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.