The External Quality Assurance Oversight Laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay

Abstract

The Interferon-gamma Enzyme-Linked ImmunoSpot (IFN-γ ELISpot) assay has been developed and used as an end-point assay in clinical trials for infectious diseases and cancer to detect the magnitude of antigen-specific immune responses. The ability to compare data generated by different laboratories across organizations is pivotal to understand the relative potency of different therapeutic and vaccine strategies. We developed an external proficiency program for the IFN-γ ELISpot assay that evaluates the laboratory performance based on five parameters: timeliness for data reporting; ability to handle cellular samples; detection of background (non-specific) responses; accuracy to consensus of the results; and precision of the measurements. Points are awarded for each criterion, and the sum of the points is used to determine a numeric and adjectival performance rating. Importantly, the evaluation of the accuracy to the consensus mean for the detection of antigen-specific responses using laboratory-specific procedures informs each laboratory and its sponsor on the degree of concordance of its results with those obtained by other laboratories. This study will ultimately provide the scientific community with information on how to organize and implement an external proficiency program to evaluate longitudinally the performance of the participating laboratories and, therefore, fulfill the requirements of the GCLP guidelines for laboratories performing end-point IFN-γ ELISpot assay for clinical trials.

Biomarkers in blood for individualization of the pharmacotherapy with immunosuppressive drugs after transplantation of solid organs

“…A weaker IFN-γ formation was associated with better renal function, allowing a milder immunosuppression [23] . Similar to the ImmuKnow ® test, the ELISPOT assay can be standardized and controlled very well [24,25] , which significantly improves the comparability of results between different laboratories. This allows for multicenter studies and increases the chances of routine use if the relevant clinical data are available.…”

Biomarker im Blut zur Individualisierung der immunsuppressiven Therapie nach der Transplantation solider Organe

“…Eine schwächere IFN-γ Bildung war mit einer besseren Nierenfunktion verbunden und erlaubte eine mildere Immunsuppression [23]. Ähnlich wie der ImmuKnow ® -Test lässt sich der ELISPOT-Test recht gut standardisieren und kontrollieren [24,25], was die Vergleichbarkeit der Ergebnisse zwischen unterschiedlichen Laboratorien enorm verbessert. Das erlaubt multizentrische Studien und vergrößert die Chancen auf einen Routineeinsatz, wenn die entsprechenden klinischen Daten aus Studien vorliegen.…”

Variability of the IFN-γ ELISpot assay in the context of proficiency testing and bridging studies

“…The Duke University Human Vaccine Institute (DHVI) has been the central laboratory of the National Institute of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) External Quality Assurance Program Oversight Laboratory (EQAPOL) through a Department of Health and Human Services contract since September 2010. As part of this program, EQAPOL has developed and run PT programs for Interferon-gamma (IFN- γ ) Enzyme-linked immunosorbent spot (ELISpot), multiparameter Intracellular Cytokine Staining (ICS) Flow Cytometry and Luminex bead-based multiplex cytokine assays (Lynch et al, 2014; Rountree et al, 2014; Sanchez et al, 2014; Staats et al, 2014). …”

“…Both IFN- γ ELISpot and ICS assays, performed to evaluate antigen-specific immune responses, have been subjected to validation procedures (Russell et al, 2003; Horton et al, 2007) according to Good Clinical Laboratory Practice (GCLP) guidelines, and the assays have been performed by EQAPOL laboratories accordingly (Sanchez et al, 2014; Staats et al, 2014). In the germinal paper that compared the performance of the ELISpot assay conducted by several different laboratory sites (Cox et al, 2006; Sanchez et al, 2014), several reagents were found to be key in the overall performance of the assay across laboratories with fetal bovine serum (FBS) being one of them (Cox et al, 2006; Janetzki et al, 2008).…”

“…In the germinal paper that compared the performance of the ELISpot assay conducted by several different laboratory sites (Cox et al, 2006; Sanchez et al, 2014), several reagents were found to be key in the overall performance of the assay across laboratories with fetal bovine serum (FBS) being one of them (Cox et al, 2006; Janetzki et al, 2008). The initial report, and subsequent experience among the laboratories, indicated that studies addressing between reagent lot-to-lot variation should be performed for cellular assays.…”