Substantial improvements in performance indicators achieved in a peripheral blood mononuclear cell cryopreservation quality assurance program using single donor samples

Abstract

Storage of high-quality cryopreserved peripheral blood mononuclear cells (PBMC) is often a requirement for multicenter clinical trials and requires a reproducibly high standard of practice. A quality assurance program (QAP) was established to assess an Australia-wide network of laboratories in the provision of high-quality PBMC (determined by yield, viability, and function), using blood taken from single donors (human immunodeficiency virus [HIV] positive and HIV negative) and shipped to each site for preparation and cryopreservation of PBMC. The aim of the QAP was to provide laboratory accreditation for participation in clinical trials and cohort studies which require preparation and cryopreservation of PBMC and to assist all laboratories to prepare PBMC with a viability of >80% and yield of >50% following thawing. Many laboratories failed to reach this standard on the initial QAP round. Interventions to improve performance included telephone interviews with the staff at each laboratory, two annual wet workshops, and direct access to a senior scientist to discuss performance following each QAP round. Performance improved substantially in the majority of sites that initially failed the QAP (P = 0.002 and P = 0.001 for viability and yield, respectively). In a minority of laboratories, there was no improvement (n = 2), while a high standard was retained at the laboratories that commenced with adequate performance (n = 3). These findings demonstrate that simple interventions and monitoring of PBMC preparation and cryopreservation from multiple laboratories can significantly improve performance and contribute to maintenance of a network of laboratories accredited for quality PBMC fractionation and cryopreservation.

FIG. 1.

Sequential assessment of viability and yield results from (A) laboratories that performed adequately throughout the QAP and (B) laboratories that either improved during the course of the QAP or failed to improve. Viabilities (upper panels) and yields (lower panels) of PBMC from HIV-positive (left panels) and HIV-negative (right panels) common donors supplied from each participating laboratory (same symbols in each panel) are shown. Dashed lines represent the required performance standards.

FIG. 2.

Summary (means and standard deviations) of viabilities (A) and viable yields (B) of PBMC prepared by the participating laboratories in each QAP round (yield data were calculated with the maximum yield being 100%). The ratio of new to total labs participating in each round is indicated. Initiatives to improve performance are indicated by arrows. The dashed lines represent the performance standards set for both viability and yield. Local donor results represent PBMC collected from an HIV-negative donor at the laboratory site.

FIG. 3.

Association between viability and viable cell yield, revealing differences in quality of thawed PBMC between QAP rounds. Linear regression and 95% confidence interval curves are shown (for data taken from the single-donor PBMC only). In the fifth QAP round, there was a significant correlation between viability and yield for the HIV-negative single-donor specimens (dashed line) (r² = 0.455; P = 0.046), but it was not significant for the HIV-positive donor specimens (solid line) (r² = 0.175; P = 0.263); however, overall significance was reached when data from both donors were combined (r² = 0.289; P = 0.022). Low yield was not associated with viability (uniformly high) in specimens from the seventh QAP round, consistent with improvements in PBMC quality from this round.

FIG. 4.

As a consequence of uniformly high cell viability in the seventh QAP PBMC specimens, there was no association between viability or yield and immune function (determined by the IFN-γ response to the CEF peptide pool, as measured by ELISPOT assay). Linear regression and 95% confidence interval curves are shown (for data taken from the single-donor PBMC only). SFC, spot-forming cells.