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. 2024 Jun 6;29(2):164-175.
doi: 10.5603/rpor.99678. eCollection 2024.

Determination of dose-response calibration curves for gamma radiation using gamma-H2AX immunofluorescence based biodosimetry

Solomon Raj Jose  1 , Peace Balasingh Timothy  2 , J Suganthy  3 , Selvamani Backianathan  2 , Soosai Manickam Amirtham  4 , Sandya Rani  5 , Rabi Singh  2
Affiliations

Determination of dose-response calibration curves for gamma radiation using gamma-H2AX immunofluorescence based biodosimetry

Solomon Raj Jose et al. Rep Pract Oncol Radiother. .

Abstract

Background: Gamma-H2AX immunofluorescence assay has gained popularity as a DNA double strand break marker. In this work, we have investigated the potential use of gamma H2AX immunofluorescence assay as a biological dosimeter for estimation of dose in our institution.

Materials and methods: Seven healthy individuals were selected for the study and the blood samples collected from the first five individuals were irradiated to low doses (0-10 cGy) and high doses (50-500 cGy) in a telecobalt unit. All the samples were processed for gamma-H2AX immunofluorescence assay and the dose-response calibration curves for low and high doses were determined. In order to validate the determined dose-response calibration curves, the blood samples obtained from the sixth and seventh subjects were delivered a test dose of 7.5 cGy and 250 cGy. In addition, time and cost required to complete the assay were also reported.

Results: The goodness of fit (R2) values was found to be 0.9829 and 0.9766 for low and high dose-response calibration curves. The time required to perform the gamma-H2AX immunofluorescence assay was found to be 7 hours and 30 minutes and the estimated cost per sample was 5000 rupees (~ 60 USD).

Conclusion: Based on this study we conclude that the individual dose-response calibration curves determined with gamma-H2AX immunofluorescence assay for both low and high dose ranges of gamma radiation can be used for biological dosimetry. Further, the gamma-H2AX immunofluorescence assay can be used as a rapid cost-effective biodosimetric tool for institutions with an existing confocal microscope facility.

Keywords: molecular biology; radiation biology; radiation oncology.

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Conflict of interest statement

Conflicts of interest: The authors declare none.

Figures

Figure 1
Figure 1
Setup for blood sample irradiation in Theratron Equinox 80C
Figure 2
Figure 2
Segregation of peripheral blood mononuclear cells (PBMCs) after irradiation. A. Appearance of buffy coat consisting of PBMCs after gradient centrifuge method; B. PBMC pellet formation after the phosphate-buffered saline (PBS) wash
Figure 3
Figure 3
Radiation induced gamma-H2AX foci for low and high dose ionizing radiation. The green fluorescence (Alex fluor 488®) indicates the presence of gamma-H2AX at the site of DNA DSBs within the cell nuclei and the blue fluorescence (DAPI) represents the cell nuclei
Figure 4
Figure 4
Mean gamma-H2AX foci per cell for low dose-response calibration curve of five individuals. [(100 cells per dose point) × 5 individuals = 500 cells in total per dose point was plotted in this graph]. The error bars represent standard deviation. Linear model was fit and the mathematical expression for the same was computed
Figure 5
Figure 5
Mean gamma-H2AX foci per cell for high dose-response calibration curve of five individuals. [(100 cells per dose point) × 5 individuals = 500 cells in total per dose point was plotted in this graph]. The error bars represent standard deviation. Linear model was fit and the mathematical expression for the same was computed
Figure 6
Figure 6
Mean gamma-H2AX foci per cell for integrated dose-response curve of five individuals. [(100 cells per dose point) × 5 individuals = 500 cells in total per dose point was plotted in this graph]. The error bars represent standard deviation. Linear-quadratic model was fit and the mathematical expression for the same was computed
Figure 7
Figure 7
A. Irradiated cells without cytospin shows the dispersion of cells with lesser number of cell nuclei per microscopic view (cytofunnel chamber loaded with 20 μL of PBMCs); B. Evenly deposited monolayer of irradiated cells after cytospin in slide A [cytofunnel chamber loaded with 10 μL of peripheral blood mononuclear cells (PBMCs)]; C. Evenly deposited monolayer with excessive amount of overlap and background of irradiated cells after cytospin in slide B (cytofunnel chamber loaded with 20 μl of PBMCs)
Figure 8
Figure 8
A. Poor differentiation of gamma-H2AX foci in irradiated cells was observed after 1-hour primary and 1/2-hour secondary antibody incubation; B. Optimal differentiation of gamma-H2AX foci was observed in irradiated cells after 2-hour primary and 1-hour secondary antibody incubation
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