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.     

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.     

Evaluation of proliferative disorders in canine bone marrow by use of flow cytometric scatter plots and monoclonal antibodies

The combination of flow cytometric scatterplot analysis and specific monoclonal antibodies was used to evaluate the lineage of cells from six dogs with proliferative disorders of bone marrow. Scatterplot analysis was used to identify mature and immature myeloid and erythroid cells. The immunophenotype of cells in the immature myeloid gate was determined by labeling cells with four monoclonal antibodies. These results were compared to results of cytologic and cytochemical evaluation. The immunophenotype of a dog with a diagnosis of myelogenous leukemia was a cluster of differentiation-18 (CD-18) positive, CD-14 negative, Thy-1 negative, and a major histocompatibility complex (MHC) class II negative. The immunophenotype of a dog with a diagnosis of myelomonocytic leukemia was CD-18 positive, CD-14 positive, Thy-1 positive, and MHC class II positive. Although this phenotype clearly differentiated myelomonocytic leukemia from myelogenous leukemia, it was similar to the immunophenotype of dogs with a diagnosis of malignant histiocytosis or hemophagocytic syndrome. The immunophenotype of two dogs with myelodysplastic syndrome was CD-18 positive and CD-14 negative. Results for Thy-1 and MHC class II were variable. As additional lineage-specific monoclonal antibodies become available, immunophenotyping should become a valuable tool for determination of the lineage of cells in canine myeloproliferative disorders.     

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.     

Canine hemophagocytic histiocytic sarcoma: a proliferative disorder of CD11d+ macrophages

Histiocytic disorders of dogs include histiocytoma, localized histiocytic sarcoma (HS), disseminated HS (malignant histocytosis), and the reactive histiocytoses: cutaneous and systemic. A common element to these diseases is proliferation of dendritic cells (DC) of either Langerhans cell (epithelial DC) or interstitial DC lineage. In this report, 17 dogs with hemophagocytic HS are described. Breeds affected included Bernese Mountain Dog (6), Golden Retriever (4), Rottweiler (3), Labrador Retriever (2), a mixed-breed dog, and a Schnauzer, which were from 2.5 to 13 years old. The dogs presented with Coombs negative responsive anemia in 16/17 dogs (94%), thrombocytopenia in 15/17 dogs (88%), hypoalbuminemia in 16/17 dogs (94%), and hypocholesterolemia in 11/16 dogs (69%). All dogs died or were euthanized. The clinical course ranged from 2 to 32 weeks (mean 7.1 weeks). Diffuse splenomegaly with ill-defined masses was consistently present. Microscopic lesions were prevalent in spleen, liver, lung, and bone marrow. Metastasis occurred by insidious intravascular invasion with minimal mass formation. Histiocytes were markedly erythrophagocytic and accompanied by foci of extramedullary hemopoiesis. Cytologically, the histiocytes varied from well differentiated to atypical, with atypia more prevalent in spleen than bone marrow. These tumors arose from splenic red pulp and bone marrow macrophages, which expressed major histocompatibility complex class II and the beta2 integrin, CD11d. They had low and/or inconsistent expression of CD1 and CD11c, which are dominantly expressed by canine nonhemophagocytic HS of DC origin. Canine histiocytic proliferative diseases now encompass proliferation of all members of the myeloid histiocytic lineage: Langerhans cells, interstitial DC, and macrophages.     

Separation of bovine bone marrow into maturation-related myeloid cell fractions

A prerequisite for studies on bovine myeloid cells in relation to maturity is a reliable separation method, in order to obtain enriched and partially purified cell fractions of different maturation stages. Since current techniques for bovine bone marrow cell isolation fall short of this requirement, a technique for fractionating bovine bone marrow using a three-layer discontinuous Percoll gradient was developed. Three maturation-dependent myeloid cell fractions were obtained at specific densities, as maturation of cells is accompanied with a progressive density increase. Early immature myeloid cells, i.e. myeloblasts and promyelocytes, were found at a density of 1.060g/ml. Late immature myeloid cells, i.e. myelocytes and metamyelocytes, were retrieved at 1.080g/ml. Bands and segmented cells, representing the mature fraction, accumulated in the high-density pellet (>1.080g/ml). Myeloid cell populations were identified in each fraction by flow cytometry based on their forward and side scatter pattern. Confirmation was provided by light microscopy of flow cytometrically sorted myeloid populations, using morphological characteristics. The developed method provides a unique tool for studying maturation-dependent functions in bovine bone marrow.     

Localized and disseminated histiocytic sarcoma of dendritic cell origin in dogs

Canine histiocytic proliferative disorders include a wide spectrum of diseases characterized by different biologic behaviors. The etiology and pathogenesis of these diseases are largely unknown. The clinicopathologic, morphologic and immunophenotypic characteristics of canine localized and disseminated histiocytic sarcoma were examined in 39 dogs. Rottweilers, Bernese Mountain Dogs, and retrievers were most commonly affected (79%). Localized histiocytic sarcomas (19 dogs) arose from a single site, and metastatic lesions were observed in draining lymph nodes. Predilection sites were subcutis and underlying tissues on extremities, but tumors occurred in other locations, including spleen, lung, brain, nasal cavity, and bone marrow. Disseminated histiocytic sarcomas (20 dogs), a multisystem disease previously described as malignant histiocytosis, primarily affected spleen, lungs, bone marrow, liver, and lymph nodes. Both localized and disseminated canine histiocytic sarcomas were composed of pleomorphic tumor cell populations. CD1+, CD4-, CD11c+, CD11d-, MHC II+, ICAM-1 +, Thy-1 +/- tumor cells were identified in all snap-frozen samples (31 dogs). This phenotype is characteristic for myeloid dendritic antigen-presenting cell lineage. Hence, canine localized and disseminated histiocytic sarcomas are likely myeloid dendritic cell sarcomas. Dendritic antigen-presenting cells are a heterogeneous cell population with regards to their ontogeny, phenotype, function, and localization. The exact sublineage of the proliferating dendritic antigen-presenting cells involved in canine histiocytic sarcomas remains to be determined. Phenotypic analysis of formalin-fixed tissues from eight dogs was limited by available markers. Morphologic features and the phenotype CD18+, CD3-, and CD79a- were the most useful criteria to indicate likely histiocytic origin.     

A retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital (1996-2004)

BACKGROUND: An 8-year retrospective study was conducted to evaluate the prevalence and the classification of canine bone marrow disorders in a clinical pathology service at a university referral hospital. ANIMALS: Dogs evaluated for bone marrow disorders at a veterinary teaching hospital. HYPOTHESIS: A better understanding of the spectrum and the prevalence of canine bone marrow disorders can be achieved with a multiyear retrospective study. METHODS: Bone marrow aspirate smears, core biopsy specimens, and case records from 717 dogs were reviewed. RESULTS: Bone marrow specimens were first categorized based on the presence or the absence of a primary bone marrow disorder. Nondysplastic and nonmalignant pathologic changes were placed into 14 subcategories. Frequently observed pathologic disorders included nonregenerative immune-mediated anemia, pure red cell aplasia, bone marrow necrosis, myelofibrosis, and hemophagocytic syndrome. Dysmyelopoiesis (n = 61) was subcategorized into myelodysplastic syndromes (n = 27), and congenital (n = 1) and secondary (n = 33) dysmyelopoiesis. One hundred twenty-six cases of neoplasia were divided into acute leukemia (n = 46), chronic leukemia (n = 7), stage 5 malignant lymphoma (n = 28), multiple myeloma (n = 25), malignant histiocytosis (n = 11), metastatic mast-cell tumor (n = 3), sarcoma (n = 5), and carcinoma (n = 1). CONCLUSIONS AND CLINICAL IMPORTANCE: This study provides a general indication of the spectrum and the prevalence of canine bone marrow disorders at a referral center in North America.     

Bone Marrow Cytological Findings in 4 Dogs and a Cat With Hemophagocytic Syndrome

Hemophagocytic syndrome or hemophagic histiocytosis was diagnosed in 4 dogs and 1 cat by evaluation of bone marrow aspirate smears. One of the dogs had a suspected infection with canine parvovirus and a confirmed infection with Salmonella spp, 2 dogs had presumptive diagnoses of myeloproliferative and lymphoproliferative disease, respectively, and 1 dog died without a diagnosis. The cat had hepatic lipidosis and lesions compatible with feline calicivirus infection. All animals had cytopenias involving 2 or more cell lines, and fragmented erythrocytes in the blood, along with mild to moderate increases in the number of macro-phages in the bone marrow. Numerous marrow macro-phages contained phagocytized hematopoietic cells. Other cytological features of the bone marrow were variable in each patient, but the degree of response in the blood was inadequate, even in those with bone marrow hyperplasia. The phagocytosis of hematopoietic elements did not appear to be caused by a primary immune disorder, but rather by the inappropriate activation of normal macrophages secondary to infectious, neoplastic, or metabolic diseases. These findings suggest that hemophagocytic syndrome may be an important factor in the development of cytopenias; the data also support the cytological evaluation of bone marrow aspirates as an aid in the diagnosis of hemophagocytic syndrome. J Vet Intern Med 1996;10:7–14. Copyright © 7996 by the American College of Veterinary Internal Medicine .     

Determination of differential cell counts in feline bone marrow by use of flow cytometry

OBJECTIVE: To evaluate the potential usefulness of 2 flow cytometric methods for determination of differential cell counts in feline bone marrow. SAMPLE POPULATION: 10 bone marrow specimens from client-owned cats. PROCEDURE: Bone marrow specimens were stained with 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluated by use of flow cytometry. Differential counts were also determined by analysis of scatterplots of forward-angle versus side-angle light scatter of unstained specimens, obtained by use of flow cytometry (scatterplot method). Results of both flow cytometric methods were compared with differential cell counts determined by manually counting 1,000 cells on slides of Wright-stained smears. RESULTS: Staining with DiOC6 resulted in identification of mature and immature erythroid and myeloid cells and lymphocytes. Use of the scatterplot method resulted in identification of mature and immature erythroid and myeloid cells and metamyelocytes. However, to identify lymphocytes by use of the scatterplot method, bone marrow specimens were first labeled with an anti-major histocompatability class-II antibody. Comparison of results of the scatterplot method with manual counts yielded higher correlation coefficients and more similar mean values than did comparison of results of the DiOC6 method. Conclusions and Clinical Relevance: The scatterplot method provided more accurate and precise results than the DiOC6 method for determination of bone marrow differential cell counts in cats by use of flow cytometry. When combined with fluorescent labeling of lymphocytes, the scatterplot method has potential to provide rapid semiquantitative assessment of bone marrow differential cell counts in cats.     

Use of the Cell-Dyn 3500 to predict leukemic cell lineage in peripheral blood of dogs and cats

Background— Morphology and cytochemistry are the foundation for classification of leukemias in dogs and cats. Advances in automated hematology instrumentation, immunophenotyping, cytogenetics, and molecular biology are significantly improving our ability to recognize and classify spontaneous myeloproliferative and lymphoproliferative disorders. Objective— The purpose of this study was to assess the utility of flow cytometry‐based light scatter patterns provided by the Cell‐Dyn 3500 (CD3500) automated hematology analyzer to predict the lineage of leukemic cells in peripheral blood of dogs and cats. Methods— Leukemic cells from 15 dogs and 6 cats were provisionally classified using an algorithm based on the CD3500 CBC output data and were subsequently phenotyped by enzyme cytochemistry, immunocytochemistry, indirect flow cytometry, and analysis of antigen receptor gene rearrangement. Results— The algorithm led to correct predictions regarding the ontogeny of the leukemic cells (erythroid/megakaryocytic potential, myeloid leukemia, monocytic leukemia, chronic granulocytic leukemia, lymphoid leukemia) in 19/21 animals. Mismatches in the WBC impedance count and the WBC optical count in conjunction with microscopic assessment of blasts in the blood were useful for predicting myeloproliferative disorders with erythroid or megakaryocytic potential. The leukocyte light scatter patterns enabled distinction among myeloid leukemias (represented by acute myelomonocytic leukemia, acute monocytic leukemia, chronic granulocytic leukemia) and lymphocytic leukemias (including acute and chronic lymphocytic leukemias). One case of acute lymphocytic leukemia was misidentified as chronic lymphocytic leukemia. Conclusions— Algorithmic analyses can be applied to data generated by the CD3500 to predict the ontogeny of leukemic cells in the peripheral blood of dogs and cats. This rapid and quantitative technique may be used to improve diagnostic decisions, expand therapeutic choices, and increase prognostic accuracy.     

Hemophagocytic syndrome in dogs: 24 cases (1996-2005)

OBJECTIVE: To determine the frequency, potential causes, and clinical and clinicopathologic features of hemophagocytic syndrome in dogs. DESIGN: Retrospective study. ANIMALS: 24 client-owned dogs. PROCEDURES: Records for dogs in which diagnostic bone marrow specimens (including an aspiration smear and core biopsy material) were obtained from 1996 to 2005 were reviewed. Inclusion criteria were presence of bicytopenia or pancytopenia in the blood and > 2% hemophagocytic macrophages in the bone marrow aspirate. RESULTS: Of 617 bone marrow specimens evaluated, evidence of hemophagocytic syndrome was detected in 24 (3.9%). The Tibetan Terrier breed was overrepresented among dogs with hemophagocytic syndrome. Clinical signs associated with hemophagocytic syndrome included fever, icterus, splenomegaly, hepatomegaly, and diarrhea. Hemophagocytic syndrome was associated with immune-mediated, infectious, and neoplastic-myelodysplastic conditions and also occurred as an idiopathic condition. Overall, dogs with infection-associated hemophagocytic syndrome had better 1-month survival rates than dogs with immune-associated and idiopathic hemophagocytic syndrome. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that hemophagocytic syndrome may occur more frequently in dogs than has previously been suspected on the basis of the paucity of reported cases. Although most dogs had definable underlying disease conditions, idiopathic hemophagocytic syndrome was also identified. Hemophagocytic syndrome of any cause is potentially life-threatening; however, the prognosis should be adjusted on the basis of the associated disease process and potential for successful treatment.     

Use of monoclonal antibodies to refine flow cytometric differential cell counting of canine bone marrow cells

OBJECTIVES: To evaluate use of monoclonal antibodies to increase accuracy of flow cytometric differential cell counting of canine bone marrow cells. SAMPLE POPULATION: Bone marrow specimens from 15 dogs. PROCEDURES: Specimens were labeled with monoclonal antibodies that detected CD18, major histocompatability antigen class-II (MHC class-II), CD14, and Thy-1. Location of fluorescent and nonfluorescent cells within gates of a template developed for canine bone marrow differential cell counting was determined, the template was revised, and 10 specimens were analyzed by use of the old and revised templates and by labeling cells with anti-MHC class-II and anti-CD14. RESULTS: Data confirmed the presumptive location of marrow subpopulations in scatter plots, permitted detection of lymphocytes and monocytemacrophages, and was used to revise the analysis template used for differential cell counting. When differential cells counts determined by the original and revised templates were compared with results of manual differential cell counts, the revised template had higher correlation coefficients and more similar mean values. Labeling cells with anti-MHC class-II and anti-CD14 permitted identification of lymphoid and monocyte-macrophages cells in bone marrow specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the revised flow cytometric analysis template combined with anti-CD14 and anti-MHC class-II antibody labeling provides reliable differential cell counts for clinical bone marrow specimens in dogs. These techniques have potential applications to clinical bone marrow examination and preclinical toxicity studies.     

Malignant histiocytosis in four dogs

The clinical and pathologic features of 4 dogs with malignant histiocytosis were evaluated. The most common clinical signs were weight loss, lethargy, lymphadenopathy, hepatosplenomegaly, and anemia. Neoplastic histiocytic infiltrates most commonly were found in the spleen, bone marrow, liver, or lymph nodes. Malignant histiocytosis was considered as a differential diagnosis for anemic dogs with lymphadenopathy and/or hepatosplenomegaly.     

Hemophagocytic syndrome in a pancytopenic simian retrovirus-infected male rhesus macaque (Macaca mulatta)

Hemophagocytic syndrome (HPS) is a macrophage hyperactivation disorder triggered by disrupted T-cell macrophage cytokine interaction. HPS has been reported in humans, dogs, cats, and cattle, and it is infrequent and poorly characterized in animals. A 16-year-old male rhesus macaque was euthanized because of severe pancytopenia, including nonregenerative anemia (hematocrit = 5.5%), neutropenia (0.29 K/μl), and thrombocytopenia (21 K/μl). Bone marrow was hypocellular with normal maturation, myeloid hypoplasia, and few megakaryocytes. There were numerous morphologically normal macrophages (12% of nucleated cells), with 6% of nucleated cells being hemophagocytic macrophages in the bone marrow. Serology was negative, but polymerase chain reaction and immunohistochemistry were positive for simian retrovirus type 2. Blood and bone marrow findings were consistent with HPS. Cytopenias are common in simian retrovirus-infected macaques, but HPS has not been reported. An association between simian retrovirus infection and HPS is undetermined, but retrovirus-associated HPS has been observed in humans.     

Hematopoietic Neoplasias in Horses: Myeloproliferative and Lymphoproliferative Disorders

Leukemia, i.e., the neoplasia of one or more cell lines of the bone marrow, although less common than in other species, it is also reported in horses. Leukemia can be classified according to the affected cells (myeloproliferative or lymphoproliferative disorders), evolution of clinical signs (acute or chronic) and the presence or lack of abnormal cells in peripheral blood (leukemic, subleukemic and aleukemic leukemia). The main myeloproliferative disorders in horses are malignant histiocytosis and myeloid leukemia, the latter being classified as monocytic and myelomonocytic, granulocytic, primary erythrocytosis or polycythemia vera and megakaryocytic leukemia. The most common lymphoproliferative disorders in horses are lymphoid leukemia, plasma cell or multiple myeloma and lymphoma. Lymphoma is the most common hematopoietic neoplasia in horses and usually involves lymphoid organs, without leukemia, although bone marrow may be affected after metastasis. Lymphoma could be classified according to the organs involved and four main clinical categories have been established: generalized-multicentric, alimentary-gastrointestinal, mediastinal-thymic-thoracic and cutaneous. The clinical signs, hematological and clinical pathological findings, results of bone marrow aspirates, involvement of other organs, prognosis and treatment, if applicable, are presented for each type of neoplasia. This paper aims to provide a guide for equine practitioners when approaching to clinical cases with suspicion of hematopoietic neoplasia.     

Malignant histiocytosis in a Bernese mountain dog presenting as a mandibular mass.

A Bernese mountain dog was evaluated because of a gingival mass, multiple abdominal masses, and a pulmonary mass. Malignant histiocytosis was diagnosed based on cytological examination of splenic and bone marrow aspirates and histological examination of a bone marrow biopsy and the gingival mass. The case demonstrates that malignant histiocytosis is difficult to diagnose due to the variety of histiocytic disorders.     

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.     

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).