Validation of an automatic comet assay analysis system integrating the curve fitting of combined comet intensity profiles.

In recent years, the single-cell gel electrophoresis (comet) assay has become a reference technique for the assessment of DNA fragmentation both in vitro and in vivo at the cellular level. In order to improve the throughput of genotoxicity screening, development of fully automated systems is clearly a must. This would allow us to increase processing time and to avoid subjectivity brought about by frequent manual settings required for the 'classical' analysis systems. To validate a fully automatic system developed in our laboratory, different experiments were conducted in vitro on murine P388D1 cells with increasing doses of ethyl methanesulfonate (up to 5 mM), thus covering a large range of DNA damage (up to 80% of DNA in the tail). The present study (1) validates our 'in house' fully automatic system versus a widely used semi-automatic commercial system for the image-analysis step, and versus the human eye for the image acquisition step, (2) shows that computing tail DNA a posteriori on the basis of a curve fitting concept that combines intensity profiles [G. Dehon, P. Bogaerts, P. Duez, L. Catoire, J. Dubois, Curve fitting of combined comet intensity profiles: a new global concept to quantify DNA damage by the comet assay, Chemom. Intell. Lab. Syst. 73 (2004) 235-243] gives results not significantly different from the 'classical' approach but is much more accurate and easy to undertake and (3) demonstrates that, with these increased performances, the number of comets to be scored can be reduced to a minimum of 20 comets per slide without sacrificing statistical reliability.

[1]  P. Olive,et al.  The comet assay: a comprehensive review. , 1995, Mutation research.

[2]  K. Linnet,et al.  Estimation of the linear relationship between the measurements of two methods with proportional errors. , 1990, Statistics in medicine.

[3]  E. Kiskinis,et al.  Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test. , 2001, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[4]  A. Collins,et al.  The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. , 1995, Mutation research.

[5]  P. Duez,et al.  Statistics of the Comet assay: a key to discriminate between genotoxic effects. , 2003, Mutagenesis.

[6]  L M Thienpont,et al.  Validity of linear regression in method comparison studies: is it limited by the statistical model or the quality of the analytical input data? , 1998, Clinical chemistry.

[7]  W Frieauff,et al.  Automatic analysis of slides processed in the Comet assay. , 2001, Mutagenesis.

[8]  P. Marquis.  Comparaisons de méthodes analytiques , 1999 .

[9]  J O Westgard.  Points of care in using statistics in method comparison studies. , 1998, Clinical chemistry.

[10]  K. O’Neill,et al.  Analysis of single-cell gel electrophoresis using laser-scanning microscopy. , 1993, Mutation research.

[11]  J Ashby,et al.  The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment. , 1995, Mutagenesis.

[12]  Tomasz Kuszewski,et al.  A cross-platform public domain PC image-analysis program for the comet assay. , 2003, Mutation research.

[13]  B. Hellman,et al.  The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay. , 1995, Mutation research.

[14]  N. Sadlej-Sosnowska,et al.  Statistical evaluation and comparison of comet assay results. , 2003, Mutation research.

[15]  B. K. Murray,et al.  Tail profile: a more accurate system for analyzing DNA damage using the Comet assay. , 2003, Mutation research.

[16]  D. Lovell,et al.  Issues related to the experimental design and subsequent statistical analysis of in vivo and in vitro comet studies. , 1999, Teratogenesis, carcinogenesis, and mutagenesis.

[17]  P. Schmezer,et al.  The effect of various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in the COMET assay. , 1994, Mutation research.

[18]  Martin M. Schumacher,et al.  Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations. , 2004, Mutagenesis.

[19]  Stig Johan Wiklund,et al.  Aspects of design and statistical analysis in the Comet assay. , 2003, Mutagenesis.

[20]  C Helma,et al.  A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay. , 2000, Mutation research.

[21]  H. Wulf,et al.  Laser scanning cytometry for comet assay analysis. , 2000, Cytometry.

[22]  V. McKelvey-Martin,et al.  Evaluation of manual and image analysis quantification of DNA damage in the alkaline comet assay. , 1997, Mutagenesis.

[23]  R. Tice,et al.  A simple technique for quantitation of low levels of DNA damage in individual cells. , 1988, Experimental cell research.

[24]  J. Mclean,et al.  Comet assay: rapid processing of multiple samples. , 2000, Mutation research.

[25]  P. Olive,et al.  DNA damage and repair in individual cells: applications of the comet assay in radiobiology. , 1999, International journal of radiation biology.

[26]  R R Tice,et al.  Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop. , 2003, Mutagenesis.

[27]  B. Rinkevich,et al.  Use of the comet assay for studying environmental genotoxicity: Comparisons between visual and image analyses , 2003, Environmental and molecular mutagenesis.

[28]  P. van Oostveldt,et al.  Miniaturizing the comet assay with 3D vertical comets , 2003, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[29]  E. Rojas,et al.  Single cell gel electrophoresis assay: methodology and applications. , 1999, Journal of chromatography. B, Biomedical sciences and applications.

[30]  E. Kiskinis,et al.  High throughput Comet assay using 96-well plates. , 2002, Mutagenesis.

[31]  H. Kobayashi.  A comparison between manual microscopic analysis and computerized image analysis in the single cell gel electrophoresis , 1995 .

[32]  M. Dusinska,et al.  Comet assay in human biomonitoring studies: Reliability, validation, and applications , 1997, Environmental and molecular mutagenesis.

[33]  A. Beckett,et al.  AKUFO AND IBARAPA. , 1965, Lancet.

[34]  G. Steel,et al.  The comet moment as a measure of DNA damage in the comet assay. , 1995, International journal of radiation biology.

[35]  A. Collins,et al.  Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells. , 1992, International journal of radiation biology.

[36]  A. Collins,et al.  The comet assay for DNA damage and repair , 2004, Molecular biotechnology.

[37]  W Böcker,et al.  Image analysis of comet assay measurements. , 1997, International journal of radiation biology.

[38]  R. Tice,et al.  Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing , 2000, Environmental and molecular mutagenesis.

[39]  J. Nygren,et al.  The comet assay: mechanisms and technical considerations. , 1996, Mutation research.

[40]  D. Altman,et al.  STATISTICAL METHODS FOR ASSESSING AGREEMENT BETWEEN TWO METHODS OF CLINICAL MEASUREMENT , 1986, The Lancet.

[41]  Philippe Bogaerts,et al.  Curve fitting of combined comet intensity profiles : a new global concept to quantify DNA damage by the comet assay , 2004 .

[42]  K. Linnet,et al.  Performance of Deming regression analysis in case of misspecified analytical error ratio in method comparison studies. , 1998, Clinical chemistry.

[43]  U. Plappert,et al.  Comparative study with the alkaline Comet assay and the chromosome aberration test. , 2003, Mutation research.

[44]  T. Lörch,et al.  New developments in automated cytogenetic imaging: unattended scoring of dicentric chromosomes, micronuclei, single cell gel electrophoresis, and fluorescence signals , 2004, Cytogenetic and Genome Research.

[45]  W Böcker,et al.  Automated comet assay analysis. , 1999, Cytometry.