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This article summarizes the current applications of flow cytometry in clinical veterinary medicine, which is largely restricted to the diagnosis of hematopoietic neoplasms (lymphomas and leukemias) of domestic dogs, cats, and horses. A brief background on the technique of flow cytometry and fundamentals of data interpretation are included. Major emphasis is placed on clinical indications for flow cytometry, principles of sample collection and submission, and awareness of diagnostic and prognostic utility. Expectations regarding both the benefits and limitations of flow cytometry in a clinical setting, and its complementary nature with other types of testing, are also reviewed.

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.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.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.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.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.

BACKGROUND: Bacterial sepsis is a relatively common, life-threatening condition with a high case fatality rate. The current primary diagnostic tools for detecting bacterial infection in fluids are bacterial culture and fluid cytology. While culture is the gold standard, it can take up to several days for results to be made available to clinicians, which can delay recognition of bacterial sepsis and negatively impact patient outcomes. OBJECTIVES: The aim of this study was to evaluate the diagnostic accuracy of cytology for detecting bacterial infection in body fluids. METHODS: We retrospectively reviewed 10 years of medical records at the Ohio State University’s Veterinary Medical Center for mammalian patients with both cytology and bacterial culture of fluid samples, including body cavity fluids (abdominal and thoracic effusion), blood, joint fluid, and CSF. The overall sensitivity and specificity of cytology relative to the reference method of bacterial culture was recorded, as well as among the subcategories of fluid type. RESULTS: The overall sensitivity and specificity of cytology for the diagnosis of sepsis were 42.6% and 93.0%, respectively. Individual sensitivities and specificities were also calculated for each fluid type. Thoracic fluid cytology had relatively high sensitivity and low specificity, in contrast to the other fluid types analyzed. CONCLUSIONS: Overall, cytology is poorly sensitive but highly specific for the detection of bacterial infection in fluid samples. The results from this study will allow a better comparison between the diagnostic accuracy of cytology and emerging diagnostic tests for the detection of bacterial sepsis in mammalian patients.

BACKGROUND The Cobas u411 Analyzer (Roche Diagnostics) is an automated, reflectance photometry-based urinalysis instrument designed for use with Roche's CHEMSTRIP 10UA technology and human urine samples. OBJECTIVE We aimed to optimize and validate the Cobas u411 Analyzer for use in canine and feline urinalysis. METHODS Patient urine samples presenting to the Clinical Pathology Laboratory at the Colorado State University Veterinary Teaching Hospital were analyzed with the Cobas u411 and by manual readings in parallel. Initially, 223 canine and 83 feline urine samples were run using the u411 factory settings. Following comparisons with manual results, and evaluation for directional bias, adjustments to the reflectance values were made in the instrument's programming. An additional 183 canine and 95 feline samples were run using the adjusted settings. Total urine protein concentrations were measured in 48 samples and used to generate receiver operating characteristic curves for the protein test pad. RESULTS Following adjustments in reflectance programming, concordance between u411 and manual results was increased by 17.7% for protein, 11.7% for ketones, and 4.5% for bilirubin. Concordances for pH, glucose, and blood were not substantially changed. Discordance for all analytes was ≤3%. Canine and feline samples had similar levels of discordance, though marginal concordance was higher in dogs for ketones, bilirubin, and blood. CONCLUSIONS Adjustments to the reflectance programming of the Cobas u411 Analyzer improved concordance with manual results for canine and feline samples. This instrument has the potential to greatly increase both efficiency and consistency of urinalysis procedures in higher throughput veterinary diagnostic laboratories.

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.

The goal of this study was to formally evaluate the administration of unlicensed, crowd-sourced antiviral GS-441524-like therapy for cats suspected to have feline infectious peritonitis (FIP), a previously fatal disease. Members of a large social media support and GS-441524-like drug distribution group were surveyed via the Internet. The survey was targeted toward owners who had treated their cats for at least 12 weeks with unlicensed GS-441524-like drugs. Of the 393 analyzed surveys which met inclusion criteria, 73.7% of owners utilizing this therapy were from the United States. Only 8.7% of owners reported receiving help from their veterinarian in administering the treatment to their cat. The mean cost of treatment was USD 4920. A majority of owners (88.2%) reported noticeable improvement in clinical signs within one week of initiating therapy. At the time of the survey, 96.7% (380 cats) were alive, with 54.0% of them considered cured and another 43.3% being monitored in the 12-week observation period. A total of 12.7% of the cats suffered a relapse of clinical signs of FIP, and 3.3% of the cats died despite GS-441524-like therapy. Reported complications were mostly related to owner administration of subcutaneous injections of the acidic GS-441525-like therapy, such as vocalization, pain, struggling, and injection-site wounds. Limitations of this study include a retrospective design, bias in case selection, reliance on owner-reported data, and inability to confirm the contents of unlicensed pharmaceuticals, however, important lessons can be learned from the experiences of these owners. While unconventional, and certainly not free from medical and legal risks, unlicensed, at-home GS-441524-like therapy, according to owner reports, can apparently offer benefits in the treatment of cats suspected of FIP.

Background Perianal (hepatoid) gland tumors are common in dogs, and the distinction between the benign and malignant forms is clinically important. Cytology of these tumors typically contains many large hepatoid cells and fewer small basal cells. Objectives The objective of this study was to determine whether the proportion of the smaller basaloid reserve cells in cytologic samples from perianal tumors correlates with malignancy. Methods Eighty-three cases of cytologically diagnosed perianal gland tumors with corresponding histopathologic sections were identified from two separate institutions and included six (7.2%) malignant tumors and 77 (92.8%) benign tumors. The proportion of basal cells from each sample was evaluated. Results No difference between these groups was found, although the study was sufficiently powered to detect an approximately 1.5-fold change in basal cell proportion. Conclusions This report found no evidence that the proportion of basal cells in canine perianal tumor cytology is an indication of the potential for malignancy. We, therefore, do not recommend citing this feature in cytologic reports or when communicating with clinicians.

BACKGROUND Lymphoma is an important disease of pet guinea pigs, although validation of immunophenotyping techniques based on cytologic or hematologic samples has not been reported. OBJECTIVE To describe an immunocytochemical method for immunophenotyping of lymphoma (as either T- or B-cell) in guinea pigs, and to validate antibodies for this purpose. METHODS Blood and tissues were obtained at the time of necropsy from laboratory guinea pigs and a privately owned dog (control) euthanized for reasons unrelated to lymphoproliferative disease. Fine-needle aspirates of enlarged peripheral lymph nodes were obtained from a case of spontaneous lymphoma in a pet guinea pig. Anti-CD3 and anti-Pax5 antibodies were validated by a combination of western blotting performed on splenic lysates of both the dog and guinea pigs, immunohistochemical studies on normal guinea pig tissues, and immunocytochemistry on normal guinea pig peripheral blood and splenic impression smears. RESULTS The antibodies bound to antigens of an appropriate size in both the dog and guinea pig splenic lysates by Western blot analysis. Immunohistochemistry and immunocytochemistry demonstrated the expected distribution of putative T- and B-lymphocytes in normal tissues, peripheral blood, and splenic impression smears. As a proof-of-principle for its clinical utility, this immunocytochemical assay was used to diagnose a B-cell phenotype in a spontaneous lymphoma case in a pet guinea pig. CONCLUSIONS Here, we validated an immunocytochemical method for immunophenotyping of lymphoma in guinea pigs as either a T- or B-cell phenotype. This enables future research into the clinical attributes of these subtypes and may ultimately improve both prognostication and therapy of lymphoma in guinea pigs.

Urothelial carcinoma, also known as transitional cell carcinoma, is the most common primary bladder tumour in dogs, and can also involve the prostate gland. Cytology is a common diagnostic tool utilized for dogs with bladder or prostate gland lesions. The objectives of this retrospective study were to compare the sensitivity and specificity of cytologic evaluation for urothelial or prostatic carcinoma between two institutions with different cytology review protocols as well as determine if certain collection methods resulted in higher cytologic accuracy. A total of 298 cases met inclusion criteria. The overall sensitivity and specificity for institution 1 were 91.8% and 50%, respectively, compared to 31.1% and 97.4%, respectively, for institution 2. When the urine sample review protocol at institution 2 was matched to that of institution 1, sensitivity and specificity were more similar to institution 1 (71.2% and 88.9%, respectively). Our results show that the sensitivity and specificity of cytology are affected by screening and review protocols implemented by different institutions. The data also demonstrate that sensitivity and specificity vary by collection method. Diagnostic catheterization had the highest performance: of the 11 cases between two institutions, it had 100% sensitivity and specificity. In contrast, examination of urine sediment not collected via diagnostic catheterization had low sensitivity and specificity that varied greatly by institution. In summary, cytologic interpretation should be undertaken with consideration given to both processing and collection protocols.

Virtual microscopy (VM) using scanned slides and imaging software is increasingly used in medical curricula alongside instruction in conventional microscopy (CM). Limited reports suggest that VM is useful in the veterinary education setting, and generally well-received by students. Whether students can apply knowledge gained through VM to practical use is unknown. Our objective was to determine whether instruction using VM, compared to CM, is a successful method of training veterinary students for the application of cytology in practice (i.e., using light microscopes). Seventy-one veterinary students from Colorado State University who attended a voluntary 3-hour cytology workshop were randomized to receive the same instruction with either VM ( n = 35) or CM ( n = 36). We compared these students to a control group ( n = 22) of students who did not attend a workshop. All students took a post-workshop assessment involving the interpretation of four cases on glass slides with CM, designed to simulate the use of cytology in general practice. Students also took an 18-question survey related to the effectiveness of the workshop, providing their opinions on cytology instruction in the curriculum and their learning preference (VM or CM). The mean assessment score of the VM group (14.18 points) was significantly higher than the control group (11.33 points, p = .003), whereas the mean of the CM group (12.77 points) was not statistically significantly different from controls ( p = .170). Not only is VM an effective method of teaching cytology to veterinary students that can be translated to a real-world case scenario, but it outperformed CM instruction in this study.

Background Feline Infectious Peritonitis (FIP) is a fatal disease of cats that can be very difficult to definitively diagnose antemortem. Multiplex fluorescent immunocytochemical (MF-ICC) assays are emerging as useful diagnostic tests in veterinary medicine, particularly for fluid samples. Objective We aimed to develop and optimize an MF-ICC assay to detect feline coronavirus within macrophages, with the primary goal of determining the allowable/recommended sample storage conditions for clinical use of this assay. Methods A feline macrophage cell line was infected with the FIP virus. Following harvest into EDTA tubes (simulating typical clinical collection of effusion), cells were stored at 4℃, 22℃, and 37℃. For each temperature condition, slides for MF-ICC were made at 0, 1, 2, 3, and 5 days post-collection. To assess the stability of immunoreactivity following fixation, freshly harvested infected cells were fixed onto slides and maintained at 4℃ for 1, 2, 4, and 12 weeks. All slides were analyzed by MF-ICC for the presence of mononuclear cells with co-expression of vimentin and coronaviral antigen. Results MF-ICC confirmed that cells tested positive for coronavirus at 4℃ through 3 days post-harvest, 22℃ through 48 hours post-harvest, and 37℃ through 24 hours post-harvest. The MF-ICC assay was successfully performed on fixed slides through the 12-week time point. This assay also demonstrated positive results on a clinical sample of abdominal fluid from a cat later confirmed to have FIP. Conclusions The MF-ICC assay described here offers a potentially specific and relatively stable antemortem diagnostic test for feline infectious peritonitis. Evaluation of this assay in clinical samples is ongoing.

Feline infectious peritonitis is a devastating, fatal disease of domestic cats caused by a pathogenic mutant virus derived from the ubiquitous feline enteric coronavirus (FECV). Infection by FECV is generally subclinical, and little is known about the mucosal immune response that controls and eliminates the virus. We investigated the mucosal immune response against FECV in an endemically infected breeding colony over a seven-month period. Thirty-three cats were grouped according to FECV seropositivity and fecal virus shedding into na&iuml, ve/immunologically quiescent, convalescent and actively infected groups. Blood, fecal samples and colon biopsies were collected to assess the mucosal and systemic immunologic and virologic profile. Results showed that cats with active FECV infections have strong systemic IgG and mucosal IgA responses that wane after virus clearance. Significant FECV-specific mucosal T cell IFN&gamma, responses were not detected in any of the three groups. A shift toward an inflammatory state in the mucosa was suggested by increased IL17:FoxP3 expression. However, no histologic abnormalities were observed, and no shifts in lymphocyte subpopulation phenotype or proliferation were noted. Together, the results suggest that control of FECV is mediated by humoral mucosal and systemic responses and that perturbations in the primary reservoir organ (colon) are minimal.

BACKGROUND Biochemistry analyzers in many high-throughput laboratories use indirect potentiometry to determine serum electrolyte concentrations, which involves a pre-analytical dilution step that may be associated with artifactual increases or decreases in electrolyte concentrations under circumstances of altered serum water fraction (SWF). Severe hypo- and hyperproteinemia, conditions that cause altered SWF, are recognized but under-emphasized causes of falsely measured serum sodium concentrations. OBJECTIVES The goals of this study were to determine the average actual SWF (SWFA ) and establish formulae to correct serum sodium concentration measured by indirect potentiometry in hypo- and hyperproteinemic cats. METHODS Serum samples from 112 feline patients were analyzed for electrolytes (measured by both indirect and direct potentiometry), total protein, albumin, triglycerides, and cholesterol. Each serum sample was also lyophilized to determine the SWFA . A feline-specific formula to estimate SWF (SWFE-FEL ) was developed and evaluated with a multivariable linear model. RESULTS The mean SWFA in this population of cats was 91.2%, which was significantly different (P

A 6-year-old, spayed female cat was presented with acute respiratory signs and pleural effusion. Computed tomography scan revealed a large, lobulated mass effect in the ventral right hemithorax with concurrent sternal lymphadenopathy. A cytologic sample of the mass contained pyogranulomatous inflammation, necrotic material, and abundant yeast structures that lacked a distinct capsule and demonstrated rare pseudohyphal forms. Fungal culture and biochemical testing identified the yeast as Cryptococcus albidus, with susceptibility to all antifungal agents tested. However, subsequent 18S PCR identified 99% homology with a strain of Cryptococcus neoformans and only 92% homology with C. albidus. The patient responded well to fluconazole therapy unlike the only known previous report of C. albidus in a cat. The unusual cytologic morphology in this case underscores the need for ancillary testing apart from microscopy for fungal identification. Though C. albidus should be considered as a potential feline pathogen, confirmation with PCR is recommended when such rare non-neoformans species are encountered.