Assessment of serum glutamate dehydrogenase activity and comparison to serum alanine aminotransferase activity in cats with increased blood total T4 concentration.

Glutamate dehydrogenase (GLDH), a key enzyme in amino acid oxidation and urea production, is mainly derived from the liver and its activity may increase with hepatocellular necrosis. Feline hyperthyroidism is associated with elevated serum activities of various enzymes, but the pattern of serum GLDH activity has not been reported, to our knowledge. Feline clinical biochemistry results from 2 commercial diagnostic laboratories were reviewed retrospectively to assess changes in serum GLDH activity in cats with significantly elevated serum total T4 concentrations, which is highly suggestive of hyperthyroidism. A total of 2,773 records were analyzed, of which 2,370 (85%) had normal total T4 (≤50 nmol/L) and 403 (15%) had increased total T4 (≥60 nmol/L) concentrations. Among cats with an increased total T4 concentration, 26.5% had increased serum GLDH activity. All cats with increased GLDH activity also had increased serum ALT activity. In 42.9% of cats, ALT activity was increased, but GLDH activity was normal. In 30.5% of cats, both serum GLDH and ALT activities were within RIs. The fold-increase of GLDH activity was almost half of the ALT fold-increase. Although serum GLDH activity increased in some cats with hyperthyroidism, serum ALT activity increased more frequently and to a greater extent.

Systemic fungal infection in a dog: a unique case in Ireland.

A three year old male entire Staffordshire bull terrier was referred to University College Dublin Veterinary Hospital, with a two week history of fever, inflammation of the right hock, lameness on the right hindlimb, peripheral lymphadenopathy and gastrointestinal signs (vomiting and diarrhoea). For the preceding three months the dog had been treated for atopic dermatitis with oral ciclosporin (5 mg/kg, PO, q 24 hours). Cytological analysis of the affected lymph nodes demonstrated fungal-like organisms predominantly contained within macrophages. Subsequent fungal culture and microscopic identification confirmed the presence of a Byssochlamys sp. This fungus is a saprophytic organism which has been associated with mycotoxin production. It has not previously been identified as a cause of systemic infection in animals or humans. Ciclosporin was discontinued, and a second generation triazole, voriconazole prescribed at a dose of 6 mg/kg for the first two doses, and continued at 3 mg/kg every 12 hours for six months. There was an excellent response. Follow-up examination five weeks after treatment was completed confirmed remission of the disease. The dog remains alive and well three years later. The present case represents an unusual fungal infection in a dog secondary to immunosuppressive therapy with ciclosporin. Such a possibility should be considered in animals presenting with signs consistent with systemic infection when receiving immunosuppressive medication.

High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas.

BACKGROUND: The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists mainly based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High content imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. OBJECTIVE: We tested the hypothesis that HCI could semi-automatize the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. METHODS: Lymphocytes were obtained from lymph nodes of six lymphoma-free and five lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T-cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: (a) cell area, (b) nuclear area, (c) nuclear displacement, (d) cytoplasmic area, (e) nucleus to cell area ratio (N/Cell), (f) nucleus to cytoplasm ratio (N/Cyt), and (g) cell roundness. RESULTS: HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences and the means for each of the samples were not overlapping between the two groups. Mean/median/SD for control versus lymphoma samples were: (a) cell area (µm(2) ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, (b) nuclear area (µm(2) ): 33.4/34.8/3.5 versus 40.2/38.5/5, (c) cytoplasm area (µm(2) ): 22.1/21/6.2 versus 40/38.4/4.9, (d) N/cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, (e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30/0.056, (f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, and (g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (P < 0.05 for all). CONCLUSION: HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype.

"High Content Imaging for the Morphometric Diagnosis and Immunophenotypic Prognosis of Canine Lymphomas"

Background: The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High Content Imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. Objective: We tested the hypothesis that HCI could semi-automatise the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. Methods: Lymphocytes were obtained from lymph nodes of 6 lymphoma-free and 5 lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T- cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: a) cell area, b) nuclear area, c) nuclear displacement, d) cytoplasmic area, e) nucleus to cell area ratio (N/Cell), f) nucleus to cytoplasm ratio (N/Cyt), g) cell roundness. Results: HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences without value overlap between groups. Mean/ median/SD for control versus lymphoma samples were: a) cell area (µm2 ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, b) nuclear area (µm2 ): 33.4/34.8/3.5 versus 40.2/38.5/5, c) cytoplasm area (µm2 ): 22.1/216.2 versus 40/38.4/4.9, d) N/Cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30//0.056, f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (p<0.05). Conclusion: HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype. © 2014 Clinical Cytometry Society.

Malignant melanoma in a grey horse: case presentation and review of equine melanoma treatment options.

A 15 year-old grey Thoroughbred gelding presented for investigation of chronic weight loss and recent onset of respiratory difficulty. Clinical examination confirmed tachypnoea with increased respiratory effort. Thoracic ultrasound examination detected pleural effusion. The dyspnoea was related to the large volume of pleural effusion and, following post-mortem examination, to the presence of a large mediastinal mass. Multiple pigmented masses, likely melanomas, were detected peri-anally. Thoracic radiography, cytological examination of the pleural fluid and a fine needle aspirate of a thoracic mass led to a presumptive diagnosis of malignant melanoma and this was confirmed at post mortem examination. Further metastatic spread to the central nervous system and right guttural pouch was also identified. In conclusion this case manifests the potential malignant behaviour of equine melanomas, and a review of proposed therapies for melanoma treatment highlights the therapeutic options and current areas of research.

Rapid, effective and user-friendly immunophenotyping of canine lymphoma using a personal flow cytometer.

BACKGROUND: Widespread use of flow cytometry for immunophenotyping in clinical veterinary medicine is limited by cost and requirement for considerable laboratory space, staff time, and expertise. The Guava EasyCyte Plus (Guava Technologies, Hayward, CA, US) is the first, personal, bench-top flow cytometer designed to address these limitations. OBJECTIVE: The aim of this study was to adapt the immunohistochemical protocol used for immunophenotyping of canine lymphoma to the personal flow cytometer for rapid, effective and user-friendly application to the diagnosis and prognosis of canine lymphoma and to demonstrate its practicality for widespread veterinary application. Performance of the personal flow cytometer for immunophenotyping T and B lymphocytes in blood and lymph nodes from normal dogs and dogs with lymphoproliferative disease, was assessed using only two monoclonal antibodies (against CD3 and CD21), and by comparison with analysis using two conventional flow cytometers. METHODS: 26 dogs with lymphoproliferative disease (23 with lymphoma, 3 with lymphocytic leukaemia) were studied along with 15 controls (2 non-lymphoma lymph nodes and 13 non-leukemic bloods. Lymphocytes were immunostained with fluorescent-labeled, monoclonal antibodies against CD3 and CD21. To assess the effectiveness of the personal flow cytometer in discrimination between T and B cell immunophenotypes, T and B cell counts for half the samples (14 blood and 11 lymph node) were also determined using the same method and conventional flow cytometers (FACSCalibur, Cyan Dako). To assess the effectiveness of the personal flow cytometer in discriminating between leukocyte types, lymphocyte differential counts were determined for 21 blood samples and compared with those from automated hematology analyzers (CELL-DYN 3500, n=11 and ADVIA 2120, n=10). Quality and sub-cellular distribution of immunostaining was assessed using fluorescence microscopy. RESULTS: The protocol for immunophenotyping took 2 to 3 hours to complete from the point of receipt of sample to reporting of immunophenotype. The personal flow cytometer differential lymphocyte counts correlated highly (n=20; r=0.97, p<0.0001) with those of automated haematology analyzers. The personal flow cytometer counts consistently, but mildly, underestimated the percentages of lymphocytes in the samples (mean bias of -5.3%.). The personal flow cytometer immunophenotype counts were indistinguishable from those of conventional flow cytometers for both peripheral blood samples (n=13; r=0.95; p<0.0001; bias of -1.1%) and lymph node aspirates (n=11,r=0.98; p<0.001; bias of 1%). All but one leukemic and one lymphomatous lymph node sample, out of 26 samples of dogs with lymphoproliferative disease analyzed, could be immunophenotyped as either B or T cells. CONCLUSIONS: We conclude that use of only 2 monoclonal antibodies is sufficient for immunophenotyping most cases of canine lymphoma by flow cytometry and enables rapid immunophenotyping. The personal flow cytometer may be as effectively used for immunophenotyping canine lymphoma as conventional flow cytometers. However, the personal flow cytometer is more accessible and user-friendly, and requires lower sample volumes.

Bacterial cholangiohepatitis in a dog.

A 9-year-old female Yorkshire terrier was presented for vomiting and diarrhea. Blood chemistry tests revealed hepatic dysfunction, cholestasis, and inflammation. Liver ultrasonography and liver biopsy were consistent with cholangiohepatitis. Fine-needle aspiration of the gallbladder revealed the presence of bacteria later identified as Clostridium spp. The cholangiohepatitis was successfully treated.

Veterinary and toxicological applications for the detection of cardiac injury using cardiac troponin.

The use of cardiac troponin (cTn), the 'gold-standard' biomarker of myocardial injury in humans, is growing in veterinary medicine and in animal safety studies, although there are differences in its application in animals. In this study six new assays for the marker were assessed in 619 animals of six different species (dog, cat, horse, cattle, rat and rabbit), in clinical and drug-safety studies. Healthy animals and clinical cases without cardiac disease served as controls. Several of the tested assays had poor analytic or diagnostic sensitivity and only one test was effective in all species and in all models of cardiac injury. This assay had the highest sensitivity and widest dynamic range, and identified cardiac injury due to anaemia, pancreatitis, uncontrolled Addison's and Cushing's disease, old age, renal disease, severe colic, lymphoma and neoplasia. Detection of the cTnI and cTnT forms correlated with loss of cardiac function in toxicity studies in rodents and rabbit. Increased serum cTnI was not found to correlate with disease aetiology or pathogenesis, but was effective in detecting, monitoring and quantifying ongoing cardiac injury. Cardiac injury, as demonstrated by elevated cTnI in blood, appears to be a common sequel to a wide variety of both primarily cardiac disease and of other diseases that do not primarily involve the cardiovascular system.