Posted on December 16, 2013 by Chris Hughes
Gentronix, specialists in genotoxicity testing, now offers in vivo Micronucleus testing of rodent blood samples using flow cytometry.
The rodent peripheral blood micronucleus test provides an assessment of the ability of a test compound to induce chromosomal damage or mis-segregation in haematopoietic cells in rodent bone marrow. During the generation of red blood cells, these progenitor stem cells differentiate to form erythroblasts, which in turn expel their nuclei to form immature red blood cells (reticulocytes, RETs). If these cells suffer chromosomal damage, nuclear membranes form around the resulting fragments, generating micronuclei. These micronuclei are not expelled with the nucleus of the erythroblast during differentiation, resulting in RETs that contain micronuclei and therefore DNA (MN-RETS).
Whilst MN-RETs from peripheral blood can be easily scored by microscopy, given the relative rarity of these events this is a time-consuming process that requires the scoring of thousands of reticulocytes per sample. Using a flow cytometric approach allows the robust & rapid enumeration of tens of thousands of RETs from a small aliquot of rodent blood. Gentronix Ltd utilises MicroFlow® technology, developed by Litron Laboratories, to perform in vivo micronucleus testing of rodent peripheral blood. This method uses a staining procedure that restricts analysis to the newly formed RETs population (using anti-CD71) and thus avoids the problems caused by splenic clearance of MN-RETs in many animal species. These immature RETs are then assessed for DNA content using a nucleic acid stain, allowing MN-RETs to be differentiated from the healthy background population.
Peripheral blood samples (~ 100µl) from in vivo studies are stabilised and shipped to Gentronix for analysis using a kit provided. Samples are then labelled for flow cytometric data collection, with typically 20,000 RETs scored for micronuclei. A study report is provided and data reported on the percentage of RETs and MN-RETs observed in each treatment group, with further statistical analysis also available. Flow cytometer standards and quality controls ensure robustness in the collection of in vivo micronucleus test data.
The flow cytometric rodent peripheral blood micronucleus test is included in the recent ICH revision to the guidelines on genotoxicity , with a requirement for restriction of analysis to the RETs population required for non-mouse in vivo studies – the offering from Gentronix meets this criteria. Results turnaround times are sample size dependent, although a typical 60 sample study completes within 2-weeks. In addition to providing this service, Gentronix is also a distributor of the Litron Laboratory kits throughout Europe and Japan