基于细胞表型异常的流式细胞术检测急性前体B细胞白血病微小残留病

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precursor—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10^4个正常骨髓细胞中可以有效检出1个precursor—B-ALL白血病细胞。

Flow Cytometric Detection of Minimal Residual Disease in Precursor-B-Acute Lymphoblastic Leukemia on the Basis of Phenotypic Aberrancies on Minor Leukemic Cell Populations

To test the European BIOMED-1 Concerted Action proposed technique to detect minimal residual disease (MRD) in the chinese patients with precursor-B-acute lymphoblastic leukemia (precursor-B-ALL) by triple-staining flow cytometry and to define both normal and aberrant phenotypic profiles of precursor B cells, a series of bone marrow samples, 35 from precursor-B-ALL (13 in newly diagnosed cases, 15 at the end of remission induction therapy and 7 at end of the consolidations), and 19 from normal controls, were immunophenotyped with the five triple-staining antibodies (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) recom-mended by the BIOMED-1 using common flow cytometric protocols. Further, with different ratios of the leukemic cells with CD34/CD38/CD19 phenotype and normal mononuclear cells, a serial dilution test was analyzed. The results showed that three major CD19(+) cell subpopulations were identified in the normal controls, representing three consecutive maturation stages. The subpopulations in the precursor-B-ALL cases disappeared and were replaced with a great number of luekemic cells which had different characteristics of phenotypes, and then they reappeared with almost same characteristics as the normal CD19(+) cells after the patients achieved complete remission. When the five triple-staining antibody combinations were used, the phenotypic aberrancies could be identified in 12/13 (92.3%) cases with newly diagnosed precursor-B-ALL, at least one triple-labeling per case at the level of 0.01% or more. The frequencies of phenotypic aberrations detected with the triple-staining were 8/13 (61.5%) for CD10/CD20/CD19, 5/13 (38.5%) for CD34/CD38/CD19, 4/13 (30.8%) for CD10/TdT/CD19, 3/13 (23.1%) for CD34/CD22/CD19, and 2/13 (15.4%) for CD34/CD45/CD19. At the end of remission induction, the phenotypic aberrancies could be detected in 5/15 (33.3%), of which, 3/8 (37.5%) cases with the leukemic phenotypes detected both at the newly diagnosis and at the end of induction. The dilution test indicated that the cells with CD34/CD38/CD19 detected by flow cytometry correlated well with the leukemic cells added (r = 0.85, P < 0.05) over 1:1 to 1:400,000. It is concluded that the flow cytometric detection of precursor-B-ALL-MRD proposed by BIOMED-1 Concerted Action were well realized in this study. The one precursor-B-ALL cell can be effectively detected out of 10(4) normal bone marrow cells.