Impact of logistical variation in sample handling on comet results in human leukocytes.
Toraason-M; Krieg-E; Singh-N
Environ Mol Mutagen 2006 Jul; 47(6):434
Epidemiological studies employing biomarkers such as DNA damage often encounter logistical restrictions in the filed that unavoidably lead to variation in the handling of biological samples. This may lead to variation in sample analysis outcome. The present study assessed potential sources of variation in comet assay results (% tail DNA and DNA tail moment) in human leukocytes arising from variations in day and time of blood draw, freezing protocols, pre-freezing duration, freezing duration, and analysis lot. Two experimental designs were used to assess variability. Under the first design, blood was drawn at the same time on three sequential days from a single individual in the laboratory where comet analyses were performed. Following blood draw, a subset of samples were exposed to 0 or 200 rads of X rays and analyzed in the comet assay as fresh blood or after being frozen for 1 month at -80 degrees C using three freezing protocols. The protocols evaluated provide both advantages and disadvantages depending on whether blood was irradiated or not. Adding 100 microliters of whole blood directly to 900 microliters of RPMI 1640 containing 20% DMSO at 4 degrees C was optimum in terms of accurately replicating the overall outcome obtained with fresh blood. Comet results from three sequential blood samples drawn over as many days and frozen for 1 month did not vary significantly regardless of whether samples were analyzed on a single day in a single analysis lot or over three sequential days in three separate analyses lots. Results also indicate that maintaining blood on ice prior to freezing increases DNA damage in time-dependent fashion with small by significant effects becoming evident at 8 hrs. Under the second design, morning and evening blood samples were acquired on the same day from 10 individuals and duplicate samples were analyzed 1 or 3 months after freezing and shipping across the US. Comet results were analyzed using a mixed model to calculate variance components and to test for effects of time of blood draw and month analysis. Time of blood draw was without significant effect. Month of analysis had a small but significant effect on % tail DNA (1 month, 50.4 vs 3 months, 48.6; P=0.044), but not on DNA tail moment (1 month, 2034 vs 3 months, 1965; P=0.313).
Humans; Exposure-methods; Bioassays; Biomarkers; Biological-monitoring; Blood-samples; Blood-tests; Blood-analysis; Blood-cells; Analytical-methods
NIOSH, 4676 Columbia Parkway, Cincinnati, OH 45226
Abstract; Conference/Symposia Proceedings
Environmental and Molecular Mutagenesis