γ-H2AX As An Indicator For The Feasibility Study Of Ionizing Radiation Dose Evaluation By Lfow Cytometry

Objective: To explore the possibility for the early stage and rapid assessing ionizingradiation damage as a biological dosimeter, a rapid and high-throughput approach wasestablished for detecting the expression levels of γ-H2AX in peripheral bloodlymphocytes by flow cytometry. This approach studied the effect of ionizing radiationon γ-H2AX content in lymphocytes, and was used for establishing the time-effect curveand dose-response curves.Methods: The peripheral blood from health donors and the geometric meanfluorescence intensity values of γ-H2AX in lymphocytes labeled withanti-γ-H2AX-antibody by using indirect immunofluorescence were analyzed by flowcytometry, and the relative fluorescence intensity was calculated. We optimized theexperimental condition and compared the intracellular effect of the geometric meanfluorescence intensity values and the relative fluorescence intensity among differentfixed liquid concentrations and fixed times, and different combination time andconcentration to anti-γ-H2AX-antibody, and different concentration to IgG-FITCantibody. The geometric mean fluorescence intensity values and the relativefluorescence intensity of γ-H2AX in intracellular effect were compared between thedifferent instruments type and parameters setting. The peripheral blood were irradiatedin vitro by60Co γ-rays, and we studied the effect of ionizing radiation on γ-H2AXcontent in lymphocytes, and the relative fluorescence intensity of γ-H2AX as anindicator was used for establishing the time-effect curve and dose-response curves andanalyzing the healthy human background and the difference between intra-individualsand inter-individuals. Results: Through the comparison of experimental results which observed the cellmorphology, there can be found2%PFA concentration are better and fixed30minwhich are similar to fixed60min in cell morphology was better than fixed15min.There was no significant difference anti-γ-H2AX-antibody binding between30min and3h. There was significant difference anti-γ-H2AX-antibody concentration between1:200and1:400, but no significant difference between1:200and1:100. There was nosignificant difference IgG-FITC antibody concentration between1:200and1:100. Thegeometric mean fluorescence intensity was obviously affected by different instrumentstype and parameters setting after irradiation with various doses, but the relativefluorescence intensity of γ-H2AX in lymphocytes did not significantly correlate withthe different instruments type and parameters setting after irradiation with various doses.The experimental results shown that the healthy human average background values was2.10±0.54, the maximum is3.13and the minimum is1.07. The results also shown thatthere was no significant difference background between male and female. The relativefluorescence intensity which was little difference among the different doses(0,2,6Gy),respectively. The time-effect curve shown that the signal increased after irradiation andreached the maximum at30min, then the signal would be gradually lost after30min.The dose-response curves shown that the expression levels of γ-H2AX in lymphocytesin a dose-dependent that increased as dose increases. And the expression levels ofγ-H2AX in lymphocytes gradually decreased with time after irradiation. The averagerelative fluorescence intensity were analyzed by irradiated after30min which from twodifferent volunteers blood, and the average relative fluorescence intensity was be usedfor establishing the dose-response curves linear regression equation wasY=0.799D+3.443(R2=0.897). There were small difference between the twovolunteers linear regression equations which was Y=0.779D+2.760(R2=0.865) andY=0.636D+3.126(R2=0.969), respectively. The average relative fluorescence intensity were analyzed by irradiated after60min which from one volunteer blood in twoindependent experimental, and the average relative fluorescence intensity was be usedfor establishing the dose-response curves linear regression equation was Y=0.903D+2.993(R2=0.910). There were small difference between the two independentexperimental which the linear regression equation was Y=0.901D+3.724(R2=0.910)and Y=1.312D+2.645(R2=0.896), respectively. The average relative fluorescenceintensity were analyzed by irradiated after60min which from two different volunteersblood, and the average relative fluorescence intensity was be used for establishing thedose-response curves linear regression equation wasY=0.743D+3.396(R2=0.843). There were small difference between the twovolunteers linear regression equations which was Y=0.935D+3.075(R2=0.900) andY=0.932D+4.334(R2=0.866), respectively. The dose-response curves of30min and60min after irradiated from different volunteers shown, the relative fluorescenceintensity value which30min after irradiated is higher than the relative fluorescenceintensity value which30min after irradiated and this result maintained the goodconsistency to the time-effect curve.Conclusion: To establish a analysis method of γ-H2AX expression levels in peripheralblood lymphocytes by flow cytometry. The relative fluorescence intensity as theindicator analyzed by flow cytometry might improve the stability and repeatability, sothis approach would be a new method to assess the radiation biodosimetry. The resultsshown healthy human background value is chose, and the γ-H2AX expression levelswere no significant difference between male and female. The results verify the smalldifferences between inter-individuals and different doses, and then shown a highstability and repeatability with the relative fluorescence intensity of γ-H2AX as theindicator. so this new approach would be a potential biological dosimeter.