Adapting the c-H2AX Assay for Automated Processing in Human Lymphocytes. 1. Technological Aspects

The immunofluorescence-based detection of c-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this wellestablished biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated highthroughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure c-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the c-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of cH2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purposebuilt robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of c-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in c-H2AX fluorescence in peripheral blood samples irradiated ex vivo with c rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify c-H2AX total fluorescence in irradiated peripheral lymphocytes. g 2011 by Radiation Research Society