High Reproducibility of ELISPOT Counts from Nine Different Laboratories
Srividya Sundararaman,
Alexey Y. Karulin,
Tameem Ansari,
Nadine BenHamouda,
Judith Gottwein,
Sreenivas Laxmanan,
Steven M. Levine,
John T. Loffredo,
Stephanie McArdle,
Christine Neudoerfl,
Diana Roen,
Karina Silina,
Mackenzie Welch,
Paul V. Lehmann
Affiliations
Srividya Sundararaman
Cellular Technology Limited, Shaker Hts. 44122, OH, USA
Alexey Y. Karulin
Cellular Technology Limited, Shaker Hts. 44122, OH, USA
Tameem Ansari
Cellular Technology Limited, Shaker Hts. 44122, OH, USA
Nadine BenHamouda
Européen Georges Pompidou, Paris 75015, France
Judith Gottwein
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
Sreenivas Laxmanan
Development Translational Medicine, Biogen Idec, Cambridge 02142, MA, USA
Steven M. Levine
Immuno-Virology Drug Discovery, Bristol-Myers Squibb, Wallingford 06492, CT, USA
John T. Loffredo
Immuno-Sciences Biology Drug Discovery, Bristol-Myers Squibb, Princeton 08543, NJ, USA
Stephanie McArdle
Nottingham Trent University, Nottingham NG118NS, UK
Christine Neudoerfl
Medizinische Hochschule Hannover 30625, Germany
Diana Roen
Pharmasan Labs, Osceola 54020, WI, USA
Karina Silina
Latvian Biomedical Research and Study Center, Riga LV1067, Latvia
Mackenzie Welch
Development Translational Medicine, Biogen Idec, Cambridge 02142, MA, USA
Paul V. Lehmann
Cellular Technology Limited, Shaker Hts. 44122, OH, USA
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.
