User vs. software-dependent variability of ELISPOT counts obtained from ten different laboratories

Introduction In each human donor’s PBMC, there is a defined number of T cells specific for any given antigen. A major goal of immune monitoring with ELISPOT is to measure this number accurately and reproducibly between different laboratories. In ELISPOT assays, cytokine spots produced by antigen-specific T cells show a broad spectrum of sizes and densities over variable background. Therefore, even experienced investigators are likely to come up with different spot counts when subjectively judging the minimal spot size/density to be counted and the maximal spot size for the cut off between single-cell-derived spots vs. those created by cell clusters. This study aims to find out whether statistics-based automated gating can harmonize spot counts obtained in different laboratories.