流式细胞术产前非侵入性定量检测胎-母出血的研究

Study on noninvasively quantifying pregnatal fetomaternal hemorrhage by flow cytometry

目的 建立产前非侵入性定量检测RhD阴性孕妇胎 母出血的流式细胞术实验方法。方法 RhD阴性红细胞与RhD阳性红细胞按一定比例混合 ,用抗 D作为一抗结合D+ 红细胞 ,以羊抗人IgGF(ab′) 2 FITC作为二抗 ,连接抗 D ,应用流式细胞术检测D+ 红细胞占D-红细胞的比例 ,建立抗 D及抗人IgGF(ab′) 2 FITC的量效关系 ,确定两者最佳使用剂量。在此实验条件下 ,进行流式细胞术检测值与实际值的相关性分析 ,判定可以检测出的D-红细胞中混有D+ 红细胞的准确出血量范围 ,应用建立的流式细胞术方法测定 9例RhD阴性孕妇外周血中的胎 母出血量。结果 抗 D的有效使用剂量为 1∶4 0 0稀释 ,10 0 μl/5× 10 6细胞 ;羊抗人IgGF(ab′) 2 FITC的有效使用剂量为 2 5 μg/5× 10 6细胞 ;流式细胞术测定比例与已知实际比例的相关系数r =0 998;准确检测的胎 母出血量为 (0 .6～ 30 )ml。 9例RhD阴性孕妇外周血中除 2例未检出D+ 细胞而无法判断外 ,其余 7例均检测出胎 母出血 ,胎 母出血量为 (1.2～ 6 .4 8)ml。结论 针对孕期发生胎 母出血反应的RhD阴性孕妇 ,可以应用流式细胞术方法在产前准确无损伤地定量检测胎 母出血量 ,指导临床预防。

Objective To establish the noninvasive method of quantifying the volume of pregnatal fetal D positive cells in the circulation of D negative pregnancies by flow cytometry (FCM).Methods D negative RBCs and D positive RBCs were mixed in accordance with the given ratio. Anti D was used as the first antibody to bind the D positive RBCs. The goat anti human IgGF(ab′) 2 FITC was taken as the second antibody to conjugate the anti D.The ratio of D positive RBCs vs D negative RBCs was determined by the FCM and the best dosage of anti D and IgGF(ab′) 2 FITC were defined. The ratio of two groups' cells measured by FCM was correlatively compared with the actual ratio given by us. The range of the volume of D positive cells in the D negative cells was calculated. 9 cases of RhD negative pregnancies were studied by FCMmethod.ResultsTheeffectivedosagesofanti DandIgGF(ab′) 2 FITCwere1∶400/100μland2.5μg/5×10 6 cells,respectively. The correlation coefficient between the ratio of two groups' cells measured by FCM and the actual ratio was 0.998.The range of fetomaternal hemorrhage (FMH) was (0.6～30) ml. Among the 9 cases, 2 cases were failed to detect the fetal D + cells. 7 cases were satisfactory to quantify the FMH by FCM,and the range of FMH was (1.2～6.48) ml. Conclusions\ The FCM method can quantify FMH accurately and noninvasively in RhD negative women who had FMH during the pregnancy, and be used to prevent hemolytic disease of the newborn(HDN) of Rh.