TY  - JOUR
A1  - Karulin, Alexey Y.
A1  - Caspell, Richard
A1  - Dittrich, Marcus
A1  - Lehmann, Paul V.
T1  - Normal distribution of CD8+ T-cell-derived ELISPOT counts within replicates justifies the reliance on parametric statistics for identifying positive responses
JF  - Cells
N2  - 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.
KW  - ELISPOT
KW  - statistics
KW  - t-Test
KW  - ANOVA
KW  - T-cells
KW  - normal distribution
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149968
VL  - 4
IS  - 1
ER  - 
TY  - JOUR
A1  - Wunsch, Marie
A1  - Caspell, Richard
A1  - Kuerten, Stefanie
A1  - Lehmann, Paul V.
A1  - Sundararaman, Srividya
T1  - Serial measurements of apoptotic cell numbers provide better acceptance criterion for PBMC quality than a single measurement prior to the T cell assay
JF  - Cells
N2  - As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.
KW  - T cell assay
KW  - apoptosis
KW  - acceptance
KW  - viability
KW  - ELISPOT
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-150213
VL  - 4
IS  - 1
ER  -