Sensitivity and variability of visual scoring in the comet assay. Results of an inter-laboratory scoring exercise with the use of silver staining.

Nineteen scorers from seven Cuban laboratories participated in this slide exercise designed to test the influence of the scorer on the accuracy, sensitivity and variability of the comet assay when a visual method of DNA damage evaluation is used. The assay was performed using human lymphocytes from a single donor exposed in vitro for 5 min at 0 degrees C to doses of 0, 5, 10, 25, 50, 100 and 200 microM of hydrogen peroxide. Each participant scored the same set of 14 coded slides with silver stained comets. The comets were classified visually into five categories according to the appearance resulting from the relative proportion of DNA in the tail. The extent of DNA damage was expressed in arbitrary units. At zero dose the median values of 12 scorers out of 19 were included between the values of the overall 25 and 75 per thousand. This proportion remains practically the same as the dose increases. The lowest dose detected by this method for the majority of scorers (11) was 10 microM. The coefficient of variation at the control dose was the highest (median value 26%), progressively declined to 20%, and starting from 25 microM, values are around 10%. The results of the exercise show the reliability of the silver staining and visual scoring for the comet method.

[1]  N. Singh,et al.  Microgels for estimation of DNA strand breaks, DNA protein crosslinks and apoptosis. , 2000, Mutation research.

[2]  V. Didenko.  In situ detection of DNA damage : methods and protocols , 2002 .

[3]  H. Rupp,et al.  The DNA 'comet assay' as a rapid screening technique to control irradiated food. , 1997, Mutation research.

[4]  K. Radack,et al.  Sources of variability in the human lymphocyte micronucleus assay: A population‐based study , 1995, Environmental and molecular mutagenesis.

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

[6]  A. Collins,et al.  Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. , 1993, Carcinogenesis.

[7]  C. Merril.  Silver staining of proteins and DNA , 1990, Nature.

[8]  J. Mclean,et al.  The use of Silver‐stained “Comets” to Visualize DNA Damage and Repair in Normal and Xeroderma pigmentosum Fibroblasts After Exposure to Simulated Solar Radiation , 2000, Photochemistry and photobiology.

[9]  D. Lloyd,et al.  Chromosomal aberrations in human lymphocytes induced in vitro by very low doses of X-rays. , 1992, International journal of radiation biology.

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

[11]  J. G. Brewen,et al.  Evaluation of radiation-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro: result of an IAEA-coordinated programme. , 1982, Mutation research.

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

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

[14]  J. Mclean,et al.  Silver-stained comet assay for detection of apoptosis. , 1999, BioTechniques.

[15]  G. Stephan,et al.  Intercomparison of translocation and dicentric frequencies between laboratories in a follow-up of the radiological accident in Estonia , 2002, International journal of radiation biology.

[16]  R E Durand,et al.  Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. , 1990, Radiation research.

[17]  H. Magnussen,et al.  Reproducibility of basal and induced DNA single-strand breaks detected by the single-cell gel electrophoresis assay in human peripheral mononuclear leukocytes , 1995, International archives of occupational and environmental health.

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

[19]  M Di Giorgio,et al.  Intercomparison in cytogenetic dosimetry among five laboratories from Latin America. , 1995, Mutation research.

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

[21]  L. Vargas-Roig,et al.  A Silver Staining Method for Single-cell Gel Assay , 2001, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

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

[23]  M. J. Silva,et al.  Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project. , 2003, Mutation research.