Fully Automatic Comet Assay Analysis with Optional User Validation and Support of Virtual Slides

The Comet Assay Extension in the Aphelion™ Imaging Software Suite provides support for the Single Cell Gel Electrophoresis (SCGE) comet assay. This simple test is used in oncology to predict the intrinsic radio-sensitivity of tumors, or for the study of individual sensitivity to genotoxic agents. It is also used in genetic toxicology to quantize DNA damage in high-risk populations (for example farmers, smokers, or workers in a controlled environment). The comet assay quantifies the ratio of simple and double DNA breaks within individual cells. It is based on the capability of DNA fragments with a negative load to migrate through an agarose gel when an electric field is applied. One of the main benefits of this assay is that it is inexpensive, as only 1000 to 10000 cells are enough to unveil a DNA break for 1010 daltons.

The assay can reveal 100 breaks per cell on average. To prepare images for further image processing, cells are placed on a microscope plate before electrophoresis. After rinsing the plates, DNA is marked with a fluorescent dye (e.g. ethidium, bromure, and acridine). The plate is then placed under a fluorescent microscope for analysis. In damaged cells, broken DNA spreads towards the anode part of the electrophoresis device, displaying a comet-like shape with an intense head and a diffused tail. The length and intensity of the comet tail vary by function of the broken DNA. Healthy and intact nuclei retain the shape of a disk. Using the software, images can be automatically segmented, and normal, damaged, and highly damaged cells (HDC) are detected and then analyzed.