流式细胞术检测RhD(-)血液中微量RhD(+)红细胞的研究

目的探讨建立流式细胞术(FCM)检测微量RhD(+)红细胞的试验方法。方法选取标准的RhD(+)和RhD(-)细胞按不同比例混合,用间接标记法一抗为IgG抗-D,二抗为FITC-抗-IgG F(ab’)2染色,应用FCM检测RhD(+)细胞的比例,并确立IgG抗-D的最佳使用剂量。在此实验条件下,作FCM检测值与实际值的相关性分析和FCM的检测重复性分析。结果抗-D的最佳使用剂量是1∶4稀释,50μl/1×106细胞;当RhD(+)细胞占RhD(-)细胞比例范围为2.5%—0.312%时,FCM测定比例与已知实际比例的相关系数(r)=0.987。RhD(+)细胞比例分别为10%、2.5%的同一测定管各重复测定10次,变异系数分别3.4%、4.9%。结论FCM检测微量RhD(+)细胞具有较好的准确度和重复性,可应用于临床检测RHD(-)患者体内的微量RhD(+)细胞。

Study on the detection of RhD(+) red blood cells mixed in D-negative blood by flow cytometry

Objective To establish a method for detection of RhD(+) red blood cells mixed in D-negative blood by flow cytometry(FCM).Method RhD(+) and RhD(-) RBCs were mixed according to predefined ratios.Cells were indirectly labeled,with IgG anti-D labeled as the first antibody,and FITC-anti-IgG F(ab')2 as the second antibody.The percentage of RhD(+) RBCs was determined by FCM,and the best dosage of IgG anti-D was also defined.The ratio of red cells in the two groups,measured by FCM,was compared with the actual ratio.The consistency of method was also evaluated.Results The effective dosage of IgG anti-D was 1∶4,and 50μl/1×106 cells.When the actual percentages of RhD(+) cell among RhD(-) cells were 2.5%-0.312%,the correlation coefficient between the percentages measured by FCM and the actual percentages was 0.987.The same tubes,containing 10% and 2.5% RhD(+) RBCs,were each tested for 10 times,and their coefficient of variation were 3.4%,and 4.9%,respectively.Conclusion The method of quantifying the RhD(+) RBCs in D-negative blood by FCM is feasible and repeatable,which deserves a further clinical application.