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

By 王璐璐, 王建中, 屈晨雪, 王卉, 刘抗援, 龚岩, 邢莹 and 伍平


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、CD45、CD10为主的两管八色抗体组合检测MRD的方法;当掺入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106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cTdT+,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患者骨髓中白血病细胞与对照组相比,均存在抗原表达异常;8c-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新方法检测骨髓中白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 (8c-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 cases 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 0.01%.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 ⅡCD45 +/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.中文核心期刊要目总览(PKU)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)05423-4303

Topics: 流式细胞术, 急性淋巴细胞白血病, 肿瘤,残余, Flow cytometry, Acute lymphoblastic leukemia, Neoplasm,residual
Publisher: 中华检验医学杂志
Year: 2012
DOI identifier: 10.3760/cma.j.issn.1009-9158.2012.05.009
OAI identifier: oai:localhost:20.500.11897/123622
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