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.     

Flow cytometric evaluation of canine bone marrow based on intracytoplasmic complexity and CD45 expression

BACKGROUND: Because of the complexity, subjectivity, time, and technical skill required for determination of manual bone marrow differential cell counts, an alternative method is needed. Several flow cytometric methods have been described, but all have limitations. OBJECTIVE: The purpose of this study was to evaluate a technique for bone marrow differential cell counting based on flow cytometric evaluation of CD45 expression and intracellular complexity (CD45 scatter plots). METHODS: Bone marrow was obtained from 15 dogs that were being evaluated for hematologic disorders. In preliminary studies, the location of bone marrow subpopulations in the CD45 scatter plots was evaluated by labeling bone marrow with lineage-specific markers. A template was developed to identify these cell populations. Gates were set to identify granulocytes, myeloblasts, monocyte/macrophages, lymphocytes, and nucleated erythroid populations. RESULTS: The CD45 labeling technique accurately quantified granulocytes, myeloblasts, erythroid precursors, and lymphocytes in canine bone marrow. Correlation coefficients with manual counts for granulocytes, myeloblasts, erythroid cells, lymphocytes, and monocyte/macrophages were 0.90, 0.89, 0.96, 0.91, and 0.54, respectively. CONCLUSIONS: The capacity of the CD45 scatter-plot technique to quantify lymphocytes and myeloblasts is an advantage over previously described techniques. The simplicity of the CD45 labeling method and the ease with which batches of samples can be analyzed makes the technique potentially applicable as a routine test in clinical and research laboratories.     

Flow cytometric evaluation of hemophagocytic disorders in canine

Background — Hemophagocytic macrophages in canine bone marrow are observed in malignant histiocytosis as well as benign hemophagocytic histiocytosis. Cytomorphologic evaluation alone may be inadequate to consistently differentiate between benign and malignant forms of hemophagocytic disorders. Objective — The purpose of this study was to evaluate the ability of flow cytometry and immunophenotyping to differentiate between benign and malignant types of hemophagocytic disorders in dogs. Methods — Blood smears and bone marrow differential cell counts were evaluated for 10 dogs with hemophagocytic disorders. Bone marrow samples were labeled with monoclonal antibodies to CD18, MCH class‐II, Thy‐1, CD14, CD3, and CD21. Using flow cytometry, forward‐angle versus side‐angle light scatter plots were analyzed and immunophenotypes were determined. Results — Scatter plots from 3 dogs with a necropsy diagnosis of malignant histiocytosis revealed 2 atypical cell clusters. One cluster contained cells of similar size or larger than immature myeloid cells and metamyelocytes. Cells in the other cluster were highly granular, with granularity similar to or greater than that of metamyelocytes. In bone marrow from dogs with malignant histiocytosis that was labeled with anti‐CD14 antibody, macrophages represented 29–48% of nucleated cells. Seven dogs had a clinical or histopathologic diagnosis of benign hemophagocytic syndrome. Three of the dogs had normal cell distribution in scatter plots. Two dogs had 2 abnormal cell clusters: 1 within the immature myeloid and metamyelocyte gates and the other with granularity similar to or greater than that of metamyelocytes. The remaining 2 dogs had an atypical cell population, mostly within the immature myeloid gate. For dogs with benign hemophagocytic syndromes, 6–17% of cells in the bone marrow were CD14 positive. Conclusions — The cellular distribution in scatter plots and the total number of macrophages in bone marrow may be useful in differentiating malignant histiocytosis from benign hemophagocytic syndromes in dogs.     

Epidemiology: Culture of bovine bone marrow progenitor cells in vitro

Summary

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400g). The use of 3% bovine leucocyte‐conditioned medium, produced by stimulation of blood lymphocytes with 4 pg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte‐conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose‐based medium requires different culture conditions, which are different from the culture conditions for human cells.     

Cytologic evaluation of bone marrow in rats: indications, methods, and normal morphology

Bone marrow examination is an important part of the evaluation of the hematopoietic system. In pharmaceutical and toxicological research, bone marrow evaluation can help determine the potential hematotoxicity or effects of new compounds on hematopoietic cells. The rat is a common research animal, and bone marrow evaluation often is performed in this species. The goal of this review is to provide clinical pathologists and researchers with an updated overview of bone marrow evaluation in rats as well as practical guidelines for methods and microscopic evaluation. Indications for bone marrow collection in a research setting, methods of collection and smear preparation, and unique morphologic features of rat bone marrow cells are discussed. A summary of published cell differential percentages for bone marrow from healthy rats and possible explanations for discrepancies in these values also are provided.     

Lesions in the thymus and bone marrow in chicks with experimentally induced chicken infectious anemia disease

One-day-old SPF chicks were inoculated with the Cux-l strain of chicken infectious anemia virus (CIAV), and the clinical development of disease and its macroscopic and microscopic alterations in the thymus and bone marrow, were observed. Tissue sections of thymus and bone marrow were stained using the streptavidin-biotin peroxidase method and examined under light microscope for evaluation of antigenic intensities in tissues. Those findings were then compared with blood parameters and ELISA results obtained through collected sera during sacrifice procedures. We sought to determine: the localization of viral antigens in thymus and bone marrow tissues after inoculation, the correlation between antigen intensities and hematologic, serologic and histopathologic findings, definitive diagnostic criteria using histopathologic and immunoperoxidase methods, and the reliability of these methods in the diagnosis of CIAV infection. For this purpose, 83, one-day-old SPF chicks were used. The birds were divided into experimental (n = 52) and control (n = 26) groups. A virus dose of TCID50 of 100,000/ml was administered intramuscularly to every bird in the experimental group. Based on the results of this study, we have suggested that clinical examination, along with macroscopic and microscopic evaluation of the thymus and bone marrow, maybe undertaken starting from day 7 post-inoculation (PI). ELISA, might be of value, as it might give consistent results starting from day 14 PI. However, the most reliable results were obtained through examination of thymus and bone marrow sections from infected birds stained by immunoperoxidase technique, as early as day 4 PI.     

沙咪珠利对小鼠精子畸形和骨髓细胞微核的影响

为评价沙咪珠利的安全性,对其进行了小鼠精子畸形和骨髓细胞微核试验研究。选择健康ICR小鼠随机分为受试物高、中、低剂量组,阳性对照组和阴性对照组,进行精子畸形试验和骨髓细胞微核试验,计算精子畸形率和微核率。结果显示,受试物各剂量组精子畸形率与骨髓细胞微核率与阴性对照组比较,差异不显著(P0.05),与阳性对照组比较,差异有统计学意义(P0.01)。表明沙咪珠利对小鼠精子和骨髓细胞微核无影响,不具有遗传毒性。     

Type of smear may influence thrombopoietic cell counts in the bone marrow of clinically healthy dogs

BACKGROUND: The expected number of thrombopoietic cells in normal canine bone marrow is poorly defined and there is no consensus on the most appropriate way to prepare cytologic smears to evaluate these cells nor on the optimum method for their quantification. OBJECTIVE: The purpose of this study was to determine total and differential counts of thrombopoietic cells in the bone marrow of clinically healthy Beagle dogs by comparing 4 different smear types and bone marrow core biopsies. METHODS: Twenty-two clinically healthy, male Beagle dogs, 10 to 12 months old, were used in the study. Following bone marrow aspiration and core biopsy from the iliac crest, Giemsa-stained smears were prepared by 4 techniques: drop-squash, particle-squash, buffy coat, and fat-layer smears. Thrombopoietic cells were counted in up to 100 low-power fields (LPF, X10 objective) in the aspiration smears and in all possible high-power fields (HPF, X40 objective) in H&E-stained biopsy sections. RESULTS: Mean total thrombopoietic cell counts were 2.76 cells/LPF (drop-squash), 1.55 cells/LPF (particle-squash), 8.05 cells/LPF (buffy coat), and 3.08 cells/LPF (fat-layer). Core biopsies yielded 5.31 cells/HPF but frequently failed to provide interpretable specimens. There was a significant difference in cell counts among the 4 smear types (P <.001). Based on evaluation of buffy coat smears, thrombopoietic cells included 1.23% megakaryoblasts, 8.77% promegakaryocytes, and 90% megakaryocytes, with a mean maturation index of 0.11. CONCLUSION: Thrombopoietic cell counts in canine bone marrow are influenced by the smear technique. Buffy coat and fat-layer smears may be useful to obtain cellular smears in hemodiluted or small aspirate samples.     

Marginal zone lymphoma in a dog

A 13-year-old spayed female American Cocker Spaniel was presented for evaluation of a cough and weight loss. Physical exam revealed generalized lymphadenopathy. The patient was diagnosed with marginal zone lymphoma (MZL) on histopathology of an extirpated lymph node. This report demonstrates an unusual case of a pleomorphic neoplastic population documented on cytologic evaluation that had moncytoid features and peripheral blood involvement; a previously undocumented IgG1 monoclonal gammopathy was also an interesting feature of this canine MZL. The patient did not undergo chemotherapy for lymphoma and was euthanized over 4 years after the initial presentation.     

Pathophysiological effects of endotoxins in ruminants

Summary

Metabolic disturbances following intravenous and intramammary administration of endotoxins in ruminants are described. In contrast to the similarity in response of blood biochemical parameters after intravenous and intramammary administrations of endotoxins, responses in plasma concentrations of enzyme activities, the thyroid hormones, cortisol, and somatotropin differ markedly. Biochemical changes in blood after endotoxin administration are predominantly dose‐dependent; thus some of the biochemical parameters ‐ especially plasma concentrations of Fe and Zn ‐ serve also to evaluate the effects of certain drugs in endotoxin models.

Changes in milk composition have been documented only after intramammary infusion of endotoxins and can partly be explained by the increased permeability of the blood/milk barrier. Appearance and production of milk returns to normal within a week after intramammary endotoxin treatment, indicating that the mammary gland is only temporarily damaged by endotoxin‐induced mastitis.     

Cytologic evaluation of benign and malignant hemophagocytic disorders in canine bone marrow

Abstract: Canine hemophagocytic disorders were studied to better understand the cytologic features that differentiate benign and malignant disease. Of 286 canine clinical bone marrow reports evaluated retrospectively, 13 (4.5%) noted at least 3% hemophagocytic macrophages. Macrophages comprised between 6% and 44% of nucleated bone marrow cells. Clinical diagnoses for dogs with hemophagocytic disorders included malignant histiocytosis (n = 2), myelodysplastic syndromes (n = 4), round cell neoplasia (n = 2), immune-mediated disorders (n = 2), and idiopathic hemophagocytic syndrome (n = 3). Differentiation of benign and malignant forms of histiocytosis was problematic. Two dogs with a diagnosis of hemophagocytic syndrome had macrophages with atypical features similar to those described for malignant histiocytosis. Furthermore, only 2 of 11 dogs with presumably benign hemophagocytic disorders had exclusively mature macrophages in bone marrow. Other dogs had variable numbers of large reticular-type cells characterized by lacy chromatin, anisocytosis, anisokaryosis, and prominent and/or multiple nucleoli. On the basis of these results, cytomorphologic evaluation of bone marrow alone may not be adequate to consistently differentiate benign and malignant forms of hemophagocytic disorders.     

Primary bone marrow T‐cell lymphoma in a Golden Retriever

A 6.2‐year‐old 28‐kg (61.7 lb) intact female Golden Retriever was referred due to persistent and multiple cytopenias noted on a routine CBC prior to a mature ovariohysterectomy procedure. The patient's physical examination was unremarkable, and staging of the thorax and abdomen identified no abnormalities. At the referral hospital, moderate hypercalcemia, borderline anemia, and neutropenia were noted. Assessment of bone marrow samples by cytology, histology, immunohistochemistry, and flow cytometry indicated a T‐cell neoplasm. The patient was treated with a multi‐agent chemotherapy protocol for 6 months, which induced remission. Nine months after diagnosis, she relapsed with recurrence of hypercalcemia, cytopenias, and clinical illness. Single‐agent anthracycline (mitoxantrone) in combination with prednisone therapy was initiated for 3 months. Two months after completion, the patient relapsed again, and palliative therapy with prednisone was elected. The patient was euthanized 16 months after diagnosis due to progressive disease. Post‐mortem histopathologic evaluation showed extensive replacement of bone marrow by neoplastic cells, and infiltrates in multiple organs. The neoplasm was diagnosed as lymphoma rather than leukemia due to the lack of abnormal circulating cells throughout the course of disease. The neoplasm was detected only in marrow at the time of initial diagnosis, and the marrow was the most extensively effaced organ at the time of death. Therefore, leukemia or stage V lymphoma was considered unlikely. In patients with a cytopenia and lack of neoplastic leukocytosis or solid tissue masses, primary bone marrow lymphoma should be considered among the differential diagnoses.     

Persistent reticulocytosis in a case of poodle macrocytosis

A healthy 14‐year‐old, male neutered, Miniature Poodle was found to have a persistent erythrocyte macrocytosis and reticulocytosis with a normal and stable HCT. The hematologic features of macrocytosis, increased Howell‐Jolly bodies, and metarubricytosis, in the absence of anemia or other cytopenias, combined with the cytologic evidence of bone marrow erythroid dysplasia, including megaloblastosis, binuclearity, increased mitotic activity, and nuclear fragmentation, are consistent with previous reports of congenital dyserythropoiesis termed poodle macrocytosis. We speculate that the additional presence of persistent reticulocytosis in the absence of an identifiable stimulus for accelerated erythropoiesis may represent a phenotypic variation of this inherited condition, and the morphologic abnormalities of the dyserythropoiesis are described.     

International recommendations for training future toxicologic pathologists participating in regulatory-type, nonclinical toxicity studies

The International Federation of Societies of Toxicologic Pathologists (IFSTP) proposes a common global framework for training future toxicologic pathologists who will support regulatory-type nonclinical toxicology studies. Trainees optimally should undertake a scientific curriculum of at least 5 years at an accredited institution leading to a clinical degree (veterinary medicine or medicine). Trainees should then obtain 4 or more years of intensive pathology practice during a residency and/or on-the-job "apprenticeship," at least 2 years of which must be focused on regulatory-type toxicologic pathology topics. Possession of a recognized pathology qualification (i.e., certification) is highly recommended. A non-clinical pathway (e.g., a graduate degree in medical biology or pathology) may be possible if medically trained pathologists are scarce, but this option is not optimal. Regular, lifelong continuing education (peer review of nonclinical studies, professional meetings, reading, short courses) will be necessary to maintain and enhance one's understanding of current toxicologic pathology knowledge, skills, and tools. This framework should provide a rigorous yet flexible way to reliably train future toxicologic pathologists to generate, interpret, integrate, and communicate data in regulatory-type, nonclinical toxicology studies.     

Histopathology of incidental findings in beagles used in toxicity studies

The purpose of our publication is to widely communicate the pictures of spontaneous findings occurring in beagles. Spontaneous arteritis occurs commonly in beagles. Frequent sites of arteritis are the heart, spleen, pancreas, epididymis and spinal cord. Morphological similarities between spontaneous and drug-induced arterial lesions may cause confusion when evaluating vascular toxicity of chemicals such as vasodilating agents. Focal and minimal inflammatory lesions are occasionally seen in the lung and may be associated with aspiration of food particles or of unknown causes. A cystic change with copious mucin production occurs occasionally in the mucosal epithelium of the gall bladder. Nesidioblastosis is seen rarely in the pancreas of beagles. C-cell complex and lymphocytic thyroiditis are common thyroid lesions. Spontaneous focal hypospermatogenesis and lobular Sertoli-cell-only seminiferous tubules occurring frequently in beagles must be distinguished from drug-induced damage of the seminiferous tubules in toxicity studies. The morphological differences of the female genital system in each cycle need to be understood; therefore, we present the normal features of the cyclic changes of the female genital organs. Further, we provide more information on spontaneous findings in beagles for exact diagnoses in toxicity studies.