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.     

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.     

Chronic granulocytic leukaemia in a dog with associated bacterial endocarditis, thrombocytopenia and preretinal and retinal haemorrhages

The clinical, haematological and pathological features of a case of chronic granulocytic leukaemia (CGL) associated with valvular bacterial endocarditis are described in a nine-year-old male Springer Spaniel. The disease was characterized clinically by lethargy, weight loss, hepatosplenomegaly, anaemia and leucocytosis. Severe thrombocytopenia resulted in preretinal and retinal haemorrhages. A diagnosis of CGL was based on the finding of a hypercellular bone marrow with increased M: E ratio and morphologically abnormal myeloid precursors. The dog terminally became leucopenic and bacteraemic. Necropsy revealed invasion of liver, spleen and bone marrow with a pleiomorphic population of myeloid blast cells, consistent with transition to a blast cell crisis.     

Visceral leishmaniasis in the German shepherd dog. II. Pathology

Three German shepherd dogs were inoculated with Leishmania chagasi and three with Leishmania donovani and the infection was followed for 82 days. All infected dogs developed splenomegaly and lymphadenomegaly. In lymph nodes there was a reduction in lymphocyte population in paracortical areas, extensive proliferation of macrophages in paracortical areas and medullary cords, follicular hyperplasia, and increased numbers of plasma cells. The spleen had decreased numbers of lymphocytes in periarteriolar lymphoid sheaths, proliferation of macrophages in these regions, follicular hyperplasia, and enlargement of the red pulp with clusters of macrophages and plasma cells. The morphology of the tonsil was similar to the lymph nodes. Clusters of macrophages, often containing Leishmania spp, were present in liver, bone marrow, lung, and the intestines. The morphologic changes in lymph nodes and spleen were suggestive of a suppressed cell-mediated immunity and an active humoral immunity. The German shepherd dog may be a useful laboratory model for the study of immunopathologic changes in visceral leishmaniasis.     

Long-term complete chimerism and stable hematopoiesis in beagles after fetal liver hematopoietic stem cell transplantation

Karyotype analysis of bone marrow cells was performed from 6 Beagles that had received sex-mismatched fetal liver hematopoietic stem cell grafts 2.4 to 6.5 years earlier. Two dogs received dog leukocyte antigen-matched fetal liver cells and 4 received dog leukocyte antigen-mismatched cells. In each dog, all 25 evaluated metaphase spreads were of donor genotype. All dogs had normal hemograms and repopulation of bone marrow and no dog developed graft-vs-host disease.     

Systemic Mastocytosis in 16 Dogs

The clinical and pathologic features of systemic mastocytosis in 16 dogs are reported. There was no apparent breed or sex predilection, and the median age at presentation was 9.5 years. In 14 of 16 cases there was a primary cutaneous mast cell tumor (MCT). When cutaneous tumor location was compared with previous reports, there was no association between location and systemic dissemination. The most common presenting signs associated with the cutaneous tumor were regional dissemination, edema, ulceration, and abscessation. They were present in 12 dogs (69%). Signs of systemic illness, including anorexia, vomiting, and diarrhea, were seen in eight dogs (50%). Other than the cutaneous tumors, the most consistent physical and radiographic abnormalities included lymphadenopathy, splenomegaly, and hepatomegaly. Eosinophilia and basophilia were seen in two and five dogs, respectively. Six dogs had increased numbers of mast cells in peripheral blood or buffy coat smears. Five of the nine dogs evaluated had increased numbers of mast cells in bone marrow aspirates. Bone marrow aspiration was superior to both peripheral blood and buffy coat smears in predicting mastocytosis. Coagulation abnormalities were seen in three of five dogs tested. Using a conventional histomorphologic grading system, 10 of 13 (77%) tumors were classified as Grade III or undifferentiated and were overrepresented when compared with previous reports of cutaneous MCTs. Eighty-eight percent of the dogs either died or were euthanatized because of their tumors. Organs commonly involved at necropsy included lymph nodes, spleen, liver, and bone marrow; four dogs had gastroduodenal ulcers.     

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.     

MYELOID NEOPLASIA IN A DOG

A 4-year-old Scotch Collie bitch was presented for examination because of hyperthermia and anaemia. Haematological examination and bone marrow biopsy led to a diagnosis of myeloid neoplasia. At autopsy there was slight enlargement of the liver, spleen and some lymph nodes. Microscopic examination revealed extensive infiltration of these tissues by neoplastic myeloid cells.     

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.     

Leishmania (Leishmania) chagasi is not vertically transmitted in dogs.

The most frequent and most important mode of human or canine visceral leishmaniasis (CVL) transmission is through the bite of infected sand flies. This study investigates Leishmania (Leishmania) chagasi vertical transmission in offspring of naturally infected dogs. Thus 63 puppies from 18 female dogs with CVL were used. Parasite presence was evaluated through parasitologic and histopathologic examination of lymphatic organs, as well as polymerase chain reaction (PCR) on samples from adults (milk, uterus, placenta, spleen, liver and bone marrow) and offspring (spleen, liver, lymph nodes and bone marrow). PCR sensitivity and specificity were calculated using a microscope as the gold standard on samples of bone marrow, spleen and liver. Specificity was 100% for all organs and sensitivity was 100% for bone marrow, 71.4% for spleen and 66.6% for liver. Bone marrow smears (n = 63), histopathology and imprint of spleen (n = 25), liver (n = 25) and lymph nodes (n = 25) were performed to evaluate congenital transmission in the 63 offspring. PCR was done on 92 samples collected from 56 of the offspring. No test performed on the offspring was positive. It was not possible to confirm vertical transmission of CVL (95% confidence interval for the observed prevalence), despite positive PCR in the placenta of seropositive adults.     

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.     

Idiopathic myelofibrosis accompanied by peritoneal extramedullary hematopoiesis presenting as refractory ascites in a dog

A 2.5‐year‐old spayed female American Pit Bull Terrier dog presented with a primary complaint of chronic refractory ascites. The dog's CBC displayed a moderate to severe macrocytic, hypochromic, nonregenerative anemia, and a moderate leukopenia as result of a moderate neutropenia and monocytopenia. Microscopic examination of the blood smear showed marked anisocytosis, mild polychromasia, mild acanthocytosis and ovalocytosis, moderate schistocytosis and poikilocytosis, and 4 metarubricytes/100 WBC. Abdominal ultrasonography revealed a homogenous, mild to moderately hyperechoic appearing liver as well as marked amounts of speckled anechoic to slightly hypoechoic peritoneal fluid. Cytology of the ascitic fluid demonstrated a sterile transudate, with evidence of a chronic inflammatory reaction as well as erythroid and myeloid precursor cells, and a few megakaryocytes with occasional micromegakaryocytes. Histologic sections of bone marrow, spleen, and liver were examined, using routine H&E stains, as well as a variety of immunohistochemistry and other special stains. Histopathology of the bone marrow and spleen revealed varying degrees of fibrosis, erythroid, and myeloid hyperplasia, as well as multiple small hyperplastic clusters of megakaryocytes. The megakaryocytes displayed many features of atypia such as increased cytoplasmic basophilia and occasional abnormal chromatin clumping with mitoses. Histopathologic examination of the liver disclosed evidence of mild extramedullary hematopoiesis. This case represents the first report of canine idiopathic myelofibrosis associated with peritoneal extramedullary hematopoiesis, resulting in refractory ascites. Although idiopathic myelofibrosis is a relatively rare condition in dogs, this case demonstrates that ascites caused by peritoneal implants of hematopoietic tissue may be the initial manifestation of myelofibrosis.     

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.     

Isolation and characterization of pediatric canine bone marrow CD34+ cells

Historically, the dog has been a valuable model for bone marrow transplantation studies, with many of the advances achieved in the dog being directly transferable to human clinical bone marrow transplantation protocols. In addition, dogs are also a source of many well-characterized homologues of human genetic diseases, making them an ideal large animal model in which to evaluate gene therapy protocols. It is generally accepted that progenitor cells for many human hematopoietic cell lineages reside in the CD34+ fraction of cells from bone marrow, cord blood, or peripheral blood. In addition, CD34+ cells are the current targets for human gene therapy of diseases involving the hematopoietic system. In this study, we have isolated and characterized highly enriched populations of canine CD34+ cells isolated from dogs 1 week to 3 months of age. Bone marrow isolated from 2- to 3-week-old dogs contained up to 18% CD34+ cells and this high percentage dropped sharply with age. In in vitro 6-day liquid suspension cultures, CD34+ cells harvested from 3-week-old dogs expanded almost two times more than those from 3-month-old dogs and the cells from younger dogs were also more responsive to human Flt-3 ligand (Flt3L). In culture, the percent and number of CD34+ cells from both ages of dogs dropped sharply between 2 and 4 days, although the number of CD34+ cells at day 6 of culture was higher for cells harvested from the younger dogs. CD34+ cells harvested from both ages of dogs had similar enrichment and depletion values in CFU-GM methylcellulose assays. Canine CD34+/Rho123lo cells expressed c-kit mRNA while the CD34+/Rhohi cells did not. When transplanted to a sub-lethally irradiated recipient, CD34+ cells from 1- to 3-week-old dogs gave rise to both myeloid and lymphoid lineages in the periphery. This study demonstrates that canine CD34+ bone marrow cells have similar in vitro and in vivo characteristics as human CD34+ cells. In addition, ontogeny-related functional differences reported for human CD34+ cells appear to exist in the dog as well, suggesting pediatric CD34+ cells may be better targets for gene transfer than adult bone marrow. The demonstration of similarities between canine and human CD34+ cells enhances the dog as a large, preclinical model to evaluate strategies for improving bone marrow transplantation protocols, for gene therapy protocols that target CD34+ cells, and to study the engraftment potential of various cell populations that may contain hematopoietic progenitor cell activity.     

Pancytopenia Secondary to Lymphoid Leukemia in Three Horses

Pancytopenia was observed in two 3-year-old geldings and one 11-year-old mare. All horses had a brief history (2 days to 4 weeks) of fever, anorexia, and depression. One of the three horses had blast cells present on a peripheral blood smear. Examination of the bone marrow showed substantial infiltration with neoplastic lymphoid cells. At necropsy, neoplastic cells were restricted to the bone marrow in one horse, present in bone marrow, liver, and spleen in the second horse, and reported in multiple tissues in the third horse, including bone marrow, kidneys, lung, myocardium and lymph nodes. The value of a bone marrow aspirate and core biopsy in the investigation of pancytopenia is highlighted. (Journal of Veterinary Internal Medicine 1993; 7:360–363. Copyright © 1993 by the American College of Veterinary Internal Medicine.)     

Blood clearance of radiolabeled gold colloid by the turkey mononuclear phagocytic system

Radiolabeled gold colloid (198Au), which has been used to assess particle clearance in mammalian species, was used to assess blood-borne particle clearance in turkeys. When turkeys 16 weeks of age were injected intravenously with these particles, there was greater than a 98% decrease in blood gamma emission from 1 minute to 6 minutes postinjection. Uptake of particles was predominantly hepatic with minor uptake by the spleen and bone marrow. Negligible uptake was observed in lung, kidney, and skeletal muscle. Autoradiography demonstrated particles within Kupffer cells of the liver, periarteriolar macrophages of the spleen, and bone-marrow macrophages. Particles could not be demonstrated within the lung or kidney. The mononuclear phagocyte system responsible for blood particle clearance in turkeys is therefore located predominantly within the liver, spleen, and bone marrow and is similar to that of rats, mice, rabbits, and dogs. Pulmonary intravascular macrophages, which have recently been described in ruminants and pigs, are not apparent in this 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.     

Ultrasound-Guided Cytology of Spleen and Liver: A Prognostic Tool in Canine Cutaneous Mast Cell Tumor

Background: In the clinical staging of cutaneous mast cell tumors (cMCT), the diagnosis of metastasis is controversial based on cytological examination of lymph nodes, spleen, liver, bone marrow, and blood.
Objectives: To define the prognostic role of ultrasound-guided cytology of spleen and liver in cMCT. The results of cytological evaluation were compared in relation with survival time.
Animals: Fifty-two client-owned dogs with a diagnosis of cMCT.
Methods: Selection of cases was based on cytological evaluation of liver and spleen to detect infiltration at distant sites. The Kaplan Meier method was used to compare survival in dogs with and without infiltration of spleen and liver (log-rank test P < .05).
Results: Ten dogs with cMCT had mast cell infiltration of spleen, liver, or both and 4 of these dogs had involvement of the regional lymph nodes. The majority of dogs had 2 or more ultrasonographically abnormal findings simultaneously in spleen and liver. Nine dogs had grade II cMCT, and 1 had grade III cMCT. Dogs with positive evidence of mast cell infiltration to spleen, liver, or both had shorter survival times (34 versus 733 days) compared with dogs negative for mast cell infiltration at distant sites.
Conclusion and Clinical Importance: Dogs with evidence of mast cell infiltration at distant sites have a shorter survival times than dogs without evidence of infiltration at distant sites. This study suggests that cytology of spleen and liver is indicated either for ultrasonographically normal or for ultrasonographically abnormal spleen and liver in dogs with cMCT.     

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.     

Morphological aspects of prenatal haematopoietic development in the cat

The appearance of haematopoietic cells and the development of haematopoiesis in certain embryonic organs of the cat were studied using light microscopic (cytological smears and paraffin embedded tissues) and transmission electron microscopic methods. Primitive erythropoiesis occurred predominantly in the yolk sac whereas definitive erythropoiesis occurred in the yolk sac, liver, bone marrow and, to a lesser extent, in the spleen. In the yolk sac, erythropoiesis was predominantly intravascular whereas in the liver and bone marrow it was usually extravascular. Granulopoiesis occurred mainly in the liver and bone marrow. In the liver it was predominantly extravascular and occurred around vessels, bile ducts and in perisinusoidal spaces. Megakaryocytopoiesis occurred in the yolk sac, liver, bone marrow and spleen. The megakaryocytic line of cells were similar to those occurring in adult cats except that in the yolk sac unusual small, mononuclear to binuclear thrombocytogenic megakaryocytes were present also. The relative contributions of the embryonic organs of the cat to haematopoiesis were similar to those described for man and certain other mammals but the results for the sites of development and the appearance of early forms in the erythrocytic, granulocytic and megakaryocytic lines varied at times from those reported for man and other mammals.