Validation of an automated immunoturbidimetric assay for feline serum amyloid A.

BACKGROUND: Serum Amyloid A (SAA) is a major acute phase protein in cats, increasing rapidly in response to various inflammatory diseases. An automated latex-enhanced immunoturbidimetric assay for human SAA (LZ-SAA, Eiken), previously validated for use in cats, has had further major modification (VET-SAA, Eiken) for specific use in veterinary diagnostic laboratories but has yet to be validated in cats. RESULTS: Intra-assay and inter-assay CVs for the VET-SAA assay ranged from 1.88-3.57% and 3.98-6.74%, respectively. Linearity under dilution was acceptable with no prozone effect observed. Limit of detection was 1.65 mg/L and limit of quantification was 6 mg/L. Haemoglobin and triglyceride showed no adverse interference, but bilirubin produced positive bias in samples with low SAA. Comparison with the LZ-SAA assay showed significant correlation with proportional bias increasing as SAA concentration increased, likely related to differing calibration standards. SAA was significantly higher in patients with inflammatory disease compared with non-inflammatory disease, and in patients with moderate to highly elevated α1-AGP compared with patients with normal α1-AGP. Improvement of the assay range may be required to fully evaluate differences between disease groups at low SAA levels. Based on ROC curve analysis, at a cut-off point of 20.1 mg/L the VET-SAA assay discriminated between inflammatory and non-inflammatory disease with sensitivity of 0.93 and specificity of 0.99. CONCLUSIONS: The automated VET-SAA assay is a robust, precise, and accurate method for measurement of feline SAA which can clearly identify patients with inflammatory disease. It should be a valuable biomarker for use in feline medicine.

Retrospective investigation of the neutrophil-to-lymphocyte ratio in dogs with pneumonia: 49 cases (2011-2016).

OBJECTIVE: To assess the utility of the neutrophil-to-lymphocyte ratio (NLR) in predicting outcome in canine pneumonia compared with routine hematological parameters and systemic inflammatory response syndrome (SIRS) scores. DESIGN: Retrospective study. SETTING: University teaching hospital. ANIMALS: Forty-nine client-owned dogs. INTERVENTIONS: None MEASUREMENTS AND MAIN RESULTS: Medical records were reviewed to identify dogs with a diagnosis of pneumonia from July 2011 to December 2016. Signalment, clinical findings, laboratory characteristics, and outcome were recorded. Inclusion criteria were a clinical and radiographic diagnosis of pneumonia, plus reference laboratory hematology at diagnosis. Cases that received steroids were excluded. Euthanized dogs were only included in statistical analysis if euthanized solely due to pneumonia severity. The NLR, total WBC count, neutrophil count, lymphocyte count, band neutrophil percent of total WBC count (%-bands), and percentage of cases diagnosed with SIRS were compared between survivors and nonsurvivors. Receiver operating characteristic curves were generated to identify optimal sensitivity and specificity cutoffs for nonsurvival to discharge. Two hundred records were retrieved; 49 cases fulfilled the inclusion criteria. Of these, 33 (67%) survived to discharge. The NLR did not differ significantly between the survivors and nonsurvivors, nor did total WBC count or neutrophil count. Survivors had a significantly lower %-bands than nonsurvivors (P < 0.001) and higher lymphocyte count (P = 0.004). The mortality rate did not differ significantly between dogs with and without SIRS. Receiver operating characteristic analysis identified a %-bands cutoff of 2.5% or higher had an 83% sensitivity and 79% specificity for nonsurvival. CONCLUSIONS: Unlike in human medicine, neither NLR nor SIRS scores predicted outcome in this cohort of dogs with pneumonia. However, survivors had a lower %-bands and higher lymphocyte count than nonsurvivors, which may be helpful prognostically in clinical cases.

A case of T-cell chronic lymphocytic leukemia progressing to Richter syndrome with central nervous system involvement in a dog.

An 8-year-old neutered Beagle dog was presented with polyuria and polydipsia. Routine clinicopathologic testing showed a significant lymphocytosis and proteinuria. Lymphocytes were of small to intermediate in size with a mature morphology. Infectious disease screening was negative. PCR for antigen receptor gene rearrangements showed a clonal T-cell receptor (TCR) rearrangement consistent with T-cell chronic lymphocytic leukemia (CLL). Bone marrow cytology showed <30% lymphocytes, while the proportion in splenic fine-needle aspirate cytology was considered increased. The dog was initially monitored but started on prednisolone and chlorambucil therapy 2 months later due to worsening clinical signs and progressive lymphocytosis. After an additional 2 weeks, the dog developed multifocal spinal pain and single-node lymphadenomegaly. Cytology of the lymph node showed a monomorphic population of large lymphoblasts consistent with lymphoma. Cytology of a cerebrospinal fluid sample also showed large lymphoblasts. PCR for antigen receptor gene rearrangement at both sites showed a clonal TCR rearrangement of the same molecular size as in the initial leukemic cells. The dog was diagnosed with a transformation of the CLL to Richter syndrome (RS) with involvement of the central nervous system (CNS). Therapy was started with L-asparaginase and an increased dose of prednisolone; however, the dog was euthanized due to progressive clinical signs. To our knowledge, this is the first report of canine RS with direct involvement of the CNS.

A metastatic secretory gastric plasmacytoma with aberrant CD3 expression in a dog.

A 10-year-old crossbred dog was presented with a 6-week history of hematemesis, melena, anorexia, and lethargy. Clinical evaluation revealed a gastric mass with a regional lymphadenomegaly as well as a monoclonal gammopathy manifesting as hyperglobulinemia. Cytologic and histopathologic analyses were consistent with a round cell neoplasm; neoplastic cells showed nuclear immunoreactivity for MUM1 and diffuse cytoplasmic reactivity for CD3. Polymerase chain reactions performed on fixed and fresh tissue identified a clonal rearrangement with an IgH primer set. An extramedullary plasmacytoma (EMP) was confirmed by cellular morphology and molecular diagnostics. Following an objective response to chemotherapy, the dog was euthanized 8 months after diagnosis, and a postmortem examination confirmed the clinical findings. This is the first reported case of a monoclonal gammopathy secondary to a gastric EMP coupled with aberrant expression of CD3 in an aggressive plasmacytic tumor, and highlights the utility of molecular diagnostics for classifying atypical hemolymphoid neoplasms.

Optimisation and validation of a PCR for antigen receptor rearrangement (PARR) assay to detect clonality in canine lymphoid malignancies.

PCR for antigen receptor gene rearrangements (PARR) analysis is being increasingly used to assist diagnosis of canine lymphoma. In this study, PARR was carried out on consecutive samples received as part of routine diagnostic practice from 271 patients: 195 with lymphoid malignancies, 53 with reactive conditions and 23 with other neoplasms. Initially, published primer sets were used but later minor primer modifications were introduced and primers were rationalised to give a PARR panel that provides a good compromise between sensitivity and cost. Results were compared to diagnoses made by histology or cytology, coupled with immunophenotyping by flow cytometry or immunohistochemistry where possible. After exclusion of 11 poor quality samples, 230/260 (88%) gave a clear result with 162/163 (99%) of samples classified as clonal and 56/67 (84%) classified as polyclonal giving results concordant with the cytological/histological diagnosis. Among 30 samples with equivocal results, 21 had clonal peaks in a polyclonal background and nine showed little amplification. These were from patients with a range of neoplastic and non-neoplastic conditions emphasising the need to interpret such results carefully in concert with other diagnostic tests. The combination of primer sets used in this study resulted in a robust, highly specific and sensitive assay for detecting clonality.

Gammaherpesviruses and canine lymphoma: no evidence for direct involvement in commonly occurring lymphomas.

Lymphoma is the most common haematopoietic malignancy in dogs, but little is known about the aetiology of this heterogeneous group of cancers. In humans, the Epstein-Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it was suggested that EBV or an EBV-like virus is circulating in dogs. We therefore investigated whether EBV, or a novel herpesvirus, is associated with canine lymphoma using both serological and molecular techniques. In an assay designed to detect antibodies to EBV viral capsid antigens, 41 % of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridization or PCR, and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. We therefore found no evidence that herpesviruses are directly involved in common types of canine lymphoma although cannot exclude the presence of an EBV-like virus in the canine population.

The retrovirus XMRV is not directly involved in the pathogenesis of common types of lymphoid malignancy.

BACKGROUND: A novel retrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been detected in prostate cancer samples and in peripheral blood mononuclear cells (PBMC) from patients with chronic fatigue syndrome. In addition, the virus has been identified in PBMCs from healthy controls. These data suggest that XMRV is circulating in the human population. XMRV is closely related to murine leukemia viruses, which cause lymphoid malignancies in mice. The aim of this study was to determine whether XMRV is directly associated with common forms of human lymphoma or leukemia. METHODS: DNA samples from 368 patients with lymphoid malignancies and 139 patients with benign lymphadenopathy or other malignant disease were screened for XMRV, using three specific and sensitive quantitative PCR assays. RESULTS: XMRV was not detected in any sample using any of the three assays. CONCLUSIONS: The data suggest that this virus is not directly involved in the pathogenesis of common types of lymphoid malignancy and that XMRV is not a prevalent blood borne infection, at least in the United Kingdom. IMPACT: There is no evidence that XMRV is associated with lymphoid malignancies, and further studies should resolve inconsistencies in results of studies examining XMRV prevalence.