γH2AX foci as a measure of DNA damage: A computational approach to automatic analysis

Abstract

The γH2AX focus assay represents a fast and sensitive approach for detection of one of the critical types of DNA damage – double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable approach due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.

Evaluation of Severe Combined Immunodeficiency and Combined Immunodeficiency Pediatric Patients on the Basis of Cellular Radiosensitivity

“…6e8, 40,44,45 Recently, we presented several analytical tools for improving the statistical and computational approaches to applying the assay for differential diagnostics in RS and non-RS biodosimetry in tissues and in the primary fibroblast skin culture model. 45,46 Here, we further refine the mathematical criteria of cellular RS. To describe the kinetics of g-H2AX foci decline, we fitted the experimental data (foci counts per nucleus; at 0, 0.5, 2, 6, 24, 48, and 72 hours after irradiation) to an empirical model that assumed the presence of two repair components, slow and fast.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…In addition, a 100× objective is required for imaging, which reduces the speed of the instrument, rendering high throughput as low throughput. Therefore, efforts have been made to improve this method, based on microscopy techniques such as imaging modalities in cell culture and in tissues,162 and computer‐assisted approaches 163. The computational approaches are supported by image analysis software such as NIH Image and its Windows counterpart ScionImage, ImageJ, HistoLab, AutoQuantX, or Image Pro, among others, which rely on different computational algorithms for foci identification or the calculation of quantitative foci parameters 164, 165, 166.…”

Suitability of the γ-H2AX Assay for Human Radiation Biodosimetry

“…While fluorescence microscope in combination with a digital camera is standard equipment in many laboratories, the availability of specialized software, especially freeware, for the analysis of γ-H2AX foci is increasing (Carpenter, 2006) (Jucha, 2010) (Ivashkevich, 2011). Image analysis implies two distinct steps: selection of the cells and detection of γ-H2AX foci; therefore it needs to load two colour-types of grey scale images: one with counterstained nuclei for cell selection and the other one with fluorescent-coupled anti-γ-H2AX for focus detection.…”

“…We do not compare it to imageJ as a CellProfiler module has been recently created to allow the user to run ImageJ macros and plugins as part of a CellProfiler image processing pipeline (http://cellprofiler.org/CPmanual/RunImageJ.html). In a recent publication, a new image cytometry program that will become freely available was presented (Ivashkevich, 2011). The details on the measurements that will be possible to obtain with this software are not yet disclosed, so we cannot fairly compare it with CellProfiler.…”