ELISPOTs produced by CD8 and CD4 cells follow log normal size distribution

Introduction Each positive well in ELISPOT assays always contain spots of variable sizes that range from micrometers up to a millimeter in diameter. Therefore, when it comes to counting ELISPOTs it is critical to decide how to set the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters. If the spot sizes produced by T cells would not follow a certain statistical distribution, the size thresholds (gates) for counting would need to be set based on subjective judgment, thus leading to variability in spot counts when parameters are set by different investigators. In contrast, if the spot sizes follow a defined statistical distribution, precise predictions can be made as to the minimal and maximal spot sizes that belong to a given population. Hence, the gates could be set automatically ensuring counting results that are independent of subjective judgments.