Establishment and multiparametric-cytogenetic validation of 60Co-gamma-ray induced, phospho-gamma-H2AX calibration curve for rapid biodosimetry and triage management during radiological emergencies.

Exposure to ionizing radiation is unavoidable to our modern developing society as its applications are widespread and increasing with societal development. The exposures may be planned as in medical applications or may be unplanned as in occupational work and radiological emergencies. Dose quantification of planned and unplanned exposures is essential to make crucial decisions for management of such exposures. This study aims to establish ex-vivo dose-response curve for 60Co-gamma-ray induced gamma-H2AX-foci by immunofluorescence using microscopy and flowcytometry with human lymphocytes. This technique has the potential to serve as a rapid tool for dose estimation and triage application during small to large scale radiological emergencies and clinical exposures. Response curves were generated for the dose range 0-4 Gy (at 1, 2, 4, 8, 16, 24, 48, 72 and 96 h of incubation after irradiation) with microscopy and 0-8 Gy (at 2, 4, 8, 16 and 24 h of incubation after irradiation) with flow cytometry. These curves can be applied for dose reconstruction when post exposure sampling is delayed up to 96 h. In order to evaluate Minimum Detection Limit (MDL) of the assay, variation of background frequency of gamma-H2AX-foci was measured in 12 volunteers. To understand the application window of the assay, gamma-H2AX foci decay kinetics has been studied up to 96 h with microscopy and response curves were generated from 1 to 96 hours post exposure. Gamma-H2AX fluorescence intensity decay kinetics was also studied up to 96 h with flow cytometry and response curves were generated from 2 to 24 hours post irradiation. Established curves were validated with dose blinded samples and also compared with standard cytogenetic assays. An inter-comparison of dose estimates was made among gamma-H2AX assay, dicentric aberrations and reciprocal translocations for application window in various dose ranges and time of blood collection after exposures.