The Center for HIV/AIDS Vaccine Immunology (CHAVI) multi-site quality assurance program for cryopreserved human peripheral blood mononuclear cells

Abstract

The Center for HIV/AIDS Vaccine Immunology (CHAVI) consortium was established to determine the host and virus factors associated with HIV transmission, infection and containment of virus replication, with the goal of advancing the development of an HIV protective vaccine. Studies to meet this goal required the use of cryopreserved Peripheral Blood Mononuclear Cell (PBMC) specimens, and therefore it was imperative that a quality assurance (QA) oversight program be developed to monitor PBMC samples obtained from study participants at multiple international sites. Nine site-affiliated laboratories in Africa and the USA collected and processed PBMCs, and cryopreserved PBMC were shipped to CHAVI repositories in Africa and the USA for long-term storage. A three-stage program was designed, based on Good Clinical Laboratory Practices (GCLP), to monitor PBMC integrity at each step of this process. The first stage evaluated the integrity of fresh PBMCs for initial viability, overall yield, and processing time at the site-affiliated laboratories (Stage 1); for the second stage, the repositories determined post-thaw viability and cell recovery of cryopreserved PBMC, received from the site-affiliated laboratories (Stage 2); the third stage assessed the long-term specimen storage at each repository (Stage 3). Overall, the CHAVI PBMC QA oversight program results highlight the relative importance of each of these stages to the ultimate goal of preserving specimen integrity from peripheral blood collection to long-term repository storage.

Keywords: Biorepository; Cryopreservation; HIV; Human clinical trials; PBMC; Vaccine.

Fig. 1. Design of the CHAVI PBMC QA Oversight Program

This schematic illustrates the shipment of PBMC from the site-affiliated laboratories to the repositories and to end user laboratories and the stages of the CHAVI PBMC QA Oversight Program: Stage 1 - PBMC Initial viability, yield, and processing time at the site-affiliated laboratories; Stage 2 - Cryopreserved PBMC viability and cell recovery after thawing at the repositories; Stage 3 - Sentinel Program for long-term storage of cryopreserved PBMCs at the repositories.

Fig. 2. Stage 2 - Cryopreserved PBMC Viability and Cell Recovery after Thawing at the Repositories

The Day 1 PBMC post-thaw viability (Panel A) and total cell recovery (Panel B) QC results from PBMC preparations collected over five years from each site-affiliated laboratory are presented. The bars represent the raw data mean +/− 95% Confidence Interval (CI) for the QC results (left axis). The circle markers on the line represent the percent of the PBMC preparations from each site-affiliated laboratory that passed the acceptance criteria (right axis). The asterisks in Panel A indicate that the viability results differed between site-affiliated laboratories based on non-overlapping model-based 95% CI boundaries of the means. The upper and lower limits of the acceptance criteria are indicated by the horizontal dotted line(s) in each of the graphs. Acceptance criteria for PBMCs: post thaw viability ≥80%; post-thaw total cell recovery 55–120%.

Fig. 3. Stage 3 - Sentinel Program for Long-Term Storage of Cryopreserved PBMCs at the Repositories

The control sample post-thaw viability (Panel A) and Day 1 total cell recovery (Panel B) QC results collected over two years from each thawing location are presented. The bars represent the raw data mean +/− 95% CI for the viability results and the model-based mean +/− 95% CI for the recovery results (left axis). The circle markers on the line represent the percent of the control sample vials from each thaw site that passed the acceptance criteria (right axis) Acceptance criteria for the control samples: post-thaw viability ≥92.9%; post-thaw recovery 57.7–120.7%.

Fig. 4. Stage 3 - Sentinel Program for Long-Term Storage of Cryopreserved PBMCs at the Repositories: Analysis of Each Repository Freezer over Time

Post-thaw viability (Panel A) and Day 1 total cell recovery (Panel B) QC results from the control sample vials stored in each repository freezer [Freezer A and B at the central repository 1 (R1 A, R1 B); Freezer A at the central repository 2 (R2 A); and Freezer A at the repository 3 (R3 A)] over two years are presented. The markers represent the raw data mean +/− 95% CI for the QC results. The asterisk in Panel B indicates that, by regression analysis, Freezer A at repository 3 control sample post-thaw recoveries changed over time (p=1.000). The upper and lower limits of the Acceptance Criteria are indicated by the horizontal dotted line(s) in each of the graphs. Acceptance criteria for the control samples: post-thaw viability ≥92.9%; post-thaw recovery 57.7–120.7%.