When Is a Spot Not a Spot?

Since 1983, it has been possible to detect molecules produced by individual cells using a laboratory assay called Elispot (short for enzyme-linked immunospot). The molecules are detected by antibodies to which an enzyme is attached. When exposed to its binding partner, the enzyme catalyzes a chemical reaction that produces a colored spot on a white membrane at the bottom of a well in a culture plate. Each spot represents one cell that produced the molecule of interest.

Elispot is used in every sector of immunology, from basic research, to diagnostic tests, to clinical trials. It’s about as ubiquitous to the field of immunology as a calculator is to accounting. Only problem is, the results of one lab’s Elispot assay do not always match the results of another lab’s—even for the same sample. Different Elispot-reading machines, it seems, count different numbers of spots.

This discrepancy was revealed about ten years ago, when a “proficiency panel,” or quality assurance program, was initiated by the Cancer Immunotherapy Consortium (CIC) of the Cancer Research Institute. The goal of the program was first to identify deficiencies in current immune assays and then establish protocols that optimize assay performance, in order to support more reliable data comparisons across institutions.

Easier said than done. It took ten years to identify the sources of variation and to develop methods to standardize assay interpretation. The final results of the program were published just this week in the journal Nature Protocols.

In order to solve the spot problem, the CIC recruited 75 panelists from 52 laboratories from various countries. Each panelist had to analyze and report back the spot counts on a sample provided by the CIC, and also fill out a detailed survey addressing their general approach to plate evaluation.

After the results were collected and analyzed, additional reviewers were brought in to make recommendations. Based on this feedback, the CIC then developed step-by-step guidelines for Elispot plate evaluation.

“Adoption of these guidelines,” the authors of the Nature Protocols paper write, “should enable a further reduction in assay variability and an increase in the reliability and comparability of results obtained by Elispot.”

Spot problem solved.

Full citation: Sylvia Janetzki, Leah Price, Helene Schroeder, Cedrik M. Britten, Marij J. P. Welters & Axel Hoos (2015). Guidelines for the automated evaluation of Elispot assays. Nature Protocols 10, 1098–1115.