Background: Naïve CD4⁺ T cells express higher levels of CD31 compared to memory CD4⁺ T cells and naïve T cells expressing CD31 are enriched in T cell receptor excision circles (TREC) and thus represent primary thymic emigrants. In this study, the timing of processing peripheral blood (PB) as well as lymphocyte separation and cryopreservation procedures were evaluated in both HIV^– and HIV⁺ participants to study the effects they may have on the CD31 expression on naïve T cell subsets by flow cytometry.

Methods:  Immunophenotyping of PB was done at 4, 24, 48, and 72 hours after blood draw on 54 participants (36 were HIV⁺).  PB was collected in a heparinized syringe, and PBMC were isolated by Ficoll-Hypaque separation. A fraction of PBMC was processed immediately for immunophenotyping, and the remaining PBMC were cryopreserved. The cryopreserved PBMC were processed for immunophenotyping after 1 week.  Staining with antibodies to CD4, CD45RO, CD27, and CD31 was performed and naïve cells were defined as CD45RO^–CD27⁺.

Results:  The mean age of all participants was 38 and the mean CD4⁺ T cell count of was 689 cells/µL. A small but statistically significant overestimation of %CD31 expression on naïve CD4 T cells was observed with delayed processing of PB.  The mean difference was +1.6 at 24 hours (p = 0.005), +3.1 at 48 hours (p <0.001), and +5.6 at 72 hours (p <0.001) for all participants.  The measurements from the cryopreserved PBMC were also significantly higher than both the PB (mean difference of +1.5, p = 0.3) and PBMC measurements (mean difference of +4.4, p = 0.002). The mean age of the HIV⁺ cohort was 45 and the mean CD4⁺ T cell count was 679 cells/µL. When HIV⁺ samples were analyzed separately, similar findings were noted with CD31 mean differences in PB of +1.7 at 24 hours (p = 0.019), +3.1 at 48 hours (p <0.001), and +5.4 at 72 hours (p <0.001). Additionally, in HIV⁺ cryopreserved PBMC, the mean differences observed from PB and freshly isolated PBMC measurements were +2.0, p = 0.3 and –4.8, p = 0.002.

Conclusions:  Delayed PB processing up to 48 hours may be acceptable for measurement of CD31 expression on naïve CD4 T cells allowing centralized processing in multi-center studies.  Cryopreservation can result in a small systematic overestimation of the proportion of naïve CD4⁺CD31⁺ cells and this should be taken into consideration when comparing freshly isolated and cryopreserved cells.