Flow Cytometric Immunophenotyping: Minimal Differences in Fresh and Cryopreserved Peripheral Blood Mononuclear Cells versus Whole Blood

<b>Background/Objectives</b>: Flow cytometry is a convenient tool in immunophenotyping for monitoring the status of immunological conditions and diseases. The aim of this study was to investigate the effect of isolation and cryopreservation by flow cytometric analysis on subpopulations of CD4⁺ T helper (Th), T regulatory (Treg), CD8⁺ T cytotoxic (Tc), CD56⁺ NK, CD19⁺ B and monocytes. Freshly isolated and cryopreserved peripheral blood mononuclear cells (PBMCs) were compared to fresh whole blood. <b>Methods</b>: Peripheral blood was collected from healthy donors and prepared for flow cytometric analysis using the same panels of antibodies throughout the study. <b>Results</b>: Comparisons between fresh (F)- and cryopreserved (C)-PBMCs showed no major differences in percentages of CD4⁺, Th1, Th2 and CD4⁺CD25⁺CD127^low Treg cells. No differences in percentage of CD8⁺ or subpopulations of naive/stem, central or effector memory cells were observed between F- and C-PBMCs. The percentage of CD56⁺ NK cells, CD19⁺ B cells or classical and nonclassical monocytes did not differ between F-and C-PBMCs either. On the contrary, whole blood had lower percentages of Th and NK cells but higher percentages of Th1, Th17, Th1Th17, Tregs, Tc and B cells compared to C-PBMCs, while it had a higher proportion of Tc compared to F-PBMCs. <b>Conclusions</b>: Flow cytometric immunophenotyping minimally differs between freshly isolated and cryopreserved PBMCs. This implies the possibility of cryostorage of cohorts for later analysis. Importantly, care must be taken when comparing results from whole blood with isolated and cryopreserved PBMCs. Collectively, these results can contribute to the standardization of flow cytometric protocols in both clinical and research settings.