流式细胞术在白血病诊断中的应用课件.ppt

1、BD FACSVantage SE lFCM的结构的结构细胞流动室；细胞流动室；光源；光源；聚光系统；聚光系统；信号检测器；信号检测器；电子计算机；电子计算机；细胞分选装置。细胞分选装置。光源细胞流动室聚光系统信号监测器l数据的存储：数据的存储：listmodel显示：显示：单参数直方图单参数直方图(histgram);：常用于分析：常用于分析DNA等；等；双参数：二维点图等（双参数：二维点图等（dot plot)；等高图：等高图：(contour)三维等高图：三维等高图：3D plotl设门（设门（gating):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

2、，不能显示两个独立参数与细胞的关系。Flow Cytometry cont.Data displayedGreen Fluorescence IntensityNumber of CellsUnstained cellsFITC-labeled cellsOne Parameter HistogramRed Fluorescence IntensityGreen Fluorescence IntensityTwo Parameter HistogramContour plot3D plot:细胞多的地方，山峰高。l前向角散射前向角散射(forward scatter FSC):与激光束平与激光

3、束平行的信号。与行的信号。与被测细胞大小有关被测细胞大小有关。l侧向角散射（侧向角散射（side scatter SSC):与激光束呈与激光束呈90的光信号。与的光信号。与细胞内结构和颗粒物质多少有关。细胞内结构和颗粒物质多少有关。l采用采用FSC、SSC组合，可区分淋巴细胞、单核组合，可区分淋巴细胞、单核细胞、粒细胞等群体。细胞、粒细胞等群体。l荧光素探针标记的细胞所发荧光。荧光素探针标记的细胞所发荧光。l荧光信号强弱、多少与细胞的抗原量、内含物荧光信号强弱、多少与细胞的抗原量、内含物多少有关。多少有关。l常用的荧光显色剂：常用的荧光显色剂：FITC：PE：藻红蛋白藻红蛋白 PerCP；PI

4、：碘化丙啶，测定碘化丙啶，测定DNA含量。含量。l临床常用三色荧光检测，现有四色分析临床常用三色荧光检测，现有四色分析。lCD45为造为造血细胞（血细胞（红系除外红系除外）的共同）的共同抗原。避抗原。避免杂质细免杂质细胞的沾染胞的沾染。CD45:淋巴细胞单核细胞成熟粒细胞原幼细胞l细胞的分化成熟、细胞内部结构及其分化抗原细胞的分化成熟、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。一个细胞的多种参数方能正确判断细胞属性。l当细胞发生克隆增殖后，可出现抗原量变、丢当细胞发生克隆增殖后，可出现抗原量变

5、、丢失和错译表达。失和错译表达。l细胞抗原的阳性率及荧光强度是同等重要的指细胞抗原的阳性率及荧光强度是同等重要的指标。标。l单个细胞，新鲜标本，抗凝保存：单个细胞，新鲜标本，抗凝保存：PB；BM；（；（有时有时BM活检标本）活检标本）LN（包括包括FNA）；）；CSF；组织浸润组织浸润FNA 或活检标本；或活检标本；l白血病细胞的抗原表达一定程度遵循正常细胞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。抗原表达的规律。l白血病细胞为肿瘤性生长，可出现正常细胞表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。达以外的异常现象，如缺失、错译表达等。l目前未发

6、现白血病特异性抗原，须多参数分析。目前未发现白血病特异性抗原，须多参数分析。l临床原临床原/幼细胞群定性比定量更为重要。幼细胞群定性比定量更为重要。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，cMPO B-cell:CD19、CD10、cCD22 CD20 T-cell:CD7、CD2、CD5、cCD3 红系：GlyA 巨核细胞：CD41、CD61 非系列特异性：CD34、DR、CD45lCD7/CD117/CD45-protocallHLA-DR/CD13/CD45lCD14/CD2/CD45lCD19/CD5/CD45lCD33/CD34

7、/CD45lCD20/CD10/CD45 CD64,l117,13,33,14,64myeloidl19,10,20B lineage 7,2,5T lineage lDR,34 immature FAB SubtypeCommon PhenotypeComments/VariationsM0DR.CD13,CD33,CD34,CD7,cMPO*Generally negative for lymphoid markersM1Similar to M0 except CD15M2DR,CD13,CD33,more CD15,lessCD34CD19(+)-t(8;21)favorable pr

8、ognosisM3DR(-)*,CD13,CD15,CD33,CD34-/+,CD2(+),DR(-)in maturing AML,consider M3.Myeloid/naturalkiller cell ALCD56(+).M4/M5DR,CD15,CD14,CD33,CD13,CD4weakCD2(+),consider M4EoM6DR,CD13-/+,CD33+/-,CD34,CD45 weakMature forms express glycophorinM7DR-/+,CD33+/-,CD34,CD41,CD61Beware of platelet.adhesionDiagn

9、osis:AML-M1Antigen Profile:positive:DR,CD13,CD34,CD38;partially positive for CD7,negative cCD3.DR,CD19,CD20,CD5(-),CD22,CD23(-),CD10(-),CD11c,CD25,CD103CD20 CD8 CD33Diagnosis:Acute megakaryoblastic leukemia(FAB M7)Antigen Profile:Positive for CD33,CD71,CD34,CD61Negative for CD56,CD10,CD2FCM检测常用参数内部参

10、数散射光测定CD2,CD3,CD5(-)CD7(-),CD4(-),CD8dim,NK marker CD16,CD57,but CD56(-)B-precursor ALL等高图：(contour)SIgD common,very bright CD22 and CD11cDR,CD15,CD14,CD33,CD13,CD4weakDR,CD19,CD20-/+,CD10+/-,CD34,TdT,sIg(-)Bright CD38,dim CD45Diagnosis:Acute myeloid leukemia with differentiation(AML M2)with feature

11、s consistent with t(8;21)Antigen Profile:Positive for CD33(dim),CD34,CD13,CD15 CD11b(partial)with CD19.Negative for CD56,CD10,CD2 72,female.A bone marrow examination was performed.Diagnosis:AML-M5Antigen Profile:Positive for CD33,CD38,CD34,HLADR,CD4(dim),CD64,CD13,CD14,CD11b and CD15.Diagnosis:Eryth

12、roleukemia(FAB M6)Antigen Profile:Blasts positive for CD33,CD71,CD34,CD38,CD13;Diagnosis:Acute megakaryoblastic leukemia(FAB M7)Antigen Profile:Positive for CD33,CD71,CD34,CD61SubtypeCommon PhenotypeComments/VariationsB-precursor ALLDR,CD19,CD20-/+,CD10+/-,CD34,TdT,sIg(-)Infants CD10(-)Multile myelo

13、id Agt(9;22)Pre-B ALLDR,CD19,CD20+/-,CD10,CD34(-),cIgM(+),TdT+/-t(1;19)-CD34(-)poor prognosis.B-ALLDR,CD19,CD20,CD22,CD24,CD10+/-,CD34(-),SIg L3T-ALLcCD3,CD5,CD7,CD1,CD2,dual CD4/8,CD10+/-CD34-/+Frequently lose T-cell Ag.SIgD common,very bright CD22 and CD11cCD45:淋巴细胞单核细胞成熟粒细胞原幼细胞Dual CD4/CD8 rare.周

14、围T-LPD常见CD表达Data Acquisition-ListmodeImmunofluorescenceB-LPD常见CD表达One Parameter Histogram2 cCD79a cCD3 cMPOCD2,CD3,CD5,CD7,CD4（-）,CD8(-)Common PhenotypeCD20 CD8 CD33Diagnosis:ALL,B-precursor type(common ALL)Antigen Profile:Positive for CD71,CD19,CD10,CD22,HLADR,CD34;dimly positive for CD33Dual CD4/C

15、D8 rare.Negative for CD56,CD10,CD2Comments/Variation巨核细胞：CD41、CD616y.Girl,Hb:100g/LWBC:10X10G/L,LC80%Plt.85X10G/LDiagnosis:ALL,B-precursor type(common ALL)Antigen Profile:Positive for CD71,CD19,CD10,CD22,HLADR,CD34;dimly positive for CD33A 15 year old girl who played for her high school soccer team

16、noted to have difficulty breathing during her workouts.A chest x-ray revealed a large mediastinal mass,and she was found to have anemia and an elevated white blood countDiagnosis:ALL,T-cell Antigen Profile:Positive for CD7,CD5,CD3,CD34,CD10;partly positive for CD4 and CD8Diagnosis:B-Precursor ALL(co

17、mmon ALL)Antigen profile:Positive for CD19,CD10,CD34,HLADR;negative for CD20,CD45,smIglAUL：仅占仅占1%典型的典型的AUL：DR+，CD34+with no lineage-specific antigens.lBiphenotypic leukemia(mixed):仅有仅有7%能严格符合此型。过度诊能严格符合此型。过度诊断是由于：断是由于：未排除非白血病细胞；未排除非白血病细胞；非特异性结合；非特异性结合；某些抗体缺乏系特异性；某些抗体缺乏系特异性；l特异性抗原为：特异性抗原为：BcCD22;TCD3

18、;MMPO;Points B-淋巴淋巴 T-淋巴淋巴 髓系髓系 2 cCD79a cCD3 cMPO CD22 TCRab cIgM TCRrd 1 CD19 CD2 CD117 CD10 CD5 CD13 CD20 CD8 CD33 0.5 TdT TdT CD14 CD7Diagnosis:Acute mixed lineage leukemia with t(4;11)Antigen Profile:One population CD19+CD22+DR+CD20-CD10-;another CD33+DR+CD64+CD14+DisorderCommon PhenotypeCommen

19、ts/VariationsCLLDR,CD19,CD20,CD5,CD22(-),CD23,CD10(-).clonal SIgM,FMC7(-)CD20 dimPLLDR,CD19,CD20.CD5(-),CD22,CD23(-),CD10(-),bright clonal SIgCD20 bright。CLL/PLL(30%幼淋细胞)MCLDR,CD19,CD20,CD5,CD22,CD23(-),CD10(-)FMC7(+)SIg bright Cyclin D1 overexpressedFCC lymphomaDR,CD19,CD20,CD5(-),CD22,C23+/-,CD10C

20、D10 negative20%HCLDR,CD19,CD20,CD5(-),CD22,CD23(-),CD10(-),CD11c,CD25,CD103SIgD common,very bright CD22 and CD11cPlasma cell dyscrasiasDR(-),CD19(-),CD20(-),CD22(-),CD38,CD45,clonal cIg,Bright CD38,dim CD45 73,woman,mild splenomegaly,WBC normal,with slight relative lymphocytosis,a PB specimen was se

21、nt for flow cytometryDiagnosis:Chronic lymphocytic leukemiaAntigen Profile:Positive for CD19,CD22,CD5,kappa,CD23,dimly positive for CD20;MCLAg profile:positive for CD19,CD5,lambda(bright),CD20（bright),FMC7.negative for CD10,CD23,CD11c.CD103.CD3.Diagnosis:CLL.Antigen Profile:Positive for CD19,CD22,CD

22、5,lambda,CD23,dimly positive for CD20;partly positive for CD11cDisorder Common Phenotype（the major features are CD3/CD4 or CD3/CD8)Comments/VariationT-PLLCD2,CD3,CD5,CD7,CD4,CD8(-)Dual CD4/CD8 rare.More aggressive than B-PLLLGL-TCD2,CD3,CD5(-)CD7(-),CD4(-),CD8dim,NK marker CD16,CD57,but CD56(-)TCR rearrangementIf CD3,CD56(+),more aggressive.LGL-NKCD2,CD16,CD56 CD3(-),CD57(-),CD8(-)Hb BPC ,hepatosplenomegaly.ATL/LCD2,CD3,CD5,CD7(-),CD4,CD8(-),DR,bright CD25T-CLLCD2,CD3,CD5,CD7,CD4（-）,CD8(-)Clonal TCR rearrahgement谢谢观看谢谢观看谢谢观看谢谢观看