Human blood PIG-A mutation and micronucleated reticulocyte flow cytometric assays: Method optimization and evaluation of intra- and inter-subject variation

We previously described flow cytometry-based methods for scoring the incidence of micronucleated reticulocytes (MN-RET) and PIG-A mutant phenotype reticulocytes (MUT RET) in rodent and human blood samples. The current report describes important methodological improvements for human blood analyses, including immunomagnetic enrichment of CD71-positive reticulocytes prior to MN-RET scoring, and procedures for storing frozen blood for later PIG-A analysis. Technical replicate variability in MN-RET and MUT RET frequencies based on blood specimens from 14 subjects, intra-subject variability based on serial blood draws from 6 subjects, and inter-subject variation based on up to 344 subjects age 0 to 73 years were quantified. Inter-subject variation explained most of the variability observed for both endpoints (≥77%), with much lower intra-subject and technical replicate variability. The relatively large degree of inter-subject variation is apparent from mean and standard deviation values for MN-RET (0.15 ± 0.10%) and MUT RET (4.7 ± 5.0 per million, after omission of two extreme outliers). The influences of age and sex on inter-subject variation were investigated, and neither factor affected MN-RET whereas both influenced MUT RET frequency. The lowest MUT RET values were observed for subjects <11 years old, and males had moderately higher frequencies than females. These results indicate that MN-RET and MUT RET are automation-compatible biomarkers of genotoxicity that bridge species of toxicological interest to include human populations. These data will be useful for appropriately designing future human studies that include these biomarkers of genotoxicity, and highlight the need for additional work aimed at identifying the sources of inter-individual variability reported herein.     

Flow cytometric analysis of micronucleated reticulocytes: Time- and dose-dependent response of known mutagens in mice, using multiple blood sampling

According to the current Organization of Economic Cooperation and Development (OECD) and International Committee on Harmonization (ICH) guidelines for the mammalian erythrocyte micronucleus (MN) test, analysis of peripheral blood reticulocytes (RETs) for the presence of micronuclei can be performed using flow cytometry. The MicroFlow PLUS method (Litron Laboratories, Rochester, NY) for MN analysis by flow cytometry is based on the binding of FITC-labeled antibodies to the CD71 transferrin receptor of immature RETs, on parallel RNA degradation, and on propidium iodide staining of DNA present as micronuclei. The objective of this study was to assess the sensitivity of this flow cytometry method to detect time- and dose-dependent induction of micronuclei in mouse peripheral blood RETs after treatment with nine chemical agents. Five known clastogens, two known aneugens, and two compounds previously reported to be inactive in the mouse bone marrow MN test were evaluated at three dose levels. Multiple blood sampling of the same animal before and at two time points after treatment was conducted. All known mutagens produced a dose-dependent increase in micronucleated reticulocytes (MN-RETs); the compounds previously shown to be inactive in the in vivo MN test were also negative using the present methodology. The highest frequency of MN-RETs was observed at 48 hr after treatment, except for 5-fluorouracil, which had its peak response at 72 hr. The results indicate that micronuclei can be measured by multiple blood sampling of the same animal before and after treatment without altering the sensitivity of the assay. The results confirm that the flow cytometric assessment of MN-RETs in mouse peripheral blood using MicroFlow PLUS is a sensitive method with high analysis throughput, and robust quality control.     

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.     

Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood

Experiments described herein were designed to evaluate the performance characteristics of a flow cytometry-based system that scores the incidence of peripheral blood micronucleated reticulocytes (MN-RETs). These procedures represent the continued refinement of a previously reported anti-CD71-based method (Dertinger et al. [1996]: Mutat Res 371:283-292), with the following modifications: incorporation of a third fluorescent label to exclude platelets from the MN-RET region, and use of a CD71-associated fluorescence thresholding technique to increase data acquisition rates. Mouse, rat, and human blood samples were analyzed using both the previously described two-color procedure (anti-CD71-FITC and propidium iodide) and a newly developed three-color technique (which adds an antiplatelet-PE antibody). The rodent specimens were also evaluated by standard microscopy procedures (acridine orange staining). Mouse blood was collected via heart puncture of vehicle- and 5-fluorouracil-treated CD-1 mice; blood samples from saline-treated Sprague-Dawley rats were collected from the tail vein and via heart puncture. Rodent blood samples were analyzed by both the two- and three-color methods. Human blood specimens, obtained via arm venipuncture from cancer patients undergoing radiation therapy, were analyzed for MN-RETs using the two-color method. Subsequently, blood samples from a single chemotherapy patient were analyzed by both the two- and three-color methods. Finally, the chemotherapy patient blood samples and blood samples from 15 healthy volunteers were evaluated at very high densities in conjunction with a CD71-associated fluorescence thresholding technique. Results of these investigations showed that data from mouse blood analyzed by the two- and three-color procedures correlated well with microscopy data (r values = 0.917 and 0.937 for the two- and three-color methods, respectively); all three methods confirmed the genotoxicity of 5-FU. Data from rat tail vein samples showed improved reproducibility with the three-color technique, but no significant difference between the two techniques was seen with the heart puncture specimens. Human blood analyzed according to the two-color procedure produced unreliable results, as platelets and platelet aggregates impacted the rare MN-RET scoring region. The three-color technique effectively overcame this problem and produced reproducible measurements that fell within expected ranges. For human blood analyses, the high cell density/CD71-thresholding technique provided significant improvements over the low-density technique, as it allowed data acquisition to occur approximately six times faster with no loss of sensitivity.     

Spontaneous and mitomycin C-induced micronuclei in peripheral blood reticulocytes from severely malnourished rats

Severe malnutrition caused by deficiencies in protein, calorie, and micronutrient intake is widely distributed throughout the world and is a particular problem in developing countries. Animal models have been useful for studying the effects of malnutrition under different experimental conditions. In this study, we have evaluated the effect of malnutrition on the frequency of spontaneous and mitomycin C (MMC)-induced micronuclei in the peripheral blood of rats measured using a flow cytometric analysis technique. Neonatal rats were experimentally malnourished during lactation and assayed at weaning (21 days of age). The malnourished rats weighed 49.2% less than well-nourished controls and had lower concentrations of serum protein, triglycerides, and cholesterol. In rats not treated with MMC, the frequency of micronucleated reticulocytes (MN-RETs) was 1.6 times greater in malnourished rats than in well-nourished rats (0.48% +/- 0.16% vs. 0.31% +/- 0.09%). The mean MN-RET frequency measured 32 hr after treatment with single i.p. doses of 0.5, 0.75, or 1.0 mg/kg of MMC was 0.60 +/- 0.10 vs. 0.84 +/- 0.14, 1.21 +/- 0.52 vs. 2.36 +/- 0.47, and 2.50 +/- 0.06 vs. 4.64 +/- 1.14 for well-nourished vs. malnourished rats, respectively. Statistical comparisons indicate significant differences between the two groups of rats at all doses tested. Malnourishment and MMC treatment had no significant effects on the frequencies of RETs or micronucleated normochromatic erythrocytes. The data indicate that protein-calorie malnutrition during lactation is associated with increased frequencies of MN-RETs, which are indicative of chromosome damage. These findings suggest that malnutrition could result in greater susceptibility to environmental damage.     

Trimethoprim-sulfamethoxazole increase micronuclei formation in peripheral blood from weanling well-nourished and malnourished rats

The combination of trimethoprim and sulfamethoxazole (TMP-SMX) is a widely used drug. In spite of this, there are few reports on its genotoxicity, and the results are controversial. Severe malnutrition is a complex condition that increases the susceptibility to infections. Consequently, drugs are extensively used in malnutrition cases. Experimental animal models have been widely used to study the effects of malnutrition. Neonatal rats were experimentally malnourished (UN) during lactation. The UN rats weighed 51.1% less than the well-nourished (WN) controls and had lower concentrations of serum protein and blood lipids. The micronucleus (MN) assay is useful for detecting chromosome damage induced by nutritional deficiencies. In vivo rodent MN assays have been widely used to screen genotoxic agents. In this study, we have evaluated the frequency of spontaneous and TMP-SMX-induced micronuclei in the peripheral blood of weanling (21 days of age) rats using a flow cytometric analysis technique. The spontaneous frequency of micronucleated reticulocytes (MN-RETs) was 2.7 times greater in the UN rats than in the WN rats. In rats that were not treated with TMP-SMX, the percentage of reticulocytes was significantly lower (41.1%) in the UN rats than the WN controls. A therapeutic dose of TMP-SMX (80 mg/kg (TMP), 400 mg/kg (SMX) for 48 hr) increased MN-RETs in the WN and in the UN rats. The data demonstrate the genotoxic effect of this drug. The results indicate that severe protein-calorie restriction and drug treatment enhance DNA damage in rat peripheral blood reticulocytes, potentially increasing the risk of negative effects on health.     

The single laser flow cytometric micronucleus test: a time course study using colchicine and urethane in rat and mouse peripheral blood and acetaldehyde in rat peripheral blood.

A single laser flow cytometric procedure to quantify micronucleus frequency in rat and mouse peripheral blood was evaluated. Reticulocytes express the transferrin receptor (also known as the CD71-defined antigen). When combined with a DNA stain, antibodies against this antigen can be used to differentially label and quantify micronucleated reticulocytes. The object of this study was to evaluate the method for rat and mouse peripheral blood using flow cytometry and compare the results obtained between two laboratories (GlaxoWellcome and Litron Laboratories). The compounds selected were the rodent carcinogens colchicine, urethane and acetaldehyde. Colchicine gives a positive response in the rat bone marrow micronucleus assay and an inconclusive result in the rat peripheral blood micronucleus assay. The latter two are both established rat carcinogens readily detected in both the bone marrow and peripheral blood micronucleus assays. In these experiments both rat and mice were treated with either colchicine or urethane and rats alone treated with acetaldehyde. After a single treatment, repeat sampling of peripheral blood was made at 0, 24, 48 and 72 h. Replicate blood samples were obtained and fixed for flow cytometric analysis at both facilities. The micronucleated reticulocyte frequency of each blood sample was determined by analysing 20 000 total reticulocytes per blood sample. The data suggest that the single laser flow cytometric procedure resulted in consistent reticulocyte and micronucleated reticulocyte frequencies between laboratories. Furthermore, these flow cytometric data compare favourably with previously published data.     

Interlaboratory assessment of mitotic index by flow cytometry confirms superior reproducibility relative to microscopic scoring

A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose‐dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland–Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Assessment of systemic genetic damage in pediatric inflammatory bowel disease

The etiology of distal site cancers in inflammatory bowel disease (IBD) is not well understood and requires further study. We investigated whether pediatric IBD patients' blood cells exhibit elevated levels of genomic damage by measuring the frequency of mutant phenotype (CD59-/CD55-) reticulocytes (MUT RET) as a reporter of PIG-A mutation, and the frequency of micronucleated reticulocytes (MN-RET) as an indicator of chromosomal damage. IBD patients (n = 18 new-onset disease, 46 established disease) were compared to age-matched controls (constipation or irritable bowel syndrome patients from the same clinic, n = 30) and young healthy adults age 19–24 (n = 25). IBD patients showed no indication of elevated MUT RET relative to controls (mean ± SD = 3.1 ± 2.3 × 10⁻⁶ vs. 3.6 ± 5.6 x 10⁻⁶, respectively). In contrast, 59 IBD patients where %MN-RET measurements were obtained, 10 exceeded the upper bound 90% tolerance interval derived from control subjects (i.e., 0.42%). Furthermore, each of the 10 IBD patients with elevated MN-RET had established disease (10/42), none were new-onset (0/17) (p = .049). Interestingly, each of the subjects with increased chromosomal damage was receiving anti-TNF based monotherapy at the time blood was collected (10/10, 100%), whereas this therapy was less common (20/32, 63%) among patients that exhibited ≤0.42% MN-RET (p = .040). The results clearly indicate the need for further work to understand whether the results presented herein are reproducible and if so, to elucidate the causative factor(s) responsible for elevated MN-RET frequencies in some IBD patients.     

In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability

The in vitro micronucleus test has received considerable attention in recent years for its use in drug safety assessment and toxicological research. The less tedious nature of the assay relative to chromosome aberration analyses is a driving force, and explains why many chemical and drug safety programs have adopted the endpoint. Development of a high-throughput micronucleus scoring system would further enhance the utility of the assay for lead optimization and other early drug development work. Although several variations of a flow cytometric (FCM) method for scoring cell-culture-derived micronuclei (MN) have been described in the literature, they have been unable to distinguish true MN from apoptotic and necrotic chromatin (Nüsse M and Marx K 1997: Mutat Res 392: 109-115). Here, we report advances to this methodology whereby a sequential staining procedure is used to differentially label these types of sub-2n particles. With the use of ethidium monoazide (EMA), the chromatin of dead and dying cells is labeled. After a photoactivation step that covalently binds EMA to chromatin, cytoplasmic membranes are digested and resulting lysates are incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and sub-2n particles that are labeled with either SYTOX or SYTOX and EMA. Preliminary studies with heat-shocked L5178Y mouse cells demonstrated that EMA stains necrotic and mid- through late-stage apoptotic cells. Importantly, the sequential labeling procedure provided reliable micronucleus enumeration, even when cultures contained high percentages of dead cells. Subsequently, experiments with the following diverse genotoxicants were performed: hydroxyurea, methyl methanesulfonate, benzo[a]pyrene, etoposide, cyclophosphamide, and vinblastine. Additionally, the nongenotoxicants sucrose, tributyltin methoxide, and dexamethasone were tested up to 5 mg/ml, or to cytotoxic concentrations. FCM data were found to correspond closely with microscopy-based measurements. Collectively, these data suggest that this sequential EMA/SYTOX staining procedure provides reliable, high-throughput enumeration of in vitro MN.     

Reticulocyte counts in laboratory animals using a manual and a flow cytometric procedure (sysmex R-3000?)

An evaluation of the flow cytometric procedure for reticulocyte counting was performed, together with a comparison to a reference manual counting method. An automated reticulocyte analyser, the Sysmex R-3000™ (Flow cytometric auramine O methods) was used to enumerate the absolute reticulocyte counts and the percentage of reticulocytes in blood obtained from rats, dogs and monkeys. Simultaneously, a maturation distribution was obtained by subdividing the reticulocytes into three maturation classes according to their fluorescence intensity. The reticulocyte counts and the maturation distributions can be determined rapidly and easily. The coefficients of variation (CV) for the within-day precision study in the reticulocyte counts of the three species were found to be 9.7%–20.5% for the micro-scopic method and 4.4%–9.4% for the flow cytometric method. A very good correlation was observed between the microscopic count and flow cytometric methods (r=0.958). In conclusion, because of high reproducibility and good agreement with the manual counting method for these species, flow cytometric analysis of reticulocytes is an attractive alternative procedure to micro-scopic enumeration in toxicological studies.     

Clinical flow cytometric reticulocyte analysis.

The flow cytometric (FCM) analysis of reticulocytes in a clinical laboratory can be accomplished using acridine orange (AO), thiazole orange (TO), auramine O, thioflavin T, pyronin Y, dimethyl-oxacarbocyanine or transferrin receptor assays. AO and TO are vital stains, show good correlation with microscopic reticulocyte determinations and, when compared to each other, give an excellent correlation. The coefficient of variation is below 5% and batch analysis on blood samples stored for 96 h further reduces manpower needs. Finally we have used the mean fluorescent intensity of TO as a reticulocyte maturity index (RMI). In bone marrow transplant patients, the RMI has been the earliest indicator of marrow engraphment. Using the RMI we have been able to define three patterns of engraphment: early, delayed and failed. Although a variety of standards will be required, the clinical FCM reticulocyte analysis promises to be the preferred and accepted method in the clinical laboratory.     

Flow cytometric scoring of micronucleated reticulocytes as a possible high‐throughput radiation biodosimeter

Micronucleated reticulocyte (MN‐RET) scoring by flow cytometry (FCM) has been used in assessment of the clastogenic effects of chemicals. However, its dose–response to acute whole body irradiation (WBI) at moderate dose rates remains to be established. We show that FCM scoring of MN‐RET in peripheral blood from male ICR mice exposed to WBI X‐ray doses of 0.5–5 Gy at a dose rate of 0.488 Gy/min exhibits a linear dose–response relationship 24, 48, and 72 hr following WBI. Parallel microscopic counting of micronucleated polychromatic erythrocytes (MN‐PCE) in bone marrow smears from the same animals showed similar linear dose–response patterns at the same time points. Indeed, MN‐RET and MN‐PCE were highly correlated at all doses and time points. In view of the speed and accuracy of this method, in addition to the small blood sample size needed for the assay, the flow cytometric protocol for MN‐RET scoring may provide a minimally‐invasive, high throughput radiation biodosimeter. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Interlaboratory Pig-a gene mutation assay trial: Studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (～20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [2011]: Mutat Res 721:163-170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN-RET) counts. 1,3-PS was found to increase Pig-a mutation and MN-RET frequencies in both 3- and 28-day study designs. While the greatest induction of MN-RETs was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one-third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated.     

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.     

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

In this study, we determined the induction and time-dependent accumulation of micronuclei in the peripheral blood of transgenic C57BL/6 p53+/- mice (p53+/- mice), FVB/N Tg.AC v-Ha-ras mice (Tg.AC mice) and their isogenic parental strains, FVB/N and C57BL/6 following inhalation exposure to benzene. Our objective was to determine the impact of p53 heterozygosity in p53+/- mice and the v-Ha-ras transgene in Tg.AC mice on micronuclei induction following exposure to inhaled benzene. A flow cytometric technique that distinguishes micronucleated red blood cells (MN-RBC) from micronucleated reticulocytes (MN-RET) was used. Mice were exposed to 0, 100 or 200 p.p.m. benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 p.p.m.x hours) to benezene: 100 p.p.m. for 6 h/day, 5 days/week, Monday to Friday (M-F); 100 p.p.m. for 10 h/day, 3 days/week, Monday, Wednesday, Friday (MWF); and 200 p.p.m. for 5 h/day, 3 days/week MWF. Significant elevations of MN-RBC and MN-RET were observed from 1 week exposure in all of the benzene-exposed groups that increased in a time-dependent manner for up to 13 weeks exposure. Fewer MN-RBC and MN-RET were induced in the 200 p.p.m. benzene exposure group than in mice exposed to 100 p.p.m. The reduction in the frequency of MN-RBC in the 200 p.p.m.x5 h benzene exposure group is probably due to metabolic saturation resulting in a lower bone marrow dose (concentration x time) than in the 100 p.p.m. exposure groups. No differences were observed in the frequency of MN-RBC or MN-RET in Tg.AC compared with the FVB/N isogenic controls. At certain time points the frequency of micronuclei was less in the heterozygous p53+/- mice than determined in the wild-type C57BL/6 isogenic parental strain. These results indicate that the heterozygous state in p53+/- mice, but not the v-Ha-ras transgene in Tg.AC mice can influence the induction of micronuclei by benzene.     

International Pig-a gene mutation assay trial (stage III): results with N-methyl-N-nitrosourea

N-methyl-N-nitrosourea (MNU) was evaluated in the in vivo Pig-a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28-day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low-dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig-a phenotypic mutation, CD59-negative reticulocytes/erythrocytes (RETs(CD592-)/ RBCs(CD592-)) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN-RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose-dependent increases in the frequency of RETs(CD592-) and RBCs(CD592-) were observed by Day 15 or 29, respectively. Dose-dependent increases were observed in %MN-RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig-a mutation assay results, consistent with previous observations with MNU and N-ethyl-N-nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.     

Quality control in the flow cytometric measurement of T-lymphocyte subsets: the multicenter AIDS cohort study experience. The Multicenter AIDS Cohort Study Group

Since 1984, the Multicenter AIDS Cohort Study (MACS) has utilized four flow cytometry laboratories to measure T-lymphocyte subset levels semiannually in a large cohort of homosexual men. This report summarizes the steps taken in the MACS laboratories to attain comparability of lymphocyte subset determinations across the centers and over time. Identical flow cytometers, monoclonal antibodies, and analytic procedures have been used, and over a period of time, the procedure for sample preparation was also standardized. Interlaboratory proficiency testing utilizing identical specimens analyzed in the four laboratories was performed to evaluate the comparability of the data among the laboratories. Our results verify that such testing can identify technical bias in flow cytometric evaluations performed at different laboratories. Temporal laboratory consistency in flow cytometric measurements was evaluated using data from each site's HIV-seronegative homosexual reference group. Both sequential 95% confidence intervals (mean +/- 2 x SEM) and the within-person standard deviations of the immune measurements were considered. Significant variation in CD3, CD4, and CD8 lymphocyte subset percentages over time in the seronegative reference population was observed. Our observations indicate that the lymphocyte subset values of this seronegative group should be used to adjust those obtained on the seropositive study participants during a particular time period, thereby allowing improved discrimination of the effects of HIV on T cells in infected individuals. The data presented are of use for designing epidemiologic and intervention studies in HIV-1-infected individuals, especially for calculating sample sizes. The methods we have used to assess the quality of data in the MACS have general application to quality control programs in flow cytometry laboratories. In particular, comparison of sequential confidence intervals and within-person standard deviations for lymphocyte subset determinations on control populations are essential to a comprehensive proficiency testing program because they permit assessment of consistency within a laboratory over time.     

Lack of hydroxyurea-associated mutagenesis in pediatric sickle cell disease patients

Hydroxyurea is approved for treating children and adults with sickle cell anemia (SCA). Despite its proven efficacy, concerns remain about its mutagenic and carcinogenic potential that hamper its widespread use. Cell culture- and animal-based investigations indicate that hydroxyurea's genotoxic effects are due to indirect clastogenicity in select cell types when high dose and time thresholds are exceeded (reviewed by Ware & Dertinger, 2021). The current study extends these preclinical observations to pediatric patients receiving hydroxyurea for treatment of SCA. First, proof-of-principle experiments with testicular cancer patients exposed to a cisplatin-based regimen validated the ability of flow cytometric blood-based micronucleated reticulocyte (MN-RET) and PIG-A mutant reticulocyte (MUT RET) assays to detect clastogenicity and gene mutations, respectively. Second, these biomarkers were measured in a cross-sectional study with 26 SCA patients receiving hydroxyurea and 13 SCA patients without exposure. Finally, a prospective study was conducted with 10 SCA patients using pretreatment blood samples and after 6 or 12 months of therapy. Cancer patients exposed to cisplatin exhibited increased MN-RET within days of exposure, while the MUT RET endpoint required more time to reach maximal levels. In SCA patients, hydroxyurea induced MN-RET in both the cross-sectional and prospective studies. However, no evidence of PIG-A gene mutation was found in hydroxyurea-treated children, despite the fact that the two assays use the same rapidly-dividing, highly-exposed cell type. Collectively, these results reinforce the complementary nature of MN-RET and MUT RET biomarkers, and indicate that hydroxyurea can be clastogenic but was not mutagenic in young patients with SCA.