| abstract |
Genotoxic chemicals are an existing wide-spread health hazard to the human population. Advances in genetic toxicology testing have made it possible to assay potential mutagens, carcinogens, teratogens and clastogens in the environment. The mouse micronucleus assay provides an example of an excellent test for genetic damage to cells. When chromosome breaks occur in the blood stem cell population, the damaged piece of chromosome remains behind as a micronucleus in the normally DNA deficient red blood cells. However, currently available manual micronucleus assays are costly, time consuming, and labor intensive. In addition, the statistics are often marginal since the number of micronucleii (MNs) in 1000 polychromatic cells are scored manually, yielding limited amounts of data. This invention discloses the means for assaying the change in micronucleated cells by high speed flow cytometry. In this procedure, cells are streamed at high speed (2500 cells/second) through a laser beam whereupon the fluorescence emission and light scatter properties of each cell are obtained. The invention discloses the procedures for dosing mice, obtaining blood samples, fixing and staining cells, configuring the flow cytometer for MN analysis, the mode of data acquistion and analysis. Internal and external quality controls are also described that are routinely used to optimize the flow cytometer conditions and permit the evaluation of the fidelity of data in real time. This process permits the analysis of 1,000,000 total cells for each sample in minutes, and it removes the subjective judgement of the manual method. In addition to vastly improved statistics, the sensitivity of the assay is significantly improved with this process. |