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.     

Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.     

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 method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation

The current report describes a newly devised method for automatically scoring the incidence of rat hepatocyte micronuclei (MNHEP) via flow cytometry, with concurrent assessments of hepatocyte proliferation—frequency of Ki‐67‐positive nuclei, and the proportion of polyploid nuclei. Proof‐of‐concept data are provided from experiments performed with 6‐week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) or quinoline (QUIN) for 3 or 14 consecutive days. Non‐perfused liver tissue was collected 4 days after cessation of treatment in the case of 3‐day studies, or 1 day after last administration in the case of 14‐day studies for processing and flow cytometric analysis. In addition to livers, blood samples were collected one day after final treatment for micronucleated reticulocyte (MN‐RET) measurements. Dose‐dependent increases in MNHEP, Ki‐67‐positive nuclei, and polyploidy were observed in 3‐ and 14‐day DEN studies. Both treatment schedules resulted in elevated %MNHEP for QUIN‐exposed rats, and while cell proliferation effects were subtle, appreciable increases to normalized liver weights were observed. Whereas DEN caused markedly higher %MNHEP when exposure was extended to two weeks, QUIN‐induced MNHEP were slightly increased with protracted dosing. Parallel microscopy‐based MNHEP frequencies were highly correlated with flow cytometry‐based measurements (four study/aggregate R² = 0.80). No increases in MN‐RET were seen in any of the four studies. Collectively, these results suggest liver micronuclei are amenable to an automated scoring technique that provides objective analyses and higher information content relative to conventional microscopy. Additional work is needed to expand the number and types of chemicals tested, identify the most advantageous treatment schedules, and test the transferability of the method. Environ. Mol. Mutagen. 59:176–187, 2018. © 2018 Wiley Periodicals, Inc.     

Flow cytometric analysis of Pig‐a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD‐1 mice

Procarbazine is a genotoxic carcinogen whose DNA‐damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD‐1 mice using a multi‐endpoint study design that scored micronucleated reticulocyte (MN‐RET) frequency and gene mutation at the Pig‐a locus. CD‐1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day. Blood samples collected on Day 3 exhibited robust induction of MN‐RETs, with the high dose group exhibiting a mean 29‐fold increase. Blood collected 15 and 30 days after treatment began was analyzed for Pig‐a mutation with a dual labeling method that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. Procarbazine significantly increased mutant reticulocyte frequencies by Day 15. Mutant erythrocyte responses were also apparent, with a peak incidence observed for the high dose group on Day 30. These results demonstrate that the complex metabolism and resulting genotoxicity of procarbazine is best evaluated in intact animal models, and show that the flow cytometric methods employed offer a means to efficiently monitor both in vivo chromosomal damage and mutation. Environ. Mol. Mutagen. 54:294–298, 2013. © 2013 Wiley Periodicals, Inc.     

Flow cytometric detection of HLA antibodies using a spectrum of microbeads

We describe here the use of HLA antigen coated beads for specificity and class determination of HLA antibodies by flow cytometry. The HLA specificity of antibodies was determined by use of beads containing eight levels of fluorescence. HLA antigens isolated from eight cultured cells were coated onto these beads so that each bead was the equivalent of one cell. By using four sets of eight beads, an equivalent of 32 cells could be examined in four test tubes. A total of 76 class I and 25 class II specificities could be determined by the 32 class I bead-panel and 32 class II bead-panel used, respectively. We noted no cross-reactivity of reactions between class I and II. The sensitivity of the test was shown to be higher than that of the standard cytotoxicity by dilution experiments and detection of additional cross-reacting antigens. By use of these coated beads, we achieved improved standardized detection of HLA antibodies. Antigen-coated beads have several advantages over the use of spleens or lymphocytes. (a) A highly selected panel of antigens can be routinely used. (b) Class I and class II antibodies can be readily distinguished from each other, even when they are present as mixtures in one serum. (c) Non-HLA antibodies are not detected because the beads do not have any other antigens than HLA on them. (d) The quantity of antigens coated on beads is more uniform than that found in cells from different individuals. (e) Beads are more convenient for storage and daily use.     

Flow cytometric testing of susceptibilities of Mycobacterium avium to amikacin, ciprofloxacin, clarithromycin and rifabutin in 24 hours

Objective   To develop a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing Mycobacterium avium organisms in drug-free and antimycobacterial agent-containing medium.
Methods   Prior to analysis by flow cytometry, all M. avium susceptibility test samples were inactivated by exposure to paraformaldehyde. The susceptibilities of 20 clinical isolates of M. avium to amikacin, ciprofloxacin, clarithromycin, and rifabutin were tested by the flow cytometric and BACTEC methods.
Results   Agreement was 97% between the results of the two methods. The results of flow cytometric susceptibility tests were available 24 h after inoculation of drug-containing medium, while the BACTEC method required 4–8 days to complete.
Conclusions   The flow cytometric assay is safe, simple and reproducible.     

A high‐throughput assay for the measurement of uropathogenic Escherichia coli attachment to urinary bladder cells

Strains of uropathogenic Escherichia coli (UPEC) are the major causative agent of urinary tract infections (UTI), the most common infectious diseases in the world. Their ability to attach and enter into cells in the urinary tract is a limiting step for their pathogenicity. Many studies are thus focussing on these key mechanisms to propose new therapeutic strategies. To facilitate such studies, we developed a fast and high‐throughput assay which makes it possible to monitor the interaction of UPEC with cultured human uroepithelial cells. This assay allows measurement of the in vitro association of fluorescently labelled clinical isolates with bladder epithelial cells using flow cytometry in a microplate format. The assay was sensitive enough to detect variations between isolates expressing different adhesins and virulence factors and the inhibitory effect of proanthocyanidins. Thus we have developed a fast and robust assay which allows us to measure variations in the adhesion properties of UPEC to human bladder cells. This novel assay will be valuable for the study of initial steps of pathogenesis in UTI and for the screening or validation of inhibitory molecules.     

Flow cytometric analysis of peripheral blood lymphocyte subset light scatter characteristics as a means of monitoring the development of rat small bowel allograft rejection.

This investigation used flow cytometry to monitor peripheral blood lymphocyte morphology after rat small bowel transplantation. Preliminary studies demonstrated that in vitro activated peripheral blood lymphocytes exhibited increased cell size and granularity as measured by flow cytometric analysis of forward (FSc) and side (SSc) light scatter characteristics. The formation of distinct 'activated' light scatter regions by such lymphoblastoid transformation occurred concomitantly with up-regulated p55IL-2R expression. Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats without immunosuppression. Animals receiving isografts served as controls. Peripheral blood lymphocyte subsets were identified using appropriate MoAbs, and the light scatter characteristics of each cell subset were determined by backgating strategies. Increased proportions of activated alpha/beta T cell receptor (TCR)-positive cells could be detected in allografted animals as early as day 2 post-transplantation. B cells showed peak activation by day 4, at which time the proportion of activated cells was over two-fold greater than that seen in untransplanted animals--few activated B cells were detected in isografted animals. Resting natural killer (NK) cell light scatter regions only partially overlap with those of resting T and B lymphocytes, but in allografted animals almost the entire NK population fell outside the resting lymphocyte gate by day 2 post-transplantation, an activation state which was maintained until day 4. These findings associate peripheral blood cell subset lymphoblastoid transformation with developing small bowel allograft rejection. Importantly, changes were detected early and prior to the onset of overt rejection. These data suggest that analysis of peripheral blood lymphocyte light scatter properties may provide an insight into in vivo immune status after small bowel transplantation.     

Fine‐needle aspiration with flow cytometric immunophenotyping for primary diagnosis of intra‐abdominal lymphomas

Cytomorphology in conjunction with immunophenotypic characterization is becoming increasingly used for the primary diagnosis of non‐Hodgkin's lymphomas (NHL). This combination is especially advantageous for the diagnosis of intra‐abdominal and intrathoracic lymphomas, since unlike superficial lesions, open biopsy of deep‐seated tissues is more invasive and more costly, and is associated with a higher risk. We report the cytologic and immunophenotypic features of intra‐abdominal NHL obtained by fine‐needle aspiration (FNA). Twenty‐two cases of intra‐abdominal lesions obtained by image‐guided FNA where flow cytometry was also performed were reviewed. Of the 22 studied cases, 7 were classified as large‐cell lymphoma, 5 as follicular center‐cell lymphoma, 2 as small noncleaved‐cell lymphoma, 2 as lymphoplasmacytoid lymphoma, one as small lymphocytic lymphoma, and one as marginal‐zone lymphoma. In the remaining 4 cases where the immunophenotypic pattern was not definitive, the cytomorphologic features were of small cleaved cells in 3 cases and of mixed small cleaved and large cells in one case. We successfully classified 9 of the 10 patients on whom histologic confirmation was obtained. The successful primary classification of most intra‐abdominal non‐Hodgkin's lymphomas can be done with a combination of cytology and flow cytometry, and this can be the initial approach in patients with deep‐seated lesions. Diagn. Cytopathol. 1999;21:98–104. © 1999 Wiley‐Liss, Inc.     

Flow cytometric 96‐well microplate‐based in vitro micronucleus assay with human TK6 cells: Protocol optimization and transferability assessment

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96‐well plate (Bryce SM et al. [2010]: Mutat Res 703:191‐199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non‐genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5‐ to 2‐cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN‐fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non‐genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints—relative survival and quantification of ethidium monoazide‐positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96‐well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity. Environ. Mol. Mutagen. 54:180–194, 2013. © 2013 Wiley Periodicals, Inc.     

Flow cytometric analysis of CFP-YFP FRET as a marker for in vivo protein-protein interaction

The technique of observing fluorescence resonance energy transfer (FRET) between a cyan fluorescent protein (CFP) fusion protein and a yellow fluorescent protein (YFP) fusion protein has been used in numerous studies as a reliable indicator of protein-protein interaction. Moreover, because CFP and YFP fusions generally retain activity and maintain their normal subcellular localizations, the detection of CFP-YFP FRET in live cells typically reflects the steady-state association of functional proteins in their native states. Although CFP-YFP FRET can also be monitored by fluorimetry and fluorescence microscopy, the ability of flow cytometry to rapidly acquire multiparameter fluorescence data on a large number of individual cells provides several important advantages. The analysis of CFP-YFP FRET on a cell-by-cell basis allows for a sensitive and highly rigorous assessment of protein interaction, and the large number of cells that can be examined by flow cytometry provides for a high degree of statistical confidence. In addition, simple, yet stringent, gating-based analyses of FRET can be performed on a flow cytometer simultaneously with data collection, allowing FRET-based cell sorting that can be used in high-throughput screens to identify interacting proteins. As a brief review of flow cytometric CFP-YFP FRET analysis, this article outlines a typical instrument configuration, describes the required controls, explains how gating is performed, and discusses the basic physical principles behind FRET as they relate to the successful design and interpretation of protein interaction experiments.     

Pig‐a gene mutation and micronucleated reticulocyte induction in rats exposed to tumorigenic doses of the leukemogenic agents chlorambucil,thiotepa, melphalan,and 1,3‐propane sultone

To evaluate whether blood‐based genotoxicity endpoints can provide temporal and dose‐response data within the low‐dose carcinogenic range that could contribute to carcinogenic mode of action (MoA) assessments, we evaluated the sensitivity of flow cytometry‐based micronucleus and Pig‐a gene mutation assays at and below tumorigenic dose rate 50 (TD50) levels. The incidence of micronucleated reticulocytes (MN‐RET) was used to evaluate chromosomal damage, and the frequency of CD59‐negative reticulocytes (RET^CD59?) and erythrocytes (RBC^CD59?) served as phenotypic reporters of mutation at the X‐linked Pig‐a gene. Several leukemogenic agents with a presumed genotoxic MoA were studied. Specifically, male Sprague Dawley rats were treated via oral gavage for 28 days with chlorambucil, thiotepa, melphalan, and 1,3‐propane sultone at doses corresponding to 0.33x, 1x, and 3x TD50, as well as at the maximum tolerated dose. Frequencies of MN‐RET were determined at Days 4 and 29, and RET^CD59? and RBC^CD59? data were collected pretreatment as well as Days 15/16, 29, and 56/57. Dose‐related increases were observed for each endpoint, and time to maximal effect was consistently: MN‐RET < RET^CD59? < RBC^CD59?. For each of the chemicals studied, the genotoxic events occurred long before tumors or preneoplastic lesions would be expected. Furthermore, in the case of Pig‐a gene mutation, the responses were observed at or below the TD50 dose for three out of the four chemicals studied. These data illustrate the potential for quantitative blood‐based analyses to provide dose‐response and temporality information that relates genetic damage to cancer induction. Environ. Mol. Mutagen. 55:299–308, 2014. © 2014 Wiley Periodicals, Inc.     

Time‐course of micronucleated erythrocytes in response to whole‐body gamma irradiation in a model mammalian species,the bank vole (Clethrionomys glareolus,Schreber)

The time course of the formation of micronucleated polychromatic (MNPCEs) and normochromatic erythrocytes (MNNCEs) in the bone marrow of the bank vole (Clethrionomys glareolus, Schreber), a model mouse‐like species, was studied using the standard micronucleus test at 0, 6, 12, 18, 24, 30, 36 and 48 hr following whole‐body acute γ‐irradiation at a dose of 0.5 Gy. Based on the existing literature on laboratory mice, it was suggested that such a dose will not have significant effect on erythroid cell proliferation in the bank vole and hence on the time course of the rise of micronucleated cells. In total, ～905,000 polychromatic (PCEs) and normochromatic erythrocytes (NCEs) from 82 adult bank voles were analyzed. Although the mean frequencies of MNNCEs were too low to allow for the correct assessment of their time course, an analysis of PCEs showed an increasing rate of MNPCE appearance at 6 hr that reached a maximum at 18–24 hr after irradiation and subsequently decreased. Because the kinetics of MNPCEs reflects the process of erythropoiesis, the current results regarding the time points of appearance of radiation‐induced MNPCEs provide the first information on the prolongation of one of the terminal stages of erythrocyte formation in bank vole specimens, namely the stage of maturation of PCEs from erythroblasts. Moreover, the observed time‐course data, as well as the low‐background frequencies of MNPCEs and characteristic level of PCEs response to radiation, showed similarities between the two model species: bank vole (this study) and laboratory mice (literature data). Environ. Mol. Mutagen. 52:50–57, 2011. © 2010 Wiley‐Liss, Inc.     

Flow cytometric analysis of platelet surface glycoproteins in the diagnosis of thirty-two Turkish patients with Glanzmann thrombasthenia: a multicenter experience

Background/aimGlanzmann thrombasthenia (GT) is a rare autosomal recessively inherited bleeding disorder characterized by the quantitative (type 1 and type 2) or qualitative (type 3) deficiency in platelet membrane glycoprotein (GP) IIb/IIIa (CD41a/CD61) fibrinogen receptors. In type 1, 2, and 3, CD41a/CD61 expression is 5%, 5%–20% and above 20%, respectively. In this study, diagnosis of GT was confirmed and subgroups were identified in 32 Turkish patients by flow cytometry analysis.Materials and methodsCD41a/CD61 expression levels in platelet-rich plasma (PRP) obtained from peripheral venous EDTA blood samples were analyzed with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). GT subgroup analysis was performed by counting 50,000 events in the BD FACSDiva Software v6.1.3 program of the instrument.ResultsIn the present study, in blood samples of 32 patients from 23 families with GT and 22 healthy controls, co-expression levels of CD41a and CD61 in PRP was analyzed. 12 out of 23 families were consistent with type 1 GT (52.2%), 4 were consistent with type 2 GT (17.4%), and 7 were consistent with type 3 GT (30.4%).ConclusionEspecially due to consanguineous marriages, GT with various glycoprotein levels may be detected. As a result of the flow cytometry analysis of the present study with the highest GT patient population in Turkey, type 1 GT patients were the most common subgroup. In the determination of the GT subgroups; especially in the detection of type 3 GT, flow cytometry is the most sensitive glycoprotein analysis method. In addition to light transmission aggregometry, CD41a/CD61 study by flow cytometer confirms diagnosis when mutation analysis cannot be performed.     

Flow cytometry methods for the study of cell‐cycle parameters of planarian stem cells

Due to their characteristic inaccessibility and low numbers, little is known about the cell‐cycle dynamics of most stem cells in vivo. A powerful, established methodology to study cell‐cycle dynamics is flow cytometry, which is used routinely to study the cell‐cycle dynamics of proliferating cells in vitro. Its use in heterogeneous mixtures of cells obtained from whole animals, however, is complicated by the relatively low abundance of cycling to non‐cycling cells. We report on flow cytometric methods that take advantage of the abundance of proliferating stem cells in the planarian Schmidtea mediterranea. The optimized protocols allow us to measure cell‐cycle dynamics and follow BrdU‐labeled cells specifically in complex mixtures of cells. These methods expand on the growing toolkit being developed to study stem cell biology in planarians, and open the door to detailed cytometric studies of a collectively totipotent population of adult stem cells in vivo. Developmental Dynamics 238:1111–1117, 2009. © 2009 Wiley‐Liss, Inc.     

Validation of Gelbond® high‐throughput alkaline and Fpg‐modified comet assay using a linear mixed model

Even if the comet assay has been widely used for decades, there is still a need for controlled studies and good mathematical models to assess the variability of the different versions of this assay and in particular to assess potential intra‐experimental variability of the high‐throughput comet assay. To address this point, we further validate a high‐throughput comet assay that uses hydrophilic polyester film (Gelbond®). Experiments were performed using human peripheral blood mononuclear cells (PBMC) either untreated or treated with different concentration of MMS (methyl methanesulfonate). A positive control for the Fpg (Formamidopyrimidine DNA glycosylase)‐modified comet assay (Ro 19‐8022 with light) was also included. To quantify the sources of variability of the assay, including intradeposit variability, instead of summarizing DNA damage on 50 cells from a deposit by the mean or median of their percentage DNA tail, we analyzed all logit‐transformed data with a linear mixed model. The main source of variation in our experimental data is between cells within the same deposit, suggesting genuine variability in the response of the cells rather than variation caused by technical treatment of cell samples. The second source of variation is the inter‐experimental variation (day‐to‐day experiment); the coefficient of this variation for the control was 13.6%. The variation between deposits in the same experiment is negligible. Moreover, there is no systematic bias because of the position of samples on the Gelbond^® film nor the position of the films in the electrophoresis tank. This high‐throughput comet assay is thus reliable for various applications. Environ. Mol. Mutagen. 59:595–602, 2018. © 2018 Wiley Periodicals, Inc.     

Further development of the rat Pig-a mutation assay: measuring rat Pig-a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes

Recent studies indicate that the Pig-a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig-a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid-specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 x 10(6) CD71-positive RETs could be evaluated by flow cytometry for Pig-a mutant frequency within 5 to 8 min. CD59-deficient RET and BME frequencies of more than 100 x 10(-6) and 80 x 10(-6) were detected 1 week after treating rats with 40 mg/kg N-ethyl-N-nitrosourea; by comparison, the frequency of CD59-deficient total RBCs in these rats was 13.2 x 10(-6). The frequency of spontaneous Pig-a mutant RETs and BMEs was less than 5 x 10(-6) and 15 x 10(-6), respectively. Since approximately 98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59-deficient Pig-a mutations in cells of erythroid lineage. Conducting concurrent Pig-a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary.     

The frequency of Pig‐a mutant red blood cells in rats exposed in utero to N‐ethyl‐N‐nitrosourea

The Pig‐a assay has been developed as a rapid sensitive measure of gene mutation in adult rats; however, no data exist on its ability to detect mutation following in utero exposures or in neonatal animals. Pregnant Sprague‐Dawley rats were treated daily on gestational days 12–18 with oral doses of 0, 6, or 12 mg/kg/day N‐ethyl‐N‐nitrosourea (ENU); following parturition, the offspring and dams were monitored over a period of 5 months for the frequency of CD59‐deficient erythrocytes as a marker of Pig‐a mutation. Significant dose‐related increases in Pig‐a mutant red blood cells (RBCs) were observed in ENU‐treated dams. However, only very weak increases in RBC Pig‐a mutant frequency (MF) were noted in offspring treated in utero with the lower ENU dose. The higher ENU dose produced extremely variable responses in the offspring as a function of age, even among littermates, ranging from a steady low or moderately high Pig‐a MF to a rapidly increasing or decreasing Pig‐a MF. The manifestation kinetics of Pig‐a mutant RBCs in the offspring suggest that the change from predominantly hepatic to predominantly bone marrow erythropoiesis that occurs during early development may have contributed to this variability. Our results indicate that using the RBC Pig‐a model for mutation detection in animals treated in utero may require analysis of multiple offspring from the same litter to account for potential “jack pot” effects, and that detection of the earliest treatment effect (i.e., in neonates using the hepatic RBC fraction) may require optimization of blood processing. Environ. Mol. Mutagen. 2012. Published 2012 Wiley Periodicals, Inc.     

Multiplex targeted high‐throughput sequencing in a series of 352 patients with congenital limb malformations

Congenital limb malformations (CLM) comprise many conditions affecting limbs and more than 150 associated genes have been reported. Due to this large heterogeneity, a high proportion of patients remains without a molecular diagnosis. In the last two decades, advances in high throughput sequencing have allowed new methodological strategies in clinical practice. Herein, we report the screening of 52 genes/regulatory sequences by multiplex high‐throughput targeted sequencing, in a series of 352 patients affected with various CLM, over a 3‐year period of time. Patients underwent a clinical triage by expert geneticists in CLM. A definitive diagnosis was achieved in 35.2% of patients, the yield varying considerably, depending on the phenotype. We identified 112 single nucleotide variants and 26 copy‐number variations, of which 52 are novel pathogenic or likely pathogenic variants. In 6% of patients, variants of uncertain significance have been found in good candidate genes. We showed that multiplex targeted high‐throughput sequencing works as an efficient and cost‐effective tool in clinical practice for molecular diagnosis of congenital limb malformations. Careful clinical evaluation of patients may maximize the yield of CLM panel testing.