采用五色流式细胞术分析白血病和淋巴瘤免疫表型方案的建立及其应用

Novel 5-color flow cytometric immunophenotyping protocol for leukemia/lymphoma and its clinical application

目的建立五色流式细胞术分析白血病和淋巴瘤免疫表型方案，并评价其临床应用价值。方法对73例急性白血病和30例淋巴瘤患者，采用五色流式免疫表型分析方案中系列敏感指标组合（CD7／CD33／CD19／CD13／CD45）进行第一轮筛选，系列特异指标（cMPO／cCD79a／cCD3／CD117／CD45）组合做确认检测，并根据确认试验结果进行第二轮检测。结果用仪器软件自动程序可方便地得到理想的五色荧光补偿值。五色流式表型分析结果显示，急性B淋巴细胞性白血病（B—ALL）中CD19阳性率100％（27／27），cCD7％阳性率92．6％（25／27），根据CD34、CD10、cμ、sIgM等表达情况可对B—ALL进行确切分期诊断；急性T淋巴细胞性白血病（T—ALL）中均表达CD7（8／8）和cCD3（8／8），根据CD2、CD1a、CD3、CD4、CD8等表达情况可对T—ALL进行确切分期；急性髓细胞性白血病（AML）中髓系细胞系列相对特异抗原cMPO表达率89．5％（34／38）、CD117为81．6％（31／38），26．3％（10／38）的AML混合表达淋系抗原CD7，结合形态学等可进行亚型辅助诊断。采用五色抗体组合并用CD19／SSC设门可对B细胞淋巴瘤进行鉴别诊断。开展五色流式表型分析节省检测的上样管数，由原来三色方案的12管减少到6管，同时可节省20％的抗体用量。结论五色流式表型分析方案提高了免疫表型分析的准确性，降低了成本，适合临床实验室开展白血病和淋巴瘤的免疫表型分析。

Objective To establish a five-color flow cytometric immunophenotyping protocol and valuate its clinical application in leukemia and lymphoma. Methods Samples from 73 cases of acute leukemia and 30 cases of lymphoma were analyzed using a comprehensive antibody panel with five-color combination. The antibody combination CD7/CD33/CD19/CD13/CD45 and MPO/cCD79a/cCD3/CD117/CD45, composed of different lineage distinctive and lineage sensitive markers of B, T and myeloid cells, were applied to determine the lineage originality of leukemia and lymphoma cells. The markers used in the second step analysis were based on the findings of the first step. Results Five-color compensation can be performed automatically and easily with the advanced digital compensation program. The results showed that the expression ratio of CD19 in B-ALL was 100% （27/27）, cCD79a positivity was 92.6% （25/27） and CDT, cCD3 was expressed in all T-ALL cases （8/8）. The B-ALL could be staged depending on the expression level of CD34, CD10, cμ, sIgM and the T-ALL could be clearly staged dependent on the expression level of CD2 ,CD1a ,CD3 ,CD4 ,CDs. The expression ratio of cMPO, CD117 was 89.5% （34/38） and 81.6% （31/38） in AML respectively, however CD7 was also expressed in 26. 3% （10/38） of AML cases. Combined with morphology, immunophenotypes could be used for diagnosing AML with subtyping. The CD19/SSC gating can be used for immunophenotyping of B cell lymphoma with differential diagnosis. The number of tubes required was significantly reduced with our panel from 12 tubes of 3-color to 6 tubes of 5-color. Implementation of the five-color protocol had resulted in 20% reduction in reagent costs. Conclusions The application of fivecolor staining protocol significantly improve the sensitivity and accuracy of measurement of immunophenotypes of acute leukemia and lymphoma. It reduces the cost and can be widely applied in the clinical laboratory diagnosis.