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Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays
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Cells 2015, 4(1), 96-111;

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

Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA
Biocenter, University Wuerzburg, Am Hubland, Würzburg 97074, Germany
Author to whom correspondence should be addressed.
Academic Editor: Alexander E. Kalyuzhny
Received: 23 October 2014 / Revised: 12 January 2015 / Accepted: 16 January 2015 / Published: 2 March 2015
(This article belongs to the Special Issue ELISPOT Research)
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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 replicates needed for detection of weak responses can be calculated using parametric statistics. View Full-Text
Keywords: ELISPOT; statistics; t-Test; ANOVA; normal distribution; T-cells ELISPOT; statistics; t-Test; ANOVA; normal distribution; T-cells

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Karulin, A.Y.; Caspell, R.; Dittrich, M.; Lehmann, P.V. Normal Distribution of CD8+ T-Cell-Derived ELISPOT Counts within Replicates Justifies the Reliance on Parametric Statistics for Identifying Positive Responses. Cells 2015, 4, 96-111.

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