Mean frequency and relative fluorescence intensity measurement of γ‐H2AX foci dose response in PBL exposed to γ‐irradiation: An inter‐ and intra‐laboratory comparison and its relevance for radiation triage

Abstract

Measurement of c-H2AX protein changes in the peripheral blood lymphocytes (PBL) of individuals exposed to ionizing radiation is a simple, sensitive, and rapid assay for radiation triage and early marker of dose estimation. The qualitative and quantitative measurements of the protein changes were examined using flow cytometry and microscopy. Whole blood and isolated lymphocytes were exposed in vitro between 0.1 and 5 Gy doses of 60 Co c-radiation at a dose rate of 1 Gy/min. Radiation induced c-H2AX foci frequency (n 5 3) and relative fluorescence intensity (n 5 7) in PBL was measured at 0.5 and 2 hrs postexposure. The observed dose response for c-H2AX foci frequency at both time points, for whole blood and isolated lymphocytes did not show any significant (P > 0.05) differences. However, when compared with c-H2AX foci frequency scored manually (microscopy), the semiautomated analysis (captured images) showed a better correlation (r 2 5 0.918) than that obtained with automated (Metafer) scoring (r 2 5 0.690). It is noteworthy to mention that, the c-H2AX foci frequency quantified using microscopy showed a dose dependent increase up to 2 Gy and the relative fluorescence intensity (RFI) measured with flow cytometry revealed an increase up to 5 Gy in the PBL exposed in vitro. Moreover, a better correlation was observed between the c-H2AX foci frequency obtained by manual scoring and RFI (r 2 5 0.910). Kinetic studies showed that the c-H2AX foci remain more or less unchanged up to 4 hrs and reduces gradually over 48 hrs of postexposure at 378C. Further, inter and intra-laboratory comparisons showed consistency in the scoring of c-H2AX foci frequency by manual and semiautomated scoring. The overall results suggest that measurement of c-H2AX (microscopy and flow cytometry) should be employed within 4 to 6 hrs for a reliable dosimetry either by sharing the work load between the laboratories or investing more manpower; however, triage can be possible even up to 48 hrs of postirradiation. V C 2015 International Society for Advancement of Cytometry

Enhancement of Cytotoxicity of Enediyne Compounds by Hyperthermia: Effects of Various Metal Complexes on Tumor Cells

“…Because accurate counting of γ-H2AX foci can be challenging due to the irregular size and morphology of foci, which may be attributed in part to cell cycle phase, we opted to also measure pan-nuclear γ-H2AX (fluorescence per nuclei, as an indication of the pan-nuclear γ-H2AX phosphorylation that occurred), especially since we noted that fluorescence was not totally confined to foci. Although the relationship between pan-nuclear γ-H2AX response and foci formation remains unclear, there is a strong correlation between the number of γ-H2AX foci per cell and total γ-H2AX fluorescence after inducing DSBs either by ionizing radiation or clastogenic drugs (20,21). Thus, other investigators have concluded that total γ-H2AX fluorescence can be used as a reliable and sensitive measurement of induction of DNA DSBs as well as DSB repair kinetics.…”

Sesamol ameliorates radiation induced DNA damage in hematopoietic system of whole body γ‐irradiated mice

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Comparison of inexperienced operators and experts in γH2A.X and 53BP1 foci assay for high-throughput biodosimetry approaches in a mass casualty incident

“…Nevertheless, in case of a large-scale accident, a huge number of samples have to be processed and evaluated. Immense man power is needed even for a highthroughput method like cH2A.X (Venkateswarlu et al 2015).…”

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