Log-normal ELISPOT spot size distribution permits counts harmonization among different laboratories

Results The analysis of antigen-elicited ELISPOT sizes for IFNg, IL-2, IL-4, IL-5 and IL-17 was found to exhibit a LogNormal distribution pattern for all 24 donors, and for all CD4 and CD8 cell-derived cytokine signatures. The significance levels were over 5% according to Kolmogorov-Smirnov test. When the spot counts were established in 10 different laboratories using gating criteria established based on Log Normal distributions, the coefficient of variation (CV) of mean spot counts between different laboratories was 6.7%. In contrast, when the participating scientists set gates based on subjective assessment, the CV of mean spot counts obtained between the different laboratories was 26.7%. Conclusions For all 24 donors, 34 antigens, and for all five cytokines studied, ELISPOTs were observed to follow log normal distribution. This statistical function permits us to set upper and lower size gates automatically with a 98% confidence. Using this statistics-based approach, ten different laboratories obtained close to identical counts, as opposed to when the gates were set subjectively by the different investigators. Harmonization of accurate and objective ELISPOT counts can be accomplished based on statistical principles.