title: High Reproducibility of ELISPOT Counts from Nine Different Laboratories.
abstract: The primary goal of immune monitoring with ELISPOT is to measure the number of T cells, specific for any antigen, accurately and reproducibly between different laboratories. In ELISPOT assays, antigen-specific T cells secrete cytokines, forming spots of different sizes on a membrane with variable background intensities. Due to the subjective nature of judging maximal and minimal spot sizes, different investigators come up with different numbers. This study aims to determine whether statistics-based, automated size-gating can harmonize the number of spot counts calculated between different laboratories. We plated PBMC at four different concentrations, 24 replicates each, in an IFN-γ ELISPOT assay with HCMV pp65 antigen. The ELISPOT plate, and an image file of the plate was counted in nine different laboratories using ImmunoSpot® Analyzers by (A) Basic Count™ relying on subjective counting parameters set by the respective investigators and (B) SmartCount™, an automated counting protocol by the ImmunoSpot® Software that uses statistics-based spot size auto-gating with spot intensity auto-thresholding. The average coefficient of variation (CV) for the mean values between independent laboratories was 26.7% when counting with Basic Count™, and 6.7% when counting with SmartCount™. Our data indicates that SmartCount™ allows harmonization of counting ELISPOT results between different laboratories and investigators.
authors.affiliation: Cellular Technology Limited, Shaker Hts. 44122, OH, USA. srividya.sundararaman@immunospot.com.
authors.forename: Srividya
authors.initials: S
authors.lastname: Sundararaman
authors.affiliation: Cellular Technology Limited, Shaker Hts. 44122, OH, USA. ayk@immunospot.com.
authors.forename: Alexey Y
authors.initials: AY
authors.lastname: Karulin
authors.affiliation: Cellular Technology Limited, Shaker Hts. 44122, OH, USA. tameem.ansari@immunospot.com.
authors.forename: Tameem
authors.initials: T
authors.lastname: Ansari
authors.affiliation: Européen Georges Pompidou, Paris 75015, France. nadine.benhamouda@egp.aphp.fr.
authors.forename: Nadine
authors.initials: N
authors.lastname: BenHamouda
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2650, Denmark. judith@gottwein.eu.
authors.forename: Judith
authors.initials: J
authors.lastname: Gottwein
authors.affiliation: Development Translational Medicine, Biogen Idec, Cambridge 02142, MA, USA. srilaxmanan@gmail.com.
authors.forename: Sreenivas
authors.initials: S
authors.lastname: Laxmanan
authors.affiliation: Immuno-Virology Drug Discovery, Bristol-Myers Squibb, Wallingford 06492, CT, USA. steven.m.levine@bms.com.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Levine
authors.affiliation: Immuno-Sciences Biology Drug Discovery, Bristol-Myers Squibb, Princeton 08543, NJ, USA. John.Loffredo@bms.com.
authors.forename: John T
authors.initials: JT
authors.lastname: Loffredo
authors.affiliation: Nottingham Trent University, Nottingham NG118NS, UK. stephanie.mcardle@ntu.ac.uk.
authors.forename: Stephanie
authors.initials: S
authors.lastname: McArdle
authors.affiliation: Medizinische Hochschule Hannover 30625, Germany. Neudoerfl.Christine@mh-hannover.de.
authors.forename: Christine
authors.initials: C
authors.lastname: Neudoerfl
authors.affiliation: Pharmasan Labs, Osceola 54020, WI, USA. diana.roen@pharmasan.com.
authors.forename: Diana
authors.initials: D
authors.lastname: Roen
authors.affiliation: Cellular Technology Limited, Shaker Hts. 44122, OH, USA. karina@biomed.lu.lv.
authors.forename: Karina
authors.initials: K
authors.lastname: Silina
authors.affiliation: Development Translational Medicine, Biogen Idec, Cambridge 02142, MA, USA. Mackenzie.Welch@biogenidec.com.
authors.forename: Mackenzie
authors.initials: M
authors.lastname: Welch
authors.affiliation: Cellular Technology Limited, Shaker Hts. 44122, OH, USA. pvl@immunospot.com.
authors.forename: Paul V
authors.initials: PV
authors.lastname: Lehmann