ANALYSIS OF DNA SINGLE-STRAND BREAKS IN HUMAN VENOUS-BLOOD - A TECHNIQUE WHICH DOES NOT REQUIRE ISOLATION OF WHITE BLOOD-CELLS

For DNA strand break analysis in human white blood cells, usually metr izoate-Ficoll centrifugation is used to isolate mononuclear cells. Thi s procedure is time-consuming and requires at least 20 mi of blood per sample. Therefore, we developed a technique which does not require is olation of white blood cells prior to DNA strand break analysis by alk aline elution (direct method). The sensitivity of this new technique w as compared to that of the standard method, which includes isolation o f mononuclear blood cells. A statistically significant increase in sen sitivity was observed using the direct method. After in vitro gamma-ir radiation of venous blood, an increase in the elution rate of 7.7 x 10 (-3) hr(-1)/Gy was detected if mononuclear blood cells were isolated c ompared to 10.5 x 10(-3) hr(-1)/Gy with the new technique (P < 0.05). incubation of venous blood with ethylene oxide for 1 hr caused an incr ease in the elution rate of 5.8 x 10(-3) hr(-1)/mM ethylene oxide for the standard and 12 x 10(-3) h(-1)/mM for the direct method (P < 0.05) . DNA single-strand breaks were detected in blood cells of 10 persons without any apparent genotoxic exposure. A mean normalized elution rat e of 1.30 +/- 0.38 (95% confidence interval) was detected in isolated mononuclear blood cells, and a similar mean normalized elution rate of 1.41 +/- 0.50 was obtained using the direct method. The difference wa s not statistically significant. Five patients treated with a combinat ion chemotherapy consisting of cyclophosphamide (750 mg/m(2) i.v.), do xorubicin (50 mg/m(2) i.v.), vincristine (1.4 mg/m(2) i.v.), and predn isolone (100 mg/m(2) p.o.) for non-Hodgkin's disease were analyzed for DNA single-strand breaks before and 16-18 hr after the application of chemotherapy. Increases in mean elution rate of 68% and 116% were det ected using the standard and the direct methods, respectively. For the direct method, only 3 mi of venous blood were sufficient for analysis of one sample, compared to 25 mi needed if mononuclear cells were iso lated, and about 4 hr of work per assay can be saved. (C) 1997 Wiley-L iss, Inc.