The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 μl of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

Lipoxygenase mRNA in rabbit reticulocytes. Its isolation, characterization and translational repression

The synthesis of the erythroid lipoxygenase, an enzyme which is of importance for the degradation of mitochondria during the maturation of reticulocytes to erythrocytes, was studied in reticulocytes from bone marrow and in density-separated fractions from peripheral blood of anemic rabbits. Lipoxygenase mRNA was enriched to about 75% by digestion of polysomes with protease K, poly(U)-Sepharose chromatography and repeated sucrose gradient centrifugation. From sucrose gradient centrifugation, electrophoresis and electron microscopy a molecular weight of about 10(6) was calculated. Synthesis of lipoxygenase is absent in erythroblasts, in very young reticulocytes obtained from bone marrow, or in the lightest fractions of reticulocytes from the peripheral blood. More mature blood reticulocytes show a considerable synthesis of the enzyme. The induction of the synthesis of the lipoxygenase seems to be initiated when reticulocytes have reached the peripheral blood. It is shown that lipoxygenase mRNA is present in reticulocytes as a translationally inactive free cytoplasmic messenger ribonucleoprotein (mRNP) particle. After deproteinization isolated mRNA obtained from masked mRNP codes for authentic lipoxygenase in a cell-free protein-synthesizing system of reticulocytes.     

Heterogeneity of globin protein synthesis in bone marrow cells of patients with homozygous beta-thalassemia from Tadzhikistan

The synthesis of globin proteins in blood reticulocytes of homozygous beta-thalassemic patients from Tadzhikistan has been previously studied. beta-thalassemia with sharp repression of beta-globin protein synthesis (alpha/beta greater than 10) has been shown to be most representative for the region. In this work, the synthesis of globin proteins has been studied in bone marrow cells of homozygous beta-thalassemic patients. Comparison of data on globin synthesis in bone marrow cells and in blood reticulocytes of the patients has revealed that in some cases the disbalance of chain synthesis in both cell types is equal. In other cases the disbalance in bone marrow cells is less than in blood cells, indicating the instability of beta-globin mRNA that is partially degrading in the process of cell maturation. Homozygous beta-thalassemic cases with low content of Hb F in blood cells (5-10%), with substantial disbalance of alpha and beta-globin synthesis and marked production of gamma-globins in bone marrow cells and in blood reticulocytes are of special interest. It has been assumed that parallel to beta-thalassemia some instability of gamma-globin proteins takes place in these patients.     

Plasma fibronectin (Fn) study in homozygous beta-thalassemia: relation to splenectomy and transfusion

The concentration of plasma Fn was determined in non-splenectomized and splenectomized patients with homozygous beta-thalassemia, before and 7-10 days after blood transfusion. The mean Fn concentration of non-splenectomized patients before transfusion did not differ from that of matched normal controls but appeared significantly decreased following blood transfusion. On the other hand, in splenectomized thalassemics, Fn levels were increased but were unrelated to transfusion. It is concluded that Fn plays some homeostatic function when RES activity of thalassemic patients is altered either as a result of splenectomy or blood transfusion.     

Biosynthesis of rat hemoglobin fractions under normal and increased erythropoiesis]

Studies of erythroid elements of hematopoietic organs and peripheric blood and bone marrow cell populations fractionated by precipitation in an albumin density gradient and synchronized by actinomycin D revealed that the rate of synthesis of individual haemoglobin fractions in the course of erythropoiesis is changed. The proliferating cells are characterized by a predominant accumulation of haemoglobin in fractions with beta c-and in the maturing cells--with beta b-chains. The radioactivity in the whole population of hematopoietic organs under phenylhydrazine anemia is mainly increased in the beta c-chains, whereas under the effects of erythropoietic factors (erythropoietin, animal sera)--that of beta b-chains is increased. In early erythroblasts the erythropoietic factors activate the formation of beta c-chains and in late ones--that of beta b-chains. Similar time dependence was observed in case of synthesis and activation of alpha b- and alpha a-chains. The specific cell responses in the erythroid series of bone marrow, spleen and blood to the effects of erythropoietic factors and anemia were established. The molecular mechanisms involved in the switch-over of the synthesis of various haemoglobin chains are discussed.     

Biological effect of human erythropoietin in transgenic mice]

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

Gall stones in sickle cell disease in the United Kingdom.

The prevalence of gall stones was studied prospectively by abdominal ultrasound examination in 131 patients with sickle cell disease aged 10-65 years. Of 95 patients with homozygous sickle cell disease, 55 (58%) had gall stones or had had a cholecystectomy. Gall stones were present in four out of 24 (17%) patients with haemoglobin S + C disease and two out of 12 (17%) with haemoglobin S beta thalassaemia. The presence of gall stones was not related to sex, geographical origin, or haematological variables and was not associated with abnormal results of liver function tests. Symptoms typical of biliary colic were reported by 32 out of 47 adult patients with gall stones, and cholecystitis or cholestasis was diagnosed in 18. Cholecystectomy was performed in 29 patients with good relief of symptoms in most cases. Postoperative complications were common, occurring in 10 of the 28 patients who could be evaluated, but not generally serious; they were considerably lessened by a preoperative exchange transfusion that reduced the haemoglobin S concentration to below 40%. It is suggested that all patients with sickle cell disease should be screened for gall stones and that elective cholecystectomy should be performed in those with symptoms or complications.     

Some selected haematological indices in Wistar rats in the vanadium-ethanol interaction

1. Wistar rats of both sexes daily received an ethanol solution of ammonium metavanadate (AMV) of 0.3 mg V/cm3 5% ethanol concentration as sole drinking liquid, for a period of 4 weeks. 2. The reference groups received for drinking aqueous AMV solution, or 5% ethanol, or water. 3. In animals drinking both water and ethanol AMV solution a decrease in the erythrocyte count and haemoglobin level was noted together with an increase of the percentage of reticulocytes and polychromatophilic erythrocytes in the peripheral blood. 4. A small rise of the percentage of polychromatophilic and orthochromatic erythroblasts was at the same time noted in the bone marrow of animals receiving ethanol AMV solution. 5. In the group of animals drinking 5% ethanol a fall of the erythrocyte count was observed and a rise of the leukocyte count, particularly of lymphocytes. 6. Substitution of water by 5% ethanol solution as solvent for AMV did not have any distinct influence on the toxicity of the tested compound.     

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

The mechanism of inactivation of the bird erythrocyte genome. III. Pathways of terminal differentiation of erythrocytes]

Under the conditions of acute phenylhydrazine anemia in pigeons, early erythroblasts develop in reticulocytes with basophilic cytoplasm directly by passing the stages of polychromatophilic and ortochromic erythroblasts. During the maximum activity of bone marrow erythropoiesis stimulated by anemia over 80% of reticulocytes develop in such a way. A hypothesis is put forward to the effect that there exist in birds two alternative pathways of erythropoiesis: the main pathway which ensures the slow repopulation of blood by erythrocytes under normal conditions and the reserve pathway which opens for a short time under anemia and ensures rapid increase of the erythrocyte titer in blood up to its normal value.     

The effect of splenectomy on bone marrow haematopoiesis in mice.

Splenectomy was performed in strain H mice. Erythrocyte macrocytosis and an increase in the reticulocyte, leucocyte and thrombocyte count were found in the peripheral blood of splenectomized animals; only the erythrocyte count fell in the first 3 weeks after splenectomy. Changes in the myelogram during the first weeks after splenectomy were characterized by an increase in the proportion of cells of the erythrocytic and lymphocytic series. The stem cell count in the bone marrow (determined after Till and McCulloch) was slightly elevated on the 8th day after splenectomy, but in subsequent weeks was rather lower than the control group values. Whatever the post-splenectomy interval at which bone marrow was taken from splenectomized mice, there was no significant difference in the transplantation effect of bone marrow cells on white and thrombocyte haematopoiesis. Bone marrow transplantation was found have a stimulant effect only on the reticulocyte count and the sooner bone marrow was collected after splenectomy, the more pronounced the effect. Changes in the myelogram and splenogram of irradiated mice to which the bone marrow cells of splenectomized mice had been transplanted were relatively small.     

Stem cells from peripheral blood and bone marrow: a comparative evaluation of the hemopoietic potential in the dog

The kinetics and pattern of hemopoietic recovery after supralethal total-body irradiation (TBI) were compared after transfusion of cryopreserved autografts derived from peripheral blood and bone marrow. Fractionated TBI was given in three doses of 6 Gy each at intervals of 48 h. Grafts of peripheral blood mononuclear cells (MNC) were collected by means of continuous-flow centrifugation and by using the mobilizing agent, dextran sulphate. Autografts were adjusted to contain equal numbers of committed progenitor cells (CFU-GM). Dogs grafted with blood-derived MNC (group A) and with MNC from bone marrow (group B) all received about 1 X 10(5) CFU-GM per kg body weight. In all dogs consistent hemopoietic engraftment was achieved. Comparing the pattern of regeneration of the granulocytes, group A dogs showed a significant regeneratory advantage over group B dogs, particularly during the first 20 days after transplantation. Lymphoid recovery was more rapid in group A until day 14. In both groups, blood lymphocytes remained below normal values beyond day 100. The regeneration patterns of the platelets and reticulocytes revealed no significant differences. These results are in agreement with the hypothesis that there are differences in the relationship between CFU-GM content and hemopoietic potential of autografts from different sources.     

B-lymphocyte differentiation in lethally irradiated and reconstituted mice. III. The influence of splenectomy on the recovery of the B-cell populations.

The recovery of the B-cell population was studied in irradiated and fetal liver-reconstituted mice. Since in irradiated and reconstituted mice the B-cell population in the spleen recovers much more rapidly than in the other lymphoid organs, we assessed the role of the spleen in the recovery of the B-cell compartment in the other organs. It was found that the absence of the spleen did not delay or diminish the recovery of the immunoglobulin (Ig)-bearing (B)-cell population in the bone marrow, lymph nodes, Peyer's patches, and peripheral blood. Throughout the recovery period the number of B lymphocytes in the lymphoid organs of splenectomized mice was even greater than in the same organs of sham-operated mice. B cells obtained from the bone marrow of splenectomized, irradiated, and reconstituted mice appeared to be fully immunocompetent, as shown by their ability to cooperate with thymocytes in an adoptive plaque-forming cell response to sheep red blood cells. The compensatory effect of the increased numbers of B cells in the bone marrow and peripheral lymphoid organs of splenectomized mice was reflected in the level of the serum immunoglobulins. Apart from a lower IgM concentration in the serum of splenectomized mice, no significant differences were found in IgG₁, IgG_2b, and IgA levels between splenectomized and sham-splenectomized mice. It is concluded that the spleen is not essential for both normal B-lymphocyte differentiation and maturation after irradiation and reconstitution.     

Bone marrow cell differential counts obtained by multidimensional flow cytometry.

Five-dimensional flow cytometric analysis of normal bone marrow aspirates was utilized to determine the frequency of neutrophils, eosinophils, monocytes, lymphocytes, nucleated erythrocytes, reticulocytes, platelets, and a cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils. Each of these bone marrow cell populations had unique features with respect to forward light scatter, orthogonal light scatter, and staining with Thiazole-Orange, LDS-751, and CD45 labeled with Phycoerythrin (PE). The identity of the cell populations was verified by sorting each of the cell populations and subsequent light microscopic examination of the cells. The frequencies of the nucleated bone marrow cell subpopulations of 50 normal donors were for neutrophils, mean 72.3%; SD +/- 5.1; 95% limits, 70.9-73.8%; eosinophils, mean 1.8%; SD +/- 1.3; 95% limits, 1.4-2.1%; monocytes, mean, 2.8%; SD +/- 1.2; 95% limits, 2.5-3.1%; lymphocytes, mean 12.1%; SD +/- 3.6; 95% limits 11.1-13.2%; nucleated erythrocytes, mean 8.9%; SD +/- 3.9; 95% limits, 7.8-10.1%; and the cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils, mean 1.6%; SD +/- 1.2; 95% limits, 1.3-1.9%. The percentage of reticulocytes in bone marrow aspirates from 50 normal donors correlated with the reticulocyte frequency in the peripheral blood of these donors. However, the mean frequency of reticulocytes was significantly greater (p < 0.0001) in bone marrow (mean 2.19%; SD +/- 0.88) than in peripheral blood (mean 1.71%; SD +/- 0.88). The technique could discriminate between immature and mature reticulocytes based on the brighter staining with both Thiazole-Orange and LDS-751 of the immature reticulocytes. This was confirmed by cell sorting of both reticulocyte populations, which revealed larger clumps of New Methylene Blue staining material in the brighter Thiazole-Orange and LDS-751 stained reticulocytes. The immature reticulocytes were present in normal bone marrow, but not in normal peripheral blood. As expected, a significantly greater frequency of nucleated cells was found in bone marrow aspirates (mean 0.85%; SD +/- 0.59) than in peripheral blood (mean 0.20%; SD +/- 0.11). The frequency of platelets was significantly lower in bone marrow (mean 1.24%; SD +/- 0.69) than in peripheral blood (mean 2.94%, SD +/- 1.14). Flow cytometric bone marrow analysis can provide clinical laboratories with a technique that generates quantitative bone marrow cell differentials and potentially can reduce the need for light microscopic examination of bone marrow smears.     

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.     

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.     

Experimental study of the organotropic properties of dactinomycin

Dactinomycin was studied pharmacologically on experimental animals. When dactinomycin was administered to the test-animals in doses close to the therapeutic ones for humans, suppression of the bone marrow blood formation was registered in spite of some increase in the number of the reticulocytes and thrombocytes in the peripheral blood and acceleration of the process of blood coagulation. In addition, the urea nitrogen blood levels increased. When the drug was administered in higher doses, suppression of the bone marrow blood formation was pronounced and the number of the leucocytes, reticulocytes and thrombocytes in the peripheral blood decreased. The rate of the blood coagulation decreased, while the biochemical values of the blood were indicative of impairement of the liver and kidney functions.