直标法流式细胞术测定人红细胞表面CD35水平的方法学研究

Detection of Human Erythrocytes CD35 by Flow Cytometry with Direct Fluorescent Antibody Technique

为研究直标法流式细胞术测定红细胞表面CD35免疫分子水平的方法,抽取健康献血员静脉血,采用直标法流式细胞术测定红细胞CD35水平,研究测试后数据分析方法的可靠性和测试前抗凝剂、标本放置时间和抗体用量等因素对测定结果的影响。结果显示:M1界值选择对CD35阳性红细胞(CD35+-E)百分率计算结果有很大影响,CD35几何平均荧光强度(CD35-GMFI)随FL2通道电压升高而增加,而CD35相对几何平均荧光强度(CD35-rGMFI)不受电压影响;EDTA-2K、肝素锂、枸橼酸钠三种抗凝剂以及标本放置72h内CD35+-E百分率和CD35-rGMFI结果均无明显差异(P>0.05);取5×105个人红细胞反应时,最适合的鼠抗人CD35单克隆抗体量为7μL。可见,CD35-rGMFI是描述红细胞表面CD35水平的最理想指标,抗凝剂、标本放置时间对测定结果无明显影响,当红细胞数量为5×105时,最佳抗体用量为7μL。

To study the methods of human erythrocytes CD35 detection by flow cytometry with direct fluorescent antibody technique, human erythrocytes immune adherence receptor, complement receptor type 1 （ CD35 ）, was detected by flow cytometry with direct fluorescent antibody technique, and the reliabilities of data analysis methods and the effects of pre-detection factors such as deeoagulants, sample deposit time and antibodies quantities on results of CD35 detection were studied. The results showed that the percentages of CD35 positive erythrocytes （CD35 ^＋-E） were affected by the choice of M1 boundary, and the geometric mean fluorescence intensity of CD35 （CD35-GMFI） increased with the elevation of FL2 channel voltage, while the relative geometric mean fluorescence intensity of CD35 （CD35-rGMFI） was not influenced by it. There were not statistically significant differences in CD35 ^＋ E percentages and CD35-rGMFIs among decoagulants （dipotassium ethylenediamine tetraacetic acid, heparin lithium and natrium citricum） or sample deposit time （0 -72h）. Seven microliters of mouse anti-human CD35 monoclonal antibody was optimal for 5 × 10^5human erythroeytes. It is thus evident that CD35-rGMFI is the most ideal index to describe the level of CD35 on erythroeytes, and there are no effects of decoagulants and sample deposit time on CD35 ^＋ -E percentages and CD35-rGMFI. Sevenmicroliters of mouse anti-human CD35 monoclonal antibody is optimal for 5 × 10^5human erythrocytes.