6533b7d6fe1ef96bd1267031

RESEARCH PRODUCT

Analysis of DNA single-strand breaks in human venous blood: a technique which does not require isolation of white blood cells.

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For DNA strand break analysis in human white blood cells, usually metrizoate-Ficoll centrifugation is used to isolate mononuclear cells. This procedure is time-consuming and requires at least 20 ml of blood per sample. Therefore, we developed a technique which does not require isolation of white blood cells prior to DNA strand break analysis by alkaline elution (direct method). The sensitivity of this new technique was compared to that of the standard method, which includes isolation of mononuclear blood cells. A statistically significant increase in sensitivity was observed using the direct method. After in vitro gamma-irradiation of venous blood, an increase in the elusion rate of 7.7 × 10−3 hr−1/Gy was detected if mononuclear blood cells were isolated compared to 10.5 × 10−3 hr−1/Gy with the new technique (P < 0.05). Incubation of venous blood with ethylene oxide for 1 hr caused an increase in the elution rate of 5.8 × 10−3 hr−1/mM ethylene oxide for the standard and 12×10−3h−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 rate 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 was not statistically significant. Five patients treated with a combination chemotherapy consisting of cyclophosphamide (750 mg/m2 i.v.), doxorubicin (50 mg/m2 i.v.), vincristine (1.4 mg/m2 i.v.), and prednisolone (100 mg/m2 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 detected using the standard and the direct methods, respectively. For the direct method, only 3 ml of venous blood were sufficient for analysis of one sample, compared to 25 ml needed if mononuclear cells were isolated, and about 4 hr of work per assay can be saved. Environ. Mol. Mutagen. 29:58–62, 1997 © 1997 Wiley-Liss, Inc.