EFFECTS IN THE COMET ASSAY OF STORAGE-CONDITIONS ON HUMAN BLOOD

The Comet assay is a rapid and sensitive method for analyzing single c ells for DNA damage. Using human lymphocytes, the assay is particularl y useful for human monitoring studies, as well as for in vitro genotox icity testing of chemicals. In such studies, it is not always possible to collect and process matched samples on the same day as the blood i s taken. It would be useful if some samples could be stored and examin ed at a different time, without loss of viability or other factors aff ecting responses. It is thus important to understand the effects of st orage conditions on blood to be used in such studies and how exposure or treatment might modify such responses. In a joint study in two labo ratories, blood was taken from various donors and stored under differe nt conditions. It was examined on day 1 (day on which sample was taken ) and days 2, 3, 4, 5, or 8 at room temperature, 4 degrees C, or -20 d egrees C. Cells were treated after storage (from day 2 onward) with bl eomycin (BLM) and ethylnitrosourea (ENU). The data were analyzed eithe r by eye (classifying cells with different categories of damage) or by using a computerized image analysis system (Kinetic Imaging Ltd., Liv erpool UK, Software Package Comet 3.0) where the tail moment, which is considered to be a sensitive measurement, has been analyzed. There wa s no loss of cell viability at 4 degrees C or room temperature up to 8 days when measured by trypan blue dye exclusion. Findings suggest tha t on days 1-4 for the untreated samples at room temperature or 4 degre es C there were no biologically meaningful changes in both the differe nt categories of cell damage and tail moment data. In treated cultures up to day 4, either at room temperature or at 4 degrees C, responses were only minimally affected and changes were considered not to be of biological significance. However, there was slightly less Variability between samples at 4 degrees C than at room temperature in one laborat ory. The reverse was true in the other. This would suggest that sample s can probably be stored up to day 4 at 4 degrees C or room temperatur e without any untoward effects. Provided samples can be processed with in this 4-day time frame, it would not seem necessary to cryopreserve samples at -196 degrees C. (C) 1997 Wiley-Liss, Inc.