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Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat.
Toxicol Sci. 2006 Nov; 94(1):92-107.TS

Abstract

We have evaluated a flow cytometric method that allows assessment of micronucleated reticulocytes (MN-RETs) in microliter quantities of peripheral blood and compared results using this assay with those of established microscopic methods of scoring bone marrow and peripheral blood from rats treated with well-characterized genotoxic agents. Young reticulocytes (RETs) are labeled with FITC-anti-CD71 (transferrin receptor) and micronuclei with propidium iodide (with RNase treatment). Red blood cells parasitized with Plasmodia serve as a calibration standard for DNA content. Microscopic scoring used acridine orange (AO) staining of methanol-fixed slides or supravital AO staining. The effect of the rat spleen on the parameters evaluated was determined by comparing age- and sex-matched normal and splenectomized rats treated with cyclophosphamide, cis-platin, or vinblastine under treatment conditions that established a steady-state frequency of MN-RETs in the bone marrow and peripheral blood compartments. The data demonstrate the sensitivity and reproducibility of the flow cytometric assay in the Sprague-Dawley rat, and comparative studies using identical blinded samples at multiple laboratories show that inter- and intra-laboratory reproducibility is much higher with the flow method than with the microscopic methods currently employed for regulatory studies. A significant effect of splenic selection against genotoxicant-induced MN-RETs was observed with each of the three scoring methodologies, despite the fact that the flow and supravital AO techniques restrict analysis to the youngest fraction of RETs. The high precision of flow-based measurements also demonstrated a slight but statistically significant level of selection against spontaneously arising MN-RET. Despite these spleen effects, assay sensitivity for blood-based analyses was maintained by the flow method as it was shown to have superior counting statistics, lower variability, and higher sensitivity than manual scoring. The data suggest that flow cytometric assessment of micronucleus induction can be integrated into routine toxicity testing, eliminating the need for a separate bioassay.

Authors+Show Affiliations

FDA National Center for Toxicological Research, Rockville, Maryland 21012, USA. jtmacgregor@earthlink.netNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Validation Study

Language

eng

PubMed ID

16888079

Citation

MacGregor, James T., et al. "Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes: II. an Efficient Method of Monitoring Chromosomal Damage in the Rat." Toxicological Sciences : an Official Journal of the Society of Toxicology, vol. 94, no. 1, 2006, pp. 92-107.
MacGregor JT, Bishop ME, McNamee JP, et al. Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat. Toxicol Sci. 2006;94(1):92-107.
MacGregor, J. T., Bishop, M. E., McNamee, J. P., Hayashi, M., Asano, N., Wakata, A., Nakajima, M., Saito, J., Aidoo, A., Moore, M. M., & Dertinger, S. D. (2006). Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat. Toxicological Sciences : an Official Journal of the Society of Toxicology, 94(1), 92-107.
MacGregor JT, et al. Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes: II. an Efficient Method of Monitoring Chromosomal Damage in the Rat. Toxicol Sci. 2006;94(1):92-107. PubMed PMID: 16888079.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat. AU - MacGregor,James T, AU - Bishop,Michelle E, AU - McNamee,James P, AU - Hayashi,Makoto, AU - Asano,Norhide, AU - Wakata,Akihiro, AU - Nakajima,Madoka, AU - Saito,Junichiro, AU - Aidoo,Anane, AU - Moore,Martha M, AU - Dertinger,Stephen D, Y1 - 2006/08/03/ PY - 2006/8/5/pubmed PY - 2007/3/14/medline PY - 2006/8/5/entrez SP - 92 EP - 107 JF - Toxicological sciences : an official journal of the Society of Toxicology JO - Toxicol Sci VL - 94 IS - 1 N2 - We have evaluated a flow cytometric method that allows assessment of micronucleated reticulocytes (MN-RETs) in microliter quantities of peripheral blood and compared results using this assay with those of established microscopic methods of scoring bone marrow and peripheral blood from rats treated with well-characterized genotoxic agents. Young reticulocytes (RETs) are labeled with FITC-anti-CD71 (transferrin receptor) and micronuclei with propidium iodide (with RNase treatment). Red blood cells parasitized with Plasmodia serve as a calibration standard for DNA content. Microscopic scoring used acridine orange (AO) staining of methanol-fixed slides or supravital AO staining. The effect of the rat spleen on the parameters evaluated was determined by comparing age- and sex-matched normal and splenectomized rats treated with cyclophosphamide, cis-platin, or vinblastine under treatment conditions that established a steady-state frequency of MN-RETs in the bone marrow and peripheral blood compartments. The data demonstrate the sensitivity and reproducibility of the flow cytometric assay in the Sprague-Dawley rat, and comparative studies using identical blinded samples at multiple laboratories show that inter- and intra-laboratory reproducibility is much higher with the flow method than with the microscopic methods currently employed for regulatory studies. A significant effect of splenic selection against genotoxicant-induced MN-RETs was observed with each of the three scoring methodologies, despite the fact that the flow and supravital AO techniques restrict analysis to the youngest fraction of RETs. The high precision of flow-based measurements also demonstrated a slight but statistically significant level of selection against spontaneously arising MN-RET. Despite these spleen effects, assay sensitivity for blood-based analyses was maintained by the flow method as it was shown to have superior counting statistics, lower variability, and higher sensitivity than manual scoring. The data suggest that flow cytometric assessment of micronucleus induction can be integrated into routine toxicity testing, eliminating the need for a separate bioassay. SN - 1096-6080 UR - https://www.unboundmedicine.com/medline/citation/16888079/Flow_cytometric_analysis_of_micronuclei_in_peripheral_blood_reticulocytes:_II__An_efficient_method_of_monitoring_chromosomal_damage_in_the_rat_ L2 - https://academic.oup.com/toxsci/article-lookup/doi/10.1093/toxsci/kfl076 DB - PRIME DP - Unbound Medicine ER -