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

Development of an in-house SARS-CoV-2 interferon-gamma ELISpot and plate reader-free spot detection method

“…Most laboratories use an automated ELISpot plate reader to detect and quantify spot-forming cells. Since these analysers are large investments and manual counting of spots likely leads to higher variability of results when compared to objective software-dependent spot detection ( Ansari et al, 2013 ), we developed an ELISpot plate reader-free but software-dependent method to detect and quantify spots. Similar to the study of Hayashi et al that assessed response to cancer vaccine immunotherapy ( Hayashi et al, 2020 ), we used the Particle Analysis tool of the open platform software FIJI in which we set a particle intensity and size threshold to distinguish distinct spots from background noise.…”