Normal Distribution of CD8+ T-Cell-Derived ELISPOT Counts within Replicates Justifies the Reliance on Parametric Statistics for Identifying Positive Responses

Cells
A Y KarulinPaul V Lehmann

Abstract

Accurate assessment of positive ELISPOT responses for low frequencies of antigen-specific T-cells is controversial. In particular, it is still unknown whether ELISPOT counts within replicate wells follow a theoretical distribution function, and thus whether high power parametric statistics can be used to discriminate between positive and negative wells. We studied experimental distributions of spot counts for up to 120 replicate wells of IFN-γ production by CD8+ T-cell responding to EBV LMP2A (426 - 434) peptide in human PBMC. The cells were tested in serial dilutions covering a wide range of average spot counts per condition, from just a few to hundreds of spots per well. Statistical analysis of the data using diagnostic Q-Q plots and the Shapiro-Wilk normality test showed that in the entire dynamic range of ELISPOT spot counts within replicate wells followed a normal distribution. This result implies that the Student t-Test and ANOVA are suited to identify positive responses. We also show experimentally that borderline responses can be reliably detected by involving more replicate wells, plating higher numbers of PBMC, addition of IL-7, or a combination of these. Furthermore, we have experimentally verified that the number of...Continue Reading

References

Jul 1, 1990·Statistics in Medicine·Y Hochberg, Y Benjamini
Mar 31, 1998·Journal of Immunological Methods·M McCutcheonE G Spack
Jun 9, 2004·Journal of Immunological Methods·Michael G HudgensM Juliana McElrath
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Oct 1, 2011·Methods in Molecular Biology·Wenji Zhang, Paul V Lehmann
Oct 1, 2011·Methods in Molecular Biology·Marcus Dittrich, Paul V Lehmann
Oct 1, 2011·Methods in Molecular Biology·Zoe MoodieCedrik M Britten

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Citations

Apr 25, 2016·Cancer Immunology, Immunotherapy : CII·Ioana MoldovanPaul V Lehmann
Jan 15, 2015·Cells·Srividya SundararamanPaul V Lehmann
Sep 13, 2017·Cells·Andrea DuechtingPaul V Lehmann
Oct 24, 2018·The Journal of Clinical Investigation·Klaudia KurandaFederico Mingozzi
Jun 16, 2021·Frontiers in Immunology·Alexander A LehmannPaul V Lehmann

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Methods Mentioned

BETA
fluorescence microscopy

Software Mentioned

XLSTAT
ImmunoSpot®
SPSS
ELISPOT

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