Flow-cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood.

A flow-cytometric assay is described that can be used to determine the frequency and the DNA content of micronucleated polychromatic (PCE) and normochromatic (NCE) erythrocytes in mouse peripheral blood. Thiazole orange was used for discrimination between PCEs and NCEs, while Hoechst 33342 was used to detect micronucleated PCEs and NCEs. Up to 70,000 polychromatic erythrocytes can be analyzed in less than 10 min. This corresponds to 150-3,000 micronucleated polychromatic erythrocytes, 90-95% of which are true events as determined with a fluorescence microscope after sorting. Using X-rays as the inducing agent in dose-response experiments, a significant increase can be registered at doses of 0.02 Gy. It seems possible that the method will also allow the detection of clastogenic effects of other inducing agents at lower doses than previously possible.     

Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry

Summary The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromsomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored using a microscope following staining with May-Grünwald-Giemsa solutions. These time-consuming, painstaking, and tedious manual evaluations are often sources of unreliability and uncertainty. Here, a fluorescence technique is presented which applies DNA and protein fluorochromes to discriminate normal anucleated erythrocytes from micronucleated erythrocytes using a fluorescence microscope. This method is particularly tailored to be applied to flow cytometric instrumentation. Data obtained manually and automatically in flow show a strong linear correlation with high significance (r=0.96) as far as the percentage of micronucleated erythrocytes as an indicator for the mutagenicity of a given drug is concerned. These results have been obtained by means of the established clastogens cyclophosphamide and mitomycin C.     

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.     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c, DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection

The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gel beads. In this way, the phospholipid content and composition of the membrane of nonparasitized cells, the erythrocyte membrane of parasitized cells and the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phospholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phospholipids in: erythrocytes from healthy monkeys, nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.     

The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: implications for cytogenetic screening

Micronucleated erythrocytes are selectively removed from the peripheral circulation of normal rats. Splenectomy prevents this selective removal. In normal rats treated daily for 20 days with 0.2 mg/kg triethylenemelamine (TEM), micronucleated normochromatic (mature) erythrocytes did not accumulate in peripheral blood. In these same animals, the frequencies of micronucleated cells among polychromatic (newly formed) erythrocytes increased from 0.21 to 5.25 per thousand in peripheral blood and from 1.75 to 31.5 per thousand in bone marrow. Since both control and induced frequencies in peripheral blood were approximately 15% of those in bone marrow, the removal appears to be equally efficient for cells containing either spontaneously occurring or clastogen-induced micronuclei. In splenectomized rats treated daily for 11 days with 0.2 mg/kg TEM, the frequency of micronucleated normochromatic erythrocytes (NCEs) in the peripheral blood rose rapidly to 9 times the control value and remained elevated for 50-55 days, indicating a life span approximately equivalent to that of normal erythrocytes. Among splenectomized rats exposed to either 0.15 mg/kg triethylenemelamine, 6.5 mg/kg cyclophosphamide, or 300 mg/kg urethane for periods exceeding the erythrocyte life span, the incidences of micronucleated NCEs in the peripheral blood rose steadily from a control value of 1.0 per thousand to maximum values of 15.0, 12.7 and 8.9 per thousand, respectively. During these extended exposures, the mean frequencies of micronucleated polychromatic erythrocytes (PCEs) in peripheral blood increased from a spontaneous value of 0.9 per thousand to 23.0, 13.0 and 6.6 per thousand, respectively, reflecting the frequencies among PCEs in the bone marrow and approximating the maximum values among NCEs in the peripheral blood. Thus, the frequency of micronucleated erythrocytes in the peripheral blood of splenectomized rats can be used as an index of both acute and cumulative chromosomal damage, while in normal rats the use of peripheral blood for cytogenetic monitoring is restricted by the selective removal of these micronucleated cells.     

Effect of residual splenic function and folate levels on the frequency of micronucleated red blood cells in splenectomized humans.

Frequencies of micronucleated erythrocytes in the peripheral blood of splenectomized individuals can be used as an index of genetic damage to erythrocyte precursor cells in the bone marrow. This is in contrast to non-splenectomized humans, whose micronucleated erythrocytes are removed by the spleen. Many subjects whose spleen has been removed surgically have residual spleen tissue and consequent residual spleen function (RSF), which can be measured by the percentage of 'pitted' peripheral red blood cells. In this study evidence of RSF was associated with decreased frequencies of micronucleated erythrocytes. Analysis of data limited to subjects with minimal spleen function suggested an inverse association between the incidence of micronucleated erythrocytes and serum folate levels that was not apparent in the absence of stringent control for RSF.     

The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood

The time-course of micronucleated polychromatic erythrocytes (MPCE) in mouse bone marrow and peripheral blood, induced by an acute 0.1 Gy dose of X-rays, was determined using flow cytometric analysis, which made frequent sampling possible and allowed use of a dose low enough not to affect erythroid cell proliferation. The frequency of MPCE (fMPCE) began to increase in the bone marrow at 10 h after irradiation and reached a maximum at 28 h after irradiation. In the peripheral blood fMPCE began to increase at 20 h after irradiation and peaked at about 40 h after irradiation. The time-course found is discussed on the basis of data on the differentiation of erythroid cells. The results indicate that the micronuclei registered in polychromatic erythrocytes may originate from lesions induced not only during the last cell cycle but also during earlier ones. After an acute dose of 1.0 Gy of X-rays the maximum fMPCE was delayed both in bone marrow and peripheral blood reflecting an effect on the cell cycle progression of erythroblasts.     

Extended exposure of adult and fetal mice to 50 Hz magnetic field does not increase the incidence of micronuclei in erythrocytes.

The flow cytometer-based micronucleus assay was used to study the effects on chromosomes in erythroid cells of CBA/Ca mice after extended exposure to 50 Hz magnetic field (MF), 14 microT, peak-to-peak (p-p). The study included two different experiments: (a) mice exposed in utero during 18 days of their prenatal stage, and (b) adult mice exposed for 18 days. In experiment (a) 35 days after exposure was terminated, peripheral blood was drawn from the mice exposed in utero to determine whether the exposure had a genotoxic effect on the pluripotent erythroid stem cells. About 200000 polychromatic erythrocytes (PCE) and 200000 normochromatic erythrocytes (NCE) were analysed from each of 20 exposed mice. The EMF exposure did not significantly change the frequency of micronucleated PCE or NCE in comparison with 20 sham-irradiated mice. There was no difference in the proportion of PCE between exposed and unexposed animals. Similarly, in experiment (b) no differences were seen between EMF exposed and unexposed adult mice when samples of peripheral blood were taken at the end of exposure and analyzed for micronuclei in PCE and NCE. The proportion of PCE was the same in both groups. The results indicate that exposure to EMF does not induce direct or indirect effects on chromosomes in erythroid cells expressed as increased levels of micronucleated erythrocytes of mice. No indications of delayed genetic effects were found.     

Hematologic values of New Zealand white rabbits determined by automated flow cytometry.

Hematologic values of peripheral blood from normal adult New Zealand White rabbits were determined by five different automated flow cytometers in use in a routine clinical hematology laboratory: Technicon H1, Coulter Counter CC540, Coulter Counter VCS, Sysmex NE8000 and Sysmex R1000. The software designed for human blood analysis was used in all instances without adaptation. The total numbers of white blood cells, red blood cells, reticulocytes and platelets were measured with high precision and accuracy. Except for hemoglobin content, concordance was excellent for all measured and calculated values among the different automated flow cytometers. Determining the white blood cell differential count was more complex. Eosinophils and lymphocytes were quantified reliably by all the automated flow cytometers used. However, the results were rejected by Technicon H1 and Sysmex NE8000 in 50% of the cases. Rabbit basophils were recognized with accuracy by Technicon H1 only. The proportion of polymorphonuclear versus mononuclear white cells was identical when measured with Technicon H1 and Coulter Counter VCS. These results show that the new generation of automated flow cytometers designed for human blood can be used with some limitations for animal studies. They allow the standardization of normal values and comparison of results among or between laboratories. They also introduce new parameters, the value of which is as yet undefined.     

Increase in micronucleated erythrocytes associated with babesiosis in Syrian golden hamsters

The effect of infection by Babesia microti, a tick-borne piroplasm endemic to the northeastern United States, on the temporal pattern of micronucleated erythrocyte frequencies in peripheral blood was investigated in male Syrian golden hamsters. Significantly greater frequencies of micronucleated erythrocytes occurred in the blood of infected hamsters from 26 to 46 days after injection with B. microti, the magnitude of which within individual hamsters correlated highly with the percentage of polychromatic erythrocytes and the extent of parasitization. These data suggest that parasitic infection and other factors which alter the rate of erythropoiesis should be considered when the micronucleus assay is used in environmental or laboratory studies of genetic toxicity.     

NASA/American Cancer Society High-Resolution Flow Cytometry Project-I

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. METHODS: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. RESULTS: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 +/- 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. CONCLUSIONS: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.     

Flow cytometry of mammalian sperm: progress in DNA and morphology measurement.

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.     

Modulation of the Host's Immune Response to Plasmodium berghei by a Parasite-Derived Immunosuppressive Factor

ABSTRACT. It is well known that Plasmodium -infected hosts are immunosuppressed, as shown by their depressed immune responsiveness to a variety of antigens. It is not known, however, whether the immune response of malaria-infected animals to the malarial parasite itself is suppressed. The availability of a noninfectious, immunosuppressive factor (ISF) derived from Plasmodium berghei -infected rat erythrocytes made it possible to investigate this question. Mice infected with P. berghei and injected with the ISF had higher levels of parasitemia and shorter survival times than control mice that were similarly infected but were treated with control material derived from noninfected rat erythrocytes or with saline solution. Conversely, mice immunized against the ISF and then infected with P. berghei had lower parasitemias and longer survival times than mice immunized with the control material or with saline solution. We conclude that immunosuppression in murine malaria affects the course of malaria infection.     

Immunological biochips for studies of human erythrocytes

Immunological microarrays (biochips) for detecting erythrocyte surface antigens, viz., blood group antigens (A, B, 0) and Rhesus system antigens (D, E, e, C, and c), are described. The biochips represent transparent plastic supports onto which 1.5-mm spots of specific immobilized antibodies (IgM) are coated in different dilutions. The volume of tested blood samples is rather small (1–2 μl). Binding of erythrocytes to antibodies immobilized on the biochips is specific and allows further morphological analysis of bound cells. Analysis of the dynamics of cell detachment from biochip spots using a microfluidic chamber at different flow rates of the washing solution showed that combination of a biochip with a microfluidic chamber is a promising approach to concentration of cells of various immunotypes even if their content in the mixture is very low.     

Density-gradient enrichment of newly-formed mouse erythrocytes. Application to the micronucleus test

To facilitate scoring micronuclei in peripheral blood erythrocytes, we have developed a centrifugation method to concentrate polychromatic and newly-formed normochromatic erythrocytes from microliter quantities of blood in a Percoll density gradient. Erythrocytes were separated into two discrete bands in a continuous gradient generated in situ in a microhematocrit capillary tube. The upper band contained white blood cells and a mixture of polychromatic and young normochromatic erythrocytes with a density of 1.080-1.082 g/ml. More than 75% of the polychromatic erythrocytes in samples of normal blood were recovered in the upper band. Older normochromatic erythrocytes migrated to the lower band. The frequency of polychromatic erythrocytes was increased from approximately 2% in whole blood to 60-80% in the upper band. After clastogen treatments, the elevated frequencies of micronuclei in the upper band polychromatic erythrocytes were similar to those in unfractionated blood. The frequencies of micronucleated normochromatic erythrocytes in the upper band were higher than those in whole blood at 48, 72 and 96 h after clastogen treatment, consistent with the expectation that the low-density normochromatic cells are newly derived from polychromatic erythrocytes. This density-gradient centrifugation technique enhances the efficiency of scoring micronuclei in the acute peripheral blood micronucleus test.     

Ion metabolism in malaria-infected erythrocytes

Malaria parasites of the genus Plasmodium spend much of their asexual life cycle inside the erythrocytes of their vertebrate hosts. Parasites presumably have to exploit metabolic and transport mechanisms to adapt themselves to the host erythrocyte's physicochemical environment. This review surveys the metabolism and transport of Ca2+, alkali cations, and H+ in malaria-infected erythrocytes. The Ca2+ content of Plasmodium-infected erythrocytes increases as the parasite matures. An increase in the influx of extracellular Ca2+ into infected erythrocytes is evident at later stages of parasite development. In infected erythrocytes, Ca2+ is almost exclusively localized in the parasite compartment and changes but little in the cytosol of the host cell. The importance of Ca2+ in supporting the growth of intraerythrocytic parasites and the invasion of erythrocytes by the merozoite has been assessed by depletion of extracellular Ca2+ with chelators, or by disturbance of the metabolism and transport of Ca2+ with a variety of Ca2+ modulators. Membranes of malaria-infected erythrocytes change their permeability to alkali cations. Hence, levels of K+ decrease and levels of Na+ increase in the cytosol of infected erythrocytes. Intraerythrocytic parasites maintain a high K+, low Na+ state, suggesting a mechanism for transporting K+ inward and Na+ outward against concentration gradients of the alkali cations across the parasite plasma membrane and/or the parasitophorous vacuole membrane (PVM). Concomitantly, P. falciparum can grow in Na(+)-enriched human erythrocytes. Experimental evidence suggests that Plasmodium possesses in its plasma membrane a proton pump which is very sensitive to orthovanadate, carbonylcyanide m-chlorophenylhydrazone, a protonophore, and dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, but is only slightly sensitive to inhibitors of bacterial and mitochondrial respiration, such as antimycin A, CN-, or N3-, and ouabain, a Na+, K(+)-ATPase inhibitor. By operating this proton pump, parasites extrude H+ and thus generate an electrochemical gradient of protons (an internal negative membrane potential and a concentration gradient of protons) across the parasite plasma membrane. The electrochemical gradient apparently drives inward movement of Ca2+ and, possibly, glucose from the cytosol of infected erythrocytes. Little is known about the transport properties of the PVM. Recent sequence studies suggest that Plasmodium contains a cation-transporting ATPase which exhibits a high homology to the Ca2(+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidative damage of mice erythrocytes infected with Plasmodium berghei

By using the model of infection with plasmodium berghei in white mice the attempt was made to explain the oxidative damages of red blood cells as a cause for haemolysis. In addition to a diminished new formation of erythrocytes there was an increased cell lysis under the impact of infection. Without any changes of the Met-Hb-percentage and of Heinz bodies an increase of GSH and GSSG could be measured. The conclusion was drawn that a damage of red blood cells caused by oxidation may occur by changing the relation of reduced tripeptides to oxidised ones.     

Micronucleated CD71-positive reticulocytes: a blood-based endpoint of cytogenetic damage in humans

The frequency of micronuclei (also known as Howell-Jolly bodies) in peripheral blood erythrocytes of humans is extremely low due to the efficiency with which the spleen sequesters and destroys these aberrant cells. In the past, this has precluded erythrocyte-based analyses from effectively measuring chromosome damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronucleated reticulocytes (MN-RET) in human blood. Differential staining of these cells was accomplished by combining the immunochemical reagent anti-CD71-FITC with a nucleic acid dye (propidium iodide plus RNase). The immunochemical reagent anti-CD42b-PE was also incorporated into the procedure in order to exclude platelets which can interfere with analysis. This analytical system was evaluated with blood samples from ten healthy volunteers, one splenectomized subject, as well as samples collected from nine cancer patients before and over the course of radio- or chemotherapy. The mean frequency of MN-RET observed for the healthy subjects was 0.09%. This value is nearly two orders of magnitude higher than frequencies observed in mature erythrocytes, and is approximately half the MN-RET frequency observed for the splenectomized subject (0.20%). This suggests that the spleen's effect on micronucleated cell incidence can be minimized by restricting analyses to the youngest (CD71-positive) fraction of reticulocytes. Furthermore, MN-RET frequencies were significantly elevated in patients undergoing cancer therapy. Collectively, these data establish that micronuclei can be quantified in human peripheral blood reticulocytes with a single-laser flow cytometer, and that these measurements reflect the level of chromosome damage which has occurred in red marrow space.