流式细胞术在白血病诊断中的应用PPT课件

流式细胞术在白血病诊断中的应用,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（cell sorting).科研用。,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。,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（dot plot) ；等高图：(contour)三维等高图：3D plot设门（gating):,Data 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。,Immunofluorescence,Flow Cytometry cont. Data displayed,Green Fluorescence Intensity,Number of Cells,Unstained cells,FITC-labeled cells,One Parameter Histogram,Contour plot,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。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。,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：,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、CD45,AL panel,CD7/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,AML常见CD表达,正常骨髓的FCM二维点图,Diagnosis: AML- M1Antigen Profile: positive : DR, CD13, CD34, CD38; partially positive for CD7,negative cCD3.,Diagnosis: 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,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: Erythroleukemia (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, CD61,ALL常见CD表达,6y. Girl, Hb: 100g/LWBC:10X10G/L,LC80%Plt. 85X10G/L,Diagnosis: ALL, B-precursor type (common ALL)Antigen Profile: Positive for CD71, CD19, CD10, CD22, HLADR, CD34; dimly positive for CD33,A 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 she was found to have anemia and an elevated white blood count,Diagnosis: ALL, T-cell Antigen Profile: Positive for CD7, CD5, CD3, CD34, CD10; partly positive for CD4 and CD8,Diagnosis: B-Precursor ALL (common ALL)Antigen profile: Positive for CD19, CD10, CD34, HLADR; negative for CD20, CD45, smIg,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；非特异性结合；某些抗体缺乏系特异性；特异性抗原为： BcCD22; TCD3; MMPO;,Scoring syetem for the definition of biphenotypic AL,Points 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 CD7,Diagnosis: Acute mixed lineage leukemia with t(4;11)Antigen Profile: One population CD19+CD22+DR+CD20-CD10-; another CD33+DR+CD64+CD14+,B-LPD常见CD表达,73 , woman,mild splenomegaly ,WBC normal,with slight relative lymphocytosis, a PB specimen was sent for flow cytometry,Diagnosis: Chronic lymphocytic leukemiaAntigen Profile: Positive for CD19, CD22, CD5, kappa, CD23, dimly positive for CD20;,MCL,Ag profile: positive for CD19,CD5,lambda(bright),CD20（bri