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The Capital Region of Denmark - a part of Copenhagen University Hospital
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High Reproducibility of ELISPOT Counts from Nine Different Laboratories

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  • 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
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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.

Original languageEnglish
JournalCells
Volume4
Issue number1
Pages (from-to)21-39
Number of pages19
DOIs
Publication statusPublished - Jan 2015

ID: 44894349