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为了建立一种基于细胞表型异常的流式细胞术以检测急性前体B细胞白血病微小残留病,对35份pre-cursor-B-ALL病人骨髓和19例正常对照骨髓应用BIOMED-1推荐的5种3色抗体组合(TdT/CD10/CD19,CD10/CD20/CD19,CD34/CD38/CD19,CD34/CD22/CD19和CD19/CD34/CD45)进行了流式免疫分型,以确定前体B细胞正常和异常抗原表型流式图形特征。在35例患者中初诊病人13例,完成诱导缓解后的病人15例,完成巩固治疗的患者7例。应用不同比例的正常骨髓单个核细胞和带有CD34/CD38/CD19阳性白血病细胞进行了系列稀释试验。结果显示:在正常对照组中流式细胞术分析显示了3群CD19阳性细胞,代表了B细胞的3个连续成熟阶段。在precursor-B-ALL患者中这3群细胞消失,代之以大量的白血病细胞,而这些白血病细胞的表型特征与正常B细胞不同。当病人获得完全缓解时,这3群细胞会重新出现,而且具有与正常CD19阳性细胞几乎相同的表型特征。用5组3色抗体组合检测病人时,初诊患者12/13(92.3%)可检出抗原表型异常,也即在0.01%的敏感性水平每个患者至少有1种抗体组合的异常。在本研究初诊病人中这些抗体组合的异常频率:CD10/CD20/CD19为8/13(61.5%);CD34/CD38/CD19为5/13(38.5%);CD10/TdT/CD19为4/13(30.8%);CD34/CD22/CD19为3/13(23.1%);CD34/CD45/CD19为2/13(15.4%)。刚获得完全缓解的患者抗原表型异常的检出率为5/15(33.3%),其中初诊和缓解时同时检出异常者3/8(37.5%)。稀释试验表明,从1∶1至1∶400000的范围,流式细胞术检出与已知加入的CD34/CD38/CD19阳性白血病细胞数有良好的线性相关(r=0.80,P<0.05)。结论:BIOMED-1协作组建议的基于细胞表型异常的流式细胞术用于precursor-B-ALL微小残留病的检测在本研究中能较好地实现。在104个正常骨髓细胞中可以有效检出1个precursor-B-ALL白血病细胞。
To establish a flow cytometer based on abnormal cell phenotypes for the detection of acute residual B cell leukemia minimal residual disease, BIOMED-1 was recommended in 35 pre-cursor-B-ALL patients with bone marrow and 19 normal control bone marrow Flow immunophenotyping was performed on the five 3-color antibody combinations (TdT / CD10 / CD19, CD10 / CD20 / CD19, CD34 / CD38 / CD19, CD34 / CD22 / CD19 and CD19 / CD34 / CD45) Cell Normal and Abnormal Antigen Phenotypes Flow Graph Features. Thirteen patients were newly diagnosed in 35 patients, 15 patients completed induction therapy and 7 patients completed consolidation therapy. Serial dilutions were performed using different ratios of normal bone marrow mononuclear cells and with CD34 / CD38 / CD19 positive leukemia cells. The results showed that in the normal control group, flow cytometry analysis revealed three CD19 positive cells, representing three consecutive maturation stages of B cells. In the precursor-B-ALL patients, the three groups of cells disappeared, replaced by a large number of leukemic cells, and these leukemic cells phenotypic characteristics and normal B cells are different. When the patient achieved complete remission, these 3 groups of cells reappeared and had almost the same phenotypic characteristics as normal CD19-positive cells. When the patients were tested on a combination of five 3-color antibodies, 12/13 (92.3%) of the newly diagnosed patients showed an abnormal phenotype of the antigen, ie at least 1 antibody combination abnormality per patient at a sensitivity of 0.01%. Abnormal frequencies of these antibody combinations were 8/13 (61.5%) for CD10 / CD20 / CD19; 5/13 (38.5%) for CD34 / CD38 / CD19; and 4/10 for CD10 / TdT / CD19 (30.8%); 3/13 (23.1%) for CD34 / CD22 / CD19 and 2/13 (15.4%) for CD34 / CD45 / CD19. The prevalence of abnormal antigen phenotype in newly acquired patients was 5/15 (33.3%), of which 3/8 (37.5%) were detected abnormally at the time of initial diagnosis and remission. Dilution tests showed a good linear correlation between flow cytometry and known addition of CD34 / CD38 / CD19 positive leukemia cells (r = 0.80, P <0.05) in the range of 1: 1 to 1: 400000. CONCLUSIONS: Flow cytometry based on cell phenotype abnormality suggested by BIOMED-1 collaborative group for the detection of precursor-B-ALL minimal residual disease can be well achieved in this study. One precursor-B-ALL leukemia cell line was successfully detected in 104 normal bone marrow cells.