八色流式细胞术检测急性B淋巴细胞白血病微小残留病方法的建立

Method establishment of minimal residual disease in B lineage acute lymphoblastic leukemia by 8- color flow cytometric detection

目的建立八色流式细胞术（8c—FCM）检测急性B淋巴细胞白血病（B-ALL）微小残留病（MRD）的方法，并进行初步验证。方法采用CD19／侧向角散色光（SSC）、CD45／10和CD34（或cTdT）联合设门，两管八色抗体组合检测B—ALL的MRD。将B—ALL细胞株Nalm－6细胞掺入正常人骨髓细胞中，使Nalm--6细胞占有核细胞比例为10．00％、1．00％、0．10％、0．01％和0．005％，采用已建立的8c—FCM，进行回收试验和重复性试验，评价检测方法的准确度和精密度。检测抗体标记Nalm-6后保存不同时间的荧光强度变化，评价各荧光抗体的稳定性。检测并分析39份骨髓细胞免疫表型，包括对照者9例，初发或复发B-ALL患者9例，B—ALL化疗或骨髓移植（BMT）后缓解期病例21例，评价8c—FCM方法检测正常骨髓B淋巴系细胞群和B—ALL细胞的免疫表型结果，以及对B—ALL的MRD检测结果，并探讨其与现用四色流式细胞术（4c．FCM）的可比性。结果建立了以CD19、CIM5、CD10为主的两管八色抗体组合检测MRD的方法；当掺人的细胞株占有核细胞比例≥0．10％时，变异系数（CV）〈2．5％，平均回收率为95．81％；当获取10。个细胞，Nalm-6细胞比例10．00％-0．01％时，检测到的Nalm-6细胞占骨髓有核细胞比例与实测值呈线性相关（r=0．99，P〈0．05），该法检测骨髓中Nalm-6细胞的灵敏度达到0．01％。Nalm-6细胞株标记抗体后随时间延长略有降低，但24h内平均荧光强度减低〈10％。应用该方法检测骨髓中CD19阳性的B淋巴系细胞，对照组呈现4个连续发育阶段，可分为4期：I期CD45low／CD10stro／CD20-／CD38stro／CD34＋或eTdT＋，Ⅱ期CD45-／CD10＋／CD20-／CD38stro／CD34＋或cTdT＋，Ⅲ期CD45＋／CD10＋／CD20-／CD38stro／CD34-或cTdT-，Ⅳ期CD45stro／CD10-／CD20＋／CD38low／CD34-或cTdT-。初发和复发B-ALL患者骨髓中自血病细胞与对照组相比，均存在抗原表达异常；8e—FCM检测B—ALL缓解组，有5例存在MRD，主要抗原表达与4c—FCM检测结果一致，5例MRD细胞的范围为0．02％-5．42％。结论本研究所建立的两管8c—FCM新方法检测骨髓中自血病细胞的重复性好、准确度高，可鉴别骨髓正常B淋巴系细胞群、B—ALL治疗缓解后骨髓造血重建的B系前体细胞与MRD细胞群；和4c-FCM比较，两管8c—FCM新方法的标本用量少，检测速度快，能够有效诊断B—ALL的MRD。

Objective To establish and evaluate the new method of 8-color flow cytometry （Se-FCM） with two tube detecting bone marrow minimal residual disease （MRD） in B lineage acute lymphoblastic leukemia （B-ALL）. Methods The MRD cells were analyzed by using two combinations of 8c-FCM antibody panels, gating with CD19/Side scatter（SSC）, CD45/CD10 and CD34（or cTdT）. Nalm-6 cell of B-ALL was mixed into normal marrow cells, with proportion of 10. 00%, 1.00%, 0. 10%, 0. 01% and 0. 005% , and recovery test and reproducibility test were carried with 8c-FCM established to value its accuracy and precision of. Fluorescence intensity was detected on different time points after marked Nalm-6 by antibodies to evaluate the fluorescent stability of the antibodies. The immunophenotyping was analyzed in 39 bone marrow specimens, including 9 cases of normal control, 9 eases of B-ALL primary or recurrent, 21 cases of complete remission （CR） after chemotherapy or bone marrow transplantation （BMT）, to evaluate the detection of normal B lymphocyte lineage, leukemia cells of B-ALL, MRD cells of B-ALL using the 8c- FCM and compare it with 4c-FCM in being. Results The method of two tube 8c-FCM with main antibodies of CD19, CD45 and CD10 to detect MRD of B-ALL was founded; CV 〈 2. 5% , average recovery rate was 95.81% , when the actual percent of Nalm-6 mixed into normal bone marrow≥0. 10% , and the percent of Nalm-6 detected and actual was linear dependent （ r = 0. 99, P 〈 0.05 ） ranged from 10.00% - 0. 01% , when 106 cells were acquired; the sensitivity of the method established could reach O. 01%. The fluorescent intensity decreased along with the time after Nalm-6 cell marked, but less than 10% in 24 hours. Using the antibody combinations and analyze strategy, the immunophenotye of B lymphocyte in normal bone marrow presented four sequential stages： Stage I CD45low/CD10stro/CD20-/CD38stro/CD34＋ or cTdT＋, Stage 11 CD45 ＋/CD10 ＋ / CD20 -/CD38stro/CD34＋ or cTdT ＋ , Stage III CD45 ＋/CD10 ＋/CD20 -/CD38stro/CD34 - or cTdT- , Stage IV CD45stro/CD10-/CD20 ＋/CD38low/CD34- or cTdT-. Antigen expressions of leukeamic cells of B-ALL primary or recurrent were different compared with control team ; there were 5 cases with MRD positive in CR team, and the main antigen expression was consistent with the results from 4c-FCM. The range of the percent of MRD cells was 0. 02% - 5.42% of the 5 cases of MRD positive. Conclusions The new method of two tube 8c-FCM established shows good reproducibility and high accuracy, and can identify normal B lymphocyte populations in bone marrow and regenerated B-precursors in CR cases with MRD cells; compared with 4c-FCM, the new method of two tube 8c-FCM with the fewer specimen is faster and efficient to diagnose MRD of B-ALL .