| Abstract: | The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4+ and CD8+ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.Utilization of cryopreserved peripheral blood mononuclear cells (PBMC) for immunologic assays has dramatically increased in recent years. Cryopreserved PBMC are particularly useful in clinical trials with low end-point frequencies because they allow the immunologic assays to be performed after the conclusion of the studies, when all the end points have already been identified (1, 14, 18). In addition, the use of cryopreserved PBMC allows all assays to be performed in a single laboratory, eliminating interlaboratory variability, which has been a confounder in some studies (15). Furthermore, if changes in immunologic parameters over time are the main outcome of the immunologic studies, interassay variability may become a confounder, too. Testing all the cryopreserved PBMC per subject at one time can eliminate this confounder.The use of cryopreserved PBMC in immunological assays poses challenges, including the availability of adequate equipment (7) and the need for technical proficiency. Assays have to be adapted and validated for the use of cryopreserved PBMC (4, 6, 9, 11, 17, 19), and the quality of the frozen cells has to be monitored (5) to ensure reliable results in functional and phenotypic assays.The Cryopreservation Working Group (WG) of the Pediatric AIDS Clinical Trials Group (PACTG), which operated between 1999 and 2006, developed a series of experiments aimed at optimizing methods of PBMC cryopreservation for assays requiring viable cells, adapting immunologic assays to cryopreserved PBMC, and establishing quality control parameters for immunologic assays with cryopreserved PBMC. The group focused on highly complex functional and phenotypic assays commonly used as outcome measures in studies that address immune suppression or reconstitution. Unlike previous studies of cryopreserved PBMC in immunologic assays, which were done at single laboratories (3, 8, 13, 17), our study presents results obtained at eight laboratories, substantiating the generalizibility of our findings. |
