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.
Highlights
The T lymphocyte system responds to encounters with viruses such as HCMV via clonal expansion of virus-specific T cells, generating effector and memory cells that can protect the host
Since the spots are of different sizes, determining the cut-off value for the smallest and largest spot size to be counted poses a challenge even to ELISPOT experienced investigators, and this judgment call will inherently be subjective
Feature of the ImmunoSpot® Software, which based on parametric statistics, determines the upper and lower spot-size gates with 99% confidence
Summary
The T lymphocyte system responds to encounters with viruses such as HCMV via clonal expansion of virus-specific T cells, generating effector and memory cells that can protect the host. The extent of this clonal expansion defines the magnitude of T cell immunity in an individual and is reflected by the frequencies of virus specific T cells in his/her blood. Multi center immunoassay proficiency panels reported significant discrepancies in determining the numbers/frequencies of antigen-specific T cells when different laboratories tested the same number of Peripheral Blood Mononuclear Cells (PBMC) from the same donors [2]. The authors of this report concluded: “The high degree in variability
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