红细胞CD55、CD59流式细胞术检测方法的若干影响因素探讨

目的探讨流式细胞仪测定红细胞表面CD55、CD59试验过程中影响检测的若干因素,建立此项目的标准操作程序。方法利用流式细胞仪对15例非血液病、11例阵发性血红蛋白尿症（PNH）的乙二胺四乙酸（EDTA）外周抗凝血进行红细胞CD55、CD59检测,观察单克隆抗体（mAb）用量、荧光素种类及反应时间对测定结果的影响。结果在未饱和抗原的情况下（〈9μL）,平均荧光强度（MFI）随抗体量的增加而升高。CD55-异硫氰酸荧光素（FITC）的阳性率和MFI均低于CD55-藻红蛋白（PE）,CD59的FITC和PE 2种荧光标记的阳性率间差异无统计学意义,但在MFI方面前者明显高于后者。反应时间对CD55-PE阳性率、CD59-FITC和CD55-PE的MFI有明显影响（P〈0.05）;3 h内2组CD59-FITC的阳性率无明显变化。结论抗体用量、荧光素种类及标记模式和温育时间对流式细胞仪检测红细胞CD55、CD59有明显的影响。本实验室在3个影响因素上分别选择12μL抗体标记1.6×105～2.8×105个红细胞、荧光素组合模式为CD59-FITC、CD55-PE双色标记和40 min的温育时间,这样既确保了反应充分又提高了结果的稳定性。

Investigation of several influential factors on flow cytometry analysis of CD55 and CD59 expression on red blood cells

Objective To study some factors that may influence the measurements of CD55 and CD59 expression on red blood cells by flow cytometry and establish the standard operation procedure.Methods Ethylene diamine tetraacetic acid（EDTA）-anticoagulated peripheral bloods from 15 patients with non-hematological disorder and 11 patients with paroxysmal nocturnal hemoglobinuria（PNH） were determined for CD55 and CD59 expression on red blood cells by flow cytometry.The various amounts of monoclonal antibody（mAb）,the fluorescein type that conjugated with mAb and the staining time were analyzed.Results The mean fluorescence intensity（MFI） increased with the increasing amount of mAb before the saturated amount of mAb（9 μL） were added.The positive rates and MFI from using anti-CD55 labeled with fluorescein isothiocyanate（FITC） were lower than those from anti-CD55-phycoerythrin（PE）.There was no statistical significance in CD59 positive rate between FITC and PE,while the higher MFI was observed significantly using anti-CD59-FITC.The staining time had a significant impact on not only the positive rate of CD55-PE but also the MFI obtained from CD59-FITC and CD55-PE（P0.05）,except the CD59-FITC positive rate between the 2 groups with 3 h.Conclusions The flow cytometry analysis of CD55 and CD59 expression on red blood cells is strongly influenced by the mAb amount,the mAb′s fluorescein type and staining time.To ensure a proper reaction and a reliable result,we recommend a two-color staining method of anti-CD59-FITC and anti-CD55-PE,red blood cells in the range from 1.6×105 to 2.8×105 stained with 12 μL of each mAb and an incubation time of 40 min.