多聚甲醛固定对流式细胞术检测人外周血淋巴细胞亚群的影响

Evaluation for impacts of paraformaldehyde fixation on detection of flow cytometry for human peripheral blood lymphocyte subsets

目的考察多聚甲醛(PFA)浓度和固定时间对流式细胞术检测人外周血T、B、NK淋巴细胞亚群百分比和平均荧光强度的影响。方法以健康人外周血为研究对象,将六色荧光抗体标记后溶血处理的细胞分别用PBS、1%PFA(10 g/L)和4%PFA(40 g/L)进行重悬或固定,用流式细胞仪在固定0、4、8、12、24、48和72 h后检测CD19-CD3^+T、CD3^+CD4^+T、CD3^+CD8^+T、CD3-CD19^+B、CD3-CD56^+NK细胞占淋巴细胞百分比,及CD45、CD3、CD4、CD8、CD19、CD56的平均荧光强度。结果与PBS对照组相比,72 h内,PFA浓度及固定时间对外周血T、B、NK淋巴细胞亚群百分比均没有影响(P均>0.05);固定8 h内,PBS对各淋巴细胞亚群平均荧光强度均无影响,1%PFA组CD4平均荧光强度下降,4%PFA组CD45、CD3、CD4、CD8平均荧光强度均下降;固定24 h后,各淋巴细胞亚群平均荧光强度均下降,同时4%PFA固定后平均荧光强度均低于1%PFA(P<0.05)。结论使用流式细胞术检测人外周血淋巴细胞亚群,细胞染色后若8 h内检测用PBS进行细胞重悬检测效果最好,PFA固定不应超过12 h,1%PFA固定效果优于4%PFA。

Objective To evaluate the impacts of different concentrations and fixation time of paraformaldehyde(PFA)on detection of the percentages and mean fluorescence intensity(MFI)for human peripheral blood lymphocyte subset,such as T,B,NK cells,by flow cytometry.Methods Peripheral blood mononuclear cells were collected from healthy adults and detected by 6 color fluorescent cytometry(FCM).The hemolytic blood cells were resuspended and fixed with PBS,1%PFA(10 g/L)of 4%PFA(40 g/L),respectively.The percentages of CD19-CD3^+T cells,CD3^+CD4^+T cells,CD3^+CD8^+T cells,CD3-CD19^+B cells and CD3-CD56^+NK cells were analyzed,and the mean fluorescence intensity(MFI)of CD45,CD3,CD4,CD8,CD19 and CD56 on the cell surface were measured by flow cytometry at different time points as 0,4,8,12,24,48 and 72 hours following PFA fixation.Results Compared with PBS controls,PFA fixation at either 1%or 4%concentrations,had no significant effect on the percentage of peripheral lymphocyte subsets as T,B,NK cells(P>0.05)in various time points within 72 hours.PBS had no effect on the MFI of each lymphocyte subgroup within 8 hours,but the MFI of CD4 in 1%PFA group and all the MFI of CD45,CD3,CD4 and CD8 in 4%PFA group were decreased.After PFA fixation for 24 hours,the MFI of the cell surface marker expressions were obviously decreased,and the MFI of the cells fixed by 4%PFA was significantly lower than those of the cells fixed by 1%PFA(P<0.05).Conclusion PBS should be the optimum resuspension for PFA fixation in the detection of lymphocyte subsets within 8 hours of cellular staining and the results of PBS resuspended cells achieved the best effects.PFA fixation may be used for cell preservation within 12 hours,and the effects of 1%PFA fixation for preserving the cell surface marker expression should be better than those of 4%PFA.