EXERCISE 8: Manual Leukocyte Count

MLAB 1315 Hematology

MANUAL WBC
LAB OBJECTIVE The tu!ent "#ll $e a$le to %e&'o&m( "#th#n _)*+ accu&acy a com%a&e! to the automate! &e ult( '#,e manual "h#te $loo! cell count u #ng the -no%ette y tem. PRINCIPLE /&ee0'lo"#ng ca%#lla&y o& "ell0m#1e! ant#coagulate! 2E3TA4 ,enou $loo! # a!!e! to a !#luent 2ammon#um o1alate4 at a %ec#'#c ,olume #n the -no%ette &e e&,o#&. The !#luent ly e the e&yth&ocyte $ut %&e e&,e leukocyte an! %latelet . The !#lute! $loo! # a!!e! to the hemacytomete& cham$e&. Cell a&e allo"e! to ettle 'o& 1* m#nute $e'o&e leukocyte an! %latelet a&e counte!. SPECIMEN E3TA0ant#coagulate! $loo! o& ca%#lla&y $loo! # %&e'e&&e!. REAGENTS, SUPPLIES AND EQUIPMENT -no%ette &e e&,o#& no. 585565855 21.78 ml o' !#luent 8 *.*) ml $loo! 9 161** !#lut#on44 Ammon#um o1alate 11.55 g So&en en: %ho %hate $u''e& 1.* g Th#me&o al *.1 g ;u&#'#e! "ate& < to 1*** mL -no%ette ca%#lla&y %#%et( )*0=L ca%ac#ty Hemacytomete& an! co,e& l#% M#c&o co%e L#nt0'&ee "#%e 2>#m ?#%e 4 Alcohol %a! Han! counte& ;et&# !# h "#th mo# t '#lte& %a%e&

LAB EXERCISES

MLAB 1315@ 1

EXERCISE 8: Manual Leukocyte Count

MLAB 1315 Hematology

Note on the Hemac tomete! The hemacytomete& count#ng cham$e& # u e! 'o& cell count#ng. It # con t&ucte! o that the !# tance $et"een the $ottom o' the co,e& l#% an! the u&'ace o' the count#ng a&ea o' the cham$e& # *.1 mm.

LAB EXERCISES

MLAB 1315@ )

EXERCISE 8: Manual Leukocyte Count

MLAB 1315 Hematology

The u&'ace o' the cham$e& conta#n t"o <ua&e &ule! a&ea e%a&ate! $y an H0 ha%e! moat. The e t"o <ua&e a&e #!ent#cal( allo"#ng the technolog# t to !u%l#cate the cell count. Each ha a total a&ea o' 7 mm) 21 mm on each #!e4. The e <ua&e a&e !#,#!e! #nto n#ne %&#ma&y <ua&e "#th an a&ea o' 1mm). The 'ou& co&ne& %&#ma&y <ua&e a&e u e! "hen count#ng leukocyte . (If the WBC is very low, you would count all 9 squares.4 The e 5 la&ge co&ne& <ua&e conta#n 1A malle& econ!a&y <ua&e ( each "#th an a&ea o' *.*5 mm). All )5 econ!a&y <ua&e o' the

cente& %&#ma&y <ua&e a&e u e! to count %latelet ( an! each o' the e )5 <ua&e # 'u&the& !#,#!e! #nto 1A malle& te&t#a&y <ua&e 2 ee '#gu&e4. Cell "h#ch l#e on LE/T an! TB; $oun!a&y l#ne o' the <ua&e a&e counte!. Cell "h#ch l#e on the RICHT an! BBTTBM $oun!a&y l#ne o' the <ua&e a&e DBT counte!. 2Th# # to make cell count tat# t#cally accu&ate.4 Hemacytomete& an! co,e& l#% houl! meet the %ec#'#cat#on o' the Dat#onal Bu&eau o' Stan!a&! an! a&e o ma&ke! $y the manu'actu&e&. A tan!a&!#Ee! co,e& l#% houl! $e u e! that ha $een g&oun! to '#t the %ec#'#c o' the hemacytomete&( en u&#ng a un#'o&m !e%th an! the&e'o&e a con tant ,olume. A &egula& co,e& l#% cannot $e u e!. QUALIT" CONTROL A no&mal cont&ol %ec#men houl! $e counte!. ;e&'o&m e t#mate! ?BC '&om ?&#ght0 ta#ne! %e&#%he&al mea& to con'#&m &e ult. DBTE: /o& th# la$ e1e&c# e( FC "#ll $e %&ec# #on check "#th automate! ?BC.

LAB EXERCISES

MLAB 1315@ 3

EXERCISE 8: Manual Leukocyte Count

MLAB 1315 Hematology

1. ). 3. 5.

PROCEDURE Refer to figure for following instructions. - #ng the %&otect#,e h#el! on the ca%#lla&y %#%ette( %unctu&e !#a%h&agm o' -no%ette &e e&,o#&. Remo,e h#el! '&om %#%ette a em$ly $y t"# t#ng. Hol!#ng %#%ette almo t ho&#Eontally( touch t#% o' %#%et to #e$$%m&'e( $loo!. ;#%et "#ll '#ll $y ca%#lla&y act#on. /#ll#ng "#ll cea e automat#cally "hen the $loo! &eache the en! o' the ca%#lla&y $o&e #n the neck o' the %#%et. ?#th a >#m ?#%e( "#%e the out #!e o' the ca%#lla&y %#%et WELL 2'&om to% o' ca%#lla&y %#%et to the $ottom o' the %#%et4 to &emo,e e1ce $loo! that "oul! #nte&'e&e "#th the !#lut#on 'acto&. A,o#! touch#ng the t#% o' the %#%et "#th a >#m ?#%e to a,o#! &emo,#ng any $loo! '&om the %#%et. ?#th one han!( <ueeEe &e e&,o#& l#ghtly to 'o&ce out ome a#& "h#le #multaneou ly ma#nta#n#ng %&e u&e on &e e&,o#&. ?#th the othe! han!( co,e& the o%en#ng o' o,e&'lo" $ack cham$e& o' %#%et "#th #n!e1 '#nge&. ;lace the %#%et ecu&ely #n neck o' the &e e&,o#&. T"# t the %#%et '#&mly #nto the &e e&,o#& neck to 'o&m a t#ght eal. Relea e %&e u&e '&om the '#& t han! on &e e&,o#&. Then &emo,e '#nge& on the o%%o #te han! '&om %#%et o%en#ng. At th# t#me negat#,e %&e u&e "#ll !&a" $loo! #nto &e e&,o#&. S<ueeEe &e e&,o#& gently t"o o& th&ee t#me to &#n e ca%#lla&y $o&e 'o&c#ng !#luent u% #nto( $ut not out o'( o,e&'lo" cham$e&( &elea #ng %&e u&e each t#me to &etu&n m#1tu&e to &e e&,o#&. ;lace #n!e1 '#nge& o,e& u%%e& o%en#ng an! gently #n,e&t e,e&al t#me to tho&oughly m#1 $loo! "#th !#luent. Let m#1tu&e tan! 1* m#nute $e'o&e cha&g#ng the hemacytomete&.to allo" the &e! cell to ly e. ?h#le the -no%ette # tan!#ng 'o& 1* m#nute ( tho!o)*h$ clean the hemacytomete& an! #t co,e& l#% "#th an alcohol %a! an! then !&y "#th a >#m ?#%e. To cha&ge the hemacytomete&( con,e&t to !&o%%e& a em$ly $y "#th!&a"#ng %#%et '&om &e e&,o#& an! &e eat#ng ecu&ely #n !e+e!,e %o #t#on. Tho&oughly m#1 the $loo!6!#luent m#1 $y "#&l#ng the a em$ly. In,e&t &e e&,o#& an! !# ca&! the '#& t 3 o& 5 !&o% o' m#1tu&e. Ca&e'ully cha&ge cleane! hemacytomete& "#th !#lute! $loo! $y gently <ueeE#ng #!e o' &e e&,o#& LAB EXERCISES MLAB 1315@ 5

5. A. G. 8. 7. 1*. 11. 1). 13. 15.

EXERCISE 8: Manual Leukocyte Count

MLAB 1315 Hematology

to e1%el content unt#l cham$e& # %&o%e&ly '#lle!. The cham$e& m),t $e com%letely '#lle! "#thout $u$$le (See A below ( $ut the $loo!6!#luent m#1tu&e m),t NOT o,e&'#ll the cham$e& (See B below .

15.

1A. 1G.

?#thout !# tu&$#ng the co,e& l#%( %lace hemacytomete& #n mo# t ;et&# !# h 'o& 1* m#nute to allo" "h#te cell to ettle #nto the ame %lane. 2Mo# tene! '#lte& %a%e& &eta&! e,a%o&at#on o' the %late! %ec#men "h#le tan!#ng.4 3o not let the hemacytomete& #t mo&e than 15 m#nute #n the ;et&# !# h $ecau e e,a%o&at#on "#ll $eg#n to occu& an! cau e e&&oneou count . ?#thout !# tu&$#ng the co,e& l#%( mount the hemacytomete& on the m#c&o co%e an! lo"e& the con!en e&. ;&oce!u&e 'o& count#ng ?BC:

A. -n!e& 1*1 magn#'#cat#on( can to en u&e e,en !# t&#$ut#on. Leukocyte a&e counte! #n THE -OUR OUTSIDE $a!*e ,.)a!e, o' count#ng cham$e&. B. Count cell ta&t#ng #n the u%%e& the le't to% la&ge co&ne& <ua&e. Mo,e to the u%%e& &#ght co&ne& <ua&e( $ottom &#ght co&ne& <ua&e( an! en! #n the $ottom le't co&ne& LAB EXERCISES MLAB 1315@ 5

EXERCISE 8: Manual Leukocyte Count

MLAB 1315 Hematology

<ua&e. C. Count all cell that touch any o' the u%%e& an! le't l#ne ( !o not count any cell that touche a lo"e& o& &#ght l#ne. 3. Calculat#on 'o& count#ng 5 la&ge <ua&e : 14 - e the a,e&age total num$e& o' cell counte! '&om $oth #!e o' the hemacytomete& (!ne side includes the total nu"ber of cells counted in four large squares and the other side includes the total nu"ber of cells counted in its four large squares . )4 /#nal cell count # &e%o&te! a the num$e& o' "h#te $loo! cell %e& m#c&ol#te&

2?BC6HL4. 34 /o&mula:
?BC6 L 9 A,e&age o' cell 1 Co&&ect#on 'o& !#lut#on Do . o' <ua&e counte! 1 Iolume o' one <ua&e

A+e!a*e o/ ce$$, 9 the a,e&age o' the total num$e& o' cell counte! #n the 'ou& la&ge <ua&e on BBTH #!e o' the hemacytomete& Co!!ect&on /o! (&$)t&on 9 the !#lut#on 'acto& (which is the reci#rocal of the blood dilution The !#lut#on "hen u #ng the ?BC -no%ette # 161**( o the !#lut#on 'acto& # 1**. N)m0e! o/ ,.)a!e, co)nte( 9 'ou& 254 Vo$)me o/ one $a!*e ,.)a!e 9 *.1 HL E1AMPLE2 ?BC6=L 9 A,g J cell counte! 1 1** J <ua&e counte! 1 *.16=L Dum$e& o' cell counte! #n #!e 1 9 )G LAB EXERCISES MLAB 1315@ A

EXERCISE 8: Manual Leukocyte Count Dum$e& o' cell counte! #n #!e ) 9 )7 A,e&age Dum$e& 9 )8 ?BC6=L 9 )8 1 1** 9 G.* 1 1*36=L 5 1 *.1

MLAB 1315 Hematology

RECORDING "OUR RESULTS - e the 'o&m %&o,#!e! to &e%o&t you& &e ult . Reco&! ?BC: counte! #n each <ua&e $e'o&e %&ocee!#ng to the ne1t <ua&e. P!ec&,&on: The a$#l#ty to &e%&o!uce a te t &e ult on the ame am%le. To !ete&m#ne #' the t"o count a&e #n clo e enough ag&eement( u e the 'ollo"#ng %&oce!u&e: )1*3A. B$ta#n the !#''e&ence 2C4 $et"een the S#!e A an! S#!e B o' the hemacytomete&. )1*3G. Calculate ) S.3. a 'ollo" :
) S. 3 . 9 ) A 8 B

)1*38. I' C # le than ) S.3.( the &e ult a&e acce%ta$le. I' g&eate&( the count mu t $e &e%eate!. E1am%le: A 9 53* B 9 5G* C 9 5*
) S . 3 . 9 ) 5G* 8 53* 9 ) 7** 9 ) 1 3* 9 A*

C 25*4 # le than ) S.3. 2A*4 an! the&e'o&e # acceptable. I' C # g&eate& than ) S.3.( the &e ult a&e not %&ec# e 2not clo e togethe& enough4 an! the count mu t $e &e%eate!. Re0clean the hemacytomete&( &e0m#1 the -no%ette !#lut#on( &e0loa! the hemacytomete&( etc. Acc)!ac : The clo ene o' a te t &e ult to the t&ue ,alue. To !ete&m#ne #' the t"o count a&e accu&ate 2"#th#n )*+ o' the automate! count4( u e the 'ollo"#ng %&oce!u&e: E1am%le: Automate! count 9 G.* 1 1*36HL. G.* 1 1*36L 0 )*+ 9 5.A 1 1*36HL G.* 1 1*36L 8 )*+ 9 8.5 1 1*36HL Acce%ta$le &ange # 5.A 0 8.5 1 1*36HL Manual count 9 A.* 1 1*36HL o manual count # acce%ta$le. REPORTING RESULTS Do&mal Ialue De"$o&n 7.*03*.* 1 1*36HL 1 "eek 5.*0)1.* 1 1*36HL 1 month 5.*017.5 1 1*36HL A01) month A.*01G.5 1 1*36HL LAB EXERCISES MLAB 1315@ G

EXERCISE 8: Manual Leukocyte Count ) yea& Ch#l!6a!ult A.)01G.* 1 1*36HL 5.801*.8 1 1*36HL

MLAB 1315 Hematology

NOTES )155). 3#luent an! $loo! houl! $e %&o%e&ly m#1e! $e'o&e '#ll#ng the hemactyomete&. )1553. The hemacytomete& mu t $e %&o%e&ly '#lle! to a,o#! e&&oneou &e ult #n manual cell count#ng. I' the cham$e& # o,e&'#lle!( a #n!#cate! $y the %&e ence o' e1ce 'lu#! #n the moat o' hemacytomete&( clean hemacytomete& an! &echa&ge cham$e&. A "#th all manual count ( the !#luent am%le mu t $e tho&oughly m#1e! $e'o&e cha&g#ng the hemacytomete&( "h#ch mu t $e %&o%e&ly '#lle!. )1555. The cham$e& mu t $e "ell0cleane! an! '&ee o' !e$&# "h#ch coul! $e m# taken 'o& leukocyte . )1555. A h#ghly ele,ate! leukocyte count 2leukocyto # 4 may make accu&ate count#ng !#''#cult. In e#the& #n tance( a econ!a&y !#lut#on houl! $e ma!e. ?hen calculat#ng the total count( a!Ku t the 'o&mula to allo" 'o& econ!a&y !#lut#on. )155A. The&e a&e %hy #olog#c ,a&#at#on to con #!e& "hen %e&'o&m#ng ?BC count . H#ghe& ?BC count 2leukocyto # 4 a&e een 'ollo"#ng e1e&c# e( emot#onal t&e ( an1#ety( an! 'oo! #ntake. Inc&ea e! leukocyte count a&e al o een #n ce&ta#n !# ea e con!#t#on uch a $acte&#al #n'ect#on ( #n'lammat#on an! leukem#a . 3ec&ea e! leukocyte count 2leuko%en#a4 a&e een #n ,#&al !# o&!e& ( &a!#at#on #n!uce! leuko%en#a( chemothe&a%y #n!uce! leuko%en#a( a%la t#c anem#a an! megalo$la t#c anem#a.

LAB EXERCISES

MLAB 1315@ 8

)155G. I' mo&e than 1* nucleate! RBC: a&e een on the !#''e&ent#al( the total leukocyte count houl! $e co&&ecte! u #ng the 'ollo"#ng calculat#on: Co&&ecte! ?BC 9 Total leukocyte count 1 1** 1** 8 J o' DRBC: 61** ?BC: on !#''e&ent#al

RE-ERENCES Ha&men#ng.( 3en# e( Cl#n#cal Hematology an! /un!amental o' Hemo ta # ( 3&! e!#t#on( %%. 5730 577. Tu&geon( Ma&y Lou# e( Cl#n#cal Hematology 0 Theo&#e an! ;&oce!u&e ( 3&! e!#t#on( %%3)*03)1.

Man)a$ WBC Re3o!t -o!m /o! La0 E'e!c&,e

Patient ID Side 1 1. 2. 3. 4. Total: Average: Difference between results: !alculate 2 S.D. for precision: Is t"e difference less t"an 2 S.D.# !alculation of result: Side 2 1. 2. 3. 4. Total:

Patient ID Side 1 1. 2. 3. 4. Total: Average: Difference between results: !alculate 2 S.D.for precision: Is t"e difference less t"an 2 S.D.# !alculation of result: Side 2 1. 2. 3. 4. Total:

$anual %&! result: Auto'ated result: ()btain fro' instructor for accurac*+ Auto'ated result range _2,-: Is 'anual result wit"in _2,- of auto'ated result#

$anual %&! result: Auto'ated result: ()btain fro' instructor for accurac*+ Auto'ated result range _2,-: Is 'anual result wit"in _2,- of auto'ated result#

STUDY QUESTIONS
.a'e Date 2 pts 1. ). Calculate the 'ollo"#ng "h#te cell count #' the !#lut#on o' the -no%ette # 161**. Sho" you& calculat#on . S#!e J1 S#!e J) S<ua&e 1 9 3* "h#te cell S<ua&e 1 9 )G "h#te cell S<ua&e ) 9 )8 "h#te cell S<ua&e ) 9 )G "h#te cell S<ua&e 3 9 )5 "h#te cell S<ua&e 3 9 )5 "h#te cell S<ua&e 5 9 )G "h#te cell S<ua&e 5 9 )5 "h#te cell

) %t

). 3.

Calculate the 'ollo"#ng "h#te cell count #' the !#lut#on o' the -no%ette # 161** an! 7 <ua&e "e&e counte!. Sho" you& calculat#on . Total cell counte! on #!e 1 9 3A Total cell counte! on #!e ) 9 3)

) %t

3. 5.

Mou a&e %e&'o&m#ng a manual "h#te $loo! cell count. Mou ha,e ma!e the !#lut#on an! ha,e '#lle! the hemacytomete&. A tat u&#naly # # $&ought to the la$o&ato&y an! you lea,e the hemacytomete& #tt#ng on the m#c&o co%e tage "h#le you %e&'o&m the -A. /#'teen m#nute late& you &etu&n to com%lete the count. Shoul! you count the !#lut#on on the hemacytomete&N ?hy o& "hy notN

1 %t

5. 5.

?hy mu t the !#lute! $loo! #t #n the -no%ette 'o& 1* m#nute $e'o&e #t # %late! on the hemacytomete&N

1 %t

5. A.

?hy mu t the loa!e! hemacytomete& #t 'o& 1* m#nute $e'o&e the manual ?BC # &ea! un!e& the m#c&o co%eN

) %t

A. G.

Mou ha,e %e&'o&me! a manual ?BC an! the count '&om the t"o #!e o' the cham$e& a&e )3) an! )A8. A&e the count clo e enough to acce%tN Sho" you& calculat#on .

) %t

G. 8.

State no&mal ?BC &e'e&ence &ange 'o&: De"$o&n LLLLLLLLLLLLLL Ch#l!6A!ultLLLLLLLLLLLLL

8 %t

8. 7.

State 'ou& ou&ce o' e&&o& an! #n!#cate #' the cell count "oul! $e 'al ely #nc&ea e! o& !ec&ea e!. A. B. C. 3.

1 %t

7. 1*. 1*. 11.

State the !#luent that # u e! 'o& -no%ette count .

1 %t

State the 'o&mula 'o& co&&ect#ng a ?BC count #' DRBC a&e %&e ent.

) %t

11.

1).

Calcul ate the co&&ec te! ?BC count 'o& the 1). 13.

'ollo"#ng: Sho" you& calculat#on. ?BC count 9 )5(***6=L J o' DRBC 9 3561** ?BC

3e'#ne leuko%en#a an! leukocyto # .

5 %t . 13. State 5 a oc#ate! con!#t#on 'o& leuko%en#a. A. B. C. 3. ) %t . 15. State ) !# ea e 2%atholog#c4 con!#t#on 'o& leukocyto # . A. B. 5 %t 15. State 5 %hy #olog#c con!#t#on that can cau e an ele,at#on #n the ?BC. A. B. C. 3. ) %t 1A. Ho" can the t"o count o' a manual ?BC $e con'#&me! on a manual hemacytomete& 2%&ec# #on4N