流式细胞术在白血病诊断中的应用.ppt

单击此处编辑母版标题样式,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,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（,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,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,Red Fluorescence Intensity,Green Fluorescence Intensity,Two Parameter Histogram,Contour plot,3,D 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,。,CD,45,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：,CD,33,、CD,13、,CD,14，,c,MPO,B-cell:CD,19,、CD,10、c,CD,22,CD,20,T-cell:CD,7,、CD,2,、CD,5、c,CD,3,红系：,GlyA,巨核细胞：,CD41、CD61,非系列特异性：,CD,34,、DR、CD,45,AL panel,CD7/CD117/CD45-protocal,HLA-DR/CD13/CD45,CD14/CD2/CD45,CD19/CD5/CD45,CD33/CD34/CD45,CD20/CD10/CD45 CD64,117,13,33,14,64myeloid,19,10,20B lineage 7,2,5T lineage,DR,34 immature,AML常见CD表达,FAB Subtype,Common Phenotype,Comments/Variations,M,0,DR.CD13,CD33,CD34,CD7,cMPO*,Generally negative for lymphoid markers,M1,Similar to M0 except CD15,M2,DR,CD13,CD33,more CD15,lessCD34,CD19(+)-t(8;21)favorable prognosis,M3,DR(-),*,CD13,CD15,CD33,CD34-/+,CD2(+),DR(-)in maturing AML,consider,M3.Myeloid/naturalkiller cell ALCD56(+).,M4/M5,DR,CD15,CD14,CD33,CD13,CD4weak,CD2(+),consider M4Eo,M6,DR,CD13-/+,CD33+/-,CD34,CD45 weak,Mature forms express glycophorin,M7,DR-/+,CD33+/-,CD34,CD41,CD61,Beware of platelet.adhesion,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,Subtype,Common Phenotype,Comments/Variations,B-precursor ALL,DR,CD19,CD20-/+,CD10+/-,CD34,TdT,sIg(-),Infants CD10(-)Multile myeloid Agt(9;22),Pre-B ALL,DR,CD19,CD20+/-,CD10,CD34(-),cIgM(+),TdT+/-,t(1;19)-CD34(-)poor prognosis.,B-ALL,DR,CD19,CD20,CD22,CD24,CD10+/-,CD34(-),SIg,L3,T-ALL,cCD3,CD5,CD7,CD1,CD2,dual CD4/8,CD10+/-CD34-/+,Frequently lose T-cell Ag.,6,y.Girl,Hb:100g/L,WBC: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,Disorder,Common Phenotype,Comments/Variations,CLL,DR,CD19,CD20,CD5,CD22(-),CD23,CD10(-,).clonal SIgM,FMC7(-),CD20 dim,PLL,DR,CD19,CD20.,CD5(-),CD22,CD23(-,),CD10(-),bright clonal SIg,CD20 bright。CLL/PLL(30%,幼淋细胞),MCL,DR,CD19,CD20,CD5,CD22,CD23(-),CD10(-)FMC7(+),SIg bright Cyclin D1 overexpressed,FCC lymphoma,DR,CD19,CD20,CD5(-),CD22,C23+/-,CD10,CD10 negative20%,HCL,DR,CD19,CD20,CD5(-),CD22,CD23(-),CD10(-),CD11c,CD25,CD103,SIgD common,very bright CD22 and CD11c,Plasma cell dyscrasias,DR(-),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 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（bright),FMC7.negative for CD10,CD23,CD11c.CD103.CD3.,Diagnosis:CLL.Antigen Profile:Positive for CD19,CD22,CD5,lambda,CD23,dimly positive for CD20;partly positive for CD11c,周围T-LPD常见CD表达,Disorder,Common Phenotype（the major features are CD3/CD4 or CD3/CD8),Comments/Variation,T-PLL,CD2,CD3,CD5,CD7,CD4,CD8(-),Dual CD4/CD8 rare.More aggressive than B-PLL,LGL-T,CD2,CD3,CD5(-)CD7(-),CD4(-),CD8dim,NK marker CD16,CD57,but CD56(-),TCR rearrangement,If CD3,CD56(+),more aggressive.,LGL-NK,CD2,CD16,CD56 CD3(-),CD57(-),CD8(-),Hb,BPC,hepatosplenomegaly.,ATL/L,CD2,CD3,CD5,CD7(-),CD4,CD8(-,),DR,bright CD25,T-CLL,CD2,CD3,CD5,CD7,CD4（-）,CD8(-),Clonal TCR rearrahgement,谢谢！,