流式细胞术在白血病诊断中的应用.

流式细胞术在白血病诊断中的应用四川大学华西医院血液科朱焕玲一简介FCM：集激光、电子、光电测量、计算机、荧光化学、单抗的高科技仪器。对处在快速直线流动状态中的单个细胞或生物颗粒进行多参数的、定量分析和分选的技术。分两类：台式机（临床型）大型机（cellsorting).科研用。BDFACSVantageSEFCM的结构FCM的结构–细胞流动室；–光源；–聚光系统；–信号检测器；–电子计算机；–细胞分选装置。光源细胞流动室聚光系统信号监测器数据的存储、显示与分析数据的存储：listmode显示：–单参数直方图(histgram);：常用于分析DNA等；–双参数：二维点图等（dotplot)；–等高图：(contour)–三维等高图：3Dplot设门（gating):DataAcquisition-ListmodeEventParam1FSParam2SSParam3FITCParam4PE1501008090255110150953110608030一定条件下，荧光强度与细胞内DNA含量成正比。只反应一个参数与细胞数量间的关系，不能显示两个独立参数与细胞的关系。Immunofluorescence•FlowCytometrycont.–DatadisplayedGreenFluorescenceIntensityNumberofCellsUnstainedcellsFITC-labeledcellsOneParameterHistogramRedFluorescenceIntensityGreenFluorescenceIntensityTwoParameterHistogramContourplot3Dplot:细胞多的地方，山峰高。FCM检测常用参数内部参数—散射光测定前向角散射(forwardscatterFSC):与激光束平行的信号。与被测细胞大小有关。侧向角散射（sidescatterSSC):与激光束呈90°的光信号。与细胞内结构和颗粒物质多少有关。采用FSC、SSC组合，可区分淋巴细胞、单核细胞、粒细胞等群体。FCM常用参数外部参数—荧光测量荧光素探针标记的细胞所发荧光。荧光信号强弱、多少与细胞的抗原量、内含物多少有关。常用的荧光显色剂：FITC：PE：藻红蛋白PerCP；PI：碘化丙啶，测定DNA含量。临床常用三色荧光检测，现有四色分析。CD45vsSSC设门CD45为造血细胞（红系除外）的共同抗原。避免杂质细胞的沾染。CD45:淋巴细胞单核细胞成熟粒细胞原幼细胞在FCM分析时特别注意：细胞的分化成熟、细胞内部结构及其分化抗原的逐渐增减是渐进过程，非突变。故同时获知一个细胞的多种参数方能正确判断细胞属性。当细胞发生克隆增殖后，可出现抗原量变、丢失和错译表达。细胞抗原的阳性率及荧光强度是同等重要的指标。FCM可采用的标本单个细胞，新鲜标本，抗凝保存：–PB；–BM；（有时BM活检标本）–LN（包括FNA）；–CSF；–组织浸润FNA或活检标本；急性白血病FCM分析白血病细胞的抗原表达一定程度遵循正常细胞抗原表达的规律。白血病细胞为肿瘤性生长，可出现正常细胞表达以外的异常现象，如缺失、错译表达等。目前未发现白血病特异性抗原，须多参数分析。临床原/幼细胞群定性比定量更为重要。抗体的选择：1.常用抗体选择：核心McAbs:确定淋系或髓系（系特异性）髓系：CD33、CD13、CD14，cMPOB-cell:CD19、CD10、cCD22CD20T-cell:CD7、CD2、CD5、cCD3红系：GlyA巨核细胞：CD41、CD61非系列特异性：CD34、DR、CD45ALpanelCD7/CD117/CD45---protocalHLA-DR/CD13/CD45CD14/CD2/CD45CD19/CD5/CD45CD33/CD34/CD45CD20/CD10/CD45CD64,117,13,33,14,64—myeloid19,10,20—Blineage7,2,5—TlineageDR,34immatureAML常见CD表达FABSubtypeCommonPhenotypeComments/VariationsM0DR.CD13,CD33,CD34,CD7,cMPO*GenerallynegativeforlymphoidmarkersM1SimilartoM0exceptCD15M2DR,CD13,CD33,moreCD15,lessCD34CD19(+)--t(8;21)favorableprognosisM3DR(-)*,CD13,CD15,CD33,CD34-/+,CD2(+),DR(-)inmaturingAML,considerM3.Myeloid/naturalkillercellALCD56(+).M4/M5DR,CD15,CD14,CD33,CD13,CD4weakCD2(+),considerM4EoM6DR,CD13-/+,CD33+/-,CD34,CD45weakMatureformsexpressglycophorinM7DR-/+,CD33+/-,CD34,CD41,CD61Bewareofplatelet.adhesion正常骨髓的FCM二维点图Diagnosis:AML-M1AntigenProfile:positive:DR,CD13,CD34,CD38;partiallypositiveforCD7,negativecCD3.Diagnosis:Acutemyeloidleukemiawithdifferentiation(AMLM2)withfeaturesconsistentwitht(8;21)AntigenProfile:PositiveforCD33(dim),CD34,CD13,CD15CD11b(partial)withCD19.NegativeforCD56,CD10,CD272,female.Abonemarrowexaminationwasperformed.Diagnosis:AML-M5AntigenProfile:PositiveforCD33,CD38,CD34,HLADR,CD4(dim),CD64,CD13,CD14,CD11bandCD15.Diagnosis:Erythroleukemia(FABM6)AntigenProfile:BlastspositiveforCD33,CD71,CD34,CD38,CD13;Diagnosis:Acutemegakaryoblasticleukemia(FABM7)AntigenProfile:PositiveforCD33,CD71,CD34,CD61ALL常见CD表达SubtypeCommonPhenotypeComments/VariationsB-precursorALLDR,CD19,CD20-/+,CD10+/-,CD34,TdT,sIg(-)InfantsCD10(-)MultilemyeloidAg—t(9;22)Pre-BALLDR,CD19,CD20+/-,CD10,CD34(-),cIgM(+),TdT+/-t(1;19)--CD34(-)—poorprognosis.B-ALLDR,CD19,CD20,CD22,CD24,CD10+/-,CD34(-),SIgL3T-ALLcCD3,CD5,CD7,CD1,CD2,dualCD4/8,CD10+/-CD34-/+FrequentlyloseT-cellAg.6y.Girl,Hb:100g/LWBC:10X10G/L,LC80%Plt.85X10G/LDiagnosis:ALL,B-precursortype(commonALL)AntigenProfile:PositiveforCD71,CD19,CD10,CD22,HLADR,CD34;dimlypositiveforCD33A15yearoldgirlwhoplayedforherhighschoolsoccerteamnotedtohavedifficultybreathingduringherworkouts.Achestx-rayrevealedalargemediastinalmass,andshewasfoundtohaveanemiaandanelevatedwhitebloodcountDiagnosis:ALL,T-cellAntigenProfile:PositiveforCD7,CD5,CD3,CD34,CD10;partlypositiveforCD4andCD8Diagnosis:B-PrecursorALL(commonALL)Antigenprofile:PositiveforCD19,CD10,CD34,HLADR;negativeforCD20,CD45,smIg未分化和双标白血病AUL：仅占1%典型的AUL：DR+，CD34+withnolineage-specificantigens.Biphenotypicleukemia(mixed):仅有7%能严格符合此型。过度诊断是由于：–未排除非白血病细胞；–非特异性结合；–某些抗体缺乏系特异性；特异性抗原为：B—cCD22;T—CD3;M—MPO;ScoringsyetemforthedefinitionofbiphenotypicALPointsB-淋巴T-淋巴髓系2cCD79acCD3cMPOCD22TCRabcIgMTCRrd1CD19CD2CD117CD10CD5CD13CD20CD8CD330.5TdTTdTCD14CD7Diagnosis:Acutemixedlineageleukemiawitht(4;11)AntigenProfile:OnepopulationCD19+CD22+DR+CD20-CD10-;anotherCD33+DR+CD64+CD14+B-LPD常见CD表达DisorderCommonPhenotypeComments/VariationsCLLDR,CD19,CD20,CD5,CD22(-),CD23,CD10(-).clonalSIgM,FMC7(-)CD20dimPLLDR,CD19,CD20.CD5(-),CD22,CD23(-),CD10(-),brightclonalSIgCD20bright。CLL/PLL(30%幼淋细胞)MCLDR,CD19,CD20,CD5,CD22,CD23(-),CD10(-)FMC7(+)SIgbrightCyclinD1overexpressedFCClymphomaDR,CD19,CD20,CD5(-),CD22,C23+/-,CD10CD10negative20%HCLDR,CD19,CD20,CD5(-),CD22,CD23(-),CD10(-),CD11c,CD25,CD103SIgDcommon,verybrightCD22andCD11cPlasmacelldyscrasiasDR(-),CD19(-),CD20(-),CD22(-),CD38,CD45,clonalcIg,BrightCD38,dimCD4573,woman,mildsplenomegaly,WBCnormal,withslightrelativelymphocytosis,aPBspecimenwassentforflowcytometryDiagnosis:ChroniclymphocyticleukemiaAntigenProfile:PositiveforCD19,CD22,CD5,kappa,CD23,dimlypositiveforCD20;MCLAgprofile:positiveforCD19,CD5,lambda(bright),CD20（bright),FMC7.negativeforCD10,CD23,CD11c.CD103