Automated gamma-H2AX focus scoring method for human lymphocytes after ionizing radiation exposure

The purpose of this study was to develop a microscopy-based foci quantification protocol in human lymphocyte capable of supplying useful data for radiation sensitivity assays. Human peripheral blood was exposed to gamma-rays and isolated lymphocytes were stained with fluorochrome-coupled anti-gamma-H2AX (Histone 2AX phosphorylation of serine 139) antibodies. Microscopy slides were automatically acquired and the resulting images were subjected to 3 focus scoring methods: manual, semi-automated and fully automated. All scoring methods were sufficiently sensitive to detect an irradiation of at least 0.05 Gy with low variation between experiments. For higher doses, both automated approaches tend to detect fewer foci than manual scoring but still obtaining a linear correlation (lowest r2 > 0.971). Compared with manual scoring on the images, the automated approaches are at least 5 times faster with minimum operator intervention needed. We can conclude that our method is able to obtain the foci score of a blood sample in less than 6 h. In addition to the foci score the programs used perform several cell and foci measurements of potential biological importance.