Development of a high-throughput γ-H2AX assay based on imaging flow cytometry

Background: Measurement of gamma-H2AX foci levels in cells provides a sensitive and reliable method for quantitation of the radiation-induced DNA damage response. The objective of the present study was to develop a rapid, high-throughput gamma-H2AX assay based on imaging flow cytometry (IFC) using the ImageStream (R)(X) Mk II (ISX) platform to evaluate DNA double strand break (DSB) repair kinetics in human peripheral blood cells after exposure to ionizing irradiation. Methods: The gamma-H2AX protocol was developed and optimized for small volumes (100 mu L) of human blood in Matrix (TM) 96-tube format. Blood cell lymphocytes were identified and captured by ISX INSPIRE (TM) software and analyzed by Data Exploration and Analysis Software. Results: Dose- and time-dependent gamma-H2AX levels corresponding to radiation exposure were measured at various time points over 24 h using the IFC system. gamma-H2AX fluorescence intensity at 1 h after exposure, increased linearly with increasing radiation dose (R-2 = 0.98) for the four human donors tested, whereas the dose response for the mean number of gamma-H2AX foci/cell was not as robust (R-2 = 0.81). Radiation-induced gamma-H2AX levels rapidly increased within 30 min and reached a maximum by similar to 1 h, after which time there was fast decline by 6 h, followed by a much slower rate of disappearance up to 24 h. A mathematical approach for quantifying DNA repair kinetics using the rate of gamma-H2AX decay (decay constant, K-dec), and yield of residual unrepaired breaks (F-res) demonstrated differences in individual repair capacity between the healthy donors. Conclusions: The results indicate that the IFC-based gamma-H2AX protocol may provide a practical and high-throughput platform for measurements of individual global DNA DSB repair capacity which can facilitate precision medicine by predicting individual radiosensitivity and risk of developing adverse effects related to radiotherapy treatment.