Measurement of Feline Alpha-1 Acid Glycoprotein in Serum and Effusion Using an ELISA Method: Analytical Validation and Diagnostic Role for Feline Infectious Peritonitis.

BACKGROUND: Alpha-1 acid glycoprotein (AGP) may support a clinical diagnosis of feline infectious peritonitis (FIP). In this study, we assessed the analytical and diagnostic performances of a novel ELISA method to measure feline AGP. METHODS: AGP was measured in sera and effusions from cats with FIP (n = 20) or with other diseases (n = 15). Precision was calculated based on the coefficient of variation (CV) of repeated testing, and accuracy was calculated by linearity under dilution (LUD). RESULTS: The test is precise (intra-assay CVs: <6.0% in individual samples, <15.0% in pooled samples; inter-assay CVs <11.0% and <15.0%) and accurate (serum LUD r2: 0.995; effusion LUD r2: 0.950) in serum and in effusions. AGP is higher in cats with FIP than in other cats in both serum (median: 1968, I-III interquartile range: 1216-3371 µg/mL and 296, 246-1963 µg/mL; p = 0.009) and effusion (1717, 1011-2379 µg/mL and 233, 165-566 µg/mL; p < 0.001). AGP discriminates FIP from other diseases (area under the receiver operating characteristic curve: serum, 0.760; effusion, 0.877), and its likelihood ratio is high (serum: 8.50 if AGP > 1590 µg/mL; effusion: 3.75 if AGP > 3780 µg/mL). CONCLUSION: This ELISA method is precise and accurate. AGP in serum and in effusions is a useful diagnostic marker for FIP.

Subcutaneous panniculitis-like T-cell lymphoma: Morphological, immunophenotypical and clonality assessment in six cats.

BACKGROUND: Primary cutaneous lymphoma represents 0.2%-3% of all feline lymphomas, with nonepitheliotropic lymphomas being the most common. In humans and dogs, subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a primary nonepitheliotropic lymphoma with a T-cell phenotype developing in the subcutis and often mimicking inflammation. OBJECTIVE: The aim of this report is to describe pathological, phenotypical and clonal features of SPTCL in cats. ANIMALS: Six cats with SPTCL were included in this study. MATERIALS AND METHODS: Skin biopsies were formalin-fixed, routinely processed and stained. Histological and immunohistochemical investigation for anti-CD18, CD204, CD79a, CD20, CD3, FeLVp27and FeLVgp70 and clonality assessment were performed. RESULTS: Four male and two female domestic shorthair cats, mean age 11.2 years, developed SPTCL in the abdominal (three), inguinal (two) and thoracic (one) regions. Variably pleomorphic neoplastic lymphoid cells were present in the panniculus in percentages, expanding the septa (six of six) and extending into fat lobules in one of six cats. Tumours were associated with elevated numbers of neutrophils (five of six), lesser macrophages (six of six) and variable necrosis (six of six). Neoplastic cells expressed CD3+ (six of six), with clonal T-cell receptor rearrangement detected in five of six cats. CONCLUSIONS AND CLINICAL RELEVANCE: This is the first description of SPTCL in cats. Lesions can be confused with panniculitis, leading to delay in diagnosis and therapy. Awareness of this neoplastic disease is relevant to avoid misdiagnoses and to gain greater knowledge about the disease in cats.

Subcutaneous mast cell tumours: A prospective multi-institutional clinicopathological and prognostic study of 43 dogs.

BACKGROUND: Canine subcutaneous mast cell tumours (ScMCTs) reportedly have a good prognosis. However, biomarkers that can be used to predict outcome are currently limited. METHODS: A multicentre prospective study was conducted to identify new prognostic markers. Dogs with a first occurrence of ScMCT were enrolled upon primary tumour removal and regional lymphadenectomy. In the absence of metastasis, dogs were monitored, while dogs with overtly metastatic lymph nodes (histological node 3, HN3) received adjuvant vinblastine. RESULTS: Forty-three dogs were enrolled: 15 (34.9%) had at least one HN3 lymph node and received vinblastine, and 28 (65.1%) were monitored. Three tumours harboured exon 8 and 9 c-kit mutations. Eight (18.6%) dogs experienced tumour progression, and five (11.6%) died of MCT-related causes. The 1- and 2-year survival rates were 90% and 77%, respectively. Variables significantly associated with an increased risk of progression included high cytograde, a mitotic count (MC) greater than 4/10 high-power fields (hpf) and Ki67-index greater than 23. An MC greater than 4/10 hpf was also associated with an increased risk of tumour-related death. LIMITATIONS: Regional rather than sentinel lymphadenectomy was performed in these dogs. Dogs were enrolled in oncology referral centres, constituting a different population compared to previous studies. CONCLUSIONS: ScMCTs have a good prognosis. However, the metastatic rate at admission was higher in this study than previously reported, and a subset of tumours were associated with a fatal outcome despite multimodal treatment. Proliferative activity and cytograding may predict more aggressive behaviour in ScMCTs.

Pre-analytical and analytical variability of reticulocyte counts in dogs.

BACKGROUND: Automated fluorescence-based haematology analysers are now available for reticulocyte enumeration in veterinary medicine, but manual counting is still largely used. This study aimed to evaluate potential sources of analytical and pre-analytical errors when performing automated and manual counts. METHODS: Automated and two-operator double-blind manual reticulocyte counts were performed on 15 blood samples. The intra-assay variation of the automated and manual counts and the interoperator variation in the manual counts were then calculated. In addition, the effects of storage were evaluated using samples refrigerated at 4°C or stored at room temperature for 2, 4, 12, 24, 48 or 72 hours after sampling. RESULTS: Intra-assay coefficients of variation were lower for automated counts than for manual counts. Comparison between automated and mean total manual reticulocyte count showed no significant differences. In both refrigerated samples and those stored at room temperature, an increase in reticulocyte count was recorded only after 72 hours. Staining artefacts occurred only in one stored sample counted manually. LIMITATIONS: The presence of cytoplasmic particles other than RNA can cause misinterpretation of cells, leading to an erroneous reticulocyte count. CONCLUSION: The use of an automated analyser is preferable for reticulocyte enumeration in dogs. Common storage conditions seem to minimally affect reticulocyte evaluation; however, it is recommended to perform the analysis as soon as possible after sampling.

Evaluation of urinary neutrophil gelatinase-associated lipocalin to detect renal tubular damage in dogs with stable myxomatous mitral valve disease.

BACKGROUND: Dogs with myxomatous mitral valve disease (MMVD) can experience progressive renal tubular damage and dysfunction. The prevalence of renal tubular damage is not known in dogs with stable MMVD. OBJECTIVE: To evaluate renal tubular damage in dogs with stable MMVD by evaluation of urinary neutrophil gelatinase-associated lipocalin (NGAL). ANIMALS: Ninety-eight MMVD dogs grouped according to the American College of Veterinary Internal Medicine (ACVIM) staging (group B1, n = 23; group B2, n = 27; group C + D, n = 48) and 46 healthy dogs. METHODS: Multicenter prospective observational study. Serum and urine chemistry including NGAL reported as uNGAL concentration (uNGAL) and normalized with urinary creatinine (uNGALC) were compared between MMVD dogs and healthy controls, and among different MMVD ACVIM stages. RESULTS: The MMVD dogs had significantly higher uNGAL and uNGALC (1204 pg/mL; range, 30-39 732 and 1816 pg/mg; range, 22-127 693, respectively) compared to healthy dogs (584 pg/mL; range, 56-4072 and 231 pg/mg; range, 15-2407, respectively; P = .002 and P < .0001, respectively). Both uNGAL and uNGALC increased with the increasing ACVIM stage (P = .001 and P < .001, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Renal tubular damage is present in dogs with stable MMVD, as measured by increased uNGAL. This tubular damage is subclinical, occurs in all stages of MMVD even in the absence of azotemia, and increases with the severity of MMVD. Reno-protective approaches to manage MMVD dogs should be explored to slow the progression of renal tubular damage in these patients.

Utility of the Ratio between Lactate Dehydrogenase (LDH) Activity and Total Nucleated Cell Counts in Effusions (LDH/TNCC Ratio) for the Diagnosis of Feline Infectious Peritonitis (FIP).

BACKGROUND: We tested the hypothesis that the ratio between lactate dehydrogenase activity (LDH) and total nucleated cell counts (TNCC) in effusions may be useful to diagnose feline infectious peritonitis (FIP). METHODS: LDH/TNCC ratio was retrospectively evaluated in 648 effusions grouped based on cytology and physicochemical analysis (step 1), on the probability of FIP estimated by additional tests on fluids (step 2) or on other biological samples (step 3, n = 471). Results of different steps were statistically compared. Receiver Operating Characteristic (ROC) curves were designed to assess whether the ratio identify the samples with FIP "probable/almost confirmed". The cut-offs with the highest positive likelihood ratio (LR+) or Youden Index (YI) or with equal sensitivity and specificity were determined. RESULTS: A high median LDH/TNCC ratio was found in FIP effusions (step1: 2.01) and with probable or almost confirmed FIP (step 2: 1.99; 2.20 respectively; step 3: 1.26; 2.30 respectively). The optimal cut-offs were 7.54 (LR+ 6.58), 0.62 (IY 0.67, sensitivity: 89.1%; specificity 77.7%), 0.72 (sensitivity and specificity: 79.2%) in step 2 and 2.27 (LR+ 10.39), 0.62 (IY 0.65, sensitivity: 82.1%; specificity 83.0%), 0.54 (sensitivity: 82.1%; specificity 81.9%) in step 3. CONCLUSIONS: a high LDH/TNCC ratio support a FIP diagnosis.

Erythrophagocytic multiple myeloma in a dog.

A 5-y-old spayed female Golden Retriever dog was referred because of severe normocytic normochromic nonregenerative anemia and thrombocytopenia. Serum analysis revealed hyperproteinemia and monoclonal or oligoclonal gammopathy. Fine-needle aspiration of the spleen revealed a highly erythrophagocytic population of neoplastic round cells, morphologically suggestive of plasma cells. After euthanasia, histologic assessment of the spleen and liver revealed an erythrophagocytic round cell tumor. Immunohistochemical analysis of the tumor population was positive for MUM1p and negative for CD3, CD20, and Iba-1, confirming the plasma cell origin of the tumor. Erythrophagocytic multiple myeloma is a very rare neoplastic condition in dogs.

Urinary cortisol-creatinine ratio in dogs with hypoadrenocorticism.

BACKGROUND: Basal serum cortisol (BSC) ≥2 µg/dL (>55 nmol/L) has high sensitivity but low specificity for hypoadrenocorticism (HA). OBJECTIVE: To determine whether the urinary corticoid:creatinine ratio (UCCR) can be used to differentiate dogs with HA from healthy dogs and those with diseases mimicking HA (DMHA). ANIMALS: Nineteen healthy dogs, 18 dogs with DMHA, and 10 dogs with HA. METHODS: Retrospective study. The UCCR was determined on urine samples from healthy dogs, dogs with DMHA, and dogs with HA. The diagnostic performance of the UCCR was assessed based on receiver operating characteristics (ROC) curves, calculating the area under the ROC curve. RESULTS: The UCCR was significantly lower in dogs with HA (0.65 × 10-6 ; range, 0.33-1.22 × 10-6 ) as compared to healthy dogs (3.38 × 10-6 ; range, 1.11-17.32 × 10-6 ) and those with DMHA (10.28 × 10-6 ; range, 2.46-78.65 × 10-6 ) (P < .0001). There was no overlap between dogs with HA and dogs with DMHA. In contrast, 1 healthy dog had a UCCR value in the range of dogs with HA. The area under the ROC curve was 0.99. A UCCR cut-off value of <1.4 yielded 100% sensitivity and 97.3% specificity in diagnosing HA. CONCLUSIONS AND CLINICAL IMPORTANCE: The UCCR seems to be a valuable and reliable screening test for HA in dogs. The greatest advantage of this test is the need for only a single urine sample.

Detection of a characteristic beta-2 peak in serum by capillary zone electrophoresis in dogs with Angiostrongylus vasorum.

BACKGROUND: Angiostrongylosis is an emerging canine parasitic disease that often causes polyclonal hyperglobulinemia. In the authors' experience, we have seen what could be a typical serum protein electrophoretic pattern characterized by a large, symmetrical beta-2 peak in dogs with angiostrongylosis. OBJECTIVES: The purpose of this study was to evaluate the frequency of this pattern in dogs infected with Angiostrongylus vasorum compared with a randomly selected canine population. METHODS: Serum protein capillary zone electrophoreses (CZEs) from dogs with angiostrongylosis were assigned to Group 1. The CZE pattern was classified as normal, nonspecific polyclonal, or polyclonal with a typical beta-2 peak. The frequency of this latter pattern in Group 1 was compared with the frequency of similar findings in a randomly selected canine sera group (Group 2). RESULTS: Groups 1 and 2 included 22 and 3687 cases, respectively. Thirteen of 22 (59.1%) dogs in Group 1 had this peak in the beta-2 region. A similar CZE pattern was observed in 43 dogs in Group 2 (1.2%). The remaining dogs in Group 1 had a nonspecific polyclonal hyperglobulinemia (six cases), a normal tracing (one case), or an ambiguous tracing (two cases). CONCLUSIONS: Dogs with angiostrongylosis commonly have polyclonal hyperglobulinemia, frequently characterized by a large and symmetrical beta-2 peak on CZE, which is uncommon in the canine population. Additional studies are needed to identify the protein composition of this peculiar beta fraction.

A retrospective study on prophylactic regional lymphadenectomy versus nodal observation only in the management of dogs with stage I, completely resected, low-grade cutaneous mast cell tumors.

BACKGROUND: While lymphadenectomy of metastatic lymph nodes (LNs) has been associated with improved outcome, the clinical utility of prophylactic lymphadenectomy in dogs with stage I cutaneous mast cell tumors (cMCTs) remains a controversial topic. To assess the therapeutic role of lymphadenectomy of uninvolved regional LNs, the long-term outcome of cMCT-bearing dogs with cytologically negative and surgically unresected regional LNs (observation only, OO) was compared with that of dogs with surgically resected and histologically negative regional LNs (prophylactic regional lymphadenectomy, PRL). RESULTS: A retrospective analysis of 64 dogs with a low-grade, completely resected stage I cMCT was performed: 35 (54.7%) dogs were subjected to OO and 29 (45.3%) underwent PRL. Dogs were monitored for a median of 813 and 763 days in the OO group and PRL group, respectively. The number of dogs undergoing MCT progression was significantly higher in the OO group (P = 0.028) and curve comparison revealed a tendency to a better time to progression in the PRL group (P = 0.058). No significant difference in survival time (P = 0.294) was observed between dogs in the OO and PRL groups. CONCLUSIONS: Our results showed that lack of immediate lymphadenectomy was associated with a higher risk for tumor progression. This preliminary judgement, reinforced by the findings that lymphadenectomy was well tolerated in all cases, and that histopathology provides the definitive assessment of the nodal pathological status, may suggest that prophylactic lymphadenectomy is indicated in the management of stage I MCTs. Larger prospective studies are warranted for generating clinical evidence of this latter hypothesis.

A case of "misplaced" Tritrichomonas foetus infection in a dog in Northern Italy.

An 8-year-old, spayed female Rottweiler dog, under immunosuppressant treatment for protein-losing enteropathy (PLE) and intestinal lymphangiectasia, was presented for anorexia, poor general conditions and episodes of diarrhea. A subcutaneous mass between the caudal abdominal mammary glands was found. A fine-needle aspiration cytology was performed and revealed the presence of inflammatory cells mixed with pear- or round-shaped microorganisms with cytomorphological features of flagellated protozoan trophozoites, belonging to Trichomonadida order. The final diagnosis of Tritrichomonas foetus infection was achieved with molecular analysis (Real-Time PCR) and sequencing. T. foetus has often been reported as a pathogenic agent in cattle and cats, and rarely reported in dogs, where its pathogenetic role is poorly understood. To the author's knowledge, this is the first case of T. foetus infection described in a location different from the gastrointestinal tract in a dog.

Cutaneous metastases of prostatic adenocarcinoma in two dogs.

Canine prostatic adenocarcinoma is an aggressive malignancy characterized by rapid growth, local invasiveness, and early metastatic spread. Metastases of prostatic cancer are generally diffuse at the time of diagnosis due to hematogenous or lymphatic spread and by direct exfoliation of neoplastic cells into the peritoneal cavity. Here we describe two dogs with prostatic adenocarcinoma and skin metastases. The first was a 12-year-old intact male German Shepherd dog that was presented with a history of chronic prostatic disease and multiple skin nodules that recently appeared on the ventral abdomen. The second was an 8-year-old intact male mixed breed dog that was referred for a neurologic examination because of a 1-month history of back pain and kyphosis of undefined origin. Cutaneous cytology of the first case was suggestive of carcinoma, and at necropsy, prostatic adenocarcinoma with metastases to the skin, spleen, liver, pancreas, kidneys, and lungs were found. In the second case, a computed tomographic examination revealed a prostatic neoplasm with inguinal, subcutaneous, and cutaneous nodular metastases. Cytology and histopathology were suggestive of primary prostatic adenocarcinoma with cutaneous and subcutaneous metastases. To the authors' knowledge, these are the first reported cases of prostatic adenocarcinoma skin metastases in dogs with cytologic descriptions.

Use of recombinant canine granulocyte-colony stimulating factor to increase leukocyte count in dogs naturally infected by canine parvovirus.

Canine parvovirus (CPV) is one of the most important cause of mortality in young dogs and no specific treatment exists. Since prolonged leukopenia greatly increases the risk of death in infected pups, strategies to counteract this decline were investigated. The outcomes of CPV naturally infected pups treated with the recombinant canine granulocyte-colony stimulating factor (rcG-CSF), in combination with the routine therapy, were compared with similarly-managed infected pups not treated with rcG-CSF. A non-randomized prospective clinical trial was performed on 62 CPV infected pups with WBC counts <3000 cells/µL and two different groups were selected based on a non-randomized approach. Group A dogs (31/62) received 5 µg/Kg of rcG-CSF daily from the hospitalization day until WBC reached the reference range (3-5 days) and group B (31/62) received 1 ml of placebo injection. All dogs in group A recovered, while five dogs in group B died. The rcG-CSF treatment demonstrated a statistically significant effect on WBC counts (p < 0.0001) and, surprisingly, also on lymphocytes and monocytes counts (p < 0.0001). There was no significant effect of treatment on neutrophil count (p = 0.5502). Although lymphocytes and monocytes are not a specific target for rcG-CSF, our study highlights that rcG-CSF is able to improve haematological parameters compared to untreated dogs and a clear increase in their number was detected, as previously described for humans treated with the homologous molecule.

Evaluation of the analytical variability of urine protein-to-creatinine ratio in cats.

BACKGROUND: Proteinuria quantification with the urinary protein-to-creatinine (UPC) ratio is part of the diagnostic process in feline patients suspected of chronic kidney disease (CKD). In affected cats, monitoring and substaging of the UPC according to the International Renal Interest Society (IRIS) guidelines is also necessary for appropriate patient management. No information is available about the possible effects of analytical variability on urinary proteins (UPs) and the UPC ratio in cats. OBJECTIVES: This study aimed to determine whether imprecision and method-dependent differences due to the two dye-binding methods, pyrogallol red-molybdate (PRM) and Coomassie brilliant blue (CBB), could affect IRIS substaging. METHODS: Urine samples were collected from proteinuric and nonproteinuric cats. Intra-assay and inter-assay repeatability were assessed with both the PRM and CBB methods. Urinary supernatants (n = 120) were tested using both methods. Agreement between the methods and concordance with sample classification according to IRIS guidelines were determined. RESULTS: On average, the PRM method yielded a higher CV (UP 8.4 ± 5.2%; UPC 9.5 ± 4.8%) than the CBB method (UP 5.6 ± 2.6%; UPC 7.2 ± 2.6%), but similar rates of misclassification were found in samples with UPC ratios close to the IRIS cut-off. Although the two methods were correlated, the CBB method tended to yield UPs and UPC ratios that were significantly higher (P < 0.0001) than that of the PRM method. The Passing-Bablok test also found constant and proportional errors between the PRM and CBB methods. Concordance in substaging samples according to IRIS was good (k coefficient = 0.62). CONCLUSIONS: The two methods were precise, but the higher UPC ratios obtained with the CBB methods might affect the interpretation using the IRIS guidelines and clinical decisions.

Cladosporium cladosporioides-complex infection in a mixed-breed dog.

A 3-year-old female mixed-breed dog was referred with a 2-day history of serious dyspnea, coughing, lethargy, anorexia, and a low-grade right anterior lameness. At presentation, the dog had an increased respiratory rate, dull heart and lung sounds, and cyanotic mucous membranes. It was hyperthermic and slightly dehydrated. Laboratory findings showed mild neutrophilia with a left shift, while serum biochemistry variables were in the normal range. However, urinalysis revealed mild proteinuria and rare erythrocytes and leukocytes on sediment examination. Thoracic radiographs showed a diffuse mixed interstitial and alveolar pattern with an air bronchogram, while appendicular radiographs showed a right humeral interrupted brush-like periosteal reaction. Thoracic ultrasonography revealed mediastinal lymph node enlargement. Cytology from a fine-needle aspirate of mediastinal lymph nodes revealed a pyogranulomatous lymphadenitis with numerous fungal hyphae. Culture on Sabouraud dextrose agar isolated dark fungal colonies with microscopic features consistent with Cladosporium spp. Sequencing of the internal transcribed spacer region identified the fungus as a species of the Cladosporium cladosporioides-complex.

Pleural lymphocyte-rich transudates in cats.

Objectives Non-chylous lymphorrhagic pleural effusions are transudative effusions with a predominance of lymphocytes; however, they do not contain chylomicrons and therefore do not have the classical milky aspect of true chylous effusion. This type of effusion has been anecdotally associated with cardiac diseases in cats, but studies are lacking. The aim of this study was to investigate the association between this type of effusion and the primary disease. Methods In this study, feline non-chylous lymphorrhagic pleural effusions were retrospectively selected from the database of the authors' institutions over a 3 year period. All cases underwent thoracic imaging, including echocardiography. Effusions classified as transudates with a predominance of lymphocytes on cytology were included. Results Thirty-three cases fulfilled the inclusion criteria: 23 (69.7%) had a concurrent cardiac disease, eight (24.2%) cases were associated with the presence of a mediastinal lymphoma or carcinoma or a thoracic mass, one case (3.0%) was a thymoma and one case (3.0%) was a sequela of a pyothorax. Conclusions and relevance Since a clear lymphatic origin of the fluid could not be demonstrated, lymphocyte-rich transudate might be considered a better designation for these kinds of effusions rather than non-chylous lymphorrhagic effusions. Although the number of cases in this preliminary study is low, the presence of a pleural lymphocyte-rich transudate in a cat should prompt the search for cardiac disease or intrathoracic neoplasia.

Measurement of proteinuria in dogs: analytic and diagnostic differences using 2 laboratory methods.

BACKGROUND: Urinary protein-to-creatinine (UPC) ratio is an early diagnostic and prognostic marker of renal disease in dogs. Pyrogallol red molybdate (PRM) and Coomassie brilliant blue (CBB) are the most popular dye-binding assays for measurement of proteinuria. Published guidelines recommend strict cut-off points to substage patients with chronic renal diseases, irrespective of the assay applied. However, analytic variability and method-dependent differences could affect substaging of patients. OBJECTIVES: The aims of this study were to analytically validate the CBB assay to evaluate possible method-dependent differences with PRM in urinary protein (UP) determination, and to assess the influence of such differences in substaging according to the International Renal Interest Society (IRIS). METHODS: Urine was collected from healthy and proteinuric dogs. Intra-assay and inter-assay repeatability (imprecision), linearity under dilution (LUD), and spiking recovery (inaccuracy) were determined for the CBB assay. Split samples were measured with PRM and CBB, and agreement between methods and concordance in classification according to IRIS guidelines was determined. RESULTS: The CBB assay was precise (< 10%) at all urine protein concentrations after excluding outliers from the intra-assay precision assay of high urine protein concentrations. Acceptable accuracy was demonstrated with both LUD and spiking recovery test. Both UP and UPC determined by CBB were significantly higher (P < .0001) than those obtained with PRM, and both a constant and proportional bias were present. Concordance of IRIS substaging was only moderate. CONCLUSIONS: The CBB is precise and accurate, but the higher UPC obtained with CBB vs PRM may affect interpretation of the IRIS guidelines.