Carol R. Tometsko

Malaria-infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry

A procedure for optimizing the configuration of flow cytometers for enumerating micronucleated erythrocytes is described. The method is based on the use of a biological model for micronucleated erythrocytes, the malaria parasite Plasmodium berghei. P. berghei endows target cells of interest (erythrocytes) with a micronucleus-like DNA content. Unlike micronuclei, parasitized red blood cells have a homogenous DNA content, and can be very prevalent in circulation. These characteristics make malaria-infected erythrocytes extremely well suited for optimizing instrument setup on a daily basis. The experiment described herein was designed to test the hypothesis that malaria-infected erythrocytes can greatly enhance the consistency with which flow cytometers are configured for micronucleus analyses, and thereby minimize intra- and interexperimental variation. Data collected over the course of several months, on two different flow cytometers, supports the premise that malaria-infected blood represents a useful biological standard which helps ensure reliable and consistent flow cytometric enumeration of rare micronucleated erythrocytes.

Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer

The extreme rarity of micronucleated reticulocytes (RETs) in the peripheral blood of non-splenectomized humans has precluded facile enumeration of these cells, as well as evaluation of this endpoint as an index of cytogenetic damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronuclei (MN) in newly formed human RETs. The procedure is based on an immunochemical reagent that differentially labels the most immature fraction of RETs from mature erythrocytes based on the expression level of the transferrin receptor (also known as CD71). The resolution of four erythrocyte populations (young RETs and mature erythrocytes, with and without MN) was achieved for human blood cells treated with phycoerythrin-conjugated anti-CD71, RNase, and either SYTOX Green or SYBR Green I nucleic acid dyes. Anti-glycophorin A labeling of erythroid cells (CyChrome conjugate) was also incorporated into the staining procedure to ensure that debris or other potential artifacts did not adversely impact the analyses. Instrument calibration procedures utilizing malaria-infected rodent erythrocytes were also developed, and are described. Using this analytical system, blood samples from 10 healthy non-splenectomized human volunteers were analyzed for micronucleus frequencies with a single-laser flow cytometer. Average micronucleus frequencies in the mature and most immature fraction of RETs were 0.016 and 0.19%, respectively. Blood samples from three healthy splenectomized volunteers were also evaluated. As expected, these samples exhibited higher micronucleus frequencies in the mature subset of erythrocytes (range 0.03-0.18%). The resulting data suggest that MN can be quantified in human erythrocyte populations with a single-laser flow cytometer, and that the frequency of MN cells in the youngest reticulocyte population approaches values expected in the absence of splenic selection against MN-erythrocytes. This high throughput system is potentially important for evaluating the value of the micronucleated reticulocyte endpoint as an index of chromosome breakage and/or chromosome segregational abnormalities in human populations.

Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study

Micronuclei (MN) are routinely enumerated in mouse peripheral blood to index genotoxicity. Recent data from the Collaborative Study Group for the Micronucleus Test (CSGMT) [CSGMT (The Collaborative Study Group for the Micronucleus Test), 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 (1998) 84-100] suggest that rat peripheral blood may also be appropriate for the enumeration of MN, if scoring is limited to the youngest fraction of reticulocytes. The experiments described herein were designed to test whether modifications to a flow cytometric scoring procedure for measuring micronucleated reticulocytes (MN-RET) in mouse peripheral blood could be extended to accurately enumerate MN in rat peripheral blood. Rats were treated with saline or one of three genotoxic agents (6-mercaptopurine, ethyl methanesulfonate or propane sultone) in an acute dosing protocol. Peripheral blood samples were subsequently collected for both microscopic and flow cytometric analysis. Micronucleus frequencies were scored in the youngest fraction of reticulocytes: scoring by microscopy was restricted to the types I and II reticulocytes based on RNA content utilizing acridine orange supravital staining; flow cytometric measurements were restricted to the youngest fraction of reticulocytes based on transferrin receptor (CD71) staining. A statistically significant dose-related increase in the incidence of MN was observed, irrespective of scoring method. A higher level of statistical discrimination between control and genotoxin-treated groups was observed for the flow cytometric data and can most likely be explained by the increased number of cells scored (10x more than microscopy) and the lower scoring variability. Together, these data suggest that (i) rat peripheral blood represents an appropriate compartment for evaluating genotoxin-induced MN when the analysis is restricted to young reticulocytes, and (ii) the measurement of MN in rat peripheral blood reticulocytes benefits from the high throughput methodology of flow cytometry.

An automated method for discriminating aneugen- vs. clastogen-induced micronuclei.

A flow cytometric (FCM) procedure for quantitating micronucleated reticulocytes in mouse peripheral blood samples was evaluated for its ability to discriminate between aneugen‐ and clastogen‐induced micronuclei (MN). In this experiment, BALB/c mice were injected with 0.9% saline, the model clastogen methyl methanesulfonate (100 mg/kg bw) or the aneugen vincristine (0.2 mg/kg bw). Peripheral blood samples were collected 48 hr after injection and were subsequently fixed and stained for flow cytometric analysis. The staining method utilized FITC‐conjugated anti‐CD71 to differentially label reticulocytes, and the nucleic acid dye propidium iodide to resolve erythrocyte populations with and without micronuclei. The frequency of micronucleated reticulocytes was determined by analyzing 10,000 total reticulocytes per blood sample. A second analysis was performed on each sample whereby the propidium iodide associated fluorescent signals of 250 MN were collected and graphed as a single‐parameter histogram. The histogram statistic “median channel” was recorded for each sample and provided a quantitative description of MN distribution according to DNA content. Cumulatively, the results of this study suggest that 1) flow cytometry can be employed to measure the incidence of MN resulting from clastogenic or aneugenic activity, and 2) MN resulting from aneugenscan be discriminated from those arising spontaneously or from clastogen treatment based on flow cytometric analysis of DNA content. Environ. Mol. Mutagen. 31:340–344, 1998.

Measurement of Cytogenetic Damage in Rodent Blood with a Single‐Laser Flow Cytometer

The in vivo rodent micronucleus test is widely utilized to screen chemicals for genotoxic activity. Double‐strand chromosome breaks or dysfunction of the mitotic spindle apparatus can lead to micronuclei formation in dividing cells. Erythrocytes have become the target population of choice, as precursor cells are continuously dividing and micronuclei are readily observable after extrusion of nuclei. The traditional method has been to stain peripheral blood or bone marrow smears and microscopically determine the frequency of micronucleated erythrocytes. Because these events are rare, the process is tedious and time consuming. This unit describes a procedure for fixing and staining rodent peripheral blood for flow cytometric enumeration. The combination of reagents provides for differential labeling and enumeration of four subpopulations: mature erythrocytes, micronucleus‐containing mature erythrocytes, young erythrocytes (reticulocytes), and micronucleus‐containing young erythrocytes. Malaria‐infected rodent erythrocytes, which closely mimic micronucleus‐containing erythrocytes, serve as a biological standard to facilitate rational and consistent equipment calibration. Keywords: flow cytometry; genotoxicity; chromosome damage; micronuclei; reticulocytes; propidium iodide; CD71‐defined antigen The in vivo rodent micronucleus test is widely utilized to screen chemicals for genotoxic activity.

Cross‐Species Genetic Toxicity Assessment Accomplished by Flow Cytometric Analysis of Blood

The formation of micronuclei in blood cells has been an established indicator of genotoxicity for decades. Standard microscopy methods are time‐consuming and lack the objectivity that fully automated methods can provide. The ability of flow cytometric technology to rapidly and objectively survey thousands of cells for micronuclei can significantly improve the value of this endpoint. In addition, since many more cells can be scored, and specific populations can be targeted, species that historically have been difficult to obtain micronucleus data from, such as humans, can now be readily evaluated using this methodology. This unit describes a procedure for fixation, staining, and analysis of blood samples using materials supplied in MicroFlow kits (Litron Laboratories) and a single‐laser flow cytometer. This methodology provides a reliable, robust assessment of chromosome damage that is used in basic science research as well as drug‐safety screening programs at large pharmaceutical and chemical companies. Curr. Protoc. Toxicol. 36:2.14.1‐2.14.15.