Proficiency tests to evaluate the impact on assay outcomes of harmonized influenza-specific Intracellular Cytokine Staining (ICS) and IFN-ɣ Enzyme-Linked ImmunoSpot (ELISpot) protocols

New publication to which our member, Center for Vaccinology (CEVAC) contributed. This research addresses the critical need for harmonized protocols in (influenza-specific) immune response assays, focusing specifically on ICS and ELISpot techniques. The lack of harmonization in these cellular immunomonitoring assays can significantly hinder the comparability of data across clinical studies and laboratories.


The magnitude and quality of cell-mediated immune responses elicited by natural infection or vaccination are commonly measured by Interferon-ɣ (IFN-ɣ) Enzyme-Linked ImmunoSpot (ELISpot) and Intracellular Cytokine Staining (ICS). To date, laboratories apply a variety of in-house procedures which leads to diverging results, complicates interlaboratory comparisons and hampers vaccine evaluations. During the FLUCOP project, efforts have been made to develop harmonized Standard Operating Procedures (SOPs) for influenza-specific IFN-ɣ ELISpot and ICS assays. Exploratory pilot studies provided information about the interlaboratory variation before harmonization efforts were initiated. Here we report the results of two proficiency tests organized to evaluate the impact of the harmonization effort on assay results and the performance of participating FLUCOP partners.

The introduction of the IFN-ɣ ELISpot SOP reduced variation of both background and stimulated responses. Post-harmonization background responses were all lower than an arbitrary threshold of 50 SFU/million cells. When stimulated with A/California and B/Phuket, a statistically significant reduction in variation (p < 0.0001) was observed and CV values were strongly reduced, from 148% to 77% for A/California and from 126% to 73% for B/Phuket. The harmonizing effect of applying an ICS SOP was also confirmed by an increased homogeneity of data obtained by the individual labs. The application of acceptance criteria on cell viability and background responses further enhanced the data homogeneity. Finally, as the same set of samples was analyzed by both the IFN-ɣ ELISpot and the ICS assays, a method comparison was performed. A clear correlation between the two methods was observed, but they cannot be considered interchangeable.

In conclusion, proficiency tests show that a limited harmonization effort consisting of the introduction of SOPs and the use of the same in vitro stimulating antigens leads to a reduction of the interlaboratory variation of IFN-ɣ ELISpot data and demonstrate that substantial improvements for the ICS assay are achieved as comparable laboratory datasets could be generated. Additional steps to further reduce the interlaboratory variation of ICS data can consist of standardized gating templates and detailed data reporting instructions as well as further efforts to harmonize reagent and instrument use.

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