Böcker W, Rolf W, Bauch T, Müller W U, Streffer C
Institut für Medizinische Strahlenbiologie, Universitätsklinikum Essen, Germany.
Cytometry. 1999 Feb 1;35(2):134-44.
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 parameters for DNA damage requires the interaction of a human observer. In this report, we describe an automated analysis system that is based on self-developed software and hardware and needs minimal human interaction.
The image analysis is divided into two parts: 1) automatic cell recognition and comet classification and 2) quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (Böcker et al.: Phys Med Biol 1997;42:1981-1992) allows for faster analysis.
Our recognition software shows a sensitivity of 95.2% and a specificity of 92.7% when tested on test samples from routine work with DNA damage 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.
A comparison of measurements made on the same samples by manual and automated analysis systems revealed that there are no significant differences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possible.
最近，“彗星试验”或“单细胞凝胶电泳试验”已被确立为检测DNA损伤和修复的一种灵敏方法。目前现有的用于量化DNA损伤各种参数的大多数软件都需要人工观察者进行交互操作。在本报告中，我们描述了一种基于自主研发的软硬件且只需最少人工交互的自动化分析系统。
图像分析分为两个部分：1）自动细胞识别和彗星分类；2）所需彗星参数的量化。图像预处理、分割和特征分类是基于数学形态学的算法开发的。为提高评估速度，我们在Windows NT操作系统（微软公司，华盛顿州雷德蒙德）下引入了数据并行处理。使用模拟实时自动聚焦装置（博克等人：《物理医学与生物学》1997年；42:1981 - 1992）可实现更快的分析。
当用低剂量辐射（0 - 2 Gy）导致DNA损伤的常规工作测试样本对我们的识别软件进行测试时，其灵敏度为95.2%，特异性为92.7%。并行的软硬件概念使我们能够在不到15分钟的时间内分析一张载玻片上的100个彗星。
对同一批样本分别采用手动和自动化分析系统进行测量的比较结果显示，二者无显著差异。剂量反应曲线的斜率和修复动力学非常相似，这表明彗星试验自动分析是可行的。
