SYSTEMATIC EVALUATION OF 106 LABORATORY REFERENCE DATA ARTICLES FROM NONDOMESTIC SPECIES PUBLISHED FROM 2014 TO 2016: ASSESSING COMPLIANCE WITH REFERENCE INTERVAL GUIDELINES.

Population-based reference intervals (RIs) are vital tools used to characterize health and disease based on laboratory values. The science and statistical basis for RI generation have evolved over the past 50 yr. Current veterinary-specific guidelines by the American Society of Veterinary Clinical Pathology exist for establishing RIs from nondomestic and wild animals. A list of 35 items that should be included during generation and publication of reference data was distilled from the currently available RI guidelines. The archives of five peer-reviewed journals were searched and 106 articles presenting laboratory reference data from nondomestic or wildlife species were identified and each reviewed by two authors to determine compliance with the list of 35 items. A compliance score was calculated as the number of articles that fulfilled the item out of the number where it would have been appropriate to fulfill the item. Most articles reported the number of reference individuals (compliance score 0.98), their partitioning demographics (compliance score 0.95), and sample collection and handling practices (compliance scores 0.97 and 0.96, respectively). Common deficiencies included omitting discussion of the validation status of the analytical methods for the species being evaluated (compliance score 0.12), documentation of use of exclusion criteria (compliance score 0.51), outlier detection (compliance score 0.43), appropriate statistical methods for the reference population (compliance score 0.34), and calculation and presentation of confidence intervals around the reference limits (compliance score 0.35). Compliance scores were not statistically different when stratified on the number of individuals in the largest and smallest evaluated group or the format of the article (full vs short format). Articles that cited RI generation guidelines fulfilled more of the required steps and provided a more complete description of their data (compliance score 0.74) than those that did not cite guidelines (compliance score 0.58). Additional attention to the science of and recommendations for RI generation is recommended to strengthen the utility of published data.

What is your diagnosis? Perifemoral mass in a cow.

A 15-year-old female Simmental cross-breed cow was presented to the Purdue University Veterinary Teaching Hospital for evaluation of a perifemoral soft tissue mass. Impression smears made from an excisional biopsy contained a population of pleomorphic mesenchymal cells with abundant, periodic acid-Schiff-positive (PAS), intracytoplasmic granular material, and rare elongated multinucleated cells consistent with strap-like cells. A second population of small round cells suggestive of lymphocytes or progenitor cells was also noted. A cytologic diagnosis of sarcoma was made, with rhabdomyosarcoma considered most likely based on the large amount of PAS-positive material (presumed to be glycogen) and the rare strap-like cells. Histopathologic sections contained an unencapsulated, densely cellular neoplasm composed of haphazardly arranged highly pleomorphic mesenchymal cells and a few small round cells. The mesenchymal cells were positive for vimentin, non-specific muscle actin, and myoglobin, and negative for phosphotungstic acid-hematoxylin, smooth muscle actin, and desmin. Glycogen granules were confirmed by transmission electron microscopy. A diagnosis of pleomorphic rhabdomyosarcoma was made. While cytologic findings may suggest rhabdomyosarcoma, cytologic features can be highly variable, and a definitive diagnosis usually requires cytochemical and immunohistochemical staining.

Effect of furosemide and furosemide-carbazochrome combination on exercise-induced pulmonary hemorrhage in Standardbred racehorses.

The objective was to quantify the effect of furosemide and carbazochrome on exercise-induced pulmonary hemorrhage (EIPH) in Standardbred horses using red blood cell count and hemoglobin concentration in bronchoalveolar lavage (BAL) fluid. Six healthy Standardbred horses with prior evidence of EIPH performed a standardized treadmill test 4 h after administration of placebo, furosemide, or furosemide-carbazochrome combination. Red blood cell (RBC) counts and hemoglobin concentrations were determined on the BAL fluid. The RBC count in BAL ranges were (2903-26,025 cells/microL), (45-24,060 cells/microL), and (905-3045 cells/microL) for placebo, furosemide, and furosemide-carbazochrome, respectively. Hemoglobin concentration ranges were (0.03-0.59 mg/mL), (0.01-0.55 mg/mL), and (0.007-0.16 mg/mL) for placebo, furosemide, and furosemide-carbazochrome groups, respectively. No significant differences were detected among treatments. However, there was great variability among horses, suggesting that a larger sample size or better selection of horses was needed.

Evaluating urinary estrogen and progesterone metabolites using dried filter paper samples and gas chromatography with tandem mass spectrometry (GC-MS/MS).

BACKGROUND: Measuring concentrations of metabolites of estradiol and progesterone in urine, instead of measuring serum concentrations, is common in research and also is used in patient care. The primary aim of this study was to demonstrate that analysis of urine samples dried on filter paper by gas chromatography with tandem mass spectrometry (GC-MS/MS) provides results similar to serum analyzed by radioimmunoassay (RIA). Secondary aims were to show that collection of four samples during the day (4-spot method) can be substituted for a 24-h collection, and that analysis of urine from dried samples is equivalent to liquid urine samples. METHODS: This prospective observational study compared results of urine and serum analyses. Urine samples from women throughout the menstrual cycle and single samples from postmenopausal women were evaluated. Urine was collected onto filter paper and dried. Dried urine was extracted, hydrolyzed, and derivatized prior to analysis by GC-MS/MS. Hormone concentrations were normalized to creatinine. Single samples were used to compare results of 24-h urine collection to the 4-spot method from a separate population of women and men. A subset of these samples were used to compare results from dried urine to liquid urine. RESULTS: The primary study showed good reliability in the comparisons between the dried urine and serum assays. During the menstrual cycles of a subset of four women, urine metabolite concentrations followed the same pattern as serum concentrations. Comparison of 4-spot to 24-h urine collections and of dried to liquid urine measurements had intraclass correlation coefficients (ICC) greater than 0.95, indicating excellent agreement. CONCLUSIONS: For estradiol and progesterone, the dried urine assay is a good surrogate for serum testing. The 4-spot method can be used instead of 24-h urine collections and dried urine results are comparable to liquid urine. The dried urine assay is useful for some clinical assessments of hormone disorders and may be useful in large epidemiologic studies due to ease of sample handling.

Assessment of platelet growth factors in supernatants from rehydrated freeze-dried equine platelets and their effects on fibroblasts in vitro.

OBJECTIVE: To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. ANIMALS: 6 clinically normal adult horses. PROCEDURES: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor beta1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS). RESULTS: Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor-stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS. CONCLUSIONS AND CLINICAL RELEVANCE: The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.

What is your diagnosis? Peritoneal fluid from an Arabian horse after colic surgery.

A 16-year-old castrated male Arabian horse was presented to the Purdue University Veterinary Teaching Hospital with a 4-hour history of colic. Initial examinations provided strong evidence for small intestinal obstruction. Abdominal surgery revealed a strangulating lipoma, and 25 feet of small intestine were resected. Postoperatively, the horse developed obstructive ileus due to adhesion formation, which required a second laparotomy. During and after surgery, the abdomen was lavaged with sodium carboxymethylcellulose (CMC). One week after the second surgery, evaluation of peritoneal fluid revealed an inflammatory exudate, with many macrophages containing amorphous to granular, pink to magenta phagocytosed material. Extracellular aggregates of the material were also observed. The material was consistent with CMC. To our knowledge, this report is the first to demonstrate the phagocytosis of CMC by peritoneal fluid macrophages.

Prevalence of reduced fibrinogen binding to platelets in a population of Thoroughbreds.

OBJECTIVE: To measure the frequency and magnitude of reduced fibrinogen binding in a population of horses from a Thoroughbred breeding farm. ANIMALS: 444 Thoroughbred horses, 1 to 27 years old, including 316 females, 72 geldings, and 56 sexually intact males. PROCEDURES: Blood was collected from horses into tubes containingacid citrate dextrose adenine, and washed platelets were examined by use of flow cytometry for their ability to bind fibrinogen. RESULTS: Data regarding fibrinogen binding to activated platelets were normally distributed, with nearly identical amounts of variation regardless of sex. In 3 horses, fibrinogen binding to platelets was reduced from 67.6% to 83.4%, compared with normal platelets, which indicated an inability of platelets to aggregate in response to thrombin (0.1 U/mL). CONCLUSIONS AND CLINICAL RELEVANCE: Platelet fibrinogen binding of the affected horses identified in this study was characteristic of a reported heritable bleeding disorder in which the reduction in fibrinogen binding correlated with prolonged bleeding times in template bleeding assays. The bleeding disorder is distinct from Glanzmann thrombasthenia, in which platelets fail to bind fibrinogen because of lack of alphallb-beta3 integrin on their surface. The prevalence of affected horses within the small sample population studied here (0.7% [n = 3]) is considerably higher than the prevalence of bleeding disorders within more genetically diverse groups.

Feline leukocyte adhesion (CD18) deficiency caused by a deletion in the integrin ß2 (ITGB2) gene.

BACKGROUND: Leukocyte adhesion deficiency (LAD) or CD18 deficiency is an autosomal recessive immunodeficiency which has been described in people, cattle, dogs, and knockout mice. OBJECTIVES: The study goals were to characterize the clinicopathologic, immunologic, and molecular genetic features of feline LAD (FLAD) in a neutered male adult Domestic Longhair cat with severe leukocytosis and recurrent infections. METHODS: Flow cytometry evaluated surface expression of CD18 on neutrophils. In vitro functional assays assessed CD18-dependent neutrophil adhesion and T-cell proliferation. Genomic DNA and cDNA were used to identify a causative mutation in the coding sequence of the integrin ß2 subunit (ITGB2) gene. RESULTS: The affected cat developed periodontitis during the first months of life followed by recurrent infections poorly responsive to antibiotic therapy, accompanied by extreme neutrophilia. Neutrophils from the proband, compared to feline controls, did not express any CD18 on the cell surface. Adhesion of affected neutrophils was severely impaired with and without phorbol-myristate-acetate activation. The proband's T-cells proliferated weakly to 1 pg but normally to 100 pg staphylococcal enterotoxin A, suggesting a CD18-independent T-cell response at higher doses. Molecular genetic analysis of the ITGB2 gene revealed a 24 bp deletion at the exon 2 to intron 2 boundary (c.46_58 + 11del), predicting premature translational termination due to abnormal splicing of exon 1 to exon 3 or 4. CONCLUSIONS: Feline LAD exhibits features similar to LAD in other species. However, clinical episodes in FLAD appeared milder allowing for an extended life expectancy under long-term antimicrobial therapy, possibly due to an alternative, CD18-independent T-cell proliferation pathway.

Investigation of a novel, heritable bleeding diathesis of Thoroughbred horses and development of a screening assay.

BACKGROUND: Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable. HYPOTHESIS: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors. ANIMALS: The study included 3 clinically normal Thoroughbred horses: the subject and her offspring. METHODS: Washed platelets were examined for their ability to (1) translocate phosphatidylserine to the outer leaflet of the platelet membrane as determined by annexin-V binding, (2) generate thrombin as assessed by the activity of the prothrombinase enzyme complex, and (3) bind fibrinogen and form aggregates as determined by flow cytometry. RESULTS: Subject and offspring platelets created procoagulant surfaces by translocating phosphatidylserine. The subject's platelets demonstrated reduced prothrombinase activity, resulting in decreased production of thrombin relative to control platelets. Subject and offspring platelets bound less fibrinogen than control platelets when stimulated with thrombin. CONCLUSIONS AND CLINICAL IMPORTANCE: The subject mare has a transmissible defect that involves reduced generation of thrombin by activated platelets, resulting in decreased aggregation and ineffective clotting. A flow cytometric assay of fibrinogen binding to washed platelets discriminates individuals with this platelet dysfunction and may be useful for discerning subclinical congenital or acquired platelet dysfunctions.

Evaluation of clinical characteristics, diagnostic test results, and outcome in horses with internal infection caused by Corynebacterium pseudotuberculosis: 30 cases (1995-2003).

OBJECTIVE: To determine clinical signs, results of diagnostic testing, and outcome in horses with internal Corynebacterium pseudotuberculosis infection. DESIGN: Retrospective study. ANIMALS: 30 horses. PROCEDURE: Information pertaining to clinical data, results of diagnostic tests, and costs of hospitalization and treatment was extracted from medical records of affected horses. RESULTS: Internal C. pseudotuberculosis infection was diagnosed on the basis of clinical signs, diagnostic imaging, and clinicopathologic data, including results of serologic tests and bacterial culture. The most common clinical signs were concurrent external abscesses, anorexia, fever, lethargy, weight loss, and signs of respiratory tract disease or abdominal pain. Clinicopathologic abnormalities included a geometric mean reciprocal serum synergistic hemolysin inhibition titer > or = 512, leukocytosis with neutrophilia, hyperglobulinemia, hyperfibrinogenemia, and anemia. Specific organ involvement was diagnosed in 27 of 30 horses. Affected organs included the liver (18 horses), lungs (12), kidneys (7), and spleen (3); multiple organs were affected in 10 horses. Treatment with antimicrobials for a median of 36 days (range, 7 to 97 days) was usually successful, yielding an overall survival rate of 71%. CONCLUSIONS AND CLINICAL RELEVANCE: Early diagnosis and long-term antimicrobial treatment were important for a successful outcome in horses with internal C. pseudotuberculosis infection. Ultrasonographic imaging was an important technique for identifying specific organs affected, aiding in obtaining samples for a definitive diagnosis, and monitoring response to treatment. Pregnant mares with internal infections are at risk for fetal loss. Preexisting chronic organ disease may be associated with a poor prognosis.

Malignant Sertoli cell tumor in the retained abdominal testis of a unilaterally cryptorchid horse.

A 13-year-old Morgan gelding was evaluated because of a mass in the caudal region of the abdomen. The horse had been presumed to be a gelding, but necropsy findings revealed a retained testis in the right retroperitoneal space. Histologically, the retained testis contained neoplastic cells; metastases were identified in the liver, spleen, lungs, and sublumbar lymph nodes. Immunohistochemical examination of the testis and metastatic tissues confirmed the diagnosis of malignant Sertoli cell tumor. Testicular neoplasms are infrequently reported in stallions. Seminomas are most commonly reported, whereas Sertoli cell tumors are considered to be rare. Typical biological behavior of Sertoli cell tumors in horses is unknown. To the authors' knowledge, there have been 2 reports of Sertoli cell tumors in horses; the tumors developed in descended testes, and 1 tumor was malignant.