Clinical data, clinicopathologic findings and outcome in dogs with amegakaryocytic thrombocytopenia and primary immune-mediated thrombocytopenia.

OBJECTIVES: The aim of this study was to identify distinguishing characteristics between dogs diagnosed with amegakaryocytic thrombocytopenia and those diagnosed with presumed primary peripheral immune-mediated thrombocytopenia. Presenting clinical and clinicopathologic data and outcomes were compared between the two groups. METHODS: Retrospective study performed on seven client-owned dogs diagnosed with amegakaryocytic thrombocytopenia and 34 client-owned dogs with primary peripheral immune-mediated thrombocytopenia. RESULTS: All dogs in the amegakaryocytic thrombocytopenia group were anaemic on presentation with a median haematocrit of 23% (range 9·4 to 36), while the primary peripheral immune-mediated thrombocytopoenia group had a median presenting haematocrit of 35% (range 10 to 53). Dogs with amegakaryocytic thrombocytopenia had a median of five (range 4 to 7) clinical signs of bleeding compared to a median of three (range 0 to 6) in the primary peripheral immune-mediated thrombocytopenia group with 86% (6 of 7) of amegakaryocytic thrombocytopenia dogs requiring a blood transfusion compared to 41% (14 of 34) of primary peripheral immune-mediated thrombocytopenia dogs. Six of the seven amegakaryocytic thrombocytopenia dogs did not survive to discharge, while only five of the 34 primary peripheral immune-mediated thrombocytopenia dogs did not survive to discharge. CLINICAL SIGNIFICANCE: The clinical presentation of dogs with amegakaryocytic thrombocytopenia and primary peripheral immune-mediated thrombocytopenia is similar, but dogs with amegakaryocytic thrombocytopenia had a more severe clinical course compared to primary peripheral immune-mediated thrombocytopenia dogs. The prognosis for dogs with amegakaryocytic thrombocytopenia is poor.

Secretoglobin and Transferrin Expression in Bronchoalveolar Lavage Fluid of Horses with Chronic Respiratory Disease.

BACKGROUND: Lower expression of secretoglobin and transferrin has been found in the bronchoalveolar lavage fluid (BALF) of a small number of horses with experimentally induced signs of recurrent airway obstruction (RAO) compared to healthy controls. HYPOTHESIS/OBJECTIVES: Secretoglobin and transferrin BALF expression will be similarly decreased in horses with naturally occurring clinical signs of RAO and in horses with experimentally induced clinical signs of RAO as compared to healthy controls and intermediate in horses with inflammatory airway disease (IAD). ANIMALS: Recurrent airway obstruction-affected and control horses were subjected to an experimental hay exposure trial to induce signs of RAO. Client-owned horses with a presumptive diagnosis of RAO and controls from the same stable environments were recruited. METHODS: Pulmonary function and BALF were evaluated from control and RAO-affected research horses during an experimental hay exposure trial (n = 5 in each group) and from client-owned horses (RAO-affected horses, n = 17; IAD-affected horses, n = 19; healthy controls, n = 5). The concentrations of secretoglobin and transferrin in BALF were assessed using Western blots. RESULTS: Naturally occurring and experimentally induced RAO horses had similar decreases in BALF transferrin expression, but secretoglobin expression was most decreased in naturally occurring RAO. Secretoglobin and transferrin expression were both lower in BALF of RAO-affected horses than in IAD-affected and control horses. CONCLUSIONS AND CLINICAL IMPORTANCE: Secretoglobin and transferrin expression is decreased in BALF of RAO-affected horses after both experimental and natural exposure. Secretoglobin and transferrin likely play clinically relevant roles in the pathophysiology of RAO, and may thus be used as biomarkers of the disease.

Environmental exposures and airway inflammation in young thoroughbred horses.

BACKGROUND: Inflammatory airway disease (IAD) in horses is a widespread, performance-limiting syndrome believed to develop in response to inhaled irritants in the barn environment. OBJECTIVES: To evaluate changes in bronchoalveolar lavage fluid (BALF) cytology and exposure to particulates, endotoxin, and ammonia during horses' first month in training. ANIMALS: Forty-nine client-owned 12- to 36-month-old Thoroughbred horses entering race training. METHODS: In this prospective cohort study, a convenience sample of horses was assigned to be fed hay from a net (n = 16), whereas the remaining horses were fed hay from the ground (n = 33). BALF was collected at enrollment and after 14 and 28 days in training. Respirable particulate, inhalable particulate, respirable endotoxin, and ammonia concentrations were measured at the breathing zone of each horse weekly. RESULTS: Median respirable particulates were significantly higher when horses were fed from hay nets than when fed hay from the ground (hay net 0.28 mg/m(3) , no hay net 0.055 mg/m(3) , P < .001). Likewise, inhalable particulate (hay net 8.3 mg/m(3) , no hay net 3.3 mg/m(3) , P = .0064) and respirable endotoxin (hay net 173.4 EU/m(3) , no hay net 59.2 EU/m(3) , P = .018) exposures were significantly higher when horses were fed from hay nets. Feeding hay from a net resulted in significantly higher BALF eosinophil proportions over time (P < .001). BALF eosinophils were significantly related to respirable particulate exposure (14 days in training rs = 0.37, P = .012, 28 days in training, rs = 0.38, P = .017). CONCLUSIONS AND CLINICAL IMPORTANCE: Pulmonary eosinophilic inflammation develops in response to respirable particulate exposure in young Thoroughbreds, indicating a potential hypersensitivity to inhaled particulate allergens.

Glycoproteomic profiling of serum peptides in canine lymphoma and transitional cell carcinoma.

Differential expression of fucosylated glycoproteins has been correlated with malignancy and metastatic potential in various types of neoplasia. Utilizing glycoproteomics techniques, changes in fucosylated serum peptides associated with naturally occurring canine lymphoma and transitional cell carcinoma (TCC) have been evaluated. In both types of neoplasia, the majority of the fucosylated peptides that changed increased with the cancer. In one lymphoma case that was examined over the course of the disease, the same fucosylated peptides that increased during pre-chemotherapy decreased during post-chemotherapy, and then subsequently increased upon recurrence of the lymphoma. When comparing all the fucosylated peptides that increased in both types of cancer, there were only two peptides in common allowing discrimination between lymphoma and TCC based on their peptide profiles. These results emphasize the prospect of glycopeptide profiling in proteomics for use in discovering a panel of non-invasive, diagnostic or prognostic biomarkers of cancer.

Morphologic, cytochemical staining, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes (Crotalus adamanteus).

OBJECTIVE: To evaluate light microscopic, cytochemical, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes. ANIMALS: 10 healthy snakes. PROCEDURE: Various stains, including Wright-Giemsa, benzidine peroxidase, Sudan black B, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, leukocyte alkaline phosphatase, periodic acid-Schiff with diastase, and toluidine blue, were used to stain leukocytes differentially on multiple blood smears. Electron microscopy also was performed. RESULTS: Lymphocytes were the most commonly observed leukocyte and could be distinguished from thrombocytes, using periodic acid-Schiff stain with diastase. Azurophils also were commonly observed; their granules stained with peroxidase. Eosinophils were not identified; however, 2 morphologic variations of heterophils were seen in the blood of all snakes and were considered the same cell type at different stages of cytoplasmic granule development. Heterophil granules were better preserved, using a one-step Wright-Giemsa method that did not require alcohol fixation prior to staining. Degranulated heterophils were observed in all preparations. CONCLUSIONS: Most leukocytes of eastern diamondback rattlesnakes can be identified easily on Wright-Giemsa-stained preparations. However, hematologic stains that do not require alcohol fixing prior to staining may be preferred for leukocyte evaluation in certain reptiles. A limited degree of heterophil maturation may continue in the blood of healthy snakes. This, along with degranulation of heterophils, may result in a variable staining pattern in this cell type, regardless of the stain used. CLINICAL RELEVANCE: Results provide baseline data for use in hematologic testing in diagnosis of disease and monitoring of treatment of sick or injured snakes.

Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles.

OBJECTIVE: To identify and characterize blood cells from free-ranging Hawaiian green turtles, Chelonia mydas. SAMPLE POPULATION: 26 green turtles from Puako on the island of Hawaii and Kaneohe Bay on the island of Oahu. PROCEDURE: Blood was examined, using light and electron microscopy and cytochemical stains that included benzidine peroxidase, chloroacetate esterase, alpha naphthyl butyrate esterase, acid phosphatase, Sudan black B, periodic acid-Schiff, and toluidine blue. RESULTS: 6 types of WBC were identified: lymphocytes, monocytes, thrombocytes, heterophils, basophils, and eosinophils (small and large). Morphologic characteristics of mononuclear cells and most granulocytes were similar to those of cells from other reptiles except that green turtles have both large and small eosinophils. CONCLUSIONS: Our classification of green turtle blood cells clarifies improper nomenclature reported previously and provides a reference for future hematologic studies in this species.

Primary encephalic plasma cell tumor in a dog.

A 5-year-old female spayed Spitz dog had a 5-week history of right head tilt, seizures, and progressive quadriplegia. Analysis of cerebrospinal fluid revealed 27,600 white blood cells per microliter with 63% mononuclear phagocytes, 27% lymphocytes, 6% neutrophils, 3% plasmacytoid cells, and 1% eosinophils, and over 2000 mg/dl protein. On contrast-enhanced magnetic resonance images, a focal 1-cm oval lesion was identified in the right ventral brainstem. There was also marked contrast enhancement of the meninges in the following areas: surrounding the brainstem, outlining cerebellar folia, along the ventral floor of the brain and extending to the falx cerebri, and partially outlining the left frontal lobe. At necropsy, the areas of contrast enhancement corresponded to the presence of compact cellular sheets of pleomorphic, anisocytotic, oval to polygonal neoplastic cells with plasmacytoid differentiation. The smaller of these plasmacytoid cells stained predominantly for cytoplasmic immunoglobulin A using immunoperoxidase methodology. Ultrastructurally, the neoplastic cells had morphologic features typical of plasma cells, with large amounts of predominantly rough endoplasmic reticulum with variably prominent Golgi formation. This is the first report of a canine primary intracranial malignant plasma cell tumor.

Acute myelogenous leukaemia in a mare.

A 5-year-old Thoroughbred mare presented with a 4 week history of weight loss, fever and leukopenia. Rectally, a large active foetus, thickened spleen and an abdominal mass were palpated. Leukopenia, mild anaemia, marked thrombocytopenia and hyperfibrinogenaemia were found. Cytology and cytochemical staining of a bone marrow aspirate supported a diagnosis of acute myelogenous leukaemia. The mare deteriorated despite medical therapy and was humanely euthanased.

Intraerythrocytic inclusions associated with iridoviral infection in a fer de lance (Bothrops moojeni) snake.

Intraerythrocytic inclusions associated with infection by an iridovirus were observed in a fer de lance (Bothrops moojeni) snake that was being evaluated for the presence of renal carcinoma. The erythrocytes contained two types of inclusions, one viral and one crystalline, usually concomitantly. The snake was markedly anemic and exhibited a marked regenerative response. Ultrastructural analysis identified the virus to be an iridovirus consistent with snake erythrocyte virus and the crystalline structures to be of a different nature than hemoglobin.

Basophilic leukemia in a dog.

Basophilic leukemia with thrombocytosis was diagnosed in a 4-year-old Shih Tzu. This diagnosis was based on cytochemical staining and cytologic examination of blood and bone marrow smears. Hydroxyurea, an inhibitor of DNA synthesis, at a dose of 50 mg/kg PO bid induced hematologic remission after 7 days of treatment. Adverse effects observed included pruritus, erythema of the ventral abdomen, generalized alopecia, and possibly, diabetes mellitus. The dog remained in remission for 21 months before becoming lethargic, at which time the owners requested euthanasia but did not allow a necropsy.

Staining and morphologic features of bone marrow hematopoietic cells in desert tortoises (Gopherus agassizii).

OBJECTIVES: To determine optimal site for collection of bone marrow from desert tortoises, and to characterize cytologic staining and morphologic features of bone marrow hematopoietic cells. ANIMALS: 16 desert tortoises. PROCEDURE: Bone marrow was obtained at necropsy from the pelvis, proximal portion of the humerus, femur, and thickened portions of the cranial to craniolateral and caudal to caudolateral margins of the carapace and plastron for histologic and cytologic examinations. Cytocentrifuged preparations of marrow cells were evaluated for reactivity to cytochemical stains. RESULTS: Histologic sections were adequate for evaluating acidophils, acidophil precursors, and erythrocyte precursors. It was difficult to differentiate among monocytes, lymphocytes, thrombocytes, and blast cells, and eosinophils could not be differentiated from heterophils. Basophils were in rare, small clusters of 3 to 12 cells. A few lymphoid follicles were found in the pelvis and long bones. Use of cytochemical staining accomplished differentiation between agranular heterophil precursors and granulated heterophils, and between granulated eosinophils and basophils. Monocytes, azurophils, and monoblasts had similar staining features. Staining of erythrocyte precursors with Sudan black B differentiated them from lymphocytes. Only a few small cells with periodic acid-Schiff-positive cytoplasm were identified as thrombocytes. Lymphocytes did not stain with any of the cytochemical stains. CONCLUSIONS: For histologic and cytologic evaluation of bone marrow hematopoietic cells, pelvis, proximal portion of the humerus, femur, and thickened portions of the peripheral cranial and caudal regions of the carapace and plastron are suitable sites to collect specimens. There are distinct cytochemical markers for heterophil, monocyte, and erythrocyte precursors, as well as later stage heterophils, eosinophils, basophils, monocytes, and azurophils.

Myelopoiesis and myeloproliferative disorders.

Myeloid cells arise from a common stem cell whose development is regulated by stimulatory and inhibitory growth factors. Pluripotential hematopoietic stem cells are most influenced by IL-3, GM-CSF, and stem cell factor while committed progenitor cells are regulated by variable concentrations of GM-CSF, G-CSF, M-CSF, IL-5, Epo, and Tpo. As a result of their common origin, a key point to remember about myeloproliferative disorders is the involvement of multiple cell lines in dysplastic and neoplastic conditions. Dysplastic changes may signal early neoplastic changes with cases progressing to acute leukemia. Myelodysplastic syndrome (MDS) is associated with anemia or multiple cytopenias, normal to hypercellular bone marrow, ineffective hematopoiesis, and less than 30% blast cells of all nucleated cells in the bone marrow. Chronic myeloid leukemias also have less than 30% blast cells of all nucleated cells in the bone marrow and are distinguished from MDS by elevated cell counts of one or more cell lines with mature forms predominating. Acute myeloid leukemias, often the end result of all myeloproliferative disorders, are recognized by equal or greater 30% blast cells of all nucleated cells in the bone marrow. Additional diagnostic information from cytochemical stains, immunohistochemical staining, and cytogenetic analysis can influence the final diagnosis when morphology alone is equivocal. In conclusion, prognosis and response to treatment are best determined by application of a uniform set of standards in evaluating hematolymphatic neoplasia. Critical to diagnosis are complete blood and bone marrow evaluations including observation for dysplastic changes and blast cell quantitation. In addition, evidence for tissue infiltration identified through cytologic or histologic evaluations of lymph node, spleen, or liver is recommended.

Amphotericin B treatment of Candida arthritis in two horses.

Infectious arthritis caused by Candida spp was diagnosed in 2 horses. Source of infection was by direct inoculation in 1 horse and was presumed to be hematogenous in the other horse. On microbial culturing of synovial fluid and synovial membrane specimens, the organisms were isolated in both horses. In both horses, the joint infections resolved after i.v. administration of amphotericin B and joint drainage; however, 1 horse was eventually euthanatized because of signs of cervical pain and progressively worsening ataxia. Fungal organisms isolated on microbial culturing of joint specimens in horses, although uncommon, should not be dismissed as contaminants, particularly if the same organism is recovered from more than 1 specimen. Successful resolution of fungal arthritis may be achieved with appropriate antifungal treatment, combined with joint drainage.

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.

Morphologic and cytochemical characteristics of blood cells from the desert tortoise (Gopherus agassizii).

Morphologic and cytochemical staining characteristics of erythrocytes, leukocytes, and thrombocytes of the desert tortoise (Gopherus agassizii) were evaluated, using blood smears prepared from 23 healthy tortoises of Kern County, Calif. Special emphasis was placed on differentiating features of the various leukocytes and thrombocytes. A variety of cytochemical stains, including benzidine peroxidase, Sudan black B, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, leukocyte alkaline phosphatase, periodic acid-Schiff, and toluidine blue were used. Heterophils had a characteristic, large, focal area of positive staining with chloroacetate esterase, alpha-naphthyl butyrate esterase, and acid phosphatase. Eosinophils stained diffusely positive with benzidine peroxidase, allowing differentiation of this leukocyte from heterophils. Thrombocytes stained focally positive with periodic acid-Schiff, allowing differentiation of these cells from lymphocytes, which stained uniformly negative. An intracytoplasmic body, commonly observed within erythrocytes, was considered ultrastructurally to represent a degenerate organelle.

Effects of human recombinant interleukin 2 on in vitro tumor cytotoxicity in dogs.

In these studies, the effects of recombinant human interleukin 2 (rHuIL-2) were examined on in vitro tumor cytotoxicity by canine blood lymphocytes obtained from peripheral vessels through use of a chromium release microcytotoxicity assay. Cytotoxic activity by lymphokine-activated killer cells was significantly increased, compared with that by untreated lymphocytes in a dose-dependent manner. The maximal effect was attained with 300,000 IU of rHuIL-2/ml. Lymphokine-activated killing also was dependent on the duration of incubation with rHuIL-2. After 1 day of rHuIL-2 incubation, cytotoxicity was significantly increased, compared with that of untreated lymphocytes. Of the 3 times examined, cytotoxicity peaked after 3 days of rHuIL-2 incubation. High levels of cytotoxic activity were still present at 7 days of incubation. Numbers of granular lymphocytes increased over the times examined. These results demonstrate functional and morphologic changes in canine peripheral blood lymphocytes obtained from peripheral vessels after incubation with rHuIL-2 in a dose- and time-dependent manner.

Prevalence of leukemic blood and bone marrow in dogs with multicentric lymphoma.

Multicentric lymphoma was diagnosed in 53 dogs. A study was performed to evaluate the prevalence of leukemic involvement in blood samples, bone marrow aspirates, and bone marrow core biopsy specimens at the time of initial diagnosis. Data indicated that 57% (30/53) of the dogs were leukemic when all materials were considered relative to the presence of cellular atypia or immaturity and abnormal tissue distribution. In the 30 leukemic dogs, detection was made in the specimens with the following frequency: 15 in blood (50%), 18 in bone marrow aspirates (60%), and 29 in bone marrow core biopsy specimens (97%). Five cases (17%) were only detected by core biopsy examination, even when dogs with bone marrow lymphocytosis of greater than 15% of nucleated cells were considered leukemic. Nondiffuse histologic colonization patterns accounted for the lack of correlation between the type of bone marrow specimens. Clinical staging for treatment response and prognosis was best determined by evaluation of concurrently obtained blood samples, bone marrow aspirates, and bone marrow core biopsy specimens.