LaboratoryHematology

LABORATORYHEMATOLOGY
Laboratoryplaysanindispensableroleinthediagnosisandmanagementofalmostall
hematologicdisorders.Thepurposehereistointroduceyouwiththecommonlaboratory
testsandexplaintheirsignificance.Someofthecommontestsinahematologylaboratory
arethefollowingbutwewilldiscussonlythefirst4tests.Othertests arediscussed
elsewhere
Completebloodcountwithdifferential
Reticulocytecount
Peripheralbloodsmearevaluation
Detectionoffetalhemoglobin
Serumandurineproteinelectrophoresis(SPEP&UPEP)
Hemoglobinelectrophoresisand/orHPLCforhemoglobin
Cytochemistry
Leukocyteimmunophenotyping
Coagulationtests

COMPLETEBLOODCOUNT:
Thisisthemostcommonofallhematologytests.AstandardCBCisreferredtothe
following tests. Some parameters are actual measurements whereas others are just
calculatedbytheanalyzerbasedonothermeasuredvalues
1. Whitebloodcell(Leukocyte)count(WBC)withmeasured
5partdifferentialcount
5partabsolutecount
2. Redbloodcell(Erythrocyte)countmeasured
3. Hemoglobinconcentrationmeasured
4. Hematocritpercentagemeasuredorcalculated
5. Meancellvolume(MCV)calculated
6. Meancellhemoglobin(MCH)calculated
7. Meancellhemoglobinconcentration(MCHC)calculated
8. Redcelldistributionwidth(RDW)calculated
9. Plateletcountmeasured
10. Meanplateletvolume(MPV)calculated

AnEDTAanticoagulatedbloodisusuallyusedforCBCdetermination.Althougha10cc
ora5ccbloodcollectiontubeisusuallyused,theamountofbloodutilizedforCBCisa
verysmallfraction(<<1cc).

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LaboratoryHematology

Any abnormality flagged by automated counter triggers a manual review of the

peripheralsmearandcellcounting.

AUTOMATEDBLOODANALYSIS:

Severalautomatedhematologyanalyzersareavailableforthispurpose.Oneofthemost
hematologyanalyzeristheonefromCoultercalledCoultercounter.Coultermethodof
cellcountingandsizingisbasedonthedetectionandmeasurementofchangesinthe
electricalresistanceproducedbycellssuspendedinaconductiveliquidandtraversinga
smallaperture.Suspendedbloodcellsarefocusedintoasinglecellstreamandpassed
throughasmallaperturewithelectrodesoneitherside.Thecellsactasdiscretenon
conductors orinsulators of current as they pass fromthe electrode andthrough the
aperture. The momentary drop or interruption of current produced by each cell is
measuredasapulse.
Cellcountisproportionaltothenumberofdetectedsignals(orpulses)
Cellsizeisproportionaltothemagnitude(amplitude)ofeachsignal

[This picture is adapted from BLOOD: Principles & Practice of Hematology by Handin et al. Lippincott Press]

Thereare2channelsofcoultercounter.
(a) No lytic agent / No shrinkage agent channel Used for RBC and platelet
evaluations(givesaccurateRBCandplateletvolumes)
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LaboratoryHematology

(b) RBClyticagent/WBCshrinkageagent UsedforWBCevaluationandHb

measurement
Eachchannelisanalyzedby3distinctaperturesfor4secondseach.Thetotaltimefor
measurementsandcalculationsis<1minute.
AtypicalCBCreportfromanormalcountisshownbelow:

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LaboratoryHematology

SomeoftheCBCreportsfromactualpatientsareshownbelow:
NEUTROPHILIA,absoluteandrelative:

EOSINOPHILIA,absoluteandrelative:

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LaboratoryHematology

LYMPHOCYTOSIS,absoluteandrelative:

PANCYTOPENIA:

WhiteBloodCell(WBC)evaluation:

WBCcountmeasuredasnumberofcells>35flremaininginthelysedchannel.
Reportedascellsx109/L

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LaboratoryHematology

The5partdifferentialandabsolutecountismadepossiblebytwomodalities
(i)
Conductivitydistinguishesamongcellsbasedoncharacteristicsofthe
nucleus,granules,andchemicalcompositionofthecells
(ii)
Lightscatteringdistinguishesamongcellsbasedoncharacteristicsofthe
cellsurface
Datageneratedbyallthreeinterrogationmodalitiesareusedtogeneratea3D
scatterplottoseparatedifferentWBCs.

RedBloodcell(RBC)evaluation:

RBCcount[M4.55.5x1012/L,F45x1012/L]ismeasuredasnumberofcells
with sizes >36 fl in the unlysed channel. WBC are also counted but these
represent<0.1%ofthecellscounted.Reportedascellsx1012/L.RBCcountis
increasedinpolycythemia&decreasedindecreasedproductionbythemarrow.
Hemoglobinconcentration [Males1318g/dl,Females1216]ismeasured
spectrophotometrically in the lysed channel. Reported as g/dl. Increased in
polycythemiaanddecreasedinanemia.
MeanCellularVolume(MCV) [80100fl]isdeterminedfrommeasuredRBC
sizedistribution(histogram)andisreflectiveoftrueRBCsize.Reportedinfl
(Femtoliter).Increasedinmacrocytosissuchasseeninmegaloblasticanemiaand
decreasedinmicrocyticanemiasuchasirondeficiencyanemiaandthalassemia.
ReticulocytesarealsolargerthanmatureRBCsandthusincreasedreticulocytes
mayartifactuallyincreasetheMCV.Inpatientswithcombinedmacrocytosis
and microcytosis as seen in combined nutritional deficiencies of vitamin
B12/folateandironmayintheendresultinafalselynormalMCV.Thatiswhy
examinationofaperipheralbloodsmearissoimportant!
Hematocrit[M42%52%,F37%48%]iscalculatedbycoultercounterbya
formula=RBCxMCV/10).Reportedin%.Itisincreasedinpolycythemiaand
decreasedinanemia.Itis,however,directlyaffectedbytheplasmavolume.In
dehydratedstates,Hctisincreasedwhereasinvolumeoverloadislow.
Meancellhemoglobin(MCH) [2833pg/cell]iscalculatedbyHb/RBCx10
andreportedinpg(picograms).Itrepresentstheamountofhemoglobinpresent
inoneredbloodcell!Itisdecreasedinhypochromia.
Meancellhemoglobinconcentration(MCHC) [3236g/dl]iscalculatedby
Hb/Hctx100andreporteding/dl.ItrepresentstheamountofHbpresentina
populationofredcellsandtakesintoaccountanyvariationinthecellsizeand
shape.AlowMCHCreflectshypochromia.
Redcelldistributionwidth(RDW)[12%15%]isthecoefficientofRBCsize
variation, which is calculated from the measured RBC size in histogram. It
provides a quantitative expression of the size spread of the RBC populations
(anisocytosis).Reportedas%.Itallowsthepresenceofmorethanonecellsize
population to be appreciated quantitatively. The RDW is often used to
provisionallydistinguishmicrocyticanemiacausedbyirondeficiency(inwhich
RDWisincreased)andthalassemia(inwhichRDWisnormal).Inmacrocytic

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LaboratoryHematology

anemia it helps in distinguishing megaloblastic anemia (in which RDW is

increased)andothermacocyticanemias(inwhichRDWisoftennormal).

Plateletevaluation:

Plateletcount[150450x109/L]isdeterminedfrommeasurementsofnumber
ofcellswithsizes220flintheunlysedchannelReportedascellsx109/L
Meanplateletvolume[68fl]ismeasuredfromahistogramandisreflectiveof
thetrueplateletvolume!Itisrarelyclinicallyused.Reportedinfl

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LaboratoryHematology

[This ptable is adapted from BLOOD: Principles & Practice of Hematology by Handin et al. Lippincott Press]

RETICULOCYTECOUNT:
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LaboratoryHematology

ReticulocytesarenewlyproducedRBCsthathaveextrudedtheirnucleibutstillshow
ribosomalRNAmaterialthatimpartsareticulatedappearancetoRBCs.Theyconstitute
about12%ofperipheralbloodpopulationandcanberecognizedasslightlylargerthan
normalRBCsandwithsomeblue/purpleappearingfinedots(ribosomalRNA)causing
polychromasia.Themostcommonmethodofhighlightingreticulocytesisasupravital
staintoprecipitateintracellularRNA.Afterstaining,athinperipheralsmearismadeand
reticulocytesarecountedbymanualmethod.Thesecellscanalsobecountedbyflow
cytometry.Reticulocytesareincreasedinanyconditioninwhichthereisincreasedoutput
ofnewRBCintheperipheralbloodsuchasaresponsetobleedingandhemolysisand
theyaredecreasedinconditionsofdecreasedproductionofRBCbythemarrow.

PERIPHERALBLOODEVALUATION:
This is animportant part ofevaluation of many hematologic disorders. A peripheral
bloodsmearismadeunderfourmainconditions:
(i)
The automated Coulter counter flagged an abnormality that triggers
furtherevaluation
(ii)
Aphysicianrequestsevaluationofperipheralbloodsmearbyapathologist
basedonclinicalhistory,and
(iii)
At a pathologists request to further evaluate any hematologic disease
process even when the CBC is normal such as suspected presence of
parasitesinthebloodandwithincellssuchasEhrlichia
(iv)
Aspartofroutinequalitycontrolmechanismbylaboratories
[Thedetaileddescriptionofaperipheralbloodsmearwillbeprovidedduringthesession
forperipheralbloodevaluationtowardtheendofthecourse]

DETECTIONOFFETALHEMOGLOBIN:
(ByKleihauerBetketest)
Detectionoffetalhemoglobin(hemoglobinF)inindividualRBCsisusefultoestablish
thediagnosisoffetomaternaltransfusionandalsointheevaluationofHbFinpatients
withhemoglobinopathies.ThistestisbasedontheprinciplethatHbFisresistantto
elutionbyanacidwhereasHbAwashesoffleavingghostsofcells.Amanualcount
yieldsthe%ofFcellspresent.

The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.