Value of a quality assessment program in optimizing cryopreservation of peripheral blood mononuclear cells in a multicenter study

Abstract

Cryopreservation of peripheral blood mononuclear cells (PBMC) allows assays of cellular function and phenotype to be performed in batches at a later time on PBMC at a central laboratory to minimize assay variability. The Multicenter AIDS Cohort Study (MACS) is an ongoing prospective study of the natural and treated history of human immunodeficiency virus (HIV) infection that stores cryopreserved PBMC from participants two times a year at four study sites. In order to ensure consistent recovery of viable PBMC after cryopreservation, a quality assessment program was implemented and conducted in the MACS over a 6-year period. Every 4 months, recently cryopreserved PBMC from HIV-1-infected and HIV-1-uninfected participants at each MACS site were thawed and evaluated. The median recoveries of viable PBMC for HIV-1-infected and -uninfected participants were 80% and 83%, respectively. Thawed PBMC from both HIV-1-infected and -uninfected participants mounted a strong proliferative response to phytohemagglutinin, with median stimulation indices of 84 and 120, respectively. Expression of the lymphocyte surface markers CD3, CD4, and CD8 by thawed PBMC was virtually identical to what was observed on cells measured in real time using whole blood from the same participants. Furthermore, despite overall excellent performance of the four participating laboratories, problems were identified that intermittently compromised the quality of cryopreserved PBMC, which could be corrected and monitored for improvement over time. Ongoing quality assessment helps laboratories improve protocols and performance on a real-time basis to ensure optimal cryopreservation of PBMC for future studies.

Fig 1

Viability and recovery of cryopreserved PBMC from 75 HIV-uninfected and 76 HIV-infected participants across the four MACS sites. The solid lines represent the median, and the dashed lines represent the mean. The box represents the 25th to 75th percentiles. The lower and upper horizontal bars represent the 10th and 90th percentiles, respectively.

Fig 2

Viable cell recovery from each vial of cryopreserved PBMC from 75 HIV-uninfected and 76 HIV-infected participants. Numbers on the x axis represent sequential times when cryopreserved PBMC from the four MACS sites were tested. The QA testing program was started in July 2004 and was performed every 4 months. The first symbol in each testing round for each of the MACS sites is the value for the HIV-uninfected participants, and the second symbol is the value for the HIV-infected participants. The dashed lines represent 75% and 100% viable cell recoveries.

Fig 3

Proliferative responses of cryopreserved PBMC (n = 28 per group) to phytohemagglutinin (PHA). The stimulation index was defined as the counts per minute in the presence of PHA divided by the counts per minute in the absence of PHA. The solid lines represent medians, and the dashed lines represent the means of the distributions. For HIV-uninfected samples the median and mean were the same. The boxes represent the 25th to 75th percentiles. The lower and upper horizontal bars indicate the 10th and 90th percentiles, respectively.

Fig 4

Correlation between the PHA-induced proliferative response of cryopreserved PBMC and the percentage of CD4⁺ (top panel) and CD8⁺ (bottom panel) T lymphocytes for 27 HIV-uninfected and 27 HIV-infected participants. Least-squares regression lines are indicated.

Fig 5

Deming regression analyses of expression of cell surface markers CD3 (top panel), CD4 (middle panel), and CD8 (bottom panel) on paired samples of fresh whole-blood and cryopreserved PBMC. A total of 54 paired blood samples (27 from HIV-uninfected and 27 from HIV-infected participants) from the 4 MACS sites were evaluated. The dashed lines represent the line of identity (y = x), and the solid lines represent the least-squares regression lines of the data.