流式细胞仪在儿童急性B淋巴细胞性白血病免疫分型中的应用(1)
【摘要】 目的 研究流式细胞仪检测儿童急性B淋巴细胞性白血病(B-ALL)免疫分型的临床价值，并探讨与预后的关系。方法 利用流式细胞仪对39例B-ALL患儿在初诊时进行免疫分型。CD45-SSC设门，在CD19、CD10、CD20和CD22中选择两个单克隆抗体组合，进行三色组合分析。髓系抗体选用CD33和CD13。根据是否伴有髓系抗原表达分为两组，并随访预后。结果 ①CD19、CD22、CD10的阳性率都超过或接近90%，分别为CD19：94.87%，CD22：92.31%，CD10：87.18%，而CD20只有15.38%的阳性率；有33.33%的患儿检测到髓系抗原(My)标志，其中以CD+138例，CD+335例；②共有6例患儿复发，两组经χ2精确概率法检验，P>0.05，差异无统计学意义。结论 ①流式细胞仪可以用于绝大多数的B-ALL患儿免疫表型的检测;②伴有髓系表达不具有提示复发的意义。
【关键词】儿童；B淋巴细胞性白血病；免疫分型；流式细胞仪
Immunophenotype of acute B-lineage lymphoblastic leukemia in children by flow cytometry
WANG Yi-lin,SUN Li-rong.The Medical Shchool Hospital of Qingdao University,Qingdao 266003,China
【Abstract】 Objective To investigate the value of detecting immunophenotypes by flow cytometry(FCM) in B-lineage acute lymphoblastic leukemia (B-ALL) and discuss the relationship between immunophenotype and outcome.Methods 39 children with B- ALL were enrolled at diagnosis and immunophenotypes were detected by FCM.Gating was performed using the CD45-SSC plot.Various combinations with two monoclonal antibodies among CD19,CD10,CD20 and CD22 were analyzed with three-color stainings.CD33 and CD13 were used as myeloid antibodies for B-ALL with myeloid markers.2 groups were divided by myeloid markers.Results ①The positive rates of CD19,CD22 and CD10 were 94.87%,92.31%,and 87.18% respectively.The positive rates of CD20 was only 15.38%.CD13 and CD33 were expressed in 33.33% children with B-ALL,in which 8 cases of CD13 positive and 5 cases of CD33 positive were included.②There are 6 patiens relapse.There were no significance between 2 groups by Fisher’s Exact Test( P>0.05).Conclusion ①Flow cytometry can be used to detect immunophenotypes in the majority of children with B-ALL.②Myeloid markers in acute B-ALL are not the risk factors of relapse.
【Key words】Children；B-lineage Leukemia；Immunophenotype；Flow cytometry
白血病免疫分型是对形态学分型的重要补充和进一步深化，随着儿童急性白血病治疗的进展，其免疫分型越来越受到重视，国际白血病MIC分型协作组认为免疫分型对每一例急性白血病都是必不可少的。为了发现儿童急性B淋巴细胞白血病(B-ALL)免疫表型的特点及与预后的关系，我们利用流式细胞仪对39例急性B淋巴细胞白血病患儿的免疫表型进行分析，并对其治疗结果动态随访。现报告如下。
1 材料和方法
1.1 病例 选择我院2002年9月至2007年1月初诊的B-ALL患儿共39例，年龄1岁4个月~12岁，中位年龄7岁，其中男27例，女12例，其中L1 10例，L2 28例，L3 1例，随访时间3~50个月，平均随访时间 16.87个月。
1.2 主要设备与试剂 流式细胞仪为美国BeckmanCoulter公司生产，仪器型号：EPICS-XL。所有单克隆抗体(McAb)均购自法国Immunotech公司。
1.3 检测方法 采用直接免疫荧光标记法，每种单抗20 μl，各加入100 μl 肝素抗凝的骨髓，室温避光，溶解红细胞，PBS洗涤，离心，加入500 μl PBS，上机检测。采用CD45-SSC设门，用Elite 4.5软件进行分析，同时以异硫氰酸荧光素(FITC)、藻红蛋白荧光素(PE)、多甲藻素叶绿蛋白(PerCP)荧光素标记的无关同型IgG1亚类作为阴性对照。不同的抗体组合及不同的患者标本都用同一设定的条件检测，收集每管中的全部细胞。
1.4 资料分析 所有样本均在EPICS- XL流式细胞仪上检测，CD45-SSC设门将被检测细胞群分为4个区域：R1(淋巴细胞门)、R2(原始细胞门)、R3(成熟粒细胞门)、R4(单核细胞、巨核细胞门)，使用Elite 4.5软件对R2门内的细胞进行分析，同时在CD19、CD10、CD20和CD22中选择两个单克隆抗体(McAb)组合，进行三色组合分析，确定免疫表型，≥20%为阳性。, 百拇医药(王懿林 孙立荣)
【关键词】儿童；B淋巴细胞性白血病；免疫分型；流式细胞仪
Immunophenotype of acute B-lineage lymphoblastic leukemia in children by flow cytometry
WANG Yi-lin,SUN Li-rong.The Medical Shchool Hospital of Qingdao University,Qingdao 266003,China
【Abstract】 Objective To investigate the value of detecting immunophenotypes by flow cytometry(FCM) in B-lineage acute lymphoblastic leukemia (B-ALL) and discuss the relationship between immunophenotype and outcome.Methods 39 children with B- ALL were enrolled at diagnosis and immunophenotypes were detected by FCM.Gating was performed using the CD45-SSC plot.Various combinations with two monoclonal antibodies among CD19,CD10,CD20 and CD22 were analyzed with three-color stainings.CD33 and CD13 were used as myeloid antibodies for B-ALL with myeloid markers.2 groups were divided by myeloid markers.Results ①The positive rates of CD19,CD22 and CD10 were 94.87%,92.31%,and 87.18% respectively.The positive rates of CD20 was only 15.38%.CD13 and CD33 were expressed in 33.33% children with B-ALL,in which 8 cases of CD13 positive and 5 cases of CD33 positive were included.②There are 6 patiens relapse.There were no significance between 2 groups by Fisher’s Exact Test( P>0.05).Conclusion ①Flow cytometry can be used to detect immunophenotypes in the majority of children with B-ALL.②Myeloid markers in acute B-ALL are not the risk factors of relapse.
【Key words】Children；B-lineage Leukemia；Immunophenotype；Flow cytometry
白血病免疫分型是对形态学分型的重要补充和进一步深化，随着儿童急性白血病治疗的进展，其免疫分型越来越受到重视，国际白血病MIC分型协作组认为免疫分型对每一例急性白血病都是必不可少的。为了发现儿童急性B淋巴细胞白血病(B-ALL)免疫表型的特点及与预后的关系，我们利用流式细胞仪对39例急性B淋巴细胞白血病患儿的免疫表型进行分析，并对其治疗结果动态随访。现报告如下。
1 材料和方法
1.1 病例 选择我院2002年9月至2007年1月初诊的B-ALL患儿共39例，年龄1岁4个月~12岁，中位年龄7岁，其中男27例，女12例，其中L1 10例，L2 28例，L3 1例，随访时间3~50个月，平均随访时间 16.87个月。
1.2 主要设备与试剂 流式细胞仪为美国BeckmanCoulter公司生产，仪器型号：EPICS-XL。所有单克隆抗体(McAb)均购自法国Immunotech公司。
1.3 检测方法 采用直接免疫荧光标记法，每种单抗20 μl，各加入100 μl 肝素抗凝的骨髓，室温避光，溶解红细胞，PBS洗涤，离心，加入500 μl PBS，上机检测。采用CD45-SSC设门，用Elite 4.5软件进行分析，同时以异硫氰酸荧光素(FITC)、藻红蛋白荧光素(PE)、多甲藻素叶绿蛋白(PerCP)荧光素标记的无关同型IgG1亚类作为阴性对照。不同的抗体组合及不同的患者标本都用同一设定的条件检测，收集每管中的全部细胞。
1.4 资料分析 所有样本均在EPICS- XL流式细胞仪上检测，CD45-SSC设门将被检测细胞群分为4个区域：R1(淋巴细胞门)、R2(原始细胞门)、R3(成熟粒细胞门)、R4(单核细胞、巨核细胞门)，使用Elite 4.5软件对R2门内的细胞进行分析，同时在CD19、CD10、CD20和CD22中选择两个单克隆抗体(McAb)组合，进行三色组合分析，确定免疫表型，≥20%为阳性。, 百拇医药(王懿林 孙立荣)