Background

 We have established Peripheral Blood Mononuclear Cells (PBMC) Repository at Duke University Medical Center [DUMC] as part of the CTC-VIMC to streamline the assessment of assay competency of various labs within the consortium and to provide characterized specimens for assay optimization and/or validation procedures. The overall aims are: 1) to acquire PBMC samples with viability ≥90% and total viable cell recovery ≥80%; 2) to select samples with a range of T cell responses to relevant virus antigens.

 

 

 Methods

 PBMCs were collected by leukopheresis procedure from 60 prescreened HIV-1 seronegative donors at DUMC, isolated by standard Ficoll-Hypaque density gradient separation, and cryopreserved using a programmable rate-controlled freezer. Cell recovery and viability were recorded and cells were screened for their functional responses to CEF, CMV, PHA and media only stimulation in IFN-γ ELISpot assay. Additionally these specimens were tested for their poly-functional adaptive T cell responses in multicolor flow cytometric panels.

 

 

 Results

 From 60 leukopaks collected at DUMC, 32 were female, 28 male with race distribution of 81% White, 12% Black, 6% Hispanic and 2% other. All PBMC specimens met our criteria for viability (mean = 94.7+ 2.3%) and total viable cell recovery (mean = 93.7+17.3%) after overnight rest. Samples exhibited a variety of responses in IFN-γ ELISpot (range of response to CEF 28-4668 SFC/106, CMV: 3-5245 SFC/106) and multicolor flow assays revealing diverse patterns of functionality.

 

 

 Conclusions

 Centrally supplied characterized specimens for assay validation/optimization and proficiency testing activities will ensure greater uniformity of data from multiple labs. To meet the need for immunologically characterized samples across our network from diverse ethnic/HLA backgrounds, we are establishing a PBMC repository in South Africa. This will allow us to characterize sets of PBMC from HIV seronegative and seropositive individuals, recognizing that potential differences in response to common reagents may exist.