Variability of the IFN-γ ELISpot assay in the context of proficiency testing and bridging studies
Wes Rountree1, Mark Berrong1, Ana M Sanchez1
1Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, USA.
Insights
A new dispersion limit of 3.3 was established for Interferon-gamma (IFN-γ) Enzyme-linked immunosorbent spot (ELISpot) assays to assess reagent lot-to-lot variability. This metric aids in ensuring reproducible cellular immune response assay results and guides optimal reagent evaluation strategies.
Area of Science:
- Immunology
- Clinical Laboratory Science
- Assay Development
Background:
- Cellular immune response assays require stringent validation under Good Clinical Laboratory Practice (GCLP) for reproducibility.
- Standard Operating Procedures (SOPs) need defined validation, pass/fail criteria, and positivity criteria.
- Limited guidance exists for longitudinal assessment of critical reagents in these assays.
Purpose of the Study:
- To establish a standardized metric for assessing within-site variability in Interferon-gamma (IFN-γ) Enzyme-linked immunosorbent spot (ELISpot) assays.
- To evaluate the utility of a calculated 'dispersion limit' for bridging studies assessing reagent lot-to-lot variations.
- To provide guidance on optimal experimental design for reagent evaluation, including replicate numbers and donor reactivity.
Main Methods:
- Proficiency testing (PT) data from six rounds of an IFN-γ ELISpot assay program were analyzed.
- A dispersion limit (variance/mean) of 3.3 was calculated for within-site variability.
- Control samples were used to assess within- (precision) and between- (accuracy) experiment variability, and simulations were performed for bridging studies.
Main Results:
- An overall dispersion limit of 3.3 was established for the ELISpot PT program's within-site variability.
- The dispersion limit demonstrated utility in assessing reagent lot-to-lot variations through bridging studies.
- Simulations suggest a minimum of six replicate wells with at least 150 spot-forming cells/well from a control donor are optimal for evaluating new reagents.
Conclusions:
- The 3.3 dispersion limit provides a robust metric for assessing both within- and between-experiment variability in IFN-γ ELISpot assays.
- This metric is valuable for determining significant lot-to-lot variations and ensuring reagent consistency.
- The findings offer practical guidance for optimizing assay validation and reagent qualification processes in clinical immunology settings.
Abstract:
Assays that assess cellular mediated immune responses performed under Good Clinical Laboratory Practice (GCLP) guidelines are required to provide specific and reproducible results. Defined validation procedures are required to establish the Standard Operating Procedure (SOP), include pass and fail criteria, as well as implement positivity criteria. However, little to no guidance is provided on how to perform longitudinal assessment of the key reagents utilized in the assay. Through the External Quality Assurance Program Oversight Laboratory (EQAPOL), an Interferon-gamma (IFN-γ) Enzyme-linked immunosorbent spot (ELISpot) assay proficiency testing program is administered. A limit of acceptable within site variability was estimated after six rounds of proficiency testing (PT). Previously, a PT send-out specific within site variability limit was calculated based on the dispersion (variance/mean) of the nine replicate wells of data. Now an overall 'dispersion limit' for the ELISpot PT program within site variability has been calculated as a dispersion of 3.3. The utility of this metric was assessed using a control sample to calculate the within (precision) and between (accuracy) experiment variability to determine if the dispersion limit could be applied to bridging studies (studies that assess lot-to-lot variations of key reagents) for comparing the accuracy of results with new lots to results with old lots. Finally, simulations were conducted to explore how this dispersion limit could provide guidance in the number of replicate wells needed for within and between experiment variability and the appropriate donor reactivity (number of antigen-specific cells) to be used for the evaluation of new reagents. Our bridging study simulations indicate using a minimum of six replicate wells of a control donor sample with reactivity of at least 150 spot forming cells per well is optimal. To determine significant lot-to-lot variations use the 3.3 dispersion limit for between and within experiment variability.
