Cryopreservation and long-term liquid nitrogen storage of peripheral blood mononuclear cells for flow cytometry analysis: effects on cell subset proportions and fluorescence intensity

The effect of cryopreservation and long-term liquid nitrogen storage on peripheral blood mononuclear cell (PBMC) subsets was prospectively analyzed using monoclonal antibodies and flow cytometry. Brief cryopreservation did not significantly alter the proportion of positively stained cells for CD3+, CD4+, CD8+, CD14+, CD16+, and CD19+ cells. A small but statistically significant increase in the proportion of positive cells was observed for HLA-DR+ and HLe-1+ cells. Brief cryopreservation was associated with a decrease in the mean fluorescence intensity (MFI) values for CD3+, CD4+, and CD8+ cells; an increase in MFI values for CD14+ and HLA-DR+ cells; and no change for CD16+, CD19+, and HLe-1+ cells. There was no significant change in the proportion of CD3+, CD4+, or CD16+ cells during 20 months of storage in liquid nitrogen. Small but statistically significant decreases in the proportion of CD8+ and CD19+ cells were observed over the same interval, and the proportion of CD14+ cells (monocytes) was highly variable. Chronologic changes in fluorescence intensity during long-term storage were observed for all cell subsets except CD16+ and CD19+ cells. Cryopreservation is a valuable technique for long-term storage of viable cells. For many laboratory applications, the small changes noted in the present study will have no practical importance. However, for clinical and epidemiological investigations encompassing large numbers of samples, statistical techniques to adjust for small changes during storage should be considered.