Diagnostic Validation of a Whole-Slide Imaging Scanner in Cytological Samples: Diagnostic Accuracy and Comparison With Light Microscopy.

Digital slides created by whole-slide imaging scanners can be evaluated by pathologists located in remote sites, but the process must be validated before this technology can be applied to routine cytological diagnosis. The aim of this study was to validate a whole-slide imaging scanner for cytological samples. Sixty cytological samples, whose diagnoses were confirmed by gold-standard examinations (histology or flow cytometry), were digitalized using a whole-slide imaging scanner. Digital slides and glass slides were examined by 3 observers with different levels of cytopathological expertise. No significant differences were noted between digital and glass slides in regard to the number of cases correctly diagnosed, or the sensitivity, specificity, or diagnostic accuracy, irrespective of the observers' expertise. The agreements between the digital slides and the gold-standard examinations were moderate to substantial, while the agreements between the glass slides and the gold-standard examinations were substantial for all 3 observers. The intraobserver agreements between digital and glass slides were substantial to almost perfect. The interobserver agreements when evaluating digital slides were moderate between observers 1 and 2 and between observers 1 and 3 while they were substantial between observers 2 and 3. In conclusion, our study demonstrated that the digital slides produced by the whole-slide imaging scanner are adequate to diagnose cytological samples and are similar among clinical pathologists with differing levels of expertise.

Canine Clitoral Carcinoma: A Clinical, Cytologic, Histopathologic, Immunohistochemical, and Ultrastructural Study.

Vaginal and vulvar tumors are uncommon in dogs. Knowledge of canine primary clitoral neoplasia is restricted to a few case reports, and only carcinomas have been reported. Cytologic and histologic features reported in the literature seem to overlap with those of canine apocrine gland anal sac adenocarcinoma (AGASA). Clinical features also recall those of canine AGASA, such as locoregional metastases and hypercalcemia of malignancy (HM). In this study, 6 cases of primary canine clitoral carcinomas (CCCs), with and without HM, were investigated by means of cytology, histopathology, electron microscopy, and immunohistochemistry for neuroendocrine markers including chromogranin A (CGA), synaptophysin (SYN), neuron-specific enolase (NSE), and S-100. In all 6 tumors, cytologic findings were consistent with malignant epithelial neoplasia of apocrine gland origin. The tumors examined were classified into 3 different histological patterns representing different degrees of differentiation: tubular, solid, and rosette type. Both CGA and SYN were mildly expressed in 2 of 6 tumors, while NSE was consistently expressed in all 6 cases. None of the tumors were S-100 positive. Transmission electron microscopy revealed electron-dense cytoplasmic granules compatible with neuroendocrine granules in all 6 cases. CCCs presented clinicopathologic features resembling AGASAs with neuroendocrine characteristics, and 2 of 6 neoplasms were considered as carcinomas with neuroendocrine differentiation and were positive for 3 neuroendocrine markers. CCCs can often present with HM, and long-term outcome is likely poor. Our study concludes that CCC seems to be a rare tumor, but it might be underestimated because of the overlapping features with AGASA. Further studies should aim to define the true incidence of this disease.

Digital Cytology in Veterinary Education: A Comprehensive Survey of Its Application and Perception among Undergraduate and Postgraduate Students.

The COVID-19 pandemic accelerated technological changes in veterinary education, particularly in clinical pathology and anatomic pathology courses transitioning from traditional methods to digital pathology (DP). This study evaluates the personal effectiveness and satisfaction, as well as the advantages and disadvantages, of DP, in particular digital cytology (DC), as a teaching method among European veterinary students, both at the undergraduate and postgraduate level, who attended digital pathology courses during and before the pandemic. A further aim is to discuss the differences between the two student groups. A Google Form survey consisting of 11 multiple-choice questions was emailed to pathology teachers and distributed to their students. Results indicated that undergraduate students showed greater digital pathology training, favouring DC as the most effective learning modality. In contrast, postgraduate students reported less digital slide training, and their preference for learning cytology was split between DC alone and DC integrated with traditional microscopy. All students experienced whole slide imaging for learning cytology slides prevalently, and they stated that DC enhanced their learning experience. While DC demonstrates personal effectiveness and satisfaction as a teaching method, it is important to not replace pathology training with light microscopy completely, as almost a third of the students indicated.

Description of a double centrifugation tube method for concentrating canine platelets.

BACKGROUND: To evaluate the efficiency of platelet-rich plasma preparations by means of a double centrifugation tube method to obtain platelet-rich canine plasma at a concentration at least 4 times higher than the baseline value and a concentration of white blood cells not exceeding twice the reference range. A complete blood count was carried out for each sample and each concentrate. Whole blood samples were collected from 12 clinically healthy dogs (consenting blood donors). Blood was processed by a double centrifugation tube method to obtain platelet concentrates, which were then analyzed by a flow cytometry haematology system for haemogram. Platelet concentration and white blood cell count were determined in all samples. RESULTS: Platelet concentration at least 4 times higher than the baseline value and a white blood cell count not exceeding twice the reference range were obtained respectively in 10 cases out of 12 (83.3%) and 11 cases out of 12 (91.6%). CONCLUSIONS: This double centrifugation tube method is a relatively simple and inexpensive method for obtaining platelet-rich canine plasma, potentially available for therapeutic use to improve the healing process.

Injection-Site Sarcoma in Three Village Weaver Birds (Ploceus cucullatus) Associated with Autogenous Yersinia pseudotuberculosis Vaccination.

Post-vaccinal sarcomas have been reported in cats and rarely in other domestic mammals, but not in birds. Three village weaver birds (Ploceus cucullatus) presented with poor flying ability and abnormal wing carriage attributable to large, unilateral pectoral masses. All had received at least one dose of autogenous Yersinia pseudotuberculosis vaccine into the affected pectoral muscle 74-408 days previously. Following euthanasia, gross post-mortem examination revealed locally invasive subcutaneous tumours extending through the sternum into the coelomic cavity. Cytology and histology revealed neoplasms of pleomorphic spindloid neoplastic cells with foci of coagulative necrosis and cavitation, sometimes containing faintly refractile non-polarizing granular material, both extracellularly and after phagocytosis by surrounding cells, including multinucleated giant cells. Immunohistochemistry in one bird supported a striated muscle cell origin. Findings of anaplastic sarcoma with intralesional foreign crystalline material resembled typical injection-site sarcomas in cats. This is the first report of presumptive vaccine-associated sarcoma in a non-mammalian species.

Performance of lymph node cytopathology in diagnosis and characterization of lymphoma in dogs.

BACKGROUND: Cytopathology is a minimally invasive and convenient diagnostic procedure, often used as a substitute for histopathology to diagnose and characterize lymphoma in dogs. OBJECTIVES: Assess the diagnostic performance of cytopathology in diagnosing lymphoma and its histopathological subtypes in dogs. ANIMALS: One-hundred and sixty-one lymph node samples from 139 dogs with enlarged peripheral lymph nodes. METHODS: Based only on cytopathology, 6 examiners independently provided the following interpretations on each sample: (a) lymphoma vs nonlymphoma; (b) grade and phenotype; and (c) World Health Organization (WHO) histopathological subtype. Histopathology and immunohistochemistry (IHC) findings were used as reference standards to evaluate diagnostic performance of cytopathology. Clinical, clinicopathologic, and imaging data also were considered in the definitive diagnosis. RESULTS: Classification accuracy for lymphoma consistently was >80% for all examiners, whereas it was >60% for low grade T-cell lymphomas, >30% for high grade B-cell lymphomas, >20% for high grade T-cell lymphomas, and <40% for low grade B-cell lymphomas. Interobserver agreement evaluated by kappa scores was 0.55 and 0.32 for identification of lymphoma cases, and of grade plus immunophenotype, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Cytopathology may result in accurate diagnosis of lymphoma, but accuracy decreases when further characterization is needed. Cytopathology represents a fundamental aid in identifying lymphoma and can be used as a screening test to predict grade and phenotype. However, these results must be confirmed using other ancillary techniques, including flow cytometry, histopathology, and immunohistochemistry (IHC).

Pathology, infectious agents and horse- and management-level risk factors associated with signs of respiratory disease in Ethiopian working horses.

BACKGROUND: Respiratory disease is a common cause for presentation of working horses to clinics in Ethiopia and a priority concern for owners. OBJECTIVES: To identify risk factors for and association of pathogens with respiratory signs in working horses. STUDY DESIGN: Unmatched case-control study. METHODS: Cases were those animals recently coughing (last 7 days) or observed with coughing, nasal discharge or altered respiration at the time of examination. A physical exam and respiratory endoscopy were performed including a tracheal wash sample to detect the presence of pathogens and serology performed on blood. An owner questionnaire was administered. Risk factors were determined using multivariable logistic regression. RESULTS: Data on 108 cases and 93 unmatched control horses were obtained. Case horses often had underlying lower airway pathology and were significantly more likely to have Streptococcus zooepidemicus detected (OR: 12.4, 95% CI: 3.6-42.4). There was no evidence of a major role for viral respiratory pathogens. Risk factors included completion of strenuous work (OR: 2.7, 95% CI: 1.2-6.3), drinking from stagnant water sources (OR: 2.3, 95% CI: 1.0-5.2) or being housed on a cobbled floor (OR: 2.0, 95% CI: 1.1-3.8). There were increased odds of respiratory disease in young and old horses in this population. MAIN LIMITATIONS: Samples for pathogen detection and cytology were only taken from the trachea. CONCLUSION: S. zooepidemicus, a common commensal, may play a role in clinical respiratory disease in this population.

A text-mining based analysis of 100,000 tumours affecting dogs and cats in the United Kingdom.

Cancer is a major reason for veterinary consultation, especially in companion animals. Cancer surveillance plays a key role in prevention but opportunities for such surveillance in companion animals are limited by the lack of suitable veterinary population health infrastructures. In this paper we describe a pathology-based animal tumour registry (PTR) developed within the Small Animal Veterinary Surveillance Network (SAVSNET) built from electronic pathology records (EPR) submitted to this network. From an original collection of 180232 free text (non-structured) EPRs reported between April 2018 and June 2019, we used specific text-mining methodologies to identify 109895 neoplasias. These data were normalized to describe both the tumour (type and location) and the animal (breed, neutering status and veterinary practice postcode). The resulting PTR, the largest of its kind for companion animals to date, is an important research resource being able to facilitate a wide array of research in areas including surveillance, clinical decision making and comparative cancer biology.

My week: Francesco Cian.

Francesco Cian is a veterinary clinical pathologist and co-manager at BattLab, a veterinary diagnostic laboratory based in Coventry.

A case of giant cell tumor of soft parts in a horse.

A 12-year-old British Warmblood mare was examined by the referring veterinarian for evaluation of a cutaneous lesion on the dorsal thorax to the right of the midline. Cytologic examination of fine-needle aspirates from the mass was supportive of a giant cell tumor of soft parts (GCTSP). Laser surgical excision and postoperative methyl aminolevulinate (MAL) photodynamic therapy (PDT) were performed. Histologic examination of the mass confirmed the cytologic diagnosis. At 8 months from surgery, no evidence of recurrence has been observed. Giant cell tumors of soft parts are rare cutaneous neoplasms, observed in several domestic species, including the horse where they commonly appear as superficial cutaneous lesions without aggressive biologic behavior. Previously classified as giant cell variant of malignant fibrous histiocytoma, these superficial tumors have now been designated as giant cell tumors of soft tissue or giant cell tumors of low malignant potential within the category of fibrohistiocytic neoplasms.

Gamma delta T-cell large granular lymphocyte lymphoma in a dog.

A 2-year and 6-month-old female neutered Labrador Retriever with Horner syndrome, megaesophagus, and a mediastinal mass was referred to the Queen Mother Hospital for Animals of the Royal Veterinary College. A large granular lymphocyte (LGL) lymphoma was diagnosed on cytology; flow cytometric analysis revealed a γδ T-cell phenotype (CD3+, CD5+, CD45+, TCRγδ+, CD4-, CD8-, CD34-, CD21-). Chemotherapy was started with a combination of lomustine, vincristine, procarbazine, and prednisolone, followed by bleyomicin. Euthanasia was elected by the owners, due to progressive deterioration and lack of quality of life, 28 days after diagnosis. This is the first cytologic and immunophenotypic characterization of a canine γδ T-cell lymphoma with LGL morphology and probably of mediastinal origin. The role of chemotherapy in delaying the disease progression remains unknown.

Use of Six Sigma Worksheets for assessment of internal and external failure costs associated with candidate quality control rules for an ADVIA 120 hematology analyzer.

BACKGROUND: Quality control (QC) validation is an essential tool in total quality management of a veterinary clinical pathology laboratory. Cost-analysis can be a valuable technique to help identify an appropriate QC procedure for the laboratory, although this has never been reported in veterinary medicine. OBJECTIVE: The aim of this study was to determine the applicability of the Six Sigma Quality Cost Worksheets in the evaluation of possible candidate QC rules identified by QC validation. METHODS: Three months of internal QC records were analyzed. EZ Rules 3 software was used to evaluate candidate QC procedures, and the costs associated with the application of different QC rules were calculated using the Six Sigma Quality Cost Worksheets. The costs associated with the current and the candidate QC rules were compared, and the amount of cost savings was calculated. RESULTS: There was a significant saving when the candidate 1-2.5s, n = 3 rule was applied instead of the currently utilized 1-2s, n = 3 rule. The savings were 75% per year (£ 8232.5) based on re-evaluating all of the patient samples in addition to the controls, and 72% per year (£ 822.4) based on re-analyzing only the control materials. The savings were also shown to change accordingly with the number of samples analyzed and with the number of daily QC procedures performed. CONCLUSIONS: These calculations demonstrated the importance of the selection of an appropriate QC procedure, and the usefulness of the Six Sigma Costs Worksheet in determining the most cost-effective rule(s) when several candidate rules are identified by QC validation.

A novel point mutation in the ß1-tubulin gene in asymptomatic macrothrombocytopenic Norfolk and Cairn Terriers.

BACKGROUND: An asymptomatic macrothrombocytopenia, phenotypically similar to asymptomatic inherited macrothrombocytopenia in Cavalier King Charles Spaniels, was described in a group of Norfolk Terriers (NT) from Northern Italy, and isolated cases were also reported in Cairn Terriers (CT). OBJECTIVES: The purpose of this work was to evaluate for the presence of a genetic defect in the ß1-tubulin gene in macrothrombocytopenic NT and CT. METHODS: Samples from 20 healthy dogs (13 NT and 7 CT) were collected at different institutions in Italy (n = 8), United Kingdom (n = 3), and United States (n = 9). Genomic DNA was harvested from EDTA-anticoagulated blood and all coding areas and exon-intron splice sites in the gene encoding ß1-tubulin were amplified and sequenced. RESULTS: Twelve dogs (9 NT and 3 CT) showed a single nucleotide polymorphism (SNP) in exon 1 at nucleotide position 5 (G5A) that would result in the change of an arginine to a histidine at amino acid position 2 (R2H). Four dogs (3 NT and one Cairn Terrier) were heterozygous for the SNP, and 4 dogs (one Norfolk Terrier and 3 CT) matched the normal canine genome. Homozygous dogs for the SNP were macrothrombocytopenic with platelet counts ranging from 19,000 to 110,000/µL. Heterozygous and normal dogs had normal platelet counts and morphology. None had the CKCS point mutation. CONCLUSIONS: The ß1-tubulin N-terminal amino acids form the nucleotide-binding domain and thus this mutation could affect GTP binding enough to influence platelet formation in homozygous but not in heterozygous dogs. The presence of macrothrombocytopenia only in homozygous affected dogs reveals an association between the SNP and the phenotype.

Stability of immunophenotypic lymphoid markers in fixed canine peripheral blood for flow cytometric analysis.

BACKGROUND: Flow cytometric analysis of blood samples for immunophenotyping lymphoproliferative diseases has become popular in veterinary medicine. Unfortunately, the use of this technique has been limited by the necessity to test samples within a short time frame after blood collection. A possible solution to this problem is the use of fixative products to preserve the stability of lymphoid antigens. OBJECTIVES: The aim of this study was to evaluate the expression of 5 lymphoid surface markers (CD3, CD4, CD8, CD21, and CD45) in blood samples collected in K3-EDTA and Cyto-Chex BCT tubes from healthy dogs. METHODS: Blood from 8 dogs was collected in K3-EDTA and Cyto-Chex BCT tubes and analyzed by flow cytometry at 6 hours, one day, 3 days, and 7 days after collection. Lymphocyte percentage, lymphocyte mor-phology, expression of lymphoid surface markers and mean fluorescence intensity (MFI) were recorded at each time point and compared. RESULTS: Lymphocyte percentage and morphology were preserved up to 3 days in samples collected in Cyto-Chex BCT, and lymphocyte percentage was mildly decreased on day 7. CD4, CD8, and CD21 were stable in Cyto-Chex BCT up to 7 days, whereas CD3 and CD45 showed a significant decrease in expression from day 3, with a decrease on average of 21% and 2.4%, respectively, on day 7. MFI was significantly decreased on day 7 for all markers except CD21. CONCLUSIONS: These findings indicate that storage of samples in Cyto-Chex BCT affects lymphoid marker expression and caution should be exercised when interpreting data produced on such samples.

Immunocytochemical demonstration of feline infectious peritonitis virus within cerebrospinal fluid macrophages.

A 4-month-old female entire domestic shorthair cat presented with an acute onset of blindness, tetraparesis and subsequent generalised seizure activity. Haematology and serum biochemistry demonstrated a moderate, poorly regenerative anaemia, hypoalbuminaemia and hyperglobulinaemia with a low albumin:globulin ratio. Serology for feline coronavirus antibody was positive with an elevated alpha-1 acid glycoprotein. Analysis of cisternal cerebrospinal fluid (CSF) demonstrated markedly elevated protein and a mixed, predominately neutrophilic pleocytosis. Immunocytochemistry for feline coronavirus was performed on the CSF, with positive staining observed inside macrophages. The cat was subsequently euthanased, and both histopathology and immunohistochemistry were consistent with a diagnosis of feline infectious peritonitis. This is the first reported use of immunocytochemistry for detection of feline coronavirus within CSF macrophages. If this test proves highly specific, as for identification of feline coronavirus within tissue or effusion macrophages, it would be strongly supportive of an ante-mortem diagnosis of feline infectious peritonitis in cats with central nervous system involvement without the need for biopsy.