Standardization and cross validation of alloreactive IFNγ ELISPOT assays within the clinical trials in organ transplantation consortium

Abstract

Emerging evidence indicates memory donor-reactive T cells are detrimental to transplant outcome and that quantifying the frequency of IFNγ-producing, donor-reactive PBMCs by ELISPOT has potential utility as an immune monitoring tool. Nonetheless, differences in assay performance among laboratories limit the ability to compare results. In an effort to standardize assays, we prepared a panel of common cellular reagent standards, developed and cross validated a standard operating procedure (SOP) for alloreactive IFNγ ELISPOT assays in several research laboratories supported by the NIH-funded Clinical Trials in Organ Transplantation (CTOT) Consortium. We demonstrate that strict adherence to the SOP and centralized data analysis results in high reproducibility with a coefficient of variance (CV) of ≈ 30%. This standardization of IFNγ ELISPOT assay will facilitate interpretation of data from multicenter transplantation research studies and provide the foundation for developing clinical laboratory testing strategies to guide therapeutic decision-making in transplant patients.

Figure 1

Alloreactive IFNγ ELISPOT assays performed without SOP. A. Representative replicate wells of IFNγ ELISPOT assays performed using aliquots of the same PBMC responders and B cell stimulators at 2 different sites according to site specific laboratory protocols. B. Quantified ELISPOT results of 4 different responder stimulator pairs as performed at 2 different laboratories.

Figure 2

Alloreactive IFNγ ELISPOT assay quality determined by multiple technical factors. A. 2 sets of IFNγ ELISPOT assays were compared using 2 different responder stimulator pairs at each of the 2 sites. Results represent means + SD of quadruplicate wells. *p<0.05. B. Representative assay wells of one responder stimulator pair without DNAse added during cell thawing showing typical artifacts (top) or with the addition of DNAse (bottom) C. Captured images of wells from the same plate allowed to dry without removal of the plastic gasket with the wells still slightly wet (top) and after removal of plastic gasket when the wells are fully dry (bottom). D. Representative replicate assay wells performed at 2 sites using the same responder stimulator pairs following the newly devised SOP.

Figure 3

Site reported results show significant variability. A. Site reported frequencies of IFNγ producing cells for each of 103 responder stimulatory pairs using common reagents, SOP at 3 different sites (each dot is the mean of triplicate wells performed at a single site). Dash is the mean value of the 3 sets of data from the 3 sites. All sites detected essentially no response for a syngeneic responder stimulator pair (top graph, first data point). B. 2 way comparisons of results among sites with correlation coefficients shown in the lower right of each graph. C. Coefficients of variance for each responder stimulator pair using site reported data.

Figure 4

Centralized data analysis reduces assay variance. A. Representative comparisons and correlation coefficients of site reported results (left, same data as shown in lower left of Fig 3C), results of analysis of digital files captured by the site from assays performed at the site but analyzed by a single core lab (middle) and results of central analysis of plates obtained from each lab (right). B. Coefficients of variance for each responder/stimulator pair as determined by each methods (only those with a response of ≥ 25 spots were included in this analysis).

Figure 5

SOPs result in concordance of strong alloresponses among sites. Frequency distribution of assays performed at each of the 3 sites but analyzed centrally of the physical plates.

Figure 6

Assays using stored clinical samples show similarly low variance. A. Representative assay wells of transplant recipient responder cells stimulated with matched donor (top) or 3^rd party donor (bottom). B. 2-way comparison of results from sites with correlation coefficient.