Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions

Abstract

The enzyme-linked immunospot (ELISPOT) assay is a powerful tool for measuring antigen-specific cellular immune responses. The ability to use frozen peripheral blood mononuclear cells (PBMC) facilitates testing samples in multicenter clinical trials; however, unreliable ELISPOT responses may result if samples are not handled properly. Exposure of frozen PBMC to suboptimal storage temperature (-20 degrees C) or repeated cycling between more optimal storage temperatures (less than -130 degrees C and -70 degrees C) reduced the quality of frozen PBMC, as assessed by cell viability and functional ELISPOT response measures. Cell viability as assessed by trypan blue dye exclusion was reduced, and the percentage of apoptotic cells, as determined by the Guava Nexin assay, was significantly increased after these events. The functional gamma interferon ELISPOT responses to phytohemagglutinin (PHA) mitogen, a CD4 T-cell-specific antigen (varicella-zoster virus), and a CD8 T-cell-specific antigen (pool containing known cytomegalovirus, Epstein-Barr virus, and influenza virus peptides) were all significantly reduced after 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). The percent viable or percent apoptotic cells after thaw, as well as the functional ELISPOT response to PHA, were all effective when applied with limits as AC for separating samples damaged during storage from valid control samples. Although 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.

FIG. 1.

Temperatures within dry ice shipping container, measured with a TempTale probe reader system.

FIG. 2.

Effect of short-term −20°C temperature storage on PBMC viability and IFN-γ ELISPOT responses. Samples were transferred from LN₂ freezer into −20°C freezer for the times indicated and then returned to the LN₂ freezer until assay testing. Averages and standard deviations for three vials tested per condition are shown for the percent viability determined by trypan blue dye exclusion (a), PHA ELISPOT response (b), and VZV ELISPOT response (c). Bars: ▪, donor M4866; □, donor M5020.

FIG. 3.

Effect of cycling the temperature of storage on PBMC viability and IFN-γ ELISPOT responses. One cycle represents transfer from an LN₂ freezer storage to −70°C freezer storage and back to LN₂ freezer storage. After the indicated number of storage cycles, samples were stored in an LN₂ freezer until assay testing. Averages and standard deviations of five vials tested per condition are shown for the percent viability by trypan blue dye exclusion (a), PHA ELISPOT response (b), and VZV ELISPOT response (c). Bars: ▪, donor M4866; □, donor M1481.

FIG. 4.

Plots of viability, IFN-γ ELISPOT response to PHA, and percent apoptosis with antigen-specific IFN-γ ELISPOT responses compared to antigen-specific IFN-γ ELISPOT response. (a) AC correlation with VZV antigen response (CD4-specific); (b) AC correlation with EPI peptide pool response (CD8-specific).

FIG. 5.

Application of trypan blue dye viability AC, with an AC limit of ≥89% viable, to assay testing with frozen PBMC after optimal LN₂ storage, 8 h exposure to −20°C storage, and storage temperature cycling. (a) Donor M3143 response to VZV antigen; (b) donor M5020 response to VZV antigen; (c) donor M3143 response to EPI peptide pool; (d) donor M5020 response to EPI peptide pool.

FIG. 6.

Application of IFN-γ ELISPOT response to PHA AC, with an AC limit of ≥1,000 SFC, to assay testing with frozen PBMC after optimal LN₂ storage, 8 h exposure to −20°C storage, and storage temperature cycling. (a) Donor M3143 response to VZV antigen; (b) donor M5020 response to VZV antigen; (c) donor M3143 response to EPI peptide pool; (d) donor M5020 response to EPI peptide pool.

FIG. 7.

Application of percent apoptosis AC, with an AC limit of ≤18%, to assay testing with frozen PBMC after optimal LN₂ storage, 8 h exposure to −20°C storage, and storage temperature cycling. (a) Donor M3143 response to VZV antigen response; (b) donor M5020 response to VZV antigen; (c) donor M3143 response to EPI peptide pool; (d) donor M5020 response to EPI peptide pool.