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Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study
D K Torous1, S D Dertinger, N E Hall
1Litron Laboratories, 1351 Mount Hope Avenue, Rochester, NY 14620, USA.
Abstract:
Micronuclei (MN) are routinely enumerated in mouse peripheral blood to index genotoxicity. Recent data from the Collaborative Study Group for the Micronucleus Test (CSGMT) [CSGMT (The Collaborative Study Group for the Micronucleus Test), Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS MMS, Environ. Mol. Mutagen. 32 (1998) 84-100] suggest that rat peripheral blood may also be appropriate for the enumeration of MN, if scoring is limited to the youngest fraction of reticulocytes. The experiments described herein were designed to test whether modifications to a flow cytometric scoring procedure for measuring micronucleated reticulocytes (MN-RET) in mouse peripheral blood could be extended to accurately enumerate MN in rat peripheral blood. Rats were treated with saline or one of three genotoxic agents (6-mercaptopurine, ethyl methanesulfonate or propane sultone) in an acute dosing protocol. Peripheral blood samples were subsequently collected for both microscopic and flow cytometric analysis. Micronucleus frequencies were scored in the youngest fraction of reticulocytes: scoring by microscopy was restricted to the types I and II reticulocytes based on RNA content utilizing acridine orange supravital staining; flow cytometric measurements were restricted to the youngest fraction of reticulocytes based on transferrin receptor (CD71) staining. A statistically significant dose-related increase in the incidence of MN was observed, irrespective of scoring method. A higher level of statistical discrimination between control and genotoxin-treated groups was observed for the flow cytometric data and can most likely be explained by the increased number of cells scored (10x more than microscopy) and the lower scoring variability. Together, these data suggest that (i) rat peripheral blood represents an appropriate compartment for evaluating genotoxin-induced MN when the analysis is restricted to young reticulocytes, and (ii) the measurement of MN in rat peripheral blood reticulocytes benefits from the high throughput methodology of flow cytometry.
Insights
Flow cytometry accurately detects genotoxicity in rat peripheral blood micronuclei (MN) within young reticulocytes. This method enhances sensitivity and efficiency for genotoxicity testing in rats.
Area of Science:
- Toxicology
- Genetics
- Biotechnology
Background:
- Micronuclei (MN) in mouse peripheral blood are standard for genotoxicity assessment.
- Recent studies suggest rat peripheral blood may be suitable for MN enumeration, focusing on young reticulocytes.
Purpose of the Study:
- To adapt and validate a flow cytometric method for enumerating micronucleated reticulocytes (MN-RET) in rat peripheral blood.
- To compare the efficacy of flow cytometry versus microscopy for MN detection in rats.
Main Methods:
- Rats were administered saline or genotoxic agents (6-mercaptopurine, ethyl methanesulfonate, propane sultone).
- Peripheral blood samples were analyzed using both microscopy (acridine orange staining) and flow cytometry (CD71 staining) to score MN in young reticulocytes.
- Flow cytometry scored significantly more cells than microscopy.
Main Results:
- A statistically significant, dose-related increase in MN incidence was observed in genotoxin-treated rats, regardless of the scoring method.
- Flow cytometry demonstrated superior statistical discrimination between control and treated groups compared to microscopy.
- Higher cell counts and lower variability contributed to flow cytometry's enhanced sensitivity.
Conclusions:
- Rat peripheral blood is a viable compartment for genotoxicity testing via MN analysis when restricted to young reticulocytes.
- Flow cytometry offers a high-throughput, sensitive, and reproducible method for measuring MN in rat peripheral blood reticulocytes, improving genotoxicity assessment.