Abstract B57: NK cell-mediated cytotoxicity detection can be miniaturized by direct imaging in assay wells using a Terasaki plate format using only 100,000 effector cells (PBMC)
Authors: Sundararaman Srividya , Karacsony Kinga , Roen Diana , Ghosh Jaya , Lehmann Paul V. 
Published: October 2015

Abstract
Introduction: Natural Killer (NK) Cell - and Antibody Dependent (ADCC) Cell - Cytotoxicity has traditionally been assessed by the release of radioactive Chromium from target cells following lysis. This assay is laborious, and requires substantial quantities of patient blood to detect minor changes in cell lysis due to the inherent background noise (spontaneous release). We have developed an assay that can visualize individual target cells to detect cytolytic activity without involving radioactivity, via high-throughput imaging in microtiter well format. We also developed a miniaturized version of this assay using Terasaki plates to measure NK activity with one tenth of the blood needed for a classic Chromium Release assay, and with less labor.
Methods: The assay we developed, Target cell Visualization Assay (TVA™) images individual fluorescence-labeled target cells. Labelled K562 tumor cells were used as targets, and peripheral blood mononuclear cells (PBMC) as effector cells. In an assay setup similar to Chromium Release Assays (CRA), constant number of labelled target cells was incubated with serially diluted effector cells. Four hours later, the cells were transferred to flat bottom microtiter plates and the number of viable tumor cells was quantitated using a plate reader; CTL ImmunoSpot® Analyzer, S6ULT-00-9000. For the miniaturized version, one tenth of effector and target cells were plated in a Terasaki plate format and imaged directly in the assay wells.
Results: Only viable target cells retain the fluorescent dye; it is lost upon cell death. When effector and target cells are mixed in various ratios, the % of target cell lysis is inversely proportional to the number of effector cells in the well. The TVA™ and CRA in a 96-well format provided equivalent results. However, the TVA™ is a non-radioactive system and can be performed with much less labor. The TVA™ assay exhibited high intermediate precision as well as inter-assay repeatability. The assay results obtained in Terasaki plates, miniaturizing the assay tenfold, paralleled those of the 96-well format.
Conclusion: The miniaturized TVA™ uses only 100,000 effector cells for eight serial dilutions, for Effector:Target ratios starting from 100:1. We have demonstrated the feasibility of detecting and assessing NK function in a non-radioactive, high-throughput capable system using a fraction of the blood/effector cells as is required by traditional CRA. This is of particular value when access to PBMC is limited, such as pediatric, geriatric and immune deficient populations. The assay readout and analysis is automated by the CTL ImmunoSpot® software providing audit trails for each test condition.
Citation Format: Srividya Sundararaman, Kinga Karacsony, Diana Roen, Jaya Ghosh, Paul V. Lehmann. NK cell-mediated cytotoxicity detection can be miniaturized by direct imaging in assay wells using a Terasaki plate format using only 100,000 effector cells (PBMC). [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B57.