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Impact of delayed blood processing on phenotypic characterization of peripheral blood mononuclear cell subsets: implications for biobanking
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1Kerckhoff Heart and Thorax Center, Cardiology - Bad Nauheim - Germany
2Justus-Liebig-University Giessen, Medical Clinic I, Cardiology and Angiology - Giessen - Germany
Background: Isolation of human peripheral blood mononuclear cells (PBMCs) is one of the most commonly performed tests for studying human immune responses in cardiovascular diseases, and the use of PBMCs in biomarker research is gaining importance. Although several studies have examined the effect of cryopreservation and subsequent thawing of PBMCs on cell phenotype and function, much less is known about the effects of processing delays.
Purpose: We investigated whether delayed processing of whole blood affects the results of subsequent immunophenotypic studies of PBMCs in order to optimize processing protocols for the detection of leukocytes.
Methods: Peripheral venous blood from 10 healthy individuals was collected into vacuum cell preparation tubes. PBMCs were isolated by centrifugation without any delay (0 h) or after a processing delay of 1, 2, 4, 8 and 12 h during which the samples were stored at room temperature. PBMCs were then cryopreserved in vapor-phase nitrogen and tested simultaneously in order to diminish inter-assay variability. Cellular yield was determined before freezing, and PBMC recovery and viability were assessed after thawing. PBMC subpopulations were characterized by flow cytometry. A fixable aqua dead cell stain and phosphatidylserine were used to distinguish viable, apoptotic, and dead PBMC subpopulations.
Results: Processing delays beyond 1 h resulted in a significantly decreased yield of viable cells after PBMC isolation (0 h vs. 12 h: 11.9×10E6 vs. 8.9×10E6 cells, p<0.001). Differences in recovery and viability of PBMCs were not significant with respect to the different delays in processing (viable cell recovery rate 0 h vs. 12 h: 68±2.2% vs. 69±2.4%, p=n.s.). Flow cytometry revealed a significant increase in B cell frequencies after processing delays beyond 1 h (0 h vs. 12 h: 6.2±0.7% vs. 9.6±1.1% of lymphocytes, p<0.001). In contrast, we observed a selective loss of phenotypically defined CD3+ T cell frequencies after processing delays of 4 to 12 h in comparison with immediately processed PBMCs. Proportions of CD4+, CD8+, and NK-like T cells were not significantly different in samples with processing delays compared with immediately processed PBMCs. Delayed processing also had no effect on monocyte subset frequencies or the percentage of apoptotic T cell subsets. However, we found significantly higher percentages of apoptotic B cells for processing delays beyond 1 h (0 h vs. 12 h: 9.4±1.2% vs. 16±1.8% of total B cells, p<0.001).
Conclusion: Our data indicate that PBMCs isolated and cryopreserved with a delay of up to 12 h after blood draw can be used in several phenotypic assays without a significant loss in cell recovery. However, specific cell subsets are significantly influenced by processing delays prior to PBMC isolation and show increased apoptosis. Therefore, the time between sample acquisition and initiation of processing should be carefully considered in study designs involving PBMC immunoassays.
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