Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Abstract

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNγ ELISPOT assay is a well-standardized and validated method for the determination of functional IFNγ-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNγ ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNγ ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning.

Validation of biomarkers to predict response to immunotherapy in cancer: Volume I — pre-analytical and analytical validation

“…Apoptotic cell contamination should be kept to a minimum [86]. Overnight resting of previously frozen samples prior to the assay has been shown to remove apoptotic cells and restore functionality [87, 88]. …”

“…Clear directions in prequalification criteria for large-batch stored materials are highly recommended (e.g., a viability cut-off to qualify control donor PBMC used as an in-study quality control). For functional cell-based assays, such as ELISpot and SCNP that require cell-preconditioning, specific validated SOPs ensuring reproducibility are necessary [88, 118]. …”

Guidelines for the automated evaluation of Elispot assays

“…Not only are apoptotic or dead cells and unwanted contaminating subpopulations of cells the leading causes of artificial signals on the plate membrane and hence causes of variability in assay results, but they also dilute the living cell population and suppress T cell functionality 14,30 . Excellent recommendations to improve the quality and composition of the tested cell populations have recently been given to the community 18,31,32 . The pros and cons of expanding cells in an assay-preceding in vitro stimulation step have also been demonstrated 33 , and the isolation of subpopulations of cells via magnetic beads is part of the methodological repertoire of many laboratories.…”

“…5. Allow the cells to rest for a minimum of 16 h at high density at 37 °C, 5% CO 2 (5 ml of cell suspension with 2 × 10 6 cells per ml in complete culture medium in a 50-ml tube permitting gas exchange) 18 .  crItIcal step Do not use tissue culture plates or flasks as monocytes will adhere, thus making it difficult to recover them for the assay where they may be needed as antigen presenters.…”

“…These improvements include an overnight resting step of previously frozen peripheral blood mononuclear cells (PBMCs) and the enumeration of apoptotic cells to obtain precise counts of the viable fraction of cells added to the assay. Both improvements have been recently reviewed elsewhere 18 .…”