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Cells 2015, 4(1), 21-39; doi:10.3390/cells4010021

High Reproducibility of ELISPOT Counts from Nine Different Laboratories

1
Cellular Technology Limited, Shaker Hts. 44122, OH, USA
2
Européen Georges Pompidou, Paris 75015, France
3
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
4
Development Translational Medicine, Biogen Idec, Cambridge 02142, MA, USA
5
Immuno-Virology Drug Discovery, Bristol-Myers Squibb, Wallingford 06492, CT, USA
6
Immuno-Sciences Biology Drug Discovery, Bristol-Myers Squibb, Princeton 08543, NJ, USA
7
Nottingham Trent University, Nottingham NG118NS, UK
8
Medizinische Hochschule Hannover 30625, Germany
9
Pharmasan Labs, Osceola 54020, WI, USA
10
Latvian Biomedical Research and Study Center, Riga LV1067, Latvia
*
Author to whom correspondence should be addressed.
Academic Editor: Alexander E. Kalyuzhny
Received: 24 October 2014 / Accepted: 26 November 2014 / Published: 9 January 2015
(This article belongs to the Special Issue ELISPOT Research)
View Full-Text   |   Download PDF [1830 KB, uploaded 9 January 2015]   |  

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. View Full-Text
Keywords: ELISPOT; Smart Count™; Log Normal distribution; harmonization ELISPOT; Smart Count™; Log Normal distribution; harmonization
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Sundararaman, S.; Karulin, A.Y.; Ansari, T.; BenHamouda, N.; Gottwein, J.; Laxmanan, S.; Levine, S.M.; Loffredo, J.T.; McArdle, S.; Neudoerfl, C.; Roen, D.; Silina, K.; Welch, M.; Lehmann, P.V. High Reproducibility of ELISPOT Counts from Nine Different Laboratories. Cells 2015, 4, 21-39.

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