High Reproducibility of ELISPOT Counts from Nine Different Laboratories

Cells

Srividya Sundararaman⁸  Alexey Y. Karulin⁸  Tameem Ansari⁸  Nadine BenHamouda¹⁰  Judith Gottwein²  Sreenivas Laxmanan⁵  Steven M. Levine⁶  John T. Loffredo⁴  Stephanie McArdle³  Christine Neudoerfl⁷  Diana Roen⁹  Karina Silina^1,8  Mackenzie Welch⁵  Paul V. Lehmann⁸ 

[1] Latvian Biomedical Research and Study Center, Riga LV1067, Latvia; E-Mail:;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; E-Mail:;Nottingham Trent University, Nottingham NG118NS, UK; E-Mail:;Immuno-Sciences Biology Drug Discovery, Bristol-Myers Squibb, Princeton 08543, NJ, USA; E-Mail:;Development Translational Medicine, Biogen Idec, Cambridge 02142, MA, USA; E-Mails:;Immuno-Virology Drug Discovery, Bristol-Myers Squibb, Wallingford 06492, CT, USA; E-Mail:;Medizinische Hochschule Hannover 30625, Germany; E-Mail:;Cellular Technology Limited, Shaker Hts. 44122, OH, USA; E-Mails:;Pharmasan Labs, Osceola 54020, WI, USA; E-Mail:;Hôpital Européen Georges Pompidou, Paris 75015, France; E-Mail:

关键词: ELISPOT;    Smart Count™;    Log Normal distribution;    harmonization;   

DOI  :  10.3390/cells4010021

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.