Detection of Anti-Erythrocyte Antibodies in Dogs with Inflammatory Bowel Disease (IBD).

Several extra-intestinal manifestations, including immune-mediated cytopenias, are reported in human inflammatory bowel disease (IBD), whereas they are poorly documented in dogs. Hypothesizing that immune-mediated subclinical anemia can occur in canine IBD, the study aim was to evaluate the erythrogram and the presence of anti-RBC antibodies in dogs with IBD. IBD was diagnosed according to the following criteria: chronic gastrointestinal signs, ruling out of extra-intestinal diseases, no improvement with diet trial, histological evidence of inflammatory infiltration, and improvement after immunosuppressant therapy. Canine Chronic Enteropathy Clinical Activity Index (CCECAI) endoscopic and histopathological scores were assessed for each dog. Twenty-five dogs were enrolled, and each dog had a CBC evaluation prior to endoscopy. The CBC was performed using laser hematology analyzer and blood smears were carefully reviewed for the presence of nucleated RBC, anisocytosis, polychromasia, and Howell-Jolly bodies. IgG and IgM anti-RBC antibodies were evaluated with flow cytometry. A high frequency of positive cases for anti-RBC antibodies in dogs with IBD (17/25 dogs) was ascertained. Approximatively 50% of dogs showed some hematologic features of RBC regeneration in addition to hematologic findings consistent with chronic inflammation. Anti-RBC antibodies and signs of erythroid regeneration may suggest possible subclinical chronic immune-mediated hemolysis that can cause anemia in dogs with IBD, together with the chronic inflammation.

Evaluation of blood and bone marrow in selected canine vector-borne diseases.

BACKGROUND: Bone marrow (BM) is a major hematopoietic organ that can harbour a variety of vector-borne pathogens; however, knowledge of BM pathological changes in dogs infected with vector-borne pathogens is limited. Thus, the aim of the present study was to assess the pathological changes in canine BM associated with natural infections by four vector-borne pathogens, as well as to determine the relationships between such changes and abnormalities of the peripheral blood. METHODS: Cytological disorders and pathological changes of the BM of 83 dogs naturally-infected with one or more of four vector-borne pathogens (i.e., Anaplasma platys, Leishmania infantum, Babesia vogeli and Hepatozoon canis) were evaluated and compared with the corresponding hematological findings. RESULTS: Dysgranulopoiesis and dysmegakaryocytopoiesis were the most frequently observed BM abnormalities in infected dogs. Erythroid suppression, and lymphocytic, monocytic and macrophage hyperplasia were also observed. Interestingly, associations between suppression and hyperplasia of specific cell lines in the marrow and corresponding changes in numbers of circulating peripheral blood cells were not observed. CONCLUSIONS: Infections with one or more of the vector-borne pathogens examined in this study should be considered as differential diagnoses for secondary dysmyelopoiesis.

Validation of spectrophotometric assays for serum paraoxonase type-1 measurement in dogs.

OBJECTIVE: To evaluate and validate 3 spectrophotometric assays for measuring serum activity of paraoxonase type-1 (PON1), an enzyme associated with high-density lipoproteins, in dogs. ANIMALS: 22 healthy adult dogs and 10 dogs with eccentrocytosis. PROCEDURES: 2 methods were adapted for use in 96-well microplates with phenyl acetate and 5-thiobutyl butyrolactonase as substrates, and 1 was adapted for use in an automated analyzer with p-nitrophenyl acetate as substrate. Blood samples were collected from all dogs, serum was harvested, and serum PON1 activity was measured with each method. RESULTS: Imprecision was low for all 3 methods, with the exception of interassay imprecision for 5-thiobutyl butyrolactonase, and results were linear across serial sample dilutions. The 3 methods were able to detect low PON1 activity when EDTA was used for blood sample collection, yielded lower PON1 values in sick dogs with eccentrocytosis than in healthy dogs, and yielded highly correlated results. CONCLUSIONS AND CLINICAL RELEVANCE: The methods described here may allow a wider use of PON1 activity as a biomarker of oxidative stress in dogs in clinical and research settings. Results of each method were robust and precise (with the exception of the interassay values for the lactonase method), and the methods were easy to set up in a laboratory.

High serum and urine lysozyme levels in a dog with acute myeloid leukemia.

A 2-year-old, female German Shepherd Dog with facial nerve paralysis was diagnosed with acute myelomonocytic leukemia based on clinical, cytologic, and immunologic findings. Proteinuria (urine proteincreatinine ratio = 1.5) occurred in the absence of renal failure. Qualitative assessment of proteinuria by sodium dodecyl sulfate-agarose gel electrophoresis revealed a broad band with a molecular weight of approximately 15 kDa that was compatible with lysozyme (LZM). A diagnosis of tubular proteinuria was made, and a chemical evaluation of LZM in serum and urine samples was performed using a turbidimetric assay. The LZM concentrations were 24.5 mg/l (reference interval: 2.5-8.0 mg/l) and 274.5 mg/l (reference interval: <2 mg/l) in serum and urine, respectively.

Serum acute phase protein concentrations in dogs with hyperadrenocorticism with and without concurrent inflammatory conditions.

BACKGROUND: Acute phase proteins (APPs) are promising markers of inflammation in dogs, because they are more sensitive than WBC counts in detecting clinical and subclinical inflammation. Endogenous corticosteroids can mask an acute phase response and make it more difficult to identify underlying inflammatory disease. OBJECTIVE: The purpose of this study was to evaluate the acute phase protein response in dogs with spontaneous hyperadrenocorticism (HAC) with and without concurrent inflammatory conditions. METHODS: Serum concentrations of C-reactive protein (CRP), haptoglobin (Hp), fibrinogen, and albumin were measured in 44 healthy adult dogs and 39 dogs with HAC; the HAC group was further divided into dogs with and without concurrent infection/inflammation. A fourth group of dogs with severe sepsis and without HAC was compared with the dogs with HAC and severe sepsis. RESULTS: Dogs with uncomplicated HAC had significantly higher Hp and fibrinogen concentrations compared with healthy control dogs (P<.001). Dogs with HAC and severe inflammatory disease also had significantly higher CRP and lower albumin concentrations than control dogs and dogs with HAC without concurrent inflammation. Dogs with sepsis but without HAC had significantly higher CRP concentrations than dogs with HAC and sepsis. CONCLUSIONS: Dogs with HAC had increases in the moderate APPs (Hp and fibrinogen), and no significant changes in CRP and albumin compared with healthy dogs. Although concurrent HAC appeared to blunt the CRP response in dogs with sepsis, increased serum CRP concentration in dogs with HAC is likely indicative of severe concurrent inflammation.

Hematologic abnormalities and flow cytometric immunophenotyping results in dogs with hematopoietic neoplasia: 210 cases (2002-2006).

BACKGROUND: Growing interest in veterinary oncohematology has facilitated the recent development and advancement of new techniques, such as flow cytometry, for immunophenotyping hematopoietic neoplasia in animals. OBJECTIVE: The aim of this retrospective study was to characterize hematologic abnormalities and flow cytometric immunophenotyping (FCI) results in cases of hematopoietic neoplasia in dogs. METHODS: Signalment, CBC data, and FCI results were obtained for 210 dogs with blood samples submitted to our laboratory. Immunophenotyping was carried out using an Epics XL-MCL flow cytometer and a panel of 10 antibodies (CD45, CD3, CD4, CD8, CD79, CD21, CD14, CD34, CD41/61, CD61). The prevalence and severity of hematologic abnormalities was determined for the different types of hematopoietic neoplasms. RESULTS: Based on cell morphology and phenotype, cases were classified as: acute lymphoblastic leukemia (ALL, n=51), acute myeloid leukemia (AML, n=33), chronic lymphocytic leukemia (CLL, n=61), and leukemic high-grade lymphoma (L-HGL, n=65). Most cases of ALL (47/51) and L-HGL (41/65) had a B-cell phenotype, while most cases of CLL (54/61) had a T-cell phenotype, with a high prevalence of the large granular lymphocyte subtype (49/61). Anemia was found in 85% of all cases and was significantly more severe in ALL and AML compared with CLL and L-HGL. Neutropenia was seen in 64-78% of acute leukemias (AML and ALL) in contrast to no cases of CLL and 11% of L-HGL. Thrombocytopenia was seen in 88-90% of acute leukemias in contrast to 15% of CLL and 40% of L-HGL. Thrombocytopenia was more prevalent (71% vs 22%) and significantly more severe in T-cell vs B-cell L-HGL. CONCLUSION: A standard CBC is useful in suggesting the type of hemoproliferative disorder and may also help to predict the phenotype, especially in cases of L-HGL.

Evaluation of EDTA hematology tubes for collection of blood samples for tests of secondary hemostasis in dogs.

OBJECTIVE: To evaluate the use of EDTA tubes for collection of blood samples for assays of secondary hemostasis in dogs. ANIMALS: 108 dogs of various ages, breeds, and sexes (19 healthy and 89 with abnormalities of secondary hemostasis). PROCEDURES: Blood samples were collected via cephalic venipuncture and transferred to sodium citrate tubes and EDTA tubes. Plasma was harvested from each type of tube for assays of concentrations of fibrinogen and D-dimer as well as prothrombin time, activated partial thromboplastin time, and antithrombin activity. Intra-assay and interassay precision and correlation coefficients for all hemostatic tests were calculated for each type of plasma sample. The effect of storage conditions on assay results for the 2 types of plasma samples was also evaluated. RESULTS: Results of hemostatic tests were highly correlated between citrated and EDTA-treated plasma samples. Intra-assay imprecision for all hemostatic tests with the exception of D-dimer concentration was < 10% for both citrated and EDTA-treated plasma samples; interassay imprecision was higher for EDTA-treated versus citrated plasma samples. Storage of plasma samples for 1 hour did not result in significantly different assay results for either type of plasma sample, but storage for 2 hours significantly affected values for EDTA-treated plasma samples. CONCLUSIONS AND CLINICAL RELEVANCE: Although evaluation of the sensitivity and specificity of hemostatic tests that use EDTA-treated plasma samples is required, EDTA may be a suitable alternative to sodium citrate as an anticoagulant for use in hemostatic testing in conditions in which tests could be performed within 1 hour after sample collection.

Vertebral polyostotic lymphoma in a young dog.

An unusual clinical presentation of lymphoma with vertebral involvement in a dog is reported. A 20-month-old intact female Golden Retriever presented with progressive paraparesis and anorexia. Complete blood count and serum biochemistry profile demonstrated pancytopenia and hypercalcemia. Ventral fusion of the lumbar vertebrae by new bony tissue deposition was evident on X-ray and CT scan. Fine needle aspiration revealed neoplastic lymphoid cells in lymph nodes and bone marrow. Histologically, vertebral bone and osteophytes, liver, bone marrow, kidney, and lymph nodes were diffusely infiltrated by neoplastic, lymphoid cells, with scant cytoplasm and round hyperchromatic nuclei. Polyostotic and medullary T-cell lymphoma with spondylosis was diagnosed. Lymphoma mainly affecting bone is uncommon in the dog. The present case differs from previously described polyostotic lymphomas in clinical signs of the disease, mainly attributable to spondylarthrosis. In addition, lymphomatous proliferation was associated with osteoproductive lesions of the vertebrae.

Artifactual changes in canine blood following storage, detected using the ADVIA 120 hematology analyzer.

BACKGROUND: Artifactual changes in blood may occur as a consequence of delayed analysis and may complicate interpretation of CBC data. OBJECTIVE: The aim of this study was to characterize artifactual changes in canine blood, due to storage, using the ADVIA 120 hematology analyzer. METHODS: Blood samples were collected into EDTA from 5 clinically healthy dogs. Within 1 hour after blood sample collection and at 12, 24, 36 and 48 hours after storage of the samples at either 4 degrees C or room temperature (approximately 24 degrees C), a CBC was done using the ADVIA 120 and multispecies software. A linear mixed model was used to statistically evaluate significant differences in values over time, compared with initial values. RESULTS: The HCT and MCV were increased significantly after 12 hours of collection at both 4 degrees C and 24 degrees C, and continued to increase through 48 hours. The MCHC initially decreased significantly at 12-24 hours and then continued to decrease through 48 hours at both temperatures. Changes in HCT, MCV, and MCHC were greater at 24 degrees C than at 4 degrees C at all time points. A significant increase in MPV and a decrease in mean platelet component concentration were observed at all time points at 24 degrees C. Samples stored at 24 degrees C for 48 hours had significantly higher percentages of normocytic-hypochromic RBCs, and macrocytic-normochromic RBCs, and lower platelet and total WBC counts. CONCLUSIONS: Delayed analysis of canine blood samples produces artifactual changes in CBC results, mainly in RBC morphology and platelet parameters, that are readily detected using the ADVIA 120. Refrigeration of specimens, even after 24 hours of storage at room temperature, is recommended to improve the accuracy of CBC results for canine blood samples.

A retrospective study of 60 cases of eccentrocytosis in the dog.

BACKGROUND: Eccentrocytes are RBCs that appear in a peripheral blood smear to have their hemoglobin shifted to one side of the cell. This abnormality, which is confined to the RBC membrane and cytoskeleton, is induced by oxidative damage. Eccentrocytes have been reported rarely in dogs and are associated with onion and garlic ingestion and the administration of oxidant drugs. OBJECTIVE: The purpose of this study was to describe the occurrence and severity of eccentrocytosis in dogs and the diseases or disorders associated with eccentrocytes. METHODS: Detailed history, and results of physical examination, CBC, biochemical and coagulation profiles, and urinalysis from all canine patients admitted during a 2.5-year period were evaluated. Eccentrocytes, when observed, were graded 1+ (few) to 4+ (many). The severity of eccentrocytosis was compared with that of anemia and reticulocytosis. RESULTS: Eccentrocytes were found in blood smears from 60 of 4251 dogs (1.4%) and were associated with mild to moderate anemia in 40 (66.6%) of the cases. Eccentrocytosis was found in 16 (26.6%) dogs with drug administration, 11 (18.3%) with presumptive onion and garlic ingestion, 8 (13.3%) with vitamin K antagonist intoxication, 7 (11.6%) with ketoacidotic diabetes, 5 (8.3%) with T-cell lymphoma, 4 (6.6%) with severe infections, 1 (1.6%) with compensated diabetes mellitus, and 8 (13.3%) with other conditions. Certain dog breeds, such as Whippet, Boxer, and English Setter, and young dogs seemed to be overrepresented. CONCLUSIONS: We describe, for the first time, associations between eccentrocytes and diabetes mellitus, T-cell lymphoma and vitamin K antagonist intoxication in dogs. The significance of eccentrocytes should not be underestimated, because they can be a signal of an oxidative process.

Use of fine needle aspirates and flow cytometry for the diagnosis, classification, and immunophenotyping of canine lymphomas.

Fifty canine lymphomas were classified cytomorphologically using the updated Kiel classification scheme. Aspirates of lymph nodes from dogs with lymphoma were stained using 5 canine-specific antibodies and 3 human-specific antibodies that cross-react with canine lymphocytes. The antibody-stained aspirates were analyzed by flow cytometry. A total of 32 (64%) of the 50 lymphomas were characterized as B-cell origin and 18 (36%) were of T-cell origin. B-cell lymphomas were identified in 12 females and 20 males with a mean age of 8.35 years. T-cell lymphomas were identified in 8 females and 10 males with a mean age of 7.9 years. A minority of the lymphomas were low-grade B-cell and T-cell lymphomas (6/50, 12% and 4/50, 8%, respectively). The most common morphologic types were high-grade centroblastic and unclassifiable plasmacytoid for B- and T-cell lymphomas (18/50, 36% and 7/50, 14%, respectively).