流式细胞术在白血病诊断中的应用课件

1、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流式细胞术在白血病诊断中的应用课件BD FACSVantage SE BD FACSVantage SE FCM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。FCM的结构FCM的结构光源细胞流动室聚光系统信号监测器光源细胞流动室聚光系统信号监测器流式细胞术在白血病诊断中的应用课件数据的存储、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（dot plot) ；等高图：(contour)三维等高图：3D plot设门（gatin

2、g):数据的存储、显示与分析数据的存储：listmodeData Acquisition - ListmodeData Acquisition - Listmode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。一定条件下，荧光强度与细胞内DNA含量成正比。Immunofluorescence Flow Cytometry cont. Data displayedGreen Fluorescence IntensityNumber of CellsUnstained cellsFITC-labeled cellsOne Para

3、meter HistogramRed Fluorescence IntensityGreen Fluorescence IntensityTwo Parameter HistogramImmunofluorescence Flow CytomContour plotContour plot3D plot:细胞多的地方，山峰高。3D plot: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(forward scatter FSC):与激光束平行的信号。与被测细胞大小有关。侧向角散射（side scatter SSC):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。

4、采用FSC、SSC组合，可区分淋巴细胞、单核细胞、粒细胞等群体。FCM检测常用参数内部参数散射光测定前向角散射(forw流式细胞术在白血病诊断中的应用课件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。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流式细胞术在白血病诊断中的应用课件CD45 vs SSC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

5、成熟粒细胞原幼细胞CD45 vs SSC设门CD45为造血细胞（红系除外）的共在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。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；FCM可采用的标本单个细胞，新鲜标本，抗凝保存：流式细胞术在白血病诊断中的应用课件流

6、式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件急性白血病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。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体的选择：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、C

7、D5、cCD3 红系：GlyA 巨核细胞：CD41、CD61 非系列特异性：CD34、DR、CD45抗体的选择：常用抗体选择：AL panelCD7/CD117/CD45-protocalHLA-DR/CD13/CD45CD14/CD2/CD45CD19/CD5/CD45CD33/CD34/CD45CD20/CD10/CD45 CD64,117,13,33,14,64myeloid19,10,20B lineage 7,2,5T lineage DR,34 immature AL panelCD7/CD117/CD45-protoAML常见CD表达FAB SubtypeCommon Pheno

8、typeComments/VariationsM0DR.CD13,CD33,CD34,CD7, cMPO*Generally negative for lymphoid markersM1Similar to M0 except CD15M2DR,CD13,CD33,more CD15,lessCD34CD19(+) -t(8;21) favorable prognosisM3DR(-)*,CD13,CD15,CD33,CD34-/+,CD2(+),DR(-) in maturing AML,consider M3.Myeloid/naturalkiller cell ALCD56(+).M4

9、/M5DR,CD15,CD14,CD33,CD13,CD4weakCD2(+),consider M4EoM6DR,CD13-/+,CD33+/-,CD34,CD45 weakMature forms express glycophorinM7DR-/+,CD33+/-,CD34,CD41,CD61Beware of platelet.adhesionAML常见CD表达FAB SubtypeCommon Phe正常骨髓的FCM二维点图正常骨髓的FCM二维点图流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件D

10、iagnosis: AML- M1Antigen Profile: positive : DR, CD13, CD34, CD38; partially positive for CD7,negative cCD3.Diagnosis: AML- M1Antigen Pro流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件DR,CD19,CD20,CD5(-),CD22,CD23(-),CD10(-),CD11c,CD25,CD103CD20 CD8 CD33Diagnosis: Acute megakaryoblastic leukemia (FAB M7)Antigen

11、Profile: Positive for CD33, CD71, CD34, CD61Negative for CD56,CD10,CD2FCM检测常用参数内部参数散射光测定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

12、 CD38,dim CD45Diagnosis: Acute myeloid leukemia with differentiation (AML M2) with features consistent with t(8;21)Antigen Profile: Positive for CD33(dim), CD34, CD13, CD15 CD11b (partial) with CD19. Negative for CD56,CD10,CD2DR,CD19,CD20,CD5(-),CD22,CD23( 72 , female . A bone marrow examination was

13、 performed. 72 , female . A bone marrow e流式细胞术在白血病诊断中的应用课件Diagnosis: AML- M5Antigen Profile: Positive for CD33, CD38,CD34,HLADR, CD4(dim), CD64, CD13,CD14, CD11b and CD15.Diagnosis: AML- M5Antigen ProDiagnosis: Erythroleukemia (FAB M6)Antigen Profile: Blasts positive for CD33, CD71, CD34, CD38, CD13

14、; Diagnosis: Erythroleukemia (FA流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件Diagnosis: Acute megakaryoblastic leukemia (FAB M7)Antigen Profile: Positive for CD33, CD71, CD34, CD61Diagnosis: Acute megakaryoblasALL常见CD表达SubtypeCommon PhenotypeComments/VariationsB-precursor ALLDR, CD19, CD20-/+, CD10+/-, CD34, T

15、dT,sIg(-)Infants CD10(-) Multile myeloid 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.ALL常见CD表达SubtypeCommon PhenotySIgD comm

16、on,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:

17、Positive for CD71, CD19, CD10, CD22, HLADR, CD34; dimly positive for CD33Dual CD4/CD8 rare.Negative for CD56,CD10,CD2Comments/Variation巨核细胞：CD41、CD616y. Girl, Hb: 100g/LWBC:10X10G/L,LC80%Plt. 85X10G/LSIgD common,very bright CD22 a流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件Diagnosis: ALL, B-precursor type (co

18、mmon ALL)Antigen Profile: Positive for CD71, CD19, CD10, CD22, HLADR, CD34; dimly positive for CD33Diagnosis: ALL, B-precursor tyA 15 year old girl who played for her high school soccer team noted to have difficulty breathing during her workouts. A chest x-ray revealed a large mediastinal mass, and

19、she was found to have anemia and an elevated white blood countA 15 year old girl who played 流式细胞术在白血病诊断中的应用课件Diagnosis: ALL, T-cell Antigen Profile: Positive for CD7, CD5, CD3, CD34, CD10; partly positive for CD4 and CD8Diagnosis: ALL, T-cell Antigen流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课

20、件流式细胞术在白血病诊断中的应用课件Diagnosis: B-Precursor ALL (common ALL)Antigen profile: Positive for CD19, CD10, CD34, HLADR; negative for CD20, CD45, smIgDiagnosis: B-Precursor ALL (co未分化和双标白血病AUL：仅占1%典型的AUL：DR+，CD34+ with no lineage-specific antigens.Biphenotypic leukemia(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

21、异性结合；某些抗体缺乏系特异性；特异性抗原为： BcCD22; TCD3; MMPO;未分化和双标白血病AUL：仅占1%Biphenotypic Scoring syetem for the definition of biphenotypic ALPoints B-淋巴 T-淋巴 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 CD7Scoring syetem for the definit流式细胞术在白血病诊断中的应用课件流式

22、细胞术在白血病诊断中的应用课件流式细胞术在白血病诊断中的应用课件Diagnosis: Acute mixed lineage leukemia with t(4;11)Antigen Profile: One population CD19+CD22+DR+CD20-CD10-; another CD33+DR+CD64+CD14+Diagnosis: Acute mixed lineageB-LPD常见CD表达DisorderCommon PhenotypeComments/VariationsCLLDR,CD19,CD20,CD5, CD22(-), CD23,CD10(-).clonal

23、 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+/-,CD10CD10 negative20% HCLDR,CD19,CD20,CD5(-),CD22,CD23(-),CD

24、10(-),CD11c,CD25,CD103SIgD common,very bright CD22 and CD11cPlasma cell dyscrasiasDR(-),CD19(-),CD20(-),CD22(-),CD38,CD45,clonal cIg,Bright CD38,dim CD45B-LPD常见CD表达DisorderCommon Phen 73 , woman,mild splenomegaly ,WBC normal,with slight relative lymphocytosis, a PB specimen was sent for flow cytometry 73 , woman,mild splenomegaly Diagnosis: Chronic lymphocytic leukemiaAntigen Profile: Positive for CD19, CD22, CD5, kappa, CD23, dimly positive for CD20; Diagnosis: Chronic lymphocyticMCLAg profile: positive for CD19,CD5,lambd