FLOW CYTOMETRY IN BIOLOGICAL DOSIMETRY

Abstract

It is generally accepted that neutrons are more effective at causing cell damage when compared to X- or gamma radiation. This study, however, indicates that for radiation-induced apoptosis this is not the case. Previously, most RBE (Relative Biological Effectiveness) values for neutrons have been calculated based on chromosome aberrations. In this study the RBE of human lymphocytes after exposure to 280keV neutrons and l37Cs gamma radiation was measured. In the work presented here, the RBE values were calculated based on radiation-induced apoptosis as the biological endpoint and were close to unity. Different markers of apoptosis were compared using flow cytometry assays and microscopy (comet assay). The apoptosis assays measured by flow cytometry were: • Annexin V conjugated with fluorescein isothiocyanate (FITC) binds to phosphatidylserine exposed on outer leaflet of the cell membrane in apoptotic cells. • The DNA specific viability dye 7-amino-actinomycin D (7- AAD), binds to the DNA and is excluded from living cells (due to an intact membrane). The membrane-permeable lipophilic cationic fluorochrome. 3,3'-dihexyloxacarbocyanine iodide (DiOCf)), accumulates in the mitochondria ofliving cells. v • A DNA intercalating dye propidium iodide (PI), binds to the DNA ofdead cells after membrane degradation. • In addition, FITC conjugated active caspase-3 antibody; caspase-3 is a protein which is activated in apoptotic cells. Data for a dose response curve were collected for two radiation qualities (280keV neutrons and l37Cs gamma radiation) over a range of acute doses (0, 0.25, 0.5, 1, 2 and 5 Gy). Kinetic studies were done to compare the time course of the apoptosis process by comparing the induction after exposure to either 280keV neutrons and 137Cs gamma radiation at different timepoints over 96 hours. Radiation induced apoptosis increased with dose, and apoptosis levels peaked between 48 hours and 72 hours depending on the assay which was related to the stage of apoptosis at the time of measurement. For five independent experiments, the two radiation qualities induced similar levels of apoptosis per unit dose. The purpose of this research was to develop and test different assays to measure apoptosis in human lymphocytes. The specific aim was to compare different radiation qualities and assign an RBE value for low energy fast-neutrons. The overall objective was to determine the sensitivity and feasibility ofthe different techniques for emergency, accidental, or clinical biological dosimetry. The caspase-3 flow cytometry assay had very good sensitivity at low doses (less than 0.25 Gy). There was a statistically significant difference between the level of apoptosis induced in unirradiated samples and lymphocytes which received 0.25 Gy of neutron or gamma radiation. However, the caspase-3 assay, when compared to other flow cytometry assays, was more time consuming and labour intensive. For speed and simplicity, the annexin V-FITC and 7-AAD assay was preferable. Both Annexin-V and 7-AAD had good dose response at low doses (0 - 0.25 Gy) but was not as statistically significant as caspase-3 at low doses. Economically, the DiOCg assay was most feasible; however DiOCe did not seem very sensitive and had the largest interdonor variation. For sensitivity, simplicity and labour requirements the Annexin V-FITC and 7-AAD assay was the most economical. Overall the comet assay, while technologically being the simplest, was the most labour intensive, as each cell must be scored visually and therefore the most prone to human error.