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Laboratory 10 1 Manual Differential

Laboratory #10: MANUAL DIFFERENTIAL Skills= 20

Obj !ti" s: 1. To determine the relative number of each type of white cell present in the blood by performing differential cell counts on normal and abnormal blood smears. 2. Calculate absolute white blood cell counts when given the total WBC count and the relative (% number of each type of WBC. !. To determine within one "ualitative unit the red cell# white cell# and platelet morphology of each of the blood smears. $. To determine within % !&% accuracy an estimate of the white cell counts and the platelet counts of each of the blood smears. '. To calculate the corrected automated WBC count for nucleated red blood cells. #ri$!i%l : ( stained smear is e)amined in order to determine the percentage of each type of leu*ocyte present and assess the erythrocyte and platelet morphology. +ncreases in any of the normal leu*ocyte types or the presence of immature leu*ocytes or erythrocytes in peripheral blood are important diagnostically in a wide variety of inflammatory disorders and leu*emia (see ,BC -orphology .ab# CBC/01 Correlation .ab# and the WBC 2 0.T 3stimates/+nclusions .ab s for e)amples . 3rythrocyte abnormalities are clinically important in various anemias. 0latelet si4e irregularities are suggestive of particular thrombocyte disorders. The WBC count is verified by estimating the count in the e)amination area of the blood smear. ( well made blood smear with proper distribution of WBCs throughout e)amination area and feather edge is essential for WBC estimation (see Blood 1mear 0reparation .ab . The platelet count is verified by estimating the number platelets on the blood smear as well. The tube must be chec*ed for clumps. +f visible clots are observed# the specimen is 56(CC30T(B.3. 37T( induced platelet satellitism falsely decreases platelet counts. ( 6a Citrate specimen can be used for the platelet count in this circumstance. ,efer to the WBC 2 0.T 3stimates/+nclusions .ab for corrective action and the calculation. S% !i& $: 0eripheral blood smear made from 37T(8anticoagulated blood. 1mears should be made within $ hours of blood collection from 37T( specimens stored at room temperature to avoid distortion of cell morphology. 5nstained smears can be stored

Laboratory 10 2 Manual Differential for indefinite periods in a dry environment# but stained smears gradually fade unless coverslipped. R a' $ts( s)%%li s( a$* +)i%& $t: 1. 2. !. $. '. 0repared slides -anual cell counter designed for differential counts -icroscope# +mmersion oil .ens paper

,)ality -o$trol: Training and e)perience in e)amining immature and abnormal cell morphology are essential. +n the clinical environment# a set of reference slides with established parameters should be established to assess the competence of an individual to perform differential and morphological identification of leu*ocytes and erythrocytes. 0articipation in a "uality assurance program continues to document the e)pertise of the hematologist in microscopy. 9uestionable or abnormal smears should be referred to a supervisor or pathologist for verification. #ro! *)r : 1. :ocus the microscope on the 1&; ob<ective (low power . 1can the smear to chec* for cell distribution# clumping# and abnormal cells. +n scanning the smear it is important to note anything unusual or irregular# such as rouleau) or ,BC clumping. 2. 3)amine the peripheral edge of the smear. The number of WBCs should 6=T e)ceed !; the number of cells in the proper e)amination area. .arge cells such as neutrophils and monocytes can be pushed to the edges. +f this occurs# the distribution of the cells is poor and the smear is unacceptable. !. +f the smear is acceptable# estimate the white cell count by counting the number of WBCs in ten (1& '&; fields# and then apply the calculation found in the WBC 2 0.T 3stimates/+nclusions .ab. This number should be within %!&% of the automated WBC count provided by the instructor or from the instrument printout. +f it is not within this range# the white cell count and the estimation should be repeated.

$. 5sing the 1&& ; oil ob<ective# e)amine the smear for platelet morphology and number. :ind a thin area where red cells are not overlapping. ( single field with a normal platelet count would contain >82? platelets on 1&&;. +n the

Laboratory 10 3 Manual Differential appropriate area# count the number of platelets on . successive fields# and then apply the formula found in the WBC 2 0.T 3stimates/+nclusions .ab. This number should be within %!&% of the actual platelet count. +f it is not within this range# the estimation should be repeated. +f platelets are clumped# include an appropriate comment from the 0latelet table in the WBC 2 0.T 3stimates/+nclusions .ab to the report form. '. To perform the differential# choose the portion of the smear where '&% of the ,BCs are slightly overlapping and '&% are individually spaced. The ,BCs should have a central pallor. @ou should use the 1&& ; ob<ective when learning cells or '&; if available. ?. Begin the count in the thin area of the slide and utili4e the battlement trac* to read the slide. A. Count each white cell seen and record on a differential cell counter# until 1&& white cells have been counted. R !or* yo)r r s)lts o$ t/ *ata s/ t. B. NR0-S +f any nucleated red cells (6,BCs are seen during the differential count# enumerate them using the n,BC button or on a separate counter. They are $ot to be included in the 1&&8cell differential count. They are reported as C6,BC/1&& WBCs and the WBC count must be corrected if there are 1. NR0-s 2 100 WBCs. The following formula is used to correct the WBC countD -orr !t * 30- !o)$t= WBC count ; 1&& n,BCs E 1&& 3)ampleD Fiven automated WBC countD 1$2&&/G. 21 n,BCs observed when doing a 1&& WBC diff (1$#2&& ) 1&& / 1&& E 21 6,BC H 11#B&&/G. WBC corrected >. 3)amine the red cell morphology in a thin area of the slide where the red cells either do not overlap or lightly overlap. They should have a central pallor. +n most cases an abnormality must be a consistent finding in order to be significant. 6ote any variations from normal and classify them according to the grading system given in the ,BC -orphology .ab.

Laboratory 10 4 Manual Differential 1&. (ll abnormal morphology (,BC# WBC# or 0.T is reported in the -orphology section of the report form for each patient/slide. +f you do not observe abnormal morphology# write InormalJ in this section. MANUAL DIFFERENTIAL SUMMAR4 1. 2. !. $. '. ?. A. 1&) scan '&; WBC estimate 1&&; 0.T estimate 1&&; 0.T morphology 1&&; WBC differential 1&&; WBC morphology 1&&; ,BC morphology

R 5 r $! "al) s vary depending on age. :or this e)ercise# the following values will be used (inside cover of te)t D - ll Ty% Total WBC ) 1&!/K. 6eutrophils % .ymphocyte % -onocyte % 3osinophil % Basophil % #ro! *)ral Not s 1. ( well8made and well8stained smear is essential to the accuracy of the differential count. The *nowledge and ability of the cell morphologist is critical to high8"uality results. 2. When in the clinical world# before reporting significant abnormalities such as blasts# malaria or other significant finding on a patientLs differential# as* a more e)perienced tech to review the smear for confirmation. +n clinical settings where a pathologist or hematologist is present# the smear is set aside for 0athologist ,eview. R "i 8 t/ 30- 9 #LT Esti&at s2I$!l)sio$s Lab a$* R0- Mor%/olo'y Lab 5or A-- o% rati$' %ro! *)r 5or r %orti$' % ri%/ ral s& ar 5i$*i$'s: 0irt/ >8!& '&8?& 2'8!' 281& &8' &81 6 &o ?81B 2'8!' ''8?' 281& &8' &81 7yr $.'81!.' !'8$' '&8?' 281& &8' &81 17 yrs a$* )% $.'811.& $&8B& 2'8!' 281& &8' &81

Laboratory 10 5 Manual Differential ;: Absol)t -o)$ts +t is also helpful to *now the actual number of each white cell type per K. of blood. This is referred to as the absol)t !o)$t: +f not reported by the instrument# absolute counts can be calculated easily using the automated WBC count and calculated as followsD Absol)t # o5 ! lls2<l H % of cell type in diff ) white cell count MM6oteDMM +mmature cells within a cell line are included in the percentage that is multiplied by the total WBC. (ie. ?% bands is added to !$% segmented neutrophils to get $&% neutrophils . (typical/reactive lymphocytes are added to normal mature lymphocytes to get the total lymphocyte percentage. $. +f disrupted cells are present such as s&)*' ! lls (bas*et cells # do not document them on the report. +n the clinical setting an albumin smear should be made if the white cell smudging e)ceeds 1281'% of the cells present. (n albumin prep is made by adding one drop of 22% bovine albumin to B drops of blood prior to preparing the wedge smear. ,BC morphology and WBC morphology must always be performed on the $o$8albumin smear. '. When the WBC is very high (N$&#&&&/K. # a 2&&8cell diff should be performed to increase the accuracy of the diff. The results are then divided by 2 and a note made on the report that 2&& white cells were counted.

?. 6ever hesitate to as* "uestions concerning morphology or the identification of cells. The differential is one of the most difficult laboratory tests to learn. +n fact# learning about cells and their morphology is a process that continues for as long as you perform differentials. A. +t is permissible to use a '&) ob<ective to perform a differentialO however *eep the following points in mindD 1 +f the WBC is increased# you should use the 1&&) to ensure that you will not s*ip cells in a crowded field# 2 I5 yo) ar /a"i$' tro)bl i* $ti5yi$' a ! ll( yo) &)st s8it!/ to t/ 100= i$ or* r to ' t a &or * tail * "i 8:


1. ,oda*# Bernadette# PematologyQClinical 0rinciples and (pplications# $ th edition# pp2&282&$. 2. Turgeon# -ary .ouise# Clinical Pematology 8 Theories and 0rocedures# !rd edition# pp!1B8!1>. !. 1eton -edical Center Pematology 0rocedure -anual $. -c*en4ie# 1hirlyn# Clinical .aboratory Pematology#2nd edition# pp.AA28AA!.

MLAB 1415: Hematology

Laboratory #10 Manual Differential

-anual 7ifferential ,eport :orm 1*illsD 2& pts.

1tudent 6ameDRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR #ati $t Na& a$* ID or Sli* N)&b r 30- Esti&at #LT Esti&at N )tro%/il > 7ateDRRRRRRRRRRRRRRRRRRRRRRRRRRRRR Ly&%/o!yt > Mo$o!yt > Eosi$o%/il > 0aso%/il > Ot/ rs > # o5 $R0-s2100 30-s

0roD -yeloD -etaD BandD 1egD Total: 0roD -yeloD -etaD BandD 1egD Total:

(typD .ymph Mor%/olo'y Total:

(typD .ymph Mor%/olo'y Total:

MLAB 1415: Hematology

Laboratory #10 Manual Differential

STUD4 ,UESTIONS (you may need to reference other labs to answer these "uestions ;7 %oi$ts 6ame RRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR 7ate RRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR 1. .ist the normal adult ranges for each cell type. ,elative ,eference ,ange in %

' pt

Cell Type 1egmented 6eutrophil .ymphocyte -onocyte 3osinophil Basophil

2 pts 2. 7escribe the procedure for estimating the white count.

2 pts !. 7escribe the procedure for estimating the platelet count.

2pts. '. Calculate the neutrophil absolute count for the following differentialD Total WBC H 1$.2 ) 1&!/K. A&% segs !% band 2&% lymphs !% monos $% eos

2 pts. ?. When performing a manual differential# you see 12 6,BCs. Pow do you report themS !pts. A. Calculate the corrected WBC given the following results. WBC H !2.& ) 1&!/K. C 6,BC/1&& WBC H 2&

MLAB 1415: Hematology

Laboratory #10 Manual Differential

2 pt

B. 7escribe the proper area where you should begin your WBC differential.

2 pt

>. 7raw a diagram of a blood smear and illustrate the path you would ta*e in performing a manual differential.

2 pt 1&. 5pon scanning a stained peripheral smear# you notice clumped platelets. There appear to be a very high number of platelets because on a!/ 5i l* you observe at least ?& platelets in multiple clumps. What would you doS

2pt 11. Fiven the following WBC count and differential# calculate the (bsolute lymphocyte count. (utomated WBC countD A.B ; 1&>/. 1egsD $?% .ymphsD 2$% -onosD 12% 3osD 1'% BasosD !%

2pt 12. ( patientLs automated platelet count is 1!2 ; 1& !/G.. 5pon slide review# the patientLs sample has rosettes of platelets surrounding the segmented neutrophils. ( 6a Citrate tube is collected on the patient. The technician gets a 1>A ; 1& !/G. platelet count on the 6a citrate tube. What is the correct platelet count to report to the physicianS

MLAB 1415: Hematology

Laboratory #10 Manual Differential

B pts 1!. +n the blan* ne)t to the term# write the number of the corresponding description (found below . RRRR(. to)ic granulation RRRRB. 7Thle bodies RRRRC. hypersegmentation RRRR7. auer rod RRRR3. smudge cell RRRR:. 0elger8Puet anomaly RRRRF. platelet satellitism RRRRP. 0y*notic neutrophil

1. 2. !. $. '. ?. A. B.

.ight blue oval inclusions composed of ,ough 3, ,eddish8blue staining inclusions found in myeloblasts Cell with ruptured cytoplasm 1egmented neutrophil with 2 lobes 1egmented neutrophil with ? lobes 1egmented neutrophil with degenerating nucleus 6eutrophil with pieces of mega*aryocyte cytoplasm encircling it (bnormally large primary granules of neutrophils