Effect of delayed cell processing and cryopreservation on immunophenotyping in multicenter population studies

Abstract

Variability induced by delayed cell processing and cell cryopreservation presents unique challenges for immunophenotyping in large population studies. We conducted a pilot study to evaluate the effect of delayed cell processing and cryopreservation on cell percentages obtained by immunophenotyping. We collected blood from 20 volunteers and compared the effect of (a) delayed cell processing up to 72 h (b) cryopreservation and (c) the combined effect of delayed cell processing and cryopreservation on immunophenotyping of 31 cell subsets that included several subsets of T, B, Natural Killer (NK) cells, monocytes and dendritic cells using both whole blood collected in EDTA tubes and peripheral blood mononuclear cells collected in CPT tubes. We found the delayed cell processing up to 72 h or cryopreservation alone did not significantly affect the percentages T cells, dendritic cells or monocytes but significantly increased the percentage of B cells and NK cells (p for trend ≤0.01) but. However combination of delayed cell processing up to 72 h and cryopreservation significantly increased the percentage of T cells as compared to cells processed immediately (p for trend <0.0001) while a delayed cell processing followed by cryopreservation decreased the percentage of NK cells (p for trend <0.0001). Total B-cells increased significantly with a 24-48 h delay in cell processing and cryopreservation but not at 72 h. The percentages of monocytes and dendritic cells remained unaffected by the combination of delayed cell processing and cryopreservation. These findings suggest that immunophenotyping of several immune cell subsets can be successfully implemented in large population studies as long as blood is processed within 48 h of biospecimen collection though some cell subsets may be more susceptible to a combination of delayed cell processing and cryopreservation.

Keywords: Cell processing; Cohort study; Cryopreservation; Immunophenotyping.

Figure 1:

Overview of study design to evaluate the effect of delay in cell processing and cryopreservation on proportion of 31 cell subsets identified by immunophenotyping.

Figure 2A:. Effect of delayed cell processing on immune cell subsets.

The figure shows average cell percentages of T cells, B cells, NK cells, monocytes and dendritic cells (DC) at time 0 (WB-D0) to average cell percentages of fresh blood processed at times 24, 48 and 72 after collection (WB-D24, WB-D48 and WB-D72 respectively). Cell percentages are expressed as percent lymphocytes for B and T cells and as percent PBMCs for DC, NK and monocytes. indicates statistically significant difference (p<0.007) in proportion of cells detected at particular time point versus WB-D0 (reference). WB-D0 = Whole blood processed immediately, WB-D24 = Whole blood processed after a delay of 24 hours, WB-D48 = Whole blood processed after a delay of 48 hours and WB-D72 = Whole blood processed after a delay of 72 hours.

Figure 2B:. Effect of cryopreservation on immune cell subsets.

The figure shows average cell percentages of T cells, B cells, NK cells, monocytes and dendritic cells for cryopreserved samples at time 0 (PBMC-D0) to average cell percentages of fresh blood at time 0 (WB-D0). Cell percentages are expressed as percent lymphocytes for B and T cells and as percent PBMC for monocytes, DC, and NK cells. indicates statistically significant difference (p<0.007) in proportion of cells detected at particular time point versus WB-D0 (reference). WB-D0 = Whole blood processed immediately, PBMC-D0 = Peripheral Blood Mononuclear Cells cryopreserved immediately.

Figure 2C:. Combined effect of delay in cell processing and cryopreservation on immune cell subsets.

The figure shows average cell percentages of T cells, B cells, NK cells, monocytes and dendritic cells for cryopreserved samples at time 24, 48 and 72 hours (PBMC-D24, PBMC-D48, PBMC-D72 respectively) to average cell percentages in whole blood processed immediately (WB-D0). Cell percentages are expressed as percent lymphocytes for B and T cells and as percent PBMCs for DC, NK and monocytes. indicates statistically significant difference (p<0.007) in proportion of cells detected at particular time point versus WB-D0 (reference). WB-D0 = Whole blood processed immediately, PBMC-D24 = Peripheral Blood Mononuclear Cells cryopreserved after a delay of 24 hours, PBMC-D48 = Peripheral Blood Mononuclear Cells cryopreserved after a delay of 48 hours and PBMC-D72 = Peripheral Blood Mononuclear Cells cryopreserved after a delay of 72 hours.