Grey eosinophils in a Miniature Schnauzer with a poorly differentiated mast cell tumor.

A 10-year-old female spayed Miniature Schnauzer was presented for investigation of an intra-nasal mass. The mass was diagnosed by histopathologic examination as an undifferentiated round cell neoplasm with an infiltrate of segmented leukocytes, interpreted as neutrophilic inflammation. The mass was treated with palliative radiation and systemic chemotherapy due to the presence of regional lymph node metastasis. During subsequent monitoring over several months, the peripheral leukocyte concentration was repeatedly within reference intervals to slightly increased with low numbers of toxic neutrophils. Four months after the initial diagnosis, there was a significant leukocytosis of 66 100 cells/µL, and 39 700 cells/µL of the leukocytes had variably mature, lobulated, and hypolobulated nuclei, and grey cytoplasm with clear vacuoles, resembling grey eosinophils. To further characterize these cells, peripheral blood smears from the patient and a canine control with eosinophilia were stained for alkaline phosphatase (AP), peroxidase, and esterase activities, and with Luxol fast blue (LFB). Histopathologic sections of the nasal mass were stained with LFB and immunohistochemically for tryptase. On blood smears, the cytoplasm of the suspected grey eosinophils stained for AP and granules stained with LFB confirmed that there was an eosinophilic lineage. Peroxidase staining was weak, and esterase staining was absent. On histopathologic sections from the nasal mass, the segmented leukocytes contained LFB-staining granules, indicating an eosinophilic infiltrate was present. Neoplastic cells expressed tryptase, which confirms a mast cell lineage. Our findings suggest that grey eosinophils might be under-recognized and interpreted incorrectly as toxic neutrophils. This report expands the canine breeds in which these eosinophils have been identified.

Analytical quality assessment and method comparison of two immunoassays for the measurement of serum cardiac Troponin I in dogs and cats.

BACKGROUND: Cardiac troponin I (cTnI) is a sensitive and specific biomarker for myocardial injury. Validation of point-of-care (POC) analyzers for cTnI measurement is valuable to the critical care setting, in which rapid results can facilitate prompt diagnoses. An immunoassay for detecting cTnI is available for the POC AIA-360 analyzer (Tosoh Bioscience), but this has not been validated using canine and feline serum. OBJECTIVES: The objectives were (a) to determine precision, accuracy, and linearity of cTnI measurement using the AIA-360 immunoassay in pooled canine and feline samples, and (b) to compare results for individual canine and feline samples with those obtained using a reference chemiluminescence method (Immulite 1000, Siemens). METHODS: Intra- and inter-assay repeatability was determined using pooled canine and feline samples, and the coefficient of variation (CV) was calculated for each. Pooled samples were also serially diluted to assess linearity. A modified spike and recovery analysis was performed by mixing pooled samples with different concentrations. Bland-Altman and Deming regression analyses were used to determine bias for individual samples, and the total observed error (TEobs ) was calculated. RESULTS: Coefficient of variation values were well within the required maximum of 20%. Linearity was demonstrated over the range of samples tested, and the recovery study showed minimal proportional inaccuracies. Although the correlation between the analyzers was excellent, there was a large mean bias due to relative proportional bias. Total observed error consequently exceeded the total allowable error (TEA ). CONCLUSION: Although, in most respects, the analyzer demonstrated adequate performance, pronounced bias contributed to the large TEobs , indicating a requirement for analyzer-specific reference intervals.

Method Comparison Using 2 Point-of-Care Meters and a Reference Analyzer for Measuring Blood Triglycerides in Psittacine Birds.

Female reproductive disorders, such as chronic egg laying, are common in captive psittacine birds. While a disease diagnosis related to reproductive disorders can often be accomplished by physical examination and diagnostic imaging, monitoring of the response to environmental modification and medical treatment is more challenging. Monitoring ideally would involve measurement of luteinizing hormone or estrogen to assess ovarian activity. However, the amount of blood required for hormone analysis is greater than the small sample size that one can collect from these birds. Additionally, the lack of reference intervals limits their use as a diagnostic tool. Because plasma triglyceride increases during sustained estrogen release from the ovary, it may be used as an alternative method for assessing ovarian activity in birds. Point-of-care (POC) analyzers for measuring lipids in human plasma use very small sample volumes and have been used for measuring triglycerides in animals, including chickens. The authors therefore performed a method comparison study with 2 POC analyzers and a reference analyzer and plasma and whole blood from psittacine birds to determine whether these meters are suitable for triglyceride measurement in a known population of psittacine birds. Correlation, Deming regression, and Bland-Altman analyses were used to assess performance, and the total observed error for each meter relative to the reference analyzer was calculated. One of the meters exhibited fair performance and, with species-specific reference intervals, is likely to be clinically useful for triglyceride measurement in psittacine birds. The other meter demonstrated poor performance with unacceptable error, and its use for this purpose is strongly discouraged.

Validation of 2 point-of-care meters for measuring triglycerides in chickens using whole blood and plasma.

Disorders of the avian reproductive tract are common, yet monitoring their resolution presents a diagnostic dilemma. Reproductive hormones such as luteinizing hormone or estrogen are the best reflection of reproductive status, but the required sample volumes and lack of reference intervals limit their clinical utility. An alternative analyte is blood triglyceride, the concentration of which rises markedly during sustained estrogen release from the ovary. Portable meters for measuring human blood triglyceride concentration offer the advantage of using minimal sample volumes, but these have not been validated for use in birds. We assessed the precision and accuracy of 2 portable meters for measuring blood triglyceride concentration in pooled whole blood and plasma from chickens ( n = 42), and performed method comparison using a reference analyzer and determined total error. Within-run repeatability was fair-to-excellent using whole blood and plasma (range: 2.5-11.5%), and between-run repeatability using plasma was similar (3.1-12.2%). The meters performed well in recovery and dilution studies in which almost all readings fell within the preset requirement of 75-125%. Correlations between each meter, using whole blood and plasma, and the reference analyzer, using plasma only, were high to very high (0.86-0.98). Bias determined by Bland-Altman analysis was similar between whole blood and plasma for each meter, yet markedly different between the meters. The calculated total observed error was consequently within our pre-set total allowable error of 25% for one meter but not the other, indicating the requirement for a meter-specific reference interval.

Sarcocystid organisms found in bile from a dog with acute hepatitis: a case report and review of intestinal and hepatobiliary Sarcocystidae infections in dogs and cats.

Sarcocystidae is a family of coccidian protozoa from the phylum Apicomplexa that includes Toxoplasma, Neospora, Sarcocystis, Hammondia, and Besnoitia spp. All species undergo a 2-host sexual and asexual cycle. In the definitive host, replication is enteroepithelial, and infection is typically asymptomatic or less commonly causes mild diarrhea. Clinical disease is most frequently observed in the intermediate host, often as an aberrant infection, and is mostly associated with neurologic, muscular, or hepatic inflammation. Here, we review the literature regarding intestinal Sarcocystidae infections in dogs and cats, with emphasis on the life cycle stages and the available diagnostic assays and their limitations. We also report the diagnostic findings for an 11-year-old dog with acute neutrophilic hepatitis, biliary protozoa, and negative biliary culture. Although Toxoplasma and Neospora IgG titers were both high, PCR for these 2 organisms was negative for bile. The organisms were identified by 18S rDNA PCR as most consistent with Hammondia, either H heydorni or H triffittae. This is the first report of presumed Hammondia organisms being found in canine bile.

Initial analytic quality assessment and method comparison of an immunoassay for adrenocorticotropic hormone measurement in equine samples.

BACKGROUND: Equine pituitary pars intermedia dysfunction (PPID) may be diagnosed by measuring baseline plasma adrenocorticotrophic hormone (ACTH). The Immulite 1000 analyzer uses an automated chemiluminescence enzyme assay, previously validated for measuring equine ACTH. Recently, an automated bench-top immunoassay analyzer (AIA-360), designed for analytes in people, became available for veterinary use. OBJECTIVES: Objectives were to evaluate analytic performance of the AIA immunoassay for measuring equine ACTH, and compare the results with those obtained by the Immulite. METHODS: Adrenocorticotrophic hormone was measured in plasma samples from 52 clinical cases. For the AIA, within- and between-run coefficients of variation (CV) were assessed, linearity and recovery studies performed, and observed total error (TEobs ) calculated. Correlation and agreement between the 2 analyzers were also evaluated. RESULTS: Within-run and between-run CV of the AIA ranged from 2.3% to 4% and 3.5% to 8%, respectively. ACTH recoveries ranged from 89.5% to 115.9%. TEobs at 26.5 pg/mL ACTH was 4.1 pg/mL. The ACTH results (median: 25.9 pg/mL; range: 4.3-276.7 pg/mL) with AIA were significantly lower (P < .0001) than with the Immulite (median: 29.9 pg/mL; range: 10.3-639.0 pg/mL). Correlation between the 2 analyzers was r = 0.882 (P < .0001), with a significant bias for the AIA of -16 pg/mL. The 2 methods were not identical within inherent imprecision. CONCLUSION: The AIA is precise for measuring ACTH in horses. Although correlation between the instruments is good, the values obtained by the immunoassays cannot be used interchangeably and should be interpreted using reference intervals established for each analyzer to avoid false negatives. Diagnostic sensitivity and specificity of the AIA-360 should be evaluated before clinical use.

Evaluation of an in-practice wet-chemistry analyzer using canine and feline serum samples.

A wet-chemistry biochemical analyzer was assessed for in-practice veterinary use. Its small size may mean a cost-effective method for low-throughput in-house biochemical analyses for first-opinion practice. The objectives of our study were to determine imprecision, total observed error, and acceptability of the analyzer for measurement of common canine and feline serum analytes, and to compare clinical sample results to those from a commercial reference analyzer. Imprecision was determined by within- and between-run repeatability for canine and feline pooled samples, and manufacturer-supplied quality control material (QCM). Total observed error (TEobs) was determined for pooled samples and QCM. Performance was assessed for canine and feline pooled samples by sigma metric determination. Agreement and errors between the in-practice and reference analyzers were determined for canine and feline clinical samples by Bland-Altman and Deming regression analyses. Within- and between-run precision was high for most analytes, and TEobs(%) was mostly lower than total allowable error. Performance based on sigma metrics was good (σ > 4) for many analytes and marginal (σ > 3) for most of the remainder. Correlation between the analyzers was very high for most canine analytes and high for most feline analytes. Between-analyzer bias was generally attributed to high constant error. The in-practice analyzer showed good overall performance, with only calcium and phosphate analyses identified as significantly problematic. Agreement for most analytes was insufficient for transposition of reference intervals, and we recommend that in-practice-specific reference intervals be established in the laboratory.

Identification of key residues that confer Rhodobacter sphaeroides LPS activity at horse TLR4/MD-2.

The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.