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.     

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.     

Histopathology of canine bone marrow in malignant lymphoproliferative disorders

Bone marrow core biopsies from 63 dogs with malignant lymphoproliferative disorders and leukemic involvement were evaluated. Multicentric lymphoma (44), multiple myeloma (8), chronic lymphocytic leukemia (9), and acute lymphoblastic leukemia (2) were found. Four distinct bone marrow histologic patterns were identified: focal (6), mixed (20), interstitial (28), and packed (9). Of those with focal or mixed patterns, 77% (20/26) had paratrabecular distribution. Stromal changes were infrequent, with 6% (4/63) having necrosis, 3% (2/63) fibrosis, and 6% (4/63) osteolysis. For each condition, the interstitial and mixed patterns were the most common presentations, while focal and packed patterns occurred less frequently. Morphologically, cells of metastatic lesions of lymphoma resembled those of primary sites. Colonization of bone marrow by various cytologic types of lymphoma was independent of the histologic patterns.     

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.     

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.     

Cytologic differentiation of benign from malignant canine mammary tumors

Cytologic and histologic examination of 91 canine mammary masses was performed by two cytologists and two histopathologists. Ten important cytologic criteria of malignancy for canine mammary tumors were identified. A cytologic grading system for differentiation of benign from malignant mammary tumors was proposed using these criteria. With this system, approximately one fourth of the malignant mammary tumors were given a concordant cytologic diagnosis. Approximately one-half of the benign masses were given a concordant cytologic diagnosis by the two cytologists. One-half of all the tumors examined were given inconclusive cytologic diagnoses by both cytologists. The cytologic identification of spindle cells did not differentiate complex and mixed mammary tumors from simple tumors. Only five of the animals studied died of mammary cancer, precluding a critical analysis of the cytologic criteria for prediction of cancer mortality.     

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.     

A retrospective study of canine pancytopenia

To better define the incidence and causes of canine pancytopenia, we retrospectively evaluated the results of complete blood counts submitted to the University of Minnesota Veterinary Teaching Hospital during a 1-year period. Pancytopenia was defined as packed cell volume < 36%, total leukocyte count < 6,000/microliter or total segmented neutrophil count < 3,000/microliter, and platelet count < 200,000/microliter. Of 4,560 complete blood counts, 110 (2.4%) samples from 51 dogs met the criteria for pancytopenia. Eleven different disease processes were identified. These included chemotherapy-associated pancytopenia (n=22), parvovirus infection (n=5), malignant histiocytosis (n=5), idiopathic aplastic anemia (n=3), sepsis (n=3), myelodysplastic syndrome (n=3), immune-mediated hematologic disease (n=3), lymphoblastic leukemia (n=2), ehrlichiosis (n=2), estrogen toxicity (n=2), and multiple myeloma (n=1). Malignant histiocytosis and idiopathic aplastic anemia occurred more frequently than was expected. Doxoruicin was the chemotherapeutic agent associated with pancytopenia. Hematologic recovery and patient survival time varied with the cause of pancytopenia; therefore, a specific diagnosis was essential for establishing prognosis. Differentiation among causes of pancytopenia requires a systemic approach that includes elimination of infectious and drug-induced causes, and examination of bone marrow aspiration and core biopsy specimens.     

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.     

Evaluation of bone marrow aspirates from multiple sites for staging of canine lymphoma and mast cell tumours

This prospective study evaluated the utility of bone marrow aspirates (BMAs) obtained from multiple sites for staging of canine lymphoma (LSA) and mast cell tumours (MCTs). Forty dogs (LSA, n = 24; MCTs, n = 16) were enrolled, but only 33 (82.5%) had diagnostic bone marrow (BM) aspirates obtained from two sites for inclusion in the study. Nineteen dogs with LSA were included, and 6 (31.6%) had BM involvement. Neoplastic lymphocytes were present in BM from both sites in all of these dogs. Fourteen dogs with MCTs were included, and 3 (21.4%) had BM involvement. Neoplastic mast cells were present at both sites in two dogs and at only one site in the third. These results indicate that BMAs from multiple sites may not be needed for accurate staging of canine LSA patients, but more studies evaluating the pattern of BM infiltration in dogs with high‐grade MCTs are warranted.     

Cytology of canine malignant histiocytosis

Cytologic features of bone marrow, tissue, and abdominal fluid in seven cases of malignant histiocytosis in dogs are described, and histopathology, hematology, and serum biochemistry of the cases are reviewed. Diagnosis of malignant histiocytosis was confirmed by tissue morphology and immunohistochemistry; neoplastic cells in all cases had positive immunoreactivity to lysozyme. This stain can be used to definitively establish the diagnosis of malignant histiocytosis on cytology specimens as well as tissue sections. Cytologic findings included numerous pleomorphic, large, discrete mononuclear cells with abundant, lightly basophilic, vacuolated, granular cytoplasm. Nuclei were round to oval to reniform with marked anisocytosis and anisokaryosis; nucleoli were prominent. Mitotic figures, often bizarre, were occasionally seen. Multinucleated giant cells and phagocytosis of erythrocytes and leukocytes were prominent features in cytologic preparations in four cases. Four dogs were anemic, five dogs were thrombocytopenic, and three dogs were hypercalcemic. Breeds affected included Doberman Pinscher (1), Golden Retriever (2), Flat Coated Retriever (3), and mixed-breed dog (1).     

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.     

Differentiating benign and malignant causes of lymphocytosis in feline bone marrow

Differentiation of benign and malignant causes of lymphocytosis in blood or bone marrow can be problematic. In the present study, reports of examinations of bone marrow from cats, submitted over an 8-year period, were reviewed to identify cats with increased numbers of small lymphocytes. Of 203 reports reviewed, 12 (5.9%) indicated increased numbers of small lymphocytes. Diagnoses for these cats included chronic lymphocytic leukemia (CLL; n = 2), pure red cell aplasia (PRCA; n = 4), immune-mediated hemolytic anemia (IMHA; n = 3), thymoma (n = 1), cholangiohepatitis (n = 1), and fever of unknown origin (n = 1). Several factors were identified that could be used to differentiate reactive lymphocytosis from CLL. Cats with CLL tended to be older, and lymphocytes were slightly larger and had cleaved or lobulated nuclei. Reactive lymphocytosis was associated with immune-mediated anemias and inflammatory diseases. In reactive lymphocytosis, the proliferating lymphocytes were organized into lymphoid aggregates in bone marrow and were predominately B cells. Alternatively, in CLL and thymoma, the proliferating lymphocytes were diffusely distributed and were predominately T cells. Therefore, differentiation of the causes of lymphocytosis should include evaluation of signalment, concurrent disease conditions, lymphocyte morphology, lymphocyte distribution in bone marrow, and immunophenotype. Cat age, presence of severe anemia, and evidence of inflammatory disease also should be considered.     

Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.     

Magnetic resonance imaging of bone marrow in the pelvis and femur of young dogs

The purpose of this study was to determine the magnetic resonance (MR) imaging characteristics of bone marrow in the pelvis and femur of normal, young dogs. Six greyhounds were imaged at 4, 8, 12, and 16 months of age. Sagittal images of the femur and dorsal images of the pelvis were obtained with T1-weighted, fast spin echo (FSE) T2-weighted, and short tau (T1) inversion recovery (STIR) sequences. On T1-weighted images areas with high signal intensity, similar to fat, included the femoral heads, mid-diaphysis of the femur, femoral condyles, and the body of the ilium. T2-weighted images were characterized by uniform intermediate signal intensity (less than fat, but greater than muscle) in the femoral head, high signal intensity, similar to fat, in the mid-diaphysis of the femur and ilial body, and intermediate to high signal intensity in the femoral condyle. By 16 months high signal intensity was seen in the diaphysis and distal metaphysis on both T1- and T2-weighted images. On STIR images the femoral head had intermediate to low signal intensity, compared with muscle. The mid-diaphysis of the femur was of low signal intensity, similar to fat, and the body of the ilium had mixed signal intensity at all ages. The femoral condyle had inhomogenous, intermediate to low signal intensity at 4 months, but was of uniform low signal intensity at 8-16 months.     

Evaluation of bone marrow cytology and stainable iron content in healthy adult llamas

Complete blood counts and sternal bone marrow aspirates were obtained from healthy adult llamas ranging in age from 2.5 to 8.4 years. Megakaryocyte numbers and erythroid and myeloid series maturation and morphology appeared similar to other mammalian species. The particles contained 50 to 75% marrow cells with the remainder composed of lipocytes and stromal cells. In samples with numerous particles and adequate cellularity, M/E ratios ranged from 0.9 to 2.9. Samples with higher white blood cell counts and fewer particles had higher M/E ratios. Eosinophils comprised 14.3% of the myeloid series which is higher than other domestic mammals. Hemosiderin granules were numerous in marrow particles.     

Quantification of thrombopoietic activity in bone marrow aspirates of dogs

The aims of the study were to investigate possible influences of different numbers of counted fields on slides, and different slides on the counting of platelet precursors, and to establish guidelines for bone marrow examination in dogs. The study was based on bone marrow slides of 131 healthy dogs. The slides were prepared by a squash technique. On each slide the number of thrombopoietic cells at different levels of maturation were counted in all fragment-containing fields. The results of a variance component model revealed no influence of different slides, in contrast to a high variance related to different fields. Between 20 and 25 low power fields (100x magnification) had to be examined to achieve an imprecision of 20%. An imprecision of 10% required the counting of 100 fields. The lowest thrombopoietic activity was seen in the bone marrow of dogs aged one to six years. The total number of platelet percursors per field was significantly higher in adult female (10.4 +/- 2.67) than in male dogs (7.84 +/- 2.04, P = 0.0008) as was the number of megakaryocytes. We recommend assessment of thrombopoiesis on the basis of 20-25 fragment-containing low power fields and that age- and sex-related differences should be considered.