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recently The body fluid mode of the XE-5000™ performs eryth- developed software package allows the data management rocyte counts (RBC), leukocyte counts (WBC), two-part system to calculate the percentage of hypochromic eryth- differentials (polymorphonuclear cells and mononuclear rocytes (%HYPO HE) and hyperchromic erythrocytes cells), and total nucleated cell counts (TC-BF) on all body (%HYPER HE). Using the pulse height (i.e., volume) for fluids (e.g., cerebrospinal, serous, synovial fluids).34,35,36 each erythrocyte counted, the data management system No pretreatment of the sample is required. Using the body generates the percentage of microcytes (%MICRO R) and fluid mode, the cell-counting time is extended to increase percentage of macrocytes (%MACRO R).31 Studies indi- the precision of the cell counts. The RBC count is deter- cate that these additional parameters, %HYPO HE and mined by impedance, and the WBC count and two-part %MICRO R, have potential use in the differentiation of the differential are determined by fluorescent flow cytometry. b@thalassemia trait from other microcytic anemias and that The data collected in these measurements are analyzed by %HYPO HE may be a useful parameter in the evaluation of specific algorithms to generate the cell counts and two-part iron status.31,32,33 differential. When the laboratory professional switches the The data analysis system analyzes all data, including instrument from the whole blood mode to the body fluid cell counts, histograms, and scattergrams. The results are mode, the instrument performs a rinse cycle to prevent car- displayed on the computer screen, printed to a hard copy ryover from previously tested whole blood specimens as (Figure 39-22), or transferred to the LIS (see Online Student well as a background check before the body fluid sample Resources Table 39-1 for reported parameters). An exten- is tested. sive flagging program with interpretive comments alerts the laboratory professional to abnormal results. Use of the flag- ging system and the observations of the scattergrams and Sysmex XN-Series™ histograms allow the laboratory professional to focus on Introduced in 2012, the XN-10™ instrument is a modular specific abnormalities when performing a peripheral blood component capable of determining the CBC, six-part leu- smear evaluation. kocyte differential (neutrophils, lymphocytes, monocytes, DIFF WBC/BASO WBC 7.98 [10^3/uL] RBC 4.34 [10^6/uL] HGB 12.1 [g/dL] HCT 35.1 [%] MCV 80.9 [fL] MCH 27.9 [pg] SSC SSC MCHC 34.5 [g/dL] PLT 217 [10^3/uL] IMI NRBC RDW-SD 37.0 [fL] RDW-CV 12.5 [%] MPV 9.8 [fL] NEUT 4.59 [10^3/uL] 57.6 [%] LYMPH 2.42 [10^3/uL] 30.3 [%] MONO 0.72 [10^3/uL] 9.0 [%] EO 0.21 [10^3/uL] 2.6 [%] BASO 0.04 [10^3/uL] 0.5 [%] NRBC [10^3/uL] [/100WBC] DC SFL IG 0.01 [10^3/uL] 0.1 [%] RET [%] [10^6/uL] RET PLT-O IRF [%] SFL SFL RBC PLT Figure 39.22 Sysmex XE-Series™ report from a normal individual. FSC RF SFL FSC FSC FSC 978 Chapter 39 eosinophils, basophils, and IGs), nucleated erythrocyte count, blasts based on analysis of the differences in these cell clus- and reticulocyte count from a single aspiration of a blood ters. Application of this algorithm improves the specificity sample. (See Table 39-1 for reported parameters.) The addi- of alert flags for atypical lymphocytes and blasts. tion of the SP-10™ slidemaker/stainer creates a complete In addition, accurate leukocyte differentials are modular workstation. To increase the productivity of a work- obtained for blood specimens with leukocyte (WBC) counts station, additional XN-10™ instruments can be added. For less than 0.5 * 103/mcL 1mL2 when performed in the low example, the XN-3000™ is a workstation with two XN-10™ WBC mode. The instrument can be programmed to auto- modular units and the SP-10™ slidemaker/stainer unit. matically perform the WBC count and leukocyte differential The XN-series™ instrument uses the same methods as in this mode if the WBC count falls below this level. In the described for the XE-series™ for determining the erythro- low WBC mode, the sample is analyzed three times longer cyte (RBC) count, hemoglobin, RBC parameters (e.g., Hct, to accumulate more cell events and evaluated in the WDF MCV), platelet count (impedance), reticulocyte count, and channel, thus improving the WBC count and differential’s reticulocyte parameters (e.g., IRF, Ret He). New methods accuracy. allow clearer discrimination of leukocytes, abnormal leu- Within the white cell nucleated (WNR) channel, the kocytes, and nucleated erythrocytes.37,38 leukocyte (WBC) count is determined and basophils and The white cell differential (WDF) channel differentiates nucleated erythrocytes are clearly differentiated from other and enumerates neutrophils and basophils, lymphocytes, leukocytes to determine the number of basophils and nucle- monocytes, eosinophils, and IGs (Figure 39-23). These cells ated erythrocytes within each blood specimen. In the WNR are classified based on their side scatter and fluorescent channel, lyse reagent lyses the erythrocytes and removes intensity, characteristics that are similar to those used for the nucleated erythrocyte’s cytoplasmic membrane. For the cell classification by the DIFF channel of the XE-series™ leukocytes, lyse reagent perforates the cytoplasmic mem- instrument. The key difference is the instrument’s new brane and alters the leukocyte’s external shape and internal algorithm, Sysmex Adaptive Flagging Algorithm, based on structure depending on the characteristics of the specific shape recognition (SAFLAS) that allows better discrimina- leukocyte (e.g., segmented neutrophil versus basophil). The tion of monocytes, lymphocytes, atypical lymphocytes, and fluorescent dye (polymethine) binds to the bare nuclei of the Monocytes IE Immature granulocytes MONO Lymphocytes Neutrophils and LYMPH Basophils NEUT & BASO EO Eosinophils DEBRIS Side scatter (SSC) Figure 39.23 WDF scattergram. Lymphocytes (LYMPH), monocytes (MONO), eosinophils (EO), neutrophils 1NEUT2 + basophils (BASO), and immature granulocytes (IG) are identified. Basophils are specifically identified in the WNR scattergram. SSC, side scatter; SFL, fluorescence intensity. Fluoresence intensity (SFL) Automation in Hematology 979 nucleated erythrocytes and enters the leukocytes to bind to PLT-F channel can be included as part of the routine mea- nucleic acids and intracytoplasmic organelles. The cells are surement for all blood specimens, or the user can program then analyzed by fluorescent flow cytometry as in the XE- it to be a reflex test and performed only when the analyzer series™ instrument to determine each cell’s forward scat- onboard rule system detects an abnormal platelet histogram ter (cell size) and fluorescent intensity (amount of bound or low platelet count (predetermined by the user). The PLT-F fluorescent dye). These data are used to determine the total channel provides an accurate platelet count when numbers leukocyte (WBC) count and generate the WNR scattergram are low or when potential interferences such as erythrocyte (Figure 39-24). Nucleated erythrocytes and basophils can be or leukocyte fragments are present. clearly delineated on this scattergram from other leukocytes and enumerated. Because the delineation between nucle- ated erythrocytes and leukocytes is clear, the total leukocyte Checkpoint 39.6 Which cellular characteristics are used to determine the IPF on (WBC) count does not require correction for their presence. the Sysmex XN-Series™ instrument? The fluorescent platelet (PLT-F) channel is a separate channel that determines the IPF and a platelet count (i.e., PLT-F count). Within this channel, the aspirated blood speci- The XN-Series™ analyzers have a body fluid module men is diluted, and a fluorescent dye (oxazine) is added to for counting erythrocytes, leukocytes, and determining a the dilution. The oxazine binds to the nucleic acid content two-part differential for all body fluids.39 The new methods of the platelet organelles and binds only diffusely to the introduced for the leukocyte count and leukocyte differential reticulocytes. The dilution is then analyzed by fluorescent have also improved the instrument’s ability to perform cell flow cytometry to determine each cell ’s forward scatter counts and cell differentials on body fluids. No pretreatment and fluorescent intensity. Using this information, the PLT-F or off-line preparation is required for body fluid analysis. scattergram is generated (Figure 39-25). Platelets are clearly The erythrocyte count (RBC-BF) is determined by imped- delineated from other cells including reticulocytes based on ance in the RBC/platelet channel, whereas the leukocyte their size and fluorescent intensity as depicted in the PLT-F count (WBC-BF), total nucleated cell count (TC-BF), and scattergram. The IPF is determined from this scattergram two-part differential (polymorphonuclear cells and mono- because these cells have high fluorescence intensities. The nuclear cells) are determined by fluorescent flow cytometry Basophils BASO NRBC WBC DEBRIS Fluorescence Intensity (SFL) Figure 39.24 WNR scattergram. Nucleated erythrocytes and basophils are clearly delineated from other leukocytes (e.g., neutrophils, lymphocytes, and monocytes). Therefore, the total WBC count does not require correction for the presence of nucleated erythrocytes. Forward Scatter (FSC) 980 Chapter 39 RBC IPF PLT-F SFL Figure 39.25 Sysmex XN-Series PLT-F scattergram showing the platelet population is clearly separated from the erythrocyte population. The immature platelet fraction (IPF) is identified within the platelet-fluorescent (PLT-F) population by its high fluorescence and larger size. FSC, forward scatter; SFL, fluorescence intensity; RBC, red blood cell. (side scatter and fluorescent intensity) in the WDF channel. The BF scattergram depicts the polymorphonuclear cells HF-BF (PMNs) and mononuclear cells (MNs) by their characteris- tic side scatter and fluorescent intensity patterns (Figure 39- 26). High-fluorescent intensity body fluid (HF-BF) cells (e.g., MN macrophages, mesothelial cells) can also be seen on this scat- tergram. The WBC-BF count represents the number of PMNs plus MNs, whereas the TC-BF count includes WBC-BF count MO-BF plus the HF-BF cells identified by the BF-WDF scattergram. Abbott CELL-DYN Sapphire® LY-BF GHOST Like other automated cell counting instruments, the CELL- PMN DYN Sapphire® uses a combination of technologies to enu- EO-BF NE-BF merate erythrocytes, leukocytes, platelets, and reticulocytes and determine a five-part leukocyte differential.40,41 Flow PLT-O cytometry, fluorescence staining, and impedance are used to make these determinations. SIDE SCATTER The instrument aspirates an aliquot of EDTA-antico- agulated blood and sends a portion of this specimen to the Figure 39.26 WDF scattergram, Sysmex XN-Series™. hemoglobin dilution cup. The hemoglobin reagent dilutes Lymphocytes, monocytes, eosinophils, and immature granulocytes the blood specimen, lyses erythrocytes, and converts free are identified. Basophils are specifically identified in the WNR hemoglobin to a single chromogen by forming a complex scattergram. FSC SFL Automation in Hematology 981 with imidazole. The hemoglobin concentration is deter- sets a discriminant line. Scatterplots and contour plots of the mined spectrophotometrically at 540 nm. A reagent blank data are used to further classify subpopulations of leuko- is used to minimize optical interferences, whereas leuko- cytes. The 0° (size) versus 7° (complexity) scatterplot allows cyte interference is minimal because the reagent destroys differentiation of neutrophils, monocytes, and lymphocytes leukocytes and cellular fragments. This cyanide-free reac- (Figure 39-28). Eosinophils are differentiated from neutro- tion shows good correlation with the cyanmethemoglobin phils in the 90°D (granularity) versus 90° (lobularity) scat- reference method. terplot (Figure 39-29). Finally, lymphocytes are separated In the WBC dilution cup assembly, the specimen is from basophils based on size and complexity. The data man- diluted for enumerating total leukocyte (WBC) count, detect- agement system analyzes these characteristics to determine ing and quantitating nucleated erythrocytes, and determin- the total WBC count and the five-part leukocyte differential. ing the five-part leukocyte differential. The preheated WBC Additional information obtained from the evaluation of this reagent dilutes the leukocytes, lyses erythrocytes, strips the dilution includes the enumeration of nucleated erythrocytes cytoplasmic membrane from nucleated erythrocytes and and identification of nonviable or fragile leukocytes, which fragile leukocytes, and stains DNA of the exposed nuclei are based on light scatter characteristics and fluorescence with propidium iodide (PI), a fluorescence dye. This dilu- intensity (i.e., DNA content). Because the nucleated eryth- tion is sent to the optical flow cell. Hydrodynamic focusing rocytes are clearly distinguished from leukocytes in this directs cells through the flow cell in single file. A solid-state dilution, the total WBC count and five-part differential are laser interacts with each intact cell or exposed nucleus to unaffected by their presence. create light scatter and fluorescence (PI excites at 488 nm Two different dilutions are prepared in the RBC/PLT and emits at 630 nm). Using multi-angle polarized scat- dilution cup assembly. One dilution represents the erythro- ter separation (MAPSS™) technology, the following light cyte/platelet dilution. The diluent dilutes the blood speci- scatter characteristics are determined as indicated: (1) 0° men and spheres the erythrocytes. Using the principles of light scatter or forward scatter that reflects cell size, (2) 7° hydrodynamic focusing and impedance, the erythrocytes light scatter that reflects cell complexity, (3) 90° light scatter and platelets are evaluated as they pass singly through the or side scatter that reflects nuclear lobularity, and (4) 90° impedance transducer from which the RBC count, erythro- depolarized light scatter (90°D) that reflects cytoplasmic cyte size

distribution curve (histogram), platelet size dis- granularity (Figure 39-27). MAPSS™ technology-generated tribution curve, and platelet count are obtained. A second data are used in different ways to classify leukocyte sub- portion of the erythrocyte/platelet dilution is sent to the opti- populations and identify certain morphologic flags.40 For cal flow cell for enumeration of these cells based on light- example, discriminant line analysis isolates and identifies scatter characteristics. The RBC count from the optical flow various cell populations. A histogram is created based on cell and the platelet count from the impedance transducer are one-dimensional data (e.g., size) or two-dimensional data used as internal quality control checks against the reported (e.g., size versus DNA content). The data management sys- RBC count from the impedance transducer and the reported tem identifies the valley between the cell populations and WBC Differential 90°D 90° 7° 0 Laser beam 0° S I Z E 7° 7° – COMPLEXITY Figure 39.28 0° (Size) vs 7° (complexity) scatterplot, CELL- Figure 39.27 Four light scatter measurements from the DYN Sapphire®. Neutrophils, monocytes, and lymphocytes are CELL-DYN Sapphire®. The 0° scatter reflects cell size; 7° scatter identified. Lymphocytes are located lower left in the scatterplot; the reflects cell complexity; 90° scatter reflects nuclear lobularity; and middle cell population is composed of monocytes; and neutrophils 90° depolarized scatter reflects cytoplasmic granularity. are located upper center in the scatterplot. 982 Chapter 39 In 2010, Abbott Diagnostics introduced a new soft- ware package for the CELL DYN Sapphire® that extends the number of RBC and reticulocyte parameters gener- ated by this instrument. The data management system 90ºD calculates these parameters from the data collected dur- ing the erythrocyte or reticulocyte enumeration and determines microcytic RBC% (%MIC), macrocytic RBC% Eosinophils (%MAC), hypochromic RBC% (%HPO), hyperchromic Neutrophils RBC% (%HPR), MCVr (reticulocyte mean cell volume), MCHr (reticulocyte mean cell hemoglobin), and CHCr 90º (reticulocyte mean cell hemoglobin concentration). These parameters have been shown to correlate well with simi- Figure 39.29 90°D (granularity) versus 90° (lobularity), CELL- lar parameters introduced with the ADVIA hematology DYN Sapphire. Eosinophils are distinguished from neutrophils. instruments (Siemens Healthcare).42 platelet count from the optical flow cell. The second dilution represents the reticulocyte dilution. The blood specimen is diluted with the isotonic diluent, and nucleic acids are stained Checkpoint 39.7 a. Which CELL-DYN scatterplot allows differentiation of eosino- with a fluorescence dye (i.e., fluorescein isothiocyanate) that phils from neutrophils? excites at 488 nm and emits at 530 nm. This dilution is sent b. The CELL-DYN Sapphire’s WBC dilution contains propidium to the optical flow cell where the number of reticulocytes is iodide, so why do the neutrophils not fluoresce? determined based on fluorescence intensity and light-scatter characteristics (i.e., 7° light scatter). From this information, a reticulocyte histogram is created for the determination of the The CELL-DYN Sapphire® can perform two additional reticulocyte count and the IRF (Figure 39-30). assays, Immuno T-cell (CD3/4/8) Assay, and ImmunoPlt™ The data management system analyzes and compiles (CD61) assay. Both represent fluorescent immunophenotyp- all data obtained from the instrument and determines the ing methods. The Immuno T-cell (CD3/4/8) Assay uses reported parameters (Table 39-1). The results are displayed monoclonal antibodies with different fluorescent labels to on a computer screen, printed to a hard copy for the labora- identify the T-cell population (i.e., fluorescein isothiocya- tory professional’s review (Figure 39-31), or transferred to nate labeled anti-CD3) and its two subpopulations, T helper the LIS. System-initiated messages and data flags alert the cells (i.e., phycoerythrin labeled anti-CD4) and T cytotoxic laboratory professional to potential abnormalities or errors cells (i.e., phycoerythrin labeled anti-CD8). Enumeration of in the results. This information is used to correlate CBC data these subpopulations is important in evaluating patients with peripheral blood morphology to improve the identifi- with immunodeficiency syndromes and monitoring certain cation and confirmation of abnormalities. therapeutic interventions. The Immuno T-cell (CD3/4/8) Assay consists of two RETC reagent tubes, CD3/CD4 and CD3/CD8. The instrument prepares the reaction mixture in each tube by adding the patient specimen and the diluent to the tube’s monoclo- nal antibodies. The instrument rocks the reagent tube gen- tly, incubates the reaction mixture for 2 minutes at room temperature, and then aspirates a portion of the reaction mixture from the tube and places it in the WBC dilution cup. WBC reagent is added to create a final dilution of 1:36, which is sent to the optical flow cell for light scatter and fluorescence intensity measurements. Fluorescence inten- sity, 0° light scatter (i.e., size), and 7° light scatter (i.e., cell complexity) are used to identify and enumerate the differ- ent lymphocyte populations. The scatterplot of 0° light scat- FL1 – RNA ter versus 7° light scatter is used to isolate the lymphocyte Figure 39.30 population for further examination. The scatterplot of CD3 Reticulocyte histogram, CELL-DYN Sapphire®. Reticulocytes are found between the two gates. The peak to the left fluorescence intensity (measured at fluorescein isothiocya- represents mature erythrocytes. x-axis, fluorescence; y-axis, relative nate’s emission wavelength of 530 nm) versus CD4 fluo- number of cells. rescence intensity (measured at phycoerythrin’s emission Automation in Hematology 983 Figure 39.31 CELL-DYN Sapphire® report depicting WBC differential. wavelength of 630 nm) determines the percentage of T prior to preparation of the assay’s reagent tubes. This CBC helper cells; likewise, the scatterplot of CD3 fluorescence also provides a check of the lymphocyte’s viability. intensity versus CD8 fluorescence intensity determines The ImmunoPlt™ (CD61) assay uses a platelet-specific the percentage of T cytotoxic cells (Figure 39-32). The total monoclonal antibody, anti-CD61, which is labeled with fluo- CD3 fluorescence intensity on a given scatterplot reflects rescein isothiocyanate. This assay is useful when a patient’s the percentage of T cells present in the patient specimen. platelet count is very low (e.g., less than 10 * 103/mcL) or Comparison of the percentage of CD3 + cells from each when leukocyte or erythrocyte fragments within the patient scatterplot can be used as an internal control. The computer specimen interfere with platelet enumeration. Because of system uses established algorithms to process the light the specificity of this assay, it provides an accurate platelet scatter and fluorescence measurements and generate the enumeration. Like the T-cell assay, the monoclonal antibody reported parameters. These parameters include total T-cell is present within a reagent tube. Patient specimen and dilu- count, percentage of T cells, absolute T helper cell count, ent are added to this tube, which are gently mixed. The percentage of T helper cells, absolute T cytotoxic cell count, reagent tube incubates for 1 minute at room temperature. At percentage of T cytotoxic cells, and the ratio of T helper to T this point, additional diluent is added to the reagent tube to cytotoxic cells. The concentration of these cells can be deter- further dilute the mixture. A portion of this reaction mixture mined because a CBC is performed on the patient specimen is aspirated into the RBC/platelet dilution cup and diluent 984 Chapter 39 CD3 CD3 CD4 CD8 Figure 39.32 The CD3 versus CD4 scatterplot is used to define the T helper lymphocyte population (i.e., CD4+ /CD3+ cell population in the upper right quadrant). The plot also depicts CD4- /CD3+ lymphocytes (i.e., T cytotoxic lymphocytes) in the upper left quadrant and CD4- /CD3- lymphocytes (i.e., primarily B lymphocytes) in the lower left quadrant. Similarly, the CD3 versus CD8 scatterplot defines these same populations: T cytotoxic lymphocytes 1CD8+ /CD3+2 in the upper right quadrant, T helper lymphocytes 1CD8- /CD3+2 in the upper left quadrant, and CD8- /CD3- (i.e., primarily B lymphocytes) in the lower left quadrant. Thus, using three fluorescent-labeled monoclonal antibodies, the two subpopulations of T lymphocytes can be clearly defined. NOTE: These are schematic scatterplots. is added to create a final dilution of 1:290. A portion of the measurement channels. The erythrocyte/platelet channel final dilution is injected into the optical flow cell for mea- determines the erythrocyte and platelet counts by light- surement. Measurements include 0° light scatter, 7° light scattering measurements obtained as diluted cells pass sin- scatter, and fluorescence intensity at 530 nm (i.e., fluorescein gly through a helium-neon laser beam. The diluent used isothiocyanate’s emission wavelength). The instrument’s for erythrocyte and platelet counts causes isovolumetric computer system uses established algorithms to process sphering of the erythrocytes and platelets. Isovolumetric the data generated by these measurements and determines sphering of erythrocytes eliminates cell volume errors due the PLT count. Because of CD61’s specificity for platelets, to variations in erythrocyte shape.46,47 The erythrocytes are other cells or cell fragments do not affect this platelet count. counted and sized by both high-angle (5–15°) and low-angle (2–3°) light-scattering measurements. Individual erythro- cyte hemoglobin concentration is determined by the high- Light-Scattering angle measurement, and cell volume is determined from the low-angle measurement; thus, the MCV and cellular Instruments hemoglobin concentration mean (CHCM) are obtained. Technicon Instruments Corporation was important in the Together, these measurements are used to generate the development of hematology instrumentation that used erythrocyte cytogram, erythrocyte histogram, and hemo- light-scattering technology to enumerate blood cells. Its globin histogram (Figure 39-33). The RDW and the hemo- first instruments were based on continuous flow analysis globin distribution width (HDW) are derived from these similar to its chemistry instruments. The Hemolog D per- histograms. formed leukocyte differentials based on continuous flow Platelets are evaluated simultaneously with the eryth- analysis and peroxidase cytochemical staining. The Tech- rocytes using both high-angle light scatter and low-angle nicon H-6000 was capable of performing a complete blood light scatter; however, these signals are amplified for cell count and five-part leukocyte differential using con- platelet enumeration. This information is used to create a plat tinuous flow analysis and an improved cytochemical stain- elet cytogram, and the actual platelet count is obtained by ing method. The Technicon H *1 was the first of a series of integrated analysis of the platelet cytogram and erythrocyte instruments that combined these principles of cell detection cytogram to include large platelets while excluding erythro- and identification with flow cytometry.43,44 The two instru- cytes, erythrocyte fragments, and erythrocyte ghosts. ments described next represent current models whose basic Within the hemoglobin channel, a portion of the EDTA- principles of cell enumeration and determination of the anticoagulated blood is mixed with the hemoglobin diluent. leukocyte differential can be traced back to methodologies The erythrocytes are lysed, and free hemoglobin is con- introduced by the Technicon Instruments Corporation. verted to cyanmethemoglobin. The concentration of cyan- methemoglobin is determined photometrically at 546 nm. Siemens Healthcare ADVIA 120 The leukocyte count and five-part leukocyte differential are obtained utilizing two different methods and two separate The ADVIA 120 is capable of performing a CBC, five-part channels, the peroxidase and the basophil/lobularity. The leukocyte differential, and reticulocyte count.45 It has five peroxidase channel identifies neutrophils, monocytes, and Automation in Hematology 985 TEST NORM ABN NORMAL RANGE UNITS WBC 8.28 ( 4.8 –10.8 ) x10.e3 /uL Name: WBCB 8.28 ( 4.4 –9.9 ) WBCP 8.18 ( 4.4 –9.9 ) RBC 4.61 ( 4.15 –5.90 ) x10.e6 /uL SS: HGB 14.0 ( 12.9 –18.0 ) g/dL HCT 40.3 ( 40.0 –53.0 ) % Location: MCV 87.4 ( 83.3 –101.7 ) fL MCH 30.3 ( 27.0 –33.6 ) pg MANUAL DIFFERENTIAL MCHC 34.6 ( 30.4 –35.7 ) g/dL SEGS META CHCM 34.0 ( 30.4 –35.7 ) g/dL BAND MYEL RDW 13.7 ( 12.6 –15.7 ) % LYMPH PRO PLT 311 ( 130 –400 ) x10.e3 /uL MONO BLAST MPV 7.7 ( 7.4 –10.4 ) fL EOS I.M.C. %NEUT 47.4 ( 25.0 –85.0 ) % BASO NRBC %LYMPH 45.0 ( 10.0 –50.0 ) % ATYP %MONO 3.4 ( 0.0 –15.0 ) % RBC MORPHOLOGY %EOS 1.1 ( 0.0 –15.0 ) % POIK POLY %BASO 0.3 ( 0.0 –3.0 ) % TARGET BURR %LUC 2.7 ( 0.0 –6.0 ) % OVAL TOXIC #NEUT 3.93 ( 1.9 –8.0 ) x10.e3 /uL TEAR DOHLE #LYMPH 3.73 ( 0.9 –5.2 ) x10.e3 /uL SCHISTO CREN #MONO 0.28 ( 0.16 –1.0 ) x10.e3 /uL OTHER #EOS 0.09 ( 0.0 –0.8 ) x10.e3 /uL COMMENTS: #BASO 0.02 ( 0.0 –0.2 ) x10.e3 /uL #LUC 0.23 ( 0 –0.4 ) x10.e3 /uL TECH’S INITIAL MPXI –0.2 LI 2.17 %BLASTS 0.2 RBC VHC RBC VOLUME BASO PLT VOL RBC HC PEROX RBC CH Figure 39.33 ADVIA 120 report from a normal individual. eosinophils by the degree of peroxidase positivity (i.e., and this information is used to create the

peroxidase cyto- increased absorption) and the amount of forward light scat- gram (Figure 39-34). Within the basophil/lobularity channel, ter. Lymphocytes and large unstained cells (LUCs) are iden- EDTA-anticoagulated blood is mixed with basophil diluent tified by the amount of forward light scatter and the fact that lyses erythrocytes and platelets and strips all leukocytes that they remain unstained by this peroxidase cytochemi- except basophils of their cytoplasm. The helium-neon laser cal–staining method. Erythrocytes are removed prior to the flow cell measures this dilution and determines the degree peroxidase staining by lytic action. The amount of forward of high-angle scatter and low-angle scatter for each cell scatter and degree of peroxidase positivity are detected as examined. This information is used to create the basophil/ the cells pass through a tungsten halogen-based flow cell, lobularity cytogram (Figure 39-35). The data management 986 Chapter 39 PEROX the basophil/lobularity cytogram, the normal cell pattern is Neutrophils referred to as the worm, with the head region representing Large mononuclear cells and the body region representing poly- Unstained morphonuclear cells as classified by their high-angle scat- Cells ter signatures. Basophils have large, low-angle scattered signatures and are located in the region above the worm (Figure 39-35). For the peroxidase cytogram, the normal cell pattern depicts the neutrophils in the upper right quadrant, Monocytes eosinophils in the lower right quadrant, and monocytes in L Y the center triangular region; lymphocytes are located adja- M cent to the y-axis in the center left quadrant and LUCs are located in the upper left quadrant of the peroxidase cytogram (Figure 39-34). The absolute count for each leukocyte popula- Eosinophils tion is obtained from the appropriate channel (e.g., absolute neutrophil count from the peroxidase channel and abso- lute basophil count from the basophil/lobularity channel). The absolute lymphocyte count is obtained by subtracting Figure 39.34 ADVIA 120 peroxidase cytogram. Neutrophils, the absolute basophil count from the absolute lymphocyte monocytes, and eosinophils are differentiated based on the strength count that was obtained from the peroxidase channel. This is of peroxidase positivity and degree of forward scatter (i.e., size) such necessary because lymphocyte and basophils cannot be dif- that neutrophils are located in the upper right quadrant, eosinophils ferentiated from each other based on analysis of the peroxi- in the lower right quadrant, and monocytes in the center triangular area. Lymphocytes lack peroxidase activity and are medium- to dase cytogram. The percentage values are calculated from small-sized cells so that they fall close to and near the center of the absolute cell counts. The total WBC count is obtained the y-axis. A fifth population of cells known as large unstained cells from both channels. The data management system compares (i.e., LUCs) fall directly above the lymphocyte population. Reactive these counts and flags the total WBC count if the results do lymphocytes are an example of large unstained cells. not compare. An advantage of this method of determining cell counts is ability to obtain accurate WBC counts and dif- system uses cluster analysis to identify individual cell pop- ferentials on samples with very low counts (WBC less than ulations within a given cytogram. Each population is identi- 0.1 * 103/mcL). fied by its position, area, and density. Thresholds are set, and The fifth channel is the reticulocyte channel where cel- the number of cells in each population is determined. For lular RNA of the reticulocytes is stained with oxazine 750, a nucleic acid dye. The helium-neon laser flow cell evaluates the erythroid cells for light-scattering and absorbance charac- BASO teristics. The high-angle light scatter reflects the hemoglobin concentration within the individual erythroid cells, and low- angle light scatter reflects the size of each cell. Reticulocytes are differentiated from mature erythrocytes based on their Basophils RNA content, which is determined by the cell’s absorbance reading. Reticulocytes have a higher absorbance compared to mature erythrocytes. In addition to the absolute reticu- Mononuclear cells locyte count and relative reticulocyte percentage, reticulo- cyte analysis includes a measure of the reticulocyte cellular Polymorphonuclear cells hemoglobin concentration (CHr™), MCVr, and the reticu- locyte cellular hemoglobin concentration mean (CHCMr). The ADVIA 120’s data management system evaluates the information from the two flow cells, determines the reported parameters, and displays the information on the computer screen (see Table 39-1 for the reported parame- ters). The results can be printed to hard copy (Figure 39-33) Figure 39.35 ADVIA 120 basophil/lobularity cytogram. or transferred to the LIS. If abnormalities are detected in Basophils are clearly differentiated on this cytogram since all cell counts, histograms, or cytograms, the instrument flags other leukocytes are stripped of their cytoplasmic membrane and are evaluated based on their nuclear characteristics only. the appropriate result(s). The flagging criteria assist the Mononuclear cells represent lymphocytes and monocytes, while laboratory professional in defining the abnormalities to be polymorphonuclear cells are neutrophils and eosinophils. reviewed by peripheral blood smear examination. Automation in Hematology 987 The ADVIA 120 and ADVIA 2120 perform CSF analy- Checkpoint 39.8 sis using a dedicated CSF mode.54 The CSF sample is first What is the similarity in the reticulocyte methods performed on mixed with the CSF assay reagent and incubated for a mini- the ADVIA 120 and XN-Series™ instruments? mum of 4 minutes (no longer than 4 hours) to sphere and fix the cells. The instrument aspirates pretreated CSF sample, and determines the cell counts and two-part differential by Siemens Healthcare ADVIA 2120 light scatter and absorbance measurements. The reported results include CSF WBC count, CSF RBC count, and two- The ADVIA 2120 represents the latest model in this series of part differential. The PMNs and MNs are reported in abso- hematology instruments. The basic principles of blood cell lute and relative percentage values. enumeration, reticulocyte enumeration, and determination of the five-part leukocyte differential are the same as those discussed in detail for the ADVIA 120. Hemoglobin is deter- Automated Digital Cell mined by a cyanide-free method. The reagent contains alka- line borate and a surfactant that lyse the erythrocytes and Morphology Instrument facilitate oxidation of heme iron, forming met-heme. The The automated blood cell–counting instruments have metheme groups are ligated by a hydroxyl ion and water reduced the number of manual peripheral blood smear molecule. The ligated metheme groups are solubilized by examinations performed in the hematology laboratory. the surfactant and the colored product is read spectropho- However, these examinations are still required when the tometrically. This cyanide-free hemoglobin method shows automated blood cell–counting instrument alerts the labo- good correlation to the ADVIA 120 hemoglobin method that ratory professional to abnormalities with suspect or user- contains cyanide.48 A disadvantage of the ADVIA 2120 and defined flags. Studies have shown that approximately ADVIA 120 is that neither is currently capable of determin- 15% of CBC results from automated blood cell–counting ing and reporting a nucleated erythrocyte count. instruments require manual microscopic examination.55,56 The ADVIA 120 and ADVIA 2120 were the first Automated digital cell morphology provides a mechanism hematology instruments to introduce extended RBC and to further reduce the number of manual peripheral blood reticulocyte parameters (Table 39-1). The percentage of smear examinations and the time required to perform the hypochromic erythrocytes (%HYPO) and percentage of examinations. hyperchromic erythrocytes (%HYPER) are obtained from the red cell erythrogram, a derivation of the erythrocyte cytogram. The percentage of microcytes (%MICRO) and CellaVision® DM96 System percentage of macrocytes (%MACRO) are derived from the The CellaVision® DM96 system is an automated digital cell erythrocyte cytogram. Studies indicate that the combined morphology identification system that includes a slide scan- use of %HYPO and CHr™ can be useful in detecting iron ning unit, slide feeder, microscope, and digital camera; Cel- deficiency in patients on hemodialysis for chronic renal dis- laVision® DM blood differential software; and a computer ease. These patients could experience a superimposed iron system.55,56,57,58 Barcode-labeled Wright-stained blood smear deficiency with anemia of chronic disease or have a func- slides are placed in an eight-slide magazine and the maga- tional iron deficiency (Chapter 12), which decreases their zine is fed into the slide scanning unit. The slide is scanned response to erythropoietin therapy. Early detection of iron at low power (10* objective) to identify potential leuko- deficiency results in appropriate therapeutic intervention cytes and the internal camera subsequently takes a digital to resolve the iron deficiency and, therefore, decrease the image of each cell at high power (50* objective). The instru- poor response rate of hemodialysis patients to erythropoi- ment performs acquisition and preclassification of cells by etin therapy.4,49,50 comparing the cell’s image to its predefined database of Based on the information provided by the two meth- leukocytes. The laboratory professional examines the digi- ods of determining WBC counts and five-part differential, tal display of the cells and each cell’s preclassification to the ADVIA 2120 calculates a new index, the delta neu- verify or modify this initial classification. The laboratory trophil index (DNI), which is defined as follows: DNI = professional can add comments as necessary. The system’s (neutrophils + eosinophils from the peroxidase channel) leukocyte classifications are segmented neutrophils, band minus (polymorphonuclear cells from the basophil/lobu- neutrophils, eosinophils, basophils, monocytes, lympho- larity channel). This index reflects the number of immature cytes, promyelocytes, myelocytes, metamyelocytes, blast granulocytes (i.e., promyelocytes, myelocytes, metamyelo- cells, variant form of lymphocytes, and plasma cells. Non- cytes) present in the sample. Recent studies reveal that the leukocyte classifications are erythroblasts, giant platelets, DNI may be useful as a prognostic indicator for sepsis or an platelet aggregation, smudge cells, and artifacts. If the Cel- early marker of disease severity in patients with sepsis.51,52,53 laVision® DM96 system cannot identify a cell, it is classified 988 Chapter 39 as “Unidentified". A 100-cell leukocyte differential is typi- rare 11+2, moderate 12+2, and many 13+2—and quanti- cally performed. However, the differential can be extended tatively as a percentage of the erythrocytes. The 21 mor- to 300 or 500 cells if necessary. phologic categories are size: anisocytosis, microcytes, In addition to the leukocyte differential, this system macrocytes; color: polychromasia, hypochromasia; shape: also identifies erythrocyte morphologic characteristics and poikilocytosis, target cells, schistocytes, helmet cells, sickle determines a platelet estimate. The erythrocyte morphol- cells, spherocytes, elliptocytes, ovalocytes, teardrop cells, ogy is performed by scanning the slide at high power (100* stomatocytes, acanthocytes, echinocytes; inclusions: baso- objective). With the CellaVision® Advanced RBC Software philic stippling, Howell-Jolly bodies, Pappenheimer bod- Application, an overview of the erythrocyte morphology ies, and parasites. reflecting an area of 8 microscopic fields is determined.59 The platelet estimate is performed by scanning the Within this overview picture, the software application slide at high power (100* objective) and creating an over- locates and classifies individual erythrocytes into one of view image that reflects an area of eight microscopic fields 21 morphologic categories and displays these results in a of view.60 This overview image is divided into nine grid high-resolution overview. The laboratory professional veri- squares and the laboratory professional counts the number fies or manually modifies each erythrocyte’s initial classifi- of platelets in each grid. The platelet estimate 1*103/mcL2 cation. Approximately 2000 erythrocytes are evaluated per is determined by multiplying the average number of plate- slide. Results for each morphologic category are reported lets per high-power field by a predetermined platelet esti- both semi-quantitatively, using four grades—absent (0), mate factor. Summary This chapter briefly reviewed the ever-increasing uses of the erythropoietic response of the bone marrow to therapy technology for the automated hematology laboratory. The for anemia and the hemoglobin content of reticulocytes blood cell–counting instruments include those using a com- (CHr™), which indicates the functional availability of iron bination of technologies such as impedance, light scatter, to the erythron and its incorporation into hemoglobin. radio frequency, and fluorescent detection to determine the The hematology instruments’ data analysis systems CBC and five-part leukocyte differentials. The two major examine the data for possible interference or abnormalities principles used by hematology instruments are impedance and if found, the laboratory professional is alerted by sus- and light scatter. The impedance principle of blood cell pect or user-defined flags. The laboratory professional uses counting is based on the increased resistance that occurs the flags to correlate the CBC data with peripheral blood when a blood cell with poor conductivity passes through an morphology and confirm the abnormalities or correct erro- electrical field. The number of pulses indicates the blood cell neous results. count, and the amplitude (i.e., height) of each

pulse is pro- To operate these instruments to their fullest poten- portional to the cell’s volume. The optical light–scattering tial, it is important that qualified laboratory professionals principle of blood cell counting is based on light-scattering evaluate the data created by the instrument’s analysis of an measurements obtained as a single blood cell passes individual cell’s characteristics. Through careful review of through a beam of light (optical or laser). Forward scatter that data, new applications of these instruments can arise is a measurement of cell size, whereas side scatter is a mea- to aid in the early detection of abnormalities. Automation sure of cell granularity or complexity. Most instruments also has increased precision and accuracy within the hematology measure reticulocytes and nucleated erythrocytes. Reticu- laboratory and shortened the amount of time needed for locyte evaluation includes not only quantitative data but analysis, but it has also increased the need for the individual also data on the immaturity of reticulocytes, which indicates laboratory professional’s interpretive skills. Automation in Hematology 989 Review Questions Level I c. calculated using the MCH and erythrocyte count 1. Which automated blood cell–counting instrument d. derived from erythrocyte cytogram uses the optical light scatter method to determine the erythrocyte count? (Objective 3) 7. Which technology does the XE-5000™ use to deter- mine a leukocyte count on a body fluid? (Objective 3) a. Unicel® DxH 800 a. Impedance b. XN-10™ c. CELL-DYN Sapphire® b. Conductivity c. Fluorescent flow cytometry d. ADVIA 2120 d. Optical density 2. Why is hydrodynamic focusing included in the 8. Which parameter does the CELL-DYN Sapphire® performance of RBC counts by several automated blood cell–counting instruments? (Objective 3) directly measure? (Objective 5) a. To ensure that only a single cell enters the detection a. Hematocrit area at any given time b. Platelet count b. To direct the beam of light onto the center of the c. Relative neutrophil percentage photodetector d. Absolute reticulocyte count c. To select the appropriate wavelength of light for analysis 9. Which automated blood cell–counting instrument does not use the analysis of fluorescence intensity d. To focus the beam of light on the detection area and light scatter to determine the reticulocyte count? (Objective 6) 3. Which parameter does the LH 780 instrument calcu- late? (Objective 5) a. XE-5000™ a. Erythrocyte count b. ADVIA 120 b. MCV c. CELL-DYN Sapphire® d. Unicel® c. Reticulocyte percentage DxH 800 d. Absolute neutrophil count 10. Which parameter does the Sysmex XE-5000™ derive from a histogram or scattergram? (Objective 5) 4. Which dye is used to stain cellular RNA for reticulo- cyte counting on the ADVIA 2120? (Objective 6) a. Relative monocyte percent a. Thiazole orange b. Erythrocyte count b. Oxazine 750 c. Mean cell volume c. New methylene blue d. Hematocrit d. Auramine-O Level II 5. Which automated blood cell–counting instrument has 1. What information is needed to create an erythrocyte a dedicated channel for nucleated erythrocyte deter- histogram? (Objective 1) mination? (Objective 3) a. Cell volume and relative cell number a. CELL-DYN Sapphire® b. Cell size and cell complexity b. XE-5000™ c. Nuclear size and cellular density c. LH 780 d. Cell forward scatter and cell side scatter d. ADVIA 2120 2. The Sysmex XE-2100™ uses all of the following 6. The ADVIA 2120’s determination of the %HYPO technologies to determine its five-part leukocyte parameter is: (Objective 5) differential except: (Objective 1) a. calculated using the CHCM and MCV a. fluorescence b. derived from hemoglobin histogram b. radio frequency 990 Chapter 39 c. optical light scatter 7. The IRF reported by the Sysmex XE-2100™ represents d. differential cell lysis the following combined parameters: (Objective 2) a. HFR and LFR 3. Using the ADVIA 2120 instrument, which leukocyte cell type is located in the body of the worm of the b. LFR and MFR basophil/lobularity cytogram? (Objective 1) c. HFR and MFR a. Basophil d. absolute and relative reticulocyte counts b. Monocyte 8. The advanced flagging system of the Sysmex c. Neutrophil XE-5000™ has the potential to: (Objective 1) d. Lymphocyte a. increase the number of peripheral blood smear reviews 4. The Coulter® LH 750 five-part leukocyte differential is determined by the analysis of cellular characteristics b. eliminate the peripheral blood smear reviews as defined by: (Objective 1) c. decrease the number of peripheral blood smear reviews a. light scatter, cytochemical staining, and radio frequency d. increase the number of errors in blast cell identification b. light scatter and radio frequency c. impedance and cytochemical staining 9. Which automated blood cell–counting instrument d. impedance, conductivity, and light scatter reports the number of immature granulocytes as part of its leukocyte differential based on analysis of its 5. The CHr™ is determined by measuring a cell’s: white cell scattergram? (Objective 1) (Objective 2) a. CELL-DYN Sapphire® a. absorbance and light scatter characteristics b. XN-Series™ b. absorbance and radio frequency characteristics c. Unicel® DxH 800 c. fluorescence intensity and impedance characteristics d. 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List the specimens appropriate for immuno- cytometer and the principles of cell analysis. phenotyping by flow cytometry. 2. Illustrate by example the clinical 7. Describe how flow cytometry can be used in applications of flow cytometry. cell quantitation. 3. Appraise the use of fluorochrome-labeled 8. Calculate and interpret the absolute CD4 antibodies in immunophenotyping by flow count. cytometry. 9. Explain how flow cytometry can be applied 4. Give examples of the clinical applications of to DNA analysis. immunophenotyping by flow cytometry and 10. List the cells positive for CD34, and explain interpret single dot plots. the purpose of a CD34 count. 5. Define clonality and identify methods for detecting a monoclonal population of cells by immunophenotyping. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare and contrast the immunopheno- malignancies by flow cytometry and typing results characteristic of chronic lym- generate potential solutions. phocytic leukemia, hairy cell leukemia, and 3. Compare and contrast the immunophe- non-Hodgkin lymphoma. notyping results characteristic of acute 2. Identify the pitfalls that can be encountered lymphoblastic leukemia (ALL) and acute in immunophenotyping mature lymphoid myeloid leukemia. 993 994 Chapter 40 4. Identify the pitfalls that can be encountered 10. Summarize the usefulness of flow in immunophenotyping acute leukemia and cytometry in evaluation of hereditary lymphoma by flow cytometry and generate immunodeficiency disorders. potential solutions. 11. Describe the role of flow cytometry in 5. Define minimal residual disease (MRD) in monitoring of systemic autoimmune hematologic malignancies and describe flow diseases. cytometric approaches to identify MRD. 12. Calculate and interpret the S phase 6. Compare and contrast the usefulness fraction and the DNA index and give of the sucrose hemolysis test, Ham test, the principle of DNA analysis by flow and flow cytometry immunophenotyp- cytometry. ing in diagnosing paroxysmal nocturnal 13. Assess the findings of flow cytom- hemoglobinuria (PNH). etry cell analysis using two or more 7. Describe the role of flow cytometry in dot plots, and select the most likely cell diagnosis of hereditary spherocytosis (HS). represented. 8. Select and explain the quality measures 14. Evaluate flow cytometry results to identify that must be enforced in quantitative flow problems and generate solutions. cytometry. 15. Summarize the uses of analysis for the 9. Compare and contrast the absolute CD4 CD34 antigen, and choose recommended count and HIV viral load in the surveillance procedures to analyze this antigen. of HIV infection. Chapter Outline Objectives—Level I and Level II 993 Principles of Flow Cytometry 995 Key Terms 994 Immunophenotyping by Flow Cytometry 998 Background Basics 995 DNA Analysis 1008 Case Study 995 Summary 1009 Overview 995 Review Questions 1009 Introduction 995 References 1011 Key Terms Acute undifferentiated leukemia Fluorochrome Immunophenotyping CD designation Forward light scatter Minimal residual disease Compensation Gating Mixed phenotype acute leukemia DNA Index (DI) Hematogone Photodetector Flow chamber Hydrodynamic focusing Side light scatter Flow Cytometry 995 Background Basics The information in this chapter builds on the concepts • Summarize the classification of malignant leukocyte learned in previous chapters. To maximize your learning disorders. (Chapters 23–28) experience, you should review these concepts before start- ing this unit of study: Level II Level I • Summarize the subtypes of malignant disorders and • Describe the cellular characteristics that differen- laboratory tests used to help classify them. (Chapter 23) tiate T and B lymphocytes; differentiate the stages • Describe the abnormality associated with paroxys- of development of granulocytes, lymphocytes, and mal nocturnal hemoglobinuria (PNH). (Chapter 17) monocytes. (Chapters 7, 8) • Outline the cell cycle. (Chapter 2) CASE STUDY Introduction We refer to this case study throughout the chapter. A flow cytometer is an instrument capable of detecting mol- ecules on the surface or inside individual particles such as Andrew, a 76-year-old male, had a complete blood cells. Isolating single particles/cells and labeling the mol- count (CBC) performed during hospitalization ecule of interest with a fluorescent marker detects molecules for pneumonia. He was found to have a WBC of (Figure 40-1). Particles/cells that possess the molecule of 76 * 103/mcL with 80% lymphocytes. interest are recognized by the emission of fluorescent light Consider the conditions that are associated following excitation. Information is acquired from many with these results and the follow-up testing that thousands of particles/cells and stored on a computer for might be necessary to confirm a diagnosis. further analysis (Figure 40-1). The current applications of flow cytometry in the clinical laboratory include immu- nophenotyping leukocytes and erythrocytes (identifying antigens using detection antibodies), counting CD4+ and Overview CD34+ cells, and analyzing DNA (Table 40-1).1 The purpose of flow cytometry is to detect and measure multiple properties so that cells or particles can be identified Principles of Flow and quantitated. This chapter introduces the principles of flow cytometry and discusses its clinical applications. First, Cytometry the method of detecting and quantitating particles/cells by flow cytometry is described. This description includes Isolation of Single Particles specimen requirements and processing and the concept Flow cytometry is performed on particles in suspension (e.g., of gating to isolate cells of interest. The remainder of the cells or nuclei). Leukocytes from peripheral blood and bone chapter addresses the uses of flow cytometry in the clini- marrow specimens are often analyzed after the removal of cal laboratory. Flow cytometry is currently used to analyze red blood cells (RBCs) by lysis. The cell suspension is aspi- individual cells for the presence of antigens (immunophe- rated and injected into a flow chamber ( Figure 40-1), the notyping), count cells with a particular immunophenotype, specimen-handling area of a flow c ytometer where cells are and quantitate deoxyribonucleic acid (DNA). Immunophe- forced into single file and directed into the path of a laser notyping is one of the tools used for diagnosing mature beam. lymphoid malignancies, lymphoblastic leukemia/lym- The flow chamber contains two columns of fluid. The phoma, acute myeloid leukemia (AML), and paroxysmal particles/cells are contained in an inner column of sam- nocturnal hemoglobinuria (PNH) and monitoring human ple fluid that is surrounded by a column of sheath fluid immunodeficiency virus (HIV) infection. The material in ( Figure 40-1). The sheath and sample fluids are main- this chapter should be studied with Chapters 26, 27, and 28 tained at different pressures and move through the flow (neoplastic disorders) to obtain a full understanding of chamber at different speeds. This gradient between the where flow cytometry fits into the diagnostic workup of sample and sheath fluid keeps the fluids separate (laminar these disorders. This chapter also introduces the application flow) and is used to control the diameter of the column of flow cytometry in DNA analysis. of sample fluid. The central column of sample fluid is 996 Chapter 40 Fluorochrome- labeled antibody staining PerCP 45 34 34 FITC 38 APC PerCP Dot plot 45 FITC 34 Sample 14 Sheath fluid 38 APC 105 Laser Hydrodynamic focusing PE 104 103 Single 102 cell Forward scatter 101 0 signal Filter -105 Photo -6 101 102 103

104 105 detector CD14 FITC-A Side light scatter PMT 105 104 103 102 101 0 -105 -6 101 102 103 104 105 CD14FITC-A Fluorscence signal Filter PMT PMT Fluorescence signal Computer analysis Figure 40.1 Flow cytometer. The cells in the specimen are stained with fluorochrome-labeled antibodies and separated into single cells that pass in front of a laser light source. Scattered and emitted light are detected with photodectors and photomultiplier tubes (PMTs), undergo computer analysis, and are displayed as dot plots. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll. narrowed to isolate single particles/cells that pass through detected using another type of photodetector called a a laser beam (hydrodynamic focusing) like a string of photomultiplier tube (PMT). beads. Laser light is focused on these single p articles/ cells and as it is scattered off them, it is measured using Light Scattering photodetectors called photodiodes. If particles/cells have fluorescent molecules attached, the laser light excites the When the laser beam interacts with a single particle/cell, molecules, which then emit light of a specific wavelength, light is scattered, but its wavelength is not altered. The amount of light scattered in different directions can be used to identify the particle/cell because it is related to the parti- Table 40.1 Applications of Flow Cytometry cle’s physical properties (size, granularity, and nuclear com- Application Examples plexity). Light scattered at a 90° angle (side light scatter) is Immunophenotyping Diagnosis and classification of mature lymphoid related to the internal complexity and granularity of the par- malignancies ticle/cell. Neutrophils produce much side scatter because Prognostic markers in chronic lymphocytic leuke- mia (CD38 and ZAP-70) of their numerous cytoplasmic granules (Figure 40-2). Diagnosis and classification of acute leukemia Light that proceeds in a forward direction (forward light Detection of minimal residual leukemia following scatter) is related to the particle’s size. Large cells produce therapy Diagnosis of PNH more forward scatter than small cells do. Therefore, light Diagnosis of hereditary spherocytosis scattering can be used to distinguish particles, and several Enumeration of T-cell subsets (e.g., CD4 counts in HIV) hematology analyzers currently use it to perform differen- Diagnosis of hereditary immunodeficiencies tial counting of leukocytes (Chapter 10). Monitoring of B-cell depletion therapy in autoim- mune disease Enumeration of CD34+ progenitor cells for transplantation Checkpoint 40.1 RNA analysis Reticulocyte counting (Chapter 11) Would a lymphocyte or monocyte have more forward light DNA analysis S phase fraction scatter? Ploidy CD45 PerCP-Cy5-5-A 38 CD45 PerCP 45 14 45 38 34 Flow Cytometry 997 Figure 40.2 Flow cytometry histograms. Data acquired using flow cytometry can be displayed in a variety of plots. The plot of forward light scatter (FSC-A) versus side light scatter (SSC-A) (top left) distinguishes granulocytes (black) with high side scatter from other cells but cannot distinguish blasts (blue) from lymphocytes (red). The plot of CD45 versus SSC-A (top right) distinguishes blasts with weak intensity CD45 expression (blue) from lymphocytes with bright intensity CD45 expression (red). Data can also be displayed as single-color histograms (lower plot) but are less useful for distinguishing cells types. Histograms generated using FACS DIVA software, BD Biosciences, San Jose, CA. Detection of Fluorochromes further antigens usually requires additional laser light sources (e.g., helium neon, emission 633 nm). Currently, In addition to light scattering, the flow cytometer can be clinical immunophenotyping studies frequently utilize two used to detect bound fluorescent markers ( fluorochromes), or three laser light sources to identify four to eight antigens which are molecules that are excited by light of one wave- on each cell analyzed (4–8 color analysis) (Table 40-2). Some length and emit light of a different wavelength (fluorescent flow cytometers used in a research setting are capable of light). Fluorochromes can be attached to the cells using detecting 18 different fluorochromes. detection antibodies or bind stoichiometrically to DNA A fluorochrome unfortunately does not emit light at (Table 40-2). Flow cytometers use light of a single wave- a single wavelength. Mirrors, filters, and photodiodes are length generated by a laser to excite fluorochromes bound used to detect the peak wavelength of light emitted from to the particle of interest. Light emitted from the fluoro- each fluorochrome. However, some overlap between the chrome is separated from the incident laser light using a light emitted from different fluorochromes usually exists. combination of filters and mirrors. A PMT then detects and For example, although the peak emissions for the two fluo- quantifies the emitted light (Figure 40-1). rochromes fluorescein isothiocyanate (FITC) and phycoery- Clinical flow cytometers usually contain an argon laser thrin (PE) are different, PE emits some light at the wavelength that generates light at 488 nm. This single wavelength is used to detect FITC. This overlap is compensated for by often used to excite three different fluorochromes, each adjusting the settings on the flow cytometer or by perform- emitting light at different wavelengths. Using three different ing a mathematical correction either before or after the data fluorochromes allows detection of three different antigens are collected. This process is called compensation. on the cell. Excitation of additional fluorochromes to detect 998 Chapter 40 Table 40.2 Example Fluorochromes Used in Flow Cytometry Fluorochrome Excitation Wavelength (nm) Detection Wavelength (nm) BD Horizon™ V450 405 450 BD Horizon™ V500 405 500 FITC 488 525 PE 488 575 PE-Cy7 488/561 774 PerCP-Cy5.5 488/675 690 Propidium iodide 488 620 APC 650 660 APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, R-phycoerythrin. PE-Cy7 is a conjugate of two fluorochromes: PE is excited at 488 nm, and the emitted signal (561 nm) excites the Cy7 component of the molecule. PerCP-Cy5.5 is a conjugate of two fluoro- chromes: PerCP is excited at 488 nm and the emitted signal (675 nm) excites the Cy5.5 component of the molecule. V450 and V500 (BD Biosciences, San Jose, CA). Although FDA-approved commercially available kits are available for some specific flow cytometric tests (e.g., Immunophenotyping by CD4 or CD34 enumeration), clinical laboratories are respon- Flow Cytometry sible for developing combinations of fluorochrome-labeled antibodies that meet other specific clinical needs (e.g., Immunophenotyping is the identification of antigens detection of mature B-cell lymphoid neoplasms). Assay using detection antibodies. Antibodies are utilized development includes identifying the properties of inter- because they bind specifically to antigens and can be est, selecting fluorochrome labeled reagents, titering all labeled with fluorochromes to provide a sensitive and reagents, and testing the assay’s performance such as pre- specific detection method. Most flow cytometric immu- cision, sensitivity, and specificity, before implementation. nophenotyping studies involve detecting cell surface anti- gens. Intracytoplasmic and intranuclear antigens can be Data Analysis detected following permeabilization of cell membranes with detergent and/or alcohol. Detection antibodies can Flow cytometry generates a large amount of data, often be either polyclonal or monoclonal. Polyclonal antibod- with at least six parameters measured for each of tens of ies are made by injecting antigen into animals (e.g., rab- thousands of particles/cells. Although semiautomated bit antihuman antibodies). The animal produces many computer analysis programs are available for some appli- antibodies that are directed against different portions cations (e.g., CD4 and CD34 enumeration), clinical labora- (epitopes) of the antigen. Therefore, the antigen can be tory professionals perform most clinical analysis manually. recognized even if some parts of it are abnormal. How- Analysis typically involves identification of the particles/ ever, polyclonal antibodies are often difficult to standard- cells of interest, including distinction from debris and per- ize and prone to nonspecific binding. haps dead cells, and characterization by evaluating all Monoclonal antibodies are directed against a single parameters measured. Analysis is performed by plotting the epitope of the antigen. They are produced in myeloma/ data on graphs that display the presence or absence of the tumor cell lines and therefore have high purity and parameter measured and the intensity of the fluorescencent reproducibility. Often several different monoclonal or signal detected ( Figure 40-2). Histograms display informa- polyclonal antibodies are available from different sup- tion from one parameter versus the number of particles/ pliers to detect a single antigen and can be given a unique cells detected with different intensity of emitted light. Two- company-specific designation. An international work- dimensional (2D) dot plots display information from two shop has been developed to systematically review anti- different parameters with each particle/cell represented as a bodies and group those recognizing the same antigen into single point on the graph. Many 2D dot plots are necessary a cluster of differentiation (CD designation). For exam- to view all the possible combinations of parameters evalu- ple, the commercially available antibodies Leu-4 and ated with a single 4–10 color tube. OKT3 recognize the same antigen and both are designated CD3. Many fluorochrome-labeled detection antibodies Checkpoint 40.2 are available commercially (Table 40-3). Combinations of A laboratory wants to identify cells that have two different anti- these antibodies are currently used in the clinical labora- gens. How many laser light sources are needed? tory to help identify cells and diseases1,2 (Appendix B Table). Flow Cytometry 999 Table 40.3 Antibodies Used for Immunophenotyping by Flow Cytometry CD Designation Normal Distribution Use CD1a Immature T cell Lymphoblastic leukemia/lymphoma CD2 T cell, NK cell Lineage of lymphoma or leukemia CD3 T cell Lineage of lymphoma or leukemia CD4 T cell Lineage of lymphoma or leukemia CD5 T cell Lineage of lymphoma/leukemia or aberrant expression on B cell SLL/CLL and mantle cell lymphoma CD7 T cell, NK cell Lineage of lymphoma or leukemia CD8 T cell Lineage of lymphoma or leukemia CD11c Monocytes, lymphoid cells Hairy cell leukemia: bright + ; SLL /CLL: dim + CD11b Neutrophils Myelodysplastic syndrome CD13 Monocytes Lineage of leukemia Myeloid cells Myelodysplastic syndrome CD14 Monocytes Lineage of leukemia CD15 Monocytes Lineage of leukemia Myeloid cells CD16 NK, NK-like T cells Large granular lymphocyte leukemia Granulocytes Myelodysplastic syndrome CD19 B cell Lineage of lymphoma or leukemia CD20 B cell Lineage of lymphoma or leukemia CD22 B cell Lineage of lymphoma or leukemia, SLL/CLL: dim + ; PLL: bright + CD23 B cell SLL/CLL + , mantle cel l - CD25 Many cell types Hairy cell leukemia bright + CD33 Monocytes Lineage of leukemia Myeloid cells CD34 Stem cells, progenitor cells Stem cells for transplantation, acute leukemia CD38 Plasma cells, some lymphocytes, monocytes, myeloid cells Plasma cell neoplasms Prognostic marker in CLL CD42 Megakaryocytes Acute megakaryocytic leukemia CD45 All leukocytes Lineage of malignancy Gating CD55 GPI-anchored protein Paroxysmal nocturnal hemoglobinuria CD56 NK, NK-like T cells Large granular lymphocyte leukemia CD57 NK, NK-like T cells Large granular lymphocyte leukemia CD59 GPI-anchored protein Paroxysmal nocturnal hemoglobinuria CD61 Megakaryocytes Acute megakaryocytic leukemia CD79a B cells (blasts to plasma cells) Lineage of lymphoma or leukemia CD103 Subset of intramucosal T cells Hairy cell leukemia Enteropathy associated T-cell lymphoma Glycophorin A Erythroid True erythroleukemia Myeloperoxidase Myeloid cells Lineage of leukemia Kappa B cell Maturity, clonality, SLL/CLL dim Lambda B cell Maturity, clonality, SLL/CLL dim CLL, chronic lymphocytic leukemia; SLL, small lymphocytic lymphoma; PLL, prolymphocytic leukemia; dim, weak intensity of emitted fluorescence; bright, strong intensity of emitted fluo- rescence; NK, natural killer; GPI, glycosyl phosphatidylinositol. 1000 Chapter 40 Checkpoint 40.3 Isolation of Cells by Gating A cell population is positive with both Leu1 and T1 monoclonal Immunophenotyping requires isolation of the cells of inter- antibodies. As a result, the cell is classified as CD5 positive. est (e.g., lymphocytes from monocytes and granulocytes). Explain. Cells can be separated during data analysis by placing an electronic gate around those with the same light-scattering or fluorescence properties (gating). For example, lympho- Specimen Requirements and cytes can usually be separated from neutrophils by their Preparation for Immunophenotyping location on the forward versus side light scatter dot plot (Figure 40-2). Fluorescence dot plots can then be set up to Immunophenotyping by flow cytometry requires a display only information obtained from cells falling within suspension of individual live cells (Table 40-4). Anti- the chosen gate. If the gate is too wide, many cell types are coagulated blood or bone marrow aspirate, body fluid included, and identifying the phenotype of the cells of inter- specimens, and fine needle aspiration samples are ideal est becomes difficult. If the gate is too narrow, some cells of for immunophenotyping by flow cytometry because they interest can be excluded. already contain cells in suspension. Leukocytes can be The forward versus side light scatter dot plot unfortu- isolated from these samples either by erythrocyte lysis nately is not capable of separating all the cells of interest. or

density gradient centrifugation. Lysis methods are For example, blasts and lymphocytes often appear in the strongly recommended because leukocytes are retained same region on the forward versus side light scatter dot in their original proportions without the risk of losing cell plot. Therefore, a different gating strategy could be required subtypes. Hematopoietic and lymphoid cells can also be for analyzing acute leukemia. Blasts often have dim CD45 isolated from fresh tissue biopsy specimens by manual expression, whereas lymphocytes have bright CD45 stain- disaggregation. ing. Therefore, blasts and lymphocytes can be distinguished Once a suspension of leukocytes without intact erythro- on a dot plot displaying CD45 intensity versus side angle cytes has been prepared, the sample is stained using fluoro- light scatter (Figure 40-2). Fluorescence staining can be used chrome-labeled antibodies. In some studies, the leukocytes for gating only if the chosen antibody (CD45) is present in are stained before the erythrocytes are lysed. The labeled each analysis tube. sample is aspirated into the flow cytometer, and the amount of scattered light and the intensity of each fluorescent sig- nal are recorded for every cell analyzed. The acquired data CASE STUDY (continued from page 995) are then displayed graphically on dot plots (Figure 40-2). Flow cytometry immunophenotyping was Populations of cells that have similar staining properties requested on Andrew. and can be highlighted with a different color are identified 1. What is the optimal specimen? (Figure 40-2). Alternatively, plots are divided into quad- rants, indicating cells labeled with one fluorochrome, the 2. Which cells are of interest and should be included other fluorochrome, or both fluorochromes. in the gate? The intensity of light emitted is determined by com- 3. What are the typical forward and side scatter parison with the known range of intensities for that anti- properties of the cells of interest? body/fluorochrome combination and divided roughly into three portions: dim or weak intensity, intermediate intensity, and bright or strong intensity (Figure 40-3). The intensity of emitted light is related to the density of Checkpoint 40.4 antigens. Explain why lymphocytes and neutrophils can be separated on a forward versus side light scatter dot plot. Table 40.4 Specimen Requirements for Immunophenotyping by Flow Cytometry Fluorescence Activated Cell Sorting Specimen Type Requirements Storage (FACS) Peripheral blood 5 mL in EDTA or heparin Less than 24 hrs, RT Bone marrow 1 mL in EDTA or heparin Less than 24 hrs, RT Fluorescence activated cell sorting (FACS) is a method for Fluids as much as possible Less than 24 hrs, sorting a heterogeneous mixture of biological cells into 4°C separate containers, one cell at a time, based on the specific Tissue Preferably 7 1 2 cm3 Less than 24 hrs, light scatter and fluorescence characteristics of each cell. 4°C Sorting is achieved by droplet formation. The basic com- RT, room temperature; EDTA, ethylenediaminetetraacetic acid. ponents of any sorter include a droplet generator, a droplet Flow Cytometry 1001 Figure 40.3 Flow cytometry histograms from analysis of peripheral blood. Cells with low side light scatter were gated and staining with a number of fluorochrome-labeled antibodies were displayed. The population of interest (red) demonstrates staining for CD19, CD20 (weak intensity), CD5, and l light chain, but lacks staining for CD10. The plots also display T cells (green) and a few polytypic B cells (blue). Histograms generated using FACS DIVA software, BD Biosciences, San Jose, CA. charging and deflecting system, a collection component and identifying a subtype of malignancy. Neoplasms are made an electronic circuitry for coordinating the timing and gen- up of a population of identical cells (clone). A clone of B eration of droplet-charging pulses. The cell suspension is lymphocytes can be recognized by uniform expression of entrained in the center of a narrow, rapidly flowing stream one immunoglobulin light chain (k or l light chain class of liquid. The flow is arranged so that there is a large sepa- restriction). In contrast, a reactive population of B lympho- ration between cells relative to their diameter. A vibrating cytes contains a mixture of cells, each with expression of mechanism causes the stream of cells to break into indi- either k or l immunoglobulin light chain. vidual droplets. The system is adjusted so that there is a low Clonality can also be detected by identifying a popu- probability of more than one cell per droplet. Just before the lation of cells that display an abnormal phenotype (aber- stream breaks into droplets, the flow passes through a fluo- rant expression of a lymphoid antigen). For example, rescence measuring station where the fluorescent character mature T lymphocytes normally express CD3, CD2, CD5, of each cell of interest is measured. and CD7. A clone of malignant T lymphocytes could lack detectable CD5 and/or CD7. Another example of Diagnosis and Classification of an abnormal phenotype is acquisition of an antigen that Mature Lymphoid Neoplasms is not normally present. For example, the T-cell marker CD5 is aberrantly expressed on a subset of B-cell malig- Flow cytometric immunophenotyping can be used to iden- nancies (small lymphocytic lymphoma/chronic lympho- tify cell lineage (B, T, or natural killer [NK] cell) and the cytic leukemia and mantle cell lymphoma) (Figure 40-3). presence of an abnormal population of lymphocytes. This Therefore, the presence of CD5+ B lymphocytes can assist information can assist in detecting malignant cells and in identifying a lymphoid malignancy and diagnosing a 1002 Chapter 40 specific subtype. Panels of antibodies are usually selected lymphoma, and Burkitt lymphoma. Therefore, it is impor- to separate the common subtypes of lymphoproliferative tant to interpret immunophenotyping data in conjunction disorders (Table 40-5). In addition, flow cytometric immu- with morphology. nophenotyping can be used to demonstrate the presence of antigens for directed therapy (for example, CD20 for Diagnosis and Classification of Acute anti-CD20 monoclonal antibody therapy), identify prog- Leukemia nostic markers such as ZAP-70 expression in chronic lym- phocytic leukemia (CLL), and identify a small population Using the World Health Organization (WHO) classifica- of abnormal cells following treatment (minimal residual tion, a diagnosis of acute leukemia requires manual dif- disease). ferential counting to identify more than 20% blasts in the peripheral blood or bone marrow (Chapter 23). Once a diagnosis of acute leukemia has been established, flow CASE STUDY (continued from page 1000) cytometry can be used to assist in identifying its subtype.3 Flow cytometry revealed the results displayed in Currently, most treatment protocols require the distinc- Figure 40-3. tion of acute lymphoblastic leukemia/lymphoma (ALL) and 4. What is the phenotype? acute myeloid leukemia (AML). The recognition of Auer rods or the presence of staining with cytochemical stains 5. Which features indicate clonality? allows the identification of AML (Chapters 23 and 26). 6. What is the diagnosis? Until the advent of immunophenotyping, all cases of acute leukemia lacking these features were assumed to be ALL. However, immunophenotyping studies have revealed that this assumption is erroneous. Some AML Although flow cytometric immunophenotyping usu- cases lack cytochemical staining and are recognized only ally provides useful information, potential pitfalls can lead by immunophenotyping (AML not otherwise specified to an incorrect diagnosis. A malignant B-cell lymphopro- [NOS] with minimal differentiation). In addition to the liferative disorder is easily overlooked if it lacks surface accurate separation of ALL from AML, flow cytometric immunoglobulin. Some lymphoid malignancies frequently phenotyping can be used to assist in identifying subtypes lack diagnostic surface antigens, including plasma cell of leukemia that have a different prognosis (e.g., T lym- neoplasms, HIV-associated lymphoma, and mediastinal phoblastic leukemia/lymphoma has a worse prognosis lymphoma. T-cell lymphoma can be difficult to detect in general than B lymphoblastic leukemia/lymphoma) because many cases do not demonstrate an abnormal or require an alternate therapeutic regimen (e.g., acute phenotype. Hodgkin lymphoma is difficult to detect by promyelocytic leukemia). flow cytometry for several reasons: the neoplastic cells are rare and lack many cell surface lymphoid antigens, and a ACUTE LYMPHOBLASTIC LEUKEMIA/LYMPHOMA single cell suspension is often difficult to produce because Immunophenotyping is essential for the diagnosis of of the presence of fibrosis. Another potential pitfall in ALL, and separation of T- and B-ALL. B-ALL makes up flow immunophenotyping is the presence of an abnor- 80% of childhood ALL. B-cell lineage is usually defined mal phenotype that is not specific for a single subtype by the presence of surface CD19 and/or cytoplasmic of lymphoid malignancy. For example, CD10 expression CD22 expression. B-cell differentiation in the normal can be seen in follicular lymphoma, diffuse large B-cell human bone marrow is accompanied by characteristic Table 40.5 Characteristic Immunophenotype of Lymphoproliferative Disorders Diagnosis CD19 CD5 CD23 CD11c CD22 CD25 sIg Chronic lymphocytic leukemia + + + +/- +w +/- +w/- Prolymphocytic leukemia + -/+ - - + +/- + Hairy cell leukemia + - -/+ +br +br + + Small lymphocytic lymphoma + + + +/- +w +/- +w/- Mantle cell lymphoma + + - - + - + Follicular lymphoma + - +/- - + - + br, bright or strong fluorescence intensity; w, weak or dim fluorescence intensity; + , antigen present; - , antigen absent; +/- variable expression (often present); -/+ , variable expression (often absent). Flow Cytometry 1003 patterns of antigen expression on consecutive stages of CD13) is found in 30–50% of cases of B-ALL. However, B-cell precursors. Immaturity of the cells is recognized the phenotype of leukemic blasts often resembles a nor- by expression of CD10 and terminal deoxynucleotidyl mal stage of maturation (e.g., precursor B cells). There- transferase (TdT), a DNA polymerase. However, this phe- fore, the presence of genetic abnormalities is a better notype overlaps with a significant proportion of normal predictor of prognosis and is used in the current WHO bone marrow precursors (hematogones) (Chapter 38). classification scheme. Therefore, recognition of leukemic blasts requires iden- tifying cells with an abnormal phenotype (Figure 40-4). During normal maturation in the bone marrow, B cells CASE STUDY (continued from page 1002) gain and lose antigens in synchrony until they acquire a mature B-cell phenotype (Chapter 8). In contrast, leuke- 7. Which of the flow cytometry results presented mic blasts demonstrate asynchronous expression of anti- in this case indicate that this is a malignancy of gens (e.g., TdT expression without CD34). In addition, mature lymphocytes (CLL), not ALL? aberrant expression of a myeloid antigen (e.g., CD33 or Figure 40.4 Aberrant antigen expression in B-ALL in comparison with normal precursor B cells. The top two plots display the typical phenotype of normal maturing bone marrow B-cell precursors (hematogones), including a population of most immature cells with expression of CD34 and CD10 but lacking CD20 (green), a population of most mature cells that are CD34- , CD10- , and CD20+ (purple) and an intermediate population with gain of CD20 expression (blue). The lower two plots display an example of B-LBL. The leukemic blasts (green) demonstrate CD34 expression and lack of CD20 staining, similar to the most immature normal B cells but demonstrate abnormal, partial CD10 expression. A few mature CD20+ , CD10- , CD34- lymphoid cells are also present. 1004 Chapter 40 T-ALL accounts for 15–20% of lymphoblastic neoplasms with a leukemic presentation, but most cases of lympho- Table 40.6 Flow Cytometric Requirements for Assigning Blast Lineage blastic lymphoma. Unlike B-cell precursors, T-cell precur- sors originate in the thymus and not in the bone marrow. Lineage Requirement T lymphoblasts often express CD3 only in the cytoplasm, Myeloid Myeloperoxidase or monocytic differentiation (CD11c, not on the cell surface and they often show similarities to CD14, CD64) cortical thymic T cells, expressing both CD4 and CD8 or T cell CD3 cytoplasmic (epsilon chain) or CD3 surface lacking both CD4 and CD8. TdT is present in more than 90% B cell Strong CD19 along with at least one other B-cell marker of cases, and CD10 can be present. This is in contrast with (CD79a, CD22 cytoplasmic, or CD10) or weak CD19 along with at least two B-cell markers (CD79a, CD22 the normal bone marrow, where there are no TdT-positive cytoplasmic, or CD10) T cells expressing cytoplasmic CD3. Most cases of T-ALL express the T-cell antigens CD1, CD2, CD3, CD5, and CD7. phenotype acute leukemia can be divided into subsets by The phenotype of T-ALL can be abnormal with loss of one the lineages represented (e.g., B/myeloid, T/myeloid). or more of these T-cell antigens. Myeloid markers are aber- However, the phenotype of the blasts and whether the anti- rantly expressed in 25–30% of

pre-T-ALL cases. In general, gens are expressed on the same or separate cells is of less the prognosis for patients with T-ALL is worse than for importance in determining the prognosis than the presence those with precursor B-ALL. of associated genetic abnormalities, such as the BCR-ABL1 or MLL gene rearrangements. The designation acute undif- Checkpoint 40.5 ferentiated leukemia requires lack of demonstrable lineage- Why is it important to do immunophenotyping in a case of ALL/ restricted antigens after comprehensive evaluation. For this LBL? evaluation, it is important to include only antigens that have specificity for a lineage. For example, the presence of CD7 ACUTE MYELOID LEUKEMIA alone does not distinguish T-ALL from AML because CD7 is The WHO classification scheme recognizes several subtypes normally present on immature T cells and is aberrantly of AML (Chapters 23, 26). Most subtypes can be identified expressed in approximately 10% of AML. In cases of leu- using morphology and/or cytogenetics. Immunophenotyp- kemia with a phenotype that does not indicate the lineage, ing is essential for the diagnosis of AML NOS, minimally other studies, including morphologic analysis, cytochemical differentiated, and is often used for the diagnosis of acute stains, molecular diagnostic studies, and cytogenetic stud- megakaryocytic leukemia. AML NOS, minimally differenti- ies, may assist. ated, is diagnosed when Auer rods are absent, cytochemical stains are negative, and there is evidence of nonlympho- Diagnosis of Myelodysplastic cytic differentiation by the expression of myeloid antigens. Syndrome (MDS) The diagnosis of acute megakaryocytic leukemia relies on Flow cytometry has also been used to identify abnormalities identification of megakaryocytic differentiation by immu- in the phenotype of myeloid populations in myelodysplas- nophenotyping (CD41, CD61) or electron microscopy that tic syndromes. A detailed knowledge of normal immu- assesses platelet peroxidase positivity with the platelet per- nophenotypes of the blasts, maturing granulocytes, and oxidase (PPO) stain. Although acute promyelocytic leuke- monocytes is necessary for evaluation of aberrant features mia characteristically lacks staining for CD34 and HLA-DR, suggestive of MDS. Granulocyte maturation is accompa- a negative CD34 and HLA-DR phenotype is not specific. nied by a continuous variation of CD13, CD11b, and CD16 Therefore, morphology in combination with cytogenetic expression as the myeloid blasts mature into promyelocytes, studies still remains the gold standard for diagnosing AML. myelocytes, and segmented neutrophils. In contrast, MDS, In addition to diagnosis and classification, flow cytometry shows aberrant maturation patterns using CD13 and CD16 can be used to demonstrate the presence of the antigens for and/or CD13 and CD11b antibody combinations. Another directed therapy, for example the presence of CD33 antigen consistently reported aberration of the maturing myeloid for anti-CD33 monoclonal antibody therapy. cells in MDS is a lower side scatter that is due to lower than ACUTE LEUKEMIA OF AMBIGUOUS LINEAGE normal granularity. Abnormal expression of lymphoid Some cases of acute leukemia do not fit neatly into the cat- lineage markers such as CD2, CD7, and CD56 is also fre- egories of ALL and AML. It can be impossible to assign quently observed in MDS. Unfortunately, none of the vari- lineage because blasts express antigens from more than ous abnormalities observed in MDS are specific enough to one lineage (mixed phenotype acute leukemia) or express reach a diagnosis, and the flow cytometric results should very few antigens (acute undifferentiated leukemia). The not be interpreted alone but used as a part of an integrated WHO classification defines the requirements for assigning diagnostic approach in combination with clinical, morpho- each lineage (Table 40-6). By using these criteria, mixed logical, and cytogenetic findings. Flow Cytometry 1005 Detection of Minimal Residual results across flow cytometric laboratories, large collabora- Disease (MRD) by Flow Cytometry tive clinical trials would not be possible. MRD detection in PCM is challenging because normal plasma cells constitute Accurate quantification of post-treatment residual disease a normal component of bone marrow cells. Recently, Con- burden is prognostically relevant in hematologic malignan- sensus Guidelines on Plasma Cell Myeloma MRD Analysis cies, including ALL, AML, CLL and plasma cell myeloma. and Reporting have been proposed to overcome the hetero- In general, achievement of complete remission (CR) after geneity between flow cytometric laboratories.5 The recent treatment is associated with superior progression-free and guidelines support the use of five initial gating parameters, overall survival. Patients who achieve a remission may have including CD38, CD138, CD45, forward, and side scatter for low-level disease that can be demonstrated in the blood or an accurate identification of the plasma cells, and recom- bone marrow only with a highly sensitive technique, such mends reporting aberrant immunophenotypic findings as as multicolor flow cytometry or PCR. These residual cells being reduced, normal, or increased when compared to nor- ultimately are responsible for clinical relapse. MRD tech- mal plasma cells. The Consensus Guidelines recommend a niques need to be sensitive (10-4 to 10-6), broadly applica- lower limit of detection of 0.001%, which requires at least ble, accurate, reliable, fast, and affordable. Multicolor flow 395 * 106 bone marrow cells measured in the analysis. cytometry can detect MRD to a sensitivity of 1:1,000,000 AML is a highly heterogenous disease from both the 110-62. In order to achieve the desired level of sensitivity, genetic and immunophenotypic point of view. The imple- 500,000 to 10,000,000 events should be collected in an MRD mentation of FC MRD assessment in AML has lagged analysis. The presence or absence of MRD at different time behind ALL and still today is mainly restricted to special- points during therapy can help to predict the outcome and ized reference laboratories. One possible strategy in AML guides further treatment. is based on the identification of “different-from-normal” MRD monitoring was first introduced for monitoring immunophenotypes, an approach similar to ALL. This of pediatric ALL, and by today, it has become a routine might be particularly useful when the original diagnostic clinical practice for both pediatric and adult ALL patients. immunophenotype is not available for comparison. MRD MRD in ALL has been proven to be the strongest prognostic detection in AML still suffers from lack of comparability of factor and allows for risk group assignments and selection results between different laboratories. It is likely that stron- of different treatment arms. Accurate and sensitive detec- ger standardization of FC MRD analysis will evolve from tion of rare ALL cells requires highly specific markers for networked centralized laboratories.6 discrimination between ALL cells and normal leukocytes. For example, expanded normal B-cell precursors (hemato- CASE STUDY (continued from page 1003) gones) in the bone marrow may interfere with detection of B-ALL because they show some immunophenotypic simi- 8. How can flow cytometry be used to assess larities to B-ALL. An important concept of multicolor flow Andrew’s response to therapy? cytometry (FC) MRD detection is the identification of aber- 9. What is the significance of minimal residual dis- rant immunophenotypes that do not overlap with normal ease (MRD) after completion of chemotherapy? leukocytes (“empty spaces” on selective informative dot- plots). A disadvantage of FC MRD is that immunostaining protocols, antibody panels and gating strategies may differ significantly between centers and represent highly subjec- Diagnosis and Surveillance of tive, expert procedures. Immunodeficiency Disorders In CLL, the gold standard of MRD detection is PCR to the immunoglobulin gene of the B-CLL cell. Molecular Immunophenotyping by flow cytometry can be used to analysis, however is not always feasible, and a four-color FC identify a deficiency of a leukocyte subset. The most fre- approach provides a rapid turnaround time and a sensitiv- quent clinical application of flow cytometry is monitoring ity comparable to that of the PCR. Peripheral blood samples the immunodeficiency acquired following HIV virus infec- appear equally or more sensitive than bone marrow sam- tion. Less frequently, immunophenotyping is used to detect ples in more than 90% of cases. Though different antibody inherited immunodeficiencies. combinations may be used with success, a large study advo- DIAGNOSIS AND SURVEILLANCE OF HIV cated the combination of CD20/CD38/CD19/CD5, CD22/ The HIV virus uses the CD4 antigen to infect T lympho- CD81/CD19/CD5, and CD43/CD79b/CD19/CD5 based on cytes and monocytes. After viral fusion, internalization, the lowest interlab variability and false-positive rate.4 replication, and dissemination, CD4+ cells are destroyed. In plasma cell myeloma (PCM), MRD evaluation The resultant decrease in CD4+ cells leads to immunode- became an essential component of disease management. ficiency. The absolute number and percentage of CD4+ T However, without the possibility of direct comparison of lymphocytes present in the peripheral blood can be used 1006 Chapter 40 to monitor the immune system. HIV surveillance is used to A precise and accurate CD4 count is essential. Results predict the disease course, decide when to start prophylactic from a patient are often compared with previous results and therapy for opportunistic infections, and determine when published diagnostic and therapeutic levels. Therefore, the to commence antiretroviral therapy. An absolute number procedure used for quantitative flow cytometry should of CD4+ T lymphocytes 200/mcL in the peripheral blood attempt to eliminate preanalytical and analytical variables. is also used to diagnose AIDS in HIV-infected individuals. Preanalytical variables include biologic variability (age, The CD4 count is determined by staining with fluores- diurnal rhythm, and medications), specimen collection, and cent-tagged antibodies against CD3 (found on T cells but storage. Analytical variables include sample preparation not monocytes) and CD4 followed by whole blood RBC and errors associated with flow cytometers and hematology lysis. The percentage of CD4+ lymphocytes is determined analyzers. The CDC publishes guidelines for standardiza- by flow cytometry. Gating is critical to avoid exclusion of tion of CD4 determination. Two levels of stabilized quality lymphocytes and contamination by cells other than lympho- control material must be run with each batch of CD4 assays. cytes such as CD4+ monocytes. The absolute CD4 count can In addition, proficiency testing for professionals is available be determined directly by flow cytometry (single platform) through College of American Pathologists (CAP) surveys. by comparing the number of CD4+ cells identified per unit The HIV viral load is another monitor of HIV infection. volume analyzed with an absolute number of highly fluores- Viral load testing often measures viral RNA in plasma. The cent beads added to the analysis tube (Figure 40-5). Alterna- CD4 count and the HIV viral load often provide complemen- tively, the CD4 count can be calculated from the percentage tary information. The CD4 count is the best indicator of the of CD4+ lymphocytes and the absolute number of lympho- balance between immune cell production and destruction cytes determined by a hematology analyzer (dual platform): and therefore indicates the risk of opportunistic infection or secondary malignancy. The HIV viral load indicates the bur- Absolute CD4 count = Absolute lymphocyte count * den of disease and therefore can be used to monitor response ,CD4+ Lymphocytes/100 to antiviral therapy and the acquisition of drug resistance. Absolute lymphocyte count = WBC count 1*109/L2 * ,Lymphocytes from WBC differential/100 Checkpoint 40.6 A patient has 10% CD4+ lymphocytes, a WBC count of 5 * 103/mcL, and 30% lymphocytes. What is the absolute CD4 count? Is this count compatible with a diagnosis of AIDS in an HIV-infected individual? FINDINGS IN PRIMARY IMMUNODEFICIENCY DISORDERS Primary immune deficiencies are rare disorders that reflect abnormalities in the development, maturation, or per- formance of different cell subsets of the immune system. Affected individuals are susceptible to repeated infections, allergies, autoimmune diseases, or malignancies. Over 200 primary immunodeficiency diseases have been clinically identified. Flow cytometry can identify various abnormali- ties that help define immunodeficiency disorders, such as the relative representation of a specific cell subset, the detection of a specific antigen, or demonstration of func- tional abnormalities. Flow cytometric findings in some of the most common primary immune deficiency disorders are summarized in Table 40-7. Figure 40.5 Flow Cytometry in Systemic CD4 enumeration. CD4+ lymphocytes are isolated by gating on cells with bright intensity CD45 and low side light scatter Autoimmune Disease (red, top left dot plot), bright intensity CD3 (blue, lower left dot plot), Flow cytometry can be helpful in the clinical monitoring of and CD4 (greenish-blue, lower right dot plot). The absolute CD4 count is determined by comparing the number of CD4+ events with certain autoimmune diseases, such as rheumatoid arthri- the number of beads counted (top right dot plot). Analysis performed tis (RA) and systemic lupus erythematosis (SLE). B-cell using BD Multitest™, BD Biosciences, San Jose, CA. depletion therapy using the anti-CD20 antibody rituximab Flow Cytometry 1007 Table 40.7 Flow Cytometry

in the Diagnosis of Major Primary Immune Deficiencies Primary Immune Deficiency Type Flow Cytometric Findings X-linked congenital agammaglobulinemia Absence of CD20+ and/or CD19+ B lymphocytes Absence of intracellular Bruton tyrosine kinase (BTK) in monocytes and platelets Severe combined immune deficiency (SCID) Wide range of defects: Adenosine deaminase deficiency: lack of T, B, NK cells Janus kinase 3 deficiency: lack of T and NK cells RAG1/2 deficiency: lack of T and B cells CD3 d or e deficiency: lack of T cells Hyper IgM syndromes Decrease of CD40 and/or CD40 ligand (CD154) expression on CD4+ T cells Wiscott Aldrich syndrome (WAS) Decreased expression of WAS protein Decrease of CD8+ T cells and increase of NK cells Chronic granulomatous disease Decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity following granulocyte activation Leukocyte adhesion deficiency type 1 (LAD1) Decreased or absent CD11a, CD11b, CD11c, and CD18 on granulocytes Autoimmune lymphoproliferative syndrome Increased levels of CD4- CD8- (double negative) T cells Low memory B cells 1CD20+ CD27+2 Familial hemophagocytic lymphohistiocytosis Defects of expression of perforin, syntaxin-11 has been effective in treating RA and other autoimmune progress has been made in standardizing CD34 flow cyto- diseases, underlying the important pathogenic role of B metric assay procedures and analysis protocols (Table 40-8, lymphocytes in these disorders. High sensitivity FC can be Table 40-9).8,9 useful in monitoring the return of B cells in the peripheral blood after B-cell depletion. Furthermore, FC can differenti- Paroxysmal Nocturnal ate between different subsets of memory B cells, including naïve 1IgD+CD27-2, unswitched memory 1IgD+ , CD27+2, Hemoglobinuria (PNH) switched memory 1IgD- CD27+2 (includes plasmablasts), PNH is a chronic hemolytic disorder caused by an acquired and double negative memory cells 1IgD-CD27-2. Recent mutation of the PIG-A gene (Chapter 17), which encodes studies on RA indicate an important role of the IgD+CD27+ an enzyme critical in the synthesis of the glycosylphos- memory cells in therapy resistance and in overall disease phatidylinositol (GPI) anchor that links many proteins to progression: patients with lower numbers of IgD memory the cell membrane. Flow cytometry can be used to detect cells at the early phase of post-rituximab repopulation had the presence of GPI-anchored membrane proteins. In addi- a much more favorable clinical response.7 tion, some flow cytometric assays assess the ability of white blood cells to bind a fluorescent-labeled modified toxin CD34 Enumeration (FLAER) that attaches through GPI anchors. Flow cytomet- ric immunophenotyping of erythrocytes and leukocytes CD34 enumeration is used to support bone marrow and is a more sensitive and specific test for diagnosing PNH peripheral blood stem cell transplantation (Chapter 29). than the sucrose hemolysis test or Ham test. Erythrocyte CD34 is an antigen restricted to multipotential hematopoi- immunophenotyping is easier to interpret than analysis of etic stem cells and early progenitors of all lineages. Hema- neutrophils but can give a false negative result if hemolysis topoietic stem cells are responsible for reconstituting bone is active or a transfusion was recently given. Cells with a marrow function following transplantation. CD34 enumera- PNH-type phenotype have also been identified in patients tion is used to determine whether the product collected for transplantation has enough cells for rapid and long-term engraftment. CD34+ cells can be collected by either bone Table 40.8 Procedural Requirements for Quantitative marrow aspiration or peripheral blood apheresis. Hemato- Flow Cytometry poietic progenitor cells are often mobilized into the periph- Standardization of procedure eral blood using a combination of cytotoxic drugs and • Patient preparation growth factors. CD34 enumeration can also be performed • Specimen handling on the peripheral blood to determine the optimal time for • Specimen preparation peripheral blood stem cell collection. CD34 determination is • Analysis (gating accuracy and purity and precision) challenging because it needs to be fast, accurate, and precise Quality control Proficiency testing even at low numbers (0.1% of cells 5 cells /mcL). Significant Documentation of personnel competency 1008 Chapter 40 permeable to allow access of the fluorescent dye to the DNA) Table 40.9 Recommendations for CD34 Enumeration and stained with a fluorochrome that binds to DNA. If the Use bright (e.g., PE) fluorochrome conjugates of class II or III monoclonal fluorescent dye (e.g., propidium iodide) binds to both DNA antibodies that detect all glycoforms of CD34. and RNA, RNA is removed by digestion. The amount of fluo- Use a vital nuclei acid dye to exclude platelets, unlysed RBCs, and debris or 7-amino actinomycin (7-AAD) to exclude dead cells during acquisition. rochrome bound is proportional to the amount of DNA. The Include CD45 to assist in detection of hematopoietic progenitor cells. DNA content varies during cell division (proliferation) and is Use a Boolean gating strategy to resolve the CD34+ HPC from abnormal in cells with numerical chromosome abnormalities irrelevant cells. (aneuploid) (Figure 40-6). Computer software uses mathe- Include the identification of cells with low levels of CD45 expression and matical formulas to calculate the cells in each phase of the cell low side scatter and include CD34 bright and dim staining populations. Acquire sufficient events to generate at least 100 CD34+ cells to ensure cycle, calculate indexes, and identify abnormal populations. a 10% precision. Proliferation with aplastic anemia and some myelodysplastic syndromes Tumors with an increased number of dividing cells often and in very small numbers in normal individuals.10 have a worse prognosis but in some instances are susceptible to therapy directed at dividing cells. The DNA content of cells Hereditary Spherocytosis (HS) can be used to determine the proportion of dividing cells. During cell division and prior to mitosis, the DNA content Hereditary spherocytosis (HS), a heterogeneous group of increases (synthesis, or S phase) until there are two copies of inherited red blood cell membrane disorders, is the most the entire genome prior to mitosis 1G2/M@phase2 (Figure 40-6, common cause of inherited chronic hemolysis in Northern Chapter 2). The S phase fraction is the proportion of all cells Europe and North America. The basis of these disorders is 1G0/G1 + S + G2/M2 that are in the S phase. The prolifera- an inborn deficiency or dysfunction of one of the red blood tive index is the proportion of all cells that are in proliferative cytoskeleton proteins (spectrin, ankyrin, band 3, and /or pro- phases 1S + G2/M2 (Table 40-10). tein 4.2) whose role is to maintain the shape and elasticity of the red blood cells. There is often a family history of chronic Ploidy anemia. Patients present with splenomegaly, increased num- bers of spherocytes on the peripheral blood smear, increased All nondividing, normal human cells contain 46 chromo- mean corpuscular hemoglobin concentration (MCHC), and somes (two each of 22 autosomes, and the sex chromosomes, hemolysis. Although the osmotic fragility test is a useful either XX or XY) and are therefore referred to as being diploid. screening method based on the increased sensitivity of HS The presence of tumor cells containing an abnormal number red blood cells to an osmotic challenge compared to normal of chromosomes is often associated with a worse progno- red blood cells, it suffers from low sensitivity and low speci- sis. The DNA Index (DI) is the DNA content of tumor cells ficity. The flow cytometry-based eosin-5-maleimide (EMA) relative to a diploid population of cells. It is calculated as binding dye test is based on the binding of EMA to band the DNA content of cells in the tumor in the G0/G1 phase 3, Rh blood group proteins, Rh glycoprotein, and CD47 on membrane molecules in red blood cells and has as a higher specificity than the osmotic fragility test. In hereditary mem- brane disorders, a reduced fluorescence intensity of EMA is observed. Another advantage of the EMA binding dye test is the ability to differentiate hereditary pyropoikilocy- tosis (HPP), hereditary elliptocytosis (HE), and HS based on a graded reduction of fluorescence intensity (HPP the lowest 6 HS 6 HE). Flow cytometry laboratories must establish their own cut-off values for distinguishing between normal and HS blood cells 1mean+/- 2SD2.11 DNA Analysis DNA analysis is usually performed on solid tumors to pro- vide prognostic information. Either fresh frozen tissue or a thick section of fixed, paraffin-embedded tissue is processed Figure 40.6 Fluorescence intensity of DNA-bound dye during to produce individual cells or nuclei. The suspension is phases of the cell cycle. Mathematical programs are used to distinguish permeabilized (the cell and/or nuclear membrane is made three areas under the curve (cell-cycle phases G0/G1, S, and G2/M). Flow Cytometry 1009 Table 40.10 Calculations for DNA Analysis by Flow Clinical Applications of DNA Cytometry Analysis S phase fraction SPF = 100 * S/1G0/G1 + S + G2/M2 The clinical utility of DNA analysis remains controversial. Proliferation index PI = 100 * 1S + G2/M2/1G0/G1 + S + G2/M2 One of the most extensively studied tumors is breast carci- DNA index DNA index = DNA content G0/G1 sample/DNA noma. A correlation exists between increased S phase frac- content G0/G1 diploid control tion and other markers of a poor prognosis (large breast tumor, the presence of metastatic disease in axillary lymph of the cell cycle relative to the DNA content of G0/G1 cells nodes, high histologic grade, and absence of steroid recep- in a diploid control. For example, diploid cells have a DI of tors). However, the main value of DNA analysis in breast 1.0. Tetraploid cells (four copies of all chromosomes) have a carcinoma can be the identification of patients who have a DI of 2.0, and cells with only one copy of each chromosome low S phase fraction and might not require chemotherapy (haploid) have a DI of 0.5. Aneuploidy is the presence of cells in addition to surgical excision. Interlaboratory variability with an abnormal DNA content that is not a multiple of the in methodology and interpretation of results has delayed DNA content of haploid cells, for example, a DI of 1.3. the routine use of this technique.12 Summary Flow cytometry is a technique that involves the analysis fluorochrome-labeled detection antibodies to identify cel- of single particles for their ability to scatter light and fluo- lular antigens. Its primary use is to classify and subtype resce. This technology is used by stand-alone instruments leukemia and lymphoma. Minimal residual disease detec- (flow cytometers) and has also been incorporated into tion in hematologic malignancies is an evolving field of other instruments such as hematology cell analyzers. Flow application with an important role in selection of therapies cytometry is used in the clinical laboratory for immunophe- and prediction of outcomes. Reticulocyte and DNA analy- notyping, reticulocyte counting (discussed in Chapters 11 sis utilize fluorescent dyes that bind to nucleotides. DNA and 39), and DNA analysis. Immunophenotyping uses analysis is used to analyze solid tumors. Review Questions Level I 3. Which of the following statements explains the use of fluorochromes in immunophenotyping by clinical 1. Which of the following is a (are) component(s) of a flow cytometry? (Objectives 3, 5) flow cytometer? (Objective 1) a. All fluorochromes emit light at the same wavelength. a. Flow chamber b. Fluorochromes bind nonspecifically to leukocytes. b. Laser light source c. The wavelength of the emitted light is the same as c. Light detectors that of the incident light. d. All of the above d. Several fluorochromes can be excited by light of a single wavelength. 2. Which of the following properties of the cells analyzed by flow cytometry is related to forward 4. Which of the following properties is (are) used for angle light scatter? (Objectives 1, 7) gating in clinical flow cytometry? (Objective 1) a. Granularity a. Forward angle light scatter b. Nuclear complexity b. Side angle light scatter c. Size c. Intensity of CD45 staining d. Shape d. All of the above 1010 Chapter 40 5. Which of the following is a (are) clinical application(s) What is the phenotype of the majority of lymphocytes of flow cytometry? (Objective 2) represented in this diagram? (Objective 4) a. DNA quantitation a. CD19+ , CD5- b. Immunophenotyping b. CD19+ , CD5+ c. Leukemia classification c. CD19- , CD5- d. All of the above d. CD19- , CD5+ 6. Which of the following flow cytometry methods is Level II the most appropriate for diagnosing and classifying 1. A 45-year-old female presented with pancytopenia. chronic lymphocytic leukemia? (Objectives 4, 5, 7) Flow cytometry performed on the peripheral blood a. CD34 enumeration revealed the following results: b. DNA quantitation c. Immunophenotyping d. RNA quantitation 7. Which of

the following is detected during immuno- phenotyping by flow cytometry? (Objective 3) a. Antibodies using detection antigens Which is the predominant cell type in the gate? b. Antibodies using detection fluorochromes (Objective 9) c. Fluorochromes using detection antibodies a. Eosinophil d. Antigens using detection antibodies b. Lymphocyte 8. Which of the following specimens is most appropriate c. Monocyte for flow cytometry immunophenotyping? d. Neutrophil (Objective 6) 2. Further flow cytometric analysis performed on the a. Formalin-fixed bone marrow aspirate clot sample described in question 1 revealed the following b. Fresh lymph node biopsy results: c. Frozen bone marrow biopsy d. One-week-old (ethylenediaminetetraacetic) EDTA anticoagulated peripheral blood 9. A peripheral blood sample needs to be shipped to a reference lab for flow cytometry immunophenotyp- ing. Which of the following procedures is most appro- priate? (Objective 6) a. Overnight delivery at room temperature b. Overnight delivery at 4°C c. Regular mail service at room temperature d. Regular mail service at 4°C What is the phenotype of the cells present? (Objective 9) 10. An 80-year-old male was found to have lymphocyto- sis. Flow cytometry of the peripheral blood revealed a. CD19+ , CD5- , CD11+ , CD25+ the following results: b. CD19+ , CD5+ , CD11+ , CD25+ c. CD19+ , CD5- , CD11c- , CD25- d. CD19- , CD5- , CD11c- , CD25- 3. Which of the following phenotypes is indicated by the k and l light chain dot plot displayed in question 2? (Objective 9) a. Absence of surface immunoglobulin b. Monoclonal l immunoglobulin light chain Flow Cytometry 1011 c. Monoclonal k immunoglobulin light chain be difficult to detect by flow cytometry cell surface d. Polyclonal immunoglobulin light chains antigen studies? (Objective 2) a. Hodgkin’s disease 4. Which of the antigens expressed in question 2 indi- cates a mature B lymphocyte phenotype? (Objective 1) b. T-cell lymphoma c. Plasma cell neoplasms a. CD5 d. All of the above b. CD11c c. CD19 8. A patient presented with circulating blasts. Flow d. Surface immunoglobulin cytometry immunophenotyping performed on the peripheral blood revealed the following phenotype: 5. Which of the following is the most likely diagnosis for CD19+ , CD10+ , surface immunoglobulin IgM k. the case described in question 2? (Objective 1) Which of the following is the most likely diagnosis? (Objective 3) a. ALL b. Chronic lymphocytic leukemia a. Burkitt lymphoma c. Hairy cell leukemia b. ALL, precursor B-cell type d. Infectious mononucleosis c. ALL, precursor T-cell type d. AML 6. Flow cytometric analysis of a lymph node fails to reveal an abnormal phenotype. The pathologist is 9. Pitfalls in diagnosing mature lymphoid malignancies convinced that the node is involved by lymphoma. by flow cytometry include: (Objective 2) A touch preparation performed from the specimen a. malignant B cells can lack surface immunoglobulin received in the laboratory contains numerous large cells that are absent from a cytocentrifuge slide b. some malignant lymphoid cells lack diagnostic made from the cell suspension prepared for staining. surface antigens Which of the following is the most likely explanation? c. neoplastic cells can be rare (Objective 4) d. all the above a. The sample received for flow cytometry does not 10. A patient was found to have circulating blasts. No represent the malignancy. Auer rods were identified, and all cytochemical stains b. The malignant cells were lost during manual were negative. Flow cytometry immunophenotyping disaggregation of cells. revealed the presence of myeloid antigens. Which c. The malignant cells were excluded from the gate. of the following is the most appropriate diagnosis? d. The malignant cells have a normal phenotype. (Objective 3) a. ALL 7. Repeat analysis of the case described in question 6 increases the number of large cells present in b. AML the suspension stained and analyzed on the flow c. Chronic lymphocytic leukemia cytometer. An abnormal phenotype is still not d. Chronic myeloid leukemia detected. Which of the following neoplasms could References 1. Givan, A. L. (2001). Principles of flow cytometry: An overview. (ERIC) update on the international harmonised approach for Methods of Cell Biology, 63, 19–50. flow cytometric residual disease monitoring in CLL. Leukemia, 27, 2. Swerdlow, S. H., Campo, E., Harris, N. L., Jaffe., E. S., Pileri, S. A., 142–149. Stein, H., . . . Vardiman, J. W. (2008). WHO Classification of 5. Arroz, M., Came, N., Lin, P., Chen, W., Yuan, C., Lagoo, A., Tumours of Haematopoietic and Lymphoid Tissues (4th ed.). Lyon, Monreal, M., . . . Paiva, B. (2016). Consensus guidelines on France: IARC Press. plasma cell myeloma minimal residual disease analysis and 3. Craig, F. E., & Foon, K. A. (2008). Flow cytometric reporting. Cytometry Part B (Clinical Cytometry), 90B, 31–39. immunophenotyping for hematologic neoplasm. Blood, 111, 6. Feller, N., van der Velden, V. H. J., Brooimans, R. A., Boeckx, N., 3941–3967. Preijers, F., Kelder, A., de Greef, I., . . . Gratama, J.W. (2013). 4. Rawstron, A. C., Bottcher, S., Letestu, R., Villamor, N., Fazi, C., Defining consensus leukemia-associated immunophenotypes for Kartsios, H., de Tute, R.M., . . . Ghia, P. (2013). Improving effi- detection of minimal residual disease in acute myeloid leukemia ciency and sensitivity: European Research Initiative in CLL in a multicenter setting. Blood Cancer Journal, 3, e129. 1012 Chapter 40 7. Dass, S., Rawstron, A. C., Vital, E. M., Henshaw, K., McGonagle, 10. Borowitz, M. J., Craig, F. E., DiGiuseppe, J. A., Illingworth, A. J., D., & Emery, P. (2008). Highly sensitive B-cell analysis predicts Rosse, W., Sutherland., D. R., . . . Richards, S. J. (2010). response to rituximab therapy in rheumatoid arthritis. Arthritis Guidelines for the diagnosis and monitoring of paroxysmal and Rheumatism, 58, 2993–2999. nocturnal hemoglobinuria and related disorders by flow 8. Gratama, J. W., Orfao, A., Barnett, D., Brando, B., Huber, A., cytometry. Cytometry Part B (Clinical Cytometry), 78, 211–230. Janossy, G., . . . Papa, S. (1998). Flow cytometric enumeration 11. Bolton-Maggs, P. H. B., Stevens, R. F., Dodd, N. J., Lamont, G., of CD34+ hematopoietic stem and progenitor cells. European Tittensor, P., & King M. J. (2004). Guidelines for the diagnosis Working Group on Clinical Cell Analysis. Cytometry, 34, and management of hereditary spherocytosis. British Journal of 128–142. Haematology, 126, 455–474. 9. Whitby, A., Whitby, L., Fletcher, M., Reilly, J. T., Sutherland, D. R., 12. Wenger, C. R., & Clark, G. M. (1998). S-phase fraction and Keeney, M., & Barnett, D. (2012). ISHAGE protocol: Are we doing breast cancer—a decade of experience. Breast Cancer Research and it correctly? Cytometry Part B (Clinical Cytometry), 82, 9–17. Treatment, 51, 255–265. Chapter 41 Chromosome Analysis of Hematopoietic and Lymphoid Disorders Kathleen S. Wilson, MD, FCAP, FACMG Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define chromosome and mitosis. use the appropriate terminology to describe 2. List the basic steps of cytogenetic analysis them. and select the most appropriate type of spec- 4. List the practical uses of cytogenetics in the imen for analysis of suspected constitutional diagnosis and prognosis of hematolymphoid and neoplastic (acquired) disorders. disorders. 3. Identify the major types of chromosome abnormalities, describe how they occur, and Objectives—Level II At the end of this unit of study, the student should be able to: 1. Describe chromosome morphology and 5. Define and compare translocation, deletion, mitosis. inversion, and isochromosome. 2. Determine which specimen type is most 6. Identify the general categories of hematopoi- appropriate for various clinical indications. etic disorders for which cytogenetic analysis 3. Describe each step of the cytogenetic harvest is useful for patient care. and banding procedure. 7. Correlate diagnostic chromosome aber- 4. Define and compare aneuploidy, nondis- rations with types of hematolymphoid junction, and anaphase lag. disorders and assess their prognostic and therapeutic implications. 1013 1014 Chapter 41 8. Assess the prognostic impact of cytogenetic 10. Correlate chromosome abnormalities with results in lymphoblastic leukemia (acute specific oncogene activation, and assess the lymphoblastic leukemia, ALL). role of the oncogene in the neoplasm. 9. Explain the clinical utility of cytogenetics in transplantation. Chapter Outline Objectives—Level I and Level II 1013 Chromosome Abnormalities 1020 Key Terms 1014 Cytogenetic Nomenclature 1022 Background Basics 1014 Cytogenetic Analysis of Hematopoietic and Lymphoid Case Study 1015 Disorders 1023 Overview 1015 Bone Marrow Transplantation 1028 Introduction 1015 Molecular Cytogenetics 1028 Chromosome Structure and Morphology 1015 Summary 1029 Mitosis 1016 Review Questions 1030 Cytogenetic Procedures 1017 References 1032 Key Terms Acquired aberration Fluorescence in situ hybridization Monosomy Acrocentric (FISH) Mosaic Aneuploid Haploid Nondisjunction Chimerism Heterologous Polymorphic variant Constitutional cytogenetic Homologous Polyploid aberration Hyperdiploid Pseudodiploid Cytogenomic microarray analysis Hypodiploid Satellite DNA (CMA) Karyotype Submetacentric Diploid Metacentric Trisomy Endomitosis Background Basics The information in this chapter builds on the concepts Level II presented in other chapters relating to cell division • Outline the classification of acute myeloid leukemia (Chapters 2, 4) and hematopoietic and lymphoid neo- and acute lymphoblastic leukemia. (Chapters 26, 27) plasms (Chapters 23–28). To maximize your learning, • Describe the various typical laboratory findings and you should review these concepts before beginning this criteria for classification of myeloproliferative neo- unit of study: plasms. (Chapter 24) Level I • Describe the typical laboratory findings and classi- fication of the myelodysplastic states. (Chapter 25) • Describe the stages of the cell cycle, particularly the steps of mitosis. (Chapter 2) • Summarize the chronic lymphoproliferative neo- plasms, list the criteria for distinction of Hodgkin’s • List the major types of neoplastic hematopoietic and disease versus non-Hodgkin lymphomas, and define lymphoid neoplasms. (Chapter 23) the major classification terminology. (Chapter 28) Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1015 acid (RNA), are composed of polynucleotides. A single CASE STUDY nucleotide consists of a phosphate, a sugar (deoxyribose We refer to this case study throughout the chapter. for DNA and ribose for RNA), and a base. The base can Gregory, a healthy 25-year-old man, has a routine be a purine (A = adenine, G = guanine) or a pyrimidine physical examination and laboratory studies for (C = cytosine and T = thymine [DNA] or U = uracil new employment. The total white blood cell (WBC) [RNA]). The bases are aligned on the polynucleotide strand count is 30,000 * 103/mcL. in a triplet code so that three bases code for a single amino Consider what conditions could result in this acid; the succession of bases in the triplet code determines clinical picture and the follow-up studies that the protein products that will result from transcription of should be done. the DNA and translation of the messenger RNA produced. Human cells have approximately 20,000 genes in each located at a specific site on a specific chromosome (the gene locus). The different possible expressions of a gene are known as alleles. For example, the gene for the ABO blood Overview group has three major alleles: A, B, and O. This chapter is designed to give the reader a background DNA exists as a double-stranded helix with the two for understanding the terminology and application of cyto- polynucleotide strands held together by hydrogen bonds genetics in the diagnosis and treatment of hematolymphoid between complementary bases so that G will bind only neoplasms (a term commonly used to encompass both hema- with C and A only with T (or U for RNA). The bonding of topoietic and lymphoid neoplasms). The chapter begins A-T and G-C is called a base pair. This double helix has a with a review of chromosome structure and morphology diameter of approximately 20 Å and is of variable length. and is followed by a summary of the procedure used to For example, the amount of DNA contained in the smallest prepare specimens for chromosome studies. Chromosomal chromosome, number 21, is composed of approximately abnormalities are discussed, and the terminology used to 50 million base pairs, whereas the largest chromosome, describe cytogenetic findings is defined. The remainder of number 1, has approximately 250 million base pairs. The the chapter discusses practical uses of cytogenetic analysis double helix coils around histone proteins resulting in a of hematolymphoid neoplasms. series of structures called nucleosomes (Figure 41-1). These nucleosomes form a superhelix with six nucleosomes per turn forming a chromatin fiber (called a solenoid) with a diameter of 250 Å. These fibers are looped back and forth Introduction on a protein-RNA scaffold to form an identifiable chro- Cytogenetics is the study of chromosome structure and matid with a diameter of 0.2@0.5 mcM (mm). After

DNA number, particularly as they relate to a normal or patho- replication, identical sister chromatids are connected logic state. In 1956, the normal number of chromosomes at the centromere, giving the final structure of a mitotic per human cell was established as 46, and since that time, chromosome. many chromosome abnormality syndromes have been reported. The use of cytogenetics has markedly improved patient diagnosis and family counseling in the field of con- stitutional aberrations, including important advances in T A prenatal diagnosis. Cytogenetic studies are also responsible G C for major advances in the field of hematolymphoid malig- T A nancies and solid tumors. Chromosome analysis of many G C malignant disorders has become a critical component for patient diagnosis and prognosis as well as for research stud- a b c d e f ies of these disorders. Figure 41.1 Chromosome morphology and ultrastructure. (a) Molecular structure of DNA with two -polynucleotide Chromosome Structure chains held together by hydrogen bonding of base pairs (T = thymine, C = cytosine, A = adenine, G = guanine) and Morphology (b) Double helical structure of DNA. (c) Coiling of double helix strand around histone proteins to produce nucleosome. (d) Superhelix of nucleosome producing chromatin fiber. (e) Coiling of chromatin Nuclear chromatin of human cells is composed of nucleic fiber to produce chromomere. (f) Final structure of chromosome acid and protein and is organized into 46 chromosomes. The consisting of two identical sister chromatids (condensed nucleic acids, deoxyribonucleic acid (DNA), and ribonucleic chromomere) held together at the centromere. 1016 Chapter 41 The centromere divides chromosomes into short (p) number 9 chromosome and the allele for blood group B on arms and long (q) arms (Figure 41-2). If a centromere is in the paternal number 9 chromosome. A heterologous pair the center of the chromosome so that the length of p = q, it (i.e., the sex chromosomes, X and Y, in a male) consists of is referred to as a metacentric chromosome (chromosomes morphologically nonidentical chromosomes that have dif- 1, 3, 16, 19, and 20). If the centromere is not in the center, ferent gene loci. so that p 6 q, the chromosome is called submetacentric (chromosomes 2, 4, 5, 6–12, 17, 18, and X), and when the centromere is located close to the end of the chromosome Mitosis so that p is very short, the chromosome is called acrocen- Cells that go through a proliferative division do so by a series tric (chromosomes 13–15, 21, 22, and Y). The area of the of stages called the cell cycle; it consists of four major phases: chromosome around the centromere contains highly repeti- G tive DNA in long clusters of tandem repeats, also known 1, S, G2, and M (mitosis) (Figure 41-3) (Chapter 2). A cell that is not dividing but is performing its designated func- as satellite DNA, which is permanently coiled tightly into tion is in interphase. Interphase begins at G1 of the cell cycle heterochromatin. Heterochromatin stains darkly and is and continues until the end of G transcriptionally inactive; euchromatic areas stain lighter 2. During G1, the nuclear chromatin is dispersed, and chromosome morphology is not and are transcriptionally active during interphase. The het- identifiable. The next phase, S, is the time of DNA synthesis. erochromatin on the end of the p arm of acrocentric chro- The DNA is replicated, and identical sister chromatids are mosomes is attached to the rest of the p arm by less tightly attached at the centromere. The S phase is followed by a coiled chromatin stalks (Figure 41-2). short resting phase, G2, after which the cell enters mitosis. A normal human cell has 46 chromosomes consisting The length of time that a cell spends in each phase is quite of 23 pairs. Chromosome pairs 1–22 are autosomes, and the variable. The average time that a cell spends in mitosis is X and Y chromosomes are sex chromosomes. Before band- estimated to be ∼45 minutes. ing techniques were developed, the chromosome numbers To study human chromosomes, the cells must be mitoti- were assigned according to the total length of the chromo- cally active. Mitosis is the process of division of somatic cells some beginning with number 1 as the longest and number by which each daughter cell ends with the same genetic 22 as the shortest. With the advent of chromosome band- composition as the parent cell. Prophase is the first stage of ing, chromosome 21 was determined to be shorter than mitosis (Figure 41-4) during which the DNA begins to coil, 22, but the designations were not changed. A homologous chromosome morphology becomes recognizable, and a pair chromosome pair consists of two morphologically identi- of cytoplasmic organelles known as centrioles, which are cal chromosomes that have identical gene loci but can have attached to the mitotic spindle, migrate to opposite poles different alleles at a given locus because one member of a of the cell. In metaphase, the DNA is tightly coiled and homologous pair is of maternal origin and the other is of the chromosomes align in the center of the cell (equatorial paternal origin. For example, a homologous pair consist- plate, metaphase plate), and the mitotic spindle apparatus ing of both chromosomes number 9 has the gene locus attaches to the kinetochores of the chromosome centromere for the ABO blood group on the long arm. An individual can inherit the allele for blood group A on the maternal Short arm Satellite Centromere Stalk Heterochromatin Long arm Submetacentric Acrocentric Figure 41.3 Diagram of the cell cycle beginning with G1, the chromosome chromosome stage in which the cell is performing its designated duties. The DNA is uncoiled and exists as 46 single chromatids. S phase is the time of Figure 41.2 Chromosome structure of a typical DNA synthesis after which the DNA is still uncoiled and consists of submetacentric chromosome and an acrocentric chromosome. The 46 chromosomes (sister chromatids joined at the centromere). G2 is short arm is known as “p” and the long arm as “q.” a resting phase followed by mitosis. Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1017 a b e c d Figure 41.4 Mitosis. (a) Interphase. Chromatin is dispersed. (b) Prophase. Chromosome structure is discernible and centrioles begin to migrate. (c) Metaphase. Chromosomes are lined up in the center, spindle fibers from the centrioles connect to the centromeres, and the nuclear membrane is not visible. (d) Anaphase. Spindle fibers contract and sister chromatids migrate to opposite poles of the cell. (e) Telophase. Chromatid migration is complete and the cytoplasmic membrane forms down the center, completing the cell division. via microtubules. Anaphase begins with the contraction of the constitutional cytogenetic aberrations (aberrations present spindle fibers pulling apart the sister chromatids so that one in every cell in a patient’s body, i.e., its constitution), periph- sister chromatid migrates to one pole of the cell and the other eral blood is the most appropriate sample because circu- migrates to the opposite pole of the cell. During telophase, the lating lymphocytes can be easily manipulated to undergo daughter nuclei begin to form at the opposite poles of the mitosis by the use of mitogens. For example, phytohemag- cell, and cytokinesis begins. At the end of telophase, cytoki- glutinin (PHA) stimulates predominantly T-lymphocytes. nesis (the division of the cytoplasm into two daughter cells) If the patient has a lymphocytopenia or if there is a suspi- completes, and each of the daughter cells then re-enters the cion that cells from different tissue types can have different G1 phase of the cell cycle. Each resultant daughter cell has karyotypes (a condition termed mosaicism), a punch-biopsy the identical genetic composition as the parent cell. of skin can be obtained for fibroblast culture. When the Meiosis is the specialized division of diploid (46 chro- clinical situation involves a spontaneous abortion (miscar- mosomes) primary gametocytes that results in each gamete riage), stillbirth, or death shortly after birth, the appropriate (oocyte and sperm) having a haploid number of chromo- sample is fetal tissue (products of conception) or autopsy- somes (23). The understanding of meiosis is critical for the acquired organ samples (lung, liver, kidney, diaphragm). study of constitutional chromosome aberrations and is not A prenatal evaluation requires culturing either amniotic discussed in this chapter. fluid cells or a chorionic villus biopsy processed by direct harvest and tissue culture. These various cultures are har- vested at the time of maximal mitotic activity, usually 72–96 Cytogenetic Procedures hours for peripheral blood and 7–21 days for amniotic fluid Specimen Preparation and fibroblast cultures. Acquired aberrations (those happening after birth Specimens submitted for cytogenetic analysis must have in a single cell) occur in neoplastic processes such as leu- viable cells capable of undergoing mitosis; the choice of kemia, lymphoma, and other tumors. Evaluation of these appropriate specimens depends on the patient’s clini- disorders requires that the neoplastic cells be sampled cal situation (Table 41-1). For the evaluation of possible directly either by peripheral blood, bone marrow, or solid 1018 Chapter 41 Table 41.1 Appropriate Specimens for Cytogenetic Analysis Clinical Situation Appropriate Specimen Type of Processing Constitutional aberrations Peripheral blood Mitogen stimulation of lymphocytes with phytohemagglutinin Skin biopsy Tissue culture Autopsy organ samples Tissue culture Products of conception Tissue culture Amniotic fluid Tissue culture Chorionic villus sample Direct harvest, tissue culture Neoplastic (acquired) aberrations Peripheral blood/bone marrow Direct harvest, unstimulated cultures; stimulated cultures of mature B-cell malignancies; all cultures from cell suspension Lymph node/spleen Direct harvest, unstimulated cultures; stimulated cultures of mature B-cell malignancies; all cultures from cell suspension Solid tumor biopsy Monolayer and suspension tissue culture tumor biopsies. These samples are harvested immediately The slide is dried and examined by phase microscopy for (direct harvest) or by short-term unstimulated cultures to appropriate spreading and number of mitotic figures. If the obtain mitoses of the neoplastic cells, not the associated first slide does not show optimal quality, the suspension can nontumor cells. be concentrated or diluted, or other manipulations can be The first step of the cytogenetic procedure is to induce performed to obtain improved chromosome morphology. cells into mitosis. Once this has been accomplished by any The remainder of the slides are then prepared and “aged” of the methods named, the cells are “harvested.” The har- for banding; this often involves heating the slides in a 60 °C vest procedure processes cells that are mitotically active to oven overnight. visualize the chromosomes. The major steps are metaphase Chromosome banding is obtained by various stain- inhibition, hypotonic incubation, and fixation. ing procedures that result in a specific pattern of dark-to- light stained bands for each homologous chromosome pair. The first chromosome banding technique was reported CASE STUDY (continued from page 1015) in 1970 with the use of quinacrine (Q), a fluorescent stain The differential WBC count shows 7% blasts, 3% that reveals a pattern of bright and dull bands (Q-bands).2 promyelocytes, 25% myelocytes, 10% metamyelo- Q-banding techniques are relatively simple; however, the cytes, 5% bands, 25% segmented neutrophils, 10% banding fades when examined microscopically with ultra- basophils, 5% eosinophils, and 10% lymphocytes. violet illumination, and the resolution of bands is not as The differential diagnosis includes leukemoid reac- detailed as are G-bands (G = Giemsa). Most laboratories tion, chronic myeloid leukemia (CML), and myelo- routinely analyze G-bands using Giemsa stain and some proliferative neoplasms other than CML. A bone form of enzyme pretreatment, usually trypsin.3 These tech- marrow aspirate is performed. niques result in a high-quality banding pattern that does not fade with microscopic examination. The pattern of 1. What is the most appropriate specimen to submit bright or dull Q-bands and dark- or pale-staining G-bands for cytogenetics, and how should it be processed? is essentially the same. Reverse banding (R-bands) yields a band pattern opposite to that of Q- and G-bands so that a pale G-band will be darkly stained with R-banding. Harvest Procedure and Banding Other banding techniques and special harvest procedures can be helpful for evaluation in certain clinical situations Mitotically active cells are stopped in metaphase by incuba- (Table 41-2). tion with agents that disrupt the spindle apparatus, most The development of fluorescently labeled DNA probes commonly colchicine or colcemid.1 The cells are then incu- for specific chromosome centromeres, whole arms, whole bated with a hypotonic solution (frequently 0.075 M KCl) chromosomes, and individual genes (fluorescence in situ that hemolyzes erythrocytes

and partially swells the nucle- hybridization [FISH]) has enhanced the study of chromo- ated cells. Fixation of the cells is then accomplished with some morphology (Chapter 42).4 Fluorescently labeled Carnoy’s fixative, 3:1 methanol:glacial acetic acid. After probes allow both specific identification of chromosomes fixation, a trial slide is prepared by putting three to four involved in structural or numerical aberrations and analysis drops of the final cell suspension onto a clean glass slide. of interphase cells. The labeled probe is hybridized directly Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1019 Table 41.2 Cytogenetic Banding Techniques and Special Procedures Type of Banding or Special Procedure Procedure Description Result Q banding Quinacrine fluorescence Reveals distinct bright and dull band patterns of homologous chromosomes A-T rich areas = bright G-C rich areas = dull G banding Giemsa stain after enzyme pretreatment Reveals distinct dark and pale band patterns of homologous chromosomes identical to Q bands A-T rich areas = dark G-C rich areas = pale R banding A-T rich areas = pale Reveals distinct pale and dark band patterns of homologous G-C rich areas = dark chromosomes reverse of Q and G bands C banding Giemsa stain after acid-alkali denaturation Stains heterochromatic pericentric regions of chromosomes 1, 9, 16, and long arm of Y chromosome NOR staining Silver stain of nucleolar organizing region Stains stalk region of acrocentric chromosomes 13, 14, 15, 21, and 22 FISH Fluorescently labeled DNA probes Depending on probe used, hybridizes with a specific chromosome pericentromeric region, arm, whole chromosome, or gene Synchronization Synchronization of cells in cell cycle with blocking Increases number of cells in mitosis agent (high-dose thymidine) High-resolution banding Cells synchronized and stopped in late prophase or Reveals chromosomes that are less condensed, more toward early metaphase the prometaphase, and banded at a more than 800 band stage Fragile site Cells cultured with folate/thymidine deprivation Reveals areas of chromosome gaps, previously used for fragile X SCE Sister chromatid exchange detected by incubation Stains sister chromatids dark or light showing areas of through two cell cycles in BrdU and stained with exchange, indicating mutagenicity fluorescent; patient and control DNA are amplified and labeled with different fluorophores and hybridized to an array with DNA probes specific for the entire genome or part of the genome (chromosome specific) CMA High-resolution chromosome analysis performed by Differential in signal intensity is indicative of copy number gain hybridization of fluorescently labeled patient DNA to or loss a microarray embedded with oligonucleotides tiled to cover the genome FISH, fluorescence in situ hybridization; NOR, nuclear organizing region; SCE, sister chromatid exchange; CMA, cytogenomic microarray analysis. to cells mounted on glass slides. This procedure preserves the morphologic information of the cell with the mutation. For hybridization to occur, however, the cells must be per- meabilized and the DNA denatured. The molecular probe is allowed to hybridize to the chromosomes in the tissue speci- men on the slide. Probe binding can be visualized using a fluorescent microscope. Chromosome Analysis Conventional cytogenetic analysis and FISH is performed microscopically. The adequacy of conventional cytoge- netic analysis depends on the mitotic rate of the cells and on banded chromosome morphology. An optimal prepa- ration has mitotic spreads with moderately long chro- mosomes, few chromosome overlaps, and good quality banding (Figure 4 1-5). A karyotype is a representation of the chromosome makeup of a cell that is constructed using Figure 41.5 Metaphase spread of chromosomes belonging to a video-computer-linked analysis system. To prepare a a single cell obtained by direct harvest of a bone marrow aspirate karyotype, the chromosomes are grouped initially by size (G-banded, 1000* magnification). 1020 Chapter 41 Figure 41.6 Karyotype of normal male cell with numbered chromosome pairs, G-banded. and centromere position and then by the specific pattern of Numerical Aberrations dark-to-light-staining bands (Figure 41-6). The number of cells analyzed per case varies according to the clinical situ- A normal human cell with 46 chromosomes is called ation. Accrediting agencies such as the College of American diploid. The word haploid designates half the number of Pathologists (CAP) have set standardized guidelines for chromosomes, 23, and n is an abbreviation for the haploid cytogenetic evaluation. number. Therefore, a cell with 2n (2 * 23) has 46 chromo- somes (diploid), and a cell with 3n has 69 chromosomes (triploid). Aneuploid refers to a chromosome count other Checkpoint 41.1 than 46 that is not a multiple of n. If a cell has more than A newborn baby boy has multiple congenital malformations, 46 chromosomes, the word hyperdiploid is used, and if a cell and a chromosome abnormality is suspected as the cause. has fewer than 46 chromosomes, it is called hypodiploid. What is the most appropriate specimen to submit for chromo- It is thought that a process of nondisjunction causes some analysis, and how should the laboratory professional pro- most numerical aberrations. It occurs during meiotic or cess the specimen? mitotic cell division when a spindle fiber from the cen- triole does not connect to the chromosome centromere or when the spindle fiber connects but does not contract Chromosome Abnormalities (Figure 41-7). This situation results in one daughter cell with an extra chromosome (trisomy) and one daughter cell Chromosome abnormalities are either numerical or struc- with a chromosome loss (monosomy). In most cases, the tural and can involve the autosomes (1–22) and/or the X and cell with the chromosome loss does not survive the next cell Y sex chromosomes. Constitutional abnormalities are pres- cycle. Another process, termed anaphase lag, results when ent at the time of birth and in all cells if they are inherited one chromatid does not completely migrate to the opposite from a parent carrier or if they occurred during gametogen- pole but lags behind and gets caught outside the nuclear esis. Constitutional aberrations can also occur in the embryo membrane (Figure 41-8), yielding one daughter cell with shortly after fertilization resulting in a mosaic: Some cells a chromosome loss and one daughter cell that is normal. have the aberration and some are normal. If the aberrations The word polyploid refers to cells that have a chromo- occur some time after birth, they are acquired, as is usually some count that is a multiple of the n. Hence, a tetraploid seen in a single cell line identifying a neoplastic clone. cell is polyploid and has a chromosome count of 4n, or 92. Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1021 Figure 41.7 Nondisjunction. During anaphase, the sister chromatids of a chromosome do not disjoin, resulting in one daughter cell with an extra chromosome (trisomy) and the other with a chromosome loss (monosomy). Endomitosis is the process that results in polyploid cells of numerical and/or structural aberrations. For example, a when there are multiple rounds of S phase (DNA synthesis) cell with a karyotype of 46, XX,+8, -21 is an abnormal cell without karyokinesis (nuclear division) or cytokinesis (cyto- but has a chromosome count of 46 and therefore is pseudo- plasmic division). The megakaryocyte is an example of a nor- diploid: it has 46 chromosomes including the two sex chro- mal polyploid cell. The word pseudodiploid is used when a mosomes, XX, but it is missing a number 21 chromosome cell has a chromosome count of 46 but is not normal because (-21), and has three number 8 chromosomes (+8). Figure 41.8 Anaphase lag. Chromatid does not complete migration, resulting in one daughter cell with a normal chromosome count and the other with a chromosome loss. 1022 Chapter 41 Structural Aberrations variant morphology that have no clinical consequence) are easily demonstrated with various banding techniques Structural chromosome aberrations result when chromo- and can be used to identify maternal versus paternal origin some breakage occurs and the repair process results in of homologous chromosomes. Some of the more common structural loss or in abnormal recombinations. Table 41-3 polymorphic variants include a pericentric inversion of lists structural aberrations with a short description and chromosome 9; variable amounts of pericentric heterochro- example nomenclature. Any of these can be seen as consti- matin on chromosomes 1, 9, and 16; and a variable amount tutional or acquired with the exception of homogeneously of heterochromatin on the long arm of the Y chromosome. staining regions and double minutes that have been seen Also, amounts of satellite material on the short arms of the only in neoplastic cells as acquired aberrations. acrocentric chromosomes can be variable. Polymorphic Variation Morphologic variations are known to occur in certain chro- Cytogenetic Nomenclature mosomes. These variations have no clinical significance An international committee promoting one standard- but, if present, will be inherited consistently through each ized nomenclature system has established the designa- generation. Polymorphic variants (chromosomes with tion of chromosome number, region, band, and karyotype Table 41.3 Examples of Structural Chromosome Aberration Nomenclature with Explanations Structural Aberration and Nomenclature Explanation Chromosome/chromatid breaks Break occurs in chromosome/chromatid and is usually repaired. Increased random breaks can be seen with toxins, radiation, and virus exposure. Dicentric (dic) example: dic(7;8)(q32;q23) Breaks occur in two chromosomes and the chromosomes—including centromeres—are repaired together resulting in a chromosome with two centromeres; the acentric (lack of a centromere) fragments are lost. Double minutes (dmin) Small acentric pieces of DNA, usually paired, indicate gene amplification. Homogeneously staining region (hsr) Region of chromosome that stains homogeneously and indicates gene amplification. example: hsr(11)(q23) Deletion (del), interstitial example: del(7) Two breaks occur in one arm and material between breaks is lost; break ends are repaired by joining (q31q32) together. Deletion, terminal example: del(7)(q32) Break occurs and acentric fragment is lost during mitosis/meiosis. Duplication (dup) example: dup(7)(q31q32) Region of a chromosome is duplicated and can be direct or inverted. Isochromosome (i) example: i(7)(p10) or i(7) Centromere splits horizontally and results in chromosome with only short or long arm material. The (q10) remaining arm of the chromosome is usually lost. Inversion (inv), paracentric example: inv(7) Two breaks occur, and the material between the breaks inverts and then is repaired. When the inversion (q21q32) does not involve the centromere, it is referred to as a paracentric inversion. Inversion, pericentric example: inv(7)(p15q21) Two breaks occur, and the material between the breaks inverts and then is repaired. When the inversion involves the centromere, the resulting chromosomal aberration is referred to as a pericentric inversion. Ring chromosome (r) example: r(7)(p21q35) Breaks occur in the short and long arms, and the broken ends are repaired together; the acentric fragments are lost. Translocation (t), balanced example: t(7;8) Breaks occur in two different chromosomes with fragments repaired (joined) to the opposite chromosome; (q32;q23) no loss of DNA occurs. Derivative (der) chromosome example: der(7) This is a structurally rearranged chromosome derived most often from two or more chromosomes. In this t(7;8)(q32;q23) example, the derivative, der (7), is the abnormal chromosome 7 that results from a translocation between chromosome 7 and 8 at the designated break points. Translocation, Robertsonian example: A unique type of translocation, breaks occur at or near the centromeres of two acrocentric chromosomes; der(14;21)(q10;q10) the centromeric regions fuse, and the short arm/satellite material is lost. The chromosome is derived (der) from the long arms of each chromosome. nuc ish (MYCN 12~50)[200] Twelve to 50 copies of MYCN found in 200 interphase nuclei evaluated by FISH. nuc ish (ABL1,BCR)x3(ABL1 con BCRx2) Three copies of ABL1 and three copies of BCR with juxtaposition of two of the ABL1 signals with two of [400] the BCR signals found in 400 interphase nuclei evaluated by FISH. 47,XY,+mar.ish der(8)(D8Z1+) An extra marker chromosome identified by FISH of metaphase preparations derived from chromosome 8 using an 8-specific alpha-satellite probe. arr(1-22,X)x2 Normal female results by cytogenomic microarray analysis. arr(1-22)x2,(X,Y)x1 Normal male results by cytogenomic microarray analysis. arr[GRCh38] 20q13.13q13.33 Cytogenomic microarray analysis shows a single copy number loss of the long arm of chromosome 20 (51001876_62375085)x1 with nucleotide designation based on human genome build GRCh38. Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1023 nomenclature. The International System for Human Cyto- for chromosomes 8 and 21 is designated: 48,XY, +8, +21. A genetic Nomenclature (ISCN) has published guidelines female cell with trisomy for chromosomes 3, 8, and 15 and including specific rules for cancer cytogenetics for use by a translocation involving chromosomes 9 and 22 is desig- clinical and research laboratories.5 The short and long arms nated 49,XX,+3, +8, t(9;22)(q34;q11.2),+15. The q34;q11.2 of each chromosome are divided into regions by major land- refers to the long

arms (q), regions 3 and 1, and bands 4 mark bands (Figure 41-9). Each region is further divided and 1.2, respectively. (See Table 41-3 for other examples of into distinct light-, intermediate-, and dark-staining bands. nomenclature.) The numbering of regions and bands begins at the centro- mere and proceeds distally to the terminal portions, pter and qter. The numbering of bands begins with the number 1 for Cytogenetic Analysis each region. To designate a specific band of a chromosome, the order is written as chromosome number, arm, region, of Hematopoietic and and band. A standardized nomenclature system also exists for Lymphoid Disorders gene names. The HUGO Gene Nomenclature Committee Cytogenetic analysis has become an essential part of the (HGNC) is the recognized body that approves a gene name diagnostic evaluation of patients with known or suspected and symbol for each known human gene. All approved neoplasms. Many chromosome aberrations ascertained symbols are stored in the HGNC database. Approved gene names and symbols are readily accessible electronically.6 by either conventional (G-banded) techniques or FISH are now considered diagnostic of or have significant prognostic The karyotype of a cell is designated first by the total implications for hematolymphoid malignancies7 and solid number of chromosomes followed by the sex chromosomes tumors.8 The cloning of genes at critical chromosome break- (XX for female and XY for male). If aberrations are present, points in various neoplasms has permitted the discovery sex chromosome abnormalities are listed first followed by of the role that these genes play in tumorigenesis and has abnormalities of autosomes listed in numerical order of the provided specific DNA sequence targets that can be used chromosomes involved. Numerical abnormalities are desig- for the molecular cytogenetic technique of FISH for patient nated by + or - before the chromosome number. Structural diagnostics. A chromosome aberration found in neoplastic abnormalities are listed by the appropriate abbreviation cells is referred to as an acquired, clonal aberration; it is an followed by the chromosomes involved in parenthesis and aberration that occurs sometime after birth and is present then by the break point band designation in parentheses. only in the neoplastic cells. Therefore, normal female and normal male karyotypes, A clone exists if numerical and/or structural aberra- respectively, are 46,XX and 46,XY. A male cell with trisomy tions are identical in at least two cells unless the abnormal- ity is a single chromosome loss (monosomy); then three cells 2 2 must have the same chromosome loss. The presence of an 1 abnormal clone is evidence of a neoplasm. Table 41-4 lists p 5 the most common uses of cytogenetic analysis in hemato- 4 lymphoid disorders. 1 3 2 Constitutional chromosome aberrations occasionally 1.2 are found when analyzing neoplastic cells. Most often these 1.1 1.1 are constitutional abnormalities that are not associated with 1 1.2 an abnormal phenotype. For example, a female patient with acute leukemia and a 47,XXX karyotype could have the +X 1 2 2 Checkpoint 41.2 q Cytogenetic studies were performed on a bone marrow speci- 1 men from a female patient with a myelodysplastic state with the following results: 3 2 3 4 1 cell — 47,XX,+8 5 1 cell — 45,XX,-20 6 5 cells — 46,XX,del(5)(q13q34),-7, +21 Figure 41.9 Diagram of bands on chromosome 7 with arm, Which of these aberrations is clonal? Why? What term would region, and band designations. The band located at the arrow is apply to the five cells? designated 7q32. Short arm, p; long arm, q. 1024 Chapter 41 are obtained by inoculating 1 million cells per milliliter Table 41.4 Present Applications of Conventional of media. The best specimen for study of lymphoma is an Cytogenetics and Molecular Cytogenetics (FISH) in Hematolymphoid Disorders involved lymph node. In general, the resolution of chro- mosomes from an unstimulated peripheral blood or bone • Confirm or establish the diagnosis of a number of leukemias and lymphomas marrow/lymph node specimen is not as good as that from • Predict response to specific agents such as tyrosine kinase inhibitors a phytohemagglutinin-stimulated study, which can reflect • Confirm or predict blast crisis of CML an inherent feature of the malignant cells. In fact, cells with • Confirm or establish remission and monitor minimal residual disease particularly poor morphology can represent the abnormal • Aid in the diagnosis and prognosis of myelodysplastic states clone, whereas the cells with better morphology can be the • Evaluate bone marrow transplant for donor versus recipient cells and remaining normal population. When working in cancer possible recurrence of original neoplasm. cytogenetics, therefore, the laboratory professional must • Evaluate clonal evolution, which portends a more aggressive phase of disease. be careful to analyze each mitotic spread and to evalu- ate the metaphase preparations with poor resolution and morphology. as an acquired aberration indicative of the malignant cells, Processing cells from lymphoma can include incuba- or the +X could represent a constitutional abnormality and tion with mitogens in addition to the unstimulated cultures. be present in all of the patient’s cells. If the karyotype is The neoplastic cells from mature B-cell lymphomas often 45,XX,-7, however, this is most likely an acquired aberra- respond to pokeweed or lipopolysaccharide antigens and tion because monosomy 7 as a constitutional aberration is to stimulation with phytohemagglutinin in the presence of not compatible with life. The presence of constitutional aber- interleukin-2. Table 41-5 provides a summary of specimen rations must be accurately interpreted and distinguished processing. from acquired clonal aberrations. This is most often accom- plished by stimulating peripheral lymphocyte analysis or occasionally by performing skin biopsy fibroblast culture Chronic Myelogenous Leukemia because these cells are not part of the neoplastic clone and aberrations that are present are constitutional. The first chromosome abnormality reported to be associated with a malignancy was described in 1960 as an abnormally small chromosome seen in patients with chronic myelog- Processing Specimens enous leukemia (CML).9 This abnormality was designated The best sample for cytogenetic analysis of hematolym- the Philadelphia or Ph1 (now designated as Ph) chromo- phoid disorders, excluding lymphomas, is a bone marrow some and was believed to be the result of a deletion of the aspirate; even when blast cells are present in the peripheral long arm of chromosome 22. With the advent of banding blood, a higher mitotic rate is usually achieved from the techniques, the abnormality was found actually to be a bone marrow sample. These cells are processed by direct balanced translocation involving chromosomes 9 and 22, harvest and/or unstimulated cultures. The overall cellu- t(9;22)(q34.1;q11.2).10 This translocation is seen in approxi- larity of the marrow aspirate or peripheral blood sample mately 90–95% of patients with CML. can vary greatly and affects the mitotic yield and chromo- Investigators attempted for some time to decipher some morphology. Therefore, it is best for the cytogenetic why a specific chromosome translocation should be so laboratory to evaluate the cell count of each specimen, closely associated with a single morphologic type of leu- usually with an automated cell counter. Optimal cultures kemia. It is now known that the proto-oncogene ABL1, Table 41.5 Appropriate Specimens and Type of Processing for Cytogenetic Analysis of Neoplastic Cells Possible Diagnosis Specimen Processing Acute leukemias (myeloid and lymphoid) Bone marrow at diagnosis 24- to 72-hour unstimulated cultures; FISH analysis with appropriate probes Myeloproliferative neoplasms Bone marrow at diagnosis; peripheral blood for FISH Unstimulated cultures Myelodysplastic syndromes evaluation to monitor minimal residual disease Therapy-related myeloid disorders Lymphoproliferative neoplasms Morphologically involved lymph node, spleen, bone Unstimulated cultures; T-cell: phytohemagglutinin marrow, peripheral blood stimulation; B-cell: pokeweed, lipopolysaccharide, PHA, and IL-2 Solid tumor Morphologically involved tumor tissue Monolayer and/or suspension cultures Effusion (pleural, peritoneal, etc.) Direct harvest, unstimulated cultures FISH, fluorescence in situ hybridization; PHA, phytohemagglutinin; IL-2, interleukin-2. Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1025 normally located at 9q34.1, is translocated and juxtaposed next to the BCR gene at 22q11.2 in the Philadelphia trans- location.11 A proto-oncogene is a normal gene involved in cell division/proliferation that has the capability of becoming an oncogene (Chapters 23, 42). The Ph trans- location results in a new chimeric gene consisting of a portion of the ABL1 from chromosome 9 and a portion of the BCR from chromosome 22. The ABL1 is activated to a functioning oncogene, and a 210 kD (kD = kiloDaltons) polypeptide product of BCR/ABL1 is present in the leuke- mic cells.12 An oncogene is involved in deregulating cell growth, proliferation, and differentiation and is responsi- ble for neoplastic proliferation usually by mutation, over- expression, or amplification. Of CML patients, 5–10% lack the classic t(9;22) and instead have a variant translocation involving at least one other chromosome in addition to chromosomes 9 and 22 or a cryptic translocation of chromosomes 9 and 22. Cryp- tic translocations are molecular genetic rearrangements that cannot be identified by conventional cytogenetics and must be evaluated by a molecular technique such as FISH Figure 41.10 Identification of the BCR/ABL1 gene (Figure 41-10) or the reverse transcriptase polymerase rearrangement with a dual color, dual fusion probe set. The ABL1 chain reaction (rtPCR) for BCR/ABL1 gene rearrangement.7 probe is labeled in red and the BCR probe in green. One normal ABL1 gene and one normal BCR gene are located on separate Patients with variant or cryptic translocations have the chromosomes. The BCR and ABL1 genes are juxtaposed on the two same clinical features and prognosis as those with the typi- chromosomes involved in the translocation, resulting in two yellow cal (9;22) translocation (Chapter 42). fusion signals. In the past, CML was difficult to treat, and patient sur- vival was poor because the t(9;22) and concurrent BCR/ ABL1 gene rearrangement occur in a pluripotent hemato- is seen in the marrow or peripheral blood, allowing more poietic stem cell (HSC) and has the potential to affect all rapid institution of appropriate treatment.14 marrow cells derived from that HSC.12 The identification of The World Health Organization (WHO) has estab- the BCR/ABL1 fusion transcript and the subsequent cloning lished a classification system for both myeloid and lym- of these genes permitted an understanding of the endog- phoid disorders.7 Regarding CML, most cases in chronic enous tyrosine kinase activity of BCR/ABL1 and prompted phase can be diagnosed from peripheral blood for the the development of an agent targeted specifically at the detection of the t(9;22) by conventional cytogenetic analysis protein product of the BCR/ABL1 gene (Chapter 24). This or the BCR-ABL1 rearrangement by molecular techniques. group of agents, called tyrosine kinase inhibitors (TKIs), Bone marrow evaluation, however, is still recommended at was the initial model for the development of genetically diagnosis to allow for provision of sufficient material for targeted cancer treatments, so-called precision medicine, all analyses and to confirm the phase of the disease. The because various types of neoplasms involve genes also accelerated phase of CML is now rare due to the efficacy with tyrosine kinase activity. The TKIs have been shown to of TKI treatment and there is now no currently accepted significantly prolong overall survival in a number of leuke- universal criteria to define it. Diagnosis of blast crisis, how- mias, lymphomas, and solid tumors and have revolution- ever, requires either a blast count of 20% or the presence of ized the treatment of these neoplasms by targeting specific blasts outside of blood or bone marrow (extramedullary genetic loci.13 accumulation).15 When CML patients enter the accelerated phase or The t(9;22) is also seen at diagnosis in approximately blast crisis, the majority have a change in the karyotype 15% of cases of acute lymphoblastic leukemia (ALL), more with chromosome aberrations in addition to the t(9;22) commonly in adults (Chapter 24). Although the transloca- (i.e., clonal evolution). The most frequently observed addi- tion appears the same cytogenetically as that seen in CML, tional aberrations are an extra Ph [+der(22)t(9;22)], an extra there is a difference in the site of breakage at the BCR locus, chromosome 8 [+8], an isochromosome for the long arm of resulting in a 190 kD protein. The cases of Ph-positive ALL chromosome 17 [i(17)(q10)], and an extra chromosome 19 must be distinguished from a patient presenting in lym- [+19]. This clonal evolution can be detected days or weeks phoid blast crisis of CML because the prognosis and treat- before the actual morphologic transformation to blast crisis ment are different. 1026 Chapter 41 and molecular genetic subgroups7 (Chapters 23, 26). Spe- CASE STUDY (continued from page

1018) cific cytogenetic aberrations now define particular subtypes Cytogenetic analysis of bone marrow shows all of AML that have targeted treatments. Although particu- of the cells to have this karyotype: 46,XY,t(9;22) lar morphologic and clinical features characterize these (q34.1;q11.2). cytogenetic entities, the primary diagnostic modality is genetic. The WHO AML classification includes the aber- 2. Is this a constitutional or acquired aberration? rations in Appendix C. Recent advances in the molecular 3. Is it a clonal aberration? characterization of AML patients with normal karyotypes including mutations in FLT3, NPM1, CEBPA, KIT, and 4. What is the significance of this finding for KMT2A have further refined the genetic classification of diagnosis? this leukemia.17 The t(8;21) and inv(16)/t(16;16) both disrupt the same transcription factor, core binding factor (CBF). The t(8;21) disrupts the a@chain of CBF that is encoded by RUNX1, and Myeloproliferative Neoplasms the inv(16)/t(16;16) disrupts the b@chain of CBF. These leu- Other Than CML kemias have relatively favorable prognoses, are more com- The other myeloproliferative neoplasms (MPN)—chronic mon in younger patients, and can have a unique type of neutrophilic leukemia, polycythemia vera, primary myelo- presentation with a particular type of disease called myeloid fibrosis, essential thrombocythemia, chronic eosinophilic sarcoma, a soft tissue infiltration by the myeloblasts. leukemia, and MPN unclassifiable—have acquired clonal The t(8;21), previously reported predominantly in cases chromosome aberrations in approximately 50–60% of cases. of AML-M2 by French–American–British (FAB) classifica- Diagnosis of these disorders now requires documented tion (AML with differentiation) occurs in 5–12% of cases absence of BCR/ABL1 gene rearrangement.7 Recurrent of AML. The protein product of the RUNX1T1/RUNX1 aberrations in these myeloproliferative neoplasms include gene rearrangement promotes leukemogenesis by block- abnormalities of 1q, +8, +9, del(13q), and del(20q). In ing myeloid cell differentiation.18 A loss of a sex chromo- addition to negative BCR/ABL1 gene rearrangement status, some, Y in the male and X in the female, is frequently seen molecular genetic evaluation for mutation of JAK2, as well with the t(8;21). FAB classification previously reported the as for MPL, CALR, and CSF3R mutations, have become stan- inv(16)/t(16;16) in cases of AML-M4eo (acute myelomono- dard of care for the diagnosis of these neoplasms.15 These cytic leukemia with increased marrow eosinophils), which occurs in 10–12% of AML.7,19,20 genetic aberrations are now being developed into models The t(15;17) is present in that will help establish prognosis and predict survival.16 acute promyelocytic leukemia (APL), previously classified as AML-M3 by FAB, and accounts for 5–8% of AML. The Acute Myeloid Leukemia translocation results in the fusion of the retinoic acid recep- tor alpha (RARA) gene at 17q21.1 with the nuclear regula- Approximately 55% of patients with de novo acute myeloid tory factor promyelocytic leukemia (PML) gene at 15q24.1. leukemia (AML) have acquired clonal chromosome aber- These patients respond to treatment with all transretinoic rations in the leukemic cells that are readily identified by acid (ATRA), which induces differentiation of the abnormal conventional cytogenetic analysis or FISH with probes spe- promyelocytes. This treatment is followed by chemother- cific for the genes involved in the rearrangement. The chro- apy. Variant aberrations also rearrange the RARA gene but mosome abnormalities found can be single, numerical, or involve different partner chromosomes (Appendix B). Iden- structural or complex. Some aberrations such as trisomy 8 tification of these variant translocations is clinically impor- occur frequently in AML but are not diagnostic for a specific tant because some resist treatment with ATRA, whereas type of leukemia. When present in the diagnostic specimen, others respond.7 APL with PML-RARA is so named to chromosome aberrations can be valuable in following the emphasize the importance of the genetic rearrangement and leukemia’s progression or regression. If the original leuke- to clarify the fact that the gene rearrangement may occur in mic cells have a clonal aberration, such as +8, a complete a complex cytogenetic rearrangement other than the t(15;17) remission sample should have only a normal karyotype. or may be cryptic.15 In subsequent samples, the presence of even one cell with The WHO also recognizes an entity of AML with +8 would indicate an early relapse, which might not be myelodysplasia (MDS)-related changes that is characterized detectable morphologically. Some patients clonally evolve, by a blast count of more than 20% in the blood or bone mar- usually indicating cytogenetic transformation to a more row, morphologic features of MDS or a history of MDS, aggressive and treatment-resistant disease. MDS-related cytogenetic abnormalities (often complex The current World Health Organization (WHO) clas- including chromosome 5 and 7 aberrations), and an absence sification system for AML focuses on recurrent cytogenetic of the aberrations seen in AML with recurrent genetic Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1027 abnormalities. These patients have a poor prognosis with pediatric cases, and the results are directly related to prog- decreased likelihood of achieving complete remission and nosis. Patients who have the most favorable prognosis have short overall survival. Therapy-related myeloid neoplasms hyperdiploid karyotypes with a chromosome count ranging occur in patients who have previously received chemother- from 54–65 with concurrent trisomies of chromosomes 4, 10, apy or radiation therapy. These cases are called secondary or and 17. Most cases of hyperdiploid ALL have other clini- therapy-related myeloid disorders and are considered as a dis- cally favorable findings such as 3–7 years of age, total leu- tinct entity by the WHO. Alkylating agent-related myeloid kocyte count less than 10 * 103/mcL, and precursor B-cell disorders occur approximately 5–6 years after treatment immunophenotype with CD10 positivity. The t(12;21) and are associated with aberrations of chromosomes 5 and (p13;q22.3) also confers a favorable prognosis. It rearranges 7. Topoisomerase II inhibitor-related myeloid disorders ETV6 at 12p13 and RUNX1 at 21q22.3, and it is a cryptic present within 2–3 years of exposure and are associated aberration, undetectable in conventional metaphase prepa- with break points at band 11q23 and concurrent KMT2A rations, because the size of the translocated segments is gene rearrangement.7,15,21 similar in size and staining intensity. To ascertain the pres- ence of this rearrangement, molecular methods, either FISH or rtPCR, must be employed. Hypodiploidy, the t(4;11) Checkpoint 41.3 (q21;q23) that rearranges the AF4 and KMT2A genes, the A 35-year-old man has acute leukemia. Cytogenetic stud- ies of the bone marrow reveal the following: 46,XY,t(15;17) t(9;22)(q34.1;q11.2) that rearranges the BCR and ABL1 genes, (q24.1;q21.1). What type of leukemia does this patient have, intrachromosomal amplification of the RUNX1 gene on and what will cytogenetic studies show after treatment if he chromosome 21, and translocations involving tyrosine achieves complete remission? kinases or cytokine receptors (BCR-ABL-like) are associated with a poor outcome. Low hypodiploidy has been found to be associated with TP53 mutations that are often constitu- Myelodysplastic Syndromes tional. The patient’s age influences the prognostic impact of MDSs are clonal neoplastic disorders of hematolymphoid these cytogenetic aberrations. ALL patients with the t(9;22) stem cells characterized by dysplasia and subsequent inef- ranging in age from 1–18 years have more favorable out- fective hematopoiesis in two or more myeloid cell comes than adults. Infants and adults with the t(4;11) have lines (Chapter 25). Two international classification systems significantly shorter overall survivals than children with the for MDS have been developed with their major prognostic same translocation. See Appendix C for some of the more commonly seen aberrations in ALL.7,15,23 variables including age, sex, morphologic evaluation, hemoglobin and ferritin levels, the presence of bone mar- row fibrosis, and the type of cytogenetic abnormality. Gen- Checkpoint 41.5 erally, good-risk cytogenetics include a normal karyotype, A 5-year-old girl has fatigue and easy bruising. A CBC shows a del(5q) as the sole abnormality, del(20q) as the sole abnor- leukocyte count of 40 * 103/mcL with 85% blasts. Bone mar- mality, and @Y. Poor-risk cytogenetics include three or more row cytogenetic studies are performed and show all cells with the chromosome aberrations and chromosome 7 abnormalities. following karyotype: 53,XX, +X, +4, +6, +10, +18, +20, +21. Intermediate risk refers to other abnormalities. These spe- What is the prognostic significance of this finding? cific cytogenetic aberrations define particular categories of MDS and play a major role in diagnosis, treatment, and management of patients with them.22 Lymphoma and Lymphoproliferative Disorders Checkpoint 41.4 In recent years, chromosome analysis in lymphoma and Five years ago, a 46-year-old woman received chemotherapy lympho-proliferative disorders has added greatly to the and radiation treatment for breast cancer. She now has pan- understanding of the importance of the gene loci involved cytopenia. Cytogenetic analysis of the bone marrow is per- in chromosome aberrations seen with specific types of formed and shows 10 of 20 cells with the following: 45,XX,del(5) lymphoma. The results of these analyses have also led to (q13q34),-7. What is the significance of this finding? the development of molecular probes and their use in the clinical laboratory. The first abnormality described in the Acute Lymphoblastic Leukemia/ lymphomas was the 14q+ seen in Burkitt lymphoma.24 Lymphoma This was later characterized as t(8;14)(q24.1;q32.3) and is seen in approximately 75% of cases of Burkitt lymphoma. Approximately 60–75% of patients with ALL have clonal Two variant translocations, t(2;8)(p11.2;q24.1) and t(8;22) acquired aberrations of the malignant cells. Cytogenetic (q24.1;q11.2), have been reported in 10–15% of Burkitt lym- findings are a crucial part of the leukemia workup for phoma. The 14q32.3 is the site of the gene for the heavy 1028 Chapter 41 chain of immunoglobulin, and the breakpoint at 8q24.1 is monitoring for minimal residual disease. Following suc- just proximal to the site of the proto-oncogene MYC. There- cessful transplantation, only donor cells should be present. fore, the MYC gene is translocated to the heavy chain locus Occasionally, 1–2% of recipient cells are detectable shortly at 14q32.3.25 This was the first demonstration of a proto- after transplant but then diminish. Recipient cells that recur oncogene that was translocated to a location known to be with a normal karyotype indicate the development of a active in B-cell lymphoma, resulting in the activation of chimerism, the presence of cells of two different genetic the oncogene. The t(2;8) results in the juxtaposition of a origins in an individual. For example, a female recipient gene for k light chain to MYC, and the t(8;22) results in the after a male-donated transplant can show 30% XY and 70% juxtaposition of a gene for l light chain to MYC. In cases XX by FISH analysis with probes for the X and Y centro- of Burkitt lymphoma that have the two variant transloca- meres, which is consistent with partial but not complete tions, the tumor cells are found to mark with surface k@chain engraftment. Treatment to promote engraftment, such as when the t(2;8) is found and with l@chain when the t(8;22) increasing the immunosuppression or giving additional is found. donor cells, can thus be employed. If the recipient mar- Many cytogenetic aberrations are now associated with row cells have an initial chromosome aberration, FISH of specific types of lymphoma and play a major role in diag- DNA probes specific for the genes that are rearranged by nosing these entities as well as having prognostic signifi- that aberration can be used to assess the degree of disease cance. Most of these translocations are known to involve burden persisting. genes that are critical for proliferation of the neoplastic cells or are involved in programmed cell death, apopto- sis. Chronic lymphocytic leukemia (CLL) patients with CASE STUDY (continued from page 1026) trisomy 12, 11q, or 17p aberrations are associated with an An allogeneic bone marrow transplant is per- atypical morphology and shorter overall survival.26 Addi- formed with Gregory’s sister as the donor. Three tional molecular mutations of potential clinical relevance months after transplant, the karyotype is 46,XX. that may be seen in conjunction with classical cytogenetic aberrations include those in TP53, NOTCH1, SF3B1, ATM, 5. What is the significance of this finding? and BIRC3.27 The t(11;14) is diagnostic of mantle cell lym- phoma, an aggressive entity that requires chemotherapy and transplantation, in the appropriate morphologic and clinical context.28 Two molecular subtypes are now rec- ognized based on IGH and SOX11 mutation status.27 The Molecular Cytogenetics presence of the t(11;18) or t(1;14) in extranodal gastric lym- Techniques are now available to evaluate not only the gross phoma of mucosa-associated lymphoid tissue (MALT), a chromosome morphology but also the individual gene com- type of marginal zone lymphoma, defines it as malignancy position. FISH and cytogenomic microarray analysis (CMA) and requires treatment with chemotherapy as opposed to are

examples of such technology. The role FISH plays in the antibiotics.29 Patients with anaplastic large cell lymphoma diagnosis and prognosis of patients with hematolymphoid bearing the t(2;5) and overexpression of the ALK gene have disorders has been previously discussed. The advantage a significantly longer survival than those patients with- of these techniques over conventional cytogenetic studies out this aberration.30 See Appendix C for the characteris- is that molecular DNA techniques do not require viable tic chromosome aberrations with the lymphoproliferative cells capable of mitotic activity. Samples of tumor cells can disorders and gene loci known to be involved in these include nondividing peripheral blood cells and paraffin- rearrangements. embedded tissue. The disadvantage of certain molecular studies, such as FISH, is that they give information about only a single molecular genetic aberration based on the Bone Marrow specific probe used, potentially missing other chromosome aberrations that might be present (Chapter 42). Transplantation The following scenario illustrates the use of the two dif- ferent techniques. A 25-year-old man is known to have CML Some of the hematolymphoid disorders presented in this and has received an allogeneic transplant from his sister. chapter are treated by bone marrow or peripheral blood The first post-transplant specimen did not have dividing stem cell transplantation, depending on the clinical situa- cells in the conventional cytogenetic preparations. FISH tion and availability of donors. The molecular cytogenetic of DNA probes that are rearranged in this leukemia, the technique of FISH, along with other molecular method- BCR and ABL1 genes, was performed and was positive in ologies, is a valuable tool in evaluating the rate of engraft- 10 of 200 interphase nuclei examined, consistent with a ment of the donor cells in opposite sex transplants and low-level persistence of disease. A second sample obtained Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1029 3 months later was sufficient for conventional cytogenetics CMA was initially used predominantly for clinical and yielded the following results: diagnostics of constitutional disorders and could detect copy gain and loss of regions that were approximately 10 kb 80%–46,XX in length.31 The technology has now evolved and can use 10%–46,XY,t(9;22)(q34.1;q11.2) single nucleotide polymorphisms (SNPs) to permit the 10%–47,XY,+8, t(9;22)(q34.1;q11.2) detection of absence of heterozygosity (AOH), the presence In the first sample, the molecular FISH studies were of only one allele at a specific chromosomal locus. AOH is critical to show the persistence of the leukemia. Cytogenet- a frequent finding in cancer specimens and can have diag- ics performed on the second sample was critical to show nostic utility. Laboratory guidelines related to the validation a cytogenetic transformation toward blast crisis with the and clinical implementation of CMA, specifically for cancer additional +8 abnormality. The molecular technique of diagnosis, have been developed.32 Although the presence of rtPCR also has significant clinical utility in diagnosing complex copy number variation in conjunction with the cur- and monitoring patients with hematolymphoid neoplasms rent lack of clinical correlation for AOH at many cytogenetic including CML (Chapter 42). loci makes interpretation challenging, CMA now plays a Cytogenomic microarray analysis (CMA) has been role in the routine molecular profiling of neoplasia, particu- widely used clinically to analyze changes in chromo- larly when used in conjunction with next-generation sequencing technology.33 some copy number (copy number variation). The greatly increased resolution that these assays provide, has revealed that the human genome has much more variability than pre- viously thought with differences found at up to thousands CASE STUDY (continued from page 1028) of loci, most of which are not clinically significant. This vari- Six months after transplant, Gregory had bone ability makes interpretation of CMA potentially challeng- marrow aspirated for analysis. The mitotic yield of ing. In addition, the microarrays used in CMA have been the specimen was not sufficient for routine cytoge- designed to analyze critical regions of the DNA for well- netic analysis. defined genetic abnormalities. Two array methodologies are now widely clinically utilized: array comparative genomic 6. What other studies that would be informative hybridization (aCGH) and in silico methods. Array CGH as to the status of the donor and recipient cells employs DNA from the patient and normal-control DNA could be performed? that are labeled using different fluorescent dyes, mixed, and 7. Five years after the transplant, cytogenetic analy- hybridized to a slide containing hundreds to thousands of sis shows the following: defined DNA probes. The color intensity ratio is analyzed along the DNA to detect regions of copy number gain or 5 cells—46,XX loss. In silico methods use only patient DNA that is also 15 cells—47,XX,+8, t(9;22)(q34;q11.2),i(17)(q10) hybridized to a microarray. Normal fluorescence intensity values have been previously established by prior determi- What is the significance of these findings? nation with a large cohort of normal controls. Summary Clinical specialists in the hematology laboratory are fre- clone if the original pretreatment sample revealed a clonal quently asked when cytogenetic studies are indicated and aberration. what specimens are appropriate to submit. For these rea- The chromosome aberrations found in hematolym- sons, it is important for these specialists to have a basic phoid disorders are present only in the malignant cells. understanding of the specimen requirements, processing, Many cytogenetic aberrations are now recognized as diag- and clinical indications for chromosome analysis. Acquired nostic of particular hematolymphoid neoplasms; for exam- clonal chromosome aberrations can be seen in most of the ple, the t(15;17) is diagnostic of APL. Identification of these hematolymphoid disorders, including acute and chronic specific aberrations with DNA probes has clinical utility for leukemias, myelodysplastic states, myeloproliferative dis- evaluating the patient’s response to treatment and degree of orders, and lymphomas. These results are used for diag- residual disease. The identification of these molecular cyto- nosis and prognosis. In addition, cytogenetics can be used genetic targets has also lead to more effective treatment for to follow the progression or regression of a malignant many hematolymphoid neoplasms. 1030 Chapter 41 Review Questions Level I 7. The ability to identify individual chromosomes depends on which of the following? (Objective 2) 1. Which of the following is the major component of chromosomes? (Objective 1) a. Banding a. Nucleotides b. Hypotonic incubation b. Enzymes c. Type of specimen c. Lipids d. Fixation d. Proteins 8. Which of the following terms is appropriate to describe a human cell that has 47 chromosomes? 2. In which stage of mitosis is the chromosome (Objective 3) morphology best observed? (Objective 1) a. Diploid a. Anaphase b. Aneuploid b. Interphase c. Polyploid c. Metaphase d. Normal d. Prophase 9. Which of the following can result in trisomy? 3. A 4-year-old boy has intellectual disability and (Objective 3) developmental delay. Which of the following would be the most appropriate specimen for chromosome a. Anaphase lag analysis? (Objective 2) b. Endomitosis a. Bone marrow c. Nondisjunction b. Skin biopsy d. Chromosome breakage c. Peripheral blood 10. Which of the following can result in an abnormal d. Lymph node amount of cellular DNA? (Objective 3) 4. A 62-year-old man has acute myeloid leukemia. a. Balanced translocation Which of the following would be the most appropri- b. Paracentric inversion ate specimen for chromosome analysis to determine c. Pericentric inversion whether acquired chromosome aberrations exist? d. Isochromosome (Objective 2) Level II a. Bone marrow b. Lymph node Use this case history for questions 1–3. c. Peripheral blood d. Skin biopsy Stanley, the patient, is a 35-year-old male who pre- sented with a severe nosebleed. He has been previ- 5. Which of the following is a basic criterion of cells that ously healthy; however, in the last three weeks, he would be processed for chromosome analysis? has noticed easy bruising, and on the day of admis- (Objective 2) sion to the hospital, he had a nosebleed that he a. Mitotic activity could not stop. Initial CBC revealed the following: b. Protein production Hb 10 g/dL (100 g/L) c. Presence of nucleolus Hct 30% (0.30 L/L) d. Presence of mitochondria MCV 85 fL 6. In the harvest procedure, cells are stopped in WBC 50 * 103/mcL (50 * 109/L) metaphase by which of the following? (Objective 2) Platelet count 20 * 103/mcL (20 * 109/L) a. Colchicine/colcemid incubation The peripheral smear showed the majority of cells b. Incubation with hypotonic solution to have a high nuclear:cytoplasmic ratio, imma- c. Fixation with Carnoy’s fixative ture chromatin, and cytoplasmic hypergranulation d. Incubation with phytohemagglutinin Chromosome Analysis of Hematopoietic and Lymphoid Disorders 1031 c. inv(16) with azurophilic granules. Preliminary cyto- d. +8 genetic analysis performed on bone aspirate revealed 75% of cells to have t(15;17)(q24.1;q21.1); 5. Which of the following genes does the chromosome 25% of cells have a normal male karyotype. rearrangement identified in question 4 involve? (Objective 9) a. BCR/ABL1 1. The most likely diagnosis is: (Objective 6) b. KMT2A a. chronic myeloid leukemia c. CBFB/MYH11 b. APL d. ETV6/RUNX1 c. anaplastic large cell lymphoma d. secondary (therapy-related) AML 6. What is the other AML with a recurrent cytogenetic aberration that involves the same transcription factor 2. The breakpoint on chromosome 17 involves which as identified in question 5? (Objective 9) gene? (Objective 9) a. t(8;21) a. ABL1 b. inv(16) b. MYC c. t(15;17) c. BCR d. 11q23 d. RARA 7. Which of the following is the most likely diagnosis? 3. The identification of the cytogenetic aberration in (Objective 6) question 2 in patients has led to their treatment with which of the following? (Objectives 5, 6) a. Akylating agent-related AML a. Interferon b. Core-binding factor leukemia b. Interleukin c. AML with myelodysplasia-related changes c. All-trans retinoic acid d. AML of ambiguous lineage d. Vitamin K 8. Which of the following genetic abnormalities is Use this case study for questions 4–7. associated with a good prognosis when it is found in ALL? (Objective 7) A 16-year-old girl has fatigue and increased bruising. A CBC shows the following: a. Chromosome count more than 54 b. t(9;22) Hb 7.0 g/dL (70 g/L) c. t(4;11) Hct 21% (0.21 L/L) d. Chromosome count less than 45 WBC 35 * 103/mcL (35 x 109/L) 9. Which of the following is associated with a poor Platelet count 60 * 103/mcL (60 * 109/L) prognosis when found in ALL? (Objective 7) Differential, bone marrow aspirate: a. Chromosome count more than 54 50% blasts with monocytoid features b. t(12; 21) 15% dysplastic eosinophils with large basophilic granules c. Normal karyotype 10% myelocytes 10% metamyelocytes d. t(9;22) 13% red blood cell precursors 10. Which of the following is associated with Burkitt 2% megakaryocytes lymphoma? (Objective 6) 4. Which of the following is most likely to be seen on a. t(8;21) chromosome analysis of the bone marrow cells? b. t(14;18) (Objective 6) c. t(12;21) a. t(8;21) d. t(8;14) b. t(15;17) 1032 Chapter 41 References 1. Arsham, M. S., Barch, M. J., & Lawce, H. J., eds. (2017). The 19. Marcucci, G., Mrozek, K., Ruppert, A. S., Maharry, K., Kolitz, J. E., AGT cytogenetics laboratory manual (4th ed.). New York: Wiley Moore, J. O., . . . 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133–145. August 7, 2017, from www.genenames.org. 22. Della Porta, M. G., Tuechler, H., Malcovati, L., Schanz, J., Sanz, 7. Swerdlow, S. H., Campo, E., Harris, N. L., Jaffe, E. S., Pileri, S. A., G., Garcia-Manero, G., . . . Cazzola, M. (2015). Validation of Stein, H., . . . Vardiman, J. W., eds. (2008). World Health Organiza- WHO classification-based prognostic scoring system (WPSS) for tion classification of tumours of haematopoietic and lymphoid tissues myelodysplastic syndromes and comparison with the revised (4th ed.). Lyon, France: IARC Press. International Prognostic Scoring System (IPSS-R): A study of the 8. Fletcher, C. D. M., Unni, K. K., & Mertens, F., eds. (2002). World International Working Group for Prognosis in Myelodysplasia Health Organization classification of tumours: Pathology and genetics (IWG-PM). Leukemia, 29(7), 1502–1503. of tumours of soft tissue and bone. Lyon, France: IARC Press. 23. Pui, C. H., & Evans, W. E. (2006). Treatment of acute lymphoblas- 9. 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Döhner, H., Stilgenbauer, S., Benner, A., Leupolt, E., Kröber, A., 239–242. Bullinger, L., . . . Lichter, P. (2000). Genomic aberrations and sur- 12. Faderl, S., Talpaz, M., Estrov, Z., O’Brien, S., Kurzrock, R., & vival in chronic lymphocytic leukemia. New England Journal of Kantarjian, H. M. (1999). The biology of chronic myeloid Medicine, 343, 1910–1916. leukemia. New England Journal of Medicine, 341, 164–172. 27. Swerdlow, S. H., Campo, E., Pileri, S. A., Harris, N., Stein, H., 13. Quan, C., Xiao, J., Liu, L., Duan, Q., Yuan, P., & Zhu, F. (2017). Pro- Siebert, R., . . . Jaffe, E. (2016). The 2016 revision of the World tein kinases as tumor biomarkers and therapeutic targets. Current Health Organization classification of lymphoid neoplasms. Blood, Pharmaceutical Design. doi: 10.2174/1381612823666170720113216. 127(20), 2375–2390. [Epub ahead of print] 28. Bertoni, F., Zucc, E., & Cavalli, F. (2004). Mantle cell lymphoma. 14. Johansson, B., Fioretos, T., & Mitelman, F. (2002). Cytogenetic and Current Opinion in Hematology, 11, 411–418. molecular genetic evolution of chronic myeloid leukemia. Acta 29. Farinha, P., & Gascoyne, R. D. (2005). Molecular pathogenesis of Haematologica, 107, 76–94. mucosa-associated lymphoid tissue lymphoma. Journal of Clinical 15. Arber, D. A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M. J., Oncology, 23, 6370–6378. Le Beau, M. M., . . . Vardiman, J. W. (2016). The 2016 revision to 30. Jaffe, E. S. (2001). Anaplastic large cell lymphoma: the shifting the World Health Organization classification of myeloid neo- sands of diagnostic hematopathology. Modern Pathology, 14, plasms and acute leukemia. Blood, 127(20), 2391–2405. 219–228. 16. Passamonti, F., Giorgino, T., Mora, B., Guglielmelli, P., Rumi, E., 31. Miller, D. T., Adam, M. P., Aradhya, S., Biesecker, L. G., Maffioli, M., . . . Vannucchi, A. M. (2017). A clinical-molecular Brothman, A. R., Carter, N. P., . . . Ledbetter, D. H. (2010). prognostic model to predict survival in patients with post poly- Consensus statement: Chromosomal microarray is a first-tier cythemia vera and post essential thrombocythemia myelofibrosis. clinical diagnostic test for individuals with developmental dis- Leukemia. doi: 10.1038/leu.2017.169. [Epub ahead of print] abilities or congenital anomalies. American Journal of Human 17. Patel, U., Luthra, R., Medeiros, J., & Patel, K. P. (2017). Diagnostic, Genetics, 86, 749–764. prognostic, and predictive utility of recurrent somatic mutations 32. Cooley, L. D., Lebo, M., Li, M. M., Slovak, M. L., & Wolff, D. J. in myeloid neoplasms. Clinical Lymphoma, Myeloma and Leukemia, (2013). American College of Medical Genetics and Genomics 17(Suppl), S62–S74. technical standards and guidelines: Microarray analysis for chro- 18. Westendorf, J., Yamamoto, C., Lenny, N., Downing, J. R., Selsted, mosome abnormalities in neoplastic disorders. Genetics in Medi- M. E., & Hiebert, S. W. (1998). The t(8;21) fusion product, AML- cine, 15(6), 484–493. 1-ETO, associates with C/EBP-alpha, inhibits C/EBP-alpha- 33. Dang, T., Wang, Y. C., & Huang, Q. J. (2014). Microarray and next- dependent transcription, and blocks granulocytic differentiation. generation sequencing to analyze gastric cancer. Asian Pacific Molecular Cell Biology, 18, 322–333. Journal of Cancer Prevention, 15(19), 8033–8039. Chapter 42 Molecular Analysis of Hematologic Diseases Sara Taylor, PhD Sally Lewis, PhD Objectives—Level II This chapter differs from others in that it has only Level II objectives. The material in this chapter is at the advanced level and requires a background in genetics. At the end of this unit, the student should be able to: 1. Define terms and appropriately use nomen- 5. Explain how B- and T-cell malignancies can clature associated with molecular pathology. be differentiated from other conditions by 2. Describe the principles and summarize molecular testing. the procedures for each common labora- 6. Select the most appropriate molecular test tory test used in molecular diagnostics of for a given patient condition or provisional hematopathology. diagnosis. 3. Describe and explain the applications of 7. Compare and contrast the various molecular molecular tests in diagnosing and managing methods with regard to diagnostic inherited disease, infectious disease, and applications and principles. cancer as they pertain to hematology and 8. Compare the advantages and disadvantages hemostasis. of molecular tests with other laboratory 4. Identify common chromosomal transloca- tests used in diagnosing and managing tions and molecular genetic abnormalities hematologic disorders. associated with chronic myeloid leukemia, acute leukemia, and lymphoma. 1033 1034 Chapter 42 Chapter Outline Objectives—Level II 1033 Clinical Applications of Molecular Diagnostics Key Terms 1034 in Hematopathology 1043 Background Basics 1034 Clinical Applications of Molecular Diagnostics Case Study 1034 in Hemostasis 1049 Overview 1035 Summary 1050 Introduction 1035 Review Questions 1050 Overview of Molecular Technologies 1036 References 1051 Key Terms Allele Genome Probe Complementary DNA (cDNA) Genotype Restriction endonuclease DNA sequencing Hybridization Southern blot Fluorescence in situ hybridization Mutation Transcription (FISH) Polymerase chain reaction Translocation Gene (PCR) Background Basics This chapter builds on concepts learned in other chapters • Describe the application of cytogenetic analysis in of this textbook. In addition, the reader should have a back- diagnosing hematologic disease. (Chapter 41) ground of genetic principles. To maximize your learning • Summarize the etiology and pathophysiology of experience, you should review the following material: leukemia and lymphoma. (Chapters 23–27) Level II • Describe the congenital aberrations that lead to hypercoagulability and venous thrombosis. • Summarize the pathophysiology and etiology of ( Chapter 34) sickle cell anemia. (Chapter 13) • Describe the role of oncogenes in the etiology and pathophysiology of cancer. (Chapter 23) CASE STUDY shows a left shift and an increased platelet count. We refer to this case study throughout the chapter. His red blood cell (RBC) morphology is described as a normocytic, normochromic cell population. Warren, a 59-year-old white male, sees his physi- His physician suspects chronic myeloid leukemia. cian with a complaint of increasing abdominal Consider the laboratory tests that should be discomfort. A physical examination reveals that performed to verify the physician’s preliminary he has an enlarged spleen. His WBC count is diagnosis. 34 * 103/mcL, and the peripheral blood smear Molecular Analysis of Hematologic Diseases 1035 Overview can be of great consequence to the organism. Changes to DNA structure can occur spontaneously (de novo) dur- Molecular diagnostics is essentially the analysis of DNA ing DNA replication, recombination, or during repair pro- and RNA at the molecular level for the purposes of eluci- cesses. Mutations can also occur as a result of exposure to dating disease etiology. DNA and RNA underlie cell func- chemicals, UV radiation, or ionizing radiation. Recently, tion, and increasingly more pathologies are attributable to epigenetic modifications to DNA have been recognized as genetic mutations. Molecular diagnostics has applications contributory to DNA expression (Chapters 2, 23). Epigenetic in the identification of infectious agents, patient stratifica- changes do not involve sequential changes to DNA but tion, drug regimen selection, toxicity avoidance, therapeutic silence or allow expression of DNA by well-recognized monitoring, and detection of predisposition to disease. mechanisms, such as DNA methylation or histone meth- Numerous scientific discoveries needed to take place ylation and/or acetylation. before the technology that permits molecular diagnosis Individuals inherit two alleles (one gene from each could develop. Most scientists would agree that Mendel’s parent). The most common version of the allele is referred work elucidating inheritance was among the earliest scien- to as the wild type (WT). If a mutation occurs in either tific contributions that lead to our understanding of genetics allele, the function of the protein for which it encodes could and that the works of Rosalind Franklin, Maurice Wilkins, be altered. The modified function could either be reduced James Watson, and Francis Crick lead to an understand- or intensified, so the gene mutation is referred to as a loss- ing of nucleic acid structure. Nucleic acid sequence and of-function or a gain-of-function mutation. Loss-of-function structure are the basis of nearly all molecular techniques (a mutations might result in impaired biological activity or review of nucleic acid structure and function is provided in cancer if the gene (or its encoded protein) lost is a tumor Chapters 2 and 41). In 1983, a technique, polymerase chain suppressor. Although it is expected that most mutations reaction (PCR), that capitalized on nucleic acid structure lead to a loss of function, it is possible that a new and was developed and revolutionized the way that medicine important function could result from the mutation. In these is practiced. Since then, an explosive increase has occurred cases, the mutation creates a new allele that produces a pro- in molecular techniques including an astonishing num- tein with new activity, referred to as gain of function. Gain- ber of variations of nucleic acid amplification, fluorescent of-function mutations to genes encoding essential proteins in situ hybridization (FISH), microarray technology, and of cell cycling, signaling, and anti-apoptosis can lead to (next-generation) DNA sequencing. Other advances have dysregulation of these essential cell processes resulting in been in the areas of miniaturization, automated nucleic malignancy (leukemias or lymphomas). When the outcome acid extraction, and automated analysis—all of which of gene deviation is cancer, the altered gene is referred to have greatly enhanced the speed, accuracy, and flexibility as an oncogene; its normal counterpart is known as a proto- of molecular diagnostic testing. This chapter describes the oncogene (Chapter 23). most commonly used molecular tests and their application Gross chromosome alterations such as deletions, inser- in selected inherited and malignant hematologic and hemo- tions, and translocations are demonstrated well with stasis disorders. karyotyping. However, subtle mutations such as single- nucleotide polymorphisms (SNPs), small deletions, and small insertions are inconspicuous aberrations in otherwise Introduction normal chromosomes that are often best elucidated with molecular techniques. Undoubtedly, the greatest contribution molecular testing The addition of molecular technologies to traditional makes to clinical diagnostics is the use of an individual’s techniques such as the interpretation of hematoxylin genetic material for rational decision making about the and eosin (H&E)–stained sections, immunohistochemi- person’s care. Thus, molecular diagnostics is essential in cal staining patterns, flow cytometry data, and clinical the development of personalized medicine. As its name information has revolutionized the practice of hemato- implies, personalized medicine utilizes each individual’s pathology. Improvements to the quality of patient care unique clinical, genetic, and environmental information to have resulted from the application of molecular methods determine a person’s susceptibility to disease, diagnose ill- to the diagnosis, prognosis, and treatment of hematologic ness, make treatment decisions, determine response, and to disease. track minimal residual disease. Today patient care is devel- oping along the lines of personalized medicine, a trend that Checkpoint 42.1 is likely to expand. Consider the role that oncogenes and tumor suppressor genes At the molecular level, mutations are alterations in

play in the development of cancer and the concept of personal- the portion of DNA molecules that constitute genes. DNA ized medicine. is a blueprint of the genome and changes to its structure 1036 Chapter 42 Overview of Molecular be performed. For formalin-fixed, paraffin-embedded (FFPE) tissue, paraffin removal could involve the use of Technologies an organic solvent such as xylene or a non-organic sol- vent method available in a kit. DNA extracted from FFPE Clinical laboratories have experienced an influx of molec- tissue has been cross-linked to proteins; therefore, high- ular technologies that were once mainly restricted to the molecular-weight DNA (intact or non-degraded DNA) is research setting or to isolated molecular diagnostic labora- not available.4 High-molecular-weight DNA is required tories. The initial molecular technologies that entered the when the particular target of interest (portion of the gene clinical laboratory in the 1980s were labor intensive and target) is large. required extensive molecular skills that most medical labo- After nucleic acid extraction, either DNA or RNA ratory scientists did not possess. Our current understanding requires an assessment of its purity and concentration. This of the molecular basis of human pathology, including the can be achieved by spectrophotometrically measuring the identification of disease causing polymorphisms related to intensity of absorbance of the nucleic acid solution at 260 prognosis or therapeutic efficacy, has expanded the menu of nm and 280 nm. A pure sample of DNA has a 260:280 ratio molecular tests offered. Additionally, the spectrum of tech- of 1.8 and is relatively free from protein contamination. An nology available to analyze genetic defects has expanded to RNA 260:280 ratio of ∼2.0 indicates a pure preparation. include many user-friendly, semi-automated methods that Certain platforms (Agilent Bioanalyzer) have automated are highly suitable for the clinical laboratory. Molecular the determination of nucleic acid concentration and purity. testing can be used in hematology and hemostasis to assist Many molecular assays require high-quality DNA prepara- in diagnosis, risk assessment, treatment optimization, and tions, and others require high-quality RNA, for example, prevention of adverse drug reactions. In the future, health reverse transcriptase polymerase chain reaction (RT-PCR; assessment and management applications can expand as described later in the section “Reverse Transcriptase PCR evidence-based medicine research confirms the clinical util- [RT-PCR]”).4 ity of these applications.1 Although many clinical labora- tories do not offer an extensive menu of molecular testing, several technologies have become standard in the assess- Nucleic Acid Amplification ment of diseases of hematological and hemostasis origin. Any method that allows for numerous copies of DNA or Typical work flow in a molecular diagnostics labora- RNA sequences to be generated is considered to be a nucleic tory begins with nucleic acid extraction (either DNA or acid amplification technique. The original, and most com- RNA) and then assessment of quality and quantity fol- monly used technique, is the polymerase chain reaction. lowed by specific molecular techniques. These techniques POLYMERASE CHAIN REACTION can include variations of amplification, probe-based, and At the core of molecular technology is the amplification sequencing methodologies. A brief description of molecular technique called the polymerase chain reaction or PCR. This methods follows. technique copies a particular segment of DNA one billion- fold. This process permits rapid, sensitive, and specific Nucleic Acid Extraction identification of a segment of DNA that can then be further tested for a disease-specific genetic defect. One important component of the molecular testing pro- The PCR technique amplifies (makes many copies of) cess is the isolation of nucleic acid, either DNA or RNA, a target sequence of DNA that lies between two regions by manual or automated methods. Various commercial of known sequence. After double-stranded DNA is dena- extraction kits are available and have been expanded to tured, short oligonucleotide primers (pieces of DNA) automated extraction platforms. High-throughput molecu- anneal (bind) to complementary DNA sequences flanking lar diagnostics laboratories require robotic systems to pro- the target. The oligonucleotides anneal based on Watson- vide high-quality nucleic acids free from impurities and Crick base pairing (A-T, C-G; Figure 42-1). After primer contamination. Several commercial platforms, including the Roche MagNA Pure LC system and the Qiagen Sym- 59. . . G G C A T C G A A T G A . . . 39 phony, are able to provide high-quality DNA or RNA.2,3 DNA can be isolated from whole blood, white blood cells, 39. . . C C G T A G C T T A C T . . . 59 peripheral blood mononuclear cells, or cultured cells, and the resultant DNA is suitable for use in downstream Figure 42.1 The structure of DNA. DNA is a double-stranded reactions.4 molecule composed of sequences of nucleotides. One strand is bound by hydrogen bonds (shown as diagonal bridges) to its Depending on the nature of the specimen, additional complementary strand. According to the rules of complementary steps could be required to extract an optimum nucleic base pairing, the nucleotide adenine (A) is complementary to acid. For fresh or frozen tissue, homogenization must thymine (T), and guanine (G) is complementary to cytosine (C). Molecular Analysis of Hematologic Diseases 1037 annealing, the enzyme DNA polymerase extends the 3′ end reactions, products are frequently electrophoresed on an of the primer using deoxynucleotides (dNTPs), including agarose gel and stained with ethidium bromide. A UV light adenosine triphosphate (ATP), cytosine triphosphate (CTP), source is used to visualize the DNA bands, which are com- guanosine triphosphate (GTP), and thymidine triphosphate pared with bands on standard DNA ladders to determine (TTP). Synthesis of the new DNA strand always occurs in approximate size.5 Other applications can include DNA the 5′@to@3′ direction because the new dNTP can be added sequencing of specific portions of the PCR product, high- only to the 3′ OH group. Under appropriate reaction con- resolution melt (HRM) analysis (Figure 42-3), or hybrid- ditions, new DNA that is complementary to the template ization techniques using a probe complementary to a strands is synthesized.5 specific DNA sequence. Reagents for the PCR reaction include the template con- tained in the sample, a DNA polymerase, primers, dNTPs, REAL-TIME POLYMERASE CHAIN REACTION (QPCR) MgCl2, and buffer. One cycle of a PCR consists of DNA Traditional PCR can be used only to detect the presence of denaturation (95°C) and primer annealing (about 55–60°C), an amplicon but cannot be used to determine exactly how followed by extension (65–75°C). The process is repeated many copies have been generated. This limitation of the for 20–40 cycles in an instrument called a thermocycler. The assay catalyzed the development of real-time, quantitative repetition allows new DNA strands to be available to serve PCR, or qPCR, that can be used to quantify the number of as template strands for subsequent cycles of amplification5 copies of a target sequence in a patient’s sample. In addi- (Figure 42-2). After 32 PCR cycles, more than one billion tion, qPCR can be used to detect the mRNA product of DNA copies of DNA have been generated. transcription. Importantly, qPCR is used in hematopathol- The products of the PCR reaction, or amplicons, are ogy to determine the proportion of mutated cells among available for qualitative detection (presence or absence normal cells in peripheral blood, otherwise known as mini- of the target) or further analysis. For traditional PCR mal residual disease (Chapter 40). 59 Target DNA 39 Add: Primers ( ) Nucleotides DNA polymerase Buffer 59 Cycle 1: Dissociate strands at 958C Hybridize primers at 558C 39 59 39 DNA synthesis at 728C 59 39 Repeated thermal cycles 1 billion copies of target DNA after 30 cycles Figure 42.2 PCR is a method of enzymatically amplifying a particular segment of DNA through a process of repeated cycles of heating, cooling, and DNA synthesis. First the target (patient) DNA is mixed with the chemicals needed for DNA synthesis. Included are two short DNA probes (called primers, shown as half arrows) designed to flank the particular segment of DNA that needs to be amplified. A thermocycler instrument is programmed to sequentially heat and cool the sample. In cycle 1, the sample is heated to 95°C to dissociate complementary strands of DNA and then is cooled to 55°C to permit binding of the short DNA probes that serve as primers for subsequent enzymatic DNA replication at 72°C. This replication generates new complementary strands to produce an exact copy of the original target DNA. In subsequent cycles, the products of previous cycles can serve as templates for DNA replication, allowing an exponential accumulation of DNA copies. After 30 cycles, which takes only several hours, approximately 1 billion copies of the target DNA have been generated. PCR, polymerase chain reaction. 1038 Chapter 42 59 39 Mismatch F1 F2 59 39 Match F1 F2 Normal patients Heterozygous patients 44 46 48 50 52 54 56 58 60 62 64 Temperature (8C) Figure 42.3 Melt curve analysis is a method used to determine whether a particular mutation is present by evaluating the temperature at which a labeled probe (shown in red) melts away from its target DNA. To accomplish this, a mixture of probe and target DNA is gradually heated until the probe melts away. Melting occurs at a low temperature if a mismatch exists but at a high temperature if there is a perfect match between the probe and target sequence. The temperature at which dissociation occurs is detected in a qPCR instrument that analyzes the interaction between a fluorochrome linked to the probe (shown in red) and another fluorochrome (shown in green) on an adjacent probe. Proximity of the red and green fluorochromes generates a yellowish signal when both probes are bound to the target, whereas the red and green fluorochromes floating apart indicate that melting has occurred. The graph depicts melt curves for a series of 20 patients who were tested for the F2 mutation; those with two normal F2 genes have a single melt peak at 60°C. In contrast, those patients having one mutant copy and one normal copy of the F2 gene have two melt peaks, one at 50°C and the other at 60°C, indicating that they are heterozygous for the mutation and at increased risk for venous thrombosis. PCR, polymerase chain reaction; qPCR, real-time PCR. Detection of qPCR products takes place during the exponential generation of amplicons (exponential phase). By detecting fluorescence in the exponential phase, a quan- titative relationship exists between the amount (copy num- ber) of the starting material in the sample and the cycle Template copy number = 50,000 number at which the amplification curve crosses a mathe- 5000 matically determined threshold. An increase in a reporter— fluorescent signal—detected in the exponential phase is 500 50 directly proportional to the number of amplicons generated 5 ( Figure 42-4). By using a known series of diluted standards, a real-time instrument is able to perform a standard curve and extrapolate quantitative results for unknown samples by reporting copy number.6 5 10 15 20 25 30 35 Several detection technologies have been developed; Cycle number SYBR Green® is the simplest. It attaches to double-stranded DNA in the minor groove and fluoresces. Therefore, when Figure 42.4 Tumor burden in acute promyelocytic leukemia double-stranded PCR products are generated and SYBR with PML-RARA can be measured using qPCR that targets the PML/RARA transcript (Chapter 26). The higher the tumor burden, Green® binds the DNA, the instrument software captures the more fusion transcripts are present, so the more template fluorescence. Other detection systems that provide more spe- cDNA is present, and the earlier the products appear during PCR cific detection of the desired amplicon have been developed. amplification cycles. Amount of PCR product Molecular Analysis of Hematologic Diseases 1039 One is the TaqMan system based on the 5′ exonuclease complimentary DNA (cDNA), copy of an RNA sequence activity of DNA polymerase and captured fluorescence of a followed by a standard PCR to amplify the target cleaved reporter dye-labeled probe.7 TaqMan technology has sequence. Reverse transcriptase PCR (RT-PCR) is used been successfully incorporated into many clinical real-time when the target nucleic acid is RNA.6 Applications include protocols to provide sensitive and specific detection. detection of RNA transcripts such as BCR/ABL1 (chronic In the clinical setting, qPCR has advantages over tra- myeloid leukemia [CML]; Chapter 24), RUNX1-RUNX1T1 ditional PCR for several reasons. The greatest advantage is (acute myeloid leukemia [AML]; Chapter 26), PML-RARA that qPCR detection is accomplished while the PCR reaction (acute

promyelocytic leukemia [APL]; C hapter 26), and takes place, eliminating the need for cumbersome, time- ETV6-RUNX1 (acute lymphoblastic leukemia [ALL]; consuming, and non-automated gel detection of traditional Chapter 27).6 PCR. In addition, for gene expression studies, results can MULTIPLEX PCR be expressed in fold differences by comparing the fluores- Use of multiple sets of primers in one PCR reaction can cent signal generated by the sample to a signal generated simultaneously detect several targets.6 Multiplex PCR can by a reaction targeting a commonly expressed gene called be used to detect multiple disease-associated mutations a housekeeping gene. within one gene such as the numerous mutations in the The qPCR technology has revolutionized the practice b@globin gene of patients with b@thalassemia. It can also be of clinical molecular diagnostics by providing closed sys- used to detect B- or T-lymphocyte clonality of plasma cell tems that are rapid and highly sensitive and can be custom- malignancies. For example, when detecting and assess- ized to numerous applications including minimal residual ing minimal residual disease in a B-cell lymphoma, an disease detection in subclinical patients. Among the many immunoglobulin heavy chain (IGH) gene clonality assay applications beyond hematopathology are viral quantita- can be used (Figure 42-5). A typical IGH assay is based tion of viral load for HIV-I and HCV, quantitation of gene on multiplex reactions targeting conserved regions within expression (both absolute and relative), pathogen detection, the variable (V), diversity (D), and joining (J) regions of microarray verification, genotyping, drug therapy efficacy, the immunoglobulin heavy chain. Finally, multiplex PCR and quality control and assay verification.7 can also be used in assays that identify multiple possible REVERSE TRANSCRIPTASE PCR (RT-PCR) break points of translocations, for example, BCR/ABL1, The enzyme reverse transcriptase, whose gene is encoded MLL/AFF1 (ALL; Chapter 27), and NPM1/RARA (APL; by the HIV-I retroviral genome, can be used to produce a Chapter 26).6 Variable Diversity Joining Constant IGH gene rearrangement Clonal B-cell tumor Normal polyclonal B lymphocytes Size of PCR product Figure 42.5 Rearrangement of the IGH gene involves random splicing of V, D, and J segments to produce a unique coding sequence. This process brings the V and J segments so close together that it becomes possible to PCR amplify across the rearranged gene by using several primers (shown as half arrows) targeting various V and J segments. In a B-cell tumor, all tumor cells contain exactly the same IGH rearrangement that was present in the original transformed B-cell from which the tumor arose. This tumor-related clonal arrangement is identified by capillary gel electrophoresis as a spike representing a single-size PCR product with each primer set. In contrast, benign tissue has normal B-lymphocytes whose polyclonal rearrangements appear as multiple different-size PCR products. V, variable; D, diversity; J, joining; PCR, polymerase chain reaction. Amount of product 1040 Chapter 42 OTHER AMPLIFICATION TECHNIQUES is washed away. Fluorescence imaging is used to visualize Numerous amplification technologies have been developed the probe. to circumvent patent obligations of PCR and offer selected FISH is a particularly important technique for visual- advantages in particular applications. Among these varia- izing chromosomal translocations in leukemias (Table 42-1). tions are the transcription-based amplification systems Allele-specific probes can be hybridized to the sample to exemplified by transcription-mediated amplification (TMA) reveal when two genes that should be found in separate, by Genprobe and nucleic acid sequence-based amplification distinct locations, have been juxtaposed. For example, in (NASBA) offered by Organon-Teknika. RNA serves as the normal cells, a probe that hybridizes to the BCR gene at typical target instead of DNA and the process is isother- 22q11.2 can be detected with a green fluorochrome, whereas mal, not requiring a thermocycler. These processes have the ABL1 gene at 9q34 can be detected by a red fluoro- been used to amplify and quantify BCR/ABL1 transcripts chrome. When translocated together, BCR/ABL1 will appear in CML.8 as a bright yellow spot (the combination of green and red Probe-based amplification methodologies amplify fluorochromes) in leukemia cells.4 specific synthetic probes that hybridize to the target of SOUTHERN BLOT interest rather than amplifying the DNA target. Included The Southern blot involves restriction digestion of sample in these methods are the ligase chain reaction (LCR) and DNA using restriction endonucleases followed by gel elec- strand displacement amplification (SDA). Sickle cell trophoresis of the products (Figure 42-6). Following dena- mutation analysis was one of the first applications of the LCR.9 turation of the double-stranded DNA, single-stranded DNA is then transferred or “blotted” to a more easily handled matrix, usually nitrocellulose or nylon. Capillary action of Hybridization Techniques moistened filter paper facilitates the transfer of DNA to the new matrix. Ultraviolet light helps to secure the DNA to FLUORESCENT IN SITU HYBRIDIZATION (FISH) Fluorescent in situ hybridization (FISH) uses a labeled the membrane. Subsequently, a labeled probe designed to probe to detect and localize specific RNA or DNA sequences hybridize to the sequence of interest is incubated with the in tissue samples. FISH relies on DNA’s ability to hybrid- membrane-bound DNA, followed by a detection method ize with a complimentary, fluorescently labeled nucleotide specific to the type of label. The Southern blot has served probe. In situ means “in the original place” in Latin. Ulti- as the gold standard to which newer and more rapid tech- mately, the location of the target sequence (DNA or RNA) nologies have been compared. Although time consuming can be detected in the cell, tissue, or chromosome. The and technically difficult, the Southern blot can distinguish sample is first fixed onto a glass slide and is treated with the single-base change found in patients with sickle cell anemia.10 chemicals to permeabilize the cells and allow probe entry. If DNA is the target of interest, it must be denatured to make ALLELE-SPECIFIC OLIGONUCLEOTIDES (ASO) it single stranded so that the probe can hybridize. A fluo- ASO technology, also called the reverse dot blot, amplifies, rescently labeled nucleic acid probe complementary to the labels, and applies (blots) the patient’s DNA in “engineered target of interest is added to the sample and excess probe dots.” The dots are constructed using short oligonucleotides Table 42.1 Genetic Abnormalities of Neoplastic Hematological Disorders That Can Be Detected by FISH Hematological Anomaly Detection by FISH AML t(8;21), t(6;9) APL t(15;17) (PML-RARA) AMML t(11;21) AMoL t(9;11) CML t(9;22), t(11;22) B-cell leukemia t(2;8), t(8;14), t(8;22), t(11;14) CLL Deletions: 11q22 (ATM), 13q14 (DLEU1/2 and RB1) and 17p13 (p53) Duplications: 6q, trisomy 12 ALL t(9;22), t(12;21), t(8;14) Multiple myeloma t(14q32) Myelodysplastic syndrome 5q- AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; AMML, acute myelomonocytic leukemia; AMoL, acute monocytic leukemia; ATM, ataxia telangiectasia mutated; CML, chronic myeloid leukemia; CLL, chronic lymphocytic leukemia; DLEU1/2, deleted in leukemia; RB1, retinoblastoma. Molecular Analysis of Hematologic Diseases 1041 Normal HBB (b-globin) gene: Probe 59 39 1.15kb 0.2 kb Southern blot Mstll Mstll Mstll Normal Carrier Affected 1.35 kb Mutated HBB (b-globin) gene: 1.15 kb Probe 59 39 1.35kb Mstll Mstll Figure 42.6 Southern blot analysis of the sickle cell mutation is accomplished by first extracting DNA and then cutting it with Mstll restriction endonuclease that cleaves DNA at a specific nucleotide sequence (vertical lines). Because that specific sequence is present many times in the human genome, Mstll cuts genomic DNA into many small fragments. The resultant DNA fragments are separated by size using gel electrophoresis, dissociated into single strands by soaking in an alkaline solution, and then transferred to a nylon membrane by a blotting procedure. To identify the fragment containing the HBB gene, the membrane is soaked in a radiolabeled DNA probe (bar) that hybridizes to a complementary segment of the HBB gene. The pattern of bands that the probe recognizes reflects the size of the corresponding restriction fragments measured in kilobases (kb). An HBB gene harboring the sickle mutation (shown as a pink circle) fails to cut with Mstll, thus altering the band pattern. In this way, a person affected by sickle cell anemia can be distinguished from a carrier (sickle trait) and from a person of normal genotype. Note that restriction endonucleases are naturally occurring enzymes that recognize and cleave specific nucleotide sequences in DNA. Each restriction endonuclease is named for the bacteria from which it was purified. For example, EcoRl is derived from Escherichia coli and cleaves DNA having the sequence 5′@GAATTC@3′, whereas Hindlll derived from Haemophilus influenza cleaves at 5′@AAGCTT@3′. In this example, Mstll cleaves the normal DNA when it recognizes the sequence 5′@CCTGAGG@3′, but it cannot cleave DNA harboring the sickle mutation 5′@CCTGTGG@3′. Mstll, Microcoleus spp. (oligos) engineered to be complementary to known tar- spotted on a slide in triplicate, incubated with fluorophore- geted sequences of WT or mutant alleles and fixed to a labeled patient sample, and read by a fluorometer.11 A vari- membrane for stability. The dot oligo hybridizes only to ety of vendors has evolved alternate detection chemistries. specific complementary sequences (blots) found in the One particular application of the microarray is for gene patient’s DNA. Numerous applications of this technology, expression profiling. Clinicians can seek to determine which involving known mutations, have been successful because genes are being differentially expressed in leukemic cells the process can be completed in one day and is suitable for compared with normal cells.11 high-throughput laboratories. The ASO process has been Microarray technology can be used to classify ALL by successful for diseases characterized by a single mutation analyzing gene expression patterns in leukemic cells and (sickle cell anemia) or only a limited number of mutations to identify expression of genes associated with prognosis such as hereditary hemochromatosis. Reverse dot-blot or to identify candidate genes for new anticancer therapies. analysis has been developed to screen many b@thalassemia BEAD ARRAY TECHNOLOGY mutations and to use in prenatal diagnosis. Allele-specific A variation of microarray technology can be used with oligonucleotide platforms have largely been replaced with bead array techniques. Probes can be immobilized on real-time PCR technology.10 beads to provide a surface for hybridization of ampli- MICROARRAY TECHNOLOGY fied samples. Numerous samples can be tested simulta- As the demand for high-throughput screening for gene neously for a series of SNPs or genotypes. Fluorescent expression has been accelerated by the quest for person- color-coding beads and labeling the sample with an alter- alized medicine, the popularity of microarray technology native fluorescent dye allow the hybridized combination has burgeoned. Microarray technology enables large-scale to be detected. Software analysis provides interpretation analysis of multiple targets simultaneously. A variety of of the patient results. Luminex Corp. has been a pioneer in microarray technologies exist, but in general, array targets bead array technology with commercial systems adapted are immobilized on a glass slide. The targets can be DNA, to genotyping for CYP2C19 related to clopidogrel (Plavix) cDNA, PCR products, RNA, or proteins. These targets are metabolism. 1042 Chapter 42 programs can be used initially to develop interpretative CASE STUDY (continued from page 1034) clinical reports comparing patient sequences with known After viewing the bone marrow differential and variations related to pathology.4 molecular results, a diagnosis of CML is made, and Warren is placed on imatinib mesylate therapy NEXT-GENERATION SEQUENCING of 450 mg daily, which he tolerates well. After 3 Next-generation sequencing (NGS) technologies have been months of treatment, Warren exhibits a complete rapidly integrated into molecular pathology, increasing the hematologic response and a partial cytogenic actionable genomic information available to pathologists response with 30% or 6 of 20 Ph+ metaphases (by and oncologists.12 These next-generation sequencing tech- FISH) and 7% International Scale (IS) by qPCR. nologies provide sequencing information for large numbers Treatment is continued, and by his 12-month of templates and have the potential to provide coverage over whole genomes with single-base pair resolution.4examination, Warren has a complete cytogenetic response of no Ph+ metaphases, and less than 0.1% Financial pressures have driven the development of IS. Imatinib treatment is discontinued, but Warren high-throughput sequencing methods that produce thou- continues to see his physician every 6 months for sands of sequences in parallel. Costs for next-generation follow up testing. sequencing are lower than with the traditional dye termina- tion methods when multiple targets are included. Numer- 1. What type of molecular diagnostic testing would ous vendors offer next-generation sequencing systems, be suitable to follow Warren’s minimal residual including Applied Biosystems’ SOLID system, Illumina’s disease (MRD)? HiSeq and MySeq systems, Life Technologies Ion TorrentTM Semiconductor Sequencing Chips, and Roches’s GS FLX+ system. Next-generation sequencing platforms can differ

in Direct DNA Sequence Analysis output, run time, read length, and accuracy. One system may be more suited for a particular genomic sequencing DNA sequencing is used to determine the nucleotide application than another. Next-generation sequencing has sequence in a segment of DNA by replicating the DNA been used to identify novel mutations in disorders such as strands and monitoring the order in which labeled nucleo- T-ALL, AML, and CML.6 tides are added to the new strands. It is particularly useful Next-generation sequencing employs massively paral- for detecting point mutations located anywhere in the DNA lel sequencing of nucleic acids to interrogate unique and segment. validated genomic alterations in hematologic malignancies, solid tumors, and other applications. The first FDA clear- Chain Termination Sequencing ance of an NGS platform was the Illumina MiSeqDx instru- Traditional DNA sequencing is based on the Sanger dide- ment and Illumina Universal kits. Universal kit reagents oxynucleotide chain termination method. This method isolate and amplify copies of genes of interest from patient involves the addition of an unusual dideoxyribonucleotide blood samples and the MiSeqDx instrument sequence and (ddNTP) that is missing the 3′ OH group necessary for analyze the genes by comparing the patient’s sequence to nucleotide addition. The ddNTPs ultimately determine the a reference genomic sequence. Differences in the sequence, sequence of a target DNA template as they are incorporated known as variants, are reported. The testing sequence for into a growing DNA strand that is complementary to the the Illumina MiSeqDx platform is diagramed in Figure 42-7 target sequence.4,6 and consists of the following steps: Current sequencing technologies can be based on the 1. Extraction and purification of DNA or RNA from a chain termination methodology and have been adapted to patient’s specimen an automated format. The automated instrument detects 2. DNA is fragmented and platform-specific adaptors are four fluorescent dyes with distinct but slightly overlapping added to the ends of the fragments to create a library wavelengths that are attached to the ddNTPs. The instru- ment produces a series of peaks called an electropherogram 3. The library is clonally amplified, captured, and that correspond to the four bases (A, T, C, G). The soft- sequenced in a flow cell generating chemiluminescent ware assigns a letter to the corresponding dye peak as that or fluorescent images that are translated into sequence ddNTP is incorporated into the growing strand of DNA that reads. Paired-end sequencing is performed to double is complementary to the template. Clinical analysis of the the sequence data and to provide the distance between sequence information is incorporated into the instrument the paired ends. software and detailed reports provide both the sequence 4. Data management and informatics analysis consists data and interpretation relative to known mutations or of base calling and associated quality scores, removal SNPs that are implicated in disease. Numerous software of low-quality sequences, alignment to a reference Molecular Analysis of Hematologic Diseases 1043 • Specimen With the advantages of interrogation of large portions of the genome comes the increased likelihood of uncover- Extraction • DNA ing variants of unknown significance. Genetic experts must establish guidelines of the reporting of these variants to the • Library Amplification/ clinician and the patient. It is predicted that whole-exome capture sequencing will eventually replace the targeted panels as • Sequencing the informatics pipeline catches up with the sequencing • Base Calling data.15 • Alignment CASE STUDY (continued from page 1042) Fill-in of missing data • Variant Calling orthogonal confirmation After 3 years of follow-up testing to track his MRD, Warren’s qPCR reveals an increase in IS to 4%. This • Annotation/Filtering/Classification is worrisome because it suggests that he is com- ing out of remission. Imatinib mesylate therapy is • Interpretation resumed, but after 2 months of treatment, Warren’s • Report IS had risen to 10%, indicating a resistance to ima- tinib treatment. Several possible point mutations in BCR/ABL1 Figure 42.7 Workflow in next-generation sequencing. can result in imatinib resistance. From Regulatory Considerations for Next Generation Sequencing Based Tests. Published by U.S. Food and Drug Administration. 2. Before increasing Warren’s imatinib dose, what type of molecular testing might Warren’s physi- cian order to help in the decision about how to sequence, and variant calling. Annotation consists of manage Warren’s disease? interpretation of the variants in relation to the patient’s phenotype. 5. Reporting of patient variants include the clinical signifi- cance of the variants.13 Clinical Applications of Analytical considerations of NGS platforms include regulatory requirements, assay validation, and inclusion of Molecular Diagnostics in appropriate reference materials, and newly identified vari- ants must be verified by Sanger sequencing. Numerous Hematopathology organizations have developed NGS guidelines, including Molecular methods are increasingly valuable for prognos- the Association for Molecular Pathology (AMP), the College tication, therapeutic response to treatment, and detection of American Pathologists (CAP), the Division of Laboratory of minimal residual disease in hematopathology. Genetic Science and Standards within the Centers for Disease Con- abnormalities can be used for predictive prognostic infor- trol and Prevention (CDC), and the American College of mation about a disease when they appear either by them- Medical Genetics (ACMG), among others.14 selves or when present with other genetic mutations (e.g., The integration of NGS into the clinical laboratory NPM1 mutations are associated with a good prognosis involves several levels of complexity, including targeted in the absence of FLT3 internal tandem duplication [ITD] resequencing of multigene panels, whole-exome sequenc- mutations, but they confer an intermediate prognosis if they ing (WES), and whole-genome sequencing (WGS). NGS of are present in conjunction with FLT3-ITD (Chapter 26).16 multi-gene “hotspot” panels has become critically impor- Mutation analysis also impacts treatment decisions. tant at many well-respected oncology centers that are diag- Tyrosine kinase inhibitors (TKIs) have been used success- nosing and treating solid tumors. These “hotspot” panels fully to treat malignancies, and they are examples of the may detect from 1,000 to 6,000 protein coding regions that use of gene mutations as potential therapeutic targets. have clinically actionable results. With multigene panels, Malignancies in which the receptor tyrosine kinase KIT has both the quality of the data and the interpretation within the a gain of function can respond favorably to TKIs, although context of the patient’s phenotype is maximized. Multiple the strength of the response depends on the type of KIT gene panels are advantageous as they avoid the necessity to mutation that is present.17 return for additional single gene tests with negative results Finally, the presence or absence of MRD has a direct and are more cost effective than when several gene tests are impact on prognosis, evaluation of relapse, and decisions ordered separately. concerning further treatment. Amplification technologies Data Analysis 1044 Chapter 42 are especially sensitive in detecting MRD because as few as straightforward. For example, only about one-half of the 0.01% of abnormal cells carrying a genetic abnormality can patients with hemophilia A display an identifiable mutation be detected. Moreover, molecular responses can be associated in the gene encoding factor VIII. The other half presumably with disease outcome. For example, detection of BCR/ABL1 harbors mutations in other genes that result in abnormal is diagnostic of CML, and a reduction of BCR/ABL1 by 3 log proteins whose downstream effect is to diminish the expres- or more is sufficient for an excellent prognosis, whereas a rise sion of viable factor VIII protein. Table 42-2 provides a list in BCR/ABL1 expression heralds the reappearance of CML.18 of some molecular tests used in clinical laboratories with The greatest value of hematopathology is likely the use a summary of some of the hematopathologies for which of molecular technologies for diagnosis of disease. In the molecular testing is available to assist the clinician with past decade, the number of identified genetic abnormali- diagnosis, prognosis, and therapeutic decision making. The ties that can serve as biomarkers for hematological disease list of diagnostic and prognostic factors is growing rapidly, has grown explosively. Identification of new biomarkers making it difficult to determine which markers provide the will undoubtedly continue to expand rapidly. It is impor- most beneficial information. It is likely that an integrated tant to note, however, that molecular testing is not always testing algorithm will emerge. Table 42.2 Molecular Testing in Hematopathologies Hematopathology Gene/Mutation/Location Molecular Technique Notes Erythrocyte hematopathologies Fanconi anemia FANCA, FANCC, FANCE, or FANCG Amplification (various chromosomes) Hemochromatosis HFE (6p22.2) Amplification Increases total body iron content; provides valu- able diagnostic, prognostic information Hereditary persistence of fetal HBB, HBG1, HBG2 on chromo- Southern blot, multiplex hemoglobin some 11 PCR Thalassemia a and b gene deletions/mutations on Amplification, sequencing, Emphasis is on screening programs to detect thal- chromosomes 16 and 11 reverse dot-blot assemia carriers in areas of high prevalence and in certain populations Hemoglobinopathy Point mutations on HBB resulting Amplification, sequencing, in hemoglobin S/C/E or HBD/HBB multiplex PCR fusion resulting in hemoglobin Lepore on chromosome 11 Polycythemia vera JAK2 (V617F) (9p24) Amplification, HRM Leukocyte hematopathologies AML CEBPa (19q13.1) Amplification, HRM, Various CEBPa mutations confer variable likeli- sequencing hood of achieving complete remissions; valuable in determining therapy NPM1 (5q35) Sequencing, HRM, Characterization of NPM1 mutations provides sub- heteroduplex analysis stantial prognostic value KIT (4q11–q12) Sequencing, ASO, HRM, Mutations are gain of function to a cellular tyrosine qPCR kinase receptor that culminates in a proliferation advantage for the cell AML and ALL FLT3 (13q12) Amplification Internal tandem duplication/point mutations result in constitutive tyrosine kinase receptor activity; increases cell proliferation Myeloproliferative neoplasms JAK2 (V617F) (9p24) Amplification, sequencing, Mutation characteristic of polycythemia vera; also ASO, HRM, qPCR seen in essential thrombocythemia and primary myelofibrosis MPL (1p34) Amplification, sequencing, HRM, qPCR ALL, AML, MDS, plasma cell NRAS/KRAS (1p13.2/12p12.1) Sequencing Activating mutations lead to proliferation advan- neoplasms tages for the affected cell CML and ALL BCR/ABL1 t(9;22)(q34;q11.2) FISH, sequencing, ASO Fusion gene confers proliferation advantage to affected cells; mutations provide information con- cerning TKI therapy Follicular lymphoma and diffuse IGH/BCL2 t(14;18)(q32;q21) FISH, qPCR, microarray Leads to overexpressed BCL-2 protein and large B-lymphoma reduced apoptosis in cells Mantle cell lymphoma CCND1/IGH t(11,14)(q13;q32) FISH Dysregulated cell-cycle progression as gene for cyclin D is brought under control of IGH promoter Molecular Analysis of Hematologic Diseases 1045 Hematopathology Gene/Mutation/Location Molecular Technique Notes Burkitt lymphoma MYC/IGH t(8;14)(q24.1;q32) FISH Dysregulated cell growth as C-MYC is brought under control of the IGH promoter Acute promyelocytic leukemia PML/RARA t(15;17)(q22;q12) RT-PCR, FISH PML/RARA rearrangement is important in APL because it is correlated with responsiveness to treatment with all-trans retinoic acid Childhood ALL ETV6/RUNX1 t(12;21)(p13;q22) Amplification Fusion gene is associated with a favorable prog- nosis and helps determine treatment Hemostasis disorders Warfarin response CYP2C9 on chromosome 10 qPCR, bead array Warfarin sensitivity VKORC1 on chromosome 16 qPCR Factor V Leiden F5, G1691A SNP on chromosome 1 Amplification Prothrombin G20210A F2, G20210 SNP on chromosome Amplification Inheritance of both G1691A and G20210A con- 11 fers at least a 20-fold increase in risk of a venous thromboembolic event Hemophilia A/B F8/F9 greater than 1,000 mutations Amplification, sequencing on X chromosome Methylenetetrahydrofolate MTHFR, C677T, and A1298C SNPs Amplification SNPs result in reduced MTHFR enzyme leading reductase deficiency on chromosome 1 to elevated homocysteine; even small elevations greatly increase thrombotic risk Von Willebrand disease VWF on chromosome 12 Due to complexity of the VWD genotype, VWD diagnosis no longer includes von Willebrand factor gene mutations ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ASO, allele-specific oligonucleotide; CML, chronic myeloid leukemia; FISH, fluorescent in situ hybridization; HRM, high-resolution melt curve analysis; MDS, myelodysplastic syndrome; qPCR, quantitative (real-time) PCR; RT-PCR, reverse transcriptase PCR; SNP, single- nucleotide polymorphism; TKI, tyrosine kinase inhibitor. liver biopsy was the gold standard for diagnosis, but more Checkpoint 42.2 recently, less invasive molecular testing, such as amplifica- List and briefly describe the four general ways in which molecu- tion methods, is gaining favor with clinicians.20 lar diagnostics is useful to clinicians in delivering care to their patients. HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (HPFH) Erythrocyte Disorders HPFH (Chapter 14) can result from large deletions of pro- moters of the g@globin genes HBG1 and HBG2. Specialized Most erythrocyte disorders for which molecular testing amplification techniques such as multiplex ligation-depen- exists are inherited disorders. A rapidly expanding list of dent probe amplification (MLPA) is used to identify the molecular testing for erythrocyte disorders is emerging in mutations.21 the clinical laboratory today. Whereas traditional diagnos- THALASSEMIA tic methods are sometimes still preferred, molecular testing The molecular basis of

thalassemia lies in mutations that can provide reliable information that aids in the diagnosis alter the genes encoding either a@ or b@chains that comprise of several erythrocytic diseases. Erythrocyte disorders in hemoglobin (Chapter 14). Non-molecular testing (mean cell which molecular testing has been useful but are not listed volume/red blood cell count ratio and hemoglobin electro- here include hemolytic anemias with membrane structural phoresis) continues to be very useful in screening for thalas- defects or enzyme deficiencies and porphyrias. semias. Molecular techniques that utilize amplification are FANCONI ANEMIA (FA) cost and time effective, although sequencing remains the Numerous genes are implicated in the development of preferred method for second-line testing.22 FA (Chapter 16); however, most people have a mutation in HEMOGLOBINOPATHIES the FANCA, FANCC, FANCE, or FANCG genes.19 Molecular Hemoglobin (Hgb) S, Hgb C, and Hgb E are abnormal genetic testing with amplification methodologies is clini- variations of Hgb A and account for a large percent of cally available for most of the genes, but increased mito- hemoglobinopathies that develop as a result of amino mycin C or diepoxybutane-induced chromosome breakage acid substitutions in the b@globin chain of hemoglobin remains the gold standard of diagnosis. ( Chapter 13). Although the abnormal proteins can be HEMOCHROMATOSIS identified with protein electrophoresis, DNA amplification Mutations in the HFE gene are seen in HFE-associated and sequencing of the b@globin gene cluster provide reliable hereditary hemochromatosis (Chapter 12). Previously, a testing methodologies.22 1046 Chapter 42 Hgb Lepore is a rare hemoglobinopathy in which a is at location 210 on the BCR gene. The fusion protein pos- fusion occurs between the d@globin and the b@globin genes, sesses constitutive tyrosine kinase activity. Tyrosine kinase HBD and HBB.23 Multiplex PCR and gel electrophoresis can inhibitors (TKIs) are an effective treatment in many cases of be used to detect the three common Hgb Lepore mutations. CML, but the BCR-ABL1 T3151 mutation imparts resistance to all currently approved TKIs. POLYCYTHEMIA VERA A somatic mutation in Janus kinase 2 (JAK2) at exon 14 CALR (19p13.2) The protein product of CALR has an causes the V617F mutation, which is found in about 97% of important function in cell proliferation and differentiation patients with polycythemia vera (Chapter 24). Although the via calcium regulation. CALR mutations, along with BCR/ single SNP mutation in exon 14 is the most common muta- ABL1 fusion, MPL, and JAK2 mutations have all been shown tion that results in polycythemia vera, many cases have a to be sufficient for development of MPNs. JAK2 exon 12 mutation. These mutations are revealed with JAK2 (9p24) The protein encoded by this gene participates PCR amplification techniques and HRM analysis.24 in the JAK/STAT signaling pathway, which transmits extra- cellular signals to the nucleus. This pathway controls the Leukocyte Disorders production of blood cells from hematopoietic stem cells. Cytogenetic analysis or karyotyping is essential for diag- Two mutations to this gene are characteristic of the MPNs nostic evaluation of hematopathologies, especially the (other than CML). Jak2V617F produces a protein that has an leukemias (Chapter 41). Many leukemic disorders result altered growth factor response. The mutation is detected in from somatic mutations in either a multipotential or lin- most polycythemia vera patients, and a significant number eage-restricted progenitor. Cell signaling and downstream of essential thrombocythemia and primary myelofibrosis growth, differentiation, and proliferation become dysregu- patients. Various gain of function mutations in the exon 12 lated as a result (Chapter 23). region of JAK2 (JAK2 exon 12 mutations) exist in JAK2V617F- Cytogenetic analysis can reveal chromosomal deviance negative MPN patients. such as translocations, insertions, inversions, deletions, and MPL (1p34) Thrombopoietin phosphorylates/activates repeats that are strong predictors of leukemia. Molecular JAK2, which then phosphorylates/activates MPL. Phos- DNA testing can reveal these same genetic abnormalities phorylated MPL activates both the JAK/STAT and the by designing nucleic acid probes that recognize the abnor- MAPL signaling pathways. Aberrant MPL activity has been mal fusion gene. Although DNA technology is more sensi- shown to be a significant factor in the development and tive than karyotyping, it is limited to testing for a specific progression of MPLs. gene mutation. On the other hand, karyotyping has the advantage of presenting the clinician a global picture of MYELODYSPLASTIC SYNDROME (MDS) the patient’s entire genetic situation. The following sec- The genetic mutations associated with the development and tions provide a compilation of molecular abnormalities and progression of MDS are extensive and variable. In recent examples of the genes commonly involved in leukocyte dis- years, the genetic mutations that contribute to the develop- orders. Investigation of these mutations generally involves ment and progression of MDS can be categorized into muta- microarray, FISH, or sequencing techniques utilized in test tions involved in transcriptional control, epigenetic control, panels. tumor suppression, cell signaling, and splicing factors (see Table 25.1).25,27 MYELOPROLIFERATIVE NEOPLASMS (MPNS) Myeloproliferative neoplasms (MPNs) are characterized by ACUTE MYELOID LEUKEMIA (AML) a clonal proliferation of mature blood cells (Chapter 24). In recent years, chromosomal abnormalities have been iden- Many mutations germane to the development, prognosti- tified in nearly half the adults presenting with AML. Some cation, and treatment of MPNs have been identified in the cytogenetic abnormalities correlate with longer remissions past decade. Mutations affecting cell signaling, cytokine whereas others are associated with a poor response to ther- receptors, cellular proliferation, differentiation, apoptosis, apy and abbreviated survival.25,28 and immunologic response are significant in the develop- ASXL1 (20q11) This gene encodes a protein that alters chro- ment of MPNs.25,26 matin structure, which may enhance transcription of some BCR/ABL1 (22q11.23/9q34.1) The function of the BCR genes and repress the transcription of others. Mutations in gene product has not been identified, but ABL1 codes for the ASXL1 gene are also associated with the development a tyrosine kinase that participates in the cell signaling of of MDS and CML. cell differentiation, cycling, and proliferation. A reciprocal CEBPa (19q13.1) The transcription factor encoded by this translocation between chromosomes 22 and 9 produces the gene promotes myelocytic maturation and differentiation. Philadelphia chromosome, which is often found in patients It also functions as a tumor suppressor. If mutated, its aber- with chronic myeloid leukemia when the major breakpoint rant effect is specific to AML. Molecular Analysis of Hematologic Diseases 1047 KIT (4q12) Receptor tyrosine kinases encoded by KIT genes TET2 (4q24) TET2 -mutant AML often displays increased mediate signaling pathways that control many important promoter methylation, which leads to speculation that TET2 cellular processes such as cell proliferation, survival, and could be useful as an epigenetic marker of loss of function migration. Normal c-KIT expression is crucial for normal of gene promoters. hematopoiesis. Although KIT mutations are not often seen TP53 (4q24) The TP53 protein appears to act as a tumor in AML, if the AML presents with a core binding factor suppressor, which is a protein that prevents (hematopietic) (CBF) mutation (as in t(8;21) or inv(16)), the incidence of cells from growing and dividing in an uncontrolled way. KIT mutations increases to nearly 30%. TP53 mutations are mostly associated with AML with com- DNMT3A (2p23) DNMT3A gene directs the synthesis of plex karyotype. DNA methyltransferase 3a. This enzyme is essential for ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) epigenetic control of the genome. DNMT3A mutations, The development of ALL is complicated. It seems that there along with FLT3 and NPM1 mutations, are the most com- is heritable genetic predisposition for the development of mon anomalies seen in AML. ALL and that acquired “second hits” are probably necessary FLT3 (13q12) FLT3 mutations in AML are of two types, as well. BCR/ABL1, ETV6/RUNX1, and MLL gene rearrange- FLT3-ITD and FLT3-TKD. FLT3 encodes a receptor tyrosine ments are thought to be crucial for the initiation of ALL. kinase that ITD mutations constitutively activate, confer- Developmental arrest of the hematopoietic cells is also fun- ring intensified cell survival and proliferation in the hema- damental to ALL progression; the mutations that result in topoietic stem cells housing the mutation. The relevance developmental constraint include PAX5, IKZF1, and EBF1. of TKD mutations is less clear. DNMT3A, NPM1, and FLT3 In addition to the initiating events and the developmental mutations are the most frequently observed mutations arrest that must occur in order for ALL to progress, there observed in AML. are many cooperating events that contribute to the devel- opment of ALL. These involve mutations to genes of the IDH1/IDH2 (2q33.3/15q26.1) Mutations to these genes are cell cycle, tumor suppressors, cytokine receptors, cell sig- gain-of-function mutations that are oncogenic. Patients with naling, and epigenetic regulators of hematopoiesis. These NPM1 mutations and wild-type FLT3 have a better prog- gene mutations are generally included as part of panels nosis if IDH mutations are present, thus identification of that investigate myelocytic and lymphocytic hematopoietic mutations in these genes may be valuable to predict patient pathologies.29,30,31 The testing can be done with microarray, outcomes. FISH, or sequencing techniques. MLL (11q23) Certain MLL alterations correspond with BCR/ABL1 (22q11.23/9q34.1) BCR and ABL genes fuse in a specific leukemic diseases (i.e., AML and ALL), as well as well-represented translocation seen in adult-onset ALL of outcomes (favorable or poor). For this reason, identifying the precursor B-cell type. As it does in CML, the fusion gene MLL alterations is crucial for rational decision making with produces a constitutively active tyrosine kinase. regard to treatment. ETV6/RUNX1 (12p13/21q22.3) ETV6 and RUNX1 are fused NPM1 (5q35.1) Nucleophosmin (NPM) protein prevents in t(12;21), one of the most common genetic abnormali- cells from growing and dividing in an uncontrolled way. ties seen in childhood ALL. Reduced differentiation and NPM1 mutations (along with DNMT3A and FLT3 muta- enhanced self-renewal of B-lineage hematopoietic progeni- tions) are the most common genetic permutations that occur tor cells accompanies ETV6/RUNX1 fusion. in AML. MLL (11q23) The MLL gene encodes a transcription protein KRAS, NRAS (12p12.1, 1p13.2) KRAS and NRAS are essential for regulating gene expression during early devel- oncogenes—mutated versions of normal proto-oncogenes. opment and hematopoiesis. The Ras protein product of the KRAS/NRAS proto-oncogenes PAX5 (9p13) PAX5 encodes an activating transcription fac- participate in the ubiquitous MAPK(ERK) signaling path- tor specific for B-cells. The gene is expressed at early, but not way. Mutated KRAS/NRAS can result in abnormal cell divi- late, stages of B-cell differentiation. sion, differentiation, and apoptosis, often leading to the development of malignant cells in both AML and ALL. IKZF1 (7p12.2) The protein expressed as a result of the activation of IKZF1 functions as a regulator of lymphocyte RUNX1/RUNX1T1 (21q22.3/8q22) RUNX1 and RUNX1T1 differentiation. are part of the t(8;21) fusion gene of AML with recurrent abnormal karyotype. Fusion of RUNX1 and RUNX1T1 EBF1 (5q33.3) Early B-cell factor 1 (EBF1) is a transcription disallows the normal formation of RUNX1:CBFa, with the factor encoded by EBF1 that is critical for B-cell lympho- result that differentiation is hindered, but proliferation pro- poiesis and function. The protein is essential for B-lineage ceeds actively. specification and commitment. 1048 Chapter 42 CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) places the CCND1 (cyclin D) gene next to the IGH promoter. Chronic lymphocytic leukemia is an indolent B-cell leuke- This genetic change triggers the transformed B-lymphocyte mia that is characterized bv a clonal expansion of B-lym- (lymphoma cell) to overproduce cyclin D1, a protein that phocytes able to reach maturity in the peripheral blood, directs progression of cells from G1 to S phase of the cell bone marrow, and lymph tissue. The indolent nature of CLL cycle (Chapters 23, 28). progression often means that patients are not in need of MYC/IGH The proto-oncogene MYC (C-MYC) encodes treatment. In patients in need of treatment, certain genetic markers can help predict treatment response.32 a nuclear protein that mediates the cellular response to growth factors. The t(8;14)(q24.1;q32) rearrangement leads DEL (13q14) This genetic lesion is one of the most fre- to dysregulated cell growth because C-MYC is brought quently encountered in CLL. The deleted region contains under control of the promoter for IGH in lymphoprolifera- microRNAs that inhibit the expression of several genes tive disorders (Chapters 27, 28). The variability in the trans- including BCL2 and CCND1. The protein products of location break point in this gene rearrangement makes FISH these genes have an inhibitory effect on cell survival; the a good choice for detection. MYC gene rearrangements help del(13q14) mutation reduces the inhibitory effect, with the differentiate between Burkitt lymphoma and diffuse large result that normal cell apoptosis is reduced. The mature B-cell lymphoma (DLBCL) since all MYC rearrangements, lymphocytic malignancies

are characterized by the inabil- including t(8;14), t(2;8), and t(8;22), can be determined. ity of affected cells to undergo a normal death procedure. TP53 (17p13.1) TP53 codes for a central regulator of DNA Checkpoint 42.3 damage response by the cell. Mutations of TP53 often mani- NRAS/KRAS mutations are seen in a wide variety of hematologi- fest with poor prognosis and chemo/immunotherapeutic cal cancers including ALL, AML, and plasma cell neoplasms. refractoriness in CLL. Explain how mutations in these genes contribute to the etiology of these diseases. NOTCH1 (9q34.3) The normal gene codes for transmem- brane receptors involved in signaling that regulates cell death, proliferation, and differentiation. Gain of function mutations result in a constitutively active protein. Mutations Infectious Diseases may predict for a lower response to anti-CD20 treatment. Molecular testing for the presence of infectious disease has an application for hematological pathologies (Table 42-3). ATM (11q22–q23) ATM gene inactivation is associated with Three of the pathogens listed, human T-lymphotropic virus a del11q karyotype. ATM helps regulate cell-cycle arrest at type 1 (HTLV1), Epstein-Barr virus (EBV), and human G1/S and G2/M checkpoints to allow repair of damaged herpes virus 8 (HHV8) have been consistently linked to DNA. The presence of an ATM deletion translates into a poor lymphoid neoplasms. About 0.1% of people infected with response to chemotherapy, although this can be improved HTLV1 eventually develop adult T-cell leukemia/lym- with the addition of rituximab to the treatment regimen. phoma. EBV is the causative agent of infectious mono- SF3B1 (2q33.1) Found mostly in patients with del11q, muta- nucleosis and is believed to be an etiologic agent in the tions of this gene co-occur with ATM deletions. The gene development of Burkitt lymphoma as well as immunodefi- codes for an RNA protein that forms part of the spliceosome. ciency-related lymphomas and many Hodgkin lymphomas. SFB1 mutations predict an aberrant response to DNA damage. HHV8 has been isolated from a majority of Karposi sarco- LYMPHOMA mas in AIDS patients and can cause other tumors such as Most lymphomas (and CLL) exhibit great variability in primary effusion lymphoma (PEL) and multicentric Castle- man’s disease (MCD).33 mutations. Most of these mutations need to be further scrutinized to determine their value in the diagnosis, prog- Molecular technology has provided new tools for nostication, and treatment of lymphomas. What follows detecting microorganisms based on the unique genetic code are some well-known translocations associated with three important lymphomas. Table 42.3 Pathogens of Hematologic Significance Detectable by Molecular Techniques IGH/BCL2 The t(14;18)(q32;q21)IGH/BCL2 is the mutation associated with follicular lymphoma (Chapter 28) in which Cytomegalovirus (CMV) Epstein-Barr virus (EBV) the anti-apoptotic gene BCL2 is brought under the control Human herpes virus 8 (HHV8) of the IGH promoter. The influence of the Ig heavy-chain Human immunodeficiency virus (HIV) promoter leads to overexpression of the BCL2 protein and Human T-lymphotropic virus type 1 (HTLV1) Malaria reduced apoptotic cell death in cells with this mutation. Mycobacteria Mycoplasma CCND1/IGH The t(11,14)(q13;q32)CCND1/IGH mutation is Parvovirus B19 found in nearly all mantle cell lymphomas. Translocation Toxoplasma Molecular Analysis of Hematologic Diseases 1049 of each species. DNA amplification strategies are most use- assays have traditionally been used to diagnose this disorder, ful because they are sensitive, specific, and rapid for detect- molecular testing has the advantage of determining whether ing pathogen-specific nucleic acid sequences. Quantitative patients are homozygous or heterozygous for the mutation. qPCR assays are useful for monitoring the level of organ- Homozygotes have more than 10 times the clotting risk of het- isms during treatment. In situ hybridization is helpful for erozygotes. Molecular testing includes PCR amplification.35 identifying lesion-specific pathogens in biopsy samples. Prothrombin G20210A Base substitutions in the prothrombin (F2) gene represent Clinical Applications of the second most prevalent hereditary defect underlying Molecular Diagnostics in thrombosis risk. An SNP is responsible for a gain of func- tion mutation that leads to excessive plasma prothrombin. Hemostasis Inheritance of both FVL and G20210A confers a significant increase in risk of a venous thromboembolic event. PCR is Molecular testing in disorders of hemostasis is proving to used to detect these mutations.35 be invaluable for diagnosing hemostasis disorders, screening potential carriers of hemostasis diseases, and making thera- Hemophilia A peutic decisions. It is beyond the scope of this chapter to pro- Hemophilia A can arise from numerous mutations, includ- vide a comprehensive review of all genetic abnormalities that ing inversions, insertions, and deletions of the factor VIII underlie disorders of hemostasis, but a review of some of the (F8) gene. Carrier testing on daughters of either hemophili- most commonly encountered molecular aberrations follows. acs or carriers and prenatal testing of a fetus can be per- formed with amplification methods.36 CYP2C9 CYP2C9 is a cytochrome P450 enzyme that is involved in Hemophilia B the metabolism of several medications. CYP2C9 metabo- More than 1,000 mutations in the factor IX gene have been lizes more than 100 therapeutic drugs including warfarin, reported. Carrier status of daughters of men with hemo- a widely prescribed anticoagulant, but its dosage is diffi- philia B and at-risk fetuses is commonly achieved using cult to predict because of widely variant patient responses. amplification techniques. Direct sequencing of the factor IX CYP2C9 genotype establishes a patient’s ability to metabo- (F9) gene is available at large reference laboratories.36 lize this medication. For example, individuals with the CYP2C9*2 or CYP2C9*3 genotypes show reduced ability to metabolize warfarin, leading to its prolonged retention Methylenetetrahydrofolate in their system. These patients require a reduced dose of Reductase (MTHFR) warfarin for safe anticlotting treatment. qPCR is used to A C 7 T missense mutation in the MTHFR gene results in determine CYP2C9 polymorphisms.34 the substitution of alanine for valine with the downstream effect of elevated levels of plasma homocysteine. Even VKORC1 small increases in homocysteine are attributed to increased This gene’s product encodes the enzyme that is responsible thrombotic risk. Molecular testing for the MTHFR mutation for reducing vitamin K 2,3-epoxide to an active form so has become readily available in the clinical laboratory. The that it can participate in the synthesis of several vitamin most common methodologies are PCR based. K-dependent coagulation factors. Fatal bleeding can be caused by vitamin K deficiency and by warfarin therapy von Willebrand Disease (VWD) because warfarin is a vitamin K antagonist. Mutations Due to the complexity of VWD genotype, the criteria in this gene are associated with deficiencies in vitamin for its diagnosis no longer include von Willebrand fac- K-dependent clotting factors. Patients harboring this muta- tor (VWF) gene mutations. The International Society of tion should be given a reduced warfarin dose. As with Thrombosis and Hemostasis maintains a database of VWF CYP2C9, molecular testing is most often by qPCR.34 polymorphisms.36 Factor V Leiden (FVL) Checkpoint 42.4 A lack of standard nomenclature means that the SNP that Explain how PCR technology can provide quantitative results. is responsible for the factor V (F5) mutation responsible for Suggest several hematologic and hemostasis applications of the development of FVL can be referred to in several ways, qPCR technology. including G1691A, Arg506Gln, and R506Q. Although clotting 1050 Chapter 42 Summary DNA technology provides a powerful new tool for labora- molecular diagnosis. All infectious organisms are suitable tory diagnosis of a wide variety of hematologic diseases targets for molecular detection because invading organisms including inherited diseases, infectious diseases, and can- have unique genomes that can be differentiated from the cer. Many of the erythrocytic and hemostatic diseases that host genome, denoting infection. are amenable to molecular diagnosis are inherited diseases The molecular methods commonly used include mul- whose mutations and genetic aberrations are discern- tiple variations of nucleic acid amplification, fluorescent in able with multiple molecular techniques. Leukemias and situ hybridization, and Southern blot analysis. Newer meth- lymphomas have chromosomal translocations or clonal ods such as DNA sequencing and microarray technology gene rearrangements that are also easily confirmed with are also gaining a foothold in clinical laboratory testing. Review Questions Level II 3. Which of the following reagents is most critical for making a PCR specific for the F5 gene mutation (FVL) Use this case history for questions 1 and 2. as opposed to a prothrombin F2 gene mutation? (Objective 2) A 38-year-old woman complained of dizziness, fatigue, and abdominal pain. On physical examination she a. Primers appeared pale, and her spleen was enlarged. Labora- b. Nucleotides tory studies revealed anemia and an elevated leukocyte c. DNA polymerase count of 69 * 103/mcL. A complete spectrum of granu- d. Buffer locytic cells from myeloblasts to neutrophils was present in the blood, and the number of basophils was increased. 4. All molecular tests that analyze specific portions of the The bone marrow could not be aspirated, but a biopsy human genome rely on the principle that: (Objective 2) revealed that the marrow was packed with myeloid ele- a. DNA is different in every cell of a particular ments. Cytogenetics could not be performed because of individual. the lack of an adequate marrow aspirate and the inabil- b. Probes bind to their complementary target ity to induce cell division in peripheral blood leukocytes. sequence through the hybridization process. Blood was submitted for molecular diagnostic testing. c. Restriction endonucleases cut sites that remain the same regardless of any mutations. 1. Which genetic defect is the most appropriate t arget d. Heat or alkaline pH can convert single-stranded for molecular testing to assist in diagnosing the DNA to double-stranded DNA. patient’s hematologic disorder? (Objective 4) a. PML/RARA 5. Which of the following assays is most appropriate for detecting a tumor-associated genetic defect that is b. BCL2/IGH present in only 0.1% of the cells in a patient sample? c. BCR/ABL1 (Objective 7) d. BCL1/IGH a. PCR 2. Is there any reason to use molecular testing for the b. Southern blot analysis patient at a later date? (Objectives 3, 6) c. Karyotyping a. No, the positive test results are definitive, and d. Immunophenotyping nothing more can be accomplished. b. Yes, the patient can be monitored to detect minimal 6. PCR differs from reverse transcriptase PCR (RT-PCR) residual disease following therapy. in the following way(s): (Objectives 2, 7) c. No, but family members should be tested for the a. Ribonucleotides rather than deoxyribonucleotides same molecular defect. are added to the reaction mixture of RT-PCR. d. Yes, the translocation break point must be b. Following amplification, PCR generates a DNA prod- sequenced to prove which genes are involved. uct, whereas RT-PCR generates an RNA product. Molecular Analysis of Hematologic Diseases 1051 c. RNA rather than DNA serves as the substrate for 9. Characteristics of next-generation sequencing include RT-PCR. which of the following? d. RT-PCR generates a protein product, whereas PCR a. The ability to sequence numerous targets generates a DNA amplicon. simultaneously 7. Immunoglobulin and T-cell receptor gene b. The possibility of detecting variants of unknown rearrangement studies can be used to: (Objective 5) significance c. The ability to sequence whole genomes a. distinguish B-cell leukemia from B-cell lymphoma d. All of the above are characteristics of next- b. determine whether a lymphoid clone is present in generation sequencing a tissue specimen c. prove that a tissue sample is benign 10. A technique that uses amplification of a patient d. detect Epstein-Barr virus in a tissue specimen sample target that is applied to a blot of wild type and mutant oligonucleotides is called a(n): 8. Which of the following is true about the molecular ( Objectives 2,7) genetics of cancer? (Objective 3) a. Southern blot a. Virtually all cancers are thought to harbor genetic b. qPCR defects. c. direct sequencing b. DNA testing can be helpful in making a cancer d. ASO diagnosis. c. The genes responsible for tumor formation are called oncogenes. d. 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Define accuracy, precision, control material, ment program and match sources of error mean, and standard deviation. to each component (e.g., pre-examination 10. Given the appropriate data, calculate the component). mean and standard deviation and create a 2. State the importance of a quality assessment quality control chart. program. 11. Interpret quality control results using estab- 3. State the importance of documentation in a lished control charts. quality assessment program. 12. Given test results, recognize complete blood 4. Describe the use of proficiency testing in count (CBC) data and/or histogram varia- the clinical laboratory, including required tions that indicate the presence of white frequency. blood cell (WBC), red blood cell (RBC), and 5. Given data, use an appropriate method platelet abnormalities. to determine the reference interval for an 13. Recognize complete blood count (CBC) data analyte. that indicate the presence of interfering 6. Define universal precautions and identify substances such as lipemia, hemolysis, and their source. icterus. 7. Demonstrate knowledge of Occupational 14. Identify coagulation test results that indicate Safety and Health Administration (OSHA) a problem with specimen integrity. standards and their application in the clini- 15. Define patient safety and the six quality cal laboratory. aims to measure and improve healthcare. 8. Given a safety data sheet (SDS), identify critical information. 1054 Quality Assessment in the Hematology Laboratory 1055 Objectives—Level II At the end of this unit of study, the student should be able to: 1. Design methods of competency testing. 7. Assess the use of patient specimens to moni- 2. Apply and interpret statistics used in tor daily quality control in hematology. method evaluation. 8. Select the appropriate actions to take when 3. Determine the components and interpret the abnormalities are detected in hematology or results of a method evaluation study. coagulation results. 4. Determine and appraise a method’s report- 9. Recommend procedures to correct for the able range. presence of lipemia, hemolysis, and icterus. 5. Interpret the Westgard rules and explain 10. Demonstrate the ability to use delta checks their use in evaluating quality control in a quality control program. results. 11. Describe how to incorporate the patient 6. Describe the use of moving averages to safety quality aims into the laboratory qual- monitor red blood cell (RBC) data. ity improvement program. Chapter Outline Objectives—Level I and Level II 1054, 1055 Quality Control 1066 Key Terms 1055 Review of Patient Results 1069 Background Basics 1056 Summary 1074 Overview 1056 Review Questions 1074 Test Coding and Reimbursement 1056 References 1076 Quality Assessment 1056 Key Terms Analytical measurement range Delta check Random variation (AMR) Diagnosis-related group (DRG) Reference interval (RI) Analytical sensitivity Health Insurance Portability and Reportable range Analytical specificity Accountability Act (HIPAA) Safety data sheet (SDS) Analytical time International Classification of Dis- Slope (b) “Blinded” preanalyzed specimen ease, 10th revision (ICD-10) Split specimen Clinical Laboratory Improvement Internal quality control program Standard deviation (SD) Amendments of 1988 (CLIA ’88) Linearity Systematic variation Clinical and Laboratory Standards Medical decision level Transcription error Institute (CLSI) Outlier Turnaround time (TAT) Competency assessment Patient safety Universal precaution Correlation coefficient (r) Proficiency testing y-intercept (a) Current Procedure Terminology Quality control (QC) limit (CPT) code Critical value 1056 Chapter 43 Background Basics The information in this chapter builds on the concepts • Summarize the characteristics of an optimally stained learned in previous chapters. To maximize your learning peripheral blood smear and give potential sources of experience, you should review these concepts before start- error. (Chapters 10, 37) ing this unit of study: • Summarize each of the screening coagulation tests used in the laboratory and give potential sources of Level I and Level II error. (Chapter 36) • Describe the specimen collection protocol for hema- • Describe the principles of cell counting used by the tology and hemostasis procedures. (Chapters 36, 37) automated hematology instruments and principles of • Summarize each of the routine hematology proce- clot detection for the automated coagulation instru- dures and give potential sources of error. (Chapter 37) ments. (Chapters 36, 39) Overview DRG code is assigned when the patient is discharged and is adjusted for severity of the condition (MS-DRG). One of the most important responsibilities of a labora- The laboratory uses the Current Procedure Terminol- tory professional is to ensure the quality of test results. ogy (CPT) code (Chapter 1)when billing for outpatient tests. To accomplish this, laboratories must establish quality The CPT code represents a number assigned to a laboratory assessment and quality control programs. These programs test (as well as medical, surgical, and other diagnostic ser- consist of guidelines designed to ensure accurate testing vices) by the American Medical Association’s CPT Editorial and reporting of results. A protocol for reviewing patient Panel. For example, the ICD-10 code for a patient with per- results to determine whether results can be reported must nicious anemia is ICD-10 D51.0 and the CPT code for intrin- be included. This chapter discusses components of these sic factor antibodies is 86340. Third-party payers review a programs. test’s CPT code and determine the medical necessity for that specific test based on the ICD code for the patient’s con- dition. Reimbursement is based on a predetermined rate Test Coding and within a geographic region for each test. Reimbursement Laboratory professionals are naturally concerned about Quality Assessment the quality of laboratory test results; an additional con- Laboratories must have an established quality assessment sideration, of necessity, is ensuring that appropriate tests program as mandated by subpart K—Quality Systems for are being ordered and coded correctly so the laboratory

Nonwaived Testing of the Clinical Laboratory Improve- can be reimbursed for the testing performed. In addition ment Amendments of 1988 (CLIA ’88).1 A laboratory’s to the order for a particular test, the health care provider quality assessment program should be designed to moni- must supply information for purposes of reimbursement tor all aspects related to testing patient specimens1 to ensure by Medicare and other third-party payers. This includes accurate testing and reporting of results from all specimens the World Health Organization’s International Classifica- submitted to the laboratory. Accrediting organizations such tion of Disease, 10th revision (ICD-10) codes that are used as the College of American Pathologists (CAP) and The Joint in most of the world. The ICD coding system is interna- Commission monitor the quality and comprehensiveness of tionally accepted as a method for coding medical diagno- assessment programs. This chapter reviews important ele- sis, conditions, and symptoms and the cause of death in ments of a comprehensive quality assessment program. all health care settings. One of its uses is to help evaluate medical necessity and determine reimbursement for tests and procedures. Basic Components Another coding system, the diagnosis-related group A common approach to developing a quality assessment pro- (DRG), was developed for Medicare as a part of the pro- gram is to divide it into three components: (1) pre-e xamination spective payment system for inpatients. Reimbursement for (previously referred to as preanalytical), which examines care of hospitalized patients by almost all payers does not all aspects affecting the test outcome occurring before the occur for each test or procedure but for the overall diag- testing procedure, (2) examination (previously referred to nosis, the DRG. There are approximately 500 DRGs. The as analytical), which incorporates all aspects affecting the Quality Assessment in the Hematology Laboratory 1057 testing procedure itself, and (3) post-examination (previ- ously referred to as postanalytical), which addresses aspects Table 43.1 Comprehensive Quality Assessment Program affecting the test outcome occurring after the testing proce- Pre-examination Patient test requisitions dure (Table 43-1). components Patient preparation Specimen collection protocol Specimen transport protocol PRE-EXAMINATION COMPONENT Specimen processing protocol The testing process begins with the test order. Laboratory Specimen acceptability and rejection criteria test requisitions, in electronic (computerized provider order Specimen storage Phlebotomy training entry [CPOE]) or paper formats, should be designed to be Examination Test method/procedure user friendly and to provide adequate patient information. components Reagents At a minimum, this information should include the patient’s Internal quality control External quality control (proficiency testing) name and unique patient identifier, age, sex, diagnosis, test Instrument maintenance to be performed, and source of the specimen. Linearity/reportable range determination Method evaluation (instrument comparison) The patient should receive appropriate information to Reference range determination or verification prepare for the tests; for example, they must abstain from Personnel requirements taking aspirin or aspirin-like medication before platelet Competency testing Continuing education function testing. The laboratory’s specimen collection pro- Post-examination Reviewing patient results cedure manual should provide this information in a format components Posting patient results easily distributed to the patient and clear enough for that Maintaining patient records Monitoring turnaround time person to understand. Administering and reviewing customer satisfaction One of the most important factors affecting a test’s out- surveys Documenting maintenance come is specimen collection3 (Chapters 36, 37). Many vari- ables enter into the specimen collection process that can room temperature if testing is to be performed within 4 affect the outcome (Table 43-2). The specimen collection pro- hours (Chapter 36). All information specific to the test to be cedure manual and a thorough educational program for the performed should be included in the specimen collection phlebotomist or the individual designated to perform the procedure manual. phlebotomy (i.e., nursing personnel) should address each potential error. In addition, staff should participate in peri- EXAMINATION COMPONENT odic continuing education addressing specimen collection The examination component addresses all factors involving problems and introducing new protocols. the testing procedure itself. A test method procedure man- When the specimen has been collected, it must be ual should be available in all laboratories. The procedures properly labeled with patient’s name, unique identifica- within this manual should follow the guidelines established tion number, collection date and time, and phlebotomist’s by the Clinical and Laboratory Standards Institute (CLSI) initials and then transported to the laboratory for process- and address each test, including its purpose, principle, spec- ing and testing. If testing cannot be done immediately, the imen requirements, reagents, quality control, step-by-step specimen should be stored properly. For example, a speci- procedure, reporting method, reference interval(s), critical men for routine coagulation testing must be centrifuged and value(s) if appropriate, interpretation of results, potential the plasma must be separated from the cells and stored at sources of error, and references.4,5 Table 43.2 Potential Sources of Errors in Specimen Collection and Their Effect on Test Outcome Source of Error Effect on Test Outcome Patient misidentification Inaccurate test results Hemolyzed specimen Dilutional effect on analytes, false increase of analytes, and decreased erythrocyte counts Failure to properly mix by inversion collection tubes that contain Clotted specimen and falsely decreased cell counts or prolonged coagulation test results anticoagulant Failure to fill collection tube properly Under- or over-anticoagulated specimen for coagulation testing with citrate tube Failure to follow the order of the drawa Cross-contamination with collection tube additives Tourniquet application longer than 1 minute Hemoconcentration of specimen Collection from an IV site Dilutional effect on analytes Time of draw Analyte dependent (e.g., hemoglobin is highest in the morning) Patient anxiety or crying Analyte dependent (e.g., increases leukocyte count) a Order of the draw refers to the suggested order in which different anticoagulant and non-anticoagulant collection tubes are filled from a single venipuncture. For example, a heparin collection tube should be filled before an EDTA collection tube to avoid contamination of the heparin collection tube with potassium EDTA (Chapter 37). 1058 Chapter 43 Under regulations to meet CLIA ‘88 legislation, each Clinical laboratories provide a service. Therefore, qual- laboratory is required to establish an internal quality con- ity assessment programs need to include assessments of trol program to monitor its testing process to ensure accu- customer (e.g., physician or patient) satisfaction and com- rate patient test results on a daily basis. Quality control is munication. A committee oversees the quality assessment addressed in more detail in the “Quality Control” section program, establishing protocols and determining changes in this chapter. In addition, each laboratory is required to that need to be made and how to implement them. As an participate in an external quality control program to assess example, the committee establishes protocols to address overall quality control, also known as proficiency test- customer complaints and other communication issues to ing6 (addressed in the “Proficiency Testing” section of this minimize customer dissatisfaction. Surveys can be used to chapter). assess customer satisfaction and identify areas that need Maintenance of analytical instruments (e.g., automated to be addressed. An important factor affecting satisfaction blood cell–counting instrument) must be performed as level is the turnaround time (TAT) for test results. Criti- directed by the manufacturer, documented, and be easily cal patient care decisions often depend on a laboratory test accessible for troubleshooting quality control problems. result. As a result, TAT problems represent a common Individuals performing the testing procedures must complaint that the committee will investigate and identify meet the personnel requirements established by CLIA ’88, appropriate action to correct specific TAT problems (e.g., which vary depending on the complexity of the testing delayed reporting of CBC results to the emergency depart- procedure. Continuing education is also required to keep ment). Investigating TAT problems is more manageable testing personnel aware of changes within the testing pro- with computerization (i.e., hospital information system cedures and the practice of the profession. [HIS] and LIS). The committee also reviews critical value records to identify problems with timely reporting of these POST-EXAMINATION COMPONENT values and recommends remedies to correct the problems. The post-examination component addresses factors that In accordance with CLIA ’88 and the Health Insurance can affect the test result and its use in patient treatment. Portability and Accountability Act (HIPAA), each labora- Procedures should be established for reviewing patient tory should establish measures to ensure confidentiality of results and identifying those results that require further patient information in each component of the quality assess- attention, such as a critical value (test result that exceeds ment program.1,7 For example, many facilities use unique its reference interval to the extent that it indicates potential identifiers rather than patient names or social security num- life-threatening condition requiring immediate attention bers to identify a specimen and its test requisition. For the by a physician) or results that do not correlate with other post-examination component, the laboratory should have test results. For example, a specimen should be retested if a policy to ensure and document that the appropriate indi- the hemoglobin (Hb) and hematocrit (Hct) do not match vidual receives electronically transmitted test results. (e.g., Hb * 3 = Hct). Finally, documented records of all aspects of the quality The laboratory should have a policy for reporting criti- assessment program should be maintained and retrieved cal values. It should identify the laboratory professional upon request. These documents provide important informa- (e.g., testing personnel or supervisor) who is responsible tion regarding the recognition of a problem, the process for notifying a health care provider of a patient’s critical used to resolve it, and the change that occurred as a result value and the specific health care provider who should of that process. receive the test result (e.g., ordering physician). The method of communication can be phone notification or automated electronic messaging. The record of notification for a critical Checkpoint 43.1 Explain the importance of each component of the quality value should include patient name, unique identification assessment program to the goal it is intended to meet. number, test result, date and time of notification, health care provider who received the test result, and laboratory profes- sional who communicated the test result. HEALTHCARE QUALITY IMPROVEMENT Automated instruments can be interfaced with the After To Err is Human was published in 2000, healthcare laboratory information system (LIS) to electronically trans- professionals added the task of improving patient safety fer patient results. The LIS can also be interfaced with the to their quality improvement plans and projects. From this hospital’s or outpatient facility’s computer system to add report a patient-centered definition of healthcare quality patient results directly to the patient’s chart. Electronic was identified: “the degree to which health services for indi- transfer of results minimizes transcription errors. Records viduals and populations increase the likelihood of desired of patient test results should be maintained within the labo- health outcomes and are consistent with current profes- ratory according to procedures established for encrypting, sional knowledge.”8 Patient safety is more than doing no archiving, and retrieving patient test results. harm or ensuring that a process is performed accurately; it Quality Assessment in the Hematology Laboratory 1059 is “freedom from accidental injury: avoidance, prevention, area that requires continuous attention in order to ensure and amelioration of adverse outcomes or injuries stemming that specimen integrity is consistently maintained. from the process of care.”8 Timely Timely healthcare is defined as “reducing waits Healthcare quality has six aims or components that can and sometimes harmful delays for both those who receive be used for measuring improvement of healthcare systems. and those who give care.”9 Laboratory professionals have Under the patient safety model, healthcare quality is safe, been measuring turn-around-time for their services for effective, efficient, timely, patient-centered, and equitable.9 decades. For the most part, this measurement has focused Laboratory professionals can both improve and demon- on the process that occurs within the walls of the labora- strate the value of laboratory services to the healthcare tory, from specimen receipt to the time when test results system by incorporating these six aims into their quality are released to the clinician or the electronic medical record. assessment program.10 Expanding the steps measured for the turn-around-time to Safe Safe healthcare is defined by the Institute of Medi- include the time when the laboratory order is placed to the cine (IOM) as “avoiding injuries to patients from the care time when the clinician makes a decision regarding the diag- that is intended to help them.”9 With respect to the care that nosis or treatment is one

that embodies the patient safety laboratory professionals provide, this means patients are not philosophy and creates opportunities to impact the delivery harmed during venipuncture to procure specimens and that of healthcare. An example of a timeliness measure includes patients are not harmed if the test results are delayed, per- measuring the time to diagnosis for a patient whose CBC formed inaccurately or the clinician does not act upon abnor- and differential first exhibits blasts or time to diagnosis for mal test results. Much of what is discussed in this chapter a patient whose screening procedures in coagulation are ensures the safety of the examination phase of the laboratory identified as a coagulopathy. testing process. However, in addition to the other five aims, Patient-Centered Patient-centered is defined as “providing errors or defects that may also impact the safety of laboratory care that is respectful of and responsive to individual prefer- services can occur in the pre- and post-examination phases. ences, needs, and values and ensuring that patient values guide all clinical decisions.”9 From a quality improvement Effective Effective healthcare is “providing services based perspective, laboratory professionals can improve their pro- on scientific knowledge to all who could benefit and refrain- cesses by asking their patients about their experiences in a ing from providing services to those not likely to benefit.”9 survey. It can be a short or long survey that is either adminis- This is the premise of evidence-based medicine. Measuring tered at the conclusion of the service the patient experiences, this aim in a quality assessment program will be more dif- such as at the conclusion of an outpatient specimen collec- ficult. It is not an aspect of the laboratory testing process tion procedure or sent to the patient at their home after their that can use a checklist or simple program. Measuring the experience. Although this is not a new concept for laboratory effectiveness of hematology laboratory services requires professionals, new questions could be asked about patients’ examining practice guidelines and implementing protocols experiences when reading their laboratory test information for using specific tests for specific situations. To improve on their electronic medical record, whether it was received effectiveness, laboratory professionals need to focus upon in a timely manner and if they understood the information.11 testing that is over- and under-utilized as well as misused and create an intervention to improve the utilization of a Equity The last quality aim is Equity, defined as “provid- specific test or set of tests. This should be followed by mea- ing care that does not vary in quality because of personal surement of how the change improved patient care.11 One characteristics such as gender, ethnicity, geographic loca- example of improving utilization is monitoring anti-factor tion, and socioeconomic status.”9 Equity is a complex aim Xa and the platelet count for patients who receive unfrac- for healthcare, one that includes access to healthcare ser- tionated heparin therapy. vices. Areas impacting equity that laboratory professionals can address through a quality assessment program include Efficient Efficient healthcare is different than timely health- providing educational materials at the appropriate reading care, efficient healthcare focuses upon reducing defects in the level and in languages other than English that meet the process that require additional work that is often repeated needs of their patient population.11 An example of educa- effort. Efficient healthcare “avoids waste, including waste of tional material that would be beneficial to patients includes equipment, supplies, ideas, and energy.”9 Efficiency in the information about venipuncture.12 clinical laboratory can be improved by reducing the num- ber of specimens that are rejected due to integrity concerns, such as clotted EDTA specimens, insufficiently filled sodium Checkpoint 43.2 Define effectiveness with respect to laboratory testing and iden- citrate tubes, or lengthy delays between collection and receipt tify one process to measure in the pre-examination and post- of specimens for coagulation studies. Quality improvement examination phases for the hematology section of the laboratory. studies for this aim are easier to develop, however this is an 1060 Chapter 43 Proficiency Testing Checkpoint 43.3 Proficiency testing is a required component of a quality How does a laboratory that has lost its certification to perform assessment program and represents an external quality con- protein C assays regain that certification? trol program that monitors the long-term accuracy of the different test systems (e.g., prothrombin time by the Diagnos- tica Stago STA R Max® instrument) through comparison to Competency Testing peer laboratories. Since the 1960s, many clinical laboratories An additional required component of a quality assessment have participated in proficiency testing surveys such as the program is a competency assessment of all personnel per- CAP survey program. In addition, CLIA ’88 mandated that forming nonwaived testing. This assessment takes place all clinical laboratories performing nonwaived testing (test- twice during the first year of employment and annually ing methodologies not on the waived test list; Chapter 37) thereafter. CLIA ’88 identified six elements of competency participate in a proficiency testing survey at least three that must be evaluated (Table 43-3) but did not clearly out- times a year.13 Failure to achieve an acceptable rating for line the exact mechanisms to use to evaluate competency.14,15 any given analyte (e.g., prothrombin time) in two of three Accrediting organizations such as The Joint Commission surveys can result in sanctions being placed on a laboratory, and CAP provide guidance to supervisors and laboratory such as delineation of a plan of corrective action for that test directors in developing assessment tools through their stan- procedure or suspension of the certification to perform that dards and checklists. Competency assessment must include test procedure. The Centers for Medicare and Medicaid Ser- all six elements as appropriate for a given test procedure. vices (CMS) issues the sanctions and penalties. To reinstate Direct observation checklists, random assignment of a test procedure, the laboratory must obtain an acceptable proficiency testing materials, or “blinded” preanalyzed spec- rating for that analyte in two consecutive proficiency test- imens can be used to evaluate elements of competency assess- ing surveys. ment. If the test procedure involves microscopic examination Laboratories contract with organizations such as CAP for evaluation and identification, prepared slides or digital or the American Proficiency Institute to provide the pro- images can be used. Criteria for each assessment tool must be ficiency testing service. A proficiency testing survey con- established to judge acceptable performance. In the case of a sists of unknown specimens of whole blood or lyophilized 100-cell leukocyte differential, an acceptable criterion might be serum/plasma representing the full range of values that based on the 95% confidence limits of the expert (e.g., hema- would be expected in patient specimens. For microscopic tology supervisor or pathologist) results. No single method procedures, the survey can include prepared slides and/or evaluates all elements of competency nor is it appropriate digital (photographic) images for evaluation and identifica- for all test procedures. Additionally, educational materials tion. These specimens are sent to the laboratory at specified (e.g., textbooks, selected journal articles, slide study sets, vid- time intervals, usually three times per year. The laboratory eotapes, computer-based instruction) should be available to should test proficiency specimens as part of a typical patient assist laboratory professionals to improve their competency. specimen run. Results are sent to the survey provider for statistical analysis. The survey provider determines the Checkpoint 43.4 target value (TV) for each test result through comparison What is an appropriate method to assess a laboratory profes- studies with peer laboratories and establishes the accept- sional’s competency in performing prothrombin time (PT) and able performance (AP) ranges based on the CLIA ’88 toler- activated partial thromboplastin time (APTT) using an automated ance limit (TL). For example, hemoglobin’s tolerance limit coagulation instrument? is 7%. If the target value for hemoglobin specimen 1 is 12.0 g/dL, the acceptable performance range is 11.2–12.8 g/dL (AP = TV { TL * TV). The survey provider notifies the Table 43.3 Six Elements of Competency Assessment laboratory and CMS of its findings. 1. Direct observation of routine patient test performance, including Each laboratory should have a comprehensive pro- patient preparation if applicable, specimen collection, handling, pro- gram to respond to an unsatisfactory result. The source of cessing, and testing the problem that caused the unsatisfactory result can be 2. Monitoring of the recording and reporting of test results 3. Review of intermediate test results or worksheets, quality control identified by checking for changes in the test procedure or records, proficiency testing results, and preventive maintenance reagents, reviewing the instrument’s maintenance log and records previous quality control results, and identifying changes 4. Direct observation of performance of instrument maintenance and function checks in testing personnel. When the problem has been identi- 5. Assessment of test performance by testing previously analyzed fied, corrective action can be taken to solve it. The labora- specimens, internally blinded specimens, or external proficiency test- tory should maintain proficiency testing survey results and ing specimens documentation of corrective action. 6. Assessment of problem-solving skills Quality Assessment in the Hematology Laboratory 1061 Method Evaluation/Instrument to new instrumentation available and other laboratories’ Comparison experiences with specific instrumentation. The in-house evaluation of each instrument is a crucial step in the selec- Selection, evaluation, and implementation of a new meth- tion process. At this time, all interested parties would have odology or instrument in the hematology/hemostasis labo- a hands-on opportunity to assess the actual performance of ratory should follow an established protocol. This section the instrument in a real-time laboratory. Thus, a more mean- discusses several important components to be included. ingful evaluation can be obtained with regard to whether the SELECTION instrument meets the laboratory’s needs. The more informa- Selecting a new methodology or instrument is a daunting tion the committee has on which to base its selection, the task. In the ideal setting, a committee should be formed to better the selection will be. Ultimately, the instrument selec- do this. For a new instrument selection, committee mem- tion is based on a particular laboratory’s needs and the cost bership can include the hematology/hemostasis supervisor, to meet them. For example, a full-service hematology labo- several laboratory professionals, LIS personnel, the qual- ratory requires an automated blood cell– counting instru- ity assessment supervisor, a biomedical engineer, and the ment capable of performing a complete blood count (CBC), laboratory director. platelet count, five-part leukocyte differential, reticulocyte This committee’s first task is to determine the desirable count, and immature reticulocyte fraction. characteristics of the new instrument.16 A needs assessment The process of selecting a new methodology or test survey could be used for this purpose; it should be com- system is similar. The selection committee must consider pleted by those individuals who will use the instrument the cost per test, reagents, reagents’ shelf-life and storage and by those who might be affected by its use. Desirable requirements, quality control program, test’s analytical sen- characteristics (Table 43-4) identified by this survey can then sitivity (ability to detect small quantities of the analyte), ana- be used to solicit proposals from vendors (e.g., sales person- lytical specificity (ability to determine only the analyte in nel for Beckman-Coulter, Siemens Healthcare Diagnostics, question), and linearity (range of concentration over which or Abbott Laboratories). the test method can be used), required instrumentation and The selection committee’s careful evaluation of the ven- equipment, analytical time, and specimen types that can dor’s proposal packet narrows the selection process to sev- be analyzed (e.g., whole blood, serum, cerebrospinal fluid). eral possible instruments. Committee members should also Both testing personnel and potential clients should be con- seek input from colleagues and the literature with regard sulted for their input during the selection process. Table 43.4 Elements of a Needs Assessment Survey Individuals to be included in a needs assessment Desirable characteristics of a hematology instrument Testing personnel Dimensions or footprint* Phlebotomists High throughput & minimal analysis time Specimen receiving personnel Interface capabilities with LIS Laboratory management personnel Compatibility to robotic tracks Biomedical engineers Wide test menu (i.e., 18 parameters or 23 parameters) LIS personnel Minimal maintenance requirements QA personnel Large reagent capacity Pathologists Long reagent shelf life Clients including: High sensitivity & specificity • Physicians Small specimen volume requirement • Physician assistants Specimen integrity checks • Nurse practitioners On-board quality control program • Nursing supervisors On-line troubleshooting • Critical care unit supervisors (e.g. ER, OR, ICU) Specific test methodologies (i.e., electrical impedance

or light scatter for cell counts) • Outreach facilities Low cost service contract User-friendly training for operators Low cost per test Test menu expansion capabilities (i.e., addition of reticulocyte count) *footprint refers to the physical space requirements for the instrument and its component parts. ER, emergency room; ICU, intensive care unit; LIS, lab.oratory information system; OR, operating room; QA, quality assurance 1062 Chapter 43 ANALYTICAL RELIABILITY still unacceptable, significant random variation exists within With the purchase of a new instrument or the introduction this method or reagent and/or testing personnel errors have of a new methodology, the laboratory must verify the perfor- affected the study. mance of the instrument and/or method through a series of Systematic variation is assessed through the methods performance studies. To verify an instrument’s analytical reli- comparison procedure, which allows comparison of patient ability, the laboratory professional must evaluate the instru- results between the new method and a method that is known ment with regard to random variation (variation resulting to be accurate (e.g., current method). Split specimens (divi- from chance) and systematic variation (variation within the sion of a single specimen into two or more aliquots) are instrument that alters results but is predictable). Precision used. The CLSI recommends that at least 40 patient speci- studies are used to assess random variation and evaluate mens be tested over at least 5 working days.18,19 To increase the reproducibility of the test method.17 To check within the reliability of this comparison, more patient specimens run precision, the laboratory professional should choose at should be used. The specimens should be random so they least two patient specimens and analyze 10–20 aliquots of represent the clinical range of specimens. Ideally, they each specimen in the same test run. These patient specimens should represent different pathologic conditions as well should have different concentration levels that correspond (i.e., results outside the reference interval). With the speci- to an analyte’s medical decision levels (concentration of an mens identified, each is split for analysis by each method. analyte indicating that medical interpretation is required for The specimens are tested in duplicate for each method, and patient care). For example, to check within run precision for replicate testing should be done during the same test run. hemoglobin, three patient s pecimens can be chosen: speci- Analysis of a given specimen should be completed within 2 men 1 Hb = 8.0 g/dL, specimen 2 Hb = 12.0 g/dL, and hours on both methods and the results documented. Before specimen 3 Hb = 19.0 g/dL. Each specimen is separated statistical analysis can be performed, the results should into 10 aliquots each of which is analyzed. For each set of be examined to determine whether any outliers exist, the data, the mean, standard deviation (SD), and coefficient of results are linear, and range of results is adequate. Detec- variation are calculated (Table 43-5). Precision can be deter- tion of outliers and determination of linearity can be made mined by applying a statistical test called the F-test or by by plotting the new method results (Y) versus the current comparing the calculated coefficient of variation (CV) to the method (X). The coefficient of determination (r2) is used to manufacturer’s CV. Within run precision is acceptable if the determine whether adequate range exists. If the results are CV is less than or equal to the manufacturer’s CV. If the CV linear and r2 is greater than or equal to 0.95, simple linear is higher than the manufacturer’s CV, the laboratory profes- regression analysis can be used. Linear regression analysis sional should check the data for outliers (data point that allows for the determination of the y-intercept (a), slope (b), falls outside the expected range for all data). Any outlier standard error of the estimate (sy/x), correlation coefficient should be discarded and the data re-evaluated. If the CV is (r), and coefficient of determination (r2) (Figure 43-1). The Table 43.5 Within Run Precision Study for Hemoglobin Determination by Daman EXCELL-16 Specimen 1 Specimen 2 Specimen 3 1 7.9 12.0 19.2 2 8.1 12.3 19.4 3 8.0 12.2 19.4 4 8.1 12.2 19.4 5 8.1 12.2 19.3 6 8.0 12.3 19.4 7 8.0 12.3 19.4 8 8.1 12.4 19.5 9 8.1 12.4 19.3 10 8.1 12.3 19.6 Mean 8.1 12.3 19.4 SD 0.07 0.11 0.11 CV 0.86% 0.89% 0.57% Manufacturer’s CV Less than 1.0% Less than 1.0% Less than 1.0% Three patient specimens were chosen, and 10 aliquots of each specimen were tested. The mean, standard deviation (SD), and coefficient of variation (CV) were determined for each patient specimen. Comparison of each calculated CV to that of the manufacturer reveals acceptable precision for the hemoglobin procedure because the calculated CV is less than the manufac- turer’s CV. This procedure demonstrates the reproducibility of this hemoglobin determination. Results are reported in g/dL. Quality Assessment in the Hematology Laboratory 1063 Regression plot concentration. The observation of a constant systematic 17 error usually indicates a calibration problem. Proportional 16 systematic errors are identified by changes in the slope. A change in the slope represents a difference between the new 15 method and the current method that is proportional to the 14 analyte’s concentration. That is, the higher the concentra- 13 tion, the greater the difference is between the two methods. 12 If there is no difference between the current method and the 11 new method, the slope is 1.00 { 5%. A proportional sys- 10 tematic error is most frequently associated with erroneous calibration. Random error can be detected by an increasing 9 standard error of the estimate. Increased dispersion of 8 results about the regression line causes an increased stan- 8 9 10 11 12 13 14 15 16 17 Automated cell-counting instrument dard error of the estimate. No standard criteria exist for the interpretation of an acceptable standard error of the esti- Y = –0.291 + 1.012x r2 = 0.998 mate. Thus, the result should be evaluated in conjunction Sy/x = 0.007 with the results of the precision studies. Figure 43.1 Linear regression analysis for comparison Checkpoint 43.5 of hemoglobin by automated cell-counting instrument (x-axis) Linear regression analysis was performed on results from a and point-of-care instrument (y-axis). Interpretation of the linear method comparison of the prothrombin time between auto- regression analysis reveals a strong relationship, r2 = 0.998, in mated coagulation instrument A and automated coagulation the hemoglobin method between the automated cell-counting instrument and the point-of-care instrument. No proportional instrument B. The following results were obtained: systematic error exists because the slope (1.012) is between 0.95 g@intercept = 0.8157 and 1.05. The y-intercept (-0.291) is slightly less than 0, which indicates a small degree of negative bias or constant systematic Slope = 0.9982 error. Most laboratories will consider this negligible. Random error Standard error of the estimate = 0.0807 also is not indicated because the standard error of the estimate (0.007) is nearly 0. Overall, this analysis demonstrates excellent What conclusions can be drawn from these results? comparison of methods. general formula for the linear regression line is y = a + bx, LINEARITY AND REPORTABLE RANGE where y is the predicted mean value of y for a given x value. DETERMINATIONS The coefficient of determination evaluates the strength of The manufacturer determines an instrument’s linearity the relationship between the two methods. For example, or analytical measurement range (AMR). For hematol- an r2 value of 0.90 for a comparison between current and ogy instruments, linearity is determined for each directly new methods means that 90% of the variability in the new measured parameter (Table 43-6). Verification of the instru- method is directly predictable from the variability in the ment’s reportable range (i.e., analytical measurement current method. Therefore, a strong relationship exists range) must be included in the method evaluation proce- between the two methods. dure for a new hematology or coagulation instrument or as The paired t test can be included as a statistical tool; one of the installation procedures for that new instrument.20 it compares the mean of the differences of test results for To verify the reportable range, the laboratory professional the two methods and determines whether a statistically sig- should select linearity check specimens (i.e., linearity check nificant difference exists between the current method and materials and/or fresh human whole blood specimens) the new method. The calculated t-value for the two sets of that span the instrument’s established linear range for each results is compared to the critical t-value from a statistical directly measured parameter. These linearity check speci- table. If the calculated t-value is less than the critical t-value, mens are then analyzed multiple times to minimize the no significant difference exists between the two methods. effect of imprecision on the linearity study’s results. Linear regression analysis is also used to detect system- After the data are examined for possible imprecision, atic (constant or proportional) and random errors. Constant they are plotted (known result versus measured result) to systematic errors are identified by a change in the y-inter- allow visual assessment of the linearity of these results. cept. A y-intercept with a value other than 0 (y 7 0 or y 6 0) The degree of nonlinearity at each level is also determined. indicates that a constant difference exists between the new This degree of nonlinearity or nonlinear error is compared method and the current method regardless of the analyte’s to the predetermined goal for nonlinear error established Point-of-care instrument 1064 Chapter 43 Table 43.6 Display Range and Analytical Measurement Range (AMR) for Abbott Cell-Dyn Emerald Instrument Parameter Unitsa Display Range AMR WBC * 103/mcL 0–100 0.4–96.1 RBC * 106/mcL 0–8 0.22–7.61 Hb g/dL 0–25 3.3–24.6 Hct % 0–80 5.3–75.6 MCV fL 0–150 48.8–115 Platelets * 103/mcL 0–1500 9–1375 WBC, white blood cell; RBC, red blood cell; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume aResults are expressed in Conventional (US) units. Courtesy of Abbott Laboratories, Abbott Park, IL. by each laboratory following the guidelines outlined in the the manufacturer’s hematology/coagulation instrument CLSI Evaluation of the Linearity of Quantitative Measurement and reagent manuals. To use these RIs, a laboratory must Procedures: A Statistical Approach.21 Alternatively, the manu- verify that the RIs are appropriate for its patient popula- facturer of the linearity check materials can provide data tion as required by CLIA ’88.20 Diversity of instrumenta- analysis for a laboratory’s linearity study. The laboratory tion, choice of reagents, and patient population served by follows the instructions supplied by the manufacturer to the laboratory influence RIs. The laboratory can choose to perform the linearity study and submits a spreadsheet with validate a manufacturer’s RIs or establish an RI. Validat- the data. The manufacturer then evaluates the data and ing an RI is less time consuming and more cost effective.22 returns a printed document that details the linearity study Once validated, the RIs can be used as representative for results. If they fall within the laboratory’s acceptable range the laboratory and its patient population. If the RIs are not for nonlinear error, the reportable range is verified. If the validated, the laboratory can choose to establish their own results do not fall within the acceptable range, the process RIs, as described below. should be repeated using more linearity check specimens Establishing an RI is an arduous task. It involves careful in the affected part of the range. If the results do not verify planning to define the criteria for subject selection, the data the instrument’s reportable range, the laboratory should acquisition process, and the data analysis (Table 43-7). In modify the reportable range to reflect the instrument’s per- the ideal situation, RIs should be established based on the formance characteristics in its current setting. In addition, The Joint Commission requires calibration verifications to be performed on hematology and coagulation Table 43.7 Recommended Procedure for the Validation instruments every 6 months if the instrument has a calibration of a Reference Interval process. The calibration verification essentially determines the 1. Select 20 donor individuals. These individuals should meet the speci- linear range as just described and requires a minimum of three fications used by the manufacturer in establishing the reference inter- val (e.g., age and sex). levels of linearity check specimens that span the analytical 2. Perform phlebotomy on the day of testing to obtain appropriate measurement range or reportable range for each parameter. specimen (e.g., EDTA anticoagulant for

whole blood or sodium citrate With the verification of the reportable range, each lab- anticoagulant for platelet-poor plasma). oratory should establish its protocol for handling results 3. Process and store specimens properly prior to testing. 4. Perform quality control procedure to ensure instrument is functioning that exceed the reportable range, either above or below. For properly. example, specimens with results that are above the report- 5. Perform specimen testing on the 20 donor specimens. Several (3–5) able range are diluted and retested, and the subsequent donor specimens should be tested each day for a period of 5–10 days. This will decrease chance introduction of bias. result is multiplied by the dilution factor to obtain the accu- 6. Check the results for outliers. Apply a statistical method such as rate result. Hematology results below the reportable range Reed/Dixon or Tukey method to detect the outlier. A detected out- require retesting and review of the peripheral blood smear lier should be discarded from the data set and replaced with a new donor individual result to obtain 20 results with no outliers. before the result is reported as being less than the lower 7. Compare the results from these 20 specimens with the manufactur- limit of the reportable range. er’s 95% reference interval (mean { 2 SD). The manufacturer’s ref- erence interval is validated when no more than 2 of the 20 specimens fall outside the reported reference interval. If 3 or 4 results fall outside Reference Interval Determination the reference interval, perform validation study on an additional 20 donor specimens that are free of outliers. If no more than 2 of the Hematology and coagulation reference intervals (RIs) are additional 20 specimens fall outside the reported reference interval, available in various recognized hematology textbooks and the reference interval is validated. Quality Assessment in the Hematology Laboratory 1065 patient population’s age and sex stratification. For reliable discovery of the human immunodeficiency virus (HIV) estimates of an RI, a minimum of 120 individuals in each and its potential transmission through exposure to age and sex category should be tested.22 One method of cat- infected blood or other body fluids, the CDC recognized egorizing age groups is by decade of life. However, simple the need for preventative guidelines to minimize poten- mathematical calculation would show an overwhelming tial exposure of health care workers to this virus. Uni- number of individuals needed for such a process. Winsten versal precautions state that health care workers should suggests a mechanism to decrease the number of individu- consider all body fluids as potentially infectious. There- als needed by dividing the patient population into four age fore, health care workers must use the appropriate per- categories23 (Table 43-8). All subjects should provide brief sonal protective equipment (PPE) when handling body histories to determine their acceptability for the study and fluids to minimize the risk of exposure to biohazardous be given the appropriate instructions to prepare for the agents such as HIV, hepatitis B, and other blood-borne blood draw. In addition, each subject must sign an institu- pathogens (Chapter 37). tional review board (IRB) approved consent form. Ideally, The CDC has updated the original guidelines and 5–10 subjects should be tested per day to minimize possible continues to do so. Current guidelines recommend that random introduction of a shift in the RI due to instrument all health care workers receive the hepatitis B vaccine.25,26 or reagent differences. Within a health care facility such as a hospital or medical When all data have been acquired, they must be ana- center, the term standard precautions is used to define the lyzed. Computer-based spreadsheets facilitate data analy- facility’s policies regarding universal precautions and infec- sis. CLSI recommends the use of percentile analysis, a tion control. nonparametric method,22 which is appropriate because the OCCUPATIONAL HEALTH AND SAFETY ADMINISTRA- analysis does not make any specific assumptions regarding TION (OSHA) STANDARDS the distribution of data points (e.g., Gaussian or non- OSHA regulates many aspects of the clinical laboratory Gaussian distribution). Using percentile analysis, the upper to ensure a safe work environment. Each clinical labora- and lower limits of the RI depend on the ranks of reference tory must meet OSHA’s standards for chemical, physical, data arranged in order of increasing values. The lower limit and fire safety. For biologic safety, OSHA implemented the identifies the estimated 2.5th percentile, and the upper Blood-borne Pathogen Standards in 1992. All OSHA stan- limit identifies the estimated 97.5th percentile, thus defin- dards require educating and training laboratory employees, ing the 95% RI. implementing an exposure control plan, and using a record- keeping mechanism. OSHA’s website (www.osha.gov) is a Checkpoint 43.6 source of additional information and details regarding these Define the term reference interval. standards. SAFETY DATA SHEETS The safety data sheet (SDS) provides safety information Laboratory Safety for laboratory professionals who use hazardous materials. The laboratory environment includes biohazards, chemical It includes pertinent safety information regarding the fol- hazards, and physical hazards. All laboratory employees lowing for a chemical: proper storage and disposal, precau- must know the requirements for performing their jobs in a tions that should be taken in handling it, potential health manner that protects them and their coworkers from these hazards associated with exposure to it, and whether the hazards. Several governmental agencies have established chemical is a fire or explosive hazard. Under the Hazard guidelines and standards for ensuring the safety of the labo- Communication Standard, or “Right to Know Law,” labora- ratory professional. tory professionals must receive training regarding the haz- ardous chemicals they work with.27 This training should UNIVERSAL PRECAUTIONS include the potential health risks associated with the chemi- The Centers for Disease Control and Prevention (CDC) cals, interpretation of SDS and chemical labels, and review introduced universal precautions in 1982.24 With the of the laboratory’s hazard communication program. SDSs must be available at all times to the laboratory Table 43.8 Winsten’s Recommended Age Categories for professionals. Reference Interval Determination 1. Newborns Checkpoint 43.7 2. Prepubertal individuals If a spill occurred when handling the CELL-DYN CN-free Sap- 3. Adult (includes postpubertal and premenopausal) phire hemoglobin reagent, what document should be used for 4. Older adults (includes males over age 60 and postmenopausal information regarding a means to safe clean-up of the spill? females) 1066 Chapter 43 Quality Control The clinical laboratory’s quality control program is a critical 6.70 component of the quality assessment program and moni- tors the testing process to ensure that reliable test results are obtained for the patient specimens, to detect potential 6.40 problems within the testing system, and to allow correction of the problem before patient results are affected. 6.10 Control Materials 0 5 10 15 20 25 30 Control materials are assayed specimens with predeter- Day mined test results. The manufacturer assigns a lot number to each batch of control material. Within a given lot number, Figure 43.2 Quality control chart for level 1 hemoglobin the assayed characteristics of the control are the same. Most control depicting control results for days 1–12. The {2 SD limits are hematology procedures use stabilized cell suspensions that 6.10–6.70 g/dL, with a mean of 6.40 g/dL. The quality control results closely match the characteristics of human whole blood. would be interpreted as being in control. The stability of cell suspensions is limited. For most com- mercially available cell suspensions, the time from a given Checkpoint 43.8 lot number’s start date to its expiration date is 4 months. To establish the control limits for a new lot number of level 1 PT For coagulation procedures (e.g., PT and APTT), lyophi- coagulation control, the following data points were collected lized control materials are used. When reconstituted, the (results are in seconds): 11.8, 11.6, 12.1, 12.0, 12.3, 12.6, 11.9, lyophilized control has behavioral characteristics similar to 12.2, 12.0, 11.5, 12.7, 12.1, 11.2, 12.3, 12.9, 13.0, 12.3, 11.9, platelet-poor citrated plasma. A given lot number for these 12.4, and 12.5. What are the {2s control limits? lyophilized controls is ordered in sufficient quantities to meet the laboratory’s testing needs for 1 year. Because a new control limit must be determined for each new lot number of control, this is advantageous, decreasing the laboratory’s Interpreting Quality Control Charts time spent on determining control limits and providing a Statistically, 95% of the control results should fall within {2 continuous monitor of the testing process over reagents and SD, and 99% should fall within {3 SD. Careful and con- laboratory personnel changes. tinual evaluation of the QC charts alerts the laboratory pro- fessional to potential problems in the testing process before Establishing Quality Control (QC) a serious breakdown in the test’s integrity occurs. By using Limits the Westgard multirule approach, the problem can be iden- tified and corrected. Quality control (QC) limits must be established for each control material before its use within the quality control WESTGARD RULES program. Standard protocol for determining quality con- James O. Westgard, Ph.D., and colleagues developed the trol limits calls for testing a new control material to col- Westgard rules to evaluate control results when two or lect a minimum of 20 data points (control measurements) more levels of control material are used.29 See Table 43-9 over 10 working days while monitoring the integrity of the for lists and definitions of the most commonly used West- testing process with the current control material.28 Initial gard rules. control limits are determined using this data. As more data EVALUATION OF QUALITY CONTROL CHARTS USING points are collected, however, the limits should be recalcu- THE WESTGARD MULTIRULE APPROACH lated using all data points to establish truly reliable control Each laboratory creates its multirule protocol for a given limits. The statistics used to establish the QC limits are the instrument by selecting a combination of Westgard rules. mean (x) and SD. Using the mean and SD, the control limits The selection depends on the acceptable level of false rejec- {1s, {2s, and {3s, are calculated. tion (rejection of a control run that is not truly out of con- The mean and control limits are then used to estab- trol) and error detection (rejection of a control run when a lish the QC chart (also known as a Levey-Jennings chart) true error is detected) and the number of control levels run (Figure 43-2). The QC chart is used to plot the control mate- on that instrument.30 The goal of the multirule protocol is rial results over time to provide a graphical display of the to minimize the chance of false rejection of a control run distribution of control results over a given period, usually and maximize the ability to detect true error. For example, 1 month. the multirule protocol for an instrument using two control HGB (g/dL) Quality Assessment in the Hematology Laboratory 1067 Table 43.9 Westgard Rules Westgard Rule Definition Type of Error 12s One control result exceeds a 2s limit. This is considered a warning of a potential out-of-control problem. 22s for across runs Two consecutive control results exceed the same 2s limit. Run Systematic should be rejected and out-of-control problem investigated. 41s for across runs Four consecutive control results exceed the same 1s limit. Run Systematic should be rejected and out-of-control problem investigated. 10x for across runs Ten consecutive control results exceed the mean in the same direc- Systematic tion (e.g., results are above the mean). Run should be rejected and out-of-control problem investigated. 41s for across runs and between control levels Two consecutive control results for two control levels exceed the Systematic 1s limit in the same direction across the last two control runs. Run should be rejected and out-of-control problem investigated. 10x for across runs and between control levels Five consecutive control results for two control levels exceed the Systematic mean in the same direction across the last five control runs. Run should be rejected and out-of-control problem investigated. 22s for within run Two consecutive control results for two control levels exceed the 2s Systematic limit in the same direction for the current control run. Run should be rejected and out-of-control problem investigated. 13s for within run One control result exceeds a 3s limit. Run should be rejected and Random out-of-control problem investigated. R4s for within run One control result exceeds +2s limit, and the other control result Random exceeds -2s limit when using two control levels. Run should be rejected and out-of-control

problem investigated. These rules can be applied “across runs” for two or more runs of a single control or both controls or “within run” for a single control or both controls. levels might be 12s/13s/22s/R4s/41s/10x. Westgard uses S Bull’s Testing Algorithm (Moving as an alternate abbreviation for SD. The 12s rule is used as a warning to indicate the possibility that a rule has been Averages) violated. If a 12s warning is observed for one of the control Moving averages (continuous statistical analysis on con- results from the current test run, the laboratory professional secutive patient erythrocyte indices by an automated cell- should evaluate the QC charts and consider previous con- counting instrument) is a method of using the erythrocyte trol results to determine whether a violation has occurred indices, mean corpuscular volume (MCV), mean corpus- (Figure 43-3). cular hemoglobin (MCH), and mean corpuscular hemo- Depending on the violation, an identified problem can globin concentration (MCHC) to monitor the instrument’s be classified as a random or systematic error. A random performance in determining the erythrocyte parameters error occurs by chance and can result from missampling or (e.g., erythrocyte count or hemoglobin).31 Bull’s testing algo- misidentifying the control. This type of error can be identi- rithm (X-B analysis) represents a calculation of the moving fied and corrected by carefully repeating the control. Sys- averages for erythrocyte indices of the patient population. It tematic errors indicate a problem within the testing system, is based on the premise that the erythrocyte indices within which can result from poor calibration, a change in reagent a patient population are stable. Therefore, moving averages or an expired reagent, expired or improperly stored control, can be used to monitor the precision and accuracy of the or deteriorating light source. Review of the daily and peri- instrument’s performance. odic maintenance logs for the instrument can help identify To establish the acceptable ranges for the moving aver- the problem. Once the problem has been identified, the cor- ages, MCV, MCH, and MCHC, the erythrocyte indices on rect solution can be implemented. 500 consecutive patient specimens are determined and the mean for each index is calculated. The acceptable range for each index is {3% of the mean; however, each laboratory should determine its acceptable range.32 These ranges are Checkpoint 43.9 entered into the instrument’s computer. The majority of After performing daily quality control on the automated hema- hematology instruments calculate the moving average from tology instrument, the laboratory professional observes a 41s violation for the hemoglobin parameter. What type of error is each group of 20 patient specimens and determine whether indicated? those moving averages fall within the acceptable range. The instrument alerts the laboratory professional if the moving 1068 Chapter 43 Low control instrument’s reproducibility over a 24-hour period as a result of the stability of cell counts in an ethylenedi- aminetetraacetic acid (EDTA)–anticoagulated specimen. 6.70 For example, four or five patient specimens are selected from the morning test run, each specimen is analyzed to determine its mean, and the specimens are separated 6.40 into two sets. The first set of separated specimens that is tested every 4 hours is used to monitor the precision of 6.10 the instrument during a 24-hour period. Using the SD and CV for these data, the instrument’s precision is evaluated to determine its acceptability. The second set of separated 0 5 10 15 20 25 30 specimens that is refrigerated for 24 hours monitors pre- Day cision from day to day. After 24 hours, the second set is analyzed to determine each specimen’s 24-hour mean. The Normal control original mean is compared to the 24-hour mean to deter- mine acceptability. Each laboratory should establish its own limits for acceptability. 12.80 Another potential use of retained patient specimens is as quality control materials for the laboratory’s second- 12.40 ary hematology instrument. Patient specimens analyzed on the primary hematology instrument, which has been 12.00 determined to be in control by purchased control materials are analyzed on the secondary instrument. The results are compared to the primary instrument’s results and, if com- 0 5 10 15 20 25 30 parable, the secondary instrument is considered in control Day during the same time interval. Figure 43.3 Quality control charts for hemoglobin controls Individual Quality Control Plan depicting violation of 22s rule. Inspection of the current run, day 27, reveals that both control materials exceed the -2S limit, indicating a Quality control requirements are delineated by accrediting violation of the 22s rule within a run. This run should be rejected and the organizations and regulatory agencies such as CAP and problem resolved before patient specimens can be run. Because the 22s CMS respectively. Beginning January 2016, clinical labora- violation indicates a systematic error, the laboratory professional should investigate the possibility of a reagent, control, or instrument problem. tories have two options to meet the quality control require- ments for nonwaived testing, following the Individualized average exceeds the acceptable range. If a moving average Quality Control Plan (IQCP) or the minimum daily QC is unacceptable, the laboratory professional should identify requirements identified in the CLIA regulations.33 IQCP the previous 20 specimens because this method of monitor- cannot be used for waived procedures, cytopathology ing moving averages of an erythrocyte index is sensitive to or anatomic pathology testing. The minimum daily QC the patient population. If the previous 20 specimens were requirement states that two levels (e.g., high and low con- patients from the renal dialysis clinic or oncology clinic, the centration) of quality control material must be performed change in moving averages could result from the patient daily for quantitative tests. QC for coagulation tests must population, not an instrument problem. A true alert would be performed every eight hours of testing. Qualitative indicate an instrumentation problem affecting one or more tests require performing a positive and negative control of the erythrocyte parameters: erythrocyte count, hemo- each day.34 globin, or hematocrit (Chapters 37, 39). Newer automated IQCP can be used if the device, instrument or proce- cell-counting instruments have expanded moving average dure includes an internal control process and if the manu- program analysis to include WBC, PLT, and RETIC param- facturer’s instructions permit less than the CLIA required eters and other RBC parameters (e.g., RBC, RDW). frequency of QC analyses.35 If both of these requirements are met, laboratory professionals can develop an IQCP. Monitoring Quality Control with There are three components to IQCP: completing a risk assessment, developing a quality control plan and imple- Patient Specimens menting quality assessment.33 IQCP does not focus entirely Patient specimens can be retained and used in conjunction upon the examination phase of laboratory testing, factors with purchased control materials to monitor a hematology that impact the pre- and post-examination phases are taken HGB (g/dL) HGB (g/dL) Quality Assessment in the Hematology Laboratory 1069 into consideration in the risk assessment, quality control plan, and quality assessment program.36 Checkpoint 43.11 Risk assessment requires examining the entire labora- In reviewing a patient’s CBC results, the laboratory professional tory testing process for errors for each analyte considered notes an MCHC of 37 g/dL. What corrective action should be taken? for IQCP for these five areas: specimen, environment, test- ing personnel, reagents, and test system.33 After the risk assessment is completed laboratory professionals develop On occasion, CBC results that are spurious (e.g., results the quality control plan. This plan delineates the type of that first seem to be accurate but on review are invalid) are quality control to be used, the number to be analyzed, the obtained. These results might not be grossly abnormal or frequency it is to be performed, and identifies acceptable flagged by the instrument’s computer, but the results do performance for the quality control program.36 Laboratory fall significantly outside the reference interval. The labora- professionals must examine the effectiveness of the quality tory professional should be alert for this possibility because control plan as a component of their quality assessment pro- spurious results can indicate a problem with the specimen gram using the same five areas included in the risk assess- itself. Before these CBC results can be reported, the specimen ment process. should be examined for potential sources of error (e.g., clots, lipemia, and agglutination). Table 43-11 reviews the spuri- Checkpoint 43.10 ous hematology results that can be encountered in the clini- What type of quality control program should the laboratory cal laboratory including the underlying problem that led to consider for its hematology and coagulation instruments? the erroneous parameter and the possible causes.39,40,2,3,4,5 USE OF DELTA CHECKS Delta checks rely on consecutive testing of a particular patient (Table 43-12). Comparison of current hematol- Review of Patient Results ogy results to the most recently reported previous result Although automated verification or autoverification of for a given patient allows the detection of certain random errors.45 patient results can be used in the hematology/hemostasis This method of error detection is termed delta laboratory, the underlying criteria for the process come from check and has been one of the greatest benefits of imple- the laboratory’s protocol for reviewing patient results.37 menting the LIS into laboratory services. Automated verification allows a faster turnaround time for Limits can be defined to determine the allowable differ- patient results. The laboratory professional is responsible ence among consecutive results of a specific test (e.g., hemo- for addressing those patient results that require special globin) over an established time interval. The limits define attention. This section describes review protocols and cer- when the LIS flags a result. The delta check difference can tain corrective actions for abnormal results in hematology be calculated either as a difference in the absolute value or and hemostasis. as a percentage of the difference. Regardless of the method used, the delta limit should be set so that true changes in Hematology test results are not flagged as delta check failures. If the time limit between comparisons and the maximum allow- Laboratory professionals use the review protocol to exam- able differences have been carefully set, correct results will ine patient data obtained from an automated cell-counting not be flagged. Therefore, the most likely causes of a delta instrument and determine whether the CBC results can be check are a specimen mislabeling or random testing error.46 reported or further action is required. In the hematology laboratory, certain tests, especially the erythrocyte indices, platelet counts, prothrombin time DETECTION OF ABNORMAL TEST RESULTS (PT), and other coagulation studies, have very little intrain- The initial identification of potential abnormalities in the dividual variation. A delta check for one of these parameters CBC results is accomplished by the instrument’s com- should result in an investigation before results are reported. puter system, which is programmed to evaluate the numerical data, histograms, and scattergrams and to gen- CORRECTION FOR INTERFERING SUBSTANCES erate suspect flags and user-defined flags (e.g., definitive The presence of lipemia, icterus, or hemolysis in the plasma flags) (Chapter 39). The numerical data, histograms, scat- of an EDTA-anticoagulated blood specimen can cause an tergrams, and alert flags provide the laboratory profes- artificial elevation of the hemoglobin because of increased sional information that can indicate the presence of absorbance of light from the instrument’s light source by interfering substances, abnormal cell morphology, or the diluted specimen (Chapter 37). The presence of inter- abnormal cells. See Table 43-10 for common abnormal fering substances is commonly detected by the application results or alert flags and corrective actions taken as a of the Rule of 3, or hemoglobin * 3 = hematocrit { 3. To result.38 correct for the presence of these substances, an aliquot of the 1070 Chapter 43 Table 43.10 Common Abnormal Results or Alert Flags and Their Corrective Actions Abnormal Result/Alert Flag Confirmation/Corrective Action Rationale Hemoglobin less than 7.0 g/dL Confirm by repeat analysis or by analysis on A hemoglobin less than 7.0 g/dL is a critical value and alternate instrument. should be confirmed before it is reported. Hemoglobin * 3 does not equal Confirm by repeat analysis, analysis on alternate This rule applies to normal individuals only and is hematocrit {3 instrument, or perform manual hematocrit. used to identify common interfering substances in hematocrit determination (MCV) or hemoglobin deter- mination (see MCHC). MCHC greater than 36 g/dL 1. Perform manual hematocrit. Poor correlation between hemoglobin and hema- 2. Check for presence of cold agglutinins, lipemia, tocrit and an MCHC greater

36 g/dL is associated or icterus. with cold agglutinins, lipemia, and icterus. Cold agglutinins cause erythrocyte clumping and result in 3. Incubate blood specimen at 37 °C for 15 minutes, falsely elevated MCV, falsely decreased RBCs, falsely mix, and analyze if cold agglutinins are present. decreased hematocrit, and MCHC greater than 36 4. Perform hemoglobin on plasma supernatant, then g/dL. Lipemia causes a falsely elevated hemoglobin subtract plasma hemoglobin from blood speci- because of the increased turbidity of specimen and men’s hemoglobin if lipemia is present. MCHC greater than 36 g/dL. MCHC greater than 36 g/dL; no Examine peripheral blood smear for presence of Spherocytes have a decreased surface area-to- correction after warming to 37 °C spherocytes. volume ratio and typically have an MCHC greater than 36 g/dL After ruling out the possibility of cold agglutinins, the presence of spherocytes should be suspected. Platelet count less than 50 * 103/mcL 1. Perform platelet estimate on peripheral blood Presence of platelet clumps or platelet satellitism smear and examine feather edge for platelet results in falsely decreased platelet counts. Plate- clumps. let satellitism and platelet clumps can be EDTA 2. Examine for grapelike cluster clumps of platelets dependent. Therefore, use of sodium citrate as the associated with EDTA-dependent agglutination. If anticoagulant circumvents the mechanism of platelet present, recollect using sodium citrate anticoagu- clumping and platelet satellitism and allows determi- lant and multiply result by 1.1 (dilution factor). nation of the platelet count. 3. Examine for platelet satellitism. Platelet count greater than Perform platelet estimate on peripheral blood smear. Poor correlation between platelet estimate and plate- 900 * 103/mcL Platelet estimate should agree within plus and minus let count could be caused by the presence of eryth- 25% of the platelet count. Examine for presence of rocyte fragments that are similar in size and volume to erythrocyte fragments. platelets. Erythrocyte fragments can cause a falsely elevated platelet count. WBC less than 2.0 * 103/mcL or greater Perform leukocyte estimate on peripheral blood Poor correlation between leukocyte estimate and leu- than 30.0 * 103/mcL smear. Leukocyte estimate should agree within plus kocyte count can be caused by instrument errors or and minus 25% of the leukocyte count. specimen collection error. MCV less than 70 fL, greater than 110 fL Review peripheral blood smear for microcytosis or Clinically significant changes in MCV are associated (adult), greater than 120 fL pediatrics macrocytosis. with nutritional deficiencies and thalassemias. Automated differential flagged Perform manual differential. Confirm presence of abnormal cells or abnormal erythrocyte morphology. Automated differential vote out Perform manual differential. Because of the presence of abnormal cells, the instrument is unable to properly classify the cells and determine the automated differential. The leukocyte differential must be obtained by per- forming a manual differential. Microcytic/fragmented RBC flags 1. Review erythrocyte morphology on peripheral Microcytic erythrocytes or erythrocyte fragments are blood smear. similar in size and volume to platelets. Therefore, the 2. Perform platelet estimate on peripheral blood presence of microcytic cells/fragments can result smear. in a falsely elevated platelet count. Poor correlation between the platelet estimate and platelet count is associated with this observation. WBC, RBC, or platelets greater than Perform a 1 2 or 1 3 dilution of the specimen with dilu- Cell counts that exceed the upper limit of their report- upper limit of established reportable ent. Run the diluted specimen and multiply the result able range require a dilution of that specimen to lower range (or + + + + + ) by the dilution factor. Report the elevated parameter the number of cells present and bring the diluted cell from the diluted specimen only. count into the instrument’s reportable range. The actual cell count is then obtained by multiplying the diluted specimen’s cell count by the dilution factor. WBC, white blood cell; RBC, red blood cell; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. well-mixed blood specimen is placed in another test tube plasma hemoglobin result from the whole blood hemo- and centrifuged at 1500 rpm for 5 minutes. A hemoglobin globin result (Hemoglobincorrected = Hemoglobinoriginal - determination is performed on the plasma (supernatant). Hemoglobinsupernatant). Alternatively, the plasma can be The corrected hemoglobin is calculated by subtracting the removed from the centrifuged specimen and replaced with Quality Assessment in the Hematology Laboratory 1071 Table 43.11 Spurious Hematology Results, Underlying Problems, and Possible Causesa, b, c Erroneous Parameter Underlying Problem Possible Cause Pseudoleukocytosis Platelet irregularities Platelet clumps, very large platelets, EDTA-induced platelet aggregation Unexpected cell type Nucleated erythrocytes (if not directly enumerated by instrument), micromegakaryocytes, megakaryocyte fragments Unlysed erythrocytes Presence of abnormal hemoglobins such as HbC, patients receiving chemotherapy, cold agglutinins Abnormal precipitates Cryoglobulinemia, cryofibrinogenemia, fibrin strands Intraerythrocytic parasites Malarial parasites Carryover between specimens in leukocyte dilution Extreme leukocytosis in previous specimen chamber Pseudoleukopenia Cell lysis Storage artifact, leukemia (especially CLL), uremia, immunosuppression Aggregates counted as single cells Presence of segmented neutrophil aggregates induced by certain anticoagulants such as EDTA Specimen collection issue Clotted specimen Pseudo-increased hemoglobin Turbidity Elevated leukocyte count, hyperlipemia, paraprotein- emia, cryoglobulinemia, cryofibrinogenemia, abnormal hemoglobin (e.g., HbS and HbC) Interference at 540 nm Carboxyhemoglobinemia Pseudo-decreased hemoglobin Interference at 540 nm Sulfhemoglobinemia Unlysed erythrocytes Presence of abnormal hemoglobins (e.g., HbC), patients receiving chemotherapy Specimen collection issue Clotted specimen Inappropriate dilution Overfilled collection tubes Pseudo-increased RBC count Other cells counted Elevated leukocyte count, elevated numbers of very large platelets Abnormal precipitates Cryoglobulinemia, cryofibrinogenemia Pseudo-decreased RBC count Aggregates counted as single cells Cold agglutinins, warm agglutinins Very small erythrocytes that fall below lower Erythrocyte fragments, microcytes (MCV less than threshold for RBC count 50 fL) Specimen collection issue In vitro hemolysis, clotted specimen Pseudo-increased MCV Other cells sized as RBCs Elevated leukocyte count Aggregates sized as single cells Cold agglutinins, warm agglutinins Osmotic swelling Hyperglycemia, hypernatremia, K2 EDTA in excess, storage at room temperature Pseudo-decreased MCV Instrument-related artifact Hypochromic erythrocytes Other cells/particles sized as RBCs Very large platelets, cryoglobulinemia, cryofibrinogenemia Osmotic shrinking Hypo-osmolar states such as hyponatremia Pseudo-increased hematocrit, automated Artificial increase in MCV Elevated leukocyte count greater than 50 * 103/mcL, hyperglycemia, hypernatremia Artificial increase in erythrocyte count Cryoglobulinemia, cryofibrinogenemia, elevated num- bers of very large platelets Pseudo-decreased hematocrit, automated Artificial reduction in MCV Hypo-osmolar states Artificial reduction in erythrocyte count Cold agglutinins, warm agglutinins, erythrocyte frag- ments, microcytes, in vitro hemolysis, clotted specimen Pseudo-increased hematocrit, manual Increased plasma trapping Polycythemia, microcytosis, presence of sickle cells, spherocytosis Centrifuge force too low, decreased centrifugation Burn victim Decreased erythrocyte flexibility Prolonged storage Pseudo-decreased hematocrit, manual Decreased plasma trapping Prolonged centrifugation, increased centrifugal force 1072 Chapter 43 Erroneous Parameter Underlying Problem Possible Cause Specimen collection issue In vitro hemolysis Technique-related issue Incomplete sealing of the microhematocrit tube Increased cell shrinkage Excess EDTA; K3 EDTA rather than K2 EDTA or Na2EDTA Pseudo-increased MCH Spuriously high hemoglobin (See hemoglobin) Spuriously low erythrocyte count (See erythrocyte count) Pseudo-decreased MCH Spuriously low hemoglobin (See hemoglobin) Spuriously high erythrocyte count (See erythrocyte count) Pseudo-increased MCHC Spuriously high hemoglobin (See hemoglobin) Spuriously low hematocrit or product of (See hematocrit) MCV * erythrocyte count Pseudo-decreased MCHC Spuriously low hemoglobin (See hemoglobin) Spuriously high hematocrit or product of (See hematocrit) MCV * erythrocyte count Pseudothrombocytosis, automated Very small erythrocytes or RBC fragments Erythrocyte fragments, microcytes (MCV less than 50 fL), microspherocytes in burn victims Cytoplasmic fragments of nucleated cells Cytoplasmic fragmentation of blasts in acute leukemia or leukemic cells in certain lymphomas Microorganisms: bacteria, fungi, or parasites Bacterial septicemia, infection with Candida sp. similar in size to platelets, presence of Plasmodium falciparum trophozoites Abnormal precipitates Cryoglobulinemia, cyrofibrinogenemia Pseudothrombocytopenia, automated Aggregates counted as single cell or aggregates EDTA-induced platelet aggregation exceed upper threshold and are not counted Platelet satellitism Platelets surround WBCs in EDTA-induced process, in particular segmented neutrophils Very large platelets that exceed upper threshold for Abnormally large platelets seen in myeloproliferative platelet count neoplasms or myelodysplastic syndromes Specimen collection issue Clotted specimen Pseudo-increased reticulocyte count, Inaccurate gating of RBCS Platelet clumps, very large platelets, WBC fragments automated Other RBC inclusions Howell-Jolly bodies, Pappenheimer bodies, Heinz bod- ies, basophilic stippling Intraerythrocytic parasites Plasmodium spp., Babesia Abnormal precipitates Cold agglutinins, paraproteinemia Autofluorescence of RBCs Porphyria, certain drugs EDTA, ethylenediaminetetraacetic acid; HbC, hemoglobin C; CLL, chronic lymphocytic leukemia; HbS, hemoglobin S; RBC, red blood cell; MCV, mean corpuscular volume; K2 EDTA, dipo- tassium ethylenediaminetetraacetic acid; K3 EDTA, tripotassium ethylenediaminetetraacetic acid; Na2 EdTA, disodium ethylenediaminetetraacetic acid; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBCs, white blood cells. aBrigden, M. L., & Dalal, B. I. (1999). Spurious and artifactual test results I: Cell counter-related abnormalities. Laboratory Medicine, 30(5), 325–334. bZandecki, M., Genevieve, F., Gerard, J., & Godon, A. (2007). Spurious counts and spurious results on haematology analysers: A review. Part 1: Platelets. International Journal of Laboratory Hematology, 29(1), 4–20. cZandecki, M., Genevieve, F., Gerard, J., & Godon, A. (2007). Spurious counts and spurious results on haematology analysers: A review. Part II: White blood cells, red blood cells, haemoglo- bin, red cell indices and reticulocytes. International Journal of Laboratory Hematology, 29(1), 21–41. Table 43.12 Investigation of a Delta Check 1. Confirm specimen identification by comparing the specimen label to the laboratory request. If the specimen appears to be labeled properly, continue. If an improperly labeled specimen is identified, notify other areas of the laboratory that may have also received an improperly labeled specimen, i.e., chemistry. 2. Retest by another method or on another instrument, if possible. 3. Review cumulative results on the patient for comparison. 4. Call nursing staff or the clinician for information. Current conditions or treatments may provide clues that explain significant changes, i.e., surgery, blood loss or transfusion, and dehydration. 5. If no reason for the delta change can be determined, it is logical to suspect improper collection or labeling of the specimen (e.g., collection near an I.V. line) and request a new specimen. 6. If there is no evidence of a specimen error, make a comment on the test report to document that the unusual result has been checked and verified. Quality Assessment in the Hematology Laboratory 1073 an equal volume of saline. The cells are resuspended in the The ratio of blood:anticoagulant is affected when a saline, and the corrected hemoglobin is obtained by per- patient’s hematocrit exceeds 55%. The specimen has less forming a hemoglobin determination on this specimen. plasma to be anticoagulated compared to a specimen with The MCH and MCHC should be recalculated using the a normal hematocrit (e.g., 45%). This results in an over- corrected hemoglobin result and initial red blood cell count anticoagulated specimen and falsely prolonged clotting and hematocrit regardless of the correction method used times. The effect on clotting times (i.e., PT and APTT) has for the hemoglobin. been observed with the use of 3.2% sodium citrate as an Certain patients develop IgM antibodies or cold agglu- anticoagulant.47,48 The correct amount of anticoagulant to tinins directed against erythrocyte antigens. As the blood use is determined by the formula given in Figure 43-4 and specimen cools, these antibodies begin to agglutinate eryth- is based on the patient’s hematocrit. rocytes. The automated cell-counting instrument evalu- ates the agglutinated erythrocytes as one cell, resulting in Checkpoint 43.12 a decreased erythrocyte count, increased MCV, decreased The laboratory professional observes that the 3.2% sodium hematocrit, and MCHC greater than 36 g/dL. Incubation citrate tube for a PT and APTT is only two-thirds full. Explain of the blood specimen at 37 °C for 15 minutes disrupts the the effect this will have on the patient’s coagulation results. antigen–antibody reaction and dissociates the agglutinated erythrocytes. The warmed specimen should be mixed thor- oughly and analyzed immediately; its results are report- INTERFERING SUBSTANCES able unless the platelet count decreases by more than Hemolyzed specimens are unacceptable for coagulation 50 * 103/mcL. Warming the specimen occasionally causes testing because thromboplastin-like substances have been loss of platelets. If this occurs, the original platelet count released, resulting in shortened clotting times. Coagula- should be reported. Some patients exhibit a strong cold tion instruments based on photo-optical detection could be agglutinin titer, and the results do not correct after extended unable to test specimens that contain interfering substances warming. In this situation, a manual hematocrit should be such as lipemia or icterus because these substances affect the performed, and the following additional results should be endpoint detection (e.g., absorbance). An electromechanical reported from the original specimen: white blood count, (Chapter 36)

or manual clot detection method should be hemoglobin, and platelet count. The other CBC parameters used to obtain accurate results from these specimens. cannot be reported. USE OF DELTA CHECKS Each hemostasis laboratory must determine appropriate Hemostasis limits for delta checks. In general, a change in the PT of plus and minus 5 seconds or in the APTT of plus and minus As in hematology, patient results for coagulation tests 15 seconds from a specimen tested in the previous 24 hours should be reviewed for accuracy. This includes ensuring could indicate a mislabeled specimen. correct procurement of specimens, checking for interfering substances, and using delta checks. OVERANTICOAGULATION/ Figure 43.4 Correction formula for proper volume of UNDER-ANTICOAGULATION anticoagulant. This correction formula is used when the patient’s hematocrit exceeds 55%. As discussed in Chapter 36, the proper ratio of (1.85 * 10-3) ~ (100 - Hct) ~ V = C blood:anticoagulant is 9:1 for the coagulation specimen. All specimen tubes should be examined visually to ensure where, C = volume of sodium citrate (mL) V = volume of whole blood (mL) that they have been properly filled. Comparison tubes can Hct = patient>s hematocrit (%) be prepared by adding water to empty collection tubes up to the expected fill level. Coagulation specimen collection Example: Paitient>s Hct = 63% tubes containing less than 90% of the expected volume must V = 5 mL be rejected because the specimen is over-anticoagulated (1.85 * 10-3) ~ (100 - 63) ~ 5 = C because of a decrease in the 9:1 ratio. (1.85 * 10-3) ~ 37 ~ 5 = 0.34 mL 1074 Chapter 43 Summary This chapter reviewed the three components of a labo- random variation, and linear regression analysis assesses ratory’s quality assessment program: pre-examination, systematic variation. A laboratory that chooses to use the examination, and post-examination. The pre-examination manufacturer’s reference intervals must validate them to component includes all aspects, such as specimen collec- be able to use them for that laboratory and patient popu- tion, handling, and test requisition that could occur before lation. A laboratory can choose to establish its own refer- testing and affect the results. The examination component ence interval. OSHA and other federal and state agencies includes all testing aspects including proficiency testing mandate safety procedures to which each laboratory must and personnel competency. Proficiency testing monitors adhere. the reliability of a laboratory’s test results by comparison The laboratory’s quality control program including the to those of its peers and provides a good indication of a use of Westgard rules and moving averages monitors a test test method’s long-term accuracy. Competency testing method’s day-to-day reliability and provides an early indi- ensures that laboratory professionals are proficient in per- cation of potential problems with it. Each laboratory creates forming, interpreting, and troubleshooting test procedures its multirule protocol for an instrument by selecting a com- within their assigned area. The post-examination includes bination of Westgard rules. aspects, such as review of patient results and turnaround Review of patient results is necessary to ensure that time, that occur after the testing is performed and could results reflect the patient’s condition. This review is a critical affect the results. An overarching focus of a laborato- component of the quality assessment program. The labora- ry’s quality assessment program is patient safety. From tory professional’s ability to recognize and take corrective selection of the correct test order to the clinician’s deci- action when abnormal patient results occur is at the heart sion based on the test result, the laboratory professional of the medical laboratory science profession. Review and impacts patient safety. recognition of abnormal patient results followed by cor- When a new instrument or method is introduced to rective action represent the final steps before a test result the laboratory, method evaluation studies must be per- is reported. The physician uses the reported test results to formed to compare the new method to the current method, make critical decisions in a patient’s diagnosis and treat- assess the new one for random and systematic variation, ment or management of disease. Therefore, a good quality and validate the reportable range. Precision studies assess assessment program directly affects patient care. Review Questions Level I 3. When validating a manufacturer’s reference interval, what is the minimum number of subject specimens 1. Which source of error represents a pre-examination that should be used? (Objective 5) factor? (Objective 1) a. 10 a. Failure to refrigerate the thromboplastin reagent b. 20 b. Failure to correct the platelet count when using sodium citrate c. 60 c. Failure to invert collection tubes properly d. 120 d. Failure to perform daily maintenance on 4. Which of the following is an OSHA recommendation to cell-counting instrument be followed in the clinical laboratory (www.osha.gov)? (Objective 7) 2. Under CLIA ’88, how frequently should proficiency testing be performed? (Objective 4) a. Gloves should be worn when working with blood but are not required when performing a a. Once a year venipuncture. b. Twice a year b. Safe needle devices should be used when perform- c. Three times a year ing a venipuncture or capillary puncture. d. Four times a year Quality Assessment in the Hematology Laboratory 1075 c. Mouth pipetting is acceptable for transferring c. Increase the likelihood of patients’ health liquid reagents (e.g., saline or deionized water). outcomes d. Handwashing is necessary only if hands become d. Prevent adverse outcomes to patients who have contaminated with blood or other body fluids. laboratory tests 5. Which critical information regarding a chemical is Level II found on its SDS? (Objective 8) 1. Which performance study in the method evaluation a. Expiration date process assesses random variation? (Objective 3) b. Lot number a. Precision study c. Storage requirements b. Method comparison study d. Intended use c. Linearity study 6. You are responsible for determining the control limits d. Calibration study for the low fibrinogen control. The data points were collected, and the mean (x = 64 mg/dL) and SD 2. Given the following linear regression results, which is (s = 3.0 mg/dL) were determined. What are the 3SD the appropriate interpretation? (Objective 2) limits? (Objective 10) • y-Intercept 0.869 a. 61.0–67.0 mg/dL • Slope 1.027 b. 58.0–70.0 mg/dL • Standard error of the estimate 0.014 c. 55.0–73.0 mg/dL a. No significant difference exists between the new d. 52.0–76.0 mg/Dl method and current method results. b. A proportional systematic error exists between the 7. How should the laboratory professional interpret the new method and current method results. PT control results for the current control run if the {2 c. A random error exists between the new method SD control limits are 11.80–14.20 seconds for level I and current method results. and 25.00–28.00 seconds for level II? (Objective 11) d. A constant systematic error exists between the new • Level I 11.6 seconds method and current method results. • Level II 24.6 seconds 3. To verify the hemoglobin reportable range on an auto- a. As acceptable mated hematology instrument, the laboratory’s first b. As unacceptable step is to: (Objective 4) 8. Which of the following parameters will be affected by a. calibrate the instrument and perform quality the presence of lipemia? (Objective 13) control b. select the statistical method for data analysis a. RBC, Hb, Hct, MCV, MCH, MCHC c. determine the laboratory’s goal for nonlinear b. RBC, Hct, MCV, MCHC error c. Hb, MCH, MCHC d. choose linearity check materials that span the d. Hct, MCV, MCHC instrument’s reportable range 9. In interpreting a patient’s CBC results, the labora- 4. After performing daily quality control on the auto- tory professional notes that the platelet count is mated hematology instrument, the laboratory profes- 25 * 103/mcL. Which of the following could be asso- sional observes a 22s violation for the hemoglobin, ciated with this finding? (Objective 12) MCH, and MCHC parameters. All other parameters a. Observation of erythrocyte fragments on the blood were in control. What type of error is indicated? smear (Objective 5) b. Presence of cryoglobulins a. Random c. Observation of platelet clumps at the feather edge b. Systematic of the blood smear 5. Regarding question 4, which is the most likely cause d. Presence of intracellular parasites such as malaria of the error? (Objective 5) 10. Which of the following best describes patient safety a. Failure to adequately mix the control materials with respect to laboratory services? (Objective 15) b. Improper storage of the control materials a. Decrease utilization of laboratory tests c. Depleted supply of lysing reagent b. Ensure that each test is performed accurately d. Expired diluting fluid 1076 Chapter 43 6. For the automated hematology instruments, moving a. Report the results to the patient’s chart averages of erythrocyte indices can be used to moni- b. Observe the specimen for the presence of tor the instrument’s performance in determining hemolysis which parameter: (Objective 6) c. Check the specimen for small clots a. leukocyte d. Examine the collection tube to determine whether b. erythrocyte it was underfilled c. platelet 9. Which source of error would a delta check detect? d. reticulocyte (Objective 10) 7. Initial interpretation of a patient’s CBC results a. Depleted reagent supply showed an MCHC of 38 g/dL. The laboratory profes- b. Improper calibration of the instrument sional warmed the specimen to 37 °C and retested it. c. Deteriorating light source No change was observed. The appropriate course of action is to: (Objective 8) d. Failure to correctly label the patient’s specimen a. observe the blood smear for the presence of 10. The hematology laboratory measures the number of spherocytes clotted EDTA specimens that arrive for testing as a b. replace the patient’s plasma with an equal volume component of their quality improvement plan. 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Development and evaluation of a logical delta check WI: Westgard Quality Corporation. for identifying erroneous blood count results in a tertiary care hos- 31. Bull, B. S., Elashoff, R. M., Heilbron, D. C., & Couperus, J. (1974). pital. Archives of Pathology and Laboratory Medicine, 139(8), 1042–1047. A study of various estimators for the derivation of quality control 47. Marlar, R. A., Potts, R. M., & Marlar, A. A. (2006). Effect on rou- procedures from patient erythrocyte indices. American Journal of tine and special coagulation testing values of citrate anticoagu- Clinical Pathology, 61(4), 473–481. lant adjustment in patients with high hematocrit values. American 32. CELL-DYN Sapphire™ operator’s manual. (2014). Abbott Park, IL: Journal of Clinical Pathology, 126(3), 400–405. Abbott Diagnostics. 48. Favaloro, E. J., Funk, D. M. A., & Lippi, G. (2012). Pre-analytical 33. Centers for Medicare and Medicaid Services. (2014). CLIA variables in coagulation testing associated with diagnostic errors IQCP, what is an IQCP? [CLIA Brochure #13]. Retrieved from in hemostasis. Laboratory Medicine, 43(2), 1–10. Appendix A Data for reference values in these tables was compiled from multiple sources. These values will vary slightly among laboratories. Laboratories should derive reference inter- vals for their population and geographic location. Table A Hematology Reference Values in Adults and Children (Hb, Hct, and RBC shown in conventional units; SI units in parentheses) RBC : 106/mcL MCHC Reticulocytes , 1absolute Age Hb g/dL (g/L) Hct % (L/L) ( : 1012 L 2 MCV (fL) MCH (pg) (g/dL) count : 103>mcL 2 Adult Male 14–17.4 42–52 4.5–5.5 80–100 28–34 32–36 0.5–2.0 (25–75) (140–174) (0.42–0.52) (4.5–5.5) Female 12.0–16.0 36–46 4.0–5.0 80–100 28–34 32–36 0.5–2.0 (25–75) (120–160) (0.36–0.46) (4.0–5.0) Critical low 6.6 g/dL, 1.7 SD 18%, 5 SD limit Critical high 19.9 g/dL, 2.7 SD 61%, 6 SD limit Birth 135–200 0.42–0.60 3.9–5.9 98–123 31–37 30–36 1.7–7.0 (220–420) 2 weeks 130–200 0.39–0.65 3.6–5.9 88–123 30–37 28–35 1.0–3.0 (45–135) (same up to 1 year) 1 month 11–17 (110–170) 33–55 3.3–5.3 91–112 29–36 28–36 (0.33–0.55) 2 months 9–13 (90–130) 28–42 3.1–4.3 84–106 27–34 28–35 (0.28–0.42) 4 months 10–13 (100–130) 32–44 3.5–5.1 76–97 25–32 29–37 (0.32–0.44) 6 months 11–14 (110–140) 31–41 3.9–5.5 68–85 24–30 33–37 (0.31–0.41) 9 months 11–14 (110–140) 32–40 4.0–5.3 70–85 25–30 32–37 (0.32–0.40) 1 year 13–14 (130–140) 33–41 4.1–5.3 71–84 24–30 32–37 (0.33–0.41) 2–6 years 11.5–13.5 34–41 3.9–5.3 75–87 24–30 31–37 (115–135) (0.34–0.41) 6–12 years 11.5–15.5 35–45 4.0–5.2 77–95 25–33 31–37 (115–155) (0.35–0.45) Reference intervals derived from combined data. Critical limits are the low and high boundaries of life-threatening values. Results that fall below the low critical limit and above the high critical limit are “panic values” or critical results that require emergency notification of physicians. These limits were derived by Dr. George Kost from a national survey of 92 institutions. SOURCE: Data from article by Kost, G.J. Critical limits for urgent clinician notification at US Medical Centers. JAMA. 1990; 263:704. 1078 Appendix A 1079 Table B Age and Race-Specific Reference Intervals for Leukocyte Count and Differentiala Birth 6 Months 4 Years Adult Adult of African Descent Total leukocyte count 1*109>L2 9.0–30.0 6.0–18.0 4.5–13.5 4.5–11.0 3.0–9.0 Segmented neutrophil: percent (%) 50–60 25–35 35–45 40–80 45–55 Absolute 1*109>L2 4.5–18.0 1.5–6.3 1.5–8.5 1.8–7.0 1.5–5.0 Band neutrophil percent (%) 5–14 0–5 0–5 0–5 0–5 Absolute 1*109>L2 0.5–4.2 0–1.0 0–0.7 0–0.7 0–0.7 Lymphocyte percent (%) 25–35 55–65 50–65 25–35 35–45 Absolute 1*109>L2 2.0–11.0 4.0–13.5 2.0–8.8 1.0–4.8 1.0–4.8 Monocyte percent (%) 2–10 2–10 2–10 2–10 2–10 Absolute 1*109>L2 0.2–3.0 0.1–2.0 0.1–1.4 0.1–0.8 0.1–0.8 Eosinophil percent (%) 0–5 0–5 0–5 0–5 0–5 Absolute 1*109>L2 0–1.5 0–0.9 0–0.7 0–0.4 0–0.4 Basophil percent (%) 0–1 0–1 0–1 0–1 0–1 Absolute 1*109>L2 0–0.6 0–0.4 0–0.3 0–0.2 0–0.2 aCompiled from multiple sources. Values may vary among sources and laboratories. Table C Other Hematology Reference Values Analyte Reference Value (SI units) Immature reticulocyte fraction (IRF) 0.09–0.31 RDW 12–14.6 Platelet count 1509400 * 103>mcl1*109>L2 MPV 6.8–10.2 fL IPF 0.00–0.04 PDW 9–15 fL Sedimentation rate 0–15 mm/hr Male 650 years 0–20 mm/hr 750 years 0–20 mm/hr Female 650 years 0–30 mm/hr 750 years Zeta sedimentation rate Male 40–52 Female 40–52 Cerebrospinal fluid Erythrocytes 0 Leukocytes 65>mcL 1080 Appendix A Table D Reference Intervals for Common Coagulation Table E Reference Intervals for Tests to Monitor Testsa,b Erythrocyte Destruction Analyte Reference Interval Analyte Age Conventional Units Ac tivated partial thromboplastin 36-40 seconds (birth to 1 week) Bilirubin 61 day 1–6 mg/dL time (APTT) 1–2 days 6–7.5 mg/dL 25–35 seconds (1 week to adult) 2–65 days 4–8 mg/dL Prothrombin time 11.9–15.1 seconds 75 days to adult 0.2–1.0 mg/dL total (high critical 0.8–1.2 INR (6 months to adult) limit 15 mg/dL with 5 SD) 2.0–3.0 INR range for anticoagulant 0–0.2 mg/dL (conjugated) therapy for venous thrombosis Haptoglobin — 2.5–3.5 INR range for anticoagulant therapy for mechanical heart valve Female 40–175 mg/dL (400–1750 mg/L) PFA–100 Establish by using normal donors Male 35–164 mg/dL (350–1640 mg/L) Thrombin time 15–19 seconds Fibrinogen 200–400 mg/dL (2.0-4.0 g/L) Reptilase time 18–22 seconds Protein S functional 55–174% Table F Reference Intervals for Iron Analytesa Protein S free 70–140% Conventional Protein S total 70–140% Analyte Age Sex Units Protein C functional 70–140% Serum iron 61 month — 60–200 mcg/dL Protein C antigen 65–150% 1 month–1 year — 15–160 mcg/dL FII 70–145% 1–17 years — 50–120 mcg/dL FV 60–150% Adult Male 65–180 mcg/dL FVII 65–140% Adult Female 50–180 mcg/dL FVIII 50–150% Serum ferritin Newborn — 25–200 mcg/L FIX 50–160% 6 months–15 years — 7–142 mcg/L FX 70–150% 715 years Male 20–300 mcg/L FXI 70–130% 715 years Female 12–200 mcg/L FXII 50–160% Iron deficient 715 years — 635 mcg>L Fa ctor II, V, VII, VIII, IX, X, XI 605 BU/mL inhibitors Iron overload 715 years — 7335 mcg>L D-dimer 6400 ng/L TIBC Adult — 250–450 mcg/dL Dil ute Russell’s viper venom time 640 seconds UIBC Adult Male 132–323 mcg/dL (DRVVT) Adult Female 144–412 mcg/dL Hexagonal phase correction 68 seconds Percent saturation Adult Male 20–50% Lupus anticoagulant negative Adult Female 15–50% Antithrombin (AT) 80–125% sTfR Adult — 8.7–28.1 nmol/L AT TfR-Ferritin index 0.63–1.8 Antigen 22–40 mg/dL aCompiled from multiple sources. Activity 80–120% Ac tivated protein C resistance 72.1 (APCR) Plasminogen 76–124 (males) 65–153% (females) Pla sminogen activator inhibitor-1 (PAI-1) Activity 78–142% Antigen 4–43 mcg/mL von Willebrand factor activity 42–139% von Willebrand factor antigen 60–150% aCompiled from multiple sources. bResults vary depending on reagent and instrument combinations used in testing. Appendix A 1081 Table G Reference Intervals for Other Common Tests to Table I Reference Intervals for Normal Osmotic Fragility Differentiate Causes of Anemiaa Sodium Analysis Conventional Units Chloride (NaCl) % Hemolysis % Hemolysis After Concentration (%) Before Incubation Incubation Hemoglobin Hb A 7 95, electrophoresis 0.20 100 95–100 Hb A2 1.5–3.7% 0.30 97–100 85–100 Hb F 0–2.0% 171 year2 0.35 90–99 75–100 0–85% (birth to 1 year) 0.40 50–95 65–100 Vitamin B12 150–450 pg/mL (decreases significantly 0.45 5–45 55–95 with age in males) 0.50 0–6 40–85 Serum folate 72.5 ng>mL (adults) 0.55 0 15–70 73.0 ng>mL (1-year-old children) 0.60 0 0–40 76.5 ng>mL (1–10-year-old children) 0.65 0 0–10 RBC folate 160–700 ng/mL (adults) 0.70 0 0–5 75–1000 ng/mL 1chi ldren 6 1 year old2 0.75 0 0 95–365 ng/mL (children 1–11 years old) Increased osmotic fragility, as occurs in the presence of spherocytes, Erythropoietin (EPO) 3–16 IU/L (SI unit) shows hemolysis beginning at 0.65–0.60% NaCl and is complete at about Methemoglobin 0.06–0.24 g/dL (9.3–37.2 mmol/L) 0.45–0.40% before incubation. Some forms of hereditary spherocytosis will not show increased osmotic fragility until the cells are incubated at 37°C for aCompiled from multiple sources. 24 hours before running this test. After incubation these abnormal cells will begin to lyse at 0.65–0.70% NaCl, and lysis is complete at about 0.55%. Table H Reference Intervals

for Porphyrins and Porphyrin Precursorsa Table J Reference Intervals for Leukocyte Alkaline Phosphatase (LAP) Scores Analyte Urine Erythrocytes Interpretation Score Uroporphyrin 4–20 mcg/day Coproporphyrin 13–180 mcg/day Normal 13–130 Porphobilinogen 62 mg>day Chronic myelogenous leukemia 0–13 ∆ Aminolevul inic acid 1.5–7.5 mg/day Pol ycythemia vera, infection, Upper range of normal or elevated myeloid metaplasia Protoporphyrin 17–77 mcg/dL of RBCs Pa roxysmal nocturnal Very low a hemoglobinuria Compiled from multiple sources. Appendix B Hematopoietic and Lymphoid Neoplasms: Immunophenotypic and Genetic Features Disease Chromosome Category Diagnosis Immunophenotype Translocation Genotypic Finding Mature B-cell Chronic lymphocytic CD19+ , CD20+dim, CD5+ , CD10- , No specific abnormality; could No specific abnormality; neoplasms leukemia/small CD23+ , FMC@7- , sIg+dim have del(13q14.3), trisomy subtypes with and without lymphocytic lymphoma 12, del(11q22-), del(17p13.1), somatic hypermutation IGHV del(6q21) t(14;19)(q32.3;19q13) that have approximate correla- tion with DNA methylation status B-cell prolymphocytic CD19+ , CD20+ , CD5+/- , CD23+/- , No specific abnormality; No specific abnormality; TP53 leukemia FMC@7+ complex karyotype and mutations frequent del(17p13.1) frequent Hairy cell leukemia CD19+ , CD10- , CD5- , CD11c+ , No specific abnormality BRAF V600E mutation in most CD25+ , CD103+ , annexin@A1+ , sIg+ Follicular lymphoma CD19+ , CD10+ , CD5- , bcl@2+ , sIg+ t(14;18)(q32.3;q21.3) IGH/BCL2 rearrangement; copy neutral absence of heterozygos- ity of 1p36 and mutations in TNFRSF14 common Mantle cell lymphoma CD19+ , CD10- , CD5, CD23+/- , t(11;14)(q13;q32.3) CCND1/IGH rearrangement; FMC@7+ , cyclin@D1+ , sIg+ molecular subtypes recognized based on IGH and SOX11 mutation status Extranodal marginal No specific phenotype Variety of associated transloca- No specific abnormality; zone lymphoma of CD19+ , CD20+ , CD5- , CD10- , sIg+ ; tions that vary in frequency BIRC3/MALT1 fusion, or mucosa associated could have plasmacytic differentiation among sites BCL10, MALT1, FOXP1; lymphoid tissue type t(11;18)(q22;q21), t(1;14) transcriptional deregulation (MALT lymphoma) (p22;q32.3), t(14;18) (q32.3;q21), t(3;14) (p14.1;q32.3), +3, +18 Lymphoplasmacytic No specific phenotype No specific abnormality; often More than 90% have MYD88 lymphoma CD19+ , CD20+ , CD5- , CD10- , sIg+ del(6q) L265P lymphoid cells and plasmacytic differentiation Diffuse large B-cell No specific phenotype No specific abnormality; could No specific abnormality; could lymphoma CD19+ , CD20+ , CD5+/- , CD10+/- have abnormalities of 3q27.3, have BCL6, BCL2, or MYC t(14;18)(q32.3;q21.3) rearrangement, often with IGH. Burkitt lymphoma CD19+ , CD10+ , CD5- , sIg+ t(8;14)(q24.1;q32.3) or MYC/IGH, IGK, or IGL; TCF3 t(8;22)(q24.1;q11.2), t(2;8) and ID3 mutations common in (p11.2;q24.1) sporadic cases Plasma cell neoplasms CD138+ , CD38+ bright, cIg+ , often FISH detects aberrations in Disease progression associated with CD56+ , CD19- 90% most commonly hyper- with TP53 mutation diploidy, del RB1 (13q14.2), IGH (14q32.3) and CCND1 (11q13.3), MAF (16q23.2), FGFR3/NSD2 (4p16.3), CCND3 (6p21.1), MAFB (20q12)), MAFA (8q24.3), CCND2 (12p13.3) 1082 Appendix B Hematopoietic and Lymphoid Neoplasms: Immunophenotypic and Genetic Features 1083 Disease Chromosome Category Diagnosis Immunophenotype Translocation Genotypic Finding Mature T-cell Peripheral T-cell No specific phenotype No specific abnormality; com- Expression of TBX21 and GATA3 neoplasms lymphoma, NOS CD3+ , CD4+/- , CD8-/+ , plex karyotypes common recurrent CD5+/- , CD7+/- Anaplastic large cell Could express T-cell antigens (e.g., t(2;5)(p23;q35.1), or t(1;2) ALK rearranged with NPM1, lymphoma CD2, CD3, CD5, CD7), but often “null” (q21.2;p23), or inv(2) TPM3, ATIC, or others phenotype lacking antigens (p23q35.1), or others Hepatosplenic T-cell CD2+ , CD3+ , CD4- , CD5- , Isochromosome 7q Rearrangement of T-cell receptor lymphoma CD7+ , CD8+ , most cases g/d TCR+ , chain genes subset a/b TCR+ Extranodal NK/T-cell CD2+ , CD56+ , CD7+/- , CD4- , Deletion of 6q recurrent No specific abnormality lymphoma, nasal type CD8- , CD16- , CD57- Subcutaneous pannicu- CD8+ , a/b TCR+ , granzyme B+ , No specific abnormality No specific abnormality litis-like T-cell lymphoma a/b TCR+ , perforin+ , TIA1+ , betaF1+ , CD56- Enteropathy-associated CD2+ , CD3+ , CD7+ , CD8+ , Gain of 9q34 or loss of TRB and TRG rearranged in T-cell lymphoma a/b TCR+ 16q12.1 more than 95% T-cell prolymphocytic No specific phenotype. inv(14)(q11.2q32.3), t(14;14) No specific abnormality; leukemia CD2+ , CD3+ , CD7+ (q11.2;q32.3), t(X;14) often TRA juxtaposed with (q28;q11.2); also, idic(8p11), TCL1A and TCL1B or MTCP1; t(8;8)(p11–12;q12), + (8q), and ATM mutation; often TP53 del(12p13) mutation T-cell large granular lym- CD3+ , usually No specific abnormality TRG rearranged in virtually all phocytic leukemia CD8+ , CD57+ , CD16+ , often with cases; STAT3 mutations in one decreased expression of CD5 or CD7 third of cases Adult T-cell leukemia/ CD2+ , CD3+ , CD4+ , No specific abnormality Multiple genes associated with lymphoma CD25+ , FoxP3+ , usually CD7- T-cell receptor signaling recurrent Sézary syndrome CD3+ , CD4+ , CD5+ , CD7+/- Complex karyotypes common No specific abnormality; often mutations in TP53, CDKN2A, and DNMT3A Precursor B lymphoblastic leuke- CD19+ , CD10, CD34+ , TdT+ , often t(9;22)(q34.1;q11.2) BCR-ABL1 majority in minor lymphoid mia/lymphoma (B LL) with aberrant expression of CD13 and/ breakpoint cluster region leading neoplasms with t(9;22)(q34.1;q11.2) or CD33 to p190 fusion protein in chil- dren, p210 in ∼50, in adults B LL with t(v;11q23.3) CD19+ , CD10- , CD15+ , TdT+ 11q23.3 breakpoint KMT2A rearranged B LL with t(12;21) CD19+ , CD10+ , CD34+ , TdT+ , t(12;21)(p13.2;q22.12) ETV6-RUNX1 (p13.2;q22.12) often with aberrant expression of CD13 Hyperdiploid B LL CD19+ , CD10+ , CD34+ , CD45- Gain of chromosomes 4, 10, Genetic gain related to (54–65 chr.) 17, 21, and X most common chromosomes involved Hypodiploid B LL (less CD19+ , CD10+ Modal chromosome number Genetic loss related to than 44 chr.) from 45 to near-haploid chromosomes involved B LL with t(5;14) CD19+ , CD10+ t(5;14)(q31.1;q32.3) IL3/IGH (q31.1;q32.3) B LL with t(1;19) CD19+ , CD10+ , Cm+ t(1;19)(q23.3;p13.3) PBX1/TCF3 (q23.3;p13.3) Acute myeloid AML with t(8;21) CD13+ , CD33+/- , MPO+ , t(8;21)(q21.3;q22.12); loss RUNX1/RUNX1T1 leukemia (q21.3;q22.12) CD34+ , HLA@Dr+ , aberrant CD19+ of sex chromosome and (AML) with blasts in addition to maturing del(9q) common as secondary recurrent granulocytes aberrations genetic abnormalities AML with inv(16) CD13+ , CD33+ , MPO+ , CD34+ , inv(16)(p13.11;q22.1) or CBFB/MYH11 (p13.11q22.1) or HLA@Dr+ blasts, often in addition to t(16;16)(p13.11;q22.1) t(16;16)(p13.11;q22.1) maturing granulocytes or monocytes Acute promyelocytic CD13+ , CD33, t(15;17)(q24.1;q21.2) Also PML/RARA leukemia MPO+ , CD34- , HLA@Dr- immature the following variants: t(11;17) Also the following variants: cells (q23.2;q21.2) ZBTB16/RARA t(5;17)(q35.1;q21.2) NPM1/RARA t(11;17)(q13.4;q21.2) NUMA1/RARA dup(17q21.2) STAT5B/RARA AML with t(9;11) CD13+ , CD33+ , MPO+ , CD34- , t(9;11)(p21.3;q23.3) KMT2A/MLLT3 (p21.3;q23.3) HLA@Dr+ blasts, in addition to monocytic cells AML with t(6;9) No specific phenotype t(6;9)(p23;q34.13) DEK/NUP214 (p22.3;q34.13) 1084 Appendix B Hematopoietic and Lymphoid Neoplasms: Immunophenotypic and Genetic Features Disease Chromosome Category Diagnosis Immunophenotype Translocation Genotypic Finding AML with inv(3) No specific phenotype Expression of inv(3)(q21.3q26.2) GATA2/MECOM (q21.3q26.2) or t(3;3) CD41 has been reported t(3;3)(q21.3;q26.2) (q21;q26.2) AML with t(1;22) CD41 and/or CD61 t(1;22)(p13.3;q13) RBM15/MKL1 (p13.3;q13) AML with mutated Usually lack CD34. Frequently express Most have normal karyotype NPM1 mutation NPM1 monocytic antigens CD14 or CD11b Occasionally +8 and del(9q) FLT3 and DNMT3A common as additional aberrations AML with mutated No specific phenotype Most have normal karyotype CEBPA mutation; favorable CEBPA CD13+ , CD34+ , HLA@DR+ with vari- Trisomies 8 and 13 prognosis requires biallelic AML with mutated able monocytic markers mutation RUNX1 Monoallelic mutation AML with No specific phenotype -5/del(5q), -7/del(7q), com- Multiple genes myelodysplasia- plex karyotype, and others related changes Therapy-related AML/ No specific phenotype del(5), del(7), complex karyo- Alkylating agent/radiation related MDS type, and others KMT2A and RUNX1 rearranged 11q23.3 and 21q22.12 (topoisomerase-II inhibitor aberrations related) AML, NOS AML with minimal HLA-DR, CD117, CD13, CD33, CD34, No specific abnormalities differentiation CD38 identified AML without maturation HLA-DR, CD117, CD13, CD33, CD34, No specific abnormalities CD14 identified AML with maturation HLA@DR+/- , CD117, CD13, CD33, No specific abnormalities CD34+/- , identified Acute myelomonocytic CD117, CD13, CD33, CD11b, Nonspecific myeloid aberra- No specific abnormalities leukemia CD14+/- tions (+8) identified Acute monoblastic/ HLA-DR, CD117, CD13, CD33, t(8;16)(p11.2;p13.3) recurrent MYST3/CREBBP monocytic leukemia CD11b, CD14 Pure erythroid leukemia HLA@DR+/- , CD117, CD13, CD33, No specific abnormalities CD71 identified Acute megakaryoblastic HLA-DR, CD117, CD33, CD34, No specific abnormalities leukemia CD41/42/61+/- identified Acute basophilic HLA-DR, CD117, CD33, CD34, t(X;6)(p11.2;q23.3) recurrent in MYB/GATA1 leukemia CD41/42/61+/- male infants Acute panmyelosis with HLA-DR, CD117, CD13, CD33, CD34, No specific abnormalities myelofibrosis CD71+/- , CD41/42/61+/- identified Myeloid Reflective of the specific myeloid cell(s) Recurrent translocations or NPM1 mutations recurrent sarcoma involved others reflective of the myeloid cells involved Myeloid CD34, CD56, CD117, CD13, CD33, Constitutional trisomy 21 GATA1 mutations common proliferations CD7, CD4, CD41, CD42, CD61 related to Down syndrome Blastic CD4, CD43, CD56, CD123, TCL1, Complex karyotype plasmacytoid CLA, CD68{ dendritic cell neoplasm Acute Acute undifferentiated Various subtypes, variable phenotype t(9;22)(q34.1;q11.2) BCR/ABL1 leukemias of leukemia t(v;11q23.3) KMT2A rearranged ambiguous lineage, MPAL with t(9;22) various types (q34.1;q11.2) MPAL with t(v;11q23.3) MPAL, B/myeloid, NOS MPAL, T/myeloid, NOS Myelopro- Chronic myeloid leuke- Maturing neutrophilic cells and t(9;22)(q34.1;q11.2), BCR-ABL1 usually in major liferative mia, BCR-ABL1 positive increased basophils Philadelphia-chromosome (Ph) breakpoint cluster region leading neoplasms Could see nonspecific aberrant anti- translocation to p210 fusion protein gen expression Disease progression associated with an extra Ph, +8, i(17q), or +19 Appendix B Hematopoietic and Lymphoid Neoplasms: Immunophenotypic and Genetic Features 1085 Disease Chromosome Category Diagnosis Immunophenotype Translocation Genotypic Finding Chronic Neutrophilic No specific abnormality identified Normal karyotype CSF3R T618I or other activating Leukemia CSF3R mutation Essential Could see nonspecific aberrant Usually no chromosomal Driver mutations: JAK2 V617F thrombocythemia antigen expression abnormalities 64.1%, CALR 15.5%, MPL W515K/L 4.3%. Other mutations in EPO or G-CSF Polycythemia vera Could see nonspecific aberrant No specific cytogenetic JAK2 V617F mutation more than antigen expression abnormality; could see +8, +9, 95% (also seen in other myelo- del(20q), del(13q), and del(9p) proliferative neoplasms). Other mutations include TET2, MPL, LKN, Bcl-XL, IDH1/2 Primary myelofibrosis No specific abnormality identified No specific chromosomal Driver mutations: JAK2 V617F abnormality 60%, CALR 30%, MPL del(20q) (23.3%); W515K/L about 5%. No driver del(13q) (18.2%); mutations (triple negative) have +8 (11.1%); worse prognosis. +9 (9.9%); Other mutations: TET2, ASXL1, trisomy 1q+ (9.7%); DNMT3A, EZH2, CBL -7/7q- (7.1%); der(6)t(1;6) (q21–q23;p21.3) Myeloproliferative neo- No specific abnormality identified No specific molecular plasm, unclassified abnormality Myeloid and Myeloid or lymphoid No specific abnormality identified. Cryptic del(4)(q12–q12), t(5;12) FIP1L1-PDGFRA fusion lymphoid neoplasms with PDG- Could see aberrant mast cell (q32;p13.2), 5q31–33 translo- PDGFRB rearrangement (ETV6- neoplasms FRA rearrangement; expression of CD2 and CD25 cations with 8p11.23 PDGFRB); variety of FGFR1 with myeloid neoplasms with fusion genes (8p11.23) eosinophilia PDGFRB rearrange- and abnor- ment; myeloid or lym- malities of phoid neoplasms with PDGFRA, FGFR1 abnormalities PDGFRB or FGFR1 Myelodysplas- Chronic myelomono- CD13, CD33, CD14, CD68 +8 and del(7q) recurrent; Ph- ASXL1 mutation 40% tic syndrome/ cytic leukemia +8 and del(7q) recurrent No specific molecular myelopro- pical chronic myeloid CD13, CD33 abnormality liferative Aty Mutations in RAS effector path- syndrome leukemia, BCR-ABL1 negative way genes Juvenile myelomono- CD13, CD33, CD14, CD68, CD64 Monosomy 7 recurrent; Ph- cytic leukemia MDS/MPN with ring No specific phenotype Majority with normal karyotypes SF3B1 mutation in more than sideroblasts and 60% thrombocytosis MDS/MPN-U No specific phenotype No specific chromosomal No specific molecular abnormality abnormality Myelo- MDS-SLD Heterogeneous: lymphoid Heterogeneous: SF3B1 and TET2 most common dysplastic antigens on myeloid cells, over- dromes MDS-MLD -5/5q- , -7/7q- , and syn or underexpression of antigens, complex karyotype most MDS-RS-SLD immature/mature antigens on mature/ frequent cytogenetic immature cells aberrations MDS-RS-MLD Normal karyotype seen in 50% MDS-EB-1 of cases MDS-EB-2 MDS-U MDS with isolated del 5(q) alone, or with 1 addi- del(5q) tional abnormality except -7 or del(7q) Appendix C 2017 WHO Classification of Hematologic, Lymphopoietic, Histiocytic/Dendritic Neoplasms I. Mature Myeloid Neoplasms Myelodysplastic/myeloproliferative neoplasm 1. Myeloproliferative Neoplasms (MPN) with ring sideroblasts and marked thrombocytosis Chronic myelogenous leukemia (CML), BCR/ABL1 4. Myelodysplastic Syndromes (MDS) positive MDS with single-lineage dysplasia (MDS-SLD) Chronic neutrophilic leukemia (CNL) MDS with ring sideroblasts (MDS-RS) Essential thrombocythemia (ET) MDS with multilineage dysplasia (MDS-MLD) Polycythemia vera (PV) MDS with excess blasts (MDS-EB) Primary myelofibrosis (PMF) MDS associated with isolated del(5q) Chronic eosinophilic leukemia, not otherwise MDS, unclassifiable (MDS, U) specified (CEL, NOS) Childhood MDS Mastocytosis Refractory cytopenia of childhood Cutaneous mastocytosis II. Acute Myeloid Leukemia (AML) and Related Systemic mastocytosis Precursor Neoplasms Mast cell sarcoma 1. AML with Recurrent Genetic Abnormalities Myeloproliferative neoplasm, unclassifiable AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 (MPN, U) AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); 2. Myeloid and Lymphoid Neoplasms with CBFB-MYH11 Eosinophilia and Gene Rearrangement Acute promyelocytic leukemia (APL) with t(15;17) Myeloid/lymphoid neoplasms with PDGFRA (q22;q12); PML-RAR rearrangement AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A Myeloid/lymphoid neoplasms

with PDGFRB AML with t(6;9)(p23;q34); DEK-NUP214 rearrangement AML with inv(3)(q21.3;q26.2) or t(3;3) (q21.3;q26.2); Myeloid/lymphoid neoplasms with FGFR1 GATA2, MECOM abnormalities AML (megakaryoblastic) with t(1;22)(p13;q13); 3. Myelodysplastic/Myeloproliferative Neoplasms RBM15-MKL1 (MDS/MPN) AML with gene mutations Chronic myelomonocytic leukemia (CMML) mutated NPM1 Atypical chronic myeloid leukemia, BCR/ABL1 biallelic mutated CEBPA negative (aCML) mutated RUNX1 Juvenile myelomonocytic leukemia (JMML) 2. AML with Myelodysplasia-Related Changes Myelodysplastic/myeloproliferative neoplasm, unclassifiable (MDS/MPN, U) 3. Therapy-Related Myeloid Neoplasms 1086 Appendix C 2017 WHO Classification of Hematologic, Lymphopoietic, Histiocytic/Dendritic Neoplasms 1087 4. AML, NOS 2. T Lymphoblastic Leukemia/Lymphoma AML with minimal differentiation V. Mature B-Cell Neoplasms AML without maturation Chronic lymphocytic leukemia/small lymphocytic AML with maturation lymphoma Acute myelomonocytic leukemia B-cell prolymphocytic leukemia Acute monoblastic/monocytic leukemia Splenic marginal zone lymphoma Pure erythroid leukemia Hairy cell leukemia Acute megakaryoblastic leukemia Splenic B-cell lymphoma/leukemia, unclassifiable Acute basophilic leukemia Lymphoplasmacytic lymphoma 5. Myeloid Proliferations Related to Down Waldenström macroglobulinemia Syndrome Heavy chain diseases Transient abnormal myelopoiesis (TAM) a@heavy chain disease Myeloid leukemia associated with Down g@heavy chain disease syndrome m@heavy chain disease 6. Blastic Plasmacytoid Dendritic Cell Neoplasm Plasma cell neoplasms 7. Myeloid Sarcoma Monoclonal gammopathy of undetermined III. Acute Leukemia of Ambiguous Lineage significance (MGUS) Acute undifferentiated leukemia Plasma cell myeloma Mixed phenotype acute leukemia with t(9;22) Solitary plasmacytoma of bone (q34;q11.2); BCR/ABL1 Extraosseous plasmacytoma Mixed phenotype acute leukemia with t(v;11q23.3); Monoclonal immunoglobulin deposition KMT2A rearranged diseases Mixed phenotype acute leukemia, B/myeloid, Extranodal marginal zone lymphoma of NOS mucosa- associate lymphoid tissue (MALT lymphoma) Mixed phenotype acute leukemia, T/myeloid, Nodal marginal zone lymphoma NOS Pediatric nodal marginal zone lymphoma Mixed phenotype acute leukemia, NOS Follicular lymphoma IV. Precursor Lymphoid Neoplasms Pediatric-type follicular lymphoma 1. B Lymphoblastic Leukemia/Lymphoma Primary cutaneous follicle center lymphoma B lymphoblastic leukemia/lymphoma, NOS Mantle cell lymphoma B lymphoblastic leukemia/lymphoma with Diffuse large B-cell lymphoma (DLBCL), NOS recurrent genetic abnormalities T-cell/histiocyte rich large B-cell lymphoma B lymphoblastic leukemia/lymphoma with t(9;22)(q34;q11.2); BCR-ABL1 Primary DLBCL of the CNS B lymphoblastic leukemia/lymphoma with Primary cutaneous DLBCL, leg type t(v;11q23.3); KMT2A rearranged EBV positive DLBCL of the elderly B lymphoblastic leukemia/lymphoma with DLBCL associated with chronic inflammation t(12;21)(p13;q22); ETV6-RUNX1 Lymphomatoid granulomatosis B lymphoblastic leukemia/lymphoma with Primary mediastinal (thymic) large B-cell lymphoma hyperdiploidy Intravascular large B-cell lymphoma B lymphoblastic leukemia/lymphoma with ALK positive large B-cell lymphoma hypodiploidy (hypodiploid ALL) Plasmablastic lymphoma B lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32); IL3-IGH HHV8-associated lymphoproliferative disorders B lymphoblastic leukemia/lymphoma Primary effusion lymphoma with t(1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1) Burkitt lymphoma 1088 Appendix C 2017 WHO Classification of Hematologic, Lymphopoietic, Histiocytic/Dendritic Neoplasms B-cell lymphoma, unclassifiable, with features Peripheral T-cell lymphoma, NOS intermediate between DLBCL and classic Hodgkin Angioimmunoblastic T-cell lymphoma lymphoma Anaplastic large cell lymphoma, ALK positive VI. Mature T and NK Cell Neoplasms Anaplastic large cell lymphoma, ALK negative T cell prolymphocytic leukemia VII. Hodgkin Lymphoma T cell large granular lymphocytic leukemia 1. Nodular lymphocyte predominant Chronic lymphoproliferative disorder of NK cells 2. Classic Aggressive NK cell leukemia Nodular sclerosis EBV positive T cell and NK cell lymphoproliferative Lymphocyte rich diseases of childhood Mixed cellularity Hydroa vacciniforme-like lymphoproliferative disorder Lymphocyte depleted Adult T cell leukemia/lymphoma VIII. Histiocytic and Dendritic Cell Neoplasms Extranodal NK/T-cell lymphoma, nasal type Histiocytic sarcoma Enteropathy-associated T cell lymphoma Langerhans cell histiocytosis Hepatosplenic T cell lymphoma Langerhans cell sarcoma Subcutaneous panniculitis-like T cell lymphoma Interdigitating dendritic cell sarcoma Mycosis fungoides Follicular dendritic cell sarcoma Sézary syndrome Fibroblastic reticular cell tumor Primary cutaneous CD30+ T cell lymphoproliferative disorders Indeterminate dendritic cell tumor Lymphomatoid papulosis Disseminated juvenile xanthogranuloma Primary cutaneous anaplastic large cell lymphoma IX. Post-Transplant Lymphoproliferative Disorders (PTLD) Primary cutaneous T cell lymphomas, rare subtypes Polymorphic PTLD Primary cutaneous CD8+ aggressive epidermotropic Monomorphic PTLD (B and T/NK cell types) cytotoxic T-cell lymphoma Classic Hodgkin lymphoma type PTLD Primary cutaneous CD4+ small/medium T-cell lymphoma Adapted from: Swerdlow, S. H., Campo, E., Harris, N. L., Jaffe, E. S., Pileri, S. A., Harald, S., & Thiele, J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC; 2017. Appendix D Hematology Procedures PROCEDURE NUMBER PROCEDURE NAME Procedure 37-2 Peripheral Blood Smear Preparation 37-1 Koehler illumination 1. Place one drop of well-mixed blood near the end of the 37-2 Peripheral blood smear preparation microscope slide. 37-3 Peripheral blood smear staining procedure 2. With the spreader slide at a 45-degree angle, draw it to 37-4 Peripheral blood smear examination the drop of blood. 37-5 Manual leukocyte count 3. Allow the entire drop of blood to spread the width of 37-6 Manual platelet count the spreader slide (i.e., the interface of spreader slide 37-7 Hemoglobin concentration determination and microscope slide). 37-8 Microhematocrit determination 4. Smoothly push the spreader slide to the opposite end 37-9 Erythrocyte sedimentation rate of the slide, pulling the blood behind it. 37-10 Reticulocyte count 5. Air dry the smear immediately. 37-11 Solubility test for hemoglobin S 6. Label the blood smear by writing into the thick end of 37-12 Osmotic fragility test the smear using a pencil or write on the frosted end of Procedure 37-1 Koehler Illumination the slide. 1. Place sample on the stage. Procedure 37-3 Peripheral Blood Smear Staining 2. Rotate 10x objective into position. 1. Place the air-dried blood smear on the staining rack. 3. Adjust brightness control to comfortable level. 2. Flood the smear with absolute methanol and fix for one 4. Adjust interpupillary distance scale so right and left eye minute. images merge into one. 3. Drain the excess absolute methanol from the smear. 5. Using coarse knob, focus with 10x objective. Adjust 4. Flood the smear with Wright's stain and leave stain in with fine focus. place for two minutes. 6. Switch to 40x objective and fine focus. 5. After two minutes, add an equal volume of the buffer 7. Switch back to 10x objective and adjust focus with eye- to the stain. piece diopters if necessary. 6. Mix buffer with stain by gently blowing. Allow smear 8. Switch to 40x objective to recheck focus. to stain for 8–10 minutes. (Optimal time will vary 9. Return to 10x objective. depending on the lot number of the stain.) 10. Close down field diaphragm to smallest opening. 7. Rinse the smear thoroughly with a stream of distilled water. 11. Bring image into sharp focus by moving condenser up or down. 8. Use a wet gauze to wipe the stain off the back of the slide. 12. Center the image of the field diaphragm using the con- 9. Allow slides to air dry. denser centering screws. Procedure 37-4 Peripheral Blood Smear Examination 13. Open field diaphragm until rim of light just disappears beyond the field. Principle 14. Adjust focus for fine detail. A well-stained peripheral blood smear is examined 15. Open condenser diaphragm completely. microscopically. The peripheral blood smear evalu- 16. Slowly close condenser diaphragm until background ation includes: a leukocyte estimate, observation for just begins to turn dark. the presence of abnormal cells, abnormal erythrocyte 1089 1090 Appendix D Hematology Procedures distribution, and erythrocyte, leukocyte, and platelet 3. On 100x magnification (10x objective): morphology, a platelet estimate, and a 100-cell leukocyte a. Scan the blood smear to assure even distribution of differential. leukocytes and observe for immature or abnormal cells. Observe for platelet clumps. Sample b. Examine for abnormal erythrocyte distribution pat- Whole blood, anticoagulated with EDTA or blood collected terns such as rouleaux or agglutination. by a capillary puncture. Blood smears should be prepared c. Locate the optimal counting area. This area extends within 3 hours of collection in EDTA. from the point where the erythrocytes are barely touching to the point where erythrocytes are slightly Reagents and Equipment overlapping. d. Determine the leukocyte estimate by counting the 1. Microscope number of leukocytes in each of five fields of view, 2. Type B immersion oil determining the average number of leukocytes 3. Lens tissue within those five fields and multiplying that num- ber by 0.2 * 103/mcL (1 WBC = 0.2 * 103/mcL). 4. Lens cleaning agent FIGURE 37-11 provides an example calculation. Compare the leukocyte estimate to the leukocyte Quality Control count. The leukocyte estimate should agree {25,. The visual examination of a peripheral blood smear is 4. On 1000x magnification (100x objective): fraught with imprecision and inaccuracies. Each labora- tory should establish a set of guidelines for the peripheral a. Determine the platelet estimate by counting the num- blood smear examination that will minimize the occurrence ber of platelets in each of five fields of view, deter- of these inherent errors. The CLSI Reference Leukocyte Dif- mining the average number of platelets within those ferential Count (Proportional) and Evaluation of Instrumental five fields and multiplying the number by the appro- Methods, 2nd edition can be used as a reference to establish priate conversion factor (1 platelet = 20 * 103/mcL the laboratory's guidelines. if capillary blood or 1 platelet = 15 * 103/mcL if At a minimum, the guidelines should include: EDTA-anticoagulated blood). 1. A definition of the terms used. FIGURE 37-12 provides an example calculation. Compare the platelet estimate to the platelet count. 2. Establishment of 95% confidence limits within The platelet estimate should agree within {25,. the l aboratory for differential performance and reporting. b. Evaluate platelet morphology. This includes obser- vation of platelet size and granularity. Normal plate- 3. Determination of guidelines for a repeat differential. lets are ∼ 294 mcm in diameter. 4. Establishment of a list of abnormalities, which if c. Evaluate erythrocyte (RBC) morphology (Chapter 10). observed on visual examination, must be reviewed by This includes observation of erythrocyte size, shape, the supervisor or clinical pathologist before results are color, and presence of inclusions. reported. 5. Circulation of unfamiliar slides among laboratory pro- 1. RBC size: Normal erythrocytes are approximately fessionals and comparison of results with established 7.0 mcm in diameter. The descriptive term is confidence limits. normocytic. Comparison of erythrocytes to the nucleus of a small lymphocyte is helpful in deter- 6. Define level of accuracy a new laboratory professional mining the erythrocyte size because the nucleus is must achieve prior to being cleared to report differen- approximately 7.0 mcm in diameter. Any change tial results. in size beyond normal variation is termed aniso- cytosis. Microcytic and macrocytic are terms used Procedure to define specific changes in size. An erythro- cyte with a diameter less than 7.0 mcm is micro- 1. Check that the slide identification (i.e., patient's name cytic, whereas an erythrocyte with a diameter and identification number) matches the information on greater than 7.0 mcm is macrocytic. The degree the collection tube or test requisition. of anisocytosis and specific change in size should 2. Observe the overall staining quality of the blood smear be reported. For example, anisocytosis can be both macroscopically and microscopically. reported in terms of slight, moderate, or marked. Appendix D Hematology Procedures 1091 2. RBC color: Erythrocytes with a normal comple- e. Evaluate leukocytes for characteristic changes in ment of hemoglobin have an area of central pal- morphology (e.g., Döhle bodies, hypersegmented lor that is approximately one-third of the cell. The neutrophils, etc.). The changes in leukocyte mor- descriptive term is normochromic. If the area of phology may indicate an underlying hematological central pallor is increased (i.e., greater than one- disorder (Chapter 21, 22). third) indicating a decreased hemoglobin level, the erythrocytes are referred to as hypochromic. Reference Interval The degree of hypochromasia should be reported in terms of slight, moderate, or marked. An addi- Refer to Appendix A, Clinical Laboratory Hematology, 4th edi- tional color change that can be observed is poly- tion for the reference intervals. chromasia or polychromatophilia. This refers to the presence of immature erythrocytes whose Comments residual RNA gives the cell's cytoplasm a blu- ish tinge. If present, the degree of polychromasia 1. The examination of a peripheral blood smear must should be noted in terms of slight, moderate, or occur within the optimal counting area for reliable marked. results to be obtained. Evaluation outside this area leads to inaccurate results. 3. RBC shapes: Normal erythrocytes are bicon- cave discs and will appear as round discs with 2. If a cell estimate doesn't agree with the cell count, this an area of central pallor on the blood smear. may indicate a random error such as a mislabeled blood Any change in shape beyond normal varia- smear. Both cell count and the cell estimate should be tion is termed poikilocytosis. This term should rechecked. always be used in conjunction with the specific 3. The

manual leukocyte count or automated leukocyte cell shapes present. Specific poikilocytes are count, if instrument doesn't perform this correction, discussed in detail in Chapter 10. Certain vari- should be corrected for the presence of nucleated red ations in the shape of erythrocytes are charac- blood cells when five or more nucleated red blood teristic of hematological disorders. For example, cells are seen per 100 leukocytes. The calculation for presence of sickle cells is associated with sickle corrected leukocyte count is provided in Figure 37-14. cell anemia. 4. An abnormal percentage of cell types requires that a 4. RBC inclusions: The observation of specific second 100-cell differential be performed on the "sec- erythrocyte inclusions may indicate an under- ond" blood smear that was prepared on this sample. If lying hematological disorder and their pres- replicate results agree, average the results. Each labo- ence should be noted. Erythrocyte inclusions ratory should establish "Guidelines for Repeat Differ- seen on the peripheral blood smear include ential." For example, repeat differential is performed if basophilic stippling, Howell-Jolly bodies, and greater than 10% reactive lymphocytes are observed on Pappenheimer bodies. The erythrocyte mor- the peripheral blood smear. phology should correlate with the erythrocyte 5. Smudge cells may be minimized by the addition of 22% indices. For example, if hypochromic, micro- human albumin to a portion of the blood sample. Add cytic erythrocytes are observed, the MCHC and one drop of albumin to five drops of blood and prepare MCV should be below their respective reference a blood smear from this suspension. intervals. 6. Sources of errors in manual differentials: d. Perform a leukocyte differential by counting and a. Slide distribution errors: With the wedge smear, classifying 100 leukocytes sequentially. Use the there is uneven distribution of the leukocytes. Larger "battlement" track for examination (Figure 37-13). cells will be found more frequently at the edges of Each leukocyte encountered must be identified and the smear and feather edge than in the center. Small placed in the appropriate category, include distorted lymphocytes will tend to be concentrated in the cen- cells only if they are clearly identifiable. Nucleated ter of the smear. To minimize errors due to distribu- erythrocytes are not included within the 100-cell tion, the manual differential should include areas of differential but are tabulated separately. The results the edge of the smear. of the differential are reported as a percentage of b. Cell-recognition errors: These errors are related to all leukocytes counted. The nucleated erythrocytes the level of expertise, motivation, concentration, are expressed per 100 leukocytes (e.g., 3 NRBC/100 and attitude of the examinee. Poorly prepared WBC). and poorly stained smears will contribute to cell 1092 Appendix D Hematology Procedures recognition errors. To overcome these errors, a cell- material (commercial control or previously assayed patient by-cell comparison with a group of observers may sample) should be analyzed for each 8 hours of patient be done or a comparison of cell-type percentage val- testing. ues with those percentage values determined by the reference method. Sample c. Statistical sampling errors: This is the largest source of error in a manual cell differential. The small sam- Whole blood, anticoagulated with EDTA or free flowing ple size provides only a rough estimate of the fre- capillary blood can be used. quencies of occurrence of cell types, especially types that are normally in low concentrations. Increasing Procedure the number of cells counted as the white cell count rises helps to lower statistical sampling errors. 1. Label the duplicate Leuko-TIC® vials with the sample identification (i.e., patient's name or control). 7. Excess anticoagulant or failure to prepare the blood smears in the allowed time period may result in anticoagulant 2. Prepare duplicate Leuko-TIC® vials as follows: changes in the leukocytes. These anticoagulant changes a. Open snap-top lid of the Leuko-TIC® vial. include cytoplasmic vacuolization, degranulation, karyor- b. Fill the end-to-end capillary with whole blood. Wipe rhexis, karyolysis, and changes in nuclear shape. excess blood from the outside of the capillary. c. Drop the end-to-end capillary into the Leuko-TIC® See references listed in Chapter 37. vial and close the snap-top lid. Gently shake the vial until all blood has left the capillary. Leave end-to- Procedure 37-5 Manual Leukocyte Count end capillary in the vial. Principle d. Prepare the duplicate Leuko-TIC® vial in the same manner. Whole blood is diluted with an acetate buffer, pH 3, solu- e. Let duplicate Leuko-TIC® vials incubate for five tion containing gentian violet stain. The acetate buffer solu- minutes to allow erythrocytes to hemolyze and leu- tion hemolyzes mature erythrocytes and the gentian violet kocyte nuclei to stain. stains the leukocyte's nucleus a light violet-blue color. 3. Clean the hemacytometer and coverglass by flooding Thus, it facilitates the counting process. The dilution cre- them with 70% alcohol. Dry thoroughly with gauze or ated using the Leuko-TIC® method is 1:20. The dilution is tissue; do not allow the alcohol to dry on the hemacy- mixed well and mounted on a hemacytometer. The cells are tometer. Be sure to remove all lint. Place the coverglass allowed to settle, and then are counted in specific areas of in position over the ruled area. the hemacytometer chamber using brightfield microscopy. The number of leukocytes is calculated per microliter (* 109/L) of 4. Charge hemacytometer—Mix diluted blood thoroughly blood. by inverting Leuko-TIC® vial to resuspend cells. a. Fill the chamber filling capillary approximately 1 2 its Reagents and Equipment length with the mixed contents of the Leuko-TIC® vial by capillary action. Close the capillary on the 1. 2 Leuko-TIC® vials containing: upper end with a gloved finger. Acetate buffer, pH = 3.0 380 mcL b. Gently wipe the tip of the chamber filling capillary Gentian violet 0.1 g/L with a kimwipe. Place the capillary tip on the edge of the ruled area of the counting chamber. Carefully 2. 2 End-to-end volume capillaries, 20 mcL charge the hemacytometer with diluted blood by 3. 2 Chamber-filling capillaries releasing pressure applied by a gloved finger to the 4. Hemacytometer with coverglass chamber filling capillary until the hemacytometer 5. Petri dish with filter paper chamber is properly filled. Using the same process, load the other side of the hemacytometer chamber 6. Hand counter with the second Leuko-TIC® vial. 7. Microscope c. Place the hemacytometer on moistened filter paper in a Petri dish, and allow to stand five min- Quality Control utes to permit the cells to settle. (Moistened filter Commercial quality control materials with established paper retards evaporation of diluted sample while control limits should be assayed. At least one quality control standing.) Appendix D Hematology Procedures 1093 5. Carefully place the hemacytometer on the microscope 2. Average the replicate leukocyte counts (i.e., leuko- stage. Perform the cell count as follows: cyte count for each Leuko-TIC® vial), if there is good precision. a. With the low power (10x) objective, locate the ruled area. With proper light adjustment, the leukocyte nuclei should appear light violet-blue in color. Example: b. Begin the counting procedure in the upper left large square (1 mm2) as seen in Figure 37-15. Use the fine Replicate leukocyte count #1 8.1 * 103/mcL adjustment knob to focus up and down as needed to Replicate leukocyte count #2 7.6 * 103/mcL identify the leukocytes. These replicate leukocyte counts demonstrate good precision (see c. Using a hand counter, count all the leukocytes in the Comment #4). upper left large square as follows: Report leukocyte count as: 7.9 * 103/mcL (8.1 * 103/mcL + 7.6 * 103/mcL/2) 1. Count the leukocytes in the first row of squares by going from left to right, then from right to left Reference Intervals in the second row; follow this pattern until all Adult rows in this square are counted. 4.5911.0 * 103/mcL Newborn 9.0930.0 * 103/mcL 2. Within each square, count all leukocytes touch- (mean = 19.5 * 103/mcL) ing the top and left-hand borders. Do not count any cells touching the bottom or right-hand borders. Comments d. Repeat this counting procedure for the other three 1. Up-to-date documents for Leuko-TIC® 1:20 blue proce- large (1 mm2) squares. Record the total count for the dure can be found at www.bioanalytic.de. four large squares. 2. Leukocyte counts should be performed within three e. Count the leukocytes in the four large squares on the hours following dilution of the blood sample. opposite side of the hemacytometer. 3. The moistened filter paper retards evaporation of diluted sample while cells settle on the hemacytometer. Calculations 4. In general, precision between replicates is demon- 1. The calculation formula for hemacytometer cell counts strated if the difference between the two leukocyte determines the number of cells within 1 microliter (1 L counts doesn't exceed 10% of the lower count. or 1 mm3) of blood (Figure 37-16). To make this deter- 5. Technical sources of error in hemacytometer cell counts mination, the total number of cells counted must be cor- are discussed in the table. rected for the initial dilution of blood and the volume of 6. Physiologic sources of error in hemacytometer cell blood used. Because the dilution of blood for a leuko- counts: cyte count is 1:20, the dilution factor is 20. The volume a. Errors resulting in false low count: of blood used is based on the area and depth of the counting area. The area counted is 4 mm2 and the depth 1. Difficulty in obtaining capillary blood—cells is 0.1 mm; therefore, the volume factor is 0.4 mm3. become diluted with interstitial fluid Total number of cells counted * dilution factor * 2. Excessive tissue trauma during collection of cap- volume factor = cells/mm3 illary blood resulting in cell clumping Volume factor = 1/area * depth 7. Presence of nucleated erythrocytes results in a falsely elevated WBC count because they are indistinguishable Example: from leukocytes. The nucleated erythrocyte's nucleus will also stain light violet-blue with the gentian blue Total number of cells (one side of 162 cells stain. A leukocyte differential must be performed to hemacytometer) distinguish between nucleated erythrocytes and leuko- Dilution 1:20 cytes. The number of nucleated erythrocytes per 100 leu- Area counted 4 mm2 kocytes is noted. The correction formula (Figure 37-14) Depth 0.1 mm is applied to the leukocyte count when 5 or more nucle- ated erythrocytes are observed during the 100-cell 162 * 20 * 1/0.4 mm3 = 8,100/mm3 (L) or 8.1 * 103/mcL differential. 1094 Appendix D Hematology Procedures Potential sources of error in hemacytometer counts 2. Prepare duplicate Thrombo-TIC® vials as follows: Source of Error Result a. Open snap-top lid of the Thrombo-TIC® vial. Improper preparation of dilution Falsely decreased or elevated b. Fill the end-to-end capillary with whole blood. Wipe Insufficient mixing of the sample Falsely decreased excess blood from the outside of the capillary. Improperly filled hemacytometer Falsely elevated c. Drop the end-to-end capillary into the Thrombo- Use of kimwipe to remove excess fluid Falsely elevated TIC® vial and close the snap-top lid. Gently shake from hemacytometer the vial until all blood has left the capillary. Leave Improper counting technique Falsely elevated end-to-end capillary in the vial. Evaporation of fluid from hemacytometer Falsely elevated Miscalculation Falsely elevated or decreased d. Prepare the duplicate Thrombo-TIC® vial in the same manner. See references listed in Chapter 37. e. Let duplicate Thrombo-TIC® vials incubate for 5 minutes to allow erythrocytes to hemolyze. Procedure 37-6 Manual Platelet Count 3. Clean two hemacytometers and two coverglasses by Principle flooding them with 70% alcohol. Dry thoroughly with gauze or tissue; do not allow the alcohol to dry on the Whole blood is diluted with a 1% oxalate buffer, pH 6, hemacytometer. Be sure to remove all lint. Place the solution. In this diluent, all erythrocytes are lysed and coverglass in position over the ruled area. platelets are disaggregated. The dilution created using the 4. Charge hemacytometer—Mix diluted blood thoroughly Thrombo-TIC® method is 1:100. The dilution is mixed well by inverting Thrombo-TIC® vial to resuspend cells. and mounted on a hemacytometer. The cells are allowed to settle, and then are counted in a specific area of the hema- a. Fill the chamber filling capillary approximately 1 2 its cytometer chamber using phase contrast microscopy. With length with the mixed contents of the Thrombo-TIC® phase microscopy, the platelets appear as dark round or vial by capillary action. Close the capillary on the oval bodies due to their high refractive index.

The number upper end with a gloved finger. of platelets is calculated per microliter (* 109/L) of blood. b. Gently wipe the tip of the chamber filling capillary with a kimwipe. Place the capillary tip on the edge Reagents and Equipment of the ruled area of the counting chamber. Carefully charge the hemacytometer with diluted blood by 1. 2 Thrombo-TIC® vials containing 1% oxalate buffer, pH = 6.0, releasing pressure applied by a gloved finger to the 990 mcL chamber filling capillary until the hemacytometer 2. 2 End-to-end volume capillaries, 10 mcL chamber is properly filled. 3. 2 Chamber-filling capillaries c. Repeat the procedure to charge the other side of the 4. Hemacytometer with coverglass hemacytometer with the same vial (Thrombo-TIC® 5. vial #1). Petri dish with filter paper 6. Hand counter d. Place the hemacytometer on moistened filter paper in a Petri dish, and allow to stand ten min- 7. Microscope utes to permit the cells to settle. (Moistened filter Quality Control paper retards evaporation of diluted sample while standing) Commercial quality control materials with established control e. Using this same procedure, charge a second hema- limits should be assayed. At least one quality control material cytometer with the second Thrombo-TIC® vial (commercial control or previously assayed patient sample) (Thrombo-TIC® vial #2). should be analyzed for each 8 hours of patient testing. 5. Carefully place the hemacytometer on the microscope Sample stage. Perform the cell count as follows: Whole blood, anticoagulated with EDTA or free flowing a. With the low power (10x) objective, locate the ruled capillary blood can be used. area and the center large square (1 mm2). Examine the entire center square for even distribution of platelets, then Procedure carefully switch to the high-dry-power objective (40x) phase contrast objective for counting platelets. 1. Label the duplicate Thrombo-TIC® vials with the With phase contrast microscopy, platelets appear as sample identification (i.e., patient's name or control). dark round or oval bodies. Appendix D Hematology Procedures 1095 b. Platelets are counted in the entire center large square Reference Intervals (1 mm2) (Figure 37-15) as follows: 1. Count platelets in the first row of squares going 1509450 * 103/mcL from left to right, then from right to left in the second row; follow this pattern until all rows are counted. Comments 2. Within each square, count all platelets touching 1. Up-to-date documents for Thrombo-TIC® 1:100 the top and left-hand borders. Do not count any procedure can be found at www.bioanalytic.de. cells touching the bottom or right-hand borders. 2. Platelet counts should be performed within three hours 3. Use the fine adjustment knob to focus up and following dilution of the blood sample. down to identify the platelets. 3. The moistened filter paper retards evaporation of c. Repeat this counting procedure for the other side of diluted sample while cells settle on the hemacytometer. the hemacytometer. 4. In general, precision between replicates is demon- d. Record the total number of platelets for the strated if the difference between the two platelet counts hemacytometer. doesn't exceed 10% of the lower count. e. Count the platelets on the second hemacytometer 5. A Wright-stained peripheral blood smear should be following the above counting procedure. examined and the platelet estimate determined to con- firm the hemacytometer platelet count. The platelet Calculations estimate should correlate with the platelet count {25,. If a discrepancy exists, this may indicate a random error 1. The calculation formula for hemacytometer cell counts such as mislabeled slide. The platelet count and periph- determines the number of cells within one microliter eral blood smear estimate should be repeated. (1 mm3) of blood (Figure 37-16). To make this determina- 6. Potential sources of error for manual platelet counts are tion, the total number of cells counted must be corrected discussed in the table. for the initial dilution of blood and the volume of blood 7. Technical sources of error in hemacytometer cell counts used. Because the dilution of blood for platelet counts is are discussed in Procedure 37-5. 1:100, the dilution factor is 100. The volume of blood used is based on the area and depth of the counting area. The Potential Source of Error for Manual Platelet Counts area counted is 2 mm2 (i.e., when diluted blood from a sin- Source of Error Result Resolution gle Thrombo-TIC® vial is used to charge a hemacytometer) Insufficient Falsely Recollect sample and thoroughly mix and the depth is 0.1 mm; therefore the volume is 0.2 mm3. anticoagulation of decreased sample immediately after collection. sample Total number of cells counted * dilution factor * Platelet clumping Falsely If capillary sample: recollect sample volume factor = cells/mm3 decreased removing 1st drop of blood and assur- ing free flowing capillary blood or recol- Volume factor = 1/area * depth lect as venous sample with EDTA. If venous sample: recollect sample Example: using sodium citrate anticoagulant. Platelet count should be corrected for dilution effect of liquid citrate Total number of cells (Thrombo-TIC® vial #1) 200 cells (di lution factor = 1.1). Dilution 1:100 Platelet satellitism Falsely See correction for platelet clumps in decreased EDTA venous sample. Area counted 2 mm2 Leukocyte Falsely No correction. Depth 0.1 mm fragmentation increased 200 * 100 * 1/0.2 mm3 = 100,000/mm3 (mcL) or 100 * 109/L (i.e., presence of hyaline bodies) 2. Average the replicate platelet counts (i.e., platelet count for each Thrombo-TIC® vial), if there is good precision. See references listed in Chapter 37. Example: Procedure 37-7 Hemoglobin Concentration Determination Replicate platelet count #1 100 * 103/mcL Principle Replicate platelet count #2 106 * 103/mcL Whole blood is diluted in cyanmethemoglobin reagent. This These replicate platelet counts demonstrate good p recision (see Comment #4). reagent hemolyzes the erythrocytes which releases hemo- Report platelet count as: globin into the solution. The ferrous ions (Fe++) of the hemo- 103 * 103/mcL (100 * 103/mcL + 106 * 103/mcL/2) globin molecules are oxidized by potassium ferricyanide to 1096 Appendix D Hematology Procedures ferric ions (Fe+++). This oxidation results in the formation of a. Label 13 * 100 mm test tubes. methemoglobin which then combines with the cyanide ions b. Using the appropriate volumetric pipets, pipet the (CN-) to form cyanmethemoglobin, a stable compound. All cyanmethemoglobin standard into the lower third hemoglobin derivatives except sulfhemoglobin are con- of each test tube. verted to cyanmethemoglobin. c. Using the 5 mL serologic pipet, add the proper When measured spectrophotometrically at 540 nm, the amount of cyanmethemoglobin reagent to each tube. absorbance of cyanmethemoglobin follows Beer-Lambert’s law and is directly proportional to the concentration of hemoglobin d. Mix contents of each tube thoroughly. in the blood. A reference (standard) curve is prepared using e. Transfer the standard solutions into matched cyanmethemoglobin standard solutions of known hemoglobin cuvets. concentrations (Figure 37-17). An unknown hemoglobin con- f. Read the % transmittance of each solution at 540 nm centration is read from the standard calibration curve. using a reagent blank. Record the results. Reagents and Equipment g. Convert % transmittance for each solution to absor- bance and record. 1. Cyanmethemoglobin standard—available commer- h. Prepare a standard hemoglobin curve using graph cially (80 mg/dL) paper. Plot the absorbance on the y-axis and the hemo- 2. Cyanmethemoglobin reagent—available commer- globin concentration on the x-axis (Figure 37-17). cially as a dry powder or liquid reagent containing 2. Hemoglobin Procedure: potassium ferricyanide (K3Fe(CN6)), potassium cya- a. Pipet 5.0 mL of cyanmethemoglobin reagent into nide (KCN), and sodium bicarbonate. Cyanmethemo- appropriately labeled 13 * 100 mm test tubes (i.e., globin reagent should be stored in a brown bottle to control or patient). prevent deterioration. b. Draw 0.020 mL of well-mixed whole blood into the 3. Test tubes, 13 * 100 mm micropipet. Carefully wipe the excess blood from the 4. Micropipet, 0.020 mL (20 mcL) outside of the pipet tip. 5. Volumetric pipets: 1 mL, 2 mL, 3 mL, 4 mL c. Expel the blood from the micropipet into the cyan- 6. Serologic pipets, 5 mL methemoglobin reagent. 7. Matched cuvets d. Rinse the micropipet several times to remove all 8. Spectrophotometer remaining blood from the pipet tip. 9. Graph paper e. Parafilm the test tube and mix the contents of the test tube thoroughly by inversion. Quality Control f. Incubate the diluted hemoglobin solution at room Commercial controls with established control limits should temperature for 10 minutes. This ensures that full be run with each hemoglobin determination. color development occurs. g. Mix the test tube thoroughly and transfer the con- Sample tents to a matched cuvet. Whole blood; either anticoagulated venous blood or capil- h. Read the % transmittance of the diluted hemoglobin lary blood. EDTA is anticoagulant of choice. solution using a reagent blank at 540 nm and record results. Procedure i. Convert % transmittance for each diluted hemoglo- 1. Preparation of a Standard Hemoglobin Curve: bin solution to absorbance and record results. Prepare duplicate dilutions of the cyanmethemoglobin j. Determine the hemoglobin concentration (g/dL) for standard representing 4.0 g/dL, 8.0 g/dL, 12.0 g/dL, each sample (i.e., control or patient) using its absor- 16.0 g/dL, 20.0 g/dL. bance reading and the standard hemoglobin curve established for that particular spectrophotometer Cyanmethemoglo- Cyanmethemoglo- and set of reagents. Tube bin Standard bin Reagent Dilution 4.0 g/dL 1 mL 4 mL 1/5 Interpretation of Results 8.0 g/dL 2 mL 3 mL 2/5 Using linear graph paper, a standard curve is prepared 12.0 g/dL 3 mL 2 mL 3/5 by plotting the concentration of each cyanmethemoglobin 16.0 g/dL 4 mL 1 mL 4/5 standard on the x-axis and its corresponding absorbance 20.0 g/dL 5 mL 0 mL on the y-axis ( Figure 37-17). A straight line that best fits the Appendix D Hematology Procedures 1097 plotted data points is drawn. The results for control and Quality Control patient samples are read from this curve using their respec- tive absorbance readings. Control material should be run periodically. The frequency is determined by each laboratory’s workload. For instance, Reference Intervals control material can be run at the beginning of each 8-hour shift. Conventional Units (g/dL) SI Units (g/L) Adult Males: 14.0–17.4 140–174 Sample Adult Females: 12.0–16.0 120–160 Whole blood, anticoagulated with EDTA or capillary blood collected in heparinized capillary tubes. Sample should Comments be centrifuged within 6 hours of collection when stored at room temperature. 1. The 80 g/dL cyanmethemoglobin standard is equiva- lent to 20.0 g/dL hemoglobin. Procedure 2. A new standard curve should be established whenever a new lot number of cyanmethemoglobin reagent is 1. Fill two capillary tubes approximately 2/3 to 3/4 full prepared, a change in spectrophotometers is made, or with well-mixed blood sample. the spectrophotometer light source is changed. 2. Seal the dry end of the capillary tube by placing it into 3. Cyanmethemoglobin reagent is unstable and deterio- the sealing clay at a 90° angle. rates upon exposure to light; therefore, it should be 3. Place the capillary tubes in the microhematocrit centri- kept in a brown bottle. Cyanmethemoglobin reagent fuge with the sealed end toward the periphery. Dupli- is stable for several months when stored at room tem- cate tubes should be opposite each other to balance the perature and protected from light. centrifuge. 4. Technical sources of error include inadequate mixing of 4. Centrifuge for 5 minutes (centrifugation time should be sample; inaccurate pipets; pipeting error; use of dirty, determined as part of the laboratory’s quality assess- scratched, or unmatched cuvets; and use of deterio- ment program for the microhematocrit determination). rated reagents. 5. Using a microhematocrit reading device, determine 5. Physiologic sources of error are described in Table 37-9. the microhematocrit. Results should be recorded to the See references listed in Chapter 37. nearest whole number. Duplicates should agree within 1 unit (1%). Procedure 37-8 Microhematocrit Determination Reference Intervals Principle Conventional Units (%) SI Units (L/L) Anticoagulated whole blood is centrifuged, and the volume Adult Males: 42–52% 0.42–0.52 occupied by the erythrocytes is expressed as a percentage of Adult Females: 36–46% 0.36–0.46 the total volume (packed cell volume, PCV). Reagents and Equipment Comments 1. OSHA requires the use of mylar-wrapped capillary 1. Capillary tubes (75 { 0.5 mm in length, tubes or plastic capillary tubes to minimize risk of cap- 1.155 { 0.085 mm in bore) illary sharps injuries. Plain tubes for anticoagulated blood 2. Each laboratory should have a “Quality Assessment Heparinized tubes for capillary blood Program for Microhematocrit Determination”. This

2. Clay program should include: 3. Microhematocrit centrifuge specifications: a. Centrifuge calibration: a. Radius should be greater than 8.0 cm 1. The centrifuge timer should be checked for b. Capable of reaching maximum speed in 30 seconds accuracy and reproducibility with a traceable c. Sustains an RCF of 10,000915,000 * g for 5 minutes stopwatch. without exceeding a temperature of 45°C 2. The centrifuge speed should be checked with a 4. Microhematocrit reading device properly calibrated tachometer. 1098 Appendix D Hematology Procedures 3. The minimum time to achieve optimal packing Potential Sources of Error in the Microhematocrit Procedure of the red cells should be checked with the fol- Source of Error Result lowing procedure: Inadequate centrifugation Falsely elevated Choose two fresh EDTA-anticoagulated blood sam- Incomplete sealing of microhematocrit tube Falsely decreased ples (one sample should have a Hct greater than Excess anticoagulant Falsely decreased 50%). Perform duplicate microhematocrit determi- Failure to read within 10 minutes after Falsely elevated nations at increasing times beginning at 2 minutes. centrifugation Centrifuge times should be increased by 30-second intervals. Record duplicate results at each time inter- See references listed in Chapter 37. val. Continue to increase centrifuge time until the result remains the same for two consecutive time Procedure 37-9 Erythrocyte Sedimentation Rate intervals. The second time interval is the minimum time for optimal packing of red cells. Principle b. Centrifuge brushes should be checked regularly and In the modified Westergren method for determining erythro- replaced when the brushes are less than half their cyte sedimentation rate (ESR), anticoagulated blood is diluted original size. with 0.85% saline and aspirated into a calibrated pipet. The 3. Inclusion of the buffy coat (leukocytes and platelets) cells are allowed to settle for a period of exactly one hour. in the microhematocrit reading will result in falsely elevated microhematocrit results. Reagents and Equipment 4. Sources of technical error for the microhematocrit pro- cedure are given in the table. 1. Westergren pipet—must meet CLSI’s specifications Overall length: 300.5 { 0.5 mm 5. Sources of physiologic error include: Internal diameter: 2.65 { 0.15 mm a. A small amount of plasma remains trapped in the External diameter: 5.5 { 0.5 mm erythrocyte portion even when the sample is prop- Uniformity of tube bore: { 0.05 mm erly centrifuged. This trapped plasma may result 2. Westergren pipet rack in a falsely elevated microhematocrit. Increased amounts of trapped plasma are associated with 3. Timer hypochromasia, macrocytosis, spherocytosis, thal- 4. 0.85% NaCl assemia, sickle cell anemia, and polycythemia. As a 5. Test tubes, 13 * 100 mm result of trapped plasma, the spun microhematocrits 6. Pipets, 2.0 and 0.5 mL are 1–3% higher than hematocrits calculated by elec- tronic counters. Quality Control b. Prolonged use of a tourniquet during sample collection will cause hemoconcentration of the Commercial quality control materials with established con- sample, which is associated with falsely increased trol limits are available for use. The frequency is determined results. by each laboratory’s workload. For instance, quality control material can be run at the beginning of each 8-hour shift. c. Difficult venipuncture or skin puncture resulting in dilution of the sample with interstitial fluid causes a falsely decreased result. Sample d. Hemolysis of the sample during its collec- Whole blood, anticoagulated with EDTA, is the sample tion or transport results in a falsely decreased of choice. The ESR should be set up within 6 hours after microhematocrit. collection. e. Dehydration resulting in a decreased plasma vol- ume will increase the microhematocrit. Procedure f. After acute blood loss, the hematocrit is not a reli- 1. Sample preparation able assessment of the degree of anemia. In this condition, the plasma volume is replaced faster than a. Pipet 0.5 mL of 0.85% NaCl into a 13 * 100 mm test erythrocyte volume. The net result is a falsely low tube. hematocrit until the plasma volume and erythrocyte b. Add 2.0 mL of well-mixed, whole blood to the test tube. volume equalize. c. Mix the test tube to obtain an even suspension. Appendix D Hematology Procedures 1099 2. Using a pipeting device, fill the Westergren pipet to number of reticulocytes present (Figure 37-20). An eryth- the “0” mark ({1 mm) with the diluted blood sam- rocyte containing two or more particles of blue-stained ple. Wipe off the outside of the pipet with gauze or material is a reticulocyte. The number of reticulocytes kimwipe. If necessary, adjust the volume of blood is expressed as a percentage of the total number of to the “0” mark. There should be no bubbles in the erythrocytes counted. blood. 3. Place the Westergren pipet in the pipet rack. Make sure Reagents and Equipment the pipet fits snugly to eliminate possible leakage and that the pipet is in a vertical position. 1. New methylene blue N 4. Set timer for one hour. New methylene blue N 1.0 g (certified by U.S. Biological Stain Commission) 5. In exactly one hour, record the distance in millimeters NaCl 0.89 g between the meniscus of the plasma and the top of the QS with distilled water to 100 mL sedimented erythrocyte column. Do not include the Filter staining solution through Whatman No. 1 filter paper prior buffy coat in the reading. The resulting distance is the to use. erythrocyte sedimentation rate in mm/hr. 2. Test tubes, 12 * 75 mm 3. Pasteur pipets Reference Interval 4. Glass slides Adult Males: 0–10 mm/hr 5. Spreader slide Adult Females: 0–20 mm/hr 6. Microscope Children: 0–10 mm/hr 7. Miller ocular disc 8. Hand counter Comments 1. Disposable Westergren pipets and dilution reservoirs Quality Control are available from several laboratory manufacturers (Figure 37-18). Two levels of commercial quality control materials with established control limits should be run periodically. The 2. Technical sources of error are given in the following frequency is determined by consideration of: (1) control table. procedures specified by the test method’s manufacturer; (2) reagent stability; (3) frequency and volume of test Potential Sources of Error in the ESR Procedure performance; (4) technique dependence of test method; Source of Error Result (5) frequency of quality control failures; and (6) competency Increased environmental Falsely elevated of testing personnel. For example, two levels of quality temperature control materials can be analyzed daily. Westergren pipet in non-vertical Falsely elevated position Vibration of ESR pipet rack Falsely elevated Sample Improper dilution of sample Falsely elevated if overdilution Whole blood anticoagulated with EDTA is recommended; Excess anticoagulant Falsely decreased however, any anticoagulant is acceptable. Free-flowing cap- illary blood can be used. See references listed in Chapter 37. Procedure 37-10 Reticulocyte Count Procedure Principle 1. Reticulocyte Staining: a. Pipet five drops of new methylene blue N solution Using a supravital stain (e.g., new methylene blue), into the labeled 12 * 75 mm test tube. residual ribosomal RNA within the polychromatophilic b. Add an equal volume (five drops) of well-mixed erythrocytes is precipitated creating the reticulocyte. An blood (control or patient sample). equal volume of stain is added to EDTA-anticoagulated blood, the dilution mixture is incubated, and a smear c. Mix gently using a pasteur pipet. is prepared. The smear is examined to determine the d. Incubate at room temperature for 10 minutes. 1100 Appendix D Hematology Procedures 2. Resuspend mixture thoroughly, and prepare 2–3 wedge Reference Interval smears. Air-dry the smears immediately by gently waving the slide. Relative Absolute 3. Label the reticulocyte smear by writing appropriate Adults: 0.5–2.5% 189158 * 103/mcL information directly into the thick end of the smear or Newborn: 1.8–8.0% 2209420 * 103/mcL write on the frosted end of the slide. 4. Reticulocyte Counting: Comments a. Using the oil immersion lens and an ocular fitted with a Miller disc (Figure 37-19), select an area of the 1. Allowing the incubation time to exceed 15 minutes smear where the erythrocytes are evenly distributed, increases the possibility of erroneous results due to the without overlapping. dye adhering to mature erythrocytes. b. Count a minimum of 112 erythrocytes (include 2. The minimum number of erythrocytes evaluated in reticulocytes) in the smaller (B) square and the the determination of the reticulocyte count should reticulocytes in the larger (A) square. When the be 1000 cells or greater. Using the Miller disc, this is minimum of 112 erythrocytes are counted in the accomplished by counting at least 112 erythrocytes in smaller (B) square, a total of 1008 erythrocytes the smaller (B) square. will have been evaluated in the sum of large 3. Technical sources of error: (A) squares. a. Inter-observer error is the greatest cause for analyti- 5. A second laboratory professional should perform cal error, in particular, the variation in what to call a a reticulocyte count on a second smear in the same reticulocyte. “Calibration” reticulocyte slides can be manner. used to assure the clinical laboratory professionals are consistent in what is called a reticulocyte. b. The number of erythrocytes counted is a second Calculations source of analytical error. The chance of error increases as the number of erythrocytes counted 1. The calculation formula for determining the percentage decreases. of reticulocytes present is: c. Other RBC inclusions (Pappenheimer bodies, Howell- Jolly bodies and Heinz bodies, Table 10-10) will be stained with new methylene blue. Howell-Jolly bod- ies and Heinz bodies may be distinguished from precipitated reticulum by their shape and staining characteristics. Heinz bodies appear as light blue green inclusion located at the periphery of the eryth- rocyte. Howell-Jolly bodies are usually one or two round deep-purple staining inclusions and are also visible on Romanowsky stains. Pappenheimer bodies 2. The absolute reticulocyte count is determined by the are indistinguishable from reticulum of reticulocytes. following calculation: If Pappenheimer bodies are suspected, a Prussian blue iron stain should be performed to verify their Absolute Reticulocyte count = Total RBC count presence. (* 106/mcL) * Relative reticulocyte , d. Whole blood-stain mixture should be resuspended prior to making the smears. Reticulocytes have a lower density than mature erythrocytes, and there- Example: fore will be located near the top during incubation. e. Poor drying or moisture may result in the presence of If the reticulocyte count is 1% and the erythrocyte count is refractive artifact on the smears. This refractive arti- 5.00 * 106/mcL, then: fact may be confused with precipitated reticulum. 1, (i.e., 0.10) • 5.00 * 106/mcL = 0.05 * 106/mcL or However, precipitated reticulum is not refractive 50 * 103/mcL and fine focus adjustment will reveal the difference. Appendix D Hematology Procedures 1101 f. Increased glucose levels may cause the reticulocytes Prepare sufficient quantity for the day by adding 5 mg to have a pale stain. sodium dithionite (Na2S2O4) to 1 mL of stock solution. g. Counterstaining with a Romanowsky-type stain is 3. Test tubes, 12 * 75 mm no longer recommended as it can obscure the pre- 4. Micropipet, 0.02 mL cipitated reticulum. 5. Micropipet tips 4. Misinterpretation can result when reporting only the percentage of reticulocytes present in the peripheral 6. Pipets, 2.0 mL blood, because the reticulocyte result is dependent on 7. Paper-board test-tube holder—reading card should the total number of erythrocytes present in the periph- have 14-point or 18-point black type in straight lines on eral blood. If the total erythrocyte count is decreased, a white background, approximately 0.5 cm apart. Tubes the reticulocyte percentage does not accurately reflect should be held 2.5 cm from the reading card. the bone marrow’s production of new erythrocytes. The correction formulas (e.g., corrected reticulocyte count and reticulocyte production index) used to avoid inter- Quality Control pretation errors due to the total erythrocyte count and A positive control (A/S) containing 30–45% HbS and a increased bone marrow stimulation are discussed in negative control (A/A) should be tested with each batch of detail in Chapter 11. patient samples. 5. A reticulocyte production index greater than 2 is associ- ated with hemolytic anemias (e.g., hereditary sphero- Sample cytosis), recent hemorrhage, and response to therapy. A reticulocyte production index less than 2 is associated Whole blood anticoagulated with EDTA, heparin, or sodium with hypoproliferative disorders (e.g., aplastic anemia) citrate is acceptable. Samples can be stored at 4°C for up to and ineffective erythropoiesis seen in megaloblastic three weeks before testing. anemias. See references listed in Chapter 37. Procedure 1. Allow reagents and samples to warm to room tempera- Procedure 37-11 Solubility Test for Hemoglobin S ture prior to performing this test. Principle 2. Pipet 2.0 mL of

working solution into a labeled 12 * 75 mm test tube. This hemoglobin S screening test is based on the relative 3. Add 0.02 mL of whole blood to the appropriately insolubility of hemoglobin S when combined with sodium labeled test tube. dithionite (i.e., sodium hydrosulfite), a reducing agent. When blood is mixed with the working solution, saponin 4. Mix the contents thoroughly. lyses the erythrocytes and hemoglobin is released. Sodium 5. Incubate the tubes for five minutes in the test tube dithionite reduces the released hemoglobin. If hemoglobin holder at room temperature. S is present, it will form liquid crystals and give a turbid 6. Read for turbidity. appearance to the solution. A transparent solution is seen with other hemoglobins that are more soluble in the reduc- ing agent. Results A positive result is indicated by a turbid suspension through Reagents and Equipment which the ruled lines are not visible. A negative result is indicated by a transparent suspension through which the 1. Stock solution: ruled lines are visible (Figure 37-22). K2HPO4, anhydrous 216 g KH2PO4, crystals 169 g Saponin 10 g Comments QS with deionized water to l liter 1. This is a qualitative test and does not distinguish (Store reagent at 4°C for 1 month) between hemoglobin S disease (S/S) and hemoglo- 2. Working solution: bin S trait (A/S). To confirm the presence of HbS and 1102 Appendix D Hematology Procedures differentiate between the two states, hemoglobin elec- area defined by the two sigmoid curves. A curve to the left trophoresis should be performed. of normal indicates increased fragility and a curve to the 2. Other abnormal hemoglobin variants are known to right decreased fragility. cause sickling and will give a positive solubility test. 100 These variants include HbC Harlem, HbS Travis, and HbC Ziguinchor. To differentiate these variants from 90 HbS, high performance liquid chromatography (HPLC) or immunoelectrophoresis fixation may be used to dif- 80 ferentiate these variants from HbS. 70 3. Technical sources of error include: 60 a. Inactive or outdated reagents b. Reagent below room temperature 50 c. Improper mixing of sample with reagent 40 d. Improper interpretation of results 30 4. Physiologic sources of error include: 20 a. Erythrocytosis, hyperglobulinemia, extreme leuko- cytosis, or hyperlipidemia may cause false positive 10 results. To correct the problem, wash erythrocytes 0 with isotonic saline and centrifuge to obtain packed 1.0 .90 .80 .70 .60 .50 .40 .30 .20 .10 0 erythrocytes. Use 0.01 mL of the packed erythrocytes Percent sodium chloride concentration to repeat the test. b. An anemic individual (6 15, hematocrit) may have If spherocytes are present, increased osmotic fragility a false negative result. To resolve this problem, use is observed due to their decreased surface area-to-volume 0.01 mL of packed erythrocytes or 0.04 mL of whole ratio and a limited ability to expand in hypotonic solutions. blood (i.e., double the blood volume) and 2.0 mL An increased osmotic fragility is associated with hemolytic working solution. anemias in which spherocytes are present, in particular, c. Recent transfusion with normal erythrocytes may hereditary spherocytosis. cause a false negative result. d. If an infant is younger than 6 months, false negatives Reagents and Equipment may occur due to low concentration of HbS because 1. Buffered NaCl stock solution, 10%: HbF will be the predominant hemoglobin. NaCl 90.0 g See references listed in Chapter 37. Na2HPO4 13.65 g NaH2PO4•2H2O 2.43 g Procedure 37-12 Osmotic Fragility Test Adjust final volume to 1 liter with distilled water. 2. Working NaCl solution, 1%: Prepare 1:10 dilution of the Principle buffered NaCl stock solution in sufficient volume for Whole blood is added to increasingly hypotonic solutions the number of tests to be performed. of buffered sodium chloride (0.85% to 0.00%), the solutions 3. Serologic pipets, 5 mL and 10 mL are incubated 30 minutes at room temperature, and centri- 4. Micro volume pipettor, 50 mcL with pipet tips fuged to obtain the supernatant. The amount of hemoly- sis at each concentration is determined by measuring the 5. Test tubes, 13 * 100 mm absorbance of the supernatants spectrophotometrically. 6. Centrifuge An osmotic fragility graph is prepared by plotting the % 7. Matched cuvets hemolysis for each solution against its concentration, and 8. Spectrophotometer the results are compared to a normal control. In normal individuals, an almost symmetrical sigmoid shaped curve is obtained. Quality Control Figure: Normal osmotic fragility curve. The osmotic A normal blood sample should always be tested with the fragility curve of a normal individual would fall within the patient sample. Percent hemolysis Appendix D Hematology Procedures 1103 Sample Abs - Ab * , Hemolysis = x s0.85, 100 Abs Whole blood anticoagulated with heparin is the recom- 0, - Abs0.85, mended sample. Where Absx is the absorbance of solution of any given osmotic strength of sodium chloride. Procedure Construction of Erythrocyte Fragility 1. Label 10 test tubes (13 * 100 mm) for each sample (i.e., Graph patient and normal control). Prepare dilutions of work- ing NaCl solution as indicated in the table, using one Plot the percent hemolysis values for each buffered saline set of labeled test tubes (e.g., patient sample set). concentration on the y-axis against the osmotic strengths of the saline diluents on the x-axis (see figure). 2. Transfer 5 mL of diluted working NaCl solution from each tube to the corresponding tube in the second set of labeled test tubes (e.g., control sample set). Reference Interval 3. Add 50 mcL of the appropriate sample (i.e., patient or Normal erythrocytes will begin to hemolyze around 0.50% normal control) to each dilution tube. Mix each test NaCl concentration and hemolysis will be complete at tube by gentle inversion. 0.30% NaCl. 4. Incubate diluted samples at room temperature for thirty (30) minutes. Comments 5. Remix each test tube by gentle inversion. Centrifuge all test tubes at 1200 g for five minutes. 1. An incubated osmotic fragility test is performed to 6. Carefully transfer the supernatant solution from each tube identify patients with mild hereditary spherocytosis in into a cuvet. Do not disrupt the centrifuged cell button. which the standard osmotic fragility test is normal. In the incubated osmotic fragility test, patient’s blood and 7. Measure the % transmittance for each supernatant solu- control blood are incubated for 24 hours at 37 °C under tion against a water blank with the spectrophotometer sterile conditions. A significantly increased osmotic set at 540 nm. fragility after incubation is characteristic of hereditary 8. Calculate the absorbance for each supernatant and spherocytosis. record results. 2. EDTA, citrate, or oxalate anticoagulants should not be used. They will alter the pH of the reaction mixtures. Preparation of the NaCl Solutions for Osmotic Fragility Test 3. Sources of error include: Tube 1% Buffered Distilled Water NaCl%, final a. Hemolyzed sample NaCl (mL) (mL) concentration b. Temperature fluctuations during incubation period 1 8.5 1.5 0.85 c. Improper preparation of dilutions 2 6.5 3.5 0.65 3 6.0 4.0 0.60 d. Chemical purity of sodium chloride solutions 4 5.5 4.5 0.55 4. If the sample is icteric or lipemic, the plasma may be 5 5.0 5.0 0.50 replaced with isotonic saline prior to testing for eryth- 6 4.5 5.5 0.45 rocyte fragility. 7 4.0 6.0 0.40 5. If the patient sample has a low hemoglobin content, 8 3.5 6.5 0.35 wash the erythrocytes of the patient and the control 9 3.0 7.0 0.30 in isotonic saline. Centrifuge the samples and remove 10 0.0 10.0 0.00 the supernatant. Resuspend both samples so that the ratio of cells to saline is the same for both samples. Calculations Perform the osmotic fragility tests on the resuspended Determine the percent hemolysis for each supernatant solu- samples. tion by substituting the absorbance value for the desired concentration in the following formula. See references listed in Chapter 37. Appendix E Answers to Review Questions CHAPTER 1 4. d 5. a 5. a 6. c 1. a 2. d Level II 7. d 8. d 3. a 1. a 9. c 4. c 2. a 10. b 5. b 3. d 6. a 4. a Level II 7. a 5. b 1. d 8. a 6. d 2. d 9. b 7. c 3. c 4. d CHAPTER 2 CHAPTER 4 5. d Level I Level I 6. b 7. c 1. b 1. b 8. a 2. c 2. c 9. a 3. c 3. d 10. c 4. c 4. a 5. a 5. d 6. a 6. a CHAPTER 6 7. b 7. b Level I 8. c 8. c 9. a 9. b 1. a 10. d 10. c 2. b 3. b Level II 4. c Level II 1. c 5. d 1. d 2. a 6. c 2. b 3. c 7. b 3. c 4. b 8. c 4. b 5. a 9. b 5. a 6. d 10. d 6. d 7. d 7. a 8. a Level II 8. d 9. b 1. d 9. b 10. c 2. b 10. b 3. a CHAPTER 5 4. c CHAPTER 3 5. b Level I 6. d Level I 1. a 7. b 1. a 2. b 8. b 2. b 3. b 9. a 3. a 4. c 10. a 1104 Appendix E Answers to Review Questions 1105 CHAPTER 7 3. d 9. b Level I 4. a 10. b 5. b 1. c 6. c CHAPTER 11 2. b 7. c 3. b Level I 8. c 4. a 9. d 1. d 5. a 10. b 2. a 6. d 3. a 7. d 4. a 8. a CHAPTER 9 5. d 9. c Level I 6. a 10. a 7. d 1. d 11. c 8. c 2. c 12. a 9. c 3. b 13. b 10. c 4. c 14. b 5. a Level II 15. d 6. d 16. c 1. c 7. c 2. d Level II 8. c 3. c 1. a 4. d 2. d Level II 5. a 3. a 1. d 6. c 4. c 2. a 7. d 5. b 3. c 8. b 6. a 4. d 9. a 7. d 5. b 10. d 8. d 6. c 11. d 9. d 12. b 10. a CHAPTER 10 11. c CHAPTER 12 12. a Level I Level I 13. d 1. b 14. d 1. b 2. c 15. a 2. a 3. b 3. c 4. b 4. c CHAPTER 8 5. b 5. d Level I 6. c 6. d 7. d 1. b 7. d 8. c 2. b 8. c 9. b 3. d 9. c 10. c 4. c 10. c 5. c Level II Level II 6. b 1. b 1. a 7. c 2. a 2. d 8. a 3. a 3. b 9. b 4. c 4. c 10. b 5. d 5. b Level II 6. c 6. c 1. c 7. c 7. b 2. b 8. d 8. a 1106 Appendix E Answers to Review Questions 9. b 8. d 9. d 10. a 9. a 10. a 11. c 10. a 12. c CHAPTER 17 CHAPTER 15 Level I CHAPTER 13 Level I 1. a Level I 2. c 1. b 3. d 1. a 2. a 4. b 2. d 3. c 5. b 3. c 4. c 6. d 4. c 5. d 7. c 5. a 6. a 8. c 6. c 7. b 9. d 7. c 8. c 10. b 8. d 9. a 9. d 10. c Level II 10. c Level II 1. c Level II 2. b 1. b 3. c 1. b 2. d 4. a 2. d 3. c 5. a 3. a 4. b 6. c 4. c 5. a 7. a 5. b 6. a 8. d 6. a 7. b 9. c 7. b 8. d 10. b 8. d 9. c 9. b 10. b CHAPTER 18 10. a Level I CHAPTER 16 CHAPTER 14 1. d Level I 2. b Level I 1. a 3. c 1. c 2. a 4. d 2. a 3. c 5. c 3. d 4. a 6. a 4. a 5. b 7. a 5. d 6. b 8. c 6. c 7. a 9. a 7. b

8. d 10. d 8. a 9. a Level II 9. c 10. c 10. b 1. c Level II 2. b Level II 1. d 3. a 1. a 2. d 4. b 2. c 3. a 5. a 3. a 4. b 6. c 4. a 5. c 7. a 5. d 6. c 8. d 6. b 7. b 9. d 7. b 8. c 10. a Appendix E Answers to Review Questions 1107 CHAPTER 19 3. a 5. b Level I 4. b 6. a 5. a 7. a 1. b 6. a 8. a 2. c 7. c 9. d 3. a 8. a 10. b 4. a 9. c 11. a 5. d 10. c 12. d 6. c 7. c Level II 13. a 8. b 1. d Level II 9. c 2. c 1. a 10. b 3. d 2. c Level II 4. b 3. d 5. c 4. c 1. b 6. a 5. a 2. b 7. b 6. b 3. a 8. d 7. c 4. a 9. b 8. b 5. d 10. a 9. d 6. d 11. c 10. b 7. c 12. a 8. a CHAPTER 24 9. c CHAPTER 22 10. d Level I Level I 1. b CHAPTER 20 1. c 2. a Level I 2. b 3. b 1. b 3. a 4. c 2. d 4. d 5. a 3. a 5. b 6. b 4. d 6. c 7. d 5. c 7. c 8. a 6. d 8. d 9. c 7. a 9. d 10. b 8. a 10. c Level II 9. c Level II 1. b 10. a 1. a 2. a Level II 2. a 3. c 1. a 3. c 4. b 2. c 4. c 5. c 3. d 5. b 6. d 4. b 6. a 7. a 5. a 7. d 8. a 6. c 8. b 9. d 7. d 9. a 10. b 8. b 10. d 9. c CHAPTER 25 10. d CHAPTER 23 Level I Level I CHAPTER 21 1. d 1. a 2. a Level I 2. a 3. d 1. b 3. b 4. a 2. d 4. b 5. c 1108 Appendix E Answers to Review Questions 6. c 4. c 8. a 7. a 5. a 9. a 8. a 6. a 10. a 9. c 7. a 10. d 8. a CHAPTER 29 11. b 9. d Level I 12. a 10. d 1. d 13. a 2. d 14. b CHAPTER 27 3. b 15. c Level I 4. c 16. d 1. b 5. b 17. d 2. d 6. a 18. c 3. c 7. a Level II 4. b 8. d 1. a 5. b 9. c 2. a 6. d 10. b 3. a 7. a Level II 4. c 8. c 1. c 5. c 9. c 2. c 6. c 10. b 3. a 7. c Level II 4. d 8. a 1. d 5. d 9. b 2. d 6. d 10. a 3. a 7. b 11. c 4. a 8. b 12. c 5. b 9. c 13. b 6. c 10. a 14. b 7. b 15. b 8. a CHAPTER 30 16. c 9. b Level I 17. c 10. a 18. c 1. d 19. a 2. c CHAPTER 28 20. c 3. d 21. d Level I 4. d 1. d 5. a CHAPTER 26 2. b 6. d 3. d 7. d Level I 4. b 8. a 1. b 5. d 9. d 2. a 6. a 10. a 3. b 7. d Level II 4. b 8. c 5. a 1. d 9. d 6. c 2. a 10. a 7. b 3. b Level II 8. d 4. a 9. d 1. a 5. b 10. b 2. c 6. a 3. a 7. c Level II 4. c 8. a 1. a 5. a 9. c 2. d 6. b 10. c 3. a 7. c 11. d Appendix E Answers to Review Questions 1109 CHAPTER 31 3. d 6. d Level I 4. b 7. d 5. a 8. c 1. b 6. c 9. a 2. c 7. d 10. a 3. c 8. d 4. c Level II 9. d 5. d 10. b 1. a 6. d 2. c 7. d Level II 3. b 8. b 1. c 4. d 9. a 2. a 5. a 10. a 3. c 6. a Level II 4. a 7. c 5. b 8. d 1. b 6. d 9. a 2. a 7. c 10. b 3. d 8. d 4. a 9. c 5. c CHAPTER 36 10. a 6. d Level I 7. a 8. b CHAPTER 34 1. b 2. c 9. c Level I 3. b 10. b 1. d 4. a CHAPTER 32 2. a 5. a 3. b 6. b Level I 4. b 7. b 1. b 5. c 8. a 2. a 6. d 9. c 3. c 7. a 10. a 4. c 8. d 11. c 5. b 9. b 12. b 6. b 10. b 13. d 7. b 8. c Level II 14. a 9. c 1. b Level II 10. a 2. a 1. c Level II 3. c 2. a 4. c 3. d 1. a 5. b 4. c 2. d 6. a 5. a 3. d 7. c 6. d 4. c 8. b 7. c 5. b 9. d 8. b 6. b 10. b 9. b 7. d 10. a 8. a 9. b CHAPTER 35 11. a 10. a Level I CHAPTER 37 CHAPTER 33 1. a 2. a Level I Level I 3. c 1. c 1. d 4. d 2. c 2. d 5. b 3. d 1110 Appendix E Answers to Review Questions 4. a 6. d 8. b 5. b 7. c 9. c 6. b 8. b 10. d 7. d 9. d Level II 8. a 10. c 9. c 1. b Level II 10. a 2. d 1. a 3. c Level II 2. b 4. c 1. b 3. c 5. c 2. d 4. d 6. a 3. b 5. a 7. b 4. b 6. d 8. a 5. a 7. c 9. d 6. b 8. c 10. d 7. a 9. b 8. c 10. a CHAPTER 42 9. a Level II 10. b CHAPTER 40 1. c Level I 2. b CHAPTER 38 3. a 1. d Level I 4. b 2. c 5. a 1. c 3. d 6. c 2. a 4. d 7. b 3. b 5. d 8. d 4. d 6. c 9. d 5. a 7. d 10. d 6. a 8. b 7. c 9. a CHAPTER 43 8. b 10. b 9. a Level I Level II 10. a 1. c 1. b Level II 2. c 2. a 3. b 1. b 3. b 4. b 2. c 4. d 5. c 3. c 5. c 6. c 4. a 6. b 7. b 5. a 7. d 8. c 6. c 8. a 9. c 7. b 9. d 10. d 8. a 10. b 9. c Level II 10. b CHAPTER 41 1. a 2. d Level I CHAPTER 39 3. c 1. a 4. b Level I 2. c 5. c 1. d 3. c 6. b 2. a 4. a 7. a 3. d 5. a 8. b 4. b 6. a 9. d 5. b 7. a 10. b Appendix F Answers to Checkpoints CHAPTER 1 Checkpoint 2-3 Checkpoint 1-1 What is the difference between a polymorphism and a What cellular component of blood can be involved in dis- mutation? orders of hemostasis? Answer Answer Polymorphism describes a change in the nucleotide sequence The platelets are involved in hemostasis. of a gene (multiple alternate copies; alleles) that occurs with a frequency of the population. Mutation describes a change Checkpoint 1-2 in the nucleotide sequence of a gene that results in an abnor- A 13-year-old female saw her physician for complaints of mality of function of that gene or gene product. a sore throat, lethargy, and swollen lymph nodes. A CBC Checkpoint 2-4 was performed with the following results: Hb 9.0 g/dL; Hct 30%; WBC 15 * 103>mcL. On the basis of these results, A cell undergoing mitosis fails to attach one of its duplicated should reflex testing be performed? chromosomes to the microtubules of the spindle apparatus during metaphase. The cell’s metaphase checkpoint mal- Answer functions and does not detect the error. What is the effect The girl has a decreased hemoglobin and increased WBC (if any) on the daughter cells produced? count. Reflex testing is suggested to help identify the cause of these abnormal results. A differential will identify the Answer types of leukocytes that are present in increased concen- If one of the paired (duplicated) sister chromatids fails to trations and give clues to the diagnosis. Further laboratory attach to the mitotic spindle during metaphase, the dupli- testing can also help identify the cause of the anemia. cated chromosomes will not separate during anaphase and telophase. If the cell does not catch this mistake and cytoki- CHAPTER 2 nesis still completes, one daughter cell will have two copies of that chromosome and the other daughter cell will have Checkpoint 2-1 none. Both cells are said to be aneuploid (i.e., have an abnor- What does the phrase lipid asymmetry mean when describ- mal number of chromosomes). ing cell membranes? Checkpoint 2-5 Answer What would be the effect on the hematopoietic system The phospholipid’s content of the inner (cytoplasmic) half homeostasis if the expanded clone of antigen-activated B of the lipid bilayer differs from the phospholipid’s content lymphocytes failed to undergo apoptosis after the antigenic of the outer (external) half of the lipid bilayer. Phospha- challenge was removed? tidylethanolamine (PE) and phosphatidylserine (PS) occur in the inner layer, whereas phosphatidylcholine (PC) and Answer sphingomyelin (SM) occur predominantly in the outer layer. The result would be an accumulation of excess lymphocytes Checkpoint 2-2 and a progressive lymphocytosis. Explain the difference between densely staining chromatin CHAPTER 3 and lighter staining chromatin when viewing blood cells under a microscope. Checkpoint 3-1 Answer Describe the bone marrow stromal location of erythrocyte, granulocyte, platelet, and lymphocyte differentiation. Densely staining chromatin represents tightly twisted or folded regions of chromatin that are transcriptionally inac- Answer tive. Lighter staining chromatin represents unwound or Erythrocytes develop in erythroblastic islands located near loosely twisted regions of chromatin that are transcription- the sinuses. The more mature cells are at the periphery of ally active. the island, and the more immature are closer to the center. 1111 1112 Appendix F Answers to Checkpoints Granulocytes are produced in nests that are near trabeculae CHAPTER 4 and arterioles but distant from venous sinuses. Platelets are Checkpoint 4-1 produced from the cytoplasm of megakaryocytes, which are located next to the vascular sinus. Hematopoietic stem cells that have initiated a differentia- tion program are sometimes described as undergoing death Checkpoint 3-2 by differentiation. Explain. Describe the process by which a blood cell moves from the Answer marrow to the vascular space. When a hematopoietic stem cell makes the commitment to Answer differentiate, it begins a maturation process that culminates New blood cells move along adventitial cells to the ablu- in what we call terminally differentiated cells (i.e., cells that minal surface of venous sinuses. Spaces between reticular have lost the capacity to divide and have a finite [limited] cells allow hematopoietic cells to contact the abluminal side life span). Thus, the process of differentiation “dooms” the of vascular endothelium, and a receptor-mediated process cell to eventual death. forces the abluminal and luminal sides of the endothelial cell to touch where the new blood cell is located. These two Checkpoint 4-2 portions of the endothelial membrane fuse and create a pore Explain the difference in the nomenclature used to label pro- through which the blood cell enters the vascular space. genitor cells from that used to label maturing cells within the hematopoietic hierarchy of cells. Checkpoint 3-3 Describe

how the spleen removes old or damaged erythro- Answer cytes from the circulation. Because progenitor cells are not morphologically recog- nizable, they are identified by the progeny they produce Answer when grown in in vitro cultures. Thus, they are identified Old or damaged erythrocytes circulating through the slow as colony-forming units (CFU) or burst-forming units (BFU) transit compartment of the red pulp encounter a toxic with the types of cells in the colony indicated by the cor- (hypoxic, hypoglycemic, acidic) environment and become responding appended letter (e.g., CFU-E, a colony of ery- identifiable to macrophages in the red pulp cords as cells throid cells). The maturing cells, which are morphologically that need to be removed from the circulation. The macro- recognizable, are described using the root of the lineage phages phagocytose these undesirable cells before they exit (e.g., erythro-) with appropriate prefix and suffix to indicate the spleen. stage of development (e.g., proerythroblast). Checkpoint 3-4 Checkpoint 4-3 Contrast primary and secondary hypersplenism and Cytokine control of hematopoiesis is said to be character- give an example of a disorder that can cause secondary ized by redundancy and pleiotropy. What does this mean? hypersplenism. Answer Answer Redundancy refers to the fact that many different cytokines Primary hypersplenism occurs when no underlying disease do the same thing (i.e., GM-CSF and IL-3 have overlapping causes the condition of hypersplenism. Secondary hyper- [nearly identical] activities. Both IL-11 and TPO stimulate splenism occurs when an underlying disorder causes the platelet production [although TPO is the more important hypersplenism. Workload splenic hypertrophy is a form of cytokine, physiologically]). Redundancy is a good thing secondary hypersplenism. It is associated with inflamma- because it assures the organism that if a mutation knocks tory and infectious diseases that increase the defensive func- out a regulatory gene, additional cytokines can take over tion of the spleen. Workload splenic hypertrophy is thought and substitute for the lost activity. At least part of the to be caused by an increase in the number of macrophages explanation for redundancy can be the “shared receptor or an increase in lymphoid cells. Blood disorders in which chains” that have been identified for certain groups of cells are intrinsically abnormal or are coated with antibody cytokines. lead to removal of these cells by the splenic macrophages. Pleiotropy refers to the fact that a single cytokine often Additional cells or metabolic by-products in the spleen has multiple activities, frequently on multiple target cells. can cause infiltrative hypertrophy. This includes disorders in which macrophages accumulate large amounts of undi- Checkpoint 4-4 gestible substances, neoplasms in which tumor cells infil- Individuals with congenital defects of the g@chain of the IL-2 trate the spleen, myelofibrosis in which the spleen contains receptor suffer from profound defects of lymphopoiesis far foci of extramedullary hematopoiesis, and congestive sple- greater than individuals with congenital defects of the chain nomegaly following liver cirrhosis. of the IL-2 receptor. Why? Appendix F Answers to Checkpoints 1113 Answer Checkpoint 5-5 The g@chain of the IL-2 receptor is shared with five other How would an increase in RBC membrane permeability cytokines, all important in regulating lymphopoiesis: affect intracellular sodium balance? IL-4, IL-7, IL-9, IL-11, and IL-15. Thus, a disabling muta- Answer tion of the g@chain results in loss of physiologic activity of all six cytokines, whereas mutations affecting the a@chain A cation pump within the membrane controls the intracellu- compromise only the physiologic activity of a single lar balance of sodium and potassium. Sodium is maintained cytokine, IL-2. at a low intracellular concentration, whereas potassium is kept concentrated inside the cell at 25 times more than the concentration of sodium. The biconcave disc shape of an CHAPTER 5 RBC is vital to the cell’s function and longevity. Maintaining Checkpoint 5-1 this shape depends on the membrane structure providing What is meant by the term erythron? an exact permeability to ions on either side of the mem- brane. Even a slight increase in membrane permeability can Answer cause an inflow of sodium, resulting in a defective mem- Erythron refers to the summation of stages of erythrocytes in brane and an alteration of shape. the marrow, peripheral blood, and within vascular areas of Checkpoint 5-6 specific organs such as the spleen. Erythropoiesis involves the entire erythron. Uncontrolled oxidation of hemoglobin results in what RBC intracellular inclusion? Checkpoint 5-2 Answer What is the first stage of red cell maturation that has vis- ible cytoplasmic evidence of hemoglobin production on a The erythrocyte normally maintains a large ratio of Romanowsky-stained smear? NADPH to NADP+ . When the RBC metabolic pathways fail to reduce oxidized hemoglobin, hemoglobin sulfhydryl Answer groups 1-SH2 are oxidized, which leads to the denatur- The basophilic normoblast stage can have patches of newly ation and precipitation of hemoglobin in the form of Heinz synthesized hemoglobin. The RNA in the young cell stains bodies. Heinz bodies attach to the inner surface of the cell dark blue, but the hemoglobin stains pink. In the next stage, membrane, decreasing cell flexibility. polychromatophilic normoblast, a mix of the blue and pink Checkpoint 5-7 stains give the cytoplasm a bluish gray, or polychromatic, staining appearance. Which erythrocyte metabolic pathway is responsible for providing the majority of cellular energy? For regulating Checkpoint 5-3 oxygen affinity? For maintaining hemoglobin iron in the Explain how a deficiency or absence of LCAT can lead to reduced state? the expansion of the surface area of the red cell membrane. Answer Answer About 90–95% of the cell’s glucose breakdown occurs in the LCAT esterifies cholesterol. Once esterified, cholesterol can- Embden–Meyerhof pathway. Two moles of ATP are gen- not return to the red cell membrane. When LCAT is defi- erated from each mole of glucose metabolized. ATP is the cient, free plasma cholesterol increases and accumulates primary energy source of the erythrocyte. The Rapoport- with the erythrocyte membrane expanding the surface area Luebering shunt produces 2,3-DPG, which affects oxygen of the cell membrane. affinity. The hexose-monophosphate shunt and methemo- Checkpoint 5-4 globin reductase pathways help maintain hemoglobin iron in the reduced state. Compare placement in the membrane and function of peripheral and integral erythrocyte membrane proteins. Checkpoint 5-8 Answer Why are there different reference intervals for hemoglobin concentration in male and female adults but not in male and Integral proteins span the entire thickness of the cell mem- female children? brane, whereas peripheral proteins are on the inner side (cytoplasmic) of the membrane. The integral proteins Answer carry the erythrocyte antigens and attach the skeletal lat- The male hormone, testosterone, stimulates erythropoiesis tice to the bilipid layer of the membrane. The peripheral indirectly by stimulating EPO production in the kidney. This proteins form a skeletal support for the membrane lipid results in an increase of about a 1–2 g in hemoglobin in males. layer. Testosterone production increases significantly in adolescence. 1114 Appendix F Answers to Checkpoints Checkpoint 5-9 Answer What would the predicted serum EPO levels be in a patient Embryonic Hb Portland, z2P2 Hb Gower 1, a2P2 Hb Gower with an anemia due to end-stage kidney disease? 2, z2g2 Fetal HbF (also found in adult), a2g2 Adult HbA, Answer a2b2, HbA2, a2d2. End-stage renal disease results in a primary decrease in Checkpoint 6-4 EPO production and, therefore, decreases serum EPO lev- A patient has an anemia caused by a shortened RBC els. Because EPO is the primary stimulator of erythropoi- life span (hemolysis); how would this affect the HbA1c esis, low levels result in marrow erythroid hypoplasia and measurement? a moderate to severe anemia. Answer Checkpoint 5-10 The red cells are being destroyed at an increased rate and Explain how oxidation of RBC cellular components can lead are being replaced with new cells that have no HbA1c. The to extravascular hemolysis. amount of HbA1c depends on a time-averaged concentra- Answer tion of glucose with older cells having more than younger cells. The HbA1c is not a good indication of glucose con- Oxidation of critical membrane proteins, lipids, and hemo- trol in this case. This same phenomenon also could occur globin can cause protein denaturation, distortion, and rigid- in patients treated with erythropoietin. Additionally, if ity of the membrane and contribute to the cell’s removal abnormal forms of hemoglobin, such as in sickle cell ane- by macrophages. Oxidation also causes clustering of band mia (HbS), are present, there is no HbA and therefore there 3 molecules, which can be a marker of senescence. is no HbA1c. Checkpoint 6-5 CHAPTER 6 What factors influence an increase in the amount of oxygen Checkpoint 6-1 delivered to tissue during an aerobic workout? Describe the quaternary structure of a molecule of hemo- Answer globin. How can a mutation in one of the globin chains at the subunit interaction site, a1b2, affect hemoglobin During an aerobic workout, a buildup of lactic acid and function? CO2 occurs in the tissues. This leads to a shift to the right in affinity of hemoglobin for oxygen, resulting in an increase Answer of oxygen delivered to the tissues. Hemoglobin is composed of four subunits, each of which Checkpoint 6-6 is a heme group nestled in a globin chain. There are two pairs of globin chains. The four subunits are held together What lab tests would help diagnose an increase in RBC by salt bonds, hydrophobic contacts, and hydrogen bonds destruction (i.e., hemolysis), and what would be the in a tetrahedral formation, giving the hemoglobin mol- expected results? ecule a nearly spherical shape. An oxygen molecule can Answer attach to each of the four subunits. In the deoxyhemo- The lab tests should include the total bilirubin, haptoglobin, globin form, 2,3-DPG combines in the molecule’s central and the hemoglobin/hematocrit. If hemolysis is significant, cavity. the indirect bilirubin would increase and the hematocrit Because considerable movement occurs between chains and hemoglobin would decrease. The haptoglobin would when ligands such as oxygen bind to a hemoglobin mol- be decreased because it removes extracellular hemoglobin ecule, mutations in globin genes can affect subunit or dimer dimers. Respiratory carbon monoxide could also be mea- pair interactions. These could result in altered oxygen affin- sured as an indicator of heme catabolism, but this is rarely ity or hemoglobin instability. done. When present in large quantity, methemalbumin and Checkpoint 6-2 hemopexin–heme complexes impart a brownish color to the What globin chains are synthesized in the adult? plasma. A Schumm’s test could be performed in addition to detect these abnormal compounds spectrophotometrically. Answer Checkpoint 6-7 The chains produced are a, b, d, g. The chains pair up to form HbA, HbA2, and HbF. A 2-year-old child was found to have 15% methemoglobin by spectral absorbance at 630 nm. What tests would you Checkpoint 6-3 suggest to help differentiate whether this is an inherited or What are the names and globin composition of the embry- acquired methemoglobinemia, and what results would you onic, fetal, and adult hemoglobins? expect with each diagnosis? Appendix F Answers to Checkpoints 1115 Answer Answer Tests that should be done include NADPH-reductase Neutrophils are increased in a bacterial infection. Basophils activity and hemoglobin electrophoresis. See Table 6-6 for are increased in an immediate hypersensitivity reaction. expected results. Eosinophils are increased in an asthmatic reaction. Checkpoint 7-6 CHAPTER 7 An adult patient’s neutrophil count and monocyte count Checkpoint 7-1 are extremely low (less than 0.5 * 103>mcL and less than An adult patient’s peripheral blood smear revealed many 0.05 * 103>mcL, respectively). What body defense mecha- myelocytes, metamyelocytes, and band forms of neutro- nism is at risk? phils. Is this a normal finding? Answer Answer Phagocytosis and/or the innate immune system is compro- No, metamyelocytes and myelocytes are not normal find- mised when the neutrophil and monocyte concentrations ings in the peripheral blood. They indicate a shift to the are decreased. left in granulocytes and can be seen in infection, inflamma- tion, leukemia, and other conditions. (See Chapter 21 for a CHAPTER 8 complete list.) Checkpoint 8-1 Checkpoint 7-2 Describe the subsets of T cells and B cells derived from the An adult patient’s WBC count is 10 * 103>mcL and there CLP. are 90% neutrophils. What is the absolute number of neutro- phils? Is this within the reference interval for neutrophils? If Answer not, what term would be used to describe it? The CLP differentiates into T, B, and NK cells under the influence of specific transcription factors. Effector T lym- Answer phocyte subsets include cells

responsible for cell-mediated The absolute count for neutrophils is 9 * 103>mcL. This cytotoxic reactions (cytotoxic T lymphocytes [CTL] or indicates an increase in neutrophils referred to as neutrophilia. cytotoxic T cells [CTC]); cells that provide helper activity Checkpoint 7-3 for B cells, macrophages, and other T cells (helper T cells 3TH4 with subsets TH1, TH2, TH17); and cells that function A patient with life-threatening recurrent infections is found to suppress other T-cell immune responses (regulatory T to have a chromosomal mutation that results in a loss of cells 3T active integrin molecules on the neutrophil surface. Why Reg4 ). There are two B lymphocyte subsets, B-1 (a minor component of B lymphocytes) and B-2 (the majority would this result in life-threatening infections? of B cells in the blood and lymphoid tissues). Each mature Answer B lymphocyte makes a specific antibody targeted against a This is known as leukocyte adhesion deficiency-1 (LAD-1), specific triggering antigen by rearranging its immunoglobu- an inability of the neutrophils to “tightly adhere” to the lin genes. B lymphocytes also can concentrate and present vascular endothelial surface. If the neutrophils cannot do antigens to T cells and are the precursors of immunoglobu- this, they have difficulty moving to the site of infection. lin-secreting plasma cells. The NK cell is a form of cytotoxic Therefore, a patient with this condition would be at risk lymphocyte that functions as part of the innate immune for infections. system. NK cells play a key role in the cytolysis of both tumor and pathogen-infected cells. They do not rearrange Checkpoint 7-4 or express T-cell receptor genes or B-cell immunoglobulin A patient has a compromised ability to utilize the oxygen- genes and thus do not express antigen-specific receptors. dependent pathway in neutrophils. What two important Checkpoint 8-2 microbial killing mechanisms could be affected? What characteristics differentiate an immature and a mature Answer B lymphocyte? Production of the respiratory burst and generation of cyto- Answer toxic reactive oxygen intermediates would be abnormal. Membrane markers are present at various stages in B lym- Checkpoint 7-5 phocyte development. B lymphocytes are identified using Indicate which of the granulocytes will be increased in the a panel of antibodies to surface antigens that correspond to following conditions: a bacterial infection, an immediate the stage of the cell’s differentiation. CD markers expressed hypersensitivity reaction, and an asthmatic reaction. by B lymphocytes and their precursors include CD10, CD19, 1116 Appendix F Answers to Checkpoints CD20, CD21, CD22, CD24, and CD38. The CD19 antigen pattern can be dispersed, and nucleoli can be visible. The is considered a “pan-B” antigen because it is found on the cytoplasm is frequently abundant, very basophilic, and earliest B lymphocyte and is retained until the latest stages foamy. The cytoplasmic membrane can have scalloped of activation. The CD10 marker, also known as the common edges because of indentations made by surrounding eryth- acute lymphoblastic leukemia antigen (CALLA), was origi- rocytes. Azurophilic granules can be present. A plasma cell nally believed to be a specific marker of leukemia cells in on a peripheral blood smear ranges in size from 14 to 20 acute lymphoblastic leukemia. It is now known that CALLA mcM and has a decreased N:C ratio. The small nucleus is is present on a small percentage ( 6.3%) of normal bone mar- eccentrically placed in the cell. The nuclear chromatin can row cells and is found only on early B lymphocyte precur- have a spoke-wheel pattern. The cytoplasm is nongranular sors and disappears as cell maturation occurs. and has a deep basophilic color. Checkpoint 8-3 Checkpoint 8-7 What cells produce immunoglobulin? Describe the struc- Describe the process of lymphocyte recirculation. ture of an immunoglobulin molecule. Answer Answer About 95% of lymphocytes are in the extravascular tissue B lymphocytes and plasma cells produce immunoglobu- of lymph nodes and spleen. These cells move continuously lin. Immunoglobulin consists of two pairs of polypeptide between the intravascular and extravascular compart- chains— two heavy and two light chains—linked together ments. They migrate from the lymph nodes into the lym- by disulfide bonds. The pairs of chains are always identical. phatic channels and into the blood via the right lymphatic Each heavy and light immunoglobulin chain consists of a duct and thoracic duct. Lymphocytes in the blood trans- variable region and a constant region. The constant region is migrate between the borders of epithelial cells or through the same for all antibodies within a given class or subclass, epithelial cells lining the blood vessels to reach extravas- but the variable region in each molecule is different. The cular tissue. variable regions of the light and heavy chains determine Checkpoint 8-8 the antibody site. Where are the majority of plasma cells found, and what is Checkpoint 8-4 their function? CD markers identify lymphocytes. What are the CD mark- Answer ers for B cell precursors and T cell precursors? Plasma cells are found primarily in lymph nodes. They can Answer also be found in the bone marrow. Their function is synthe- The CD markers for B cell precursors are CD34, CD19, sis and secretion of antibodies. CD10, CD20, and CD24. The CD markers for T cell precur- sors are CD34, CD44, CD7, CD3, CD4, and CD8. CHAPTER 9 Checkpoint 8-5 Checkpoint 9-1 A young adult patient has a WBC count of 10 * 103>mcL What would be the effect on the platelet count if a patient with 80% lymphocytes. The blood smear reveals 70% reac- had a mutation in the gene for thrombopoietin that resulted tive lymphocytes and 10% nonreactive lymphocytes. What in the gene’s inability to code for functional mRNA? is the absolute concentration of total lymphocytes and Answer reactive lymphocytes? What is a probable cause of these findings? The platelet count would be decreased (due to lack of TPO) because thrombopoietin is the growth factor for Answer megakaryocytes. The counts indicate 8 * 103>mcL total lymphocytes and Checkpoint 9-2 7 * 103>mcL reactive lymphocytes. A viral infection is a probable cause of these results. (See Chapter 22 for specific What is the relationship between megakaryocyte ploidy causes of increased reactive lymphocytes.) level and eventual number of platelets produced from that megakaryocyte? Checkpoint 8-6 Answer How would you morphologically differentiate a reactive lymphocyte from a plasma cell on a peripheral blood smear? Endomitosis and the resulting polyploidy are likely needed to produce the building blocks necessary for thrombopoi- Answer esis. These processes provide a means to generate the neces- A reactive lymphocyte’s size is usually increased, 16 to 30 sary proteins, lipids, granules, and cytoplasmic area for the mcM, and the N:C ratio is decreased. The nuclear chromatin DMS required for platelet release. Appendix F Answers to Checkpoints 1117 Checkpoint 9-3 Checkpoint 10-3 A patient receiving chemotherapy has a post-chemother- A CBC was performed on a blood specimen from a 15-year- apy platelet count of 75 * 103>mcL, with 24% reticulated old female. The results were: Hb 13 g/dL, Hct 40%, RBC platelets. Should the clinician be concerned about the low 4.5 * 106>mcL, WBC 15 * 103>mcL. The differential revealed platelet count? 70% segmented neutrophils, 25% lymphocytes, 4% mono- Answer cytes, 1% eosinophils. Calculate the indices and absolute WBC differential counts. Are any of these parameters out- Although the platelet count is low, an increased reticulated side the reference intervals for this patient? If so, which platelet count after chemotherapy correlates with platelet ones? recovery. The reference interval for reticulated platelets is 5–10%. Answer Checkpoint 9-4 The RBC indices are within the reference range for this patient 1MCV = 88.8 fL, MCH = 28.8 pg, MCHC = 32.5 g>dL2. Platelets have several physiological functions; what are The WBC count is increased. The differential shows a nor- their functions in hemostasis and the immune response? mal relative count for all four WBC types but an increase in Answer the absolute neutrophil count 110.5 * 103>mcL2. In hemostasis, platelets form the primary hemostatic plug Checkpoint 10-4 and assist with secondary hemostatic plug formation A patient has an MCV of 130 fL and an RDW of 14.5. Review and repair of vascular damage. In the immune response, of the blood smear reveals anisocytosis. Explain the discrep- platelets assist in both the innate and adaptive immune ancy between the blood smear finding and RDW. Why is systems. examination of an erythrocyte histogram/cytogram helpful in this case? CHAPTER 10 Answer Checkpoint 10-1 The RDW reflects the ratio of the standard deviation of cell A courier service delivers a tube of blood from an outpatient volume and the MCV. An increased standard deviation with drawing station across town. The requisition for the sample an increased MCV can have an RDW in the reference inter- is for a CBC. The blood is in a blue top vacutainer tube val. Review of the histogram will show an increased base if (containing 3.2% sodium citrate) and was transported in an anisocytosis is present. insulated box containing dry ice. What are the next steps in processing this sample? Checkpoint 10-5 Answer Results from a CBC include MCV 63 fL, MCH 16.7 pg, and MCHC 26.5 g/dL. Describe these cells. The tube of blood should have been collected in a tube con- taining EDTA (purple-topped tube) instead of sodium citrate Answer (blue-topped tube). Because the sample is in a container of These cells would be microcytic and hypochromic. dry ice, it is either frozen or was frozen before arrival in Checkpoint 10-6 your lab and as such, the cells have lysed. This sample is not appropriate to be analyzed for a complete blood count. Review of a peripheral blood smear reveals significant num- The drawing station should be contacted, reminded of the bers of codocytes and echinocytes. Describe the morphol- proper collection and handling of CBC specimens, and the ogy of these cells. Should you report this on the laboratory patient should be redrawn. report? Why or why not? Checkpoint 10-2 Answer The results on a blood specimen drawn from a patient in Codocytes are also called target cells. They look like a tar- the doctor’s office and transported to the hospital laboratory get with the bull’s eye of hemoglobin concentrated in the were: Hb 15 g/dL, Hct 35%, and RBC 2.8 * 106>mcL. Should center of the cell and a thin rim of hemoglobin next to the these results be reported? What should be the next step? membrane. Although both can be artifactual findings, when there is a significant number of these cells present, it should Answer be reported. Echinocytes have spiny processes equally No, the results should not be reported. The RBC parameters spaced around the cell membrane. Target cells can be seen do not meet the rule of 3 and should be investigated. The in liver disease, thalassemia, iron deficiency, and a variety laboratory professional should find out when the blood was of other anemias. Echinocytes can be seen in liver disease, drawn and how it was stored. The blood sample should be PK deficiency, and other pathologic conditions. To differ- checked for hemolysis. entiate whether echinocytes are the result of in vivo or in 1118 Appendix F Answers to Checkpoints vitro conditions, a wet preparation should be examined. If CHAPTER 11 the echinocytes are not present in the wet preparation, their Checkpoint 11-1 presence is an artifact. Explain why a 30-year-old female who smokes a pack of Checkpoint 10-7 cigarettes a day and lives in the Rocky Mountains can be Increased polychromasia is reported on a blood smear. diagnosed with anemia when her hemoglobin is 12 g/dL. What is polychromasia, and what other hematologic assay Answer will reflect the presence of polychromasia? A hemoglobin of 12 g/dL is within the reference inter- Answer val for females. However, the reference interval must be Polychromasia is the term that refers to the presence of poly- adjusted upward for individuals who smoke (+ 0.3 g/dL) chromatophilic erythrocytes. They stain with a bluish tinge and who live at high altitudes (+ 12 g/dL for altitudes of with Romanowsky stains. This blue color is the result of the 9000–9999 feet). presence of residual RNA in the cytoplasm. These cells are Checkpoint 11-2 most likely reticulocytes. The presence of polychromasia is usually reflected in an increased reticulocyte count. Is it possible to have an increased relative reticulocyte count but an absolute reticulocyte count in the reference range? Explain. Checkpoint 10-8 Answer

A medical laboratory scientist reports the presence of How- ell-Jolly bodies in the erythrocytes on a blood smear. What Yes. If the patient is very anemic, the ratio of reticulo- is the composition of these inclusions, and how can he be cytes to total number of erythrocytes is not a very accu- sure they are not Pappenheimer bodies or Heinz bodies? rate indicator of bone marrow production. For example, a reticulocyte count in a patient with a decreased erythrocyte Answer count can be calculated to be an increased percentage of Howell-Jolly bodies are DNA remnants found in RBCs reticulocytes. However, calculating the absolute number before removal by the spleen’s macrophages. They are of reticulocytes will reveal that this number is actually in thought to be the remains of single chromosomes that failed the reference interval. Of course, a reticulocyte count in to attach to the spindle apparatus during mitosis. They usu- the reference interval in the presence of anemia is not an ally occur singly in cells and rarely as two per cell. They are adequate response. round and stain dark blue with Romanowsky stains. Checkpoint 11-3 Pappenheimer bodies are mitochondrial or lysosomal remnants containing iron and protein. They are usually What laboratory test is the least invasive and most cost found as clusters of small blue-staining granules with an effective to evaluate erythrocyte production in the presence iron stain instead of singly as Howell-Jolly bodies. Pappen- of anemia? heimer bodies stain blue, but Howell-Jolly bodies stain red. Answer Heinz bodies usually occur as single round masses The reticulocyte count is an informative index of erythro- near the cell membrane. Heinz bodies do not stain well poietic activity. It requires a peripheral blood specimen and with Romanowsky stains and require supravital stains or is easy to perform. Calculation of the absolute reticulocyte phase contrast microscopy for visualization. Heinz bodies count or reticulocyte production index (RPI) provides more are aggregated denatured hemoglobin. information. The immature reticulocyte fraction (IRF) is Checkpoint 10-9 provided by some instruments and is very helpful in pro- The following data is found on the CBC of a newborn male. viding a reticulocyte maturity index. Hb = 19.5 g>dL1195 g>L2; Hct = 59,10.59 L/L2; Checkpoint 11-4 RBC = 6.5 * 106>mcL; WBC = 17.6 103>mcL Explain why classification of anemia is important, and give the categories of the morphologic and functional Calculate the MCV and MCHC. Evaluate all of the data classifications. for abnormalities. Answer Answer The classification of anemia is important to help the phy- MCV = 59>6.50 * 10 = 90.7 fL sician narrow the pathophysiology of the anemia and MCHC = 195>59 = 33.0 g> subsequently give the appropriate therapy. The classifi- L cation system uses laboratory tests to categorize the ane- All of the CBC data and the calculated indices are normal mias, but the physician also uses patient history, physical for a newborn male. The Hgb, Hct, RBC, and WBC would examination, and symptoms to arrive at a diagnosis. The all be abnormally increased for an adult. morphologic classification subgroups the anemias by Appendix F Answers to Checkpoints 1119 MCV: macrocytic; microcytic, hypochromic; normocytic, Checkpoint 12-5 and normochromic. The functional classification includes What is the risk of population genetic screening for proliferation defects, maturation defects, and survival HH? What is the benefit of population genetic screening defects. for HH? CHAPTER 12 Answer Checkpoint 12-1 The risks are that health insurance companies can dis- criminate against individuals who have the mutated gene In iron-deficiency anemia, would you expect synthesis of and that all of those who test positive might not develop ALAS2 to increase or decrease? Explain. the disease because disease penetrance has not yet been Answer determined. The benefit is that individuals who have the You would expect the synthesis of ALAS2 to decrease. The mutated gene can be identified early before they develop mRNA of ALAS2 has an iron responsive element (IRE) in clinical signs of the disease. Therapy by phlebotomy can the 5′ UTR. When iron is scarce, the iron regulatory protein then prevent chronic disease associated with HH. (IRP) binds to the IRE preventing the assembly of the ini- tiation factors and resulting in decreased translation of the CHAPTER 13 mRNA. Checkpoint 13-1 Checkpoint 12-2 Why is it not possible for all structural hemoglobin variants A patient’s iron studies revealed serum iron 100 mcg/dL to be identified by hemoglobin electrophoresis? and TIBC 360 mcg/dL. Calculate the percent saturation and Answer UIBC. Are these values normal or abnormal? Not all point mutations in the hemoglobin molecule change Answer its electrical charge. If the charge is not changed, the variant hemoglobin will travel as a normal one. , saturation = Serum iron>TIBC * 100 Checkpoint 13-2 100>360 * 100 = 27.8, saturation What does the term silent carrier mean when referring to a UIBC = TIBC - Serum iron hemoglobinopathy? 360 - 100 = 260 mcg>dL Answer These results are normal. The patient has an abnormal hemoglobin, but this does not Checkpoint 12-3 cause any symptoms of disease. A 30-year-old female and a 25-year-old male both had Checkpoint 13-3 bleeding ulcers. Assume that they acquired the ulcer at the The mutation in HbJ-Capetown, a92, Arg S Gln, stabilizes same time, were losing about the same amount of blood, hemoglobin in the R state (Chapter 6). What functional had equal amounts of storage iron to begin with, and were effect does this have on the hemoglobin molecule? taking in about 15 mg of dietary iron each day. Would you expect that the woman and man would develop ID at the Answer same time? Explain. It increases oxygen affinity. The R structure is the oxygenated form of hemoglobin, whereas the T form is deoxygenated. Answer The woman would probably develop ID sooner because Checkpoint 13-4 she is also losing about 20 mg of iron per month through Why do newborns with sickle cell anemia not experience menstruation. episodes of vaso-occlusive crisis? Checkpoint 12-4 Answer How does the peripheral blood picture in ACD differ from The predominant hemoglobin in newborns is HbF, which that seen in IDA? does not sickle. HbS does not become the predominant hemoglobin until after the first 6 months of life. Answer In IDA, the erythrocytes are usually microcytic and hypo- Checkpoint 13-5 chromic. In ACD, the erythrocytes are usually normocytic, Outline the treatment options for a patient with HbS dis- normochromic; normocytic, hypochromic; or in long-stand- ease, pneumonia, and vaso-occlusive crisis. Discuss how ing cases, microcytic, hypochromic. each would affect the patient’s clinical condition. 1120 Appendix F Answers to Checkpoints Answer Checkpoint 13-9 In the short term, the patient’s pain would be managed and The red cell morphology in HbE disease and b@thalassemia he would be hydrated. He would be given antibiotics and are similar: microcytic, hypochromic anemia with target respiratory therapy for the pneumonia. Long-term therapy cells. What laboratory test(s) could differentiate these two might include transfusions to reduce symptoms of anemia. conditions? He might be placed on hydroxyurea therapy to increase Answer the amount of HbF, thus reducing the frequency of vaso- occlusive crises. If he has not suffered severe organ dam- Hemoglobin electrophoresis. HbE shows an abnormal band, age and a compatible donor can be found, he might be a but b-thalassemia has no abnormal hemoglobins present. candidate for bone marrow transplant to correct the genetic Checkpoint 13-10 defect causing sickle cell disease. a. Explain why patients with an unstable hemoglo- Checkpoint 13-6 bin variant usually experience acute hemolysis only after administration of certain drugs or with A child’s parents both have sickle cell trait. The physi- infections. cian orders a hemoglobin electrophoresis on the child. Results of electrophoresis on cellulose acetate at pH 8.4 b. A patient is suspected of having a congenital Heinz body hemolytic anemia but hemoglobin electrophore- show 65% HbS, 30% HbA, 3% HbF, 2% HbA2. Explain sis is normal. Why is it necessary to perform additional these results, and suggest further testing that could help tests? in diagnosis. Answer Answer a. The hemoglobin becomes unstable when the normal In sickle cell trait, the amount of HbS is usually less than red blood cell environment changes to a more acidic or 50%, whereas in sickle cell anemia, it is typically greater hypoxic environment. This can occur when a patient is than 90%. The presence of some HbA suggests sickle taking medication or has an infection. cell trait in this patient. The patient could have inherited b. The abnormal hemoglobin can have an electropho- another abnormal hemoglobin gene such as thalassemia. retic mobility identical to that of normal hemoglo- Electrophoresis at acid pH should be performed to elimi- bin, thereby masking the presence of the abnormal nate the possibility of hemoglobins with similar mobilities hemoglobin. at alkaline pH. DNA testing can provide evidence of thalas- semia. A microcytic hypochromic anemia is also a clue to Checkpoint 13-11 the presence of thalassemia. Why should red cell enzyme assays and hemoglobin elec- Checkpoint 13-7 trophoresis both be performed on a patient with congenital cyanosis? What is the functional abnormality of HbC and HbS? Why do these two abnormal hemoglobins have the same altered Answer functions? The defect can result either from inheritance of HbM, which Answer could be detected by hemoglobin electrophoresis, or from a The HbS and HbC have decreased solubility and tend to defect in the methemoglobin reduction system, which can crystallize. They both have a point mutation in the b6 posi- be detected by enzyme analysis. tion where a nonpolar amino acid replaces glutamine. Checkpoint 13-8 CHAPTER 14 A 13-year-old black female had a routine physical. Her CBC Checkpoint 14-1 was normal, but the differential revealed many target cells. Differentiate the etiology of thalassemias and hemo - Hemoglobin electrophoresis revealed a band that migrated globino pathies. like HbS on cellulose acetate at pH 8.4. Her hemoglobin Answer solubility test was negative. Explain the results and suggest a follow-up test to determine a diagnosis. Thalassemia results from deletional or nondeletional mutations in one or more globin genes that reduce or Answer eliminate synthesis of the corresponding globin chain(s). The patient can have another abnormal hemoglobin that Hemoglobinopathy is a qualitative hemoglobin disorder migrates in a manner similar to HbS such as HbD and caused by a point mutation in a globin gene, usually HbG. Electrophoresis on citrate agar at pH 6.0 should be the b gene that results in an amino acid substitution in performed to allow distinction of this hemoglobin from the corresponding globin chain and alters hemoglobin HbS. stability and function. Appendix F Answers to Checkpoints 1121 Checkpoint 14-2 Answer What are the most common genetic mutations associated b@Thalassemia major is characterized by an absence or sig- with a@thalassemia? nificant reduction in the synthesis of b@genes. Although the synthesis of b@gene begins at approximately the third Answer trimester of fetal development, quantities sufficient to Gene deletions. assemble significant amounts of HbA do not occur until Checkpoint 14-3 approximately 6 months of age. Therefore, the lack of pro- duction of b@chains and the resultant lack of HbA does Why do a- and b@thalassemia result in more clinically severe not manifest until 6 months of age when HbA synthesis is disease than other types of thalassemia? expected. The major hemoglobin at birth is HbF. Answer Checkpoint 14-7 HbA is composed of a- and b@chains and constitutes 97% In b-thalassemia, what erythrocyte parameter on the CBC of adult hemoglobin. Thus, a reduction in either a@chains or differs significantly from that found in iron deficiency? b@chains decreases the concentration of the most abundant normal adult hemoglobin. The other globin chains are associ- Answer ated with hemoglobins that are either low in concentration The erythrocyte count in thalassemia is normal or high for in adult blood or are designed to function in embryonic or the degree of anemia, but in iron deficiency, it is decreased. fetal life. Both iron deficiency and thalassemia are characterized by Checkpoint 14-4 microcytic, hypochromic anemia with target cells. Which of the three normal adult hemoglobins would be Checkpoint 14-8 affected in hydrops fetalis? Why is gdb@thalassemia more severe than db@thalassemia and CS-thalassemia? Answer Answer Hydrops fetalis occurs when there is a deletion in all four a@genes; therefore, all three normal adult hemoglobins, All normal adult hemoglobin contains a@chains with either HbA, HbA g@, (HbF), d@, or b@ (HbA) chains. A

reduction or absence of 2, and HbF are affected because each contains a@chain. all three chains in gdb@thalassemia decreases the production of all three adult hemoglobins. gb@thalassemia can partially Checkpoint 14-5 compensate by producing g@chains (HbF). Patients with CS Compare oxygen-binding characteristics of HbH relative to thalassemia bear some normal a@genes and can therefore HbA and myoglobin. make all adult hemoglobins, albeit at a reduced rate. Answer Checkpoint 14-9 The allosteric interactions of the two a@chains and the two In combination disorders of structural Hb variants and thalas- b@chains confer on HbA cooperative oxygen-binding char- semia, why is a@thalassemia inherited with sickle cell trait less acteristics called heme/heme interaction (Chapter 6). As a severe than b@thalassemia coexpressed with sickle cell trait? deoxygenated HbA molecule binds an oxygen molecule, Answer hemoglobin incurs an increased affinity for oxygen at the other The severity of symptoms associated with HbS is related three available sites. Oxygen affinity continues to increase with to the concentration of HbS. The combination of one HbS each subsequent molecule bound. By virtue of the binding of b@gene and one b@thalassemia gene results in little to no hydrogen atoms and 2,3-DPG to oxyhemoglobin in the tis- HbA formation because both of the b@genes are mutated. sues, HbA releases oxygen molecules in an incremental fash- In contrast, an a@gene mutation reduces the number of ion regulated by the oxygen, carbon dioxide, and hydrogen a@chains that can combine with bS, reducing the amount ion concentrations in the tissue. The incremental binding and of the abnormal structural hemoglobin (HbS) and thus release of oxygen is expressed as a sigmoidal oxygen dissocia- reducing the symptoms associated with the abnormal tion curve. HbH, by virtue of its four b@chain composition, hemoglobin. does not exhibit heme/heme interaction and therefore func- tions as four separate subunits with identical oxygen affini- Checkpoint 14-10 ties. The oxygen dissociation curve is therefore expressed as Which laboratory tests should be performed first to differ- a hyperbolic curve similar to that of monomeric myoglobin. entiate thalassemia and iron deficiency? Checkpoint 14-6 Answer Why are the symptoms of b thalassemia major delayed until The most cost-efficient and diagnostically effective studies approximately the sixth month of life? should be performed first. In this case, iron studies should 1122 Appendix F Answers to Checkpoints be done first because iron deficiency is the most common Answer cause of microcytic hypochromic anemia. The iron stud- Macro-ovalocytes, Howell-Jolly bodies, and hypersegmen- ies include serum ferritin, serum iron, TIBC, and percent tation of neutrophils. saturation. If the iron studies do not reveal iron deficiency, hemoglobin electrophoresis should be performed. Checkpoint 15-5 Hal had small bowel resection due to carcinoma. Explain CHAPTER 15 why he is at high risk for folate deficiency. Checkpoint 15-1 Answer Explain why patients with cobalamin or folate deficiency Dietary folate must be deconjugated in the small intes- have megaloblastic maturation. tine for absorption to occur. Intestinal villi in the small Answer intestine contain receptors for folate binding and absorp- tion into the body. An individual with a small bowel The megaloblastic anemias are the result of abnormal DNA resection would require ongoing folate therapy because synthesis (a nuclear maturation defect). As a result, the absorption is impaired. Pharmacological doses of folate delayed nuclear development prevents cell division. RNA are given periodically intramuscularly to maintain synthesis and cytoplasmic maturation are not affected. The normal levels. result is production of large cells with nuclear cytoplasmic asynchrony. The basis for the nonmegaloblastic anemias can Checkpoint 15-6 be related to an increase in membrane lipids. What is the most common cause of folate deficiency, and in Checkpoint 15-2 what groups of individuals is it usually found? Patients with megaloblastic anemia often present with a yel- Answer low or waxy pallor. What is the diagnostic significance of Nutritional insufficiency is the most common cause of folate this clinical symptom? deficiency. Body stores of folate are sufficient to maintain Answer minimal body requirements for only 3–6 months. Folate deficiency develops more rapidly than cobalamin (vitamin Ineffective erythropoiesis in the marrow results from B nuclear/cytoplasmic asynchrony of cell development as a 12) deficiency. Once a negative folate balance occurs, serum folate values can begin to decrease within 2 weeks. In devel- result of the lack of thymidine and a diminished DNA syn- oped countries, the elderly, alcoholics, and pregnant women thesis. The abnormal cells are hemolyzed in the marrow. If are considered high-risk groups for folate deficiency. intramedullary hemolysis is significant, all classic signs of hemolysis will accompany the megaloblastosis. Unconju- Checkpoint 15-7 gated bilirubin levels of plasma increase and result in jaun- A patient has the following results: cobalamin 50 pg/mL, dice, which results in a yellow and waxy skin pallor. serum folate 4 ng/dL, RBC folate 100 ng/mL. Interpret Checkpoint 15-3 these results. Why are abnormalities of leukocytes and platelets present Answer in megaloblastic anemia? Normal serum cobalamin (vitamin B12) values range from Answer 247–800 pg/ml in adults and 160–1200 pg/ml in newborns. Granulocytes and platelets can also show changes evident Normal serum reference interval for serum folate is 2.5–20 of abnormal hematopoiesis (nuclear maturation defect). ng/ml and for RBC folate is 160–700 ng/ml. These results Hypersegmented neutrophils (more than five nuclear lobes) indicate the patient is cobalamin deficient with an accom- can be found in megaloblastic anemia even in the absence panying decrease in RBC folate. Serum folate is normal. of macrocytosis. This finding is considered highly sensitive The RBC and serum folate are not good indicators of folate and specific for megaloblastic anemia. Giant metamyelo- status in cobalamin deficiency; a deficiency of cobalamin cytes and bands with loose, open chromatin in the nuclei impairs methionine synthesis and leads to the accumula- are diagnostic. The myelocytes show poor granulation as tion of methyl-THF in the serum. In addition, cobalamin is do more mature stages. Megakaryocytes can be decreased, required for normal transfer of methyl-THF to the cells and normal, or increased. Maturation, however, is distinctly for conjugating the folate to keep it in the cell. Thus, serum abnormal. Some larger than normal forms with separation folate can be falsely increased and erythrocyte folate can of nuclear lobes and nuclear fragments can be found. be falsely decreased in cobalamin deficiency. In this case, the patient appears to have a cobalamin deficiency. Even Checkpoint 15-4 though the RBC folate is low, it is not a good indicator of What abnormal morphological findings on a stained blood folate deficiency. Homocysteine and MMA tests can help smear compose the triad in megaloblastic anemia? define the deficiency. Appendix F Answers to Checkpoints 1123 Checkpoint 15-8 Answer Explain why there is a megaloblastic anemia in transcobala- A specific test that measures the increased excretion of min deficiency when the serum cobalamin concentration is methylmalonic acid (MMA) in the urine indirectly indi- normal. cates decreases in cobalamin concentration. Homocysteine Answer increases in the plasma of patients with cobalamin or folate deficiency. Monitoring serum levels can serve as an early Although transcobalamin is only about 5% saturated, it is detector of cobalamin deficiency. Many recent studies have the primary transport protein for cobalamin. Transcobala- concluded that MMA and homocysteine are the most sensi- min binds 90% of the newly absorbed cobalamin. Con- tive and specific indicators of cobalamin deficiency. genital deficiency of transcobalamin produces a severe megaloblastic anemia in infancy. However, serum cobala- Checkpoint 15-12 min concentration in this condition is normal. Which clinical type of CDA gives a positive Ham test result Checkpoint 15-9 and presents with a normoblastic marrow? Explain why severe cobalamin deficiency sometimes pres- Answer ents with neurological disease. With CDA-II, bone marrow precursors are normoblastic but Answer are typically astnucleated with up to seven nuclei. Type II is distinguished by a positive acidified serum test (Ham test) In cobalamin deficiency, a defect in degradation of propi- but a negative sucrose hemolysis test. In the Ham test, only onyl CoA to methylmalonyl CoA and, finally, to succinyl about 30% of normal sera are effective in lysing CDA II cells. CoA occurs. As propionyl CoA accumulates, it is used as a primer for fatty acid synthesis replacing the usual primer Checkpoint 15-13 acetyl CoA. It is probable that demyelination (destruction, What are the causes of macrocytosis seen in alcoholism? removal, or loss of the lipid substance that forms a myelin sheath around the axons of nerve fibers), a characteristic Answer finding in cobalamin deficiency, is a result of this errone- Alcohol abuse is one of the most common causes of non- ous fatty acid synthesis. Because of the defective fatty acid anemic macrocytosis. The macrocytosis associated with degradation, a critical feature of demyelination in cobala- alcoholism is usually astfactorial and can be megaloblas- min deficiency is neurological disease. Peripheral nerves tic. In up to 60% of cases, anemia does not accompany are most often affected, presenting as motor and sensory macrocytosis. neuropathy. The brain and spinal cord can also be affected Checkpoint 15-14 leading to dementia, spastic paralysis, and other serious neurological disturbances. What are three clinical or laboratory findings (in addition to assessing the bone marrow) that can distinguish a non- Checkpoint 15-10 megaloblastic macrocytic anemia from a megaloblastic Why is pernicious anemia considered an autoimmune anemia? disorder? Answer Answer A normoblastic macrocytic anemia does not present with Pernicious anemia is often associated with autoimmune pancytopenia. Granulocytes and platelet counts are usu- disease. Antibodies directed against the patient’s own cells ally normal. Hypersegmented neutrophils are not found or proteins are found. Autoantibodies found in pernicious in nonmegaloblastic macrocytosis, and the abnormal RBC anemia (PA) are of two types, blocking and binding. Block- morphology is less pronounced. In addition, symptoms ing antibodies prevent the formation of the intrinsic cobala- commonly found in megaloblastic anemia such as jaundice, min complex, and binding antibodies prevent attachment glossitis, and neuropathy are absent in nonmegaloblastic and absorption of cobalamin into the ileum mucosal epi- anemia. thelial cell. Antibodies against intrinsic factor are detected in up to 75% of PA patients. The majority of PA patients CHAPTER 16 demonstrate antibodies against the parietal cells in the floor Checkpoint 16-1 of the stomach. Patients with PA can have one or both types of antibodies. An anemic patient has a (corrected) reticulocyte count of 1.5%, hemoglobin of 10.0 g>dl1100 g>L2, hematocrit of 30% Checkpoint 15-11 10.30 L>L2, total neutrophil count of 30 * 103>mcL, and a What two lab tests are the most specific indicators of cobala- platelet count of 0.4 * 103>mcL. Is it likely that this patient min deficiency? has aplastic anemia? 1124 Appendix F Answers to Checkpoints Answer red cells increases if the membrane is faulty as is the case of the This patient’s current laboratory results do not meet the cri- red cells seen in hereditary spherocytosis. The spherocytes in teria for a diagnosis of severe aplastic anemia. It is possible HS are lacking membrane proteins needed for normal red cell that a bone marrow analysis will be ordered to determine permeability and integrity. This problem leads to an increased the cause of the pancytopenia. If the bone marrow is hypo- need for glucose and ATP. Hemolysis can be delayed by add- plastic, the patient’s laboratory tests can be monitored for ing glucose prior to incubation as spherocytes can utilize this further decreases in cell counts. extra glucose and remain intact for a longer period of time. Checkpoint 16-2 Checkpoint 17-3 A pancytopenic patient has a presumptive diagnosis of Why do the elliptocytes in HE demonstrate normal osmotic aplastic anemia. While performing a smear review, you fragility? observe the presence of several types of poikilocytes, dys- Answer morphic neutrophils, and large agranular platelets. Do these The elliptocytes in HE demonstrate a normal osmotic fragil- findings support the presumptive diagnosis? If not, which ity curve because the defect does not lead to a change in the disorder(s) would be more likely for this patient? surface-area-to-volume ratio of the erythrocyte. The ellipto- Answer cyte can take on as much fluid as a normal cell and lyse at No, these findings are not consistent with a diagnosis of the same concentration of hypotonic saline as a normal cell. aplastic anemia. Usually morphology of cells in all three cell Checkpoint 17-4 lines is normal in patients with aplastic anemia. The presence Interpret the results of the following thermal sensitivity test: of dysmorphic erythrocytes, neutrophils, and platelets sug- Patient’s erythrocytes

Marked erythrocyte fragmentation after gests the pancytopenic patient described here has a different 10-minute incubation at 46°C clinical condition. Myelodysplastic syndrome is one possibil- normal control no significant change in erythrocyte morphology ity, and a bone marrow exam is indicated for confirmation. after 10-minute incubation at 46°C. Answer CHAPTER 17 The patient’s erythrocytes are demonstrating an increased Checkpoint 17-1 sensitivity to heat by fragmenting at 46°C. normal erythro- List various factors related to changes in the erythrocyte cytes do not fragment until heated to 49–50°C. Hereditary that can lead to a decrease or increase in the MCV in heredi- pyropoikilocytosis is an inherited disorder that demon- tary spherocytosis. strates erythrocytes that are sensitive to heat. Answer Checkpoint 17-5 Erythrocyte factors that can cause a change in the MCV Explain why the osmotic fragility is decreased in DHS. include formation of spherocytes, presence of polychroma- Answer sia (reticulocytosis) and depletion of iron, folate or cobala- The erythrocytes in DHS are dehydrated. They can take on min needed to form hemoglobin. Many densely stained more water than a normal erythrocyte and display complete spherocytes on the peripheral smear can cause a decrease in lysis at a lower concentration of hypotonic saline than nor- the MCV. Increased polychromasia can lead to an increase mal erythrocytes. in the MCV. Chronic hemolysis can lead to depleted iron Checkpoint 17-6 stores and folate/cobalamin levels. Low iron stores will lead to microcytic erythrocytes and thus a decreased MCV; Determine the type of erythrocyte membrane disorder pres- depleted folate/cobalamin levels will lead to the formation ent based on the following lab results: reticulocyte count: of macrocytes and an increased MCV. 4%; osmotic fragility: increased; autohemolysis: increased; bilirubin: increased; peripheral smear: 30% stomatocytes Checkpoint 17-2 present. Explain why a patient with hereditary spherocytosis will Answer demonstrate increased hemolysis in an autohemolysis test and why adding glucose prior to incubation will lead to a The erythrocyte membrane disorder that best fits these lab normal rate of hemolysis in these patients. results is hereditary overhydrated stomatocytosis. key features include a large percentage of stomatocytes and an increased Answer osmotic fragility test. The patient’s bilirubin level is elevated, The autohemolysis test determines whether a patient’s red which signifies increased erythrocyte hemolysis. Other dis- cells can remain intact when incubated for 24 hours at 37°C. orders presenting with an increased number of stomatocytes It is a measure of red cell membrane integrity as well as the include acute alcoholism, liver disease, and cardiovascular adequacy of enzymes involved in glycolysis. Hemolysis of the disease. These disorders, however, show little hemolysis. Appendix F Answers to Checkpoints 1125 Checkpoint 17-7 Checkpoint 18-2 Compare the membrane lipid abnormalities seen in LCAT Oxidant compounds are harmful because they result in the deficiency, spur cell anemia, and abetalipoproteinemia, and production of toxic peroxides or other oxygen radicals that explain how they result in hemolysis of the cell. overwhelm the body’s natural mechanisms to scavenge Answer them. Why is the protection against oxidants easily com- promised in G6PD deficiency? Spur cell anemia: increase in serum lipoproteins, increase in membrane cholesterol, but normal membrane concentration Answer of phospholipid. As the ratio of the membrane cholesterol Protection from oxidants is compromised in G6PD defi- to phospholipid increases, the cell is flattened and loses ciency because the enzyme cannot supply enough NADPH deformability. After repeated splenic passages, portions of to keep glutathione in the reduced state. Reduced glutathi- membrane fragment and the cell acquires the spur shape, one is the cell’s primary protection from oxidants. resulting in eventual hemolysis. Abetalipoproteinemia: absence of serum b@lipoprotein, Checkpoint 18-3 low serum cholesterol, triglycerides, phospholipids. There Erythrocyte morphology should always be examined is an increase in the ratio of cholesterol to phospholipid. carefully. The ability to pick up subtle clues regarding Erythrocyte membrane cholesterol is normal, but sphin- the cause of a disease process can be acquired from a gomyelin is increased. Membrane fluidity is decreased comprehensive evaluation of abnormal erythrocyte mor- because of increased sphingomyelin. As the cell ages, the phology. How is this likely to aid the diagnosis of G6PD degree of distortion increases, resulting in hemolysis. deficiency? LCAT deficiency: LCAT catalyzes formation of choles- Answer terol esters from cholesterol. In LCAT deficiency, increased Careful evaluation of a blood smear and reporting of bite levels of cholesterol, phospholipids, and triglycerides in cells alert the health care practitioner to the mechanism serum occur. High-density lipoproteins decrease. Increased of cell destruction. When the mechanism of destruction is serum cholesterol results in increased cholesterol in the considered, this leads to the selection of more diagnosti- erythrocyte. Target cells are formed with high cholesterol cally effective definitive tests to find the root cause of the levels. Hemolysis is mild. anemia. Checkpoint 17-8 Checkpoint 18-4 Explain why immunophenotyping with CD14, CD55, and CD59 is used to establish a diagnosis of PNH. What are the differentiating characteristics of PK and G6PD deficiencies found on the peripheral blood smear? Answer Answer PNH is characterized by a deficiency of GPI necessary to link a variety of proteins to the cell membrane. CD55, CD59, and PK deficiency has echinocytes and irregularly contracted CD14 are GPI linked proteins. CD55 and CD59 are found on cells; G6PD deficiency has bite cells and spherocytes. After neutrophils. CD59 is found on erythrocytes, and CD14 is found splenectomy, additional cell abnormalities can be found. on monocytes. These proteins are linked to the cell membrane by GPI. It is recommended that at least two different antibod- CHAPTER 19 ies be used to detect different kinds of GPI anchored proteins. Checkpoint 19-1 CHAPTER 18 What are the three major categories of immune hemolytic anemia, and how is antibody production stimulated in each Checkpoint 18-1 type? Transfusion of red blood cells in a patient with chronic non- spherocytic, hemolytic anemia as the result of an erythro- Answer cyte enzyme deficiency does not reverse or prevent the Autoimmune: alteration of self-antigen or immune system recipient’s condition. However, a transfusion does help regulation dysfunction to raise the patient’s hemoglobin. If tests are performed to Alloimmune: response to foreign erythrocyte antigen quantitate the enzyme after a transfusion, the results can be Drug induced: immune response to epitope on drug in com- within the normal reference intervals. Explain. bination with erythrocyte antigen Answer Checkpoint 19-2 Presence of transfused cells contributes to a falsely normal Explain how the class of immunoglobulin, amount of anti- test result because these cells are normal and contain a nor- body bound, and thermal reactivity of the antibody affect mal amount of the enzyme. hemolysis. 1126 Appendix F Answers to Checkpoints Answer SLE, chronic inflammatory diseases, and viral/bacterial IgM is able to activate complement efficiently, causing infections. intra-vascular hemolysis. IgG is less able to activate com- Checkpoint 19-6 plement, and the coated cells are removed in the spleen What is the DAT pattern in WAIHA? Explain why sphero- (extravascular) via interaction with Fc receptors on splenic cytes are commonly seen in WAIHA. macrophages. Because a minimum amount of antibody molecules Answer must be bound for hemolysis to occur, the more antibody The most common pattern is reaction with: molecules on the cell, the more likely it is that hemolysis Polyspecific AHG positive will occur. anti-IgG positive IgG antibodies typically have optimal reactivity at 37°C anti-C3 negative (body temperature) whereas IgM antibodies have an opti- In some cases, the anti-C3 is positive. mal thermal amplitude of 20–25°C. Spherocytes are commonly seen because the erythro- Checkpoint 19-3 cyte is coated with IgG, and as it passes through the spleen, Compare the mechanisms of IgG-mediated hemolysis with the splenic macrophages (with Fc receptors) interact with those of IgM-mediated hemolysis. the cell and remove portions of the erythrocyte membrane. As more portions are removed, the cell becomes round Answer (spherocyte). IgM-mediated hemolysis has two possible outcomes. The Checkpoint 19-7 first occurs when the IgM molecule fixes complement and the complement cascade goes to completion (C1–C9). This Describe the mechanism of cell destruction in CAS. results in intravascular hemolysis. If, however, the IgM Answer molecule fixes complement but the cascade does not go to The antibody in CAS is usually an IgM that is capable of completion, the C3b molecules that remain on the erythro- activating complement and causing intravascular hemoly- cytes interact with C3b receptors on macrophages, resulting sis. The IgM activates complement in the cooler portions in extravascular hemolysis. of the circulation. As the erythrocytes move into warmer In IgG-mediated hemolysis, complement is infrequently parts of the circulation, IgM leaves the cell, and complement activated and most hemolysis is extravascular. The Fc por- cascade goes through C9. tion of the attached IgG molecule reacts with Fc receptors on In other instances, the IgM antibody fixes complement, splenic macrophages. The cell can be completely phagocy- but the cascade does not go to completion. Cells are then tized by the macrophage, or portions of the membrane can destroyed via the C3b receptors on macrophages. be removed resulting in spherocytes. Checkpoint 19-4 Checkpoint 19-8 Explain why the MCV, MCH, and MCHC can be falsely Compare the purpose of the DAT and the IAT, and state the increased when blood from someone with CAS is tested type of specimen used for each test. using an autoated cell counter. Answer Answer DAT is used to detect patient erythrocytes that have been coated with antibody or complement fragments in vivo. If the blood is cool, the IgM antibodies cause agglutina- The specimen used is patient erythrocytes collected in tion of the cells. When these clumps of cells are measured EDTA anticoagulant. The IAT is used to detect the presence for MCV, each clump is measured as a single cell and thus of antibodies in the patient’s serum. The specimen used is appears macrocytic. EDTA plasma or serum. The hematocrit (which is calculated) is incorrect because of the incorrect erythrocyte count. Hemoglobin is Checkpoint 19-5 correct because this parameter is determined by lysis of the What are the clinical findings and the immune stimuli for cells. The erythrocyte count is falsely decreased because the WAIHA? clumps are counted as one cell. Answer MCH, which is derived from the hemoglobin and eryth- rocyte count, then is falsely elevated as is the MCHC, which Clinical findings: symptoms related to anemia including is derived from the hemoglobin and hematocrit values. weakness, jaundice, and pallor. Idiopathic WAIHA has no identifiable cause. Secondary WAIHA is associated Checkpoint 19-9 with disease such as lymphoproliferative diseases includ- Compare the DAT findings and the antibody specificity in ing CLL, neoplasms, and autoimmune disorders such as WAIHA and CAS. Appendix F Answers to Checkpoints 1127 Answer Acute Delayed WAIHA CAS DAT: Positive but may be weak Positive (can take because of intravascular 12–24 hours) Polyspecific AHG: + + hemolysis; sometimes Anti-IgG: + - negative Anti-C3: - or + + Visible hemoglobin: Positive Negative Antibody specificity: directed to a complex Rh antigen molecule anti-I Checkpoint 19-14 Checkpoint 19-10 Compare the pathophysiology and clinical findings of ABO- Compare the antibody specificity and the confirmatory test HDFN and Rh-HDFN. for PCH and CAS. Answer Answer ABO-HDFN: This usually occurs when the mother is group PCH CAS O and the baby is group A or B. Antibody specificity: anti-P anti-I Pathophysiology: The naturally occurring IgG anti-A, anti-B, Confirmatory test: Donath-Landsteiner test Cold agglutinin test or anti-A,B is able to cross the placenta and coats the fetal Checkpoint 19-11 cells. It does not require previous sensitization. Compare the different types of drug-induced hemolysis Clinical findings: Generally mild anemia (or none) is present including the type of hemolysis, the drug usually associ- in newborn. The patient can present with jaundice 24–48 ated with the mechanism, and the DAT profile. hours after birth, but kernicterus resulting from high levels of bilirubin is rare. Answer Rh-HDFN: This occurs when the mother is Rh negative and Drug Immune the baby is Rh positive. Adsorption Complex Autoantibody Pathophysiology: Mother has been previously sensitized to Hemolysis Extravascular Intravascular Extravascular the Rh (D) antigen through previous pregnancy or transfu- Drug profile: Penicillin Quinidine Aldomet DAT sion. As the fetal erythrocytes containing the D antigen (Rh Poly Positive Positive Positive positive) enter maternal circulation, they serve as a second- Anti-IgG Positive negative Positive ary immune stimulus and increase the titer of anti-D. This Anti-C3 negative Positive negative is an IgG-class antibody that crosses the placenta and coats Checkpoint 19-12 the baby’s cells.

Compare the underlying mechanisms, pathophysiology, Clinical findings: Anemia can be mild to severe. The baby can and clinical symptoms of an acute hemolytic transfusion become jaundiced in the first 24 hours after birth. Hepato- reaction with a delayed one. splenomegaly can be present. As the level of unconjugated bilirubin rises because of increased hemolysis of erythro- Answer cytes, it can reach toxic levels and deposit bilirubin in the Acute Delayed brain causing kernicterus. Mechanism: IgM (usually ABO system) IgG (usually kidd system) Intravascular hemolysis Extravascular hemolysis Checkpoint 19-15 Pathophysiology: naturally occurring; Immune stimulated; Compare the laboratory findings including the peripheral ABO antibodies react Secondary exposure to blood smear and the DAT for newborns with ABO-HDFN immediately with f oreign foreign antigen causes antigen on transfused cells antibody titer to increase and those with Rh-HDFN. Clinical Chills, fever, hypotension, Malaise, fever, drop in symptoms: difficulty in breathing, hemoglobin, dark or red Answer increased pulse rate urine, usually one week post-transfusion ABO-HDFN Rh-HDFN Checkpoint 19-13 DAT Weakly positive with poly- Positive with polyspecific specific AHG and anti-IgG AHG and anti-IgG Compare the characteristic laboratory findings in acute Blood Spherocytes (severe Macrocytic, normochromic hemolytic transfusion reactions and in delayed transfusion smear cases) (rare spherocyte) Polychromatophilia Polychromatophilia reactions. Nucleated erythrocytes Nucleated erythrocytes (severe cases) Increased Increased reticulocytes Answer reticulocytes Increased leukocytes with Required tests are clerical check, DAT, and comparison for shift to the left visible hemoglobin between the pretransfusion and post- Hemoglobin Normal to slight decrease Moderate to severe decrease transfusion specimens. Bilirubin Slight increase Moderate to significant increase 1128 Appendix F Answers to Checkpoints CHAPTER 20 thrombocytopenia. The entire range of symptoms is rarely Checkpoint 20-1 seen. What abnormal erythrocytes characterize microangiopathic Checkpoint 20-6 hemolytic anemia? How are these cells formed? Explain how DIC can be differentiated from TTP and HUS Answer based on coagulation tests. The abnormal erythrocytes are schistocytes. As erythrocytes Answer are forced through the fibrin deposits in the blood vessels, In DIC, the PT, APTT, and thrombin time are prolonged. the membranes can be sliced open. When the membrane There is an increase in fibrin degradation products and reseals itself, abnormal forms such as the schistocytes result. fibrinogen is decreased. In HUS and TTP, the PT is usually Checkpoint 20-2 normal or only slightly prolonged; the APTT is normal. Any increase in fibrin degradation products is slight. What are the two types of HUS, and what organisms or diseases are most commonly associated with each type? Checkpoint 20-7 Answer Why do malaria and babesiosis result in anemia? Diarrhea positive HUS is due primarily to Shiga-like toxin Answer I and II from Escherichia coli O157:H7 but is occasionally Both organisms (Plasmodium sp. and Babesia sp.) are because of Shiga toxin of Shigella dysenteriae. intraerythrocytic parasites. After they have completed Diarrhea negative HUS is caused by Streptococcus part of the life cycle within an erythrocyte, the organism pneumoniae. causes the cell to rupture, resulting in intravascular hemo- Checkpoint 20-3 lysis. High levels of parasitemia and chronic infection result in anemia. Some cells containing the organisms can Explain how infection with E. coli O157:H7 results in intra- also be removed in the spleen, resulting in extravascular vascular hemolysis. hemolysis. Answer The Shiga-like toxins of E. coli O157:H7 damage the intesti- CHAPTER 21 nal mucosa, which allows toxin and inflammatory media- Checkpoint 21-1 tors to enter the circulation. Toxins attach to receptors on An adult patient’s total leukocyte count is 5.0 * 103>mcL. the endothelial cells of the vessels. Platelet aggregating There are 60% segmented neutrophils, 35% lymphocytes, substances are released and cause platelet activation and and 5% monocytes on the differential. Calculate the abso- formation of thrombi. Erythrocytes are damaged as they lute number of each cell type. Is each of these relative and pass through the thrombi in the microvasculature resulting absolute cell counts normal or abnormal? in intravascular hemolysis. Answer Checkpoint 20-4 Absolute counts: What are the typical erythrocyte morphology and coagula- Neutrophils 3.0 * 103>mcL tion test results in children with HUS? Lymphocytes 1.65 * 103>mcL Answer Monocytes 0.25 * 103>mcL Schistocytes, spherocytes, and burr cells can be present. All of the cell counts are normal. If the total leukocyte There can be polychromasia. The PT can be normal or count and the relative differential percentages are normal, slightly prolonged, the APTT is normal, and the fibrin split the absolute counts are also normal. products are slightly elevated. Checkpoint 21-2 Checkpoint 20-5 How can CML be distinguished from a leukemoid reaction? How does the clinical presentation of TTP differ from that Answer of HUS? How is it similar? In CML, the white cell count is usually higher and there are Answer more immature cells. Eosinophils and basophils are often The conditions share four traits: thrombocytopenia, cen- elevated in CML but not in a leukemoid reaction. Other cell tral nervous system abnormalities, renal dysfunction, and counts (RBC and platelet) are more likely to be abnormal in presence of schistocytes. The degree of renal dysfunction in CML. The LAP (low in CML and high or normal in leuke- HUS is more severe; in TTP, the neurologic symptoms are moid reaction) and Philadelphia chromosome (positive in more severe. The diagnosis of TTP requires only two crite- CML and negative in leukemoid reaction) can be helpful. ria: microangiopathic hemolytic anemia (schistocytes) and Patient demographic and symptoms follow: CML is usually Appendix F Answers to Checkpoints 1129 a disease of middle age adults and presents with systemic Answer symptoms such as enlarged spleen and nodes and bone Toxic granulation and vacuoles are associated with infec- pain because of bone marrow expansion; leukemoid reac- tion. Thus, you should correlate these findings with other tion from a reactive process usually presents with fever or signs of infection. A high WBC count and a shift to the left symptoms related only to the primary condition. can be present in an infectious state. The patient’s history is Checkpoint 21-3 also important. A comparison to other patients’ blood smears What is the difference between a leukemoid reaction and a stained with the same stain is helpful if it is suspected that the leukoerythroblastic reaction? large dark granules result from precipitated stain. If it results from staining artifact, the toxic granulation is present on Answer other patient blood smears as well. The time the blood was A leukemoid reaction occurs when a leukocytosis with a collected should be checked; blood stored in EDTA might shift to the left in circulating granulocytes occurs. It is a tran- show vacuoles in PMNs. sient condition that happens in response to a stimulus such Checkpoint 21-7 as infection or inflammation. A leukoerythroblastic reac- tion is characterized by a shift to the left in granulocytes Why are the basophil and eosinophil counts important when and nucleated RBCs in the peripheral blood. The total WBC assessing the benign or neoplastic nature of a disorder? count can be increased, normal, or decreased. A leukoeryth- Answer roblastic reaction is found in myeloproliferative conditions Basophils and eosinophils are rarely increased in benign and leukemia. disorders but are commonly increased in neoplastic dis- Checkpoint 21-4 orders. Evaluated with other hematologic data and the How can the correct white cell count be determined when patient’s clinical history, the eosinophil and basophil count neutrophils clump in the presence of EDTA? can provide important clues to the disorder assessment. Answer CHAPTER 22 If neutrophils clump in the presence of EDTA, the blood Checkpoint 22-1 for the white count and differential must be collected with- out EDTA. Manual or automated counts can be performed A patient with lymphocytosis showing reactive lymphocyte using finger stick or capillary collection. Blood collected in morphology with large, basophilic cells, fine chromatin, and a different anticoagulant also can be used for selected tests. a visible nucleolus has a negative infectious mononucleosis Heparin provides an accurate white cell count, but distorts serologic test. What is a possible cause for this altered lym- the erythrocytes. Citrate can be used if corrections are made phocyte morphology? for the dilutional effects of the increased volume of antico- Answer agulant in a blue top tube. Causes of reactive lymphocyte morphology are other viral Checkpoint 21-5 infections, particularly hepatitis or cytomegalovirus, and reac- Describe the difference between hypersegmented and hypo- tions to drugs or toxoplasmosis. A child younger than 10 years segmented neutrophils and pyknotic nuclei. In what condi- of age cannot produce heterophil antibodies in increased tions are each seen? quantities and thus may yield a negative serologic test when infected with EBV. Some adults do not produce a positive Answer serologic test either even though they are infected with EBV. Hypersegmented neutrophils have six or more nuclear seg- ments and are associated with megaloblastic anemia. Checkpoint 22-2 Hyposegmented neutrophils are mature cells with one or Why is lymphocytopenia a concern if there is no accompa- two nuclear segments and are associated with Pelger-Huët nying leukopenia? anomaly and some leukocyte malignancies. Be aware that Answer bands, metamyelocytes, and myelocytes have one or two A decrease in lymphocytes impairs the body’s immune segments as well but are immature cells. Pyknotic nuclei are response and makes the patient more susceptible to disease. smooth nuclear fragments seen when the cell is dying and disintegrating. Disintegrating cells can be seen in blood but Checkpoint 22-3 more often are found in infectious body fluids. Why does infection with HIV result in an increased chance Checkpoint 21-6 for opportunistic infections? Explain how you can determine whether toxic granulation Answer and vacuoles in the neutrophils are due to the patient’s con- HIV-1 gains access to cells through the CD4 protein receptor dition or to artifact. on the cell surface. Some T lymphocytes, monocytes, and 1130 Appendix F Answers to Checkpoints macrophages express this receptor. Once in the cell, HIV-1 mutations, requiring loss of both alleles. Explain this differ- destroys the T lymphocyte, compromising the cell-mediated ence in behavior of the gene products. immune system and allowing opportunistic infections to Answer cause disease. The products of proto-oncogenes are usually proteins that Checkpoint 22-4 are important in regulating cell growth, differentiation, or Would you expect female carriers of X-linked SCIDS to be apoptosis in their wild-type state. Mutations in a single more susceptible to infection than the normal population? allele of a proto-oncogene can activate it to an oncogene and Why or why not? result in enhanced cell growth or in defective differentiation or apoptosis whenever the aberrant protein is produced. In Answer contrast, tumor suppressor genes produce protein products No, all of their lymphocytes have the normal X chromosome that inhibit cell growth or promote apoptosis. Because the activated. human genome carries two copies of all genes carried on Checkpoint 22-5 the autosomes, a mutation of one tumor suppressor allele resulting in a loss of function leaves the other allele produc- What laboratory findings suggest WAS in a child, and how ing a wild-type protein product. Because both alleles must be is the diagnosis confirmed? mutated before the cell experiences complete loss of that pro- Answer tein effect, these mutations function in a recessive manner. Thrombocytopenia, lack of blood group reverse typing, Checkpoint 23-4 decreased IgM levels but increased IgE and IgA levels sug- A cell contains a mutation that blocks expression of p16. gest WAS. Diagnosis for WAS is substantiated by flow cytom- What is the effect (if any) on the daughter cells produced? etry and molecular analysis. A positive PCR test revealing the gene mutation confirms the presence of the disease. Answer The G1 checkpoint in cell-cycle regulation checks for DNA CHAPTER 23 damage and prevents progression into S phase if DNA is damaged. Without a functional p16 protein, passage from Checkpoint 23-1 G1 to S phase progresses without regard to the state of the A patient has 50% monoblasts in the bone marrow. Which genomic DNA and any mutations in the DNA are passed precursor cell could be the cancer-initiating cell? on to all daughter cells. Answer Checkpoint 23-5 The cancer-initiating cell (leukemic stem cell) is one that Does a 3-year-old child with Down syndrome have an already had the capacity to self-renew or has recently increased risk of developing leukemia? Why or why not? acquired that capacity. Therefore, a patient could have 50% Answer monoblasts in the bone marrow because of abnormal pro- Yes, some individuals with congenital abnormalities asso- liferation of the HSC, the

MPP, or the CMP (each of which ciated with karyotypic abnormalities have a markedly could be the cancer-initiating cell). The CLP differentiates increased risk of developing acute leukemias. Because indi- into lymphocytes and could not be the cancer-initiating cell. viduals with Down syndrome have an extra chromosome Checkpoint 23-2 21, there is an increased probability for chromosomal trans- A 62-year-old male presents with an elevated leukocyte locations to occur, and every gene found on chromosome 21 count, mild anemia, and a slightly decreased platelet count. will be expressed at 150% of normal (three copies instead of His physician suspects leukemia. Explain why the erythro- two copies of each allele). cytes and platelets are affected. Checkpoint 23-6 Answer Why is finding Auer rods an important factor in the diag- Leukemia is a stem cell disorder that involves all cell progeny. nosis of leukemia? If the patient has the suspected leukemia, he would experi- Answer ence an unregulated production of neoplastic cells. As the Auer rods can be found in the blast cells and promyelocytes of neoplastic cell population increases, the concentration of nor- some acute myeloid leukemias. Finding them can help estab- mal cells, such as the platelets and erythrocytes, decreases. lish the diagnosis because these unique, pink-staining inclu- Checkpoint 23-3 sions are not found in ALL. Auer rods are fused lysosomes. Mutations in proto-oncogenes predisposing to malignancy Checkpoint 23-7 are said to be dominant mutations, whereas mutations in A patient has 35% blasts in the bone marrow. They do not tumor suppressor genes are said to behave as recessive show any specific morphologic characteristics that will Appendix F Answers to Checkpoints 1131 allow them to be classified according to cell lineage. What Answer are the next steps that the clinical laboratory scientist should The BCR/ABL1 fusion gene occurs in CML. take with this specimen? Checkpoint 24-6 Answer Is a patient who has a platelet count of 846 * 103>mcL, Cytochemistry can help establish cell lineage as myeloid splenomegaly, and abnormal platelet function tests of or lymphoid. An immunologic analysis provides informa- hyperaggregation likely to have reactive or essential throm- tion on what specific membrane antigens are on the blast bocytosis? Explain. cells. By using a panel of monoclonal antibodies, the cells’ lineage can usually be determined because specific surface Answer markers are characteristically found on each particular cell This patient is likely to have essential thrombocyto- line. Genetic analysis can also be performed because some sis. Patients with reactive thrombocytosis do not usu- cytogenetic chromosome karyotypes are commonly found ally have platelet counts this high and rarely higher than in certain types of leukemia. 1000 * 103>mcL, nor would they have splenomegaly or thrombotic complications. CHAPTER 24 Checkpoint 24-7 Checkpoint 24-1 Renal tumors can produce an inappropriate amount of EPO, In essential thrombocythemia, all hematopoietic lines have resulting in what type of polycythemia? increased cell proliferation. Which lineage has the greatest Answer increase? Secondary polycythemia or increase of red cell mass from Answer an identifiable cause. The megakaryocytic cell line is most increased in essential Checkpoint 24-8 thrombocythemia. Which of these conditions—iron deficiency, smoking, Checkpoint 24-2 emphysema, pregnancy, dehydration—are associated with A patient has the CML phenotype, but the genetic karyo- an absolute increase in RCM? type does not show the Ph chromosome. If this is truly a Answer CML, what should molecular analysis show? Smoking, emphysema, and pregnancy are conditions in Answer which additional red cells are needed to prevent a physi- Molecular analysis would show a BCR/ABL1 gene rear- ologic anemia. Iron deficiency prevents the formation of rangement in a new fused hybrid gene. additional red cells. Dehydration causes a relative red cell Checkpoint 24-3 mass increase because of decreased plasma volume. Describe the peripheral blood differential of a CML patient. Checkpoint 24-9 Answer What growth factors are primarily responsible for stimulat- ing fibrogenesis in the bone marrow? There is a shift to the left in granulocytes with high numbers of myelocytes and promyelocytes. If blasts are present, they Answer compose less than 20% of cells. Platelet-derived growth factor, transforming growth factor- Checkpoint 24-4 b, and epidermal growth factor are increased and associated with marrow fibrosis. What clinical, peripheral blood, and genetic features dif- ferentiate CML from an infectious process? Checkpoint 24-10 Answer What erythrocyte morphologic feature is a hallmark for myelofibrosis? An extreme peripheral blood leukocytosis—segmented neutrophils with a left shift showing many myelocytes, Answer some promyelocytes, and blasts—are found in CML. Age The finding of dacryocytes (teardrop erythrocytes) is a hall- of the patient and absence of infection, low LAP score, and mark for myelofibrosis with myeloid metaplasia. presence of the Philadelphia chromosome or BCR/ABL1 translocation all differentiate the hallmarks of CML. CHAPTER 25 Checkpoint 24-5 Checkpoint 25-1 What is the most important feature that separates all other How does the pathogenesis of primary MDS differ from forms of MPNs from CML? secondary MDS? 1132 Appendix F Answers to Checkpoints Answer Checkpoint 25-5 Patients with secondary MDS have been exposed to a known Why is it important to correctly identify the number of genotoxic event. These include environmental chemicals, blasts when evaluating the peripheral blood or bone mar- such as benzene and radiation therapy or to various che- row smear of a patient suspected of having MDS? motherapeutic agents (alkylating agents or topoisomerase Answer II inhibitors). Patients that present with primary MDS have The number of blasts is used to classify the MDS into differ- no known history of exposure to genotoxic agents. ent subtypes and to predict prognosis and treatment. It is Checkpoint 25-2 also used to differentiate MDS from acute leukemia. How do epigenetics contribute to the pathogenesis of MDS? Checkpoint 25-6 Answer When a diagnosis of MDS is considered for a patient, why Epigenetic changes turn on the expression of normally must other causes of reactive cytopenias be ruled out? What silenced genes (oncogenes) or turn off the expression of type of testing helps determine the diagnosis of MDS? genes involved in normal cellular processes (tumor sup- Answer pressor genes). Epigenetic alterations do not change the It is important to identify the cause of cytopenias and/or dys- sequence of the DNA but act through DNA methylation plasia so that the patient can be appropriately treated. Cyto- (silencing) or through histone acetylation (activating). penias caused by drugs, alcohol, dietary deficiencies (folate, Histone methylation can be either inhibitory or activating, cobalamin, iron), and so on should be identified and treated depending on the gene. accordingly. Moreover, the patient’s prognosis will depend Checkpoint 25-3 on the appropriate diagnosis. Cytogenetic and molecular How does the typical peripheral blood picture in MDS dif- testing can provide the diagnostic criteria for MDS. fer from that in aplastic anemia (Chapter 16)? Checkpoint 25-7 Answer How do the laboratory findings of aCML, BCR/ABL1 differ The typical blood picture in aplastic anemia is pancyto- from those observed in CML (Chapter 24)? penia, a significant decrease in all three cell populations Answer (Chapter 16). In MDS, cytopenia is present in the periph- The most notable difference is that a diagnosis of CML eral blood in one cell line or more. Pancytopenia has been requires detection of the BCR>ABL1- fusion gene. Addition- estimated to occur in only 15% of cases of MDS. Both MDS ally, the WBC count is increased in both conditions, but CML and aplastic anemia can be macrocytic. MDS exhibits more tends to present with a higher WBC count (25975 * 103>mcL frequent qualitative abnormalities indicating dyspoiesis, in early diagnosed CML vs. greater than 13 * 103>mcL in such as anisocytosis, poikilocytosis, basophilic stippling, aCML, BCR/ABL1–). Patients with CML are more likely to Howell-Jolly bodies, and nucleated RBCs. The neutrophils present with basophilia and eosinophilia whereas these are often show abnormal granulation and pseudo-Pelger-Huët mostly absent in patients with aCML, BCR/ABL1– cells. Giant and hypogranular platelets may be seen. The bone marrow is the best way to differentiate the two syn- CHAPTER 26 dromes. A hypoplastic bone marrow is typical in aplastic Checkpoint 26-1 anemia. MDS usually shows a hypercellular marrow, but if it is hypocellular, it can be difficult to differentiate from What results would you expect to find on the CBC and dif- aplastic anemia. ferential in a suspected case of AL? Checkpoint 25-4 Answer Why is serum cobalamin, serum folate level, or bone mar- You would expect a normocytic, normochromic anemia; row iron stain important in diagnosing MDS? decrease in platelets with some large forms present; neu- tropenia; monocytosis; and on the differential blast cells, an Answer increase in eosinophils and basophils. Some features of the blood picture in MDS resemble the Checkpoint 26-2 megaloblastic anemias (macrocytosis, megaloblastoid Explain why molecular analysis is not performed on all sus- changes in erythrocytic precursor cells). A normal serum pected cases of acute leukemia. cobalamin and/ or folate level rule out megaloblastic ane- mia. An iron stain on the bone marrow allows assessment Answer of the presence of ring sideroblasts, which may be seen in The molecular aberrations are not known for all cases of AL; MDS-SLD and MDS-MLD and are necessary for a diagnosis thus, probes are not available. Those in which an aberration of MDS-RS. is commonly present and can be probed for are AML with Appendix F Answers to Checkpoints 1133 recurrent genetic abnormalities [AML with t(8;21), inv(16), Answer t(15;17), t(9;11), t(6;9), inv(3), t(1;22)] and mutated nPM1 or This is an AML without maturation. There are greater than CEBPA. 90 myeloblasts, and greater than 3% are positive with MPO. Checkpoint 26-3 AML with differentiation has less than 90% myeloblasts and AML minimally differentiated is negative with MPO. What does recurrent genetic abnormality mean in the con- text of AML, and what is the general outcome of these Checkpoint 26-8 abnormalities? Describe the peripheral blood picture for a patient with Answer pure erythroid leukemia? Recurrent genetic abnormality suggests that the AML com- Answer monly presents with a specific alteration to the DNA in the The peripheral blood will show anemia with poikilocytosis neoplastic cells. This is frequently a reciprocal translocation and anisocytosis. A large number of dysplastic nucleated and results in the expression of oncogenic fusion genes that erythrocytes with megaloblastoid nuclei and/or bi- or mul- have gained a new function. tinucleation may be present. The cytoplasm of these cells Checkpoint 26-4 will frequently contain vacuoles. Myeloblasts can be seen in the peripheral blood as well. Why is it important to perform molecular studies on patients with AML with t(15;17)? Checkpoint 26-9 Answer Predict the peripheral blood picture of a patient on antifo- late chemotherapy. The abnormality t(15;17), PML>RARa is diagnostic of APL. Those with this translocation respond to therapy with all Answer transretinoic acid. If the translocation disappears after ther- There would be a megaloblastic blood picture including apy and then reappears, relapse can be predicted. Also, the pancytopenia with a macrocytic anemia. fusion gene product is not always detectable by cytogenetics. Checkpoint 26-5 CHAPTER 27 Would the FLT3, NPM1, and CEBPA genes be classified as Checkpoint 27-1 tumor suppressor or proto-oncogenes? Compare the typical age groups in which AML and ALL Answer are found. Both NPM1 and CEBPA proteins demonstrate a loss of func- Answer tion when they are mutated in AML. Therefore, these genes The AMLs are characteristically found in adults in contrast are considered tumor suppressor genes in the context of to the ALLs, which are typically found in children. The AML. On the other hand, FLT3 gains a new function when AML minimally differentiated is most common in adults it is mutated and is therefore considered an oncogene when and infants. mutated in AML. Checkpoint 27-2 Checkpoint 26-6 Explain the difference between ALL and LBL. What hematologic features help distinguish AML mini- mally differentiated from AML without maturation? Answer Answer ALL is a lymphoid neoplasm that primarily involves the peripheral blood and bone marrow. LBL presents as a solid Cytochemical stains in AML minimally differentiated are tumorous mass in nodal or extranodal tissue. It is thought negative by morphology and light microscopy cytochemical that ALL and LBL are different clinical presentations of the stains. In AML without maturation, less than 3% of blasts same disease. have a positive reaction with myeloperoxidase and/or Sudan black B and/or have Auer rods. Myeloid antigens, CD13 and Checkpoint 27-3 CD33, can be detected by immunophenotyping in both. Why is it necessary to immunophenotype the lympho- Checkpoint

26-7 blasts in ALL if they have been identified as lymphoblasts morphologically? A patient with AML has a peripheral blood differential that includes 91% myeloblasts, 4% promyelocytes, 3% granulo- Answer cytes, and 2% monocytes, and 30% of the blasts are posi- The lymphoblasts should be immunophenotyped to deter- tive with MPO. Which category of AML is the most likely mine whether they are of T or B cell origin. This information diagnosis? Explain. has treatment and prognostic implications. 1134 Appendix F Answers to Checkpoints Checkpoint 27-4 Answer A patient has 50% blasts in his bone marrow. Immuno- Staging determines the extent and distribution of dis- phenotyping is CD19 positive, but CD20, CD2, CD10, ease; grading separates classifications of lymphoma and and CD7 negative. What additional testing can be help- often uses a combination of morphology, phenotype, and ful to distinguish the immunologic subgroup of this genotype. leukemia? Checkpoint 28-3 Answer Chronic lymphoid malignancies compose a heterogeneous Cytochemistry results indicate that these appear to be group. What characteristics allow these malignancies to be lympho-blasts. The cells have the B cell antigen CD19 but grouped together? are negative for B cell markers CD10 and CD20. Cells are Answer negative for the T cell markers CD2 and CD7. This leukemia can be a very early progenitor B ALL. Testing for the CD34 The chronic lymphoid leukemic malignancies are grouped marker and the presence of TdT would be helpful. These together because the malignant cell is a mature lympho- are markers of very early cells. Molecular testing for the cyte found in the blood and bone marrow (leukemic rearrangement of the immunoglobulin genes can also help distribution). determine whether these are B cells. Checkpoint 28-4 Checkpoint 27-5 Name and describe the cell that is characteristic of Hodgkin A 3-year-old patient has 45% lymphoblasts in the bone lymphoma. marrow. If the cells tested positive for CD19, CD10, and Answer CD34, what is the most likely immunologic subgroup? Why The Reed-Sternberg cell characteristically distinguishes should cytogenetics be performed on this patient? Hodgkin lymphoma from non-Hodgkin lymphoma. It Answer has two or more nuclear lobes containing inclusion-like These markers suggest the early pre-B immunologic sub- nucleoli and an area of perinucleolar clearing (owl’s eye group. Cytogenetics is important in ALL to provide treat- appearance). ment and prognostic information. Checkpoint 28-5 Checkpoint 27-6 What clinical finding differentiates multiple myeloma from A patient with acute leukemia has two morphologi- other plasma cell neoplasms? cally different types of blasts. One population is positive Answer for CD7 and CD2. The other is positive for CD33 and CD13. What is the most appropriate classification of this The presence of multiple lytic bone lesions differentiates leukemia? multiple myeloma from other plasma cell neoplasms. Answer This is most likely an acute leukemia with lineage hetero- CHAPTER 29 geneity. Because the lineage-associated markers are found Checkpoint 29-1 on two different sets of blasts, it is most likely a bilineage A physician is evaluating a 28-year-old patient with a his- acute leukemia. The blasts appear by immunophenotype to tory of acute lymphoblastic leukemia for PBSC transplanta- be myeloblasts and T lymphoblasts. tion. The laboratory professional found circulating leukemic blasts in the peripheral blood. Is this patient a candidate for CHAPTER 28 an autologous PBSC transplant? Checkpoint 28-1 Answer How does the BCL-2 gene rearrangement differ from most Presence of circulating blasts in a patient who is being eval- other oncogenes? uated for the autologous PBSC is not a good sign. Because Answer PBSCs are collected from the person’s peripheral blood by The BCL-2/ gene rearrangement leads to persistence of cells apheresis technology, the stem cell product in this patient because of an inhibition of apoptosis rather than uncon- would definitely contain leukemic blasts. It would be trolled cell proliferation. almost impossible to separate these leukemic blasts from the normal stem cells even by purging. Therefore, autolo- Checkpoint 28-2 gous PBSC transplant is not an option for this patient, at How does staging differ from grading in characterizing the least at this point. The search for the matched allogeneic lymphoid neoplasms? stem cell donor should be made. Appendix F Answers to Checkpoints 1135 Checkpoint 29-2 CMV-seronegative patient, the preference is given to the What would be the best form of transplant for a patient donor who is seronegative for CMV. The rationale is that with CML who needs an SCT, and what antigen type needs CMV infection can be fatal in the peritransplant setting. to be matched? However, if the CMV-seronegative donor is not available but a matched CMV-seropositive donor is available, trans- Answer plant can still be performed. Most transplant physicians CML is a stem cell disorder, which means the patient’s own believe that a CMV-seronegative recipient should receive stem cells are affected by the disease. Therefore, the best CMV-seronegative cellular blood components if the stem form of transplant for this patient is an allogeneic stem cells’ donor is also CMV-seronegative. If the CMV-negative cell transplant for which the patient and donor should be blood component is not available, transfusing a leukocyte- matched for HLA-A, B, and DR antigens. ABO antigens are reduced filtered component can effectively prevent the tested for both donor and patient; however, the matching is transmission of CMV disease. not required for the allogeneic stem cell transplant. Checkpoint 29-3 CHAPTER 30 You receive a peripheral blood specimen in the hematol- Checkpoint 30-1 ogy laboratory with a request for a mononuclear cell count To obtain a sample of CSF for analysis, the needle must be and analysis of CD34+ cells. Without any other information, inserted into what area of the central nervous system? why should you consider this a STAT request? Answer Answer Subarachnoid space. The volume of cells necessary to achieve a successful engraftment can be determined indirectly by the MNC Checkpoint 30-2 count or the CD34+ count. A clinical decision can be made A 32-year-old woman has right-side chest pain and short- to collect more cells, depending on these counts. The timing ness of breath that has worsened over a two-week period. of the collection of the stem cells also can be determined Chest radiologic studies reveal a right pleural effusion, and based on these counts. a thoracentesis is performed. The pleural fluid specimen on a cytocentrifuged, Wright-stained slide reveals cells like that Checkpoint 29-4 seen in Figure 30-10. What is the best interpretation of this A physician wants to evaluate the engraftment on a male finding? If there is a strong concern that this can represent a patient who received SCT from his brother 4 months ago. low-grade lymphoma, what would be the best way to deter- What laboratory tests should be performed to make this mine whether these are benign or malignant lymphocytes? assessment? Answer Answer These cells are benign lymphocytes with the artifact of a To evaluate the long-term engraftment of HSCs, various lab- pale staining area resembling nucleoli. These are not blasts oratory methods have evolved. Currently, the most widely because of the mature chromatin. Immunophenotyping by used tests are in situ hybridization (ISH) with sex chromo- flow cytometry of the pleural fluid would be the best way some and typing of variable number tandem repeat (VNTR) to determine whether this is a benign or malignant popula- polymorphism by DNA amplification. The ISH is applicable tion of lymphocytes. when the donor and recipient are of different sex. In this case, the VNTR polymorphism by DNA amplification to Checkpoint 30-3 distinguish donor and recipient cells must be performed. What are the best features to use in determining whether Checkpoint 29-5 tissue cells are benign or malignant when examining a cyto- centrifuged, Wright-stained slide of a body fluid specimen? A CMV-seronegative patient requires SCT. Two HLA- matched donors are available. Is it important to know the Answer stem cell donor’s CMV status? If the stem cell donor is CMV Nuclear features are the best to use to distinguish benign seronegative and the patient requires red cell transfusion and malignant cells. These include nuclear membrane during the peritransplant period, what blood components (smooth or irregular), chromatin pattern (regular or irregu- (in terms of CMV status) would you select for this patient? lar distribution), and nucleoli (present or absent, nucleolar Answer membrane smooth or irregular). The CMV-seropositive person (i.e., a subject who shows Checkpoint 30-4 serologic evidence of prior CMV exposure) can be an A wife finds her 47-year-old husband comatose at home. SCT donor to a CMV-seronegative recipient. How- During examination in the emergency room, a spinal tap ever, if two HLA-compatible donors are available for a is performed and grossly bloody spinal fluid is obtained. 1136 Appendix F Answers to Checkpoints The total RBC count in the first tube is the same as that Checkpoint 31-5 in the third tube. A cytocentrifuged, Wright-stained slide Your finger is still bleeding at this point, but the platelets shows findings like that seen in Figure 30-44. What is the are aggregating to form the primary hemostatic plug. Let’s most appropriate interpretation of these findings? review the key events: Answer a. To what do platelets first adhere? There has been a true subarachnoid hemorrhage. b. What bridge and what platelet membrane receptor are needed for platelet adhesion? Checkpoint 30-5 c. What bridge and what platelet membrane receptor are A 57-year-old man has an acutely swollen, painful, red- needed for platelets to attach to one another? dened joint in his left great toe. Joint fluid is aspirated, and d. What is the attachment of platelets to one another a photo-micrograph is taken (Figure 30-61). This picture called? is taken with polarized light using a quartz compensator. What is the most appropriate interpretation of this finding? Answer Answer a. Collagen Both monosodium urate and calcium pyrophosphate crys- b. Von Willebrand factor and glycoprotein Ib/IX tals are present. c. Fibrinogen and glycoprotein IIb/IIIA d. Platelet aggregation CHAPTER 31 Checkpoint 31-6 Checkpoint 31-1 Your finger has now stopped bleeding. Outline the steps of Think about the last time that you injured your finger with primary hemostasis that have occurred. a paper cut. Did your finger bleed immediately? If not, what might have prevented immediate bleeding? Answer The blood vessels have undergone vasoconstriction and Answer are by now possibly beginning to dilate. Platelet adhesion, No, vasoconstriction of the blood vessels prevented it. platelet secretion, platelet aggregation, and formation of the Checkpoint 31-2 primary hemostatic plug have occurred. What actions of the endothelial cells prevent clotting from occurring within the blood vessels? CHAPTER 32 Answer Checkpoint 32-1 The endothelial cells’ negatively charged surface repels clot- What is the major distinction between the so-called extrinsic ting factors and platelets in the normal peripheral blood and intrinsic pathways? circulation. They synthesize heparan sulfate, TFPI, and thrombomodulin, which inhibit fibrin formation. They syn- Answer thesize PGI2, NO, and ADPase, which inhibit platelet activa- Both pathways require enzymes and protein cofactors origi- tion. They synthesize tPA and uPA, which control fibrinolysis. nally present in plasma; however, the extrinsic pathway also requires an activator (tissue factor) that is not found in the Checkpoint 31-3 blood under normal circumstances. If a patient inherited a mutation of the gene for glycoprotein IIIa that resulted in its absence, what two platelet antigens Checkpoint 32-2 would be decreased or absent? Will a vitamin K-deficient patient produce any of the vita- Answer min K-dependent factors? Why is vitamin K so vital to the formation of coagulation complexes? The HPA-1 and HPA-4 would be decreased or absent. Checkpoint 31-4 Answer If a patient with Bernard-Soulier syndrome or VWD cut a A patient who is vitamin-k deficient still synthesizes the finger, would you expect bleeding to stop as fast as it does proteins but fails to attach the extra carboxyl group to the when you cut your own finger? Why or why not? g-carbon of glutamic acid residues in the GLA domains of the protein, which is required for full functional activ- Answer ity of the proteins. The g@carboxy form of the proteins is No, because their platelets would be unable to adhere to required for CA++ interaction with phospholipid surfaces, collagen so the primary hemostatic plug would take longer which is required for forming the coagulation activation to form to halt the bleeding. complexes. Appendix F Answers to Checkpoints 1137 Checkpoint 32-3 result is thrombosis; if fibrinolysis is excessive, the result is Why are the domains of the

serine proteases involved in hemorrhage. blood clotting so important in the hemostatic mechanism? Checkpoint 32-8 Answer How are the PLN degradation products of fibrinogen and The catalytic domain of the protease cleaves the substrate(s) fibrin different? of this protease. The various noncatalytic domains of the Answer serine proteases contain the regulatory elements of the pro- Plasmin cleaves at the same place on molecules of either teins and are responsible for conferring the specificity of fibrin or fibrinogen (at the coiled regions, midway between activation and activity of each enzyme. They bind calcium the terminal D domains and the C-central domain). In fibrin- and promote interaction with phospholipids, cofactors, ogen, this produces separate D and E fragments. However, receptors, and substrates. because fibrin monomers have been covalently cross-linked Checkpoint 32-4 by FXIIIa, complexes of various combinations (particularly Which components of the intrinsic pathway are believed to the so-called D-dimer) are formed. The presence of D-dimer be essential for in vivo hemostasis? confirms that both the procoagulant system (thrombin) and the fibrinolytic system (plasmin) have been activated. Answer Checkpoint 32-9 Factors IX, VIII, and possibly XI are the components. Factor XII, prekallikrein, and HMW kininogen are not essential for Why are naturally occurring inhibitors important in the normal in vivo hemostasis. hemostatic mechanism? Checkpoint 32-5 Answer Historically, major importance for initiating coagulation Naturally occurring inhibitors help control the activity of was assigned to either the intrinsic or extrinsic pathway. the coagulation and fibrinolytic proteases. They are inactive What are some observations that suggest that the classic when distant from a site of vessel damage, helping to limit concepts were not accurate? clot formation to areas of vessel injury. They are essential in preventing unwarranted initiation or excessive amplifica- Answer tion of the coagulation cascade. Thrombin can activate factor XI, bypassing the need for contact activation by factor XII/kallikrein/HK. Factor IX CHAPTER 33 can be activated by factor VIIa as well as factor XIa (again, Checkpoint 33-1 bypassing the need for contact activation). Thus, initiation of coagulation by tissue factor/factor VII is sufficient to initiate Assume that you are the clinical laboratory professional col- activation of both pathways. However, full escalation of the lecting a blood specimen from a patient with a suspected coagulation system requires proteins from both pathways. bleeding disorder. You noticed petechiae and several bruises on the patient’s arm. What screening tests would likely have Checkpoint 32-6 been ordered? What results of these tests would you expect What are the three steps in the formation of an insoluble (normal or abnormal) in this patient? fibrin clot? Answer Answer A platelet count, prothrombin time, and activated partial They are (1) proteolytic cleavage of fibrinopeptides A and B thromboplastin time would likely be ordered as screening by thrombin, forming a fibrin monomer, (2) spontaneous tests. The presence of petechiae indicates a platelet abnormal- polymerization of fibrin monomers to form fibrin polymers, ity, the most common of which is thrombocytopenia. There- and (3) stabilization of the fibrin polymers by FXIIIa. fore, you would expect the platelet count to be decreased. Checkpoint 32-7 The prothrombin and activated partial thromboplastin times are normal in platelet abnormalities that are not complicated Why is the process of fibrinolysis a vital part of the hemo- or accompanied by abnormalities of fibrin formation. static mechanism, and why must it be closely regulated and controlled? Checkpoint 33-2 Answer List and describe the platelet changes you should look for when examining a stained blood smear microscopically. Fibrinolysis is needed to restore the blood vessel struc- ture and function to normal when a fibrin clot is no longer Answer needed. It is essential to balance the activity of the proco- You should evaluate platelet quantity by performing a plate- agulant proteins. If activity of fibrinolysis is deficient, the let estimate. You should also look for changes in platelet 1138 Appendix F Answers to Checkpoints size (large or giant platelets), granulation (hypogranular or Answer agranular), and distribution (clumping). The defect is in platelet adhesion because ristocetin evalu- Checkpoint 33-3 ates adhesion. Two clinical conditions that would produce A 6-year-old boy was brought to his pediatrician because these results are BSS and VWD. To differentiate the two, per- the mother noticed small pinkish spots on his legs. Exami- form platelet aggregation testing with Ristocetin + vWF. In nation discovered several bruises on his arms and legs. BSS the results will remain abnormal. In VWD the results Laboratory tests were ordered. His platelet count was will correct to normal because of vWF present. 20 * 103>mcL, and the PT and APTT were within normal Checkpoint 33-7 limits. The complete blood count (CBC) was normal except Why are the bleeding time test and closure time abnormal for the low platelet count. The boy had no previous history for up to 7 days following ingestion of aspirin? of bleeding. The mother said she had noted the spots after he was given the hepatitis vaccine. What is the most prob- Answer able type of thrombocytopenia? Should other coagulation Aspirin inhibits the platelet enzyme cyclooxygenase, which tests be performed at this time? is necessary for production of thromboxane A2. TXA2 is nec- essary in the activated platelet for secretion of granule con- Answer tents and, therefore, the function of the platelets is impaired. The most probable type of thrombocytopenia experienced The defective platelets continue to circulate for their normal by this patient is an immune type of increased destruction, life span, which is about 10 days. Because they are circu- which is reportedly associated with viral infections in some lating, the bone marrow is not stimulated to produce new children. Other coagulation tests are not necessary. platelets to replace the defective ones. Checkpoint 33-4 CHAPTER 34 What is the pathophysiology of thrombocytopenia in mega- loblastic anemia? Checkpoint 34-1 a. Why do patients with type 1 VWD have 25–50% of Answer VWF in their plasma? Thrombocytopenia occurs in megaloblastic anemia because b. Why do they have a corresponding decrease in FVIII of ineffective production of all myeloid lineages in the bone in their plasma? marrow. Answer Checkpoint 33-5 a. Type 1 VWD is inherited as an autosomal dominant Explain why primary thrombocytosis is often associated characteristic. Patients have symptoms with abnormali- with abnormal platelet function, whereas secondary throm- ties of only one gene. The second gene is still functional. bocytosis is not. Theoretically, 50% of VWF would be present but other Answer variations such as the blood type O can result in less than 50% in some patients. Primary thrombocytosis is associated with the myelo- b. Patients with type 1 VWD have a corresponding proliferative disorders, which are clonal disorders of the decrease in FVIII in their plasma because FVIII requires pluripotential stem cell. The abnormal clone grows autono- the presence of VWF to be present in the plasma. mously and not in response to normal regulatory factors. It is likely that abnormalities in platelet function are acquired Checkpoint 34-2 along with the ability to grow autonomously. In secondary A patient with VWD has an equal decrease in FVIII activity, thrombocytosis, the platelets are increased because of the VWF:RCo, and VWF:Ag assay. normal regulatory routes in response to a need for more a. Is this a likely indication that there is a true decrease in platelets. the amount of VWF, or is it more likely to indicate that the patient has a type of VWD that is characterized by a Checkpoint 33-6 functional abnormality of VWF? Support your answer. A patient is having laboratory tests to evaluate for a bleed- b. What type of VWD is most likely in a patient with these ing phenotype suggestive of a defect of primary hemostasis. laboratory results? Platelet aggregation test results indicate normal aggregation with thrombin, epinephrine, and ADP but defective agglu- Answer tination with ristocetin. What aspect of platelet function is a. A true quantitative decrease in the amount of VWF defective? name two clinical conditions that would produce occurs because the tests for function and immunologic these test results and describe laboratory testing that could activity are both decreased. be performed to differentiate the two. b. These results are characteristic of VWD, type 1. Appendix F Answers to Checkpoints 1139 Checkpoint 34-3 Answer What abbreviation is acceptable for: All laboratory screening tests depend on fibrin formation a. the antigenic properties of VWF? in the test container and are not influenced by whether or b. the functional activity of FVIII? not the fibrin was covalently cross-linked. FXIII functions c. the complex of FVIII and VWF? after fibrin formation to stabilize the fibrin in vivo. It is not necessary to produce the end-point (fibrin formation) in the Answer in vitro assays. a. VWF:Ag b. F-VIII:C CHAPTER 35 c. F-VIII/VWF complex Checkpoint 35-1 Checkpoint 34-4 A patient has low levels of tPA and elevated levels of PAI-1. Why would this result in hypercoagulability in this patient? Referring to Table 34-3, explain why the platelet func- tion tests are abnormal in VWD but not in FVIII or FIX Answer deficiencies. This combination of low levels of tPA and increased levels Answer of PAI-1 would result in a decrease in fibrinolytic activity and could lead to formation of a thrombus because the deli- Platelet function tests are abnormal in VWD because VWF cate balance between clot-promoting factors (procoagulant is necessary for platelets to adhere to collagen. This affects influences) and clot-inhibiting factors (anticoagulants and the closure time, the bleeding time, and platelet aggregation fibrinolysis) has been disturbed, and the clot-promoting fac- studies with ristocetin. Ristocetin takes the place of collagen tors dominate the clinical picture. in the platelet aggregation test system. In the absence of VWF, platelets do not adhere and do not aggregate. VWF Checkpoint 35-2 is not needed for platelets to aggregate with other agonists. Why are both clotting and immunologic assays for PS, PC, FVIII and FIX deficiencies have a decrease in these coagula- and AT necessary when evaluating an individual for inher- tion proteins that affects fibrin formation, but platelets are ited thrombophilia associated with these proteins? normal, and VWF is present. Answer Checkpoint 34-5 Inherited defects of the hemostatic proteins involved in Explain why the thrombin time is abnormal in patients with familial thrombophilia can be either quantitative or quali- afibrinogenemia and dysfibrinogenemia. tative. Immunologic assays for total protein fail to diag- Answer nose qualitative defects (dysfunctional proteins), which typically give normal antigen levels but reduced functional The thrombin time depends on the adequate conversion of levels. Functional assays are generally considered the bet- soluble fibrinogen to fibrin. Patients with afibrinogenemia ter screening assay if a familial thrombophilia is suspected. have no fibrinogen to convert to fibrin, and patients with dysfibrinogenemia have fibrinogen with an abnormal abil- Checkpoint 35-3 ity to convert to fibrin. A patient was being evaluated for inherited thrombophilia. Checkpoint 34-6 The results for the clotting assay for APC resistance were positive, but the FVL molecular assay was negative. Are Explain why the prothrombin time but not the APTT is pro- these results inconsistent? longed in FVII deficiency. Answer Answer No, they are not inconsistent because ∼ 10, of patients Tissue thromboplastin activates FVII, which is the first step with APCR have a mutation other than the FVL mutation. in fibrin formation via the extrinsic pathway. The reagent for the prothrombin time is tissue thromboplastin; therefore, Checkpoint 35-4 when the FVII is deficient, fibrin formation is delayed. In Why is thrombotic disease associated with hereditary the APTT, tissue thromboplastin is not present in the test thrombophilia considered a multigene (or multi-risk fac- system. Instead, fibrin is formed by activating the contact tor) disease? factor system and FVII is bypassed. Answer Checkpoint 34-7 Individuals who have inherited a thrombophilia gene gen- Explain why the laboratory screening tests are normal in erally do not experience thrombotic episodes unless they patients with FXIII deficiency. have a second genetic or acquired predisposing factor. Thus, 1140 Appendix F Answers to Checkpoints many individuals who carry thrombophilia genes do not because functional assays detect both quantitative and have acute thrombotic events and can be diagnosed only qualitative deficiencies. If immunologic assays are used for when family screening is done for another family member screening, they would detect only quantitative deficien- who has had a thrombosis. cies, not qualitative defects (dysfunctional proteins) that Checkpoint 35-5 typically give normal antigen levels but reduced functional

levels. Functional assays are generally considered the bet- A patient was being evaluated for HIT. The ELISA assay for ter screening assay if a familial thrombophilia is suspected. HIT IgG was positive, and the platelet SRA was negative. Should this patient’s physician be concerned? Why or why Checkpoint 35-8 not? Why should heparin therapy overlap initiation of oral anti- Answer coagulant therapy when treating a patient with an acute thrombosis? The physician should not be concerned. Most patients on heparin form antibodies to the heparin–PF4 complex. Answer However, only those antibodies capable of binding to and Oral anticoagulants (vitamin-K antagonists) decrease func- activating platelets are of concern for causing clinical symp- tional levels of procoagulant proteins FII, FVII, FIX, and FX, toms of HIT. as well as the anticoagulant proteins PC and PS. However, Checkpoint 35-6 this action is not immediate (nor is the anticoagulant effect of heparin), and the decrease in activity of each of these a. Why is thrombocytopenia usually present in a patient proteins varies, depending on the half-life of each in the with DIC? circulation. Because PC has a relatively short half-life, clear- b. Which hemostasis laboratory screening tests (PT and ance of biologically active molecules (presynthesized before APTT), if any, will the following affect: oral anticoagulants were begun) occurs more quickly for Decreased FV PC than for the procoagulant protein factors FII, FIX, and Decreased FVIII FX. Thus, for the first few days after initiating coumadin Decreased fibrinogen therapy, there is an imbalance between clot-inhibiting influ- Decreased antithrombin ences (PC) and clot-promoting influences (FII, FVII, and c. Which laboratory test results would distinguish DIC FX). The net effect is a period of hypercoagulability before from hemophilia A? the full anticoagulant effect of coumadin is realized. For a patient who already has a hemostatic system that is pre- Answer sumably not in balance, this additional imbalance between a. During DIC, activation of the coagulation system leads clot-promoting and clot-inhibiting factors could further to consumption of clotting factors, inhibitors, fibrino- aggravate it. Thus, heparin anticoagulant therapy should be lytic components, and platelets (thrombocytopenia) at continued through the first 4–5 days of oral anticoagulant a rate faster than they can be synthesized. therapy until the full effect of coumadin is achieved. b. Decreased FV (PT and APTT) Decreased FVIII (APTT) CHAPTER 36 Decreased fibrinogen (PT and APTT) Checkpoint 36-1 Decreased antithrombin (neither) Why have most clinical laboratories switched from 3.8% to c. In hemophilia A, the platelet count, platelet function, 3.2% sodium citrate for specimen collection in coagulation TT, PT, and all fibrinolysis assays will be normal, while studies? the APTT will be prolonged and decreased levels of FVIII will be observed. On the other hand, in DIC, Answer the platelet count will be decreased (functional assays The use of 3.2% sodium citrate minimizes the chances of will be normal), the TT, PT, and APTT will be pro- over-anticoagulating a specimen because of a high hemato- longed, D-dimer will be increased (FVIII levels may be crit or an inadequately filled collection tube. decreased due to consumption). Checkpoint 36-2 Checkpoint 35-7 What affect does aspirin taken daily for a heart condition Why are functional assays recommended when screening a have on a patient’s platelet functional studies? Explain. patient suspected of having a familial thrombophilic defect? Answer Answer Aspirin irreversibly inhibits cyclooxygenase, which is Functional assays are recommended when screening a responsible for thromboxane A2 synthesis. Thromboxane patient suspected of having a familial thrombophilic defect A2 is required for platelet aggregation; therefore, platelet Appendix F Answers to Checkpoints 1141 aggregation and thus, primary hemostasis are abnormal. Checkpoint 36-7 Platelet aggregation with collagen is absent. Aggregation In diagnostic laboratory evaluation of a hypercoagulable with ADP and epinephrine shows only a primary wave. state such as antithrombin deficiency, why is it important to Aggregation with ristocetin is normal. There is a suppressed determine the functional activity of the coagulation protein? response with arachidonic acid. Answer Checkpoint 36-3 Several hypercoagulable states are characterized by a nor- Routine coagulation testing was performed on a properly mal antigenic level of protein but have decreased functional collected and processed specimen. The PT result was pro- activity. In other words, the hypercoagulable state is the longed, but APTT was normal. What is the best interpreta- result of a dysfunctional coagulation protein. Assessing tion of these results? only the antigenic level would miss the diagnosis. Answer Checkpoint 36-8 These results indicate an FVII deficiency. The PT evaluates A patient has a venous thrombotic episode and is to be factors in the extrinsic and common pathways, and the treated appropriately. When should the blood sample be APTT evaluates factors in the intrinsic and common path- drawn to perform a thrombotic workup to diagnose an ways. Because only the PT is prolonged, the factor defi- acquired or congenital deficiency? Why? ciency must be in the extrinsic pathway. Answer Checkpoint 36-4 To properly diagnose a thrombotic deficiency, the patient Given the following coagulation test results, what is the must be off treatment. If the sample is drawn too early, the appropriate follow-up or reflex test? consumptive process that occurred can produce decreased PT Normal levels of coagulation proteins as well as conflicting results APTT Prolonged because of anticoagulation treatment and possible supple- Mixing studies No correction mental treatment with FFP. This can lead to misdiagnosis of a deficiency in one or more factors. If the patient is tested Answer while hospitalized and the results conflict, repeat studies The appropriate follow-up or reflex test is the platelet neu- should then be performed. Family studies also should be tralization procedure, dilute Staclot LA, or Russell’s viper considered. venom time. Each of these tests would identify the presence of lupus-like anticoagulant. Checkpoint 36-9 What is the appropriate interpretation of the following Checkpoint 36-5 coagulation test results? What is the function of the factor-deficient substrate reagent and the reference plasma in a factor assay? D-dimer: Elevated PAI-1 activity: Increased Answer Plasminogen activity: Decreased The factor deficient substrate reagent ensures that all coagu- lation factors except the coagulation factor to be assayed are Answer at optimal activity levels. In this way, only the activity level The D-dimer is a specific marker for plasmin degradation of a single coagulation factor is evaluated. Reference plasma of fibrin clots. The increased PAI-1 also suggests defective contains a known activity level for each coagulation factor fibrinolysis. and is used to establish the factor activity curve from which Plasminogen is activated to plasmin by tPA. Decreased the patient’s factor activity can be determined (similar to a levels of plasminogen are found in DIC and liver disease. standard curve). The D-dimer test together with the abnormalities in Checkpoint 36-6 proteins involved in fibrinolysis (increased consumption) suggests DIC. Why are excess lipids able to neutralize lupuslike anticoagulants? Checkpoint 36-10 Answer A patient has a PT of 21.5 seconds (producing an INR of 2.2). In this case, the patient is not on oral coagulation. What is Platelet membrane phospholipids are exposed when the explanation for not reporting the INR? platelets rupture. Because lupus-like anticoagulants are antiphospholipid antibodies, they bind to the exposed Answer phospholipids and no longer interfere with the test reagent The INR reflects the standardization of the thromboplas- phospholipids. tin reagent for a certain PT to a standardized reference 1142 Appendix F Answers to Checkpoints thromboplastin using the same type of instrumentation. It Answer does not reflect the level of deficiency present in the sample. The laboratory professional should try increasing the angle Therefore, only the PT should be reported for patients who of the spreader slide and increasing the speed. This will are not on oral anticoagulants. achieve a thicker blood smear. Checkpoint 37-6 CHAPTER 37 Which component of Wright stain is responsible for staining Checkpoint 37-1 hemoglobin within erythrocytes? Contrast the mechanisms of anticoagulation for EDTA and Answer heparin. Eosin, the acidic dye, binds to basic groups on the hemo- Answer globin molecule. EDTA prevents blood coagulation by chelating calcium. Checkpoint 37-7 Because calcium is an important component of several If the erythrocytes appear bluish gray and the leukocyte enzymatic reactions in the coagulation cascade, its removal nuclei are black, how would you correct this problem? blocks fibrin formation. Heparin prevents blood coagula- tion by enhancing antithrombin activity. Antithrombin, in Answer turn, inhibits thrombin and blocks fibrin formation. Two common causes for excessive blue appearance are pro- Checkpoint 37-2 longed staining and use of a buffer or stain with pH that is too alkaline. Therefore, try decreasing the staining time to What are three steps a phlebotomist should take to mini- see whether the problem resolves or check the pH of the mize the risk of exposure to blood-borne pathogens when buffer and stain to verify whether one or both are too alka- performing a venipuncture? line. If the pH is the problem, make the appropriate adjust- Answer ments to lower the pH or try a new bottle of reagent. Steps to minimize exposure to blood-borne pathogens Checkpoint 37-8 include (1) wearing gloves, (2) using goggles, (3) per- In a platelet estimate, 76 platelets were observed in five fields forming venipuncture with a safe needle tube holder, (4) on a stained blood smear. Calculate the platelet estimate. If disposing of the needle and holder in a biohazard sharps the platelet count was 189 * 103>mcL1189 * 109>L2, would container, and (5) washing the hands following the removal these results correlate? Assume that the specimen was col- of gloves. lected with EDTA. Checkpoint 37-3 Answer What is the total magnification when a 10* objective is com- The platelet estimate is 228 * 103>mcL. Yes, the estimate is bined with a 10* eyepiece? within 25% of the platelet count (189 * 103>mcL * 0.25 = Answer 47 * 103>mcL). The total magnification is 100* . Checkpoint 37-9 Checkpoint 37-4 a. A manual platelet count was performed on an EDTA- anticoagulated blood specimen using the standard How does the examination of a specimen by phase-contrast dilution. The number of platelets counted from the first microscopy differ from that of bright-field microscopy? diluted vial was 125 and from the second diluted vial Answer was 131. What is this patient’s platelet count? b. What are two possible physiologic causes or mecha- (1) Bright-field microscopy requires a stained sample to nisms that could lead to a decrease in this patient’s examine cellular details; phase-contrast microscopy allows platelet count? examination of unstained cells. (2) Phase-contrast micros- copy requires a condenser with an annular ring and objec- Answer tive with phase ring. These components must be matched. a. The manual platelet count is calculated as follows: The plate- Bright-field microscopy uses a standard condenser and let count from the first diluted vial is 125 * 100>0.2 = objectives. 62.5 * 103>mcL; the platelet count from the second diluted vial is 131 * 100>0.2 = 65.5 * 103>mcL. The Checkpoint 37-5 patient’s platelet count equals (62.5 * 103>mcL + 65.5 * A laboratory professional consistently prepares thin blood 103>mcL)>2, or 64.0 * 103>mcL. smears resulting in minimal counting area. What would you Note: Each diluted vial was used to charge both ruled suggest this individual do to improve the quality of these areas of the hemacytometer so that the area counted blood smears? was 2 mm3. Appendix F Answers to Checkpoints 1143 b. This patient’s decreased platelet count can be the result b-thalassemia major because it is characterized by an uneven of decreased production of platelets in the bone mar- distribution of HbF within its erythrocyte population. row (e.g., aplastic anemia) or increased destruction of the platelets (e.g., immune thrombocytopenia.) Checkpoint 37-14 A patient’s osmotic fragility test shows beginning hemolysis Checkpoint 37-10 at 0.60% NaCl and complete hemolysis at 0.50% NaCl. How Given the following erythrocyte data, what is the expected should these results be interpreted? erythrocyte morphology? Hemoglobin = 6.2 g>dL; hematocrit = 28,; erythrocyte count = 4.10 * 106> Answer mcL. These results indicate increased osmotic fragility that is Answer associated with hereditary spherocytosis. Based on the erythrocyte data, the erythrocyte indices are Checkpoint 37-15 MCHC = 22 g>dL; MCV = 68 fL; MCH = 15 pg. These indices are lower than their respective reference intervals. A patient was recently diagnosed with a hypochromic, Therefore, the expected erythrocyte morphology would be microcytic anemia. Additional laboratory testing revealed the presence of hypochromic microcytic erythrocytes. EPO 1.5

mIU/mL and sTfR 15.8 nmol/L. Which anemia is consistent with these results? Checkpoint 37-11 Answer Morphologic evaluation of a Wright-stained peripheral blood smear reveals the presence of Pappenheimer bodies. These results are consistent with anemia of chronic inflam- How does this affect a reticulocyte count to be performed mation (anemia of chronic disease). A normal sTfR level but on the same blood specimen? How would you confirm the decreased EPO is a characteristic finding in this anemia. presence of Pappenheimer bodies? Checkpoint 37-16 Answer A pathology resident is evaluating the peripheral blood The presence of Pappenheimer bodies could falsely elevate smear of a 40-year-old female. The resident suspects CML. the reticulocyte count because both Pappenheimer bodies To confirm this suspicion, the resident ordered an LAP and reticulum stain with new methylene blue. Prussian blue score. Do you think that the LAP score will be helpful in stain is used to confirm the presence of Pappenheimer bod- making the correct diagnosis? ies. It stains iron but not reticulum. Answer Checkpoint 37-12 Yes, the LAP score can be used to differentiate CML from a What is the basis for hemoglobin separation in the hemo- leukemoid reaction because the LAP score in patients with globin electrophoresis procedures? CML is usually decreased. However, patients with CML in blast crisis with high numbers of circulating blasts can have Answer an increased score. Therefore, LAP scores should always be The net charge of hemoglobin molecules can be altered interpreted in conjunction with the morphologic examina- by the buffer’s pH. When placed in an electrical field, the tion of the sample. hemoglobin molecules will separate based on their net Checkpoint 37-17 charge. For example, at pH 8.6, hemoglobin molecules have a net negative charge with hemoglobin A possessing the A 30-year-old male presented to the emergency room strongest net negative charge and traveling the fastest. complaining of fatigue, weakness, and gum bleeding. The CBC showed anemia, thrombocytopenia, and a WBC of Checkpoint 37-13 30 * 103>mcL. The peripheral blood smear revealed numer- What are the advantages of using flow cytometry to quan- ous intermediate to large size blasts with fine nuclear chro- titate HbF? matin and abundant cytoplasm. The bone marrow biopsy Answer contained 60% blasts. Careful inspection detected no Auer rods. Several cytochemical stains were then performed. The Flow cytometry allows the evaluation of large numbers of blasts failed to stain with myeloperoxidase, SBB, and specific erythrocytes, thus increasing the significance of the HbF esterase; however, the majority of blasts reacted intensely result. In addition, the HbF concentration within individ- with a@naphthyl esterase. Incubation with sodium fluoride ual erythrocytes is observed to demonstrate the distribution inhibited the staining seen with a@naphthyl esterase. What of HbF within the patient’s erythrocyte population. Flow type of leukemia do you think this patient has? cytometry can also be used to demonstrate HbF concen- tration in polychromatophilic erythrocytes (i.e., reticulo- Answer cytes) and nucleated erythrocytes. These findings can be a@Naphthyl esterase stains primarily monocytes and its precur- useful in diagnosing certain erythrocyte disorders such as sors; therefore, it is useful in the diagnosis of acute leukemias 1144 Appendix F Answers to Checkpoints with a monocytic component such as acute monocytic and peripheral blood smear revealed numerous myelocytes. A myelomonocytic leukemias. However, other bone marrow bone marrow biopsy was performed. The bone marrow elements can also show staining with a@naphthyl esterase. differential was reported as follows: blasts 1%, myelocytes Therefore, the fluoride inhibition test is recommended. In this 35%, metamyelocytes 20%, bands 10%, segmented neutro- test, sodium fluoride is added to the a@naphthyl esterase incu- phils 20%, eosinophils 1%, basophils 3%, pronormoblasts bation mix. Sodium fluoride inhibits the monocytic enzyme. 1%, basophilic normoblasts 3%, polychromatophilic nor- In our patient, the blasts failed to stain in the mixture contain- moblasts 2%, orthochromatic normoblasts 4%, lymphocytes ing the fluoride, confirming the monocytic differentiation. The 0%, and monocytes 0%. What is the M:E ratio? predominance of staining with a@naphthyl esterase and the Answer absence of staining with myeloperoxidase, Sudan black B, and specific esterase indicate that this patient most likely has acute The myeloid precursors include blasts, myelocytes, meta- monocytic leukemia (AMoL). myelocytes, bands, segmented neutrophils, eosinophils, and basophils. Thus, the total number of myeloid cells is Checkpoint 37-18 90 11 + 35 + 20 + 10 + 20 + 1 + 32. The nucleated red A pathologist is reviewing the slides from the pleural fluid cell precursors include pronormoblasts, basophilic nor- of a 50-year-old male who has hepatosplenomegaly and moblasts, polychromatic normoblasts, and orthochro- minimal lymphadenopathy. It is not clear whether the matic normoblasts. The sum of erythroid precursors is mononuclear cells are mature lymphoma cells or leukemic 10 11 + 2 + 3 + 42, which produces an M:E ratio of 9:1. blasts. There is not enough material for flow cytometry to This elevated M:E ratio with the clinical history and periph- do immunophenotyping, but there is enough to make a few eral blood findings suggests chronic myelogenous leukemia. extra cytospin smears. Which stain do you think would be Checkpoint 38-3 useful in this case? A pathologist is evaluating a bone marrow biopsy from a Answer patient suspected of having a myelodysplastic syndrome. The indirect immunofluorescence technique for TdT is use- Evaluation of the peripheral blood reveals both hypochro- ful as it can be performed on cytospin smears. TdT is a DNA mic and normochromic red cells. The bone marrow aspirate polymerase found in the nuclei of immature lymphocytes shows dysplastic (abnormal cell development) changes in and is present in the majority of ALL cases. TdT is a primi- the red cell precursors. The pathologist ordered an iron stain tive cell marker and is of value in distinguishing ALL from on the particle crush preparation. Why is the iron stain use- malignant lymphoma. It is also helpful in defining leukemic ful in this case? cells in body fluids. Answer CHAPTER 38 The pathologist is most probably looking for the presence of ring sideroblasts, nucleated red cells in which one-third Checkpoint 38-1 of the nucleus is encircled by iron granules. The presence A 25-year-old male was recently diagnosed with acute lym- of ring sideroblasts is diagnostic for sideroblastic anemia phocytic leukemia (ALL) and received chemotherapy. It is and/or myelodysplastic syndrome. However, ring sid- 21 days after the chemotherapy. A bone marrow biopsy has eroblasts can also be seen in myeloproliferative disorders, been scheduled to determine whether residual leukemia is megaloblastic anemia, and alcoholism as well as following present. His platelet count is 10 * 109>L. Does this patient chemotherapy. have a significant risk of bleeding? Is it necessary to can- cel the procedure until the platelet count is greater than CHAPTER 39 50 * 109>L150 * 103>mcL2? Checkpoint 39-1 Answer What is the basis of the impedance principle for cell Bone marrow biopsies can be performed on thrombocy- counting? topenic patients. Applying pressure at the biopsy site and Answer having the patient lie on his or her back for 20–30 minutes can control the local bleeding seen in thrombocytopenic The impedance principle is based on the fact that blood patients. Evaluating this patient’s marrow is more impor- cells are poorly conductive particles. Increased resistance is tant than delaying because of the risk of local bleeding. observed when a poorly conductive particle passes through an electrical field. The increased resistance results in a pulse Checkpoint 38-2 as the cell goes through an aperature through which a cur- A 60-year-old male complained to his local physician of rent is flowing. The number of pulses indicates the blood abdominal fullness. The CBC showed an elevated leuko- cell count and the amplitude (i.e., height) of each pulse is cyte count with absolute neutrophilia and basophilia. The proportional to the volume of the cell. Appendix F Answers to Checkpoints 1145 Checkpoint 39-2 and five light scatter characteristics is used to determine the Why is lytic agent added to the leukocyte dilution? five-part leukocyte differential. Answer Checkpoint 39-6 First, the lytic agent lyses the erythrocytes and eliminates Which cellular characteristics are used to determine the IPF their interference with the WBC count (i.e., causing a falsely on the Sysmex XN-Series® instrument? elevated WBC count). The lytic agent also perforates the Answer leukocyte’s membrane and shrinks the membrane around Within the fluorescent platelet channel (PLT-F), the imma- the nucleus. This allows the instrument to measure each ture platelet fraction is isolated based on the size of the leukocyte’s cell volume and create a WBC histogram. immature platelets and their fluorescence intensity. The Finally, the lytic agent converts the released hemoglobin to fluorescence intensity is caused by oxazine binding to the cyanmethemoglobin for the measurement of hemoglobin nucleic acids within platelet organelles. concentration. Checkpoint 39-7 Checkpoint 39-3 a. Which CELL-DYn scatterplot allows differentiation of Explain how the Coulter® LH 780 determines the five-part eosinophils from neutrophils? leukocyte differential. b. The CELL-DYn Sapphire’s WBC dilution contains Answer propidium iodide, so why do the neutrophils not fluoresce? The Coulter® LH 780 uses impedance, conductivity, and light scatter to determine the leukocyte differential. Imped- Answer ance identifies each cell’s volume and is traditionally used a. The 90°D versus 90° scatterplot allows differentiation to generate a three-part differential. With the additional data of eosinophils from neutrophils based on their different generated for each cell by conductivity (i.e., cell’s internal granularity and lobularity characteristics. conductivity) and light scatter (i.e., cell’s size and granu- b. Propidium iodide binds specifically to DNA within larity) and the use of contour gating to analyze the data in the cell but is unable to pass through intact cell mem- three-dimensional space, the five-part leukocyte differential branes. As a result, the neutrophils and other intact leu- can be determined (i.e., neutrophil, lymphocyte, monocyte, kocytes do not fluoresce because the propidium iodide eosinophil, and basophil). is unable to enter the cell and bind to its DNA. Checkpoint 39-4 Checkpoint 39-8 The following results were obtained from a blood specimen What is the similarity in the reticulocyte methods performed analyzed by the Unicel® DxH 800: NRBC# = 2.7 * 103>mcL on the ADVIA 120 and XE-SeriesTM instruments? and corrected WBC = 18.1 * 103>mcL. What was the Answer uncorrected WBC count? Both methods use oxazine for the detection of residual Answer RNA. They both also use flow cytometric analysis, which Because the corrected WBC count is the uncorrected WBC is common to all instruments discussed in this chapter. count, the absolute number of nucleated red blood cells (NRBC#), the uncorrected WBC count is 20.8 * 103>mcL. CHAPTER 40 Checkpoint 39-5 Checkpoint 40-1 How does the determination of the five-part leukocyte dif- Would a lymphocyte or monocyte have more forward light ferential for the Unicel® DxH 800 instrument and the Sys- scatter? mex XE-Series® instrument differ? Answer Answer Forward light scatter depends on cell size; larger cells have The Sysmex XE-Series® instrument uses fluorescent stain- more forward scatter. A monocyte is larger than a lympho- ing of nucleic acids and forward/side-scatter characteris- cyte and would have more forward light scatter. tics to determine lymphocytes, monocytes, eosinophils, Checkpoint 40-2 and neutrophils + basophils. It separates basophils from A laboratory wants to identify cells that have two different the neutrophils in a second dilution using a specific lyse antigens. How many laser light sources are needed? reagent that leaves the basophils intact. The light scatter characteristics of the basophils versus other leukocytes Answer allows clear delineation of basophils. For the Unicel® DxH One laser light source is needed because a single wave- 800 instrument, a combination of impedance, conductivity, length can excite up to three fluorochromes. 1146 Appendix F Answers to Checkpoints Checkpoint 40-3 1 cell — 47,XX, +8 A cell population is positive with both Leu1 and T1 mono- 1 cell — 45,XX, -20 clonal antibodies. As a result, the cell is classified as CD5 5 cells — 46,XX,del(5)(q13q34),-7, +21 positive. Explain. Which of these aberrations is clonal? Why? What term Answer would apply to the five cells? The Leu1 and T1 antibodies detect the same antigen. This Answer antigen has been given the cluster of differentiation desig- The clonal aberrations are the del(5), -7, +21. A clone exists nation CD5. if numerical and/or structural abnormalities are found in at Checkpoint 40-4 least 2 cells. The +8 and -20 and are seen in only one cell each and are therefore nonclonal. The five abnormal cells

Explain why lymphocytes and neutrophils can be separated would be referred to as pseudodiploid. on a forward versus side light scatter dot plot. Checkpoint 41-3 Answer A 35-year-old man has acute leukemia. Cytogenetic stud- Granulocytes have more side light scatter than lymphocytes ies of the bone marrow reveal the following: 46,XY,t(15;17) because of their granularity. (q24.1;q21.1). What type of leukemia does this patient have, Checkpoint 40-5 and what will cytogenetic studies show after treatment if he Why is it important to do immunophenotyping in a case of achieves complete remission? ALL/LBL? Answer Answer The t(15;17) is diagnostic for acute promyelocytic leukemia, Immunophenotyping is necessary to differentiate T-ALL AML-M3. If the patient achieved complete remission, cyto- from B-ALL and mature B-ALL from precursor B-ALL. genetic analysis of bone marrow cells would show a normal Treatment and prognosis in these different subtypes differ. male karyotype. Checkpoint 40-6 Checkpoint 41-4 A patient has a WBC count of 5 * 103>mcL, and 30% lym- Five years ago, a 46-year-old woman received chemother- phocytes; 10% of the lymphocytes are CD4+ . What is the apy and radiation treatment for breast cancer. She now has absolute CD4 count? Is this count compatible with a diag- pancytopenia. Cytogenetic analysis of the bone marrow nosis of AIDS in an HIV-infected individual? is performed and shows 10 of 20 cells with the following: 45,XX,del(5) (q13q34), -7. What is the significance of this Answer finding? 15 * 103>mcL2 * 0.30 = 1.5 * 103>mcL1150>mcL2 total lym phocytes. 1.5 * 103>mcL * 0.10 = 0.15 * 103>mcL Answer 115 mcL2 CD4+ lymphocytes. A CD4 count 6200>mcL is These results show clonal acquired aberrations indicative diagnostic of AIDS in an HIV-infected individual. of a neoplastic cell line. The del(5) and -7 are consistent with a secondary myelodysplastic state/acute leukemia. CHAPTER 41 Checkpoint 41-5 Checkpoint 41-1 A 5-year-old girl has fatigue and easy bruising. A A newborn baby boy has astple congenital malformations, CBC shows a leukocyte count of 40 * 103>mcL with and a chromosome abnormality is suspected as the cause. 85% blasts. Bone marrow cytogenetic studies are per- What is the most appropriate specimen to submit for chro- formed and show all cells with the following karyotype: mosome analysis, and how should the laboratory profes- 53,XX, +X, +4, +6, +10, +18, +20, +21. What is the prog- sional process the specimen? nostic significance of this finding? Answer Answer The most appropriate specimen to determine the presence of This karyotype in a pediatric patient with acute lympho- a constitutional aberration is peripheral blood. The specimen blastic leukemia would correlate with a good prognosis. should be processed by culture stimulated with phytohemag- glutinin to stimulate mitosis of the circulating lymphocytes. CHAPTER 42 Checkpoint 41-2 Checkpoint 42-1 Cytogenetic studies were performed on a bone marrow Consider the role that oncogenes and tumor suppressor specimen from a female patient with a myelodysplastic genes play in the development of cancer and the concept of state with the following results: personalized medicine. Appendix F Answers to Checkpoints 1147 Answer Answer If a mutation occurs in either of an individual’s alleles, Real-time PCR technology coupled the PCR amplification it is possible that the function of the protein for which it process with fluorescent detection and eliminates the need encodes will be altered—either lost or exaggerated. Either for post-PCR processing. Detection of PCR florescence in abnormal effect could dysregulate cell metabolism so that the exponential phase shows a quantitative relationship cancer results. normal alleles that become mutated in a way between the amount (copy number) of the starting material that contributes to the development of cancer are known as in the sample and the cycle number at which the amplifica- proto-oncogenes. Their nomenclature changes to oncogenes tion curve crosses a mathematically determined threshold. if they become altered and begin producing cancer-induc- An increase in the reporter dye fluorescent signal is directly ing proteins. Tumor suppressor genes do just that: encode proportional to the number of amplicons generated. Detec- proteins that suppress cell proliferation. tion systems include SYBR Green®, which binds to double- Personalized medicine is a model of health care deliv- stranded DNA and labels any amplicon product. Other ery in which decisions about such delivery are made based variations of detection systems have been developed to on each person’s unique clinical, environmental, and provide more specific detection of the desired target ampli- genetic information. Because these variables are different con than the nonspecific SYBR Green dye. One particular for everyone, the pathophysiology, stratification, prognos- detection system, the TaqMan system, is based on the 5′ tication, and optimal treatment are assumed to be unique exonuclease activity of DNA polymerase and captured fluo- to the individual. Personalized medicine is really about rescence of a cleaved reporter dye-labeled probe. numerous developing therapeutic decision making that is unique and real-time PCR (qPCR) applications are shown in Table 42-2. individual to the patient. Checkpoint 42-2 CHAPTER 43 List and briefly describe the four general ways in which Checkpoint 43-1 molecular diagnostics is useful to clinicians in delivering Explain the importance of each component of the quality care to their patients. assessment program to the goal it is intended to meet. Answer Answer Molecular diagnostics testing can be used to help establish The pre-examination component is critical for a quality an initial diagnosis by identifying DNA mutations that have assessment (QA) program because the test outcome is only been correlated to pathologies. Molecular diagnostics can as good as the specimen from which it is determined. The aid in the prognostication of the patient, again by identify- QA program must ensure proper collection and handling of ing mutations that have been correlated to either favorable specimens before testing. The examination component repre- or unfavorable outcomes. Often, treatment decisions are sents all factors that affect the testing system. The test result made based on the information about the patient’s genetic will be affected if a breakdown in the testing system occurs makeup, and finally after treatment, the patient can be because of the lack of a proper QA program. A breakdown followed for MRD to determine whether he or she is con- in the QA program within the post-examination component tinuing to be in remission or whether a reoccurrence of the affects the recording/reporting of the test result. This may pathology is being established. delay appropriate treatment for the patient. Checkpoint 42-3 Checkpoint 43-2 NRAS/KRAS mutations are seen in a wide variety of hema- Define effectiveness with respect to laboratory testing and tological cancers including ALL, AML, and plasma cell neo- identify one process to measure in the pre-examination and plasms. Explain how mutations in these genes contribute to post-examination phases for the hematology section of the the etiology of these diseases. laboratory. Answer Answer Mutations of NRAS/KRAS often underlie disrupted RAF- Effectiveness can be defined as evidence-based laboratory MEK-ERK signaling, a major cell-signaling path that cul- medicine. With respect to hematology it means that hema- minates in changes in transcription. Activating mutations tology procedures, such as CBCs or coagulation procedures in both these genes produce a proliferation advantage in such as prothrombin times, are performed for patients when affected cells, one of the major attributes of malignancies. their clinical signs and symptoms warrant the procedure. Checkpoint 42-4 It also means that these procedures are not requested and Explain how PCR technology can provide quantitative performed more than clinically necessary. An example of results. Suggest several hematologic and hemostasis appli- overutilization would be requesting and performing a daily cations of qPCR technology. CBC for an inpatient who is not actively bleeding. 1148 Appendix F Answers to Checkpoints An example of a metric for measurement in the pre- Checkpoint 43-6 examination phase is frequency of CBC ordered on inpa- Define the term reference interval. tients who have a stable hematocrit. A metric for the post-examination phase is frequency of Answer follow-up testing for abnormal coagulation screening pro- For a given analyte, the reference interval represents the usual cedures (PT and APTT). results for a healthy population. For example, the reference interval for hemoglobin in the adult male population is Checkpoint 43-3 14.0–17.4 g/dL. How does a laboratory that has lost its certification to per- form protein C assays regain that certification? Checkpoint 43-7 If a spill occurred when handling the CELL-DYN Sapphire Answer CN-free hemoglobin reagent, what document should be The laboratory professional should determine the reason for used for information regarding a means to safe clean-up the failure, correct the problem, and document the process. of the spill? Following the corrective action, successful participation in Answer two consecutive proficiency-testing surveys for protein C would result in the laboratory’s recertification to perform Safety data sheet protein C assays. Checkpoint 43-8 Checkpoint 43-4 To establish the control limits for a new lot number of level What is an appropriate method to assess a laboratory pro- 1 PT coagulation control, the following data points were fessional’s competency in performing prothrombin time collected (results are in seconds): 11.8, 11.6, 12.1, 12.0, 12.3, (PT) and activated partial thromboplastin time (APTT) 12.6, 11.9, 12.2, 12.0, 11.5, 12.7, 12.1, 11.2, 12.3, 12.9, 13.0, 12.3, using an automated coagulation instrument? 11.9, 12.4, and 12.5. What are the 2s control limits? Answer Answer A combination of direct observation checklist and blinded The first step to determine the control limits for these results preanalyzed samples provides an appropriate method of is to calculate the mean and standard deviation. Based on assessing competency in performing PT and APTT. these data points, the mean is 12.2 seconds and the standard deviation is 0.50. Therefore, the 2s control limits would be Checkpoint 43-5 11.20–13.20 seconds. Linear regression analysis was performed on results from a Checkpoint 43-9 method comparison of the prothrombin time between auto- After performing daily quality control on the automated mated coagulation instrument and automated coagulation hematology instrument, the laboratory professional instrument. observes a 41s violation for the hemoglobin parameter. What The following results were obtained: type of error is indicated? y@intercept = 0.8157 Answer Slope = 0.9982 The 41s violation indicates a systematic error that can result Standard error of the estimate = 0.0807 from an expired lysing reagent or a deteriorating hemoglo- bin lamp. What conclusions can be drawn from these results? Checkpoint 43-10 Answer What type of quality control program should the labo- Based on the linear regression results, a constant differ- ratory consider for its hematology and coagulation ence occurs between the automated instrument A and the instruments? automated instrument B because the y-intercept is more than zero. That is, the automated instrument B gives con- Answer sistently higher results than the automated instrument A. For coagulation instruments two levels of QC material must Therefore, a constant systematic error exists. The slope be analyzed every eight hours of testing for each coagula- between 0.95 and 1.05 indicates no proportional system- tion test performed. atic error in the two methods. There is no random error An IQCP can be developed for the hematology instru- because the standard error of estimate is nearly zero, ment if there is an internal control process and the instru- indicating very little dispersion of results about the linear ment manufacturer’s instructions permit less than the regression line. CLIA-required frequency of QC analyses. Appendix F Answers to Checkpoints 1149 Checkpoint 43-11 can quickly identify this problem. If cold agglutinins are In reviewing a patient’s CBC results, the laboratory profes- present, the sample is warmed to 37 °C for 15 minutes to sional notes an MCHC of 37 g/dL. What corrective action dissociate the agglutinated cells prior to analysis to obtain should be taken? accurate results. If other possibilities are ruled out, the peripheral blood smear is examined for the presence of Answer spherocytes. no corrective action is needed if spherocytes The laboratory professional must first determine if the are present. MCHC is falsely elevated. The presence of cold agglutinins, Checkpoint 43-12 lipemia, icteria, or spherocytes in a patient sample has been The laboratory professional observes that the 3.2% sodium associated with an MCHC 7 36 fL. Lipemia and icteria are citrate tube for a PT and APTT is only two-thirds full. substances that cause falsely elevated hemoglobin and con- Explain the effect this will have on the patient’s coagula- sequently falsely elevated MCHC. Centrifuging the sample tion results. and examining the plasma can identify these substances. If either

is present, the saline replacement procedure is Answer performed to obtain an accurate hemoglobin and MCHC. If the sample collection tube is only two-thirds full, excess If cold agglutinins are suspected, the hematocrit will be anti-coagulant is present and the plasma is over-antico- falsely decreased (i.e., MCV * RBC = Hct), and conse- agulated. Therefore, the coagulation results are falsely quently, the MCHC falsely elevated. A manual hematocrit prolonged. Appendix G Answers to Case Study Questions CHAPTER 1 Question Question 2. What conditions can cause this bone marrow finding? 1. If Aaron was diagnosed with otitis media, what cellular Explanation component(s) in his blood would be playing a central Conditions associated with an increased cellularity of bone role in fighting this infection? marrow include anemia and leukemia. Explanation Question The leukocytes, or white blood cells, are the cells that are 3. What do you think is the cause of the splenomegaly? central in fighting infection. Explanation Question The splenomegaly could be the result of extramedullary 2. Aaron’s physician ordered a CBC. The results are hematopoiesis. Malignant cells could be proliferating in Hb 11.5 g/dL; Hct 34%. What parameters, if any, are the spleen. outside the reference intervals? Why do you have to Question take Aaron’s age into account when evaluating these 4. Why might the peripheral blood reveal changes associ- results? ated with hyposplenism when the spleen is enlarged? Explanation Explanation It is important to consider Aaron’s age because the reference Tumor cells can incapacitate the spleen, causing functional intervals for blood cell concentrations are different in chil- hyposplenism. dren of various ages and are different from those in adults. Question The hemoglobin and hematocrit are within the reference interval for this patient’s age. 5. What might explain the lymphadenopathy? Chapter 1 Case Summary: Aaron had clinical signs of Explanation infection and a past history of ear infections. The CBC The malignant lymphocytic cells could be proliferating in results revealed a high WBC count consistent with an infec- the lymph nodes causing enlargement. tious process. The diagnosis of otitis media usually can be Chapter 3 Case Summary: Francine had an acute lympho- made by history and physical examination. Laboratory tests cytic leukemia. This is a type of leukemia that is character- are not required. ized by a malignant proliferation of immature lymphocytic cells. The hemoglobin is decreased. The WBC is normal, but CHAPTER 3 the platelets are markedly decreased. Question 1. Determine which blood cell parameters are abnormal. CHAPTER 5 Describe Francine’s bone marrow as normal, hyper- Question plastic, or hypoplastic. 1. Predict Stephen’s reticulocyte count: low, normal, or Explanation increased. Refer to the tables on the inside cover of the book. Explanation The hemoglobin is decreased. The WBC is normal, From the erythrocyte count, Hb, and Hct, we know the but the platelets are markedly decreased. The bone mar- patient is moderately anemic. The increase of polychro- row is hyperplastic. Normal bone marrow cellularity is matic erythrocytes on a blood smear suggests an increased 650,. number of reticulocytes. 1150 Appendix G Answers to Case Study Questions 1151 Question The amount of iron in these pools is regulated by proteins 2. What cellular mechanism results in hemolysis due to a involved in the transcription and translation of ALAS, fer- deficiency in G6PD? ritin and transferrin receptors. In an iron deficient state, the synthesis of ALAS and ferritin is decreased and synthesis Explanation of transferrin receptors is increased. In the hexose monophosphate shunt, the reduction of NADPH Question and glutathione depends on the enzyme G6PD. When this enzyme is deficient, hemoglobin denatures under oxidant 2. What was the rationale for giving Jerry the iron? stress, and intracellular hemoglobin precipitates form. Explanation Question Because Jerry had lost the blood through bleeding, he also 3. Explain how Heinz body inclusions cause damage to lost a substantial amount of iron. Even if body storage of the erythrocyte membrane. iron is normal, iron supplements are often given, in this case to provide the iron needed for rapid and increased hemo- Explanation globin synthesis. Hemoglobin precipitates known as Heinz bodies form along Question the inner surface of the erythrocyte membrane. This results in a loss of membrane flexibility, cell lysis, and splenic 3. Explain why Jerry may have these symptoms. trapping. Explanation Question Jerry’s hemoglobin was very low, which means that his tis- 4. Would you predict Stephen’s serum erythropoietin lev- sues were not getting the oxygen they needed. This leads els to be low, normal, or increased? Why? to a decrease in metabolic activity and, consequently, a decrease in energy. Pallor is a classic sign of anemia because Explanation blood is preferentially circulated to critical areas of the body In hemolytic anemia, the loss of erythrocytes results in a including the brain, heart, and so on. The skin’s blood sup- systemic decrease in cellular oxygen tension. This stimu- ply decreases, causing a loss of the pinkish color of the skin, lates EPO production from the kidneys, which in turn stim- especially apparent in Caucasians. ulates erythropoiesis in the marrow. Question Question 4. Explain why Jerry may have had more energy after the 5. Explain why Stephen has a low haptoglobin value. transfusions. Explanation Explanation The oxidant stress caused by the malarial drug primaquine The transfusions boosted Jerry’s hemoglobin level and, was out of balance because of the patient’s erythrocyte defi- thus, increased the amount of oxygen that could be trans- ciency of G6PD. This resulted in hemoglobin precipitation ported to the tissues for critical metabolic processes. (Heinz bodies) and cell destruction. Some of the hemoglobin is released into the peripheral blood and is bound by hapto- Chapter 6 Case Summary: Jerry lost a substantial globin. The haptoglobin reserves become depleted quickly. amount of blood from the fractures and surgery. If his hemoglobin had been normal before the accident (14–16 Chapter 5 Case Summary: This is a case of a 28-year-old g/dL), he lost about one-half of the volume of his blood. Caucasian male of Italian descent with an acute hemolytic With the loss of this much blood, he also lost a substan- anemia. An initial diagnosis of malaria was presumptively tial amount of iron. Although he was given iron supple- made. The patient, however, was negative for malaria. He ments, it will take time for his hemoglobin to reach normal was eventually diagnosed as having glucose-6-phosphate again. He had symptoms of anemia with lethargy and pal- dehydrogenase (G6PD) deficiency and a hemolytic anemia lor. They result from a loss of hemoglobin and, hence, a induced by the antimalarial drug primaquine. decrease in the amount of oxygen delivered to the tissue. The blood transfusions will bring his hemoglobin concen- CHAPTER 6 tration up more rapidly and give his body the energy it Question needs to repair itself. 1. If Jerry is iron deficient, what is the effect on synthesis of ALAS, transferrin receptor, and ferritin? CHAPTER 7 Explanation Question Iron may be either in the metabolic pool (for heme syn- 1. Are any of these results outside the reference interval? thesis) or the storage pool (hemosiderin and ferritin). If yes, which one(s)? 1152 Appendix G Answers to Case Study Questions Explanation Explanation The WBC count is above the reference interval. T lymphocytes mature in the thymus under the influence of Question cytokines produced by the thymic microenvironment. 2. If this were a newborn, would you change your evalu- Question ation? If so, why? 3. Calculate the absolute concentration of lymphocytes. Explanation Is this increased, decreased, or within the reference interval? Newborns have a higher reference interval for WBC; there- fore, if this were a newborn, the WBC count would fall Explanation within the reference interval. The absolute concentration is 8.4 * 103/mcL. This is within the reference range (4.0913.5 * 103/mcL) for a 6-month-old Question child. 3. Are any of the WBC concentrations outside the refer- ence interval (relative or absolute)? Question 4. If this were a 30-year-old man, would this be consid- Explanation ered normal or abnormal? Explain. No, all results (both percentages and absolute values) are within the reference interval. Explanation This is abnormal, or increased, for an adult. The reference Question interval for adults is 1.094.8 * 103/mcL. 4. Is there a need for reflex testing on Harry? Explain your answer. Question 5. Is there a need for concern regarding the infant’s Explanation results? Explain your answer. Although Harry’s WBC count is slightly above the refer- Explanation ence interval, his absolute individual white cell numbers are within it. Given the fact that he has no symptoms, the physi- Infants and children have higher lymphocyte concentrations cal examination was normal, and no abnormal cells were than adults. Because this infant’s results are within the refer- noted on the blood smear, there is probably no need to do ence range for his age group and he has no history of illness, additional testing. The reference interval is usually set by cal- there is no apparent reason to question the lab results. culating the mean and adding and subtracting two standard Chapter 8 Case Summary: The laboratory results on this deviations. This range includes 95% of normal individuals. baby are normal for his age. It may appear that there is a About 5% of individuals have a result outside this interval lymphocytosis, but this lymphocyte count—both relative but are still normal. This could be the case with Harry. and absolute—are within the reference range for his age. Chapter 7 Case Summary: Harry had a physical as a pre- requisite for buying a life insurance policy. Except for the CHAPTER 9 WBC count, his results were within the reference interval Question for his sex and age. 1. Is the patient’s platelet count normal or abnormal? CHAPTER 8 Explanation Question The platelet count is increased. The reference interval is 1509400 * 103/mcL. 1. What class of lymphocytes would you expect to make up the majority of peripheral blood lymphocytes in this Question child and which subclass CD marker is present on the 2. Why do you think the spleen is enlarged? majority of these cells? Explanation Explanation About one-third of the platelets are sequestered in the Most lymphocytes in the peripheral blood are T lymphocytes. spleen. Because this patient’s platelet count is very high, the Because there is no history of illness in this child, you would mass of platelets in the spleen is probably very increased, expect the majority of lymphocytes to be T lymphocytes. From leading to splenomegaly. 60–80% of peripheral blood T lymphocytes are CD4+ cells. Question Question 3. A bone marrow was ordered. What cells would you expect 2. Where does this class of lymphocytes differentiate? to see increased? Why? Would this affect the M:E ratio? Appendix G Answers to Case Study Questions 1153 Explanation Question You would expect the megakaryocytes to be increased 2. Evaluate the calculated Hct, MCH, and MCHC as com- because these cells are the precursors of platelets and the pared with the reference intervals for a 48-year-old platelets are very increased. This will not affect the M:E ratio female. in the bone marrow because megakaryocytes and mega- Explanation karyoblasts are not included in this ratio. The hematocrit is lower than expected, and the MCH and Question MCHC are much higher than the reference interval for this patient. 4. What cytokine normally stimulates the production of these cells? What is the receptor for this cytokine? Question Explanation 3. What should the laboratory professional report about Thrombopoietin (TPO) is the cytokine that stimulates the the RBCs in the critical area of this smear? proliferation of megakaryocytes. The receptor for TPO is Explanation c-mpl (CD110). RBC agglutination is present. Question Question 5. The patient was diagnosed with essential thrombocythe- 4. Which results of the CBC might be affected by the find- mia. What is the pathophysiology of this disorder? ings on the smear? Explanation Explanation Essential thrombocythemia is a neoplastic disorder that The abnormality in this case is most likely the presence belongs to the group of disorders called myeloproliferative dis- of a cold agglutinin. Agglutination of the RBCs causes a orders (Chapter 24). It is associated with acquired mutations falsely low RBC count, falsely increased MCV, falsely low of protein involved in the TPO receptor-signaling pathway, calculated Hct, and falsely increased calculated MCH and which results in increased proliferation of megakaryocytes. MCHC. The failure of the rule of 3

also indicates a mismatch Chapter 9 Case Summary: This patient has a decreased between the hemoglobin and hematocrit. hemoglobin, normal WBC count, and a significant increase in Question platelets. A bone marrow examination was ordered to evalu- ate the production of platelets. The examination indicated 5. Explain why the abnormal values in the CBC occurred that her megakaryocytes were increased. Molecular testing in this case. showed the presence of the JAK2 (V617F) mutation, which Explanation codes for a mutated protein kinase receptor that is involved Each RBC agglutinate containing several cells was counted in the cell signaling pathway. This mutation is associated as one cell. This falsely decreased the RBC count. Each with about 50% of patients with essential thrombocythemia, agglutinate appeared to the instrument as a large cell, a myeloproliferative neoplasm associated with an increase in and this falsely elevated the MCV. Because the RBC count platelets. The diagnosis is essential thrombocythemia. decreased more than the MCV increased, the calculated Hct (MCV * RBC/10) was falsely decreased. The decreased CHAPTER 10 RBC count falsely elevated the calculated MCH, and the Question falsely decreased hematocrit falsely increased the calculated MCHC. 1. Calculate the Hct, MCH, and MCHC from the initial results. Question Explanation 6. Predict the RDW value for the RBCs from this patient The indices are Hct 19%, MCH 47 pg, and MCHC 43.2 g/dL. as increased or normal. Calculation Patient data Reference Interval (conventional) MCV (fL) * RBC (* 106/mcL) 110 * 1.73 36–46 % Hct , = = 19, 10 10 Hb (g/dL) 8.2 28–34 pg MCHc = * 10 * 10 = 47 pg RBC (*106/mcL) 1.73 Hb (g/dL) 8.2 32–36 g/dL MCHCd = * 100 * 100 = 43.2 g/dL Hct (,) 19 1154 Appendix G Answers to Case Study Questions Explanation Question The RDW should be increased because of the increase in 7. Calculate the Hematocrit, MCH, and MCHC on the the variation in the RBC volume. The agglutinates falsely warmed specimen. How have they changed? elevate the RDW value. Explanation The indices are Hct 24%, MCH 31.2 pg, and MCHC 34.2 g/dL. Calculation Patient data Reference Interval (conventional) MCV (fL) * RBC (* 106/mcL) 91 * 2.63 36–46 % Hct , = = 24, 10 10 Hb (g/dL) 34 pg MCH = * 10 8.2 28– RBC (*106/mcL) * 10 = 31.2 pg 2.63 Hb (g/dL) 32–36 g/dL MCHC = * 100 8.2 Hct (,) * 100 = 34.2 g/dL 24 The hematocrit increased and the MCH and MCHC have decreased. Question 8. Explain what may have happened in this case? the clumps of RBCs are analyzed as one cell, thus decreas- ing the RBC count and increasing the MCV. The presence Explanation of cold agglutinins can be verified by examining the blood Warming the blood at 37 °C will usually cause the aggluti- smear. The RBC parameters that use the RBC, MCV, and nation to disappear. The antigen antibody reaction occurs calculated hematocrit will be in error. The specimen needs only at temperatures below 37 °C. Dispersion of the aggluti- to be warmed and rerun. nates corrects the RBC count, MCV, and calculated Hct. The MCH and MCHC calculations are now accurate because the CHAPTER 11 RBC and Hct values are correct. Question Chapter 10 Case Summary: This patient had a cold agglu- 1. Calculate the erythrocyte indices. Does the information tinin, which causes the RBCs to clump together. When given suggest acute or chronic blood loss? What is the blood is analyzed on an automated hematology instrument, significance of the RDW? Explanation The indices are MCV 113 fL, MCH 43.7 pg, and MCHC 38.8 g/dL. Calculation Patient data Reference Interval ( conventional) Hct (,) 8 80–100 fL MCV = * 10 * 10 = 113 fL RBC (*106/mcL) 0.71 Hb (g/dL) 3.1 28–34 pg MCH = * 10 * 10 = 43.7 pg RBC (*106/mcL) 0.71 Hb (g/dL) 3.1 32–36 g/dL MCHC = * 100 * 100 = 38.8 g/dL Hct (,) 8 The case history suggests chronic blood loss. The hemoglobin is very low, and the patient probably would be in shock if he had lost this much blood suddenly. The RDW suggests notable anisocytosis. Question 2. Calculate his absolute reticulocyte count. Is this increased, decreased, or normal? Explanation The absolute reticulocyte count is 156 * 103/mcL. Calculation Patient data Reference Interval (conventional) Absolute reticulocyte (* 103/mcL) = RBC (* 106/mcL) * 0.71 * 0.22 = 0.156 * 106/mcL 25975 * 103/mcL reticulocyte count (,) = 156 * 103/mcL This reticulocyte count is increased. Appendix G Answers to Case Study Questions 1155 Question Explanation 3. George’s blood smear revealed marked spherocytosis. Loss of blood can lead to anemia and an increase in eryth- Explain the importance of this finding. ropoiesis in the bone marrow. Chronic bleeding can lead to Explanation iron deficiency. Infection can stimulate an increase in leuko- cytosis, especially of the neutrophils or lymphocytes. Spherocytes are cells that have lost membrane. They are significant in this case because they indicate a hemolytic Question anemia. 2. How would you describe his anemia morphologically? Question Explanation 4. Explain George’s abnormal indices. MCV = 63 fL, MCH = 19.5 pg. The anemia is microcytic, Explanation hypochromic. The indices— MCV 113 fL, MCH 43.6 pg, and MCHC 38.8 Question g/dL—are all elevated. The increased MCV could be the 3. Calculate the percentage of saturation. result of the high reticulocyte count. The MCH is elevated because of the presence of these large cells that are able to Explanation hold more hemoglobin than a smaller cell. The MCHC is Serum iron/TIBC = 4,. The serum iron is low, and the elevated because of the marked spherocytosis. TIBC increased resulting in a low percentage of saturation. Question Question 5. Classify George’s anemia morphologically and 4. Is this value normal, decreased, or increased? functionally. Explanation Explanation Less than 15% saturation is decreased. George’s anemia is morphologically classified as macro- Question cytic. Functionally, it is classified as a survival defect. It appears that an immune hemolytic process is destroying 5. What disease, if any, is suggested by this value? the cells. The bone marrow has increased production of cells Explanation as the increased reticulocyte count indicates. Iron deficiency (ID) is suggested. The percentage of satura- Chapter 11 Case Summary: This patient had a severe ane- tion of transferrin is decreased in ID, usually to less than mia as revealed by the red cell indices (macrocytic with an 10%. elevated MCHC). The anemia developed slowly over time, Question which gave his body the opportunity to adapt to a low hemoglobin level. The probability of shock and death of a 6. How do the iron study results of our patient help in patient who had lost this much blood quickly would have differentiating the diagnosis of ID from ACD? been high. The yellowness of the eyes suggests a high bili- Explanation rubin concentration, which is typical of a hemolytic anemia. In ACD, the total body iron is normal to increased. In ACD, This patient had a test that revealed antibodies and comple- the serum iron is low, and the TIBC and percentage of sat- ment on his red blood cells, which supports the diagnosis uration are normal or decreased. In ID, total body iron is of an immune hemolytic anemia. The presence of sphero- decreased. In ID the serum iron is low, TIBC is increased, cytes supports this diagnosis because they indicate that the and percentage of saturation is low. spleen has removed antibody/antigen complexes from the cell membrane. Note that the MCHC is increased, which Question is typical of spherocytes. The high reticulocyte count and 7. What additional iron test, that was not performed, presence of polychromatophilic erythrocytes indicate that would be most helpful in this case? the bone marrow is responding appropriately by increasing output of erythrocytes. The high reticulocyte count is prob- Explanation ably responsible for the increased MCV. Serum ferritin is a good indicator of iron stores and is less invasive than a bone marrow. CHAPTER 12 Question Question 8. Do the iron studies in Jose (serum iron 18 mcg/dL, 1. How can these conditions affect the hematopoietic TIBC 425 mcg/dL) suggest sideroblastic anemia? system? Explain. 1156 Appendix G Answers to Case Study Questions Explanation Question No, iron studies in this patient reveal a lack of total body 5. What physiological condition does this patient have iron. Sideroblastic anemia has a defect in the incorporation that may lead to sickling of his erythrocytes? of iron into the porphyrin ring. Iron accumulates in the red Explanation cell and macrophage. Thus, the total body iron is increased in sideroblastic anemia. This patient has fever, suggesting infection or inflammation. The chest radiograph indicated pneumonia. It is possible Question that the infection is causing hypoxia and other physiological 9. Do Jose’s laboratory test results and clinical history alterations that promote sickling. indicate that a bone marrow examination is necessary Question to make a diagnosis? 6. What is the cause of this patient’s pain and acute distress? Explanation Explanation No, adequate information is present from other laboratory The patient is experiencing a vaso-occlusive crisis as a result tests. The CBC and iron studies give important clues to a of the sickling of erythrocytes in the microvasculature. (Phy- diagnosis of ID anemia. A bone marrow could be performed sicians refer to this as a “pain” crisis.) It is possible he is also in difficult cases but is usually not necessary. experiencing acute chest syndrome. Chapter 12 Case Summary: This patient is suffering from IDA as the result of chronic blood loss from the genital- Question urinary tract. He has the typical blood picture of microcytic, 7. Why might Shane be more susceptible to pneumonia hypochromic erythrocytes. His iron studies reveal a lack of than an individual without sickle cell disease? total body iron. The serum iron and percentage of transfer- Explanation rin saturation are low. It is likely that he has functional asplenia as a result of repeated sickling episodes in the spleen. Without a func- CHAPTER 13 tioning spleen, he is more susceptible to certain bacterial Question infections. Often sickle cell patients are treated with pro- phylactic antibiotics to prevent infections. 1. Identify a laboratory test needed to determine Shane’s hemoglobinopathy. Question Explanation 8. Which of the patient’s hematologic test results are con- sistent with a diagnosis of sickle cell anemia? Hemoglobin electrophoresis is needed to identify a hemoglobinopathy. Explanation His hemoglobin is markedly reduced and typical for sickle Question cell disease. Also, leukocytes and platelets are increased, 2. What is the abnormal hemoglobin causing this patient’s which are common findings. The presence of sickle cells disease? and other findings on the blood smear is consistent with a Explanation diagnosis of sickle cell disease. HbS. Question Question 9. What does the presence of polychromatophilic erythro- cytes signify? 3. Is the patient heterozygous or homozygous for the disorder? Explanation Polychromatophilic erythrocytes are actually reticulocytes. Explanation This indicates that the bone marrow is attempting to com- We can assume that the patient is homozygous because of pensate for the deficit of erythrocytes in the peripheral the very high concentration of HbS present and lack of HbA. blood by releasing these slightly immature cells. Question Question 4. What is this disorder called? 10. Why is the absolute neutrophil count elevated? Explanation Explanation This disorder is referred to as sickle cell disease or sickle cell It is elevated because bacterial infection is associated with anemia. neutrophilia. Appendix G Answers to Case Study Questions 1157 Question Question 11. What is the significance of ovalocytes on the blood 3. Name three disorders that frequently present with smear? the same poikilocyte that dominates in this peripheral Explanation blood smear. These cells are probably irreversibly sickled cells. Explanation Question Thalassemia Hemoglobinopathy 12. What is the significance of Howell-Jolly bodies on the Iron-deficiency anemia smear? Explanation Question The patient’s spleen is not functional and is incapable of 4. List two additional lab tests that would help confirm removing these inclusions from the erythrocytes. the diagnosis and predict the results of each. Question Explanation 13. What is the significance of the patient’s elevated LD? Hb Hb Solubility Disorder Electrophoresis Test Iron Panel Explanation Thalassemia (a) HbH Negative Normal Lactic dehydrogenase is an enzyme found in high concen- Hb Bart’s tration in erythrocytes. Elevated LD levels are associated T

HbA, A2, and F with increased hemolysis of erythrocytes, a typical finding in sickle cell disease. Thalassemia (b) Hb Bart’s Negative Normal T HbA Chapter 13 Case Summary: This patient, previously c HbA2 and HbF diagnosed with a hemoglobinopathy, was admitted to Hemoglobin- HbS or HbC or Positive with Normal the hospital with symptoms of vaso-occlusive crisis. Test- opathy HbE, etc. some (HbS, ing revealed he had pneumonia and sickle cell disease. etc.) The infection was probably responsible for precipitating T HbA the crisis. Iron deficiency Normal Negative T Serum anemia iron CHAPTER 14 T Ferritin Question T, saturation 1. Based on the indices, classify the anemia T BM iron morphologically. c TIBC Explanation It is microcytic and hypochromic: Question 5. Is the hemoglobin electrophoresis normal or abnormal? MCV = 69 fL MCH = 21 pg Explanation MCHC = 29.2 g/dL It is abnormal. The MCV is below the lower limit of normal (80 fL), indi- Question cating the presence of microcytic erythrocytes. The MCHC 6. If abnormal, list hemoglobins that are elevated, is the best indicator of hemoglobin content and is below decreased, or abnormally present. the lower limit of normal (32 g/dL), suggesting hypochro- masia. The below normal MCH (627 pg) corroborates the Explanation decreased erythrocyte hemoglobin content. Elevated Decreased Abnormal Question None HbA Hb Bart’s 2. Name the dominant poikilocyte observed in this HbF HbH peripheral blood smear. HbA2 Explanation Question The dominant poikilocytes are target cells. 7. If abnormal, which globin chains are decreased? 1158 Appendix G Answers to Case Study Questions Explanation HbA2, and HbF, suggest an a@thalassemia. The presence of a@chains and all a@chain9containing hemoglobins (HbA, F, the abnormal hemoglobins, HbH, and Hb Bart’s, rule out b@ and A2) are decreased. and g@thalassemia, respectively. The severity of the symp- toms, the inherited nature of the disease, the early age of Question onset, the microcytic, hypochromic peripheral blood picture 8. If abnormal, which globin chains are produced in with target cells, and the presence of HbH and Hb Bart’s on excess? Hb electrophoresis indicate a severe form of a@thalassemia Explanation called HbH disease. b@chains composing HbH and g@chains composing Hb Bart’s Chapter 14 Case Summary: John had the typical symp- are elevated, ruling out a b@, g@, db@, and gdb@thalassemia. toms of anemia. His CBC revealed a microcytic, hypo- chromic anemia. Tests for iron deficiency were negative. Question Hemoglobin electrophoresis was abnormal with the pres- 9. Is the iron panel normal or abnormal? ence of hemoglobins H and Bart’s. These hemoglobins indi- Explanation cate a deficiency of a@chains. The presence of some HbA, HbA2, and HbF indicate that some a@chains are being pro- It is normal. duced. This suggests the presence of a@thalassemia. The par- Question ents also exhibit symptoms of anemia and should be tested 10. If the iron test results are abnormal, list those outside to determine whether they have a form of a@thalassemia. the normal range and indicate whether they are ele- This will help confirm the child’s diagnosis. vated or decreased. CHAPTER 15 Explanation Question They are normal and so not applicable. 1. What is the morphologic classification of the patient’s Question anemia? 11. If abnormal, state the disorder(s) consistent with the abnormal iron panel. Explanation The patient’s anemia is macrocytic, normochromic. Macro- Explanation cytic is the classification when the MCV 7 100 fL and since An abnormal iron panel usually presents with a pattern the MCHC is 32.7 g/dL (reference interval = 32936 g/dL) consistent with one of the iron metabolism disorders (i.e., the term normochromic is used. iron deficiency, lead poisoning, anemia of chronic disease, sideroblastic anemia). A normal iron panel, as is the case Question here, rules out iron deficiency anemia. 2. Based on the information obtained so far, what is the most likely defect? Question 12. Given all the data supplied, what is the definitive diag- Explanation nosis of John’s anemia? There is a vitamin B12 (cobalamin) deficiency. The patient was admitted with signs of a moderate hemolytic anemia Explanation and neurological symptoms. Her CBC shows she has a A microcytic, hypochromic anemia with target cells sug- macrocytic anemia. Her leukocyte and platelet counts gests a thalassemia, hemoglobinopathy, or iron-deficiency are at the lower end of the normal range, indicating a anemia. The other microcytic, hypochromic anemias involv- developing pancytopenia. The blood smear revealed ing abnormal iron metabolism are also possibilities but usu- hypersegmented neutrophils, ovalocytes, and Howell- ally do not present with significant numbers of target cells. Jolly bodies. Her bilirubin values support a diagnosis The normal iron panel rules out iron deficiency anemia and of hemolysis. On a differential diagnosis, neurological the other disorders of iron metabolism. The negative hemo- symptoms typically accompany cobalamin deficiency globin solubility rules out some hemoglobinopathies. The rather than a folate deficiency. Based on her serum cobal- abnormal hemoglobin electrophoresis confirms the diag- amin and folate results, she can be definitively diagnosed nosis by further ruling out iron deficiency (which shows as having a megaloblastic anemia that is from a cobala- a normal Hb electrophoresis) and hemoglobinopathies by min deficiency. absence of structural hemoglobin variants that have an amino acid substitution (i.e., HbS, HbC, HbE). The decreased Question concentration of all three a@containing hemoglobins, HbA, 3. What is the significance of the AST/ALT results? Appendix G Answers to Case Study Questions 1159 Explanation Explanation The results indicate no liver disease. These are both liver The CBC is an important screening test for all anemias. enzymes. Because they are normal, liver disease is ruled out as a source of jaundice or the macrocytic anemia. Question 2. Justify the selection of laboratory screening tests based Question on Rachael’s clinical signs and symptoms. 4. What further testing can be done to obtain a definitive Explanation diagnosis? It is important to know the patient’s hemoglobin and hema- Explanation tocrit because anemia may be one cause of her weakness Testing for methylmalonic acid (MMA) and homocys- and shortness of breath. A low platelet count could explain teine are helpful to identify a cobalamin deficiency. Both the presence of petechiae and bruises. are increased in cobalamin deficiency. To help determine whether the deficiency results from an absence of intrinsic Question factor, tests for intrinsic factor antibodies, the Schilling test, 3. Evaluate the relationship between Rachael’s age and and/or gastric analysis can be done. A common cause of the likelihood of having aplastic anemia. cobalamin deficiency in a patient with no previous gastro- Explanation intestinal history is pernicious anemia. Because approximately 25% of cases occur in persons younger Question than age 20, it is possible she has aplastic anemia. Overall, 5. What is this patient’s definitive diagnosis? however, the incidence of aplastic anemia is quite low. Explanation Question She has pernicious anemia. The diagnosis of pernicious ane- 4. If aplastic anemia is present, would you expect her to have mia is supported by the finding of intrinsic factor–blocking an idiopathic or secondary form? Explain your answer. antibodies in her blood. Explanation Question It is difficult to estimate whether she has an idiopathic or 6. What would you predict this patient’s reticulocyte secondary form without a more complete history. Idio- count to be? pathic forms are more common in this age group, however. Approximately 50–70% of all cases of aplastic anemia can- Explanation not be linked to a specific cause. The patient’s reticulocyte count would probably be normal to decreased. Megaloblastic anemia results from ineffective Question erythropoiesis with intramedullary hemolysis, which blunts 5. What aspect of this patient’s history may be associated the number of reticulocytes in the marrow storage pool. In with the occurrence of aplastic anemia? addition, polychromasia was not found on the patient’s Explanation blood smear, which is a sign of reticulocytosis. Our knowledge of the previous infection with hepatitis is Chapter 15 Case Summary: This is the case of a 36-year- helpful. Some data suggest an association between aplasia old female with a megaloblastic anemia. An initial diagnosis and infections with viruses. of moderate anemia, jaundice, and neurological compli- cations was made. Based on the patient’s laboratory test Question results, she was diagnosed as having a vitamin B12 (cobala- 6. Is it likely that Rachael has a constitutional form of min) deficiency. The Intrinsic-factor-blocking antibodies aplastic anemia? Explain your answer. demonstrated that the deficiency resulted from the absence of intrinsic factor. This patient can be diagnosed with a Explanation megaloblastic anemia because of a lack of intrinsic factor. It is unlikely that a constitutional form of aplastic anemia The patient is suffering from the adult form of pernicious would be detected at such an advanced age. Fanconi’s ane- anemia with demonstrated anti-intrinsic factor antibodies. mia is first observed in much younger children who typi- cally have other congenital abnormalities. CHAPTER 16 Question Question 7. Correlate Rachael’s clinical findings of weakness and 1. Select laboratory tests appropriate for screening for shortness of breath as well as petechial hemorrhages aplastic anemia. and bruises with her laboratory screening results. 1160 Appendix G Answers to Case Study Questions Explanation Question The patient’s weakness and shortness of breath are related 10. Classify the morphologic type of anemia. to the degree of anemia indicated by her decreased hemo- Explanation globin and hematocrit. Petechiae and bruising are caused by her severe thrombocytopenia. Recurrent fevers suggest The patient’s MCV is 100 fL and MCHC is 29. Depending infection. She is severely neutropenic and, therefore, at high on the laboratory’s reference range for MCV, the RBCs may risk for infection. be normocytic or macrocytic. The anemia is normochromic. This finding is consistent with aplastic anemia. Question Question 8. Evaluate each of Rachael’s laboratory results by com- 11. Calculate the absolute lymphocyte count. Are her lym- paring them to reference intervals. phocytes truly elevated as suggested by the relative Explanation lymphocyte count? All CBC parameters are below reference range for a person Explanation of this age (13 years old). The absolute lymphocyte count is 1.1 * 103/mcL, which is slightly decreased. The relative lymphocyte count (94%) is Question very high because the patient is severely neutropenic. Thus, 9. Which of the patient’s routine laboratory results are examining the relative count without considering the total consistent with those expected for aplastic anemia? leukocyte count can be very misleading. Explanation Question All are consistent with expected results for patients with 12. Correct the reticulocyte count. Why is this step important? aplastic anemia. Explanation The patient’s corrected reticulocyte count is 0.4%, which is below reference interval. Calculation Patient data Reference Interval (conventional) Patient hematocrit 24 0.5–2% Corrected reticulocyte count = * , Reticulocyte * 0.7 = 0.4 , Normal hematocrit 45 All reticulocyte counts need to be corrected when anemia is present in order to assess the bone marrow’s degree of com- pensation for the anemia. Question 13. Calculate the absolute reticulocyte count. Explanation The patient’s absolute reticulocyte count is 10 * 103/mcL. Calculation Patient data Reference Interval (conventional) Absolute reticulocyte (* 103/mcL) = RBC (* 106/mcL) * 2.42 * 0.04 = 0.01 * 106/mcL 25975 * 103/mcL reticulocyte count (,) = 10 * 103/mcL The low absolute reticulocyte count is consistent with a diagnosis of aplastic anemia. Question Explanation 14. Compare these results with those expected for a person If malignant cells were present in the marrow, a diagno- with aplastic anemia. sis of metastatic disease or lymphoma rather than aplastic Explanation anemia would have been likely. Bone marrows of patients with leukemias and myelodysplastic syndromes typically A markedly hypocellular marrow is consistent with a diag- are hyperplastic with increased numbers of hematopoietic nosis of aplastic anemia. The hematopoietic material was blasts present. insufficient to aspirate an adequate sample. Question Question 15. Interpret the significance of the lack of malignant cells 16. Suggest a means of improving the validity of bone mar- and hematopoietic blasts. row examination results for this patient. Appendix G Answers to Case Study Questions 1161 Explanation Question When aplastic anemia is suspected, it might be advisable to 18. Predict a treatment regimen. sample multiple areas of the bone marrow. Explanation Question Treatment would consist of supportive therapy using blood components. A platelet transfusion would probably be 17. Appraise the prognosis for Rachael. ordered immediately. If the aplasia did not resolve, a bone Explanation marrow transplant would be considered. Prognosis is rather poor for patients with aplastic anemia, Question including Rachael, unless a compatible donor can be found 19. What other

hematologic conditions must be ruled out for bone marrow transplant. for this patient? Explanation Other causes of pancytopenia of the peripheral blood include myelodysplastic syndromes (MDS) and megaloblastic anemia. Calculation Patient data Reference Interval (conventional) Hct (,) 29.2 80–100 Fl MCV = * 10 * 10 = 73 fL RBC (*106/mcL) 4 Hb (g/dL) 10.8 28–34 pg MCH = * 10 * 10 = 27 pg RBC (*106/mcL) 4.0 Hb (g/dL) 10.8 32–36 g/Dl MCHC = * 100 * 100 = 37 g/dL Hct (,) 29.2 Question Question 20. What laboratory test is most beneficial in differentiating 2. Based on the calculated indices, describe the patient’s aplastic anemia from these other disorders? Compare red blood cells. the expected results for aplastic anemia with those of Explanation the other disorders. The patient’s erythrocytes are microcytic (MCV 6 80), Explanation hyperchromic. The term hyperchromic is used sparingly. The Serum vitamin B12 and folic acid levels could be used to term signifies that the erythrocytes have too much hemo- rule out anemia from a deficiency of one of these nutrients. globin when in essence that is not true. Instead, these eryth- However, a bone marrow examination is essential to make rocytes have lost part of their membrane, which causes a a diagnosis in this case. If the patient had megaloblastic decrease in the surface-area-to-volume ratio. This changes anemia, megaloblastic changes would be evident in the the erythrocyte from a discocyte to a spherocyte. The only hematopoietic cells. Myelodysplastic changes and increased erythrocyte that will reflect an MCHC of 736 g/dL is the numbers of myeloblasts would support a diagnosis of MDS. spherocyte. Chapter 16 Case Summary: Rachael had symptoms of Question anemia and a bleeding disorder when she first saw her phy- 3. What additional lab tests should be ordered? sician. Her past medical history was significant in that she had recently recovered from viral hepatitis. Her laboratory Explanation results revealed pancytopenia and a low reticulocyte count. This patient has a hemolytic type of anemia with eryth- Bone marrow examination showed hypoplasia. These find- rocytes that are microcytic. Erythrocyte morphology ings suggest acquired aplastic anemia associated with past on the peripheral smear revealed elliptocytes, sphe- viral infection. rocytes, and fragmented erythrocytes. It is important to know whether this problem is the result of a stimu- CHAPTER 17 lated immune system (antibodies present). An AHG test Question should be ordered to determine whether this is true. If 1. Calculate the erythrocyte indices. the AHG is negative, the patient could have an inher- ited erythrocyte membrane defect. Lab tests that could Explanation be used to differentiate the erythrocyte membrane dis- The indices are Hct 24%, MCH 31.2 pg, and MCHC 34.2 orders include the osmotic fragility test and the thermal g/dL. sensitivity test. 1162 Appendix G Answers to Case Study Questions Question Explanation 4. Interpret the results of the osmotic fragility test. The initial test for G6PD was normal for two reasons. First, Explanation the older, more deficient erythrocytes were selectively destroyed during the hemolytic crisis, leaving younger The patient’s erythrocytes lysed at a higher NaCl concentra- cells with more normally functioning enzyme. Second, the tion than the control cells. This signifies that the patient’s patient had been transfused, and therefore any blood sam- erythrocytes cannot take on as much water as normal cells, ple would not be representative of his own cells but would and, thus, they are said to have increased osmotic fragility. contain donor erythrocytes with normal amounts of G6PD. Question Question 5. What do the results of the thermal sensitivity test reveal 3. What was the precipitating cause of the patient’s about the patient’s erythrocytes? anemia? Explanation Explanation The results of the thermal sensitivity test reveal that the The patient developed anemia because of his treatment with patient’s erythrocytes are abnormally heat sensitive. Nor- the oxidant drug primaquine. Oxidant damage to hemoglo- mal erythrocytes will not fragment until heated to 49–50 °C. bin from lack of reducing power supply by G6PD in the hex- Question ose monophosphate shunt leads to erythrocyte destruction. 6. What disorder is suggested by the patient’s lab Chapter 18 Case Summary: A 25-year-old African Ameri- findings? can male experienced fever, chills, and general malaise 3 days Explanation after receiving prophylactic primaquine. Laboratory testing revealed that he was anemic and his bilirubin was increased. The patient has a hemolytic anemia that is not a result of In addition, his haptoglobin was decreased. Review of the a stimulated immune system. The erythrocyte morphol- blood smear revealed bite cells and spherocytes. A Heinz ogy reveals microcytosis, spherocytes, elliptocytes, tear- body stain was positive. Based on these results, a hemolytic drop cells, and micropoikilocytes. The osmotic fragility is anemia was suspected. He received two units of packed red increased, and the thermal sensitivity test shows that the blood cells. A test for G6PD was performed and results were erythrocytes are heat sensitive. These findings suggest that borderline low. Follow-up testing revealed a low G6PD. the patient has hereditary pyropoikilocytosis. Chapter 17 Case Summary: Jack is a 12-year-old with a CHAPTER 19 lifelong history of hemolytic crises, suggesting a hereditary Question condition. Laboratory results reveal a microcytic, hyper- 1. What are some reasons that this person may have a low chromic anemia. The blood smear shows a variety of poi- hemoglobin value? kilocytes that suggest hemolysis. The osmotic fragility test is increased. The test that is most helpful in this case is ther- Explanation mal sensitivity. The cells are heat sensitive, which with the Low hemoglobin could be the result of iron deficiency, other laboratory results and clinical history suggests heredi- occult bleeding, or extravascular hemolysis. tary pyropoikilocytosis. Question CHAPTER 18 2. What is the significance of the spherocytes? Question Explanation 1. What test should be considered after finding bite cells They indicate damage to the cell membrane and extravas- on a blood smear? cular hemolysis. Explanation Question The appearance of bite cells on a blood smear suggests 3. Based on these results, what do you suspect is going on physical damage to the RBCs as the spleen attempts in Nancy’s blood? Explain. removal of Heinz bodies. Therefore, a Heinz body stain Explanation can be employed to reveal their presence as a cause of the Some type of immune-mediated hemolysis is occurring anemia. from the presence of IgG on the erythrocyte. Question Question 2. Why was the initial G6PD test result normal and the 4. What type of antibody appears to be present in Nancy? repeat test abnormal? Explain. Appendix G Answers to Case Study Questions 1163 Explanation with SLE often develop not only antibodies against nuclear It is an autoantibody. The patient’s serum is reacting with components of all cells in the body but also to erythrocytes, her own cells, which indicates that the antigen is present as demonstrated in this patient. Treatment would begin on her own cells. initially with steroids, and if the patient were nonrespon- sive, other modalities would be considered. Transfusion is Question discouraged unless the hemoglobin value drops to a very 5. What is the relationship of the patient’s primary dis- low level. ease, systemic lupus erythematosus, and her anemia? CHAPTER 20 Explanation SLE is an autoimmune disease in which the patient often Question develops a WAIHA secondary to the disease. 1. What are some conditions that result in the presence of schistocytes? Question Explanation 6. How would knowing that the patient had not been transfused in the last several months help you decide Some conditions are disseminated intravascular coagula- on the underlying cause of the antibody? tion, hemolytic uremic syndrome, thrombotic thrombocy- topenic purpura, mechanical trauma from artificial heart Explanation valves, malignant hypertension, and burns (thermal injury). If the patient had been transfused, the foreign antigens on Question the transfused cells could have stimulated an alloantibody. However, the alloantibody would not be reacting with the 2. What is the significance of these results? patient’s own cells. Explanation Question The platelet count indicates a thrombocytopenia, which correlates with the patient’s increased tendency to bruis- 7. What would you tell the clinician about giving a ing. Her reticulocyte count was elevated and indicates a transfusion? response by the bone marrow. Explanation Question Transfusions are not indicated in WAIHA because the trans- 3. Why might the clinician order coagulation tests? fused cells contain an antigen that will react with the anti- body and be destroyed just as the patient’s own cells are Explanation destroyed. The presence of unexplained bruises may indicate an abnor- mality in the hemostatic mechanism. This can be screened Question for by coagulation tests. 8. What kind of therapy might be used? Question Explanation 4. What do these findings indicate about the underlying The most common therapy is the use of corticosteroids, problem? which depress the immune reaction. If the patient does not respond to this drug, other cytotoxic drugs or splenectomy Explanation can be indicated. The PT and APTT showed slightly prolonged levels, and the Chapter 19 Case Summary: This patient’s data dem- fibrinogen was only slightly decreased. This rules out DIC onstrate reactions seen in warm autoimmune hemolytic because DIC would have greatly prolonged PT and APTT anemia secondary to systemic lupus erythematosus. Sphe- results, decreased fibrinogen, and increased FDP. rocytes indicate extravascular hemolysis, and reticulocy- Question tosis suggests compensation by increased bone marrow 5. Based on these results, what is the most likely condition production of erythrocytes. The positive DAT (anti-IgG) is associated with these clinical and laboratory results? a clue that the cell destruction is immune mediated. Her Explain. serum reacts with all antibody screening cells and panel cells after addition of antihuman globulin. The diagnosis Explanation of systemic lupus erythematosus (SLE)—an autoimmune It is most likely TTP. The patient’s age, normal urinary vol- disease characteristically diagnosed in women between the ume, and lack of diarrheal prodrome help rule out HUS. ages of 24 and 40—provides additional support for the sec- The neurological symptoms in concert with the other labo- ondary nature of the immune-mediated hemolysis. Patients ratory values point to TTP. 1164 Appendix G Answers to Case Study Questions Question Explanation 6. What therapy might be used? Pelger-Huët anomaly is benign, and the neutrophils appear Explanation to function normally. The greatest significance of this disor- der is that it be recognized and cells not mistakenly identi- Plasma exchange with either fresh frozen plasma (FFP) or fied as bands, leading to an incorrect diagnosis of infection. with cryo-poor FFP can be used. In this case, the patient had additional tests (the cultures) Chapter 20 Case Summary: This case demonstrates the performed that probably were not necessary had the condi- sequence of testing that might be used to determine the tion been diagnosed previously. cause of microangiopathic hemolytic anemia. The presence Question of schistocytes may be seen in a number of conditions, but additional laboratory tests such as platelet count and coagu- 5. Why is the white count elevated? lation studies will help in identifying the underlying cause. Explanation In addition, medical history such as age, diarrheal episodes, pregnancy status, or other physical conditions (acquired or Pelger-Huët does not typically present with leukocytosis inherited) will help in differential diagnosis and in deciding unless it is accompanied by another condition. The white a sequence of additional testing. In this case, age and lack count is probably increased because of tissue damage from of diarrheal prodrome help rule out HUS. The low platelet the accident trauma and/or the surgery. Also, although the count but normal coagulation tests help rule out DIC. The patient’s hemoglobin is only low normal, it is likely that a most likely condition is TTP. The lack of an identifiable pre- young, previously healthy man had a higher value before cipitating condition and the adult age onset indicate that the accident. He could have lost blood from the accident this is most likely of the single episode, nonrecurring type. and/or during the surgery. Acute blood loss and surgery are associated with leukocytosis. CHAPTER 21 Chapter 21 Case Summary: This trauma patient had sur- Question gery to repair bone fractures. After surgery, his WBC count 1. What results, if any, are abnormal? was elevated with a predominance of bands. The platelet count and hemoglobin were also below the reference range Explanation for his age and sex. Although a bacterial infection was sus- The white blood count and number of bands

are elevated. pected based on the CBC, cultures were negative. Despite the high WBC count and shift to the left, there were no toxic Question changes of the leukocytes. After further review, it was deter- 2. What is the most likely reason for these results? mined that the patient had Pelger-Huët anomaly. The bands Explanation were actually mature neutrophils whose nucleus failed to segment. The most likely reason for an elevated white count and left shift on a trauma patient who has had emergency surgery is CHAPTER 22 a bacterial infection acquired either from the original trauma or during surgery. Leukocytosis without toxic changes pres- Question ent or a left shift can occur in postsurgical patients. 1. Does this patient have a leukocytosis, leukopenia, or neither? Explain. Question 3. Given the leukocyte morphology and cultures, what Explanation additional condition must now be considered? No, she does not. The WBC count is within the reference Explanation interval. Because the cultures are all negative and there are no other Question indications of a reactive process, such as toxic granulation, 2. Does this patient have an abnormal lymphocyte count? Döhle bodies, or vacuoles, it is unlikely the patient has an Explain. infection. Pelger-Huët anomaly is a benign inherited con- dition in which the neutrophils have hyposegmentation. Explanation The nuclei are shaped like dumbbells or wire-rimmed eye- The absolute lymphocyte count is 7.6 * 103/mcL * 0.04 = glasses and have very condensed chromatin. 0.30 * 103/mcL. This is abnormally low because the refer- ence interval for this age is 2.5916.5 * 103/mcL. Question 4. Explain the clinical significance of the nuclear anomaly Question described in Dennis. 3. What is the absolute lymphocyte count? Appendix G Answers to Case Study Questions 1165 Explanation investigation suggested an immune deficiency disorder At 54 days after birth, the absolute lymphocyte count is only and led to additional laboratory tests. These tests revealed 12.3 * 103/mcL * 0.01 = 0.12 lymphocytes * 103/mcL, a high levels of erythrocyte adenosine and deoxyadenosine, condition of lymphocytopenia. indicating a deficiency in adenosine deaminase. This defi- ciency occurs from a gene deletion resulting in an autoso- Question mal inherited severe combined immunodeficiency. Normal 4. What possible causes exist for these opportunistic immune function is restored when (1) toxic metabolites are infections? cleared, (2) a normal adenosine deaminase gene is inserted into the patient’s lymphocytes, or (3) the patient has a bone Explanation marrow transplant. Two causes of immunodeficiency are suspected with oppor- tunistic infection and severe lymphocytopenia: (1) AIDS and (2) severe combined immunodeficiency syndrome. CHAPTER 23 Question Question 5. Is this child more likely to have a congenital or acquired 1. Given the patient’s laboratory results, would this most immune deficiency? likely be considered an acute or chronic leukemia? Explain. Explanation Explanation A congenital immune deficiency is more probable because the child has had recurrent infections and lymphocytopenia It is likely chronic leukemia. The peripheral blood shows an since birth. accumulation of mature lymphocytic cells with an increased white blood cell count. Question Question 6. If she has a congenital immune deficiency, is it more likely she has X-linked or autosomal SCIDS? 2. What group of leukemia (cell lineage) is suggested by the patient’s blood cell differential results? Explanation Explanation Autosomal SCIDS is more likely. Females who carry the abnormal X-linked SCIDS gene have normal immunity. Lymphocytic leukemia is suggested. The patient exhibits Only normal X chromosomes are found in the lymphocytes an increased percentage of mature lymphocytes with an of females who carry the abnormal X-linked SCIDS gene. increased white blood cell count and a lack of myelocytic cells. Question Question 7. Are the lymphocytes more likely to be morphologically heterogeneous or homogeneous? Why? 3. What would you expect the blast count on the bone marrow to be? Explanation Explanation They are more likely homogeneous. The congenital immune deficiency disorders are characterized by normal appearing It would be 620, under the WHO definition. In acute leu- lymphocytes, but the lymphocytes are either defective or kemias, blasts constitute more than 20% of the nonerythroid decreased in concentration or both. Heterogeneous lympho- marrow nucleated cells; in MDS, MPN, and chronic leuke- cytes are normally reactive lymphocytes that are stimulated mia, blasts compose less than 20% of the marrow cells. by infectious agents. Question Question 4. Would you expect Agnes to survive more than 3 years 8. What confirmatory test is indicated? or succumb fairly quickly after treatment? Explanation Explanation A test to determine a decreased adenosine deaminase level She should survive more than 3 years. Chronic leukemias or increased purine metabolites of deoxyadenosine tri- progress slowly, and the course of the disease is measured phosphate and deoxyguanosine triphosphate confirms an in years rather than months as is typical for the acute enzyme deficiency. leukemias. Chapter 22 Case Summary: This young infant has had Question recurrent infections since birth. Her WBC was normal, 5. Is Agnes a suitable candidate for a bone marrow trans- but there was an absolute lymphocytopenia. Further plant? Why or why not? 1166 Appendix G Answers to Case Study Questions Explanation Agnes is not a candidate for bone marrow transplant. Per- She is not. The highest rate of success with bone marrow manent remission in CLL is rare. Treatment is conservative transplants has occurred in those younger than 40 years and usually reserved for patients with more aggressive of age in a first remission with a closely matched donor. forms of the disease. Because this patient is 72 years old and CLL patients usu- Chapter 23 Case Summary: This patient has chronic lym- ally do well with supportive therapy, it is unlikely that she phocytic leukemia. The patient is elderly and had been in would be a candidate for a bone marrow transplant. good health, and the symptoms of the leukemia had pro- Question gressed slowly. The predominant cell present on the periph- 6. What types of treatment are available for our patient eral blood smear is a mature lymphocyte. The patient’s Agnes? prognosis should be good, with a life span of 5 years or more with supportive therapy. Explanation Chemotherapy, radiation, bone marrow transplant, immuno- CHAPTER 24 therapy, epigenetic therapy, molecular therapies that target the genetic mutation, and stem cell transplant are avail- Question able for treatment of leukemia. As stated in Checkpoint 5, 1. What are Roger’s MCV and MCHC? Explanation The MCV is 97 fL and the MCHC is 33 g/dL Calculation Patient data Reference Interval (conventional) Hct (,) 35 80–100 fL MCV = * 10 * 10 = 97 fL RBC (*106/mcL) 3.6 Hb (g/dL) 11.6 32–36 g/dL MCHC = * 100 * 100 = 33 g/dL Hct (,) 35 Question proliferation and reticulin fibrosis in the bone marrow. Dry 2. How would you classify his anemia morphologically? taps can also result from improper placement of the needle into the marrow cavity, a marrow that is hypocellular, or Explanation one that is packed tightly with neoplastic cells. The patient has a normochromic, normocytic anemia. Question Question 6. What characteristic peripheral blood morphologies cor- 3. Based on Roger’s history and current laboratory data, relate with the bone marrow picture and physical exam? what other tests should be performed? Explanation Explanation You would expect to see dacryocytes and should look for Because of the hepatosplenomegaly and laboratory results, micromegakaryocytes in the peripheral blood. a bone marrow and Philadelphia chromosome analysis should be considered. BCR/ABL1 gene rearrangement is Question also necessary. 7. What is he most likely explanation for the increased Question splenomegaly? 4. What diagnoses do these results suggest? Explanation Explanation The progressive bone marrow fibrosis causes anemia, All MPNs (CML, essential thrombocytosis, polycythemia resulting in extramedullary hematopoiesis in the spleen and vera, or PMF) can produce a leukocytosis and fibrotic bone liver. This increases splenic blood flow, red cell pooling, and marrow. enlargement of the spleen. Question Question 5. Give a reason for the unsuccessful, dry-tap, bone mar- 8. What are possible outcomes of this disorder? row aspiration. Explanation Explanation PMF can slow progressive fibrosis or evolve into polycy- Increased fibrosis in the bone marrow makes aspira- themia vera, essential thrombocythemia, CML, or acute tion difficult. Excess PDGF and TGF-b stimulate collagen leukemia. Appendix G Answers to Case Study Questions 1167 Chapter 24 Case Summary: The clinical findings of Explanation abdominal discomfort, increasing splenomegaly, weak- The MCV is 106 fL. ness, and bone pain are consistent with chronic myelopro- The peripheral blood findings help to rule out megaloblas- liferative neoplasms. The laboratory findings of increased tic anemia: hyposegmentation of neutrophils rather than uric acid, left shift, increased platelet count, difficult bone hypersegmentation, WBC count slightly elevated rather than marrow aspiration, dacryocytes on peripheral blood smear, decreased as is typical in megaloblastic anemia, and left shift and cytogenetic abnormalities are all characteristic of pri- with blasts that is not characteristic of megaloblastic anemia. mary myelofibrosis (PMF). Malignant stem cells give rise to abnormal leukocytes, erythrocytes, and megakaryocytes. Reference Interval The megakaryocytes secrete excessive PDGFs that stimu- Calculation Patient data (conventional) late fibroblasts to lay down excessive reticulin and colla- Hct (,) 17 80–100 fL gen in the bone marrow cavity. This fibrosis leads to the MCV = * 10 * 10 = 97 fL RBC (*106/mcL) 1.6 extramedullary hematopoiesis causing splenomegaly and hepatomegaly. Question In this case, the peripheral blood film with a leukoeryth- 5. What features of the differential resemble chronic roblastic picture of immature myeloid cells, nucleated red myelocytic leukemia (CML)? What helps to distinguish blood cells, and dacryocytes should rule out causes of mar- this case from CML? row infiltration by nonhematopoietic tumor cells, granulo- mas, or fungus. Explanation The diagnosis of this case is PMF. Myelofibrosis can Increased eosinophils and all stages of granulocytes in the evolve into any of the chronic myeloproliferative neo- peripheral blood are found in both MDS and CML. How- plasms or acute leukemia. A moderate leukocytosis (WBC ever, the WBC count is too low for typical CML; the RBC 30.0 * 109/L) leukoerythroblastosis, dry tap, and lack of and platelet counts are not usually decreased to this extent the Philadelphia chromosome distinguish PMF from the in CML. other myeloproliferative neoplasms. Question 6. Which of the hematopoietic lineages exhibit dyshema- CHAPTER 25 topoiesis in the bone marrow? Question Explanation 1. In what cell lineages is cytopenia present? All three cell lineages (myeloid, erythroid, and megakaryo- Explanation cytic) exhibit dyshematopoiesis in the bone marrow. Cytopenia is present in the erythrocytic and megakaryo- Question cytic lineages (RBCs and platelets are decreased). 7. How would you classify the bone marrow cellularity? Question Explanation 2. What abnormalities are present in the differential? The bone marrow shows increased cellularity for a person Explanation 65 years old; normal cellularity is approximately 35%. The abnormalities present in the differential are a left Question shift, including metamyelocytes, myelocytes, promyelo- 8. What does the M:E ratio indicate? cytes, and a few blasts as well as a marked increase in eosinophils. Explanation The M:E ratio indicates myeloid hyperplasia; a normal M:E Question ratio is 3:1 or 4:1. 3. What evidence of dyspoiesis is seen in the leukocyte morphology? Question 9. Identify at least two features of the bone marrow that Explanation are compatible with a diagnosis of MDS. Hyposegmentation and hypogranulation in the leukocyte morphology are evidence of dyspoiesis. Explanation Features in the bone marrow compatible with a diagnosis Question of MDS are increased blasts in the bone marrow but 620,; 4. Calculate the MCV. What peripheral blood findings are dyserythropoiesis, dysleukopoiesis, dysmegakaryopoiesis, helpful to rule out megaloblastic anemia? and hypercellularity. 1168 Appendix G Answers to Case Study Questions Question Question 10. What chemistry tests would be helpful to rule out meg- 2. Based on the presenting data, what additional testing aloblastic anemia? might be of value? Explanation Explanation Testing for cobalamin and folate levels would be helpful to A bone marrow should be performed. Other tests could rule out megaloblastic anemia; values should be normal to include cytogenetics and molecular testing for the increased, not decreased as would be seen in megaloblastic PML/RAR@a translocation. anemia. Early megaloblastic changes might be detected by Question testing for methylmalonic acid (MMA) and homocysteine 3. Based on the peripheral blood examination, what cyto- levels. These components are intermediaries in folate and chemical stain results would you expect to find on Jona- cobalamin metabolism and are elevated early in functional than’s

neoplastic cells? vitamin deficiencies. Explanation Question Myeloperoxidase, Sudan black B, and specific esterase 11. What is the most likely MDS subgroup, and on what should be positive because these cells are myeloid in origin. criteria is the answer based? Question Explanation 4. If the cells from Jonathan’s bone marrow were immuno- The most likely MDS subgroup is MDS-EB-1 based upon phenotyped, which of these—CD13, CD33, CD20, CD2, the following criteria: 65, blasts in the peripheral blood, CD7, CD10, CD19—would you expect to be positive? 5–9% blasts in the bone marrow, cytopenia in two cell lin- Explanation eages, and qualitative abnormalities in all lineages. Because these are promyelocytes, the cells should be posi- Question tive for CD13 and CD33. The CD34 is generally negative in 12. Using the International Prognostic Scoring System APL. CD2, CD7, CD10, and CD19 are lymphocyte markers (IPSS), what is the prognosis for this patient? and should be negative. Question Explanation 5. Based on the morphology and cytochemical staining of Use of the IPSS-R on this patient is that platelets 650 = 1.0; these cells, what is the most likely WHO classification? Hemoglobin 68 = 1.5; 9% blasts in bone = 2.0; com- plex karyotype with multiple abnormalities = 4.0. Explanation Total = 76 = Very High risk. It most likely belongs to the group acute myeloid leuke- mias with recurrent genetic abnormalities and the subgroup Chapter 25 Case Summary: Hancock had bicytopenia acute promyelocytic leukemia. (anemia and thrombocytopenia) with a left shift and dys- plastic erythrocyte and granulocyte features. The bone Question marrow was hypercellular with an increased M:E ratio. 6. What are at least two recurrent genetic mutations seen There was trilineage dysplasia. Based on the WHO criteria in this type of presentation? for subgrouping MDS, he most probably has MDS-EB-1. The neutrophilic cells show marked hyposegmentation Explanation and hypogranulation. RBC morphology included aniso- Recurrent genetic mutations include, PML@RARa, cytosis and poikilocytosis, teardrop cells, ovalocytes, and NuMA@RARa, PLZF@RARa, NPM1@RARa, PLZF@RARa, schistocytes. STAT5b@RARa. Question CHAPTER 26 7. What chromosome abnormality is associated with this Question leukemia? 1. What clues do you have that this patient may have an Explanation acute leukemia? The t(15;17) karyotype is diagnostic of acute promyelocytic Explanation leukemia. At the molecular level, the PML/RAR@a is found. Anemia, increased WBC, decreased platelets, and, most sig- Question nificantly, blasts and promyelocytes on the peripheral blood 8. What is the major complication associated with this smear indicate this. leukemia? Appendix G Answers to Case Study Questions 1169 Explanation Explanation Disseminated intravascular coagulation (DIC) is common CALLA-positive ALL is the most common ALL found in because of the release of large numbers of granules from the children in the western world, and it has the most favorable promyelocytes, which have procoagulant activity. outcome of the immunologic subtypes. Question Chapter 27 Case Summary: Dan had nonspecific but typi- 9. If this patient were treated with all-transretinoic acid cal symptoms and clinical findings of an acute lymphocytic and 2 weeks later the blood count was repeated, what leukemia. His CBC revealed anemia, thrombocytopenia, would you expect to find? and leukocytosis. The differential showed that the majority of peripheral blood leukocytes were lymphoblasts. A bone Explanation marrow, immunophenotyping, and cytogenetics should be The peripheral blood count would be increased because performed for treatment and prognostic information. the promyelocytes mature into granulocytes with this therapy. CHAPTER 28 Chapter 26 Case Summary: The patient experienced Question bleeding symptoms (easy bruising, gingival bleeding, and petechiae) suggesting a thrombocytopenia and the CBC 1. What is the differential diagnosis? revealed a leukocytosis. The differential was abnormal with Explanation blasts present. A bone marrow was performed and tested in The differential diagnosis for a lymphocytosis composed the laboratory and revealed the t(15;17) karyotype and the of mature cells includes all chronic leukemia lymphoprolif- PML/RAR@a fusion gene; this translocation is diagnostic of erative disorders discussed: chronic lymphocytic leukemia, acute promyelocytic leukemia. prolymphocytic leukemia, hairy cell leukemia, large granu- lar lymphocyte leukemia, Sézary syndrome, and circulating CHAPTER 27 lymphoma. Both the nuclear irregularities and history of Question lymphadenopathy suggest circulating lymphoma. 1. Based upon these data, what would be the initial inter- Question pretation of Dan’s presentation? 2. What studies could be performed to establish the Explanation diagnosis? These results are characteristic of acute lymphoblastic Explanation leukemia. Flow cytometry immunophenotyping performed on Question the peripheral blood could help to establish a diagnosis. Chronic lymphocytic leukemia, hairy cell leukemia, large 2. What tests should be used as the initial follow up in this granular lymphocyte leukemia, and Sézary syndrome have case? a characteristic immunophenotype. Circulating lymphoma Explanation can either have a generic, monoclonal B cell phenotype or A bone marrow test should be performed and the periph- express antigens specific for the subtype of lymphoma, eral blood specimen should be sent for immunophenotyp- such as CD10 expression in follicular lymphoma. The case ing to determine the T- or B-cell lineage of the blasts. described was CD10 positive. This information and the appearance of the lymphocytes suggest the presence of Question non-Hodgkin lymphoma, follicular type. Biopsy of a lymph 3. If the flow cytometry pattern showed a positive CD10, node would be of value in confirming this interpretation. what would the classification of this acute leukemia be? Question Explanation 3. What is the cause of the lymphadenopathy? Is this pro- The CD10 antigen is found on a pre-B acute leukemia and is cess low grade or high grade? called common acute lymphocytic leukemia antigen, or CALLA. This identifies a subgroup of ALL known as common ALL Explanation or CALLA-positive ALL. The lymphadenopathy results from the presence of non- Hodgkin lymphoma. Neoplastic lymphoid cells have Question replaced the lymph node’s normal cells. The lack of tan- 4. In this situation, would the therapeutic outcome be con- gible body macrophages and mitotic figures distinguishes sidered favorable or bleak? Why? the neoplastic nodules from benign germinal centers. 1170 Appendix G Answers to Case Study Questions Question blasts, he is a candidate for a stem cell transplant (if the 4. Is this process low grade or high grade? donor is immediately available). The patient has AML, so an allogeneic SCT is an appropriate option. Explanation This non-Hodgkin lymphoma has characteristics of a low- Question grade lymphoma: nodular growth, small cells, lack of apop- 3. What testing should Brandon undergo to proceed with tosis (tangible body macrophages), and absence of mitotic the transplant? figures. The diagnosis of follicular lymphoma, grade 1 was Explanation established. HLA typing should be performed on the patient, and the Question search for HLA-matched siblings should be done. 5. What is the diagnosis? Question Explanation 4. An HLA-matched sibling has agreed to be a donor for The diagnosis is diffuse large B-cell lymphoma. Brandon. Should the source of stem cells be peripheral blood or bone marrow? Why? Question Explanation 6. What is the relationship of this disease to the previous diagnosis? To harvest bone marrow stem cells, the donor has to undergo general anesthesia while stem cells from periph- Explanation eral blood can be collected by apheresis. In addition, studies This disease probably represents transformation from the have shown that the PBSCs engraft earlier than the bone previously diagnosed chronic lymphocytic leukemia. marrow stem cells and have less degree of GVHD. Given Chapter 28 Case Summary: Julia, a 56-year-old female, these facts, peripheral blood should be the source of stem had painless lymphadenopathy. Her laboratory results cells. revealed a leukocytosis with a relative and absolute lym- Question phocytosis. The lymphocytes were mature with clumped 5. PBSCs were collected by apheresis. Enumeration of chromatin and irregular nuclear outline. The lymphocytes CD34 count indicates that the total CD34 positive cells were CD10 positive, characteristic of circulating lymphoma collected are 6 * 106/kg. Is this an adequate dose for cells. A biopsy of the lymph node showed infiltration with SCT? neoplastic lymphocytes. The diagnosis of low-grade, non- Hodgkin lymphoma, follicular type was made. She received Explanation chemotherapy. Two years later, she returned with expand- Yes, for allogeneic SCT, the total CD34+ cells should be ing lymph nodes in her neck. A biopsy was performed, and more than 2 * 106/kg. a diagnosis of diffuse large cell lymphoma was made. The second lymphoma was probably a transformation of the Question previous low-grade lymphoma. 6. Stem cells were collected and frozen for Brandon. Does he need to undergo any form of therapy before the CHAPTER 29 transplant? Question Explanation 1. Is Brandon a candidate for SCT? Yes, the patient should receive myeloablative chemotherapy and/or irradiation. Explanation Yes, these findings indicate that he could have a relapse of Question leukemia. 7. Brandon received stem cells from his HLA-matched sibling that were successfully engrafted. Three months Question later, he developed diarrhea, skin rash, and jaundice. 2. If yes, what form of transplant is required for him? What could be the possible cause for this? Explanation Explanation Circulating blasts in the peripheral blood are a poor prog- Because the patient received allogeneic SCT, the presence nostic sign and indicate that this patient is probably having of diarrhea, skin rash, and jaundice most likely represents a relapse of his leukemia. A bone marrow examination is GVHD. However, opportunistic infections and drug reac- definitely indicated. If this patient has residual leukemic tions should also be considered. Appendix G Answers to Case Study Questions 1171 Chapter 29 Case Summary: Brandon, a 35-year-old male Question (weight 80 kg) was recently diagnosed with AML with 6. What is the most appropriate interpretation of these maturation and received induction chemotherapy. Day findings? 21 after chemotherapy, the bone marrow reveals no evi- dence of residual leukemia. Two weeks later, circulating Explanation blasts were seen in the peripheral blood. He was evalu- Most of the infection is resolved and the tissue cells show ated for a stem cell transplant. He was given a PBSCT changes suggestive of malignancy, most likely an adenocar- from a sibling and developed what appeared to be GVHD cinoma. This needs to be verified by cytology. after 3 months. Question 7. What is the most appropriate interpretation of these CHAPTER 30 cells? Question Explanation 1. Is this a transudate or exudate? These cells initially appear as mesothelial; however, the CSF Explanation does not have mesothelial cells, and the patient is an adult This is an exudate because the protein fluid/serum = 0.75, with no history of recent brain surgery or CSF shunt or res- the fluid LD/serum LD = 0.80, and the WBC is 71000/mcL. ervoir. Therefore, this most likely represents a metastatic carcinoma. This should be verified by cytology. Question Chapter 30 Case Summary: Radiologic studies showed 2. Is this a chylous fluid? a large effusion in the right pleural cavity. A thoracentesis Explanation was performed, and 1 L of thick, yellow fluid was aspi- No, this is not chylous because the predominant cell is not rated. Laboratory studies showed a total protein of 4.5 g/dL a lymphocyte. Because of its appearance, it could be consid- (serum = 6 g/dL), lactate dehydrogenase 40 U/L ered pseudochylous. (serum = 50 U/L), and total leukocyte count of 20,000/ mcL with 90% segmented neutrophils, 10% histiocytes, Question and many degenerating cells. The laboratory analysis sug- 3. What would be an appropriate next step to determine gests a metastatic carcinoma. This should be verified by whether the material seen is debris or true organisms? cytology. Explanation CHAPTER 31 The quickest and easiest procedure is to make another slide and perform a Gram stain. If there is not a sufficient amount Question of fluid remaining, a Gram stain can be performed on the 1. Does Michael have a defect in primary or secondary original unstained slide. The material seen is degenerating hemostasis? neutrophils. Explanation Question He has a defect in primary hemostasis as a result of 4. Some of the large tissue cells showed features of degen- thrombocytopenia. eration and suspicious for malignancy. How should Question this be interpreted? 2. If Michael were given TPO, how would you expect his Explanation bone marrow and peripheral blood picture to change? In fluids that have findings of severe infection, the reactive Explanation changes of the mesothelial cells can be striking, and caution should be taken not to overinterpret them. Also, degenera- Because TPO influences megakaryocytes to proliferate, you tive changes can simulate changes of malignancy with what would expect to see an increase in megakaryocytes in the

appears to be an irregular nuclear membrane. bone marrow and an increase in platelets in the peripheral blood. Question Question 5. Is this an exudate or transudate? 3. The physician explained to Michael that the reddish- Explanation purple spots on his legs and ankles were tiny pinpoint This is an exudate because the protein fluid/serum = 0.78, hemorrhages into the skin. Explain the relationship of the fluid LD/serum LD = 0.83, and the WBC is 71000/mcL. these hemorrhages to the platelet count. 1172 Appendix G Answers to Case Study Questions Explanation Explanation The platelet count is severely decreased. Platelets play an This patient’s bleeding history began when he was just 18 essential role in maintaining vascular integrity. With throm- months old and has occurred regularly since then. Nose- bocytopenia the intercellular contacts between vascular bleeds begin spontaneously (in the absence of trauma), and endothelial cells disassemble, allowing blood to leak from the patient has a history of easy bruisability. His brother also small vessels. had a bleeding diathesis and died of intracranial hemor- rhage at age 10. Question 4. What is the most likely cause of Michael’s pancytopenia? Question Explanation 4. Are the nosebleeds significant in considering a diagnosis? Acute leukemia is associated with thrombocytopenia and anemia. The WBC could be normal or increased. Explanation Treatment for leukemia includes chemotherapy, which Nosebleeds in the absence of trauma are significant; destroys not only leukemic cells but also normal hema- although they often indicate a platelet defect, platelet topoietic cells. screening tests in this patient were normal. Question Question 5. Why would the administration of growth factors such 5. What coagulation factors are included in the extrinsic as EPO and TPO be considered in this case? pathway? Explanation Explanation EPO and TPO stimulate the proliferation of erythroblasts FVII and tissue factor are included. and megakaryoblasts, respectively, in the bone marrow. Question This increases the number of red blood cells and platelets in the peripheral blood. 6. What factors are included in the intrinsic pathway? Chapter 31 Case Summary: Michael was diagnosed with Explanation ALL and received chemotherapy as a part of his treatment FXII, FXI, prekallikrein, high-molecular-weight kininogen, regimen. His CBC showed pancytopenia after several treat- FIX, and FVIII are included. ments. He had pinpoint hemorrhages on his legs and ankles Question as a result of the severe thrombocytopenia. 7. What factors are included in the common pathway? CHAPTER 32 Explanation Question FX, FV, prothrombin, and fibrinogen are included. 1. What do the results of the screening tests (platelet Question count, bleeding time, prothrombin time [PT], and acti- 8. An abnormal PT and a normal APTT would indicate a vated partial thromboplastin time [APTT]) indicate? problem with which factor? Explanation Explanation Screening tests indicate that the platelet compartment (pri- There is a problem with FVII. mary hemostasis) is likely normal. The problem probably is a defect of the plasma proteins (secondary hemostasis). Question Question 9. Does any evidence exist to indicate a problem with Shawn’s fibrinolytic system? 2. What component of the hemostatic mechanism is most likely affected? Explanation Explanation No evidence is present. A normal APTT and a prolonged PT indicate a defect of the Question extrinsic pathway. 10. Why were liver function tests done on Shawn? Question Explanation 3. What evidence indicates that Shawn has a hereditary They were done to rule out an acquired deficiency of coagu- bleeding disorder? lation proteins. Appendix G Answers to Case Study Questions 1173 Question Explanation 11. What is the significance of normal results in a patient The patient most likely has a disorder of primary hemo- with hemostatic disease? stasis. The presence of petechiae and excess bleeding from Explanation superficial cuts are seen in primary hemostatic disorders and are unlikely in disorders of secondary hemostasis. Deficiency of one or more coagulation factors in the absence of liver disease or overt clinical thrombosis suggests a Question genetic deficiency of the factor being evaluated. 3. Explain how these laboratory tests confirm that the patient’s bleeding is related to a problem in primary Question hemostasis rather than secondary hemostasis. 12. Do these findings explain the patient’s bleeding history? Explanation Most disorders of secondary hemostasis are ruled out when Explanation the results of the prothrombin time and activated partial Yes, the boy has a homozygous FVII deficiency, and his par- thromboplastin time are within the reference intervals (see ents are heterozygous. Because homozygosity for FVII is Table E, Appendix A). The patient’s platelet count is slightly rare, the family history proved to be significant. The nose- decreased. bleeds (more commonly associated with platelet dysfunc- tion) are probably the result of the reduction of thrombin Question generation upon hemostatic activation and the resultant 4. Is the bleeding more likely related to problems with the loss of thrombin-catalyzed platelet activation. vascular system or with platelets? Why? Chapter 32 Case Summary: Shawn, a 10-year-old boy, had Explanation recurrent nosebleeds and anemia. Epistaxis began when he Both hereditary and acquired vascular disorders have char- was about 18 months old. The nosebleeds occurred one or acteristic abnormalities that are not included in the history. two times a month and began spontaneously. He bruised Therefore, we assume them to be absent. A platelet problem easily. The family history indicated that the boy’s grand- is most likely. parents were cousins. Shawn had a brother who died of Question intracranial hemorrhage at 10 years of age. Laboratory test- ing revealed a prolonged PT but an APTT in the reference 5. Is this profuse bleeding with the presence of petechiae range. This suggests a defect in one of the coagulation fac- consistent with the platelet count? Why? tors in the extrinsic pathway (FVII). Explanation Profuse bleeding and petechiae are not usually evident until CHAPTER 33 the platelet count is below 50 * 103/mcL. Therefore, the Question amount of bleeding is not consistent with the platelet count. 1. Which laboratory tests, hematology, and hemostasis Question would likely be ordered immediately and which would 6. What additional testing would be helpful in identifying be most informative in interpreting the cause of the the cause of the patient’s excess bleeding? patient’s bleeding? Explanation Explanation The patient should be questioned about recent ingestion Platelet count, prothrombin time, and activated partial of drugs that affect platelet function, for example, aspirin thromboplastin time are the screening tests ordered most or aspirin-containing products. In the absence of a history often. These tests will detect the presence of a problem of drug ingestion, platelet aggregation testing to identify either in the platelet numbers or in fibrin formation in most abnormal platelet function would likely be ordered. patients with hemorrhagic symptoms. The platelet count would probably be most informative because the patient’s Question symptoms are consistent with a defect in primary hemosta- 7. Is there a possibility that the patient’s platelet count is sis. However, the defect may not be in platelet quantity but spuriously increased or decreased? Why? rather in platelet function, so platelet function tests may be suggested as a follow-up. Explanation This is unlikely because the patient’s peripheral smear report Question does not indicate platelet satellitism, platelet agglutination, 2. Does the patient most likely have a disorder of primary or the presence of abnormally small erythrocytes or other fea- or of secondary hemostasis? Why? tures that could cause a spuriously increased platelet count. 1174 Appendix G Answers to Case Study Questions Question Explanation 8. Is Mohammad’s problem more likely acquired or inher- The type of bleeding in this patient indicates delayed bleed- ited? Why? ing and joint bleeding (hemarthrosis). Explanation Question The patient’s problem is most likely inherited because he 2. Does this history seem to be typical of a platelet disor- has had bleeding problems all of his life. der or of a coagulation factor disorder? Why? Question Explanation 9. What is the significance of these platelet function tests? These bleeding symptoms are characteristic of coagulation factor disorders (disorders of secondary hemostasis) rather Explanation than platelet disorders (disorders of primary hemostasis). The closure time was prolonged, which indicates abnormal Question platelet function. The platelet aggregation studies with risto- cetin are abnormal, but aggregation is normal with other ago- 3. What type of inheritance is most probably present in nists. Abnormal aggregation with ristocetin is characteristic this family? of either von Willebrand disease or Bernard-Soulier disease. Explanation Question X-linked inheritance is most probable in this family. 10. What is Mohammad’s most likely condition? Question Explanation 4. Is this history typical for a patient with von Willebrand disease? Why? The patient’s platelet aggregation studies were not corrected with the addition of von Willebrand factor. This indicates Explanation that the problem is unlikely to be von Willebrand disease. No, the inheritance pattern seems to be X-linked. The inheritance Because the patient had large platelets on the blood smear of VWD is usually autosomal dominant, although its inheri- and typical platelet aggregation studies, the most likely tance of the severe form is described as autosomal recessive. diagnosis is Bernard-Soulier disease. Question Question 5. What could have caused the patient’s great uncle to 11. What additional testing would be considered? have acquired an HIV infection? Explanation Explanation Additional testing would include DNA studies performed The uncle’s HIV infection could have been acquired prior to in a research facility to determine the molecular abnormal- 1984 from preparations of FVIII concentrate that he received ity or flow cytometry to demonstrate reduction of the GPIb/ to control bleeding (prior to current protocols that include a IX complex on the platelet membrane. viral-inactivation step in the production of the concentrate). Chapter 33 Case Summary: Mohammed experienced Question increased bleeding from superficial wounds acquired from 6. Name the coagulation factor deficiencies that are pos- an automobile accident. Petechiae were also noted. These sible with these laboratory results. symptoms most likely indicate a platelet disorder. His labo- Explanation ratory tests confirmed this. His platelet count was slightly When the APTT is abnormal and the PT is normal, the pos- decreased but not so low as to cause his bleeding symp- sible deficiencies are FVIII, FIX, FXI, FXII, prekallikrein, and toms. His closure time was prolonged, which indicated a high molecular weight kininogen. platelet function disorder. Coagulation factor screening tests were normal. Platelet aggregation studies showed abnor- Question mal aggregation with ristocetin that was not corrected by 7. What is the most likely factor deficiency? Why? VWF. This confirmed that his disorder was Bernard-Soulier syndrome. Explanation The most likely factor deficiency is FVIII because it is the CHAPTER 34 most common. Question Question 1. What term is used to describe the type of bleeding from 8. What do the results of the APTT on the mixture of minor cuts that the mother is describing? Scott’s plasma with normal plasma indicate? Appendix G Answers to Case Study Questions 1175 Explanation Question The results of the mixing studies indicate that normal 2. What other possibilities could explain the thrombotic plasma corrected the patient’s result. This indicates a factor event in this patient? deficiency rather than an inhibitor. Explanation Question She was initially tested twice because testing during an 9. What test should be performed next? acute thrombotic episode or while taking Coumadin is not as informative as testing after the episode has resolved Explanation or when the patient is no longer taking anticoagulants. An FVIII assay should be performed next. Unfortunately, based on the available tests, neither set of Question tests helped establish a diagnosis. Additional causes of hereditary thrombophilia could include activated protein 10. If an FVIII assay was performed and the results were C resistance; prothrombin mutation G20210A; hyperhomo- less than 1 U/dL, what would be the most likely diag- cysteine; deficiencies of heparin cofactor II, TFPI, t-PA, or nosis, given all other lab testing results and the patient FXII; and elevated fibrinogen, FVIII, or PAI-1. history? Explanation Question 3. Why is Andrea at higher risk for a thrombotic event Molecular studies for the inversion mutation in intron 22 than her mother or her two sisters? should be done for all patients with severe FVIII deficiency. Explanation Question Specific molecular assays of the family were subsequently 11. What therapy is indicated for this patient? done, and they revealed both the FVLeiden and prothrom- Explanation bin G20210A mutations. The fact that both these mutations Therapy for FVIII deficiency includes heat-inactivated FVIII were found in the same family probably accounts for the concentrates

and recombinant FVIII. strong family history. The patient had inherited two differ- ent genetic mutations, both of which carry an increased risk Question of thrombosis. In addition, she had several other clinical 12. What complications from the therapy are possible? risk factors, probably contributing to her thrombotic event. Because the mother and sisters had a single genetic muta- Explanation tion, their risk of thrombosis was probably less than that of A complication of therapy that occurs in patients with Andrea. The patient was taken off oral contraceptives and hemophilia A, particularly those with the inversion muta- put back on Coumadin. tion in intron 22, is the development of an inhibitor. Chapter 35 Case Summary: Andrea is a 37-year-old Chapter 34 Case Summary: Scott was seen in the ER for female who was diagnosed with deep vein thrombosis of severe bleeding in the knee after a slight fall. Males on the the leg. She is dehydrated and is taking an oral contracep- maternal side of the family have a family history of similar tive and Coumadin. Her family history revealed that pater- bleeding. A prothrombin time and thrombin time on Scott nal uncles and maternal cousins had thrombotic episodes. were normal, but the APTT was prolonged. The APTT was Testing for AT, PC, PS, fibrinogen, D-dimer, and plasmin- corrected when patient’s plasma was mixed with normal ogen was performed during the DVT episode and again plasma, suggesting a factor deficiency. An FVIII assay 6 months after she had discontinued Coumadin. All tests showed a severe decrease. Scott was diagnosed with hemo- were within the reference interval. Several years later, she philia A. was tested and found to be heterozygous for both FVLeiden and prothrombin G20210A. CHAPTER 35 Question CHAPTER 38 1. What risk factors, if any, are revealed in the patient’s Question history? 1. Is a bone marrow evaluation indicated for this patient? Explanation Why or why not? Andrea had several risk factors for thrombosis including Explanation dehydration and oral contraceptive use. There is also a CBC data indicate that the patient is pancytopenic. Presence strong family history of thrombosis, suggesting a hereditary of pancytopenia in a young patient is worrisome. The com- component in the etiology of her acute thrombotic event. mon causes of pancytopenia in a young patient include HIV 1176 Appendix G Answers to Case Study Questions infection, alcoholism, use of medications, and B12 and folic sample could be used if it were processed within 24 hours of acid deficiency. Presence of teardrop cells with pancytopenia being drawn. Other anticoagulants (e.g., heparin and ACD) indicates marrow fibrosis or marrow infiltration by leuke- are adequate. mia, lymphoma, or granulomas. Bone marrow evaluation for this patient is definitely indicated. The patient does not have Question lymphadenopathy, hepatomegaly, or splenomegaly, so the 2. Which cells are of interest and should be included in the chance of lymphoma involving the marrow is very unlikely. gate? Question Explanation 2. How should the marrow evaluation proceed? The cells of interest are the lymphocytes. Explanation Question Fortunately, the laboratory professional made several touch 3. What are the typical forward and side scatter properties imprints from the biopsy. There is no reason for doing cyto- of the cells of interest? chemical stains on the aspirate smear if a good-quality and Explanation good number of touch imprint slides are available, espe- Lymphocytes have low side scatter (granularity) and vari- cially if the amount of aspirate is low. These touch imprints able forward scatter (size). were of good quality and showed many blasts. The touch imprints were stained with cytochemical stains. No differ- Question entiation was present among these blasts. The cells stained 4. What is the phenotype? consistent with myeloblasts. The hemodiluted marrow was sent for flow cytometry and cytogenetics. The aspirate can Explanation be sent for flow cytometry because flow is more sensitive in The majority of the lymphocytes have the following abnor- detecting the lineage of blasts. Flow cytometry was positive mal phenotype: CD45+ , CD19+ , CD20+ , CD23+ , CD5+ , for myeloid markers and negative for lymphoid markers. lambda+ , and kappa- . The remainder of the aspirate was sent for cytogenetics with the hope that some cells might be able to grow and show Question any specific cytogenetic abnormality. 5. Which features indicate clonality? Question Explanation 3. Can a diagnosis be made from tests on the aspirate Expression of only one immunoglobulin light chain rather than waiting for the biopsy slides? (lambda, but not kappa) and the aberrant expression of CD5 indicate a clonal population of B cells. Explanation Yes, the presence of many blasts in the bone marrow aspirate Question smears and on the touch imprints indicates acute leukemia. 6. What is the diagnosis? The lineage of blasts can be determined based on cytochem- Explanation ical stains and/or flow cytometry. Bone marrow biopsy in this case would not provide any added information. The diagnosis is chronic lymphocytic leukemia, B-cell (CLL), or small lymphocytic lymphoma (SLL). CD5-positive B cells Chapter 38 Case Summary: Robert was pancytopenic. are characteristic of CLL, SLL, and mantle cell lymphoma Clinical history and further laboratory tests revealed no (MCL). CD23 expression is found in CLL and SLL but not cause for this. A bone marrow aspirate and a biopsy were MCL. Although lymphoma primarily involves the lymph obtained. Evaluation revealed the presence of many blasts. nodes and other lymphoid organs, there could be second- The diagnosis based on bone marrow testing was acute ary involvement of the peripheral blood and bone marrow. myelocytic leukemia. Question CHAPTER 40 7. Which of the flow cytometry results presented in this case indicate that this is a malignancy of mature lym- Question phocytes (chronic lymphocytic leukemia), not acute 1. What is the optimal specimen? lymphoblastic leukemia? Explanation Explanation The CBC revealed peripheral blood lymphocytosis. There- The strong expression of CD45, presence of surface immu- fore, flow cytometry of the peripheral blood is appropri- noglobulin, and absence of CD34 and CD10 expression indi- ate. An EDTA-anticoagulated specimen is ideal. The CBC cate a mature B lymphocyte phenotype. Appendix G Answers to Case Study Questions 1177 Question Explanation 8. How can flow cytometry be used to assess Andrew’s This is a clonal aberration because it is present in more than response to therapy? one cell (in this case, it was present in 100% of cells ana- Explanation lyzed, which is typical for CML). Flow cytometry can be used to quantify the number of cir- Question culating malignant cells of hematologic origin, including 4. What is the significance of this finding for the diagnosis? ALL, AML, CLL and plasma cell myeloma. During and after therapy, multicolor flow cytometry can help to determine if Explanation residual malignant cells (minimal residual disease [MRD]) The finding of the t(9;22) confirms the diagnosis of CML. persist in a patient. The residual cells are ultimately respon- sible for clinical relapse. The presence or absence of MRD Question at different time points during therapy can help to predict 5. Three months after transplant, the karyotype is 46XX. the outcome and guides further treatment for the patient. What is the significance of this finding? Question Explanation 9. What is the significance of minimal residual disease This finding confirms the engraftment of female donor cells (MRD) after completion of chemotherapy? in the recipient bone marrow. Although no cells with the Explanation t(9;22) are found by cytogenetic methods, molecular studies might show the presence of the BCR gene rearrangement After therapy has ended for a patient, if malignant cells because these techniques are more sensitive than routine are detected by flow cytometry or molecular methods, the cytogenetics. likelihood of relapse is greatly increased. MRD is a strong prognostic indicator for patients. Question Chapter 40 Case Summary: The case is an example of 6. What other studies that would be informative as to chronic lymphocytic leukemia involving the peripheral the status of the donor and recipient cells could be blood. It demonstrates the technique of gating the cells of performed? interest, determining a phenotype, and using of a character- Explanation istic phenotype in establishing a diagnosis of a subtype of FISH analysis can be performed on the interphase nuclei lymphoid malignancy, chronic lymphocytic leukemia (CLL) using probes for the X and Y chromosome to demonstrate or small lymphocytic lymphoma (SLL). the sex of the cells present. In addition, probes for the t(9;22) can be used if male cells are present to determine CHAPTER 41 whether they are leukemic cells. Molecular studies can also Question be obtained for the BCR gene rearrangement. 1. What is the most appropriate specimen to submit for Question cytogenetic analysis, and how should it be processed? 7. What is the significance of these findings? Explanation The most appropriate specimen is a bone marrow sample. If Explanation the bone marrow can not be aspirated, peripheral blood can The cytogenetic results performed at 5 years post-transplant be used but sometimes does not yield mitosis. The process- show not only a relapse of the CML, but also a cytogenetic ing must be performed by direct harvest and/or unstimu- transformation indicative of pending blast crisis as evi- lated cultures. denced by the +8 and i(17q) that were not seen in the origi- nal leukemic cells. Question Chapter 41 Case Summary: Gregory had leukocytosis 2. Is this a constitutional or acquired aberration? and a left shift when he was initially seen. The differential Explanation diagnosis was important to determine whether this abnor- This is an acquired aberration that is present only in the mal blood picture was the result of a benign or neoplastic hematopoietic cells—RBC precursors, WBC precursors, process. Cytogenetic studies revealed an acquired clonal megakaryocytes, and some lymphocytes. aberration, t(9;22), diagnostic of CML. Gregory had a bone marrow transplant with his sister’s donated cells. Five years Question later, cytogenetic studies revealed a relapse and impending 3. Is it a clonal aberration? blast crisis. 1178 Appendix G Answers to Case Study Questions CHAPTER 42 Chapter 42 Case Summary: Warren has test results that Question suggest CML or a reactive leukocytosis. However, a reac- tive leukocytosis does not usually have thrombocytosis, 1. What type of molecular diagnostic testing would be nor is an enlarged spleen characteristic. Any molecular suitable to follow Warren’s MRD? analysis that recognizes the t(9;22) translocation that is Explanation characteristic of chronic myelogenous leukemia would RQ-PCR testing of Warren’s peripheral blood is a highly be useful in diagnosis. Additionally, the presence of BCR- accurate and sensitive test of the presence of the BCR-ABL1 ABL1 RNA transcripts in the leukocytes of the peripheral translocation and is suitable. blood could be assessed. At this point, FISH analysis of the patient’s bone marrow aspirate could be performed with Question labeled oligonucleotides that recognize the t(9;22) trans- 2. Before increasing Warren’s imatinib dose, what kind location characteristic of chronic myelogenous leukemia. of molecular testing might Warren’s physician order FISH testing can analyze both dividing (metaphase) and to help in the decision about how to manage Warren’s nondividing (interphase) cells, and it is especially helpful disease? in recognizing chromosomal translocations. In addition to FISH testing, the presence of BCR-ABL1 RNA transcripts Explanation in the leukocytes of the peripheral blood can be analyzed Mutation analysis with direct sequencing could be per- with the real-time quantitative polymerase chain reaction formed to reveal breakpoints in Warren’s BCR-ABL1 gene (RQ-PCR) technique. After 3 years, Warren’s test results to determine whether he is exhibiting a common breakpoint showed an increase in Ph+ cells. This could indicate that mutation associated with Imatinib resistance. Warren’s phy- he has developed a resistance to Imatinib. Additional test- sician might consider several therapy options for patients ing should be performed to see whether he has developed exhibiting Imatinib refractoriness. additional mutations that confer resistance to Imatinib. Glossary Abetalipoproteinemia (hereditary acanthocytosis) rare, ADAMTS-13 See a disintegrin-like and metalloprotease with autosomal recessive disorder characterized by the absence of thrombospondin. serum b@lipoprotein low serum cholesterol, low triglyceride, and Adaptive immune response interaction of the T lymphocyte, B low phospholipid and an increase in the ratio of cholesterol to lymphocyte, and macrophage in a series of events that allows the phospholipid. body to attack and eliminate foreign antigens. Acanthocyte abnormally shaped erythrocyte with spicules of vary- ADCC See Antibody-dependent cell cytotoxicity. ing length irregularly distributed over the cell membrane’s outer surface; has no central area of pallor. Also

known as spur cell. Adipocyte cell whose cytoplasm is largely replaced with a single fat vacuole; fat cell. Achlorhydria absence of hydrochloric acid in stomach gastric secretions. A disintegrin-like and metalloprotease with thrombospondin type motif (ADAMTS-13) metalloprotease enzyme responsible Acquired aberration chromosome abnormality (either numerical or for cleavage of the ultralarge multimers of von Willebrand factor structural) that occurs at some time after birth and involves only one (VWF) released from endothelial cells into the VWF multimer sizes cell line. normally found in the circulation. Mutations in or deficiencies of Acquired immune deficiency syndrome (AIDS) disease caused this enzyme is a risk for thrombosis. by infection with human immunodeficiency virus type I (HIV-1) Advanced HIV disease stage of infection with the retrovirus HIV that selectively infects helper T lymphocytes (CD4+ ), causing rapid in which there are decreased CD4+ T lymphs and the presence of depletion of these cells. This causes a deficiency in cell-mediated conditions that require antiretroviral therapy. immunity. Patients have repeated infections with multiple oppor- tunistic organisms and an increase in malignancies. Afibrinogenemia condition in which coagulation factor I, also called Acquired inhibitor See Circulating inhibitor (anticoagulant). fibrinogen, is absent in the peripheral blood. It can be caused by a mutation in the gene controlling the production of this plasma Acrocentric having a chromosome that has the centromere close to protein or by an acquired condition in which the plasma protein is the terminal end so that the short arm is much shorter than the long pathologically converted to fibrin. arm. The short arm consists only of a stalk and a small amount of DNA called a satellite. Agglutinate clumping of erythrocytes as a result of interactions between membrane antigens and specific antibodies. Acrocyanosis See Raynaud’s phenomenon. Agglutination process of erythrocyte clumping. Activated partial thromboplastin time (APTT) screening test used to detect deficiencies in the intrinsic and common pathway of the Aggregating reagent chemical substance (agonist) that promotes coagulation cascade. platelet activation and aggregation by attaching to a receptor on the platelet’s surface. Activated lymphocyte See Reactive lymphocyte. Aggregation attachment of platelets to other platelets. When Activated protein C resistance (APCR) hemostatic condition in platelets are activated, they undergo shape change and expose the which activated protein C is not able to inactivate F-V, which could fibrinogen receptor GPIIb/GPIIIa. The binding of fibrinogen to cause or contribute to thrombosis. In most cases, it is caused by a this receptor links adjacent, activated platelets through fibrinogen mutation in F-V in which Arg 506 is replaced with Gln (F-VLeiden). bridges. Platelet aggregation requires Ca++. Acute leukemia (AL) malignant hematopoietic stem cell disorder Agonist chemical substance that can attach to a platelet characterized by unregulated proliferation and a block in matura- membrane receptor and activate platelets, causing them to tion of a mutated stem cell or progenitor cell resulting in accumula- aggregate (e.g., collagen, ADP). It is used in the laboratory to test tion of immature and nonfunctional hematopoietic cells in the bone platelet f unction using a platelet aggregometer or platelet function marrow, peripheral blood, and other organs. analyzer. Acute lymphocytic leukemia (ALL) malignant disorder character- a@granule (aG) See Alpha (a) granule. ized by unregulated proliferation and a block in the maturation of a mutated lymphoid progenitor cell resulting in the accumulation of Agranulocytosis absence of white blood cells in the peripheral lymphoid cells in the bone marrow. Peripheral blood smear reveals blood. the presence of many undifferentiated or minimally differentiated Alder-Reilly anomaly benign condition characterized by the pres- cells. ence of functionally normal leukocytes with large purplish granules Acute myeloid leukemia (AML) malignant myeloproliferative in their cytoplasm when stained with a Romanowsky stain. disorder characterized by unregulated proliferation and a Aleukemic leukemia disorder in which abnormal malignant cells block in the maturation of a mutated hematopoietic stem cell or are found only in the bone marrow. myeloid p rogenitor cell resulting in the accumulation of primarily undifferentiated or minimally differentiated myeloid cells in the Allele one of two or more genes that correspond to the same trait bone marrow and peripheral blood. and occupy the same position on paired chromosomes. Acute phase reactant plasma protein that rises rapidly in response Alloantibody blood protein produced in one individual in response to inflammation, infection, or tissue injury. to the antigens of another individual of the same species. Acute undifferentiated leukemia (AUL) critical disorder in which Allogeneic pertaining to an allograft in which the donor and host the morphology, cytochemistry, and immunophenotype of the belong to the same species but are not genetically identical. proliferating blasts lack sufficient information to classify them as Allogeneic stem cell transplantation process of moving stem cells myeloid or lymphoid in origin. between genetically dissimilar animals of the same species. 1179 1180 Glossary Alloimmune hemolytic anemia hemolytic disorder generated Anti-factor Xa testing chromogenic assay that is designed to when blood cells from one person are infused into a genetically measure and monitor heparinoids or direct oral anticoagulants. unrelated person. The recipient’s lymphocytes recognize antigens Antigen any foreign substance that evokes antibody production on the infused donor cells as foreign, stimulating the production of (an immune response) and reacts specifically with that antibody. antibodies that react with donor cells and cause hemolysis. Antigen-dependent lymphopoiesis development of immunocom- Alpha (a) granule small particle of platelet storage containing petent lymphocytes into effector T and B lymphocytes that mediate a variety of proteins that are released into an area after platelet the immune response through production of lymphokines and activation. antibodies. The process, which occurs in secondary lymphoid tissue, Alpha-2 antiplasmin serine protease inhibitor responsible for is initiated when mature lymphocytes come into contact with an inactivating plasmin. Participates in fibrinolysis and degradation of antigen. various other proteins. Antigen-independent lymphopoiesis development of lymphoid Analytical measurement range instrument’s linearity field. stem cells into immunocompetent T and B lymphocytes (virgin Analytical sensitivity ability to detect small amounts of an analyte. lymphocytes). This process occurs in the primary lymphoid tissue under the regulation of hematopoietic growth factors. Analytical specificity ability to detect only the analyte in question. Antigen-presenting cell (APC) macrophage in the immune Analytical time period between entering a specimen into the test response; the macrophage phagocytizes substances foreign to the system and reporting the result by the instrument. host and presents the foreign substance’s antigenic determinants on Anaplastic large cell lymphoma (ALCL) subtype of T/NK cell its membrane to antigen-dependent T lymphocytes. neoplasm characterized by large bizarre anaplastic cells that can Antihuman globulin (AHG) protein used in a laboratory procedure resemble Reed-Sternberg and Hodgkin variant cells; cells are usually designed to detect the presence of antibodies directed against positive for the CD30 antigen but usually lack CD15 expression and erythrocyte antigens on the erythrocyte membrane. evidence of Epstein-Barr virus infection; often positive for leukocyte common antigen (LCA) and epithelial membrane antigen (EMA). Antiphospholipid antibody (aPL) immunoglobulin produced in response to an antigenic substance and directed against antigens Anemia disorder characterized by a decrease in the normal concen- that consist of a negatively charged phospholipid. Clinically impor- tration of hemoglobin or erythrocytes; can be caused by increased tant antiphospholipid immunoglobulins include anticardiolipin erythrocyte loss or decreased erythrocyte production and can result antibody (ACA) and lupus anticoagulant (LA). In some individuals, in hypoxia. these immunoglobulins are associated with thrombosis and other Anemia of chronic disease abnormal state caused by decrease hemostatic defects. in hemoglobin that accompanies many chronic conditions such Antiphospholipid antibody syndrome (APS) clinical condition as rheumatoid arthritis. It is characterized by low serum iron but characterized by the presence of high titers of antiphospholipid normal iron stores. There is a block in iron release from macrophag- immunoglobulin, thrombocytopenia, and recurrent arterial and es for iron recycling mediated by hepcidin produced in response to venous thromboses, often affecting young males. IL-6, an inflammatory cytokine. Also called anemia of inflammation. Antithrombotic agent any drug that reduces the formation of blood Anemia of inflammation (AI) other term for anemia of chronic disease. clots (thrombi) either for prevention or treatment. Aneuploid used to describe a condition in which the number of Aperture small opening with electrodes located on either side chromosomes per cell does not equal a multiple of the haploid through which blood cells are drawn into an electronic cell counter. number, n; for example, in human cells, a chromosome count of 45, Electrical resistance is detected as the cell passes through the aperture. 47, 48, and so on. Apheresis separation, removal, or withdrawal of whole blood from Anisocytosis general variation in erythrocyte size. the donor or patient and separated into its components, one of Annexin II endothelial cell receptor for tPA that functions as a which is retained; the remaining constituents are recombined and coreceptor for both tPA and PLG. Binding and activation of tPA and returned to the individual. PLG help maintain a fibrinolytic potential on undamaged vascular Apixaban (Eliquis) anticoagulant used in the treatment of venous surfaces. thromboembolic events; a direct factor Xa inhibitor. Antibody immunoglobulin produced in response to an antigenic Aplasia failure of hematopoietic cells to generate and develop in the substance. bone marrow. Antibody-dependent cell cytotoxicity (ADCC) mechanism of the Aplastic anemia disorder characterized by peripheral blood pancy- recognition and lysis of organisms by NK cells through binding IgG topenia and hypocellular bone marrow; considered a hematopoietic to the NK cell CD16 receptor. Any target organism coated with IgG stem cell disorder. can be bound to NK cells and lysed. Monocytes, macrophages, and neutrophils also have this receptor and act in a similar manner. Aplastic crisis abrupt, transient cessation of erythropoiesis that occurs in some hemolytic anemias and infections. Anticardiolipin antibody (ACA) negatively charged antibody associated with the presence of arterial and venous thromboem- Apoferritin cellular protein that combines with iron to form ferritin; bolism, thrombocytopenia, first-trimester fetal loss, stroke, and found attached only to iron, not in the free form. immunological issues such as systemic lupus erythematosus; may Apoptosis programmed cell death resulting from activation of a be detected by solid-phase ELISA procedures. predetermined sequence of intracellular events; “cell suicide.” Anticoagulant chemical substance added to whole blood to prevent Apotransferrin iron transport protein without iron attached. When it from clotting. Depending on its type, in vitro clotting is prevented the protein has iron attached, it is referred to as transferrin. by the removal of calcium (EDTA) or the inhibition of the serine pro- teases such as thrombin (heparin). Medications or other substances APTT See Activated partial thromboplastin time. such as unfractionated heparin, low-molecular-weight heparins, Arachidonic acid (AA) unsaturated essential fatty acid, usually pentasaccharides, direct thrombin inhibitors, vitamin K antagonists, attached to the second carbon of the glycerol backbone of and direct oral anticoagulants that are used therapeutically to pre- phospholipids, released by phospholipase A2; a precursor of prosta- vent the blood from clotting. glandins and thromboxanes. Glossary 1181 Arachnoid mater delicate membrane that covers the central nervous specific granules. The cell is 9915 mcM (mm) in diameter. Also called system; middle layer of the meninges. stab or unsegmented neutrophil. Artificial oxygen carrier (AOC) a class of manufactured blood Barr body (drumstick) chromatin body; inactive X chromosome that that has two groups including hemoglobin-based oxygen carriers appears as an appendage of the neutrophil nucleus but is not visible (HBOCs) in solution and perfluorocarbons (PFCs). The HBOCs in every neutrophil. consist of purified human or bovine hemoglobin and recombinant Basophil mature granulocytic cell characterized by the presence of hemoglobin. The oxygen dissociation curve of HBOCs is similar large readily stained granules that are purple blue or purple black to that of native human blood. Hemoglobin tests based on colori- with Romanowsky stain. The cell is 10914 mcM (mm) in diameter, metric analysis could give erroneous results. PFCs are fluorinated and the nucleus is segmented. Granules are cytochemically posi- hydrocarbons with high gas-dissolving capacity. They do not mix in tive with periodic acid-Schiff (PAS) and peroxidase and contain aqueous solution and must be emulsified. In contrast to HBOCs, a histamine and heparin peroxidase. This granulocytic cell constitutes linear relationship exists between PO2 and oxygen content in PFCs. less than 0.2 * 103/mcL or 0–1% of peripheral blood leukocytes, Thus, relatively high O2 partial pressure is required to maximize functions as a mediator of inflammatory responses, and has recep- delivery of O2 by PFCs. tors for IgE. Ascites effusion and accumulation of fluid in the peritoneal cavity. Basophilia increased

concentration of circulating basophils. Ascitic fluid liquid substance that abnormally collects in the perito- Basophilic normoblast nucleated precursor of the erythrocyte that neal cavity of the abdomen. is derived from a pronormoblast. The cell is 10916 mcM (mm) in Atypical chronic myeloid leukemia (aCML, BCR/ABL1−) variant diameter. The nuclear chromatin is coarser than the pronormoblast, condition of MDS/MPN characterized by primary involvement of and nucleoli are usually absent. Cytoplasm is more abundant, and it the neutrophil series with leukocytosis involving dysplastic imma- stains deeply. The cell matures to a polychromatophilic normoblast. ture and mature neutrophils. Multilineage dysplasia is common. Also called prorubricyte. Does not have the BCR/ABL1 gene mutation. Basophilic stippling presence of precipitating ribonucleoproteins Atypical lymphocyte See Reactive lymphocyte. and mitochondrial remnants that compose erythrocyte inclusions. Auer rod reddish-blue staining needlelike inclusion within the cyto- Observed on Romanowsky-stained blood smears as diffuse or plasm of leukemic myeloblasts that occur as a result of abnormal punctate bluish-black granules in toxic states such as drug (lead) cytoplasmic granule formation. Their presence on a Romanowsky- exposure. Diffuse, fine basophilic stippling can occur as an artifact. stained smear is helpful in differentiating acute myeloid leukemia BCL-2 gene hereditary unit on chromosome 18 producing bcl-2 pro- from acute lymphoblastic leukemia. tein. The translocation t(14;18) found in follicular lymphoma leads Autoantibody immunoglobulin produced in response to an anti- to bcl-2 overexpression and inhibition of lymphocyte cell death. genic substance in the blood capable of reacting with the subject’s Beer-Lambert’s law formula for the mathematical basis for color- own antigens. imetry. The equation is A = C * L * K. A is absorbance, C is the Autocrine See Autocrine signaling. concentration of the colored substance, L is the depth of the solution through which the light travels, and K is a constant. Autocrine signaling process that produces a signal that acts on the same cell that produced it. Bence-Jones proteinuria excessive immunoglobulin light chain found in the urine from patients with multiple myeloma. Autohemolysis process of destruction of the subject’s own erythro- cytes by hemolytic agents in the subject’s blood. Benign nonmalignant tissue formed from highly organized, differ- entiated cells that do not spread or invade surrounding tissue. Autoimmune hemolytic anemia (AIHA) condition that results when individuals produce antibodies against their own erythro- Bernard-Soulier syndrome rare autosomal-recessive hereditary cytes. The antibodies are usually against high-incidence antigens. platelet disorder characterized by a moderate to severe thrombocy- topenia, giant platelets, and abnormal platelet function. The defect Autologous derived from self. is a quantitative decrease or abnormal function of the GPIb/IX/V Autologous stem cell transplantation infusion of a person’s own complex resulting in the platelets’ inability to adhere to collagen. stem cells. Bethesda inhibitor assay used to determine levels of antibodies to Autosome chromosome that does not contain genes for sex differen- the presence of infused factor levels, particularly in patients with tiation; in humans, chromosome pairs 1–22. hemophilia A. Autosplenectomy extensive splenic damage secondary to infarction; Bethesda titer measurement of the quantity of an inhibitor or anti- often seen in older children and adults with sickle cell anemia. body; expressed as Bethesda units per mL (BU/mL). Azurophilic granule small particle (primary) within myelocytic BFU-E See Burst-forming unit–erythroid. leukocytes that has a predilection for the aniline component of a Bilineage acute leukemia malignant blood cell condition that is Romanowsky-type stain. This particle appears bluish-purple or characterized by the presence of two separate populations of malig- bluish-black when observed microscopically on a stained blood nant cells, one of which phenotypes as lymphoid and the other as smear. It first appears in the promyelocyte. myeloid. B cell acute lymphoblastic leukemia (B-ALL) immunologic type of Bilirubin breakdown product of the heme portion of the hemo- cancer of the blood and bone marrow in which the neoplastic cell is globin molecule. Initial steps in the degradation of hemoglobin a B lymphoid cell; it has subtypes. result in a lipid-soluble form (unconjugated or indirect bilirubin) B cell receptor (BCR) specific entity on the B lymphocyte membrane that travels in the blood stream to the liver. There it is converted that reacts with antigen. into a water-soluble form (conjugated or direct bilirubin) that can be B lymphoid cell antigen CD marker that when present is useful in excreted into the bile. differentiating a B cell ALL from other types of acute leukemia. Bioavailability degree and rate at which a free drug is available to Band neutrophil cell type that is immediate precursor of the produce its effect. mature granulocyte. It can be found in either the bone marrow or Biphasic antibody immunoglobulin that binds to erythrocytes at peripheral blood. The nucleus is elongated, and nuclear chroma- room temperature or below and causes hemolysis when the blood tin is condensed. The cytoplasm stains pink, and there are many warms to 37°C. 1182 Glossary Biphenotypic acute leukemia malignant blood cell disorder in Buffy coat layer of white blood cells and platelets that lies between which there is an unregulated proliferation and block in maturation the plasma and erythrocytes in centrifuged blood sample. of mutated progenitor cells that have myeloid and lymphoid mark- Burkitt cell lymphoblast that is found in Burkitt’s lymphoma. ers on the same population of neoplastic cells. Burst-forming unit erythroid committed red blood cell progenitor Birefringence quality of a substance that can change the direction that develops into the unipotential CFU-E stem cell. It is relatively of light rays that are directed at a substance; can be used to identify insensitive to EPO except in high concentrations. IL3 and GM-CSF crystals. stimulates it to enter the cell cycle. 2,3-bisphosphoglycerate (2,3-BPG) product of the glycolytic pathway Butt cell circulating neoplastic lymphocyte with a deep indentation that affects the oxygen affinity of hemoglobin. It serves in the biochem- (cleft) of the nuclear membrane. Butt cells can be seen when follicu- ical feedback system that regulates the amount of oxygen released to lar lymphoma involves the peripheral blood. the tissues. As the concentration of 2,3-BPG increases, hemoglobin’s affinity for oxygen decreases and more oxygen is released to the tissue. Cabot ring reddish-violet erythrocyte inclusion resembling the Also referred to as 2,3-diphosphoglycerate (2,3-DPG). figure 8 on Romanowsky-stained blood smears that can be found in some cases of severe anemia. Bite cell erythrocyte with a portion of the cell missing; seen in G6PD deficiency and drug-induced oxidant hemolysis; produced when the Cancer-initiating cell cell from which cancer originally emerges; spleen macrophages remove Heinz bodies from the cell. sometimes called the cell of origin, it has the capacity for unlimited self-renewal. Blast crisis evolution of a chronic hematopoietic neoplasm to an acute leukemia. Various genetic changes in the leukemic stem cells Cancer stem cell cell within a tumor that is capable of extensive accompany this terminal stage of the disease. proliferation in vitro (in colony-forming assays) and in vivo (in transplantation models). It is believed to be rare within a tumor but Blast transformation complex cell process that occurs after con- has infinite proliferative potential that drives the formation and tact and binding of an antigen to the antigen receptors of T- and growth of tumors. B- immunocompetent lymphocytes; result is the clonal amplification of cells responsible for the expression of immunity to that specific Carboxyhemoglobin compound formed when hemoglobin is antigen. exposed to carbon monoxide; it is incapable of oxygen transport. Bleeding time and PFA 100 screening test that measures platelet Carboxylation addition of a carboxyl group to coagulation factors function. in the prothrombin group (II, VII, IX, X, proteins C, S, Z) in the liver; it is necessary for the factor to become functional. Vitamin K is Blinded preanalyzed sample previously tested specimen that is required for this reaction. integrated randomly into a specimen run and possesses no identify- ing feature (e.g., number or designation) to indicate that it differs Cardiac tamponade critical clinical condition in which the pericar- from current patient specimens. Only the individual who selected dial sac fills with fluid and restricts the heartbeat and venous return and relabeled it as part of a quality control program can identify to the heart. them. Caspase cysteine protease responsible for cell alterations in Blister cell cell with a clear area next to the membrane on one side; apoptosis. thought to be formed when the phagocyte removes a Heinz body in Catalytic domain area in a protein that is common to all serine pro- the cell and is seen in G6PD deficiency. teases involved in blood clotting. Cleavage of a peptide bond occurs Blood coagulation formation of a blood clot, usually considered a here and converts the proenzyme to its active form. normal process. CD designation name of a cluster of differentiation of a group of Blood island cluster of cells in the yolk sac of the human embryo monoclonal antibodies recognizing the same protein marker antigen that gives rise to yolk sac erythroblasts. on a cell. The antibodies are used to classify cell types and stages of maturation. Bohr effect result of pH on hemoglobin-oxygen affinity; serves as one of the most important buffer systems in the body. As the H+ Cell cycle biochemical and morphological stages a cell passes concentration in tissues increases and the affinity of hemoglobin for through leading up to its division; includes G1, S, G2, and oxygen decreases, oxygen unloading is permitted. M phases. Bone marrow aspirate fluid withdrawn from the bone marrow by Cell-cycle checkpoint place in the cell cycle at which its progress aspiration using a special needle (e.g., Jamshidi needle) and syringe. can be halted until conditions are suitable for the cell to proceed to The fluid represents the specialized soft tissue that fills the medul- the next stage. lary cavities between the bone trabeculae. Its examination is useful Cell-mediated immunity resistant response mediated by T lympho- in evaluating hematopoietic cellular morphology, distribution, and cytes that requires interaction between histocompatible T lympho- development; observing for presence of abnormal cells; and estimat- cytes and macrophages with antigen. At least three important T ing cellularity. lymphocyte subsets are involved: helper, regulatory and cytotoxic. Bone marrow trephine biopsy removal of a small piece of a soft Cellular hemoglobin concentration mean (CHCM) erythrocyte highly vascular modified connective tissue within the core of bone index that represents the average hemoglobin concentration of indi- that contains hematopoietic tissue, fat, and trabecula. Examination vidual cells analyzed. It is derived from the hemoglobin histogram. of the trephine biopsy is useful in observing the tissue’s architecture Interference with the hemoglobin determination resulting from and cellularity and allows interpretation of the spatial relationships turbidity or lipemia can be identified by comparing it to the MCHC. of bone, fat, and marrow cellularity. Central nervous system (CNS) part of the nervous system that Bordetella pertussis gram-negative aerobic coccobacilli that causes consists of the brain and spinal cord. whooping cough. The hematologic picture in whooping cough is leukocytosis with lymphocytosis. The lymphocytes are small cells Centriole cytoplasmic organelle that is the point of origin for the with folded nuclei. contractile protein known as spindle fiber. Bronchoalveolar lavage sample of fluid obtained by a diagnostic Centromere primary constriction that attaches sister chromatids in a procedure of infusing and removing a sterile saline solution into the chromosome, dividing them into long and short arms. alveolar and bronchial airspaces of the lung via a bronchoscope. It is Cerebrospinal fluid (CSF) liquid substance normally produced to performed to detect diseases of the lower respiratory tract. protect the brain and spinal cord. Produced by the choroid plexus Glossary 1183 cells and absorbed by the arachnoid pia, it circulates in the suba- Chronic lymphocytic leukemia (CLL) disorder characterized by rachnoid space. a neoplastic growth of lymphoid cells in the bone marrow and an Ceruloplasmin ferroxidase that converts Fe++ to Fe+++ for b inding extreme elevation of these cells in the peripheral blood. It is charac- to transferrin. Export of iron from nonintestinal cells, including terized by leukocytosis, less than 20% blasts, and a predominance of macrophages, requires this protein. mature lymphoid cells; classified by WHO as a mature B lymphoid neoplasm. CFU-E See Colony-forming unit-erythroid. Chronic idiopathic myelofibrosis (CIMF) myeloproliferative CH50 functional hemolytic titration assay to measure lysis, the neoplasm characterized by excessive proliferation of all cell lines as endpoint of complement activation. It measures the amount of well as progressive bone marrow fibrosis and blood cell production patient serum required to lyse 50% of a

standardized concentration at sites other than the bone marrow, such as the liver and spleen. of antibody-sensitized sheep red blood cells. Because all comple- Also called agnogenic myeloid metaplasia, myelofibrosis with myeloid ment proteins are required for lysis to occur, any single complement metaplasia, and primary myelofibrosis. factor deficiency causes a negative reaction (no lysis). Chronic myelogenous leukemia (CML) neoplasm characterized by Charcot–Leyden crystal material formed from eosinophil granules a neoplastic growth of primarily myeloid cells in the bone marrow that is found in tissues with large numbers of eosinophils. and an extreme elevation of these cells in the peripheral blood. The Chediak–Higashi anomaly multisystem disorder inherited in two phases of the disease are chronic and blast crisis. The chronic an autosomal recessive fashion and characterized by recurrent phase has less than 20% blasts in the bone marrow or peripheral infections, hepatosplenomegaly, partial albinism, and central blood, whereas the blast crisis phase has more than 20% blasts. Indi- nervous system (CNS) abnormalities; neutrophil chemotaxis and viduals with this disease have the BCR/ABL1 translocation, which killing of organisms is impaired. Giant cytoplasmic granular inclu- codes for a unique P210 protein. Also referred to as chronic granulo- sions are in leukocytes and platelets. cytic leukemia (CGL). Chemokine cytokine with chemotactic activity. Chronic myelomonocytic leukemia (CMML) subgroup of the myelodysplastic syndromes characterized by anemia and a vari- Chemotaxin chemical messenger that causes migration of cells in able total leukocyte count. An absolute monocytosis more than one direction. Also called chemokine. 1 * 103/mcL is present; immature erythrocytes and granulocytes Chemotaxis migration in response to a chemical signal or can be present. There are less than 5% blasts in the peripheral blood. stimulation. The bone marrow is hypercellular with proliferation of abnormal Chimerism state of being when cells from two different zygotes are myelocytes, promonocytes, and monoblasts, and there are less than expressed in one individual. 20% blasts. Chloride shift phenomenon in which a plasma chlorine ion diffuses Chronic neutrophilic leukemia (CNL) myeloproliferative neoplasm into the erythrocyte when a free bicarbonate ion diffuses from the (MPN) characterized by a sustained increase in neutrophils in the erythrocyte into the plasma. peripheral blood with a slight shift to the left. The Ph chromosome and BCR/ABL1 translocation are absent. Cholecystitis inflammation of the gallbladder. Chronic nonspherocytic hemolytic anemia group of chronic condi- Cholelithiasis formation of calculi or bile stones in the gallbladder tions marked by a deficiency of red blood cells or of hemoglobin in or bile duct. the blood and premature erythrocyte destruction. Spherocytes are CHr reticulocyte hemoglobin content provided by Advia 120 and not readily found, which is helpful when differentiating these condi- 2120 System by Bayer Diagnostics; analogous to the MCH index of tions from hereditary spherocytosis. erythrocytes. Chylous body effusion that has a milky, opaque appearance from Chromatid structure of DNA during G0 and G1 of the cell cycle. the presence of lymph fluid and chylomicrons. After S phase, DNA has been replicated, and the chromosome Circulating inhibitor (anticoagulant) acquired pathologic protein, consists of two parallel, identical chromatids held together at the primarily immunoglobulins (IgG or IgM) with antibody specificity centromere. toward a factor involved in fibrin formation. Circulating inhibitors Chromogenic assay spectrophotometric measurement of an that interfere with the factor’s activity are associated with a number enzyme’s activity based on the release of a colored pigment fol- of conditions, such as hemophilia, autoimmune diseases, malignan- lowing enzymatic cleavage of the pigment-producing substrate cies, certain drugs, and viral infections. (chromogen). Circulating leukocyte pool population of neutrophils actively circu- Chromosome nuclear structure seen during mitosis and meio- lating within the peripheral blood stream. sis consisting of supercoiled DNA with histone and nonhistone Circulating pool See Circulating leukocyte pool. proteins; consists of two identical (sister) chromatids attached at the Clinical and Laboratory Standards Institute (CLSI) volunteer- centromere. driven organization that promotes the development and use of Chronic basophilic leukemia rare myeloproliferative neoplasm voluntary laboratory standards and guidelines; its mission is “to (MPN). It is characterized by an extreme increase in basophils in develop best practices in clinical and laboratory testing and promote the peripheral blood. The cell of origin is the common m yeloid their use throughout the world using a consensus-driven process that progenitor cell or the CFU-Baso, a bipotential progenitor balances the viewpoints of industry, government, and the healthcare cell capable of differentiating into either basophil or mast cell professions.” (http://www.clsi.org/Content/NavigationMenu/ lineages. AboutCLSI/VisionMissionandValues/Vision_Mission_Value.htm). Chronic eosinophilic leukemia, not otherwise specified (CEL- Clinical Laboratory Improvement Amendments of 1988 (CLIA NOS) clonal myeloproliferative neoplasm that presents with ’88) regulations that mandate standards in clinical laboratory eosinophilia of 1.5 * 103/mcL or greater in blood that is not classi- operations and testing signed into federal law in 1988. fied as another neoplastic condition and does not have the PDGFRA, Clonal hematopoiesis of indeterminate potential (CHIP) PDGFRB, or FGFR1 mutations. analogous to monoclonal gammopathy of undetermined Chronic idiopathic thrombocytopenic purpura (ITP) immune form significance; a condition in which there is a somatic mutation found of thrombocytopenia that occurs most often in young adults and in hematopoietic cells that leads to clonal expansion of the abnormal lasts longer than 6 months. cell; there is no cytopenia or dysplasia typical of MDS. 1184 Glossary Clonal hypereosinophilia progenitor cell disorder of eosinophils Colony-forming unit visible aggregation (in vitro) of cells that that is classified as either myeloid and lymphoid neoplasms with developed from a single stem cell. eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 Colony-forming unit-erythroid (CFU-E) unipotential stem cell or as chronic eosinophilic leukemia, not otherwise specified derived from the BFU-E. It has a high concentration of EPO- (CEL-NOS). membrane receptors and with EPO stimulation transforms into a Clonality presence of identical cells derived from a single progeni- pronormoblast, the earliest recognizable erythroid precursor. tor. It can be detected by identifying only one of the immunoglobu- Colony-stimulating factor cytokine that stimulates the growth of lin light chains (k or l) on B cells or the presence of a population of immature leukocytes in the bone marrow. cells with a common phenotype. Column chromatography laboratory separation method based on Clonogenic ability of a cell to form a clone. the differential distribution of a liquid or gaseous sample (mobile Clot extravascular coagulation whether occurring in vitro or in phase) that flows through a sequence of specific substance (station- blood shed into the tissues or body cavities. ary phase). Depending on the chemical characteristics of the station- ary phase, the substance of interest can bind to the stationary phase Clot retraction cohesion of a fibrin clot that requires adequate, func- and remain in the column or directly pass through the column and tionally normal platelets. Retraction of the clot occurs over a period remain in the mobile phase. If the substance remains in the column, a of time and results in the expression of serum and a firm mass of second mobile phase (elution buffer) is used to release the substance cells and fibrin. from the stationary phase and allow it to pass through the column. Cluster analysis method of classifying floating thresholds of large Commitment state of two cells derived from the same precursor cell group of specific cell populations based on size and staining or each of which takes a separate route of development. absorption characteristics. It uses an instrument that can accom- modate for shifts in abnormal cell populations from one sample to Committed/progenitor cell parent or ancestor organism that dif- another sample. ferentiates into one cell line. Cluster of differentiation (CD) designation that refers to unique Common pathway one of the three interacting pathways in the cell surface proteins that are each assigned a different number; coagulation cascade. The common pathway includes three rate- several different antibodies that recognize these unique antigens limiting steps: (1) activation of factor X by the intrinsic and extrinsic are available from different companies and often are given a pathways, (2) conversion of prothrombin to thrombin by activated unique company-specific designation (e.g., Leu-4 and OKT3); factor X, and (3) cleavage of fibrinogen to fibrin. these antibodies that recognize the same antigen are grouped Comparative genomic hybridization (CGH) assay that can be used into a cluster of differentiation and assigned a specific, unique to analyze changes in chromosome copy number (copy number number (e.g., CD3) by an international group. Cellular e xpression variants) and critical regions of the DNA for well-defined genetic of a CD protein may not be specific to one cell or one lineage; abnormalities. however, CD proteins are useful for the characterization of cell phenotypes. Compensated hemolytic disease disorder in which the erythro- cyte life span is decreased but the bone marrow is able to increase CLSI See Clinical and Laboratory Standards Institute. erythropoiesis enough to compensate for the decrease; anemia does Coagulation factor soluble inert plasma protein that interacts to not develop. form fibrin after an injury. Compensation process of adjusting the settings on the flow Cobalamin cobalt-containing complex that is common to all sub- cytometer or performing a mathematical correction for overlap of groups of the vitamin B light emitted by several fluorochromes either before or after the data 12 group. are collected. Codocyte See Target cell. Competency assessment mechanism for determining the requisite Codon sequence of three nucleotides that encodes a particular ability that personnel have to perform a given laboratory procedure. amino acid. It includes recognition of specimen collection errors, interpretation Coefficient of determination (r2) statistic that represents the square of test results to detect possible instrument or specimen problems of the correlation coefficient. It is a measure of the strength of the and of quality control results, investigation of instrument or speci- relationship between two data sets. men problems, and proper reporting of results. Coefficient of variation relative standard deviation or standard Complement any one of the 11 serum proteins that causes lysis of deviation expressed as a percentage of the mean for a set of data. the cell membrane when sequentially activated. Cofactor coagulation component that functions together with other Complementary DNA synthetic DNA transcribed from an RNA coagulation factors. It is required for the conversion of specific template by the enzyme reverse transcriptase. Also known as cDNA. zymogens to the active enzyme form. Complete blood count (CBC) hematology screening test that Coincidence in an electronic cell counter, a phenomenon when two includes the white blood cell (WBC) count, red blood cell (RBC) or more cells cross the sensing zone at the same time and are count, hemoglobin, hematocrit, and, often, platelet count. It can also evaluated as only one cell. include red cell indices. Cold agglutinin disease See Cold agglutinin syndrome. Compression syndrome condition of altered physiological function of an organ or tissue because of impingement by an abnormal mass. Cold agglutinin syndrome (CAS) condition associated with the presence of cold-reacting autoantibodies (IgM) directed against Compound heterozygote individual possessing two different abnor- erythrocyte surface antigens. This causes clumping of the red cells at mal alleles of a gene. room or lower temperatures. Conditioning regimen treatment process in which high-dose Collagen major protein of the white fibers of connective tissue, chemotherapy and/or irradiation are given to a patient before stem cartilage, and bone. It is exposed to circulating platelets and acts as a cell transplantation. substrate for the adhesion and activation of platelets. Congenital present at birth. Collagen binding assay procedure used to differentiate VWD type Congenital aberration chromosome abnormality (either numerical 2A and 2B from type 2M. Test primarily used to help differentiate or structural) that is present at the time of birth in all cell lines or in VWD variants. several cell lines in the case of mosaicism. Glossary 1185 Congenital amegakaryocytic thrombocytopenia (CAMT) condi- Cross-reacting material positive (CRM+) description of function- tion present at birth with decreased marrow megakaryocytes and ally defective clotting factor that can be identified by immunologic decreased platelets in the peripheral blood which eventually converts means. into bone marrow failure and aplastic anemia. Most cases are caused Cross-reacting material reduced (CRMR) description of defective by mutations of the gene for the thrombopoietin receptor (c-mpl). clotting factor that is identified by an equal decline of both func- Congenital Heinz body hemolytic anemia inherited disorder char- tional and immunological assays. acterized by anemia resulting from decreased erythrocyte life span. Cryoprecipitate preparation of proteins containing fibrinogen, von Erythrocyte hemolysis results from the precipitation of hemoglobin Willebrand factor, and factor

VIII prepared by freezing and thawing in the form of Heinz bodies, which damage the cell membrane and plasma; used for replacement therapy in patients with hemophilia A cause cell rigidity. and von Willebrand disease. Congenital thrombocytopenia with radioulnar synostosis Cryopreserved preserved cell stored at very low temperatures while (CTRUS) disorder present at birth that presents with decreased maintaining cell viability. marrow megakaryocytes and decreased platelets in the peripheral blood, which eventually converts into bone marrow failure and Cryosupernatant product that lacks large VWF multimers that are aplastic anemia. Most cases are caused by mutations within the present in fresh frozen plasma yet still contains the VWF-cleaving HOXA11 gene (which codes for a regulatory protein involved in the protease missing in thrombotic thrombocytopenic purpura (TTP) development of hematopoietic and bone tissue). patients. Consolidation therapy second phase of cancer chemotherapy whose Culling spleen’s filtration and destruction of senescent/damaged function is to damage or kill those malignant cells that were not red cells. destroyed during the induction phase. Current Procedure Terminology (CPT) coding system in which Constitutional aberration genetic abnormality present in every cell numbers are assigned to laboratory tests (as well as medical, surgi- in a patient’s body. cal, and other diagnostic services) by the American Medical Associa- tion’s CPT Editorial Panel. Used for billing and record keeping. Constitutional cytogenetic aberration genetic abnormality found in every cell of the body. Cyanosis bluish color of the skin and mucous membranes that develops as a result of excess deoxygenated hemoglobin in the blood. Consumption coagulopathy condition resulting from activation of the coagulation system in which the clotting factors, inhibitors, Cyclins/Cdk kinase protein that regulates the transition between the fibrinolytic components, and platelets are consumed faster than they various phases of the cell cycle. can be synthesized. Also known as disseminated intravascular coagula- Cytochemistry chemical staining procedure used to identify various tion (DIC). constituents (enzymes and proteins) within white blood cells. It is Contact group coagulation factors in the intrinsic pathway involved useful in differentiating blasts in acute leukemia, especially when with the initial activation of the coagulation system including factors morphologic differentiation on Romanowsky-stained smears is XII, XI, prekallikrein, and high-molecular-weight kininogen. Activa- impossible. tion of the factors requires contact with a negatively charged surface. Cytogenetic remission absence of recognized cytogenetic abnormal- Continuous flow analysis automated method of analyzing blood ities associated with a given neoplastic disease (previously identified cells that allows measurement of cellular characteristics such as in a patient) after therapy. individual cells that flow singly through a laser beam. Cytogenomic microarray analysis molecular genetic assessment Contour gating subclassification of cell populations based on two technique that can be used to examine gene composition. characteristics such as size (x-axis) and nuclear density (y-axis) and Cytokine protein produced by many cell types; it modulates the the frequency (z-axis) of that characterized cell type. This infor- function of other cell types. The group includes interleukins, colony- mation is used to create a three-dimensional plot and to draw a stimulating factors, and interferons. line along the valley between two peaks to separate the two cell populations. Cytomegalovirus (CMV) herpes virus that replicates only in human cells; has a widespread distribution and is spread by close contact Correlation coefficient (r) multiplier or factor that measures the with an infected person. distribution of data about the estimated linear regression line. Cytoplasm protoplasm of a cell outside the nucleus. Coverglass smear thin film of blood prepared by placing a drop of blood in the center of one cover glass and then placing a second cover Cytotoxic T cell (Cytotoxic T lymphocyte) effector cell produced glass on top of the blood at a 45° angle to the first cover glass. The from T immunoblasts. two cover glasses are pulled apart, creating two cover glass smears. Dabigatran (Pradaxa) anti-IIa inhibitor used in treating patients Critical area (optimal counting area) region of the blood smear in with atrial fibrillation, and other hypercoagulable states. It is an which erythrocytes are just touching but not overlapping; used for orally administered direct oral anticoagulant. morphologic evaluation and identification of cells. Dacryocyte type of poikilocyte that is shaped like a teardrop; also Critical limit critical high and low value for a test result. Values called a dacrocyte or teardrop. See Teardrop. above or below these values pose a life-threatening situation and are DcytB duodenal cytochrome–B reductase; ferric reductase that termed panic values. decreases ferric iron to the ferrous state at the enterocyte brush border. Critical value laboratory result that reflects a life-threatening situ- D-dimer cross-linked fibrin degradation product that is the result ation determined by a physician and for which an intervention is of plasmin’s proteolytic activity on a fibrin clot. The presence of possible. D-dimers is specific for fibrinolysis. Crossover reciprocal exchange of genetic material between chro- Decay-accelerating factor regulating complement protein found on matids; normally occurs in meiosis to increase the diversity of the cell membranes that accelerates decay (dissociation) of membrane- species. bound complement (C3bBb). Its absence leads to excessive sensitiv- Cross-reacting material negative (CRM−) description of defective ity of these cells to complement lysis. clotting factor that can be identified by both abnormal functional Deep vein thrombosis (DVT) formation of a blood clot in the deep and immunologic tests. veins (usually a leg vein). 1186 Glossary Degranulation process in phagocytosis by which the neutrophil thrombin diluted with normal human plasma and a set of calibra- granules fuse with the phagosome and release their contents. tors specific for the medication being tested. Dehydrated hereditary stomatocytosis (DHS) horizontal red blood 2,3-diphosphoglycerate (2,3-DPG) See 2,3-bisphosphoglycerate cell membrane defect in which the cell is abnormally permeable to (2,3-BPG). cations; net loss of intracellular K+ exceeds the passive Na+ influx, Diploid number of chromosomes in somatic cells that are 2n. For and net intracellular cation and water content are thus decreased; human cells, 2n = 46. the cell dehydrates and appears targeted, contracted, and spiculated. Also known as hereditary xerocytosis. Direct antiglobulin test (DAT) laboratory test used to detect the presence of antibody and/or complement that is attached to the Delayed bleeding symptom of severe coagulation factor disorders erythrocyte. It uses antibody directed against human immunoglobulin in which a wound bleeds a second time after initial stoppage. It and/or complement. Also called the antihuman globulin (AHG) test. occurs because the primary hemostatic plug is not adequately stabilized by the formation of fibrin. Direct oral anticoagulants (DOACs) medications that specifically target the enzymatic activity of thrombin (FIIa) and factor Xa, used Delta check comparison of current hematology results to the most to prevent the development of blood clots. recently reported previous result for a given patient; helps detect certain random errors. Disseminated intravascular coagulation (DIC) complex condition in which the normal clotting process is altered (resulting in systemic Delta (d) storage pool disease autosomal dominant disorder charac- rather than localized activation) by an underlying condition. Result- terized by a decrease in dense granules in the platelets. ing complications can include thrombotic occlusion of vessels, Demarcation membrane system cytoplasmic membrane system in bleeding, and ultimately organ failure. DIC is initiated by multiple the megakaryocyte that separates small areas of the cell’s cytoplasm triggers, most involving damage to the endothelial lining of vessels. that eventually become platelets. DMT1 integral membrane protein that transports ferrous iron across Demargination movement of neutrophils that are loosely attached the apical enterocyte plasma membrane. to endothelial cells lining the blood vessels into the circulation; can DNA (deoxyribonucleic acid) blueprint that cells use to catalog, cause a pseudo-neutrophilia. express, and propagate information; the fundamental substance Demyelination destruction, removal, or loss of the lipid substance of heredity that is carried from one generation to the next. It is a that forms a myelin sheath around the axons of nerve fibers; a char- double-stranded molecule composed of complementary nucleotide acteristic finding in vitamin B12 deficiency. sequences. The two strands of the substance are held together by hydrogen bonds formed according to the following rules of comple- Dense granule small particle in platelet that stores nonmetabolic mentary nucleotide pairing: G bonds with C; A bonds with T; other ADP, calcium, and serotonin as well as other compounds released combinations cannot bond. from activated platelets. DNA index (DI) DNA content of tumor cells relative to a diploid Dense tubular system (DTS) complex made up of membranes in the population of cells. It is calculated as the DNA content of cells in platelet that originate from the smooth endoplasmic reticulum of the the tumor in the G0/G1 phase of the cell cycle relative to the DNA megakaryocyte. It is one of the storage sites for calcium ions within content of G0/G1 cells in a diploid control. platelets, and its channels do not connect with the platelet’s surface. DNA sequencing determining the nucleotide string in a segment of Densitometer instrument that can be used to measure the density of the blueprint that cells use to catalog, express, and propagate infor- materials using different wavelengths of light and filters. mation by replicating its strands and monitoring the order in which Densitometry laboratory testing method that determines the pattern labeled nucleotides are added to the new strands. and concentration of protein fractions separated by electrophoresis Döhle body oval aggregate of rough endoplasmic reticulum that by measuring the amount of light absorbed by each dye-bound stains light gray-blue (with Romanowsky stain) found within the protein fraction as it passes a slit through which light is transmitted. cytoplasm of neutrophils and eosinophils; associated with severe The amount of light absorbed (optical density) is directly propor- bacterial infection, pregnancy, burns, cancer, aplastic anemia, and tional to the protein’s concentration. toxic states. Deoxyhemoglobin hemoglobin without oxygen. Donath-Landsteiner antibody biphasic IgG associated with par- Diagnosis Related Group (DRG) code system of about 500 medical oxysmal cold hemoglobinuria; reacts with erythrocytes in capillar- conditions developed for Medicare as a part of the prospective pay- ies at temperatures below 15 °C and fixes complement to the cell ment system for inpatients. Reimbursement for care of hospitalized membrane. Upon warming, the terminal complement components patients by almost all payers is not for each test or procedure but for on erythrocytes are activated, causing cell hemolysis. the overall diagnosis. It is assigned when the patient is discharged Double heterozygous having two different mutated genes that and is severity adjusted (MS-DRG). result in combination disorders. In the hemoglobinopathies, the Diamond-Blackfan anemia (DBA) congenital, progressive eryth- most common structural hemoglobins inherited with thalassemia rocyte hypoplasia that occurs in very young children. There is no are HbC, HbS, and HbE. leukopenia or thrombocytopenia. Downey cell outdated term used to describe morphologic variations Diapedeses of the reactive lymphocyte. passage of blood cells through the unruptured capillary wall. For leukocytes, this involves active locomotion. Drepanocyte (sickle cell) elongated, crescent-shaped erythrocytes with pointed ends. They form when hemoglobin S is present within the Diapedesis See Diapedeses. erythrocyte, deoxygenates, and polymerizes. At low oxygen tension, Differentiation discernment of the appearance of various properties low pH, and increased temperature, deoxy-HbS molecules polymerize in cells that were initially equivalent. into rigid aggregates and the cell assumes the “sickled shape.” Dilute Russell viper venom time (dRVVT) test recommended as Drug-induced hemolysis rupture or destruction of red blood cells the screening test for detection of lupus anticoagulant; Russell viper that occurs when a drug attaches to the erythrocyte membrane and venom (RVV) activates FX, resulting in clot formation. If LAs/aPLs are alters it in some way. present, the patient’s dRVVT is longer than that of the normal control. Drug-induced hemolytic anemia hemolytic disorder precipitated Dilute thrombin time assay that is used to test for the presence of by ingestion of certain drugs. The process can be immune mediated dabigatran, an anti-FIIa inhibitor. This is accomplished by using or nonimmune mediated. Glossary 1187 Drumstick See Barr body. Endothelial cell flat cells that line the cavities of the blood and Dry tap result of a bone marrow procedure when bone marrow lymphatic vessels, heart, and other related body cavities. cannot be aspirated. This situation can be caused by inadequate Endothelial cell protein C receptor (EPCR) molecule on the technique or alterations in the bone marrow architecture such as membrane of endothelial cells of larger vessels that binds and extensive fibrosis or very increased cellularity. immobilizes protein C, augmenting its activation by the thrombin: Dura mater dense membrane covering the central nervous system;

thrombomodulin complex. outermost layer of the meninges. End point detection place where the completion of a reaction is Dutcher body intranuclear membrane-bound inclusion found in marked by some change such as formation of a clot or color change. plasma cells. The body stains with periodic acid-Schiff (PAS), indi- Engraftment process of infusing stem cells into the bone marrow cating that the tissue contains glycogen or glycoprotein. It appears microenvironment resulting in hematopoietic recovery. as finely distributed chromatin, nucleoli, or intranuclear inclusions. Enzyme protein that catalyzes a specific biochemical reaction but is Dysfibrinogenemia hereditary condition in which the fibrinogen not itself altered in the process. molecule has a structural alteration. Enzyme-linked immunosorbent assay analysis used to test for pro- Dyshematopoiesis abnormal formation and/or development of teins using immunologic principles; one use in coagulation testing is blood cells within the bone marrow leading to qualitative morpho- to detect antibodies against heparin–PF4 complexes. logic abnormalities in one or more cell lineages. Eosinophil mature granulocyte cell characterized by the presence Dyspepsia symptom caused by abnormalities in the process of of large acidophilic granules that are pink to orange pink with digestion. Romanowsky stains. The cell is 12917 mcM (mm) in diameter, Dysplasia abnormal cell development. and the nucleus has two to three lobes. Granules contain acid phosphatase, glucuronidase cathepsins, ribonuclease, arylsulfatase, Dyspoiesis abnormal development of blood cells frequently charac- peroxidase, phospholipids, and basic proteins. Eosinophils have a terized by asynchrony in nuclear to cytoplasmic maturation and/or concentration of less than 0.45 * 103/mcL in the peripheral blood. abnormal granule development. The cell membrane has receptors for IgE and histamine. Ecarin clotting time (ECT) meizothrombin generation test that can Epigenetics related to heritable changes in gene expression not be used to measure the activity of the direct thrombin inhibitors caused by changes in DNA sequence. such as agatroban but is likely to be useful with the introduction of the new oral direct thrombin inhibitors. Epistaxis hemorrhage from the nose. Ecchymoses bruise (bluish-black discoloration of the skin) that is Epitope structural portion of an antigen that reacts with a specific larger than 3 mm in diameter caused by bleeding from arterioles antibody. Also called antigenic determinant. into subcutaneous tissues without disruption of intact skin. Epstein-Barr virus (EBV) infectious agent that attaches to the mem- Echinocyte spiculated erythrocyte with short, equally spaced brane surface of B lymphocytes by a specific receptor designated projections over the entire outer surface of the cell. CD21. Edematous marked by the swelling of body tissues from the Error detection identification of a laboratory’s multirule quality accumulation of tissue fluid. control procedure to detect a true mistake in the testing system and Effector lymphocyte antigen-stimulated white blood cell that reject the control run. mediates the efferent arm of the immune response. Erythroblast nucleated erythrocyte precursor in the bone marrow. If Effector T lymphocyte T lymphocyte that has encountered normal, it can be referred to as normoblast. antigen and undergone blast transformation; morphologically Erythroblastic island composite of erythroid cells in the bone mar- indistinguishable from original T lymphocyte. row that surrounds a central macrophage. These cell groups are Efficacy ability to produce the desired effect (e.g., anticoagulation). usually disrupted when bone marrow smears are made but can be found in erythroid hyperplasia. The least mature cells are closest to Effusion abnormal accumulation of fluid. the center and the more mature cells are on the periphery. The cen- Egress action of going out or exiting; describes the exit of blood cells tral macrophage is thought to transfer iron to the developing cells. from the blood to the tissue. Erythroblastosis fetalis hemolytic anemia in newborns caused by ELISA See Enzyme-linked immunosorbent assay. an antigen–antibody reaction when fetal-maternal blood group Elliptocyte abnormally shaped erythrocyte that is an oval to elon- incompatibility involves the Rh factor or ABO blood groups. gated ellipsoid with a central area of pallor and hemoglobin at both Antibody from the mother traverses the placenta and attaches to ends. Also known as ovalocyte, pencil cell, and cigar cell. antigens on the fetal cells. Embolism blockage of an artery by a piece of blood clot or other Erythrocyte red blood cell (RBC) that has matured to the non- foreign matter, resulting in obstruction of blood flow to the tissues. nucleated stage. The cell is about 7 mcM (mm) in diameter and con- tains the respiratory pigment hemoglobin, which readily combines Embolus piece of blockage or other foreign matter that circulates with oxygen to form oxyhemoglobin. The cell develops from the in the blood stream and usually becomes lodged in a small vessel pluripotential stem cell in the bone marrow under the influence of obstructing blood flow. the hematopoietic growth factor erythropoietin and is released to the Endomitosis round of nuclear DNA synthesis without nuclear or peripheral blood as a reticulocyte. Its average life span is about 120 cytoplasmic division. days after which it is removed by cells in the mononuclear–phago- Endoplasmic reticulum (ER) cytoplasmic organelle in eukaryotic cyte system. The average concentration is about 5 * 106/mcL for cells that consists of a network of interconnected tubes and flat- males and 4.5 * 106/mcL for females. tened membranous sacs. If the ER has ribosomes attached, it is Erythrocytosis abnormal increase in the number of circulating known as granular or rough endoplasmic reticulum (RER), and if erythrocytes as measured by the erythrocyte count, hemoglobin, or ribosomes are not attached, it is known as smooth endoplasmic hematocrit. reticulum (SER). Erythroferrone (ERFE) erythroid regulator induced by erythropoi- Endosteum membrane that lines the bone medullary cavity that etic stimulation and secreted by differentiating erythroblasts; acts on contains the bone marrow. the liver to suppress hepcidin synthesis and increase iron delivery 1188 Glossary from dietary absorption and stores; functions as a hormone linking Factor VIII/VWF complex plasma form of VWF associated with erythropoiesis and iron regulation. FVIII. Erythron summation of the stages of erythrocytes in the marrow or False rejection failure of a control run that is not truly out of control peripheral blood and within vascular areas of specific organs such as whose result falls outside the control limits or violates a Westgard the spleen. rule; is caused by the inherent imprecision of the test method. Erythrophagocytosis phagocytosis of an erythrocyte by a histio- Fanconi anemia (FA) autosomal recessive disorder characterized by cyte that can be seen within its cytoplasm as a pink globule or, if chromosomal instability including breakage, gaps, rearrangements, digested, a clear vacuole on stained bone marrow or peripheral exchanges, and duplications. Patients have a complex assortment blood smears. of congenital anomalies in addition to a progressive bone marrow Erythropoiesis formation and maturation of erythrocytes in the hypoplasia and an increased predisposition to develop leukemia bone marrow. It is under the influence of the hematopoietic growth and cancer. factor erythropoietin. Favism sensitivity to a species of bean, Vicia faba; commonly found Erythropoietin hormone secreted by the kidney that regulates eryth- in Sicily and Sardinia in individuals who have inherited glucose- rocyte production by stimulating the stem cells of the bone marrow 6-phosphate dehydrogenase (G6PD) deficiency. Its characteristics to mature into erythrocytes. Its primary effect is on the committed are fever, acute hemolytic anemia, vomiting, and diarrhea after stem cell CFU-E. ingestion of the bean or inhalation of the plant pollen. Essential thrombocythemia (ET) myeloproliferative neoplasm Fee for service payment method for health care in which consumers primarily affecting the megakaryocytic lineage in the bone marrow. choose their own health care providers and the provider determines There is extreme thrombocytosis in the blood (minimum: greater the fees for the services. The patient or a third-party payer pay the than 450 * 103/mcL, usually greater than 1000 * 103/mcL). Also fees. called primary thrombocythemia, hemorrhagic thrombocythemia, and Ferritin iron-phosphorus-protein that forms a compound when megakaryocytic leukemia. iron complexes with the protein apoferritin. It is a storage form of Euchromatin region of the chromosome that contains genetically iron found primarily in the bone marrow, spleen, and liver. Small active DNA, is light staining, and replicates early in the cell cycle’s S amounts can be found in the peripheral blood proportional to that phase. See Heterochromatin. found in the bone marrow. Evan’s syndrome condition characterized by a warm autoimmune Ferroportin protein that transports ferrous iron across the basolat- hemolytic anemia and concurrent severe thrombocytopenia. eral membrane of enterocytes. The only known cellular exporter of iron, it is involved in the membrane transport of iron from mac- Exchange transfusion simultaneous withdrawal of blood and infu- rophages and hepatocytes. Also known as IREG1. sion with compatible blood. Fibrin degradation product (FDP) breakdown product of fibrin Exon protein-coding DNA sequence of a gene. or fibrinogen that is produced when plasmin’s proteolytic action Extracellular matrix noncellular component of the hematopoietic cleaves this molecule. The four main products are fragments X, Y, D, microenvironment in the bone marrow. and E. The presence of fibrin degradation products indicates either fibrinolysis or fibrinogenolysis. Extramedullary erythropoiesis red blood cell production occurring outside the bone marrow. Fibrin monomer structure resulting when thrombin cleaves the A and B fibrinopeptides from the a@ and b@chains of fibrinogen. Extramedullary hematopoiesis formation and development of blood cells at a site other than the bone marrow. Fibrinogen group assemblage of coagulation factors activated by thrombin and consumed during the formation of fibrin and there- Extravascular occurring outside of the blood vessels. fore absent from serum. It includes factors I, V, VIII, and XIII; also Extrinsic pathway one of the three interacting coagulation factor called consumable group. sequences in the coagulation cascade initiated when tissue Fibrinolysis breakdown of fibrin. factor comes into contact with blood and forms a complex with FVII. The complex activates FX. The term extrinsic is used because the Fibrin polymer complex of covalently bonded fibrin monomers. The sequence of factor activation requires tissue factor, a factor outside bonds between glutamine and lysine residues are formed between of blood. terminal domains of g@chains and polar appendages of a@chains of Extrinsic Xase complex neighboring residues. complex of tissue factor and FVIIa that forms when a vessel is injured and serves to activate FX. Fibronectin extracellular-matrix glycoprotein capable of binding Exudate effusion formed by increased vascular permeability and/or heparin. decreased lymphatic resorption; indicates a true pathologic state in Fibrosis abnormal formation of fibrous tissue. the anatomic region, usually either infection or tumor. Flame cell abnormal plasma cell with reddish-purple cytoplasm. FAB classification See French-American-British classification. The red tinge is caused by the presence of a glycoprotein and the Factor V Leiden (FVL) abnormal coagulation protein formed by a purple of ribosomes. single point mutation of the FV gene (F5), involving replacement Flow chamber specimen-handling area of a flow cytometer where of Arg 506 with Gln(FVR506Q) that makes the mutant FVa molecule cells are forced into single file and directed in front of the laser resistant to inactivation by APC by altering an APC cleavage site beam. and leads to thrombophilia; FV serves as a cofactor in the APC Fluorescence in situ hybridization (FISH) technique in which inactivation of FVIIIa. FVL is a much less effective cofactor in this whole chromosomes (metaphase or interphase) are hybridized to inactivation of FVIIIa, which contributes to the thrombophilic state. a complementary probe that is labeled with a fluorochrome and Factor VIII: C assay method that determines the amount of FVIII. visualized by microscopy. Factor VIII concentrate lyophilized preparation of condensed FVIII Fluorochrome molecule excited by light of one wavelength and used for replacement therapy of FVIII in patients with hemophilia A. emits light of a different wavelength. Factor VIII inhibitor IgG immunoglobulin with antibody specific- Folate large group of compounds whose parent substance is folic ity to FVIII that inactivates the factor. The antibodies are time and acid; composed of three parts: pteridine, p-amino-benzoic acid, and temperature dependent. It is associated with hemophilia. chain of glutamic acid residues. The function of the active form, Glossary 1189 tetrahydrofolate (THF), is to transfer one-carbon compounds from 46 chromosomes including 22 pairs of autosomes numbered 1–22 donor molecules to acceptor molecules in intermediary metabolism. and the two sex chromosomes. Folic acid parent substance of folates. Genomics study of all nucleotide sequences, including structural Fondaparinux sodium (pentasaccharide) synthetic heparinoid that units of heredity, regulatory sequences, and noncoding DNA inhibits thrombin generation by selectively inhibiting factor Xa. segments in an organism’s chromosomes. Forward light scatter laser light spread in a forward

direction in a Genotype hereditary constitution of an individual, often referring to flow cytometer; is related to particle size (e.g., large cells produce a particular DNA locus. more forward spread than smaller cells). Germinal center lightly staining core of a lymphoid follicle where B Free erythrocyte protoporphyrin (FEP) protoporphyrin within cell activation occurs. an erythrocyte that is not complexed with iron. Its concentration Germline cell lineage that pass their genetic material to progeny increases in iron-deficient states. In the absence of iron, erythrocyte Glanzmann thrombasthenia rare hereditary platelet disorder protoporphyrin combines with zinc to form zinc protoporphyrin (ZPP). characterized by a genetic mutation in one of the genes coding for French-American-British (FAB) classification system for the glycoproteins IIb or IIIa and resulting in the inability of platelets hematopoietic disorders based on cell lineage as determined by the to aggregate. morphology and results of cytochemical stains; has been replaced by Global testing analyzing the entire hemostatic process including the WHO 2008 classification. coagulation, anticoagulant effects, fibrin formation and stabilization, Fresh frozen plasma (FFP) colorless fluid portion of blood that is clot retraction, and fibrinolysis on a whole blood sample by use of a frozen at -15°C or colder within 6 hours of collection; formed by specialized instrument. removal of all cellular components. Globin protein portion of the hemoglobin molecule. F-test statistical tool used to compare features of two or more sets of Glossitis inflammation of the tongue. data. Glucose-6-phosphate-dehydrogenase (G6PD) enzyme within Functional anemia decrease in hemoglobin concentration accompa- erythrocytes that is important in carbohydrate metabolism. It dehy- nied by a decrease in oxygen delivery to the tissues. drogenates glucose-6-phosphate to form 6-phosphogluconate in the Functional hyposplenism reduced splenic function not caused by hexose monophosphate shunt. This reaction produces NADPH from the loss of splenic tissue but by the accumulation of cells seques- NADP and provides reducing power to the erythrocyte, protecting tered in the spleen. the cell from oxidant injury. Functional iron-deficiency form of iron restricted erythropoiesis in Glutathione tripeptide that takes up and gives up hydrogen and which iron stores are sufficient but in the presence of acute increased prevents oxidant damage to the hemoglobin molecule. A deficiency erythropoietic demand, enough iron cannot be mobilized from stor- of it is associated with hemolytic anemia. age sites to meet the demand of developing erythroblasts. Glycocalin portion of glycoprotein Ib of the platelet membrane g@Carboxylation post-translational modification of a protein that external to the platelet surface; contains binding sites for von adds an extra carboxyl group (COOH) to the g@carbon of the glu- Willebrand factor and thrombin. tamic acid residues; requires reduced vitamin K; the prothrombin Glycocalyx amorphous coat of glycoproteins and mucopolysac- group of coagulation factors as well as protein C, S, and Z require charides covering the surface of cells, particularly the platelets and this conversion to become active. endothelial cells. Gammopathy abnormal condition with an increase in serum Glycolysis anaerobic conversion of glucose to lactate and pyruvic immunoglobulins. acid resulting in the production of energy (ATP). Gating in flow cytometry, the process of isolating cells with the Glycoprotein Ib substance on the platelet surface that contains the same light-scattering or fluorescence properties by electronically receptor for von Willebrand factor and is critical for initial adhesion placing a gate around them. of platelets to collagen after an injury. GDF15 growth differentiation factor 15; a member of the bone mor- Glycoprotein IIb/IIIa complex of membrane proteins on the platelet phogenetic protein (BMP) family; may be released by erythroblasts surface that is functional only after activation by agonists and then under stress and apoptosis; suppresses hepcidin synthesis; increased becomes a receptor for fibrinogen and von Willebrand factor. It is during ineffective erythropoiesis. essential for platelet aggregation. Gene functional segment of DNA that serves as a template for RNA Glycosylated hemoglobin (HbA1c) hemoglobin that has transcription and protein translation. Regulatory sequences control glucose irreversibly attached to the terminal amino acid of the its expression so that only a small fraction of the estimated 100,000 b@chains. units is ever transcribed by a given cell. Golgi apparatus cytoplasmic organelle composed of flattened Gene cluster group of closely linked units of heredity that can be sacs or cisternae arranged in stacks. It functions to concentrate affected as a group. and package the products of secretory cells. It does not stain with Gene promoter DNA sequence that RNA polymerase binds to in Romanowsky stains and appears as a clear area usually adjacent to order to begin transcription of a unit of heredity. the nucleus. Gene rearrangement process in which segments of DNA are cut Gower hemoglobin embryonic intracellular erythrocyte protein that and spliced to produce new DNA ones. During normal lymphocyte is detectable in the yolk sac for up to 8 weeks of gestation; composed development, reorganization of the immunoglobulin and the T cell of two zeta (z) chains and two epsilon (e) chains. receptor DNA results in new sequences that encode the antibody Graft-versus-host disease (GVHD) tissue injury secondary to and surface antigen receptor proteins necessary for immune HLA-mismatch grafts resulting from immunocompetent donor T function. lymphocytes that recognize HLA antigens on the host cells and initi- Gene therapy treatment that places a normally functioning unit of ate a secondary inflammatory response. heredity into an affected individual’s appropriate target cell. Graft versus leukemia (GVL) favorable effect seen when immuno- Genome total aggregate of inherited genetic material that in competent donor T cells present in the allograft destroy the recipi- humans consists of 3 billion base pairs of DNA divided among ent’s leukemic cells. 1190 Glossary Granulocytopenia decrease in neutrophils below the reference Hematopoietic stem cell (HSC) blood precursor cell capable of giv- range (1.8 * 103/mcL). ing rise to all lineages of blood cells. Granulocytosis increase in neutrophils above the reference range Heme nonprotein portion of hemoglobin and myoglobin that (7.0 * 103/mcL), usually seen in bacterial infections, inflammation, contains iron nestled in a hydrophobic pocket of a porphyrin ring metabolic intoxication, drug intoxication, and tissue necrosis. (ferroprotoporphyrin); responsible for the characteristic color of Granulomatous distinctive pattern of chronic reaction in which the hemoglobin. predominant cell type is an activated macrophage with epithelial- Hemochromatosis clinical condition resulting from abnormal iron like (epithelioid) appearance. metabolism characterized by accumulation of iron deposits in body Gray platelet syndrome (a−storage pool disease) rare hereditary tissues. disorder characterized by the lack of a@granules of thrombocytes. Hemoconcentration increased concentration of blood components Hairy cell neoplastic cell of hairy cell leukemia characterized by from loss of plasma from the blood. circumferential, cytoplasmic, hair-like projections. Hemoglobin intracellular erythrocyte protein that is responsible Ham test specific laboratory test for paroxysmal nocturnal hemo- for transporting oxygen and carbon dioxide between the lungs and globinuria (PNH). When erythrocytes from a patient with PNH are body tissues. incubated in acidified serum, the cells lyse as the result of comple- Hemoglobin distribution width measure of the distribution of ment activation. Also called acid-serum lysis test. intracellular erythrocyte protein within an erythrocyte population. It Haploid having an n number of chromosomes in a gamete; consist- is derived from the hemoglobin histogram generated by the Bayer/ ing of one of each of the autosomes and one of the sex chromo- Technicon instruments. somes. For human cells, it is 23; n = 23. Hemoglobin electrophoresis method of identifying intracellular Haplotype one of the two alleles at a genetic locus. erythrocyte proteins based on differences in their electrical charges. Haptoglobin serum a2@globulin glycoprotein that transports free Hemoglobinemia presence of hemoglobin in the plasma. plasma hemoglobin to the liver. Hemoglobinopathy disease that results from an inherited abnor- HDFN See Hemolytic disease of the fetus and newborn. mality of the structure or synthesis of the globin portion of the hemoglobin molecule. Health Insurance Portability and Accountability Act (HIPAA) law that mandates health care entities to establish measures that ensure Hemoglobinuria presence of free hemoglobin in the urine. the confidentiality of patient information. Hemojuvelin (HJV) glycosylphosphatidylinositol-anchored protein Heinz body substance in the erythrocyte composed of denatured that has been shown to regulate hepcidin expression. or precipitated hemoglobin; appears as purple-staining body on Hemolysis destruction of erythrocytes resulting in the release of supravitally stained (crystal violet) blood smears. hemoglobin. In hemolytic anemia, the premature destruction of HELLP syndrome See Hemolysis, elevated liver enzymes and low erythrocytes. platelet (HELLP) syndrome. Hemolysis, elevated liver enzymes and low platelet (HELLP) Helmet cell abnormally shaped erythrocyte with one or several syndrome obstetric disorder characterized by hemolysis (H), ele- notches and projections on either end that look like horns. The shape vated liver enzymes (EL), and a low platelet count (LP); its etiology is caused by trauma to the erythrocyte. Also called keratocyte and and pathogenesis are not well understood. It can cause microangio- horn-shaped cell. pathic hemolytic anemia. Helper T cell effector lymphocyte subset that provides helper Hemolytic anemia disorder characterized by a decreased erythro- activity for B cells, macrophages, and other T cells; subsets cyte concentration from premature destruction of the erythrocyte. include Th1, Th2, TFH, Th9, Th17 Hemolytic disease of the fetus and newborn (HDFN) alloimmune Hematocrit packed cell volume of erythrocytes in a given volume disease characterized by fetal red blood cell destruction as a result of of blood following centrifugation expressed as a percentage of total incompatibility between maternal and fetal blood groups. blood volume or as a liter of erythrocytes per liter of blood (L/L). Hemolytic transfusion reaction one of two types of interaction Also referred to as packed cell volume (PCV). between foreign (nonself) erythrocyte antigens and plasma antibod- Hematogone precursor B lymphocyte present normally in the bone ies from a transfusion of blood: immediate (within 24 hours) and marrow. delayed (occurring 2 to 14 days after transfusion). Hematologic remission in a patient treated for a hematologic neo- Hemolytic uremic syndrome (HUS) disorder characterized by a plasm, it is the state of absence of neoplastic cells in the peripheral combination of microangiopathic hemolytic anemia, acute renal blood and bone marrow and the return to normal levels of hemato- failure, and thrombocytopenia. logic parameters. Hemopexin plasma glycoprotein (b@globin) that binds the heme Hematology study of formed cellular blood elements. molecule in plasma in the absence of haptoglobin. Hematoma localized collection of blood under the skin or in other Hemophilia A sex-linked (X-linked) hereditary hemorrhagic organs caused by a break in a blood vessel wall. disorder caused by a genetic mutation of the gene coding for coagulation FVIII. Hematopoiesis production and development of blood cells normal- ly occurring in the bone marrow under the influence of hematopoi- Hemophilia B sex-linked (X-linked) hereditary hemorrhagic disorder etic growth factors. caused by a genetic mutation of the gene coding for coagulation FIX. Hematopoietic microenvironment specialized, localized surround- Hemorrhage loss of a large amount of blood either internally or ing in hematopoietic organs that supports the development of externally. hematopoietic cells. Hemorrhagic disease of the newborn severe bleeding disorder in Hematopoietic progenitor cell blood precursor cell developmental- the first week of life caused by deficiency of vitamin K. ly located between stem cells and the morphologically recognizable Hemosiderin water insoluble, heterogeneous iron–protein complex blood precursor cells; includes multilineage and unilineage found primarily in the cytoplasm of cells (normoblasts and histio- cell types. cytes in the bone marrow, liver, and spleen); the major long-term Glossary 1191 storage form of iron. It is readily visible microscopically in unstained Heterochromatin region of the chromosome that contains genetical- tissue specimens as irregular aggregates of golden yellow to brown ly inactive DNA, is dark staining, and replicates late in the S phase granules. It can be visualized with Prussian-blue stain as blue gran- of the cell cycle. ules that are normally distributed randomly or diffused. Heterologous related to morphologically nonidentical chromosomes Hemosiderinuria presence of iron-containing protein in the urine that have different gene loci. as a result of intravascular hemolysis and disintegration of renal Heterophile antibody immunoglobulin that can react against a tubular cells. heterologous antigen that did not stimulate the production of immu- Hemostasis localized, controlled process that arrests bleeding after noglobulin. In infectious mononucleosis, this immunoglobulin is in an injury. response to infection with Epstein-Barr virus and reacts with sheep, Heparin polysaccharide that inhibits coagulation of blood by pre- horse, and beef erythrocytes. venting thrombin from cleaving fibrinogen to form fibrin; commer- Heterozygous related to different genes at a gene locus. cially available in the form of a sodium salt for

therapeutic use as an Hexose-monophosphate shunt (HMP) metabolic pathway that anticoagulant. converts glucose-6-phosphate to pentose phosphate. This pathway Heparin-associated thrombocytopenia (HAT) abnormal drop in the couples oxidative metabolism with the reduction of nicotinamide number of platelets associated with specific anticoagulant (heparin) adenine dinucleotide-phosphate (NADPH) and glutathione, which therapy in some patients from a nonimmune-mediated direct plate- provides the cell with reducing power and prevents injury by let activation effect. oxidants. Heparin-induced thrombocytopenia (HIT) abnormal drop in the HFE See human hemochromatosis protein. number of blood cells involved in forming blood clots associated Histogram graphical representation of the number of cells within a with anticoagulant (heparin) therapy in some patients resulting defined parameter such as size. from an immune-mediated destruction of these cells from drug (heparin)-dependent platelet-activating IgG antibodies produced HIV-I See human immunodeficiency virus type I. against the platelet factor 4 (PF4)-heparin complex. Hodgkin lymphoma malignancy that most often arises in lymph Hepcidin master iron-regulating protein that regulates iron recy- nodes and is characterized by the presence of Reed-Sternberg cells cling/balance via interaction with ferroportin; a negative regulator and variants with a background of varying numbers of benign lym- of intestinal iron absorption. phocytes, plasma cells, histiocytes, and eosinophils. The malignant cell’s origin is still controversial. Hephaestin protein that facilitates export of iron across the baso- lateral enterocyte membrane and oxidizes Fe++ iron to Fe+++ for Homologous pertaining to two morphologically identical chro- binding to apotransferrin. mosomes that have identical gene loci but can have different gene alleles because one member of the pair is of maternal origin and the Hereditary elliptocytosis autosomal-dominant condition character- other is of paternal origin. ized by the presence of increased numbers of elongated and oval erythrocytes. The abnormal shape results from a horizontal interac- Homozygous pertaining to identical genes at a gene locus. tion defect with abnormal spectrin, deficiency, or defect in band 4.1 Horizontal interaction side-by-side interrelationship involving the or deficiency of glycophorin C and abnormal band 3. proteins of the erythrocyte membrane. Hereditary erythroblastic multinuclearity with positive acidified Howell-Jolly body erythrocyte inclusion composed of nuclear rem- serum test (HEMPAS) congenital erythrocytic multinuclear- nants (DNA). On Romanowsky-stained blood smears, it appears as a ity with positive acidified serum lysis that is type II congenital dark purple spherical granule usually near the cell’s periphery. It is dyserythropoietic anemia (CDA), which is characterized by both commonly associated with megaloblastic anemia and splenectomy. abnormal and ineffective erythropoiesis. Type II is distinguished by Human hemochromatosis protein (HFE) transmembrane protein a positive acidified serum test but a negative sucrose hemolysis test. that associates with b2@microglobulin. It binds to the transferrin Hereditary pyropoikilocytosis (HPP) rare but severe hemolytic receptor (TfR) on cells. It is involved in regulating iron absorption anemia inherited as an autosomal recessive disorder. It is character- and uptake. Mutations are associated with a decrease in hepcidin, ized by marked erythrocyte fragmentation. The defect is most likely resulting in hemochromatosis. a spectrin abnormality in the erythrocyte cytoskeleton. Human immunodeficiency virus type I virus that causes acquired Hereditary spherocytosis chronic hemolytic anemia caused by an immunodeficiency syndrome (AIDS). inherited erythrocyte membrane disorder. The vertical interaction Human platelet antigen (HPA) unique alloantigens associated with defect is most commonly the result of a combined spectrin and the surface of the blood cells involved in clotting. ankyrin deficiency. The defect causes membrane instability and progressive membrane loss. Secondary to membrane loss, cells Humoral immunity protection imparted as a result of B lymphocyte become spherocytes and are prematurely destroyed in the spleen. activation. The B lymphocyte differentiates to a plasma cell that pro- The condition is usually inherited as an autosomal dominant duces antibodies specific to the antigen that stimulated the response. trait. Hybridization process in which one nucleotide strand binds to Hereditary stomatocytosis rare hemolytic anemia inherited in an another strand by forming hydrogen bonds between complemen- autosomal dominant fashion. The erythrocyte membrane is abnor- tary nucleotides. mally permeable to cations. The cell becomes overhydrated, result- Hydrodynamic focusing technique that allows cells/particles to ing in the appearance of stomatocytes or dehydration resulting in flow in a single column because of differences in the pressures of the appearance of targeted or contracted and spiculated cells. Also two columns of fluid in a flow chamber of a flow cytometer. The called hereditary hydrocytosis. See overhydrated hereditary stomatocyto- particles are contained in an inner column of sample fluid sur- sis, OHS; dehydrated hereditary stomatocytosis, DHS. rounded by a column of stream sheath fluid. The gradient between Hereditary xerocytosis congenital disorder in which the erythro- the sample and sheath fluid keeps them separate (laminar flow) and cyte is abnormally permeable to sodium and potassium with an is used to control the diameter of the column of sample fluid. The increased potassium efflux. The erythrocyte becomes dehydrated central column of sample fluid is narrowed to isolate single cells that and appears as either a target or spiculated cell. The cell is rigid pass through a laser beam like a string of beads. and becomes trapped in the spleen. Now referred to as dehydrated Hydrops fetalis genetically determined hemolytic disease (thalas- hereditary stomatocytosis (DHS). semia) resulting in production of an abnormal hemoglobin 1192 Glossary (hemoglobin Bart’s, g4) that is unable to carry oxygen. No alpha (a) Idiopathic pertaining to disorders or diseases in which the patho- globin a@chains are synthesized. There is gross edema of the entire genesis is unknown. body. Idiopathic cytopenia of undetermined significance (ICUS) condi- Hypercoagulable condition (either inherited or acquired) in which tion in patients with unexplainable, persistent cytopenia, but no there is an abnormally increased tendency toward blood clotting abnormal marrow morphology and no known MDS-associated (coagulation). somatic mutation or abnormal karyotype. Hypercoagulable state condition associated with an imbalance Idiopathic thrombocytopenic purpura (ITP) See Immune between clot-promoting and clot-inhibiting factors. leading to an thrombocytopenia. increased risk of developing thrombosis. Immature platelet fraction (IPF) modern parameter of hematology Hyperdiploid having more than 2n chromosomes per cell; for instruments that measures young reticulated platelets in the periph- human cells, this would be greater than 46. eral blood and is reported as a percentage of the total platelet count. Hypereosinophilia increase in the concentration of eosinophils in Immature reticulocyte fraction (IRF) index of reticulocyte maturity the peripheral blood greater than 0.5 * 103/mcL associated with provided by flow cytometry that can be helpful in evaluating bone parasitic infection, allergic conditions, hypersensitivity reactions, marrow erythropoietic response to anemia, monitoring anemia, and cancer, and chronic inflammatory states. evaluating response to therapy. Hypereosinophilic syndrome persistent blood eosinophilia Immune hemolytic anemia (IHA) disorder caused by premature, greater than 1.5 * 103/mcL with tissue infiltration, absence of immune-mediated destruction of erythrocytes. Diagnosis is con- clonal genetic aberrations, and no apparent cause of the increase firmed by the demonstration of immunoglobulin (antibodies) and/ in eosinophils. or complement on the erythrocytes. Hyperhomocysteinemia medical condition characterized by Immune response (IR) body’s defense mechanism that includes elevated level of homocysteine in the blood as a result of impaired producing antibodies to foreign antigens. homocysteine metabolism. It can be due to acquired or congenital Immune thrombocytopenia (ITP) autoimmune disorder in which causes. It is associated with premature atherosclerosis and arterial autoreactive antibodies bind to platelets, shortening the platelet life thrombosis. span. Platelets are decreased. Also referred to as idiopathic thrombocy- Hyperplasia enlargement of an organ or tissue caused by an topenic purpura. increase in the number of cells. This can result from an increase in Immune tolerance immune regulatory mechanisms that govern the number of cells replicating, an increase in the rate of replica- response to self-antigens. tion, or prolonged survival of cells. The cells usually maintain normal size, shape, and function. The stimulus for the prolifera- Immunoblast T or B lymphocyte that is mitotically active as a result tion can be acute injury, chronic irritation, or prolonged, increased of stimulation by an antigen. The cell is morphologically character- hormonal stimulation. In hematology, a hyperplastic bone marrow ized by a large nucleus with prominent nucleoli, a fine chromatin is one in which the proportion of hematopoietic cells to fat cells is pattern, and an abundant, deeply basophilic cytoplasm. increased. Immunocompetent having the ability to respond to stimulation by Hypersplenism disorder characterized by enlargement of the spleen an antigen. and pancytopenia in the presence of a hyperactive bone marrow. Immunoglobulin (Ig) protein produced by B lymphocytes and Hypocellularity decreased state of hematopoietic precursors in the plasma cells. It reacts with antigen and consists of two pairs of bone marrow. polypeptide chains: two heavy and two light chains linked together by disulfide bonds. Also called antibody. Hypochromic cell pertaining to reduced coloration in erythrocytes with an enlarged area of pallor caused by a decrease in the cell’s Immunohistochemical stain dye applied using immunologic prin- hemoglobin content. The mean corpuscular hemoglobin concen- ciples and techniques to study cells and tissues. Usually a labeled tration (MCHC) and mean corpuscular hemoglobin (MCH) are antibody is used to detect antigens (markers) on a cell. decreased. Immunophenotyping identifying antigens using detection Hypodiploid having less than 2n of chromosomes per cell; for antibodies. human cells, this would be less than 46. Immunosuppressed repressed ability to produce antibodies to Hypoferremia condition of low serum iron; found in iron-deficiency antigens. anemia and anemia of chronic disease. Immunotherapy form of treatment in which different immune cells Hypofibrinogenemia condition in which there is an abnormally are manipulated in vivo or in vitro and later infused to alter the low fibrinogen level in the peripheral blood. It can be caused by a immune function of other cells. mutation in the gene controlling the production of fibrinogen or an Indirect antiglobulin test (IAT) laboratory examination used to acquired condition in which fibrinogen is pathologically converted detect the presence of serum antibodies against specific erythrocyte to fibrin. antigens. Hypogammaglobulinemia condition associated with a decrease in Induction therapy initial phase of cancer treatment using chemical resistance to infection as a result of decreased g@globulins (immuno- substances. Its function is to rapidly drop the tumor burden and globulins) in the blood. induce a remission to a normal state. Hypoplasia condition of underdeveloped tissue or organ usually Ineffective erythropoiesis premature death of red blood cells in the caused by a decrease in the number of cells. This bone marrow type bone marrow, preventing release into circulation. is one in which the proportion of hematopoietic cells to fat cells is decreased. Infectious lymphocytosis condition found in young children. The most striking hematologic finding is a leukocytosis of Hypoproliferative having decreased production of any cell type. 40950 * 103/mcL with 60–97% small, normal-appearing lympho- Hypoxia deficiency of oxygen to the cells. cytes. It is thought to be a reactive immune response to a viral Hypoxia-inducible factor-2 (HIF-2) transcription factor that infection and is no longer considered a unique disease. regulates erythropoietin synthesis and a spectrum of other hypoxic Infectious mononucleosis self-limiting lymphoproliferative disease responses; activated under hypoxic conditions. caused by infection with Epstein-Barr virus (EBV). The usually Glossary 1193 increased leukocyte count is related to an absolute lymphocytosis. The IRP binding affinity for the IRE is determined by the amount of Various forms of reactive lymphocytes are present. Serologic tests to cellular iron; the IRP binds to the IRE region when iron is scarce and detect the presence of heterophile antibodies are helpful in differen- dissociates when it is plentiful. When bound, the IRP modulates the tiating this disease from more serious diseases. Also known as the translation of the mRNA. The translation of the proteins involved in kissing disease. iron metabolism including ferritin, ferroportin, ALAS2, transferrin, Innate immune response body’s first reaction to common classes of and DMT1 is regulated by this mechanism. invading pathogens. It is rapid but limited. The leukocyte receptors Iron-responsive element-binding protein (IRE-BP) protein that that participate in it are always available and do not require cell binds to a stem-loop structure of some mRNAs which is known as activation to be expressed. Once a pathogen is recognized, effec- the iron-responsive element (IRE). The IRE-BP binding affinity for the tor cells can attack, engulf, and kill it. Neutrophils, monocytes, and IRE is determined by the amount of cellular iron and is involved in macrophages play a major role in the innate immune system. the regulation of translation of proteins involved in iron metabolism. In situ hybridization detection of specific DNA or RNA sequences Iron-restricted erythropoiesis production of erythrocytes

when in tissue sections or cell preparations using a labeled complementary there is a reduced supply of iron for erythropoiesis even though the nucleic acid sequence or probe. level of iron stores is usually replete; a condition in which there is Integral protein protein embedded between phospholipids within a insufficient iron to insert into the protoporphyrin ring to form heme; cell membrane. also referred to as iron-restricted erythropoiesis. Internal quality control program system designed to verify the Irreversibly sickled cell (ISC) rigid cell that has been exposed validity of laboratory test results as part of the daily laboratory to repeated sickling events and cannot revert to a normal discoid operations. Typically, it is monitored using Levy-Jennings plots and shape. It is ovoid or boat shaped and has a high MCHC and low Westgard rules. MCV. International Classification of Disease code method for indicating Ischemia relating to the deficiency of blood supply to a tissue medical diagnosis, conditions, symptoms, and cause of death in all caused by constriction of the vessel or blockage of the blood flow health care settings; accepted worldwide. One of its uses is to help through it. evaluate medical necessity and determine reimbursement for tests Isoelectric focusing technique of moving charged particles through and procedures. a support medium with a continuous pH gradient. Individual pro- International normalized ratio (INR) method of reporting pro- teins move until they reach the pH that is equal to their isoelectric thrombin time results when monitoring long-term oral anticoagu- point. lant therapy. Results are independent of the reagents and methods Isopropanol precipitation technique that identifies the presence of used. unstable hemoglobins because of their insolubility in isopropanol as International sensitivity index (ISI) list of values provided by the compared to normal hemoglobins. manufacturer of thromboplastin reagents indicating the responsive- Isovolumetric sphering method employed by the Bayer/Technicon ness of the particular lot of reagent compared to the international instruments in which a specific buffered diluent is used to sphere reference thromboplastin. and fix the blood cells without altering their volume. Intrinsic coagulation pathway one of the three interacting sequenc- JAK/STAT signaling system used by hematopoietic cells to relay es of coagulation factors in the coagulation cascade. The intrinsic transmissions from the cell membrane receptors to the nucleus to passage sequence is initiated by exposure of the contact coagula- activate or inhibit gene expression. tion factors (FXII, FXI, prekallikrein, and high-molecular-weight Janus kinase 2 gene (JAK2) heredity unit that codes for a tyrosine kininogen) with vessel subendothelial tissue. The intrinsic sequence kinase closely associated with cytokine receptors. A gain in function activates FX. mutation that gives the cell a proliferative advantage is common in Intrinsic factor (IF) glycoprotein secreted by the parietal cells of the myeloproliferative disorders, especially polycythemia vera. the stomach that is necessary for binding and absorption of dietary Jaundice condition characterized by yellowing of skin, mucous cobalamin (vitamin B12). membranes, and the whites of the eye caused by accumulation of Intrinsic pathway See Intrinsic coagulation pathway. bilirubin. Intrinsic Xase complex network of FIXa, FVIIIa, phospholipid, and Juvenile myelomonocytic leukemia (JMML) clonal hematopoi- calcium that assembles on membrane surfaces. etic neoplasm of childhood characterized by proliferation of the Intron DNA base sequence that interrupts the protein coding granulocytic and monocytic lineages. There is a peripheral blood sequence of a gene; this sequence is transcribed into RNA but is cut monocytosis more than 1 * 103/mcL with less than 20% blasts and out of the message before it is translated into protein. promonocytes. Classified as an MDS/MPN disorder by the WHO 2008 classification. Iron-deficiency anemia (IDA) microcytic, hypochromic disorder caused by a deficiency of total body iron, resulting in iron-restricted Juxtacrine contact-dependent signaling used to describe a type of erythropoiesis. This is the last stage of the disorder when all labora- paracrine cell action in which a cell produces a cytokine but does tory tests for iron status become abnormal. not secrete it. The cytokine remains bound to the cell membrane and requires another cell to have direct cell-to-cell contact in order to Iron refractory iron-deficiency anemia (IRIDA) iron-deficiency achieve the desired effect. anemia that does not respond to oral iron therapy. It is an autosomal recessive disorder caused by a mutation in the gene (TMPRSS6) Karyolysis destruction of the nucleus. that encodes transmembrane protease, serine6, also known as Karyorrhexis disintegration of the nucleus resulting in the irregular matriptase-2 (MT-2); can activate hepcidin transcription. distribution of chromatin fragments within the cytoplasm. Iron regulatory protein (IRP) proteins that control iron metabolism Karyotype systematic display of a cell’s chromosomes that deter- and post-transcriptionally regulate the expression of the major iron mine the number of chromosomes present and their morphology. homeostasis genes. Keratocyte abnormally shaped erythrocyte with one or several Iron-responsive element (IRE) stem-loop-stem structure in either notches and projections on either end that look like horns. The shape the 5′ or 3′ noncoding regions of mRNA recognized and bound is caused by trauma to the erythrocyte. Also called helmet cells and by iron-binding proteins (IRE-BP or iron-regulatory protein [IRP]). horn-shaped cells. 1194 Glossary Kernicterus toxic buildup of bilirubin in brain tissue; associated Leukocytosis increase in WBCs in the peripheral blood; WBC count with hyperbilirubinemia. more than 11 * 103/mcL. Knizocyte abnormally shaped erythrocyte that appears on stained Leukoerythroblastic reaction response characterized by the pres- smears as a cell with a dark, stick-shaped portion of hemoglobin in ence of nucleated erythrocytes and a shift to the left in neutrophils in the center and a pale area on either end. The cell has more than two the peripheral blood; often associated with myelophthisis. concavities. Leukopenia decrease in leukocytes less than 4.5 * 103/mcL. L&H/popcorn cell See Popcorn cell. Leukopoiesis formation and development of white blood cells. Lacunar cell neoplastic variant found in NS Hodgkin lymphoma Linearity range of concentration over which a test method can be characterized by abundant pale-staining cytoplasm, cytoplasmic used without modifying (i.e., diluting) the specimen. clearing, and delicate, multilobated nuclei. Linearity check material commercially available matter with known Lamellar body count densely packed layer of phospholipid secreted concentrations of analytes that do not contain interfering substances by type II alveolar cells; is the storage package for surfactant in or conditions. It is used to determine the linearity of an instrument amniotic fluid. The number of lamellar bodies present in the amni- or a method. otic fluid is proportional to the amount of surfactant available. Linear regression analysis statistical tool used to determine a single Large granular lymphocyte (LGL) null cell with a low nuclear-to- line through a data set that describes the relationship between two cytoplasmic ratio, pale blue cytoplasm, and azurophilic granules. It methods, X and Y. General equation is Y = a + bx, where a denotes does not adhere to surfaces or phagocytose. the y-intercept; b is the slope; and Y is the predicted mean value of Y Latex immunoassay (LIA) test immunoturbidimetric analysis using for a given x value. microlatex particles coated with specific antibodies. In the presence of Linkage analysis process of following the inheritance pattern of the antigen to be tested, the particles agglutinate, producing an adsorp- a particular gene in a family based on its tendency to be inherited tion of light proportional to the antigen level present in the sample. with another locus on the same chromosome. LDL receptor-like protein (LRP receptor) receiver on hepatocytes Locus specific position on the chromosome. that removes plasmin/antiplasmin, plasminogen activator/plasmi- Low-molecular-weight heparin (LMWH) heparin molecule of nogen activator inhibitor, and thrombin/antithrombin complexes M.W. 2–12 kilodaltons (2,000–12,000 daltons); used as thrombolytic from the circulation. therapy in acute thrombotic events. Lecithin cholesterol acyltransferase (LCAT) deficiency rare auto- LRP receptor See LDL receptor-like protein. somal disorder that affects the metabolism of high-density lipopro- teins; characterized by the deficiency of an enzyme that catalyzes the Lupus anticoagulant See Lupus-like anticoagulant. formation of cholesterol esters from cholesterol. Its onset is usually Lupus-like anticoagulant circulating substance that occurs sponta- during young adulthood. neously in patients with a variety of conditions (originally found in Leptocyte abnormally shaped erythrocyte that is thin and flat with those with lupus erythematosus) and directed against phospholipid hemoglobin at the periphery. It is usually cup shaped. components of the reagents used in laboratory tests for clotting fac- tors. See Antiphospholipid antibody. Leukemia progressive, malignant disease of the hematopoietic sys- tem characterized by unregulated, clonal proliferation of hematopoi- Lymphadenopathy abnormal enlargement of lymph nodes. etic stem cells; generally classified as chronic or acute and lymphoid Lymphoblast lymphocytic precursor cell found in the bone m arrow. The or myelogenous. The malignant cells eventually replace normal cell is 10920 mcM (mm) in diameter and has a high nuclear:cytoplasmic cells. ratio. The nucleus has a fine (lacy) chromatin p attern with one or two Leukemia of ambiguous lineage condition in which neoplastic cells nucleoli. The cytoplasm is agranular and scant and stains deep blue cannot be defined by one lineage. Either no lineage-specific antigens with Romanowsky stain. The cell contains terminal deoxynucleotidyl can be identified or the blasts express antigens of more than one transferase (TdT) but no peroxidase, lipid, or esterase. lineage. Lymphocyte mature leukocyte with variable size, depending on Leukemic hiatus gap in the normal maturation pyramid of cells the state of cellular activity and amount of cytoplasm. The nucleus with many blasts and some mature forms but very few intermediate is usually round with condensed chromatin and stains deep, dark maturational stages. Eventually, the immature neoplastic cells fill purple with Romanowsky stains. The cytoplasm stains a light blue. the bone marrow and spill over into the peripheral blood, producing Nucleoli are usually not visible. A few azurophilic granules can be leukocytosis (e.g., acute leukemia). present. These cells interact in a series of events that allow the body to attack and eliminate foreign antigen. They have a peripheral blood Leukemic stem cell (LSC) rare cell that has infinite proliferative concentration in adults from 1.0 to 4.8 * 103/mcL (20–40% of leuko- potential and drives the formation and growth of tumors. cytes). The concentration in children less than 10 years old is higher. Leukemogenesis process of developing a leukemic disease. Lymphocytic leukemoid reaction response characterized by an Leukemoid reaction transient, responsive condition resulting from increased lymphocyte count with the presence of reactive or certain types of infections or tumors characterized by an increase in immature appearing lymphocytes. It is associated with the total leukocyte count to more than 25 * 103/mcL and a shift to whooping cough, chickenpox, infectious mononucleosis, the left in leukocytes (usually granulocytes). infectious lymphocytosis, and tuberculosis. Leukocyte white blood cell (WBC) of which there are five types: Lymphocytopenia condition that has less than 1.0 * 103/mcL neutrophil, eosinophil, basophil, lymphocyte, and monocyte. The of a type of white blood cell known as a lymphocyte. Also called function of these cells is to defend against infection and tissue lymphopenia. damage. The normal reference range for total leukocytes in periph- Lymphocytosis increase in peripheral blood concentration of eral blood is 4.5911.0 * 103/mcL. a type of white blood cell known as a lymphocyte (more than Leukocyte alkaline phosphatase (LAP) test that identifies the 4.8 * 103/mcL in adults or more than 9 * 103/mcL in children). amount of enzyme present within the specific (secondary) granules Lymphoepithelial lesion region damaged by injury or disease such of granulocytes (from the myelocyte stage onward). It is useful in as infiltration of epithelium by groups of lymphocytes. Infiltration distinguishing leukemoid reaction/reactive neutrophilia (high LAP) of mucosal epithelium by neoplastic lymphocytes is characteristic of from chronic myelogenous leukemia (low LAP). MALT lymphoma. Glossary 1195 Lymphoid follicle spherical mass of B cells within lymphatic tissue. of disorders associated with abnormalities of the MYH9 gene Lymphokine substance released by sensitized lymphocytes and (nonmuscle myosin gene). responsible for activation of macrophages and other lymphocytes. Mean cell hemoglobin (MCH) indicator of the average weight of Lymphokine-activated killer (LAK) cells subpopulation of periph- the intracellular erythrocyte protein, hemoglobin, in individual eral blood mononuclear cells (natural killer cells) that have been erythrocytes reported in picograms. The reference interval for MCH harvested from a patient, expanded and activated with IL-2 ex vivo, is 28–34 pg. This parameter is calculated from the hemoglobin and and re-infused into the patient to induce an enhanced antitumor erythrocyte count: cytolytic response. Hb (g/dL) Lymphoma malignant proliferation of a type of white blood cell MCH (pg) =

RBC count ( * 1012/L) known as a lymphocyte. Most cases arise in lymph nodes, but it can begin at many extranodal sites; classified as to B or T cell and low, Hb (g/dL) * 10 MCH (pg) = intermediate, or high grade. RBC count ( * 106/mcL) Lymphoma classification process of dividing (grading) the disease Mean cell hemoglobin concentration (MCHC) measure of the aver- into groups, each with a similar clinical course and response to treat- age mass of the protein hemoglobin within erythrocytes in grams ment. Current schemes use a combination of morphologic appear- per deciliter to the volume of erythrocytes in which it is contained. ance, phenotype, and genotype. The reference interval is 32–36 g/dL. The MCHC is useful when evaluating erythrocyte hemoglobin content on a stained smear. This Lyonization one X chromosome in each female cell is inactivated parameter correlates with the extent of chromasia the stained cells randomly and remains inactive throughout all subsequent divisions exhibit; calculated from the hemoglobin and hematocrit: of that cell. Hb (g/L) Lyophilized freeze-dried serum or plasma sample that can be recon- MCHC (g/dL) = stituted with a diluent, typically distilled or deionized water. Hct (L/L) * 10 Lysosomal granule small particle containing lysosomal enzymes. Hb (g/dL) * 100 MCHC (g/dL) = Lysosomal storage disorder disruption of membrane-bound Hct (%) sac characterized by defects in various enzymes (including Mean cell volume (MCV) average volume of -individual erythro- glucosidases, lipases, proteases, and nucleases) that are involved cytes reported in femtoliters. The reference interval is 80–100 fL. in degradative processes leading to the accumulation of either This parameter is useful in evaluating erythrocyte morphology on a nondegraded substrates or catabolic products that are unable to be stained blood smear. The MCV usually correlates with the diameter transported out of the lysosome. of the erythrocytes observed microscopically and can be calculated Lysosome membrane-bound sac in the cytoplasm that contains from the hematocrit and erythrocyte count: various hydrolytic enzymes. Hct (%) * 10 MCV (fL) = Macrocyte abnormally large erythrocyte (greater than 100 fL). RBC count (* 106/mcL) Macro-ovalocyte abnormally large erythrocyte with an oval shape Hct (L/L) * 1000 characteristically seen in megaloblastic anemia. MCV (fL) = RBC count (* 1012/L) Macrophage large—10–20 mcM (mm)—tissue cell derived from Mean platelet volume (MPV) average cell volume of a platelet monocytes; it secretes a variety of products that influence the func- population; analogous to the MCV of erythrocytes. tion of other cells and plays a major role in both nonspecific and specific immune responses. Medical decision level concentration of an analyte indicating that medical intervention is required for proper patient care. Maintenance chemotherapy third and final phase of cancer treat- ment that uses chemical agents to prevent the repair and/or return Medullary hematopoiesis blood cell production and development of the malignant clone, thus allowing the normal immune system to in the bone marrow. clear away all remaining disease. Megakaryoblast progenitor cell committed to the megakaryocyte Malignant neoplastic with potential to metastasize. lineage; develops into the megakaryocyte. Malignant neoplasm clone of identical, anaplastic Megakaryocyte large cell found within the bone marrow charac- (de-d ifferentiated), proliferating cells; can metastasize. terized by the presence of large or multiple nuclei and abundant cytoplasm. It produces the blood platelets. Marginating pool population of neutrophils attached to or margin- ated along the vessel walls and not actively circulating. This is about Megaloblastic asynchronous maturation of any nucleated cell one-half of the total pool of neutrophils in the vessels. type characterized by delayed nuclear development in comparison to the cytoplasmic development. The abnormal cells are large and Mastocytosis disorder caused by heterogeneous group of mast cell characteristically found in pernicious anemia and other diseases characterized by the abnormal proliferation of mast cells in megaloblastic anemias. one or more organ systems. Two major groups of mast cell disorders are cutaneous and systemic. It is suggested that mast cell disorders Membrane inhibitor of reactive lysis (MIRL) regulatory protein be classified as myeloproliferative disorders. (CD59) on normal cell membranes responsible for preventing ampli- fication of complement activation; prevents interaction between Maturation process of attaining complete development of the cell. C8 and C9. Deficiency in PNH is due to the lack of a glycolipid Mature neoplasm usually a chronic condition in which the peripher- anchor (glycosyl-phosphatidyl inositol) that attaches it to the cell al blood contains an increased total WBC and mature forms of cells. membrane. Maturing cell committed cell that assumes the morphologic char- Memory cell long-lived T or B lymphocyte produced by anti- acteristics of its lineage. It makes up the majority of hematopoietic gen stimulation of naïve lymphocytes that produce a rapid and precursor cells. enhanced immune response to the initial and later exposure to the May-Hegglin anomaly (MHA) inherited disorder in which platelet stimulating antigen. production is decreased; characterized by a moderate macrothrom- Meninges (singular: meninx) three membranes covering the brain bocytopenia and Döhle-like inclusions in leukocytes; one of a group and spinal cord. 1196 Glossary Mesenchymal stem cell (MSC) multipotent bone marrow stromal cells diagnosis requires exclusion of other plasma cell and lymphoid that can differentiate into bone, cartilage, and fat cells (adipocytes). malignancies. Metacentric having a chromosome that has the centromere near Monocyte mature leukocyte found in bone marrow or peripheral center so that the short arm and long arms are equal in length. blood. Its morphology depends on its activity. The cell ranges in size Metamyelocyte granulocytic precursor cell normally found in from 12930 mcM (mm) with an average of 18 mcM. The blue-gray the bone marrow. The cell is 10915 mcM (mm) in diameter. The cytoplasm is evenly dispersed with fine dustlike granules. Of the cytoplasm stains pinkish and there is a predominance of specific two types of granules, one contains peroxidase, acid phosphatase, granules. The nucleus is indented with a kidney bean shape, and the and arylsulfatase. Less is known about the content of the other nuclear chromatin is condensed and stains dark purple. granule. The nuclear chromatin is loose and linear, forming a lacy pattern. The nucleus is often irregular in shape. Methemoglobin form of hemoglobin with iron that has been oxidized to the ferric state; (Fe+++) and is incapable of combining with oxygen. Monocyte–macrophage system See Mononuclear phagocyte (MNP) system. Methemoglobinemia condition with the intracellular erythrocyte protein iron in the ferric state; this affects the oxygen affinity of the Monocytopenia disorder characterized by a decrease in the concen- molecule, which cannot combine with oxygen. tration of a class of circulating white blood cells known as monocytes (less than 0.1 * 103/mcL). Methemoglobin reductase pathway metabolic reaction that uses methemoglobin reductase and NADH to maintain heme iron in the Monocytosis increase in the concentration of circulating white blood reduced state (Fe++). cells known as monocytes (greater than 0.8 * 103/mcL). Microangiopathic hemolytic anemia (MAHA) process in which the Mononuclear phagocyte (MNP) system collection of white blood erythrocyte is damaged by prosthetic devices or lesions of the small cells known as monocytes and macrophages found both intravas- blood vessels. cularly and extravascularly. It plays a major role in initiating and regulating the immune response. Microcyte abnormally small erythrocyte. The MCV is typically decreased. Monosomy condition having one daughter cell with a missing chro- mosome (one copy instead of two). Microenvironment unique surrounding in the bone marrow where orderly proliferation and differentiation of precursor cells take place. Morulae basophilic, irregularly shaped granular, cytoplasmic inclu- sions found in leukocytes in an infectious disease called ehrlichiosis. Micromegakaryocyte small, abnormal cell sometimes found in the peripheral blood in MDS and the myeloproliferative syndromes. Mosaic pattern that occurs in the embryo shortly after fertilization, resulting in congenital aberrations in some cells and some normal Microparticle (MP) membrane-bound vesicle produced by blebbing cells. of the parent cell’s membrane released in response to apoptosis or cell activation. Mott cell pathologic plasma cell whose cytoplasm is filled with colorless globules that most often contain immunoglobulin (Russell Microtubule cylindric structure—20–27 mcM (mm) in diameter— bodies) and forms as a result of accumulation of material in the composed of protein subunits. It is a part of the cytoskeleton helping RER, SER, or Golgi complex caused by an obstruction of secretion. some cells maintain shape. It increases during mitosis and forms The cell is associated with chronic plasmocyte hyperplasia, parasitic the mitotic spindle fibers and assists in transporting substances in infection, and malignant tumors. Also called grape cell. different directions. In the platelet, a band of tubules located on the circumference is thought to be essential for maintaining the disc Multimer analysis testing that determines the structure of VWF shape in the resting state. multimers. Minimal residual disease See Minimum residual disease. Multiple myeloma plasma cell malignancy characterized by increased plasma proteins. Minimum residual disease condition with a combination of nega- tive “traditional” tests (peripheral blood and bone marrow blast Mutation any change in the nucleotide sequence of DNA. When count and cytogenetics) and positive molecular tests for the presence large sequences of nucleotides are missing, the alteration is referred of malignant cells. to as a deletion. Mitotic pool population of cells within the bone marrow that is Myeloblast first microscopically identifiable granulocyte pre- capable of DNA synthesis. Also called proliferating pool. cursor normally found in the bone marrow. The cell is large (15920 mcM [mm]) with a high nuclear-cytoplasmic ratio. The Mixed lineage acute leukemia critical blood cell malignancy that nucleus has a fine chromatin pattern with a nucleoli. There is has both myeloid and lymphoid populations present or blasts that moderate amount of blue, agranular cytoplasm. possess myeloid and lymphoid markers on the same cell. Myelocyte granulocytic precursor cell normally found in the bone Mixed phenotype acute leukemia See Mixed lineage acute leukemia. marrow that is 12918 mcM (mm) in diameter with a pinkish granular Molecular remission absence of detectable genetic abnormalities cytoplasm. Both primary and secondary granules are present. using PCR or related molecular technologies in patients who had Myelodysplastic syndrome (MDS) type of blood cell neoplasm char- identifiable abnormalities before therapy. This is the most sensitive acterized by a group of primary neoplastic pluripotential stem cell test for detecting minimal residual disease. disorders with one or more cytopenias in the peripheral blood and Monoblast precursor cell found in bone marrow. It is about prominent maturation abnormalities (dysplasia) in the bone marrow. 14918 mcM (mm) in diameter with abundant agranular, blue-gray Myelodysplastic syndrome with excess blasts-1 (MDS-EB-1) cytoplasm. The nucleus can be folded or indented. The chromatin is subgroup of MDS characterized by cytopenia occurring in one to finely dispersed, and several nucleoli are visible. The cell has non- three lineages and conspicuous qualitative abnormalities in one to specific esterase activity that is inhibited by sodium fluoride. This three lineages. Evidence of dysgranulopoiesis is often prominent. cell will develop into monocytes. The bone marrow has 5–9% blasts, and the peripheral blood has Monoclonal gammopathy alteration in immunoglobulin pro- less than 5% blasts. duction characterized by an increase in one specific class of Myelodysplastic syndrome with excess blasts-2 (MDS-EB-2) s ubgroup immunoglobulin. of MDS characterized by cytopenia occurring in one to three lineages Monoclonal gammopathy of undetermined significance and conspicuous qualitative abnormalities in one to three lineages. Evi- (MGUS) condition characterized by a low level of serum dence of dysgranulopoiesis is often prominent. The bone marrow has monoclonal protein without evidence of an overt neoplasm. Its 10–19% blasts, and the peripheral blood has 5–19% blasts. Glossary 1197 Myelodysplastic syndrome with multilineage dysplasia (MDS- Neutrophil extracellular trap (NET) extracellular web-like struc- MLD) subgroup of MDS characterized by dysplastic features in at ture, composed of DNA and microbicidal proteins from neutrophils, least 10% of the cells in two or more cell lineages, cytopenia in one that functions to trap and kill microbes independent of phagocytic to three lineages, with less than 5% blasts in the bone marrow and uptake. less than 1% blasts in the peripheral blood. Neutrophilia increase in neutrophil concentration (greater than Myelodysplastic syndrome with ring sideroblasts (MDS-RS) 7.0 * 103/mcL). It can be seen in bacterial infections, inflammation, subgroup of the WHO classification of MDS; characterized by 5-15% metabolic intoxication, drug intoxication, and tissue necrosis. ring sideroblasts; if there are between 5 and 15% ring sideroblasts Nijmegen-Bethesda titer assay modification of the original among the erythroid precursors, the SF3B1 mutation must be present Bethesda method for measuring factor VIII inhibitors. for inclusion in

MDS-RS. If there are at least 15% ring sideroblasts, the presence of mutated SF3B1 becomes irrelevant since the presence Nomogram chart that displays the relationship between numerical of at least 15% ring sideroblasts qualifies the syndrome as MDS-RS. variables; three parallel scales are used for different variables; when a straight line connects two values, the third can be read from the Myelodysplastic syndrome with single lineage dysplasia (MDS- point intersected by the line; used in determining heparin dosing. SLD) subgroup of MDS characterized by isolated cytopenia or bicytopenia accompanied by unilineage dysplasia. There are less Nondisjunction error in segregation that occurs in mitosis or meio- than 1% blasts in the peripheral blood and less than 5% blasts in the sis so that sister chromatids do not disjoin. A spindle fiber malfunc- bone marrow. tion results in one daughter cell with an extra chromosome (trisomy) and one daughter cell with a missing chromosome (monosomy). Myelodysplastic/myeloproliferative neoplasm category of disor- ders that is included in the WHO classification but not in the FAB Nonspecific esterase a@naphthyl esterase enzyme present in mono- classification system. It includes clonal hematopoietic disorders that cytic cells, megakaryocytes, hairy cells, plasma cells, T lymphocytes, have some clinical, laboratory, or morphologic findings of both a and macrophages. Can be identified by stains using either acetate or myelodysplastic syndrome (MDS) and a chronic myeloproliferative butyrate as a substrate at alkaline pH. neoplasm (MPN). Nonspecific granule large, blue-black organelle found in promye- Myeloid associated antigen CD marker that when present is useful locytes. It has a phospholipid membrane and stains positive for in differentiating AML from other types of acute leukemia. peroxidase. Myeloid-to-erythroid ratio (M:E ratio) proportion of granulocytes Non-thrombocytopenic purpura condition in which platelets are and their precursors to nucleated erythroid precursors derived from normal in number but purple discoloration is present; is considered performing a differential count on bone marrow nucleated hemat- to be caused by damage to the blood vessels. opoietic cells. Monocytes and lymphocytes are not included. The Nonthrombogenic pertaining to the inhibition of blood clot forma- normal ratio is usually between 1.5:1 and 3.5:1, reflecting a predomi- tion; absence of clot-promoting (thrombogenic) characteristics/ nance of myeloid elements. activities. Myeloperoxidase (MPO) enzyme present in the primary granules of Non-transferrin bound iron (NTBI) iron bound to albumin or other myeloid cells including neutrophils, eosinophils, and monocytes. low molecular weight molecules; this occurs when the concentration Myelophthisis displacement of normal hematopoietic tissue in bone of plasma iron exceeds transferrin binding capacity. marrow by fibrosis, leukemia, or metastatic cancer cells. Normal pooled plasma platelet-poor, colorless fluid part of blood Myeloproliferative neoplasm (MPN) group of neoplastic clonal collected from at least 20 individuals for coagulation testing. PT and disorders characterized by excess proliferation of one or more cell APTT should give results within the laboratory’s reference interval. types in the bone marrow. Formerly referred to as myeloproliferative The fluid is pooled and used in mixing studies to differentiate a disorders (MPD). circulating inhibitor from a factor deficiency. Natural killer (NK) cell type of lymphoid cell that has the capacity Normoblast nucleated erythrocyte precursor in the bone marrow. for spontaneous, non-MHC-restricted cytotoxicity for various target Also known as erythroblast. cells. It possesses CD16 (the FcgIII receptor for IgG) and CD56. NK Normochromic refers to the color (“-chromic”) of erythrocytes on cells constitute about 15% of the circulating lymphocytes in the a peripheral blood smear; coloration due to hemoglobin covers peripheral blood; it is involved in several activities such as resist- approximately two-thirds of the cell, with a center that is paler in ance to viral infections, regulation of hematopoiesis, and process coloration and approximately one-third of cell; may be reflected by a against tumor cells. mean cell hemoglobin concentration of 32–36 g/dL. Natural killer T (NKT) cell small subpopulation of lymphocytes Normocytic characterized by normal size blood cells; describes that express both T lymphocyte receptors (TCR) and some surface erythrocytes that have a mean corpuscular volume (MCV) within molecules characteristic of natural killer cells. the reference range (80–100 fL). Necrosis pathologic cell death resulting from irreversible damage; Nuclear-cytoplasmic asynchrony used to describe blood cell matu- “cell murder.” ration in which the cellular nucleus matures more slowly than the Neonatal alloimmune thrombocytopenia (NAIT) deficiency of cytoplasm, suggesting a disturbance in coordination. As a result, the platelets in the blood caused by immune destruction of these cells nucleus takes the appearance of one associated with a younger cell that occurs in newborns; results from the transfer of maternal than its cytoplasmic development indicates. This is a characteristic alloantibodies. of megaloblastic anemias. Neoplasm abnormal formation of new tissue (such as a tumor) that Nuclear-to-cytoplasmic ratio (N:C ratio) proportion of the volume serves no useful purpose; can be benign or malignant. of the cell nucleus to the volume of the cell’s cytoplasm; usually estimated as the proportion of the diameter of the nucleus to the Nephelometry technique used to measure the concentration of par- diameter of the cytoplasm. In immature hematopoietic cells, the N:C ticles in a solution by analysis of the light scattered by the particles. ratio is usually higher than in more mature cells. As the cell matures, Neutropenia decrease in neutrophils (less than 1.8 * 103/mcL). the nucleus condenses and the cytoplasm expands. Neutrophil mature white blood cell with a segmented nucleus and Nucleolus (pl: nucleoli) spherical body within the nucleus in which granular cytoplasm. This cell constitutes the majority of circu- ribosomes are produced. It is not visible in cells that are not synthe- lating leukocytes. The absolute number varies between 1.8 and sizing proteins or that are not in mitosis or meiosis. It stains a lighter 7.0 * 109/L. Also called granulocyte or seg. blue than the nucleus with Romanowsky stains. 1198 Glossary Nucleotide basic building block of DNA composed of nitrogen p53 gene hereditary unit that normally functions as an antioncogene base (A = adenine, T = thymine, G = guanine, or C = cytosine) by preventing proliferation of DNA-damaged cells and unwanted attached to a sugar (deoxyribose) and a phosphate molecule. DNA amplification and promoting apoptosis of these damaged cells. Nucleus (pl: nuclei) characteristic structure in the eukaryotic cell When mutated, it can lose its tumor-suppressive effect. that contains chromosomes and nucleoli; stains deep bluish-purple Paired t-test statistical tool used to compare the difference between with Romanowsky stain. It is separated from the cytoplasm by two paired data sets. Use of pairs determines whether a statistically a nuclear envelope. In young, immature hematopoietic cells, its significant difference exists between the two data sets. material is open and dispersed in a lacy pattern. As the cell becomes Pancytopenia marked decrease of all lineages of blood cells in the mature, the structural material condenses and appears structureless. peripheral blood. Null cell See Large granular lymphocytes. Panhypercellular increase in all blood cells in the peripheral blood. Oncogene mutated gene that leads to the transformation of a Panic value test result that is above or below the critical limit and normal cell to a cancer cell. Most are altered forms of normal could pose a life-threatening situation that should be repeated to hereditary units that function to regulate cell growth and confirm the results. If confirmed, the results should be immediately differentiation. Its normal counterpart is known as a proto-oncogene. communicated to the physician and properly documented. Open canalicular system (OCS) membrane system in the platelet Panmyelosis panhypercellularity in the bone marrow. forming twisted channels that lead from the platelet surface to the interior of the platelet; is a remnant of the demarcation membrane Pappenheimer body iron-containing particle in mature erythro- system of the megakaryocyte. Also called surface connected canalicular cytes. On Romanowsky stain, it is visible near the periphery of the system (SCCS). cell and often occurs in clusters. Opportunistic organism cell that is usually part of the normal flora Paracrine type of cell signaling when signals produced by one cell but can cause disease if there is a significant change in host resist- act on an adjacent cell, typically over short distances. ance or within the organism itself. Paroxysmal cold hemoglobinuria (PCH) autoimmune hemolytic Opsonin antibody or complement that coats microorganisms or anemia characterized by lysis of erythrocytes and hematuria upon other particulate matter found within the blood stream so that the exposure to cold. foreign material can be more readily recognized and phagocytized Paroxysmal nocturnal hemoglobinuria (PNH) stem cell disease by leukocytes. in which the erythrocyte membrane is abnormal, making the cell Oral anticoagulant type of drug taken by mouth (e.g., Coumadin, more susceptible to hemolysis by complement. There is a lack of warfarin) that prevents coagulation by inhibiting the activity of vita- decay-accelerating factor (DAF) and MIRL on the membrane that min K, which is required for the synthesis of functional prothrombin are normally responsible for preventing amplification of complement group coagulation factors. activation. DAF and MIRL deficiency results from the lack of glycosyl phosphatidyl inositol (GPI), a membrane glycolipid that Oral anticoagulant therapy drug treatment taken by mouth to serves to attach (anchor) proteins to the cell membrane. prevent coagulation. Intravascular hemolysis is intermittent. Orthochromatic normoblast nucleated precursor of the erythrocyte Passenger lymphocyte syndrome (PLS) condition that causes that develops from the polychromatophilic normoblast normally immune hemolytic state following a solid organ, bone marrow, or found in the bone marrow. It is the last nucleated stage of erythro- stem cell transplant. The donor B lymphocytes transplanted with cyte development. the organ or the bone marrow produce antibodies against recipi- Osmotic fragility laboratory procedure to evaluate the ability of ent’s blood group antigens. Hemolysis is primarily caused by ABO erythrocytes to withstand different salt concentrations; depends on incompatibility between donor and recipient (Group O donor and the erythrocyte’s membrane, volume, surface area, and functional Group A or B recipient). Although ABO is the most frequent antigen state. system involved, Rh, Kell, Kidd, or other blood group systems can be involved. Osteoblast cell involved in formation of calcified bone. Patient safety ensuring that patients are not harmed as a result of a Osteoclast cell involved in resorption and remodeling of calcified healthcare system encounter by improving the system using the six bone. quality aims: safe, effective, efficient, timely, patient-centered and Outlier data point that falls outside the expected range for all data; equitable. is not considered to be part of the population that was sampled. Pathogen-associated molecular pattern (PAMP) structure shared by Ovalocyte erythrocyte that is oval in shape and can be seen in many different pathogens or common alterations that the pathogen patients with megaloblastic and other anemias. makes to the body’s cells. Overhydrated hereditary stomatocytosis (OHS) form of hereditary Pattern recognition receptor (PRR) leukocyte surface responder for deformation of red blood cells in which the red cell membrane is PAMP. abnormally permeable to both Na+ and K+. The net gain of Na+ ions Pelger-Huët anomaly (PHA) inherited benign condition charac- is higher than the net loss of K+ ions because the capacity of the terized by the presence of functionally normal neutrophils with a cation pump (fueled by ATP derived from glycolysis) to maintain bilobed or round nucleus. Cells with the bilobed appearance are normal intracellular osmolality is exceeded. As the pump fails, the called pince-nez cells. intracellular concentration of cations increases, water enters the cell, and the overhydrated cells appear to have a mouthlike area of pallor Percent (%) hypochromic red cells percent of total red cells that are and are called stomatocytes. Also known as hereditary hydrocytosis. hypochromic reported by some automated hematology analyzers. Oxygen affinity ability of hemoglobin to bind and release oxygen. It Percent saturation portion of transferrin that is complexed with iron. is decreased by an increase in CO2, acid, and heat and increased by Pericardial cavity body space that contains the heart. a rise in pO2. Pericardium membrane that lines the pericardial cavity. Oxyhemoglobin compound formed when the erythrocyte protein Periodic acid-Schiff (PAS) stain used to identify and differentiate that transports respiratory gases combines with oxygen. blood cells. The stainable component is primarily glycogen. Mature P50 value partial pressure of oxygen at which 50% of hemoglobin is granulocytes, platelets, megakaryocytes, and monocytes are stained saturated with oxygen. with PAS. Glossary 1199 Peripheral membrane protein compound of amino acids attached Plasmacytoid lymphocyte intermediate cell in immunoblast to the cell membrane by ionic or hydrogen bonds but is outside the development between

the B cell and the plasma cell. It has mor- lipid framework of the membrane. phologic similarity to the lymphocyte but has marked cytoplasmic Peritoneal cavity space between the inside abdominal wall and out- basophilia similar to that of plasma cells. It is occasionally seen in side of the stomach, small and large intestines, liver, superior aspect the peripheral blood of patients with viral infection. of the bladder, and uterus. Plasmacytoma localized, tumorous collection of clonal plasma cells. Peritoneum lining of the peritoneal cavity. The disease prognosis is related to its location. Plasmacytosis presence of plasma cells in the peripheral blood or an Pernicious anemia (PA) megaloblastic anemia resulting from a lack excess of plasma cells in the bone marrow. of intrinsic factor needed to absorb cobalamin (vitamin B12) from the gut. Plasmin proteolytic enzyme with trypsin-like specificity that digests fibrin or fibrinogen as well as other coagulation factors; formed from Persistent polyclonal B cell lymphocytosis rare disorder found pri- plasminogen. marily in the female adult smoker. There is a polyclonal expression of lymphocytes but often there is genetic evidence of a malignant Plasminogen inactive precursor of the enzyme plasmin; b@globulin, process. Hematologic findings are normal except for lymphocytosis single-chain glycoprotein that circulates in the blood as a zymogen. and the presence of binucleated lymphocytes. There is a polyclonal Large amounts are absorbed with the fibrin mass during clot forma- increase in serum IgM but low IgG and IgA levels. Bone marrow tion; it is activated by intrinsic and extrinsic activators to form plasmin. examination reveals lymphocytic infiltrates. Plasminogen activator protein of the fibrinolytic system that con- Petechiae small, pinhead-size purple spots caused by blood escap- verts a precursor protein plasminogen to plasmin. ing from capillaries into intact skin. These are associated with Plasminogen activator inhibitor-1 (PAI-1) primary inhibitor of tissue platelet and vascular disorders. plasminogen activator (t-PA) and urokinase-like plasminogen activator Phagocytosis process of cells engulfing and destroying a foreign (tcu-PA) released from platelet a@granules during platelet activation. particle through active cell membrane invagination. Plasminogen activator inhibitor-2 (PAI-2) inhibitor of tissue and Phagolysosome digestive vacuole (secondary lysosome) formed by urokinase-like plasminogen activator. Its secretion is stimulated by the fusion of lysosomes and a phagosome. The hydrolytic enzymes endotoxin and phorbol esters. Increased levels impair fibrinolysis of the lysosome digest the phagocytosed material. and are associated with thrombosis. Phagosome formation of an isolated vacuole within the process of Platelet round or oval disc-shaped structure in the peripheral blood opsonization. formed from the cytoplasm of megakaryocytes in the bone marrow. It plays an important role in primary hemostasis by adhering to the Pharmacokinetics branch of pharmacology that performs a quanti- ruptured blood vessel wall and aggregating to form a plug over tative study of a drug’s disposition in the body over time. the injured area. It is also important in secondary hemostasis by Phase microscopy type of light microscope used to count platelets; providing phospholipids important for the activation of coagulation places an annular diaphragm below or in the substage condenser proteins. The normal reference interval is 1509400 * 103/mcL. and a phase-shifting element in the rear focal plane of the objective. Platelet activation stimulation of the cell involved in primary This causes alterations in the phases of light rays and increases the hemostasis (platelets) that occurs when agonists bind to its surface contrast between the cell and its surroundings. and transmit signals to the cell’s interior. Activated platelets form Phenotype physical manifestation of an individual’s genotype, often aggregates known as the primary platelet plug. referring to a particular genetic locus. Platelet adhesion ability of the cell involved in primary hemostasis Photodetector device used to measure/detect light scattered off or to attach to collagen fibers or other nonplatelet surfaces. emitted from particles. Platelet aggregation interaction of the cells involved in primary Photomultiplier tube light detector used in flow cytometers and hemostasis with one another to form a clumped mass; can occur in other analytical instruments. vitro or in vivo. Pia mater thin membrane directly covering the central nervous Platelet clump aggregation of cells involved in primary hemostasis; system; middle layer of the meninges. can occur when blood is collected by capillary puncture (from cell activation) and when blood is collected in EDTA anticoagulant (the Pica perversion of appetite that leads to bizarre eating practices; a result of unmasking cell antigens that can react with antibodies in clinical finding in some individuals with iron-deficiency anemia. the serum). Pitting removing abnormal inclusions from erythrocytes by the Platelet distribution width (PDW) coefficient of variation of platelet spleen. volume distribution; analogous to RDW. PIVKA See Protein induced by vitamin K absence or antagonist. Platelet factor 4 protein present in the platelet a@granules; is capable Plasma liquid component of blood. of neutralizing heparin. Plasma cell transformed, fully differentiated B lymphocyte normally Plateletpheresis procedure in which cells involved in primary found in the bone marrow and medullary cords of lymph nodes. hemostasis are removed from the circulation. It can be seen in the circulation in certain infections and disorders Platelet-poor plasma (PPP) citrated liquid blood component con- associated with increased serum g@globulins. It is characterized by taining less than 15 * 103/mcL platelets; used for the majority of the presence of an eccentric nucleus containing condensed, deeply coagulation tests; prepared by centrifugation of citrated whole blood staining chromatin and deep basophilic cytoplasm. The large Golgi at a minimum RCF of 1000 * g for 15 minutes. apparatus next to the nucleus does not stain, leaving an obvious Platelet procoagulant activity property of platelet that enables clear paranuclear area. The organism has the PC-1 membrane anti- activated coagulation factors and cofactors to adhere to the cell’s gen and cytoplasmic immunoglobulin. surface during the formation of fibrin. Plasma cell neoplasm malignant disorder of the immunoglobulin Platelet-rich plasma (PRP) citrated blood component containing secreting, fully differentiated B lymphocyte. approximately 200 * 109/L platelets fused in aggregation studies. Plasma exchange removal of patient plasma and replacement with It is prepared by centrifugation of citrated whole blood at an RCF of donor plasma. 150 * g for 10 minutes. 1200 Glossary Platelet satellitism adherence of primary platelets to neutrophil Polyploid having a number of chromosomes per cell that is a multi- membranes in vitro; can occur when blood is collected in EDTA ple of n (23) other than 1n or 2n (e.g., 3n[69], 4n[92]). anticoagulant. Polyploidy pertaining to cells and organisms that contain more than Platelet secretion release of the contents of the platelet’s a@granules two paired (homologous) sets of chromosomes. and dense bodies during platelet activation. Popcorn cell (L&H cell) neoplastic cell found in LP Hodgkin Platelet-type-pseudo VWD disorder characterized by an increased lymphoma characterized by a delicate multilobated nucleus and affinity of the platelet GPIb/IX receptor for VWF, resulting in multiple, small nucleoli. It has a B cell phenotype: LCA+ (leukocyte spontaneous binding of the large VWF multimers to the platelet. It common antigen), CD20+ , CD15. resembles VWD clinically and often presents with similar laboratory Porphyria group of inherited disorders characterized by a block test results but is not associated with genetic mutations involving in porphyrin synthesis caused by a defect in one or more of the the VWF gene and thus is not considered “true” VWD. enzymes in the heme synthesis pathway. It causes a buildup of Pleocytosis abnormally increased number of cells in the cerebrospi- compounds of heme precursors that accumulate in tissues and are nal fluid (CSF). excreted in large amounts in the urine and/or feces. The two forms are hepatic and erythropoietic. Only the erythropoietic types affect Pleura lining of the cavities between the chest wall and the lungs. the erythrocytes. Plethora excess of blood. Porphyrin any of various compounds of highly unsaturated tetrapy- Pleural cavity space between the chest wall and the lungs. rrole ring bonded by 4 methane ( ¬ CH ¬ ) bridges; is metabolically active only when chelated. Substituents occupy each of the eight Plumbism lead poisoning. peripheral positions on the 4-pyrrole rings. The type and order of Pluripotential cell cell that differentiates into many different cell these substituents determine the type of compound. lines. It has the potential to self-renew, proliferate, and differentiate Portland hemoglobin embryonic intracellular erythrocyte protein into erythrocytic, myelocytic, monocytic, lymphocytic, and mega- in the yolk sac that is detectable up to 8 weeks of gestation. It is karyocytic blood cell lineages. composed of two zeta (z) and two gamma (g) chains. Poikilocytosis presence of erythrocytes with variations in shape. Postmitotic pool compartment of neutrophils including metamye- Point-of-care testing laboratory examination performed at a site locytes, bands, and segmented ones in the bone marrow that are not close to the patient. capable of mitosis. Cells spend about 5–7 days in this compartment before being released to the peripheral blood. Also called maturation- Point-of-care (POC) instrument device designed for analytical test- storage pool. ing of patient specimens outside the laboratory setting (e.g., at home or physician’s office). Post-translational modification process occurring in eukaryotic cells that change the protein product resulting from the ribosomal trans- Polychromatophilia quality of being stainable with more than one lation; it can involve the addition of sugar groups (glycosylation) portion of the stain; commonly used to describe erythrocytes that and phosphate groups (phosphorylation) or other modifications to stain with a grayish or bluish tinge with Romanowsky stains from amino acids (e.g., gamma carboxylation of coagulation proteins). residual RNA, which takes up the blue portion of the dye. Precursor neoplasm acute abnormal tissue derived from a mutated Polychromatophilic erythrocyte red blood cell with a bluish tinge precursor cell and represented by an increased number of blasts in when stained with Romanowsky stain; contains residual RNA. If the peripheral blood and bone marrow. stained with new methylene blue, the cell shows reticulum and is identified as a reticulocyte. Primary aggregation earliest association of platelets in a reversible combination. Polyclonal derived from different cell clones. Primary fibrinogenolysis clinical situation that occurs when exces- Polyclonal gammopathy alteration in immunoglobulin production sive quantities of plasminogen activators are released into the blood characterized by an increase in immunoglobulins of more than one in the absence of fibrin clot formation. Excess plasmin degrades class. fibrinogen and the clotting factors, leading to a potentially danger- Polycythemia condition associated with increased erythrocyte ous hemorrhagic condition. count. Primary hemostasis initial arrest of bleeding that occurs with blood Polycythemia vera (PV) myeloproliferative neoplasm in which there vessel–platelet interaction. is an increased proliferation of erythroid cells. Primary hemostatic plug platelet aggregate that initially halts blood Polymerase chain reaction procedure for copying a specific DNA flow from an injured vessel. sequence many times. Primary myelofibrosis (PMF) neoplastic clonal hematopoietic stem Polymorphic being genetically variable. cell disorder with splenomegaly, leukoerythroblastosis, extramed- ullary hematopoiesis (myeloid metaplasia), and progressive bone Polymorphic variant version of the form and structure of a portion marrow fibrosis; classified as a myeloproliferative neoplasm by the of a chromosome that has no clinical consequence. World Health Organization. Also known as myelofibrosis with myeloid Polymorphism condition in which alternate copies (alleles) of a metaplasia. gene are present. Primary thrombocytosis disease caused by an increase in platelets Polymorphonuclear having the form and structure of the nucleus of that is not secondary to another condition. It usually refers to the the granulocyte in which the nucleus is segmented. increase in platelets that can occur in neoplastic disorders. Polymorphonuclear neutrophil (PMN) mature granulocyte found Probe tool for identifying a particular nucleotide sequence of inter- in bone marrow and peripheral blood that primarily functions as est. It is composed of a nucleotide sequence that is complementary defense against foreign antigens. The nucleus is segmented into to the sequence of interest and is therefore capable of hybridizing to two or more lobes. The cytoplasm stains pinkish, and specific the sequence. Probes are labeled in a way that is detectable, such as granules are abundant. This is the most numerous leukocyte by radioactivity. in the peripheral blood (1.897.0 * 103/mcL) and is active in Procoagulant inert precursor of a natural substance necessary for phagocytosis and killing microorganisms. Also called segmented blood clotting or a property of anything that favors formation of a neutrophil or seg. blood clot. Glossary 1201 Proficiency testing examining unknown specimens from an external Prourokinase immature, single-chain form of enzyme prepared source (e.g., College of American Pathologists) to monitor the qual- from urine and by recombinant DNA techniques;

can be activated to ity of a given laboratory’s test results. a two-chain form by plasmin. Progenitor cell parent or ancestor cell that differentiates into mature, Pseudochylous fluid that appears chylous because of the pres- functional cells. ence of many inflammatory cells; does not contain lymph fluid or Prolymphocyte immediate precursor of the lymphocyte; normally chylomicrons. found in bone marrow. It is slightly smaller than the lymphoblast Pseudodiploid cell that has a chromosome count of 2n (46) but with and has a lower nuclear-to-cytoplasmic ratio. The nuclear chromatin a combination of numerical and/or structural aberrations (e.g., 46, is somewhat clumped, and nucleoli are usually present. The XY, -5, -7, 2D8, 2D21). cytoplasm stains light blue and is agranular. Pseudo-neutrophilia increase in the concentration of neutrophils Promonocyte monocytic precursor cell found in the bone marrow; is in the peripheral blood (more than 7.0 * 103/mcL) occurring as a 14918 mcM (mm) in diameter with abundant blue-gray cytoplasm. result of cells from the marginating pool entering the circulating Fine azurophilic granules can be present. The nucleus is often pool. The response is immediate but transient. This redistribution irregular and deeply indented. The chromatin is finely dispersed of cells accompanies vigorous exercise, epinephrine administration, and stains a light purple blue. Nucleoli can be present. Cytochemi- anesthesia, convulsion, and anxiety states. Also called immediate or cally, the cells stain positive for nonspecific esterase, peroxidase, acid shift neutrophilia. phosphatase, and arylsulfatase. The cell matures to a monocyte. Pseudo–Pelger-Huët cell unusually acquired condition in which Promyelocyte granulocytic precursor cell normally found in the neutrophils display a hyposegmented nucleus. Unlike the real Pelger- bone marrow; is 15921 mcM (mm) in diameter. The cytoplasm is Huët anomaly, this cell’s nucleus contains a significant amount of basophilic, and the nucleus is quite large. The nuclear chromatin is euchromatin and stains more lightly. A critical differentiation point lacy, staining a light purple blue. Several nucleoli are visible. The is that all neutrophils are equally affected in the genetic form of distinguishing feature is the presence of large blue-black primary Pelger-Huët anomaly, but only a fraction are hyposegmented cells in (azurophilic) granules that have a phospholipid membrane that the acquired state. It is associated with MDS and MPD and can also stains with Sudan black B. The granules contain acid phosphatase, be found after treatment for leukemias. myeloperoxidase, acid hydrolases, lysozyme, sulfated mucopolysac- charides, and other basic proteins. The cell matures to a myelocyte. Pulmonary embolism (PE) obstruction of the pulmonary artery Also called progranulocyte. or one of its branches by a clot or foreign material that has been dislodged from another area by the blood current. Pronormoblast precursor cell of the erythrocyte. The cell is derived from the pluripotential stem cell and is found in the bone marrow. Pure red cell aplasia (PRCA) anemia with selective decrease in The cell is 12920 mcM (mm) in diameter and has a high nuclear- erythrocyte precursors in the marrow. cytoplasmic ratio. The cytoplasm is deeply basophilic with Romanowsky Purging cleansing that removes undesirable cells that are present in stains. The nuclear chromatin is fine and has one or more nucleoli. the blood or bone marrow products. This cell matures to a basophilic normoblast. Also called rubriblast. Purpura (1) purple discoloration of the skin caused by petechiae Proplatelet group of platelets comprising a long, slender protrusion and/or ecchymoses; (2) diverse group of disorders characterized by of megakaryocyte cytoplasm released from mature megakaryocytes. the presence of petechiae and ecchymoses. It breaks up into individual platelets. Pyknotic pertaining to degeneration of the cell’s nucleus in which Protein induced by vitamin K absence or antagonists factor that the chromatin condenses to a solid, structureless mass and shrinks. is the nonfunctional form of the prothrombin group of coagulation factors. It is synthesized in the liver in the absence of vitamin K and Quality control (QC) limit expected range of results used to deter- lacks the carboxyl (COOH) group necessary for binding the factor to mine whether a test method is in control and to minimize the chance a phospholipid surface (acarboxy form). of inaccurate results. If the test method is out of control, an interven- tion is required to reconcile the inaccuracy. Proteasome eukaryotic assembly of proteins that degrades other proteins. Quebec platelet disorder storage pool abnormality of platelets caused by aberrant proteolysis of a@granule proteins resulting from Proteomics study of the structure and function of proteins in a cell increased levels of urinary-type plasminogen activator. or tissue at a specific time under certain predefined conditions; includes information on how the proteins interact with each other Quiescence (G0) phase in a cell that has exited the cell cycle and is inside cells. in a nonproliferative state. Prothrombinase complex complex formed by coagulation factors Relaxed (R) structure conformational change in hemoglobin that Xa and V, calcium, and phospholipid; activates prothrombin to occurs as the molecule takes up oxygen. thrombin. Radial immunodiffusion technique in which an antibody is incor- Prothrombin group assemblage of coagulation factors that are porated into an agarose gel into whose wells the antigen is placed. vitamin K dependent for synthesis of their functional forms and that The antigen is quantitated by the size of a precipitin ring that forms require calcium for binding to a phospholipid surface. It includes as the antigen diffuses from a sample well into the gel. factors II, VII, IX, and X. Also known as vitamin K–dependent factor. Random access capability of an automated hematology instru- Prothrombin time (PT) screening test used to detect deficiencies in ment to process specimens independently of one another; can be the extrinsic and common pathway of the coagulation cascade and programmed to run individual tests (e.g., Hb or platelet counts) or to monitor the effectiveness of oral anticoagulant therapy. a panel of tests (e.g., CBC with reticulocyte count) without operator intervention. Prothrombin time ratio proportion calculation derived by dividing the patient’s prothrombin time result by midpoint of the labora- Random variation fluctuation within an instrument or test method tory’s normal range and to calculate the International Normalized caused by chance. It can be either positive or negative in direction Ratio (INR). and affects precision. Proto-oncogene normal hereditary unit that has the potential Rapoport-Luebering shunt metabolic pathway in which 2,3-bispho- to cause cancer when it mutates to become a dominant-acting sphoglycerate (2,3-BPG) is synthesized from 1,3-bisphosphoglycer- oncogene. It is normally involved in regulating the cell cycle, cell ate. 2,3-BPG facilitates the release of oxygen from hemoglobin in the differentiation and maturation, and apoptosis. erythrocyte. Also referred to as 2,3-diphosphoglycerate (DPG). 1202 Glossary Raynaud’s phenomenon secondary disorder resulting from vaso- Relaxed (R) structure conformation of hemoglobin when it com- arterial spasms in the extremities of the body when exposed to bines with oxygen; high oxygen affinity conformation. the cold. It is characterized by blanching of the skin followed by Remission diminution of disease symptoms. cyanosis and finally redness when the affected area is warmed. Also referred to as acrocyanosis. Replication process by which DNA is copied during cell division by the enzyme DNA polymerase, which recognizes single-stranded RBC indices measurements that help classify erythrocytes as to DNA and fills in the appropriate complementary nucleotides to their size and hemoglobin content. The values for hemoglobin, produce double-stranded DNA. Synthesis is initiated at a free 5′ hematocrit, and erythrocyte are used to calculate the three indices: end where double-stranded DNA lies adjacent to single-stranded mean corpuscular volume (MCV), mean corpuscular hemoglobin DNA, and replication proceeds in the 5′ direction. In the laboratory, concentration (MCHC), and mean corpuscular hemoglobin (MCH). the DNA duplication can be induced as a means of copying DNA They give a clue as to what the erythrocytes should look like on a sequences as exploited in the polymerase chain reaction. stained blood film. Reportable range span defined by a minimum value and a maxi- Reactive lymphocyte antigen-stimulated white blood cell that mum value of calibration material. exhibits a variety of morphologic features. It is usually larger and has a more basophilic cytoplasm than the resting cell and has an Restriction point (R) stage that occurs in late G1 that occurs when irregular shape. Its nucleus is often elongated and irregular with a cell cycle progression becomes autonomous. finer chromatin pattern than that of the resting lymphocyte. Often Reticular cell one of the three major types of cells in the bone this cell is increased in viral infections. Also called virocyte, or marrow stroma. It also is found in the spleen and lymph nodes. stimulated, transformed, atypical, activated, or leukocytoid lymphocyte. These cells branch to form reticular fibers. Also called fibroblasts. Reactive lymphocytosis self-limiting, reactive process resulting in Reticulated platelet young, immature platelet with residual RNA an increase in lymphocytes that occurs in response to an infection or circulating in the peripheral blood. The number of reticulated plate- inflammatory condition. Both T and B types are commonly affected, lets reflects the activity of megakaryopoiesis in the bone marrow. but their function remains normal. Reticulocyte first non-nucleated stage of erythrocyte development Reactive neutrophilia increase in the concentration of peripheral in the bone marrow. It contains RNA that is visualized as granules blood neutrophils (more than 7.0 * 103/mcL) as a result of reaction or filaments within the cell when stained supravitally with new to a physiologic or pathologic process. methylene blue. Normally, this stage constitutes approximately 1% of the circulating erythrocyte population. Reagent blank measurement of absorbance from a substance alone that eliminates a false increase in sample absorbance caused by the Reticulocyte hemoglobin (RET-He) reticulocyte hemoglobin report- substance’s color. ed by the Sysmex automated hematology analyzers; analogous to the MCH of mature erythrocytes. Recombinant factor VIIa (rFVIIa) form of blood factor VII that has been manufactured via laboratory method of genetic technology. Reticulocyte production index (RPI) calculated value that is an indicator of bone marrow response in anemia. Its calculation Recombination-activating genes 1 and 2 (RAG-1, RAG-2) enzyme corrects the reticulocyte count for the presence of marrow components of the V(D)J recombinase complex expressed in devel- reticulocytes in the peripheral blood. It is calculated as follows: oping B and T cells, which are critical for DNA recombination events that form functional immunoglobulin and T cell receptor genes. RPI = (Patient hematocrit [L/L] , 0.45 [L/L] * reticulocyte Red blood cell (RBC) cellular element of blood that carries the protein count [,]) * (1 , maturation time of shift reticulocytes) hemoglobin. Its function is to carry oxygen and carbon dioxide Reticulocytosis presence of excess reticulocytes in the peripheral between the lungs and the tissues. It does not have a nucleus and blood. develops in the bone marrow from the hematopoietic stem cell. Rh null disease disorder associated with the lack of the Rh antigen When released to the peripheral blood from the bone marrow, it has on erythrocytes. a lifespan of about 120 days. Also called an erythrocyte. Ribosomes cellular particle composed of ribonucleic acid (RNA) Red cell distribution width (RDW) coefficient of variation of the and protein whose function is to synthesize polypeptide chains from MCV; a calculated index provided by hematology analyzers to help amino acids. The sequence of amino acids in the chains is specified identify anisocytosis (standard deviation of MCV * 100/mean by the genetic code of messenger RNA. This particle appears singly MCV) with a reference interval of 11.5–14.5%. or in reversibly dissociable units and can be free in the cytoplasm Red thrombi blood clots formed in the veins where flow is slower or attached to endoplasmic reticulum. The cytoplasm of blood cells than in other areas; primarily composed of red blood cells trapped that contain a high concentration of ribosomes stains bluish purple in the fibrin mesh with relatively few platelets and leukocytes. with Romanowsky stains. Reed-Sternberg (R-S) cell cell found in the classic form of Hodg- Richter’s transformation change from CLL to another disease, kin lymphoma; characterized by a multilobated nucleus and large usually large B cell lymphoma. inclusion-like nucleoli. Ring sideroblast erythroblast with abnormal deposition of excess Reference interval (RI) test value range considered to be normal iron within mitochondria resulting in a ring formation around the and generally determined to include 95% of the normal population. nucleus. Reflex test follow-up examination performed as the results of Ristocetin aggregating reagent that specifically evaluates VWF screening tests. interaction with glycoprotein Ib on platelets. Refractive index degree to which a transparent

object will deflect a Ristocetin cofactor property of the aggregating reagent when it acts light ray from a straight path. as a cofactor in the ristocetin cofactor assay that screens for binding of VWF to platelet GPIb. When this reagent is added to a suspension Refractory pertaining to disorders or diseases that do not respond of platelets in plasma that contains VWF, it promotes the binding of readily to therapy. VWF to platelet membrane GPIb, causing platelet aggregation at a Regulatory T lymphocyte (TReg) subpopulation of T lymphocytes rate depending on the concentration of VWF and reagent. that suppress the immune response and maintain peripheral toler- Ristocetin-induced platelet aggregation (RIPA) measures ability of ance to self-antigens (i.e., suppress autoimmunity); this subpopula- patient’s VWF to bind to normal platelets, inducing platelet combin- tion primarily includes CD4+ , CD25+ , and FoxP@3+ . ing in a mass in a platelet aggregation assay. Glossary 1203 Rivaroxaban (Xarelto) anti-FXa direct oral anticoagulant (DOAC) Secondary (reactive) thrombocytosis disorder in which platelet used to prevent thrombosis in a variety of conditions. concentration in the blood increases in response to stimulation by RNA (ribonucleic acid) single-stranded molecule composed of another condition. ribonucleotides (A, C, G, and U) produced by transcription of genes Secretion energy-dependent discharge or release of products from a DNA template. This molecule in turn serves as a template for usually from glands in the body after stimulation of the platelets by protein translation. agonists; also, the product that is discharged or released. Romanowsky-type stain any dye consisting of methylene blue Self-renewal property of regenerating the same cells. and its oxidation products and eosin Y or eosin B. Wright’s stain, a Semen fluid in the male that contains spermatozoa and is dis- Romanowsky-type stain, is commonly used to stain hematopoietic charged at ejaculation. cells. Sequestration crisis sudden splenic pooling of sickled erythrocytes Rouleaux erythrocyte distribution characterized by being stacked that can cause a large decrease in erythrocyte mass within a few like a roll of coins. This is the result of abnormal coating of the cell’s hours. surface with increased plasma proteins, decreasing the zeta potential between cells. Serine protease family of enzymes that cleave peptide bonds in proteins including thrombin, factors VIIa, IXa, Xa, XIa, Rule of three in general, the formula is XIIa, and the digestive enzymes chymotrypsin and trypsin. It hemoglobin * 3 = hematocrit that is used as a quick mathematical selectively hydrolyzes arginine- or lysine-containing peptide check on the accuracy of the measurement of hemoglobin and hema- bonds of other zymogens, converting them to serine proteases. tocrit. It works only with normochromic, normocytic erythrocytes. If Each involved in the coagulation cascade is highly specific for its the calculated hematocrit does not agree within {3, of the meas- substrate. ured hematocrit, investigation for an error or instrument malfunc- tion should be conducted. The patient also can have a disorder that Serpin family of proteins with similar structures that inhibit target needs further investigation. molecules by forming a 1:1 stoichiometric complex. Russell body globule filled with immunoglobulin found in patho- Serum transferrin receptor (sTfR) extracellular portion of the pro- logic plasma cells called Mott cells. See Mott cell. tein of the b@globulin group receiver that is cleaved by proteolytic enzymes as the erythrocytes mature and is released into the blood. Russell’s viper venom poisonous substance that possesses thrombo- When erythropoiesis increases, the sTfR increase. plastin-like activity and activates factor X. Severe combined immunodeficiency syndrome (SCIDS) most Safety data sheet (SDS) document that provides safety information severe form of a heterogeneous group of disorders in which for clinical laboratory professionals who use hazardous materials; there is a lack of ability to mount an immune response. The includes pertinent safety information regarding the proper storage disease can be inherited either as a sex-linked trait or as an and disposal of a chemical, precautions that should be taken in han- autosomal-recessive trait. dling it, potential health hazards associated with exposure to it, and whether it is a fire or explosive hazard. Sézary cell circulating neoplastic cell found in patients with Sézary syndrome; it is a malignant mature memory helper T lymphocyte Satellite DNA nucleic acid containing many tandem repeats. which is characterized by a very convoluted (cerebriform) nuclear Morphologically, it appears as a small ball-like structure making outline. up the short arm of acrocentric chromosomes. It is the locus of the nucleolar organizing region. Shift neutrophilia See Pseudo-neutrophilia. Scatterplot dot-plot histogram of two cellular characteristics. Shift to the left (left shift) appearance of increased numbers of Together, the two chara cteristics allow definition of the leuko- immature leukocytes in the peripheral blood. cyte subpopulations. Sickle cell (drepanocyte) elongated crescent-shaped erythrocyte Schilling test definitive analysis used in distinguishing cobalamin with pointed ends whose formation may be observed in wet prepa- (vitamin B12) deficiency caused by malabsorption, dietary deficiency, rations or in stained blood smears from patients with sickle cell or absence of IF. It measures the amount of an oral dose of radioac- anemia. These cells are formed when the HbS becomes deoxygen- tively labeled crystalline vitamin B12 that is absorbed in the gut and ated and polymerizes into rigid aggregates. excreted in the urine. Sickle cell anemia genetically determined disorder in which hemo- Schistocyte fragment of an erythrocyte that can have a variety of globin S is inherited in the homozygous state, (bsbs). No hemoglobin shapes including triangle, helmet, and comma. A is present. Hemoglobins S, F, and A2 are present. Scott syndrome rare platelet disorder characterized by abnormal Sickle cell trait genetically determined disorder in which hemo- Ca++ induced phospholipids scrambling in which plate- globin S is inherited in the heterozygous state. The patient has one let membranes fail to support plasma procoagulant protein normal b@globin gene and one bs@globin gene. Both hemoglobins A activation. and S are present as well as hemoglobins F and A2. Sea blue histiocytosis syndrome rare inherited disorder character- Side light scatter laser light distributed at a 90° angle caused by ized by splenomegaly and thrombocytopenia. Sea blue-staining a particle’s internal complexity and granularity (e.g., neutrophils macrophages are found in the liver, spleen, and bone marrow. The produce much side spread because of their numerous cytoplasmic cell is large (in diameter) with a dense eccentric nucleus and cyto- granules). plasm that contains blue or blue-green granules. In most patients, Sideroblast erythroblast that contains stainable iron granules. the course of the disease is benign. Sideroblastic anemia form of congenital and acquired disorders Secondary fibrinolysis clinical condition characterized by exces- characterized by an increase in total body iron, the presence of ring sive fibrinolytic activity in response to disseminated intravascular sideroblasts in the bone marrow, hypochromic erythrocytes in the clotting. peripheral blood, and deficiency of hemoglobin and/or erythro- Secondary hemostasis formation of fibrin that stabilizes a primary cytes. The acquired idiopathic form is classified as a myelodysplastic platelet plug. syndrome (MDS). Secondary hemostatic plug primary platelet aggregate that has Siderocyte erythrocyte that contains stainable iron granules. been stabilized by fibrin formation during secondary hemostasis. Sideropenic lack of iron. 1204 Glossary Sideropenic anemia blood disorder caused by insufficient iron for Starry sky appearance morphologic pattern characteristic of erythropoiesis resulting in a deficiency of hemoglobin. high-grade lymphoma produced by numerous tingible body Single nucleotide polymorphism (SNP) change in which a single macrophages (stars) and a diffuse sheet of neoplastic cells (sky). base in the DNA differs from the usual base at that position. Stomatocyte abnormal erythrocyte shape characterized by a slitlike Slope (b) angle or direction of the regression line with respect to the area of central pallor and a uniconcave, cup shape. x- and y-axes that is used to identify the presence of proportional Streptokinase bacterial enzyme derived from group C-beta hemo- systematic error. lytic streptococci; the enzyme activates plasminogen to plasmin and Small lymphocytic lymphoma (SLL) condition identical to CLL is used as a thrombolytic agent in the treatment of thrombosis. except that it primarily involves the lymph nodes instead of the Stress erythropoiesis acutely increased erythrocyte production in bone marrow and peripheral blood. The two disorders appear to response to increased demand such as in hemolytic anemia. belong to one disease entity with differing clinical manifestations. Stroma extracellular matrix or microenvironment that supports Smooth endoplasmic reticulum (SER) See Endoplasmic reticulum. hematopoietic cell proliferation in the bone marrow. Smudge cell leukocyte whose cytoplasmic membrane has ruptured, Stromal cell element of the hematopoietic microenvironment in the leaving a bare nucleus. Increased numbers are observed in lym- red portion of bone marrow. phoproliferative disorders such as chronic lymphocytic leukemia. The ruptured leukocyte can also be seen in reactive lymphocytosis Submetacentric chromosome that has the centromere positioned off and in other neoplasms. An increased number of these leuko- center so that one arm is shorter than the other arm. cytes can result in a falsely decreased WBC count and erroneous Sucrose hemolysis test screening examination to identify eryth- differential. rocytes that are abnormally sensitive to complement lysis. When Specific esterase enzyme that hydrolyzes particular esters; can be erythrocytes, serum, and sucrose are incubated together, cells abnor- detected in cells with a stain using naphthol AS-D chloroacetate mally sensitive to complement lyse. The test is used to screen for at an acid pH; is considered specific for the granulocytic series, paroxysmal nocturnal hemoglobinuria. Also called sugar-water test. and is present in mast cells; used to differentiate monoblasts and Sulfhemoglobin stable compound formed when a sulfur atom myeloblasts combines with each of the four heme groups of the intracellular Specificity ability of a test method to determine only the analyte erythrocyte oxygen carrying protein; when this protein is combined meant to be detected or measured. with sulfur, it is incapable of carrying oxygen. Specimen run interval, period of time, or number of specimens for Supernatant clear liquid remaining on top of a solution after cen- which the accuracy and precision of the laboratory procedure is trifugation of the particulate matter. expected to remain stable. Supravital stain agent used to color cells or tissues while they are Spectrin predominant peripheral membrane protein composed of still living. dimeric chains a and b that associate to form tetramers; found in Syngeneic in transplantation biology, having identical genotypes. the erythrocyte cytoskeleton. Syngeneic stem cell transplantation medical process of moving Spent phase stage in polycythemia vera in which after a period of stem cells between genetically identical twins. 2–10 years, the patient can develop bone marrow failure accom- panied by an increase in splenomegaly. Anemia and bleeding can Synovium continuous membrane that lines the bony, cartilaginous, be the primary clinical findings, secondary to a decreased platelet and connective tissue surfaces of a joint. count and decreasing hematocrit. This phase is often a transition Systemic mastocytosis abnormal clonal proliferation of mast cells to AML. in at least one extracutaneous organ with or without evidence of Sperm male gamete formed in the seminiferous tubules. Also skin lesions; the bone marrow is almost always involved but the known as spermatozoa. peripheral blood rarely has significant numbers of circulating mast cells.; laboratory studies that demonstrate a mutation of the c-Kit Spherocyte abnormally round erythrocyte with dense hemoglobin proto-oncogene and an aberrant immunophenotype are needed to content (increased MCHC) and no central area of pallor because it make the diagnosis. has lost its biconcave shape. Systematic variation difference within an instrument or test method Splenectomy removal of the spleen. that occurs in one direction, can be predicted, and affects accuracy. Splenomegaly abnormal enlargement of the spleen. TH17 lymphocytes subpopulation of CD4+ T cells identified for Split specimen division of a single specimen into two or more ali- their production of IL-17, this subpopulation of cells induce an quots for testing that uses two or more instruments within the same inflammatory response and aid in host defense against extracellular time period or retesting the sample at another time. pathogens. Spur cell anemia acquired hemolytic condition associated with Target cell abnormally shaped erythrocyte that has decreased severe hepatocellular disease such as cirrhosis in which serum osmotic fragility and appears as a target with a bull’s-eye center lipoproteins increase, leading to an excess of erythrocyte membrane mass of hemoglobin surrounded by an achromic ring and an outer cholesterol. The total phospholipid content of the membrane, how- ring of hemoglobin. Also called Mexican hat cell and codocyte. ever, is normal. The predominant poikilocyte is an erythrocyte with Tartrate-resistant

acid phosphatase (TRAP) acid phosphatase irregular points and no area of central pallor (acanthocytes). whose activity is not inhibited following tartrate incubation; found Spurious fake, misleading, not what it purports to be. in hairy cells of hairy cell leukemia. Stab See Band neutrophil. T cell acute lymphoblastic leukemia (ALL) neoplasm of the white Stage phase of a neoplastic disease defined by the extent and distri- blood cells, and immunologic subgroup of ALL. The neoplastic cells bution of disease. Determining the phase of disease usually involves are precursor cells of the lymphocytic lineage with T cell antigens. radiologic studies, peripheral blood examination, bone marrow aspi- T cell receptor (TCR) antigen receiver on immunocompetent T ration, and biopsy. lymphocytes. Standard deviation (SD) distribution of a set of data about the T lymphoid cell antigens cluster of differentiation (CD) markers mean. that characterize the T cell lineage. Glossary 1205 Teardrop (dacryocytes) erythrocyte that is elongated at one end to Thrombopoietin cytokine that regulates the maturation of mega- form a teardrop or pear-shaped cell; can form after erythrocytes with karyocytes and the production of platelets. cellular inclusions have transversed the spleen. The erythrocyte can- Thrombosis formation or presence of a clot, usually considered to not return to its original shape because it has either been stretched be under abnormal conditions within a blood vessel. beyond its limits of deformability or has been in the abnormal shape too long. Thrombotic microangiopathy (TMA) condition that results in thrombosis in capillaries and arterioles, due to an endothelial injury. Telangiectasia condition characterized by persistent dilation of It may be seen in association with thrombocytopenia, anemia, pur- superficially located veins. pura, and renal failure. Tense (T) structure conformational change in the makeup of a Thrombotic thrombocytopenic purpura (TTP) acute disorder char- hemoglobin molecule that occurs when oxygen is released from acterized by microangiopathic anemia, decreased number of plate- hemoglobin. lets, and renal failure as well as neurological symptoms. It is caused Terminal deoxynucleotidyl transferase (TdT) DNA polymerase by decreased activity of ADAMTS-13, resulting in the presence of found in pre-B and pre-T lymphoid precursor cells, which adds ultralarge VWF molecules in circulation and platelet agglutination. nucleotides to V, D, and J segments during DNA recombination events that form functional immunoglobulin and T cell receptor Thromboxane one of two compounds: designated A2 and B2. genes; is responsible for junctional diversity. Thromboxane A2 is synthesized by platelets and is an inducer of platelet aggregation and platelet release functions and is a vasocon- TH17 cell See TH17 lymphocyte. strictor; it is very unstable and is hydrolyzed to thromboxane B2. Thalassemia group of genetically determined microcytic, hypochro- Thrombus blood clot within the vascular system. mic anemias resulting from a decrease in synthesis of one or more globin chains in the hemoglobin molecule. The disorder can occur TIBC See Total iron-binding capacity. in the homozygous or heterozygous state. Heterozygotes can be Tingible body macrophage tissue cell of the immune system phago- asymptomatic or have a mild disease, but homozygotes typically cytosing, engulfing, and usually destroying fragments of dying cells. have a severe, often fatal, disease. Thalassemia occurs most fre- It is found in areas of extensive apoptosis (reactive germinal centers quently in populations from the Mediterranean area and Southeast and high-grade lymphoma). Asia. Tissue factor coagulation component present on subvascular cells Therapeutic range level of a drug that is beneficial but not toxic to that forms a complex with factor VII when the vessel is ruptured. the individual. This complex activates factor X and is an integral protein of the cell Threshold limit level above which voltage pulses of particles are membrane. counted on an automated hematology analyzer. Adjusting the Tissue factor pathway initiation of coagulation associated with the threshold limit allows different types of cells to be counted. exposure of flowing blood to tissue factor (TF), the cellular receptor Thrombin activatable fibrinolysis inhibitor (TAFI) protein and cofactor for factor VIIa (FVIIa), begins the coagulation cascade that inhibits fibrinolysis when activated by the thrombin- that leads to clot formation. thrombomodulin complex. Tissue factor pathway inhibitor (TFPI) coagulation inhibitor Thrombocyte See Platelet. protein that blocks the activity of both FVIIa and FXa by forming a quaternary complex of TFPI-FXa-TF-FVIIa. Thrombocytopenia deficiency of platelets in the blood; decrease in the number of platelets in the peripheral blood below the reference Tissue homeostasis maintenance of an adequate number of cells to range for an individual laboratory (usually less than 150 * 103/mcL). carry out the organism’s functions. It is controlled by cell prolifera- tion, cell differentiation, and cell death (apotosis). Thrombocytopenia with absent radii (TAR) inherited condition characterized by isolated hypoplasia of the megakaryocytic lineage, Tissue-plasminogen activator (tPA) serine protease that activates thrombocytopenia, and bilateral radial aplasia. plasminogen to plasmin. It forms a bimolecular complex with fibrin, increasing the catalytic efficiency of tPA for plasminogen activation. Thrombocytosis disorder in which the body produces too many platelets (thrombocytes) that play an important role in blood clot- T lymphoid cell antigen CD marker that when present is unique to ting; the number of platelets in the peripheral blood is above the and useful in differentiating a T cell from a B cell; used in differenti- reference range for an individual laboratory (usually greater than ating TALL from other types of acute leukemia. 400 * 103/mcL). Total iron-binding capacity (TIBC) Maximum amount of iron able Thromboembolism (TE) blockage of a small blood vessel by a to be bound in the serum; is measured by transferrin. Each gram blood clot (or part of a clot) that was formed elsewhere and moved of transferrin binds 1.4 mg of iron. Enough transferrin is present in through blood vessels until reaching a smaller vessel and blocking plasma to bind 253@435 mcg (mg) of iron per deciliter of plasma. further blood flow. Toxic granule large, dark blue-black primary particle in the cyto- Thrombogenic tendency to clot; to promote the formation of a clot. plasm of neutrophils present in certain infectious states; usually seen in conjunction with Döhle bodies. Thrombolytic therapy use of drugs to dissolve a blood clot or thrombus in vivo in a variety of conditions. Toxoplasmosis condition that results from infection with Toxoplasma gondii; can be asymptomatic or symptoms can resemble infectious Thrombomodulin (TM) integral membrane glycoprotein found on mononucleosis. It is characterized by a leukocytosis with relative endothelial cells that forms a 1:1 complex with thrombin; named lymphocytosis or rarely an absolute lymphocytosis and the presence for its ability to alter the activity of the proteolytic enzyme throm- of reactive lymphocytes. bin from procoagulant to anticoagulant so that it loses its ability to cleave fibrinogen to fibrin and to activate FV, FVIII, or platelets but Trabecula projection of calcified bone extending from cortical bone instead rapidly activates PC in the presence of Ca++. into the marrow space; provides support for marrow cells. Thrombophilia tendency to form blood clots abnormally. Also Transcription synthesis of RNA from a DNA template. referred to as hypercoagulability. Transcription error mistake made in reporting test results when Thrombophlebitis thrombosis within a vein that is accompanied by copying results manually from the instrument to a paper or elec- an inflammatory response, pain, and redness of the area. tronically by typing them into a computer. 1206 Glossary Transcription factor (TF) protein that controls the switching on or uPAR See Urokinase-type plasminogen activator. off the transcription of genes. It binds to regulatory regions in the Urokinase-type plasminogen activator (uPA) enzyme found in genome and helps control gene expression. urine and plasma that activates plasminogen to plasmin; is used as a Transferrin (Tf) plasma b1@globulin responsible for binding iron and thrombolytic agent in the treatment of thrombosis. transporting it in the bloodstream. Each gram can bind 1.25 mg of Urokinase-type plasminogen activator receptor (uPAR) endothelial iron. The capacity of transferrin to bind iron is functionally meas- cell membrane receiver that binds uPA and facilitates plasminogen ured as the total iron-binding capacity (TIBC). activation. Transferrin receptor 1 (TfR1) transmembrane glycoprotein dimer Variable number tandem repeat any of several identical DNA with two identical subunits, each of which can bind a molecule of segments lying one after the other in a sequence duplicated in a transferrin; present on virtually all cells but found in high numbers genome; can vary among different individuals. on cells with high iron requirements. Vascular permeability property of endothelial cells of blood vessels Transferrin receptor 2 (TfR2) transmembrane glycoprotein found that selectively allows for exchange of gases, nutrients, and waste predominantly on hepatocytes, duodenal crypt cells, and erythroid products. cells; it interacts with HFE to regulate hepcidin synthesis and thus has a role in regulating total body iron homeostasis. Vasculitis inflammation of a blood vessel. Transferrin saturation amount of plasma b1@globulin that is Vasoconstriction narrowing of the lumen of blood vessels that complexed with iron. Calculated by serum iron/TIBC * 100 = , occurs immediately after an injury to the vessel. saturation; is normally about one-third saturated with iron. Vaso-occlusive crisis acute event caused by spontaneous blockage Transglutaminase activity of clotting FXIII that promotes the forma- of microvasculature by rigid sickle cells; can be triggered by infec- tion of stable covalent cross-links between glutamine and lysine tion, dehydration, decreased oxygen pressure, or slow blood flow residues on strands of fibrin; FXIIIa is the only coagulation protein but often occurs without a known cause. with this activity. Vertical interaction up-and-down interaction involving the skeletal Translation step in protein biosynthesis in which synthesis of a lattice and proteins of the erythrocyte membrane that stabilizes the protein from an RNA template occurs. lipid bilayer membrane. Translocation abnormal chromosomal rearrangement in which part Viral load number of copies of HIV-1 RNA that indicates a patient’s of one chromosome breaks off and becomes attached to another one. status usually reported in virus particles per milliliter. The site of juxtaposition between the two chromosomes is referred Viscosity resistance to flow; physical property that depends on the to as the break point. friction of component molecules in a substance as they pass one Transudate effusion that is formed as the result of increased hydro- another. static pressure or decreased osmotic pressure; does not indicate a Vitamin K deficiency bleeding (VKDB) symptomatic VK deficiency true pathologic state in the anatomic region. in newborns that results in bleeding; formerly, called hemorrhagic Trisomy condition in which one daughter cell has an extra chromo- disease of the newborn (HDN). some (three copies instead of two). Vitamin K dependent having to do with a coagulation factor that Tumor suppressor gene hereditary unit whose protein products requires vitamin K to be functional; such a protein includes the function to inhibit the growth of normal cells. Both alleles of a tumor prothrombin group coagulation factors, protein C, protein S, and suppressor hereditary unit must be altered to lose the function of a protein Z tumor suppressor unit of heredity. Vitamin K-dependent factor See Prothrombin group. Turbidimetric clot detection measurement of the cloudiness of a Vitronectin serum or extracellular-matrix glycoprotein capable of fluid caused by suspension of particles. binding heparin. Turnaround time (TAT) period between collecting a specimen and von Willebrand disease (VWD) autosomal dominant hereditary reporting a test result. bleeding disorder that lacks von Willebrand factor (VWF), which Tyrosine kinase protein compound that regulates metabolic path- is needed for platelets to adhere to collagen. Platelet aggregation is ways and serves as receptor for growth factors. abnormal with ristocetin as is the bleeding time. The APTT can be prolonged because of a decrease in the FVIII molecule secondary to Ubiquitin protein found in all eukaryotic cells that becomes a decrease in VWF. covalently attached to certain residues of other proteins and tags a protein for intracellular proteolytic destruction. von Willebrand disease Type 1 (classic VWD) coagulation abnor- mality characterized by a quantitative decrease of structurally UIBC See Unsaturated iron-binding capacity normal VWF. Unfractionated heparin (UFH) heterogeneous mixture of sulfated von Willebrand disease Type 2 coagulation abnormality character- glycosaminoglycans (range 5–30 kilodaltons; 5,000–30,000 daltons) ized by a qualitative disorder of VWF; has four possible subtypes: obtained from extraction of porcine intestinal mucosa or bovine 2A, 2B, 2M, and 2N. lung. von Willebrand disease Type 3 coagulation abnormality character- Unique identification number figure assigned to a patient admitted ized by a severe, rare quantitative deficiency of VWF. to the hospital used to identify her or him; printed on the patient’s arm band. von Willebrand factor

(VWF) plasma component that is a molecule of multimers; needed for platelets to adhere to collagen via platelet Universal precaution preventative guideline such as requiring glycoprotein Ib. It is synthesized in megakaryocytes and endothelial health care workers to use gloves when handling any bodily fluid to cells and is noncovalently linked to FVIII in plasma. minimize the workers’ potential exposure to blood-borne pathogens. von Willebrand factor (VWF) activity (VWF:RCo; VWF:A) test Unsaturated iron-binding capacity (UIBC) portion of transferrin that determines the ability of VWF to function in platelet adhe- that is not complexed with iron (TIBC - serum iron = UIBC). sion. VWF:RCo measures the ability of the patient’s VWF to Untranslated region (UTRs) portion of DNA on a chromosome support agglutination of normal platelets by ristocetin in a platelet whose bases are not involved in protein synthesis. aggregometer. Glossary 1207 von Willebrand factor (VWF) antigen immunologic property of the White thrombus blood clot formed in arteries where blood flow is VWF that allows the protein to be identified and measured. rapid; is composed primarily of platelets and fibrin with relatively von Willebrand factor (VWF):Ag assay immunologic test that deter- few leukocytes and erythrocytes. mines the quantity of VWF protein. Wiskott-Aldrich syndrome/X-linked thrombocytopenia (WAS/ von Willebrand factor (VWF) multimer VWF molecule consisting XLT) disorder of small platelets, thrombocytopenia, and severe of multiple copies of identical subunits linked together. immune dysregulation; involves mutations of the WAS gene, which manifests as isolated thrombocytopenia without immune VWF multimers assay test in which the structure of VWF multimers is dysfunction. determined by electrophoresis using 1% agarose gels in the presence of sodium dodecyl sulfate (SDS); used to establish the subtype of VWD. World Health Organization (WHO) classification grouping system for neoplastic blood disorders that uses the test results of cytoge- Waldenström macroglobulinemia combination of lymphoplasma- netics, molecular genetics, and flow cytometry together with cell cytic lymphoma (LPL) with bone marrow involvement and an IgM morphology to identify and classify hematopoietic and lymphoid monoclonal paraprotein. neoplasms. Warm autoimmune hemolytic anemia condition resulting from X-linked thrombocytopenia (XLT) disorder involving mutations the presence of IgG autoantibodies that are reactive at 37°C with of the WAS gene, which manifests as isolated thrombocytopenia antigens on subject’s erythrocytes. The antibody–antigen complex without immune dysfunction. on the cell membrane sensitizes the erythrocyte, which is removed in the spleen or liver. X-linked thrombocytopenia with thalassemia (XLTT) X-linked inherited thrombocytopenia that can occur with or without anemia Wedge smear blood preparation on a glass microscope slide by as a result of mutations in the gene for the transcription factor placing a drop of blood at one end and using a second slide to pull GATA-1 combined with thalassemia. the blood the length of the slide. y-intercept point where the regression line intersects the y-axis; is White blood cell (WBC) cellular component of blood with five used to identify the presence of constant systematic error. major types: neutrophils, eosinophils, basophils, lymphocytes, and monocytes. It develops from the hematopoietic stem cell under the Zygosity state of being homozygous or heterozygous for a gene. influence of cytokines (growth factors) and functions to defend the Zymogen inactive precursor that can be converted to the active form body against foreign antigens such as bacteria. The reference inter- by an enzyme, alkali, or acid; its inert coagulation factors are zymo- val is 4.5911.0 * 103/mcL. Also called leukocyte. gens. Also called proenzyme. This page intentionally left blank Index A Acute basophilic leukemia (ABL), Acute myeloid leukemias (AML), not Abbott CELL-DYN Sapphire, 980–84 600–601 otherwise specified, 596–97 Abetalipoproteinemia, 375, 376, 1125 Acute chest syndrome, 275 Acute myeloid leukemias (AML), Abnormal bleeding, 770–72 Acute leukemias (AL), 585, 1087, WHO classification of, 591–601 Abnormal erythrocyte nucleotide 1168 acute panmyelosis with metabolism, 395 of ambiguous lineage, 616–17, 1004 myelofibrosis, 601 Abnormal tyrosine kinase (TK) bilineage, 616 with BCR-ABL1, 595–96 protein, 504 diagnosis and classification of, 1002 with biallelic mutations of CEBPA, 595 ABO blood group, 839 with lineage heterogeneity, 616–17 FLT3 and, 596 ABO-HDFN, 1127 Acute lymphoblastic leukemia (ALL), with INV(16)(P13.1;Q22) OR ABO incompatibility, 422 508, 608–18, 688, 1047 T(16;16)(P13.1;Q22); Acanthocytes, 73, 187 bone marrow, 610 CBFBMYH11, 592 Acanthocytosis, 375–77 central nervous system with INV(3)(Q21.3;Q26.2) OR Acarboxy form, 738 involvement, 609 T(3;3)(Q21.3;Q26.2); GATA2, Achlorhydria, 334 clinical presentation, 505, 608–9 MECOM, 594–95 Acid elution, for hemoglobin F, 929–30 cytogenetic analysis of, 612, 614, with maturation, 597–601 Acid phosphatase and tartrate- 615, 1027 with minimal differentiation, 597 resistant acid phosphatase immunophenotyping, 611–12 with mutated NPM1, 595 (TRAP), 937–38 laboratory evaluation, 609–11 with mutated RUNX1, 596 Acquired aberration, 1017 peripheral blood, 609–10 with myelodysplasia-related Acquired acute pure red cell aplasia, therapy, 617 changes, 596 356–57 tissue involved in, 610–11 myeloid proliferations related to Acquired autoimmune hemolytic Acute lymphoblastic leukemia (ALL), down syndrome, 601 anemia (AIHA), 369 classification of myeloid sarcoma, 601 Acquired disorders, of vascular of acute leukemias of ambiguous not otherwise specified, 596–97 system, 773–74 lineage, 588, 608 with PML-RARA, 592–94 Acquired immune deficiency syndrome cell lineage, 587–91 with recurrent genetic (AIDS), 150, 477, 483–85 FAB classification, 507, 519 abnormalities, 591 Acquired nonfunctional hemoglobins, WHO classification, 612–17 with T(6;9)(P23;Q34.1); DEK- 104–6 Acute lymphocytic leukemias (ALL), NUP214, 594 Acquired pathologic inhibitors, 819 1002–4 therapy-related myeloid Acquired sideroblastic anemia, 249–51 Acute megakaryoblastic leukemia neoplasms, 596 Acquired thrombohemorrhagic (AMkL), 600 with T(9;11)(P21.3;Q23.3); MLLT3- conditions, 841–53 Acute monoblastic leukemia, 599–600 KMT2A, 592 acquired fibrinolytic defects, 842 Acute monocytic leukemia, 599–600 with T(1;22)(P13.3;Q13.32); RBM15- antiphospholipid antibodies/ Acute myelocytic leukemia. See Acute MKL1, 595 antiphospholipid syndrome, myeloid leukemias (AML) with T(8;21)(Q22;Q22.1); RUNX1- 842–44 Acute myelogenous leukemia. See Acute RUNX1T1, 591–92 hematologic disorders, 853 myeloid leukemias (AML) without maturation, 597 heparin-induced Acute myeloid leukemias (AML), 503, Acute myelomonocytic leukemia thrombocytopenia, 844–46 583–602, 934, 1002, 1046–47, (AMML), 598, 689 malignancy, 852 1086–87. See also Acute Acute neutrophilia, 454 postoperative state and trauma, 853 myeloid leukemias (AML), Acute panmyelosis with pregnancy and oral contraceptives, WHO classification of myelofibrosis, 601 852–53 bone marrow, 586–87, 591, 592 Acute phase reactants, 924 thrombotic microangiopathies, cell lineage, identifying, 587–91 Acute promyelocytic leukemia (APL), 846–52 classification, 587–601 592–94 Acquired von Willebrand syndrome cytochemistry, 588 Acute splenic sequestration, 275 (AVWS), 806 cytogenetic analysis, 1026–27 Acute undifferentiated leukemia Acrocentric chromosomes, 1016 cytogenetic analysis of, 588, 591 (AUL), 616, 1004 Activated partial thromboplastin time diagnosis and classification of, 1004 ADAMTS-13, 436, 439, 744, 846, 882 (APTT), 6, 435, 437, 533, 734, etiology and pathophysiology, 585 Adaptive immune response (adaptive 771, 816, 868, 875–76, 893–94 immunophenotyping, 588–90 IR), 133 Activated protein C resistance laboratory evaluation, 585–87 adhesion molecules, 158–59 (APCR), 838, 889 peripheral blood, 585–86 defined, 114 Acute, acquired hemolytic anemia, 390 therapy, 601 lymphocytes, 140 1209 1210 Index Adducin, 76 Amyloidosis, 774 hemoglobin count, 206–7 Adenocarcinoma, 684 Analytical measurement range (AMR), leukocyte and platelet Adenosine deaminase (ADA) 1063 abnormalities, 210 deficiency, 486 Analytical reliability, 1062 reticulocyte count, 207–8 Adenosine diphosphate (ADP) Analytical sensitivity, 1061 Anemia of chronic disease (ACD), 227, receptor antagonists, 858 Analytical specificity, 1061 236 Adenosine triphosphate (ATP), 217, Anaphase, 17 bone marrow, 247 385–86, 1037 Anaphase lag, 1020 clinical presentation, 246 Adherence, neutrophil, 120–22 Anaplasma phagocytophilum, 462 conditions associated with, 246 Adhesion molecules, 121, 122, 158–59 Anaplastic large cell lymphoma etiology, 245 Adipocytes, 31 (ALCL), 636, 638 laboratory evaluation, 246–47 Adult hemoglobin (HbA), 95–96, 99 Anemia, 200–220, 917 pathophysiology, 245–46 Advanced HIV disease (AHD), 484 abnormal hemoglobin peripheral blood, 246–47 Aerobic workout, 1114 concentrations, 203 therapy, 247 Aeromonas hydrophila, 379 adaptations to, 203–4 Anemia of inflammation (AI). See Afibrinogenemia, 813 aplastic, 348–56 Anemia of chronic disease African Americans defined, 202 (ACD) anemia in, 207 development of, 202–3 Anemias of disordered iron CBC variations in, 194 fanconi, 1045 metabolism Agglutination, 178, 182, 406 functional, 202 iron-deficiency anemia, 240–45 Aggregate, 872 functional iron-deficiency, 202 iron, laboratory assessment of, Aging, lymphocyte function and, 159 hemolytic, 215–16, 364–79 (See also 238–40 Agonist, 720, 872 Hemolytic anemia) iron metabolism, 227–38 a@granules (aG), 168, 716–17 iron-deficiency, 227 Aneuploid, 1020, 1111 Agranulocytosis, 120, 456 macrocytic, 213 Angioimmunoblastic T cell lymphoma AIDS. See Acquired immune megaloblastic, 215, 319–43 (AITL), 638 deficiency syndrome (AIDS) microcytic, hypochromic, 212–13 Animal venoms, 442 a@interferon (IFN@a), 166 normocytic, normochromic, 213 Anion exchange protein 1 (AEP1), 74 ALAS, 249 oxygenated blood flow, increase in, Anisocytosis, 184–85 Alcoholism 204 Ankyrin, 76 acquired sideroblastic anemia from, oxygen utilization by tissue, Annexin II (A2), 751 250 increase in, 204 Antibiotics, 793 liver disease, 340–41 pernicious, 334–35 Antibody screen, 406 macrocytic anemia without sickle cell, 271–79 Anticoagulants, 910–11 megaloblastosis, 340–41 sideropenic, 227 Anticoagulant therapy, 854–58, 891 platelet dysfunction from, 793 spur cell, 341, 376 antiplatelet therapy, 858 Alder-Reilly anomaly, 462 Anemia associated with abnormal heparin, 855–56 Algorithms, 7 heme synthesis, 248–53 heparin therapy monitoring, 893–94 Alkali denaturation, 930 Anemia, classification of, 211–20 new, 857 Alleles, 14, 296, 1035 functional, 213–18 oral, 856, 892 Allele-specific oligonucleotide (ASO), maturation defects, 214–15 Antigenic determinants, 133 1040–41 by MCV and RDW, 218 Antigen-presenting cells (APCs), 133, Allele-specific oligonucleotides (ASO), morphologic, 211–13 156 1040–41 proliferation defects, 213–14 Antihemophilic factor, 741–42 Allogeneic stem cell transplantation survival defects, 215–18 Antihuman globulin (AHG) test, 216, (HSCT), 529, 648–49 using red cell distribution width, 218 371, 405 Alloimmune hemolytic anemia, 417–22 Anemia, diagnosis of, 204–11 Antinuclear antibody (ANA), 542 Alloimmune thrombocytopenia, 780 history, 204 Antiphospholipid antibodies (APLs), a2@antiplasmin (AP), 754 important information for, 205 820, 842–44, 882–83 activity, 885 laboratory evaluation (see Anemia, Antiphospholipid syndrome (APS), a1@antitrypsin, 760 laboratory evaluation) 842–44 a@macroglobulin, 754–55 laboratory investigation (See Antiplatelet therapy, 858 a2@macroglobulin, 759–60 Anemia, laboratory Antithrombin (AT), 755 a@naphthl acetate esterase (ANAE), 936 evaluation) deficiency, 834–35 a@naphthyl esterase, 1143–44 laboratory testing schemas, 218–20 II, 834 a@storage pool disease, 791 physical examination, 204–5 III, 887 a@thalassemia major (hydrops fetalis), Anemia, laboratory evaluation Antithymocyte globulin (ATG), 349, 354 298 blood smear examination, 209–10 Anti-Xa assay, 894 a@thalassemia minor, 300–301 bone marrow examination, 211 Aorta-gonads-mesonephros (AGM), 30 Alpha hemoglobin-stabilizing protein differential diagnosis and, 211 Apertures, 964 (AHSP), 94 erythrocyte count, 206–7 Apheresis, 651 Alveolar macrophages, 131 erythrocyte destruction, 210–11 Aplasia, 348 Ambiguous lineage, 1087 erythrocyte indices, 207 Aplastic anemia, 348–56, 782, 1159 Amplification techniques, 1040 hematocrit count, 206–7 chemical agents, 350 Index 1211 classification and etiology, 349–52 Autologous bone marrow, 511 Benign neoplasms, defined, 498 clinical presentation, 352–53 Autologous stem cell transplantation, Bernard-Soulier syndrome (BSS), 784, drug inhibition, 350 511, 649–50 788–89 epidemiology, 348 Automated blood cell-counting Bethesda titer, 819 idiopathic, 349–50 instruments, 963–87 Bethesda Titer assay, 882 infectious agents, 351 impedance instruments, 963–84 Bilineage acute leukemia, 616 inherited, 351–52 light-scattering instruments, 984–87 Bilirubin, 102 ionizing radiation, 351 Automated cell counts, 674 Biochemical inhibitors, 755–60 laboratory evaluation, 353–54 Automated digital cell morphology Biphasic antibody, 931 metabolic causes of, 351 instrument, 987–88 Biphenotypic acute leukemia, 616 pancytopenia and, differentiating Automated hemostasis analyzer Birbeck granules, 468 from other causes of, 355–56 methodologies, 895–97 Birefringence, 690–93 pathophysiology, 348–49 Automation in hematology/ Bite cells, 391 prognosis and therapy, 354–55 hemostasis, 918, 961–88 Blackfan-Diamond syndrome, 357–58 Aplastic crises, 274 automated blood cell-counting Blackwater fever, 441 Apoferritin, 232 instruments, 963–84 Blast crisis classification, 522, 528, Apoptosis, 15, 20–23 Autosomal dominant inheritance, 565–67 cancer and, 503–4 802–7 Blastogenesis, 153 cardinal features of, 20 Autosomal recessive disorders, 122, Blast transformation (blastogenesis), caspases and, 21–22 812–16 153 diseases associated with increased/ Autosomal SCID, 486–87 Bleeding disorders, 769–73 decreased, 20, 21 Autosplenectomy, 38, 274–75 Bleeding risk, 771 hematopoietic system and, 22–23 Azurophilic granules, 115 Bleeding time, 871–72 inhibitors and initiators/inducers “Blinded” preanalyzed specimens, of, 21 B 1060 molecular regulation of, 21 Babesia microti, 441 Blister cell, 189, 391 necrosis versus, 21 Babesiosis, 441 Blood cell concentration, reference role of Bcl-2 proteins, 22, 23 Bacterial infection intervals for, 4–5 tissue homeostasis, 20–23 neutrophilia, 454–55 Blood cell egress, 34 Apoptosome, 23 sickle cell anemia, 275 Blood clots Apotransferrin, 230 Band, 76 bone marrow biopsy, 952–53 Arachidonic acid (AA), 723 Banding, 1018–19 Blood components, 4–6 Arachnoid mater, 668 Band neutrophils, 117, 118 Blood composition, 4 Arterial thrombi, 829–30 Barr body, 118 Blood flow, 35–36, 755 microparticles in, 831–32 Bartonella bacilliformis, 441 Blood islands, 30 Artificially induced purpura, 774 Bartonellosis, 441–42 Blood loss, iron-deficiency anemia Artificial oxygen carriers (AOC), 101 Basal coagulation, 761 and, 240–41 Ascites, 667 Basophilia, 129, 466, 677 Blood smear examination for anemia, Ascitic fluid, 667 Basophilic myelocyte, 129 209–10 Ascorbate cyanide test, 392 Basophilic normoblast, 71 anisocytosis, 184–85 Aspirin, 792, 858 Basophilic stippling, 191–92 erythrocyte distribution on stained Ataxia-telangiectasia (AT), 489 Basophils, 112, 128–30, 677 smears, variation in, 184 Atypical chronic myeloid leukemia B cell acute lymphoblastic leukemia erythrocyte

inclusions, 191, 192 (aCML), 520, 575 (B-cell ALL), 609 poikilocytosis, 184, 187 Atypical CML (aCML), 520 B cell lymphomas, 626–28 variation in hemoglobin (color), 190 Atypical HUS (aHUS), 433–36 diffuse large, 633 Blood urea nitrogen (BUN), 435 Atypical lymphocytes, 476 non-Hodgkin, 625 Blood vessels, 707–9 Auer rods, 506, 587, 597, 1130 prolymphocytic leukemia, 626, 628 B lymphoblastic leukemia/ Autocrine signals, 53 B cell receptor (BCR), 141 lymphoma, 612–15 Autohemolysis test, for hereditary BCL-2 gene, 625 B-lymphoblastic lymphoma (B-LBL), spherocytosis, 370–71 BCL-2/ gene rearrangement, 1134 613 Autoimmune hemolytic anemia Bcl-2 proteins, 22, 23 B lymphocyte, 1115–16 (AIHA), 401 BCR/ABL1 translocation, 1025, 1026 B lymphocytes, 140, 143–47, 155–56 cold, 411–14 BCR/AB1 mutation, 509 antigen receptor, 144–45 mixed-type, 402, 415 BCR/ACL1 fusion oncogene, 524 cell development, 155–56 negative direct antiglobulin test in, BCR/ACL1 translocation, 523 developmental stages, 145–47 407 BCR gene, 524 immunoglobulin, 144–45, 156 paroxysmal cold hemoglobinuria, Bead array technology, 1041 immunoglobulin gene 402, 414–15 Beckman-Coulter Unicel DxH 800, rearrangement, 145 warm, 402, 408–11 968–77 immunologic maturation, 143 Autoimmune myelofibrosis, 542 Beer-Lambert’s law, 922 membrane markers, 143–44 Autoimmune neutropenia (AIN), 458 Bence-Jones proteinuria, 634 subclasses, 147 Autoimmune thrombocytopenia, Benign cold agglutinations, 413 B lymphoid cell antigens, 588 777–78 Benign lymphoid aggregates, 953–54 B/myeloid leukemia, 616 1212 Index Body fluids differential count, 950–51 Catalytic domain, 738 amniotic, 693–94 iron stores, 954–55 CD (cluster of differentiation), 45, 114, bronchoalveolar lavage, 670 myeloid to erythroid ratio, 951–52 116, 121, 508, 574, 588, 613, cell counting, 672–75 touch imprints, 952 614, 616, 995, 999, 1001, 1002, cerebrospinal, 668–70, 686–88 Bone marrow studies, 955–57 1004, 1005, 1007, 1082–85 hematologic analysis of, 670–93 cytochemical stains, 957 CD designation, 998 joint, 670, 688–90 cytogenetics, 955 CD34 enumeration, 1007 malignant cells in, 680–83 flow cytometry, 955 CD4 lymphocytes, 480 morphologic findings in, 664–700 molecular genetics, 955–56 CD8 lymphocytes, 480 nucleated cell differential and, special requirements for, 956 CD34 + progenitor, 45 674–93 triaging bone marrow samples for, CD4+ T cells, 156–57 pleural, pericardial, and peritoneal, 956 CD8+ T CELLS, 157 674, 683–86 Bone marrow transplantation, CellaVision® DM96 system, 987–88 semen, 695–98 cytogenetic analysis of, 1028 Cell cycle, 16–19, 1016 synovial, 670 Bordetella pertussis, 476, 480 cyclins (Cdks), 17–18 types of, 666–70 Bothrops atrox, 879 kinase activity, 18 Bohr effect, 99 Bright-field microscopy, 914–15 molecular regulation of, 17 Bone marrow, 164 Bronchoalveolar lavage (BAL), 670, 675 phases of, 16–17 acute lymphoblastic leukemia, 610 Bruisability, 770 Cell cycle checkpoints acute myeloid leukemias, 586–87, Bruisability, easy, 774 cancer and, 503 591, 592 Bruton’s tyrosine kinase (BTK), 488 in tissue homeostasis, 18–19 anemia, examination for, 211 Bruton type X-linked Cell death, 20–22 anemia of chronic disease, 247 agammaglobulinemia, 504 Cell enumeration by hemacytometer, aplastic anemia, 353, 354 b@thalassemia, 1121 921–22 aspiration, 949 b@thalassemia intermedia, 307 Cell lineage, identifying, 567, 587–91 blood cell egress, 34 b@thalassemia major, 303–6 Cell-mediated immunity (CMI), 140, chronic myeloid leukemia, 526 b@thalassemia minima, 307–8 156–57, 484 core biopsy, 949–50 b@thalassemia minor, 306–7 Cell membrane, 11–12 decreased production of, 456–57 Bull’s testing algorithm (moving carbohydrates, 12 essential thrombocythemia, 533 averages), 1067–68 lipids, 11 extramedullary hematopoiesis, 34 Burkitt lymphoma, 625, 634 proteins, 12 hematopoietic compartment, 33 Burr cells, 189 Cell structure, 11–13 hyperplasia, 33 Burst-forming unit-erythroid (BFU-E), Cell turnover rate, 119 infectious mononucleosis, 478 48, 58, 68, 74 Cellular hemoglobin concentration iron deficiency, 244 Burst-promoting activity (BPA), 68 mean (CHCM), 984 macrophages in, 31 Butt cells, 629 Cellular homeostasis medullary hematopoiesis, 34 abnormal tissue homeostasis and megaloblastic anemia, 325–26 C cancer, 23 myelodysplastic syndromes, 563–64 Cabot rings, 192 cell structure, 11–13 neutrophils, 119–20 Calcium, 76, 723 genetic information, 13–15 polycythemia vera, 538, 540 Calibrated automated thrombogram tissue homeostasis, 15–23 primary myelofibrosis, 543 (CAT), 886–87 Cellular immunity, 477 replacement of normal, 783 Cancer, abnormal tissue homeostasis, Centers for Disease Control and sickle cell anemia, 276 23, 500–504 Prevention (CDC), 1065 sideroblastic anemia, 252 apoptosis, 503–4 AIDS defined by, 483–84 stroma, 31–32 cell cycle checkpoints, 503 Central nervous system (CNS), 666, 668 transplantation, 485, 511, 601, 651 epigenetics, 502 Cerebrospinal fluid (CSF), 668–70, 674, vasculature, 31 oncogenes, 500–501 686–88 warm autoimmune hemolytic tumor suppressor genes, 502 Ceruloplasmin, 229 anemia, 410 Cancer-initiating cell, 500, 1130 C1-esterase inhibitor, 760 Bone marrow examination, 945–58 Cancer stem cells, 500 Chain termination sequencing, 1042 indications for evaluation, 947 Candida, 461 Charcot-Leyden crystals, 127, 465 morphologic interpretation of, Candida albicans, 680 Chédiak-Higashi syndrome (CHS), 462 950–55 Candida tropicalis, 680 Chemical agents, aplastic anemia and, procedure, 948–49 Capillaries, 929 350 processing for, 949–50 Capillary electrophoresis (CE), 269 Chemokines, 121 report, 957 Capillary puncture, 913–14 Chemotaxis, 121, 123 Bone marrow, morphological Carbohydrates, 12 Chemotherapy, 457, 510–11, 534, interpretation of Carbon dioxide transport, 100–101 538–39 aspirate, 950–52 Carboxyhemoglobin, 106 Children, CBC variations in, 194 benign lymphoid aggregates vs. Carboxyl group (COOH), 737 Chimerism, 654, 1028 malignant lymphoma, 953–54 Cardiopulmonary bypass surgery, 793 Chloride shift, 100 bone marrow cellularity, 952–53 Cartwheel arrangement, 154 Chloroacetate esterase, 935–36 bone marrow particle, 952–53 Caspases, 21–22 Chloromas, 525 Index 1213 Cholecystitis, 369 Chroni myeloid/granulocytic Colony-forming unit-granulocyte, Cholelithiasis, 369 leukemia (CML/CGL), 522 erythroid, macrophage, and Chromatic aberration, 915 Chylous, 672 megakaryocyte (CFU-GEMM), Chromatin, 13 Cigar cells, 189 49, 50, 52, 54 Chromogenic assay, 894 Ciliocytophthoria, 693 Colony-forming units (CFU), 648 Chromogenic detection methods, 896 Circulating anticoagulants, 819 Colony-stimulating factors (CSF), 52 Chromosomes Circulating pool (CP), 120 Column chromatography, 929 aberration nomenclature, 1022 Citrated whole blood, 871 Commitment, 44 abnormalities, 1020–22 Classical Hodgkin lymphoma (CHL), Common acute lymphoblastic leukemia acrocentric, 1016 640–41 antigen (CALLA), 144, 528 analysis of, 955, 1013–29 Clinical and Laboratory Standards Common acute lymphocytic leukemia heterologous, 1016 Institute (CLSI), 118, 209, 1057 antigen (CALLA), 1169 homologous, 1016 Clinical Laboratory Improvement Common ALL cell, 613 metacentric, 1016 Amendments of 1988 (CLIA Common lymphoid progenitor (CLP) structure and morphology, 1015–16 1988)., 1056 cell, 48, 52, 141, 648, 1115 submetacentric, 1016 Clonal hematopoiesis of Common myeloid progenitor (CMP) Chronic acquired pure red cell aplasia, indeterminate potential cell, 50–52, 115, 141, 165, 648 357 (CHIP), 557 Common myeloid progenitor (CMP) Chronic eosinophilic leukemia (CEL), Clonal hypereosinophilia, 545–48 cell, 48 520 Clonal (neoplastic) hypereosinophilia, Common pathway, 734, 745–49 Chronic eosinophilic leukemia, not 465 Compensated hemolytic disease, 215 otherwise specified (CEL- Clonality, 625 Compensation, 997 NOS), 545, 547–48 Clonogenic, defined, 653 Competency assessment, 1060 Chronic granulocytic leukemia (CGL), Clostridium perfringens, 442 Complement-mediated hemolysis, 522 Clostridium welchii, 106 404–5 Chronic granulomatous disease. Clot retraction, 726 Complete blood count (CBC), 6, 176 See Chronic myelogenous Clots. See Blood clots examination phase, 177–93 leukemia (CML) Cluster analysis, 986 iron, 239 Chronic lymphocytic leukemia (CLL), Cluster of differentiation (CD), 114 phases, 177 408, 481, 503, 1048 CMV-seronegative patient, 1135 physiologic variation in hematologic Chronic myelocytic leukemia (CML), Coagulation parameters, 194–95 522, 1167 basal, 761 post-examination phase, 194 Chronic myelogenous leukemia mechanism, 734 pre-examination phase, 177 (CML), 455, 504 physiologic pathway of, 760–62 variations in, 194–95 cytogenetic analysis, 1024–25 proteins, 735, 737–38 Complimentary DNA (cDNA), 1039 Chronic myeloid leukemia (CML), Coagulation cascade, 738–49 Compound heterozygote, 270–71 522–30. See Chronic common pathway, 745–49 Compound microscope, 914–15 myelogenous leukemia (CML) extrinsic pathway, 744–45 Compression syndromes, 295 atypical (aCML), 520 intrinsic pathway, 738–44 Compression ultrasonography (CUS), BCR/ACL1 translocation, 523 phospholipid membranes, complex 831 bone marrow, 526 formation on, 738 Computerized tomography (CT), 831 clinical presentation, 525 Coagulation factors, 733 Conditioning regimen, 654 differential diagnosis, 529–30 defined, 733 Congenital amegakaryocytic disease progression and disorders of, 812–16 thrombocytopenia (CAMT), chromosome/molecular elevated levels of, 841 352, 783–84 mutations, 524–25 properties of, 735–36 Congenital deficiencies/disorder etiology and pathophysiology, specific, 878–79 congenital immune, 485–89 523–25 Coated platelets, 726 Heinz body hemolytic anemias, 271 laboratory evaluation, 525–27 Cobalamin. See Vitamin B12 neutropenia, 457 peripheral blood, 525–26 Cobalamin deficiency, 1123, 1158 orotic aciduria, 339 Philadelphia (Ph) chromosome in CobaSorb test, 337–38 Congenital dyserythropoietic anemia acute leukemias, 523, 525 Codocytes, 187–88 (CDA), 339, 355 terminal phase, 527–28 Coincidence, 968 Congenital nonspherocytic hemolytic therapy, 528–29 Cold agglutinin disease, 917 anemia, 390 Chronic myelomonocytic leukemia Cold agglutin syndrome (CAS), 411, Congenital thrombocytopenia (CMML), 524, 541, 574–75 415. See also Cold autoimmune with radioulnar synostosis Chronic myeloproliferative disease, hemolytic anemia (CTRUS), 783 unclassified (CMPD-U), 545 Cold autoimmune hemolytic anemia, Congential erythropoietic porphyria Chronic neutrophilia, 454 411–14 (CEP), 256–58 Chronic neutrophilic leukemia (CNL), Collagen-binding assays, 882 Consolidation therapy, 510, 617 530–31 Collection tubes, 911 Constitutional cytogenetic Chronic nonspherocytic hemolytic Colony-forming unit (CFU), 50 aberrations, 1017 anemia, 390 Colony-forming unit-erythroid Consumption coagulopathy, 817, 848 Chronic renal failure, 792 (CFU-E), 48, 68 Contact group, 735, 736 1214 Index Contact plasminogen activator (PA) Cytokine regulation, 56 vitamin B12 deficiency, 335 pathway, 750 Cytokines, 52–57. See also Growth Differentiation Continuous flow analysis, 984 factors (GFs) basophils, 129 Contour gating, 966 Cytokinesis, 17 cluster of, 114 Control materials, 1066 Cytomegalovirus (CMV), 479–80, 656 defined, 19, 44 Coombs test. See Direct antiglobulin Cytometry, flow, 875, 955, 993–1009 eosinophils, 126–27 test (DAT) Cytopenia, 1167 monocytes, 130–31 Core-binding-factor (CBF), 591 Cytoplasm, 12–13 neutrophils, 115–18 Core biopsy, 949–50 basophilic normoblast, 71 Diffuse large B-cell lymphoma, 633 Correlation coefficient, 1062 orthochromic normoblast, 71 DiGeorge syndrome, 488 Cortex, of lymph nodes, 38 polychromatophilic normoblast, 71 Dihydrofolate (DHF), 328 Corticosteroids, 410–11, 438, 456 pronormoblast, 69 Dihydrorhodamine 123 (DHR123) Coulter® LH 780, 1145 Cytoplasmic abnormalities, 460–62 assay, 463 Coulter LH series, 963–68 Cytoplasmic maturation, 167 Dilute Russell viper venom time test Coverglass smear, 917 Cytoplasmic vacuoles, 460, 461 (dRVVT), 882, 883 CpG islands, 19 Cytoreductive therapy, 617 Dilutional thrombocytopenia, 784 C-reactive protein (CRP), 830 Cytosine triphosphate (CTP), 1037 Diploid, 1008, 1020 Creatine phosphokinase (CK), 442 Cytotoxic T cells (CTC), 142, 1115 Diploid state, 296 Critical area, 183 Cytotoxic T lymphocytes (CTL), 23, Dipyridamole, 858 Critical limits, 194 142, 1115 Direct antiglobulin test (DAT), 216, Critical value, 194, 1058 Cytotyoxic drugs, 411 386, 406, 413, 441 Crossover, 310–11 negative, in AIHA, 407 Cross-reacting material negative D positive, in normal individuals, 407 (CRM- ), 801 Dacryocytes, 76, 187, 188 for warm autoimmune hemolytic Cross-reacting material positive Damage-associated molecular anemia, 410 (CRM+ ), 801 patterns (DAMPs), 114 Direct DNA sequence analysis, 1042 Cross-reacting material reduced d@aminolevulinic acid synthase enzyme Direct oral anticoagulants (DOACs), (CRMR), 801 (ALAS), 249 875, 892–93 Cryopreservation and storage of Danazol, 411 Direct thrombin inhibitor (DTI) hematopoietic stem cells, 652 Dawn of neutrophilia, 117 therapy monitoring, 894–95 Cryosupernatant, 437 db@thalassemia, 308 Discocyte, 184 Cryptococcus, 680 D-dimer, 884–85 Disseminated intravascular CSF2, 59 Death signals, 22 coagulation (DIC), 418, 437–38, CSF3, 59 Decay-accelerating factor (DAF), 377 592, 782, 817, 848–52, 877, 1169 Culling, 36 Deep vein thrombosis (DVT), 830, 884 Distal histidines (E7), 94 Current Procedure Terminology (CPT) Definitive erythropoiesis, 30 Divalent metal transporter1 (DMT1), code, 1056 Degranulation, 123–25 229 Cushing syndrome, 773 Dehydrated hereditary stomatocytosis DMT1, 229, 253 CXCL12, 166 (DHS), 374 DNA, 13, 995, 1036–37 Cyanosis, 78, 104 Delta checks, 193, 1069 analysis, 1008–9 Cyclic AMP (cAMP) pathway, 724 Demarcation membrane system clinical applications of analysis, 1009 Cyclin-dependent kinases, 17–18 (DMS), 168 complimentary, 1039 Cyclins (Cdks), 17–18 Demargination, 454 Index (DI), 1008 CYP2C9, 1049 Demyelination, 333–34 replication, 13–14 Cytoadhesion molecules, 61 Dense granules (DGs), 168, 716 satellite, 1016 Cytochemical analysis, 508 deficiencies of, 791 sequencing, 1042 Cytochemical staining, 392, 934–39 Densely staining chromatin, 1111 synthesis, 333 Cytochemistry, 611 Dense tubular system (DTS), 168, 717–18 transcription, 1037 defined, 508 Densitometer, 928 Döhle bodies, 460 in identifying cell lineage, 588 Deoxyhemoglobin, 96 Donath-Landsteiner (D-L) antibody, Cytogenetic nomenclature, 1022–23 Deoxyribonucleic acid. See DNA 414, 415 Cytogenetic procedures, 1017–20 Deoxyuridine monophosphate Donath-Landsteiner test, 931–33 Cytogenetics, 509 (dUMP), 328, 330 Double heterozygous inheritance, 303 acute lymphoblastic leukemia, 612, D + HUS/D - HUS. See Hemolytic Down syndrome, 504, 587, 601, 1130 614, 615 uremic syndrome (HUS) Drepanocytes, 187, 188 acute myeloid leukemias, 588, 591 Diagnosis-related group (DRG), 1056 Drug-induced thrombocytopenia, 781 bone marrow, 955 Diagnostic gene rearrangement, 593–94 Drug inhibition myelodysplastic syndromes, 558–60 Diamond-Blackfan syndrome (DBA), aplastic anemia and, 350 remission, 510 357–58 folic acid deficiency and, 330 T lymphoblastic lymphoma Diapedese, 4 hemolysis, 402, 442–43 (T-LBL), 615 Diapedesis, 121 hemolytic anemias, 415–17 Cytogenomic microarray analysis Diet megaloblastic anemia and, 338, 339 (CMA), 1028, 1029 folic acid deficiency and, 330 platelet dysfunction, 792–93 Cytokine receptors, 57–58 iron-deficiency anemia and, 240 Drumstick (Barr body), 118 Index 1215 Dry tap, 949 Epidermal growth factor (EGF) recumbant forms of human d@storage pool disease, 791 domain, 541 (rHuEPO), 82 Duodenal cytochrome B (DcytB), 229 Epigenetics, 19, 502, 510, 511, 536 regulation of erythrocyte Dura mater, 668 Epistaxis, 775 production, 80–82 Dutcher bodies, 635 Epitopes, 133 Erythropoietin receptors (EPO-R), 68 Dye reduction test, 392 E-prostaglandins (PGE), 56 Escherichia coli, 431, 433, 1128 Dyserythropoiesis, 563–64

Epstein-Barr virus (EBV), 351, 356, Esherichia coli, 847 Dysfibrinogenemia, 813–14, 840 476, 625, 634, 1048 Essential thrombocythemia (ET), 531–34 Dysgranulopoiesis, 564 Erythroblastic islands (EI), 33, 62, 71–72 clinical presentation, 532 Dyshematopoiesis, 355, 1167 Erythroblast macrophage protein differential diagnosis, 534 Dyskeratosis congenita (DC), 352 (EMP), 71 laboratory evaluation, 532–33 Dysmegakaryopoiesis, 564 Erythroblastosis fetalis, 421 pathophysiology, 535–37 Dysmyelopoietic syndrome, 555 Erythroblasts, 68 prognosis and therapy, 534 Dyspepsia, 322 Erythrocyte, 283 synonyms for, 531 Dysplasia, 555, 586 Erythrocyte iron turnover (EIT) rate, 240 Ethnic groups, CBC variations in, Dyspoiesis, 555 Erythrocyte protoporphyrin (EP) 194–95 studies, 244 Ethylenediaminetetraacetic acid E Erythrocytes, 3, 44, 66–83, 1111, 1126 (EDTA), 177, 458, 671, 910, 954 Early-acting (multilineage) concentration, 80 Euglobulin clot lysis, 885 hematopoietic growth factors, count, 178–79, 207 Excess bleeding, 770 54–55 destruction, 82, 210–11 Exchange transfusion, 373–74 Easy bruising syndrome, 774 disorders, 1045–46 Exercise-induced hemoglobinuria, 439 Ecchymoses, 770 inclusions, 191, 192 Exogenous activators, 752 Echinocytes, 187, 189 indices, 179–81, 207, 923–24 Exons, 14 Edematous, 913 kinetics, 80–82 Extracellular matrix (ECM), 61 Effector T lymphocytes, 142 manual count, 922 Extramedullary erythropoiesis, 295 Effusion fluids, 667 maturation, 69–72 Extramedullary hematopoiesis, 34 Egress, 454 membrane, 72–77 Extranodal NK/T cell lymphoma, Ehrlichia, 461 metabolism, 77–80 nasal type, 639 Ehrlichiosis, 461 morphology, 184 Extranodal T and NK cell lymphomas, Elderly, CBC variations in, 194–95 in myelodysplastic syndromes, 561 638–39 Electromechanical clot detection normochromic, 184 Extravascular destruction, 82, 102–3 systems, 895 polychromatophilic, 191 Extravascular hemolysis, 215–16 Electrophoresis, 269, 298, 928–29 regulation of production, 80–82 Extrinsic pathway, 734, 744–45 Elliptocytes, 187, 189 shape, abnormalities in, 184–85 Extrinsic Xase, 738 Embolism, 829 size, abnormalities in, 184 Exudate, 668 Embolus, 829 survival studies, 211 Embryonic hemoglobins, 95 variation in color, 191 F Endocrine abnormalities, 359 Erythrocyte sedimentation rate (ESR), FAB classification, 507, 519 Endomitosis, 166–67, 562, 1021, 1116 924–26 Factor eight inhibitor bypassing Endoplasmic reticulum (ER), 131 Erythroferrone (ERFE), 235, 236 activity (FEIBA), 819 Endosteum, 31 Erythroid, 348 Factor replacement therapy, 811 Endothelial cells, 709–11 niches, 62 Factors Endothelial Protein C Receptor Erythroid burst-forming units I, 734, 736 (EPCR), 758–59 (BFU-E), 48, 58, 68, 74 II, 736, 750, 814 Endothelium-derived relaxing factor Erythroid colony forming units IIa, 735, 736 (EDRF), 101, 709 (CFU-E), 48, 68 IIIa, 735, 736, 744 Engraftment, hematopoietic, 647, 654 Erythroid-maturing cells, 68–69 IX, 741, 807, 819 Enteropathy-associated T cell Erythroid progenitor cells, 68 IXa, 757, 760 lymphoma (EATL), 639 Erythron, 68, 1113 tissue, 744–45 Enzyme deficiencies, megaloblastic Erythrophagocytosis, 132 V, 745, 814 anemia, 338 Erythropoiesis, 30, 56, 68 VII, 745 Enzyme-linked immunosorbent assay stimulated, 342 VIII, 735, 736, 741–42, 806, 815–16, (ELISA) techniques, 458, 841, Erythropoiesis-stimulating agents 819, 839, 880, 881 881–82, 883, 884, 890, 891, 933 (ESAs), 248 VIIIa, 761 Eosinophil cationic protein (ECP), 127 Erythropoietic protoporphyria (EPP), vitamin K-dependent clotting, 816 Eosinophil chemotactic factor 257–58 von Willebrand, 742–44 (ECF), 128 Erythropoietin, 236 X, 745, 757, 814–15 Eosinophil-derived neurotoxin (EDN), Erythropoietin (EPO), 69, 357, 933 X activation, 745 127 characteristics of, 81 XI, 736, 815 Eosinophilia, 128, 677, 1086 erythroid maturation, 69 XII, 736, 774, 841 Eosinophilic myelocyte, 126–27 hematopoietic growth factors, 55 XIII, 748–49, 815, 880 Eosinophil peroxidase (EPO), 127 inappropriate increase in, 539 Factor V Leiden (FVL), 831, 838, 889, Eosinophils, 112, 126–28, 677 receptors, 68 1049 1216 Index False neutropenia, 458 DNA analysis, 1008–9 primary myelofibrosis, 543–44 Familial polycythemia, 539 immunophenotyping by, 998–1008 susceptibility, oncogenes and, 504 Fanconi anemia (FA), 351–52, 782, 1045 MRD, 1005 Genome/genomics, 13, 1035 Fas Ligand, 20 myelodysplastic syndromes, 567 Genotype, 297, 1041 Fatty acid degradation, defective, principles of, 995–98 Germinal centers, 35, 38 333–34 in systemic autoimmune disease, Germline configuration, 143 Favism, 390 1006–7 Giant platelets, 169 F cells, 96 Fluorescence activated cell sorting GLA domain, 737 Ferritin, 95, 232–33 (FACS), 1000–1001 Glanzmann thrombasthenia (GT), 789 Ferrokinetics, 240 Fluorescence in situ hybridization Global testing, 886 Ferroportin 1, 229, 253, 254 (FISH), 524, 558, 612, 627, 631, Globin, 90 Fetal hemoglobin (HbF), 95, 99, 929 635, 654, 1018, 1019, 1025, Globin chain synthesis, 91–94 Fibrin degradation products, 1028, 1029, 1035, 1040 Glossitis, 323 752–53 Fluorescent-activated cell sorting Glucocorticoids, 120 Fibrinogen, 747–48 (FACS), 45 Glucocorticoid therapy, 773 disorders, 840–41 Fluorescent-labeled inactive toxin Glucose-6-phosphate (G6P), 391 g@chain gene, 840 aerolysin (FLAER) test, 379 Glucose-6-phosphate dehydrogenase Fibrinogen group, 735–36 Fluorescent spot test, 391–92 (G6PD), 385, 416 Fibrinogenolysis, primary, 817 Fluoride inhibition, 936 clinical presentation, 390–91 Fibrinolysis, 5, 1137 Fluorochromes, detection of, 997–98 deficiency, 386 activators of, 751–52 Folates, 327–31 deficiency, females with, 389 defined, 749 Folate trap, 329 differential diagnosis, 392 inhibitors of, 753–55 Folic acid, 322–23 etiology, 387 Fibrinolysis, secondary hemostasis deficiency, 329–31 laboratory evaluation, 391–92 and, 731–64 metabolism, 329 mutant classes, 389 coagulation cascade, 738–49 requirements, 329 pathophysiology, 387–89 coagulation mechanism, 734 structure and function, 327–28 quantitation of, 392 fibrinolytic system, 749–55 Follicular lymphoma (FL), 629–30 therapy, 392–93 hemostasis, physiological control Formalin-fixed, paraffin-embedded variants, 389 of, 755–60 (FFPE) tissue, 1036 Glucose phosphate isomerase physiologic hemostasis, 760–62 Formiminoglutamic acid (FIGLU), deficiency (GPI), 395 procoagulant factors, 734–38 327, 328 Glutahione (GSH), 385 Fibrinolytic protein inhibitors, 816 Forward light scatter, 996 Glycocalyx, 712 Fibrinolytic system Frataxin, 238 Glycolysis, 77 activators of, 751–52 French, American, British (FAB) Glycolytic pathway, 77, 385–86 components of the, 749–50 classification. See FAB Glycoprotein Ib (GPIb), 713–14 disorders, 841 classification Glycoprotein IIb/IIIa (GPIIb/IIIa), fibrin degradation, 752–53 Fresh frozen plasma (FFP), 437, 887, 1164 714–15 inhibitors of, 753–55 Functional anemia, 202 Glycosaminoglycans (GAGs), 757 introduction, 749 Functional hyposplenism, 294 Glycosylated hemoglobin, 96 laboratory investigation of, 884–85, Functional iron deficiency (FID), 248 Glycosyl-phosphatidyl inositol (GPI), 886–87 Functional iron-deficiency anemia, 202 377 plasmin, 750–51 Golgi apparatus, 115, 117 plasminogen, 750–51 G Golgi apparatus, 32, 33 plasminogen and plasmin, 750–51 Gaisböck’s syndrome, 539 G6PD deficiency, 1151 properties of, 750 Galectin-10, 127 GPIIb/IIIa, 714–15 Fibrinopeptides, release of, 747 Gastric analysis, 336–37 Graft failure, 656 Fibrin polymers, 748 Gating, isolation of cells of interest by, Graft rejection in hematopoietic stem Fibrin stabilization, 748 1000 cell transplantation, 649, 650 Fibroblasts, 61 Gaucher disease, 467 Graft-versus-host disease (GVHD), Fibronectin (Fn), 61 g@carboxylation, 737 648, 656 Fibrosis, myelodysplastic syndromes G-CSF, 34 Graft-versus-leukemia (GVL), 648 with, 571 gdb@thalassemia, 308 Granules Field curvature aberrations, 915 Gender, CBC variations in, 194–95 a@granules, 716–17 Fitzgerald factor, 816 Gene clusters, 292 dense, 168, 716, 791 5-aminolevulinate synthase (ALAS), Gene expression, control of, 14 Granulocyte-colony stimulating factor 91 Gene rearrangement, 1048, 1086 (G-CSF), 34, 511, 512 Flame cells, 481, 635 Genes, 1035 Granulocyte monocyte-colony Flaujac factor, 816 Gene therapy, 657–58, 811–12 stimulating factor (GM-CSF), Flow chamber, 995 Genetic analysis, 509 34, 55, 456, 512 Flow cytometry, 875, 955, 993–1009, Genetic information, 13–15 Granulocyte, monocyte progenitor 1143, 1177 Genetics (GMP), 48, 130 data analysis, 998 molecular, 955–56 Granulocytes, 1112 defined, 995 polycythemia vera, 540 Granulocytopenia, 59, 120 Index 1217 Granulocytosis, 120 erythrocyte sedimentation rate, Hematopoietic organs, structure and Granulomatous diseases, 952 924–26, 1098–99 function of, 28–39 Granulopoiesis, 56 hematocrit, 923 Hematopoietic precursor cells (HPCs), Gray platelet syndrome (GPS), 791 hemoglobin concentration, 922–23 51 Growth differentiation factor-15 hemoglobin concentration Hematopoietic progenitor cells, 44 (GDF15), 235, 236 determination, 1095–97 Hematopoietic stem cells (HSCs), 499 Growth factors (GFs), 52, 501. See also Koehler illumination, 1089 CD34 enumeration by flow Cytokines manual leukocyte count, 1092–94 cytometry, 652–53 characteristics of, 53 manual platelet count, 1094–95 cell culture for colony forming clinical use of, 59 microhematocrit determination, units, 653 early-acting (multilineage), 54–55 1097–98 collection target for, 653–54 functions, 53 microscopy and microscope, 914–17 cryopreservation and storage of, 652 hematopoietic, 512 osmotic fragility test, 1102–3 hematopoietic engraftment, 647, 654 indirect-acting, 56 peripheral blood smear, 917–20 hematopoietic precursor cells and, later-acting (lineage-restricted), peripheral blood smear 30, 44 55–56 examination, 1089–92 infusion of, 652 receptor superfamily, 57 peripheral blood smear molecular regulators of, 48 vascular endothelial, 716 preparation, 1089 origin and differentiation of, 647–48 Guanosine triphosphate (GTP), 1037 peripheral blood smear staining purging, 651–52 procedure, 1089 quantitation of, 652–53 H phlebotomy, specimen collection, umbilical cord blood, 648, 650 Haemophilus influenzae, 37, 275, 414 910–14 Hematopoietic stem cell Hairy cell leukemia (HCL), 628–29 reflex, 927–39 transplantation, 511, 645–60 Hairy cells, 628 reticulocyte count, 926–27, 1099– allogeneic stem cell transplantation, Half-staggered array, 748 1101 528, 648–49 Hallmark cell, 638 routine, 917–27 autologous stem cell Haploid, 1020 solubility test for hemoglobin, 927 transplantation, 649–50 Haplotypes, 300 solubility test for hemoglobin S, availability and success of, 657–58 Haptocorrins (HC), 333 1101–2 clinical laboratory professional’s Haptoglobin (Hp), 103, 419 Hematology reference values, 1079 role in, 654–55 Haptoglobin–hemoglobin (HpHb) in adults and children, 1078 complications associated with, complex, 103 Hematology results, review of, 1069–73 656–57 Harvest procedure, 1018–19 Hematolymphoid neoplasms, 1015 conditioning regimen for, 654 Hashimoto’s thyroiditis, 632 Hematoma, 770 gene therapy, 657–58 HBB gene, 271–72 Hematopathology, clinical graft-versus-host disease and graft- Healthcare quality improvement, applications of molecular versus-leukemia effect, 655–56 1058–59 diagnostics, 1043–45 types of, 648–50 Health Insurance Portability and Hematopoiesis umbilical cord blood stem cells Accountability Act (HIPAA), characteristics, 44 versus marrow stem cell 1058 cytokine receptors, 57–58 donors, 650 Heat denaturation test, 930–31 cytokines and control of umbilical cord stem cell Heinz bodies, 75, 78, 192, 271, 388, 1151 hematopoiesis, 52–57 transplantation, 650 stain, 931 cytokines regulation, lineage Hematopoietic system, 22–23 Helicopter pylori, 632 specific, 56 Hematopoietic tissue, 30–39 HELLP syndrome, 432, 438, 832 defined, 4, 44 Heme, 90–91, 102, 257, 258 Helmet cells, 189 development of, hematopoietic organ Heme-heme interaction, 98, 1121 Helper T cells, 142 structure/function and, 30 Hemochromatosis (HFE), 253–55, 1045 Hemacytometer, cell enumeration by, hematopoietic growth factors, 52–57 Hemoconcentration, 912 921–22 hematopoietic microenvironment, Hemodialysis, 248 Hemapoietic microenvironment 59–62 Hemoglobin, 4, 87–106, 1114 niches, 61–62 hematopoietic precursor cells, 44–51 A2, 929 Hematocrit, 4–5, 178, 206–7, 923 negative regulators of, 56 acquired nonfunctional, 104–6 Hematogones, 951, 1003 signaling pathways, 58 adult, 91, 95–96, 99 Hematologic disorders, 792, 853 transcription factors, 58–59 allosteric property of, 97–99 Hematologic remission, 510 Hematopoietic growth factors, 59, 60 binding, 100–101 Hematology, 1069–73 Hematopoietic/lymphoid disorders, catabolism, 102–4 automation in, 918, 961–88 chromosome analysis of concentration, 89, 922–23 defined, 3 chromosome abnormalities, 1020–22 count, 178–79, 206–7 screening tests, 6 chromosome structure and distribution width (HDW), 984 Hematology procedures, 905–42 morphology, 1015–16 electrophoresis, 269, 928–29 cell enumeration by cytogenetics, 1023–28 embryonic, 91, 95 hemacytometer, 921–22 mitosis, 1016–17 F, acid elution for, 929–30 equipment, 911–12 Hematopoietic microenvironment fetal, 91, 95, 99 erythrocyte indices, 923–24 (HM), 59–62 function, 96–101 1218 Index Hemoglobin (Continued) laboratory findings, 215 prenatal period, 420–21 glycosylated, 96 sites of destruction, 215–16 Rh immune globulin, 422 heat denaturation test, 930–31 sources of defect, 217 Rh incompatibility, 421–22 mean cell, 178, 179 Hemolytic anemia, enzyme therapy, 422 nitric oxide and, 101 deficiencies, 383–95 Hemolytic transfusion reactions, ontogeny of, 95–96 abnormal erythrocyte nucleotide 418–19 regulation of synthesis, 94–95 metabolism, 395 Hemolytic uremic syndrome (HUS), S, 927 clinical/laboratory evaluation in, 431–36, 438, 782, 846–48 solubility and polymerization, 386 Hemonectin, 61 272–73 diagnosis, 387 Hemopexin, 104, 419 structure of, 89–90 glucose-6-phosphate Hemophilia synthesis, 94–95 dehydrogenase deficiency, A, 807, 1049 variation in, 190 387–93 B, 807, 1049 Hemoglobin-based oxygen carriers glycolytic pathway, 385–86 carrier detection and prenatal (HBOCs), 101 hexose monophosphate shunt, 385 diagnosis, 810–11 Hemoglobin C disease, 279 other, 393–95 clinical presentations, 808–9 Hemoglobin Constant Spring, 292, pyruvate kinase deficiency, historical background, 807–8 298, 308–9 393–94 inheritance characteristics, 808 Hemoglobinemia, 215, 435 Hemolytic anemia, membrane defects, laboratory evaluation, 809–10 Hemoglobin Lepore, 292, 310–11 364–79 pathophysiology of bleeding, 808 Hemoglobinopathies, 265–85, 1045–46 acanthocytosis, 376 therapy for, 811–12 hemoglobin C disease, 279 hereditary elliptocytosis, 371–73 Hemorrhagic disease of the newborn hemoglobin D disease, 280–81 hereditary pyropoikilocytosis, (HDN), 818–19 hemoglobin E disease, 281 373–74 Hemorrhagic thrombocythemia, 531 hemoglobin H disease, 297–300 hereditary spherocytosis, 367–71 Hemosiderin, 95, 233 hemoglobin S/C disease, 279–80 hereditary stomatocytosis Hemosiderinuria, 103, 211 hemoglobin variants with altered syndromes, 374–75 Hemostasis, 4, 5 oxygen affinity, 283–84 lipid composition abnormalities, clinical applications of molecular sickle cell anemia, 271–79 366–67 diagnostics in, 1049 structural hemoglobin variants, paroxysmal nocturnal control of, 755–60 268–71 hemoglobinuria, 377–79 instrumentation, 895–97 thalassemias versus, 267, 292 skeletal protein abnormalities, in the neonate, 820 unstable hemoglobin variants, 366–67 in the newborn, 762 281–83 Hemolytic anemia, nonimmune patient results, 1073 Hemoglobinopathy, 1120 defects, 429–43 physiologic, 760–62 Hemoglobinuria, 215, 414 animal venoms, 442 in platelets, 170 Hemoglobin variants caused by antagonists in the blood, point-of-care instruments, 897 with altered oxygen affinity, 283–84 440–43 tests of, 533 identification, 268 caused by physical injury to Hemostasis, primary, 704–27 structural, 268–71 erythrocyte, 431–40 defined, 706 unstable, 281–83 disseminated intravascular hemostasis, platelets in, 711–26 Hemojuvelin (HJV), 235 coagulation, 437–38 laboratory investigation of, 871–75, Hemolysis, 215. See also Immune exercise-induced hemoglobinuria, 886–87 hemolytic anemia

(IHA) 439 platelet disorders, 767–93 complement-mediated, 404–5 HELLP syndrome, 432, 438 vascular system, 707–11 drug-induced, 402 hemolytic uremic syndrome, Hemostasis, secondary, 731–64. See IgG-mediated, 404 432–36 also Fibrinolysis, secondary IgM-mediated, 405 infectious agents, 440–42 hemostasis and sites and factors affecting, 403 malignant hypertension, 438–39 autosomal dominant inheritance, Hemolysis, elevated liver enzymes microangiopathic hemolytic 802–7 and low platelet counts anemia, 443 autosomal recessive disorders, (HELLP) syndrome, 432, 438 thermal injury, 439 812–16 Hemolytic anemia. See also Immune thrombotic thrombocytopenic disorders of, 798–821 hemolytic anemia (IHA) purpura, 434, 436–37 disseminated intravascular acute, acquired, 390 Hemolytic disease of the fetus and coagulation, 817 chronic nonspherocytic, 390 newborn (HDFN), 417, fibrin formation, protein disorders classification of, 217 419–22, 456 of, 800 drug-induced, 415–17 ABO incompatibility, 422 laboratory investigation of, 875–84, erythrocyte survival studies, 211 caused by ABO and Rh(D), 886–87 extravascular hemolysis, 215–16 comparison of, 420 platelets and, 725–26 extrinsic defects, 217–18 clinical presentation, 421 screening tests, 875–77 hereditary nonspherocytic, 386, 390 laboratory evaluation, 421–22 von Willebrand factor, laboratory intravascular hemolysis, 215 pathophysiology, 420–21 evaluation of, 804–6 intrinsic defects, 217 postnatal period, 421 x-linked recessive disorders, 807–12 Index 1219 Heparin, 855–56 Histone deacetylases (HDAC), 503 Hyperplasia, bone marrow, 33 cofactor II deficiency, 839 Histone deacetylases inhibitors, 503 Hypersegmentation, 459 low-molecular-weight, 845, 893 Histone modifications, 19–20 Hypersegmented neutrophils, 118, 1129 therapy monitory, 893–94 Histoplasma, 461 Hypersplenism, 37, 355, 458, 784 unfractionated, 845, 893 HIV virus infection. See Human Hypervolemia, 203 Heparin-associated immunodeficiency virus (HIV) Hypocellularity, 348 thrombocytopenia, 781 HLA-DR antigens, 45 Hypochromic cells, 190–91 Heparin cofactor II (HCII), 757 Hodgkin lymphomas, 507, 625, Hypodiploid, 1020 Heparin-induced thrombocytopenia 640–42, 1088 Hypodysfibrinogenemias, 813 (HIT), 781, 844–46 Hodgkin’s disease, 534 Hypofibrinogenemia, 813 tests for diagnosis of, 871 Holo-transferrin, 230 Hypogammaglobulinemia, 156 Hepatosplenic T cell lymphoma Homeostasis, iron, 234 Hypoplasia, 348 (HSTCL), 638 Homocysteine, 336 Hypoplastic myelodysplastic Hepcidin, 234, 235, 239 Homologous chromosomes, 1016 syndromes (MDS), 355, 571 inhibition, 235 Homozygotes, 310 Hypoproliferative anemias, 346–60 synthesis, 236 Homozygous state, 297, 311 aplastic anemia, 348–56 Hephaestin, 229 Horizontal interactions, 366 endocrine abnormalities, 359 Hereditary disorders Howell-Jolly bodies, 192 pure red cell aplasia, 356–58 of platelet function, 787–92 HUGO Gene Nomenclature renal disease, 358–59 of secondary hemostasus, 802–20 Committee (HGNC), 1023 Hyposegmented neutrophils, 1129 of vascular system, 773 Human granulocytic anaplasmosis Hypothyroidism, 342 Hereditary elliptocytosis (HE), 371–73 (HGA), 462 Hypovolemia, 203 Hereditary erythroblastic Human hemochromatosis protein, 235 Hypoxia, 104, 236, 386 multinuclearity with Human herpes virus 8 (HHV8), 1048 Hypoxia-inducible factor 2a (HIF@2a), positive acidified serum test Human immunodeficiency virus 236 (HEMPAS), 339 (HIV), 483, 504. See also Hypoxic environment, in utero, 80 Hereditary hemochromatosis, 253–55 Acquired immune deficiency Hereditary hydrocytosis, 374 syndrome (AIDS) I Hereditary nonspherocytic hemolytic HIV viral load, 485 Idiopathic cytopenia of undetermined anemia, 386, 390 Human monocyte ehrlichiosis (HME), significance (ICUS), 558 Hereditary persistence of fetal 461 Idiopathic, defined, 350 hemoglobin (HPFH), 309–10, Human platelet antigen (HPA), 715 Idiopathic hypereosinophilia, 465 1045 Human T cell leukemia/lymphoma Idiopathic hypereosinophilic Hereditary pyropoikilocytosis (HPP), virus (HTLV-I, II, V), 504 syndrome (I-HES), 548 373–74 Human T-lymphotropic virus type 1 Idiopathic thrombocytosis, 531 Hereditary sideroblastic anemia, 249 (HTLV1), 1048 IgG-mediated hemolysis, 404 Hereditary spherocytosis (HS), Humoral immunity, 140, 155–56, 484 IgM-mediated hemolysis, 405, 1126 367–71, 1008, 1124 Humoral regulation, 165–66 Imatinib mesylate (Gleevec), 528–29 Hereditary stomatocytosis syndromes, Hybridization, 1037, 1040–41 Immature B cell, 146 374–75 allele-specific oligonucleotides, Immature lymphocytes, 151–53 Hereditary xerocytosis, 374 1040–41 Immature platelet fraction (IPF), 776 Heterogeneous nuclear bead array technology, 1041 Immature reticulocyte fraction (IRF), ribonucleoprotein-E1 fluorescence in situ, 1040 208 (hnRNP-E1), 329 microarray technology, 1041 Immediate neutrophilia, 453–54 Heterologous chromosomes, 1016 Southern blot, 1040 Immune deficiency disorders, 483–89 Heterophile antibodies, 478 Hydrodynamic focusing, 966, 996 Immune hemolytic anemia (IHA), Heterozygotes, 310 Hydrops fetalis, 297, 298 399–423, 1125. See also Heterozygous state, 297, 311 Hydroxyurea (HU), 277–78, 531 Hemolysis Hexagonal phospholipids, 883–84 Hyperchromic, 1161 agglutinins in, characteristics of, 402 Hexokinase (HK) deficiency, 395 Hyperchromic cell population, 180, 190 alloimmune hemolytic anemia, Hexose monophosphate (HMP) shunt, Hypercoagulable, defined, 828 417–22 77–78, 123, 385, 393 Hypercoagulable state, 887–90 autoimmune hemolytic anemia, High-dose intravenous Hyperdiploid, 1020 407–17 immunoglobulin (IVIG), 411 Hypereosinophilia, 464–65 classification of, 402–3 High iron Fe (HFE), 235 primary, 465 laboratory identification of, 405–7 High-molecular-weight kininogen reactive (secondary), 464–65 Immune-mediated destruction, 776–78 (HK), 740–41 Hypereosinophilic syndrome (HES), Immune neutropenia, 457–58 High-performance liquid 465, 545 Immune response (IR), 140. See also chromatography (HPLC), 269 Hypergranular APL, 592 Adaptive immune response Histiocytes, 131 Hyperhomocysteinemia (HC), 831, (adaptive IR) Histocompatibility complex, 147–48 839–40 cell-mediated, 140, 156–57, 484 Histocytoses, 468 Hyper IgE syndrome (HIES), 489 humoral, 140, 155–56, 484 Histogram, 964 Hyperleukocytosis, 594 immunoblasts, 153 Histone code, 19 Hyperplasia, 303 memory cells, 154 1220 Index Immune thrombocytopenia (ITP), Interleukin 2 (IL-2), 57 pathophysiology, 241 777–78 Interleukin 3 (IL-3), 55, 57, 128, 712 peripheral blood, 243 Immune tolerance, 408 Interleukin 5 (IL-5), 128 therapy, 245 Immunoblast, 152, 153 Interleukin 5 (IL-5), 57 Iron homeostasis, 234–35 Immunodeficiency disorders, diagnosis Interleukin 6 (IL-6), 55, 522 Iron, laboratory assessment of, 238–40 and surveillance of, 1005–6 Interleukin 6 (IL-6), 57 Iron metabolism, 95, 227–38 Immunoglobulin (Ig), 144–45, 1116 Interleukin 7 (IL-7), 57 absorption, 228–30 Immunohistochemical stains, 952 Interleukin 9 (IL-9), 57 dietary iron, 228–29 Immunologic analysis, 508 Interleukin 11 (IL-11), 55 distribution, 228 Immunologic detection methods, 896 Interleukin 11 (IL-11), 57 homeostasis, 234–35 Immunologic dysfunction, oncogenes Interleukin 15 (IL-15), 57 iron requirements, 238 and, 504 Interleukin 21 (IL-21), 57 in mitrochondria, 238 Immunophenotyping, 508, 588–90 Interleukins, 52 storage, 232–33 acute lymphoblastic leukemias, Internal quality control program, 1058 transport, 230–32 611–12, 613 International Classification of Disease, Iron refractory iron-deficiency anemia antibodies used for, 999 10th revision (ICD-10) codes, (IRIDA), 247–48 by flow cytometry, 998–1008 1056 clinical presentation, 247 specimen requirements and International Committee for diagnostic algorithm, 247 preparation for, 859 Standardization in pathophysiology, 247 T lymphoblastic lymphoma Hematology, 211 therapy, 248 (T-LBL), 615 International normalized ratio (INR), Iron-regulatory protein (IRP), 234, 237 Immunosuppressed individuals, 478 891 Iron-responsive element (IRE), 237, Immunosuppressive therapy (IST), 354 International Prognostic Scoring 1119 Impedance instruments, 963 System (IPSS), 572, 1168 Iron-responsive element-binding Indices, erythrocytes, 179–81, 207 International sensitivity index (ISI), protein (IRP-BP), 237 Indirect-acting hematopoietic growth 892 Iron studies, bone marrow, 238–39 factors, 56 International Society on Thrombosis Irreversibly sickled cells (ISC), 273 Indirect antiglobulin test (IAT), 406–7 (ISTH), 883 Ischemia, 273, 774 Individualized Quality Control Plan International System for Human Isoelectric focusing, 269, 927 (IQCP), 1068–69 Cytogenetic Nomenclature Isopropanol precipitation, 931 Induction therapy, 510, 617 (ISCN), 1023 Isotype switching, 146 Ineffective erythropoiesis, 72, 291 Interpreting quality control charts, Isovolumetric sphering, 984 Ineffective hematopoiesis, 557 1066 Infants/children, iron requirements Intracellular bridges, 742 J in, 238 Intracellular organisms, 461–62 JAK-STAT signaling pathway, 58, 59 Infectious agents Intracellular pathogens, 156 Janus kinase 2 (JAK2) gene, 59, 81, 509, aplastic anemia and, 351 Intravascular destruction, 82, 103–4 521, 531–33, 536, 539, 540, 543, hemolysis, 440–42 Intravascular hemolysis, 215 1026, 1046 Infectious diseases, 1048–49 Intravenous antiplatelet agents, 858 Joint fluid, 670 Infectious mononucleosis, 476–79 Intrinsic factor (IF), 323 Juvenile myelomonocytic leukemia Infinity optical system, 915 Intrinsic factor blocking antibody (JMML), 575–76 Inherited functional abnormalities, (IFBA), 336 Juvenile pernicious anemia, 335 462–64 Intrinsic pathway, 734, 738–44 Juxtacrine signals, 53 Inhibitors Intrinsic Xase, 738 acquired, 819 Introns, 14 K biochemical, 755–60 Ionizing radiation, aplastic anemia Kaposi’s sarcoma, 483, 682 C1-esterase, 760 and, 351 Karyokinesis, 17 of fibrinolysis, 753–55 Iron balance Karyotype, 558–59, 1019 fibrinolytic protein, 816 intracellular, 237–38 Keratocytes, 187, 189 hemophilia, 812 physiological regulation of, 233–38 Kernicterus, 421 identification of, 882–84 Iron-deficiency anemia (IDA), 227, Kinins, 125–26 naturally occurring, 755 230, 240–45, 565 Kit ligand (KL), 54 plasminogen activator, 754, 890 blood loss, 240–41 Klebsiella pneumoniae, 414 of single factors, 819 bone marrow, 244 Knizocytes, 187, 189–90 thrombin-activatable fibrinolysis clinical presentation, 241–42 Koehler illumination, 916–17 (TAF), 754, 890 dietary deficiency, 240 Kostmann’s syndrome, 58 tissue factor pathway, 755 erythrocyte protoporphyrin studies, Kupffer cells, 37, 131 Innate immune response (innate IR), 244 114 etiology, 240–41 L Integral proteins, 74 historical aspects, 240 Laboratory safety, 1065 Intercellular adhesion molecules iron studies, 243–44 Laboratory tests (ICAM), 121 laboratory evaluation, 242–44 anticoagulant therapy, 891 Interfering substances, 1073 malabsorption, 241 fibrinolytic system, 884–85, 886–87 Interferon (IFN), 133 obesity, 241 hemostasis instrumentation, 895–97 Index 1221 hypercoagulable state, 887–90 mixed phenotype acute, 616 Low-molecular-weight heparin in investigation of hematologic Philadelphia (Ph) chromosome in (LMWH), 845, 893 problem, 6 acute, 523, 525 Loxosceles reclusa, 442 for iron, 238–40 small lymphocytic, 626–28 LRP receptor (LDL receptor-related molecular markers of hemostatic T-cell large granular lymphocytic, protein), 754 activation, 891 639 Lupus anticoagulant (LA), 820, 842, primary hemostasis, 871–75 T-cell prolymphocytic, 639 882–83 regulations, 909–10 Leukemia inhibitory factor, 57 Lymphadenopathy, 39, 1169 secondary hemostasis, 875–84 Leukemic T and NK cell lymphomas, Lymph nodes, 38–39 for specific factor deficiency, 877–82 639–40 Lymphoblast, 151 specimen collection and processing, Leukemogenesis, 504–5 Lymphoblastic leukemia (LL) 869–71 Leukemoid reaction, 455 diagnosis and classification of, value of, 6–7 Leukocyte adhesion deficiency (LAD), 1002–4 for von Willebrand factor, 880–82 122, 464 Lymphocyte-depleted HL, 641 Lactic dehydrogenase (LD), 414, 506, Leukocyte adhesion deficiency-1 Lymphocyte recirculation, 1116 565, 1157 (LAD-1), 1115 Lymphocytes, 677 Lacunar cells, 641 Leukocyte alkaline phosphatase activated, morphology of, 153–54 Lamellar body counts (LBCs), 693–94 (LAP), 937 adhesion molecules of adaptive Lancets, 912 scores, 1081 immune response, 158–59 Langerhans cell histiocytoses (LCH), Leukocyte disorders, 1046–48 aging and function, 159 468 Leukocytes, 3, 112 antigen receptors of, 143 Langerhans cells, 131 abnormalities, 210 B cells, 155–56 Large granular lymphocytes (LGLs), basophils, 128–30 disorders, nonmalignant, 473–90 151 concentration of, in peripheral distribution, concentration, and Later-acting (lineage-restricted) blood, 113–14 kinetics, 154 growth factors, 55–56 count, 178–79 function, 154–59 Lead poisoning, 249–50 defined, 112 identification and morphology, Lecithin-cholesterol acyl transferase eosinophils, 126–28 151–54 (LCAT), 73, 1113, 1125 function of, 114 immature, morphology of, 151–53 Lecithin-cholesterol acyl transferase manual count, 921–22 immunoblast, 152, 153 (LCAT) deficiency, 377 monocytes, 130–33 large granular, 151 Left shift, 454 in myelodysplastic syndromes, lymphoblast, 151 Lens aberration, 915 561–62 mature, 142 Leptocytes, 188, 190 neutrophils, 115–26 metabolism, 159 Leukemia, 498, 625 surface markers, 114 morphology, 152 acute basophilic, 600–601 Leukocytosis, 114, 452, 456, 537 plasma cell, 152, 154 acute lymphoblastic, 505, 508, 688, Leukoerythroblastic reaction, 455–56 plasmacytoid lymphocyte, 153, 154 1027 Leukopenia, 114, 453 prolymphocyte, 151 acute lymphocytic, 1002 L&H cells, 640 reactive, 153 acute megakaryoblastic, 600 Light scattering, 996, 997 sequestration and destruction, acute monoblastic, 599–600 Light-scattering instruments, 984–87 482–83 acute monocytic, 599–600 Limits, quality control, 1066 Lymphocytic leukemoid reaction, 481 acute myeloid, 503, 505, 508, 583– Lin, 45, 49 Lymphocytoid plasma cell, 153 602, 1004, 1026–27 Lineage-specific cytokine regulation, Lymphocytopenia, 154, 482–89 acute myelomonocytic, 598, 689 56 Lymphocytosis, 154, 475–82 acute panmyelosis with Linearity, 1061 Bordetella pertussis, 476, 480 myelofibrosis, 601 Lipid asymmetry, 1111 conditions associated with, 476 acute promyelocytic, 592 Lipid composition, 73, 74 cytomegalovirus, 479–80 acute undifferentiated, 1004 Lipid composition abnormalities, infectious mononucleosis, 476–79 of ambiguous lineage, 588, 608 366–67 persistent, 481–82 atypical chronic myeloid, 520, 575 Lipid-derived mediators, 127 plasmacytosis, 481 B-cell prolymphocytic, 628 Lipids, 11 reactive, 480–81 bilineage acute, 616 Lipoprotein receptor-related protein Lymphoepithelial lesion, 632 biphenotypic acute, 616 (LRP), 754 Lymphoid antigen–positive ALL, 616 chronic eosinophilic, 520, 545 Liver clearance, 755 Lymphoid disorders. See chronic granulocytic, 522 Liver disease, 817–18 Hematopoietic/lymphoid chronic lymphocytic, 503, 626–28 in alcoholic individuals, 341–42 disorders, chromosome chronic myelogenous, 504 causes and characteristics of analysis of chronic myeloid, 520, 522–30 anemia in, 341–42 Lymphoid follicle, 35 chronic myelomonocytic, 524 nonmegaloblastic macrocytic Lymphoid malignancies chronic myeloproliferative, 546 anemia and, 340 diagnosis and classification of, chronic neutrophilic, 520, 530–34 LMAN1 (L-mannose-1), 816 625–26, 1001–2 hairy cell, 628 Long-term repopulating cells (LTR), 45 environmental factors, 625 Juvenile myelomonocytic, 575–76 genetic factors, 624 1222 Index Lymphoid neoplasms, 507, 546–47 mucosa-associated lymphoid Megakaryocyte hypoplasia Lymphoid niches, 62 tissue, 625 syndromes, 782 Lymphoid-primed multipotential mucosa-associated lymphoid tissue Megakaryocytes, 48 progenitor (LMPP), 141 lymphoma, 632 developmental stages of, 167 Lymphokine-activated killer (LAK) peripheral T cell or natural killer growth and development factor, cells, 158 cell lymphoma, 637–38 166–69 Lymphoma malignancies. See also small lymphocytic lymphoma, lineage, 50 Malignant lymphomas 626–28 microenvironment, 166 chronic leukemic, 626–28 Waldenström macroglobulinemia, peripheral blood platelets, 169 mature, 626–36 633 production regulation, 165–66 Lymphomas, 499, 625, 1048 Malignant neoplasms, defined, 498 progenitor cell compartment, 165 cytogenetic analysis of, 1027–28 Mantle cell lymphoma (MCL), 630–31 Megakaryocytic hypoplasia, 348 defined, 610 Manual cell counts, 673–74 Megakaryocytic niches, 62 diagnosis and classification of, March hemoglobinuria, 439 Megakaryocytopoiesis, 56 1002–4 Marginating pool (MP), 120 Megakaryopoiesis, 165–69 Lymphoplasmacytic lymphoma (LPL), Marrow fibrosis, conditions associated Megaloblastic anemia, 213, 215, 632–33 with, 544

319–43, 1122, 1123 Lymphopoiesis, 140–42 Masson’s trichrome stain, 939 bone marrow, 325–26 antigen-dependent and- Mast cell disease, 466, 549 clinical presentation, 323–24 independent, 141–42 Mast cell growth factor (MCGF), 54 congenital deficiencies, 339 cytokines in, 141 Mast cells, 129, 677 drugs, 338 ontogeny of, 140–41 Mastocytosis, 466, 548–49 enzyme deficiencies, 338 transcriptional regulation of, 141 Matched unrelated donor (MUD) folic acid, 322–23, 327–31 Lymphoproliferative disease, 408 transplant, 649 hematopoietic diseases, other, 340 cytogenetic analysis of, 1027–28 Matriptase-2 (MT-2), 235, 247 laboratory values in, 325 Lyonization, 389 Maturation-inducing domain, 58 neutropenia, 457 Lysis, 885 Maturation/maturing cells, 44 other laboratory findings, 326–27 Lysomal storage disorders, 467, 468 acute myeloid leukemias, 597–601 peripheral blood, 324–25 Lysophospholipase, 127 basophils, 129 therapy, 338 Lysosomes, 15, 716 defects in functional classification vitamin B12, 331–38 Lytic agent, 1145 of anemia, 214–15 Megaloblastic maturation, 1122 Lytic state, 857 eosinophils, 126–27 Membrane attack complex (MAC), monocytes, 130–31 377, 379, 404, 433 M neutrophils, 115–18 Membrane inhibitor of reactive lysis Macrocytes, 186, 209, 210 storage pool, 119 (MIRL), 377 Macrocytic anemia, 213 Maturation/maturing cells, 50 Membrane permeability, 76–77 Macrocytic anemia without Mature B cell, 146 Membrane receptors, 713 megaloblastosis, 340–42 Mature lymphocytes, 142 Memory cells, 147, 154 Macrocytosis, 1123 Mature neoplasms, 498 Memory T cells, 157 Macrophage disorders, 466–68 Mature neutrophil reserve, 119 Meningeal membranes, 668 Macrophage inflammatory Mature RBC, 70 Meninges, 668 protein@1a (MIP@1a), 57 May-Hegglin anomaly (MHA), Menstruation, daily iron loss in, 238 Macrophages, 31–32, 131 462–63, 784 M:E ratio, 1167 Maintenance chemotherapy, 510, 617 Mean cell hemoglobin (MCH), 923 Mesenchymal stem cells (MSCs), 32 Major basic protein (MBP), 127 Mean cell (corpuscular) hemoglobin Mesothelial cells, 683–86 Major breakpoint cluster region (MCH), 89, 412 Messenger ribonucleic acid (mRNA), (MBC-R) gene, 523 Mean cell (corpuscular) hemoglobin 14, 95, 169 Major histocompatibility complex concentration (MCHC), 180, Metacentric chromosome, 1016 (MHC), 648 412, 436, 923 Metamyelocyte, 117–18 Malabsorption, 241, 330, 335 Mean cell (corpuscular) hemoglobulin Metaphase, 17 Malarial parasites, 440–41 (MCH), 179 Methemoglobin, 104–5 Malignancy, 852 Mean cell volume (MCV), 179, 923, Methemoglobinemia, 104–5, 284 Malignant cells, in body fluids, 680–83 1124 Methemooglobin reductase pathway, Malignant hypertension, 438–39 Mean corpuscular hemoglobin 78 Malignant lymphomas concentration (MCHC), 1067 Methylation, 19 benign lymphoid aggregates versus, Mean platelet volume (MPV), 170, 965 Methylene tetrahydrofolate reductase 953–54 Mean reticulocyte volume (MRV), 971 (MTHFR), 839, 840, 1049 Burkitt lymphoma, 634 Medical decision levels, 1062 Methylmalonic acid (MMA), 326–28, diffuse large B cell lymphoma, 633 Medulla, of lymph nodes, 38 331, 336, 1123, 1159 follicular lymphoma, 629–30 Medullary hematopoiesis, 34 Methyl trap, 329 Hodgkin lymphomas, 640–42 Medulloblastoma, 688 Michael’s pancytopenia, 1172 lymphoplasmacytic lymphoma, Megakaryoblast, 165–67 Microangiopathic hemolytic anemia 632–33 Megakaryocyte, erythroid progenitor (MAHA), 431–39 mantle cell lymphoma, 630–31 (MkEP), 48 Microarray technology, 1041 Index 1223 Microcytes, 186 Morphologic findings in body fluids, variants of, 571 Microcytic, hypochromic anemia, 664–700 Myelofibrosis with myeloid 212–13 Morulae, 461 metaplasia (MMM), 520 Microenvironment, megakaryocyte, M phase, 17 Myeloid, 348 166 Mpl-ligand, 56 Myeloid antigen–positive ALL, 616 Microfilaments, 716 Mucosa-associated lymphoid tissue Myeloid neoplasms, 506, 546, 1086 Microglial cells, 131 (MALT), 38–39, 625, 632 Myeloid proliferations related to Microgranular APL variant, 592–93 Multiplate® platelet function analyzer, down syndrome, 601 Micromegakaryocytes, 562–63 874–75 Myeloid sarcoma, 601, 1026 Microorganisms, 680 Multiple myeloma, 917 Myeloid-to-ethyroid ratio (M:E ratio), Microparticles (MPs), 831–32 Multiplex polymerase chain reaction 951 Micro-RNAs (miRNAs), 113 (PCR), 1039 Myeloperoxidase (MPO), 588, 934 Microscopes, 914–17 Multipotential hematopoietic stem deficiency, 463–64 Microscopy, 914–17 cells, 49, 52 Myelophthisic anemia, 355 bright-field, 914–15 Multipotential precursors, 45 Myelophthisis, 355, 456 Koehler illumination, 916–17 Mutation, 14, 1111 Myeloproliferative disorders, 1153 phase-contrast, 915–16 Mutations, 1035 Myeloproliferative disorders (MPDs) preventative maintenance, 917 Mycoplasma pneumoniae, 411 chronic myeloid leukemia, 522–30 Microtubules (MTs), 716 Myeloblasts, 115–17, 565–66 chronic neutrophilic leukemia, Migration, neutrophil, 123 Myelocytes, 116, 117 530–31 Minimal residual disease (MRD), 510, Myelodysplastic/myeloproliferative classification of, 520–21 611, 1002, 1037, 1177 neoplasm (MDS/MPN), clonal hypereosinophilia, 545–48 flow cytometry, 1005 574–76, 576, 1086, 1132 differential features of, 521 Miotic pool, 119 Myelodysplastic syndrome (MDS), essential thrombocythemia, 531–34 Mitosis, 1016–17 249, 355, 555–77, 1046, 1086 general features of, 521–22 Mixed cellularity HL, 641 blast cell classification, 565–67 mast cell disease, 549 Mixed phenotype acute leukemia bone marrow, 563–64 neoplasms, 517–49 (MPAL), 616, 1004 classification of, 567–68 pathophysiology, 521 Mixed-type autoimmune hemolytic clinical presentation, 560 polycythemia vera, 520, 535–40 anemia, 402, 415 cytogenetic analysis of, 1027 primary myelofibrosis, 540–44 Mixing studies, 877–78 cytogenics and molecular testing, unclassifiable (MPN, U), 545 MLL gene, 592 558–60 Myeloproliferative neoplasms (MPNs), Molecular analysis, 509, 1036–43 diagnosis of, 1004 505, 517–49, 1046, 1086 acute lymphoblastic leukemia, 612 differential diagnosis, 571–72 cytogenetic analysis, 1026 cancer, molecular basis of, 500–504 dyserythropoiesis, 563–64 molecular diagnostics, laboratory dysgranulopoiesis, 564 N methods in, 392 dysmegakaryopoiesis, 564 Naive lymphocytes, 141 Molecular cytogenetics, 533, 1028–29 erythrocytes, 561 Nasal NK/T cell lymphomas, 639 Molecular genetics, 955–56 with excess blasts, 569–70 National Health and Nutrition Molecular remission, 509 with fibrosis, 571 Examination Survey Molecular-targeted therapy, 511 hematologic abnormalities in, 561 (NHANES III), 206 Molecular targets, 534 hypoplastic, 571 National Marrow Donor Program Monoblasts, 130 incidence, 560 (NMDP), 649 Monoclonal B lymphocytosis (MBL), with isolated del(5q), 570 Natural killer (NK) cells, 23, 140, 150, 627 laboratory evaluation, 560–65 157–58, 404, 617 Monoclonal gammopathies of leukocytes, 561–62 Natural killer T (NKT) cells, 150 undetermined significance micromegakaryocytes, 562–63 Necrosis, 20, 774 (MGUS), 636 molecular diagnostics, 564–65 apoptosis versus, 21 Monoclonal gammopathy, 156 with multilineage dysplasia, 569 cardinal features of, 20 Monocyte colony-stimulating factor oncogenes, 560 Needles, 911–12 (M-CSF), 55 oncogenes in, 504 Negative feedback inhibition, 755 Monocyte/macrophage disorders, pathophysiology, 557–60 Neisseria meningitidis, 275 466–68 peripheral blood, 560–63 Neonatal alloimmune Monocyte-macrophage system, 130 platelets in, 562 thrombocytopenia (NAT), 780 Monocytes, 130–33 prognosis, 572 Neonatal disorders Monocytopenia, 467 proliferation abnormalities, 560 hyperbilirubinemia, 390–91 Monocytosis, 466–67 ring sideroblasts, 569, 576 polycythemia, 539 Monoferric transferrin, 230 with single lineage dysplasia, Neoplasms, 496–513 Mononuclear phagocyte (MNP) 568–69 benign, defined, 498 system, 101, 130 subgroups of, 568–70 classification of, 506–7 Monopoiesis, 56 therapy, 573 clinical presentation, 505 Monosodium urate (MSU) crystals, therapy-related, 571 defined, 498 690–91 tumor suppressor genes, 560 diagnosing and classifying, 507–9 Monosomy, 1020 unclassifiable, 570, 576 epidemiology, 505 1224 Index Neoplasms (Continued) Nocturnal paroxysmal in myelodysplastic syndromes, 560 etiology/pathophysiology (See also hemoglobinuria (PNH), 351, proto-, 501 Oncogenes) 377–79, 392 somatic mutation, 504 hematolymphoid, 1015 Nodal T and NK cell lymphomas, translocations, molecular basis of laboratory evaluation, 506 637–38 cancer, 500–501 leukemias, 498 Nodular lymphocyte-predominant viral infection, 504 lymphoid, 507 Hodgkin lymphoma (NLPHL), Oncostatin M (OSM), 57 malignant, defined, 498 640 1,3-bisphosphoglycerate (1,3 BPG), 78 myelodysplastic syndromes, 507 Nodular sclerosis, 641 Open canalicular system (OCS), 717 myeloid, 506 Nomenclature Opportunistic organisms, 483 myeloproliferative disorders, 506, cytogenetic, 1022–23 Optimal counting area, 917 507, 517–49 of hemoglobin, 270–71 Oral anticoagulants, 856, 1140 pathophysiology, 499–505 Nomogram, 893 Oral anticoagulant therapy, 891 plasma cell, 634–36 Noncatalytic domains, 738 Oral contraceptives, 852–53 prognosis, 509–12 Nonclonal (reactive) Oral vitamin K antagonist therapy, Nephelometry, 895 hypereosinophilia, 464–65 891–92 Neural tube defects (NTD), 329 Nondifferentiating cell division, 46 Organelles, 12 Neutropenia, 120, 456–58 Nondisjunction, 1020 Organelle zone, 716–17 Neutrophil disorders, 453–64 Non-Hodgkin lymphoma (NHL), 625, Orotic aciduria, 339 bacterial infection, 454–55 1170 Orthochromic normoblast, 71 leukemoid reaction, 455 Non-Langerhans histiocytoses (non- Osmotic fragility test, 370, 931 leukoerythroblastic reaction, LCH), 468 Osteoblastic niche, 47 455–56 Non-Parenteral Anticoagulation, 893–94 Osteoblasts, 32, 61 physiologic leukocytosis, 456 Nonsegmented neutrophils (stab), 118 Osteoclasts, 32 qualitative or morphologic, 458–64 Nonspecific reactive changes, 678–80 Outliers, 1062 quantitative, 453–58 Nonsteroidal anti-inflammatory drugs Ovalocytes, 189 reactive chronic neutrophilia, 453 (NSAIDs), 793, 858 Overhydrated herditary stimulated bone marrow states, 456 Nonthrombocytopenic purpura, 773 stomatocytosis (OHS), 374 tissue destruction/injury, Nonthrombogenic blood vessels, 710 Oxygen affinity, 96 inflammation, metabolic Non-transferrin bound iron (NTBI), 231 of HbS, 272 disorders, 455 Nonwaived tests, 910 hemoglobin variants with altered, Neutrophil extracellular traps (NETs), Normoblasts, 69 99–100 125, 126, 170 Normochromic cell population, 180 Oxygenated blood flow, in anemia, 204 Neutrophilia, 117, 120, 453–56, 1115 Normochromic erythrocytes, 184 Oxygen-dependent microbicidal Neutrophil oxidative burst assay, 463 Normocytic anemia, 180 activity, 123–25 Neutrophils, 115–26 Normocytic erythrocytes, 184 Oxygen dissociation curve (ODC), 97 band, 113, 116–18 Normocytic, normochromic anemia, Oxygen-independent granule bone marrow, 119–20 213 proteins, 125 concentration, distribution, and N-terminal disulfide knot (N-DSK), 748 Oxygen transport, 96–100 kinetics, 119–20 Nuclear abnormalities, 458–60 Oxygen utilization by tissue, in differentiation, maturation, and Nuclear-cytoplasmic asynchrony, 322 anemia, 204 morphology, 115–18 Nuclear envelope, 13 Oxyhemoglobin, 96 function, 120–26 Nucleated cell differential, 674–93 granule contents of, 117 Nucleic acid P hypersegmented, 118, 324–25 amplification, 1036–40 Packed red blood cells (PRBCs), 305 metamyelocyte, 117–18 extraction, 1036 Pancytopenia, 214, 348, 355–56 myeloblast, 115–17 Nucleoli, 13 Panmyelosis, 520 myelocytes, 116, 117 Nucleus, 13 Pappenheimer bodies, 192, 193, 251 nonsegmented, 118 basophilic normoblast, 71 Paracrine signals, 53 peripheral blood, 120 orthochromic normoblast, 71 Paraproteins, 773–74 promyelocyte/progranulocyte, 116, polychromatophilic normoblast, 71 Parietal membrane, 667 117 pronormoblast, 69, 71 Paroxysmal cold hemoglobinuria segmented, 116, 118 Numerical aberrations, 1020–22 (PCH), 402, 414–15 tissue, 120 Donath-Landsteiner test for, 931–33 Newborns, CBC variations in, 194 O Paroxysmal nocturnal hemoglobinuria Next-generation sequencing (NGS), Obesity, 241 (PNH), 351, 377–79, 392, 853, 1042–43 Occupational health and safety 1125 Nicotinamide-adenine dinucleotide administration (OSHA) flow cytometry, 1007–8 (NADH), 77–78 standards, 1065 Parvovirus, 274, 356 Niemann-Pick disease, 467–68 Old/damaged erythrocytes, 1112 Passenger lymphocyte syndrome Nijmegen-Bethesda assay, 884 Oncogenes (PLS), 417, 649, 656 Nitric oxide, 101, 273 genetic susceptibility, 504 Pathogen-associated molecular Nitroblue tetrazolium slide test (NBT), immunologic dysfunction, 504 patterns (PAMPs), 114, 123, 125, 463 miscellaneous factors, 505 142–43 Index 1225 Patient samples, monitoring quality Peritoneal cavity, 667 Plasminogen (PLG), 750–51, 890 control with, 1068 Peritoneal fluid, 674, 683–86 activator, 750, 754 Patient specimens, 1068 Peritoneum, 667 deficiency, 841 Pattern recognition receptors (PRRs), Peritransplant infections, 656–57 tissue plasminogen activator, 841 114, 142 Perl’s Prussian blue stain, 71 Plasminogen activator inhibitor Pelger-Huët anomaly, 458–59, 542, Pernicious anemia (PA), 334–35, 1123 (PAI-1), 753–54, 890 596, 600 Peroxisomes, 717 Plasmodium falciparum, 440, 441 Pencil cells, 189 Persistent polyclonal B-cell Platelet-activating factor (PAF), 711, 717 Perfluorocarbons (PFC), 101 lymphocytosis (PPBL), 481–82 Platelet activation, laboratory markers Periarteriolar lymphatic sheath Petechiae, 770 of, 891 (PALS), 35 Peyer’s patches, 39 Platelet-derived growth factor Pericardial cavity, 668 PFA-100®, 200®, 872 (PDGF), 522 Pericardial fluid, 674, 683–86 Phagocytosis, 123, 124 Platelet-derived growth factor Pericardium, 668 Pharmacokinetics, 893 receptor a, 546 Periodic acid-Schiff (PAS) stain, 936–37 Phase-contrast microscopy, 915–16 Platelet disorders, 767–93 Peripheral blood Phenotype, 300, 536–37 acquired, 792–93 acute lymphoblastic leukemia, 609–10 Philadelphia (Ph) chromosome in bleeding, 769–73 acute myeloid leukemias, 585–86 acute leukemias, 523, 525 hereditary, 787–92 anemia of chronic disease, 246–47 Phlebotomy safety, 914 qualitative (functional), 787–93 chronic myeloid leukemia, 525–26 Phlebotomy, sample collection, 538 quantitative, 775–87 essential thrombocythemia, 532–33 Phlebotomy, specimen collection, Platelet distribution width (PDW), 181 hemoglobin Lepore, 311 910–14 Platelet function analyzers (PFA), 872–75 hemoglobin S, 313 Phosphoenolpyruvate (PEP), 393 Platelet-poor plasma (PPP), 871 hemoglobin variants, unstable, 282 Phosphofructokinase (PFK) deficiency, Platelet release reaction, 720 hereditary elliptocytosis, 373 395 Platelet-rich plasma (PRP), 871–73 hereditary pyropoikilocytosis, 374 Phosphoglycerokinase (PGK) Platelets, 3, 163–71 infectious mononucleosis, 477–78 deficiency, 395 abnormalities, 210, 533 iron deficiency, 243 Phosphoinositide pathway, 723 activation, 719–20, 726 megaloblastic anemia, 324–25 Phospholipase C, 723 adhesion, 718, 787–89 myelodysplastic syndromes, 560–63 Phospholipids, 73 aggregation, 721–22, 789–90 in neutrophils, 120 Photodetectors, 996 aggregometry, 872–75 pancytopenia, 351, 353 Photomultiplier tube (PMT), 996 agonists, 720 platelets, 169–70 Physiologic anemia of the newborn, 80 alloantigens, 715 polycythemia vera, 537–38 Physiologic hemostasis, 760–62 clumps, 919 primary myelofibrosis, 542–43 Physiologic plasminogen (PLG) coated, 726 sickle cell anemia, 276 activators, 750 count, 170 sideroblastic anemia, 251–52 Pia mater, 668 distribution width, 170 thalassemia, 295 Pica syndrome, 241 factor 3, 726 warm autoimmune hemolytic PIGA gene, 377 function, 170, 718–26 anemia, 409–10 Pince-nez cells, 459 giant, 169 Peripheral blood count (PBC), 176 Pitting, 36 in hemostasis, 170, 711–26 Peripheral blood smears, 181–93 PIVKA (protein induced by vitamin k immune responses, 170 abnormalities detected on, 183, 920 absence or antagonists), 738 indices, 170, 181 anisocytosis, 184–85 Plasma, 4 manual count, 922 automated method, 918 cells, 152, 154 mean platelet volume, 181 erythrocyte inclusions, 191, 192 components of, 4 megakaryocyte, 165–69 erythrocyte morphology, 184–85 exchange, 411, 437 metabolic activity, 717 examination, 919–20 fresh frozen, 437, 887 morphology, 169–70 high-power magnification, 183–93 membrane, 713 in myelodysplastic syndromes, 562 low-power magnification, 182–83 platelet-poor, 871 peripheral blood, 169–70 lymphocytes, 185 platelet-rich, 871–73 platelet distribution width, 181 neutrophils, 185 pooled normal, 877 plug, 724 platelets, 169–70 transport, 100 procoagulation activity, 726 poikilocytosis, 184, 187 Plasma cell myeloma, 634–35 production of, 165, 168, 169 preparation, 917–18 Plasma cell neoplasms, 634–36 quantitative

evaluation, 170 staining, 918–19 Plasma cells, 1116 release of, 720–21 variation in hemoglobin (color), 190 Plasmacytoid lymphocyte, 153, 154 reticulated, 170, 975 Peripheral membrane proteins, 75 Plasmacytoma, 636 satellitism, 169, 183, 919 Peripheral proteins, 74, 75 Plasmacytosis, 481 satellitosis, 169 Peripheral T-cell lymphoma, not Plasmapheresis, 411 secretion, 720–21, 790–91 otherwise specified, 637–38 Plasmin (PLN), 750–51 shape change, 720 Peripheral T cell or natural killer cell systemic effects of, 753 stress, 169 lymphoma (T/NK), 637–38 Plasmin–antiplasmin (PAP) structure of, 712–18 Peripheral zone, 712–13 complex, 890 white blood cells, 183–84 1226 Index Platelet satellitism, 169 Preleukemia, 505, 555. See also Protein tyrosine phosphatases (PTPs), Platelet type-pseudo-VWD, 806 Myelodysplastic syndrome 58 Pleiotropy, 1112 (MDS) Protein Z–dependent protease Pleocytosis, 668 Prenatal diagnosis, of hemophilia, inhibitor (zpi), 760 Plethora, 537, 539 810–11 Prothrombinase complexes, 738 Pleural cavities, 667 Preventative maintenance, in Prothrombin group, 735 Pleural fluid, 674, 683–86 microscopy, 917 Prothrombin time (PT), 6, 745–46, 875 Ploidy, 1008–9 Primary eosinophilia, 465 deficiency, 814 Plumbism, 249 Primary fibrinogenolysis, 817 G20210A, 838–39, 889, 1049 Pluripotential hematopoietic stem Primary graft failure, 656 gene mutation 20210, 838 cells, 141 Primary hemostasis, 5 time (PT), 435, 533, 771 Pneumocystis jirovecii, 670 Primary hemostasis plug. See also Protofibrils, 748 Poikilocytosis, 184, 187 Hemostasis, primary Proto-oncogenes, 23, 501 Point-of-care (POC) instruments, 897 defined, 706 Protopod, 151 Point-of-care testing (POCT), 909 formation of, 718–26 Proximal histidine (F8), 94 Polychromasia, 1118 Primary hypersplenism, 1112 Prussian blue stain for iron, 954–55 Polychromatophilic erythrocytes, 68, Primary lymphoid tissues, 30 Pseudochylous, 672 70, 191, 1156 Primary myelofibrosis (PMF), 540–44 Pseudodiploid, 1021 Polychromatophilic normoblast, 71 Primary throbocythemia, 531 Pseudo-neutropenia, 458 Polyclonal gammopathy, 156 Primary thrombocytosis, 531, 785 Pseudo-neutrophilia, 454 Polycythemia, 535, 917 Primitive erythropoiesis, 30 Pseudopolycythemia, 539 Polycythemia vera (PV), 520, 535–40, Pro-B cell, 145 Pteroylmonoglutamate (PteGlu), 327 1046 Probe, 1037 Pulmonary embolism (PE), 830 bone marrow, 538, 540 Procoagulation factors, 734–38 Pure erythroid leukemia (PEL), 600 classification of, 535 Proficiency testing, 1060 Pure red cell aplasia, 348, 356–58 clinical presentation, 537 Profuse bleeding, 1173 Purging, of hematopoietic stem cells, differential diagnosis, 539 Progenitor cells, 44, 48–50, 56, 61, 165, 651–52 erythropoietin measurement, 540 648 Purine nucleoside phosphorylase genetics, 538, 540 Proliferating pool, 119 (PNP), 486 laboratory differentiation of, 540 Proliferating progenitor cells, 166 Purpura laboratory evaluation, 537–38 Proliferation, 16–19, 1008 associated with dysproteinemias, other laboratory evaluation, 538 Proliferation defects, in functional 773–74 pathophysiology, 535–37 classification of anemia, 213–14 defined, 770 peripheral blood, 537–38 Proliferation-inducing domain, 58 due to decreased connective tissue, prognosis and therapy, 538–39 Proliferative index, 1008 773 Polymerase chain reaction (PCR), 462, Prolonged prothrombin time (PPT), due to vasculitis, 774 632, 1035–37 437 fulminans, 774 Polymerization, 272–73, 748 Prolymphocytes, 151, 626 mechanical, 774 Polymorphic, defined, 648 Promonocytes, 130 miscellaneous causes of, 774 Polymorphic variants, 1022 Promyelocytes, 116, 117, 119, 566 nonthrombocytopenic, 773 Polymorphism, 14, 654, 1111 Pronormoblasts, 68–71 post-transfusion, 781 Polymorphonuclear, 112 Prophase, 17 senile, 773 Polymorphonuclear neutrophil (PMN), Proplatelets, 168, 169 thrombotic thrombocytopenic, 782, 1115 Proteasome, 15 846 Polyploid, 167, 1020 Protein degradation, 15 P50 value, 299 Pooled normal plasma (PNP), 877 Protein folding, 14–15 Pyknotic nucleus, 459–60 Popcorn cells (L&H cells), 640 Proteins, 12 Pyridoxine therapy, 253 Porphobilinogen deaminase (PBGD), acarboxy, 738 Pyrimidine 5'-nucleotidase (P5N), 395 94 activated protein C resistance, 838 Pyruvate kinase (PK), 385 Porphyrias, 227, 248, 255–58 anion exchange, 74 deficiency, 393–94 Porphyrin, 90 Bence-Jones, 634 Positive feedback amplification, 755 C, 835–37, 888–89 Q Postoperative state, 853 C (PC), 757–59 Qualitative fluorescent spot test, Postoperative trauma, 853 composition, in erythrocyte 391–92 Post-transfusion purpura, 781 membrane, 74–76 Qualitative platelet disorders, 787–93 Post-translational modifications, 15 C-reactive, 830 Qualitative platelet evaluation, 170 Post-transplant lymphoproliferative integral, 74–75 Quality assessment, in hematology disorders (PTLD), 1088 in iron homeostasis, 234 laboratory, 1054–74 Pre-B cells, 145 peripheral, 74, 75 analytical reliability, 1062–63 Precursor neoplasms, 498 peripheral membrane, 75 Clinical Laboratory Improvement Pregnancy, iron requirements during, S, 837, 889 Amendments of 1988, 1056 238 S (PS), 757–59 competency assessment, 1060 Prekallikrein (PK), 740 Z, 760 examination component, 1057–58 screening test, 880 Protein tyrosine kinases (PTKs), 58 linear regression analysis, 1063 Index 1227 method evaluation/instrument destruction, 273 Rhodamine 123, 45 comparison, 1061–64 mature, 70 Ribonucleic acid (RNA), 13, 55, 251, 963 patient results, review of (See Red cell distribution width (RDW), Richter’s transformation, 627 Hematology results, review of) 180, 241, 586 Ring sideroblasts, 252, 566–67, 955 post-examination component, 1058 classification of anemia with, 218 Ristocetin, 873 pre-examination component, 1057 Red cell mass (RCM), 535 Ristocetin cofactor (RCo) assay, 881 proficiency testing, 1060 Red cell membrane skeleton, 75 Ristocetin-induced platelet reference interval determination, Red thrombi, 830 agglutination test (RIPA), 806 1064–65 Redundancy, 53, 1112 Rituximab (monoclonal anti-CD20), safety, 1065 Reed-Sternberg (R-S) cell, 640, 682 411 selection, 1061 Reference interval, 1148, 1151–52 RNA, 13 test coding and reimbursement, Reference intervals (RIs), 872, 921, RNA Processing, 14 1056 1064–65 Romanowsky-type stains, 169, 182, turnaround time, 1058 for common coagulation tests, 1080 191, 918 Quality control (QC), in hematology determination, 1064–65 ROTEM®, 886 laboratory, 1066–69 differentiate causes of anemiaa, 1081 Rough endoplasmic reticulum (RER), Bull’s testing algorithm (moving for iron analytes, 1080 153, 481 average), 1067–68 for leukocyte alkaline phosphatase Rouleaux, 183, 635, 919 charts, interpretation of, 1066–67 (LAP) scores, 1081 Routine hematology procedures, control materials, 1066 for leukocyte count and 917–27 correction for interfering differentials, 1079 Rubriblast, 69 substances, 1069–70, 1073 for normal osmotic fragility, 1081 Rule of three, 178, 923 individual quality control plan, for porphyrins and porphyrin 1068–69 precursors, 1081 S limits, establishing, 1066 for tests to monitor erythrocyte Safety data sheet (SDS), 1065 monitoring with patient samples, destruction, 1080 Safety, in hematology laboratory, 1065 1068 Reference plasma, 1141 Sandhoff disease, 468 Quantitation, of hemoglobin F, 929 Reflex hematology procedures, 927–39 Satellite DNA, 1016 Quantitative fibrinogen assay, 876–77 Reflex testing, 7, 113 Satellitism, 183 Quantitative platelet disorders, 775–87 Refractive index, 915 Scatterplots, 968 Quaternary structure of a protein, 15 Refractory anemia with ring Schistocytes, 76, 188, 190 Quebec platelet disorder, 791 sideroblasts (RARS), 249 Scott syndrome, 791 Quiescence (G0), 17 Refractory cytopenia of childhood, 570 Scramblase, 791 Regulatory T cells, 142 Screening tests, for secondary R Reimbursement, 1056 hemostasis, 875–77 Random variation, 1062 Reiter’s disease, 690 Scurvy, 773 Rapoport-Luebering shunt, 78, 79 Relapse, 617 Sea-blue histiocytosis syndrome, 468 Raynaud’s phenomenon, 412 Relative polycythemia, 539–40 Secondary graft failure, 656 RBC indices, 6 Relaxed (R) structure, 96 Secondary hemochromatosis, 255 RB gene, 502 Renal disease, 358–59 Secondary hemostasis, 5 Reactive chronic neutrophilia, 453 Reportable range, 1063 Secondary hemostasis plug, 706. See Reactive (secondary) eosinophilia, Reptilase time (RT), 879–80 also Hemostasis, secondary 464–65 Respiratory burst, 125 Secondary hypersplenism, 1112 Reactive lymphocytes, 153, 476 Respiratory burst oxidase, 123 Secondary lymphoid tissues, 30 Reactive lymphocytosis, 480–81 Restriction endonucleases, 1040 Secondary polycythemia, 539 Reactive oxygen species (ROS), 78, 303 Restriction point (R), 17 Secondary thrombocytosis, 786 Reactive thrombocytosis, 786 Reticular cells, 31 Segmented neutrophils, 116, 118 Reagent blank, 966 Reticulated platelets, 170, 776, 975 Semen, 695–8 Real-time polymerase chain reaction Reticulin, 506 Senile purpura, 773 (QPCR), 1037–39 Reticulin stain, 939 Sequestrian crisis, 275 Receptor editing, 146 Reticulocytes, 68, 80, 81, 1159 Serine proteases, 737 Receptor functional domains, 58 count, 180–81, 207–8, 926–27 Serologic tests, for infectious Receptor serine kinases (RSKs), 58 hemoglobin content, 209 mononucleosis, 478 Receptor tyrosine kinases (RTKs), 57, immaturity, quantitation of, 208–9 Serotonin release assay (SRA), 845 58 mean cell volume, 179 Serous fluids, 667–68 Recombinant FVIIa (rFVIIa), 790 production index (RPI), 208 Serpin, 753 Recombination-activating gene 1 stress, 208 Serum, 4 (Rag1), 143 Reverse transcriptase PCR (RT-PCR), Serum-ascites albumin gradient Recombination-activating gene 2 1039 (SAGG), 668 (Rag2), 143 Rheostat, 22 Serum/soluble transferrin receptors Recurrent genetic abnormalities, 591 Rh-HDFN, 1127 (sTfRs), 232 Recurrent genetic abnormality, 1133 Rh immunoglobulin (RhIG), 422 Severe combined immunodeficiency Red blood cells (RBCs), 3. See also Rh incompatibility, 421–22 syndrome (SCID), 57, 486–87, Erythrocytes Rh null disease, 375 624 1228 Index Sex-linked agammaglobulinemia, Siderocytes, 71, 193, 233 Stem cell transplantation (SCT). See 488–89 Sideropenic anemia, 227 Hematopoietic stem cell Sex-linked SCID, 486–87 Siderosomes, 233 transplantation Sex-linked sideroblastic anemia Siemens Healthcare ADVIA 120, Stimulated bone marrow states, 456 (XLSA), 249 984–86 Stimulated erythropoiesis, 342 Sézary cells, 639 Siemens Healthcare ADVIA 2120, 987 Stomatocytes, 188, 190 Sézary’s syndrome, 639–40 Signaling pathways, 58 Storage, 232–33 Shift reticulocytes, 208 Signal transducers, 501 Streptococcus pneumoniae, 37, 275, 434, Shift to the left, 454 Silent carrier, 1119 435 Shiga toxin–producing E. Coli Silent carrier a@thalassemia, 301 Streptokinase (SK), 752, 890 hemolytic uremic syndrome Single nucleotide polymorphism Stress erythrocytosis, 539 (Stec-Hus), 432–33 (SNP), 14 Stress erythropoiesis, 230 Shigella dysenteriae type 1, 847 Sjögren’s syndrome, 632, 842 Stress platelets, 169 Short-term repopulating cells (STRs), Skeletal protein abnormalities, 366–67 Stress reticulocytes, 208 45 Slope, 1062 Stroma, 31–32 Shwachman-Diamond Syndrome Small-cell carcinoma, 684–85 Stromal cell-derived factor-1 (SDF-1), (SDS), 352 Small lymphocytic lymphoma (SLL), 166 Sickle cell anemia, 271–79 626–28 Stromal cells, 61 acute chest syndrome, 275 Smudge cells, 626, 919 Structural aberrations, 1022 acute splenic sequestration, 275 Solubility Structural zone, 715–16 anemia, 273–74 hemoglobin S, 927 Subarachnoid hemorrhage (SAH), 668 bacterial infection, 275 test for hemoglobin, 277 Subcutaneous panniculitis-like T cell bone marrow, 276 Soluble transferrin receptor, 933–34 lymphoma, 639 clinical presentation, 273–75 Somatic mutation, oncogenes and, 504 Submetacentric chromosomes, 1016 hemoglobin electrophoresis, 276–77 Somatic recombination, 143 Substrate, 737 iron overload, 275 Southern blot, 1040 Sudan Black B (SBB), 934–35 irreversibly sickled cells, 273 Specific factor inhibitor assay, 884 Sulfhemoglobin, 105–6 laboratory evaluation, 276–77 Specimens, collecting and processing, Supernatants, 931 oxygen affinity of HbS, 272 869–71, 1024 Supravital stain, 926 pathophysiology, 271–73 Spectrin, 76 Surface folate receptor@a, 329 peripheral blood, 276 Spent phase, 537 Surface markers, leukocyte, 114 RBC destruction, 273 Sperm, 696 Survival defects in functional sickle cell trait, 278 count, 696 classification of anemia, 215–18 sickling test, 277 vitality, 696 Synergism, 53 solubility and polymerization of S phase, 17 Syngeneic, 647 hemoglobin, 272–73 fraction, 1008 Synovial fluid, 670, 674–75 solubility test, 277 Spherical aberration, 915 Synovium, 670 therapy, 277–78 Spherocytes, 188, 190 Syringes, 912 vaso-occlusion, 273 Spleen, 35–38 Sysmex Automated Hematology vaso-occlusive crisis, 274–75 architecture, 35 Analyzers, 209 Sickle cell disease, 101, 278–79 blood flow, 35–36 Sysmex XE-series, 971–77 Sickle cell disorder, 278–79 function, 36–37 Sysmex XE-Series® instrument, 1145 Sickle cells, 188. See also Drepanocytes hypersplenism, 37 Sysmex XN-series, 977–80 Sickle cell trait, 278, 1120 splenectomy, 37–38 Sysmex XN-Series® instrument, 1145 Side light scatter, 996 Splenectomy, 37–38, 411 Systematic variation, 1062 Sideroblastic anemia (SA), 227 Splenic sequestration, increased, 784 Systemic lupus erythrematosus (SLE), bone marrow, 252 Split specimens, 1062 542 clinical presentation, 251 Spoke wheel arrangement, 154 Systemic mastocytosis (SM), 548, 549 d@aminolevulinic acid synthase Spur cell anemia, 341, 376, 1125 enzyme, 249 Spur cells, 187 T erythroid form of, 249 Spurious, 771 Target cells, 187–88, 1117 laboratory evaluation, 251–52 Spurious polycythemia, 539 Tartrate-resistant acid phosphatase molecular studies, 252 Stage, 626 (TRAP), 937–38 peripheral blood, 251–52 Standard deviation (SD), 1062 Tay-Sachs disease, 468 sex-linked, 249 “Starry sky” appearance, 634 T cell acute lymphoblastic leukemia therapy, 253 Stem cell disorders, 456–57. See also (T-cell ALL), 609 Sideroblastic anemias (SAs), 248–52 Hematopoietic stem cells T-cell large granular lymphocytic acquired, 249–52 (See Acquired (HSCs) (T-LGL) leukemia, 639 sideroblastic anemia) Stem cell factors (SCFs), 52, 54–55, 128 T-cell prolymphocytic leukemia classification, 248–49 Stem cell inhibitor (SCI), 57 (T-PLL), 639 hereditary, 249 Stem cells, 44 T cell receptor (TCR), 141, 148 nonerythroid form of, 249 compartment, 45–46 T-cell receptor (TCR), 484 pathophysiology, 249–51 niches, 46–48, 61–62 Teardrops, 188. See Dacryocytes Sideroblasts, 71, 193 phenotype, 45 Technologies, molecular, 1036–43 Index 1229 Telomerase reverse transcriptase Thermal injury, 439 Thrombotic thrombocytopenic (TERT), 349 The WHO Laboratory Manual for the purpura (TTP), 434, 436–37, Telomerase RNA template (TERC), 349 Examination and Processing of 782, 846 Telophase, 17 Human Semen, 695 Thrombus, 828 Tense (T) structure, 96 Threshold limits, 964 formation, 829–32 Terminal deoxynucleotidyl transferase Thrombin Thymidine triphosphate (TTP), 1037 (TdT), 143, 611, 681, 938 generation, 745–46 Thymocytes, 147 Terminally differentiated cells, generation test (TGT), 887 Thymus, 34–35 44, 1112 time (TT), 437, 830, 876, 894 Tingible body macrophages, 629 Test coding and reimbursement, Thrombin-activatable fibrinolysis Tissue destruction/injury, 1056 inhibitor (TAFI), 746, inflammation, metabolic Thalassemia, 289–315, 1045, 1120 754, 890 disorders, associated with b-thalassemia,301–8 Thrombocytes, 3 neutrophilia, 455 clinical presentation, 294–95, 306 Thrombocythemia, 534 Tissue factor pathway, 759–61 combination disorders, 311–13 Thrombocytopenia, 169, 170, 775–85, Tissue factor pathway inhibitor db, 308 1138 (TFPI), 755, 759 differential diagnosis of, 313–14 autoimmune, 777–78 variant, 839 gdb, 308 conditions with multiple Tissue homeostasis, 15–23 genetic defects in, 293 mechanisms of, 784–85 defined, 11 Hemoglobin Constant Spring, 292, dilutional,

784 differentiation, 19–20 308–9 drug-induced, 781 proliferation, 16–19 Hemoglobin Lepore, 292, 310–11 heparin-associated, 781 Tissue homeostasis, 43 hemoglobinopathies versus, heparin-induced, 781, 844–46 Tissue hypoxia, in secondary 267, 292 hereditary, 783 polycythemia, 539 hereditary persistence of fetal immune, 777–78 Tissue, neutrophil, 120 hemoglobin, 309–10 neonatal alloimmune, 780 Tissue plasminogen activator (tPA), laboratory evaluation, 295, 306 platelets, increased destruction of, 841, 890 other thalassemias, characteristics 776–82 Tissue-type plasminogen activator of, 309 splenic sequestration, increased, (tPA), 740, 751 pathophysiology, 293–94, 306 784 T lymphoblastic lymphoma (T-LBL), 615 a@thalassemia trait, 296–301 Thrombocytopenia with absent radii T lymphocyte, natural killer cell treatment, 295–96, 306–7 (TAR), 783 progenitor (TNKP), 48 types of, 292–93 Thrombocytosis, 534, 785–86 T lymphocytes, 140, 147–50, 156–58 Thalassemia, alpha (a), 296–301 causes of, 786 antigen receptor, 148 affected alleles, 296–97 primary, 785 cytotoxic, 23 affected individuals, 297 reactive, 786 developmental stages, 148–49 characteristics of, 297 secondary, 786 major histocompatibility complex, clinical presentation, 298 Thromboelastography (TEG), 886 147–48 etiology, 296 Thromboembolic disease, 841 membrane markers, 148 genotypes, 297 Thromboembolism (TE), 829 natural killer cell, 150 hemoglobin H disease, 297–300 Thrombogenic blood vessels, 710 subclasses, 149–50 laboratory results, 298 Thrombolytic therapy, 854, 857–58 T lymphoid cell antigens, 588 major (hydrops fetalis), 298 Thrombomodulin (TM), 758 TMARSS6 gene, 247 minor, 300–301 Thrombophilia, 826–59 T/myeloid leukemia, 616 pathophysiology, 298 acquired, 841–53 Toluidine blue, 938 silent carrier, 301 anticoagulant therapy, 851–58 Topoisomerase II inhibitors, 558 Thalassemia, beta (b), 301–8 defined, 828, 832 Total blood neutrophil pool, 120 affected individuals, 303 genetic risk factors, 839–41 Total iron-binding capacity (TIBC), characteristics of, 302 hereditary, 833–39 230, 238, 565 clinical presentation, 304 thrombosis, laboratory testing Touch imprints, 952 genetics, 301–2 in patients with suspected, Tourniquet, 912 intermedia, 307 853–54 Toxic granules, 460, 461 laboratory evaluation, 304–5 thrombus formation, 829–32 Toxoplasma gondii, 479 major, 303–6 Thrombophlebitis, 830 Toxoplasmosis, 479 minima, 307–8 Thrombopoiesis, 56, 169 Trabeculae, 30 minor, 306–7 Thrombopoietin (TPO), 56, 165–66, Transcobalamin, 1123 pathophysiology, 303–4 522, 712, 1153 Transcobalamin I, II, or III, 332, 333 treatment, 305–6 Thrombopoietin (TPO) receptor, 45 Transcription, 1037 Therapeutic plasma exchange, 411 Thromboregulation, 709 errors, 1058 Therapeutic ranges, 892 Thrombosis, 706, 828, 853–54, 890 factors (TFs), 14, 58–59, 501 Therapy-related myelodysplasia Thrombotic microangiopathic anemia Transcription-ready status, 19 (t-MDS), 571 (TMA), 431–39 Transferrin (T), 72, 230 Therapy-related myeloid disorders, Thrombotic microangiopathies receptor, 231 594, 1027 (TMA), 846–52 saturation, 230 1230 Index Transferrin receptor 1 (TfR1), 231 Vasculature, of bone marrow, 31 Wedge smear, 182, 917 Transferrin receptor 2 (TfR2), 231 Vasculitis, 774 Westgard Rules, 1066–67 Transforming growth Vasoconstriction, 755 White blood cells (WBCs), 3, 979. See factor@b (TGF - b), 56, 166 Vaso-occlusion, sickle cell anemia, 273 also Leukocytes Transglutaminase, 737 Vaso-occlusive crisis, 274–75 morphologic abnormalities of, 185 Transient erythroblastopenia of Venipuncture, 912–13 and platelets, 183–84 childhood (TEC), 356–58 Venous thrombi, 830–31 White thrombi, 829 Translational regulation, 20 microparticles in, 831–32 WHO classification, 587 Translocations, 1035 Venous thromboembolism (VTE), 830, acute leukemia, 1087 Transplant-associated microangiopathy 887 for acute leukemias, 1004 (TA-TAM), 439 VERIFYNOW accumetrics assay for acute lymphoblastic leukemias, Transudate, 668 system, 872 612–17 Triacidic stain, discovery of, 112 Vertical interactions, 366 acute myeloid leukemia (AML), Triosephosphateisomerase (TPI) Viral infection, oncogenes and, 504 1086–87 deficiency, 395 Viral load, 485 for acute myeloid leukemias, Trisomy, 1020 Visceral membrane, 667 591–601, 1026 Tropomodulin, 76 Viscosity, 971 for hematopoietic and Tropomyosin, 76 Vitamin B12, 322–23, 331–38 lymphopoietic neoplasms, Tube holders, 912 deficiency, 333–38 498 Tumor necrosis factor (TNF), 20, 68, metabolism, 332–33 histiocytic and dendritic cell 119, 349 requirements, 333 neoplasms, 1088 Tumor suppressor genes, 23, 502, 560 structure and function, 331–32 for HIV, 484 Turbidimetric clot detection, 896 Vitamin K deficiency bleeding Hodgkin lymphoma, 1088 Turnaround time (TAT), 909, 1058 (VKDB), 817 for lymphoid malignancies, 626 Twisted gastrulation protein homolog Vitamin K-dependent clotting factors, for mast cell disease, 549 1 (TWSG1), 235, 236 816 mature B-cell neoplasms, 1087–88 2,3-bisphosphoglycerate (2,3 BPG), 98 Vitamin K-dependent coagulation mature myeloid neoplasms, 1086 2,3-diphosphoglycerate (2,3-DPG), 204 proteins, 735, 737–38 mature T and NK cell neoplasms, Tyrosine kinase inhibitors (TKIs), 528, VKORC1, 1049 1088 1025 von Willebrand disease (VWD), 789, for myelodysplastic syndromes, 802–7, 1049 558, 568, 570, 572 U classification, 803 for myeloproliferative disorders, 519 Ubiquitin, 15 clinical presentation, 803–4 for myeloproliferative neoplasms, 520 Ubiquitin proteasome system, 15 collagen-binding assays for, 882 for neoplasms, 507 Ultralarge VWF multimers (ULVWF), inheritance characteristics, 803 for oral anticoagulant therapy, 892 744 laboratory evaluation, 804–6 precursor lymphoid neoplasms, Umbilical cord blood, 651 prenatal diagnosis, 807 1087 Umbilical cord stem cell related clinical syndromes, 806–7 PTLD, 1088 transplantation, 650 therapy for, 807 Whole blood (WB) aggregation, Under-anticoagulation/ von Willebrand factor, 802–3 873–74 overanticoagulation, 1073 von Willebrand factor (vWF), 436, 522, Williams factor, 816 Unfractionated heparin (UFH), 845, 533, 537, 710, 742–44, 802–3 Wiskott-Aldrich syndrome (WAS), 893 activity, 880 487–88, 624, 784 Unique identification number, 912 antigen (VWF:Ag), 803 Wolman’s disease, 468 Universal precautions, 1065 antigen assay, 881–82 World Health Organization (WHO), Unsaturated iron-binding capacity laboratory tests for, 804–6, 880–82 389 (UIBC), 230–31 multimers, 805 Wright-Giemsa stain, 299, 300, 305, Untranslated regions (UTRs), 14 ristocetin cofactor activity 311, 313, 323, 325, 326, 919 uPA receptor (uPAR), 752 (VWF:RCo), 805, 881 Upshaw-Shulman syndrome, 846 von Willebrand Factor Activity X Urinary-type plasminogen activator (VWF:A), 880 X chromatin body (Barr body), 118 (uPA), 740, 752 von Willebrand factor multiplex, 806 X-linked lymphoproliferative disorder Urokinase (UK), 751 von Willebrand factor multiplex (XLP), 477 Uropod, 151 activity assay, 806 X-linked recessive disorders, 807–12 VWF antigen assay (VWF:Ag), 881–82 X-linked thrombocytopenia (XLT), 783 V VWF multimer analysis, 806 X-linked thrombocytopenia with Vascular endothelial cells (VECs), 120, VWF multimeter analysis, 882 thalassemia (XLTT), 783 122 Vascular endothelial growth factor W Y (VEGF), 716 Waldenström macroglobulinemia, 633 y-intercept, 1062 Vascular niche, 47, 166 Warfarin/Coumadin, 738 Vascular system Warm autoimmune hemolytic anemia Z disorders of, 772–73 (WAIHA), 402, 408–11, 1126 Zygosity state, 297 primary hemostasis, 711–26 WBC differential, 113, 181 Zymogens, 734

Current Topics in Behavioral Neurosciences Series Editors: Mark Geyer, La Jolla, CA, USA Bart Ellenbroek, Wellington, New Zealand Charles Marsden, Nottingham, UK About this series Current Topics in Behavioral Neurosciences provides critical and comprehensive discussions of the most significant areas of behavioral neuroscience research, written by leading international authorities. Each volume offers an informative and contemporary account of its subject, making it an unrivalled reference source. Titles in this series are available in both print and electronic formats. With the development of new methodologies for brain imaging, genetic and genomic analyses, molecular engineering of mutant animals, novel routes for drug delivery, and sophisticated cross-species behavioral assessments, it is now possible to study behavior relevant to psychiatric and neurological diseases and disorders on the physiological level. The Behavioral Neurosciences series focuses on ‘‘transla- tional medicine’’ and cutting-edge technologies. Preclinical and clinical trials for the development of new diagnostics and therapeutics as well as prevention efforts are covered whenever possible. . Jo C. Neill l Jayashri Kulkarni Editors Biological Basis of Sex Differences in Psychopharmacology Editors Prof. Dr. Jo C. Neill Prof. Dr. Jayashri Kulkarni School of Pharmacy Monash Alfred Psychiatry Research Centre University of Bradford The Alfred Hospital and Monash University Bradford BD7 1DP Commercial Road United Kingdom Prahran, Victoria j.c.neill@bradford.ac.uk Australia j.kulkarni@alfred.org.au ISSN 1866-3370 e-ISSN 1866-3389 ISBN 978-3-642-20005-2 e-ISBN 978-3-642-20006-9 DOI 10.1007/978-3-642-20006-9 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2011931783 # Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Cover illustration: Artistic representation of oscillatory synchrony and timing of neurons in networks by Gyorgy Buzsaki Cover design: deblik, Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Preface This volume attempts to answer the question of how sex influences brain and behaviour. It brings together experts in this field, psychiatrists and other mental health care professionals with preclinical researchers to review the latest work in this area and give a thorough overview of how males and females are different in terms of brain function and behaviour. This is followed by a clinical perspective, applying brain biology to explain why some illnesses are gender specific and how gonadal steroids are involved in the aetiology and symptomatology of psychiatric diseases and may be modulated to provide new therapeutic approaches to mental illnesses. Appreciation and improved understanding of sex differences will certain- ly lead to improvements in the diagnosis, tailored treatment approaches and hence outcomes for people suffering with mental disorders. This volume will be essential reading for all health care professionals. Animal behavioural models of efficacy studies in pharmacology generally use male rodents. However, the presentation of sex differences in behaviour under- scores the need to include female animals in basic research studies. Similarly, gender differences in the presentation, treatment and outcomes of mental illnesses are often overlooked. The biological basis of sex differences and its application in clinical disorders are important issues that are highlighted by the research discussed in this volume. The volume is divided into two sections, the first deals with the importance of recognising and studying sex differences in brain function and behaviour in animal models. The second section is dedicated to the consideration of biological sex differences in the presentation of aspects of mental illnesses such as schizophrenia, bipolar disorder, depression and anorexia nervosa. The two sections are inter- related and provide an integrated approach with animal research informing the human application in considering the biological basis of sex differences in psycho- pharmacology. The volume starts with an overview by Professor Kay Marshall (a reproductive endocrinologist who has been persuaded to study the brain!) This chapter gives an important introduction into the mechanisms by which gonadal steroids produce their effects which explains how sex differences come about. The second chapter by Berend Olivier and colleagues explores how sex matters for rats. They provide an v vi Preface overview of rodent sexual behaviour and how to measure this in the laboratory. The chapter has a particular emphasis on the role of serotonin (5-HT) on sexual activity and on sexual dimorphism in response to serotonergic agents. This is clearly of much relevance as drugs such as SSRIs cause sexual dysfunction in the clinic, and it is essential to model this appropriately in the laboratory. In a later clinical chapter on the impact of sex on antidepressants, John Sramek and colleagues detail clinical trial work on this important area. An investigation into sex differences and the effect of gonadal steroids on cognitive function in rodents is provided by Jane Sutcliffe, which is of particular importance as cognitive dysfunction occurs in many psychiatric illnesses such as depression, ADHD and PTSD. In schizophrenia most notably this remains an unmet clinical need, with emphasis on the development of new therapies for cognitive and other symptoms of this illness. Implications for the aetiology of schizophrenia are explored by Veena Kumari in her chapter dealing with human sensorimotor gating. Chapter 9 written by Anita Riecher-Rossler and Jayashri Kulkarni, and Chapter 10 by Angelika Wieck present the very important role that oestrogen plays as a key neuroprotective agent, and its impact on the timing and gender differences in illness presentation. The possibility of using hormone modulation as a new treatment approach is also discussed with respect to psychotic disorders. Chapter 4 by Dai Mitsushima illustrates that in rodents neurotransmitters show sexual dimorphism, and that neurotransmitter release is affected by gonadectomy with a focus on acetylcholine, again of particular importance for cognition. A subsequent chapter by Justin Anker and Marilyn Carroll deals with the very important topic of drug dependence. They show that females are more sensitive than males to the reinforcing effects, and less sensitive to withdrawal effects, of certain drugs of abuse, making them more vulnerable to drug dependence which can be effectively modelled in animals. The translation from animals to humans here is impressive with female rats showing greater propensity for drug self- administration and relapse in animal models. The authors go on to demonstrate how these effects in females may be mediated by gonadal steroid hormones, in particular oestrogen and progesterone (which is important, as it is not all about oestrogen, as Kay Marshall explains in her opening chapter). They discuss possible mecha- nisms including oestrogen receptors and their interaction with the mesolimbic dopamine system with the emphasis on addiction to psychostimulants such as cocaine. Applying this framework, a novel proposal for the noted sex differences in anorexia nervosa is described in a chapter by Charlotte Keating, with applicability for new thinking about the aetiology of this severe and female dominant eating disorder. Stress is of course an important feature of human lives, including our response to drugs of abuse and Christina Dalla and her colleagues cover this topic in some depth in their chapter. Men and women differ in their vulnerability to stress and stress- related psychiatric disorders such as depression. The authors explore sex differ- ences in the response to acute and chronic stress in several animal models in some detail. Male and female rodents differ in their reactivity and adaptation to various stressors, and the authors demonstrate the link between this and differences in the Preface vii neuroendocrine system and its interaction with neurotransmitter systems, such as serotonin and dopamine. The final preclinical chapter provides an elegant review by Elizabeth Tunbridge and Paul Harrison into sex differences in the catechol-O- methyltransferase (COMT) gene. The gene encodes an enzyme that metabolises catechol compounds including dopamine, and the authors explain how sexual dimorphism in this gene and its interaction with oestrogen impacts on psychiatric disease states. Many of the preclinical studies suggest that sex-specific interven- tions may be a beneficial approach when treating patients, and understanding the sex differences in developmental disorders experienced early in life is an important area detailed by Bruce Tonge and colleagues in a clinical chapter that also proposes treatment strategies for early psychiatric presentations. In summary, sex differences are observed in humans and animals in brain function and behaviour and in the response to illness. Men and women have different advantages in many aspects of behaviour particularly cognitive function which is a key component of many psychiatric disorders. Indeed, there is both clinical and preclinical evidence to support a role for the sex steroids in modulating performance in certain cognitive domains. At present, these interactions are com- plex and the underlying mechanisms have yet to be elucidated. Once these relation- ships are understood, there is potential for more effective therapeutic exploitation. This volume covers in some depth many illnesses in which sex differences and gonadal steroids are important in terms of aetiology, symptomatology, progression and treatment. It is the first volume to successfully achieve this and will be of considerable importance to workers in all aspects of mental illness. Bradford, UK Jo C. Neill Melbourne, Australia Jayashri Kulkarni . Contents Introduction to the Interaction Between Gonadal Steroids and the Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Kay M. Marshall Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Berend Olivier, Johnny S.W. Chan, Eelke M. Snoeren, Jocelien D.A. Olivier, Jan G. Veening, Christiaan H. Vinkers, Marcel D. Waldinger, and Ronald S. Oosting Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor Subtypes and Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Jane Suzanne Sutcliffe Sex Differences in the Septo-Hippocampal Cholinergic System in Rats: Behavioral Consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Dai Mitsushima Females Are More Vulnerable to Drug Abuse than Males: Evidence from Preclinical Studies and the Role of Ovarian Hormones . . . . . . . . . . . . . . . 73 Justin J. Anker and Marilyn E. Carroll Sex Differences in Response to Stress and Expression of Depressive-Like Behaviours in the Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Christina Dalla, Pothitos M. Pitychoutis, Nikolaos Kokras, and Zeta Papadopoulou-Daifoti Importance of the COMT Gene for Sex Differences in Brain Function and Predisposition to Psychiatric Disorders . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . 119 Elizabeth M. Tunbridge and Paul J. Harrison ix x Contents Sex Differences and Hormonal Influences in Human Sensorimotor Gating: Implications for Schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Veena Kumari Estrogens and Gonadal Function in Schizophrenia and Related Psychoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Anita Riecher-Rössler and Jayashri Kulkarni Oestradiol and Psychosis: Clinical Findings and Biological Mechanisms . . 173 Angelika Wieck Sex Differences Precipitating Anorexia Nervosa in Females: The Estrogen Paradox and a Novel Framework for Targeting Sex-Specific Neurocircuits and Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Charlotte Keating Gender Differences in Neurodevelopmental Disorders: Autism and Fragile X Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Nicole J. Rinehart, Kim M. Cornish, and Bruce J. Tonge The Impact of Gender on Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 John J. Sramek and Neal R. Cutler Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Contributors Justin J. Anker Department of Psychiatry, University of Minnesota, MMC 392, MN 55455 Minneapolis, USA, anke0022@umn.edu Marilyn E. Carroll Department of Psychiatry, University of Minnesota, MMC 392, Minneapolis, MN 55455 USA, anke0022@umn.edu Johnny S. W. Chan Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, j.s.w.chan@uu.nl Kim M. Cornish Centre for Developmental Psychiatry and Psychology, School of Psychology and Psychiatry, Faculty of Medicine, Nursing and Health Sciences, Melbourne, VIC, Australia Neal R. Cutler Worldwide Clinical Trials, Inc., 401 N. Maple Drive, Beverly Hills, CA 90210 USA, neal.cutler@wwctrials.com Christina Dalla Department of Pharmacology, Medical School, University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece, cdalla@med.uoa.gr Paul J. Harrison Department of Psychiatry, University of Oxford, Neurosciences Building, Warneford Hospital, Oxford, OX3 7JX, UK, paul.harrison@psych.ox.ac.uk xi xii Contributors Charlotte Keating Monash Alfred Psychiatry Research Centre (MAPrc), The Alfred Hospital, 1st floor, Old Baker Building, Commercial Road, Prahran, VIC 3181 Australia, char- lottekeating1@gmail.com Nikolaos Kokras Department of Pharmacology, Medical School, University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece, NKokras@med.uoa.gr Jayashri Kulkarni Monash Alfred Psychiatry Research Centre, The Alfred Hospital and School of Psychology and Psychiatry, Monash University, Commercial Road, Melbourne, VIC 3004 Australia, jayashri.kulkarni@med.monash.edu.au Veena Kumari Department of Psychology, Institute of Psychiatry, King’s College London, De Crespigny Park, London SE5 8AF, UK, veena.kumari@kcl.ac.uk Kay M. Marshall School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK, k.m. marshall@bradford.ac.uk Dai Mitsushima Department of Physiology, Yokohama City University Graduate School of Med- icine, 3-9 Fukuura Kanazawaku, Yokohama 236-0004, Japan, dm650314@med. yokohama-cu.ac.jp Berend Olivier Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, b.olivier@uu.nl Jocelien D. A. Olivier Department Clinical Neuroscience, Division of Psychiatry, Karolinska Institu- tet, KFC Novum Level 6, Exp 617 Lab 614, 14157 Huddinge, Sweden, jocelien. olivier@ki.se Ronald S. Oosting Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, r.s.oosting@uu.nl Contributors xiii Zeta Papadopoulou-Daifoti Department of Pharmacology, Medical School, University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece, zdaifoti@med.uoa.gr Pothitos M. Pitychoutis Department of Pharmacology, Medical School, University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece, PPitychoutis@med.uoa.gr Anita Riecher-Rössler Psychiatric University Clinic Basel, University Psychiatric Outpatient Depart- ment, c/o University Hospital Basel, Petersgraben 4, Basel, 4031 Switzerland, anita.riecher@upkbs.ch Nicole J. Rinehart Centre for Developmental Psychiatry and Psychology, School of Psychology and Psychology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, VIC Australia Eelke M. Snoeren Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, e.m.snoeren@uu.nl John J. Sramek Worldwide Clinical Trials, Inc., 401 N. Maple Drive, Beverly Hills, CA 90210 USA, jsramek@wwctrials.com Jane Suzanne Sutcliffe Maccine Pte Ltd, 10, Science Park Road, 01-05 The Alpha, Singapore Science Park II, Singapore 117684 Singapore, Jane.Sutcliffe@Maccine.com Bruce J. Tonge Centre for Developmental Psychiatry and Psychology, School of Psychology and Psychology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, VIC, Australia, bruce.tonge@monash.edu Elizabeth M. Tunbridge Department of Psychiatry, University of Oxford, Neurosciences Building, Warneford Hospital, Oxford, OX3 7JX, UK, elizabeth.tunbridge@psych.ox.ac.uk xiv Contributors Jan G. Veening Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, j.g.veening@uu.nl Christiaan H. Vinkers Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, c.h.vinkers@uu.nl Marcel D. Waldinger Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, md@demon.waldinger.nl Angelika Wieck Laureate House, Wythenshawe Hospital, Manchester Mental Health and Social Care Trust, University of Manchester, Southmoor Road, Manchester M239LT, UK, angelika.wieck@manchester.ac.uk Introduction to the Interaction Between Gonadal Steroids and the Central Nervous System Kay M. Marshall Contents 1 Hormone Levels in the CNS and Role of Aromatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.1 Oestrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.2 Progesterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.3 Androgen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Abstract The sex steroids are frequently referred to as the gonadal steroids and are erroneously assumed to be exclusively linked to the ovaries in women or the testes in men and the functions of the reproductive tract. This chapter will provide an overview of some of the extragonadal effects of these hormones, focusing on the central nervous system, and the mechanisms of hormone action. Hormone synthesis and metabolism within the CNS will be discussed with particular focus on the role of aromatase. Sex steroids exert many of their effects via intracellular receptors and these genomic responses tend to be slow in onset, however, some responses to steroids occur more quickly and are mediated via membrane receptors and involve interactions with many different transduction pathways to produce a diverse array of responses. These complexities do pose challenges but also offer opportunity for novel approaches for therapeutic exploitation as the pharmacological tools with which to modulate systems become increasingly available. Keywords Androgen
Mechanism of action
Oestrogen
Progesterone K.M. Marshall School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK e-mail: k.m.marshall@bradford.ac.uk J.C. Neill and J. Kulkarni (eds.), Biological Basis of Sex Differences in Psychopharmacology, 1 Current Topics in Behavioral Neurosciences 8, DOI 10.1007/7854_2011_136 # Springer‐Verlag Berlin Heidelberg 2011, published online 5 June 2011 2 K.M. Marshall The sex steroids are commonly referred to as the gonadal steroids and are erroneously assumed to be exclusively linked to the ovaries inwomen or to the testes inmen and to the functions of the reproductive tract (Chabbert Buffet et al. 1998; Genazzani et al. 2002; Mooradian et al. 1987). The sex steroids themselves are not gender specific; for example, testosterone has several key functions inwomen; testosterone imbalance has been linked to depression in women (Rohr 2002) and oestrogen promotes hippocam- pal neurogenesis inmale rats (Bowers et al. 2010). These steroids can be synthesised at extragonadal sites and have effects that are outside those obviously linked with reproductive processes. This chapter will provide an overview of some of the extra- gonadal effects of these hormones, focusing on the central nervous system (CNS), and themechanisms of hormone action. The examples citedwill be somewhat oestrogeno- centric as this reflects the perhaps disproportionate amount of research time spent on oestrogen (a simple Medline search for 2010 suggests that twice as many papers were published on oestrogen than on testosterone). However, progesterone and testosterone and their related steroidal products should not be overlooked as it is often the balance of these hormones and/or their metabolites with one another that will determine the

overall response. For example, progesterone can reverse the effects of oestrogen on dendritic spine density and on brain-derived neurotrophic factor (Murphy and Segal 2000; Aguirre et al. 2010). In addition, all responses mediated by sex steroids will in turn be influenced by chronobiology beginning in utero (Pilgrim and Hutchison 1994) and including the lifting of the hypothalamic–pituitary block that facilitates the onset of puberty. The reactivation of the hypothalamic gonadotrophin-releasing hormone (GnRH) secretory system (which can be stimulated by noradrenaline and glutamate and inhibited by GABA) results in the establishment of reproductive rhythms. In women, these can be disrupted by pregnancy, where the magnitude of the hormonal changes far exceed those occurring during the menstrual cycle (for detailed review, see Brunton and Russell 2010), and end at menopause or in the male, the increasingly recognised, although less clearly demarked andropause (Keenan and Veldhuis 2009). The timing of these biological life events has considerable impact on, and importance for, long-term health. 1 Hormone Levels in the CNS and Role of Aromatase For a recent review of steroid hormone biosynthesis, see Gilep et al. (2011). The extent to which peripheral levels of either endogenous or exogenous circulating hormones reflect levels in the CNS is still subject to scrutiny. It is known that the sex steroids can be synthesised in the brain; for example, progesterone is synthe- sised by glial cells (Garcia-Segura and Melcangi 2006). A recent study by Caruso et al. (2010) measured levels of sex steroids in plasma and in the CNS (cerebellum, cerebral cortex and spinal cord) in intact and gonadectomised male and female rats and found that after gonadectomy, changes in the CNS did not necessarily reflect the situation in the plasma. In addition to this, there is the further complication of identifying which hormone is the active moiety. For example, there are several forms of endogenous oestrogen namely: 17b-estradiol (E2), which is the most Introduction to the Interaction Between Gonadal Steroids and the Central Nervous System 3 active and has the highest receptor affinity; estrone (E1), which is a less active product of oxidation of E2; estriol (E3), which can be produced from either of the former or from the androgen androstenedione. E3 is produced in abundance during pregnancy [it has been postulated that this happens to protect the developing CNS in the foetus (Reyes-Romero 2001)] but after the menopause levels do not really change and are similar to those found in men. 17a-estradiol, another form of endogenous oestrogen, has lower receptor affinity, but it is known to be synthesised locally in rodent brain (Simpkins et al. 1997; Levin-Allerhand et al. 2002). How- ever, Nguyen et al. (2011) have recently reported measuring all of these oestrogens after derivitisation (using liquid chromatography separation, electrospray ionisation and tandem mass spectrometry, ESI-MS/MS) in human cerebrospinal fluid taken from trauma patients and E2 was found to predominate. This may represent assay or sample limitation; alternatively it could reflect another species difference and steroidogenesis does differ between species (Gilep et al. 2011). The synthetic pathways and resultant products (including metabolites, some of which retain biological activity) are determined by the types of steroidogenic cytochromeP450s (CYPs) and the dehydrogenase enzymes. In addition to inter-species variation, there is also some intra-species variation as the CYPs are subject to polymorphisms which may influence hormone as well as drug metabolism. Hormone metabolism may also explain some of the apparently paradoxical responses observed; for example, when the endogenous metabolite 2-methoxyestradiol is formed, it may possess some of the antioxidant properties of E2 but unlike E2 it cannot protect hippocampal neurones from insult by kainic acid (Picazo et al. 2003). Furthermore, E2 metabolism can lead to redox cycling and free radical formation and neural damage can ensue (Liehr and Roy 1990; Picazo et al. 2003). In terms of variation between peripheral versus central steroid levels, dehydroe- pianstrosterone (DHEA) is a good example as DHEA can be detected in brain tissue of many species, including humans, at levels that exceed those in the periphery (Baulieu and Robel 1996; Tunbridge and Harrison 2011). DHEA is a substrate common to both oestrogen and androgen biosynthesis, and it appears that astrocytes from different brain areas can metabolise DHEA differentially; for example, hypo- thalamic cells are more active in producing E2 than similar cells from the cerebral cortex (Zwain and Yen 1999). However, there are some metabolic consistencies, as characterisation of the 5a-reductase-3a-hydroxysteroid dehydrogenase complex (which is key to androgen metabolism) in human brain samples has indicated no sex-specific differences and no differences over time (Steckelbroeck et al. 2001), which may suggest a non-reproductive role such as catabolism of neurotoxic steroids. Oestrogens can also be metabolised in the brain as indicated by high levels of 2- and 4-hydroxyoestrogens (the catecholoestrogens) which are metabolised further by catecholeamine-O-methyltransferases, an enzyme which itself is subject to oestrogenic influence and polymorphisms (Harrison and Tunbridge 2008). The role of cytochrome P450 aromatase which is the protein product of gene Cyp 19 also needs to be taken into account when considering the level of steroid hormones in the brain. This enzyme is responsible for converting androgens (C19 products) to oestrogens (C18 products), namely testosterone to E2 and 4 K.M. Marshall androstenedione to E1; thus it plays a key role in regulating the androgen–oestrogen balance. Aromatase is present at extragonadal sites including the breast (where its role and inhibition, using aromatase inhibitors or AIs, have been most extensively studied; for more details, see Furr 2006) and the brain where it appears to be concentrated in the preoptic area, ventro-medial hypothalamus and the bed nucleus striae terminalis (Balthazart et al. 2003). In terms of clinical relevance, an example would be the expression of aromatase by astrocytes following injury (Garcia- Segura et al. 2003). Aromatase is subject to different regulatory controls; for example, in the gonads cAMP is stimulatory but in neural tissues it is inhibitory (Lephart 1996). Interestingly in rat brain, androgen is not only the substrate for aromatase, but dihydrotestosterone can, like oestradiol, also induce its activity (Roselli and Resko 1993). This regulation can be slow (as would be expected if control is via genomic mechanisms) or fast (suggesting non-genomic mechanisms). The presence of phosphorylation consensus sites on the enzymewould correlate with the latter mechanism (Balthazart and Ball 2006). Neurotransmitters such as dopamine and glutamate can inhibit the activity of aromatase as can kainate and NMDA (Balthazart et al. 2003). Other considerations when attempting to correlate levels of sex steroids with function could arise from the fact that only free, that is hormone unbound to plasma proteins such as albumin or more specifically in the case of the sex hormones, sex hormone-binding globulin (SHBG), hormone is active (the presence or absence of this globulin should also be considered when using animal models and extrapolat- ing to the human). However, SHBG seems to have biological activity via a membrane receptor coupled to a cAMP pathway (Nakhla et al. 2009), and it may even be a marker for the integrity of the blood–brain barrier (Gustafson et al. 2007). 2 Mechanisms of Action Sex steroids can act as ligand-activated transcription factors when they exert their effects via a genomic mechanism, and the response is slow in onset to allow for the eventual translation of message (see Fig. 1). 2.1 Oestrogen In the case of oestrogen, these receptors are known as ERa and ERb (Green et al. 1986; Kuiper et al. 1996), and for progesterone there are also two forms namely PR-A and PR-B. These receptors are part of the nuclear/steroid receptor superfamily (see Mangelsdorf et al. 1995). Oestrogen is also acknowledged to exert some of its actions via membrane receptors as discussed below. Responses mediated by these receptors tend to be slow in onset to allow for subsequent modulation of gene transcription (Tsai and O’Malley 1994). ERa and ERb share considerable sequence homology at the DNA (96%) and ligand (56%) (Weiser et al. 2008) binding domains and, in vitro at least, ERa is a stronger Introduction to the Interaction Between Gonadal Steroids and the Central Nervous System 5 H Plasma Sex hormone membrane enters cells by diffusion H Shedding H of co-repressors Nuclear membrane Hormone Conformational change binds to and homo-or hetero- receptor Recruitment dimerisation of co-activators Binding to hormone H response element on chromatin H Transcription RESPONSE Fig. 1 Diagramatic representation of a sex hormone entering a target cell and binding to its receptor. The sex hormone (H) diffuses into the cell and binds to its intranuclear receptor, co- activator and co-repressor proteins are then recruited or shed (respectively) from the complex as it dimerises (this can either be with an identical complex, eg another ERa to form a homo-dimer or with a simlar complex eg an ERb to form a hetero-dimer). This results in the remodelling or activation of the complex to facilitate its binding to the hormone response element of the chromatin which will in turn induce transcription and the response to the hormone (Beato and Sanchez-Pacheco 1996) transcriptional activator (Delaunay et al. 2000). Effective transcription does require the recruitment of steroid receptor co-activators (SRCs) as shown in Fig. 1. These molecules influence several processes that follow ligand receptor interaction such as phosphorylation and thus modulate transcription (O’Malley 2006). They may also be involved in the aetiology of disease including neurological disorders (Lonard et al. 2007). The distribution of ERa and ERb has been extensively studied in different species (see Österlund and Hurd 2001; Weiser et al. 2008). ERa and ERb differ not only in their distribution in the brain but also in their ligand-binding ability (Damdimopoulos et al. 2008) and roles (Bodo and Rissman 2006). A pertinent example could be oestrogenic protection against insult by NMDA in the hippocampus, an activity which is mediated by ERb and induction of BDNF, a response inhibited by progesterone (Aguirre et al. 2010). The potential for selective agonism or antagonism at receptor subtypes has been exploited therapeutically by, for example, the selective oestrogen receptor 6 K.M. Marshall modulators (SERMs) and the selective oestrogen receptor down-regulators (SERDs) such as tamoxifen and faslodex respectively, which have been used extensively in the management of breast cancer. Indeed, since the publication of the findings from the Women’s Health Initiative (WHI) in the USA and in the UK, the Million Women Study (MWS) (for more information and resultant publications go to http://www.nhlbi.nih.gov/whi/references.htm and http://www.millionwomen- study.org/publications/ respectively), which in many respects were controversial not least with respect to the findings in relation to neuroprotection, the use of agents like the SERMs or even more selective compounds is likely to increase as hormone replacement regimens with better side-effect profiles are sought to manage the consequences of the menopause as women can now expect to live approximately a third of their lives in an oestrogen-deficient state. The central effects of the SERMS have yet to be fully evaluated, but they do have potential as tools and therapies (Arevalo et al. 2011) as summarised in Fig. 2. SERMs Classical ERs ER-independent mechanisms MAPK PI3K/Akt CREB NF-kB Telomersse sctivity Antioodant effects Syneptic Bcl-2 Bax transmission Proinflammatory Seladin-1 cytokines Excitotodic Mood and cognition Oxdative neuronal death and stress and apoptoels Inflammation Neuronal survival Fig. 2 Summary of the molecular mechanisms involved in the neuroprotective effects of SERMs. SERMs act in the nervous system through classical ERs or by ER-independent mechanisms and activate a variety of signalling molecules, including MAPK, PI3K, Akt, CREB and NF-kB. These molecules, in turn, trigger different coordinated mechanisms to promote neuronal survival and regulate mood and cognition (Journal of Molecular Endocrinology (2011) 46, 111–119) Introduction to the Interaction Between Gonadal Steroids and the Central Nervous System 7 In addition to the intracellular receptors, ERa and ERb, there are also mem- brane receptors which are as yet less well defined. One such receptor is called GPR30 and is a 7 transmembrane domain G-protein-coupled structure (Lappano et al. 2010). Most of the work on this receptor has been done in relation to breast cancer (for review, see Maggiolini and Picard 2010), but GPR30 is expressed in the hypothalamic– pituitary axis, hippocampus and substantia nigra (Brailoiu et al. 2007). In the neo-cortex, a membrane-associated ER (ER-X) has been reported which mediates the activation of mitogen-activated protein kinase (MAPK), and 17aE2 appears to be the preferred ligand (Toran-Allerand 2004). The MAPK pathway can evoke the transcription of cAMP response element (CREB). 17bE2 can

also interact with the phosphotidylinositol-3-kinase (P13K) pathway resulting in Akt (protein kinase B) activation which inhibits proapop- totic proteins. E2 can also affect the monoaminergic systems as it can influence synthesis and degradation of dopamine, noradrenaline and 5-hydroxytryptamine aswell as regulating monoamine reuptake transporters and second messengers. Findings from a study in female rats by Lubbers et al. (2010) indicate that SERMs may have the potential to manipulate monoamines selectively allowing somemodulation of cognition and affec- tive function. Oestrogens have several other central properties including (Amantea et al. 2005): an antioxidant effect principally due to direct free radical scavenging; anti- inflammatory action via suppression of interleukin-1b induction of COX-2 (for review and information on structure–activity relationships, see Prokal and Simpkins 2007). Oestrogen can also rapidly induce nitric oxide synthase activity in cortical neurones including in the hippocampus (Mannella et al. 2009). 2.2 Progesterone The progesterone receptor also has two known subtypes namely PR-A and PR-B, the former being a truncated form of PR-B. PR-A dominantly represses the transcriptional activity mediated by PR-B (Giangrande and McDonnell 1999). These receptors are distributed in the human brain (Bezdickova et al. 2007) including in the hippocampus and frontal cortex (for review of form and function, see Brinton et al. 2008). Increased expression of PR-A can increase responsiveness to oestrogen via ERa (Mesiano 2001). Membrane receptors for progesterone, coupled to G-proteins, have also been identified that are associated with more rapid actions of the hormone (Thomas 2008; Dressing et al. 2011). Interestingly, unlikewith PR-A and PR-B,many of the synthetic progestogens (see below) do not bind to the membrane receptor. Progesterone, like oestrogen, has effects beyond those well recognised in the hypothalamus. Progesterone can also activate the MAPK and ERK signalling pathways which, can via CREB, lead to up-regulation of bcl-2 in hippocampal neurones (Nilsen and Brinton 2002). Progesterone has been found to up-regulate BDNF in murine models of cerebral ischaemia (Coughlan et al. 2009) and may play a role in maintenance of the integrity of the blood–brain barrier (Ishrat et al. 2010). 8 K.M. Marshall A small clinical trial involving patients with traumatic brain injuries showed that the use of progesterone was not harmful and it may indeed have some beneficial effects (Wright et al. 2007). Phase III trials are now underway. Selective agents for progesterone receptors have also been used experimentally to investigate further the role of progesterone. Therapeutic application of these agents will follow, and one agent, ulipristal acetate (ellaOne), was licenced in May 2009 in the UK for emergency contraception within 120 h (5 days) of unprotected intercourse or contraceptive failure. Ulipristal is an orally active synthetic selective progesterone receptormodulator (SPRM)with high affinity for PR-A. However, in animal studies it also has affinity for the glucocorticoid receptor, and in animals antiglucocorticoid effects have been seen. However, these antiglucocorticoid effects have not been seen in humans to-date and this may be indicative of real species differences. A variety of synthetic progestogens are used in hormone replacement therapy and contraception, as progesterone itself has poor oral bioavailability. Not all progesto- gens have the same pharmacological profile (Hapgood et al. 2004), and these differ- ences have implications for their usage. Two of the most widely used synthetic progestogens are medroxyprogesterone acetate and norethisterone. These are used as the progestogenic component of an HRT regimen in combination with oestrogen but have been shown to increase the risk of breast cancer in long-term HRT users (MillionWomen Study 2003;Women’sHealth Initiative 2002). Structurally,medrox- yprogesterone acetate is more similar to natural progesterone than norethisterone. The metabolism of these two compounds is also different, as medroxyprogesterone acetate is the major progestogenic compound rather than its metabolites. In contrast, the metabolites of norethisterone (a first generation progestagen) exhibit significant activity in addition to a wide range of non-progestogenic actions. Norethisterone also binds to SHBG, whereas medroxyprogesterone acetate does not. The most notable difference in steroid receptor-binding affinity between the two synthetic progestogens and endogenous progesterone is that, although all the compounds have affinity for the mineralocorticoid receptor, only the natural compound has antagonist activity. MPA is the most potent of the three in terms of glucocorticoid activity and correspondingly it has the highest affinity for the glucocorticoid receptor, but this relationship is complicated further by the fact that the resultant effect appears to be dependent on glucocorticoid receptor density (Hapgood et al. 2004). Unlike endoge- nous progesterone, bothMPAand the 19-nortestosterone derivatives also have affinity for the androgen receptor (but MPA has less intrinsic activity), although this is decreased in the third-generation compounds such as desogestrel. Such off-target pharmacology influences the side-effect profiles of these synthetic analogues. In addition, natural progesterone and its metabolites allopregnanolone and pregnanolone have high affinity for GABAA receptors (Paul and Purdy 1992), and progesterone can decrease glutamic acid decarboxylase (GAD) and so attenuate GABA synthesis (Wallis and Luttge 1980). The GABAergic system can also be influenced by the androgen metabolite 5a-androstane-3a,17b-diol (3a-Adiol), which is a metabolite of the potent androgen dihydrotestosterone (DHT) which is formed from testosterone by the action of 5a-reductase, and can modulate GABAA receptors (Reddy 2004). 3a-Adiol is in turn metabolised by CYP7B1 which is Introduction to the Interaction Between Gonadal Steroids and the Central Nervous System 9 expressed in the hippocampus, and this metabolic route may, therefore, indirectly influence GABAA pathways (Pettersson et al. 2009). 2.3 Androgen As with oestrogen and progesterone, it is recognised that androgens may also contribute to CNS physiology and pathology outside the control of reproduction. The two most important androgens are testosterone which is transformed within the CNS by 5a-reductase to 5a-dihydrotestosterone (DHT) (testosterone can also undergo aromatisation to estradiol as discussed earlier), and they mediate their effects, like oestrogen and progesterone, via a ligand-activated transcription factor, namely the androgen receptor (AR). Much of the knowledge of the AR has been derived from work done in the field of prostate cancer (Powell et al. 2004). In the CNS, mapping of AR distribution has revealed that there is a high level of co-localisation with ERs (Patchev et al. 2004). Overlap also occurs with respect to AR expression as in, for example, the male rat forebrain oestrogens up-regulate expression. Androgen withdrawal (via castration) can increase expression in some brain areas (preoptic nucleus) but have no effect in others (amygdala); these effects are also influenced by age (Kumar and Thakur 2004), and that perinatal or even prenatal androgen exposure may be important (Goren and Kruijver 2002). There- fore, there appears to be age-dependent, hormone and tissue-specific control of the AR. The influence of androgens outside reproduction is less clear than for oestrogen and progesterone; however, high levels of AR mRNA have been detected in human hippocampus (Beyenberg et al. 2000). There is evidence to suggest that testosterone and DHT can, like oestrogen, influence neuronal plasticity (Matsumoto and Prins 2002) and dendritic spine density (Leranth et al. 2004). Androgens may also reduce neuronal cell death after exposure to various insults, such as b-amyloid (Pike 2001) and oxidative stress (Ahlbom et al. 2001). These effects could be mediated via AR activation of MAPK/ERK signalling pathway (Nguyen et al. 2005). Androgens may also be involved in the regulation of several proteins involved in axonal regeneration such as neuritin and tubulin, and there is evidence that androgen action is potentiated by BDNF (Fargo et al. 2009). The complexities of these mechanisms may allow for selective activation of signalling pathways, and selective androgen receptor mod- ulators (SARMs) are being developed to specifically manipulate certain effects; as yet these are limited to the periphery to improve outcomes in the management of prostatic disease, osteoporosis and muscle wasting (Gao and Dalton 2007). 3 Conclusion The effects of the sex steroids in the brain, begin in utero (Swaab 2007), are complex, and the final response is dependent not only on receptor up- or down- regulation but also on a series of coordinated metabolic events and cross-talk 10 K.M. Marshall between receptor signalling pathways. 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Oosting Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2 Serotonin and Sexual Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3 Serotonin, Serotonergic Receptors and Male Sexual Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.2 SSRIs and Male Sexual Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.3 Acute and Chronic SSRI Administration in Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.4 SERT-KO Rats and Male Sexual Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.5 Serotonin Receptor Agonists and Antagonists and Ejaculation in Male Rats . . . . . . . . 23 3.6 Animal Models of Premature and Retarded Ejaculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.7 Studies with Rats Displaying Hyposexual Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.8 Studies with Rats Displaying Hypersexual Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.9 Conclusion: Serotonin and Male Sexual Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4 Serotonin, Serotonergic Receptors and Female Sexual Behaviour . . . . . . . . . . .

. . . . . . . . . . . . . . 27 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.2 SERT-KO Rats and Female Sexual Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Abstract Serotonin plays an important role in both male and female sexual beha- viour. In general, reduction of 5-HT function facilitates, whereas enhancement inhibits sexual behaviour. Most fundamental research on the involvement of 5-HT in sex has been performed in rats. Selective serotonin reuptake inhibitors (SSRIs) B. Olivier (*), J.S.W. Chan, E.M. Snoeren, J.G. Veening, C.H. Vinkers, M.D. Waldinger, and R.S. Oosting Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht University, Sorbonnelaan 16, 3584 CAUtrecht, The Netherlands e-mail: b.olivier@uu.nl; j.s.w.chan@uu.nl; j.g.veening@uu.nl; c.h.vinkers@uu.nl; md@demon. waldinger.nl; r.s.oosting@uu.nl J.D.A. Olivier Division of Psychiatry Dept. Clinical Neuroscience, Karolinska Institutet, KFC Novum Level 6, Exp 617 Lab 614, SE - 14157 Huddinge, Sweden e-mail: jocelien.olivier@ki.se J.C. Neill and J. Kulkarni (eds.), Biological Basis of Sex Differences in Psychopharmacology, 15 Current Topics in Behavioral Neurosciences 8, DOI 10.1007/7854_2010_116 # Springer‐Verlag Berlin Heidelberg 2011, published online 4 March 2011 16 B. Olivier et al. have comparable effects on male and female sexual behaviour in rats; they inhibit it but only after chronic administration. Activation of the 5-HT1A receptor facilitates sexual behaviour inmale rats but inhibits sexual behaviour in female rats, suggesting a differential role for 5-HT1A receptors in male and female rats. Research on sexual behaviour in rats with null mutations in the serotonin transporter (SERT) indicated also a differential role for 5-HT1A receptors in male and female sexual behaviour. Evidence exists that different pools of 5-HT1A receptors have differential roles in various parts of the cascade of sexual events occurring during sexual interactions. Roles for other 5-HT receptors are less well defined although 5-HT1B, 5-HT2A/B and 5-HT7 receptors seem to be involved. Identification of putative differential or comparable roles in female and male sexual activities requires more research. Keywords 5-HT
5-HT1A receptor
5-HTT
8-OH-DPAT
Gender
Hypersexual behaviour
Hyposexual behaviour
Paroxetine
Premature ejaculation
Retarded ejaculation
Serotonin
Serotonin receptor knockout rat
Serotonin transporter
SERT polymorphism
Sexual behaviour
SSRI
WAY100635 1 Introduction Sexual behaviour in rodents (and we strictly focus on the rat) happens when animals reach adulthood and engage in behaviours that result in the joining of a male and female, ending in copulation, with the intent to reproduce. The female rat’s sexual behaviour is dependent on the reproductive cycle, whereas the male’s sexual behaviour is not. The female’s sexual behaviour is strongly dependent on peripheral gonadal steroids that have both peripheral and central nervous system (CNS) effects. Steroids act on the brain to induce sexual receptivity and all associated behaviours (proceptive, receptive and pacing behaviours). Quite some work has been performed to delineate the neural circuitry and neurochemistry of female behaviour, especially from lordosis, a behaviour that is evoked by external stimuli, normally, a male rat. Lordosis behaviour is only observed when the female is hormonally (or naturally) primed (oestradiol + progesterone) and the circuitry involves sensory, brain, spinal cord and motoric activation. In the CNS, the ventromedial nucleus of the hypothalamus (VMH), the preoptic area (POA), the midbrain central gray (MCG) and two areas in the spinal cord (cervical and lumbar) are the key structures. All structures contain oestrogen receptors that seem essential for the final integrative performance of full sexual behaviour. Many neurotransmit- ter systems in the CNS regulate or modulate (aspects of) sexual behaviour, includ- ing serotonin. There is strong evidence that the serotonergic modulation of sexual behaviours mainly occurs at the level of the VMH and POA. In male rodents (rat), testosterone (T) acts during development to promote genital development and organization of the CNS neural circuitry. In adulthood, the neural circuitry along with the appropriate sensory and motoric systems controls the male’s sexual motivation and performance. Male rat’s sexual behaviour includes penile erection, sexual motivation and mating behaviour. All can be Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin 17 studied while observing the mating behaviour of a male rat in direct interaction with a receptive female. In such an interaction, the male approaches the female, sniffs her and starts mounting (the female displays lordosis). The male displays a series of mounts and intromissions that end in ejaculation. After an ejaculation, the male displays for some time sexual quiescence followed by the next series of mounts and intromissions, leading again to ejaculation, and so on. In the male rat, the testicular secretion of T occurs throughout the year, although pulsatile patterning occurs over the day. Seasonal variations in behavioural responsiveness to T of male rats have not been found, making the male (and female) rat ideal experimental animals to study the neural mechanisms of, and neurotransmitter involvement in, sexual behaviour. The neural systems involved in male sexual behaviour seem to involve many structures that are also involved in female sexual behaviour, although clear differ- ences are also notable. The POA and the bed nucleus of the stria terminalis (BNST) are core structures via which T acts to activate male sexual behaviour. In particular, the POA seems an integrative structure in coordinating the actions of T on both motivational and consummatory aspects of male sexual behaviour. Several neuro- chemical systems, including peptidergic, dopaminergic and serotonergic systems, play a role in mediating sexual behaviour. 2 Serotonin and Sexual Behaviour The focus of this chapter is the role of serotonin in male and female sexual behaviour in the rat. There is hardly any research performed on gender differences in the development and adult functioning of the 5-HT system in the brain and spinal cord. Seeing the overlap, but also the divergence of the various neural structures and hormonal receptor systems in the male and female rat CNS, it may be difficult to predict the effects of psychopharmacological treatment with serotonergic ligands on male and female sexual behaviour. Serotonergic psychopharmacology in humans is rather limited; only the selec- tive serotonin reuptake inhibitors (SSRI) are selective serotonergic drugs exten- sively used in patients, whereas most other drugs with some serotonergic profile exert inherently other mechanisms like dopamine D2 receptor antagonism (olanza- pine, risperidone, buspirone). In the latter case, it is often impossible to purely deduct the specific contribution of the serotonergic component on the putative effects on sexual behaviour or sexual dysfunctions induced. SSRIs are widely used to treat depression both in human males and females and are notoriously implicated (Zemishlany and Weizman 2008) in inducing sexual disturbances (Kennedy and Rizvi 2009; Balon 2006). However, a complicating factor is that major depression per se is often (if not always) associated with sexual disturbances (e.g. in libido, motivation, erection: Kendurkar and Kaur 2008; Kennedy and Rizvi 2009). SSRIs enhance serotonergic neurotransmission which is generally believed to inhibit sexual behaviour, both in males and females (Zemishlany and Weizman 2008; Williams et al. 2006; Kennedy and Rizvi 2009; Kendurkar and Kaur 2008). This is confirmed by various studies showing that SSRI antidepressants induce 18 B. Olivier et al. sexual disturbances, in addition to already present dysfunctions due to the underly- ing depression, in both males and females (Cyranowski et al. 2004; Regitz-Zagrosek et al. 2008). No studies in humans have looked into the brainmechanisms underlying the SSRI-induced sexual dysfunction and putative gender differences. While it is still assumed that high extracellular 5-HT levels (e.g. after SSRI treatment) are needed to promote antidepressant activity, the disadvantage is the directly associated decrease in sexual behaviours. The emerging pattern seems to indicate that SSRIs, which enhance serotonergic neurotransmission in the brain, have similar inhibitory effects in human males and females. In line with the latter notion is the finding (Sugden et al. 2009) that gene expression for 5 serotonergic genes (including 5-HTT) did not differ between genders in postmortem human brains. 3 Serotonin, Serotonergic Receptors and Male Sexual Behaviour 3.1 Introduction The importance of 5-HT in male sexual behaviour has been demonstrated by numerous studies showing that, for instance, lesions of the brainstem raphé nuclei (Albinsson et al. 1996) and 5-HT depletion (Tagliamonte et al. 1969) facilitate sexual behaviour. On the other hand, administration of 5-hydroxytryptophan, the direct precursor of 5-HT, 5-HT itself and 5-HT releasers such as MDMA and fenfluramine, inhibits sexual behaviour (Ahlenius et al. 1980; Dornan et al. 1991; Foreman et al. 1992; Gonzales et al. 1982). Altogether these findings suggest that a decrease in 5-HT neurotransmission may be involved in facilitation, whereas an increase in 5-HT neurotransmission may result in inhibition of male sexual behaviour. 3.2 SSRIs and Male Sexual Behaviour The frequently reported sexual effects of SSRIs in men demonstrate an important role of 5-HT in human ejaculatory behaviour. In several human studies we and others have demonstrated that SSRIs including paroxetine, sertraline and fluoxetine are able to delay ejaculation in premature ejaculation (for review, see Waldinger 2002; De Jong et al. 2006). Moreover, these studies show that SSRIs exert only a minimal ejaculation delay in the first week that is often not clinically relevant. A clinically relevant ejaculation delay occurs gradually after 2–3 weeks of daily treatment. Interestingly, despite the putative similar underlying mechanism of action of SSRIs – briefly, preventing the reuptake of 5-HT, thereby elevating 5-HT levels – not all SSRIs delay ejaculation to the same extent. In humans, the tricyclic antidepressant, clomipramine and the SSRI, paroxetine have stronger ejaculation-delaying effects after 4–6 weeks of daily treatment than other SSRIs (Waldinger et al. 1998, 2001a, b). Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin 19 3.3 Acute and Chronic SSRI Administration in Male Rats Analogous to the human situation, in male rats a distinction can be made between the effects of acute and chronic SSRI administration on ejaculation. Acute admin- istration of various SSRIs, such as citalopram, paroxetine, sertraline, fluoxetine and fluvoxamine, did not or marginally delay ejaculation (Mos et al. 1999; Ahlenius and Larsson 1999; Matuszcyk et al. 1998). On the other hand, chronic administration of fluoxetine (Matuszcyk et al. 1998; Cantor et al. 1999; Frank et al. 2000) and paroxetine (Waldinger et al. 2001a, b) delayed ejaculation in male rats. Nonethe- less, as in humans, not all SSRIs potently delay ejaculation after chronic adminis- tration in male rats: fluvoxamine slightly affected some aspects of copulatory behaviour, but did not affect ejaculation (Waldinger et al. 2001a, b; De Jong et al. 2005a). It is unclear why the various SSRIs differ in their ability to delay ejaculation after chronic administration. The delay in onset of the therapeutic effect of SSRIs in depression and anxiety disorders has been related to adaptive changes of serotonergic

autoreceptors (Haddjeri et al. 1998; Le Poul et al. 2000), and it is conceivable that the ejaculation-delaying effects of various SSRIs are due to differential adaptive changes of 5-HT receptors. An example of the effects of an SSRI antidepressant (paroxetine) in male rat sexual behaviour is shown in Fig. 1. The effect is clearly seen in the number of ejaculations per 30-min test in sexually trained animals. Acutely (Day 1: 30 min after injection) paroxetine does not inhibit sexual behaviour whereas after 7 (sub- chronic; 5 and 10 mg/kg) or 14 days treatment (chronic; 2.5, 5.0 and 10.0 mg/kg) ejaculation frequency Fig. 1 The mean number of 3.5 vehicle ejaculations  SEM of male 3.0 2.5mg/kg rat groups treated with 2.5 5mg/kg vehicle or different doses of 2.0 10mg/kg the SSRI paroxetine (2.5, 5.0 1.5 and 10.0 mg/kg IP) is given * after acute (30 min pre- 1.0 treatment), sub-chronic ** ** 0.5 (7 days; once daily) and 0.0 chronic (14 days: once daily) acute subchron chronic washout treatment. One week after treatment cessation of treatment 1st ejaculation latency (washout), sexual behaviour 2000 was again measured but now vehicle without any treatment. Sexual 2.5mg/kg * behaviour tests were run on ** 5mg/kg days 1, 7, 14 and 21 and 10mg/kg consisted of a 30-min test in 1000 * which a male rat had free * access to a female that was hormonally brought into oestrus (method: Chan et al. 0 2010). *p < 0.05 compared acute subchron chronic washout to vehicle treatment ejaculation latency (s) ejaculation frequency (#) 20 B. Olivier et al. paroxetine strongly (and dose dependently) reduces sexual behaviour. The effect is reversible as animals return to their pre-testing level 1 week after cessation of treatment. A similar picture emerges for the first ejaculation latency that is not affected acutely, but is dose-dependently enhanced after 7 days and 14 days of treatment, and returns to baseline 1 week after cessation of treatment. Ahlenius and Larsson (1999) have studied the mechanism of SSRI-induced delay of ejaculation in more detail and showed that acute treatment with citalopram did not affect ejaculatory behaviour. Co-administration of the 5-HT1A receptor antagonist WAY-100635 with citalopram strongly delayed ejaculation latencies, suggesting 5-HT1A receptor involvement in the effect of citalopram on ejaculation. De Jong et al. (2005a, b) also showed that citalopram, acutely or chronically, while not inhibiting sexual behaviour itself, when combined with a sexually inactive dose of WAY100635 completely abolished sexual behaviour. We studied this phenomenon further and confirmed earlier findings (Looney et al. 2005) that a dose as low as 0.01 mg/kg of WAY100635 facilitated the behaviourally inactive acute 10 mg/kg paroxetine dose and led to strong inhibition of male sexual behaviour (Fig. 2). The data suggest that the inhibitory action of SSRIs after (sub) chronic treatment are related to changes at certain 5-HT1A receptors after long-term treatment. ejaculation frequency 3 2 1 * Fig. 2 Sexually trained male 0 * rats were acutely injected 1 0 ve h eh 0 10 + v ve h + + AR R R1 with saline or 10 mg/kg Y P PA ve h A AY + PA + + paroxetine (IP; 30 min before W h Y Y 0.0 3W 0.3 ve A A W 3W testing) immediately 0.0 3 0. followed by an injection of 1st ejaculation latency either saline or a dose (0.03 2000 and 0.3 mg/kg IP) of the * * 5-HT1A receptor antagonist WAY100635. During an ensuing sexual behaviour test 1000 of 30 min, the sexual behaviour of the male was scored. In the figure, the mean number of 0 ejaculations  SEM is given. h 0 PAR paroxetine, WAY ve + ve h eh 0 0 v R1 R1 1 WAY100635, VEH vehicle. h + + PA A PAR ve AY AY + + P + *p < 0.05 compared to 3W W h 0.0 0.3 ve AY AY 3W .3W vehicle 0.0 0 ejaculation latency (s) ejaculation frequency (#) Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin 21 Subsequently, it was found that the ejaculation-delaying effects of the combina- tion of citalopram and WAY100635 could be fully blocked by a selective 5-HT1B receptor antagonist, suggesting a role for this receptor subtype in the delay of ejaculation (Hillegaart and Ahlenius 1998). Interestingly, a previous study from the same laboratory also suggested a role of the 5-HT1B receptor in the delay of ejaculation. In this study, it was shown that the 5-HT1B receptor agonist anpirtoline dose-dependently delayed ejaculation in rats (Hillegaart and Ahlenius 1998). 3.4 SERT-KO Rats and Male Sexual Behaviour In humans, the SERT plays a prominent role in the homeostasis of serotonergic neurotransmission. Polymorphisms in the promoter region of the SERT influence the activity of SERT, and the two length alleles (S and L allele) have functional consequences for the function of the 5-HT system (Murphy and Lesch et al. 2008). L and S (LL > LS > SS) generate allele-dependent 5-HT activity with associated functional consequences (Lesch et al. 2008). Rats do not possess such promoter length polymorphisms but genetic knockout of the SERT gene might generate rat models of the S-allele versions of the human SERT. Therefore, SERT/ and SERT+/ can be compared to wild-type (SERT+/+) male rats and their sexual behaviour studied (Chan et al. 2011). It was expected, in analogy to treatment with chronic SSRI treatment, that SERT/ and SERT+/ rats would display a lowered sexual behaviour compared to SERT+/+ rats. All rats (30 per genotype) were trained up to seven times (once weekly a test of 30 min) and gene knockout rats indeed showed lower sexual performance than wild-type rats. On average the mean number of ejaculations at week 7 was 1.6 for SERT+/+, 1.1 for SERT+/ and 0.7 for SERT/ rats (Fig. 3), a significant decrease 2.0 1.5 WT HET 1.0 * KO * 0.5 0.0 1 2 3 4 5 6 7 Successive weekly 30-min sexual test Fig. 3 Development of sexual behaviour (mean number of ejaculations/test) in male wild-type (SERT+/+, WT), heterozygous (SERT+/, HET) and homozygous (SERT/, KO) rats tested weekly over 7 weeks in a sexual behaviour test of 30 min with an oestrus female. *p < 0.05 compared to WT animals Ejaculations per 30 min (mean) 22 B. Olivier et al. Fig. 4 Effects of the 5-HT1A receptor agonist 8-OH-DPAT (s.c.) on ejaculation frequency over a 30-min test (a), latency to first ejaculation (b), first ejaculatory series mounts (c) and first ejaculatory series intromissions (d) of SERT+/+ (+/+) and SERT/ (/) animals. *p < 0.05 compared to wild type (+/+); a: p < 0.05 compared to vehicle treatment for the homozygote gene knockout. The heterozygote KO was not different from the wild type. Next, the 5-HT1A/7 receptor agonist +/8-OH-DPAT was tested. 5-HT1A stimula- tion has pro-sexual activities in rats which also occur in the three genotypes. Although the basal level of sexual behaviour (number of ejaculations, ejaculation latency, post- ejaculatory latency) in the SERT/ is lower than in the other two genotypes (Fig. 4), the stimulant effect of 8-OH-DPAT in all three genotypes is similar, indicating that 5- HT1A receptors mediating this effect have not changed [(de)sensitized]. The 5-HT1A receptor antagonist WAY100635 had no effects in the WT and heterozygote rats but had a dose-dependent inhibitory effect in the SERT-KO (Fig. 5), suggesting that a different pool of 5-HT1A receptors is involved in its action and that these receptors appear sensitized in the SERT-KO. Remarkably, the heterozygous SERT+/ rats did in no way differ from the WT rats. Heterozygous SERT-KO rats have intermediate enhanced extracellular 5-HT levels compared to WT and SERT-KO (SERT/ > SERT+/ > SERT+/+). Apparently, like the effective dose of SSRIs that need to occupy at least 80% of the SERTs before antidepressant efficacy is observed (Kugaya et al. 2003), the SERT+/ still has sufficient SERT capacity (50%) left to show undisturbed sexual behaviour. To summarize, the sexual side effects of SSRIs are still not fully understood. Nevertheless, some recent findings and genetic evidence suggest that adaptive changes in the 5-HT system and probably its interactions with neuroendocrine systems (De Jong et al. 2007) may be responsible for their sexual effects. Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin 23 Fig. 5 Effects of WAY100635 (IP) on ejaculation frequency over 30-min test (a), latency to first ejaculation (b), first ejaculatory series mounts (c) and first ejaculatory series intromissions (d) of wild-type (+/+) and serotonin transporter knockout (/) animals. *p < 0.05 compared to wild type (+/+); a: p < 0.05 compared to vehicle treatment 3.5 Serotonin Receptor Agonists and Antagonists and Ejaculation in Male Rats As described above, activation of 5-HT1B receptors has been associated with delaying ejaculation in male rats. 5-HT2 receptors are also implicated in modulation of sexual activity, e.g. shown by the 5-HT2A/2C receptor agonist DOI-induced inhibition of sexual behaviour (Klint and Larsson 1995). On the other hand, several other studies have shown that 5-HT2A/2C receptor agonists generally inhibit sexual behaviour by decreasing the number of animals that initiated copulation, but do not affect ejaculation latencies in animals that do initiate copulation (Ahlenius and Larsson 1998; Klint et al. 1992; Watson and Gorzalka 1991). Thus, it appears that 5-HT2 receptors in general inhibit sexual behaviour, but their precise role in the regulation of ejaculation is not entirely clear. A facilitatory role on ejaculation has been ascribed to activation of 5-HT1A receptors, and various selective agonists for this receptor, such as 8-OH-DPAT (Ahlenius and Larsson 1990), FG-5893 (Andersson and Larsson 1994) and flesi- noxan (Haensel and Slob 1997; Mos et al. 1991), potently facilitate sexual beha- viour and decrease ejaculation latencies. Nevertheless, the underlying mechanisms of the facilitatory effects of 5-HT1A receptor agonists are still unclear. A possibility for the mechanism of action may be activation of presynaptic 5-HT1A receptors that 24 B. Olivier et al. Table 1 Mean number of ejaculations, mounts and intromissions and ejaculation latency (in seconds) for sexually naı̈ve male rats during a 15-min test with a sexually active, oestrus female Drug (route) Dose (mg/kg) EF MF IF EL (s) 8-OH-DPAT (SC) 0 0.1 10.5 8.3 869 0.1 1.5a 6.5a 7.6 351a 0.2 1.9a 3.5a 5.1 187a 0.4 1.7a 1.1a 1.1a 238a Flesinoxan (IP) 0 0.3 13.9 12.2 854 0.1 1.0a 7.9 13.4 636a 0.3 1.3a 5.5a 9.8 459a 1.0 1.8a 3.3a 8.2 281a Buspirone (IP) 0 0.3 10.3 9.9 860 3.0 1.2a 7.0 11.3 502a 10.0 0.1 0.3a 1.8a 849 Ipsapirone (IP) 3.0 0.9(a) 7.9 11.3 502a 10.0 1.5a 10.9 12.2 636a All data are depicted as means EF ejaculation frequency,MFmount frequency, IF intromission frequency, EL ejaculation latency aSignificantly different (p < 0.05) from the corresponding vehicle (0 mg/kg) dose will lead to an inhibition of 5-HT neuronal firing and consequently results in facilitation of sexual behaviour as described above. Alternatively, activation of postsynaptic 5-HT1A receptors may result in facilitation of sexual behaviour. Evidence for a postsynaptic mechanism of action is provided by studies demon- strating that injection of 8-OH-DPAT directly into the medial preoptic area potently facilitated sexual behaviour and lowered ejaculatory threshold (Matuszewich et al. 1999). Administration of 5-HT1A receptor antagonists does not lead to any change in sexual behaviour (Ahlenius and Larsson 1999; De Jong et al. 2005a; Sura et al. 2001). Moreover, the effects of 5-HT1A receptor agonists can be antagonized by 5-HT1A receptor antagonists. When 5-HT1A receptor antagonists are combined with SSRIs (after acute or chronic administration), the inhibitory action of SSRIs is facilitated indicating a role for the 5-HT1A receptor in the inhibitory action of SSRIs in male sexual behaviour (De Jong et al. 2005a, b; Table 1) 3.6 Animal Models of Premature and Retarded Ejaculation Most of our current understanding of the anatomy and neurobiology of sexual behaviour is based on animal studies using rats that are sexually experienced and display normal sexual behaviour. Interestingly, the comparable ejaculation- delaying effects of SSRIs in humans and rats suggest high translational validity with regard to the regulation of ejaculation. Nevertheless, face validity is low when one tries to extend results obtained in rats that display normal sexual behaviour to dysfunction such as premature and retarded or even (an)-ejaculation. Over the last decades, several groups have studied rats that display hyposexual behaviour and are referred to, by different investigators, as sexually inactive, sluggish, impotent or

Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin 25 Fig. 6 More than 1,900 male rats were tested over a period of 5 years and trained weekly for 4 weeks in a sex test of 30 min against a female rat brought into behavioural oestrus. The graph represents the number of animals that displayed: 0, 1, 2, 3, 4 or 5 ejaculations during the last training test. Animals with 0 or 1 ejaculations/test were depicted as “slow” or “sluggish”; animals with two to three ejaculations/test as “normal” and animals with more than three ejaculations/test as “fast” non-copulating rats. Recent findings suggest the presence of neurobiological differ- ences associated with the hyposexual behaviour that these rats display. On the other hand, hypersexual behaviour can also be provoked pharmacologically. However, there are only few studies that have studied rats that are hypersexual by nature. Thus, investigating animals that do not display normal sexual behaviour may help understanding of the underlying neurobiological mechanisms and hopefully will provide further insight in the aetiology of ejaculatory dysfunction. In our laboratory, we have found (Pattij et al. 2005; Olivier et al. 2005) that male outbred Wistar rats display sexual “endophenotypes”. In subsequent cohorts of 100–120 male rats, we consistently found rats that display a very low (0–1), normal (2–3) or high (4–5) number of ejaculations in 30-min tests with a receptive female even after four to eight training tests. The behaviour of these males seems very stable, and we suggest the low performing animals as putative model for delayed ejaculation in humans and the high performing rats as model for premature ejacu- lation (Pattij et al. 2005; Olivier et al. 2006). Figure 6 shows the distribution of these “endophenotypic” sexual phenotypes in 1,982 male rats we tested thus far. These various endophenotypes are now the subject of pharmacological studies. 3.7 Studies with Rats Displaying Hyposexual Behaviour It was already demonstrated in early experiments in the 1940s that rats reared in isolation are either not capable to achieve ejaculation or remain sexually inactive, after repeated exposure to a receptive female (Beach 1942). In contrast, rats that were reared in groups with either same-sex or hetero-sex cage mates did not show these clear deficits in copulatory behaviour. Importantly, in most but not all of the 26 B. Olivier et al. isolation-reared males, sexual performance gradually improved with experience. These early findings suggest that experience and learning play an important role in rat copulatory performance, but apparently do not exclusively determine the ability to successfully copulate until ejaculation. In early studies focussing on rats dis- playing different levels of sexual performance, in our laboratory we have tried to create hyposexual behaviour in male rats by manipulating the level of sexual experience (Mos et al. 1990). To this end, we have studied the sexual behaviour of 278 sexually naı̈ve male Wistar rats in 15-min tests with an oestrus female. From those 278 males, 23 showed no sexual activity at all, i.e. no intromissions and maximally one mount was scored during the test. From the remaining 255 rats, 211 displayed sexual activity, but failed to ejaculate during the test. The average ejaculation latency of the 44 ejaculating males was 620  28 s. If sexually naı̈ve male rats were treated with 5-HT1A receptor agonists, these males performed quite well (Table 1). In particular, the two full 5-HT1A receptor agonists ()-8-OH- DPAT and flesinoxan enhanced sexual behaviour to the level of sexually experi- enced male rats. The partial 5-HT1A receptor agonists buspirone and ipsapirone also facilitated sexual activity. These findings indicate that naı̈ve male rats are able to perform sexual activities reminiscent of sexually “experienced” rats in a very short time interval. Apparently, sexually naı̈ve rats may be influenced by certain factors that can be overcome by treatment with psychoactive drugs, at least 5-HT1A receptor agonists and (not shown here) a2-adrenoceptor antagonists like yohimbine and idazoxan (Mos et al. 1990, 1991). These pharmacological studies strongly suggest that neurobiological mechan- isms underlie the differences observed in basal sexual behaviour. 3.8 Studies with Rats Displaying Hypersexual Behaviour In contrast to studies focussing on rats that are hyposexual by nature, reports of rats that are hypersexual by nature are scarce. Nevertheless, numerous studies have indicated that a variety of selective pharmacological compounds, neurotransmitters and neuropeptides may facilitate sexual behaviour (Bitran and Hull 1987; Argiolas 1999). Most interesting are those studies in which male rat sexual behaviour is potently facilitated and in which the behaviour shares some of the characteristics of human premature ejaculation. Indeed, some of the clinical symptoms of premature ejaculation can be evoked pharmacologically in male rats. For instance, various selective 5-HT1A receptor agonists have been shown to potently decrease ejacula- tion latencies and intromission and mount frequencies. Apart from selective 5-HT1A receptor agonists, a selective dopamine D2 receptor agonist SND-919 (Ferrari and Giuliani 1994) has also been shown to decrease ejaculation latencies in rats, although its effects were much less pronounced compared to the effects of 5-HT1A receptor agonists. Not only can pharmacological manipulations facilitate ejaculatory behaviour, but “tactile” stimulation, such as shock and tail-pinching (Barfield and Sachs 1968; Wang and Hull 1980), also facilitate ejaculatory behaviour. Presumably these Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin 27 facilitatory effects are mediated by activation of the brain dopaminergic system (Leyton and Stewart 1996). 3.9 Conclusion: Serotonin and Male Sexual Behaviour Research in humans and rats has indicated that modulating 5-HT levels in the CNS changes ejaculatory thresholds and associated sexual behaviour. Activation of 5-HT1A receptors and blockade of 5-HT2C receptors facilitates sexual behaviour, whereas activation of 5-HT1B and 5-HT2A receptors inhibits it. SSRIs, which facilitate serotonin neurotransmission, inhibit sexual behaviour but only after chronic administration or genetic inactivation of the SERT gene. There is a paucity of data on the putative role of other 5-HT receptors in the modulation of male sexual behaviour. 4 Serotonin, Serotonergic Receptors and Female Sexual Behaviour 4.1 Introduction The pharmacology of sexual behaviour in females is rather restricted compared to males. The majority of work has focused on one aspect of it: the lordosis reflex. Female sexual behaviour consists of attractivity, proceptivity and receptivity. Attractivity reflects behaviour, smell and sounds by the female that attract the male and most often leads to proceptive behaviour of the female, including solici- tation, hopping and darting. Receptivity is reflected in the lordosis reflex required for successful copulation. Beach (1948) introduced the lordosis quotient (LQ ¼ lordosis to mount ratio X 100) reflecting the lordotic response of the female to a mounting male. The LQ is the most frequently used parameter when studying effects of hormones and drugs on female sexual behaviour (cf. Uphouse 2000; Uphouse and Guptarak 2010). The lordosis reflex (arching of the back, elevation of the rump, dorsoflexion of the tail and extension of the neck) is a very stereotyped posture in response to a mounting male (Pfaff 1999). The tactile stimulation stimulates cutaneous receptors in the flank, rump, tail base and perineum, which feed their information to the brain where primarily areas in the hypothalamus (notably the VMH) are crucial in the control of lordosis. Oestrogen (Era) receptor activation is required to induce the lordosis reflex, and there is a minimum amount of circulating oestrogen needed to reach a certain lordosis threshold. Moreover, a latent period (minimally 16 h) is needed for receptivity development. Normally, both oestrogen and progesterone are used to optimally organize the libido reflex, but progesterone is not needed if the oestrogen dose is extra high. Adding proges- terone reduces the amount of oestrogen needed to induce lordosis behaviour. 28 B. Olivier et al. Pharmacological studies often use submaximal oestrogen (or progesterone) doses in ovariectomized females which produce submaximal lordosis quotients and generate a model that can be pharmacologically manipulated. Early studies showed that reduction of monoamine levels in the brain (e.g. by pCPA or reserpine) activated lordosis in suboptimally oestrogen-primed ovariectomized rats, while activation of 5-HT function inhibits it (for review, see Uphouse 2000; Uphouse and Guptarak 2010). With the emerging availability of selective 5-HT receptor ligands more specific studies could be performed, but still serotonergic psychopharmacology has been mainly restricted to 5-HT1A and 5-HT2 receptors. Activation of 5-HT1A receptors leads to inhibition of the lordosis reflex in hormonally suboptimally and optimally primed female rats (Ahlenius et al. 1986; Mendelson and Gorzalka 1986). Work from Uphouse’s group (Uphouse 2000) has found that the underlying mechanism of this inhibition is mediated via postsynaptic 5-HT1A receptors in the hypothalamus, specifically, although not exclusively, in the VMH. Blocking of these 5-HT1A receptors, however, did not lead to facilitation of the lordosis reflex which also does not happen after systemic administration of 5-HT1A receptor antagonists (Uphouse 2000), a finding we confirmed in our laboratory (see SERT-KO data later). The role of 5-HT1B receptors in lordosis is somewhat disputed (Uphouse and Guptarak 2010). Notwithstanding the limited evidence and lack of selective agonists, data suggest that activation of presynaptic 5-HT1B receptors facilitates lordosis (Mendelson 1992), whereas blockade of 5-HT1B receptors inhibits it (Uphouse et al. 2009). Activation of 5-HT2A/2C receptors (e.g. by DOI) facilitates lordosis in subopti- mally primed rats (Mendelson and Gorzalka 1990), whereas 5-HT2A/2C receptor antagonists inhibit it (Hunter et al. 1985; Mendelson and Gorzalka 1985). These effects seem also to be mediated in the hypothalamus probably in close interaction with those mediated by 5-HT1A receptors (Uphouse 2000; Uphouse and Guptarak 2010). 5-HT3 receptors do not play an important role in female sexual behaviour; the few studies reported (for overview, see Uphouse and Guptarak 2010) do not point to central 5-HT3 receptors as a primary target. Similarly, an inhibitory role in lordosis of 5-HT7 receptors has been suggested (Siddiqui et al. 2007), but these data are much linked to 5-HT1A receptor modulation and research involving selective 5-HT7 receptor agonists is required. As SSRIs are reported to induce a high incidence of sexual disturbance in human females (Balon 2006; Montgomery et al. 2002), it is relatively surprising that only a few studies have been performed in rats. Acute treatment with SSRIs reduces lordosis in hormonally primed ovariectomized rats (Frye et al. 2003; Sarkar et al. 2008). Because sexual side effects of SSRIs in humans are particu- larly disturbing after chronic administration, animal studies using chronic SSRIs are particularly relevant. Matuszcyk et al. (1998) found that chronic fluoxetine reduced sexual behaviour in female rats. This and other studies (Maswood et al. 2008; Uphouse and Guptarak 2010) are complicated by the fact that natural cycling females were used and fluoxetine affected the cycle, at least in a large number of the animals. A better strategy would be to chronically treat Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin 29 ovariectomized female rats with an SSRI, prime them with a dose of oestrogen and progesterone to induce lordosis and to test the effects of the SSRI in this model. Sarkar et al. (2008) found, using this paradigm, that fluoxetine acutely reduced lordosis but this effect was attenuated after sub-chronic fluoxetine administration, suggesting that some tolerance for the sexual inhibitory effect of the SSRI had occurred. 4.2 SERT-KO Rats and Female Sexual Behaviour An alternative way to study the role of the SERT in female sexual behaviour is using genetically modified animals, in this case the SERT-KO rat made by ENU mutagenesis (Smits et al. 2006). Female Wistar intact rats were tested in a paced mating design where sexually experienced males were restricted to one side of a cage, whereas the female (brought into behavioural oestrus by a high dose of oestradiol) could spend time on both sides of a divider which allowed passage of the female (but not the male) through a couple of openings in the divider. Figure 7 shows that mutant SERT genotypes (SERT+/ and SERT/) were not different from wild types (SERT+/+) in any aspect of proceptive or receptive behaviour over three consecutive tests of 30 min. This indicates that permanent absence of the serotonin transporter has no influence on female sexual behaviour under normal conditions. Treatment with a 5-HT1A receptor agonist (+/8-OH-DPAT) dose- dependently reduced proceptive behaviours (b) in all three genotypes, but in the Fig. 7 Effects of three doses of 8-OH-DPAT (0.01, 0.1 and 1 mg/kg, SC) and one dose of WAY100635 (0.1 mg/kg, IP) on ejaculation frequency over 30-min test

(a), latency to first ejaculation (b), first ejaculatory series mounts (c) and first ejaculatory series intromissions (d) of SERT+/+ (+/+) and SERT/ (/) animals. *p < 0.05 compared to wild type (+/+) 30 B. Olivier et al. a b # Total darts and hops Time spent in male compartment (s) basal levels in intact females basal levels in intact females 125 1400 SERT+/+ 1200 SERT+/+ 100 SERT+/- 1000 SERT+/- SERT-/- SERT-/- 75 800 50 600 400 25 200 0 0 Test 1 Test 2 Test 3 Test 1 Test 2 Test 3 c d Lordosis quotient Lordosis score basal levels in intact females 3 basal levels in intact females 125 100 2 75 50 1 25 0 0 SERT+/+ SERT+/- SERT-/- SERT+/+ SERT+/- SERT-/- Fig. 8 In a paced mating situation (Snoeren et al. 2010) female wild-type (SERT+/+), heterozy- gous (SERT+/) and homozygous (SERT/) rats were brought into behavioural oestrus by hormonal priming and tested against a sexually experienced male rat. Females could pace the behaviour and stay in- or outside the male compartment. The number of proceptive [hopping and darting (a)] and receptive behaviours [Lordosis quotient and Lordosis score (c and d)] and the time spent in the male compartment (b) were measured SERT-KO the dose–response curve clearly shifted to the right, indicative of a desensitized 5-HT1A receptor (Fig. 8). However, time spent with the male was not affected (a), showing that the decreased proceptive behaviour was not caused by a diminished interaction with the male. Treatment with a 5-HT1A receptor antago- nist (WAY100635) did not affect any behaviour alone [(c) and (d)], whereas a selected dose of WAY100635 (0.1 mg/kg IP) was able to antagonize the 8-OH- DPAT-induced reduction in proceptive behaviour (f). Apparently, normal female sexual behaviour is not dependent on the functional status of 5-HT1A receptors, but when challenged 5-HT1A receptors appear desensitized in homozygous, but not heterozygous SERT-KO rats (Fig. 9). 5 Conclusions The neurotransmitter serotonin clearly plays a role in male and female sexual behaviour (Table 2). Lowering serotonergic function seems to facilitate and enhancing it to inhibit sexual behaviour. The availability of blockers of the # lordosis responses/ # male behaviors Average score of lordosis response Differences in Sexual Behaviour in Male and Female Rodents: Role of Serotonin 31 a b Time spent in male compartment (s) # Total darts and hops ±8-OH-DPAT SERT+/+ ±8-OH-DPAT 1400 SERT+/- 125 1200 SERT-/- SERT+/+ 100 * SERT+/- 1000 SERT-/- 800 75 * 600 50 400 25 200 0 0 Vehicle 0.03 mg/kg 0.1 mg/kg 0.3 mg/kg 1 mg/kg Vehicle 0.03 mg/kg 0.1 mg/kg 0.3 mg/kg 1 mg/kg ± 8-OH-DPAT ± 8-OH-DPAT c Time spent in male compartment (s) d # Total darts and hops WAY100635 WAY100635 SERT+/+ SERT+/+ 1400 125 SERT+/- SERT+/- 1200 SERT-/- 100 SERT-/- 1000 800 75 600 50 400 25 200 0 0 Vehicle 0.1 mg/kg 0.3 mg/kg 1 mg/kg Vehicle 0.1 mg/kg 0.3 mg/kg 1 mg/kg WAY100635 WAY100635 e f Time spent in male compartment (s) #Total dart sandhops ± 8-OH-DPAT and WAY100635 ±8-OH-DPAT and WAY100635 1400 SERT+/+ SERT+/+ 125 SERT+/- 1200 SERT+/- SERT-/- SERT-/- 100 1000 * 800 75 600 50 * 400 25 200 0 0 Vehicle Vehicle 0.3 mg/kg±8-OH-DPAT 0.3 mg/kg±8-OH-DPAT 0.3 mg/kg WAY100635 0.3 mg/kg WAY100635 ±8-OH-DPAT + WAY100635 ±8-OH-DPAT + WAY100635 Fig. 9 Female wild-type (SERT+/+), heterozygous serotonin transporter knockout (SERT+/) and homozygous serotonin transporter knockout (SERT/) rats brought into behavioural oestrus were treated with the 5-HT1A receptor agonist +/8-OH-DPAT (a); the 5-HT1A receptor antagonist WAY100639 (b) or a combination of selected doses of 8-OH-DPAT (0.3 mg/kg) and WAY100639 (0.3 mg/kg) (c). The left part of each figure shows the time spent by the female in the male compartment; the right part the number of proceptive behaviours (hopping and darting) performed by the female during the test. The test was performed using a paced mating design in which the male and female were separated by a perforated wall that could be crossed by the female but not by the male. The female decides whether she wants to spend time with the male and receive mounts, intromissions and ejaculations. *p < 0.05 compared to wild type 32 B. Olivier et al. Table 2 Summary of the effects of various serotonergic ligands on male and female sexual behaviour in rats after acute or chronic treatment Target/ligand Treatment Male sexual Female sexual behaviour behaviour SERT/SSRI Acute ¼ ¼ SERT/SSRI Chronic # nd 5-HT1A R agonist Acute " # 5-HT1A R agonist Chronic " nd 5-HT1A R antagonist Acute ¼ ¼ 5-HT1A R antagonist Chronic nd nd 5-HT1B R agonist Acute # " 5-HT1B R agonist Chronic nd nd 5-HT1B R antagonist Acute ¼ # 5-HT1B R antagonist Chronic nd nd 5-HT2A/C R agonist Acute # " 5-HT2A/C R agonist Chronic nd nd 5-HT2A/C R antagonist Acute # # 5-HT2A/C R antagonist Chronic nd nd 5-HT7 R agonist Acute nd # 5-HT7 R agonist Chronic ¼ nd 5-HT7 R antagonist Acute ¼ ¼ 5-HT7 R antagonist Chronic ¼ nd ¼ not affected, nd not determined, " enhanced, # lowered, R receptor, SSRI selective serotonin reuptake inhibitor, SERT serotonin transporter serotonin transporter and ligands for various serotonergic receptors has led to studies on male and female rat sexual behaviour that shed light on the contribu- tions of individual receptors/transporter in male and female sexual function. SSRIs, blocking the SERT, generally lead to inhibition (after chronic treatment) of male and female sexual behaviour in agreement with the theory that enhance- ment of serotonergic function inhibits sexual behaviour. 5-HT1A receptor activa- tion facilitates male ejaculatory behaviour but inhibits female lordosis behaviour, suggesting an opposing role for this receptor in males and females. Clear-cut roles for other serotonergic receptors are less developed and need considerable research efforts. Genetic manipulation of the SERT in rats indicated a differential influence of the absence of the SERT in male and female sexual behaviour; KO males, but not females, had lower baseline sexual activities. 5-HT1A receptors were not desensitized in male KO, but were desensitized in females, indicating a differ- ential role of various 5-HT1A receptor pools in male and female sexual behaviour. 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Adv Psychosom Med 29:89–106 Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor Subtypes and Glutamate Jane Suzanne Sutcliffe Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2 In vivo Evidence for Gender Differences in Cognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.1 Object Recognition Tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.2 Spatial Maze Paradigms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.3 Social Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.4 Classical and Operant Conditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3 Female Advantage Under Stressful Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4 Mechanisms of Sex Hormone Action for Cognition? The Oestrogen Perspective . . . . . . . . . 48 4.1 Hippocampal Architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.2 Oestrogen Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4.3 Neurotransmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4.4 Glutamate and Oestrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 5 Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Abstract Interest in the influence of sex hormones within the central nervous system is a rapidly expanding area of research. A considerable amount of evidence has recently been obtained to support an important role of the gonadal steroids in cognitive processing. Not only are distinct and complementary behavioural pheno- types evident for each gender, in the case of the female but they are also reliant upon hormonal status. Gender influences and hormonal status are thus paramount and should encourage the development of more hypothesis-driven research strategies to understand gender differences in both normal behaviour and where this is altered in neuropsychiatric disorders. Keywords Cognition
In vivo
Oestrogen
Rat
Sex differences J.S. Sutcliffe Maccine Pte Ltd, 10 Science Park Road, #01-05 The Alpha, Singapore Science Park II, Singapore 117684, Singapore e-mail: Jane.Sutcliffe@Maccine.com J.C. Neill and J. Kulkarni (eds.), Biological Basis of Sex Differences in Psychopharmacology, 37 Current Topics in Behavioral Neurosciences 8, DOI 10.1007/7854_2011_120, # Springer‐Verlag Berlin Heidelberg 2011, published online 2 March 2011 38 J.S. Sutcliffe 1 Introduction Men and women differ not only in their physical attributes and reproductive function but also in many other characteristics including cognitive abilities and intellectual problem solving skills. For the past few decades, it has been ideologically fashionable to insist that these behavioural differences are minimal; research into sex differences in cognition has often been neglected and for many years, the male animal has been the standard gender used for behavioural research. Historically, female subjects have not been studied due to perceived problems inherent with oestrous cycle monitoring and also since it is assumed that their behaviour will be similar to those of a male. Strangely enough, evidence from human studies illustrates that men are actually more variable on tests of cognitive score and intellectual

performance (Sahay and Hen 2007). Sex differences cannot be considered irrelevant as there are indisputable, and well-established gender differences in metabolism, neuroanatomy, endocrinology, biochemistry and behaviour (Cahill 2006). Moreover, sex differences in many behavioural paradigms have been reported since the early days of the last century (Baker 1987; Corey 1930). One of the most important discoveries regarding gender differences is concerned with the innate differences between the anatomy and neurophysiology of the male and female brain. Differences begin during early development due to a combination of genetic and hormonal events and persist throughout the lifespan of an individual [for a comprehensive review on the origins of gender differences, see (Wilson and Davies 2007)]. The imprinting of the female brain is not dependent upon androgen-oestrogen influence but for male differentia- tion, androgens must be present (Pilgrim and Reisert 1992). Research during the past decade confirms that gonadal steroids, such as oestro- gen, progesterone and testosterone, have the ability to influence the structural properties of the brain regions that sub-serve learning and memory. One of the most comprehensively characterised regions of the brain is the hippocampus, which has been shown to display different structural changes in response to different gonadal hormones. Evaluation of brain structure, function and chemistry over the course of the menstrual cycle as well as across the life span in women is critical to understanding sex differences in both normal and aberrant behaviour. This chapter provides an overview of common cognitive paradigms with respect to inherent sex-specific abilities and the impact of cyclic or manipulated hormonal changes on learning and memory in male and female rats. Mechanisms by which these gender specific abilities are purported to arise are also discussed with particular relevance to oestrogen receptors (ERs) and glutamate. 2 In vivo Evidence for Gender Differences in Cognition The relationship between gonadal hormones and cognition remains immensely complex and depends on many factors, which require unique consideration such as the target brain structures and the recruited memory systems for that particular Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor 39 cognitive challenge. Effects of the gonadal hormones and inherent sex differences on cognitive behaviour are not immediately obvious since gender differences are investigated across multiple laboratories, under different environmental conditions, with different strains of animals and sex differences are rarely discussed. Through- out this section, gender differences in popular cognitive paradigms are discussed and concisely compiled in Table 1. 2.1 Object Recognition Tasks During the past few years, the ethologically relevant novel object recognition (NOR) and object displacement (OD) paradigms have enjoyed much scientific interest. These paradigms depend entirely on the rat’s natural preference for novelty (objects and location), there is no requirement for rule specific learning and thus, there is a logistical advantage whereby the inherent variability during rule acquisi- tion and undue stress is avoided. There is also good evidence to suggest that the NOR task is more sensitive to recognition memory impairments than other tasks such as the Delayed Non-Matching to Sample (DNMS) task (Nemanic et al. 2004; Pascalis et al. 2004). The NOR consists of a familiarisation phase, where two identical objects are presented to the subject, an inter-trial delay (which can be manipulated) and a test phase where one familiar and one novel object are presented to the subject (Ennaceur and Delacour 1988). Similar to the NOR task, the OD task consists of a familiarisation phase with presentation of two identical objects, which progresses, after an inter-trial delay, onto the test phase where one of these objects now occupies a new location with respect to the previous trial. Compared to incremental learning tasks using multiple learning trials, the NOR and OD tasks allow the investigation of drug effects on different stages of memory formation and recollection. To assess the influence of a compound on object encoding, it may be administered prior to the sample trial. The compound in question can also be administered intermediately (during the inter-trial interval) to assess the influence of drug effects on consolidation of object information. Thus, object recognition tasks are fast becoming a powerful ethologically relevant, scientific tool. Recent research using object recognition tasks has provided clear evidence of gender-specific abilities (Sutcliffe et al. 2007). Both genders show a progressive decline when longer inter-trial intervals are experienced for both the NOR and the OD tasks; however, gender-specific cognitive abilities are evident. Female hooded- Lister rats have been shown to display a sustained object recognition memory when compared to male rats during this task when the length of memory retention is challenged. Recent studies (Sutcliffe et al. 2007) have demonstrated a clear female advantage during the NOR paradigm, where female hooded-Lister rats exhibit memory retention at inter-trial intervals of up to 3 h compared with 30 min in their male counterparts. The converse is true for the OD task, where male hooded- Lister rats exhibit a preference for the displaced object at an inter-trial interval of 3 h compared to only 30 min when compared to female hooded-Lister rats 40 J.S. Sutcliffe Table 1 Summary of gender differences in common behavioural paradigms Cognitive Gender bias summary Oestrous cycle influence References paradigm NOR Females! Males Cyclic performance, Sutcliffe et al. (2007), Inconsistencies on improved King et al. (2004), actual length of performance with Ghi et al. (1999), memory retention high oestrogen and Frye et al. (2007), across research detrimental Walf et al. (2006), groups influence of Inagaki et al. (2010) ovariectomy which and Aubele et al. is reversed by (2008) hormone replacement OD Females Males Cyclic performance, Sutcliffe et al. (2007) improved and Frye et al. performance with (2007) high oestrogen RAM/MWM Small male advantage Negative effect of rising Jonasson (2005), Bucci but inconsistent oestrogen across the et al. (1995) and findings, often no oestrous cycle Faraji et al. (2010) sex differences observed ZT Females Males Not reported Faraji et al. (2010) Social recognition Young; Females! High oestrogen at pro- Markham and Juraska Males oestrous results in (2007), Sánchez- Aged; Females¼Males extended memory Andrade and retention. Kendrick (2011) and Detrimental Hlináck (1993) influence of ovariectomy, which is reversed by hormone replacement Eyeblink Females! Males Detrimental influence of Dalla et al. (2009), conditioning during acquisition ovariectomy, which Wood and Shors only. Evidence is reversed by (1998) and Leuner suggests more supraphysiological et al. (2004) females reach levels of hormone experimental replacement criterion and retain the knowledge of the task longer than males Fear conditioning Females Males Ovariectomy results in Maren et al. (1994), male-like Pryce et al. (1999) performance ability and Gupta et al. (2001) FR1 Females!Male during No effect of Dalla et al. (2008), acquisition ovariectomy Shors et al. (2007), Beatty and Beatty (1970) and Van Oyen et al. (1981) (continued) Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor 41 Table 1 (continued) Cognitive Gender bias summary Oestrous cycle influence References paradigm FR2 Females!Male during No effect of Dalla et al. (2008), acquisition ovariectomy Shors et al. (2007), Beatty and Beatty (1970) and Van Oyen et al. (1981) DRL/Lever Simple tasks Females Ovariectomy results in van Haaren et al. (1990), pressing Males male-like van Hest et al. " Complexity Females performance ability (1987), Beatty ! Male (1973), Roth et al. (2004) and Lynch et al. (2002) Self-administration Faster acquisition in Ovariectomy results in Fattore et al. (2007, females male-like 2009), Roth et al. performance ability (2004) and Lynch et al. (2002) (Fig. 1a–d) (Sutcliffe et al. 2007). Conversely, other research groups have demon- strated male hooded-Lister rats retain the preference for the novel object up to a 2 h inter-trial interval (King et al. 2004). Gender differences within the NOR paradigm have also been confirmed in the Wistar rat, females again exhibit superior perfor- mance during longer inter-trial intervals (90 min) when compared to their male counterparts (60 min) (Ghi et al. 1999). Cyclic variations in sex hormones also exert a significant impact on performance during object recognition tasks. Female rats demonstrated a distinct variability in cognitive performance during the OD task throughout the oestrous cycle with enhanced object recognition during pro-oestrous or oestrous when compared to di-oestrous when oestrogen levels are at their lowest (Sutcliffe et al. 2007; Frye et al. 2007). No cyclic alteration in performance was observed during the NOR paradigm at a 1 h inter-trial interval (Sutcliffe et al. 2007), but a significant influence of the oestrous cycle during the NOR has been reported in female Long-Evans rats when the inter-trial interval is increased to a 4 h inter-trial interval, which was concomitant with increases in serum estradiol, progesterone and 3 alpha- hydroxy-5 alpha-pregnan-20-one (Walf et al. 2006). Further supporting the hypothesis that the sex steroids play an important role in cognition, ovariectomy produces a robust deficit in the NOR and OD paradigms, which is reversed with ovarian steroid replacement regimens (Inagaki et al. 2010; Frye et al. 2007. The cognitive deficit due to ovariectomy is a robust finding across research groups, in our laboratory sexually mature, ovariectomised animals were shown to significantly identify the novel from familiar object after an inter-trial interval of 1 h on weeks 1 and 2 but not weeks 3–6 and week 40 following surgery in comparison with sham-operated animals who retained recognition ability. Furthermore, ovariectomised animals receiving sub-cutaneous hormone replacement consisting of oestrogen alone or oestrogen in combination with a progestin (450 mg/kg 17-b-estradiol propionate once per week plus medroxyprogesterone 17-acetate or levonorgestrel at 15 mg/kg 42 J.S. Sutcliffe a b 40 Familiar ** 35 Novel 50 Familiar ** Novel 30 40 ** * 25 *** * 30 20 ** 15 20 10 10 5 0 0 0.050.5 1 2 3 4 5 24 48 0.050.5 1 2 3 4 5 24 48 Inter-trial Interval (h) Inter-trial Interval(h) c d Stationary 30 Displaced 40 ** Stationary 35 ** Displaced ** ** 30 ** ** 20 25 20 10 15 10 5 0 0 0.050.5 1 2 3 4 5 24 48 0.050.5 1 2 3 4 5 24 48 Inter-trial Interval (h) Inter-trial Interval (h) e f Familiar 20 Familiar Novel 30 * Novel *** 25 ** 15 * * 20 ** 10 15 10 5 5 0 0 1 2 3 4 5 6 40 V E EM EL Weeks Post Ovariectomy Hormone Regime Fig. 1 Exploration times of gonadally intact mature female (a, c) and male (b, d) hooded-Lister rats during the retention trial in the NOR task (where one familiar and one novel object are presented to the subjects, a and b) and during the OD task (where two familiar, identical objects are presented to the subject but one object is displaced compared to the acquisition trial, c and d). Figures 1e and f illustrate the robust impact of ovariectomy (e) and continuous 16 week hormone replacement (f) – V (vehicle 1 ml/kg s.c), E (450 mg/kg 17-b-estradiol propionate s.c once per week), EM (E plus 15 mg/kg medroxyprogesterone 17-acetate s.c once every second week) and EL (E plus 15 mg/kg levonorgestrel s.c once every second week) during the NOR task with a 1h inter-trial interval. *p < 0.05, **p < 0.01 represent significant differences between the time spent exploring the familiar compared to the novel object. **p < 0.05, **p < 0.01, ***p < 0.001 represent significant differences between the time spent exploring the displaced object compared to the stationary object. Data are expressed as the mean  S.E.M. and were analysed at each time point using paired sample’s t-tests (n ¼ 6) once every second week) for 15 weeks immediately after ovariectomy sustained NOR but not OD performance at a 1 h inter-trial interval. Delayed (initiation of hormone replacement at 13 weeks post-ovariectomy) and intermittent (initiation of treatment on weeks 1–6 resuming on 13–18) hormone replacement regimes Exploration Time (s) Exploration Time (s) Exploration Time (s) Exploration Time (s) Exploration Time (s) Exploration Time (s) Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor 43 highlighted the benefit of initiating hormone replacement immediately following ovariectomy using a 1 h inter-trial interval in the NOR task. However, when the inter-trial interval was increased to 3 h, only the oestrogen alone intermittent group was able to significantly differentiate between the novel and familiar object (Fig. 1e, f. Sutcliffe et al., unpublished findings). Interestingly, gonadectomised (GDX) mature male Sprague-Dawley rats explore a novel and familiar object equally during the

NOR task, and these adverse effects of gonadectomy are attenuated by supplement- ing GDX animals with testosterone-propionate but not 17-b-estradiol (Aubele et al. 2008). Endogenous or exogenous oestrogen replacement is consistently reported to be beneficial during the NOR task for females, a finding which is not reported in males, suggesting innate activational differences by which oestrogen acts within the brain. 2.2 Spatial Maze Paradigms The existence of gender differences in rodent models of spatial learning and memory is a prominent yet controversial and often contested topic in the literature. A variety of studies have revealed a male superiority during performance on spatial tasks such as the Radial Arm Maze (RAM) developed by Olton and Samuelson (Olton and Samuelson 1976) and the Morris Water Maze (MWM) developed by Roger Morris (Morris 1981). In the MWM task, subjects will swim in a pool of water to identify/locate a hidden platform employing the topographical relation- ships among the distal visual cues, pool wall and goal location. Similar to the MWM, subjects completing the RAMmust find the locations of food rewards at the end of some of the maze arms and retrieve this information for successive trials. The RAM has primarily been adopted to assess spatial working memory but is also adaptable to recruit working and reference memory simultaneously. During both the standard 8-arm version and the 17-arm version of this task, gender differences have not always been reliably demonstrated with some inconsistent findings (Jonasson 2005). A direct comparison between male and female Long Evans rats at 6 months of age in the MWM task has concluded that there are no sex differences in place learning ability. Furthermore, search accuracy on probe trials, when the platform was unavailable, was also equivalent for the male and female groups. A recent review illustrates that, while there is a distinct male advantage on spatial tasks there is a lack of reliable replication across different laboratories which may be due to the strain of animal used, variations in stage of the oestrous cycle and stress levels whilst under test (Jonasson 2005). Fisher, Long Evans and Sprague-Dawley rats have been shown to yield the largest and most robust male advantage during spatial cognition tasks, while the same tasks employed in Wistar rats denote only a small male advantage when compared to female performance (Bucci et al. 1995). Interestingly, the promising development of a new paradigm – the dry-land ziggurat task (ZT) may provide the research community with more robust and consistent 44 J.S. Sutcliffe gender-related results. The ZT consists of an open field containing 16 identical ziggurats (pyramid-shaped towers) positioned equal distances apart. One ziggurat is baited with a food reward and the rat must navigate through the open field to retrieve the food reward using a combination of distal and/or proximal cues. The ZT relies on the ability of the test subject to acquire and recall the location of the baited ziggurat and this is tested in consecutive training sessions of eight trials per day for 10 days. The location of the baited ziggurat is changed every second day, requiring the rats to learn a total of five different locations. Indices of learning and memory are based on several parameters, including latency to find the target, distance travelled, the number of visits to non-baited ziggurats (errors) and the number of returns. A recent study directly compared the performance of male and female Long-Evans rats in the wet-land MWT with the dry-land ZT. While males and females did not display significant differences in the traditional measures of spatial navigation within the MWT, they displayed a robust, male biased, sex difference in all measures of the ZT indicating task-specific gender differences in spatial performance. Taken together these findings suggest that males and females may employ different learning strategies in the MWT and ZT and that the latter task provides a more favourable task for assessing gender differences in spatial memory in rats (Faraji et al. 2010). Fluctuations in sex hormones (e.g., oestrogen, progesterone and testosterone) will undoubtedly cause a shift in male and female performance. Varying levels of oestrogen results in variations in spatial learning and memory so that, when tested across the oestrous cycle females perform as well as males on days of low oestrogen but poorly when levels rise (Frye 1995; Warren and Juraska 1997). When female Long Evans rats are tested at a single point during the oestrous cycle, females in oestrous outperform those during the pro-oestrous phase (Warren and Juraska 1997). Conversely, other studies have found limited or no cyclical variations in performance (Healy et al. 1999; Bucci et al. 1995; Berry et al. 1997), indicating that retention for spatial information may be preserved despite morphological alterations in hippocampal dendritic spine density in the normally cycling female rat. Interestingly, administration of chronic oestrogen to ovariectomised rats enhanced spatial memory during the RAM task (Luine et al. 1998) and age- associated decline in sex steroid levels have been found to more profoundly impair spatial working memory in female rats in comparison with their male counterparts (Markowska 1999). Taken together, this evidence suggests a pivotal role for oestrogen for mnemonic abilities. Variations in stress levels of the animals whilst under testing conditions may also play a critical role in gender-specific abilities during spatial paradigms. A growing body of literature demonstrates that certain aspects of spatial mnemonic function are dependent upon a stress response pattern, which is shown to be sexually dimorphic. The deleterious influence of chronic stress evident in male subjects in numerous spatial tasks, displayed as an impaired performance is not evident in female rats and this effect is thought to be mediated by oestrogen (to be discussed in more detail later in this chapter, Sect. 3). Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor 45 2.3 Social Recognition Social recognition memory, vitally important for social interaction and the establish- ment of dominance hierarchies, is the ability to discriminate between unfamiliar and familiar conspecifics. In rodents, the task utilises chemosensory cues present in the anogenital region, which composes an “olfactory signature”. Similar to the NOR task, the amount of time one individual spends investigating another may be evaluated in the laboratory. Young (3–5 months) adult female rats have been shown to discriminate between novel and familiar juveniles for longer intervals than males (a 120 min compared to a 90 min interval), although aged male and female rats (16.5–19.5 months) have been shown to display the same social discrimination memory abilities (Markham and Juraska 2007). The superior memory ability of females was not found to be preserved during ageing, discrimination between a novel and familiar juvenile was abolished after a 120 min interval. More recently, the influence of gender and the oestrous cycle has been evaluated on the formation and long-term (24 h)maintenance of social recognitionmemory inmice with a focus on the respective involvement of a- and b-oestrogen receptors. Female wild-type animals were able to successfully form memories during all stages of their oestrous cycle however, only when learning occurred during proestrus (when oestro- gen levels are highest) was the memory retained for a period of 24 h. The acyclic, a- receptor knockout female mice demonstrated impairments in both the formation and the maintenance of social recognition memory, whereas b-receptor female knockouts showed no significant deficits and exhibited the same proestrus-dependent retention of memory at 24 h akin to the wild-typemice. To investigate gender differences, male a- and b-oestrogen receptor knockout mice were also evaluated in the same paradigm demonstrating similar results to the females. The male a-receptor knockouts had normal memory formation and only exhibited the 24 h memory retention deficit, indicating a greater female dependence on a-receptor expression for memory forma- tion during this specific task (Sánchez-Andrade and Kendrick 2011). Historically, the behavioural phenomenon of social recognition has been exten- sively studied in males. Insights into the impact of hormonal states on recognition memory (towards a juvenile male) in females have demonstrated that in young adult female rats social recognition memory is negatively affected 3 weeks after ovariectomy and restored with oestrogen replacement, co-incidentally, similar those deficits observed during the NOR task (Fig. 1e, f. Sutcliffe et al., unpublished findings. Complementing these findings it was further observed that 6 weeks after the termination of oestrogen replacement recognition memory was once again impaired (Hlináck 1993). 2.4 Classical and Operant Conditioning The classical, hippocampal-dependent, trace eyeblink conditioning paradigm is viewed as an associative task in which the animal is presented with a conditioned 46 J.S. Sutcliffe stimulus (normally white noise) followed closely by an aversive eyelid stimulation, the result of which causes the animal to blink (an unconditioned response). As the animal learns, the unconditioned response becomes a conditioned response (i.e., the animal learns to predict the eyelid stimulation in advance). Prior to training, age- matched male and female Sprague-Dawley rats express similar levels of spontane- ous blinking; however, females learn to anticipate the onset of the unconditioned stimulus and thus learn to time the conditioned response (i.e. eyeblink) sooner than males, this response being most evident on the first day of training. At the end of training, there are no gender differences but interestingly, more females than males reach a criterion of 60% conditioned responses (7 out of 8 females compared to 7 out of 10 males reaching the same criterion) (Dalla et al. 2009). Perhaps not surprisingly, re-exposure of trained animals to the conditioned stimulus some weeks later elicits a higher percentage conditioned response in females compared to the males suggesting that the female rats have learnt and retained the rules of this task better than the males (Dalla et al. 2009). Further demonstrating oestrogen sensitivity in this task, removal of the ovaries prevents the sex differences in performance (Wood and Shors 1998) but enhanced conditioned responding is displayed in ovariectomised female rats following two injections of 40 mg estradiol 24 h apart, albeit at supraphysiological doses which produced plasma estradiol levels of greater than 250 pg/mL (Leuner et al. 2004). Sex differences have also been reported in another type of classical conditioning referred to as fear conditioning. Typically, the animal is trained to associate a cue or a context (e.g., a tone) with an aversive stimulus (i.e. a footshock). Re-exposure to the same cue or context results in the animal “freezing” or to express an enhanced startle reflex in anticipation of the aversive stimulus. A male bias is typically observed during cue fear conditioning since male rats acquire the association between the cue and foot shock quicker than females (Maren et al. 1994). The ability of males to outperform females within this paradigm is also a consistent finding in three strains of laboratory rat, the Wistar, Fischer and Lewis (Pryce et al. 1999). Castration elicits no effect on the conditioned response (Anagnostaras et al. 1998); in contrast, ovariectomy results in female rats displaying comparable levels of fear to males (Gupta et al. 2001). Although in classical fear conditioning studies males have outperformed the females, during more complex avoidance operant tasks the opposite is true. During an operant task, the animal must make an overt response in order to learn, which is often to escape an aversive stimulus – normally a mild foot shock. During the one- way avoidance task (FR1), the animal must learn to pass through the door way of a shuttle box once to avoid a mild footshock (FR1). This task is often learnt within a day and it has been observed that female rats will learn this task sooner than the males (Dalla et al. 2008; Shors et al. 2007). When the task difficulty is increased to the two-way avoidance task (FR2) where the animals must learn to pass through the doorway twice to terminate the footshock, more striking gender differences have been observed (Dalla et al. 2008; Shors et al. 2007). Females acquire the FR2 task within the first few trials but males require more trials and in some cases never learn the rule (Dalla et al. 2008). It would appear that female rats respond actively Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor 47 to aversive stimuli, whereas males exhibit behavioural inhibition with passive reactions and freezing. Ovary removal does not prevent the gender difference in operant conditioning (Beatty and Beatty 1970; Van Oyen et al. 1981); however, cyclic oestrogen and progesterone

influence conditioned avoidance behaviour and escape latencies as evidenced by enhanced performance during pro-oestrous when oestrogen levels are at their highest (Sfikakis et al. 1978). Another form of operant conditioning which has shown sexual dimorphism is differential-reinforcement-of-low-rate of responding (DRL), a task in which the animal must learn to press a lever for a reward (often food). Irrespective of reward receipt, male animals have shown better performance when compared to females during acquisition of instrumental responding, which is contributed to a higher level of interaction with the lever (van Haaren et al. 1990). On the contrary, when the task difficulty is more complex and the animals have to systematically increase the number of times they press the lever to receive the reward, females outperform the males (van Hest et al. 1987) – although incentive motivation for the reward could be a driving factor. Gender differences in differential reinforcement paradigms are also reliant upon the activational effects of the gonadal hormones, since they are not observed during puberty and are abolished with ovariectomy (Beatty 1973). Finally, sex differences are also observed in stimulant self-administration para- digms. Female rats are consistently reported to be more sensitive to the reinforcing actions of stimulants and acquire stable, high levels of drug self-administration of low doses of cocaine, methamphetamine, opioids and nicotine at a faster rate than males [for comprehensive reviews see (Fattore et al. 2009; Roth et al. 2004; Lynch et al. 2002)]. Ovariectomy results in slower cannabinoid self-administration acquisition when compared to cycling Long-Evans and Lister-hooded rats. Intrigu- ingly, in these rat strains ovariectomy decreased cannabinoid intake to the same level as those shown by males (Fattore et al. 2007, 2009), suggesting that the ovarian hormones play a crucial role in these responses to cannabinoids. 3 Female Advantage Under Stressful Conditions The limbic region, critical for processing information, shows plasticity under chronic stress with important gender-related differences (Eichenbaum et al. 2007; Lipton and Eichenbaum 2008). Chronic stress has repeatedly been shown to impair spatial learning and memory in male subjects yet produces different outcomes in females, thus gender is gaining recognition as an important variable acting as a mitigating factor or fundamental aetiology influencing stress-related disorders. Male rats exposed to chronic stress (6 h daily restraint for 21 days) perform poorly on tasks where normally a common male advantage is observed such as the RAM (Luine et al. 1993, 1994; Bowman et al. 2003), Y-maze (Conrad et al. 1996; Wright and Conrad 2005) and the MWM (Markowska 1999). In an almost opposite outcome, stressed female rats perform and complete the maze task with fewer 48 J.S. Sutcliffe errors and more correct choices (30% enhancement) when compared to unstressed females (Bowman et al. 2003). Not only does this female advantage influence classical tasks for spatial memory, stress has also been demonstrated to enhance female rat performance during the object placement task at an ITI of 2.5 h and 4 h (Beck and Luine 2002), where unstressed females are unable to perform this task at ITIs of greater than 1 h. Dendritic retraction of the CA3 region of the hippocampus has been implicated in the observed sexual dimorphisms under stressful situations. Chronic stress administered to cycling female rats has been shown to result in mild (Galea et al. 1997) or no (McLaughlin et al. 2010) dendritic retraction in the CA3 region. In ovariectomised rats, chronic stress produces drastic CA3 dendritic retraction (McLaughlin et al. 2010); however, spatial learning and memory remains func- tional and is even facilitated in ovariectomised and cycling chronically stressed female rats. This disconnection between the CA3 dendritic retractions and spatial memory in females may be attributed to the ovarian hormones. Indeed, within the CA3 region, females express more ERb immunoreactivity when compared to the CA3 region in male rats (Zhang et al. 2002), implying that oestrogens may be the key neuroprotective agent to prevent stress-induced CA3 dendritic attrition. 4 Mechanisms of Sex Hormone Action for Cognition? The Oestrogen Perspective Much evidence now supports a role for the interplay of many neurotransmitter systems, neuroplasticity and oestrogen with respect to understanding cognitive abilities. Many of oestrogen’s actions in the brain are attributed to the activation of the classical ERs a and b (ERa and ERb, respectively) and their subsequent impact on synaptic density and morphology, which is concomitant with alterations in learning and memory. 4.1 Hippocampal Architecture The medial temporal lobe system, including the hippocampal formation (entorhinal cortex, dentate gyrus, areas CA1-4 and subiculum), amygdale, and the parahippocam- pal cortices are considered to serve as a declarative memory system. The hippocampal formation, along with the frontal cortex, is an extensively explored area of the brain with regard to cognitive competence. This region has been investigated by means of excitotoxic, ablation and radiofrequency lesions, transient neuronal inactivation (lidocaine), lesions, or transections of its connections, by pharmacological N-methyl- D-Aspartate (NMDA) receptor blockade and even via genetic inactivation of the CA1- NMDA receptors (Dere et al. 2007) pre-clinically – evidence of which is supported by Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor 49 human lesion studies (Zola-Morgan et al. 1986). Cytotoxic and radio-frequency lesions to the hippocampus have been shown to result in an object recognition impairment after inter-trial intervals of 5 and 10 min, 1, 4 and 24 h (Ainge et al. 2006; Clark et al. 2000; Mumby et al. 2002). However, the size of the lesion to the hippocampus (>75%) remains important for object recognition memory (Broadbent et al. 2004). Oestrogen-associated changes (direct or indirect) within the hippocampus impact upon neural pathways, acting to alter performance in specific tasks. In the hippocampus of the female rat,Woolley and colleagues (Woolley et al. 1990;Woolley and McEwen 1992, 1994; Woolley et al. 1997; Woolley 1998) discovered that CA1 spine density naturally fluctuates across the female rat oestrous cycle, peaking with an increase of 32% during pro-oestrus when oestrogen levels are at a maximum com- pared to the di-oestrus phase. Subsequent studies have shown that oestrogen replace- ment in the ovariectomised female rat increases CA1 spine density (Woolley and McEwen 1992, 1993; Woolley 1998; Birzniece et al. 2006) through NMDA receptor- mediated mechanisms (Woolley and McEwen 1994; Woolley et al. 1997) and ERb activation (Liu et al. 2008). This research may provide important mechanistic evi- dence for the female advantage in object and social recognition tasks; furthermore, neuroprotection is observed when oestrogen is administered prior to the NMDA antagonist, PCP (Sutcliffe et al. 2008). This important relationship between glutamate, NMDA and oestrogen is discussed in more detail in Sects. 4.3 and 4.4. 4.2 Oestrogen Receptors Sex differences exist in the majority of brain regions including many involved in cognitive function such as the hippocampus, amygdala and neocortex (Juraska 1991). In many cases, these gender differences are not evident in overt anatomical structure, but in functional dimensions (e.g., a brain region may differ between the sexes in aspects of neurotransmitter function). The majority of research has investigated the effect of the steroid oestrogen on cognitive function and ERs have been found in several areas of the brain including the amygdala, cerebral cortex, cerebellum and hippocampus (Shughrue and Merchenthaler 2000; Tsutsui et al. 2004). ERs are important not only for the sexual differentiation of the brain but also are known to exert receptor-mediated functions on a number of beha- vioural functions. While the existence of ERa has been long known (Jensen et al. 2010), the discovery of ERb has been much more recent (Kuiper et al. 1996). Of particular interest to the present topic is the hippocampus which is known to play a significant role in working and spatial memory (Jarrard 1993) and which expresses both forms (a and b) of the ER (Cahill 2006; Frye 1995; Birzniece et al. 2006); however, their distribution does not completely overlap (Milner et al. 2001, 2005). ERb is the predominant ER in the cerebral cortex and the hippo- campus. Other areas within the brain which are confirmed as supporting mne- monic processes include the frontal cortex and the striatum, and it has become clear that there are strong interconnections (or loops) between the hippocampus 50 J.S. Sutcliffe Table 2 Distribution and gender differences in ERb immunoreactivity in the Wistar rat brain Region of interest Female Male CA1 +/ +/ CA2 +/ +/ CA3 ++ + CA4 ++ + Dentate gyrus ++ + Endopiriform nucleus +++ +++ Medial septal nucleus +++ ++ Purkinje cells +++ +++ Lateral and medial amygdaloid ++ + +++, high; ++, moderate, +, low and +/, weak. Source: Shughrue et al. (2000) and frontal cortex (Vertes 2006). In the frontal cortex of male and female rats, each ER isoform has been shown to display its own unique, selective distribution (Zhang et al. 2002; Kritzer 2002). Expression of ERb in the medial mammillary nucleus (a limbic area often associated with the hypothalamus) was only detected in the male rat brain. The female rat brain shows a higher predominance of ERb immunoreactivity (Table 2) in the medial septal nucleus (an area which receives reciprocal connections from the olfactory bulb, hippocampus, amygdala and hypothalamus), pyramidal cells of the CA3 and CA4, the dentate gyrus and the lateral amygdaloid nucleus (Shughrue et al. 2000). Whether such gender differ- ences in anatomical expression occur across species and in man has yet to be shown but the development of novel ER molecules as therapeutic agents remains an exciting prospect, especially considering the growing body of literature sug- gesting ERb modulation regulates neuroplasticity and cognition (Liu et al. 2008; Choleris et al. 2008; Walf et al. 2008; Rhodes and Frye 2006). 4.3 Neurotransmission The ability of oestradiol to induce and increase dendritic spine density is suggested to be due to the reduction of GABA inhibition by oestradiol in the hippocampal area (Murphy et al. 1998; Weaver et al. 1997). Furthermore, this oestrogen-induced increase in spine density is positively correlated with increases in NMDA receptor binding and sensitivity (Daniel and Dohanich 2001) providing one possible mechanism for the ability of oestrogen to improve cognitive function, especially when administered prior to an NMDA receptor antagonist such as PCP (Sutcliffe et al. 2008). Ovariectomy results in significant decreases in choline acetyltransferase (ChAT) activity and high affinity choline uptake (HACU) in the rat basal forebrain, hippocampal formation and cerebral cortex beyond normal ageing (Gibbs et al. 2002), which can be reversed by acute treatment with physiological levels of oestrogen [10 mg/kg oestradiol benzoate (Luine 1985; O’Malley et al. 1987) and Female Rats Are Smarter than Males: Influence of Test, Oestrogen Receptor 51 17-b-estradiol silastic capsules (Singh et al. 1994)]. Thus, oestrogen (exogenous or endogenous) may play an important role in the maintenance or regeneration of healthy cholinergic projections to the hippocampus and prefrontal cortex, which, in turn may be correlated with the enhanced cognitive performance observed during some cognitive paradigms when compared to males. One of the mechanisms by which oestrogen may enhance cognitive function is the modulation of the produc- tion and release of acetylcholine (ACh), which is shown to enhance the amplitude of synaptic potentials following long-term potentiation in regions such as the dentate gyrus, CA1, piriform cortex, and neocortex. This effect most likely occurs either through an indirect action on NMDA receptors, which are shown to be more abundant in the CA1 region of the female hippocampus compared to the male. Work in understanding how oestrogen affects cognition has concentrated on the oestrogen-mediated changes in hippocampal spine density and synaptogenesis favouring an NMDA-dependent mechanism of GABAergic disinhibition of pyramidal neurons mediated through non-genomic actions. Recent reports indicate that septal cholinergic inputs are required for this oestrogen-mediated enhancement of work- ing memory and NMDA receptor binding in the CA1 region of the hippocampus (Daniel and Dohanich 2001) and oestrogen-mediated changes in hippocampal spine density in rats (Lam and Leranth 2003), thereby linking these mechanisms. 4.4 Glutamate and Oestrogen Glutamate is the predominant excitatory amino acid neurotransmitter in the cortical, hippocampal and hypothalamic areas of the brain (Brann 1995) and glutamate receptors are considered to be responsible for the glutamate-mediated post-synaptic excitation of neural cells – important for neural transmission, memory consolidation through synaptic plasticity and accordingly learning. Synergistic interactions between glutamate and the gonadal steroids may underlie multiple limbic roles. The two primary glutamate receptors are NMDA and a-amino-3-hydroxy-5-methyl-4-isoxa- zolepropionic acid (AMPA), named after their preferred agonists, and it is through the NMDA

and AMPA receptors that glutamate may be neurotoxic. Glutamate toxicity may be predominantly mediated by the NMDA subclass of glutamate receptors (Choi et al. 1988). Administration of the non-competitive NMDA receptor antagonist, PCP, has reliably been shown to result in cognitive deficits in the rat (Neill et al. 2010) after both acute and sub-chronic administration, such deficits have been reversed with both the atypical antipsychotics (Grayson et al. 2007) and after pre-treatment with oestradiol-benzoate (Sutcliffe et al. 2008). Protective effects of oestrogens against glutamate toxicity have been described in cultured hippocampal neurons (Goodman et al. 1996). The toxicity experienced by each hippocampal culture was attenuated following a 2 h pre-treatment with 100 nM to 10 mM 17-b Estradiol, estriol or progesterone. This protective influence of oestrogen is further confirmed by the finding that a 24 h pre-treatment with 15–50 nM of 17-b Estradiol significantly decreased the lactate dehydrogenase efflux from primary cortical neurons exposed 52 J.S. Sutcliffe to glutamate for 5 min (Singer et al. 1996, 1999). Moreover, it was demonstrated that the selective ER modulator, tamoxifen, blocked the protective effects of oestrogen, thus suggesting that classical ER activations are required for oestrogen neuroprotec- tion against glutamate toxicity. Accumulating evidence suggests a sexually dimorphic vulnerability to neurological insults with the ER receptors conveying differential protective capacities (Bryant and Dorsa 2010). Accordingly, a therapeutic opportunity exists for oestrogen modulators, not only in the acute reversal of disease-induced cognitive deficits but also for long-term neuroprotection in neurodegenerative disease. 5 Summary and Conclusions Gender differences in cognition are evident in both pre-clinical models and in the clinic. Gender differences in learning vary as a function of the demands of the task and this review has primarily focussed on those where a female bias is observed. However, there are paradigms in which males excel such as those which require spatial navigation. In recent years, research in the area surrounding hormonal function in the central nervous system has increased with new discoveries and technology aiding the advance of knowledge regarding the effects of hormones on neurotransmission and consequent interactions within the brain. 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J Neurosci 6(10):2950–2967 Sex Differences in the Septo-Hippocampal Cholinergic System in Rats: Behavioral Consequences Dai Mitsushima Contents 1 General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 2 Physiological Role of ACh in the Hippocampus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3 Monitoring of In Vivo ACh Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 4 ACh Release in the Hippocampus Is Time-Dependent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 5 Sex Differences in ACh Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 6 Neural Control of Septo-Hippocampal Cholinergic Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 7 Circulating Sex Steroids Activate ACh Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 8 Sexual Differentiation Produces the Sex-Specific Activational Effect . . . . . . . . . . . . . . . . . . . . 64 9 Interaction with Environmental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 10 A Possible Treatment Strategy for Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 11 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Abstract The hippocampus is processing temporal and spatial information in particular contexts or episodes. Using freely moving rats, we monitored extracellu- lar levels of acetylcholine (ACh), a critical neurotransmitter activating hippocam- pal circuits. We found that the ACh release in the dorsal hippocampus increases during the period of learning or exploration, exhibiting a sex-specific 24-h release profile. Moreover, neonatal increase in circulating androgen not only androgenizes behavioral and hormonal features, but also produces male-type ACh release profile after the development. The results suggest neonatal sexual differentiation of septo- hippocampal cholinergic system. Environmental conditions (such as stress, housing or food) of animals further affected the ACh release. Although recent advances of neuroscience successfully revealed molecular/ cellular mechanism of learning and memory, most research were performed using D. Mitsushima Department of Physiology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura Kanazawaku, Yokohama 236-0004, Japan e-mail: dm650314@med.yokohama-cu.ac.jp J.C. Neill and J. Kulkarni (eds.), Biological Basis of Sex Differences in Psychopharmacology, 57 Current Topics in Behavioral Neurosciences 8, DOI 10.1007/7854_2010_95, # Springer‐Verlag Berlin Heidelberg 2010, published online 5 October 2010 58 D. Mitsushima male animals at specific time period. Sex-specific or time-dependent hippocampal functions are still largely unknown. Keywords Acetylcholine
Androgen
Diurnal rhythm
Estrogen
Learning and memory
Sex difference 1 General Introduction The hippocampus is a part of the limbic system and is a brain structure critically involved in learning and memory. The hippocampus processes temporal and spatial information within specific episodes (Komorowski et al. 2009; Gelbard-Sagiv et al. 2008). In freely moving animals, acetylcholine (ACh) release in the hippocampus increases during learning or exploration, showing a temporal 24-h release profile. ACh release changes with spontaneous movement (Day et al. 1991; Mitsushima et al. 1996) that stimulates electrical activity of cholinergic neurons in the basal forebrain (Buzsáki et al. 1988). Moreover, voluntary running enhances learning in mice (van Praag et al. 1999), while a restriction of exploratory behavior impairs ACh release and learning (Mitsushima et al. 1998, 2001). In this review, we focused on in vivo ACh release in the hippocampus to improve our understanding of the role of sexual dimorphism and temporal effects on hippocampal function. 2 Physiological Role of ACh in the Hippocampus A number of studies suggest that ACh plays an important role in orchestrating major hippocampal functions (Fig. 1). In behavioral studies, ACh release increases during learning (Ragozzino et al. 1996; Stancampiano et al. 1999; Hironaka et al. 2001) and is positively correlated with learning performance (Gold 2003; Parent and Baxter 2004). Bilateral injections of scopolamine into the dorsal hippocampus impair spatial learning ability (Herrera-Morales et al. 2007), suggesting that mus- carinic ACh receptors mediate the formation of spatial memory. At the network level, ACh generates a theta rhythm (Lee et al. 1994) that modulates the induction of long-term potentiation (LTP) in hippocampal CA1 neurons (Hyman et al. 2003). Studies exploring a genetic deficiency of muscarinic ACh receptors (M1 or M2) further show the impairment of LTP in the CA1 region (Seeger et al. 2004; Shinoe et al. 2005). At the cellular level, both pyramidal and nonpyramidal neurons in the hippocampal CA1 area receive direct cholinergic afferents mediated by muscarinic receptors (Cole and Nicoll 1983; Markram and Segal 1990; Widmer et al. 2006). In vitro studies showed that bath application of carbachol, a cholin- ergic agonist, induces LTP in CA1 pyramidal neurons without electrical stimulus, Sex Differences in the Septo-Hippocampal Cholinergic System in Rats 59 Hippocampus LTP induction Theta oscillation Neurogenesis ACh release septo-hippocampal cholinergic neurons Fig. 1 Schematic illustration of septo-hippocampal cholinergic neurons. The released ACh activates major hippocampal functions. ACh, acetylcholine. LTP, long-term potentiation suggesting that ACh in the hippocampus plays a principal role in the synaptic plasticity of the CA1 pyramidal neurons (Auerbach

and Segal 1996). Further- more, a recent study revealed an intracellular mechanism of ACh: focal activa- tion of muscarinic ACh receptors in one CA1 pyramidal neuron induces Ca2þ release from inositol 1,4,5-trisphosphate-sensitive stores to induce LTP (Fernández de Sevilla et al. 2008). Furthermore, not only is ACh critically involved in synaptic plasticity, but ACh release in the hippocampus is also responsible for neurogenesis in the dentate gyrus. Thus, neurotoxic lesions of forebrain cholinergic neurons or long- term scopolamine treatment significantly decreases the number of newborn cells in the dentate gyrus, approximately 90% of those were also positive for the neuron- specific marker NeuN (Mohapel et al. 2005; Kotani et al. 2006). 3 Monitoring of In Vivo ACh Release Cholinergic neurons within the basal forebrain provide the major projection to the neocortex and hippocampus (Mesulam et al. 1983). Cortical regions receive cho- linergic inputs mainly from the nucleus basalis magnocellularis (NBM) or the diagonal band of Broca, whereas the hippocampus receives cholinergic inputs mostly from the medial septum and horizontal limb of the diagonal band of Broca (Mesulam et al. 1983). Because the cholinergic projections are necessary to maintain learning and memory (Perry et al. 1999, Sarter and Parikh 2005), we hypothesized that in vivo monitoring of ACh release in the hippocampus is neces- sary to elucidate learning function. To measure ACh release, we have performed in vivo microdialysis studies in freely moving male rats. Briefly, a microdialysis probe with a semipermeable membrane (1.0 mm in length) was inserted into a specific brain area via a surgically pre-implanted guide cannula. We perfused the 60 D. Mitsushima Injection pump Artificial Internal ACh assay CSF standard Auto injector Computer 1 HPLC system Slip ring Eicom Interface Locomotor activity Freely Computer 2 moving rat Animex Interface Fig. 2 Experimental setup of in vivo microdialysis system. In order to evaluate the activational effect of sex hormones on ACh release, we simultaneously measured spontaneous locomotor activity in the same subject inside of the membrane with artificial cerebrospinal fluid, and assayed ACh in dialysates using a high-performance liquid chromatography system. As a result, we were successful in determining an in vivo ACh release profile in selected brain areas in freely moving rats (Fig. 2). 4 ACh Release in the Hippocampus Is Time-Dependent Using this in vivo measuring system, we showed a temporal 24-h profile of ACh release in the hippocampus. ACh release was episodically observed during the dark phase, but the episodic release was not frequently observed during the light phase (Mitsushima et al. 1998; Masuda et al. 2005). Simultaneous monitor- ing of spontaneous behavior revealed that the temporal pattern of ACh release is highly correlated with spontaneous movement in freely moving rats (Day et al. 1991; Mizuno et al. 1991; Mitsushima et al. 1996). Since a restriction of explor- atory behavior reduces ACh levels and also spatial learning (Mitsushima et al. 1998, 2001), episodic spontaneous behaviors may activate ACh release. In Sex Differences in the Septo-Hippocampal Cholinergic System in Rats 61 addition, spontaneous behavior stimulates electrical activity of cholinergic neu- rons in the basal forebrain (Buzsáki et al. 1988). Moreover, voluntary running enhances neurogenesis, spatial learning, and synaptic plasticity in mice (van Praag et al. 1999). Interestingly, this daily change is quite similar to the daily rhythm in hippocampal mitogen activated protein kinase (MAPK) activity and cAMP: phosphorylated extracellular signal-regulated (ERK) protein, GTP-bound Ras protein, and cAMP in the hippocampus show clear daily changes in male mice (Eckel-Mahan et al. 2008). Although the time-resolution of molecular changes may be low at present, it would be of interest to elucidate the intracellular signaling change with spontaneous behavior in future. 5 Sex Differences in ACh Release We first reported sex-specific ACh release in the hippocampus in 2003 (Mitsushima et al. 2003a). Gonadally intact male rats consistently show a greater ACh release in the hippocampus compared with diestrous or proestrous female rats, suggesting a sexually dimorphic septo-hippocampal cholinergic system. Moreover, we found that sex-dependent ACh release also shows a time-dependent 24-h profile: ACh release in the hippocampus was relatively similar in the light phase, but consistently lower in female compared with male rats in the dark phase (Masuda et al. 2005). Although ACh release clearly showed a daily rhythm in female rats, females exhibited smaller amplitude of daily change than males. However, it is necessary to rule out the possibility that the sex difference in ACh release reflects the differences in spontaneous locomotor activity levels. By simultaneous monitoring of ACh levels and spontaneous locomotor activity, we revealed a real sex difference in the “ACh release property” (Fig. 3, Mitsushima et al. 2009): males showed higher ACh release than females while displaying similar levels of behavioral activity. Although female rats showed slightly higher overall spontaneous activity than intact male rats, male rats showed higher ACh release than female rats. Simple linear regression analysis was used to evaluate the relationship between ACh levels and spontaneous locomotor activity (Fig. 3). Pearson’s correlation coefficient (r) or slope of the best fit line was calculated for each rat, and sex difference was evaluated using ANOVA. We found that the data from intact males had a steep slope of fit line, while the data from females had a gentle slope. These results suggest that sex-specific ACh release is not due to the change in spontaneous behavior, but due to actual differences in the ACh release property in gonadally intact rats (Mitsushima et al. 2009). To analyze the sex difference in the septo-hippocampal cholinergic neurons, we performed immunocytochemistry. Stereological analysis showed that no sex dif- ference was observed in the number of choline acetyltransferase immunoreactive (ChAT-ir) cells in the medial septum or horizontal limb of diagonal band (Takase et al. 2009). Since the number of septo-hippocampal cholinergic neurons does not appear to be involved in the sex difference in ACh release in the hippocampus, we 62 D. Mitsushima Male #102 Female #175 0.6 0.4 0.2 correlation (r) = 0.817 correlation (r) = 0.776 slope = 19.3 x 10–4 slope = 5.3 x 10–4 0.0 0 100 200 300 0 100 200 300 Locomotor Activity (counts/20min) Fig. 3 Sex specific ACh release property in behaving rats. Representative data from a male (#102) and a female (#175) rat were shown. Simple linear regression analysis revealed a sex-specific “ACh release property.” Male rats showed higher ACh release than females undergoing similar behavioral activity levels. Although both sexes showed a high correlation, male rats showed a steeper slope than female rats. (see Mitsushima et al. 2009) hypothesized that sex-specific neural circuits or substance(s) may control the endogenous release. 6 Neural Control of Septo-Hippocampal Cholinergic Neurons Neurotransmitters may be involved in expression of the sex difference in ACh release. For instance, dopaminergic neurons in the ventral tegmental area (A10) have been shown to control septo-hippocampal cholinergic neurons through the A10-septal dopaminergic pathway in male rats (Swanson 1982; Nilsson et al. 1992; Yanai et al. 1993). A neuroanatomical study suggested that dopamine D2 receptors rather than D1 receptors mediate the dopaminergic control of septo-hippocampal cholinergic neurons (Weiner et al. 1991). It has been shown that opiatergic neu- rons also control septo-hippocampal cholinergic neurons in male rats (Mizuno and Kimura 1996); the injection of naloxone, a m opioid receptor antagonist, into the medial septum markedly increased ACh release in the hippocampus, while a m opioid receptor agonist decreased its release (Mizuno and Kimura 1996). In con- trast, GABA seems to inhibit septo-hippocampal cholinergic neurons; the injection of muscimol, a GABA receptor agonist, into the medial septum decreased ACh release in the hippocampus, while the injection of bicuculline, a GABA receptor antagonist, increased it (Moor et al. 1998). Although the neural systems are still unknown for female rats, it seems likely that neural control of septo-hippocampal ACh (pmol/20min) Sex Differences in the Septo-Hippocampal Cholinergic System in Rats 63 cholinergic neurons is involved in the expression of sex differences in ACh release. It will be important to investigate these neural systems in female rats in future studies. 7 Circulating Sex Steroids Activate ACh Release Not only neurotransmitters, but also circulating sex steroids, may regulate cholin- ergic neurons. In fact, neuroanatomical studies have demonstrated that, in intact male and female rats, a number of dopaminergic neurons in the A10 region have androgen receptor immunoreactivity (Kritzer 1997) and 45–60% of choliner- gic neurons in the medial septum have estrogen receptor a immunoreactivity (Miettinen et al. 2002; Mufson et al. 1999). Taken together with the fact that female rats show a greater circulating estrogen concentration than male rats (Shors et al. 2001; Mitsushima et al. 2003b) and male rats show a greater circulating androgen concentration than female rats (Falvo et al. 1974; Rush and Blake 1982), it is possible that cholinergic neurons are affected by sex steroids differently in male and female rats. The activational effects of sex steroids on cholinergic neurons have been sug- gested by previous neuroanatomical and neurochemical findings. For example, male gonadectomy decreases the density of cholinergic fibers in the dorsal hippo- campus, while testosterone replacement in gonadectomized male rats maintains fiber density (Nakamura et al. 2002). Also, estradiol increases the induction of choline acetyltransferase in the basal forebrain in gonadectomized female rats (Luine et al. 1986; McEwen and Alves 1999). A previous in vitro study demon- strated that estradiol treatment increases both high affinity choline uptake and ACh synthesis in basal forebrain neurons (Pongrac et al. 2004). Furthermore, we recently reported an activational effect of sex steroids on the maintenance of stress-induced ACh release in the dorsal hippocampus in immobilized rats (Mitsushima et al. 2008). These findings suggest the activational effect of sex steroids on ACh release in the dorsal hippocampus, and we presented conclusive evidence of activational effects on dynamic ACh changes in behaving animals. To analyze the precise effects of sex steroids on ACh release, we simultaneously analyzed ACh release and spontaneous locomotor activity to determine the precise effect of sex steroids. Simultaneous analysis revealed that gonadectomy severely impaired ACh release without affecting spontaneous locomotor activity levels. Moreover, the activational effect on ACh release was apparent, especially during the active period, i.e., the dark phase, but not during the rest period, the light phase (Fig. 4 and Mitsushima et al. 2009). Our results provide the first evidence that the sex-specific 24-h profile of ACh release is highly dependent on the presence of sex steroids. Moreover, we found that after gonadectomy, the positive correlation between ACh release and locomotor activity levels was severely impaired, suggesting that hippocampal function may not always be activated at low sex steroid levels (Mitsushima et al. 2009). This therefore suggests that learning impairment in gonad- ectomized rats (Gibbs and Pfaff 1992; Daniel et al. 1997; Kritzer et al. 2001; 64 D. Mitsushima Males Gdx Males Gdx+T Males Gdx+E Males 0.6 0.4 0.2 0.0 12 18 0 6 12 12 18 0 6 12 12 18 0 6 12 12 18 0 6 12 Females Gdx Females Gdx+T Females Gdx+E Females 0.6 0.4 0.2 . 0.0 12 18 0 6 12 12 18 0 6 12 12 18 0 6 12 12 18 0 6 12 Clock time (h) Fig. 4 ACh release in the hippocampus is time-dependent, sex-specific, and hormone-dependent. Experiments were performed 2 weeks after gonadectomy or steroid replacement. Gdx, gonadec- tomized. +T, testosterone-priming. +E, estradiol-priming. The number of animals was 6–8 in each group. 19–5 h is the dark phase, shown as black bars on the x axes. (see Mitsushima et al. 2009) Markowska and Savonenko 2002; Luine et al. 2003) may be due to insufficient activation of hippocampus at the appropriate time. Because the replacement of sex- specific steroids restored the high positive correlation between ACh release and activity levels, the correlation appears to depend on the presence of sex steroids. These results suggest that circulating sex steroids strengthen the coupling between spontaneous behavior and ACh release (Mitsushima et al. 2009). 8 Sexual Differentiation Produces the Sex-Specific Activational Effect The activational effect of sex steroids was sex-specific (Fig. 4). Testosterone replacement in gonadectomized female rats failed to increase ACh release to levels seen in gonadectomized testosterone-primed male rats. Similarly, estradiol replace- ment was unable to restore ACh release in gonadectomized male rats. Moreover, estradiol consistently increases N-methyl-D-aspartate receptor binding and spine density in the CA1 area of gonadectomized female rats, although the treatment fails to increase these same parameters in gonadectomized male

rats (Romeo et al. 2005; Parducz et al. 2006). These results suggest that sex-specific steroids are important for maintaining hippocampal function. Based on our data, we hypothesized that the action of sex-specific steroids is due to neonatal sexual differentiation rather than the activational effects of sex steroids in adult rats. Moreover, in the latest study, we found that neonatal androgenization in females increased ACh release to resemble that of normal males without affecting spontaneous activity levels (Mitsushima ACh release (pmol / 20 min) Sex Differences in the Septo-Hippocampal Cholinergic System in Rats 65 et al. 2009). These results indicate an organizational effect on sex-specific ACh release in behaving rats, and support currently accepted theories of sexual differentiation. Because testosterone can be aromatized to estradiol in the forebrain, neonatal sex steroids activate both estrogen and androgen receptors (McEwen 1981). In our study, both testosterone and estradiol treatment in neonatal female pups masculi- nized ACh release profile in adults, suggesting an estrogen receptor-mediated masculinization of septo-hippocampal cholinergic systems (Mitsushima et al. 2009). These results are consistent with the previous finding that testosterone or estradiol treatment in neonatal female pups improves their adult spatial perfor- mance, whereas neonatal gonadectomy in male pups impairs the performance (Williams and Meck 1991). In contrast, dihydrotestosterone treatment failed to masculinize the ACh release profile. Although dihydrotestosterone has been classi- cally considered as a prototypical androgen receptor agonist, a metabolite of dihydrotestosterone, 3b-diol, has a higher affinity for estrogen receptor b (Lund et al. 2006). Therefore, dihydrotestosterone and its metabolites may stimulate both androgen receptor and estrogen receptor b, whereas estradiol stimulates estrogen receptors a and b. Considering the action of sex steroids and their metabolites, estrogen receptor a may mediate the organizational effect on the septo-hippocam- pal cholinergic system. 9 Interaction with Environmental Conditions Various environmental conditions may interact with the activational effects of sex steroids. First, we reported an interaction between stress and sex steroids. Although sex steroids did not show activational effects on baseline levels of ACh release, sex steroids clearly activated the immobility stress-induced ACh release response. In addition, we found that the contributing sex hormone effect to maintain the ACh release response was sex-specific: testosterone enhanced the ACh release response in male rats, while estradiol maintained the response in females (Mitsushima et al. 2008). Second, we reported an interaction between the light/dark cycle and sex steroids. Although sex steroids slightly enhanced ACh release during the light phase, the activational effects were much stronger during the dark phase (Fig. 4). Considering the fact that the time-dependent activational effect was also sex- specific and hormone-dependent, environmental conditions seem to have compli- cated interactions with sex steroids (Mitsushima et al. 2009). Some other environmental effects may affect the basal forebrain cholinergic system. Environmental conditions, such as complex or restricted (Brown 1968; Smith 1972), enriched or impoverished (Greenough et al. 1972), social or isolated conditions (Hymovitch 1952; Juraska et al. 1984; Seymoure et al. 1996), seem to affect spatial learning ability in a sex-specific manner. For example, male rats exhibited superior performance in learning maze tests compared with female rats if they were housed socially (Einon 1980). But if they were housed in isolation, 66 D. Mitsushima female rats exhibited a performance superior to that of male rats (Einon 1980). Although few studies were performed on the relationship between the sex-specific environmental effects and ACh release in the brain, we have reported that 4-day housing in a small cage attenuates the ACh release in the hippocampus in male rats (Mitsushima et al. 1998), but not in female rats (Masuda et al. 2005). Taken together, these results suggest that housing conditions contribute to the sex differ- ence in ACh release and spatial learning ability. Feeding conditions after weaning also affect spatial learning ability. If fed pelleted diet (i.e., standard laboratory diet), male rats show performance superior to that of female rats (Beatty 1984; Williams and Meck 1991). But when fed powdered diet, female rats, but not male rats, showed improved performance (Endo et al. 1994; Takase et al. 2005a). In our study, it was found that feeding with powdered diet after weaning increased ACh release in the hippocampus in female rats, but not in male rats (Takase et al. 2005b). A 24-h ACh release in female rats fed powdered diet was as high as that in male rats fed either powdered or pelleted diet, showing no sex difference. Since feeding with powdered diet improved spatial learning ability in female rats (Endo et al. 1994), the increase in the ACh release in the hippocampus in female rats fed powdered diet may partly contribute to this effect. Our findings provide evidence that environmental conditions such as housing or feeding may play a role in sex-specific hippocampal function. 10 A Possible Treatment Strategy for Alzheimer’s Disease Activational effects of sex steroids are very important in humans, since circulating sex steroid levels decline with age. A reduction in ACh synthesis is known as a common feature of Alzheimer’s disease (Coyle et al. 1983), afflicting more than 18 million people worldwide (Ferri et al. 2005; Mount and Downtown 2006). The disease is the most common form of dementia (Cummings 2004) and is frequently accompanied by insomnia, poor concentration, and day/night confusion (McCurry et al. 2004; Starkstein et al. 2005). The centrally active acetylcholinesterase inhibi- tor (donepezil) is effective in not only mild, but also moderate to severe cases (Petersen et al. 2005; Winblad et al. 2006), proving the importance of endogenous ACh in humans. In addition, women are twice as likely to develop the disease (Swaab and Hofman 1995), and estradiol seems to play a protective role (Zandi et al. 2002; Norbury et al. 2007). A recent study using single photon emission tomography showed that estrogen replacement therapy in healthy postmenopausal women increases muscarinic M1/M4 receptor binding in the hippocampus (Norbury et al. 2007). Conversely in men, testosterone but not estradiol seems to play a protective role (Moffat et al. 2004; Rosario et al. 2004) and testosterone supple- mentation clearly improved hippocampal-dependent learning deficits in men with Alzheimer’s disease (Cherrier et al. 2005). These results suggest a sex-specific activational effect of gonadal steroids on the cholinergic system in humans. Thus, there are many similarities between the rat model and the human studies, supporting Sex Differences in the Septo-Hippocampal Cholinergic System in Rats 67 the idea that gonadal steroid replacement therapy or an increase in bioavailability is beneficial when there is a subthreshold level of the hormone. Based on the neonatal sexual differentiation of the septo-hippocampal cholinergic system, we may have to search for sex-specific clinical strategies for Alzheimer’s disease. 11 Conclusions Gonadally intact male rats consistently show a greater ACh release in the hippo- campus compared with diestrous or proestrous female rats. The activational effects of sex steroids are important for sex-specific ACh release in the hippocampus, since impaired ACh release in gonadectomized rats does not show sex-specific effects. Neonatal treatment with either testosterone or estradiol clearly increased ACh release in female rats, suggesting neonatal sex differentiation of septo-hippocampal cholinergic systems. Moreover, environmental effects on the basal forebrain cho- linergic system seem to be sex-specific; housing in a small cage attenuated ACh release in male rats only, while feeding with powdered diet after sexual maturation increases ACh release in female rats only. 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Carroll Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 2 Menstrual Cycle and Hormonal Effects on Responses to Drugs of Abuse: Clinical Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3 Sex and Ovarian Hormones Influence Drug Seeking and Drug Taking: Preclinical Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 3.1 Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 3.2 Escalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 3.3 Extinction/Reinstatement (Relapse) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 4 Neurobiological Basis of Sex Differences and EST Effects in Drug Seeking . . . . . . . . . . . 82 4.1 Brain Dimorphism and Sex Differences in Drug Addiction . . . . . . . . . . . . . . . . . . . . . . . 83 4.2 Role of DA . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 4.3 Progestins’ Influence on the DA System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 4.4 Gamma-Aminobutyric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 4.5 HPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Abstract Human and animal research indicates the presence of sex differences in drug abuse. These data suggest that females, compared to males, are more vulnera- ble to key phases of the addiction process that mark transitions in drug use such as initiation, drug bingeing, and relapse. Recent data indicate that the female gonadal hormone estrogen may facilitate drug abuse in women. For example, phases of the menstrual cycle when estrogen levels are high are associated with enhanced positive subjective measures following cocaine and amphetamine administration in women. Furthermore, in animal research, the administration of estrogen increases drug taking and facilitates the acquisition, escalation, and reinstatement of cocaine-seeking behavior. Neurobiological data suggest that estrogen may J.J. Anker (*) and M.E. Carroll Department of Psychiatry, University of Minnesota, MMC 392, Minneapolis, MN 55455, USA e-mail: anke0022@umn.edu J.C. Neill and J. Kulkarni (eds.), Biological Basis of Sex Differences in Psychopharmacology, 73 Current Topics in Behavioral Neurosciences 8, DOI 10.1007/7854_2010_93, # Springer‐Verlag Berlin Heidelberg 2010, published online 12 October 2010 74 J.J. Anker and M.E. Carroll facilitate drug taking by interacting with reward- and stress-related systems. This chapter discusses sex differences in and hormonal effects on drug-seeking beha- viors in animal models of drug abuse. The neurobiological basis of these differ- ences and effects are also discussed. Keywords Drug abuse
Estrogen
Progesterone
Rats
Sex differences
Sex hormones 1 Introduction Historically, drug abuse has been considered a male disease. Research from several areas including epidemiology, behavioral pharmacology, and neuroscience has taken a male-centric approach when studying factors and/or treatments that influ- ence drug abuse. This approach has led to neglect of factors underlying drug abuse in women such as ovarian hormones. In fact, over the centuries, the predominant sex that abused drugs has varied from female to male, depending on cultural conditions (Kornetsky 2007). However, in recent years, epidemiological research has shown that females are catching up and exceeding males in their drug use, particularly among younger populations. Thus, an important direction for current research is to acknowledge that sex is a vulnerability factor in drug abuse and to study the neurobiological basis for this trend and its implications for drug abuse treatment (Ashley et al. 2003; Marsh et al. 2004). Clinical and preclinical research indicates that females, compared to males, exhibit greater vulnerability toward drug abuse at stages of the addiction process that mark transitions in drug use. These stages include drug initiation, bingeing, withdrawal, and relapse and may be modeled in animals using acquisition, escalation, withdrawal/extinction, and reinstatement procedures, (Carroll et al. 2009a). Clinical reports indicate that women are more likely than men to initiate drug use at an earlier age (Chen and Kandel 2002), engage in binge-like patterns of drug intake (Becker and Hu 2008; Brady and Randall 1999; Lynch et al. 2002; Mann et al. 2005; Randall et al. 1999), report greater difficulty in quitting (Becker and Hu 2008; Carpenter et al. 2006; Lynch et al. 2002), exhibit greater drug craving (Robbins et al. 1999), relapse (Ignjatova and Raleva 2009), and resume higher levels of drug use following relapse (Gallop et al. 2007). One area in which males exceed females in the phases of drug abuse is during withdrawal where males experience more severe withdrawal effects than females (Carroll et al. 2009b; Perry et al. 2008). Thus, elevated drug use in females may be due not only to their greater sensitivity to rewarding effects but also to their resilience to the negative effects of drugs. This differential sensitivity to the rewarding and aversive aspects of drug use has parallels in other addiction-prone and -resistant phenotypes (Carroll et al. 2009a), and it is an emerging area of research that should yield interesting developments. Females Are More Vulnerable to Drug Abuse than Males 75 Animal models of drug abuse add further support to enhanced female vulnera- bility across most stages of the addiction process. Female rats acquire drug self- administration at a faster rate, they exhibit greater binge-like patterns of drug intake, and they are more vulnerable to relapse of drug-seeking behavior (for reviews, see Carroll and Anker 2009; Carroll et al. 2009a; Lynch et al. 2002). The animal data suggest that females may be more vulnerable than males due to an underlying biological predisposition related to ovarian hormones or developmental/ organizational differences in male and female neurobiology (Becker and Hu 2008). As a consequence, it is important to identify biological vulnerability factors that contribute to the onset and progression of drug addiction in women. A growing number of findings suggest that the biological basis for increased drug abuse vulnerability in women may be attributed to female gonadal hormones (Becker and Hu 2008; Carroll et al. 2004; Festa and Quinones-Jenab 2004; Lynch et al. 2002; Terner and de Wit 2006). More specifically, enhanced drug-seeking and subjective effects in women are associated with higher levels of endogenous estro- gen (EST) (Evans 2007; Terner and deWit 2006). Further work supports this and has demonstrated that endogenous or exogenous EST (e.g., estradiol benzoate, 17b-estradiol) facilitates the acquisition, escalation, and reinstatement of cocaine- seeking behavior in female rats (for a review, see Carroll and Anker 2010). Preclini- cal work further suggests that EST’s potentiating effects on drug-related responses may involve activation of the EST-receptor subtype b (ER-b), whereas ER-a had little influence on drug seeking (Larson and Carroll 2007). In contrast to the potentiating effects of EST, progesterone (PROG), another female gonadal hormone, attenuates responses to drugs of abuse in both humans and animals. This chapter emphasizes sex and hormonal influences across several phases of the human drug abuse process that are represented by animal models. Particular attention is given to the effects of EST on drug-related responses; however, as PROG often opposes EST’s behavioral effects, its influence on drug-seeking behavior is also important to consider. The final section of this chapter discusses possible neurobiological mechanisms underlying sex differences and EST’s effects on addiction-related behaviors. EST’s interactions within the mesolimbic dopamine (DA) system, the hypothalamic-pituitary-adrenal (HPA) axis, and the involvement of ER-b receptors are considered. Since a majority of research on sex differences and hormonal influences on drug abuse involves psychomotor stimulants, this drug class is the primary focus. 2 Menstrual Cycle and Hormonal Effects on Responses to Drugs of Abuse: Clinical Evidence Natural fluctuations of hormones during the menstrual cycle correspond to differences in the physiological and subjective effects of some stimulant drugs 76 J.J. Anker and M.E. Carroll (Evans 2007; Terner and de Wit 2006). Cardiovascular and/or positive subjective responses to cocaine (Evans and Foltin 2006; Evans et al. 2002; Sofuoglu et al. 1999) and amphetamine (Justice and de Wit 1999, 2000; White et al. 2002), but not nicotine (Terner and de Wit 2006), were enhanced during the EST-dominant follicular phase of the menstrual cycle compared with the luteal phase (when EST levels are low) in women. These results were also extended to measures of stress reactivity and craving elicited by cocaine-related stimuli in cocaine- dependent women (Sinha et al. 2007). In this study, women in the follicular phase showed higher systolic/diastolic blood pressure measures, and they scored higher on self-reported measures of anxiety and drug craving following presen- tations of stressful or drug-related stimuli than women during the midluteal phase (Sinha et al. 2007). The finding with stress-induced craving is especially important given that clinical and preclinical reports implicate stress as a primary factor in drug bingeing and relapse (Covington et al. 2005; Sinha 2008), and stress is associated with heightened drug abuse vulnerability in women (Fox and Sinha 2009). Taken together, these results indicated that the phase of the menstrual cycle in which EST levels were highest was associated with high positive affective responses to cocaine and enhanced cue- and stress-induced cocaine craving. Thus, EST may operate as a vulnerability factor that facilitates the positive and aversive aspects of cocaine abuse in women. Others have found no differences in the physiological and subjective responses to cocaine during the female menstrual cycle (Lukas et al. 1996; Mendelson et al. 1999). However, methodological differences related to the dose and route of cocaine administra- tion may account for the discrepancy in results. Compared to results with stimulants, behavioral/subjective responses to other drugs of abuse do not consistently vary with the phase of the menstrual cycle in humans. Subjective measures were insensitive to menstrual cycle effects following alcohol (Freitag and Adesso 1993; Hay et al. 1984; Holdstock and de Wit 2000; Nyberg et al. 2004; Sutker et al. 1987), nicotine (Allen et al. 1999, 2004; Pomerleau et al. 1992, 2000; Snively et al. 2000), marijuana (Lex et al. 1984), and opioid (Gear et al. 1996) administration in women. In contrast to the results with EST, PROG has an opposite effect on subjective measures following drug administration (for review, see Evans 2007). Women treated with PROG showed a decrease in the positive-subjective effects of smoked (Evans and Foltin 2006; Sofuoglu et al. 2002) and iv (Sofuoglu et al. 2004) cocaine compared with placebo-treated controls. In addition, high circulating plasma levels of PROG were associated with decreased craving following a stress- or drug-related cue in cocaine-dependent women (Sinha et al. 2007). Overall, results from clinical studies indicate that the female gonadal hormone, EST, may be associated with sex differences in cocaine abuse, as phases of the menstrual cycle associated with heightened EST corresponded to increases in positive-subjective measures following cocaine. In contrast, PROG had an attenu- ating effect on these measures and suppressed stress- and cue-induced drug craving. Females Are More Vulnerable to Drug Abuse than Males 77 3 Sex and Ovarian Hormones Influence Drug Seeking and Drug Taking: Preclinical Evidence Preclinical studies corroborate clinical findings and confirm the importance of sex differences and hormonal influences during key phases of the addiction process as they are modeled in animals. In animal research, the self-administration paradigm is considered a valid model of human drug addiction (Panlilio et al. 2007), as subjects have control over their self-administration of the drug. Animal models of drug self- administration allow a controlled longitudinal approach to the study of factors that predict drug abuse in addition to potential treatments during critical transition phases of the drug abuse process (Carroll et al. 2009a). Phases to be discussed are acquisition (initiation) of drug use, maintenance of steady drug intake, bingeing or escalation of drug intake, extinction (withdrawal),

and reinstatement (relapse). The following section discusses sex differences and the effects of EST with regard to these important transition phases of drug addiction. Results from these studies are summarized in Table 1. 3.1 Acquisition In animal research, acquisition or the initiation of drug self-administration is measured using several techniques that primarily involve automatic (e.g., autoshap- ing) or experimenter-administered priming infusions of drug prior to, or at the beginning of, each self-administration session. Animals achieve acquisition criteria once they earn a predefined number of self-administered drug infusions. 3.1.1 Sex Differences Similar to clinical findings (Chen and Kandel 2002), females acquired drug self- administration faster than male rats across a wide range of drugs including canna- binoids (Fattore et al. 2007), cocaine (Jackson et al. 2006; Lynch 2008; Lynch and Carroll 1999b), methamphetamine (Roth and Carroll 2004a), nicotine (Chaudhri et al. 2005), and heroin (Carroll et al. 2002; Lynch and Carroll 1999b). Research with monkeys also confirmed that females acquired PCP self-administration more successfully than males, with 100% of the females acquiring compared to only 36.4% of the males (Carroll et al. 2000). Together, these results indicated that females had increased vulnerability to initiate drug use compared with males. 3.1.2 Hormonal Influences Preclinical work implicates EST in sex differences in the acquisition of drug abuse. Two methods are involved in investigating the contributions of EST and PROG in 78 J.J. Anker and M.E. Carroll Table 1 Summary of the sex differences and estrogen effects on behavioral responses to drugs of abuse across phases of the drug abuse process: animal models Independent variable Dependent Drug Finding Reference measure Sex differences Acquisition PCP F > M Carroll et al. (2000) (monkey) Escalation PCP F > M Carroll et al. (2005) Sex differences (rat) Acquisition Cannabinoids F > M Fattore et al. (2007) Cocaine F > M Lynch and Carroll (1999b), Lynch (2008), Jackson et al. (2006) Methamphetamine F > M Roth and Carroll (2004a) Nicotine F > M Chaudhri et al. (2005) Heroin F > M Lynch and Carroll (1999b), Carroll et al. (2002) Escalation Cocaine F > M Lynch et al. (2000), Lynch and Taylor (2004, 2005), Roth and Carroll (2004b) Extinction Cocaine F > M Lynch and Carroll (2000), Kippin et al. (2005), Lynch et al. (2005), Kerstetter et al. (2008), Perry et al. (2008) Cocaine-primed Cocaine F > M Lynch and Carroll (2000), Kerstetter et al. (2008), Anker et al. reinstatement (2009) Stress-induced Cocaine F > M Anker and Carroll (2010) reinstatement Cue-induced Cocaine M < F Fuchs et al. (2005) reinstatement Systemic estrogen Acquisition Cocaine OVX-E > OVX-V Lynch et al. (2001), Jackson et al. (2006) administration OVX-E > OVX-E+P Jackson et al. (2006) Heroin OVX-E > OVX-V Roth et al. (2002) Escalation Cocaine OVX-E > OVX-V Lynch and Taylor (2005), Larson et al. (2007) OVX-E > OVX-E+P Larson et al. (2007) Cocaine-primed Cocaine OVX-E > OVX-V Larson et al. (2005), Anker et al. (2007), Larson and Carroll reinstatement (2007) OVX-E > OVX-E+P Anker et al. (2007) OVX-ERbeta > OVX- Larson and Carroll (2007) ERalpha, OVX-V F Female, M Male, E Estrogen, V vehicle, P PROG, OVX ovariectomy Females Are More Vulnerable to Drug Abuse than Males 79 animal models of drug abuse. The first involves comparing drug-seeking behavior across different phases of the ovarian hormone cycle, and the second involves depleting naturally occurring levels of hormones through ovariectomy (OVX), administering EST and/or PROG, measuring the addiction-related response, and comparing this response to gonadally intact sham (SH)-operated controls. The acquisition rates for iv cocaine self-administration were reduced in OVX female rats compared with SH-operated females (Jackson et al. 2006), whereas the administration of EST in OVX females facilitated the acquisition of cocaine (Jackson et al. 2006; Lynch et al. 2001) and heroin (Roth et al. 2002) self- administration. The injection of the EST receptor antagonist, tamoxifen, blocked EST’s facilitation of cocaine acquisition in OVX female rats (Lynch et al. 2001). In contrast to EST, PROG had an opposite effect on the acquisition of drug self- administration. For example, Jackson et al. (2006) showed that PROG treatment blocked the effects of EST on the acquisition of cocaine self-administration in OVX female rats. Together, the results suggest that EST and PROG have opposite roles during the initiation of drug self-administration in females. 3.2 Escalation The transition from steady to dysregulated drug consumption characterizes the escalation phase of the drug abuse process (Ahmed and Koob 1998, 1999; Lynch and Carroll 1999a). In humans, escalation represents out-of-control drug bingeing that is linked to overdose and death (Kalivas and Volkow 2005). Females, com- pared to males, are more susceptible to binge-like drug intake (Brady and Randall 1999; Mann et al. 2005; Randall et al. 1999). Thus, it is important to use animal models to identify factors that contribute to the development of this critical aspect of drug addiction in women. Animal models offer a means to examine sex differ- ences and hormonal influences on binge-like drug intake. 3.2.1 Sex Differences Drug bingeing is modeled in animals using an extended-access procedure. In these studies, long access (LgA) to a self-administered drug (e.g., 6 h) results in increased drug intake over subsequent days (Ahmed and Koob 1999). In a study by Roth and Carroll (2004b), female rats escalated cocaine intake to a greater extent than males during LgA. Furthermore, females responded significantly more for iv infusions of cocaine at lower doses (Roth and Carroll 2004b). Similar results were reported with rhesus monkeys self-administering PCP (Carroll et al. 2005) providing cross- species evidence for sex differences. Females and males did not differ in PCP intake under a short-access condition (ShA) (3 h); however, females exceeded males in mg/kg PCP intake when access was extended to 6 h. Dose–response functions under a ShA progressive-ratio (PR) schedule before and after the LgA 80 J.J. Anker and M.E. Carroll condition indicated both groups experienced a rightward shift in their dose– response curves following LgA and that females (vs. males) consumed more PCP across several concentrations under the post-LgA PR condition (Carroll et al. 2005). Escalation of drug intake has also been modeled in animals using a dose self- selection procedure in which animals achieve a preferred dose of drug by responding on two levers that respectively increase or decrease the infusion duration (Lynch and Carroll 2001; Lynch et al. 1998). Female rats exhibited greater dysregulation of drug intake compared with males as determined by a lower correlation between the interdose interval and the preceding dose size, and females responded on the dose-increasing lever more than males (Lynch et al. 2000). Another method for assessing excessive drug intake in animal models is the discrete-trials procedure that allows two to four 10-min trials/h during self- administration. In a study by Lynch and Taylor (2004), male and female rats self- administered similar amounts of cocaine under ShA FR 1 and PR schedules. However, when rats were subsequently placed on the discrete-trials procedure for 7 days, females self-administered significantly more cocaine than males and showed greater disruption in diurnal self-administration patterns (Lynch and Taylor 2005). When performance under the PR schedule was reassessed 10 days following the discrete-trials procedure, females surpassed males in cocaine infusions. 3.2.2 Hormonal Influences In a study by Larson et al. (2007), the effects of EST and PROG were examined on cocaine self-administration under ShA and LgA conditions. Five groups were compared: OVX-VEH, OVX-EST, OVX-EST+PROG, SH-VEH, and SH-PROG. Prior to LgA, all groups exhibited similar levels of ShA cocaine intake, a finding consistent with previous studies (Cain et al. 2004; Larson et al. 2005; Lynch and Carroll 2000; Roth and Carroll 2004b). However, when access was extended to 6 h/day (LgA), groups SH-VEH, OVX-EST, and OVX-VEH escalated cocaine intake, whereas the PROG-treated groups (SH-PROG, OVX-EST+PROG) did not. Furthermore, OVX EST-treated rats escalated their drug intake more rapidly and self-administered more cocaine during LgA than OVX-VEH rats. Thus, exoge- nously administered EST facilitated the escalation of cocaine intake, whereas PROG attenuated it. Similar results have been reported with the 24 h/day discrete-trials procedure. Lynch and Taylor (2005) demonstrated that OVX rats treated with VEH earned fewer cocaine infusions under the discrete-trials procedure when compared with SH-operated controls, and EST enhanced drug intake in OVX female rats relative to VEH-treated controls to levels of the SH-operated rats (Lynch and Taylor 2005). Taken together, these results suggest that EST is involved in enhanced escalation of drug taking in females relative to males. Females Are More Vulnerable to Drug Abuse than Males 81 3.3 Extinction/Reinstatement (Relapse) Relapse is one of the most difficult aspects of drug abuse to treat due to the craving and other withdrawal effects that result in its high rate of recurrence (McKay and Weiss 2001). As previously mentioned, women are especially vulnerable to drug abuse relapse following a period of abstinence (Ignjatova and Raleva 2009), and once relapse occurs, women are prone to consume excessive amounts of drug (Gallop et al. 2007). Relapse is modeled in animals using the reinstatement proce- dure. Typically, animals are allowed to self-administer a drug for several days (usually 10–14 days). Drug solutions are then removed or replaced with saline, and animals subsequently extinguish their responding for the drug over the next 2–3 weeks. Subsequently, a priming stimulus consisting of the drug, a drug- associated cue, or a physical (e.g., shock) or chemical (e.g., yohimbine) stressor is introduced. Subsequent responding on the device previously associated with the drug delivery following one or more of the priming stimuli is considered a measure of reinstatement and a predictor of relapse in humans (Katz and Higgins 2003; Shaham et al. 2003). Results from animal reinstatement studies confirm female vulnerability during this critical stage of the drug abuse process and implicate EST in the facilitation of these measures. The following section discusses these findings. 3.3.1 Sex Differences Several studies have demonstrated sex differences in the extinction and reinstate- ment of drug-seeking behavior. During extinction, female (vs. male) rats show greater resistance to extinguishing responding that was previously maintained by iv cocaine infusions (Anker and Carroll 2010; Kerstetter et al. 2008; Kippin et al. 2005; Lynch and Carroll 2000; Lynch et al. 2005; Perry et al. 2008). Elevated responding in female rats relative to males extends to the reinstatement phase as well; however, this depends on the type of reinstatement stimulus used. Female (vs. male) rats responded more on a lever previously associated with cocaine self- administration than male rats following a priming injection of cocaine (Anker et al. 2009; Kerstetter et al. 2008; Lynch and Carroll 2000) and after 1, 14, 60, and 180 days of cocaine withdrawal (Kerstetter et al. 2008). In another study, Anker and Carroll (2010) showed that females reinstated significantly more than males follow- ing an injection of the pharmacological stressor, yohimbine. This is especially pertinent, as previous clinical studies indicate that female cocaine addicts are more vulnerable to stress-induced relapse than male cocaine addicts (Fox and Sinha 2009). 3.3.2 Hormonal Influences During the estrus phase, female rats were more resistant to extinction of lever pressing previously reinforced with cocaine than during any other phases of the rat 82 J.J. Anker and M.E. Carroll estrous cycle (Feltenstein and See 2007; Kerstetter et al. 2008). Furthermore, systemic injection of EST enhanced the cocaine-primed reinstatement responding in OVX female rats relative to OVX rats treated with VEH (Anker et al. 2007; Larson and Carroll 2007; Larson et al. 2005). Larson and Carroll (2007) examined the effects of ER-a and ER-b on cocaine-seeking behavior under a reinstatement procedure. Following extinction of lever pressing, OVX rats received acute sys- temic injections of EST (ER-a and ER-b agonist), the ER-a agonist propyl- pyrazole-triol (PPT), or the ER-b agonist diarylpropionitrile (DPN). They were then tested on cocaine-primed reinstatement of cocaine seeking. The results indi- cated that EST- and DPN-treated OVX rats reinstated significantly more than OVX rats treated with VEH, while there were no differences in reinstatement responding in OVX rats treated with PPT compared with those treated with VEH. Thus, EST may facilitate reinstatement responding via activation of ER-b. In contrast to the results with EST, PROG and its metabolite, allopregnanolone (ALLO), have opposite effects on animal models of relapse. Increases in plasma PROG levels in freely cycling female rats were associated with decreased reinstatement responding following a cocaine priming injection (Feltenstein and See 2007). In addition, systemic

injections of PROG in SH- and EST-treated OVX female rats attenuated reinstatement responding relative to SH rats treated with VEH and OVX rats treated with EST alone (Anker et al. 2007). The suppression of reinstatement responding by PROG may be attributed to its metabolism into ALLO. In a follow-up study, coadministering finasteride, a 5-a reductase inhibi- tor that prevents the conversion of PROG into ALLO, blocked PROG’s attenuat- ing effects on cocaine-primed reinstatement (Anker et al. 2009). Taken together, these results indicate that female gonadal hormones are involved in susceptibility toward (EST) and protection against (PROG) relapse of cocaine seeking in females. 4 Neurobiological Basis of Sex Differences and EST Effects in Drug Seeking Drugs interact with motivational systems that regulate survival behaviors, and drug and nondrug stimuli activate common neurobiological systems (Spanagel and Weiss 1999; Wise 1996). General reward-mediated responding involves the inter- action of several neuronal systems within the ventral and midbrain areas of the brain that contain the nucleus accumbens (NA) and the ventral tegmental area, collectively referred to as the mesolimbic reward pathway. This section discusses male/female brain dimorphism and the interaction between female sex hormones, and neurotransmitter systems within this motivational pathway, with respect to drug abuse. Most of this research has centered on stimulants, and consequently this drug class will be the focus. Females Are More Vulnerable to Drug Abuse than Males 83 4.1 Brain Dimorphism and Sex Differences in Drug Addiction Sexual dimorphism in areas of the brain involved in motivation and/or hormone- DA system interactions may play a key role in sex differences in drug abuse. These topics have been reviewed elsewhere (Becker 2009) and are briefly covered here. Masculinization of the brain occurs early during maturation (perinatal period) and is largely attributed to the gonadal hormone testosterone (Becker 2009; McCarthy et al. 1997), while feminization occurs in the absence of testosterone. Gonadectomy during this early period of brain sexualization decreased amphetamine-induced DA increases that occurred during adulthood in female rats, while it had no effect on adult males (Becker and Ramirez 1981a). Thus, it is hypothesized that sexual differentiation of key components of the DA system during periadolescence may later sensitize rats to the facilitating effects of EST and contribute to the sex differences in the reinforcing effects of stimulants (Becker 2009). Morphological differences in areas of the brain that regulate cocaine craving are also observed between sexes in adult humans. For example, men and women differ in the relative size of mesolimbic and mesocortical structures that are implicated in responses to drugs of abuse such as the cerebral cortex (Rabinowicz et al. 1999), medial amygdala (Mizukami et al. 1983), and the hippocampus (Fattore et al. 2008; Filipek et al. 1994). 4.2 Role of DA 4.2.1 Sex Differences Several lines of research indicate that sex differences and the influence of EST affect neurotransmitter systems that operate in the mesolimbic reward pathway to regulate the abuse-related effects of stimulants. In the striatum, there are clear sex differences in baseline DA tone and activation following exposure to drugs of abuse. Striatal D1 DA receptors decrease while D2 receptors increase cocaine- seeking behavior (Becker and Hu 2008; Self et al. 1996). Interestingly, there are approximately 10% more striatal D1 DA receptors in male rats compared to females (Andersen et al. 1997), which may explain why females outperform males on several measures of cocaine seeking. However, there are reportedly no sex differ- ences in D2 receptor densities in striatal regions in humans (Farde et al. 1995; Munro et al. 2006). There are also sex differences in extracellular striatal DA concentrations. For example, basal (Castner et al. 1993) and K+-stimulated (Walker et al. 2000) DA concentrations are greater in female rats compared to males, a finding that may be due to differential affinity for the DA transporter in presynaptic terminals (Walker et al. 2000, 2006). Protein kinase A (PKA) signaling has been shown to alter DA transmission within the mesolimbic reward pathway and is implicated in drug abuse (Nairn et al. 2004; Nestler 2005). In a study by Lynch 84 J.J. Anker and M.E. Carroll et al. (2007), females exhibited higher levels of PKA-mediated phosphorylization of DARPP-32 (DA- and cyclic AMP-regulated phosphoprotein) in the striatum and NA (Nazarian et al. 2009; Zhou et al. 2009), while Nazarian et al. (2009) reported similar results with PKA protein levels in the NA. Administration of stimulants enhances sex differences in dopaminergic activa- tion in the mesolimbic pathway, and this may also lead to subsequent sex differ- ences in drug reinforcement. For example, females exhibited increased striatal DA following amphetamine than males (Becker and Cha 1989; Becker and Ramirez 1981b), and they were more sensitive to the facilitating effects of cocaine on electrically stimulated DA release than males (Walker et al. 2006). Males and females also differed in activation of the DARPP-32 pathway in the NA following cocaine administration (females < males) (Lynch et al. 2007; Zhou et al. 2009). Striatal DA levels were greater in females than males following the administration of other drugs of abuse. For example, using in vivo microdialysis, Blanchard and Glick (1995) demonstrated that mesolimbic DA levels in the NA were greater in female rats following administration of low-to-moderate doses of alcohol, and female rats consumed more alcohol at these doses than male rats (Blanchard and Glick 1995; Blanchard et al. 1993). Females also exhibited an increased number of DA transporters in the NA following repeated injections of intravenous nicotine (Harrod et al. 2004). 4.2.2 Estrous Cycle and EST Sex differences in the activation of the mesolimbic DA system have been attributed to circulating hormones. Several studies have demonstrated that EST treatment enhances striatal DA release (Becker 1990a,b, 1999; Becker and Ramirez 1981a; Dazzi et al. 2007; McEwen and Alves 1999; Zhang et al. 2008) and induces conditioned place preference when injected in large doses (Frye and Rhodes 2006) in OVX rats relative to VEH-treated controls. Striatal DA levels are also significantly higher in gonadally intact females compared to OVX female rats (Becker and Beer 1986; Becker et al. 1984; Becker and Ramirez 1981a), suggesting that the absence of EST may decrease DA levels. This may explain why a lack of EST, due to natural fluctuations or pharmacological and/or surgical manipulation (Anker et al. 2007; Larson and Carroll 2007; Larson et al. 2005, 2007; Lynch et al. 2001), leads to attenuated cocaine seeking. Ligand-bound EST receptors regulate the transcription of proteins involved in the DA system (Jones and Miller 2008). Indeed, D2 receptor densities in the striatum and other areas of the brain implicated in addiction vary across the estrous cycle in rats. They are greater following natural elevations of EST or following systemic EST administration (Bazzett and Becker 1994; Czoty et al. 2009; Di Paolo et al. 1988; Pazos et al. 1985; Zhou et al. 2002) and decrease significantly within 2 weeks following OVX (Le Saux et al. 2006). In contrast, in one study using positron emission tomography, D2 receptor concentrations were significantly lower Females Are More Vulnerable to Drug Abuse than Males 85 during the EST-dominant follicular compared to the luteal phase of the menstrual cycle. There is also evidence indicating that intracellular DA activity changes across the estrous cycle in female rats, and this may also contribute to cycle-dependent alterations in responses to drugs of abuse. Weiner et al. (2009) demonstrated that phosphorylated DARPP-32 levels in female rats were significantly lower during the estrus phase compared to all other phases of the estrous cycle. This result was explained as a consequence of heightened DA levels in the NA during the estrus phase, and lower DARPP-32 levels reflected a compensatory mechanism to stabi- lize excessive DA concentrations (Weiner et al. 2009). Research also indicates hormone cycle mediation of dopaminergic responses to stimulants. For example, amphetamine-stimulated DA release in striatal tissue was increased during the estrus phase of the estrous cycle as determined using in vitro infusion (Becker and Ramirez 1981b), microdialysis (Becker and Cha 1989), and voltammetry (Becker 1990b). Several studies also implicate EST in modulating stimulant-induced dopaminergic activity. For example, EST treatment in OVX rats promoted DA neuronal sensitivity to cocaine, while DA neurons in OVX rats treated with VEH produced no change (Zhang et al. 2008). In another study, the induction of DA by cocaine- and amphetamine-regulated transcript (CART), a protein the regulates mesolimbic function in response to stimulants (Kuhar et al. 2005), was enhanced by EST administration in OVX rats relative to OVX rats treated with VEH (Shieh and Yang 2008). Furthermore, this effect was attrib- uted to an intracellular mechanism as only administration of EST that was perme- able to cellular membranes facilitated CART-induced DA turnover (Shieh and Yang 2008). EST treatment also facilitated nicotine-evoked DA release in the striatum in female, but not male rats (Dluzen and Anderson 1997), suggesting that the effects of EST on stimulant-induced DA are sex specific. 4.2.3 Estrogen Receptor Subtype b Facilitation by EST on the reinforcing effects of cocaine may be attributed to the interaction between ER-b and DA neurotransmission in the mesolimbic pathway. ER-b is found in DA neurons (Laflamme et al. 1998) and has been shown to influence DA receptor expression and neurotransmission (Morissette et al. 2008; Schultz et al. 2009) in the mesolimbic DA pathway. Furthermore, administration of the ER-b agonist DPN, but not the ER-a agonist PPT, reversed OVX-induced decreases in D2 receptors and DA turnover within the striatum and NA core (Le Saux and Di Paolo 2006). ER-b also regulates cocaine-seeking behaviors. As previously noted, administration of the ER-b agonist DPN, but not the ER-a agonist PPT, enhanced cocaine-primed reinstatement (Larson and Carroll 2007) and amphetamine-induced CPP (Silverman and Koenig 2007) in OVX female rats. Also, administration of tamoxifen, a partial antagonist at ER-a, but pure antagonist at ER-b, reduced EST’s-facilitating effects on the acquisition of cocaine 86 J.J. Anker and M.E. Carroll self-administration in EST-treated OVX female rats (Lynch et al. 2001). The contribution of ER-b to the rewarding effects of cocaine may be attributed to an intracellular mechanism involving downregulation of the regulator of G-protein signaling (RGS) 9-2. RGS9-2 regulates intracellular D2 receptor activity and is highly localized in the NA following chronic cocaine exposure (Rahman et al. 2003; Wood 2007). Mice lacking this gene show enhanced responsiveness to cocaine (Rahman et al. 2003). Silverman and Koenig (2007) demonstrated that administration with an ER-b agonist, but not an ER-a agonist, reduced RGS9- 2 expression in the core of the NA, a structure also implicated in cocaine-induced reinstatement (Ping et al. 2008), and enhanced amphetamine-induced CPP in OVX female rats. Together, these results suggest that EST enhancement of stimulant- related behaviors involves an interaction between ER-b and the mesolimbic DA pathway via intracellular transcription-related mechanisms that influence D2 recep- tor activity. 4.3 Progestins’ Influence on the DA System Far less work has been conducted on the effects of progestins on the mesolimbic DA pathway. However, the PROG metabolite, ALLO, attenuated stress-induced increases in DA (Dazzi et al. 2002), and altered DA release in the striatum and NA (Barrot et al. 1999; Jaworska-Feil et al. 1998; Laconi et al. 2007; Rouge-Pont et al. 2002). PROG also modulate DA levels in the striatum; however, results were equivocal and dependent on the time of testing, manner of administration, and presence or absence of EST (Dluzen and Ramirez 1984; 1987a,b; Fernandez-Ruiz et al. 1989). Thus, PROG and ALLO may interact with DA systems to influence drug-seeking behavior, but further work is needed to substantiate this. 4.4 Gamma-Aminobutyric Acid An additional mechanism that may underlie EST and PROG’s influence on cocaine seeking may be related to gamma-aminobutyric acid (GABA) neurotrans- mission. The facilitation and inhibition of GABA receptor neurotransmission resulted in the suppression and enhancement of mesolimbic DA, respectively (Tam and Roth 1985, 1990). Decreased GABA release was also associated with increased DA mediated behavior such as drug seeking in rats (Caille and Parsons 2004; Tang et al. 2005), while the administration of GABA receptor agonists decreased cocaine seeking (Campbell et al. 1999, 2002). This suggests that decreased GABA is associated with heightened vulnerability to cocaine-seeking behavior, whereas increased GABA may attenuate this behavior. Previous work indicated that EST inhibited activation of medium spiny GABAergic neurons in the striatum (Mermelstein et al. 1996), increased striatal GABA release (Hu et al. Females Are More Vulnerable to Drug Abuse than Males

87 2006), enhanced DA metabolism and turnover (Di Paolo et al. 1985; Shimizu and Bray 1993), and enhanced stimulant-elicited DA release in the striatum (Becker 1990a,b; Becker and Beer 1986; Castner et al. 1993). Conversely, PROG and its metabolites promoted striatal-GABA activity (Schumacher et al. 1989a,b) and decreased DA in the striatum (Dazzi et al. 2002; Dluzen and Ramirez 1987b; Jaworska-Feil et al. 1998; Laconi et al. 2007; Shimizu and Bray 1993). Thus, the different effects of EST and ALLO on cocaine seeking may be explained by their opposite effects on GABA and/or DA neurotransmission in areas of the brain that regulate drug seeking. 4.5 HPA Stress is a major contributor to drug abuse (Fox and Sinha 2009), and activation of the stress system, the hypothalamic-pituitary-adrenal (HPA) axis, is associated with enhanced drug reward (Goeders 2002a,b). Preclinical work indicates that EST potentiated the release of CRF (Patchev et al. 1995; Swanson and Simmons 1989) and increased adrenocorticotropic hormone and corticosterone (Burgess and Handa 1992), which led to increased HPA activity (Dallman et al. 2004). These effects also extended to cocaine-induced HPA activation. In a study by Niyomchai and colleagues (2005), EST increased cocaine-induced corticosterone levels relative to VEH-treated controls. Behavioral studies provided further support for the role of EST on stress-related responses. EST facilitated fear-potentiated startle in OVX female rats relative to OVX rats treated with VEH, while PROG attenuated this facilitation (Hiroi and Neumaier 2006; Toufexis et al. 2004). Interestingly, ALLO attenuated HPA activation (Drugan et al. 1993; Frye et al. 2006; Owens et al. 1992; Patchev et al. 1994; Purdy et al. 1991), and it blocked stress-induced reinstatement in rats (Anker and Carroll, 2010). Further work is needed to examine the interaction between EST, the effects of stress, and behavior associated with drug abuse. Overall, the neurobiological findings indicate that there are sex differences in DA receptor densities, intracellular DA neuronal activity, and extracellular DA levels in areas of the brain implicated in drug abuse vulnerability. Results presented in this chapter implicate EST in these differences possibly through its interaction with the ER-b. EST also decreases striatal GABA activation, and that may facilitate DA activation leading to increased sensitivity to the rewarding effects of drugs of abuse. Further, EST facilitates neurobiological and behavioral substrates of stress, which is a primary vulnerability factor in drug abuse. In contrast to EST, PROG and its metabolite ALLO exert an opposite effect on DA neurotransmission and poten- tiate GABA receptors and HPA activation that may lead to an attenuation of drug reinforcement. In conclusion, the results indicate that females are more vulnerable to drug abuse than males during almost all of the critical phases of drug abuse: initiation, maintenance of rewarding effects, escalation of intake, extinction/craving, and 88 J.J. Anker and M.E. Carroll reinstatement/relapse. Females are also more responsive than males to a wide range of behavioral and pharmacological interventions that reduce drug taking and drug seeking. In contrast, males exhibited greater withdrawal effects than females, suggesting they are more sensitive to the aversive effects of drugs, while females are more responsive to the rewarding effects. These differences in responsiveness to the positive and negative effects of drugs are an emerging area of interest for medication development and other treatment approaches for drug abuse. 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