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Determining exposure dose for ionizing radiation using dicentric chromosomes from chromosome soup
Yogev Yehezkel1; yogev6235@gmail.com
Prof. Ariela Burg1, Dr. Inna Levitsky1, Dr. Raphael Gonen2 1Sami Shamoon College of Engineering, Beer-Sheva 2Nuclear Research Center Negev
Over the last decade, radiology and nuclear technologies have developed significantly, however, along with their benefits, we now face increasing risks of radiological and nuclear incidentsRapid identification and measurement of exposure to ionizing radiation is crucial during mass casualty radiological incidents. By sorting casualties based on their levels of radiation dosage, essential medical treatment can be given in a timely manner, also preventing further medical complications. As such, there is need of an efficient and reliable tool that measures the exposure dose of ionizing radiation.
This research is based on the dicentric chromosome assay )DCA( method, which is considered a gold standard for biological dosimetry. It involves counting the number of dicentric chromosomes and using a calibration curve to determine the radiation dose. Quantification is carried out using cells in the metaphase stage, obtained after performing several key steps: collecting a peripheral blood sample; stopping lymphocytic cell divisions; synchronizing the cells with the start of mitosis; and stopping cell divisions in the metaphase stage. The chromosomes are stained and examined under a microscope using common methods. The DCA involves growing lymphocyte cells and sorting metaphases, making it a time-consuming process. The main objective of this research is the development of a convenient, inexpensive, and time-saving dicentric-like assay for the easy determination of exposure to ionizing radiation. Additionally, the construction of a calibration curve shows the correlation between the number of dicentric chromosomes counted and different doses of radiation. During the initial stage of the study, various lysis solutions were examined for use in the extraction of chromosomes from cellular contents and to obtain a chromosome soup. Next, chromosome spreads were prepared on slides by the adsorption of chromosomes released from metaphasic cells using the optimal lysis solution selected.
For adsorption and optimal drying, two methods were tested—“overnight adsorption“ and “chromosome spin.“ Moreover, a manual method for quantifying dicentric chromosomes was developed; this new technique relies on the use of an epifluorescence microscope and a glass cover with a mesh pattern, allowing for a continuous and organized count. To reduce the time required for photographing and quantifying chromosomes manually, a semi-automatic confocal microscope was used )with manual definition of the area to be photographed plus automatic photo acquisition(. Once there were appropriate conditions for preparing a chromosome spread and the counting methods had been established, radiation experiments were conducted on blood samples from different donors. Gamma radiation from a Co-60 source was used, with a radiation dose rate of 0.27 Gy/min. Lymphocyte cells were cultured from the blood samples and metaphase synchronization was conducted. The chromosomes were extracted to prepare chromosome spreads and photographed using the confocal microscope.
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