六色流式细胞术用于儿童急性B系淋巴细胞白血病相关免疫表型的检测

目的比较六色流式细胞术与三色流式细胞术在急性B系淋巴细胞白血病(B-ALL)中检测出白血病相关免疫表型(LAIP)的差异。方法利用两组六色抗体组合CD20/CD34/CD10/CD22/CD45/CD19、CD7/CD5/CD13/CD33/CD45/CD19对25例三色流式免疫分型诊断为B-ALL的初诊患儿骨髓标本进行检测,通过FSC/SSC、CD45/SSC、CD19/SSC设门,在多个双参数二维点图上对幼稚细胞群、CD19+细胞群进行分析。结果三色流式免疫分型使用23种抗体共检测出CD19strong、CD10strong、CD34strong、CD22strong、CD19+/CD45-/dim+、CD19+/CD34+/CD38-、CD19+/CD13+、CD19+/CD15+、CD19+/CD33+9种LAIP。25例患儿中23例检测出LAIP,其中1个LAIP 4例,2个LAIP 8例,3个LAIP 5例,4个LAIP 3例,5个LAIP 3例。六色流式细胞术使用10种抗体共检测出CD10strong、CD34strong、CD22strong、CD45-/dim+、CD19+/CD13+、CD19+/CD33+、CD19+/CD34+/CD20+、CD19+/CD34+/CD22++、19+/CD34+/CD10dim+、19+/CD45-/dim+/CD34-/dim+、19+/CD10-/CD20-/CD34-11种LAIP。25例中23例检测出LAIP,其中1个LAIP 4例,2个LAIP 5例,3个LAIP 3例,4个LAIP 6例,5个LAIP 5例。结论儿童B-ALL患者具有LAIP者比例较高,适用于运用流式细胞术对B-ALL患儿进行化疗骨髓缓解(CR)后微小残留病(MRD)检测。六色流式细胞术使用的抗体总数较少,但通过FSC/SSC、SSC/CD45、SSC/CD19设门,对幼稚细胞群、CD19+细胞群在多个双参数点图上进行分析,能发现比三色流式细胞术更多的LAIP,并且六色流式细胞术可在同一试管中同时检测6种抗原,能根据患儿初诊时免疫分型结果进行灵活调整,更有利于进行CR后MRD的检测。

The detection of leukemia-associated immune phenotypes by six-color flow cytometry in pediatric B-lineage acute lymphoblastic leukemia

Objective To compare the differences between three-color and six-color flow cytometry(FCM) on detecting leukemia-associated immune phenotype(LAIP) in pediatric B-lineage acute lymphoblastic leukemia(B-ALL).Methods Twenty-five patients diagnosed as B-ALL by three-color FCM were detected by two groups of six-color antibody combinations: CD20/CD34/CD10/CD22/CD45/CD19 and CD7/CD5/CD13/CD33/CD45/CD19.Naive cell populations and CD19+ cell populations were analyzed on multiparametric two-dimensional point maps through FSC/SSC,CD45/SSC and CD19/SSC gating.Results Nine LAIPs were detected using 23 antibodies by three-color FCM,including CD19strong,CD10strong,CD34strong,CD22strong,CD45dim+/-,CD19+/CD34+/CD38-,CD19+/CD13+,CD19+/CD15+ and CD19+/CD33+.Twenty-three cases(23/25) had LAIPs detected,of which,4 cases had 1 LAIP,8 cases had 2 LAIPs,4 cases had 3 LAIPs,3 cases had 4 LAIPs and 3 had 5 LAIPs.By contrast,11 LAIPs were detected using 10 antibodies by six-color FCM,including CD10strong,CD34strong,CD22strong,CD45-/dim,CD19+/CD13+,CD19+/CD33+,CD19+/CD34+/CD20+,CD19+/CD34+/CD22++,19+/CD34+/CD10dim+,19+/CD45-/dim+/CD34-/dim+ and 19+/CD10-/CD20-/CD34-.Twenty-three cases(23/25) had LAIPs detected,of which,4 cases had 1 LAIP,5 cases had 2 LAIPs,3 cases had 3 LAIPs,6 cases had 4 LAIPs and 5 had 5 LAIPs.Conclusions High proportions of LAIPs can be detected by FCM in B-ALL patients.Though fewer number of antibodies was used,more LAIPs were detected by six-color FCM than those by three-color FCM.Besides,six-color FCM can detect six antibodies at the same time in one tube,and adjust the antibody combination flexibly,which makes MRD detection more reasonable and convenient.