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P ! P - C ! P C ! ! P ! 3 LD E B B ) &EBB)' 2 2 E B &EBB)' 5 " !0# N " !0# N " !# % !0# 0 ,!"# !# % !0# 0 ,!"# " ! 8* ! 6 FDG FDG 1 C C ! ! P P % C 5 C ! ! P P / C 2 C ! ! P ! 4 C ! * ! ! P - C P ( !7 8 C778 ?75 C4 4 64 2 4' 7 ? ? 4 8 * JL5 00 018 ! #0 +7 4B8 7378 . 8 778 38 %7375 G3 4 ' - 7 3 - A A8 J!""L5 0 +(8 ! 1! )7 48 778 - 8 ?7F78 3 2 8 A778 A . 8 37 75 $ 4' 2 4 - 7 ? 4 = . 4 8 !(J)L5 +!0 ++8 +((! 7 8 ?78 C8 75 9 2 - 24 ! J- L7 A 3 - A- -- 8 "J0L5 #)0 #18 ! #1 M CN "7 8 %75 2 2 4 5 . 4 .' .7 %5 !0 3 2 =4 - 2 4 2 4 = .7 = 8 78 8 8 8 $ 8 +(() #7 8 %78 C8 D8 448 8 .8 ?5 C4 2 ' 2 4 4 5 - 4 7 %5 % 3 2 4 2 4 7 = 8 &78 --7 )!+ )!07 C4' 4 8 ?-8 2 4 8 D7D78 ! 1# 07 8 78 ?7=75 4 2 4 - 2 24 ' 422 7 %5 4 2 4 8 --7 !#0 !0!7 = 8 ?7 =78 F8 A7 -8 48 8 8 737 9 %78 37:7 3 - 8 ! 1 17 8 F7C75 4-48 8 4 ' 7 - -- 2 U V ' 7 - 8 !0J+L5 !0) !1(8 +((! 7 8 7G78 8 9778 -8 =7F75 - 2 422 - 7 - 8 !(5 ++18 ! 1# !(7 8 7G78 &. 8 7 78 8 778 3 8 7 78 8 7 ?75 $' . 4 2 4 7 ? - 8 +#J!L5 ! "8 ! 1 !!7 8 7G78 8 ?7 78 C .8 =778 . 8 7 ?78 .8 :7375 ' - 2 4 422 7 3 - 8 !(JL5 1+) 1)18 ! ! !+7 8 975 7 %5 45 . 2[ D 4 8 --7 1 !(7 = $ 8 C78 8 7 4 8 &. / 8 8 +((+ 5/ !)7 8 978 F 8 &78 9 8 78 .8 78 8 =78 $ 8 C75 9 - 2 H4 3 ' 4 2 2' 4 2 4 24 -7 G24 8 !(0J"L5 ) 0 (+8 +(( M $N !7 48 78 2 8 78 8 5 - 2 2 ' 4 . 2 4 7 %5 & 4 48 --7 )" #7 = 47 &8 C8 4- \ 8 +(() !"7 48 78 68 378 F8 $78 D8 7$78 A 8 78 8 A75 4 - 2 2 H4 2 24 2 - ' 447 ? 4 = . 4 8 !(JL5 + )(18 +((! !#7 . 8 75 34 - 4 4 47 ? 8 +00 +18 ! ! !07 48 G78 8 5 44 -2247 %5 44 8 --7 +) +#(7 = 8 78 .8 7 [8 F8 8 G 4 .B 8 ! # !17 48 7 78 9 8 ?7 C75 4 4 2 ' - 24 2 - -5 2 2 ' - 4 ' 7 - 8 !#J0L5 #00 # 8 +((( ! 7 3 8 ?7 ?78 8 3778 G8 ?75 2 ' 4 7 - 8 "JL5 +" +"08 ! 1 +(7 3 $7 7 ?78 ?75 2 4 7 3 ' - -8 "5 !08 ! 1" +!7 3 2 8 A775 4-4 4 - 2 - 2 422 7 4 $ 8 !"J"L5 ##0 #0+8 ! 1+ ++7 3 8 778 8 A778 S4 8 7 ?75 4 4 - 5 F - X 8 0+J! +L5 " 08 ! 0 +)7 3 8 37A78 4 8 775 2 24 2 4 7 3 - A A8 J+!L5 !#( !#8 ! 10 +7 3 8 =7:75 C4 2 7 %5 4 8 --7 )#0 !+7 = A . 8 3778 ?78 C778 %%%8 --7 )#0 !+7 -8 8 8 8 8 8 F7 7 4 3 - 8 ! ( +"7 348 A775 2 7 34 - - ' 7 3 - A A8 +(15 )( ) 8 ! 1# +#7 9. 8 ?78 CB-8 A78 38 75 2 4 7 - 2 H4 7 ? ? 4 8 1!J)L5 +( +#8 ! +07 9. 8 ?78 CB-8 A78 38 75 S4 -- 2 - 4 4 4 7 ? ? 4 8 01JL5 " ) #((8 ! # +17 98 75 A 8 8 4 7 %5 4 2 4 8 --7 )" #7 = 98 78 -- )" #7 -8 ?7 7 --' 3 - 8 ! 1) $ 54 + 7 9 7C78 3 $78 $775 : ' 2 4 6 7 % 3 4 5 +"" +1!8 ! )(7 93 4 778 775 -7 38 !!5 +!" ++08 ! "# )!7 98 7C75 4 2 4 4 6 7 4 3 & 8 )5 !"! !""(8 ! #) )+7 =8 75 9 2 - 422 7 3 - A A8 J+"L5 # 08 ! ( ))7 =8 78 $8 $7 75 - - 2 24' 422 7 = ' =8 78 $8 $7 78 -- !1! +(+7 -8 8 D 8 8 48 8 8 C 8 ! ) )7 =8 78 $8 $7 75 2 -' 422 5 ' - ' -7 = =8 78 $8 $7 78 --7 !"" !1(7 -8 8 D 8 8 48 8 8 C' 8 ! ) )"7 = 8 F7C78 -8 7 78 $ 8 775 2 4 2 44 7 3 - A A8 !")5 !(# !+(8 ! 1( )#7 =-8 A7=78 .B8 7=78 C8 7$78 C8 ?7 78 ?78 8 97$75 5 - A 2 -24 4 7 A 8 !10J+L5 0 1!8 ! ) )07 =48 =78 A[ 8 75 -4247 %5 44 8 --7 +#! +0+7 = 8 78 .8 78 -- +#! +0+7 [8 F8 8 G .B 8 ! # )17 C 8 78 48 78 38 75 . 422 5 2 4 2 7 %5 4227 = $B 8 978 $ B8 78 8 78 --7 +#! +#"7 8 =8 ! 0 ) 7 C 8 ?78 A 8 775 - 4-4 -7 %5 4 2 2 - 4
7 &. 8 48 +(() (7 C8 ?7 78 8 F778 ?8 7=78 8 C778 %%%5 $ 4 - 7 . 422 2 7 ? ' - 8 )J!L5 ) #8 ! 11 !7 C 8 A7 ?78 . 8 D7 78 $B8 7 75 4 2 -4B ' - 4 2 7 ? ? 4 8 0J0L5 !()+ !()08 ! + +7 $W8 75 9 44 - 7 % "7 94 ]' 7 .B G2 4 8 --7 !)+ !)#7 8 8 &. / 8 - 8 ! 11 )7 $8 ?78 8 75 32 2 4 7 - 8 +J)L5 +1 )(+8 ! " M $N *7 $8 375 T\-47 3 T C38 ))5 "! 07 8 =- 2H4 C8 ! 11 "7 $8 378 & 228 A7F75 32 2 4 6 7 3 - A A8 J((L5 #" 0#8 +((+ #7 $ 8 C78 = 4 8 8 B8 C5 - 2 2 4 2 -4 2 4 6 7 ? 4 = . 4 8 )5 !!! !+18 ! 5- 07 $ 8 775 9 4 4- 2 4- 4 4- -7 ? - 48 0J+L5 !(#8 ! ) 17 $ 8 775 -4 24 5 7 ? - 4 8 )J!L5 ++ ))8 ! " * 7 $ 48 978 8 ?7 78 48 ?78 48 78 : 8 7375 C 4 ' 422 4-47 ' - - 4 3 7 3 - A A8 )(5 01 1)8 ! "(7 8 78 $ B8 78 A8 75 = 4 2 2 4 ' - - 4 4 5 - - 4 7 ? 4 = . 4 8 1J!L5 ## 08 ! "!7 8 78 8 5 A 8 A 4 B-7 = 78 --7 ))) )07 [8 ?8 G 4 C8 +((+ "!7 8 78 8 78 8 75 ' 2 - . 7 - 7 ? ? 4 '+((# "+7 8 78 8 78 8 78 8 778 8 75 ' 4- - 2 4 4 2 -4 5 - - - 4 7 ? 4 = . 4 8 !)J!L5 " !+8 +((* ")7 8 78 8 D8 . 5 A 7 %5 4' 44 7 = 8 78 D4 8 78 .8 78 --7 !* !#07 [8 F8 8 G 4 .B 8 ! ( "7 8 78 F $75 - 422 7 %5 A 422 7 = 4 8 F78 ?78 --7 !+ !" 7 8 -8 8 8 8 4 8 D 8 48 8 8 F' .8 F F8 ! # ""7 8 D78 C44 8 78 8 78 D 8 /75 A - 2 422 7 ' 7 3 - A A8 +"5 + #8 ! ( "#7 4-8 7 ?78 3 2 8 A775 2 4 5 - 2 8 8 4 4 7 ? 4 = . 4 8 1J#L5 "" "#8 ! "07 .8 A78 48 ?778 48 $7 ?78 8 ?75 2 4' 4 4 24 - H47 - 8 !+5 0+08 ! 11 "17 8 78 C8 78 8 $75 W A 4 B-7 %5 45 . 2[ D 4 7 = $ 8 C78 8 78 4 8 &. / 8 $ : 8 )!( )!#8 +((+ " 7 8 A7 78 8 &7$78 : $48 A778 F 8 :7 75 . #' 2 .'4- 4 2 422 5 - 2 4 47 ? 4 = . 4 8 J"L5 )0) )1!8 +((( #(7 4 8 778 8 7378 8 375 9 - 2 4-- 4 45 9 J 2 8 4 L M ' $ 51 N7 G-- = 3 $ 4- JG=3$L7 ? % 8 + J#L5 #(+ #(18 ! # #!7 % 8 A78 $8 78 8 75 =- 2 -4 247 %' 2 2 ))1 247 - 8 ##J"L5 ) " ) 08 ! " #+7 ? 8 78 8 75 - 2 2 4 247 G24 8 10J!!L5 #0 0)8 ! 1* M $N #)7 ? 8 A78 D8 78 8 D78 $B8 A78 [8 7=75 32 ' - 2 2 24 2 - 447 %5 4 2 4 7 = 8 ?78 F8 A778 --7 ))( ))7 -8 8 48 8 737 9 %78 37:7 3 - 8 ! 1 #7 ? 8 C7F78 $ 8 37=78 D8 A7 78 $ 48 A7=75 -4 4 2 4 - 7 ? - 8 ! J"L5 +1 )8 ! ! #"7 ? 8 C7F78 ? 8 37 75 24 64 2 4 7 3 - A A8 J!0)L5 !!0 !+8 ! 1) ##7 ? 8 C7F78 D8 A7 78 $ 8 37=75 4 - .' 7 - 2 422 - 7 - A8 !1J L5 #) 0"8 ! 1 #07 D 8 7 %78 A 8 778 3 8 7 ?78 :8 &7&78 8 ?7D78 8 7A75 & ' B 2 7 ? 4 = . 4 8 !J)L5 +0 +1"8 +((" #17 D8 F7 78 48 ?75 -4 4 -7 ? - 4 8 !!J+L5 !+ !"!8 +(() # 7 D 8 78 B8 378 $228 75 - 4 2 ' -2 H4 ' 2'2 H4 2 2 4225 F A 3422 % 7 3 ? - 8 !)J+L5 1 +8 +(() 0(7 D 8 78 $228 78 8 78 & 8 75 - 4 2 '-2 H4 2 2 4 2 4 7 F 4 % % JF%L7 ? - 8 +#J#L5 0#* 00+8 ! 1 0!7 D6 ' 8 78 C8 778 66 8 ?778 --8 75 - 2 2 . 4 - 7 ? - 8 J+L5 !(8 ! 1 0+7 D48 78 - 8 ?7F78 4. 8 $778 3 2 8 A775 - 4' 24 2 4 - 7 ? ? 4 8 1#'JL5 #1( #1 8 +(( 0)7 D8 A778 ? 8 C7F78 ? 8 37 75 4 . 7 3 - 8 !JL5 !0 )"8 ! " 07 T%8 ?7378 F8 A7 C78 3 8 7 78 8 97F78 8 7$75 2' H4 2 2 - 24 2 4 7 ? ? 4 8 0 J"L5 0)1 018 ! 0 0"7 8 78 98 D78 =8 75 A - 4 2 4 2 27 A 9 J L8 !1JL5 1! * !8 ! 00 55 0#7 \ 38 C ?75 A4 2 4 5 -' 4 2 - 7 %5 4 - 7 = F8 $78 48 78 --7 ++! +)(7 8 8 &. / 8 8 D 8 8 8 8 - 8 8 - 8 ! 007 8 C75 5 7 ? ? 4 8 ! 5 #1) #1 8 ! )0 017 8 78 8 5 - - $ 4 7 %5 44 7 = 8 78 --7 +)0 +0!7 [8 ?8 G' 4 C8 +((+ 0 7 --8 78 9778 8 C775 - 2 4 24 5 - 4 7 %5 4 5 2 7 = 8 C7 78 %%%8 C48 C7 78 .8 A7 ?78 --7 "(! "!17 A 8 2 - 4 8 ! + 1(7 8 %7D78 $228 78 D 8 75 - 2 '-2 H4 2 2 4 2 2 4 5 F ' 2 4 JFL 7 3 8 J1L5 00! 0018 +((! 1!7 4 8 7 ?75 2 B 4 7 3 - 7 22 2 -4 4 7 44 ' 2 6 -47 7 - 4--8 !! 5 ! " 8 ! # 1+7 228 7F78 $8 $7 78 8 75 4- 4'- ' - 2 4 7 ? - 8 +)J!L5 ) 18 ! " 1)7 48 &7978 = 8 F7C78 8 A775 - - - 7 ' 2 7 ? ? 4 8 ""J0L5 !)"! !)##8 ! 0) 17 8 C778 %%%8 8 9778 + 8 8 ?7 75 2 4 7 3 - 8 !(JL5 01) 0118 ! ! 1"7 \8 978 D-28 ?7 C78 $ B8 78 A 8 78 8 C78 F8 $75 A4 2 ' - 2 ' - 2 4227 +7 32' 2 4227 A 3 - A- -- 8 0 J0L5 ")+ "*!8 ! ) M CN 1#7 \8 978 A 8 78 A 8 &78 \ 8 378 F8 $75 3 - ' 5 4 4 7 %5 4 - 7 = F8 $78 48 78 --7 !#) !0!7 8
8 &. / 8 ' 8 D 8 8 8 8 - 8 8 - 8 ! 107 8 378 A8 A7978 8 7 ?75 2 4 7 %5 "1 4 2 2 - 4 7 8 32 8 ! ! 117 8 37978 48 7 78 8 78 $-8 75 - %% 5 4 - - 4- 422 7 - 8 !J#L5 "") "#"8 ! 1 1 7 8 7C75 4 8 +17 = 4 8 34 8 ! 0+ $ 52 (7 4 8 7D78 F 8 :7=78 C 8 $7D75 A 422 . 7 ? ? 4 8 ##J)L5 +0 + 8 ! 1 !7 [8 75 - 2 2 24 2 7 %5 ' 4 2 2 7 = [8 78 >.8 78 8 A78 F' 8 78 --7 !!1 !+"7 8 8 8 &. / 8 - ': 8 ! 11 +7 48 7 78 : 4 8 3778 &8 7 ?75 2 - 2 2 - 7 ? 4 = . 4 8 J"L5 )1+ )1"8 +((( )7 & %%8 3775 9- - 4 247 %7 32 4' 7 ? ? 4 8 "+J#L5 !(00 !(1 8 ! 0( 7 & %%8 3775 C 4' 2 2 - 4 245 -4- 47 ? 4 = . 4 8 !!JL5 )1 ((8 +((+ "7 & %%8 3775 C4 2 . 2 ' 4 47 ? 48 )5 !!(8 ! #) #7 & %%8 3775 C4 2 2 7 3 - A A8 "15 ) "(8 ! #1 07 & %%8 3775 %- 7 3 - A A8 !0)5 0( 008 ! 1) 17 & %%8 3775 & 4 2 7 ? 8 !0+5 !((# !(!!8 ! #( 7 & %%8 3778 C 8 37A75 %2 -4 2 2 4 2 4 2 4 7 - - 7 ? ? 4 8 #+J#L5 1 0 (18 ! 1( !((7 & %%8 3775 9 7 %5 4 4 7 = & %%8 3778 --7 +0* )#+7 -8 8 8 8 8 8 F7 7 4' 3 - 8 ! ( !(!7 & %%8 3775 C47 %5 4 4 7 = & %%8 3778 --7 )#) +(7 -8 8 8 8 8 8 F7 7 4 3 - 8 ! ( !(+7 & %%8 3775 $ 4 - 7 %5 4 4 7 = & %%8 3778 --7 !) +0!7 -8 8 8 8 8 ' 8 F7 7 4 3 - 8 ! ( !()7 &8 7 ?75 -47 - 3 & 8 !1J)L5 ) )8 ! 10 !(7 &8 7 ?75 4 - 4 5 4 2 2 4 7 - 8 J!L5 !0 +!8 ! ) !("7 &8 7 ?75 $9 5 4 2 4 -7 ' - 8 J!L5 ++ +)8 ! ) !("7& \'? 8 78 8 75 4 4 2 4 5 - 2 7 %5 F8 $78 48 7 J 7L5 4 - 8 --7 +) +"(7 8 8 - 8 ! !(#7 4 8 A7=75 2 -7 ? ? 4 8 ) 5 0"+ 0"08 ! #! !(07 8 975 32 2 422 7 3 - A A8 J+"L5 1! 1#8 ! ( 23 !(17 A 8 78 D.B8 ?7 ?75 5 7 %5 9 2 4 5 7 = % 8 ?78 F8 $7A78 --7 + " )! 7 -8 -- F @ F8 ! !( 7 A8 75 4 2 2 B 4 7 ? A4' 8 JL5 ! ) ! #8 ! 0" !!(7 A 8 7 78 4 978 48 78 8 D7 78 8 778 B8 75 4--4 - 4 - 5 8 8 - 4 7 &4 4 8 "5 0 "!8 ! 0 !!!7 A 3 8 77=778 A 8 A7A78 8 D7&78 8 7G78 C ' A7 ?78 $8 $7 78 $ 7$78 % 8 ?778 .8 :7378 ?778 Y48 ?7975 B 2 2 4 24' 7 ? 4 = . 4 8 )5 )0 )"+8 ! !!+7 A8 7 %78 A 8 ?778 A 8 78 8 7 %75 2 ' 4 2 4 -7 8 )!J L5 !! ! !! 18 ! 0# !!)7 A 8 D7=78 4 ' 8 =78 68 &78 48 /75 9 - 2 4 - 7 3 A8 JL5 !) ! 8 ! ! !!7 A 8 37 75 C4 2 47 =- 2' 7 ? ? 4 8 1(J)L5 0# 18 ! 1 !!"7 A . 8 3778 ?78 F $7A78 / 4 8 97375 %64 2 4' ? 7 %5 C4 47 = A . 8 377 ?78 $ 9778 4' B8 A7F78 8 ?7978 --7 !)! !!7 -8 &. / 8 --' A8 ! # !!"7A . 8 3778 ?78 F8 775 %64 4 ? 7 %5 C' 4 47 = A . 8 377 ?78 $8 9778 4 B8 A7F78 ' 8 ?7978 --7 !!" !0!7 -8 &. / 8 -- A8 ! # !!#7 A .8 37A78 8 978 4 8 F7F75 - 45 ' '4 4 7 ? ? 4 8 #(J!L5 ! !#8 ! 01 !!07 8 A778 8 :75 9 - 2 4 7 ? ? 4 8 #"JL5 "# #(8 ! 1) !!17 8 7$78 8 78 $8 375 4 4 7 32 - 7 - 8 +#J!(L5 ( !8 ! 0 M $N !! 7 8 ?7 C78 .8 A7 ?75 32 - 2 2 4 7 3 - A A8 + !5 0 ! 8 ! ) !+(7 48 C78 C 8 78 4 8 978 4H48 978 F8 $78 8 975 $ 4 - 2 4 ' . 4-4 2 4227 A4 2 4 4 2 1( 4 7 ? ? 4 8 1#J)L5 )11 ) "8 +(( !+!7 8 7 ?75 - 2 2 422 4 ' 7 %5 4 - 7 = 8 7 ?78 --7 +(! +(07 ' -8 -- F F8 +(() !++7 8 7 ?75 J ' L 7 %5 4 ' - 7 = 8 7 ?78 --7 !!" !)+7 &. / 8 7 48 C' 8 4 8 ^8 38 8 8 8 3 8 8 $ 2 8 &. 98 8 ?48 - 8 8 8 8 $.'8 ! * !+)7 8 7 ?75 4- 48 - 2 4 7 %5 4 - 7 = 8 7 ?78 --7 !0 !#"7 -8 ' 8 &. / 8 8 4 8 D 8 8 8 -- F F8 +(() !+7 - 8 ?7F78 3 2 8 A778 T9 8 7F78 8 7=78 A . 8 37 75 A - 2 . 4 - 7 ? 4 =' . 4 8 J#L5 "(0 "!)8 +((( !+"7 4B8 $78 D48 778 D48 C78 $8 75 - 2 - 5 2 2 47 D 4 - 4 ' 8 1J"L5 +0( +08 +((( !+#7 8 :78 -8 7978 =8 ?7 78 ?75 - 2 4 7 %5 9 2 4 5 7 = % 8 ?78 F8 $7A78 --7 "! 0#7 -8 8 &. / 8 8 4 8 D 8 8 8 -- F F8 ! 1 !+07 8 7378 8 C778 %%%5 -- - 2 4 2 ' 4 2 4 4 ' 7 ? ? 4 8 0!JL5 "(# "!)8 ! 1 !+17 B48 78 A 8 /78 48 378 948 ?7 78 8 C75 - 2 4--4 7 A% "" - . 422 - ' 7 - 8 #0J)L5 +#* +#18 ! # !+ 7 8 ?7978 8 &7378 8 7 75 4 - 5 424 - 2 2 7 3 - A A8 +15 !!! !! 8 ! #) !)(7 G 228 7D78 8 ?7F75 32 - 2 4225 - ' 8 8 7 ? - 4 8 "JL5 !1) ! !8 ! 0 !)!7 : 8 3778 ?78 ? 8 7 78 F 8 78 ? 8 9775 2 2 - 2 -4 47 4 2 !(( 4 7 ? ? 4 8 0#J#L5 "! "8 ! * !)+7 : 8 ?778 8 378 48 78 A . 8 3778 ?8 D775 3422 - 5 - 8 2 8 2 7 ? ? 4 8 1# 4-- +5 )" (8 +(( !))7 F8 $75 9 2 4 2 H4 2 2 4 7 %5 C - 3 2 =4 - 2 4 2 4 = .8
--7 "! ""7 8 ! 10 !)7 F8 $78 8 A78 48 78 D 4 8 75 - 4 2 - 4 7 ? - 8 !J#L5 0"# 0#(8 ! !)"7 F8 F778 8 ?7 78 F 8 7 75 - . 4 2 4 7 %5 4 2 4 7 = 8 ?7 =78 F8 A778 --7 ! "7 8 37:7 8 ! 1* !)#7 F 8 78 8 778 8 ?7 ?75 =- . & 4 ' -7 %5 4 2 4 7 = 8 ?7=78 F8 A778 --7 ++* ++17 -8 8 737 9 %78 37:7 3 - 8 ! 1* 2 !)07 F 28 =7 78 3 8 ?7378 9 8 D75 4 4 2 4 4 2 4 7 - 8 !!J"L5 #(( #(08 ! " !)17 F 28 A7=78 = 8 F7C75 4 2 44 ' -7 - 3 & 8 +0J)L5 0) 1!8 ! # !) 7 F 8 7F78 3 2 8 A775 4 24 2 4 - 7 ? ? 4 8 00J L5 !)( !)#8 ! " !(7 / 48 D78 8 A75 9 2 - 7 %5 9 2 4 5 = % 8 ?78 F8 $7A78 --7 !" ! (7 -8 8 &. / 8 8 4 8 D 8 8 8 -- F F8 ! !!7 /8 =778 A--8 7 ?78 8 78 $8 375 3 ' 4 7 ? 4 = . 4 8 !J+L5 !+1 !))8 +((" !+7 Y8 78 $8 378 8 ?75 S4 2 4 2 422 . 7 % A 8 ))J)L5 !#) !0(8 ! 1 !)7 Y 8 978 9 8 :7:75 3 4 - 24 2 -47 - 8 "J!L5 #( #"8 ! 0* !*7 $ 378 D4 A75 % 4-4 2 2 4-4 4' 7 ? ? 4 J L8 0)5 )1 ) 8 ! ! !"7 $ 7 ?78 $ &75 - 4 5 2 2 2 4 7 ? ? 4 8 1+ 5 # 08 +((! !#7 78 . A78 75 = - 4 - ' 2 4 7 ? - 8 !05 #* #18 ! 1 !07 C A778 ?75 % 2 7 %5 & 2 2 7 F F8 - ")7 8 ! 1( !17 - 2 9778 = F7 ?75 4 2 - 2 2 7 3 -8 !)(5 !0"8 ! 01 ! 7 34 A775 4 4 2 2 447 ? ? 4 8 "1' 5 )!)8 ! 0# & , ' - ! ) 3'6 ) + H4 4 - 44' 44 00 28 4 ' 4 # - ! " 4 64 ) 0) 4 6 ! ! - + -4 ! 2 - !! 0) 2 A%% #! -4 4 10 4 = . 4 !"" J=L +++ - - !"" - 2'4 +++ 4 #" - ++0 !!0 4 ++1 4 24 !(#8 !(08 !!! !+0 !( '32 !)! 4 !!0 !)) 3 T !10 !)) - J3L 422 +) !)0 22 +* 24 - !)" 3 ' 4 ! 44 - !)0 ' '' !)) +(* -- !)+ ' -2 +(+ 4 42 2 !)! H4 +(" '- 24 !)! 4 +(* - 24 !)! +# 4 !#0 422 - +!8 !#( 4 !#0 !#+ 0) 2 -8 2 )" 9 2 8 4 - 2 )" J9L +!) 2/ & !2 4 ++( 24' !+)8 !)( ++( 2 B 4 !) 9 E - ++! - !) ++! !) - 4 ++! 24 2 422 +# - !#) 3'6 ) 4 # $9 0" 4 " 4 * 8 ")8 "# 4 0) 2 "* #* ") - 4 - 44' "# 00 - - - "( 4 "1 2 ! " 8 24 !1 -- 24 !!0 !#+ !!0 24 !1 0" - - ' 4 !"" - 8 !"0 24 !"( 2 4 )) 0 3 )* 2 4'-8 24 !+ 4 - !+ $ 0" 24 '- 24 !+) 4 !(#8 !(08 !!! - ! 0 !( " 4 !!0 ' 0 -- !!0 1( !!0 1( 2 4'- !+ 4 !#0 !1 4 !#0 !1 4 44 !"0 !"( 44 248 H4 2 - ' '- !+) !( 448 H4 2 - !( 4 !!( 4 !0 - !+# %$8 - "" -- 4 !+ - 08 1 - 4 !! 2 !"0 -4 !) 2 4 "* 4 ' !+! 648 4 ) ) !7 24 64 8 3'6 !() 4 - ) #+ - 0" . % !1# 4 #" . % !10 #* . %%% !1 4 #1 - 4 0# 4 0# 4 0# 422 !"1 4 4 0# !" 4 #" +1 4 # 4 ## 4 24 !!( !1* 4 24 !0 4 ") 4 - #! 4 #!8 #1 4 - 0+ 6 2 !0 44 - !1 6 !1* 4 ! 44 !1) 4 ! & ' 0" T% +# 0) 0) - 24 !+# 0) 2 2 - )" -4 4 10 - 2 )" 4 0) ) 0" 2 $9 0" * 0 !#" $ 0" 4 8 2 !# ' 0 2 . 1* 1( 2 H4 +") 1( 2 4 H4 ' ' ( +"# 4 ## & ' 0" 0) -4 # -' +( S4 0" -- 4 24 !+ UV !11 0) )# - 4- 0! 2 )# - 2 %$ "" 2- & !2 - ## -4 24 !) - 4 0# 3'6 64 !() 4 0# - !0 4 0# - !1( 4 4 0# !1! - 4 24 !! !1( -4 # H4 2 - 44 24 !( C')# +)) 3 4 +( S4 0" C')#! 4 +)" H4 +# C')#! +)* 4 H4 +1 C')#+ 4 +)1 S49 +! C')#+ +)0 C')#+ +! : +7( +! 4 !) 4 ! ! 4 - - ! +8 ! * J9%L +) ! )8 ! +" 4 - ! ! - 4 JL ++1 4 " ++ 4 !0! )# 2 !0 2 )# B !0+ 4 - ) 2 !0" 4 32 - 4 2 !0" %4 422 2 2 !0! JL ++ . !0) #+ . 2 4 !0) 4 6 ! . - 2 !0+ 44 . 2 !0+ H4 4 - 00 422 H4 ' 2'2 4 - 4 - 00 JA3'SL +## 4 !"0 422 !0 4-4 +1 - +) 4- 4 4- - 2 +* J3L !"! 24 +# 4--4 " - - !08 ! 4--4 4 - + A . +" A% + 2 +" 4- )( 4 ' 24 !+! * * UV !11 -4 * 2 * ) !7 21 )! G3 4 +!+ 4 #" +# +1 -B +1 + : ++ 8 4-4 +1 - !"0 '- !+ 4 # 48 4 8 4' JG L " 4 2 +# - 0" F'94- +# G "
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Fourth Edition Clinical Laboratory Hematology Shirlyn B. McKenzie, PhD, MLS(ASCP)CM, SH(ASCP)CM Medical Laboratory Sciences, Department of Health Sciences, School of Health Professions UT Health San Antonio Kristin Landis-Piwowar, PhD, MLS(ASCP)CM Clinical and Diagnostic Sciences, School of Health Sciences Oakland University J. Lynne Williams, PhD, MT(ASCP) Clinical and Diagnostic Sciences, School of Health Sciences Oakland University Courseware Portfolio Manager, Health Sciences: John Goucher Editorial Assistant: Cara Schaurer Managing Content Producer: Melissa Bashe Content Producer: Michael Giacobbe Design Coordinator: Mary Siener Vice President of Sales and Marketing: David Gesell Vice President, Director of Marketing: Brad Parkins Director, Digital Studio: Amy Peltier Digital Project Manager: Ellen Viganola Full-Service Project Management and Composition: Pearson CSC Full-Service Project Manager: Pearson CSC, Dan Knott Manufacturing Buyer: Maura Zaldivar-Garcia, LSC Communications, Inc. Cover Designer: Pearson CSC Copyright © 2020 by Pearson. All rights reserved. Manufactured in the United States of America. 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Library of Congress Cataloging-in-Publication Data Names: McKenzie, Shirlyn B., author. \| Landis-Piwowar, Kristin, author. \| Williams, Joanne Lynne, 1949- author. Title: Clinical laboratory hematology / Shirlyn B. McKenzie, Kristin Landis-Piwowar, J. Lynne Williams. Other titles: Pearson clinical laboratory science series. Description: Fourth edition. \| Hoboken : Pearson, [2020] \| Series: Pearson’s clinical laboratory science series \| Includes bibliographical references and index. Identifiers: LCCN 2019010608\| ISBN 9780134709390 \| ISBN 013470939X Subjects: \| MESH: Clinical Laboratory Techniques \| Hematology—methods \| Hematologic Diseases—diagnosis \| Hematopoietic System—physiology Classification: LCC RB45 \| NLM WH 25 \| DDC 616.1/5075—dc23 LC record available at https://lccn.loc.gov/2019010608 ISBN-13: 978-0-134-70939-0 ISBN-10: 0-134-70939-X To my family, the wind beneath my wings, Gary, Scott, Shawn, Belynda, and Dora; my special grandchildren Lauren, Kristen, Weston, Waylon, and Wyatt; to the memory of my parents, George and Helen Olson. —Shirlyn B. McKenzie To Theron and Kaia, you sustain my every breath and to Todd, you are my rock. —Kristin R. Landis-Piwowar For my mother, Mary Williams, who gave her children roots as well as wings; for Lee, Laurie, Roger, and Richard, who sustain my roots; for Dulaney, Corie, Chris, Ava, and Holden, whom I love as my own; and to the memory of my father, David Williams. —J. Lynne Williams iii This page intentionally left blank Contents Foreword xvii Development of Hematopoiesis 30 Preface xviii Hematopoietic Tissue 30 Bone Marrow 30 Section One Thymus 34 Introduction to Hematology 1 Spleen 35 Lymph Nodes 38 1 Summary • Review Questions • References Introduction 2 Overview 3 4 Hematopoiesis 42 Introduction 3 Composition of Blood 4 Overview 43 Reference Intervals for Blood Cell Concentration 4 Introduction 43 Hemostasis 5 Hematopoiesis 44 Blood Component Therapy 5 Hematopoietic Precursor Cells 44 Investigation of a Hematologic Problem 6 Cytokines and the Control of Hematopoiesis 52 Cytokine Receptors, Signaling Pathways, The Value of Laboratory Testing 6 and Transcription Factors 57 Summary • Review Questions • References Hematopoietic Microenvironment 59 Components of the Hematopoietic 2 Cellular Homeostasis 9 Microenvironment 61 Hematopoietic Microenvironment Overview 10 Niches 61 Introduction 11 Summary • Review Questions • References Review of Cell Structure 11 Cell Membrane 11 Cytoplasm 12 5 The Erythrocyte 66 Nucleus 13 Overview 67 Review of the Flow of Genetic Information 13 Introduction 68 DNA Replication 13 Erythropoiesis and Red Blood Cell Maturation 68 Transcription 14 Erythroid Progenitor Cells 68 Translation 14 Erythroid-Maturing Cells 68 Protein Degradation 15 Characteristics of Cell Maturation 69 Tissue Homeostasis: Proliferation, Differentiation, Erythroblastic Islands 72 and Apoptosis 15 Erythrocyte Membrane 72 Proliferation: The Cell Cycle 16 Membrane Composition 72 Differentiation 19 Lipid Composition 73 Apoptosis 20 Protein Composition 74 Abnormal Tissue Homeostasis and Cancer 23 Membrane Permeability 76 Summary • Review Questions • References Erythrocyte Metabolism 77 Glycolytic Pathway 77 Section Two Hexose Monophosphate (HMP) Shunt 77 The Hematopoietic System 27 Methemoglobin Reductase Pathway 78 Rapoport-Luebering Shunt 78 3 Structure and Function Erythrocyte Kinetics 80 of Hematopoietic Organs 28 Erythrocyte Concentration 80 Regulation of Erythrocyte Production 80 Overview 29 Erythrocyte Destruction 82 Introduction 29 Summary • Review Questions • References v vi Contents 6 Hemoglobin 87 8 Lymphocytes 138 Overview 89 Overview 140 Introduction 89 Introduction 140 Hemoglobin Structure 89 Lymphopoiesis 140 Hemoglobin Synthesis 90 Ontogeny of Lymphopoiesis 140 Heme 90 Transcriptional Regulation of Lymphopoiesis 141 Globin Chain Synthesis 91 Cytokines in Lymphopoiesis 141 Regulation of Hemoglobin Synthesis 94 Antigen-Dependent and -Independent Ontogeny of Hemoglobin 95 Lymphopoiesis 141 Embryonic Hemoglobins 95 Mature Lymphocytes 142 Fetal Hemoglobin 95 Lineage Differentiation 142 Adult Hemoglobins 95 B Lymphocytes 143 Glycosylated Hemoglobin 96 T Lymphocytes 147 Hemoglobin Function 96 Natural Killer Cells 150 Oxygen Transport 96 Natural Killer T (NKT) Cells 150 Carbon Dioxide Transport 100 Lymphocyte Identification and Morphology 151 Nitric Oxide and Hemoglobin 101 Morphologic Classification of Immature Lymphocytes 151 Artificial Oxygen Carriers 101 Morphology of Activated Lymphocytes 153 Hemoglobin Catabolism 102 Lymphocyte Distribution, Concentration, and Kinetics 154 Extravascular Destruction 102 Lymphocyte Function 154 Intravascular Destruction 103 B Lymphocytes (Humoral Immunity) 155 Acquired Nonfunctional Hemoglobins 104 T Lymphocytes (Cell-Mediated Immunity) 156 Methemoglobin 104 Natural Killer Cells 157 Sulfhemoglobin 105 Adhesion Molecules of the Adaptive Immune Response 158 Carboxyhemoglobin 106 Aging and Lymphocyte Function 159 Summary • Review Questions • References Lymphocyte Metabolism 159 Summary • Review Questions • References 7 Granulocytes and Monocytes 110 Overview 112 9 The Platelet 163 Introduction 112 Overview 164 Granulopoiesis and Monocytopoiesis 112 Introduction 164 Leukocyte Concentration in the Peripheral Blood 113 Megakaryocytes 165 Leukocyte Surface Markers 114 Megakaryopoiesis 165 Leukocyte Function 114 Thrombopoiesis 169 Neutrophils 115 Peripheral Blood Platelets 169 Differentiation, Maturation, and Morphology 115 Platelet Morphology 169 Distribution, Concentration, and Kinetics 119 Quantitative Platelet Evaluation 170 Function 120 Platelet Function 170 Eosinophils 126 Summary • Review Questions • References Differentiation, Maturation, and Morphology 126 Distribution, Concentration, and Kinetics 127 10 The Complete Blood Count and Function 128 Peripheral Blood Smear Evaluation 174 Basophils 128 Differentiation, Maturation, and Morphology 129 Overview 176 Distribution, Concentration, and Kinetics 129 Introduction 176 Function 129 Pre-Examination Phase of the CBC 177 Monocytes 130 Examination Phase of the CBC 177 Differentiation, Maturation, and Morphology 130 Automated Results 177 Distribution, Concentration, and Kinetics 132 The Peripheral Blood Smear 181 Function 132 Clinical Laboratory Professional’s Review Summary • Review Questions • References of CBC Data 193 Contents vii Post-Examination Phase of the CBC 194 Laboratory Evaluation 242 Physiologic Variation in Hematologic Parameters 194 Therapy 245 CBC Variations in Newborns and Children 194 Anemia of Chronic Disease 245 CBC Variations Between Ethnic Groups Etiology 245 and Sexes, in Elderly People, and Pathophysiology 245 by Geographic Location 194 Clinical Presentation 246 Summary • Review Questions • References Laboratory Evaluation 246 Therapy 247 Section Three Iron Refractory Iron-Deficiency Anemia (Irida) 247 The Anemias 199 Pathophysiology 247 Clinical Presentation 247 11 Laboratory Evaluation 247 Introduction to Anemia 200 Therapy 248 Overview 202 Functional Iron Deficiency (Fid) 248 Introduction 202 Etiology 248 How Anemia Develops 202 Laboratory Evaluation 248 Interpretation of Abnormal Hemoglobin Therapy 248 Concentrations 203 Anemias Associated with Abnormal Heme Synthesis 248 Adaptations to Anemia 203 Sideroblastic Anemias 248 Increase in Oxygenated Blood Flow 204 Therapy 253 Increase in Oxygen Utilization by Tissue 204 Hemochromatosis 253 Diagnosis of Anemia 204 Hereditary Hemochromatosis 253 History 204 Secondary Hemochromatosis 255 Clinical Presentation 204 Treatment 255 Laboratory Evaluation 206 Porphyrias 255 Classification of Anemias 211 Etiology 255 Morphologic Classification 211 Pathophysiology 256 Functional Classification 213 Clinical Presentation 258 Classification Using the Red Cell Distribution Laboratory Evaluation 258 Width 218 Prognosis and Therapy 258 Laboratory Testing Schemas for Anemia Diagnosis 218 Summary • Review Questions • References Summary • Review Questions • References 13 Hemoglobinopathies: Qualitative 12 Anemias of Disordered Regulation Defects 265 of Iron Metabolism and Heme Overview 267 Synthesis 224 Introduction 267 Overview 226 Structural Hemoglobin Variants 268 Introduction 227 Identification of Hemoglobin Variants 268 Iron Metabolism 227 Methods of Analysis 269 Distribution 228 Nomenclature 270 Absorption 228 Pathophysiology 271 Transport 230 Sickle Cell Anemia 271 Storage 232 Pathophysiology 271 Physiological Regulation of Iron Balance 233 Clinical Presentation 273 Iron Requirements 238 Laboratory Evaluation 276 Laboratory Assessment of Iron 238 Therapy 277 Iron Studies 238 Sickle Cell Trait 278 Iron-Deficiency Anemia 240 Other Sickling Disorders 278 Historical Aspects 240 Hemoglobin C Disease 279 Etiology 240 Hemoglobin S/C Disease 279 Pathophysiology 241 Hemoglobin D 280 Clinical Presentation 241 Hemoglobin E 281 viii Contents Unstable Hemoglobin Variants 281 Megaloblastic Anemia 323 Pathophysiology 281 Clinical Presentation 323 Clinical Presentation 282 Laboratory Evaluation 324 Laboratory Evaluation 282 Folate 327 Therapy 283 Cobalamin (Vitamin B12) 331 Hemoglobin Variants With Altered Oxygen Affinity 283 Other Megaloblastic Anemias 338 Hemoglobin Variants with Increased Macrocytic Anemia Without Megaloblastosis 340 Oxygen Affinity 283 Alcoholism 340 Hemoglobin Variants with Decreased Liver Disease 341 Oxygen Affinity 283 Stimulated Erythropoiesis 342 Methemoglobinemias 284 Hypothyroidism 342 Summary • Review Questions • References Summary • Review Questions • References 14 Thalassemia 289 16 Hypoproliferative Anemias 346 Overview 291 Introduction 291 Overview 348 Thalassemia versus Hemoglobinopathy 292 Introduction 348 Types of Thalassemia 292 Aplastic Anemia 348 Genetic Defects in Thalassemia 293 Epidemiology 348 Pathophysiology 293 Pathophysiology 348 Clinical Presentation 294 Classification and Etiology 349 Laboratory Evaluation 295 Clinical Presentation 352 Treatment 295 Laboratory Evaluation 353 a@Thalassemia 296 Prognosis and Therapy 354 General Considerations 296 Differentiation of Aplastic Anemia from other a@Thalassemia Major (a0/a0 or a@thal@1/a@thal@1); Causes of Pancytopenia 355 Hydrops Fetalis 298 Pure Red Cell Aplasia 356 Hemoglobin H Disease (a0/a+ or a@thal@1/a@thal@2 298 Acute Acquired Pure Red Cell Aplasia 356 a@Thalassemia Minor (a+/a+ or a@thal@2/a@thal@2; Chronic Acquired Pure Red Cell Aplasia 357 a0/a or a@thal@1/normal) 300 Diamond-Blackfan Syndrome 357 Silent Carrier (a+/a or a@thal@2/normal) 301 Other Hypoproliferative Anemias 358 b@Thalassemia 301 Renal Disease 358 General Considerations 301 Endocrine Abnormalities 359 b@Thalassemia Major (b0/b0, b0/b+, b+/b+) 303 Summary • Review Questions • References b@Thalassemia Minor (b0/b or b+/b) 306 b@Thalassemia Intermedia (b+/b+, b0/b+, b0/b) 307 b@Thalassemia Minima (bSC/b) 307 17 Hemolytic Anemia: Membrane Other Thalassemias and Thalassemia-Like Conditions 308 Defects 364 dbThalassemia 308 gdbThalassemia 308 Overview 366 Hemoglobin Constant Spring 308 Introduction 366 Hereditary Persistence of Fetal Hemoglobin (HPFH) 309 Skeletal Protein Abnormalities 366 Hemoglobin Lepore 310 Vertical Interactions 366 Combination Disorders 311 Horizontal Interactions 366 Differential Diagnosis of Thalassemia 313 Lipid Composition Abnormalities 366 Summary • Review Questions • References Hereditary Spherocytosis 367 Pathophysiology 368 15 Megaloblastic and Nonmegaloblastic Clinical Presentation 369 Macrocytic Anemias 319 Laboratory Evaluation 369 Identification of Deficient/Defective Overview 321 Membrane Protein 371 Introduction 321 Therapy 371 Contents ix Hereditary Elliptocytosis 371 Clinical Presentation 393 Pathophysiology 371 Laboratory Evaluation 394 Clinical Presentation 372 Therapy 394 Laboratory Evaluation 372 Other Enzyme Deficiencies in the Glycolytic Pathway 394 Therapy 373 Abnormal Erythrocyte Nucleotide Metabolism 395 Hereditary Pyropoikilocytosis (HPP) 373 Summary • Review Questions • References Pathophysiology 373 Clinical Presentation 373 Laboratory Evaluation 374 19 Hemolytic Anemia: Immune Therapy 374 Anemias 399 Hereditary Stomatocytosis Syndromes 374 Overview 401 Pathophysiology 375 Introduction 401 Laboratory Evaluation 375 Classification Of Immune Hemolytic Anemias 401 Therapy 375 Sites And Factors That Affect Hemolysis 403 Abnormal Membrane Lipid Composition: Mechanisms Of Hemolysis 404 Acanthocytosis 375 IgG-Mediated Hemolysis 404 Spur Cell Anemia 376 Complement-Mediated Hemolysis 404 Abetalipoproteinemia (Hereditary Acanthocytosis) 376 IgM-Mediated Hemolysis 405 Lecithin-Cholesterol Acyl Transferase (LCAT) Laboratory Identification of Sensitized Red Cells 405 Deficiency 377 Direct Antiglobulin Test 406 Rare Forms 377 Indirect Antiglobulin Test 406 Paroxysmal Nocturnal Hemoglobinuria (PNH) 377 Negative DAT in AIHA 407 Pathophysiology 377 Positive DAT in Normal Individuals 407 Clinical Presentation 378 Autoimmune Hemolytic Anemias (AIHA) 407 Laboratory Evaluation 378 Warm Autoimmune Hemolytic Anemia 408 Therapy 379 Laboratory Evaluation 409 Summary • Review Questions • References Cold Autoimmune Hemolytic Anemia 411 Paroxysmal Cold Hemoglobinuria 414 18 Hemolytic Anemia: Enzyme Mixed-Type AIHA 415 Deficiencies 383 Drug-Induced Hemolytic Anemias 415 Alloimmune Hemolytic Anemia 417 Overview 385 Hemolytic Transfusion Reactions 418 Introduction 385 Hemolytic Disease of the Fetus Hexose Monophosphate Shunt 385
and Newborn (HDFN) 419 Glycolytic Pathway 385 Summary • Review Questions • References Clinical and Laboratory Evaluation in Erythrocyte Enzyme Deficiencies 386 Diagnosis 387 20 Hemolytic Anemia: Nonimmune Glucose-6-Phosphate Dehydrogenase Deficiency 387 Defects 429 Etiology 387 Overview 431 Pathophysiology 387 Introduction 431 G6PD Variants 389 Hemolytic Anemia Caused by Physical Injury to Females with G6PD Deficiency 389 the Erythrocyte 431 Clinical Presentation 390 Thrombotic Microangiopathic Anemia (TMA) 431 Laboratory Evaluation 391 Other Erythrocyte Physical Trauma Resulting Differential Diagnosis 392 in Hemolytic Anemia 439 Therapy 392 Hemolytic Anemias Caused by Antagonists in Other Defects and Deficiencies of the HMP Shunt the Blood 440 and GSH Metabolism 393 Infectious Agents 440 Pyruvate Kinase (Pk) Deficiency 393 Animal Venoms 442 Etiology 393 Chemicals and Drugs 442 Pathophysiology 393 Summary • Review Questions • References x Contents Section Four Pathophysiology 499 Cancer Stem Cells 500 Nonmalignant Disorders Molecular Basis of Cancer 500 of Leukocytes 449 Leukemogenesis 504 Epidemiology 505 21 Nonmalignant Disorders Clinical Presentation 505 of Leukocytes: Granulocytes Laboratory Evaluation 506 and Monocytes 450 Hematopoietic Neoplasm Classification 506 Laboratory Procedures for Diagnosing and Overview 452 Classifying Neoplasms 507 Introduction 452 Cytochemical Analysis 508 Neutrophil Disorders 453 Immunologic Analysis 508 Quantitative Disorders 453 Genetic Analysis 509 Qualitative or Morphologic Abnormalities 458 Prognosis and Treatment of Neoplastic Disorders 509 Eosinophil Disorders 464 Prognosis 509 Nonclonal (Reactive) Hypereosinophilia 464 Treatment 510 Clonal (Neoplastic) Hypereosinophilia 465 Summary • Review Questions • References Idiopathic Hypereosinophilia 465 Basophil and Mast Cell Disorders 466 24 Myeloproliferative Neoplasms 517 Monocyte/Macrophage Disorders 466 Quantitative Disorders 466 Overview 519 Qualitative Disorders 467 Introduction 519 Summary • Review Questions • References Classification 520 Pathophysiology 521 22 General Features 521 Nonmalignant Lymphocyte Chronic Myeloid Leukemia (CML) 522 Disorders 473 Etiology and Pathophysiology 523 Overview 475 Clinical Presentation 525 Introduction 475 Laboratory Evaluation 525 Lymphocytosis 475 Terminal Phase 527 Infectious Mononucleosis 476 Therapy 528 Toxoplasmosis 479 Differential Diagnosis 529 Cytomegalovirus 479 Chronic Neutrophilic Leukemia (CNL) 530 Bordetella Pertussis 480 Etiology and Pathophysiology 530 Reactive Lymphocytosis 480 Clinical Presentation 530 Plasmacytosis 481 Laboratory Evaluation 530 Persistent Polyclonal B-Cell Lymphocytosis 481 Therapy 531 Lymphocytopenia 482 Differential Diagnosis 531 Lymphocyte Sequestration and Destruction 482 Essential Thrombocythemia (ET) 531 Immune Deficiency Disorders 483 Etiology and Pathophysiology 531 Summary • Review Questions • References Clinical Presentation 532 Laboratory Evaluation 532 Section Five Prognosis and Therapy 534 Differential Diagnosis 534 Neoplastic Hematologic Polycythemia Vera (PV) 535 Disorders 495 Classification 535 Etiology and Pathophysiology 535 23 Introduction to Hematopoietic Clinical Presentation 537 Neoplasms 496 Laboratory Evaluation 537 Prognosis and Therapy 538 Overview 498 Differential Diagnosis 539 Introduction 498 Relative Polycythemia 539 Contents xi Primary Myelofibrosis (PMF) 540 Therapy 573 Etiology and Pathophysiology 541 Myelodysplastic/Myeloproliferative Neoplasms Clinical Presentation 542 (MDS/MPNS) 574 Laboratory Evaluation 542 Chronic Myelomonocytic Leukemia (CMML) 574 Prognosis and Therapy 544 Atypical Chronic Myeloid Leukemia (aCML, Differential Diagnosis 544 BCR/ABL1−) 575 Myeloproliferative Neoplasm, Unclassifiable (MPN, U) 545 Juvenile Myelomonocytic Leukemia 575 Clonal Hypereosinophilia 545 MDS/MPN with Ring Sideroblasts and Thrombocytosis (MDS/MPN-RS-T) 576 Myeloid and Lymphoid Neoplasms Associated with Eosinophilia and PDGFRA, PDGFRB, Myelodysplastic/Myeloproliferative Neoplasm, or FGFR1 Mutations 546 Unclassifiable (MDS/MPN, U) 576 Chronic Eosinophilic Leukemia, Not Otherwise Summary • Review Questions • References Specified (CEL-NOS) 547 Idiopathic Hypereosinophilic Syndrome (I-HES) 548 26 Acute Myeloid Leukemias 583 Mast Cell Disease (Mastocytosis) 548 Overview 585 Summary • Review Questions • References Introduction 585 25 Etiology And Pathophysiology 585 Myelodysplastic Syndromes 555 Laboratory Evaluation 585 Overview 557 Peripheral Blood 585 Introduction 557 Bone Marrow 586 Pathophysiology 557 Other Laboratory Evaluation 587 Cytogenetics, Epigenetics, and Single Gene Classification 587 Mutations 558 Identification of Cell Lineage 587 Proliferation Abnormalities 560 WHO Classification of AML 591 Incidence 560 Therapy 601 Clinical Presentation 560 Summary • Review Questions • References Laboratory Evaluation 560 Peripheral Blood 560 27 Precursor Lymphoid Neoplasms 606 Bone Marrow 563 Overview 608 Molecular Diagnostics 564 Introduction 608 Additional Laboratory Evaluation 565 Etiology and Pathophysiology 608 Blast and Precursor Cell Classification 565 Myeloblasts 565 Clinical Presentation 608 Promyelocytes 566 Laboratory Evaluation 609 Ring Sideroblasts 566 Peripheral Blood 609 Immunological Identification of Blasts 567 Bone Marrow 610 Tissue Involvement 610 Classification 567 Other Laboratory Evaluation 611 Description of MDS Subgroups 568 Identification of Cell Lineage 611 MDS with Single Lineage Dysplasia (MDS-SLD) 568 Morphology and Cytochemistry 611 MDS with Ring Sideroblasts (MDS-RS) 569 Terminal Deoxynucleotidyl Transferase (TdT) 611 MDS with Multilineage Dysplasia (MDS-MLD) 569 Immunophenotyping 611 MDS with Excess Blasts (MDS-EB) 569 Cytogenetic Analysis 612 MDS with Isolated del(5q) 570 Molecular Analysis 612 MDS, Unclassifiable (MDS-U) 570 WHO Classification 612 Refractory Cytopenia of Childhood 570 B Lymphoblastic Leukemia/Lymphoma 612 Variables of MDS Subgroups 571 T Lymphoblastic Leukemia/Lymphoma 615 Hypoplastic MDS 571 Acute Leukemias of Ambiguous Lineage 616 MDS with Fibrosis 571 Natural Killer Cell Lymphoblastic Therapy-Related Myelodysplasia 571 Leukemia/Lymphoma 617 Differential Diagnosis 571 Therapy 617 Prognosis 572 Summary • Review Questions • References xii Contents 28 Mature Lymphoid Neoplasms 622 Cryopreservation and Storage of Hematopoietic Stem Cells 652 Overview 624 Infusion of Hematopoietic Stem Cells 652 Introduction 624 Quantitation of Hematopoietic Stem Cells 652 Etiology and Pathophysiology 624 Determination of Mononuclear Cell Count 652 Acquired Genetic Factors 624 CD34 Enumeration by Flow Cytometry 652 Inherited Genetic Factors 624 Cell Culture for Colony Forming Units 653 Environmental Factors 625 Collection Target for Stem Cells 653 Diagnosis and WHO Classification 625 Hematopoietic Engraftment 654 Laboratory Evaluation 625 Evidence of Initial Engraftment 654 Prognosis 625 Evidence of Long-Term Engraftment 654 Therapy 625 Role of the Clinical Laboratory Professional Staging 626 in Stem Cell Transplantation 654 Mature B Cell Neoplasms 626 Graft-Versus-Host Disease and Graft-Versus-Leukemia Chronic Lymphocytic Leukemia/Small Effect 655 Lymphocytic Lymphoma 626 Complications Associated with Stem Cell B Cell Prolymphocytic Leukemia 628 Transplantation 656 Hairy Cell Leukemia 628 Early Complications 656 Follicular Lymphoma 629 Late Complications 657 Mantle Cell Lymphoma (MCL) 630 Increased Availability and Success of Stem Cell Extranodal Marginal Zone Lymphoma Transplantation 657 of Mucosa-Associated Lymphoid Tissue 632 Gene Therapy 657 Lymphoplasmacytic Lymphoma 632 Summary • Review Questions • References Diffuse Large B Cell Lymphoma 633 Burkitt Lymphoma 634 Section Six Plasma Cell Neoplasms 634 Mature T and NK Cell Neoplasms 636 Body Fluids 663 Nodal T and NK Cell Lymphomas 637 Extranodal T and NK Cell Lymphomas 638 30 Morphologic Analysis of Body Leukemic T and NK Cell Lymphomas 639 Fluids in the Hematology Laboratory 664 Hodgkin Lymphoma (HL) 640 Nodular Lymphocyte-Predominant Hodgkin Overview 666 Lymphoma (NLPHL) 640 Introduction 666 Classical Hodgkin Lymphoma (CHL) 640 Types of Body Fluids 666 Summary • Review Questions • References Serous Fluids 667 Cerebrospinal Fluid 668 29 Hematopoietic Stem Cell Synovial Fluid 670 Bronchoalveolar Lavage (BAL) 670 Transplantation 645 Hematologic Analysis of Body Fluids 670 Overview 647 Specimen Collection and Handling 670 Introduction 647 Physical Characteristics 671 Origin and Differentiation of Hematopoietic Cell Counting 672 Stem Cells 647 Nucleated Cell Differential 675 Sources of Hematopoietic Stem Cells and Types Analysis of Other Fluids 693 of Stem Cell Transplants 648 BAL Fluid 693 Allogeneic Stem Cell Transplantation 648 Amniotic Fluid Lamellar Body Counts 693 Autologous Stem Cell Transplantation 649 Semen Analysis 695 Umbilical Cord Blood Stem Cell Transplantation 650 Summary • Review Questions • References Collection and Processing of Hematopoietic Stem Cells 650 Section Seven Bone Marrow 651 Peripheral Blood 651 Hemostasis 703 Umbilical Cord Blood 651 Purging 651 31 Primary Hemostasis 704 Contents xiii Overview 706 Clinical Manifestations of Secondary Hemostasis Introduction 706 Bleeding Disorders 770 Role of the Vascular System 707 Nonspecific Bleeding 770 Structure of Blood Vessels 707 Evaluation of a Patient with Abnormal Bleeding 770 Functions of Blood Vessels in Hemostasis 709 Laboratory Evaluation of Abnormal Bleeding 771 Functions of Endothelial Cells 709 Hereditary Vascular System Disorders 773 Platelets in Hemostasis 711 Acquired Vascular System Disorders 773 Platelet Structure 712 Purpura Resulting from Decreased Connective Tissue 773 Platelet Functions 718 Purpura Associated with Dysproteinemias 773 Physiologic Controls of Platelet Activation Purpura Resulting from Vasculitis 774 and Aggregation 726 Miscellaneous Causes of Purpura 774 Summary • Review Questions • References Quantitative Platelet Disorders 775 Thrombocytopenia 775 32 Secondary Hemostasis Thrombocytosis 785 and Fibrinolysis 731 Artifacts in the Quantitative Measurement of Platelets 786 Overview 733 Qualitative (Functional) Platelet Disorders 787 Introduction 733 Hereditary Disorders of Platelet Function 787 The Coagulation Mechanism 734 Acquired Disorders of Platelet Function 792 Procoagulant Factors 734 Summary • Review Questions • References Properties of the Blood Coagulation Factors 735 Mechanism of Action 737 34 Disorders of Secondary Hemostasis 798 Vitamin K–Dependent Coagulation Proteins 737 Structure of the Blood Coagulation Proteins 738 Overview 800 Coagulation Cascade 738 Introduction 800 Complex Formation on Phospholipid Surfaces 738 Bleeding Characteristics 800 The Intrinsic Pathway 738 Laboratory Evaluation 801 The Extrinsic Pathway 744 Hereditary Disorders of Secondary Hemostasis 802 The Common Pathway 745 Autosomal Dominant Inheritance – von Willebrand Fibrinolytic System 749 Disease 802 Components of the Fibrinolytic System 749 X-Linked Recessive Disorders 807 Plasminogen (PLG) and Plasmin (PLN) 750 Autosomal Recessive Disorders 812 Activators of Fibrinolysis 751 Acquired Disorders of Hemostasis Associated Fibrin Degradation 752 with Bleeding 817 Inhibitors of Fibrinolysis 753 Common Bleeding Disorders in the Neonate 820 Summary • Review Questions • References Control of Hemostasis 755 Blood Flow 755 Liver Clearance 755 35 Thrombophilia 826 Positive Feedback Amplification 755 Overview 828 Negative Feedback Inhibition 755 Biochemical Inhibitors 755 Introduction 828 Physiologic Hemostasis 760 Thrombus Formation 829 The Tissue Factor Pathway 760 Arterial Thrombi 829 Basal Coagulation 761 Venous Thrombi 830 Hemostasis in the Newborn 762 Microparticles in Arterial and Venous Thrombosis 831 Summary • Review Questions • References Thrombophilia 832 Hereditary Thrombophilia 833 33 Other Potential Genetic Risk Factors 839 Disorders of Primary Hemostasis 767 Acquired Thrombohemorrhagic Conditions 841 Overview 769 Laboratory Testing in Patients with Suspected Introduction 769 Thrombosis 853 Diagnosis of Bleeding Disorders 769 Anticoagulant Therapy 854 Clinical Manifestations of Primary Hemostasis Heparin 855 Bleeding Disorders 770 Oral Anticoagulants 856 xiv Contents New Oral Anticoagulants (NOAC)/Direct Oral Section Eight Anticoagulants (DOAC) 857 Thrombolytic Therapy 857 Hematology Procedures 905 Antiplatelet Therapy 858 Summary • Review Questions • References 37 Hematology Procedures 906 36 Overview 909 Hemostasis: Laboratory Testing Introduction 909 and Instrumentation 866 Laboratory Testing Regulations 909 Overview 868 Specimen Collection: Phlebotomy 910 Introduction 869 Anticoagulants 910 Specimen Collection and Processing 869 Equipment 911 Specimen Collection 869 Venipuncture 912 Specimen Processing 871 Capillary Puncture 913 Laboratory Investigation of Primary Hemostasis 871 Phlebotomy Safety 914 Bleeding Time 871 Microscopy: The Microscope 914 Platelet Function Analyzers 872 Bright-Field Microscopy 914 Phase-Contrast Microscopy 915 Laboratory Investigation of Secondary Hemostasis 875 Koehler Illumination 916 Screening Tests 875 Preventative Maintenance 917 Tests to Identify a Specific Factor Deficiency 877 Identification of Inhibitors 882 Peripheral Blood Smear Preparation 917 Manual Method 917 Laboratory Investigation of the Fibrinolytic System 884 Automated Method 918 D-Dimer 884 Fibrin Degradation Products 885 Peripheral Blood Smear Staining 918 Euglobulin Clot Lysis 885 Peripheral Blood Smear Examination 919 a Cell Enumeration by Hemacytometer 921 2@Antiplasmin Activity 885 Laboratory Investigation of Hemostasis (Primary, Manual Leukocyte Count 921 Secondary, or Fibrinolytic Pathway) Using Global Assays 886 Manual Erythrocyte Count 922 Thromboelastography (TEG) 886 Manual Platelet Count 922 ROTEM® (TEM) 886 Hemoglobin Concentration 922 Calibrated Automated Thrombogram (CAT) 886 Hematocrit 923 Laboratory Investigation of Hereditary and Acquired Erythrocyte Indices 923 Thrombophiilias (Hypercoagulable States) 887 Erythrocyte Sedimentation Rate (ESR) 924 Antithrombin (AT) 887 Reticulocyte Count 926 Protein C 888 Solubility Test for Hemoglobin S 927 Protein S 889 Hemoglobin Electrophoresis 928 Activated Protein C Resistance (APCR) 889 Quantitation Of Hemoglobin A2 929 Prothrombin G20210A 889 Acid Elution for Hemoglobin F 929 Additional Testing for Thrombosis 890 Quantitation of Hemoglobin F 930 Molecular Markers of Hemostatic Activation 891 Alkali Denaturation 930 Laboratory Evaluation: Assessment for Pharmaceutical Other Methods 930 Intervention of Hemostasis 891 Heat Denaturation Test for Unstable Hemoglobin 930 Oral Vitamin K Antagonist Therapy and the Prothrombin Time–INR Value 891 Heinz Body Stain 931 Direct Oral Anticoagulants (DOACs) 892 Osmotic Fragility Test 931 Non-Parenteral Anticoagulation 893 Donath-Landsteiner Test for Paroxysmal Cold Direct Thrombin Inhibitor (DTI) Therapy Hemoglobinuria (PCH) 931 Monitoring 894 Erythropoietin 933 Hemostasis Instrumentation 895 Soluble Transferrin Receptor 933 Evolution of Hemostasis Testing 895 Cytochemical Stains 934 Automated Hemostasis Analyzer Methodologies 895 Myeloperoxidase 934 Point-of-Care Hemostasis Instrumentation 897 Sudan Black B 934 Summary • Review Questions • References Chloroacetate Esterase 935 Contents xv a@Naphthyl Esterase (Nonspecific Esterase) 936 40 Flow Cytometry 993 Periodic Acid-Schiff 936 Leukocyte Alkaline Phosphatase 937 Overview 995 Acid Phosphatase and Tartrate-Resistant Acid Introduction 995 Phosphatase (TRAP) 937 Principles of Flow Cytometry 995 Terminal Deoxynucleotidyl Transferase
938 Isolation of Single Particles 995 Toluidine Blue 938 Light Scattering 996 Reticulin Stain and Masson’s Trichrome Stain 939 Detection of Fluorochromes 997 Summary • Review Questions • References Data Analysis 998 Immunophenotyping by Flow Cytometry 998 38 Bone Marrow Examination 945 Specimen Requirements and Preparation for Immunophenotyping 1000 Overview 947 Isolation of Cells by Gating 1000 Introduction 947 Fluorescence Activated Cell Sorting (FACS) 1000 Indications for Bone Marrow Evaluation 947 Diagnosis and Classification of Mature Bone Marrow Procedure 948 Lymphoid Neoplasms 1001 Bone Marrow Processing for Examination 949 Diagnosis and Classification of Acute Leukemia 1002 Bone Marrow Aspirate 949 Diagnosis of Myelodysplastic Syndrome (MDS) 1004 Bone Marrow Core Biopsy 949 Detection of Minimal Residual Disease (MRD) by Flow Cytometry 1005 Morphologic Interpretation of Bone Marrow 950 Diagnosis and Surveillance of Immunodeficiency Bone Marrow Aspirate 950 Disorders 1005 Bone Marrow Touch Imprints 952 Flow Cytometry in Systemic Autoimmune Disease 1006 Bone Marrow Clot and Particle Preparation, CD34 Enumeration 1007 Sections, and Core Biopsy 952 Paroxysmal Nocturnal Hemoglobinuria (PNH) 1007 Benign Lymphoid Aggregates versus Malignant Lymphoma 953 Hereditary Spherocytosis (HS) 1008 Bone Marrow Iron Stores 954 DNA Analysis 1008 Special Studies on Bone Marrow 955 Proliferation 1008 Flow Cytometry 955 Ploidy 1008 Cytogenetics 955 Clinical Applications of DNA Analysis 1009 Molecular Genetics 955 Summary • Review Questions • References Cytochemical Stains 957 Bone Marrow Report 957 41 Chromosome Analysis of Summary • Review Questions • References Hematopoietic and Lymphoid Disorders 1013 39 Automation in Hematology 961 Overview 1015 Overview 962 Introduction 1015 Introduction 962 Chromosome Structure and Morphology 1015 Automated Blood Cell–Counting Instruments 963 Mitosis 1016 Impedance Instruments 963 Cytogenetic Procedures 1017 Coulter® LH Series 963 Specimen Preparation 1017 Beckman-Coulter Unicel® DxH 800 968 Harvest Procedure and Banding 1018 Sysmex XE-Series™ 971 Chromosome Analysis 1019 Sysmex XN-Series™ 977 Chromosome Abnormalities 1020 Abbott CELL-DYN Sapphire® 980 Numerical Aberrations 1020 Light-Scattering Instruments 984 Structural Aberrations 1022 Siemens Healthcare ADVIA 120 984 Polymorphic Variation 1022 Siemens Healthcare ADVIA 2120 987 Cytogenetic Nomenclature 1022 Automated Digital Cell Morphology Cytogenetic Analysis of Hematopoietic Instrument 987 and Lymphoid Disorders 1023 CellaVision® DM96 System 987 Processing Specimens 1024 Summary • Review Questions • References Chronic Myelogenous Leukemia 1024 xvi Contents Myeloproliferative Neoplasms Other Than CML 1026 Quality Assessment 1056 Acute Myeloid Leukemia 1026 Basic Components 1056 Myelodysplastic Syndromes 1027 Proficiency Testing 1060 Acute Lymphoblastic Leukemia/Lymphoma 1027 Competency Testing 1060 Lymphoma and Lymphoproliferative Disorders 1027 Method Evaluation/Instrument Comparison 1061 Bone Marrow Transplantation 1028 Reference Interval Determination 1064 Molecular Cytogenetics 1028 Laboratory Safety 1065 Summary • Review Questions • References Quality Control 1066 Control Materials 1066 42 Molecular Analysis Establishing Quality Control (QC) Limits 1066 Interpreting Quality Control Charts 1066 of Hematologic Diseases 1033 Bull’s Testing Algorithm (Moving Averages) 1067 Overview 1035 Monitoring Quality Control with Patient Introduction 1035 Specimens 1068 Overview of Molecular Technologies 1036 Individual Quality Control Plan 1068 Nucleic Acid Extraction 1036 Review of Patient Results 1069 Nucleic Acid Amplification 1036 Hematology 1069 Hybridization Techniques 1040 Hemostasis 1073 Direct DNA Sequence Analysis 1042 Summary • Review Questions • References Chain Termination Sequencing 1042 Clinical Applications of Molecular Diagnostics Appendices in Hematopathology 1043 Erythrocyte Disorders 1045 APPENDIX A 1078 Leukocyte Disorders 1046 APPENDIX B: Hematopoietic and Lymphoid Infectious Diseases 1048 Neoplasms: Immunophenotypic and Genetic Features 1082 Clinical Applications of Molecular Diagnostics APPENDIX C: 2017 WHO Classification of in Hemostasis 1049 Hematologic, Lymphopoietic, Histiocytic/Dendritic Neoplasms 1086 CYP2C9 1049 VKORC1 1049 APPENDIX D: Hematology Procedures 1089 Factor V Leiden (FVL) 1049 APPENDIX E: Answers to Review Questions 1104 Prothrombin G20210A 1049 APPENDIX F: Answers to Checkpoints 1111 Hemophilia A 1049 APPENDIX G: Answers to Case Study Questions 1150 Hemophilia B 1049 Methylenetetrahydrofolate Reductase (MTHFR) 1049 Glossary 1179 von Willebrand Disease (VWD) 1049 Summary • Review Questions • References Index 1209 Section Nine Quality Assessment 1053 43 Quality Assessment in the Hematology Laboratory 1054 Overview 1056 Test Coding and Reimbursement 1056 Foreword Clinical Laboratory Hematology is part of Pearson’s Clinical Laboratory Science (CLS) series of products, which is designed to balance theory and practical applications in a way that is engaging and useful to students. The authors of and contributors to Clinical Laboratory Hematology present highly detailed technical information and real-life case studies that will help learners envision themselves as members of the health care team, providing the laboratory services specific to hematology that assist in patient care. The mixture of theo- retical and practical information relating to hematology provided in this text allows learners to analyze and synthesize this information and, ultimately, to answer questions and solve problems and cases. Additional instructional resources are available at www.pearson.com Elizabeth A. Gockel-Blessing, PhD, MT(ASCP), CLS(NCA) Clinical Laboratory Science Series Editor Pearson Health Science Vice Chair & Associate Professor Department of Clinical Laboratory Science Doisy College of Health Sciences Saint Louis University xvii Preface Revel ™ for Clinical Laboratory Hematology takes on Organization a new face as a digital textbook. Revel is Pearson’s newest way of delivering our respected content. We believe that students must have a thorough knowledge of Fully digital and highly engaging, Revel offers an immersive normal hematopoiesis and cell processes to understand the learning experience designed for the way today’s students pathophysiology of hematologic/hemostatic diseases, evalu- read, think, and learn. Enlivening course content with media ate and correlate laboratory test results, and ensure the appro- interactives and assessments, Revel empowers educators priate utilization of the laboratory in diagnosis and patient to increase engagement with the course, and to better con- follow-up. Thus, this book is organized so that the first nine nect with students. To our knowledge this is the first book chapters give the students a comprehensive base of knowl- written for medical laboratory technician (MLT) and medi- edge about blood cell proliferation, maturation, and differen- cal laboratory science (MLS) students in this format. Pearson tiation and the processes that control hematopoiesis. Section performed in-depth analysis to determine the learning pref- One (Chapters 1–2) includes an introduction to hematology erences of our students and teaching methods of instructors. and hematopoiesis, including cell morphology and the cell The results indicated that many students prefer to read and cycle and its regulation. This introduction includes a descrip- study using digital resources. Furthermore, on-line instruc- tion of cellular processes at the molecular level, which could tion is now commonplace. Based on this knowledge, the be new material for some students and a basic review for oth- decision was made to update to the fourth edition using the ers. The reader might want to review these chapters before Revel platform. The focus of the book remains the same; it is beginning a study of neoplastic disorders (Chapters 23–28). a comprehensive resource that MLT and MLS students can Section Two (Chapters 3–10) includes chapters on normal use in all their hematology courses. Laboratory practitioners hematopoiesis, including a description of the structure and will find the book a welcome resource to help them keep up function of hematopoietic tissue and organs, erythropoiesis, with advances in the field. Although the book is primarily leukopoiesis, thrombopoiesis, and hemoglobin. Hemoglo- written for clinical laboratory students and practitioners, it bin synthesis, function, and breakdown are discussed in also is suited for use by students and practitioners in other Chapter 6. The chapter on leukocytes is divided into two health care professions including pathology, medicine, phy- separate chapters: granulocytes/monocytes (Chapter 7) and sician assistant, and nursing. Great effort has been put in by lymphocytes (Chapter 8). An introductory chapter on plate- our authors to ensure this edition is thoroughly updated to lets (Chapter 9) completes the discussion of normal blood include the latest in advances in laboratory medicine. Each cells. Details of platelet function and physiology are found chapter has a similar format that makes it easy for readers to in Section Seven, Chapter 31. Rounding out this section, “The find information on each topic. The digital format makes it Complete Blood Count and Peripheral Blood Smear Exami- convenient to instantly find word definitions, cell images, and nation” (Chapter 10) describes the information that can be other information as needed. An image atlas, test bank, pow- gained about blood cells from these frequently ordered labo- erpoint presentation and instructor’s manual are available for ratory tests. Most of the remaining chapters refer to the tests instructors who adopt the book for their classes. In summary, that are described in this chapter. the book is not just a book but a package of learning tools. The next three sections include discussions of hemato- You will note that we have added a new editor, Dr. logic disorders. Section Three (Chapters 11–20) begins with Kristin Piwowar-Landis. Kristin was a consulting editor on an introduction to anemia (Chapter 11). We combined the the previous edition. Based on her outstanding writing and introduction to anemia and the introduction to hemolytic editing skills as well as thorough knowledge of hematol- anemia into one chapter because many anemias have a hemo- ogy with a focus on hematopoietic genetics, she was invited lytic component. This chapter is followed by chapters on the to serve as an editor in this edition with Dr. McKenzie as various anemias. Each anemia is discussed in the following her mentor. The ultimate goal is for Kristin to replace Dr. manner: introduction, etiology, pathophysiology, clinical McKenzie who wants to retire from the responsibilities of presentation, laboratory presentation, and therapy. This for- primary editor in future editions. It has been a pleasure to mat helps readers understand what laboratory tests can help mentor her through the book writing process. Dr. Lynne in diagnosis and how to interpret the results of these tests. Williams continues as an editor in this edition. Lynne’s in- Section Four (Chapters 21 and 22) covers the nonmalignant depth knowledge in hemostasis as well as cellular biology disorders of leukocytes. Section Five ( Chapters 23–29) is a shines through in the chapters she authors. study of hematopoietic neoplasms. This section begins with xviii Preface xix an overview of these disorders to help students understand is in an ideal position to assist physicians in interpreting the classification, terminology, and pathophysiology of neo- laboratory test results and choosing the best reflex tests to plasms and the laboratory’s role in diagnosis and therapy. As arrive at a diagnosis or evaluate therapy. Many laborato- a part of this section, we included a chapter on stem cell trans- ries develop algorithms to assist in these tasks. This text plantation (Chapter 29) because it is a frequently used therapy includes several algorithms that some laboratories use. for these neoplasms and the laboratory plays a critical role in harvesting the stem cells and preparing them for transplant. Molecular studies are becoming a major diagnostic tool for Suitable for all Levels of Learning neoplastic disorders and are discussed within each chapter The book is designed for both MLT and MLS students. as well as in the chapter devoted to molecular diagnostics Using only one textbook for both levels is beneficial and (Chapter 42). Some instructors might prefer to cover Section economic for laboratory science programs that offer both Eight, the study of bone marrow (Chapter 38), flow cytometry levels of instruction. It also is helpful for programs that have (Chapter 40), cytogenetics (Chapter 41), and molecular diag- developed articulated MLT to MLS curricula. The MLS pro- nostics (Chapter 42) before teaching Section Five or integrate gram can be confident of the MLT’s knowledge in hema- this material with Section Five. Some hematology courses tology without doing a time-consuming analysis of the do not include these topics, or instructors might not want to MLT course. In addition, this book is expected to be a great cover them in the depth presented in this book. resource for students in on-line courses and for instructors Section Six (Chapter 30) is a study of body fluids from a who teach using this format. hematologic perspective and thus includes a large number Objectives are divided into two levels: Level I (basic) of photographs of cells found in body fluids. Discussions and Level II (advanced). MLT instructors who reviewed the of semen analysis and amniotic fluid lamellar body counts objectives for this text generally agreed that most Level I are included. Not all hematology courses include this topic, objectives are appropriate for the MLT body of knowledge. but the chapter is written in such a way that it can be used They also indicated
that some Level II objectives are appro- separately in a body fluid course. priate for MLTs. MLS students should be able to meet both Section Seven (Chapters 31–36) is a study of hemosta- Level I and Level II objectives in most cases. If the MLS sis. Chapters on normal hemostasis include primary and program has two levels of hematology courses—Level I and secondary hemostasis and fibrinolysis. They are followed Level II—this book can be used for both. by three chapters on disorders of hemostasis. Chapter 36 All instructors, regardless of discipline or level, need describes the testing procedures for hemostasis, includ- to communicate to their students what is expected of them. ing information on automation. This chapter describes an They might want their students to find the information in extensive collection of coagulation procedures. the text that allows them to satisfy selected objectives, or Section Eight (Chapters 37–42) includes chapters on test they might assign particular sections to read. If not assigned procedures that help in the diagnosis of hematologic dis- specific sections to read, the MLT students may read more orders. Automation in hematology is included in Chapter than expected, which is not a bad thing! 39. Chapter 42 is designed to introduce molecular proce- The design of the text is such that each chapter is dures and their use in detecting various hematologic and divided into modules and each objective is identified with hemostatic disorders. A background in genetics is suggested the module that addresses it. These objectives are divided before students begin this chapter. into Level 1 and Level 2. There are two levels of review Section Nine (Chapter 43) is a thorough discussion of questions at the end of each module and chapter that are quality assessment in the hematology laboratory. Problems matched to the two levels of objectives. Case Study ques- discussed include common abnormal results, errors, and tions and the Checkpoints are included within each mod- alert flags. Corrective action to take to resolve these prob- ule and are appropriate for the information in that module lems is described. Several excellent tables help to quickly or another previous module. Checkpoints and case study find needed information. We suggest that these tables be questions are not delineated by level. Students should use read early in the course of study because they can be used these valuable resources to assess their understanding of periodically when attempting to interpret and correlate the material. laboratory test results. Chapter 10 refers the reader to these We recognize that there are many approaches to orga- tables because it discusses interpretation of test results and nizing a hematology course and that not all instructors teach abnormalities in the CBC. in the same topic sequence or at the same depth. Thus, we Appendices collect additional information including encourage instructors to use the book by selecting appro- step-by-step procedures for some hematology testing, and priate chapters and objectives for their students based on reference tables. their course goals. Each program should assess what con- The text emphasizes the effective, efficient, and ethical tent fits its particular curriculum. The layout of the book use of laboratory tests. The clinical laboratory professional is such that instructors can select the sequence of chapters xx Preface in an order that fits their course design, which might not they just read. They are questions that require students necessarily be the sequence in the book. However, we rec- to pause along the way to recall or apply information ommend that the course begin with Sections One and Two covered in preceding sections. The answers are pro- and that the chapters “Introduction to Anemia” and “Intro- vided after the student submits their answer. duction to Hematopoietic Neoplasms” be studied before • A Summary concludes the text portion of each chapter the individual chapters that follow on these topics. The to help students bring all the material together. Background Basics sections help the instructor determine • Review Questions appear at the end of each chapter. which concepts students should master before beginning The two sets of questions, Level I and Level II, are ref- each chapter. This feature helps instructors customize their erenced and organized to correspond to the Level I courses. Some hematology courses might not include some and Level II objectives. Answers are provided in the chapters on subjects such as molecular techniques, cytoge- Appendix. netics, flow cytometry, and body fluids but they might be helpful in other courses. • Image Atlas As a note, this text uses “mc” as an abbreviation for The page design features a number of enhancements “micro”, which replaces m. Thus, abbreviations of mcg, intended to aid the learning process. mcL, mcM replace those that use the Greek letter “mu” (mg, mL, mM). • Figures and tables are used liberally to help students organize and conceptualize information. This is espe- Unique Pedagogical Features cially important for visual learners. • Microphotographs are displayed liberally in the book As in the past, the text has a number of unique pedagogi- and are typical of those found in a particular disease cal features to help the students assimilate, organize, and or disorder. Students should be aware that cell varia- understand the information. Each chapter begins with a tions occur and that blood and bone marrow findings group of components intended to set the stage for the con- do not always mimic those found in textbooks. Because tent to follow. there is so much variation in the morphology of normal • The Objectives comprise two levels: Level I for basic or and abnormal cells, we added a Flash Card review of essential information and Level II for more advanced additional cells at the end of many chapters. The legend information. Each instructor should guide students to for each microphotograph gives the original magnifica- the appropriate level to meet course expectations. tion but sometimes the image was zoomed to enhance • The Key Terms feature alerts students to important detail. terms used in the chapter and found in the glossary. With this digital version, these terms are provided as Appendices links within the chapter, giving the student the defini- • Appendix A contains tables of reference intervals for tion within the glossary. common hematology test results. • The Background Basics component alerts students to • The table in Appendix B was extensively revised and material that they should have learned or reviewed updated consistent with the WHO 2017 classifica- before starting the chapter. In most cases, this feature tion of hematopoietic and lymphoid tissue through links readers to previous chapters to help them find the a collaborative effort of several authors (Drs. Kath- material if they want to review it. leen Wilson, Katalan Keleman, Sara Taylor, and Tim • The Case Study is a running scenario that first appears Randolph). It lists hematopoietic neoplasms with the at the beginning of a chapter, giving a patient’s clinical following information on each: immunophenotype and laboratory information that is related to the chap- using CD markers, cytogenetic abnormalities, and ter content. It is meant to focus the students’ attention genotypic findings. This table provides a ready refer- on the chapter subject matter. At appropriate places ence for information from the chapters in Section Five throughout the chapter additional information on the (Neoplastic Hematologic Disorders) and Section 8 case is provided, such as additional laboratory test (Hematology Procedures). results, followed by questions that relate to the mate- • Appendix C is a comprehensive classification of hema- rial presented in preceding sections. The answers are topoietic, lymphopoietic, and histiocytic/dendritic provided after the student submits the answers. neoplasms using the updated 2017 WHO classification. • The Overview gives readers an idea of the chapter con- • Appendix D is a collection of common laboratory pro- tent and organization. cedures that are linked from Chapter 37 where the pro- • Checkpoints are integrated throughout the chapter to cedure is discussed. These can be printed and used in help the student determine if they understand what hematology laboratory courses. Preface xxi • Appendix E provides the answers to the multiple reviewers and users of the previous editions provided help- choice questions that appear at the end of each chapter. ful suggestions that were incorporated into the chapters. • Appendix F provides the answers to the Checkpoint Dave Falleur, Diana Cochran-Black, Muneez Esani, Sara questions that appear throughout each chapter. Wagner, Holly Weinberg, and Linda Whaley had important roles in reviewing select chapters. We offer our thanks to this • Appendix G provides the answers to the Case Study group who ensured a quality textbook for a wide audience. questions that appear throughout each chapter. Mark Cohen from Pearson was responsible for the cre- A Complete Teaching and Learning ation of the first edition of this text. His keen insights into developing a unique textbook design with pedagogical Package enhancements helped Clinical Laboratory Hematology become A variety of ancillary materials designed to help instructors a leading textbook in the field of clinical laboratory science. be more efficient and effective and students more successful Thank you, Mark. Thank you, Pearson, for having faith in complements this book. us to publish a fourth edition in digital format. Thank you An Instructor’s Resource Center is available upon for creating and providing the special team of experts to help adoption of the text and gives the instructor access to a us accomplish this task. We recognize that the job is not over number of powerful tools in an electronic format. The fol- but will require the efforts of sales and marketing to ensure lowing materials are downloadable: widespread use and adoption. John Goucher had the fore- sight to develop the fourth edition and for the background • The TestGen feature includes questions to allow work that identified and justified the need for a digital ver- instructors to design customized quizzes and exams. sion. He had faith in us and provided support and encour- Download the TestGen desktop application and test agement for another edition of Clinical Laboratory Hematology. bank, choose questions that align to your textbook, and Michael Giacobbe was the Pearson man behind the generate your test — it’s that easy! scenes who kept the entire process moving forward. He • The PowerPoint Lectures tool contains key discussion also on-boarded authors for support materials including points and color images for each chapter. This feature PowerPoints, test questions, and the instructor’s manual. provides dynamic, fully designed, integrated lectures This group of author educators, Elizabeth Warning, MS, that are ready to use, allowing instructors to custom- MLS(ASCP)CM, University of Cincinnati; Joshua J. Can- ize the materials to meet their specific course needs. non, MS, MLS(ASCP)CM, Thomas Jefferson University; and These ready-made lectures will save instructors time Holly Weinberg, BS, MLS(ASCP)CM, contributed behind the and allow an easy transition into using Clinical Labora- scenes to enhance the instructors’ use of this book. Thank tory Hematology. you all for your timely assistance. Ellen Viganola, Digital • The Image Library feature contains all of the images Project Manager for Pearson did a great job transferring the from the text. Instructors have permission to copy and manuscript from print to digital. paste these images into PowerPoint lectures, printed Development editor, Barbara Price was our daily con- documents, or website as long as they are using Clinical tact who kept us on track even though it meant multiple Laboratory Hematology as their course textbook. deadline revisions. This was especially challenging as we • The Instructor’s Resource Manual tool in Word for- moved from a paper copy to a digital version of the book. mats can be accessed. Her gentle prodding was evident and appreciated. Her edit- ing was superb. Dan Knott, Editorial Project Manager, and Acknowledgments Prathiba Rajagopal, Senior Project Manager, both from SPI Global, kept track of the many people, files and technical Writing a textbook is a complicated task that requires a team elements involved in bringing the book to the digital realm. of dedicated authors, editors, copy editors, artists, permis- Sara Wagner and John Landis contributed countless sion researchers, educators, practitioners, content reviewers, hours of scanning slides and taking pictures to produce an project and program managers, and many other individuals incredible image atlas that accompanies this fourth edition. behind the scenes. The team that Pearson and the editors put John Landis lead
the compiling, organizing, and editing of together to make the fourth edition of this book an excellent the atlas images and as well as other microphotographs in hematology and hemostasis resource for students and health the book. care practitioners worked tirelessly over several years to Maggie Sera carefully reviewed and edited the refer- bring the project to completion. Dr. Kristin Landis-Piwowar ences in this edition. Her attention to detail and sleuthing envisioned how the mechanics of this new digital “book” skills were imperative to ensuring that we presented our could lend to a pioneering form of medical laboratory sci- citations properly. ence education. The new and returning authors ensured that their chapters were up to date and accurate. Content SBM, KLP, JLW xxii Preface I have enjoyed the unique opportunity to edit four edi- I extend a special thank you to my colleagues in the tions of Clinical Laboratory Hematology with Pearson. As Clinical and Diagnostic Sciences program at Oakland Uni- our knowledge in hematology has expanded, many new versity—Dr. Sumit Dinda, Dr. Bekah Martin, Lisa DeCeun- tests have been developed to help diagnose hematologic dis- inck, Terese Trost, and Bill Van Dyke, and our many eases. Likewise, the number of authors and editors needed part-time instructors—who kept the programs moving to cover this material has increased. My thanks go out to forward while we were working on this new edition and all hematologists who have contributed over the years to to the CDS students of the past 2 years who tolerated a dis- make this text a leader for MLS/MLT education and prac- tracted and often absent-minded professor. To all my former tice. I am privileged to work with my brilliant coeditors, students: You have been my inspiration to try to create a Lynne and Kristin. Thank you to Kristin for the superb job meaningful and useful book to support your educational you have done in creating, editing, and authoring, especially endeavors. But special thank you to my co-editors, Dr. Shir- the image atlas. I couldn’t have found a better replacement. lyn McKenzie, for the privilege of accompanying you on this Thank you to my colleague and friend Dr. J. Lynne Williams wonderful journey through these three editions together, for her dedication to this process in the last several editions. and Dr. Kristin Landis-Piwowar, who has been a major Her sharp eyes, superb writing talent and keen mind are guiding force and visionary for this new e-edition book. essential traits for an editor. We have similar philosophies about teaching hematology and often discussed how to best JLW present the information in this book and make it better. During the time this book was under development, my Reviewers professional life took over many hours of my personal life. Many thanks to my husband and best friend Gary for his sup- LINDA L. BREIWICK, BS, CLS(NCA), MT(ASCP) port, sacrifices, and understanding during some very stressful Program Director, Medical Laboratory Technology times so this edition could become a reality. My sincerest grat- Shoreline Community College itude to my parents, George and Helen Olson, who instilled Seattle, WA in me the confidence that I could accomplish anything I set my heart to. This mind-set has stuck with me through life, LINDA COMEAUX, BS, CLS(NCA) especially in this task. I hope that through example I have Vice-President for Instruction provided the same to my children and grandchildren. Red Rocks Community College Lakewood, CO SBM MONA GLEYSTEEN, MS, CLS(NCA) Program Director, Medical Laboratory Technician I am thankful to those who helped to build my fire. My Program father, John Landis, guided me into medical laboratory sci- Lake Area Technical Institute ence and became my hematology professor. My fascination Watertown, SD with blood cells was sparked by my dad. Lynne Williams provided me the opportunity of an academic position and ANNA SWANN, MS, MLS (ASCP)CM encouraged me to participate in the third edition of this book; The University of Southern Mississippi Lynne has kindled my professional work. As digital natives, Hattiesburg, MS my students have taught me how they learn in the technol- ogy era. They inspired my vision and pursuit of a novel Contributors means to teach hematology through technology; these past students are my energizing air. The foundation of this book is GRACE B. ATHAS, PhD the ingenuity of Shirlyn McKenzie who has dedicated her life Assistant Professor, Department of Pathology to educating generations of budding hematologists. Because Louisiana State University, School of Medicine she entrusted me as editor, Shirlyn is my flame. New Orleans, LA I am also thankful to those who sustained my fire. My Chapter 16 husband, Todd, and my children, Theron and Kaia, are the glowing embers that supported my fire when my profes- CHERYL BURNS, MS, MLS(ASCP)CM sional endeavors intersected with my personal life. And my Distinguished Teaching Professor mother, whose empowering spirit, eternal energy, and help- Department of Health Sciences, Medical Laboratory ing hands keep my blaze roaring. I wouldn’t be half of who Sciences I am without my family. School of Health Professions My gratitude is endless to you all. University of Texas Health Science Center at San Antonio San Antonio, TX KLP Chapters 37, 39, 43 Preface xxiii MICHELLE BUTINA, PhD, MLS(ASCP)CM Texas Tech University Health Science Center, School of Associate Professor, Department of Pathology, Anatomy, Health Professions and Laboratory Medicine Lubbock, TX West Virginia University, School of Medicine Chapters 5, 15 Morgantown, WV KATALIN KELEMEN, MD, PhD Chapter 9 Consultant, Department of Pathology and Laboratory DIANA L. COCHRAN-BLACK, DR PH, MLS(ASCP)CM, Medicine SH(ASCP)CM Associate Professor of Pathology Associate Professor and Chair, Medical Laboratory Sciences Mayo Clinic Arizona Program Phoenix, AZ Wichita State University Chapters 28, 40 Wichita, KS Chapter 17 GIDEON H. LABINER, MS, MLS(ASCP)CM Associate Professor, Medical Laboratory Science FIONA E. CRAIG, MD Program Professor of Pathology, Division of Hematopathology University of Cincinnati Department of Laboratory Medicine and Pathology Cincinnati, OH Mayo Clinic Arizona Chapter 23 Phoenix, AZ Chapters 28, 40 JOHN H. LANDIS, MS, MLS(ASCP) Professor Emeritus, Ferris State University AAMIR EHSAN, MD Canadian Lakes, Michigan Chief, Pathology and Laboratory Medicine Adjunct Professor, University of Cincinnati South Texas Veterans Health Care System Chapter 10 Associate Professor, Department of Pathology University of Texas Health Science Center at San Antonio KRISTIN LANDIS-PIWOWAR, PhD, MLS(ASCP)CM San Antonio, TX Associate Professor, Clinical and Diagnostic Sciences Chapters 29, 38 Associate Dean, School of Health Sciences, Oakland University MUNEEZA ESANI, PhD, MPH, MHA, MT(ASCP) Rochester, MI Assistant Professor, Clinical Laboratory Science Chapters 2, 7, 10, 21 University of Texas Medical Branch Galveston, TX SALLY S. LEWIS, PhD, MLS (ASCP), HTL, MB Chapter 12 Associate Dean and Professor, College of Health Sciences and Human Services DEBORAH E. FOX, PhD, MT (ASCP) Tarleton State University Associate Professor and Director, Medical Laboratory Stephenville, TX Science Program Chapter 42 Franciscan Missionaries of Our Lady University Baton Rouge, LA DAVID L. MCGLASSON, MS, MLS(ASCP)CM Chapter 13 Retired Clinical Research Scientist 59th Clinical Research Division ROBERT C. GOSSELIN, CLS Laboratory Services Hemophilia Treatment Center JBSA Lackland, TX Division of Hematology/Oncology Chapter 36 UC Davis Health System Sacramento, CA SHIRLYN B. MCKENZIE, PhD, MLS(ASCP)CM, SHCM Chapter 36 Distinguished Teaching Professor Professor Emeritus and Chair Emeritus KRISTLE HABERICHTER, DO Department of Health Sciences, Division of Medical Anatomic and Clinical Pathology Laboratory Sciences Grand Traverse Pathology, PLLC School of Health Professions Traverse City, MI University of Texas Health Science Center at Chapter 38 San Antonio JOEL D. HUBBARD, PhD, MLS(ASCP) San Antonio, TX Associate Professor, Laboratory Sciences and Primary Care Chapters 1, 6, 11, 12 xxiv Preface ROSLYN WOFFORD MCQUEEN, PhD, CCRC, BROOKE L. SOLBERG, PhD, MLS(ASCP)CM MT(ASCP), SH Associate Professor and Chair, Department of Medical Clinical Research Coordinator Laboratory Science Hurley Medical Center University of North Dakota School of Medicine and Health Flint, MI Sciences Chapter 22 Grand Forks, ND CATHERINE N. OTTO, PhD, MBA, MLS(ASCP)CM Chapters 33, 34 Associate Professor, Department of Clinical Laboratory JEAN SPARKS PhD, MT(ASCP) and Medical Imaging Sciences Associate Professor, Department of Life Sciences School of Health Professions Texas A&M University-Corpus Christi Rutgers, The State University of New Jersey Corpus Christi, TX Newark, NJ Chapter 18 Chapter 11, 43 SARA TAYLOR, PhD, MLS(ASCP), MBCM KEILA POULSEN, BS, MLS(ASCP)CM, H, SH Associate Professor, Medical Laboratory Science and Public Hematology and Histology Supervisor Health Eastern Idaho Regional Medical Center Tarleton State University Idaho Falls, ID Fort Worth, TX Chapter 10 Chapters 25, 42 TIM R. RANDOLPH, PhD, MT(ASCP) SARA L. WAGNER, MLS (ASCP)CM Associate Professor, Department of Clinical Health Sciences Instructor of Hematology Saint Louis University Beaumont Health School of Medical Laboratory Science St. Louis, MO Royal Oak, MI Chapters 14, 24 Chapters 10, 37, 39 KYLE B. RIDING, PhD, MLS(ASCP) JERELYN WALTERS, MLS(ASCP), SH Assistant Professor of Medicine Technical Supervisor, Esoteric Testing, ACL Laboratories University of Central Florida Milwaukee, WI Orlando, FL Chapter 30 Chapters 26, 27 LINDA WHALEY, MLS STACEY ROBINSON, MS, MLS(ASCP)CM, SHCM Technical Specialist, Stem Cell Laboratory Supervisor, Clinical Microscopy Cardinal Bernard Cancer Center Walter Reed National Military Medical Center University Medical Center Bethesda, MD Loyola University Chapters 5, 15 Chicago, IL ANNETTE SCHLUETER, MD, PhD Chapter 29 Clinical Professor, Department of Pathology J. LYNNE WILLIAMS, PhD, MT(ASCP) University of Iowa Professor and Chair, Clinical and Diagnostic Sciences Iowa City, IA Oakland University, School of Health Sciences Chapter 3 Rochester, MI LINDA SMITH, PhD, MLS(ASCP)CM, BB Chapters 4, 8, 31, 32, 35 Professor Emeritus, Department of Health Sciences, KATHLEEN S. WILSON, MD, FCAP, FACMG Division of Medical Laboratory Sciences Professor, Department of Pathology and The McDermott University of Texas Health San Antonio Center for Human Growth and Development San Antonio, TX Director, Cytogenomic Microarray Analysis Laboratory Chapters 19, 20 University of Texas Southwestern Medical Center JULIE K. SODER, MS, MLS(ASCP)CM Dallas, TX Assistant Professor, Department of Clinical Laboratory Chapter 41 Science ANDREA C. YUNES, MD University of Texas Medical Branch Audie L. Murphy Memorial VA Hospital Galveston, TX San Antonio, TX Chapter 13 Chapter 29, 38 Preface xxv Credits Chapter 14 Figures 14-4a,b; 14-5; 14-8; 14-9; 14-11; 14-12; 14-13; 14-14 Shirlyn McKenzie. Table 14-8 Data from Chapter 1 Table 1-1 Courtesy of Linda Smith. Maria Stella Figueiredo (2015) The Compound state: Chapter 2 Figure 2-1 From “The fluid mosaic model of the Hbs/Beta. Thalassemia, Rev Brass Hemtol Hemotes 37(3). structure of cell membranes” by Singer S J & Nicolson G L 150–152; Ngo DA et.al.(2011) Fetal H eameoglobin levels in Science, Volume 175, Number 46, pp. 720–31. Published and haematological characteristics of compound heterozy- by Elsevier, © 1972. gotes for haemoglobin and deletional hereditary persis- tence of fetal haemoglobin, 156:259–264; Fucharoen S, Chapter 3 Figure 3-2 Used by permission of Dr. Corey et.al. (2003) Interaction of haemoglobin and several forms Parlet. Figure 3-4 a,b Annette Schlueter. of thalassemia in combidian families 88(10) 1092–1098. Chapter 5 Figure 5-2 Shirlyn McKenzie. Figure 5-3 Kristin Chapter 15 Figure 15-1b Kristin Landis-Piwowar. Landis-Piwowar. Figure 5-4 Based on Clinical Expression Figures 15-1a; 15-03; 15-04a,b; 15-11 Shirlyn McKenzie. and Laboratory Detection of Red Cell Membrane Protein Mutations by J. Palek and P. Jarolim in SEMINARS IN Chapter 16 Figure 16-1 Shirlyn McKenzie. HEMATOLOGY 30(4): Chapter 17 Figure 17-1 Pearson Education, Inc. Figures 17-2; 249–283, October 1993. Published by W.B./Saunders Co., 17-4; 17-5; 17-6; 17-7; 17-8; 17-9 Shirlyn McKenzie. an imprint of Elsevier Health Science Journals. Table 5-1 Chapter 18 Figures 18-2; 18-4; 18-6 Shirlyn McKenzie. images Shirlyn McKenzie. Chapter 19 Figures 19-4; 19-5 Shirlyn McKenzie. Chapter 6 Figure 6-8 MCMURRY, JOHN; CASTELLION, MARY E.; BALLENTINE, DAVID S.; FUNDEMENTALS OF Chapter 20 Figures 20-2; 20-3; F20-4a-c Shirlyn McKenzie. GENERAL, O RGANIC, AND BIOLOGICAL CHEMISTRY, Figure 20-5 John H. Landis. 5th ED.,© 2007. Reprinted and Elctronically reproduced by Chapter 21 Figures 21-1; 21-2; 21-3a,b; 21-4; 21-5; 21-7; permission of Pearson Education, Inc., Upper Saddle River, 21-8; 21-9; 21-10a,b; 21-11; 21-12; 21-13; 21-14; 21-15; 21-16; New Jersey. Figure 6-9 Based on Principles of Biochemistry, 21-17 Shirlyn McKenzie. Figure 21-6 John H. Landis. 4E by H. R. Horton, L. A. Moran, K. G. Scrimgeour and Chapter 22 Figures 22-1; 22-2; 22-3; 22-4; 22-5 Shirlyn M. D. Perry. Published by Pearson Education, Inc., © 2006. McKenzie. Table 22-6 From MMWR Recommendations Chapter 7 Figures 7-1; 7-2; 7-3; 7-4; 7-10; 7-11a-c; 7-12; 7-13; and Reports: Past Volume: 63(RR-03):1–10. Published by Table 7-3 images Shirlyn McKenzie. Centers for Disease Control and Prevention. Chapter 8 Table 8-3 images Shirlyn McKenzie. Chapter 23
Figures 23-2; 23-3 Shirlyn McKenzie. Chapter 9 Figures 9-1; 9-2a,b; 9-3a,b; 9-4; 9-5 Shirlyn Chapter 24 Figures 24-2; 24-5; 24-6; 24-7; 24-8; 24-9; 24-10; McKenzie. 24-12; 24-13; 24-14 Shirlyn McKenzie. Table 24-14 Data from Chapter 10 Figures 10-1; 10-2; 10-4a; 10-5a-c; 10-7; 10-9; Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, 10-11; 10-13; LeBeau MM, Bloomfield CD, Cazzola M, and Vardiman 10-14; 10-15; Table 10-10 images 1-4 Shirlyn McKenzie. JW. The 2016 revision to the World Health Organization Figure 10-3 Roche Diagnostics Hematology. Figures 10- classification of myeloid neoplasms and acute leukemia. 4b,c; 10-12 John H. Landis. Figures 10-6; 10-8a,b; 10-10; Blood. 2016;127(20);2391–2405. Table 10-7 images; Table 10-10 image 5; Case Study images Chapter 25 Figures 25-1; 25-2; 25-3; 25-4; 25-5; 25-6; 25-7; Kristin Landis-Piwowar. 25-8; 25-09a,b; 25-11a,b; 25-12 Shirlyn McKenzie. Chapter 11 Figure 11-1 Shirlyn McKenzie. Figure 11-2 Roche Chapter 26 Figures 26-2a,b; 26-3; 26-4; 26-5; 26-6; 26-7a,b; Diagnostics Hematology. Tables 11-3, 11-4, 11-5, 11-6 From 26-08a,b Shirlyn McKenzie. Morbidity And Mortality Weekly Report. Published by Chapter 27 Figures 27-1a,b; 27-2a,b Shirlyn McKenzie. Centers for Disease Control. Chapter 28 Figures 28-2; 28-5d; 28-7; 28-11a; 28-12a,b; 28-13 Chapter 12 Figures 12-11; 12-13a,b; 12-14; 12-15; 12-17 Fiona Craig. Figures 28-1a,b; 28-3a,b; 28-4a-d; 28-5a-c; Shirlyn McKenzie. 28-6a,b; 28-8; 28-9a-c; 28-10a,b; 28-11b-d; 28-14; 28-15a-c; Chapter 13 Table 13-1 Data from Huisman, T.H., Carver, Shirlyn McKenzie. M.F., Baysal, E., & Efremov, G. D. (2017). et al. A Database of Chapter 29 Figures 29-1; 29-2a,b Dr. Aamir Ehsan. Human Hemoglobin Variants and Thalassemias [Data file]. Retrieved from http://globin.cse.psu.edu/globin/hbvar/ Chapter 30 Table 30-2 Data from Morgenstern LB, menu.html. Figures 13-3, 13-4, 13-5, 13-w6, 13-7 Shirlyn Luna-Gonzales H, Huber JC et al. Worst headache and McKenzie. subarachnoid h emorrhage: p rospective, modern comput- ed tomography and spinal fluid a nalysis. Ann Emerg Med. 1998;32:297–304; Julia-Sanchis ML. Rapid differential xxvi Preface diagnosis between subarachnoid hemorrhage and Distribution Parameters – (1) RDW – SD (2) RDW traumatic lumbar puncture by D-dimer assay. Clin Chem. (CV), Technical Bulletin 9617. Copyright © 1983 by 2007;53:993. Figures 30-5; 30-6; 30-7; 30-59a,b; 30-60; 30-62; Beckman Coulter, Inc. Figures 39-11; 39-12; 39-13; 39-14 30-63; 30-64; 30-65; 30-66; 30-67 Jerelyn Walters. Figures 30- From Advancements in Technology: WBC Differential 8 through 30-31; 30-33 through 30-58; 30-68 through 30-71 Methodology, Technical Bulletin 9403. Copyright © 2009 Shirlyn McKenzie. by Beckman Coulter, Inc. Figures 39-9; 39-16; 39-17; 39-18; Chapter 31 Figures 31-7a-d Shirlyn McKenzie. 39-21; 39-25; 39-26 Shirlyn McKenzie. Chapter 33 Figures 33-02a-c; 33-7 Shirlyn McKenzie. Chapter 41 Figures 41-5; 41-6; 41-10 Shirlyn McKenzie. Figure 41-4 NiMedia/Shutterstock. Chapter 37 Figures 37-1; 37-2; 37-3; 37-4; 37-5; 37-9; 37-18 Cheryl Burn. Figures 37-10; 37-20; 37-22; 37-25; 37-30 Chapter 43 Table 43-6 From Abbott Laboratories. Used by Shirlyn McKenzie. Figures 37-6 Tracey C. Webb. permission of Abbott Laboratories. Figures 37-29; 37-31; 37-32; 37-33; 37-34; 37-35; 37-36; 37-37 Appendix C Modified with permission from Swerdlow, Dr. Aamir Ehsan. Figures 37-23; 37-24 Courtesy of Helena SH, Campo, E, Harris, NL, Jaffe, ES, Pileri, SA, Stein, H, Laboratories. Thiele, J, Arber DA, Hasserjian RP, Le Beau MM, Orazi Chapter 38 Figures 38-2; 38-3; 38-4; 38-5a-c; 38-6; 38-7; 38-8 A, Siebert R. World Health Organization Classification Shirlyn McKenzie. of Tumours of Haematopoietic and Lymphoid Tissues, revised 4th edition. IARC, Lyon, 2017. Chapter 39 Figure 39-1 From Seminar and Case Studies: The Automated Differential by Pierre R. Copyright © 1985 Glossary Clinical and Laboratory Standards Institute by Beckman Coulter, Inc. Figures 39-2; 39-3; 39-6 From (CLSI) entry is Copyright by Clinical and Laboratory Significant Advances in Hematology. Copyright © 1983 Standards Institute. by Beckman Coulter, Inc. Figure 39-4 From Red Cell Section One Introduction to Hematology 1 Chapter 1 Introduction Shirlyn B. McKenzie, PhD Objectives—Level I and Level II At the end of this unit of study, the student should be able to: 1. Compare the reference intervals for 6. Define hemostasis and describe the result of hemoglobin, hematocrit, erythrocytes, and an upset in the hemostatic process. leukocytes in infants, children, and adults. 7. Identify hematology and hemostasis 2. Identify the function of erythrocytes, screening tests. leukocytes, and platelets. 8. List the three components of laboratory 3. Describe the composition of blood. testing and correlate errors with each 4. Explain the causes of change in the steady component. state of blood components. 9. Define value-based health care and give an 5. Describe reflex testing, and identify the example of how the laboratory can assist in laboratory’s role in designing reflex testing building the value agenda. protocols. Chapter Outline Objectives—Level I and Level II 2 Hemostasis 5 Key Terms 3 Blood Component Therapy 5 Background Basics 3 Investigation of a Hematologic Problem 6 Case Study 3 The Value of Laboratory Testing 6 Overview 3 Summary 7 Introduction 3 Review Questions 7 Composition of Blood 4 References 8 Reference Intervals for Blood Cell Concentration 4 2 Introduction 3 Key Terms Activated partial thromboplastin Hematopoiesis RBC indices time (APTT) Hemoglobin Red blood cell (RBC) Complete blood count (CBC) Hemostasis Reflex test Diapedese Leukocyte Serum Erythrocyte Plasma Thrombocyte Hematocrit Platelet White blood cell (WBC) Hematology Prothrombin time (PT) Background Basics Students should complete courses in biology and physiology before beginning this study of hematology. platelets (phlegm); and a layer of yellowish serum (yellow CASE STUDY bile).1 Health and disease were thought to occur as a result We refer to this case study throughout the chapter. of an upset in the equilibrium of these humors. Aaron, a 2-year-old male, was seen by his pediatrician The cellular composition of blood was not recognized because he had a fever of 102–104 °F over the past until the invention of the microscope. With the help of a 24 hours. Aaron was lethargic. Before this, he had crude magnifying device that consisted of a biconvex been in good health except for two episodes of otitis. lens, Antonie van Leeuwenhoek (1632–1723) accurately Consider why the pediatrician might order described and measured the red blood cells (also known laboratory tests and how this child’s condition as RBCs or erythrocytes). The discovery of white blood might affect the composition of his blood. cells (also known as WBCs or leukocytes) and platelets (also known as thrombocytes) followed after microscope lenses were improved. As a supplement to these categorical observations of Overview blood cells, Karl Vierordt, in 1852, published the first quan- titative results of blood cell analysis.2 His procedures for Hematology is the study of blood and blood-forming quantification were tedious and time consuming. After organs. The hematology laboratory is one of the busiest several years, many others attempted to correlate blood cell areas of the clinical laboratory. Even small, limited-service counts with various disease states. laboratories usually offer hematology tests. This chapter is Improved methods of blood examination in the 1920s an introduction to the composition of blood and the test- and the increased knowledge of blood physiology and ing performed in the hematology laboratory to identify the blood-forming organs in the 1930s allowed anemias and presence and cause of disease. other blood disorders to be studied on a rational basis. In some cases, the pathophysiology of hematopoietic dis- orders was realized only after the patient responded to Introduction experimental therapy. Contrary to early hematologists, modern hematologists Blood has been considered the essence of life for centu- recognize that alterations in the components of blood are ries. One of the Hippocratic writings from about 400 b.c. the result of disease, not a primary cause of it. Under nor- describes the body as being a composite of four humors: mal conditions, the production of blood cells in the bone black bile, blood, phlegm, and yellow bile. It is thought marrow, their release to the peripheral blood, and their that the theory of the four humors came from the observa- survival are highly regulated to maintain a steady state of tion that four distinct layers form as blood clots in vitro: a morphologically normal cells. Quantitative and qualitative dark-red, almost black, jellylike clot (black bile); a thin layer hematologic abnormalities can result when an imbalance of oxygenated red cells (blood); a layer of white cells and occurs in this steady state. 4 Chapter 1 Composition of Blood enzymes, can indicate an abnormal increase in cell destruc- tion in a specific organ. Blood is composed of a liquid called plasma and of cellular Blood cells are produced and develop in the bone elements, including leukocytes, platelets, and erythrocytes. marrow. This process is known as hematopoiesis. After blood coagulates, the resulting liquid component is Undifferentiated hematopoietic stem cells (precursor cells) called serum. The normal adult has about 6 liters of this proliferate and differentiate under the influence of proteins vital fluid, which composes from 7% to 8% of the total body that affect their function (cytokines). When the cell reaches weight. Plasma makes up about 55% of the blood volume; maturity, it is released into the peripheral blood. about 45% of the volume is composed of erythrocytes, and Each of the three cellular constituents of blood has 1% of the volume is composed of leukocytes and platelets. specific functions. Erythrocytes contain the vital protein Variations in the quantity of these blood elements are often hemoglobin, which is responsible for transport of oxygen the first sign of disease occurring in body tissues. Changes from the lungs to the body tissues. Erythrocytes also facili- in diseased tissue may be detected by laboratory tests that tate the transport of carbon dioxide from the tissues back measure deviations from normal in blood constituents. to the lungs. The five major types of leukocytes are neutro- Hematology is primarily the study of the formed cellular phils, eosinophils, basophils, lymphocytes, and monocytes. elements of the blood. Each type of leukocyte has a role in defending the body The principal component of plasma is water, which against foreign pathogens such as bacteria and viruses. contains dissolved ions, proteins, carbohydrates, fats, hor- Platelets are necessary for maintaining hemostasis. Blood mones, vitamins, and enzymes. The principal ions neces- cells circulate through blood vessels, which are distributed sary for normal cell function include calcium, sodium, throughout every body tissue. Erythrocytes and platelets potassium, chloride, magnesium, and hydrogen. The main generally carry out their functions without leaving the ves- protein constituent of plasma is albumin, which is the most sels, but leukocytes diapedese (pass through intact vessel important component in maintaining osmotic pressure. walls) to tissues where they defend against invading foreign Albumin also acts as a carrier molecule, transporting com- pathogens. pounds such as bilirubin and heme. Other blood proteins carry vitamins, minerals, and lipids. Immunoglobulins, syn- thesized by lymphocytes, and complement are specialized CASE STUDY (continued from page 3) blood proteins involved in immune defense. The coagula- tion proteins responsible for hemostasis (arrest of bleed- 1. If Aaron was diagnosed with otitis media, what ing) circulate in the blood as inactive enzymes until they cellular component(s) in his blood would be are needed for the coagulation process. An upset in the bal- playing a central role in fighting this infection? ance of these dissolved plasma constituents can indicate a disease in other body tissues. Blood plasma also acts as a transport medium for cell nutrients and metabolites; for example, the blood transports hormones manufactured in one tissue to target tissue in Reference Intervals for other parts of the body. Albumin transports bilirubin, the main catabolic residue of hemoglobin, from the spleen to Blood Cell Concentration the liver for excretion. Blood urea nitrogen, a nitrogenous Physiologic differences in the concentration of c ellular waste product, is carried to the kidneys for filtration and elements can occur according to race, age, sex, and excretion. Increased concentration of these normal catabo- geographic location; pathologic changes in specific blood lites can indicate either increased cellular metabolism or a cell concentrations can occur as the result of disease or defect in the organ responsible for their excretion. For exam- injury. The greatest differences in reference intervals occur ple, in liver disease, the bilirubin level in blood increases between newborns and adults. In general, newborns because the liver is unable to function normally and clear have a higher erythrocyte concentration than any other the bilirubin. In hemolytic anemia, however, the bilirubin age group. The erythrocytes are also larger than those of concentration can rise because of the increased metabolism adults. In the 6 months after birth, erythrocytes gradually of hemoglobin that exceeds the
ability of a normal liver to decrease in number and then slowly increase. Hemoglobin clear bilirubin. and erythrocyte counts increase in children between the When body cells die, they release their cellular constitu- ages of 5 and 17. The leukocyte concentration is high at ents into surrounding tissue. Eventually, some of these con- birth but decreases after the first year of life. A common stituents reach the blood. Many constituents of body cells finding in young children is an absolute and relative lym- are specific for the cell’s particular function; thus, increased phocytosis (increase in lymphocytes). After puberty, males concentration of these constituents in the blood, especially have higher hemoglobin, hematocrit (packed red blood Introduction 5 cell volume in whole blood), and erythrocyte levels than system’s ability to form a barrier (blood clot or thrombus) females. The hemoglobin level decreases slightly after age to prevent excessive blood loss when the vessel is trauma- 70 in males. This is thought to be due to the decrease in tized, limit the barrier to the site of injury, and dissolve the testosterone. Appendix D, Tables A through J give hemato- thrombus to ensure normal blood flow when the vessel is logic reference intervals for various age groups and by sex repaired. Hemostasis occurs in stages called primary and if appropriate. secondary hemostasis and fibrinolysis (breakdown of fibrin). Each individual laboratory must determine reference These stages are the result of interaction of platelets, blood intervals of hematologic values to account for the p hysiologic vessels, and proteins circulating in the blood. An upset in differences of a population in a specific geographical area. any of the stages can result in bleeding or abnormal blood Reference intervals for a hematologic parameter are deter- clotting (thrombosis). Laboratory testing for abnormali- mined by calculating the mean {2 standard deviations for ties in hemostasis is usually performed in the hematology a group of healthy individuals. This interval represents the section of the laboratory; occasionally, hemostasis test- reference interval for 95% of normal individuals. A value ing is performed in a separate specialized section of the just below or just above this interval is not necessarily laboratory. abnormal; normal and abnormal overlap. Statistical prob- ability indicates that about 5% of normal individuals will Checkpoint 1.1 fall outside the {2 standard deviation range. The further a What cellular component of blood can be involved in disorders value falls from the reference interval, however, the more of hemostasis? likely the value is to be abnormal. CASE STUDY (continued from page 4) Blood Component Therapy Aaron’s physician ordered a complete blood count (CBC). The results are Hb 11.5 g/dL; Hct 34%. Blood components can be used in therapy for various hematologic and nonhematologic disorders. Whole blood 2. What parameters, if any, are outside the reference collected from donors can be separated into various cel- intervals? Why do you have to take Aaron’s age lular and fluid components. Only the specific blood com- into account when evaluating these results? ponent (i.e., platelets for thrombocytopenia or erythrocytes for anemia) needed by the patient will be administered. In addition, the components can be specially prepared for Hemostasis the patient’s specific needs (i.e., washed erythrocytes for patients with IgA deficiency to reduce the risk of anaphy- Hemostasis is the property of the circulation that main- lactic reactions). Table 1-1 lists the various components that tains blood as a fluid within the blood vessels and the can be prepared for specific uses. Table 1.1 Blood Components and Their Uses Component Name Composition Primary Use Whole blood Red blood cells and plasma Not used routinely; can be used in selected trauma, autologous transfusions, and neonatal situations; increases oxygen-carrying capacity and volume Packed red blood cells (PRBCs) PRBCs Used in individuals with symptomatic anemia to increase oxygen-carrying capability PRBCs, washed PRBCs; plasma with most leukocytes Used for individuals with repeated allergic reactions to components and platelets removed containing plasma and for IgA-deficient individuals with anaphylactic reactions to products containing plasma PRBCs, leukoreduced PRBCs; WBC removed Used to decrease the risk of febrile nonhemolytic transfusion reaction, HLA sensitization, and cytomegalovirus (CMV) transmission PRBCs, frozen, deglycerolized PRBCs frozen in cryroprotective agent, Used for individuals with rare blood groups (autologous donation) thawed, washed PRBCs, irradiated PRBCs with lymphocytes inactivated Used to reduce the risk of graft-versus-host disease Platelets, pooleda 4–6 units of random donor platelets Used to increase platelet count and decrease bleeding when there is a deficiency or abnormal function of platelets Platelets, singlea donor (pheresis) Equivalent of 4–6 donor platelets Used to treat patients refractory to random platelet transfusion or to collected from single donor increase platelet count due to a deficiency or abnormal function of platelets (Continued ) 6 Chapter 1 Table 1.1 Blood Components and Their Uses (Continued) Component Name Composition Primary Use Fresh frozen plasma (FFP) Plasma with all stable and labile coag- Used to treat patients with multiple coagulation factor deficiencies; ulation factors; frozen within 8 hours of disseminated intravascular coagulation (DIC); used with packed RBC in collection of unit of blood multiple transfusions Cryoprecipitated AHFb Concentrated FVIII, fibrinogen, FXIII, Used to treat patients with hypofibrinogenemia, hemophilia A, von von Willebrand factor Willebrand’s disease, FXIII deficiency Plasma, cryo-poor Plasma remaining after cryo removed Used to treat thrombotic thrombocytopenic purpura (TTP) Liquid plasma Plasma not frozen within 8 hours of Used in patients with deficiency of stable coagulation factor(s) and for collection volume replacement Granulocytes Granulocytes Used to treat the neutropenic patient who is septic and unresponsive to antimicrobials and who has chance of marrow recovery a Platelets can also be leukoreduced or irradiated. See PRBC for reasons. b Cryoprecipitated antihemophilic factor. Courtesy of Linda Smith, Ph.D., MLS(ASCP)CM; adapted from the circular of information for the use of human blood and blood components. Prepared jointly by the American Association of Blood Banks, America’s Blood Centers, and the American Red Cross (2002). Investigation of a direct further testing. The CBC can also include a WBC dif- ferential. This procedure enumerates the five types of WBCs Hematologic Problem and reports each as a percentage of the total WBC count. A differential is especially helpful if the WBC count is abnor- Laboratory testing is divided into three components: pre- mal. When the count is abnormal, the differential identifies examination, examination, and post-examination (formerly which cell type is abnormally increased or decreased and known as preanalytical, analytical, postanalytical). The pre- determines whether immature and/or abnormal forms are examination component includes all aspects that occur prior present, thus providing a clue to diagnosis. The morphol- to the testing procedure that affect the test outcome such ogy of RBCs and platelets is also studied as a routine part as phlebotomy technique and transport and storage of the of the differential and reported if abnormal. The assessment specimen after it is drawn but before the test is run. The of RBC morphology can provide key information to a differ- examination phase refers to all aspects affecting the test pro- ential diagnosis and help guide the selection of additional cedure. The post-examination component includes all aspects tests for a definitive diagnosis.3 after the testing is completed such as reporting of results If a hemostasis problem is suspected, the screening tests and execution of appropriate clinical responses. These three include the platelet count, prothrombin time (PT), and acti- aspects of testing are the backbone of a quality assessment vated partial thromboplastin time (APTT) (Chapter 36). program. See Chapters 10 and 43 for a detailed description The PT and APTT tests involve adding calcium and throm- of these three phases. boplastin or partial thromboplastin to a sample of citrated A physician’s investigation of a hematologic problem plasma and determining the time it takes to form a clot. includes taking a medical history and performing a physical These tests provide clues that guide the choice of follow-up examination. Clues provided by this preliminary investiga- tests to help identify the problem. tion help guide the physician’s choice of laboratory tests to help confirm the diagnosis. The challenge is to select appro- priate tests that contribute to a cost-effective and efficient diagnosis. Laboratory testing usually begins with screening The Value of Laboratory tests; based on results of these tests, more specific tests are ordered. The same tests can be ordered again on follow-up Testing to track disease progression, evaluate treatment, identify The “value agenda” of health care advocates is achieving side effects and complications, or assist in prognosis. the best outcomes at minimal cost without sacrificing qual- Hematology screening tests include the complete blood ity.4 In 2011, the Centers for Medicare and Medicaid Services count (CBC), which quantifies the WBCs, RBCs, hemoglo- (CMS) announced a reimbursement model intended to bin, hematocrit, and platelets, and the RBC indices (Chapter increase the accountability for achieving the best patient 10). The indices are calculated from the results of the hemo- outcomes at minimal cost without sacrificing quality of globin, RBC count, and hematocrit to define the size and care.5 The focus is on population health management versus hemoglobin content of RBCs. The indices are important a fee for service based on volume. By 2014, 20% of Medicare parameters used to differentiate causes of anemia and help reimbursement shifted to value-based payment models that Introduction 7 directly link reimbursement to the health and well-being of than others in helping diagnose or follow effectiveness of patients. The goal is to have 50% of Medicare payments in treatment. Readers are urged to use the reflex testing and these value-based payment models by 2018. The laboratory algorithm concepts in their thought processes when study- can play a major role in creating and advancing the value ing the laboratory investigation of a disease. agenda by improving clinical outcomes through the use of In an effort to help the student gain the knowledge appropriate laboratory testing while helping payers hold to perform these functions, in this text each hematologic down costs (economic outcomes). disorder is discussed in the following order: etiology, if Follow-up testing that is done based on results of known, pathophysiology, clinical presentation, labora- screening tests is referred to as reflex testing. These test- tory evaluation, and therapy. The reader should consider ing protocols are sometimes referred to as algorithms. which laboratory tests provide the information necessary Follow-up tests can include not only hematologic tests to identify the cause of the disorder based on the suspected but also chemical, immunologic, microbiologic, and/ disorder’s pathophysiology. Although it is unusual for the or molecular a nalysis. As scientists learn more about the physician to provide a patient history or diagnosis to the pathophysiology and t reatment of hematologic disease laboratory when ordering tests, this information is often and hemostasis, the number of tests designed to assist in crucial to direct investigation and assist in interpretation of diagnosis expands and, without testing guidelines, the the test results. In any case, if laboratory professionals need cost can increase due to inappropriate and unnecessary more patient information to appropriately perform testing, test selection. Errors in selection of the most appropriate they should obtain the patient’s chart or call the physician. laboratory tests and interpretation of results can result in misdiagnosis or treatment errors and is a major source of poor patient outcomes. Laboratory p rofessionals can assist in promoting good patient outcomes by assisting physi- Checkpoint 1.2 cians and patient care teams in selecting the most efficient A 13-year-old female saw her physician for complaints of a sore throat, lethargy, and swollen lymph nodes. A CBC was and effective testing strategies6,7,8 through development of performed with the following results: Hb 9.0 g/dL; Hct 30%; test ordering protocols and assisting in interpretation of WBC 15 * 103/mcL. On the basis of these results, should test results.9 Furthermore, validation studies of algorithms reflex testing be performed? may help determine if a particular testing protocol is better Summary Hematology is the study of the cellular components of can order reflex tests if one or more of the CBC parameters blood: erythrocytes, leukocytes, and platelets. Physiological are outside the reference interval. Platelet count, PT, and changes in the concentrations of these cells occur from APTT are screening tests for disorders of hemostasis. infancy until adulthood. Diseases can upset the steady Changes in the health care system focus on containing state concentration of these parameters. A CBC is usually costs while maintaining quality of care.
The laboratory’s performed as a screening test to determine whether there role in this system is to work with physicians to optimize are quantitative abnormalities in blood cells. The physician utilization of laboratory testing. Review Questions Level I and Level II 2. Which cells are important in transporting oxygen and carbon dioxide between the lungs and body tissues? 1. In which group of individuals would you expect to (Objective 2) find the highest reference intervals for hemoglobin, hematocrit, and erythrocyte count? (Objective 1) a. Platelets a. Newborns b. Leukocytes b. Males older than 12 years of age c. Thrombocytes c. Females older than 17 years of age d. Erythrocytes d. Children between 1 and 5 years of age 8 Chapter 1 3. Forty-five percent of the volume of blood is normally c. Second test by a different instrument composed of: (Objective 3) d. Standing orders for all inpatients a. erythrocytes 7. Screening tests used to evaluate the hemostasis sys- b. leukocytes tem include: (Objective 6) c. platelets a. PT and APTT d. plasma b. CBC 4. Alterations in the concentration of blood cells gener- c. hemoglobin ally are the result of: (Objective 4) d. WBC count a. laboratory error 8. A patient blood specimen is stored in a car for 2 b. amount of exercise before blood draw hours with the outside temperature of 95 °F. This is c. a disease process an example of error in which component of testing? d. variations in analytical equipment (Objective 8) a. Pre-examination 5. Leukocytes are necessary for: (Objective 2) b. Examination a. hemostasis c. Post-examination b. defense against foreign pathogens c. oxygen transport 9. The value of laboratory medicine can be increased by: d. excretion of cellular metabolites (Objective 9) a. providing raw data to payers when asked 6. Laboratories can use which type of testing to help b. prompting physicians to use a testing algorithm for direct the physician’s selection of appropriate testing anemia diagnosis after screening tests are performed? (Objective 5) c. focusing on fee for service based on increasing test- a. Reflexive based on results of screening tests ing volume. b. Manual repeat of abnormal results d. not interfering with physician test ordering References 1. Wintrobe, M. M. (1980). Blood: Pure and eloquent. New York: 6. Hernandez, J. S. (2003). Cost-effectiveness of laboratory testing. McGraw-Hill. Archives of Pathology and Laboratory Medicine, 127, 440–445. 2. Vierordt, K. (1852). Zahlungen der Blutkorperchen des Menschen. 7. From, P., & Barak, M. (2012). Cessation of dipstick urinalysis Archives of Physiology Heilk, 11, 327. reflex testing and physician ordering behavior. American Journal 3. Ford, J. (2013). Red blood cell morphology. International Journal of of Clinical Pathology, 137(3), 486–489. Laboratory Hematology, 35(3), 351–357. 8. Feldman, L. S., Shihab, H. M., Thiemann, D., Yeh, H. C., Ardolino, 4. Hillary, W., Justin, G. L., Bharat, M., & Jitendra, M. (2016). Value M., Mandell, S., & Brotman, D. J. (2013). Impact of providing fee based healthcare. Advances in Management, 9(1), 1–8. data on laboratory test ordering. JAMA Internal Medicine, 173(10), 5. Center for Medicare and Medicaid Services (CMS). Department 903–908. of Health and Human Services. Final Rule. (2011). Medicare 9. Dighe, A. S., Makar, R. S., & Lewandrowski, K. B. (2007). program; Hospital inpatient value-based purchasing program. Medicolegal liability in laboratory medicine. Seminars in Federal Register, 76(88), 26489–26547. Diagnostic Pathology, 24(2), 98–107. Chapter 2 Cellular Homeostasis Kristin Landis-Piwowar, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Describe the location, morphology, and 5. Define R (restriction point) and its role in function of subcellular organelles of a cell. cell-cycle regulation. 2. Describe the lipid asymmetry found in the 6. Define apoptosis and explain its role in plasma membrane of most hematopoietic normal human physiology. cells. 7. Classify and give examples of the major 3. Differentiate DNA replication, transcription, categories of initiators and inhibitors of translation, and DNA repair. apoptosis. 4. Differentiate the parts of the mammalian 8. List the major events regulated by apoptosis cell cycle. in hematopoiesis. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Explain the significance of SNPs, introns, 4. Define the two major classes of CKIs exons, UTRs, and post-translational protein (cyclin-dependent kinase inhibitors) and modifications. describe their function. 2. List the components and explain the 5. Compare the function of cell-cycle function of the ubiquitin-proteasome checkpoints in cell-cycle regulation. system. 6. Describe/illustrate the roles of p53 and pRb 3. Define cyclins and Cdks and their role in in cell-cycle regulation. cell-cycle regulation; describe the associated 7. Propose how abnormalities of cell-cycle Cdk partners and function of cyclins D, E, regulatory mechanisms can lead to A, and B. malignancy. 9 10 Chapter 2 8. Define caspases and explain their role in 12. Give examples of diseases associated apoptosis. with increased apoptosis and inhibited 9. Differentiate the extrinsic and intrinsic (decreased) apoptosis. pathways of cellular apoptosis. 13. Define epigenetics, and give examples of 10. Define and contrast the roles of pro- epigenetic changes associated with gene apoptotic and anti-apoptotic members of silencing. the Bcl-2 family of proteins. 14. Differentiate, using morphologic 11. Describe apoptotic regulatory observations, the processes of necrotic cell mechanisms. death and apoptotic cell death. Chapter Outline Objectives—Level I and Level II 9 Tissue Homeostasis: Proliferation, Differentiation, Overview 10 and Apoptosis 15 Introduction 11 Abnormal Tissue Homeostasis and Cancer 23 Review of Cell Structure 11 Summary 23 Review of the Flow of Genetic Review Questions 24 Information 13 References 26 Key Terms Apoptosis Intron Quiescence Caspase Mutation Restriction point (R) Cell cycle Necrosis Single-nucleotide polymorphism (SNP) Cell-cycle checkpoint Polymorphism Tissue homeostasis Cyclins/Cdk Post-translational modification Transcription factor (TF) Epigenetics Proteomics Tumor suppressor gene Exon Proteasome Ubiquitin Genome/genomics Proto-oncogene Untranslated region (UTR) Background Basics Level I and Level II organelles. They should have an understanding of Students should have a solid foundation in basic cell the segments composing a cell cycle (interphase and biology principles, including the component parts of mitosis) and the processes that take place during each a cell and the structure and function of cytoplasmic stage. Overview chapters and thus may be of value to some users. The chapter begins with a review of the basic components and Not all hematology courses include the material in this cellular processes of a normal cell and presents the concept chapter. It is a review of basic principles of cell and molec- of tissue homeostasis. Cellular processes that maintain ular biology and homeostasis, which provide the founda- tissue homeostasis—cell proliferation, cell differentia- tion for understanding many pathologic abnormalities tion, and cell death—are discussed at the functional and underlying the hematologic disorders in subsequent molecular level. Cellular Homeostasis 11 Introduction of ions, nutrients, and communication signals between the cytoplasm and its extracellular milieu and thus determines The maintenance of an adequate number of cells to carry the interrelationships of the cell with its surroundings. out the functions of an organism is referred to as tissue homeostasis. It depends on the careful regulation of several LIPIDS cellular processes, including cellular proliferation, c ellular The plasma membrane consists of a complex, ordered array differentiation, and cell death (apoptosis). A t horough of lipids and proteins that serve as the interface between understanding of cell structural components as well as the the cell and its environment (Figure 2-1b). The lipids have processes of cell division and cell death allows us to under- their polar (hydrophilic) head groups directed toward the stand not only the normal (physiologic) regulation of the outside and inside of the cell and their long-chain (hydro- cells of the blood, but also disease processes in which these phobic) hydrocarbon tails directed inward. Although the events become dysregulated (e.g., cancer). plasma membrane has traditionally been described as a “fluid mosaic” structure,1 it is in fact highly ordered with asymmetric distribution of both membrane lipids and pro- teins. The lipid and protein compositions of the outside and Review of Cell Structure inside of the membrane differ from one another in ways that A cell is an intricate, complex structure consisting of a reflect the different functions performed at the membrane’s membrane-bound aqueous solution of proteins, carbo- two surfaces. hydrates, fats, inorganic materials, and nucleic acids. The Four major phospholipids are found in the plasma nucleus, bound by a double layer of membrane, controls membrane: phosphatidylethanolamine (PE), phosphatidyl- and directs the development, function, and division of the serine (PS), phosphatidylcholine (PC), and sphingomyelin cell. The cytoplasm, where most of the cell’s metabolic reac- (SM). (Web Figure 2-1). Most blood cells have an asym- tions take place, surrounds the nucleus and is bound by metric distribution of these phospholipids with PE and PS the cell membrane. The cytoplasm contains highly ordered occurring in the inner layer of the lipid bilayer and PC and organelles, which are membrane-bound components with SM occurring predominantly in the outer layer. The mem- specific functions (Figure 2-1a). The different types of organ- brane lipids can freely diffuse laterally throughout their elles and the quantity of each depend on the function of the own half of the bilayer, or they can flip-flop from one side cell and its state of maturation. of the bilayer to the other in response to certain stimuli, as occurs in platelets when activated. Membrane lipids Cell Membrane including phospholipids, cholesterol, lipoproteins, and lipopolysaccharides contribute to the basic framework of The outer boundary of the cell, the plasma membrane, is cell membranes and account for the cell’s high permeability often considered a barrier between the cell and its environ- to lipid-soluble substances. Different mixtures of lipids are ment. In fact, it functions to allow the regulated passage found in the membranes of different types of cells. Nucleus Nucleolus Smooth Vacuole endoplasmic reticulum Integral globular Hydrophilic Golgi proteins polar head apparatus Lysosome Rough endoplasmic reticulum Polysome Centrioles Lipid Nuclear pore bilayer Nuclear envelope Hydrophobic Mitochondrion hydrocarbon tail a b Figure 2.1 (a) Drawing of a cell depicting the various organelles. (b) The fluid mosaic membrane model proposed by Singer and Nicholson (1972). 12 Chapter 2 PROTEINS of the membrane. Functions of the carbohydrate moieties Although lipids are responsible for the basic structure of include specific binding, cell-to-cell recognition, and cell the plasma membrane, proteins carry out most of the mem- adhesion. The sugar groups are added to the lipid or protein brane’s specific functions. The proteins of the membrane molecules in the lumen of the Golgi apparatus after synthe- provide selective permeability and transport of specific sub- sis by the endoplasmic reticulum. Many of the glycoprotein stances, structural stability, enzymatic catalysis, and func- transmembrane proteins serve as receptors for extracellular tions in cell-to-cell recognition. molecules such as growth factors. The binding of the spe- The membrane proteins are divided into two general cific ligand to a receptor can result in transduction of a sig- groups: integral (transmembrane) proteins and peripheral nal to the cell’s interior without passage of the extracellular proteins. The peripheral proteins are located on either the molecule through the membrane (see discussion of cytokine cytoplasmic or the extracellular half of the lipid bilayer. regulation of hematopoiesis in Chapter 4). Some of the integral proteins span the entire lipid bilayer; other integral proteins only partially penetrate the mem- Cytoplasm brane. Some membrane-spanning proteins traverse the membrane once (e.g., erythrocyte glycophorin A) while oth- The cytoplasm, or cytosol, is where the metabolic activities ers cross multiple times (e.g., erythrocyte band 3, the cation of the cell including protein synthesis, growth, motility, and transporter). In some cells, such as erythrocytes, peripheral phagocytosis take place. The structural components, called proteins on the cytoplasmic side of the membrane form a organelles, include the mitochondria, lysosomes, endoplas- lattice network that functions as a cellular cytoskeleton, mic reticulum (ER), Golgi apparatus, ribosomes, granules, imparting order on the membrane (Chapter 5). microtubules, and microfilaments (Table 2-1). Organelles and other cellular inclusions lie within the cytoplasmic CARBOHYDRATES matrix. The composition of the cytoplasm depends on cell Carbohydrates linked to membrane lipids (glycolipids) or lineage and degree of cell maturity. The appearance of cyto- proteins (glycoproteins) can extend from the outer surface plasm in fixed, stained blood cells is important in evaluating Table 2.1 Cellular Organelles Structure Composition Function Ribosomes RNA + proteins Assemble amino acids into protein “Free” Scattered in the cytoplasm Synthesis of protein destined to remain in
cytosol Linked by mRNA-forming polyribosomes “Fixed” Ribosomes bound to outer surface of rough ER Synthesis of protein destined for export from the cell ER Interconnecting membrane-bound tubules and vesicles Synthesis and transport of lipid and protein Rough ER Studded on outer surface with ribosomes Abundant in cells synthesizing secretory protein; protein transported to Golgi Smooth ER Lacks attached ribosomes Lipid synthesis, detoxification, synthesis of steroid hormones Golgi apparatus Stacks of flattened membranes located in juxtanuclear region Protein from rough ER is sorted, modified (e.g., glycosylated), and packaged; forms lysosomes Lysosomes Membrane-bound sac containing catalase, peroxidase, other Destruction of phagocytosed material (extracellular proteins, metabolic enzymes cells) and cellular organelles (autophagy) Peroxisome Membrane-bound sacs containing hydrolytic enzymes Catabolism of long-chain fatty acids; detoxification of toxic substances Mitochondria Double-membrane organelle; inner folds (cristae) house Oxidative phosphorylation (ATP production) abundant in enzymes of aerobic metabolism metabolically active cells Cytoskeleton Microfilaments, intermediate filaments, and microtubules Gives cell shape, provides strength, and enables movement of cellular structures Microfilaments Fine filaments (5–9 nm); polymers of actin Control shape and surface movement of most cells Intermediate filaments Ropelike fibers (∼10 nm); composed of a number of fibrous Provide cells with mechanical strength proteins Microtubules Hollow cylinders (∼25 mm); composed of protein tubulin Important in maintaining cell shape and organization of organelles; form spindle apparatus during mitosis Centrosome “Cell center”; includes centrioles Microtubule-organizing center; forms poles of mitotic spindle during anaphase Centrioles Two cylindrical structures arranged at right angles to each Enable movement of chromosomes during cell division; other; consist of nine groups of three microtubules self-replicate prior to cell division ER, endoplasmic reticulum. Cellular Homeostasis 13 the morphology, classifying the cell, and determining the RNA and proteins and is believed to be important in RNA stage of differentiation. Immature or transcriptionally active synthesis. The nucleolus of very young blood cells is easily blood cells stained with Romanowsky stains (Chapter 37) seen with brightfield microscopy on stained smears. have very basophilic (blue) cytoplasm due to the large quantity of ribonucleic acid (RNA) they contain. Checkpoint 2.2 Explain the difference between densely staining chromatin and Checkpoint 2.1 lighter staining chromatin when viewing blood cells under a What does the phrase lipid asymmetry mean when describing microscope. cell membranes? Nucleus Review of the Flow of The nucleus contains the genetic material, deoxyribonucleic Genetic Information acid (DNA), responsible for the regulation of all cellular Hematopoietic cells must divide in order to maintain a functions. The nuclear material, chromatin, consists of long critical mass within hematopoietic tissues and, ultimately, polymers of nucleotide subunits (DNA) and associated to supply the body with blood cells. The most basic structural proteins (histones) packaged into chromosomes. function of cell division is the accurate duplication of The genome is the total genetic information stored in all of DNA and the segregation of chromosomes into two genet- an organism’s chromosomes. ically identical daughter cells. Through the process of NUCLEAR ENVELOPE transcription, a portion of the DNA code, called a “gene,” The nuclear envelope is a double membrane ( phospholipid is copied into an RNA sequence. If a gene provides the bilayer) that surrounds the nuclear contents. The outer information to produce a protein (most genes provide membrane (cytoplasmic side) is continuous with ER such p rotein-coding information), then the transcribed that contains ribosomes. The nuclear membranes are RNA will be used to produce the amino acid sequence permeable to small nonpolar molecules only. The nuclear of a protein through the process of translation. Once a envelope is interrupted by channels, or nuclear pores, that protein is no longer needed in a cell, or if it is damaged, function as selective gates for the bidirectional movement it must be degraded. of molecules. These nuclear pores provide a means of communication between nucleus and cytoplasm, export- DNA Replication ing newly assembled ribonucleic acids while importing DNA is a molecule comprised of two chains or “strands” proteins such as transcription factors and DNA repair of complementary nucleotide subunits. DNA replication enzymes. is a process regulated by numerous proteins and enzymes CHROMATIN in which the two DNA strands must separate and serve as The appearance of chromatin varies, presumably depending the template for the synthesis of daughter DNA strands on activity. The dispersed, lightly stained portions of chro- that are accurate replicas of the original DNA molecule. matin (euchromatin) are generally considered to represent However, DNA replication is not always error-free. It unwound or loosely twisted regions of chromatin that are is estimated that about 0.01% of the 6 billion base pairs transcriptionally active. The condensed, more deeply stain- are copied incorrectly during each DNA replication.2 ing chromatin (heterochromatin) is believed to represent Fortunately, the process of DNA replication is coupled tightly twisted or folded regions of chromatin strands that with DNA repair systems to ensure that errors in DNA are transcriptionally inactive. The ratio of euchromatin to replication are corrected. heterochromatin depends on cell activity, with the younger DNA REPAIR or more active cells having more euchromatin and a finer In addition to correcting copying errors, DNA repair sys- chromatin appearance microscopically. tems correct DNA damage that might have occurred by The fundamental subunit of chromatin is the nucleo- other events. If DNA replication errors cannot be cor- some, a beadlike segment of chromosome composed of about rected, the cell may undergo apoptosis (discussed later); 180 base pairs of DNA wrapped around a histone protein. this is believed to be the underlying basis for the large The linear array of successive nucleosomes gives chromatin degree of ineffective erythropoiesis in megaloblastic ane- a “beads-on-a-string” appearance in electron micrographs. mias (Chapter 15). However, if the cell does not activate NUCLEOLI apoptosis, the failure of DNA repair often contributes to The nuclei of most active cells contain one to several pale the acquired mutations that result in the development of a staining nucleoli. The nucleolus (singular) consists of malignancy (Chapter 23). 14 Chapter 2 MUTATIONS AND POLYMORPHISMS When miscopied DNA is not corrected, a mutation (or new Checkpoint 2.3 polymorphism) can occur. Often, the word mutation is used What is the difference between a polymorphism and a mutation? only to describe a deleterious change in a gene (e.g., the bs globin mutation in sickle cell anemia [Chapter 13]). However, CONTROL OF GENE EXPRESSION not every alteration in DNA produces an abnormality. The Many signals that regulate genes come from outside the term polymorphism is used to describe the presence of mul- cell (e.g., cytokine control of hematopoiesis; Chapter 4). tiple alternate copies (alleles) of a gene and generally, the The external molecule or ligand (cytokine) binds to its term is used if the change in DNA sequence does not result specific receptor on the surface of the cell. The binding in a functional abnormality. of ligand to receptor activates the receptor and initiates a A region of DNA that differs in only a single DNA cell-signaling pathway that conveys the activation signal nucleotide is called a single-nucleotide polymorphism from the receptor to the nucleus. The end result is that a (SNP). SNPs are found at approximately 1 in every 1000 base protein called a transcription factor (TF) interacts with pairs in the human genome (resulting in about 2.5 m illion the DNA and either activates or represses transcription of SNPs in the entire genome of a cell). To be considered a the target gene(s). true polymorphism, a SNP must occur with a frequency of The term “gene expression” refers to the production more than 1% in the general population. If the alteration is of a gene’s functional product (often protein). Control of known to be the cause of a disease, the nucleotide change is gene expression is a complex process that is regulated in considered to be a mutation rather than a SNP. both time (e.g., during certain developmental stages) and location (e.g., tissue-specific gene expression). In order for Transcription transcription of a gene to take place, a sequence of DNA called the promoter is bound by a TF. Some TFs can activate All RNA is produced by means of DNA transcription, a pro- and/or repress gene expression, depending on the specific cess that shares similarities with DNA replication. In brief, targeted gene. Often TFs are tissue specific, such as GATA-1, when a gene is to be transcribed, the two DNA strands must a known erythroid-specific TF that regulates expression of be separated and one strand serves as the template for the glycophorin and globin chains in developing cells of the generation of an RNA molecule. The newly generated RNA erythroid lineage3 (Chapter 6). sequence is an exact replica of a gene’s DNA code. Genes In addition to the basic on/off function of the pro- that encode for proteins produce messenger RNA (mRNA) moter region, there are additional layers of control of gene that will later undergo translation into a sequence of amino expression. Some genes have response elements called acids (protein). enhancer elements or silencer elements, which are nucleo- RNA PROCESSING tide sequences that can amplify or suppress gene expres- Most genes are composed of stretches of nucleotides that are sion, respectively.2 These response elements influence gene organized into segments called exons and are interrupted expression by binding specific regulatory proteins (tran- by intervening sequences called introns. The exons contain scriptional activators, transcriptional repressors). the nucleotide sequences that correspond to the information for the final protein product, while the nucleotide base pairs Translation of the introns do not code for protein. Once an mRNA is produced and processed, its nucleotide When the DNA of a gene is transcribed into RNA, sequence can be used as information to generate a strand the entire sequence of exons and introns is used to gen- of amino acids. When linked together, this strand of amino erate a premessenger RNA. Subsequently, the nucleotides acids is called a polypeptide and will form part or all of a corresponding to the intron sequences are removed functional protein. Free ribosomes in the cytoplasm or those (spliced out) and the exons are ligated together. This that make up rough ER are the sites of mRNA translation process results in a shorter, mature mRNA. Several into proteins. A coordinated series of events ensures that inherited hematologic diseases, such as some of the thal- the mRNA is pulled through the active site of the ribosome. assemias, result from mutations that derange mRNA Transfer RNAs (tRNAs) deliver the amino acids in the cor- splicing (Chapter 14). rect sequence to generate the polypeptide chain. For a pro- A mature mRNA is flanked on its extreme ends (5′ and tein to properly function, additional events such as folding 3′ ends) by sequences that will not be translated into protein. and post-translational modifications are required. These ends are called untranslated regions (UTRs). The UTRs influence mRNA stability and the efficiency of mRNA PROTEIN FOLDING translation into protein and they contribute to the regulation The primary structure of a protein is defined by its amino of other cellular proteins, including those involved in iron acid sequence. Depending on the primary amino acid metabolism in developing erythrocytes (Chapter 12). sequence, the protein is subsequently folded into specific Cellular Homeostasis 15 structures (secondary structures). The two major second- Ub ary protein structures are a@helices and b@pleated sheets. The tertiary structure of a protein refers to its unique three-dimensional shape determined by additional folding E1 Ub E2 Ub of secondary structures. Sometimes, appropriately folded protein monomers are assembled with other proteins to form multi-subunit complexes. The quaternary struc- ture of a protein refers to the assembly of independently synthesized polypeptide chains into a multimeric protein E2 Ub (e.g., the a2b2 tetramer, which constitutes hemoglobin A; E3 Chapter 6). Target Target POST-TRANSLATIONAL MODIFICATIONS Changes in protein structure that occur after the protein Ub Ub is produced by translation on the ribosome are called Ub Ub post-translational modifications. These modifications Ub Ub can occur shortly after translation to mediate proper Ub Ub folding or after folding in order to impart the biologi- Ub cal activity of the protein. Post-translational modifica- 26S Proteosome tions include the addition of nonprotein groups (such as sugars or lipids), modification of existing amino acids Figure 2.2 Ubiquitin proteasome system. Ubiquitin-activating (such as the g@carboxylation of glutamic acid residues on enzyme (E1) activates ubiquitin (Ub), which is then transferred to certain coagulation proteins; Chapter 32) or cleavage of the
Ub-conjugating enzyme (E2); Ub ligase (E3) functions in target the initial polypeptide product resulting in a multichain substrate recognition; it brings together the target and E2-Ub and molecule. then catalyzes the transfer of Ub from E2-Ub to the target. Once As proteins exit the rough ER, they may be accompa- a target has become multi-ubiquitinated, it is directed to the 26S nied by molecules that facilitate their transfer to the Golgi proteosome for degradation. apparatus. During transport through the Golgi, additional processing of the protein can occur. A mutation in one of these transfer molecules, ERGIC-53, is the cause of the Tissue Homeostasis: hemostatic disorder Combined Factor V and VIII defi- ciency (Chapter 34). Proliferation, Differentiation, If a mutation alters the primary amino acid sequence, it can result in a protein that fails to function. Mutations and Apoptosis can occur in a critical functional amino acid or can cause Tissue homeostasis refers to the maintenance of an adequate the substitution of a different amino acid that prevents number of cells to carry out the organism’s functions. In the the protein from folding into its proper three-dimensional human body, somatic cells (including blood cells) generally structure. Improperly folded proteins are marked for undergo one of three possible fates: destruction and are degraded via the ubiquitin protea- some system. 1. Proliferation by mitotic cell division 2. Differentiation and acquisition of specialized Protein Degradation functions 3. Death and elimination from the body Cells contain two major mechanisms for the degradation of proteins (proteolysis): lysosomes and the ubiqui- Cell proliferation is required to replace cells lost to ter- tin proteasome system. The ubiquitin proteasome sys- minal differentiation, or cell death. Differentiation provides tem accounts for the vast majority of proteolysis and a variety of cells, each of which is capable of executing spe- functions in the cytoplasm of most cells. It is respon- cific and specialized functions. Cell death, in the form of sible for disposing of damaged or misfolded proteins apoptosis, is an active process that the cell itself can initiate. and regulates cellular processes.4 Proteins destined for Apoptosis is physiologically as important as cell proliferation destruction are tagged with a small polypeptide called and differentiation in controlling the overall homeostasis of ubiquitin (Figure 2-2). Ubiquitin-labeled proteins are various tissues. When any of these three cellular processes then transferred to an ATP-dependent protease complex malfunctions or the processes become unbalanced, the con- (the proteasome) and through which proteins are chan- sequence may be tissue a trophy, f unctional insufficiency, or neled for destruction. excessive growth/neoplasia (cancer; Chapters 23–28). 16 Chapter 2 Proliferation: The Cell Cycle Gap 2 (G2), and mitosis (M) (Figure 2-3). These phases are carefully regulated by a variety of proteins. Cell division is required throughout the life of all eukary- otes. When a cell is stimulated to divide, it goes through a PHASES series of well-defined stages called the cell cycle, which is The interval between successive mitoses is called interphase. divided into four phases: Gap 1 (G1), DNA synthesis (S), During interphase, the cell synthesizes molecules, increases G0 M G2 G1 S a R Spindle Pair of sister pole chromatids bodies Nuclear Spindle envelope microtubules Interphase Prophase Prometaphase Sister chromatids Telophase Anaphase Metaphase Cytokinesis Daughter cells b Figure 2.3 The four phases of the cell cycle: G1, S, G2, and M. (a) G0 represents the state of quiescence when a cell is withdrawn from the cell cycle. R represents the restriction point—the point in the cell cycle after which the cell is no longer dependent on extracellular signals but can complete the cycle in the absence of mitogenic stimuli. (b) The stages of mitosis. During G1, S, and G2, the nucleus is in interphase. During interphase, the spindle pole bodies duplicate and nucleate cytoplasmic microtubules. In prophase, the chromatin condenses into visible chromosome fibers; the nuclear membrane starts to break down. By prometaphase, the nuclear envelope has disappeared, and each pole body is organizing a set of microtubules. The two sets of microtubules constitute the spindle. At this stage, each condensed chromosome consists of two duplicate DNA molecules called sister chromatids. Each chromatid has a centromere and an associated structure called a kinetchore at which microtubules attach. At metaphase, all the chromosomes are balanced halfway between the poles in a plane called the metaphase plate. After all the chromosomes are aligned, the sister chromatids separate (the beginning of anaphase). The chromosomes (now single chromatids) move along the microtubule bundles to the poles. At telophase, the chromosomes have moved to the poles, and the poles are separated by their maximum distance. The nuclear envelope reforms. At cytokinesis, the cytoplasm pinches to give two separate cells. Cellular Homeostasis 17 in size, and duplicates its components in preparation for the requires the enzymatic activities of kinase proteins (cyclin- next mitosis. dependent kinases [Cdks]) that phosphorylate t arget Not all of the cells in the body are actively dividing molecules important for cell cycle control. A Cdk must be (i.e., actively engaged in the cell cycle). Cells can exit the complexed with a regulatory protein called cyclin (hence cell cycle at G1 and enter a nonproliferative phase called the name, cyclin-dependent kinase) in order to function. G0, or quiescence (Figure 2-3). In response to a specific The concentration of the different cyclin proteins that regu- stimulus (often in the form of proteins called growth fac- late the cell cycle rises and falls at specific times during the tors), quiescent cells can exit G0 and re-enter the cell cycle cell cycle. Sequential activation of specific cyclin/Cdk com- at the level of G1. Some cells, such as terminally differ- plexes drive the cell from one cell-cycle phase to the next as entiated neutrophils, have irreversibly exited the cell summarized in Table 2-2. Each complex in turn phosphory- cycle during differentiation and are locked in G0. Other lates key substrates and facilitates progression through the cells, such as hematopoietic stem cells or antigen-specific cell cycle (Figure 2-4a). memory lymphocytes, p rimarily reside in G0 but can be When a mammalian cell receives external signals induced to return to G1 and begin cycling with appropriate (growth factors and/or hormones) that trigger the cell to stimulation. G1 is a gap period in which the cell prepares the enzymes and proteins required for DNA replication in S phase. The conditions must be suitable (sufficient nutrients Table 2.2 Cell Cycle Regulatory Proteins or growth factors) for a cell to progress through G1 and Cell-Cycle Phase Cyclin Partner Cdk pass what is called the restriction point (R) (Figure 2-3a). G1 D1, D2, D3 Cdk4, Cdk6 R defines a point in the cell cycle after which the cell no G1/S E Cdk2 longer depends on extracellular signals but is committed to S A Cdk2 completing that cell cycle independent of further stimulation G2/M A Cdk1 (i.e., cell-cycle completion becomes autonomous).5 Cells M B Cdk1 then transit across the G1/S boundary into S phase. After the DNA has been replicated in S phase, G2 is a second gap phase, during which time DNA is checked for replication errors or damage. Upon the completion of interphase, the Cyclin B Cyclin Cdk1 D1, D2, D3 onset of mitosis commences.5 The physical process of cell division (M phase, or mito- M Cdk4, 6 G1 sis) includes a series of morphologically recognizable stages G2 (Figure 2-3b). During mitosis, chromosomes condense (pro- Cyclin E phase) and align on a microtubular spindle (metaphase), and S Cdk2 sister chromatids segregate to opposite poles of the cell (anaphase and telophase). The latter stages of mitosis result Cyclin A in both nuclear division (karyokinesis) and cytoplasmic Cdk2 separation (cytokinesis). a MOLECULAR REGULATION Cell-cycle cyclin patterns The fundamental task of the cell cycle is to faithfully repli- cate DNA once during S phase and to distribute identical E A B1, B2 copies of each chromosome to each daughter cell during M phase. Progression through the cell cycle normally ensures D that this takes place, so that cells do not initiate mitosis before DNA duplication is completed, do not attempt to segregate sister chromatids until all chromosome pairs are aligned on the mitotic spindle at metaphase, and do not reduplicate their chromosomes (reinitiate S phase) before G1 S G2 M b the paired chromatids have been separated at the previ- ous mitosis. Failure to regulate this process results in the Figure 2.4 Cell-cycle phases and regulatory proteins. aneuploidy (abnormal chromosome number) that is seen (a) The phases of the cell cycle with the major regulatory cyclin/Cdk in many malignancies. complexes depicted for each. (b) The levels of the various cyclin proteins during the cell cycle. The cyclins rise and fall in a periodic CYCLINS AND CYCLIN-DEPENDENT KINASES The fashion, thus controlling the cyclin-dependent kinases and their transition between the various phases of the cell cycle activities. 18 Chapter 2 proliferate the result is an increase of one (or more) of the D p21, p27, p57 cyclins of which there are three: D1, D2, D3. Cyclin D com- plexes with Cdk4 or Cdk6 and phosphorylates target mol- ecules required for G1 S S progression. The D cyclins are unique in that they are synthesized in response to growth Cyclin D1 factor stimulation and remain high in concentration as long Cyclin D2 Cyclin E Cyclin A Cyclin B as the mitotic stimulus is present (Figure 2-3b). During mid Cyclin D3 to late G1, cyclin E increases and binds with Cdk2. The cyclin G0 E/Cdk2 complex is required for the G1 S S transition. Once the cell enters S phase, cyclin E degrades rapidly, and cyclin G1 S G2 M A activates Cdk2. Cyclin A/Cdk2 is required for the onset of DNA synthesis and progression through S phase. Toward Cdk4 the end of S phase, cyclin A starts to activate another kinase, Cdk2 Cdk2 Cdk1 Cdk6 Cdk1, which signals the completion of S phase and the onset of G2. Cyclin B (which begins to increase during S and G2) takes over from cyclin A as the activating partner of Cdk1, and cyclin B/Cdk1 controls the onset, sequence of events, p15, p16, p18, p19 and the completion of mitosis. Cyclin B must be destroyed for the cell to exit mitosis and for cytokinesis to take place. Figure 2.5 Cdk inhibitors. There are two families of cyclin- REGULATION OF CDK ACTIVITY Control of Cdk activ- dependent kinase inhibitors. The first group of inhibitors, including p21, p27, and p57, possesses a wider spectrum of inhibitory activity, ity is somewhat unique in that protein levels of the enzyme inhibiting the G1 as well as S phase cyclin/Cdk complexes (cyclin (kinase) subunit remain constant throughout the cell cycle. D/Cdk4/6, cyclin E/Cdk2, and cyclin A/Cdk2). The second group The cell cycle is controlled by altering the availability of includes p15, p16, p18, and p19 and inhibits only D-type cyclin/ the regulatory cofactor (the cyclins) through periodic (and Cdk4 or Cdk6 complexes. # indicates inhibition of the pathway. cell-cycle phase-specific) synthesis and degradation of the appropriate cyclin via the ubiquitin proteasome system6 (Figure 2-4b). The periodic accumulation of different phase and checks for damaged or unreplicated DNA; it cyclins determines the sequential rise and fall of kinase can block mitosis if any is found. The metaphase checkpoint activities, which in turn regulates the events of cell-cycle (also called the mitotic-spindle checkpoint) functions to ensure progression. that all chromosomes are properly aligned on the spindle Another level of regulation involves two groups of pro- apparatus prior to initiating chromosomal separation and teins that function as inhibitors of Cdks and cyclin/Cdk segregation at anaphase. complexes7 (Figure 2-5). The first Cdk inhibitor identified If defects are detected at any of these checkpoints, the was p21; other Cdk inhibitors with structural and functional cell cycle is stopped and repair pathways are initiated, or if similarities to p21 include p27 and p57. The “p” indicates the damage is severe and/or irreparable, apoptosis can be that they are proteins of the indicated molecular mass in triggered (see the section “Apoptosis”). For example, the kilodaltons (e.g., p21 is a protein of molecular weight of proteins ATM (ataxia-telangiectasia mutated) and ATR (AT 21,000 daltons). These three inhibitors are considered “uni- and RAD-3-related) detect and are activated by damaged versal” because they bind multiple cyclin/Cdk complexes DNA. These proteins help to activate p53, a transcription (cyclin D/Cdk4/6, cyclin E/Cdk2, and cyclin
A/Cdk2). A factor that can either activate or inhibit gene expression, second group of inhibitors is a family of structurally related depending on the target gene,9 and in turn, p53 halts cell- proteins that include p15, p16, p18, and p19. These inhibi- cycle progression, allowing time for DNA repair to take tors are more restricted in their inhibitory activity (inhibit place, or triggers apoptosis if repair is not possible. p53 only Cdk4 and Cdk6) and induce cell-cycle arrest in G1.7 is an important component of both the G1 and the G2/M checkpoints. When p53 is mutated, an important cell- CHECKPOINTS cycle checkpoint is lost. In fact, p53 is the most commonly Cell proliferation requires cell-cycle checkpoints to monitor mutated gene in human malignancies.9 events at critical points in the cell cycle and, if necessary, Another critical cell-cycle regulator is Rb, a protein halt the cycle’s progression.8 Four major cell-cycle check- that is present throughout the cell cycle, although its points have been described. The G1 checkpoint checks for phosphorylation state changes markedly at different DNA damage and prevents progression into S phase if the phases (Figure 2-6).10 In its hypophosphorylated (active) genomic DNA is damaged. The S-phase checkpoint moni- state, Rb inhibits cell-cycle progression by binding the tors the accuracy of DNA replication. The G2/M checkpoint transcription factor, E2F. When growth factors induce activa- also monitors the accuracy of DNA replication during S tion of cyclin D/Cdk4/6, the Rb protein is phosphorylated, Cellular Homeostasis 19 Mitogenic Cytokine/Growth Factor activated and inactivated, resulting in a c hanging l andscape of mRNAs and proteins that drive the differentiation Cytoplasmic signaling pathways process. In addition to the role of transcription factors (discussed previously), epigenetics and mRNA interference Increase cyclin D1 are important in the regulation of differentiation. EPIGENETICS Cdk4/D1 Epigenetics (meaning “on top of genetics”) refers to sta- ble changes in gene expression that are not the result of a change in the genetic code and that can be passed from one Rb-P cell to its progeny. Epigenetic changes play an important Rb/E2F E2F role in normal development and differentiation. They are associated with both the “silencing” of genes and chromatin condensation into heterochromatin and with the “activa- tion” of genes.11 G1 M S METHYLATION One of the most common epigenetic changes found in the human genome involves the meth- ylation of certain cytosine nucleotides (CM) within genes Figure 2.6 The role of the retinoblastoma susceptibility gene and/or their promoter regions.11 Cytosine nucleotides product (Rb) in regulation of the cell cycle. Stimulation of a cell found adjacent to a guanine nucleotide, the so-called CpG with mitogens or growth factors induces synthesis of the D-type dinucleotide, are particularly susceptible to methylation, cyclins. Activation of G1 phase kinase activity (cyclin D/Cdk4/6) and are often found in close proximity, called CpG islands. phosphorylates a number of intracellular substrates including the Rb protein. In the hypophosphorylated (active) state, Rb binds and CGATCGATCGAT S CMGATCMGATCMGAT sequesters the transcription factor E2F, rendering it inactive. When cyclin D/Cdk4 or Cdk6 phosphorylates Rb, it releases E2F, which These methylations or epigenetic changes become then moves to the nucleus, and initiates transcription of genes incorporated into the genetic/epigenetic regulatory mech- required for cell-cycle progression (including the genes for cyclin E anisms of the cell, are conserved during subsequent cell and cyclin A). T indicates stimulation of the pathway; # indicates divisions, and play a significant role in cellular differentia- inhibition of the pathway. tion pathways. The methylation of CpG dinucleotides is a reversible process. Approximately 70–75% of CpG dinucleo- resulting in the inactivation of Rb, the release of the active tides in the human genome are methylated.12 CpG islands E2F transcription factor, and the activation and expression are often clustered in and around the promoter regions of of genes required for cell-cycle progression. Cells that lack genes. The unmethylated state of a gene’s promoter region functional Rb protein have deregulation of cell-cycle genes indicates a transcription-ready status and is seen in genes and cell proliferation, sometimes resulting in malignancy. actively being transcribed into mRNA. Typically, methyla- tion of a promoter region is associated with gene silencing and is part of the normal terminal differentiation process Checkpoint 2.4 seen in many diverse tissue types. A cell undergoing mitosis fails to attach one of its duplicated chromosomes to the microtubules of the spindle apparatus dur- HISTONE MODIFICATIONS Extensive information also ing metaphase. The cell’s metaphase checkpoint malfunctions can be encoded in the protein component of the chroma- and does not detect the error. What is the effect (if any) on the tin in what is called the histone code. Modifications of the daughter cells produced? histone proteins include lysine acetylation, serine phos- phorylation, and lysine and arginine methylation.13 These Differentiation modifications can also be passed from one cell generation to the next during cell division and play an important role in Differentiation is the process that generates the diverse cell the complex system responsible for regulating euchromatin populations found throughout the body. Since all cells in to heterochromatin transitions. Hypo-acetylated histones the human body contain identical genetic information, the bind tightly to the phosphate backbone of DNA and help appearance of distinct characteristics in a cell population is maintain chromatin in an inactive, silent state. Acetylated dictated by the transcription of specific protein coding genes histones maintain a more relaxed chromatin structure that and the translation of that genetic information into func- allow gene transcription to occur.13 tional proteins. As differentiation progresses within a given Both DNA methylation and histone hypoacetylation tissue or cell lineage, different genes will be sequentially promote chromatin condensation and gene silencing. As 20 Chapter 2 cells go through a particular differentiation program, the Quiescence DNA methylation patterns and histone acetylation/deacet- ylation patterns change as successive genes are activated Senescence Cell cycle Terminal differentiation and deactivated. Neoplastic transformation TRANSLATIONAL REGULATION Apoptosis Genomic expression within a cell is controlled not only Proliferation at the level of gene transcription but also by post-tran- scriptional events that affect mRNA stability. Interfering Figure 2.7 Alternative fates for a cell induced to enter the with the ability of a mRNA (RNA interference) to be cell cycle. translated can also modify the expression of genes.2 Two forms of RNA are involved in regulating mRNA translation, micro-RNA (miRNA) and small interfering proliferation and cell death. It also occurs at defined times RNA (siRNA). Both mi- and siRNAs bind to specific and locations during development. mRNAs, which functionally interferes with the ability In adults, apoptosis plays a role in tissue h omeostasis; of the target mRNA to be translated or causes degrada- homeostasis generally balances generation of new cells tion of the target mRNA. In either case, “gene silencing” with the loss of terminally differentiated cells. Apopto- results. sis is responsible for the elimination of excess cells such as expanded clones of lymphocytes following immune Apoptosis stimulation (Chapter 8), or excess neutrophils following a bacterial challenge (Chapter 7). As a defense mechanism, Cells stimulated to enter the cell cycle can experience apoptosis is used to remove unwanted and potentially dan- outcomes other than proliferation (Figure 2-7). Cells can gerous cells such as self-reactive lymphocytes, cells infected undergo senescence in which they are permanently growth by viruses, and tumor cells. Diverse forms of cellular dam- arrested and no longer respond to mitogenic stimuli. They age can also trigger apoptotic death including DNA damage can also become terminally differentiated (committed) into or errors of DNA replication. Similarly, intracellular protein specialized cell types. Uncontrolled cell cycling is a charac- aggregates or misfolded proteins can stimulate apoptosis teristic feature of neoplastic cells. Finally, cells can exit at (e.g., the apoptotic death of erythroblasts in b@thalassemia any phase of the cell cycle by undergoing programmed cell major triggered by aggregates of a globin chains [Chapter death (apoptosis). 14]). In addition to the beneficial effects of programmed cell Cells can die by either necrosis or apoptosis. Necrotic death, the inappropriate activation of apoptosis can cause or death is induced by lethal chemical, biological, or physical contribute to a variety of diseases14 (Table 2-4). events (extracellular assault; Table 2-3). Such a death is Apoptosis is initiated by three major types of stimuli analogous to “cell murder.” In contrast, apoptosis, or (Table 2-5): “ programmed cell death,” is a self-induced death program 1. Deprivation of survival factors (growth factor with- regulated by the cell itself (“cell suicide”). drawal or loss of attachment to extracellular matrix) Apoptosis plays an essential role in the d evelopment and homeostasis of all multicellular organisms.14,15 2. Signals by “death” cytokines through apoptotic “death” Apoptosis helps maintain a constant organ size in tis- receptors (tumor necrosis factor [TNF], Fas ligand) sues that undergo continuous renewal, balancing cell 3. Cell-damaging stress Table 2.3 Cardinal Features of Apoptosis and Necrosis Feature Necrosis Apoptosis Stimuli Toxins, severe hypoxia, massive insult, Physiologic and pathologic conditions without ATP depletion conditions of ATP depletion Energy requirement None ATP dependent Histology Cellular swelling; disruption of organelles; Cellular shrinkage; chromatin condensation; fragmentation into death of patches of tissue apoptotic bodies; death of single isolated cells Plasma membrane Lysed Intact, blebbed with molecular alterations (loss of phospholipid asymmetry) Phagocytosis of dead cells Immigrant phagocytes Neighboring cells Tissue reaction Inflammation No inflammation Cellular Homeostasis 21 Table 2.4 Diseases Associated with Increased and cells pinch off as apoptotic bodies that are phagocytized Decreased Apoptosis by neighboring cells or macrophages. Thus, apoptosis is a very efficient process by which the body can remove Increased Apoptosis Decreased Apoptosis a population of cells at a given time or in response to a AIDS Cancer Neurodegenerative disorders given stimulus without the activation of an inflammatory • Follicular lymphomas • Alzheimer’s disease response. • Other leukemias/lymphomas • Parkinson’s disease • Carcinomas with p53 mutations MOLECULAR REGULATION • Amyotrophic lateral • Hormone-dependent tumors sclerosis Apoptosis is a tightly regulated physiologic process. (breast, prostate, ovarian) • Retinitis pigmentosa Apoptosis is initiated by a variety of events and leads to Autoimmune disorders Myelodysplastic syndromes two major death pathways, “extrinsic” and “intrinsic.” • Systemic lupus erythematosus Aplastic anemia Ischemic injury • Other autoimmune diseases The extrinsic pathway is triggered by extracellular signals (“death cytokines”) and transmitted through “death recep- • Myocardial infarction Viral infections • Stroke • Herpes viruses tors” on the surface of the cell (Figure 2-8). The intrinsic • Reperfusion injury • Poxviruses pathway is a mitochondria-dependent pathway initiated Toxin-induced liver disease • Adenoviruses by intracellular signals in response to stress, exposure to cytotoxic agents, DNA damage, or radiation. Both pathways involve a group of proteins called caspases and the Bcl-2 family of proteins. Table 2.5 Inhibitors and Initiators/Inducers of Apoptosis CASPASES The cellular events responsible for apoptotic Inhibitors Initiators/Inducers cell death are directed by caspases,15 a family of cysteine pro- Presence of survival factors Deprivation of survival factors teases that cleave after aspartic acid amino acids in a protein • Growth factors • Growth factor withdrawal and are responsible for the orderly dismantling of the cell • Extracellular matrix • Loss of matrix attachment • Interleukins Death cytokines • Estrogens, androgens • TNF Death Receptor/Death Cytokine Viral products that block apoptosis • Fas ligand Apoptotic Pathway • Cowpox virus CrmA Cell-damaging stress • Epstein Barr virus BHRF-1 • Bacterial toxins Binding of death cytokine to cell receptor Pharmacologic inhibitors • Viral infections Anti-apoptotic proteins (Bcl-2, • Oxidants Bcl@XL, Mcl-1) • Glucocorticoids Caspase recruitment • Cytotoxic drugs • Radiation therapy Pro-apoptotic proteins (BCL@Xs, Bcl-2 Bid, Bad, Bax) Activation of initiator caspases family Bcl-2 Activation of effector caspases Conversely, apoptosis is inhibited by growth-promoting family cytokines and interaction with appropriate extracellular envi- ronmental stimuli. Cleavage of crucial cellular proteins NECROSIS VERSUS APOPTOSIS When a cell is damaged, the plasma membrane often loses Cell death its ability to regulate cation fluxes, resulting in the accumu- lation of Na+, Ca++, and water (Table 2-3). Consequently, Figure 2.8 The apoptotic pathway triggered by death the necrotic cell exhibits a swollen morphology. The organ- cytokine binding to death receptors. Activation of a death receptor elles also accumulate cations and water, swell, and ulti- by binding of death cytokine results in the recruitment of specific mately lyse. The rupture of the cytoplasmic membrane adapter proteins and activation of initiator caspases. Activated and organelles releases cytoplasmic components (includ- initiator caspases can then proceed to activate downstream ing proteases and lysozymes) into the surrounding tissue, effector
caspases that mediate the cleavage of various cellular triggering an inflammatory response. In contrast, apop- proteins during apoptosis. The contribution of the Bcl-2 family of pro-apoptotic and anti-apoptotic proteins in determining whether tosis is characterized by cellular shrinking rather than activation of initiator caspases will proceed through to activation swelling, with condensation of both the cytoplasm and of effector caspases is depicted. T indicates stimulation of the the nucleus. Apoptotic cells do not lyse, but portions of the pathway; # indicates inhibition of the pathway. 22 Chapter 2 undergoing apoptosis. Caspases are found in healthy cells Death cytokine as zymogens (inactive forms of enzymes) and only express Receptor their protease activity upon activation. A hierarchical rela- tionship exists among the apoptotic caspases. Early-acting, Loss of Cell death survival cytokines initiator caspases are recruited and activated in response to signal apoptotic stimuli. Initiator caspases activate downstream effector caspases, which in turn, orchestrate the cell’s death Genotoxic damage (Figure 2-8). Activation of caspases in apoptosis does not PCD On/Off lead to indiscriminate proteolytic degradation but to spe- p53 cific cleavage of key substrates including proteins involved Bax Death Bax in cell structure, cell-cycle regulation, transcription, transla- checkpoint tion, DNA repair, and RNA splicing (Table 2-6). BCL-2 PROTEINS The Bcl-2 family of proteins includes PCD both pro-apoptotic and anti-apoptotic members and On/Off constitutes a critical intracellular checkpoint regulating Death Death apoptosis.15 The founding member, Bcl-2, was a protein checkpoint substrates originally cloned from B-cell lymphomas with a character- istic c hromosomal translocation (t[14;18]; Chapter 28). Since Past that initial discovery, several additional proteins related to the point of no return Bcl-2 have been identified (thus, the Bcl-2 family). Some Bcl-2 proteins promote (pro-apoptotic) whereas others oppose (anti-apoptotic) apoptosis. The Bcl-2 family of pro- teins is localized at or near the mitochondrial membranes Figure 2.9 Model of cell death checkpoints. Following delivery and constitutes an intracellular checkpoint of apoptosis. of a cell death signal (genotoxic damage, loss of survival cytokines, or Bcl-2 proteins determine whether or not initiator caspases presence of death cytokines), the ratio of pro-apoptotic components are activated (Figure 2-8). (Bax and related molecules) versus anti-apoptotic components The relative levels of anti-apoptotic and pro-apoptotic (Bcl-2 and related molecules) determines whether the death Bcl-2 family members constitute a rheostat for apoptosis. program will continue to completion. A preponderance of Bax:Bax homodimers promotes continuation of the process while Bax–Bcl-2 This rheostat is regulated by different associations between heterodimers shuts it down. PCD, programmed cell death (apoptosis). anti-apoptotic and pro-apoptotic proteins, all of which share similar structural regions that allow them to bind each other. Bax, a pro-apoptotic member, can associate with cell death (Chapter 4). Cytokines and components of the itself (Figure 2-9); Bax:Bax homodimers promote apoptosis. extracellular matrix provide cell survival signals that sup- They induce permeabilization of the mitochondrial mem- press apoptosis in hematopoietic cells. Various molecules brane and release of proteins, including cytochrome-c, and serve as inhibitors of apoptosis (IAPS) and function to sup- activation of the caspase cascade. When Bcl-2 is increased, press apoptosis, allowing survival of hematopoietic cells. Bax:Bcl-2 heterodimers form and repress apoptosis. It is Apoptosis plays several essential roles in hematopoiesis, the overall ratio of various death agonists (Bax and related including: proteins) to death antagonists (Bcl-2 and related proteins) • Lymphocyte selection, eliminating those that are non- and their interactions with each other that determine the functional or autoreactive (Chapter 8) susceptibility of a cell to a death stimulus15 (Figure 2-10). • Eliminating excess cells when expanded numbers of APOPTOSIS AND THE HEMATOPOIETIC SYSTEM mature cells are no longer needed (i.e., elimination of Apoptosis is important in the hematopoietic system. The white blood cells following an infection/inflammatory default cellular status of hematopoietic precursor cells is response) Table 2.6 Caspase Substrates Cleaved During Apoptosis Nuclear Proteins Involved In: Structural Proteins Involved In: Regulatory Proteins DNA repair Nuclear organization Translation Transcriptional regulation Cell adhesion Cell cycle RNA processing Transmembrane receptors Signaling pathways DNA fragmentation Calcium ion transport Bax Bcl-2 Cellular Homeostasis 23 Bcl-Xs Bid Endoplasmic reticulum Bim Bad Bak Ca++ Apaf-1 Bax C C release Apoptotic Signal C Procaspase-9 Bcl-2 Cytochrome c Apoptosome Effector release (consisting of Apaf-1, caspases Bcl-X Caspase-9, Cytochrome-c) L Mcl-1 Mitochondria Figure 2.10 Bcl-2-related proteins and control of apoptosis. Pro-apoptotic (blue ovals) and anti-apoptotic (pink rectangles) Bcl-2-related proteins interact in response to an apoptotic signal. If the pro-apoptotic signals prevail, cytochrome-c (yellow circle) is released from the mitochondria, binds to an adapter protein (Apaf-1), and recruits an initiator caspase (procaspase-9); the resulting caspase-activating assembly, the apoptosome, is associated with the intrinsic pathway of apoptosis. • The mechanism of cytotoxic killing by cytotoxic T-lymphocytes and natural killer cells Abnormal Tissue • Through caspase activation, platelet production and Homeostasis and Cancer release from mature megakaryocytes, and the final Scattered throughout the human genome are genes that stages of erythrocyte maturation (chromatin conden- sation and organelle removal)16,17 have the potential to cause cancer (proto-oncogenes) and other genes that have the power to block it (tumor Dysregulation of apoptosis also contributes to suppressor genes). Many of the proteins that are hematologic disorders. Apoptosis is increased in the encoded by proto-oncogenes and tumor suppressor myelodysplastic syndromes and tends to be decreased genes regulate normal cell processes (Chapter 23). In in the acute leukemias, which may explain the pancy- normal cells, proto-oncogenes encode proteins that pro- topenias and leukocytosis, respectively, seen in those mote the initiation of DNA replication, regulate cellular disorders (Chapters 25–27). differentiation, and/or inhibit apoptosis. Conversely, tumor suppressor genes function in opposition to Checkpoint 2.5 proto-oncogenes. Thus, mutations in proto-oncogenes What would be the effect on the hematopoietic system result in growth-promoting activity, while mutations in homeostasis if the expanded clone of antigen-activated tumor suppressor genes inactivate growth suppression; B lymphocytes failed to undergo apoptosis after the antigenic in either case, mutations in these genes contribute to challenge was removed? tumor development. Summary The cell is an intricate, complex structure surrounded by a endoplasmic reticulum, the Golgi apparatus, lysosomes, phospholipid bilayer that is embedded with integral pro- mitochondria, microfilaments, and microtubules. The teins and carbohydrates. Some proteins function as recep- nucleus contains the genetic material, DNA, which regu- tors that transmit messages to the cell’s nucleus. Within lates all cell functions. the cell is the cytoplasm with numerous organelles and The cell cycle is a highly ordered process that results the nucleus. The cellular organelles include ribosomes, in the accurate duplication and transmission of genetic 24 Chapter 2 information from one cell generation to the next. The cell necrosis. Apoptosis plays important roles in the develop- cycle is divided into four stages: M phase (in which cell ment of the organism, in controlling the number of vari- division or mitosis takes place), S phase (during which ous types of cells, and as a defense mechanism to eliminate DNA synthesis occurs), and two gap phases, G1 and G2. G0 unwanted and potentially dangerous cells. Apoptosis is an refers to quiescent cells that are temporarily or permanently active process initiated by the cell and results in the orderly out of cycle. The normal cell depends on external stimuli dismantling of cellular constituents. Apoptosis is directed (growth factors) to move it out of G0 and through G1. The by cysteine proteases called caspases. Pro-apoptotic and anti- cell cycle is regulated by a series of protein kinases (Cdks) apoptotic proteins (Bcl-2 family members) and specific pro- whose activity is controlled by complexing with a regula- tein inhibitors (IAPs, or inhibitors of apoptosis) regulate this tory partner (cyclin). Different cyclins with their associated process. Apoptosis is triggered by loss of survival factors (and activated) Cdks function at specific stages of the cell (survival cytokines or extracellular matrix components), cycle. Kinase activity is further modulated by both activat- presence of death cytokines, or cell-damaging stress. ing and inactivating phosphorylation of kinase subunits The various processes that govern tissue homeosta- and by specific cell-cycle kinase (Cdk) inhibitors. A series sis—proliferation, differentiation, cytokine regulation, and of checkpoint controls or surveillance systems functions to apoptosis—are highly ordered and tightly regulated. When ensure the integrity of the process. the regulation of these processes malfunctions, the result Cells use the process of programmed cell death, or can be deregulated cell production. Mutations or epigen- apoptosis, as well as proliferation to maintain tissue homeo- etic changes that alter the structure or function of the genes stasis. Apoptosis is a unique form of cell death that can be that regulate these processes can result in uncontrolled cell morphologically and biochemically distinguished from growth and malignancy. Review Questions Level I c. Prophase 1. Selective cellular permeability and structural stability d. Metaphase are provided by: (Objective 1) 5. Cells that have exited the cell cycle and entered a a. membrane lipids nonproliferative phase are said to be in: b. membrane proteins (Objective 4) c. ribosomes a. quiescence d. the nucleus b. interphase c. G 2. Rough endoplasmic reticulum is important in: 1 ( Objective 1) d. G2 a. synthesizing lipid 6. The generation of an RNA from DNA is termed: b. synthesizing hormones (Objective 3) c. synthesizing and assembling proteins a. transcription d. phagocytosis b. promotion c. RNA processing 3. Chromatin is composed of DNA that is wrapped around ______ proteins: (Objective 1) d. translation a. nucleolus 7. The point in the cell cycle at which cell proliferation b. proteome (cycling) no longer depends on extracellular signals c. histone is: (Objective 5) d. hormone a. G1 b. R 4. Condensation of chromosomes occurs during which c. G phase of mitosis? (Objective 4) 2 d. M a. Anaphase b. Telophase Cellular Homeostasis 25 8. Programmed cell death (cell suicide) is also known as: 4. The purpose of the S-phase checkpoint is to: (Objective 6) (Objective 5) a. necrosis a. generate the DNA replication machinery b. senescence b. monitor the accuracy of DNA replication c. apoptosis c. regulate the formation of the mitotic spindle d. terminal differentiation d. assess the extracellular environment 9. All of the following are considered initiators of 5. Overexpression of the p21 protein would have what apoptosis except: (Objective 7) effect on the cell cycle of proliferating cells? (Objective 4) a. estrogens a. Decrease cell-cycle progression b. death cytokines b. Increase cell-cycle progression c. loss of matrix attachment c. Trigger apoptosis d. cell-damaging stress d. None 10. Which of the following events in hematopoiesis is 6. The protein responsible for binding the transcrip- regulated by apoptosis? (Objective 8) tion factors E2F and blocking cell-cycle progression beyond the restriction point (R) is: (Objective 6) a. Terminal differentiation of white blood cells a. p53 b. Progression through the cell cycle b. p15 c. Proliferation of red blood cells following hemorrhage c. p21 d. Removal of excess lymphocytes following immune d. Rb stimulation 7. Apoptotic cell death is characterized by all of the Level II following except: (Objective 14) 1. UTRs are regions of mRNA that: (Objective 1) a. triggering an inflammatory response b. condensation of chromatin a. represent variations of the genetic sequence of a gene in different individuals c. loss of membrane phospholipid asymmetry b. represent the regions of the gene that are d. condensation of the cytoplasm and cell shrinkage transcribed 8. The components of apoptosis directly responsible c. contain the splice sites for mRNA processing for dismantling the cell during the programmed cell d. influence the stability of mRNA and translation of death process are: (Objective 8) protein a. Bcl-2 family members 2. The main function of the ubiquitin proteasome system b. IAPs is to: (Objective 2) c. initiator caspases a. assist in the three-dimensional folding of d. effector caspases polypeptides into their tertiary structure 9. A predominance of Bax-Bax homodimers has what b. degrade unwanted or damaged polypeptides effect on apoptosis? (Objective 11) c. facilitate transfer of polypeptides from the endoplasmic reticulum to the Golgi a. Inhibits initiator caspases d. direct post-translational modifications of b. Promotes activation of effector caspases proteins c. Activates death receptors on the cell surface d. Neutralizes IAPs 3. The kinase complex responsible for passage through and exit from mitosis is composed of: 10. Which of the following are associated with gene (Objective 3) silencing? (Objective 13) a. cyclin A/Cdk2 a. DNA (CpG) methylation and histone acetylation b. cyclin D/Cdk4 b. DNA (CpG) methylation and histone deacetylation c. cyclin B/Cdk1 c. unmethylated CpG and histone acetylation d. cyclin E/Cdk2 d. unmethylated CpG
and histone deacetylation 26 Chapter 2 References 1. Singer, S. J., & Nicholson, G. L. (1972). The fluid mosaic model of J. Anastasi, eds. Hematology: Basic principles and procedures the structure of cell membranes. Science, 175, 720–731. (6th ed., pp. 147–157). New York: Churchill Livingstone. 2. Wagner, A. J., Berliner, N., & Benz, E. J. (2013). Anatomy 10. Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle and physiology of the gene. In: R. Hoffman, E. J. Benz, L. E. control. Cell, 81, 323–330. Silberstein, H. Heslop, J. Weitz, & J. Anastasi, eds. Hematology: 11. Jorde, L. B. (2010). Genomics and Epigenetics. In: J. T. Prchal, K. Basic principles and procedures (6th ed., pp. 3–15). New York: Kaushansky, M. A. Lichtman, T. J. Kipps, & U. Seligsohn, eds. Churchill Livingston. Williams hematology (8th ed.). New York: McGraw-Hill. 3. Papayannopoulou, T., & Migliaccio, A. R. (2013). Biology of 12. Esteller, M., Corn, P. G., Baylin, S. B., & Herman, J. G. (2001). erythropoiesis, erythroid-differentiation, and maturation. In: A gene hypermethylation profile of human cancer. Cancer R. Hoffman, E. J. Benz, L.E. Silberstein, H. Heslop, J. Weitz, & Research, 61, 3225-8. J. Anastasi, eds. Hematology: Basic principles and procedures 13. Burger, M. (2005). Hyperacetylated chromatin domains: lessons (6th ed., pp. 258–279). New York: Churchill Livingstone. from heterochromatin. Journal of Biological Chemistry, 280, 4. Kaufman, R. J., & Popolo, L. (2013). Protein synthesis, processing, 21689–21692. and trafficking. In: R. Hoffman, E. J. Benz, L. E. Silberstein, 14. Hetts, S. W. (1998). To die or not to die: an overview of apoptosis H. Heslop, J. Weitz, & J. Anastasi, eds. Hematology: Basic principles and its role in disease. Journal of the American Medical Association, and procedures (6th ed., pp. 35–47). New York: Churchill Livingstone. 279, 300–307. 5. Pardee, A. B. (1989). G1 events and regulation of cell proliferation. 15. Danial, N. N., & Hockenbery, D. M. (2013). Cell death. In: R. Science, 246, 603–608. Hoffman, E. J. Benz, L. E. Silberstein, H. Heslop, J. Weitz, & J. 6. King, R. W., Deshaies, R. J., Peters, J. M., & Kirschner, M. W. Anastasi, eds. Hematology: Basic principles and procedures (1996). How proteolysis drives the cell cycle. Science, 274, (6th ed., pp. 158–169). New York: Churchill Livingstone. 1652–1659. 16. Kaluzhny, Y., & Ravid K. (2004). Role of apoptotic processes in 7. Sherr, C. J., & Roberts, J. M. (1995). Inhibitors of mammalian G-1 platelet biogenesis. Acta Haematologica, 111, 67–77. cyclin dependent kinases. Genes Develop, 9, 1149–1163. 17. Kolbus, A., Pilat, S., Huszk, A., Deiner, E., Stengl, G., Beug, H., & 8. Nurse, P. (1997). Checkpoint pathways come of age. Cell, 91, 865–867. Baccarini, M. (2002). Raf-1 antagonizes erythroid differentiation 9. Lee, W. M., & Dang, C. V. (2013). Control of cell division. by restraining caspase activation. Journal of Experimental Medicine, In: R. Hoffman, E. J. Benz, L. E. Silberstein, H. Heslop, J. Weitz, & 196, 1347–1353. Section Two The Hematopoietic System 27 Chapter 3 Structure and Function of Hematopoietic Organs Annette J. Schlueter, MD, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Identify the sites of hematopoiesis during 3. Explain the difference between primary and embryonic and fetal development, child- secondary lymphoid tissue. hood, and adulthood. 4. Describe the function of bone marrow, 2. Identify organ/tissue sites in which each spleen, lymph nodes, and thymus. hematopoietic cell type differentiates. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Associate physical findings (hypersplen- 4. Create a sketch of the structure of bone mar- ism, lymphadenopathy) with the presence of row, spleen, lymph nodes, and thymus that hematologic disease. shows the location of hematopoietic cells. 2. Assess the pathophysiologic changes that 5. Differentiate between primitive and defini- lead to bone marrow hyperplasia or extra- tive erythropoiesis. medullary hematopoiesis. 3. Identify sites of extramedullary hematopoiesis. 28 Structure and Function of Hematopoietic Organs 29 Chapter Outline Objectives—Level I and Level II 28 Development of Hematopoiesis 30 Key Terms 29 Hematopoietic Tissue 30 Background Basics 29 Summary 39 Case Study 29 Review Questions 39 Overview 29 References 40 Introduction 29 Key Terms Adipocyte Germinal center Osteoblast Blood island Hematopoietic stem cell (HSC) Osteoclast Culling Hyperplasia Pitting Endosteum Lymphoid follicle Reticular cell Erythroblastic island Medullary hematopoiesis Stroma Extramedullary hematopoiesis Mesenchymal stem cell (MSC) Trabecula Background Basics The information in this chapter builds on the con- Level I and Level II cepts learned in the first chapter. A basic anatomy • List the types of blood cells and give their basic functions and physiology course would also be helpful. To • Define hemoglobin and explain its function maximize your learning experience, you should review these concepts before starting this unit of • Locate the tables in the book that give the reference study: intervals for blood cells and hemoglobin embryo to the adult. Each tissue’s histologic structure and CASE STUDY its function in hematopoiesis are discussed. Abnormalities We refer to this case throughout this chapter. in hematopoiesis that are associated with histologic and Francine, a 10-year-old female, was brought to her functional changes in these tissues are briefly described. pediatrician for complaints of lethargy and leg pain. Physical examination revealed splenomegaly and lymphadenopathy. A complete blood count Introduction was ordered with the following results: Hb 8 g/dL; Cellular proliferation, differentiation, and maturation of WBC 6.5 * 109/L; platelets 21 * 109/L. blood cells take place in the hematopoietic tissue, which in Refer to the tables on the front inside cover of the adult consists primarily of bone marrow, although some the book and determine which blood cell param- lymphocyte development also takes place in the spleen and eters, if any, are abnormal. thymus. Mature cells are released to the peripheral blood and can live out their lifespan in the blood or take up residence in the spleen, lymph nodes, or other tissues. The link between Overview the bone marrow and blood cell production was not estab- lished until it was recognized that blood formation was a con- This chapter includes a description of the tissues involved tinuous process. Before 1850, it was believed that blood cells in the production and maturation of blood cells. It begins formed in the fetus were viable until the host’s death and that with a look at blood cell production sequentially from the there was no need for a continuous source of new elements. 30 Chapter 3 Development of Bone marrow Hematopoiesis Liver Hematopoiesis begins as early as the 19th day after fertil- Yolk sac ization in an extraembryonic location, the yolk sac of the AGM human embryo.1 The cells made in the yolk sac include erythrocytes and a few macrophages and megakaryocytes Spleen (precursors of platelets).2 The ability to make erythrocytes is important because the embryo must be able to transport 1 2 3 4 5 6 7 8 Birth oxygen to developing tissue early in gestation. Shortly there- Months gestation after, at about 4 weeks of gestation, intraembryonic hema- topoiesis begins in the aorta-gonad-mesonephros (AGM) Figure 3.1 Location of hematopoiesis during fetal region located in the ventral lumen of the developing aorta. development. At birth, most blood cell production is limited to the This region has the ability to make a wider range of hema- marrow. topoietic cells, including lymphocytes, than those made in the yolk sac.3 Erythrocyte production from the yolk sac is bone marrow before erythropoiesis, which does not transi- called primitive erythropoiesis. Hemoglobin in these cells is tion until the end of gestation. The thymus becomes the not typical of that seen in later developing erythroblasts, major site of T-lymphocyte (a subclass of lymphocytes) pro- they do not differentiate from self-renewing hematopoietic duction during fetal development and continues to be active stem cells (HSCs), and they complete their maturation in throughout the neonatal period and childhood. As is true the circulation rather than in an organ.4 The yolk sac eryth- for erythrocytes in the yolk sac, the first T-cells to develop roblasts arise from clusters of cells called blood islands and differ from their adult counterparts. They use a different set are closely related to development of endothelium, the cells of genes to make the T-cell receptor, which the T-cell uses lining blood vessels.5 Definitive erythropoiesis begins with the to recognize and react to foreign substances8 (Chapter 8). formation of self-renewing HSC in the AGM. The HSC is Lymph nodes and the spleen continue as an important site the common precursor cell for all developing hematopoietic of late B-cell differentiation throughout life. cells and is characterized by its ability to proliferate with- out differentiation (“self-renewal”). Primitive erythroblasts have a megaloblastic appearance (large cells with coarse Hematopoietic Tissue clumped chromatin; Chapter 15). The hemoglobin in these The adult hematopoietic system includes tissues and organs cells consists of the embryonic varieties, Gower 1, Gower 2, involved in the proliferation, maturation, and destruction and Portland6 (Chapter 6). of blood cells. These organs and tissues include the bone At about the third month of fetal life, the liver becomes marrow, thymus, spleen, and lymph nodes. Bone marrow the chief site of blood cell production by which time the is the site of myeloid, erythroid, and megakaryocyte as well yolk sac and AGM have discontinued their role in hema- as early stages of lymphoid cell development. The thymus, topoiesis. The liver continues to produce a high proportion spleen, and lymph nodes are primarily sites of later lym- of erythroid cells, but myeloid and lymphoid cells begin phoid cell development. The tissues in which lymphoid cell to appear in greater numbers,7 indicating the beginning development occurs are divided into primary and second- of a transition to adult patterns of hematopoiesis in which ary lymphoid tissue. Primary lymphoid tissues (bone marrow myeloid differentiation predominates over erythroid differ- and thymus) are those in which T- and B-cells develop from entiation. Hemoglobin F production replaces the embryonic nonfunctional precursors into cells capable of responding to hemoglobins during this period. foreign antigens (immunocompetent cells). Secondary lym- As fetal development progresses, hematopoiesis also phoid tissues (spleen and lymph nodes) are those in which begins to a lesser degree in the spleen, kidney, thymus, immunocompetent T- and B-cells further divide and dif- and lymph nodes. Erythroid and myeloid cell production ferentiate into effector cells and memory cells in response as well as early B-lymphocyte (a subclass of lymphocytes) to antigens (Chapter 8). development gradually shifts from these sites to bone mar- row during fetal and neonatal life as the hollow cavities within the bones are formed. The bone marrow becomes the Bone Marrow primary site of hematopoiesis at about the seventh month Blood-forming tissue located between the trabeculae of of gestation and continues as the primary source of blood spongy bone is known as bone marrow. (Trabecula refers to production after birth and throughout adult life (Figure 3-1). a projection of bone extending from cortical bone into the Granulocyte and megakaryocyte production shifts to the marrow space; it provides support for marrow cells.) This Hematopoiesis Structure and Function of Hematopoietic Organs 31 major hematopoietic organ is a cellular, highly vascular- Reticular cells (a type of fibroblast) send long cytoplasmic ized, loose connective tissue. It is composed of two major processes into the stroma. They are an abundant source of compartments: the vascular and the endosteal. The vascular CXCL12 (SDF-1), which is critical for maintaining an HSC compartment is composed of the bone marrow arteries and pool in the marrow.11 These cells also produce reticular veins, stromal cells, and hematopoietic cells (Figure 3-2). fibers, which contribute to the three-dimensional support- The endosteal compartment is primarily the site of bone ing network that holds hematopoietic elements as well as remodeling but also contains HSC. bone marrow vasculature. The fibers can be visualized with light microscopy and after silver staining. Reticular cells are VASCULATURE alkaline phosphatase positive. The vascular supply of bone marrow is served by two arterial sources, a nutrient artery and a periosteal artery, STROMA that enter the bone through small holes, the bone foram- The bone marrow stroma (supporting tissue in the vascu- ina. Blood is drained from the marrow via the central vein lar compartment) provides a favorable microenvironment (Figure 3-3). The nutrient artery branches around the cen- for sustained proliferation of hematopoietic cells, forming a tral sinus that spans the marrow cavity.
Arterioles radiate meshwork that creates a three-dimensional scaffolding for outward from the nutrient artery to the endosteum (the them.12 Stromal cellular components also provide cytokines inner lining of the cortical bone), giving rise to capillar- that regulate hematopoiesis (Chapter 4). The stroma is com- ies that merge with capillaries from periosteal arteries to posed of three major cell types: macrophages, lymphocytes, form sinuses within the marrow. The sinuses, lined by sin- and adipocytes (fat cells). gle endothelial cells and supported on the abluminal side Macrophages serve two major functions in the bone (away from the luminal surface) by adventitial reticular marrow: phagocytosis and secretion of hematopoietic cyto- cells, ultimately gather into wider collecting sinuses, which kines (proteins secreted by a cell; these proteins modulate open into the central longitudinal vein.9 The central longi- the function of another cell). Macrophages phagocytize tudinal vein continues through the length of the marrow the extruded nuclei of maturing erythrocytes, B-cells that and exits through the foramen where the nutrient artery have not differentiated properly, and differentiating cells entered. Nerve fibers surrounding marrow arteries regu- that die during development. Some macrophages serve as late blood flow into the bone marrow, which in turn con- the center of the erythroblastic islands as discussed in the trols hematopoietic progenitor release into the circulation.10 section “Hematopoietic Cells.” Macrophages also provide Bone Endosteum Lymphoid aggregate HSCs Blood vessel Adipocytes Granulocytic Osteoclast nest Megakaryocyte Osteoblasts Erythroblastic island Figure 3.2 Schematic drawing of a section of bone marrow. Figure courtesy of Dr. Corey Parlet. 32 Chapter 3 Periosteal Central Nutrient artery vein artery Periosteum Osseous bone Endosteum Periosteal capillaries Sinus Marrow Central sinus Figure 3.3 Diagram of the microcirculation of bone marrow. The major arterial supply to the marrow is from periosteal capillaries and capillary branches of the nutrient artery that have traversed the bony enclosure of the marrow through the bone foramina. The capillaries join with the venous sinuses as they reenter the marrow. The sinuses gather into wider collecting sinuses that then open into the central longitudinal vein (central sinus). many colony-stimulating factors (cytokines that stimulate Osteoblasts and osteoclasts are found in the endos- the growth and development of immature hematopoietic teum (internal surface of calcified bone). These cells can be cells) needed for the development of myeloid lineage cells. dislodged during bone marrow biopsy and can be found Macrophages stain acid phosphatase positive. in the specimen with hematopoietic cells. Osteoblasts Lymphocytes that reside in the bone marrow, particu- differentiate from MSCs; osteoclasts differentiate from larly T-cells, also influence hematopoiesis by secretion of HSCs.14,16 cytokines. When appropriately activated, they help myeloid Osteoblasts are involved in the formation of calci- cells become fully mature.13 fied bone and produce cytokines that can positively or Adipocytes are cells whose cytoplasm is largely replaced negatively regulate HSC activity.17 They are large cells with a single fat vacuole. They differentiate from mesenchy- (up to 30 mcM (mm in diameter) that resemble plasma mal stem cells (MSCs), and their production is inversely cells except that the perinuclear halo (Golgi apparatus) proportional to osteoblast formation.14 MSCs are multipotent is detached from the nuclear membrane and, in Wright- stromal stem cells that can differentiate into bone, cartilage, stained specimens, appears as a light area away from the and fat cells. Adipocytes mechanically control the volume nucleus (Figure 3-4a). In addition, the cytoplasm may be of bone marrow in which active hematopoiesis takes place. less basophilic, and the nucleus has a finer chromatin pat- They also provide steroids and other cytokines that influ- tern than plasma cells. Osteoblasts are normally found in ence hematopoiesis and maintain osseous bone integrity.15,16 groups and are more commonly seen in children and in The proportion of bone marrow composed of adipo- metabolic bone diseases. The cells are alkaline phospha- cytes changes with age. For the first 4 years of life, nearly tase positive. all marrow cavities are composed of hematopoietic cells, Osteoclasts are cells related to macrophages that are or red marrow. After 4 years of age, adipocytes or yellow involved in resorption and remodeling of calcified bone. marrow gradually replaces the red marrow in the shafts of Up to 100 mcM in diameter, they are even larger than osteo- long bones. By the age of 25 years, hematopoiesis is limited blasts. The cells are multinucleated, form from fusion of acti- to the marrow of the skull, ribs, sternum, scapulae, clavi- vated monocytes, and have granular cytoplasm that can be cles, vertebrae, pelvis, upper half of the sacrum, and proxi- either acidophilic or basophilic. They resemble megakaryo- mal ends of the long bones. The distribution of red:yellow cytes except that the nuclei are usually discrete (whereas the marrow in these bones is about 1:1. The fraction of red megakaryocyte has a single, large multilobulated nucleus) marrow in these areas continues to decrease with aging. and often contain nucleoli (Figure 3-4b). Structure and Function of Hematopoietic Organs 33 a b Figure 3.4 (a) Osteoblast; arrows point to the Golgi apparatus (perinuclear halo). (b) Osteoclast in bone marrow aspirate (Wright-Giemsa stain; 1000* magnification). HEMATOPOIETIC CELLS These cells are arranged in distinct niches within the vascu- Checkpoint 3.1 lar compartment of the marrow cavity. Erythroblasts con- Describe the bone marrow stromal location of erythrocyte, stitute 25–30% of the marrow cells and are produced near granulocyte, platelet, and lymphocyte differentiation. the venous sinuses. They develop in erythroblastic islands composed of a single macrophage surrounded by eryth- roblasts in varying states of maturation. The macrophage occurs in many anemias and leukemias, must be compen- cytoplasm extends out to surround the erythroblasts. Dur- sated for by a change in the space-occupying adipocytes. ing this close association, the macrophages regulate eryth- Normal red marrow can respond to stimuli and increase ropoiesis by secreting various cytokines. The macrophages its activity to several times the normal rate. As a result, the also phagocytize nuclei extruded from erythroblasts.18 The red marrow becomes hyperplastic and replaces portions of least mature cells are closest to the center of the island, and the fatty marrow. Bone marrow hyperplasia (an excessive the more mature cells are at the periphery. proliferation of normal cells) accompanies all conditions The location of leukocyte development differs depend- with increased or ineffective hematopoiesis. The degree of ing on the cell type. Granulocytes are produced in nests hyperplasia is related to the severity and duration of the close to the trabeculae and arterioles and are relatively pathologic state. Acute blood loss can cause erythropoietic distant from the venous sinuses. These nests are not quite tissue to temporarily replace fatty tissue; severe chronic as apparent morphologically as are erythroblastic islands. anemia can cause erythropoiesis to be so intense that it Megakaryocytes are very large, polyploid cells (DNA con- not only replaces fatty marrow but also erodes the bone’s tent more than 2N) that produce platelets from their cyto- internal surface. In malignant diseases that invade or origi- plasm. They are located adjacent to the vascular sinus.19 nate in the bone marrow such as leukemia, proliferating Cytoplasmic processes of the megakaryocyte form long abnormal cells can replace both normal hematopoietic proplatelet processes that pinch off to form platelets. Lym- tissue and fat. phocytes are normally produced in lymphoid aggregates In contrast, the hematopoietic tissue can become inac- located near arterioles. Lymphoid progenitor cells can tive or hypoplastic (a condition in which the hematopoi- leave the bone marrow and travel to the thymus where etic cells in bone marrow decrease). Fat cells then increase, they mature into T-lymphocytes. Some remain in the bone providing a cushion for the marrow. Environmental factors marrow where they mature into B-lymphocytes. Some such as chemicals and toxins can suppress hematopoiesis B-cells return to the bone marrow after being activated in whereas other types of hypoplasia can be genetically deter- the spleen or lymph node. Activated B-cells transform into mined (Chapter 16). Myeloproliferative disease, which plasma cells, which can reside in the bone marrow and begins as a hypercellular disease, frequently terminates in produce antibody. a state of aplasia (absence of hematopoietic tissue in bone Bone forms a rigid compartment for the marrow. marrow) in which fibrous tissue replaces hematopoietic Thus, any change in volume of the hematopoietic tissue, as tissue (Chapter 25). 34 Chapter 3 CASE STUDY (continued from page 29) Checkpoint 3.2 Microscopic examination of a stained blood smear Describe the process by which a blood cell moves from the marrow to the vascular space. from Francine revealed a predominance of very young blood cells (blasts) in the peripheral blood. These cells are normally found only in the bone marrow. Subsequently, she had bone marrow EXTRAMEDULLARY HEMATOPOIESIS aspirated for examination. This revealed 100% cel- Hematopoiesis in the bone marrow is called medullary lularity (red marrow) with a predominance of the hematopoiesis. Extramedullary hematopoiesis denotes same type of blasts as those found in the peripheral blood cell production in hematopoietic tissue other than blood. bone marrow. In certain hematologic disorders, when 1. Describe Francine’s bone marrow as normal, hyperplasia of the marrow cannot meet the physiologic hyperplastic, or hypoplastic. blood needs of the tissues, extramedullary hematopoiesis can occur in the hematopoietic organs that were active in 2. What conditions can cause this bone marrow the fetus, principally the liver and spleen. Organomegaly finding? frequently accompanies significant hematopoietic activity at these sites. BLOOD CELL EGRESS Thymus Special properties of the maturing hematopoietic cell and of The thymus is a lymphopoietic organ located in the upper the venous sinus wall are important in migration of blood part of the anterior mediastinum. It is a bilobular organ cells from the bone marrow to the circulation.20 These cells demarcated into an outer cortex and central medulla. The must migrate between reticular cells but through endothelial cortex is densely packed with small lymphocytes (thymo- cells to reach the circulation. As cell traffic across the sinus cytes), cortical epithelial cells, and a few macrophages. The increases, the reticular cells contract, creating a less continu- medulla is less cellular and contains more mature thymo- ous layer over the abluminal sinus wall. When the reticular cytes mixed with medullary epithelial cells, dendritic cells, cell layer contracts, it creates compartments between the and macrophages (Figure 3-5). reticular cell layer and the endothelial layer where mature The primary purpose of the thymus is to serve as a cells accumulate and can interact with sites on the sinus compartment in which T-lymphocytes mature.23 Precur- endothelial surface. sor T-cells leave the bone marrow and enter the thymus The new blood cell interacts with the abluminal endo- through arterioles in the cortex. As they travel through the thelial membrane by a receptor-mediated process, forcing cortex and the medulla, they interact with epithelial cells the abluminal membrane into contact with the luminal and dendritic cells, which provide signals to ensure that endothelial membrane. The two membranes fuse, and T-cells can recognize foreign antigen but not self-antigen. under pressure from the passing cell, they separate, creat- They also undergo rapid proliferation. Only about 3% of the ing a pore through which the hematopoietic cell enters the cells generated in the thymus successfully exit the medulla lumen of the sinus. These pores are only 2–3 mcM in diam- as mature T-cells; the rest die by apoptosis and are removed eter; thus, blood cells must have the ability to deform so by thymic macrophages. The thymus is responsible for sup- that they can pass through the sinusoidal lining. Progressive plying the T-dependent areas of lymph nodes, spleen, and increases in deformability and motility have been noted as other peripheral lymphoid tissue with immunocompetent granulocytes mature from the myeloblast to the segmented T-lymphocytes. granulocyte stage, facilitating the movement of cells into The thymus is a well-developed organ at birth and the sinus lumen. continues to increase in size until puberty. After puberty, Many soluble factors are important in regulating the however, it begins to atrophy until old age when it release of blood cells from bone marrow, including granulo- becomes barely recognizable. This atrophy may be driven cyte-colony stimulating factor (G-CSF/CSF 3), granulocyte by increased steroid levels beginning in puberty and monocyte-colony stimulating factor (GM-CSF/CSF 2), and decreased growth factor levels in adults.24,25 The atrophied a large number of chemokines21,22 (Chapter 4). Some of these thymus is still capable of producing some new T-cells if molecules are used clinically to increase circulating granulo- the peripheral
pool becomes depleted as occurs after the cytes or release HSCs into the circulation to obtain granulo- lymphoid irradiation that accompanies bone marrow cytes for transfusion or stem cells for transplantation. transplantation.27 Structure and Function of Hematopoietic Organs 35 Macrophage Cortical epithelial cell Thymocyle precursors Subcapsular cortex Immature Medullary thymocytes epithelial cell Cortex Capsule Hassall’s corpuscles Medulla Macrophage Mature thymocytes Dendritic cell Figure 3.5 A schematic drawing of the thymus. See text for the role of the various cell types. Hassall’s corpuscles are collections of epithelial cells that may be involved in the development of certain T-cell subsets (regulatory T-cells) in the thymus. Spleen phagocytic macrophages. The immune response is initiated in the white pulp. Germinal centers are surrounded by man- The spleen is located in the upper-left quadrant of the abdo- tle zones containing small B-cells. In some cases of height- men beneath the diaphragm and to the left of the stomach. ened immunologic activity, the white pulp can increase to After several emergency splenectomies were performed occupy half the volume of the spleen (it is normally …20,). without causing permanent harm to the patients, it was rec- The red pulp contains sinuses and cords. The sinuses ognized that the spleen was not essential to life. However, are dilated vascular spaces for venous blood. The pulp’s red it does play a role in filtering foreign substances and old color is caused by the presence of large numbers of eryth- erythrocytes from the circulation, storage of platelets, and rocytes in the sinuses. The cords are composed of masses immune defense. of reticular tissue and macrophages that lie between the ARCHITECTURE sinuses. The cords of the red pulp provide zones for platelet Enclosed by a capsule of connective tissue, the spleen con- storage and destruction of damaged blood cells. tains the largest collection of lymphocytes and macrophages in the body (Figure 3-6). These cells, together with a reticu- BLOOD FLOW lar meshwork, are organized into two zones: white pulp The spleen is richly supplied with blood. It receives 5% of and red pulp. the total cardiac output, a blood volume of 300 mL/minute. The white pulp, a visible grayish-white zone, is com- Blood enters the spleen through the splenic artery, which posed of lymphocytes and is located around a central branches into many central arteries. Vessel branches can artery.28 The area closest to the artery, which contains many terminate in the white pulp or red pulp. Blood entering the T-cells as well as macrophages and dendritic cells, is termed spleen can follow either the rapid transit pathway (closed the periarteriolar lymphatic sheath (PALS). Peripheral to this circulation) or the slow transit pathway (open circulation). area are B-cells arranged into follicles (a sphere of B-cells The rapid transit pathway is a relatively unobstructed route within lymphatic tissue). Activated B-cells are found in by which blood enters the sinuses in red pulp from the specialized follicular areas called germinal centers, which arteries and passes directly to the venous collecting system. appear as lightly stained areas in the center of a lymphoid In contrast, blood entering the slow transit pathway moves follicle. The germinal centers consist of a mixture of B-lym- sluggishly through a circuitous route of macrophage-lined phocytes, follicular dendritic cells, a few plasma cells, and cords before it gains access to the venous sinuses. Plasma 36 Chapter 3 Trabecular artery Central artery Germinal center Follicle Follicle Mantle layer Periarteriolar lymphatic sheath (T-cell zone) Germinal center Splenic sinus Trabecular vein Red pulp cord Trabecular Marginal Germinal Capsule artery zone center Figure 3.6 A schematic drawing of splenic tissue. See text for an explanation of the splenic tissue architecture. The periarteriolar lymphatic sheath contains many T-cells, macrophages, and dendritic cells. B-cells are arranged into follicles. Activated B-cells are in the germinal centers. in the cords freely enters the sinuses, but erythrocytes or damaged erythrocytes. Slow passage through a macro- meet resistance at the sinus wall where they must squeeze phage-rich route allows the phagocytic cells to remove these through the tiny openings. This skimming of the plasma old or damaged, less deformable erythrocytes before or dur- from blood cells sharply increases the hematocrit in the ing their squeeze through the 3 mcM pores to the venous cords. Sluggish blood flow and continued erythrocyte met- sinuses. Normal erythrocytes withstand this adverse envi- abolic activity in the cords result in a splenic environment ronment and eventually reenter the circulation. that is hypoxic, acidic, and hypoglycemic. Hypoxia and hypoglycemia occur as erythrocytes utilize available oxy- Checkpoint 3.3 gen and glucose, and metabolic byproducts create the acidic Describe how the spleen removes old or damaged erythrocytes environment. Blood leaves the spleen through the splenic from the circulation. vein, which drains into the portal circulation. Liver disease can reduce blood flow out of the spleen and thereby lead to Pitting refers to the spleen’s ability to “pluck out” par- splenic enlargement. ticles from intact erythrocytes without destroying them. FUNCTION Blood cells coated with antibody are susceptible to pitting Blood that empties into the cords of the red pulp takes the by macrophages. The macrophage removes the antigen– slow transit pathway, which is very important to splenic antibody complex and the attached blood cell membrane. function including culling, pitting, and storing blood cells. The pinched-off cell membrane can reseal itself, but the cell The discriminatory filtering and destruction of senescent cannot synthesize lipids and proteins for new membrane (aged) or damaged red cells by the spleen is termed cull- due to its lack of cellular organelles. Therefore, extensive ing. Cells entering the spleen through the slow transit path- pitting causes a reduced surface-area-to-volume ratio, way become concentrated in the hypoglycemic, hypoxic resulting in the formation of spherocytes (erythrocytes that cords of the red pulp—a hazardous environment for aged have no area of central pallor on stained blood smears). The Structure and Function of Hematopoietic Organs 37 presence of spherocytes on a blood film is evidence that the lymphoid cells can result from prolonged infection. Several erythrocyte has undergone membrane assault in the spleen. blood disorders can cause splenomegaly. In these disorders, The white pulp of the spleen is an important line of intrinsically abnormal blood cells or cells coated with anti- defense in blood-borne infections because of its rich supply body are removed from circulation in large numbers (e.g., of lymphocytes and phagocytic cells (macrophages) and the hereditary spherocytosis, immune thrombocytopenic pur- slow transit circulation through these areas. Blood-borne pura; Chapters 17 and 19). antigens are forced into close contact with macrophages Infiltration of the spleen with additional cells or met- (functioning as antigen-presenting cells) and lymphocytes abolic byproducts can also cause hypersplenism. Such allowing for recognition of the antigen as foreign and lead- conditions include disorders in which the macrophages ing to phagocytosis, T- and B-cell activation, and antibody accumulate large quantities of undigestible substances; formation. some of these disorders, such as Gaucher’s disease, will be The spleen’s immunologic function is probably less discussed later (Chapter 21). Neoplasms in which the malig- important in the well-developed adult immune system nant cells occupy much of the splenic volume can cause than in the still-developing immune system of the child. splenomegaly. If the tumor cells incapacitate the spleen, Young children who undergo splenectomy may develop the peripheral blood will show evidence of hyposplenism overwhelming, often fatal, infections with encapsulated (similar to the findings after splenectomy). Congestive sple- organisms such as Streptococcus pneumoniae and Haemophilus nomegaly can occur following liver cirrhosis with portal influenzae. This can also be a rare complication of splenec- hypertension or congestive heart failure when blood that tomy in adults. does not flow easily through the liver is rerouted through The red pulp cords of the spleen act as a reservoir for the spleen. platelets, sequestering approximately one-third of the cir- culating platelet mass. Massive enlargement of the spleen (splenomegaly) can result in a pooling of 80–90% of the Checkpoint 3.4 platelets, producing peripheral blood thrombocytopenia. Contrast primary and secondary hypersplenism and give an Removal of the spleen results in a transient thrombocytosis example of a disorder that can cause secondary hypersplenism. with a return to normal platelet concentrations in about 10 days. SPLENECTOMY HYPERSPLENISM Splenectomy can relieve the effects of hypersplenism; how- In a number of conditions, the spleen can become enlarged ever, this procedure is not always advisable, especially when and, through exaggeration of its normal activities of filter- the spleen is performing a constructive role such as produc- ing and phagocytosing, cause anemia, leukopenia, throm- ing antibody or filtering protozoa or bacteria. Splenectomy bocytopenia, or combinations of cytopenias. A diagnosis of appears to be most beneficial in patients with hereditary hypersplenism is made when three conditions are met: (1) or acquired conditions in which erythrocytes or platelets the presence of anemia, leukopenia, or thrombocytopenia in are undergoing increased destruction, such as hemolytic the peripheral blood, (2) the existence of a cellular or hyper- disorders or immune thrombocytopenia. The blood cells plastic bone marrow corresponding to the peripheral blood are still abnormal after splenectomy, but the major site of cytopenias, and (3) the occurrence of splenomegaly. The cor- their destruction is removed. Consequently, the cells have a rection of cytopenias following splenectomy confirms the more normal life span. Splenectomy results in characteristic diagnosis. erythrocyte abnormalities that experienced clinical labora- Hypersplenism has been categorized into two types: tory professionals can note easily on blood smears. After primary and secondary. Primary hypersplenism is said to splenectomy, the erythrocytes often contain inclusions (e.g., occur when no underlying disease can be identified. The Howell Jolly bodies, Pappenheimer bodies), and abnormal spleen functions abnormally and causes the cytopenia(s). shapes can be seen (Chapter 10). This type of hypersplenism is very rare. Secondary hyper- The lifespan of healthy erythrocytes is not increased splenism occurs in those cases in which an underlying after splenectomy. Other organs, primarily the liver, assume disorder causes the splenic abnormalities. Secondary the culling function. Blood flow through the liver also is hypersplenism has many and varied causes. Hypersplen- slowed by passage through sinusoids, which are lined with ism can occur secondary to compensatory (or workload) specialized macrophages called Kupffer cells. These macro- hypertrophy of this organ. Inflammatory and infectious phages can perform functions similar to those of phagocytes diseases are thought to cause splenomegaly by an increase in the splenic cords. Even when a spleen is present, the liver, in the spleen’s immune defense functions. For example, because of its larger blood flow, is responsible for removing an increase in clearing particulate matter can lead to an most of the particulate matter of the blood. The liver, how- increase in number of macrophages, and hyperplasia of ever, is not as effective as the spleen in filtering abnormal 38 Chapter 3 erythrocytes, probably because of the relatively rapid flow or chains along the larger lymphatic vessels. Fluid from of blood past hepatic macrophages. the lymphatic vessels enters the lymph node through affer- Acquired hyposplenism is a complication of sickle cell ent lymphatic vessels and exit through efferent lymphatic anemia. The spleen’s acidic, hypoxic, hypoglycemic envi- vessels. Lymph nodes contain an outer cortex and an inner ronment leads to enhanced sickling of the erythrocytes in medulla (Figure 3-7). Fibrous trabeculae extend inward from the spleen. This leads to blockage of the blood vessels and the capsule to form irregular compartments within the cor- infarcts of the surrounding tissue. The tissue damage is pro- tex. Lymphocytes enter the lymph node from the blood by gressive and leads to functional splenectomy (also referred migrating across specialized endothelium of post-capillary to as autosplenectomy) (Chapter 13). venules (high endothelial venules) in the cortex. The cortex contains B-cell follicles surrounded by T-lymphocytes and macrophages. Similar to the spleen, some follicles contain CASE STUDY (continued from page 34) areas of activated B-cells known as germinal centers. A stimu- Francine was diagnosed as having leukemia. lated node can have many germinal centers, but a resting node contains follicles with small resting lymphocytes and 3. What do you think is the cause of the macrophages. The medulla, which surrounds the efferent splenomegaly? lymphatics, consists of cords of B-cells, plasma cells, and 4. Why might the peripheral blood reveal changes macrophages that lie between sinusoids. associated with hyposplenism when the spleen Lymph nodes act as filters to remove foreign particles is enlarged? from lymph by resident dendritic
cells and macrophages. In addition, dendritic cells activated in the tissues can travel via the lymphatics to the lymph nodes. Dendritic cells in turn stimulate T- and B-cells. Stimulated B-cells move from Lymph Nodes the germinal centers to the medulla where they reside as The lymphatic system is composed of lymph nodes and plasma cells and secrete antibody. Thus, lymph nodes pro- lymphatic vessels that drain into the left and right lym- vide immune defense against pathogens in virtually all phatic ducts. Lymph is formed as a filtrate of blood plasma tissues. that escapes into connective tissue. The lymphatic vessels MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) originate as lymphatic capillaries in connective tissue spaces Collections of loosely organized aggregates of lympho- throughout the body, collect the lymph, and carry it toward cytes found throughout the body in association with the lymphatic ducts near the neck where the fluid reenters mucosal surfaces are mucosa-associated lymphoid tissue the blood. The bean-shaped lymph nodes occur in groups (MALT).29 Its basic organization is similar to lymph nodes Lymphatic capillary (efferent) Capsule Artery Vein Medulla Cortex (plasma cells) Medullary sinus Follicle Interfollicular area (T lymphocytes) Cortical sinus Germinal center (B lymphocytes) Lymphatic capillary (afferent) Figure 3.7 A schematic drawing of a lymph node. Note the location of T- and B-lymphocyte populations. Structure and Function of Hematopoietic Organs 39 in that T- and B-cell–rich areas can be identified but are not or malignant transformation of lymphocytes or macro- as clearly demarcated as in lymph nodes. The medulla is phages. Alternatively, lymph node enlargement can occur not present as a separate structure, and no fibrous capsule because of metastatic tumors that originate in extranodal can be identified. In the intestine, some of these aggregates sites. are known as Peyer’s patches. Tonsils and the appendix also are part of MALT. Its function is to trap antigens that are crossing mucosal surfaces and initiate immune responses CASE STUDY (continued from page 38) rapidly. Francine had lymphadenopathy. The leukemia was LYMPHADENOPATHY diagnosed as a leukemia of lymphocytic cells. Lymph nodes can become enlarged (lymphadenopathy) by expansion of the tissue within the node due to inflam- 5. What might explain the lymphadenopathy? mation, prolonged immune response to infectious agents, Summary Hematopoiesis occurs in several different locations during complete their differentiation in the bone marrow. T-cells human development. The major locations include the yolk finish most of their differentiation in the thymus and sec- sac, aorta-gonad-mesonephros (AGM) region, liver, bone ondary lymphoid tissues. B-cells can respond to antigens marrow, and thymus. Further differentiation of lympho- by the time they leave the bone marrow but differentiate cytes also occurs in the spleen and lymph nodes. In the further into antibody-secreting plasma cells in the spleen adult, the bone marrow is the location of hematopoietic and lymph nodes. The spleen removes senescent or abnor- stem cells and thus is ultimately responsible for initiat- mal erythrocytes and particulate matter from erythrocytes. ing all hematopoiesis. The bone marrow stroma (support- The spleen can become enlarged and through exaggera- ing tissue) provides a microenvironment for proliferation tion of its normal functions cause cytopenias (hypersplen- and differentiation of hematopoietic cells (red marrow). ism). Lymph nodes contribute to immune defense by The stroma consists of macrophages, reticular cells, and initiating immune responses to foreign particles found adipocytes. The adipocytes form the yellow marrow and in lymph. Lymph nodes can become enlarged due to an mechanically control the volume of hematopoietic tis- immune response to infectious agents or malignant tumor sue. Myeloid cells, platelets, and erythrocytes essentially (lymphadenopathy). Review Questions Level I 3. Peyer’s patches are structurally most closely related to the: (Objective 3) 1. B-cells develop or differentiate in all of the following tissues except: (Objective 2) a. lymph node a. thymus b. spleen b. bone marrow c. thymus c. spleen d. liver d. lymph nodes 4. All of the following are functions of bone marrow stroma except that it: (Objective 4) 2. Lack of a spleen results in: (Objective 4) a. controls the volume of marrow available for a. younger circulating erythrocytes hematopoiesis b. granular inclusions in erythrocytes b. provides structural support for marrow elements c. pitting of erythrocytes c. secretes growth factors for hematopoiesis d. spherocytes d. provides an exit route from marrow for mature blood cells 40 Chapter 3 5. Which site of early hematopoiesis is extraembryonic? c. definitive erythropoiesis (Objective 1) d. primitive erythropoiesis a. yolk sac 4. Extramedullary hematopoiesis in the adult is most b. liver often accompanied by: (Objectives 1, 2, 3) c. AGM a. splenomegaly d. spleen b. liver atrophy Level II c. leukocytosis 1. A common site of adult extramedullary hematopoiesis d. hyposplenism is the: (Objective 3) 5. Culling and pitting of erythrocytes in the circulation a. liver takes place in the: (Objective 4) b. thymus a. germinal centers of lymph nodes c. lymph node b. cords of red pulp of the spleen d. yolk sac c. cortex of the thymus 2. A patient has infectious mononucleosis. His cervical d. sinuses of the bone marrow lymph nodes are enlarged. This is most likely due to: 6. Primitive erythropoiesis: (Objective 5) (Objective 2) a. occurs primarily in the liver a. an immune response to an infectious agent b. originates from a self-renewing hematopoietic stem cell b. a malignant tumor c. utilizes the same hemoglobin genes as adults c. extramedullary hematopoiesis d. involves the formation of blood islands d. presence of macrophages containing undigestible substances 7. Erythroblastic islands are composed of erythrocyte precursors and: (Objective 4) 3. Gower I, Gower 2, and Portland hemoglobins in erythroblasts characterize: (Objective 5) a. megakaryocytes b. lymphocytes a. erythropoiesis in the spleen c. macrophages b. erythropoiesis in the bone marrow d. adipocytes References 1. Palis, J., & Yoder, M. C. (2001). Yolk-sac hematopoiesis: the first 8. Elliott, J. F., Rock, E. P., Patten, P. A., Davis, M. M., & Chien, Y. H. blood cells of mouse and man. Exp Hematol., 29, 927–936. (1988). The adult T-cell receptor delta-chain is diverse and dis- 2. Baron, M. H. (2003). Embryonic origins of mammalian hemato- tinct from that of fetal thymocytes. Nature, 331, 627–631. poiesis. Exp Hematol., 31, 1160-1169. 9. Iversen, P. O. (1997). Blood flow to the haemopoietic bone mar- 3. Marcos, M. A., Godin, I., Cumano, A., Morales, S., Garcia-Porrero, row. Acta Physiol Scand., 159, 269–276. J. A., Dieterlen-Lievre, F., & Gaspar, M. L. (1994). Developmental 10. Afran, A. M., Broome, C. S., Nicholls, S. E., Whetton, A. D., & events from hemopoietic stem cells to B-cell populations and Ig Miyan, J. A. (1997). Bone marrow innervation regulates cellular repertoires. Immunol Rev., 137, 155–171. retention in the murine hematopoietic system. Br J Haematol., 98, 4. Ottersbach, K., Smith, A., & Wood, A. (2009). Ontogeny of hae- 569–577. matopoiesis: recent advances and open questions. Br J Haematol., 11. Sugiyama, T., Kohara, H., Noda, M., & Nagasawa, T. (2006). 148, 343–355. Maintenance of the hematopoietic stem cell pool by CXCL12- 5. Pardanaud, L., Luton, D., Prigent, M., Bourcheix, L.M., Catala, CXCR4 chemokine signaling in bone marrow stromal cell niches. M., & Dieterlen-Lievre, F. (1996). Two distinct endothelial lin- Immunity, 25, 977–988. eages in ontogeny, one of them related to hemopoiesis. Develop- 12. Birbrair, A., & Frenette, P. S. (2016). Niche heterogeneity in the ment, 122, 1363–1371. bone marrow. Ann NY Acad Sci, 1370, 82-96. 6. Farace, M. G., Brown, B. A., Raschella, G., Alexander, J., Gambari, 13. Monteiro, J. P., Benjamin, A., Costa, E. S., Barcinski, M. A., & R., Fantoni, A., . . . Edgell, M. H. (1984). The mouse beta h1 gene Bonomo, A. (2005). Normal hematopoiesis is maintained by acti- codes for the z chain of embryonic hemoglobin. J Biol Chem., 259, vated bone marrow CD4+ T cells. Blood, 105, 1484–1491. 7123–7128. 14. Muruganadan, S., Roman, A. A., & Sinal, C. J. (2009). Adipocyte 7. Chang, Y., Paige, C. J., & Wu, G.E. (1992). Enumeration and char- differentiation of bone marrow-derived mesenchymal stem cells: acterization of DJH structures in mouse fetal liver. EMBO J., 11, cross talk with the osteoblastogenic program. Cell Mol Life Sci., 66, 1891–1899. 236–253. Structure and Function of Hematopoietic Organs 41 15. Rickard, D. J., Subramaniam, M., & Spelsberg, T. C. (1999). Molec- independent of an effect on adhesion: comparison with granulo- ular and cellular mechanisms of estrogen action on the skeleton. J cyte macrophage colony stimulating factor (GM-CSF). Br J Hae- Cell Biochem., Suppl 32–33, 123–132. matol., 94, 40–47. 16. Moriyama, Y., & Fisher, J. W. (1975). Effects of testosterone and 23. Laurence, A. D. J. (2005). Location, movement and survival: the erythropoietin on erythroid colony formation in human bone role of chemokines in haematopoiesis and malignancy. Br J Hae- marrow cultures. Blood, 45, 665–670. matol., 132, 255–267. 17. Lampiasi, N., Russo, R., & Zito, F. (2016). The alternative faces of 24. von Boehmer, H. (2014) The thymus in immunity and malig- macrophage generate osteoclasts. Biomed Res Int. [Article ID 9089610]. nancy. Cancer Immunol Res, 2, 592-597. 18. Bianco, P., Sacchetti, B., & Riminucci, M. (2011). Osteoprogenitors 25. Zoller, A. L., & Kersh, G. J. (2006). Estrogen induces thymic and the hematopoietic microenvironment. Best Pract Res Clin Hae- atrophy by eliminating early thymic progenitors and inhibit- matol., 24, 37–47. ing proliferation of beta-selected thymocytes. J Immunol., 176, 19. Yu, V. W. C., & Scadden, D. T. (2016) Heterogeneity of the bone 7371–7378. marrow niche. Curr Opin Hematol., 23, 331-338. 26. Garcia-Suarez, O., Perez-Perez, M., Germana, A., Estaban, I., & 20. Lichtman, M. A., Chamberlain, J. K., Simon, W., & Santillo, P. A. Germana, G. (2003). Involvement of growth factors in thymic (1978). Parasinusoidal location of megakaryocytes in marrow: a involution. Microsc Res Tech., 62, 514–523. determinant of platelet release. Am J Hematol., 4, 303–312. 27. Douek, D. C., & Koup, R. A. (2000). Evidence for thymic function 21. Lichtman, M. A., Packman, C. H., & Costine, L. S. (1989). Blood in the elderly. Vaccine, 16, 1638–1641. cell formation: The Role of the Hematopoietic Microenvironment. Clif- 28. Steiniger, B. S. (2015). Human spleen microanatomy: why mice ton, NJ: Humana Press. do not suffice. Immunology, 145, 334-346. 22. Yong, K. (1996). Granulocyte colony-stimulating factor (G-CSF) 29. MacDonald, T. T. (2003). The mucosal immune system. Parasite increases neutrophil migration across vascular endothelium Immunol., 25, 235–246. Chapter 4 Hematopoiesis J. Lynne Williams, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Describe the basic concepts of cell 4. List the general characteristics of growth differentiation and maturation. factors and identify the major examples 2. Compare and contrast the categories of of early acting (multilineage), later acting hematopoietic precursor cells: hematopoietic (lineage restricted), and indirect acting stem cells, hematopoietic progenitor cells, growth factors. and maturing cells, including proliferation 5. Compare and contrast paracrine, autocrine, and differentiation potential, morphology, and juxtacrine regulation. and population size. 6. List examples of negative regulators of 3. Describe the hierarchy of hematopoietic hematopoiesis. precursor cells and the relationships of the 7. Define hematopoietic microenvironment. various blood cell lineages to each other (including the concept of colony-forming unit [CFU]). Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare and contrast the phenotypic 4. Summarize the concept of signal characteristics differentiating the transduction pathways. hematopoietic stem cells and progenitor cells. 5. Explain the roles of transcription factors 2. Identify the key cytokines required for in the regulation of hematopoiesis and lineage-specific regulation. differentiation. 3. Describe the structure and role of growth 6. Outline current clinical uses of cytokines. factor receptors. 42 Hematopoiesis 43 7. Identify and describe the cellular 8. List and explain the proposed mechanisms and extracellular components of the used to regulate hematopoietic stem/ hematopoietic microenvironment. progenitor cell proliferation/differentiation. Chapter Outline Objectives—Level I and Level II 42 Hematopoiesis 44 Key Terms 43 Hematopoietic Microenvironment 59 Background Basics 43 Summary 62 Overview 43 Review Questions 62 Introduction 43 References 64 Key Terms Autocrine Hematopoietic microenvironment Maturing cell Commitment (HM) Paracrine Cytokine Hematopoietic progenitor cell Progenitor cell Differentiation Hematopoietic stem cell Stromal cell Extracellular matrix (ECM) Juxtacrine Transcription factor (TF) Hematopoiesis Maturation Background Basics The information in this chapter will build on the concepts • Describe the cell cycle and the molecules that regulate learned in previous chapters. To maximize your learn- it. (Chapter 2) ing experience, you should review these concepts before • Describe apoptosis
and the roles of caspases and the starting this unit of study: Bcl-2 family of proteins in the process. (Chapter 2) Level I and Level II • Summarize the structure of the bone marrow, including the concepts of vascular and endosteal • Identify the component parts of a cell, including compartments. (Chapter 3) the structure and function of cellular organelles. (Chapter 2) Overview presented with a summary of the signaling pathways and transcription factors activated by receptor-cytokine binding. This chapter begins with an introduction to the concepts Finally, the hematopoietic microenvironment is described of cellular commitment and differentiation in the hema- and its role in hematopoiesis summarized. topoietic system. The characteristics that have historically defined the hematopoietic precursor cells and the cytokines that regulate the development of these precursor cells are discussed. New information on the development and dif- Introduction ferentiation of hematopoietic precursor cells and proposed The maintenance of an adequate number of cells to carry “models” of a differentiation hierarchy are presented. out the functions of the organism is referred to as tissue The structure and function of the cytokine receptors are homeostasis. It depends on a careful balance between 44 Chapter 4 cellular proliferation, cellular differentiation, and cell death platelets to about 4 months for erythrocytes. As a result, (apoptosis). The hematopoietic system presents a challenge these cells are described as terminally differentiated. The con- when considering the homeostasis of the circulating blood stant death of mature, functional blood cells by apoptosis because the majority of circulating cells are postmitotic cells means that new cells must be produced to replace those that are relatively short lived. Thus, circulating blood cells that are removed either as a consequence of performing are intrinsically incapable of providing their replacements their biologic functions (e.g., platelets in hemostasis, gran- when they reach the end of their life spans. Hematopoiesis ulocytes in host defense) or through cellular senescence or is the process responsible for the replacement of circulat- “old age” (erythrocytes). The replacement of circulating, ing blood cells; it depends on the proliferation of precursor terminally differentiated cells depends on the function of cells in the bone marrow that still retain mitotic capability. less differentiated hematopoietic precursor cells that still This process is governed by a multitude of cytokines (both retain significant proliferative capabilities. These hemato- stimulating and inhibitory growth factors) and takes place poietic precursor cells, located primarily in the bone mar- in a specialized microenvironment uniquely suited to regu- row in adults, consist of a hierarchy of cells with enormous late the process. proliferation potential. They maintain a daily production of approximately 2 * 1011 red blood cells (RBCs) and 1 * 1011 (each) white blood cells (WBCs) and platelets for Hematopoiesis an individual’s lifetime.2 In addition, acute stress (blood loss or infection) can result in a rapid, efficient, and specific Cell proliferation and programmed cell death ( apoptosis) increase in production over baseline of the particular cell work together to provide an adequate number of cells lineage needed (up to a 10-fold increase in cell production). (Chapter 2). Differentiation is responsible for g enerating For example, acute blood loss results in a specific increased the diverse cell populations that provide the specialized production of erythrocytes while a bacterial infection results functions needed by the organism. Differentiation is defined in an increased production of phagocytic cells (granulocytes as the appearance of different properties in cells that were and monocytes). initially equivalent. Because all cells of an organism carry the same genetic information, differentiation (or the appearance of specific characteristics) occurs by the progressive restric- Hematopoietic Precursor Cells tion of other potential developmental programs available to The pioneering work of Till and McCulloch began to the cell. Commitment is defined as the instance when two define the hierarchy of hematopoietic precursor cells using cells derived from the same precursor take a separate route in vivo clonal assays.3 They were the first to demonstrate of development.1 Commitment “assigns the program,” and the existence of hematopoietic stem cells with the ability the maturation process executes it (maturation includes all to regenerate all types of blood lineages. It was not until phenomena that begin with commitment and end when the the development of in vitro clonal assays, however, that cell has all its characteristics).1 a model of blood cell production began to evolve.3,4,5,6,7 Hematopoiesis, the development of all the different Hematopoietic precursor cells can be divided into three blood cell lineages, has two striking characteristics: the vari- cellular compartments defined by their relative maturity: ety of distinct blood cell types produced and the relatively hematopoietic stem cells, hematopoietic progenitor cells, brief life span of the individual cells. The cells circulating in and maturing cells (Table 4-1). The nomenclature used to the peripheral blood are mature and, with the exception of define these various compartments over the past 20 years lymphocytes, are generally incapable of mitosis. They also has lacked uniformity. Although there is general agree- have a limited life span from days for granulocytes and ment on the designations stem cells and progenitor cells, Table 4.1 Comparison of Hematopoietic Precursor Cells Stem Cells Progenitor Cells Maturing Cells About 0.5% of total hematopoietic precursor cells 3% of total hematopoietic precursor cells Greater than 95% of total hematopoietic precursor cells Multilineage differentiation potential Restricted developmental potential Committed (unipotential) transit population (multipotential S unipotential) Quiescent cell population—population size stable Population amplified by proliferation Population amplified by proliferation Population maintained by self-renewal Transit population without true self-renewal Proliferative sequence complete before full maturation Not morphologically recognizable Not morphologically recognizable Morphologically recognizable Measured by functional clonal assays in vivo and Measured by clonal assays in vitro Measured by morphologic analysis; cell counting in vitro differentials Hematopoiesis 45 various authors have called the third category precursor that have begun to differentiate. There is no unique surface cells,8 maturing cells,9 or morphologically recognizable marker that definitively identifies an HSC. precursor cells.10 In this chapter, we use the term precursor CD34 is a 110 kDa glycoprotein expressed by human to include all cells antecedent to the mature cells in each lin- HSCs and early progenitor cells, as well as vascular endo- eage and the term maturing cells to include those precursor thelial cells.13 Expression of CD34 is lost as cells mature cells within each lineage that are morphologically identifi- beyond the progenitor cell compartment. Thy-1 (CD90) is able under the microscope. The prevailing model describ- a membrane glycoprotein originally discovered as a thy- ing these three cellular compartments of precursor cells is mocyte antigen involved in T-lymphocyte adhesion to presented below. stromal cells; it is also an important marker in conjunction with CD34 for HSC identification.14,15 CD49f is the integrin STEM CELLS a6 subunit polypeptide, important in cell adhesion.15 CD133 All hematopoiesis derives from a pool of undifferentiated is a transmembrane protein associated with human hema- cells, hematopoietic stem cells (HSCs), which give rise to all topoietic stem cells and progenitor cells.16 CD38 is a 45 kDa hematopoietic lineages by the processes of proliferation and glycoprotein considered to be an early myeloid differentia- differentiation.11 The stem cell compartment is the smallest tion antigen. Lin- (lineage negative) refers to the absence of the hematopoietic precursor compartments, constituting of known differentiation markers or antigens present on only about 0.5% of the total marrow nucleated cells. How- lineage-restricted progenitors (Table 4-2). The HLA-DR ever, these rare cells are capable of regenerating the entire antigens are a component of the human major histocom- hematopoietic system. Thus, they are defined as multipoten- patibility complex antigens. Rhodamine123 (Rho123) is a tial precursors because they maintain the capacity to give rise fluorescent supravital dye that is taken up by cells.15 HSCs to all lineages of blood cells. The other defining characteris- have high levels of pumps capable of effluxing dyes (and tic of stem cells is their high self-renewal capacity (i.e., they drugs). They transport the dye out of the cells and display can give rise to daughter stem cells that are exact replicas of low-intensity staining for Rho123 (Rh123Lo). Thus, the mul- the parent cell). Despite their responsibility for generating tipotential stem cells capable of long-term hematopoietic the entire hematopoietic system, at any one time the major- reconstitution are found in the population of cells that con- ity (more than 95%) of stem cells are not dividing, most are tain no lineage-specific antigens, CD38, or HLA-DR anti- withdrawn from the cell cycle or quiescent (G0 phase of the cell cycle; Chapter 2).8 gens but express CD34, Thy-1, CD49f, CD133, SCF-R, and TPO-R and are largely quiescent. Stem Cell Phenotype Stem cells are not morphologically The Stem Cell Compartment HSCs constitute a broadly recognizable. Primitive stem cells, isolated by fluorescent- heterogeneous population of cells, in terms of both their activated cell sorting (FACS), are mononuclear cells very self-renewal and differentiation attributes. An “age hier- similar in appearance to small lymphocytes. Because stem archy” has been described based on the time it takes for cells are not morphologically identifiable, they are defined transplanted marrow cells to repopulate a lethally irradi- functionally by their ability to reconstitute both lymphoid ated animal and the duration of resultant hematopoiesis. and myeloid hematopoiesis when transplanted into a recipi- Thus, the terms long-term repopulating cells (LTRs; Rho123Lo) ent animal. In mice, the existence of the true HSC has been and short-term repopulating cells (STRs; Rho123Hi) are used. unequivocally demonstrated by the occasional successful The STR cells are further along the hematopoietic devel- transplant using single purified stem cells, thus providing opmental pathway, are more likely to be proliferating, and direct proof that single cells capable of sustaining lifelong hematopoiesis do exist.12 have decreased self-renewal potential2 (Figure 4-1). STR There are no accurate quantita- cells cannot sustain hematopoiesis for the recipient animal’s tive assays for human HSCs, and practical and ethical dif- lifetime but are more important in blood formation for the ficulties limit an effective in vivo assay for human stem first few months after HSC transplantation.17 cells. However, a number of characteristics have been used to define their phenotype. These include surface mark- ers, defined by monoclonal antibody binding to the cell Table 4.2 Lineage-Specific Markers Used in Purification membrane, which can be used in cell-separation protocols, of HSC resulting in a relatively high degree of purity of HSCs. The Lineage Specific Biomarker currently proposed phenotype of the human HSC is: Erythrocytes Glycophorin A CD34+Thy1+CD49f+CD133+CD38-Lin-HLADR-Rh123Lo Megakaryocytes Glycoprotein (GP) IIb/IIIa In addition, HSCs are positive for the receptor for stem Neutrophils CD13, CD15, CD33 cell factor (SCF-R/c-kit, CD117) and the thrombopoietin Monocytes and macrophages CD11b, CD14 (TPO) receptor, TPO-R, (Mpl, CD110). CD34, SCF-R, and B lymphocytes CD10, CD19, CD20 TPO-R are not found exclusively on HSCs but also on cells T lymphocytes CD3, CD4, CD5, CD8, CD38 46 Chapter 4 Hemangioblast Vascular endothelium LTR- HSC STR- HSC CLP CMP Apoptosis Figure 4.1 Derivation and fates of hematopoietic stem cells (HSCs). Hemangioblasts are precursor cells giving rise to both HSCs and vascular endothelium during embryonic development. LTR (long-term repopulating) HSC and STR (short-term repopulating) HSC refer to the length of time these HSC subpopulations take to repopulate depleted hematopoietic tissue and the duration of hematopoiesis arising from each. LTR cells are developmentally more primitive than STR cells. HSCs undergoing mitosis have three possible fates: self-renewal, commitment to differentiation (becoming common lymphoid progenitors [CLP] or common myeloid progenitors [CMP]), or apoptosis. These cell-fate decisions are highly regulated and involve specific transcription factors. The process of self-renewal is a nondifferentiating cell pool of primitive stem cells. An HSC that underwent a cell division and ensures that the stem cell population is main- division resulting in two differentiating cells would deplete tained throughout an individual’s lifetime. It is associated the stem cell pool. Thus, the HSC must carefully regulate with elevated levels of telomerase (which prevents replica- the simultaneous processes of expansion (self-renewal) and tive senescence, an irreversible cease in proliferation after differentiation in order to prevent hematopoietic failure or a finite number of cell divisions) and Bcl-2 (which prevents uncontrolled stem cell expansion. HSCs normally maintain apoptosis)18 (Chapter 2). Humans are estimated to have this balance by a process of asymmetric cell division in only about 2 * 104 HSCs.2 This small group of cells is which one daughter cell retains all properties of the parent able to sustain tremendous hematopoietic cell production cell (self-renewal), while the
other daughter cell undergoes through the division of only a small fraction of its mem- differentiation.16 bers, keeping the remainder of the stem cells in reserve. The size of the stem cell compartment is relatively stable under Stem Cell Niches Specific microenvironments within the homeostatic conditions. In a stem cell compartment that bone marrow (BM) regulate the state and function of hema- remains stable in size but supplies differentiating cells, a cell topoietic stem and progenitor cells. HSCs reside in unique must be added to the HSC compartment by proliferation “stem cell niches” in the BM, where HSCs are retained (self-renewal) for each cell that leaves by the process of dif- via adhesion molecules and membrane-bound cytokines. ferentiation. An HSC undergoing mitosis that results in two Interactions between HSCs and BM stromal cells help regu- daughter HSCs (symmetric cell division) would increase the late and balance the processes of quiescence, self-renewal, Hematopoiesis 47 and differentiation.17,21 The niche provides both a physi- The regulation of stem cell fate—whether to remain cal anchor for the HSCs and factors that regulate HSC quiescent, self-renew, initiate differentiation, or die—is number and function (see later section “Hematopoietic complex and still not fully understood. It is regulated by Microenvironment”). both cell-intrinsic functions and regulatory signals provided There are two important HSC niches.19 An osteoblastic by the HSC niche. Internal cell factors regulating HSC fate niche, found adjacent to the endosteal surface, supports and include proteins such as SCL (product of stem cell leukemia maintains HSC quiescence and/or self-renewal.18 The sec- gene), LMO2 (Lim-only protein 2), and the transcription fac- ond is a vascular niche, located near the BM sinusoidal endo- tors GATA2, AML1, and MYB.15,17 Abnormal upregulation thelial cells, which provides signals for proliferation and of many of these factors is seen in acute leukemias and lym- differentiation19 (Chapter 3). Apoptosis, or programmed phomas (Chapters 26, 27, 42). cell death, can be triggered if the appropriate cytokines The osteoblasts in the osteoblastic niche play an impor- or microenvironment is not available to sustain the HSCs tant role in regulation of HSC number and function via acti- (Figure 4-1). Figure 4-1 also depicts the hemangioblast, vation of external ligand-receptor signal pathways between which is a multipotential precursor capable of producing these two cells 15,19 (Figure 4-2, Table 4-3). HSC quies- both HSCs and vascular endothelium.20 cence is maintained through interaction with osteoblasts, Jagged; Delta Notch Wnt Frizzled Sonic Hedgehog Patched Bone Angiopoitin-1 Tie-2 SDF-1/CXCL12 CXCR4 SCF C-kit Osteoblast Hematopoietic stem cell Figure 4.2 The osteoblastic HSC niche. The interactions between the endosteal osteoblasts and hematopoietic stem cells are depicted. The first three pairs represent ligand (Jagged, Wnt, Sonic Hedgehog)—receptor (Notch, Frizzled, Patched) signaling pathways. The last three pairs represent growth factor (angiopoitin-1, SDF-1, SCF)—receptor (Tie-2, CXCR4, C-kit) interactions. These interactions are thought to determine HSC self-renewal, quiescence, and differentiation. HSC, hematopoietic stem cell; SCF, stem cell factor; C-kit, stem cell factor receptor. Table 4.3 Molecular Regulators of Hematopoietic Stem Cell (HSC) Fate HSC Receptor Proteins Osteoblast Ligands Function Notch proteins Jagged, delta proteins Promote HSC self-renewal; blockade of differentiation Frizzled proteins (Wnt receptors) Wnt proteins Promote HSC self-renewal and expansion Patched proteins (Shh receptors) Sonic hedgehog (Shh) Promote mitosis and initiation of differentiation Tie-2 Angiopoietin-1 Promote HSC quiescence CXCR4 SDF1/CXCL12 Promote survival, proliferation of HSC C-kit SCF Promote survival, proliferation, and differentiation of HSC 48 Chapter 4 molecules in the hematopoietic microenvironment, and downregulate or silence HSC-associated genes, the promis- cytokines that have an inhibitory effect on hematopoiesis cuous gene expression is reduced; genes of the lineage to (see the section “Negative Regulators of Hematopoiesis”). which the cell has committed are upregulated while the Regulation of the cell cycle determines the HSC choice expression of genes associated with alternate lineages is between quiescence and proliferation. As an example, silenced (epigenetic regulation; Chapter 2). Lineage- specific TGF@b, a negative regulator of hematopoiesis, upregulates transcription factors play essential roles in this process the cell-cycle inhibitor p21 (Chapter 2) to help maintain the (see the section “Transcription Factors”). quiescent status of HSCs.21 The HPC compartment is larger than the HSC compart- Recently a number of additional factors important in ment, constituting about 3% of the total nucleated hemato- the regulation of HSC function have been described.15,18 poietic cells. HPCs do not possess self-renewal ability; in These proteins are important regulators of HSC quies- general, their process of cell division is linked to differen- cence, self-renewal, and induction of differentiation within tiation. They are, in essence, transit cells said to be on a the endosteal HSC niche (Figure 4-2, Table 4-3). There is “suicide” maturation pathway (because full maturation and significant interest in understanding how these factors regu- differentiation result in a terminally differentiated cell with a late self-renewal. A more complete understanding of this finite life span). Like the HSC, HPCs are not morphologi- process will allow the development of novel therapeutic cally identifiable but are functionally defined based on the approaches for the treatment of hematologic malignancies. mature progeny that they produce. The first branching point in the developmental pro- PROGENITOR CELLS cess gives rise to the earliest differentiating daughter cells The prevailing model of hematopoiesis is a hierarchy of the HSC, which are slightly more restricted in differen- of precursor cells with the multipotent HSC at the apex. tiation potential. One group of daughter cells—the com- These cells are described as having full self-renewal abili- mon lymphoid progenitor (CLP) cell—is a precursor capable ties and give rise to all subsequent hematopoietic precursor of giving rise to all cells of the lymphoid system.24 The cells (Figure 4-3). Upon commitment to differentiation, the other group—the common myeloid progenitor (CMP) cell—is stem cell enters the next compartment, the hematopoietic composed of daughter cells restricted to producing cells of progenitor cell (HPC) compartment. Hematopoietic cell the myeloid system (the cell lineages of bone marrow).25 differentiation occurs in a step-by-step process, producing These cells are multipotent progenitors, and give rise to multipotent and lineage-restricted HPCs. The HPC com- all the lineage-committed cells of the hematopoietic sys- partment thus includes all precursor cells developmentally tem. The phenotypes for the various levels of HPC are located between HSCs and the morphologically recogniz- described in Table 4-4. able precursor cells. Following additional differentiation steps, the CLP To meet the cell demands imposed on the hematopoi- gives rise to B cell progenitors (BCP) and the T lymphocyte, etic system, some stem cells from the HSC compartment natural killer (NK) cell progenitor (TNKP). The TNKP is the initiate differentiation. Initially the daughter cells arising common precursor for T cells, NK (natural killer) cells, and from undifferentiated stem cells retain the potential to lymphoid dendritic cells.26 generate cells of all hematopoietic lineages (multipotential The CMP gives rise to at least six different lines of cel- progenitor cells [MPPs]; Figure 4-3). As these cells con- lular differentiation, producing committed progenitors tinue to divide, they generate populations of intermediate for neutrophils (NP), monocytes (MP), eosinophils (EoP), lineage-restricted differentiating cells that lack the capacity basophils (BaP), erythrocytes (EP), and megakaryocytes/ to self-renew and are gradually more limited in differentia- platelets (MkP). However, layers of functionally defined tion options,24 gradually become restricted in differentiation cells have been described between the CMP and the com- potential to a single cell lineage (unilineage or committed mitted progenitor cells. Neutrophils and monocytes are progenitor cells). derived from a common bi-potential progenitor cell, the The molecular mechanisms that HSCs utilize to control granulocyte, monocyte progenitor (GMP), which ultimately whether they will self-renew or differentiate upon mitosis gives rise to lineage-restricted progenitor cells (NP and remain unresolved. The transition from an HSC to a com- MP).28 Similarly, EPs and MkPs appeared to be derived from mitted progenitor correlates with the downregulation of a common bi-potential progenitor cell, the megakaryocyte, HSC-associated genes via gene silencing and the upregula- erythroid progenitor (MkEP).21,30 tion or activation of lineage-specific genes.22 Multipotential Within some lineages, subpopulations of committed stem and progenitor cells simultaneously express low lev- progenitor cells were identified. Committed erythroid pro- els of many different genes characteristic of multiple dif- genitors are designated as erythroid burst-forming units ferent, discreet lineages (e.g., transcription factors, cytokine (BFU-E) and erythroid colony forming units (CFU-E) with receptors).23 This so-called promiscuous gene expression is the BFU-E being the more primitive precursor cell anteced- characteristic of most multipotent cells. As developing cells ent to the CFU-E. A similar BFU-Mk/CFU-Mk hierarchy Hematopoiesis 49 B lymphocyte BCP NK cell NKP T lymphocyte TNKP TCP CLP Dendritic cell Monocytes MP HSC MPP Neutrophils GMP NP Basophils BaP CMP CFU-GEMM Mast cells Eosinophils EoP RBCs EMkP EP Platelets MkP Figure 4.3 Prevailing model of the differentiation of blood cells from a multipotential stem cell. The multipotential hematopoietic stem cell (HSC), multipotential progenitor cell (MPP), common myeloid progenitor (CMP), and the colony-forming unit granulocyte, erythrocyte, macrophage, and megakaryocyte (CFU-GEMM) have the potential to differentiate into one of several cell lineages and are therefore multilineage precursor cells. The granulocyte, monocyte progenitor (GMP); megakaryocyte, erythroid progenitor (MEP); and T-lymphocyte, natural killer cell progenitor (TNKP) are bipotential progenitors. The committed (unilineage) progenitors—NP (neutrophil); MP (monocyte); EoP (eosinophil); BP (basophil); EP (erythrocyte); and MkP (megakaryocyte)—differentiate into only one cell lineage. The mature blood cells are found in the peripheral blood. The common lymphoid progenitor cell (CLP) can differentiate into T cell (TCP) or B cell (BCP) progenitors, natural killer progenitors (NKP), or lymphoid dendritic cells. Table 4.4 Phenotype of Hematopoietic Precursor Cells HSC CD34+Thy1+ CD49f+CD133+CD38-Lin-HLADR-Rh123Lo, SCF@R+, TPO@R+ CLP CD34+, Lin-, IL7R+, Thy@1-, SCFR1o CMP CD34+, Lin-, IL7R-, SCFR+ GMP CD34+, SCFR+, FcgRHi, CD33+, CD13+ MkEP CD34-, SCFR+, FcgRLo, CD33-, CD13- HSC, hematopoietic stem cell; Lin-, lineage markers negative; Rh123Lo, negative for supravital dye Rhodamine123; SCF-R, stem cell factor receptor/c-Kit; TPO-R, thrombopoietin receptor/ Mpl; CLP, common lymphoid progenitor; IL7-R, IL-7 receptor; CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MkEP, megakaryocytic erythroid progenitor; FcgR, receptor for Fc component of IgG g chain. 50 Chapter 4 was described for the megakaryocyte lineage.31 Each com- and cytoplasmic morphologic characteristics that can be mitted progenitor cell differentiates into morphologi- used to classify their lineage and stage of development. cally identifiable precursors of its respective lineage (e.g., A unique nomenclature is used to categorize these maturing EP S proerythroblasts, NP S myeloblasts). cells morphologically. Generally, the earliest morphologi- Both multipotential and unipotential HPCs can be cally recognizable cell of each lineage is identified by the assayed by their ability to form colonies of cells in semi- suffix blast with the lineage indicated (e.g., lymphoblast solid media in vitro and were originally described as [lymphocytes], myeloblast [granulocytes], or megakaryo- colony- forming units (CFUs) with the appropriate lineage(s) blast [megakaryocytes/platelets]). Additional differentia- appended. For example, a CFU-GEMM would be a progeni- tion stages are indicated by prefixes or qualifying adjectives tor cell capable of giving rise to a mixture of all myeloid (e.g., proerythroblast, basophilic erythroblast). A complete lineages: granulocytic, erythrocytic, monocytic, and mega- discussion of the stages of maturing cells of each lineage can karyocytic. HPCs are mitotically more active than stem cells be found in the appropriate chapters (Chapter 5, erythro- and are capable of expanding the size of the HPC compart- cytes; Chapter 7, granulocytes; Chapter 8, lymphocytes; ment by proliferation in response to increased needs of the Chapter 9, megakaryocytes/platelets). body. Thus, the HPC compartment consists of a potentially amplifying population of cells as opposed to the stable size of the HSC compartment. Checkpoint 4.1 Hematopoietic stem cells that have initiated a differentiation MATURING CELLS program are sometimes described as undergoing death by After a series of amplifying cell divisions, the committed differentiation. Explain. precursor undergoes a further change when the cell takes on the morphologic characteristics of its lineage. Maturing cells constitute the majority of hematopoietic precursor REVISED HEMATOPOIETIC PRECURSOR CELL MODEL cells; proliferation and amplification boost these cells to New data suggest that lineage fate may be determined earlier greater than 95% of the total precursor cell pool. In general, than thought, perhaps as early as within the HSC compart- the capacity to proliferate is lost before full maturation of ment (Figure 4-4). Most studies concur that the populations these cells is complete. They exhibit recognizable nuclear of cells phenotypically identified as MPP, CMP, and even HSC Lineage-biased Lineage-restricted progenitors Phenotypic Phenotypic CLP CMPs B cells T cells Myeloid Erythroid Granulocytes/ Megakaryocyte Dendritic cell Monocytes
a Figure 4.4 Proposed (revised) models of lineage determination in hematopoiesis. (a) In contrast to the prevailing model in which the multipotential progenitor (MPP) cell gives rise to the CMP and CLP, new evidence suggests hematopoietic stem cells (HSC) may be lineage biased and directly generate a pool of progenitors that are committed to lineages upstream of the CMP stage (phenotypically defined by surface markers). (b) Additionally, erythroid cells can be derived from a more immature CMP within the MPP population, leaving a proportion of myeloid cells derived from progenitors that produce neither erythrocytes nor lymphoid cells. (c) Megakaryocytes may bypass the CMP and be directly derived from HSCs. HSC, hematopoietic stem cell; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; MkP, megakaryocytic progenitor; EP, erythroid progenitor; MP, monocytic progenitor; NP, neutrophil progenitor. Hematopoiesis 51 HSC CLP CMP NP MP MkP EP B cells T cells Neutrophils Monocytes Myeloid Megakaryocyte Erythroid Dendritic cell b HSC CLP CMP NP MP MkP EP B cells T cells Neutrophils Monocytes Myeloid Erythroid Megakaryocyte c Dendritic cell Figure 4.4 Continued. HSC by cell surface markers, are highly heterogeneous popu- Under normal steady-state physiological conditions, lations of cells, and include subpopulations with unilineage the majority of hematopoietic precursor cells (HSC and myeloid, erythroid, and megakaryocytic potential. Addition- HPC) are retained in the bone marrow. A small population ally, several studies reported self-renewing, lineage-restricted of HSC and HPC, however, can be found circulating in the progenitors, generated directly from HSCs32,33,34(Figure 4-4a). peripheral blood. The number of circulating HSCs/HPCs Other groups have reported an “earlier” branch for erythroid can be increased by the infusion of various cytokines, differentiation35 (Figure 4-4b), or megakaryocytic differentia- enabling the collection of “mobilized” peripheral blood tion36 and that these progenitors may even be derived from HSCs/HPCs for transplantation purposes rather than from HSCs without progressing through conventional MPPs and a direct bone marrow harvest (Chapter 29). CMPs (Figure 4-4c). These new findings are challenging the currently accepted model of hematopoietic development, Checkpoint 4.2 and which subsets of precursor cells have self-renewal capa- Explain the difference in the nomenclature used to label pro- bilities and which do not. Undoubtedly this is an area of genitor cells from that used to label maturing cells within the hematology that is unsettled, and in which new findings are hematopoietic hierarchy of cells. constantly challenging what had been an accepted “theory.” 52 Chapter 4 Cytokines and the Control of The first identified growth factors (GFs) were described Hematopoiesis as colony-stimulating factors (CSFs) because they supported the growth of hematopoietic colonies in in vitro cultures. The regulation of HSC and HPC differentiation and expansion Subsequently, as additional cytokines were discovered, the is critical because it determines the concentration of the vari- nomenclature was changed to interleukins. When a new ous lineages in the marrow and eventually in the peripheral cytokine is discovered, the initial description is based on blood. Specific glycoproteins called hematopoietic growth factors, its biologic properties; when the amino acid sequence has or cytokines (Figure 4-5), govern hematopoietic precursor been defined it is assigned an interleukin number. The sys- cell survival, self-renewal, proliferation, and differentiation. tem has some exceptions and inconsistencies, however. For Growth factor control of hematopoiesis is an extraordinarily historic reasons, some cytokines retain their original names complex and highly efficient intercellular molecular commu- (e.g., EPO, TPO). The initial research into the biologic activi- nication system that allows coordinated increases in the pro- ties of other cytokines focused on activities outside hema- duction and functional activity of appropriate hematopoietic topoietic regulation, and their original names have been cell types without expansion of irrelevant ones. retained (e.g., kit-ligand [KL], also called stem cell factor IL21, 22, 24,25,26 B lymphocyte BCP SCF, S DF21 IL212, IL215 NK cell , FL, IL27 NKP IL27, FL, SCF IL22, 24, 210, 212 T lymphocyte TNKP TCP IL21, 24, 27, T 7 1 CLP PO, SCF, FL Dendritic cell FL, IL 2 F2 F, SD FL, IL24 GM-CSF SC Monocytes TPO, FL IL23, Gm-CSF, M-CSF GM MP -CSF, M-CSF SCF, IL21 HSC MPP IL23, IL26 3 Neutrophils CF, IL2 IL23, GM-CSF, G-CSF GM-CSF, G-CSF IL211 GMP -C SF FL, S SCF, NP I GM L23, IL24 SCF, IL23 Basophils BaP SCF, IL23 Mast cells CMP GM-CSF, IL25 Eosinophils EoP RBCs EPO EPO IL23, GM-CSF EMkP SC EP F G , I M L2 - I C 3 L S 2 F 6, IL Platelets 21 TPO, IL26, 211 1 MkP Figure 4.5 The multipotential hematopoietic stem cell (HSC) gives rise to erythrocytes, platelets, monocytes, macrophages, granulocytes, and lymphoid cells. Under stimulation from selective growth factors, stem cell factor (SCF), Flt ligand (FL), and interleukins (IL), the HSC in quiescence (G0) enters the cell cycle (G1) and initiates differentiation, first becoming a multipotential progenitor cell (MPP). The MPP differentiates to the common myeloid progenitor cell (CMP) and, subsequently, to the colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte (CFU-GEMM). The CFU-GEMM then differentiates into granulocytes, erythrocytes, monocytes, and megakaryocytes under the influence of specific growth factors, erythropoietin (EPO), thrombopoietin (TPO), colony-stimulating factor-2 (CSF-2, Granulocyte- Monocyte-CSF), colony-stimulating factor-3 (CSF-3, Granulocyte-CSF), colony-stimulating factor-1 (CSF-1, Monocyte-CSF), and interleukin-5 (IL-5). Different combinations of hematopoietic cytokines regulate the differentiation of HSCs into the common lymphoid progenitor cell (CLP) and subsequently into B and T lymphocytes, natural killer (NK) cells, and lymphoid dendritic cells. SDF-1, stromal cell derived factor-1. 5 2 3, IL F, IL2 M-C S SCF, G 3 F, I L2 SC FCS GM - FL , S CF , T PO GM-C SF 1, 23, IL2 26, 211 Hematopoiesis 53 [SCF]; Flt3 ligand [FL]). At least 37 interleukins have been restricted to target cells of the appropriate responding cell isolated and characterized to date. lineage(s). Because many precursor cells respond to more than one cytokine, they express receptors for multiple GFs. GROWTH FACTOR FUNCTIONS Some GFs influence hematopoiesis directly by binding to The growth of hematopoietic precursor cells requires the receptors on precursor cells and inducing the appropriate continuous presence of GFs. If the relevant GFs are with- response (survival, proliferation, differentiation). Other GFs drawn, the cells die within hours by the process of apop- influence the process indirectly by binding to receptors on tosis (programmed cell death; Chapter 2). Thus, the first accessory cells, which in turn respond by releasing other effect of some GFs is to promote cell survival by suppress- direct-acting cytokines. Some GFs trigger cell division and ing apoptosis. Second, GFs promote proliferation. Hema- differentiation, and others support survival without induc- topoietic cells are intrinsically incapable of unstimulated ing proliferation. cell division. All cell division or proliferation depends on Hematopoietic regulatory cytokines interact in a highly stimulation by appropriate regulatory cytokines. Addition- ordered, interdependent network, creating a complex cell- ally, GFs control and regulate the process of differentiation, to-cell communication system. Individual GFs by them- which ultimately produces the mature functional cells from selves are poor stimulators of colony growth; the control of their multipotential progenitor cell precursors. Finally, GFs hematopoiesis generally involves the interplay of at least that induce proliferation of precursor cells sometimes have several GFs. Some GFs act synergistically with other cyto- the capacity to enhance the functional activity of the termi- kines (synergism occurs when the net effect of two or more nally differentiated progeny of these precursor cells. events is greater than the sum of the individual effects). CHARACTERISTICS OF GROWTH FACTORS Many cytokines have overlapping activities (redundancy). Although many different cytokines have been identified The cytokine network often exhibits signal amplifica- as hematopoietic growth factors, some share a number of tion circuits including autocrine, paracrine, and juxtacrine characteristics (Table 4-5). GFs are produced by multiple mechanisms of stimulation/amplification (Figure 4-6). cell types, including monocytes, macrophages, activated T Autocrine signals are produced by and act on the same lymphocytes, fibroblasts, endothelial cells, osteoblasts, and cell. Paracrine signals are produced by one cell and act on adipocytes (bone marrow stromal cells). Except for eryth- an adjacent cell, typically over short distances. Juxtacrine ropoietin (EPO), most GFs are produced by stromal cells signals represent a specialized type of paracrine signaling in in the hematopoietic microenvironment. EPO production is which the cytokine is not secreted by the cell that produced atypical of most lymphohematopoietic GFs in that EPO is it but remains membrane bound, necessitating direct pro- produced mainly in the kidney, is released into the periph- ducer cell–target cell contact to achieve the desired effect. eral blood, and is carried to the bone marrow where it regu- In contrast, endocrine signals (classic hormones) typically lates RBC production. As such, it is the only true hormone act over fairly long distances. The majority of cytokines (endocrine cytokine); the majority of the other cytokines regulating hematopoiesis exert their effects via paracrine exert their effects on cells in the local environment where or juxtacrine interactions. they are produced. Often, a single stromal cell source can GF requirements change during the differentiation produce multiple cytokines. process so that the cytokines/GFs needed by HSCs and Most GFs are not lineage specific; each GF has multiple early multipotential HPCs differ from the GF requirements functions, and most act on more than one cell type (i.e., they of the later, lineage-restricted progenitors and the matur- are pleiotrophic) (Table 4-5 and Table 4-6). Cytokines must be ing precursor cells. These are described as early-acting bound to surface receptors on their target cells to express (multilineage) GFs and later-acting (lineage-restricted) their activity. They interact with membrane receptors GFs, respectively. GFs and their receptors share a number of Table 4.5 Characteristics of Hematopoietic Growth Factors (GFs) Production Function Mechanism of Action Stromal cells in the hematopoietic microenvironment Inhibit apoptosis; promote proliferation; regulate Interact with membrane receptors restricted to (e.g., endotheial cells, fibrobasts, osteoblasts) differentiation cells of appropriate lineage Accessory cells (e.g., monocytes, macrophages, Pleiotrophy: Individual GFs have multiple biologic GF signaling alters the expression, activity, or T-lymphocytes) activities localization of transcription factors Renal interstitial fibroblasts (produce EPO) Redundancy: Many different GFs have similar or identical activities Synergy: Individual GFs are poor stimulators of colony growth; control of hematopoiesis involves the interplay of several GFs 54 Chapter 4 Table 4.6 Hematopoietic Growth Factors (GFs), Their Sources, and Targets/Actions GF Chromosome Source Major Target Cells/Actions EPO 7 Kidney (liver) Erythroid precursor cells CSF-1 (M-CSF) 1 Monocytes, macrophages, BM stromal cells Monocytes, macrophages, osteoclasts CSF-2 (GM-CSF) 5 T lymphocytes, BM stromal cells, macrophages Granulocytes, monocytes, eosinophils, erythroid precursor cells, megakaryocytes, HPCs, DCs CSF-3 (G-CSF) 17 Monocytes, macrophages, BM stromal cells Granulocytes, early HPC IL-1 2 Monocytes, macrophages, dendritic cells Monocytes, ECs, fibroblasts, lymphocytes, PMNs, early HPC IL-2 4 Activated TH1 lymphocytes Proliferation and activation of T, B, and NK lymphocytes IL-3 5 Activated T lymphocytes, mast cells Myeloid HPC, mast cells IL-4 5 Activated TH2 lymphocytes Stimulate TH2 suppress TH1 B lymphocytes, mast cells, basophils, fibroblasts IL-5 5 Activated TH2 lymphocytes, mast cells Eosinophils, B cells, cytotoxic T lymphocytes IL-6 7 Macrophages, TH2 lymphocytes, B cells Early HPCs, T and B lymphocytes; megakaryocytes; myeloma cells IL-7 8 Stromal cells (BM and thymus) Pre-T, pre-B lymphocytes, NK cells IL-8 4 Monocytes, macrophages, ECs Chemotaxis of granulocytes (chemokine) IL-9 5 Activated TH2 cells T and B cells, early erythroid precursor cells, mast cells IL-10 1 TH2 cells, monocytes, macrophages, activated B lymphocytes, mast cells, TH2 inhibit TH1 B cells lymphocytes IL-11 19 BM stromal cells B lymphocytes, megakaryocytes, early HPC IL-12 3,5 Monocytes, macrophages, B lymphocytes, TH1 cells, NK cells T lymphocytes IL-13 5 TH2 lymphocytes, basophils Isotype switching of B lymphocytes; inhibit c ytotoxic and inflammatory functions of monocytes and macrophages IL-14 16 T lymphocytes Activated B lymphocytes IL-15 4 Monocytes, macrophages, ECs, fibroblasts T lymphocytes (CTLs), NK cells (LAK), costimulator for B lymphocytes IL-16 15 T lymphocytes, eosinophils, epithelial cells Chemotactic for CD4+ T lymphocytes IL-17 2 Activated TH17 lymphocytes Induces cytokine production by stromal cells IL-18 7 Macrophages, keratinocytes Induces IFN production by TH1, NK cells SCF/KL 12 Fibroblasts, ECs, stromal cells Stem cells, early HPCs, basophils and mast cells, melanocytes, germ cells FL 19 Stromal cells, monocytes, macrophages, Stem cells, HPCs, B & T precursor lymphocytes, T lymphocytes DC precursors TPO 3 Stromal cells, hepatocytes, kidney Megakaryocytes, HSCs NK, natural killer cells; BM, bone marrow; HPC, hematopoietic progenitor cells; DCs, dendritic cells; ECs, endothelial cells; PMNs, neutrophils; activated T cells, T cells activated by antigens, mitogens, or cytokines;
CTLs, cytotoxic T lymphocytes; LAK, lymphokine activated killer cells; T cells, T lymphocytes; B cells, B lymphocytes structural features, perhaps explaining some of the observed several cell lineages, additional factors are necessary in functional redundancies. Most GFs have been cloned and many instances for the production of mature cells in these characterized, and recombinant proteins are available; cer- lineages (Figure 4-4). tain of these GFs have been shown to have important clini- Stem Cell Factor (SCF) and Flt3 Ligand (FL) Stem cell factor cal applications. (SCF) (also known as kit ligand [KL] or mast cell growth Early-Acting (Multilineage) Growth Factors Several factor [MCGF]) suppresses apoptosis of HSCs and pro- GFs have direct effects on multipotential precursor cells motes the proliferation and differentiation of stem cells, and thus are capable of inducing cell production within multilineage progenitor cells, and some committed pro- several lineages. Early-acting cytokines primarily affect genitor cells (CFU-GEMM, GMP, MkP,BFU-E). SCF also proliferation of these noncommitted progenitor cells. promotes the survival, proliferation, and differentiation These include SCF, FL, IL-3, CSF-2 (GM-CSF), IL-6, and of mast cell precursors and has functional activity outside IL-11. Although these factors can initiate proliferation in the hematopoietic system (melanocyte development and Hematopoiesis 55 Endocrine signaling Endocrine cell Target cell Bloodstream Autocrine Paracrine Juxtacrine signaling signaling signaling Figure 4.6 Mechanisms of cytokine regulation. Autocrine signals are produced by and act on the same cell. Paracrine signals are produced by one cell and act on an adjacent cell, typically over short distances. A juxtacrine signal is a specialized type of paracrine signaling in which the cytokine is not secreted by the producing cell but remains membrane bound, necessitating direct cell–cell contact to achieve the desired effect. In contrast, endocrine signals (classic hormones) typically act over fairly long distances. gametogenesis). Flt3 ligand (FL) increases recruitment of vivo. IL-6 also stimulates the production of hepcidin, a reg- primitive HSCs/HPCs into the cell cycle and inhibits apop- ulator of iron absorption (Chapter 12). tosis.37 In contrast to SCF, FL has little effect on unilineage Later-Acting (Lineage-Restricted) Growth Factors The EP, MCP, or EoP but is a potent stimulator of granulocytic/ growth factors included in this group tend to have a nar- monocytic, B lymphocytic, and dendritic cell proliferation rower spectrum of influence and function primarily to and differentiation. FL and SCF have similar protein struc- induce maturation along a specific lineage. Most are not tures and share some common characteristics. Both cyto- lineage specific, however, but instead demonstrate a pre- kines can be found as either membrane-bound or soluble dominant effect on the committed progenitor cell of a sin- forms, although the membrane-bound form appears more gle lineage, inducing differentiation of these more mature important, physiologically, in stimulating hematopoiesis; cells. These growth factors include the granulocyte colony- thus, both operate primarily through juxtacrine interac- stimulating factor CSF3 (G-CSF, granulocytes), the mono- tions.38 Neither cytokine has independent proliferation- cyte colony-stimulating factor CSF1 (M-CSF, monocytes), inducing activity, but both act synergistically with IL-3, erythropoietin (EPO) (erythrocytes), thrombopoietin (TPO) CSF2, CSF3, and other cytokines to promote early progeni- (megakaryocytes and platelets), IL-5 (eosinophils), and the tor cell proliferation. interleukins important in lymphopoiesis (IL-2, -4, -7, -10, Interleukin 3 and GM-CSF (CSF2) Interleukin 3 (IL-3) was -12, -13, -14, -15). one of the earliest recognized multipotential growth fac- EPO is the only cytokine to function as a true hor- tors that directly affects multilineage progenitor cells and mone because it is produced primarily in the kidneys and early committed progenitors such as BFU-E. IL-3 also has travels via the circulation to the bone marrow to influ- indirect actions and can induce the expression of other ence erythrocyte production. It is expressed primarily by cytokines. GM-CSF (CSF2) is a multipotential GF that stim- hepatocytes in embryonic life and by cells of the kidney ulates clonal growth of all lineages except basophils. GM- (and to a lesser extent, the liver) in adult life. Its release CSF also activates the functional activity of most mature is regulated by the body’s oxygen needs and is induced phagocytes including neutrophils, macrophages, and by hypoxia (Chapter 5). EPO stimulates survival, growth, eosinophils. and differentiation of erythroid progenitor cells (with its major effect on EP). It also stimulates proliferation and Interleukin 6 and Interleukin 11 Interleukin 6 (IL-6) and ribonucleic acid (RNA) and protein synthesis in erythroid- interleukin 11 (IL-11) are pleiotropic cytokines with overlap- maturing cells. Reticulocytes and mature erythrocytes do ping growth stimulatory effects on myeloid and lymphoid not have receptors for EPO and thus are not influenced by cells as well as on primitive multilineage cells.39,40 Each this cytokine. cytokine rarely acts alone but functions synergistically with CSF3, CSF1, and IL-5 stimulate the proliferation of IL-3, SCF, and other cytokines in supporting hematopoiesis. granulocyte, monocyte/macrophage, and eosinophil pro- Both cytokines have significant effects on megakaryocyto- genitor cells, respectively. All three also influence the func- poiesis and platelet production.41 Both mediate the acute tion of mature cells of their respective lineages, increasing phase response of hepatocytes and are major pyrogens in migration, phagocytosis, and metabolic activities and 56 Chapter 4 augmenting prolongation of their life spans. CSF1 also spleen (Chapter 8). Multiple GFs play a role in T and B regulates the production of osteoclasts, and IL-5 stimulates lymphocyte growth and development, most of which act lymphocyte development. synergistically (Figure 4-5). TPO, also known as mpl-ligand, is the major physiologic regulator of megakaryocyte proliferation and platelet pro- Checkpoint 4.3 duction. In vitro, TPO primes mature platelets to respond Cytokine control of hematopoiesis is said to be characterized by to aggregation-inducing stimuli and increases the platelet redundancy and pleiotrophy. What does this mean? release reaction.42 TPO also synergizes with a variety of other GFs (SCF, IL-3, FL) to inhibit apoptosis and promote Negative Regulators of Hematopoiesis In addition to the maintenance of HSCs/HPCs. cytokines that function as positive regulators of hema- Indirect-Acting Growth Factors Some cytokines that reg- topoiesis, a second group of polypeptides that inhibit ulate hematopoiesis do so indirectly by inducing acces- cellular proliferation exists (Table 4-7). Either decreas- sory cells to release direct-acting factors. An example ing production of stimulating factors or increasing fac- is IL-1, which has no colony-stimulating activity itself. tors that inhibit cell growth can limit the proliferation of However, when administered in vivo, it induces neu- hematopoietic precursor cells. A homeostatic network of trophilic leukocytosis by promoting the release of other counteracting growth inhibitors is secreted in response direct-acting cytokines from accessory cells. to growth promoting factors, which normally limit cell proliferation after growth stimuli. Some of these negative Lineage-Specific Cytokine Regulation regulators of hematopoiesis (e.g., transforming growth Erythropoiesis In the erythroid lineage, EPs give rise factor b [TGF@b]) may contribute to the quiescent state to two distinct types of erythroid colonies in culture of stem cells and early progenitor cells.26 Several nega- (Chapter 5). A primitive progenitor cell, the BFU-E, is rela- tive regulators have been shown to upregulate cell-cycle tively insensitive to EPO and forms large colonies after 14 inhibitors such as p16 and p21 (Chapter 2). Others may days in the form of bursts. Production of BFU-E colonies oppose the actions of positive regulators that act on these is supported by IL-3 or CSF2. CFU-E colonies depend pri- same cells. Whether or not precursor cells synthesize marily on EPO. The CFU-E are the descendants of BFU-E DNA and proliferate depends on a balance between these and subsequently give rise to the first recognizable eryth- opposing influences. rocyte precursor, the pronormoblast. Other cytokines The interferons and TGF@b suppress hematopoietic reported to influence production of red cells include IL-9, progenitor cells by inhibiting proliferation or inducing IL-11, and SCF. However, EPO is the pivotal factor that programmed cell death. Tumor necrosis factor a(TNF@a) functions to prevent apoptosis and induce proliferation/ directly suppresses colony growth of CMP, GMP, and BFU-E, differentiation of committed erythroid progenitor cells and and E-prostaglandins (PGEs) suppress granulopoiesis their progeny. and monopoiesis by inhibiting GMP, NP, and MP. Acidic Granulopoiesis and Monopoiesis Granulocytes and mono- ferritins and lactoferrin are products of mature neutro- cytes are derived from a common bipotential progenitor phils that inhibit hematopoiesis via feedback regulation. cell, the GMP, derived from the CMP. Acting synergisti- Di-hydroxyvitamin D3 (Di-OH Vitamin D3), classically cally with CSF2 and/or IL-3, specific GFs for granulo- associated with the stimulation of bone formation, also cytes and monocytes support the differentiation pathway functions to inhibit myelopoiesis. Additionally, cellu- of each lineage. CSF1 supports monocyte differentiation, lar components of the immune system, including T cells and CSF3 induces neutrophilic granulocyte differentiation. and NK cells, can function as negative regulators of Eosinophils and basophils also are derived from the CMP hematopoiesis. under the influence of growth factors IL-5 and IL-3/IL-4, respectively. Table 4.7 Negative Regulators of Hematopoiesis Megakaryocytopoiesis/Thrombopoiesis Platelets are derived from megakaryocytes, which are ultimately progeny of the Neutrophil Cytokines Products Cells Other MkEP. MkP are induced to proliferate and differentiate into megakaryocytes by several cytokines. However, the cyto- Interferons Acidic isoferritins T cells kines that induce the greatest increase in platelet production TGF@b Lactoferrin NK cells Di-OH PGEs vitamin are IL-11 and TPO. D3 Lymphopoiesis The growth and development of lymphoid TNF@a cells from the CLP occurs in multiple anatomic locations SCI (MIP@1a) including the bone marrow, thymus, lymph nodes, and PGEs, E-prostaglandins. Hematopoiesis 57 Stem cell inhibitor (SCI), also known as macrophage L L inflammatory protein-1a (MIP@1a), is believed to be a primary negative regulator of stem cell proliferation.43 It is a local- L L acting juxtacrine cytokine present in the stromal microenvi- Membrane ronment, which functions to maintain quiescent stem cells in the G0 phase of the cell cycle. Transmembrane receptor Dimerization A Cytokine Receptors, Signaling Cellular Pathways, and Transcription Factors B response Cytokines must bind to surface receptors on their target cells to express their activity. They interact with membrane C receptors restricted to cells of the appropriate lineage. Cells also need a mechanism to transfer signals from extracellular stimuli (cytokines) into appropriate intracellular responses. (Gene activation, Silencing) Binding of a cytokine (ligand) to its specific receptor trans- Nucleus duces an intracellular signal through which the particu- lar survival, proliferation, or differentiation responses are initiated. The intracellular portion of the receptor binds to associated intracellular molecules that activate signaling Figure 4.7 A model for the transfer of signals from extracellular pathways. These signaling molecules translocate to the stimuli (cytokines) into appropriate intracellular responses. The binding of a cytokine or ligand (L) to its cognate receptor generally nucleus, recruit appropriate transcription factors, and acti- induces receptor dimerization, the activation of a cascade of vate or silence gene transcription (Figure 4-7). Ultimately, downstream-signaling molecules (A-, B-, C-signal transduction changes in protein synthesis lead to alterations in cell prolif- pathways) that converge on the nucleus to induce or repress eration or other modifications of cellular response induced cytokine-specific genes. The result is an alteration of transcription, by the cytokine involved. RNA processing, translation, or the cellular metabolic machinery. CYTOKINE RECEPTORS Many receptors for hematopoietic cytokines have been configured as a heterodimer or homodimer. The receptors characterized and can be grouped according to certain for many GF receptors in this large group share peptide structural characteristics.44 Some cytokine receptors, includ- subunits with other receptors.17,45 The three major sub- ing the receptors for EPO, CSF3, and TPO, are homodimers groups are: (i.e., they consist of two identical subunits). Other receptors 1. IL-3, IL-5, and CSF2 receptors: have unique cytokine- are heterodimers or heterotrimers, consisting of different specific a chains but share a common signal-transduc- polypeptide subunits (the receptors for most of the other ing b chain (the bc family). hematopoietic cytokines). 2. IL-6 and IL-11: similarly have cytokine-specific a chains Receptors With Intrinsic Tyrosine Kinase Domains These and share a common signal-transducing b chain called receptors, called receptor tyrosine kinases (RTKs), are trans- GP130. GP130 is also a subunit of the receptors for sev- membrane proteins with cytoplasmic regions that con- eral other cytokines, including LIF (leukemia inhibitory tain a tyrosine kinase catalytic site or domain. When factor) and OSM (oncostatin M). GF binds to the receptor, the receptor chains dimerize, 3. The receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21: enhancing the catalytic activity of the kinase domain have unique, cytokine-specific a chains and share a and activating intracellular signaling pathways directly. common signaling g chain. IL-2 and IL-15 are
actually Included in this group are the receptors for CSF1, SCF, trimeric structures and share a common b subunit as and FL. well. Inherited abnormalities of the shared g chain gene are responsible for the X-linked form of severe com- Hematopoietic Growth Factor Receptor Superfamily The bined immunodeficiency (SCID) (Chapter 22). receptors for the majority of hematopoietic GFs do not pos- sess intrinsic kinase activity. Cytokine binding and receptor The functional redundancy seen in the cytokine regula- activation induce the docking of cytoplasmic molecules, tion of hematopoiesis (i.e., the fact that multiple GFs often which do have kinase activity, leading to phosphorylation have overlapping activities) may be partly explained by the of cellular substrates. All of these receptors are multichain sharing of common receptor signaling subunits. For exam- transmembrane proteins that promote signal transduction ple, IL-3 and GM-CSF have very similar spectra of biologic (i.e., phosphorylation of target cellular proteins) when activities and share a common b subunit. 58 Chapter 4 Receptor Functional Domains Most receptors have dis- crete functional domains in the cytoplasmic region of one Checkpoint 4.4 or more of the receptor chains. Thus, mutations disrupt- Individuals with congenital defects of the g chain of the IL-2 ing a discrete domain of the receptor can disrupt part, receptor suffer from profound defects of lymphopoiesis far greater than individuals with congenital defects of the a chain but not all, of the functions of that receptor. Kostmann’s of the IL-2 receptor. Why? syndrome (congenital agranulocytosis) is a rare disorder characterized by a profound absolute neutropenia with a maturation arrest of precursor cells at the promyelocyte/ Receptors that do not have intrinsic kinase activity myelocyte stage. Erythropoiesis and thrombopoiesis are recruit cytoplasmic proteins to their intracellular “tails” normal. In some patients, molecular studies have revealed and induce the association and assembly of multisubunit a mutation of the CSF3 receptor that disrupts a terminal protein complexes that generate the enzymatic (phosphory- maturation-inducing domain but leaves intact a subtermi- lation) activity. The recruited proteins are termed protein nal proliferation-inducing domain.46 These patients sustain tyrosine kinases (PTKs). Most hematopoietic receptors sig- proliferation of neutrophilic progenitor and early matur- nal through the Janus family of PTKs, called JAKs. Once ing cells, but fail to complete final maturation of cells in activated, the JAK kinases recruit molecules that relay the this lineage. Similarly, some individuals with previously signal, often including members of the STAT family of unexplained primary erythrocytosis (i.e., not secondary transcription factors (Signal Transducers and Activators to smoking, high altitude, or increased EPO levels) have a of Transcription); this pathway is referred to as the JAK- mutation affecting the EPO-receptor (EPO-R).47 The EPO-R STAT signaling pathway. Four different JAK kinases and also has been shown to have two separate domains in the about 10 different STAT proteins have been identified. Dif- cytoplasmic region of the receptor: The domain closest to ferent JAK and STAT proteins are involved in activation of the membrane constitutes a positive control domain pro- the various hematopoietic lineages. Once STAT proteins are moting proliferation, and the terminal, negative control phosphorylated by activated JAK kinases, they dimerize, domain slows down the intracellular signaling from the translocate to the nucleus, bind to cytokine-specific DNA receptor. In some patients with familial erythrocytosis, a sequences, and activate (or inhibit) specific gene expres- sion 47,48 mutation results in the generation of a truncated receptor (Figure 4-8). Abnormalities of the erythrocyte that lacks the terminal negative control domain, thus result- JAK-STAT signaling pathway are the major cause of poly- ing in enhanced responsiveness of target cells (BFU-E and cythemia vera (Chapter 24). EP) to the growth stimulatory effects of EPO and a (benign) TRANSCRIPTION FACTORS erythrocytosis. A cell’s phenotype and function are determined by the genes expressed in that cell. Thus, different gene expres- SIGNALING PATHWAYS sion patterns regulate hematopoietic differentiation. The Cells use a variety of “signal transduction pathways” to growth factors that maintain hematopoiesis are not thought transfer signals from the cytokine receptor into an appro- to be “instructive” for the pathway of differentiation but to priate response. These are initiated by a ligand (cytokine) be “permissive” for cell viability and proliferation.49 The binding to its specific receptor followed by the activation components that actually establish the patterns of gene of “downstream signaling molecules,” which ultimately expression associated with lineage differentiation are the converge on the nucleus to modulate transcription, RNA nuclear transcription factors (TFs). Cell-fate decisions are processing, the protein synthetic machinery (translation), controlled by the integrated effects of signaling pathways the cellular metabolic machinery, or cytoskeletal-dependent initiated by external cytokines and internal transcription functions47 (Chapter 2; Figure 4-7). The signaling cascades factors.24 that are activated can involve the formation of multiprotein TFs are DNA binding proteins that interact with the complexes, proteolytic cascades, and/or phosphorylation/ regulatory promoter regions of their target genes. The dephosphorylation reactions. effect of a particular TF can be either gene expression or Protein phosphorylation is often an important part gene suppression, depending on the additional molecules of the signaling response from cell-surface receptors (coactivators or corepressors) recruited to the gene pro- involved in hematopoiesis. Receptors that contain intrin- moter region upon TF binding. Different TFs are restricted sic kinase (or phosphatase) activity are identified by the in their expression to particular lineages and to particular target amino acid to be phosphorylated or dephosphory- differentiation stages within one or more lineages. TFs lated as receptor tyrosine kinases (RTKs), receptor serine associated with the activation of a particular lineage-spe- kinases (RSKs), or receptor protein tyrosine phosphatases cific differentiation program often simultaneously inhibit (PTPs).47 Ligands activate these receptors by promoting alternate lineage-specific transcription factors.50 Interest- receptor oligomerization and activation of their cytoplas- ingly, more than half of the hematopoietic transcription mic kinase domains. factors identified have been shown to be dysregulated in Hematopoiesis 59 EPO Table 4.8 Transcription Factors in Hematopoietic Lineage Differentiation EPO Hematopoietic EPO-R Lineage Transcription Factors Erythroid/ GATA1, FOG1, Gfi-1b, and Fli1 megakaryocytic Myeloid PU.1, C/EBPa, C/EBPe, Gfil, Egr1 and RARa Lymphoid PU.1, Ikaros, E2A, EBF, PAX5, Notch1 and GATA3 P JAK2 JAK2 P P STAT5 P P CLINICAL USE OF HEMATOPOIETIC GROWTH STAT5 P P FACTORS JA The cloning and characterization of genes encoding the K2 STAT5 STAT5 hematopoietic GFs have allowed scientists to produce these cytokines in large quantity using recombinant DNA tech- Nucleus nology. As a result, GFs can be used in therapeutic regimens for hematopoietic disorders (Table 4-9). Some cytokines approved by the Food and Drug Administration for clini- cal use include CSF3 and CSF2 (used to accelerate recovery from granulocytopenia), EPO (for treatment of anemia of Figure 4.8 Cytokine receptor-JAK-STAT model of signal various etiologies), IL-11 (for treatment of thrombocytope- transduction. Cytokine (e.g., EPO) interaction with its specific nia), the interferons (IFNa, IFNb, and IFNg used to treat receptor (EPO-R) leads to receptor dimerization and activation of a number of malignant and nonmalignant disorders), and JAK kinases associated with the activated receptor. Activated JAK IL-2 (for treatment of metastatic renal cell cancer and mela- kinases mediate autophosphorylation as well as phosphorylation of the receptor, which then serves as a docking site for signal noma). In vitro studies show that cytokines used in combi- transducers and activators of transcription (STATs). These STATs nation often show synergy in terms of their biologic effects. are phosphorylated, dissociate from the receptor, dimerize, and Consequently, the use of combinations of growth factors is translocate to the nucleus where they activate gene transcription. P, being evaluated clinically, often with dramatic results.52 phosphorylated protein. hematologic malignancies (translocations, point muta- tions of TF genes)51 (Chapters 23–28). Thus, the impaired Hematopoietic differentiation seen in leukemia is likely due to abnor- malities of critical, discrete pathways of transcriptional Microenvironment control. Hematopoiesis is normally confined to certain organs and Different TFs are functional at different points in hema- tissues (Chapter 3). The proliferation and maturation of topoietic differentiation.51 Four TFs (or proteins that inter- hematopoietic precursor cells take place within a microen- act with them) have been identified as important in early vironment that provides the appropriate milieu for these embryonic HSCs, and all have been associated with vari- events.53,54 Patients undergoing bone marrow transplants ous hematopoietic malignancies. They include SCL/TAL1, receive donor cells by intravenous infusion; the cells “home” AML1/Runx1, KMT2A, and LMO2. Other TFs involved in to and initiate significant hematopoiesis only in the recipi- either stem cell self-renewal or differentiation include HOX ent’s bone marrow. No biologically significant hematopoietic A9, TEL, Bmi1, and Gfi1. activity occurs in nonhematopoietic organs. For successful Although certain TFs are associated with lineage-spe- engraftment, HSCs require an appropriate microenviron- cific differentiation pathways, many are also expressed, ment, which presumably has specific properties that make it a usually at much lower levels, in hematopoietic progenitor unique site for stem cell renewal, growth, and differentiation. cells that are not yet committed to a specific differentiation The term hematopoietic microenvironment (HM) refers pathway. This simultaneous expression of TFs for different to the localized environment in the hematopoietic organs that lineages is thought to explain the progenitor cell’s poten- is crucial for the development of hematopoietic cells and tial for multilineage development.24 Once a differentiation maintains the hematopoietic system throughout the individu- decision has been made (commitment), upregulation of TFs al’s lifetime. The HM includes cellular elements and extracel- for one lineage and downregulation or antagonism of the lular components including matrix proteins and regulatory others occur. TFs that specify the various hematopoietic lin- cytokines (Table 4-10, Figure 4-9). The HM provides homing eages are listed in Table 4-8.51 and adhesive interactions important for the colocalization of 60 Chapter 4 Table 4.9 Clinical Applications of Hematopoietic Growth Factors Growth Factor Clinical Applications EPO Stimulation of erythropoiesis in a variety of anemias CSF3 and CSF2 Recovery from treatment-induced myelosuppression IL-3, CSF2, EPO Therapy of myelodysplastic syndromes IL-2, IFN@a, IFN@b; TGF@b antagonists Treatment for various malignancies IL-3, CSF3, CSF2, FL Priming of bone marrow for donation IL-1, IL-3, IL-6, IL-11, CSF3, LIF Stimulation of malignant cells to differentiate (variable results) IL-1, IL-6 Enhancement of the acute phase response IL-2, IL-15 (and other lymphocyte-stimulating growth factors) Enhancement of the immune system G-CSF, CSF2, EPO, IL-11 Stimulation of marrow recovery in BM transplantation IL-3, CSF3, CSF2 Treatment in bone marrow failure Table 4.10 Hematopoietic Microenvironment Cellular (stroma) Extracellular Components Function Components Function Adipocytes, endothelial Expression of homing receptors Soluble factors (cytokines and Regulation of hematopoietic stem/ cells, fibroblasts, CAR Production of soluble growth and growth factors) progenitor cell differentiation and cells, osteoblasts, T cells, differentiation factors Extracellular matrix (ECM) expansion macrophages Production of integral membrane proteins Collagen Structural support that function as juxtacrine regulators (SCF, Glycosaminoglycans (heparan-, Cell-to-cell interactions; localization FL, SCI) chondroitin-, dermatan-sulfate) of growth factors Production of ECM components Cytoadhesion molecules Adhesion of hematopoietic precursors to ECM proteins Negative Positive Macrophage CSF3 CSF2 IFN IL-3 TNF IL-1 HSC TGFb IL-6 MIP-1a Stromal IL-11 cell SCF CAM FL Extracellular Endothelial cell matrix Figure 4.9 A model for regulation of hematopoietic precursor cells in the bone marrow microenvironment. The hematopoietic stem cell (HSC) attaches to bone marrow stromal cells via specific receptors and ligands. The HSC is then influenced by both positive and negative regulatory growth factors. CAM, cell adhesion molecule; SCF, stem cell factor; FL, Flt3-ligand; IFN, interferon; TNF, tumor necrosis factor; TGF@b, transforming growth factor b; MIP@1a, macrophage inflammatory protein@1a [stem cell inhibitor]; CSF3, colony-stimulating factor 1; CSF2, CSF 2; IL, interleukin. Hematopoiesis 61 stem cells, progenitor cells, and growth-regulatory proteins different cytokines to proliferate or differentiate. Specialized within the marrow cavity. These are achieved via cell-cell, stromal cells produce extracellular matrix components and cell-cytokine, and cell-extracellular matrix interactions. hematopoietic cytokines that are conducive for the commit- ment and/or differentiation of precursor cells of a specific Components of the Hematopoietic hematopoietic lineage. These interactions likely contribute Microenvironment to the tight regulation of precursor cell differentiation and proliferation.54,55 CELLULAR COMPONENTS The cellular elements of the HM are referred to as hematopoi- ADHESION TO THE MICROENVIRONMENT etic stromal cells and accessory cells. Stromal cells include One of the important determinants of the geographic local- adipocytes (fat cells), endothelial cells, fibroblasts, CXCL12 ization of hematopoiesis appears to be the presence of mem- abundant reticular (CAR) cells, and osteoblasts. Accessory brane receptors on hematopoietic precursors for stromal cells cells include T lymphocytes, monocytes, and macrophages. and ECM proteins. A number of ligand-receptor interac- The
stromal cells’ capacity to support hematopoiesis derives tions are important in retaining HSCs in the marrow space. from a number of characteristics. These cells express hom- These include SDF-1 (stromal-derived factor 1, also known as ing receptors, although the exact mechanisms involved in CXCL12) and its receptor CXCR4, SCF and its receptor SCFR mediating the homing of hematopoietic cells are unclear. (c-kit), integrins (VLA-4) interacting with their ligands VCAM- 1, and hyaluronic acid interacting with its receptor CD44.20 They also produce the various components constituting the extracellular matrix of the HM. Both stromal cells and acces- Fibronectin is a large, adhesive glycoprotein that binds sory cells synthesize and secrete soluble growth and differ- cells, growth factors, and ECM components. HSC/HPC and entiation factors and negative regulators as well as a number developing erythroblasts have fibronectin receptors (FnR) on of membrane-bound cytokines that function as juxtacrine their surface membrane. As developing erythroblasts mature regulators of hematopoiesis (e.g., SCF, FL, SCI). Many of the to the reticulocyte stage, they lose their FnR; loss of attach- secreted cytokines bind the extracellular matrix, which con- ment via fibronectin facilitates the egress of reticulocytes centrates these factors within the HM, keeping them adja- and erythrocytes from the erythroblastic islands in the bone cent to the developing hematopoietic precursor cells. marrow. Likewise, hemonectin is an adhesive glycoprotein found in the microenvironment that interacts with hemonec- EXTRACELLULAR MATRIX tin receptors (HnR) on HSCs, HPCs, and granulocytes and The stromal cells produce and secrete the extracellular is important for the attachment and retention of these cells matrix (ECM), which provides the adhesive interactions in the marrow. Loss of HnR by developing granulocytes and important for the colocalization of HSCs, HPCs, and the loss of adhesion to ECM mediates release of mature granulo- growth-regulatory proteins. The ECM is composed of colla- cytes to the circulation. Adhesive interactions between HSCs, gens, glycoproteins, glycosaminoglycans, and cytoadhesion HPCs, and the ECM function to help hold the hematopoietic molecules (e.g., fibronectin, hyaluronan, tenascin). Varia- precursor cells in microenvironmental niches, bringing cells tions in the type and relative amounts of these components into close proximity with growth-regulatory cytokines that produce the characteristic properties of ECMs in different are also bound and held by the ECM.52 tissues. Collagen provides the structural support for the other components. Glycosaminoglycans (heparan-sulfate, STEM CELL NICHE chondroitin-sulfate, dermatan-sulfate) play a role in cell– As discussed in the section “Stem Cells,” the quiescent state cell interactions, helping to mediate progenitor-cell binding of stem cells is controlled by their localization in osteoblas- to the stroma. They also serve to bind and localize cytokines tic niches that block their responsiveness to differentiation- in the vicinity of the hematopoietic cells. Cytoadhesion mol- inducing signals (Figures 4-2 and 4-9, Table 4-3). Stromal ecules important in hematopoietic cell localization include cells produce cell-surface–associated (juxtacrine) factors the b1 integrins (VLA@4/a4b1, VLA@5/a5b1) found on hema- that restrain HSC differentiation. Removal of HSCs from topoietic cells binding to ligands VCAM-1 and fibronectin this niche results in a cascade of differentiation events. on marrow stromal cells or in the HM. A major role of stromal tissue in the regulation of hema- topoiesis thus may be to safeguard and ensure stem cell Hematopoietic Microenvironment maintenance. Hematopoietic stem cells removed from Niches their marrow environment do not retain their “stemness” for more than a few weeks when cultured in vitro in the Within the hematopoietic bone marrow, precursor cells of absence of stromal cells. Inevitably, they differentiate into different lineages and at different stages of differentiation progenitor cells and mature cells of the various lineages and can be found in distinct areas throughout the marrow space. thus undergo “death by differentiation.” Precursor cells at various stages of differentiation can inter- The HSC niche is hypoxic.54 The hypoxia of the HSC act with different ECM components and can be induced by niche may protect HSCs from oxidative stress potentially 62 Chapter 4 associated with aerobic metabolism. In addition, hypoxia ERYTHROID NICHES may promote cell-cycle quiescence (an HSC characteristic), Erythropoiesis occurs in unique anatomical configurations which can protect the HSC pool from excess proliferation. called erythroblastic islands (Chapter 5). Some of them are located adjacent to the marrow sinusoids, and others are LYMPHOID NICHES scattered throughout the bone marrow cavity. The bone marrow is the site of B-cell lymphopoiesis (Chapter 8). The less mature developing B cells are located MEGAKARYOCYTIC NICHES closer to the endosteal surface with the more differentiated Megakaryocytes tend to localize near the marrow sinusoidal cells nearer the sinusoidal endothelial cells.54 Naïve recir- endothelial cells where they are positioned to release platelets culating B and T cells are also located in the perisinusoidal into the intravascular sinusoidal space. There is evidence that space in close proximity to dendritic cells. The majority of megakaryocyte localization within a specific vascular micro- long-lived memory T cells reside in the bone marrow in environment, mediated by specific cytokines, is necessary for close contact with IL-7 secreting stromal cells.54 megakaryocyte maturation and platelet production.54 Summary Hematopoiesis is the production of the various types or lin- cytokine usually exerts more than one biologic effect). eages of blood cells. Mature, terminally differentiated blood These cytokines interact with their target cell by means of cells derive from mitotically active precursor cells found unique transmembrane receptors responsible for generat- primarily in the bone marrow in adults. Hematopoietic pre- ing the intracellular signals that govern proliferation and cursor cells include multipotential hematopoietic stem cells, differentiation. hematopoietic progenitor cells (multilineage and unilin- Hematopoiesis takes place in a unique microenviron- eage), and maturing (morphologically recognizable) cells. ment in the marrow consisting of stromal cells and extra- Hematopoietic growth factors or cytokines (colony- cellular matrix, which plays a vital role in controlling stimulating factors and interleukins) stimulate hema- hematopoiesis. Specialized stromal cells produce extracel- topoietic precursor cells to proliferate and differentiate. lular matrix components and hematopoietic cytokines that Cytokine control of hematopoiesis is characterized by promote the commitment and/or differentiation of precur- redundancy (more than one cytokine is capable of exert- sor cells of a specific hematopoietic lineage, resulting in ing the same effect on the system) and pleiotrophy (a given lineage-specific niches within the bone marrow. Review Questions Level I b. erythrocytes and monocytes 1. Self-renewal and multipotential differentiation are c. eosinophils and megakaryocytes characteristics of: (Objective 2) d. erythrocytes and megakaryocytes a. mature cells 4. All hematopoietic cells are derived from the CMP b. stem cells except: (Objective 3) c. progenitor cells a. lymphocytes d. maturing cells b. platelets 2. Precursor cells that are morphologically recognizable c. eosinophils are found in the: (Objective 2) d. erythrocytes a. stem cell compartment b. progenitor cell compartment 5. The following cell that is most sensitive to erythropoietin is: (Objective 4) c. maturing cell compartment d. differentiating cell compartment a. reticulocyte b. CMP 3. The MEP gives rise to: (Objective 3) c. BFU-E a. eosinophils and megakaryocytes d. EP Hematopoiesis 63 6. All of the following are considered “early acting, b. FcRg multilineage” cytokines except: (Objective 4) c. CD33 a. IL-5 d. CD13 b. CSF2 c. SCF 3. All of the following are important regulators of granulopoiesis except: (Objective 2) d. IL-3 a. CSF2 7. Pleiotrophy refers to: (Objective 4) b. FL a. multiple different cells that can produce the same c. IL-2 cytokine d. IL-3 b. a cytokine with multiple biologic activities c. multiple cytokines that can induce the same 4. The major cytokine important for eosinophil cellular effect differentiation is: (Objective 2) d. a cytokine that can be produced by multiple a. IL-3 different tissues b. IL-5 8. Cytokine regulation in which the cytokine is not c. IL-7 secreted by the producing cell but remains membrane d. IL-11 bound, necessitating direct cell–cell contact to achieve the desired effect is: (Objective 5) 5. Which of the following growth factor receptors share a. paracrine a common b chain? (Objective 3) b. endocrine a. IL-3 and CSF2 c. juxtacrine b. TPO and EPO d. autocrine c. IL-2 and IL-3 9. All of the following are thought to be negative d. CSF3 and CSF2 regulators of hematopoiesis except: (Objective 6) 6. Cytokine receptors that lack an intrinsic kinase a. TGF@b domain generally signal: (Objective 4) b. SCF a. through an intrinsic phosphatase domain c. TNF b. by recruiting membrane-embedded kinases d. MIP@1a c. through an intrinsic protease domain 10. The hematopoietic microenvironment is composed of: d. by recruiting cytoplasmic kinases (Objective 7) a. hepatocytes and extrahepatic matrix 7. The function of the JAK-STAT pathway in hematopoiesis is to: (Objective 4) b. osteoblasts and osteoclasts c. marrow stromal cells and extracellular matrix a. localize cytokines in the hematopoietic microenvironment d. hepatocytes and splenic macrophages b. generate homing receptors for stem and progenitor Level II cells 1. Hematopoietic stem cells are characterized by all of c. produce cytoadhesion molecules to retain the following markers except: (Objective 1) precursor cells in the marrow a. CD34+ d. function as a signal transduction pathway for cytokine-activated receptors b. Lin- c. HLA@DR+ 8. The stromal elements of the hematopoietic d. Rhodamine 123Lo microenvironment include all of the following except: (Objective 7) 2. The major molecular marker that differentiates CLP a. B lymphocytes from CMP is: (Objective 1) b. adipocytes a. IL7-R 64 Chapter 4 c. fibroblasts 10. The role of the osteoblastic stem cell “niche” in the d. osteoblasts bone marrow is thought to be to: (Objective 7) 9. Which of the following cytoadhesion molecules plays a. protect hematopoietic precursor cells from the lytic an important role in retaining erythroid-developing cells action of osteoclasts in the bone marrow microenvironment? (Objective 7) b. provide nourishment (oxygen, nutrients) to developing precursor cells a. Hemonectin c. regulate the quiescent state of stem cells blocking b. Fibronectin differentiation-inducing signals c. Thrombospondin d. produce cytoadhesion molecules important for d. Glycosaminoglycans homing to the marrow References 1. Bessis, M. (1977). Blood smears reinterpreted (G. Brecher, Trans.). 15. Rossmann, M. P., Orkin, S. H., & Chute, J. P. (2018). Hematopoi- Berlin, Germany: Springer-Verlag. etic stem cell biology. In: R. Hoffman, E. J. Benz Jr., L. E. Silber- 2. Kaushansky, K. (2016). Hematopoietic stem cells, progenitors, and stein, H. E. Heslop, J. I. Weitz, & J. Anastasi, eds. Hematology: cytokines. In K. Kaushansky, M. A. Lichtman, J. T. Prchal, M. M. Basic principles and practice (7th ed., pp. 95–110). Philadelphia, PA: Levi, O. W. Press, L. J. Burns, et al. (Eds.), Williams hematology Elsevier. (9th ed., pp. 257–278). New York, NY: McGraw-Hill Education. 16. Li, Z. (2013). CD133: A stem cell biomarker and beyond. 3. Till, J. E., & McCulloch, E. A. (2011). A direct measurement of Experimental Hematology and Oncology, 2(1), 17. doi: the radiation sensitivity of normal mouse bone marrow cells. 10.1186/2162-3619-2-17 Radiation Research, 175(2), 145–149. 17. Shaheen, M., & Broxmeyer, H. E. (2009). The humoral regulation 4. Bradley, T. R., & Matcalf, D. (1966). The growth of mouse bone of hematopoiesis. In: R. Hoffman, E. J. Benz, S. J. Shattil, B. Furie, marrow cells in vitro. Australian Journal of Experimental Biology L. E. Silberstein, & P. McGlave, eds. Hematology: Basic principles and Medical Sciences, 44(3), 287–299. and practice (5th ed., pp. 253–275). New York, NY: Churchill 5. Silver, R. K., & Ersley, A. J. (1974). The action of erythropoietin on Livingstone Elsevier. erythroid cells in vitro. Scandinavian Journal of Haematology, 13(5), 18. Schoemans, H., & Verfaillie, C. (2009). Cellular biology of 338–351. hematopoiesis. In: R. Hoffman, E. J. Benz Jr., S. J. Shattil, B. Furie, 6. Metcalf, D., MacDonald, H. R., Odartchenko, N., & Sordat, B. (1975). L. E. Silberstein, & P. McGlave, eds. Hematology: Basic principles Growth of mouse megakaryocyte colonies in vitro. Proceedings of and practice (5th ed., pp. 200–212). Philadelphia, PA: Churchill the National Academy of Sciences of the United States of America, 72(5), Livingstone Elsevier. 1744–1748. 19. Yoder, M. C. (2009). Overview of stem cell biology. In: R. Hoffman, 7. Vainchenker, W., Bouguet, J., Guichard, J., & Breton-Gorius, E. J. Benz Jr., S. J. Shattil, B. Furie, L. E. Silberstein, & P. McGlave, J. (1979). Megakaryocyte colony formation from human bone eds. Hematology: Basic principles and practice (5th ed., pp. 187–199). marrow precursors. Blood, 54(4), 940–945. Philadelphia, PA: Churchill Livingstone Elsevier. 8. Williams, D. A. (2000). Stem cell model of hematopoiesis. In: 20. Gur-Cohen, S., Golan, K., Lapid, K., Canaani, J., Kollet, O., R. Hoffman, ed. Hematology: Basic principles and practice (3rd ed.
Lapidot, T., & Anastasi, J. (2013). Dynamic interactions between pp. 126–138). London, England: Churchill Livingstone. hematopoietic stem and progenitor cells and the bone marrow: 9. Lord, B. I., & Testa, N. G. (1988). The hemopoietic system: Current biology of stem cell homing and mobilization. In: Structure and regulation. In: N. G. Testa & R. P. Gale, eds. R. Hoffman, E. J. Benz Jr., L. E. Silberstein, H. E. Heslop, & Hematopoiesis: Long-term effects of chemotherapy and radiation J. I. Weitz, eds. Hematology: Basic principles and practice (6th ed., (Vol. 8, pp. 1–26). New York, NY: Marcel Dekker. pp. 117–125). Philadelphia, PA: Elsevier. 10. Bagby, G. C. (1994). Hematopoiesis. In: G. Stamatoyannopoulos, A. 21. Boulais, P. E., & Frenette, P. S. (2015). Making sense of hemato- W. Nienhuis, P. W. Majerus, & H. Varmus, eds. The molecular basis poietic stem cell niches. Blood, 125(17), 2621–2529. doi: 10.1182/ of blood diseases (2nd ed., pp.71-103). Philadelphia, PA: Saunders. blood-2014-09-570192 11. Prchal, J. T., Throckmorton, D. W., Carroll, A. J., III, Fuson, E. W., 22. Kiel, M. J., Yilmaz, O. H., Iwashita, T., Yilmaz, O. H., Terhorst, C., Gams, R. A., & Prchal, J. F. (1978). A common progenitor for & Morrison, S. J. (2005). SLAM family receptors distinguish human myeloid and lymphoid cells. Nature, 274(5671), 590–591. hematopoietic stem and progenitor cells and reveal endothe- 12. Bhatia, M., Wang, J. C., Kapp, U., Bonnet, D., & Dick, J. E. (1997). lial niches for stem cells. Cell, 121(7), 1109–1121. doi: 10.1016/j. Purification of primitive human hematopoietic cells capable of cell.2005.05.026 repopulating immune-deficient mice. Proceedings of the National 23. Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J. C., & Keller, Academy of Sciences of the United States of America, 94(10), 5320–5325. G. (1998). A common precursor for hematopoietic and endothelial 13. Berenson, R. J., Andrews, R. G., Bensinger, W. I., Kalamasz, D., cells. Development (Cambridge, England), 125(4), 725–732. Knitter, G., Buckner, C. D., & Bernstein, I. D. (1988). Antigen 24. Zhu, J., & Emerson, S. G. (2002). Hematopoietic cytokines, CD34 marrow cells engraft lethally irradiated baboons. Journal transcription factors and lineage commitment. Oncogene, 21(21), of Clinical Investigation, 81(3), 951–955. doi: 10.1172/JCI113409 3295–3313. doi: 10.1038/sj.onc.1205318 14. Majeti, R., Park, C. Y., & Weissman, I. L. (2007). Identification 25. Terskikh, A. V., Miyamoto, T., Chang, C., Diatchenko, L., & of a hierarchy of multipotent hematopoietic progenitors in Weissman, I. L. (2003). Gene expression analysis of purified human cord blood. Cell Stem Cell, 1(6), 635–645. doi: 10.1016/ hematopoietic stem cells. Blood, 102(1), 94–101. doi: 10.1182/ j.stem.2007.10.001 blood-2002-08-2509 Hematopoiesis 65 26. Krause, D. S. (2002). Regulation of hematopoietic stem cell fate. reconstitution in transplant mice: Acceleration of recovery of Oncogene, 21(21), 3262–3269. doi: 10.1038/sj.onc.1205316 peripheral blood neutrophils and platelets. Blood, 81(1), 27–34. 27. Galy, A., Travis, M., Cen, D., & Chen, B. (1995). Human T, B, 42. Toombs, C. F., Young, C. H., Glaspy, J. A., & Varnum, B. C. (1995). natural killer, and dendritic cells arise from a common bone Megakaryocyte growth and development factor (MGDF) moder- marrow progenitor cell subset. Immunity, 3(4), 459–473. ately enhances in-vitro platelet aggregation. Thrombosis Research, doi: 10.1016/1074-7613(95)90175-2 80(1), 23–33. doi: 10.1016/0049-3848(95)00147-J 28. Akashi, K., Traver, D., Miyamoto, T., & Weissman, I. L. (2000). 43. Graham, G. J., Wright, E. G., Hewick, R., Wolpe, S. D., Wilkie, N. M., Clonogenic common myeloid progenitor that gives Donaldson, D., . . . Pragnell, I. B. (1990). Identification and charac- rise to all myeloid lineages. Nature, 404(6774), 193–197. terization of an inhibitor of haemopoietic stem cell proliferation. doi: 10.1038/35004599 Nature, 344(6265), 442–444. doi: 10.1038/344442a0 29. Ikawa, T., Kawamoto, H., Fujimoto, S. Fujimoto, S., & Katsura, Y. 44. Shaheen, M., & Broxmeyer, H. E. (2009). The humoral regula- (1999). Commitment of common T/Natural killer (NK) pro- tion of hematopoiesis. In: R. Hoffman, E. J. Benz Jr., S. J. Shattil, genitors to unipotent T and NK progenitors in the murine fetal B. Furie, L. E. Silberstein, & P. McGlave, eds. Hematology: Basic thymus revealed by a single progenitor assay. Journal of Experi- principles and practice (5th ed., pp. 253–275). Philadelphia, PA: mental Medicine, 190(11), 1617. Churchill Livingstone Elsevier. 30. McDonald, T. P., & Sullivan, P. S. (1993). Megakaryocytic 45. Dong, F., Hoefsloof, L. H., Schelen, A. M., Broeders, C. A., and erythrocytic cell lines share a common precursor cell. Meijer, Y., Veerman, A. J., . . . Lowenberg, B. (1994). Identification Experimental Hematology, 21(10), 1316–1320. of a nonsense mutation in the granulocyte-colony-stimulating 31. Long, M. W., Gragowski, L. L., Heffner, C. H., & Boxer, L. A. factor receptor in severe congenital neutropenia. Proceedings of the (1985). Phorbol diesters stimulate the development of an early National Academy of Sciences of the United States of America, 91(10), murine progenitor cell: The burst-forming unit-megakaryocyte. 4480–4484. Journal of Clinical Investigation, 76(2), 431–438. doi: 10.1172/ 46. De la Chapelle, A., Traskelin, A. L., & Juvonen, E. (1993). JCI111990 Truncated erythropoietin receptor causes dominantly inherited 32. Yamamoto, R., Morita, Y., Ooehara, J., Hamanaka, S., Onodera, benign human erythrocytosis. Proceedings of the National Academy M., Rudolph, K. L., . . . Nakauchi, H. (2013). Clonal analysis of Sciences of the United States of America, 90(10), 4495–4499. unveils self-renewing lineage-restricted progenitors generated 47. Neel. B.G., & Carpenter, C. L. (2009). Regulation of cellular directly from hematopoietic stem cells. Cell, 154(5), 1112–1126. response. In: R. Hoffman, E. J. Benz Jr., S. J. Shattil, B. Furie, doi: 10.1016/j.cell.2013.08.007 L. E. Silberstein, & P. McGlave, eds. Hematology: Basic principles 33. Mercier, F. E., & Scadden, D. T. (2015). Not all created equal: and practice (5th ed., pp. 49–60). Philadelphia, PA: Churchill Lineage hard-wiring in the production of blood. Cell, 163(7), Livingstone Elsevier. 1568–1570. doi: 10.1016/j.cell.2015.12.013 48. Rane, S. G., & Reddy, E. P. (2002). JAKs, STATs and Src kinases 34. Paul, F., Arkin, Y., Giladi, A., Jaitin, D. A., Kenigsberg, E., Keren- in hematopoiesis. Oncogene, 21(21), 3334–3358. doi: 10.1038/ Shaul, H., . . . Amit, I. (2015). Transcriptional heterogeneity and sj.onc.1205398 lineage commitment in myeloid progenitors. Cell, 163(7), 49. Orkin, S. H. (2000). Diversification of haematopoietic stem 1663–1677. doi: 10.1016/j.cell.2015.11.013 cells to specific lineages. Nature Reviews. Genetics., 1(1), 57–64. 35. Perie, L., Duffy, K. R., Kol, L., De Boer, R. J., & Schumacher, T. N. doi: 10.1038/35049577 (2015). The branching point in erythro-myeloid differentiation. 50. Cantor, A. B., & Orkin, S. H. (2002). Transcriptional regulation of Cell, 163(7), 1655–1662. doi: 10.1016/j.cell.2015.11.059 erythropoiesis: An affair involving multiple partners. Oncogene, 36. Notta, F., Zandi, S., Takayama, N., Dobson, S., Gan, O. I., Wilson, 21(21), 3368–3376. doi: 10.1038/sj.onc.1205326 G., . . . Dick, J. E. (2016). Distinct routes of lineage development 51. Dahl, R., & Hromas, R. (2009). Transcription factors in normal reshape the human blood hierarchy across ontogeny. Science, and malignant hematopoiesis. In: R. Hoffman, E. J. Benz 351(6269), 126–127. doi: 10.1126/science.aab2116 Jr., S. J. Shattil, B. Furie, L. E. Silberstein, & P. McGlave, eds. 37. Veiby, O. P., Jacobsen, F. W., Cui, L., Lyman, S. D., & Jacobsen, Hematology: Basic principles and practice (5th ed., pp. 213–230). S. E. (1996). The Flt3 ligand promotes the survival of primitive Philadelphia, PA: Churchill Livingstone Elsevier. hemopoietic progenitor cells with myeloid as well as B lymphoid 52. Rowe, J. M., & Avivi, I. (2009). Clinical use of hematopoietic potential: Suppression of apoptosis and counteraction by growth factors. In: R. Hoffman, E. J. Benz Jr., S. J. Shattil, B. Furie, TNF-alpha and TGF-beta. Journal of Immunology, 157(7), L. E. Silberstein, & P. McGlave, eds. Hematology: Basic principles 2953–2960. and practice (5th ed., pp. 907–920). Philadelphia, PA: Churchill 38. Flanagan, J. G., Chan, D. C., & Leder, P. (1991). Transmembrane Livingstone Elsevier. form of the kit ligand growth factor is determined by alternative 53. Schoemans, H., & Verfaillie, C. (2000). Cellular biology of splicing and is missing in the Sld mutant. Cell, 64(5), 1025–1035. hematopoiesis. In: R. Hoffman, E. J. Benz, S. J. Shattil, B. Furie, doi: 10.1016/0092-8674(91)90326-T L. E. Silberstein, P. McGlave, & H. Heslop, eds. Hematology: Basic 39. Kopf, M., Ramsay, A., Brombacher, F., Baumann, H., Freer, G., principles and practice (5th ed., pp. 200–212). Philadelphia, PA: Galanos, C., . . . Kohler, G. (1995). Pleiotropic defects of IL-6 Churchill Livingstone Elsevier. deficient mice including early hematopoiesis, T and B cell 54. Scadden, D., & Silberstein, L. (2018). Hematopoietic function, and acute-phase responses. Annals of the New York microenvironment. In: R. Hoffman, E. J. Benz Jr., L. E. Silberstein, Academy of Sciences, 762, 308–318. doi: 10.1111/j.1749-6632.1995. H. E. Heslop, J. I. Weitz, & J. Anastasi, eds. Hematology: tb32335.x Basic principles and practice (7th ed., pp. 119–126). Philadelphia, 40. Musashi, M., Clark, S. C., Sudo, T., Urdal, D. L., & Ogawa, M. PA: Elsevier. (1991). Synergistic interactions between interleukin-11 and inter- 55. Quesenberry, P. L., Crittenden, R. B., Lowry, P., Kittler, E. W., leukin-4 in support of proliferation of primitive hematopoietic Rao, S., Peters, S., . . . Stewart, E. M. (1994). In vitro and in vivo progenitors of mice. Blood, 78(6), 1448–1451. studies of stromal niches. Blood Cells, 20(1), 97–104. 41. Du, X. X., Neben, T., Goldman, S., & Williams, D. A. (1993). Effects of recombinant human interleukin-11 on hematopoietic Chapter 5 The Erythrocyte Joel Hubbard, PhD Stacey Robinson, MS Objectives—Level I At the end of this unit of study, the student should be able to: 1. List and describe the stages of erythrocyte 5. Describe the function of the erythrocyte maturation in the marrow from youngest to membrane. most mature cells. 6. Name the energy substrate of the 2. Explain the maturation process of reticulo- erythrocyte. cytes and the cellular changes that take place. 7. Define and differentiate intravascular and 3. Identify the reference interval for extravascular red cell destruction. reticulocytes. 8. State the average dimensions and life span 4. Explain the function of erythropoietin and of the normal erythrocyte. include the origin of production, bone mar- 9. Describe the function of 2,3-BPG and its row effects, and normal values. relationship to the erythrocyte. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Summarize the mechanisms involved in the 4. Compare and contrast three pathways of regulation of erythrocyte production. erythrocyte metabolism and identify key 2. Describe the structure of the erythrocyte intermediates as well as the relationship of membrane, including general dimensions each to erythrocyte survival and longevity. and features; assess the function of the major 5. Generalize the metabolic and catabolic changes membrane components. within the erythrocyte over time that “label” 3. Explain the mechanisms used by the eryth- the erythrocyte for removal by the spleen. rocyte to regulate permeability to cations, 6. Predict the effects of increased and anions, glucose, and water. decreased erythropoietin levels in the blood. 66 The Erythrocyte 67 Chapter Outline Objectives—Level I and Level II 66 Erythrocyte Metabolism 77 Key Terms 67 Erythrocyte Kinetics 80 Background Basics 67 Erythrocyte Destruction 82 Case Study 67 Summary 83 Overview 67 Review Questions 83 Introduction 68 Disclaimer 85 Erythropoiesis and Red Blood Cell Maturation 68 References 85 Erythrocyte Membrane 72 Key Terms Acanthocyte Erythropoiesis Normoblast BFU-E Erythropoietin Peripheral membrane protein CFU-E Glycolysis Polychromatophilic erythrocyte Cyanosis Heinz body Reticulocyte Erythroblast Hypoxia Spectrin Erythron Integral protein Background Basics The information in this chapter builds on the concepts • Give the functional description of the erythroid mar- learned in previous chapters. To maximize your learning row (Chapter 3). experience, you should review these concepts before start- ing this unit of study: Level II • List and describe the function of specific growth factors Level I important in erythrocyte development (Chapter 4). • Describe the process of cell differentiation and matu- • Describe the structure and function of the spleen and ration, regulation, and the function of growth factors; bone marrow (Chapter 3). describe cell organelles and their function (Chapters 2, 4). CASE STUDY while traveling abroad. Blood smears examined We refer to this case study throughout the chapter. for malaria, however, resulted in a negative diagnosis. Stephen, a 28-year-old Caucasian male of Ital- Consider what additional laboratory tests could ian descent, became progressively ill following a help identify the cause of Stephen’s anemia. safari vacation to West Africa. The patient arrived at the emergency room (ER) for evaluation fol- lowing several days of fever, chills, and malaise. The advent of hemoglobinuria prompted the Overview patient to seek emergency aid. A clinical history and physical examination supported a diagnosis This chapter is a
study of the erythrocyte. It begins with of anemia. Because Stephen had recently returned a description of erythrocyte production and maturation. from a malarial endemic area, the physician first This is followed by an account of the erythrocyte membrane suspected malaria even though the patient had composition and function, cell metabolism, and kinetics of been on primaquine preventive drug therapy cell production. The chapter concludes with a description of the destruction of the senescent cell. 68 Chapter 5 Introduction IL-3, GM-CSF EPO The erythrocyte (red blood cell, RBC) was one of the first Recognizable microscopic elements recognized and described after the HSC BFU-E CFU-E cells discovery of the microscope.1 RBCs play a vital role in phys- iology, carrying oxygen from the lungs to the tissues where it is utilized in oxidative metabolism. An insufficient num- Stem cells Committed progenitor cells Maturing cells ber of RBCs results in a condition called anemia, which is characterized by inadequate tissue oxygenation. An excess Figure 5.1 Erythroid maturation. Erythrocyte development number of circulating RBCs is called erythrocytosis, a condi- proceeds through three levels of maturation beginning with the multipotential hematopoietic stem cell (HSC), maturing tion that has no adverse effect on pulmonary gas exchange. into committed progenitor cells BFU-E and CFU-E, and into morphologically recognizable cells. IL-3 and GM-CSF are the primary cytokines that affect maturation of BFU-E. EPO primarily Erythropoiesis and Red affects the CFU-E and developing normoblasts. Blood Cell Maturation within 7 days of culture.2 CFU-Es have a high concentration Erythron refers to the totality of all stages of erythrocyte of EPO membrane receptors; hence, they respond to lower development, including precursor cells in the marrow and EPO concentrations than do BFU-Es. The CFU-E is the imme- mature cells in the peripheral blood and vascular areas of diate precursor of the earliest morphologically recognizable specific organs such as the spleen. Erythropoiesis, or the erythroid precursor, the pronormoblast. Figure 5-1 shows the production of erythrocytes, is normally an orderly process relationship of the various hematopoietic progenitor cells to through which the peripheral concentration of erythrocytes the cytokines that affect their maturation. is maintained at a steady state. The life cycle of erythrocytes BFU-Es are positive for CD34 (CD34+), a progenitor cell includes stimulation of lineage commitment and matura- marker, and have a high proliferative potential but a low rate tion of precursor cells in the marrow by erythropoietin (the of cycling. As they mature to the CFU-E stage, they lose CD34 major cytokine regulating erythropoiesis), a circulating life expression but begin to express surface proteins character- span for mature cells of approximately 100–120 days, and istic of the erythroid lineage including glycophorin A, Rh the destruction of senescent cells by mononuclear phago- antigens, and in a subset of CFU-E, the ABH and Ii antigens.2 cytic cells in the liver, spleen, and bone marrow. Additional cytokines shown to have a positive effect on erythrocyte precursor proliferation include stem cell fac- Erythroid Progenitor Cells tor (SCF), thrombopoietin, and IL-11; tumor necrosis fac- tor-a (TNF a), transforming growth factor-b (TGF b), and Red cell production begins with the hematopoietic stem interferon-g (INFg) have a negative effect on erythropoiesis. cell (HSC) (Chapter 4). Stem cell differentiation is induced by microenvironmental influences to produce a committed erythroid progenitor cell. The committed (unipotential) ery- Erythroid-Maturing Cells throid progenitor cell compartment consists of two popula- Erythroid-maturing cells include those precursor cells in tions of cells, neither identifiable microscopically but defined the bone marrow that are morphologically identifiable. by their behavior in cell culture systems: the burst-forming Nucleated erythrocyte precursors in the bone marrow are unit-erythroid (BFU-E) and colony-forming unit-erythroid collectively called erythroblasts. If the maturation sequence (CFU-E). The BFU-E progenitor cells proliferate under the is “normal,” the cells are often called normoblasts. Young influence of what was originally called “burst-promoting erythrocytes with residual RNA but without a nucleus are activity” (BPA), now known to be the cytokine IL-3 or GM- referred to as reticulocytes (polychromatophilic erythro- CSF/CSF2, released by local microenvironmental stromal cytes). Bone marrow normoblast maturation occurs in an cells. BFU-Es have a low concentration of erythropoietin orderly and well-defined sequence under normal condi- (EPO) receptors and thus are relatively insensitive to EPO tions encompassing six morphologically defined stages. except in high concentrations. The BFU-Es are defined as pro- The process involves a gradual decrease in cell size together genitor cells that give rise to a “burst” or multifocal colony with progressive condensation of the nuclear chromatin and of cells in an in vitro colony assay in 10–14 days. The colony eventual expulsion of the pyknotic nucleus. The cytoplasm consists of several hundred to several thousand cells recog- in the younger normoblasts stains deeply basophilic due nizable as hemoglobin-containing RBC precursors. Matura- to the abundance of RNA (Figure 5-2). As the cell matures, tion of the BFU-E gives rise to the CFU-E progenitor cell. An there is an increase in hemoglobin production, which is individual CFU-E, which undergoes only a few cell divisions, acidophilic, and the cytoplasm takes on a gray to pink or gives rise to a single, discrete colony of 8–64 identifiable cells salmon color (Figures 5-2 and 5-3). Cytokine regulation of erythropoiesis The Erythrocyte 69 relatively small number of erythroid precursors in the mar- row in comparison to the large circulating erythrocyte mass. Erythropoietin (EPO) is the only cytokine important in regulating the final stages of erythroid maturation (the b maturing cells). A number of hormones are known to have some erythropoietic effect, however. The most notable is the erythropoietic effect of androgens, which was exploited for a the treatment of various anemias before the development of recombinant EPO. Androgens appear to both stimulate EPO secretion and directly affect the erythropoietic marrow, which partially explains the difference in hemoglobin concentra- tions according to sex and age. Other hormones that have Figure 5.2 Pronormoblast in the center (arrow, a). Note varying (although minor) effects on erythropoiesis include the large N:C ratio, presence of nucleoli, and lacy chromatin. thyroid hormones, adrenal cortical hormones, and growth The cytoplasm is deep blue with a light area next to the nucleus. hormone.3 Anemic patients with hypopituitarism, hypothy- Also note above at about 1 o’clock the polychromatophilic normoblast (arrow, b) (Bone marrow; Wright-Giemsa stain, 1000* roidism, and adrenocortical insufficiency show an increase in magnification). erythrocyte concentration when the appropriate deficient hormone is administered. The reduction of EPO in hypothy- roidism could partially be the result of the reduced demand for cellular oxygen by metabolically hypoactive tissue. Checkpoint 5.1 What is meant by the term erythron? Basophilic normoblast (singular) Characteristics of Cell Maturation Although the stages of erythrocyte maturation are usually described in a steplike fashion, the actual maturation is a gradual and continuous process (Table 5-1). Some normo- blasts might not conform to all criteria for a particular stage, and a judgment must be made when identifying those cells. The more experience one has in examining the blood and bone marrow, the easier it becomes to make these judgments. Figure 5.3 Developing normoblasts. At left center is a basophilic normoblast; below this are three polychromatophilic PRONORMOBLAST normoblasts (bone marrow; Wright-Giemsa stain, 1000* The earliest morphologically recognizable erythrocyte pre- magnification). cursor is the pronormoblast. Each pronormoblast produces between 8 and 32 mature erythrocytes (a total of 3–5 cell Two terminology systems have been used in the past to divisions during the maturation sequence). This cell is the describe erythrocyte precursors. One uses the word normo- largest of the normoblast series, from 20 to 25 mcM (mm) blast and the other rubriblast. This chapter uses the normo- in diameter with a high nuclear-to-cytoplasmic ratio (N:C). blast terminology; the rubriblast terminology is outdated and The nucleus occupies 80% or more of the cell. This cell is rarely used today. The stages in order from most immature rather rare in the bone marrow. to mature cell are BFU-E, CFU-E, pronormoblast, basophilic Cytoplasm The cytoplasm contains a large number of normoblast, polychromatophilic normoblast, orthochromatic ribosomes and stains deeply basophilic. A pale area next normoblast, reticulocyte, and erythrocyte (Table 5-1). to the nucleus is sometimes apparent. This represents the The normoblasts generally spend from 5 to 7 days in the Golgi apparatus, which does not take up the dyes of the proliferating and maturing compartment of the bone marrow. Romanowsky stain. Small amounts of hemoglobin are pres- After reaching the reticulocyte stage, there are an additional ent (less than 1% of total cytoplasmic protein) but are not 2–3 days of maturation, the first 1–2 days of which are spent in visible by light microscopy. the marrow before the cell is released to the peripheral blood. The mature erythrocyte remains in the circulation for approx- Nucleus The nucleus is large, taking up most of the cell imately 120 days. This lengthy life span accounts for the volume, and stains bluish-purple. The chromatin is in a fine 70 Chapter 5 Table 5.1 Morphologic Characteristics of Erythroid Precursors Cell Type (% of nucle- Cytoplasmic Nuclear Image (Wright stain) Size N/C Ratio ated cells in BM) Characteristics Characteristics Pronormoblast (1%) 20-25 mcM High (8:1) Small to moderate 1–3 faint nucleoli; amount of deep blue reddish purple cytoplasm; pale area color; homo- next to nucleus may be geneous, lacy seen chromatin Basophilic normoblast 16-18 mcM Moderate (6:1) Deep blue-purple color; Indistinct nucleoli; (1–3%) occasionally small coarsening chro- patches of pink; irregu- matin; deep pur- lar cell borders can be plish-blue color present; perinuclear halo can be apparent Polychromatophilic nor- 12-15 mcM Low (4:1) Abundant; gray-blue Chromatin moblast (13–30%) color irregular and coarsely clumped, eccentric Orthochromic normoblast 10-15 mcM Low (1:2) Pink-salmon color; can Small; dense; (1–4%) have varying degrees of pyknotic; round basophilia or bizarre shape; eccentric Reticulocyte (new methy- 7-10 mcM — Punctate purplish-blue No nucleus lene blue stain) inclusions Polychromatophilic Polychromatophilic (dif- No nucleus erythrocytes fusely basophilic) Mature RBC 7-8 mcM — Salmon color No nucleus Note: Wright-Giemsa stain except where noted for reticulocyte The Erythrocyte 71 linear network often described as lacy. The pronormoblast these cells demonstrate motility with protraction and chromatin has a coarser appearance and stains darker than retraction of cytoplasmic projections.4 These movements the chromatin of a white cell blast. The nucleus contains prepare for ejection of the nucleus, which usually occurs from one to three faint nucleoli. while the erythroblast is still part of the erythroblastic BASOPHILIC NORMOBLAST island (EI; see the section “Erythroblastic Islands”). Proper This cell is similar to the pronormoblast except that it is enucleation requires interaction between erythroblasts and usually smaller (16–18 mcM in diameter) and has a slightly the macrophage of the EI, mediated by a protein called decreased N:C ratio. The nucleus occupies approximately erythroblast macrophage protein (EMP). 2 Alternatively, the three-fourths of the cell volume. Because these cells are nucleus can be lost as the cell passes through the wall of a actively dividing, it is possible to find a basophilic normo- marrow sinus. The expelled nucleus is engulfed by a mar- blast (in G2, prior to mitosis) that is larger than the pronor- row macrophage. moblast (in G1). RETICULOCYTE Cytoplasm The cytoplasm is still deeply basophilic, often After the nucleus is extruded, the cell is known as a reticu- more so than that of the previous stage due to the increased locyte. When the nucleus is first extruded, the cell has an number of ribosomes. However, in late basophilic normo- irregular lobulated or puckered shape. The cell is remod- blasts, the presence of varying amounts of hemoglobin can eled, eliminating excess membrane and gradually acquiring cause the cell to take on a lighter blue hue or show scattered its final biconcave shape while it completes its maturation pink areas. program.5 The reticulocyte has residual RNA and mitochondria in Nucleus The chromatin is coarser than that of the pronor- the cytoplasm, which give the young cell a bluish tinge with moblast. The dark violet heterochromatin interspersed with Romanowsky stains; thus, the cell is appropriately described the lighter-staining euchromatin can give the chromatin a as a diffusely basophilic erythrocyte or polychromatophilic wheel-spoke appearance. A few small masses of clumped erythrocyte. About 80% of the cell’s hemoglobin is made chromatin can be seen along the rim of the nuclear mem- during the normoblast stages with the remaining 20% made brane. Nucleoli usually are not apparent. during the reticulocyte stage. The reticulocyte is slightly POLYCHROMATOPHILIC NORMOBLAST larger, 7–10 mcM, than
a mature erythrocyte. These cells The cell is about 12–15 mcM in diameter with a decreased can be identified in vitro by reaction with supravital stains, N:C ratio due to continued condensation of the nuclear new methylene blue N, or brilliant cresyl blue, which cause chromatin. This is the last stage capable of mitosis. precipitation of the RNA and mitochondria. This supravital staining method identifies the reticulocytes by the presence Cytoplasm The most characteristic change in the cell at of punctate purplish-blue inclusions (Chapter 37). Nor- this stage is the presence of abundant gray-blue cytoplasm. mally, reticulocytes compose 0.5–2.0% (absolute concentra- The staining properties of the cytoplasm are due to the tion 25 - 75 * 109/L) of peripheral blood erythrocytes in synthesis of large amounts of hemoglobin and decreasing a nonanemic adult. The absolute concentration of reticulo- numbers of ribosomes. The cell derives its name, polychro- cytes is calculated by multiplying the percent of reticulo- matophilic, from this characteristic cytoplasmic feature. cytes by the RBC count. When reticulocytes are increased, Nucleus The nuclear chromatin is irregular and coarsely an increased number of polychromatophilic erythrocytes clumped due to increasing aggregation of nuclear (polychromasia) can be seen on the Romanowsky-stained heterochromatin. peripheral blood smear. Reticulocytes can contain small amounts of iron dis- ORTHOCHROMIC NORMOBLAST persed throughout the cytoplasm, which can be identified This cell is about 10–15 mcM in diameter with a low N:C with Perl’s Prussian blue stain. Erythrocytes with identified ratio. iron are called siderocytes. Nucleated RBC precursors that stain Cytoplasm After the final mitotic division of the eryth- with Prussian blue are called sideroblasts. The spleen is respon- ropoietic precursors, the concentration of hemoglobin sible for removal of these iron-containing granules, and the increases within the erythroblast. The cytoplasm is pre- normal mature circulating cell is devoid of granular dominantly pink or salmon color but retains a tinge of blue inclusions. due to residual ribosomes. Checkpoint 5.2 Nucleus The nuclear chromatin is heavily condensed. What is the first stage of red cell maturation that has visible cyto- Toward the end of this stage, the nucleus is structureless plasmic evidence of hemoglobin production on a Romanowsky- (pyknotic) or fragmented. The nucleus is often eccentric or stained smear? even partially extruded. Using phase-contrast microscopy, 72 Chapter 5 becomes sufficiently mature for nuclear expulsion, cytoad- CASE STUDY (continued from page 67) hesion molecules forming macrophage/erythroblast and In addition to malaria screening, the ER physician erythroblast/matrix attachments become downregulated, also ordered a CBC with the following results: or competing molecules can block attachment.6 As a result, WBC 14 * 109/L Differential the cell detaches from the EI, passes through a pore in the cytoplasm of endothelial cells lining the marrow sinuses, RBC 3.10 * 1012/L Segs 70% and enters the circulation.4 The central macrophage phago- Hb 9.2 g/dL (92 Bands 11% cytizes the nucleus, which is extruded from the orthochro- g/L) matic erythroblast before the cell leaves the bone marrow. Hct 28% (0.28 L/L) Metas 2% Any defective erythroblasts produced during the process of MCV 93 fL Lymphs 13% erythropoiesis (“ineffective erythropoiesis”) also are phago- MCH 30.6 pg/dL Monos 2% cytized by the macrophage. Surrounding the EI are fibroblasts, macrophages, and MCHC 32.5 g/dL Eos 2% endothelial cells, which provide the optimal microenviron- PLT 230 * 109/L NRBCs/100 18 ment for terminal erythroid maturation. The EI is a fragile WBCs structure and is usually disrupted by the process of marrow RBC aspiration. However, maturing erythroblasts with adherent Morphology macrophage cytoplasmic fragments may be seen on marrow Anisocytosis 3+ aspirate smears. Intact erythroblastic islands can occasion- Poikilocytosis 2+ ally be seen in marrow core biopsies.4 Spherocytosis 1+ Schistocytes 1+ Polychromasia 2+ Erythrocyte Membrane 1. Predict Stephen’s reticulocyte count: low, nor- The red cell membrane is essential for erythrocyte devel- mal, or increased. opment and function. Developing erythroblasts have membrane receptors for EPO and transferrin (the plasma transport protein for iron), which are required during eryth- ERYTHROCYTE ropoiesis. The erythrocyte membrane selectively seques- The mature erythrocyte is a biconcave disc about 7–8 ters vital components (e.g., organic phosphates such as mcM in diameter and 80–100 fL in volume. It stains pink 2,3-BPG) and lets metabolic waste products (lactate, pyru- to orange because of hemoglobin, the acidophilic protein vate) escape. The membrane helps regulate metabolism that fills the cell (28–34 pg/cell). Mature erythrocytes lack by reversibly binding and inactivating many glycolytic the cellular organelles (ribosomes, mitochondria) and enzymes. It carefully balances exchange of bicarbonate and enzymes necessary to synthesize new lipid and protein. chloride ions, which aids in the transfer of carbon diox- Thus, extensive damage to the cell membrane cannot be ide from tissues to lungs and balances cation and water repaired, and the spleen will cull the damaged cell from concentrations to maintain erythrocyte ionic composition. circulation. Finally, in association with the “membrane skeleton,” the erythrocyte membrane provides the red cell with the dual Erythroblastic Islands characteristics of strength and flexibility needed to survive in the circulation. Erythropoiesis occurs in distinctive histologic configura- tions called erythroblastic islands (EIs) that consist of con- centric circles of developing erythroblasts and reticulocytes Membrane Composition clustered around a central macrophage sometimes referred The erythrocyte membrane is a phospholipid bilayer-pro- to as a “nurse cell” (Chapter 3). The central macrophage tein complex composed of ∼52, protein, 40% lipid, and sends out slender cytoplasmic processes that maintain 8% carbohydrate7 (Table 5-2). The chemical structure and direct contact with each erythroblast. An array of adhesion composition control the membrane functions (e.g., trans- molecules has been identified on developing erythroblasts port, durability/strength, flexibility) and determine the that mediate interactions with other erythroblasts, mac- membrane’s antigenic properties. Any defect in structure rophages, and bone marrow extracellular matrix, includ- or alteration in chemical composition can alter erythro- ing fibronectin.6 The maturing erythroblast moves along cyte membrane functions and lead to the cell’s premature a cytoplasmic extension of the macrophage. As the cell death (Chapter 17). The Erythrocyte 73 membrane fluidity. Exchange between phospholipids of the Table 5.2 Erythrocyte Membrane Composition membrane and plasma can occur, especially with the phos- Lipids and Unesterified cholesterol pholipids of the outer bilayer leaflet. glycolipids Phospholipids (∼45,) Cholesterol and glycolipids are intercalated between Phosphatidylinositol /PI the phospholipids in the membrane bilayer.8 Cholesterol Phosphatidylethanolamine/PE is present in approximately equal proportions on both Phosphatidylserine/PS Phosphatidylcholine/PC (lecithin) sides of the lipid bilayer and affects the surface area of the Sphingomyelin/SM cell and membrane permeability. Membrane cholesterol Lysophospholipids (lysophosphatidylcholine [LPC], exists in equilibrium with unesterified (free) cholesterol of lysophosphatidylethanolamine [LPE]) plasma lipoproteins. In the plasma, cholesterol is partially Glycolipids converted to esterified cholesterol by the action of lecithin- Proteins and Integral proteins cholesterol acyl transferase (LCAT). Esterified plasma cho- glycoproteins Glycophorins A, B, C, D, E (carry antigens on (∼55,) lesterol cannot exchange with the red cell membrane. When exterior of membrane) Band 3 (attaches skeletal lattice to membrane lipid LCAT is absent (congenital LCAT deficiency or hepatocel- bilayer; anion exchange channel) lular disease), free plasma cholesterol increases, resulting in Peripheral proteins (form membrane skeletal lattice accumulation of cholesterol within erythrocyte membranes and attach it to membrane) and RBC membrane surface area expansion. An excess of Spectrin (a and b polypeptides) cell membrane due to proportional increases in cholesterol Actin (band 5) and phospholipids, maintaining the normal ratio, results Ankyrin (band 2.1) Band 4.2 in the formation of macrocodocytes (large target cells). An Band 4.1 increase in the cholesterol-to-phospholipid ratio, however, Adducin (band 2.9) decreases the membrane fluidity and results in the forma- Band 4.9 (dematin) tion of acanthocytes (Chapter 11). These cells have reduced Tropomyosin (band 7) survival as compared with normal RBCs. Tropomodulin (band 5) The shape of the red cell can also be altered by expansions of the separate lipid bilayers relative to each other. Processes Lipid Composition that expand the bilayer’s outer leaflet (or contract the inner) result in the formation of membrane spicules, producing echi- Approximately 95% of the lipid content of the membrane nocytes (see Figure 5-4). Conversely, expansion of the inner consists of equal amounts of unesterified cholesterol and leaflet of the bilayer leads to invagination of the membrane and phospholipids. The remaining lipids are free fatty acids (FAs) the formation of stomatocytes (cup-shaped cells) (Figure 5-4). and glycolipids (e.g., globoside). Mature erythrocytes depend Reticulocytes normally contain more membrane lipid on lipid exchange with the plasma and fatty acid acylation and cholesterol than do mature erythrocytes. This excess for phospholipid repair and renewal during their life span. lipid material is removed from the reticulocytes during the The overall structure of the membrane is that of a phos- final stages of maturation in the circulation by the splenic pholipid bilayer with the phospholipid molecules arranged macrophages. Splenectomized patients can have cells with with polar heads exposed at the cytoplasmic and plasma an abnormal accumulation of cholesterol and/or other lipids membrane surfaces and their hydrophobic fatty acid side in the membrane, which will present as target cells, acantho- chains directed to the interior of the bilayer. The major cytes, and/or echinocytes on the blood smear. (Alterations phospholipids are phosphatidylcholine (PC), phosphati- of red cell shape are described in Chapters 10 and 11.) dylethanolamine (PE), phosphatidylserine (PS), phospha- A small portion of membrane lipids consists of glyco- tidylinositol (PI), and sphingomyelin (SM) (Table 5-2). The lipids in the form of glycosphingolipid. Red cell glycolipids phospholipids are asymmetrically distributed within the are located entirely in the external half of the lipid bilayer membrane bilayer.8 The choline phospholipids (PC, SM) with their carbohydrate portions extending into the plasma. are concentrated in the outer half of the bilayer, and the These glycolipids are responsible for some antigenic proper- amino phospholipids (PE, PS, PI) are largely confined to ties of the red cell membrane (they carry the ABH, Lewis, the inner half. Although there is transmembrane diffusion and P blood group antigens). of the phospholipids from areas of higher concentration to the bilayer leaflet of lower concentration, the asym- metry is maintained by an ATP-dependent transport sys- Checkpoint 5.3 tem, the aminophospholipid translocase (also nicknamed Explain how a deficiency or absence of LCAT can lead to the “flippase”).9 Considerable evidence exists that the mobil- expansion of the surface area of the red cell membrane. ity of phospholipids within the membrane contributes to 74 Chapter 5 Normal discocyte Expansion of the outer leaflet Echinocytes Expansion of the inner leaflet Stomatocytosis Figure 5.4 Model of discocyte-echinocyte and discocyte-stomatocyte transformation. RBC shape is determined by the ratio of the surface areas of the two hemileaflets of the lipid bilayer. Preferential accumulation of compounds in the outer leaflet of the lipid bilayer causes expansion and results in RBC crenation and echinocytosis; expansion of the inner leaflet of the bilayer results in invagination of the membrane and stomatocytosis. SOURCE: Based on Clinical Expression and Laboratory Detection of Red Cell Membrane Protein Mutations by J. Palek and P. Jarolim in SEMINARS IN HEMATOL- OGY, 30(4), 249–283, October 1993. Published by W.B./Saunders Co., an imprint of Elsevier Health Science Journals. Protein Composition (GPA, GPB, GPC)—are made up of three domains: the cyto- plasmic, the hydrophobic, which spans the bilayer, and the Erythrocyte membrane proteins are of two types: integral extracellular on the exterior surface of the membrane.7 The and peripheral (Figure 5-5). Integral proteins penetrate or tra- extracellular domain is heavily glycosylated and is respon- verse the lipid bilayer and interact with the hydrophobic lipid sible for most of the negative surface charge (zeta potential) core of the membrane. In contrast, peripheral proteins do not that keeps red cells from sticking to each other and to the penetrate the lipid bilayer but interact with integral proteins vessel wall. They also carry various red cell antigens (MN, or lipids at the membrane surface. In the red cell, the major Ss, U, Gerbich antigens).8 GPC also plays a role in attach- peripheral proteins are on the cytoplasmic side of the mem- ing the skeletal protein network located on the cytoplasmic brane attached to membrane lipids or integral proteins by ionic side of the membrane to the lipid bilayer. The glycophorins and hydrogen bonds. Both types of membrane proteins are are synthesized early in erythroid differentiation (GPC is synthesized during erythroblast development. The proteins of found in BFU-E) and thus serve as lineage-specific markers the red cell have been studied by lysing the cell, extracting the for erythrocytic differentiation. proteins,
and analyzing them by polyacrylamide gel electro- Band 3, also known as the anion exchange protein 1 (AE1), phoresis in sodium dodecyl sulfate (SDS-PAGE). The proteins, is the major integral protein of the red cell with more than separated according to molecular weight, were identified by 1 million copies per cell. Band 3 is the transport channel for number according to their migration during electrophoresis the chloride-bicarbonate exchange, which occurs during the with the larger proteins (which migrated the shortest distance) transport of CO2 from the tissues back to the lungs (Chapter beginning the numbering sequence. 6). Like most transport channels, band 3 spans the mem- Integral proteins include transport proteins and the brane multiple times (12–14). Anion exchange is thought to glycophorins. The three major glycophorins—A, B, and C occur by a “ping-pong” mechanism. An intracellular anion The Erythrocyte 75 RBC Skeletal protein Red cell membrane organization lattice Band 3 Glycophorin C Spectrin a b Band 4.2 Band 4.1 Band b a 4.9 Ankyrin Adducin Spectrin tetramer Spectrin dimer Actin oligomer Tropomyosin Tropomodulin Horizontal interactions Figure 5.5 Model of the organization of the erythrocyte membrane showing the peripheral and integral proteins and lipids. Spectrin is the predominant protein of the skeletal protein lattice. Spectrin dimers join head to head to form spectrin tetramers. At the tail end, spectrin tetramers come together at the junctional complex. This complex is composed of actin oligomer and stabilized by tropomyosin, which sits in the groove of the actin filaments. The actin oligomer is capped on one end by tropomodulin and on the other by adducin. Band 4.9 (dematin) binds to actin and bundles actin filaments. Spectrin is attached to actin by band 4.1, which also attaches the skeletal lattice to the lipid membrane via its interaction with glycophorin C (minor attachment site). Ankyrin links the skeletal protein network to the inner side of the lipid bilayer via band 3. Band 4.2 interacts with ankyrin and band 3 (major attachment site). enters the transport channel and is translocated outward transporter proteins (glucose transporter, urea transporter, and released. The channel remains in the outward confor- Na+/K+@ATPase, Ca++@ATPase, Mg++@ATPase), some red mation until an extracellular anion enters and triggers the cell antigens (Rh, Kell), various receptors (transferrin, insu- reverse cycle.8 lin, etc.), and decay-accelerating factor (DAF; Chapter 17). In addition to its role as an anion exchanger, band 3 is a Peripheral membrane proteins are found primarily major binding site for a variety of enzymes and cytoplasmic on the cytoplasmic face of the erythrocyte membrane and membrane components.8 The cytoplasmic domain of band include enzymes and structural proteins (Table 5-2). The 3 binds glycolytic enzymes, regulating their activity; thus, structural proteins are organized into a two-dimensional band 3 serves as a regulator of red cell glycolysis. Band 3 lattice network directly laminating the inner side of the also binds hemoglobin at its cytoplasmic domain with membrane lipid bilayer called the red cell membrane skel- intact hemoglobin binding weakly but partially denatured eton.13 The horizontal interactions of this lattice are parallel hemoglobin (Heinz bodies) binding more avidly. Binding to the membrane’s plane and serve as a skeletal support for of hemoglobin to band 3 is thought to play a role in eryth- the membrane lipid layer. The vertical interactions of the rocyte senescence. Band 3 is also important in attaching the lattice are perpendicular to the membrane’s plane and serve skeletal protein network to the lipid bilayer by binding to to attach the skeletal lattice network to the lipid layer of the the skeletal proteins ankyrin and band 4.2.10 The Ii blood membrane. The skeletal proteins give red cell membranes group antigens are carried on the carbohydrate component their viscoelastic properties and contribute to cell shape, of the red cell band 3 protein.11,12 deformability, and membrane stability. Defects in this cyto- The red cell membrane contains more than 100 addi- skeleton are associated with abnormal cell shape, decreased tional integral proteins.8 These include all of the various stability, and hemolytic anemia (Chapter 17). Vertical inrteractions 76 Chapter 5 Spectrin, the predominant skeletal protein, exists as a of structural integrity and deformability. The 7 mcM eryth- heterodimer of two large chains (a and b). The two chains asso- rocyte must be flexible enough to squeeze through the tiny ciate in a side-by-side, antiparallel arrangement (N-terminal capillary openings, particularly in the splenic vasculature of a chain associated with C-terminal of b chain). The ab (∼3 mcM). At the same time, cells must be able to withstand heterodimers form a slender, twisted, highly flexible molecule the rigors of the turbulent circulation as they travel through- ∼100 nm in length. Spectrin heterodimers in turn self-associate out the body. Deformability of the red cell is due to three head to head to form tetramers and some larger oligomers.8 unique cellular characteristics:15,18 Spectrin is described as functioning like a spring. The spectrin • Its biconcave shape (large surface area-to-volume ratio) tetramers are tightly coiled in vivo with an end-to-end dis- tance of only ∼76 nm (these could have an extended length of • The viscosity of its internal contents (the “solution” of ∼200 nm).14 These coiled tetramers can extend reversibly hemoglobin) when the membrane is stretched but cannot exceed their • The unique viscoelastic properties of the erythrocyte maximum extended length (200 nm) without rupturing. membrane Ankyrin is a large protein that serves as the high-affinity Red cells have an “elastic extension” ability, primarily binding site for the attachment of spectrin to the inner mem- due to the elasticity of coiled spectrin tetramers and associa- brane surface. Ankyrin binds spectrin near the region involved tion–dissociation of skeletal proteins. As a result, the cells in dimer–tetramer associations. In turn, ankyrin is bound with can resume a normal shape after being distorted by an high affinity to the cytoplasmic portion of band 3 (the anchor external applied force. However, application of large or pro- for the membrane skeleton).15 Band 4.2 binds to ankyrin and longed forces can result in reorganization of the cytoskeletal band 3, strengthening their interaction and helping to bind the proteins, producing a permanent deformation (e.g., dacryo- skeleton to the lipid bilayer at its major attachment point.10,16 cytes) or, if the force is excessive, fragmentation (e.g., schis- Red cell actin is functionally similar to actin in other tocytes).18 In addition to being a major component of cells. Red cell actin is organized into short, double-helical erythrocyte deformability, the membrane skeleton is the protofilaments of 12–14 actin monomers. These short fila- principal determinant of erythrocyte stability. The propor- ments are stabilized by their interactions with other pro- tion of spectrin dimers versus tetramers (or higher oligo- teins of the red cell skeleton including tropomodulin, adducin, mers) is a key factor influencing membrane stability.19 tropomyosin, and band 4.9. Spectrin dimers bind to actin Higher proportions of dimers result in increased fragility, filaments near the tail end of the spectrin dimer. Band 4.1 and higher proportions of tetramers and oligomers result in interacts with spectrin and actin and with GPC in the over- stabilization. Also, interaction of the cytoskeleton with the lying lipid bilayer. It serves to stabilize the otherwise weak lipid bilayer and integral membrane proteins stabilizes the interaction between spectrin and actin and is necessary for cell membrane. If the bilayer uncouples from the skeleton, normal membrane stability.8 This complex of spectrin, actin, portions of lipid-rich membrane will be released in the form tropomodulin, tropomyosin, adducin, band 4.9, and band of microvesicles, resulting in a decrease in the surface area- 4.1 serves as the secondary attachment point for the red cell to-volume ratio and the formation of spherocytes (Chapter skeleton, binding to GPC of the membrane. 17).20 The skeletal proteins are in a continuous disassocia- tion–association equilibrium with each other (e.g., spectrin Checkpoint 5.4 dimer-tetramer interconversions) and with their attach- Compare placement in the membrane and function of peripheral ment sites. This equilibrium occurs in response to various and integral erythrocyte membrane proteins. physical and chemical stimuli that affect the erythrocytes as they journey throughout the body. Calcium also influences the red cell cytoskeleton. Most intracellular calcium (80%) is found in association with the erythrocyte membrane. Membrane Permeability Calcium is normally maintained at a low intracellular The red cell membrane is freely permeable to water concentration by the activity of an ATP-fueled Ca++ pump (exchanged by a water channel protein)21 and to anions (Ca++@ATPase). Elevated Ca++ levels induce membrane pro- (exchanged by the anion transport protein, band 3). In tein cross-linking.17 This cross-linking essentially acts as a contrast, the red cell membrane is nearly impermeable to fixative, stabilizing red cell shape and reducing the cell’s monovalent and divalent cations. Glucose is taken up by a deformability. For example, the abnormal erythrocyte shape glucose transporter in a process that does not require ATP of irreversibly sickled cells (Chapter 13) can be produced by nor insulin.22 calcium-induced irreversible cross-linking and alteration of The cations Na+, K+, Ca++, and Mg++ are maintained the cytoskeletal proteins. in the erythrocyte at levels much different than those in The erythrocyte membrane together with the membrane plasma (Table 5-3). Erythrocyte osmotic equilibrium is nor- skeleton is responsible for the dual cellular characteristics mally maintained by both the selective (low) permeability The Erythrocyte 77 2. Hemoglobin iron in the reduced state Table 5.3 Concentration of Cations in the Erythrocyte versus Plasma 3. Reduced sulfhydryl groups in hemoglobin and other proteins Erythrocyte Cation Plasma (mmol/L) (mmol/L) 4. Red cell membrane integrity and deformability Sodium (Na+) 5.4–7.0 135–145 The most important metabolic pathways in the mature Potassium (K+) 98–106 3.6–5.0 erythrocyte are linked to glucose metabolism (Table 5-4). Calcium (Ca++) 0.0059–0.019 2.1–2.6 Because the red cell lacks a citric acid cycle (due to the lack Magnesium (Mg++) 3.06 0.65–1.05 of mitochondria), it is limited to obtaining energy (ATP) solely by anaerobic glycolysis. Glucose enters the red cell through a membrane-associated glucose carrier in a process that does not require ATP or insulin. of the membrane to cations and cation pumps located in the cell membrane. To maintain low intracellular Na+ and Ca++ and high K+ concentrations (relative to plasma concentra- Glycolytic Pathway tions), the red cell utilizes two cation pumps, both of which The erythrocyte obtains its energy in the form of ATP from use intracellular ATP as an energy source. glucose breakdown in the glycolytic pathway, formerly The Na+/K+ cation pump hydrolyzes one mole of ATP known as the Embden-Meyerhof pathway (Figure 5-6). in the expulsion of 3Na+ and the uptake of 2K+. This nor- About 90–95% of the cell’s glucose consumption is metab- mally balances the passive “leaks” of each cation along its olized by this pathway. Normal erythrocytes do not store respective concentration gradient between plasma and cyto- glycogen and depend entirely on plasma glucose for gly- plasm. Calcium plays a role in maintaining low membrane colysis. Glucose is metabolized by this pathway to lactate permeability to Na+ and K+. An increase in intracellular or pyruvate, producing a net gain of 2 moles of ATP per Ca++ allows Na+ and K+ to move in the direction of their mole of glucose. If reduced nicotinamide-adenine dinucleo- concentration gradients.8 Increased intracellular Ca++ also tide (NADH) is available in the cell, pyruvate is reduced activates the Gárdos channel, which causes selective loss of to lactate. The lactate or pyruvate formed is transported K+ and, consequently, water, resulting in dehydration. Low from the cell to the plasma and metabolized elsewhere in intracellular Ca++ is maintained by a Ca++@ATPase pump. the body. The Ca++ pump depends on magnesium to maintain its ATP is necessary to maintain erythrocyte shape, flex- transport function. Although Mg++ is necessary for active ibility, and membrane integrity and to regulate intracellular extrusion of Ca++ from the cell, Mg++ is not transported cation concentration (see previous discussion in the section across the cell membrane in the process. “Membrane -Permeability”). Increased osmotic fragility is If erythrocyte membrane permeability to cations noted in cells with abnormal cation permeability and/or increases or the cation pumps fail (either due to decreased decreased ATP production. Upon the exhaustion of glucose, glucose for generation of ATP via glycolysis or decreased ATP for the cation pumps is no longer available, and cells ATP), Na+ accumulates in the cells in excess of K+ loss. The cannot maintain normal intracellular cation concentrations. result is an increase in intracellular monovalent
cations The cells become sodium and calcium loaded and potas- and water, cell swelling, and, ultimately, osmotic sium depleted. The cell accumulates water and changes hemolysis. from a biconcave disc to a sphere and is removed from the circulation. Checkpoint 5.5 How would an increase in RBC membrane permeability affect Hexose Monophosphate (HMP) intracellular sodium balance? Shunt Not all of the glucose metabolized by the red cell go through Erythrocyte Metabolism the direct glycolytic pathway. Of cellular glucose, 5–10% enters the oxidative HMP shunt, an ancillary system for Although the binding, transport, and release of O2 and CO2 producing reducing substances (Figure 5-6). Glucose- are passive processes not requiring energy, various energy- 6-phosphate is oxidized by the enzyme glucose-6-phos- dependent metabolic processes that are essential to eryth- phate dehydrogenase (G6PD) in the first step of the HMP rocyte viability occur. Energy is required by the erythrocyte shunt. In the process NADP+ is reduced to nicotinaminde- to maintain: adenine dinucleotide phosphate NADPH. 1. The cation pumps, moving cations against electrochem- Glutathione is highly concentrated in the erythrocyte ical gradients and is important in protecting the cell from oxidant damage 78 Chapter 5 Table 5.4 Role of Metabolic Pathways in the Erythrocyte Metabolic Pathway Key Enzymes Function Hematopathology Glycolytic pathway Phosphofructokinase (PFK) Produces ATP accounting for 90% Hemolytic anemia Pyruvate kinase (PK) of glucose consumption in RBC Hereditary PK deficiency Hexose-monophosphate shunt Glutathione reductase (GR) Provides NADPH and glutathione Hemolytic anemia Glucose-6-phosphate dehydroge- to reduce oxidants that would shift Hereditary G6PD deficiency nase (G6PD) the balance of oxyhemoglobin to Glutathione reductase deficiency methemoglobin Hemoglobinopathies Rapoport-Luebering BPG-synthase Controls the amount of 2,3-BPG Hypoxia produced, which in turn affects the oxygen affinity of hemoglobin Methemoglobin reductase Methemoglobin reductase Protects hemoglobin from oxidation Hemolytic anemia via NADH (from glycolytic pathway) Hypoxia and methemoglobin reductase by reactive oxygen species (ROSs) produced during oxy- reduced (ferrous) state, Fe++ (Figure 5-6). Methemoglobin gen transport and by other oxidants such as chemicals and (hemoglobin with iron in the oxidized ferric state, Fe+++) is drugs. In the process of reducing oxidants, glutathione itself generated simultaneously with the oxidative compounds is oxidized. (Reduced glutathione is referred to as GSH, discussed earlier as O2 dissociates from the heme iron. Met- and the oxidized form is referred to as GSSG.) NADPH hemoglobin cannot bind oxygen. The enzyme methemoglo- produced by the HMP shunt converts GSSG back to GSH, bin reductase (also known as NADH diaphorase, or the form necessary to maintain hemoglobin in the reduced cytochrome b5) with NADH produced by the glycolytic functional state. The erythrocyte normally maintains a large pathway functions to reduce the ferric iron in methemoglo- ratio of NADPH to NADP+. When the HMP shunt is defec- bin, converting it back to ferrous hemoglobin. In the absence tive, hemoglobin sulfhydryl groups (-SH) are oxidized, of this system, the 2% of methemoglobin formed daily even- which leads to denaturation and precipitation of hemoglo- tually builds up to 20–40%, severely limiting the blood’s bin in the form of a Heinz body. Heinz bodies attach to the oxygen-carrying capacity. Certain oxidant drugs can inter- inner surface of the cell membrane, decreasing cell flexibil- fere with methemoglobin reductase and cause even higher ity. Macrophages in the spleen remove them from the cell levels of methemoglobin. This results in cyanosis (a bluish together with a portion of the membrane. These bodies can discoloration of the skin due to an increased concentration be visualized with supravital stains (Chapter 37). If large of deoxyhemoglobin in the blood). portions of the membrane are damaged in this manner, the whole cell may be removed. This commonly occurs in patients with G6PD deficiency (Chapter 18). Checkpoint 5.6 GSH also is responsible for maintaining reduced -SH Uncontrolled oxidation of hemoglobin results in what RBC intra- groups of cytoskeletal proteins and membrane lipids. cellular inclusion? Decreased GSH leads to oxidative injury of membrane protein -SH groups, compromising protein function and resulting in “leaky” cell membranes. Cellular depletion of ATP can then Rapoport-Luebering Shunt occur due to increased consumption of energy by the cation pumps. The Rapoport-Luebering shunt is a part of the glycolytic Ascorbic acid, or vitamin C, is also an important pathway (Figure 5-6), which bypasses the formation of antioxidant in the erythrocyte as it consumes oxygen 3-phosphoglycerate and ATP from 1,3-bisphosphoglycerate free radicals and helps preserve alpha-tocopherol (vita- (1,3-BPG). Instead, 1,3-BPG forms 2,3-BPG (also known as min E, another important antioxidant) in membrane 2,3-diphosphoglycerate [2,3-DPG]) catalyzed by BPG mutase. lipoproteins.23 Therefore, the erythrocyte sacrifices one of its two ATP-pro- ducing steps in order to form 2,3-BPG. When hemoglobin binds 2,3-BPG, oxygen release is facilitated (i.e., binding 2,3- Methemoglobin Reductase Pathway BPG causes a decrease in hemoglobin affinity for oxygen). The methemoglobin reductase pathway, an offshoot of the Thus, 2,3-BPG plays an important role in regulating oxygen glycolytic pathway, is essential to maintain heme iron in the delivery to the tissues (Chapter 6). The Erythrocyte 79 Glycolytic pathway Hexose monophosphate pathway H2O2 H2O GP Glucose ATP GSH GSSG Hexokinase GR ADP NADP NADPH G6P 6–PG G6PD PI 6PDG F6P PP ATP PFK ADP Fructose 1, 6-biP Aldolase Glyceraldehyde 3P NAD Methemoglobin reductase + H Methemoglobin Methemoglobin G3PD reductase NADH Methemoglobin reductase Hemoglobin pathway Rapoport-Luebering shunt 1,3 BPG Mutase ADP 2,3 BPG BPG synthase PGK ATP Phosphatase 3 PG 2 PG Enolase PEP ADP PK ATP Pyruvate LD Lactate Figure 5.6 Erythrocyte metabolic pathways. The glycolytic pathway is the major source of energy for the erythrocyte through production of ATP. The hexose-monophosphate pathway is important for reducing oxidants by coupling oxidative metabolism with pyridine nucleotide (NADP) and glutathione (GSSG) reduction. The methemoglobin reductase pathway supports methemoglobin reduction. The Rapoport- Luebering Shunt produces 2,3-BPG, which alters hemoglobin-oxygen affinity. G6P, glucose- 6-phosphate; PI, glucose-6-phosphate isomerase; F6P, fructose-6-phosphate; PFK, 6-phosphofructokinase; fructose 1,6-biP, fructose 1,6-bisphosphate; Glyceraldehyde 3P, glyceraldehyde 3-phosphate; G3PD, glyceraldehyde 3-phosphate dehydrogenase; 1,3 BPG, 1, 3-bisphosphoglycerate; PGK, phosphoglycerate kinase; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; PK, pyruvate kinase; LD, lactate dehydrogenase; GP, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione reduced; GSSG, glutathione oxidized; G6PD, glucose-6-phosphate dehydrogenase; 6-PG, 6-phosphogluconate; 6PDG, 6-phosphodehydrogenase gluconate; PP, pentose phosphate. 80 Chapter 5 g/dL) at birth are followed by a gradual decrease that con- CASE STUDY (continued from page 72) tinues until about the second or third month of extrauterine Stephen was admitted for identification and treat- life. At this time, red blood cell and hemoglobin values fall to ment of the anemia. More lab tests were ordered 3.194.3 * 1012/L and 9.0913 g/dL, respectively.25 This eryth- with the following results: rocyte decrease in infancy is sometimes called physiologic ane- Patient Reference mia of the newborn, the result of a temporary cessation of bone Interval marrow erythropoiesis after birth due to a low concentration of EPO. EPO levels are high in the fetus due to the relatively Total bilirubin 4.8 mg/dL 0.1–1.2 hypoxic environment in utero and the high oxygen affinity of Direct bilirubin 1.6 mg/dL 0.1–1.0 hemoglobin F (fetal hemoglobin). After birth, however, when Haptoglobin 25 mg/dL 35–165 the lungs replace the placenta as the means of providing oxy- Hemoglobin gen, the arterial blood oxygen saturation rises from ∼45, to electrophoresis ∼95,. EPO cannot be detected in the infant’s plasma from HbA 98% More than about the first week of extrauterine life until the second or 95% third month. Reticulocytes reflect the bone marrow activity Hb-F 1% Less than during this time. At birth and for the next few days, the mean 2% reticulocyte count is high (1.8–8.0%). Within a week, the count Hb-A2 1% 1.5–3.7% drops and remains low (less than 1%) until about the second month of life, at which time EPO levels rise again (when the Heinz body stain Positive Negative hemoglobin levels fall to ∼12 g/dL). This corresponds to the Fluorescent spot test for Positive Negative recovery from “physiologic anemia of the newborn.” G6PD deficiency Males have a higher erythrocyte concentration than 2. What cellular mechanism results in hemolysis females after puberty due to the presence of testosterone. due to a deficiency in G6PD? Before puberty and after “male menopause,” males and females have comparable erythrocyte levels.26,27 Testosterone 3. Explain how Heinz body inclusions cause dam- stimulates renal and extrarenal EPO production and directly age to the erythrocyte membrane. enhances differentiation of marrow stem and progenitor cells.28 Individuals living at high altitudes have a higher mean erythrocyte concentration than those living at sea level. Decreases in the partial pressure of atmospheric oxygen at high Checkpoint 5.7 altitudes result in a physiologic increase in erythrocytes in the Which erythrocyte metabolic pathway is responsible for provid- body’s attempt to provide adequate tissue oxygenation. ing the majority of cellular energy? For regulating oxygen affinity? For maintaining hemoglobin iron in the reduced state? Checkpoint 5.8 Why are there different reference intervals for hemoglobin con- Erythrocyte Kinetics centration in male and female adults but not in male and female children? In the late 1800s, it was observed that individuals living at high altitudes had an increase in erythrocytes, which was attributed to an acquired adjustment to the reduced atmo- Regulation of Erythrocyte Production spheric pressure of oxygen.24 Over the following decades, it was discovered that the stimulation of erythropoiesis in The body can regulate the number of circulating erythro- the bone marrow in response to decreased oxygen levels cytes by changing the rate of cell production in the marrow was the result of a hormone, erythropoietin (EPO), that and/or the rate of cell release from the marrow. Delivery of is released into the peripheral blood by renal tissue in erythrocytes to the circulation is normally well balanced to response to hypoxia. Hormonal control of red blood cell match the rate of erythrocyte destruction, which does not mass is closely regulated and is normally maintained in a vary significantly under steady-state conditions. Impaired steady state within narrow limits. oxygen delivery to the tissues and low intracellular oxygen tension (PO2) trigger increased EPO release and increased Erythrocyte Concentration erythrocyte production by the marrow. Conditions that stimulate erythropoiesis include anemia, cardiac or pul- The normal erythrocyte concentration varies with sex, monary disorders, abnormal hemoglobins, and high alti- age, and geographic location. A high erythrocyte count tude. Erythropoiesis is influenced by a number of cytokines 3.995.9 * 1012 L) and hemoglobin concentration (13.5–20 including SCF, IL-3, GM-CSF/CSF2, and EPO (Chapter 4). The Erythrocyte 81 However, EPO is the principal cytokine essential for termi- action is stimulation of committed progenitor cells, primar- nal erythrocyte maturation. ily the CFU-E, to survive, proliferate, and differentiate (see EPO is a thermostable renal glycoprotein hormone the section “Erythropoiesis and Red Blood Cell Maturation” with a molecular weight of about 34,000 daltons. Renal earlier in this chapter). A subset of BFU-E has EPO-R but in cortical interstitial cells secrete EPO in response to cellu- small number, and BFU-Es are largely unresponsive to the lar hypoxia.29 This feedback control of erythropoiesis is the effects of EPO. Thus, under conditions of EPO stimulation, mechanism by which the body maintains optimal erythro- the primary elements of the erythroid precursor cells that cyte mass for tissue oxygenation. Plasma levels of EPO are respond are the CFU-Es and early normoblasts. However, constant when the hemoglobin concentration is within the acute demands for erythropoiesis with extremely high EPO reference interval but increase steeply when the hemoglobin levels can stimulate the BFU-E. When this occurs, the char- decreases below 12 g/dL.30 EPO is also produced by extra- acteristics of the resulting erythrocytes include an increase in renal sources, including marrow macrophages and stromal mean corpuscular volume (MCV) and an increase in i anti- cells, which likely contribute to steady-state erythropoiesis.2 gen and HbF concentration.2 EPO-Rs on the cell membrane However, under conditions of tissue hypoxia, oxygen sen- increase as the BFU-E matures to the CFU-E and gradually sors in the kidneys trigger the release of renal EPO, result- decrease as the normoblasts mature. The EPO-R is absent on ing in an increased stimulus for erythropoiesis. reticulocytes. Other effects of EPO are described in Table 5-5.38 EPO has been defined in biologic terms to have an activ- A major way by which EPO increases RBC production ity of ∼130,000 IU/mg of protein.31 Normal plasma contains is by preventing apoptosis. Erythropoiesis is maintained from 3.1 to 16.5 IU of EPO per L of serum.32 EPO can also by a finely tuned balance between the positive signals be
found in the urine at concentrations proportional to that generated by EPO and negative signals from death recep- found in the plasma33 (Table 5-5). In anemia, EPO plasma tor ligands and inhibitory cytokines (Chapters 2, 4). Ery- levels are related to both hemoglobin concentration and the throid progenitors differ in their sensitivity to EPO; some pathophysiology of the anemia. For example, patients with progenitors require much less EPO than others to survive pure erythrocyte aplasia (Chapter 16) have plasma EPO levels and mature to reticulocytes.39 Progenitors with increased significantly higher than patients with iron deficiency anemia sensitivity to EPO are thought to provide RBC production or megaloblastic anemia even though hemoglobin concentra- when EPO levels are normal or decreased. Progenitors that tion in all three types of anemia may be similar. Plasma EPO require high concentrations of EPO die of apoptosis under levels reflect not only EPO production but also its disappear- these conditions. Progenitors requiring high concentrations ance from the blood and/or utilization by the bone marrow of EPO, however, will be rescued from apoptosis when EPO (i.e., uptake by EPO-receptor-bearing cells in the marrow). concentrations are elevated as occurs in anemia, thus pro- Patients with renal disease and nephrectomized patients viding increased erythrocytes under these conditions. are usually severely anemic, but they continue to make The EPO-R exists in the membrane as a homodimer and some erythrocytes and produce limited amounts of EPO in lacks intrinsic kinase activity. However, the cytoplasmic tail response to hypoxia. In addition to the production of EPO by of the receptor recruits and binds cytoplasmic kinases, Janus marrow macrophages and stromal cells, hepatocytes act as an kinase 2 (JAK-2), which are activated when EPO binds to extrarenal source of EPO, but normally account for less than 15% of the total EPO production in humans.34 The adult liver appears to require a more severe hypoxic stimulus for EPO Table 5.5 Characteristics of Erythropoietin production than the kidney. The liver is the major site of EPO production during fetal development, but at birth, a gradual General Characteristics shift from hepatic to renal production of EPO occurs.35 Composition Glycoprotein Increased EPO secretion is due to de novo synthesis of Stimulus for synthesis Cellular hypoxia EPO rather than release of preformed stores. The hypoxia- Origin Kidneys 80–90% induced increase of EPO is due to both increased gene Liver less than 15% transcription mediated by the transcription factor hypoxia- Reference interval Serum 3.1–16.5 IU/L inducible factor-1 (HIF-1), and stabilization of EPO mes- Functions senger RNA.36,37 Under hypoxic conditions, HIF-1 binds to Stimulates BFU-E and CFU-E to divide and mature DNA regulatory sequences (hypoxia-responsive element Increases rate of mRNA and protein (hemoglobin) synthesis [HRE]) in the EPO gene, activating transcription. Under con- Decreases normoblast maturation time ditions of normal oxygen concentration, HIF-1 is degraded by a hydroxylase enzyme that requires oxygen for activity, Increases rate of enucleation (extrusion of nucleus) resulting in a decreased production of EPO mRNA.38 Stimulates early release of bone marrow reticulocytes (shift reticulocytes) EPO exerts its action by binding to specific receptors Response to Anemia (EPO-R) on erythropoietin-responsive cells. EPO’s major Generally increased except in anemia of renal disease 82 Chapter 5 the EPO-R (see Figure 4-7). At least four different signaling in oxidation of critical membrane proteins, lipids, and hemo- pathways are activated by this EPO/EPO-R/JAK-2 interac- globin, loss of the ability to maintain cell shape and deform- tion. Abnormal interactions and/or function of these com- ability, and loss of membrane integrity, all of which contribute ponents have been linked to familial forms of erythrocytosis to the cell’s removal.42,43,44 Oxidative damage also causes and certain myeloproliferative disorders (Chapter 24). clustering of band 3 molecules, which can be a senescence- The normal bone marrow can increase erythropoiesis identifying feature. The glucose supply in the spleen is low, 5- to 10-fold in response to increased EPO stimulation if limiting the energy-producing process of glycolysis within sufficient iron is available. Erythropoiesis is affected (and the erythrocyte. Aged erythrocytes can quickly deplete their limited) by serum iron levels and by transferrin satura- cellular level of ATP, resulting in limited ability to maintain tion40 (Chapter 6). In hemolytic anemia, a readily available osmotic equilibrium via the energy-dependent cation pumps. supply of iron is recycled from erythrocytes destroyed in Additionally, aged erythrocytes accumulate IgG (an immu- vivo that results in a sustained ∼6@fold increase in eryth- noglobulin) on their membrane. Splenic macrophages have ropoiesis. The rate of erythropoiesis in blood loss anemia receptors for this IgG, which can enhance recognition of aged during which iron is lost from the body, however, depends cells. The exposure of phosphatidylserine (PS) on the outer more on preexisting iron stores. In this case, the rate of eryth- leaflet of the erythrocyte membrane (normally concentrated ropoiesis usually does not exceed 2.5 times normal unless on the inner leaflet of the membrane) is another signal that large parenteral or oral doses of iron are administered. allows macrophages to recognize senescent erythrocytes.45 A number of tumors have been reported to cause an This is the only major difference between senescent and non- increase in erythropoietin production. Stimulation of the senescent erythrocytes that has been clearly documented.46 hypothalamus can cause an increased release of EPO from Any combination of these events could contribute to the trap- the kidneys, explaining the association of polycythemia and ping of erythrocytes in the vasculature of the spleen and their cerebellar tumors. The serum EPO level increases dramati- removal by splenic macrophages. cally in patients undergoing chemotherapy for leukemia as The chromatin condensation and mitochondrial destruc- well as other cancers in response to marrow suppression by tion that occurs during erythrocyte production parallel chemotherapeutic agents.40 changes seen in apoptotic cells, as does the PS externalization The production of synthetic hematopoietic growth fac- seen in erythrocyte senescence. The parallels with apoptosis tors using recombinant DNA technology has revolutionized have led some researchers to speculate that erythrocyte matu- the management of patients with some anemias. Several ration and senescence represent “apoptosis in slow motion.”47 recombinant forms of human EPO (rHuEPO) are available Erythrocyte removal by the spleen, bone marrow, and and are commonly used for treatment of the anemia associ- liver is referred to as extravascular destruction. This pathway ated with end-stage renal disease and chemotherapy as well accounts for about 90% of aged erythrocyte destruction. It as HIV-related anemia.31,41 is the most efficient method of cell removal, conserving and recycling essential erythrocyte components such as amino acids and iron. (Hemoglobin catabolism is covered in detail CASE STUDY (continued from page 80) in Chapter 6.) Most extravascular destruction of erythro- cytes takes place in the macrophages of the spleen. The 4. Would you predict Stephen’s serum erythropoi- spleen’s architecture with its torturous circulation, sluggish etin levels to be low, normal, or increased? Why? blood flow, and relative hypoxic and hypoglycemic envi- ronment makes it well suited for culling aged erythrocytes and trapping those cells that have minimal defects (Chapter 3). In contrast to the macrophages in the spleen, the liver Checkpoint 5.9 macrophages lack the ability to detect cells with minimal What would the predicted serum EPO levels be in a patient with defects. However, because the liver receives 35% of the car- an anemia due to end-stage kidney disease? diac output (the spleen receives 5%) it can be more efficient in removing cells it recognizes as abnormal. Erythrocyte Destruction Severe trauma to the RBC that damages the cell’s struc- tural integrity and leads to a breach in the cell membrane Red blood cell destruction is normally the result of senescence. results in intravascular cell lysis and release of hemoglobin Several theories have been proposed to explain the under- directly into the blood (intravascular destruction). Only about lying pathology of senescent red cells. Erythrocyte aging is 10% of erythrocyte destruction occurs in this manner. characterized by a decline in certain cellular enzyme systems, Released hemoglobin is bound by the plasma proteins hap- including glycolytic enzymes and enzymes needed for main- toglobin and hemopexin and is transported to the liver tenance of redox status. This in turn leads to decreased ATP where the hemoglobin is catabolized similar to the process production and loss of adequate reducing systems, resulting in extravascular hemolysis. The Erythrocyte 83 Checkpoint 5.10 CASE STUDY (continued from page 82) Explain how oxidation of RBC cellular components can lead to extravascular hemolysis. 5. Stephen’s haptoglobin level is 25 mg/dL. Explain why he has a low haptoglobin value. Summary Erythrocytes are derived from the unipotent committed Although to date we know a lot about the structure of the progenitor cells BFU-E and CFU-E. Morphologic devel- red cell membrane, there is still a great deal to be learned opmental stages of the erythroid cell include (in order of about the physiology of the membrane’s skeleton, the struc- increasing maturity) the pronormoblast, basophilic nor- ture of spectrin in situ, the way spectrin and other proteins moblast, polychromatophilic normoblast, orthochromatic bind to actin, and how the membrane is assembled.48 The normoblast, reticulocyte, and erythrocyte. Erythropoietin, normal erythrocyte life span is 100–120 days. a hormone produced in renal tissues, stimulates erythro- The erythrocyte derives its energy and reducing power poiesis and is responsible for maintaining a steady-state from glycolysis and ancillary pathways. The glycolytic erythrocyte mass. Erythropoietin stimulates survival and pathway provides ATP to help the cell maintain erythrocyte differentiation of erythroid progenitor cells, increases the shape, flexibility, and membrane integrity through regula- rate of erythropoiesis, and induces early release of reticulo- tion of intracellular cation permeability. The HMP shunt cytes from the marrow. provides reducing power to protect the cell from permanent The erythrocyte concentration varies with sex, age, and oxidative injury. The methemoglobin reductase pathway geographic location. Higher concentrations are found in helps reduce heme iron from the ferric (Fe+++) to the fer- males (after puberty) and newborns and at high altitudes. rous (Fe++) state. The Rapoport-Luebering shunt facilitates Decreases below the reference interval result in a condition oxygen delivery to the tissue by producing 2,3-BPG. called anemia. Destruction of aged erythrocytes occurs primarily in The erythrocyte membrane is a lipid-protein bilayer the macrophages of the spleen and liver through processes complex that is important for maintaining cellular deform- known as extravascular and intravascular destruction. Extra- ability and selective permeability. As the cell ages, the mem- vascular destruction of the erythrocyte is the major physi- brane is reduced in surface area relative to cell volume, and ological pathway for aged or abnormal erythrocyte removal the cell becomes more rigid and is culled in the spleen. (splenic and hepatic macrophages). Review Questions Level I 3. An increase in 2,3-BPG occurs at high altitude in an effort to: (Objective 9) 1. The earliest recognizable erythroid precursor on a Wright-stained smear of the bone marrow is: (Objec- a. increase oxygen affinity of hemoglobin tive 1) b. decrease oxygen affinity of hemoglobin a. pronormoblast c. decrease the concentration of methemoglobin b. basophilic normoblast d. protect the cell from oxidant damage c. CFU-E 4. The erythrocyte life span is most directly determined d. BFU-E by: (Objective 5) 2. This renal hormone stimulates erythropoiesis in the a. spleen size bone marrow: (Objective 4) b. serum haptoglobin level a. IL-1 c. membrane deformability b. erythropoietin d. cell size and shape c. granulopoietin d. thrombopoietin 84 Chapter 5 5. Which of the following depicts the normal sequence Level II of erythroid maturation? (Objective 1) 1. Results of a CBC revealed a MCHC of 40 g/dL. What a. Pronormoblast S basophilic normoblast S poly- characteristic of the RBC will this affect? (Objective 2) chromatic normoblast S orthochromic normo- blast S a. Oxygen affinity reticulocyte b. Cell metabolism b. Pronormoblast S polychromatic normoblast S orthochromic normoblast S basophilic normo- c. Membrane permeability blast S reticulocyte d. Cell deformability c. Basophilic normoblast S polychromatic normo- blast S reticulocyte S orthochromic normoblast 2. If the erythrocyte cation pump fails because of inad- S pronormoblast equate generation of ATP, the result is: (Objective 3) d. Orthochromic normoblast S basophilic normo- a. decreased osmotic fragility due to formation of blast S reticulocyte S polychromatic normo- target cell blast S pronormoblast b. formation of echinocytes due to influx of potassium 6. The primary effector (cause) of increased erythrocyte c. cell crenation due to efflux of water and sodium production, or erythropoiesis, is: (Objective 4) d. cell swelling due to influx
of water and cations a. supply of iron b. rate of bilirubin production 3. As a person ascends to high altitudes, the increased c. tissue hypoxia activity of the Rapoport-Luebering pathway: (Objec- tive 4) d. rate of EPO secretion a. results in precipitation of hemoglobin as Heinz 7. An increase in the reticulocyte count should be bodies accompanied by: (Objective 2) b. has no effect on oxygen delivery to tissues a. a shift to the left in the Hb@O2 dissociation curve c. results in increased release of oxygen to tissues b. abnormal maturation of normoblasts in the bone d. results in decreased release of oxygen to tissues marrow c. an increase in total and direct serum bilirubin 4. A newborn has a hemoglobin level of 16.0 g/dL at birth. Two months later, a CBC indicates a hemo- d. polychromasia on the Wright's-stained blood globin concentration of 11.0 g/dL. The difference smear in hemoglobin concentration is most likely due to: 8. What property of the normal erythrocyte membrane (Objective 1) allows the 7-mcM cell to squeeze through 3-mcM fen- a. chronic blood loss estrations in the spleen? (Objective 5) b. inherited anemia a. Fluidity c. increased intravascular hemolysis b. Elasticity d. physiologic anemia of the newborn c. Permeability d. Deformability 5. A 50-year-old patient had a splenectomy after a car accident that damaged her spleen. She had a CBC 9. An increase of erythrocyte membrane rigidity would performed at her 6-week postsurgical checkup. Many be predicted to have what effect? (Objective 5) target cells were identified on the blood smear. This finding is most likely: (Objective 2) a. Increase in erythropoietin production b. Increase in cell volume a. an indication of liver disease c. Decrease in cell life span b. a loss of RBC membrane peripheral proteins d. Decrease in reticulocytosis c. an abnormal protein to phospholipid ratio of the RBC membrane 10. Extravascular erythrocyte destruction occurs in: d. an accumulation of cholesterol and phospholipid (Objective 7) in the RBC membrane a. the bloodstream b. macrophages in the spleen c. the lymph nodes d. bone marrow sinuses The Erythrocyte 85 6. Which of the following is necessary to maintain c. abnormal RBC membrane permeability reduced levels of methemoglobin in the erythrocyte? d. RBC fragility due to accumulation of intracellular (Objective 4) calcium a. Vitamin B6 9. A laboratory professional finds evidence of Heinz b. NADH bodies in the erythrocytes of a 30-year-old male. This c. 2,3-BPG is evidence of: (Objective 4) d. Lactate a. increased oxidant concentration in the cell 7. A patient lost about 1500 mL of blood during surgery b. decreased hemoglobin-oxygen affinity but was not given blood transfusions. His hemoglobin c. decreased production of ATP before surgery was in the reference range. The most d. decreased stability of the cell membrane likely finding 3 days later would be: (Objective 1, 6) a. increase in total bilirubin 10. A 65-year-old female presents with an anemia of 3 weeks’ duration. In addition to a decrease in her b. increase in indirect bilirubin hemoglobin and hematocrit, she has a reticulocyte c. increase in erythropoietin count of 6% and 3+ polychromasia on her blood d. increased haptoglobin smear. Based on these preliminary findings, what serum erythropoietin result is expected? (Objective 6) 8. A patient with kidney disease has a hemoglobin of 8 a. Decreased g/dL. This is most likely associated with: (Objective 6) b. Normal c. Increased a. decreased EPO production d. No correlation b. increased intravascular hemolysis Disclaimer The views expressed in this chapter are those of the author the Army/Navy/Air Force, Department of Defense, or and do not reflect the official policy of the Department of U.S. Government. References 1. Beutler, E. (1980). The red cell: A tiny dynamo. In M. M. Win- 6. Chasis, J. A., & Mohandas, N. (2008). Erythroblastic islands: trobe (Ed.), Blood, pure and eloquent (pp. 141–168). New York: Niches for erythropoiesis. Blood, 112(3), 470–478. doi: http://doi. McGraw-Hill. org/10.1182/blood-2008-03-077883. 2. Papayannopoulou, T., & Migliaccio, A. R. (2013). Biology of 7. Mohandas, N. (1995). The red cell membrane. In R. Hoffman, E. J. erythropoiesis, erythroid differentiation, and maturation. Benz, S. J. Shattil, B. Furie, H. J. Cohen, & L. E. Silberstein (Eds.), In: R. Hoffman, E. J. Benz, L. E. Silberstein, H. E. Heslop, & Hematology: Basic principles and practice (pp. 264–269). Philadel- J. I. Weitz, & J. Anastasi, eds. Hematology: Basic principles and phia: Elsevier Churchill Livingstone. practice (6th ed., pp. 258–279). Philadelphia: Elsevier Saunders. 8. Lux, S. E., & Palek, J. (1995). Disorders of the red cell mem- 3. Gregg, X. T. (2015). Erythropoietic effects of endocrine disorders. brane. In R. I. Handin, S. E. Lux, & T. P. Stossel (Eds.), Blood In: K. Kaushansky, M. A. Lichtman, J.T. Prchal, M. M. Levi, O. W. principles and practice of hematology (pp. 1701–1818). Philadelphia: Press, L. J. Burns, & M. Caligiuri, eds. Williams hematology Lippincott. (9th ed.). New York: McGraw-Hill. Retrieved from http://access- 9. Devaux, P. F. (1992). Protein involvement in transmembrane lipid medicine.mhmedical.com.proxy.libraries.rutgers.edu/content. asymmetry. Annual Review of Biophysics and Biomolecular Structure, aspx?bookid=1581&Sectionid=94304032. 21(1), 417–439. 4. Narla, M. (2015). Structure and composition of the erythrocyte. 10. Butler, J., Mohandas, N., & Waugh, R. E. (2008). Integral Pro- In: K. Kaushansky, M. A. Lichtman, J. T. Prchal, M. M. Levi, O. W. tein Linkage and the Bilayer-Skeletal Separation Energy in Red Press, L. J. Burns, & M. Caligiuri, eds. Williams hematology Blood Cells. Biophysical Journal, 95(4), 1826–1836. doi: http://doi. (9th ed.). New York: McGraw-Hill. Retrieved from http://access- org/10.1529/biophysj.108.129163. medicine.mhmedical.com.proxy.libraries.rutgers.edu/content.asp 11. Fukuda, M., Dell, A., & Fukuda, M. N. (1984). Structure of fetal bookid=1581&Sectionid=94303279. lactosaminoglycan: The carbohydrate moiety of Band 3 isolated 5. Liu, J., Guo, X., Mohandas, N., Chasis, J. A., & An, X. (2010). from human umbilical cord erythrocytes. Journal of Biological Membrane remodeling during reticulocyte maturation. Chemistry, 259(8), 4782–4791. Blood, 115(10), 2021–2027. doi: http://doi.org/10.1182/ 12. Fukuda, M., Dell, A., Oates, J. E., & Fukuda, M. N. (1984). Struc- blood-2009-08-241182. ture of branched lactosaminoglycan, the carbohydrate moiety 86 Chapter 5 of band 3 isolated from adult human erythrocytes. Journal of Erythropoietin in the general population: Reference ranges and clin- Biological Chemistry, 259(13), 8260–8273. ical, biochemical and genetic correlates. PloS One, 10(4), e0125215. 13. Platt, O. S. (1988). Inherited disorders of red cell membrane pro- 33. Marsden, J. T. (2006). Erythropoietin-measurement and clinical teins. In R. L. Nagel (Ed.), Genetically abnormal red cells. applications. Annals of Clinical Biochemistry, 43(2), 97–104. Boca Raton, FL: CRC Press. (pp. 663-680). 34. Hodges, V. M., Rainey, S., Lappin, T. R., & Maxwell, A. P. (2007). 14. Vertessy, B. G., & Steck, T. L. (1989). Elasticity of the human red Pathophysiology of anemia and erythrocytosis. Critical Reviews in cell membrane skeleton. Effects of temperature and denaturants. Oncology/Hematology, 64(2), 139–158. Biophysical Journal, 55(2), 255–262. 35. Zanjani, E. D., Ascensao, J. L., McGlave, P. B., Banisadre, M., & 15. Bennett, V., & Stenbuck, P. J. (1979). The membrane attachment Ash, R. C. (1981). Studies on the liver to kidney switch of erythro- protein for spectrin is associated with band 3 in human erythro- poietin production. Journal of Clinical Investigation, 67(4), 1183. cyte membranes. Nature, 280(5722), 468–473. 36. Nangaku, M., & Eckardt, K. U. (2007). Hypoxia and the HIF 16. Cohen, C. M., Dotimas, E., & Korsgren, C. (1993). Human eryth- system in kidney disease. Journal of Molecular Medicine, 85(12), rocyte membrane protein band 4.2 (pallidin). Seminars in 1325–1330. Hematology, 30(2), 119–137. 37. Semenza, G. L., & Wang, G. L. (1992). A nuclear factor induced 17. Lorand, L., Siefring Jr, G. E., & Lowe-Krentz, L. (1979). Enzymatic by hypoxia via de novo protein synthesis binds to the human basis of membrane stiffening in human erythroyctes. Seminars in erythropoietin gene enhancer at a site required for transcriptional Hematology, 16(1), 65. activation. Molecular and Cellular Biology, 12(12), 5447–5454. 18. Mohandas, N., & Chasis, J. A. (1993). Red blood cell deform- 38. Unger, E. F., Thompson, A. M., Blank, M. J., & Temple, R. (2010). ability, membrane material properties and shape: Regulation by Erythropoiesis-stimulating agents—time for a reevaluation. transmembrane, skeletal and cytosolic proteins and lipids. New England Journal of Medicine, 362(3), 189–192. Seminars in Hematology, 30(3), 171–192. 39. Kelley, L. L., Koury, M. J., Bondurant, M. C., Koury, S. T., Sawyer, 19. Liu, S. C., & Palek, J. (1980). Spectrin tetramer–dimer equilibrium S. T., & Wickrema, A. (1993). Survival or death of individual pro- and the stability of erythrocyte membrane skeletons. Nature, erythroblasts results from differing erythropoietin sensitivities: 285(5766), 586–588. A mechanism for controlled rates of erythrocyte production. 20. Liu, S. C., & Derick, L. H. (1992). Molecular anatomy of the red Blood, 82(8), 2340–2352. blood cell membrane skeleton: structure-function relationships. 40. Sawabe, Y., Takiguchi, Y., Kikuno, K., Iseki, T., Ito, J., Iida, S., . . . Seminars in Hematology, 29(4) 231–243. Yonemitsu, H. (1998). Changes in levels of serum erythropoietin, 21. Zeidel, M. L., Ambudkar, S. V., Smith, B. L., & Agre, P. (1992). serum iron and unsaturated iron binding capacity during chemo- Reconstitution of functional water channels in liposomes con- therapy for lung cancer. Japanese Journal of Clinical Oncology, 28(3), taining purified red cell CHIP28 protein. Biochemistry, 31(33), 182–186. 7436–7440. 41. Kimmel, P. L., Greer, J. W., Milam, R. A., & Thamer, M. (2000). 22. Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Trends in erythropoietin therapy in the US dialysis population: Morris, H. R., . . . Lodish, H. F. (1985). Sequence and structure of a 1995 to 1998. Seminars in Nephrology, 20(4), 335–344. human glucose transporter. Science, 229(4717), 941–945. 42. Suzuki, T., & Dale, G. L. (1988). Senescent erythrocytes: Isolation 23. May, J. M. (1998). Ascorbate function and metabolism in the of in vivo aged cells and their biochemical characteristics. human erythrocyte. Frontiers in Bioscience, 3, d1–d10. Proceedings of the National Academy of Sciences, 85(5), 1647–1651. 24. Viault, F. (1890). Sur l'augmentation considérable du nombre des 43. Zimran, A., Forman, L., Suzuki, T., Dale, G. L., & Beutler, E. globules rouges dans le sang chez les habitants des hauts pla- (1990). In vivo aging of red cell enzymes: Study of biotinylated teaux de l'Amérique du Sud [High Altitude Physiology]. Comptes red blood cells in rabbits. American Journal of Hematology, 33(4), Rendus de l’Académie des Sciences, 111, 917–918. 249–254. 25. Bao, W. H., Dalferes, E. R., Srinivasan, S. R., Webber, L. S., & 44. Steinberg, M. H., Benz, E. J., Adewoye, A. H., & Ebert, B. L. Berenson, G. S. (1993). Normative distribution of complete blood (2013). Pathobiology of the human erythrocyte and its hemoglo- count from early childhood through adolescence: The Bogalusa bins. In: R. Hoffman, E. J. Benz, L. E. Silberstein, H. E. Heslop, J. Heart Study. Preventive Medicine, 22(6), 825–837. I. Weitz, & J. Anastasi, eds. Hematology: Basic principles and practice 26. Cavalieri, T. A., Chopra, A., & Bryman, P. N. (1992). When out- (6th ed., pp. 406–417). Philadelphia: Elsevier Saunders. side the norm is normal: Interpreting lab data in the aged. Geriat- 45. Boas, F. E., Forman, L., & Beutler, E. (1998). Phosphatidylserine rics, 47(5), 66–70. exposure and red cell viability in red cell aging and in hemolytic 27. Kosower, N. S. (1993). Altered properties of erythrocytes in the anemia. Proceedings of the National Academy of Sciences, 95(6), aged. American Journal of Hematology, 42(3), 241–247. 3077–3081. 28. Besa, E. C. (1994). Hematologic effects of androgens revisited: An 46. Thiagarajan P., & Prchal, J. T. (2015). Erythrocyte Turnover. In: K. alternative therapy in various hematologic conditions. Seminars Kaushansky, M. A. Lichtman, J. T. Prchal, M. M. Levi, O. W. Press, in Hematology, 31(2), 134–145. L. J. Burns, & M. Caligiuri, eds. Williams hematology (9th ed.). 29. Bachmann, S., Le Hir, M., & Eckardt, K. U. (1993). Co-localization New York: McGraw-Hill. Retrieved from http://accessmedicine. of erythropoietin mRNA and ecto-5'-nucleotidase immuno- mhmedical.com.proxy.libraries.rutgers.edu/content.aspx?bookid reactivity in peritubular cells of rat renal cortex indicates that =1581&Sectionid=94303531. fibroblasts produce erythropoietin. Journal of Histochemistry and 47. Gottlieb, R. A. (2010). Apoptosis. In: K. Kaushansky, M. A. Cytochemistry, 41(3), 335–341. Lichtman, E. Beutler, T. J. Kipps, U. Seligsohn, & J. T. Prchal, 30. Gabrilove, J. (2000). Overview: Erythropoiesis, anemia, and the eds. Williams hematology (8th ed., pp. 161–167). New York: impact of erythropoietin. Seminars in Hematology, 37, 1–3. McGraw-Hill. 31. Jelkmann, W. (2007). Erythropoietin after a century of research: 48. Lux, S. E. (2016). Anatomy of the red cell membrane skeleton: Younger than ever. European
Journal of Haematology, 78(3), Unanswered questions. Blood, 127(2), 187–199. 183–205. 32. Beverborg, N. G., Verweij, N., Klip, I. T., van der Wal, H. H., Voors, A. A., van Veldhuisen, D. J., . . . van der Meer, P. (2015). Chapter 6 Hemoglobin Shirlyn B. McKenzie, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Diagram the quaternary structure of a 6. Define hemoglobin reference intervals hemoglobin molecule, identifying the 7. Explain how the fine balance of hemoglobin heme ring, globin chains, and iron. concentration is maintained. 2. Assemble a diagram of fetal and adult hemoglo- 8. Compare HbA with HbA1c and explain what bin molecules with appropriate globin chains. an increased concentration of HbA1c means. 3. Explain how pH, temperature, 2,3-BPG, 9. Diagram and describe the mechanism of and PO2 affect the oxygen dissociation extravascular erythrocyte destruction and curve (ODC). hemoglobin catabolism and name laboratory 4. List the types of hemoglobin normally tests that can be used to evaluate it. found in adults and newborns and give 10. Diagram and describe the mechanism of their approximate concentration. intravascular erythrocyte destruction and 5. Summarize hemoglobin’s function in hemoglobin catabolism and name laboratory gaseous transport. tests that can be used to evaluate it. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Construct a diagram to show the synthesis 4. Given information on pH, 2,3-BPG, CO2, of a hemoglobin molecule. temperature, and HbF concentration: 2. Describe the ontogeny of hemoglobin types; interpret the ODC, and translate it into the contrast differences in oxygen affinity physiologic effect on oxygen delivery. of HbF and HbA and relate them to the 5. Contrast the structures and functions of structure of the molecule. relaxed and tense hemoglobin and propose 3. Explain the molecular control of heme synthesis. how these structures affect gaseous transport. 87 88 Chapter 6 6. Describe how abnormal hemoglobins are 9. Explain the role of hemoglobin in the acquired, and select a method by which they nitrogen oxide-control of blood flow and can be detected in the laboratory. vessel homeostasis. 7. Assess the oxygen affinity of abnormal, 10. Compare and contrast erythrocyte acquired hemoglobins and reason how this extravascular destruction with intravascular affects oxygen transport. destruction and identify which process is 8. Compare and contrast the exchange of O2, dominant given laboratory results. CO2, H+, and Cl- at the level of capillaries and 11. Identify the breakdown products of the lungs. hemoglobin and determine how the body conserves and recycles essential components. Chapter Outline Objectives—Level I and Level II 87 Regulation of Hemoglobin Synthesis 94 Key Terms 88 Ontogeny of Hemoglobin 95 Background Basics 88 Hemoglobin Function 96 Case Study 89 Hemoglobin Catabolism 102 Overview 89 Acquired Nonfunctional Hemoglobins 104 Introduction 89 Summary 106 Hemoglobin Structure 89 Review Questions 107 Hemoglobin Synthesis 90 References 108 Key Terms Artificial oxygen carrier (AOC) Ferritin Oxygen affinity Bilirubin Haptoglobin Oxyhemoglobin Bohr effect Heme Methemoglobin Carboxyhemoglobin Hemopexin Relaxed (R) structure Chloride shift Hemosiderin Sulfhemoglobin Cyanosis Hemosiderinuria Tense (T) structure Deoxyhemoglobin Hypoxia Background Basics The information in this chapter builds on the concepts • Describe the site of erythropoiesis (Chapter 4). learned in previous chapters. To maximize your learn- ing experience, you should review these concepts before Level II starting this unit of study: • Describe the metabolic pathways present in the mature erythrocyte, and explain their role in main- Level I taining viability of the erythrocyte (Chapter 5). • List and describe the stages of erythrocyte matura- • Summarize the development of hematopoiesis from tion (Chapter 5). the embryonic stage to the adult (Chapter 4). • Summarize the role of erythropoietin in erythropoi- esis (Chapter 5). Hemoglobin 89 in the membrane that alter its permeability or alterations of CASE STUDY the cell’s enzyme systems can lead to changes in the struc- We refer to this case study throughout the chapter. ture and/or function of the hemoglobin molecule and affect Jerry, a 44-year-old male, arrived in the emergency the capacity of this protein to deliver oxygen (Chapter 5). room by ambulance after a bicycle accident. Exami- Although a small amount of hemoglobin is synthesized nation revealed multiple fractures of the femur. He as early as the pronormoblast stage, most hemoglobin is was otherwise healthy. The next day, he was taken synthesized in the polychromatophilic normoblast stage. to surgery to repair the fractures. After surgery, his In total, 75–80% of the cell’s hemoglobin is made before hemoglobin was 7 g/dL. He refused blood transfu- the extrusion of the nucleus. Because the reticulocyte does sions and was discharged 6 days later. Jerry called not have a nucleus, it cannot make new RNA for protein his doctor within days of being discharged and synthesis. However, residual RNA and mitochondria in the told him that he had difficulty walking around the reticulocyte enable the cell to make the remaining 20–25% house on crutches because of shortness of breath of the cell’s hemoglobin. The mature erythrocyte contains and lack of stamina. no nucleus, ribosomes, or mitochondria and is unable to Consider why Jerry’s hemoglobin decreased synthesize new protein. after surgery and how this could be related to his Hemoglobin concentration in the body is the result of a current symptoms. fine balance between production and destruction of eryth- rocytes. The normal hemoglobin concentration in an adult male is about 15 g/dL with a total blood volume of about 5 L (50 dL). Therefore, the total body mass of hemoglobin is approximately 750 g: Overview 15 g/dL * 50 dL = 750 g This chapter describes the synthesis and structure of hemo- Because the normal erythrocyte life span is about globin and factors that regulate its production. It compares 120 days, 1 120 of the total amount of hemoglobin is lost each the different types of hemoglobin produced according to day through removal of senescent erythrocytes. Thus, an developmental stage, considers the function of hemoglobin equivalent amount must be synthesized each day to main- in gaseous exchange, and analyzes factors that affect this tain a steady-state concentration. This amounts to approxi- function. The chapter also discusses structure, formation, mately 6 g of new hemoglobin per day: and laboratory detection of abnormal hemoglobins. The catabolism of hemoglobin in extra- and intravascular deg- 750 g (amount of hemoglobin lost radation is also discussed. = 6.25 g/day 120 days and synthesized each day) If we divide the total amount of hemoglobin synthe- Introduction sized each day (6.25 g) by the mean amount of hemoglobin in a red cell (MCH, about 30 pg), we can determine the daily Hemoglobin is a highly specialized intracellular erythrocyte production of new red blood cells: protein responsible for transporting oxygen from the lungs to tissue for oxidative metabolism and facilitating carbon 6.25 g/day 1012 pg * = 2 * 1011 cells/day dioxide transport from the tissue to the lungs. Each gram of 30 pg/cell g hemoglobin can carry 1.34 mL of oxygen. It is also a trans- porter of nitric oxide (NO), which modulates vascular tone. Hemoglobin occupies approximately 33% of the vol- Hemoglobin Structure ume of the erythrocyte and accounts for 90% of the cell’s Hemoglobin is a large tetrameric molecule, molecular dry weight. Each cell contains between 28 and 34 pg of weight 66.7 kD, composed of four globular protein sub- hemoglobin, a concentration close to the solubility limit units (Figure 6-1). Each of the four subunits contains a heme of hemoglobin. This concentration is measured by cell group and a globin chain. Heme, the prosthetic group of analyzers and reported as mean corpuscular hemoglobin hemoglobin, is a tetrapyrrole ring with ferrous iron located (MCH). In anemic states, the erythrocyte may contain less in the center of the ring. Hemoglobin structure is described hemoglobin (decreased MCH) and/or the individual may in Table 6-1 (Figure 6-2). Each heme subunit can carry one have fewer than normal erythrocytes present, both of which molecule of oxygen bound to the central ferrous iron; thus, result in a decrease of the blood’s oxygen-carrying capacity. each hemoglobin molecule can carry four molecules of The erythrocyte’s membrane and its metabolic path- oxygen. ways are responsible for protecting and maintaining the The composition of the globin chains is responsible hemoglobin molecule in its functional state. Abnormalities for the different functional and physical properties of 90 Chapter 6 chains (a or Z) combines with a pair of non-a-chains (e, b, a2 d, or g) to form the various types of hemoglobin (Table 6-2). a1 The arrangement of each globin chain is similar. Each a- Heme and Z-chain has 141 amino acids, and each e, b, d, and Globin g-chain has 146 amino acids. The b- chain is composed of Chain eight a-helical segments separated by seven short, nonheli- cal segments, and the a-chain has seven a-helical segments. The helical segments are lettered A–H, starting at the amino end of the chain. The amino acids of the globin chains are identified by their helix location and amino acid number (e.g., F8 is the eighth amino acid in the F helix). Amino acids between helices are identified by amino acid number and the letters of the two helices (e.g., EF3). The nonhelical seg- b ments allow the chains to fold upon themselves. b 2 1 The four subunits of hemoglobin, each consisting of a heme group surrounded by a globin chain, are held together by salt bonds, hydrophobic contacts, and hydrogen bonds Figure 6.1 Hemoglobin is a molecule composed of four in a tetrahedral formation giving the hemoglobin molecule polypeptide subunits. Each subunit has a globin chain with a heme nestled in a hydrophobic crevice that protects the iron from being a nearly spherical shape. When ligands such as oxygen oxidized. There are four different types of globin chains:a, b, d, g. bind to hemoglobin, the number and stringency of inter- Two a- chains and two non-a occur in identical pairs to form a subunit contacts change and the shape of the molecule tetramer. The types of globin chains present determine the type of changes. Mutations in the primary structure of globin hemoglobin. Depicted here is hemoglobin A, consisting of two chains can affect subunit or dimer pair interactions and a- and two b-chains. The contacts between the a, b-chains in a dimer (i.e., a1b1) are extensive and allow little movement. The thus alter hemoglobin-oxygen affinity or the molecule’s contacts between the dimer pairs (i.e., a1b2, a2b1), however, are stability. smaller and allow conformational change of the molecule as it goes from oxyhemoglobin to deoxyhemoglobin. Checkpoint 6.1 Describe the quaternary structure of a molecule of hemoglobin. How can a mutation in one of the globin chains at the subunit Table 6.1 The Structure of Hemoglobin interaction site, a1b2, affect hemoglobin function? Structure Conformational Description Primary Sequence of individual amino acids held together by peptide bonds in the globin chains; is critical to stability and function of molecule; determines the Hemoglobin Synthesis overall structure Secondary Arrangement of the amino acids resulting from Heme hydrogen bonding between the peptide bonds of Heme is an iron-chelated porphyrin ring that functions as the amino acids next to or near each other (75% of the chain is in the form of an a-helix; each chain a prosthetic group (nonamino acid component) of a pro- consists of 7 or 8 a-helix segments, labeled A S H, tein. The porphyrin ring, protoporphyrin IX, is composed separated by n onhelical [pleated] segments that do not participate in forming the a-helix but allow the of a flat tetrapyrrole ring (four linked pyrrole rings) with polypeptide to fold on itself) ferrous iron (Fe++) inserted into the center. (Porphyrins are Tertiary Folding superimposed on the helical and pleated metabolically active only when chelated.) Ferrous ions have domains; forms the heme hydrophobic pocket within globin chains and places polar (hydrophilic) six electron pairs per atom. In heme, four of these electron residues on the exterior of the molecule; this tertiary pairs are coordinately bound to the N atoms of each of the structure changes upon ligand binding four pyrrole rings. In hemoglobin, one of the two remaining Quaternary Relationship of the four protein subunits to one electron pairs (fifth) is coordinately bound with the N of the another; quaternary structural changes that occur upon ligand binding result from the tertiary changes proximal histidine (F8)
of the globin chain, and the other pair (sixth) is the binding site for molecular oxygen. In the deoxygenated state, the sixth electron pair is occupied by a hemoglobin. Two types of globin chains are produced: water molecule. Iron must be in the ferrous (Fe++) state for alpha-like (alpha [a], zeta [Z]), and non-alpha (epsilon [e], oxygen binding to occur. Ferric iron (Fe+++), which has lost beta [b], delta [d], gamma [g]). The tetrameric hemoglobin an electron, cannot serve as an oxygen carrier because the molecule consists of two pairs of unlike chains: two identi- sixth potential binding site (electron pair) for oxygen is no cal a-like and two identical non-a-chains. A pair of a-like longer available. Hemoglobin 91 Ala Ser Leu Asp Arg Cys Yal Pro Lys Phe Tyr Thr Leu His Lys Asn a Primary Asp Leu Arg structure Ser Ala Cys Yal Pro Thr Tyr Phe Lys Leu His Lys Asn b Secondary Polypeptide structure (globin) Heme group c Tertiary structure Fe++ Heme d Quaternary structure Figure 6.2 The structure of hemoglobin. (a) Primary structure is the sequence of amino acids. (b) Secondary structure is the coiled a-helix and b-pleated sheet formed by hydrogen bonding between the peptide bonds in the chain. (c) Tertiary structure is the folding of the molecule into a three-dimensional structure. (d) Quartenary structure is the combination of the four polypeptide subunits, each of which contains a heme group, into a larger protein. Table 6.2 A (CoA) to form 5-aminolevulinic acid (ALA). This reac- Normal Types of Hemoglobin According to Developmental Stage tion occurs in the presence of the cofactor pyridoxal phos- phate and the enzyme 5-aminolevulinate synthase (ALAS). Developmental Globin Reference Type This first reaction is the rate-limiting step in the synthesis Stage Chains Interval of heme and occurs only when the cell has an adequate Embryonic Gower 1 Z2e2 — supply of iron1 (Chapter 12). Synthesis continues through Gower 2 a2e2 — a series of steps in the cytoplasm, eventually forming cop- Portland Z2g2 — roporphyrinogen. Coproporphyrinogen then reenters the Fetal HbF a2g2 90–95% before birth mitochondria and is further modified to form the protopor- 50–85% at birth phyrin IX ring (Figure 6-4). The final step, also occurring HbA a2b2 10–40% at birth in the mitochondria, is the chelation of iron with protopor- HbA2 a2d2 less than 1% at birth phyrin IX catalyzed by ferrochelatase (FECH) to form heme More than year old HbF a2g2 Less than 2% (Figure 6-5). Heme then leaves the mitochondria to combine HbA a with a globin chain in the cytoplasm. 2b2 More than 95% HbA2 a2d2 Less than 3.5% Adult HbA a2b2 More than 95% Globin Chain Synthesis HbA2 a2d2 1.5–3.7% Globin chain synthesis is directed by genes in two clusters HbF a2g2 Less than 2% on chromosomes 11 and 16 (Figure 6-6). These genes pro- a, alpha; b, beta; g, gamma; d, delta; Z, zeta; e, epsilon. duce the seven different types of globin chains: zeta, alpha, epsilon, gamma-A, gamma-G, delta, and beta (a, Z, e, gA, gG, Heme synthesis includes seven reactions that occur in d, b). Two are found only in embryonic hemoglobins (Z, e). two different cell compartments: the mitochondria and the The genes for the Z-chain (the fetal equivalent of the a-chain) cytosol (Figure 6-3). Synthesis begins in the mitochondria and a-chain are located on the short arm of chromosome 16 with the condensation of glycine and succinyl coenzyme (the a gene cluster). The Z-chain is synthesized very early 92 Chapter 6 Mitochondria Cytosol COO2 CH2 COO2 d–ALAS COO2 1 CH2 NH3 CH2 COO2 CH2 CH2 ALA dehydrase COO2 CH2 C S CoA C O 2H2O CH2 CH2 O glycine CH2 Succinyl CoA NH + 3 CH2 N d–ALA NH 1 H M V 3 Porphobilinogen (PBG) M N Porphobilinogen deaminase M 4NH3 (uroporphyrinogen I synthase) N Fe N P N V Hydroxymethylbilane Heme Uroporphyrinogen III H2O cosynthelase P M 2H+ Uroporphyrinogen I ferrocheletaso A P Fe++ M V A N H A NH HN M N H M P H P NH HN N P H V N P A Protoporphyrin IX Uroporphyrinogen III P M Uroporphyrinogen 4CO Protoporphyrinogen 2 decarboxylase 3H oxidase 2 M V M P M N H M Coproporphyrinogen M N H M NH HN oxidase NH HN P H V P H P N 2CO2 + 4H+ N P M P M Protoporphyrinogen IX Coproporphyrinogen III Figure 6.3 The synthesis of heme begins in the mitochondria with the condensation of glycine and succinyl CoA to form d-aminolevulinic acid (d-ALA). The reaction is catalyzed by ALA-synthase. Then d-ALA leaves the mitochondria to form a pyrrole ring, porphobilinogen. Four pyrroles combine to form a linear tetrapyrrole, hydroxymethybilane. The tetrapyrrole cyclizes to form uroporphyrinogen. Decarboxylation of the side chains of uroprophyrinogen forms corproporhyrinogen. The final reactions, the formation of protoporphyrin IX and insertion of iron into the ring, occur in the mitochondria. Incorporation of iron into the protoporphyrin ring is catalyzed by ferrochelatase. in embryonic development, but after 8–12 weeks, Z-chain gG, and the other directs the production of a g-chain with synthesis is replaced by a-chain synthesis. There are two alanine at position 136, gA. The gG-chain synthesis predomi- a-loci (a-1, a-2), both of which transcribe mRNA for a-chain nates before birth (3:1), but gG- and gA-chain syntheses are synthesis. The protein product from each locus is structur- equal (1:1) in adults. The next two genes on chromosome ally identical. 11, d and b, are switched on to a small degree when the The non-a-globin genes are arranged in linear fashion g-genes are activated, but they are not fully activated until in order of activation on chromosome 11 (the non-a-gene g-chain synthesis diminishes at about 35 weeks of gestation. cluster). The e-gene the first non-a-gene to be activated, is The rate of synthesis of the d-chain is only 1 140 that of the located toward the 5′ end of chromosome 11; during embry- b-chain, due to differences in the promoter regions of the onic development, e-chain synthesis is switched off, and the two genes. After birth, most cells produce predominantly two g-genes are activated. One g-gene directs the produc- a- and b- chains for the formation of HbA, the major adult tion of a g-chain with glycine at amino acid position 136, hemoglobin (97%). Hemoglobin 93 Mitochondria Cytoplasm ALA dehydrase Succinyl CoA ALAS2 ALA PBG and Succinyl glycine CoA synthase, HMB synthase pyridoxal phosphate Hydroxymthyblane Coproporphyrinogen III Heme Ferrochelatase Urocarboxylase Protoporphyrin IX Uroporphyrinogen II Protoporphyrinogen oxidase Protoporphyrinogen III Coproporphyrinogen oxidase Coproporphyrinogen III Figure 6.4 Synthesis of heme. It begins in the mitochondria with the condensation of glycine and succinyl CoA catalyzed by 5-aminolevulinate synthase 2 (ALAS2) and succinyl CoA synthase and co-factor pyridoxal phosphate. The product, 5-aminolevulinate (ALA), leaves the mitochondria to form the pyrrole ring, porphobilinogen (PBG). The combination of four pyrroles to form a linear tetrapyrrole (hydroxymethylbilane), the cyclizing of the linear form to uroporphyrinogen III catalyzed by interaction between HMB synthase and uroporphyrinogen III cosynthase, and the decarboxylation of the side chains to form coproporphyrinogen occur in the cytoplasm. The final reactions, the formation of protoporphyrin IX, and the insertion of iron into the protoporphyrin ring occur in the mitochondria. CH2 H CH3 C H3C CH2 N N HC Fe CH N N H CH3 C H HOOC COOH Hemoglobin Heme structure Figure 6.5 The hemoglobin molecule on the left reveals the quartenary structure of hemoglobin with four protein chains, each folded around a heme molecule. On the right is a heme molecule. Heme is composed of a flat tetrapyrrole ligand (porphyrin) and iron. The iron has six coordinate sites. Nitrogen atoms of porphyrin occupy four coordination sites in a square planar arrangement around the iron. Iron in the ferrous state has two other coordinate sites, one of which is occupied by the N of the proximal histidine (F8) of globin and one with molecular oxygen or H2O. The synthesis of globin peptide chains occurs on poly- formed a-chain-heme subunit and a non-a-chain-heme ribosomes in the cytoplasm of developing erythroblasts subunit combine spontaneously, facilitated by electrostatic (Figure 6-7). Globin chains are released from the polyribo- attraction, to form a dimer (e.g., ab). Charge differences somes and combine with heme molecules released from the exist among the non-a-globin chains. This promotes a hier- mitochondria. The globin chains are folded to create a hydro- archy of affinity of these chains for the a-globin chains. The phobic pocket near the exterior surface of the chain between b-globin chain has the greatest affinity for a-globin chains the E and F helices. Heme is inserted into this hydrophobic followed by g- and d- chains. Then two dimers combine to pocket where it is readily accessible to oxygen. A newly form the tetrameric hemoglobin molecule (e.g., a2b2). The 94 Chapter 6 Chromosome 16 Chromosome 11 G A Globin G A chain Figure 6.6 The genes for the globin chains are located on chromosomes 11 and 16. The Z-chain appears to be the embryonic equivalent of the a-chain, both of which are located on chromosome 16. Note that a-gene the is duplicated. The other globin genes are located on chromosome 11. a Globin b proximal histidine is bonded with the heme iron. The iron mRNA is protected in the reduced ferrous state in this hydrophobic pocket. The exterior of the chain is hydrophilic, which NH makes the molecule soluble. 2 1 NH2 Fe Heme Fe Mitochondria Mitochondria Checkpoint 6.2 1 2 What globin chains are synthesized in the adult? Fe Fe a b 1 2 Fe Regulation of Hemoglobin a b Encounter complex Fe Synthesis Balanced synthesis of globin chains and heme is important Fe to the survival of the erythrocyte because hemoglobin tet- a b Stable ab dimer ramers are soluble, but individual components of hemoglo- Fe bin such as unpaired globin chains, protoporphyrin, and iron are not. Normally, the production of a-globin subunits, non-a globin subunits, and heme are nearly equal. This indicates that tight regulatory mechanisms exist, control- b Fe ling the production of hemoglobin. Hemoglobin synthesis a2b2 Tetramer a a is regulated by several mechanisms including: Fe b • Activity and concentration of the erythroid enzyme 5-aminolevulinate synthase (ALAS2) • Activity of porphobilinogen deaminase (PBGD) Figure 6.7 Assembly of hemoglobin. The a- and b- globin polypeptides are translated from their respective mRNAs. Upon • Concentration of iron heme binding, the protein folds into its native three-dimensional • Regulation of globin chain synthesis structure. The binding of a- and b- hemoglobin subunits to each other is facilitated by electrostatic attraction. An unstable The first step in the heme synthetic pathway, catalyzed intermediate encounter complex can rearrange to form the stable by ALAS, is the rate-limiting step in heme synthesis and ab dimer. Two dimers combine to form the functional a2b2 tetramer. takes place in the mitochondria. ALAS is synthesized on ribosomes in the cytosol, and must be imported into the protein alpha hemoglobin-stabilizing protein (AHSP) plays mitochondria to catalyze the reaction. This mitochondrial an important role in coordinating heme and globin assem- import can be inhibited by high concentrations of free bly. AHSP binds free a- chains and stabilizes their structure, heme.1 Heme also inhibits uptake of iron from transferrin increasing their affinity for b- chains. This accelerates the into the cell. When iron is scarce, the synthesis of ALAS is formation of hemoglobin tetramers.1 decreased. The heme is positioned between two histidines of the Iron entering the developing erythroblast can be in globin chain, the proximal (F8) and distal (E7) histidines. The either the pool available for metabolic processes (heme Hemoglobin 95 synthesis) or the storage pool (ferritin and hemosiderin). The synthesis of different globin chains occurs in The amount of iron in these pools is regulated by pro- sequence depending on the developmental stage. This teins that control transcription and translation of proteins switching appears to be due to the sequential activation involved in heme synthesis and the formation of ferritin as and then inactivation of transcription among the a- and well as transferrin receptors.1 Iron metabolism is discussed non-a-globin gene clusters. Globin gene expression is also in detail in Chapter 12. affected by cellular, microenvironmental, and humoral The expression of globin genes occurs only in erythroid influences that affect the proliferation and differentiation cells during a narrow period of differentiation. Synthesis of
of stem cells. The developmental control of the perinatal globin chains begins in the pronormoblast and continues switch from HbF to HbA synthesis appears to be intrinsic to until the reticulocyte loses remnants of mRNA. The rate of the erythroid cell.3 The progenitor cells are gradually repro- globin synthesis is governed primarily by the rate at which grammed during the perinatal period, leading to a switch- the DNA is transcribed to mRNA, but it is also modified by ing from g-chain production to predominantly b-chain the processing of globin pre-mRNA to mRNA, the transla- production. Two hemoglobin switches occur from early tion of mRNA to protein, and the stability of globin mRNA. embryogenesis to birth. The initial switch is from embryonic The individual globin genes have separate promoter regions to fetal globins and the second from fetal to adult globins.4 available for activation at variable times during embryonic Studies of the developmental switch that silences g-globin and fetal development. In addition, the b-gene cluster has reveals that several transcription factors including TR2/ a locus control region (LCR), located upstream (5′) of the TR4, MYB, BCL11A, GATA1, and KLF1, play a major role.5 genes, which plays an important role in regulating the Epigenetic modifications of globin DNA also modulate entire gene cluster. The a-gene cluster has a similar control globin-gene expression (Chapter 2). DNA methylation of region that is thought to have a similar function. embryonic and fetal globin genes reduces gene expression,5 Heme plays an important role in controlling the syn- while DNA methyltransferase (DNMT1) inhibitors lead to thesis of globin chains. It stimulates globin synthesis by demethylation of g-globin promoters and induces expres- inactivating a heme-regulated inhibitor (HRI) of transla- sion of HbF. Transcription factors MYB and BCL11A coop- tion. Without heme, the inhibitor accumulates.2 The HRI erate with DNMT1 to repress expression of embryonic and prevents the accumulation of excessive unfolded proteins. fetal b-like globin genes.6,7 Acetylation of globin genes also Heme also has a positive effect on globin transcription by promotes gene transcription and induces HbF.4 binding to a globin transcriptional repressor. A slight excess of a-chain mRNA is produced, but the mRNA of b-chains Embryonic Hemoglobins is more efficiently translated, resulting in almost equal syn- thesis of a- and b-chains. Embryonic erythropoiesis is associated with the produc- tion of the embryonic hemoglobins (Gower 1, Gower 2, and Portland) that are synthesized in succession as globin CASE STUDY (continued from page 89) synthesis switches from Z S a and from d S g in the first Jerry’s doctor gave him iron supplements to take trimester of gestation. Embryonic hemoglobins are made every day. from the combination of pairs of embryonic globin chains, Z and e (Z2e2) or embryonic chains in combination with a- 1. If Jerry is iron deficient, what is the effect of this and g-chains (a2e2, Z2g2). These primitive hemoglobins are deficient state on the synthesis of ALAS, transfer- detectable during early hematopoiesis in the yolk sac and rin receptor, and ferritin? liver and are not usually detectable after the third month 2. What was the rationale for giving Jerry the iron? of gestation. Fetal Hemoglobin As embryonic erythropoiesis shifts to fetal erythropoiesis, Ontogeny of Hemoglobin hemoglobin F (HbF; a2g2) becomes the predominant hemo- globin formed during liver and bone marrow erythropoiesis The type of hemoglobin is determined by its composition in the fetus. HbF composes 90–95% of the total hemoglobin of globin chains (Table 6-2). Individual globin chains are production in the fetus until about 34–36 weeks of gestation. expressed at different levels in developing erythroblasts of At birth, the infant has 50–85% HbF. the human embryo, fetus, and adult. Some hemoglobins (Gower 1, Gower 2, Portland) occur only in the embryonic stage of development. HbF is the predominant hemoglobin Adult Hemoglobins in the fetus and newborn, and hemoglobin A is the pre- The fetal to adult shift in erythropoiesis reflects transcrip- dominant hemoglobin after 1 year of age. tion of the b- globin chain. In adults, hemoglobin A (HbA; 96 Chapter 6 a2b2) is the major hemoglobin. Although HbA is found as proportional to the average blood glucose level over the early as 9 weeks gestation, b-chain synthesis occurs at a low previous 2–3 months. Average levels of HbA1c are 7.5% in level until the third trimester of pregnancy. b- chain syn- diabetics and 3.5% in normal individuals. thesis steadily increases from gestational week 30 onward but does not exceed g-chain synthesis until after birth. After Checkpoint 6.4 birth, the percentage of HbA continues to increase with the A patient has an anemia caused by a shortened RBC life span infant’s age until normal adult levels (more than 95%) are (hemolysis); how would this affect the HbA1c measurement? reached by the end of the first year of life. HbF production constitutes less than 2% of the total hemoglobin of adults. In normal adults, most, if not all, HbF is restricted to a few erythrocytes, referred to as F cells. Hemoglobin Function F cells constitute 2–5% of adult RBCs, and from 13 to 25% of The function of hemoglobin is to transport and exchange the hemoglobin within each F cell is HbF. The switch from respiratory gases. The air we breathe is a mixture of HbF to HbA after birth is incomplete and in part reversible. nitrogen, oxygen, water, and carbon dioxide. Each of the For example, patients with hemoglobinopathies or severe gases contributes to the atmospheric pressure (measured anemia can have increased levels of HbF, often proportion- in mmHg) in proportion to its concentration. The par- ate to the decrease in HbA. In bone marrow recovering from tial pressure each gas exerts is referred to as P (e.g., PO2) suppression and in some neoplastic hematologic diseases, and determines the rate of diffusion of that gas across the HbF levels often rise. alveolar-capillary membrane. Arterialized blood leaves the HbA2 (a2d2) appears late in fetal life, composes less than lungs with a PO2 of 100 mmHg and a PCO2 of 40 mmHg. In 1% of the total hemoglobin at birth, and reaches normal adult comparison, the PO2 of interstitial fluid in tissues is about values (1.5–3.7%) after one year. The d-gene locus is tran- 40 mmHg, and the PCO2 is about 45 mmHg. Thus, when scribed very inefficiently compared with the b-locus due to blood reaches the tissues, oxygen diffuses out of the blood changes in the promoter region of the d-gene that is recognized to the tissues and CO2 diffuses into the blood from tissue. by erythroid-specific transcription factors (e.g., GATA-1). The amount of dissolved O2 and CO2 that the plasma can HbA2 has a slightly higher oxygen affinity than HbA; other- carry is limited. Most O2 and CO2 diffuse into the erythro- wise, the two hemoglobins have similar or identical ligand cyte to be transported to tissue or lungs. binding curves, Bohr effect, and response to 2,3-BPG. Oxygen Transport Checkpoint 6.3 Hemoglobin with bound oxygen is called oxyhemoglobin; What are the names and globin composition of the embryonic, hemoglobin without oxygen is called deoxyhemoglobin. fetal, and adult hemoglobins? The amount of oxygen bound to hemoglobin and released to tissues depends not only on the PO2 and PCO2 but also Glycosylated Hemoglobin on the affinity of Hb for O2. The ease with which hemoglo- bin binds and releases oxygen is known as oxygen affinity. Prolonged exposure of hemoglobin to chemically active Hemoglobin affinity for oxygen determines the proportion compounds in the blood can result in modifications to the of oxygen released to the tissues or loaded onto the cell at hemoglobin molecule. HbA1 is a minor component of nor- a given oxygen pressure (PO2). Increased oxygen affinity mal adult hemoglobin (HbA) that has been modified post- means that the hemoglobin has a high affinity for oxygen, translationally; a component has been added to (usually) will bind oxygen more avidly, and does not readily give the N terminus of the b-chain. Also known as “fast hemo- it up; decreased oxygen affinity means the hemoglobin globin” or glycated hemoglobins, HbA1 consists of three has a low affinity for oxygen and releases its oxygen more subgroups: HbA1a, HbA1b, and HbA1c. The clinically most readily. important subgroup of HbA1 is HbA1c, which is produced Oxyhemoglobin and deoxyhemoglobin have differ- throughout the erythrocyte’s life and is proportional to the ent three-dimensional configurations. In the unliganded or concentration of blood glucose. Older erythrocytes typi- deoxy state, the tetramer is stabilized by intersubunit salt cally contain more HbA1c than younger erythrocytes, hav- bridges and is in the tense (T) structure or state. In oxyhe- ing been exposed to plasma glucose for a longer period. moglobin, the salt bridges are broken, and the molecule is in However, if young cells are exposed to extremely high con- the relaxed (R) structure or state. The change in conforma- centrations of glucose (greater than 400 mg/dL) for several tion of hemoglobin (from T to R) occurs because of a coor- hours, the concentration of HbA1c increases. dinated series of changes in the quaternary structure of the Measurement of HbA1c is routinely used as an indicator tetramer as the subunits bind oxygen (see the section “The of control of blood glucose levels in diabetics because it is Allosteric Property of Hemoglobin”). The T configuration Hemoglobin 97 is a low oxygen-affinity conformation, and the R state is a in the capillary beds of tissues. This is physiologically of high oxygen-affinity conformation. significant importance, for it allows the overall transfer of Oxygen affinity of hemoglobin is usually expressed oxygen from the lungs to the tissues with relatively small as the PO2 at which 50% of the hemoglobin is saturated changes in PO2. The ODC shows that the oxygen satura- with oxygen (P50). The P50 in humans is normally about tion of hemoglobin drops from about 100% in the arteries to 26 mmHg. If hemoglobin-oxygen saturation is plotted ver- about 75% in the veins. This indicates that hemoglobin gives sus the partial pressure of oxygen (PO2), a sigmoid-shaped up about 25% of its oxygen to the tissues. When the curve is (S-shaped) curve results. This is referred to as the oxygen shifted to the right, the P50 is increased, indicating that the dissociation curve (ODC) (Figure 6-8). The shape of the curve oxygen affinity has decreased. This results in the release of reflects subunit interactions between the four subunits of more oxygen to the tissues. When the curve is shifted to the hemoglobin (heme–heme interaction or cooperativity). left, the P50 is decreased, indicating that oxygen affinity has Monomeric molecules such as myoglobin have a hyperbolic increased. In this case, less oxygen is released to the tissues. ODC indicating no cooperativity of oxygen binding. The sigmoid-shaped curve of hemoglobin dissociation indicates that the deoxyhemoglobin tetramer is slow to take up an O2 CASE STUDY (continued from page 95) molecule, but binding one molecule of O2 to hemoglobin Jerry was lethargic and pale and was having prob- facilitates the binding of additional O2. Thus, the “appetite” lems with activities of daily living. of hemoglobin for oxygen grows with the addition of each oxygen molecule. 3. Explain why Jerry could have these symptoms. The shape of the curve has certain physiologic advan- tages. The “flattened” top of the S reflects the fact that more than 90% saturation of hemoglobin still occurs over a broad THE ALLOSTERIC PROPERTY OF HEMOGLOBIN range of PO2. This enables us to survive and function in The sigmoid shape of the ODC is primarily due to heme– conditions of lower oxygen availability, such as living (or heme interactions (described below). However, the relative skiing) at high altitudes. Note that the steepest part of the position of the curve (shifted right or left) is due to other curve occurs at oxygen tensions found in tissues. This allows variables. the release of large amounts of oxygen from hemoglobin Hemoglobin is an allosteric protein, meaning that its during the small physiologic changes in PO2 encountered structure (conformation) and function are affected by other 100 80 Increased H+ Increased CO2 60 Increased temperature Increased 2.3-BPG 40 Normal conditions 20 Decreased H+ Decreased CO2 Decreased temperature Decreased 2.3-BPG 0 20 40 60 80 100 120 O2 partial pressure (mm Hg) Figure 6.8 The oxygen affinity of hemoglobin is depicted by the oxygen dissociation curve (ODC). The fractional saturation of hemoglobin (y
axis) is plotted against the concentration of oxygen measured as the PO2 (x axis). At a pH of 7.4 and an oxygen tension (PO2) of 26 mmHg, hemoglobin is 50% saturated with oxygen (red line). The curve shifts in response to temperature, CO2, O2, 2,3-BPG concentration, and pH. When the curve shifts left (light blue line), there is increased affinity of Hb for O2. When the curve shifts right (dark blue line), there is decreased affinity of Hb for O2. “FIGURE 29.12” from Fundamentals of General, Organic and Biological Chemistry, 5E by John McMurry, Mary E. Castellion, and David S. Ballantine. Copyright © 2007 by Pearson Education. Reprinted and electronically reproduced by permission of Pearson Education, Inc., Upper Saddle River, New Jersey. Percent saturation of Hb by O2 98 Chapter 6 molecules. The primary allosteric regulator of hemoglobin heme–heme interaction. This interaction of the heme groups is 2,3-bisphosphoglycerate (2,3-BPG; also referred to as is the result of movements within the tetramer triggered 2,3-diphosphoglycerate [2,3-DPG]). A byproduct of the gly- by the uptake of a molecule of oxygen by one of the heme colytic pathway (Chapter 5), 2,3-BPG, is present at almost groups. equimolar amounts with hemoglobin in erythrocytes. In the In the deoxygenated state, the heme iron is 0.4–0.6 Å presence of physiologic concentrations of 2,3-BPG, the P50 of out of the plane of the porphyrin ring because the iron hemoglobin is about 26 mmHg. In the absence of 2,3-BPG, atom is too large to align within the plane. The iron is dis- the P50 of hemoglobin is 10 mmHg, indicating a very high placed toward the proximal histidine of the globin chain to oxygen affinity. Thus, in the absence of 2,3-BPG, little oxy- which it is linked by a coordinate bond. Fully deoxygenated gen is released to the tissues. hemoglobin (T state) has a low oxygen affinity, and load- Protons 1H+2, CO2, and organic phosphates (2,3-BPG) ing the first oxygen onto the tetramer does not occur easily. are all allosteric effectors of hemoglobin that preferentially On binding of an oxygen molecule, the atomic diameter of bind to deoxyhemoglobin, forming salt bridges within and iron becomes smaller due to changes in the distribution of between the globin chains and stabilizing the deoxyhe- electrons, and the iron moves into the plane of the porphy- moglobin (T) structure. The ratio in which 2,3-BPG binds rin ring, pulling the histidine of the globin chain with it to deoxyhemoglobin is 1:1. The binding site for 2,3-BPG (Figure 6-10). These slight changes in the tertiary structure is in a central cavity of the hemoglobin tetramer between of the molecule near the heme group result in a large shift the b-globin chains. It binds to positive charges on both in the quaternary structure, altering the bonds and con- b-chains, thereby crosslinking the chains and stabilizing tacts between chains and weakening the intersubunit salt the quaternary structure of deoxyhemoglobin (Figure 6-9). bridges. Likewise, loading a second O2 onto the tetramer Hemoglobin also binds oxygen allosterically. Oxygen while it is still in the T conformation does not occur eas- binds to hemoglobin in a 4:1 ratio because one molecule of ily. However, the iron atom of the second heme is likewise O2 binds to each of the four heme groups of the tetramer. shifted, further destabilizing the salt bridges. While loading The binding of oxygen by a hemoglobin molecule depends the third O2 onto hemoglobin, the salt bridges are broken, on the interaction of the four heme groups, referred to as and the hemoglobin molecule shifts from the T to the R 2,3-Bisphosphoglycerate Figure 6.9 2,3-BPG binds in the central cavity of deoxyhemoglobin. This cavity is lined with positively charged groups on the beta chains that interact electrostatically with the negative charges on 2,3-BPG. The a-globin chains are in yellow, the b-chains are in blue, and the heme prosthetic groups in red. SOURCE: Based on Principles of Biochemistry, 4E by H. R. Horton, L. A. Moran, K. G. Scrimgeour and M. D. Perry. Published by Pearson Education, Inc., © 2006. Hemoglobin 99 Table 6.3 Factors That Affect Hemoglobin–Oxygen Affinity Increase Affinity Decrease Affinity cO2 cCO2 TCO2 cH+ TH+ c Temperature T Temperature c 2,3-BPG T 2,3-BPG Key: c = increased; T = decreased Porphyrin Fe++ Porphyrin plane plane temperature affect hemoglobin–oxygen affinity (Table 6-3 and Figure 6-8). Several physiologic mechanisms of oxygen delivery can O2 be explained by the hemoglobin–2,3-BPG interaction. When going from sea level to high altitudes the body adapts to Figure 6.10 Changes in the conformation of hemoglobin the decreased PO2 by releasing more oxygen to the tissues. occur when the molecule takes up O2 In the deoxyhemoglobin state, This adaptation is mediated by increases of 2,3-BPG in the the heme iron of a hemoglobin subunit is below the porphyrin plane erythrocyte, usually noted within 36 hours of ascent. EPO (green). On uptake of an O2 molecule, the iron decreases in diameter and moves into the plane of the porphyrin ring, pulling the proximal and erythrocyte mass also increase as a part of the body’s histidine with it (yellow). The helix containing the histidine also shifts, adaptive mechanism to decreased PO2 but this adaptation disrupting ion pairs that link the subunits. 2,3-BPG is expelled, and can take several days to improve tissue oxygenation.8 the remaining subunits can combine with O2 more readily. Fetal hemoglobin (HbF) has a higher oxygen affin- ity compared with adult hemoglobin, HbA. The g-globin configuration, pulling the b-chains together. Consequently, chain has a serine residue at the helical H21 position. In the size of the central cavity between the b-chains decreases, the b-globin chain, a histidine residue occupies this posi- and 2,3-BPG is expelled. In the high oxygen–affinity R con- tion. This change results in weak binding of 2,3-BPG and formation, the third and fourth O2 molecules are added eas- increased oxygen affinity in HbF. The more efficient binding ily. The structural changes within successive heme subunits of 2,3-BPG to HbA facilitates the transfer of oxygen from the facilitate binding the oxygen by the remaining heme sub- maternal (HbA) to the fetal (HbF) circulation. units because fewer subunit crosslinks need to be broken Rapidly metabolizing tissue (e.g., during exercise) to bind subsequent oxygen molecules. Thus, hemoglobin produces CO2 and acid 1H+2 as well as heat. These factors performs like a “mini-lung,” changing shape as it takes up decrease the oxygen affinity of hemoglobin and promote and releases O2 to the tissue. the release of oxygen to the tissue. In the alveolar capillar- Oxygen interacts weakly with heme iron, and the two ies of the lungs, the high PO2 and low PCO2 drive off the can dissociate easily. As O2 is released by hemoglobin in CO2 in the blood and reduce H+ concentration, promoting the tissues, the heme pockets narrow and restrict entry of the uptake of O2 by hemoglobin (increasing oxygen affin- O2, and the space between the b-chains widens and 2,3- ity). Thus, PO2, PCO2, and H+ facilitate the transport and BPG binds again in the central cavity. Thus, as 2,3-BPG con- exchange of respiratory gases. centration increases, the T configuration of hemoglobin is The effect of pH on hemoglobin–oxygen affinity is favored and the oxygen affinity decreases. known as the Bohr effect and is an example of the acid–base This cooperative binding of oxygen makes hemoglobin equilibrium of hemoglobin that is one of the most important a very efficient oxygen transporter. Cooperativity ensures buffer systems of the body. A molecule of hemoglobin can that once a hemoglobin tetramer begins to accept oxygen, accept H+ when it releases a molecule of oxygen. Deoxyhe- it is promptly fully oxygenated. In the process of oxygen moglobin accepts and holds the H+ better than oxyhemo- release to the tissues, the same general principle is followed. globin. In the tissues, the H+ concentration is higher because Individual hemoglobin molecules are generally either fully of the presence of lactic acid and CO2. When blood reaches deoxygenated or fully oxygenated. Only a small portion of the tissues, hemoglobin’s affinity for oxygen is decreased the molecules exists in a partially oxygenated state. by the high H+ concentration, thereby permitting the more ADJUSTMENTS IN HEMOGLOBIN–OXYGEN AFFINITY efficient unloading of oxygen at these sites. Variations in environmental conditions or physiological Hb # 4O2 + 2H+ H Hb # 2H+ + 4O2 demand for oxygen result in changes in erythrocyte and plasma parameters that directly affect hemoglobin–oxygen Thus, proton binding facilitates O2 release and helps affinity. In particular, PO2, pH 1H+2, PCO2, 2,3-BPG, and minimize changes in the hydrogen ion concentration of the 100 Chapter 6 blood when tissue metabolism is releasing CO2 and lactic when carbon dioxide diffuses from the plasma into the acid. Up to 75% of the hemoglobin oxygen can be released erythrocyte. In the presence of the erythrocyte enzyme if needed (as in strenuous exercise) as the erythrocytes pass carbonic anhydrase (CA), CO2 reacts with water to form through the capillaries. carbonic acid 1H2CO32. H2O + CO2 d CA S H2CO3 Checkpoint 6.5 What factors influence an increase in the amount of oxygen Subsequently, hydrogen ion and bicarbonate are delivered to tissue during an aerobic workout? liberated from carbonic acid and the H+ is accepted by deoxyhemoglobin: HHb Carbon Dioxide Transport c After diffusing into the blood from the tissues, carbon diox- H2CO3 — CA ¡ H+ + HCO - ide is carried to the lungs by three separate mechanisms: 3 dissolution in the plasma, as HCO - 3 in solution, and bind- The bicarbonate ions do not remain in the RBC because ing to the N-terminal amino acids of hemoglobin (carbami- the cell can hold only a small amount of bicarbonate. Thus, nohemoglobin) (Table 6-4; Figure 6-11). the free bicarbonate diffuses out of the erythrocyte into the PLASMA TRANSPORT plasma. The cell cannot tolerate a loss in negative ions, so A small amount of carbon dioxide is dissolved in the plasma in exchange for the loss of bicarbonate, Cl- diffuses into and carried to the lungs. There it diffuses out of the plasma the cell from the plasma, a phenomenon called the chloride and is expired. shift. This occurs via the anion exchange channel (band 3) in the erythrocyte membrane (Chapter 5). The bicarbonate CARBONIC ACID combines with Na+ 1NaHCO32 in the plasma and is carried Most of the carbon dioxide transported by the blood is in to the lungs where the PCO2 is low. There the bicarbonate the form of bicarbonate ions 1HCO- 32, which are produced diffuses back into the erythrocyte, is rapidly converted back into CO2 and H2O, and is expired. Table 6.4 Carbon Dioxide Transport in Blood HEMOGLOBIN BINDING Approximately 23% of the total CO2 exchanged by the Mechanism Percent of Transportation erythrocyte in respiration is through carbaminohemoglo- Dissolved in plasma 7 bin. Deoxyhemoglobin directly binds 0.4 moles of CO2 per Formation of carbonic acid, H2CO3 70 mole of hemoglobin. Carbon dioxide reacts with uncharged Bound to Hb 23 N-terminal amino groups of the four globin chains to form Arterial blood Lungs Venous blood Red blood cell Cells Red blood cell O2 (in) O2 1 HHb HbO2 1 H1 CO2 CO2 1 H2O H2CO3 1 (in) HCO 2 HCO 2 3 3 HHb 1 CO2 HHbCO2 H1 1HCO 2 2 3 HCO3 CO CO2 1 H 2 2O H2CO3 (out) 2 Cl2 O2 CO O2 1 HHb HbO2 2 1 HHb HHbCO Cl 2 plasma (out) Cl2 Cl2 a From lungs to arterial blood b From blood to cell Figure 6.11 Transport of oxygen and carbon dioxide in the erythrocyte is depicted. (a) In the lungs, O2 and HCO - 3 enter the red blood cell. O 2 combines with Hb, releasing H+. HCO - 3 combines with H+ to form H2CO3, which dissociates into H2O and CO2, and CO2 is expired. To maintain electrolyte balance, at the same time that HCO - 3 flows into the red blood cells, Cl- flows out (the reverse chloride shift). The cell membrane anion-exchange protein (band 3) controls this ion exchange. Carbaminohemoglobin (HHbCO2) releases CO2 in the lungs (where the PCO2 decreases) and is expired. The HHb releases the H+ as Hb takes up oxygen. (b) CO2 diffuses from the tissue into the venous blood and then into the erythrocyte. Within the erythrocyte, CO2 reacts with water to form bicarbonic acid, H2CO3.
The bicarbonic acid dissociates into a bicarbonate ion 1HCO - 3 2 and a proton 1H+2. The HCO - 3 leaves the cell and enters the plasma. In exchange, chloride 1C1-2 from the plasma enters the erythrocyte (chloride shift). The proton facilitates the dissociation of oxygen from oxyhemoglobin (HbO2) through the Bohr effect. When O2 enters the tissues, the H+ is taken up by deoxyhemoglobin. Hemoglobin 101 carbaminohemoglobin. At the lungs, the plasma PCO2 hemorrhagic shock (such as in military conflicts or massive decreases, and the CO2 bound to hemoglobin is released civilian casualties) or when transfusion of allogenic blood is and diffuses out of the erythrocyte to the plasma. It then is not an acceptable option (such as treatment of a Jehovah’s expired as it enters the alveolar air space. Witness patient). AOCs are also useful in the prehospital environment and in the developing world where donor blood is not readily available. No AOC has been approved CASE STUDY (continued from page 97) by the Food and Drug Administration (FDA) for use in the After a week at home, Jerry called his doctor, who United States.11,12 sent him back to the hospital where he was given Two groups of AOCs are the most developed alterna- 2 units of packed red cells. Within a day, he had tives to RBC donor units: acellular hemoglobin-based oxy- more energy. gen carriers (HBOCs) in solution and perfluorocarbon (PFC) 4. Explain why Jerry would have had more energy emulsions. The HBOCs consist of purified human or bovine after the transfusions. hemoglobin or recombinant hemoglobin. The hemoglobin is altered chemically or genetically or is microencapsulated to decrease oxygen affinity, to make its ODC similar to that of native human blood and to prevent its breakdown into Nitric Oxide and Hemoglobin dimers that have significant nephrotic toxicity.13 The HBOCs have a relatively short half-life (usually about 24 hours), Nitric oxide (NO) is a critical component for the mainte- which requires repetitive infusions. Adverse side effects of nance of blood vessel homeostasis. NO derived its name as the artificial oxygen carriers are related to NO scavenging the endothelium-derived relaxing factor (EDRF) because of and inactivation by the free hemoglobin as well as endothe- its ability to relax smooth muscle and dilate blood vessels.9 lin (a vasoconstrictor) release and sensitization of peripheral It is important in other aspects of normal vessel physiol- adrenergic receptors.14 They induce hypertension and lead ogy as well as inhibition of platelet activation. NO is pro- to low cardiac output.15 Most acellular HBOCs have short duced in the endothelium from arginine by the action of circulatory half-lives, limiting their utility. A meta-analysis NO synthase. NO can diffuse from the plasma across the study published in 2008 revealed a 30% increased risk of erythrocyte membrane where it is picked up by oxyhe- mortality and a 2.7-fold increase in myocardial infarction moglobin. Reaction with oxyhemoglobin destroys the NO associated with the use of HBOCs.16 Although the study and forms methemoglobin and nitrate, a process known as was shown to be flawed, following the report, develop- dioxygenation. ment of HBOCs decreased and some clinical trials stopped HbO2 + NO S MetHb + NO - 3 prematurely. However, development of second-generation HBOCs has continued. Reaction of NO and hemoglobin is limited because of PFCs are fluorinated hydrocarbons with high gas- hemoglobin compartmentalization in the erythrocyte, slow dissolving capacity. They do not mix in aqueous solution diffusion of NO across the RBC membrane, and the laminar and must be emulsified. In contrast to HBOCs, a linear blood flow that pushes the erythrocytes inward away from relationship between PO2 and oxygen content in PFCs the vessel endothelium where the NO is concentrated.10 The exists. This means that relatively high O2 partial pres- rate of reaction of NO with cell-free hemoglobin is increased sure is required to maximize delivery of O2 by PFCs. The by at least 1000-fold. This extracellular reaction is responsi- PFC droplets are taken up by the mononuclear phagocyte ble for complications such as vasoconstriction and increase (MNP) system, broken down, bound to blood lipids, trans- of blood pressure that are encountered when using artificial ported to the lungs, and exhaled.14 Clinical trials of PFCs hemoglobin-based oxygen carriers in solution. The reaction were terminated due to adverse effects, but the search for also appears to be responsible for complications (e.g., high alternatives to PFCs continues. Current PFC development blood pressure) that accompany some hemolytic anemias is aimed at producing stable emulsions of smaller size, such as sickle cell disease. which may improve biological distribution and clearance properties. Artificial Oxygen Carriers Research is underway to develop novel synthetic AOCs, For about 30 years there has been substantial effort to including microparticle, nanoparticle, and stem cell–based develop a blood substitute (artificial oxygen carrier [AOC]) oxygen carriers.17 to help meet the demand for blood and avoid complica- Hemoglobin in solution imparts color to plasma. Thus, tions associated with acquiring and storing whole blood for it might not be possible to perform laboratory tests based transfusions. AOCs are also useful when donor RBC units on colorimetric analysis of patients receiving these products are unavailable in trauma cases for treatment of severe because measurements could give erroneous results. 102 Chapter 6 Hemoglobin Catabolism normal erythrocyte aging process is conserved and reuti- lized. The globin portion of the hemoglobin molecule is When the erythrocyte is removed from circulation by mac- broken down and recycled into the amino acid pool. rophages (extravascular hemolysis) or is lysed in the blood Heme, the porphyrin ring, is further catabolized by stream (intravascular hemolysis), hemoglobin is released the macrophage and eventually excreted in the feces. The and catabolized. a-methane bridge of the porphyrin ring is cleaved, produc- ing a molecule of carbon monoxide and the linear tetrapyr- Extravascular Destruction role biliverdin. Carbon monoxide is released to the blood stream, carried to the lungs, and expired. The biliverdin In extravascular hemolysis, erythrocyte removal by macro- is rapidly reduced within the cell to bilirubin. Released phages in the spleen, bone marrow, and liver conserves and from the macrophage, bilirubin is bound by plasma albu- recycles essential erythrocyte components such as amino min and carried to the liver (this is called unconjugated or acids and iron (Figure 6-12). Most extravascular destruction “indirect” bilirubin). Upon uptake by the liver, bilirubin is of erythrocytes takes place in the macrophages of the spleen. conjugated with two molecules of bilirubin glucuronide by Within the macrophage, the hemoglobin molecule is the enzyme bilirubin UDP-glucuronyltransferase present in broken down into heme, iron, and globin. Iron and globin the endoplasmic reticulum of the hepatocyte. Once conju- (a polypeptide) are conserved and reused for new hemoglo- gated, bilirubin becomes polar and lipid insoluble. Bilirubin bin or other protein synthesis. Heme iron can be stored as diglucuronide (called conjugated or “direct” bilirubin) is ferritin or hemosiderin within the macrophage or released excreted into the bile, eventually reaching the intestinal tract to the iron transport protein, transferrin, for delivery to where intestinal bacterial flora converts it into urobilinogen. developing normoblasts in the bone marrow. This endog- Most urobilinogen is excreted in the feces where it is quickly enous iron exchange is responsible for about 80% of the iron oxidized to urobilin or stercobilin. However, 10–20% of the passing through the transferrin pool. Thus, iron from the urobilinogen is reabsorbed from the gut back to the plasma. Extravascular hemoglobin degradation Hemoglobin Heme + Globin Plasma protein and amino acid pool Macrophage Lungs Biliverdin + CO + Fe Transferrin + Fe Bone marrow Blood Bilirubin Plasma albumin Bilirubin-Albumin (unconjugated) Liver Bilirubin diglucuronide (conjugated) Bile duct to duodenum Urobilinogen Blood Kidney Stool Urobilinogen (urine) Urobilinogen + Stercobilinogen Figure 6.12 Most hemoglobin degradation occurs within the macrophages of the spleen. The globin and iron portions of the molecule are conserved and reutilized. Heme is reduced to bilirubin, eventually degraded to urobilinogen, and excreted in the feces. Thus, indirect indicators of erythrocyte destruction include the blood bilirubin level and urobilinogen concentration in the urine. Hemoglobin 103 The reabsorbed urobilinogen is either excreted in urine or to maintain plasma haptoglobin levels. Haptoglobin, how- returned to the gut via an enterohepatic cycle. In liver dis- ever, is an acute-phase reactant, and increased concentra- ease, the enterohepatic cycle is impaired and an increased tions can be found in inflammatory, infectious, or neoplastic amount of urobilinogen is excreted in the urine. conditions. (An acute phase reactant is a protein whose plasma concentration increases in response to inflamma- Intravascular Destruction tion and serves a function in the immune response.) There- fore, patients with hemolytic anemia (anemia caused by The small amount of hemoglobin released into the periph- increased destruction of erythrocytes) accompanied by an eral blood circulation through intravascular erythrocyte underlying infectious or inflammatory process can have breakdown undergoes dissociation into ab dimers, which normal haptoglobin levels. are quickly bound to the plasma a-glycoprotein haptoglobin When haptoglobin is depleted, as in severe hemolysis, (Hp) in a 1:1 ratio (Figure 6-13). The haptoglobin– free ab hemoglobin dimers can be filtered by the kidney and hemoglobin (HpHb) complex is too large to be filtered by reabsorbed by the proximal tubular cells. ab dimers passing the kidney, so haptoglobin carries hemoglobin dimers in through the kidney more than the reabsorption capabilities the blood to the liver. Hepatocytes, which have haptoglobin of the tubular cells appear in the urine as free hemoglobin. receptors, take up the HpHb and process it in a manner like Dimers reabsorbed by the tubular cells are catabolized to that of hemoglobin released by extravascular destruction. bilirubin and iron, both of which can reenter the plasma The HpHb complex is cleared very rapidly from the pool. However, some iron remains in the tubular cell and bloodstream with a T1/2 disappearance rate of 10–30 min- is complexed to storage proteins forming ferritin and utes. The haptoglobin concentration can be depleted very hemosiderin. Eventually, tubular cells loaded with iron are rapidly in acute hemolytic states because the liver is unable sloughed off and excreted in the urine ( hemosiderinuria). Intravascular hemoglobin degradation Free Hb in blood Haptoglobin Hb-haptoglobin Liver (catabolism same as extravascular) Hb in excess of haptoglobin ab dimers Methemoglobin Kidney Globin Amino acid pool Tubular reabsorption Heme (Fe***) Urine Urine hemoglobin hemosiderin Hemopexin Hemopexin-heme Albumin Methemalbumin Albumin Heme RE cells in liver Figure 6.13 When the erythrocyte is destroyed within the vascular system, hemoglobin is released directly into the blood. Normally, the free hemoglobin quickly complexes with haptoglobin, and the complex is degraded in the liver. In severe hemolytic states, haptoglobin can become depleted, and free hemoglobin dimers are filtered by the kidney. In addition, with haptoglobin depletion, some hemoglobin is quickly oxidized to methemoglobin and dissociates into heme and globin. The heme combines with hemopexin or albumin and is cleared by the liver. Globin is recycled into the amino acid pool and bound to either hemopexin or albumin for eventual degradation in the liver. 104 Chapter 6 The iron inclusions can be visualized with the Prussian blue hypoxia and/or cyanosis (Table 6-5). Hypoxia is a condition stain. Thus, the presence hemosiderinuria is a sign of recent in which there is an inadequate amount of oxygen at the tis- increased intravascular hemolysis. sue level. Hypoxemia is an inadequate amount of oxygen in Hemoglobin not excreted by the kidney or bound to the blood; arterial PO2 less than 80 mmHg. Cyanosis refers haptoglobin is either cleared directly by hepatic uptake or to a bluish or slate-gray color of the skin due to the presence oxidized to methemoglobin. Heme dissociates from methe- of more than 5 g/dL of deoxyhemoglobin in the blood. moglobin and avidly binds to a b-globutlin glycoprotein, hemopexin. Hemopexin is synthesized in the liver and Methemoglobin combines with heme in a 1:1 ratio. The hemopexin–heme complex is cleared from the plasma slowly with a T Methemoglobin is hemoglobin with iron in the ferric 1/2 disappearance of 7–8 hours. When hemopexin becomes 1Fe+++2 state and is incapable of combining with oxygen. depleted, the dissociated oxidized heme combines with Methemoglobin not only decreases the oxygen-carrying plasma albumin in a 1:1 ratio to form methemalbumin. capacity of blood but also results in an increase in oxygen Methemalbumin clearance by the liver is also very slow. affinity of the remaining normal hemoglobin. This results In fact, methemalbumin may be only a temporary carrier
in an even higher deficit of O2 delivery. Normally, methe- for heme until more hemopexin or haptoglobin becomes moglobin composes less than 3% of the total hemoglobin in available. Heme is transferred from methemalbumin to adults.15 At this concentration, the abnormal pigment is not hemopexin for clearance by the liver as it becomes avail- harmful because the reduction in oxygen-carrying capacity able. When present in large quantity, methemalbumin and of the blood is insignificant. hemopexin–heme complexes impart a brownish color to Clinically important methemoglobinemia can be due to the plasma. The Schumm’s test is designed to detect these the following (Table 6-6): abnormal compounds spectrophotometrically. 1. Deficiencies of enzymes that reduce Fe+++-hemoglobin to Fe++-hemoglobin; of these, the most important, accounting for more than 60% of the reduction of Checkpoint 6.6 methemoglobin, is NADH methemoglobin reductase What lab tests would help diagnose an increase in RBC destruc- tion (i.e., hemolysis), and what would be the expected results? (Table 6-7) (Chapter 18). 2. Globin chain mutations that stabilize heme iron in the Fe+++ state (hemoglobin M; Chapter 13). This structural variant of hemoglobin is characterized by amino acid Acquired Nonfunctional substitutions in the globin chains near the heme pocket that stabilize the iron in the oxidized Fe+++ Hemoglobins state. 3. Exposure to toxic substances that oxidize hemoglobin The acquired, nonfunctional hemoglobins are hemoglobins and overwhelm the normal reducing capacity of the that have been altered post-translationally to produce mole- cell. Increased levels of methemoglobin are formed cules with compromised oxygen transport, thereby causing when an individual is exposed to certain oxidizing Table 6.5 Abnormal Acquired Hemoglobins Hemoglobin Acquired Change Abnormal Function Lab Detection Methemoglobin Hb iron in ferric state Cannot combine with oxygen Demonstration of maximal absorption band at wave length of 630 nm; chocolate color blood Sulfhemoglobin Sulfur combined with hemoglobin 1 100 oxygen affinity of HbA Absorption band at 620 nm Carboxyhemoglobin Carbon monoxide combined with Affinity for carbon monoxide is 200 times Absorption band at 541 nm hemoglobin higher than for oxygen Table 6.6 Differentiation of Types of Methemoglobinemia Cause of Methemoglobinemia Inherited/Acquired Enzyme Activity Hb Electrophoresis Decreased enzyme activity Inherited Decreased Normal Presence of hemoglobin M Inherited Normal Abnormal Exposure to oxidants Acquired Normal Normal Hemoglobin 105 can be chocolate brown in color when compared with a Table 6.7 Erythrocyte Systems Responsible for normal blood specimen, and the color does not change to Methemoglobin Reduction red upon exposure to oxygen.18 Differentiation of acquired Rank in Order types from hereditary types of methemoglobin requires of Decreasing System assay of NADH methemoglobin reductase and hemoglo- Methemoglobin Reduction bin electrophoresis (Table 6-7). Enzyme activity is reduced only in hereditary NADH-methemoglobin reductase defi- First NADH methemoglobin reductase (also known as cytochrome b5 methemoglobin reductase, diaph- ciency, and hemoglobin electrophoresis is abnormal only orase I, DPNH-diaphorase, DPNH dehydrogenase in HbM disease. Acquired methemoglobinemia shows I, NADH dehydrogenase, NADH methemoglobin- ferrocyanide reductase) normal enzyme activity and a normal electrophoresis Second Ascorbic acid pattern. Third Glutathione In the presence of methemoglobinemia, oxygen satu- Fourth NADPH methemoglobin reductase ration obtained by a cutaneous pulse oximeter (fractional oxyhemoglobin, FhbO2) can be lower than the oxygen satu- ration reported from a blood gas analysis. This is because chemicals or drugs. Even small amounts of these chem- FhbO2 is calculated as the amount of oxyhemoglobin com- icals and drugs can cause oxidation of large amounts pared with the total hemoglobin (oxyhemoglobin, deoxyhe- of hemoglobin. If the offending agent is removed, moglobin, methemoglobin, and other inactive hemoglobin methemoglobinemia returns to normal levels within forms) whereas oxygen saturation in a blood gas analy- 24–48 hours. sis is the amount of oxyhemoglobin compared with the total amount of hemoglobin able to combine with oxygen Infants are more susceptible to methemoglobin produc- (oxyhemoglobin plus deoxyhemoglobin). FhbO2 and oxy- tion than adults because HbF is more readily converted to gen saturation are the same if no abnormal hemoglobin is methemoglobin and because infants’ erythrocytes are defi- present.19 cient in reducing enzymes. Exposure to certain drugs or chemicals that increase oxidation of hemoglobin or water high in nitrates can cause methemoglobinemia in infants. Sulfhemoglobin Color crayons containing aniline can cause methemoglo- Sulfhemoglobin is a stable compound formed when a sul- binemia if ingested. fur atom combines with the heme group of hemoglobin. Cyanosis develops when methemoglobin levels exceed The sulfur atom binds to a pyrrole carbon at the periphery 10% (more than 1.5 g/dL) hypoxia is produced at levels of the porphyrin ring. Sulfuration of heme groups results in exceeding 30–40%. Toxic levels of methemoglobin can be a drastically right-shifted oxygenation dissociation curve, reduced by medical treatment with methylene blue or which renders the heme groups ineffective for oxygen ascorbic acid, which speeds up reduction by NADPH- transport. This appears to be because even half-sulfurated, reducing enzymes. The NADPH reductase system requires half-oxygen–liganded tetramers exist in the T configuration G6PD and therefore this method of treatment is not effec- (the low oxygen-affinity form) of hemoglobin. Although the tive in patients with G6PD deficiency. In some cases of heme iron is in the ferrous state, sulfhemoglobin binds to severe methemoglobinemia, exchange transfusions are oxygen with an affinity only one-hundredth that of normal helpful. hemoglobin. Thus, oxygen delivery to the tissues can be In the congenital methemoglobinemias, cyanosis is compromised if there is an increase in this abnormal hemo- observed from birth, and methemoglobin levels reach globin. The bright green sulfhemoglobin compound is so 10–20%. Normal hemoglobin’s oxygen affinity is increased stable that the erythrocyte carries it until the cell is removed in these children, resulting in increased erythropoiesis and from circulation. Ascorbic acid or methylene blue cannot subsequently higher than normal hemoglobin levels and reduce it; however, sulfhemoglobin can combine with car- erythrocytosis. Even in the homozygous state, individu- bon monoxide to form carboxysulfhemoglobin. Normal als with HbM or defects in the reducing systems rarely levels of sulfhemoglobin do not exceed 2.2%. Cyanosis is have methemoglobin levels of more than 25% and are produced at levels exceeding 3–4%. usually asymptomatic except for mild cyanosis. They do Sulfhemoglobin has been associated with occupa- not usually require treatment. However, cyanosis can be tional exposure to sulfur compounds, environmental improved by treatment with methylene blue or ascor- exposure to polluted air, and exposure to certain drugs. bic acid. Laboratory diagnosis of methemoglobinemia Sulfhemoglobinemia is formed during the oxidative dena- involves demonstration of a maximum absorbance band turation of hemoglobin and can accompany methemoglo- at a wavelength of 630 nm at pH 7.0–7.4. The blood sample binemia, especially in certain drug- or chemical-induced 106 Chapter 6 hemoglobinopathies. Sulfhemoglobin is formed on expo- Blood normally carries small amounts of carboxyhe- sure of blood to trinitroluene, acetanilid, phenacetin, and moglobin formed from the carbon monoxide produced sulfonamides. It also is elevated in severe constipation during heme catabolism. The level of carboxyhemoglobin and in bacteremia with Clostridium welchii. Diagnosis of varies depending on individuals’ smoking habits and their sulfhemoglobinemia is made spectrophotometrically by environment. City dwellers have higher levels than country demonstrating an absorption band at 620 nm. Confirma- dwellers because of the carbon monoxide produced from tion testing is done by isoelectric focusing. This is the only automobiles and industrial pollutants in cities. abnormal hemoglobin pigment not measured by the cyan- Acute carboxyhemoglobinemia causes irreversible tis- methemoglobin method, which is used to measure hemo- sue damage and death from anoxia. Chronic carboxyhemo- globin concentration. globinemia is characterized by increased oxygen affinity and polycythemia. In severe cases of carbon monoxide Carboxyhemoglobin poisoning, patients can be treated in hyperbaric oxygen chambers. Carboxyhemoglobin is formed when hemoglobin is Carboxyhemoglobin is commonly measured in whole exposed to carbon monoxide. Hemoglobin’s affinity for car- blood by a spectrophotometric method. Sodium hydrosul- bon monoxide is more than 200 times higher than its affinity fite reduces hemoglobin to deoxyhemoglobin, and the for oxygen. Carboxyhemoglobin is incapable of transport- absorbances of the hemolysate are measured at 555 and ing oxygen because CO occupies the same ligand-binding 541 nm. Carboxyhemoglobin has a greater absorbance at position as O2. As is the case with methemoglobinemia, car- 541 nm. boxyhemoglobin has a significant impact on oxygen deliv- ery because it destroys the molecule’s cooperativity. CO also has a pronounced effect on the oxygen dissociation curve, shifting it to the left, resulting in increased affinity and a Checkpoint 6.7 decreased release of O2 by remaining normal hemoglobin A 2-year-old child was found to have 15% methemoglobin by spectral absorbance at 630 nm. What tests would you sug- molecules. Elevated levels of carboxyhemoglobin impart a gest to help differentiate whether this is an inherited or acquired cherry red color to the blood and skin. However, elevated methemoglobinemia, and what results would you expect with levels of it together with elevated levels of deoxyhemoglo- each diagnosis? bin can give blood a purple-pink color. Summary Hemoglobin is the intracellular protein of erythrocytes curve that has shifted to the right reflects decreased oxy- responsible for transport of oxygen from the lungs to the gen affinity; when it has shifted to the left, oxygen affin- tissues. A fine balance between production and destruction ity has increased. Increased CO2, heat, and acid decrease of erythrocytes serves to maintain a steady-state hemoglo- oxygen affinity; high O2 concentrations increase oxygen bin concentration. affinity. Hemoglobin is a globular protein composed of four Hemoglobin is an allosteric protein, which means that subunits. Each subunit contains a porphyrin ring with an other molecules affect hemoglobin structure and function. iron molecule (heme) and a globin chain. The four globin In particular, the uptake of 2,3-BPG or oxygen can cause chains are arranged in identical pairs, each composed of conformational changes in the molecule. The structure of two different globin chains (e.g., a2b2). Hemoglobin syn- deoxyhemoglobin is known as the T structure and that of thesis is controlled by iron concentration within the cell, oxyhemoglobin is known as the R structure. synthesis and activity of the first enzyme in the heme syn- When hemoglobin is exposed to oxidants or other thetic pathway (ALAS), activity of PBGD, and globin chain compounds, the molecule can be altered, which can com- synthesis. promise its ability to carry oxygen. High concentrations The oxygen affinity of hemoglobin depends on PO2, of these abnormal hemoglobins can cause hypoxia and pH, PCO2, 2,3-BPG, and temperature. Hemoglobin–oxy- cyanosis, which can be detected by spectrophotometric gen affinity can be graphically depicted by the ODC. A methods. Hemoglobin 107 Review Questions Level I c. unaffected in those who are physically fit 1. Which of the following types of hemoglobin is the d. affected only if the duration is more than 1 hour major component of adult hemoglobin? (Objective 4) 8. Which of the following is considered a normal a. HbA hemoglobin concentration in an adult male? b. HbF (Objective 6) c. HbA2 a. 11.0 g/dL d. Hb Portland b. 21.0 g/dL 2. One of the most important buffer systems of the body c. 15.0 g/dL is the: (Objective 5) d. 9.0 g/dL a. chloride shift 9. Haptoglobin can become depleted in: (Objective 10) b. Bohr effect a. inflammatory conditions c. heme–heme interaction b. intravascular hemolysis d. ODC c. infectious diseases 3. When iron is depleted from the developing d. kidney disease erythrocyte, the: (Objective 7) 10. A patient with an anemia due to increased a. synthesis of heme is increased extravascular hemolysis would likely present with b. activity of ALAS is decreased which of the following lab results? (Objective 9) c. formation of globin chains stops a. Increased haptoglobin d. heme synthesis is not affected b. Hemoglobinuria 4. When the H+ concentration in blood increases, the c. Normal hemoglobin and hematocrit oxygen affinity of hemoglobin: (Objective 3) d. Increased serum bilirubin a. increases b. is unaffected Level II c. decreases 1. Which of the following hemoglobins is not found in d. cannot be measured the normal adult? (Objective 2) a. a2b2 5. Which of the following is the correct molecular structure of hemoglobin? (Objective 1) b. a2g2 c. a2d2 a. Four heme groups, two iron, two globin chains d. a2e2 b. Two heme groups, two iron, four globin chains c. Two heme groups, four iron, four globin chains 2. Which of the following is the major hemoglobin in the d. Four heme groups, four iron, four globin chains newborn? (Objective 2) a. a2b6. 2,3-BPG combines with which type of hemoglobin? 2 (Objectives 3, 5) b.
a2g2 c. a a. Oxyhemoglobin 2d2 d. a2eb. Relaxed structure of hemoglobin 2 c. Deoxyhemoglobin 3. A 2-year-old patient who had been cyanotic since d. ab dimer birth was seen by a pediatrician. Blood was drawn for analysis of NADH methemoglobin reductase and 7. During exercise, the oxygen affinity of hemoglobin is: results were normal. What follow-up test would you (Objective 3) suggest to the physician? (Objective 6) a. increased in males but not females a. Hemoglobin electrophoresis b. decreased due to production of heat and lactic acid b. Bone marrow aspiration and examination 108 Chapter 6 c. Haptoglobin and sulfhemoglobin determination 7. When iron in the cell is replete, the translation of d. Glycosylated hemoglobin measurement by column ferritin mRNA is: (Objective 3) chromatography a. decreased 4. A 25-year-old male was found unconscious in a car b. increased with the motor running. Blood was drawn and sent c. unaffected to the chemistry lab for spectral analysis. The blood d. variable was cherry red in color. Which hemoglobin should be tested for? (Objective 6) 8. An aerobics instructor just finished an hour of instruction. Blood is drawn from her for a research a. Sulfhemoglobin study, and the oxygen dissociation is measured. b. Methemoglobin What would you expect to find? (Objective 4) c. Carboxyhemoglobin a. A shift to the left d. Oxyhemoglobin b. A shift to the right 5. The oxygen dissociation curve in a case of chronic car- c. No shift boxyhemoglobin poisoning would show: (Objective 7) d. An increased oxygen affinity a. a shift to the right 9. In the lungs, a hemoglobin molecule takes up two b. a shift to the left oxygen molecules. What effect will this have on the c. a normal curve hemoglobin molecule? (Objectives 5, 8) d. decreased oxygen affinity a. It will increase oxygen affinity. 6. A college student from Louisiana vacationed in b. It will narrow the heme pockets blocking entry of Colorado for spring break. He arrived at Keystone additional oxygen. Resort on the first day. The second day, he was c. The hemoglobin molecule will take on the tense nauseated and had a headache. He went to the structure. medical clinic at the resort and was told he had d. The center cavity will expand, and 2,3-BPG will altitude sickness. The doctor told him to rest for enter. another 24 hours before participating in any activities. What is the most likely reason he will overcome this 10. An anemic patient has hemosiderinuria, increased condition in the next 24 hours? (Objective 4) serum bilirubin, and decreased haptoglobin. This is an indication that there is: (Objective 10) a. His level of HbF will increase to help release more oxygen to the tissues. a. increased intravascular hemolysis b. The amount of carboxyhemoglobin will decrease to b. decreased extravascular hemolysis normal levels. c. hemolysis accompanied by renal disease c. The levels of ATP in his blood will reach maximal d. a defect in the Rapoport-Leubering pathway levels. d. The level of 2,3-BPG will increase and, in turn, decrease oxygen affinity. References 1. Rouault, T. A. (2006). The role of iron regulatory proteins in J. I. Weitz, eds. Hematology: Basic principles and practice. mammalian iron homeostasis and disease. Nature Chemical (pp. 406–417). Philadelphia: Elsevier Churchill Livingstone. Biology, 2(8), 406–414. 4. Bauer, D. E., & Orkin, S.H. (2011). Update on fetal hemoglobin 2. Quigley, J. G., Means Jr., R. T., & Glader, B. (2014). The birth, life, gene regulation in hemoglobinopathies. Current Opinion in and death of red blood cells. Erythropoiesis the mature red blood Pediatrics, 23(1), 1–8. cell, and cell destruction. In: A. F. List, B. Glader, D. A. Arber, F. 5. Pace, B. S., Liu, L., & Makala, L.H. (2015). Cell signaling Paraskevas, G. M. Rodgers, & J. Foerster, eds. Wintrobe’s clinical pathways involved in drug-mediated fetal hemoglobin hematology. (pp. 88–124). Philadelphia: Wolters Kluwer induction: Strategies to treat sickle cell disease. Experimental Health/Lippincott Williams & Wilkins. Biology and Medicine, 240, 1050–1064. 3. Steinberg, M. H., Benz, E. J., Adewoye, A. H., & Ebert, B. L. (2013). 6. Roosjen, M., McColl, B., Kao, B., Gearing, L. J., Blewitt, M. E., & Pathobiology of the human erythrocyte and its hemoglobins. In: Vadolas, J. (2014). Transcriptional regulators Myb and BCL11A R. Hoffman, E. J. Benz Jr., L. E. Silberstein, H. E. Heslop, & interplay with DNA methyltransferase 1 in developmental Hemoglobin 109 silencing of embryonic and fetal B-like globin genes. FASEB 13. Henkel-Hanke, T., & Oleck, M. (2007). Artificial oxygen carriers: Journal, 28(4), 1610–1620. A current review. AANA Journal, 75(3), 205. 7. Bauer, D. E., & Orkin, S. H. (2015). Hemoglobin switching’s 14. Spahn, D. (1999). Blood substitutes artificial oxygen carriers: surprise: The versatile transcription factor BCL11A is a master Perfluorocarbon emulsions. Critical Care, 3(5), R93. repressor of fetal hemoglobin. Current Opinion in Genetics and 15. Chen, J. Y., Scerbo, M., & Kramer, G. (2009). A review of blood Development, 33, 62–70. substitutes: Examining the history, clinical trial results, and ethics 8. Andoh-Duku, A. & Rafeq, S. (2017). High-altitude sickness. In of hemoglobin-based oxygen carriers. Clinics, 64(8), 803–813. F.F. Ferri (Ed.), Ferri’s clinical advisor (pp. 590–591). Philadelphia: 16. Natanson, C., Kern, S. J., Lurie, P., Banks, S. M., & Wolfe, S. M. Elsevier Churchill Livingstone. (2008). Cell-free hemoglobin-based blood substitutes and risk of 9. Schechter, A. N., & Gladwin, M. T. (2003). Hemoglobin and the myocardial infarction and death: A meta-analysis. Journal of the paracrine and endocrine functions of nitric oxide. New England American Medical Association, 299(19), 2304–2312. Journal of Medicine, 348(15), 1483. 17. Tao, Z., & Ghoroghchian, P. P. (2014). Microparticle, nanoparticle, 10. Kim-Shapiro, D. B., Schechter, A. N., & Gladwin, M. T. (2006). and stem cell-based oxygen carriers as advanced blood substi- Unraveling the reactions of nitric oxide, nitrite, and hemoglobin tutes. Trends in Biotechnology, 32(9), 466–473. in physiology and therapeutics. Arteriosclerosis, Thrombosis, and 18. Benz Jr., E. J., & Ebert, B. L. (2013). Hemoglobin variants Vascular Biology, 26(4), 697–705. associated with hemolytic anemia, altered oxygen affinity, 11. Mer, M., Hodgson, E., Wallis, L., Jacobson, B., Levien, L., and methemoglobinemias. In R. Hoffman, E. J. Benz Jr., Snyman, J., et al. (2016). Hemoglobin glutamer-250 (bovine) in L. E. Silberstein, H. E. Heslop, & J. I. Weitz (Eds.), Hematology: South Africa: Consensus usage guidelines from clinician experts Basic principles and practice. (Chapter 41). Philadelphia: Elsevier who have treated patients. Transfusion, 56(10), 2631–2636. Churchill Livingstone. 12. Castro, C. I., & Briceno, J. C. (2010). Perfluorocarbon-based 19. Wentworth, P., Roy, M., & Wilson, B. (1999). Clinical pathology oxygen carriers: Review of products and trials. Artificial Organs, rounds: Toxic methemoglobinemia in a 2-year-old child. 34(8), 622–634. Laboratory Medicine, 30(30), 311–315. Chapter 7 Granulocytes and Monocytes Kristin Landis-Piwowar, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Identify terms associated with increases and 6. Summarize the processes of neutrophil decreases in granulocytes and monocytes. migration, phagocytosis, and neutrophil extracellular traps. 2. Differentiate morphological features of the gran- ulocyte and monocyte precursors found in the 7. List the adult reference intervals for the proliferative compartment of the bone marrow. granulocytes and monocytes found in the peripheral blood. 3. Describe the development, including distin- guishing maturation and cell features, of the 8. Calculate absolute cell counts from data granulocytic and monocytic-macrophage cell provided. lineages. 9. Differentiate and interpret absolute values 4. Describe and differentiate the morphologic and relative values of cell count data. and other distinguishing cell features of 10. List causes/conditions that increase or each of the granulocytes and monocytes decrease absolute numbers of individual found in the peripheral blood. granulocytes and monocytes found in the 5. Explain the function of each type of granu- peripheral blood. locyte and monocyte found in the peripheral 11. Compare and contrast pediatric and newborn blood. reference intervals with adult reference intervals. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Summarize the kinetics of the granulocytic 2. Describe the processes that permit and monocytic-macrophage cell lineages. neutrophils to leave the peripheral blood circulation and move to a site of infection 110 Granulocytes and Monocytes 111 and propose how defects in these processes 4. Explain the physiological events that alter affect the body’s defense mechanism. the number of circulating granulocytes and 3. Compare and contrast the immunologic fea- monocytes in the peripheral blood. tures and functions of each of the granulocytes 5. Correlate the laboratory data that pertain and monocytes found in the peripheral blood. to granulocytes and monocytes with the clinical information for a patient. Chapter Outline Objectives—Level I and Level II 110 Eosinophils 126 Key Terms 111 Basophils 128 Background Basics 111 Monocytes 130 Case Study 112 Summary 133 Overview 112 Review Questions 134 Introduction 112 References 137 Neutrophils 115 Key Terms Agranulocytosis Erythrophagocytosis Neutrophil extracellular traps Azurophilic granule Granulocytosis (NETs) Charcot-Leyden crystal Leukocytosis Neutrophilia Chemokine Leukopenia Opsonin Chemotaxis Marginating pool (MP) Pathogen-associated molecular pat- Circulating pool (CP) Monocyte-macrophage system tern (PAMP) Cluster of differentiation (CD) Mononuclear phagocyte (MNP) Pattern recognition receptor (PRR) Degranulation system Phagocytosis Diapedesis Neutropenia Polymorphonuclear Drumstick (Barr body) Background Basics In addition to the basics from previous chapters, it is helpful • Summarize the function of growth factors and the to have a general understanding of immunology (immune hierarchy of hematopoiesis. (Chapter 4) system and function); biochemistry (proteins, carbohy- • Describe the function of the hematopoietic organs. drates, lipids); algebra; and the use of percentages, ratios, (Chapter 3) proportions, and the metric system. To maximize your learning experience, you should Level II review these concepts from previous chapters before start- • List the growth factors and identify their function in ing this unit of study: leukocyte differentiation and maturation. (Chapter 4) Level I • Describe the structure of the hematopoietic organs. (Chapter 3) • Identify components of the cell and describe their function. (Chapter 2) 112 Chapter 7 acidic dye (eosin), and acidic cellular elements react with CASE STUDY the basic dye (methylene blue). The eosinophil contains We refer to this case study throughout the chapter. large amounts of basic protein in its granules that react Harry, a 30-year-old male in good physical con- with the eosin dye—hence the name eosinophil—whereas dition, had a routine physical examination as a the basophil has granules that are acidic and react with the requirement for purchasing a life insurance policy. basic dye, methylene blue—hence the name basophil. The A CBC was ordered with the following results: Hb neutrophil reacts with both acid and basic components of 15.5 g/dL, Hct 47%, RBC count 5.2 * 106/mcL, the stain, giving the cell cytoplasm a clear or tan to pink- platelet count 172 * 103/mcL, and WBC count ish appearance with pink to violet stained granules. The 12 * 103/mcL. nuclear DNA and cytoplasmic RNA of cells are acidic and Consider how you could explain these results pick up the basic stain, methylene blue. in a healthy male. The eosinophil, basophil, and neutrophil are polymor- phonuclear (their nuclei have many lobes) and because their cytoplasm contains many granules, they are classi- fied as granulocytes. Monocytes are mononuclear cells and Overview contain small numbers of fine granules in a bluish-gray cytoplasm. The terms leukocyte and white blood cell (WBC) are the syn- Leukocytes perform their function of host defense, pri- onymous names given to the nucleated blood cells that marily in the tissues, where they fight infection by two sepa- are involved in the defense against foreign pathogens or rate but interrelated events: the innate immune response antigens. Leukocytes develop from the multipotential (phagocytosis) and the adaptive immune response. The hematopoietic stem cell in the bone marrow. In the pres- innate immune response is mediated by granulocytes and ence of infection or inflammation, leukocytes can increase monocytes and the adaptive immune response is mediated in number and can display morphologic changes. Thus, an by monocytes and lymphocytes (Chapter 8). Eosinophils important screening test for a wide variety of conditions and basophils also mediate allergic and hypersensitivity is the leukocyte count, more commonly referred to as the reactions. WBC count (Chapter 10). Leukocytes are classified as gran- ulocytes (neutrophils, eosinophils, basophils), monocytes, and lymphocytes. This chapter is a study of the normal dif- Granulopoiesis and Monocytopoiesis ferentiation and maturation of granulocytes and the non- Leukocytes develop from multipotential hematopoietic granulocytic monocyte. Each of these cells is discussed in
stem cells (HSCs) in the bone marrow. Upon specific hema- terms of production, cell morphology, concentration in the topoietic growth factor or cytokine stimulation, the stem peripheral blood, and function. Lymphocytes are discussed cell proliferates or commits to differentiate (Chapter 4). The in Chapter 8. differentiated daughter cells of the HSC are the common myeloid progenitor (CMP) cell and the common lymphoid progenitor (CLP). The CMP is restricted to producing cells Introduction of the myeloid system and gives rise to the committed pre- cursor cells for the neutrophilic, eosinophilic, basophilic, With the exception of T lymphocytes, leukocyte precursors and monocytic lineages, whereas the common lymphoid proliferate, differentiate, and mature in the bone marrow. progenitor (CLP) cell gives rise to committed precursor cells Mature leukocytes are released into the peripheral blood for T, B, and natural killer (NK) lymphocytes2 (Chapter 4). where they circulate briefly until they move into the tissues In order for the CMP to differentiate into specific in response to stimulation. The neutrophil, eosinophil, baso- myeloid cells (i.e., a neutrophil vs. a monocyte), it responds phil, monocyte, and lymphocyte are leukocytes normally to various cytokines through an assortment of receptors. found in the peripheral blood of children and adults. These receptors become restricted in their expression as Leukocytes are nearly colorless in an unstained blood the cell commits to a specific lineage. Cytokines that act on smear—hence the term leuko-, meaning “white.” The era of early progenitors of granulocytes and monocytes include morphologic hematology began in 1877 with Paul Ehrlich’s the interleukins IL-1, IL-3, and IL-6, stem cell factor (SCF), discovery of a triacidic stain that allowed for the differ- FLT3 ligand, granulocyte colony-stimulating factor (G-CSF entiation of leukocytes on fixed blood smears.1 The stain or CSF-3), granulocyte monocyte colony-stimulating fac- used today is called Wright’s stain. This stain is composed tor (GM-CSF or CSF-2), and monocyte colony-stimulating of methylene blue and eosin to color the cellular compo- factor (M-CSF or CSF-1). Cytokines not only contribute nents of blood and bone marrow that are spread on glass to differentiation, but also to promoting survival and slides (Chapter 37). Basic cellular elements react with the proliferation. Granulocytes and Monocytes 113 In addition to cytokine-mediated cellular differentia- associated with diseases and disorders will be discussed in tion, micro-RNAs (miRNAs) also contribute to the process of subsequent chapters on leukocytes. granulo- and monopoiesis. MiRNAs are short (18–24 nucle- otides long), noncoding RNAs that regulate gene expres- sion (Chapter 2). They do so by binding and destabilizing CASE STUDY (continued from page 112) mRNA targets or blocking mRNA translation, thus limiting Harry’s CBC results were Hb 15.5 g/dL, Hct the particular genes that can be expressed in the differenti- 47%, RBC count 5.2 * 106/mcL, platelet count ating cell and helping to determine lineage development. 175 * 103/mcL, and WBC count 12 * 103/mcL. Once early granulocyte and monocyte progenitors dif- ferentiate and mature into functional cells, they may be 1. Are any of these results outside the reference released into the peripheral blood or remain in the bone interval? If so, which one(s)? marrow storage pool until needed. 2. If this were a newborn, would you change your evaluation? If so, why? Leukocyte Concentration in the Peripheral Blood An individual’s age and various physiologic and pathologic Neutrophils comprise the largest portion of WBCs in conditions predominantly affect the WBC count. The total peripheral blood followed by lymphocytes (Chapter 8), WBC count is high at birth, ranging from 9 to 30 * 103/mcL monocytes, eosinophils, and basophils, respectively. In an (Table 7-1). A few immature granulocytic cells (myelocytes, adult, neutrophils make up 40–80% of total leukocytes. metamyelocytes) can be seen in the circulation during the At birth, the neutrophil concentration is about 50–60%; first few days of life. However, immature leukocytes are not this level drops to about 30% by 4–6 months of age. After present in the peripheral blood after this age except in cer- 4 years of age, the concentration of neutrophils gradually tain diseases. Within the first week after birth, the leukocyte increases until adult values are reached at about 6 years of count drops to 5921 * 103/mcL. A gradual decline contin- age (1.897.0 x 103/mcL) (Table 7-2). Most peripheral blood ues until about 8 years of age at which time the leukocyte neutrophils are mature segmented forms. However, up to concentration averages 8 * 103/mcL. Adult values average 5% of the less mature, nonsegmented forms, called neutro- from 4.5 to 11.0 * 103/mcL and generally do not vary by phil bands, can be seen in normal specimens. Most variations age or decline with age.3 in the total WBC count are due to an increase or decrease In addition to age, physiologic and pathological events in neutrophils. affect the concentrations of leukocytes. Pregnancy, time of Monocytes usually compose 2–10% (0.190.8 * 103/mcL) day, and an individual’s activity level affect the WBC con- of circulating leukocytes. Occasionally, reactive lympho- centration. Infections and immune-regulated responses cytes (Chapter 8) resemble monocytes in morphology, cause significant changes in leukocytes. Many other patho- posing classification difficulty even for the experienced logic disorders can also cause quantitative and/or qualita- hematologist. Monocytes are functionally more similar to tive changes in white cells. Considerable heterogeneity in the granulocytes than to the nongranulocytic lymphocyte. leukocyte concentration has been found among racial, eth- Peripheral blood eosinophil concentrations are main- nic, and sex subgroups, suggesting the need for unique ref- tained at 0–5% (up to 0.4 * 103/mcL) throughout life. It erence intervals for specific populations.4 Thus, when WBC is possible that no eosinophils will be seen on a 100-cell counts are evaluated, the patient’s age, and possibly race/ differential. However, careful scanning of the entire smear ethnicity and sex, provide useful information. It also is help- should reveal an occasional eosinophil. ful to assess the accuracy of cell counts by correlating them Basophils are the least plentiful cells in the peripheral with the patient’s previous cell counts and clinical history. blood, 0–1% (up to 0.2 * 103/mcL). It is common to find no Additional testing, called reflex testing, can be indicated as basophils on a 100-cell differential. The finding of an abso- a result of abnormalities in the WBC count. Changes lute basophilia (greater than 0.2 * 103/mcL), however, is very important because it can indicate the presence of a hematologic malignancy. An altered concentration of all leukocyte types or, more Table 7.1 WBC Count Intervals by Age commonly, an alteration in one specific type of leukocyte Age Reference Interval can cause an increase or decrease in the total WBC count. Birth For this reason, an abnormal total WBC count should be fol- 9930 * 103/mcL lowed by a leukocyte differential count (commonly referred Childhood 4.5913.5 * 103/mcL to as a WBC differential, or simply diff; Chapter 10). A man- Adults 4.5911.0 * 103/mcL ual WBC differential is performed by enumerating each 114 Chapter 7 stages of development. Other markers are expressed only Table 7.2 Granulocyte and Monocyte Numbers in the after the cell has been stimulated and thus can be used as Peripheral Blood of Adults indicators of cell activation. CD markers are helpful in dif- WBC Reference Interval ferentiating neoplastic hematologic disorders (Chapter 23). Neutrophil 1.897.0 * 103/mcL Monocytes 0.190.8 * 103/mcL Leukocyte Function Eosinophils 090.4 * 103/mcL The primary function of leukocytes is to protect the host Basophils 090.2 * 103/mcL from infectious agents or pathogens by employing defense mechanisms called the innate (natural) and/or the adaptive leukocyte type within a total of 100 leukocytes on a stained (acquired) immune systems. The innate immune response blood smear using a microscope. The differential results (innate IR) is the body’s first response to invading pathogens. are reported as the percentage of each cell type counted. To Many pathogens (bacteria, fungi, and some viruses) carry accurately interpret whether an increase or decrease in cell molecular structures called pathogen-associated molecu- types exists, however, it is necessary to calculate the absolute lar patterns (PAMPs). Examples of PAMPs include bacte- concentration using the results of the WBC count and the rial lipopolysaccharide, fungal mannans, and viral RNA or differential (relative concentration) in the following manner: DNA. Leukocytes use pattern recognition receptors (PRRs) that interact with PAMPs.5 PRRs may be transmembrane Differential count (in decimal form) * WBC count proteins that function to activate the leukocyte, while other * (103/mcL) = Absolute cell count (* 103/mcL) PRRs are soluble serum proteins that act as opsonins to help The application of this calculation is emphasized in the recognize or neutralize pathogens. Once a pathogen has been following example. Two different blood specimens from two recognized, leukocytes can attack, engulf, and kill it. Neutro- different patients were found to have a relative neutrophil phils, monocytes, and macrophages play a major role in the concentration of 85%. The total WBC count in one patient innate immune system. The innate IR is rapid but limited. was 3 * 103/mcL and in the other was 9 * 103/mcL. The adaptive immune response (adaptive IR) is initi- The relative neutrophil concentration on both specimens ated in lymphoid tissue where pathogens encounter lym- appears elevated (reference interval is 40–80%); however, phocytes, the major cells involved in this response. This calculation of the absolute concentration (reference interval IR is slower to develop than the innate IR, but it provides 1.897.0 * 103/mcL) shows that only one specimen has an long-lasting immunity (memory) against the pathogen with absolute increase in neutrophils, whereas the other is within which it interacts. The adaptive IR is discussed in more the reference interval: detail in Chapter 8. In addition to its role in protection against infections, 0.85 * (3 * 103/mcL) = 2.6 * 103/mcL the cells of the innate immune system possess mechanisms (within the reference interval) to recognize the products of damaged and dead host cells, 0.85 * (9 * 103/mcL) = 7.7 * 103/mcL (increased) eliminating those cells and initiating tissue repair. These substances are called damage-associated molecular patterns A normal leukocyte count does not rule out the pres- (DAMPs) and include stress-associated heat shock proteins, ence of disease, but leukocytosis (an increase in leukocytes) crystals, and nuclear proteins.5 or leukopenia (a decrease in leukocytes) is an important clue to disease processes and deserves further investiga- tion, including a leukocyte differential count to identify the concentration of the different types of leukocytes. CASE STUDY (continued from page 113) The WBC differential performed on the specimen Leukocyte Surface Markers from Harry had the following results: WBCs 12.0 * 103/mcL Leukocytes and other cells express a variety of molecules on their surfaces that can be used as markers to help identify Neutrophils 58% the cell’s lineage as well as subsets within the lineage. These Lymphocytes 32% markers can be identified by reactions with specific antibod- Monocytes 6% ies, a process called immunophenotyping (Chapter 40). A Eosinophils 3% nomenclature system using the term cluster of differentia- Basophils 1% tion (CD), followed by a number, identifies the molecule recognized by the antibody. In addition to using CD mark- 3. Are any of the WBC concentrations outside the ers to identify cell lineage, they are used to identify stages reference interval (relative or absolute)? of maturation since they are transiently expressed at specific Granulocytes and Monocytes 115 Neutrophils molecules of physiologic importance. The biosynthesis of the granule content is primarily determined by activation or inhi- Neutrophils are the most numerous leukocyte in the periph- bition of transcription factors at certain time points during eral blood. They are easily identified on Romanowsky- neutrophil development. Leukopoiesis is a process that gen- stained peripheral blood smears as cells with a segmented erates 195 * 109 cells per hour or 1011 cells per day.6,7 How- nucleus and fine pink to lavender granules. ever, the marrow has the capacity to significantly increase the neutrophil production over this baseline level in response to Differentiation, Maturation, and infectious or inflammatory stimuli. The morphology of the Morphology stages of maturation is discussed in the following sections. When lineage commitment to the CMP has occurred, matu- MYELOBLAST ration begins. Myeloid cells go through a unique matura- The myeloblast (Table 7-3, Figure 7-1 and Figure 7-3) is tion process, described in Figure 4-5 in Chapter 4. the earliest morphologically recognizable precursor of Neutrophilic production is primarily regulated by three the myeloid lineage. The myeloblast size
varies from cytokines, IL-3, GM-CSF, and G-CSF. GM-CSF and G-CSF 14–20 mcM (mm) in diameter, and it has a high nuclear to also regulate survival and functional activity of mature neutrophils. The neutrophil undergoes six morphologi- cally identifiable stages in the process of maturation. The a stages from the first morphologically identifiable cell to the mature segmented neutrophil include (1) myeloblast, (2) promyelocyte, (3) myelocyte, (4) metamyelocyte, (5) band or nonsegmented neutrophil, and (6) segmented neutrophil, also referred to as the polymorphonuclear neutrophil (PMN). During the maturation process, progressive morpho- logical changes occur in the nucleus. The nucleoli disappear, the chromatin condenses, and the once round nuclear mass b indents and eventually segments. These nuclear changes are accompanied by distinct cytoplasmic changes. The scanty, agranular, basophilic cytoplasm of the earliest stage Figure 7.2 In the center are a myelocyte (a) and a is gradually replaced by pink-to-tan-staining granular cyto- promyelocyte (b). Note the changes in the nucleus and cytoplasm. plasm in the mature differentiated stage (Figures 7-1 and 7-2, The myelocyte has a clear area next to the nucleus, which represents the Golgi apparatus. Note the azurophilic granules in the Table 7-3). The four subsets of granules/organelles (pri- promyelocyte. Also present are two bands and in the top right corner mary, secondary, secretory, tertiary) are produced at specific a metamyelocyte. Orthochromatic normoblasts are present (bone times during neutrophil development and contain specific marrow, Wright-Giemsa stain, 1000* magnification). a b Figure 7.3 (a) Indicates a pronormoblast and (b) indicates a myeloblast. Note that the myeloblast has more lacy, lighter-staining Figure 7.1 Stages of neutrophil development. Compare the chromatin with distinct nucleoli and bluish cytoplasm whereas chromatin pattern of the nucleus and the cytoplasmic changes the pronormoblast chromatin is more smudged with indistinct in the various stages. From left: a very early band, myelocyte, nucleoli and very deep blue-purple cytoplasm. Also pictured promyelocyte, myeloblast, and very early band; above the are bands, metamyelocyte, myelocytes, basophilic normoblast, myeloblast are two segmented neutrophils (bone marrow; Wright- polychromatophilic normoblast, and orthochromatic normoblast Giemsa stain; 1000* magnification). (bone marrow, Wright-Giemsa stain; 1000* magnification). 116 Chapter 7 Table 7.3 Characteristics of Cells in the Maturation Stages of the Neutrophil Cell Stage (% in bone N:C Ratio; CD Maturation Figure Nucleus Cytoplasm Granules marrow) Size (mcM) Markers Transit Time Myeloblast (0.2–1.5) Round or oval; delicate, Light blue High; 14–20 Absent CD13, ~1 day lacy chromatin; nucleoli CD33, CD34, CD162 Promyelocyte (2–4) Round or oval, Deep blue High but less Large, reddish-purple CD13, 1–3 days chromatin lacy but than myeloblast; (azurophilic) primary or CD 15, more condensed than 15–21 nonspecific granules CD33, blast; nucleoli present CD162 Myelocyte (8–16) Round to oval; Light blue, more Decreased from Small pinkish-red CD15, 1–5 days chromatin more mature shows tan to promyelocyte; specific granules; CD18, condensed; nucleoli pink 12–18 azurophilic granules; CD162 usually absent secretory vesicles Metamyelocyte (9–25) Chromatin condensed; Pinkish-tan Decreased 10–18 Predominance of small CD15, 0.5–4 days stains dark purple; pinkish-lavender specific CD55, kidney bean shape granules; some azurophilic CD162 to oval granules present; secretory vesicles Band ( nonsegmented) Chromatin Pink to tan to clear Decreased Abundant small, pinkish CD15, 0.5–4 days (9–15) condensed at ends lavender specific granules; CD55, of horseshoe-shaped some azurophilic granules CD65, nucleus; stains dark present; secretory vesicles; CD162 purple tertiary granules Segmented Neutrophil Nucleus segmented Pink or tan to clear Decreased As in band CD13, 1–5 days (polymorphonuclear) into 2–4 lobes; CD15, chromatin condensed; CD65, stains deep purple/black CD162 Granulocytes and Monocytes 117 cytoplasmic (N:C) ratio. The nucleus is usually round or early myelocyte has a rather basophilic cytoplasm, whereas oval and contains a delicate, lacy, evenly stained chromatin. the later, more mature myelocyte, has a more tan to pink One to five nucleoli are visible. The small amount of cyto- cytoplasm as the cell begins to lose cytoplasmic RNA. plasm is agranular, staining from deep blue to a lighter blue. The hallmark for the myelocyte stage is the appearance A distinct unstained area adjacent to the nucleus represent- of specific or secondary granules. Synthesis of peroxidase- ing the Golgi apparatus can be seen. Myeloblasts can stain positive primary granules is halted, and the cell switches faintly positive for peroxidase and esterase enzymes and to synthesis of peroxidase-negative secondary granules. for lipids (Sudan black B; Chapter 37) although granules Secondary granules are detected first near the nucleus in are not evident by light microscopy. Staining reactions with the Golgi apparatus. This has sometimes been referred to as peroxidase and esterase help differentiate myeloblasts from the dawn of neutrophilia. These neutrophilic secondary gran- monoblasts and lymphoblasts. CD markers also aid in iden- ules are small and sandlike with a pinkish-red to pinkish- tifying the lineage of blasts (Chapters 37 and 43). Myeloblast lavender tint. Like the primary granules, a phospholipid CD markers include CD33, CD13, CD34, and CD162.7 membrane surrounds the secondary granules. Large pri- mary azurophilic granules can still be apparent, but their PROMYELOCYTE concentration decreases with each successive cell division The promyelocyte (also called progranulocyte; Table 7-3, because their synthesis has ceased. Their ability to pick up Figures 7-1 and 7-2) varies in size from 15–21 mcM. The stain also decreases with successive mitotic divisions. See nucleus is still quite large, and the N:C ratio is high. The Table 7-4 for a partial list of secondary granule contents.8 nuclear chromatin structure, although coarser than that of Secretory vesicles are scattered throughout the cyto- the myeloblast, is still open and rather lacy, staining purple plasm of myelocytes, metamyelocytes, band neutrophils, to dark blue. The color of the nucleus varies somewhat and segmented neutrophils2 (Table 7-4). Secretory vesicles depending on the stain used, and several nucleoli can still are formed by endocytosis in the later stages of neutrophil be visible. The basophilic cytoplasm is similar to that of the maturation and contain plasma proteins including albumin. myeloblast but is differentiated by the presence of promi- When neutrophils are stimulated, the cytoplasmic secretory nent, reddish-purple primary granules, also called nonspe- vesicles fuse with the plasma membrane to increase the neu- cific or azurophilic granules, which are synthesized during trophil surface membrane and expression of adhesion and this stage. The primary granules are surrounded by a phos- chemotactic receptors. pholipid membrane and contain peroxidase and a number of antimicrobial compounds. See Table 7-4 for a list of the METAMYELOCYTE contents of primary granules. The metamyelocyte (Table 7-3, Figures 7-1 and 7-3) varies in MYELOCYTE size from 10–18 mcM in diameter and is not capable of cell The myelocyte (Table 7-3, Figures 7-1 through 7-3) varies in division. Nuclear indentation that gives the nucleus a kid- size from 12–18 mcM. The nucleus is reduced in size (as is ney bean shape can be a characteristic that differentiates a the N:C ratio) due to nuclear chromatin condensation and metamyelocyte from a myelocyte, but nuclear shape is vari- appears more darkly stained than the chromatin of the pro- able and is not the most reliable identifying feature. Care myelocyte. Nucleoli can be seen in the early myelocyte but should be taken to review other cellular features such as the are usually indistinct. The myelocyte nucleus can be round, degree of the chromatin clumping, color of the cytoplasm, oval, slightly flattened on one side, or slightly indented.8 The predominant granules present, and the cell size. The nuclear clear light area next to the nucleus, representing the Golgi chromatin is coarse and clumped and stains dark purple. apparatus, can still be seen. The myelocyte goes through Nucleoli are not visible. The cytoplasm has a predominance two to three cell divisions; this is the last stage of the matu- of secondary and secretory granules. The ratio of second- ration process in which the cell is capable of mitosis. The ary to primary granules is about 2:1. The metamyelocyte’s Table 7.4 Neutrophil Granule Contents Primary Granules Secondary Granules Secretory Vesicles Tertiary Granules Myeloperoxidase Lactoferrin Alkaline phosphatase Gelatinase Lysozyme Lysozyme Complement receptor 1 Lysozyme Cathepsin G, B, and D Histaminase Cytochrome b558 Defensins (group of cationic proteins) Collagenase Bactericidal permeability increasing protein Gelatinase (BPI) Esterase N Heparinase Elastase 118 Chapter 7 cytoplasm resembles the color of the cytoplasm of a fully included with the total neutrophil count by the hematology mature neutrophil (pinkish-tan). Tertiary or gelatinase- instrument.9 containing granules are synthesized mainly during the The cytoplasm of the mature neutrophil stains a pink metamyelocyte and band neutrophil stages.8 or tan to clear color. It contains many secondary and ter- tiary granules and secretory vesicles. Primary granules are BAND NEUTROPHIL present, but they lose staining quality and therefore might The band neutrophil, also called nonsegmented neutrophil not be readily evident. The ratio of secondary to primary or stab cell, is slightly smaller in diameter than the meta- granules remains about 2:1. myelocyte. The metamyelocyte becomes a band when the Neutrophil granules contain protein, lipids, and car- indentation of the nucleus is more than half the diameter of bohydrates. Many of the proteins (enzymes) have already the hypothetical round nucleus (Table 7-3 and Figure 7-1). been discussed. About one-third of the lipids in neutrophils The indentation gives the nucleus a horseshoe shape. The consist of phospholipids that are present in the plasma chromatin displays increased condensation at either end of membrane or membranes of the various granules. Most the nucleus. The cytoplasm appears pink to tan, resembling of the nonphospholipids are cholesterol and triglycerides. both the previous stage and the fully mature segmented Although cytoplasmic nonmembrane lipid bodies can also forms. The band neutrophil is the first stage that normally be found in neutrophils, their role in cell function is unclear. is found in the peripheral blood. All four types of granules Lipid material is likewise found in neutrophil precursors. (primary, secondary, secretory, tertiary) can be found at this Carbohydrate in the form of glycogen is also found in neu- stage, but primary granules are not usually differentiated trophils and some myeloid precursors. Neutrophils use with Wright stain in band neutrophils. glycogen to obtain energy by glycolysis when required to SEGMENTED NEUTROPHIL function in hypoxic conditions (e.g., an abscess site). CD Although similar in size to the band form, the neutrophil, markers on the neutrophil include CD13, CD15, CD16, is recognized, as its name implies, by a segmented nucleus CD11b/CD18, and CD33.7 with two or more lobes connected by a thin nuclear fila- In normal females with two X chromosomes or males ment (Table 7-3, Figure 7-1). The chromatin is condensed with XXY chromosomes (Klinefelter syndrome), one X chro- and stains a deep purple black. Most neutrophils have three mosome is randomly inactivated in each somatic cell of the or four nuclear lobes, but a range of two to five lobes is pos- embryo and remains inactive in all daughter cells produced sible. Fewer than three lobes are considered hyposegmented. from that cell. The inactive X chromosome appears as an A cell with more than five lobes is considered abnormal appendage of the neutrophil nucleus and is called a drumstick and referred to as hypersegmented. Observing three or more (Barr body) or an X chromatin body (Figure 7-4). The number of five-lobed neutrophils in a 100-cell differential is usually chromatin bodies detected in the neutrophil is one less than considered pathologic (megaloblastic anemia; Chapter 15). the number of X chromosomes present; however, chromatin Nuclear lobes are often touching or superimposed on one bodies are not visible in every neutrophil. The X chromatin another, sometimes making it difficult to differentiate the bodies can be identified in 2–3% of the circulating neutrophils cell as a band or a neutrophil. of 46, XX females, and Klinefelter males (47, XXY).8 Individual laboratories and agencies such as the Clini- cal and Laboratory Standards Institute (CLSI) have outlined criteria for differentiating bands from neutrophils when performing manual differentials.9 A band is defined as hav- ing a nucleus with a connecting strip or isthmus with par- allel sides and having width enough to reveal two distinct margins with nuclear chromatin material visible between the margins. If a margin of a given lobe can be traced as a definite and continuing line from one side across the isth- mus to the other side, a filament is assumed to be present although it is not visible. If a laboratory professional is not sure whether a neutrophil is a band form or a segmented form, it is
arbitrarily classified as a segmented neutrophil. From a traditional clinical viewpoint, determining whether young forms of neutrophils (band forms and younger) are increased may be useful.8 However, differentials per- formed by automated hematology instruments do not dif- Figure 7.4 The segmented neutrophil on the right has an X ferentiate between band and segmented neutrophils. Band chromatin body (arrow) (peripheral blood, Wright-Giemsa stain, neutrophils are fully functional phagocytes and often are 1000* magnification). Granulocytes and Monocytes 119 Checkpoint 7.1 Bone marrow An adult patient’s peripheral blood smear revealed many myelo- cytes, metamyelocytes, and band forms of neutrophils. Is this Stem cell pool a normal finding? • Hematopoietic stem cells • Progenitor cells Distribution, Concentration, and Kinetics Mitotic pool (3–6 days) The kinetics of a group of cells—their production, distribu- • Myeloblasts tion, and destruction—is also described as the cell turnover • Promyelocytes rate. For the neutrophil, kinetics follows the movement of • Myelocytes the cell through a series of interconnected compartments: the bone marrow (site of proliferation, differentiation, and maturation), the peripheral blood (where they circulate for a Postmitotic/storage pool few hours), and the tissues (where they perform their func- (5–7 days) tion of host defense). • Metamyelocytes • Bands BONE MARROW • Segmented neutrophils Neutrophils in the bone marrow are derived from the stem cell pool and can be divided into two pools: the mitotic pool and the postmitotic pool (Figure 7-5). The mitotic pool, also Peripheral blood (<10 hours) called the proliferating pool, includes cells capable of DNA CP synthesis: myeloblasts, promyelocytes, and myelocytes. Cells spend about 3–6 days in this proliferating pool and MP undergo four to five cell divisions. Although two to three of these divisions occur in the myelocyte stage, the number of cell divisions at each stage is variable. The postmitotic pool, also known as the maturation and storage pool, includes Tissues (1–5 days) metamyelocytes, bands, and segmented neutrophils. Cells • Segmented neutrophils spend about 5–7 days in this compartment before they are released to the peripheral blood. However, during infec- tions, the myelocyte-to-blood transit time can be as short as 2 days. The number of cells in the postmitotic storage pool Figure 7.5 Neutrophils are produced from stem cells in the bone marrow and spend about 1–2 weeks in this maturation is almost three times that of the mitotic pool.10 compartment. Most neutrophils are released to the peripheral blood The largest compartment of neutrophils is found within as segmented forms. When the demand for these cells is increased, the bone marrow and is referred to as the mature neutrophil more immature forms can be released. One-half of the neutrophils in reserve. The number of neutrophils circulating in the periph- the peripheral blood are in the marginating pool (MP); the other half eral blood, the blood compartment, is about one-third the are in the circulating pool (CP). Neutrophils spend less than 10 hours in the blood before marginating and exiting randomly to tissue. size of the bone marrow compartment. Once precursor cells have matured in the bone marrow, they are released into the peripheral blood. Normally, the input of neutrophils from of several mechanisms: (1) acceleration of maturation, (2) the bone marrow to the peripheral blood equals the out- skipped cellular divisions, and (3) early release of cells from put of neutrophils from the blood to the tissues, maintain- the marrow. ing a relative steady-state blood concentration. However, The mechanisms regulating the production and release when the demand for neutrophils increases, as in infectious of neutrophils from the bone marrow to the peripheral states, the neutrophil concentration in the peripheral blood blood are not completely understood but likely include can increase quickly when cells are released from the bone a feedback loop between the circulating neutrophils and marrow storage (reserve) pool. Depending on the strength the bone marrow. In normal conditions, this mediator is and duration of the stimulus, the marrow myeloid precur- likely G-CSF, produced by marrow stromal cells and mac- sor cells (GMP, CFU-G) also can be induced to proliferate rophages. Inflammatory cytokines such as IL-1 and tumor and differentiate to form additional neutrophils. The tran- necrosis factor (TNF) are important in causing an increase sit time between development in the bone marrow and in the neutrophil concentration in pathologic conditions by release to the peripheral blood can be decreased as a result inducing the macrophage to increase the release of G-CSF 120 Chapter 7 and GM-CSF. The vascular endothelial cells (VECs) that NEUTROPHIL KINETICS form the inner lining of blood vessels also generate cyto- Neutrophils constitute the majority of circulating leuko- kines that govern activation and recruitment of leukocytes. cytes. A number of physiologic and pathologic variations Endothelial cells can be important in recruiting neutrophils affect the concentration of circulating leukocytes. Patho- in the earliest phases of inflammation and injury. logic causes of changes in leukocyte numbers are discussed The release mechanism of the bone marrow storage pool in subsequent chapters on white cell disorders including is selective in normal, steady-state kinetics, releasing only Chapters 21–28. segmented neutrophils and a few band neutrophils. The Alteration in the concentration of peripheral blood leu- mechanisms controlling this regulated release are not fully kocytes is often the first sign of an underlying pathology. understood. The release is partially regulated by the small Granulocytopenia (granulocytes less than 2.0 * 103/mcL) pore size in the vascular endothelium lining the bone marrow defines a decrease in all types of granulocytes (i.e., eosin- sinusoids and by the mature segmented neutrophil’s ability ophils, basophils, neutrophils). Neutropenia is a more to deform enough to squeeze through the narrow opening. specific term denoting a decrease in only neutrophils. Neu- Immature cells are larger and less deformable and cannot tropenia in adults exists if the absolute neutrophil count penetrate the small pores; however, when an increased falls below 1.8 * 103/mcL. The condition of absence of demand for neutrophils exists, a higher proportion of less granulocytes is called agranulocytosis, and the patient is mature neutrophils is released into the peripheral blood. at high risk of developing an infection. Granulocytosis is Glucocorticoids, endotoxin (bacterial lipopolysaccharide), a term used to denote an increase in all granulocytes. Neu- and G-CSF can increase neutrophil release from the marrow. trophilia is a more specific term indicating an increase in neutrophils. Neutrophilia in adults occurs when the abso- PERIPHERAL BLOOD lute concentration of neutrophils exceeds 7.0 * 103/mcL. Neutrophils are released from the bone marrow to the This condition is most often the result of the body’s reactive peripheral blood, but not all neutrophils are circulating response to bacterial infection, metabolic intoxication, drug freely at the same time. About half the total blood neutro- intoxication, or tissue necrosis. phil pool is temporarily marginated along the vessel walls Although the WBC count and absolute neutrophil and is called the marginating pool (MP), whereas the other count are used to evaluate neutrophil production, they half is freely circulating and is referred to as the circulat- reflect a transient moment in overall neutrophil kinetics and ing pool (CP).6 Thus, if all marginated neutrophils were to do not provide accurate, quantitative information on the circulate freely, the total neutrophil count would double. rate of production or destruction, status of marrow reserves, Marginating neutrophils roll on the endothelial surface at or abnormalities in cell distribution in the tissues. a slow rate caused by a loose binding interaction between selectin adhesion proteins on neutrophils (L-selectin) and the L-selectin ligand on endothelial cells. The two pools are in equilibrium and rapidly and freely exchange neutro- Checkpoint 7.2 An adult patient’s WBC count is 10 * 103/mcL and there are phils. Stimulants such as strenuous exercise, epinephrine, or 90% neutrophils. What is the absolute number of neutrophils? stress can induce a shift from the MP to the CP, temporar- Is this within the reference interval for neutrophils? If not, what ily increasing the neutrophil count. The average neutrophil term would be used to describe it? circulates about 7.5 hours in the blood before diapedesing (transendothelial migration) to the tissues, although a few die of apoptosis while in the circulation. These are occa- sionally seen as “necrobiotic neutrophils” with a pyknotic Function nucleus on the peripheral blood smear (Chapter 10). To be effective in its role in host defense, the neutrophil must move to the site of the foreign agent, engulf it, and TISSUES destroy it. Thus, neutrophils function primarily in the tis- Most neutrophils move into the tissues from the MP in sues where microbial invasion typically occurs. Monocytes- response to chemotactic stimulation (see section on Neutro- macrophages help in this process but are slower to arrive at phil Function). In the tissues, the neutrophil is either destroyed the site. The four steps in the innate immune response can by trauma (cell necrosis) or lives until programmed cell death, be described as adherence, migration (chemotaxis), phago- apoptosis, occurs (Chapter 2). Neutrophils that do not receive cytosis, and bacterial killing. activation signals generally die within 1–2 days. However, elevated levels of GM-CSF or G-CSF associated with an infec- ADHERENCE tion or inflammatory process can prolong the neutrophil life Neutrophils flow freely along the vascular endothelium span to 3–5 days by blocking apoptosis. Senescent or apop- when neither the neutrophil nor VEC is activated. Neutro- totic neutrophils are phagocytosed by macrophages.10 phil adherence and migration to the site of infection begin Granulocytes and Monocytes 121 with a series of interactions between the neutrophils and (cytokines and chemokines). The initial result is activation VEC when these cells are activated by a variety of inflam- of all three classes of CAMs. The neutrophil-endothelial cell matory mediators (cytokines). adhesion and migration process11 can be divided into four Several different families of cell adhesion molecules stages: (1) activation of VEC, (2) activation of neutrophils, (CAM) and their ligands play a major role in the adherence (3) binding of neutrophils to inner vessel linings, and (4) process. Adhesion molecules include (Table 7-5): transendothelial migration (Figure 7-6). • b2 (CD18) family of leukocyte integrins and their Stage 1 involves the activation of VECs that allows for ligands (immunoglobulin-like CAM) a loose association of VECs with neutrophils. Inflammatory cytokines induce VECs to express of E- and P-selectins and • Selectins and their ligands L-selectin ligand. E- and P-selectin molecules on the VEC • Intercellular adhesion molecules (ICAMs) surface interact with their ligands on the neutrophil. Addi- Adhesion molecules and their ligands located on the tionally, L-selectin, which is constitutively present in the leukocytes and VEC act together to induce activation- neutrophil membrane, interacts with its L-selectin ligands dependent adhesion events. They are critical for every step that are upregulated on the surface of the activated VEC. of neutrophil recruitment to sites of tissue injury including These interactions induce the neutrophil to transiently asso- margination along vessel walls, diapedesis (passage of cells ciate and dissociate with the VEC, causing the neutrophil to through intact vessel walls), and chemotaxis (migration in “roll” on the VEC surface. The rolling neutrophils are thus response to a chemical stimulation). Adhesion molecules situated to respond to additional signals from chemoattrac- are transmembrane proteins with three domains: extracel- tants (chemotactic substances—chemical messengers that lular, transmembrane, and intracellular. A ligand’s binding cause directional migration of cells along a concentration to the CAM extracellular domain on a neutrophil sends gradient) generated by infectious agents or an inflamma- tory response.11 a signal across the membrane to the cell’s interior, which activates secondary messengers within the cell. These sec- Stage 2 is the activation of neutrophils. Chemokines ondary messengers affect calcium flux, NADPH oxidase (cytokines with chemotactic activity) or other chemoat- activity, cytoskeleton assembly, and phagocytosis. tractants bind to the endothelial cell surface where they Neutrophils and endothelial cells are transformed from interact with the loosely bound neutrophils and result in a basal state to an activated state by inflammatory mediators activation of neutrophil integrins. Chemoattractants are Table 7.5 Adhesion Molecules Important in Leukocyte–Endothelial Cell Interactions Molecules CD Designation Expressed By Counter-Receptor/Ligand 1. b2@Integrins/neutrophils CD11a/CD18 Activated leukocytes ICAM-1 on VEC (CD54) aLb2 (LFA-1) ICAM-2 on VEC (CD102) ICAM-3 (CD50) aL b2 (Mac-1) CD11b/CD18 Activated leukocytes ICAM-1 on EC iC3b, fibrinogen, factor X axb2 (p150, 95) CD11c/CD18 Activated leukocytes iC3b, fibrinogen 2. Selectins L-selectins CD62L Leukocytes Sialylated carbohydrates; PSGL-1 E-selectin CD62E Activated VEC Sialylated carbohydrates (SLex) and L-selectin on activated leukocytes P-selectin CD62P Activated VEC and
platelets PSGL-1; sialylated carbohydrates 3. Immunoglobulin supergene family ICAM-1 CD54 VEC LFA-1 (CD11a-CD18) ICAM-2 CD102 VEC LFA-1 (CD11a-CD18) ICAM-3 CD50 Neutrophils VEC PECAM-1 CD31 VEC CD31, avb3 LFA-2 CD2 T lymphocytes LFA-3 (CD58) on EC LFA-3 CD58 VEC CD2 on T lymphocytes VCAM-1 CD106 VEC VLA-4 (CD49d) on monocytes, lymphocytes, eosinophils, basophils LFA, leukocyte function-related antigen; CD, cluster of differentiation; VEC, vascular endothellial cell; ICAM, intercellular adhesion moleecules; PSGL-1, P selectin glcoprotein ligand-1. 122 Chapter 7 Leukocyte “rolling” Adhesion integrins selectin mediated and lg-like-mediated Diapedesis Stage 1 Cytokines Stage 2 Stage 3 Stage 4 tissue Chemoattractants PMN b2 integrin RBC Blood vessel VEC receptor (for L- VEC E-, P-selectin selectin) PMN-receptor (for E-, P-selectin) ICAM Basement membrane L-selectin of vessel wall Figure 7.6 Adhesion of neutrophils to the vascular endothelium and eventual migration of neutrophils into the tissue occur as a result of activation of endothelial cells (EC) and neutrophils by exposure to chemoattractants. When the cells are activated, they are induced to express adhesion molecules. These transmembrane molecules send a signal across the membrane to the interior of the cell when they attach to their receptor. The process occurs in four stages: In Stage 1, E-, P-selectin and L-selectin (VEC) receptor are expressed on activated EC. The neutrophil’s L-selectin and receptors for E-, P-selectin cause the neutrophil to attach loosely to the EC and roll along the endothelium. Neutrophils in Stage 2 are activated by the presence of chemoattractants in the local environment and express the b2@integrins. These chemoattractants also activate the ECs. The neutrophils in Stage 3 attach to the activated ECs via the attachment of their b2@integrins to ICAMs of the EC, resulting in a firmer attachment than in Stage 1 and halting the rolling of the neutrophil. The neutrophil in Stage 4 migrates through the endothelium and basement membrane (diapedesis) to the area of inflammation. They move in the direction of the chemoattractants (chemotaxis). released by tissue cells, microorganisms, and activated activated (discussed in the section, “Bacterial Killing and/ VEC and include specific and nonspecific proinflam- or Digestion”). matory mediators such as C5a (complement activation Stage 4 involves the transendothelial migration phase peptide), bacterial products, lipid mediators (e.g., platelet- that occurs when neutrophils move through the vessel activating factor [PAF]), and chemokines. Upon neutro- wall at the borders of VECs by the process of diapedesis. phil activation, activation-dependent adhesion receptors As neutrophils pass out of the vessel and into the tissue, including the b2@integrin molecules are expressed. Leuko- VECs modify their cell-to-cell adherent junctions. The neu- cyte plasma membranes have at least three b2@integrins: trophils use pseudopods to squeeze between endothelial CD11a/CD18 (leukocyte function associated antigen-1 cells, leaving the vascular space and passing through the [LFA-1]), CD11b/CD18 (Mac-1, also known as the comple- subendothelial basement membranes and periendothelial ment receptor 3 [CR3]), and CD11c/CD18. Each has an a cells. Subendothelial basement membranes are presumably subunit (CD11a, CD11b, CD11c) noncovalently linked to eroded by the secretion of the neutrophil enzymes gelatin- a b subunit (CD18).10 The neutrophil molecule, L-selectin, ase B and elastase from neutrophil granules. Migration is is downregulated at this time. enhanced when IL-1 and/or TNF activate the VEC.10 Stage 3 involves arrest of neutrophil rolling because An autosomal recessive disorder, leukocyte adhesion the activated neutrophils are more tightly bound to the deficiency type-I (LAD-I), has partial or total absence of VECs. Expression of activation-dependent b2@integrin expression of the b2@integrins on leukocyte membranes, adhesion molecules on neutrophils mediates firm adher- often due to a mutation of the CD18 gene. This results in ence to ICAMs expressed by activated VECs near the site of absence of leukocyte adhesion to the VEC as well as lack infection or inflammation. This induces a cytoskeletal and of mobility and migration into the tissues and can result in morphologic change in the leukocyte required for cellular life-threatening bacterial infections.12 An inability to synthe- migration. Stage 3 ends with a strong, sustained attach- size the E- and L-selectin ligand CD15s is seen in leukocyte ment of the leukocyte to the VEC. At this time, leukocyte adhesion deficiency type 2 (LAD-2) and also results in reoc- NADPH oxidase membrane complexes are assembled and curring infections (Chapter 21). Granulocytes and Monocytes 123 MIGRATION Two well-characterized opsonins are immunoglobulin Once in the tissues, neutrophil migration (chemotaxis) is G (IgG) and complement component C3b (see Chapter guided by chemoattractant gradients. Neutrophils continue 8 for a description of IgG structure). The antibody IgG their migration through the extravascular tissue, moving by binds to the microorganism/particle by means of its Fab directed ameboid motion toward the infected site. Locomo- region, while the Fc region of the antibody attaches to tion of neutrophils (and other leukocytes) is a process of three classes of Fc receptors on the neutrophil membrane “crawling,” not “swimming.” During locomotion along a (FcgRI3CD644 , FcgRII3CD324 , FcgRIII3CD164). Thus, the chemotactic gradient, the neutrophils acquire a characteris- antibody forms a connecting link between the microorgan- tic asymmetric shape, a process made possible by alterations ism/particle and the neutrophil. The neutrophil also has of the cytoskeleton triggered by neutrophil activation. There receptors for activated complement component C3b (CR1/ is an extension of a broad pseudopodium (protopod) at the CD35, CR3/CD11b/CD18, CR4/CD11c/CD18). Some bac- anterior of the cell (containing the nucleus and organelles) teria with polysaccharide capsules avoid recognition, thus and a narrow knoblike tail (uropod) at the rear of the cell. reducing the effectiveness of phagocytosis. Neutrophil migration through the tissues requires b1 and b2 Following recognition and attachment, the particle integrins with the continuous formation of new adhesive is surrounded by neutrophil cytoplasmic extensions or contacts at the cell front and detachment from the adhesive pseudopods (Figure 7-7). As the pseudopods touch, they substrate at the rear of the cell. Chemotaxis is induced by a fuse, encompassing the particle within a phagosome that is variety of chemoattractant molecules including bacterial for- bound by the cytoplasmic membrane turned “inside out.” myl peptides (fMLP), C5a, IL-8, and PAF, many of the same A plasma membrane–bound oxidase is activated during molecules that activated the neutrophil during Stage 2.11 ingestion, which plays an important role in microbicidal activities. Degranulation Bacterial killing and/or digestion follows Checkpoint 7.3 ingestion of particles. After formation of the phagosome, A patient with life-threatening recurrent infections is found to neutrophil granules migrate toward and fuse with the have a chromosomal mutation that results in a loss of active phagosome membrane, discharging their granule contents integrin molecules on the neutrophil surface. Why would this into the phagocytic vesicle ( degranulation), forming a pha- result in life-threatening infections? golysosome. The microbicidal and digestive proteins con- tained within both primary and secondary granules, which are normally sequestered from the cytoplasm of the neu- MICROBIAL KILLING: INTRACELLULAR trophil, are thus selectively released and activated within a After arriving at the site of infection, phagocytosis by neu- membrane-enclosed system. trophils can begin. Monocytes and macrophages also arrive Microbicidal mechanisms that follow ingestion can at the site of injury and continue to accumulate and con- be divided into oxygen-dependent/oxidative or oxygen- tribute to the inflammatory process. Phagocytes must rec- independent/nonoxidative activities (Table 7-6). ognize the pathogen as foreign before attachment occurs and phagocytosis is initiated (Figure 7-7). Once a pathogen Oxygen-Dependent Killing Oxygen-dependent microbici- is recognized, ingestion of the particle, fusion of the neu- dal activity is most important physiologically. Phagocyto- trophil granules with the phagosome (degranulation), and sis is accompanied by an energy-dependent “respiratory finally the process of bacterial killing and digestion occur. burst” that generates oxidizing compounds produced from Phagocytosis is an active process that requires a large partial oxygen reduction. The respiratory burst is described expenditure of energy by the cells. The energy required for as including a significant increase in glycolysis, a 2–3-fold phagocytosis can result from anaerobic glycolysis or aerobic increase in oxygen consumption, a 2–10-fold increase in hex- processes. ose monophosphate (HMP) shunt activity and generation The principal factor in determining whether phago- of NADPH, and the production of a series of reactive oxy- cytosis can occur is the physical nature of the surfaces of gen species (ROS).13 The oxidizing compounds—ROS—are both the foreign particle and the phagocytic cell. Phago- important agents in killing ingested organisms. The enzyme cytes recognize unique molecular characteristics of the activity needed to generate ROS is provided by NADPH pathogen’s surface (PAMPs) and bind to the invading oxidase, also known as respiratory burst oxidase. In resting organism via specific PRR (see the earlier section, “Leu- cells, NADPH oxidase is found as separate components of kocyte Function”). Some pathogens are recognized by the plasma membrane (gp91phox, p22phox, Rap1) and intra- the process of opsonization (the coating of a particle cellular stores (p47phox, p67phox).7 When phagocytosis takes with a soluble factor that enhances the recognition pro- place, the plasma membrane is internalized so that what cess). Enhancement of phagocytosis through the pro- was originally the outer plasma membrane surface is now cess of opsonization speeds the ingestion of particles. the lining of the phagocytic vesicle and faces the interior 124 Chapter 7 Pseudopodia 2 Pathogen 1 3 Engulfment Recognition and binding 7 Formation of phagocytic vacuole Phagosome Lysosome 4 Exocytosis 6 5 Killing and digestion Phagolysosome fusion Figure 7.7 Phagocytosis begins with (1) recognition and attachment of the pathogen to the neutrophil (or macrophage). The pathogen is then internalized (2), forming a phagocytic vacuole (phagosome [3]). Next, a primary granule (lysosome) fuses with the vacuole (4), forming a phagolysosome (5). The granule releases its contents (6) into the vacuole to help kill and digest the microbe (degranulation). This is followed by extrusion of undigested vacuole contents from the neutrophil (exocytosis [7]). Table 7.6 Neutrophil Antimicrobial Systems Oxygen Dependent Oxygen Independent • Myeloperoxidase independent • Acid pH of phagosome: antibacterial; enhances some oxygen-dependent antimi- Hydrogen peroxide (H crobial mechanisms and other enzymes 2O2) Superoxide anion (O - 2 ) • Lysozyme (primary and secondary granules): hydrolyzes cell wall of some bacteria; digests killed microbes Hydroxyl radicals (OH-) • Lactoferrin (secondary granules): binds iron necessary for bacterial growth; directly Singlet oxygen (1O2) bactericidal • Myeloperoxidase dependent (forms oxidized halogens) • BPI (bactericidal permeability-inducing protein; primary granules); Coat microbes; alters cell permeability • Defensins: small cationic peptides; broad spectrum of bactericidal activity • Collagenase (secondary granules): degrades microbe macromolecules • Hydrolases (primary granules): digests microbe of the phagosome. When the resting cell is exposed to any location, the NADPH oxidase generates and pours ROS into of a wide variety of activating stimuli, activated NADPH the phagosome. oxidase is assembled from the cytoplasmic and membrane- Once assembled, the oxidase produces superoxide associated subunits at the phagosome membrane. From this anion (O - 2 ) and NADP+. The NADP+ activates the HMP Granulocytes and Monocytes 125 shunt (Chapter 5), generating more NADPH. O - 2 is further matrices. Neutrophils are not resistant to the toxic effects of metabolized to produce additional ROS with increasing the oxidants they secrete and thus have high mortality dur- microbicidal potency. ing any sustained inflammatory response.14 NADPH oxidase Microbial Killing: Neutrophil Extracellular Traps (NETs) In 2 NADPH + 2 O2 S 2 NADP+ + 2 O - 2 + 2 H+ addition to the process of phagocytosis and intracellu- lar degranulation, neutrophils use a second mechanism Superoxide oxidase 2 H+ + O - 2 + O - 2 S H2O2 + to destroy microorganisms. First discovered in 2004, O2 neutrophil extracellular traps (NETs) are extracellular web- H 2O2 + O - 2 S 2 OH- + 1O2 like structures, composed of DNA and proteins, that func- The activated oxidase can be detected in the laboratory tion to eradicate microbes independent of phagocytosis.15 by a nitroblue tetrazolium (NBT) test, cytochrome reduc- The neutrophil’s DNA comprises the entrapping matrix and tion, or chemiluminescence test. carries proteins found in primary granules (elastase, cathep- The second oxygen-dependent microbicidal system sin G and myeloperoxidase), secondary granules (lactofer- involves the neutrophil’s primary granule enzyme myelo- rin), and cytoplasm.16 peroxidase (MPO). Myeloperoxidase catalyzes the interac- NETs’ activation and release occurs through one of two tion of hydrogen peroxide produced during the respiratory pathways that are collectively called NETosis (Figure 7-8). burst with halide ions (e.g., chloride/Cl-) giving rise to The first form of NETosis is a slow process that is initatied oxidized halogens (e.g., hypochlorous acid/HOCl) that through chromatin decondensation (removal of histone increase bacterial killing.13 proteins;
Chapter 2). The nuclear envelope is disassem- M H2O2 + Cl- SPO OCl- bled, allowing for the chromatin to enter the cytoplasm. + H2O Depolarization of the cell causes the granule contents to be The oxidants generated by the respiratory burst have released intracellularly and to mix with the chromatin. The potent microbicidal activity against a wide variety of plasma membrane then ruptures and allows for the NETs microorganisms such as bacteria, fungi, and multicellular to be released extracellularly. This process can take several and unicellular parasites. However, the phagocyte and sur- hours and results in death of the neutrophil. The second rounding tissues are also susceptible to damage. To detoxify form of NETosis (non-lytic) occurs without neutrophil cell the oxidant radicals, phagocytes use a variety of mecha- death. It entails expulsion of nuclear chromatin along with nisms such as superoxide dismutase, catalase, and a variety degranulation. The chromatin and granule contents assem- of other antioxidants. ble extracellularly, leaving a cytoplasmic remnant that can In patients with chronic granulomatous disease, neu- continue to ingest and destroy microorganisms.16 trophils are missing one of the components of the NADPH NETs not only trap, neutralize, and kill bacteria, fungi, oxidase complex and therefore fail to produce the respi- viruses, and parasites, but also prevent the spread of ratory burst. They are still capable of eliminating infec- microbes. They primarily function in the tissues, but can tion caused by strains of bacteria susceptible to killing by also form in the vasculature during sepsis. In the presence oxygen-independent mechanisms, but this antimicrobial of bacteria, intravascular NET formation is regulated by system is not very effective alone. Often these patients die platelets that bind to and activate neutrophils to form NETs. of multiple infections with bacteria resistant to the killing These intravascular NETs can trap circulating bacteria to actions of these granule proteins (Chapter 21). prevent further spreading.16 NETs also have pathogenic potential. Excess NET for- Oxygen-Independent Killing Oxygen-independent granule mation is damaging to tissues, particularly in fungal lung proteins present in primary, secondary, and tertiary gran- infections and during sepsis. Additionally, NETs have been ules of neutrophils can successfully kill and degrade many implicated in vaso-occlusion and appear to have a role strains of both gram-negative and gram-positive bacteria. in some autoimmune disorders.16 Research regarding the See Table 7-6 for a list of the most important nonoxygen- understanding of NETs is ongoing. dependent antimicrobial proteins of neutrophils. Initially, the pH within the phagolysosome decreases and inhibits Other Functions In addition to their primary functions of bacterial growth, but this alone is insufficient to kill most phagocytosis and killing of microorganisms, neutrophils microbes. Acidic conditions, however, can enhance the interact in other physiologic processes. Neutrophils stimu- activity of some granule proteins such as hydrolases and late coagulation by releasing a substance that activates lactoferrin, which perform optimally at low pH. In the prekallikrein to kallikrein, which in turn cleaves kinins from extracellular environment, microorganisms that escape high-molecular-weight kininogen. Kinins are responsible phagocytosis are also subject to killing by reactive oxygen for vascular dilation and increased vessel permeability. metabolites such as H2O2 that form from the O - 2 secreted by Kinins are also chemotactic molecules that attract neutro- active plasma membrane NADPH complexes into the tissue phils to sites of inflammation. Neutrophils initially activate 126 Chapter 7 1a 2a 3a 1b 2b Figure 7.8 The two pathways of NETosis. (1.a.) The first form of NETosis is a slow process that begins when the nuclear envelope breaks down and chromatin decondenses. (2.a.) The chromatin enters the cytoplasm and complexes with the cell’s granules. (3.a.) Lastly, the plasma membrane breaks down, releasing the NETs extracellularly. The neutrophil does not survive. (1.b.) Non-lytic NETosis is a quicker process and begins with extracellular release of the nuclear chromatin and degranulation. (2.b.) The result is an anuclear phagocytic cytoplasm that is capable of phagocytosis (the neutrophil “survives”). kinin production, but as the cells accumulate, they break of the eosinophil appear. Granule formation begins in down kinins. Neutrophils also secrete interleukin-1 (IL-1), the promyelocyte with small primary granules that lack a pyrogen that acts on the hypothalamus to produce fever. the crystalloid core of the specific granules. The first two stages (eosinophilic myeloblast and promyelocyte) will not be described because they are morphologically identical to Checkpoint 7.4 the neutrophilic myeloblast and promyelocyte. A patient has a compromised ability to utilize the oxygen-depen- dent pathway in neutrophils. What two important microbial kill- EOSINOPHILIC MYELOCYTE TO MATURE EOSINOPHIL ing mechanisms could be affected? The eosinophilic myelocyte contains large, eosin-staining, crystalloid granules. Maturation from the myelocyte to the metamyelocyte, band, and segmented eosinophil stage is Eosinophils similar to that described for the neutrophils with gradual nuclear indentation and segmentation. No appreciable The eosinophil originates from the IL-5–responsive CD34+ change occurs in the cytoplasm in these later stages of myeloid progenitor cells (CFU-Eo). Cytokines that influence development. The reddish orange spherical granules are proliferation and differentiation of the eosinophil lineage larger than neutrophilic granules, uniform in size, and include GM-CSF, IL-3, and IL-5.17 However, it is now recog- evenly distributed throughout the cell. Because of the low nized that IL-5, released largely by activated TH2 lympho- percentage of eosinophils in the bone marrow, differentiat- cytes and in small amounts by eosinophils, mast cells, NK ing the eosinophil into its maturational stages (e.g., eosino- cells, and natural killer T (NKT) cells, has relative lineage philic myelocyte) serves no useful purpose when the count specificity for eosinophils and is the major cytokine required is normal. Bone marrow maturation and storage time are for eosinophil production and terminal differentiation.18 about 9 days. The mature eosinophil (Figure 7-9) is 12–15 mcM in Differentiation, Maturation, and diameter. The nucleus usually has no more than two or three lobes, and the cytoplasm is completely filled with Morphology granules. The mature eosinophil contains three types of The eosinophil undergoes a morphologic maturation simi- granules: primary granules, small granules, and specific or lar to the neutrophil with the same six stages of maturation secondary granules. identified. However, it is not possible to morphologically Eosinophils express CD9, CD-11a, 11b, 11c, and CD13 differentiate eosinophilic precursors from neutrophilic pre- molecules on their cytoplasmic membrane. These molecules cursors with the light microscope until the myelocyte stage. function in antigen presentation, VEC adhesion, and trans- At this stage, the typical acidophilic crystalloid granules migration into the tissues, respectively. Additionally, the Granulocytes and Monocytes 127 with eosinophilic inflammatory reactions.20 Small granules contain the enzymes acid phosphatase and arylsulphatase but are not well characterized. Specific granules are the primary source of the cytotoxic and proinflammatory properties of the eosinophil.18 Specific granules are large, bound by a phospholipid membrane, and have a central crystalloid core surrounded by a matrix. These granules contain four major proteins: major basic protein (MBP), eosinophil cationic protein (ECP), eosino- phil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN) (Table 7-7). The MBP is located in the crystalloid core; the other three proteins are found in the granule matrix. The crystalloid core also appears to store a number of proin- flammatory cytokines such as IL-2, IL-4, and GM-CSF, and the matrix contains IL-5 and TNF@a. The eosinophil has Figure 7.9 Eosinophil (peripheral blood, Wright-Giemsa stain, the capacity to synthesize and elaborate a number of other 1000* magnification). cytokines as well. The eosinophil’s capacity to produce cytokines has led to increased interest into the eosinophil’s role as an effector cell in allergic inflammation. In addition primary receptors that impart the unique functional fea- to granules, the eosinophil, like the neutrophil, contains a tures of eosinophils are interleukin-5 receptor subunit@a number of lipid bodies that increase during eosinophil acti- (3IL@5Ra4 responsible for proliferation, activation, and vation in vitro.17,18 survival), CC-chemokine receptor 3 ([CCR3] promoting chemotaxis in response to eotaxins), and sialic acid-binding immunoglobulin-like lectin 8 ([SIGLEC8] whose signaling Distribution, Concentration, and induces apoptosis).18,19 Kinetics EOSINOPHILIC GRANULES Eosinophils in adults have a concentration in the peripheral Primary granules contain Charcot–Leyden crystal proteins blood less than or equal to 0.40 x 103/mcL. The cell shows (also called galectin-10) that possess lysophospholipase a diurnal variation with highest concentration in the morn- activity. The Charcot–Leyden crystal proteins are found in a ing and the lowest concentration in the evening.21 Eosino- variety of tissues, body fluids, and secretions in association philia in adults is defined as greater than 0.40 x 103/mcL Table 7.7 Major Constituents of Eosinophil Granules Protein Characteristics Major basic protein (MBP) Cytotoxic for protozoans and helminth parasites Stimulates release of histamine from mast cells and basophils Neutralizes mast cell and basophil heparin Eosinophil cationic protein (ECP) Capable of killing mammalian and nonmammalian cells Stimulates release of histamine from mast cells and basophils Inhibits T lymphocyte proliferation Activates plasminogen Enhances mucus production in the bronchi Stimulates glycosaminoglycan production by fibroblasts Eosinophil-derived neurotoxin (EDN) Can provoke cerebral and cerebellar dysfunction in animals Inhibits T-cell responses Eosinophil peroxidase (EPO) Combines with H2O2 and halide ions to produce a potent bactericidal and helminthicidal action Cytotoxic for tumor and host cells Stimulates histamine release and degranulation of mast cells Diminishes roles of other inflammatory cells by inactivating leukotrienes Lysophospholipase Forms Charcot-Leyden crystals Miscellaneous enzymes Phospholipase D: inactivates mast cell PAF Arylsulphatase: inactivates mast cell (leukotriene D4) Histaminase: neutralizes mast cell histamine Acid phosphatase, catalase, nonspecific esterases Lipid-derived mediators Promote smooth muscle contraction and mucus secretion and inhibit mast cell degranulation PAF; thromboxane B2 128 Chapter 7 and is associated with allergic diseases, parasitic infec- from basophils and mast cells (eosinophil chemotactic fac- tions, toxic reactions, gastrointestinal diseases, respiratory tor [ECF]), lymphokines from sensitized lymphocytes, and tract disorders, neoplastic disorders, and other conditions allergy-related antigen–antibody complexes are strongly (Chapter 21). Eosinophilia is T-cell dependent because T cells chemotactic for eosinophils. Eosinophils express Fc recep- are the predominant source of IL-5. Persistent eosinophilia tors for IgE, the immunoglobulin that is prevalent in the is seen in hypereosinophilic syndrome, a myeloproliferative response to parasitic infections and mediates activation of disorder (Chapter 24). eosinophil killing mechanisms. The cytokines IL-3, IL-5, Very little is known about the kinetics of eosinophils. and GM-CSF promote the adherence of eosinophils to VEC; Most of the body’s eosinophil population lies in connective transendothelial migration is 10 times higher in the pres- tissue below the epithelial layer in tissues that are exposed ence of these cytokines. to the external environment such as the nasal passages, Eosinophils have a b2 integrin-independent mecha- lung, skin, gastrointestinal tract,22 and urinary tract. These nism for recruitment into the tissues that appears to be cells spend about 18 hours in the peripheral blood before modulated by the eosinophil adhesion receptor, VLA-4, and migrating to the tissues where they can live for several its ligand VCAM-1, found on VECs that have been activated weeks. Once in the tissues, eosinophils do not re-enter the by IL-1, TNF, or IL-4. Changes in eosinophil adhesion mol- circulation.18 ecule expression occur during eosinophil migration. This implies that dynamic changes in cell adhesion molecules Function are involved in cell recruitment to areas of inflammation. The eosinophil liberates substances that can neutralize The cellular arm of the adaptive immune system (T lym- mast cell and basophil products, thereby down modulating phocytes; Chapter 8) influences eosinophil production and the allergic response (Table 7-7). Increasing evidence sug- function.23,5 Eosinophils are pro-inflammatory cells associ- gests a direct correlation between the degree of eosinophilia ated with allergic diseases, parasitic infections, and chronic and severity of inflammatory diseases, such as asthma, in inflammation. Their major role is host defense against hel- which eosinophil activation and degranulation contribute minth parasites via a complex interaction of eosinophils, to the characteristic features of mucous production, bron- the adaptive immune system, and parasite. The eosinophil choconstriction, and tissue remodeling.18 In inflammatory adheres to the organism and releases its granule contents conditions, the cytotoxic potential of eosinophils is turned onto the surface of the parasite via exocytosis. A number against the host’s own tissue.24 of eosinophil proteins, including MBP, ECP, and EPO, are highly toxic for larval parasites. Eosinophils are also capa- ble of phagocytizing bacteria (although less efficiently than neutrophils and macrophages) and have been shown to Basophils function as antigen-presenting cells.25 Basophils (Figure 7-10) originate from the CD34+ myeloid Eosinophils respond weakly to IL-3, IL-5, and GM-CSF progenitors in the bone marrow. IL-3 is the main cytokine as chemotaxins, but IL-5 synthesized by T lymphocytes has
involved in human basophil growth and differentiation, but been shown to strongly prime eosinophils for a chemotactic GM-CSF, stem cell factor (SCF), IL-4, and IL-5 can also be response to PAF, leukotriene B4, or IL-8. Products released involved.26 a b Figure 7.10 (a) Basophil (peripheral blood, Wright-Giemsa stain, 1000* magnification). (b) Basophil with washed out granules (Wright- Giemsa stain, staining artifact). Granulocytes and Monocytes 129 Differentiation, Maturation, and the bone marrow and tissues but are not found in periph- Morphology eral blood. Mast cells have proliferative potential and live for several weeks to months. At times, differentiating the Basophils undergo a maturation process similar to that mast cell and the basophil precursors in the bone mar- described for the neutrophil. The first recognizable stage row is difficult although some differences exist (Table 7-8). is the promyelocyte, although this stage is very difficult to The mast cell nucleus is round and surrounded by a dense differentiate from the promyelocyte of the neutrophil or population of granules. The mast cell granules contain acid eosinophil. As with eosinophils and neutrophils, the vari- phosphatase, protease, and alkaline phosphatase. Mast ous stages of the maturing basophil are characterized by a cells have a membrane antigen profile similar to that of gradual indentation and segmentation of the nucleus. macrophages. BASOPHILIC MYELOCYTE TO MATURE BASOPHIL The basophilic myelocyte, metamyelocyte, band, and seg- Distribution, Concentration, and mented form are easily differentiated from other granulo- Kinetics cytes by the presence of the large purple-black granules unevenly distributed throughout the cytoplasm. The gran- Basophils’ maturation in the bone marrow requires 2.5–7 ules are described as metachromatic and contain histamine, days before they are released into circulation. In the periph- heparin, cathepsin G, major basic protein, and lysophos- eral blood, they number less than 0.2 * 103/mcL (less than pholipase.26 The mature basophil ranges in size from 10–15 1% of the total leukocytes). Basophilia in adults is defined as mcM and has a segmented nucleus and many purple gran- greater than 0.2 * 109/L in the peripheral blood. Basophils ules obscuring both the background of the cytoplasm and are end-stage cells incapable of proliferation and spend only the nucleus. Basophil granules contain peroxidase and are hours in the peripheral blood. positive with the PAS cytochemical reaction. The gran- ules are water-soluble and can dissolve on a well-rinsed Function Wright-stained smear, resulting in clear areas within the Both basophils and mast cells function as mediators of cytoplasm. Usually a few deep-purple–staining granules inflammatory responses, especially those of immediate remain to aid in the identification of the cell. Basophils hypersensitivity reactions such as asthma, urticaria, allergic express CD9, CD11a, and CD13 molecules on their cyto- rhinitis, and anaphylaxis. These cells have membrane recep- plasmic membrane. tors for IgE (FcεR). When IgE attaches to the receptor, the MAST CELL cell is activated and degranulation is initiated. Degranula- The relationship between basophils and mast cells contin- tion releases enzymes that are vasoactive, bronchoconstric- ues to be investigated. Basophils and mast cells represent tive, and chemotactic (especially for eosinophils). This distinct, terminally differentiated cells, separately derived release of mediators initiates the classic clinical signs of from the CD34+ common myeloid progenitor cell. Distinct immediate hypersensitivity reactions. These cells can syn- committed progenitor cells (CFU-Ba and CFU-MC) have thesize more granules after degranulation occurs. Basophils been identified for each lineage.26 Mast cells are found in and mast cells express CD40 ligand (CD40L) that interacts Table 7.8 Comparison of the Characteristics of Basophils and Mast Cells Characteristics Basophils Mast Cells Origin Hematopoietic stem cell Hematopoietic stem cell Site of maturation Bone marrow Connective or mucosal tissue Proliferative potential No Yes Life span Days Weeks to months Size Small Large Nucleus Segmented Round Granules Few, small (peroxidase positive) Many, large (acid phosphatase, alkaline phosphatase positive) Key cytokine regulating development IL-3 SCF Surface receptors: IL-3-R Present Absent c-kit (SCF-R) Absent Present IgE receptor (FcPR) Present Present 130 Chapter 7 with CD40 molecules expressed on B lymphocytes. In con- PROMONOCYTE junction with IL-4, the interaction of B lymphocyte CD40 The promonocyte (Figure 7-11b) is an intermediate form and basophil CD40L can induce IgE synthesis by B lympho- between the monoblast and the monocyte. The promono- cytes. Thus, basophils can play an important role in induc- cyte is usually the first stage to develop morphologic ing and maintaining allergic reactions.26 characteristics that allow it to be clearly differentiated as a monocyte precursor by light microscopy. The cell is large, 12–20 mcM in diameter. The nucleus is most often Checkpoint 7.5 irregular and indented with a fine chromatin network. Indicate which of the granulocytes will be increased in the fol- Nuclear chromatin is coarser than the monoblast, and lowing conditions: a bacterial infection, an immediate hypersen- nucleoli can be present. The promonocyte’s cytoplasm sitivity reaction, and an asthmatic reaction. is abundant with a blue-gray color; azurophilic granules can be present. Cytochemical stains for nonspecific ester- ase, peroxidase, acid phosphatase, and arylsulfatase are Monocytes positive. The monocyte is produced in the bone marrow from a MONOCYTE bipotential progenitor cell, the GMP, which is capable of Mature monocytes (Figure 7-11c) range in size from 12–20 producing either mature monocytes or neutrophils. The mcM with an average size of 18 mcM, making them the differentiation and proliferation of GMP into monocytes largest mature cells in peripheral blood. The nucleus is fre- depend on the action of GM-CSF, IL-3, and M-CSF. The quently horseshoe- or bean-shaped and possesses numer- primary role of monocytes is host defense and this role is ous folds, giving it the appearance of brainlike convolutions fulfilled in the tissues. Monocytes continue to differentiate or chewed gum. The chromatin is loose and linear, forming in the tissues, transforming into macrophages. Monocytes a lacy pattern in comparison to the clumped dense chro- and macrophages can be stimulated by T lymphocytes and matin of mature lymphocytes or granulocytes. Monocytes, endotoxin to liberate endogenous M-CSF, which can be one however, are sometimes difficult to distinguish from large mechanism for the monocytosis associated with some infec- lymphocytes, especially in reactive states when there are tions. M-CSF also activates the secretory and phagocytic many reactive lymphocytes. The monocyte cytoplasm has activity of monocytes and macrophages.27 Monocytes and variable morphologic characteristics depending on its activ- macrophages make up the monocyte-macrophage system, ity. The cell adheres to glass and “spreads” or sends out also called the mononuclear phagocyte (MNP) system. numerous pseudopods, resulting in a wide variation of size and shape on blood smears. The blue-gray cytoplasm Differentiation, Maturation, and is evenly dispersed with fine, dustlike membrane-bound granules, which give the cell cytoplasm the appearance Morphology of ground glass. Vacuoles are frequently observed in the The morphologically recognizable monocyte precursors in cytoplasm. the bone marrow are the monoblast and the promonocyte. Electron-microscopic cytochemistry reveals two types These cells are present in a very low concentration in normal of granules present in monocytes. One type contains peroxi- bone marrow and are found in abundance only in leukemic dase, acid phosphatase, and arylsulfatase, suggesting that processes involving the MNP system. The monoblast of the these granules are similar to the lysosomes (primary azu- marrow cannot be morphologically distinguished from rophilic granules) of neutrophils. Less is known about the the myeloblast by light microscopy unless proliferation content of the other type of granule except that they do not of the monocytic series is marked as occurs in monocytic contain alkaline phosphatase and are therefore dissimilar leukemia. Because myeloblasts and monoblasts are indis- to specific granules of neutrophils.28 The lipid membrane tinguishable by light microscopy, immunophenotyping of the granules stains faintly with Sudan black B. Many CD and cytochemical stains (Chapters 23, 26, 40) are frequently markers including CD11b/CD18, CD13, CD14, and CD15 used to help differentiate myeloblasts and monoblasts in are expressed by monocytes. suspected cases of leukemia. MACROPHAGE MONOBLAST The monocyte leaves the blood and enters the tissues where The monoblast (Figure 7-11a) nucleus is most often ovoid or it matures into a macrophage (Figure 7-12). The transition round but can be folded or indented. Monoblasts are large from monocyte to macrophage is characterized by progres- (12–20 mcM in diameter). The pale blue-purple nuclear sive cellular enlargement, reaching a size of 15–80 mcM. chromatin is finely dispersed (lacy), and several nucleoli The nucleus becomes round with a reticular (netlike) are easily identified. The monoblast has abundant agranular appearance, nucleoli appear, and the cytoplasm appears blue-gray cytoplasm. blue-gray with irregular edges and many vacuoles present. Granulocytes and Monocytes 131 a b c Figure 7.11 Stages of monocyte maturation: (a) monoblast: Note lacy chromatin, nucleoli, and high N:C ratio; (b) promonocyte: The chromatin is somewhat coarser and the amount of cytoplasm is increased; (c) monocyte: The nucleus is lacier than that of a neutrophil or lymphocyte and is irregular in shape ([a, b]: bone marrow, Wright-Giemsa stain, 1000* magnification. [c]: peripheral blood, Wright-Giemsa stain, 1000* magnification). As it matures, the macrophage loses peroxidase, but the in lipid metabolism. These cells can live for months in the amount of endoplasmic reticulum (ER), lysosomes, and tissues. Macrophages do not normally re-enter the blood, mitochondria all increase. In addition, distinct granules but in areas of inflammation, some can gain access to the are noted in the maturing macrophage and are found to lymph, eventually entering the circulation. contain lysosomal hydrolases. Macrophages acquire the Tissue macrophages, also known as histiocytes, develop expression of CD68, a glycoprotein that can be important different cytochemical and morphologic characteristics that depend on the site of maturation and habitation in tissue. These cells are widely distributed in the body and have been given specific names depending on their anatomic location. For example, macrophages in the liver are known as Kupffer cells, those in the lung as alveolar macrophages, those in the skin as Langerhans cells, and those in the brain as microglial cells. The osteoclasts in the bone are also of MNP derivation.29 Macrophages can proliferate in the tissues, especially in areas of inflammation, thereby increasing the number of cells at these sites. Occasionally, two or more macrophages fuse to produce giant multinucleated cells. This occurs in chronic inflammatory states and granulomatous lesions where many macrophages are tightly packed together. Figure 7.12 Arrow indicates a macrophage. Note the Fusion also occurs when particulate matter is too large for numerous vacuoles and cellular debris (bone marrow, Wright- one cell to ingest or when two cells simultaneously ingest Giemsa stain, 1000* magnification). a particle. 132 Chapter 7 Distribution, Concentration, and ingestion occurs in a manner similar to that of neutrophils Kinetics (Figure 7-7). Primary lysosomes fuse with the phago- some, releasing hydrolytic enzymes and other microbici- Before maturing into monocytes, the promonocyte under- dal substances, the most powerful of which are products goes two or three divisions. Bone marrow transit time is of oxygen metabolism—superoxide anion (O - 2 ), hydroxy about 54 hours. In contrast to the large neutrophil storage radical (OH-), singlet oxygen (1O2), and hydrogen peroxide pool, there is no significant reserve pool of monocytes in (H2O2):generated in a reaction analogous to the neutro- the bone marrow. Most monocytes are released within a phil respiratory burst. day after their maturation from promonocytes. Monocytes CELLULAR SCAVENGING diapedese into the tissue from the peripheral blood in a ran- In addition to microbicidal activity, activated macro- dom manner after circulating for an average transit time of about 8 hours.27 phages also attach to tumor cells and kill them by a direct cytotoxic effect. If the tumor cell has immunoglobulin Similar to neutrophils, the total vascular monocyte pool attached, the macrophage Fc receptor attaches to the Fc consists of a marginated pool and a circulating pool. How- portion of the immunoglobulin and exerts a lytic effect on ever, unlike neutrophils, the marginating pool is about three the tumor cell. times the size of the circulating pool. Monocytes in the cir- culating peripheral blood number about 0.190.8 * Monocytes in the blood ingest activated clotting fac- 103/mcL tors, thus limiting the coagulation process. They also in the normal adult, or about 2–10% of the total leukocytes. ingest denatured protein and antigen–antibody com- Children have a slightly higher concentration. Monocytosis plexes. Macrophages lining the blood vessels remove (increase in monocytes) in adults occurs when the absolute monocyte count is greater than 0.8 * toxic substances from the blood, preventing their escape 103/mcL. into tissues. The macrophages of
the spleen are impor- tant in removing aged erythrocytes from the blood; they Function conserve the iron of hemoglobin by either storing it for Monocytes and macrophages are active in both the innate future use or releasing it to transferrin for use by devel- and adaptive IR. Macrophages are important scavengers oping erythroblasts in the marrow (Chapter 6). By virtue that phagocytize microbes, cellular debris, aging cells, and of their Fc receptor, the splenic macrophages also remove other particulate matter. In addition to their phagocytic cells sensitized with antibody. In autoimmune hemolytic function, they secrete a variety of substances that affect the anemias or in autoimmune thrombocytopenia, the spleen function of other cells, especially lymphocytes. Lympho- is sometimes removed to prevent premature destruction cytes in turn secrete soluble products (lymphokines) that of these antibody-coated cells in an attempt to alleviate the modulate monocytic functions. resulting cytopenias. For a variety of reasons, sometimes unknown, erythro- BACTERIAL KILLING cytes in some pathologic conditions are randomly phagocy- Monocytes and macrophages ingest and kill microorganisms. tosed and destroyed by monocytes and macrophages in the They are particularly effective in inhibiting the growth of blood and bone marrow (erythrophagocytosis) (Figure 7-13). intracellular microorganisms, a process that first requires Erythrophagocytosis is readily identified when the ingested monocyte activation. Activation results in the production erythrocytes still contain hemoglobin. At times, erythrocyte of many large azurophilic granules, enhanced phagocytosis, digestion can be inferred by finding ghost spheres within and an increase in the activity of the HMP shunt. Monocyte the macrophage. activation occurs in the presence of lymphokines produced by T lymphocytes. Killing by activated monocytes is non- specific (i.e., the secretions from Listeria-sensitized T cells activate a killing mechanism in monocytes not only to Listeria but also to other microorganisms). Activation can also occur as the result of the actions of other substances on monocytes such as endotoxins and naturally occurring opsonins. Monocytes/macrophages have some ability to bind directly to microorganisms via PAMP and PRR (see the section, “Leukocyte Function”), but binding is enhanced if the microorganism has been opsonized by complement or immunoglobulin. Macrophages possess receptors for the Fc component of IgG and for the complement compo- Figure 7.13 Erythrophagocytosis by a monocyte (peripheral nent C3b. Following attachment to an opsonized organism, blood, Wright-Giemsa stain, 1000* magnification). Granulocytes and Monocytes 133 ADAPTIVE IMMUNE RESPONSE metabolites (e.g., leukotrienes, prostaglandins) inhibit the In addition to its role in pathogen control and tissue function of activated lymphocytes. Activated lymphocytes homeostasis, the MNP plays a major role in initiating and in turn secrete lymphokines that regulate the function of regulating the adaptive IR.28 Macrophages phagocytize and macrophages. For these interdependent reactions to occur degrade both soluble and particulate substances that are between the macrophage and lymphocyte, the two cell pop- foreign to the host. They process the degraded substances, ulations must express compatible MHC antigens. generating fragments containing antigenic determinants or In addition to IL-1, macrophages release a variety of epitopes that are bound by MHC molecules on the macro- substances that are involved in host defense or that can phage membrane and presented to T lymphocytes. Thus, affect the function of other cells. Other secretory products monocytes and macrophages can function as antigen- involved in host defense include lysozyme, complement presenting cells (APCs) in the adaptive immune system. components, and IFN (an antiviral compound). Secreted In addition to antigen presentation, the macrophage pro- substances that modulate other cells include hematopoietic duces a number of cytokines that regulate the adaptive IR growth factors (e.g., G-CSF, M-CSF, GM-CSF), substances as well as the inflammatory response. Antigen-specific T that stimulate the growth of new capillaries (angiogenic lymphocyte proliferation requires antigen presentation in cytokines), factors that stimulate and suppress the activity context with cell surface MHC antigens and stimulation of lymphocytes, chemotactic substances for neutrophils, with soluble mediators such as IL-1 and IL-2. T lympho- and a substance that stimulates the hepatocyte to secrete cytes respond to foreign antigens only when the antigens fibrinogen. After death, activated macrophages also release are displayed on APCs that have the same MHC phenotype enzymes such as collagenase, elastase, and neutral protein- as the lymphocyte. ase that hydrolyze tissue components. Macrophages stimulate the proliferation and differ- entiation of lymphocytes through secretion of cytokines. Checkpoint 7.6 They secrete IL-1, which stimulates T lymphocytes to An adult patient’s neutrophil count and monocyte count secrete interleukin-2 (IL-2), a growth factor that stimulates are extremely low (less than 0.5 * 103/mcL and less than the proliferation of other T lymphocytes. In addition, IL-2 0.05 * 103/mcL) respectively). What body defense mecha- acts in synergy with interferon (IFN) to activate macro- nism is at risk? phages. When released from macrophages, arachidonic Summary Leukocytes include five morphologically and functionally result of an increase or decrease in all cell types or, more distinct types of nucleated blood cells: neutrophils, eosino- commonly, in just one cell type. When the WBC count is phils, basophils, monocytes, and lymphocytes, all of which abnormal, a differential should be performed to determine develop from the multipotential hematopoietic stem cells which cell type is increased or decreased. Both the absolute in the bone marrow. Under the influence of hematopoietic and relative (%) cell type concentrations should be deter- growth factors, the HSC matures into terminally differen- mined and reported. tiated cells. These cells leave the bone marrow and enter Leukocytes serve as the defenders of the body against the circulation. The two pools of neutrophils in the blood foreign invaders and noninfectious challenges by participat- are circulating and marginated pools. About one-half of the ing in phagocytosis (innate IR) and the adaptive IR. Leu- neutrophils are in each pool. Monocytes also exist in two kocytes are attracted to sites of inflammation, infection, or pools, but the marginating pool is about three times the size tissue injury by chemoattractants and leave the circulation of the circulating pool. Leukocytes generally circulate only a using special adhesion molecules and ligands located on matter of hours in the peripheral blood before diapedesing the leukocytes and endothelial cells of the vessel walls. The into the tissues. Normally, only mature forms are found in neutrophil-endothelial cell adhesion and migration process the peripheral blood but immature forms can be seen in involves four stages: (1) activation of VEC, (2) activation of newborns and in a variety of diseases. The adult reference neutrophils, (3) binding of neutrophils to inner vessel lin- interval for total WBC count is 4.5911 * 103/mcL. New- ings, and (4) transendothelial migration. Neutrophils and borns have higher counts than adults (9930 * 103/mcL). monocytes are active in phagocytosis and development of In the circulation, neutrophils are the most numerous cells the innate IR. Eosinophils function in defending the body followed by lymphocytes, monocytes, eosinophils, and against parasites and are also involved in allergic reac- basophils. An increase or decrease in leukocytes can be the tions and chronic inflammation. Basophils are involved in 134 Chapter 7 allergic reactions by releasing histamine and heparin when especially that of lymphocytes. Monocytes, also referred activated via the binding of IgE to membrane Fc receptors. to as antigen-processing (or presenting) cells, play a major Monocytes function as phagocytes and secrete a role in initiating and regulating the adaptive immune variety of cytokines that affect the function of other cells, response. Review Questions Level I 6. A(n) __________ has cytoplasm with a ground glass appearance while a __________ contains cytoplasm 1. Leukocytosis can be defined as an increase in: that is pinkish to clear in color. (Objective 4) (Objective 1) a. eosinophil; neutrophil a. neutrophils, monocytes, and macrophages b. monocyte; lymphocyte b. neutrophils, eosinophils, erythrocytes, and c. lymphocyte; basophil basophils d. monocyte; neutrophil c. neutrophils, eosinophils, basophils, monocytes, and lymphocytes 7. Basophils and mast cells have receptors for which d. neutrophils, eosinophils, basophils, monocytes, immunoglobulin? (Objectives 3, 5) lymphocytes, and megakaryocytes a. IgA b. IgG 2. The hallmark of differentiating myelocytes from pro- myelocytes morphologically is the visual identifica- c. IgM tion of what in the myelocytes? (Objective 2) d. IgE a. Primary granules 8. The total WBC count for an adult is 13.1 * 103/mcL b. Secondary granules with a differential count that reveals 20% eosinophils. c. Loss of nucleoli This represents a: (Objectives 1, 7, 8, 9) d. Pink cytoplasm a. relative and absolute eosinophilia b. relative eosinophilia and normal absolute count 3. Primary granules first appear in the: (Objective 2) c. normal relative count and absolute eosinophilia a. myeloblast d. normal eosinophil count b. promyelocyte 9. An absolute neutrophilia is most likely to be associ- c. myelocyte ated with a(n): (Objectives 1, 10) d. band a. allergic response 4. The eosinophil’s primary function is to: (Objective 5) b. parasitic infection a. protect the host from helminth parasites c. bacterial infection b. protect the host from autoimmune destruction d. viral infection c. secrete cytokines to attract monocytes to the site of 10. Routine hematological analysis was performed on a infection 1-day-old baby. The WBC count was 21.3 * 103/mcL. d. secrete cytokines to attract lymphocytes to the site This finding represents a(n): (Objectives 1, 11) of infection a. normal leukocyte count 5. Leukocyte migration to the tissues is regulated by b. absolute leukocytosis leukocyte-endothelial cell recognition that requires: c. relative leukopenia (Objective 6) d. absolute leukopenia a. interaction of adhesion molecules and their receptors 11. Which of the following leukocytes are most likely to b. activation of membrane oxidase resemble the morphology of a monocyte? (Objective 4) c. leukocyte degranulation a. Neutrophils d. hematopoietic growth factors b. Basophils Granulocytes and Monocytes 135 c. Reactive lymphocytes c. 8–10 days d. Eosinophils d. 10 years 12. Monocytes function in the innate immune response 2. The following cells are found in the granulocytic by their ability to __________ and function in the proliferating pool (mitotic pool) of the marrow: adaptive immune response by __________ and (Objective 1) __________. (Objective 5) a. multipotential stem cells a. phagocytose; antigen presentation; cytokine b. unipotential progenitor cells secretion c. monoblasts, myeloblasts, and macrophages b. degranulate; erythrophagocytosis; diapedesis d. myeloblasts, promyelocytes, and myelocytes c. stimulate T cells; secretion of IgM; remove helminths 3. What crystal proteins play a role in the cytotoxic d. secrete cytokines; chemotaxis; degranulation and proinflammatory properties of eosinophils? (Objective 3) 13. Patients with chronic granulomatous disease lack the a. Charcot-Leyden ability to produce a neutrophilic respiratory burst and often die of bacterial infections. Which phase of b. IgD and IgG phagocytosis is disrupted? (Objective 6) c. NETs a. Ingestion of bacteria d. Esterases b. Bacterial cell killing 4. An individual who has a mutation in the CD18 c. Neutrophilic degranulation gene that results in absence of the b2@integrin on the d. Bacterial cell recognition leukocyte membrane will likely have: (Objective 2) a. severe allergic reactions 14. What would be the major effect on the body of a severe monocytopenia? (Objectives 1, 5) b. leukocytosis with neutrophilia c. life-threatening bacterial infections a. Increased risk of parasitic infections d. a defect in phagocytosis b. Increased risk of bacterial infections c. Increased risk of viral infections 5. A patient was seen in the ER for symptoms of d. Decreased risk of allergic reactions appendicitis. A complete blood count was ordered. The WBC count was 20 * 103/mcL and the 15. If a neutrophil lacked the ability to produce L-selectin, differential revealed 60% segmented neutrophils, which of its functions or abilities would be disrupted? 15% bands, 20% lymphocytes, 4% monocytes, and (Objective 6) 1% eosinophils. All other parameters were within the reference interval. These results are most likely due a. Phagocytosis to: (Objectives 1, 4, 5) b. Degranulation a. release of neutrophils from the bone marrow prolif- c. Cytokine secretion erating/mitotic compartment d. Margination b. release of neutrophils from the bone marrow matu- 16. Neutrophil extracellular traps (NETs) use what two ration/postmitotic compartment substances to ensnare and kill microorganisms? c. a shift in the marginated pool of neutrophils in the (Objective 6) peripheral blood compartment a. Actin and cytokines d. a shift in the circulating pool of neutrophils in the peripheral blood compartment b. Plasma membrane and CD proteins c. DNA and granule contents 6. Segmented neutrophils are more capable of egressing d. Collagen and antibodies from the bone marrow into the peripheral blood than myelocytes because: (Objective 2) Level II a. mature neutrophils more easily deform through 1. The average cell turnover rate
for granulocytes and the small pore diameter in endothelial cells lining monocytes in the peripheral blood is: (Objective 1) the marrow sinusoids b. mature neutrophils secrete a cytokine that enables a. hours them to adhere to endothelial cells of the marrow b. 24 hours sinusoids 136 Chapter 7 c. there is a higher concentration of mature neutro- 11. A gene defect that reduces the ability of M-CSF to be phils in the bone marrow but myelocytes are rare produced in sufficient quantity would result d. only neutrophils possess the secondary granules in a: (Objectives 1, 4) required for chemotaxis a. monocytosis 7. A 30-year-old healthy male needed a CBC as part of b. neutrophilia his physical examination for purchasing a life insur- c. monocytopenia ance policy. He decided to combine his daily 5-mile d. basophilia run with his appointment to get his blood drawn. He ran 3 miles to the laboratory, had his blood drawn, 12. While neutrophils predominantly function in the and returned home. His WBC count was increased tissues, they can function as phagocytic cells in the to 15 * 103/mcL, but all other CBC parameters were blood stream during which of the following? normal. What is the most likely explanation for the (Objectives 3, 4) increased WBC count? (Objectives 1, 5) a. When bacteria are present in the blood a. He has a bacterial or viral infection. b. During times of intense physical activity b. He has a leukemia with leukemic cells present. c. During an allergic response c. Nucleated RBCs have entered the peripheral blood. d. During a viral infection d. The marginating neutrophil pool entered the circulating pool. 13. An adult male presents to the emergency department after experiencing severe nausea and vomiting. The 8. If the neutrophil count was determined before and total WBC count for the patient is 7.8 * 103/mcL with after the run in the patient in question 7, what would a differential count that reveals 10% monocytes. The be the most likely results? (Objective 5) laboratory data for this patient represent a: (Objective 5) a. Absolute neutrophilia before and relative a. relative and absolute monocytosis neutrophilia after b. relative monocytosis and normal absolute count b. Relative neutrophilia before and absolute c. normal relative count and absolute monocytosis neutrophilia after d. normal monocyte count c. Neutropenia before and absolute neutrophilia after d. Increased neutrophils after in comparison to before 14. During a severe bacterial infection, the concentration of neutrophils will decrease in which of the following 9. An absence of the E- and P-selectin receptors on locations? (Objective 1) neutrophils results in: (Objective 2) a. The site of infection a. increased movement of neutrophils from the b. The tissues circulation into the tissues c. The proliferating pool b. increased neutrophil response to chemotaxins d. The storage pool c. arrest of neutrophil rolling on the VEC surface d. inability of the neutrophil to interact with the VEC 15. A genetic alteration that produces improperly formed Fc receptors on the surface of neutrophils would have 10. Monocytes contribute to a proper functioning which of the following effects? (Objective 3) adaptive immune response by: (Objective 3) a. The neutrophil would not recognize IgG-coated a. processing and presenting foreign antigens to bacterial cells. lymphocytes b. The neutrophil would not be able to phagocytize b. removing red blood cells from the circulation viral particles. c. removing lymphocytes from the circulation c. Activated complement could not bind to the neu- d. secreting substances that neutralize inflammatory trophil receptors. products of eosinophils d. Phagocytosis could not take place at all. Granulocytes and Monocytes 137 References 1. Wintrobe, M. M. ed. (1980). Blood, pure and eloquent (2nd ed., pp. 16. Papayannopoulos, V. (2017). Neutrophil extracellular traps in 19–22, 428–431). New York: McGraw-Hill. immunity and disease. Nature Reviews Immunology. 2. Mangel, M., & Bonsali, M. B. (2013) Geriatric hematology. Stem doi: 10.1038/nri.2017.105 cell biology is population biology: Differentiation of hematopoi- 17. Jacobsen, E. A., Taranova, A. G., Lee, N. A., & Lee, J. J. (2007). etic multipotent progenitors to common lymphoid and myeloid Eosinophils: Singularly destructive effector cells or purveyors progenitors. Theoretical Biology and Medical Modelling, 10(5), 1–14. of immunoregulation? Journal of Allergy and Clinical Immunology, doi: 10.1186/1742-4682-10-5 119(6), 1313–1320. doi: 10.1016/j.jaci.2007.03.043 3. Lim, E., Cembrowski, G., Cembrowski, M., & Clarke, G. (2010). 18. Rosenberg, H. F., Dyer, K. D., & Foster, P. S. (2013). Eosinophils: Race-specific WBC and neutrophil count reference intervals. Changing perspectives in health and disease. Nature Reviews International Journal of Laboratory Hematology, 32(6, Pt. 2), 590–597. Immunology, 13, 9–22. doi: 10.1038/nri3341 doi: 10.1111/j.1751-553x.2010.01223.x 19. Ackerman, S. J., Liu, L., Kwatia, M. A., Savage, M. P., Leonidas, 4. Beutler, B. (2016). Innate Immunity. In: K. Kaushansky, J. T. Prchal, D. D., Swaminathan, G. J., & Acharya, K. R. (2002). Charcot- O. W. Press, M. A. Lichtman, M. Levi, L. J. Burns, & M. Caligiuri, Leyden crystal protein (galectin-10) is not a dual function galectin eds. Williams hematology (9th ed., pp. 293–306). with lysophospholipase activity but binds a l ysophospholipase New York: McGraw-Hill Education. inhibitor in a novel structural fashion. Journal of Biological 5. Abbas, A. K., Lichtman, A. H., & Pillai, S. (2015). Innate Chemistry, 277(17), 14859–14868. doi: 10.1074/jbc.m200221200 immunity. In: Cellular and molecular immunology (8th ed., Student 20. Gorczyca, W., Sun, Z., Cronin, W., Li, X., Mau, S., & Tugulea, S. Consult, pp. 51–86). Philadelphia: Elsevier. (2011). Immunophenotypic pattern of myeloid populations by 6. Cowland, J. B., & Borregaard, N. (2016). Granulopoiesis and flow cytometry analysis. Methods in Cell Biology, 103, 221–266. granules of human neutrophils. Immunological Reviews, 273(1), 21. Wardlaw, A. J. (2015). Eosinophils and related disorders. In: K. 11-28. doi: 10.1111/imr.12440 Kaushansky, J. T. Prchal, O. W. Press, M. A. Lichtman, M. Levi, 7. Khanna-Gupta, A., & Berliner, N. (2018). Granulocytopoiesis L. J. Burns, & M. Caligiuri, eds. Williams hematology (9th ed., pp. and monocytopoiesis. In: R. Hoffman (Author) & E. J. Benz, L. 947–964). New York: McGraw-Hill Professional. E. Silberstein, H. Heslop, J. Weitz, & J. Anastasi, eds. Hematology: 22. Powell, N., Walker, M. M., & Talley, N. J. (2010). Gastrointestinal Basic principles and practice (7th ed., pp. 321–333). Philadelphia: eosinophils in health, disease and functional disorders. Nature Elsevier. Reviews Gastroenterology and Hepatology, 7(3), 146–156. 8. Smith, C. W. (2016). Structure and composition of neutrophils, doi: 10.1038/nrgastro.2010.5 eosinophils, and basophils. In: K. Kaushansky, J. T. Prchal, O. W. 23. Linch, S. N., & Gold, J. A. (2011). The role of eosinophils in non- Press, M. A. Lichtman, M. Levi, L. J. Burns, & M. Caligiuri, eds. parasitic infections. Endocrine, Metabolic and Immune Disorders - Williams hematology (9th ed., pp. 925–938). New York: McGraw- Drug Targets, 11(2), 165–172. doi: 10.2174/187153011795564188 Hill Education. 24. Jacobsen, E. A., Helmers, R. A., Lee, J. J., & Lee, N. A. (2012). The 9. Koepke, J. A. (2007). Reference leukocyte (WBC) differential count expanding role(s) of eosinophils in health and disease. Blood, (proportional) and evaluation of instrumental methods: Approved 120(19), 3882-3890. doi: 10.1182/blood-2012-06-330845 standard (2nd ed.). Wayne, PA: Clinical and Laboratory Standards 25. Wang, H., Ghiran, I., Matthaei, K., & Weller, P. F. (2007). Airway Institute. eosinophils: Allergic inflammation recruited professional anti- 10. Baehner, R. L. (2005) Normal phagocyte structure and function. gen-presenting cells. Journal of Immunology, 179(11), 7585–7592. In: R. Hoffman, E. J. Benz Jr, S. J. Shattil, H. Heslop, J. Weitz, & 26. Befus, A. D., & Denburg, J. A. (2009). Basophilic leukocytes: Mast J. Anastasi, eds. Hematology: Basic principles and practice (4th ed., cells and basophils. In: J. P. Greer, J. Foerster, G. M. Rodgers, 737–762). Philadelphia: Elsevier. F. Paraskevas, B. Glader, & D. A. Arber, eds. Wintrobe’s clinical 11. Rot, A., Massberg, S., Khandoga, A. G., & Von Andrian, U. H. hematology (12th ed., Vol. 1, pp. 236–248). Philadelphia: Lippincott (2018). Hematopoietic cell trafficking and chemokines. In: R. Williams & Wilkins. Hoffman, E. J. Benz Jr., L. E. Silberstein, H. Heslop, J. Weitz, J. 27. Douglas, S. D., & Douglas, A. G. (2016). Structure, receptors, and Anastasi, eds. Hematology: Basic principles and practice (7th ed., functions of monocytes and macrophages. In: K. Kaushansky, pp. 135–143). Philadelphia: Elsevier. J. T. Prchal, O. W. Press, M. A. Lichtman, M. Levi, L. J. Burns, & 12. Rosenzweig, S. D., & Holland, S. M. (2011). Recent insights into M. Caligiuri, eds. Williams hematology (9th ed., pp. 1045–1074). the pathobiology of innate immune deficiencies. Current Allergy New York: McGraw-Hill Education. and Asthma Reports, 11(5), 369–377. doi: 10.1007/s11882-011-0212-9 28. Douglas S. D., & Douglas A. G. (2016). Morphology of monocytes 13. Borregaard, N. (2017). Disorders of neutrophil function. In: M. A. and macrophages. In: K. Kaushansky, J. T. Prchal, O. W. Press, Lichtman, K. Kaushansky, J. T. Prchal, M. M. Levi, L. J. Burns, & M. A. Lichtman, M. Levi, L. J. Burns, & M. Caligiuri, eds. Williams J. O. Armitage, eds. Williams manual of hematology (9th ed., pp. hematology (9th ed., p. 989). New York: McGraw-Hill Education. 165–174). New York: McGraw-Hill Education. 29. Jordan, M. B., & Filipovich, A. H. (2018). Histiocytic disorders. In: 14. Geiszt, M., & Leto, T. L. (2004). The Nox family of NAD(P)H oxi- R. Hoffman (Author) & E. J. Benz, L. E. Silberstein, H. Heslop, dases: Host defense and beyond. Journal of Biological Chemistry, J. Weitz, & J. Anastasi, eds. Hematology: Basic principles and 279(50), 51715-51718. doi: 10.1074/jbc.r400024200 practice (7th ed., pp. 724–739). Philadelphia: Elsevier. 15. Brinkmann, V. (2004). Neutrophil extracellular traps kill bacteria. Science, 303(5663), 1532–1535. doi: 10.1126/science.1092385 Chapter 8 Lymphocytes Lynne Williams, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Describe the function of lymphocytes 5. Outline and describe the development (T cells, B cells, NK cells). of lymphocytes including 2. Summarize the distribution and state the distinguishing maturation and concentration of lymphocytes in peripheral morphologic features of cells of the blood. lymphocytic lineage. 3. List the age-related reference intervals for 6. Describe the morphology of the activated or peripheral blood lymphocytes. reactive lymphocyte. 4. List causes/conditions associated with 7. Define antigen-presenting cell and an increase or decrease in the absolute identify cells that have this functional numbers of lymphocytes found in the capability. peripheral blood. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Summarize the kinetics of the lymphocytic 4. Compare and contrast the immunologic lineage. features and functions of the various types of lymphocytes found in the peripheral 2. Outline and describe the hierarchy of lym- blood (B cells; CD8+ T cells; CD4+ T cells, phocyte development. including subsets). 3. Describe and compare T and B lymphocyte 5. Summarize molecular characteristics used developmental stages. to differentiate lymphocyte subtypes. 138 Lymphocytes 139 6. Describe the synthesis of immunoglobulin 7. Differentiate between polyclonal and including heavy and light chain gene monoclonal gammopathies and describe rearrangement. each type in relationship to a patient’s clinical condition. Chapter Outline Objectives—Level I and Level II 138 Lymphocyte Identification and Key Terms 139 Morphology 151 Background Basics 139 Lymphocyte Distribution, Concentration, and Case Study 140 Kinetics 154 Overview 140 Lymphocyte Function 154 Introduction 140 Summary 159 Lymphopoiesis 140 Review Questions 160 Lineage Differentiation 142 References 161 Key Terms Adaptive immune response Immune response (IR) Natural killer (NK) cell Antigen-presenting cell (APC) Immunoblast Natural killer T (NKT) cell B cell receptor (BCR) Immunoglobulin (Ig) Plasma cell Blast transformation Innate immune response Plasmacytoid lymphocyte Cell-mediated immunity Large granular Reactive lymphocyte Cytokine lymphocyte (LGL) Recombination-activating gene 1 Cytotoxic T cell, cytotoxic Lymphocytopenia and 2 (Rag1/Rag2) T lymphocyte (CTC/CTL) Lymphocytosis Regulatory T lymphocyte (TReg) Effector T lymphocyte Lymphokine-activated killer cell T cell receptor (TCR) Helper T cell (Th) (LAK) Terminal deoxynucleotidyltransfer- Humoral immunity Memory cell ase (TdT) Background Basics In addition to information from previous chapters, a • Summarize the function of growth factors, the basic understanding of immunology (immune system hierarchy of hematopoiesis, and the concept of the and function) and biochemistry (proteins, carbohydrates, CD nomenclature. (Chapter 4) lipids) is helpful for understanding the concepts in this • Describe the structure and function of the chapter. To maximize your learning experience, you hematopoietic organs. (Chapter 3) should review these concepts from previous chapters before starting this unit of study: Level II • List the growth factors and identify their func- Level I tion in leukocyte differentiation and maturation. • Identify cell components and describe their func- (Chapter 4) tion. (Chapter
2) 140 Chapter 8 are the major cells of the adaptive immune response. Their CASE STUDY primary functions are to recognize and react with specific We refer to this case study throughout the chapter. antigens, work with macrophages to eliminate pathogens, A 6-month-old male infant was seen by his and provide long-lasting immunity to previously encoun- pediatrician for his 6-month checkup. His mother tered pathogens. The NK cells have characteristics distinctly reported that he had no health problems. A CBC different from those of T and B lymphocytes and are actu- was ordered with the following results: WBC ally effector cells of innate immunity. count 14.0 * 103/mcL, RBC count 4.5 * 106/mcL, B lymphocytes are the primary effector cells for the Hb 12.5 g/dL: Hct 37.5%; differential: segmented adaptive humoral immune response (i.e., the production neutrophils 37%; lymphocytes 60%; monocytes 3%. of antibodies). To accomplish effective humoral immu- Evaluate these results as you study this chapter. nity, the B lymphocyte must be activated and differentiate to a plasma cell. T lymphocytes are primarily involved in cell-mediated immunity (CMI), which requires interaction among macrophages, dendritic cells (DC), T lymphocytes, and antigens. CMI is independent of antibody production Overview by B lymphocytes. B and T lymphocytes consist of several Lymphocyte precursors develop from the pluripotential distinct subpopulations with different phenotypic markers hematopoietic stem cell in the bone marrow. They differen- and functions. There are two subsets of B cells (B-1 and B-2) tiate into several functionally different types of lymphocytes and at least three important functional subsets of T lympho- (T, B, natural killer [NK] cells), all of which are involved in cytes: helper T lymphocytes (Th), cytotoxic T lymphocytes an immune response (IR). This chapter is a study of the (CTL, TC), and regulatory T lymphocytes (TReg). Effec- normal differentiation and maturation of these cells includ- tive immunocompetence (ability to generate an adaptive ing morphology, concentration, and function. The synthesis immune response) depends on a balance and interaction and structure of immunoglobulins, lymphocyte receptors, between B and T lymphocytes. and cell antigens are described with attention to the use of these markers in identifying lymphocyte subpopulations. Lymphopoiesis Introduction Lymphopoiesis is the process whereby the cellular compo- nents of the immune system (T, B, and NK cells) are pro- For many years after its discovery, the lymphocyte was con- duced during hematopoietic differentiation (Chapter 4). sidered an insignificant component of blood and lymph. Since 1960, however, major advances in immunology have identified lymphocytes as the principal effector cells of Ontogeny of Lymphopoiesis the adaptive immune response. In contrast to nonspecific During embryogenesis, the onset of lymphopoiesis occurs innate immunity that involves granulocytes and monocytes later than the initiation of myelopoiesis and erythropoiesis. and occurs rapidly as the body’s initial response to patho- Lymphocytes are not found in the early embryonic sites of gens, adaptive immunity develops more slowly as it gener- hematopoiesis (e.g., extraembryonic yolk sac, AGM region), ates a response to eliminate specific pathogens. Adaptive but rather, are first seen around week 8–10 in the fetal immunity involves lymphocytes, macrophages, and other liver.2,3 These early lymphocytes, pre-B cells, possess immu- antigen-presenting cells and results in immunologic mem- noglobulin M (IgM) in their cytoplasm and by 10–12 weeks ory, by which the lymphocytes are primed to respond rap- gestation, IgM is present on their cell surfaces.4 Mature T idly to antigens on subsequent encounters. The two types cells are present in the fetal thymus by 13–16 weeks gesta- of adaptive immune responses are humoral immunity and tion.5 Functional NK cells can be detected in the fetal liver cell-mediated immunity, which function to eliminate dif- by 9–10 weeks gestation, while dendritic-like cells are pro- ferent types of infectious pathogens as well as substances duced at all stages of embryonic and fetal hematopoiesis.4,6 considered foreign to the body (e.g., cancer cells).1 Like production of other hematopoietic lineages, lym- Lymphocytes include subpopulations of cells with phopoiesis occurs in the fetal liver before birth and largely various origins, life spans, and function. A lymphocyte’s in the bone marrow after birth. Fetal liver–derived lym- life can span several years or the cell can die within hours phoid precursors give rise to lymphocytes with distinctly or days. There are three major types of lymphocytes that different characteristics than those derived from the bone are morphologically identical but immunologically and marrow. Fetal liver–derived B cells are mainly B-1 cells functionally diverse: T lymphocytes, B lymphocytes, and and fetal liver–derived T cells are primarily gd T cells (see natural killer (NK) cells. T lymphocytes and B lymphocytes the section “Lymphocyte Function”). These B and T cells Lymphocytes 141 generated during fetal development generally have less Transcriptional Regulation of diverse antigen receptors than cells derived from bone mar- row HSCs.7 Lymphopoiesis In adults, the lymphoid cells arise from the pluripo- Commitment to the specific lymphocytic lineage (T, B, NK) tential hematopoietic stem cell (HSC) found in the bone is associated with specific transcription regulators (tran- marrow. The earliest events in lymphoid lineage commit- scription factors [TF]) that influence a CLP to assume a B ment are less understood than the more terminal events cell, T cell, or NK cell fate7,10 (Table 8-1). in lymphoid differentiation, and are still controversial. For several decades the HSCs were thought to give rise Cytokines in Lymphopoiesis to committed progenitor cells: the common lymphoid Many cytokines and cytokine pathways regulate lymphoid progenitor cell (CLP) and the common myeloid progeni- development, differentiation, and function. A complete tor cell (CMP), the first branch point in lineage commit- description of cytokine regulation of lymphopoiesis is too ment (Chapter 4). The CLP was defined as a cell that can complex for a full description in this chapter. However, differentiate and mature under the inductive influence of the cytokines associated with the common gamma chain selective microenvironments and cytokines into lympho- (gc) family of cytokine receptors (Chapter 4) is of particu- cytic subpopulations of T cells, B cells, NK cells, and some lar importance in lymphopoiesis. The gc subunit is found dendritic cells (Figure 4-3). in six different cytokine receptors—IL-2, -4, -7, -9, -15, and Recently, a lymphoid-primed multipotential progenitor -21—all of which play essential, although variable, roles in (LMPP) has been described, which has full lymphoid lymphopoiesis.10 Individuals with mutations in the gene for (T, B, and NK) developmental potential and limited the gc cytokine receptor subunit have an X-linked severe myeloid (mostly monocytic) potential8 (Figure 8-1). combined immunodeficiency disorder (X-SCID). Primitive multilymphoid progenitors with little or no myeloid or erythroid potential have also been i solated (the classical “CLP”).9 The CLP (CD10+ ) cell is now Antigen-Dependent and -Independent described as having predominantly B and some NK and T Lymphopoiesis potential and devoid of myeloid potential. Early m arkers T and B lymphopoiesis can be divided into two distinct of lymphoid commitment include CD10+ , CD7+ , and phases: antigen-independent lymphopoiesis and antigen- CD45RA+ (CD34+LinnegCD38+CD45RA+CD10+ ). dependent lymphopoiesis (Figure 8-2). Antigen-independent lymphopoiesis takes place within the primary lymphoid tissue (liver in the fetus and bone marrow in the adult for HSC/MPP B cells; thymus for T cells in both the fetus and the adult). Antigen-independent lymphopoiesis begins with the CLP LMPP and results in the formation of immunocompetent T and B lymphocytes (referred to as naive lymphocytes because they Myeloid Lymphoid have not yet reacted with antigens). These cells exit the pri- mary lymphoid tissue and migrate to secondary lymphoid GMP CLP tissue (spleen, lymph nodes, gut-associated lymphoid tis- sue) where the antigen-dependent phase of lymphopoi- esis takes place. Antigen-dependent lymphopoiesis begins with the recognition of and the interaction with antigens by specific antigen receptors on the surface of the immu- MEP GMP nocompetent T and B lymphocytes (T cell receptor [TCR] DC B NK T and B cell receptor [BCR]). These receptors are not encoded Figure 8.1 Human lymphoid progenitor pathways. Populations with multilineage potential capable of giving rise to all blood cell Table 8.1 Transcription Factor Regulation of Lymphoid lineages include HSC/MPP (hematopoietic stem cells/multipotential Differentiation progenitors). The LMPP (lymphoid multipotential progenitor) has full lymphoid (T, B, and NK cell development potential) and limited Transcription Factors Lymphoid Lineage Regulating Differentiation myeloid (mostly monocytic) potential. The CLP (common lymphoid progenitor) cell has predominantly B, some NK and T potential, Early lymphoid progenitor cells Ikaros, Pu.1, E2A and is almost devoid of myeloid potential. CMP, common T lymphocytes GATA-3, TCF1, Bcl-11b myeloid progenitor; GMP, granulocyte-macrophage progenitor; B lymphocytes EBF, E2A, Pu.1, Pax-5 MEP, megakaryocyte-erythroid progenitor; DC, dendritic cell; B, B lymphocyte; NK, natural killer cell; T, T lymphocyte. Natural Killer lymphocytes Id2, Ikaros, E4BP4, Eomes, Tbet 142 Chapter 8 Peripheral blood Primary lymphoid Secondary lymphoid tissue tissue and peripheral blood Lymph nodes Pre-T cells T lymphocytes Immunoblast Activated Cell-mediated (CD4 and CD8) T lymphocytes and immunity T memory cells Thymus HSC Pre-B Antigen Pre-NK CLP LD cells CMP B lymphocytes Immunoblast Plasma cells Humoral and B memory immunity cells (antibody production) Immuno-incompetent Immunocompetent Activated lymphocytes lymphocytes lymphocytes Figure 8.2 Lymphocytes originate from the common lymphoid progenitor cell/CLP (derived from the pluripotential hematopoietic stem cell/HSC) in the bone marrow. Lymphocyte progenitors that mature in the thymus become T lymphocytes, and those that mature in the bone marrow become B lymphocytes, natural killer cells (NK), or lymphoid-derived dendritic cells (LD). Three morphologic stages can be identified in this development to T and B cells: lymphoblast, prolymphocyte, and lymphocyte. On encounter with antigen, these immunocompetent T and B lymphocytes undergo blast transformation, usually in the lymph nodes, to form effector lymphocytes. The B lymphocytes eventually emerge as plasma cells. Effector T lymphocytes, however, are often morphologically indistinguishable from the original naive T lymphocytes. The recognizable morphologic stages of blast transformation include reactive lymphocytes, immunoblasts, plasmacytoid lymphocytes (B cells), and plasma cells (B cells). Flow cytometry indicates that some morphologic stages (i.e., small lymphocytes) can represent several stages of immunologic maturation. by conventional genes but are derived from rearrangement c oncentrate and present antigens to T cells and are the pre- of DNA segments in the respective gene loci to create bil- cursors of immunoglobulin-secreting plasma cells. The NK lions of different versions of the basic receptor.1 As a result, cell is a form of cytotoxic lymphocyte that functions as part these receptors are highly variable and pathogen-specific. of the innate immune system. NK cells play a key role in the Interaction with antigen results in the formation of effector cytolysis of both tumor and pathogen-infected cells. They T and B lymphocytes, which undergo subsequent changes do not rearrange or express T cell receptor genes or B cell in phenotype and functional capacity. These effector cells immunoglobulin genes and thus do not express antigen- mediate a pathogen-specific adaptive immune response. specific receptors. Mature Lymphocytes Checkpoint 8.1 Effector T lymphocyte subsets include cells responsible Describe the subsets of T cells and B cells derived from the CLP. for cell-mediated cytotoxic reactions (Tc, cytotoxic T lym- phocytes [CTL] or cytotoxic T cells [CTC]); cells that pro- vide helper activity for B cells, macrophages, and other T cells (helper T cells [Th] with subsets Th1, Th2, TFH, Lineage Differentiation Th9, Th17); and cells that function to suppress other T cell The cells of the innate immune system (neutrophils, mono- immune responses (regulatory T cells [TReg]). There are two cytes, macrophages) recognize pathogens using a limited B lymphocyte subsets, B-1 (a minor component of B lym- number of receptors encoded in the genome as discreet genes, phocytes) and B-2 (the majority of B cells in the blood and which recognize molecules unique to human pathogens and lymphoid tissues). Each mature B lymphocyte makes a spe- thus recognizable as “non-self.” One group of receptors, cific antibody targeted against a specific triggering antigen called pattern recognition receptors (PRRs), function to recog- by rearranging its immunoglobulin genes. They also can nize molecules referred to as pathogen-associated molecular Lymphocytes 143 patterns (PAMPs; Chapter 7). In contrast, in the adaptive bases to the broken DNA ends, contributing to additional immune response, lymphocytes recognize pathogens using receptor diversity. The broken DNA ends are ligated by the receptors (BCRs/TCRs) that are not inherited as functional same double-stranded break repair process that all cells use to genes, but are made during lymphopoiesis. It is estimated repair damaged DNA. This process of gene rearrangement is that an individual has Ú107 different B cell
and T cell recep- known as somatic recombination [orV(D)J rearrangement] and is tors, each expressed by a small subset of lymphocytes.7 Only the principle source of the great diversity of pathogen recep- those cells bearing receptors that recognize a particular tors that exists for lymphocytes.7 This process also plays a role pathogen will be activated to effector cells when encounter- in defining populations of precursor lymphocytes during the ing that particular pathogen. The molecule, virus particle, or first phase of lymphopoiesis in the primary lymphoid organs. cell structure that is recognized by the BCR/TCR is called its corresponding antigen, and the receptors themselves are referred to as the antigen receptors of the lymphocytes. B Lymphocytes The diversity of the BCRs/TCRs (Ú107) does not require B lymphocyte precursors arise in the bone marrow from the a large number of distinct antigen receptor genes. Rather, anti- CLP derived from hematopoietic stem cells. The B lympho- gen receptor diversity arises due to a unique genetic p rocess cyte precursors undergo the antigen-independent phase of in which different parts of the polypeptides that make up their development entirely within the bone marrow and exit the receptors are encoded by separate gene s egments called as naive B lymphocytes. B lymphocytes compose 15–30% of V, D, and J. The gene regions for the BCR and TCR poly- peripheral blood lymphocytes. peptides have arrays of many different V, D, and J gene seg- ments. The chromosomal regions containing these V, D, and B LYMPHOCYTE MEMBRANE MARKERS J segments are in a disconnected form (called the germline B lymphocytes can be identified and differentiated from configuration) and cannot be transcribed and translated into T lymphocytes by the presence of surface membrane protein in most of the cells of the body. During lymphopoie- immunoglobulin and CD antigens/markers. Surface mem- sis, however, one of each segment (as appropriate) is brought brane immunoglobulin and CD markers can be detected by together by breaking and rejoining the DNA and eliminat- fluorescein-conjugated antisera to the proteins, using flow ing intervening regions, resulting in a functional gene. DNA cytometry (Chapter 40). cleavage is mediated by two l ymphocyte-specific proteins Membrane markers are present at various stages in B lym- (recombination-activating genes 1 and 2 [Rag1/Rag2]), phocyte development. B lymphocytes are identified using a which together with additional accessory proteins produce panel of antibodies to surface antigens that corresponds to the V(D)J recombinase activity. The lymphoid-specific enzyme the stage of the cell’s differentiation (Figure 8-3). CD markers terminal deoxynucleotidyltransferase (TdT) randomly adds expressed by B lymphocytes and their precursors include Lymph nodes/ Peripheral bone Bone marrow blood marrow Mature Plasmacytoid Plasma HSC CLP Pro-B *Pre-B B B cell cell CD34 CD34 CD34 TdT HLA-DR HLA-DR CD38 c-Kit c-Kit TdT HLA-DR CD19 CD19 CD138 Flt-3 CD19 CD10 CD22 CD20 sIg- CD22(c) CD19 CD24 CD22 clg+ HLA-DR CD22(s)+/- CD20 CD24 CD10 CD24 CD21 CD21+/- CD20 CD45Ra CD45Ra Cm slgM, lgD slgM, G or A CD45Ra (BCR) (BCR) Pre-BCR Figure 8.3 Immunologic maturation of the B lymphocyte from the pluripotential hematopoietic stem cell (HSC) to the plasma cell. Specific antigens (CD) that appear sequentially on the developing cell can define each maturation stage. The drawing shows selected differentiation antigens. Stem cells, pro-B lymphocytes, and pre-B lymphocytes are normally found in the bone marrow. The mature B lymphocyte is found in the peripheral blood. When stimulated by antigens, the B lymphocytes undergo maturation to plasma cells in the lymph nodes or bone marrow. Cm, cytoplasmic m chains; sIg, surface membrane immunoglobulin; cIg, cytoplasmic immunoglobulin; (c), cytoplasmic expression; (s), surface expression; CD, cluster of differentiation; BCR, B cell receptor; c-Kit, stem cell factor receptor (also called CD117). 144 Chapter 8 CD10, CD19, CD20, CD21, CD22, CD24, CD38, and HLA-DR B LYMPHOCYTE ANTIGEN RECEPTOR (BCR) (most of which are not unique to B cells, but are expressed B cells recognize and interact with specific antigens via their on a variety of cell types). The CD19 antigen is considered a antigen-specific BCR (Figure 8-4a). The BCR consists of an “pan-B” antigen because it is found on the earliest B lympho- immunoglobulin (Ig) molecule identical to that produced by cyte and is retained until the latest stages of activation. The the mature B lymphocyte/plasma cell. The complete BCR CD10 marker, also known as the common acute lymphoblas- includes the immunoglobulin molecule with two accessory tic leukemia antigen (CALLA), was originally believed to be molecules, Ig a and Igb, which function as signaling mol- a specific marker of l eukemic cells in acute lymphoblastic ecules during B cell activation.7 leukemia. It is now known that CALLA is present on a small Immunoglobulin (Ig) is a unique molecule produced percentage (less than 3%) of normal bone marrow cells, is by B lymphocytes and plasma cells. Ig, also known in found only on early B lymphocyte precursors, and disap- its secreted form as antibody, consists of two pairs of pears as cell maturation progresses. polypeptide chains: two heavy chains (HC) and two light m Heavy chain BCR k or l Light chain lga lgb Cell membrane a m Heavy chain Pre-BCR Vpre B Surrogate l-5 light chain (cLC) lga lgb Cell membrane b Figure 8.4 (a) BCR. The B cell receptor (BCR) consists of an immunoglobulin molecule with two heavy chains (HC) and two light chains (LC) complexed with accessory molecules, Iga and Igb, needed for cell signaling. (b) Pre-BCR. During development, before the cells are fully mature, a “pre-BCR” is made, using a surrogate LC composed of two molecules (Vpre B and l@5) complexed with a m HC and the Iga and Igb accessory molecules. Lymphocytes 145 chains (LC) linked together by disulfide bonds (Figure 8-5). IMMUNOGLOBULIN GENE REARRANGEMENT The number and arrangement of these bonds are specific The Ig molecule that will eventually be produced by mature for the various Ig classes and subclasses. There are five B cells and plasma cells serves as part of the BCR of B cells types of HC, a, d, e, g, and m, which determine the class and undergoes the V(D)J rearrangement discussed previ- of the antibody (IgA, IgD, IgE, IgG, IgM, respectively). ously. The initiation of Ig gene rearrangement is one of the Although each B cell precursor has two sets of HC genes earliest features allowing the cell to be recognized as a B (one on each chromosome 14), only one encodes the HC cell precursor and is a marker of commitment to the B cell protein in any given cell. Thus, within a given Ig molecule, lineage. HC and LC gene rearrangement occurs at different the two HC are always identical. There are two types of stages of B cell development (see the section “B Lymphocyte LC, k and l, and as with the HC, the two LC within an Ig Developmental Stages”). Rearrangement of the HC locus molecule are always identical. Either k- or l@chain can be involves random selection of a coding sequence from each found in association with any of the various HC, forming of three groups of DNA segments (V, D, J) and recombina- the five major classes of antibodies. Each of the classes and tion to form a variety of unique antibody specificities. The subclasses of Ig has distinct physical and biologic proper- first step is splicing a D and J segment (DJ); subsequently ties (Web Table 8-1). a V segment is added to the fused DJ, forming the VDJ Each heavy and light Ig chain consists of a variable sequence that codes for the variable region of the heavy region and a constant region. The constant region is the chain (VH) (Figure 8-6). The k and l LC also rearrange gene same for all antibodies within a given class or subclass, but segments for VL, but LC-variable gene segments include the variable region in each Ig molecule differs. The constant only a V and J segment (no D segment). region mediates effector functions such as complement acti- B LYMPHOCYTE DEVELOPMENTAL STAGES vation. Together, the variable regions of the light (VL) and Differentiation of the developmental stages for both B and T heavy (VH) chains determine the antibody-combining site. lymphocytes is based on the appearance of specific molecu- Somatic recombination (or gene rearrangement) of gene seg- lar changes as well as membrane molecules. Distinct stages ments of the heavy chain and light chain variable regions of B cells are defined using monoclonal antibodies to iden- allows a diverse repertoire of Ig specificity for antigens. tify the presence of CD markers on the cell surface as well as molecular evidence of Ig gene rearrangement. Some CD markers appear at a very early developmental stage of the Antigen-combining cell and disappear with maturity; other CD markers appear sites on more mature cells (Figure 8-3). Early antigen-independent stages of B lymphopoiesis occur in the bone marrow where marrow stromal cells VH VH provide a specialized microenvironment for B cells at vari- VL V Hinge L C ous stages of maturation. Cytokines important in the early H1 region phases of B cell development include SCF, Flt-3 ligand (FL), Light CL -s-s- CL Fab chain -s-s- - -s-s- s-s- fragment and SDF-1.11 Pro-B to Pre-B Cells The earliest committed B cell precur- sor is the pro-B cell (Figure 8-2).12 It is characterized by the Heavy Fc presence of CD34 (an early hematopoietic cell marker), chain fragment CD19 (pan-B marker), CD10 (CALLA), and TdT. During the pro-B stage, HC rearrangement of the m heavy chain of IgM takes place. Only about half of the total number of developing pro-B cells successfully makes a functional HC; the other half dies of apoptosis in the marrow. Basic structure of immunoglobulin When a successful m heavy chain rearrangement is completed, free cytoplasmic m chains (Cm) can be found Figure 8.5 Schematic drawing of an IgG molecule. The four and the cell becomes an early (large) pre-B cell. Pre-B cells peptide chains include two light and two heavy chains. Disulfide are characterized by the loss of CD34, persistence of CD19 bonds between the chains are indicated by -s-s-. The variable and CD10 (CALLA), and the appearance of CD20, CD22, domains are indicated by V and the constant regions by C. The and CD24. Pre-B cells produce a “pre-BCR” on the cell sur- variable regions have variable amino acid sequences depending on face consisting of the m HC complexed with a surrogate the antibody specificity, and the constant regions have a constant sequence among immunoglobulins of the same class. The two light chain (because true LC are not yet being produced), heavy chains (H) determine the class of immunoglobulin, in this case and the accessory molecules Ig a and Ig b (Figure 8-4b). for IgG. The two light chains can be either k or l. Once formed, the pre-BCR delivers to the cell signals that CCHH33 CCHH22 CCHH33 CCHH22 C H1 146 Chapter 8 N = ~100 V1 V2 V3. VN D1 D2. .D22 D23 J1 J2 J3 J4 J5 J6 Cm Cd Cg . D-J joining N = ~100 V1 V2 V3. VN D1. .D8 J3 J4 J5 J6 Cm Cd Cg . V-D joining V1. .V15 V16 D8 J3 J4 J5 J6 Cm Cd Cg . RNA transcription and splicing V16 D8 J3 Cm Translation IgM heavy chain protein Figure 8.6 During B cell differentiation, the immunoglobulin (Ig) heavy chain (HC) gene rearranges to produce a unique coding sequence that determines antibody specificity. This occurs through a process of splicing and deletion whereby 1 of 23 diversity (D) regions is juxtaposed with 1 of 6 joining (J) regions and then with 1 of about 100 variable (V) regions. Finally, constant (C) region splicing determines Ig isotype (IgM, IgD,). In this example, V, D, J, and Cm segments are sequentially spliced together to generate a nucleic acid sequence that encodes IgM HC proteins. These HC proteins complex with k or l light chain proteins (that are also encoded by rearranged genes) to produce a functional Ig molecule. Each developing B cell has different Ig gene rearrangements, so a population of normal B cells is characterized by polyclonal Ig genes. The diversity of these genes and their encoded immunoglobulins permits immune recognition of many different antigens. promote survival, proliferation, and continued maturation.7 B lymphocytes are not capable of reacting with antigens Toward the end of the pre-B
cell stage, LC gene rearrange- until they develop both IgM and IgD on their surface (i.e., ment occurs; it is characterized by loss of TdT and persis- mature B cell stage). The immunocompetent but naive cells tence of CD-19, 20, 22, and 24. About 85% of the pre-B cell leave the marrow, circulate through the blood, and enter the population makes a successful rearrangement of the LC.12 peripheral lymphoid tissues where the antigen-dependent phase of B cell development occurs. Mature B cells enter the Immature to Naive Mature B Cells When successful rear- germinal centers (follicles) of lymph nodes, the germinal rangement of the LC locus is complete and the cell expresses centers of the spleen, and other secondary lymphoid tissue either a km or a lm IgM, the molecule is transported to the where they undergo further differentiation into effector B cell surface to form a functional BCR (surface IgM [sIgM] cells (plasma cells) in response to encounters with specific plus Ig a and Ig b). The cell is now an immature B cell; it antigens. is positive for CD-19, 20, 21, 22, and 24. The immunocom- petent but naive mature B cell is characterized by the dual expression of both m (IgM) and d (IgD) BCR on the cell Checkpoint 8.2 surface. If immature or mature B cells recognize and bind What characteristics differentiate an immature and a mature B self-antigen in the bone marrow with high avidity, the cells lymphocyte? either undergo a process called receptor editing (in which B cells alter their light chain to eliminate self-reactivity), or cell death (apoptosis) occurs. During antigen-independent Final Maturation to Effector B Cells After encountering the lymphopoiesis in the marrow, the processes of both prolif- antigen recognized by the specific BCR, some activated B eration and apoptotic cell death are significant. This elimi- cells undergo isotype switching of the constant region of the nates those cells with unsuccessful or nonfunctional Ig gene heavy chain gene, converting to IgG, IgA, or IgE BCR. Most rearrangement as well as those that produce autoreactive mature B lymphocytes possess only one class of immuno- antigen receptors. globulin on their membrane. Lymphocytes 147 If mature, immunocompetent B cells fail either to enter and maturation of developing T cells. These stimuli include a lymphoid follicle or to encounter and be stimulated by direct physical interaction with cortical and medullary epi- specific antigens, the cells die (by apoptosis) within several thelial cells and dendritic cells as well as cytokines includ- days to several weeks. However, encounter with specific ing IL-7, SCF, FL, and SDF-1.11 Developing T cells in the antigen causes the naive B cell to undergo proliferation thymus are called thymocytes. Lymphopoiesis at this stage (clonal expansion) and differentiation into plasmacytoid correlates to the antigen-independent stage of T lymphocyte lymphocytes and finally into antibody-secreting plasma cells. development. Some of the progeny of this clonal expansion will become The historic viewpoint was that the thymus functions long-lived memory cells, which have the capacity to respond in T lymphopoiesis primarily during fetal life and the first rapidly to subsequent exposures to the same antigen. few years after birth and that the T lymphoid system was nearly fully developed at birth. If the thymus does not prop- B CELL SUBCLASSES erly develop in the fetus (DiGeorge syndrome; Chapter 22), Two subclasses of B lymphocytes are detectable in adults. the result is the absence of T lymphocytes as well as severe B-1 lymphocytes represent a minority subset of B cells impairment of adaptive immunity. Surgical removal of the (only about 5% of the total B cells) and are produced from thymus after birth, however, does not significantly impair the HSCs that are primarily active in the fetal liver during immunologic defense. By one year after birth, the lymphoid prenatal hematopoiesis. B-1 lymphocytes are distinguished tissue in the thymus begins to be gradually replaced by from other B cells by a number of characteristics: (1) they fat (called involution of the thymus).7,14 Although the aging have a limited diversity of antigen receptors, (2) they human thymus maintains total size, parenchymal tissue are found primarily in pleural, peritoneal, and mucosal atrophies dramatically. While thymic function declines sig- sites, (3) they spontaneously secrete “natural” antibodies, nificantly with age (by five-fold at age 35 years), the T lym- (4) they recognize common bacterial polysaccharides and phocyte pool continues to be replenished by the residual other carbohydrate antigens, and are thought to provide functioning thymus throughout life.15 defense against environmental flora, and (5) they have a unique phenotype of CD20+CD27+CD43+ .13 They are MAJOR HISTOCOMPATIBILITY COMPLEX the primary source of the “naturally occurring” anti-A and Effective functioning of T lymphocytes in the adaptive IR anti-B antibodies characteristic of the ABO blood group requires interaction with “histocompatible” macrophages, system. The population of B-1 cells arises early in ontog- dendritic cells, B lymphocytes, and other infected host eny and they cannot be generated later in life; B-1 cells are cells. Recognition and interaction between T cells and other maintained in adults largely by the division (self-renewal) cells of the body are achieved via specialized proteins, of existing B-1 cells. the major histocompatibility complex (MHC) molecules The majority of B lymphocytes are sometimes called or antigens. The MHC molecules in humans are called B-2 cells. B-2 lymphocytes are the major B cells produced the human leukocyte antigens (HLAs; Table 8-2). The MHC in the bone marrow after birth and are found primarily in locus on chromosome 6 contains two groups of HLA genes the circulation and secondary lymphoid tissues. The origin important for T cell function, Class I and Class II, which of B-1 and B-2 cells has been controversial, but evidence encode two types of structurally distinct proteins. There suggests that they represent separate B cell lineages, both are three major Class I gene loci—HLA-A, HLA-B, HLA- arising from the CLP, with an ontological switch from B-1 to C—and three major Class II gene loci—HLA-DP, HLA-DQ, B-2 production occurring around the time of birth.13 and HLA-DR. Class I MHC molecules (HLA-A, HLA-B, HLA-C proteins) are found on essentially all nucleated Checkpoint 8.3 cells. MHC Class II molecules (DP, DQ, DR proteins) are What cells produce immunoglobulin? Describe the structure of found on B lymphocytes, dendritic cells, macrophages, and an immunoglobulin molecule. activated T lymphocytes. The HLA molecules function to present antigens to T lymphocytes and must be recognized T Lymphocytes Table 8.2 Major Histocompatibility Complex Antigens Some bone marrow lymphoid precursor cells migrate to Class I Class II (Ia antigens) Class III the thymus (primary lymphoid organ) where they prolifer- HLA-A HLA-DP Complement components ate and differentiate to acquire cellular characteristics of T HLA-B HLA-DQ Cytokines lymphocytes. The dominant cell type that migrates from the bone marrow and seeds the thymus to initiate thymopoiesis HLA-C HLA-DR has yet to be determined (HSC, LMPP, CLP). The thymic Minor Ags: Minor Ags: environment provides stimuli required for the proliferation HLA-E, -F, -G HLA-DM, -DO 148 Chapter 8 by the T cell (i.e., “histocompatible”) to elicit an immune of distinct TCR polypeptides: TCR with g and d chains ( gd response (see “Lymphocyte Function”). The MHC Class III T cells) and TCR with a and b chains (ab T cells). More than genes code for components of the complement system and 90% of T lymphocytes are ab T cells.14,16 The organization some selected cytokines. of the TCR chains is similar to that of the BCR immuno- globulin chains. Each TCR chain is composed of a variable T LYMPHOCYTE MEMBRANE MARKERS region that recognizes and binds an antigen and a constant CD surface markers used to differentiate developmental region that anchors the TCR to the cell membrane. Similar stages of T precursor cells include CD2, CD3, CD5, CD7, to the H and L chains of the BCR, the variable (V) region CD25, and CD4 and/or CD8. is made from three DNA segments—V, D, and J in the b T LYMPHOCYTE ANTIGEN RECEPTOR and d chains and V and J segments in the a and g chains. The T lymphocyte antigen receptor, also called the T cell A complete V region (i.e., VDJ or VJ) is joined to a constant receptor (TCR), differs from the BCR in that it is a het- (C) segment to form the individual TCR polypeptides. The erodimer of two peptide chains linked by a disulfide bond random rearrangement and recombination of various V, D, (Figure 8-7). Two subsets of T cells are defined by expression and J segments and subsequent joining with a C segment provide the antigenic diversity of TCR, allowing the T lym- phocyte to recognize many different antigens. The ab and gd TCR are expressed in the T cell membrane as a molecular complex with several other transmembrane molecules, including the CD3 complex (consisting of three APC polypeptides, g, d, and e in the form of d:e and g:e heterodi- mers) and a j@chain homodimer (CD247) (Figure 8-6). These auxiliary molecules (CD3 and CD247) with the gdTCR or abTCR mediate intracellular signaling when antigen binds MHC CD4 Class II to the TCR.17 Ag Ag T LYMPHOCYTE DEVELOPMENTAL STAGES Commitment to the T cell lineage depends on cytokines a b produced by the thymic microenvironment including IL-7 and tumor necrosis factor@a (TNF@a). Although the exact S S V lymphoid progenitor cell that migrates to the thymus is S S unclear, acquisition of the cell surface marker CD1 iden- tifies a precursor cell committed to T lineage differen- tiation.18,19 Differentiation of the various developmental S S C S S stages for T lymphocytes parallels that for B lymphocytes and is defined by the presence of CD markers on the cell surface as well as molecular evidence of TCR gene CD3 S S CD3 z-polypeptide rearrangement (Figure 8-8). The major developmental dimer stages are pro-T cell, pre-T cell (both of which are double negative [DN]), double positive (DP) and single positive e e (SP) thymocyte, and T cell. g d Pro-T to Pre-T Cell to Double Positive Thymocyte The ear- T lymphocyte liest committed T cell precursor, a pro-T cell, is CD34+ and contains germline configuration of the TCR a@, b@, g@ and d@gene loci. These cells are CD25+ (IL-2 receptor a@ chain), CD44+ (thymic homing receptor), TdT+ , and lack Figure 8.7 CD4 and CD8 (i.e., “double negative” cells). During the The T lymphocyte has an antigen receptor (TCR) on its surface that is composed of two peptide chains linked by a pro-T stage, rearrangement of the b@chain locus occurs. If disulfide bond. This receptor is expressed in a complex with CD3 ( g:e the rearrangement is successful, the b@chain is expressed and d:e polypeptide heterodimers) and a z polypeptide homodimer. in the cytoplasm (cb) toward the end of the pro-T stage. This TCR is in close proximity to a major histocompatibility complex These immature thymocytes are found in the cortex of the (MHC)–restricted co-receptor (CD4 or CD8) that recognizes the thymus and migrate through the cortex to the medulla as appropriate MHC molecule on antigen-presenting cells (APCs). The helper T lymphocyte co-receptor (CD4) shown here recognizes Class maturation proceeds. II MHC molecules, and the cytotoxic T lymphocyte’s co-receptor During the pre-T cell stage, the TCR b@chain appears on (CD8) recognizes Class I MHC molecules. the cell surface, linked with a surrogate a@chain(preTa), the Lymphocytes 149 Bone marrow Thymus Intrathymic Post- Peripheral (cortical) thymic blood HSC CLP T/NK Pro-T Pre-T DP SP CD4+/TH CD34 CD34 CD34 CD34 CD34+/- CD1a CD7 c-Kit c-Kit c-Kit c-Kit TdT TdT CD2 Flt 3 IL-7R TdT CD7 CD7 CD5 TdT CD44 CD44 CD38 CD3 CD3 HLA-DR CD25 CD2 CD4 CD7 CD3 CD5 TCR/CD3 CD38 CD2 CD4,8 Pre-TCR SP CD8+/Tc CD7 CD2 CD5 CD3 CD8 TCR/C3 Figure 8.8 Immunologic maturation of the T lymphocyte from the common lymphoid progenitor cell (CLP) to the peripheral blood CD4 + and CD8 + lymphocytes, showing selected identification markers. Monoclonal antibodies and molecular studies for TCR gene rearrangements have identified at least four intrathymic stages of maturation before the cells are released to the peripheral blood as mature T lymphocytes. These include pro-T cells, pre-T cells, double positive (DP), and single positive (SP) cells. Differentiation into either CD4 + or CD8 + lymphocytes occurs at the last intrathymic stage of maturation. CD, cluster of differentiation; TCR, T cell receptor, TdT, terminyldeoxynucleotidyltransferase; IL7R, interleukin
7 receptor; c-Kit, stem cell factor receptor (or CD117). CD3 complex, and z chains to form the pre-TCR. Signals ensures that they are tolerant (e.g., do not respond) to most from the pre-TCR support survival and proliferation (clonal self-antigens. Intrathymic proliferation and death for poten- expansion) of the pre-T thymocytes, initiate recombination tial T lymphocytes is high with about 95% of the cells pro- of the TCR a@chain, and induce expression of both CD4 and duced undergoing apoptosis. Consequently, only a small CD8, forming double positive (DP) thymocytes (late pre-T cell). portion of T precursor cells developing in the thymus leave When a functional a@chain has been produced, it as immunocompetent, naive T lymphocytes. replaces the surrogate a@chain and pairs with the b@chain to form the abTCR that associates with the CD3 complex Double positive to Single Positive Thymocytes to Mature T and z@chains. Subsequently, the DP thymocyte that success- Lymphocytes Differentiation into single positive CD4+ or fully undergoes selection (see “Positive and Negative Selec- CD8+ thymocytes is determined by whether an individual tion”) proceeds to differentiate into either single positive thymocyte’s TCR recognizes antigen bound to MHC Class CD4+ CD8 - or CD4- CD8+ T cells. At this point, the cells II or MHC Class I molecules, respectively. This is primarily are immunocompetent but naive T cells. mediated by thymic epithelial cells during the process of positive selection.17 Positive and Negative Selection Thymocytes must survive Single positive cells leave the thymus and enter the two selection processes during development in the thymus. circulation to complete final maturation. In the periphery, Mature T cells must be able to recognize foreign peptides encounters with the antigen specifically recognized by the presented by “self” MHC molecules during the immune unique TCR result in activation and generation of appro- response. DP thymocytes expressing abTCR encounter priate effector T lymphocytes and memory T cells (antigen- self-peptides displayed by self-MHC molecules on corti- dependent phase of lymphopoiesis). CD4+ CD8- cells cal epithelial cells (the only peptides normally present in become helper T cells (Th), and CD4- CD8 + cells become the thymic cortex). In one selection process, thymocytes cytotoxic T cells (CTC or TC). with TCRs that recognize and bind weakly to self-antigens presented by self-MHC are stimulated to survive (positive T LYMPHOCYTE SUBCLASSES selection). Cells with TCRs that fail to recognize self-MHC T lymphocytes constitute about 70% of total peripheral blood molecules die by apoptosis. In a second selection process, lymphocytes, and about 60–80% of peripheral blood T lym- thymocytes whose TCRs bind strongly to self-antigens are phocytes are CD4 cells (T4). CD4 cells are also the predomi- deleted by apoptosis (negative selection) or differentiate nant T lymphocytes found in the lymph nodes. CD8 cells (T8) into TReg cells; this second selection process functions to pre- make up only 35% of peripheral blood T lymphocytes but vent autoimmune reactions. Positive selection ensures that are the predominant T lymphocyte found in the bone mar- T cells are self-MHC restricted whereas negative selection row. The normal T4@to@T8 ratio in circulating blood is thus 150 Chapter 8 about 2:1. The balance between T4 and T8 is critical for normal the major cytokines regulating NK cell differentiation and activity of the immune system. This ratio can decrease in viral development (Figure 8-9). infections, immune deficiency states, and acquired immune Immature NK cells are characterized by high levels of deficiency syndrome (AIDS) and can increase in disorders CD56, lack CD16, and have low cytolytic activity. Mature such as acute graft versus host disease, scleroderma, and mul- NK cells have low levels of CD56, high levels of CD16, and tiple sclerosis. In the fetal thymus, the first TCR gene rear- potent lytic activity.22 CD56brightCD16- NK cells are pres- rangement involves the g and d loci. Less than 10% of double ent in the endometrium in early pregnancy, where they are negative thymocytes mature to gdTCR T cells after birth. thought to facilitate embryonic implantation and modulate the maternal immune response against embryo antigens, Natural Killer Cells among other functions.23 NK cells constitute 5–20% of the circulating lympho- The third population of lymphoid cells, natural killer (NK) cytes in the blood and spleen. Most NK cells are short lived cells, were first identified as cytotoxic cells capable of killing with life spans from a few days to a few weeks, although tumor cells and virus-infected cells in the absence of immune some may persist for months after viral exposure.20 sensitization.20 NK cells are actually effector cells of innate immunity, not adaptive immunity, and thus are sometimes Natural Killer T (NKT) Cells referred to as innate lymphoid cells.21 Their CD markers are not characteristic of either T or B cells, and they do not rearrange Natural killer T (NKT) cells possess characteristics of both either the BCR or TCR gene loci. Thus, target cell recognition NK and T cells. They express ab or gd TCRs and carry NK sur- by NK cells is different from that of CD8+ CTLs. They have a face markers. However, unlike CD8+ cytotoxic T cells, NKT diversity of receptors that allow them to recognize appropri- cell cytotoxic activities are not MHC-restricted, and they do ate target cells and destroy them. NK cells express CD56 and not recognize peptides presented by antigen-presenting cells. CD16 (the Fcg receptor III for IgG, FcgRIIIa). The CD16 marker Their TCR-independent cytolytic activity resembles that of NK cells.7,21 is also found on neutrophils and some macrophages. NK cells express some T cell markers on their cell membranes (up to 50% of NK cells can have weak expression of CD8 and CD2), but they lack CD3, CD4, and the TCR complex.20 CASE STUDY (continued from page 140) NK cells originate in the bone marrow from the CLP 1. What class of lymphocytes would you expect to (Figure 8-9). Although some NK cell differentiation can make up the majority of peripheral blood lym- occur in the thymus, NK differentiation does not require phocytes in this child and which subclass CD the thymus. Most NK differentiation occurs in secondary marker is present on the majority of these cells? lymphoid tissues. NK cells and T cells have a close devel- opmental relationship with evidence that the immediate 2. Where does this class of lymphocytes differen- precursor of an NK-committed progenitor is a bipoten- tiate? tial CFU-T/NK.18 IL-15, Flt-3 ligand, SCF, IL7, and IL3 are NK Immature Immature Mature HSC CLP T/NK progenitor NK lytic NK NK CD34 CD34 CD34 IL-2R IL-2R IL-2R IL-2R c-Kit CD10 CD10 IL-7R CD2 CD2 CD2 Flt-3 IL-7R Flt-3 CD161 CD161 CD161 IL-7R c-Kit IL-15R IL-15R CD94 CD94 c-Kit c-Kit c-Kit IL-15R CD56 CD16 Bone marrow Peripheral blood Figure 8.9 Natural killer (NK) cell maturation pathway. Immunologic maturation of NK cells from the pluripotential hematopoietic stem cell (HSC) and common lymphoid progenitor cell (CLP) to the mature NK cell. Each stage of maturation can be defined by specific antigens (CD) and/or the presence of cytokine receptors that appear on the developing cell. NK progenitors and immature cells are found in the bone marrow, and mature NK cells are found in the peripheral blood. CD, cluster of differentiation; IL7R, IL-7 receptor; c-Kit, stem cell factor receptor (or CD117). Lymphocytes 151 Lymphocyte Identification PROLYMPHOCYTE The prolymphocyte is difficult to distinguish in normal and Morphology bone marrow specimens (Table 8-3). The prolymphocyte is slightly smaller than the lymphoblast with a lower N:C Morphologic criteria cannot be used to differentiate ratio. The nuclear chromatin is clumped but more finely between T, B, and NK lymphocytes. When there is a need dispersed than that of the lymphocyte. Nucleoli are usually to distinguish between them, monoclonal antibodies and present, and the cytoplasm is light blue and agranular. flow cytometry for specific CD molecules are most often used. Typically, it is necessary to screen for co-expression LYMPHOCYTE of two or more cell surface proteins to define a functional The mature lymphocyte has wide size variability, 7–16 mcM subset of lymphocytes. Other phenotypic features of lym- when flattened on a glass slide. Size primarily depends on phocytes that allow them to be identified as T lymphocytes the amount of cytoplasm present (Table 8-3). Small lympho- or B lymphocytes include cytochemical staining properties cytes range in size from 7–10 mcM. The nucleus is about the and gene rearrangement of the TCR and BCR gene loci. T size of an erythrocyte and occupies about 90% of the cell lymphocytes contain nonspecific esterase, b@glucuronidase area; the chromatin is deeply condensed, staining a dark and N-acetyl b@glucosamidase and a punctuate pattern of purple (characteristic of non-proliferating cells). Nucleoli, acid phosphatase positivity (Chapter 37). The esterase and although always present, are rarely visible with the light acid phosphatase stains for B lymphocytes are either nega- microscope. If seen, they appear as small, light areas within tive or have a minimal scattered granular pattern of positiv- the nucleus. A narrow rim of sky blue cytoplasm surrounds ity. Large granular lymphocytes have a dispersed granular the nucleus. A few azurophilic granules and vacuoles may reaction pattern for acid phosphatase. be present. Lymphocytes are motile and can show a pecu- liar hand mirror shape on stained blood smears with the Checkpoint 8.4 nucleus in the rounded anterior portion (protopod) trailed CD markers identify lymphocytes. What are the CD markers for by an elongated section of cytoplasm known as the uropod. B cell precursors and T cell precursors? Cells that are morphologically small lymphocytes represent a variety of functional subsets of lymphocytes including immunocompetent naive cells, differentiated effector cells, Morphologic Classification of and memory T and B lymphocytes. Functionally, small lym- Immature Lymphocytes phocytes are “resting” cells in G0 of the cell cycle (i.e., they are not actively dividing). However, in response to binding Three morphologic “stages” of lymphoid maturation or acti- antigen, activation of both T and B cells results in the trans- vation are recognized: lymphoblast, prolymphocyte, and formation of small resting lymphocytes into large prolifer- lymphocyte. The morphologic changes that occur during ating cells with abundant basophilic cytoplasm, irregularly differentiation or activation are shared by the major lym- condensed nuclear chromatin, and occasional nucleoli. phocyte subgroups; thus T, B, and NK lymphocytes and Large lymphocytes are heterogeneous in appearance their precursors are indistinguishable by morphologic and range in size from 11 to 16 mcM in diameter. The criteria. nucleus can be slightly larger than in the small lympho- LYMPHOBLAST cyte, but the difference in cell size is mainly attributable to The lymphoblast (Table 8-3) is about 10–18 mcM (mm) in a larger amount of cytoplasm. The cytoplasm can be lighter diameter with a high N:C ratio. The nuclear chromatin is blue with peripheral basophilia or darker than the cyto- lacy and fine but appears more smudged or densely stained plasm of small lymphocytes. Azurophilic granules may be than that of myeloblasts. One or two well-defined pale blue present; if prominent, the cell is described as a large gran- nucleoli are visible. The nuclear membrane is dense, and a ular lymphocyte. These granules differ from those of the perinuclear clear zone may be seen; it has less basophilic myelocytic cells in that they are peroxidase negative. The agranular cytoplasm than other white cell blasts. Whereas nuclear chromatin can appear similar to that of the small subtle morphologic differences exist, lymphoblasts are usu- lymphocyte or more dispersed, and the nucleus can be ally morphologically indistinguishable from myeloblasts. slightly indented. Like small lymphocytes, these large cells Cytochemical stains can be used to help identify their lym- probably represent a diversity of functional subsets. Large phoid origin (Chapter 37). Unlike myeloblasts, lympho- lymphocytes are T or B cells that have encountered antigen blasts stain negative for peroxidase, lipid, and esterase but and have moved out of G0. contain acid phosphatase and sometimes deposits of gly- Normally, about 3% of blood lymphocytes are large cogen (PAS+ ). Both T and B lymphoblasts may contain the granular lymphocytes (LGLs). These cells consist of a DNA polymerase, TdT, depending on the developmental mixed population of both NK cells (CD3−) and some stage. activated cytotoxic T lymphocytes (CTL, CD3+ , CD8+ ).24 152 Chapter 8 Table 8.3 Lymphocyte Morphology Cell Name Shape/Size/N:C Ratio Nucleus Cytoplasm Lymphoblasts Shape Shape Scant amount, usually Round to oval Round to ovoid, centrally located; can be slightly agranular Size indented, clefted, or folded Occasional azurophilic granules 10–18 mcM
Nucleoli and vacuoles N:C Ratio One or more can be present 6:1–4:1 Chromatin Lacey or coarsely granular with distinct parachromatin Prolymphocyte Shape Shape Moderate amount of Round to oval Round to ovoid, centrally located cytoplasm, occasional Size Nucleoli azurophilic granules 12–20 mcM Single prominent N:C Ratio Chromatin 5:1–3:1 Condensed with indistinct parachromatin Small lymphocyte Shape Shape Scant amount of blue, Round to oval Round to ovoid, slightly indented or notched agranular cytoplasm Size Nucleoli 7–10 mcM Generally, not visible or can be inconspicuous N:C Ratio Chromatin 5:1–2:1 Diffusely dense; blocks of heterochromatin Large lymphocyte Shape Shape Increased amount of blue Round to oval Round to ovoid, slightly indented or notched cytoplasm (may have vari- Size Nucleoli able number of pink, coarse, 11–16 mcM Generally, not visible, can be inconspicuous azurophilic granules; consid- N:C Ratio Chromatin ered large granular lymphocyte) 4:1–2:1 Diffusely dense; blocks of heterochromatin Reactive lymphocyte Shape Shape Variable amount, may be dark Large and irregular Round, ovoid, notched, or indented blue; periphery may be light Size Nucleoli blue; occasional vacuole; 16–30 mcM Can be visible azurophilic granules can be N:C Ratio Chromatin present 3:1–2:1 Variable Dense as in a mature lymph but also can appear immature as in a blast Immunoblast Shape Shape Moderate amount; deep blue Round to oval Large, round, central Size Nucleoli 12–25 mcM Prominent N:C Ratio Chromatin 4:1–3:1 Fine Plasmacytoid lymphocyte Shape Shape Moderate amount, deeply Round to oval Round, central, or slightly eccentric basophilic Size Nucleoli 15–20 mcM Single visible N:C Ratio Chromatin 4:1–3:1 Less clumped and coarse than in plasma cell Plasma cell Shape Shape Moderate to abundant, deeply Round to oval Round, eccentrically placed basophilic; paranuclear Golgi Size Nucleoli complex stains poorly and 9–20 mcM Not present appears as a lighter area Chromatin Coarse, blocklike radial masses Lymphocytes 153 A significant proportion of NK cells are agranular and The reactive lymphocyte has also been referred to as indistinguishable from other lymphocytes. Other NK cells a stimulated, transformed, atypical, activated, or variant lym- have an LGL morphology, but not all LGLs are NK cells.24 phocyte. However, the word atypical carries the connotation LGLs are medium- to large-size lymphocytes with a round of abnormal, and therefore some hematologists prefer not or indented nucleus and abundant pale blue cytoplasm con- to use the term to describe normal lymphocytes in various taining coarse pink granules (Table 8-3). The granules contain stages of antigenic stimulation. lysosomal enzymes as well as proteins required for cytotoxic A few reactive lymphocytes can be seen in the blood of function. These include the pore-forming protein perforin healthy individuals, but they are found in increased num- and granzymes (serine proteases with pro-apoptotic activity). bers during viral infections. For this reason, the reactive lymphocyte has also been called a virocyte. Morphology of Activated IMMUNOBLAST Lymphocytes The immunoblast (Table 8-3) is the next stage in blast transformation. The cell is large, 12–25 mcM in size, and During their development into immunocompetent T and is characterized by prominent nucleoli and a fine nuclear B lymphocytes, the cells acquire specific TCR and BCR chromatin pattern (but coarser than that of other leukocyte receptors that endow them with antigen specificity. During blasts). The large nucleus is usually central and stains a pur- the antigen-dependent phase of lymphocyte development, ple-blue color. The abundant cytoplasm stains an intense contact and binding of this specific antigen to receptors on blue color due to the high density of polyribosomes. These immunocompetent T and B lymphocytes begin a complex are cells that are preparing for or engaged in mitosis. The sequence of cellular events known as blast transformation immunoblast proliferates, increasing the pool of cells pro- (blastogenesis). The end result is the clonal amplification grammed to respond to the initial stimulating antigen. of cells responsible for the expression of immunity to that These antigen-specific daughter cells (effector lymphocytes) specific antigen. Usually occurring within the lymph node, mature into cells that mediate the effector functions of the this series of events includes cell enlargement, an increase immune response. in the rough endoplasmic reticulum (RER), enlargement of The daughter cells of B immunoblasts, which medi- the nucleolus, dispersal of chromatin, an increase in DNA ate humoral immunity, are plasmacytoid lymphocytes and synthesis and mitosis. These transformed cells, called immu- plasma cells (Table 8-3). Humoral immunity is the produc- noblasts or large lymphocytes, have the option to differen- tion of antibodies by activated B lymphocytes stimulated tiate into terminally differentiated effector cells capable of by antigen. mediating the immune response or long-lived memory cells. The morphologically identifiable forms of antigen-stimu- Checkpoint 8.5 lated lymphocytes include the reactive lymphocyte, reactive A young adult patient has a WBC count of 10 * 103/mcL immunoblast, plasmacytoid lymphocyte, and plasma cell. with 80% lymphocytes. The blood smear reveals 70% Reactive lymphocytes and immunoblasts can be either reactive lymphocytes and 10% nonreactive lymphocytes. T or B lymphocytes, which can be determined only by cell What is the absolute concentration of total lymphocytes and marker studies (flow cytometry; Chapter 40). reactive lymphocytes? What is a probable cause of these REACTIVE LYMPHOCYTE findings? The reactive lymphocyte (Table 8-3) can exhibit a variety of morphologic features, including one or more of the following: PLASMACYTOID LYMPHOCYTE • It is increased in size (16–30 mcM) with a decreased The plasmacytoid lymphocyte (also referred to as N:C ratio. lymphocytoid plasma cell; Table 8-3) is believed to be an inter- • The nucleus can be round but is more frequently elon- mediate cell between the B lymphocyte and the plasma cell. gated, stretched, or irregular. It gains its descriptive name from its morphologic similarity to the lymphocyte but has marked cytoplasmic basophilia • The chromatin becomes more dispersed, staining similar to that of plasma cells. The plasmacytoid lympho- lighter than the chromatin of a resting lymphocyte, and cyte ranges in size from 15 to 20 mcM. The nuclear chro- nucleoli may be seen. matin is less clumped than that of a plasma cell, and it may • There usually is an increase in diffuse or localized baso- have a single visible nucleolus. The nucleus is central or philia of the cytoplasm, and azurophilic granules and/ slightly eccentric, and the cytoplasm is deeply basophilic. or vacuoles may be present. The cell has some cytoplasmic immunoglobulin (cIg) as • The cytoplasmic membrane may be indented by sur- well as surface membrane immunoglobulin (sIg). This cell rounding erythrocytes, which gives the cell a s calloped is occasionally seen in the peripheral blood of patients with edge. viral infections. 154 Chapter 8 PLASMA CELLS Lymphocytopenia, a decrease in lymphocytes, occurs Plasma cells (Table 8-2) are round or slightly oval with a in adults when the absolute number of lymphocytes is less 9–20 mcM diameter. The nucleus is eccentrically placed than 1.0 * 103/mcL. Lymphocytes are the second most and contains coarse blocklike radial masses of chromatin, numerous intravascular leukocyte in adults. However, often referred to as the cartwheel or spoke wheel arrange- peripheral blood lymphocytes comprise only about 5% of ment. Nucleoli are not present. The paranuclear G olgi the total body lymphocyte pool; 95% are located in extra- complex is obvious and surrounded by deeply baso- vascular tissue of the lymph nodes and spleen. Movement philic cytoplasm. The cytoplasm stains red with pyro- of lymphocytes between the intravascular and extravascu- nine (pyroninophilic) because of the high RNA content. lar compartments is continuous. Lymphocytes from lymph The RER is well developed, and the cytoplasm enlarges nodes enter the lymphatic channels and gain entry to the because of the production of large amounts of immuno- blood as the lymph drains into the right lymphatic duct globulin. Membrane sIg is absent, but azurophilic gran- and the thoracic duct. When stimulated by antigen, lym- ules and rodlike crystal inclusions of cytoplasmic Ig may phocytes migrate to specific areas of lymphoid tissue where be present. they undergo proliferation and transformation into effec- The reactive lymphocyte is commonly found in tor cells. To reach extravascular tissue, lymphocytes most the blood during viral infection; the immunoblast, often transmigrate between the borders of endothelial cells plasmacytoid lymphocyte, and plasma cell usually are lining the vessels (paracellular transmigration). Less often, found only in lymph nodes and other secondary lymphoid lymphocytes move through endothelial cells (transcellular tissue. D uring intense stimulation of the immune system, migration) rather than between them.25 however, these transformed cells can be found in the Lymphocytes leave and re-enter the blood (recirculate) peripheral blood. many times during their life spans. About 80% of lympho- In contrast to the progeny of the B immunoblast, effec- cytes in the peripheral blood are “long lived” memory cells tor cells produced from the T immunoblast (cytotoxic with a life span ranging from a few months to many years. T cells, or T helper cells) are often morphologically indis- These cells spend most of their lives in a prolonged, inter- tinguishable from the original unstimulated lymphocytes, mitotic, G0 phase. The remaining 20% of the lymphocytes or appear as LGL. live from a few hours to about 5 days. The majority of these short-lived, rapidly turning over lymphocytes are immuno- MEMORY CELLS A number of the T and B immunoblast daughter cells alter- competent, naive B and T lymphocytes. natively form T and B memory cells. Memory cells are morphologically similar to the resting small lymphocytes, CASE STUDY (continued from page 150) retain the ability to react with the stimulating antigen, and are capable of eliciting a rapid secondary immune response 3. Calculate the absolute concentration of lympho- when challenged again by the same antigen. cytes. Is this increased, decreased, or within the reference interval? Checkpoint 8.6 4. If this were a 30-year-old male, would this be How would you morphologically differentiate a reactive lympho- considered normal or abnormal? Explain. cyte from a plasma cell on a peripheral blood smear? Checkpoint 8.7 Lymphocyte Distribution, Describe the process of lymphocyte recirculation. Concentration, and Kinetics Lymphocytes have a peripheral blood concentration in adults ranging from 1.094.8 * 103/mcL. At birth, the mean Lymphocyte Function lymphocyte count is 5.5 * 103/mcL This value rises to a T lymphocytes, B lymphocytes, dendritic cells, and macro- mean of 7 * 103/mcL in the next 6 months and remains phages interact in a series of events (the adaptive immune at that level until approximately age 4. A gradual decrease response) that allows the body to attack and eliminate in lymphocytes is noted from 4 years of age until reach- foreign pathogens. Cytokines play an important role in ing adult reference interval values in the second decade of the activation and regulation of the immune response. life. Lymphocytosis, an increase in lymphocytes, occurs in Cytokines are produced by a variety of cell types includ- adults when the absolute number of lymphocytes is greater ing T lymphocytes, macrophages, dendritic cells, NK than 4.8 * 103/mcL. cells, and B lymphocytes. These cytokines influence the Lymphocytes 155 function of all of the cellular components involved in the intracellular microbes (before they infect cells). The antibod- innate and adaptive immune responses (Table 8-4). Most ies neutralize and eliminate infectious microbes and micro- cytokines work synergistically in inducing their physiologic bial toxins. Antibodies generally are produced by plasma response (Chapter 4). cells in the lymphoid organs as well as bone marrow and circulate throughout the body in the blood. IgG antibodies B Lymphocytes (Humoral Immunity) coat microbes and promote phagocytosis (opsonization) by the binding of the Ig Fc region to Fcg receptors (FcgR@I, II, When naive B cells encounter antigen in the secondary lym- III) on phagocytes. IgG, IgM, and IgA can activate comple- phoid tissues, they are induced to proliferate, r esulting in ment, which generates opsonizing molecules also capable clonal expansion producing large numbers of B cells with the of promoting phagocytosis by binding to complement same BCR capable of recognizing that particular antigen. This receptors (CR1-4). IgE binds antigen and triggers mast cell process is followed by differentiation of the antigen-activated degranulation, evoking immediate hypersensitivity reac- B cells into memory B cells and antibody-secreting plasma tions (Chapter 7). cells (the antigen-dependent phase of B lymphopoiesis). Antigen binding to and activation of naive B cells EFFECTOR CELL DEVELOPMENT results in a primary antibody response, whereas antibody Antigen activation of B lymphocytes results in the mor- activation of memory B cells results in a secondary anti- phologic transformation from reactive
lymphocytes to body response. The secondary response develops more immunoblasts, to plasmacytoid lymphocytes, and finally rapidly than does the primary and is associated with pre- to plasma cells in the lymph nodes. Plasma cells are thus dominantly IgG antibody production, whereas the pri- the fully activated terminal effector cell of the B lymphocyte mary response is associated with primarily production of lineage. Their primary function is synthesis and secretion of IgM antibodies. antibody of the same isotype (Ig class) and idiotype (anti- B cell activation and production of antibodies function gen specificity) as the BCR of its precursor B cell. Plasma primarily to defend against extracellular bacteria, fungi, and cells have large quantities of cytoplasmic immunoglobulin Table 8.4 Soluble Mediators with Effects on Immune System Cells Cytokine Action MIF Prevents migration of macrophages from site of inflammation SCF, Flt3 ligand (FL) Synergizes with other cytokines to promote proliferation and differentiation of early HSC/HPC; early T and B cell development IFNg Inhibits intracellular viral multiplication; inhibits proliferation of TH2 cells; activates macrophages IL-2 Induces proliferation and activation of lymphocytes (T cells, NK cells) IL-3 Multilineage colony-stimulating factor; acts in synergy with other cytokines to stimulate proliferation of hematopoietic cells; has a negative effect on lymphoid progenitors IL-4 Stimulates proliferation of B lymphocytes IL-5 Stimulates proliferation/differentiation of B lymphocytes and eosinophils IL-6 Stimulates proliferation/differentiation of B lymphocytes IL-7 Stimulates proliferation/differentiation of both B and T cell progenitors; stimulates proliferation, survival of naive T cells IL-9 Stimulates proliferation of TH cells; potentiates antibody production by B cells; activates mast cells IL-10 Inhibits production of IL2 and IFNg by TH1 cells; suppresses macrophage function IL-12 Stimulates T cells and NK cells; promotes development of TH1 cells IL-13 Stimulates B cell growth and differentiation; inhibits production of proinflammatory cytokines IL-14 Stimulates proliferation of activated B cells IL-15 Activates T cells, NK cells IL-16 Serves as a chemoattractant of CD4 + lymphocytes IL-17 Induces endothelial cells, epithelial cells, fibroblasts to secrete other cytokines, including IL-6, IL-8, GM-CSF, EPO, IL-1 IFN@a, @b Activate macrophages, granulocytes, CTL MIP@1a Responsible for chemotaxis, respiratory burst TNF@a Inhibits hematopoiesis HSC, hematopoietic stem cell; HPC, hematopoietic progenitor cell; IFN, interferon; MIF, migration inhibitory factor; MIP, microphage inflammatory protein; SCF, stem cell factor; TNF, tumor necrosis factor. 156 Chapter 8 (cIg) but contain little or no surface immunoglobulin (sIg). instances, the TCR will recognize only antigens presented In contrast, the B cells from which plasma cells are derived by self-MHC molecules. are SIg+ but CIg-. Binding to a target cell requires the TCR and the T cell Plasma cells are not normally present in the peripheral coreceptor (CD4 or CD8) to recognize and bind with the blood or lymph and constitute less than 4% of the cells in antigen/MHC. MHC Class I molecules (found on all cells the bone marrow. Most plasma cells are found in the med- of the body) form a complex with peptides derived from ullary cords of lymph nodes although with intense stimu- intracellular pathogens and present these antigens to CD8+ lation of the immune system (e.g., in rubeola, infectious T cells (i.e., CD8+ cells are MHC Class I restricted). Class mononucleosis, toxoplasmosis, syphilis, tuberculosis, mul- II MHC molecules complex with peptides derived from tiple myeloma) they can be found in the peripheral blood. phagocytosed extracellular pathogens that are degraded in intracellular vesicles and present them to CD4+ T cells IMMUNOGLOBULIN PATHOLOGIES (CD4+ cells are MHC Class II restricted). Aberrations in Ig production include hypogammaglobu- linemia, polyclonal gammopathy, and monoclonal gammopa- ANTIGEN-PRESENTING CELLS thy. Each class of Ig has a specific electrical charge that The term antigen-presenting cell (APC) is used to iden- permits migration in an electrical field. Thus, altera- tify specialized cells that process and display antigens to T tions of Ig p roduction can be detected by serum protein lymphocytes. The most effective APC for activating naive electrophoresis. Hypogammaglobulinemia is a decrease T cells is the dendritic cell (DC).17,26 The DC also provides in the total concentration of Ig. Polyclonal gammopathies costimulatory signals and cytokines required for T cell dif- result in an increase in Ig of more than one class (polyclonal ferentiation into effector cells. In return, the T cells secrete antibodies) and are frequently seen in viral or bacterial cytokines (e.g., IFNg) that activate the DC. This positive infections. Monoclonal gammopathies arise from one clone feedback loop maximizes the immune response. of cells and are characterized by an increase in one specific DCs are found in skin, respiratory and gastrointestinal class of Ig with identical heavy and light chains. This type epithelia, and connective tissues, the usual portals of entry of alteration is usually the result of unregulated prolifera- to the body for pathogens. DCs arise from bone marrow tion (neoplastic) of a clone of plasma cells (Chapter 28). precursors, and the majority of DC are related to mono- A balance between production of heavy and light chains nuclear phagocytes (conventional DC). They are distin- normally ensures there is no excess of one or the other. guished by their extensive membranous spiny projections. Neoplastic diseases of plasma cells, however, can result in A minor subset of DCs is referred to as plasmacytoid DCs, unbalanced chain production; excesses of light chains or which resemble plasma cells morphologically but have the heavy chains can sometimes be found in both serum and functional properties of DCs and produce large amounts urine (i.e., LC disease, HC disease). of IFNg.27 Other types of cells can function as APC.17,26 Macro- Checkpoint 8.8 phages can present antigens derived from phagocytosed Where are the majority of plasma cells found, and what is their microbes to effector Th cells and memory cells. B lympho- function? cytes can internalize protein antigens, degrade them, and present peptides to CD4+ T follicular helper (TFH) cells. All nucleated cells can become infected with intracellular pathogens and can present peptides derived from pathogen T Lymphocytes (Cell-Mediated antigens to CD8+ cells, activating a cytolytic response. Immunity) CD4+ T CELLS T lymphocytes confer protection against both intracellu- The six types of CD4+ effector cells—Th1, Th2, TFH, Th9, lar and extracellular pathogens. In addition to eradicating Th17, and Th22—produce different cytokines when acti- microbes, T cells secrete cytokines, which activate other vated and are effective against different types of infectious cells such as macrophages and B lymphocytes. Unlike B pathogens.26,28,29 Different cytokines that are produced by cells, which can recognize and interact with intact patho- immune cells (APCs, NK cells, macrophages, and mast gens/antigens, T cells recognize only short peptide frag- cells) at the site of the immune response drive the develop- ments derived from protein antigens that are bound by ment of these CD4+ subsets. See Table 8-5 for a summary MHC molecules on the surface of the individual’s own cells. of the CD4+ T cell subsets initiating cytokines, cytokines Thus, T lymphocytes are said to be “MHC-restricted” in produced, and effector functions. terms of their immune response. Some T cells must inter- The first two Th subsets identified were named act with other cells of the immune system, such as macro- Th1 and Th2. Differentiation of activated naive CD4+ phages, dendritic cells, and B lymphocytes. Other T cells T cells to Th1 effectors occurs in response to intracellular must be able to interact with any infected host cell. In both viruses and bacteria (e.g., Listeria, mycobacteria), whereas Lymphocytes 157 Table 8.5 CD4 + T Cell Subset Functions Type Stimulating Cytokine Effector Cytokines Functions Detrimental Effects Th1 IL-12 IFNg, IL-2, IL-12, TNFa, TNFb Targets intracellular pathogens, activates Autoimmunity, organ transplant IFNg macrophages, promotes production of rejection, DTH IgG2 Th2 IL-4 IL-4, IL-5, IL-10, IL-13, TGFb Targets extracellular pathogens, acti- IgE-mediated allergies and vates eosinophils and mast cells, pro- asthma motes B cell production of IgE TFH IL-16, IL-21, IL-23 IL-4, IL-10, IL-21, IFNg Ag-specific B cell differentiation into plasma cells and memory cells Th17 IL-1, IL-6 IL-17, IL-21 IL-22 Combats extracellular microbes; recruits Arthritis encephalomyelitis neutrophils Th9 IL-2, IL-4, TGFb IL-9, IL-10, IL-21 Tumor immunity in melanoma Allergic reactions Th22 IL-6, TNFa IL-22, TNFa Epidermal immunity, wound healing Autoimmunity TReg IL2, TGFb IL10, TGFb Inhibits immune response; promotes Systemic inflammatory disease self-tolerance Th2 differentiation occurs in response to helminthes and granzymes and other granule contents to enter. Granzymes allergens. Each of these subsets produces cytokines that activate the target cell apoptotic pathway, inducing target promote its own development and can suppress the devel- cell death. Cytotoxicity can also be mediated through opment of the other. CTL surface molecules including Fas ligand, membrane- Subsequently identified Th subsets were named for associated TNF, or TNF-related apoptosis-inducing ligand the primary cytokine produced or by the location of their (TRAIL) interacting with death-inducing receptors on target function. Activated Th17 cells provide protection against cell membranes (Chapter 2). pathogens by attracting neutrophils and other inflamma- MEMORY T CELLS tory cells to the site of infection.26,28,30 TFH cells are special- In addition to generation of various effector cell subsets, ized to assist in the activation and differentiation of naive B T cell–mediated immune responses generate memory cells and antibody production in lymphoid follicles.26,28 Th9 T cells specific for that antigen that can persist for years.26 cells have been implicated in the development of allergic Memory cells survive in a quiescent state (G0) after anti- reactions, and are thought to play a role in tumor immu- gen is eliminated yet can produce a larger and more rapid nity in myeloma.28,31 Th22 cells are involved in epidermal response on re-exposure to the antigen than naive cells. The immunity.29,32 ability to survive for extended periods of time is due to the A seventh subset of CD4+ T cells are TReg cells, which upregulation of antiapoptotic proteins in memory cells, are important in suppressing immune responses and main- including Bcl-2 and Bcl@XL (Chapter 2). taining self-tolerance.26,29 In the past, lymphocytes capable of regulating the immune response were described as T sup- pressor cells (Ts) and were thought to be CD8+ cells. It is Natural Killer Cells now recognized that immune suppressor function belongs NK cells are effector cells of innate immunity whose main to a unique subset of lymphocytes, the TReg cells. TReg cells function is to kill infected cells and to activate macrophages are generated when developing thymocytes recognize and to destroy phagocytosed microbes.7,35 NK cells are capable strongly bind self-antigens in the thymus (thymus derived; of spontaneous (direct) cytotoxicity for various target cells, tTReg) and when self- and foreign antigens are recognized in primarily cells infected with viruses and intracellular peripheral lymphoid organs (peripherally derived, pTReg). microbes and some tumor cells. They have similar effector CD8+ T CELLS functions as CD8+ CTLs, but their cytotoxicity is non-MHC Activation of naive CD8+ cells and differentiation into restricted (they do not require interaction with self-MHC CTLs requires both antigen presentation by MHC Class I molecules on the target cells). molecules and secondary signals, sometimes provided by NK cells can recognize and attack pathogens with CD4+ Th cells.26,34 As CD8+ T cells differentiate into effector attached IgG via their IgG receptor (CD16) (antibody- CTLs, they acquire the machinery to perform target cell kill- dependent cell-mediated cytotoxicity [ADCC]). In addition, ing and develop cytoplasmic granules containing perforin they express a variety of receptors that recognize cell surface and granzymes that function to kill other cells. On contact proteins that are altered when a cell is infected (“activat- with their target, perforin released from the granules intro- ing receptors”). For example, NKG2D is a receptor on all duces a hole in the target cell membrane, allowing activated NK cells that recognizes a family of glycoproteins related to 158 Chapter 8 MHC Class I proteins (MICA, MICB, ULBP1-ULBP6). These NK cells are called lymphokine-activated killer (LAK) proteins are expressed at low levels or are absent on healthy cells. NK cells produce a variety of cytokines, including cells, but are up-regulated or induced on viral-infected or G-CSF, GM-CSF, IL-5, TNF, IFNg, and TGFb. Thus they transformed cells. NK cells also contain “inhibitory recep- are able to stimulate and inhibit hematopoiesis, and acti- tors” that recognize MHC Class I molecules of autologous vate cells of both the innate and adaptive immune sys- cells and block NK cell activation, thus protecting healthy tems20 (Chapter 4). self-cells from an NK attack. The activity of NK cells is care- fully regulated and is activated by signals from
these vari- Adhesion Molecules of the Adaptive ous activating and inhibitory receptors, listed in Table 8-6. Like CTLs, NK cells rely primarily on the perforin– Immune Response granzyme system and Fas-Fas-ligand to kill their targets.26,34 Adhesion receptors are important in the migration of lym- Because NK cytotoxicity is spontaneous (does not require phocytes between the blood and lymphoid tissues and for clonal expansion as do CTLs), NK cells can kill viral-infected the interaction between immune cells during an immune cells before antigen-specific CTLs are fully active, early in response (Table 8-7). Most adhesion molecules important the viral infection, as part of innate immunity. for adaptive immune system function belong to either the A number of cytokines, including IL-2, IL-15, IL-12, integrin or immunoglobulin-like families of cyto-adhesion IL-18, and TNF, activate NK cells. These cytokine-activated molecules. Table 8.6 Regulation of NK Activity Source of Activating Signal Activating Signal Mode of Action Cytokines IFNa/ b Use stress molecules that serve to signal NK cells in the presence of viral pathogens IL12 Are produced by macrophages for NK cell stimulation IL-2, IL-15 Induce NK proliferation, cytokine synthesis and secretion CD16 (Fcg receptor IIIa) Fc portion of IgG Allows NK cells to target cells against which a humoral response has been mounted and lyse the cells by antibody-dependent cell-mediated cytotoxicity (ADCC) Activating and inhibitory Variety of receptors that bind ligands on both Either activate or suppress NK cell cytolytic activity receptors endogenous and exogenous target cells Activating KIR: 3DS1, 2DS1, 2DS2 CD159/NKG2-B,-C,-D,-E,-F,-G NKp30 (CD337), NKp44 (CD336), NKp46 (CD335) DNAM (CD226) Inhibiting KIR-2DL1, -2DL2, -2DL3, -2DL4, -3DL1, -3DL2 CD94:NKG2A KIR, killer cell immunoglobulin-like receptor. Table 8.7 Adhesion Molecules Involved in the Interaction of Lymphocytes with Other Cells in the Immune Response Adhesion Molecule CD Designation Expressed By Ligand Function LFA-1 aL/ b2 CD11a/CD18 Activated leukocytes ICAM-1, 2, 3 on Mediates interaction of lymphocytes with other cells and EC; APC with endothelium; provides co-stimulatory signal for cell activation adb2 CD2 CD11d/CD18 Activated VCAM-1, ICAM-3 Promotes adhesion to leukocytes and to EC lymphocytes LFA-2 CD2 T lymphocytes, LFA-3 (CD58) on Promotes adherence to EC and APC; mediates activation monocytes EC; APC of T lymphocytes CD4 CD4 TH lymphocytes Class II MHC Enhances adhesion of TH lymphocytes to APC, mediates activation of TH lymphocytes (Continued) Lymphocytes 159 Table 8.7 Adhesion Molecules Involved in the Interaction of Lymphocytes with Other Cells in the Immune Response (Continued) Adhesion Molecule CD Designation Expressed By Ligand Function CD8 CD8 CTL Class I MHC Enhances adhesion and activation of CTL to APC VLA1, VLA2, VLA3 CD49a, CD49b, Lymphocytes (VLA-4 VLA4 ligand; Increases adhesion of cells in area of inflammation VLA4, VLA5, VLA6 CD49c, CD49d, also on monocytes) VCAM-1 on EC; CD49e, CD49f ECM proteins APC, antigen-presenting cell; EC, endothelial cell; CD, cluster of differentiation; EM, extracellular matrix; LFA, leukocyte function–related antigen; ICAM, intercellular cytoadhesion molecule; MHC, major histocompatibility complex antigens; VCAM, vascular cell adhesion; VLA, very late-appearing antigen. Aging and Lymphocyte Function Lymphocyte Metabolism It is now fairly well established that immunocompetence Lymphocytes contain all enzymes of the glycolytic and declines with age.36 By middle age, the thymus has atro- tricarboxylic acid cycle. Glucose enters the cell through phied to about 15% of its maximum size. Although the total facilitated diffusion and is catabolized to produce ATP number of T cells does not decline significantly, the num- through oxidative phosphorylation. The generated ATP is ber of new naive T cells being produced does. As the naive used for recirculation and locomotion as well as replace- T cell population declines, there is a higher proportion of ment of lipids and proteins and the maintenance of ionic memory T cells, many of which gradually reach replicative equilibrium. The hexose monophosphate shunt provides senescence (the inability to proliferate) as one ages. The only a fraction of the needed energy, but it is important for net effect is an immune system that does not respond as purine and pyrimidine synthesis required for DNA replica- quickly or as efficiently to new antigenic challenges. As a tion and mitosis as well as reducing capability associated result, one’s ability to fight off infections declines. Other with production of NADPH. observations include an increase in cancers associated with aging, which may be partly explained by a declining ability CASE STUDY (continued from page 154) to detect and correct defective cells. Also, the aging immune system is less able to distinguish self from non-self, which 5. Is there a need for concern regarding the infant’s results in a higher frequency of autoantibody production results? Explain your answer. with age. Summary The three major types of lymphocytes are T, B, and NK cells. and dendritic cells phagocytize antigens, generating critical The lymphoid precursor cell from the bone marrow matures antigenic fragments that are presented to T lymphocytes in and acquires cellular and molecular characteristics of a T, B, a complex with MHC molecules. The T lymphocytes bind or NK lymphocyte. T and B lymphocytes have antigen-inde- to the antigens and the MHC molecules on the surface pendent and antigen-dependent phases of maturation. In the of the antigen-presenting cell by means of the TCR and antigen-independent maturation process, immunocompetent either the CD4 or CD8 co-receptor. Cytokines released by B lymphocytes develop in the bone marrow and T lympho- the antigen-presenting cell and the T lymphocyte activate cytes develop in the thymus. In the antigen-dependent matu- each other reciprocally. There are at least three major types ration phase, these immunocompetent lymphocytes interact of T lymphocytes: CD4+ helper T lymphocytes (Th), TReg, with antigen and mature into effector T and B lymphocytes. and CD8+ CTLs. The B lymphocyte’s functional activity The immunocompetent cells undergo a series of cellular includes differentiation into effector plasma cells and the events in response to encounters with antigens known as blast synthesis and secretion of antibodies. Surface immuno- transformation. The end result is a clonal amplification of lym- globulin serves as the B lymphocyte receptor. phocyte effector cells responsible for immunity to the specific NK cells appear morphologically as LGLs and are antigen that stimulated transformation as well as the genera- cytotoxic lymphocytes that function in the innate immune tion of long-lived memory cells. Reactive lymphocytes are the response. Cells morphologically identified as LGLs consist most common morphologically identifiable form of antigen- of two distinct populations categorized by the presence or stimulated lymphocyte found in the peripheral blood. absence of CD3. Natural killer cells are CD3- (NK-LGL), T and B lymphocytes have separate but related func- and activated CTLs are CD3+ (T-LGL). The cytotoxicity of tions in the immune response. Monocytes/macrophages NK cells is not MHC restricted. 160 Chapter 8 Review Questions Level I 7. An 80% lymphocyte count with a total WBC count of 4.4 * 109/mcL on an adult indicates a: 1. The function of the lymphocyte is: (Objective 1) (Objectives 2, 3) a. phagocytosis a. relative and absolute decrease in lymphocytes b. antigen recognition b. relative decrease in lymphocytes but an absolute c. hypersensitivity number within the reference interval d. allergic response c. relative increase in lymphocytes but an absolute number within the reference interval 2. Which description best fits the plasma cell? (Objectives 5, 6) d. relative and absolute increase in lymphocytes a. High N:C ratio, nuclear chromatin lacy and fine, nucleoli present, agranular cytoplasm 8. Lymphocyte concentrations in the peripheral b. Nucleus eccentric, chromatin in a cartwheel blood are highest at what age level? arrangement, deeply basophilic cytoplasm (Objectives 2, 3) c. Decreased N:C ratio, nucleus irregular, abundant a. 6 months cytoplasm indented by erythrocytes b. 5 years d. Round nucleus, abundant cytoplasm, cytoplasm c. 25 years contains large pink granules d. 75 years 3. All of the following can function as an 9. Which of the following is a characteristic of antigen-presenting cell except: (Objective 7) B lymphocytes? (Objective 1) a. dendritic cells a. Regulate immune response b. neutrophils b. Synthesize antibody c. macrophages c. Secrete cytokines d. B lymphocytes d. Are majority of peripheral blood 4. A cell on a Wright’s stain peripheral blood smear lymphocytes was observed with a stretched or irregular nucleus; occasional nucleoli were present. The cytoplasm was 10. Which of the following are cells of the innate abundant and scalloped around the erythrocytes. This immune system? (Objective 1) description best fits the: (Objective 6) a. Plasma cells a. plasma cell b. Natural killer cells b. lymphoblast c. B lymphocytes c. reactive lymph d. T lymphocytes d. monocyte Level II 5. The first immunoglobulin heavy chain produced in 1. Pre-B cells are characterized by the presence of: the maturing B lymphocyte is: (Objective 5) (Objective 3) a. a a. CD4 antigen b. b b. CD8 antigen c. m c. CD19 d. g d. CD3 6. The adult reference interval for peripheral blood 2. A major function of CD4+ lymphocytes is: lymphocytes is: (Objective 3) (Objectives 4, 5) a. 0.290.8 * 109/mcL a. to produce immunoglobulin b. 1.094.8 * 109/mcL b. cell-mediated immunity c. 2.097.0 * 109/mcL c. to phagocytize microbes d. 3.9910.6 * 109/mcL d. nonspecific cytotoxic function Lymphocytes 161 3. The main function of natural killer cells is: (Objective 4) c. polyclonal antibody a. cell-mediated immunity d. M protein b. antibody production 7. A patient who is immunosuppressed would most c. regulation of the immune response likely have: (Objective 4) d. cytotoxicity in innate immunity a. an increase in CD4+ lymphocytes 4. The most likely explanation for a patient who has b. lymphocytosis and eosinophilia a WBC count of 16 * 109/mcL with many reactive c. a decrease in CD4+ lymphs lymphocytes and a few immunoblasts present is: d. an increase in the T cell to B cell ratio (Objective 4) 8. Interleukin 4 plays a role in: (Objectives 2, 3) a. a heightened immune response b. early leukemia or lymphoma a. inducing proliferation and activation of lymphocytes c. the presence of immunodeficiency b. enhancing T lymphocytes cell survival d. qualitatively abnormal lymphocytes c. stimulating proliferation of B lymphocytes 5. A patient has lymphocytic leukemia with 60% d. activating macrophages and granulocytes lymphoblasts in the peripheral blood. The best way for the clinical laboratory professional to d etermine 9. KIR molecules are found on which type of whether these are T or B lymphoblasts is to: lymphocyte? (Objective 4) (Objective 5) a. B lymphocytes a. do a TdT stain on the blood b. T lymphocytes b. determine the CD surface markers on the blasts by c. TReg cells flow cytometry d. NK cells c. do a molecular analysis to find oncogenes d. send the peripheral blood specimen to 10. CD surface markers including CD2, CD3, CD5, and cytogenetics CD7 are associated with which subclass of lympho- cytes? (Objective 5) 6. A patient diagnosed with infectious mononucleosis a. B lymphocytes would most likely produce: (Objective 7) b. T lymphocytes a. monoclonal antibody c. TReg cells b. paraprotein d. NK cells References 1. Parham, P., & Janeway, C. (2015). Elements of the immune system 6. Toivanen, P., Uksila, J., Leino, A., Lassila, O., Hirvonen, T., & and their roles in defense. In The immune system (4th ed., Ruuskanen, O. (1981). Development of mitogen responding pp. 1–28). New York: Garland Science. T cells and natural killer cells in the human fetus. 2. Tavian, M., & Peault, B. (2005). Embryonic development Immunological Reviews, 57(1), 89–105. doi: 10.1111/j.1600- of the human hematopoietic system. International Journal 065x.1981.tb00443.x of Developmental Biology, 49(2–3), 243–250. doi: 10.1387/ 7. Abbas, A. K., Lichtman, A. H., & Pillai, S., eds. (2018). ijdb.041957mt Lymphocyte development and antigen receptor gene 3. Tavian, M., Robin, C., Coulombel, L., & Peault, B. (2001). The rearrangement. Cellular and molecular immunology (9th ed., pp. human embryo, but not its yolk sac, generates lympho-myeloid 179–208). Philadelphia: Elsevier. stem cells: Mapping multipotent hematopoietic cell fate in 8. Adolfsson, J., Mansson, R., Buzza-Vidas, N., Hultquist, A., Liuba, intraembryonic mesoderm. Immunity, 15(3), 487–496. K., Jensen, C., T . . . . Jacobsen, S. E. (2005). Identification of Flt3+ 4. Dorshkind, K., & Montecino-Rodriguez, E. (2007). Fetal B-cell lympho-myeloid stem cells lacking erythro-megakaryocytic lymphopoiesis and the emergence of B-1-cell potential. Nature potential: A revised road map for adult blood lineage Reviews Immunology, 7(3), 213–219. doi: 10.1038/nri2019 commitment. Cell, 121(2), 295–306. 5. Renda, M., Fecarotta, E., Dieli, F., Markling, L., Westgren, 9. Hoebeke, I., Smedt, M. D., Stolz, F., Pike-Overzet, K., Staal, F. M., Damiani, G., . . . Maggio, A.
(2000). Evidence of alloreactive J., Plum, J., & Leclercq, G. (2006). T-, B- and NK-lymphoid, but T lymphocytes in fetal liver: Implications for fetal hematopoietic not myeloid cells arise from human CD34 CD38–CD7 common stem cell transplantation. Bone Marrow Transplantation, 25(2), lymphoid progenitors expressing lymphoid-specific genes. 135–141. doi: 10.1038/sj.bmt.1702108 Leukemia, 21(2), 311–319. doi: 10.1038/sj.leu.2404488 162 Chapter 8 10. Seet, C. S., & Crooks, G. M. (2015). Lymphopoiesis. In: K. 24. Paraskevas, F. (2014). T lymphocytes and natural killer cells. Kaushansky, J. T. Prchal, O. W. Press, M. A. Lichtman, M. Levi, L. In J. P. Greer, J. Foerster, G. M. Rodgers, F. Paraskevas, B. Glader, J. Burns, & M.A. Caligiuri, eds. Williams hematology (9th ed., D. A. Arber, et al. (Eds.), Wintrobe’s clinical hematology (13th ed., pp. 1149–1158). New York: McGraw-Hill Education. pp. 279–312). Philadelphia: Lippincott, Willams & Wilkins. 11. Shaheen, M., & Broxmeyer, H. E. (2009). The humoral regulation 25. Abbas, A. K., Lichtman, A. H., & Pillai, S. (Eds). (2012). Leukocyte of hematopoiesis. In: R. Hoffman, E. J. Benz, S. J. Shattil, B.Furie, migration into tissues. Cellular and molecular immunology (7th ed., L.E. Silberstein, P. McGlave, & H. Heslop, eds. Hematology: pp. 37–53). Philadelphia: Elsevier Saunders. Basic principles and practice (5th ed., pp. 253–275). Philadelphia: 26. Parham, P., & Janeway, C. (2015). Cell mediated immunity. In The Churchill Livingstone. immune system (4th ed., pp. 199–230). New York: Garland Science. 12. Parham, P., & Janeway, C. (2015). The development of B 27. Dhodapkar, M., Mackall, C. L., & Steinman, R. M. (2015). lymphocytes. In: The immune system (4th ed., pp. 149–176). New Dendritic cells and adaptive immunity. In: K. Kaushansky, J. T. York: Garland Science. Prchal, O. W. Press, M. A. Lichtman, M. Levi, & L. J. Burns, eds. 13. Rothstein, T. L., Griffin, D. O., Holodick, N. E., Quach, T. D., & Williams hematology (9th ed., pp. 307–314). New York: McGraw- Kaku, H. (2013). Human B-1 cells take the stage. Hill Education. Annals of the New York Academy of Sciences, 1285(1), 97–114. 28. McClanahan, F., & Gribben, J. (2015). Functions of T lymphocytes: doi: 10.1111/nyas.12137 T-cell receptors for antigen. In: K. Kaushansky, J. T. Prchal, 14. Parham, P., & Janeway, C. (2015). The development of T O. W. Press, M. A. Lichtman, M. Levi, & L. J. Burns, eds. Williams lymphocytes. In: The immune system (4th ed., pp. 177–197). New hematology (9th ed., pp. 1175–1188). New York: York: Garland Science. McGraw-Hill Education. 15. Douek, D. C., McFarland, R. D., Keiser, P. H., Gage, E. A., Massey, 29. Eyerich, S., Eyerich, K., Pennino, D., Carbone, T., Nasorri, F., J. M., Haynes, B. F . . . . Koup, R. A. (1998). Changes in thymic Pallotta, S., . . . Cavani, A. (2009). Th22 cells represent a distinct function with age and during treatment of HIV infection. Nature, human T cell subset involved in epidermal immunity and 396(6712), 690–695. remodeling. Journal of Clinical Investigation, 119, 3573–3585. 16. Mccoy, K. L. (2009). Programmed B and T cell development. 30. Koenders, M. I., & Berg, W. B. (2010). Translational mini-review Nutrition Reviews, 56(1). doi: 10.1111/j.1753-4887.1998.tb01639.x series on Th17 cells: Are T helper 17 cells really pathogenic in 17. Parham, P., & Janeway, C. (2015). Antigen recognition by T autoimmunity? Clinical and Experimental Immunology, 159(2), lymphocytes. In: The immune system (4th ed., pp. 113–147). New 131–136. doi: 10.1111/j.1365-2249.2009.04039.x York: Garland Science. 31. Schmitt, E., Klein, M., & Bopp, T. (2014). Th9 cells, new players 18. Sanchez, M. J., Muench, M. O., Roncarolo, M. G., Lanier, L. L., & in adaptive immunity. Trends in Immunology, 35(2), 61–68. doi: Phillips, J. H. (1994). Identification of a common T/natural killer 10.1016/j.it.2013.10.004 cell progenitor in human fetal thymus. Journal of Experimental 32. Azizi, G., Yazdani, R., & Mirshafiey A., (2015). Th22 cells in Medicine, 180(2), 569–576. doi: 10.1084/jem.180.2.569 autoimmunity: A review of current knowledge. European Annals 19. Spits, H. (2002). Development of alphabeta T cells in the human of Allergy and Clinical Immunology, 47(4), 108–117. thymus. Nature Reviews Immunology, 2(10), 760–772. 33. Abbas, A. K., Lichtman, A. H., Pillai, S. (Eds.). (2018). 20. Trinchieri, G., Childs, R. W., & Lanier, L. L. (2015). Function of Immunologic tolerance and autoimmunity Cellular and molecular natural killer cells. In: K. Kaushansky, J. T. Prchal, O. W. Press, M. immunology (9th ed., pp. 325–350). Philadelphia: Elsevier. A. Lichtman, M. Levi, & L. J. Burns, eds. Williams hematology (9th 34. Abbas, A. K., Lichtman, A. H., & Pillai, S. (Eds.). (2012). Effector ed., pp. 1189–1194). New York: McGraw-Hill Education. mechanisms of cell-mediated immunity. Cellular and molecular 21. Spits, H., Artis, D., Colonna, M., Diefenbach, A., Santo, J. P., immunology (7th ed., pp. 225–242). Philadelphia: Elsevier Eberl, G., . . . Vivier, E. (2013). Innate lymphoid cells — a proposal Saunders. for uniform nomenclature. Nature Reviews Immunology, 13(2), 35. Shresta, S., Pham, C. T., Thomas, D. A., Graubert, T. A., & Ley, 145–149. doi: 10.1038/nri3365 T. J. (1998). How do cytotoxic lymphocytes kill their targets? 22. Caligiuri, M. A. (2008). Human natural killer cells. Blood, 112(3), Current Opinion in Immunology, 10(5), 581–587. doi: 10.1016/ 461–469. s0952-7915(98)80227-6 23. Kitaya, K. (2008). Accumulation of uterine CD16(–) natural killer 36. Ershler, W. B., Artz, A. S., & Kanapuru, B. (2015). Hematology in (NK) cells: Friends, foes, or Jekyll-and-Hyde relationship for the older persons. In: K. Kaushansky, J. T. Prchal, O. W. Press, M. A. conceptus? Immunological Investigations, 37(5–6), 467–481. doi: Lichtman, M. Levi, & L. J. Burns, eds. Williams hematology (9th 10.1080/08820130802191292 ed., pp. 129–144). New York: McGraw-Hill Education. Chapter 9 The Platelet Michelle Butina, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Name and describe the cell that is the 4. Describe the normal morphology and precursor of platelets in the bone marrow. number of platelets on a stained peripheral 2. Define endomitosis and polyploidy. blood smear and state the normal life span and concentration of platelets in the blood. 3. Identify the major cytokines regulating platelet production. 5. Summarize the function of platelets in primary hemostasis. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Describe the development of megakaryocytes 4. Describe MPV and PDW and their in the bone marrow to include the stem cell significance in the clinical evaluation and progenitor cell compartment and the of patients. recognizable morphologic stages. 5. Explain the significance of megathrombocytes 2. Define proplatelet and describe the (giant platelets) and reticulated platelets. mechanism of platelet release from the 6. Describe the role of platelets in hemostasis marrow to peripheral blood. and the immune response. 3. Describe the effect of thrombopoietin (TPO) on megakaryocytic cells. 163 164 Chapter 9 Chapter Outline Objectives—Level I and Level II 163 Megakaryocytes 165 Key Terms 164 Peripheral Blood Platelets 169 Background Basics 164 Summary 171 Case Study 164 Review Questions 171 Overview 164 References 173 Introduction 164 Key Terms a-granule (aG) Endomitosis Polyploid Demarcation membrane system Mean platelet volume (MPV) Proplatelet (DMS) Megakaryoblast Reticulated platelet Dense granule (DG) Megakaryocyte Thrombopoietin (TPO) Dense tubular system (DTS) Platelet distribution width (PDW) Background Basics The information in this chapter builds on the concepts Level II learned in previous chapters. To maximize your learning • Summarize the hierarchy of stem and progenitor experience, you should review these concepts before cells in the bone marrow and the growth factors starting this unit of study: that direct the proliferation and maturation of blood cells. (Chapter 4) Level I • Describe the bone marrow production of blood cells. (Chapters 3, 4) It describes the evaluation of peripheral blood platelets, CASE STUDY including normal physiologic and pathologic alterations We refer to this case study throughout the chapter. that can be seen. In addition, an overview of the functions A 70-year-old female saw her physician for of platelets is introduced. A complete description of the role symptoms of dizziness and headache. She also of platelets in hemostasis is detailed in the “Hemostasis” had tingling in the hands and feet. Physical exam section of Chapter 31. revealed an enlarged spleen. A CBC was ordered. The hemoglobin was 11.5 g/dL, WBC count was 10 * 109>L, and platelet count was 950 * 109>L. Introduction Compare these results to reference intervals. Platelets, small anucleate cytoplasmic fragments, were once simply considered “blood dust.”1 Studies over the past 150 years have now shown them to play an essential Overview role in hemostasis (the process that maintains the fluid- ity of blood and arrests bleeding after a vascular injury; This chapter is an introduction to platelets and details the Chapter 31). In addition, platelets aid in maintaining vas- formation of platelets from bone marrow megakaryocytes cular endothelial cell integrity and repairing vascular dam- and their release from the marrow into the peripheral blood. age, and are involved in immune responses. Platelets are The Platelet 165 derived from precursor cells, megakaryocytes, in the bone MEGAKARYOCYTE PROGENITOR CELL marrow through a complex biologic process unique to this COMPARTMENT hematopoietic lineage. Platelets are sometimes referred to MkP are actively proliferating cells responsible for expand- as “thrombocytes,” but this term is more appropriately ing megakaryocyte numbers and proliferate in response applied to the nucleated hemostatic cells found in lower to several hematopoietic cytokines. Research studies in vertebrates, including fish. which MkP were cultured in vitro have led to a better understanding of the development of these cells. Research- ers have defined a hierarchy of megakaryocyte progenitor Megakaryocytes cells that develop through stages identified as BFU-Mk and CFU-Mk.2,3 Like other hematopoietic progenitor cells, these In healthy adults, the bone marrow produces ∼1 * 1011 cells are not morphologically distinguishable. Rather, sur- platelets per day. This baseline platelet production can face antigens and growth characteristics differentiate the increase 10- to 20-fold in times of increased demand and even two stages. The BFU-Mk is CD34+, HLA@DR-, c@Kit+ (SCF further under the stimulation of thrombopoiesis-stimulating receptor), whereas the CFU-Mk is positive for all three drugs.1 Platelet production depends on the proliferation and antigens. In culture, BFU-Mk produce multifocal colonies maturation of precursor megakaryocytes in the bone mar- (bursts) of 100–500 megakaryocytes (six to nine cell divi- row, a complex process of megakaryopoiesis (megakaryocyte sions) whereas the CFU-Mk produce single colonies of 4–32 proliferation) and thrombopoiesis (platelet maturation and megakaryocytes (two to five cell divisions). The transit time release). Megakaryocytes are rare cells within the bone mar- from megakaryocyte progenitor cell to release of platelets row and comprise only ∼0.1% of the total nucleated cells. into the circulation is from 4 to 7 days.2 Megakaryopoiesis REGULATION OF MEGAKARYOCYTE PRODUCTION A regulatory process maintains an adequate number of Platelets are derived in the bone marrow from the common platelets in the peripheral blood. The bone marrow’s pro- myeloid progenitor (CMP) and other progenitor cells com- duction of platelets can increase or decrease according to mitted to megakaryocytic development (megakaryocyte the body’s needs. The major stimulus regulating production progenitor/MkP) (Figure 9-1). The earliest morphologically is the platelet mass in the circulating blood plus the mega- identifiable platelet precursor cell is called a megakaryoblast. karyocyte mass in the bone marrow.4 Several cytokines and Platelets are fragments released from the cytoplasm of growth factors affect megakaryocyte development. mature megakaryocytes. The cells of the megakaryocytic lineage include actively proliferating progenitor cells and Humoral Regulation The primary regulator of megakaryo- postmitotic (nonproliferating) megakaryocytes undergoing cyte and platelet development is thrombopoietin (TPO), maturational development. which influences all stages of megakaryocyte production Bone marrow Peripheral blood Stem and progenitor cells Morphologically recognizable megakaryocytes Platelets BFU-Mk CFU-Mk Stage I Stage II Stage III Stage IV DMS Granule Ploidy 2N 4N 8N, 16N, 32N, 64N Proliferation Mitosis Endomitosis SCF, FL, Spleen IL-6, IL-11 Humoral GM-CSF and IL-3 factors Thrombopoietin Figure 9.1 Megakaryopoiesis. Progenitor cells become committed to the megakaryocyte lineage. Committed progenitor stages are BFU-Mk and CFU-Mk, which can undergo mitosis. Megakaryoblasts stop mitosis and undergo a series of endomitoses until nuclear maturation is complete at 8N to 64N ploidy levels. Cytoplasmic maturation occurs at ploidy levels of 8N or higher. Morphologic features of each stage of cytoplasmic maturation are described in the text. Growth factors Flt ligand, SCF, GM-CSF, and IL-3 influence proliferation of the stem and progenitor cells. Thrombopoietin influences all stages of megakaryocyte production. IL-6 and IL-11 also support megakaryocyte production, primarily maturation. Marrow sinus 166 Chapter 9 from early progenitor cells to
the release of mature plate- progenitor stages of megakaryocytes to proliferate.2,3 Inter- lets from the bone marrow.4 TPO stimulates megakaryo- leukin-6 and -11 also affect megakaryocyte development, cyte survival and proliferation alone and in combination particularly the maturation phases, but only when they with other cytokines.5 TPO also plays an important role in work in synergy and with other cytokines.2 In addition to hematopoietic stem cell survival, self-renewal, and expan- the positive regulators of megakaryopoiesis, several sub- sion (Chapter 4). TPO is produced in the liver, kidneys, and stances downregulate megakaryocyte development. Trans- spleen and possibly in the bone marrow in patients who forming growth factor-b 1TGF-b2 and a-interferon 1IFN-a2 have low platelet counts.6 Thrombopoietin is structurally both inhibit megakaryopoiesis.2 related to the erythrocyte growth factor, erythropoietin. The presence of TPO had been suspected since the CASE STUDY (continued from page 164) 1950s, but it was not until 1994 that several research groups first isolated it.7 The gene for TPO was cloned shortly after 1. Is the patient’s platelet count normal or abnormal? its discovery.8 By 1997, TPO could be produced by recom- 2. Why do you think the spleen is enlarged? binant DNA techniques and soon entered clinical trials for treating patients with thrombocytopenia. However, recom- binanat TPO stimulated the production of TPO-neutralizing antibodies.2 The second-generation thrombopoietin mimet- Checkpoint 9.1 ics (small peptide or organic molecules that bind to and activate What would be the effect on the platelet count if a patient had the TPO receptor) do not stimulate TPO neutralizing anti- a mutation in the gene for thrombopoietin that resulted in the bodies. Two thrombopoietin agonists have received FDA gene’s inability to code for functional mRNA? approval (romiplostin, eltrombopag) and are used today in the treatment of chronic immune thrombocytopenia and Megakaryocyte Microenvironment Like cells of other interferon-induced thrombocytopenia.9,10 hematopoietic lineages, megakaryocytes interact closely Thrombopoietin levels are largely controlled by platelet with marrow stromal cells. Megakaryocytes are found pri- mass, thus plasma TPO levels vary inversely with platelet marily in the vascular niche (Chapters 3 and 4) where they counts. Production of TPO (primarily liver and to a lesser physically attach to endothelial cells lining the sinusoidal extent the kidney) is relatively consistent from day to day. vessels. Marrow stromal cells produce both positive and Once TPO is released into the bloodstream, it binds to its negative regulators of megakaryocyte growth. Stromal cells receptor on circulating platelets (c-Mpl, CD110) and is bear ligands for Notch proteins, thought to play a critical internalized and degraded. Thus, the amount of TPO not role in influencing the lineage choice between erythropoi- taken up by circulating platelets will remain in the blood, esis and megakaryopoiesis.14 Stromal cell-derived factor-1 available to stimulate marrow progenitors and increase (SDF-1) (also known as CXCL12) contributes to megakaryo- megakaryopoiesis. Consumption of TPO is determined by poiesis by augmenting TPO-induced megakaryocyte the total quantity of c-Mp1 receptors present on circulat- growth and endothelial adhesion within the marrow ing platelets. In a state of thrombocytosis, a higher platelet microenvironment.15,16 mass of c-Mp1 receptors will result in greater consump- STAGES OF MEGAKARYOCYTE DEVELOPMENT tion of TPO by circulating platelets, leading to reduction Platelets are fragments formed from the cytoplasm of of residual-free TPO and thus reduced megakaryopoiesis. mature megakaryocytes. Proliferating progenitor cells Conversely, in a state of thrombocytopenia, a lower platelet develop into nonproliferating (post-mitotic) megakaryo- mass of c- Mp1 receptors will result in less consumption of TPO, leading to an increase of megakaryopoiesis.11,12 cytes. Characteristics of megakaryocyte development How- critical for thrombopoiesis include endomitosis and cyto- ever, TPO synthesis can also be inducible, particularly in plasmic maturation. Endomitosis allows polyploidization times of severe thrombocytopenia, when TPO mRNA levels can rise.13 of megakaryocytes, which is important for efficient plate- let production. The earliest identifiable cell of this lineage The effects of TPO in patients with low platelet numbers is the megakaryoblast. Although not easily recognizable are to increase the number of megakaryocytes in the bone morphologically, they can be identified cytochemically by marrow, the size and DNA content (ploidy level) of mega- their expression of megakaryocyte-specific markers, such as karyocytes, and the rate of maturation of the megakaryo- glycoprotein IIb/IIIa or platelet peroxidase. cytes.2 The number of circulating platelets can increase from 10 to 20 times the baseline level by administration of TPO. Endomitosis The megakaryoblast undergoes a matura- Other cytokines and growth factors also influence tion sequence that differs from that of other marrow lin- megakaryocyte and platelet development. Similar to their eages in that nuclear maturation takes place first and is action on other myeloid cell lineages, interleukin-3 (IL-3), largely complete before cytoplasmic maturation begins. GM-CSF, stem cell factor, and Flt ligand influence the Following an initial series of proliferative (mitotic) cell The Platelet 167 divisions during BFU-Mk and CFU-Mk development, the and chromatin pattern of the nucleus. The four stages are precursor cells begin a unique nuclear maturation process described in Table 9-1. consisting of a series of endomitoses. Endomitosis is a Figure 9-2 shows an early (Stage II) megakaryocyte unique form of mitosis in which the cell’s DNA content rep- and a mature (Stage IV) megakaryocyte. The nucleus trans- licates, but cell division (cytokinesis) and nuclear division forms from a single (round) lobe with fine chromatin and (karyokinesis) do not take place.17 Endomitosis is incom- visible nucleoli to lobulated with coarse chromatin and no plete mitosis caused by a block in anaphase.18 visible nucleoli. In general, as the megakaryocyte matures, Repetitive cycles of endomitosis result in megakaryo- the cytoplasm increases in volume and changes from baso- cytes that become polyploid with the increased DNA con- philic, nongranular, and scant in the blast stage (Stage I) tent contained within a single nuclear envelope. Polyploid to completely granular and acidophilic in the mature cells contain exact multiples of the normal DNA content stage. (normally 2N) and can range from 4N to 64N or higher. Endomitosis begins in megakaryoblasts and is completed by the end of stage II megakaryocytes. The 8N stage is gen- Table 9.1 Developmental Stages of Megakaryocytes erally the first morphologically recognizable stage on a bone marrow smear because by this stage, the megakaryocytes Name Characteristics are becoming significantly larger than the other cells in the Stage I Megakaryoblast 6–24 mcM diameter bone marrow. The 16N stage is the most common ploidy Scant basophilic cytoplasm stage in adult humans. No visible granules Round nucleus The purpose of endomitosis is for nuclear maturation Visible nucleoli and enlargement of the megakaryocyte cytoplasm. The Stage II Promegakaryocyte 14–30 mcM diameter cytoplasm generates the proteins and lipids necessary to (basophilic Increased cytoplasm, primarily produce sufficient membrane for the packaging of future megakaryocyte) basophilic platelets.19 Few visible azurophilic cytoplasmic granules Cytoplasmic Maturation Cytoplasmic maturation can be Indented or bilobed nucleus initiated at nuclear ploidy levels of 8N or more, but the Beginning of demarcation mem- stage at which maturation occurs varies from cell to cell. branes (visible with electron microscopy) In general, nuclear maturation (ploidization) ceases when Stage III Granular 25–50 mcM diameter cytoplasmic maturation begins. Cytoplasmic maturation megakaryocyte Numerous cytoplasmic granules includes an increase in size, an increase in number of cyto- Abundant acidophilic cytoplasm skeletal elements, organelles, and granules, and develop- Large, multilobed nucleus ment of a complex membrane system—all of which are No visible nucleoli necessary for platelet production. Stage IV Mature 40–100 mcM diameter Megakaryocyte development occurs as a continuum megakaryocyte Abundant acidophilic, very granular but is arbitrarily divided into four stages as described on cytoplasm Romanowsky-stained bone marrow smears. The major cri- Demarcation zones present teria differentiating these stages include the quantity and Multilobulated nucleus characteristics of the cytoplasm and the size, lobulation, No visible nucleoli a b Figure 9.2 (a) Early megakaryocyte. (b) Mature megakaryocyte (bone marrow; Wright-Giemsa stain; 1000 * magnification). 168 Chapter 9 Developing megakaryocytes produce two types of platelet-specific secretory granules, A-granules (AGs) that are abundant and large, and dense granules (DGs) that are fewer in number and smaller than aGs. The cytoplasm in the early stages (Stage I and II) can have a few granules appear- ing in the region of the Golgi apparatus, whereas the cyto- plasm of the mature cell (Stage IV) appears completely filled with these azurophilic granules. The differential morphol- ogy and content of these granules is detailed in Chapter 31. a In addition to granules, the cytoplasm of a maturing megakaryocyte develops an internal membrane system of channels called the demarcation membrane system (DMS). The DMS is not visible by light microscopy but is first seen in electron micrographs at the promegakaryocyte stage. It is derived by invagination of the megakaryocyte’s outer membrane and eventually develops into a highly branched, interconnected system of channels that maintain Demarcation open communication with the extracellular space. As the membranes DMS becomes extensive, small areas of the megakaryocyte Expelled nucleus cytoplasm are compartmentalized (Figure 9-3a). These areas eventually become the platelets, and the DMS is thought to provide the material for the formation of proplatelet pro- b cesses (see “Thrombopoiesis”).20 The separated cytoplas- mic areas can be seen on the edges of the cell pictured in Figure 9-3b. Megakaryocytes also produce a second exten- Figure 9.3 (a) Mature megakaryocyte showing future platelets. (b) Platelet release from mature megakaryocyte. Outward extrusion sive membranous system, the dense tubular system (DTS), of cytoplasm, eventually breaking up into individual platelets which, unlike the DMS, does not communicate with the cell delineated by demarcation membrane system (arrow). Expelled surface. nucleus is phagocytized by marrow macrophages. 2N 4N 8N 16N 32N 1 Bone marrow 2 3 4 Blood Figure 9.4 Platelet production. Developing megakaryocytes generally undergo nuclear maturation (ploidization) prior to cytoplasmic development. Megakaryocytes at any ploidy level 8N or higher can undergo cytoplasmic maturation and platelet production. Platelets are first released between the endothelial cells of the marrow sinuses as proplatelets. Proplatelets break into mature platelets and are released into the peripheral blood. Maturation Amplification The Platelet 169 In the practical day-to-day evaluation of bone marrow specimens, distinguishing the maturation stages of mega- Peripheral Blood Platelets karyocytes is not necessary. It is, however, important to rec- Platelets are the smallest of the circulating hematologic ele- ognize a cell as being of the megakaryocyte lineage. ments. They are not truly “cells” but are membrane-bound anucleate fragments of cytoplasm derived from precursor Checkpoint 9.2 cells in the bone marrow, the megakaryocytes. Platelets What is the relationship between megakaryocyte ploidy are short-lived and circulate in the peripheral blood for level and eventual number of platelets produced from that 7–10 days; nonviable or aged platelets are removed by the megakaryocyte? spleen and liver. Platelet Morphology Thrombopoiesis On a Romanowsky-stained peripheral blood smear, platelets The primary site of megakaryocyte development (mega- appear as small, anucleate lavender-blue or colorless bod- karyopoiesis) and platelet production (thrombopoiesis) is ies with reddish-purple (azurophilic) granules (Figure 9-5). in the bone marrow. Mature megakaryocytes are typically They are generally 2–3 mcM (mm) in diameter, ∼0.5 mcM situated near the abluminal surface of the marrow sinus thick, and round to oval (discoid) in shape. Ultrastructur- endothelial cells and shed platelets directly into the mar- ally, mature platelets lack nuclear material and contain only row sinuses. Each megakaryocyte is estimated to give rise to remnants of a Golgi complex, a relatively small number of 1000–3000 platelets, depending on the ploidy of the parent ribosomes, and a small amount of mRNA.24 megakaryocyte.21 Megakaryocytes of ploidy of 8N or higher In improperly prepared peripheral blood smears, plate- can produce platelets (Figure 9-4). lets can appear in aggregates in some areas and appear Platelets form by fragmentation of megakaryocyte cyto- decreased or absent in others. These aggregates of platelets plasm. They appear to be released from membrane exten- are typically seen in the “feathered edge” of the peripheral sions of megakaryocytes in groups called proplatelets, blood smear. Aggregated platelets can be seen when the which are long slender protrusions of megakaryocyte cyto- blood is not mixed well with the anticoagulant after col- plasm (Figure 9-3b). Each megakaryocyte extrudes multiple lecting the sample, and platelet activation is initiated in cytoplasmic extensions between endothelial cells into the vitro. Platelets can also appear decreased due to platelet vascular sinuses as proplatelets, which then break up into satellitosis (platelet satellitism) when they adhere to neutro- individual platelets under the shear stress of the flowing phils (Chapter 10). blood. The megakaryocyte nucleus remains
in the marrow Unusually large platelets—megathrombocytes or giant and is engulfed by the marrow macrophages. Localized platelets (more than 5 mcM diameter)—are sometimes seen, apoptosis (caspase activation) is thought to play a role in particularly in myeloproliferative disorders (Chapter 24) the final stages of platelet formation and release.22,23 or during recovery from severe thrombocytopenias. Occa- Whole intact megakaryocytes occasionally are released sionally, a platelet overlies an erythrocyte on a peripheral from the marrow, circulate in the peripheral blood, and blood smear and can be mistaken for an erythrocyte inclu- become trapped in capillary beds in the spleen and lungs. sion. Differentiation is usually relatively easy because the These cells also can release platelets to the peripheral blood, although their contribution to total platelet production is thought to be only 7–15%.1 CASE STUDY (continued from page 166) 3. A bone marrow examination was ordered. What cells would you expect to see increased? Why? Would this affect the M:E ratio? 4. What cytokine normally stimulates the produc- tion of these cells? What is the receptor for this cytokine? 5. The patient was diagnosed with essential throm- bocythemia. What is the pathophysiology of this disorder? Figure 9.5 Peripheral blood smear. The arrows point to platelets (Wright-Giemsa stain; 1000* magnification). 170 Chapter 9 superimposed platelet has a halo surrounding it and dis- Platelet Function plays the classic morphologic characteristics of a normal platelet (e.g., azurophilic granules). Classically, platelets were first known for their role in hemostasis, the controlled process that arrests vascular Quantitative Platelet Evaluation bleeding after an injury. In addition, they have other rec- ognized functions in hemostasis, including their contribu- PLATELET COUNT tions to vascular integrity and vessel wall repair following Platelets circulate at a concentration of 1509400 * 109>L. injury. For over a century after their discovery, they were This number is an under-representation of the total num- considered mere mediators of hemostasis. However, plate- ber of platelets because normally only two-thirds of periph- lets are far more complex than originally thought. More eral blood platelets circulate and one-third is sequestered recently, studies have demonstrated that platelets also in the spleen. These two pools of platelets are in constant have important functions in inflammation and immune equilibrium. In the presence of hypersplenism or spleno- responses.26,27 megaly, the percentage of sequestered platelets can increase substantially, causing peripheral blood thrombocytopenia. HEMOSTASIS Platelet counts tend to be higher in women than men and Platelets are involved in several aspects of hemostasis, decline in both sexes after age 60.25 including maintaining vascular integrity, forming the pri- mary hemostatic plug, providing a surface for fibrin gen- PLATELET INDICES eration and secondary hemostasis, and promoting repair of Platelet indices (mean platelet volume [MPV] and p latelet the injured tissues. distribution width [PDW]) are parameters available on Platelets play a vital role in forming the primary some automated hematology analyzers (Chapter 10). hemostatic plug. Platelets normally circulate in the blood These indices correlate, to some degree, with platelet acti- vessels as discoid cell fragments that do not interact with vation and function. The MPV (reference interval 6.8–10.2 other platelets or cells. However, when there is blood vessel fL) generally shows an inverse correlation with platelet injury the platelet responses include adhesion (attachment) count. As a result, there is a relatively stable platelet mass to the vessel wall, activation that leads to shape change, (platelet count X MPV). The variation in platelet size can be release/secretion of granules, and formation of aggregates described by the PDW, a parameter analogous to the red that result in a hemostatic plug that seals the opening of the cell distribution width (RDW; Chapter 10). The reference vessel wall (Chapter 31). interval for the PDW is 9–15 fL. IMMUNE RESPONSE RETICULATED PLATELETS Platelet immune responses are diverse and contribute both Platelets newly released from the bone marrow contain to the innate and adaptive immune systems.28 Platelets higher amounts of RNA than mature platelets, similar to adhere to and interact with various leukocytes, including newly released reticulocytes (Chapter 5). These newly lymphocytes, neutrophils, monocytes, macrophages, and released immature platelets are called reticulated platelets dendritic cells.29 They have the capacity to synthesize and can be quantitated on some automated hematology cytokines, chemokines, and inflammatory mediators and instruments (Chapters 10 and 39). Analysis of reticulated thus modulate both inflammatory and immune platelets (sometimes referred to as the immature platelet responses.28 They form heterotypic aggregates with leuko- fraction) provides an estimate of bone marrow cytes and bacteria and secrete antimicrobial peptides.30,31 megakaryopoiesis. Activated platelets interact with neutrophils and trigger neutrophil extracellular trap (NET) formation, thus CASE STUDY (continued from page 169) enhancing the innate immune response.32,33 Platelets con- tribute to the adaptive (acquired) immune system by 6. Is the patient’s platelet count normal or abnormal? assisting with the trafficking, activation, and differentia- 7. Why do you think the spleen is enlarged? tion of all lymphocyte subpopulations, including T-lym- phocytes, B-lymphocytes, and natural killer cells.34 Checkpoint 9.3 A patient receiving chemotherapy has a postchemotherapy Checkpoint 9.4 platelet count of 75 * 109>L, with 24% reticulated platelets. Platelets have several physiologic functions, what are their func- Should the clinician be concerned about the low platelet count? tions in hemostasis and the immune response? The Platelet 171 Summary Peripheral blood platelets are produced by the process of One-third of the platelets entering the peripheral blood cytoplasmic fragmentation of precursor cells (megakaryo- from the marrow are sequestered in the spleen, and two- cytes) in the bone marrow. Megakaryocyte development is thirds freely circulate. Platelet parameters thought to have characterized by a unique form of mitosis (endomitosis) in clinical relevance in evaluating patients include the platelet which DNA synthesis occurs in the absence of cytoplasmic count, the mean platelet volume, the platelet distribution or nuclear division, resulting in large, polyploid cells. Four width, and the percentage of reticulated platelets, different maturation stages of developing megakaryocytes The classical function of platelets is the formation of a have been identified, but differentiation and quantitation hemostatic plug during primary hemostasis, which is the of these stages is generally not considered clinically signifi- first phase in the process of arresting bleeding. Platelets also cant. A number of cytokines control megakaryocyte and participate in secondary hemostasis and fibrinolysis (other platelet production; however, thrombopoietin is the major stages of hemostasis) as well. Newer research has led to the regulator of these processes. On average, 1000–3000 plate- discovery that platelets participate in both the innate and lets are shed from a single megakaryocyte. adaptive immune responses. Review Questions Level I c. They have abundant acidophilic cytoplasm filled with azurophilic granules and a single, large, 1. What bone marrow cell is the precursor of platelets? lobulated nucleus. (Objective 1) d. They are about the size of a marrow a. Myeloblast macrophage with a high N:C ratio and b. Erythroblast granular cytoplasm. c. Endothelial cell d. Megakaryocyte 5. The major cytokine responsible for regulating both megakaryocyte and platelet production is: 2. What is a reasonable reference interval for platelets in (Objective 3) the peripheral blood? (Objective 4) a. TPO a. 10920 * 109>L b. IL-11 b. 1509200 * 1012>L c. SCF c. 1509400 * 109>L d. IL-6 d. 4009600 * 109>L 6. Endomitosis is defined as: (Objective 2) 3. Which of the following best describes the normal morphology of platelets on a peripheral blood smear? a. cell proliferation endogenous to the bone marrow (Objective 4) b. fragmentation of megakaryocyte cytoplasm to produce platelets a. They are larger than erythrocytes. c. fusion of megakaryocyte progenitor cells to b. They are filled with azurophilic granules. produce polyploidy megakaryocytes c. They are light blue in color without granules. d. rounds of DNA synthesis without nuclear division d. They have large nuclei. or cellular division 4. Which of the following best describe the a ppearance 7. All of the following are considered functions of of mature megakaryocytes in the bone marrow? platelets except: (Objective 5) (Objective 1) a. forming hemostatic plug a. They are large cells with scanty basophilic cytoplasm (high N:C ratio). b. vascular wall repair b. They are large cells with multiple discrete nuclei, c. phagocytosis of bacteria basophilic cytoplasm. d. assist in immune response 172 Chapter 9 8. What is the normal life span of platelets in the periph- one week, the platelet count remained low, and eral blood? (Objective 4) the number of reticulated platelets was above the reference interval for that parameter. This would a. 8 hours likely indicate that the: (Objective 5) b. 1 day a. patient’s bone marrow is failing to initiate recovery c. 10 days of the thrombocytopenia d. 100 days b. patient is a candidate for erythropoietin Level II c. patient should receive a blood transfusion 1. Which of the following statements best describes d. the patient’s marrow is showing early signs of the role of the platelet parameter MPV in evaluating recovery of the thrombocytopenia patients? (Objective 4) 5. What effect does thrombopoietin have on the a. MPV is a theoretical construct and we do not yet megakaryocyte? (Objective 3) have the capacity to measure it. a. It decreases the ploidy level and number of b. MPV is measurable but not on current laboratory platelets formed. hematology instrumentation. b. It increases size, DNA ploidy level, and rate of c. MPV can be measured, but its clinical significance maturation. is unknown. c. It induces the release of megakaryocytes to the d. MPV can be measured and may contribute to the peripheral blood. differential diagnosis of platelet disorders. d. It decreases the rate of endomitosis and increases 2. Place the following megakaryocyte precursor cells in ploidy level. the correct developmental sequence: (Objective 1) 6. What is the effect of a significant number of giant a. BFU-Mk S CFU-Mk S megakaryoblast S platelets (megathrombocytes) resulting in an granular megakaryocyte increased MPV on the peripheral blood platelet b. CFU-Mk S BFU-Mk S basophilic count? (Objective 4) megakaryocyte S granular megakaryocyte a. It would have no effect on the platelet count. c. BFU-Mk S CFU-Mk S basophilic megakaryocyte S megakaryoblast b. It would generally be associated with an increased platelet count. d. CFU-Mk S BFU-Mk S megakaryoblast S basophilic megakaryocyte c. It would generally be associated with a decreased platelet count. 3. Which of the following is believed to be the d. It would result in an unreliable reading on the mechanism of platelet formation and release by automated instrument and require a manual megakaryocytes? (Objectives 1, 2) platelet count. a. Dissolution of the megakaryocyte cell membrane 7. Which statement concerning TPO levels is true? and release of preformed platelets (Objective 3) b. Release of individual reticulated platelets from the cytoplasm a. The higher the platelet count the less free TPO in the plasma. c. Formation of proplatelet processes, which subsequently segment into individual platelets b. When the platelet count decreases, there is less TPO to bind to megakaryocytes in the bone d. Apoptotic cell death of the megakaryocyte and the marrow. disintegration of the cytoplasm as platelets c. TPO levels are stable and unrelated to the platelet 4. A patient receiving chemotherapy experienced severe count. bone marrow suppression and anemia, leukopenia, d. TPO has no effect on the baseline number of and thrombocytopenia as a result. After about platelets in the peripheral blood. The Platelet 173 References 1. Kaushansky, K. (2016). Megakaryopoiesis and thrombopoiesis. 17. Cramer, E. M. (1999). Megakaryocyte structure and function. In: K. Kaushansky, M. A. Lichtman, J. T. Prchal, M. M. Levi, Current Opinion in Hematology, 6(5), 354–361. O. W. Press, L. J. Burns, M. Caligiuri, eds. Williams hematology 18. Vitrat, N., Cohen-Solal, K., Pique, C., Le Couedic, J.P., Norol, F., (13th ed., pp. 1815–1828). New York: McGraw-Hill. Larsen, A. K., . . . Debili, N. (1998). Endomitosis of human mega- 2. Cantor, A. B. (2013). Thrombopoiesis. In: R. Hoffman (Author) & karyocytes are due to abortive mitosis. Blood, 91(10), 3711–3723. E. J. Benz, L. E. Silberstein, H. Heslop, J. Weitz, & J. Anastasi, 19. Machlus, K. R., & Italiano, J. E. (2013). The incredible journey: eds. Hematology: Basic principles and practice (6th ed., From megakaryocyte development to platelet formation. Journal pp. 292–306). Philadelphia: Elsevier. of Cell Biology, 201(6), 785–796. 3. Gewirtz, A. M. (1986). Human megakaryocytopoiesis. Seminars in 20. Italiano, J. E., & Shivdasani, R. A. (2003). Megakaryocytes Hematology, 23(1), 27–42. and beyond: The birth of platelets. Journal of Thrombosis and 4. Wendling, F.
(1999). Thrombopoietin: Its role from early hemato- Haemostasis, 1(6), 1174–1182. poiesis to platelet production. Haematologica, 84(2), 158–166. 21. Harker, L. A., & Finch, C. A. (1969). Thrombokinetics in man. 5. Broudy, V. C., Lin, N. L., & Kaushansky, K. (1995). Thrombopoi- Journal of Clinical Investigation, 48(6), 963–974. etin (c-mpl ligand) acts synergistically with erythropoietin, stem 22. Li, J., & Kuter, D. J. (2001). The end is just the beginning: cell factor, and interleukin-11 to enhance murine megakaryocyte Megakaryocyte apoptosis and platelet release. International colony growth and increases megakaryocyte ploidy in vitro. Journal of Hematology, 74(4), 365–374. Blood, 85, 1719–1726. 23. De Botton, S., Sabri, S., Daugas, E., Zermati, Y., Guidotti, J. E., 6. Sungaran, R., Markovic, B., & Chong, B. H. (1997). Localization Hermine, O., . . . Debili, N. (2002). Platelet formation is the and regulation of thrombopoietin mRNa expression in human consequence of caspase activation within megakaryocytes. Blood, kidney, liver, bone marrow, and spleen using in situ hybridiza- 100(4), 1310–1317. tion. Blood, 89(1), 101–107. 24. Smyth, S. S., Whiteheart, S., Italiano, J. E., Bray, P. & Coller, B. S. 7. Kaushansky, K. (1995). Thrombopoietin: The primary regulator of Platelet morphology, biochemistry, and function. In: platelet production. Blood, 86(2), 419–431. K. Kaushansky, K. Lichtman, J. T. Prchal, M. M. Levi, O. W. 8. Lok, S., Kaushansky, K., Holly, R. D., Kuijper, J. L., Lofton-Day, C., Press, L. J. Burns, M. Caligiuri, eds. Williams hematology (13th ed., Oort, P. J., . . . Kramer, J. M. (1994). Cloning and expression pp. 1829–1914). New York: McGraw-Hill. of murine thrombopoietin cDNA and stimulation of platelet 25. Bennett, J. S., & Abrams, C. S. (2012). Differential diagnosis of production in vivo. Nature, 369(6481), 565–568. thrombocytopenia. In: V. J. Marder, W. C. Aird, J. S. Bennett, S. 9. Kuter, D. J. (2009). Thrombopoietin and thrombopoietic mimetics Schulman, & G. C. White II, eds. Hemostasis and thrombosis: Basic in the treatment of thrombocytopenia. Annual Review of Medicine, principles and clinical practice (6th ed., pp. 757–762). Philadelphia: 60, 193–206. Lippincott, Williams & Wilkins. 10. Wei, P. (2011). Thrombopoietin factors. Cancer Treatment and 26. Li, C., Li, J., Li, Y., Lang, S., Yougbare, I., Zhu, G., . . . Ni, H. (2012). Research, 157, 75–93. Crosstalk between platelets and the immune system: Old s ystems 11. Kuter, D. J., & Rosenberg, R. D. (1995). The reciprocal relationship with new discoveries. Advances in Hematology, 2012, 384685. of thrombopoietin (c-mpl ligand) to changes in the platelet mass Retrieved from http://ezproxy.uky.edu/login?url=http:// during busulfan-induced thrombocytopenia in the rabbit. Blood, search.proquest.com.ezproxy.uky.edu/docview/1080653782? 85(10), 2720–2730. accountid=11836. 12. Emmons, R. V., Reid, D. M., Cohen, R. L., Meng, G., Young, N. S., 27. Morrell, C. N., Aggrey, A. A., Chapman, L. M., & Modjeski, K. L. Dunbar, C. E., & Shulman, N. R. (1996). Human thrombopoietin (2014). Emerging roles for platelets as immune and inflammatory levels are high when thrombocytopenia is due to megakaryocyte cells. Blood, 123(18), 2759–2767. deficiency and low when due to increased platelet destruction. 28. Herter, J. M., Rossaint, J., & Zarbock, A. (2013). Platelets Blood, 87, 4068–4071. in inflammation and immunity. Journal of Thrombosis and 13. McCarty, J. M., Sprugel, K. H., Fox, N. E., Sabath, D. E., & Haemostasis, 12, 764–775. Kaushansky, K. (1995). Murine thrombopoietin mRNA levels are 29. Duerschmied, D., Bode, C., & Ahrens, I. (2014). Immune modulated by platelet count. Blood, 86, 3668–3675. functions of platelets. Thrombosis and Haemostasis, 112, 678–691. 14. Lam, L. T., Ronchini, C., Norton, J., Capobianco, A. J., & 30. Hui, H., Fuller, K., Erber, W. N., & Linden, M. D. (2015). Bresnick, E. H. (2000). Suppression of erythroid but not Measurement of monocyte-platelet aggregates by imaging flow megakaryocytic differentiation of human K562 erythroleukemic cytometry. Cytometry, 87, 273–278. cells by notch-1. Journal of Biological Chemistry, 275, 19676–19684. 31. Kerrigan, S. W. (2015). The expanding field of platelet-bacterial 15. Hodohara, K., Fujii, N., Yamamoto, N., & Kaushansky, K. interconnections. Platelets, 26, 293–301. (2000). Stromal cell-derived factor-1 (SDF-1) acts together with 32. Rondina, M. T., Weyrich, A. S., & Zimmerman, G. A. (2013). thrombopoietin to enhance the development of megakaryocytic Platelets as cellular effectors of inflammation in vascular progenitor cells (CFU-MK). Blood, 95(3), 769–775. diseases. Circulation Research, 112, 1506–1519. 16. Wang, J. F., Liu, Z. Y., & Groopman, J. E. (1998). The alpha- 33. Semple, J. W., Italiano, J. E., & Freedman, J. (2011). Platelets and chemokine receptor CXCR4 is expressed on the megakaryocytic the immune continuum. Nature Reviews Immunology, 11, 264–274. lineage from progenitor to platelets and modulates migration 34. Nailin, L. (2008) Platelet-lymphocyte cross-talk. Journal of and adhesion. Blood, 92(3), 756–764. Leukocyte Biology, 83, 1069–1078. Chapter 10 The Complete Blood Count and Peripheral Blood Smear Evaluation Kristin Landis-Piwowar, PhD John Landis, MS Keila Poulsen, BS, was an author for the third edition of this text and much of the information was retained. Objectives—Level I At the end of this unit of study, the student should be able to: 1. List the parameters typically included in the using low and high power magnification complete blood count (CBC). microscope settings. 2. Describe how to properly identify a patient 8. Define poikilocytosis and anisocytosis, and prior to blood collection. describe and identify specific poikilocytes 3. List the pre-examination precautions that and anisocytes. must be observed to produce quality results 9. Classify erythrocytes morphologically based when performing a CBC. on erythrocyte indices. 4. List the typical units of measure for 10. Recognize non-malignant variations in reporting the WBC, RBC, PLT, Hb, Hct, WBCs. and reticulocyte count. 11. Given the relative reticulocyte count 5. Define the terms MCV, MCH, MCHC, RDW, and RBC count, calculate the absolute MPV, and PDW. reticulocyte count. 6. Given the hemoglobin, hematocrit, and RBC 12. Identify erythrocyte inclusions; count, calculate the RBC indices. describe their composition and staining 7. Describe the process of evaluating characteristics. the peripheral blood smear including 13. Recognize abnormal variation in macroscopically and microscopically erythrocyte distribution on stained smears. 174 The Complete Blood Count and Peripheral Blood Smear Evaluation 175 14. Correlate polychromatophilia on a blood 16. Describe the role of the laboratory smear with other laboratory results of professional in the post-examination erythrocyte production and destruction. phase of the CBC. 15. Describe the role of the laboratory 17. Describe the variations in the CBC reference professional in the examination phase intervals found in African Americans, of the CBC. newborns, and elderly people. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Interpret RDW results. 5. Correlate CBC and reticulocyte results with 2. Assess bone marrow response to findings on a blood smear and troubleshoot anemia given CBC and reticulocyte any discrepancies. results. 6. Select methods for differentiating 3. Assess bone marrow response to erythrocyte inclusions. thrombocytopenia given CBC and 7. Evaluate distribution of erythrocytes on reticulated platelet results. stained smears, and describe how the 4. Identify poikilocytes on a blood smear, distribution could affect the CBC results. describe their mechanism of formation, 8. Correlate the age of a patient and the t ypical and correlate them with pathological changes in CBC results compared with a conditions. normal adult CBC. Chapter Outline Objectives—Level I and Level II 174 Examination Phase of the CBC 177 Key Terms 175 Post-Examination Phase of the CBC 194 Background Basics 176 Physiologic Variation in Hematologic Case Study 176 Parameters 194 Overview 176 Summary 195 Introduction 176 Review Questions 195 Pre-Examination Phase of the CBC 177 References 197 Key Terms Acanthocyte Critical value Hematocrit Agglutination Dacryocyte Howell-Jolly body Anisocytosis Delta check Hypochromic cell Basophilic stippling Drepanocyte Immature platelet fraction (IPF) Cabot ring Echinocyte Keratocyte Codocyte Elliptocyte Knizocyte Critical area Heinz body Leptocyte Critical limit Helmet cell Macrocyte 176 Chapter 10 Mean cell hemoglobin (MCH) Ovalocyte Pappenheimer body Schistocyte Mean cell hemoglobin Platelet distribution width (PDW) Sickle cell concentration (MCHC) Poikilocytosis Sideroblast Mean cell volume (MCV) Red cell distribution width (RDW) Siderocyte Mean platelet volume (MPV) Reflex testing Spherocyte Microcyte Reticulated platelets Stomatocyte Normochromic Rouleaux Target cell Normocytic Rule of three Teardrop Background Basics The information in this chapter builds on the concepts • Identify the reference intervals for each subclass of learned in Chapters 4–9. In addition to the basics from leukocytes and for platelets. (Chapters 7–9) previous chapters, it is helpful to have a basic under- standing of algebra, the use of percentages, ratios and Level II proportions, statistics, and the metric system. To maxi- • Correlate the relationships between various mize your learning experience, you should review these infectious agents and absolute and relative leukocyte concepts before starting this unit of study: counts. (Chapters 7, 8) • Predict the effects of increased and decreased Level I erythropoietin in the blood. (Chapters 5, 6) • Describe the basic concepts of cell differentiation and • Summarize the role of thrombopoietin in platelet maturation. (Chapter 4) production. (Chapter 9) • Identify the reference intervals for erythrocytes, reticulocytes, and hemoglobin. (Chapters 5, 6) prepared blood smear. Finally, a brief synopsis of the post- CASE STUDY examination characterization of the complete blood count We refer to this case study throughout the chapter. is included. The classification of disease as it is determined A 48-year-old woman was admitted to a hospital from laboratory findings is detailed in subsequent chapters because of wheezing, cough, fatigue, and d ifficulty of this book. breathing. Her history included diabetes and hypertension. Her CBC data showed a h emoglobin of 8.2 g/dL, RBC count of 73 * 106>mcL, and Introduction MCV = 109.8 fL. Her WBC count and platelet The performance of a complete blood count (CBC) has count were within the reference intervals. three phases: pre-examination (before testing), e xamination Consider the actions that the clinical laboratory (testing), and post-examination (reporting) (Table 10-1). professional should take. The pre-examination phase includes the proper identifica- tion of the patient and proper collection and handling of the specimen. The specimen is analyzed in the examination Overview phase. The CBC (sometimes referred to as the peripheral blood count) is a primary screening test that provides infor- This chapter unites the classes and characteristics of blood mation regarding the cellular components of the blood as cells that were described in Chapters 4–9 into a clinically rel- they circulate in the peripheral blood. The concentration evant “blood picture” of those cells as they circulate in the of leukocytes (white blood cell [WBC]), erythrocytes (red peripheral blood. A brief discussion of the pre-examination blood cell [RBC]), and platelets (PLT) as well as a categori- steps that provide the foundation for any routine hemato- zation of the different WBC subsets is included. Additional logic evaluation precedes a more in-depth examination of information regarding RBCs is also integrated into the blood cell counts and composition and visual features on a CBC report and includes, at minimum, the concentration The Complete Blood Count and Peripheral Blood Smear Evaluation 177 Proper test sample transport is of great concern within Table 10.1 Phases of the CBC a clinical facility and is even more important when samples Pre-examination Patient identification are brought in from locations outside the facility. Samples Specimen collection, handling, and transport from outside facilities must be delivered in a manner that Examination Automated results complies with limited temperature variations and time Evaluation and analysis of peripheral blood smear restraints. Freezing or excessive heat will damage the blood Post-examination Interpretation of data cells and render analysis invalid. Although microscopic Reporting results evaluation of a blood smear is best when the slide is pre- pared within 3 hours of collection, the instrument analysis can be delayed for 6–8 hours without deterioration of the of hemoglobin and the packed cell volume of RBCs, called data.3 Some parameters are valid for up to 24 hours after the hematocrit. Finally, a CBC provides the RBC indices that sample collection if properly stored. Often the hematology are used to depict the cell volume and the total weight and instrument’s manufacturer provides the recommended concentration of hemoglobin in an average RBC. The CBC time points for performing the automated CBC to ensure can be performed by automated and/or manual methods, that the analysis produces data for a patient of the high- which are described in detail in Chapters 37 and 39. The est quality. The information generated from manual and post-examination phase includes reporting and interpret- instrument analysis is only as good as the specimen that is ing the data.
Based on the information collected from the tested. Therefore, the pre-examination stage of testing is of CBC, the laboratory professional can provide d iagnostic primary importance. Sources of error in sample collection information or meaningful recommendations for any and their effect on test outcome are detailed in Table 43-2 follow-up testing (reflex testing) to the physician that sup- in Chapter 43. port quality patient care. Checkpoint 10.1 A courier service delivers a tube of blood from an outpatient Pre-Examination Phase drawing station across town. The requisition for the sample is for a CBC. The blood is in a blue top vacutainer tube (contain- of the CBC ing 3.2% sodium citrate) and was transported in an insulated box containing dry ice. What are the next steps in processing The pre-examination phase of the CBC encompasses this sample? patient identification, blood collection, and specimen han- dling (Chapters 37 and 43). Briefly, patient identification includes the patient’s name and a second identifier that can be a hospital number but more commonly is the patient’s Examination Phase date of birth. This documentation must be available from the patient at the time of the blood collection. of the CBC Once the patient has been properly identified, the labo- ratory professional performs a procedure called a phlebot- Automated Results omy to collect the blood sample. The phlebotomist must be Laboratory professionals use automated instruments to well acquainted with the various collection devices, their determine the CBC information for the majority of patient safety features and requirements, and the anticoagulants or samples (Chapter 39). Proper instrument preparation additives within the sample collection tubes (Chapter 37). includes quality control and assessment to confirm the This individual should have a thorough knowledge of the normal function of the instrument. Commercial controls, phlebotomy procedure safety issues including methods to patient controls, or moving averages are used to deter- prevent exposure to blood-borne pathogens. mine the analytical reliability of the automated instru- Although other blood collection tube additives can be ment (Chapter 43). Once the analytical reliability of the used for hematologic analysis, almost all specimens for a instrument has been confirmed, examination of patient routine CBC are collected in a purple-/lavender-top sample samples can begin. Throughout the examination phase, lab- collection tube that contains ethylenediaminetetraacetic acid oratory professionals must follow safety protocols designed (EDTA). Dipotassium (K2) EDTA provides the best morpho- to minimize risk of exposure to biohazards, chemical haz- logic preservation of blood cells and prevents coagulation of ards, and physical hazards and dispose of biological waste the blood specimen.1 The anticoagulated specimen allows a appropriately (Chapter 43). laboratory professional to generate multiple blood smears Each hematology instrument uses different but from one tube of blood, a technique that must be performed overlapping technologies (e.g., impedance, optical within 3 hours of blood collection.2 light scattering) to generate data on the RBCs, WBCs, 178 Chapter 10 and platelets produced by the hematopoietic system international system of units (Systeme International Units/ (Table 10-2). Because other organs and organ systems also SI units), the WBC, RBC, and PLT counts are reported as affect hematopoiesis and the circulating blood cells, a myr- the number of cells per liter; using “conventional” units iad of disease states can be evaluated from CBC data to (traditionally used in this country prior to the introduction determine diagnosis, treatment, and prognosis for a patient. of SI units) the WBCs and PLTs are reported as thousands of In addition to the typical CBC parameters, hematology cells per microliter (103>mcL); and the RBCs are reported instruments generate scatterplots and histograms (refer to as millions of cells per microliter (106>mcL). The hemato- Chapter 39 for detailed discussion) that laboratory profes- crit measures the volume the RBCs occupy within whole sionals interpret. Each scatterplot and histogram contains blood and is reported as a percentage (%) or as the volume information about the cell populations, interfering sub- of RBCs in liters per volume of whole blood in liters (L/L) stances, and instrument function and therefore serve as using SI units (Chapter 37). In automated analyzers, the forms of quality control for instrument and specimen integ- hematocrit is usually calculated from the measured MCV rity. For example, the laboratory professional consults the and RBC count using the following formula (Chapter 39): scatterplots and histograms to assess the volume of granu- MCV (fL) * RBC count (1012>L) locytes (increased volume indicates immaturity or nuclear Hct (L>L) = hypersegmentation whereas decreased volume of lympho- 1000 cytes can indicate chronic lymphocytic leukemia). The scat- Hct (%) = MCV (fL) RBC count (106>mcL) terplots and histograms are also helpful in assessing RBC 10 parameters that can be affected by conditions such as cold The hemoglobin is measured spectrophotometrically agglutination (RBC clumping at temperatures below body after it has been released from lysed erythrocytes. It is temperature) and a severely elevated WBC count. Tables reported in grams per deciliter or grams per liter. 43-8 and 43-9 in Chapter 43 provide detailed information The laboratory professional interprets the accuracy of on abnormal and spurious results, underlying problems, the RBC count, hematocrit, and hemoglobin values using a causes, and corrective actions. Hematology reference inter- quick mathematical check called the rule of three. Simply, vals for the various age groups can be found on the inside the RBC count 3 = hemoglobin * 3 = hematocrit (%). of the front cover of this textbook. If the calculated values do not agree within {3% of the measured values, a measurement error or instrument mal- LEUKOCYTE COUNT, ERYTHROCYTE COUNT, PLATELET function could have occurred, or the patient could have a COUNT, HEMATOCRIT, AND HEMOGLOBIN pathology that requires investigation. Potential underlying The leukocyte count (WBC count), erythrocyte count problems and interfering substances associated with such a (RBC count), platelet count (PLT count), hematocrit (Hct), mismatch are described in Tables 43-8 and 43-9 in C hapter 43. and hemoglobin (Hb) are determined using automated It is important to recognize that the rule of three only works instrumentation (see Table 10-2 and Chapter 39). Using the when the RBCs are normal in size and hemoglobin content. Table 10.2 Parameters and Reference Intervals of a Typical Adult CBCa White blood cell (WBC) count 4.5911.0 * 103>mcL (4.5911.0 * 109>L) WBC Differential Relative (%) Absolute * 103>mcL (109>L) Red blood cell (RBC) count 4.095.5 106>mcL14.095.5 * 1012>L2 Neutrophils 40–80 1.8–7.0 Hemoglobin (Hb) 12.0–17.4 g/dL (120–174 g/L) Lymphocytes 25–35 1.0–4.8 Hematocrit (Hct) 36–52% (0.36–0.52 L/L) Monocytes 2–10 0.1–0.8 Mean cell volume (MCV) 80–100 fL Eosinophils 0–5 0–0.4 Mean cell hemoglobin (MCH) 28–34 pg Basophils 0–1 0–0.2 Mean cell hemoglobin 32–36 g/dL concentration (MCHC) Red cell distribution width (RDW) 11.5–14.5% Reticulocyte count 0.5–2.0% Relative (%) 25975 * 103>mcL (25975 * 109>L) Absolute (109>L) Platelet (PLT) count 1509400 103>mcL (1509400 * 109>L) Mean platelet volume (MPV) 6.8–10.2 fL Platelet distribution width (PDW) 9–15 Fl aMale and female reference intervals are combined. For age- and sex-specific reference intervals, see the front cover of this textbook. Additional parameters are dependent upon instrumentation (Chapter 39). Data are shown as conventional units, with the International system of units in parentheses (SI units). The Complete Blood Count and Peripheral Blood Smear Evaluation 179 A diurnal variation in blood cell concentration occurs in which the value for the WBC count is lowest in Table 10.3 Classification of Erythrocytes Based on MCV the morning and highest in the afternoon, whereas the Terminology Description RBC count, Hct, and Hb are just the opposite: higher values Normocytic 80.0–100.0 fL are observed in the morning.4 Microcytic Red cells with a reduced volume (less than 80 fL) The Hb (Macrocytic) Red cells with an increased volume (greater than Checkpoint 10.2 100 fL) The results on a blood specimen drawn from a patient in the Anisocytosis Increased variation in the range of red cell sizes doctor’s office and transported to the hospital laboratory were: Hb 15 g/dL, Hct 35%, and RBC 2.8 * 106> mcL. Should these results be reported? What should be the next step? EXAMPLE ERYTHROCYTE INDICES The erythrocyte indices help classify the erythrocytes A patient has an Hct of 45% and an RBC count of 5.0 * 106> mcL. by their size and hemoglobin content (Chapter 37). Hemoglobin, hematocrit, and erythrocyte count values 0.45 * 1000 90.0 fL = are used to calculate the three indices: mean cell volume 5 (MCV), mean cell hemoglobin (MCH), and mean cell 45 * 10 90.0 fL = hemoglobin concentration (MCHC) (Table 10-2). When 5 The value, 90.0 fL, indicates that the cell has a volume calculating the indices, it is important to note that the con- that falls within the reference interval (80–100 fL) and is version factors used in the formulas vary depending on the therefore classified as normocytic. use of conventional units or SI units for hemoglobin and hematocrit. Table 37-9 in Chapter 37 provides both con- version factors. These indices suggest how the RBCs will appear microscopically and provide significant diagnostic information (most commonly for the diagnosis of anemias). Mean Cell Hemoglobin The MCH is a measurement of the average weight of hemoglobin in picograms, 10-12 Laboratory professionals correlate the indices with the Hct, g, in Hb, and RBC count to ensure that technical problems are individual erythrocytes. The MCH is calculated from the identified when they occur (see Tables 43-8 and 43-9 in hemoglobin and erythrocyte count. Chapter 43 for factors that falsely increase or decrease the Hb (g>dL) RBC indices). MCH (pg) = RBC (1012>L) Mean Cell Volume The MCV denotes the average vol- Hb (g>dL) 10 MCH (pg) = ume of individual erythrocytes and is expressed in RBC (106>mcL) femtoliters (fL, 10-15L). It is measured by automated instrumentation (Chapter 39) and can be calculated from the Hct and RBC count. EXAMPLE Hct (L>L) 1000 MCV (fL) = RBC count (* 1012>L) A patient has an Hb concentration of 15 g/dL and an RBC count of 5.0 * 106> mcL. Hct (%) * 1000 MCV (fL) = RBC count (106>mcL>10) 15 10 30 pg = 5 The MCV is used to classify cells as normocytic, The value, 30.0 pg, indicates that the RBCs contain an microcytic, or macrocytic (Table 10-3) and usually cor- average weight of hemoglobin that is within the reference relates with the appearance of cells on stained blood interval (28.0–34.0 pg). smears (i.e., cells with an increased MCV appear larger [macrocytic], and cells with a decreased MCV appear smaller [microcytic]). However, it must be remembered The MCH does not take into account the size of a cell; it that the MCV is a measurement of volume, whereas esti- should not be interpreted without taking into consideration mation of the size of flattened cells on a blood smear is the MCV since the MCH varies in a direct linear relationship a measurement of cell diameter. Cell diameter and cell with the MCV. Cells with less volume typically contain less volume are not the same and may not always correlate hemoglobin while cells with larger volume typically contain with each other. more hemoglobin. 180 Chapter 10 Mean Cell Hemoglobin Concentration The MCHC indi- Red Cell Distribution Width Because the MCV repre- cates the concentration of hemoglobin in the general cell sents an average of erythrocyte volume, it is less r eliable in population and is described by the suffix -chromia, meaning describing the erythrocyte population when c onsiderable color (Table 10-4). Cells can be classified morphologically variation in erythrocyte volume/size (anisocytosis) occurs. as hypochromic if the area of central pallor is greater than The red cell distribution width (RDW) (Table 10-2) is the 1/3 of the cell size. The term hyperchromic should be used coefficient of variation of the MCV and may be referred sparingly (if ever). to as the RDW-coefficient of variation (RDW-CV). The for- The only erythrocyte that is hyperchromic with mula for the RDW-CV, a calculated index from hematology an MCHC of more than 36.0 g/dL is the spherocyte. instruments that helps identify anisocytosis, follows: Spherocytes have a decreased surface-to-volume ratio due 1 standard deviation (SD) of MCV to a loss of membrane but have not lost an appreciable RDW - CV = * 100 MCV amount of their hemoglobin. Spherocytes (discussed later in this chapter and in Chapters 17 and 19) usually have a Increased RDW values (more than 14.5%) indicate normal or only slightly
decreased volume (MCV), but on a an increase in the heterogeneity of erythrocyte size. No stained smear, they cannot flatten as much as normal eryth- known abnormalities are represented by a decreased RDW. rocytes because of a decreased surface area and increased Caution must be used in interpreting the RDW-CV rigidity. Spherocytes, therefore, often appear to have a because it reflects the ratio of the standard deviation of cell smaller diameter than normal cells, and there will be no cen- volume and the MCV. An increased SD (heterogeneous cell tral pallor present. On the other hand, codocytes (discussed population) with a high MCV can give a normal RDW-CV. later in this chapter and in Chapter 11) can appear larger Conversely, a normal standard deviation (homogenous cell due to an increased diameter, but the MCV is often normal. population) with a low MCV can give an increased RDW-CV. Generally, abnormalities in the MCV are clues to disease Examination of the erythrocyte histogram and stained blood processes that involve or affect the hematopoietic system. smear gives clues as to the accuracy of the RDW-CV in these The MCHC is the ratio of hemoglobin mass to the vol- cases. When the standard deviation is increased, indicat- ume in which it is contained (i.e., average concentration ing a true variability in cell size, the base of the erythrocyte of hemoglobin in a deciliter of erythrocytes, expressed in histogram is broader than usual. Because of this interpreta- g/dL). The MCHC is calculated from the Hb and Hct. tion issue, automated instruments often report the RDW-CV and RDW-standard deviation (RDW-SD). The RDW-SD is Hb (g>dL) * 100 MCHC (g>dL) = directly measured and not affected by the MCV (detailed Hct (%) information in Chapter 39). H (g>L) MCHC (g> b dL) = Hct (L>L) * 10 CASE STUDY (continued from page 176) Table 10.4 Classification of Erythrocytes Based on 1. Calculate the Hct, MCH, and MCHC from the MCHC initial results. Normochromic 32.0–36.0 g/dL 2. Evaluate the calculated Hct, MCH, and MCHC Hypochromic Less than 32.0 g/dL as compared with the reference intervals for a Hyperchromic More than 36.0 g/dL 48-year-old female. RETICULOCYTE COUNT EXAMPLE Immature, anuclear erythrocytes containing organelles and residual ribosomes for hemoglobin synthesis are known as A patient has an Hb concentration of 15 g/dL (150 g/L) and reticulocytes (Chapter 5). These cells usually spend 2–3 days an Hct of 45% (0.45 L/L). in the bone marrow and an additional day in the periph- eral blood before their RNA is degraded and they become 15 * 100 33.3 g>dL = 45 mature erythrocytes. The peripheral blood reticulocyte 150 count indicates the degree of effective bone marrow activity 33.3 g>dL = 0.45 * 10 and is one of the most useful and cost-effective laboratory The value, 33.3 g/dL, reveals that the cells contain a tests in monitoring response to therapy and pathophysiol- normal concentration of hemoglobin (32.09 36.0 g> dL) and ogy of anemia (Chapters 11 and 37). are therefore normochromic. The absolute reticulocyte count is a more informa- tive index of erythropoietic activity than the relative The Complete Blood Count and Peripheral Blood Smear Evaluation 181 reticulocyte count (Table 10-2). When reported as a percent- Platelet Distribution Width The variation in platelet size, age, the reticulocyte count does not indicate the relation- seen in the peripheral blood, can be described by the PDW, ship between the peripheral blood erythrocyte mass and a parameter analogous to the red cell distribution width the number of reticulocytes being produced. The reticulo- (RDW). The PDW is not a Food and Drug Administration– cyte count reported as a percentage can appear increased approved parameter for any of the hematology analyzers at because of either an increase in the number of reticulocytes the time of this book’s publication. in the circulation or a decrease in the number of total RBCs. Reticulated Platelets Some automated hematology instru- Therefore, it is recommended that in addition to the per- ments detect young or immature platelets called reticulated centage of reticulocytes, laboratory professionals report platelets. These reticulated platelets are analogous to RBC the absolute reticulocyte count to provide a more useful reticulocytes and are assessed by flow cytometry using estimate of reticulocyte production. Automated analyzers RNA-binding fluorescent dyes (Chapter 39). Typically, 0–4% can provide the absolute count; or it can be calculated when of all circulating platelets are reticulated6 and represent the using manual methods for reticulocytes: immature platelet fraction (IPF). Analysis of reticulated Absolute reticulocyte (103>mcL) = platelets provides a non-invasive estimate of bone marrow RBC count 1106>mcL2 * Reticulocyte (%) megakaryocytopoiesis (Chapter 9). WBC DIFFERENTIAL The WBC differential is an analysis and enumeration of the EXAMPLE various subtypes of WBCs (Chapter 7). An altered concen- tration of one specific type of leukocyte most commonly A patient has an RBC count of 3.5 * 106>mcL and a 10% causes an increase or decrease in the total WBC count. reticulocyte count. For this reason, an abnormal total WBC count should be 3.5 * 106>mcL * 0.1 = 0.35 * 106>mcL or 350 * 103> followed by a WBC differential, also known as a diff. The mcL WBC differential (Table 10-2) is performed by automated The value 350 * 103>mcL represents an increase in instruments or manually. A detailed discussion of the auto- reticulocyte production since the reference interval upper mated WBC differential is found in Chapter 39. The dif- value is 75 * 103>mcL (Table 10-2). ferential results are reported as the percentage of each cell type counted. To accurately interpret whether an increase or decrease in cell types exists, the absolute concentration PLATELET INDICES of each cell type is calculated using the results of the WBC The laboratory professional uses both the platelet count count and the differential (see Chapter 7 for the calculation and platelet indices to assess thrombopoiesis and patho- for absolute WBC values). logic c onditions related to platelets (Chapters 9 and 33). A decreased platelet count generally represents decreased thrombopoiesis, increased platelet destruction, or platelet Checkpoint 10.3 consumption. In contrast, reactive or malignant condi- A CBC was performed on a blood specimen from a 15-year-old tions can cause an increase in the platelet count. The plate- female. The results were: Hb 13 g/dL, Hct 40%, RBC let indices help classify the platelets by their morphology 4.5 * 106> mcL, WBC 15 * 103>mcL. The differential revealed (mean platelet volume [MPV]) and size variation (platelet 70% segmented neutrophils, 25% lymphocytes, 4% mono- distribution width [PDW]) and correlate, to some degree, cytes, 1% eosinophils. Calculate the indices and absolute WBC differential counts. Are any of these parameters outside the with platelet activation and function. In healthy individuals, reference intervals for this patient? If so, which ones? there is an inverse relationship between the PLT count and the MPV. Mean Platelet Volume The MPV is similar to the MCV for erythrocytes because it represents the average volume of The Peripheral Blood Smear individual platelets and usually correlates with the appear- Each testing location and institution sets the parameters ance of platelets on stained blood smears (i.e., an increased that trigger the necessity of a manual morphologic exami- MPV will correlate with large platelets). The MPV is nation of the peripheral smear, but generally a peripheral measured by automated instrumentation (Chapter 39). blood smear is prepared for microscopic examination when Because the MPV increases with time in vitro, it is impor- CBC values obtained from an automated instrument differ tant to note the length of time from sampling to analysis from what is considered normal (Table 10-2). Instructions on when analyzing MPV results.5 The MPV may not be how to prepare and stain a peripheral blood smear are dis- reported for diagnostic purposes since the results depend cussed in Chapter 37. A laboratory professional reviews the on the sample age. blood smear for overall quality, the morphology of white 182 Chapter 10 blood cells, red blood cells, and platelets, and performs a of WBCs (i.e., WBC estimate) under low power (either 100* WBC differential. The peripheral smear is correlated with or 400* magnification) and correlates the WBC estimate to the parameters reported by the instrument as the culminat- the WBC count (Figure 37-6 in Chapter 37). ing interpretation of the CBC. On a well-made blood smear, the erythrocytes are Briefly, a “wedge smear” is made by placing a small evenly distributed and well separated on the feathered edge drop of blood at one end of a microscope slide and spreading of the smear. Stacking or aggregating of cells is associated that drop to create a thin smear or “film” of blood. After the with certain pathologic states (Chapter 11 and Table 10-6). blood has dried, the slide is stained using a Romanowsky- In the presence of IgM antibodies (cold agglutinins) directed type stain (Chapter 37). A well-made and properly stained against erythrocyte antigens, erythrocytes can agglutinate, blood smear is required for accurate interpretation of the forming irregular clusters of varying sizes (Figure 10-1). CBC. The slide is examined macroscopically and micro- This agglutination forms irregular, grapelike clusters that scopically to ensure that the blood was spread and stained are readily differentiated from rouleaux. On automated properly. The optimal blood smear is pinkish purple in color and transitions to a feathered edge (summarized in Tables 37-2 and 37-4 in Chapter 37). Information such as Table 10.6 Abnormalities in Erythrocyte Arrangement RBC agglutination (appears as a grainy blood smear), lip- Associated Physiologic idemia (represented as holes within the smear), and plasma Terminology Description State cell myeloma (bluish-colored smear) may be suspected Agglutination Irregular clumps of Due to antigen–antibody during this important macroscopic evaluation of the blood red blood cells interaction smear and should be noted by the laboratory professional Rouleaux Red blood cells Usually associated with before moving on to the microscopic evaluation. This mac- arranged in rolls or abnormal or increased plasma roscopic assessment determines whether the blood smear stacks proteins (red blood cells can be dispersed by mixing cells with is acceptable for microscopic analysis (described in the next saline) section). LOW-POWER MAGNIFICATION The laboratory professional first assesses the general appearance and distribution of WBCs, RBCs, and platelets using a 10* objective (100* magnification; Table 10-5) for low power magnification. The properties of a well-made smear are described in Table 37-4 in Chapter 37. A high concentration of WBCs at the furthest edge of the smear (the feathered edge) indicates poor cell distribution and is sufficient evidence that a new smear should be made. In addition, very large or abnormal WBCs are often pushed to the outer edges of the smear. Cells that have ruptured are called smudge cells. These are often B lymphocytes and their presence is characteristic of pathological conditions such as Figure 10.1 This blood smear is from a patient with cold chronic lymphocytic leukemia (Chapter 28). The labora- agglutinin disease. Notice the clumping of the erythrocytes tory professional also performs an estimate of the number (peripheral blood; Wright-Giemsa stain; 1000* magnification). Table 10.5 Summary of the Microscopic Peripheral Blood Smear Examination 10* Objective (100* 40* Objective (400* 100* Objective (1000* magnification) magnification) magnification) WBCs Scan for abnormal or large cells Perform WBC estimate Evaluate leukocyte morphology Smudge cells Perform 100-cell differential RBCs Scan for rouleaux and agglutination Determine the critical area Evaluate erythrocyte morphology: Assess size, shape, color, pres- ence of inclusions PLTs Scan for clumps and satellitism Perform platelet estimate Evaluate platelet morphology: Assess size and granularity The Complete Blood Count and Peripheral Blood Smear Evaluation 183 hematology analyzers, a CBC with an elevated MCV and due to improper mixing following the venipuncture). In low RBC count but a normal hemoglobin suggests the pres- either case, a new sample should be obtained from the ence of cold-reacting erythrocyte agglutinins. In addition, patient and a new smear should be made. Other common the calculated hematocrit will be falsely decreased and the sources of error that warrant morphologic examination MCH and MCHC will be falsely increased. The effect of of a peripheral blood smear can be found in Chapters 37 cold agglutinins is overcome by keeping the blood at 37°C. and 43. When performing blood counts, the diluting fluid must also The final task at low power magnification is to determine be kept at 37°C. the critical area of the smear that will be used to perform
the Rouleaux is an alignment of erythrocytes one on top morphologic examination of cells. This critical area is usu- of another resembling a stack of coins (Figure 10-2). This ally identified using the 40* objective (400* magnification) phenomenon occurs normally when blood is collected and and is characterized by the proximity of RBCs to each other allowed to stand in tubes. It can also be seen in the thick (the area of the smear in which very few RBCs overlap or portion of blood smears. In certain pathologic states that touch and are generally distributed in a uniform manner). are accompanied by an increase in fibrinogen or globulins, At high power magnification, the critical area is used to rouleaux becomes marked and is readily seen in the feath- evaluate RBC morphology (Figure 10-4a) and perform the ered edge of blood smears. When the erythrocyte assumes WBC differential and platelet estimate. abnormal shapes, such as sickled forms, rouleaux forma- HIGH-POWER MAGNIFICATION tion is inhibited. Rouleaux is also inhibited when eryth- Following the quick, yet important, scan of the blood smear rocytes are suspended in saline. The presence of rouleaux on low magnification, the laboratory professional evalu- or agglutination are possible indications that a new smear ates the smear on high power, often at 1000* magnifica- should be made. Usually the presence of rouleaux on the tion. Ultimately, interpretation of the microscopic findings peripheral smear is not associated with false RBC param- from the peripheral blood smear and correlation, or lack eters (Hct, MCV, MCH, MCHC) from the instrumentation thereof, with the automated instrument report is made for analysis. all parameters of the CBC. The platelets must also be evaluated using low power magnification. The manner in which the platelets have White Blood Cells and Platelets A WBC differential is spread on the slide is checked because platelet clumps7 performed in which 100 cells are observed and classified can be pushed to the outer edge of the smear, and in some to determine the relative number of leukocytes as a per- cases, platelets can adhere to neutrophils (a phenomenon centage (Chapter 7) and to identify the presence of mor- called satellitism; Figure 10-3).8 This can result in falsely phologic abnormalities (Table 10-7 and Chapters 21 and decreased estimation of the platelet count. Platelet clumps 22). To perform a manual WBC differential, a total of 100 and satellitism can be eliminated using sodium citrate as leukocytes are viewed on a stained blood smear and each an anticoagulant (Table 37-6 in Chapter 37). Finally, fibrin leukocyte subtype is classified. Additionally, a platelet strands can be observed in this scan of the blood smear, estimate is performed (Figure 37-7 in Chapter 37) and indicating that the blood sample was coagulated (likely compared with the instrument-generated platelet count; Figure 10.2 The erythrocytes are stacked on top of one another like a stack of coins (rouleaux). This blood smear is from a patient with plasma cell myeloma, a malignant plasma cell Figure 10.3 Platelet satellitism. The neutrophil in the center disorder. The cells stack because of the large amount of protein is surrounded by platelets (peripheral blood; Wright-Giemsa stain; (immunoglobulin) in the plasma (peripheral blood; Wright-Giemsa 1000* magnification). stain; 1000* magnification). Image courtesy of Constitution Medical, Inc., Westborough, MA. All rights reserved. 184 Chapter 10 a b c Figure 10.4 (a) Normocytic, normochromic erythrocytes. Compare the size of the cells to the nucleus of the lymphocytes. (b) The erythrocytes are microcytic (much smaller than the lymphocyte nucleus). (c) The erythrocytes are macrocytic (much larger than the lymphocyte nucleus) (peripheral blood; Wright-Giemsa stain; 1000* magnification). the morphology of the platelets is noted. Generally, with by the closeness occurring between the two concave por- a normal platelet count, there should be 8–20 platelets per tions of the membrane when the cell becomes flattened on high-powered field or one platelet present for every ∼20 a glass slide. Normally the area of pallor occupies about erythrocytes. Finally, the RBC morphology is assessed for one-third the diameter of the cell. size, shape, and color using either the 40* or 50* objec- Anisocytosis denotes a nonspecific variation in the tive and compared with the instrument report for the RBC size of the cells. Some variation in size is normal because indices. To evaluate abnormalities including inclusions, the of the variation in age of the erythrocytes with younger laboratory professional should review the slide with the cells being larger and older cells smaller (Figures 10-5a and 100* objective (1000* magnification). 10-5b). Poikilocytosis is the general term used to describe a nonspecific variation in the shape of erythrocytes Erythrocyte Morphology The erythrocyte is sometimes (Figures 10-5b and 10-5c). It is important to note that some called a discocyte because of its biconcave shape. On a abnormal morphology can be artifactual because of poorly Romanowsky-stained blood smear, the erythrocyte appears made or improperly stained smears. as a disc with an area of central pallor surrounded by a rim of pink-staining hemoglobin (the center stains lighter in Anisocytosis Anisocytosis can be detected by examining color compared with the rim). The area of pallor is caused the blood smear and/or by reviewing the MCV and RDW The Complete Blood Count and Peripheral Blood Smear Evaluation 185 Table 10.7 Morphologic Abnormalities of WBCs (Nonmalignant)a Neutrophil cytoplasmic Dohle bodies Toxic granulation Vacuoles Intracellular organisms morphology Associated disease state May Hegglin anomaly, Infection Infection Infection infection Image Neutrophil nuclear Bilobed or round nuclei Nuclear hypersegmentation Pyknotic nuclei morphology Associated disease state Pelger-Huet anomaly Megaloblastic anemia Necrobiotic (dying) neutrophil Image Lymphocyte morphology Reactive cells Plasmacytoid Associated disease state Viral infection Infection Image aSee Chapters 21 and 22. (discussed earlier in this chapter). Normal erythrocytes have a diameter of about 798 mcM(mm) and an MCV of Checkpoint 10.4 80–100 fL. If the majority of cells are larger than normal, A patient has an MCV of 130 fL and an RDW of 14.5. they are macrocytic; if smaller than normal, they are Review of the blood smear reveals anisocytosis. Explain the discrepancy between the blood smear finding and RDW. Why microcytic (Table 10-3). If there is a significant variation is examination of an erythrocyte histogram/cytogram helpful in size with microcytic, normocytic, and macrocytic cells in this case? present, the MCV can fall within the reference interval because it is an average of cell volume. In this case, the RDW is helpful. An RDW of greater than 14.5% suggests that the erythrocytes are heterogeneous in size, which CASE STUDY (continued from page 180) makes the MCV less reliable. Microscopic examination of the cells is especially helpful when the RDW is elevated. Because of the abnormal values found in the CBC, To evaluate erythrocyte size microscopically, the cells are the laboratory professional made a blood smear compared with the nucleus of a normal small lympho- and performed a WBC differential and complete cyte. Normocytic erythrocytes are about the same size evaluation of the RBCs and platelets. The primary as the lymphocyte nucleus (Figure 10-4a). Figures 10-5a abnormal finding is seen in the following smear and 10-5b show erythrocytes with a marked degree of image. anisocytosis. (Continued) 186 Chapter 10 Microcytes Microcytes are erythrocytes with a diameter of 3. What should the laboratory professional report less than 7.0 mcM and are present when the MCV is less about the RBCs in the critical area of this smear? than 80 fL (Figure 10-4b). The cell is usually hypochro- 4. Which results of the CBC might be affected by mic but can be normochromic. Microcytes in the shape of the findings on the smear? spheres (microspherocytes) can appear hyperchromic. 5. Explain why the abnormal values in the CBC Macrocytes Macrocytes are larger than normal e rythrocytes occurred in this case. with a diameter greater than 8.0 mcM and are present when 6. Predict the RDW value for the RBCs from this the MCV is more than 100 fL (Figure 10-4c). The cell usu- patient as increased or normal. ally contains an adequate amount of hemoglobin resulting in a normal MCHC and normal to increased MCH. Young erythrocytes are normally larger than mature erythrocytes, but within a day of entering the blood stream, the spleen grooms them to a normal size. When the reticulocyte count is increased, the MCV can be increased because reticulo- cytes are larger than normal erythrocytes. Checkpoint 10.5 Results from a CBC include MCV 63 fL, MCH 16.7 pg, and MCHC 26.5 g/dL. Describe these cells. 1 2 a b 4 3 c Figure 10.5 (a) Erythrocytes with anisocytosis. (b) Erythrocytes with marked anisocytosis and poikilocytosis. Arrows point to a schistocyte (1) and an acanthocyte (2). (c) Poikilocytosis with acanthocytes, helmet cell, elliptocytes, echinocytes, schistocytes (3), and spherocytes (4). There is also anisocytosis with microcytes and macrocytes. At least two of the macrocytes are polychromatophilic (peripheral blood; Wright-Giemsa stain; 1000* magnification). The Complete Blood Count and Peripheral Blood Smear Evaluation 187 Poikilocytosis Most laboratories report only significant Acanthocytes Acanthocytes, also called spur cells, are poikilocytosis. The stained smear should be reviewed while small spherical cells with irregular thornlike projections keeping in mind the overall context of the laboratory results (Figure 10-5b, Table 10-8). Often the projections have and the significance of the reported findings. To determine small bulblike tips. Acanthocytes do not have an area of the significance of and to decide whether to report poikilo- central pallor. These cells have membranes with free cho- cytes, the following should be considered: lesterol accumulating preferentially in the outer bilayer of the membrane leading to decreased fluidity. Remodel- 1. Will it assist in differential diagnosis of the disease? ing by the spleen results in spheroidal cells with irregular 2. Will it make a difference in the management of the surface projections. These cells are readily trapped in the patient? spleen. 3. Is the dominant poikilocyte significant in this setting? Codocytes Codocytes, also called target cells, are thin, 4. Does the specific constellation of findings indicate a bell-shaped cells with an increased surface area-to-volume particular pathologic state? ratio (Table 10-8, Figure 10-6). On stained blood smears, the Figure 10-4a illustrates normal erythrocytes, and cells have the appearance of a target with a bull’s-eye in Figure 10-5b and 10-5c illustrate poikilocytosis. The fol- the center. An achromic zone and a thin outer ring of pink- lowing is a description of specific types of poikilocytes staining hemoglobin surround the bull’s eye. The typical (Table 10-8). appearance of these cells is discernible only in the area of Table 10.8 Erythrocyte Morphologies Drawing of Terminology Synonyms Description Associated Disease States Morphology Poikilocytosis — Increased variation in the shape of See disease states associated with red cells specific poikilocytes on this table Acanthocyte Spur cell Red cells with spicules of varying Abetalipoproteinemia; alcoholic (spike) length irregularly distributed over the liver disease; disorders of lipid surface; no area of pallor metabolism; post splenectomy; fat malabsorption; retinitis pigmentosa (Chapters 15, 17) Codocyte Target cell Thin, bell-shaped, with increased Hemoglobinopathies; thalassemias; (bell) surface area-to-volume ratio; on obstructive liver disease; iron stained blood smears, appears as a deficiency anemia; splenectomy; target with a central bull’s-eye, sur- renal disease; LCAT deficiency rounded by achromic zone and outer (Chapters 12, 14, 15) ring of hemoglobin Dacryocyte Teardrop Round cell with a single elongated or Myelophthisic anemias; primary (tear) pointed extremity; may be microcytic myelofibrosis (PMF); thalassemias and/or hypochromic (Chapter 14) Drepanocyte Sickle cell Contain polymerized hemoglobin Sickle cell disorders (Chapter 13) (sickle) showing various shapes: sickle-, crescent-, or boat-shaped Echinocyte Burr cell; Spiculated red cells with short Liver disease; uremia; pyruvate (sea urchin) crenated cell equally spaced projections over the kinase deficiency; peptic ulcers; entire surface cancer of stomach; heparin therapy (Chapters 15, 18) Elliptocyte Ovalocyte; Oval to elongated ellipsoid cell with Hereditary elliptocytosis; iron (oval) pencil cell; area of central pallor and hemoglobin deficiency anemia; thalassemia; cigar cell at both ends anemia associated with leukemia (Chapters 12, 14, 17) Keratocyte Helmet cell; Red cells with one or several notches Microangiopathic hemolytic (horn) horn-shaped cell with projections that look like horns anemias; heart-valve hemolysis; on either end Heinz-body hemolytic anemia; glomerulonephritis; cavernous hemangiomas (Chapters 13, 20) Knizocyte — RBC with more than two concavities; Conditions in which spherocytes on stained blood smears has a dark are found (Chapter 17) band of hemoglobin across the cen- ter with a pale area on either side (Continued) 188 Chapter 10 Table
10.8 Continued Drawing of Terminology Synonyms Description Associated Disease States Morphology Leptocyte Thin cell Thin, flat cell with hemoglobin at Thalassemia; iron deficiency (thin) periphery; usually cup-shaped, MCV anemia; hemoglobinopathies; liver is decreased but cell diameter is disease (Chapters 13, 14, 15, 17) normal Schistocyte Schizocyte; Fragments of red cells; variety of Microangiopathic hemolytic (cut) fragmented cell shapes including triangles, commas; anemias; heart-valve hemolysis; microcytic disseminated intravascular coagulation; severe burns; uremia (Chapter 20) Spherocyte — Spherocytic red cells with dense Hereditary spherocytosis; hemoglobin content (hyperchro- immune hemolytic anemias; matic); lack an area of central pallor severe burns; transfusion with ABO incompatibility; Heinz-body hemolytic anemias (Chapters 13, 17, 19, 20) Stomatocyte Mouth cell; Uniconcave red cells with the shape Hereditary stomatocytosis; (mouth) cup form; of a very thick cup; on stained blood spherocytosis; alcoholic cirrhosis; mushroom cap smears cells have an oval or slitlike anemia associated with Rh area of central pallor null disease; lead intoxication; neoplasms (Chapters 17, 20) 1 3 2 Figure 10.6 Codocytes, also called target cells (peripheral Figure 10.7 Note the presence of dacryocytes (teardrops) (1). blood; Wright-Giemsa stain; 1000* magnification). Note also the echinocytes (2), acanthocytes, and spherocytes (3), (peripheral blood; Wright-Giemsa stain; 1000* magnification). the slide where the cells are well separated but not in the extreme outer-feathered edge where all cells are flattened. membrane skeleton or has remained in the abnormal shape Target cells can appear as artifacts when smears are made in for too long. a high-humidity environment or when a wet smear is blown Drepanocytes (Sickle Cells) Sickle cells, also called drepa- dry rather than fan dried. nocytes, are elongated, crescent-shaped erythrocytes with Dacryocytes Dacryocytes, also called teardrops, are eryth- pointed ends (Table 10-8, Chapter 13). Some forms have more rocytes that are elongated at one end to form a teardrop rounded ends with a flat rather than concave side. These or pear-shaped cell (Table 10-8, Figure 10-7). The teardrop modified forms of sickle shape may be capable of reversing morphology can form after erythrocytes containing cellu- to the normal discocyte. Sickle cell formation can be observed lar inclusions have traversed the spleen. Erythrocytes with in stained blood smears from patients with sickle cell ane- inclusions are more rigid in the area of the inclusion, and mia. The hemoglobin within the cell is abnormal and polym- this portion of the cell has more difficulty passing through erizes into rods at decreased oxygen tension or decreased the splenic filter than the rest of the cell. As splenic mac- pH. The cell first transforms into a holly leaf shape and as rophages attempt to remove this rigid inclusion, the cell the hemoglobin polymerization continues, it transforms into is stretched into an abnormal shape. The teardrop can- a sickle-shaped cell (Table 10-8) with increased mechanical not return to its original shape because the cell either has fragility. Some holly-leaf forms can be observed on stained been stretched beyond the limits of deformability of the blood smears in addition to the typical sickle shape. The Complete Blood Count and Peripheral Blood Smear Evaluation 189 Echinocytes Echinocytes, also called burr cells, are grooms (removes) the membrane spines; in this circum- usually smaller than normal erythrocytes with regu- stance, the cell cannot revert to a normal shape. lar, spinelike projections on their surface (Table 10-8, Elliptocytes Elliptocytes, also called pencil cells and cigar Figure 10-5c). Their presence is most often an artifact on cells, vary from elongated oval shapes (ovalocytes) to elon- stained blood smears because of the “glass effect” of the gated rodlike cells (Table 10-8, Figure 10-8). Some laboratory slide. The glass releases basic substances that raise the pH professionals may use the terms elliptocytes and ovalocytes of the medium surrounding the cell and induce echinocyte interchangeably, whereas others may use distinct guide- formation. Plasma provides a buffering effect on the cells, lines to delineate the two morphologies. True elliptocytes and for this reason, blood films made from whole blood have parallel sides and a central area of biconcavity with may show only certain areas of echinocyte transformation. hemoglobin concentrated at both ends (Figure 10-8a). Ellip- To determine the in vivo or in vitro nature of echinocytes, tocytes are formed after the erythrocyte matures and leaves a wet preparation can be made in which a drop of blood is the bone marrow because reticulocytes and young eryth- enclosed between two plastic cover slips and the unstained rocytes in patients with elliptocytosis are normal in shape. individual erythrocytes are observed. If no echinocytes The mechanism of formation is not known but is assumed are present in the wet preparation but were noted on the to involve alterations of the erythrocyte membrane skele- stained blood smears, the cell abnormality occurred as an ton (Chapter 17). Elliptocytes are the predominant shape of in vitro artifact. erythrocytes in hereditary elliptocytosis. On the other hand, Echinocytes can appear in blood that has been stored ovalocytes are fatter on one end than the other and appear at 4°C for several days. Consequently, blood specimens to have an egg shape (Figure 10-8b). Ovalocytes are formed from patients receiving transfusions can have echinocytes in a manner similar to elliptocytes. if blood is taken from the patient immediately after transfu- sion; however, after a few minutes, the buffering action of Keratocytes Keratocytes, also called helmet cells, have patient’s plasma causes the transfused echinocyte to resume a concavity on one side and two hornlike protrusions on a normal discoid shape. either end (Table 10-10, Chapter 18). Keratocytes are pro- For true “in vivo” echinocytes, the characteristic duced when a fibrin strand impales an erythrocyte. The two appearance is not related to tonicity of the medium in halves of the erythrocyte hang over the strand as saddle- which the cells are suspended. The shape change is instead bags; the membranes of the touching sides fuse, produc- thought to result from an increase in the area of the outer ing a vacuole-like inclusion on one side. This cell with an leaflet of the lipid bilayer as compared with the inner layer. eccentric vacuole is called a blister cell. The vacuole bursts, Echinocyte formation is reversible (i.e., the cell can revert to leaving a notch with two spicules on the ends. a discocyte); however, an echinocyte can eventually assume Knizocytes Knizocytes are cells with more than two con- the shape of a spherocyte, presumably because the spleen cavities (Table 10-8). This cell’s appearance on stained blood a b Figure 10.8 Note the morphologic differences between (a) elliptocytes and (b) ovalocytes (peripheral blood; Wright-Giemsa stain; 1000* magnification). 190 Chapter 10 smears can vary depending upon how the cell comes to rest slitlike (mouthlike) area of pallor. Normal discocytes can be on the flat surface; however, most knizocytes have a dark- transformed under certain conditions to stomatocytes and, staining band across the center with a pale area on either eventually, to spherostomatocytes. The stomatocyte shape is side surrounded by a rim of pink-staining hemoglobin. The reversible, but the spherostomatocyte is not. Cationic drugs mechanism of formation is unknown. and low pH cause a gradual loss of biconcavity leading to the stomatocyte and eventually the formation of a sphere. Leptocytes Leptocytes are thin, flat cells with normal or Stomatocytosis is the opposite of echinocytosis; the shape larger than normal diameter (Table 10-8). Although the change in stomatocytosis is thought to be the result of an cell’s diameter is normal or increased, its volume is usu- increase in the lipid content or area of the inner leaflet of ally decreased. The cells have an increased surface area- the membrane lipid bilayer. Stomatocytes also can appear to-volume ratio either as a result of decreased hemoglobin as an artifact on stained blood smears; thus, care should be content or increased surface area. The leptocyte is usually used in identifying them. cup-shaped like stomatocytes, but the cup has little depth. Target cells can be formed from leptocytes on dried blood smears when the depth of the cup increases. Checkpoint 10.6 Review of a peripheral blood smear reveals significant num- Schistocytes Schistocytes are erythrocyte fragments caused bers of codocytes and echinocytes. Describe the morphology by mechanical damage to the cell (Table 10-8, Figures 10-5b of these cells. Should you report this on the laboratory report? and 10-5c). They appear in a variety of shapes such as tri- Why or why not? angular, comma, and helmet-shaped. Because schistocytes are fragments of erythrocytes, they are usually microcytic. Variation in Hemoglobin (Color) Normal erythrocytes have They maintain normal deformability, but their survival in an MCH of approximately 30 pg. However, the MCHC is the peripheral blood is reduced. The fragments can assume a better indicator of chromia or color of erythrocytes on a spherical shape and hemolyze or can be removed in the Romanowsky-stained smears. Normally, on stained smears, spleen. the erythrocyte has an area of central pallor approximately Spherocytes Spherocytes (Table 10-8, Figure 10-5c) are one-third the diameter of the cell (Figure 10-4a). In certain erythrocytes that have lost their biconcavity because of a conditions, RBCs contain less hemoglobin than normal and decreased surface area-to-volume ratio. On stained blood appear to have a larger than normal central pallor (hypo- smears, the spherocyte appears as a densely stained sphere chromia; Figure 10-10). On the other hand, the only erythro- lacking an area of central pallor. Although the cell often cyte that contains more hemoglobin than normal in relation appears microcytic on stained blood smears, the volume to its volume is the spherocyte (Figure 10-11). (MCV) is usually normal. The spherocyte is the only Hypochromic Cells Hypochromic cells are poorly hemoglo- erythrocyte that can be called hyperchromic because of an binized erythrocytes with an exaggerated area of central increased MCHC. pallor (greater than one-third the diameter of the cell) on Stomatocytes In wet preparations, stomatocytes, or mouth cells, appear as small cup-shaped uniconcave discs (Table 10-8, Figure 10-9). Upon staining, these cells exhibit a Figure 10.10 Microcytic, hypochromic erythrocytes. Compare the size of the erythrocytes with the nucleus of the lymphocyte. Normocytic cells are about the same size as the nucleus. There is only a thin rim of hemoglobin around the periphery Figure 10.9 Stomatocytes. Note the slitlike area of pallor of the cells, indicating that they are hypochromic (peripheral blood; (peripheral blood; Wright-Giemsa stain; 1000* magnification). Wright-Giemsa stain; 1000* magnification). The Complete Blood Count and Peripheral Blood Smear Evaluation 191 Romanowsky-stained blood smears. Although occasion- ally normocytic, hypochromic cells are usually microcytic 2 (Figure 10-10). Hypochromic cells are the result of decreased or impaired hemoglobin synthesis (Table 10-9). When visu- alizing a blood smear, correlating the automated findings from hematology analyzers to the appearance of cells is 1 important. In the case of hypochromia, the MCHC value will be decreased. Polychromatophilic Erythrocytes Polychromatophilic eryth- rocytes (reticulocytes) are usually larger than normal cells with a bluish tinge on Romanowsky-stained blood smears (Figure 10-11). The bluish tinge is caused by the presence of Figure 10.11 The large erythrocytes with a bluish tinge are residual RNA in the cytoplasm (Table 10-9). Large numbers polychromatophilic erythrocytes, which are larger than the more of these cells are associated with decreased erythrocyte sur- mature erythrocytes. Note also the spherocytes (peripheral blood; Wright-Giemsa stain; 1000* magnification). vival or hemorrhage and an erythroid hyperplastic marrow (Table 10-9). A supravital stain such as new methylene blue or bril- liant cresyl blue must be used to definitively identify the presence of reticulocytes (Figure 10-12 and Table 10-10). Although automated methods for reticulocyte enumeration are available on some hematology instruments, many labo- ratories use a manual method (Chapter 37). Test results are expressed as a percentage of reticulocytes in relation to the total RBC count (relative count) or as the absolute number (see “Reticulocyte Count”). The automated method assesses greater than 30,000 RBCs and is therefore more precise than the manual method (assesses only 1,000 RBCs) and more accurate when the reticulocyte count is very low. Checkpoint 10.7 Increased polychromasia is reported on a blood smear. What is polychromasia, and what other hematologic assay will reflect the presence of polychromasia? Figure 10.12 The erythrocytes with the particulate inclusions are reticulocytes. The inclusions represent reticulum that stains with the supravital stain brilliant cresyl blue (peripheral blood; 1000* Erythrocyte Inclusions Erythrocytes do not normally con- magnification). tain any particulate inclusions. When present, inclusions can help direct further investigation because they are
associ- across their entire cell area (Table 10-10). The granules can ated with certain disease states. Descriptions of erythrocyte vary in size and distribution from small and diffuse to coarse inclusions as they appear on Romanowsky-stained blood and punctate. The granules, which are composed of aggre- smears, unless otherwise stated, are listed in Table 10-10. gated ribosomes, are sometimes associated with mitochon- Basophilic Stippling Erythrocytes with basophilic stippling dria and siderosomes. Basophilic stippling is not believed are cells with bluish-black granular inclusions distributed to be present in living cells; instead, stippling probably is Table 10.9 Variations in Erythrocyte Color Terminology Description Associated Physiological or Disease States Hypochromia Decreased concentration of hemoglobin in the red cell May be present in iron-deficiency anemia, thalassemia, and Red cells have an increased area of central pallor (greater other anemias associated with a defect in hemoglobin produc- than one-third the diameter of cell) tion (Chapters 12–14) Polychromasia Young red cells containing residual RNA Found in increased numbers in hemolytic anemias, newborns, Stain a pinkish-gray to pinkish-blue color on Wright-stained recovery from acute hemorrhage (Chapters 16–20) blood smears Usually appear slightly larger than mature red cells 192 Chapter 10 Table 10.10 Erythrocyte Inclusions Terminology Description Associated Disease States Image Basophilic stippling Round or irregularly shaped granules of Lead poisoning; anemias associ- variable number and size, distributed ated with abnormal hemoglobin throughout the RBC synthesis; thalassemia (Chapters Composed of aggregates of ribosomes 14, 20) (RNA) and mitochondria Stain bluish black with Wright stain Cabot rings Appear as a figure-8, ring, or incomplete ring Severe anemias; dyserythropoiesis Thought to be composed of the microtubules (Chapter 25) of the mitotic spindle Stain reddish violet with Wright stain Howell-Jolly bodies Small, round bodies composed of DNA Post splenectomy; megaloblastic usually located eccentrically in the red cell anemias; some hemolytic anemias; Usually occurs singly, rarely more than two functional asplenia; severe anemia per cell (Chapter 15) Stains dark purple with Wright stain Heinz bodies Bodies composed of denatured or G6PD deficiency; unstable precipitated hemoglobin hemoglobin disorders; oxidizing Not visible on Wright-stained blood smears drugs or toxins; post splenectomy (Chapters 13, 18) With supravital stain appear as purple, round-shaped bodies of varying size, usually close to the cell membrane Can also be observed with phase microscopy on wet preparations Pappenheimer bodies Clusters of granules containing iron that are Sideroblastic anemia; thalassemia; (siderotic granules) usually found at the periphery of the cell other severe anemias Visible with Prussian blue stain and Wright (Chapters 12, 14) stain produced during preparation of the blood smear or during Howell-Jolly bodies are associated with nuclear matura- the staining process.9 Electron microscopy has not shown an tion abnormalities. They are thought to occur as a result of intracellular structure similar to basophilic stippling. Cells an individual chromosome failing to attach to the spindle dried slowly or stained rapidly can demonstrate fine, dif- apparatus during mitosis, and, thus, it is not included in fuse stippling as an artifact. Pathologic basophilic stippling the reformed nucleus. When the nucleus is extruded, the is more coarse and punctate. Howell-Jolly body is left behind (until removed by splenic macrophages). Cabot Rings Cabot rings are reddish-violet erythrocytic inclusions usually occurring in the formation of a figure Heinz Bodies Heinz bodies do not stain with Romanowsky eight or oval ring (Table 10-10). Cabot rings are thought to stains but can be visualized with supravital stains or with be remnants of spindle fibers, which form during mitosis. phase microscopy of the living cell. They appear as 2–3 They occur in severe anemias and in dyserythropoiesis. mcM round masses lying just under or attached to the cell membrane. Heinz bodies are composed of aggregated dena- Howell-Jolly Bodies Howell-Jolly bodies are dark purple or tured hemoglobin. violet spherical granules in the erythrocyte (Table 10-10). These inclusions are nuclear (DNA) fragments usually Iron Inclusions Particulate iron molecules can be detected occurring singly in cells, rarely more than two per cell. in erythrocytic cells in both normal and abnormal The Complete Blood Count and Peripheral Blood Smear Evaluation 193 conditions. Intracellular siderotic granules represent Clinical Laboratory Professional’s iron that has not been incorporated into hemoglobin. Pappenheimer bodies are damaged secondary lysosomes Review of CBC Data and mitochondria variable in their composition of iron The role of the laboratory professional is to analyze and and protein10 (Table 10-10). This type of inclusion appears interpret the data generated by the automated hematology as clusters of small granules in erythrocytes and erythro- analyzer and the manual, peripheral blood smear review. blasts. Romanowsky stains reveal Pappenheimer bodies by The interpretation is essentially a correlation of the various staining the protein matrix of the granules whereas Prus- components of the CBC in order to identify the likelihood of sian blue is responsible for staining the iron portion of the abnormal results, pathology, and discrepancies in the gener- granules. Pappenheimer bodies occur only in pathologic ated data. The hematology instrument or laboratory infor- states. mation system produces alerts that include delta checks or RBCs that contain stainable iron granules are called that indicate the presence of interfering substances, both of sideroblasts if they contain a nucleus (erythroblasts; which the laboratory professional must resolve. Figure 10-13) or siderocytes if they are are non-nucleated, Delta checks compare a patient’s current clinical values mature erythrocytes (Figure 10-14). Sideroblasts and sid- for a test with previous values. This type of quality control erocytes can be identified with a Prussian blue iron stain can detect sudden changes in a patient’s physiology or can that stains iron aggregates blue. About 20–60% of all eryth- be useful in identifying instrument error or misidentified roblasts in the marrow contain iron that can be visualized patient samples. Delta checks are particularly important in with Prussian blue stain. This number decreases in some diagnosis and in monitoring therapy. Abnormal results and pathologic states and can be markedly increased in others. the presence of interfering substances (e.g., lipemia, hemo- Reticulocytes and erythrocytes in the peripheral blood do lysis) must also be noted and corrected. For a complete list not normally contain stainable iron aggregates unless the of abnormal test results, use of delta checks, and correc- patient has been splenectomized. tion for interfering substances, see Tables 43-7 and 43-8 in Chapter 43. In the event that the data can be correlated for diagnosis, the laboratory professional should be able to rec- Checkpoint 10.8 ommend subsequent testing to the patient’s physician. A medical laboratory scientist reports the presence of Howell- Jolly bodies in the erythrocytes on a blood smear. What is the composition of these inclusions, and how can he be sure they CASE STUDY (continued from page 185) are not Pappenheimer bodies or Heinz bodies? The laboratory professional warmed the EDTA blood sample from this patient at 37°C for 15 minutes and then reanalyzed the CBC on the analyzer and made a new smear. The new smear appeared as in the following figure. The new values were as follows: hemoglobin of 8.2 g/dL (82 g/L), RBC count of 2.63 * 106>mcL, and MCV of 91 fL. Figure 10.13 Erythroblasts containing iron granules are called sideroblasts. The granules stain blue with Prussian blue stain (peripheral blood; 1000* magnification). 8. Calculate the hematocrit, MCH, and MCHC on the warmed specimen. How have they changed? Figure 10.14 Eythrocytes containing iron granules are called siderocytes. The granules stain blue with Prussian blue stain 9. Explain what could have happened in this case. (peripheral blood; 1000* magnification). 194 Chapter 10 Post-Examination Phase The highest normal hemoglobin, hematocrit, and erythrocyte counts are observed at birth. In the neonate, of the CBC erythrocytes are macrocytic, and the reticulocyte count is 2–6%. During the first week of life, nucleated erythro- After acquisition of the sample, testing, and collecting and cytes—as many as 10 per 100 leukocytes—can be present reviewing the data, the CBC report is made available to in the peripheral blood. For approximately 2 months after the ordering physician. The steps of the post-examination birth, a gradual decrease in the hemoglobin, hematocrit, include reporting the data through a laboratory information and erythrocyte counts occurs. The mean MCV of neo- system or by other means of physician notification as well nates is 108 fL and decreases to a mean of 77 fL between as recognition and reporting of critical values. the ages of 6 months and 2 years (Figure 10-15). The mean Routine and stat (immediate) orders are generally MCV increases to 80.0 fL by age 5 but does not reach the reported through laboratory information systems; however, adult mean of 90 fL until approximately 18 years of age. The some reports can be faxed and paper reports can be used. MCH changes in parallel to the MCV throughout infancy A reference interval for each parameter of the CBC accom- and childhood, whereas the MCHC remains constant and panies each report, as do two identifiers for the patient and within the adult reference interval.11 A difference in erythro- the name of the ordering physician. Some reporting sys- cyte values between sexes is noted at puberty with females tems include flags for abnormal results indicating lower or having lower values than males. higher than expected values. Within the CBC report, the laboratory professional must be aware of the critical limits that represent the critical Checkpoint 10.9 low and high values for a CBC parameter. When values fall The following data is found on the CBC of a newborn male. above or below the critical limit threshold, they are termed Hb = 19.5 g>dL1195 g/L2; Hct = 59%(0.59 L/L) critical values and potentially pose a life-threatening risk RBC = 6.5 * 106>mcL; WBC = 17.6 * 103>mcL to the patient. The assay generating critical values must be repeated to confirm the result, phoned to the patient’s phy- Calculate the MCV and MCHC. Evaluate all of the data for sician, and then properly documented. Each clinical facil- abnormalities. ity determines its critical limits and critical values and the means by which to report those values. CBC Variations Between Ethnic Groups and Sexes, in Elderly People, Physiologic Variation in and by Geographic Location Hematologic Parameters Studies of hematology parameters among different popu- lations have revealed that African Americans have lower The normal values for hematologic parameters vary hematocrit levels, lower hemoglobin values (about 0.8 g/dL depending on age, sex, race, ethnicity, and geographic area. lower), and lower MCVs than Caucasians.12 Although Thus, it is important that this information is available when iron deficiency and the high prevalence of a@thalassemia reviewing data. CBC Variations in Newborns and Children Because various parameters of the CBC are dramatically dif- ferent in newborns compared with adults, the patient’s age must be considered when evaluating a patient’s blood pic- ture. In premature and term infants, the total WBC count is generally much higher than adults (mean WBC of approxi- mately 25 * 103>mcL) (Chapter 7). Granulocyte numbers are increased in premature and term infants and drop within the first 72 hours. In the premature infant, immature WBCs may be found in the peripheral circulation for two weeks. Lymphocytes are the predominant cell until age 4 or 5. After age 4–5, the lymphocytes and granulocytes are present in about equal numbers. After age 8, the reference Figure 10.15 Peripheral blood from a newborn; note the intervals for WBC subsets are similar to those of adults. macrocytic erythrocytes (Wright-Giemsa stain; 1000* magnification). The Complete Blood Count and Peripheral Blood Smear Evaluation 195 in African Americans (about 30%), may contribute to the in the MCV can occur. The bone marrow appears normal lower hemoglobin and MCV values, the differences nar- with normal hematopoietic precursor cells.14,15 Although rowed but persisted even when those who tested posi- anemia does not occur due to aging, it becomes more preva- tive for a@thalassemia and iron deficiency were eliminated lent as the population ages.15 The most common causes of from the test group. The WBC count is lower in African decreased hemoglobin in elderly people are iron deficiency, Americans primarily due to lower granulocyte counts, but Vitamin B12 deficiency, anemia of chronic inflammation, kid- lymphocyte counts are higher.12 ney disease, and decreased testosterone level. Variations in When comparing hematologic profiles by sex, men the CBC are also associated with malignancies that occur at a show a statistically higher Hb, Hct, RBC, and WBC than higher incidence in older adults. Chronic
lymphocytic leuke- women.13 On the other hand, women have higher plate- mia is the most common leukemia in the Western world and let counts than men. Differences in the MCV and MCH are is documented with a mean age of 72.16 Likewise, the inci- minor with slightly lower values in women. Smoking and dence of lymphomas and plasma cell myeloma is increased high alcohol intake have a significant effect on the Hb, Hct, in geriatric populations, and myelodysplastic syndromes MCV, MCH, and WBC. and acute leukemias have a higher incidence after age 60.17 Although bone marrow cellularity decreases with age, The reference interval for Hb, Hct, and RBC should in the absence of disease, the WBC, hemoglobin, platelet be adjusted upward for those living at high altitudes to count, and differential are maintained at adult reference account for the normal physiologic response to the lower intervals in adult populations older than 65.14 A slight drop partial pressure of oxygen. Summary The initial hematologic analysis of a patient is called the hemoglobin, hematocrit, indices), and PLTs (enumeration complete blood count (CBC), and the process used to deter- and volume). A peripheral blood smear may be prepared mine it consists of three general phases: pre-examination, for the laboratory professional to evaluate the circulating examination, and post-examination. The purpose of the cells within a patient. Accurate evaluation of the peripheral CBC pre-examination phase is to ensure the highest quality blood smear can be one of the most difficult skills that the sample for analysis. Best practices in patient identification, laboratory professional will master because of the numer- blood collection, and specimen handling should be fol- ous variations in WBC, RBC, and PLT morphology. lowed for a seamless transition to the examination phase. Finally, the CBC must be interpreted and reported to the The individual parameters of the CBC are determined requisitioning physician in the post-examination phase. CBC by automated and/or manual hematologic analysis in parameters that significantly differ from normal values (either the examination phase. Whole blood analysis is reported high or low) must be reported with immediacy. The CBC is as individual parts consisting of WBCs (enumeration of used to classify hematologic diseases as well as several other total WBCs and individual classes), RBCs (enumeration, pathologies and will be discussed in subsequent chapters. Review Questions Level I a. Siderotic granules 1. Which of the following are the correct units for report- b. Heinz bodies ing the absolute RBC count using the SI system? c. Howell-Jolly bodies (Objective 4) d. Basophilic stippling a. 109 > L b. 1012 > 3. Which of the following RBC indices indicates how L filled the average RBC is with hemoglobin in terms of c. 103 > mcL weight per unit volume? (Objective 5) d. 109 > fL a. MCV b. MCH 2. Which erythrocyte inclusions are composed of DNA and stain blue on Romanowsky stains? c. MCHC (Objective 11) d. RDW 196 Chapter 10 4. A blood smear reveals uneven distribution of red 10. A routine CBC is to be performed on a blood sample blood cells, and the red blood cells appear to be that arrives in the laboratory from an outside clinic stacked together like a stack of coins. How would you 4 hours after it is drawn. The sample is frozen. The describe this distribution? (Objective 12) sample is: (Objective 3) a. Agglutination a. acceptable b. Rouleaux b. acceptable but must be warmed before performing c. Anisocytosis the CBC d. Poikilocytosis c. unacceptable due to improper sample temperature d. unacceptable due to improper time restraints 5. How would you classify the red cell population with the following indices: MCV 110 fL, MCH 38 pg, Level II MCHC 33 g/dL? (Objective 9) 1. A 53-year-old patient had a hemoglobin of 7.0 g/dL. a. Normocytic, normochromic The reticulocyte count is 15%. Which of the b. Macrocytic, normochromic following would you expect on the blood smear? (Objective 4) c. Microcytic, normochromic d. Microcytic, hypochromic a. Poikilocytes b. Polychromatophilia 6. If the cell population in question 5 were homoge- c. Agglutination neous (absence of anisocytosis), the RDW might show: (Objective 5) d. Howell-Jolly bodies a. false increase 2. Numerous schistocytes in the patient in question 1 b. false decrease were identified on the blood smear. How could this finding affect the RDW? (Objectives 1, 4) c. normal reference interval d. true increase a. Increase it b. Decrease it 7. A peripheral blood smear that has an erythrocyte c. Have no effect mixture of macrocytes, microcytes, and normocytes present can best be described as: (Objective 8) d. Invalidate it a. poikilocytosis 3. Some of the RBCs on a patient’s smear contain b. polychromatophilia numerous small blue inclusions. What should be done next to determine what to report about the c. megaloblastosis inclusions? (Objective 5) d. anisocytosis a. Perform an iron stain for identification of 8. Which of the following erythrocyte inclusions cannot siderocytes be stained and visualized with Romanowsky stains? b. Perform a screen for sickle cell anemia (Objective 11) c. Use new methylene blue stain to confirm an a. Pappenheimer bodies increase in reticulocytes b. Howell-Jolly bodies d. Check the CBC data for an indication of the c. Heinz bodies presence of Howell-Jolly bodies d. Basophilic stippling 4. The RDW is found to be 19.5% on a patient. Which of the following should you find increased on the 9. If there is an increase in macrocytic, polychromato- smear? (Objective 1) philic erythrocytes on the Romanowsky-stained blood smear, which laboratory test result would correlate a. Macrocytosis with this? (Objective 13) b. Microcytosis a. Platelet count c. Anisocytosis b. Reticulocyte count d. Poikilocytosis c. Leukocyte count d. MCHC The Complete Blood Count and Peripheral Blood Smear Evaluation 197 5. Rouleaux is found on a smear of a patient with c. Is responding to the treatment for the anemia plasma cell myeloma. How will this affect the CBC d. Has a decreased RBC count results? (Objective 6) a. RBC count will be decreased. 8. The presence of dacryocytes on a peripheral blood b. Hematocrit will be increased. smear is most likely suggestive of which of the following? (Objective 3) c. MCHC will be increased. d. There will be no effect. a. Artifact b. Young RBCs 6. A CBC is ordered on a 3-day-old infant with a fever c. RBC destruction of 100°F. The laboratory professional notes nucle- d. Splenic removal of RBC inclusions ated RBCs on the peripheral blood smear but is not alarmed by this finding. Why not? (Objective 4) 9. The MCHC result is extremely elevated in a patient’s a. Nucleated RBCs are a common occurrence during CBC results. Which of the following is a likely cause infection. of this result? (Objective 4) b. The fever promoted an increase in RBC production. a. Microcytic RBCs c. Nucleated RBCs are commonly observed in the b. Agglutination of the RBCs first 7 days of life. c. Increased hemoglobin and RBC count d. The nucleated RBCs are likely to be artifacts. d. High RDW 7. A CBC is performed for a patient who has been 10. Which of the following poikilocytes are frequently treated for anemia. The laboratory professional notes artifacts, not a pathologic finding? (Objective 3) an increase in polychromatophilic macrocytes. Which of the following is speculated about the patient from a. Drepanocytes the peripheral blood smear? (Objective 2) b. Echinocytes a. Responding to an infection c. Spherocytes b. Needs further treatment for the anemia d. Schistocytes References 1. Hadley, G. G., & Larson, N. L. (1953). Use of sequestrene as an 10. Glassy, E. R. (1998). Color atlas of hematology: An illustrated field anticoagulant. American Journal of Clinical Pathology, 23, 613–618. guide based on proficiency testing. Northfield, IL: CAP. 2. Kennedy, J. B., Maehara, K. T., & Baker, A. M. (1981). Cell and 11. Proytcheva, M. A. (2009). Issues in neonatal cellular analysis. platelet stability in disodium and tripotassium edta. American American Journal of Clinical Pathology, 131, 560–573. Journal of Medical Technology, 47, 89–93. 12. Beutler, E., & West, C. (2005). Hematologic differences between 3. NCCLS. (2004). Procedures for the handling and processing of blood African Americans and whites: the roles of iron deficiency and specimens [NCCLS Document No. H18-A3]. Wayne, PA: NCCLS. on hemoglobin levels and mean corpuscular volume. Blood, 106, 4. Sennels, H. P., Jorgensen, H. L., Hansen, A. L., Goetze, J. P., & 740–745. Fahrenkrug, J. (2011). Diurnal variation of hematology param- 13. Tsang, C. W., Lazarus, R., Smith, W., Mitchell, P., Koutts, J., & eters in healthy young males: the Bispebjerg study of diurnal Burnett, L. (1998). Hematological indices in an older population variations. Scandinavian Journal of Clinical and Laboratory Investiga- sample: derivation of healthy reference values. Clinical Chemistry, tion, 71, 532–541. 44, 96–101. 5. Leader, A., Pereg, D., & Lishner, M. (2012). Are platelet volumne 14. Ershler, W. B., & Longo, D. L. (2010). Hematology in older persons. indicies of clinical use?: A multidisciplinary review. Annals of In: M. A. Lichtman, T. J. Kipps, U. Seligsohn, K. Kaushansky, & Medicine, 44, 805–816. J. T. Prchal, eds. Williams hematology (8th ed.). New York: 6. Hoffmann, J., van den Broek, N., & Curvers, J. (2013) Reference McGraw-Hill. intervals of reticulated platelets and other platelet parameters 15. Khier, F., & Haddad, R. Y. (2010). Anemia in the elderly. Disease-a- and their associations. Archives of Pathology and Laboratory Month, 56, 456–467. Medicine, 137, 1635–1640. doi: 10.5858/arpa.2012-0624-OA. 16. Friese, C. R., Earle, C. C., Magazu, L. S., Brown, J. R., Neville, B. A., 7. Sinha, S. K., Mandal, P. K., & Mallick, J. (2011). Hevelone, N. D., . . . Abel, G. A. (2011). Timeliness and quality of Pseudothrombocytopenia—a caveat. Journal of the Indian Medical diagnostic care for Medicare recipients with chronic lymphocytic Association, 109, 476–478. leukemia. Cancer, 117, 1470–1477. 8. Padayatty, J., Grigoropoulos, N., Gilligan, D., & Follows, G. (2010). 17. Ma, X., Does, M., Raza, A., & Mayne, S. T. (2007). M yelodysplastic Platelet satellitism. European Journal of Haematology, 84, 366. syndromes: Incidence and survival in the United States. Cancer, 9. Bessis, M. (1973). The erythrocytic series. In Living blood cells and 109, 1536–1542. their ultrastructure (pp. 194–245). Berlin: Springer-Verlag. This page intentionally left blank Section Three The Anemias 199 Chapter 11 Introduction to Anemia Shirlyn B. McKenzie, PhD Catherine N. Otto, PhD, MBA Objectives—Level I Upon completion of this chapter, the student should be able to: 1. Calculate the reticulocyte production index mechanisms of anemia and provide from reticulocyte results, hematocrit, and expected results. RBC count. 6. Define hemolysis and reconcile a normal 2. Identify laboratory tests used to evaluate hemoglobin concentration in compensated erythrocyte destruction and production. hemolytic disease. 3. Given CBC and RPI results, categorize 7. Assess laboratory results in intravascular an anemia according to morphologic and extravascular hemolysis. classification. 8. Summarize the clinical findings associated 4. Correlate polychromatophilia on a blood with anemia. smear with other laboratory results of eryth- 9. Explain the difference between intrinsic and rocyte production and destruction. extrinsic erythrocyte defects. 5. List the laboratory tests that can be used to help identify the pathophysiological Objectives—Level II Upon completion of this chapter, the student should be able to: 1. Relate adaptations to anemia with patient 4. Assess bone marrow response to anemia symptoms. given CBC, absolute reticulocyte count, 2. Correlate patient history and clinical symp- immature reticulocyte fraction, and reticulo- toms with laboratory results in anemia. cyte hemoglobin. 3. Evaluate clinical findings of hemolytic ane- 5. Assess and interpret bone marrow findings mia and differentiate those associated with in the presence of anemia. acute and chronic disease. 200 Introduction to Anemia 201 6. Compare the sensitivity and specificity as explain how laboratory results can be used they relate to tests used to screen and con- to differentiate. firm a differential diagnosis of anemia. 10. Recommend tests that could be necessary to 7. Compare the morphologic and functional make a diagnosis of hemolytic disease. classification of anemia. 11. Choose appropriate reflex tests to deter- 8. Given laboratory results and clinical find- mine anemia classification and evaluate the ings, classify an anemia in terms of mor- results. phology and pathophysiologic mechanism. 12. Explain the association of anisocytosis and 9. Compare and contrast the processes of intra- variation in erythrocyte chromia with dis- vascular and extravascular hemolysis and ease states. Chapter Outline Objectives—Level I and Introduction 202 Classification of Anemias 211 Level
II 200 How Anemia Develops 202 Summary 220 Key Terms 201 Interpretation of Abnormal Hemo- Review Questions 221 Background Basics 201 globin Concentrations 203 References 223 Case Study 202 Adaptations to Anemia 203 Overview 202 Diagnosis of Anemia 204 Key Terms Anemia Hemosiderinuria CHr Immature reticulocyte fraction (IRF) Compensated hemolytic disease Megaloblastic Functional iron-deficiency Pancytopenia Hemoglobinemia Percent hypochromic red cells Hemoglobinuria Reticulocyte hemoglobin (RET-He) Hemolysis Reticulocyte production index (RPI) Background Basics The information in this chapter builds on the concepts • Summarize the role of hemoglobin in gaseous trans- learned in previous chapters. To maximize your learning port. (Chapter 6) experience, you should review these concepts before start- • Discuss the appearance of a normal bone marrow ing this unit of study: and list reasons that a bone marrow examination Level I could be necessary. (Chapter 38) • Describe and recognize abnormal variation in eryth- • Diagram the process of intravascular and extravas- rocyte morphology and distribution on stained cular hemolysis. (Chapter 5) smears. (Chapter 10) • Calculate the reticulocyte count (absolute and rela- • Describe the production, maturation, and destruc- tive). (Chapter 10) tion of blood cells and explain how the balance • Calculate RBC indices and classify erythrocytes between erythrocyte production and destruction is based on results. (Chapter 10) maintained; describe the normal erythrocyte concen- • Discuss the principle and give reference intervals tration and appearance. (Chapters 3, 5) for the following tests: hemoglobin, hematocrit, 202 Chapter 11 reticulocyte count, erythrocyte count, and erythro- • Correlate CBC results with findings on the peripheral cyte indices; calculate indices. (Chapters 10, 37) blood smear and other laboratory test results; deter- mine the validity and accuracy of CBC results and sug- Level II gest corrective action when necessary. (Chapters 10, 37) • Correlate peripheral blood findings with bone mar- • Review the erythrocyte membrane structure. row appearance. (Chapter 38) (Chapter 5) For example, if a patient experiencing iron-deficiency ane- CASE STUDY mia due to chronic blood loss were given iron or a blood We refer to this case study throughout the chapter. transfusion, the hemoglobin level might temporarily rise; George, a 50-year-old male, visited his doctor when however, if the cause of the deficiency is not isolated and he noted that the whites of his eyes appeared yel- treated, serious complications of the primary disease (cause low and that he had dark urine. His CBC revealed of blood loss) could develop, and the anemia would prob- a hemoglobin of 3.1 g/dL. ably return after ceasing treatment. Thus, it is necessary to Given this clinical description, consider what identify and understand the etiology and pathogenesis of laboratory tests should be ordered to assist in an anemia to institute correct treatment. diagnosis. How Anemia Develops To understand how anemia develops, it is necessary to Overview understand normal erythrocyte kinetics. Total erythrocyte mass (M) in the steady state is equal to the number of new This chapter is a general introduction to anemia. It begins erythrocytes produced per day (P) times the erythrocyte life with a description of how anemia develops and the body’s span (S), which is normally about 100–120 days. adaptations to a decrease in hemoglobin. The emphasis of the chapter is on the laboratory investigation of anemia. M = P * S This includes discussion of screening tests used to diagnose anemia and other more specific tests used to identify the Mass Production Survival etiology and pathophysiology of the anemia. The chapter Thus, the average 70 kg man with 2 liters of erythro- concludes with a description of the morphologic and func- cytes must produce 20 mL of new erythrocytes each day tional classification schemes of anemia and the use of labo- to replace the 20 mL normally lost due to cell senescence. ratory tests to correctly classify an anemia. The hemolytic 2000 mL (M) anemias are discussed in more depth than the other func- = 20 mL/day (P) 100 days (S) tional classifications as these anemias have several possible subclassifications that help in understanding the laboratory From this formula, it is clear that if the survival time investigation used to elucidate the pathophysiology. of the erythrocyte is decreased by one-half, as can occur in hemolysis or hemorrhage, the bone marrow must double production to maintain mass at 2000 mL. Introduction 2000 mL (M) = 40 mL/day (P) Anemia is functionally defined as a decrease in the com- 50 days (S) petence of blood to carry oxygen to tissues, thereby caus- New erythrocytes are released as reticulocytes. Thus, ing tissue hypoxia. In clinical medicine, the word refers to a an increase in the absolute reticulocyte count in the periph- decrease in the normal concentration of hemoglobin and/or eral blood is a result of the increased production of cells. erythrocytes. It is one of the most common problems encoun- The marrow can compensate for decreased survival in this tered in clinical medicine. However, anemia is not a disease manner until production is increased to a level 5–8 times but the expression of an underlying disorder or disease; it is normal, which is the maximal functional capacity of the mar- an important clinical marker of a disorder that could be basic row. The increase in erythropoiesis is limited by the amount or sometimes more complex. Therefore, once the diagnosis of of iron that can be mobilized for hemoglobin synthesis. anemia is made, the physician must determine its exact cause. The term functional iron-deficiency is used when the total Treating anemia without identifying its cause could not body iron is adequate but cannot be mobilized fast enough only be ineffective but also lead to more serious problems. for the needed increase in erythropoiesis, a condition that Introduction to Anemia 203 often occurs as a result of treatment with erythropoiesis- dependent on the altitude.2 The hemoglobin reference inter- stimulating agents.1 Thus, if all necessary raw materials for val at high altitudes is higher than the reference interval at cell synthesis are readily available, erythrocyte life span can lower altitudes. Therefore, signs of anemia at high altitudes decrease to about 18 days before marrow compensation is can occur at higher hemoglobin concentrations than at sea inadequate and anemia develops. If, however, bone mar- level. Cigarette smoking has a similar effect. The hemoglo- row production of erythrocytes does not adequately increase bin and hematocrit reference interval for cigarette smokers when the erythrocyte survival is decreased, the erythrocyte is higher than for nonsmokers.3 In most cases, the physi- mass cannot be maintained and anemia develops. There is cian integrates the patient’s clinical findings with laboratory no mechanism for increasing erythrocyte life span to help test results to correctly diagnose the illness. The examples accommodate for an inadequate bone marrow response. mentioned serve to emphasize the fact that when making Anemia can develop if (1) erythrocyte loss or destruction a diagnosis of anemia, the physician depends not only on exceeds the maximal capacity of bone marrow erythrocyte laboratory test results but also considers patient history, production or (2) the bone marrow erythrocyte production is physical examination, and symptoms. impaired. Anemia can be classified according to these prin- ciples (functional classification) based on laboratory test results, which aid the physician in diagnosis. The functional Adaptations to Anemia classification includes survival defects, proliferation defects, and maturation defects (which have a high degree of ineffec- Signs and symptoms of anemia range from slight fatigue tive erythropoiesis). A morphologic classification is also pos- or barely noticeable physiologic changes to life-threatening sible based on the erythrocyte indices and reticulocyte count. reactions depending on: These classifications are discussed later in this chapter. • Rate of onset • Severity of blood loss Interpretation of Abnormal • Ability of the body to adapt Hemoglobin Concentrations With rapid loss of blood as occurs in acute hemorrhage, clinical manifestations are related to hypovolemia and vary Diagnosis of anemia is usually made after the discovery with the amount of blood lost. A normal person can lose up of a decreased hemoglobin concentration from the CBC to 1000 mL, or 20%, of total blood volume and not exhibit results (Chapter 10). Hemoglobin is the carrier protein of oxy- clinical signs of the loss at rest, but tachycardia is common gen; thus, it is expected that a decrease in its concentration with mild exercise.4 Severe blood loss of 1500–2000 mL or is accompanied by a decrease in oxygen delivery to tissues. 30–40% of total blood volume leads to circulatory collapse Screening for anemia generally relies on the relative and shock. Death is imminent if the acute loss reaches 50% hemoglobin concentration and hematocrit (grams of Hb of total blood volume (2500 mL). per deciliter of whole blood or liters of RBCs per liter of Slowly developing anemias can show an equally severe whole blood). However, the hemoglobin or hematocrit can drop in hemoglobin as is seen in acute blood loss, but the be misleading as changes in these parameters can reflect threat of shock or death is not usually present. The reason altered plasma volume and not a change in the RBC mass. for this apparent discrepancy is that in slowly developing • In hypervolemia, the total blood volume increases. This anemias, the body has several adaptive mechanisms that is primarily caused by a plasma volume increase while allow organs to function at hemoglobin levels of up to 50% the erythrocyte mass remains stable. In this case, the less than normal. The adaptive mechanisms are of two types: hemoglobin/hematocrit concentration is dispropor- an increase in the oxygenated blood flow to the tissues and tionately low relative to the red cell mass. an increase in oxygen utilization by the tissues (Table 11-1). • In hypovolemia, such as occurs in dehydration, a decrease in plasma volume relative to the RBC mass Table 11.1 Adaptations to Anemia occurs. As a result, the hemoglobin/hematocrit can be high or normal relative to the RBC mass. Increase in oxygenated Increase in oxygen utilization blood flow by tissues In acute blood loss, both the plasma volume and RBC • Increase in respiration rate and • Increase in 2,3-BPG in mass are decreased, resulting in a normal hemoglobin mea- deepen inspiration erythrocytes surement initially. As the plasma volume increases to re- • Increase in cardiac rate • Decreased oxygen affinity of establish total blood volume for adequate cardiac function, hemoglobin in tissues due to Bohr effect the hemoglobin concentration decreases. Diagnosis of anemia can require an upward adjust- • Increase in cardiac output ment of hemoglobin and hematocrit reference intervals • Increase in circulation rate 204 Chapter 11 Increase in Oxygenated Blood Flow essential to improve patient outcomes. The diagnosis of anemia and determination of its cause are made by using Increasing the cardiac rate, cardiac output, and circula- a combination of information received from patient his- tion rate can increase oxygenated blood flow to the tissues. tory, physical examination, and laboratory investigation Oxygen uptake in the alveoli of the lungs is increased by (Table 11-2). deepening the amount of inspiration and increasing the res- piration rate. In anemia, decreased blood viscosity due to the decrease in erythrocytes and decrease in peripheral resis- History tance help to increase the circulation rate, delivering oxygen The patient’s history including symptoms can reveal some to tissues at an increased rate. Blood flow to the vital organs, important clues as to the cause of the anemia. Information the heart and brain, can preferentially increase whereas flow solicited by the physician should include dietary habits, to tissues with low oxygen requirements and normally high medications taken, possible exposure to chemicals or tox- blood supply such as skin and the kidneys decreases. ins, and description and duration of the symptoms. The most common complaint is tiredness. Muscle weakness and Increase in Oxygen Utilization fatigue develop when there is not enough oxygen available by Tissue to burn fuel for the production of energy. Severe drops in hemoglobin can lead to a variety An important compensatory mechanism at the cellular level of additional symptoms. When oxygen to the brain is that allows the tissue to extract more oxygen from hemo- decreased, headache, vertigo, and syncope can occur. Dys- globin involves an increase in 2,3-BPG (2,3-bisphosphogly- pnea and palpitations from exertion, or occasionally while cerate—also known as 2,3-diphosphoglycerate/2,3-DPG) within at rest, are not uncommon complaints. The patient should the erythrocytes (Chapter 6). An increase in erythrocyte 2,3-
be questioned as to any overt signs of blood loss, such as BPG permits the tissues to extract more oxygen from the hematuria, hematemesis, and bloody or black stools. Stud- blood even though the PO2 remains constant; this shifts the ies of anemia in the elderly reveal that in this population, oxygen dissociation curve to the right. It is not clear exactly anemia is associated with a decline in physical performance, how anemia stimulates this increase in cellular 2,3-BPG. increased cognitive impairment, falls, frailty, hospitaliza- Another adaptive mechanism at the cellular level tions, and mortality.5,6 Family history can help define the involves the Bohr effect. The scarcity of oxygen causes rarer hereditary types of hematologic disorders. For exam- anaerobic glycolysis by muscles and other tissue, which ple, sickle cell anemia and thalassemia are frequently mani- produces a buildup of lactic acid. In addition, H+ is gener- fested to some degree in several members of the immediate ated from carbonic acid (H2CO3) formed during the trans- family. port of CO2 from the tissues to the lungs (Chapter 6). This acidosis decreases hemoglobin’s affinity for oxygen in the Clinical Presentation capillaries, thus causing release of more oxygen to the tis- Physical examination of the patient helps the physi- sues and shifting the oxygen dissociation curve to the right. cian detect the adverse effects of a long-standing anemia Even with these physiologic adaptations, different ane- (Table 11-2). Signs of anemia are associated with decreased mic patients respond differently to similar changes in hemo- hemoglobin levels and in hemolytic anemias with increased globin levels. The extent of the physiologic adaptations is hemoglobin catabolism and erythropoiesis. General physi- influenced by: cal findings include the following: 1. Severity of the anemia • Changes in epithelial tissue from oxygen deprivation 2. Competency of the cardiovascular and respiratory systems are noted in some patients. Sites commonly examined 3. Oxygen requirements of the individual (physical and for pallor in clinical settings are conjunctiva, nailbed, metabolic activity) palm, and tongue. The presence of pallor, particularly 4. Duration of the anemia conjunctival pallor, has been shown to be a cost-effec- 5. Disease or condition that caused the anemia tive and feasible method to screen for moderate or severe anemia in a variety of settings.7,8 6. Presence and severity of coexisting disease • Hypotension can accompany significant decreases in blood volume. Diagnosis of Anemia • Heart abnormalities can occur as a result of the increased cardiac workload associated with the physi- Anemia can impair an individual’s ability to carry on ologic adaptations to anemia. Cardiac problems usually activities of daily living and decrease the individual’s occur only with chronic or severe anemia (hemoglobin quality of life. Thus, accurate diagnosis and treatment are less than 7 g/dL). Introduction to Anemia 205 Table 11.2 Important Information for Evaluating a Patient for Anemia Diagnosis of anemia and determination of its cause requires information obtained from the patient history, physical examination, and laboratory data. Signs of Anemia Obtained by Physical Patient History Examination Laboratory Investigation • Dietary habits • Skin pallor • Erythrocyte count • Medications • Pale conjunctiva • Hemoglobin • Exposure to chemicals and toxins • Koilonychia • Hematocrit • Symptoms and their duration • Hypotension • Erythrocyte indices: MCV, MCH, MCHC • Fatigue • Jaundice • Reticulocyte count, reticulocyte production • Muscle weakness • Smooth tongue index (RPI), corrected reticulocyte count, CHr or Ret-He, IRF • Headache • Neurological dysfunction • Blood smear examination • Vertigo • Hepatomegaly • Leukocyte and platelet quantitative and qualita- • Syncope • Splenomegaly tive examination • Dyspnea • Bone deformities in congenital anemias • Peripheral blood smear evaluation for presence • Palpitations • Gallstones of spherocytes, schistocytes and other poikilo- • Dark or red urine • Extramedullary hematopoietic masses cytes, and erythrocyte inclusions • Previous record of abnormal blood examination • Tests to measure erythrocyte destruction depending on other information available: • Family history of abnormal blood examination • Serum bilirubin • Haptoglobin • Hemopexin • Lactate dehydrogenase • Methemalbumin • Urine hemosiderin • Fecal and urine urobilinogen • Blood in urine • Expired CO • Bone marrow examination (depending on results of other laboratory tests and patient clinical data) • Organomegaly of the spleen and liver are of primary particular type of anemia. These include koilonychia in importance in establishing the extent of involvement iron deficiency and a smooth tongue in megaloblastic of the hematopoietic system in the production and anemia. Hemolytic anemias are associated with jaundice destruction of erythrocytes. Massive splenomegaly and dark or red urine (if intravascular hemolysis is pres- is characteristic of some hereditary chronic anemias. ent). Gallstones consisting primarily of bilirubin are com- Splenic hypertrophy is a constant finding in hemolytic mon in congenital and other chronic hemolytic anemias. anemias with extravascular hemolysis such as in some Extramedullary hematopoietic masses can be found in autoimmune hemolytic anemias when the spleen is the hereditary hemoglobinopathies, some of which are the primary site of destruction of antibody-sensitized thought to be extrusions of the marrow cavity through erythrocytes. thinned bone cortex. Small colonies of erythrocytes also • Expansion of the bone marrow, consequently thinning can be found in the spleen, liver, lymph nodes, and peri- cortical bone and widening the spaces between inner nephric tissue. These masses can cause pressure symptoms and outer tables of bone, is present in chronic severe on adjacent organs.9 hemolytic anemias. In children, this expansion is evi- In addition to determining the extent of anemic mani- dent as skeletal abnormalities. These bone changes can festations, physical examination helps to establish the result in spontaneous fractures and osteoarthropathy.9 underlying disease process causing the anemia. Some disor- ders associated with anemia include chronic diseases such • Anemia can occur secondarily to a defect in hemosta- as rheumatoid arthritis as well as malignancies, gastrointes- sis. The presence of bruises, ecchymoses, and petechiae tinal lesions, kidney disease, parasitic infection, and liver indicates that the platelets may be involved in the dis- dysfunction. Anemia in pregnancy is common. The anemia order that is producing the anemia. can be due to a variety of underlying conditions includ- In addition to these general physical findings asso- ing iron or folate deficiency, inflammatory conditions, and ciated with anemia, findings can be associated with a hemodilution.10 206 Chapter 11 Laboratory Evaluation Table 11.3 Hemoglobin (Hb) and Hematocrit (Hct) After the physical examination and patient history, a health Cutoffs for a Diagnosis of Anemia in Children, Nonpregnant care provider who suspects the patient has anemia orders Females, and Males laboratory tests (Table 11-2). Initially, screening tests are Age (yrs) by Sex Hb (g/dL) Hct (%) performed to determine whether anemia is present and to Both Sexes evaluate erythrocyte production and destruction/loss. The 1–1.9 11.0 33.0 initial screening test is the complete blood count (CBC), 2–4.9 11.2 34.0 which includes red blood cell (RBC) count, hemoglobin, 5–7.9 11.4 34.5 hematocrit, RBC indices, white blood cell (WBC) count, 8–11.9 11.6 35.0 platelet count, and, depending on instrumentation, the differential count (Chapter 10). Depending on these test Female results, additional tests such as the reticulocyte count, bili- 12–14.9 11.8 35.5 rubin, and microscopic review of the blood smear for abnor- 15–17.9 12.0 36.0 mal cell morphology may be suggested. In addition, the 18 and above 12.0 36.0 urine and stool can be examined for the presence of blood. Male When combined with the information from the history and 12–14.9 12.3 37.0 physical examination of the patient, results of these tests 15–17.9 12.6 38.0 can give insight to the cause of the anemia. These routine 18 and above 13.6 41.0 tests can be followed by a protocol of specific diagnostic Based on fifth percentile values from the Second National Health and Nutrition Examina- tion survey conducted after excluding persons with a higher likelihood of iron deficiency. tests that help establish the etiology and pathophysiology Centers for Disease Control (CDC). (1989). CDC criteria for anemia in children and child- of the anemia. These specific tests will be discussed in the bearing-aged women. Morbidity and Mortality Weekly Report, 38(22), 400–404. appropriate chapters on anemia. ERYTHROCYTE COUNT, HEMATOCRIT, AND Table 11.4 Altitude Adjustments for Hemoglobin (Hb) and HEMOGLOBIN Hematocrit (Hct) Cutoffs for a Diagnosis of Anemia The erythrocyte count, hematocrit, and hemoglobin are Altitude (ft) Hb (g/dL) Hct (%) determined to screen for the presence of anemia. In a clinic or physician’s office with limited resources, screening may Less than 3000 — — be limited to either the hematocrit or hemoglobin. If one of 3000–3999 +0.2 +0.5 these screening tests is abnormal, it is helpful to calculate 4000–4999 +0.3 +1.0 the red cell indices. 5000–5999 +0.5 +1.5 A decreased concentration in one or more of these 6000–6999 +0.7 +2.0 parameters, based on the individual’s age and sex, should 7000–7999 +1.0 +3.0 be followed by other laboratory tests to help establish cri- 8000–8999 +1.3 +4.0 teria for diagnosis. The Centers for Disease Control and 9000–9999 +1.6 +5.0 Prevention (CDC) recommended cutoff values for a diag- More than 10,000 +2.0 +6.0 nosis of anemia according to age and sex are provided Centers for Disease Control (CDC). (1989). CDC criteria for anemia in children and child- bearing-aged women. Morbidity and Mortality Weekly Report, 38(22), 400–404. in Table 11-3. Upward adjustments for these cutoff values should be utilized for individuals living at high altitudes and for those who smoke. There is a direct dose–response Table 11.5 Adjustments for Hemoglobin (Hb) and relationship between the amount smoked and the hemo- Hematocrit (Hct) Cutoffs for a Diagnosis of Anemia in Smokers globin level.3 The CDC recommended adjustments are included in Tables 11-4 and 11-5. Hemoglobin and hemato- Characteristic Hb (g/dL) Hct (%) crit values also vary in pregnancy, with a gradual decrease Nonsmoker — — in the first two trimesters and a rise during the third tri- Smoker (all) +0.3 +1.0 mester (Table 11-6). 1 291 pack/day +0.3 +1.0 Although anemia in elderly persons is prevalent, 1–2 packs/day +0.5 +1.5 it should not be considered a normal part of aging. The More than 2 packs/ +0.7 +2.0 third National Health and Nutrition Examination Survey day (NHANES III, 1988–94) studied a group of noninstitu- Centers for Disease Control (CDC). (1989). CDC criteria for anemia in children and child- bearing-aged women. Morbidity and Mortality Weekly Report, 38(22), 400–404. tionalized older individuals and found that after age 65, the prevalence of anemia rose to 11% in men and 10.2% over 85 years of age (26% of men and 20% of women). In in women.11,12 The prevalence for those in nursing homes this group, one-third was due to blood loss/nutritional is higher.6,13 The highest prevalence of anemia is in those deficiency, one-third was due to anemia of chronic disease, Introduction to Anemia 207 Table 11.6 Hemoglobin Cutoffs for a Diagnosis of Anemia in Pregnancy by Week and Trimestera Gestation (weeks)/Trimester 12/1† 16/2 20/2† 24/2 28/3 32/3† 36/3 40/Term Mean Hb (g/dL) 12.2 11.8 11.6 11.6 11.8 12.1 12.5 12.9 5th percentile Hb 11.0 10.6 10.5 10.5 10.7 11.0 11.4 11.9 values (g/dL) Equivalent 5th percen- 33.0 32.0 32.0 32.0 32.0 33.0 34.0 36.0 tile Hct values (%)† a Based on pooled data from four European surveys of healthy women taking iron supplements. Hb values adapted for the trimester-specific cutoffs. † Hematocrit. From Centers for Disease Control (CDC). (1989). CDC criteria for anemia in children and childbearing-aged women. Morbidity and Mortality Weekly Report, 38(22), 400–404. inflammation, or chronic renal failure, and one-third was unexplained. The unexplained anemia may be due to multi- CASE STUDY (continued from page 202) ple causes. Even when allowing for a difference in reference George’s only complaint was dark urine and the interval, the prevalence of anemia in African Americans yellow color of his eyes. His CBC results were over 65 years of age is three times higher than in Cauca- hemoglobin 31 g/L (3.1 g/dL), hematocrit 8%, RBC sians. Prevalence in Mexican Americans is similar to that count 0.71 * 106/mcL, RDW 21.6, reticulocyte in Caucasians. count 22%. Calculate the erythrocyte indices. Variations in hemoglobin also are reported to occur as a result of blood-collection techniques. Hemoglobin values 1. Does this information suggest acute or chronic are about 0.7 g/dL higher if the patient’s blood is obtained blood loss? What is the significance of the RDW? while the individual is in an upright position rather than supine. Prolonged vasoconstriction by the
tourniquet can cause hemoconcentration of the sample and elevate the hemoglobin value. Checkpoint 11.1 Explain why a 30-year-old female who smokes a pack of ciga- ERYTHROCYTE INDICES rettes a day and lives in the Rocky Mountains can be diagnosed Because abnormal morphology is characteristic of dis- with anemia when her hemoglobin is 12 g/dL. tinct types of anemia, the erythrocyte indices (MCV, MCH, MCHC, RDW) give important clues as to the pathophysiology of the anemia and thus help to direct RETICULOCYTE COUNT reflex testing (Chapters 10, 37). For instance, micro- The peripheral blood reticulocyte count indicates the cytic hypochromic cells are highly suggestive of iron- degree of effective bone marrow erythropoietic activity deficiency anemia, whereas macrocytic normochromic and is one of the most useful and cost-effective labora- cells are associated with vitamin B12 or folate deficiency. tory tests in monitoring anemia and response to therapy The indices are used in the morphologic classification of (Chapters 10, 37). It is also helpful in directing the initial anemia. investigation of anemia that assists in classification of ane- Several manufacturers of hematology analyzers have mia. Increased numbers of polychromatophilic erythrocytes added two new red cell parameters that provide valuable on Romanowsky-stained smears indicates an increased information for evaluating the iron status of red cells. These reticulocyte count. new parameters calculate the percentage of hypochromic The reticulocyte count is commonly performed on and hyperchromic red cells. The percent hypochromic red an automated hematology instrument using a variety of cells indicates the availability of iron during red cell pro- methodologies including fluorescent flow cytometry or duction and maturation in the previous 3 months. Erythro- scattered light with methylene blue staining, depending on cytes are hypochromic before they become microcytic the manufacturer. These automated instruments provide during iron-deficient erythropoiesis.14 Estimates of hypo- a higher degree of accuracy, precision, and standardiza- chromic red cells are clinically useful to assess patients who tion than the manual reticulocyte counts. The availability are being treated with erythropoiesis-stimulating agents, of automated hematology instruments that can perform especially those who have kidney diseases.14 Although reticulocyte counts has significantly decreased the fre- physiologically a red cell cannot contain an excess of hemo- quency in which a manual reticulocyte method is used globin, these newer instruments determine the percentage (Chapter 39).15 In both automated and manual methods, retic- of hyperchromic red cells, which reflects the presence of ulocyte test results are expressed as the percentage of reticulo- spherocytes. cytes in relation to total RBC count or as the absolute number. 208 Chapter 11 The reference interval varies among laboratories and the procedure used, but is about 0.5–2.0% or 25975 * 103/mcL Table 11.7 Correlation of the Hematocrit with Reticulocyte Maturation Time in the Peripheral Blood for manual reticulocyte counts. Because of a lack of stan- dardization, there is no single reference interval for Reticulocyte Maturation reticulocyte parameters determined by flow cytometry.15 Hematocrit (L/L) Time (days) Laboratories need to determine reference intervals for their Greater than or equal to 0.35 1.0 own instrument and method. Greater than or equal to 0.25–0.35 1.5 The corrected reticulocyte count is a means to adjust the Greater than or equal to 0.15–0.25 2.0 reticulocyte count in proportion to the severity of anemia. In Less than or equal to 0.15 2.5 this procedure, the percentage of reticulocytes is multiplied by the ratio of the patient’s hematocrit to an average age- blood when the need for granulocytes is increased. To cor- and sex-appropriate normal hematocrit. rect for the prolongation of maturation of these circulating Patient hematocrit * shift reticulocytes, and the degree of anemia, the reticu- , Reticulocyte Normal hematocrit locyte production index (RPI) is calculated by using the = corrected reticulocyte count following formula: For practical purposes, the corrected reticulocyte count Patient>s hematocrit or preferably absolute reticulocyte count is used to assess 0.45 L/L (normal hematocrit) the degree of erythropoiesis in anemic patients.15 In patients Reticulocyte count (,) with anemia, a corrected reticulocyte count less than 2% or * = RPI Reticulocyte maturation time (days) an absolute reticulocyte count less than 75 * 103/mcL is associated with hypoproliferative anemias, whereas a cor- For example, if a patient with a 0.25 L/L hematocrit had rected count greater than 2% or an absolute count greater a 15% reticulocyte count, the RPI would be: than 100 * 103/mcL is associated with an appropriate response to blood loss and hemolytic anemias.16 Counts 0.25 L/L 15, * = 4.2 RPI between 75 and 100 * 103/mcL should be interpreted while 0.45 L/L 2.0 considering factors such as the severity of the anemia and The RPI is a good indicator of the adequacy of the other clinical information. bone marrow response in anemia. Generally speaking, an RPI greater than 2 indicates an appropriate bone marrow response, whereas an RPI less than 2 indicates an inad- Checkpoint 11.2 equate compensatory bone marrow response (hypopro- Is it possible to have an increased relative reticulocyte count but liferation) or an ineffective bone marrow response. When an absolute reticulocyte count in the reference interval? Explain. utilized in this way, the reticulocyte count provides direc- tion for the course of investigation concerning anemia eti- ology and pathophysiology. Enumeration of absolute and QUANTITATION OF RETICULOCYTE IMMATURITY relative reticulocyte counts is more accurate using an auto- Under normal physiologic conditions when there is no mated hematology analyzer. If these analyzers are available anemia, the reticulocytes are released into the peripheral and the absolute reticulocyte count is performed, the cor- blood where they spend another day maturing to the rected reticulocyte and RPI calculations do not have to be erythrocyte. When the need for erythrocytes in the circula- calculated. Analyzers with the capability to count absolute tion increases, the bone marrow releases reticulocytes ear- numbers of reticulocytes also evaluate their maturity level. lier than normal. These more immature reticulocytes are Reticulocyte maturity level can be classified based on semi- called stress reticulocytes or shift reticulocytes. They appear as quantitative assessment of RNA concentration within the large polychromatophilic cells on the Romanowsky-stained maturing erythrocyte. Younger reticulocytes contain more blood smear. It takes longer for these reticulocytes to mature RNA than more mature reticulocytes. The methods vary in the peripheral blood because the bone marrow matura- and include measurement of fluorescence, absorbance, or tion time is added to the peripheral blood maturation time light scatter of stained cells. The term used for this index is (Table 11-7). The more severe the anemia, the earlier the the immature reticulocyte fraction (IRF) to reflect the least reticulocyte is released. In a stimulated marrow, hemato- mature fraction of reticulocytes. Reference intervals vary by crit levels of 35%, 25%, and 15% (0.35, 0.25, 0.15 L/L) are manufacturer.15 associated with early reticulocyte release and a prolonga- The IRF can be helpful in evaluating bone marrow tion of the reticulocyte maturation in peripheral blood to erythropoietic response to anemia, monitoring anemia, and approximately 1.5, 2.0, and 2.5 days, respectively. This is evaluating response to therapy. In anemia, an increased IRF similar to the left shift in granulocytes seen in peripheral generally indicates an adequate erythropoietic response, Introduction to Anemia 209 whereas a normal or subnormal IRF reflects an inadequate or include schistocytes, dacryocytes, spherocytes, acanthocytes, no response to the anemia.14,15 As the bone marrow increases and marked erythrocyte shape abnormalities in normocytic production of erythrocytes, an observable increase in the anemia without evidence of hemolysis. If artifactual mor- IRF occurs before an increase in the reticulocyte count or an phology is suspected, the erythrocytes should be examined in increase in hemoglobin, hematocrit, or RBC count. After a wet preparation. If the abnormal morphology is present in bone marrow transplantation, an increased IRF has been this preparation, the possibility of artifacts can be eliminated observed as one of the first signs of cell recovery. In patients (Chapter 10). receiving human recombinant erythropoietin (rHuEPO) Characterization of predominant erythrocyte size or iron therapy for anemia, an increased IRF can indicate and variation in size is helpful in morphologic classifica- increased erythropoietic activity or a response to the therapy. tion of anemia. As with poikilocytes, anisocytes are some- The Clinical Laboratory Standards Institute (CLSI) rec- times associated with particular pathologic conditions ommends that the IRF index replace the RPI.15 Laboratories (Table 11-8). Anisocytosis can be detected by examining the that do not have instruments that measure this parameter blood smear and/or by reviewing the MCV, histograms, can use the RPI. cytograms, and RDW. Another component of automated reticulocyte analysis Microcytes (MCV less than 80 fL) are usually hypo- is the reticulocyte hemoglobin parameter. Reticulocyte chromic (decreased MCHC and MCH) but can be nor- hemoglobin is a measure of the hemoglobin content of retic- mochromic or hyperchromic if spherocytes are present ulocytes, which reflects the availability of functional iron for (Figure 11-1). Microcytes are usually associated with ane- the cell and the incorporation of iron into hemoglobin over mias characterized by defective hemoglobin formation the last several days. Thus, reticulocyte hemoglobin indi- (Table 11-8). cates response or lack of response to iron therapy. The Advia 120 and Bayer 2120 by Siemens-Healthcare directly measure the hemoglobin content of reticulocytes, reported as CHr Table 11.8 Diseases Associated with Variation and (analogous to the MCH of erythrocytes) as well as the mean Abnormalities in Erythrocyte Size reticulocyte cell hemoglobin concentration (CHCMr; analo- Terminology Associated Disease States gous to the MCHC of erythrocytes). The Sysmex Automated Anisocytosis Anemias associated with an Hematology Analyzers that perform reticulocyte counts increased RDW (see Table 11-14) have an equivalent CHr parameter called the reticulocyte Microcytosis Iron-deficiency anemia; thalas- hemoglobin (RET-He), which is measured using the for- MCV less than 80 fL semia; sideroblastic anemia, long- standing anemia of chronic disease ward scatter and side fluorescence of the reticulocytes and Macrocytosis Megaloblastic anemias; hemolytic a proprietary algorithm. A recent study found that the refer- MCV greater than 100 fL anemia with reticulocytosis; recov- ence intervals for RBC and reticulocyte hemoglobin in pico- ery from acute hemorrhage; liver grams for the two methods are comparable.15 disease; asplenia; aplastic anemia; myelodysplasia; endocrinopathies; alcoholism CASE STUDY (continued from page 207) George’s RBC count is 0.7 * 106/mcL and his reticulocyte count is 22%. 2. Calculate his absolute reticulocyte count. Is this count increased, decreased, or normal? BLOOD SMEAR EXAMINATION Although doing so is not always necessary, reviewing the stained blood smear assists in diagnosing the type of ane- mia.17 Various pathological conditions intrinsic or extrinsic to the cell can alter the erythrocyte’s normal morphology. Careful examination of a stained blood smear reveals these morphologic aberrations. Descriptions of specific poikilo- Figure 11.1 Microcytic, hypochromic erythrocytes. Compare cytes and the disorders with which they are associated are the size of the erythrocytes with the nucleus of the lymphocyte. Normocytic cells are about the same size as the nucleus. There included in Chapter 10. Some shapes and sizes are particu- is only a thin rim of hemoglobin around the periphery of the red larly characteristic of specific underlying hematologic disor- blood cells, indicating they are hypochromic. Note the elliptocytes ders or malignancies (Table 10-8). Abnormal shapes to report (Peripheral blood; Wright-Giemsa stain; 1000* magnification). 210 Chapter 11 Macrocytes (MCV greater than 100 fL) usually contain Other abnormalities found on stained blood smears an adequate amount of hemoglobin (normal MCHC and that can assist in diagnosis and classification of anemia normal to increased MCH; Figure 11-2). These cells are asso- include variations in erythrocyte color (Table 10-9), pres- ciated with impaired DNA synthesis as occurs in vitamin ence of erythrocyte inclusions (Table 10-10), and abnormal B12 or folate deficiency as well as other diseases (Table 11-8). distribution of erythrocytes (Table 10-6). Anemias associated with reticulocytosis, such as hemolytic anemias, can have an increased MCV as young reticulocytes LEUKOCYTE AND PLATELET ABNORMALITIES are generally larger than mature erythrocytes. Some nutritional deficiencies, stem cell disorders, and bone marrow abnormalities affect the production, function, and/ or morphology of all hematopoietic cells; thus, evaluation CASE STUDY (continued from page 209) of the quantity and morphology of leukocytes and plate- George’s blood smear revealed marked lets can supply additional important data as to the cause spherocytosis. of anemia. 3. Explain the importance of this finding. TESTS FOR ERYTHROCYTE DESTRUCTION Tests of erythrocyte destruction are important in evaluat- 4. Explain George’s abnormal indices. ing erythrocyte survival (Table 11-9). If the hemoglobin concentration is stable over at least
several days in an ane- mic patient, the measurements of erythrocyte production including marrow cellularity and RPI are indirect measure- ments of erythrocyte destruction. Serum unconjugated bili- rubin is primarily derived from hemoglobin catabolism; its concentration in the absence of hepatobiliary disease can yield further information concerning erythrokinetics. Increased unconjugated bilirubin indicates increased hemo- globin catabolism, either intravascular or extravascular. Conversely, anemias that are due to chronic and acute blood loss and hypoproliferative anemias are associated with nor- mal or decreased serum bilirubin because the number of erythrocytes catabolized is decreased. Cytoplasmic matu- ration abnormalities can also be accompanied by normal to Figure 11.2 decreased serum bilirubin even though erythrocyte destruc- Macrocytic erythrocytes. Compare the size of the erythrocytes with the nucleus of the lymphocyte. Spherocytes tion is increased. This happens because insufficient heme are present (Peripheral blood; Wright-Giemsa stain; 1000* is being synthesized (hypochromic cells), and less heme is magnification). being catabolized. Unconjugated bilirubin levels are usually Image courtesy of Constitution Medical Inc., 2012. All rights reserved. no more than 4 mg/dL. If more than 4 mg/dL it implies a Table 11.9 Common Laboratory Findings Reflecting Increased/Decreased Production and Destruction of Erythrocytes Increased Bone Marrow Production Decreased Bone Marrow Production of Erythrocytes Increased Erythrocyte Destruction of Erythrocytes Reticulocytosis Anemia Anemia Greater than 100 * 103/mcL; Presence of spherocytes, schistocytes, and/or Decreased reticulocytes less than RPI greater than 2 other poikilocytes 25 * 103/ mcL; RPI less than 2 Increased IRF Positive direct antihuman test (DAT) corrected reticulocyte count less than 2%, Leukocytosis Decreased haptoglobin and hemopexin decreased IRF Nucleated erythrocytes in the peripheral blood Decreased glycosylated hemoglobin Erythroid hypoplasia in the bone marrow; Polychromasia of erythrocytes on Romanowsky- Increased fecal and urine urobilinogen increased M:E ratio stained blood smears Increased bilirubin (unconjugated) Normoblastic erythroid hyperplasia in the bone Hemoglobinemiaa marrow Hemoglobinuriaa Hemosiderinuriaa Methemoglobinemiaa Increased serum LD Increased expired CO aAssociated only with intravascular hemolysis. IRF, immature reticulocyte fraction; LD, lactic dehydrogenase; RPI, reticulocyte production index; CO, carbon monoxide. Introduction to Anemia 211 concomitant impaired liver function.18 Thus, the bilirubin expected life spans from 0–100 days. Taking these facts into level should always be interpreted together with the degree consideration, the normal T1 2 with this method has been and type of anemia and hepatic function. It has been sug- determined to be 25–32 days. A steady state is necessary gested that too many variables affect serum bilirubin levels for accurate interpretation of erythrocyte survival studies to make it a reliable measurement of RBC destruction. because blood loss or transfusions can alter the data sig- Other laboratory tests can be used to evaluate eryth- nificantly. Labeled erythrocytes in this method are also rocyte turnover or blood loss (Table 11-9). Hemosiderin in useful in determining the sites of erythrocyte destruction. urine (hemosiderinuria), decreased plasma haptoglobin The amount of radioactivity taken up by an organ can be and hemopexin (as a result of increased consumption), and measured by scanning the body for 51Cr deposition and is increased methemalbumin are associated with increased proportional to the number of erythrocytes destroyed there. intravascular hemolysis. Certain biochemical constituents DIFFERENTIAL DIAGNOSIS OF ANEMIA BASED ON that are concentrated in blood cells are released to the periph- LABORATORY TESTS eral blood as the cell lyses, and these constituents indicate The choice of laboratory tests for the differential diagnosis of the degree of cellular destruction. In anemias associated with anemia should depend on the test’s specificity and sensitivity. ineffective erythropoiesis or hemolysis, these biochemical A highly sensitive test will likely be positive when the disor- constituents will be increased in the blood. The most com- der is present. A highly specific test will be negative when the monly measured constituents include uric acid, the main disorder is not present. Highly sensitive tests are good for end product of purine metabolism, and lactate dehydroge- screening for the disorder, and highly specific tests are good nase (LD), an enzyme that is present in the cell cytoplasm. for confirming the diagnosis of the disorder. Laboratory pro- BONE MARROW fessionals should keep this in mind when creating algorithms Bone marrow evaluation usually is not necessary to deter- for anemia testing. Operating characteristics for some tests mine the cause of an anemia. However, it can provide sup- used in diagnosing anemia can be found in the literature. plemental diagnostic information in anemic patients when other laboratory tests are not conclusive. For example, bone marrow evaluation in hypoproliferative anemias can reveal Checkpoint 11.3 What laboratory test is the least invasive and most cost effective myelodysplasia or infiltration of the marrow with malig- to evaluate erythrocyte production in the presence of anemia? nant cells or granulomas. Erythroid hyperplasia of the bone marrow with decreased amounts of fat is more pronounced in hemolytic anemia than in any of the nonhemolytic ane- mias. Consequently, the myeloid-to-erythroid ratio (M:E) is Classification of Anemias decreased (reference interval is 1.5–3.3; mean 2.3). The purpose of the classification of anemias is to assist the ERYTHROCYTE SURVIVAL STUDIES physician in identifying the cause by using laboratory test Erythrocyte survival studies are helpful in defining a hemo- results in addition to other clinical data. The classification lytic process in which erythrocyte survival is only mildly also is useful to laboratory professionals when they correlate decreased. In mild hemolysis, laboratory findings typical various test results for accuracy and make suggestions for of extravascular or intravascular hemolysis can be absent. additional reflex testing. Although specific diagnosis is the Survival studies give insight into the rate and mechanism ultimate goal of any anemia classification system, it must be of hemolysis. To study erythrocyte survival, a sample of the kept in mind that anemia frequently develops from more than patient’s blood is removed and labeled in vitro with trace one mechanism, complicating correlation and interpretation amounts of radionuclide. The most common label for eryth- of laboratory test results. In addition, complicating factors can rocytes, and that recommended by the International Com- alter the typical findings of a specific anemia. For example, mittee for Standardization in Hematology, is radioactive pre-existing iron deficiency can inhibit the reticulocytosis that chromium (51Cr). The chromium penetrates the erythrocytes normally accompanies acute blood loss or mask the macro- and remains trapped there. This labeled sample is injected cytic features of folic acid deficiency. In these cases, laboratory intravenously into the patient. To determine the erythrocyte test results can depend on which mechanism predominates. survival pattern, small samples of the patient’s blood are Anemias can be classified by either morphology (morphologic assayed at specific time intervals for radioactivity levels. classification) or pathophysiology (functional classification). The erythrocyte life span is expressed as the time it takes for blood radioactivity to decrease by one-half (T1 2 51Cr) Morphologic Classification starting 24 hours after injection. About 1% of the 51Cr is normally eluted from surviving cells daily. In addition, only Anemias can be initially classified morphologically accord- 1% of the labeled cells can be expected to have a life span ing to the average red cell size and hemoglobin concentra- of 100–120 days because only 1% of the total erythrocyte tion as indicated by the erythrocyte indices (Figure 11-3). mass is replaced each day. The remaining labeled cells have This morphologic classification is helpful because MCV, 212 Chapter 11 Survival defect - Hemolysis n 2 - Hemorrhage ha 2%) er t an reat Nuclear maturation defect g ter th ea (megaloblastic) (gr less than 2 - B Macrocytic RPI 12 deficiency - Folate deficiency (corrected (less than 2%) - Drug induced reticulocyte ( l l e e s - Congenital count) s s s t t h h a a n n - Myelodysplasia 2 2%) Nonmegaloblastic (proliferation, maturation, and/or survival defects) - Chronic liver disease - Alcohol abuse - Endocrinopathy - Aplastic anemia an 2 Survival defect er th %) reat - Hemolysis g /Hct r than 2 Hb RBC count Normocytic (greate - Hemorrhage RPI RBC indices normochromic Proliferation defect (corrected Morphology le - Marrow damage or reticulocyte ( s le s s t s h t a replacement or suppression count) h n a n 2 2% by drugs/toxins ) - Stem cell defects - Chronic renal disease - Endocrine hypofunction - Chronic infections or inflammation - Liver disease - Malignancy Cytoplasmic maturation defect ed ecreas - Iron deficiency D Microcytic Serum Cytoplasmic maturation defect hypochromic ferritin No I r n m c al - Chronic disease reased - Thalassemia - Hemoglobinopathies - Sideroblastic anemia - Lead intoxication - Porphyrias Figure 11.3 Classification of anemias using function and morphology reflexed by RPI and/or serum ferritin (Corrected reticulocyte count is in parentheses). MCH, and MCHC are determined when anemia is diag- or RPI, corrected reticulocyte count, CHr, and % hypochro- nosed, and certain causes of anemia are characteristically mic cells (% hypo),15 yields even more meaningful informa- associated with specific erythrocyte size (large, small, or tion. Serum iron studies are most helpful in identifying the normal) and hemoglobin content (normal or abnormal). The pathophysiology of microcytic anemias because the IRF or general categories of a morphologic classification include RPI is variable in these cases. Patient history and physical macrocytic, normochromic; normocytic, normochromic; examination are essential for a differential diagnosis within and microcytic, hypochromic. given classifications. The diagnosis in complicated cases can usually be facili- MICROCYTIC, HYPOCHROMIC ANEMIA tated by microscopic review of the blood smear for addi- Microcytic, hypochromic anemias are associated with defec- tional key morphologic information. This information should tive hemoglobin synthesis. Serum iron studies and occa- include the following red cell findings, if present: dominant sionally hemoglobin electrophoresis are usually adequate poikilocyte, presence of a dual population of cells, inclu- to differentiate the causes of these anemias (Chapters 12, sions, chromia, and arrangement (rouleaux or agglutination). 14). There are five main causes of microcytic anemia: thalas- A morphologic assessment of anemia is not sufficient; semia, anemia of chronic disease, iron deficiency (ID), lead determining the etiology and pathophysiology of anemia poisoning, and congenital sideroblastic anemia. (These five through additional laboratory tests, beginning with the IRF causes are sometimes referred to clinically by the acronym Introduction to Anemia 213 TAILS.)17 Of these causes, iron deficiency, anemia of chronic Functional Classification disease (ACD; also known as anemia of inflammation [AI]), and thalassemia are most common. The morphology of AI Because the normal bone marrow compensatory response is unremarkable but in conjunction with the CHr, percent to decreased peripheral blood hemoglobin levels is an hypochromic red cells, ferritin, and percent transferrin increase in erythrocyte production, persistent anemia can saturation, it usually can be diagnosed.15 Iron deficiency be expected as the result of three pathophysiologic mecha- is characterized by elliptocytosis, anisocytosis, target cells, nisms: (1) a proliferation defect (decreased production), decreased serum ferritin, and percent transferrin saturation. (2) a maturation defect, (ineffective erythropoiesis) or (3) Thalassemia, an inherited condition, demonstrates target a survival defect (increased destruction). These are con- cells and basophilic stippling on the blood smear. The most sidered to be the three functional classifications of anemia useful indices to discriminate between iron deficiency and (Figure 11-4). The functional classification uses the absolute thalassemia are the MCV, MCH, RBC, and RDW.19 The RBC reticulocyte count, corrected reticulocyte count, IRF or RPI, count in thalassemia is high even in the presence of anemia. and/or serum iron studies to categorize an anemia. Prolif- This characteristic is useful in differentiating iron-deficiency eration and maturation defects usually have a normal or anemia from thalassemia using the Mentzer Index, based on decreased IRF and/or RPI less than 2 and corrected reticu- the MCV and RBC. locyte count less than 2% survival defects are characterized by an increased IRF and/or RPI greater than 2 and cor- Mentzer Index20 rected reticulocyte count greater than 2%. Some anemias MCV/RBC (*106) = greater than 14 (iron deficiency) can be grouped into more than one functional subgroup (e.g., thalassemia) because they have characteristics of each = 12914 (indeterminant) (i.e., maturation and survival defects). = less than 12 (thalassemia trait) Although some anemias can be the result of several mechanisms, one mechanism is usually dominant. The ini- Hemoglobin electrophoresis and iron studies such as tial step in approaching an anemic patient is the identifi- ferritin and serum iron and % transferrin saturation are cation of this dominant mechanism. If the functional and helpful in differentiating thalassemia from other causes of morphologic classifications of anemia
are combined, the microcytosis. result is a classification using the reticulocyte count, iron MACROCYTIC ANEMIA studies, and morphology of the erythrocyte (Figure 11-3). Macrocytic anemias are associated with hemolytic anemias If an anemia does not fit into any of these categories, it is (RPI greater than 2), nuclear maturation defects (megalo- probably multifactorial. blastic anemia, RPI less than 2), or nonmegaloblastic ane- PROLIFERATION DEFECTS mia (RPI less than 2). Diagnostic features of megaloblastic Proliferation defects are characterized by decreased pro- anemia such as hypersegmented granulocytes, Howell-Jolly liferation, maturation, and release rates of erythrocytes in bodies, and oval macrocytes can be found on peripheral response to anemia (Figure 11-4). The most characteristic blood smears and supported by low vitamin B12 or folic acid laboratory findings of proliferation defects are normocytic, levels (Chapter 15). Hemolytic anemias with an increased normochromic erythrocytes, decreased absolute reticulocyte MCV due to reticulocytosis can usually be diagnosed using count, decreased corrected reticulocyte count and IRF, and other laboratory tests (the direct antiglobulin test [DAT] is RPI less than 2, signifying a marrow output of reticulocytes essential) and reviewing the blood smear. Nonmegaloblas- inadequate for the degree of anemia. Serum bilirubin levels tic macrocytic anemias have various causes, but clinical are normal or decreased because of the decrease in cell pro- symptoms, history, and other laboratory tests are usually duction. The bone marrow is hypocellular with normal or sufficient to arrive at a diagnosis. increased iron stores. Decreased proliferation can be caused NORMOCYTIC, NORMOCHROMIC ANEMIA by inappropriate erythropoietin production or production Many anemias have normal RBC morphology. Most nor- of cytokines that inhibit erythropoiesis. This trophic basis is mocytic anemias are due to decreased survival (blood loss responsible for the anemias associated with malignancies, or hemolysis) or hypoproliferation. The hypoproliferative chronic renal disease, chronic inflammation,21 and certain anemias are characterized by an RPI less than 2 and sur- endocrinopathies. vival defects with an RPI greater than 2. The hypoprolif- Conversely, erythropoietic-stimulating mechanisms erative anemias are differentiated by a hypocellular bone can be normal, but the bone marrow can fail to respond to marrow with normal or increased M:E ratio. Hemolytic the stimulus appropriately. This failure can occur when the anemias have a hypercellular bone marrow with decreased bone marrow is infiltrated with fibrous, neoplastic, or gran- M:E ratios. Evaluation of hemolytic anemias should include ulomatous tissue or when chemicals, drugs, or radiation blood smear analysis and the DAT to help determine the have damaged the marrow. Differentiating these causes of etiology and pathophysiology. hypoproliferation is possible by observing whether all cell 214 Chapter 11 Bone marrow Peripheral blood Tissue Erythroid progenitor cell Macrophage Normoblasts Erythrocytes (RBCs) Bilirubin Proliferation defects Maturation defects Survival defects Liver Bone marrow damage Nuclear (pancytopenia) Hemolysis • Radiation • Megaloblastic • Hereditary intrinsic defects of RBCs • Chemicals (folate/B12 deficiency) • RBC membrane disorders • Drugs • RBC enzyme deficiencies Conjugated Cytoplasmic (hemoglobin, • Hemoglobinopathies bilirubin Bone marrow infiltration erythrocytes only) • Thalassemia by fibrous, neoplastic, • Fe deficiency (heme) • Extrinsic assault on RBCs granulomatous tissue • Thalassemia (globin) • Autoimmune reaction GI tract • Drug-induced reaction HSC or Trophic Basis • Transfusion reaction • Inappropriate erythropoietin • Malignancy Hemorrhage Stool • Some endocrinopathies • Internal • Aplastic anemia • External • Myelodysplastic anemia Figure 11.4 Functional classification of anemia. Anemia can be due to proliferation, maturation, and/or survival defects. Proliferation and maturation defects are due to defective erythropoiesis in the bone marrow. Survival defects are caused by increased destruction of erythrocytes. This destruction can occur in the bone marrow if the cells are intrinsically abnormal and/or in the peripheral circulation. Destruction of cells in the bone marrow is called ineffective erythropoiesis. In some cases, an anemia can be due to several causes such as in thalassemia. Although thalassemia is classified as a maturation defect, it also is characterized by ineffective erythropoiesis in which the abnormal cells are destroyed before they reach the peripheral blood. Thus, there also is a survival defect. lineages are affected or only the erythrocytes are involved. abnormal cells (Figure 11-4). The erythrocytes are macrocytic If the proliferation defect is due to inappropriate erythro- in nuclear defects and microcytic in cytoplasmic defects. poietin production, decreased proliferation is limited to Despite the abnormal maturation process, the marrow the erythrocytic lineage. In contrast, marrow damage or attempts to increase production of erythrocytes, resulting in infiltration is characterized by hypoplasia of all hemato- bone marrow erythroid hyperplasia. However, because these poietic cells in the bone marrow, producing pancytopenia cells are often intrinsically abnormal, many are destroyed (a decrease in all blood cells) in the peripheral blood. In before they can be released to the peripheral blood (ineffec- addition, poikilocytosis and a leukoerythroblastic periph- tive erythropoiesis). Because many of the abnormal erythro- eral blood picture, presumably caused by damage of the cytes are not released to the peripheral blood, the corrected normal sinusoidal barrier, usually accompany marrow reticulocyte count, the absolute reticulocyte concentration, infiltration. and IRF are decreased and the RPI is less than 2. Poikilocytes Many proliferative defects also are associated with indicative of abnormal erythropoiesis are frequently present decreased erythrocyte lifespan; however, survival is only in direct proportion to the severity of the anemia. moderately decreased and could easily be compensated for Abnormal hemoglobin production causes cytoplasmic by a normally functioning marrow or a normal cytokine maturation defects. Therefore, the defect is limited to the ery- stimulus. Hypoproliferation is rarely caused by an abnor- throid lineage. Hemoglobin production can be impaired due mality in the hematopoietic stem cells (HSC) or committed to one or more of the following: limited iron supply, defective erythroid progenitor cells (EP), resulting in pure red cell iron utilization, decreased globin synthesis, and defective por- aplasia (decreased EP) or aplastic anemia (decreased HSC). phyrin (heme) synthesis. Most erythrocytes produced in asso- MATURATION DEFECTS ciation with cytoplasmic maturation defects are microcytic and Maturation defects disrupt the orderly process of either hypochromic with a variable degree of poikilocytosis. These nuclear or cytoplasmic development producing qualitatively anemias are best differentiated using iron studies (Chapter 12). Introduction to Anemia 215 Because all developing hematopoietic cells have nuclei, erythrocyte membrane damage because of membrane loss to nuclear maturation defects affect all hematopoietic cell lin- phagocytes in the spleen. Generally, the erythrocyte popula- eages and probably other body cells as well. As a result, the tion in survival defects is normocytic and normochromic as peripheral blood can reflect not only anemia but also pancy- defined by RBC indices. It is possible, however, that macro- topenia with characteristic morphologic changes apparent cytosis can prevail, depending on the degree of reticulocyto- in all cell lineages. The distinctive morphologic changes in sis, or that microcytosis can predominate, depending on the cells are collectively termed megaloblastic (delayed nuclear number of schistocytes or microspherocytes. development in comparison to cytoplasmic development). Sites of Destruction in Hemolytic Anemia Hemolysis can SURVIVAL DEFECTS occur within the circulation (intravascular) or within the Survival defects are the result of premature loss of circulating macrophages of the spleen, liver, or bone marrow (extra- erythrocytes either by hemorrhage or hemolysis (Figure 11-4). vascular). In some cases, depending on the degree of dam- Hemolysis is the premature destruction of erythrocytes. In age to the cell, destruction occurs both intravascularly and this type of defect, bone marrow proliferation increases and extravascularly. The results of laboratory tests can provide maturation is orderly. The absolute reticulocyte concentra- important clues to the hemolytic process17,18 (Figure 11-5). tion and IRF are increased, the corrected reticulocyte count Intravascular hemolysis can be caused by (1) the activa- is greater than 2% and the RPI is typically greater than 2. The tion of complement on the erythrocyte’s membrane, (2) blood film reflects this increased erythropoietic activity by physical or mechanical trauma to the erythrocyte, or (3) the the presence of polychromatophilic macrocytes. If the bone presence of soluble toxic substances in the erythrocyte’s marrow is able to compensate for the decreased erythrocyte environment (Table 11-11). life span by increasing production at the same rate as the When the erythrocyte is hemolyzed intravascularly, cells are lost or hemolyzed, anemia does not develop. This free hemoglobin is released into the plasma and binds to condition is referred to as compensated hemolytic disease, haptoglobin. The haptoglobin–hemoglobin complex is which can rapidly develop into anemia if (1) erythrocyte taken to the liver and cleared by macrophages via C163 destruction accelerates beyond the compensatory capacity receptors to be catabolized (Chapter 5).18 The hemoglobin/ of the bone marrow (hemolytic crises) or (2) the marrow sud- haptoglobin complex is degraded by lysosomes, leading to denly stops producing erythrocytes (aplastic crises). a decrease in haptoglobin. When haptoglobin is depleted, Hemolytic anemia can be classified based on the cause hemopexin (another plasma protein) complexes with heme of the shortened erythrocyte survival, as either intrinsic or and takes it to the liver to be catabolized. Hemopexin is extrinsic to the erythrocyte (Table 11-10). Intrinsic refers to quickly depleted when the complex is cleared faster than hereditary abnormalities of the erythrocyte itself, whereas the liver can synthesize hemopexin. A decrease in hemo- extrinsic refers to an antagonist in the red cell’s environ- pexin is secondary to a reduction in haptoglobin. ment that causes injury to the erythrocyte. Other possible Laboratory findings of intravascular hemolysis include classifications of hemolytic anemias include mode of onset, hemoglobinemia, hemoglobinuria, hemosiderinuria, met- site of hemolysis, and predominant poikilocyte present in hemoglobinemia, decreased haptoglobin, and decreased peripheral blood (Table 11-10). Many anemias, although hemopexin. Serum lactic dehydrogenase (LD) can increase not primarily hemolytic, have a hemolytic component. to as much as four to five times the upper limit of the refer- These include the hemoglobinopathies, thalassemia, iron- ence interval. LD can be an early and sensitive indicator of deficiency anemia, and megaloblastic anemia. intravascular hemolysis because the erythrocyte releases LD In contrast to poikilocytes that are formed in the bone into the plasma in intravascular hemolysis, and it is cleared marrow as a result of dyserythropoiesis typical of prolifera- from the plasma more slowly than hemoglobin.20 tion and maturation defects, poikilocytes of a survival defect Extravascular hemolysis is more common than intravas- are formed after the cell leaves the marrow. The most common cular hemolysis. Extravascular hemolysis that results in poikilocytes are schistocytes and spherocytes. The schistocyte premature erythrocyte destruction occurs when phagocytes is the result of intravascular mechanical trauma to the cell, such in the tissues remove erythrocytes from circulation. Hemo- as a shearing by fibrin strands or damage by passing through globin is not released directly to the plasma, so there is no abnormal capillaries. Spherocytes indicate extravascular hemoglobinemia, hemoglobinuria, or hemosiderinuria. Table 11.10 Possible Classifications of Hemolytic Anemia Based on Pathophysiology, Etiology, and/or Laboratory Findings Source of Defect Mode of Onset Site of Hemolysis Predominant Poikilocyte Intrinsic to red cell (intracellular) Inherited Extravascular Spherocyte Extrinsic to red cell (extracellular) Acquired Extravascular or intravascular Schistocyte depending on extent of cell damage 216 Chapter 11 Patient’s and family medical history and clinical examination Congenital Acquired (i) Acute or chronic hemolytic anemia causes causes (ii) Intra- or extravascular hemolysis (iii) Extrahematological signs Blood smear Direct antiglobulin analysis test Positive Negative RBC morphological Unremarkable abnormalities morphology (spherocytes, elliptocytes, Immune hemolytic CD55/59 ovalocytes, stomatocytes) anemia (i) AIHA (ii) DHTR (recent transfusion) Negative Positive RBC enzymopathies Schistocytes PNH RBC membrance defects/ CDA Acute hemolysis Chronic hemolysis (spherocytes, elliptocytes, (i) Pentose phosphate (i) Glycolysis ovalocytes, stomatocytes) shunt (ii) Nucleotide metabolism Negative Positive Infective/toxic Mechanical causes; Wilson disease hemolysis Figure 11.5 Diagnostic flowchart for hemolytic diseases. If the diagnostic flowchart turns negative for congenital hemolytic anemia, reconsider acquired causes and vice versa. RBC: red blood cells; AIHA: autoimmune hemolytic anemia; DHTR: delayed hemolytic transfusion reactions; CDA: congenital dyserythropoietic anemia; PNH: paroxysmal nocturnal hemoglobinuria. Table 11.11 Anemias Characterized by Intravascular Hemolysis Activation of Complement on the Physical or Mechanical Trauma to the Toxic Microenvironment of the Erythrocyte Membrane Erythrocyte Erythrocyte Paroxysmal nocturnal hemoglobinuria Microangiopathic hemolytic anemia Bacterial infections Paroxysmal cold hemoglobinuria Abnormalities of the heart and great vessels Plasmodium falciparum infection Some transfusion reactions Disseminated intravascular coagulation Venoms Some autoimmune hemolytic anemias Arsine poisoning Acute drug reaction in G6PD deficiency Intravenous administration of distilled water Thermal injury G6PD, glucose-6-phosphate dehydrogenase. Hemoglobin is degraded to heme, iron, and globin within the circulation by phagocytes. The antihuman globulin the phagocyte. See Chapter 6 for a description of
the process (AHG) test (the direct antiglobulin test, DAT) is helpful in of the breakdown of the hemoglobin molecule. LD may be identifying erythrocytes sensitized with antibodies and/or increased in extravascular hemolysis, but only slightly com- complement. pared to intravascular hemolysis. Bone marrow macrophages are responsible for the Laboratory findings in hemolytic anemias associated removal of maturing precursor cells that are intrinsically with extravascular hemolysis are measurements of the prod- abnormal (ineffective erythropoiesis). Many hemolytic ane- ucts of heme catabolism. These findings include increases in mias that are associated with inherited defects of the eryth- expired carbon monoxide, carboxyhemoglobin, serum biliru- rocyte membrane, hemoglobin, and intracellular enzymes bin (especially the unconjugated fraction), and both urine and have some degree of ineffective erythropoiesis. Although fecal urobilinogen. In severe or chronic extravascular hemoly- in most cases many of the abnormal cells never enter the sis, haptoglobin and hemopexin levels also can be decreased. peripheral blood, a significant number do gain access to the Antibodies directed against the erythrocyte commonly circulation. These cells are not physiologically equipped to cause hemolytic anemia associated with extravascular withstand the assaults of the peripheral circulation and are hemolysis. Antibody and complement attached to the cell damaged. The hepatic or splenic macrophages then remove membrane make the erythrocyte a target for removal from the damaged cells (Table 11-12). Introduction to Anemia 217 Source of Defect in Hemolytic Anemias Hemolytic anemias can factors can be involved, the initiating event in hemolysis be classified as intrinsic or extrinsic according to the cause is considered to be the intrinsic erythrocyte abnormality. of the shortened erythrocyte survival (Table 11-13). Refer to These intrinsic abnormalities include: the earlier explanation of intrinsic and extrinsic. • Structural defects of the erythrocyte membrane that can Intrinsic Defects. With few exceptions, intrinsic defects are cause the membrane to become abnormally permeable, hereditary. The site of hemolysis in intrinsic defects is usu- rigid, or unstable and easily fragmented (Chapters 5, 17) ally extravascular. In some cases, intrinsic defects render the • Structurally abnormal hemoglobins that result in hemo- cell more susceptible than normal cells to damage by envi- globin insolubility or instability (Chapter 13) ronmental (extracellular) factors. Although extracellular • Deficiencies of erythrocyte enzymes necessary for maintaining hemoglobin and membrane sulfhydryl groups in the reduced state or for maintaining ade- Table 11.12 Anemias Characterized by Extravascular quate levels of adenosine triphosphate (ATP) for cation Hemolysis exchange (Chapter 18) Origin Anemias Extrinsic Defects. Extrinsic defects are usually acquired, Inherited erythrocyte defects Thalassemia and hemolysis can be either intravascular or extravascular. Hemoglobinopathies The erythrocytes, as innocent bystanders, are damaged by Enzyme deficiencies chemical, mechanical, or physical agents (Chapters 19, 20). Membrane disorders Substances in the circulation can be toxic to the cell and cause Acquired erythrocyte defects Megaloblastic anemia direct cell hemolysis or alter the cell membrane, leading to Spur cell anemia removal of the cell in the spleen. Trauma to the erythrocyte Vitamin E deficiency in newborns in the circulation can cause the cell to fragment, producing Immunohemolytic anemias Autoimmune striking abnormalities on the blood smear. Immune-medi- Drug induced ated destruction can occur when antibodies and/or comple- Some transfusion reactions ment attach to the erythrocytes resulting in their removal by Table 11.13 Classification of Hemolytic Anemias Based on Underlying Defect Classification Underlying Defect Examples Intrinsic (inherited) Membrane defects Hereditary spherocytosis Hereditary elliptocytosis Hereditary pyropoikilocytosis Overhydrated hereditary stomatocytosis Dehydrated hereditary stomatocytosis Paroxysmal nocturnal hemoglobinuria (acquired) Enzyme disorders Glycolytic pathway enzyme deficiencies Hexose-monophosphate shunt enzyme deficiencies Abnormal hemoglobins Thalassemia Structural hemoglobin variants (e.g., sickle cell anemia) Extrinsic (acquired) Antagonistic plasma factors Chemicals, drugs Animal venoms Infectious agents Plasma lipid abnormalities Intracellular parasites Splenomegaly Traumatic physical cell injury Microcirculation lesions Thermal injury March hemoglobinuria Immune-mediated cell destruction Autoimmune Alloimmune Drug induced 218 Chapter 11 macrophages in the spleen or liver. The DAT is an essential in these individuals before anemia develops or before test to determine if immune-mediated destruction is present. abnormal cells can be identified on the smear. 3. The RDW is normal after acute hemorrhage if iron sup- Classification Using the Red Cell plies are adequate. Distribution Width 4. Uncompensated hemolytic anemias have a high RDW, whereas compensated hemolytic states have a normal The RDW reflects defective regulation in erythrocyte homeo- RDW. stasis that involves impaired erythropoiesis as well as red blood cell survival.22 Studies have shown that an increase in 5. Anemias due to nutritional deficiencies tend to have the RDW is characteristic of some anemias. When the RDW greater increases in the RDW than anemias caused by is combined with the MCV, classification of the anemia is genetic or primary bone marrow disorders.22 facilitated. When used with the reticulocyte count, the diag- 6. Overlap in the RDW/MCV classification exists espe- nosis can be further clarified. However, an increased RDW cially in the anemia of chronic disease (anemia of is common in a variety of acute and chronic disorders. Thus, inflammation).22 to facilitate its use in classifying anemia, a thorough clini- cal evaluation of the patient is essential. One of the technical Checkpoint 11.4 issues in assessing the RDW is that it is highly dependent Explain why classification of anemia is important, and give the on the analyzer used. Furthermore it should be recognized categories of the morphologic and functional classifications. that an increased RDW is associated with aging, black ethnic- ity, physical exercise, and EPO deficiency/hyporesponsive- ness.22 It has been suggested that the classification of anemias Laboratory Testing Schemas for use the terms heterogeneous (increased RDW) and homogeneous Anemia Diagnosis (normal RDW) in conjunction with the descriptive morpho- logic terms microcytic, normocytic, and macrocytic (e.g., homo- Knowledge of the functional and morphologic classification geneous macrocytic, heterogeneous macrocytic; Table 11-14). of anemias is necessary to design a cost-effective laboratory Studies of anemic individuals provided the following testing approach that aids in specific diagnosis. Only appro- information regarding the relation between categories of priate tests that help identify the cause of anemia should be anemia and RDW.23,24 performed on the patient’s laboratory workup. Guidelines within ICD codes for reimbursement of laboratory tests by 1. Hypoproliferative anemias have a normal RDW regard- third-party payers make it clear that test ordering must be less of the MCV. rationally based. Figure 11-6 shows general schemas of labo- 2. Maturational defect anemias (excluding the rare hered- ratory testing that are useful in diagnosing anemias. It itary types) have an increased RDW regardless of the should be remembered that the physician always takes the MCV or the degree of anemia. The RDW is increased patient’s clinical history and performs the physical Table 11.14 Classification of Anemias by MCV and RDW MCV Normal RDW (homogeneous) Increased RDW (heterogeneous) Normal Acute hemorrhage Immune hemolytic anemia Splenic pooling Early iron, B12, or folate deficiency Anemia of chronic disease Dimorphic anemia (e.g., folate and iron deficiency) Chronic leukemia Sideroblastic anemia Anemia of renal disease Myelofibrosis Sickle cell anemia/trait Chronic liver disease Myelodysplastic syndrome Transfusions Decreased Heterozygous thalassemia Iron deficiency Anemia of chronic disease Homozygous thalassemia Hemoglobin E trait Hb S/Bthal Hb H disease Hemolytic anemia with schistocytes (macroangiopathic hemolytic anemia) Increased Chronic liver disease Immune hemolytic anemia with marked reticulocytosis Aplastic anemia B12 or folate deficiency Chemotherapy CLL with high lymph count Alcohol ingestion Cytotoxic chemotherapy Antiviral medications Chronic liver disease Myelodysplastic syndrome Hereditary spherocytosis Introduction to Anemia 219 MACROCYTIC ANEMIA Reticulocyte count RPI 7 2 RPI 6 2 Smear for schistocytes/ Serum B12 and folate microspherocytes Absent Present Decreased Normal/high WBC and Platelet Hemorrhage Hemolysis Vitamin B12 counts or folic acid deficiency Decreased Normal/slightly Intravascular Extravascular decreased – T haptoglobin – c indirect bilirubin Bone marrow – hemoglobinemia – c urine urobilinogen – Aplastic anemia – hemosidinurea – Myelodysplasia – hemoglobinurea Drug inhibition Liver function studies Abnormal Normal – Liver disease – Endocrine a – Alcoholism studies Figure 11.6a These schema are a general guide to the laboratory work-up of anemia. The red cell indices and the appearance of the cells on a stained blood smear will give the first important clue to morphologic classification. The results of the next tests in the schema, the RPI and iron studies, will give clues to the pathophysiologic mechanisms involved. Note that most causes of anemia can be determined without performing a bone marrow examination. This procedure is usually reserved for those anemias that appear to be caused by a stem cell defect, marrow damage, or marrow replacement. MICROCYTIC HYPOCHROMIC ANEMIA Serum Ferritin Normal or Increased Decreased Iron deficiency Serum iron, TIBC Serum iron decreased Serum iron decreased Serum iron increased Serum iron normal TIBC Normal/Decreased TIBC increased TIBC normal/slight decrease TIBC normal Saturation 7 10% Saturation 6 10% Saturation 7 70% Free erythrocyte Chronic disease Sideroblastic Iron Deficiency protoporphyrin Anemia Normal Increased Hemoglobin Lead electrophoresis level Abnormal Increased b Thalassemia, Lead toxicity Hemoglobin E Figure 11.6b These schema are a general guide to the laboratory work-up of anemia. The red cell indices and the appearance of the cells on a stained blood smear will give the first important clue to morphologic classification. The results of the next tests in the schema, the RPI and iron studies, will give clues to the pathophysiologic mechanisms involved. Note that most causes of anemia can be determined without performing a bone marrow examination. This procedure is usually reserved for those anemias that appear to be caused by a stem cell defect, marrow damage, or marrow replacement. 220 Chapter 11 NORMOCYTIC NORMOCHROMIC ANEMIA Reticulocyte count RPI 7 2 RPI 6 2 Blood Smear for schistocytes/ WBC and platelet count microspherocytes Absent Present Decreased Normal Hemorrhage Hemolysis Bone marrow Serum iron/TIBC – Marrow damage – Marrow replacement – Stem cell defect Intravascular Extravascular Decreased Normal – T haptoglobin – c indirect bilirubin – hemoglobinemia – c urine urobilinogen – hemosidinurea Renal Function – hemoglobinurea Chronic Disease Studies Abnormal Normal – Renal Disease Bone marrow – Pure red cell c aplasia Figure 11.6c These schema are a general guide to the laboratory work-up of anemia. The red cell indices and the appearance of the cells on a stained blood smear will give the first important clue to morphologic classification. The results of the next tests in the schema, the RPI and iron studies, will give clues to the pathophysiologic mechanisms involved. Note that most causes of anemia can be determined without performing a bone marrow examination. This procedure is usually reserved for those anemias that appear to be caused by a stem cell defect, marrow damage, or marrow replacement. examination before beginning a laboratory workup. The information gained in these ways can eliminate the need for CASE STUDY (continued from page 210) some tests and/or suggest additional tests. These schemas 5. Classify George’s anemia morphologically and will gain more meaning as you read the following chapters functionally. on each group of anemias. Summary Anemia is a decrease in the ability of blood to carry adequate count, and blood smear examination. More specific tests amounts of oxygen to the tissue resulting from an insufficient can be performed based on the results of these routine tests. concentration of hemoglobin. Diagnosis of anemia is made The erythrocyte indices can be used to determine the with a combination of information from the patient history, size and hemoglobin content of erythrocytes. Because some physical examination, and laboratory investigation. Initially, anemias are characterized by specific erythrocyte morphol- routine laboratory tests are performed to determine the ogy, the indices are helpful in initially classifying the anemia. presence of anemia and to evaluate erythrocyte production The manual reticulocyte count is routinely reported in and destruction. These tests can include erythrocyte count, relative terms: the number of reticulocytes per erythrocytes hemoglobin, hematocrit, erythrocyte indices, reticulocyte expressed in percent. Automated reticulocyte counts are Introduction to Anemia 221 reported in both relative and absolute terms. More informa- examined for cellularity, cellular structure, M:E ratio, tion is available from the absolute count, corrected reticu- and iron stores. locyte count, IRF, RPI, and CHr (RET-He). Generally, the Anemias are generally classified by a functional or reticulocyte count in an anemic patient should be increased morphologic scheme or by a combination of the two. The greater than 100 * 103/mcL if the bone marrow is increas- morphologic classification includes three general categories ing production of erythrocytes. Absolute counts less than based on erythrocyte indices: normocytic, normochromic; 25 * 103/mcL in anemic patients is indicative of a hypop- macrocytic, normochromic;
and microcytic, hypochromic. roliferative response. The functional classification uses the IRF or RPI and serum Examination of the blood film is helpful in assessing iron studies to classify the anemias according to pathophys- anisocytosis and poikilocytosis. Anisocytosis is a variation iology: proliferation defect, maturation defect, and survival in erythrocyte size and is calculated and expressed as the defect. Each of these classifications has subclassifications. red cell distribution width (RDW) on some automated cell Some anemias such as thalassemia can fall into more than counters. It is not uncommon to find a variety of cell sizes one functional classification group because they have char- in some anemias. Poikilocytosis is a variation in cell shape. acteristics of both (i.e., hemolytic and maturation). These Specific shapes give clues to the cause of anemia. classifications help the laboratory professional and physi- Bone marrow examination is indicated if labora- cian design a cost-effective approach to laboratory testing tory tests give inconclusive results. Bone marrow is to reach a specific diagnosis. Review Questions Level I 5. How would you classify the cell population with the following indices: MCV 70 fL, MCH 25 pg, MCHC 1. Which of the following is characteristic of severe 30 g/dL? (Objective 3) intravascular hemolysis? (Objective 7) a. Normocytic, normochromic a. decreased bilirubin b. Macrocytic, normochromic b. increased hemopexin c. Microcytic, normochromic c. decreased urobilinogen d. Microcytic, hypochromic d. decreased haptoglobin 6. Which of the following is a hemolytic anemia that 2. A patient with anemia has an RPI of 2.3 with an would be classified as an extrinsic defect? (Objective 9) MCV of 103 fL. How would you classify this anemia? (Objective 3) a. Immune hemolytic anemia a. Macrocytic b. Anemia caused by a membrane defect b. Normocytic c. Anemia associated with a deficiency of an erythro- cyte enzyme c. Microcytic d. Anemia associated with a structurally abnormal d. Maturation defect hemoglobin that is unstable 3. Which of the following tests will give information 7. Which of the following indicates that compen- about rate of erythrocyte production? (Objective 2) sated hemolytic disease is present in a patient with a. RPI increased erythrocyte destruction? (Objective 6) b. Serum bilirubin a. Increased carboxyhemoglobin c. Serum ferritin b. Decreased hemoglobin d. MCV c. Increased hemoglobin 4. A patient has the following results: RBC count, d. Normal hemoglobin 2.5 * 106/mcL and hemoglobin 5.3 g/dL, hematocrit 8. Which of the following is most typically found in a 17%, reticulocyte count 1%. What are the absolute hemolytic anemia? (Objective 5) reticulocyte count and RPI? (Objective 1) a. Reticulocytopenia a. Absolute count, 25 * 103/mcL; RPI, 0.19 b. Decreased IRF b. Absolute count, 250 * 103/mcL; RPI, 0.15 c. RPI greater than 2 c. Absolute count, 170 * 103/mcL; RPI, 0.38 d. Increased M:E ratio in bone marrow d. Absolute count, 100 * 103/mcL; RPI, 1.5 222 Chapter 11 9. Upon examination of the stained blood smear from a 4. A patient has anemia, decreased haptoglobin, hemo- patient with anemia and jaundice, the laboratory pro- siderinurea, and hemoglobinuria. The reticulocyte fessional noted many schistocytes. This is an indica- count is 10%. How would you classify this anemia? tion of: (Objective 9) (Objectives 8, 9) a. an extrinsic defect a. Hypoproliferative b. extravascular hemolysis b. Extravascular hemolytic c. intravascular hemolysis c. Maturation defect d. inherited anemia d. Survival defect 10. The clinical finding common in a patient with hemo- 5. In an untreated anemia caused by hemorrhage, what lytic anemia is: (Objective 8) would you expect to find in the laboratory investiga- tion? (Objective 2) a. pica b. kidney stones a. Presence of polychromatophilic macrocytes on the peripheral blood smear c. jaundice b. Hypoplastic bone marrow with nuclear maturation d. lymph node enlargement abnormalities Level II c. Megaloblastosis in the bone marrow and pancyto- penia in the peripheral blood 1. What follow-up test is most appropriate to determine the cause of anemia in a patient with the following d. Decreased IRF and RPI results: RBC count 2.5 * 106/mcL, hemoglobin 5.3 g/ 6. Bone marrow examination in a patient with a hemo- dL, hematocrit 17%, reticulocyte count 1%? globin of 80 g/L reveals hypercellularity with a (Objective 11) decreased M:E ratio and normal appearing erythro- a. IRF cytic precursors. This is an indication that the anemia b. Absolute reticulocyte count is likely due to a: (Objective 5) c. Serum ferritin a. proliferation defect d. RPI b. nuclear maturation defect c. survival defect 2. What is the functional classification of the anemia in the preceding question if the serum ferritin is d. cytoplasmic maturation defect decreased? (Objective 8) 7. Which of the following is an adaptation to anemia that a. Proliferation defect tends to increase blood flow to tissues? (Objective 1) b. Survival defect a. Decrease in 2,3-DPG c. Nuclear maturation defect b. Shallow inspiration d. Cytoplasmic maturation defect c. Decreased respiratory rate 3. Why might the serum bilirubin results be misleading d. Increased heart rate as an indicator of erythrocyte destruction inpatients 8. Which of the following findings is characteristic of the with microcytic hypochromic anemias? (Objective 2) bone marrow in a hemolytic anemia? (Objective 5) a. The liver is not excreting the bilirubin due to liver a. Increased M:E ratio failure. b. Erythroid hyperplasia b. The cells are being produced at a lower rate than normal. c. Increased amount of fat c. Hypochromic cells do not release much hemoglo- d. Hypoplasia bin; hence, less bilirubin is formed. 9. A 4-year-old boy has severe anemia. His x-rays reveal d. The cells are not destroyed as fast in individuals thinning cortical bone, and he had splenomegaly. This with microcytic anemia so the bilirubin will be diagnosis and clinical symptoms indicate: (Objective 3) falsely decreased. a. a chronic hemolytic process b. an acute hemolytic anemia c. intravascular hemolysis d. extravascular hemolysis Introduction to Anemia 223 10. A patient has a hemoglobin of 9.0 g/dL and a reticu- c. Urinalysis locyte count of 20%. A bone marrow examination d. AHG (DAT) test revealed a decreased M:E ratio and 70% cellularity. How would you describe this marrow? (Objective 4) 12. What laboratory test result is suggestive of an intra- a. Aplasia vascular hemolytic process? (Objectives 9, 11) b. Erythrocytic hypoplasia a. Decreased LDH c. Dysplasia b. Decreased haptoglobin d. Erythrocytic hyperplasia c. Decreased reticulocyte count d. Decreased bilirubin 11. A patient is suspected of having an autoimmune hemolytic anemia. Many spherocytes are present on the blood smear, and the reticulocyte count is 20%. What test should be performed to determine whether this is an autoimmune process? (Objective 10) a. Serum bilirubin b. Serum LD References 1. Camaschella, C. (2015). Iron-deficiency anemia. New England Jour- 13. Gaskell, H., Derry, S., Moore, R. A., & McQuay, H. J. (2008). nal of Medicine, 372(19), 1832–1843. doi: 10.1056/NEJMra1401038 Prevalence of anaemia in older persons: Systematic review. 2. World Health Organization. (2011). Haemoglobin concentrations for BMC Geriatrics, 8, 1. doi: 10.1186/1471-23181-8-1 the diagnosis of anaemia and assessment of severity. Geneva: Author. 14. Buttarello, M. (2016). Laboratory diagnosis of anemia: Are the 3. Milman, N., & Pedersen, A. N. (2009). Blood haemoglobin con- old and new red cell parameters useful in classification and centrations are higher in smokers and heavy alcohol consumers treatment, how? International Journal of Laboratory Hematology, than in non-smokers and abstainers: Should we adjust the refer- 38(Suppl. 1), 123–132. doi: 10.1111/ijlh.12500 ence range? Annals of Hematology, 88(7), 687–694. doi: 10.1007/ 15. Piva, E., Brugnara, C., Spolaore, F., & Plebani, M. (2015). Clinical s00277-008-0647-9 utility of reticulocyte parameters. Clinics in Laboratory Medicine, 4. Hillman, R. S., & Hershko, C. (2007). Acute blood loss anemia. In: 35(1), 133–163. doi: 10.1016/j.cll.2014.10.004 M. A. Lichtman, E. Beutler, T. J. Kipps, U. Seligsohn, K. Kaushan- 16. Mark, P. W. (2012). Approach to anemia in the adult and child. sky, J. T. Prchal, eds. Williams hematology (p. 767–71). New York: In: R. Hoffman, E. J. Benz Jr, L. E. Silberstein, H. Heslop, J. Weitz, McGraw-Hill. J. Anastasi, eds. Hematology: Basic principles and practice (6th ed., pp. 418–426). Philadelphia: Elsevier, Churchill Livingstone. 5. Pang, W. W., & Schrier, S. L. (2012). Anemia in the elderly. Current Opinions in Hematology, 19, 133–140. doi: 10.1097/ 17. Ford, J. (2013). Red blood cell morphology. International Journal of MOH.0b013e3283522471 Laboratory Hematology, 35(3), 351–357. doi: 10.1111/ijlh.12082 18. Barcellini, W., & Fattizzo, B. (2015). Clinical applications of 6. Sahin, S., Tasar, P. T., Simsek, H., Çicek, Z., Eskiizmirli, H., hemolytic markers in the differential diagnosis and management Aykar, F. S., . . . Akcicek, F. (2016). Prevalence of anemia and of hemolytic anemia. Disease Markers, 3015. doi: http://dx.doi. malnutrition and their association in elderly nursing home org/10.1155/2015/635670. residents. Aging Clinical and Experimental Research, 28(5), 857–862. doi: 10.1007/s40520-015-0490-5 19. Eldibany, M. M., Totonchi, K. F., Joseph, N. J., & Rhone, D. (1999). Usefulness of certain red blood cell indices in diagnosing and dif- 7. Butt, Z., Ashfaq, U., Sherazi, S. H., Jan, N. U., & Shahbaz, U. ferentiating thalassemia trait from iron-deficiency anemia. Ameri- (2010). Diagnostic accuracy of "pallor" for detecting mild and can Journal of Clinical Pathology, 111(5), 676–682. severe anaemia in hospitalized patients. Journal of the Pakistan 20. Means, R. T. Jr., & Glader, B. (2014). Anemia: General consider- Medical Association, 60(9), 762–765. ations. In: A. P. Greer, I. A. Arber, B. Glader, A. F. List, R. T. Means 8. Kalntri, A., Karambelkar, M., Joshi, R., Kalantri, S., & Jajoo, U. Jr., F. Paraskevas, eds. Wintrobe’s clinical hematology (2010). Accuracy and reliability of paollor for detecting anaemia: (pp. 587–616). Philadelphia: Wolters Kluwer/Lippincott, A hospital-based diagnostic accuracy study. PLoS ONE, 5(1), Williams & Wilkins. e8545. doi: 10.1371/journal.pone.0008545 21. Masson, C. (2011). Rheumatoid anemia. Joint, Bone, Spine: Revue 9. Tabbara, I. A. (1992). Hemolytic anemias: Diagnosis and manage- Du Rhumatisme, 78(2), 131–137. doi: 10.1016/j.jbspin.2010.05.017 ment. Medical Clinics of North America, 76(3), 649–668. 22. Salvagno, G. L., Sanchis-Gomar, F., Picanza, A., & Lippi, G. 10. Goonewardene, M., Shehata, M., & Hamad, A. (2012). Anaemia (2015). Red blood cell distribution width: A simple parameter in pregnancy. Best Practice and Research. Clinical Obstetrics and with multiple clinical applications. Critical Reviews in Clinical Lab- Gynaecology, 26(1), 3–24. doi: 10.1016/j.bpobgyn.2011.10.010 oratory Sciences, 52(2), 86–105. doi: 10.3109/10408363.2014.992064 11. Guralnik, J. M., Eisenstaedt, R. S., Ferrucci, L., Klein, H. G., & 23. Cunha, B. A., Nausheen, S., & Szalda, D. (2007). Pulmonary com- Woodman, R. C. (2004). Prevalence of anemia in persons 65 years plications of babesiosis: case report and literature review. and older in the United States: Evidence for a high rate of unex- European Journal of Clinical Microbiology and Infectious Diseases, plained anemia. Blood, 104, 2263–2268. 26(7), 505–508. 12. Goodnough, L.T., & Schrier, S. L. (2014). Evaluation and manage- 24. Bessman, J. D., Gilmer, P. J., & Gardner, F. H. (1983). Improved ment of anemia in the elderly. American Journal of Hematology, 89, classification of anemias by MCV and RDW. American Journal of 88–96. doi: 10.1002/ajh.23598. Clinical Pathology, 80(3), 322–326. Chapter 12 Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis Shirlyn B. McKenzie, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define sideropenic as it relates to anemia. iron-deficiency anemia (IDA), anemia of 2. Diagram the transport of iron from chronic disease (ACD), lead poisoning, and ingestion to incorporation into heme. sideroblastic anemia. 3. Define the following terms and explain their 6. Explain the etiology and pathophysiology role in iron metabolism: transferrin, hemosiderin, of iron-deficiency anemia, anemia of ferritin, total iron-binding capacity (TIBC). chronic disease, and sideroblastic anemia. 4. Describe physiologic, environmental, and pathologic conditions associated with iron 7. Define hemochromatosis. deficiency. 8. Calculate transferrin saturation and 5. Compare and contrast the typical blood unsaturated iron-binding capacity (UIBC) features and iron studies associated with given serum iron and TIBC. Objectives—Level II At the end of this unit of study, the student should be able to: 1. List the three stages of iron deficiency, and serum ferritin, and RBC morphology in the define characteristic RBC morphology of three stages of iron deficiency. each stage. 3. Describe the function of the proteins involved 2. Compare and contrast iron stores, in iron metabolism including hepcidin, hemoglobin, serum iron, TIBC, saturation, HFE, transferrin receptor (TfR), hemojuvelin 224 Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 225 (HJV), divalent metal transporter 1 (DMT1), 14. Explain the significance of finding duodenal cytochrome–B reductase microcytic
anemia in the presence of lead (DCytB), hephaestin, hypoxia-inducible poisoning, and suggest reflex testing that factor-2a (HIF-2a), ferroportin 1, and erythro- would help determine an accurate diagnosis. ferrone (ERFE). 15. Explain how lead poisoning and alcohol 4. Explain the molecular control of total body affect erythropoiesis and their relationship iron (systemic) and cellular iron. to sideroblastic anemia, and recognize the 5. Describe how genetic defects in the iron abnormal peripheral blood and clinical metabolism proteins can affect the iron features that can be associated with these homeostasis in the body. disorders. 6. Contrast the basic defects in iron-deficiency 16. Discuss the treatment for iron-deficiency anemia, sideroblastic anemia, and anemia anemia, sideroblastic anemia, and anemia of chronic disease, and describe how each of chronic disease and expected labora- defect affects hemoglobin synthesis. tory findings associated with successful therapy. 7. Recognize the clinical features associated with iron deficiency. 17. Differentiate primary (hereditary) and secondary hemochromatosis and summa- 8. Correlate the following laboratory features rize typical results of iron studies in these with iron-deficiency anemia, anemia of conditions. chronic disease, and sideroblastic anemia: erythrocyte morphology and protoporphy- 18. Describe the genetic abnormalities rin studies, iron studies, and bone marrow. and pathophysiology of hereditary hemochromatosis, and identify the screen- 9. Select laboratory tests, and discuss ing and diagnostic tests for this disease. test results that help differentiate iron-deficiency anemia, anemia of chronic 19. Describe the basic defect in porphyria and disease, and sideroblastic anemia. its effect on the blood. 10. Summarize the results of bone marrow 20. Develop a reflex-testing pathway for an analysis in sideroblastic anemia and anemia efficient and cost-effective diagnosis when of chronic disease, and contrast them with microcytic and/or hypochromic cells are those found in iron-deficiency anemia. present. 11. Outline the classification of sideroblastic 21. Evaluate laboratory test results, and use anemias, and describe the differentiating them to identify the etiology and patho- feature of the hereditary type. physiology of the anemias that have a defective heme synthesis component. 12. Describe the relationship of the anemias associated with alcoholism and malignant 22. Describe the etiology and pathophysiol- disease to sideroblastic anemia. ogy of iron-refractory iron deficiency anemia (IRIDA) and compare the asso- 13. Describe the role of molecular diagnostics in ciated l aboratory results with those of hereditary sideroblastic anemia. iron-deficiency anemia. Chapter Outline Objectives—Level I and Level II 224 Introduction 227 Key Terms 226 Iron Metabolism 227 Background Basics 226 Laboratory Assessment of Iron 238 Case Study 226 Iron-Deficiency Anemia 240 Overview 226 Anemia of Chronic Disease 245 226 Chapter 12 Iron Refractory Iron-Deficiency Anemia (IRIDA) 247 Porphyrias 255 Functional Iron Deficiency (FID) 248 Summary 259 Anemias Associated with Abnormal Heme Review Questions 260 Synthesis 248 References 262 Hemochromatosis 253 Key Terms Anemia of chronic disease (ACD) Hepcidin Pica Anemia of inflammation (AI) Hephaestin Plumbism Apoferritin Human hemochromatosis protein Porphyria Apotransferrin (high iron Fe [HFE]) Serum transferrin receptor (sTfR) Ceruloplasmin Hypoferremia Sideroblastic anemia DcytB Hypoxia-inducible Sideropenic anemia DMT1 factor-2A (HIF-2A) Stress erythropoiesis Erythroferrone (ERFE) Iron-deficiency anemia (IDA) Total iron-binding capacity (TIBC) Ferritin Iron refractory iron-deficiency Transferrin (Tf) Ferroportin 1 anemia (IRIDA) Transferrin receptor 1 (TfR1) Functional iron deficiency (FID) Iron regulatory protein (IRP) Transferrin receptor 2 (TfR2) GDF15 Iron responsive element (IRE) Transferrin saturation Hemochromatosis Iron-restricted erythropoiesis Unsaturated iron-binding capacity Hemojuvelin (HJV) Non-transferrin bound iron (NTBI) (UIBC) Hemosiderin Pappenheimer body Background Basics The information in this chapter builds on the concepts Level II learned in previous chapters. To maximize your l earning • Function, structure, and synthesis of hemo- experience, you should review these concepts before globin: Diagram the synthesis of heme and starting this unit of study: explain the role of iron in hemoglobin synthesis. (Chapter 6) Level I • Erythrocyte destruction: Diagram degrada- • Diagnosis of anemia: List the laboratory tests used tion of hemoglobin when the erythrocyte is to diagnose and classify anemias, and identify destroyed and interpret laboratory tests asso- abnormal test values. (Chapters 10, 11) ciated with increased erythrocyte destruction. • Classification of anemia: Outline the morphologic (Chapters 5, 11) and functional classification of anemias. (Chapter 11) CASE STUDY Overview We refer to this case study throughout the chapter. This chapter includes a discussion of a group of anemias associated with defective hemoglobin synthesis due to Jose, an 83-year-old male, was admitted to a local faulty regulation of iron metabolism or porphyrin bio- hospital with recurrent urinary tract bleeding and synthesis. The discussion begins with a detailed descrip- an infection associated with prostatitis. tion of iron metabolism and laboratory tests used to 1. How can these conditions affect the hematopoi- assess the body’s iron concentration. This is followed etic system? by a description of the specific anemias included in this group—iron-d eficiency anemia, anemia of chronic Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 227 disease, and sideroblastic anemia. Hemochromatosis is referred to as iron-deficiency anemia (IDA). Iron deficiency also discussed even though it is usually not character- primarily affects the erythrocyte and developing central ized by anemia. In hemochromatosis, regulation of iron nervous system. Sideropenic anemia caused by inadequate absorption and/or transport is abnormal, and results of iron intake or absorption or increased blood loss responds iron studies must be differentiated from those found in to iron therapy given either orally, or less commonly, sideroblastic anemia. The rare porphyrias are discussed parenterally. briefly because porphyrin is an integral component in Anemia also can result from defective regulation of iron the synthesis of heme. metabolism. In these anemias, there is adequate or excess stores of iron but sufficient iron cannot be mobilized for the synthesis of hemoglobin (functional iron deficiency). The Introduction anemia of chronic disease (ACD), also referred to as the anemia of inflammation (AI), is included in this group, Defective hemoglobin production can be due to distur- and is characterized by iron retention in the macrophages, bances in either heme or globin synthesis (Table 12-1). thus making iron unavailable to the erythrocyte for heme The result of these disturbances is an erythrocyte cyto- synthesis. plasmic maturation defect often reflected by a microcytic, Defects in porphyrin synthesis involve the enzymes hypochromic anemia. Defective heme synthesis is caused required for heme synthesis. The defect can affect the inser- by abnormalities of iron homeostasis (deficiency and/or tion of iron into the porphyrin ring to form heme. These metabolism) or rarely by defective porphyrin metabolism conditions include primary and secondary sideroblastic (Figure 12-1). Defective globin synthesis is a result of anemias. The porphyrias are included in this chapter, deletions or mutations of globin genes. Globin gene dele- although except for erythropoietic porphyria, they are not tions that result in decreased synthesis of globin are known generally characterized by the presence of anemia. as thalassemia (Chapter 14). Mutations that result in struc- Hemochromatosis is a disorder that is characterized turally abnormal globin chains are known as hemoglobin- by progressive iron overload with deposits of excess iron opathies, which are not usually associated with microcytic in parenchymal tissue resulting in tissue damage. Anemia hypochromic erythrocytes (Chapter 13). is not present but iron studies are markedly abnormal. Anemia characterized by deficient iron for h emoglobin Hereditary forms are due to mutations in genes involved synthesis is known as sideropenic anemia, more commonly in regulation of iron metabolism. Table 12.1 Causes of Defective Hemoglobin Production Iron Metabolism That Could Result in a Microcytic Hypochromic Anemia Every cell in the body requires iron. It has vital roles in Defects in Globin Synthesis oxidative metabolism, cellular growth and proliferation, Defects in Heme Synthesis (Thalassemias) and oxygen transport and storage. Iron must be bound Abnormal iron metabolism Globin gene deletions to protein compounds to fulfill these functions. Iron in • Iron deficiency Globin gene mutations inorganic compounds or in an ionized form is potentially • Defective iron utilization dangerous. If the amount of iron exceeds the body’s capac- Defective porphyrin metabolism ity for transport and storage in the protein-bound form, iron Heme synthesis disturbed Globin synthesis abnormal Iron IDA ACD ACD Thalassemia EPO Heme Hemoglobin Globin Globin gene mutations ACD Cytokines SA porphyrias Heme enzymes Figure 12.1 Sites of defective hemoglobin synthesis that can result in anemia. Some of these anemias are hereditary or congenital and others are acquired. ACD, anemia of chronic disease; IDA, iron-deficiency anemia; SA, sideroblastic anemia; EPO, erythropoietin; //, defective or disturbed. 228 Chapter 12 toxicity can develop, causing damage to cells and a poten- Iron is found primarily in erythroblasts, erythrocytes, tially lethal condition. Conversely, if too little iron is avail- macrophages, hepatocytes, and enterocytes. Hemoglobin able, the synthesis of physiologically active iron compounds constitutes the major fraction of body iron (functional iron) is limited, and critical metabolic processes are inhibited. with a concentration of about 1 mg iron/mL erythrocytes. Iron cannot freely diffuse across membranes but Iron in hemoglobin remains in the erythrocyte until the requires special transport involving a variety of proteins. cell is removed from the circulation. Hemoglobin released Enterocytes (absorptive cells at the luminal [apical] sur- from the erythrocyte is then degraded in the macrophages face of the duodenum), hepatocytes, and macrophages can of the spleen and liver, releasing iron. Approximately import and export iron. On the other hand, erythrocyte pre- 85% of this iron from degraded hemoglobin is promptly cursors use most, if not all, of the imported iron and do not recycled from the macrophage to the plasma where it is export it. Important advances in our understanding of iron bound to the transport protein, transferrin, and delivered metabolism are the result of the discovery of genes and pro- to developing erythroblasts in the bone marrow for heme teins that participate in regulating iron homeostasis. As the synthesis. The macrophages recycle 10–20 times more iron roles of the proteins are discovered, the pathophysiology of than is absorbed in the gut. Thus, iron recycling provides disorders involving iron metabolism is revealed. most of the marrow’s daily iron requirement for erythro- poiesis (Chapter 5). Distribution Iron in hepatocytes and intestinal enterocytes is stored and utilized as needed to maintain iron homeostasis. The Iron-containing compounds in the body are one of two hepatocytes store iron that can be released and utilized types: (1) functional compounds that serve in metabolic when the amount of iron in the plasma is not sufficient to functions (hemoglobin, myoglobin) or enzymatic func- support erythropoiesis. Enterocytes that absorb dietary iron tions (cytochromes, cytochrome oxygenase, catalase, can either export it to the plasma or store it. peroxidase) and (2) compounds that serve as transport proteins (transferrin, transferrin receptor) or storage depots for iron (ferritin and hemosiderin) (Table 12-2). Absorption A poorly understood iron compartment is the intracellu- Total body iron homeostasis depends on balancing and lar “labile pool.” Iron leaves the plasma and enters the linking the absorption of iron by the enterocytes of the intracellular fluid compartment for a brief time before it is duodenum with total body requirements. No significant incorporated into cellular components (heme or enzymes) mechanism exists to effect iron loss. Factors influencing iron or storage compounds. This labile pool is believed to be absorption are listed in Table 12-3. the chelatable iron pool (see the section “Therapy for ENTEROCYTE UPTAKE OF DIETARY IRON Hemochromatosis”). The total iron concentration in the Dietary iron exists in two forms: nonheme iron (ionic or body is 40–50 mg of iron/kg of body weight. Men usually ferric form, Fe+ + + ) present in vegetables and whole grains have higher amounts than women. Table 12.2 Composition and Distribution of Iron in Adults Iron Content, Male Iron Content, Female (mg Iron/Kg Body (mg Iron/Kg Body Compound Weight) Weight) Percent of Total Iron Functional iron Hemoglobin 31 28 60–75.0 Myoglobin 5 4 3.5 Other tissue iron Less than 1 Less than 1 0.2 • Heme enzymes (cytochromes, c atalases, peroxidases) • Nonheme enzymes (iron-sulfur p roteins, metalloflavoproteins, ribonucleotide reductase) Transport Transferrin Less than 1 Less than 1 0.1 Storage Ferritin About 8 About 4 10–20 Hemosiderin About 4 About 2 5–10 Labile pool About 1 About 1 2 Total iron About 50 About 40 Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 229 Table 12.3 Factors Affecting Iron Absorption Decreased Absorption Variable Absorption Increased Absorption Iron bioavailabilty Intraluminal factors Systemic conditions • Hematopoietic (erythropoi- • Low hemoglobin • Diet • Inflammatory bowel disease • Inflammation etic) activity of bone marrow: concentration directly related to absorption • Macrophage recycling
• Parasites • Infection • Hypoxia • Tissue iron stores: iron • Toxins absorption is inversely related • Intestinal motility to storage iron • Decreased absorptive surface area and heme iron (ferrous form, Fe+ + ) present primarily in red meats in the form of hemoglobin. Nonheme iron is the most Fe11 Fe111 common form ingested worldwide; heme iron is more com- mon in Western countries (providing about 10–15% of daily DCytB iron requirements). The ferric complexes from nonheme sources are not easily absorbed. Gastric acid solubilizes this form of iron and provides an acidic environment around the apical brush border of the enterocytes. This low pH facili- tates the transport of iron across the enterocyte membrane DMT1 by membrane transporters. The ferric iron is reduced to the Ferritin ferrous state at the enterocyte brush border by the enzyme, Fe11 Duodenal cytochrome b (DcytB), a ferric reductase. The fer- Intestinal rous iron is then transported across the enterocyte apical lumen plasma membrane by divalent metal transporter1 (DMT1), Enterocyte Tissue an integral membrane protein. DMT1 also transports the divalent forms of manganese, lead, zinc, cobalt, and copper Ferroportin across the enterocyte membrane (Figure 12-2). This mem- Hephaestin brane iron transport depends on the inward transport of protons. Heme iron is more readily absorbed than nonheme Blood Fe11 Fe111 Transferrin iron, but the mechanism of absorption is less well under- stood. The low pH and proteolytic enzymes of the stom- Figure 12.2 The absorption of nonheme iron in the intestine. ach act to release heme from hemoproteins. Heme uptake Most nonheme iron in the diet is in the ferric iron form (Fe+++). When by enterocytes is most likely via the heme carrier protein 1 the Fe+++ reaches the intestine and comes into contact with the (HCP1) together with the proton coupled folate transporter cells lining the gut (enterocytes), the iron is reduced to the ferrous (PCFT) in receptor-mediated endocytosis. Once inside the form (Fe++) by a reductase, DCytB, located at the apical enterocyte cell, iron is released from heme by heme oxygenase. The membrane. The Fe++ then can be transported across the membrane iron then enters the same iron pool as the nonheme iron.1,2,3 by DMT1. Ferroportin 1 transports the iron across the enterocyte basolateral membrane, a process thought to be facilitated by EXPORT OF IRON FROM ENTEROCYTE TO PLASMA hephaestin. Hephaestin is an oxidase that oxidizes the iron to Fe+++, In the enterocyte, the iron can be stored as ferritin or trans- the form that combines with transferrin. Some iron can remain in the cell as ferritin, depending on the systemic iron balance. ported across the basolateral membrane into the plasma. The iron stored as ferritin is lost when the enterocyte is sloughed off into the intestinal tract. Iron transport across Thus, it is not surprising that copper deficiency is associ- the basolateral membrane is via the basolateral transporter ated with abnormal iron metabolism. Export of iron from protein ferroportin 1. Ferroportin 1, an integral membrane non-intestinal cells, including macrophages, requires ceru- protein, transports ferrous ions and is the only known loplasmin, which also converts Fe+ + to Fe+ + + for binding cellular exporter of iron. Export is facilitated by the fer- to transferrin. roxidase, hephaestin (a homologue of the plasma protein ceruloplasmin). Hephaestin oxidizes the Fe+ + to Fe+ + + , ABSORPTION VERSUS STORAGE the form of iron required for binding to apotransferrin in There appears to be a predetermined set point of iron stores the blood. Hephaestin is a copper-containing ferroxidase that results in a negative correlation between the amount of that requires adequate amounts of copper for its function. iron absorbed and the amount of iron stored. The efficiency 230 Chapter 12 of intestinal absorption of iron increases in response to plasma and is reused. Transferrin is a single polypeptide accelerated erythropoietic activity and depletion of body chain composed of two homologous lobes, each of which iron stores. Bleeding, hypoxia, or hemolysis results in contains a single iron-binding site. The binding of a ferric accelerated erythrocyte production (stress erythropoiesis) iron to either binding site is random. If only one transferrin and enhanced absorption of iron. However, increased iron lobe binds an iron molecule, it is termed monoferric transfer- uptake in extravascular hemolytic anemias and anemias rin; if both sites are occupied, it is diferric transferrin (also associated with a high degree of ineffective erythropoiesis called holo-transferrin). Transferrin without iron is called can lead to an excess accumulation of iron in various organs apotransferrin. because the body does not lose the iron from erythrocytes Each gram of transferrin binds 1.4 mg of iron. Enough hemolyzed in vivo. Conversely, diminished erythropoiesis transferrin is generally present in plasma to bind 250–450 as occurs in starvation decreases the absorption of iron. mcg (mg) of iron per deciliter of plasma. This is referred to Iron-deficiency anemia (IDA) from a lack of dietary as the total iron-binding capacity (TIBC). iron is usually treated with daily oral doses of ferrous salts. The efficiency of absorption of this therapeutic iron is great- IRON CONCENTRATION IN THE BLOOD est during the initial treatment period when body stores The concentration of iron in the blood is primarily deter- are depleted. Increased absorption occurs up to 6 months mined by the amount of iron released from macrophages after hemoglobin values return to normal or until iron stores to plasma transferrin and the amount extracted from the are replenished. Absorption also increases 10–20% in early plasma by developing erythroid precursor cells. The stages of developing iron deficiency (ID). serum iron concentration is about 50–180 mcg/dL and almost all (95%) of this iron is complexed with transfer- Transport rin; thus, transferrin is about one-third saturated with iron (serum iron/TIBC * 100 = , transferrin saturation). Transferrin (Tf) is a plasma iron transport protein that Clinically, the transferrin saturation is a good indicator of mediates iron exchange between tissues (Figure 12-3). It the amount of iron available for erythropoiesis. The reserve is not lost in delivering iron to the cells but returns to the iron-binding capacity of transferrin (transferrin without Transferrin Fe Fe111 Stomach Fe11 Fe11 Heme Erythroblast RBC Bone marrow Transferrin Fe111 Fe111 Other body cells Duodenum Fe11 Fe111 Fe111 Fe Ferritin Ferritin Enterocyte Hemosiderin Feces Blood vessel Liver, spleen Figure 12.3 Iron is absorbed by the enterocytes in the gut as ferrous iron. It can be stored in the enterocyte as ferritin, or it can leave the cell as oxidized ferric iron and be transported in the blood by transferrin. Transferrin can deliver iron to developing erythroblasts in the bone marrow, other body cells, or macrophages in the liver or spleen. Transferrin is reutilized after it delivers iron to the cells. Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 231 bound iron) is referred to as the serum unsaturated iron- per cell is a function of cellular iron requirements. Cells binding capacity (UIBC) (TIBC - serum iron = UIBC). with high iron requirements have high numbers of TfR1. The majority of transferrin-bound iron is delivered to the Erythroid precursors, especially intermediate erythro- developing bone marrow erythroblasts for use in hemoglo- blasts that are rapidly synthesizing hemoglobin, have high bin synthesis. Iron in excess of physiologic requirements is numbers of transferrin receptors, about 800,000 per cell. deposited in tissues (primarily the liver) for storage. The TfR1 is a transmembrane glycoprotein dimer with two Only a small amount of transferrin-bound iron is identical subunits, each of which can bind a molecule of derived from iron absorbed by enterocytes. Most of the transferrin. A homologous protein, transferrin receptor 2 iron bound to transferrin is recycled from the monocyte- (TfR2), is more limited in expression and can also bind macrophage system. The major flow of iron in the body is transferrin but with less affinity than TfR1. TfR2 is found from macrophages to erythroid marrow, to erythrocytes, predominantly on hepatocytes, duodenal crypt cells, as and back to macrophages when the senescent erythrocyte is well as erythroid cells. TfR2 interacts with HFE to regu- removed and degraded by liver, bone marrow, and splenic late hepcidin synthesis and thus has a role in regulating macrophages. Recovered iron from hemoglobin catabolism total body iron homeostasis (see subsequent discussion, in the monocyte-macrophage system enters the plasma and “Systemic Iron Balance, HFE”). is again bound to transferrin for transfer back to the bone Iron enters the cell in an energy- and tempera- marrow (Figure 12-4). In disorders associated with intra- ture-dependent process. After transferrin binds to its vascular hemolysis in which hemoglobin is released into receptor, the transferrin–TfR complex clusters with other the plasma, plasma hemoglobin combines with haptoglo- transferrin–TfR complexes on the cell membrane, and the bin (Chapter 6). The haptoglobin–hemoglobin complex is membrane invaginates and reseals, forming an endosome taken into the macrophage via the cell’s hemoglobin scav- with the complex inside (endocytosis) (Figure 12-5). In enger receptor, CD163. the acidic endosome, iron is released from transferrin and In contrast to serum ferritin, transferrin is a negative transported into the cytoplasm via DMT1 transport pro- acute phase reactant (levels decrease during the acute phase tein present in the endosomal membrane. The endosome response). Increased levels are found during pregnancy and with the apotransferrin and TfR is transported back to the in estrogen therapy use. cell surface. The apotransferrin is released, making both Lactoferrin also functions as an iron transport protein it and the receptor available for recycling. When the con- but is found primarily in tissue fluid and cells. It has anti- centration of plasma iron exceeds the transferrin binding microbial properties and is important in protecting the body capacity, the excess iron binds to low-molecular-weight from infection. molecules such as citrate or albumin. This non-transferrin bound iron (NTBI) can be taken up by tissues that have CELLULAR UPTAKE OF IRON membrane transporters for NTBI such as tissues in the Transferrin releases iron to cells on specific receptor sites pancreas, heart, and liver. referred to as transferrin receptor 1 (TfR1). These recep- Cells release the extracellular portion of their transfer- tors are expressed on virtually all cells, but the number rin receptors through proteolytic cleavage as they mature. Daily iron cycle Circulating RBC Erythroid marrow Hepatocytes Bone marrow macrophage Iron Other body cells 1 mg 1 mg (storage, Mucosal cells in duodenum myoglobin, (absorption) Loss respiratory enzymes) Figure 12.4 The daily iron cycle. Most iron is recycled from the erythrocytes to macrophages in the bone marrow. Macrophages release iron as needed to the developing erythroid cells in the bone marrow. Only a small amount of iron is lost from the body through loss of i ron- containing cells. To maintain iron balance, a similar amount of iron is absorbed from the duodenum. 232 Chapter 12 Fe 1 Iron binds to apotransferrin Fe to form diferric transferrin Fe Fe Apotransferrin F 2 e Plasma diferric transferrin 8 binds to transferrin receptors Apotransferrin is released on cell surface from receptor in neutral Receptor pH environment 3 Cell membrane invaginates with transferrin–receptor complexes inside H1 7 Iron is available for Endosome with use or storage 4 Cell membrane fuses, apotransferrin–receptor forming an endosome complexes is transported to cell surface DMT1 Ferritin H1 H1 6 In acidic (holo-transferrin) 5 environment, iron Endosome fuses with H1 is released from transferrin acidic vesicle and is transferred to the Acidic vesicle cytoplasm by DMT1. Figure 12.5 Cellular iron supply and storage. (1) Iron binds to apotransferrin in the plasma forming monoferric or diferric transferrin. (2) Transferrin binds to transferrin receptors on the cell surface (3). The transferrin–receptor complex enters the cell and the cell membrane fuses forming an endosome (4). An acidic vesicle fuses with the endosome (5) which results in the release of iron (6). The apotransferrin–receptor complex is transported back to the cell surface (7) where the apotransferrin is released from the receptor and enters the plasma for reutilization (8). These cleaved receptors, referred to as serum or soluble through bleeding. Depletion of these storage compounds transferrin receptors (sTfR), are found in the blood in pro- reflects an excess iron loss over what is absorbed. portion to the erythrocyte mass. With increased erythropoi- esis, including ineffective erythropoiesis, the concentration FERRITIN of sTfR in the plasma increases. Ferritin consists of a spherical protein shell that can store up to 4500 molecules of Fe+ + + iron. Ferritin
is 17–33% iron by Storage weight; without iron, it is called apoferritin. Apoferritin is a multimer composed of 24 subunits arranged to form a hollow In steady state erythropoiesis, most of the iron that is taken sphere. There are two types of subunit polypeptides, heavy up by erythroid precursors is incorporated into heme (H) and light (L). The H polypeptide has ferroxidase activity, within the mitochondria. Most of the remainder is stored but the L form does not. The proportion of H and L subunits in as ferritin and hemosiderin (Table 12-4). The primary iron ferritin varies by cell type. Ferritins in the heart, placenta, and storage depot is the liver. Storage iron provides a readily erythrocytes are rich in the H subunit, and ferritins in the iron available iron supply in the event of increased iron loss storage sites, such as the liver and spleen, are rich in L subunits. Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 233 Table 12.4 Storage Forms of Iron Iron Form Role Laboratory Analysis Reference Interval Ferritin Primary storage form of soluble iron; readily Serum ferritin levels 20–300 mcg/L males released for heme synthesis 12–200 mcg/L females Hemosiderin Partially degraded storage form of iron; slow Bone marrow estimation using Prussian 20–60% sideroblasts in bone marrow release blue stain Ferritin acts as the primary storage compound for the Hemosiderin can be estimated on bone marrow tissue body’s iron and is readily available for erythropoiesis. It sections. Bone marrow macrophages contain hemosiderin controls the amount of iron released for cellular activity if body iron stores are normal or increased. Hemosid- and, by binding the iron, protects the cellular constituents erin appears as yellow to brown refractile pigment on from oxidative damage catalyzed by free ferrous ions. Fer- unstained marrow or liver specimens. On Prussian blue- ritin is found in the bone marrow, liver, and spleen, usually stained specimens, the iron appears as blue intracellular within membrane-bound vesicles called siderosomes. Mature particles. Stores can be graded from 0 to 4+ or as mark- erythrocytes usually do not contain iron particles because edly reduced, normal, or increased (Chapter 38). Normally, after hemoglobin synthesis is complete, any excess iron in from 20–60% of the erythroblasts contain stainable iron the cell is removed by splenic macrophages. deposits. Erythroblasts with siderotic granules are called Ferritin is a water-soluble form of storage iron that can- sideroblasts. If siderotic granules remain after enucleation, not be visualized by light microscopy on unstained speci- the cell is called a siderocyte. mens but sometimes can be detected using Prussian blue stain (when clustered in siderosomes). It also can be visual- Physiological Regulation of Iron ized by electron microscopy. Ferritin is primarily an intra- Balance cellular protein, but small amounts enter the blood through active secretion or cell lysis (serum ferritin). Secreted ferritin Body iron is stringently conserved by recycling so that daily differs from intracellular ferritin in that it is glycosylated absorption and loss are small. Total body iron lost through and relatively iron poor. The amount of serum ferritin par- secretions of urine, sweat, bile, and desquamation of cells allels the concentration of storage iron in the body. There- lining the gastrointestinal tract amounts to about 1 mg/day. fore, serum ferritin concentration is used as an index of iron Normal erythrocyte aging results in destruction of 20–25 mL stores: 1 ng/mL of serum ferritin indicates about 8 mg of of erythrocytes/day (releasing about 20–25 mg iron), but storage iron. Serum ferritin does not exhibit diurnal varia- most of this hemoglobin iron is scavenged and reused by tions as are seen with serum iron levels. developing erythroblasts. Thus, the total daily requirement The patient’s comorbidities must be considered when for new iron is about 1 mg (Chapter 5). interpreting serum ferritin levels. Ferritin is not a reliable indi- Because there is no physiological route for e xcretion cator of iron stores in the presence of inflammation or tissue of excess iron, the major regulation of total body iron damage because it is an acute phase reactant and thus increases depends on accurate sensing of systemic iron and in these conditions. However, if serum ferritin is decreased, it adjusting iron absorption and retention a ccording invariably means that iron stores are low or depleted. to needs. ID can occur if dietary intake of iron is not adequate, if absorption is impaired, or if there is HEMOSIDERIN increased loss of iron through bleeding. Iron overload Hemosiderin is a heterogeneous aggregate of carbohy- can occur if absorption abnormally increases or if the drate, lipid, protein, and iron; up to 50% of its weight is individual receives blood transfusions or iron injections iron. Hemosiderin is found primarily in macrophages and (Table 12-5). Iron absorption is regulated by systemic is formed by the partial degradation of ferritin. At high lev- signals and intracellular mechanisms. els of cellular iron, ferritin forms aggregates that are taken up by lysosomes and degraded, forming hemosiderin. The ratio of ferritin to hemosiderin varies with the total body iron concentration. At lower cellular iron concentrations, Table 12.5 Causes of Iron Deficiency and Iron Overload ferritin predominates, but at higher concentrations, the Causes of Iron Deficiency Causes of Iron Overload majority of storage iron exists as hemosiderin. Iron from • Increased requirement/demand • Increased absorption of iron hemosiderin is released slowly and is not readily avail- due to blood loss, rapid growth, • Multiple transfusions able for cellular metabolism. Iron binding in the form of treatment with EPO • Iron injections hemosiderin also probably keeps iron from harming cellular • Inadequate diet • Genetic mutations constituents. • Malabsorption 234 Chapter 12 SYSTEMIC IRON BALANCE decreases iron absorption into the body by binding to fer- Iron homeostasis is accomplished by the interaction of iron roportin 1 (the main cellular iron exporter in mammals) and with proteins that aide in its absorption, retention, export, inducing its ubiquitination, internalization, and degrada- and transport -2a\|(Table 12-6). The liver is the major site tion. Thus, hepcidin blocks basolateral iron export from the of expression of most of these iron-regulatory proteins, the enterocyte as well as iron export from the macrophages and primary storage depot for iron, and plays a central role in hepatocytes. the regulation of total body iron homeostasis by synthesiz- When hepcidin is increased, ferroportin 1 is depleted ing hepcidin (see below) in response to multiple signals. from cell membranes and iron is retained in iron exporting Thus, the liver has been called the command central of iron cells, resulting in hypoferremia (decrease in serum iron). homeostasis. The proteins involved in iron absorption and When hepcidin is suppressed there is stabilization of release can be upregulated or downregulated depending on ferroportin 1 at the cell membrane, promoting release total body iron status. of iron from macrophages and hepatocytes, and result- Hepcidin, a peptide hormone synthesized in the liver, ing in hyperferremia (increased serum iron). Because is the master iron-regulating hormone whose expression ferroportin 1 determines whether iron is delivered to is inversely related to total body iron demand. It regulates the plasma or remains within the enterocyte, hepatocyte, how much iron is absorbed and released into the plasma or macrophage, it plays a vital role in iron homeosta- from enterocytes, hepatocytes, and macrophages. Hepcidin sis. Hepcidin’s interaction with ferroportin 1 is thought Table 12.6 Proteins Involved in Iron Homeostasis Protein Metabolic Role Bone morphogenetic protein (BMP) Receptor that binds BMP6 on hepatocytes; signals upregulation of hepcidin through SMAD signaling pathway BMP6 BMP binding protein synthesized by hepatocytes when iron levels increase and triggers hepcidin expression Ceruloplasmin Copper containing ferroxidase; plays same role in iron export from macrophages and other non-gut tissues as hephaestin does in enterocytes Divalent metal transporter1 (DMT1) Transports iron across the enterocyte apical plasma membrane; transports iron across endosome into cytoplasm of cells Duodenal cytochrome b (DCytB) Reduces Fe+++ iron to Fe++ at enterocyte apical border Erythroferron Erythroid regulator of hepcidin expression in response to EPO; necessary for rapid hepcidin suppression during stress erythropoiesis Ferroportin 1 Exports cellular iron from enterocytes, macrophages, and hepatocytes Growth differentiation factor-15 (GDF-15) Member of the transforming growth factor-b superfamily released from erythroblasts and other cells under stress and apoptosis; suppresses hepcidin synthesis; increased during ineffective erythropoiesis Hemojuvelin (HJV) Regulates hepcidin expression; acts as a coreceptor for BMP on hepatocytes Hepcidin Master iron-regulating protein; regulates iron recycling/balance via interaction with ferroportin 1; negative regulator of intestinal iron absorption; additional proteins influence synthesis; suppressed in conditions of accelerated erythropoiesis Hephaestin Facilitates cellular export of iron by ferroportin 1; oxidizes Fe++ iron to Fe+++ for binding to apotransferrin Human hemochromatosis protein (high iron Fe [HFE]) Interacts with TfR1 and TfR2 to control hepcidin expression; competes with holotransferrin for transferrin receptor1 Hypoxia inducible factor-2a (HIF 2a) Transcription factor involved in oxygen-dependent gene regulation; regulates EPO production and expression of DMT, DcytB, transferrin, TfR1, ceruloplasmin, and heme-oxygenase-1 Interleukin-6 (IL-6) Cytokine that binds to IL-6 receptor on hepatocytes; activates JAK/STAT cell signaling pathway to promote synthesis of hepcidin Matriptase-2 (MT-2) Cleaves HJV on hepatocytes; inhibits BMP and SMAD-mediated hepcidin synthesis Transferrin (Tf) Transports iron found in blood; diferric transferrin binds to TfR1 and can displace HFE from TfR1 when level of diferric transferrin increases Transferrin receptor1 (TfR1) Binds diferric-transferrin to the cell for internalization of iron; can form molecular complex with HFE Transferrin receptor2 (TfR2) Forms molecular complex with HFE to regulate hepatic hepcidin expression; can bind transferrin; HFE-TfR2-Tf complex triggers the ERK/MAPK signaling Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 235 to be the major mechanism that controls systemic iron increases, as in iron-loading conditions, it displaces HFE homeostasis.3 from TfR1. The free HFE can then interact with TfR2, Hepcidin expression is regulated by four conditions: which can also bind transferrin. This movement of HFE iron stores, erythropoiesis, hypoxia, and inflammation/ between TfR1 and TfR2 serves as a means of communica- infection.4 Hepcidin synthesis is induced by iron excess tion between the two receptors. HFE complexed with TfR2 and inhibited by a lack of iron. Plasma iron concentration and transferrin interacts with a second protein complex that is sensed by cell transferrin receptor complexes, TfR1/HFE involves the bone morphogenetic protein (BMP) receptor and TfR2/diferricTf/HFE (Figure 12-6). and a membrane-anchored BMP coreceptor, hemojuvelin (HJV). Together, BMP and HJV bind the BMP6 ligand, Increased Hepcidin Expression Human hemochromatosis which activates the BMP receptor.6,7 The activated recep- protein (high iron Fe [HFE]) is a transmembrane protein tor signals the SMAD pathway, which upregulates hepcidin found primarily in hepatocytes, hematopoietic cells, and synthesis. Thus, HFE acts as an iron sensor and is involved crypt cells of the duodenum. HFE can bind to both TfR1 in regulating iron absorption and uptake by modulating and TfR2 on cells.5 In the basal state of iron metabolism, hepcidin synthesis.2,5,8 HFE and TfR1 exist as a complex on the cell membrane. Diferric transferrin and HFE compete for binding to TfR1, HEPCIDIN INHIBITION Matriptase-2 (MT-2) acts as but TfR1 has a stronger binding affinity for diferric transfer- a negative hepcidin regulator. It cleaves HJV and thus rin than for HFE. Thus, when the level of diferric transferrin decreases the HJV concentration on the cell membrane. This impairs BMP receptor activation and decreases hepci- din synthesis. Mutations of HFE, HJV, and TfR2 are associ- HoloTf ated with hereditary hemochromatosis (a condition of total body iron overload).9 On the other hand, mutations of MT-2 are associated with increased hepcidin expression and iron- IL-6 HJV deficient erythropoiesis. H MT F 2 The body’s response to insufficient tissue oxygen deliv- BMP E 6 TFR 2 TFR 1 ery is an increase in EPO by the kidney and an increase in BMP JAK receptor erythropoiesis. An increase in erythropoietic activity results receptor in a decrease in hepcidin synthesis, allowing the absorption SMAD of more iron in the intestine and increased release of iron from JAK-STAT pathway enterocytes, macrophages, and h epatocytes. The repressive pathway effect of erythropoiesis on hepcidin s ynthesis is especially noted when erythroid precursors massively expand but Nucleus Hepcidin undergo apoptosis before maturing to erythrocytes (inef- fective erythropoiesis). Although EPO is the primary stimu- lus for increased erythropoiesis and erythropoietic activity is inversely related to hepcidin production, EPO does not have a direct effect on hepcidin production. Rather, dur- ing stress erythropoiesis, erythroid precursors in the bone marrow produce circulating factors
in response to high Figure 12.6 A model for molecular regulation of hepcidin. levels of EPO and these factors regulate hepcidin produc- Hepcidin is regulated by at least two pathways: the iron-sensing pathway and the inflammatory/infection pathway. The iron-sensing tion. Several of these erythroid regulators have been identi- pathway involves the interaction of HFE with TfR1 and TfR2. Both fied and include growth differentiation factor-15 (GDF15), receptors can bind HFE and diferric transferrin (holoTf). When erythroferrone (ERFE), and twisted gastrulation protein the concentration of holoTf increases, it displaces HFE from TfR1 homolog 1 (TWSG1)10,11,12,13 (Figure 12-7). GDF15 is pro- and both HFE and holoTf bind to TfR2. The HFE/TfR2/holoTf duced by late-stage erythroblasts and appears to suppress complex then interacts with a second protein complex (the bone hepcidin expression during i neffective erythropoiesis. ERFE morphogenetic protein [BMP] receptor, its ligand BMP6, and HJV coreceptor) to signal upregulation of hepcidin through the cell helps to ensure an iron supply to developing erythroblasts signaling SMAD pathway. Matriptase-2 (MT-2), a transmembrane during stress erythropoiesis by targeting hepatocytes in serine protease, degrades HJV, impairing BMP receptor activation and the liver and suppressing hepcidin synthesis. Thus, GDF15 decreasing hepcidin synthesis. The amount of MT-2 on hepatocytes is and ERFE act as hormones that link erythropoiesis and iron acutely increased in iron deficiency, decreasing hepcidin production. metabolism. However, it is not clear if they play a major role Hepcidin transcription is increased in the presence of inflammation/ infection. The cytokine interleukin-6 (IL-6) binds to the IL-6 membrane in baseline erythropoiesis. The role of TWSG1 in physiologi- receptor. This activates the receptor and signals hepcidin transcription cal or pathological suppression of hepcidin has not been through the JAK-STAT cell signaling pathway. defined. 236 Chapter 12 Stress Erythropoiesis Bone marrow Increased EPO erythroblasts Liver Increased ERFE, hepatocytes GDF-15, TWSG1 Decreased hepcidin Figure 12.7 During stress erythropoiesis, increased EPO causes erythroid progenitor cells in the bone marrow to produce increased levels of the circulatory proteins ERFE, GDF15, and TWSG1, which act through unknown membrane receptors to suppress the expression of hepcidin by hepatocytes, possibly through the JAK/STAT signaling pathways. EPO, erythropoietin; ERFE, erythroferrone; GDF-15, growth differentiation factor-15; TWSG1, twisted gastrulation protein homolog 1. HYPOXIA Hypoxia stimulates erythropoietin (EPO) Infection and Inflammation Hepcidin synthesis is affected production in the kidney, resulting in increased by not only iron status, hypoxia (potentially), and eryth- erythropoiesis, which increases the demand for iron. ropoietic activity, but also infection and inflammation. Hypoxia-inducible factor 2A 1HIF-2A 2 is a transcription Bacteria-activated macrophages and neutrophils synthe- factor that regulates EPO synthesis as well as a spectrum size hepcidin but at lower levels than hepatocytes. In the of other hypoxic responses14 (Figure 12-8). HIF-2a sig- presence of inflammation, the cytokine interleukin-6 (IL-6) naling is activated under hypoxic conditions. HIF-2a induces synthesis of hepcidin (through transcriptional regu- binds to specific DNA consensus sequences referred to lation of the JAK-STAT signaling pathway; Figure 12-6) (see as hypoxia-response elements (HREs) and induces tran- Chapter 4, Signaling Pathways and Transcription Factors). scription of oxygen-sensitive genes. In oxygenated cells, The increase in hepcidin results in hypoferremia. Because HIF-2a undergoes degradation. In addition to regulating hepcidin induces iron retention in the macrophages (traps EPO synthesis, HIF-2a plays a role in iron metabolism intracellular iron) and most iron used in erythropoiesis by directly regulating (increasing the synthesis of) DMT1 comes from iron recycled from the macrophage, hypofer- and DcytB, resulting in increased iron absorption in the remia can develop rapidly in the presence of inflammation gut.15 Other genes regulated by HIF-2a are ferroportin or infection even though total body iron may be normal 1, transferrin, TfR1, ceruloplasmin, and heme-oxygen- or even increased. This appears to be the pathologic basis ase-1.16 It is unclear if HIF-2a plays a role in expression for the anemia of chronic disease (ACD) (see “Anemia of of hepcidin. Chronic Disease”). Increased Increased Iron erythropoiesis Absorption DMT1 Hypoxia Kidney EPO DcytB HIF-2a Oxygen sensitive genes Figure 12.8 Hypoxia is the primary regulator of EPO and thus erythropoiesis. The hypoxia-inducible factor@2a (HIF@2a) system modulates iron metabolism and erythropoiesis via transcription factors that are regulated by oxygen. In normoxia, HIF@2a is degraded but in hypoxia, HIF@2a is stabilized and binds to hypoxia-response elements, which control the transcription of oxygen-sensitive genes including EPO, DMT1, and DcytB. Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 237 Thus, although hepcidin is the major iron-regulating decreased in the absence of iron. The mRNAs of ferritin, fer- hormone, additional proteins are involved in iron homeo- roportin 1, and ALA synthase 2 (ALAS2) fall into this group. stasis by their influence on hepcidin synthesis or function. If the IRE is in the 3′ UTR of the mRNA, binding of the IRP stabilizes the mRNA that would otherwise be digested/ INTRACELLULAR IRON BALANCE degraded (Figure 12-10). Thus, translation increases when Cytosolic iron concentrations are sensed by iron regula- iron is scarce. The mRNAs of TfR and DMT1 fall into this tory proteins (IRPs), which post-transcriptionally control second group. expression of iron regulatory genes. The IRPs recognize and Thus, IRP regulates cellular iron by coordinating syn- bind to specific non-coding sequences on mRNA known as thesis of ferritin and TfR in opposite directions. The level iron responsive elements (IREs). These IREs have similar of TfR expression reflects the cell’s need for iron and is an RNA stem-loop-stem structures in either the 5′ or 3′ non- important factor in hemoglobin synthesis. When the cell coding regions. The binding affinity of IRP for the IRE is needs more iron, TfRs increase to maximize the amount determined by the amount of cellular iron: IRP binds to of iron incorporated into the cell and ferritin formation the IRE when iron is scarce and dissociates when iron is decreases. On the other hand, when cells have adequate or plentiful. excess iron, the ferritin levels rise and transferrin receptors When bound to the IRE of mRNA, the IRP modulates decrease. Regulation of the ALAS2 gene coordinates the the translation of the mRNA. The IRP regulates translation synthesis of porphyrin (heme) with iron availability. in one of two ways, depending on the location of the IRE in In addition to the regulation of iron levels by binding of the mRNA. IRP binding to IRE in the 5′ untranslated region IRPs to IRE, another regulatory mechanism is related to the (UTR) of the mRNA results in the disruption of translation hypoxia pathway signaled by HIF-2a. In iron-deficient mice, by preventing the assembly of initiation factors at the ini- IRP1 in the kidneys binds with high affinity to the mRNA of tiator site (Figure 12-9). Because binding occurs when iron HIF-2a. This results in repression of HIF-2a and decreased is scarce, translation of proteins with IRE in the 5′ UTR is production of EPO and suppression of erythropoiesis, thus IRP Initiation factor IRP Endonuclease IRE mRNA 59 AAAAAAA 39 mRNA IRE Iron is scarce so IRP binds to IRE of mRNA, preventing formation of the initiation 59 AAAA 39 complex and translation Iron is scarce, which allows IRP to bind IRE; binding prevents endonuclease from digesting mRNA IRP Fe IRP IRP mRNA 59 AAAAAAA 39 Fe Fe mRNA Iron is replete and binds to the IRP, 59 AAAA 39 preventing its binding to IRE and allowing assembly of initiation factors; translation Iron is replete and binds to IRP; this can take place allows endonuclease to cleave the mRNA Figure 12.9 Regulation of iron at the cellular level. A stem-loop-stem structure in the 5′ noncoding region, referred Figure 12.10 Regulation of iron at the cellular level. A stem- to as the iron-responsive element (IRE), is present in some mRNA. loop-stem structure in the 3′ non-coding region, referred to as the Binding of iron-regulatory protein (IRP) to the IRE prevents initiation iron-responsive element (IRE), is present in some mRNA. Binding of of translation. When the level of cellular iron is replete, the iron binds iron-regulatory protein (IRP) to the IRE stabilizes the mRNA. When to IRP, preventing IRP binding to the IRE and allowing assembly of cellular iron is replete, the iron binds to the IRP and prevents it initiation factors at the initiation site; this allows translation to take from binding to the IRE. This results in degradation of the mRNA by place. The translation of the mRNAs for ferritin, ferroportin 1, and endonuclease. The translation of the mRNAs of TfR and DMT1 is ALAS2 are regulated in this fashion. AAAAAAA, poly-adenine tail. regulated in this fashion. AAAAAAA, poly-adenine trail. 238 Chapter 12 decreasing the drain of diminishing iron supply in the sys- depleted quickly in an infant whose primary dietary intake temic pool at the expense of other tissues that need iron.12,13 is unfortified cow’s milk. Extraction of iron from human breast milk is very efficient, enabling infants to acquire a large proportion of the iron present. Premature infants are Checkpoint 12.1 at an even higher risk of rapid iron depletion because much In iron-deficiency anemia, would you expect synthesis of ALAS2 of the placental transfer of iron occurs in the last trimester of to increase or decrease? Explain. pregnancy, and they have a faster rate of postnatal growth than full-term infants. In one study the prevalence of ID at 1 year corrected age of preterm very-low-birth-weight IRON METABOLISM IN THE MITOCHONDRIA infants was 45%.19 It is recommended that full-term infants Most iron entering erythroid cells is routed to the mitochon- begin iron supplements no later than 4 months of age and dria for hemoglobin synthesis and iron-sulfur (Fe-S) cluster that low-birth-weight infants begin no later than 2 months (iron containing functional group) assembly. Mitochondrial of age. ferritin serves as an iron storage molecule and is highly Iron requirements are also high in childhood, especially expressed in tissue with numerous mitochondria. Frataxin in 1- to 2-year-olds. Globally, about 25% of preschool chil- is the protein in the mitochondrial matrix that is thought to dren have iron-deficiency anemia.20 play a role in mitochondrial iron export and storage.17 Iron Requirements Laboratory Assessment of Normally, humans maintain a relatively constant body concentration of iron throughout their life. This is accom- Iron plished by establishing a positive iron balance during grow- Clinically useful indicators of iron status change sequen- ing years and maintaining an equilibrium between loss and tially as body iron changes from replete to deficiency or absorption in adult life. Humans are unable to excrete iron; overload. Additionally, coexisting conditions can affect test therefore, the rate of absorption and loss of iron must be results. Because no single indicator or combination of indi- balanced to avoid ID or excess. cators reveals true body iron status in all circumstances, it FACTORS THAT INCREASE FE REQUIREMENTS is recommended that multiple parameters be used to assess Normal physiologic factors that increase the daily iron status.21 Laboratory testing to determine iron status requirement for iron include menstruation, pregnancy, includes a CBC with reticulocyte indices, measurement of and growth. Pathologic factors that increase the need for serum iron, total iron-binding capacity (TIBC), calculation iron are discussed in the next section on iron-deficiency of the percent saturation of transferrin, serum ferritin, and anemia. serum transferrin receptor (sTfR) (for reference intervals see Appendix A). An indirect assessment of iron availability is Menstruation The average daily iron loss in menstruating provided by the zinc protoporphyrin (ZPP) assay. females is 0.6–2.5% more than in men and nonmenstruating women.18 To maintain total body iron balance, menstruating females must absorb about 2 mg of iron daily. Iron Studies Transferrin can be measured as a protein by immunochemi- Pregnancy The daily iron requirement during pregnancy cal methods, but because the percent saturation (with iron) is about 3.4 mg per day; if spread out as a daily average is helpful in the differential diagnosis of anemia, it is usually over the 3 trimesters, it equates to about 1000 mg per preg- measured functionally as the maximum amount of iron able nancy. The fetus accumulates about 250 mg of iron from to be bound in the serum (TIBC). The measured serum iron maternal stores via the placenta; added to this, is the iron and TIBC are used to calculate the percent saturation. requirement for increased maternal blood volume and iron As a general rule, changes in the quantity of total loss at delivery due to bleeding. Thus, a single pregnancy body storage iron
are accompanied by fluctuations in the without supplemental iron could exhaust the mother’s iron serum iron and TIBC. As storage iron increases, serum stores. iron increases and TIBC decreases; conversely, if storage Infancy/Childhood In infancy, rapid growth of body size iron decreases or is absent, serum iron decreases and TIBC and hemoglobin mass requires more iron in proportion to increases. A transferrin saturation below 15% is an indicator food intake than at any other time of life. During the first of ID, whereas a saturation above 50% suggests iron over- 6 months of life, an infant synthesizes about 50 g of new load and possibly hemochromatosis. hemoglobin. In addition, iron is needed for tissue growth. Ferritin can be measured in the serum, where its At birth, normal iron stores of 30 mg are adequate to see concentration is roughly proportional to the amount of the infant through the first 4–5 months of life but can be storage iron in the body. Generally, serum ferritin levels less Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 239 than 2 mcg/L indicate depletion of iron stores while levels more than 1000 mcg/L indicate iron overload. Decreased Checkpoint 12.2 serum ferritin levels can be the first indication of develop- A patient’s iron studies revealed serum iron 100 mcg/dL and ing IDA and is 89% sensitive for the detection of ID.22 Serum TIBC 360 mcg/dL. Calculate the percent saturation and UIBC. Are these values normal or abnormal? ferritin levels decrease before the exhaustion of mobilizable iron stores, whereas abnormalities in the TIBC and serum iron may become detectable only after iron stores are depleted. Ferritin is an acute phase reactant, so care should COMPLETE BLOOD COUNT (CBC) be used in interpreting serum ferritin levels in infectious or The normocytic, normochromic red blood cells are gradu- inflammatory states. Concomitant ID can be masked if other ally replaced by microcytic, hypochromic cells in ID. The tests of iron status are not considered. Refer to Table 12-7 for time it takes for abnormal cells to replace the normal popu- the variations in tissue iron in various disease states. lation depends on the extent of the demand for iron and Small amounts of TfR can be identified in serum (sTfR) the amount of iron available. The RDW increases as the by sensitive immunoassay techniques. The level of circulat- microcytic cells replace the normocytic cells. Some hema- ing sTfR mirrors the amount of cellular receptor. The sTfR is tology analyzers report the proportion of hypochromic inversely proportional to the amount of body iron because cells, which, based on current evidence, is the best diag- cellular receptor synthesis increases when cells lack suffi- nostic test for functional iron deficiency.23,24 Analyzers may cient iron. The majority of sTfR is derived from the erythroid also give the reticulocyte hemoglobin content (CHr), mean cells in the bone marrow; the concentration is directly cellular hemoglobin of reticulocytes (MCHr), or reticulo- proportional to erythroid activity and parallels the reticu- cyte hemoglobin equivalent (CHret) (Chapter 11). The CHr locyte count. The level of sTfR is not affected by concurrent measures the functional availability of iron during hemo- disease states as is serum ferritin. Circulating sTfR increases globin synthesis in the erythrocyte. Since the life span of in iron-deficiency anemia but not anemia of chronic disease the reticulocyte is about 4 days, a reduced CHr (less than 28 and thus is useful in the differential diagnosis. pg) provides an early indication of absence of bone marrow When iron is not available for incorporation into the iron stores and iron-restricted erythropoiesis before anemia protoporphyrin ring to form heme or heme synthesis is dis- develops. turbed, zinc can be incorporated into the ring as an alternate HEPCIDIN protoporphyrin ligand forming zinc protoporphyrin (ZPP). Hepcidin is the primary controller of iron supply and stor- As a result, excess protoporphyrin in the form of ZPP can age because of its effect on iron absorption and ferropor- accumulate in the cell. The ZPP formed during RBC devel- tin 1. Although hepcidin measurement has the potential to opment persists for the life of the cell and thus reflects iron increase diagnostic accuracy of iron-metabolism disorders, supply over the preceding weeks. ZPP can be detected by a simple, inexpensive, widely available test has not been measuring fluorescence in the blood. developed. Table 12.7 Iron Status Parameters in Patients with Various Diseases Serum Zinc Serum Ferritin Transferrin Proto- Disease MCV Iron Saturation (%) (mcg/L) Receptor porphyrin Hepcidin§ CHr (pg) Iron deficiency Normal Low More than 16 Less than 30 Increased Normal Low Low, less than 25 Iron-deficiency anemia Low Low Low, less than 16 Less than 10 Increased Increased Very low Low Iron refractory iron- Very low Low Low, less than 10 Variable Increased Increased Normal to Low deficiency anemia (IRIDA) increased Functional iron-deficiency Normal* Low to Low to normal Normal Increased Increased Low Low∞ (FID)† normal Anemia of chronic disease Normal or low Low Low to normal Normal to Low to Increased Increased Low (anemia of inflammation) increased normal Thalassemia minor Low Normal Normal Normal Normal to Normal to ND Low increased increased CHr, reticulocyte hemoglobin content; ND, not determined; §research is ongoing to determine hepcidin’s value as diagnostic tool; *useful at diagnosis and assessing trends; †the percentage of hypochromic cells is best for identifying FID; ∞less than 29 pg predicts FID in patients receiving erythropoietin-stimulating agent therapy. 240 Chapter 12 FERROKINETICS risk of developing IDA. Malnutrition is associated not only Quantitative measurement of internal iron exchange (ferro- with decreased iron intake but also with decreased intake kinetics) is useful in understanding the pathophysiology of of other essential nutrients including folate. Thus, causes of certain erythropoietic disorders. Ferrokinetic studies moni- anemia associated with malnutrition can be multifactorial. tor the movement of radioactively labeled iron (59Fe) from the plasma to the bone marrow and its subsequent uptake into developing erythroblasts. Plasma iron is labeled by Historical Aspects intravenous injection of a trace amount of 59Fe. The labeled In the United States between 1870 and 1920, chlorosis, a iron binds to transferrin for transport. Its clearance from word used to describe the condition of ID, was so common the plasma can be followed by counting the radioactivity in young women that it was believed that every female had that remains in the plasma at intervals up to 90 minutes. some form of the disease during puberty.26 The word chlo- The rate at which iron leaves the plasma is called the plasma rosis was coined because of the greenish tinge of the skin iron turnover (PIT) rate, and its primary determinant is tis- in these patients. These chlorotic girls were found to have sue need. The PIT is a good indicator of total erythropoiesis decreased numbers of red cells and an increase in the pro- and correlates well with the erythroid cellularity of bone portion of serum to cells in their blood. Some of the chlorotic marrow. girls were also noted to have unusual appetites, craving The amount of iron used for effective hemoglobin syn- substances such as chalk and bugs. Some physicians linked thesis can also be measured by determining the amount of chlorosis to dietary habits; others implicated menstruation 59Fe incorporated into circulating erythrocytes over time. as a possible cause of the disease because chlorosis affected Normal erythrocyte utilization is 70 to 90% of the injected girls in puberty but not boys. 59Fe by day 10 to 14. This is termed the erythrocyte iron turnover (EIT) rate. The EIT is a good measure of effective erythropoiesis and correlates with the reticulocyte produc- Etiology tion index. ID can occur because of normal or pathologic conditions The normal discrepancy between the rate at which iron that result in an increased demand for iron, m alabsorption, leaves the plasma (PIT = 0.7 mg/day/dL) and the rate at or poor diet. In malabsorption or with an iron-deficient which it moves from marrow to circulating erythrocytes diet, iron stores can become depleted over a period of (EIT = 0.56 mg/day/dL) suggests that the red cell utiliza- years. With an increase in the demand for iron, iron deple- tion (RCU) of iron is less than 100%. Some of the labeled tion can occur more rapidly, sometimes over a period of iron can enter the liver or bone marrow macrophages. In months. addition, 5 to 10% of bone marrow iron is involved in inef- In most developed countries, inadequate dietary intake fective erythropoiesis, causing a loss of the labeled iron by of iron is rarely the cause of anemia (except in infancy, preg- intramedullary destruction of abnormal erythrocytes. nancy, and adolescence). Diet and socioeconomic status, A rapid or increased PIT coupled with a normal or however, are factors in the development of ID in children. increased RCU indicates increased erythropoiesis. A nor- The average adult male in the United States ingests mal to increased PIT coupled with a decreased RCU many times more iron than is required. It would take an indicates ineffective erythropoiesis, and a decreased PIT adult male about 8 years to develop IDA if he absorbed no with corresponding decreased RCU indicates decreased iron during that period of time. ID in adult males is almost erythropoiesis. always due to chronic blood loss from the gastrointestinal Ferrokinetic studies can be valuable in locating sites of or genitourinary tracts. For each milliliter of blood lost, a medullary and extramedullary erythropoiesis by counting loss of about 0.5 mg of iron occurs. Gastrointestinal lesions surface radioactivity over the liver, spleen, and sacrum. leading to blood loss include peptic ulcers, hiatal hernia, malignancies, alcoholic gastritis, excessive salicylate inges- tion, hookworm infestation, and hemorrhoids. The cor- Iron-Deficiency Anemia relation of ID and GI lesions in elderly patients is high. Genitourinary tract blood loss occurs less frequently and Iron deficiency is the most common nutritional deficiency in can result from lesions within the genitourinary system. In the world, affecting one-half the world’s children and mil- women of child-bearing age, iron loss through menstruation lions of adults.25 It affects 8–10% of children in the United is the most frequent cause of ID.27 Blood loss is less often the States. It is prevalent in countries where grain is the main- result of intravascular hemolysis. If haptoglobin becomes stay of the diet or meat is scarce. Unfortunately, these are depleted, the free circulating hemoglobin dissociates into generally countries where hookworm infestation is endemic. dimers, is filtered by the kidneys, and appears in the urine The combination of decreased availability of dietary iron (hemoglobinuria). This results in a loss of iron, the amount and chronic blood loss from parasitic infection increases the of which is proportional to the amount of hemoglobin in the Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 241 urine. Some of the hemoglobin is reabsorbed in the renal • Stage 1 During iron depletion, iron stores are exhausted tubules, resulting in the deposition of iron as hemosiderin as indicated by a decrease in serum ferritin. There is in renal tubular cells and eventual sloughing of these cells no anemia and erythrocyte morphology is normal, but into the urine (hemosiderinuria). the red cell distribution width (RDW) is frequently Malabsorption is an uncommon cause of ID except elevated. The abnormal RDW may be the first hema- in malabsorption syndromes (such as sprue), after tologic indication of a developing ID in the nonanemic gastrectomy, in atrophic gastritis, and in achlorhydria. patient. Gastrectomy results in impaired iron absorption due to In hospitalized patients, the RDW is not as specific, and the absence of gastric juice, which helps to solubilize the serum ferritin is not as sensitive in detecting ID. and reduce dietary iron into the more easily absorbed Hospitalized patients have a high incidence of other ferrous form. In addition, with the loss of the reservoir diseases that can affect these parameters. function of the stomach, nutrients can transit rapidly • Stage 2 The second stage of ID is characterized by through the duodenum, allowing insufficient time for iron-deficient erythropoiesis. There is insufficient iron absorption. iron to insert into the protoporphyrin ring to form Obesity is associated with an increase in hepcidin, heme. As a result, the protoporphyrin accumulates in iron depletion, and ID.28 Thus obesity plays a role in the cell and complexes with zinc to form ZPP. Bone modifying iron regulation. The Third
National Health marrow sideroblasts are absent; macrophage iron is and Nutritional Exam survey revealed that overweight not seen. children and adolescents are twice as likely to be ID than those of normal weight. Serum iron is also lower in over- Anemia and hypochromia are still not detectable, but weight women but not in overweight men.29 Iron status the erythrocytes can become slightly microcytic. The following diet-induced weight loss or bariatric surgery CHr, which measures the functional availability of iron requires further study. during hemoglobin synthesis, can decrease even when anemia is absent (Chapter 11). Checkpoint 12.3 • Stage 3 A long-standing negative iron flow eventu- A 30-year-old female and a 25-year-old male both had bleed- ally leads to the last stage of iron deficiency: IDA. ing ulcers. Assume that they acquired the ulcer at the same Blood loss can significantly shorten the time for time, were losing about the same amount of blood, had equal this stage to develop. All laboratory tests for iron amounts of storage iron to begin with, and were taking in about status become markedly abnormal. The most signifi- 15 mg of dietary iron each day. Would you expect that the cant finding is the classic microcytic hypochromic woman and man would develop ID at the same time? Explain. anemia. It is apparent, then, that when microcytic hypochromic IDA is frequently accompanied by atrophic g astritis anemia due to ID is present, the situation represents the and achlorhydria, but it is not known whether a chlorhydria advanced stage of severely deficient total body iron. and gastritis are causes of iron malabsorption or the ID is a cause of atrophic gastritis and hence achlorhydria. In patients with gastritis and achlorhydria, therapy with oral Clinical Presentation iron may be ineffective due to poor iron absorption. The onset of IDA is insidious, usually occurring over a period of months to years. Early ID stages usually show Pathophysiology no clinical manifestations, but as anemia develops, clinical symptoms appear. In addition to symptoms of anemia, a Defined as a diminished total body iron content, ID devel- variety of other abnormalities can occur due to a decrease ops in sequential stages during a period of negative iron or absence of iron-containing enzymes in various tissues. balance (losing more iron than is absorbed in the gut). These These include koilonychia (concavity of nails), oral mani- stages are commonly referred to as: festations including dry mouth, glossitis, and burning • Iron depletion sensation of oral mucosa,30 pharyngeal webs, muscle dys- • Iron-deficient erythropoiesis function, inability to regulate body temperature when cold or stressed, and gastritis. • IDA A curious manifestation of ID is the pica syndrome. Thus, ID can range in severity from reduced iron stores Pica is an eating disorder with unusual cravings for ingest- with no functional effect (Stages 1 and 2) to severe anemia ing non-nutritional items. The most common dysphagias with deficiencies of tissue iron-containing enzymes (Stage described in patients with ID include ice-eating (phago- 3). Laboratory evaluation of iron status is helpful in defin- phagia), dirt/clay-eating (geophagia), and starch-eating ing these 3 stages (Table 12-8). (amylophagia). 242 Chapter 12 Table 12.8 Laboratory Test Profile of Fe Status in Developing Iron Deficiency Normal Stage 1: Stage 2: Iron-deficient Stage 3: Iron- Iron depletion erythropoiesis (IDE) deficiency anemia (IDA) Storage iron (serum ferritin) Threshold for IDE Transport iron (% transferrin saturation) Functional iron (hemoglobin) Stage 1 Storage Iron Parameter Reference Interval Depletion; No Anemia Stage 2 IDE Stage 3 IDA Ferritin (mcg/L) Male 20–300 Decreased Decreased Decreased Female 12–200 sTfR (nmol/L) 8.7–28.1 Normal Increased Increased TfR-F Index 0.63–1.80 Increased Increased Increased Reticulocyte hemoglobin Normal Decreased Decreased Decreased content (Ret-He or CHr)* Serum iron (mcg/dL) Male 65–180 Normal Decreased Decreased Female 50–180 TIBC (mcg/dL) 250–450 Normal or slight increase Increased Increased Saturation % Male 20–45 Normal Decreased Decreased Female 15–45 ZPP (mcg/dL) whole blood Male 1–27 Normal Increased Increased corrected to volume of packed Female 11–45 red cells of 0.35 L/L Male hemoglobin (g/L) 140–174 Normal Normal Decreased Female hemoglobin (g/L) 120–160 Normal Normal Decreased RBC morphology Normocytic, normochromic Normocytic, normochromic Normocytic, normochromic Microcytic, hypochromic sTfR, serum transferrin receptor; TfR-F, transferrin receptor-ferritin; TIBC, total iron-binding capacity; ZPP, zinc protoporphyrin; Ret-He or CHr is the first peripheral blood marker that becomes abnormal in ID. Iron-deficient infants perform worse in tests of men- when an iron-deficient person is exposed to toxic metals tal and motor development than do nonanemic infants. such as lead, cadmium, and plutonium. There is speculation that untreated ID at this stage of human d evelopment has long-lasting effects on the central Laboratory Evaluation nervous system. Symptoms reported to occur in iron-defi- Practice guidelines for diagnosis and therapy of iron cient children include irritability, loss of memory, and dif- deficiency are inconsistent. However, laboratory tests are ficulties learning. Deficiencies of the immune system have essential for an accurate diagnosis and in evaluation of been attributed to iron-related impairment of host defense therapy. More recently, a guideline for the laboratory diag- mechanisms. nosis of functional iron deficiency was published for use In the absence of iron in the gut, other metals are in patients with chronic kidney disease and in other con- absorbed in increased amounts. This can be significant ditions treated with erythropoiesis stimulating agents.23 Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 243 PERIPHERAL BLOOD Platelets may be normal, increased, or decreased. The blood picture in well-developed ID is microcytic (MCV Thrombocytosis frequently accompanies ID, and has been 55–74 fL) and hypochromic (MCHC 22–31 g/dL, MCH proposed to be related to ID caused by chronic blood loss. 14–26 pg) (Figure 12-11). Because ID develops progres- Thrombocytopenia may occur in patients with severe or sively, any gradation between the well-developed micro- long-standing anemia, especially if accompanied by folate cytic hypochromic iron-deficient blood picture and normal deficiency. Platelet numeric abnormalities may be corrected can occur. MCV and MCH are not diagnostic parameters with treatment that replenishes iron stores. but are useful in monitoring trends of ID over a period of time.23 Microcytosis and anisocytosis, characterized by an CASE STUDY (continued from page 226) increased RDW, are usually the first morphologic signs, developing even before anemia (Chapters 10, 11). The blood Jose, the 83-year-old patient, had a CBC upon film demonstrates progressive poikilocytosis. The most fre- admission. The results were: quent poikilocytes are target cells (codocytes), elliptocytes, RBC 4.15 106/mcL and dacryocytes (teardrop cells). Hb 8.1 g/dL The typical blood picture can be masked if the iron- Hct 26% deficient patient has a concurrent vitamin B12 or folate Platelets 174 * 103 deficiency (causes of macrocytic anemia). In these cases, /mcL microcytosis may become apparent only after vitamin WBC 2.8 * 103/mcL B12/folic acid replacement therapy. 2. How would you describe his anemia Both the relative and absolute number of reticulocytes morphologically? can be normal or even slightly increased, but the reticu- locyte count is decreased relative to the severity of the anemia with a reticulocyte production index (RPI) of less than 2. A decreased CHr and an increase in percentage of IRON STUDIES hypochromic red cells is an early indicator of iron-restricted Iron studies on iron-deficient patients help establish the erythropoiesis in nonanemic individuals. In patients on diagnosis. The serum iron is decreased, usually less than 30 chronic hemodialysis, the CHr has been shown to be supe- mcg/dL, the TIBC is increased, and transferrin saturation rior to the presence of hypochromic red cells in assessing is decreased to less than 15%. Serum iron concentration has the adequacy of iron for erythropoiesis. a diurnal variation with highest levels in the morning, so The leukocyte count is usually normal but can increase sampling time is an important consideration. because of chronic marrow stimulation in long-standing The serum ferritin level is decreased in all stages of ID cases or after hemorrhage. With concomitant hookworm and can be the first indication of a developing ID state. Serum infestation, eosinophilia can be present. ferritin is generally considered the single best test to detect ID. Once serum ferritin levels fall below 12 mcg/L, the lev- els no longer correlate with storage iron because stores are exhausted. Serum ferritin is an important test to differentiate IDA from other microcytic hypochromic anemias. Levels are normal to increased in ACD unless complicated by ID and increased in sideroblastic anemia and thalassemia. Because serum ferritin is an acute phase reactant, the lower limit of the reference interval for this parameter may need to be adjusted to detect ID in some patient popula- tions (Table 12-9). To detect ID in patients with concomitant ACD, inflammation, infection, pregnancy, and a wide range of other medical problems, the lower limit of serum fer- ritin should be raised above 12 mcg/L.31,32 It has also been suggested that the threshold level of serum ferritin for a diagnosis of ID in the aged subject be raised because serum ferritin levels rise with age. The sTfR assay has proved useful in detecting and dif- Figure 12.11 ferentiating IDA and ACD. Patients with ID have a mean Microcytic, hypochromic anemia of iron deficiency. Compare size of RBCs to nucleus of small lymphocyte in sTfR level over twice (13.91 + 4.63 mg/L) that of normal center. Note the anisocytosis and poikilocytosis (peripheral blood; individuals (5.36 + 0.82 mg/L). Conversely, patients with Wright-Giemsa stain; 1000* magnification). chronic disease and acute infection (ACD) have mean levels 244 Chapter 12 Table 12.9 Serum Ferritin Cutoff Levels for Detecting Iron Table 12.10 Conditions Associated with Increased Deficiency in Patients with Other Diseases and Conditions Erythrocyte Protoporphyrin (EP) Upper Serum Ferritin Cutoff • Anemia of chronic disease Disease Levels for a Diagnosis of ID • Iron-deficiency anemia Chronic renal disease (absolute ID) 100 mg/L • Lead poisoning • Erythropoietic protoporphyria Chronic renal disease on EPO 1009800 mg/L therapy* (functional ID) • Some sideroblastic anemias Anemia of chronic disease 50 mg/L • Conditions with markedly increased levels of erythropoiesis • Thalassemia Rheumatoid arthritis 70 mg/L Other medical problems 100 mg/L Pregnancy 30 mg/L In patients with concurrent lead poisoning, the ZPP Reference interval: cannot be used to distinguish ID and thalassemia because Males 209300 mg/L lead inhibits ferrochelatase, the enzyme needed to incorpo- rate iron into the protoporphyrin ring. Consequently, the Females 129200 mg/L free erythrocyte porphyrin complexes with zinc, and ZPP is *Functional iron deficiency is present when iron saturation is 20–50%, serum ferritin increased in lead poisoning whether or not iron is available. 1009800 mg/L, and the patient responds to intravenous iron therapy with an increase in hemoglobin and/or a decrease in requirement for EPO. almost identical to those of normal individuals. A combina- CASE STUDY (continued from page 243) tion of sTfR and serum ferritin can be used to calculate the Reflex testing for anemia on Jose followed based sTfR-F index: on the CBC results. The following test results were sTfR-F Index = sTfR/log ferritin obtained: Reticulocyte count 2.6% This index can improve detection of subclinical iron- deficient states in healthy individuals.33,34 An index of more Serum iron 18 mcg/dL than 1.8 indicates depletion of iron stores. TIBC 425 mcg/dL Combinations of serum ferritin, MCV, TIBC, percent 3. Calculate percent saturation. saturation, and sTfR can eliminate the need for costly, incon- venient, and painful bone marrow examination to assess 4. Is this value normal, decreased, or increased? iron stores in patients with inflammation or chronic disease 5. What disease, if any, does this value suggest? and in early stages of ID.35,36 ERYTHROCYTE PROTOPORPHYRIN (EP) STUDIES The ZPP measurement correlates inversely with serum fer- BONE MARROW ritin concentration and is more cost effective. It can detect The bone marrow shows mild to moderate erythroid hyper- iron depletion before anemia develops and thus is a good plasia with a decreased M:E ratio. Total cellularity is often screening tool for the early stages of ID. Because the number moderately increased. This increase in marrow erythropoi- of nonanemic toddlers with iron depletion is significant and etic activity without a corresponding increase in peripheral ID has a detrimental effect on development in children, ZPP blood reticulocytes suggests an ineffective erythropoietic rather than the hematocrit should be used as the screen- component. With appropriate iron therapy, the erythroid ing test for ID.37 ZPP is also more diagnostically efficient hyperplasia initially increases and then returns to nor- than serum iron or serum ferritin in screening for
ID in the mal. A common finding (not exclusive to IDA) is the pres- presence of infection or inflammation and in hospitalized ence of poorly hemoglobinized erythroblasts with scanty patients with microcytic anemia.38,39 Thus, ZPP can be a irregular (ragged) cytoplasm. This morphology is most valuable screening assay for these populations. evident at the polychromatophilic stage. Erythroid nuclear The level of ZPP is useful also as a screening test to abnormalities are sometimes present and can resemble the differentiate ID and thalassemia, the two most common changes found in dyserythropoietic anemia. These changes causes of microcytic hypochromic anemia. Whereas ZPP include budding, karyorrhexis, nuclear fragmentation, and may be elevated in thalassemia, the increase in ID is 3 to 4 multinuclearity. times higher than in thalasssemia.40 When laboratory analy- Stains for iron reveal an absence of hemosiderin in the sis reveals a high ZPP combined with a high RDW, ID is macrophages, a consistent finding in ID. Sideroblasts are strongly suggested.41 The RDW is typically normal or only markedly reduced or absent. Evaluation of iron stores using slightly elevated in thalassemia trait. Table 12-10 lists other serum iron studies eliminates the need for bone marrow conditions associated with increased EP levels. examination in most cases. Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 245 Therapy practice. If the hemoglobin concentration is decreased, a therapeutic trial of iron may be initiated in this population. Once the cause of the anemia has been established, the treat- If anemia persists after 1 month of therapy, further evalua- ment approach is to address the underlying disorder (e.g., tion is necessary (Figure 12-12). bleeding ulcer), administer iron, and observe the response. The anemia is usually corrected by the oral administration of ferrous sulfate. Parenteral iron therapy, which is more dangerous and expensive than oral iron therapy, is indi- Anemia of Chronic Disease cated only rarely for unusual circumstances. Intravenous ACD is an iron-sequestration syndrome characterized by iron dextran is often required for patients with chronic renal inadequate iron availability for erythropoiesis due to high disease who are receiving therapy with recombinant human hepcidin levels. It is the most common anemia other than erythropoietin (rHuEPO) to maintain a two- to three-fold IDA. It accounts for the anemia in more than one-third of increase in the rate of erythrocyte production.42 anemic hospitalized patients without blood loss. Anemia Iron-deficient patients treated with iron experience is present in up to 50% of patients with malignant solid a return of strength, appetite, and a feeling of well-being tumors and is often the clue that leads to a diagnosis of within 3–5 days, whereas the anemia is not alleviated for cancer. The most common anemia in these patients is ACD. weeks. The dysphagias also are corrected before the anemia. A response to iron therapy is defined as an increase Etiology of 1 gm hemoglobin in 1 month. Because reticulocytes are new red blood cells just released from the bone marrow, ACD, also called anemia of inflammation or infection (AI) is reticulocyte counts and the immature reticulocyte fraction usually defined as the anemia that occurs in patients with (IRF) give a snapshot of recent red blood cell produc- chronic infections, chronic inflammatory disorders, trauma, tion (Chapter 11). Reticulocyte response to iron therapy organ failure, or neoplastic disorders not due to bleeding, begins about the 3rd day after the start of therapy, peaks at hemolysis, or marrow involvement (Table 12-11). ACD is about the 9th to 10th day (4–10% reticulocytes), and declines characterized by low serum iron but normal iron stores. The thereafter. An increase in CHr (Chapter 11) is an early anemia appears to be a specific entity and does not relate to indicator of the availability of functional iron and its incor- any nutritional deficiency. Anemias associated with renal, poration into hemoglobin over the previous several days. endocrine, or hepatic insufficiency are usually excluded The CHr begins to increase well in advance of an increase from ACD. in reticulocytes and hemoglobin.43,44 If therapy is success- ful, the hemoglobin should rise until levels within refer- Pathophysiology ence intervals are established, usually within 6–10 weeks. The causes of ACD are multifactorial. Most of the patho- To restore iron stores, extended therapy with small amounts physiology is linked to inflammatory cytokines. ACD of iron salts may be required (usually for 6 months) after the is characterized by hypoferremia, decreased transferrin hemoglobin has returned to normal. (decreased TIBC), increased serum ferritin, and increased Because of the high prevalence of ID in toddlers, screen- iron in bone marrow macrophages. This suggests a block ing all 1-year-olds for ID and/or anemia is a well-accepted in the mobilization of iron from macrophages for recycling to the bone marrow erythroblasts. This block results in Children (1–2 years old) limited availability of iron for erythropoiesis, referred to as iron-restricted erythropoiesis. Because m acrophages Decreased Hb or MCV or CHr recycle 10–20 times more iron than is absorbed by entero- cytes, any changes in iron flux through the macrophages Therapy with iron affect iron balance more rapidly than changes in iron absorption and transport by enterocytes. Absorption of iron in the intestine is decreased, which can lead to ID 2 weeks later over time. Macrophage iron release is blocked by increased hep- No increase in Hb, CHr or IRF Increase in Hb, CHr or IRF cidin produced in response to IL-6 and BMP ligands bind- ing to their membrane receptors. The cytokines upregulate Further evaluation (iron studies, Continue iron therapy the transcription factors STAT and SMAD, which activate microcytic: hypochromic ratio) the hepcidin gene promoter and increase hepcidin pro- duction. Hepcidin also plays a role in the body’s innate Figure 12.12 The screening process for all 1-year-olds for ID immune system, which is designed to sequester iron from and/or anemia. pathogens and thus restrict their growth. 246 Chapter 12 Table 12.11 Conditions Associated with Anemia of Chronic Disorders Chronic Infections (after 1–2 months of sustained infection) Chronic, Non-infectious Inflammation Other Pulmonary infections Rheumatoid arthritis Malignant diseases Subacute bacterial endocarditis Rheumatic fever Alcoholic liver disease Pelvic inflammatory disease Systemic lupus erythematosus Congestive heart failure Osteomyelitis Sterile abscess Thrombophlebitis Chronic urinary tract infection Regional enteritis Ischemic heart disease Chronic fungal disease Ulcerative colitis Severe tissue trauma Tuberculosis Thermal injury Fractures Other contributing factors to ACD are the negative Laboratory Evaluation impact of inflammatory cytokines on survival, prolifera- tion, and differentiation of erythroid progenitor cells and an Many of the laboratory test results are nonspecific in ACD, impaired response to EPO.3,45,46,47 The shortened erythrocyte but with clinical signs and primary diagnosis, they help survival seen in ACD is a result of nonspecific macrophage to establish the ACD diagnosis. This is important because activation, hemolytic factors elaborated by tumors, vascu- ACD does not usually require therapy for the anemia. lar factors, and the presence of bacterial toxins capable of PERIPHERAL BLOOD hemolyzing erythrocytes. Of all mechanisms described, the A mild anemia with a hemoglobin of not less than 9.0 g/ block in release of iron from macrophages due to increased dL and hematocrit of not less than 27% is characteristic. hepcidin appears to be the most significant in the patho- Erythrocytes are usually normocytic (MCV more than genesis of ACD. 85 fL) and normochromic, but can present as normocytic, hypochromic, or in long-standing cases, microcytic and Clinical Presentation hypochromic (Figure 12-13). The reticulocyte production The signs and symptoms of ACD are usually those associ- Checkpoint 12.4 ated with the underlying disorder. Rarely severe, the degree How does the peripheral blood picture in ACD differ from that of the anemia roughly correlates with the activity of the seen in IDA? underlying disease. a b Figure 12.13 (a) Blood film from a patient with anemia of chronic disease. Hb 9.6 g/dL; MCV 76 fL; MCH 24 pg; MCHC 30.6 g/dL. The erythrocytes appear microcytic (peripheral blood; Wright-Giemsa stain; 1000* magnification). (b) Bone marrow from same patient in (a) stained with Prussian blue. Note the macrophages with abundant blue staining iron (bone marrow; Prussian blue stain; 1000* magnification). Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 247 index is less than 2. The white blood cell count and platelet count are normal unless they are altered due to the primary Iron Refractory Iron- disease state. Deficiency Anemia (Irida) Iron studies show decreased serum iron (10–65 mcg/ dL), decreased to normal TIBC (100–300 mcg/dL), nor- Iron refractory iron-deficiency anemia (IRIDA) is a mal to low transferrin saturation (10–20%), and normal to rare inherited form of abnormal systemic iron regulation increased serum ferritin. Serum ferritin can be helpful in in which both the absorption and utilization of iron is distinguishing this anemia from IDA. Although serum iron impaired.49 is low in both anemias, serum ferritin, which reflects body iron stores, is normal or increased in ACD and low in IDA. Pathophysiology Because ACD is characterized by iron-deficient erythropoi- IRIDA is an autosomal recessive disorder caused by esis, the ZPP levels are increased. The CHr is low. mutations in the TMARSS6 gene, which encodes trans- It is important to remember that ferritin is an acute membrane protease, serine 6, also known as matriptase-2 phase reactant and is very often increased in inflamma- (MT-2).50 Binding of BMP6 ligand to the BMP membrane tory conditions. Therefore, in ACD serum ferritin may be receptor complex activates SMAD proteins, which trans- normal even if concurrent ID exists. If serum ferritin falls locate to the nucleus and activate hepcidin transcrip- in the interval of 20–100 mcg/L in ACD, another means of tion (Figure 12-6). MT-2 inhibits the BMP cell signaling assessing iron should be considered, such as sTfR assay, pathway by cleaving HJV, a BMP coreceptor on the cell sTfR-ferritin index, or bone marrow examination.48 Serum membrane. This inhibition of BMP signaling leads to TfR is high in IDA but normal in uncomplicated ACD. The decreased hepcidin production, thus preventing hepcidin sTfR-ferritin index is increased in ID and decreased in ACD. over-expression. When mutated, MT-2 is unable to down- regulate BMP signaling, leading to a chronic increase in CASE STUDY (continued from page 244) hepcidin (hyperhepcidinemia).51 This results in systemic ID because of decreased iron absorption in the gut. The 6. How do the patient’s iron study results help in high hepcidin level also results in a block in release of differentiating the diagnosis of ID from ACD? iron from macrophages and hepatocytes for erythropoi- 7. What additional iron test, that was not per- esis, resulting in decreased plasma iron (hypoferremia) formed, would be most helpful in this case? and severe IDA. Treatment with oral iron is not successful (iron refractory). Clinical Presentation BONE MARROW The bone marrow usually shows an increased M:E ratio Most patients present in childhood with a lifelong his- because of a decrease in erythrocyte precursors. The propor- tory of moderate to severe microcytic anemia. They have tion of younger erythroblasts increases. Poor hemoglobin demonstrated abnormal iron absorption following oral production is apparent, especially in the polychromatophilic iron therapy. Iron utilization is also abnormal as demon- erythroblasts. The proportion of sideroblasts decreases to strated by an incomplete and transient response to par- less than 30%; however, the macrophages appear to have enteral iron. increased amounts of hemosiderin in the form of coarse iron aggregates (Figure 12-10b). This finding helps to distinguish Laboratory Evaluation ACD from IDA. In IDA, macrophage iron and sideroblasts are absent. A diagnostic algorithm for IRIDA has been developed.50 Bone marrow examination is usually not necessary for It begins with laboratory findings to establish ID. This distinguishing ACD from IDA. An algorithm using sTfR, includes microcytic anemia (MCV 45-65fL; hemoglobin 6–9 sTfR/log ferritin index, the MCV, serum ferritin, and iron g/dL) and hypoferremia with very low transferrin satura- saturation can correctly differentiate and classify most cases tion (less than 5%). There is normal to borderline low fer- of microcytosis due to ACD and IDA. ritin levels and increased hepcidin. Other causes of acquired iron deficiency should be excluded. This is followed by an assessment of the patient’s response to a trial of oral iron Therapy therapy and/or to parenteral iron therapy. No hematologi- Anemia can be alleviated by successful treatment of the cal response to oral iron therapy and an incomplete/tran- underlying disease. Anemia is usually mild and nonprogres- sient response to parenteral iron therapy follows an oral sive; thus, transfusion is rarely warranted
except in older iron challenge. The diagnostic test for IRIDA is sequencing patients with vascular disease and circulatory insufficiency. of the TMPRSS6 gene. 248 Chapter 12 Therapy useful variables include red cell ZPP concentration and serum ferritin. However, serum ferritin values as high as Therapy for IRIDA includes parenteral iron supplementa- 1200 mcg/L do not rule out FID. Although some patients tion. It may improve the anemia but does not usually com- with high ferritin values may respond to IV iron therapy, pletely correct it. Repeated dosing raises concern of iron iron overload is a concern with this therapy. There is no overload. Effective therapy may require manipulation that recommended highest ferritin value at which it is safe to will lower the hepcidin level.49 give IV iron therapy. The percentage transferrin saturation may be useful in monitoring a response to ESA and/or iron therapy in chronic kidney disease. Functional Iron Deficiency (FID) Anemias Associated with Functional iron deficiency (FID) is a form of iron-restricted erythropoiesis; it refers to a condition in which iron stores Abnormal Heme Synthesis are sufficient but in the presence of acutely increased eryth- ropoietic demands, sufficient iron cannot be mobilized from These anemias are associated with defects in enzymes of stores for erythropoiesis.49 This may occur when patients the heme biosynthetic pathway leading to abnormal heme are treated with erythropoiesis-stimulating agents (ESAs) synthesis. Iron incorporation into the protoporphyrin ring such as rHuEPO or when there is an increased demand for to form heme can be blocked. In contrast to IDA, the posi- erythrocytes such as occurs in hemolytic anemias or hemor- tive iron balance in these anemias can lead to an increase rhage (stress erythropoiesis). Intravenous iron therapy may in iron stores predominantly in the spleen, liver, and bone improve responsiveness to ESA. marrow. Serum ferritin levels greater than 300 mcg/L in the male and greater than 200 mcg/L in the female indicate Etiology increased iron stores. The conditions discussed in this section include sidero- Anemia is a common finding in patients with chronic kid- blastic anemia and the porphyrias. Lead poisoning is also ney disease. The causes are varied, but the most impor- included because of its pathophysiologic relationship to tant factor is a loss or decrease in EPO production by the these anemias through a block in heme synthesis. diseased kidneys. Thus, hemodialysis patients are often given rHuEPO to increase erythropoiesis. Lack of an ade- Sideroblastic Anemias quate response to EPO therapy is primarily due to FID.51 However, if the patient is given intravenous iron injections Mutations that affect the first enzymatic step in heme together with rHuEPO, the hematopoietic response may be synthesis, the formation of ALA, result in sideroblastic ane- enhanced. FID is also a complication of treatment with other mia (Chapter 6). Mutations in the subsequent steps of heme erythropoiesis-stimulating agents. synthesis result in metabolic disorders called porphyrias. Sideroblastic anemia (SA) is the result of diverse clinical and Laboratory Evaluation biochemical manifestations that reflect multiple underlying hereditary, congenital, or acquired pathogenic mechanisms. Although the MCV and MCH are useful parameters for However, all types are characterized by (1) an increase in diagnosis, they are not helpful in assessing acute changes total body iron, (2) the presence of ring sideroblasts in the in iron availability in patients receiving therapy with ESAs. bone marrow, and (3) hypochromic anemia. The best variables for identifying FID are the reticulocyte ETIOLOGY hemoglobin content (CHr) and the percentage of hypochro- The classification of SA is arbitrary at best, and many dif- mic cells. The CHr is a real-time (48-hour) parameter, while ferent schemes of classification exist. The one in Table 12-12 the percentage of hypochromic cells is a time-averaged is among the most descriptive and separates those that are marker of iron-restricted erythropoiesis. A CHr less than 29 inherited and those that are acquired. pg and a reticulocyte-hemoglobin (Ret-He) less than 25 pg predicts FID in patients on ESA therapy. A Ret-He less than 25 pg is also suggestive of ID. Table 12.12 Classification of Sideroblastic Anemia Therapy Hereditary Acquired • Sex linked • Idiopathic refractory sideroblastic anemia A Ret-He result less than 30.6 pg has the best predictive • Autosomal recessive (IRSA) or refractory anemia with ring value for a response to IV iron therapy in patients with sideroblasts (RARS) chronic kidney disease undergoing hemodialysis. Other • Secondary to malignancy, drugs, toxins, lead Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 249 The most common form of hereditary SA is due to a The activity of ALAS2 depends on the presence of pyr- defective X-linked recessive gene. Although carrier females idoxal phosphate. This cofactor binds to the enzyme and often show a dimorphic population of both morphologi- is crucial for its stability, maintenance of a conformation cally abnormal and normal erythrocytes, they rarely have optimal for substrate binding and product release, and its anemia. Affected males demonstrate the typical SA findings. catalytic activity. In 17 of the ALAS2 mutations, a partial In rare instances, both sexes are equally affected, implying to complete clinical response to pharmacologic doses of the presence of another hereditary form that is transmitted pyridoxine occurs. Excess pyridoxine possibly enhances in an autosomal recessive manner. In hereditary SA, ane- the abnormal ALAS enzyme activity by stabilizing it after mia can become apparent in infancy but most commonly synthesis. In the pyridoxine-refractory sex-linked SA, the appears in young adulthood. Occasionally, symptoms do mutation appears to affect the processing of ALAS2 pre- not occur until age 60. mRNA (which terminates ALAS2 translation prematurely) The acquired forms of sideroblastic anemia are more or abolishes its enzymatic function. common than the hereditary forms. The acquired forms are Significant ineffective erythropoiesis is characterized classified according to whether the basis of the anemia is by bone marrow erythroid hyperplasia. This increased unknown (idiopathic) or is secondary to an underlying dis- erythropoietic activity results in increased iron absorption ease or toxin (secondary type). The idiopathic form, refrac- in the gut. Iron overload can be significant and can lead to tory anemia with ring sideroblasts (RARS), can affect either complications such as cardiac failure and diabetes. sex in adult life. It is included in a group of acquired stem Other hereditary forms of SA have been described but cell disorders called myelodysplastic syndromes (Chapter 25) are less common than the sex-linked type. that have a tendency to terminate in acute leukemia. The Acquired Sideroblastic Anemia Acquired SA can be cat- acquired secondary SAs are associated with m alignancy, egorized as refractory anemia with ring sideroblasts (RARS) drugs, or other toxic substances. In this type, once the and those secondary to drugs or toxins. SA secondary to underlying disorder is effectively treated or the toxin drugs or toxins is the result of the interference of the drugs removed, the anemia abates. or toxins with the activity of heme enzymes. Lead and alco- PATHOPHYSIOLOGY hol are the most common causes of this form of SA. Studies of patients with SA have shown disturbances of Lead Poisoning Lead poisoning (plumbism) has been the enzymes regulating heme synthesis. Ring sideroblasts recognized for centuries. In children, it generally results are specific findings in these heme enzyme abnormalities. from ingestion of flaked lead-based paint (lead was Ring sideroblasts form from an accumulation of nonfer- removed from paint sold after 1978). Children from lower ritin iron in the mitochondria that encircle the erythro- socioeconomic backgrounds are at increased risk; those blast nucleus. The mitochondria eventually rupture as between 1 and 3 years of age are at greatest risk. Clinically, they become iron laden. When stained with Prussian lead toxicity in children is associated with hyperactivity, blue, the iron appears as blue punctate deposits circling low IQ, concentration disorders, hearing loss, and impaired the nucleus (ring sideroblasts). Iron within erythroblasts growth and development. Lead can also cause permanent is normally deposited diffusely throughout the cytoplasm neurological damage. In adults, lead poisoning is primar- in siderosomes. ily the result of inhalation of lead or lead compounds from Hereditary Sideroblastic Anemia The most common form industrial processes. There is growing evidence that low of hereditary SA is sex linked and due to an abnormal levels of lead can cause adverse health effects in adults as d-aminolevulinic acid synthase enzyme (ALAS). well as children. It is the most common disease of toxic There are two different forms of ALAS, the nonery- environmental origin in the United States. throid or hepatic form (ALAS1) and the erythroid form Many states require laboratories and physicians to (ALAS2). Nonerythroid ALAS1 is encoded by a gene (chro- report elevated blood lead levels to the state health depart- mosomal region 3p21) expressed in all tissues. The erythroid ment, which can report to the Centers for Disease Con- form, ALAS2, is encoded by a gene on the X chromosome trol and Prevention (CDC). Over the last 30 years, blood (Xp21-q21). ALAS2 is the first and rate-limiting enzyme in lead levels reported in the United States have continued heme synthesis. to decline.52 However, in 2014, almost 9,000 children were Sex-linked SA (XLSA) is due to an abnormal ALAS2 reported to have blood lead levels greater than 10 mcg/dL gene that is the result of heterogeneous mutations in the and in 2015, hundreds of thousands of children under age 6 catalytic domain of the protein. More than 22 different were reported to have blood lead levels greater than 5 mcg/ mutations have been described. The mutations are located dL, the level at which the CDC recommends public health in exons 5–11 with most being single-base mutations affect- actions be initiated (38 states reporting).53 Children insured ing the site at which the enzyme binds the cofactor pyri- by Medicaid are required to have blood lead level testing doxal 5′-phosphate. at ages 1 and 2 but many are not screened, so the number 250 Chapter 12 having increased lead levels may actually be higher. The and megaloblastosis (Chapter 15). Alcohol has a direct effect CDC maintains that there is no safe blood level of lead in on folate metabolism, interfering with absorption, storage, children.54 Many labs will send off any sample for confirma- and release. The presence of a dimorphic erythrocyte pop- tory testing that is positive on a screening test. ulation and siderocytes in the peripheral blood are clues Lead serves no physiological purpose. Although lead to a diagnosis of SA. Alcohol is believed to interfere with poisoning consistently shortens the erythrocyte life span, the some enzymes of hemoglobin synthesis including inhibi- anemia accompanying plumbism is not primarily the result tion of the synthesis of pyridoxal phosphate and of activity of hemolysis but of a marked abnormality in heme synthe- of uroporphyrinogen decarboxylase and ferrochelatase but sis. Once ingested, lead passes through the blood to the bone enhancement of activity of d-ALAS. Alcoholics with a poor marrow where it accumulates in the mitochondria of eryth- diet can also have an inadequate intake of pyridoxine. Alco- roblasts and inhibits cellular enzymes involved in heme hol is directly toxic to hematologic cells as evidenced by the synthesis. The heme enzymes most sensitive to lead inhi- frequent presence of vacuoles in bone marrow precursor bition are d-aminolevulinic acid dehydrase (d-ALA-D) and cells, thrombocytopenia, and granulocytopenia. ferrocheletase (heme synthase) (Chapter 6). Other enzymes Associated with Malignancy Ring sideroblasts can be found in can be affected at higher lead concentrations. Thus, the syn- diseases other than sideroblastic anemia, including hema- thesis of heme is disturbed at the conversion of d-ALA to tologic malignancies (e.g., leukemia, malignant histiocy- porphobilinogen (catalyzed by d-ALA-D); urine excretion tosis, multiple myeloma, lymphoma) (Table 12-15). Some of d-ALA increases as a result. Incorporation of iron into protoporphyrin to form heme (which uses the enzyme ferro- chelatase) is also disrupted. The effect of lead on ferrochela- Table 12.13 Mechanisms of Anemia in Chronic tase is competitive inhibition with iron; iron accumulates in Alcoholism the cell, and EP (in the form of ZPP) is strikingly increased. Category Mechanism Microcytic, hypochromic anemia is not characteris- Megaloblastosis Folate deficiency tic of elevated lead levels in most children. The presence IDA Chronic or acute blood loss of a microcytic anemia in plumbism is most likely due to complications of ID or to the coexistence of a-thalassemia Sideroblastic anemia Toxic effects of alcohol on enzymes needed for heme synthesis trait.55,56,57 One study found that 33%
of African Ameri- Hemolytic anemias can children with lead poisoning and microcytosis had Chronic hemolysis Splenic sequestration a-thalassemia trait.55 Coexistent ID and lead poisoning put Spurr cell anemia Severe liver disease, splenomegaly, children at a higher risk for developing even more serious and jaundice complications because children absorb larger portions of Transient hemolytic anemia Portal hypertension and acute con- lead in iron-deficient states and the competitive inhibition gestive splenomegaly of ferrochelatase by lead is even greater in the absence of iron. Thus, it is critical to make a diagnosis of ID when it coexists with lead poisoning. Table 12.14 Incidence and Causes of Anemia in Study of 121 Hospitalized Alcoholics73 Although the ZPP measurement has been used to screen for lead in the past, it is no longer recommended as an Number of Associated Causes appropriate screening tool because ZPP does not rise until Causes of as Defined by lead levels reach approximately 20 mcg/dL and lead lev- Anemia Proportion Laboratory Findings Percentage els below this can impair neuropsychologic development. One 45% ACD 81% Screening generally should be done by direct lead measure- Two 37% Megaloblastic changes 34 in BM ments. ZPP cannot be used to differentiate IDA and thalas- Three 18% Ringed sideroblasts 23 semia in the presence of coexistent lead poisoning because of the increase in erythrocyte protoporphyrin caused by lead. Iron deficiency 13 ACD = Anemia of Chronic Disease; BM = bone marrow Alcoholism Anemia is a common finding in hospitalized chronic alcoholics. Megaloblastic and sideroblastic ane- mia occur in the majority, but anemia in this population Table 12.15 Diseases Associated with the Presence of can have many other causes including ACD, IDA, acute Ringed Sideroblasts blood loss, and chronic hemolysis58 (Table 12-13). Less than • Sideroblastic anemia one-half of patients have an isolated cause for the anemia • Myelodysplastic syndromes: refractory anemia with ringed sideroblasts (Table 12-14). Studies show that sideroblastic anemia is • Megaloblastic anemia particularly common among alcoholics with a poor diet and • Primary bone marrow disorders can be associated with a concomitant decrease in folic acid • Chemotherapy Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 251 investigators believe that the presence of ring sideroblasts in these disorders suggests that the malignancy can result Table 12.16 Laboratory Findings in Sideroblastic Anemia from an abnormal clone of pluripotential stem cells that Peripheral Blood Increased serum iron, serum ferritin, and affects the erythroid as well as other cell lineages. Occasion- transferrin percent saturation ally ring sideroblasts appear in the bone marrow following Dual populations of erythrocytes that may be hypochromic and normochromic or treatment of malignant disease (e.g., multiple myeloma, macrocytic and microcytic Hodgkin’s disease). The appearance of ring sideroblasts Pappenheimer bodies in erythrocytes after treatment is considered a poor prognostic sign because Normal or increased platelets these cases almost always terminate in acute leukemia. Bone Marrow Ring sideroblasts Molecular Diagnostics Gene mutations in ALAS2 (predominantly) CLINICAL PRESENTATION or SLC25A38, ABCB, or glutaredoxine5 In patients with acquired sideroblastic anemias secondary to drugs or malignancy, the manifestations of the under- lying disorder dominate. Patients with hereditary sidero- blastic anemia or RARS, however, generally show primary signs and symptoms of anemia. In hereditary sideroblastic anemias, most patients also exhibit signs associated with iron overload including hepatomegaly, splenomegaly, and diabetes. In the latter stages of the disease, cardiac function can be affected. LABORATORY EVALUATION Refer to Table 12-14 for laboratory findings in sideroblastic anemia. Peripheral Blood The anemia is usually moderate to severe. A dimorphic picture of normochromic and hypo- chromic cells is characteristically seen in inherited and Figure 12.14 Blood film from a patient with sideroblastic anemia. Two populations of erythrocytes are present: hypochromic acquired secondary forms of SA (Figure 12-14). Dual popu- and normochromic. This is the dimorphic blood picture typical lations of macrocytes and microcytes or normocytes can be of sideroblastic anemia. Anisocytosis is present with microcytes, found. Hypochromic macrocytes are especially prevalent in macrocytes, and normocytes. Note also the numerous inclusions RARS, whereas hypochromic microcytes are more common (Pappenheimer bodies) (peripheral blood; Wright-Giemsa stain; in the hereditary form of SA. Macrocytes are also common 1000* magnification). in SA associated with alcoholism. If a dimorphic erythrocyte population is present, the MCV, MCH, and MCHC may be normal because these is a particularly characteristic feature of lead poisoning parameters represent an average of all erythrocytes, thus (Figure 12-15). The punctate stippling occurs in the reticu- emphasizing the need for careful examination of CBC locyte and is found in developing erythroblasts. The gran- parameters and the blood smear. The RDW and erythrocyte ules in these stippled erythroblasts contain free ionized histogram are useful in detecting these dual populations. iron and the hemoglobin production in the cells is grossly The RDW is increased, and the RBC histogram shows two deficient. Many stippled cells in the peripheral blood in peaks representing the dual population. lead poisoning are actually siderocytes. Other abnormalities of erythrocytes are often seen. Other laboratory test results can be abnormal. Even Poikilocytosis and target cells may be present. Erythro- though the bone marrow is usually hyperplastic, the retic- cytes, may contain Pappenheimer bodies (iron depos- ulocyte production index is less than 2, indicating that the its) (Chapters 10, 37). When Pappenheimer bodies are anemia has an ineffective erythropoietic component. Other present, reticulocyte counts must be performed carefully indications of ineffective erythropoiesis include a slightly because both RNA and Pappenheimer bodies take up increased serum bilirubin, (usually less than 2.0 mg/dL), supravital stains. However, Pappenheimer bodies stain decreased haptoglobin, and increased lactate dehydroge- with both Romanowsky and Prussian blue stains, whereas nase (LD). Iron studies show increased serum iron, normal reticulated RNA does not stain with either. Nucleated or decreased TIBC with increased saturation levels (some- erythrocytes are rarely present. times reaching 100%), and increased serum ferritin. Leu- Basophilic stippling can be seen in any of the SAs. How- kocyte and platelet counts are usually normal but may be ever, coarse punctate basophilic stippling, resulting from decreased. Thrombocytosis is found in about one-third of aggregated ribosomes and degenerating mitochrondria, patients. 252 Chapter 12 MCV, peripheral blood smear evaluation, serum ferritin Hypersegmented Serum ferritin PMNs <20 ng/mL or >3% Normal macroovalocytes or increased IDA TIBC or MCV > 110fL sTfR increased Serum iron sTfR TIBC increased and Serum iron serum iron normal/ TIBC decreased increased normal/low and sTfR Figure 12.15 Peripheral blood smear from a patient with lead normal poisoning. Note the heavy basophilic stippling in the erythrocyte Probable folic acid Further (peripheral blood; Wright-Giemsa stain; 1000* magnification). deficiency evaluation In alcoholics, the direct and indirect effects of alcohol Figure 12.16 An algorithm for diagnosis of anemia in alcoholics. complicate the interpretation of laboratory tests used to diagnose anemia, including MCV, serum iron, TIBC, serum ferritin, and red cell and serum folate (Figure 12-16). Even in the absence of megaloblastosis and anemia, the cells are frequently macrocytic (MCV 100–110 fL). Serum iron levels are increased during drinking episodes but return to normal after cessation. CASE STUDY (continued from page 247) 8. Do the iron studies in Jose (serum iron 18 mcg/ dL, TIBC 425 mcg/dL) suggest SA? Explain. Bone Marrow Bone marrow changes include erythroid hyperplasia often accompanied by various degrees of mega- loblastosis. The megaloblastosis is sometimes responsive to folate, which indicates the presence of a complicating folate Figure 12.17 Ring sideroblasts (bone marrow; Prussian blue stain; 1000* magnification). deficiency. Erythroblasts appear poorly hemoglobinized with scanty, irregular cytoplasm. Macrophages contain increased amounts of storage iron. Ring sideroblasts con- mutations. This can be done by studying genomic DNA stitute more than 40% of the erythroblasts (Figure 12-17). or cDNA (from RNA) from reticulocytes59,60 (Chapter 42). Ring sideroblasts are erythroblasts with iron granules that Mutations in other genes are also associated with sidero- encircle one-third or more of the nucleus. The siderotic blastic anemia but are not as common as ALAS2 mutations. granules are larger than those found in normal sideroblasts. These include SLC25A38, ABCB7, and glutaredoxine5.61 In hereditary SA, the abnormal granules occur primarily If a mutation is found, other family members should be in the later stages of erythroblast development (e.g., poly- tested because even if anemia is not present, there is a risk chromatophilic and orthochromatic erythroblast stages). In of iron overload. Molecular studies also allow a distinction the idiopathic and secondary forms, the abnormal granules between hereditary SA and RARS. occur beginning with the earlier erythroblast stages. Ring sideroblasts must be present for an SA diagnosis; however, it is important to recognize that other disease enti- CASE STUDY (continued from page 252) ties can have ring sideroblasts present without being SA. 9. Do Jose’s laboratory test results and clinical Molecular Studies If hereditary sex-linked SA is suspected history indicate that a bone marrow examination based on erythrocyte morphology and iron studies, the is necessary to make a diagnosis? patient should be tested for the presence of ALAS2 gene Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 253 Therapy saturation of greater than 45% is often recommended as the value that indicates further investigation is necessary. Pyridoxine therapy is generally attempted for patients Hemochromatosis is classified as hereditary or with hereditary SA; less than 50% experience a return to secondary. The hereditary form is caused by genetic muta- normal hemoglobin levels with therapy, although some tions, while the secondary type is associated with other have a partial response. In some patients, iron overload hematologic diseases and a variety of other conditions decreases responsiveness to pyridoxine, but when the iron (Table 12-17). load is reduced by phlebotomy, hemoglobin concentration increases.60 Folic acid is administered to those with mega- loblastic features. Although the anemia can be treated, iron Hereditary Hemochromatosis overload is the primary complication of SA. Sometimes ETIOLOGY AND PATHOPHYSIOLOGY the risk of hemochromatosis is reduced by the removal of Hereditary hemochromatosis (HH) is characterized by excess body iron through phlebotomy or chelation therapy. increased iron absorption in the gut and progressive iron Iron overload is monitored by iron studies (percent satura- overload. It is a genetic disorder with a prevalence of 1 in tion of transferrin, serum ferritin). Female carriers should 200 to 250 persons. One in ten Caucasians in the United also be monitored for iron overload. Some patients live for States is a carrier. It is the most common genetic abnormal- many years tolerating their anemia well; others die because ity in those with a European ancestry. Most of the heredi- of complications of iron overload, infections, or bone mar- tary forms of iron overload involve a deficiency of hepcidin row failure. either as a result of a mutation involving the hepcidin gene Secondary SA resulting from a disease, toxin, or drug (HAMP) or mutations of genes whose products regulate may be corrected by successful treatment of the disease or hepcidin expression (HFE, TfR2, HJV, DMT1, transferrin).62 by elimination of the toxin/drug. The result is an increase in absorption of iron in the intes- tine and/or uncontrolled release of iron from macrophages and duodenal enterocytes into the plasma iron pool. Iron Hemochromatosis in excess of that needed for erythropoiesis and myoglobin production deposits in hepatocytes and parenchymal tis- Hemochromatosis describes the clinical disorder that sue cells, resulting in an increase in total body iron stores. results in parenchymal tissue damage from progressive iron Excess iron deposits interfere with the normal function of overload. Hemochromatosis typically is not associated with these cells. This situation has potentially fatal consequences, anemia but is included in this chapter because iron stud- especially with regard to cardiac and hepatic function. Most ies are markedly abnormal in patients with this disorder. mutations in genes that affect the regulation of iron metabo- Hemochromatosis should be considered when symptoms lism are recessive disorders (Table 12-18). are present, when there is a family history of the disorder, The most common form of HH in populations of Euro- or when iron studies suggest iron overload. Iron overload is pean origin is due to mutations in the HFE gene. Mutations said to exist when serum ferritin is greater than 200 mcg/L result in an autosomal recessive disorder of low penetrance in premenopausal women or greater than 300 mcg/L in with adult onset. Clinical disease is more common in males men and postmenopausal women. A persistent transferrin than females. The HFE gene is mapped to chromosome Table 12.17 Causes
of Iron Overload Hereditary Secondary • Classical hemochromatosis: HFE–associated mutations (type I) • Anemias with ineffective erythropoiesis (e.g., sickle cell anemia, t halassemia, • Juvenile hemochromatosis (type 2) refractory anemias with erythroid hypercellular bone marrow, X-linked sideroblastic anemia) • Hemojuvelin (HJV) mutations (type 2A) • Chronic transfusions • Hepcidin (HAMP) mutations (type 2B) • Chronic liver disease • Transferrin-receptor 2 deficiency (type 3) • Viral hepatitis • Ferroportin 1 deficiency (type 4) • Alcoholism • DMT1 mutations • Insulin-resistance-associated hepatic iron overload • Congenital atransferrinemia • Dietary iron overload • Aceruloplasminemia • Medicinal iron ingestion • Overload from injections or ingestion of iron supplements • African dietary iron overload • Porphyria cutanea tarda 254 Chapter 12 Table 12.18 Summary of Hereditary (Primary) and Secondary Iron Overload Disorders Disorder Defect/Mutation Fe Absorption Fe Deposition Site(s) Plasma Iron Ferritin Hereditary Hereditary hemochro- HFE gene, TfR2 gene Increased Parenchymal tissue Increased Increased matosis (autosomal recessive) Juvenile hemochromato- HJV gene, HAMP Increased Parenchymal tissue Increased Increased sis (autosomal recessive) (hepcidin) gene, HFE and TfR2 genes Hereditary hemochromatosis (autosomal dominant) (a) Ferroportin-1 SLC40A1 (ferroportin-1) Normal or low but Predominantly deposi- Normal but increases Increased associated with impaired gene defective export of iron tion in macrophages with progression of iron export from macrophages disease (b) Ferroportin-1 SLC40A1 gene Normal but increased Parenchymal tissue: Increased Increased associated with hepcidin export of iron from heart, liver, pancreas, resistance duodenal enterocytes other organs Secondary Iron-loading Erythropoietic drive Increased Macrophage Increased Increased anemias (ineffective and possibly GDF-15, erythropoiesis; e.g., TWSG1, and/or ERFE thalassemia, XLSA) suppress hepcidin synthesis Anemia of chronic IL-6 increases expression Decreased Macrophage Decreased Normal to disease (ACD) of hepcidin, which binds increased ferroportin and traps iron in macrophage Transfusion induced Gain of 1 mg iron/mL Normal Parenchymal tissue Increased Increased erythrocytes transfused 6p21,3. The most common mutation is cys282 S tyrosine: Mutations in the ferroportin-1 gene can decrease the C282Y.63 Less common are the mutations his63 S asp: amount of functional ferroportin-1 on the cell surface, H63D; and ser65 S cys: S65C. Clinical signs of the dis- blocking iron export and resulting in retention and accu- ease can be found in homozygous C282Y and compound mulation of iron in macrophages. This type of mutation heterozygosity for C282Y and H63D. The mechanism by produces only minor clinical manifestations. Alternatively, which mutations in the HFE protein affect iron metabolism mutations can cause resistance of ferroportin-1 to internal- is unclear but can involve interactions of HFE with other ization and degradation induced by hepcidin. The result proteins in addition to TfR (see earlier discussion of “Sys- is loss of control of iron export from enterocytes and mac- temic Iron Balance, HFE”). Decreased hepatic expression rophages, which leads to iron overload in parenchymal of hepcidin in HFE-related HH suggests that the mutation cells of the liver and other organs. Ferroportin-1 mutations affects upstream regulation of hepcidin. About 3–4 mg of are inherited as dominant disorders. Aceruloplasminemia iron a day is absorbed from the GI tract as compared with affects expression of ferroportin-1 on the cell surface leading the usual 1–2 mg, resulting in an accumulation of about to ferroportin degradation. 0.5–1.0 gm/year. The capacity of cells to store iron by com- plexing with apoferritin is exceeded, and “free” intracellu- CLINICAL PRESENTATION lar iron accumulates. This free iron facilitates the buildup Clinical penetrance of the most common form of HH, HFE- of reactive oxygen species that cause cell injury and cell mutation, is incomplete with only 1 in 5,000 affected individu- death and leads to organ failure. Most of the excess iron als having clinical symptoms. About 81% of symptomatic is deposited in the liver. Mutations of the TfR2 gene result patients have the homozygous C282Y genotype. Asymptomatic in a disease that is clinically indistinguishable from HFE- patients who are homozygous for C282Y or carry the H63D/ associated HH. C282Y genotype should have their serum iron and ferritin Other less common forms of HH are due to muta- levels monitored because they are at risk for developing iron tions in other genes involved in iron sensing and regula- storage disease. Clinical findings include chronic fatigue, tion, but all appear to result in deregulation of hepcidin arthralgia, infertility, impotence, cardiac disease, diabetes, and/ expression. or cirrhosis. Hyperpigmentation of the skin is also found. Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 255 LABORATORY EVALUATION is suggested that following initial venesection therapy to Screening tests for hemochromatosis usually include per- deplete iron stores, iron status be monitored annually by cent saturation of transferrin and serum ferritin. The crite- serum ferritin levels. rion for a diagnosis of hemochromatosis is usually greater Treatment of secondary hemochromatosis sometimes than or equal to 45% transferrin saturation. Saturation often includes iron chelation (e.g., deferoxamine) to bind iron approaches 100%. Adding serum ferritin analysis increases and enable urinary excretion. Only a very small amount specificity for HH; iron overload is suggested when the of iron is available for chelation (from the labile pool) at serum ferritin is elevated. Although an increase in transfer- any given time; thus, continuous infusion of the chelator rin saturation is sensitive for hemochromatosis, DNA test- over a long period of time is required to remove this iron. ing to identify the mutated gene is necessary for a definitive Chelation therapy is the primary form of treatment for diagnosis. Biopsy of the liver and/or bone marrow usually transfusion-induced hemochromatosis. Recently oral iron is unnecessary. Liver enzymes are often elevated. Anemia chelators (deferiprone, deferasirox) have received FDA is not characteristic except in aceruloplasminemia (when a approval, and are easier to use than the infused chelator normocytic, normochromic anemia is present) and in DMT1 deferoxamine. mutations (when a severe microcytic, hypochromic anemia is present).64 Checkpoint 12.5 Screening for HH is controversial. Some recommend What is the risk of population genetic screening for HH? What is population screening with transferrin saturation testing and the benefit of population genetic screening for HH? suggest that laboratories include transferrin saturation in their routine lab panels.65 Genetic screening is still costly for population screening, and unresolved ethical issues includ- Porphyrias ing patient privacy, counseling, and insurance concerns Heme is an iron-chelated porphyrin ring. Its biosynthesis exist. With the recognition of the low penetrance of clini- occurs in most cells, but the major sites of synthesis are ery- cal disease (less than 1%), interest in large-scale screening throid cells of the bone marrow and hepatocytes. Thus, the has largely disappeared. It is recommended, however, that activity of the enzymes catalyzing porphyrin metabolism is both symptomatic and asymptomatic first-degree relatives highest in these cells. of those with HH be tested with genetic screening because transferrin saturation will not detect carriers.66 The goal of screening and identifying the disease early is to allow treat- Etiology ment with phlebotomy before the onset of clinical disease. The porphyrias represent a group of inherited disorders characterized by a block in porphyrin synthesis due to Secondary Hemochromatosis specific enzyme deficiencies. As a result, porphyrin precursors behind the enzyme defect accumulate in tis- Secondary hemochromatosis is associated with a number sues, and large amounts are excreted in the urine and/ of conditions including anemias that have an ineffective or feces. These excess porphyrin deposits cause most erythropoiesis component and increased iron absorption. of the symptoms and clinical findings associated with These include b-thalassemia, congenital dyserythropoietic porphyria. The most common findings include photo- anemia, and X-linked sideroblastic anemia. An increase in sensitivity, abdominal pain, and neuropathy (motor dys- GDF-15, TWSG1, and erythroferrone associated with inef- function, sensory loss, mental disturbances). However, fective erythropoiesis may be responsible for suppressing usually adequate production of heme for hemoglobin hepcidin synthesis, leading to an increase in iron absorption synthesis occurs. in these conditions. Iron overload often develops in patients Although rare, the porphyrias have received wide rec- who have transfusion-dependent anemias such as sickle cell ognition and stimulated interest because the disease affected disease and thalassemia. Iron overload can also be found the royal families of England and Scotland, especially those in chronic liver disease. Serum iron studies are abnormal descended from Mary, Queen of Scots. Both George III and in 40–50% of patients with chronic viral hepatitis, alcoholic his granddaughter, Princess Charlotte, were thought to be liver disease, and nonalcoholic steatohepatitis. Alcohol dis- afflicted with porphyria. rupts normal iron metabolism and results in excess iron Two forms of porphyria (erythropoietic and hepatic) deposition in the liver in about one-third of alcoholics. are described, differentiated by the primary site (bone Treatment marrow or liver) of defective porphyrin metabolism (Table 12-19).67 Only the erythropoietic porphyrias affect Phlebotomy is usually the treatment for HH. Each unit the erythrocytes. Therefore, these porphyrias are discussed of blood removes about 250 mg of iron from the body. It here. 256 Chapter 12 Table 12.19 Classification and Characteristics of the Porphyrias Metabolites in Excess Mode of Genes Erythroid Tissue Porphyria Inheritance Involved Enzymatic Defect Urine Feces Cells Source Erythropoietic Congenital Autosomal UROS Uroporphyrinogen III Uroporphyrin I, Coproporphyrin Uroporphyrin I, Erythropoietic erythropoietic recessive synthase coproporphyrin I I, uroporphyrin I coproporphyrin I porphyria (CEP) Erythropoietic Autosomal FECH Ferrocheletase (heme Normal Protoporphyrin Protoporphyrin Erythropoietic protoporphyria (EPP) recessive (FECH ALAS2 synthase) and mutation); d-aminolevulinate occasionally X-linked (ALAS2 synthase2 hepatic mutation) Hepatic and Erythropoietic Hepatoerythropoietic Recessive UROD Uroporphyrinogen Uroporphyrin Uroporphyrin ZPP Hepatic and porphyria (HEP) decarboxylase erythropoietic Hepatic Acute intermittent Autosomal HMBS Porphobilinogen d-ALA, Sometimes Normal Hepatic porphyria (AIP) dominant deaminase porphobilinogen coproporphyrin and protoporphyrin Hereditary Autosomal CPOX Coproporphyrinogen Coproporphyrin Coproporphyrin Normal Hepatic coproporphyria dominant oxidase III, uroporphyrin III (HCP) (in attack) Variegate porphyria Autosomal PPOX Protoporphyrinogen Porphobilinogen, Protoporphyrin, Normal Hepatic (PV) dominant oxidase d-ALA, uro- coproporphyrin porphyrin, coproporphyrin Porphyria cutanea Autosomal UROD Uroporphyrinogen Uroporphyrin I, Protoporphyrin, Normal Hepatic tarda (PCT) dominant decarboxylase uroporphyrin III, coproporphyrin coproporphyrin (slight) ALA dehydratase Autosomal ALAD ALA dehydratase d-ALA, copro- — Normal Hepatic deficiency porphyria recessive porphyrin III (ADP) Pathophysiology intermediates are formed. If for some reason excessive amounts of porphyrinogens are produced, the corre- The erythropoietic porphyrias result from an abnormal- sponding oxidized porphyrin compounds also increase. ity of the enzymes in the heme biosynthetic pathway Porphyrins are tetrapyrroles and resonating compounds within the erythroblasts of the bone marrow. At least two (contain alternating single and double bonds); erythro- types exist: congenital erythropoietic porphyria (CEP) cytes that contain these substances show red fluorescence and erythropoietic protoporphyria (EPP). They are clas- with UV light. sified according to the particular enzyme defect and the excessive porphyrin intermediates produced. Although CONGENITAL ERYTHROPOIETIC PORPHYRIA CEP is associated with hemolytic anemia, anemia in EPP Congenital erythropoietic porphyria (CEP; Gunther ’s is rare. disease) is characterized by the presence of excessive Porphyrins are functionless products produced by amounts of type I porphyrins: uroporphyrin I (UroI) and the irreversible oxidation of type I and type III porphy- coproporphyrin I (CoproI). A defect in u roporphyrinogen rinogens. Porphyrinogens of the type III series are the III synthase results from point mutations in the gene, precursors of heme, whereas the type I isomers do not shifting the porphobilinogen into the functionless UroI produce any useful metabolites and cannot be used in isomer (Figure 12-18). However, enough UroIII is pro- heme synthesis (Chapter 6). In normal heme synthesis, duced to generate adequate amounts of heme. This type I and III isomers are formed in a 1:10,000 ratio. suggests that a deficiency of uroporphyrinogen III Normally, most porphyrinogens are readily converted synthase is not the only abnormality. Another possibility to heme, and very small amounts of the porphyrin for the excessive amounts of UroI isomer is hyperactivity Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 257 Glycine + Succinyl coenzyme A ALA synthetase d-Aminolevulinic acid ALA dehydrase Porphobilinogen (uroporphyrinogen I Acute intermittant porphyria synthase) (uroporphyrinogen I synthase) + Congenital erythropoietic porphyria Uroporphyrin I Uroporphyrinogen I (uroporphyrinogen III synthase) Porphyria cutanea tarda (uroporphyrinogen decarboxylase) Uroporphyrinogen III Uroporphyrin III Coproporphyrin I Coproporphyrinogen I (uroporphyrinogen decarboxylase) Coproporphyrinogen III Coproporphyrin III Hereditary coproporphyria (coproporphyrinogen oxidase) Protoporphyrinogen III Variegate porphyria (protoporphyrinogen oxidase) Protoporphyrin IX Erythropoietic protoporphyria (ferrochelatase) Heme Figure 12.18 The formation of heme from glycine and succinyl-coenzyme A involves the production of porphyrinogen intermediates. Normally, only very small amounts of the series I isomers are formed. However, in a group of congenital disorders called porphyrias, there is a block in porphyrin metabolism due to a defect in one or more of the enzymes involved in porphyrin metabolism, and a
build-up of porphyrin intermediates, depending on which enzyme is involved. In congenital erythropoietic porphyria, there is an abnormality in conversion of porphobilinogen to uroporphyrinogen III, and large amounts of the functionless series I isomers are formed. These isomers are oxidized to porphyrin and accumulate in the tissues. Another form of porphyria, erythropoietic protoporphyria, is characterized by an accumulation of protoporphyrin due to an abnormality of the ferrocheletase enzyme. Other porphyrias noted in this figure are classified as hepatic because the site of abnormal metabolism is the liver. of uroporphyrinogen I synthase. Validation of either of ERYTHROPOIETIC PROTOPORPHYRIA these hypotheses requires purification, characterization, Erythropoietic protoporphyria (EPP) is an autosomal and accurate measurement of these enzymes. recessive disorder that results from mutations in the The excess porphyrins are deposited in body tissues ferrochelatase gene. The mutations markedly reduce and excreted in urine and feces. Intense fluorescence with the activity of the ferrochelatase enzyme, resulting in ultraviolet light can verify their presence. The cause of the an accumulation of protoporphyrin, the immediate hemolytic anemia that accompanies CEP is unclear but precursor of heme (Figure 12-18). Adequate amounts of is thought to be associated with the excessive porphyrin heme are produced, however, and no anemia is present. deposits within erythrocytes. The finding that normal eryth- Excess protoporphyrin IX builds up in the cell, can leak rocytes infused into CEP patients have a normal life span into skin dermal capillaries, and can be found in the skin, supports this. The erythrocytes in CEP may be subject to liver, blood, and feces. Due to its insolubility in water, photohemolysis as they pass through the dermal capillar- protoporphyrin is not present in the urine. This porphyria ies exposed to UV light. The erythrocytes show increased is thought to be of both erythropoietic and hepatic photohemolysis in vitro, but whether this occurs in vivo is origin.68 An X-linked form was recently discovered that uncertain. is clinically indistinguishable from EPP.69 In this form, 258 Chapter 12 mutations in the ALAS2 gene result in gain of function in Serum iron and storage iron are usually normal. Hap- the ALAS2 enzyme. toglobin is absent, and unconjugated bilirubin as well as urinary and fecal urobilinogen are increased. Clinical Presentation Large amounts of uroporphyrin I and coproporphyrin I are excreted in the urine and feces. These isomers are also CEP is a rare autosomal recessive disease with ∼130 cases found in the plasma and in erythrocytes. reported. EPP is inherited as an autosomal recessive trait.69 About 300 cases of EPP have been reported, but the actual ERYTHROPOIETIC PROTOPORPHYRIA rate of occurrence is probably masked due to the subtlety The blood and bone marrow in EPP usually reveal no of the clinical signs and the absence of colored porphyrins abnormalities on routine examination; however, under UV in the urine. light, the cytoplasm of erythroblasts fluoresces intensely. The first signs of CEP occur in infancy. The urine is The erythrocytes, plasma, and feces contain large amounts colored pink to reddish brown, depending on the amount of protoporphyrin; protoporphyrin is not found in the of uroporphyrin excreted. This is usually first noted as a urine. The protoporphyrin in erythrocytes is free (FEP), pink stain on the infant’s diaper. The excess porphyrins not bound to zinc as in IDA and lead poisoning. The FEP is in the skin create an extreme photosensitivity to sunlight. higher than in other disorders associated with an increase Vesicular or bullous eruptions appear on uncovered areas in erythrocyte protoporphyrin levels. This block in heme shortly after exposure to sunlight. The lesions heal slowly synthesis, which occurs in the reaction just prior to inser- and can become infected. Repeated eruptions and skin tion of iron into the porphyrin ring, would be expected injury cause scarring and can lead to severe mutilation to cause an accumulation of iron within the erythroblasts. of the face, ears, and hands. The excess porphyrin stains The fact that this iron buildup does not occur has not been the teeth a brown discoloration. Under UV light, the teeth explained. fluoresce bright red. Hypertrichosis affects the entire body but is especially present in exposed areas. The hair can be blond and downy or dark and coarse. Splenomegaly Prognosis and Therapy is a consistent finding and is usually progressive with Individuals with CEP do not usually survive beyond the the disease. A mild to severe hemolytic anemia is pres- fifth decade of life. Attempts to decrease the excess por- ent with erythrocyte life span decreased to as little as 18 phyrins have been unsuccessful, but the quality of life for days. Patients with CEP do not exhibit the abdominal pain CEP patients has improved by minimizing the scarring or neurologic and psychotic signs associated with some and mutilation with effective dermatologic treatment. hepatic porphyrias. Avoiding exposure to sunlight is critical. Splenectomy has Clinical signs of EPP are more subtle, and its course is sometimes resulted in a decrease of porphyrin production relatively mild in comparison with CEP. Photosensitivity is and helped ameliorate the hemolytic anemia. Evidence for not severe, and scarring is usually absent. Sunlight exposure long-term success with splenectomy, however, is question- leads to erythema and urticaria. Protoporphyrin accumu- able. Blood transfusion in conjunction with administration lates in the erythrocytes, which causes them to fluoresce of chelators to reduce iron overload suppresses erythropoi- intensely, but there is no hemolytic anemia. Occasionally esis and decreases or eliminates symptoms. Bone marrow hepatic damage occurs. transplantation is suggested in severe phenotypes because the predominant site of porphyrin production is the bone Laboratory Evaluation marrow. Treatment of EPP is to protect the skin from sunlight and CONGENITAL ERYTHROPOIETIC PORPHYRIA minimize the toxic effects of protoporphyrin on the liver. In The peripheral blood in CEP exhibits a mild to severe nor- most patients with EPP, high doses of d-carotene improve mocytic anemia with anisocytosis and poikilocytosis. The tolerance to sunlight. Blood transfusions and hematin can be blood smear reveals significant polychromatophilia and used to suppress erythropoiesis. Splenectomy may be help- nucleated erythrocytes. The erythrocytes fluoresce with ful if hemolysis and splenomegaly are prominent. Chole- UV light. styramine can promote excretion of liver protoporphyrin.69 The bone marrow in CEP shows erythroid hyper- Genes of the heme biosynthetic pathway have been plasia. A large portion of the erythroblasts demonstrates mapped, and mutations associated with porphyrias have been intense fluorescence with UV light. The fluorescence is identified. Gene therapy may be an option for porphyrias in localized principally in the nuclei. The fact that not all the future.70 This type of therapy involves inserting a func- erythroblasts fluoresce suggests that two populations of tional gene into specific hematopoietic or hepatic stem cells of erythrocytes exist, one of which is normal. the patient, restoring normal heme synthesis pathways. Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 259 Summary Hemoglobin synthesis requires adequate production of Abnormal porphyrin metabolism results in SA and por- heme and globin. Inadequate amounts of either can result in phyrias. SA is due to defective porphyrin synthesis and a anemia—usually microcytic, hypochromic anemia. Defects block in the insertion of iron into the porphyrin ring to form in heme synthesis could be due to either faulty iron or por- heme. The erythrocyte population in SA characteristically phyrin metabolism (Table 12-20). contains cells that are normochromic and hypochromic (dual Many proteins, including hepcidin, ferroportin 1, population). The bone marrow has ring sideroblasts. Heredi- hephaestin, DMT1, DCytB, HFE, GDF-15, HIF-2a, eryth- tary SA is due to defective ALAS2, the enzyme in the first step roferrone, TWSG1, TfR1, TfR2, HJV, and transferrin, play of heme synthesis. SA can also occur because of the effects a role in iron homeostasis. The anemias with a faulty iron of drugs or toxins on enzymes involved in heme synthesis. metabolic component include IDA and ACD. IDA is due Iron studies are helpful in differentiating these disor- to inadequate amounts of iron for heme synthesis; ID usu- ders. Serum iron is decreased in IDA and ACD; it is nor- ally occurs because of blood loss or a nutritional deficiency mal to increased in SA. The serum transferrin is increased of iron. ACD has several pathophysiologic mechanisms and saturation decreased if total body iron is decreased, related to cytokines produced as a result of inflammation whereas if storage iron is normal or increased, the serum or infection. The major mechanism is a block in reutiliza- transferrin is normal or decreased with normal or increased tion of macrophage iron due to an increase in hepcidin. The saturation. Serum ferritin is a reliable indicator of iron erythrocytes are normocytic, normochromic, but in long- stores except in the presence of inflammation or infection standing anemia, they can be microcytic, hypochromic. when it can be falsely increased. Serum ferritin is decreased Table 12.20 Summary of Conditions Associated with Abnormal Heme Synthesis and/or Regulation of Iron Metabolism/Utilization Condition Etiology CBC Iron Studies Other Iron-deficiency Inadequate iron due to Microcytic, hypochromic, Decreased serum iron, serum Increased ZPP (not used to evaluate anemia deficient dietary intake; anisocytosis, poikilocytosis, ferritin, and percent saturation if possibility of concurrent lead decreased absorption; increase in percentage of of transferrin; increased TIBC poisoning); decreased r eticulocyte increased loss hypochromic cells and sTfR hemoglobin (CHr, MCHr, RET-He); increased sTfR-F index Anemia of chronic Impaired iron release from Normocytic, normochromic; Decreased serum iron and Increased ZPP, decreased sTfR-F disease macrophages due to in long-standing cases can low to normal TIBC; low/ index increased hepcidin induced be microcytic, hypochromic normal transferrin saturation; by cytokines; inhibition increased or normal serum of EPO production and ferritin; sTfR normal impaired erythropoiesis; shortened erythrocyte survival Sideroblastic Defect in enzymes needed Dual population of Increased serum iron and Bone marrow shows ring anemia for heme synthesis; can be normochromic and transferrin saturation; normal sideroblasts; peripheral blood eryth- hereditary or acquired (sec- hypochromic erythrocytes or decreased TIBC; increased rocytes can contain Pappenheimer ondary to drugs/toxins) and macrocytic, microcytic serum ferritin bodies or macrocytic, normocytic erythrocytes Hemochromatosis Hereditary: genetic No anemia or characteristic Increased serum iron; Molecular testing to identify mutated (no anemia) mutations resulting in peripheral blood cell transferrin saturation (Ú45,) gene is suggested if transferrin defect/deficiency of morphology except in and increased serum ferritin saturation is more than 45% ferroportin or hepcidin; DMT1 and ferroportin -1 acquired: transfusion, iron mutations injections, chronic liver disease Porphyrias Block in porphyrin synthesis CEP: normocytic hemolytic Storage iron and serum iron CEP: increased uroporphyrin I and (anemia not due to defect in enzymes anemia; anisocytosis normal coproporphyrin I excreted in urine characteristic) for heme synthesis; CEP and poikilocytosis; and feces, plasma, and erythrocytes; and EPP affect erythrocytes polychromatophilia; series III isomers also increased nucleated RBCs EPP: no abnormalities CEP, EPP: normoblasts demonstrate intense fluorescence with UV light 260 Chapter 12 in ID and normal or increased in SA, ACD, and hemochro- iron metabolism can also cause HH. These mutations are matosis. The serum transferrin receptor assay is useful in believed to affect the synthesis or function of hepcidin. differentiating ID and ACD. Levels are increased in ID and Serum iron studies reflect the excessive iron overload normal in ACD. with increased serum ferritin and a very high saturation Hemochromatosis is a disorder characterized by of transferrin. total body iron excess. Anemia is not a characteristic of Porphyria is a heterogeneous group of hereditary this disorder, but it is included in this chapter to help disorders due to a block in porphyrin synthesis. The the reader compare and differentiate iron study results defect involves one of the critical enzymes in the with those associated with defective heme synthesis. porphyrin metabolic pathway. Two forms, CEP and EPP, Hemochromatosis can be caused by a genetic defect or have an erythropoietic component. Erythrocytes have chronic transfusions. The most common hereditary form very high levels of free erythrocyte protoporphyrin, and of hemochromatosis (HH) is due to a mutation of the HFE excess porphyrins are deposited in tissues and excreted gene. Mutations in several other genes associated with in feces/urine. Review Questions Level I c. cytokine inhibition of erythropoiesis 1. What is the iron transport protein called? d. defect in enzymes regulating heme (Objective 3) synthesis a. Ferritin 6. Anemia(s) characterized by defective heme synthesis b. Transferrin includes: (Objective 6) c. Hemosiderin a. hemochromatosis d. Albumin b. megaloblastic anemia 2. The term sideropenic is most closely associated with c. thalassemia which anemia?
(Objective 1) d. sideroblastic anemia a. Iron deficiency 7. The most common cause of ID in middle-aged men is: b. Sideroblastic (Objective 6) c. Lead poisoning a. inadequate iron in the diet d. Anemia of chronic disease b. cancer 3. Microcytic, hypochromic erythrocytes are most char- c. prescription drugs acteristic of which anemia? (Objective 5) d. chronic bleeding a. Megaloblastic 8. Which of the following is most often associated b. Lead poisoning with the presence of Pappenheimer bodies? c. Iron deficiency (Objective 5) d. Anemia of chronic disease a. Lead poisoning b. ID 4. Which of the following individuals is most likely to require an increased intake of iron? (Objective 4) c. Sideroblastic anemia d. Anemia of chronic disease a. Adult male b. Menopausal female 9. Which of the following best describes hemochromato- c. Mother of three preschool children sis? (Objective 7) d. Seventy-five-year-old male a. Decrease in serum iron b. Increase in TIBC 5. The basic defect in sideroblastic anemia is: (Objective 6) c. Increase in total body iron d. Increase in hepcidin a. inadequate iron intake b. inadequate absorption of iron in the gut Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 261 10. A patient with anemia of chronic disease would be 2. Which laboratory result(s) is (are) most useful in distin- expected to have which set of laboratory test results? guishing this patient’s anemia from IDA? (Objective 9) (Objective 5) a. Bone marrow a. MCV decreased, serum iron increased, serum ferri- b. Mean cell volume tin increased, TIBC and percent saturation increased c. Hemoglobin b. MCV normal, serum iron increased, serum ferritin d. Iron studies decreased, TIBC and percent saturation decreased c. MCV normal, serum iron decreased, serum ferritin 3. From the results of these laboratory studies, how would increased, TIBC and percent saturation decreased you describe the patient’s red blood cells? (Objective 8) d. MCV decreased, serum iron decreased, serum a. Microcytic, hypochromic ferritin decreased, TIBC and percent saturation b. Dual population of microcytes and normocytes decreased c. Macrocytic, normochromic Level II d. Normocytic, normochromic 4. A bone marrow examination was performed and sections were stained with Prussian blue. Numerous Use this history for questions 1–4. sideroblasts were present with a large number of ring sideroblasts. What is the most probable cause of this A 75-year-old male experiencing mental confusion anemia? (Objectives 6, 8, 10) and fatigue was seen by his physician. Laboratory tests were ordered: a. Poor diet RBC 3.3 * 106/mcL b. Chronic blood loss Hb 9.3 g/dL c. Abnormality of ALAS2 Hct 29% d. Increased iron requirement PLT 168 * 103/mcL 5. A hematocrit is not recommended to screen for iron deficiency in children because: (Objectives 1, 2, 8, 9) WBC 4.0 * 103/mcL Differential a. it is not sensitive enough to pick up anemia in children Segmented neutrophils 60% b. iron deficiency can be present without the presence Band neutrophils 9% of anemia Lymphocytes 25% c. high levels of lead will affect the hematocrit Monocytes 3% accuracy Eosinophils 3% d. serum ferritin is more cost effective RBC morphology: Anisocytosis with microcytic, 6. If a child with lead poisoning also had a significant hypochromic RBCs and normocytic, normochro- microcytic, hypochromic anemia, what complicat- mic RBCs present ing pathology/pathologies should be considered? Laboratory data for anemia workup: (Objective 14) Reticulocyte count 1.0% a. Iron deficiency Serum iron 274 mcg/dL b. Thalassemia Total iron-binding capacity 285 mcg/dL c. Iron deficiency and thalassemia (TIBC) d. Thalassemia and sideroblastic anemia 7. What laboratory test is best for screening for iron deficiency in a population of 1- to 3-year-old children 1. Which anemia of defective heme synthesis is associated who have a high incidence of elevated blood lead with this type of red cell morphology? (Objective 8) levels? (Objective 14) a. Sideroblastic anemia a. Hematocrit b. Anemia of chronic disease b. Serum ferritin c. Iron-deficiency anemia c. Serum iron d. Erythropoietic porphyria d. ZPP 262 Chapter 12 8. A 2-year-old child was tested for blood lead level. The 10. In regard to question 9, what test should you result was 25 mcg/dL. He also had microcytic, hypo- recommend be done reflexively on patients with an chromic anemia. The child’s parents were questioned, abnormal screening test? (Objective 18) and it was determined that the source of lead was a. Molecular test for HFE gene a painted crib in his day care center. The child was enrolled in another day care center. Follow-up testing b. Serum iron revealed that the blood lead level was within normal c. Serum ferritin limits but the microcytic, h ypochromic anemia was d. % Saturation still present. Which f ollow-up test(s) would you rec- ommend to help identify the etiology of this anemia? 11. Which protein regulates iron absorption in the (Objectives 9,14,15) gut and transport of iron from cells to the plasma? a. Serum iron, serum ferritin, TIBC, % saturation (Objective 3) b. Hemoglobin electrophoresis a. DcytB c. Molecular diagnostic testing for sideroblastic b. DMT1 anemia c. Hepcidin d. Molecular diagnostic testing for hemosiderosis d. Hephaestin 9. A health maintenance organization (HMO) has a contract for laboratory testing services with your 12. What effect would a high degree of ineffective laboratory. The HMO has decided to screen its members erythropoiesis have on iron metabolism? for hereditary hemochromatosis. Which l aboratory test (Objective 4) will you recommend for this screening? (Objective 18) a. Decreased absorption of iron in the intestine a. Serum iron b. Decreased transport of iron across the basolateral b. % Transferrin saturation membrane of enterocytes c. Serum ferritin c. Decreased hepcidin synthesis d. Molecular test for mutated HFE gene d. Increased serum transferrin saturation References 1. Qiu, A., Jansen, M., Sakaris, A., Min, S. H., Chattopadhyay, S., 9. Kawabata, H., Fleming, R. E., Gui, D., Moon, S. Y., Saitoh, T., Tsai, E., . . . Goldman, I. D. (2006). Identification of an intestinal O’Kelly, J., . . . Koeffler, H. P. (2005). Expression of hepcidin is folate transporter and the molecular basis for hereditary folate down-regulated in TfR2 mutant mice manifesting a phenotype of malabsorption. Cell, 127(5), 917–928. hereditary hemochromatosis. Blood, 105(1), 376–381. 2. Anderson, G. J., Frazer, D. M., & McLaren, G. D. (2009). Iron 10. Tanno, T., Bhanu, N. V., Oneal, P. A., Goh, S. H., Staker, P., Lee, absorption and metabolism. Current Opinion in Gastroenterology, Y. T., . . . Eling, T. E. (2007). High levels of GDF15 in thalassemia 25(2), 129–135. suppress expression of the iron regulatory protein hepcidin. 3. Silva, B., & Faustino, P. (2015). An overview of molecular basis Nature Medicine, 13(9), 1096–1101. of iron metabolism regulation and the associated pathologies. 11. Kautz, L., Jung, G., Valore, E. V., Rivella, S., Nemeth, E., & Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, Ganz, T. (2014). Identification of erythroferrone as an erythroid 1852(7), 1347–1359. regulator of iron metabolism. Nature Genetics, 46(7), 678. 4. Nicolas, G., Chauvet, C., Viatte, L., Danan, J. L., Bigard, X., 12. Kautz, L., & Nemeth, E. (2014). Molecular liaisons between Devaux, I., . . . Vaulont, S. (2002). The gene encoding the iron erythropoiesis and iron metabolism. Blood, 124(4), 479–482. regulatory peptide hepcidin is regulated by anemia, hypoxia, and 13. Kim, A., & Nemeth, E. (2015). New insights into iron regulation inflammation. Journal of Clinical Investigation, 110(7), 1037–1044. and erythropoiesis. Current Opinion in Hematology, 22(3), 199–205. 5. Schmidt, P. J., Toran, P. T., Giannetti, A. M., Bjorkman, P. J., & 14. Zhang, Y., Wang, L., Dey, S., Alnaeeli, M., Suresh, S., Rogers, Andrews, N. C. (2008). The transferrin receptor modulates H., . . . Noguchi, C. T. (2014). Erythropoietin action in stress Hfe-dependent regulation of hepcidin expression. Cell response, tissue maintenance and metabolism. International Metabolism, 7(3), 205–214. Journal of MolecularSciences, 15(6), 10296–10333. 6. Ganz, T., & Nemeth, E. (2012). Hepcidin and iron homeostasis. 15. Haase, V. H. (2013). Regulation of erythropoiesis by hypoxia- Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, inducible factors. Blood Reviews, 27(1), 41–53. 1823(9), 1434–1443. 16. Wilkinson, N., & Pantopoulos, K. (2013). IRP1 regulates 7. Malyszko, J. (2009). Hemojuvelin: the hepcidin story continues. erythropoiesis and systemic iron homeostasis by controlling Kidney and Blood Pressure Research, 32(2), 71–76. HIF2a mRNA translation. Blood, 122(9), 1658–1668. 8. Goswami, T., & Andrews, N. C. (2006). Hereditary 17. Mühlenhoff, U., Gerber, J., Richhardt, N., & Lill, R. (2003). hemochromatosis protein, HFE, interaction with transferrin Components involved in assembly and dislocation of iron–sulfur receptor 2 suggests a molecular mechanism for mammalian iron clusters on the scaffold protein Isu1p. EMBO Journal, 22(18), sensing. Journal of Biological Chemistry, 281(39), 28494–28498. 4815–4825. Anemias of Disordered Regulation of Iron Metabolism and Heme Synthesis 263 18. Killip, S., Bennett, J. M., & Chambers, M. D. (2007). Iron 37. Rettmer, R. L., Carlson, T. H., Origenes, M. L., Jack, R. M., & deficiency anemia. American Family Physician, 75(5), 671–678. Labbé, R. F. (1999). Zinc protoporphyrin/heme ratio for diagnosis 19. Ferri, C., Procianoy, R. S., & Silveira, R. C. (2014). Prevalence and of preanemic iron deficiency. Pediatrics, 104(3), e37. risk factors for iron-deficiency anemia in very-low-birth-weight 38. Wong, S. S., Qutishat, A. S., Lange, J., Gornet, T. G., & Buja, L. preterm infants at 1 year of corrected age. Journal of Tropical M. (1996). Detection of iron-deficiency anemia in hospitalized Pediatrics, 60, 53–60. patients by zinc protoporphyrin. Clinica Chimica Acta, 244(1), 20. Domellöf, M. (2013). Iron and other micronutrient deficiencies in 91–101. low-birthweight infants. Nestlé Nutrition Institute workshop series, 39. Labbé, R. F., & Dewanji, A. (2004). Iron assessment tests: 74, 197–206. Transferrin receptor vis-a-vis zinc protoporphyrin. Clinical 21. Clark, S. F. (2009). Iron deficiency anemia: diagnosis and Biochemistry, 37(3), 165–174. management. Current Opinion in Gastroenterology, 25(2), 122–128. 40. Harthoorn-Lasthuizen, E. J., Lindemans, J., & Langenhuijsen, M. M. 22. Radtke, H., Meyer, T., Kalus, U., Röcker, L., Salama, A., (1998). Combined use of erythrocyte zinc protoporphyrin and mean Kiesewetter, H., & Latza, R. (2005). Rapid identification of iron corpuscular volume in differentiation of thalassemia from iron deficiency in blood donors with red cell indexes provided by deficiency anemia. European Journal of Haematology, 60(4), 245–251. Advia 120. Transfusion, 45(1), 5–10. 41. Junca, J., Flores, A., Roy, C., Alberti, R., & Milla, F. (1990). Red 23. Thomas, D. W., Hinchliffe, R. F., Briggs, C., Macdougall, I. C., cell distribution width, free erythrocyte protoporphyrin, and Littlewood, T., & Cavill, I. (2013). Guideline for the laboratory England-Fraser index in the differential diagnosis of microcytosis diagnosis of functional iron deficiency. British Journal of due to iron deficiency or beta-thalassemia trait: A study of 200 Haematology, 161(5), 639–648. cases of microcytic anemia. Hematologic Pathology, 5(1), 33–36. 24. Buttarello, M. (2016). Laboratory diagnosis of anemia: Are the 42. Case, G. (1998). Maintaining iron balance with total-dose infusion old and new red cell parameters useful in classification and of intravenous iron dextran. Nephrology Nursing Journal, 25(1), 65. treatment, how? International Journal of Laboratory Hematology, 43. Cullen, P., Söffker, J., Höpfl, M., Bremer, C., Schlaghecken, R., 38(Suppl1), 123-132. Mehrens, T., . . . Schaefer, R. M. (1999). Hypochromic red cells 25. Powers, J. M., & Buchanan, G. R. (2014). Diagnosis and and reticulocyte haemglobin content as markers of iron-deficient management of iron deficiency anemia. Hematology/Oncology erythropoiesis in patients undergoing chronic haemodialysis. Clinics of North America, 28(4), 729–745. Nephrology Dialysis Transplantation, 14(3), 659–665. 26. Brumberg, J. J. (1982). Chlorotic girls, 1870–1920: A historical 44. Chang, C. C., & Kass, L. (1997). Clinical significance of immature perspective on female adolescence. Child Development, 1468–1477. reticulocyte fraction determined by automated reticulocyte 27. Napolitano, M., Dolce, A., Celenza, G., Grandone, E., Perilli, counting. American Journal of Clinical Pathology, 108(1), 69–73. M. G., Siragusa, S., . . . Mariani, G. (2014). Iron-dependent 45. Krantz, S. B. (1994). Pathogenesis and treatment of the anemia of erythropoiesis in women with excessive menstrual blood losses chronic disease. American Journal of the Medical Sciences, 307(5), and women with normal menses. Annals of Hematology, 93(4), 353–359. 557–563. 46. Means, R. T., Jr. (1994). Clinical application of recombinant 28. Tussing-Humphreys, L., Pustacioglu, C., Nemeth, E., & erythropoietin in the anemia of chronic disease. Hematology/ Braunschweig, C. (2012). Rethinking iron regulation and Oncology Clinics of North America, 8(5), 933–944. assessment in iron deficiency, anemia of chronic disease, and 47. Theurl, I., Aigner, E., Theurl, M., Nairz, M., Seifert, M., Schroll, obesity: Introducing hepcidin. Journal of the Academy of Nutrition A., . . . Wroblewski, V. J. (2009). Regulation of iron
homeostasis in and Dietetics, 112(3), 391–400. anemia of chronic disease and iron deficiency anemia: diagnostic 29. Micozzi, M. S., Albanes, D., & Stevens, R. G. (1989). Relation and therapeutic implications. Blood, 113(21), 5277–5286. of body size and composition to clinical biochemical and 48. Jain, S., Narayan, S., Chandra, J., Sharma, S., Jain, S., & Malhan, hematologic indices in US men and women. American Journal of P. (2010). Evaluation of serum transferrin receptor and sTfR Clinical Nutrition, 50(6), 1276–1281. ferritin indices in diagnosing and differentiating iron deficiency 30. Wu, Y. C., Wang, Y. P., Chang, J. Y. F., Cheng, S. J., Chen, H. M., & anemia from anemia of chronic disease. Indian Journal of Sun, A. (2014). Oral manifestations and blood profile in patients Pediatrics, 77(2), 179–183. with iron deficiency anemia. Journal of the Formosan Medical 49. Camaschella, C. (2015). Iron-deficiency anemia. New England Association, 113(2), 83–87. Journal of Medicine, 2015(372), 1832–1843. 31. Van Vranken, M. (2010). Evaluation of microcytosis. American 50. Heeney, M. M., & Finberg, K. E. (2014). Iron-refractory iron Family Physician, 82(9), 1117–1122. deficiency anemia (IRIDA). Hematology/Oncology Clinics of North 32. Fernández-Rodríguez, A. M., Guindeo-Casasús, M. C., Molero- America, 28(4), 637–652. Labarta, T., Domínguez-Cabrera, C., Hortal-Cascón, L., Pérez- 51. Finberg, K. E. (2013). Striking the target in iron overload Borges, P., . . . Palop-Cubillo, L. (1999). Diagnosis of iron disorders. Journal of Clinical Investigation, 123(4), 1424–1427. deficiency in chronic renal failure. American Journal of Kidney 52. Centers for Disease Control and Prevention. CDC’s national Diseases, 34(3), 508–513. surveillance data (1997–2015). Retrieved October 14, 2016, from 33. Suominen, P., Punnonen, K., Rajamäki, A., & Irjala, K. (1998). www.cdc.gov/nceh/lead/data/national.htm. Serum transferrin receptor and transferrin receptor-ferritin index 53. Centers for Disease Control and Prevention. Childhood lead identify healthy subjects with subclinical iron deficits. Blood, poisoning data, statistics and surveillance. Retrieved October 14, 92(8), 2934–2939. 2016, from www.cdc.gov/nceh/lead/data/national.htm. 34. Punnonen, K., Irjala, K., & Rajamäki, A. (1997). Serum transferrin 54. Centers for Disease Control and Prevention. What do parents receptor and its ratio to serum ferritin in the diagnosis of iron need to know to protect their children? Retrieved January 30, deficiency. Blood, 89(3), 1052–1057. 2018, from https://www.cdc.gov/nceh/lead/acclpp/blood_ 35. Mulherin, D., Skelly, M., Saunders, A., McCarthy, D., lead_levels.htm O’Donoghue, D., Fitzgerald, O., . . . Mulcahy, H. (1996). The 55. Bhambhani, K., & Aronow, R. (1990). Lead poisoning and diagnosis of iron deficiency in patients with rheumatoid arthritis thalassemia trait or iron deficiency: The value of the red blood and anemia: An algorithm using simple laboratory measures. cell distribution width. American Journal of Diseases of Children, Journal of Rheumatology, 23(2), 237–240. 144(11), 1231–1233. 36. Ahluwalia, N. (1998). Diagnostic utility of serum transferrin 56. Carraccio, C. L., Bergman, G. E., & Daley, B. P. (1987). Combined receptors measurement in assessing iron status. Nutrition Reviews, Iron Deficiency and Lead Poisoning in Children Effect on FEP 56(5), 133–141. Levels. Clinical Pediatrics, 26(12), 644–647. 264 Chapter 12 57. Clark, M., Royal, J., & Seeler, R. (1988). Interaction of iron transporter-1: Functional properties, physiological roles and deficiency and lead and the hematologic findings in children therapeutics. Current Topics Membranes, 70, 169–214. with severe lead poisoning. Pediatrics, 81(2), 247–254. 64. Cogswell, M. E., Burke, W., McDonnell, S. M., & Franks, A. 58. Savage, D., & Lindenbaum, J. (1986). Anemia in alcoholics. L. (1999). Screening for hemochromatosis: A public health Medicine, 65(5), 322–338. perspective. American Journal of Preventive Medicine, 16(2), 59. May, A., & Bishop, D. F. (1998). The molecular biology and 134–140. pyridoxine responsiveness of X-linked sideroblastic anaemia. 65. Olynyk, J. K. (1999). Hereditary haemochromatosis: Diagnosis Haematologica, 83(1), 56–70. and management in the gene era. Liver, 19(2), 73–80. 60. Cotter, P. D., May, A., Li, L., Al-Sabah, A. I., Fitzsimons, E. J., 66. Besur, S., Hou, W., Schmeltzer, P., & Bonkovsky, H. L. (2014). Cazzola, M., & Bishop, D. F. (1999). Four new mutations in the Clinically important features of porphyrin and heme metabolism erythroid-specific 5-aminolevulinate synthase (ALAS2) gene and the porphyrias. Metabolites, 4(4), 977–1006. causing X-linked sideroblastic anemia: Increased pyridoxine 67. de Verneuil, H., Ged, C., Boulechfar, S., & Moreau-Gaudry, F. (1995). responsiveness after removal of iron overload by phlebotomy Porphyrias: Animal models and prospects for cellular and gene and coinheritance of hereditary hemochromatosis. Blood, 93(5), therapy. Journal of Bioenergetics and Biomembranes, 27(2), 239–248. 1757–1769. 68. Mascaro, J. M. (1992). Porphyrias in children. Pediatric 61. Yun, S., & Vincelette, N. D. (2015). Update on iron metabolism Dermatology, 9(4), 371–372. and molecular perspective of common genetic and acquired 69. Fuller, S. J., & Wiley, J. S. (2013). Heme biosynthesis and its disorder, hemochromatosis. Critical Reviews in Oncology/ disorders. In: R. Hoffman, E. J. Benz, L. E. Silberstein, H. Heslop, Hematology, 95(1), 12–25. J. Weitz, & J. Anastasia, eds. Hematology: Basic principles and 62. Crownover, B. K., & Covey, C. J. (2013). Hereditary practice (pp. 457–472). New York: Elsevier. hemochromatosis. American Family Physician, 87(3). 183-190. 70. D’Avola, D., & Gonzalez Aseguinolaza, G. (2016). Prospect and 63. Shawki, A., Knight, P. B., Maliken, B. D., Niespodzany, E. J., progress of gene therapy in acute intermittent porphyria. Expert & Mackenzie, B. (2012). H (+ )@coupled divalent metal-ion Opinion on Orphan Drugs, 4(7), 711-717. Chapter 13 Hemoglobinopathies: Qualitative Defects Deborah Fox, PhD Julie Soder, MS Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define hemoglobinopathy. 6. Associate laboratory analyses with their use in 2. Explain the basis of defects resulting in the detecting and identifying hemoglobinopathies. production of variant hemoglobins. 7. Recognize and identify abnormal laboratory test results, including peripheral blood find- 3. Explain the basis of the hemoglobin ings and screening and confirmatory tests, electrophoresis method in identifying typically associated with homozygous and variant hemoglobins. heterozygous conditions involving sickle 4. Describe the epidemiology of hemoglobin (HbS), HbC, HbD, and HbE and sickle cell anemia (SCA) and other compound heterozygous conditions involv- hemoglobinopathies. ing HbS and other variant hemoglobins. 5. Identify the globin chain defects causing 8. List major clinical findings typically associ- SCA, hemoglobin C (HbC) disease, and ated with the hemoglobinopathies listed in hemoglobin E (HbE) disease. Objective 7. Objectives—Level II At the end of this unit of study, the student should be able to: 1. Compare the synthesis and concentration 3. Compare and contrast the pathophysiol- of variant hemoglobins in homozygous and ogy of common hemoglobin variants in heterozygous conditions. terms of altered solubility, function, and 2. Compare the prevalence of hemoglobins S, stability. C, D, and E. 265 266 Chapter 13 4. Analyze the structure of the hemoglo- 8. Select, evaluate, and interpret tests used bin molecule in sickle cell anemia (SCA) in detecting and identifying abnormal and relate it to the pathophysiology of the hemoglobins. disease. 9. Design a laboratory-testing algorithm for 5. Contrast clinical findings in persons who are optimizing tests used in detecting and iden- homozygous and heterozygous for hemo- tifying variant hemoglobins. globins S, C, D, and E and in those who have 10. Evaluate laboratory test results and medical combined heterozygosities for these variant history of a clinical case for a patient with a hemoglobins. hemoglobinopathy and suggest a possible 6. Identify and explain the basis of current diagnosis. therapies for SCA. 11. Explain the physiologic abnormality resulting 7. Evaluate and interpret mobility patterns in unstable hemoglobins, methemoglobin- obtained on cellulose acetate, citrate agar emia, and hemoglobin variants with increased gel, alkaline and acid hemoglobin electro- or decreased oxygen affinity. phoresis when structurally abnormal hemo- 12. Interpret laboratory findings associated with globins are present. the disorders in Objective 11. Chapter Outline Objectives—Level I and Level II 265 Hemoglobin S/C Disease 279 Key Terms 266 Hemoglobin D 280 Background Basics 266 Hemoglobin E 281 Case Study 267 Unstable Hemoglobin Variants 281 Overview 267 Hemoglobin Variants with Altered Oxygen Introduction 267 Affinity 283 Structural Hemoglobin Variants 268 Summary 285 Sickle Cell Anemia 271 Review Questions 285 Hemoglobin C Disease 279 References 287 Key Terms Aplastic crisis Hemoglobinopathy Sequestration crisis Autosplenectomy Irreversibly sickled cell (ISC) Thalassemia Compound heterozygote Ischemic Vaso-occlusive crisis Hemoglobin electrophoresis Methemoglobinemia Background Basics The information in this chapter builds on the concepts Level I learned in previous chapters. To maximize your learning • Describe erythrocyte metabolism and erythrocyte experience, you should review these concepts before start- destruction. (Chapter 5) ing this unit of study: Hemoglobinopathies: Qualitative Defects 267 • Describe the structure and function of hemoglobin; Level II list variant hemoglobins. (Chapter 6) • Explain the genetic control of globin chain synthe- • Name and describe basic laboratory procedures used sis. (Chapter 6) to screen for and assess anemia. (Chapters 10, 11, 37) • Describe the ontogeny of hemoglobin types. • Recognize abnormal values and results for basic (Chapter 6) hematologic procedures. (Chapters 10, 11, 37) • Describe the process of neutrophil adhesion to • Describe the classification systems of a nemias. vascular endothelium. (Chapter 7) (Chapter 11) CASE STUDY Introduction We refer to this case throughout the chapter. Clinical diseases that result from a genetically determined abnormality of the structure or synthesis of the hemoglobin Shane, a 16-year-old African American male with molecule are called hemoglobinopathies. The abnormality a previously diagnosed hemoglobinopathy, was is associated with the globin chains; the heme portion of the admitted to the hospital complaining of severe molecule is normal. The globin abnormality can be either a pain in his knees and back. Two of his four siblings qualitative defect in the globin chain (structural abnormal- have the same disorder. He has been admitted to ity) or a quantitative defect in globin synthesis. the hospital on numerous occasions throughout his Qualitatively abnormal hemoglobin molecules arise life for complications of his disease. Physical exam- from genetic mutations in the coding region of a globin ination reveals a thin male in acute distress, com- gene, resulting in amino acid deletions or substitutions plaining of severe pain. A head, eyes, ears, nose, in the globin protein chain. These mutations cause struc- and throat exam is positive for corkscrew vessels of tural variation in one of the globin chain classes (structural the schlerae, schleral icterus, and small, ill-defined, hemoglobin variants). The nomenclature of these disorders mobile (shotty) cervical lymph nodes. Abdominal is discussed in the later section “Nomenclature.” The most exam revealed no splenomegaly, hepatomegaly, common clinical disorder of this type of mutation is sickle tenderness, or masses. Vital signs included temper- cell anemia. ature 37.8 °C blood pressure 95/70, and pulse 82. The quantitative globin disorders result from various Blood was drawn for laboratory tests, and a chest genetic defects that reduce synthesis of structurally normal radiograph and MRI of the head were ordered. globin chains. The quantitative disorders are known col- Consider whether the patient’s current condi- lectively as thalassemias. tion is likely to be related to his previous diagnosis Clinical disorders are also associated with combination and what the laboratory’s role is now. defects involving both structural defects and quantitative deficiencies of hemoglobin. Compound heterozygotes pos- sess two different abnormal alleles of a gene coding for glo- Overview bin chains. An example is thalassemia in combination with sickle cell (Chapter 14). When hemoglobin’s molecular structure is altered, the mol- Because of the globin chain defects, hemoglobinopa- ecule’s function, stability, and/or solubility can change, thies can be associated with a chronic hemolytic anemia or often resulting in anemia and other clinical consequences. can be asymptomatic. Clinical expression of the hemoglo- Laboratory screening tests for hemoglobin variants are binopathy varies, depending on the class of globin chain based on the altered characteristics of the hemoglobin mole- involved (a, b, d, or g), the severity of hemolysis, and the cule. If screening tests are abnormal, reflex tests are required compensatory production of other normal globin chains. to confirm the presence of a variant hemoglobin. The labo- Some of the hemoglobinopathies produce no clinical signs ratory’s role is not limited to detecting variant hemoglo- or symptoms of disease and are identified only through bins but extends to monitoring a patient’s condition over population studies specifically designed to reveal “silent” the course of an illness and during treatment. This chapter carriers. As discovery of silent carriers increases, the inci- discusses the most common variant hemoglobins includ- dence of these genetic disorders is proving to be much ing epidemiology, pathophysiology, clinical presentation, higher than originally thought. laboratory evaluation, and treatment. Emphasis is on the Hemoglobinopathies are believed to be one of the most
correlation of clinical history and symptoms with labora- common hereditary diseases in humans worldwide.1 More tory tests and interpretation of test results. than 330,000 infants worldwide are born each year with 268 Chapter 13 inherited hemoglobin disorders such as sickle cell and thal- the globin gene loci. Hemoglobin F (g@chain) is expressed assemia.2 Many affected children live in low-income coun- to a significant extent only during fetal development; there- tries, and many die before age 5. fore, variant F hemoglobins are unlikely to be detectable Hemoglobinopathies are found worldwide but occur after 3–6 months of age. Mutations affecting the g@chain most commonly in African blacks and ethnic groups from locus with the potential to produce a significant clini- the Mediterranean basin and Southeast Asia. The geo- cal abnormality would likely cause fetal death. At birth graphic locations where the quantitative and qualitative and in the first few months of life, as b@chain synthesis hemoglobin disorders are found frequently overlap; thus, becomes predominant, residual abnormal HbF is masked it is not uncommon for individuals to have both a struc- by the increasing concentration of HbA in children car- tural hemoglobin variant and a form of thalassemia. This rying mutations of the g@chain, producing milder clinical could partly explain the extreme variation in clinical find- presentations. ings associated with hemoglobinopathies. Because HbA2 (d@chain) is usually a minor component This chapter discusses the structural hemoglobin vari- of the total hemoglobin in adults, HbA2 variants are also ants and Chapter 14 discusses the thalassemias and combi- unlikely to cause clinical complications and are discov- nation disorders. ered “accidentally” during laboratory evaluation for other purposes. Two copies of the a@chain gene are found on each of Structural Hemoglobin the number 16 chromosomes, resulting in a total of four genes. Thus, a mutation of a single a@locus resulting in a Variants variant a@chain@containing hemoglobin produces only a small amount of the abnormal hemoglobin and is less likely The largest group of hemoglobinopathies results from an to cause clinically significant disease. inherited structural change in one of the globin chains; The b@gene, which is found in a single copy on each however, rate of synthesis of the abnormal chain usually is chromosome 11, is most likely to be associated with a clini- not significantly impaired. Any of the globin chain classes, cal phenotype when mutated because the b@globin chain is a, b, d, or g, can be affected. a component of HbA, the major adult hemoglobin. Sickle cell anemia (SCA), caused by the most common structural hemoglobin variant, was reported by James Her- Identification of Hemoglobin rick of Chicago in 1910.3 He described the typical crescent- shaped sickled erythrocytes in a young black student from Variants the West Indies. Following this initial report, additional Most structural hemoglobin variants result from a single cases of the disease were described, and the clinical pattern amino acid substitution or deletion in the globin polypep- of SCA was established. The pathophysiologic aspects of tide chain. Most structural variants result in no clinical or the disease, however, remained a mystery until Linus Paul- hematologic abnormality and have been discovered only ing in 1949 discovered the altered electrophoretic mobility by population studies or family studies. Mutations result of the hemoglobin in patients with sickle cell disease.4 The in clinical manifestations only when the solubility, stabil- molecule’s altered electrical charge was ascribed to a molec- ity, or function (oxygen affinity) of the hemoglobin mol- ular abnormality of the globin chain. ecule is altered. These phenotypic variants produce both More than 1200 abnormal hemoglobins have since clinical and hematologic abnormalities of varying severity, been identified.5 The number of identified b@chain muta- depending on the nature and site of the mutation. Labo- tions exceeds the number identified for a@, d@, or g@chains ratory tests designed to detect and identify hemoglobin (Table 13-1). Part of the explanation for this distribution variants are based on the molecule’s altered structure or likely resides in the genetics and phenotypic expression of function. Table 13.1 Structural Variants of Globin Genes Globin Polypeptide Gene Number of Variants a1@ and/or a2@globin gene(s) 478 b@globin gene 610 d@globin gene 72 Ag@globin gene 38 Gg@globin gene 60 Hemoglobinopathies: Qualitative Defects 269 Methods of Analysis Migration Patterns for Hemoglobin Electrophoresis Controls Hemoglobin carries an electrical charge resulting from the presence of ionized carboxyl (COO-) and protonated (H+ Migration of ) Hemoglobin on amino (NH + 3 ) groups. The type (net positive, net nega- Cellulose acetate pH 8.4 or Alkaline tive) and strength of the charge depend on both the amino agarose gel acid sequence of globin chains in the hemoglobin molecule Origin A2 S F A Barts and the pH of the surrounding medium. Many amino acid C D H E G substitutions alter the molecule’s electrophoretic charge, O Lepore enabling the detection of a structural hemoglobin variant by Migration of hemoglobin electrophoresis. Some substitutions can cause Hemoglobin on identical changes in the net charge of the molecule; thus, two Citrate pH 6.2 or Acid agarose gel different mutant hemoglobins can have identical electropho- retic mobility. Other substitutions do not alter the charge of F A S Origin C D the globin chain; the variant hemoglobin migrates identically E, A2, Lepore to the “normal” globin chain and does not form an abnor- G O mal band on an electrophoresis gel. However, by varying Patient the medium and pH of the procedure, many clinically sig- Migration of nificant hemoglobins can be detected and identified. Meth- Hemoglobin on ods for performing hemoglobin electrophoresis, examples of Cellulose acetate pH 8.4 or Alkaline electrophoretic patterns, and more complete discussions of agarose gel the tests that follow are included in Chapter 37. The most common clinically symptomatic hemoglo- Figure 13.1 Electrophoretic patterns of hemoglobins. The top binopathies involve abnormalities of the b@globin chain, pattern shows the migration of control bands for cellulose acetate at pH 8.4 and alkaline hemoglobin electrophoresis. The middle resulting in a decrease or absence of HbA (a2b2) and some- pattern demonstrates the migration pattern for citrate agar gel pH times an increase in HbF (a2g2) and/or HbA2 (a2d2). A band 6.2 and acid hemoglobin electrophoresis. Controls include normal representing the mutant hemoglobin may also be present. and variant hemoglobins. Note that some hemoglobins that move Typically, an elevation in HbF and/or HbA2 is the clue to together on cellulose and alkaline gel can be separated on citrate the presence of a hemoglobinopathy, although HbF can be and acid gel. The bottom pattern shows an electrophoretic pattern of elevated in other hematologic disorders as well. HbF con- a patient with normal hemoglobin using cellulose acetate (pH 8.4) or alkaline hemoglobin electrophoresis. centrations that are greater than 10% can be measured by electrophoresis and densitometry. Smaller but significant increases in HbF can be measured more accurately by alkali and mimic that migration pattern of cellulose acetate and denaturation and other methods. HbF distribution among citrate agar (pH 6.2) respectfully. Figure 13-1 shows the the erythrocyte population is evaluated by acid elution electrophoretic mobility of normal and abnormal hemo- tests. These tests are based on the resistance of HbF to alkali globins using cellulose acetate (pH 8.4) and alkaline hemo- and acid treatment compared to other hemoglobins. globin electrophoresis as well as citrate agar gel (pH 6.2) Results of laboratory tests provide essential informa- and acid hemoglobin electrophoresis. Some laboratories tion when a hemoglobinopathy is suspected. The hemoglo- use advanced testing methods such as isoelectric focusing bin concentration from the CBC indicates whether anemia (IEF), high-performance liquid chromatography (HPLC), is present. The RDW and erythrocyte indices should be capillary electrophoresis (CE), or molecular methods.7 evaluated to help distinguish hemoglobinopathies from These laboratory methods are suitable for testing large thalassemia, which usually causes a microcytic, hypochro- numbers of individuals for hemoglobinopathies and offer mic anemia. A review of the blood smear may reveal specific improved resolution and identification of certain hemoglo- poikilocytes characteristic of a particular hemoglobinopa- bin variants over results obtained with electrophoresis.6 thy, such as sickle cells found in SCA. HPLC has the advantage over electrophoresis of quanti- An important next step in the investigation of hemo- fying low concentrations of HbA2 and HbF.7 All states in globin variants is to separate and quantify the hemoglobin the United States mandate newborn screening programs fractions HbA, HbA2, HbF, and possible variants.6 Several for sickle cell disease.7 Half of labs participating in high- options for laboratory analysis are available. Traditional volume testing of newborn screens use IEF as their primary electrophoresis on cellulose acetate (pH 8.4) and citrate detection method, while the other half use HPLC or mass agar gel (pH 6.2) are still in use. Other electrophoresis spectrometry methods.10 Unfortunately, these methods options in use include alkaline agarose electrophoresis may be unavailable for testing residents of developing and and acid agarose electrophoresis that are less cumbersome low-income countries. 270 Chapter 13 Other traditional tests for abnormal hemoglobins are (e.g., Hb Fort Worth). It also became apparent that some based on altered physical properties of the structural vari- variants with the same letter designation (same electro- ants. These include solubility tests, heat precipitation tests, phoretic mobility) had different structural variations. If the and tests for Heinz bodies. The uses of these methods are hemoglobin has the electrophoretic mobility of a previously included in the following discussion of the correspond- lettered hemoglobin, that letter is used in addition to the ing specific structural variants. Procedures are included in geographic area (e.g., HbG Honolulu). Chapter 37. A standardized hemoglobin nomenclature has been Techniques are also available to identify the specific recommended for use. All variants should be given a scien- molecular defect of hemoglobin disorders.11 These tech- tific designation as well as a common name. The scientific niques are discussed in Chapter 42. The polymerase chain designation includes the following: (1) the mutated globin reaction (PCR) has been incorporated into diagnostic proce- chain, (2) the position of the affected amino acid, (3) the heli- dures for identifying point mutations because it enhances cal position of the mutation, and (4) the amino acid substi- sensitivity and reduces the amount of DNA and time tution. If the mutation affects amino acids between helices, required for analysis. Prenatal diagnosis can be carried out the number of the amino acid and the letters of the two in the first trimester of pregnancy using DNA obtained from bracketing helices are used. For example, HbS is designated chorionic villus sampling. In cases of hemoglobinopathies b6 (A3) Glu S Val. The mutation is in the b@chain affect- caused by known common mutations, such as SCA, the fetal ing the amino acid in the sixth position of the completed DNA can be analyzed directly. If prenatal diagnosis is polypeptide located in the A3 helix position. The amino desired when the exact mutation is not known, the parents’ acid valine is substituted for glutamic acid. Hemoglobins DNA can be analyzed first (e.g., restriction fragment length with amino acid deletions include the word missing after polymorphism [RFLP]) to help identify the presence of a the amino acid and helix designation (e.g., b56959 [D7–E3] mutation (Chapter 42). missing). The advantage of the helical designation is that amino acid substitutions in the same helix can lead to simi- lar functional and structural alterations of the hemoglobin Checkpoint 13.1 molecule, allowing a better understanding of the clinical Why is it not possible for all structural hemoglobin variants to be manifestations of each. identified by hemoglobin electrophoresis? Not all globin chain mutations cause symptoms of disease; thus, many go undetected. Only those that cause clinical symptoms are likely to be brought to a physician’s CASE STUDY (continued from page 267) attention. If an individual is homozygous for the gene cod- ing for a structural b@globin mutant, no HbA is produced, 1. Identify a laboratory test needed to determine and the term disease or anemia is used to describe the specific Shane’s hemoglobinopathy. disorder (e.g., sickle cell anemia). If one of the genes coding for the b@chain is normal and the other b@gene codes for a structural variant, both HbA and the abnormal hemoglobin Nomenclature are produced, and the word trait is used to describe the het- erozygous disorder (e.g., sickle cell trait). The first abnormal hemoglobin discovered was called With the most common b@chain hemoglobin variants hemoglobin S (HbS) because it was associated with
crescent (HbS, HbC), the abnormal hemoglobin usually accounts for (sickle)–shaped erythrocytes (S for sickle). Subsequently, less than 50% of the total hemoglobin in the trait form, other hemoglobin variants were discovered and were given whereas in the homozygous state of disease, the abnormal successive letters of the alphabet according to electropho- hemoglobin usually constitutes 90–95% of the total hemo- retic mobility beginning with the letter C. The letter A was globin. This is explained by the effect of the abnormal glo- already being used to describe the normal adult hemoglo- bin chain on the formation of the hemoglobin tetramers. The bin, HbA. The letter B was not used to avoid confusion normal a@chain has a net positive charge; the normal with the ABO blood group system. The letter F had been b@chain (bA) has a negative charge, and ab dimers form ini- designated to describe fetal hemoglobin, HbF. The letter M tially through positive-negative electrostatic interactions. was given to those hemoglobins that tended to form met- b@chain mutants with a lesser negative charge than bA form hemoglobin (HbM). ab dimers more slowly than do bA (and, conversely, muta- As more variants were discovered, it was recognized tions that increased the negative charge of the b@globin that the alphabetical system was not sufficient and a dif- chain would form ab dimers more rapidly). Both bS and bC ferent nomenclature system was needed. Thus, subse- mutations cause a net reduction of the b@chain negative quent hemoglobins were given common names according charge and form abS or abC dimers more slowly than abA to the geographic area in which they were discovered dimers. As a result, heterozygotes have about 60% HbA and Hemoglobinopathies: Qualitative Defects 271 about 35-40% HbS or HbC. If HbS or HbC constitutes more ALTERED STABILITY than 50% of the total hemoglobin in a heterozygote, the Amino acid substitutions that reduce the stability of the patient could have inherited two different abnormal hemo- hemoglobin tetramer result in unstable hemoglobins. The globin genes (compound heterozygote) or a form of thalas- mutations usually disrupt hydrogen bonding or hydropho- semia in combination with the structural hemoglobin bic interactions that retain the heme component within the variant (Chapter 14). heme-binding pocket of the globin chain or that hold the tetramer together. The result is a weakening of the binding of heme to globin and detachment of the heme or the dis- Checkpoint 13.2 ruption of the integrity of hemoglobin’s tetrameric struc- What does the term silent carrier mean when referring to a ture. Consequently, hemoglobin denatures, aggregates, and hemoglobinopathy? precipitates as Heinz bodies. Clinically, the unstable hemo- globin variants are sometimes known as congenital Heinz body hemolytic anemias. In addition to altering the molecule’s CASE STUDY (continued from page 270) stability, disruption of normal conformation also can affect Results of hemoglobin electrophoresis were 90% the molecule’s function. HbS, 9% HbF, and 1% HbA2. 2. What is the abnormal hemoglobin causing Checkpoint 13.3 Shane’s disease? The mutation in HbJ-Capetown, a92, Arg S Gln, stabilizes 3. Is Shane heterozygous or homozygous for the hemoglobin in the R state (Chapter 6). What functional effect disorder? does this have on the hemoglobin molecule? 4. What is this disorder called? Sickle Cell Anemia Pathophysiology Worldwide, SCA is the most common symptomatic hemo- globinopathy with greatest prevalence in tropical Africa The structural hemoglobin variants cause symptoms if the (Table 13-2). Gene frequency in equatorial Africa can exceed amino acid substitution occurs at a critical site within the 20%. The sickle cell gene is also common in areas around molecule. Mutations that cause clinical signs of disease the Mediterranean, the Middle East, India, Nepal, and in affect the solubility, function (oxygen-affinity), and/or sta- geographic regions in which there has been migration from bility of the hemoglobin molecule. endemic areas, such as North, Central, and South America.12 ALTERED SOLUBILITY Sickle cell anemia occurs in 0.3–1.3%, and sickle cell trait If a nonpolar amino acid is substituted for a polar residue occurs in 8–10% of African Americans. near the molecule’s surface, the solubility of the hemoglobin It is interesting to note that geographic areas with the molecule can be affected. Hemoglobin S and hemoglobin C highest frequency of sickle cell genes are also areas where are examples of this type of substitution. In the deoxygen- infection with Plasmodium falciparum is common. This cor- ated state, the HbS molecule polymerizes into insoluble, relation strongly suggests that HbS in heterozygotes con- rigid aggregates. Most surface substitutions, however, do fers a selective advantage against fatal malarial infections, not affect the tertiary structure, heme function, or subunit resulting in an increase in the gene frequency. Children with interactions and are therefore innocuous. sickle cell trait are infected with the malarial parasite, but the parasite counts remain low. It has been suggested that ALTERED FUNCTION resistance to malaria occurs because parasitized cells sickle Some amino acid substitutions can affect the oxygen affinity more readily, leading to sequestration and phagocytosis of of hemoglobin by stabilizing heme iron in the ferric state, the infected cell by the spleen. Other undefined factors could producing methemoglobin, which cannot reversibly bind also contribute to reduced malarial susceptibility in individ- oxygen (Chapter 6). Hemoglobins M and Chesapeake are uals with HbS.13 Epidemiological data suggest there could examples. Mutations within the subunit interface, a1b2, be similar selective advantages to HbE and HbC.11 Molecu- can affect the allosteric properties of the molecule, lead- lar evidence indicates that the identical sickle mutation arose ing to increased or decreased oxygen affinity. Considerable independently in these geographic areas at least five times. movement occurs at the a1b2 contact region on oxygenation, which triggers these allosteric interactions (Chapter 6). High oxygen-affinity hemoglobin variants produce congenital Pathophysiology erythrocytosis whereas decreased oxygen-affinity variants Sickle cell anemia is caused by a mutation (GAG S GTG produce pseudoanemia and cyanosis. conversion) in the HBB gene (hemoglobin b gene). This 272 Chapter 13 Table 13.2 Summary of the Most Common Hemoglobin Variants Peripheral Blood, Hb Present on Hb Homozygotes Electrophoresis Mutation Lys Geographic Distribution HbS Normocytic, normochromic anemia; Homozygous: HbS, F, A2 b6(A3) Glu S Val Tropical Africa and Mediterranean reticulocytosis; poikilocytosis with Heterozygous: HbA, S, F, A2 areas, Middle East, India, Nepal sickled and boat-shaped cells HbE Microcytic, hypochromic anemia; Homozygous: HbE, F, A2 b26(B8) Glu S Lys Burma, Thailand, Cambodia, Malay- target cells Heterozygous: HbA, E, F, A2 sia, Indonesia HbC Normocytic, normochromic anemia Homozygous: HbC, F, A2 b6(A3) Glu S Lys West Africa with reticulocytosis; poikilocytosis Heterozygous: HbA, C, F, A2 with folded, irregularly contracted cells; target cells mutation results in the substitution of nonpolar valine for before significant polymerization and cell distortion occur. polar glutamic acid at the sixth amino acid position in the The length of delay depends highly on temperature, pH, A3 helix of the b@chain-b6 (A3) Glu S Val (see the previ- ionic strength, and oxygen tension in the cell’s environ- ous “Nomenclature” section for explanation)—and pro- ment. Hypoxia, acidosis, hypertonicity, and temperatures duces the mutant hemoglobin, HbS (a tetramer of abS/abS higher than 37 °C promote deoxygenation and the forma- and sometimes referred to as simply, SS). The amino acid tion of HbS polymers. The spleen, kidney, retina, and bone substitution is on the surface of the molecule, producing a marrow provide a sufficiently hypoxic, acidotic, and hyper- net decrease in negative charge; hence, it changes the mol- tonic microenvironment to promote HbS polymerization ecule’s electrophoretic mobility. and sickling. Sickling (delay time) also depends on intracellular SOLUBILITY AND POLYMERIZATION OF HEMOGLOBIN S hemoglobin composition (the proportion of HbA, HbS, The oxygen affinity of HbS differs from that of HbA, HbA2, and HbF present) as well as total hemoglobin con- resulting in important physiologic changes in vivo. HbS centration (MCHC). Increased levels of non-S hemoglobins, has decreased oxygen affinity, and the 2,3-BPG level of such as HbF, increase the delay time for polymerization, homozygotes is increased. This decreased oxygen affinity, presumably by interfering with the HbS polymerization depicted by a shift to the right in the oxygen dissociation process, thus lengthening red cell survival time. Altering curve (Chapter 6), facilitates the release of more oxygen to erythrocyte contents by increasing the concentration of HbF the tissues. This phenomenon increases the concentration of forms the basis of an important treatment for SCA. The deoxyhemoglobin S, promoting the formation of sickle cells. delay time is also inversely related to the total hemoglobin The solubility of HbS in the deoxygenated state is mark- concentration. The more concentrated the hemoglobin solu- edly reduced, producing a tendency for deoxyhemoglobin tion is within the cell (the higher the MCHC), the shorter is S molecules to polymerize into rigid aggregates when the the delay time and the greater is the potential for HbS aggre- oxygen saturation of hemoglobin falls below 85%. The cells gates to form. Using this concept, attempts are made to treat may assume a crescent (sickle) shape, depending on the the disease by hydrating the cells, which would decrease extent of polymerization of the HbS molecules. Polymeriza- the MCHC and prevent sickling. tion is generally complete at about 38% oxygen saturation, With repeated cycles of deoxygenation, polymerization, but is reversible on reoxygenation. and sickling, disruption of cation homeostasis and the phos- Polymerization is time dependent. A time delay occurs pholipid membrane occur. Cation disturbance results in an between deoxygenation and the formation of a signifi- increase in intracellular Ca++ and loss of K+ and water from cant amount of HbS polymers. The development of sig- the cell. This cellular dehydration increases the MCHC, nificant polymerization and red cell distortion takes about predisposing the cell to sickling on subsequent deoxygen- 2-4 minutes. The delay time for polymerization is impor- ation. In addition, the increased MCHC is associated with tant in considering the overall clinical consequences of HbS. an increase in cytoplasmic viscosity and a decrease in cell Even though most red cells contain some sickle hemoglobin deformability. HbS is also prone to oxidation. Oxidation polymer at the oxygen concentration in venous blood, most of membrane proteins and lipids weakens critical skeletal cells do not sickle during their journey through the circu- associations. Repeated sickling tends to decouple the lipid lation because they reach the lungs and are reoxygenated bilayer from the membrane skeleton, and loss of membrane Hemoglobinopathies: Qualitative Defects 273 phospholipid asymmetry occurs. The aggregates also dam- the coagulation cascade, upregulation of cell adhesion mol- age the erythrocyte membrane directly, and with the weak- ecules, and degradation of nitric oxide (NO).19 Red blood ened cytoskeleton, the cell’s fragility increases. cell hemolysis and inflammation are the primary stimuli of these processes. Increased levels of cell free hemoglobin IRREVERSIBLY SICKLED CELLS cause consumption of NO, a component vital for blood ves- The sickled erythrocyte can return to a normal biconcave sel maintenance (Chapter 6). Endothelial cell dysfunction shape upon reoxygenation of the hemoglobin; however, with increased red cell adherence are a consequence of low as described above, with repeated cycles of sickling, the NO levels. erythrocyte membrane undergoes changes that cause it to Inflammatory stimuli activate vascular endothelium become leaky and rigid. After repeated sickling episodes, by upregulating the production of vasoconstrictors such as the cells become irreversibly sickled cells (ISCs), which are endothelin-1 and augmenting the nuclear factor (NF)@kB locked in a sickle shape whether oxygenated or deoxygen- pathway. Increased NF@kB activation and the production of ated (i.e., regardless of polymerization state of the hemo- proinflammatory cytokines such as TNFa and IL@1b from globin molecules). This is due to abnormal interactions of activated monocytes induce expression of selectins and red cell skeletal proteins, most likely caused by oxidative intercellular adhesion molecules (ICAM-1) (Chapter 7) on damage and increased Ca++ concentrations. The ISC is the VEC and the b2@integrin molecule present on neutrophils. result of permanent alterations of the submembrane skeletal Circulating neutrophils roll across vascular endothelium and lattice.14 From 5–50% of the circulating erythrocytes in sickle become marginated as they attach via interactions between cell anemia are ISCs. This varies from person to person but such adhesion molecules. Additional b2@integrins expressed is relatively constant for a given individual. The ISCs have on the leading edge of adherent neutrophils capture ISCs, a very high MCHC and a low MCV. They are ovoid or boat triggering the formation of a cell mass which obstructs the shaped with a smooth outline and lack the
spicules charac- venous blood flow (Figure 13-2). Aggregates may develop teristic of deoxygenated sickled cells. into complex heterocellular groupings involving activated The ISCs are removed by mononuclear phagocytes in neutrophils, sickle cells, platelets, and possibly monocytes. the spleen, liver, or bone marrow. ISCs account for most, During this process, invariant NKT (iNKT) cells produce if not all, of the sickle forms on a peripheral blood smear additional cytokines and amplify the inflammatory reaction (most reversible sickle cells regain a discocyte shape when surrounding ischemia-reperfusion, causing further injury. the blood is exposed to air while making the slide).15,16 RED BLOOD CELL (RBC) DESTRUCTION The primary cause of anemia in sickle cell anemia is extra- Clinical Presentation vascular hemolysis. Erythrocyte survival depends on The first clinical signs of sickle cell anemia appear at about intracellular HbF concentration and degree of membrane 6 months of age when the concentration of HbS predomi- damage.17 Changes in the erythrocyte membrane resulting nates over HbF. Clinical manifestations result from chronic in increased fragility coupled with Heinz body formation hemolytic anemia, vaso-occlusion of the microvasculature, from denatured HbS lead to increased membrane shedding overwhelming infections, and acute splenic sequestration. (vesiculation), a decreased cell surface area, and the removal of the cell by the mononuclear phagocyte system.18 The life ANEMIA span of circulating HbS erythrocytes can decrease to as few A moderate to severe chronic anemia as the result of extra- as 14 days. The sluggish blood flow and the hypoglycemic, vascular hemolysis is characteristic of the disease. Gall- hypoxic environment of the spleen promotes HbS polymer- stones, a complication of any chronic hemolytic disorder, ization and sickling, further slowing blood circulation in the are commonly found due to cholestasis and increased bili- splenic cords and enhancing phagocytosis of erythrocytes rubin turnover. Folate deficiency due to increased erythro- containing HbS. Eventually, however, the spleen loses its cyte turnover can further exacerbate the anemia, producing functional capacity as repeated ischemic crises (see “Vaso- megaloblastosis (Chapter 15). Over time, iron overload and Occlusive Crisis”) lead to splenic tissue necrosis and atro- resultant complications, can develop. phy. With splenic atrophy, other cells of the mononuclear Hemodynamic changes occur in an attempt to com- phagocyte system in the liver and bone marrow take over pensate for the tissue oxygen deficit; as a result, symptoms the destruction of these abnormal cells. of cardiac overload including cardiac hypertrophy, cardiac enlargement, and eventually congestive heart failure are VASO-OCCLUSION frequent complications of the disease. Sickle cell vaso-occlusion results from a complex multicellu- The hyperplastic bone marrow, secondary to chronic lar and multistep process that involves not only entrapment hemolysis, is accompanied by bone changes, such as thin- of sickle cells, but also increased activation of neutrophils, ning of cortices and a “hair-on-end” appearance in x-rays vascular endothelial cells (VECs), platelets, monocytes and of the skull. Hyperplasia results from a futile attempt by the 274 Chapter 13 Sickle cell entrapment Leukocyte rolling Adhesion PMN receptor E-, P−selection for E-, P−selectin L-selectin Bz-integrin Multicellular VEC receptor aggregate ICAM (for L−selectin) Figure 13.2 An illustration depicting the multicellular complex formed between sickle cells, activated neutrophils, vascular endothelial cells, and platelets, which block a vessel and precipitate a vaso-occlusive crisis. marrow to compensate for premature erythrocyte destruc- deoxyhemoglobin S, expanding the blockaded region. If tion. Conversely, aplastic crises can accompany or follow severe, lack of oxygen can cause local tissue necrosis. Vaso- viral, bacterial, and mycoplasmal infections. This temporary occlusion occurs more often in tissues prone to vascular cessation of erythropoiesis in the face of chronic hemolysis stasis (spleen, marrow, retina, kidney). leads to an acute worsening of the anemia. The aplasia can The blockage of the microvasculature by cellular aggre- last from a few days to a week and because of the signifi- gates accounts for most of the clinical signs of sickle cell cantly decreased red cell life span, can induce a catastrophic anemia. The occlusions do not occur continuously but spo- fall in the hemoglobin concentration. Increasing evidence radically, causing acute signs of distress. These episodes are suggests that many cases of aplasia occur because of infec- called vaso-occlusive crises and serve as the hallmark for tion with human parvovirus B19. Parvovirus also causes a SCA since they are the most frequent causes of hospital- cessation of erythropoiesis in normal individuals, but with ization. The crises can be triggered by infection, decreased a normal RBC life span, normal blood cell production in atmospheric oxygen pressure, acidosis, dehydration, or these individuals is restored before any clinically significant slow blood flow, but frequently they occur without any changes in erythrocyte concentration take place. known cause. The occlusions are accompanied by pain, VASO-OCCLUSIVE CRISIS low-grade fever, organ dysfunction, and tissue necro- HbS cells are poorly deformable. Sickled cells have diffi- sis. The episodes generally last for 4–5 days and subside culty squeezing through small capillaries, and consequently, spontaneously. the rigid cells tend to aggregate in the microvasculature, Recurrent occlusive episodes can lead to infarctions increasing vascular stasis. Erythrocytes behind the block- of tissue of the genitourinary tract, liver, bone, lung, and age release their oxygen to the surrounding hypoxic tis- spleen. The chronic organ damage is accompanied by organ sue, deoxygenate, polymerize, and sickle, increasing the dysfunction. Although splenomegaly is present in early plug’s size. Erythrocytes from nearby capillaries are also childhood, repeated splenic infarctions eventually result in forced to feed the oxygen-deprived tissue around the block- splenic fibrosis and calcifications (usually by age 4 or 5). age. These cells then form additional aggregates of rigid This organ damage, secondary to infarction, is known Hemoglobinopathies: Qualitative Defects 275 as autosplenectomy. As a result, splenomegaly is rare in ACUTE SPLENIC SEQUESTRATION adults with this disease. Aseptic necrosis of the head of the In young children, sudden splenic pooling of sickled femur is common. Dactylitis, a painful symmetrical swell- erythrocytes (sequestration crisis) can cause a massive ing of the hands and feet (hand-foot syndrome) caused by decrease in erythrocyte mass within a few hours. Throm- infarction of the metacarpals and metatarsals, is often the bocytopenia can also occur. Hypovolemia and shock fol- first sign of the disease in infants. Recurrent priapism is a low. At one time, splenic sequestration was the leading characteristic, painful complication in which males expe- cause of death in infants with sickle cell anemia. Early rience an unwanted, persistent erection of the penis that diagnosis, instruction of parents in detecting an enlarg- occasionally requires surgical intervention. ing spleen, and rapid intervention with transfusion have The slow flow of blood in occlusive areas can lead to decreased morbidity and mortality associated with splenic thrombosis. Thrombosis of the cerebral arteries resulting sequestration. in stroke is common. Magnetic resonance imaging shows ACUTE CHEST SYNDROME evidence of subclinical cerebral infarction in 20–30% of This illness resembling pneumonia is the most common children with sickle cell anemia. If the arterioles of the cause of death in children with sickle cell disease and the eye are affected, blindness can occur. Chronic leg ulcers, second most common cause of hospitalization.22 Clinical found also in other hemolytic anemias, can occur at any findings include cough, fever, chest pain, dyspnea, chills, age. The ulcers appear without any known injury. These wheezing, and pulmonary infiltrates. Hemoglobin concen- painful sores do not readily respond to treatment and can tration and oxygen saturation decrease.23 The etiology of take months to heal. acute chest syndrome is varied and complex. In children, Placental infarctions in pregnant women with sickle an infectious agent can often be identified. Other possible cell disease can be a hazard to the fetus. Maternal anemia causes include pulmonary edema from overhydration, fat often becomes more severe during pregnancy. In addition, embolism from infarcted bone marrow, and hypoventilation other clinical findings can be exacerbated during preg- due to pain from rib infarcts or from excessive use of nar- nancy, endangering the life of both the mother and the cotic analgesics (e.g., opiate intoxication) to combat pain.24 fetus. The long-term effects of recurrent episodes of acute chest BACTERIAL INFECTION syndrome are unclear. Overwhelming bacterial infection is a common cause of IRON OVERLOAD death in young patients, and fever is treated as a medical Excessive iron storage (hemosiderosis) is observed as a emergency. The risk of septicemia from encapsulated micro- complication for some patients due to the use of transfu- organisms, such as Streptococcus pneumoniae and Haemophi- sion therapy, erythropoiesis-induced increase in iron lus influenzae, is extremely high in children with SCA who absorption, and an overall increase in longevity for have not received vaccines against these organisms. Bacte- patients with SCA. Hepatic fibrosis and cirrhosis related rial pneumonia is the most common infection, but meningi- to hemosiderosis can occur. The serum ferritin assay is tis is also prevalent. The reasons for this increased useful for monitoring storage iron levels in patients with susceptibility to infection are not fully understood but could SCA and can indicate the need for iron chelation be related to functional asplenia, impaired opsonization, therapy. defective neutrophil response, and abnormal complement activation.20 The spleen is particularly important for host defense in the young. Prophylactic penicillin should be given to children with sickle cell anemia before 2 months of age to reduce morbidity and mortality from invasive pneu- CASE STUDY (continued from page 271) mococcal disease.21 The pneumococcal, meningococcal, and The chest radiograph showed consolidation in H. influenzae type b vaccines protect patients from infections the left lower lobe, indicating that Shane has with S. pneumoniae, Neisseria meningitidis, and H. influenzae, pneumonia. reducing the incidence of infection. Children with sickle cell 5. What physiological conditions does Shane have anemia are also routinely immunized for hepatitis B and that could lead to sickling of his erythrocytes? seasonal influenza. 6. What is the cause of Shane’s pain and acute distress? 7. Why might Shane be more susceptible to pneu- Checkpoint 13.4 monia than an individual without sickle cell Why do newborns with sickle cell anemia not experience epi- disease? sodes of vaso-occlusive crisis? 276 Chapter 13 Laboratory Evaluation CASE STUDY (continued from page 275) PERIPHERAL BLOOD Admission CBC laboratory data on Shane A normocytic, normochromic anemia is characteristic of included: sickle cell anemia; however, with marked reticulocytosis, the anemia can appear macrocytic (Figure 13-3). Reticulo- WBC 16.4 * 103/mcL cytosis from 10 to 20% is typical. The hemoglobin ranges RBC 2.5 * 106/mcL from 6 to 10 g/dL (60–100 g/L) and the hematocrit from Hb 7.8 g/dL 18 to 30% (0.18–0.30 L/L). A calculated hematocrit from an Hct 24% electronic cell counter is more reliable than a centrifuged microhematocrit because excessive plasma trapped by PLT 467 * 106/mcL sickled cells in centrifuged specimens falsely elevates the Differential: Segs 76%, bands 10%, lymphs manual hematocrit. 9%, monos 3%, eos 1%, basos 1%; RBC morphol- The Cooperative Study of Sickle Cell Disease revealed ogy: Sickle cells 3+ , target cells 1+ , ovalocytes 1+ , that individuals homozygous for HbS have higher steady- polychromasia, 3 NRBC/100 WBCs, Howell-Jolly state leukocyte counts than do normal individuals, espe- bodies cially children less than 10 years of age.25 Platelet counts 8. Which of Shane’s hematologic test results are are also frequently higher than normal. After the age of 40, consistent with a diagnosis of sickle cell anemia? the hemoglobin concentration, reticulocyte count, leukocyte count, and platelet count decrease.26 9. What does the presence of polychromatophilic The blood smear shows variable anisocytosis with erythrocytes signify? polychromatophilic macrocytes and variable poikilocy- 10. Why is the absolute neutrophil count elevated? tosis with the presence of sickled cells and target cells. 11. What is the significance of ovalocytes on the Nucleated erythrocytes can usually be found. The RDW blood smear? is increased. During and following a hemolytic crisis, the RDW increases linearly with increases in reticulocytes.27 12. What is the significance of Howell-Jolly bodies If the patient is not experiencing a crisis, sickled cells may on the smear? not be present. In older children and adults, signs of splenic hypofunc- tion are apparent on the peripheral blood smear with the presence of basophilic stippling, Howell-Jolly bodies, sid- erocytes, and poikilocytes. times normal. If the patient is deficient in folic acid, mega- loblastosis can be seen. Iron stores are most often increased BONE MARROW but can be diminished if hematuria is excessive. Bone mar- Bone marrow aspiration shows erythroid hyperplasia, row examination is not usually performed because it
yields reflecting the attempt of the bone marrow to compensate for no definitive diagnostic information. chronic hemolysis. Erythrocyte production increases to 4–5 HEMOGLOBIN ELECTROPHORESIS The presence of HbS is often confirmed by hemoglobin electrophoresis, although other methods for its detection can be used. Electrophoresis on cellulose acetate (pH 8.4) and alkaline agarose shows 80–95% HbS (Table 13-3). HbF ranges from 5–20%. High levels of HbF (25–35%) can indicate compound heterozygosity for HbS and heredi- tary persistence of fetal hemoglobin (Chapter 14). HbA2 is normal. Newborns with SCA have 60–80% HbF with the remainder HbS. In infants less than 3 months of age with small amounts of HbS, electrophoresis on citrate agar gel (pH 6.2) or acid agarose gel permits more reli- able separation of HbF from both HbA and HbS. Citrate agar gel and acid agarose electrophoresis is also useful in separating HbD and HbG from HbS. These nonsickling Figure 13.3 Hemoglobin S disease (SCA). Note abnormal hemoglobins migrate with HbS on alkaline electropho- boat-shaped, sickled, and ovoid erythrocytes (peripheral blood; resis but migrate with HbA on agar gel electrophoresis Wright-Giemsa stain; 1000* magnification). at acid pH. Hemoglobinopathies: Qualitative Defects 277 Table 13.3 Hemoglobin Electrophoresis Results in Clinical Conditions Hemoglobin (%) Condition Beta Chains Present A A2 F S C Normal adult bA 97 3 Less than 1 0 0 Normal neonate bA 20–25 Less than 1 75–80 0 0 Sickle cell anemia (Hb bS 0 N 5–20 80–95 0 SS) Sickle cell trait (Hb SA) bA, bS 50–65 N N 35–45 0 Hemoglobin C disease bC 0 N Less than 7 0 More than 90 Hemoglobin C trait bA, bC 60–70 N N 0 30–40 Hemoglobin S/C bS, bC 0 ? ? More than 50 Less than 50 disease SOLUBILITY TEST The solubility test is a rapid test for detecting HbS in the CASE STUDY (continued from page 276) heterozygous or homozygous state. In severe anemia, the The LD level was reported as 1260 U/L (reference amount of HbS can be too low to be accurately detected, and interval 75–200 U/L). an altered procedure using increased blood volume may be required. This test should not be used as a screening test for 13. What is the significance of Shane’s elevated LD? infants less than 3 months because the high HbF level and low HbS concentration in this age group may cause false- negative results. Unstable hemoglobins can give a false positive test if many Heinz bodies are present. Other rare Therapy hemoglobin variants (e.g., HbC Harlem, HbH) can also give Disease management and treatment for patients with SCA positive tests. False positive tests can occur with elevated is extensive and multifaceted and frequent monitoring of plasma proteins and lipids.28 patient status using laboratory assays is needed. Treatment SICKLING TEST includes both immediate and prophylactic or long-term Another confirmatory test that is performed less often is the approaches. Immediate transfusion therapy may be ordered sodium metabisulfite slide test for sickling. This test is posi- for complications of SCA, including stroke, vaso-occlusion tive in both sickle cell anemia and sickle cell trait. in other organs, splenic sequestration, acute chest syndrome, aplastic crises, and others. Transfusion with units from OTHER DIAGNOSTIC TESTS normal donors dilutes the amount of HbS present, restores IEF in agar gels, CE, and HPLC can be used to identify oxygenation of tissues, and temporarily suppresses eryth- abnormal hemoglobins such as HbS. IEF allows separa- ropoiesis of new cells containing HbS. Simple transfusion or tion of HbS from HbD and HbG.9 Preferred methods for exchange transfusion may be used, often with units that are prenatal screening and diagnosis use DNA-based anal- screened for HbS and fully (or partially) antigen matched.29 ysis (polymerase chain reaction) to detect point muta- Preoperative transfusion is helpful in preventing the com- tions in globin gene sequences. DNA testing provides plications of anesthesia-induced sickling. Long-term trans- a genotype diagnosis and eliminates the need for later fusion therapy can be useful in preventing complications neonatal testing when the phenotype results can be of sickle cell anemia but is somewhat controversial due to inconclusive. The molecular techniques are discussed in potential side effects and lack of definitive treatment guide- Chapter 42. lines. Transfusion therapy is useful in prevention of stroke OTHER LABORATORY FINDINGS and other complications in children with SCA.30 Complica- Other laboratory findings are less specific. The hemolytic tions of chronic transfusion therapy include transmission nature of the disease causes indirect bilirubin to increase, of blood-borne diseases, hyperviscosity, alloimmunization, haptoglobin to decrease, and uric acid and serum lactic expense, inconvenience, and iron overload. dehydrogenase (LD) to increase. Serum ferritin is typically Various pharmacologic agents have been used to reduce increased. These tests offer no diagnostic information on intracellular sickling by increasing the level of HbF. Of these, sickle cell anemia but may be performed to evaluate com- only hydroxyurea (HU) has FDA approval for preventing plicating conditions. vaso-occlusive crises in patients with sickle cell anemia. 278 Chapter 13 HU is widely used and elevates HbF in most HbS-containing Sickle Cell Trait erythrocytes by activating genes controlling g@globin chain production. HU functions by targeting multiple components Sickle cell trait (SCT) is a heterozygous state and typically the of SCD pathophysiology, including increasing NO, reducing patient has one normal b@gene and one bS-gene (sometimes red cell adhesion to endothelium, increasing red cell hydra- abbreviated as SA). The hemoglobin tetramers formed consist of abA/abA tion, decreasing neutrophil counts/activation, and lowering , abS/abS, and abS/abA. Sickle cell trait is not as platelet levels.19 Children and adults respond well to HU, severe a disorder as sickle cell anemia because the pre/sence experiencing fewer vaso-occlusive crises and hospitaliza- of HbA or other non-S hemoglobins interferes with the process tions. Because HU is an antineoplastic and antimetabolite of HbS polymerization, preventing sickling under most phys- drug with significant side effects of neutropenia, reticulo- iologic conditions. However, sickle cell trait cells can sickle cytopenia, and thrombocytopenia, monitoring cell counts under very low oxygen tension (∼15 mmHg). Although some biweekly is recommended. HU is also a potential teratogen, hemoglobins other than HbS (especially HbC, HbD, HbE) so contraceptive precautions are advised. HU does not seem can be incorporated into the polymer of deoxyhemoglobin S, to produce serious irreversible toxicity and is not associated the presence of molecules of these hemoglobins intermixed with long-term adverse effects on growth or development with the molecules of HbS creates a weakened structure and in children.31 No evidence of long-term risk of malignancy is decreases the degree of polymerization. Because HbA consti- known. Other pharmacologic agents under investigation for tutes more than 50% of the total hemoglobin, sickle cell trait treating SCA include anti-adhesive agents, anti-inflammatory rarely results in clinical symptoms and physical examinations agents, anti-sickling agents, anticoagulants, antiplatelet are normal. However, it is important to diagnose sickle cell agents, and drugs that increase NO levels.32,33 trait because, statistically, one of four children born to parents Additional aspects of treatment and patient manage- each of whom has the trait will have sickle cell anemia and ment of SCA include prophylactic penicillin, folic acid, two of four will have the trait. immunizations, hydration, and analgesics for pain. Complications of splenic infarction and renal papillary Hematopoietic stem cell transplantation affords the necrosis have occasionally been reported in affected indi- potential for the cure of sickle cell disease. Results of mul- viduals subjected to extreme and prolonged hypoxia such ticenter case series show disease-free survival rates rang- as flying at high altitude in unpressurized aircraft or follow- ing from 80–85% with the best outcomes obtained when ing general anesthesia. Additional adverse outcomes associ- children received stem cells from HLA-identical siblings. ated with SCT include chronic kidney disease, hematuria, Risk of complications, including graft versus host disease impaired ability to concentrate urine, urinary tract infections, and neurological problems, is high for patients with sickle and enhanced risk for venous thrombosis. Exertional rhab- cell disease undergoing stem cell transplantation, especially domyolysis may result in sudden death in military personnel when nonmyeloablative approaches or cells from unrelated and athletes. A rare, but serious association with renal med- ullary carcinoma exists almost exclusively in SCT patients.37 donors are used.34 Candidates for stem cell transplantation include children with severe complications of SCA who Hematologic parameters, including hemoglobin con- have matched sibling donors. Furthermore, stem cell trans- centration, are normal. No sickled cells or other abnormali- plantation is extremely expensive and can be beyond the ties are observed on smears. Sickling can be induced with financial means of many patients. the sodium metabisulfite test, however, and the solubility Gene therapy, in which a patient’s defective stem cells test is also positive. Hemoglobin electrophoresis results are genetically manipulated and returned to the patient, is show 50–65% HbA, 35–45% HbS, normal HbF, and normal in the clinical trial stage. Gene addition can be accomplished or slightly increased HbA2. If HbA constitutes less than by transduction of hematopoietic stem cells (HSCs) with 50% of the total hemoglobin in sickle cell trait, the patient viral vector carrying human b@globin, g@globin, or hybrid is probably heterozygous for another hemoglobinopathy b/g@globin. Correction of the b@globin gene in HSCs via such as thalassemia. repair of the bS mutation to bA and induction of g globin (Hb F) expression through the use of clustered regularly Other Sickling Disorders interspaced short palindromic repeats (CRISPR) is Sickle cell disease/sickle cell disorder (SCD) are terms used to approaching clinical trials.35,36 describe clinical conditions in which erythrocyte sickling occurs due to the presence of HbS. Compound heterozy- gous conditions (genotypes with two different hemoglobin mutations, heterozygous for each) may be considered SCD Checkpoint 13.5 with variable clinical consequences. Some but not all the Outline the treatment options for a patient with HbS disease, pneumonia, and vaso-occlusive crisis. Discuss how each would clinical problems typical of sickle cell anemia apply to other affect the patient’s clinical condition. SCD. Hydroxyurea and transfusion therapies could be needed, depending on disease severity. The combination of Hemoglobinopathies: Qualitative Defects 279 bS with some forms of b thalassemia can be quite severe, requiring patient management and therapies comparable to those of sickle cell anemia. Hemoglobin electrophoresis and other methods including IEF and HPLC are needed to con- firm the diagnosis of a combined disorder involving HbS. See “Hemoglobin S/C Disease” and Chapter 14 for addi- tional information on combined disorders. Checkpoint 13.6 A child’s parents both have sickle cell trait. The physician orders a hemoglobin electrophoresis on the child. Results of electro- phoresis on cellulose acetate at pH 8.4 show 65% HbS, 30% Figure 13.4 Hemoglobin C disease. Note the cell in the top HbA, 3% HbF, 2% HbA2. Explain these results, and suggest center (arrow) with an HbC crystal and target cells (peripheral blood; further testing that could help in diagnosis. Wright-Giemsa stain; 1000* magnification). Hemoglobin C Disease On cellulose acetate (pH 8.4) and alkaline agarose gel, HbC migrates with HbA2, HbE, and HbO-Arab. HbC can be Hemoglobin C, the second hemoglobinopathy to be rec- separated from these other hemoglobins by agar gel electro- ognized, is the third most prevalent hemoglobin variant phoresis at an acid pH (Figure 13-1). For individuals with worldwide.12 The first cases of HbC were discovered in the HbC disease, electrophoresis on citrate agar gel (pH 6.2) and combined heterozygous state with HbS; this is not surpris- acid agarose gel demonstrates more than 90% HbC with a ing because both hemoglobinopathies are prevalent in the slight increase in HbF (not more than 7%). same geographic area. Hemoglobin C is found predomi- Hemoglobin C trait (sometimes referred to as CA) is nantly in West African blacks in whom the incidence of the asymptomatic. No hematologic abnormalities are produced trait can reach 17–28% of the population. From 2–3% of Afri- except that target cells are noted on blood smears. Mild can Americans carry the trait, and 0.02% have the disease. hypochromia can be present. About 60–70% of the hemo- Hemoglobin C is produced when lysine is substi- globin is HbA (abA/abA) and 30–40% is HbC or Hb CA tuted for glutamic acid at the sixth position (A3) in the (abC/abC or abC/abA). b@chain 3b6 (A3) Glu S Lys4 . The mutation in the HBB gene is in the same position as the HbS mutation. Because the substitution is a nonpolar amino acid for a polar amino acid as with HbS, hemoglobin solubility
decreases. Hemoglobin S/C Disease Intraerythrocytic crystals of oxygenated HbC can be found In hemoglobin S/C disease, both b@chains are structur- in the red cells, especially in splenectomized individuals. ally abnormal. One b@gene codes for bS@chains, and the HbF inhibits the formation of crystals. Crystal formation other gene codes for bC@chains. Tetramers composed of is enhanced when cells are dehydrated or in hypertonic abC/abC, abS/abS, and abS/abC are formed; thus, HbA solutions. Erythrocytes with crystals become rigid and is absent. The combined heterozygous state for HbS and are trapped and destroyed in the spleen, thereby reducing HbC results in a disease less severe then homozygous HbS erythrocyte life span to 30–55 days. but more severe than homozygous HbC. Hb S/C disease is Hemoglobin C disease is the result of abC/abC hemo- considered a sickling disorder. The concentration of hemo- globin tetramers. It is usually asymptomatic, but patients globin in individual erythrocytes (MCHC) is increased, occasionally experience joint and abdominal pain. In con- and the concentration of HbS is more than in sickle cell trast to sickle cell anemia, the spleen is most often enlarged. trait. The percentage of HbS is higher than that of HbC Variable hemolysis results in a mild to moderate anemia. because the bC@globin has a less negative charge than the The hemoglobin ranges from 8–10 g/dL (80–120 g/L) bS@globin; thus, the cell does not form abC@dimers as readily and the hematocrit from 25–35% (0.25–0.35 L/L). Anemia as abS@dimers. The presence of HbC makes the HbS/C cells is accompanied by a slight to moderate increase in reticu- more prone to sickling than cells that contain HbA/HbS locytes. The stained blood smear contains small cells that because HbC molecules participate in the polymerization appear to be folded and irregularly contracted as well as process with HbS molecules more easily than do HbA mol- many target cells (Figure 13-4). Intracellular hemoglobin ecules. Increased erythrocyte rigidity is noted at oxygen crystals can be found if the smear has been dried slowly. tensions less than 50 mm Hg. In addition, cells contain- Microspherocytes are occasionally present. ing HbS/C have formation of aggregates of intracellular 280 Chapter 13 HbC crystals.38 Thus, in HbS/C disease, both sickling and crystal formation contribute to the pathophysiology of the Hemoglobin D disease. Hemoglobin D (HbD) is the result of several molecular The clinical signs and symptoms of the disease are like variants of which the identical variants HbD Punjab and those of mild sickle cell anemia, and treatment can be indi- HbD Los Angeles (b1213GH44 Glu S Gln) are the most cated. Because of the poorly deformable red cells, patients common. HbD migrates with HbS on hemoglobin elec- can develop vaso-occlusive crises leading to the complica- trophoresis at alkaline pH; however, the HbD molecules tions associated with this pathology. A notable difference do not sickle and have normal solubility properties. HbD from sickle cell anemia, however, is that in HbS/C disease, variants are found in various ethnic groups including splenomegaly is prominent. Indians and African Americans. An uncommon a@chain Mild to moderate normocytic, normochromic anemia is variant, HbG Philadelphia, is also included in this group present. The hematocrit is usually more than 25% (0.25 due to its similar electrophoretic properties and must be L/L), and the hemoglobin concentration is between 10 and considered when red cell indices are normal. The rarely 14 g/dL (100 and 140 g/L). The higher hemoglobin concen- observed HbD homozygous state is associated with a tration does not necessarily mean that hemolysis is less mild hemolytic anemia and the presence of target cells, severe than in sickle cell anemia; it could be that the higher but the heterozygous state is asymptomatic. In homozy- oxygen affinity of HbS/C cells results in higher erythropoi- gous HbD, electrophoresis on cellulose acetate (pH 8.4) or etin levels. Peripheral blood smears reveal many target cells alkaline agarose gel demonstrates about 95% HbD with (up to 85%), folded cells, and boat-shaped cells but rarely the same electrophoretic mobility as HbS. Electropho- sickled forms (Figure 13-5). Typical HbC crystals are rarely resis on citrate agar (pH 6.0) or acid agarose gel allows found. Some erythrocytes contain a single eccentrically separation of HbS and HbD. At acid pH, HbD migrates located, densely stained, round mass of hemoglobin that with HbA. Differentiation of HbD from HbS can also be makes part of the cell appear empty. These cells have been achieved by IEF due to its high resolution of migrating referred to as billiard-ball cells.38 Anisocytosis and poikilocy- hemoglobin or by HPLC’s hemoglobin-specific retention tosis range from mild to severe. Small, dense, misshapen time.9 cells, some with crystals of various shapes jutting out at Although rare, the combined heterozygous state of angles, have been referred to as HbSC poikilocytes.39 Hemo- HbD and HbS exists. HbD molecules can interact with HbS globin electrophoresis shows a higher concentration of HbS molecules, producing aggregates of deoxyhemoglobin (Fig- than HbC. HbF can be increased up to 7%. No HbA is found ure 13-6). This produces a relatively mild form of sickle cell due to the absence of normal b@chains. anemia. HbD is also found in combination with b@thalassemia, resulting in a more serious clinical condition than when HbD is combined with a normal b allele. Com- pound heterozygotes can be recognized by results of elec- trophoresis and erythrocyte indices. Figure 13.5 Hemoglobin S/C disease. Notice the elongated cells, the cell with hemoglobin contracted to one side of the cell (billiard ball cell; arrow), and boat-shaped cells. The small contracted cells are typical of those seen in hemoglobin C disease (peripheral blood; Wright-Giemsa stain; 1000* magnification). Figure 13.6 A blood film from a patient with hemoglobin Checkpoint 13.7 D/S. Homozygous HbD does not usually cause anemia, but What is the functional abnormality of HbC and HbS? Why when combined with HbS, it potentiates the aggregating of do these two abnormal hemoglobins have the same altered deoxyhemoglobin and sickling of erythrocytes, producing a mild functions? sickle cell anemia. Notice the boat-shaped cells and target cells (peripheral blood; Wright-Giemsa stain; 1000* magnification). Hemoglobinopathies: Qualitative Defects 281 electrophoresis, HbE migrates with HbA2, HbC, and HbO- Checkpoint 13.8 Arab. On agar gel at an acid pH, HbE migrates with HbA. A 13-year-old black female had a routine physical. Her CBC HbE trait is asymptomatic, and hematologic parame- was normal, but the differential revealed many target cells. ters are normal except for slight microcytosis. Hemoglobin Hemoglobin electrophoresis revealed a band that migrated like electrophoresis at alkaline pH shows about 35–45% HbE. HbS on cellulose acetate at pH 8.4. Her hemoglobin solubility test was negative. Explain the results and suggest a follow-up The remainder is HbA with normal HbA2 and HbF. test to determine a diagnosis. Combined heterozygosity for HbE and forms of thalas- semia are commonly observed in some population groups. The degree of clinical severity is variable and depends on specific genotype. See Chapter 14. Hemoglobin E Hemoglobin E is the second most prevalent hemoglobinop- athy worldwide.12 It is most often encountered in individu- Checkpoint 13.9 The red cell morphology in HbE disease and b@thalassemia are als from southeast Asia. The trait has reached frequencies similar: microcytic, hypochromic anemia with target cells. What of almost 50% in areas of Thailand. It is estimated that laboratory test(s) could differentiate these two conditions? 15–30% of immigrants from southeast Asia living in North America have HbE with the highest frequencies occurring in those from Cambodia and Laos. Although found mainly in Asians, the HbE trait can also occur in blacks. Unstable Hemoglobin Hemoglobin E is the result of a substitution of lysine for glutamic acid in the b@chain (b263B84 Glu S Lys). The Variants hemoglobin is slightly unstable when subjected to oxidant Unstable hemoglobins can result from structurally abnor- stress. Also, the nucleotide substitution creates a potential mal globin chains, and more than 140 unstable variants new splicing sequence so that some of the mRNA may be have been described. The abnormal chains contain amino improperly processed. As a result, synthesis of the abnormal acid mutations at critical internal portions of the chains, b@chain is decreased, and HbE trait and disease have some which affect the molecular stability.40 The disorders are thalassemia-like characteristics including an increased ratio characterized by denaturation and precipitation of the of a:non@a@chain synthesis and a@chain excess (Chapter 14). abnormal hemoglobin in the form of Heinz bodies, caus- The oxygen dissociation curve (Chapter 6 ) is shifted to the ing cell rigidity, membrane damage, and subsequent right, indicating that HbE has decreased oxygen affinity. erythrocyte hemolysis. Although hemoglobin denatur- Homozygous HbE is characterized by the presence ation and hemolysis can occur spontaneously, symptoms of a mild, asymptomatic, microcytic hypochromic anemia associated with acute hemolysis usually occur after drug (like thalassemia) with decreased erythrocyte survival administration, infection, or other events that change the (Figure 13-7). Target cells are prominently observed on hemoglobin molecule’s normal environment. Clinical con- smears. Electrophoresis demonstrates mostly HbE (90% ditions associated with unstable variants are known as or more) with the remainder HbA2 and HbF. On alkaline unstable hemoglobin disorders or congenital Heinz body hemo- lytic anemias. Pathophysiology Most affected individuals with unstable hemoglobins are heterozygous. Most unstable hemoglobins are inherited as autosomal-dominant disorders, but a large number arise from spontaneous mutations with no evidence of hemoglo- bin instability in parents or other family members. Unstable hemoglobin variants can result from a variety of globin chain amino acid substitutions or deletions that disrupt the stability of the globin subunit or hemoglobin tetramer, or the binding of heme to globin. Such mutations resulting in unstable hemoglobin variants include: Figure 13.7 A blood film from a patient with homozygous hemoglobin E (HbEE). Note the microcytosis (compare red cell size 1. Mutation of a globin amino acid that is involved in con- to that of the nucleus of the lymphocyte) and prominent target cells tact with the heme group or that results in a tendency to (peripheral blood; Wright-Giemsa stain; 1000* magnification). dissociate heme from the abnormal globin chains 282 Chapter 13 2. Replacement of nonpolar by polar amino acids at the to the tissues and allowing patients to function at a lower interior of the molecule resulting in a distortion of the hemoglobin concentration. When the oxygen affinity of an folding of the globin chain unstable hemoglobin is decreased, the reticulocyte count 3. Deletion or insertion of additional amino acids, par- is not increased as much as would be expected for the ticularly in the helical regions of the molecule, creating degree of anemia relative to an uncomplicated hemolytic instability anemia. Most clinical findings occur as the result of increased 4. Mutations of amino acids at intersubunit contacts erythrocyte hemolysis. Jaundice and splenomegaly are (especially the a1b1 contact points), creating instability common when there is chronic extravascular hemolysis and a tendency to dissociate into monomers as well as a high incidence of cholelithiasis. Cyanosis can 5. Replacement of a hydrophobic residue with a more result from the formation of sulfhemoglobin and methe- hydrophilic amino acid in the hydrophobic pocket, moglobin that accompanies hemoglobin denaturation. disrupting the conformation of the hydrophobic heme Weakness and jaundice frequently follow the administra- cleft tion of oxidant drugs, which increase the hemoglobin’s Unstable hemoglobin denatures and precipitates as instability. Acute hemolysis can also be accompanied by Heinz bodies, which attach to the inner surface of the mem- the excretion of dark urine due to the presence of dipyr- brane, thereby decreasing cell deformability. The inclusions roles in the urine. are pitted by macrophages in the spleen, leaving the cell with less hemoglobin and decreased membrane. This pro- cess leads to rigid cells and their premature destruction in Laboratory Evaluation the spleen. PERIPHERAL BLOOD In addition to altering the molecule’s stability, disrup- The anemia of congenital hemolytic anemia involving tion of the normal conformation also can affect the mol- unstable hemoglobin variants is usually normocytic and ecule’s function. Many of the unstable hemoglobins also normochromic. Occasionally, MCV and MCH are slightly have altered oxygen affinity. If the amino acid substitution decreased because of the removal of Heinz bodies from is strategically located to affect the oxygen-binding site, erythrocytes and loss of hemoglobin in the spleen. The oxygen affinity can be increased or decreased. In addition, reticulocyte count is typically increased. The blood smear some unstable hemoglobin variants tend to spontaneously can show basophilic stippling,
pitted cells (bite cells), and oxidize to methemoglobin. small contracted cells. If splenic function is efficient, Heinz bodies are not Clinical Presentation detectable in peripheral blood cells. Heinz bodies can be found in the peripheral blood following splenec- Congenital hemolytic anemia indicative of an unstable tomy; however, this finding is not specific for unstable hemoglobin disorder requires further investigation to hemoglobin disorders. Heinz bodies can also be seen in establish a definitive diagnosis. Family history is extremely erythrocyte enzyme abnormalities that permit oxidation important in defining the hereditary nature of the disease. of hemoglobin, in thalassemia, and after administration Patient history can also provide information about the of oxidant drugs in normal individuals. Heinz bodies nature of events such as infection or drug administration of unstable hemoglobins can be generated in vitro by that precipitate acute hemolytic episodes. The severity of incubation of the erythrocytes with brilliant cresyl blue the disorder can range from asymptomatic to a chronic or other redox agents (Chapter 37). These intracellular severe hemolytic anemia. inclusions cannot be observed on smears stained with The severity of the anemia depends on the degree of Wright’s stain. instability of the hemoglobin tetramer and the change, if any, in oxygen affinity. For example, Hb Köln is an unsta- OTHER LABORATORY FINDINGS ble hemoglobin (b@chain mutation) that also demonstrates Many unstable hemoglobins identified to date have the increased oxygen affinity. Hemoglobins with increased same charge and electrophoretic mobility as normal oxygen affinity (hemoglobin-oxygen dissociation curve hemoglobin. Only about 45% can be identified by electro- is shifted left) release less oxygen to the tissues, which phoresis. Hemoglobin A2 and HbF are sometimes results in relatively higher levels of tissue hypoxia and of increased, which could suggest the presence of an abnor- erythropoietin as well as higher hematocrits than expected mal hemoglobin when it is not detected by its electropho- relative to the severity of hemolysis. Conversely, unstable retic pattern. More definitive identification of unstable hemoglobins with decreased oxygen affinity (e.g., Hb hemoglobin variants may require HPLC analysis of glo- Hammersmith) have a hemoglobin-oxygen dissociation bin chains, or genetic testing for globin gene curve that is shifted right, increasing the oxygen delivery abnormalities. Hemoglobinopathies: Qualitative Defects 283 The high-oxygen-affinity hemoglobins bind oxygen Checkpoint 13.10 more readily than normal and retain more oxygen at lower a. Explain why patients with an unstable hemoglobin variant PO2 levels, which results in a shift to the left of the oxygen usually experience acute hemolysis only after administration dissociation curve. The P50, the PO2 at which hemoglobin of certain drugs or with infections. is 50% saturated, is decreased to 12–18 mm Hg, meaning b. A patient is suspected of having a congenital Heinz body that less oxygen is released to tissues at the tissue PO2 hemolytic anemia but hemoglobin electrophoresis is normal. of ∼26 mm Hg. The resulting tissue hypoxia stimulates Why is it necessary to perform additional tests? erythropoietin release and, subsequently, formation of a compensatory increased erythrocyte mass. In addition to the primary effect of increasing oxygen affinity, secondary Therapy effects of the mutations, including hemoglobin instability, reduced Bohr effect, and reduced cooperativity of the oxy- A wide variation in the clinical presentation of patients gen binding, can also occur.40 with unstable hemoglobin variants exists, and many do not Erythrocyte counts and hematocrit levels are increased, require therapy. Splenectomy can be performed if hemoly- and hemoglobin levels are increased to about 20 g/dL sis is severe. Patients are advised to avoid oxidizing drugs, (200 g/L). Other hematologic parameters are normal as well which can precipitate a hemolytic episode. as levels of 2,3-BPG. About half of the hemoglobin variants have an altered electrophoretic mobility, enabling diagno- Hemoglobin Variants With sis by cellulose acetate or alkaline agarose electrophore- sis. Diagnosis is established by measuring oxygen affinity Altered Oxygen Affinity (determining the P50 of the patient’s hemoglobin) as well as DNA sequencing of the globin genes, which allows for quick Amino acid substitutions in the globin chains near the heme and definite identification of the variant hemoglobin.41 pocket can affect the hemoglobin’s ability to carry oxygen Individuals with these hemoglobin variants are asymp- by preventing heme from binding to the globin chain or by tomatic. A ruddy complexion is occasionally apparent stabilizing iron in the oxidized ferric state. Other substitu- because of the erythrocytosis. A family history of erythro- tions that affect oxygen affinity include those near the a1b2 cytosis can suggest the presence of a high-oxygen-affinity contacts, at the C terminal end of the b@chain, and near the hemoglobin variant. The importance of identifying its pres- 2,3-BPG binding site. These are critical sites involved in the ence is for the differential diagnosis and exclusion of other allosteric properties of hemoglobin and/or in the physi- causes of erythrocytosis and the avoidance of unnecessary ologic regulation of hemoglobin affinity for oxygen. Either and expensive diagnostic and therapeutic interventions. the a@ or b@chain can be affected, but most identified substi- tutions are associated with the b@chain. Hemoglobin Variants with Decreased Increased oxygen affinity results in congenital erythro- Oxygen Affinity cytosis, a compensatory mechanism for the reduced release Low oxygen-affinity hemoglobins result from mutations of oxygen to the tissues. Hemoglobinopathies causing that stabilize or favor the deoxygenated (T) conformation permanent methemoglobin formation are associated with of the hemoglobin molecule or destabilize the oxygenated pseudocyanosis, whereas cyanosis characterizes hemoglo- (R) form. These mutations impair oxygen binding or reduce bins with decreased oxygen affinity. heme–heme subunit interactions (cooperativity). Low oxygen-affinity hemoglobins are often associated with Hemoglobin Variants with Increased mutations involving the a1b2 contact points, which are Oxygen Affinity involved in intramolecular movement as hemoglobin goes from the oxy (R) to the deoxy (T) state. The low-affinity High-affinity hemoglobins are inherited as autosomal dom- variants result in a right-shifted oxygen dissociation curve. inant traits. All such hemoglobins discovered have been in Most low oxygen-affinity hemoglobins possess enough the heterozygous state. The variants can result from amino oxygen affinity to become fully saturated in the lungs but acid substitutions that involve the a1b2 contacts. Mutations at the capillary PO2 in the peripheral tissues, they deliver alter the quaternary structure of hemoglobin to favor the R higher than normal amounts of oxygen (i.e., become more (oxy) state by either stabilizing this conformation or desta- unsaturated). Two physiologic effects result: bilizing the T (deoxy) state. Other substitutions affect the C-terminal end of the b@chain, which is important in main- • Because oxygen delivery is so efficient, oxygen require- taining the stability of the T form. A few substitutions affect ments of the tissues can be met by lower than normal the 2,3-BPG binding sites, which alter oxygen affinity when hemoglobin concentration, producing a pseudoanemia bound to hemoglobin. (hemoglobin lower than “normal”). 284 Chapter 13 • The amount of deoxygenated hemoglobin in the cap- methemoglobinemia appear to be cyanotic, but unlike truly illaries and veins is increased, resulting in cyanosis. cyanotic people, arterial blood PO2 levels are usually nor- However, no adverse clinical effect is associated with mal (i.e., pseudocyanosis). this cyanosis. Patients are asymptomatic and require Hemoglobin M does not always separate from HbA at no treatment. an alkaline pH. Hemoglobin electrophoresis on agar gel at pH 7.1 of a blood sample containing HbM reveals a brown Diagnosis requires determining an increase in P50 or band (HbM) running anodal to a red band (HbA). The pat- identifying hemoglobin variants by performing HPLC or tern can appear sharper with IEF. Oxidizing the erythro- DNA sequencing. cyte hemolysate with ferricyanide before electrophoresis reveals a sharp separation of congenital HbM and met- Methemoglobinemias hemoglobin formed from HbA. Methemoglobin formed Methemoglobin is hemoglobin with iron oxidized to from HbA by oxidation of iron has no change in molecular the ferric state, which cannot carry oxygen. Methemo- charge and therefore has the same electrophoretic mobility globinemia is a clinical condition that occurs when as HbA. methemoglobin encompasses more than 1% of the hemo- Although HbM can be detected by spectral abnor- globin (Chapter 6). This condition can occur because of malities of the hemoglobin (absorption peaks at ∼630 and either acquired or inherited defects in the methemoglobin 502 nm), methemoglobin formed in NADH-diaphorase reductase system or because of the presence of a structur- deficiency has a normal absorption spectrum. In addition, ally abnormal globin chain. In the latter case, the defect methemoglobin formed as the result of NADH-diaphorase results in increased formation of methemoglobin by ren- deficiency can be readily reduced by incubation of blood dering the molecule relatively resistant to reduction by with methylene blue whereas methemoglobin caused by a methemoglobin reductase. structural hemoglobin variant such as HbM does not reduce This structural variant of hemoglobin is called hemo- with the methylene blue. To confirm NADH-diaphorase globin M (HbM). Ten variants have been described; all deficiency, a quantitative assay of the enzyme activity is variants have been found only in the heterozygous state. necessary. Refer to Table 13-4 for a summary of differentia- Most HbM variants are produced by a tyrosine substitu- tion of types of methemoglobinemia (see also Chapter 6, tion for the proximal or distal histidine in the heme pocket Table 6-6). of the a@ or b@chains. Tyrosine forms a covalent link with No treatment for the HbM structural variants exists heme iron, stabilizing the iron in the ferric state. The stabi- because the abnormal hemoglobin resists reduction. How- lized, oxidized iron is relatively resistant to reduction by ever, most patients are asymptomatic and require no man- the methemoglobin reductase pathway. If the substitution agement. Most patients have methemoglobin levels below occurs in the a@chain, cyanosis (or pseudocyanosis) is pres- the level at which symptoms of oxygen deprivation occur. ent from birth because the a@chain is a component of HbF For acquired drug- and chemical-induced methemoglobin- (a2g2), the major hemoglobin at birth. If the substitution emia, reduction to oxyhemoglobin can be achieved by intra- occurs in the b@chain, cyanosis does not occur until about venous administration of methylene blue.42 the sixth month after birth when HbA (a2b2) becomes the major hemoglobin. The presence of methemoglobin imparts a brown- Checkpoint 13.11 ish color to the blood. Except for this abnormal color, no Why should red cell enzyme assays and hemoglobin electropho- other hematologic abnormality is present because methe- resis both be performed on a patient with congenital cyanosis? moglobin levels are rarely higher than 30%. Patients with Table 13.4 Causes and Characteristics of Methemoglobinemia NADH-Diaphorase Reduction with Methylene Type Cause Electrophoretic Pattern Activity Blue Acquired Hb oxidized at a rate exceeding Normal Normal Yes capacity of methemoglobin reduc- tase system Congenital Recessive Defect in methemoglobin reduc- Normal Decreased Yes tase system Dominant Structural abnormality of hemoglo- HbM variant Normal No bin (HbM) Hemoglobinopathies: Qualitative Defects 285 Summary The hemoglobinopathies are a group of chronic hemolytic deoxygenated, forming rigid aggregates and reduced anemias caused by qualitative defects in the globin chains of erythrocyte deformability. The rigid cells aggregate in the hemoglobin. Quantitative defects in globin chain synthesis microvasculature, resulting in tissue hypoxia. In HbC dis- (thalassemias) are discussed in Chapter 14. ease, the erythrocytes become trapped and destroyed in the Qualitative defects are due to genetic mutations that spleen when intracellular crystals of HbC form. Generally, cause a structural change in the globin chain. Although these variants produce a normocytic, normochromic ane- the mutation can affect any of the globin chain classes, mia with anisocytosis and poikilocytosis characteristic for most clinically significant mutations affect the b@chain. The the specific hemoglobinopathy. The blood smear in HbS mutation can affect the hemoglobin molecule’s solubility, disease can show sickled forms and target cells. HbC dis- stability, or function (oxygen affinity). Hemoglobin elec- ease has numerous target cells and irregularly contracted trophoresis can be used to detect those mutants in which cells, and the erythrocytes occasionally contain HbC crys- amino acid mutations cause a change in the hemoglobin tals. In the United States, both HbS and HbC are found molecule’s electrophoretic mobility. However, not all vari- in highest frequency among African Americans. Hemo- ants can be detected and identified by electrophoresis, globin E is especially prevalent in people from southeast and other methods such as HPLC, IEF, or CE can be used. Asia. HbE disease is characterized by a mild, asymptomatic Other tests detect alterations in hemoglobin solubility microcytic anemia. and/or stability or oxygen affinity. Molecular techniques Unstable hemoglobin variants produce congenital
Heinz are available to help definitively diagnose hemoglobinopa- body hemolytic anemia. The hemoglobin denatures in the thies but are not always necessary for routine diagnostic form of Heinz bodies, decreasing cell deformability and purposes. resulting in membrane damage and premature destruction. In the United States, the most common structural vari- Hemoglobin variants with increased or decreased oxygen ants are HbS and HbC. Both are characterized by decreased affinity can result in erythrocytosis or cyanosis, respectively. hemoglobin solubility and can be detected electropho- Hemoglobin variants resulting in methemoglobin forma- retically. Hemoglobin S has decreased solubility when tion are associated with pseudocyanosis. Review Questions Level I 4. Hemoglobin level 5. Chemistry assays including bilirubin, ferritin, and 1. A patient has been previously diagnosed as being others heterozygous for hemoglobin D. What is an important a. 1 and 5 only identifying characteristic of this hemoglobin variant? (Objective 7) b. 2 and 4 only c. 1, 3, and 4 only a. It migrates with HbS on hemoglobin electrophore- sis at alkaline pH. d. all of the above b. Sickled RBCs are typically observed on smears. 3. Which part of the standard CBC provides the most c. Hemoglobin electrophoresis at alkaline pH is useful screening information when differentiating needed for its separation from another common between a patient diagnosis of sickle cell anemia ver- variant. sus thalassemia? (Objective 6) d. It causes severe hemolytic anemia with many tar- a. hemoglobin concentration alone get cells in heterozygotes. b. hemoglobin and hematocrit together 2. Which of the following laboratory assays are useful in c. erythrocyte indices recognizing and confirming a diagnosis for patients d. total WBC count and differential with hemoglobinopathies? (Objective 6) 1. Hemoglobin electrophoresis 4. Which homozygous hemoglobinopathy is most con- sistent with the following laboratory test results? 2. RBC indices (Objective 7) 3. RBC morphology 286 Chapter 13 Hemoglobin level mildly decreased, MCV 9. The presence of many sickled erythrocytes on a decreased, target cells and hypochromia observed peripheral blood smear is most likely to be found in a on smear, a large band in the A2 region on alkaline person who is: (Objective 7) electrophoresis a. heterozygous for HbC a. hemoglobin C b. homozygous for HbC b. hemoglobin D c. heterozygous for HbS c. hemoglobin E d. homozygous for HbS d. hemoglobin S 10. A hemoglobin electrophoresis showed 49% HbS, 42% 5. Which of the following is a clinical condition whose HbC, 6% HbF, and 3% HbA2. These results are consis- associated laboratory findings are seen in children tent with a diagnosis of: (Objective 7) with sickle cell anemia? (Objectives 7, 8) a. sickle cell anemia a. vaso-occlusive crisis denoted by the presence of b. sickle cell trait many sickled RBCs on smears c. hemoglobin S/C disease b. erythrocyte aplasia denoted by a rapid increase in d. hemoglobin C trait hemoglobin c. bacterial infection denoted by the appearance of Level II Howell-Jolly bodies on smears d. thrombosis denoted by an increase in WBCs and 1. In which U.S. population group would you expect to abnormal differential observe the lowest prevalence of clinically significant hemoglobinopathies? (Objective 2) 6. Hemoglobinopathies are clinical diseases that result from genetically determined abnormalities of the a. African Americans hemoglobin molecule and include those involving: b. Caucasian Americans (Objective 1) c. Immigrants from southeast Asia 1. heme structure d. Immigrants from the Mediterranean basin 2. decreased heme synthesis 2. An African American 2-year-old is seen in an outpa- 3. globin chain structure tient clinic with signs and symptoms consistent with a 4. reduced globin chain synthesis severe hemoglobinopathy. Based on this presentation, a. 1 and 3 only this child is most likely homozygous for which hemo- globin variant? (Objective 5) b. 1 and 2 only c. 3 and 4 only a. HbC d. all of the above b. HbD c. HbE 7. Amino acid substitutions on globin chains often alter d. HbS the hemoglobin molecule’s charge and mobility. This is the principle of which test for identifying hemoglo- 3. A patient with a long history of anemia is referred to a bins? (Objective 3) hematologist for diagnosis. This patient recently was a. HbA2 quantitation treated with a drug for an unrelated condition, but the therapy was discontinued following an acute episode of b. HbF quantitation severe anemia. Other family members also appear to suf- c. hemoglobin electrophoresis fer from a similar condition. The hematologist suspects d. solubility test the patient has an unstable hemoglobin variant and asks for test suggestions. What laboratory results would best 8. Tests that quantitate HbF and HbA2 are useful in support the preliminary diagnosis? (Objective 10) detecting hemoglobinopathies because: (Objective 6) a. Heinz bodies observed upon staining, increased a. they are the only valid tests available for this reticulocytes, bite cells on peripheral blood smear purpose b. decreased reticulocytes, decreased indices, target b. they are routinely performed in most laboratories cells on smears c. HbF and HbA2 are typically decreased in c. Howell-Jolly bodies and sickle cells observed on hemoglobinopathies smears d. HbF and HbA2 are typically increased in d. increased WBC concentration with abnormal hemoglobinopathies differential Hemoglobinopathies: Qualitative Defects 287 4. What is an advanced testing method suitable for 7. HbS is an example of a hemoglobin with a globin detecting and identifying hemoglobin variants that chain mutation that alters hemoglobin: (Objective 3) you might select for your high-volume laboratory in a large, urban medical center? (Objective 9) a. function b. solubility a. hemoglobin electrophoresis performed at alkaline pH only c. stability b. hemoglobin solubility test d. oxygen binding c. high-performance liquid chromatography 8. Cells containing large amounts of HbS sickle when d. hemoglobin electrophoresis performed at acid pH which of the following conditions occur? (Objective 4) only a. high oxygen tension and acidosis 5. An asymptomatic patient presents with persistent b. hypoxia and alkalosis erythrocytosis. The hematologist has ruled out poly- c. temperatures less than 37 °C and alkalosis cythemia vera and other causes. What is an additional d. temperatures more than 37 °C and hypoxia explanation for the erythrocytosis, and what labora- tory assay might be useful in this case? (Objective 11) 9. A hemoglobin electrophoresis on cellulose acetate at a. an unstable hemoglobin variant identified by per- pH 8.4 is performed on a 2-year-old African Ameri- forming a Heinz body stain can patient with severe anemia. An abnormal band b. a high-affinity hemoglobin variant identified by appears halfway between the HbA and HbA2 posi- isoelectric focusing tions on the strip. The HbF level is 10%, and the HbA2 level is normal. No HbA is present. What is the most c. a crystalizable hemoglobin variant demonstrated by observing hemoglobin C crystals on a smear likely identity of the abnormal hemoglobin? (Objective 7) d. a quantitative decrease in globin chain production indicated by observing an increased level of HbF a. HbC b. HbS 6. Rank the following hemoglobinopathies based on c. HbD quantity of HbS starting with the lowest amount. (Objective 1) d. HbE 1. adult with sickle cell disease 10. The presence of HbE disease in an adult is best 2. adult with sickle cell trait confirmed using which routine laboratory test? 3. neonate with sickle cell disease (Objective 8) a. 3, 2, 1 a. hemoglobin electrophoresis b. 2, 3, 1 b. CBC and peripheral blood smear c. 1, 2, 3 c. solubility test d. 2, 1, 3 d. PCR for molecular defect References 1. Greene D, Vaughn C, Crews B, et al. (2015). Advances in detec- carrier identification and prenatal diagnosis of the haemoglo- tion of hemoglobinopathies. Clinica Chimica Acta, 439, 50–57. binopathies. Eur J Hum Genet. 2015;23(4):426–437. doi:10.1038/ doi:10.1016/j.cca.2014.10.006 ejhg.2014.131 2. Modell B, Darlison M. Global epidemiology of haemoglobin dis- 7. Abou-Diwan C, Young AN, Molinaro RJ. Hemoglobinopathies orders and derived service indicators. Bull World Health Organ. and clinical laboratory testing. Med Lab Obs. 2009 41:8–18. 2008;86:480–487. 8. Houtovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as 3. Herrick JB. Peculiar elongated and sickle-shaped red blood cor- a diagnostic tool for hemoglobin variants and hemoglobinopa- puscles in a case of severe anemia. Trans Assoc Am Physicians. thies: a study of 60,000 samples in a clinical diagnostics labora- 1910;25:553–61. tory. Clin Chem. 2004:50:1736–47. 4. Pauling L, Itano HA, Singer SJ, et al. Sickle cell anemia: A molec- 9. U.S. Preventive Services Task Force. Screening for sickle cell ular disease. Science. 1949;110:543–48. disease in newborns: U.S. Preventive Services Task Force recom- 5. Huisman TH, Carver MF, Baysal E, et al. A database of mendation statement. Rockville (MD): Agency for Healthcare human hemoglobin variants and thalassemias. http:// globin. Research and Quality (AHRQ); 2007 (available at www.guideline. cse.psu.edu/globin/hbvar/menu.html (accessed March gov; accessed March 14, 2014). 2, 2017). 10. Association of Public Health Laboratories. Hemoglobinopathies: 6. Traeger-Synodinos J, Harteveld C, Old J, et al. EMQN best Current practices for screening, confirmation and follow-up. practice guidelines for molecular and haematology m ethods for SilverSpring (MD): Centers for Disease Control and Prevention 288 Chapter 13 (CDC); 2015 (available at www.cdc.gov/ncbddd/sicklecell/ 26. Morris J, Dunn D, Beckford M, et al. The haematology of homo- documents/nbs_hemoglobinopathy-testing_122015.pdf; accessed zygous sickle cell disease after the age of 40 years. Br J Haematol. April 4, 2017) 1991;77:382–85. 11. Breveglieri G, Finotti A, Borgatti M, et al. Recent patents 27. el Sayed HL, Tawfik ZM. Red cell profile in normal and sickle cell and technology transfer for molecular diagnosis of beta- diseased children. J Egypt Soc Parasitol. 1994;24:147–54. thalassemia and other hemoglobinopathies. Expert Opin 28. SAS Sickle Cell Test [package insert]. San Antonio, Texas: SA Sci- Ther Pat. 2015;25(12):1453–1476 doi:10.1517/13543776.2015. entific, Inc.; 2001. 1090427 29. Quirolo K. How do I transfuse patients with sickle cell disease? 12. Flint J, Harding RM, Boyce AJ, et al. The population g enetics Transfusion. 2010;50:1881–86. of the haemoglobinopathies. Baillieres Clin Haematol. 30. Miller ST, Wright E, Abboud M. Impact of chronic transfu- 1998;11:1–51. sion on incidence of pain and acute chest syndrome during the 13. Bunyaratvej A, Butthep P, Sae-Ung N, et al. Reduced Stroke Prevention Trial (STOP) in sickle-cell anemia. J Pediatr. deformability of thalassemic erythrocytes and erythrocytes 2001;139:785–89. with abnormal hemoglobins and relation with susceptibility to 31. Trompeter S, Roberts I. Haemoglobin F modulation in childhood Plasmodium falciparum invasion. Blood. 1992;79:2460–63. sickle cell disease. Br J Haematology. 2008;144:308–16. 14. Liu SC, Derick LH, Palek J. Dependence of the permanent 32. Ataga KI, Stocker J. The trials and hopes for drug development in sickle cell disease. Br J Haematol. 2015: 170: 768–780. deformation of red blood cell membranes on spectrin dimer-tetramer equilibrium: implication for permanent 33. Telen MJ. Beyond hydroxyurea: new and old drugs in the pipe- line for sickle cell disease. Blood. 2016;127:810–819. membrane deformation of irreversibly sickled cells. Blood. 1993;81:522–28. 34. Thompson AA. Advances in the management of sickle cell dis- ease. Pediatr Blood Cancer. 2005;46:533–39. 15. Ballas SK, Smith ED. Red blood cell changes during the evolution 35. Walters MC. Stem cell therapy for sickle cells disease: trans- of the sickle cell painful crisis. Blood. 1992;79:2154–63. plantation and gene therapy. Hematology 2005. Washington DC: 16. Mackie LH, Hochmuth RM. The influence of oxygen tension, American Society for Hematology. 2005:66–73 temperature, and hemoglobin concentration on the rheologic 36. Hoban MD, Orkin, SH, Bauer DE. Genetic treatment of a molecu- properties of sickle erythrocytes. Blood. 1990;76:1256–61. lar disorder: gene therapy approaches to sickle cell disease. Blood. 17. Steinberg MH. Pathophysiology of sickle cell disease. Baillieres 2016;127:839–848. Clin Haematol. 1998;11:163–84. 37. Key NS, Connes, P, Derebail, VK. Negative implications of sickle 18. Liu SC, Yi SJ, Mehta JR, et al. Red cell membrane remodeling in cell trait in high income countries: from the football field to the sickle cell anemia. J Clin Invest. 1996;97:29–36. laboratory. Br J Haematol. 2015;170(1):5–14. 19. Zhang D, Xu C, Manwani, D, Frenette PS. Neutrophils, platelets, 38. Lawrence C, Fabry ME, Nagel RL. The unique red cell heteroge- and inflammatory pathways at the nexus of sickle cell disease neity of SC disease: crystal formation, dense reticulocytes, and pathophysiology. Blood. 2016;127:801–809. unusual morphology. Blood. 1991;78:2104–12. 20. Boghossian SH, Nash G, Dormandy J, et al. Abnormal neutrophil 39. Bain BJ. Blood film features of sickle cell–haemoglobin C disease. adhesion in sickle cell anaemia and crisis: relationship to blood Br J Haematol. 1993;83:516–18. rheology. Br J Haematol. 1991;78:437–41. 40. Steinberg MH. Hemoglobins with altered oxygen affinity, unsta- 21. Meier, ER, Miller, JL. Sickle Cell Disease in Children. Drugs. 2012; ble hemoglobins, M-hemoglobins,
and dyshemoglobinemias. 72:895–906. In: Greer JP, Foester J, Rodgers GM, et al., eds. Wintrobe’s Clinical 22. Ballas SK. Sickle cell disease: Clinical management. Baillieres Clin Hematology, 13th ed. Philadelphia; Wolters Kluwer Health/ Lippincott Williams & Wilkins; 2014. Haematol. 1998;11:185–14. 41. Orvain C, Joly P, Pissard S, et al. Diagnostic approach to hemo- 23. Quinn CT, Buchanan GR. The acute chest syndrome of sickle cell globins with high oxygen affinity: Experience from France disease. J Pediatr. 1999;135:416–22. and Belgium and review of the literature. Ann Biol Clin (Paris). 24. Paul RN, Castro O, Aggarwal A, Oneal PA. Acute chest 2017;75(1):39–51 syndrome: sickle cell disease. Eur J Haematol. 87: 191–207. 42. Khan K, Kuppaswamy B. Cetacaine induced methemoglobin- 25. West MS, Wethers D, Smith J, et al. Laboratory profile of sickle emia: Overview of analysis and treatment strategies. W V Med J. cell disease: a cross-sectional analysis. The cooperative study of 2013;109(3):24–26. sickle cell disease. J Clin Epidemiol. 1992;45:893–909. Chapter 14 Thalassemia Tim R. Randolph, PhD Objectives—Level I At the end of this unit of study, the student should be able to: 1. Define thalassemia. c. Basic pathophysiology 2. Differentiate thalassemias from d. Symptoms hemoglobinopathies based on definition e. Laboratory results including blood and basic pathophysiology. cell morphology and hemoglobin electrophoresis 3. Describe the typical peripheral blood morphology associated with thalassemia. 6. For each of the six genotypes of b@thalassemia describe the: 4. Compare and contrast the etiology of a- and a. Individuals affected b@thalassemia. b. Basic pathophysiology 5. For each of the four genotypes of c. Symptoms a@thalassemia, describe the: d. Laboratory results including blood a. Number of affected alleles cell morphology and hemoglobin b. Individuals affected electrophoresis Objectives—Level II At the end of this unit of study, the student should be able to: 1. List and describe five primary genetic 4. For all four genotypes of a@thalassemia: defects found in thalassemias. a. Correlate all three nomenclature systems: 2. Compare and contrast a@ and b@thalassemia. genotype, genotype description, and phenotype. 3. Correlate the outcomes in hemoglobin b. Explain advanced pathophysiology. synthesis resulting from the five genetic c. Describe treatment and prognosis. defects in thalassemia. 289 290 Chapter 14 5. For all four phenotypes of b@thalassemia: c. Hemoglobin Constant Spring a. List expected genotypes. d. Hereditary persistence of fetal hemoglobin b. Explain advanced pathophysiology. (HPFH) c. Describe treatment and prognosis. e. Hemoglobin Lepore f. Thalassemia/hemoglobinopathy 6. Correlate clinical severities of both a@ and combination disorders b@thalassemia with their respective genotypes. 8. Differentiate iron deficiency-anemia and 7. Compare and contrast other thalassemia HPFH from thalassemia based on results and thalassemia-like conditions including: of laboratory tests and clinical findings. a. db@thalassemia b. gdb@thalassemia Chapter Outline Objectives—Level I and Level II 289 b@Thalassemia 301 Key Terms 290 Other Thalassemias and Thalassemia-Like Background Basics 290 Conditions 308 Case Study 291 Differential Diagnosis of Thalassemia 313 Overview 291 Summary 315 Introduction 291 Review Questions 315 a@Thalassemia 296 References 317 Key Terms Allele Functional hyposplenism Ineffective erythropoiesis Compression syndrome Gene cluster P50 value Crossover Genotype Phenotype Diploid Haplotype Zygosity Double heterozygous Heterozygous Extramedullary erythropoiesis Homozygous Background Basics The information in this chapter builds on concepts pre- • Interpret routine laboratory tests, such as CBC and sented in previous chapters. To maximize your learning differential, and apply both normal and abnormal experience, you should review the following concepts results in the diagnosis of anemias. (Chapters 10, before beginning this unit of study: 11, 37) • Describe the basic structure and function of hemo- Level I globin and identify the globin chain composition of • Describe the pathophysiology of hemoglobinopa- normal hemoglobin types. (Chapter 6) thies. (Chapter 13) • Describe the extravascular destruction of erythro- • Describe the morphologic and functional classification cytes and degradation of hemoglobin. (Chapters 5, 6) of anemias and the associated lab tests. (Chapter 11) Thalassemia 291 Level II • Interpret hemoglobin migration patterns on electro- • Summarize the synthesis and molecular structure phoresis, acid elution of fetal hemoglobins, hemo- of hemoglobin and correlate alterations in structure globin solubility tests, and iron panels to distinguish with function; describe globin chain synthesis in iron metabolism disorders and hemoglobinopathies utero and throughout life. (Chapters 3, 6) from thalassemias. (Chapters 12, 13, 37) unbalanced synthesis of a@ and non@a@globin chains and CASE STUDY ineffective erythropoiesis. Symptoms include anemia, hepa- We refer to this case throughout the chapter. tosplenomegaly, infections, gallstones, and bone deformities John is a 4-year-old boy who frequently complains of that alter facial features and result in pathologic fractures. weakness, fatigue, and dyspnea. The family moved The thalassemias are classified according to the affected to the United States from Greece before the child’s globin chain. Clinically, the most important thalassemias birth. Both parents experienced fatigue from time to involve the a@ and b@chains because they are components of time but never consulted a physician. Consider the hemoglobin A, which makes up 97% of normal adult hemo- types of anemia most often found at this age and the globin. a@Thalassemia results from decreased or absent pro- laboratory tests that could help establish a diagnosis. duction of a@globin chains, and b@thalassemia is caused by What is the significance, if any, of knowing the decreased or absent production of b@globin chains. Reduction parents’ background and medical history? in the synthesis of the d@chain, a component of hemoglobin A2, can occur but is not associated with anemia. Severe impair- ment of Z@, e@, or g@globin synthesis is usually lethal in utero. Thomas Cooley offered the first clinical description of Overview thalassemia in Detroit in 1925.2 At that time, thalassemia was thought to be a rare disorder restricted to Mediterranean eth- This chapter discusses a group of hereditary anemias col- nicities. Dr. Cooley’s work broadened our understanding of lectively called thalassemia. It begins with a general descrip- the nationalities that potentially could be affected by thalas- tion of thalassemia, including the genetic defects and types, semia and suggested that the disease was hemolytic in nature. pathophysiology, and clinical and laboratory findings. Sub- By 1960, it was apparent that the thalassemias composed a sequently, the following are discussed for each type: patho- heterogeneous group of genetic disorders. With the advent of physiology, clinical presentation, laboratory evaluation, molecular biology, many groups of researchers have widely treatment, and prognosis. Other thalassemia-like conditions studied thalassemias. Methods developed in the last 25 years are described and compared and contrasted with thalas- have enabled researchers to measure the quantity of globin semia. The chapter concludes by describing the laboratory chains synthesized and identify specific genetic mutations. differential diagnosis of types of thalassemia and other dis- Thalassemia is now recognized as one of the most com- orders that have similar peripheral blood morphology. mon genetic disorders affecting the world’s population. Approximately 1–5% of people are thought to be carriers of b@thalassemia.3 It is estimated that between 100,000 and Introduction 200,000 individuals worldwide are born each year with severe forms of thalassemia, and approximately 60,000 of those have Thalassemia constitutes a family of inherited disorders b@thalassemia.3 In North America, about 20% of immigrants in which mutations in one or more of the globin genes of from Southeast Asia and 6–11% of African Americans have hemoglobin cause decreased or absent synthesis of the detectable a@thalassemia. Many more are silent carriers. About corresponding globin chains. More than 600 unique muta- 6% of individuals of Mediterranean ancestry, 5% of Southeast tions have been described among this diverse group of Asians, and 0.8% of African Americans have b@thalassemia disorders.1 Consequences of the mutation depend on the along with people from the Middle East and India.4 particular chain affected and the amount of globin chain Thalassemia is a major health problem in countries produced. Limited availability of globin chains results in where these disorders are prevalent. Prevention is seen as a reduction in the assembly of hemoglobin. Patients with an essential part in the management of the problem. Thus, mild genetic defects are generally asymptomatic. Patients many of these countries now have large screening and with more severe defects present with symptoms that result education programs to detect carriers. This has drastically from one or more of the following: decreased production reduced the number of individuals born with both homo- of normal hemoglobin, synthesis of abnormal hemoglobins, zygous and heterozygous forms of the disease. 292 Chapter 14 Thalassemia versus Types of Thalassemia Hemoglobinopathy Because six different normal globin genes exist (a, b, g, The system of categorizing thalassemias and hemoglobin- d, e, Z), at least six versions of thalassemia are possible. In opathies differs among hematologists. Some use hemo- addition, deletions can occur to entire gene clusters, con- globinopathy as a disease category to encompass both currently affecting more than one globin chain. Of the six structural variants of hemoglobin (e.g., sickle cell anemia) normal globin genes, e and Z are normally synthesized only and thalassemias. Others categorize only the structural vari- early during embryonic development, and g is produced ants as hemoglobinopathies and describe thalassemias as in high amounts from approximately the third trimester of a separate disease entity. In this text, we refer to the two pregnancy until birth. Shortly before birth, g@chain synthesis diseases as separate entities. begins to decrease but can still be detected in low amounts in In the preceding chapter, hemoglobinopathies were adult life in some people. The three remaining globin chains defined as qualitative defects in the structure of globin (a, b, and d) along with the g@chains are considered nor- chains resulting in production of abnormal hemoglobin mal adult globin chains and combine to form hemoglobin molecules. Thalassemias, on the other hand, are typically A (a2b2), hemoglobin A2 (a2d2), and hemoglobin F (a2g2), quantitative disorders of hemoglobin synthesis that pro- respectively. Approximately 97% of normal adult hemoglo- duce reduced amounts of normal hemoglobin. bin is HbA; thus, a deficiency of either a@ or b@chains affects The different clinical presentations of hemoglobinopa- hemoglobin A assembly, reducing HbA concentration and thies and thalassemia are a direct result of the differences in affecting the blood’s oxygen-carrying capacity. the types of mutations encountered in these disease states. Two major types of classical thalassemia, a@thalassemia Most hemoglobinopathies result predominantly from a and b@thalassemia, have been described. When synthesis point mutation within a globin gene that is translated into of the a@chain is impaired, the disease is a@thalassemia. a globin chain containing a single amino acid substitution. When synthesis of the b@chain is affected, the disease is Hemoglobin types containing these mutations may be pro- b@thalassemia. There have been reports of d@thalassemia, duced in normal quantity, but the globin chains, and thus but its occurrence is rare and it is not clinically significant the hemoglobin molecules, are structurally abnormal. They because the d@chain is a component of the minor hemoglobin may be unstable or have abnormal function. In contrast, HbA2, which comprises only about 2.5% of total hemoglo- thalassemias result from both deletional and nondeletional bin. Combinations of gene deletions such as db@thalassemia mutations in globin genes that reduce or eliminate the syn- or gdb@thalassemia occur but are rare. The synthesis of all of thesis of the corresponding globin chain. This results in the the indicated chains is reduced. assembly of inadequate amounts of normal hemoglobin and Occasionally synthesis of a structural globin variant a reduced oxygen-carrying capacity of the blood. Unlike decreases globin chain synthesis, producing the clinical hemoglobinopathies, the amino acid sequence of the chain, picture of thalassemia (thalassemic hemoglobinopathy). if produced, is usually normal, and the chain is assembled These structural variants include those hemoglobins with into the appropriate hemoglobin, albeit in reduced amounts. abnormally long or short globin chains (e.g., hemoglobin In some of the less common thalassemias, the globin chains Constant Spring) as well as variants with a point mutation can be lengthened or truncated (Table 14-1). (e.g., hemoglobin E). Hemoglobin Lepore is a hemoglobin variant in which the non@a@globin chains are not only struc- Checkpoint 14.1 turally abnormal but also ineffectively synthesized. Because Differentiate the etiology of thalassemias and hemoglobinopathies. of their clinical similarity to thalassemias, these particular structural variants are discussed in this section. Table 14.1 Comparison of Hemoglobinopathies and Thalassemias Erythrocyte Abnormal Hb Solubility Reticulocyte Disease RBC Count Indices Ancestry Morphology Hb Test Count Hemoglobinopathy T Normocytic, Target cells, sickle HbS, HbC, + in HbS, African, c c normochromic cells (in HbS), HbC HbE, etc. Hb Bart, and Mediterranean, crystals (in HbC), HbCHarlem
Middle Eastern, others Southeast Asian Thalassemia c Compared with Microcytic, Target cells, HbH (b4), Negative Mediterranean, c what is expected hypochromic basophilic Hb Bart (g4) Southeast Asian, for the Hb level stippling African T , slight decrease; c , slight increase; c c , moderate increase; Hb, hemoglobin; RBC, red blood cell; + , positive. Thalassemia 293 A variant of b@thalassemia known as hereditary per- sistence of fetal hemoglobin (HPFH) is characterized Checkpoint 14.2 by continued production of increased amounts of HbF What are the most common genetic mutations associated with throughout life. This disorder is characterized by a failure a@thalassemia? in the switch of g@chain production to b@chain production after birth. In homozygotes, 100% of circulating hemoglo- bin is HbF. Pathophysiology Genetic Defects in Thalassemia Normally, equal amounts of a@ and b@chains are synthe- sized by the maturing erythrocyte, resulting in a b@chain Nearly all thalassemic mutations fall into one of five cat- to a@chain ratio of 1:1. In a@ and b@thalassemia, synthesis of egories of genetic lesions: gene deletion, promoter muta- one of these chains is decreased or absent, resulting in an tion, nonsense mutation, mutated termination (stop) codon, excess of the other chain. If the a@chain is affected, there is and splice site mutation (Table 14-2). Regardless of the type an excess of b@chains, and if the b@chain is affected, there is of mutation encountered, the results are the same; the glo- an excess of a@chains. This unbalanced synthesis of chains bin chain encoded by the mutated globin gene is absent, contributes substantially to the pathophysiology in thalas- reduced in concentration, or occasionally somewhat longer semia and produces several effects, all of which contribute or shorter than normal. to anemia: (1) a decrease in total erythrocyte hemoglobin If all globin genes of a single type of globin chain production, (2) ineffective erythropoiesis, and (3) chronic are affected, the corresponding hemoglobin is absent. hemolysis. Deleted genes are most common in a@thalassemia, while The exact nature of the contribution of unbalanced chain mutated genes are more common in the b@thalassemias.5 production depends on which globin chain accumulates in b@thalassemia mutations that completely block b@chain excess and on the amount of that excess. There is no com- production (b0) are caused by initiation codon, nonsense, pensatory downward adjustment of production of one glo- frameshift, and splicing mutations and in some cases, bin chain when its partner globin subunit is not synthesized gene deletion. In contrast, mutations that reduce, but not normally. Although excess b@ and g@chains can combine as completely block, b@chain production (b+) are caused by tetramers (b4, g4) to form abnormal hemoglobins, a@chains mutations in the promoter region, the polyadenylated sig- do not. Excess a@chains are highly insoluble and precipitate nal sequence, untranslated regions, or at splice sites.4 The within the cell. The precipitates (Fessas bodies) bind to the degree of reduction in globin chain production is a direct cell membrane, causing membrane damage (which leads to reflection of the type of mutation encountered and parallels apoptosis) and decreased erythrocyte deformability.4 Mac- the severity of the clinical disorder (Table 14-2). rophages may destroy the precipitate-filled developing Table 14.2 Five Common Genetic Defects in Thalassemia Mutation Type Thalassemia Encountered Effect on Gene Effect on Globin Chain Deletion (large) Predominantly a@thalassemia, some Loss of gene Absence of production b@thalassemia Promoter Predominantly b@thalassemia Impaired transcription Reduced or absent production Nonsense Predominantly b@thalassemia In frame substitution Amino acid change Frame shift Amino acid changes distal to shift Longer or shorter globin chains Stop codon Predominantly b@thalassemia Convert stop codon to amino acid Slightly lengthened globin chain codon (retained) Significantly lengthened globin chain (degraded) Splice site Predominantly b@thalassemia Create new splice sites Slightly shortened globin chain (retained) Significantly shortened globin chain (degraded) Loss of splice sites Slightly lengthened globin chain (retained) Significantly lengthened globin chain (degraded) Unaltered globin chain 294 Chapter 14 erythroblasts in the bone marrow, resulting in a large degree (Table 14-3). The combination of reduced HbA synthesis, of ineffective erythropoiesis. Cells that survive the marrow ineffective erythropoiesis, and hemolysis results in ane- environment and enter the circulation also contain precipi- mia. The severity of anemia varies widely, depending on tates that are subsequently pitted and/or removed by the the specific genetic mutation and number of genes affected. spleen, causing chronic extravascular hemolysis. Hypoxia from anemia is exacerbated in some cases by the Excess b@chains can combine to form hemoglobin presence of abnormal hemoglobins that have a high oxy- molecules containing four b@chains, called hemoglobin H gen affinity (HbH and Hb Bart). These hemoglobins do not (HbH, b4). This hemoglobin has a high oxygen affinity and release oxygen readily to the tissues. is also unstable. Thus, it is a poor transporter of oxygen. In Chronic hemolysis has several adverse effects. Spleno- the fetus, when a@chains are decreased, excess g@chains can megaly is frequently present because the spleen is a major combine to form hemoglobin molecules with four g@chains, site of extravascular hemolysis. Occasionally, the spleen can called hemoglobin Bart (Hb Bart, g4). This hemoglobin also become overburdened by the process of erythrocyte destruc- has a very high oxygen affinity. tion resulting in functional hyposplenism. In this case, the Without stable hemoglobin formation, iron accumulates spleen’s function as a secondary lymphoid tissue is com- within the body. Furthermore, in patients who are not on a promised, leading to an increase in infections (Chapter 7). transfusion regimen, erythropoiesis, anemia, and hypoxia Chronic hemolysis can also result in the formation of gall- downregulate hepcidin, resulting in excess iron absorption stones formed from the large amounts of bilirubin excreted and excess release of stored iron from macrophages.6 When by the liver. The chronic demand for erythrocytes also has blood iron exceeds the capacity of transferrin to bind and adverse effects. The bone marrow responds by increasing detoxify it, the free iron acts as a catalyst to form oxygen erythropoiesis, resulting in erythroid hyperplasia, and in radicals that lead to oxidative cell damage. This results in some of the more severe thalassemias, bone marrow expan- multiple organ damage and increased mortality.4 Oxidative sion and thinning of calcified bone. Consequently, patients damage to the RBC membrane lipids can also cause throm- develop skeletal abnormalities and pathologic fractures. bosis, especially in patients with splenectomies.7 The increased iron demand needed to support the eryth- ropoietic activity stimulates the increased absorption of Clinical Presentation iron, more than the amount required for erythropoiesis (Chapter 12). This additional iron is not effectively incorpo- The clinical presentation of thalassemia is related to ane- rated into hemoglobin, so it accumulates in macrophages mia, chronic hemolysis, and ineffective erythropoiesis in the bone marrow, liver, and spleen. As this process Table 14.3 Clinical and Laboratory Findings Associated with Thalassemia Clinical Presentation Pathophysiology Laboratory Finding Anemia/hypoxia Decreased hemoglobin production/erythropoiesis T /N RBC count, T hemoglobin, T hematocrit Ineffective erythropoiesis Microcytic/hypochromic RBCs Presence of high-affinity hemoglobins (HbH and Hb Bart) T MCV, T MCH, T MCHC Increased extravascular hemolysis c Reticulocyte count Anisocytosis and poikilocytosis Target cells, basophilic stippling, nucleated RBCs BM erythroid hyperplasia c RDW Abnormal hemoglobin electrophoresis Splenomegaly/hemolysis Splenic removal of abnormal erythrocytes c Bilirubin Ineffective erythropoiesis T Haptoglobin Gallstones Increased intravascular and extravascular hemolysis c Bilirubin Skeletal abnormalities Expansion of bone marrow BM erythroid hyperplasia Pathologic fractures Thinning of calcified bone Iron toxicity Iron overload Multiple transfusions c Prussian blue staining in BM Increased iron absorption c Serum iron/ferritin and T TIBC BM, bone marrow; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cell; RDW, red cell distribu- tion width; TIBC, total iron-binding capacity. Thalassemia 295 continues, iron eventually accumulates in parenchymal cells poikilocytosis are common. Precipitates of excess chains of various organs and adversely affects organ function. Iron or unstable hemoglobin may be visualized with supravital toxicity commonly affects such organs as the liver, pituitary, stains. Reticulocytes and bilirubin are usually increased heart, and bone, resulting in dysfunctions such as cirrho- due to the chronic hemolysis, whereas haptoglobin may sis, hypogonadism and growth failure, arrhythmias and be decreased, depending on the degree of intravascular cardiomyopathies, and pathologic fractures, respectively.8 hemolysis. The additional iron introduced through transfusion therapy Hemoglobin electrophoresis is always indicated if can exacerbate iron overload. Ineffective erythropoiesis in thalassemia is suspected. HbA is usually decreased. HbF the bone marrow can be accompanied by extramedullary and HbA2 are increased in b@thalassemia but decreased in erythropoiesis in the liver and spleen. Extramedullary a@thalassemia. Hemoglobin Bart and HbH may also be pres- erythropoiesis can produce masses large enough to cause ent in some of the a@thalassemia syndromes. compression syndromes (Table 14-3). Bone marrow studies are not necessary for diagnosis Pregnant women with thalassemia have physiologi- but when performed show marked erythroid hyperplasia. cal demands that impact the developing fetus to a greater Erythroblasts appear abnormal with very little cytoplasm, extent than the mother. A developing fetus can experience uneven cytoplasmic membranes, and striking basophilic diminished growth, premature birth, and even intrauter- stippling. Prussian blue stain reveals an abundance of iron ine death if the mother’s oxygen concentration falls below and occasionally a few ringed sideroblasts. Phagocytic 70 mm Hg. Pregnant women who present with a hemoglo- “foam” cells similar to Gaucher cells have been reported bin level of between 9 and 10 g/dL (90–100 g/L) around in the more severe forms of the disease. This morphology the time of delivery are often given a blood transfusion to results from partially digested red cell membrane lipids improve oxygen delivery. To avoid iron overload caused by associated with intense ineffective erythropoiesis. the combination of transfusion therapy and increased iron In areas of high prevalence of thalassemia and in cer- absorption, pregnant women can be given deferoxamine (an tain populations, screening programs to detect thalassemia iron chelator used to help eliminate excess iron) during and carriers have been developed. With the development of after the transfusion.9 capillary sequencing techniques, nucleic acid–based methods can be used to detect mutations. Given the large number of mutations present in the human globin genes, Checkpoint 14.3 a small panel of probes for mutations found in a specific Why do a@ and b@thalassemia result in more clinically severe ethnic group are initially screened for together with the disease than other types of thalassemia? wild-type allele. Using this approach, identification of the mutation(s) is achieved more than 90% of the time. In cases where the mutation(s) are not identified, a second Laboratory Evaluation round of multiplex screening can be performed using a Peripheral blood findings provide clues to the disease panel of more rare mutations; this is successful in most (Table 14-3). Thalassemias are characterized by microcytic, of the remaining 10% of cases. Gene sequencing can be hypochromic anemia with a decrease in MCV, MCH, and performed to identify mutations that evade both rounds usually MCHC. The erythrocyte count is often normal or of screening.12 slightly decreased, but increased relative to the hemoglobin Nucleic acid–based methods may also be used for pre- and hematocrit levels. The Mentzer Index can be helpful natal diagnosis of thalassemias and hemoglobinopathies. in differentiating thalassemia from iron-deficiency anemia Extracellular fetal DNA or cells can be detected in the (a common cause of microcytic hypochromic anemia)10 maternal circulation. For families electing in-vitro fertiliza- (Chapter 12). The index is calculated by dividing the MCV tion, preimplantation analysis can be performed.12,13 (fL) by the RBC count (* 106/mcL). If the result is less than 13, diagnosis favors thalassemia whereas if the result Treatment is greater than 13, diagnosis favors iron deficiency. The There are two broad approaches to treatment: maintenance RDW can be increased or within the reference interval. In therapy and curative therapy. iron deficiency, the RDW is most often increased (greater than 15).11 In practice, the CBC, Mentzer Index, and RDW CURATIVE THERAPY should not be used as the only parameters to differentiate While stem cell transplant remains the only curative treat- the two pathologies. In microcytic hypochromic anemia, ment, it is limited to a small percentage of patients due iron studies (e.g., serum ferritin) should be performed to cost, availability of compatible donors, and associ- routinely. Target cells and microcytosis usually are pres- ated risks.14 Cord blood transplants from a related donor ent even in cases without anemia. Basophilic stippling and show promise for a high cure rate but data are conflict- nucleated erythrocytes may be present. Anisocytosis and
ing for cord blood transplants from unrelated donors.15 296 Chapter 14 Therefore, maintenance therapy remains the mainstay will allow for its combination with excess a@chains and for treatment and thalassemia mutational status dictates reduce a@chain precipitation and the associated hemolysis. whether patients are transfusion dependent, non-transfu- The induction of g@chain synthesis for fetal hemoglobin sion-dependent, or require no therapy. production using 5-azacididine, decitabine, and butyrate derivatives is showing promise in clinical trials but is still MAINTENANCE THERAPY under investigation for b@thalassemia. Transfusion-Dependent Thalassemia Transfusion-depen- dent thalassemia is defined as patients requiring ongoing transfusion therapy for survival and/or quality of life; these patients are clinically classified as thalassemia major. Hyper- a@Thalassemia transfusion therapy is designed to correct the anemia and reduce sequelae associated with ineffective erythropoiesis General Considerations and hemolysis. Hypertransfusion suppresses erythropoiesis a@Thalassemia is a group of four disorders characterized and increases hepcidin, which inhibits gastrointestinal iron by decreased synthesis of a@chains. Although each is dis- absorption. Suppression of erythropoiesis and inhibition cussed separately, features common to each type are pre- of iron absorption will ameliorate complications like bone sented here. changes, iron excess, hepatosplenomegaly, and failure to ETIOLOGY thrive. For individuals receiving hypertransfusion therapy, In the human genome, two a@genes are located on each the outcome can be near normal growth and development.16 chromosome 16, totaling four a@genes in the diploid state Although standard of care for transfusion therapy in (Figure 14-1). Mutations can affect one or more of the the United States differs between institutions, recommenda- a@genes, resulting in four discrete clinical severities. A patient tions include immunization against hepatitis B, RBC antigen in whom all four a@genes are deleted produces no a@chains, testing, and serum IgA level determination.4 Hypertransfu- a condition referred to as hydrops fetalis. When three of the sion therapy should involve the use of leukoreduced and four a@genes are deleted, the disorder is known as hemoglobin antigen-matched RBCs. A hemoglobin measurement should H (HbH) disease. The deletion of two a@genes is known as be performed prior to each transfusion. The goal is to main- a@thalassemia minor, and the deletion of a single a@gene is tain a hemoglobin level of not less than 10 g/dL. This prac- known as silent carrier. Although less common, nondeletional tice should continue until the patient has completed growth mutations and mutations that produce unstable a@chains and development through the second decade of life.17 are also found in a@thalassemia. The outcome is usually the Non-Transfusion-Dependent Thalassemia Non-transfusion- same as that of a deletion mutation, a reduction in a@chains dependent thalassemia (NTDT) is defined as patients and in the corresponding a@containing hemoglobins. with symptomatic thalassemia that do not require ongo- AFFECTED ALLELES ing transfusion therapy for survival. However, they may The amount of a@chains synthesized is somewhat pro- receive transfusions for short periods to manage infections, portional to the number of affected alleles. However, surgery, pregnancy, or situations producing acute blood sometimes erythrocytes produce higher concentrations of loss. These patients are classified as thalassemia intermedia a@chains than the number of affected alleles would predict. (HbH disease and b@thalassemia intermedia) and include certain compound heterozygotes like HbE/b@thalassemia. Although transfusions are not required for survival, several z cz ca2 ca1 a2 a1 clinical complications like iron overload and hypercoagula- Maternal bility can occur resulting from ineffective erythropoiesis and 59 39 allele peripheral hemolysis. Ineffective erythropoiesis downregu- 1 2 lates hepcidin, which increases iron absorption and release z cz ca2 ca1 a2 a1 from macrophages. High liver iron levels are associated Paternal with a significantly higher risk of developing thrombosis, 59 39 allele 3 4 pulmonary hypertension, hypothyroidism, hypogonadism, z = zeta cz = psi zeta ca1 = psi alpha 1 osteoporosis, and renal tubular dysfunction.18,19 Complications of NTDT can be managed by splenec- a2 = alpha 2 ca2 = psi alpha 2 a1 = alpha 1 tomy, iron chelation therapy, and drugs to induce g@chain production.20 Splenectomy will lessen the anemia and Figure 14.1 A short section of chromosome 16 showing the reduce the platelet count associated with thrombotic risk. 5′ to 3′ orientation of three functional genes Z, a2, and a1 along with three pseudogenes cZ, ca2, and ca1. Pseudogenes are the result Iron chelation therapy will slow progression of iron accu- of partial gene duplications but are not expressed. There are two mulation in the liver and the associated tissue toxicities. In functional a@genes on each chromosome; the a2@gene expresses the case of b@thalassemias, increasing g@chain production 2 to 3 times as much protein product as the a1@gene. Thalassemia 297 There are two main reasons for this phenomenon. First, the GENOTYPES two a@genes on each chromosome 16 are designated as a1 Three nomenclature systems—genotypic, genotypic descrip- and a2, with the a2@gene positioned upstream (5′) of the tion, and phenotypic—classify the a@thalassemias into five a1@gene (Figure 14-1). The a2@gene produces two to three discrete categories. The addition of the normal genotype pro- times the amount of mRNA as the a1@gene.21 Therefore, a duces a total of six possibilities. The genotypic system desig- deletion of the a2@gene would reduce a@chain production nates deleted genes as (- ) and unaffected genes as (a). The to a greater degree than would a deletion of the a1@gene. genotypic description system combines the zygosity state, Second, the erythropoietic system has an internal mecha- homozygous or heterozygous, with either a gene symbol (a0 nism designed to stimulate increased production of a@chains or a+) or a nominal descriptor (a@thal@1 or a@thal@2) to des- from the unaffected genes to compensate for deletions, thus ignate the number of deleted a@genes on each chromosome. lessening the net reduction of a@chains. Both a@thal@1 and a0 indicate the deletion of both a@genes on the same chromosome (cis deletion) (- , - ). a@thal@2 AFFECTED INDIVIDUALS and a+ refer to one deleted and one unaffected a@gene a@Thalassemia is found primarily in people of Mediterra- on a given chromosome (- , a). The phenotypic system nean, Asian, and African ancestry. In particular, it is com- describes four clinical types, hydrops fetalis, hemoglobin monly seen in blacks, Indians, Chinese, and Middle Eastern H disease, a@thalassemia minor, and silent carrier with the people, with blacks usually expressing a milder version of a@thalassemia minor type exhibiting two clinical severities the disease. The reason patients of African descent tend to (Table 14-4). About 15–20% of patients have a nondeletional present with a milder version of a@thalassemia is because mutation of the a@thalassemia gene similar to those found in the deletion in this ethnic group usually involves the lower- b@thalassemia, designated as aT, that functions to reduce but producing a1@gene. not eliminate a@chain production from that gene. Table 14.4 Characteristics of a@Thalassemia Genotypic Hematologic Hemoglobins Genotype Phenotype Severity Description Findings Present (- -/- -) Homozygous a@thal@1 Hydrops fetalis Marked anemia Fatal Hb Bart (80–90%) a0/a0 Microcytic/hypochromic Hb Portland (10–20%) RBCs c c c Anisopoikilocytosis c NRBC (- -/-a) Heterozygous Hemoglobin H disease Moderate to marked Chronic, moderately Birth = Hb Bart; a0/a+ a@thal@1/a@thal@2 anemia severe hemolytic anemia Adult = HbH Microcytic/hypochromic RBCs Target cells Basophilic stippling Poikilocytosis (- -/aa) Heterozygous a@Thalassemia minor Slight anemia Mild to moderate Birth = Hb Bart; a0/a a@thal@1 Microcytic/hypochromic Adult = normal RBCs Target cells Basophilic stippling Poikilocytosis (-a/-a) Homozygous a@Thalassemia minor Slight anemia Mild Birth = Hb Bart; a+/a+ a@thal@2 Microcytic/hypochromic Adult = normal RBCs Target cells Basophilic stippling Poikilocytosis (-a/aa) Heterozygous Silent carrier Normocytic or slightly Normal Normal a+/a a@thal@2 microcytic RBCs (aa/aa) Normal None Normal Normal Normal c c c , marked increase; c , slight increase; thal, thalassemia. 298 Chapter 14 a@Thalassemia Major (a0/a0 or C G O D a@thal@1/a@thal@1 ); Hydrops Fetalis E S CA CS A2 Lepore F A Portland Barts H The most severe form of a@thalassemia, a@thalassemia major, Hydrops involves the deletion of all four a@genes (- -/- - ). Both fetalis parents of the thalassemia patient must have a@thalassemia Hb H to have a child with hydrops fetalis because both a@genes (neonate) on each parental chromosome inherited by the child are Hb H deleted (a@thal@1; - , - ). a@Thalassemia major is found (adult) almost exclusively in Asians because that is the major ethnic Hb H/ group that carries the a@thal@1 allele. CS a-Thal PATHOPHYSIOLOGY minor (neonates Because all four a@genes are deleted in hydrops fetalis, no only) Silent carrier physiologically useful hemoglobins can be synthesized CA, Carbonic anhydrase beyond the embryonic stage. Therefore, this disorder is CS, Constant Spring incompatible with life, and infants are either stillborn or die within hours of birth. In the absence of a@chains, eryth- Figure 14.2 Hemoglobin electrophoresis on cellulose rocytes assemble hemoglobin using the Z@, e@, g@, d@, and acetate or agarose at pH 8.4 is helpful in distinguishing the b@chains available. Therefore, embryonic hemoglobins type of thalassemia and in differentiating thalassemias from and abnormal hemoglobin tetramers involving globinopathies. In a@thalassemias, there is a reduction in g@chains hemo a@containing hemoglobins (HbA, HbA2, and HbF) proportional to (Hb Bart, g4) are produced. Hb Bart has a very high oxy- the number of deleted a@genes and in the more severe cases, the gen affinity and no Bohr effect (Chapter 6). Therefore, this emergence of non@a@containing hemoglobins (HbH and Hb Bart). hemoglobin cannot supply tissues with sufficient oxygen to sustain life, and the developing infant usually dies of hypoxia and congestive heart failure in utero. Hemoglo- Hemoglobin H Disease (a0/a+ or bin Gower 1 and Portland, although normally absent fol- a@thal@1/a@thal@2 lowing the first trimester, continue to be synthesized until HbH disease, a symptomatic but nonfatal type of birth in a0/a0 thalassemia because they do not contain a@thalassemia, was the first type to be described in 1956. a@chains. It occurs when three of the four a@genes are deleted CLINICAL PRESENTATION (- , -/- , a). Although gene deletion is the primary mecha- Infants who survive until birth exhibit significant physical nism in HbH disease, there are at least seven nondeletional abnormalities upon routine exam. The babies are under- forms that are more severe. Hemoglobin Constant Spring weight and edematous with a distended abdomen. The liver is the most common, others include Hb Paksé, Quong Sze, and often the spleen are enlarged due to extramedullary and Suan Dok.21 African Americans seldom present with hematopoiesis. There is massive bone marrow hyperplasia. HbH disease because they rarely express a deletion of two Hemolysis in the fetus is severe and there is extensive depo- a@genes on the same chromosome.22 sition of hemosiderin. This disorder usually results when two parents, one LABORATORY EVALUATION with heterozygous a@thal@1 (- -/aa) and the other with the Laboratory results confirm the clinical observation of severe heterozygous a@thal@2 (-a/aa) genotype, bear children.13 anemia with hemoglobin values ranging from 3 to 10 g/dL All children from a patient with HbH disease will have a (30–100 g/L) and erythrocytes that are markedly microcytic type of a@thalassemia, the severity of which depends on the and hypochromic. Hemoglobin electrophoresis on cellulose allele inherited and the other parent’s genotype. acetate or agarose at alkaline pH shows 80–90% Hb Bart PATHOPHYSIOLOGY and 10–20% Hb Portland; HbH is sometimes also detect- The dramatic reduction in a@chain synthesis (25–30% of nor- able. HbA, HbA2, and HbF are absent due to the lack of mal) results in a decrease in the assembly of HbA, HbA2, a@chain production (Figure 14-2). and HbF. In addition, a decrease in a@chains creates a rela- tive excess of b@chains, which assemble to form b@chain tet- Checkpoint 14.4 ramers called HbH. g@chains also are produced in excess of Which of the three normal adult hemoglobins would be affected a@chains, especially at birth, and combine to form g@chain in hydrops fetalis? tetramers or Hb Bart. These tetramers are unstable and pre- cipitate intracellularly, leading to ineffective erythropoiesis Thalassemia 299 and chronic hemolysis. This leads to anemia, gall stones, 100 iron overload, and hypercoagulability. Erythrocyte pro- genitors increase proliferation to compensate, resulting HbH Myoglobin Hb A in osteoporosis, bone deformities, and extramedullary 75 hematopoiesis.22 P50 is the partial pressure of oxygen at which the hemoglobin HbH is thermolabile, unstable, and tends to precipitate tested is 50% saturated. It is a inside erythrocytes triggering chronic hemolytic anemia. Its measure of the binding affinity oxygen affinity is 10 times that of HbA, reducing oxygen 50 of the hemoglobin. A higher P50 indicates a lower binding affinity delivery to the tissues. Its high oxygen affinity is attributed and a greater ability
to release to the lack of heme–heme interaction and absence of the oxygen to tissues. Bohr effect (Chapter 6). This increased oxygen affinity is 25 HbH – P50 = 6 mm Hg reflected in the lower P50 value of HbH relative to HbA and Myoglobin – P50 = 12 mm Hg myoglobin (Figure 14-3). HbA – P50 = 26 mm Hg Hemoglobin H may also occur as an acquired defect in 26 erythroleukemia and other myeloproliferative neoplasms. 0 However, the clinical manifestations and hematological 0 6 12 25 50 75 100 abnormalities of these acquired disorders make it possible Partial pressure of oxygen (mm Hg) to distinguish them from congenital HbH disease. Acquired HbH is probably due to a defect that prohibits the transcrip- Figure 14.3 The Hb dissociation curve illustrates the relative binding affinities of HbA, HbH, and myoglobin using the P50 value. tion of the a@gene. The monomeric myoglobin molecule lacks heme–heme interactions, causing it to bind oxygen tightly, decreasing the P50 value relative to HbA. The P50 value is even lower for HbH, indicating an even Checkpoint 14.5 stronger affinity for oxygen estimated to be 10 times more than the Compare oxygen-binding characteristics of HbH relative to HbA oxygen affinity of HbA. and myoglobin. similar to those found in b@thalassemia major, and there is relatively little ineffective erythropoiesis. CLINICAL PRESENTATION Symptoms are related to anemia and chronic hemolysis. LABORATORY EVALUATION Hemoglobin H disease shows a wide variation from mild to Hemoglobin H disease is characterized by a microcytic, severe anemia, which worsens during pregnancy, in infec- hypochromic anemia with hemoglobin levels usually rang- tious states, and during administration of oxidant drugs. ing from 8 to 10 g/dL (Figure 14-4a). Reticulocytes are mod- Splenomegaly and, less often, hepatomegaly are present. erately increased to 5–10%, and nucleated red blood cells Less than half of affected patients exhibit skeletal changes are observed on the peripheral blood smear. a b Figure 14.4 (a) Peripheral blood smear from a patient with HbH disease. Note the microcytic, hypochromic anemia with target cells (Wright-Giemsa stain; 1000* magnification). (b) Peripheral blood from patient in Figure 14-4a after incubation with brilliant cresyl blue. Notice the cells that have dimples and look like golf balls. These are the cells with precipitated HbH (Brilliant cresyl blue stain; 1000* magnification). % Hb saturation 300 Chapter 14 Hemoglobin electrophoresis of affected neonates shows which result in the deletion of both a@genes on the same about 25% Hb Bart with decreased levels of HbA, HbA2, chromosome.21,23 and HbF. Shortly before birth, b@chains begin to replace g@chains, and HbH eventually replaces Hb Bart. Hemoglo- PATHOPHYSIOLOGY bin H, a fast-migrating hemoglobin at alkaline pH, consti- Although a measurable decrease in the production of tutes 2–40% of the hemoglobin in adults with HbH disease. a@containing hemoglobins occurs, the unaffected a@globin HbA2 is decreased to about 1.5%, but HbF is normal. A trace genes are able to direct synthesis of a@globin chains to a of Hb Bart can be demonstrated in approximately 10% of greater than normal degree and therefore partially compen- affected adults with remaining hemoglobin being HbA sate for the deleted genes. Only minor changes occur in the (Figure 14-2). Other laboratory tests are available to assess erythrocyte count, indices, hemoglobin electrophoresis pat- patients with HbH disease. Hemoglobin H inclusions are terns, and red cell morphology. easily found upon incubation of blood with brilliant cre- CLINICAL PRESENTATION syl blue (Figure 14-4b). These inclusions tend to cover the Patients with a@thalassemia trait are asymptomatic with a inside of the plasma membrane, giving the pitted appear- mild anemia and are often diagnosed incidentally or when ance of a golf ball. being evaluated for family studies. This mild phenotype is TREATMENT AND PROGNOSIS the reason that this form is called thalassemia minor. Treatment for patients with HbH disease is variable but LABORATORY EVALUATION in severe cases can involve long-term transfusion ther- The most demonstrable laboratory abnormalities are apy and splenectomy. Regular transfusions minimize observed in the newborn. The presence of 5–6% Hb Bart the stunting of growth and the other consequences of in neonates can be helpful in diagnosing this condition.21 chronic severe anemia. Iron chelation therapy with defer- Three months after birth, Hb Bart decreases to undetectable oxamine avoids iron overload and the effects of iron tox- levels and hemoglobin electrophoresis becomes normal. icity. Deferasirox (licensed in the United States in 2006) The only persistent hematological abnormality thereafter and deferiprone (licensed in 2011) are oral chelators that is a mild microcytic, hypochromic anemia. serve as an alternative to deferoxamine.11 While they have In adult patients, hemoglobin levels are above 10 g/dL significant dose-related sequelae including neutropenia, (100 g/L), and the erythrocyte count is above 5 * 106/mcL. arthropathy, and agranulocytosis, the oral administration The peripheral blood film usually demonstrates significant may be preferable to the continuous, prolonged (24 hour) microcytosis with an MCV of 60–70 fL with few target cells intravascular administration required for deferoxamine. (Figure 14-5). Occasional cells can exhibit HbH inclusions The increased and ineffective erythropoiesis places high after incubation with brilliant cresyl blue. demands on the bone marrow requiring folate supplemen- In some cases, a@thalassemia may be masked by iron- tation to avoid a deficiency.21 Early treatment is necessary deficiency anemia. Persistence of microcytes following to prevent the typical clinical manifestations of thalas- successful treatment of iron deficiency is suggestive of thal- semia. With supportive care and behavioral interven- assemia, but further investigation is needed for conclusive tions, patients with HbH disease experience a normal life diagnosis. expectancy. For patients in whom transfusion and chela- tion therapy are not efficacious, a bone marrow transplant could be indicated.21 a@Thalassemia Minor (a+/a+ or a@thal@2/a@thal@2; a0/a or a@thal@1/normal) The a@thalassemia trait (homozygous a@thal@2 or a@thal@1 trait) occurs when two of the four a@genes, either on the same (cis) or opposite (trans) chromosomes, are missing. The condition is found in all geographic locations.1 In Afri- can Americans, the homozygous a@thal@2 form is the most common presentation. In individuals of Southeast Asian or Mediterranean descent, genetic testing has identified at Figure 14.5 A peripheral blood smear from a patient with a@thalassemia trait. The hemoglobin is 15 g/dL, RBC count least nine haplotypes (groups of alleles of different genes 6.4 * 106/mcL, and MCV 69.4 fL. The high RBC count with on the same chromosome that are closely linked and usually microcytosis is typical of thalassemia minor or trait (Peripheral inherited together), representing different mutations, all of blood; Wright-Giemsa stain; 1000* magnification). Thalassemia 301 TREATMENT AND PROGNOSIS These patients are usually asymptomatic, have a normal 3. Name three disorders that frequently present life span, and do not require medical intervention for their with the same poikilocyte that dominates in this thalassemia. peripheral blood smear. 4. List two additional lab tests that would help to Silent Carrier (a+/a or confirm the diagnosis and predict the results of a@thal@2/normal) each. The silent carrier version of a@thalassemia (a@thal@2 trait) is missing only one of four functioning a@genes. More than 25% of African Americans have been shown to express a deletion of one a@gene.24 b@Thalassemia PATHOPHYSIOLOGY/CLINICAL PRESENTATION/ General Considerations LABORATORY EVALUATION As with a@thalassemias, some features of b@thalassemias In the silent carrier state, the three remaining a@genes direct are common to all forms of the disease. The genetics of the synthesis of an adequate number of a@chains for normal b@thalassemia and the individuals affected with the disor- hemoglobin synthesis. This carrier state is asymptomatic der are presented before the discussion of its various forms. and benign, but adults can present with a borderline nor- mal MCV of around 78–80 fL.25 In affected infants, 1–2% GENETICS Hb Bart may be found at birth but cannot be detected after Whereas a total of four a@globin genes results in four major three months of age. The only definitive diagnostic test for genotypes of a@thalassemia, there are only two b@globin thalassemias in adults with one or two gene deletions is genes, one located on each chromosome 11 (Figure 14-6). globin gene analysis. If the prominent type of mutation found in b@thalassemia were also deletional, one would expect two severities, the TREATMENT AND PROGNOSIS severe homozygote and the mild heterozygote. However, Patients with the silent carrier phenotype require no treat- in b@thalassemia most mutations are nondeletional, result- ment and have a normal life span. ing in a near continuum of clinical severities. Two classi- fication systems are currently used for this diverse group of diseases. The genotypic system classifies b@thalassemia CASE STUDY (continued from page 291) patients into six genotypes based on zygosity and the The parents took John, the 4-year-old Greek degree of alteration of the b@genes, and the phenotypic sys- patient, to a pediatrician for a checkup. A CBC tem divides patients into four categories based on the sever- ordered had the following results: ity of clinical symptoms. In the genotypic system, all b@gene mutations are cat- CBC Differential egorized into two groups based on the impact of the muta- WBC 11.4 * 103/mcL Segs 55% tion on b@globin production. The two gene varieties are RBC 1.7 * 106/mcL Bands 1% termed b+ and b0. The b+@gene mutation causes a partial block in b@chain synthesis, and the b0 Hb 8.3 g/dL Lymphs 36% @gene mutation results Hct 24% Mono 7% MCV 69 fL Eos 1% e Gg Ag cb d b Maternal MCH 21 pg Moderate 5' 3' allele MCHC 29.2 g/dL poikilocytosis, 1 Plt 172 * 103/mcL polychroma- e Gg Ag cb d b sia, and many Paternal target cells; 5' 3' allele few teardrop 2 e, epsilon Gg, G gamma Ag, A gamma cells cb, psi beta d, delta b, beta 1. Based on the indices, classify the anemia morphologically. Figure 14.6 Chromosome 11 is the location of four types 2. Name the dominant poikilocyte observed in this of globin genes (e, g, d, b) The 5′ to 3′ orientation of the genes is depicted. There is one gene for e, d, and b and two g@genes. A peripheral blood smear. b@pseudogene (cb) has been identified but does not express protein product. 302 Chapter 14 in a complete absence of b@chain synthesis from that allele. gene transcription, RNA processing, mRNA translation, and In addition, a minimally affected b@allele called silent car- post-translational integrity of the protein.25 Within a given rier (bSC) has been identified. It is found only in the most population, a few genetic lesions account for most of the benign version of b@thalassemia. In the heterozygous state, b@thalassemia mutations. For instance, in Greece, five muta- hematological features are normal; it is clinically benign. tions account for 87% of the gene defects.26 Diagnosis can be made only by finding a slight imbalance The phenotypic classification recognizes four groups of of a@/non@a@chain synthesis.9 When the three gene designa- patients categorized by the clinical severity of symptoms, tions, b0, b+, and bSC are combined with the normal allele medical interventions, and prognoses. The four groups, listed (b), and the two possible zygosity patterns (homozygous in order from most severe to least severe, are b@thalassemia and heterozygous) are considered, eight possible genotypes major, b@thalassemia intermedia, b@thalassemia minor, and (b0/b0, b0/b+, b+/b+, b0/b, b+/b, b/b, b/bSC, bSC/bSC) b@thalassemia minima (Table 14-5). emerge (Table 14-5). The phenotypic classification does not accurately reflect b@thalassemia is the result of several different types the genetic description of the disease. However, a disadvan- of molecular defects. More than 350 mutations resulting tage to the genotypic system is that patients with identi- in partial to complete absence of b@gene expression have cal genotype designations can express b@thalassemia that been described, but only 20 mutations account for 80% of is phenotypically diverse. For instance, a severe form of the diagnosed b@thalassemias.1,4 b@thalassemia is rarely b+/b+@thalassemia (Mediterranean form) is characterized by due to deletion of the structural gene as is the case in the an increase in HbF (50–90%) and a normal or only slightly a@thalassemias. Most defects in b@thalassemia are point elevated HbA2, whereas a milder form of b+/b+@thalassemia mutations in regions of the DNA that control b@gene expres- (black form) has 20–40% HbF with normal or elevated HbA2, sion. These types of mutations can affect gene expression and the remainder HbA. For this reason, some clinicians ranging from minor reductions in b@globin production to prefer the phenotypic system that more closely parallels complete absence of synthesis. Mutations can affect any
symptoms and better predicts clinical interventions neces- step in the pathway of globin gene expression, including sary for appropriate management of the patient. Table 14.5 Characteristics of b@Thalassemia RBC Genotype Zygosity Phenotype RBC Count Hb Electrophoresis Severity Morphology b0/b0 Homozygous Major Relative c c c c No A, c A2, c c Severe Target cells F b0/b+ Double heterozygous Major Relative c c c c T T Severe Target cells A, c A2, c c F Intermedia c c Target cells T A, c A2, c c F Moderate b+/b+ Homozygous Major Relative c c c c T T Severe Target cells A, cA2, c c F Intermedia c c T Moderate Target cells A, c A2, c c F b0/b Heterozygous Intermedia Relative c c c T Moderate Target cells A, c A2, c F Minor c T Mild Target cells A, c A2, c F b+/b Heterozygous Minor Relative c c T Mild Target cells A, c A2, c F bSC/bSC Homozygous Mild Intermedia Relative c c T Mild Target cells A, c A2, c F bSC/b Heterozygous Minima Normal { Normal Normal Target cells b/b Homozygous Normal Normal Normal Normal Normal c , slight increase; c c , moderate increase; c c c , marked increase; +/= , occasionally seen; T , slight decrease; T T , moderate decrease; A, HbA; A2, HbA2; F, HbF; bSC, silent carrier. Thalassemia 303 AFFECTED INDIVIDUALS • Reduced HbA The most severe mutation (b0) is found more frequently in • Compensatory production of other hemoglobins the Mediterranean regions—specifically in northern Italy, • Ineffective erythropoiesis with hemolysis Greece, Algeria, Saudi Arabia—and Southeast Asia. Mul- tiple severities of the b+ mutation have been described, and • Erythroid hyperplasia are found in different ethnic populations. More severe ver- A dramatic reduction in HbA compromises the blood’s sions are observed in the Mediterranean region, the Middle oxygen-carrying capacity. Other non@b@containing hemo- East, the Indian subcontinent, and Southeast Asia; milder globins, HbF and HbA2, are increased. HbF has a higher versions are generally found in patients of African descent.25 affinity for oxygen than HbA (Chapter 6). The result is to exacerbate the already compromised oxygen delivery to b@Thalassemia Major tissues. (b0/b0, b0/b+, b+/b+) In b@thalassemia major reduced synthesis of b@chains results in an excess of free a@chains and a b@to@a chain b@Thalassemia major, also referred to as Cooley’s anemia, is ratio of less than 0.25. The excess free a@chains cannot caused by a homozygous (b0/b0, b+b+) or double hetero- form hemoglobin tetramers, so they precipitate within zygous (b0b+) inheritance of abnormal b@genes resulting in the cell, damaging the cell membrane and leading to marked reduction or absence of b@chain synthesis. As can be chronic hemolysis.27 The accumulating a@chains contain seen in Table 14-5, two of these three genotypes (b0b+, b+b+) free iron and hemichromes that generate reactive oxygen can also present as the milder b@thalassemia intermedia species (ROS). The ROS damage hemoglobin as well as the phenotype that will be discussed subsequently. membrane proteins and lipids, decreasing membrane sta- bility.28,29 PATHOPHYSIOLOGY Oxidation of membrane Band 3 produces clus- The dramatic reduction or absence of b@chain synthesis tering of the proteins, creating new antigens on the cell affects the production of HbA. The symptoms that result surface that bind IgG and complement.30 Many IgG- and from b@thalassemia major begin to manifest in infants complement-sensitized erythrocytes in the bone marrow approximately 6 months after birth. Other non@b@containing are destroyed by binding to marrow macrophages via Fc hemoglobins, HbA2 and HbF, are increased in partial com- receptors, resulting in phagocytosis and a large degree of pensation for the decreased HbA levels. hemolysis. The pathophysiologic mechanisms that result from a The majority of the hemolysis occurs within the bone lack of b@chain production can be classified into four cat- marrow primarily at the polychromatophilic erythroblast egories (Figure 14-7): stage of erythroid development, resulting in ineffective Mutated b–gene (s) Normal a, g, d genes Excess a–chains Normal synthesis of a–chains Hb A Reduced synthesis of b–chains Enhanced synthesis of g–, d–chains Precipitation of a–chains Hb F, Hb A2 Oxygen affinity Reduced oxygen delivery Damaged RBC membranes & apoptosis ANEMIA Iron P.B. circulation Bone marrow: Phagocytosis EPO Toxicity Ineffective erythropoiesis B.M. expansion Splenic Hepatosplenomegaly Fractures hemolysis Mongoloid facies Figure 14.7 In b@thalassemia major, the decreased synthesis of b@chains reduces the production of HbA and increases the production of non@b@chain containing hemoglobins (HbA2 and HbF). Excess a@chains form insoluble precipitates inside erythrocytes, damaging the membranes and reducing RBC lifespan through splenic sequestration and ineffective erythropoiesis. All of these factors contribute to a reduced oxygen delivery to the tissues resulting in anemia and hypoxia. The compensatory erythroid hyperplasia in the bone marrow expands the marrow cavity, thinning the cortical bone, resulting in pathologic fractures and mongoloid facial features. 304 Chapter 14 erythropoiesis.31 The ineffective erythropoiesis is due to activation of apoptotic mechanisms by the precipitated a@globin chains and damaged cellular components.32,33 If the patient has inherited a@thalassemia with b@thalassemia, the symptoms associated with hemolysis may be reduced because the relative excess of a@chains is reduced by the co-inherited a@thalassemia genes. The combination of reduced HbA, increased HbF, ineffective erythropoiesis, and chronic hemolysis results in significant anemia. The body attempts to compensate by stimulating erythropoi- esis. The resulting erythroid hyperplasia causes bone mar- row expansion and thinning of calcified bone. Increased erythropoietic activity decreases hepcidin synthesis, which also stimulates the absorption of more iron in the gut, lead- ing to iron toxicity. Ineffective erythropoiesis in the bone marrow can be accompanied by extramedullary hemato- poiesis in the liver and spleen, often producing hepato- splenomegaly (Figure 14-7). Checkpoint 14.6 Why are the symptoms of b@thalassemia major delayed until approximately the sixth month of life? CLINICAL PRESENTATION Figure 14.8 Increased erythropoiesis in the bone marrow Early symptoms of b@thalassemia first observed in infants of patients with b@thalassemia major expands the marrow cavity include irritability, pallor, and a failure to thrive and gain producing the typical “hair-on-end” appearance as seen on this radiograph of the skull of a boy with b@thalassemia. weight, beginning at about 6 months of age. Diarrhea, fever, and an enlarged abdomen are also common findings. If therapy does not begin during early childhood, the clinical appetite. Infection is a common cause of death. Folic acid picture of severe thalassemia develops within a few years. deficiency can develop as a consequence of increased utili- Severe anemia is the clinical condition responsible for zation by the hyperplastic marrow. many problems experienced by these children. The anemia places a tremendous burden on the cardiovascular system LABORATORY EVALUATION as it attempts to maintain tissue perfusion. Constant high The hemoglobin level can be as low as 2-3 g/dL (20–30 g/L) output of blood usually results in cardiac failure in the first in the more severe forms of the disease. The anemia is decade of life and is the major cause of death in untreated markedly microcytic and hypochromic with an MCV of less children. Growth is retarded, and a bronze pigmentation than 67 fL34 and a markedly reduced MCH and MCHC. The of the skin is notable. Chronic hemolysis often produces peripheral blood smear shows marked anisocytosis and gallstones, gout, and icterus. poikilocytosis (Figure 14-9). Precipitates of a@chains can Bone changes accompany the hyperplastic marrow. be visualized with methyl violet stain. Variable basophilic Marrow cavities enlarge in every bone, expanding the bone stippling and polychromasia are noted. Reticulocytes are and producing characteristic bossing of the skull, facial not increased to the degree expected for the severity of the deformities, and “hair-on-end” appearance of the skull on anemia because of the high degree of ineffective erythro- x-ray (Figure 14-8). The thinning cortical bone in long bones poiesis. Nucleated erythrocytes are almost always found, can lead to pathologic fractures. and the RDW may be normal or increased.35 Secondary Extramedullary hematopoiesis may occur in the liver leukopenia and thrombocytopenia can occur because these and spleen and occasionally elsewhere in the body. The components also become trapped in the enlarged spleen. spleen can become massively enlarged and congested with Chronic hemolysis is reflected by increased unconjugated abnormal erythrocytes. bilirubin. Urine can appear dark brown from the presence Other clinical findings are associated with the body’s of dipyrroles. attempt to increase erythrocyte production. Features that Bone marrow studies are not usually necessary for suggest an altered metabolism include fever, lethargy, diagnosis but when performed show marked erythroid weakened musculature, decreased body fat, and decreased hyperplasia with an M:E ratio of 1:10 or less. Thalassemia 305 Definitive diagnosis of b@thalassemia can be made by demonstration of a b@to@a@chain ratio of less than 0.25. Molec- ular techniques demonstrating specific genetic mutations can also define the presence of thalassemia (Chapter 42). TREATMENT Most children with b@thalassemia major participate in a regular transfusion program, which prolongs life into at least the second or third decade and allows normal develop- ment and growth patterns. Initial treatment protocols were mainly palliative and designed to maintain a hemoglobin level of approximately 7–8 g/dL (70–80 g/L), but evidence Figure 14.9 Peripheral blood smear from a patient with of erythroid expansion and increased iron absorption per- b@thalassemia major showing marked anisopoikilocytosis. Target sisted. Clinical evidence suggests that the more aggressive cells, schistocytes, teardrops, and ovalocytes are the major hypertransfusion programs (maintaining a hemoglobin poikilocytes observed. A nucleated RBC is also present (Wright- level of 9–10.5 g/dL) offer the highest quality of life with- Giemsa stain; 1000* magnification). out significant sequelae.38 The objectives of hypertransfu- sion programs are to minimize the anemia, reduce excess Hemoglobin electrophoresis performed on cord blood iron absorption, and suppress ineffective erythropoiesis. samples provides evidence of deficient b@chain production The large doses of iron received with these transfusions, at birth. Although normal cord blood contains about 20% however, lead to tissue damage from iron overload similar HbA, cord blood from infants with b@thalassemia major has to that seen in hereditary hemochromatosis. Iron-chelating less than 2% HbA.14 In adults, hemoglobin electrophoresis agents such as deferoxamine, deferiprone and deferasirox are given to decrease the deposition of iron in the tissues.39,40 shows variable results, depending on the thalassemia alleles inherited. Absence of HbA, 90% HbF, and low, normal, or Splenectomy can be performed in an attempt to decrease increased HbA2 is characteristic of b0/b0@thalassemia.36 The hemolysis and prolong red cell survival. However, splenec- other genotypes, b0/b+ and b+/b+, show variable HbA, tomy is usually reserved for patients older than 5 years of but the majority of the hemoglobin is HbF with normal age who receive more than 200 mL of packed red blood to increased HbA 37 2 (Figure 14-10). The increased HbF in cells (PRBCs)/kg/yr and have leukopenia, thrombocyto- thalassemia is thought to be due to the expansion of a sub- penia, splenomegaly pain, or iron overload with chelation population of erythrocytes that have the ability to synthe- therapy.39 Risks associated with splenectomy include sepsis, thrombosis, and possibly pulmonary hypertension.41,42 size g@chains. The distribution of HbF among erythrocytes is heterogeneous. Bone marrow transplants (BMT) have been attempted in an effort to provide the individual with stem cells capa- ble of producing normal erythrocytes. Although BMT is an C G option for thalassemia patients with a suitable donor, this O D technology is not widely used.23 BMT can be replaced with E S CA CS A2 Lepore F A Portland Bart's H stem cell transplants (SCT) that yield less than 10% mortal- ity, minimal morbidity, and less infertility issues.43 Clinical b-thal major efficacy is controversial with the highest risk factors being a b-thal lack of engraftment, or graft-versus-host disease. Event-free major (med) survival is less than 70% for BMT and between 80 and 90% b-thal for SCT in patients with b@thalassemia who receive HLA- intermediate identical, related BMT.43,44 The success rate is lower for b-thal adults with more heavily-iron-overloaded disease. It may minor be difficult to find HLA-matched related donors, and suc- b-thal cess is much lower (ranging from 20 to 76%) using matched, minima unrelated donors.45 Haploidentical mother-to-child trans- CA, Carbonic anhydrase plantation may be an option for children without a matched CS, Constant Spring med, Mediterranean donor.45 Cord blood transplants are being investigated and show promise.15 With a typical life expectancy of around Figure 14.10 Hemoglobin electrophoresis pattern from 50 years when patients follow recommended transfusion patients with b@thalassemia shows a reduction in b@containing HbA and chelation protocols, supportive
therapy must be con- and an increase in non@b@containing HbA2 and HbF. sidered over curative therapy unless the patient is young, 306 Chapter 14 fit, and considered for transplant prior to developing thal- heterozygous b+ patient, the thalassemic gene will also con- assemia-related sequalae (hepatomegaly, portal fibrosis, tribute to b@chain production. iron-overload).4 CLINICAL PRESENTATION Drugs to treat leukemia are being used to induce the The heterozygote appears to be asymptomatic except in re-expression of latent g@genes that would combine with periods of stress that can occur during pregnancy and with the excess a@chains and produce HbF.46 The two major risk infections. Under such conditions, a moderate microcytic factors for this treatment approach include failure of g@chain anemia can develop. Concomitant folate deficiency can pro- stimulation and induction of malignancy from the antileu- duce a macrocytic anemia. kemic drugs. There is continuing interest in gene therapy approaches LABORATORY EVALUATION in which autologous stem cells are harvested and the b@gene The anemia in b@thalassemia minor is mild with hemoglo- complex transfected using viral vectors and transplanted bin values in the range of 9–14 g/dL with the mean value back into the host. However, several complications prevent for women being 10.9 g/dL and for men 12.9 g/dL.25 The widespread use. Human globin genes are large and compli- erythrocyte count is increased (greater than 5 * 106/mcL) cated, making the finding of adequate viral vectors difficult. for what is expected at the given hemoglobin concentra- In clinical trials of gene therapy for severe immunodefi- tion. The condition is usually discovered incidentally dur- ciency diseases, some patients have developed leukemias ing testing for unrelated symptoms or during family study when the transfected genes insert next to or within critical workups. hematopoiesis control genes. The first thalassemia patient The erythrocytes are microcytic (MCV = 55-70 fL) and to receive gene therapy was reported in 2007.47 hypochromic (MCHC = 29-33 g/dL) or sometimes nor- The use of induced pluripotent stem cells (iPS) shows mochromic with an MCH that is usually less than 22 pg. The promise for hematopoietic and genetic disorders in the degree of microcytosis, as indicated by the MCV, is directly future. Somatic skin fibroblasts can be harvested and repro- related to the severity of the anemia.27 Although the anemia grammed into iPSs by inducing the expression of transcrip- is mild, the peripheral blood smear shows variable aniso- tion factors essential for maintaining pluripotency in early cytosis and poikilocytosis with target cells and basophilic embryos and embryonic stem cells.48 When grown in cul- stippling. Nucleated red blood cells are not usually found, ture, these cells could be induced to differentiate into multi- but anemic patients can have a slightly elevated reticulocyte potent hematopoietic stem cells and then autotransplanted. count. Bone marrow shows slight erythroid hyperplasia and Although significant obstacles must be overcome, cor- erythroblasts poorly filled with hemoglobin. rection of the molecular defect with gene therapy is thought Hemoglobin electrophoresis demonstrates an increase to be achievable in the future. in HbA2 of 3.5–7.0% with a mean of 5.5%. Newborns have a normal HbA2 concentration of 0.27 { 0.02,.25 HbF is nor- PROGNOSIS mal in approximately half of the patients and increased in Untreated patients generally expire during the first or the other half (Figure 14-10). If HbF exceeds 5%, however, second decade of life. Patients enrolled in a hypertransfu- the individual has probably inherited an HPFH gene (see sion program with chelation therapy can extend their life “Hereditary Persistence of Fetal Hemoglobin”) in addition expectancy by at least a decade.49 Usually in the second to the b@thalassemia gene. Vital stains to detect Heinz bod- decade of life, endocrine disorders (e.g., diabetes) and ies are usually negative. In summary, b@thalassemia minor hepatic and cardiac disturbances develop from excessive is indicated when the MCH is less than 27 pg and the HbA2 deposits of iron in these tissues if chelation therapy has not is more than 3.5%. been successful. Nucleic acid–based techniques can be performed to identify the type of mutation present and validate the het- b@Thalassemia Minor (b0/b or b+/b ) erozygous inheritance pattern but are of limited diagnostic value. Such information, however, can be helpful in coun- b@thalassemia minor results from the heterozygous inher- seling prospective parents with b@thalassemia minor. itance of either a b+@ or b0@gene with one normal b@gene (Table 14-5). No major clinical difference seems to exist in the expression between the two thalassemia genes in the Checkpoint 14.7 heterozygous state. About 1% of African Americans are het- In b@thalassemia, what erythrocyte parameter on the CBC dif- erozygous for b@thalassemia. fers significantly from that found in iron deficiency? PATHOPHYSIOLOGY The normal b@gene directs synthesis of sufficient amounts TREATMENT AND PROGNOSIS of b@chains to synthesize enough HbA for nearly normal Patients generally do not require treatment if they maintain oxygen delivery and erythrocyte survival. In the case of a good health and nutrition. They are generally asymptomatic Thalassemia 307 except during periods of physiologic stress, and they have a normal life expectancy. b@Thalassemia Intermedia (b+/b+, b0/b+, b0/b) All three patterns of inheritance—homozygous, double het- erozygous, and heterozygous—can produce b@thalassemia intermedia (Table 14-5). The homozygous and double het- erozygous forms represent a mutation in both b@alleles, resulting in a moderate reduction in b@chain synthesis. Patients who inherit a mutation of one b@gene in conjunction with a normal b@gene occasionally exhibit clinical symp- Figure 14.11 Patients with b@thalassemia minor show toms significant enough to be classified as b@thalassemia minimal morphologic abnormalities to include microcytosis with target cells. The CBC for the patient with this blood smear was: Hb intermedia rather than b@thalassemia minor. 11.1 g/dL; RBC count 5.2 * 106/mcL; MCV 61 fL; MCH 20.2 pg; Patients with b@thalassemia who co-inherit MCHC 33 g/L (Wright-Giemsa stain; 1000* magnification). a@thalassemia or HPFH can actually experience milder symptoms as compared with those with pure b@thalassemia. In both cases, excess free a@chain accumulation and precipi- Target cells are the predominant poikilocytes observed. tation are reduced, decreasing the ineffective erythropoiesis Basophilic stippling and nucleated red blood cells are also and extravascular hemolysis responsible for much of the present. The bone marrow shows hypochromic erythro- pathology. In the case of co-inherited b@thalassemia and blasts in the context of erythroid hyperplasia. However, HPFH, the overexpressed g@chains combine with the exces- bone marrow examination is not needed for diagnosis. sive a@chains to produce HbF, which reduces a@chain excess Hemoglobin electrophoresis patterns in patients with and precipitation. the more severe forms of b@thalassemia intermedia (b0b+ and b+b+) are nearly indistinguishable from those observed CLINICAL PRESENTATION in the milder forms of b@thalassemia major. Patients express Patients with b@thalassemia intermedia present with elevated HbA2 (5–10%) and HbF (30–75%) with the remain- symptoms of clinical severity intermediate between severe der being HbA. Milder versions of b@thalassemia interme- b@thalassemia major and mild b@thalassemia minor. The dia produce lower HbA2 (3.2,) and HbF levels (1.5–12.0%) b0/b+@genotype produces the greatest reduction in b@chain (Figure 14-10). Although hemoglobin electrophoresis is synthesis but has a variable clinical presentation; some helpful in diagnosing b@thalassemia intermedia, differen- patients have symptoms of b@thalassemia major, whereas tiation from b@thalassemia major and minor is a clinical others have a milder clinical phenotype. Patients who inherit decision. the b0/b@genotype generally exhibit mild symptoms. The cri- terion for b@thalassemia intermedia is the ability to maintain TREATMENT AND PROGNOSIS a hemoglobin level associated with a comfortable survival Splenomegaly is common. Functional hyposplenism leads without requiring regular transfusions.17 The need for trans- to infections requiring regular interventions with antibiotic fusions is defined by the quality of life, not the hemoglobin therapy. Chelation therapy may be warranted to combat level per se. Some patients with a hemoglobin level of 7 g/dL iron overload, which tends to develop later than in patients can be relatively symptom free, but others with a hemoglobin with b@thalassemia major. Most patients have a normal life of 9 g/dL can have clinical symptoms related to ineffective span. erythropoiesis. Clinical symptoms often intensify during periods of physiological stress as with pregnancy and infec- b@Thalassemia Minima (bSC/b) tion and can require short-term transfusion therapy. Even in b@Thalassemia minima is a form of asymptomatic the absence of regular transfusions, some patients develop b@thalassemia, exhibits no major laboratory abnormalities, progressive iron overload and require iron-chelation therapy. and is only defined by a mildly imbalanced a@ to non@a glo- LABORATORY EVALUATION bin chain synthesis ratio. The disorder is usually discovered The CBC reflects a moderate microcytic hypochromic serendipitously during family studies. The gene has been anemia with a hemoglobin value range of 7 to 10 g/dL given the designation bSC for silent carrier. The genotype (Figure 14-11). In milder cases, patients express only a slight used to describe a patient with b@thalassemia minima is reduction in hemoglobin values. The erythrocyte count is bSC/b. Homozygosity for bSC or combination of the silent disproportionately higher than the hemoglobin values and allele with other b@thal genes results in a mild form of thal- often approaches normal. assemia intermedia. 308 Chapter 14 structural hemoglobin disorders such as sickle cell anemia CASE STUDY (continued from page 301) and HbC disease (Table 14-6). Additional tests were performed on John’s blood to determine the cause of his anemia. db@Thalassemia Hemoglo- Electro- Iron d@, b@thalassemia (db@thalassemia) is a rare thalassemia bin phoresis Panel observed primarily in patients of Greek, African, Italian, HbA 66% Serum 92 mcg/dL and Arabian ancestry whose production of both b@ and iron d@chains is affected. The db@mutation can be categorized into HbA2 1% TIBC 310 mcg/dL two genotypes, db0 and db+. The db0 designation indicates a complete lack of synthesis of both b@ and d@chains from HbF 1% Serum 88 ng/mL a given chromosome, whereas the db+@genotype indicates ferritin a reduction in b@ and d@chain synthesis. The absence of b@ Hb Bart 8% Iron 33% and d@chains is most often due to deletion of the structural saturation b@ and d@gene complex. One or both of the g@genes remain, HbH 24% resulting in 100% HbF in homozygous db0/db0.25 However, increased g@chain production fails to fully compensate for 5. Is the hemoglobin electrophoresis normal or the loss of b@chain production, resulting in a@chain excess, abnormal? a thalassemic phenotype and anemia. 6. If abnormal, list hemoglobins that are elevated, In db@thalassemia, the g@chain synthesis is less than in decreased, or abnormally present. HPFH but more than in homozygous b0@thalassemia. Clini- cally, the disease is classified as thalassemia intermedia and 7. If abnormal, which globin chain(s) is (are) rarely requires blood transfusions except in cases of physi- decreased? ological stress such as pregnancy or infection. However, 8. If abnormal, which globin chains are produced because of the impaired hemoglobin synthesis, most patients in excess? with db@thalassemia have a mild hypochromic, microcytic anemia. Patients have slight hepatosplenomegaly and some 9. Is the iron panel normal or abnormal? bone changes associated with chronic erythroid hyperpla- 10. If the iron tests are abnormal, list those outside sia. Hemolysis probably contributes to the anemia because the reference interval and indicate whether they both reticulocytes and bilirubin are elevated. are elevated or decreased. The heterozygous form of db@thalassemia (db0/b) is not identified with any specific clinical finding. There is no 11. If the iron tests are abnormal, state the disorder(s) anemia or splenomegaly. The hematological picture, how- consistent with the abnormal iron panel. ever, is similar to that of b@thalassemia minor with micro- 12. Given all the data supplied, what is the definitive cytic, hypochromic erythrocytes. HbA2 is normal or slightly diagnosis of John’s anemia? decreased, whereas HbF is increased to 5–20%. HbA is usu- ally less than 90% (Table 14-6). gdb@Thalassemia Other Thalassemias This rare form of thalassemia (g@, d@, b@ thalassemia has sev- eral variants and is characterized by deletion or inactivation and Thalassemia-Like of the entire b@gene complex.33 Deletion of the g@, d@, and b@genes would result in the absence of all normal adult hemo- Conditions globin production from that chromosome. Therefore, only the heterozygous state has been encountered because a homozy- In theory, any globin gene can be mutated, resulting in a gous condition would be incompatible with life. Although reduction in the synthesis of globin chains and the corre- neonates have severe hemolytic anemia, as the children grow, sponding hemoglobin. Individuals have been observed the disease evolves to a mild form of b@thalassemia. with deficiencies in each of the normal adult globin chains. However, the thalassemias that involve
globin chains other than a and b are relatively benign in their clinical course Hemoglobin Constant Spring because they are not constituents of the major adult hemo- Hemoglobin Constant Spring (HbCS) is a hemoglobin tet- globin, HbA. Thalassemias have been observed involv- ramer formed from the combination of two structurally ing more than one globin gene and in combination with abnormal a@chains, each elongated by 31 amino acids at Thalassemia 309 Table 14.6 Characteristics of Other Thalassemias and Thalassemia-Like Conditions Disorder Defect Ancestry CBC/diff Hb Electrophoresis Other db@thalassemia homo- Deletion of db@gene Greek, African, Micro/hypo RBCs 100% HbF* Thalassemia intermedia zygous (db0/db0) complex Italian, Arabian Hb 10–12 g/dL heterozygous Same as No anemia T HbA, N@T HbA2, c Thalassemia minor (db0/b) homozygous HbF gdb@thalassemia Deletion or inactivation Mediterranean Birth: Marked anemia; THbA, THbA2, T HbF Thalassemia minor heterozygous of b@gene complex regions Adult: Slight anemia (gdb0/b) homozygous Same as Unable to observe No adult Hb Incompatible with life (gdb0/gdb0) heterozygous Constant Spring Long a@chain, mutated Thailand Micro/hypo RBCs, Hb Bart at birth, c c homozygous stop codon, reduced Hb 9–11 g/dL, c HbCS, THbA, N@HbA2 (aaCS/aaCS) synthesis reticulocytes and HbF heterozygous Same as Normal c HbCS Can be seen with a@thal2 (aaCS/aa) homozygous trait HPFH homozygous Deletion or inactivation Greek, Swiss, Mild micro/hypo RBCs, 100% HbF Two variants— pancellular, (db0/db0) of db@gene complex black no anemia, c c RBCs, heterocellular Hb 14–18 g/dL Asymptomatic heterozygous (db0/b) Same as Near normal c HbF, THbA, THbA2 Asymptomatic homozygous Hb Lepore db@hybrid chain European Marked micro/hypo No HbA, No HbA2, c c Thalassemia major or homozygous RBCs, Hb 4–11 g/dL, Hb Lepore, c c HbF intermedia (dbLepore/dbLepore) c c c anisopoikilocytosis heterozygous Same as Slight micro/hypo RBCs, T HbA, THbA2 c Hb Thalassemia minor (dbLepore/b) homozygous Hb 12 g/dL Lepore * Not all d/b0 thalassemias are deletional. Some nondeletional gene mutations produce some d and b chains and thus will show some HbA and HbA2, but the major hemoglobin is HbF. c c c , marked increase; c c , moderate increase; c , slight increase; T T , moderate decrease; T , slight decrease; N, normal. the carboxyl-terminal end, and two normal b@chains. This Heterozygotes show no hematological abnormalities, genetic mutation is common in Thailand. The chromosome but a small amount of HbCS (0.2–1.7%) can be found on with the aCS gene carries one normal a@gene. Thus, the electrophoresis. HbA2 and HbF are normal with the remain- homozygous HbCS individual has two normal a@genes, one der being HbA (Figure 14-2). on each chromosome, and the heterozygous HbCS carrier In some areas, the coexistence of HbCS with has three normal a@genes. a@thalassemia trait (- -/aaCS) can be found. The clinical The elongated a@chains of HbCS are thought to result findings are similar to those of HbH disease. Hemoglobin from a mutation of the a@chain termination codon by a single electrophoresis characteristically shows HbA, HbH, Hb base substitution.50 The abnormal a@chains are synthesized Bart, HbA2, and 1.5–2.5% HbCS51 (Figure 14-2; Table 14-6). at very low levels (about 1% of the output compared with a normal a gene) because of reduced stability of the elongated mRNA. The result is an overall deficiency of a@chain synthe- Checkpoint 14.8 sis producing an a@thalassemia@like phenotype. Why is gdb@thalassemia more severe than db@thalassemia and CS-thalassemia? CLINICAL PRESENTATION The homozygous state is phenotypically similar to a@thalassemia minor. A slight anemia accompanied by mild jaundice and splenomegaly is typical. Heterozygotes show Hereditary Persistence of Fetal no clinical abnormalities. Hemoglobin (HPFH) LABORATORY EVALUATION Hereditary persistence of fetal hemoglobin (HPFH) is actu- In homozygotes, clinical findings are similar to a relatively ally a group of heterogeneous disorders caused by either mild form of HbH disease with a mild microcytic hypochro- the absence of d@ and b@chain synthesis or a loss of suppres- mic anemia. Hemoglobin electrophoresis demonstrates the sion of the g@globin gene, resulting in an increased g@chain presence of Hb Bart at birth. In homozygous adults, HbCS production into adult life. In homozygotes, the result is an makes up 5–7% of the hemoglobin, and HbA2 and HbF are absence of HbA and HbA2 with 100% of hemoglobin pro- normal. The remainder of hemoglobin is HbA (Figure 14-2). duction being HbF. Hemoglobin F production continues at 310 Chapter 14 high levels throughout life, preventing the clinical symp- toms and hematological abnormalities associated with thal- Table 14.7 Characteristics of HPFH Variants assemia. The condition occurs in 0.1% of African Americans. Types of g@chains Distribution of HbF HPFH Type Produced in Erythrocytes GENETICS Black Gg@ and Ag@ Pancellular HPFH is characterized by either deletion or inactivation of the b@ and d@structural gene complex, mutations in Swiss Gg@ and Ag@ Heterocellular the g@globin gene promoter region affecting the binding Greek Primarily Ag@ Pancellular of transcription factors,52,53 or mutation of gene inhibitor proteins.54,55,56 Four potential mechanisms that explain the b@thalassemias with homozygous b0@thalassemia at one end increase in g@globin gene expression include: where the lack of b@chain synthesis is poorly compensated 1. Removes the d and b promoters and relieves competi- for by g@chain production and with pancellular HPFH at tion with the g@gene promoters for transcription factor the other end where the lack of b@chain synthesis is almost activation OR completely compensated for by g@chain production. 2. removes the g@gene inhibitor–binding sites; HOMOZYGOTES 3. mutations might juxtapose b@gene enhancer sequences Homozygous HPFH is asymptomatic, including no evi- to g@genes, facilitating activation of g@genes; and dence of abnormal growth patterns or splenomegaly. The abundant g@chains combine with the normal a@chains to 4. mutated transcription factors increase activation or produce HbF. inhibit suppression of the g@globin gene.4 Erythrocytosis ranging from 6 to 7 * 106/mcL occurs Most a@chains combine with the available g@chains as the result of the higher oxygen affinity of HbF as com- to produce HbF. Consequently, no accumulation and no pared with HbA. Corresponding high hemoglobin levels precipitation of excess a@chains occur. In HPFH, increased from 14.8 to 18.2 g/dL are also typical in HPFH. Erythro- production of g@chains with the corresponding elevations cytes are microcytic and slightly hypochromic with a mean of HbF compensates for the reduction in HbA and HbA2 MCV of 75 fL and a mean MCH of 25.0 pg. There is a mild synthesis and differentiates it from db@thalassemia in which degree of anisocytosis and poikilocytosis. The reticulocyte only modest elevations in HbF are observed. count range is 1 to 2%. It is doubtful that this disorder has any significant degree of hemolysis because the reticulocyte VARIANTS count, bilirubin, and haptoglobin levels are normal. Electro- HPFH can be categorized into two major groups, pancel- phoresis demonstrates 100% HbF. lular and heterocellular, based on the distribution of HbF in erythrocytes. The HbF distribution patterns can be visu- HETEROZYGOTES alized using the acid elution stain developed by Kleihauer Heterozygous HPFH is usually found incidentally through and Betke. Pancellular refers to the observance of HbF in family studies. Patients present with a slightly elevated most of the erythrocytes; in the heterocellular version, HbF erythrocyte count with the corresponding elevation of the is concentrated in a small subset of erythrocytes. hematocrit and a slightly decreased MCH (27 pg). HbF Several different types of HPFH have been described: is 10–30% of the total hemoglobin. HbA2 is decreased to the black, Greek, and Swiss types. In the black and Swiss 1–2% and the remainder is HbA. In the presence of iron types, both Gg@ and Ag@chains are produced in approxi- deficiency, HbF levels are lower (Table 14-6). mately equal amounts. The Greek form is characterized by production of both Gg@ and Ag@chains, but most HbF is Hemoglobin Lepore made up of the Ag@chains. Both the black and Greek types have the characteristic pancellular distribution pattern of Hemoglobin Lepore was first described in 1958 as a struc- HbF in erythrocytes. The Swiss form exhibits a hetero- tural hemoglobin variant with hematological changes and cellular distribution of HbF, which results from a heredi- clinical manifestations resembling those of thalassemia.57 tary increase (3%) in the number of HbF-containing cells The disorder is widely distributed throughout the world (F cells)25 (Table 14-7). but is especially common in middle and eastern Europe. When present, the pancellular distribution of HbF in GENETICS erythrocytes helps to distinguish this disorder from other In Hb Lepore, the non@a@chain is a d/b@globin hybrid disorders associated with an increase in HbF. Most other in which the N-terminal end of a d@chain is fused to the disorders with elevated HbF levels present with the hetero- C-terminal end of a b@chain. The variant hybrid genes are cellular distribution pattern. thought to occur during meiosis from an aberrant cross- It has been suggested that the various categories of over event resulting in recombination of misaligned d@ and HPFH actually represent a continuum of a spectrum of b@genes on separate chromosomes. The result of the unequal Thalassemia 311 crossover event is two fusion genes, the d/b@Lepore and the b/d@anti@Lepore genes. The d/b@Lepore fusion gene is tran- scribed and translated into the d/b@fusion globin chain, two of which combine with two a@chains to form Hb Lepore. The chromosome containing the b/d@anti@Lepore fusion gene still contains intact b@ and d@genes that are synthesized normally to form HbA and HbA2, respectively. Because the recombination event occurred in the germ cells, the newly formed chromosomes become a permanent part of the family’s gene pool. Hb Lepore is stable and has normal functional properties except for a slight increase in oxygen affinity, however it is associated with a thalassemic pheno- Figure 14.12 Peripheral blood smear from a patient with type (see “Pathophysiology” below). hemoglobin Lepore. Note the anisocytosis, poikilocytosis, and microcytosis (Wright-Giemsa stain; 1000* magnification). PATHOPHYSIOLOGY The pathophysiology of hemoglobin Lepore is similar to that Combination Disorders of b@thalassemia. No intact b@gene is present on the chromo- Occasionally an individual is doubly heterozygous for a some carrying the Lepore fusion gene, so b@chain synthesis structural hemoglobin variant and thalassemia, inheriting is absent. The Hb Lepore gene is under the influence of the one of each of the two abnormalities from each parent. The d@gene promoter, which limits synthesis of the Lepore hybrid most common structural hemoglobin variants involved in chain to approximately 2.5% of normal b@chain production. combination disorders are HbS, HbC, and HbE. When a Thus, the abnormal Lepore chains are inadequately synthe- structural variant is inherited with a b@thalassemia gene, sized, leading to an excess of a@chains. In the homozygous the severity of the combination disorder depends on the state, no normal b@chains or d@chains would be synthesized type of b@gene mutation. Patients expressing the b0@gene to combine with the a@chains being produced, thus, no HbA produce no HbA and experience moderate to severe symp- or HbA2. The more severely affected children can be transfu- toms. The b+@gene produces some b@chains, resulting sion dependent and develop complications of hemosidero- in HbA synthesis and few to no thalassemia symptoms. sis. The combination of ineffective erythropoiesis, decreased The most common example is HbS/b@thalassemia, which HbA, increased oxygen affinity of HbF, and chronic extra- has been reported in patients of African, Greek, Turkish, vascular hemolysis produces a microcytic hypochromic ane- Indian, North American, Mediterranean, and Romanian mia that is classified clinically as b@thalassemia major in the ancestry.58 Three clinical severities have been identified: more severe cases and as b@thalassemia intermedia in the HbS/b0@Type 1 (severe), HbS/b+@Type 1 (moderate), and remaining cases. As with b@thalassemia major, symptoms HbS/b+@Type 2 (asymptomatic). Differentiating this com- emerge within the first few years of life. bination disorder from sickle cell disease (HbSS) and trait Patients with heterozygous Hb Lepore are asymptom- (HbAS) is sometimes difficult, but a comparison of b:a ratio atic and classified clinically as b@thalassemia minor. The can be helpful (Table 14-8). In sickle cell disorders, the b:a blood picture is similar to that seen in b@thalassemia minor. ratio is approximately 1:1, whereas it is closer to 0.5:1 in LABORATORY EVALUATION HbS/b@thalassemia.59 In the homozygous state, hematologic findings are similar to Combination disorders are more complex when the those of b@thalassemia major. There is no detectable HbA or structural hemoglobin gene mutation is on the b@gene A2 on hemoglobin electrophoresis, Hb Lepore ranges from and the thalassemia mutation involves the a@gene because 8 to 30%, and the remainder consists of HbF. Hemoglobin different chromosomes are involved. These patients can electrophoresis must be interpreted with caution because Hb be either