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.
García
Omar
O
Centro de Protección e Higiene de las Radiaciones, Calle 20, No. 4113 e/ 41 y 47, Playa, CP, 11300 La Habana, Cuba. omar@cphr.edu.cu
Mandina
Tania
T
Lamadrid
Ana I
AI
Diaz
Adriana
A
Remigio
Antonia
A
Gonzalez
Yanela
Y
Piloto
Janet
J
Gonzalez
Jorge E
JE
Alvarez
Aimeé
A
D020552:Comet Assay / Q000592:standards*; D004249:DNA Damage; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016622:Silver Staining