ToxSci Advance Access originally published online on August 3, 2006
Toxicological Sciences 2006 94(1):83-91; doi:10.1093/toxsci/kfl075
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Published by Oxford University Press 2006.
Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes: I. Intra- and Interlaboratory Comparison with Microscopic Scoring
* Litron Laboratories, Rochester, New York 14623
U.S. Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas 72079
Health Canada, Ottawa, Ontario, Canada K1A 0L2
National Institute of Health Sciences, Tokyo 158-8501, Japan
¶ Nitto Denko Corporation, Osaka 567-8680, Japan
|| An-Pyo Center, Shizuoka 437-1213, Japan
||| Astellas Pharma Inc., Tokyo 174-8511, Japan
|||| U.S. Food and Drug Administration, National Center for Toxicological Research, Rockville, Maryland 21012
1To whom correspondence should be sent at the present address: Toxicology Consulting Services, 201 Nomini Drive, Arnold, MD 21012. Fax: 410-975-0481. E-mail: jtmacgregor{at}earthlink.net.
Received February 2, 2006; accepted July 31, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Accumulating evidence suggests that reticulocytes (RETs) in the peripheral blood of rats may represent a suitable cell population for use in the micronucleus assay, despite the ability of the rat spleen to selectively remove micronucleated erythrocytes from the peripheral circulation. To evaluate the analytical performance of a previously described flow cytometric method (Torous et al., 2003, Toxicol. Sci. 74, 309–314) that may allow this assay to be conducted using peripheral blood in lieu of bone marrow sampling, we compared the sensitivity and performance characteristics of the flow cytometric technique with two established microscopy-based scoring methods. Peripheral blood samples from single Sprague-Dawley rats treated for 6 days with either vehicle or cyclophosphamide were prepared in replicate for scoring by the three methods at different laboratories. These blood-based measurements were compared to those derived from bone marrow specimens from the same animals, stained with acridine orange, and scored by microscopy. Through the analysis of replicate specimens, inter- and intralaboratory variability were evaluated for each method. Scoring reproducibility over time was also evaluated. These data support the premise that rat RETs harvested from peripheral blood are a suitable cell population to assess genotoxicant-induced micronucleus formation. The interlaboratory comparison provides evidence of the general robustness of the micronucleus endpoint using different analytical approaches. Furthermore, data presented herein demonstrate a clear advantage of flow cytometry–based scoring over microscopy—significantly lower inter- and intralaboratory variation and higher statistical sensitivity.
Key Words: flow cytometric analysis; reticulocytes; micronucleus test; CD71.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
The in vivo rodent erythrocyte micronucleus (MN) test is widely used in research and regulatory safety assessment to evaluate the potential of chemical and physical agents to cause chromosomal damage. Historically, MN studies based on rat peripheral blood have been avoided as it has been assumed that the efficiency by which the rat spleen filters out erythrocytes with intracellular inclusions would reduce assay sensitivity (Hayashi et al., 2000
; Wakata et al., 1998). However, accumulated data suggest that peripheral blood from intact rats can be used effectively to detect chemical-induced genotoxicity (Abramsson-Zetterberg et al., 1999; Asanami et al., 1995; Hamada et al., 2001; Hayashi et al., 1992; Hynes et al., 2002; Romagna and Staniforth, 1989; Torous et al., 2000, 2003; Wakata et al., 1998). Thus, it appears that MN studies using peripheral blood sampling in the rat have the potential to substitute for labor-intensive, bone marrow–based tests. In addition, the ability to use low-volume blood samples will facilitate integration of the assay into routine toxicology and/or pharmacokinetic studies and may make it unnecessary to conduct separate assays for the evaluation of chromosomal damage (Asanami et al., 1995; Hamada et al., 2001; MacGregor et al., 1995; Wakata et al., 1998). Before rat blood–based MN assays gain wider acceptance, especially in the context of regulatory testing requirements, additional information that allows direct comparisons between bone marrow and blood data is needed. Furthermore, the performance characteristics of the most widely utilized scoring techniques require further study. The experiments described herein were designed to address these issues of analytical performance by directly comparing values in blood and bone marrow obtained at different laboratories with three widely used methodologies, comparing values derived from two microscopy-based methods with a flow cytometry–based method that incorporates a calibration standard.
For each of the three scoring techniques, at least three proficient laboratories received replicate, coded samples for reticulocyte (RET) and MN-RET scoring. Proficiency was assumed based on the high level of training that has occurred at these laboratories (L1, L2, L3, L5, L6, and L7) and/or the frequency with which they contribute in vivo rodent MN data for regulatory submission purposes (L9, L10, and L11). See Table 1 for more detailed information regarding collaborating laboratories.
|
Data presented herein describe the performance characteristics of the three scoring methods evaluated, address the sensitivity of the rat peripheral blood compartment for detecting genotoxicant-induced micronuclei, and support recommendations concerning the minimum number of rat blood RET that should be evaluated for micronuclei.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Chemicals and other reagents.
Cyclophosphamide (CP) (CAS No. 6055-19-2) was purchased from Sigma, St Louis, MO. Acridine orange (AO)–coated slides used for supravital staining, prepared according to the method of Hayashi et al. (1990)
, were provided by the National Institute of Health Sciences, Japan. Flow cytometry reagents, including fixed malaria-infected rat blood (malaria biostandard) were from Rat MicroFlowPLUS Kits contributed by Litron Laboratories (available from Litron Laboratories, Rochester, NY and BD Biosciences PharMingen, San Diego, CA).
Animals and treatment regimens.
Animal studies were conducted in compliance with guidelines of the National Research Council (1996) "Guide for the Care and Use of Laboratory Animals" and were approved by the appropriate Institutional Animal Care and Use Committees. Two female Sprague-Dawley rats, 4- to 5-weeks old, were purchased from Taconic, Germantown, NY. Animals were housed singly and were assigned randomly to treatment groups. The animals were acclimated for approximately 2 weeks before the experiment was initiated. Food and water were available ad libitum throughout the acclimation and experimentation periods. One rat was treated via oral gavage with distilled water, and the other rat was treated by the same route with 10 mg CP/kg/day for 6 consecutive days.
Blood/bone marrow sample collection and storage.
Each day, before vehicle or CP treatment, low-volume blood samples (approximately 100 µl) were collected from the tail vein using a 26.5-gauge needle and syringe after a brief warming period under a heat lamp. These samples were fixed for flow cytometric analysis of RET and MN-RET frequencies according to procedures described in the Rat MicroFlowPLUS manual (v020213). Fixed samples were stored at – 85°C until analysis. Approximately 24 h after the last administration of vehicle or CP, blood samples were collected into tubes containing heparin solution (500 USP units heparin per milliliter of phosphate buffered saline) as follows: into a small tube containing 75 µl heparin solution, blood was collected until a final volume of approximately 750 µl was obtained; into a second tube containing 5 ml heparin solution, approximately 1 ml blood was collected. To tubes with the 750 µl blood suspension, an equal volume of heat-inactivated fetal bovine serum (FBS) was added. These FBS-diluted suspensions were used to prepare replicate AO-supravital (SV) slides (8 µl per slide) according to the method of Hayashi et al. (1990, 1992). These slides were frozen, shipped to collaborating SV-AO laboratories on dry ice, and stored frozen until analysis. FBS-diluted blood suspensions were also used to prepare slides for conventional acridine orange staining of methanol-fixed smears (MeOH-AO) staining (5 µl per slide). These blood smears were prepared by drawing the cell suspensions behind a second slide with smoothed edges (a "spreader slide"). These smears were allowed to air dry and were then fixed with absolute methanol for 10 min. The slides were stored in a slide box until they were shipped to collaborating MeOH-AO laboratories for MN scoring according to their standard operating procedures. Replicate bone marrow slides were prepared as smears, air dried, methanol fixed, and shipped similarly. These bone marrow cells were harvested from two femurs per rat, whereby both ends of each femur were cut and its contents flushed with 1 ml FBS. The cells were centrifuged at approximately at 1100 rpm for 5 min and then resuspended with approximately 600 µl FBS. As with the peripheral blood, 5 µl of cell suspension was applied to each slide. The 6 ml heparinized peripheral blood suspensions were fixed with ultracold methanol according to procedures described in the Rat MicroFlowPLUS manual (v020213) in order to preserve cells for flow cytometric analysis. These cell suspensions were stored at – 85°C until analysis or shipment on dry ice to collaborating flow cytometry laboratories.
The samples obtained were divided into three identical pools, and replicate samples of each pool were provided to participating laboratories with three separate codes. Thus, laboratories received triplicate samples of each condition, but were not aware that they were from an identical pool. Thus, the analyses conducted allow assessment of both intralaboratory variability of replicate analysis of identical samples and interlaboratory variability of the same analysis. Each laboratory also conducted analysis of each of these pools on three separate occasions, allowing assessment of variability of analysis over time.
Standard acridine orange slide scoring (MeOH-AO).
Blood and bone marrow smears were scored using the MeOH-AO scoring technique at the Food and Drug Administration-National Center for Toxicological Research laboratory (L1) and three contract testing laboratories (L9, L10, and L11). Methanol fixation leads to a diffuse distribution of RNA, and erythrocytes are classified as normochromatic or as RETs based on the presence or absence of RNA-associated fluorescence. This technique is not well suited for visually classifying subpopulations of RETs. RET frequencies were determined by inspecting 500 or 1000 total erythrocytes per bone marrow or blood sample, respectively. MN-RET incidence was determined by inspecting 2000 RETs per sample. At L1, micronuclei were defined by the criteria of Schmid (1976) with the added requirements that they exhibit the characteristic yellow to yellow-green fluorescence characteristic of AO staining and that they exhibit the smooth boundary expected from a membrane-bound body. Laboratories L9, L10, and L11 were instructed to follow the standard operating procedures they use for regulatory submissions to support new drug or food additive development. Thus, the acquisition of data by these facilities allows for comparisons with three highly experienced contract laboratories under conditions associated with regulatory testing.
Supravital acridine orange slide scoring (SV-AO).
Laboratories L5, L6, and L7 scored peripheral blood samples using the SV-AO scoring technique. This staining procedure aggregates RNA, leading to punctate staining patterns. These staining characteristics allow RET to be classified into four age cohorts: Type I (youngest) through Type IV (oldest) RETs as described by Hayashi et al. (1990, 1992). The frequency of MN-RETs was determined by analyzing 2000 Type I and Type II RETs (L5 and L7) or 2000 Type I RETs (L6). An index of cytotoxicity was obtained by inspecting at least 400 RETs and calculating the percentage of Type I and Type II RET among total RETs (L5 and L7) or the percentage of Type I RETs among Type I and Type II RETs (L6). AO-coated slides were purchased from TOYOBO (Osaka, Japan). Supravitally stained triplicate slides were frozen and sent to the Japanese reference laboratory (Nitto Denko) with dry ice. Each set of slides was also sent to two other laboratories for replicate scoring by fluorescence microscopy.
Flow cytometry–based scoring.
Methanol-fixed blood samples were washed and labeled for flow cytometric analysis by L1, L2, and L3 according to procedures described in the Rat MicroFlowPLUS Kit (v020213). Samples were analyzed with 488-nm capable instruments (FACSCalibur, FACSort, and FACScan, all from Becton Dickinson, San Jose, CA). Anti-CD71-FITC and propidium iodide fluorescence signals were detected in the FL1 and FL3 channels, respectively (stock filter sets). Calibration of the flow cytometers for the MN scoring application, across laboratories and between experiments within each laboratory, was accomplished by staining Plasmodium berghei–infected rat blood (malaria biostandards) in parallel with test samples on each day of analysis (Dertinger et al., 2000; Tometsko et al., 1993; Torous et al., 2001). By adjusting voltages applied to the photomultiplier tube, it was possible to standardize the FL3 fluorescence channel into which erythrocytes with single (MN like) parasites fell. In this manner, analysis regions were consistent across laboratories and between experiments. Flow cytometry–based MN-RET measurements reported herein are based on an immature fraction of peripheral blood RETs (approximately the youngest 30–50% of propidium iodide–positive erythrocytes, based on CD71 expression level; Torous et al., 2001, 2003). This is thought to be analogous to scoring the youngest (Types I and II) RETs using the SV-AO method, which may be beneficial in view of reports which have suggested that the influence of rat spleen filtration function can be minimized by scoring the younger RETs (Abramsson-Zetterberg et al., 1999; Hayashi et al., 1992; Hynes et al., 2002; Torous et al., 2000, 2003). Data were acquired with CellQuest software (v3.3, BD-Immunocytometry Systems, San Jose, CA), with the stop mode set so that 20,000 high CD71-expressing RETs were analyzed per blood sample. The number of mature (CD71 negative) erythrocytes was determined concurrently, providing an index of cytotoxicity (%RETs).
Calculations.
All calculations were performed with Excel (Office X for Mac or Microsoft Office Excel 2002 for XP Windows Professional, Microsoft Corp., Seattle, WA). The incidences of MN-RETs are expressed as frequency percent. The percentage of RETs among total erythrocytes was measured by the flow cytometric and MeOH-AO laboratories and served as an index of bone marrow cytotoxicity. The three SV-AO laboratories used percentage of RETs in different stages of maturity as an index of toxicity; therefore, these indices are not directly comparable to those obtained by the flow cytometric and MeOH-AO microscopy laboratories. Percent coefficient of variance values (%CV, i.e., standard deviation (SD) as percent of the mean) were used to describe intralaboratory variability associated with multiple readings of replicate samples and also interlaboratory variation of vehicle control and CP-induced MN-RET measurements that were pooled according to scoring method.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Confirmation of Steady State
RET and MN-RET measurements obtained from the daily low-volume blood specimens were analyzed to confirm that the MN-RET frequency of the vehicle-treated animal was stable over the duration of the experiment and that CP treatment caused the MN-RET frequency to increase to a steady-state level of approximately 10-fold the control frequency (Fig. 1). Since the frequency of MN-RETs was at steady state in both cases, the values in bone marrow and peripheral blood should be directly comparable—that is, expected to be equal in the absence of selective removal of MN-RETs from blood or methodological differences in measurement. Thus, the samples collected in this manner allow the direct comparisons between measurements in the bone marrow and blood compartments that follow. The use of large samples from a single treated and a single control rat allows differences in methodology and scoring laboratory to be assessed independently of sample variation.
|
The dose of CP (10 mg/kg/day) had a moderate effect on erythropoiesis, as indicated by the decline in RET frequency (terminal day specimen showed a greater than 50% decrease from pretreatment value; see Fig. 1, panel a). This level of bone marrow cytotoxicity is well within the range of target toxicity recommended by current regulatory guidances (i.e.,
80%, see Organisation for Economic Cooperation and Development, 1997, Guideline 474; U.S. Food and Drug Administration, 2000. To illustrate the nature and source of the flow cytometry–based data described above, bivariate fluorescence intensity plots are provided (Fig. 2). Note the appearance of micronuclei on Day 1 in the very youngest (highest anti-CD71-FITC fluorescence) RETs (Panel b) and the more uniform distribution among RETs after a steady state has been reached on Day 3 of treatment (Panel c). Panel (d) illustrates the use of the malaria biostandard, with distinct fluorescence intensities corresponding to inclusion of one, two, or three parasites. This allows the instrument settings to be standardized to the DNA content of the parasite, which is controlled biologically to a quantity similar to that in an average MN. For research purposes, the regions may be adjusted to allow measurements in different age populations of RETs and/or micronuclei with different DNA contents. For analytical purposes, the standard can be used to achieve comparable instrument performance across time within a laboratory or across different instruments in different laboratories.
|
Intra- and Interlaboratory Variability
Replicate bone marrow and/or peripheral blood specimens obtained after 6 consecutive days of treatment were provided to each collaborating laboratory. As noted above, the frequencies of MN-RETs were at steady state and therefore not changing as a function of time. Each laboratory received three separately coded samples from each of the high and low MN-RET frequency pools but were not aware that the three separately coded samples were identical. Tabular values are the means of the values of the three separately coded samples.
Most laboratories detected a reduction in %RET for the CP-treated rat (see Table 2). However, this was somewhat variable across microscopy-based laboratories, especially when the MeOH-AO technique was used to evaluate bone marrow specimens. In two of the three laboratories that scored both bone marrow and peripheral blood, peripheral blood measurements demonstrated greater CP-associated reduction of %RETs than in bone marrow. Intra- and interlaboratory %CV values for the replicate RET analyses are presented in Table 2. Flow cytometric measurements were more consistent within and across laboratories than microscopic scoring. For instance, vehicle control specimens' %CV for pooled laboratory MeOH-AO/bone marrow data was 13.6%, while the corresponding blood-based analyses for flow cytometric, SV-AO, and MeOH-AO techniques were 1.93, 6.5, and 12.5%, respectively.
|
The interlaboratory %CV values for MN-RET determinations and the intralaboratory %CV values for the triplicate blinded analyses conducted within each laboratory are provided in Table 3. The flow cytometric analyses demonstrate superior intra- and interlaboratory consistency relative to both microscopy-based methods. %CV values for MN-RET measurements performed on vehicle control blood specimens pooled across like-method laboratories were 26.5, 94.3, and 85.6% for the flow cytometric, SV-AO, and MeOH-AO methods, respectively, and 80.5% for MeOH-AO scored bone marrow. Analogous %CV values for CP blood samples were 7.6, 24.7, and 48.2%, respectively, and 29.1% for MeOH-AO scored bone marrow.
|
Fold difference values based on each laboratory's average MN-RET frequencies, as well as for like-method pooled data, are also presented (Table 3). It was somewhat surprising that the fold difference in MN-RETs between vehicle and CP-associated blood specimens, as well as absolute MN-RET frequencies, were no higher with the flow cytometric or SV-AO techniques than with the conventional MeOH-AO method as it has been reported that restriction of MN analysis to an immature RET cohort based on RNA content or CD71 expression levels could reduce, if not eliminate, the influence of the spleen's erythrophagocytotic activity (Abramsson-Zetterberg et al., 1999
; Hayashi et al., 1992). Splenic activity and its effects on assay sensitivity for blood-based analyses have been investigated thoroughly, and these data are discussed in a companion paper that appears in this issue (MacGregor et al.).
Intralaboratory Variability Across Time
In addition to the inter- and intralaboratory analyses, an evaluation of scoring reproducibility over time was studied. This was accomplished by having flow cytometry laboratories analyze coded peripheral blood specimens on three or four different occasions, while triplicate vehicle and CP bone marrow slides were submitted to L9, L10, and L11 laboratories for analysis on two separate occasions. Reagents were prepared separately for each day of analysis. The resulting repeat-analysis RET data are presented in Figure 3 and demonstrate higher reproducibility for the multiple flow cytometric analyses compared to MeOH-AO.
|
As with RET enumeration, repeat-analysis MN-RET microscopy data were also quite variable. For instance, laboratories using the MeOH-AO method reported average CP-induced values that differed from their original mean reading by 19.4, 50.9, 58.4, and 8.6% (L1, L9, L10, and L11, respectively; see Fig. 4). Repeat-analysis MN-RET data generated by the flow cytometric technique were considerably more reproducible as average values were all within 11% of the originally reported mean frequencies. It should also be noted that the fourth flow cytometric analyses by L2 was performed more than 2 years after blood fixation, demonstrating this procedure's compatibility with long-term storage of fixed blood specimens.
|
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Distribution of replicate bone marrow and blood specimens obtained from single rats that were first shown to exhibit steady-state spontaneous or genotoxicant-induced MN-RET frequencies were used to assess inter- and intralaboratory scoring variability using two widely used microscopic and one flow cytometric procedure. These results demonstrate that the quantification of MN-RETs benefits from an objective flow cytometry–based method of data acquisition. The flow cytometric method provides better reproducibility, and the high throughput capability allows interrogation of tens of thousands of RETs per specimen. Enhanced scoring precision is important as it is necessary to offset the spleen-dependent loss of dynamic range observed in peripheral blood relative to bone marrow—a phenomenon that was observed in this as well as other reports (MacGregor et al., this issue; Wakata et al., 1998
). A recent report by Torous et al. (2006) delineates the consequential improvements to assay power when the number of cells scored per specimen is increased and supports this view.
Beyond overcoming lower genotoxicant-induced MN-RET frequencies in blood relative to bone marrow, further incentive for automating rat peripheral blood MN-RET measurements comes from a recent recommendation of the In Vivo MN Assay Expert Group of the International Working Group on Genetic Toxicology Testing (IWGT; Hayashi et al., in press). Specifically, IWGT has recommended that a sufficient number of RETs should be scored to ensure that the MN-RET counting error is kept below the level of interanimal variability. This allows the sensitivity of the experiment to be limited by the variability of spontaneous MN-RET frequency among animals, rather than being limited by the statistical variation of count. Based on the flow cytometric scoring of 20,000 peripheral blood RETs from each of the 15 control animals from the three experiments reported in the MacGregor et al. companion paper in this issue (laboratory L2, the reference laboratory), we find that the mean incidence of MN-RET ± 1 SD is 0.11% ± 0.045. This is a 41% CV. Poisson distribution theory allows us to calculate that 6 MN-RETs per animal must be scored to limit counting error to this level of variation (SD of the 
). At a spontaneous MN-RET frequency of approximately 0.1%, this means that an average of 6000 RETs per individual need to be scored for micronuclei in order to achieve a CV that is at or below the interanimal variance. This is a significantly higher number of RETs per animal than required to be scored under the current OECD MN assay guideline (which recommends scoring 2000 RETs per animal) and is difficult to achieve by manual microscopic scoring.
In conclusion, the data presented herein and in the companion paper that follows support the growing consensus that rat peripheral blood can be used to perform in vivo MN tests more effectively than the standard bone marrow–based assay. The ability of the described automated scoring procedure to greatly enhance the precision of MN-RET measurements overcomes the somewhat attenuated genotoxicant-induced frequencies observed in peripheral blood relative to bone marrow. This conclusion is supported by experiments described in the accompanying paper whereby intact and splenectomized rats were treated with diverse genotoxicants (MacGregor et al., this issue).
|
ACKNOWLEDGMENTS |
|---|
This work was supported by an intercenter research award from the Food and Drug Administration Office of Science and Health Coordination to J.T.M. The contents are the sole responsibility of the authors and do not necessarily represent the official views of the U.S. Food and Drug Administration, the National Institute of Health Sciences, Japan, or Health Canada. We would like to thank Nikki Hall for her expert assistance with bleeding and dosing procedures. Thanks also to Puntipa Kwanyuen and Dr Neal Cariello, two researchers who were early adopters of the flow cytometric technique described herein. Their experiences with an early version of the method led to the development of malaria-infected erythrocytes as daily calibration standards. Conflict of interest disclosure: Litron Laboratories holds patents pertaining to flow cytometric enumeration of micronucleated erythrocyte populations and also sells kits that facilitate these analyses. The authors express their appreciation for the cooperation and contributions of the staff of the 3 contract laboratories that participated in these studies: Drs Robert Young and Rama Gudi of BioReliance, Rockville, MD; Dr Gregory Erexson of Covance, Inc., Vienna, VA; and Dr John Mirsalis and Edward Riccio of SRI International, Menlo Park, CA.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Abramsson-Zetterberg L, Grawe J, Zetterberg G. (1999) The micronucleus test in rat erythrocytes from bone marrow, spleen and peripheral blood: The response to low doses of ionizing radiation, cyclophosphamide and vincristine determined by flow cytometry. Mutat. Res 423:113–124.[Web of Science][Medline]
Asanami S, Shimono K, Sawamoto O, Kurisu K, Uejima M. (1995) The suitability of rat peripheral blood in subchronic studies for the micronucleus assay. Mutat. Res 347:73–78.[CrossRef][Web of Science][Medline]
Dertinger SD, Camphausen K, MacGregor JT, Bishop ME, Torous DK, Avlasevich S, Cairns S, Tometsko CR, Menard C, Muanza T, et al. (2004) Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood. Environ. Mol. Mutagen 44:427–435.[CrossRef][Web of Science][Medline]
Dertinger SD, Torous DK, Hall NE, Tometsko CR, Gasiewicz TA. (2000) Malaria-infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry. Mutat. Res 464:195–200.[Web of Science][Medline]
Hamada S, Sutou S, Morita T, Wakata A, Asanami S, Hosoya S, Ozawa S, Kondo K, Nakajima M, Shimada H, et al. (2001) 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). Environ. Mol. Mutagen 37:93–110.[CrossRef][Web of Science][Medline]
Hayashi M, Kodama Y, Awogi T, Suzuki T, Asita AO, Sufuni T. (1992) The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats. Mutat. Res 278:209–213.[CrossRef][Web of Science][Medline]
Hayashi M, MacGregor JT, Gatehouse DG, Adler I-D, Blakey DH, Dertinger SD, Krishna G, Morita T, Russo A, Sutou S. (2000) In vivo rodent erythrocyte micronucleus assay: Aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring. A report from the International Workshop on Genotoxicity Test Procedures (IWGTP). Environ. Mol. Mutagen 35:234–252.[CrossRef][Web of Science][Medline]
Hayashi M, MacGregor JT, Gatehouse DG, Blakey DH, Dertinger SD, Abramsson-Zetterberg L, Krishna G, Morita T, Russo A, Asano N, 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. Environ. Mol. Mutagen (in press).
Hayashi M, Morita T, Kodama Y, Sofuni T, Ishidate M Jr. (1990) The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides. Mutat. Res 245:245–249.[CrossRef][Web of Science][Medline]
Hynes GM, Torous DK, Tometsko CR, Burlingson B, Gatehouse DG. (2002) The single laser flow cytometric micronucleus test: A time course study using colchicines and urethane in rat and mouse peripheral blood and acetaldehyde in rat peripheral blood. Mutagenesis 17:15–23.
MacGregor JT, Tucker JD, Eastmond DA, Wyrobek AJ. (1995) Integration of cytogenetic assays with toxicology studies. Environ. Mol. Mutagen 25:328–337.[Web of Science][Medline]
MacGregor JT, Wehr CM, Hiatt RA, Peters B, Tucker JD, Langlois RG, Jacob RA, Jenson RH, Yager JW, Shigenaga MK, et al. (1997) "Spontaneous" genetic damage in man: Evaluation of interindividual variability, relationship among markers of damage, and influence of nutritional status. Mutat. Res 377:125–135.[Web of Science][Medline]
Organisation for Economic Cooperation and Development (OECD) (1997). OECD Guideline 474. Guideline for the testing of chemicals. Mammalian erythrocyte micronucleus test.
Romagna F and Staniforth CD. (1989) The automated bone marrow micronucleus test. Mutat. Res 213:91–104.[Web of Science][Medline]
Schmid W. (1976) The micronucleus test for cytogenetic analysis. In Hollaender A (Ed.). Chemical Mutagens: Principles and Methods for their Detection(Plenum Press, New York) Vol. 4: pp. 31–53.
Tometsko AM, Torous DK, Dertinger SD. (1993) Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model. Mutat. Res 292:129–135.[CrossRef][Web of Science][Medline]
Torous D, Asano N, Tometsko C, Sugunan S, Dertinger S, Morita T, Hayashi M. (2006) Performance of flow cytometric analysis for the micronucleus assay—A reconstruction model using serial dilutions of malaria infected cells with normal mouse peripheral blood. Mutagenesis 21:11–13.
Torous DK, Dertinger S, Hall N, Tometsko C. (2000) Enumeration of micronucleated reticulocytes in rat peripheral blood: A flow cytometric study. Mutat. Res 465:91–99.[Web of Science][Medline]
Torous DK, Hall NE, Dertinger SD, Diehl M, Illi-Love AH, Cederbrant K, Sandelin K, Bolcsfoldi G, Ferguson LR, Pearson A, et al. (2001) Flow cytometric enumeration of micronucleated reticulocytes: High transferability among 14 laboratories. Environ. Mol. Mutagen 38:59–68.[CrossRef][Web of Science][Medline]
Torous DK, Hall NE, Murante FG, Gleason SE, Tometsko CR, Dertinger SD. (2003) Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy. Toxicol. Sci 74:309–314.
U.S. Food and Drug Administration (2000) Office of Food Additive Safety, Redbook (2000). Toxicological Principals for the Safety Assessment of Food Ingredients. Available at: http://www.cfsan.fda.gov/
redbook/red-toca.html. Updated on November 2003. U.S. Food and Drug Administration, Washington, D.C.
Wakata A, Miyamae Y, Sato S, Suzuki T, Morita T, Asano N, Awogi T, Kondo K, Hayashi M. (1998) 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 32:84–100.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. E. Hotchkiss, M. E. Bishop, S. D. Dertinger, W. Slikker Jr, M. M. Moore, and J. T. MacGregor Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes IV: An Index of Chromosomal Damage in the Rhesus Monkey (Macaca mulatta) Toxicol. Sci., April 1, 2008; 102(2): 352 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Harper, S. D. Dertinger, M. E. Bishop, A. M. Lynch, M. Lorenzo, M. Saylor, and J. T. MacGregor Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes III. An Efficient Method of Monitoring Chromosomal Damage in the Beagle Dog Toxicol. Sci., December 1, 2007; 100(2): 406 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Vikram, P. Ramarao, and G. Jena Prior bleeding enhances the sensitivity of peripheral blood and bone marrow micronucleus tests in rats Mutagenesis, July 1, 2007; 22(4): 287 - 291. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||