Abstract
BackgroundCryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors.ResultsUsing CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p ≤ 0.001). The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p ≤ 0.001). Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation) and tetramer staining (p < 0.001). Roughly similar sensitivity and specificity were observed between the three assays and between shipped and cryopreserved samples for each assay.ConclusionWe conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.
Highlights
Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and enzymelinked immunospot (ELISPOT)
Fresh versus cryopreserved assay correlations PBMC derived from leukapheresis of CMV seropositive and CMV seronegative donors were analyzed as fresh samples or frozen samples
To be sure that this was not based upon the correlation of the negative results alone, the statistics were recalculated on CMV seropositive data only, and the correlation coefficient remained 0.9 (95% C.I.: 0.7–1.0)
Summary
Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. An increasing number of experimental vaccines are being developed for diseases in which cellular immunity is likely to be required for protection [1]. These include HIV [2], hepatitis C [3], malaria [4], and cancer [5]. In these settings, immunological monitoring of antigen-specific T cell responses is likely to be an important part of vaccine assessment [6,7,8,9,10]. It is likely that inducing a particular level of antigen-specific T cell response to a vaccine will be necessary, though perhaps not sufficient, for vaccine efficacy of either prophylactic or therapeutic vaccines [9,10]
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