γH2AX-based Fluorescence Immunoassay Visualization And Quantification Of DNA Damage After CT In Human Peripheral Blood Mononuclear Cells

Part one: An experimental study in vitro onγH2AX focus assay to quantify the DNA damage induced in peripheral blood mononuclear cells after CTPurpose: To prospectively determine ifγH2AX (phosphorylated form of H2AX histone variant)-based visualization and quantification of DNA damage induced in peripheral blood mononuclear cells (PBMCs) can be used to estimate the radiation dose received after computed tomography by in vitro study, comprehend the correlation between the dose and the inducedγH2AX foci initially.Materials and Methods: Sixty-seven people were recruited prospectively; After written informed patient consent were obtained, blood samples were taken from the antecubital vein of all volunteers, and collected into vacuum tubes containing lithium heparin. The blood samples (in tubes) from each volunteer were exposed to different radiation doses with GE LightSpeed VCT scanner in vitro. 5 minutes later, blood samples were isolated, fixed, and stained forγH2AX expression. TheγH2AX focus yields were determined with fluorescence microscopy, and the radiation doses delivered during CT as dose-length product––DLP. Data were analyzed by using Kruskal–Wallis test, independent–samples t test, linear correlation and regression method. Results: We found a significantly higher number ofγH2AX foci than control check in blood samples 5 minutes after the last exposure to ionizing radiation at CT(dose threshold exceeds 199.64 mGy?cm), and the focus increase after CT depended linearly on the DLP. The regression equation model is: Y=0.1917X+0.1248, there were statistical difference(P=0.000 R2=0.906).Conclusion: The DNA damage of PBMCs after CT in vitro is always followed by the formation ofγH2AX, its yields and CT radiation doses are thought to be high correlates.Part two: Preliminary clinical study of theγH2AX foci levels after in vivo and in vitro CT irradiation in peripheral blood mononuclear cellsPurpose: To determine whetherγH2AX yield analysis can be used as a sensitive biologic dosimeter of human exposure to low-level radiation through comparing H2AX phosphorylation in the blood cells between in vivo and in vitro, and explore its prospect.Materials and Methods: Fifteen patients scheduled to undergo diagnostic CT were recruited and prospectively entered into the study. After written informed patient consent were obtained, blood samples (4ml per patient) were taken from the antecubital vein of all patients before they underwent CT, and collected into vacuum tubes containing lithium heparin(2ml per tube). The blood samples split into two groups: control check (ck) and in vitro. The ck group was incubated at 37°C. While the in vitro group was fixed on patient's body surface of central exposure field during the CT scanning, then taken out and incubated at 37°C. 5 minutes later, another blood samples (2ml per patient) were taken from the antecubital vein of all patients named as in vivo group. All of the blood samples were isolated, fixed, and stained forγH2AX expression. TheγH2AX focus yields were determined with fluorescence microscopy, making a comparative analysis. Data were analyzed by using paired–samples t test, linear correlation and regression method.Results: TheγH2AX focus yields increase 5 minutes after the last exposure to ionizing radiation at CT depended linearly on the DLP in both in vivo and in vitro groups. Furthermore, the PBMCs exposed in vitro represent a higher yields ofγH2AX focus than in vitro on the same condition of DLP(P=0.000<0.05).Conclusion:γH2AX focus assay's peculiarly sensitivity and its demonstrated applicability to CT examinations allow the investigation of people who are exposed to well defined radiation doses. It is a novel quantitative biomarker. Thus, the assessment ofγH2AX foci can serve as a biologically relevant biomarker for exposure and provides an exciting perspective to low levels of ionising radiation.