The micronucleus (MN) assay is used to evaluate test articles for their ability to cause DNA damage. This damage can either be seen as actual breaks in a chromosome (clastogens), or as the loss of an entire chromosome (aneugens). If a chromosome break or loss occurs, a micro-nucleus (i.e., a ‘small’ nucleus) is formed. Treating precusor cells (stem cells) with a chemical causing DNA damage will result in more micronuclei in the daughter cells.

The Creation Of Micronucleated Cells

These micronuclei can be clearly seen when their DNA is stained in an otherwise DNA-deficient population. Measuring MN in Red Blood Cells (RBCs) is ideal because RBCs expel their main nucleus, leaving behind only the micronuclei.

Erythrocytes: Young Versus Old

In order to see the effects of a compound in the peripheral blood, it is first necessary to differentiate between newly created RBCs and those that have been in the blood stream for some time. Micronuclei detected in the youngest cells (Reticulocytes, or RETs) are indicative of recent damage. On the other hand, MN found in mature cells (Normochromatic erythrocytes, or NCEs) indicate damage that was caused at an earlier time.

The young RETs differ from mature NCEs in several ways. RETs have a high RNA content, as well as specific cell surface markers, each of which is degraded as the cells mature. Rather than detecting RETs by their RNA content, monoclonal antibodies can be used to specifically label cell surface markers of these young cells.

In terms of short-term studies, the youngest cells (RETs) are the most appropriate population to focus, and the one suggested by regulatory agencies. With the MicroFlow process, micronuclei are scored in both the RET and NCE populations.