Agreement Between Cytology and Histopathology for Regional Lymph Node Metastasis in Dogs With Melanocytic Neoplasms.

Melanocytic neoplasms are common in dogs and frequently occur within the oral cavity or in haired skin. The behavior of melanocytic neoplasms is variable and depends on tumor location, size, and histopathologic features. This study compared cytopathology and histopathology of 32 lymph nodes from 27 dogs diagnosed with melanocytic neoplasms. Agreement between the original cytology report, cytology slide review, original histopathology report, and histopathology slide review was determined for each lymph node. A subset of lymph nodes was subjected to immunohistochemistry (Melan-A) and additional histochemical stains/techniques (Prussian blue, bleach) to assist in differentiation of melanocytes and melanophages. Agreement ranged from slight to fair for each of the variables evaluated with weighted kappa (κw) or kappa (κ) analysis (original cytology vs cytology review κw = 0.24; original cytology vs original histopathology κw = 0.007; original cytology vs histopathology review κw = 0.23; cytology review vs original histopathology κw = 0.008; cytology review vs histopathology review κw = 0.006; and original histopathology vs histopathology review κ = 0.18). The diagnoses (metastatic, equivocal, or negative for metastasis) of the original report and slide review for both cytology and histopathology were not significantly correlated with survival in this population of patients. Overall, agreement between cytology and histopathology was poor even with a single clinical or anatomic pathologist performing slide review. Consensus between routine cytology and histopathology for staging of lymph nodes in patients with melanocytic neoplasms is poor and does not correlate with survival.

Agreement between lacrimal fluid and serum for detecting urea nitrogen and creatinine in dogs.

OBJECTIVE: To determine whether urea nitrogen and creatinine levels differ in lacrimal fluid (LF) and serum (SER) in nonazotemic (control) and azotemic dogs and whether there is an agreement between LF and SER. METHODS: A prospective observational study was performed at the Auburn University Small Animal Teaching Hospital between May 2023 and March 2024. Forty control and 38 azotemic dogs were enrolled. Twenty microliters of LF per eye was collected with microcapillary tubes, and 3 mL of blood was drawn. Bland-Altman plot and intraclass correlation coefficient (ICC) were used to evaluate the agreement between LF and SER. RESULTS: There was good agreement between LF and SER levels of urea nitrogen in the control group (Bland-Altman plot mean bias of -0.8108 ± 2.407 mg/dL; ICC of 0.874 [95% CI, 0.773 to 0.934]) and the azotemic group (Bland-Altman plot mean bias of -9.681 ± 23.89 mg/dL; ICC of 0.82 [95% CI, 0.658 to 0.906]). There was poor agreement between LF and SER concentrations for creatinine in the control and azotemic groups, with only 26 dogs with creatinine detectable in LF. CONCLUSIONS: Lacrimal fluid and SER concentrations of urea nitrogen showed good agreement in both the control and azotemic groups, whereas poor agreement was found for creatinine in both groups. CLINICAL RELEVANCE: Measurement of urea nitrogen in LF may provide an alternative to blood for diagnosing uremia. However, additional research is necessary before substituting LF for SER.

Platelet Function and Therapeutic Applications in Dogs: Current Status and Future Prospects.

Significant progress has been made in the functional characterization of canine platelets in the last two decades. The role of canine platelets in hemostasis includes their adhesion to the subendothelium, activation, and aggregation, leading to primary clot formation at the site of injury. Studies on canine platelet function and advancements in laboratory testing have improved the diagnosis and understanding of platelet-related disorders as well as the knowledge of the mechanisms behind these diseases. This review focuses on the most recent discoveries in canine platelet structure, function, and disorders; and discusses the efficacy of various tests in the diagnosis of platelet-related disorders. With the relatively recent discovery of angiogenetic and reparative effects of growth factors found in platelets, this review also summarizes the use of canine platelet-rich plasma (PRP) alone or in association with stem cells in regenerative therapy. The characterization of proteomic and lipidomic profiles and development of platelet gene therapy in veterinary species are areas of future study with potential for major therapeutic benefits.

In vitro effect of blood cell counts on multiple-electrode impedance aggregometry in dogs.

OBJECTIVE To assess the effect of decreased platelet and WBC counts on platelet aggregation as measured by a multiple-electrode impedance aggregometer in dogs. ANIMALS 24 healthy dogs. PROCEDURES From each dog, 9 mL of blood was collected into a 10-mL syringe that contained 1 mL of 4% sodium citrate solution to yield a 10-mL sample with a 1:9 citrate-to-blood ratio. Each sample was then divided into unmanipulated and manipulated aliquots with progressively depleted buffy-coat fractions such that 2 to 3 blood samples were evaluated per dog. The Hct for manipulated aliquots was adjusted with autologous plasma so that it was within 2% of the Hct for the unmanipulated aliquot for each dog. All samples were analyzed in duplicate with a multiple-electrode impedance aggregometer following the addition of ADP as a platelet agonist. The respective effects of platelet count, plateletcrit, Hct, and WBC count on platelet aggregation area under the curve (AUC), aggregation, and velocity were analyzed with linear mixed models. RESULTS WBC count was positively associated with platelet AUC, aggregation, and velocity; blood samples with leukopenia had a lower AUC, aggregation, and velocity than samples with WBC counts within the reference range. Platelet count, plateletcrit, and Hct did not have an independent effect on AUC, aggregation, or velocity. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that WBC count was positively associated with platelet aggregation when ADP was used to activate canine blood samples for impedance aggregometry. That finding may be clinically relevant and needs to be confirmed by in vivo studies.

Comparison of the gene encoding, and the predicted amino acid composition of, platelet membrane receptor subunit glycoprotein Ibα in members of the family Felidae.

BACKGROUND: There is minimal information regarding platelet receptors in the family Felidae. Comparative studies assist with identifying amino acids critical for protein structure and function. OBJECTIVE: The purpose of the study was to compare the gene encoding, and the predicted amino acid composition of, platelet membrane receptor subunit GPIbα in Felidae family members. METHODS: Genomic DNA samples isolated from whole blood of 13 domestic cats and 50 big cats representing 8 different species were subjected to PCR using primers designed to flank the coding region of GPIbα in overlapping fashion. PCR products were separated via electrophoresis on agarose gels, and extracted products were submitted for sequencing. DNA sequences were used to predict the length and amino acid composition of the protein. RESULTS: Varying protein lengths were predicted in Felidae family members which were primarily due to polymorphisms in the variable number of tandem repeats region encoding the macroglycopeptide region of GPIbα. Other areas of the gene and predicted amino acid compositions were fairly conserved when compared to human sequences and between Felidae family members. CONCLUSION: Various polymorphisms within GPIbα, including length variants encoding the macroglycopeptide region, were identified in members of the family Felidae. More studies are needed to determine if a correlation exists between various polymorphisms and predisposition for hemorrhage or thrombosis as suggested in people.

A novel form of macrothrombocytopenia in Akita dogs.

Blood samples from 3 unrelated Akita dogs with a common history of persistent macrothrombocytopenia in the absence of clinical bleeding were sent to the Auburn University College of Veterinary Medicine (AUCVM) Clinical Pathology Laboratory for evaluation. Due to low platelet counts, one Akita dog had been treated with corticosteroids for presumed immune-mediated platelet destruction, and one Akita dog was treated with doxycycline for one month for presumed infection by a tick-borne agent. In spite of treatment, platelet counts remained low in both dogs. Given the absence of abnormal bleeding in all 3 dogs and lack of response to treatment in 2, congenital macrothrombocytopenia was suspected. Interestingly, platelets from all 3 dogs exhibited a consistent elongated platelet morphology. There were no morphologic abnormalities observed in other cell lines. While there have been anecdotal reports of a possible inherited macrothrombocytopenia in Akita dogs, scientific studies have not been done to verify these reports. This manuscript represents the first case report describing what is likely a congenital macrothrombocytopenia in Akita dogs based on persistently low platelet counts in the absence of clinical signs, and characterized by a unique platelet morphology.

Basset Hound thrombopathia in a 4-month-old female Ba-Shar (Sharp Asset).

A 3-month-old female Basset Hound-Shar Pei mix puppy (Ba-Shar or Sharp Asset) presented with oral bleeding due to a cracked molar. On physical exam, an aural hematoma was also noted that the owner indicated was chronic. The puppy was hospitalized for over 24 h until the bleeding was brought under control. At 4 months of age, the puppy again presented with oral bleeding due to loss of deciduous teeth and was hospitalized until bleeding was controlled. Coagulation screening tests, platelet numbers, and von Willebrand Factor antigen levels were within reference limits. Based on the presence of platelet-type bleeding in the face of normal screening test results, samples were submitted for DNA testing for Basset Hound thrombopathia. The puppy tested as affected for the calcium and diacylglycerol regulated guanine nucleotide exchange factor I (CalDAG-GEFI) mutation causing this disorder. This is the first time thrombopathia has been diagnosed in a "designer" breed.

Abnormal coagulation factor VIII transcript in a Tennessee Walking Horse colt with hemophilia A.

Hemophilia A is an X-chromosome-linked disorder caused by a deficiency in factor VIII (FVIII). Although foals have been diagnosed with hemophilia A based on deficiency in FVIII activity, causative gene mutations have not been identified. The genomic DNA and cDNA encoding FVIII of a Tennesee Walking Horse colt affected with hemophilia A and the genomic DNA of his dam and a normal unrelated horse were analyzed with no splice site or coding sequence abnormalities identified in any of the horses. Polymerase chain reactions (PCR) were then performed on hepatic cDNA from the affected colt and an unrelated normal horse, and no product was obtained for the sequence between and including exon 1 and exon 2 in the affected colt. Based on these results, suspected mutations were identified in the noncoding region of FVIII (intron 1), and genomic sequencing of intron 1 in the dam and the affected colt suggested maternal inheritance.

Disseminated lymphoma with large granular lymphocyte morphology diagnosed in a horse via abdominal fluid and transtracheal wash cytology.

A 22-year-old Tennessee Walking Horse mare was presented to the Auburn University Large Animal Teaching Hospital with a 3-day history of lethargy, anorexia, and mild signs of colic. The mare had a several-month history of weight loss and refractory cough. Physical examination revealed an increased respiratory rate, and crackles and wheezes were heard on thoracic auscultation. Thoracic ultrasonographic examination showed disseminated, minor, bilateral comet tail-like lesions on the parietal pleural surfaces. Abdominal ultrasonographic examination was unremarkable. Trans-rectal palpation revealed a firm small colon impaction with concomitant diarrhea. Laboratory data were characterized by a very pronounced acute inflammatory leukogram with severe neutropenia and significant left shift, evidence of hepatocellular damage/necrosis, cholestasis, and possibly mixed metabolic alkalosis and acidosis. On cytologic evaluation of a peritoneal fluid sample, there were many large granular lymphocytes (LGL). Large numbers of LGL were also observed on cytologic examination of a subsequent transtracheal wash. The final cytologic interpretation was disseminated lymphoma with LGL morphology. Due to worsening of the clinical signs and poor prognosis, the mare was euthanized. On necropsy and in histopathologic examination, disseminated lymphoma with LGL morphology was noted in a mesenteric lymph node, lungs, liver, spleen, kidneys, and right dorsal colon. Lymphoma with LGL morphology is rarely diagnosed in the horse. This report provides unique cytologic findings of a case of disseminated lymphoma with LGL morphology in a horse, confirmed with histopathologic evaluation.

Biomarkers for disease progression and AAV therapeutic efficacy in feline Sandhoff disease.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are progressive neurodegenerative disorders that are caused by a mutation in the enzyme ß-N-acetylhexosaminidase (Hex). Due to the recent emergence of novel experimental treatments, biomarker development has become particularly relevant in GM2 gangliosidosis as an objective means to measure therapeutic efficacy. Here we describe blood, cerebrospinal fluid (CSF), magnetic resonance imaging (MRI), and electrodiagnostic methods for evaluating disease progression in the feline SD model and application of these approaches to assess AAV-mediated gene therapy. SD cats were treated by intracranial injections of the thalami combined with either the deep cerebellar nuclei or a single lateral ventricle using AAVrh8 vectors encoding feline Hex. Significantly altered in untreated SD cats, blood and CSF based biomarkers were largely normalized after AAV gene therapy. Also reduced after treatment were expansion of the lysosomal compartment in peripheral blood mononuclear cells and elevated activity of secondary lysosomal enzymes. MRI changes characteristic of the gangliosidoses were documented in SD cats and normalized after AAV gene therapy. The minimally invasive biomarkers reported herein should be useful to assess disease progression of untreated SD patients and those in future clinical trials.

Platelet count and plateletcrit in Cavalier King Charles Spaniels and Greyhounds using the Advia 120 and 2120.

BACKGROUND: Models for the regulation of thrombopoiesis predict that platelet mass is the biologically regulated variable, yet clinical evaluations of thrombopoiesis are often based on platelet number. When thrombocytopenia and variation in platelet size occur concurrently, platelet count may poorly estimate total platelet mass, confounding clinical decision making. OBJECTIVE: We hypothesized that plateletcrit (PCT) provides clinically important information when platelet number is an incomplete representation of platelet mass, such as in genetic macrothrombocytopenia. METHODS: We retrospectively compared platelet count and PCTs with general reference intervals for 4 groups of dogs: sick Cavalier King Charles Spaniels (CKCS) and Greyhounds, presented for nonhematopoietic disease to the University of Minnesota (measured using an Advia 2120) and Auburn University Teaching Hospitals (measured using an Advia 120) over a 3-year period. RESULTS: A canine PCT reference interval of 0.129-0.403% was established. None of the 4 sample groups had significantly more individuals below the reference interval for plateletcrit. For platelet count, only the 2 CKCS groups had significantly more individuals below the reference interval than predicted. CONCLUSION: Use of the PCT as determined by the Advia 120/2120 appeared to avoid overestimation of low platelet mass in sick CKCS in a clinical setting. In contrast, the PCT performed similarly to the platelet count in evaluation of platelet mass in sick Greyhounds. Evaluation of the PCT should be considered in other conditions associated with increased mean platelet volume.

Two novel missense mutations associated with hemophilia A in a family of Boxers, and a German Shepherd dog.

BACKGROUND: Hemophilia A is an X-linked disorder caused by a deficiency in coagulation factor VIII. Over 2300 unique mutations in the gene-encoding factor VIII have been documented in people, but limited information is known in dogs. An 11-week-old male Boxer and a 5-year-old male German Shepherd were diagnosed with hemophilia A based on diminished factor VIII activity. OBJECTIVE: The purpose of the study was to identify genetic mutations associated with hemophilia A in both dogs. METHODS: Genomic DNA was isolated from EDTA blood samples from the affected German Shepherd and Boxer, the Boxer's dam, 3 female siblings, and one asymptomatic male sibling. Primers were designed in noncoding regions to amplify the 26 exons of the factor VIII gene via PCR. RESULTS: The affected Boxer sequence revealed a single nucleotide change, cytosine to guanine, at nucleotide position 1412 (1412C>G) in exon 10. The change is predicted to result in the substitution of arginine for proline at amino acid 471 (P471R) in the A2 domain of factor VIII. The dam and female siblings were carriers, the male sibling did not have the mutation. The German Shepherd dog had a single nucleotide change of a guanine to adenine at position 1643 (1643G>A) in exon 11, predicting the substitution of tyrosine for cysteine at amino acid 548 (C548Y) in the A2 domain. CONCLUSIONS: Here we document 2 mutations associated with canine hemophilia A associated with < 1% factor VIII activity, similar to that in people. Another related Boxer with the P471R mutation was later identified.

Cytologic findings from a benign giant cell tumor of the tendon sheath in a dog.

A 6-year-old male neutered Australian Shepherd dog was presented for evaluation of a subcutaneous mass on the plantar aspect of the proximal left metatarsus. Fine-needle aspirate smears contained numerous plump spindle cells and large multinucleated cells amongst a considerable amount of pink extracellular matrix. Histopathologic diagnosis of the tissue obtained during initial biopsy and eventual surgical cytoreduction of the mass was a benign giant cell tumor of the tendon sheath (GCTTS). Immunohistochemically, the synovioblastic neoplastic cells were diffusely strongly positive for vimentin and S-100, were multifocally moderately positive for cytokeratin AE1/3, and were negative for CD18, muscle-specific actin (MSA), and melanoma-associated antigen (mutated) 1 (MUM-1). The dog recovered from surgery and underwent definitive radiation therapy to treat the local residual disease. Eight months later, the mass had not recurred. The diagnosis of GCTTS in this case supports previously published reports describing GCTTS as a relevant disease entity in dogs, and provides the first documentation of cytologic findings with this tumor. Further investigation is needed to correlate pathologic features with clinical behavior and response to therapy in dogs.

Evaluation and clinical application of platelet function testing in small animal practice.

Tests that evaluate many aspects of platelet function have been applied in both human and veterinary medicine for the monitoring of treatment with platelet function inhibitors and for detection of platelet function abnormalities (inherited or acquired). Interspecies variation in the response to various platelet agonists is an important consideration when methods that have been developed for people are applied in other species. At the present time, many of these assays are not readily available in standard veterinary practice. Advanced platelet function testing for veterinary patients is offered at select academic institutions. Discussion with a specialist is recommended when considering the use of these tests, and the relative strengths and limitations of each assay should be considered in the interpretation of test results.