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Clin. Vaccine Immunol. doi:10.1128/CVI.00435-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Establishing Acceptance Criteria for Cell-Mediated Immunity Assays Using Frozen Peripheral Blood Mononuclear Cells Stored Under Optimal and Suboptimal Conditions

Vaccine & Biologics Research, Vaccine Biometrics, Merck Research Laboratories, 770 Sumneytown Pike, Merck and Co., Inc., West Point, PA 19486

^* To whom correspondence should be addressed. Email: jeffrey_smith2{at}merck.com.

	Abstract

The ELISPOT assay is a powerful tool for measuring antigen specific cellular immune responses. The ability to use frozen peripheral blood mononuclear cells (PBMCs) facilitates testing samples in multi-center clinical trials; however unreliable ELISPOT responses may result if samples are not handled properly. Exposure of frozen PBMCs to suboptimal storage temperature (-20°C) or repeated cycling between more optimal storage temperatures (<-130°C and -70°C) reduced the quality of frozen PBMCs as assessed by cell viability and functional ELISPOT response measures. Cell viability assessed by Trypan blue dye exclusion was reduced and the percentage of apoptotic cells, by the Guava Nexin^TM assay, was significantly increased following these events. The functional IFN- ELISPOT responses to PHA mitogen, a CD4 T cell specific antigen (varicella zoster virus [VZV]), and a CD8 T cell specific antigen (pool containing known CMV, EBV, and influenza virus peptides) were all significantly reduced following suboptimal storage events. However, for a given suboptimal storage event, the magnitude of the reduction varied between individuals and even among aliquots within an individual bleed, indicating the need for sample specific acceptance criteria (AC). Percent viable or percent apoptotic cells after thaw as well as functional ELISPOT response to PHA were all effective when applied with limits as acceptance criteria for separating samples damaged during storage from valid control samples. While all three AC measures could be effectively applied, the apoptosis AC limit applied was best for separating samples that could respond to antigenic stimulation from samples that could not effectively respond.