In Depth
In Vivo MicroFlow Kits

The in vivo micronucleus test is widely used in rodents to evaluate compounds for aneugenic and clastogenic potential. In the past, micronuclei (MN) were scored in peripheral blood or bone marrow through microscopic inspection of erythrocytes (also known as red blood cells or RBCs). While it is often simple to identify RBCs that contain MN using microscopy, the scarcity of these events makes scoring tedious and time-consuming. For this reason, thousands of cells must be evaluated to achieve a scoring error that is less than the level of inter-animal variation [Kissling et al., 2007].

In Vivo MicroFlow, a flow cytometric technique, allows users to analyze enough cells to sufficiently reduce scoring error, even in species with low spontaneous frequencies. This system also has the following benefits:

• Results in a fraction of the time compared to microscopy
• High potential for integration into other toxicology studies
• Reduce inter-lab and inter-experiment variability using biological standards for instrument calibration
• Able to be measured across species
• Validated method (rodent only)
• Accepted by regulatory agencies (rodent only)

The MicroFlow method involves fixed blood or bone marrow specimens being incubated with anti-CD71-FITC, RNase, and anti-CD61-PE. Anti-CD71 differentiates reticulocytes (RETs) from mature RBCs. RNase, in conjunction with propidium iodide, is used to distinguish those RBCs with MN from those without, based on DNA content. Anti-CD61 prevents platelets from interfering with MN scoring. Four populations of erythrocytes can be identified: mature and immature erythrocytes, with and without MN. See next column.

In Vivo MicroFlow kits address a chief technical hurdle associated with reliable MN scoring. The use of fixed malaria-infected blood as biological standards provides consistent configuration of instrument settings, which is not present in other systems [Tometsko et al., 1993; Dertinger et al., 2000]. Malaria parasites mimic MN within RBCs, and the use of these biological standards controls intra- and inter-laboratory variability. This consistent configuration of instrument settings has been demonstrated in several international validation studies [Torous et al., 2005; Dertinger et al., 2006].

RBCs containing MN are not actively eliminated from the circulation of mice by the spleen. The same cannot be said for most species of toxicological interest [MacGregor et al., 1980]. The MicroFlow method overcomes this challenge by restricting analysis to the youngest RBCs. For instance, several species who have spleens that filter MN-RBCs have shown significantly elevated MN-RET frequencies in subjects who have been exposed to genotoxic agents [Abramsson-Zetterberg et al., 2000; MacGregor et al., 2006; Harper et al., 2007; Dertinger et al., 2007; Hotchkiss et al., 2008]. This method allows reduction of animal usage, since it facilitates integration of a genetox endpoint into key toxicology studies, the majority of which are conducted with species other than mice.

Unpublished data has also demonstrated that scoring many thousands of RETs per rat blood sample could be used to establish a No Observable Effects Level (NOEL) for the alkylating agent methyl methanesulfonate. Additionally, other data show that clastogens can be distinguished from aneugens by the fluorescence intensity of their MN. Using this flow cytometric method allows researchers to simultaneously determine a chemical’s Mode of Action along with its genotoxicity.

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