@article{KarulinKaracsonyZhangetal.2015,
  author    = {Karulin, Alexey Y. and Karacsony, Kinga and Zhang, Wenji and Targoni, Oleg S. and Moldova, Ioana and Dittrich, Marcus and Sundararaman, Srividya and Lehmann, Paul V.},
  title     = {ELISPOTs produced by CD8 and CD4 cells follow Log Normal size distribution permitting objective counting},
  series = {Cells},
  volume    = {4},
  journal   = {Cells},
  number    = {1},
  doi       = {10.3390/cells4010056},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149648},
  pages     = {56-70},
  year      = {2015},
  abstract  = {Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7\% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.},
  language  = {en}
}
@article{WunschCaspellKuertenetal.2015,
  author    = {Wunsch, Marie and Caspell, Richard and Kuerten, Stefanie and Lehmann, Paul V. and Sundararaman, Srividya},
  title     = {Serial measurements of apoptotic cell numbers provide better acceptance criterion for PBMC quality than a single measurement prior to the T cell assay},
  series = {Cells},
  volume    = {4},
  journal   = {Cells},
  number    = {1},
  doi       = {10.3390/cells4010040},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150213},
  pages     = {40-55},
  year      = {2015},
  abstract  = {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.},
  language  = {en}
}