Lysis of whole blood in vitro causes DNA strand breaks in human lymphocytes

DNA damage in lymphocytes, as measured by alkaline single cell gel electrop horesis (pH 12.7), is greatly increased by the concurrent lysis of whole bl ood in both freshly isolated samples and in PHA-stimulated cultures over a period of 7 days. Further, there is a marked progressive increase in DNA da mage with time in PHA-stimulated lymphocytes cultured in whole blood even w hen the lymphocytes are separated before analysis; no such increase is seen in lymphocytes cultured alone. This indicates that there are components in whole blood that can cause DNA damage in lymphocytes, with granulocytes an d lysis of red blood cells likely candidates. The DNA damage is greatly red uced in granulocyte-depleted whole blood cultures, but even in these signif icant increases are seen at later sampling times. Consequently, careful sam ple preparation is of paramount importance if the Comet assay is to be succ essfully used to assess DNA damage in human peripheral blood lymphocytes. F urther, the progressive increase in DNA damage in whole blood cultures may influence other methods using lymphocytes for population biomonitoring and may be significant for in vitro genotoxicity testing.