Flow cytometric scoring of micronucleated erythrocytes: an efficient platform for assessing in vivo cytogenetic damage.

The relative simplicity of the micronucleated erythrocyte endpoint has made it amenable to automated scoring approaches. Flow cytometry is one such scoring platform that has been employed successfully. This review describes the evolution and properties of flow cytometry-based scoring of micronucleated erythrocytes. The methodology has become widely applied to rodent blood specimens and the high throughput nature of the technology provides a number of advantages over manual microscopic scoring. For instance, the ability to efficiently survey many dose levels and many more cells per specimen relative to microscopy benefits studies that are designed to identify no observable effect levels or lowest observable effect levels. Furthermore, flow cytometry makes it practical to study species with low spontaneous reticulocyte (RET) counts and micronucleus (MN) frequencies, thereby facilitating integration of blood-based micronucleated reticulocyte (MN-RET) frequency measurements into experiments conducted across species of toxicological interest. This capability enhances genotoxicity assessments that have historically been made in dedicated MN tests performed in one species. Importantly, the feasibility of using MN-RET frequencies in blood from humans as an index of genetic damage in bone marrow opens a critical area of application that had not been practical previously. We conclude with recommendations for additional work that is needed to more fully realise the potential of flow cytometric in vivo MN scoring.

[1]  S. Dertinger,et al.  Simple and reliable enumeration of micronucleated reticulocytes with a single-laser flow cytometer. , 1996, Mutation research.

[2]  R. Schlegel,et al.  The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: implications for cytogenetic screening. , 1984, Mutation research.

[3]  F Romagna,et al.  The automated bone marrow micronucleus test. , 1989, Mutation research.

[4]  S. Dertinger,et al.  Analysis of micronucleated cells by flow cytometry. 2. Evaluating the accuracy of high-speed scoring. , 1993, Mutation research.

[5]  A. Wakata,et al.  Evaluation of the rodent micronucleus assay by a 28‐day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)–Mammalian Mutagenicity Study Group (MMS) , 2001, Environmental and molecular mutagenesis.

[6]  J. S. Wassom,et al.  Origins of genetic toxicology and the environmental Mutagen Society , 1989, Environmental and molecular mutagenesis.

[7]  P. Chang,et al.  Impact of p53 status on heavy-ion radiation-induced micronuclei in circulating erythrocytes. , 2000, Mutation research.

[8]  B. Ames,et al.  A simple assay for frequency of chromosome breaks and loss (micronuclei) by flow cytometry of human reticulocytes , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  W. Schmid,et al.  The micronucleus test. Methodological aspects. , 1973, Mutation research.

[10]  T. Abe,et al.  Micronuclei in human bone-marrow cells: evaluation of the micronucleus test using human leukemia patients treated with antileukemic agents. , 1984, Mutation research.

[11]  Raffaella Corvi,et al.  Reduction of use of animals in regulatory genotoxicity testing: Identification and implementation opportunities-Report from an ECVAM workshop. , 2009, Mutation research.

[12]  R. Hiatt,et al.  Micronucleated erythrocytes as an index of cytogenetic damage in humans: demographic and dietary factors associated with micronucleated erythrocytes in splenectomized subjects. , 1990, Cancer research.

[13]  Yuhchyau Chen,et al.  Automated human blood micronucleated reticulocyte measurements for rapid assessment of chromosomal damage. , 2007, Mutation research.

[14]  B. P. Eynon,et al.  'Spontaneous' genetic damage in man: evaluation of interindividual variability, relationship among markers of damage, and influence of nutritional status. , 1997, Mutation research.

[15]  K. O’Neill,et al.  Analysis of single-cell gel electrophoresis using laser-scanning microscopy. , 1993, Mutation research.

[16]  Hong Wang,et al.  Lack of DNA Polymerase θ (POLQ) Radiosensitizes Bone Marrow Stromal Cells In Vitro and Increases Reticulocyte Micronuclei after Total-Body Irradiation , 2009, Radiation research.

[17]  Yuhchyau Chen,et al.  Micronucleated CD71-positive reticulocytes: a blood-based endpoint of cytogenetic damage in humans. , 2003, Mutation research.

[18]  R. Schlegel,et al.  Assessment of cytogenetic damage by quantitation of micronuclei in human peripheral blood erythrocytes. , 1986, Cancer research.

[19]  P. Harbach,et al.  Interlaboratory validation of a CD71‐based flow cytometric method (Microflow®) for the scoring of micronucleated reticulocytes in mouse peripheral blood , 2005, Environmental and molecular mutagenesis.

[20]  Makoto Hayashi,et al.  Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS·MMS , 1998, Environmental and molecular mutagenesis.

[21]  M. Kirsch‐Volders,et al.  Flow cytometry peripheral blood micronucleus test in vivo: Determination of potential thresholds for aneuploidy induced by spindle poisons , 2009, Environmental and molecular mutagenesis.

[22]  J. Tucker,et al.  Effects of diet and folate on levels of micronucleated polychromatic erythrocytes. , 1993, Mutation research.

[23]  J. Heddle,et al.  The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. , 1976, Mutation research.

[24]  J. T. Macgregor,et al.  Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test. , 1980, Environmental mutagenesis.

[25]  T. Sofuni,et al.  An application of Acridine Orange fluorescent staining to the micronucleus test. , 1983, Mutation research.

[26]  F. S. Williamson,et al.  The relative biological efficiency of single doses of fast neutrons and gamma-rays on Vicia faba roots and the effect of oxygen. Part II. Chromosone damage: the production of micronuclei. , 1959, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[27]  K. Witt,et al.  Elevated frequencies of micronucleated erythrocytes in infants exposed to zidovudine in utero and postpartum to prevent mother‐to‐child transmission of HIV , 2007, Environmental and molecular mutagenesis.

[28]  J. T. Macgregor,et al.  Flow cytometric analysis of micronuclei in peripheral blood reticulocytes III. An efficient method of monitoring chromosomal damage in the beagle dog. , 2007, Toxicological sciences : an official journal of the Society of Toxicology.

[29]  M. Fenech,et al.  Flow cytometric enumeration of micronucleated reticulocytes: High transferability among 14 laboratories , 2001, Environmental and molecular mutagenesis.

[30]  R. Baker,et al.  Initiating carcinogen, triethylenemelamine, induces micronuclei in skin target cells , 1989, Environmental and molecular mutagenesis.

[31]  S. Dertinger,et al.  Analysis of micronucleated cells by flow cytometry. 3. Advanced technology for detecting clastogenic activity. , 1993, Mutation research.

[32]  M. Fenech,et al.  HUman MicroNucleus project: international database comparison for results with the cytokinesis‐block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei , 2001, Environmental and molecular mutagenesis.

[33]  Draft OECD Guideline for the Testing of Chemicals , 2006 .

[34]  S. Dertinger,et al.  Quantitative analysis of Howell-Jolly bodies in children with sickle cell disease. , 2004, Experimental hematology.

[35]  B. Bridges.  Mutagen – Induced Chromosome Damage in Man. , 1979 .

[36]  W. Slikker,et al.  Flow cytometric analysis of micronuclei in peripheral blood reticulocytes IV: an index of chromosomal damage in the rhesus monkey (Macaca mulatta). , 2008, Toxicological sciences : an official journal of the Society of Toxicology.

[37]  S. Dertinger,et al.  Analysis of micronucleated cells by flow cytometry. 4. Kinetic analysis of cytogenetic damage in blood. , 1995, Mutation research.

[38]  M. Stöhr,et al.  Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry , 2004, Histochemistry.

[39]  L. Abramsson-Zetterberg,et al.  Spontaneous and radiation-induced micronuclei in erythrocytes from four species of wild rodents: a comparison with CBA mice. , 1997, Mutation research.

[40]  J. T. Macgregor,et al.  Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability. , 2007, Mutation research.

[41]  B. Ames,et al.  Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[42]  S. Dertinger,et al.  Practical threshold for micronucleated reticulocyte induction observed for low doses of mitomycin C, Ara-C and colchicine. , 2006, Mutagenesis.

[43]  R. Sram,et al.  Relationship between experimental results in mammals and man. I. Cytogenetic analysis of bone marrow injury induced by a single dose of cyclophosphamide. , 1975, Mutation research.

[44]  P. Vanparys,et al.  Flow cytometric analysis of micronucleated reticulocytes: Time‐ and dose‐dependent response of known mutagens in mice, using multiple blood sampling , 2005, Environmental and molecular mutagenesis.

[45]  Raymond R Tice,et al.  Comparison of flow cytometry- and microscopy-based methods for measuring micronucleated reticulocyte frequencies in rodents treated with nongenotoxic and genotoxic chemicals. , 2008, Mutation research.

[46]  M. Gallegos-Arreola,et al.  Spontaneous micronuclei in peripheral blood erythrocytes from 54 animal species (mammals, reptiles and birds): part two. , 2000, Mutation research.

[47]  B. Vessby,et al.  The impact of folate status and folic acid supplementation on the micronucleus frequency in human erythrocytes. , 2006, Mutation research.

[48]  J. Cantú,et al.  Micronucleated erythrocytes in splenectomized patients with and without chemotherapy. , 1996, Mutation research.

[49]  J. French,et al.  Induction and time-dependent accumulation of micronuclei in peripheral blood of transgenic p53+/- mice, Tg.AC (v-Ha-ras) and parental wild-type (C57BL/6 and FVB/N) mice exposed to benzene by inhalation. , 2001, Mutagenesis.

[50]  Junichiro Saito,et al.  Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[51]  M. Bergqvist,et al.  Human cytogenetic biomonitoring using flow‐cytometric analysis of micronuclei in transferrin‐positive immature peripheral blood reticulocytes , 2000, Environmental and molecular mutagenesis.

[52]  Stephen D Dertinger,et al.  In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test. , 2007, Mutation research.

[53]  K. Morimoto,et al.  Effect of gene polymorphisms and ethanol consumption on micronucleus frequency in human reticulocytes: a preliminary study , 2010, Environmental health and preventive medicine.

[54]  R. Mittelstaedt,et al.  Frequency of Hprt mutant lymphocytes and micronucleated erythrocytes in p53‐haplodeficient mice treated perinatally with AZT and AZT in combination with 3TC , 2007, Environmental and molecular mutagenesis.

[55]  T. Sofuni,et al.  Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow. , 1984, Mutation research.

[56]  H. Norppa,et al.  Flow cytometric micronucleus test with mouse peripheral erythrocytes. , 1992, Mutagenesis.

[57]  Csgmt.  The Collaborative Study Group for the Micronucleus Test : Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining : The summary report of the 5th collaborative study by CSGMT/JEMS MMS , 1992 .

[58]  T. Sofuni,et al.  The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides. , 1990, Mutation research.

[59]  S. Dertinger,et al.  Malaria-infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry. , 2000, Mutation research.

[60]  Csgmt.  Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: the summary report of the 5th collaborative study by CSGMT/JEMS.MMS. The Collaborative Study Group for the Micronucleus Test. , 1992, Mutation research.

[61]  C. Auerbach.  Abnormal Segregation after Chemical Treatment of Drosophila. , 1947, Genetics.

[62]  R. Schlegel,et al.  Micronuclei in circulating erythrocytes: a rapid screen for chromosomal damage during routine toxicity testing in mice. , 1983, Developments in toxicology and environmental science.

[63]  W. Choy,et al.  Primate Micronucleus Study of L‐Selenomethionine , 1989, Environmental and molecular mutagenesis.

[64]  M. Fenech,et al.  Reflections on the development of micronucleus assays. , 2011, Mutagenesis.

[65]  A. Nyfors,et al.  Cytogenetic effects of methotrexate on human cells in vivo: comparison between results obtained by chromosome studies on bone-marrow cells and blood lymphocytes and by the micronucleus test. , 1979, Mutation research.

[66]  J A Heddle,et al.  A rapid in vivo test for chromosomal damage. , 1973, Mutation research.

[67]  C. Reiners,et al.  Evaluation of the reticulocyte micronucleus assay in patients treated with radioiodine for thyroid cancer. , 2005, Mutation research.

[68]  M. Fenech,et al.  The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. , 1993, Mutation research.

[69]  S. Dertinger,et al.  Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model. , 1993, Mutation research.

[70]  M. Shelby,et al.  Comparison of results from mouse bone marrow chromosome aberration and micronucleus tests , 1995, Environmental and molecular mutagenesis.

[71]  J. T. Macgregor,et al.  In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring , 2000, Environmental and Molecular Mutagenesis.

[72]  S. Dertinger,et al.  Assessment of genotoxicity associated with hydroxyurea therapy in children with sickle cell anemia. , 2009, Mutation research.

[73]  W. Schmid,et al.  Trenimon-induced chromosomal damage in bone-marrow cells of six mammalian species, evaluated by the micronucleus test. , 1971, Mutation research.

[74]  R R Tice,et al.  In vivo rodent erythrocyte micronucleus assay. , 1994, Mutation research.

[75]  Leslie Recio,et al.  Investigation of the low-dose response in the in vivo induction of micronuclei and adducts by acrylamide. , 2009, Toxicological sciences : an official journal of the Society of Toxicology.

[76]  J. Grawé,et al.  Flow-cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood. , 1992, Cytometry.

[77]  S. Dertinger,et al.  An automated method for discriminating aneugen‐ vs. clastogen‐induced micronuclei , 1998, Environmental and molecular mutagenesis.

[78]  K. Camphausen,et al.  Three‐color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood , 2004, Environmental and molecular mutagenesis.