ACVIM consensus statement on the diagnosis of immune thrombocytopenia in dogs and cats.

Immune thrombocytopenia (ITP) is the most common acquired primary hemostatic disorder in dogs. Immune thrombocytopenia less commonly affects cats but is an important cause of mortality and treatment-associated morbidity in both species. Immune thrombocytopenia remains a diagnosis of exclusion for which diagnostic guidelines are lacking. Primary, or non-associative, ITP refers to autoimmune platelet destruction. Secondary, or associative, ITP arises in response to an underlying disease trigger. However, evidence for which comorbidities serve as ITP triggers has not been systematically evaluated. To identify key diagnostic steps for ITP and important comorbidities associated with secondary ITP, we developed 12 Population Evaluation/Exposure Comparison Outcome (PECO) format questions. These questions were addressed by evidence evaluators utilizing a literature pool of 287 articles identified by the panelists using a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations that then were integrated by diagnosis and comorbidity domain chairs. The revised PECO responses underwent a Delphi survey process to reach consensus on final guidelines. A combination of panel expertise and PECO responses were employed to develop algorithms for diagnosis of ITP in dogs and cats, which also underwent 4 iterations of Delphi review. Comorbidity evidence evaluators employed an integrated measure of evidence (IME) tool to determine evidence quality for each comorbidity; IME values combined with evidence summaries for each comorbidity were integrated to develop ITP screening recommendations, which also were subjected to Delphi review. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The final consensus statement provides clinical guidelines for the diagnosis of, and underlying disease screening for, ITP in dogs and cats. The systematic consensus process identified numerous knowledge gaps that should guide future studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Treatment of Immune Thrombocytopenia.

Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Rectally obtained fecal samples can be used for fecal occult blood testing in dogs, and fecal immunochemical tests do not detect canine or feline blood.

OBJECTIVE: The first objective was to determine if the sample collection method (naturally voided vs digital rectal examination collection) affected fecal occult blood test (FOBT) results. The second objective was to assess the ability of human fecal hemoglobin immunochemical tests to detect canine and feline blood. ANIMALS: 308 privately owned dogs, healthy and sick. METHODS: Guaiac FOBTs were performed on paired voided and rectally obtained canine fecal samples. The kappa statistic was used to assess agreement between the 2 collection methods, and a multivariate regression model was used to identify factors associated with a positive FOBT. Two fecal immunochemical tests (FITs; Hemosure One Step and OC-Light S) were tested with serially diluted human, canine, and feline blood. RESULTS: Voided and rectally obtained samples showed strong FOB-positivity agreement (k = 0.80), with 92.5% concordance and only 13/308 dogs negative on void but positive on rectal. Multivariate analysis showed dogs with gastrointestinal disease (P = .0008, rectal; P = .0001, void) were more likely and heavier dogs (P = .0037, rectal; P = .0022 void) were less likely to test FOBT positive. Health status, fasting status, NSAID use, and age were associated with FOBT results on univariate, but not multivariate, analysis. FITs did not detect canine or feline blood at any concentration while human blood performed as expected. CLINICAL RELEVANCE: Rectally obtained fecal samples can be reliably used for FOBTs. Human FITs may not be suitable for companion animals, but evaluation of other available tests is needed.

A prospective cohort study to identify clinical diagnostic and prognostic markers of primary immune thrombocytopenia in dogs.

BACKGROUND: Primary immune thrombocytopenia (pITP) in dogs presents a diagnostic challenge, and clinical markers of severity are lacking. OBJECTIVES: Identify clinicopathologic features that differentiate pITP from secondary ITP (sITP) and markers related to bleeding severity, transfusion, and survival of dogs with pITP. ANIMALS: Ninety-eight thrombocytopenic dogs (58 pITP and 40 sITP). METHODS: Client-owned dogs with platelet counts <50 000/µL were enrolled in a prospective, multi-institution cohort study. History and treatment information, through a maximum of 7 days, was recorded on standard data forms. Bleeding severity was scored daily using a bleeding assessment tool (DOGiBAT). At-admission blood samples were collected for CBC, biochemistry, C-reactive protein concentration, and coagulation panels, and to measure platelet surface-associated immunoglobulin G (PSAIg) and expression of platelet membrane proteins and phospholipids. Dogs with evidence of coincident disease were classified as sITP. RESULTS: No definitive pITP diagnostic test was found. However, pITP cases were characterized by lower platelet counts, D dimer concentrations, and platelet membrane protein expression than sITP cases. Differentiation between pITP and sITP was further enhanced using logistic regression modeling combining patient sex, coagulation profile, platelet count, D dimer, and PSAIg. A second model of pITP severity indicated that low hematocrit and high BUN concentration were associated with non-survival. Low hematocrit at admission, but not platelet count or DOGiBAT score, was associated with transfusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Pending validation studies, models constructed from at-admission clinicopathologic findings may improve differentiation of pITP from sITP and identify the most severe pITP cases at the time of presentation.

Guidelines for resident training in veterinary clinical pathology. IV: Laboratory quality management-Teaching domains, competencies, and suggested learning outcomes.

BACKGROUND: The 2019 ASVCP Education Committee Forum for Discussion, presented at the annual ASVCP/ACVP meeting, identified a need to develop recommendations for teaching laboratory quality management principles in veterinary clinical pathology residency training programs. OBJECTIVES: To present a competency-based framework for teaching laboratory quality management principles in veterinary clinical pathology residency training programs, including entrustable professional activities (EPAs), domains of competence, individual competencies, and learning outcomes. METHODS: A joint subcommittee of the ASVCP Quality Assurance and Laboratory Standards (QALS) and Education Committees executed this project. A draft guideline version was reviewed by the ASVCP membership and shared with selected ACVP committees in early 2022, and a final version was voted upon by the full QALS and Education Committees in late 2022. RESULTS: Eleven domains of competence with relevant individual competencies were identified. In addition, suggested learning outcomes and resource lists were developed. Domains and individual competencies were mapped to six EPAs. CONCLUSIONS: This guideline presents a framework for teaching principles of laboratory quality management in veterinary clinical pathology residency training programs and was designed to be comprehensive yet practical. Guidance on pedagogical terms and possible routes of implementation are included. Recommendations herein aim to improve and support resident training but may require gradual implementation, as programs phase in necessary expertise and resources. Future directions include the development of learning milestones and assessments and consideration of how recommendations intersect with the American College of Veterinary Pathologists training program accreditation and certifying examination.

Suspected primary bone marrow T-cell lymphoid neoplasia causing paraneoplastic hypercalcemia in 11 dogs (2014-2021).

OBJECTIVE: To describe the clinical findings and outcome in hypercalcemic dogs that were diagnosed with T-cell lymphoid neoplasia by bone marrow evaluation. ANIMALS: 11 client-owned dogs, identified retrospectively through 2 diagnostic laboratories between 2014 and 2021. CLINICAL PRESENTATION: Cases presented with hypercalcemia and lacked overt evidence of lymphoid neoplasia in the blood or nonmedullary tissues. T-cell lymphoid neoplasia was diagnosed once the bone marrow was investigated, using a variable combination of cytology, histology, and flow cytometry. RESULTS: The median age at presentation was 5.7 years (range, 4.0 to 8.6 years). All cases were large-breed dogs, and 4 of 11 cases were Golden Retrievers. Dogs presented most commonly for polyuria and polydipsia (72%). Eight cases had neutropenia, and 10 of 11 dogs had reported thrombocytopenia. In all cases, flow cytometry identified an expansion of neoplastic small- to intermediate-sized T cells in the bone marrow that expressed low-class-II major histocompatibility complex. Neoplastic T cells in 10 of 11 cases expressed CD4. Treatments ranged from prednisone alone to multiagent chemotherapy. The median overall survival time was 260 days (range, 25 to 792 days). CLINICAL RELEVANCE: T-cell lymphoid neoplasia diagnosed via bone marrow evaluation that may represent a unique bone marrow T-cell neoplastic entity should be considered in hypercalcemic dogs with isolated cytopenias that lack peripheral lymphocytosis, lymphadenopathy, and organomegaly. Clinical outcome in these cases was variable, which may be related to nonstandardized treatments, but a subset of patients had prolonged survival.

Retrospective evaluation of the incidence of gastrointestinal bleeding in dogs receiving ophthalmic nonsteroidal anti-inflammatory drugs.

OBJECTIVES: To report the incidence of gastrointestinal (GI) bleeding and associated risk factors in a population of dogs receiving ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs). ANIMAL STUDIED: Medical records of dogs prescribed ophthalmic NSAIDs (cases), dogs receiving systemic NSAIDs alone and dogs receiving systemic prednisone alone (controls). PROCEDURES: Data were collected retrospectively from the medical records of 204 dogs prescribed ophthalmic NSAIDs (diclofenac, ketorolac, or flurbiprofen), which were subdivided based on if they received any concurrent systemic NSAIDs or glucocorticoids, 136 dogs receiving a systemic NSAID (carprofen or meloxicam) alone, and 151 dogs receiving a systemic glucocorticoid (prednisone) alone at a referral hospital from 2015 to 2019. RESULTS: Gastrointestinal bleeds developed in 8/79 (10.1%) of topical NSAID-only cases, 10/136 (7.4%) of systemic NSAID controls, and 14/151 (9.3%) of systemic glucocorticoid controls, with no significant difference between the three groups (p = .6103). There were no significant differences in GI bleed rates between cases treated with ketorolac, diclofenac, or flurbiprofen (p = .160), although severe GI bleeding was only seen in ketorolac-treated dogs. Presence of a known concurrent risk factor for GI bleeding was significantly associated with the development of GI bleed in dogs on ophthalmic NSAIDs (p = .032). CONCLUSIONS: Dogs treated with ophthalmic NSAIDs developed GI bleeding at a frequency comparable to dogs receiving systemic NSAIDs or systemic glucocorticoids alone, suggesting that dogs receiving ophthalmic NSAIDs may be at increased risk of GI bleeding.

Oculosystemic pneumocystosis in 2 sibling Chihuahuas.

Sibling female and male Chihuahuas were evaluated for a 9-month history of tachypnea that failed to respond to fenbendazole, doxycycline, amoxicillin-clavulanate, and prednisone. Physical examination identified tachypnea, hyperpnea, and harsh bronchovesicular lung sounds. Fundic examination disclosed diffuse chorioretinitis, manifested as multifocal chorioretinal granulomas in the female dog and occasional chorioretinal scars in the male dog. Thoracic radiographs indicated moderate to severe interstitial to broncho-interstitial infiltrates in both dogs. Serum and urine antigen and antibody testing in the female dog failed to identify infectious agents, but cytologic assessment of hepatic lymph node, liver, and splenic aspirates identified Pneumocystis trophozoites. Infection was confirmed in both dogs by 28S rRNA PCR sequencing from multiple tissue samples. The female dog responded well to trimethoprim-sulfamethoxazole, but the male dog was euthanized because of liver failure, presumably related to antimicrobial treatment.

Preliminary evaluation of a flow cytometric assay with microsphere controls for the detection of platelet-bound antibodies in canine immune thrombocytopenia.

BACKGROUND: Canine immune thrombocytopenia (ITP) ranges from a mild to severe bleeding disorder, and platelet counts do not reliably predict clinical disease course. The detection of platelet autoantibodies may further define the disease phenotype, but variability in assay configurations and a lack of well-characterized controls limit the diagnostic utility of anti-platelet antibody assays. OBJECTIVES: We aimed to develop control reagents to facilitate the characterization of canine platelet surface-associated immunoglobulin (PSAIg) in flow cytometric assays. METHODS: Silica microspheres were coated with canine IgG and IgM to assess the reactivity of goat and rabbit origin anti-canine immunoglobulin reagents. They were also used as positive controls in the PSAIg assay. Preliminary assay evaluation and determination of sample stability used PRP isolated from seven healthy dogs and 26 dogs newly diagnosed with thrombocytopenia. RESULTS: Blood sample stability was established for up to a 48-hour storage time. The conjugated positive control microspheres demonstrated stable fluorescent labeling over a 2-year observation period. Rabbit and goat origin anti-dog IgM fluorescent antibody labels reacted nonspecifically with canine IgG. Rabbit origin anti-dog IgG antibody demonstrated greater class specificity for canine IgG than a goat origin antibody. Thrombocytopenic dogs had a broad range of membrane-bound immunoglobulin. Median PSAIgG for dogs with primary or secondary ITP (18.4%, 34.1%, respectively) were significantly higher than controls (3.8%, P < .05). CONCLUSIONS: The described assay reagents and procedures provide positive controls and allow consistent thresholding to define a positive test result, suitable for any flow cytometer. A rabbit anti-dog IgG fluorescent label demonstrated specificity for canine IgG and was useful for the detection of PSAIgG in thrombocytopenic dogs.

American Society for Veterinary Clinical Pathology-Recommended Clinical Pathology Competencies for Graduating Veterinarians.

Given the move toward competency-based veterinary education and the subsequent reevaluation of veterinary curricula, there is a need for specialties to provide guidance to veterinary college administrators and educators on the core knowledge and skills pertaining to their specialty to ensure their inclusion in revised or redesigned curricula. The American Society for Veterinary Clinical Pathology (ASVCP) Education Committee sought to create a list of competencies specific to clinical pathology expected of graduating veterinarians. The stimulus for this project was the American Veterinary Medical Association Council on Education Standards of Accreditation for Colleges of Veterinary Medicine, further driven by the 2018 publication of the Association of American Veterinary Medical Colleges Competency-Based Veterinary Education Working Group framework. The recommendations made in this document are the culmination of the 2016 ASVCP Education Forum for Discussion, multiple remote subcommittee communications, and feedback obtained from ASVCP membership. The final framework includes 8 clinical pathology-focused domains of competence with 20 clinical pathology competencies and 61 clinical pathology illustrative sub-competencies. The clinical pathology-focused domains of competence are: the pre-analytical phase of testing, laboratory medicine and instrumentation, principles of test selection and interpretation, hematology and hemostasis, chemistry, endocrinology, urinalysis, and cytology. These are not intended to replace the nine established AAVMC domains of competence with supportive competencies and illustrative sub-competencies but to guide institutions for how clinical pathology aligns within the competency-based veterinary education (CBVE) framework for the practice-ready veterinary graduate. This clinical pathology competency framework may prove useful and empowering during discussions of curriculum revisions and redesigns.

Prostaglandin EP4 receptor mRNA expression in canine lymphoma.

Canine lymphoma (LSA) is a diverse, aggressive malignancy initiated by a variety of factors. Understanding those factors could help identify potential treatment options. Chronic inflammation drives lymphoma in human medicine and is suspected to play a role in veterinary medicine. The exact mechanisms, however, have not been elucidated. Upregulation of the cyclooxygenase enzymes, and subsequently prostaglandins, potentially play a stimulatory role. Prostaglandins work through one of four EP receptors (EP1-EP4) and the effects mediated through EP4R specifically are thought to be the primary drivers of cancer development. In human T-cell LSA, overexpression of EP4R has been found and appears to protect LSA cells from apoptosis. The role of EP4R in human B-cell LSA is more nuanced. This study aims to evaluate the mRNA expression of the EP4R gene (ptger4) in canine B-cell and T-cell LSA. Archived canine lymph nodes with histologically confirmed B-cell and T-cell LSA, and reactive lymph nodes, were evaluated for EP4R mRNA expression using a novel RNA in situ hybridization technique (RNAscope). Quantification of RNAscope signals was completed with an advanced digital pathology image analysis system (HALO). Results were reported as copy number, H-score, and percent tumour cell expression of EP4R mRNA. All reactive, B-cell LSA, and T-cell LSA lymph nodes expressed EP4R mRNA. The mRNA copy number, H-score, and percent tumour cell expression of EP4R were higher in B-cell (p < .003) and T-cell (p < .001) LSA samples compared to reactive lymph node samples. There were no differences between B-cell LSA and T-cell LSA.

Characterization of recessed vulvas in dogs.

OBJECTIVE: To determine the prevalence of vulvar recession in a large population of dogs and to compare the reproductive and physical differences between dogs with and without recessed vulvas. ANIMALS: 250 female dogs presenting to a tertiary referral institution. PROCEDURES: Female dogs > 6 months of age presenting to a tertiary referral institution were enrolled. At enrollment, a full medical history was obtained with particular emphasis on the presence of lower urinary tract (LUT) disease in the 3 months prior to presentation. All dogs underwent a full physical examination including perivulvar cytologic examination and scoring of the degree of perivulvar skin coverage on the basis of an 8-point scale. Dogs with scores of ≥ 7 were classified as having recessed vulvas. When available, urinalysis data were also included. RESULTS: Recessed vulvas were identified in 36 of 250 (14%) dogs. Dogs with recessed vulvas had significantly higher body condition scores and body weights than unaffected dogs. In addition, recessed vulvas were more common in spayed than sexually intact dogs. Dogs spayed at ≤ 1 year of age were almost 3 times as likely to have vulvar recession, compared with dogs spayed at > 1 year of age. No significant difference was identified between affected and unaffected dogs with respect to the prevalence of LUT signs, urinary tract infections, or perivulvar dermatitis. CONCLUSIONS AND CLINICAL RELEVANCE: Although recessed vulvas were relatively common in dogs, they did not appear to be associated with an increased risk of LUT disease or perivulvar dermatitis.

Comparison of curvilinear-array (microconvex) and phased-array transducers for ultrasonography of the lungs in dogs.

OBJECTIVES: To compare the use of curvilinear-array (microconvex) and phased-array transducers for ultrasonographic examination of the lungs in dogs. ANIMALS: 13 client-owned dogs with left-sided congestive heart failure. PROCEDURES: In a prospective methods comparison study, 24 ultrasonographic examinations of the lungs (4 sites/hemithorax) were performed with both curvilinear-array and phased-array transducers at 3 clinical time points. Two observers independently assessed the number of B lines (scored per site and in total), number of sites strongly positive for B lines (ie, those with > 3 B lines/site), and image quality (scored on a 5-point scale). Analyses included assessment of interobserver agreement with κ analysis, comparison of quality scores between transducers with mixed-effects modeling, and investigation of agreement and bias for B-line data and quality scores between transducers with Passing-Bablok regression. RESULTS: Interobserver agreement for total B-line scores and number of strong-positive sites was excellent (κ > 0.80) for both transducers. There was no evidence of analytic bias for the number of B lines or strong-positive sites between transducers. Interobserver agreement for image quality scores was moderate (κ, 0.498 and 0.517 for the curvilinear-array and phased-array transducers, respectively). Both observers consistently assigned higher-quality scores to curvilinear-array images than to phased-array images. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated both curvilinear-array (microconvex) and phased-array transducers can be used by experienced sonographers to obtain diagnostic ultrasonographic images of the lungs in dogs with acute or resolving left-sided congestive heart failure and suggested the former transducer may be preferred, particularly to aid identification of anatomic landmarks for orientation.

Chemical and microbiological stability of diluted ceftazidime in three different solutions under three storage temperatures over a 28 day period.

BACKGROUND: Ceftazidime (CAZ) solutions are being used based on anecdotal reports for otitis externa complicated by multidrug-resistant Pseudomonas aeruginosa (MDR PA). The chemical and microbiological stability of these proposed compounded solutions have not been evaluated, and likely are affected by the diluent and storage duration or temperature. HYPOTHESIS/OBJECTIVES: Compounded CAZ solutions would show variable degradation dependent on diluent, time and temperature. The antimicrobial activity of the solutions would reflect changes in concentration and not alterations to the chemical compound. METHODS AND MATERIALS: Ceftazidime was compounded with 100 mL 0.9% sodium chloride (NA+CAZ), 118 mL Triz-EDTA Aqueous flush (TE+CAZ) and 125 mL Douxo Micellar Solution (MI+CAZ). Aliquots of the solutions were stored at 25°C, 4°C and -20°C for 28 days. High-performance liquid chromatography was used to analyse CAZ recovery from compounded solutions at weekly intervals. A modified broth dilution technique was utilised to assess minimum inhibitory concentration (MIC) to monitor antimicrobial activity against a reference PA strain. RESULTS: Temperature, duration of storage and diluent each had independent effects on the chemical stability of CAZ. CAZ concentrations decreased over time as well as with increased temperature. NA+CAZ solutions exhibited the least degradation compared to the other solutions. The MIC for PA was most consistent for NA+CAZ solutions regardless of storage temperature and duration of storage. CONCLUSIONS AND CLINICAL IMPORTANCE: Chemical and microbiological stability of compounded CAZ solutions varied by diluent, storage temperature and duration of storage. Dilution in NA resulted in the lowest variation in stability over 28 days when stored at refrigerated or frozen temperatures compared to other diluents.

Preliminary Investigation of Bovine Whole Blood Xenotransfusion as a Therapeutic Modality for the Treatment of Anemia in Goats.

Anemia requiring whole blood transfusion for appropriate treatment is a common clinical presentation of caprine patients to veterinary practitioners; however, identifying suitable blood donors in goat herds can be challenging. In other veterinary species, the practice of xenotransfusion, where blood from 1 species is transfused to another, is used in emergency settings. Due to their ability to donate large volumes of whole blood, cattle could be an ideal source for xenotransfusion of goats. In this study 2 healthy goats were transfused with bovine whole blood. The goats were then monitored for adverse effects and the presence of bovine erythrocyte post-xenotransfusion. Afterward, 15 caprine-bovine combinations were evaluated for compatibility via cross-matching. Both goats tolerated xenotransfusion, although transient reactions were observed. Of the 15 cross-match combinations, 11 of the major cross matches were compatible, and all minor cross matches were also compatible. While future work is necessary to refine this technique, xenotransfusion of goats with cattle blood may be a therapeutic modality for the treatment of caprine anemia.

Analysis of gene expression of prostaglandin EP4 receptor in canine osteosarcoma.

In many human cancers, the expression of the prostaglandin receptor EP4 (EP4R) is associated with the development of malignancy and a poor prognosis. The expression of EP4R has not yet been evaluated in canine tumors. The objective of this study was to characterize the messenger RNA (mRNA) expression of EP4R in canine osteosarcoma (OSA). Gene expression of EP4R was evaluated using RNA in-situ hybridization (RNAscope). In all canine OSA samples evaluated, strong universal positive expression of EP4R was identified. Gene expression was significantly higher in OSA tissue samples than in normal nasal turbinate bone, possibly implicating EP4R in the pathogenesis of canine OSA.

Dans de nombreux cancers humains, l'expression du récepteur des prostaglandines EP4 (EP4R) est associée au développement d'une malignité et à un mauvais pronostic. L'expression d'EP4R n'a pas encore été évaluée dans les tumeurs canines. L'objectif de cette étude était de caractériser l'expression de l'ARN messager (ARNm) de l'EP4R dans l'ostéosarcome canin (OSA). L'expression génique de l'EP4R a été évaluée en utilisant l'hybridation in situ d'ARN (RNAscope). Dans tous les échantillons canins OSA évalués, une forte expression positive généralisée d'EP4R a été identifiée. L'expression génique était significativement plus élevée dans les échantillons de tissus OSA que dans l'os normal du cornet nasal, ce qui impliquait peut-être EP4R dans la pathogenèse de l'OSA canin.(Traduit par Docteur Serge Messier).

Utility of point-of-care lung ultrasound for monitoring cardiogenic pulmonary edema in dogs.

BACKGROUND: Point-of-care lung ultrasound (LUS) is an effective tool to diagnose left-sided congestive heart failure (L-CHF) in dogs via detection of ultrasound artifacts (B-lines) caused by increased lung water. HYPOTHESIS/OBJECTIVES: To determine whether LUS can be used to monitor resolution of cardiogenic pulmonary edema in dogs, and to compare LUS to other indicators of L-CHF control. ANIMALS: Twenty-five client-owned dogs hospitalized for treatment of first-onset L-CHF. METHODS: Protocolized LUS, thoracic radiographs (TXR), and plasma N-terminal pro-B-type natriuretic peptide were performed at hospital admission, hospital discharge, and recheck examinations. Lung ultrasound findings were compared between timepoints and to other clinical measures of L-CHF. RESULTS: From time of hospital admission to discharge (mean 19.6 hours), median number of LUS sites strongly positive for B-lines (>3 B-lines per site) decreased from 5 (range, 1-8) to 1 (range, 0-5; P < .001), and median total B-line score decreased from 37 (range, 6-74) to 5 (range, 0-32; P = .002). Lung ultrasound indices remained improved at first recheck (P < .001). Number of strong positive sites correlated positively with respiratory rate (r = 0.52, P = .008) and TXR edema score (r = 0.51, P = .009) at hospital admission. Patterns of edema resolution differed between LUS and TXR, with cranial quadrants showing more significant reduction in B-lines compared to TXR edema score (80% vs 29% reduction, respectively; P = .003). CONCLUSIONS AND CLINICAL IMPORTANCE: Lung ultrasound could be a useful tool for monitoring resolution of pulmonary edema in dogs with L-CHF.

Neutrophil extracellular traps in stored canine red blood cell units.

BACKGROUND: Neutrophil extracellular traps (NETs), webs of DNA and citrullinated histones extruded from activated neutrophils cause transfusion-related acute lung injury. Supernatants of stored red blood cell (RBC) units might promote NETosis in neutrophils from the units or from transfusion recipients. HYPOTHESES: (1) NETs form during storage of canine RBC, (2) leukoreduction (LR) before storage of RBC reduces NETosis, and (3) supernatant from stored, nonleukoreduced (NLR) RBC units induces NETosis in healthy canine neutrophils modeling transfusion recipients. ANIMALS: Six healthy purpose-bred research dogs were utilized for blood donation. METHODS: Prospective controlled study. RBC units were collected from each dog, aseptically divided into 2 equal subunits, 1 of which was leukoreduced, and stored for 42 days. Stored units were sampled biweekly for quantification of NET markers citrullinated histone H3 (Western blot) and cell-free DNA (cfDNA) (DNA dye binding). Unit supernatants were applied ex vivo to canine neutrophils and extracellular DNA release representing NETosis was assessed. RESULTS: Markers of NETs increased during RBC storage (cfDNA P < .0001 and citrullinated H3 P = .0002) and were higher in NLR than LR units (day 42 LR cfDNA 0.34 ± 0.82 ng/mL vs day 42 NLR 1361.07 ± 741.00 ng/mL, P < .0001; day 42 LR citrullinated H3 0.19 ± 0.13 AU vs NLR 0.57 ± 0.34 AU, P = .007). Isolated neutrophils did not form NETs when exposed to stored canine RBC supernatant. CONCLUSIONS AND CLINICAL IMPORTANCE: NETosis occurs in stored canine NLR RBC units, and is attenuated by LR before storage. NETs might be mediators of transfusion reactions.

Gene expression of prostaglandin EP4 receptor in three canine carcinomas.

BACKGROUND: Chronic inflammation mediated by the cyclooxygenase enzymes, specifically their product prostaglandin E2 (PGE2), can result in the development of cancer. PGE2 promotes cell proliferation, apoptosis, and angiogenesis through interaction with its specific receptors (EP1 receptor - EP4 receptor [EP1R-EP4R]). In multiple human cancers, the expression of EP4R is associated with the development of malignancy and a poor prognosis. The expression of EP4R has not yet been evaluated in canine tumors. The aim of this study was to characterize the mRNA gene expression of EP4R (ptger4) in canine squamous cell carcinoma (SCC), apocrine gland anal sac adenocarcinoma (AGASACA), and transitional cell carcinoma (TCC). Archived tumor samples of canine cutaneous SCC (n = 9), AGASACA (n = 9), and TCC (n = 9), and matched archived normal tissue controls were evaluated for mRNA expression of canine EP4R using RNA in situ hybridization (RNAscope®). Quantification of RNAscope® signals in tissue sections was completed with an advanced digital pathology image analysis system (HALO). Data was expressed as copy number, H-index, and percent tumor cell expression of EP4R. RESULTS: In all canine SCC, AGASACA, and TCC samples evaluated, strong universal positive expression of EP4R was identified. For SCC and AGASACA, mRNA EP4R expression was statistically higher than that of their respective normal tissues. The TCC tissues displayed significantly less mRNA EP4R expression when compared to normal bladder mucosa. CONCLUSIONS: These results confirm the mRNA expression of canine EP4R in all tumor types evaluated, with SCC and AGASACA displaying the highest expression, and TCC displaying the lowest expression. This study also represents the first reported veterinary evaluation of EP4R expression using the novel in situ hybridization technique, RNAscope®.

Performance of a Nageotte hemocytometer method and a flow cytometric assay for residual leukocyte quantification in leukoreduced canine packed red blood cells.

OBJECTIVE: To evaluate the performances of a manual Nageotte hemocytometer method and commercial fluorescent bead-based flow cytometric assay for quantifying [rWBC] in leukoreduced canine packed red blood cell (pRBC) units. DESIGN: Prospective study. Five, commercially purchased, double leukoreduced canine pRBC units were spiked with canine leukocytes to create 6 pRBC standards with the following [rWBC]: < 0.1, 0.375, 1.5, 3.0, 6.0, and 24.0 WBC/µL. [rWBC] of each pRBC standard was measured with the Nageotte hemocytometer and flow cytometric techniques. Limit of detection (LoD), linearity, and bias were determined for each method. For each standard, accuracy and precision were calculated; the cumulative accuracy and mean precision for measurements between the LoD and 24.0 WBC/µL were also determined. SETTING: University veterinary blood bank and clinical pathology laboratory. MEASUREMENTS AND MAIN RESULTS: The Nageotte hemocytometer method had an LoD = 1.48 WBC/µL, inadequate linearity (R2  = 0.92), and a significant negative proportional bias (slope best-fit line = 0.52 ± 0.03). Between [rWBC] 1.5-24 WBC/µL, the technique demonstrated poor cumulative accuracy (6.7%) but acceptable mean precision (17.3%). Relative to a 2 rWBC/µL threshold, at 1.5 WBC/µL the method was inaccurate (6.7%) with acceptable precision (16.6%). The flow cytometric assay had an LoD = 1.3 WBC/µL, acceptable linearity (R2  = 0.99), and a mild positive proportional bias (slope best-fit line = 1.11 ± 0.01). The technique had acceptable cumulative accuracy (80%) and mean precision (10.7%) for measuring [rWBC] between 1.5 and 24 WBC/µL. At 1.5 WBC/µL, this method was acceptably accurate (86.7%) and precise (16.0%). CONCLUSIONS: The flow cytometric assay demonstrated acceptable performance for quantification of [rWBC] in leukoreduced canine pRBC units. The Nageotte hemocytometer method should be used cautiously due to poor accuracy and significant negative bias.