流式细胞术检测红细胞相关抗体方法

Detection of the erythrocyte associated immunoglobulin by flow cytometry

目的:建立以全血为标本的流式细胞仪(FCM)检测抗红细胞相关抗体方法,探讨实验过程中各因素对检测结果的影响。方法:多参数、半定量FCM分析。结果:在细胞数稳定在(1.6～2.0)×105/μl时,稀释度为2∶1的抗-DIgG抗体即可使红细胞表面抗原饱和。随着加入的人抗-DIgG浓度逐渐增加,红细胞阳性率无变化;EDTA二钾、肝素钠和柠檬酸钠这3种抗凝剂之间各项指标差异无统计学意义;不同反应温度对阳性模型与二抗的反应无影响;抗体与样本反应时间在组内各时间段之间的比较中,反应时间>5min时平均荧光强度(MFI)、阳性率均无统计学差异;标本放置在室温与4℃下储存时,各时间段阳性细胞比例无明显差异,而标本放置时间超过2h后,MFI降低,标本在室温和4℃下存储24h内各时间段各项指标相比均无明显差异;二抗稀释度在≥1∶1时MFI即可达平台期;红细胞表面IgG抗体的参考范围:阳性细胞比例<0.33%,MFI<27.7;FCM法的重复性高。结论:全血法FCMFSC-SSC可特异地设置细胞门,无需指定特殊抗凝剂,不同反应温度对阳性模型与二抗的反应没有影响。反应时间>5min即可,存储温度在25℃及4℃时标本均应在2h内进行检测。二抗稀释度在1∶1时即可饱和红细胞表面抗体。FCM法的重复性较高。

Objective To establish a method by flow cytometry(FCM) for detection of the erythrocyte associated immunoglobulin(EAIg), and to study the factors affecting the flow cytometric analysis. Methods Multiple parameters and semi-quantitative flow cytometric analysis were used. Taken IgG as a representative, the IgG positive red blood cell model was set up and the steps of EAIg detection were established. Results There was no significant difference on the mean fluorescence intensity(MFI) in 3 kinds of anticoagulants -edetic acid-K2, heparin sodium and sodium citrate. The MFI of sample-antibody reaction at 4℃ was significantly lower than that at room temperature. With the time of sample and antibodies' reaction, the MFI increased and there was no significant difference after 20 min of the reaction; FCM method was sensitive for the detection of EAIg. When the sample was stored at room temperature within 8 hours or at 4℃ within 24 hours, the MFIs were not significantly different from those over 2 hours. Conclusions The method does not need a special anticoagulant. It had better for sample and antibody reaction at room temperature, and the suitable reaction time is 20-40 min. The sample should be detected within 2 hours no matter at which temperature the sample is stored at room temperature or at 4℃.