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Normal distribution of CD8+ T-cell-derived ELISPOT counts within replicates justifies the reliance on parametric statistics for identifying positive responses

Please always quote using this URN: urn:nbn:de:bvb:20-opus-149968
  • 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 wereAccurate 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.show moreshow less

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Metadaten
Author: Alexey Y. Karulin, Richard Caspell, Marcus Dittrich, Paul V. Lehmann
URN:urn:nbn:de:bvb:20-opus-149968
Document Type:Journal article
Faculties:Fakultät für Biologie / Theodor-Boveri-Institut für Biowissenschaften
Language:English
Parent Title (English):Cells
Year of Completion:2015
Volume:4
Issue:1
Pagenumber:96-111
Source:Cells 2015, 4(1), 96-111. DOI: 10.3390/cells4010096
DOI:https://doi.org/10.3390/cells4010096
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=PMC4381212
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Tag:ANOVA; ELISPOT; T-cells; normal distribution; statistics; t-Test
Release Date:2019/01/23
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International