Testingofbonemarrowcytology(2)---Ⅱ.Thecontent,methodandclinicalsignificanceofthebonemarrowcytomorphologicexamination---Thetestofbonemarrowslidesiscarriedoutasfollows:1.lowpowerscan(1)makesureifthematerial-drawingandthefilmpreparationofbonemarrowaspirationissatisfied:Thespecimenshouldbeconsistedof3parts:thehead,thebodyandthetail.---(2)judgetheextentofbonemarrowhyperplasia:Theextentofbonemarrowhyperplasiaisreflectedbytheamountofkaryocytesinbonemarrowgenerally.Observetheratioofkaryocytetomatureerythrocyteunderlowpower.---Theextentofbonemarrowhyperplasiaispositedusingthe5gradesclassification.---(3)observemegakaryocytes:Fourprincipalaspectsaboutmegakaryocytesshouldbeinspected:1)amount2)degreeofmaturity3)functionofgeneratingplatelets4)shapeofmegakaryocytesandplatelets---(4)checkwhetherabnormalcellsexist:AbnormalhistiocyteMetastaticcarcinomacellGauchercellNiemann-Pickcell……Onceabnormalcellswerefound,theyshouldbeinspectedunderoilimmersion.---2.oilimmersionassayChoosetheboundariesofthefilm’scoatingbodyandtail.(1)countthekaryocytesdifferentially:Calculatetheratioofthegranulocytetotheerythrocyte(G:E).(2)observethemorphologyofcells:Includingtheshapeofmatureerythrocyteandwhetherspecialabnormalcellandparasiticagentsabound.---3.theclinicalsignificanceoftheresultTheextentofbonemarrowhyperplasia:(1)extremelyactivehyperplasiaReflectthehyperfunctionofbonemarrowhematogenesisConditions:leukemia,esp.chronicmyelocyticleukemia(CML)---(2)themanifestlyactivehyperplasiaReflectthevitalityofbonemarrowhematogenesisConditions:hyperplasticanemia,leukemia,proliferatingbonemarrowdiseases,idiopathicthrombocytopenicpurpura(ITP),hypersplenia…---(3)theactivehyperplasiaReflectthebasicallynormalconditionofbonemarrowhematogenesisConditions:normalbonemarrow,hyperplasticanemia,chronicaplasticanemiawithlocalcompensatoryhyperplasia---(4)thedecreasedhyperplasiaReflectthedecreasedfunctionofbonemarrowhematogenesisConditions:chronicaplasticanemia,granulocytopenia,agranulemia,myelofibrosis…---(5)remarkablydecreasedhyperplasiaReflectthenonfunctionofbonemarrowhematogenesisConditions:acuteaplasticanemia,bonemarrownecrosis…---------TheratioofcellsineachseriesateachdevelopingphaseinbonemarrowGranulocyteseriesAlmost50%-60%ofkaryocytesaregranulocytes.Withcellmaturity,theratioofeachdevelopingphaseincreased.Myeloblast<1%,promyelocyte<5%Myelocytes+metamyelocytes<15%Stabgranulocytes>segmentedgranulocytesMostoftheeosinophilicgranulocytesandthebasophilicgranulocytesareusuallymatureinbonemarrow.eosinophilicgranulocytes<5%basophilicgranulocytes<1%---(2)ErythrocyteseriesNormoblasts≈20%Pronormoblast<1%Basophilicnormoblast<5%Polychromaticnormoblast≈10%Orthochromaticnormoblast≈10%G:Eisdefinedastheratioofthepercentageofgranulocyteseriestoerythrocyteseries.Referencevalue:(2-4):1---(3)LymphocyteseriesThepercentageofcellsoflymphocyteseriesisapproximately20%ofkaryocytes.Higherinchildhood,itevengoesupto40%.Maturelymphocytes:majorityLymphoblastsandprolymphocytes:rare---(4)Monocyteseries<4%,almostallofthecellsarematuremonocytes.(5)PlasmacyteseriesUsu.<2%,mainlymatureplasmacytes.---(6)MegakaryocytesystemInafilmpreparationof1.5cm×3.0cm,thenormalvalueisdefinedas7-35megakaryocytescanbeseen.Granularmegakaryocytesandthrombocytogenousmegakaryocytes:commonlyseenMegakaryoblasts:seldombeseenAmongthemegakaryocytesystem:Promegakaryocyte<5%Granularmegakaryocytes10%-27%thrombocytogenousmegakaryocytes44%-60%Nakedmegakaryocyte8%-30%(7)OthercellsAfewnon-hemapoieticcellscanbeseen,e.g.reticulocytes,endotheliocytes,tissuebasophiliccells,etc.---Clinicalsignificanceofchangesinratioofeachseries---Changesofratioofgranulocytesanderythrocytes(G:E)reasondiseasesnormalG:EnormalbonemarrowGandEincreasedequallyerythroleukemiaGandEdecreasedequallyaplasticanemiahemotopoieticsystemdiseases,inwhichbothGandEarebasicallyinvariablemultiplemyeloma,bonemarrowmetastatictumor,ITP,etc.G:E↑(>5:1)G↑orE↓AML,CML,acutepyogenicinfection,neutrophilicleukemoidreaction,purefragilityoferythrocytes-aplasticanemiaG:E↓(<2:1)G↓agranulemiaE↑hyperplasticanemia,polycythemiavera,secondaryerythrocytosis,etc.---ChangediseasesGranulocyteseries↑Granulocyticleukemia,acuteinflammation,infectiousdiseases,neutrophilicleukemoidreaction↓Aplasticanemia,agranulemia,granulocytopeniaErythrocyteseries↑Hyperplasticanemia,megaloblasticanemia,DiGuglielmosyndrome↓AplasticanemiaLymphocyteseriesAbsolutelyincreaseALL,CLL,malignantlymphoma,infectiouslymphocytosis,infectiousmonocytosis,otherviralinfection,lymphocyticleukemoidreactionRelativelyincreaseAplasticanemia,

agranulemia,granulocytopenia---ChangediseasesMonocyteseries↑Hematologicalsystemdiseases,infectiousdiseases,rheumaticdiseases,malignanttumor,hepaticcirrhosis,drugreactionPlasmacyteseries↑MM,plasmacyticleukemia,macroglobulinemia,heavychaindisease,reactiveplasmacytosis,aplasticanemia,agranulemiaMegakaryocyticseries↑ITP,Evanssyndrome,proliferatingbonemarrowdiseases,hypersplenia,megakaryocyticleukemia↓Aplasticanemia,acuteleukemia,somadiseaseswithbonemarrowinfiltrationordestroy,acuteinfection,chemicalormedicinepoisoning,radiationdisease---Ⅲ.CytochemicalstainingofthebloodcellThecytochemicalstainingisakindofassay,basedoncellmorphology,whichuseschemicalreactionprinciples,stainingbonemarrowslidesaccordingtoacertainprocedure,andatlastobservingthecytochemistrycompositionanditschangesasseenonthemicroscope.Itcanbeusedintheidentificationofcategoryofbloodcells.---1.Peroxidasestaining(POX)Result:noblueorblackparticlesinthecytoplasm:(-)Tinyanddiffuselydistributedgranulesinthecytoplasm:weaklypositiveThickanddensegranules:stronglypositiveSignificance:identifythecategoryofacuteleukemiaAcutemyeloblasticleukemia:stronglypositiveAcutemonocyticleukemia:weaklypositiveor(-)Acutelymphoblasticleukemia:(-)POXismostvaluableintheidentificationofAMLandALL.---2.SudanblackBstaining(SB)Akindoffat-solublestain,canmaketheintracytoplasmiclipidturntobrownish-blackordeepblackparticles.Result:almostthesameasPOX.Granulocyteseries:na?ve→mature,thepositivereactionbecomesstrongerMonocyteseries:weakly(+)Lymphocyteseries:

(-)Significance:thesameasPOX.---3.Neutrophilalkalinephosphatasestaining(NAP)Result:ALPmainlyexistsinthematureneutrophilicgranulocyte(staborsegmented),othercells→(-)Positiveresult:graytobrownish-blackparticlesinthecytoplasmTheintensityofresponsecanbedividedinto5grades:-,1+,2+,3+,4+.Theresultispresentedasthepercentageofthepositivecellsandthescore.ReferencevalueAdult:positiveratioofNAP:10%-40%,thescore:40-80pointsSignificance:acutefesteredinfectiousdiseases:remarkably↑,viralinfections:normalofslightly↓CML:remarkably↓,usu.score0,leukemoidreaction:extremely↑AML↓,ALL↑,acutemonocyticleukemia:normalor↓Aplasticanemia↑,PNH↓---4.Acidphosphatasestaining(ACP)Result:positivereaction:brownish-blackparticlesinthecytoplasm.Significance:assistinthediagnosisofhairycellleukemia:hairycell(+)orstrongly(+),theactivitycannotbeinhibitedbyL-tartaricacid.IdentifyTlymphocyteandBlymphocyte:Tcell(+),Bcell(-).Gaucher’sdisease(+),Niemann-Pickdisease(-)monocyte(-),histiocyte(-),reticularcell(-),megakaryocyte(-).---5.NaphtholAS-Dchloroacetateesterasestaining(AS-DNCE)Alsocalledspecificesterase(SE),granulocyteesteraseResult:positivereaction:redsedimentinthecytoplasm.SEmainlyexistsingranulocyteseries,butmicroleukoblast(-)orweakly(+),frompromyelocytetomatureneutrophilicgranulocyte(+),promyelocyte:strongly(+),activityweakenedwiththematurityofthecells.Eosinophilicgranulocyte,lymphocyte,plasmacyte,normoblast:(-)Monocyte(-)orweakly(+)Significance:AML:strongly(+),acutemonocyticleukemiaandALL(-)Acutemyeloblastic-monocyticleukemia:cellsofgranulocyteseries(+),cellsofmonocyteseries(-)---6.Alpha-naphtholacctateesterasestaining(α-NAE)Alsocallednonspecificesterase(NSE)ormonocyteesteraseResult:gray-blackorbrownish-blacksedimentinthecytoplasm.NSEmainlyexistsinmonocyteseries.Monoblast(-)orweakly(+),promonocyteandmonocyte(+),granulocyteseries(-)orweakly(+),lymphocyte(-)Significance:identifytheacutemonocyticleukemiaandAML.NSEInhibitedbyNaFAcutemonocyticleukemiaStrongly(+)yesAcutemyeloblasticleukemia(-)orweakly(+)no---7.StainingforglycogenAlsocalledtheperiodicacid-Schiff’sreaction(PAS)Result:

positive:thecytoplasmstainsred,redsubstancecanbegranular,block-likeordiffusedred.Thedegreeofpositivereactioncanbedividedinto4grades:strongly(+),(+),weakly(+)and(-).Myeloblast(-),frompromyeloblasttosegmentedneutrophilicgranulocyte(+),thepositivestrengthenedwiththematurityofcells.Monocyte:weakly(+),lymphocyte(-)orweakly(+)Normoblastanderythrocyte(-)megakaryocyteandplatelet(+),withthematurityofcells,positivereactionstrengthened---Significance:normoblastoferythremicmyelosisorerythroleukemia:

strongly(+)7.StainingforglycogenDiseaseCellPASShapeofpositivesubstanceAMLMicroleukoblast(-)orweakly(+)Fine,granularofdiffusedsalmonpinkALLLymphoblastandyounglymphocyte(+)Coarsegranular-likeorchunk-likeAcutemonocyteleukemiaMonoblast(+)WidespreadandhomogeneousredorfinegranulesGauchercell:strongly(+),adenocarcinomacell:strongly(+)---8.IronstainingAlsocalledPrussianbluereactionResult:(1)Extracellularferritin:observetheferritininsidemonocyte-phagocyteseriesinthebonemarrowparvule(outsidethenormoblast).Positive:lightbluish-greenandhomogeneousshapelessorpearl-like,coarsegrain-like,blueordeepblue,orblue-blackblock-likesubstance.Thepositivereactioncanbedividedinto5grades:-,1+,2+,3+,4+.---(2)Intracellularferritin:theferritininnormoblast.Sideroblast:1to5blueandtinyferritingranulessurroundingthenucleusofnormalnormoblast(mainlyorthochromaticnormoblast)Crico-sideroblast:onesideroblastwithover10thickanddeepstainedferritingranules,whichissurroundingthenucleusforover2/3ofthenucleonicperimeter.8.Ironstaining---Referencevalue(1)extracellularferritin:1+~2+,mostly2+(2)intracellularferritin:20%~90%,theaverageis65%Significance:(1)irondeficiencyanemia:extracellularferritin(-),percentageofsideroblast↓,ofterbelow15%,even0%.(2)non-irondeficiencyanemia:includingthalassemia,sideroblasticanemia,hemolyticanemia,megaloblasticanemia,aplasticanemia,displacementanemia,theextracellularferritin↑,often3+~4+(3)sideroblasticanemia:sideroblast↑,crico-sideroblastconstitutesover15%ofthenormoblast.8.Ironstaining------Ⅳ.GroupingaccordingtocellularimmunologyAlsocalledcellimmunitymark(phenotype)assay.Itmakesuseofmonoclonalantibodyandimmunologytechniquestotestthespecificantigenonthesurfaceofcytomembraneand/orinthecytoplasm.---1.Assaymethods(1)Immunofluorescencemethod(2)immunoenzymaticstaining1)APAAP(alkaliphosphataseanti-alkaliphosphatase)2)PAP(peroxydaseanti-peroxydase)3)ABC(avidin-bioepiderm-multienzymecomplex)---2.Theapplicationofcellimmunophenotypingrecognitionofcellsindifferentseries(2)IdentifydifferentlymphocytesMembraneantigenMatureTlymphocyteCD2,CD3,CD4,CD7,CD8MatureBlymphocyteCD19,CD20,CD22NKcellCD16,CD56MembraneantigenMedullarysystemCD13,CD14,CD15,CD32,CD33,CD65,CD91,CD156,CD166LymphocyteseriesCD1,CD2,CD3,CD4,CD5,CD7,CD8,CD9,CD10,CD19,CD20,CD52,CD77MegakaryocyteandplateletCD41,CD42,CD61ErythrocyteseriesGlycophorinAorB---(3)TesttheTlymphocytesubgroup(4)Identifycellsatvariousstagesofdifferentiation(5)Identifycellsindifferentfunctionalstates(6)Isolationandresearchcellsindifferentseriesandstagesofdifferentiation(7)Immunophenotypicanalysisofvariouskindsofleukemiccells(8)TestoftheleukemictinyresidueMembraneantigenHemopoieticstemcellCD34+,CD39+,Lin-HemopoieticstemcelltransformtomedullarysystemCD34+,CD33+ProgenitorcellofTlymphocyteseriesCD34+,TDT+,CD10+,CD7+ProgenitorcellofBlymphocyteseriesCD34+,CD19+HighexpressionNoexpressionMemoryTcellCD45ROCD45RAActiveTcellCD45RO,CD25,CD2,CD54,CD58CD45RA---3.Theimmunophenotypiccharacteristicsofacuteleukemiacell(1)TheimmunophenotypiccharacteristicsofcellsinALLandAML---(2)TheimmunophenotypiccharacteristicsofALL(3)TheimmunophenotypiccharacteristicsofAML---Ⅴ.CytogeneticanalysisChromosomeanalysisTheabnormalcloneofchromosome