International Guidelines for Veterinary Tumor Pathology: A Call to Action.

Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.

Mitotic Figures-Normal, Atypical, and Imposters: A Guide to Identification.

Counting mitotic figures (MF) in hematoxylin and eosin-stained histologic sections is an integral part of the diagnostic pathologist's tumor evaluation. The mitotic count (MC) is used alone or as part of a grading scheme for assessment of prognosis and clinical decisions. Determining MCs is subjective, somewhat laborious, and has interobserver variation. Proposals for standardizing this parameter in the veterinary field are limited to terminology (use of the term MC) and area (MC is counted in an area measuring 2.37 mm2). Digital imaging techniques are now commonplace and widely used among veterinary pathologists, and field of view area can be easily calculated with digital imaging software. In addition to standardizing the methods of counting MF, the morphologic characteristics of MF and distinguishing atypical mitotic figures (AMF) versus mitotic-like figures (MLF) need to be defined. This article provides morphologic criteria for MF identification and for distinguishing normal phases of MF from AMF and MLF. Pertinent features of digital microscopy and application of computational pathology (CPATH) methods are discussed. Correct identification of MF will improve MC consistency, reproducibility, and accuracy obtained from manual (glass slide or whole-slide imaging) and CPATH approaches.

Hematologic, prostaglandin F2α -metabolite, serum amyloid A, and serum iron changes in horses with experimentally induced endotoxemia.

BACKGROUND: Endotoxemia is a common and severe disease of horses. Most previous studies have monitored changes caused by a bolus dose of endotoxin over short time periods. OBJECTIVES: We aimed to describe inflammatory responses to endotoxin with inflammatory and hematologic markers monitored over a longer time than has been performed in the past using more prolonged endotoxin exposures. METHODS: Escherichia coli O55:B5 endotoxin was administered as a 6-hour continuous intravenous infusion of lipopolysaccharide (LPS) to eight horses. Blood cell counts, and prostaglandin F2α -metabolite (PGM), serum amyloid A (SAA), and serum total iron concentrations were monitored for up to 3 or 6 days. RESULTS: An immediate and severe decrease in neutrophils and monocytes occurred in all horses, which subsequently changed to a moderate to strong neutrophilia and monocytosis that persisted for more than 78 hours postinfusion (PI) of LPS. Lymphocyte and eosinophil numbers decreased gradually and then normalized after 66- and 78-hours PI, respectively. Mild to moderate, biphasic thrombocytopenia occurred. A pronounced, transient increase in PGM occurred between 1 and 7 hours, peaking at 2 hours. Serum amyloid A began to increase after 6 hours PI and remained elevated after 72 hours PI. Serum iron was decreased between 6 and 48 hours. The clinical signs were most prominent during the first 24 hours PI and subsided within 48 hours PI. CONCLUSIONS: Neutrophilia, monocytoses, and high SAA concentrations were present in horses even after the clinical signs had subsided. Serum iron normalized before SAA. Knowledge of these findings is imperative when interpreting laboratory results in horses with possible endotoxin exposure.

Hepatozoon canis infection causing a strong monocytosis with intra-monocytic gamonts and leading to erroneous leukocyte determinations.

In this case report, a Swedish flat-coated retriever was diagnosed with an extensive Hepatozoon canis infection. The dog had a prominent monocytosis (14.0 × 109 /L) with H canis gamonts detected in most monocytes, but none were found in the neutrophils. On the hematology system ADVIA 2120 peroxidase (PEROX) cytogram, most leukocytes were seen as a distinct cell population above the lymphocytes, which indicated that most of the cells were larger than lymphocytes and had weak myeloperoxidase staining. This distinct cell cluster appeared to be of a single cell type but was incorrectly divided by the ADVIA 2120 into lymphocytes, monocytes, and large unstained cells (LUC). The total leukocyte counts on the ADVIA 2120 WBC basophil (BASO) channel were much higher than that on the WBC PEROX count. The WBC BASO cytogram appeared abnormal with two parallel cell populations, so the BASO WBC count was considered erroneous. Polymerase chain reaction and DNA sequencing verified H canis infection. The dog was treated with subcutaneous imidocarb dipropionate (6 mg/kg) injections every other week. Post-treatment hematology analyses indicated that the percentage of parasitized leukocytes decreased from 40% to 5% about 4 weeks after the start of treatment and were not found in any monocytes 6 weeks after the beginning of the treatment. In conclusion, H canis infection in this dog was associated with a strong monocytosis, and gamonts were present in many monocytes, which caused aberrant automated leukocyte counts to occur.

Massive uric acid crystalluria and cylinduria in a dog after l-asparaginase treatment for lymphoma.

A 10-year-old golden retriever bitch was treated for diarrhea and vomiting that lasted about 1 month without a specific diagnosis until a hepatic biopsy provided a histopathologic diagnosis of lymphoma. The dog was referred to the Swedish University of Agricultural Science and treated with one dose of l-asparaginase. The day after chemotherapy, the urine was dark yellow, very turbid, and had large amounts of small amorphous crystals and many casts made of similar appearing material identified by infrared spectroscopy to be 100% uric acid dihydrate. Serum uric acid was elevated at 224 µmol/L (RI 0-59). The dog's illness became worse after chemotherapy. Lymphoma treatment was not continued, and the dog was euthanized 9 days after the l-asparaginase treatment. Among other problems were persistent proteinuria with a urine protein-to-creatinine ratio of 2.3 and severe hypoalbuminemia. Serum protein electrophoresis performed 3 weeks prior to chemotherapy indicated hyperproteinemia (total protein 78 g/L) having a biclonal gammopathy with 35 g/L ß-2 globulins and 11 g/L γ globulins. Despite prominent cylinduria and crystalluria, the patient did not develop azotemia or isosthenuria.

Measurement of serum C-reactive protein concentration for discriminating between suppurative arthritis and osteoarthritis in dogs.

BACKGROUND: In a dog with joint pain, it is important to determine whether it has suppurative joint disease, characterized by exudation of neutrophils in the synovial fluid, or not, as this affects choice of diagnostic tests and treatments. The aim of this study was to evaluate whether measurement of serum C-reactive protein (CRP) concentration could be used to discriminate between dogs with suppurative arthritis and osteoarthritis (OA). Furthermore, the concentrations of serum and synovial fluid interleukin (IL) 6 concentrations were measured in dogs with joint disease and in healthy dogs, and were correlated to serum CRP concentrations. METHODS: Dogs with joint pain were enrolled prospectively and were classified to have suppurative arthritis or OA based on synovial fluid analysis and radiographic/arthroscopic findings. Healthy Beagles were enrolled as a comparative group. CRP and IL-6 concentrations were measured with canine-specific immunoassays. The performance of CRP concentration in discriminating between dogs with suppurative arthritis and OA was evaluated using a previously established clinical decision limit for CRP (20 mg/l), and by receiver operator characteristic (ROC) curve and logistic regression analysis. Comparisons of CRP and IL-6 concentrations between groups were performed using t-tests, and correlations by Spearman rank correlation coefficients. RESULTS: Samples were obtained from 31 dogs with suppurative arthritis, 34 dogs with OA, and 17 healthy dogs. Sixty-two out of 65 dogs with joint disease were correctly classified using the clinical decision limit for CRP. Evaluation of ROC curve and regression analysis indicated that serum CRP concentrations could discriminate between suppurative arthritis and OA. Dogs with suppurative arthritis had higher serum CRP and serum and synovial fluid IL-6 concentrations compared to dogs with OA (p < 0.001). Dogs with OA had higher synovial fluid IL-6 concentrations (p < 0.001), but not higher serum CRP (p = 0.29) or serum IL-6 (p = 0.07) concentrations, compared to healthy dogs. There was a positive correlation between synovial fluid IL-6 and serum CRP concentrations (rs = 0.733, p < 0.001), and between serum IL-6 and serum CRP concentrations (rs = 0.729, p < 0.001). CONCLUSION: CRP concentration was found to discriminate well between dogs with suppurative arthritis and OA.

Time-related changes in equine neutrophils after experimental endotoxemia: myeloperoxidase staining, size, and numbers.

BACKGROUND: Neutrophil myeloperoxidase content is determined by the Advia 2120 hematology system by staining characteristics. Changes in myeloperoxidase staining are shown by location of neutrophils on Advia peroxidase dot plots and as myeloperoxidase index (MPXI). Significant changes in MPXI have been reported during severe inflammation in horses, dogs, and people but conclusions were inconsistent. OBJECTIVES: Infusion of endotoxin was used to initiate an inflammatory stimulus under controlled conditions and over a longer time period than in previous studies to document kinetics of changes in neutrophil numbers, morphology, and myeloperoxidase staining. Identification of consistent time-related changes may allow better interpretation of changes in neutrophil characteristics during inflammation. MATERIALS: Five Standardbred trotting horses received an intravenous infusion over a 6-hour period with Escherichia coli endotoxin. Neutrophil count, MPXI, neutrophil characteristics in Advia 2120 Perox dot plots and neutrophil morphology in blood smears were monitored with repeated sampling for up to 10 days. RESULTS: Endotoxin infusion immediately caused severe neutropenia which converted to neutrophilia 14 hours after start of endotoxin infusion. Neutrophilia was still present 78 hours after start of infusion. Large "giant" neutrophils first appeared in blood smears and Advia Perox dot plots after 36-48 hours. A marked and consistent decrease in MPXI was seen in all horses 6 days (150 hours) after endotoxin exposure. CONCLUSIONS: Endotoxemia caused prominent, time-related changes in equine neutrophil characteristics including emergence of giant neutrophils and markedly decreased MPXI several days after endotoxin infusion.

Reducing error in feline platelet enumeration by addition of Iloprost to blood specimens: comparison to prostaglandin E1 and EDTA.

BACKGROUND: Prostaglandin E1 (PGE1) and Iloprost inhibit platelet aggregation and should prevent or minimize preanalytic error with feline platelet enumeration. OBJECTIVES: The objective was to compare the relative effectiveness in reducing errors in platelet enumeration by adding Iloprost to feline EDTA blood specimens in comparison to adding PGE1 or EDTA alone. In addition, a grading system for platelet aggregation in blood smears was evaluated for effectiveness in predicting prominent errors and compared to ADVIA's PLT-CLM flag. Finally, the use of plateletcrit in feline blood with platelet aggregation was evaluated. METHODS: Blood specimens from 35 cats were included. Blood was collected into EDTA tubes with or without Iloprost or PGE1, and was rapidly mixed. Platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), and platelet flags were determined with an ADVIA 2120. Manual PLT was performed with a Leucoplate stain. PLT was determined by an IDEXX VetAutoread hematology analyzer (QBC). RESULTS: Neither addition of Iloprost nor PGE1 to EDTA blood specimens completely prevented platelet aggregation. Iloprost-treated specimens had the least severe aggregation. PGE1 was better than EDTA alone. Significant errors in PLT results were consistently identified by the grading system. ADVIA's PLT-CL flag usually predicted significant errors in PLT. QBC PLT results showed high imprecision. Manual PLT error was smaller than ADVIA PLT in EDTA specimens with aggregation. CONCLUSIONS: Adding Iloprost to feline blood specimens improved platelet enumeration accuracy. A grading system for severity of platelet aggregation and usually the ADVIA's PLT-CL alarm predicted specimens with significant errors in platelet enumeration.

Validation of a commercially available automated canine-specific immunoturbidimetric method for measuring canine C-reactive protein.

BACKGROUND: Measurement of C-reactive protein (CRP) is used for diagnosing and monitoring systemic inflammatory disease in canine patients. An automated human immunoturbidimetric assay has been validated for measuring canine CRP, but cross-reactivity with canine CRP is unpredictable. OBJECTIVE: The purpose of the study was to validate a new automated canine-specific immunoturbidimetric CRP method (Gentian cCRP). METHODS: Studies of imprecision, accuracy, prozone effect, interference, limit of quantification, and stability under different storage conditions were performed. The new method was compared with a human CRP assay previously validated for canine CRP determination. Samples from 40 healthy dogs were analyzed to establish a reference interval. RESULTS: Total imprecision was < 2.4% for 4 tested serum pools analyzed twice daily over 10 days. The method was linear under dilution, and no prozone effect was detected at a concentration of 1200 mg/L. Recovery after spiking serum with purified canine CRP at 2 different concentrations was 123% and 116%, respectively. No interference from hemoglobin or triglycerides (10 g/L) was detected. CRP was stable for 14 days at 4°C and 22°C. In the method comparison study, there was good agreement between the validated human CRP assay and the new canine-specific assay. Healthy dogs had CRP concentrations that were less than the limit of quantification of the Gentian cCRP method (6.8 mg/L). CONCLUSIONS: The new canine-specific immunoturbidimetric CRP assay is a reliable and rapid method for measuring canine CRP, suitable for clinical use due to the option for an automated assay.

Comparison of a Schmidt and Haensch refractometer and an Atago PAL-USG Cat refractometer for determination of urine specific gravity in dogs and cats.

BACKGROUND: The performance of a digital Atago PAL-USG Cat refractometer (Atago) was compared with a Schmidt and Haensch, Goldberg type refractometer (S+H). MATERIALS AND METHODS: Specific gravity of 47 canine and feline urine samples was determined with both refractometers and the results were compared with Passing-Bablok and Bland-Altman plots. In addition, the specific gravity of dilutions of 10% glucose, 10% NaCl, and 3% albumin solutions was determined and compared with expected values. RESULTS: Both refractometers consistently reported 1.000 with distilled water. The correlation between both refractometers based on Passing-Bablok plots of 47 urine samples was excellent (r = .99), but, in the Bland-Altman plots, there was a significant, proportional, negative error for the Atago readouts. This was also illustrated by the fact that 10 urine samples with an S+H result of > 1.030 were read out between 1.023 and 1.028 by Atago. Schmidt and Haensch results of various glucose solutions matched exactly expected values, but Atago results were lower. Likewise, S+H results with diluted NaCl solutions were closer to expected results than Atago results. In contrast, Atago results with dilutions of 3% albumin were closer to expected results than S+H results. DISCUSSION: The Atago refractometer reported lower specific gravity results in urine and standard solutions of glucose and NaCl, which could adversely affect clinical decisions concerning normal renal function based on solute concentrations determined in canine and feline urine samples.

Theileria annae in a young Swedish dog.

A severe regenerative anemia was detected in a 12-week-old mixed breed puppy in Sweden. A small protozoan parasite was observed in erythrocytes on a blood smear. It was initially suspected to be Babesia gibsoni based on its size and because B. gibsoni was previously recorded in Sweden. Surprisingly, specific polymerase chain reaction analysis identified the protozoan as Theileria annae. T. annae is endemic in Northwest Spain, is very uncommonly reported elsewhere and has never been recorded in Scandinavia. T. annae has been identified in dogs used for dog fighting, and it is thought to be transmitted by dog bites. This puppy was a mixed pit bull terrier. Pit bull terriers are sometimes used for dog fighting. T. annae has been reported to be transmitted vertically, and in light of the puppy's age, this transmission was suspected in the present case.

Enumeration of feline platelets in ethylenediamine tetra-acetic acid anticoagulated blood with the ADVIA 2120 system and two manual methods: Leucoplate and Thrombo-TIC.

A manual method (Thrombo-TIC; Bioanalytic GmbH, Umkirch/Freiburg, Germany) was advertised to disaggregate platelet clumps and to make human platelets spherical to improve platelet enumeration. The current study's hypothesis was that this method would perform better than current methods for feline blood anticoagulated with ethylenediamine tetra-acetic acid (EDTA), which often contains platelet aggregates. Platelet concentrations (PLTs) were determined in 21 feline blood samples by 3 methods. Thrombo-TIC was compared to the manual method (Leucoplate; Sobioda, Montbonnot-Saint-Martin, France) currently used in the authors' laboratory along with an ADVIA 2120 (Siemens AG, Eschborn, Germany) optical platelet concentration. Feline blood samples often contained platelet aggregates. ADVIA flagged for platelet aggregates in 11 of the 21 feline blood samples, and examination of blood smear revealed platelet aggregates in 14 of the 21 samples. The hemocytometers displayed more platelet aggregates with the Thrombo-TIC method than with Leucoplate. The method giving the greatest PLT was considered most accurate. The Leucoplate median PLT (238 × 10(9)/1) was greater than Thrombo-TIC (202 × 10(9)/1) or ADVIA (157 × 10(9)/1). Intra-assay precision was determined for the 3 methods using the 21 feline blood samples. Median Thrombo-TIC and Leucoplate precision (7.4% and 7.3% coefficient of variation [CV], respectively) were similar and not much worse than ADVIA (5.9% CV). The Thrombo-TIC method did not appear to perform better than the current manual method (Leucoplate). Leucoplate appeared least affected by platelet aggregation in feline blood. The ADVIA automated PLT appeared to be most negatively affected by platelet aggregation. The Thrombo-TIC method did not appear to prevent platelet aggregation in feline EDTA blood samples and, thus, is not recommended for such use.

Cytologic appearance of retinal cells included in a fine-needle aspirate of a meningioma around the optic nerve of a dog.

A 6-year-old Wirehair Dachshund had a meningioma around the optic nerve that caused exophthalmos. A benign mesenchymal tumor was suspected based on the cytologic pattern of a fine-needle aspirate, and a meningioma was diagnosed by histopathologic examination. In addition to the meningioma cells, the cytologic smears included groups of cells from apparently 4 layers of normal retina. In particular, uniform rod-shaped structures in the cytologic sample could suggest rod-shaped bacteria, but these structures were identified as cylindrical outer segments of photoreceptor rod cells. Other retinal structures recognized included pigmented epithelial layer cells with their uniquely formed pigment granules, the characteristic bi-lobed, cleaved nuclei from the outer nuclear layer, and nerve tissue likely from the outer plexiform layer of the retina.

Validation of Advia plateletcrit for assessing platelet mass in dogs, including Cavalier King Charles spaniels.

BACKGROUND: Determination of the plateletcrit (PCT) is the most effective way to evaluate platelet mass in dogs, such as Cavalier King Charles spaniel (CKCS) dogs, with macrothrombocytopenia. The IDEXX VetAutoread hematology analyzer, which performs quantitative buffy coat (QBC) analysis, has been validated to determine platelet mass in CKCS dogs. The Advia 2120 reports a PCT, but the validity of this value has not been evaluated for dogs with macrothrombocytopenia. OBJECTIVES: The goal of this study was to validate MPV and PCT determined by the Advia 2120 in dogs, including CKCS dogs, comparing values with those obtained from QBC analysis. METHODS: Advia PCT was compared with QBC results from 43 CKCS dogs and 15 dogs of other breeds in one study. Advia PCT, platelet count, and MPV were evaluated to identify biologic patterns in 31 clinically healthy CKCS dogs and 66 dogs of 3 other breeds and to generate values used for comparisons. RESULTS: Advia PCT agreed well with QBC results in general, but had a negative bias and appeared to underestimate PCT in CKCS dogs with the lowest PCTs. Advia PCT and MPV results followed expected biologic patterns in CKCS dogs and dogs of other breeds with MPVs being highest in dogs with the lowest platelet counts. CONCLUSIONS: Advia 2120 PCT and MPV satisfactorily identified changes in platelet mass and size in CKCS dogs, but PCTs were lower than expected, especially in CKCS dogs with the lowest PCTs, when compared with QBC results.