Human cytogenetic biomonitoring using flow-cytometric analysis of micronuclei in transferrin-positive immature...

We have developed a method to isolate and analyze nascent human reticulocytes in peripheral blood for the presence of micronuclei (MN). For a very short time peripheral reticulocytes show residual expression of the transferrin receptor. Using immunomagnetic separation of cells expressing the transferrin receptor, a population of immature reticulocytes (Trf-Ret) was isolated from peripheral blood. In humans, the spleen actively removes micronucleated erythrocytes but during the short lifetime of the isolated Trf-Ret only a fraction (less than about 20%) of the MN-containing reticulocytes will have been eliminated. Cells were stained with the fluorescent dyes Thiazole Orange for RNA and Hoechst 33342 for DNA and analyzed by flow cytometry and fluorescence microscopy. Baseline frequencies of MN-Trf-Ret on a group of healthy donors were found to be 1.1% for males and 1.4% for females; however, the gender difference was not significant. The frequency of MN-Trf-Ret in the studied group increased with age, and was dependent on blood group. In three donors studied over 4 months, the baseline level remained stable. In cancer patients treated with radiation or chemotherapy, the frequency of MN-Trf-Ret increased 10- to 20-fold after 1-4 days, depending on the treatment. A high correlation between flow and manual analysis of MN-Trf-Ret was seen. We believe the method has a high potential as a sensitive and rapid method for biological monitoring in presumed exposed groups and individuals.