Anna Hillström

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels

BACKGROUND Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKSC than methods directly enumerating platelet numbers. OBJECTIVES The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs. METHODS Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample. RESULTS None of the 27 CKCS dogs had thrombocytopenia (defined as <164 x 10(9) platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum. CONCLUSIONS The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass.

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.

Evaluation of cytologic findings in feline conjunctivitis

BACKGROUND Cytologic examination of smears prepared from ocular swabs of conjunctiva from cats with conjunctivitis permits identification of the type of inflammation and possibly specific microorganisms. Results of studies of the diagnostic utility of cytology for detection of infectious causes of feline conjunctivitis have been inconsistent. OBJECTIVES The objectives of this study were to describe cytologic findings in cats with conjunctivitis and to compare those findings with results of PCR analysis for feline herpesvirus (FHV-1), Chlamydophila felis (C felis), and Mycoplasma felis (M felis). METHODS Conjunctival smears from 88 cats with conjunctivitis and 10 healthy control cats were stained with a Romanowsky stain and evaluated for the type of inflammation and evidence of an infectious agent. PCR analysis for FHV-1, C felis, and M felis was performed. RESULTS Infectious agents identified by PCR analysis were FHV-1 in 9 cats (10%), C felis in 8 cats (9%), and M felis in 6 cats (7%). Inclusions interpreted as chlamydial inclusions were found in all cytologic smears from cats positive for C felis by PCR analysis and in 3 PCR-negative cats. Inclusions interpreted as Mycoplasma organisms were found in 3 of 6 cats that were PCR-positive for M felis and in 1 PCR-negative cat. FHV-1 inclusion bodies were not detected on cytologic examination. CONCLUSIONS Cytologic examination can be diagnostic for C felis infection when many typical inclusions are present. Cytologic examination was unreliable in diagnosing M felis infection, and viral inclusions of FHV-1 were not found in specimens stained with Romanowsky stains.

Evaluation of an in-clinic Serum Amyloid A (SAA) assay and assessment of the effects of storage on SAA samples

BackgroundAn in-clinic assay for equine serum amyloid A (SAA) analysis, Equinostic EVA1, was evaluated for use in a clinical setting. Stability of SAA in serum samples was determined.MethodsIntra- and inter- assay variation of the in-clinic method was determined. The in-clinic method (EVA1) results were compared to a reference method (Eiken LZ SAA) with 62 patient samples. For samples with SAA concentrations within the assay range of EVA1 (10-270 mg/L), differences between the methods were evaluated in a difference plot. Linearity under dilution was evaluated in two samples. Stability of SAA in three serum pools stored at 4°C and approximately 22°C was evaluated with the reference method day 0, 1, 2, 4, 7, 17 and analysed with a two-way ANOVA.ResultsThe imprecision (coefficient of variation, CV) for the in-clinic method was acceptable at higher SAA concentrations with CV values of 7,3-12%, but poor at low SAA concentrations with CV values of 27% and 37% for intra- and inter-assay variation respectively. Recovery after dilution was 50-138%. The in-clinic assay and the reference method identified equally well horses with low (<10 mg/L) and high (>270 mg/L) SAA concentrations. Within the assay range of the in-clinic method, 10-270 mg/L, the difference between the two methods was slightly higher than could be explained by the inherent imprecision of the assays. There were no significant changes of serum SAA concentrations during storage.ConclusionsThe in-clinic assay identified horses with SAA concentrations of <10 mg/L and >270 mg/L in a similar way as the reference method, and provided an estimate of the SAA concentration in the range of 10-270 mg/L. The imprecision of the in-clinic method was acceptable at high SAA concentrations but not at low concentrations. Dilution of samples gave inconsistent results. SAA was stable both at room temperature and refrigerated, and thus samples may be stored before analysis with the reference method.

Validation and application of a canine-specific automated high-sensitivity C-reactive protein assay

Measurement of low concentrations of C-reactive protein (CRP) in dogs has previously been performed with nonautomated assays. The aim of this study was to validate an automated high-sensitivity CRP (hsCRP) assay, developed by modifying a routinely used canine-specific immunoturbidimetric CRP test (cCRP). Imprecision, linearity under dilution, limit of blank (LOB), limit of detection (LOD), and limit of quantification (LOQ) were determined for the hsCRP test, as well as the presence of prozone effect and interferences. The imprecision, measured as intra-assay variation, was ≤2.7%. The assay was acceptably linear under dilution. An analytically relevant prozone effect was present for samples with CRP concentration >150 mg/L, and there were mild interferences from hemolysis and lipemia. The LOB, LOD, and LOQ were 0.10 mg/L, 0.22 mg/L, and 0.50 mg/L, respectively. A method comparison study with a canine-specific enzyme-linked immunosorbent assay (ELISA) was performed, showing poor agreement between the hsCRP test and the ELISA. An additional aim of the study was to apply the hsCRP test to clinical research samples. Serum samples from 7 dogs undergoing ovariohysterectomy were collected pre- and postoperatively, and CRP was measured with both the cCRP and hsCRP assay. The expected postoperative increase in CRP was detected earlier with the hsCRP test, compared with the cCRP test. The hsCRP assay was further applied on samples from 6 lean and 9 overweight dogs. There was no significant difference in CRP concentration between the groups (P = 0.06). In conclusion, the hsCRP test had acceptable analytical performance, and the assay was successfully applied to clinical research samples.

Hereditary Phosphofructokinase Deficiency in Wachtelhunds

Hereditary phosphofructokinase (PFK) deficiency was diagnosed in two Wachtelhund dogs and suspected in three related Wachtelhund dogs with exercise intolerance, hemolytic anemia, and pigmenturia. Severe, persistent reticulocytosis in light of only mild anemia together with hemoglobinuria after strenuous exercise suggested PFK deficiency. Low erythrocyte PFK activity together with low 2,3-diphosphoglycerate concentrations and a high hemoglobin-oxygen affinity confirmed the diagnosis. The PFK deficiency is due to a single missense mutation in the muscle-type PFK M-PFK gene in English springer and American cocker spaniels, whippets, and mixed-breed dogs; however, these PFK-deficient Wachtelhunds do not have the same PFK mutation.

Biological variation of 20 analytes measured in serum from clinically healthy domestic cats

The applications of data on biological variation include assessment of the utility of population-based reference intervals, evaluation of the significance of change in serial results, and setting of analytical quality specifications. We investigated the biological variation of 19 biochemistry analytes and total T4, measured in serum from 7 clinically healthy domestic cats sampled once weekly for 5 weeks. Samples were frozen and analyzed in random order in the same analytical run. Results were analyzed for outliers, and the components of variance, subsequently generated by restricted maximum likelihood, were used to determine within-subject and between-subject variation (CVI and CVG, respectively), as well as analytical variation (CVA) for each analyte. Indices of individuality, reference change values, and analytical performance goals were calculated. The smallest CVI and CVG were found for calcium, chloride, and sodium, whereas the largest values were calculated for bile acids. Nine analytes (albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, cholesterol, creatinine, phosphate [phosphorus], total protein, total T4) demonstrated high individuality, indicating limited utility of population-based reference intervals. Individuality was low, and population-based reference intervals were thereby considered appropriate for 5 analytes (bile acids, calcium, fructosamine, glucose, potassium). The intermediate individuality observed for 4 analytes (creatine kinase, iron, magnesium, urea) indicated that population-based reference intervals should be used with caution.

Serum concentrations of C-reactive protein (CRP) in lean and overweight dogs

Obesity in dogs has been associated with both increased and decreased blood C-reactive protein (CRP) concentrations.

Serum C-reactive protein concentrations in Nova Scotia Duck Tolling Retrievers with immune-mediated rheumatic disease

Nova Scotia Duck Tolling Retrievers (NSDTRs) are a dog breed often affected by immune-mediated rheumatic disease (IMRD), a disorder characterised by chronic stiffness and joint pain. Most, but not all, dogs with IMRD, have antinuclear antibodies (ANA), which are also commonly present in the autoimmune disease systemic lupus erythematosus (SLE). The clinical and diagnostic findings of IMRD indicate that it is an SLE-related disorder. C-reactive protein (CRP), an acute phase protein, is a quantitative marker of inflammation for many diseases and is used for diagnosing and monitoring systemic inflammation in both humans and dogs. However, in human SLE, CRP concentrations are often elevated but correlate poorly with disease activity; they can be low in individual patients with active disease. The aim of the study was to investigate CRP in a group of NSDTRs with the SLE-related disorder IMRD. The hypothesis was that CRP concentrations would be increased in dogs with IMRD compared to healthy dogs, but that the increase would be mild. Serum CRP concentrations were measured in 18 IMRD-affected NSDTRs and 19 healthy control NSDTRs using two different canine-specific CRP assays. Dogs with IMRD and ANA had higher CRP concentrations than the control dogs, but the concentrations were below the clinical decision limit for systemic inflammation for most of the IMRD dogs. These results indicate that CRP concentrations were increased in dogs with IMRD and ANA, but the increase was mild, similar to what has been observed in human SLE.

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.