The use of immunomagnetic separation of erythrocytes in the in vivo flow cytometer-based micronucleus assay.

The use of sensitive test systems makes it possible to detect weakly genotoxic chemicals and to better define the shape of dose-response relationships, which make it easier to interpret the mechanism behind possible effects. In this study we have refined the flow cytometer-based micronucleus assay by use of a cytometer equipped with two lasers. Since micronucleated young polychromatic erythrocytes, MNPCE, are very few in number among the cells in peripheral blood, about one or two out of 100,000 erythrocytes, there is always a risk that other cells, doublets or crystals, by mistake will be classified as a MNPCE. With immunomagnetic separation of the very youngest erythrocytes - which are transferrin-positive (Trf+Ret) - prior to analysis, we have obtained an almost pure (>98%) Trf+Ret-population. To clarify whether this separation of cells prior to analysis increases the sensitivity of the already sensitive and further refined flow cytometer-based micronucleus assay, we studied the dose-response towards benzo(a)pyrene, B[a]P in the low-dose region, 0-30mg/kgbw. Thirty FVB mice were intraperitoneally injected with B[a]P. From the same blood samples collected from these mice, cells were prepared in the two different ways and analyzed in the flow-cytometer equipped with two lasers. The lowest dose of B[a]P that can be reliably determined without being overwhelmed by the estimated error was about the same for the two methods, about 7mg/kgbw, i.e. the immunomagnetic separation did not increase the sensitivity. A second study with BalbC mice strengthens the result obtained with the FVB mice. Prior to the low-dose study the optimal sampling time for the two methods was determined. In this case, the water-solouble chemical acrylamide was used. The time courses obtained show almost the same shape of the curves, with a maximum of fMNPCE and fMNTrf+Ret at about 40-50h after exposure.