A simple assay for frequency of chromosome breaks and loss (micronuclei) by flow cytometry of human reticulocytes

Abstract

Exposure to environmental stress, such as radiation, poor nutrition, or smoking, can cause hazardous lesions in DNA, including double-strand breaks. In red blood cells, a DNA fragment or lagging chromosome forms a micronucleus when left behind after the main nucleus is extruded to form the mature reticulocyte during erythropoiesis. Reticulocytes with micronuclei in human peripheral blood are not generally available for analysis because the spleen removes aberrant cells. We have developed a simple and rapid method to isolate and analyze immature reticulocytes in the peripheral blood for the presence of micronuclei before these cells are removed by the spleen. This method applies single-laser flow-cytometry to measure micronuclei in an enriched transferrin-positive reticulocyte population. Abramsson-Zetterberg et al. (Abramsson-Zetterberg, L., Zetterberg, G., Bergqvist, M., and Grawe, J. Environ. Mol. Mutagen. 36, 22-31, 2000) have described a method to measure micronuclei in an enriched reticulocyte population using a dual-laser flow cytometry. Unlike the beads used in their magnetic-immunoseparation procedures, the beads used in this study do not require a prelabeling step and are compatible with the flow cell, sparing the need to release the cells from the beads and avoiding the potentially confounding DNase-treatment step. Dertinger et al. (Dertinger, S. D., Torous, D. K., Hall, N. E., Murante, F. G., Gleason, S. E., Miller, R. K., and Tometsko, C. R. Mutat. Res. 515, 3-14, 2002; Dertinger, S. D., Chen, Y., Miller, R. K., Brewer, K. J., Smudzin, T., Torous, D. K., Hall, N. E., Olvany, K. A., Murante, F. G., and Tometsko, C. R. (2003) Mutat. Res. 542, 77-87, 2003) further improved the scoring of micronuclei to enable the use of bench-top instruments in analyzing samples of unenriched reticulocyte-populations. The present method is distinct from flow cytometric assays, such as reported by Dertinger et al., which enable scoring of limited numbers of reticulocytes per sample and require lengthy data acquisition times. We assessed DNA damage in smokers using this novel flow-cytometry based micronuclei-assay. The results show that this assay can effectively detect micronuclei in human blood samples. This method, unlike available micronuclei assays, allows rapid evaluation of a large number of cells and therefore should prove to be useful in monitoring of human populations for genetic damage.