Precise and cost-effective micronucleus analysis made easy with the icroFlow Micronucleus Analysis Kit
Dorothea K. Torous Carol R. Tometsko
Litron Laboratories, Rochester, NY
Mark J. Dycaico
The icroFlow Micronucleus Analysis Kit provides researchers with the necessary reagents and supplies to fix peripheral blood samples on site. The kits cost includes subsequent micronucleus (MN) analyses by high-throughput flow cytometry (FCM) at Litron Laboratories. MN data are easily obtained with more precision and less cost compared to conventional methods. The kit requires no special equipment and can be used for routine analyses of peripheral blood samples from any mouse or rat strain. Additionally, by combining this FCM micronucleus assay with Stratagenes Big Blue Transgenic Rodent Mutagenesis Assay*, researchers can concurrently evaluate the clastogenic and mutagenic activities of a test compound within the same animal.
The micronucleus assay1,2,3 is an important component of genetic toxicology screening programs throughout the world. Micronuclei are known to arise from acentric chromosome fragments and lagging whole chromosomes.4,5 Thus, the micronucleus endpoint is capable of detecting clastogenic agents, which cause chromosome breaks, as well as aneugens, which affect the spindle apparatus and result in the loss of whole chromosomes. Erythrocytes are particularly well suited for evaluating MN events, since the nucleus of the erythroblast is expelled a few hours after the last mitosis. Consequently, micronuclei are easily recognized in this population, which is otherwise devoid of DNA.
Scientists at Litron have made significant advances with MN flow c ytometric methodology, where they are able to count MN in both total red blood cell (RBC) and immature reticulocyte (RET) populations.6,7,8 Their patented method** is based on the observation that RETs express the transferrin receptor (i.e., the CD71-defined antigen), and fluorescent antibodies directed against these surface markers can differentially label immature erythrocytes.9,10 When fixed blood cells are treated with ribonuclease and antitransferrin receptor antibody (FITC-conjugate), and then stained with propidium iodide, normochromatic erythrocytes (NCEs) and RETswith and without MNare clearly resolved (Figure 1).
For researchers who do not have access to flow cytometric equipment or expertise, Stratagene and Litron now provide the icroFlow Micronucleus Analysis Kit to obtain genotoxicity data. The kit assembles all the quality-controlled supplies and reagents, allowing researchers worldwide the ability to collect peripheral blood samples and send then to Litron for FCM micronucleus analysis. Fixing the cells is technically simple (Figure 2) and requires no special equipment. Once the fixed cells are received at Litron, MN frequencies are quickly measured with ease, precision, and sensitivity unmatched by conventional microscopic-scoring techniques.
Figure 3 demonstrates the rel iability of the kits FCM-based method to score clastogen-induced MN. Four treatment groups, consisting of five female and five male BALB/c mice, were given 0.9% saline and a dose response of potassium chromate (K2CrO4) (12.5, 25, and 50 mg/kg body weight) via intraperitoneal injections. Chemicals were delivered in a volume of 25 ml/kg body weight at times 0, 24, and 48 hours. Twenty four hours after the last injection, peripheral blood samples were collected and fixed according to the manual.
Flow cytometric analysis was performed at Litron as previously described.7 When 10,000 total RETs were acquired, the MN-RET frequency was determined. Reticulocyte frequencies were also calculated as an indication of stem cell toxicity. The dual-labeling method used in this study resolved MN-RETs from other erythrocyte populations (Figure 1). Achieving this high resolution facilitated quantitative determination of MN-RET and RET frequencies.
Figure 3 shows a dose-dependent increase in MN-RET frequencies; the control group was low, compared to the K2CrO4-treated animals (0.28, 0.41, 0.43, and 0.91%, respectively). These data show similar trends in MN-RET frequencies reported by other laboratories using conventional microscopy-based methods.11 Reticulocyte frequencies were also quantitated to measure stem cell toxicity. No substantial toxicity was evident at any of the doses tested.
The flow cytometric procedure employed in the kit is used routinely at Litron Laboratories with excellent results. While the procedure critically depends on fixing the cells reliably and consistently, the method developed for the kit is both technically simple and highly repr oducible. The kit contains a detailed fixing protocol, fixing reagents, and a shipping container that holds up to 50 samples; the price includes analysis for 50 samples performed at Litron using their patented FCM-based method.
After sending blood samples for analysis, a minimum of 10,000 RETs per blood sample are examined for MN, compared to 1,000 to 2,000 using microscopic-scoring techniques. Concurrently, micronuclei are scored in the total RBC population, representing an index of subchronic/chronic treatments. The frequency of RETs is determined as an indication of stem cell toxicity. Results are returned in the form of a GLP report within seven days from receipt of the samples.
The MN endpoint continues to grow in popularity as a genotoxic screen to identify chromosome-damaging agents. Compared to conventional MN methods, the icroFlow Micronucleus Analysis Kit offers more MN data in less time and at a lower cost and with higher precision. When used in conjunction with Stratagenes Big Blue Transgenic Rodent Mutagenesis Assay, a test compound or treatment can be assessed for both mutagenic and clastogenic/aneugenic potential within the same animal.
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* U.S. Patent Nos. 5,347,075 and 5,589,155 and patents pending;
European Patent No. 289121; Japanese Patent No. 2618973.
** U.S. Patent No. 5,229,265 and patent pending.