Spot size and morphology frequently allow researchers to distinguish cytokine production by different cell types within mixed cell populations. For example, when IL-10 production by human peripheral blood mononuclear cells (PBMC) is measured in ELISPOT assays, most of the “antigen-induced” spots are not T cell derived (as would be expected), but are produced by macrophages in response to LPS contamination of the antigen. Such macrophage-derived IL-10 spots are considerably smaller than the IL-10 spots generated by antigen-specific T cells. While the LPS-induced macrophage-derived spots provide no information on specific immunity, the antigen-induced T-cell-derived IL-10 spots do, since they indicate the presence of T regulatory cells. In order to measure the latter, the former need to be excluded from the counting results by setting appropriate size thresholds. ELISPOT image analysis must therefore be capable of distinguishing different spot sizes and morphologies to provide information relevant for T cell diagnostics.
The antigen dose affects the cytokine secretion rate of T cells. Stimulation of a T cell clone with a high dose of the nominal antigenic peptide induces stronger cytokine production in the individual T-lymphocytes (that is, it triggers larger and/or denser spots) than does the stimulation of the same clone with low dose peptide. Therefore, when stimulated with a single antigen dose, as is frequently the case in ELIPSOT assays, high-avidity T-lymphocytes within the PBMC produce larger spots than low-avidity clones. Confirming this notion, increased T cell costimulation was shown to result in increased per cell productivity. In diseases such as HIV, the per cell cytokine productivity can be reduced, resulting in smaller spots. One advantage of ELISPOT assays is their ability to determine whether decreased net cytokine production in disease states is caused by a decreased number of cytokine-secreting T cells or from reduced per cell productivity by unchanged frequencies of T cells. In order to compensate for physiological and pathological variations in per cell productivity, ELISPOT image analysis tools must therefore be versatile, with the ability to permit fine-tuning of the image processing parameters.