Myelopoiesis and marrow adherent cells in estradiol-treated mice

Myelopoiesis and marrow adherent cells were evaluated in C57Bl/6J mice at two and four weeks after treatment with 0.1 mg 17 beta-estradiol cyclopentylpropionate. Estradiol-treated mice were lymphopenic and eosinopenic at two and four weeks; in addition, neutropenia occurred at four weeks. Numbers of lymphoid, granulocytic, and erythroid cells were decreased in the marrow at two and four weeks. The numbers of granulocyte-macrophage and fibroblast colony-forming units in the humeral marrow were also decreased at two and four weeks. However, the hematopoietic ability of marrow adherent cells was unchanged in estradiol-treated mice. Thymic cortical atrophy, metaphyseal osteosclerosis, and neutrophilic infiltration of the uterus occurred in estradiol-treated mice.     

Effect of chronic lead exposure on the canine bone marrow

The effect of chronic oral led acetate administration on canine bone marrow was studied. Two dogs (group 1) were used as controls, 4 dogs (group 2) were given 2 mg of lead/kg of body weight daily, and 4 dogs (group 3) were given 5 mg of lead/kg daily. After a 7-day stabilizaion period, lead dosing was conducted for 91 days (13 weeks), after which half of each group was treated with calcium ethylenediaminetetraacetic acid. All dogs were then observed for another 28 days (4 weeks). Blood lead values and bone marrow cellular changes were monitored once a week during the 126 days (18 weeks) of study. Lead-dosed dogs had lower weight gains than the controls. Clinical signs of toxicosis were observed after 6 weeks in one dog in group 3. Anorexia, body weight loss, CNS depression, muscular weakness, and trembling were seen. Blood lead concentrations increased in all group 2 and 3 dogs. Lead caused increases in bone marrow segmented neutrophils and myeloid series cells, and increased myeloid:erythroid ratios. Blood lead concentrations and myeloid:erythroid ratios decreased after cessation of lead administration.     

Characterization of fibroblast colony-forming units in bone marrow from healthy cats

The adherent cell layer of bone marrow from healthy cats was characterized in vitro, and the mean fibroblast colony-forming unit (CFU-F) was determined. The majority (82%) of the cells in the adherent cell layer were spindle-shaped fibroblastic cells. These cells were weakly positive for acid phosphatase activity and negative for alpha-naphthyl butyrate esterase and alkaline phosphatase activities. They did not phagocytose latex beads. The remaining cells (18%) in the adherent cell layer resembled macrophages. They were strongly positive for acid phosphatase and alpha-naphthyl butyrate esterase activities, and they phagocytosed latex beads. The mean CFU-F per 10(6) mononuclear cells in bone marrow from healthy kittens and adult cats was 62 and 65, respectively. The CFU-F assay was linear over a range of 0.25 to 1.25 x 10(6) bone marrow mononuclear cells cultured. Variation in the feline CFU-F assay was similar to that reported for the human CFU-F assay. Bone marrow collections repeated at 1-month intervals (from the same bone) did not affect CFU-F concentration. A difference was not observed between CFU-F cultured from the feline humerus or femur. Bone marrow adherent cells in cats resembled those described for other species. Results of the feline CFU-F assay were consistent and repeatable and were similar to those reported for other species.     

Influence of age on myelogram in dogs

The influence of age on the myelogram and on the different indices (ratios) was studied on bone marrow aspirates of 131 healthy dogs which were divided into different age groups (A [4-6 months] to G [> 8 years]). Myelograms were obtained by performing a differential count on 1000 cells. Age dependent differences were particularly seen in the 4 to 6 month old dogs. These dogs had significantly higher percentages of immature erythroid cells (p < 0.01) than dogs of the other age groups and, consequently, the lowest proportion of myeloid cells. This fact was also reflected by a lower quotient of number of myeloid cells/number of erythroid cells (M:E-ratio) and increased erythroid maturation index (I:ME-ratio). In addition, the proportions of monocytes and lymphocytes were increased and the number of plasma cells was decreased in juvenile dogs. The results in young dogs which were different from the values of adult animals reflect the increased requirements of red cell mass during the time of growth and ontogenesis of the immune system. These findings have to be considered in the interpretation of bone marrow films of young dogs.     

Suppression of feline bone marrow fibroblast colony-forming units by feline leukemia virus

Bone marrow fibroblast colony-forming units (CFU-F) were evaluated in cats experimentally infected with feline leukemia virus (FeLV). Cats that developed persistent viral infection and anemia (progressor cats) had a progressive decrease in the number of CFU-F at 2, 4, 6, 8, and 10 weeks after inoculation with FeLV. This suppression of CFU-F number in progressor cats ranged from 16 to 44% of the preinoculation CFU-F value. Cats that did not develop persistent viral infection or anemia (regressor cats) had decreased numbers of CFU-F (24% of the preinoculation CFU-F value) at 2 weeks after inoculation, but normal CFU-F numbers at 4, 6, 8, and 10 weeks after inoculation. In vitro incubation of bone marrow mononuclear cells from healthy cats with the 15,000-dalton envelope protein of FeLV resulted in decreased number of CFU-F (21% of that of untreated cultures). The number of CFU-F from bone marrow mononuclear cells incubated with the 27,000-dalton core protein of FeLV was similar to that from untreated cultures.     

Cytologic evaluation of primary and secondary myelodysplastic syndromes in the dog

Myelodysplastic syndromes are a heterogeneous group of acquired primary and secondary alterations of hematopoietic stem cells that result in cytopenias in blood and cytologic features of dysplasia in blood and/or bone marrow. To better understand the cytologic features that would permit differentiation of primary and secondary forms of myelodysplasia, we reviewed 267 consecutive bone marrow reports from dogs. These reports indicated that 34 dogs (12.7%) had dysgranulopoiesis, dyserythropoiesis, and/or dysthrombopoiesis in >10% of granulopoietic cells, erythroid cells, and/or megakaryocytes, respectively. Thirteen dogs had primary myelodysplastic syndromes, and 21 had secondary myelodysplastic syndromes. Of the 13 dogs with primary myelodysplasia, 4 were subclassified as myelodysplastic syndrome with refractory anemia (MDS-RA), and 9 were subclassified as myelodysplastic syndrome with excess blasts (MDS-EB). Secondary conditions associated with dysplasia in the bone marrow included malignant lymphoma (n = 5), myelofibrosis (n = 3), immune-mediated thrombocytopenia (n = 4), immune-mediated hemolytic anemia (n = 5), multiple myeloma with melphalan administration (n = 1), pyometra with estrogen administration (n = 1), polycythemia vera (n = 1), and thrombopathia (n = 1). MDS-RA was characterized by <5% myeloblasts in bone marrow, normal granulocyte maturation ratio, increased erythroid maturation ratio, and dysplastic changes in >15% of erythroid cells. MSD-EB was characterized by >/=5% myeloblasts in bone marrow, high granulocyte maturation and erythroid maturation ratios, >/=32% dysplastic granulocytes, and the presence of small atypical immature myeloid cells. Secondary myelodysplastic syndromes were characterized by <5% myeloblasts in bone marrow, variable granulocyte maturation and erythroid maturation ratios, and variable dysplastic features. These results indicate that morphology alone cannot be used to distinguish primary and secondary myelodysplastic syndromes in dogs.     

An appraisal of bone marrow biopsy in assessment of sick dogs.

Dogs were classified into a number of disease categories according to hematological, cytological and serochemical changes. Aspiration and core bone marrow biopsies were examined in 128 dogs in the various disease categories and compared to marrow samples in 36 dogs which appeared clinically normal. Differential cell counts on bone marrow smears were examined in relation to the blood variables in all animals. Blood and bone marrow data (group means) were compared among the normal and disease groups. Anemia, responsive and poorly responsive was the most frequent blood abnormality. Most dogs in the thrombocytopenia group had increased numbers of megakaryocytes in the marrow but two dogs had a marked decrease. The frequency of serious alteration of marrow production of the erythroid, myeloid and megakaryocytic series was less than anticipated. Marrow hemopoiesis was not significantly compromised in dogs with lymphoma or in dogs with other types of cancer. Bone marrow examination was necessary for the diagnosis of myelofibrosis and pancytopenia and was very helpful in the groups with insufficient change in the blood to permit a definitive diagnosis to be made. The myeloid-erythroid ratio was a useful indicator of marrow response while the erythroid maturation index and the myeloid maturation index were useful for identification of altered patterns of maturation (ineffective hemopoiesis). The reticulocyte response in absolute numbers is the most efficient and clinically relevant measure of erythroid response.     

Cephalosporin-induced changes in the ultrastructure of canine bone marrow

Fourteen healthy dogs were given 540 to 840 mg/kg of cefazedone (Refosporen) intravenously for up to 4 months or until peripheral blood cell count were depressed. Within 6 to 10 weeks treated dogs developed pancytopenia (5/14), thrombocytopenia (11/14), moderate to severe neutropenia (8/14), and/or normocytic anemia with erythroblastemia (8/14). Ultrastructural changes in bone marrow of severely cytopenic dogs included mitochondrial damage in hematopoietic and nonhematopoietic cells, thickening of endosteal bone lining layers, increased adventitial coverage of vascular sinuses, and an increased number of active macrophages. Swollen, ruptured mitochondria were in erythroid, granulocytic, and megakaryocytic cells, and, to a lesser extent, in macrophages, reticular endothelial, and bone lining cells. Maturation arrest was evident in both erythroid and granulocytic cell lines. There was also evidence of ineffective erythropoiesis and granulopoiesis. None of these changes were observed in bone marrow of controls, treated dogs that did not develop cytopenia, or dogs allowed to recover after cessation of dosing.     

Reference values for the bone marrow aspirates in adult dogs

Reference ranges for each cell type and six different ratios (M : E ratio = quotient of number of myeloid cells/erythroid precursors; different maturation ratios) were calculated for bone marrow aspirates of adult dogs. These values were based on 2.5 and 97.5% percentiles of differentials of 1000 cells in bone marrow aspirates of 92 healthy 1-8-year-old dogs. The results of intact male and female dogs were compared. No distinct sex-related differences were found (P > 0.05). A wide physiological range was observed in almost all bone marrow cells resulting in broad reference ranges for the ratios. The clinically relevant M : E ratio varied between 0.45 and 2.87. The accurate cytological examination of bone marrow based on the reference ranges presented in this study requires preparation of high-quality bone marrow films with minimal blood contamination.     

The morphological effects of vincristine sulfate on canine bone marrow cells

This study documents the morphologic changes observed in the bone marrow aspirate biopsies from dogs 6 and 24 hours after receiving a single therapeutic dose (0.025 mg/kg) of vincristine sulfate (Oncovin: Eli Lilly & Co., Indianapolis, Ind.) intravenously. The most striking cytologic changes were observed in the erythroid cell line. Abnormalities included increased numbers of mitotic figures, abnormal nuclear configurations, and fragmented nuclei. Erythroid cells in metaphase were prominent in marrow samples collected 6 hours post-vincristine, accounting for a mean of 27% of all erythroid precursors. Fragmented nuclei and atypical nuclear configurations were seen in low numbers (mean = 7%) of erythroid cells from these animals. In contrast, marrow collected from dogs 24 hours post-vincristine exhibited low numbers (mean = 1%) of erythroid cells in metaphase, but erythroid cells with atypical nuclear configurations and fragmented nuctei accounted for a mean of 41% of the erythroid cells present. Less dramatic increases in the number of mitotic non-erythroid cells were seen 6 hours post-vincristine (mean = 5% of non-erythroid cells) and 24 hours post-vincristine (mean = 1% of non-erythroid cells). Only rare nuclear fragmentation was observed in these cell lines. Significant alterations in megakaryocytes and myeloid to erythroid (M:E) ratios were not observed in samples taken 6 hours post-vincristine; however, M:E ratios were considerably higher in three of the four samples taken from dogs 24 hours post-vincristine. Similar time-related changes were observed in four clinical cases in which bone marrow aspirates were performed after vincristine administration.     

Reproducible cloning assays for in vitro growth of canine hematopoietic progenitor cells and their potential applications in investigative hematotoxicity

A variety of in vitro cloning assays have been used for studying hematopoiesis in mice and human beings. However, these techniques have had limited use in dogs, a species used extensively as a model for hematopoietic research, particularly hematotoxicity. We have adopted cloning assays for in vitro growth of canine colony-forming unit-erythroid (CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitor cells, using modified microplasma clot and soft agar culture systems, respectively. Marrow mononuclear cells separated by density-gradient centrifugation were added to the aforementioned culture systems. Erythroid colonies were stimulated with sheep plasma erythropoietin and incubated at 37 C in 5% CO2 for 2 days. The CFU-E colonies were fixed with 5% glutaraldehyde, stained with benzidine, counted, and expressed as a mean of 8 replicates. The CFU-GM colonies were stimulated with pooled serum from endotoxin-treated dogs and incubated for 8 days at 37 C in 10% CO2. Using an inverted microscope, the CFU-GM colonies were counted and expressed as a mean of 6 replicates. The number of colonies was proportional to the plated cell concentrations. The addition of 10% autologous serum to CFU-GM cultures increased the number of colonies by 80 to 100%, but markedly reduced the size and number of CFU-E colonies. The marrow cloning capacity among dogs of comparable age was similar, and little variation was noticed when bone marrow cells from the same dogs were cultured repeatedly over a period of 3 to 4 months. We concluded that these cloning assays are fast, reliable, and reproducible and that they allow quantitative determination of canine hematopoietic progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bone marrow contamination of canine cerebrospinal fluid

Bone marrow cells were identified in cytocentrifuge preparations of cerebrospinal fluid (CSF) obtained by lumbar punctures from two dogs. CSF analyses were characterized by normal or increased total cell counts, normal or increased total protein concentration and the presence of erythrocytes. Hematopoietic cells present included both erythroid and myeloid precursors and, in one case, an erythroblastic island was seen. Peripheral blood smear examination confirmed that the hematopoietic cells observed were not the result of blood contamination. Neither case was associated with a difficult tap procedure, nor with a specific disease process. Contamination may have resulted from actual marrow penetration or from extramedullary hematopoiesis in the dura mater. Accurate interpretation of CSF cytology requires the consideration of possible bone marrow contamination when hematopoietic cells are observed.     

Flow cytometric evaluation of canine bone marrow differential cell counts

Abstract: Three flow cytometric techniques were evaluated for determination of differential cell counts on canine clinical bone marrow specimens. Techniques included staining bone marrow specimens with 2'7'-dichlo-rofluorescein (DCF) or 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluation of forward-angle light scatter vs. side-angle light scatter plots. Flow cytometric evaluation of bone marrow cells stained with DCF failed to separate bone marrow cells into distinct cell populations. Staining with DiOC6 resulted in separation of bone marrow cells into populations of mature and immature erythroid cells, mature and immature myeloid cells, and lymphocytes. The scatter plot method resulted in identification of mature and immature erythroid cells, immature myeloid cells, metamyelocytes, and bands and segmenters. Lymphocytes could not be differentiated from mature erythroid cells by the scatter plot method. When the results of the DiOC6 method and the scatter plot method were compared with manual bone marrow differential cell counts, the scatter plot method had more similar mean values and higher correlation coefficients. The scatter plot method has the potential of providing rapid semiquantitative assessment of bone marrow differential cell counts in dogs for specimens that contain low numbers of lymphocytes.     

Normal haematopoiesis,cellular components and stainable iron content in the bone marrow of camels (Camelus dromedarius)

Bone marrow samples were collected from the ribs of 20 healthy adult male Iranian camels (Camelus dromedarius). The bone marrow smears were stained using Wright's stain. Blood samples were collected at the same time for routine haematological examination. The development and morphology of the blood cells in the bone marrow of the camels were similar to those of other domestic species. The mean myeloid/erythroid ratio was 1.21, the mean erythroid percentage was 42.7% and the mean myeloid percentage was 52.0%. The mean percentage of other cells was 5.3%. The mean percentage of eosinophils in the myeloid series was higher than in other domestic species. Iron stores, estimated from Perl's stain, ranged from scant (1+) to moderate (3+) but most samples had 2+ iron content. All peripheral blood results were within reference ranges.     

Chloramphenicol toxicosis in cats

Six cats were given chloramphenicol orally at the dose level of 120/mg/kg/day in 3 divided doses for 14 days and were then observed for another 3 weeks after treatment. Five other cats were used as untreated controls for the first 14 days and subsequently were given 60 mg of chloramphenicol/kg/day for 21 days. Clinical signs of toxicosis, which were more severe in cats given the higher dose level, included central nervous system depression, dehydration, reduced food intake, body weight loss, sporadic diarrhea, and vomiting. In cats given the higher dose level, chloramphenicol caused reversible marrow suppression, with marrow hypoplasia, maturation arrest of erythroid cells, and inhibition of mitotic activity, and caused vacuolation of lymphocytes and of early myeloid and erythroid cells. Significant changes were evident in bone marrow after treatment for 1 week and in peripheral blood at the end of the 2nd week. Hematologic changes included decreased numbers of neutrophils, lymphocytes, reticulocytes, and platelets. In cats given the lower dose level, changes in blood and bone marrow were similar but less severe.     

Comparison of biological activity and safety of recombinant canine erythropoietin with that of recombinant human erythropoietin in clinically normal dogs.

OBJECTIVE: To determine whether recombinant canine erythropoietin (rcEPO) stimulates erythropoiesis in dogs without causing the immunogenicity problem (ie, erythroid hypoplasia) associated with recombinant human erythropoietin (rhEPO). ANIMALS: 13 clinically normal dogs. PROCEDURE: Dogs were randomly assigned to 2 groups; 1 group (n = 6) received rhEPO, whereas the other group (7) received rcEPO. Both groups received SC injections of diluent for 4 weeks before initiating treatment with erythropoietin (100 U/kg of body weight, SC, 3 times/wk). Hematocrit and absolute reticulocyte count were monitored weekly, CBC were done monthly, and bone marrow aspirates for cytologic evaluation were obtained before and at 4, 8, 16, and 24 weeks during treatment. RESULTS: Weekly mean Hct and absolute reticulocyte count increased in both groups of dogs during the first 2 weeks of treatment. For dogs receiving rhEPO, precipitous decreases in reticulocyte number and more gradual decreases in Hct were associated with development of erythroid hypoplasia. Dogs receiving rhEPO developed erythroid hypoplasia by week 4 (n = 4), 8 (1), or 16 (1). With cessation of rhEPO treatment after diagnosis of erythroid hypoplasia, RBC production recovered 5 to 11 weeks (median, 7 weeks) later. In contrast, rcEPO treatment caused sustained increases in Hct and reticulocytosis. None of the dogs receiving rcEPO developed erythroid hypoplasia. CONCLUSIONS: rcEPO stimulated erythrocyte production in clinically normal dogs during a 24-week period without causing the erythroid hypoplasia encountered in rhEPO-treated dogs. CLINICAL RELEVANCE: Because rcEPO did not cause erythroid hypoplasia, rcEPO may represent an improved option, compared with rhEPO, for treatment of erythropoietin-dependent anemia in dogs.     

Myelofibrosis in the dog: three case reports

Myelofibrosis has been reported infrequently in dogs. The pathological features of three dogs with this disease are described. All three animals were presented clinically with non-regenerative anaemia. In two dogs, the neoplastic disorder was unaccompanied by other myeloproliferative disease and was characterized histologically by extensive replacement of normal bone marrow elements by proliferating reticulum cells and myeloid metaplasia of the enlarged liver and spleen. In one animal, focal myeloid metaplasia were also present in the lungs. In the third dog, myelofibrosis was accompanied by erythraemic myelosis and in this animal immature erythroid cells were present in blood, bone marrow, liver and spleen.     

Studies on vitamin E and selenium deficiency in young pigs. I. Hematological and biochemical changes.

Pigs which were deficient in vitamin E and/or selenium had the following parameters weekly determined from six to 13 weeks of age: Packed cell volume, hemoglobin concentration, red cell and white cell counts, red cell indices, reticulocyte count, serum iron, serum total iron binding capacity, myeloid: erythroid ratio, serum glutamic-oxaloacetic transaminase and creatine phosphokinase activities and body weight. Except for the myeloid:erythroid ratio and serum creatine phosphokinase activity, these parameters were not found to be significantly affected by either vitamin E deficiency, selenium deficiency or deficiency of both. The myeloid:erythroid ratio was increased (p less than 0.01) in association with selenium deficiency, which tends to indicate decreased erythropoiesis but was not reflected in the peripheral red cell picture. Evidence of dyserythropoiesis was not found to be a significant feature in serial bone marrow aspiration biopsies of vitamin E and/or selenium deficient pigs. Even if the serum glutamic-oxaloacetic transaminase activities were not found to be significantly affected by either vitamin E deficiency, selenium deficiency or deficiency in both as compared to replete animals, a few animals, especially in the group deficient in both vitamin E and selenium, presented quite marked transient increases of serum glutamic-oxaloacetic transaminase activity which was interpreted to reflect the occurrence of acute episodes of hepatosis dietetica. Serum creatine phosphokinase activities were found to be increased in association with vitamin E deficiency (p less than 0.01), selenium deficiency (less than 0.05) and the interaction was also significant (p less than 0.01). It was concluded that the serum creatine phosphokinase activity increases reflect the occurrence of subclinical muscular dystrophy and that vitamin E and selenium deficiencies have marked additive effects in the induction of skeletal muscular dystrophy.     

Bovine granulocyte/macrophage and erythroid colony culture: characteristics of the colonies and the assay systems.

Bovine bone marrow granulocyte/macrophage colonies were cultured in vitro in methyl cellulose and in plasma clots using bovine endotoxin-stimulated serum as a source of colony stimulating activity. The endotoxin-stimulated serum was four times as potent as the control serum in the methyl cellulose cultures. No significant increase in the number of colony forming units was observed when bovine marrow cells were maintained in suspension cultures for various periods prior to plating in methyl cellulose. The percentage of glass/plastic adherent cells in bovine marrow cells was observed to be 43% +/- 12 (SD). Benzidine positive erythroid colonies appeared in plasma clot cultures on day 4 and disappeared by day 9. No second population of erythroid colonies appeared either as a function of time or as a function of erythropoietin concentration. The optimum erythropoietin concentration for bovine erythroid cultures was found to be 1.0 unit/mL. A significant difference was observed between animals in their marrow capacity to produce erythroid colonies in culture but no significant difference was observed within individual animals over a period of three months.     

Blood and Bone Marrow Studies in Cattle Feeding on Brassica Species

Daily administration of 40—60 kg rape for 9 weeks to 8 cows produced no changes in the red blood picture. On the other hand a reduction of the myeloid: erythroid ratio and the maturity ratio of the erythroid cells of the bone marrow was found. This is interpreted as a sign of increased erythropoiesis. The Brassica-induced anaemia found in other investigations is assumed on several grounds to be haemolytic. The present study confirms this assumption, the increased breakdown of erythrocytes being entirely compensated by an increased erythropoiesis. The difference between the present and earlier reports, in which anaemia was found after a shorter time of feeding on roughly the same quantities of rape, is discussed, one possible explanation being that differences exist in rhodanid content between Brassica species.