VACUTAINER CPT and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples

Abstract

Background: For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV+) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV+ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNgamma expression in CD8+ and CD8- T cells, cellular viability, and cellular recovery.

Results: The results indicate that cryopreserved PBMC samples tested for CMV- and HIV-specific interferon-gamma (IFNgamma) expression performed equivalent to the respective fresh PBMC processed under both collection conditions. Compared to fresh PBMC, the viability was significantly lower for cryopreserved PBMC derived using Ficoll, although it was never less than 90%. There were no significant differences in the IFNgamma response, viability, or recovery between cryopreserved PBMC derived by Ficoll and by CPT.

Conclusion: These data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV+ PBMC, as well as a viable alternative to Ficoll gradient separation.

Figure 1

Study design. Viability, recovery, and intracellular cytokine staining of activated PBMC were compared for fresh and cryopreserved HIV⁺ PBMC processed by different methods according to the flow chart depicted here.

Figure 2

Viability of CPT-processed PBMC is not adversely affected by cryopreservation. The viability (percent of live cells) of fresh and cryopreserved PBMC was compared based on processing method. Significant difference (p) was determined by the Wilcoxon signed rank test. Each filled circle represents an individual donor and each donor is represented by the same color for each of the conditions tested. The "+" represents the median of all the donors in the category.

Figure 3

Generally equivalent PBMC yields are obtained regardless of processing method and no significant differences are seen in cryopreserved PBMC recovery. The number (cells/ml) of viable "fresh" PBMC recovered after processing are depicted in graph A. Graph B depicts the percentage of thawed, viable PBMC recovered from the number originally frozen down. In these two graphs, each filled circle represents an individual donor and each donor is represented by the same color for each of the conditions tested. The "+" represents the median of all the donors in the category. The Wilcoxon signed rank test was used to determine significance.

Figure 4

Processing method does not adversely impact functional response. IFNγ expression by CD8⁺ and CD8^- T Cells, in response to 6 hour activation with either CMVpp65 peptide mix (A and C) or HIVp55 peptide mix (B and D), was used as a read-out of functional ability of the PBMC. Data displayed have been corrected for the unstimulated expression of cytokine. For FACS analysis, a lymphocyte (forward vs. side scatter) gate, and a CD3+CD8+ gate (A and B) or a CD3+CD8- gate (C and D) were applied. Significant difference (p) was determined by the Wilcoxon signed rank test. Each filled circle represents an individual donor and each donor is represented by the same color for each of the conditions tested. The "+" represents the median of all the donors in the category.

Figure 5

Antigen-specific stimulation of T cells results in cytokine expression. Intracellular staining with IFNγ FITC/CD69 PE/CD8 PerCPCy5.5/CD3 APC by Ficoll or CPT-processed PBMC from one representative HIV⁺ donor. The numbers displayed on the dot-plots represent the percentage of CD3⁺ and CD8⁺ lymphocytes that are expressing IFNγ and CD69 in response to antigen-specific stimulation or unstimulated control.