Automated comet assay analysis

Background: Recently the "comet assay" or "single-cell gel electrophoresis assay" has been established as a sensitive method for the detection of DNA damage and repair. Most of the software now available to quantify various p arameters for DNA damage requires the interaction of a human observer. In t his report, we describe an automated analysis sg stem that is based on self -developed software and hardware and needs minimal human interaction. Methods: The image analysis is divided into two parts: 1) automatic cell re cognition and comet classification and 2) quantification of desired comet p arameters. Image preprocessing, segmentation, and feature classification we re developed with algorithms based on mathematical morphology. To enhance e valuation speed, we have introduced parallel processing of data under the W indows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (Bocker et al.: Phys Med Biol 1997;42:198 1-1992) allows for faster analysis. Results: Our recognition software shows a sensitivity of 95.2% and a specif icity of 92.7% when tested on test samples from routine work with DNA damag e by low-dose radiation (0-2 Gy) The parallel hardware and software concept enables us to analyze 100 comets on one slide in less than 15 min. Conclusions: A comparison of measurements made on the same samples by manua l and automated analysis systems revealed that there are no significant dif ferences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possib le. (C) 1999 Wiley-Liss, Inc.