急性白血病患者八色流式免疫表型分析起始管方案的建立

Establishment of the Orientation Tube Panel for 8-color Flow Cytometric Immunophenotyping for Patients with Acute Leukemia

目的建立急性白血病(AL)患者八色流式免疫表型分析起始管方案。方法用胞膜CD3(CD3)、CD19、CD10、CD34、CD45、胞浆CD79a(cCD79a)、髓过氧化物酶(MPO)和胞浆CD3(cCD3)等8种抗体建立八色流式染色方案。膜表面抗体直接染色;膜内抗体经固定破膜,再染色后上机检测。将3个血小板减少患者骨髓标本分别进行抗体的单色染色和缺一色染色;最后对17例确诊的AL初发患者标本进行检测。结果用单色染色来确定染色方案中各抗体的检测电压及荧光补偿;缺一色染色中,阳性细胞群较单色染色变化均<10%,表明方案中的各抗体相互作用小。17例AL初发患者中,6例急性B淋巴细胞白血病原始细胞均为CD34和CD19阳性,5例cCD79a阳性和4例CD10阳性;4例急性T淋巴细胞白血病患者均为cCD3阳性;6例急性髓细胞白血病均为CD34和MPO阳性;1例B+T混合表型AL患者CD34、cCD3、CD19、cCD79a及CD10均为阳性,MPO和CD3为阴性,此检测方案能够确定各类AL的细胞类型。结论建立了AL患者八色流式免疫表型分析起始管方案,操作简便快速,适用于临床检测。

Objective To develop the orientation tube panel for 8-color flow cytometric immunophenotyping for patients with acute leukemia（AL）.Methods Using 8 antibodies including membrane CD3,CD19,CD10,CD34,CD45,cytoplasmic CD79a（cCD79a）,MPO and cytoplasmic CD3（cCD3）,we developed the orientation tube panel for 8-color flow cytometric immunophenotyping for patients with AL.For antibodies of membrane antigens,cells were directly stained;for antibodies of cytoplasmic antigens,cells were fixed and permeabilized,and then stained before acquisition.Three samples from patients with thrombocytopenia were single-stained and stained with lack of 1 of 8 antibodies.Seventeen patients with confirmed AL at diagnosis were immunophenotyped by the orientation tube panel at last.Results By single-staining,detector voltages and compensations were determined.In staining lacking 1 of 8 antibodies,positive cell populations changed less than 10% compared with single-staining,which indicated that antibodies in this panel had little interactions.In the 17 patients with primary AL,blasts of 6 B acute lymphoblastic leukemia（B-ALL） were CD34 and CD19 postive among whom 5 had postitive cCD79a and 4 had positive CD10;all 4 T acute lymphoblastic leukemia（T-ALL） patients were cCD3 positive;6 acute myelocytic leukemia（AML） patients were CD34 and MPO positive;one B＋T mixed phenotype acute leukemia（MPAL） had positive CD34,cCD3,CD19,cCD79a and CD10,and negative MPO and CD3.These indicated that the orientation tube panel could determine cell lineages of AL.Conclusions The orientation tube panel for 8-color flow cytometric immunophenotyping for patients with AL has been developed.The panel is simple and rapid,and can be used for clinical detection.