br Conclusions br Conflict of interest statement br Acknowle

Conclusions
Conflict of interest statement
Acknowledgments This research was funded by São Paulo Research Foundation (FAPESP) (ProcessN. 2012/08621-1). Thanks to F.J.H. Blázquez and the staff of the Laboratório de Anatomia Microscópica e Imuno Histoquímica (LAMIH − FMVZ − USP) for the permission to use their optical equipment, and D.N. Palermo for his technical assistance during epifluorescence microscope imaging.
Introduction The analysis of DNA damage is carried out in many fields of medicine such as toxicology, pharmacogenomics, oncology, human epidemiology and biomonitoring. DNA damage occurs due to continuous exposure of IL-18, human recombinant protein to exogenous and endogenous agents. The DNA breaks thus formed can block DNA replication and transcription, and if they are not repaired or repaired incorrectly, it may lead to Alzheimer\'s disease, Parkinson\'s disease, cancer, premature aging, diabetes mellitus, mental illness, heart diseases etc. DNA damage analysis provides valuable information regarding early biological effects of hazardous chemicals and important information about the different stages of diseases. This information could be used by clinicians and pathologists for planning the treatment and determining the best course of intervention. Hence the analysis of DNA damage has great significance in clinical research and there is a clear demand for an accurate, fast and sensitive method to detect DNA damage [1]. Comet assay is a widely accepted method for assessing DNA damage in individual cells. It can be used to measure single-strand or double-strand DNA breaks, alkali labile sites, DNA crosslinks, base/base-pair damages and apoptotic cells. This method was introduced by Ostling and Johanson [2] in 1984, in which they used neutral conditions to measure DNA single strand breaks. Subsequently, in 1988 a modified version was introduced by Singh et al. [3], which used alkaline conditions that improved its reproducibility and specificity. In comet assay, during electrophoresis the negatively charged fragments of DNA migrate out from the nucleus, move towards the anode and form a comet like structure, and hence the name comet assay. Such a comet can have head and tail regions [4]. Depending upon the level of DNA damage the comet will have different sizes and shapes. The DNA damage can be quantified by measuring percentage DNA in tail, tail length and tail moment [5], [6]. Even-though the tail moment is considered as the most important parameter for DNA damage analysis, the percentage DNA in tail is preferred, as it is both informative and very easy to interpret [4]. The percentage DNA in tail is determined by comet scoring. The comets are scored in three different ways: (1) Manual method, (2) Semi automated method and (3) Fully automated method. In semi automated and automated methods, image analysis is based on computer programs. Manual methods [7] require skilled operators. But still visual scoring is usually used as a benchmark to validate the results obtained through automated algorithms. Semi automated methods were developed by Rivest et al. [10], Helma and Uhl [8] and Konca et al. [9]. CASP [8], [9] and CometScore are two semi automated open source software packages available in the internet. The disadvantages of semi automated software packages are their low throughput and lack of reproducibility of results. Over and above, the requirement of experts in different stages makes the technique user dependent, tedious and time consuming process. Fully automated systems were developed by Bocker et al. [13], Frieauff et al. [14], Dehon et al. [15], [16], Sansone et al. [17], Gonzalez et al. [18] and Gyori et al. [12]. Most of these methods were developed for fluorescent stained images except the methods proposed by Gonzalez et al. and Gyori et al. Comet assay image analysis based on fluorescent images requires high quality fluorescence microscope and the cells should be photographed and analysed immediately as the slides cannot be stored for a long period.