The COMET assay is recognized as a rapid and sensitive method in quantifying 
radiation induced DNA damage. We investigated the distorting influence of 
endogenous, assay-inherent factors onto base (single cell level) and primary 
outcome measures (experimental/slide level), such as olive tail moment (OTM) and 
percentage DNA in the tail (%tail-DNA). From 2003 to 2008, we performed the 
assay on lymphocytes isolated from the blood samples of 355 lung cancer 
patients, 170 controls, and 610 relatives, as well as one single reference 
individual, repeated 170 times. In total, the data from 10,016 single 
experiments containing around 1,750,000 cells have been included in this study. 
This is the first time that the endogenous variability of the COMET assay has 
been validated systematically on such a huge data set over a 5 year period. 
Assuming that the reference sample reflects assay specific white noise, we 
estimated a proportion of 7-95% of the variability of the outcome measures due 
to assay variation (white noise) depending on parameter, exposure level, and 
study group. The proportion of white noise was largest for the initial radiation 
damage. The specific endogenous factors considered attribute to 14.8% of the 
total variability in the primary outcome measurements of the OTM and 6.9% of the 
%tail-DNA. OTM turns out to be a sensitive parameter to detect variation, but is 
also more susceptible to disturbance caused by endogenous factors than 
%tail-DNA. To reduce the experimental variability in COMET assays, we recommend 
a highly standardized operation protocol as well as inspecting and/or adjusting 
the primary outcome measures according to endogenous factors before calculating 
secondary outcome measures, e.g. DNA repair capacity (DRC) or testing 
statistical inference. A human reference (HR) sample is also useful to inspect 
homogeneity in the temporal progression of long lasting investigations, but not 
for calibrating primary outcome measurements.