation con- surement of sperm motility and count. Suggested methods sisting of secretions from glands and containing the male to liquefy abnormal semen include treatment with a proteo- mature gamete sperm, also called spermatozoa. The routine lytic enzyme such as chymotrypsin or bromelain. laboratory should perform a basic battery of tests on the semen that includes volume measurement, appearance, liq- Viscosity Viscosity should be measured after the sample uefaction time, viscosity, pH, sperm motility, sperm count has liquefied. Semen drawn into a disposable transfer pipet and morphology evaluation, and, when indicated, sperm should slowly dispense in discrete drops if viscosity is nor- viability. The more esoteric tests used in diagnosing and mal. Semen with abnormal viscosity forms a thread at least treating infertility should be performed in an andrology 2 cm in length when dispensed from the pipet. Viscosity is laboratory and are not covered in this section. usually reported as normal or increased. Historically, methods and reference intervals for semen pH Semen pH should be measured with a pH paper or analysis have varied widely. To address this problem, the indicator strip with a range of 6.0–10.0, ideally at 30 minutes World Health Organization (WHO) published a manual of but acceptable up to 1-hour post ejaculation. A pH meter standard methods in 1989, The WHO Laboratory Manual for can also be used. Whether the laboratory uses the 30-m inute the Examination and Processing of Human Semen. The manual or 1-hour limit, all samples should be tested within the same is now in its fifth edition and is considered a global standard time constraint. Normal semen is alkaline and has a pH 7.2 for semen analysis. Methods discussed here are based on or higher. the WHO Laboratory Manual, fifth edition.46 MICROSCOPIC ANALYSIS PHYSICAL AND CHEMICAL PROPERTIES After the physical and chemical properties of the semen Samples for routine semen analysis should be collected have been analyzed and recorded, slides are made to after a period of abstinence of a minimum of 2 days to a examine the fluid microscopically. In addition to the sperm maximum of 7 days. The recommended method of collec- count, the sperm are examined for motility, viability, and tion is by masturbation. Samples collected in ordinary con- morphology. doms are not acceptable. Condoms acceptable for semen analysis are commercially available. Samples should be Motility Motility of the sperm should be assessed after collected into a clean, wide-mouth container made of liquefaction ideally at 30 minutes, but acceptably at 1-hour glass or plastic. Anecdotal information suggests that some post ejaculation. Again, all samples should be tested at a plastics can have a toxic effect on semen, resulting in a consistent time after ejaculation. The semen is mixed well decreased motility assessment. Plastic containers provided by inversion and a standard volume such as 10 mcL or one by the laboratory should be tested to verify nontoxicity drop from a standard transfer pipet placed onto a clean by performing split sample analysis (glass and plastic) microscope slide. A standard coverslip (e.g., 22 * 22 mm for motility on several high sperm count samples. Best for a 10-mcL volume) is added and the sample allowed to practice should require all variables—volume of sample spread on the slide. The goal of the standard sample volume received in the laboratory, method of collection, type of and standard coverslip is to achieve an approximate depth container, and days of abstinence—to be included in the of 20 mcM between the slide and coverslip. A depth of less reported results. than 20 mcM hinders the movement of the spermatozoa. A method for calculating the depth based on sample vol- Volume and Appearance The lower reference interval for ume and coverslip size can be found in the WHO Laboratory semen volume is 1.5 mL. The appearance of the semen Manual.46 sample should be reported. Normal semen has a homog- Motility should be determined using 400* magnifica- enous gray-opalescent appearance and a report of “normal” tion. Two hundred sperm are counted in random fields and is sufficient. A sample that is “abnormal” in color can be classified as: reported as such but should be described. A red or brown- tinged sample usually indicates the presence of blood. Jaun- • Progressive (moves forward latterly or in a straight dice, urine contamination, and some vitamins and drugs line) can result in a yellow color. • Nonprogressive (movement but no progression in any particular direction) Liquefaction Normal semen is a congealed substance when ejaculated and begins to liquefy within minutes. The • Nonmotile (no movement at all) liquefaction allows the sperm to become motile. Total liq- The process is repeated on another 200 sperm, and the uefaction should occur in 15–60 minutes and the sample percentage for each category is averaged. The average 696 Chapter 30 percentage for each of the three motility grades is reported. and the usual counting rules can be applied with the The lower reference limit for total motility (progressive and caveat that the only information that matters is where the nonprogressive) is 40%. The lower reference limit for pro- sperm head lies in regard to which sperm to include in gressive motility is 32%. the count. The position of the tail is irrelevant. A typical When examining the slide for sperm motility, abnormal dilution used for counting sperm on a hemocytometer is agglutination of the sperm should be noted if present. Agglu- 1:20. Dilutions might need to be adjusted for very high or tination refers to motile sperm sticking to each other, head very low counts with a goal of counting at least 200 sperm to head, tail to tail, or in a mixed array. Agglutinated sperm in the chamber to reduce sampling errors. Counts should are often robustly motile, appearing as a shaking clump of be done in duplicate as well as in a duplicate dilution. The sperm with little true motility. Motile spermatozoa that are standard formula for determining hemocytometer counts agglutinated in noticeable clumps should be reported. is used, but the standard formula calculates the result per microliter, whereas sperm counts are reported per millili- Vitality Sperm vitality (also known as viability) is a mea- |
ter. The calculated result from the hemocytometer must be surement of the percentage of live spermatozoa. A nigrosin- multiplied by 1000 to convert the microliter result to a per eosin stain is used to identify nonvital sperm, which have milliliter result. damaged cell membranes that allow the eosin stain to enter Newer dedicated and disposable sperm-counting and stain the sperm head pink. The intact cell membranes chambers such as the Microcell™ and Cell-Vu™ are com- of vital sperm prevent the dye from entering leaving the mercially available that simplify and standardize the dilut- sperm head unstained. The nigrosin provides a dark back- ing and counting process. ground, so the vital sperm appear white (Figure 30-66). Regardless of the method used to perform the count, The vitality stain should be performed on all samples that the sperm need to be immobilized before counting. Meth- have less than normal progressive motility. WHO suggests ods for immobilizing sperm include heating an aliquot at performing the stain on samples with less than 40% pro- 56°C for 5 minutes or adding a drop of formalin to a small gressive motility. After staining the specimen and making a amount of saline or other diluting fluid used to make the smear, 100 sperm are counted for vitality at 400* . The count counting dilution. An important step in performing any should be performed in duplicate. The average percentage type of manual sperm count is to ensure that the semen of vital sperm (unstained forms) is reported; the lower refer- sample is well mixed before a portion is removed for dilu- ence limit is 58%. tion or counting. Only intact sperm—those with a head and Sperm Count Sperm counts should be performed only tail—are included in the count. The appearance of numer- after the semen sample has liquefied. Traditionally, sperm ous unattached tails should be noted. Automated methods counts have been performed using the hemocytometer, for performing semen analysis are recently available (SQA- V™ and QwikCheck™); they can perform sperm motility, count, and morphology. Although most laboratories iden- tify the number of sperm per mL as the “sperm count,” WHO considers this the “sperm concentration.” Accord- ing to WHO, total sperm number is the total number of sperm in the entire ejaculate and is calculated by multiply- ing the sperm concentration (number per milliliter) by the semen volume. The lower reference limit for sperm count is 15 * 106/mL. Morphology Slides for morphologic examination of sperm should be made after liquefying the sample. A drop of well- mixed semen should be placed on a slide and a second slide used to make a pull-type smear. Slides should be made in duplicate. If the sperm count is very low, a portion of the sample can be concentrated by centrifugation before slide preparation. The slides should be allowed to dry before staining. Papanicolaou, Shorr, Diff-Quik, and Wright’s stain are acceptable staining methods. Figure 30.66 Nigrosine-eosin stain for sperm vitality showing When visualized by light microscopy stained sperma- an unstained vital (live) sperm (arrow) and a pink-stained nonvital tozoa consist of a head, neck (mid-piece), and tail (principal sperm. piece). The head has a light-staining area at the top called Morphologic Analysis of Body Fluids in the Hematology Laboratory 697 Table 30.10 Criteria for Normal Sperm Morphology Head Mid-piece Tail Smooth and generally oval Slender Uniform in diameter Length 4.095.0 mcM (mM) Approximately same length as the head Thinner than the mid-piece Width 2.5–3.5 mcM Axially attached to the head Approximately 10 times the length of Length to width ratio 1.3–1.8 Residual cytoplasm less than one-third the size the head (45 mcM) Acrosome occupies 40–70% of the head May be looped back on itself but no sharp angles. Acrosome contains no large vacuoles and not more than two small vacuoles occupying less than 20% of the head Post acrosome occupies 30–60% and contains no vacuoles the acrosome and a darker staining area at the bottom called tail—should be noted. The lower reference limit for normal the post acrosomal area. The head is attached to the tail by the forms is 4%. neck or mid-piece. With the Papanicolaou stain, the sperm Other cellular material that can be seen in stained head is stained pale blue in the acrosomal region and darker semen slides include WBCs, spermatocytes, and spermatids. blue in the post acrosomal region. The mid-piece and the Appearance of more than a few WBCs in each microscopic tail are stained a pinkish color. Excess residual cytoplasm, field should be noted. Spermatocytes and spermatids are usually located behind the head and around the mid-piece, precursors of the mature sperm and often look like nucle- is stained pink. ated RBCs on stained preps (Figure 30-71). If more than a Various schemes have been used for assessing sperm rare spermatid is seen, it should be noted on the report. morphology, described as “liberal” (MacLeod and GOLD) and “strict” (Tygerberg, Kruger). WHO defines morphology according to strict criteria. Refer to Table 30-10 for criteria for a morphologically normal sperm. Both the head and tail must be normal for a sperm to be considered normal (Figure 30-67). Abnormali- ties of the sperm head include size and shape defects as well as excess vacuoles (Figure 30-68). Abnormalities of the mid-piece include defects in thickness, asymmetrical attachment to the head, and sharp kinks or bends (Figure 30-69). Tail abnormalities include defects in length, width, and shape (sharp bends, coils) (Figure 30-70). Question- able or borderline forms should be considered abnormal. Any apparent predominant abnormality—head, neck, or Figure 30.68 Sperm with morphologic abnormality of the head: tapered head. Head Neck/mid-piece Tail Figure 30.69 Sperm with morphologic abnormality of the Figure 30.67 Normal sperm, Papanicolaou stain. neck/midpiece: bent neck. 698 Chapter 30 Figure 30.70 Sperm with morphologic abnormality of the tail: Figure 30.71 Spermatid in semen. coiled tail. Summary The fluids discussed in this chapter are those commonly these fluids include WBCs, tissue cells, and malignant seen in the routine clinical laboratory. Serous fluids cells. Examination of cellular morphology in body fluids, include pleural, peritoneal, and pericardial fluids. Other a critically important procedure for hematology labora- fluids examined are synovial fluids, CSF, amniotic fluid, tories, includes not only a differential leukocyte count semen, and BAL. In pathologic conditions, the amount of but also a possible demonstration of diagnostic findings, serous and synovial fluid (effusion) can increase. An effu- such as micro-organisms and malignant cells. The cyto- sion can be a transudate or an exudate. Traditional crite- centrifuge-prepared Wright-stained slide yields excellent ria for differentiating transudates from exudates include morphology of cells and can significantly aid in making specific gravity measurements and leukocyte counts. The a timely diagnosis of patients with effusions of unknown current criterion for pleural effusions, known as Light’s etiology. The hematology laboratory must take an active criteria, is to use serum and fluid measurements for protein role in correlating morphologic findings with any addi- and LDH. Chylous effusions are milky and result from tional studies performed, such as special stains, cytology, leakage of lymphatic vessels. Common cell types seen in and cultures. Review Questions Level I 3. Anatomically, where are the visceral pleura located? (Objective 1) 1. The type(s) of body fluids other than peripheral blood frequently sent to the hematology laboratory can a. Innermost aspect of the abdominal wall include which of the following? (Objective 1) b. Outermost portion of the heart a. Cerebrospinal fluid c. Innermost aspect of the chest wall b. Pleural fluid d. Outermost portion of the lung c. Pericardial fluid 4. Which of the following cell types seen in d. All of the above cerebrospinal fluid can be considered normal, not a sign of pathologic disease? (Objective 4) 2. The pleura, pericardium, and peritoneum are composed of what type of cell? (Objective 4) a. Arachnoid cells a. White blood cell b. Choroid plexus cells b. Epithelial cell c. Ependymal cells c. Mesothelial cell d. All of the above d. Squamous cell Morphologic Analysis of Body Fluids in the Hematology Laboratory 699 5. An LBC between 19,000–40,000 is: (Objective 10) 2. Which of the following cell types is responsible for the production of cerebrospinal fluid? (Objective 2) a. indeterminant b. an indication of fetal lung maturity a. Choroid plexus c. an indication of fetal lung immaturity b. Arachnoid d. the high reference level c. Neutrophil d. Mesothelial 6. Which of the following can be an artifact(s) on a cyto- centrifuged, Wright-stained slide? (Objective 5) 3. A 25-year-old woman develops a left-sided pleural effusion while recovering from bacterial pneumonia. a. Hypersegmentation of neutrophils A thoracentesis is performed, and the following labo- b. Stain precipitate ratory results are reported: c. Cytoplasmic projections of lymphocytes d. All of the above Serum Fluid Protein 6.5 g/dL 5 g/dL 7. The sperm concentration is reported as: (Objective 9) LD 75 U/L 60 U/L a. number per microliter b. number per liter These results would be best interpreted as which of c. number per deciliter the following? (Objective 3) d. number per milliliter a. Chylous 8. A CSF that appears orange should be described as: b. Exudate (Objective 2) c. Pseudochylous a. xanthrochromic d. Transudate b. hazy 4. A 56-year-old woman has a 3-week history of c. bloody abdominal pain. A peritoneal effusion is found. d. an old and unacceptable sample After paracentesis, a sample is sent to the laboratory. Examination of the cytocentrifuged, Wright-stained 9. Malignant tissue cells have which of the following slide shows clusters of large cells that have morphologic feature characteristic(s)? (Objective 7) abundant cytoplasm, smooth nuclear membranes, evenly distributed chromatin, and no nucleoli. An a. Irregular nuclear membrane occasional multinucleated cell is found with the b. Evenly distributed chromatin same features. These cells most likely represent: c. Prominent, irregular nucleoli (Objective 6) d. A and C only a. reactive mesothelial cells b. adenocarcinoma cells 10. True pathogenicity versus in vitro contamination of the body fluid specimen can be demonstrated by: c. small-cell carcinoma (Objective 6) d. ependymal cells a. finding the organisms intracellularly 5. A 60-year-old man who has smoked cigarettes b. staining the specimen purple with Wright stain since he was 16 years old has left-sided chest pain. c. staining the specimen red with Gram’s stain A thoracentesis sample is sent to the laboratory. d. finding the organisms in cytocentrifuged The cytocentrifuged, Wright-stained slide specimens examination reveals clusters of large cells that have irregular, jagged nuclear membranes, prominent Level II and irregular nucleoli, and unevenly distributed chromatin. These cells most likely represent: 1. A specimen labeled “ascites” is sent to the laboratory. (Objective 6) What type of procedure was used to obtain this fluid? (Objective 1) a. reactive mesothelial cells a. Thoracentesis b. adenocarcinoma cells b. Lumbar puncture c. large-cell lymphoma c. Pericardial aspiration d. reactive histiocytes d. Paracentesis 700 Chapter 30 6. Referring to the case in question 5, the protein, LD, RBC 100 * 109/L and other studies would most likely reveal that this is a(n): (Objective 3) WBC 0.10 * 109/L a. exudate Differential WBC 80% segmented neutrophils 15% lymphocytes b. chylous fluid 5% monocytes c. transudate d. pseudochylous fluid Which of the following would be the most specific finding in this patient for a true CNS 7. A 65-year-old woman has a painful, swollen elbow. hemorrhage versus traumatic spinal tap? A joint aspiration is performed, and a sample is sent (Objectives 7, 10) to the laboratory. A cytocentrifuged slide is examined with polarized light and a quartz c ompensator, a. Crenated red blood cells revealing birefringent crystals intracellular and b. Xanthochromia of the supernatant extracellular; they are needlelike in appearance c. Erythrophagocytosis by histiocytes and are yellow when oriented parallel to the quartz d. WBC count of 0.1 * 109 compensator. These crystals would be best identified /L as which of the following? (Objective 8) 10. Referring to the patient in question 9, what is the a. Calcium pyrophosphate significance of the WBC count of 0.1 * 109/L? b. Cholesterol (Objectives 2, 4, 10) c. Monosodium urate a. It is diagnostic for bacterial meningitis. d. Steroids b. It is diagnostic for early viral meningitis. 8. Referring to the case in question 7, which of the c. It is expected for the amount of hemorrhage. following is the most likely diagnosis? (Objective 8) d. It is diagnostic for fungal meningitis. a. Gout arthritis 11. A 45-year-old man has |
pleural effusions on the right b. Pseudo gout arthritis and left side. A right-sided thoracentesis is performed, c. Rheumatoid arthritis and a sample is sent to the laboratory. The fluid- d. Systemic lupus erythematosus serum protein ratio is 0.3, the fluid-to-serum LD ratio is 0.4, and the total nucleated cell count is low. Which 9. A 55-year-old man is brought to the emergency room of the following best describes this fluid? in a comatose state. He was found at home by his son (Objective 3) and appears to have fallen from a ladder. A spinal tap is performed and reveals the following: a. Chylous b. Exudate Color Red c. Pseudochylous Appearance Bloody d. Transudate References 1. Galagan, K. A., Blomberg, D., Cornbleet, P. J., & Glassy, E. F. (2006). diagnosis and management by laboratory methods (22nd ed., Color atlas of body fluids: An illustrated field guide based on proficiency pp. 480–510). Philadelphia: Elsevier/Saunders. testing. Northfield, IL: College of American Pathologists. 6. Morgenstern, L. B., Luna-Gonzales, H., Huber J. C., Wong, S. S., 2. Kjeldsberg, C. R., & Knight, J. A. (1993). Body fluids: Laboratory Uthman, M. O., Gurian, J. H., . . . , Grotta J. C. (1998). Worst examination of amniotic, cerebrospinal, seminal, serous and synovial headache and subarachnoid hemorrhage: Prospective, modern fluids. Chicago: American Society of Clinical Pathology. computed tomography and spinal fluid analysis. Annals of 3. Light, R. W. (2011). Pleural effusion. The Medical Clinics of North Emergency Medicine, 32(3 Pt 1), 297–304. America. 95(6), 1055-10070. 7. Arora, S., Swadron, S., & Dissanayake, V. (2007). Describing 4. McGibbon, A., Chen, G. I., Peltekian, K. M., & Van Zanten, S. V. cerebrospinal fluid red blood cell counts in patients with (2007). An evidence-based manual for abdominal paracentesis. subarachnoid hemorrhage. Annals of Emergency Medicine, 50, S51. Digestive Diseases and Sciences, 52(1), 3307–3315. doi: 10.1007/ 8. Heasley, D. C., Mohamed, M. A., & Yousem, D. M. (2005). s10620-007-9805-5 Clearing of red blood cells in lumbar puncture does not rule out 5. Karcher, D. S., & McPherson, R. A. (2011). Cerebrospinal, ruptured aneurysm in patients with suspected subarachnoid synovial, serous body fluids and alternative specimens. hemorrhage but negative head CT findings. American Journal of In: R. A. McPherson & M. R. Pincus, eds. Henry’s clinical Neuroradiology, 26(4), 820–824. Morphologic Analysis of Body Fluids in the Hematology Laboratory 701 9. Juliá-Sanchis, M. L., Estela-Burriel, P. L., Lirón-Hernández, F. J., & 27. College of American Pathologists. (2018). Cerebrospinal fluid Guerrero-Espejo, A. (2007). Rapid differential diagnosis between and body fluid cell identification. In Hematology and clinical subarachnoid hemorrhage and traumatic lumbar puncture microscopy glossary. Available from http://www.cap.org/ by D-dimer assay. Clinical Chemistry, 53(5), 993. doi: 10.1373/ ShowProperty?nodePath=/UCMCon/Contribution%20Folders/ clinchem.2007.085613 WebContent/pdf/hematology-glossary.pdf. 10. Travis, W. D., Colby, T. V., Koss, M. N., Rosado-de-Christenson, 28. Mitchell, M. A., Horneffer, M. D., & Standiford, T. J. (1993). M. L., Muller, N. L., & King, T. E. J. (2007). Handling and Multiple myeloma complicated by restrictive cardiomyopathy analysis of bronchoalveolar lavage and lung biopsy speci- and cardiac tamponade. CHEST Journal, 103(3), 946–947. mens with approach to pattens of lung injury. ARP Atlases, 1, 29. Nador, R. G., Cesarman, E., Chadburn, A., Dawson, D. B., 17–47. Ansari, M. Q., Sald, J., & Knowles, D. M. (1996). Primary 11. Heron, M., Grutters, J. C., ten Dam-Molenkamp, K. M., effusion lymphoma: A distinct clinicopathologic entity Hijdra, D., van Heugten-Roeling, A., Claessen, A. M. E., & associated with the Kaposi’s sarcoma-associated herpes virus. van Velzen-Blad, H. (2012). Bronchoalveolar lavage cell pattern Blood, 88(2), 645–656. from healthy human lung. Clinical and Experimental Immunology, 30. Knowles, D., & Chadburn, A. (2001). Lymphadenopathy and the 167(3), 523–531. doi: 10.1111/j.1365-2249.2011.04529.x lymphoid neoplasms associated with the acquired immune defi- 12. Walker, H. K., Hall, W. D., & Hurst, J. W. (1990). Clinical methods ciency syndrome. In: D. Knowles, ed. Neoplastic Hematopathology (3rd ed.). Boston: Butterworths. (2nd ed., pp.987-1090). Philadelphia: Lippincott Williams and 13. Sheaff, M.T., & Singh, N. (2013). Cytopathology: An introduction. Wilkins. London: Springer-Verlag. 31. Isaacson, P. G., Campo, E., & Harris, N. L. (2008). Large 14. Perry, J. J., Sivilotti, M. L., Stiell, I. G., Wells, G. A., Raymond, J., B-cell lymphoma arising in HHV8-associated multicentric Mortensen, M., & Symington, C. (2006). Should spectrophotom- Castleman disease. In: S. Swerdlow, E. Campo, N. L. Harris, etry be used to identify xanthochromia in the cerebrospinal fluid E. S. Jaffe, S. A. Pileri, H. Stein, . . . J. W. Vardiman, eds. World of alert patients suspected of having subarachnoid hemorrhage? Health Organization Classification of Tumours of Haematopoietic Stroke, 37(10), 2467–2472. and Lymphoid Tissues (4th ed. pp.258-260) Lyon, France: IARC 15. Horn, K. D., & Penchansky, L. (1999). Chylous pleural effusions Press. simulating leukemic infiltrate associated with t horacoabdominal 32. Olopade, O. I., & Ultmann, J. E. (1991). Malignant effusions. disease and surgery in infants. American Journal of Clinical CA: A Cancer Journal for Clinicians, 41, 166–179. Pathology, 111(1), 99–104. 33. Clare, N., & Rone, R., (1986) Detection of malignancy in body 16. Mundt, L. A., & Shanahan, K., eds. (2011). Graff’s Textbook of fluids. Laboratory Medicine, 17, 147–150. Routine Urinalysis and Body Fluids (2nd ed.). Philadelphia, PA: 34. Kendall, B., Dunn, C., & Solanki, P. (1997). A comparison of Lippincott Williams & Williams. the effectiveness of malignancy detection in body fluid 17. Steele, R., Marmer, D. J., O’Brien, M. D., Tyson, S. T., Steele, C. examination by the cytopathology and hematology R. (1986). Leukocyte survival in cerebral spinal fluid. Journal of laboratories. Archives of Pathology and Laboratory Medicine, Clinical Microbiology, 23(5), 965–966. 121(9), 976-979. 18. Geborek, P., Truedsson, L., Sandberg, M., & Wollheim, F. A. (1987). 35. Wittchow, R., Laszewski, M., Walker, W., & Dick, F. (1992). Para- Hyaluronidase treatment of synovial fluid affects the recovery of nuclear blue inclusions in metastatic undifferentiated small cell mononuclear cells and their in vitro immunoglobulin synthesis. carcinoma in the bone marrow. Modern Pathology: An Official Clinical and Experimental Rheumatology, 5(3), 255. Journal of the United States and Canadian Academy of Pathology, 5(5), 19. Galagan K. A. (2012, June). Q&A. CAP Today, 97. 555–558. 20. Galagan, K. A. (2012, January). Q&A. CAP Today, 80. 36. Odom, L. F., Wilson, H., Cullen, J., Bank, J., Blake, M., & 21. Danise, P., Maconi, M., Rovetti, A., Avino, D., Di Palma, A., Jamieson, B. (1990). Significance of blasts in low-cell-count Gerardo Pirofalo, M., & Esposito, C. (2013). Cell counting of cerebrospinal fluid specimens from children with acute body fluids: Comparison between three automated haematology lymphoblastic leukemia. Cancer, 66(8), 1748–1754. analysers and the manual microscope method. International Journal 37. Li, C. Y., & Yam, L. T. (1990). Blast transformation in chronic of Laboratory Hematology, 35(6), 608–613. myeloid leukemia with synovial involvement. Acta Cytologica, 22. Williams, J. E., Walters, J., & Kabb, K. (2011). Gaining efficiency 35(5), 543–545. in the laboratory-automated body fluid cell counts: Evaluation of 38. O’Connell, J. X. (2000). Pathology of the synovium. A merican the body fluid application on the Sysmex XE-5000 Hematology Journal of Clinical Pathology, 114(5), 773–784. doi: 10.1309/ Analyzer. Lab Medicine, 42, 395. LWW3-5XK0-FKG9-HDRK 23. Fleming, C., Brouwer, R., Lindemans, J., & de Jonge, R. (2012). 39. Judkins, S. W., & Cornbleet, P. J. (1997). Synovial fluid crystal Validation of the body fluid module on the new Sysmex analysis. Laboratory Medicine, 28, 774–779. XN-1000 for counting blood cells in cerebrospinal fluid and other 40. Rabinovitch, A., & Cornbleet, P. J. (1994). Body fluid microscopy body fluids. Clinical Chemistry and Laboratory Medicine, 50(10), in US laboratories: Data from two College of American 1791–1798. Pathologists surveys, with practice recommendations. Archives 24. Shmerling, R. H., Delbanco, T. L., Tosteson, A. N., & Trentham, D. E. of Pathology and Laboratory Medicine, 118(1), 13–17. (1990). Synovial fluid tests: what should be ordered? Journal of the 41. Lu, J., Gronowski, A. M., & Eby, C. (2008). Lamellar body counts American Medical Association, 264(8), 1009–1014. performed on automated hematology analyzers to assess fetal 25. Freemont, A. J., Denton, J., Chuck, A., Holt, P. J., & Davies, lung maturity. Laboratory Medicine, 39, 419. M. (1991). Diagnostic value of synovial fluid microscopy: A 42. Besnard, A. E., Wirjosoekarto, S. A., Broeze, K. A., Opmeer, B. C., & reassessment and rationalisation. Annals of the Rheumatic Diseases, Mol, B. W. (2013). Lecithin/sphingomyelin ratio and lamellar 50(2), 101–107. body count for fetal lung maturity: A meta-analysis. European 26. Meyer, K. C., Raghu, G., Baughman, R. P., Brown, K. K., Journal of Obstetrics and Gynecology and Reproductive Biology, 169(2), Costabel, U., Du Bois, R. M., & Rottoli, P. (2012). An official 177–183. American Thoracic Society clinical practice guideline: The clinical 43. Szallasi, A., Gronowski, A. M., & Eby, C. S. (2003). Lamel- utility of bronchoalveolar lavage cellular analysis in interstitial lar body count in amniotic fluid: A comparative study of four lung disease. American Journal of Respiratory and Critical Care different hematology analyzers. Clinical Chemistry, 49(6 Pt 1), Medicine, 185(9), 1004–1014. 994–997. 702 Chapter 30 44. Clinical Laboratory Standards Institute. (2011). Assessment of Validation of lamellar body counts using three hematology fetal lung maturity by the Lamellar body count: Approved guide- analyzers. American Journal of Clinical Pathology, 134(3), 420–428. line (CLSI Document No. C58-A). Wayne, PA: Author. 46. World Health Organization. (2010). WHO laboratory manual for 45. Lockwood, C. M., Crompton, J. C., Riley, J. K., Landeros, K., the examination and processing of human semen (5th ed.). Geneva: Dietzen, D. J., Grenache, D. G., & Gronowski, A. M. (2010). Author. Section Seven Hemostasis 703 Chapter 31 Primary Hemostasis J. Lynne Williams, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define hemostasis, blood coagulation, and 5. Name the three types of blood vessels and thrombosis. explain the general roles of the vasculature 2. Explain the general interaction of and normal endothelial cells in aiding and the systems involved in maintaining preventing the activation of the hemostatic hemostasis. system. 3. Distinguish the events that occur in primary 6. Identify and define the steps in the normal hemostasis from those that occur in second- sequence of events of platelet activation fol- ary hemostasis. lowing injury to the endothelium. 4. Differentiate the primary hemostatic plug 7. Describe the role of the primary hemostatic from the secondary hemostatic plug. plug in the cessation of bleeding. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Identify key histological features of each 3. Outline steps platelets undergo in forming type of blood vessel, and explain the the primary hemostatic plug, including the metabolic functions of endothelial cells in biochemical mediators necessary for platelet hemostasis. adhesion, platelet aggregation, and platelet 2. Identify three key substances stored in secretion. platelet dense granules and five key 4. Correlate various platelet ultrastructural substances stored in platelet a@granules and features with their functions. explain the role of each in hemostasis. 704 Primary Hemostasis 705 5. Identify platelet agonists and predict their 7. Correlate activation with changes in effect on platelet function. the platelet ultrastructure and 6. Describe the biochemical roles of the biochemistry. secreted contents of the platelet granules in 8. Describe the roles of the platelet in hemostasis. secondary hemostasis. Chapter Outline Objectives—Level I and Level II 704 Role of the Vascular System 707 Key Terms 705 Platelets in Hemostasis 711 Background Basics 705 Summary 727 Case Study 706 Review Questions 727 Overview 706 References 729 Introduction 706 Key Terms Agonist Glycoprotein IIb/IIIa complex Platelet procoagulant activity A@granule (AG) (GPIIb/IIIa) Platelet secretion (platelet release Arachidonic acid (AA) Hemostasis reaction) Clot retraction Human platelet antigen (HPA) Primary hemostatic plug Dense granule (DG) Non-thrombogenic Secondary hemostatic plug Dense tubular system Open canalicular system (OCS) Thrombogenic (DTS) Platelet activation Thrombopoietin (TPO) Glycocalyx Platelet adhesion Thromboregulation Glycoprotein Ib (GPIb) Platelet aggregation Thrombosis Background Basics The information in this chapter builds on the concepts Level II learned in previous chapters. To maximize your learn- • Summarize the hierarchy of stem and progenitor ing experience, you should review these concepts before cells in the bone marrow and the growth factors starting this unit of study: that direct the proliferation and maturation of blood cells. (Chapter 4) Level I • Describe the general biology of integrins and other • Describe the bone marrow production of blood adhesion molecules. (Chapter 7) cells. (Chapters 3, 4) • Summarize the development of megakaryocytes • Identify the marrow precursor of peripheral blood in the bone marrow |
and the mechanism of release of platelets. (Chapter 9) platelets into the peripheral blood. (Chapter 9) • State the normal lifespan of platelets in the periph- eral blood. (Chapter 9) 706 Chapter 31 under normal circumstances and the system’s ability to CASE STUDY prevent excessive blood loss upon injury. H emostasis We refer to this case study throughout the chapter. is from the Greek words heme, meaning “blood,” and Michael, a 20-year-old male with acute lym- stasis, meaning “to halt.” The barrier mass formed to phoblastic leukemia (ALL), is receiving chemo- limit blood loss is known as the hemostatic plug, blood therapy. Two weeks after his second treatment, he clot, or thrombus. H emostasis requires the interaction notices small reddish-purple spots on his lower of three compartments: the blood vessels, the platelets, legs and ankles. A CBC reveals Hb 9 g/dL; WBC and a group of soluble plasma proteins, the coagula- 2 * 103/mcL; platelets 19 * 103/mcL. tion factors. Blood coagulation (“clotting”) is the mech- Consider what could be responsible for the anism that transforms the fluid plasma into a gel by pancytopenia and the potential consequences of converting the soluble protein fibrinogen to the insolu- this condition. ble form, fibrin. Maintaining blood fluidity requires an intact v ascular endothelium, quiescent (inactive) blood platelets, and inactive plasma procoagulant proteins. On the other hand, control of bleeding requires rapid activation of platelets and plasma proteins to prevent Overview exsanguination. Hemostasis occurs in stages called primary hemostasis, This chapter is the first of a section describing the pro- secondary hemostasis, and fibrinolysis. During primary hemo- cesses involved when blood clots in response to vascular stasis, the platelets interact with the injured vessels and with injury. The actions of the blood vessels and platelets in one another. This interaction results in a mass of platelets hemostasis are collectively called primary hemostasis; the known as the primary hemostatic plug, which temporarily actions of the protein coagulation factors are called second- arrests bleeding, but is fragile and easily dislodged from ary hemostasis. This chapter discusses primary hemostasis the vessel wall. and describes the physical structure and the functions of Subsequently, insoluble strands of fibrin become the blood vessels in hemostasis. It then discusses the role deposited on and within the primary platelet plug to of platelets in hemostasis, including structure and func- reinforce and stabilize it and to allow the wound to heal tions of p latelets. Chapter 32 describes secondary hemo- without further blood loss. Generation of fibrin consti- stasis (fibrin formation) and fibrinolysis. Chapters 33 tutes the stage of secondary hemostasis. Fibrin is formed and 34 d iscuss the disorders of hemostasis that result in by a series of complex biochemical reactions involving bleeding, and Chapter 35 discusses disorders that result soluble plasma proteins (coagulation factors) as they in thrombosis. interact with the injured blood vessels and the platelet plug (Chapter 32). The fibrin-reinforced plug, or throm- bus, is called the secondary hemostatic plug. The blood Introduction has changed from a liquid to a semisolid gel at the site of injury (Figure 31-1). Blood flows through a closed system of vessels called the After the wound has healed, additional components circulatory system. The blood vessels and their constitu- of the hemostatic system break down and remove the clot ents are critical in controlling the physiologic functions during the fibrinolytic stage. Physiologic and biochemical and integrity of the circulatory system. A traumatic injury, inhibitors control all phases and components of the hemo- such as a cut to the finger, severs vessels, resulting in bleed- static system. ing. To minimize blood loss, the normally inert circulating Injury also can occur to intact, unsevered blood vessels. platelets and dissolved plasma proteins mobilize to form In this case, blood clot formation may occur on an interior a clot, an insoluble mass or structural barrier (thrombus), surface of the damaged vessel wall and result in the abnor- which occludes the injured vessel and prevents further loss mal condition thrombosis. of blood. The clot’s formation is limited to the area of injury In summary, hemostasis occurs because of the interac- so that normal circulation is maintained in vessels else- tion of the blood vessels, platelets, and certain plasma pro- where in the body. In other words, the hemostatic system teins (Table 31-1). Plasma proteins involved in hemostasis is activated when and where it is needed. This s ystem pro- include those that form fibrin, those that are involved with vides a continuous surveillance mechanism that prevents fibrinolysis, and those that inhibit all stages of the process. plasma and cells from leaking into the tissues in normal Defects in one hemostatic compartment can be overcome by circumstances. effective utilization of the other two; defects in two of the Hemostasis is the property of the circulation that three compartments generally result in pathologic hemo- maintains blood as a fluid within the blood vessels stasis and bleeding. Primary Hemostasis 707 Role of the Vascular System The vascular system forms an extensive distribution sys- tem for blood, carrying nutrients to all the body’s cells and tissues and transporting waste products for disposal. To perform this function, the system must provide an unin- terrupted flow of blood, a non-leaking circuit, and main- tain blood in a fluid state. The vascular system consists of Vascular injury three types of blood vessels: arteries, veins, and capillaries (Table 31-2). The arteries carry blood from the heart to the Primary hemostasis capillaries. The veins return blood from the capillaries to the heart. The vessels in which hemostasis occurs are pri- marily the smallest veins (venules) and, to a lesser degree, the smallest arteries (arterioles). Structure of Blood Vessels The structure of all blood vessels is similar (Figure 31-2), Platelet plug consisting of a central cavity, the lumen, through which the blood flows. The lumen is lined with a continuous, single Secondary hemostasis layer of flattened endothelial cells that separate the blood Table 31.2 Structure and Functions of Blood Vessels of the Microcirculation Structural Blood Vessel Characteristics Functions Arterioles 20–200 mcM in Serve as site of hemostatic diameter activity following injury Fibrin–platelet plug Basement membrane Regulate blood pressure by change in diameter and chemical mediators Figure 31.1 Stages of hemostasis after vascular injury. Primary components: Perform endothelial cell Bleeding occurs after an injury to a blood vessel. The hemostatic Smooth muscle cells hemostatic functions Fibroblasts system is activated to prevent excessive blood loss. Hemostasis Thicker wall of collagen occurs in two stages: (1) primary hemostasis when the platelets and extracellular matrix aggregate at the site of the injury and form the platelet plug (primary Venules 20–200 mcM in Serve as major site hemostatic plug) and (2) secondary hemostasis when fibrin develops diameter of hemostatic activity to strengthen the platelet plug forming the fibrin–platelet plug following traumatic injury (secondary hemostatic plug). Basement membrane Exchange nutrients, oxygen, and waste products Primary components: Regulate vascular Few smooth muscle permeability cells CASE STUDY (continued from page 706) Few fibroblasts Perform endothelial cell Thin layer of collagen hemostatic functions 1. Does Michael have a defect in primary or sec- and extracellular matrix ondary hemostasis? Minimal elastin fibers Capillaries 5–10 mcM in diameter Provide hemostatic effects by endothelial cell function Primary components: Exchange nutrients, Endothelial cell oxygen, and waste monolayer, surrounding products Table 31.1 pericytes Components of Hemostasis Basement membrane • Vasculature • Cellular Elements • Proteins No smooth muscle Arterioles Endothelial cells Fibrin-forming proteins cells Venules Platelets Fibrinolytic proteins No collagen fibers, Capillaries Inhibitors elastin fibers 708 Chapter 31 • Collagen types IV and V • Elastin • Mucopolysaccharides • Laminin • Fibronectin • von Willebrand factor • Vitronectin • Thrombospondin • Tissue plasminogen activator (tPA) • Plasminogen a Arteriole activator inhibitor Venule (PAI-1) • Collagenase • Heparan sulfate • PGI2 External elastic Internal elastic membrane membrane Tunica intima Tunica media Tunica adventitia Lumen Smooth muscle cells Endothelial Lumen cell Junction Basement membrane • Collagen • Proteoglycans Fibroblast • Reticular fibers • Elastin fibers Collagen type 1 Elastin fibers b Capillary Basement Cytoplasm of membrane endothelial cell Glycocalyx Abluminal surface Lumen Luminal Nucleus of surface endothelial cell Figure 31.2 Structure and function of the blood vessel wall, comparing and contrasting arterioles, venules, and capillaries. (a) Arterioles and venules. Endothelial cells, smooth muscle cells, and fibroblasts synthesize and secrete the components of the subendothelial matrix of connective tissue and the basement membrane proteins. The wall of arterioles is primarily composed of smooth muscle cells and contains elastin in contrast to the venule, which has only sparse smooth muscle cells. The media of arterioles is the most prominent layer; the adventitia layer is the largest in venules. (b) A capillary consists primarily of endothelial cells and basement membrane with very little connective tissue. from the underlying prothrombotic tissues and provide a layer, the tunica intima, consists of a single layer of endo- protective environment for the blood’s cellular components. thelial cells, the basement membrane, and subendothelial Endothelial cells are attached to a basement membrane that connective tissue that holds them together. consists of a unique form of collagen embedded in a matrix The middle layer, the tunica media, is thicker in arter- of adhesive proteins. ies than in veins. It consists of an internal elastic lamina, The walls of arteries and veins consist of three layers of which separates the intima and the media, variable layers tissue that vary in thickness and composition, depending on of smooth muscle cells, and an external elastic lamina that the size and type of vessel (Figure 31-2a).1,2 The innermost separates the media from the adventitia. In arteries, smooth Primary Hemostasis 709 muscle cells predominate in the tunica media and are sur- After an injury, the damaged vessels initiate hemo- rounded by loose connective tissue primarily consisting stasis. Their first response to injury is constriction or of collagen, reticular fibers, and proteoglycans. The tunica narrowing of the lumen of the arterioles to minimize media of veins contains only a few smooth muscle cells and the flow of blood into the wound area and the escape of fewer elastin fibers. In response to a variety of physiologic blood from the wound site. Vasoconstriction also brings stimuli, the smooth muscle cells of the tunica media can the hemostatic components of the blood (the platelets and contract and expand, which constricts or dilates the lumen the plasma proteins) closer to the vessel wall, facilitating of the vessel. their interactions. Vasoconstriction occurs immediately The tunica adventitia, the outer coat, is composed and lasts a short time. of collagen bundles and the vasovasorum, a network of The mechanisms of vasoconstriction are complex. It is microvessels that supplies oxygen and nutrients to the cells caused in part by neurogenic factors and in part by several of the media and adventitia. A few fibroblasts are embed- regulatory substances that interact with receptors on the ded in this layer; these fibroblasts synthesize and secrete surface of cells of the blood vessel wall. These include sero- fibers and other components of the matrix. tonin and thromboxane A2 (TXA2), both products of acti- The smallest of the blood vessels, the capillary vated platelets, and endothelin-1 produced by endothelial (Figure 31-2b), connects the arterioles and venules. Cap- cells.1 illaries collectively compose by far the largest surface In contrast, healthy intact endothelial cells synthesize area of all types of blood vessels. They are approximately and secrete prostaglandin PGI2, also called prostacyclin, and 5–10 mcM in diameter, just large enough for single blood nitric oxide (NO), also called endothelium derived relaxing cells to traverse. Capillaries consist of a layer of endothe- factor (EDRF). Both counteract vasoconstriction by causing lial cells on an underlying basement membrane and are vasodilation of the arterioles.3 Both also inhibit activation surrounded by a discontinuous layer of contractile cells and recruitment of platelets. called pericytes. The endothelial cells communicate with surrounding pericytes through gap junctions.1 The tissue Checkpoint 31.1 beneath a capillary’s basement membrane is sparse and Think about the last time that you injured your finger with a contains no smooth muscle cells. paper cut. Did your finger bleed immediately? If not, what might The endothelial cells (EC) in the various types of vessels have prevented immediate bleeding? are responsible for different physiological functions. The EC lining of venules is primarily responsible for mediat- ing adhesion and emigration of leukocytes into the tissues, Functions of Endothelial |
Cells as part of the immune response. Arteriolar endothelium is Endothelial cells lining the blood vessel lumen provide an important for the regulation of vasomotor tone. important dynamic interface between flowing blood and the vessel wall. They are the principal components regu- Functions of Blood Vessels in lating many vascular functions, including some functions Hemostasis that are hemostatic and others that are non-hemostatic in nature (Table 31-3). Endothelial cells play an impor- Blood vessels and their endothelial lining play a critical role tant role in thromboregulation, a process in which the in the regulation of hemostasis, including the maintenance cells of the vessel wall interact with cells and proteins of of blood fluidity, the cessation of bleeding upon injury, and the blood to facilitate or inhibit thrombus formation.3 the prevention of thrombotic (occlusive) events. The endothelial lining of normal, healthy blood vessels is Table 31.3 Functions of Endothelial Cells Component/Characteristic Function Hemostatic Functions Non-thrombogenic/antithrombotic Negatively charged surface Repels platelets and hemostatic proteins Heparan sulfate (HS) Inhibits fibrin formation (cofactor for antithrombin) Thrombomodulin (TM) Binds thrombin, enhances activation of protein C Endothelial protein C receptor (EPCR) Binds protein C; facilitates PC activation Tissue factor pathway inhibitor (TFPI) Binds TF/FVIIa/FXa complex; inhibits extrinsic pathway of coagulation Prostacyclin (PGl2) Promotes vasodilation; inhibits platelet activation (Continued ) 710 Chapter 31 Table 31.3 Functions of Endothelial Cells (Continued ) Component/Characteristic Function Tissue plasminogen activator (tPA) Activates fibrinolytic system Annexin A2 (tPA receptor) (A2) Binds tPA and plasminogen; activates fibrinolysis Urinary-type plasminogen activator receptor (uPAR) Binds uPA and plasminogen; activates fibrinolysis Nitric oxide (NO) Promotes vasodilation; inhibits platelet recruitment and accumulation ADPase (CD39) Degrades ATP and ADP to AMP and adenosine Thrombogenic Endothelin (ET) Promotes vasoconstriction von Willebrand factor (vWF; production and processing) Carries factor VIII (FVIII) in plasma; facilitates platelet adhesion Tissue factor (TF) Initiates fibrin formation, activates FVII Plasminogen activator inhibitor (PAI-1) Inhibits activation of fibrinolytic system Non-hemostatic Functions Selective blood/tissue barrier Keeps cells and macromolecules in vessels; allows nutrient and gas exchange Processing of blood-borne antigens Contributes to cellular immunity Synthesis and secretion of connective tissue: Basement membrane collagen Provides backup protection for endothelial cells Collagen of the matrix Induces platelet adhesion Elastin Mediates vasodilation and vasoconstriction Fibronectin Binds cells to one another Laminin Contributes to platelet adhesion after injury Vitronectin Binds cells to one another; possibly promotes platelet adhesion Thrombospondin Binds cells to one another; possibly promotes platelet adhesion non-thrombogenic, antithrombotic, and profibrinolytic, When vascular injury occurs and bleeding starts, preventing i nappropriate activation of hemostasis. platelets and coagulation proteins in the plasma physically Damaged endothelial cells become thrombogenic and contact exposed subendothelial tissues. Interactions then antifibrinolytic, promoting the formation of a thrombus or occur between the vessel wall, platelets, and hemostatic blood clot (Figure 31-3). These competing interactions regu- proteins in the plasma to form a thrombus, or blood clot, late blood fluidity. to stop the bleed. In the presence of normal endothelium, a non-reac- The amount of blood lost from a vessel depends tive environment, platelets and coagulation proteins on the size and type of the damaged vessel, as well as are inert. Both physiologic and biochemical interactions the efficacy of the hemostatic mechanism. Hemostasis provide this non-reactive environment.4 Physiologically, is most effective in arterioles and venules. Capillaries the surface of the endothelial cell is negatively charged lack the tissue layers beyond the basement membrane and repels negatively charged circulating proteins and and do not effectively contribute to hemostasis. When platelets. Biochemically, a wide variety of substances larger vessels are severed, hemostatic plug formation are synthesized and secreted by the endothelial cells, takes longer and might not be sufficient to stop bleed- which contribute to the non-reactive environment. These ing. The pressure within arteries is much higher than in include substances that inhibit vasoconstriction and plate- veins, and the blood flow can be so rapid in larger arter- let function, inhibit coagulation, and activate fibrinolysis ies that clotting cannot occur effectively (e.g., within the (Table 31-3). upper aorta). Damaged endothelium becomes thrombogenic, pro- ducing substances that activate platelets and coagulation proteins and inhibit fibrinolysis. Damaged endothelial cells Checkpoint 31.2 activate platelet function and/or vasoconstriction, promote What actions of the endothelial cells prevent clotting from coagulation, and inhibit fibrinolysis. See Chapter 32 for a occurring within the blood vessels? discussion of the functions of these components. Primary Hemostasis 711 FVa Resting EC FVIIIa APC PS FIIa, FXa FIXa Platelets Protein AT FIIa C HS TM EPCR TFPI uPAR ADPase A2 NO PGI2 tPA, uPA Inhibition of platelet activation; Inhibition of coagulation Activation of fibrinolysis vasodilation FX FXa Activated EC FVIIa 1 Ca11 TF TF tPA TXA Collagen PAF 2 IV, V VWF ET PAI-1 Activation of platelet Activation of Inhibition of fibrinolysis adhesion, aggregation, coagulation and vasoconstriction Figure 31.3 Antithrombotic characteristics of resting endothelium versus the prothrombotic effects of damaged or activated endothelium. Resting endothelium provides an environment that inhibits activation of hemostasis. This includes secretion of substances that (1) inhibit platelet activation (PGI2, NO, ADPase); (2) inhibit coagulation (heparan sulfate as a cofactor for AT; TM for activation of protein C, which inactivates activated FVa and FVIIIa; and TFPI), and EPCR; and (3) activate fibrinolysis (tPA, A2, uPA, uPAR). When endothelium is damaged, it secretes substances that (1) activate platelets (TXA2, PAF, ET) and bind them to the vessel wall (VWF), (2) activate coagulation (TF, which initiates formation of fibrin), and (3) inhibit fibrinolysis (PAI-1). EC, endothelial cells; ET, endothelin; PGI2, prostacyclin; NO, nitric oxide; HS, heparin sulfate; AT, antithrombin; TM, thrombomodulin; APC, activated protein C; F IIa, factor IIa (thrombin); EPCR, endothelial cell protein C receptor; PS, protein S; TXA2, thromboxane A2; tPA, tissue type plasminogen activator; A2, annexin A2; uPA, urinary-type plasminogen activator; uPAR, uPA receptor; PAF, platelet activating factor; TF, tissue factor; TFPI, tissue factor pathway inhibitor; VWF, von Willebrand factor; PAI-1, plasminogen activator inhibitor-1; S , stimulation; ¡ inhibition. Platelets in Hemostasis As described in Chapter 9, platelets are anucleate fragments of megakaryocyte cytoplasm released into The second major component of the hemostatic system is the circulation as proplatelets. They have a life span of the blood platelet. Bizzozero first established the plate- 7–10 days in the circulation if they are not con- let’s role in hemostasis in 1882.5 He noted that they were sumed in the process of hemostatic activation sooner. clumped together as part of thrombi in the mesenteric ves- Megakaryocyte differentiation and maintenance of sels of rabbits and guinea pigs. peripheral blood p latelet numbers are primarily regulated 712 Chapter 31 by thrombopoietin (TPO). Additional cytokines, includ- Table 31-4). The components of each region have specific ing stem cell factor (SCF), interleukin (IL)-3, GM-CSF, functions in activated platelets and are discussed in the fol- IL-6, and IL-11 also influence platelet production. lowing sections. PERIPHERAL ZONE CASE STUDY (continued from page 707) The platelet’s peripheral zone consists of a phospholipid 2. If Michael were given TPO, how would you membrane covered on the exterior by a fluffy surface expect his bone marrow and peripheral blood coat and on the interior by a thin submembranous region picture to change? between the cytoplasmic membrane and the next layer. Glycocalyx The surface coat, or glycocalyx, is thicker on platelets than on most other cells (about 14–20 nm). It consists of glycolipids, membrane glycoproteins, proteins, Platelet Structure mucopolysaccharides, and adsorbed plasma proteins, including coagulation factor V (FV), VWF, and fibrino- Circulating “resting” (unactivated) platelets are disc- gen (Chapters 32 and 33). The glycocalyx is responsible shaped anucleate cell fragments with smooth surfaces. for the platelet surface’s negative charge and is found on Unlike the exterior surfaces of erythrocytes and leukocytes, the surface membrane of the interior channels as well. The the surface membranes of platelets have several indenta- negative charge is thought to help prevent platelets from tions or openings resembling holes in a sponge. They are interacting with each other and with negatively charged the openings to membranous channels that extend deep endothelial cells under normal physiologic conditions. into the interior of the cells. Some surface proteins are receptors for substances that Circulating platelets repel one another and the sur- cause platelet activation. faces of the endothelial cells that line the interior lumen of blood vessels. After an injury, many changes affecting Plasma Membrane The cytoplasmic membrane has a typi- the platelet morphology and biochemistry occur, caus- cal trilaminar structure of a bilayer of phospholipids and ing the platelets to become “activated” after which they embedded cholesterol and integral proteins. It functions to interact with the vessel wall and other platelets to form maintain cytoplasmic integrity and to mediate interactions the primary hemostatic plug. To understand the activa- between platelets and the vasculature and plasma proteins. tion process, the ultrastructure of normal platelets must The membrane is formed from the demarcation membrane be considered. system of the parent megakaryocyte. The surface membrane The platelet ultrastructure is divided into four arbi- of platelets invaginates to give rise to an elaborate system trary regions or zones: peripheral zone, structural zone, of channels, the surface-connected open canalicular system organelle zone, and membrane systems6 (Figure 31-4 and (OCS), which extends throughout the platelet cytoplasm. Organelle zone α- Dense Mitochondrion granule Lysosome granule Glycogen Peripheral zone Membrane Glycocalyx Membrane systems Structural zone Open canalicular Actin (G and system F forms) Microtubules Dense tubular system Submembranous cytoskeleton Myosin Figure 31.4 Diagram of platelet ultrastructure. Primary Hemostasis 713 negatively charged PLs to the platelet surface, facilitating Table 31.4 Platelet Ultrastructure and Functions their interactions with plasma procoagulant proteins. Zone and Component Function/Role Membrane Receptors The cytoplasmic membrane contains Peripheral zone Adhesion and aggregation integral proteins that are receptors for stimuli involved in • Glycocalyx Asymmetric arrangement; source platelet function, including receptors for VWF, fibrinogen, Proteins, glycoproteins, of arachidonic acid collagen, thrombin, fibronectin, and vitronectin (Table 31-5). mucopolysaccharides Approximately 30 proteins have been identified as platelet • Phospholipid bilayer membrane glycoproteins, and a nomenclature system has Phospholipids been developed using the acronym GP for glycoprotein and • Integral proteins Adhesion and aggregation a roman numeral from I to IX according to electrophoretic • Glycoproteins Ib/IX, IIb/IIIa, GPIa/ IIa, GPVI migration by decreasing molecular weight. Many of these Enzymes Activation receptors are integrins, which are heterodimeric proteins Structural zone Structure and support identified by their a and b subunits. Those receptors can • Microtubules (tubulin) be referred to by either nomenclature. Glycoprotein recep- • Cytoskeletal network tors mediate the interaction of platelets with their exter- • Microfilaments (actin) nal environment. Many of these receptors interact on the • Intermediate filaments (filamin/ cytoplasmic side of the membrane with components of the actin-binding protein, talin, platelet cytoskeleton and contribute to the maintenance of vimetin) the discoid shape of the resting platelet. Organelle zone Storage and secretion • Granules Non-protein mediators (ADP, GPIB/IX Glycoprotein Ib (GPIb) consists of two sub- Dense granules ATP, and serotonin) units, GPIba and GPIbb, and is found in the platelet mem- a@granules Protein mediators brane as a complex with GPIX (CD42a) and GPV (CD42d) Lysosomes Enzymes in a ratio of 2:2:1 (Figure 31-5a).8 GPIb together with GPIX Peroxisomes Lipid metabolism is the major platelet receptor for VWF. The function of • Mitochondria Oxidative energy metabolism GPV is not known, and no clinical bleeding problems • Glycogen are associated with its absence. GPV is not required for Membrane systems Storage and secretion either membrane expression of the GPIb/IX complex or for • Open canalicular system • Secretion of granule contents interaction between GPIb/IX and VWF. The exact function • Dense tubular system • Storage of calcium of GPIX is also unknown, but it is required for efficient An asymmetrical arrangement of the membrane phos- Table 31.5 Platelet Receptors pholipids (PLs) is an important factor in the function of Receptor(s) Nomenclature: platelets. The negatively charged PLs (phosphatidylserine [PS], phosphatidylinositol [PI], phosphatidylethanolamine Ligand Glycoprotein Integrin CD [PE]) are predominately found on the inner half of the lipid Collagen GPIa/IIa a2b1 CD49b/CD29 bilayer, whereas neutral PLs (phosphatidylcholine [PC] GPIIb/IIIa, aIIbb3 CD41/CD61 and sphingomyelin [SM]) are largely found on the outer GPIV CD36 half of the bilayer.7 The phospholipid asymmetry is main- GPVI tained in resting platelets by an ATP-dependent amino Fibrinogen GPIIb/IIIa, aIIbb3 CD41/CD61 phospholipid translocase (“flippase”) that actively pumps Vitronectin receptor aVb3 CD51/CD61 PE and PS from the outer to the inner leaflet.7 Two other Fibronectin GPIc/IIa a5b1 CD49e/CD29 phospholipid-translocating enzymes are found in platelets, |
GPIIb/IIIa aIIbb3 CD41/CD61 an ATP-dependant “floppase” that transports neutral PLs Thrombospondin aVb3 CD51/CD61 from the inner to the outer surface, and a lipid “scramblase” GPIV CD36 that rapidly induces random bidirectional movement of all Vitronectin aVb3 CD51/CD61 lipids in the membrane. GPIIb/IIIa, aIIbb3 CD41/CD61 The negatively charged PLs accelerate several steps in von Willebrand GPIb/IX/V CD42a–d the plasma protein coagulation sequence. The placement of factor GPIIb/IIIa aIIbb3 CD41/CD61 these negatively charged PLs on the platelet’s inner mem- brane separates them from the plasma coagulation proteins Laminin GPIc/IIa a6b1 CD49e/CD29 and prevents inappropriate coagulation. However, during GPIa/IIa a2b1 CD49b/CD29 platelet activation, scramblase causes the movement of the GP, glycoprotein; CD, cluster of differentiation 714 Chapter 31 Glycocalicin Ristocetin binding area portion Thrombin N terminal von Willebrand factor binding area GPIbb GPIba Exterior surface Cytoplasmic side GPIX C-terminal GPV Actin binding protein a (Filamin) Fibrinogen Ca11 g-chain HPA-3 antigen Fibrinogen N terminal binding site GPllbb HPA-1 antigen GPlIla Exterior surface GPllba C terminal Actin Cytoplasmic side b binding area Figure 31.5 Structure of platelet membrane glycoproteins. (a) Glycoprotein Ib is composed of an a@ and a b@chain. Both span the phospholipid bilayer. The a@chain is larger and contains the binding sites for thrombin, ristocetin, and VWF. Actin-binding protein (filamin) is attached to the cytoplasmic side. Glycoprotein IX and glycoprotein V associate with glycoprotein Ib in the platelet membrane. (b) Glycoproteins IIb and IIIa associate in a complex after platelet activation. Binding sites for fibrinogen as well as platelet-specific antigens are present. Cytoplasmic portions of each component have binding areas for actin. surface expression of GPIb.6 Rare bleeding disorders are fragment (normal plasma levels of 1–3 mg/L). Plasma levels associated with abnormalities of GPIb and with GPIX. The of glycocalicin correlate with platelet production and can complex (also known as CD42a–d) is referred to in this text be used to differentiate thrombocytopenia resulting from as GPIb/IX. decreased platelet production (low plasma glycocalicin) The GPIba polypeptide is larger than the GPIbb poly- from that resulting from increased platelet destruction (ele- peptide and contains the binding sites for VWF, throm- vated glycocalicin).9,10 The remainder of GPIba is associated bin, and ristocetin (used in the platelet aggregation test with the GPIbb chain and spans the phospholipid bilayer. described in Chapter 36). Other ligands for GPIb/IX On the cytoplasmic side, both the a and b portions are asso- include P selectin, found on activated platelets and endo- ciated with the actin-binding protein, filamin. Each platelet thelial cells, and the integrin aMb2, found on leukocytes. contains approximately 25,000 GPIb/IX complexes.11 GPIb/ Interactions with these ligands help recruit leukocytes to IX functions in the platelet adhesion process (see the section activated platelets at sites of injury.9 The binding sites are “Platelet Adhesion”). located on the major extracellular portion of GPIb called glycocalicin. The glycocalicin portion of the chain extends GPIIB/IIIA Glycoproteins IIb and IIIa associate in the from the platelet surface and can be cleaved by proteolytic membrane, forming a non-covalently associated glyco- enzymes including thrombin, a disintegrin, and metallo- protein IIb/IIIa (GPIIb/IIIa) complex, which is the major protease 10 (ADAM10) and ADAM17, yielding a soluble plasma membrane receptor for fibrinogen (Figure 31-5b). Primary Hemostasis 715 The GPIIb/IIIa complex also binds other subendothelial activating platelets. It has been proposed that the two adhesive proteins such as VWF, thrombospondin, vitro- receptors work synergistically; GPIa/IIa supports adhe- nectin, and fibronectin. Each platelet has approximately sion, and GPVI supports activation.14 Other platelet mem- 80,000–100,000 copies of this receptor on its external mem- brane proteins that can bind collagen include GPIIb/IIIa brane, making it the most numerous of the platelet recep- and GPIV.15 tors. There are another 20,000–40,000 copies present within platelets on the internal membranes of the a@granules, dense Platelet Alloantigens Polymorphisms (nucleotide sequence granules, and OCS.12 These internal receptors translocate differences) in several of the major platelet glycoproteins have to the plasma membrane when platelets are activated and been described. These polymorphisms are immunogenic, undergo secretion.13 and the different inherited alleles thus produce unique Glycoprotein IIb, the larger of the two subunits, is a alloantigens associated with the platelet surface, desig- two-chain protein. The a@chain is embedded in the phos- nated as human platelet antigens (HPA). Polymorphisms pholipid bilayer, and the b@chain protrudes from the plate- within the GPIIIa polypeptide result in a number of dif- let surface. Glycoprotein IIIa is a single chain polypeptide ferent platelet alloantigens, including HPA-1, HPA-4, and and is associated with the GPIIb portion that lies within others. Polymorphisms within the GPIIb polypeptide result the phospholipid bilayer. The cytoplasmic tails of the two in the HPA-3 platelet alloantigens (formerly called Bak and proteins are associated with actin and myosin in the platelet Lek). Platelet alloantigens have also been associated with cytoskeleton. Like many integrins, the GPIIb/IIIa complex polymorphisms of GPIa and GPIba polypeptides16 and in resting platelets is in an inactive form and has low affinity have been implicated in neonatal alloimmune thrombocy- for binding fibrinogen in solution. When agonists activate topenia (NAIT) and immune thrombocytopenia purpura platelets, the GPIIb/IIIa complex is converted to a high- (ITP); Studies are ongoing to determine whether these poly- affinity ligand-binding conformation required for platelet morphisms correlate with either bleeding or thrombotic aggregation (described in the section “Platelet Aggrega- tendencies.6 tion”)13 (Figure 31-6). The GPIIb/IIIa receptors in a gran- ules (described in the section “Organelle Zone”) appear to Checkpoint 31.3 cycle from and to the plasma membrane, which seems to be If a patient inherited a mutation of the gene for glycoprotein IIIa responsible for the uptake of fibrinogen from plasma into that resulted in its absence, what two platelet antigens would megakaryocytes and platelets.6 be decreased or absent? Collagen Receptors Collagen is one of the most important platelet activators, and several platelet glycoproteins func- STRUCTURAL ZONE tion as collagen receptors. GPIa/IIa, like GPIIb/IIIa, must The discoid shape of resting platelets is maintained by a be activated to exhibit high affinity binding to collagen. specialized cytoskeleton, composed of the components of GPVI also functions as a collagen receptor, and although the structural zone. The platelet cytoskeleton is remark- it has weaker affinity for collagen, it is more effective in ably similar to the erythrocyte cytoskeleton (Chapter 5). FIB GPIIb GP IIla GPIIb GP IIla a b Figure 31.6 Activation of GPIIb/IIIa. (a) Inactive GPIIb/IIIa as found in inert platelets: complex is bent or folded in an inactive conformation and has low affinity for fibrinogen in solution. (b) Activated form of GPIIb/IIIa displays significantly enhanced fibrinogen-binding affinity: activated GPIIb-IIIa exists as an “open” conformation, with the receptor “head” extended and the two “tails” separated further apart. 716 Chapter 31 The structural zone consists of microtubules and submem- Dense Granules Platelets contain 3–8 electron-dense branous and cytoplasmic networks of microfilaments and organelles, each 20–30 nm in diameter. These dense intermediate filaments. Functionally, the structural zone sup- granules (DGs) are named because they appear denser in ports the plasma membrane, stabilizes the shape and integ- electron micrographs than the other types of granules rity of the resting platelet, and provides a means of shape (due to their high Ca++ content). These granules contain change when the platelet is activated. mediators of platelet function and hemostasis that are not proteins: ADP, ATP, and other nucleotides as well Microtubules Resting platelets have a microtubule coil, as phosphate compounds, calcium ions, and serotonin which completely surrounds the circumference of the (Table 31-6). The ADP in the DG is known as the non- platelet just below the plasma membrane, stabilizing the metabolic or storage pool ADP to distinguish it from meta- discoid shape of the resting platelet. Microtubules (MT) are bolic ADP found in the cytoplasm. The ratio of ADP:ATP the largest of the cytoskeletal filaments (25-nm diameter) in the DG is higher than that found in the cytoplasm and are composed primarily of the protein tubulin. Tubu- (3:2 versus 1:8 in the cytoplasm).6 The metabolic pool of lin subunits are in dynamic equilibrium with assembled ATP/ADP provides energy for normal platelet metabo- MTs, indicating ongoing assembly and disassembly of MTs. lism, whereas the storage pool is important in platelet acti- This dynamic process plays a role in the shape change that vation reactions. Serotonin is taken up from the plasma occurs during platelet activation.6 and stored in the DGs. ADP and serotonin released from Microfilaments and Intermediate Filaments The submem- the DGs during platelet a ctivation provide a positive branous protein network consists of structural proteins feedback mechanism to recruit and activate additional (intermediate filaments) that form a cytoskeleton support- platelets in forming the primary h emostatic plug. ing the plasma membrane. Actin, the most abundant pro- A@Granules The most numerous of the four types of gran- tein in platelets, accounts for 15–20% of the total protein ules are the A@granules 1AGs 2 , numbering about 50–80 per content. Actin is also in a dynamic equilibrium between platelet. They contain a variety of bioactive substances monomeric (globular, G form) and polymeric (filamentous, that can be divided into two major groups (Table 31-7): F form) configurations. The spectrin-actin filament network One group consists of proteins similar to hemostatic pro- in platelets binds to and helps stabilize the platelet receptors teins (coagulation factors, inhibitors) found in the plasma. in the membrane. Upon platelet activation, the submembra- Some, such as VWF, are synthesized in the megakaryocyte nous microfilaments and intermediate filaments reorganize, as the platelets develop. Others, such as fibrinogen, are polymerize, and form the characteristic pseudopods seen in endocytosed and packaged in the aG by megakaryocytes activated platelets. during thrombopoiesis and by platelets in the circulation.17 Actin also is part of a network of structural support The second group includes proteins with a variety of func- proteins dispersed throughout the cytoplasm. In the cyto- tions. Some are found exclusively in platelets (e.g., platelet plasm, actin is associated with actin-binding proteins (e.g., factor 4 and b@thromboglobulin) and their presence in the filamin, talin) and with myosin and several other contractile plasma can be used as a marker of in vivo platelet activa- proteins similar to those of smooth muscle. Unlike smooth tion. aGs contain both proangiogenic proteins (e.g., vascu- muscle, in which the ratio of actin:myosin is about 7:1, the lar endothelial growth factor [VEGF]) and antiangiogenic platelet ratio is about 100:1. This network also undergoes factors (e.g., endostatin).6 They contain factors that affect significant changes in structure and location when platelets the growth and gene expression of smooth muscle cells in become activated, resulting in the central contraction of MT the blood vessel wall (e.g., platelet-derived growth factor), and the relocation of organelles during platelet activation which play a major role in vessel repair following injury. and shape change. Plasminogen activator inhibitor (PAI) is present in platelet ORGANELLE ZONE The organelle zone is located beneath the MT layer and consists of mitochondria, glycogen particles (which sup- Table 31.6 Composition and Functions of Platelet Dense port the platelet’s metabolic activities), and four types Granule Contents of granules dispersed within the cytoplasm: dense gran- ules, a@granules, lysosomes, and peroxisomes. Platelets serve Component Role as storage sites for a variety of molecules (proteins and ADP (non-metabolic) Agonist for platelets other substances) essential for platelet function, inflam- ATP (non-metabolic) Agonist for cells other than platelets; activates Ca++ influx channel in plasma membrane mation, cell proliferation, vascular tone, fibrinolysis, and wound healing. Generally, mature platelets have Polyphosphates Acceleration of factor V activation relatively few ribosomes and little rough endoplasmic Calcium Platelet activation reticulum. Serotonin Vasoconstriction; platelet agonist Primary Hemostasis 717 Table 31.7 Composition and Functions of Platelet a@Granule Proteins Protein Role Group I—Hemostatic Proteins • Fibrinogen • Platelet aggregation; conversion to fibrin • FV, FXI, FXIII • Fibrin formation • Protein S, tissue factor pathway inhibitor, a1 protease inhibitor, • Inhibition of coagulation C1 inhibitor, a2@macroglobulin • von Willebrand factor • Platelet adhesion; carrier of FVIII in plasma • Plasminogen • Conversion to plasmin (fibrinolysis) • Plasminogen activator inhibitor-1, a2@plasmin inhibitor • Fibrinolytic inhibitors Group II—Non-hemostatic Proteins Platelet specific • b@thromboglobulin Chemoattractant for neutrophils, fibroblasts; neutralizer of heparin • Platelet factor 4 Neutralizer of heparin; weak neutrophil and fibroblast chemoattractant • Multimerin Complex with factor V Mitogenic factors • PDGF, TGFb, EGF, IGF Promotion of regrowth of smooth muscle cells (wound repair) • VEGF Angiogenic factor Miscellaneous • Albumin, immunoglobulins Unknown • Thrombospondin, fibronectin, vitronectin Adhesive glycoproteins PDGF, platelet-derived growth factor |
(PDGF)’ TGFb), transforming growth factor@b; EGF, epidermal growth factor; IGF, insulin-like growth factor; VEGF, vascular endothelial growth factor. aGs in addition to being synthesized by endothelial cells. MEMBRANE SYSTEMS Platelet aGs also contain a number of adhesive proteins The fourth structural zone of the platelet is composed of including fibronectin, VWF, vitronectin, and thrombospon- two systems of membranes: the open canalicular system din. Platelets contain distinct subpopulations of aGs, which and the dense tubular system. are thought to undergo differential release during platelet Open Canalicular System (OCS) The surface-connected activation. For example, fibrinogen and VWF are localized open canalicular system (OCS) is an elaborate, intercon- in separate aGs.6 nected series of channels leading from the platelet sur- Lysosomes and Peroxisomes Lysosomes contain several face to its interior. The OCS originates as invaginations hydrolytic enzymes and are similar to the lysosomes of the plasma membrane and serves several functions in found in other cells. Platelets can secrete lysosomal platelets. It provides for both the entry of external sub- enzymes when activated, but the release is slower and stances into the interior of platelets and a route for the less complete than release from a and dense granules. release of granule contents to the outside. The OCS also Thus, release is usually seen only with stronger platelet represents an extensive internal store of membranes. Acti- agonists. With lysosomal secretion, proteins present in vated platelets undergoing shape change, pseudopod lysosomal membranes (lysosome-associated membrane formation, and spreading require a significant increase proteins [LAMP-1, LAMP-2, LAMP-3]) appear on the in surface membrane compared with resting platelets, plasma membrane and serve as markers of high-level which OCS membranes provide. Because the OCS mem- platelet activation.18 The peroxisomes are involved in branes contain many of the same glycoproteins as the sur- fatty acid oxidation, and the synthesis of platelet activat- face membrane, it also serves as a source for additional ing factor (PAF).6 plasma membrane glycoprotein receptors during platelet activation.6 Platelet Metabolic Activity Platelets have fairly significant stores of glycogen, which can be hydrolyzed to glucose for Dense Tubular System (DTS) The dense tubular system energy. They also take up glucose from their surrounding (DTS) originates from the residual endoplasmic reticulum medium. Platelets contain all of the necessary enzymes for of the megakaryocyte and do not connect with the platelet’s the glycolytic and tricarboxylic acid cycles and for glycogen surface. The DTS is a storage site for ionized calcium within synthesis and degradation. Platelet activation is associated platelets analogous to the sarcoplasmic reticulum of muscle with marked increases in both glycolysis and oxidative ATP cells and releases Ca++ when p latelets are activated.19 The production. concentration of calcium ions within the platelet cytoplasm 718 Chapter 31 is important in regulating platelet metabolism and acti- components of the vessel walls that are normally hidden vation. The DTS is also a major site of prostaglandin and from circulating platelets. The subendothelium contains a thromboxane synthesis.20 large number of adhesive proteins with which the platelets can interact via their specific membrane receptors. The first Platelet Functions step in primary hemostatic plug formation, platelet adhe- sion, is the attachment of platelets to collagen and other Platelets are involved in several aspects of hemostasis, components of the subendothelium. Platelets contain two including maintaining vascular integrity, forming the pri- collagen receptors: GPIa/IIa and GPVI, which mediate mary hemostatic plug, providing a surface for fibrin genera- direct collagen binding at low shear rates. Platelet adhe- tion and secondary hemostatic plug formation, and finally sion to collagen at high shear rates, however, requires the promoting repair of the injured tissues. They also contribute presence of VWF and the platelet VWF receptor, GPIb/IX. substantially to inflammation and both innate and adaptive Both endothelial cells and megakaryocytes synthe- immune responses by interacting with leukocytes. Platelet– size VWF, which is stored in intracellular organelles, leukocyte interactions, in fact, may be important in the ini- the Weibel-Palade bodies of endothelial cells and aG tiation of coagulation and secondary hemostasis. of platelets. VWF is secreted into the plasma as well as deposited in the subendothelium where it is bound to col- MAINTENANCE OF VASCULAR INTEGRITY lagen (Chapter 33). VWF is a multimeric protein made up Even in the absence of physical trauma to a vessel, platelets of a series of identical subunits, each containing binding play an important role in maintaining vascular integrity, sites for GPIb/IX on the platelet surface and for collagen which requires functional and physical contacts between in the subendothelium. Circulating (soluble) VWF will platelets and the endothelium.21,22 Platelet granules con- not bind to GPIb/IX on circulating platelets. However, tain proangiogenic cytokines, growth factors, and other high shear rates cause conformational changes in immo- barrier-stabilizing molecules, which are released upon bilized VWF, and platelet adhesion is initiated by platelets platelet activation. These cytokines function to stabilize an intercellular adhesion protein complex between the vas- cular endothelial cells. When the circulating platelets are low (thrombocytopenia), the adjacent intercellular junc- tions disassemble, resulting in leakage of cells and fluid into the surrounding tissue.21 FORMATION OF THE PRIMARY HEMOSTATIC PLUG When vessel injury occurs, the platelets react by forming the primary hemostatic plug. By sticking first to exposed a b collagen and other components of the subendothelium and then to each other, the platelets form a mass that mechani- cally fills defects in the vessel lining and limits the loss of blood from the injury site. SE Platelets circulating in blood vessels do not interact with P other platelets or other cell types. Circulating platelets are disc shaped and inert in the environment of normal endothe- EC lium (Figure 31-7a). This is largely the result of endothelial release of prostacyclin (PGI2) and nitric oxide (NO), which inhibit platelet activation, and the presence of an ADPase P (CD39), which degrades ADP, a potent platelet activator. Injury to the blood vessels causes a change in the nor- mal environment, and in response, activation of platelets. The primary hemostatic plug is the result of the transfor- c d mation of the platelets from an inactive to an active state. The formation of a platelet plug requires several platelet Figure 31.7 Stages of platelet activation. (a) “Resting,” activation events, including adhesion, contraction or shape disc-shaped platelet. (b) Partially activated platelet (upper left) and change, secretion, and aggregation (Figure 31-8). fully activated platelet (spiny sphere—lower right). (c) Aggregate of activated platelets. (d) Platelets (P) adherent to exposed Platelet Adhesion The major function of platelets is to subendothelium (SE). EC, intact endothelial cells. seal defects in the vascular network. The initial stimu- Figure courtesy of Dr. Marion Barnhart, Wayne State University School lus for platelet activation is exposure of subendothelial of Medicine. Primary Hemostasis 719 interacting with VWF immobilized on collagen.23,24 VWF can also bind to the platelet GPIIb/IIIa receptor. Platelet interaction with subendothelial VWF is somewhat tran- Tissue injury sient and insufficient to result in stable adhesion by itself. Firm adhesion requires the synergistic interaction of sev- eral platelet receptors. Initial interaction with VWF slows the circulating platelets and facilitates their subsequent interaction with collagen via the GPIa/IIa and GPVI col- lagen receptors. GPIa/IIa has a higher affinity for collagen, but GPVI is a more effective platelet activator. The result of their interaction is firm adhesion and platelet activa- Platelet tion.11 GPVI activation induces ADP release from the DG adhesion and synthesis of TXA2 from arachidonic acid.14 (subendothelial collagen) Although the tissues have numerous other adhesion molecules including laminin, vitronectin, and fibronectin and the platelet surface has receptors for these adhesive proteins (Table 31-5), their contributions to platelet adhe- sion in vivo remain uncertain. Platelets preferentially bind to collagen under conditions of high shear rates. Shape The GPIb/IX receptor is functionally available in the change resting platelet, and binding does not require platelet activa- tion or Ca++. Once its ligand (VWF) becomes available with exposure of the subendothelium, adhesion occurs. Adhe- sion is a passive, non-energy-requiring process and is poten- tially reversible in its early stages. Many platelets adhere in a similar manner until a monolayer of platelets covers the exposed surface of the subendothelium (Figure 31-7d). Platelet aggregation Much of what is known about this phase of platelet function has been learned from studying patients with two diseases in which platelets fail to adhere properly: Bernard- Soulier syndrome and von Willebrand disease (VWD). Patients with Bernard-Soulier syndrome have mutations in one of the genes of the GPIb/IX complex that causes either decreased amounts of the receptor complex on the platelet surface or abnormal function of the complex (Chapter 33). Secretion Patients with VWD have mutations in the gene encoding VWF (Chapter 34). Checkpoint 31.4 If a patient with Bernard-Soulier syndrome or VWD cut a finger, would you expect bleeding to stop as fast as it does when you cut your own finger? Why or why not? Primary hemostatic plug Platelet Activation Adhesion of platelets to subendothelial components triggers a series of morphologic and functional changes known as platelet activation. Activation is a com- plex process that includes changes in metabolic biochem- Figure 31.8 Diagram of platelets forming the primary istry, platelet morphology (shape), surface receptors, and hemostatic plug. Tissue injury causes platelets to adhere to membrane phospholipid orientation. Key activation out- subendothelial structures including collagen. Shape change, comes are the generation of active GPIIb/IIIa receptors for secretion of granule contents, and aggregation follow. Additional fibrinogen binding, the secretion of the contents of the plate- platelets become activated by the secreted substances and clump together, eventually forming a mechanical barrier that halts the flow let granules into the surrounding tissue, and the formation of blood from the wound. of platelet aggregates. Only activated platelets are able to 720 Chapter 31 proceed with the subsequent steps in forming the primary microtubule coil is dismantled, reorganized, and contracted hemostatic plug. Once activated, the platelet response into the center of the cell, concentrating the platelet organ- becomes self-perpetuating and irreversible. Through strict elles in the center and causing the change to a spherical control mechanisms, platelet activation remains localized rather than flattened cell.7 This brings the platelet granules to the injured area. Activation of platelets is summarized into closer proximity to the OCS, facilitating secretion. The schematically in Figure 31-8. protrusive force for developing pseudopods is the result of actin polymerization and is an energy-requiring process. Platelet Agonists A number of substances have been After platelets adhere to the subendothelium, they undergo shown to stimulate platelets and to activate them. An agent variable degrees of “spreading,” resulting in the develop- that induces platelet activation is called an agonist. Each ment of broad lamellipodia rather than spike-like pseudo- agonist binds to the platelet surface at its specific platelet pods.26 As a result of the shape change, each platelet has a receptor. A signal is transmitted (transduced) internally by larger membrane surface area for biochemical reactions and the receptor, and a series of reactions in the interior of the a larger area for contact with the injured tissue and other platelet leads to subsequent platelet responses. The signal platelets. Also, the reorganized actin cytoskeleton provides transduction mechanisms in the platelet are similar to those a basis for “contraction” between platelets and between of other cells. platelets and the fibrin strands produced during secondary Some agonists (Table 31-8) are generated by the plate- hemostasis (clot retraction). lets themselves and some by other cells or molecules at the The cytoskeletal rearrangements resulting in shape injury site. Some agonists are normally present within cells change and spreading alter membrane glycoproteins. and are released when the cells are injured or activated; oth- GPIb/IX receptors are moved from the platelet surface to ers are synthesized de novo with activation. Agonists are the OCS and internalized, whereas active GPIIb/IIIa recep- often grouped as “strong” or “weak.” Strong agonists (e.g., tors increase in density on the platelet surface as they are collagen, thrombin) can activate the full range of platelet mobilized from aG, DG, and OCS membranes.27 The result functions themselves (shape change, secretion, aggrega- is to convert the activated platelet from an “adhesion” state tion). Weak agonists initiate platelet activation but require to an “aggregation” state. platelet release of ADP and/or synthesis and release of Shape change leads to succeeding responses if the stim- endogenous TXA2 (i.e., activation of the platelet cyclooxy- ulus from the agonists is strong enough. In the absence of genase |
pathway) to complete activation through secretion a sufficiently strong stimulus, the platelet may return to its and aggregation. Thrombin is the most potent activator of platelets in vivo.25 original discoid shape. Platelet Secretion (Release) Following the centralization of Platelet Shape Change Activation of platelets by a num- organelles by the contractile process responsible for shape ber of agonists is accompanied by a change in morphology change, platelets begin to discharge granule contents into from flattened disc-shaped cells to spiny spheres with long the surrounding area, a process known as platelet secretion projections from the surface called pseudopods or filapodia or platelet release reaction. Secretion is an energy-depen- (Figure 31-7b and Figure 31-9). Shape change involves reor- dent process requiring ATP. The OCS membrane fuses with ganization of cytoskeletal proteins in the structural zone membranes of granules that have been centralized deep including the microtubules, submembranous cytoskeletal within the platelets’ interior during platelet shape change, proteins, and actin and myosin cytoplasmic filaments. The and the granule contents are then extruded through the OCS to the platelet’s exterior. Alternatively, some secretion may occur by direct fusion with the plasma membrane.7Table 31.8 Platelet Agonists and Their Platelet Receptors Secretion provides a positive feedback mechanism in Agonists Agonist Receptors platelet activation. Some of the substances released from the Platelet-derived agonists platelet granules (e.g., serotonin, ADP) function as agonists • ADP P2Y1, P2Y that stimulate membrane receptors on additional platelets. 12 • Serotonin 5HT2A The binding of secreted ligands to platelet receptors repeats • Platelet-activating factor (PAF) PAF receptor (PAFR) itself, resulting in the recruitment of additional layers of • Thromboxane A2 (TXA2) Thromboxane receptor (TP) platelets and ultimately the formation of a platelet plug. Other (non-platelet-derived) agonists These positive feedback mechanisms ensure an adequate • Collagen* • GPIa/IIa, GPVI hemostatic response. • Thrombin* • Protease-activated receptor • Epinephrine (PAR)1, PAR4 Roles of the Granule Contents The DGs release their con- • a2@adrenergic receptors tents into the surrounding tissue (Table 31-6). The release of ADP is considered to be of primary importance because * Strong agonists (full activation of platelet does not require ADP release or cyclooxygenase activity). ADP functions as an agonist, producing a positive feedback Primary Hemostasis 721 Granule Agonist secretion Active GPIIb/IIIa appears Shape change Microtubules contract Membrane Internal lipids biochemical reorganized changes Actin/Myosin network Figure 31.9 Platelet shape change after stimulation by an agonist. Pseudopods develop on the platelet surface and contain a network of actin and myosin; the microtubule circumferential ring contracts; the membrane phospholipids are activated; activated glycoprotein IIb/IIIa receptors appear; internal biochemical changes occur; and granule secretion follows. mechanism in the continued stimulation and recruitment of mitogens and are thought to contribute to healing of the additional platelets. The ADP receptors important for plate- injured tissue.7 let activation are designated P2Y1 and P2Y12. 28 The plate- FV, VWF, and fibrinogen are proteins in the aG that are let inhibitory drugs clopidogrel, prasugrel, and ticagrelor similar to hemostatic proteins of the plasma. FV can func- function by blocking the P2Y12 ADP receptor. Stimulation by tion as a receptor on the platelet surface for hemostatic pro- ADP results in increased cytoplasmic calcium, more platelet teins (FXa and prothrombin; Chapter 32) and is a cofactor activation and DG content release, and the activation of the in the process of fibrin formation. Platelets contain about fibrinogen receptor GPIIb/IIIa. 20% of the FV found in whole blood,29 which, like fibrino- DGs release calcium extracellularly with the ADP. This gen, appears to be taken up from plasma.30 The functions calcium is non-metabolic and is not involved in the internal of VWF and fibrinogen have been discussed previously. stimulation processes. The extruded calcium is believed to PAI-1 and a2@antiplasminrelease from platelets seem to provide a high concentration outside the platelets neces- protect newly formed clots from lysis. When released from sary for fibrinogen attachment and for other enzymatic the platelet granules, these various hemostatic proteins can reactions that take place on the platelet exterior surface. serve as an additional source of these proteins for second- Refer to Table 31-6 for other DG components and their ary hemostasis. functions. Platelets contain distinct subpopulations of aG, which The aGs contain more than 300 proteins (see Table 31-7 contain different constituents. These subpopulations can for a partial list of aG components). Some are specific for undergo differential release of their cargo during activa- the platelet, and others are similar to hemostatic proteins tion. For example, platelets contain both proangiogenic found in the plasma. Platelet-specific proteins include proteins (VEGF) and antiangiogenic proteins (endostatin) platelet factor 4 (PF4) and b@thromboglobulin (bTG). Both contained in different populations of aG. Different agonists have heparin-neutralizing activity, although bTG does not are thought to differentially induce degranulation from the bind heparin as avidly as PF4. Heparin is an anticoagulant two subpopulations according to physiologic need.6 used to treat patients who are hypercoagulable or who are at increased risk of thrombosis (Chapter 35). PF4 performs Platelet Aggregation During platelet adhesion, collagen many actions including chemotactic activity for neutrophils, binding to GPVI triggers intracellular signaling that results monocytes, and fibroblasts. bTG, which is actually a family in activation of GPIIb/IIIa in the platelet membrane, which of proteins, is also a chemoattractant for fibroblasts and can then binds soluble fibrinogen. The active form of GPIIb/IIIa promote wound healing. complex appears soon after platelet activation with any ago- Thrombospondin constitutes about 20% of the protein nist. Resting platelets do not express a functional GPIIb/IIIa released from the platelet aG. It is also synthesized by other complex and are unable to bind fibrinogen. This prevents cells, including endothelial cells, and is found in the extra- unactivated platelets from aggregating as they circulate cellular connective tissue. It may function to help stabilize in the plasma. With the development of the high-affinity, the aggregated platelets.11 ligand-binding form of GPIIb/IIIa, the platelet is function- Platelets contain a number of growth factors, includ- ally able to undergo platelet aggregation.7 ing platelet-derived growth factor (PDGF), transforming Platelet aggregation is the attachment of platelets to growth factor b (TGF@b), epidermal growth factor (EGF), one another (Figure 31-7c). Newly arriving platelets flow- VEGF, and insulin-like growth factor (IGF). All function as ing into the area become activated by contact with agonists 722 Chapter 31 such as ADP and TXA2 (released by the initial adherent platelets do not aggregate in response to various agonists and activated platelets), products from the damaged tis- in platelet aggregation tests (Chapter 36). Patients who sue and endothelial cells, and thrombin (a procoagulant have decreased levels of fibrinogen have abnormal platelet enzyme generated by tissue factor/FVIIa acting on pro- aggregation as well (Chapter 33). Other adhesive proteins thrombin) (Chapter 32). With activation, the new platelets such as VWF, thrombospondin, and fibronectin can also undergo shape change and exposure of their active GPIIb/ bind to the GPIIb/IIIa receptor, but their roles in platelet IIIa sites. The fibrinogen bound to activated platelets serves aggregation in vivo are unclear. as a bridge, cross-linking GPIIb/IIIa molecules on two adja- Within in vitro test systems, aggregation occurs in two cent platelets. phases, primary and secondary aggregation (Chapter 36). Fibrinogen is able to link two platelets because of its During primary aggregation, platelets adhere loosely to unique molecular structure (Chapter 32). Briefly, it is a one another. If the stimulus by agonists is weak, primary dimeric molecule composed of two each of three polypeptide aggregation is reversible. Secondary aggregation follows chains: Aa, Bb, and g. One fibrinogen molecule attaches to and results in irreversible platelet aggregation. It begins as the GPIIb/IIIa receptors on two adjacent platelets via bind- the platelets start to release their own ADP and other gran- ing sites at the carboxy terminal end of each of the g@chains ule contents and to synthesize and release TXA2 (see the (Figure 31-10).31 The a@chains also contain sequences for section “Arachidonate Pathway”). The released substances platelet binding. Approximately 40,000–50,000 molecules of act as additional agonists, which supplement the platelet fibrinogen are bound to each activated platelet. Fibrinogen stimulation. If platelets are unable to release ADP and/or binding is irreversible after 10–30 minutes. Ca++ is needed to synthesize TXA2 secondary aggregation does not occur, for platelet aggregation to occur, whereas platelet adhesion and the platelets disaggregate. In general, the in vivo result does not require Ca++. Fibrinogen and Ca++ are supplied in such situations increases the time to stop bleeding from by both the plasma and internal platelet storage sites (aG, a wound. DG, and DTS), which provide high concentrations of both constituents in the injured area. Checkpoint 31.5 A study of patients with Glanzmann thrombasthe- Your finger is still bleeding at this point, but the platelets are nia (Chapter 33) demonstrated the importance of GPIIb/ aggregating to form the primary hemostatic plug. Let’s review IIIa receptors in platelet aggregation. Persons with this the key events: rare disease lack functional GPIIb/IIIa receptors, and their a. To what do platelets first adhere? b. What bridge and what platelet membrane receptor are needed for platelet adhesion? c. What bridge and what platelet membrane receptor are Agonist needed for platelets to attach to one another? Platelet IIb IIb-IIIa d. What is the attachment of platelets to one another called? Ca11 Biochemistry of Platelet Activation Platelets become activated after agonists bind to receptors on the platelet Low affinity IIIa surface, initiating signaling events within the platelets. receptor These signaling events eventually lead to the reorganiza- High affinity receptor tion of the platelet cytoskeleton, granule secretion, and aggregation. G PROTEINS Actions of most of the familiar agonists D E D ( collagen, ADP, thrombin, epinephrine, TXA2, arachidonic acid) and their receptors are linked with guanine nucleo- Fibrinogen (DED) tide-binding (G) proteins on the inner leaflet of the plate- Link to Link to another another let phospholipid membrane.28 G proteins are molecular platelet platelet switches that are inactive when GDP is bound but active after GTP is bound and function to facilitate the response Figure 31.10 Platelet aggregation. Agonist stimulation in the to extracellular stimuli. When the platelet adheres to colla- presence of Ca++ causes high-affinity fibrinogen receptors, activated gen, G proteins are activated and, in turn, activate enzymes glycoprotein IIb/IIIa, to form on the platelet surface. Fibrinogen binds in the platelet membrane and cytoplasm. Some activated horizontally to two platelets by peptide sequences at the terminal end of its g@and a@chains in the D domains, one g@chain to GPIIb/ enzymes cleave specific membrane phospholipids. The IIIa receptors on each platelet. Fibrinogen thus becomes a bridge resulting lipid products are “second messengers” that trans- between the two platelets. mit the signal to interior parts of the cells. Primary Hemostasis 723 Phospholipase C and the Phosphoinositide Pathway One Resting platelet important platelet activation pathway involves activation of the enzyme phospholipase C (PLC), which hydrolyzes [Ca11] DTS phospholipids between the glycerol backbone and the phos- 1027 M Ca11 phate moiety (Figure 31-11). Activated G proteins activate Pump ADP Ca11 ATP PLC, which cleaves phosphatidyl inositol bisphosphate (PIP2) into two important products critical in platelet func- tion, inositol triphosphate (IP3) and diacylglycerol. PIP Ca11 2 is the doubly phosphorylated product of the membrane lipid phosphatidyl inositol (PI) produced by PI kinase. The Agonist cleavage products released from PIP2 by PLC function to Activated platelet mobilize calcium ions from storage sites in the DTS (IP3) and to activate a kinase enzyme, protein kinase C/PKC P2X1 IP (diacylglycerol). The increased cytoplasmic Ca++ levels are 3 DTS Ca11 [Ca11 important in granule fusion and the release reaction and the ] .1026 M activation of a number of signaling enzymes and proteins Ca11 involved in cytoskeletal reorganization. Activated PKC in turn phosphorylates proteins, contributing to dense granule secretion and fibrinogen receptor activation.28 Figure 31.12 Ca++ regulation in platelets. Resting platelets maintain low levels of cytoplasmic Ca++ via active uptake by the DTS Role of Ca++ Platelet activation is accompanied by an and active extrusion from the cell, probably via a Ca++ pump. These increase in the cytosolic-free Ca++ concentration. Ca++ mechanisms counter a passive diffusion of Ca++ into the platelet serves as an intracellular second messenger and affects along a concentration gradient. With activation, platelets increase intracellular cytoplasmic Ca++ due to release from the DTS (by IP3) enzyme activity and protein–protein interactions, induc- and increase Ca++ influx (probably mediated via ATP and the P2X1 ing a number of platelet activation |
events. Resting plate- receptor). DTS, dense tubular system. lets have very low levels of ionic calcium in the cytoplasm (Figure 31-12). Many cellular enzyme systems that are inac- tive at the Ca++ concentration in resting platelets become activated by the increase in Ca++ that occurs with plate- arachidonic acid (AA) from membrane phospholipids (PC let activation. These enzymes include phospholipase A2 and PE). AA serves as a precursor of a variety of regulatory (PLA2), phospholipase C (PLC), PI kinase, and myosin substances, including prostaglandins and leukotrienes. In light chain kinase ([MLCK], important in the assembly of the platelet, thromboxane A2 (TXA2) is synthesized from the contractile mechanism responsible for platelet shape AA by the enzymes cyclo-oxygenase and thromboxane change). The increase in calcium ions results from both the synthase. In endothelial cells, prostacyclin (PGI2) is synthe- release from internal stores (by IP3 and DAG) and the influx sized from AA by cyclo-oxygenase and prostacyclin syn- from outside the cell, probably via the P2X1 receptor acti- thase (Figure 31-13). vated by ATP binding. A direct relationship exists between TXA2 is a potent platelet agonist and can stimulate the amount of cytoplasmic-free calcium and the extent of platelet activation and secretion upon binding to its recep- platelet stimulation. tor (TP; Table 31-8). Platelet activation by “weak” agonists Arachidonate Pathway Phospholipase A2 (PLA2) is acti- does not produce platelet secretion if TXA2 synthesis is vated by the increase in cytoplasmic calcium and hydrolyzes blocked, which seriously impairs subsequent steps in Activated Activation of G proteins PLC IP3 Binds DTS releases sequestered Ca11 PIP2 DAG Activates PKC Protein phosphorylation Granule secretion GPIIb/IIIa activation Figure 31.11 Phospholipase C/phosphoinositide pathway. Activated G proteins activate PLC. Activated PLC cleaves PIP2, releasing IP3 and DAG. IP3, in turn triggers release of Ca++ from the DTS. DAG activates PKC, inducing granule secretion and activation of GP IIb/IIIa, the fibrinogen receptor. PLC, phospholipase C; PIP2, phosphatidyl inositol bisphosphate; IP3, inositol triphosphate; DAG, diacylglycerol; DTS, dense tubular system; PKC, protein kinase C. 724 Chapter 31 Collagen Thrombin Arachidonic acid C R Phospholipases COOH C Free C R PO arachidonic acid 4 202 Membrane phospholipids (Site of aspirin Cyclo-oxygenase inhibition) PGG2 (PGH2) Thromboxane Prostacyclin synthetase synthetase Thromboxane Prostacyclin A2 (PGI2) in endothelial cells Spontaneous Thromboxane B2 Figure 31.13 Biochemical pathways of TXA2 formation in the platelet. Stimulation of platelet membranes (both intracellular granule and cytoplasmic membranes) by agonists (e.g., collagen, thrombin, arachidonic acid) liberates arachidonic acid from membrane phospholipids. Cyclo-oxygenase incorporates two molecules of oxygen, forming prostaglandin PGG2 (PGH2 in the reduced form). Thromboxane synthetase converts PGG2 to TXA2, and TXA2 is spontaneously converted to inactive thromboxane B2. Alternatively, PGG2 can be converted to PGI2, a powerful platelet inhibitor, in endothelial cells. TXA2, thromboxane A2; PGI2, prostacyclin. platelet function. Aspirin (acetylsalicylic acid) irreversibly Platelet Plug The platelets eventually form a barrier (the inhibits cyclooxygenase and prevents affected platelets primary hemostatic plug) that seals the injury and prevents from synthesizing TXA2 (Chapter 33). Because aspirin’s further blood loss. When the primary hemostatic plug is inhibition of cyclooxygenase is irreversible, the effect lasts formed, the bleeding stops. The time for bleeding to cease for the lifetime of the platelets exposed. TXA2 is released depends on the depth of the injury and the size of the blood from activated platelets and enhances vasoconstriction and vessel involved. Superficial wounds in which only capil- functions as a platelet agonist, perpetuating the activation laries and small vessels are affected usually stop bleeding process. A significant part of TXA2-induced platelet activa- within 10 minutes. tion is via secreted ADP. TXA2 is a labile compound quickly converted into an inert form, TXB2, shortly after its synthe- sis (T1/2 ∼ 30 seconds).32 Checkpoint 31.6 Your finger has now stopped bleeding. Outline the steps of pri- mary hemostasis that have occurred. Cyclic AMP (cAMP) Pathway cAMP is an important nega- tive regulator of platelet activation (Figure 31-14). cAMP inhibits shape change, platelet secretion, and integrin acti- Adenyl cyclase Phosphodiesterase vation (conversion of GPIIb/IIIa to an active form). PGI2 ATP, ADP cAMP AMP from endothelial cells inhibits platelet activation by stimu- lating adenyl cyclase, increasing cAMP levels. This is one PGI2 ADP Dipyridamole way to limit and localize the formation of the primary hemostatic plug.7 ADP functions as a platelet agonist by Figure 31-14 Role of cAMP in platelets. cAMP is a inhibiting adenyl cyclase, thus lowering cAMP levels and negative regulator of platelet function, inhibiting various steps of permitting platelet activation. The platelet inhibitory drug, platelet activation. Adenyl cyclase converts ATP or ADP to cAMP. dipyridamole, inhibits phosphodiesterase, the enzyme Subsequently, phosphodiesterase degrades cAMP to inactive AMP. responsible for the degradation of cAMP to AMP, thus sta- PGI2, a platelet-activation antagonist, activates adenyl cyclase, whereas ADP, a potent platelet agonist, inhibits adenyl cyclase. bilizing platelet cAMP levels and inhibiting platelet activa- Dipyridamole, a platelet inhibitory drug, functions by inhibiting tion.28 Platelet activation and biochemical reactions leading phosphodiesterase, thereby stabilizing cAMP and preventing platelet to aggregation are summarized in Figures 31-13 and 31-15. activation. cAMP, cyclic AMP; S , stimulation; : , inhibition. Primary Hemostasis 725 FIB IIb IIIa A A R R Gp IP3 PLC Ca11 Phospholipase A2 PIP2 Dense DAG Arachidonic acid tubule Ca11 Thromboxane A2 PKC Ca11 Xa Va Granule Procoagulant activity Secretion Figure 31.15 Schematic diagram showing platelet biochemical changes after activation. An agonist (A) binds to a receptor (R) on the platelet. The GPIIb/IIIa complex appears soon after platelet activation with an agonist. G-proteins (Gp) on the inner part of the phospholipid membrane (linked with R) become activated and in turn activate PLC, which cleaves PIP2 to form IP3 and DAG. Subsequently, Ca++ is mobilized into the cytoplasm from the DTS. Many enzymes are activated by the increase in Ca++, including phospholipase A2. Arachidonic acid released from the membrane phospholipids is converted to thromboxane, which stimulates platelet secretion. DAG activates PKC, which contributes to granule secretion. Coagulation factors (e.g., FXa, FVa) bind to the platelet surface leading to fibrin formation. GP IIb, IIIa, GPIIa/IIIa receptor; FIB, fibrinogen; DAG, diacylglycerol; Gp, guanine nucleotide binding protein (G protein); IP3, inositol-3-phosphate; PLC, phospholipase C; PKC, protein kinase C; PIP2, phosphatidylinositol bisphosphate. secondary hemostasis. Activated platelets accelerate throm- CASE STUDY (continued from page 712) bin formation, a function known as platelet procoagulant 3. The physician explained to Michael that the activity. Fibrin-forming proteins (coagulation factors) bind reddish-purple spots on his legs and ankles to the surface of activated platelets by binding either to spe- were tiny pinpoint hemorrhages into the skin. cific receptors on platelets or non-specifically to negatively Explain the relationship of these hemorrhages to charged phospholipids.33 The resulting platelet-coagulation the platelet count. factor interactions result in formation of the secondary hemostatic plug. The primary platelet plug is relatively unstable and is easily dislodged. During secondary hemostasis, fibrin PLATELETS AND SECONDARY HEMOSTASIS forms between and around the aggregated platelets. The final aspect of platelet activation involves the changes Platelets enhance the fibrin-forming processes by mecha- in the platelet membrane that allow platelets to function in nisms explained in Chapter 32. The proteins that interact 726 Chapter 31 enzymatically to form fibrin must be assembled on a lipid Clot retraction is believed to occur by the association of surface where the reactions take place. This localizes coagu- adjacent platelet pseudopods with each other and the fibrin lation and the formation of fibrin to the area of the develop- strands. Actin and other contractile proteins within the pseu- ing thrombus. It also protects coagulation enzymes from dopods cause the platelets to retract. A comparable phenom- inactivation by inhibitors. The membrane phospholipids enon is believed to occur in vivo, consolidating the thrombus of activated platelets are the primary source of this lipid into a cohesive mass of platelets and fibrin that seals the surface. wounded vessel and prevents further blood loss. Contrac- Fibrin-forming proteins do not bind to resting platelet tion can also decrease thrombolysis efficiency and enhance surfaces in the circulation. In resting platelets, the negatively wound healing.35 The stabilized platelet–fibrin mass remains charged phospholipids (PS and PE) are almost exclusively in place until fibroblast repair of the wound results in per- found in the inner half of the membrane bilayer. During the manent healing, and fibrinolysis dissolves the mass. activation of platelets, the membrane phospholipids flip- flop, and the negatively charged phospholipids move to the Physiologic Controls of Platelet outer leaflet via a Ca++@activated scramblase enzyme that reverses the asymmetric distribution of phospholipids.25 Activation and Aggregation This phospholipid rearrangement provides the phospho- Several inhibitory mechanisms balance platelet activation lipid surface needed for coagulation factors to bind, become and prevent excessive platelet deposition (Table 31-9). activated, and initiate fibrin formation. Platelet factor 3 activ- Platelet contact with agonists is minimized because of the ity is an older name for this platelet procoagulant activity. endothelial cell (EC) barrier. The flowing blood produces The fibrin-stabilized platelet plug is the secondary hemo- a dilutional effect, removing platelet agonists, loosely static plug. associated platelets, and coagulation proteins from the Recent evidence suggests that only a subpopulation of developing platelet aggregate. ECs produce NO and PGI2, activated platelets actually produces platelet procoagulant two potent inhibitors of platelet activation. ECs also have activity. Platelets that are simultaneously stimulated with ADPase that cleaves ATP and ADP to AMP, limiting the thrombin and collagen show higher levels of membrane- agonist effects of ADP. Physiologic controls that limit bound activated coagulation proteins than are observed thrombin activity (Chapter 32) also limit platelet aggrega- with only a single agonist (called coated platelets). Only tion because thrombin is a potent platelet activator. Many about 30% of the total platelet population are coated plate- agonists have short half-lives (T½) that limit their effects, lets with optimal agonist activation, suggesting they may and platelets can become desensitized to stimulation by be the subpopulation of platelets that actually provides the some agonists. Intraplatelet calcium levels are tightly con- procoagulant surface for secondary hemostasis to occur.7,34 trolled as is the availability of the active form of the plate- The entire platelet–fibrin mass then contracts to a let receptor GPIIb/IIIa. firmer, more cohesive clot. This process is called clot retraction. An adequate number of functional platelets are needed for clot retraction, a process that can be observed in CASE STUDY (continued from page 725) a test tube of blood when no anticoagulant is added. After 4. What is the most likely cause of Michael’s blood is placed into the test tube, the formation of fibrin pancytopenia? results in a network or mesh of fibrin strands that extends throughout, trapping essentially all of the blood cells and 5. Why would the administration of growth factors serum. The mass of fibrin and trapped cells contracts over such as erythropoietin (EPO) and TPO be consid- a period of 2–24 hours, extruding much of the serum from ered in this case? the fibrin mass. Table 31.9 Physiologic Controls of Platelet Activation and Aggregation Physical Controls Chemical and Protein Controls Platelet Structural Controls • Endothelial cell barrier limits contact with agonists • Endothelial cell production of: • Regulation of intracellular 3Ca++ 4 • Flowing blood dilutes agonists • NO • Inability of “resting” GPIIb/IIIa to bind fibrinogen • PGI2 S ccAMP; • Limited duration of agonist receptor activity • ADPase • Antithrombin • Short agonist T½ Primary Hemostasis 727 Summary After an injury, blood coagulates as the result of a series of secreting various biochemical mediators that affect all of complex biochemical reactions called hemostasis. Its purpose the subsequent steps of hemostasis. is to temporarily re-establish continuity of injured vessels The roles of the platelets are to adhere to the injured to minimize loss of blood. The components of hemostasis areas of the blood vessel walls and to aggregate by attach- are found in the blood (platelets and plasma proteins) and ing to one another and by secreting substances stored in in the tissues that compose the blood vessel walls. All of the the platelet granules. The secreted substances help to attract components are inert until activated by vessel injury. and activate new platelets that are added to the aggregate Hemostasis occurs in phases called primary hemostasis, and that help the growth of new tissue to permanently secondary hemostasis, and fibrinolysis. This chapter discussed heal the wound. The surface of the aggregated platelets is the primary hemostasis phase during which the blood |
ves- required for the reactions of secondary hemostasis. Several sels and the platelets cooperatively form an aggregate of inhibitory mechanisms including blood flow, which dilutes platelets that mechanically fills the openings in the injured platelet agonists and coagulation proteins, and inhibitors blood vessels and stops bleeding from the wound site. of platelet activation produced by endothelial cells control The injured blood vessels contribute by constricting and platelet activation and formation of platelet plugs. Review Questions Level I c. Formation of the primary hemostatic plug by aggregated platelets 1. The definition of hemostasis includes the: (Objective 1) d. Completion of fibrin formation and formation of the secondary hemostatic plug a. process of maintaining the body temperature b. termination of bleeding following a traumatic 5. What is the first step in platelet function after an injury injury? (Objective 6) c. process of forming a hematoma a. Fibrin formation d. regulation of kidney function b. Release of ADP c. Platelet aggregation 2. Which of the following is the primary element that prevents blood from clotting inside blood vessels? d. Platelet adhesion to collagen (Objective 5) 6. What is the process of platelets binding to one a. Fibrinogen another? (Objective 6) b. Capillaries a. Platelet secretion c. Endothelial cells b. Fibrin formation d. Platelets c. Platelet adhesion 3. What is the action of the blood vessels in hemostasis d. Platelet aggregation immediately after an injury? (Objective 5) 7. Each of the following is involved in hemostasis except: a. Thrombosis (Objective 2) b. Aggregation a. vasoconstriction by the blood vessels c. Vasoconstriction b. adhesion and aggregation by the platelets d. Vasodilation c. fibrin formation by proteins in the plasma and platelets 4. Which of the following has happened when a cut finger initially stops bleeding? (Objectives 6, 7) d. regulation of blood pressure a. Vasoconstriction of vessels proximal to the cut b. Vasodilation of vessels distal to the cut 728 Chapter 31 8. An abnormal condition in which a blood clot forms on 5. The formation of thromboxane A2 in the activated the interior of a blood vessel is known as: (Objective 1) platelet: (Objective 6) a. secondary hemostasis a. is needed for platelets to adhere to collagen b. thrombosis b. is caused by the a@granule proteins c. primary hemostasis c. requires the enzyme cyclooxygenase d. fibrinolysis d. occurs via a pathway involving VWF 9. The generation of fibrin by a series of reactions of 6. The function of microtubules in the resting platelet is procoagulant proteins is called: (Objective 3) to: (Objective 4) a. secondary hemostasis a. keep a high level of calcium in the cytoplasm b. primary hemostasis b. store and sequester calcium c. fibrinolysis c. provide a negative charge on the platelet surface d. hemolysis d. maintain the disc shape 10. The formation of a primary hemostatic plug results 7. The contents of the platelet granules are released from from a reaction between: (Objective 4) the platelet: (Objective 3) a. platelets and subendothelial tissue a. through the open membrane system after fusion b. platelets and coagulation proteins with the granules c. coagulation proteins and subendothelial tissue b. through the microtubules after fusion with the granules d. coagulation proteins and VWF c. by disintegration of the platelet plasma Level II membrane d. by the mitochondria 1. What are the cells that line the central cavity of all blood vessels and related tissues called? (Objective 1) 8. Which of the following is true about the relationship a. Epithelial cells between ADP and platelets? (Objective 5) b. Endothelial cells a. ADP is necessary for platelet adhesion. c. Capillaries b. ADP released from the dense granules is required d. Smooth muscle cells for adequate platelet aggregation. c. ADP is synthesized in the platelet from arachidonic 2. Platelet dense granules are storage organelles acid. for which of the following that are released after d. ADP is released from the platelets’ a@granules. activation? (Objective 2) a. Calcium, ADP, and serotonin 9. The process by which a platelet agonist causes a change in shape of the platelet from a disc to a spiny b. Fibrinogen, glycoprotein Ib, and VWF sphere is associated with platelet: (Objective 5) c. ADP, thromboxane A2, and FV a. adhesion d. Proteolytic enzymes, ATP, and FVIII b. aggregation 3. Which of the following is the platelet receptor needed c. activation for platelet adhesion? (Objective 3) d. release a. Glycoprotein IIb/IIIa 10. Which of the following statements correctly describes b. Actin the role of cAMP in platelet function? (Objective 7) c. VWF a. Elevated cAMP induces platelet aggregation. d. Glycoprotein Ib/IX b. Elevated cAMP inhibits platelet activation. 4. The platelet glycoprotein IIb/IIIa complex is: c. Elevated cAMP activates cyclooxygenase and TXA2 (Objective 3) production. a. a membrane receptor for fibrinogen d. Decreased cAMP inhibits Ca++ release from the b. secreted from the dense granules DTS. c. secreted by endothelial cells d. also called actin Primary Hemostasis 729 References 1. Karsan, A., & Harlan, J. M. (2013). The blood vessel wall. In: R. 15. Abrams, C. S., & Plow, E. F. (2013). Molecular basis for platelet Hoffman, E. J. Benz Jr., L. E. Silberstein, H. Heslop, J. Anastasi, function. In: R. Hoffman, E. J. Benz Jr., L. E. Silberstein, H. & J. Weitz, eds. Hematology: Basic principles and practice Heslop, J. Weitz, & J. Anastasi, eds. Hematology: Basic principles (6th ed., pp. 1784–1796) Philadelphia: Churchill Livingstone/ and practice (6th ed., pp. 1809–1820). Philadelphia: Churchill Elsevier. Livingstone/Elsevier. 2. Aird, W. C. (2013). Overview of vascular biology. In: V. J. Marder, 16. Kunicki, T. J., & Nugent, D. J. (2013). Platelet glycoprotein W. C. Aird, J. S. Bennett, S. Shulman, & G.C. White, eds. polymorphisms and relationship to function and Hemostasis and thrombosis: Basic principles and clinical practice immunogenicity. In: V. J. Marder, W. C. Aird, J. S. Bennett, S. (6th ed., pp. 493–497). Philadelphia: Lippincott Williams & Wilkins. Shulman, & G. C.White, eds. Hemostasis and thrombosis: Basic 3. Hajjar, K. A., Marcus, A. J., & Muller, W. (2016). Vascular principles and clinical practice (6th ed., pp. 393–439). Philadelphia: function in hemostasis. In: K. Kaushansky, M. A. Lichtman, Lippincott Williams & Wilkins. J. T. Prchal, M. M. Levi, O. W. Press, L. J. Burns, & M. Caligiuri, 17. Cramer, E. M. (1999). Megakaryocyte structure and function. eds. Williams hematology (9th ed.). New York: McGraw-Hill. booki Current Opinion in Hematology, 6(5) 354–361. d=1581§ionid=108080655. Accessed February 08, 2018. 18. Abrams, C., Shattil, S. J. (1991). Immunological detection of 4. Krisinger, M. J., & Conway, E. M. (2013). Role of endothelium in activated platelets in clinical disorders. Thrombosis Haemostasis, hemostasis. In: V. J. Marder, W. C. Aird, J. S. Bennett, S. Shulman, 65(5), 467–473. & G.C. White, eds. Hemostasis and thrombosis: Basic principles and 19. Menashi, S., Davis, C., & Crawford, N. (1982). Calcium clinical practice (6th ed., pp. 569–578). Philadelphia: Lippincott uptake associated with an intracellular membrane fraction Williams & Wilkins. prepared from human blood platelets by high-voltage, free- 5. Bizzozero, J. (1882). Ueber einen neuen Formbestandtheil flow electrophoresis. FEBS Letters, 140(2), 298–302. doi: des Blutes und dessen Rolle bei der Thrombose und der 10.1016/0014-5793(82)80918-6 Blutgerinnung. Archiv für Pathologische Anatomie und Physiologie 20. Gerrard, J. M., White, J. G., Rao, G. H., & Townsend, D. (1976). und für Klinische Medicin, 90(2), 261–332. doi: 10.1007/bf01931360 Localization of platelet prostaglandin production in the platelet 6. Smyth, S. S., Whiteheart, S., Italiano, J. E., Bray, P., & Coller, B. dense tubular system. American Journal of Pathology, 83(2), S. (2016). Platelet morphology, biochemistry, and function. In: K. 283–298. Kaushansky, M. A. Lichtman, J. T. Prchal, M. M. Levi, O. W. Press, 21. Nachman, R. L., & Rafii, S. (2008). Platelets, petechiae, and L. J. Burns, & M. Caligiuri, eds. Williams hematology (9th ed.) New preservation of the vascular wall. New England Journal of Medicine, York: McGraw-Hill. bookid=1581§ionid=108078506. 359(12), 1261–1270. 7. Walsh, P. N. (2013). The role of platelets in blood coagulation. In: 22. Brass, L. F., Zhu, L., Stalker, T. J. (2008). Novel therapeutic targets V. J. Marder, W. C. Aird, J. S. Bennett, S. Shulman, & G.C. White, at the platelet vascular interface. Arteriosclerosis, Thrombosis and eds. Hemostasis and thrombosis: Basic principles and clinical practice Vascular Biology, 28, s43-50. (6th ed., pp. 468–474). Philadelphia: Lippincott Williams & 23. Roth, G. J. (1991). Developing relationships: Arterial platelet Wilkins. adhesion, glycoprotein Ib, and leucine-rich glycoproteins. Blood, 8. Berndt, M. C., & Andrews, R. K. (2013). Major platelet 77(1), 5–19. glycoproteins: Platelet glycoprotein Ib-IX-V. In: V. J. Marder, W. 24. Andrews, R. K., Shen, Y., Gardiner, E. E., Dong, J. F., López, J. C. Aird, J. S. Bennett, S. Shulman, & G.C. White, eds. Hemostasis A., & Berndt, M. C. (1999). The glycoprotein Ib-IX-V complex in and thrombosis: Basic principles and clinical practice (6th ed., pp. platelet adhesion and signaling. Thrombosis and Haemostasis, 82, 382–385). Philadelphia: Lippincott Williams & Wilkins. 357–364. 9. Steinberg, M. H., Kelton, J. G., & Coller, B. S. (1987). Plasma 25. Bennett, J. S. (2012). Overview of megakaryocyte and platelet glycocalicin. New England Journal of Medicine, 317(17), 1037–1042. biology. In: V. J. Marder, W. C. Aird, J. S. Bennett, S. Shulman, & doi: 10.1056/nejm198710223171701 G. C. White, eds. Hemostasis and thrombosis: Basic principles and 10. Beer, J.H., Buchi, L., & Steiner, B. (19914). Glycocalicin: a new clinical practice (6th ed., pp. 341–348) Philadelphia: Lippincott assay – the normal plasma levels and its potential usefulness in Williams & Wilkins. selected diseases. Blood, 83(3), 691-702. 26. Hartwig, J. H., Barkalow, K., Azim, A., & Italiano, J. (1999). The 11. Savage, B., & Ruggeri, Z. M. (2013). The basis for platelet elegant platelet: Signals controlling actin assembly. Thrombosis adhesion. In: V. J. Marder, W. C. Aird, J. S. Bennett, S. Shulman, and Haemostasis, 82(2), 392–398. & G.C. White, eds. Hemostasis and thrombosis: Basic principles and 27. George, J. N., Pickett, E. B., Saucerman, S., McEver, R. P., clinical practice (6th ed. pp. 400–409). Philadelphia: Lippincott Kunicki, T. J., Kieffer, N., & Newman, P. J. (1986). Platelet Williams & Wilkins. surface glycoproteins: Studies on resting and activated platelets 12. Calverley, D. C., Maness, L. J. (2004). Platelet function in and platelet membrane microparticles in normal subjects, hemostasis and thrombosis. In: W. C. Wang, J. N. Lukens, G. and observations in patients during adult respiratory distress R. Lee, J. Foerster, J. Lukens, F. Paraskevas, . . . G. M. Rodgers. syndrome and cardiac surgery. Journal of Clinical Investigation, (2004). Wintrobe’s clinical hematology (11th ed., pp. 651–676). 78(2), 340. Philadelphia: Lippincott Williams & Wilkins. 28. Abrams, C. S., & Brass, L. F. (2012). Platelet signal transduction. 13. Ye, F., & Ginsberg, M. H. (2013). Major platelet glycoproteins: In: V. J. Marder, W. C. Aird, J. S. Bennett, S. Shulman, & G. C. integrin aIIbb3 (GPIIbIIIa). In: V. J. Marder, W. C. Aird, J. S. White, eds. Hemostasis and thrombosis: Basic principles and clinical Bennett, S. Shulman, & G.C. White, eds. Hemostasis and thrombosis: practice (6th ed., pp. 449–461). Philadelphia: Lippincott Williams Basic principles and clinical practice (6th ed., pp. 386–392). & Wilkins. Philadelphia: Lippincott Williams & Wilkins. 29. Tracy, P. B., Eide, L. L., Bowie, E. J., & Mann, K. G. (1982). 14. Watson, S. P., Farndale. R. W., Moroi, M. (2013). Platelet collagen Radioimmunoassay of factor V in human plasma and platelets. receptors. In: V. J. Marder, W. C. Aird, J. S. Bennett, S. Shulman, & Blood, 60(1), 59–63. G.C. White, eds. Hemostasis and thrombosis: Basic principles and 30. Camire, R. M., Pollak, E. S., Kaushansky, K., & Tracy, P. B. (1998). clinical practice (6th ed., pp. 420–430). Philadelphia: Lippincott Secretable human platelet-derived factor V originates from the Williams & Wilkins. plasma pool. Blood, 92(9), 3035–3041. 730 Chapter 31 31. Hawiger, J. (1995). Adhesive ends of fibrinogen and its practice (6th ed., pp. 468–474). Philadelphia: Lippincott Williams antiadhesive peptides: The end of a saga? Seminars in Hematology, & Wilkins. 32, 99–109. 34. Bouchard, B. A., Silveira, J. R., & Tracy, P. B. (2013). Interactions 32. Grosser, T., & Fitzgerald, G. A. (2012). Platelet prostanoid between platelets and the coagulation system. In: A. D. metabolism. In: V. J. Marder, W. C. Aird, J. S. Bennett, S. Shulman, Michelson, ed. Platelets (pp. 425–451). San Diego, CA: Academic & G. C. White, eds. Hemostasis and thrombosis: Basic principles and Press. https://doi.org/10.1016/B978-0-12-387837-3.00021-3 clinical practice (6th ed., pp. 462–467). Philadelphia: Lippincott 35. Kunitada, S., FitzGerald, G. A., & Fitzgerald, D. J. |
(1992). Williams & Wilkins. Inhibition of clot lysis and decreased binding of tissue-type 33. Walsh, P. N. (2012). The role of platelets in blood coagulation. plasminogen activator as a consequence of clot retraction. Blood, In: V. J. Marder, W. C. Aird, J. S. Bennett, S. Shulman, & G. C. 79(6), 1420–1427. White, eds. Hemostasis and thrombosis: Basic principles and clinical Chapter 32 Secondary Hemostasis and Fibrinolysis J. Lynne Williams, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define hemostasis and identify the three concepts of intrinsic, extrinsic, and common physiologic compartments involved in the pathways. hemostatic mechanism. 8. Identify the factors involved in contact 2. Differentiate primary and secondary activation. hemostasis. 9. Diagram the physiologic pathway of blood 3. List the coagulation factors using roman coagulation. numerals and determine how each factor is 10. Define fibrinolysis, identify the major compo- evaluated in lab testing. nents of the fibrinolytic system, and explain 4. Classify the coagulation factors in groups why fibrinolysis is a necessary component of and discuss their characteristics. hemostasis. 5. Evaluate the importance of vitamin K in 11. List the fragments resulting from fibrino- hemostasis. lytic degradation; compare and contrast the 6. Describe the mechanism of action of the fragments resulting from the degradation of coagulation proteins. fibrinogen and fibrin. 7. Diagram the sequence of reactions in the 12. List the major biochemical inhibitors that coagulation cascade according to the historic regulate secondary hemostasis. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Explain the interactions of the three physio- 2. Describe the domain structure of the logic compartments involved in hemostasis. coagulation factors, determine how this 731 732 Chapter 32 structure affects the action of the serine 7. Compare and contrast physiologic and proteases, and explain the significance of systemic fibrinolysis. the non-catalytic regions. 8. Describe the physiologic controls of 3. Summarize the formation of complexes on hemostasis including blood flow, feedback a phospholipid surface and explain the sig- inhibition, liver clearance, and inhibitors nificance of these complexes to hemostasis. (antithrombin, TFPI, protein C, protein S, 4. Integrate the role of contact factors with and TAFI) other systems (complement activation, 9. Evaluate a case study from a patient with a fibrinolysis, inflammation). defect in hemostasis and, using the medical 5. Describe the multiple roles of thrombin in history and laboratory results, determine hemostasis. the diagnosis. 6. Explain the physiologic functions of ADAMTS-13, A2, LRP, EPCR, and uPAR. Chapter Outline Objectives—Level I and Level II 731 Coagulation Cascade 738 Key Terms 732 Fibrinolytic System 749 Background Basics 733 Control of Hemostasis 755 Case Study 733 Physiologic Hemostasis 760 Overview 733 Hemostasis in the Newborn 762 Introduction 733 Summary 762 The Coagulation Mechanism 734 Review Questions 763 Procoagulant Factors 734 References 764 Key Terms ADAMTS-13 Intrinsic Xase complex Thrombomodulin (TM) Annexin II (A2) LRP receptor (LDL receptor-related Tissue factor pathway Coagulation factor protein) Tissue factor pathway inhibitor Common pathway Plasminogen activator (PA) (TFPI) Contact group Plasminogen activator inhibitor Tissue-type plasminogen activator Endothelial cell protein C receptor (PAI) (tPA) (EPCR) PIVKA Transglutaminase Extrinsic pathway Prothrombinase complex Urinary-type plasminogen activa- Extrinsic Xase complex Prothrombin group tor (uPA) Fibrin degradation product (FDP) Serine protease UPAR (uPA receptor) Fibrinogen group Serpin Vitamin K dependent Fibrinolysis Thrombin-activatable fibrinolysis Zymogen inhibitor (TAFI) g@Carboxylation Intrinsic pathway Secondary Hemostasis and Fibrinolysis 733 Background Basics The information in this chapter builds on the concepts Level II learned in previous chapters. To maximize your learning • Describe the structure of the platelet phospholipid experience, you should review these concepts before membrane. (Chapter 31) starting this unit of study: • Review protein structure, protein domains, and prop- erties of enzymes. (Chapter 2 and previous chemistry Level I and biology courses) • Define and summarize the events in primary hemostasis. (Chapter 31) CASE STUDY Introduction We refer to this case throughout the chapter. Hemostasis is a carefully balanced process by which the Shawn, a 10-year-old boy, saw his physician for body maintains blood in a fluid state within the vascula- recurrent nosebleeds and anemia. History revealed ture and prevents loss of blood from the vascular system that the epistaxis began when he was about 18 upon injury. Although often thought of as “clot” formation, months old. The nosebleeds occurred one or two hemostasis also includes fibrin (clot) dissolution and vessel times a month and began spontaneously. His repair. Hemostasis involves a series of complex and highly mother reported that he seemed to bruise easily. regulated events linking platelets, vascular endothelial cells, No lesion was found in the nose. No history of and coagulation proteins (coagulation factors). The interac- drugs was noted. The family history indicated that tion of these three components at the cellular and molecular the boy’s grandparents were cousins. Shawn had a levels determines whether the equilibrium between bleed- brother who died of intracranial hemorrhage at 10 ing (hemorrhage) and clotting (thrombosis) is maintained years of age. Parents and all other relatives showed (Figure 32-1). The delicate balance between bleeding and no bleeding problems. clotting requires both amplification reactions to ensure Factor XIIa Coagulation factors Factor XIa Overview Phospholipids Activators Kallikrein Ca11 tPA uPA Hemostasis requires the interaction of platelets, blood vessels, and coagulation proteins. The previous chapter described primary hemostasis (platelets and blood vessels). This chapter introduces the contributions of plasma protein Clotting Fibrinolysis systems to secondary hemostasis. It begins with an over- view of hemostasis and discusses the distinctions between primary and secondary hemostasis. The coagulation pro- AT teins are presented in related groups for initial discussion Protein C Protein S PAI-1 of general properties, mechanisms of action, and protein TFP I PAI-2 structure. The chapter describes the functional interactions a2-Macroglobulin a2-Antiplasmin Inhibitors a1-Antitrypsin a2-Macroglobulin of these procoagulant proteins from the classical perspec- C1-Inhibitor TAFI tive of intrinsic, extrinsic, and common pathways. The cur- Heparin cofactor II rent concept of in vivo blood coagulation initiated through a cell-based system is discussed. The chapter presents the Figure 32.1 The coagulation system. Activators and inhibitors fibrinolytic system (components, activators, inhibitors) of clotting and fibrinolysis keep the coagulation system in balance. and defines the products of fibrinolytic degradation. The Clotting occurs when blood vessels are damaged and activators of contributions of the various components to the control of coagulation factors are exposed or released. Clotting is controlled because fibrinolysis is initiated in response to clotting activation. hemostasis are summarized, including physiologic pro- Inhibitors of both clotting and fibrinolysis serve to bring the system cesses (blood flow, liver clearance), positive and negative back into balance. An imbalance in the activation or inhibition of feedback mechanisms, and natural inhibitors. either clotting or fibrinolysis causes thrombosis or bleeding. 734 Chapter 32 sufficient activity when needed and important regulatory reactions to ensure that excess activity does not occur. Table 32.1 Coagulation Factors in Intrinsic, Extrinsic, and Common Pathways As discussed in Chapter 31, hemostasis consists of two stages: primary and secondary. Primary hemostasis, the for- Intrinsic Pathway Extrinsic Pathway Common Pathway mation of the unstable platelet plug, is the first response to vas- Prekallikrien (PK) VII X cular injury. Secondary hemostasis, the reinforcement of the High-molecular-weight III, Tissue factor (TF) V unstable platelet plug with chemically stable fibrin, follows kininogen (HK) and includes a series of interdependent, enzyme-mediated XII II reactions. The endpoint of these reactions is the generation XI I of thrombin, the enzyme that transforms the soluble protein IX fibrinogen to insoluble fibrin to stabilize the platelet plug. VIII The process of fibrin formation is normally well balanced and controlled, limiting it to the area of vessel injury. This and interpreting the in vitro clotting assays used to evaluate localization prevents widespread or systemic coagulation coagulation. The activated partial thromboplastin time activation. The procoagulant reactions are amplified to the (APTT) measures the intrinsic and common pathways, appropriate degree, and natural inhibitors limit the proteo- whereas the prothrombin time (PT) measures the extrinsic lytic activity of the activated clotting factors. Negative feed- and common pathways. back reactions also control fibrin formation: large amounts of thrombin destroy coagulation cofactors in the rate-limiting steps of its own production. The platelet–fibrin plug seals Checkpoint 32.1 the injured vessel preventing blood loss, enabling the vessel What is the major distinction between the so-called extrinsic to begin to repair itself, and allowing fibrin ultimately to be and intrinsic pathways? digested by plasmin, an enzyme of the fibrinolytic system. Hemostasis is complex, involving many interrelating components and control mechanisms. Although no part of the system acts alone, each part is discussed individually, CASE STUDY (continued from page 733) and then the concepts are integrated into a cohesive theory Screening tests for evaluating hemostasis were of hemostasis. done on Shawn. He was found to have a platelet count of 242 * 103/mcL The PT was 29.5 seconds (with a control of 12.0 seconds; this is prolonged). The Coagulation The APTT was 31.0 seconds (with a control of 29.4 seconds, this is normal). Liver function tests Mechanism were normal. 1. What do the results of the screening tests The reactions involved in coagulation were initially (platelet count, PT, and APTT) indicate? described as occurring in a cascade1 or waterfall-like2 fash- ion in which circulating, inactive coagulation factor pre- 2. What component of the hemostatic mechanism cursors, or zymogens, are sequentially activated to their is most likely affected? enzyme forms. Originally, this process was thought to be initiated by two different pathways, as diagrammed in Figure 32-2. We now know that this separation into two pathways does not reflect in vivo coagulation, as the two pathways cannot operate independently.3,4 The intrinsic Procoagulant Factors pathway requires enzymes and protein cofactors that are all Coagulation factors are the procoagulant proteins involved in present in blood. The extrinsic pathway requires enzymes hemostasis. The International Committee on Nomenclature and protein cofactors present in blood as well as an activa- of Blood Coagulation Factors has assigned roman numerals tor—tissue factor—not found in blood under normal con- I through XIII to some factors.5 Each roman numeral was ditions. Both converge in a third path called the common assigned according to the order of discovery of the respective pathway to generate the fibrin clot. The traditional analysis factor, not its place in the reaction sequence. Each factor has of the coagulation process assigns each of the coagulation one or more common names or synonyms in addition to the factors to one of these pathways as indicated in Table 32-1. roman numeral designation, although the common names Although the model of intrinsic, extrinsic, and common are rarely used today (Table 32-2). The letter a following the pathways does not represent in vivo coagulation, the con- roman numeral indicates the activated form of the factor (i.e., cept of the three pathways is invaluable in understanding it is no longer a zymogen but has active coagulant activity). Secondary Hemostasis and Fibrinolysis 735 Intrinsic Extrinsic Contact Vessel injury XII XIIa TF 1 VII XI XIa Ca11 Common IX X IXa 1 VIIIa Tissue factor PL 1 Ca11 Ca11 1 VIIa Intrinsic Xase Extrinsic Xase complex Xa complex Xa 1 Va Prothrombinase PL 1 Ca11 complex XIII Prothrombin Thrombin XIIIa Fibrinogen Fibrin Crosslinked fibrin Figure 32.2 A simplified version of the coagulation cascade showing the cascade or waterfall-like sequence of reactions. The tan boxes indicate complex formation. PL, platelet phospholipid; TF, tissue factor. There are, however, several exceptions to this terminology. Properties of the Blood Coagulation In its activated form, factor II (prothrombin) is preferentially known as thrombin rather than FIIa. When fibrinogen (FI) is Factors cleaved by thrombin, it is preferentially called fibrin. Tissue Similarities among the structural and functional properties factor (TF) was assigned FIII, and calcium was assigned FIV, of the coagulation factors permit division into three groups: but these roman numeral designations are seldom used. FVI the prothrombin group, the fibrinogen group, and the con- is no longer included in the coagulation sequence because tact group (Table 32-3). it was originally assigned to a procoagulant activity subse- PROTHROMBIN GROUP quently found to be activated FVa. High-molecular-weight The prothrombin group of procoagulant proteins includes kininogen (HK) and prekallikrein (PK) were never assigned prothrombin (FII) and factors VII, IX, and X. These factors roman numerals. have a molecular mass ranging from 50 to 100 kilodaltons Most coagulation factors were discovered when physi- (kDa). |
The factors in the prothrombin group are synthe- cians saw patients with a lifelong history of bleeding problems. sized in the liver, and all contain the g@carboxyglutamic acid Studies of the affected patients’ blood revealed that certain (GLA)–rich region called the GLA-domain that is critical for proteins were functionally deficient. These proteins have been the calcium-binding properties of these proteins. The pro- isolated and characterized as to their composition (including thrombin group is also referred to as vitamin K dependent full amino acid sequences), biochemical functions, and the because its members require vitamin K to be synthesized as chromosomal location of their genes, providing useful infor- functional proteins (see the section “Vitamin K–Dependent mation for understanding hereditary problems (Table 32-2). Coagulation Proteins”). These zymogens circulate in an inactive state and require proteolytic activation to function CASE STUDY (continued from page 734) as an active protease. Other vitamin K–dependent proteins include protein C (PC), protein S (PS), and protein Z (PZ) 3. What evidence indicates that Shawn has a hered- (see the section “Biochemical Inhibitors”). itary bleeding disorder? FIBRINOGEN GROUP 4. Are the nosebleeds significant in considering the The fibrinogen group includes fibrinogen (FI) and FV, diagnosis? FVIII, and FXIII. Thrombin cleaves all four proteins dur- ing coagulation. These proteins have the highest molecular 736 Chapter 32 Table 32.2 Summary of the Properties of Coagulation Factors Circulating Form (Molecular weight in Chromosome (gene Factor Common Name(s) kilodaltons [kDa]) T1 (hours) Role symbol) 2 I Fibrinogen 6-chain glycoprotein 72–120 Substrate; fibrin precursor 4q23-q32 (FGA, FGB, (340 kDa) FGG) II Prothrombin Single-chain glycoprotein 60–70 Serine protease; thrombin precursor 11p11.2 (F2) (72 kDa) III Tissue factor Single-chain, transmembrane NA Cofactor in extrinsic Xase; complex 1p21–22 (F3) glycoprotein (47 kDa) not found in circulation IV Calcium Element NA Cofactor in some coagulation — reactions V Proaccelerin Single-chain glycoprotein 12–36 Cofactor in prothrombinase complex 1q23 (F5) (330 kDa) VII Proconvertin, stable factor Single-chain glycoprotein 3–6 Serine protease; constituent of 13q34 (F7) (50 kDa) extrinsic Xase complex VIII Antihemophilic factor Heterodimer glycoprotein 8–12 Complex with VWF in the circulation; Xq28 (F8) (170–280 kDa) cofactor in intrinsic Xase complex VWF von Willebrand factor Multimeric glycoprotein; 8–12 Platelet adhesion; stabilization of 12p13.3 (VWF) subunit (500-20,000 kDa) circulating FVIII IX Plasma thromboplastin Single-chain glycoprotein 18–24 Serine protease; constituent of Xq27.1 (F9) component (55 kDa) intrinsic Xase complex X Stuart factor Two-chain glycoprotein 34–40 Serine protease; constituent of 13q34 (F10) (59 kDa) prothrombinase complex XI Plasma thromboplastin Two-chain glycoprotein 60-80 Serine protease; contact factor 4q35 (F11) antecedent (143 kDa) XII Hageman factor Single-chain glycoprotein 60 Serine protease; contact factor 5q35.3 (F12) (80 kDa) XIII Fibrin-stabilizing factor Heterotetramer glycoprotein 240 Transglutaminase; fibrin stabilizer A-chain: 6p25.1 (F13A1); (320 kDa) B-chain: 1q31.3 (F13B) HK Fitzgerald factor, Williams Single-chain glycoprotein 150 Cofactor; complexed with PK and 3 (KNG1) factor, Flaujeac factor (120 kDa) FXI; contact factor PK Fletcher factor Single-chain glycoprotein 35 Serine protease complexed with HK; 4q35.2 (KLKB1) (88 kDa) contact factor T1 , biologic half-life; HK, high-molecular-weight kininogen; PK, prekallikrein. 2 weights of all the factors ranging from 170 to 340 kDa. the exception of FXI, the contact factors do not appear to They are not found in serum because they are consumed play an essential role in hemostasis in vivo.6 However, this during clotting. group of factors is integrally related to other physiologic systems and provides a key link between inflammation, CONTACT GROUP and coagulation. In addition to coagulation, the activated The contact group includes FXI and FXII, PK, and HK. contact factors can activate the fibrinolytic, kinin, and com- These proteins have molecular weights ranging from 80 to plement systems, playing an important role in the inflam- 120 kDa. The contact group is involved in the initial acti- matory response. These factors are not fully consumed vation of the intrinsic pathway in vitro and requires con- during clotting and thus are found in serum (in reduced tact with a negatively charged surface for activation. With amounts). Table 32.3 Coagulation Factor Groups Based on Physical Characteristics Contact Group Prothrombin Group Fibrinogen Group Characteristics Requires contact with a negatively Requires vitamin K for synthesis; needs Large molecules; absent from serum charged surface for activation Ca++ to bind to a phospholipid surface (consumed in clotting) Factors/Proteins included XII, XI, PK, HK II, VII, IX, X, PC, PS, PZ I, V, VIII, XIII PC, protein C; PS, protein S; PZ, protein Z. Secondary Hemostasis and Fibrinolysis 737 Mechanism of Action FUNCTION OF VITAMIN K Vitamin K is a fat-soluble vitamin found in green leafy The coagulation cascade consists of a series of reactions in vegetables, fish, and liver. Some gram-negative intestinal which limited proteolysis activates zymogens. Most of the bacteria also synthesize it. Vitamin K is necessary for the newly generated proteases have low enzymatic activity G-carboxylation of glutamic acid residues clustered in a until they bind to specific cofactors on appropriate phos- region of the proteins called the GLA domain. The addition pholipid surfaces. Thus, the coagulation proteins can be of an extra carboxyl group (COOH) to the g@carbon of the grouped functionally as cofactors, substrates, or enzymes. glutamic acid residues 1g@carboxylation2 is a post-transla- The enzymes can be further grouped as either serine prote- tional modification of the protein carried out by a specific ases or a transglutaminase. g@glutamyl carboxylase in the endoplasmic reticulum (ER) COFACTORS of the liver. The g@carboxylation is essential for Ca++ bind- FV and FVIII function as cofactors for activated coagulation ing, which induces a conformational change in the pro- proteases FXa and FIXa, respectively. The activated forms of tein, and enables the factor to bind to negatively charged these proteins, FVa and FVIIIa, have no enzymatic activity of phospholipids exposed on activated cell membranes (e.g., their own. HK functions as a cofactor for the contact activa- exposed phosphotidyl serine on activated platelets).3,4 The tion phase of coagulation (FXIIa and FXIa), PS is a cofactor carboxylase requires the reduced form of vitamin K (hydro- for activated PC (see the section “Biochemical Inhibitors”), quinone form) for its action. The carboxylation reaction and TF is a cofactor for FVIIa. Each protease has some activ- converts reduced vitamin K to the epoxide form, which is ity in the absence of its cofactor, but interaction with its converted back to the reduced form by the enzyme vitamin cofactor significantly enhances proteolytic function. K epoxide reductase. Conversion back to the reduced form allows recycling of the vitamin K. Vitamin K antagonist SUBSTRATE drugs such as warfarin/Coumadin inhibit the activity of Fibrinogen is classified as a substrate: it is acted upon by the the vitamin K epoxide reductase and prevent recycling of enzyme thrombin. It is the only coagulation protein that, as vitamin K back to the reduced form, thus preventing fur- a substrate, does not participate in furthering the coagula- ther g@carboxylation. Warfarin/Coumadin overdoses can be tion cascade. reversed by vitamin K administration (Figure 32-3). ENZYMES ACARBOXY PROTEINS The coagulation proteins that have enzymatic activity are In the absence of vitamin K, the liver synthesizes the proteins, secreted as zymogens, which are proenzymes or inactive which can be found in the plasma. However, they are non- precursors that must be modified to become active. Activa- functional because they lack the g@COOH groups necessary tion can involve either (1) a conformational change of the for Ca++@phospholipid binding. The vitamin K–dependent molecule or (2) proteolytic cleavage of one or more specific peptide bond(s). The coagulation zymogens are activated in a cascade-like sequence. Initially, a small number of zymo- Warfarin Epoxide reductase gens are activated, and each activated protease sequentially activates the next zymogen in the cascade, resulting in the H Vitamin K Vitamin K hydroquinone epoxide H amplification of the initial stimulus. H2N C COOH H2N C COOH Members of the family of serine proteases, including Carboxylase thrombin and factors VIIa, IXa, Xa, XIa, and XIIa, have a CH2 CH2 functional serine in their active sites and a common catalytic CH2 CH mechanism. They selectively cleave arginine or lysine peptide Oxygen CO2 bonds in their substrates. Each serine protease involved in the COOH HOOC COOH coagulation cascade is highly specific for its substrate(s). FXIIIa is the only coagulation enzyme with transgluta- Glutamic acid g-carboxy Glutamic acid minase activity. It catalyzes the formation of peptide bonds between glutamine and lysine residues on fibrin, forming Figure 32.3 The vitamin K–dependent g@carboxylation of stable covalent cross-links within the fibrin meshwork. glutamic acid. The coagulation factors in the prothrombin group must undergo this post-ribosomal carboxylation of glutamic acid Vitamin K–Dependent Coagulation residues in order to become functional. A specific carboxylase converts glutamic acid residues to g@carboxyglutamic acid residues Proteins in a reaction requiring oxygen, carbon dioxide, and reduced vitamin K (hydroquinone). In the process, reduced vitamin K is converted to Seven of the blood coagulation proteins, including pro- an epoxide and must be recycled to its reduced form by the enzyme thrombin, factors VII, IX, and X, and PC, PS, and PZ, require epoxide reductase. This latter reaction is blocked by vitamin K vitamin K for synthesis of the functional protein. antagonist drugs such as warfarin or Coumadin. 738 Chapter 32 factors lacking the COOH modification have previously binding. When bound, Ca++ mediates the association of been referred to as PIVKA (protein induced by vitamin k a coagulation factor with a phospholipid surface, thus absence or antagonists) or des@g@carboxy form. The Interna- restricting coagulation to the site of injury. tional Committee on Thrombosis and Haemostasis recom- The epidermal growth factor (EGF) domain, finger domain, and mends using the term acarboxy form. Warfarin/Coumadin, apple domains are thought to be involved in binding to cofactors, which inhibits the g@carboxylation process, results in the activators, or substrates. The kringle domain is a lysine-binding production of inactive (acarboxy) proteins by the liver. The site responsible for the affinity of certain proteins (plasmino- nonfunctional and functional forms of the factors are identi- gen, plasmin, tissue plasminogen activator) for fibrin. cal with respect to amino acid composition and antigenic determinants. Checkpoint 32.3 Why are the domains of the serine proteases involved in blood Checkpoint 32.2 clotting so important in the hemostatic mechanism? Will a vitamin K–deficient patient produce any of the vitamin K–dependent factors? Why is vitamin K so vital to the formation of coagulation complexes? Coagulation Cascade Structure of the Blood Coagulation Complex Formation on Phospholipid Proteins Surfaces The blood coagulation proteins are made up of multiple Most of the coagulation reactions occur on cell surface mem- functional units called domains, classified as catalytic or non- branes. Clotting factors bind to the phospholipid membrane catalytic, depending on their function.7 Catalytic domains of activated cells, forming a complex including enzyme, contain the active site of the enzyme and are involved in substrate, and cofactor.8 Subendothelial tissue exposed activating other protein substrates. N on-catalytic domains when blood vessel injury occurs and the activated platelet contain regulatory elements. Many of the proteins involved surface provide the critical phospholipids for coagulation in in hemostasis contain similar s tructural domains and are vivo. The phospholipid surface serves to localize the reac- believed to be derived from common ancestral genes. tion to the site of injury and increase the rate of activation CATALYTIC DOMAIN by several orders of magnitude.3 In addition, the reactions The catalytic domain of the serine proteases involved in are generally protected from inhibitors in these cell surface– blood clotting is highly homologous to the enzyme tryp- associated complexes. sin. This domain’s main function is to convert an inactive Three procoagulant complexes, each named for its zymogen to an active enzyme by cleaving a peptide bond physiologic substrate, are formed during the process of in its target substrate, a process known as zymogen activation coagulation. The extrinsic Xase (extrinsic tenase), intrin- by limited proteolysis. sic Xase (intrinsic tenase), and prothrombinase complexes assemble on the phospholipid membrane (Figure 32-4; NON-CATALYTIC DOMAINS Table 32-4). The extrinsic Xase is formed when TF, an inte- The non-catalytic domains give each coagulation factor gral membrane lipoprotein, is exposed to blood when ves- its own unique identity and substrate specificity. These sel injury occurs. TF binds FVII or FVIIa in the presence of domains are regulatory segments serving to bind calcium Ca++, giving rise to the FVIIa/TF complex that activates FX. and promoting interaction with phospholipids, cofactors, The intrinsic Xase is formed on membrane surfaces when receptors, and substrates. Some of the |
more common types FIXa and FVIIIa bind to phospholipid in the presence of of non-catalytic domains include the signal peptide, propep- Ca++. This complex also activates FX to FXa. In a similar tide, g@carboxyglutamic acid-rich (GLA) domain, epidermal way, FXa and FVa bind to negatively charged membranes growth factor domain, apple domain, finger domain, and in the presence of Ca++ to form the prothrombinase com- kringle domain. plex. This complex converts prothrombin, also bound to the The signal peptide is a short domain that permits trans- membrane, to thrombin. The rate of prothrombin activa- location of the protein to the endoplasmic reticulum for tion by the prothrombinase complex is about 300,000 times processing after synthesis. Vitamin K–dependent pro- faster than activation by FXa alone.9 teins have a propeptide between the signal peptide and the GLA domain, which contains the recognition site that directs g@carboxylation of the vitamin K–dependent The Intrinsic Pathway proteins after synthesis. The GLA domain contains 9–13 The “intrinsic pathway” was the first coagulation path- g@carboxyglutamic acid residues and is essential for Ca++ way described. It is more accurately called the contact Secondary Hemostasis and Fibrinolysis 739 Extrinsic Xase Intrinsic Xase TF VIIIa H VIIa VIIIa X L X IX IXa IX IXa Va H Va L II X Xa Xa X Xa TM Prothrombinase IIa PC II IIa PC APC Protein case Figure 32.4 Schematic illustration of the coagulation complexes forming on a phospholipid surface. The vitamin K–dependent proteases (FVIIa, FIXa, FXa, and thrombin) are shown associated with their cofactors (tissue factor [TF], FVIIIa, FVa, and thrombomodulin [TM], respectively) and substrates (FIX, FX, prothrombin, and protein C [PC], respectively) on the membrane surface. APC, activated protein C. system or pathway. The coagulation factors involved in this have no apparent clinical bleeding even after significant pathway (contact factors) assemble on a surface. trauma or surgery. Thus, it is unlikely that these factors CONTACT FACTORS play an important role in hemostasis in vivo. They do, how- The intrinsic pathway (contact pathway) is initiated when ever, contribute significantly to fibrinolysis, inflammation, the four contact factors—FXII, FXI, PK, and HK—are acti- complement activation, angiogenesis, and kinin formation. vated, an event that occurs after exposure to and adsorbtion About 50% of patients with FXI deficiency have clinically on negatively charged surfaces such as glass, kaolin, celite, evident bleeding abnormalities, suggesting that FXI may and ellagic acid. Activation of these factors does not require be an important accessory to blood coagulation, but it is Ca++; thus, in vitro activation (or “preactivation”) can occur probably not essential. in citrated patient plasma samples stored in glass tubes for Interestingly, contact factors can activate ex vivo when prolonged periods before testing. In spite of a markedly blood comes in contact with non-biologic surfaces, such as prolonged APTT, patients deficient in FXII, PK, and HK extracorporeal circulation systems used in cardiopulmonary Table 32.4 Coagulation Protease-Cofactor Complexes Protease Cofactor Substrate Cellular Location Factor VIIa Tissue Factor Factor IX Many Cells Factor IXa Factor VIIIa Factor X Platelets Factor Xa Factor Va Prothrombin Platelets Thrombin Thrombomodulin Protein C Endothelium Activated Protein C Protein S Factors Va, VIIIa Endothelium 740 Chapter 32 bypass surgery. This ex vivo contact can result in a sys- FXI temic inflammatory response in patients undergoing this FXIIa HK type of surgery.10 Attention recently has been refocused on the contact system. A number of studies now indicate that FXIa although it is not essential for hemostasis at sites of vessel In addition to activating PK to kallikrein, FXIIa has other injury, it does in fact contribute to pathologic thrombus for- enzymatic functions in hemostasis. As the first enzyme in mation4,10 (Chapter 35). the intrinsic pathway, FXIIa converts FXI to its active form, Factor XII (FXII) FXII (Hageman factor) is synthesized in FXIa, in a reaction that requires HK as a cofactor. Recent the liver and circulates in the blood as a single-chain zymo- evidence, however, suggests that FXIIa-dependent activa- gen. Single-chain FXII is proteolytically converted into the tion of FXI is not essential for normal hemostasis. two-chain active serine protease FXIIa by kallikrein, plasmin FXIIa also activates the fibrinolytic and complement (PLN), or “autoactivation.” Autoactivation of FXII occurs by systems. FXIIa, FXIa, and kallikrein can activate plasmino- contact with negatively charged surfaces, which creates a gen directly, although much less efficiently than tissue-type conformational change in the molecule that induces a limited plasminogen activator (tPA) or urinary-type plasminogen amount of proteolytic activity in FXII. Many non-physiologic activator (uPA) (see the section “Physiologic Plasminogen substances (glass, kaolin, celite, ellagic acid)11 have been Activators”) (Figure 32-5). FXIIa also activates the first com- associated with in vitro FXII autoactivation and are used as ponent of the complement cascade (C-1). activators in the reagents for the APTT test. The nature of the Prekallikrein (PK) PK predominantly circulates in plasma physiologic surfaces responsible for autoactivation in vivo is bound to HK (75% bound, 25% free).12 FXIIa activates PK still in question but is most likely negatively charged cellular to plasma kallikrein, which in turn can induce reciprocal membranes, platelet polyphosphate, microparticles derived activation of FXII and initiation of “intrinsic” coagula- from erythrocytes or platelets, or subendothelial structures tion. Kallikrein can also activate the kinin, fibrinolytic, and (collagen, basement membrane). complement systems. Plasma kallikrein cleaves bradykinin Bound FXIIa cleaves PK to kallikrein, which with HK (BK) from HK. Kallikrein directly activates plasminogen as a cofactor can then reciprocally proteolytically activate to plasmin and converts prourokinase (single chain uPA surface-bound FXII to FXIIa. The generation of FXIIa and [scuPA]) to uPA, which in turn can activate plasminogen. kallikrein by reciprocal activation amplifies these reactions. Plasmin subsequently can activate the first and third com- FXIIa can be further cleaved by kallikrein, yielding smaller ponents of the complement cascade (Figure 32-5). Plasma FXII fragments (FXIIf) that have little or no procoagulant kallikrein also serves as a chemoattractant and activator for activity.12 neutrophils and monocytes, facilitating the inflammatory PK response. Autoactivation FXII FXIIa High-Molecular-Weight Kininogen (HK) HK serves as a FXII FXIIa FXIIf Kallikrein nonenzymatic cofactor in the activation of the contact fac- tors, and is the source of BK. HK accelerates the rate of HK HK surface-dependent activation of FXII and of PK activation Figure 32.5 Role of contact factors in physiologic systems. Secondary Hemostasis and Fibrinolysis 741 by FXIIa. HK binds to platelets, endothelial cells, and FXIa granulocytes, assembling the other components of the con- ∂ca++ tact activation pathway. Two forms of kininogen, HK and Activation low-molecular-weight kininogen, are found in plasma. FIX ¡ FIXa + Peptide The liver produces both forms by alternative splicing of a single gene. HK, the preferred substrate for kallikrein, When activated, FIXa forms a complex (intrinsic Xase) is a single-chain glycoprotein that can be cleaved by kal- with cofactor FVIIIa and Ca++ on the surface of activated likrein into a two-chain molecule with the release of a platelets to activate FX (Figure 32-6). small peptide, BK, a potent bioactive molecule. BK has FIXa/FVIIIa/Ca++ many functions, including increasing vessel permeability, dilating small vessels, increasing blood flow, contracting activated platelet surface smooth muscle, and causing pain. BK also contributes to ∂ endothelial cell profibrinolytic, antithrombotic, and anti- FX ¡ FXa platelet properties by stimulating endothelial cell prosta- cyclin 1PGI22, nitric oxide (NO), and tPA synthesis and The extrinsic pathway complex FVIIa/TF also activates secretion (Chapter 31). FIX by cleavage at the same amino acids in zymogen FIX (see the section “The Extrinsic Pathway”). This extrinsic Factor XI (FXI) In the contact pathway, FXI is activated to pathway for FIX activation bypasses the contact activation the serine protease FXIa by FXIIa and cofactor HK. factors. FXIIa, HK FXI ¡ FXIa FIXa (but not FIX) binds to phospholipid surfaces on activated platelets in the presence of calcium via its GLA Like PK, FXI circulates in the plasma primarily as a domain; it does not bind to resting platelets. It is one of complex with HK. FXI is unique among the coagulation two coagulation proteins (FVIII and FIX) whose genes are zymogens because it contains two identical polypeptide encoded on the X chromosome. The severe bleeding that chains, each containing a catalytic site. Activation involves results from a deficiency of FIX (Hemophilia B) indicates cleavage of an internal peptide bond in each of the poly- that it plays a critical role in blood coagulation. peptide chains, resulting in a four-chain activated protease (FXIa). In addition to activation by FXIIa, both thrombin Factor VIII (FVIII) FVIII (also called antihemophilic fac- and FXIa itself can activate FXI, in a positive feedback tor) is synthesized primarily in the liver by sinusoidal endo- reaction. Thrombin is likely the physiologically relevant in thelial cells (with a small amount of extrahepatic synthesis vivo activator of FXI with minimal need for activation by from endothelial cells of many tissues),4 and circulates in FXIIa. Both FXI and thrombin bind to the surface of acti- plasma in association with von Willebrand factor (VWF). vated platelets where in vivo activation most likely occurs. Each protein in this complex is under separate genetic HK is thought to mediate binding of FXI to the negatively charged surface, binding to platelet GPIb, and to platelet apolipoprotein E receptor 2 (ApoER2).4 The preferred sub- FXa heavy strate for FXIa is FIX, and its interaction with and activation chain of FIX likely also occurs on the surface of activated plate- FIXa FX FXa lets.13 Although patients with deficiencies of other contact heavy heavy light chain factors (FXII, PK, HK) do not have bleeding problems, chain chain about 50% of patients with an FXI deficiency experience FIXa FX abnormal bleeding after surgery or injury. Plasma levels light chain light chain of FXI are not the only determinant of whether bleeding Ca11 FVlIIa Ca11 occurs because patients who have bleeding problems and those who do not can have similar plasma concentrations Platelet of FXI.13 OTHER FACTORS IN THE INTRINSIC PATHWAY Factor IX (FIX) FIX (also called Christmas factor) is a single-chain vitamin K–dependent zymogen containing 12 g@carboxyglutamic acid residues. In the intrinsic path- Figure 32.6 The complex formed by the sequential activation way, FXIa activates FIX in the presence of Ca++ and does of the intrinsic pathway (FIXa, FVIIIa, Ca++) activates FX. The cofactor, FVIIIa, binds to the platelet phospholipids and orients not require any other cofactor. Activation is associated with FIXa and FX to enhance activation of FX. FIXa and FX are bound cleavage of two bonds in FIX, releasing an activation pep- to the platelet surface via Ca++ bridges. Spikes on platelet indicate tide and a two-chain molecule, FIXa. activated platelet. 742 Chapter 32 control and has distinct biologic functions and immuno- by weak electrostatic interactions, readily dissociates. As a logic properties. Binding to VWF stabilizes FVIII and pro- result, FVIII is labile, and its activity is rapidly lost at room tects it from inhibition or degradation. FVIII circulates in the temperature. plasma in an inactive form until it is activated by thrombin Thrombin activation of FVIIIa dissociates FVIIIa from (or FXa), yielding FVIIIa, which serves as a cofactor for the the protective influence of VWF, permitting FVIIIa interaction FIXa activation of FX. with the activated platelet surface via its C domains.14 FVIIIa FVIII is synthesized as a large precursor protein con- has no enzymatic activity of its own, but as part of the intrinsic sisting of the linear array of A1:A2:B:A3:C1:C2 domains Xase complex functions as a cofactor for FIXa. Together, FVIIIa (Figure 32-7). The A domains are involved in protein–protein and FIXa activate FX (Figure 32-4), increasing the catalytic acti- interactions, the B domain is removed during activation, vation of FX about 106@fold. However, large amounts of FXa and the C domains are involved in binding to phospholipid and thrombin destroy the procoagulant function of FVIIIa. surfaces.14 On secretion from the endothelial cell to the Like FIX, the gene for FVIII is located on the X chromo- blood, FVIII is cleaved at two sites within the B domain, some, and FVIII is critical for normal blood coagulation. A releasing part of the B domain and a heterodimer composed deficiency of FVIII results in Hemophilia A. of a heavy chain (A1:A2 and the remaining part of the B domain) and a light chain (A3:C1:C2 domains). Von Willebrand Factor (VWF) VWF is a large multimeric In the plasma, FVIII associates with VWF (Figure 32-8), glycoprotein synthesized |
Subsets and Splits