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

dc.contributor.author Rountree, Wes
dc.contributor.author Berrong, Mark
dc.contributor.author Sanchez, Ana M
dc.contributor.author Denny, Thomas N
dc.contributor.author Ferrari, Guido
dc.coverage.spatial Netherlands
dc.date.accessioned 2016-06-01T14:46:38Z
dc.date.issued 2016-06
dc.identifier http://www.ncbi.nlm.nih.gov/pubmed/27021273
dc.identifier S0022-1759(16)30053-9
dc.identifier.uri https://hdl.handle.net/10161/12062
dc.description.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.
dc.language eng
dc.publisher Elsevier BV
dc.relation.ispartof J Immunol Methods
dc.relation.isversionof 10.1016/j.jim.2016.03.004
dc.subject Bridging study
dc.subject ELISpot
dc.subject EQAPOL
dc.subject Poisson
dc.subject Proficiency testing
dc.subject Variability
dc.title Variability of the IFN-γ ELISpot assay in the context of proficiency testing and bridging studies.
dc.type Journal article
duke.contributor.id Denny, Thomas N|0400543
duke.contributor.id Ferrari, Guido|0108355
pubs.author-url http://www.ncbi.nlm.nih.gov/pubmed/27021273
pubs.begin-page 69
pubs.end-page 76
pubs.organisational-group Basic Science Departments
pubs.organisational-group Clinical Science Departments
pubs.organisational-group Duke
pubs.organisational-group Duke Human Vaccine Institute
pubs.organisational-group Global Health Institute
pubs.organisational-group Institutes and Centers
pubs.organisational-group Institutes and Provost's Academic Units
pubs.organisational-group Medicine
pubs.organisational-group Medicine, Duke Human Vaccine Institute
pubs.organisational-group Molecular Genetics and Microbiology
pubs.organisational-group School of Medicine
pubs.organisational-group Staff
pubs.organisational-group Surgery
pubs.organisational-group Surgery, Surgical Sciences
pubs.organisational-group University Institutes and Centers
pubs.publication-status Published
pubs.volume 433
dc.identifier.eissn 1872-7905
duke.contributor.orcid Ferrari, Guido|0000-0001-7747-3349


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