Investigation On the Use of Comet Assay for the Measurement of High Dose Radiation
Presentations
PO-GePV-T-430 (Sunday, 7/25/2021) [Eastern Time (GMT-4)]
Purpose: To standardize the comet assay as a biodosimetric tool for estimating high levels of radiation dose.
Methods: 10 ml blood from two healthy individuals were collected using venepuncture method in EDTA containers under aseptic conditions. The blood samples were irradiated for known doses of 15, 25 and 35 Gy in Co⁶⁰ teletherapy unit. After irradiation, the samples were gently overlaid on histopaque 1077 and were centrifuged. The buffy coat of lymphocytes were mixed with 1% low melting point agarose. The mixture was gently smeared over a frosted slide and allowed to dry for 15 minutes. The slides were immersed into lysis solution for 12 hours with pH adjusted to 9.5. After lysis, the slides were washed with neutral buffer and were subjected to neutral electrophoresis using TAE buffer for 30 minutes at 25 V. Fluorescence microscope was used to capture images which were stained using propidium iodide (2.5 μg/ml). Comet assay parameters such as tail DNA % and OTM (Olive Tail Moment) were the intensity values were measured using Fiji software.
Results: The arbitrary units of tail DNA % were 38.36 ±3.40, 53.42 ±1.95, 60.34 ±4.24 and OTM values were 11.41±0.8, 17.14±0.62, 29.35 ±2.05 for 15, 25 and 35 Gy respectively. For each dose point the control values were subtracted in order to avoid inter individual effects. The dose (Gy) vs tail DNA % and OTM showed a correlation with R² value of 0.9563 and 0.9583.
Conclusion: Comet assay allows the estimation of DNA damage in single cell level and can be used as a potential biodosimeter to estimate high levels of radiation dose above 15 Gy thereby overcoming the demerit of gold standard dicentric chromosomal assay which can only measure doses up to 6 Gy.
Funding Support, Disclosures, and Conflict of Interest: 1. This work was supported by Internal fluid research grant from Christian Medical College Vellore. 2. This work is a part of Ph.D. Thesis of The Tamil Nadu Dr. M.G.R. Medical University, Chennai, India.
ePosters
Keywords
Not Applicable / None Entered.
Taxonomy
Not Applicable / None Entered.
Contact Email