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
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1.解放军总医院第五医学中心临床研究管理中心;2.中国医学科学院药用植物研究所

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宁波市医学品牌学科(PPXK2018-06);宁波市公益科技项目 (2019C50070);宁波市自然基金(2019A610378)


Impact evaluation of paraformaldehyde fixation on flow cytometry detection for human peripheral blood lymphocyte subsets
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Research Center for Clinical and Translational Medicine,Fifth Medical Center,General hospital of Chinese PLA

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    摘要:

    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 (10g/L)和4% PFA (40g/L)进行重悬或固定,应用流式细胞仪在固定0 h、4 h、8 h、12 h、24 h、48 h和72 h后检测CD19ˉCD3+ T、CD3+CD4+ T、CD3+CD8+ T、CD3ˉCD19+ B、CD3ˉCD56+ NK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小时内,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明显影响(P>0.05);固定8h内,PBS对各淋巴细胞亚群平均荧光强度均无明显影响,1% PFA组CD4平均荧光强度显著下降,4% PFA组CD45、CD3、CD4、CD8平均荧光强度均显著下降;固定24h后,各淋巴细胞亚群平均荧光强度均显著下降,同时4% PFA固定后平均荧光强度均明显低于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8h内检测用PBS进行细胞重悬检测效果最好,PFA固定不应超过12h,1% PFA固定效果优于4% PFA。

    Abstract:

    Objective This study designed to evaluate the impact of different concentration and fixation time of paraformaldehyde (PFA) on detection of human peripheral blood lymphocyte subset by flow cytometry. Methods Peripheral blood mononuclear cells were collected from health adults, and detected by 6 color FCM. The hemolyzed cells were resuspended or fixed with PBS, 1% PFA (10g/L) and 4% PFA (40g/L), respectively. The percentage of CD19ˉCD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells, CD3ˉCD19+ B cells, CD3ˉCD56+ NK cells and mean fluorescence intensity of CD45, CD3, CD4, CD8, CD19 and CD56 were analyzed by flow cytometry at different fixation time points across 72 hours. Results Within 72 hours, PFA fixation, at either 1% or 4% concentrations, was had no significant effect on the percentage of peripheral lymphocyte subsets (P>0.05). After fixation for 24 hours, MFI of the marker expression among 1% or 4% PFA obviously decreased, and the MFI of 4% PFA fixation was significantly lower than 1% PFA (P<0.05). Conclusion In this study, we have identified PBS as the best resuspension method for lymphocyte subsets detection within 8 hours. PFA fixation could be used for cell preservation within 8 to 12 hours, and 1% PFA was better at preserving the marker expression than 4% PFA.

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杨锐创,邢小燕,屈蒙蒙,毕京峰,胡燕,柴燕涛,孙慧伟,冯帆,王志杰,侯俊.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[J].临床检验杂志,2020,(10):729-733

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  • 收稿日期:2020-07-29
  • 最后修改日期:2020-11-11
  • 录用日期:2020-11-09
  • 在线发布日期: 2021-03-24
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