Validation study of canine serum cortisol measurement with the Immulite 2000 Xpi cortisol immunoassay

We report the results of validation of canine serum cortisol determination with the Immulite 2000 Xpi cortisol immunoassay (Siemens), with characterization of precision (CV), accuracy (spiking-recovery [SR] bias), and observed total error (TEo = bias + 2CV) across the reportable range, specifically at the most common interpretation thresholds for dynamic testing. Imprecision increased at increasing rate with decreasing serum cortisol concentration and bias was low, resulting in increasing TEo with decreasing serum cortisol concentration. Inter-laboratory comparison study allowed for determination of range-based bias (RB) and average bias (AB). At 38.6 and 552 nmol/L (1.4 and 20 μg/dL), between-run CV was 10% and 7.5%, respectively, and TEo ~30% and ~20%, respectively (TEo remained similar regardless of the considered bias: SR, RB, or AB). These analytical performance parameters should be considered in the interpretation of results and for future expert consensus discussions to determine recommendations for allowable total error (TEa). Importantly, the commonly used thresholds for interpretation of results were determined ~40 y ago with different methods of measurements and computation, hence updating is desirable. Quality control material (QCM) had between-run imprecision of 4% for QCM1 and 7% for QCM2; the bias was minimal for both levels. Acceptable QC rules are heavily dependent on the desired TEa for the QCM system (TEaQCM), itself limited by the desired clinical TEa. At low TEaQCM (20–33%), almost no rules were acceptable, whereas at high TEaQCM (50%), almost all rules were acceptable; further investigation is needed to determine which TEaQCM can be guaranteed by simple QC rules.

Comparison of a Point-of-Care Analyzer With a Chemiluminescent Immunoassay for Serum Progesterone Measurement in Breeding Management of the Bitch

Accurate serum progesterone measurements for timing bitches during breeding management is critical for reproductive practice, especially as artificial insemination has become routine to facilitate breeding of animals that are geographically or temporally separated. To measure serum progesterone, chemiluminescent immunoassay (CLIA) has replaced radioimmunoassay as the current standard in the bitch due to its high correlation and increased practicality. In January 2019, a colorimetric point-of-care (POC) immunoassay for quantitative in-clinic canine serum progesterone measurements in <30 min was released. This study provides an independent comparison of the POC (Catalyst One, IDEXX) to the current industry standard, CLIA (Immulite-2000, Siemens). To assess inter-assay imprecision of POC and agreement of the POC and CLIA results, 100 canine serum samples were analyzed on three analyzers (POC-1, POC-2, and CLIA), of which, 74 (POC-1) and 75 (POC-2) results were within POCs' reportable range of 0.2–20 ng/mL and included in the study. To assess intra-assay imprecision, pooled canine serum samples at low (L1), intermediate (L2), and high (L3) progesterone concentrations were analyzed ten times each on POC-1 and CLIA. Relative to CLIA, POC values showed good correlation (POC-1, r2 = 0.9366; POC-2, r2 = 0.9438, P < 0.0001) and significant positive proportional bias at values >2 ng/mL. The POC inter-assay coefficients of variation (CVs) were 13.2% (0.2–2.9 ng/mL, 0.6–9.2 nmol/L, L1), 10.0% (3.0–9.9 ng/mL, 9.5–31.5 nmol/L, L2), 7.1% (10.0–20.0 ng/mL, 31.8–63.6 nmol/L, L3), and 11.2% (all samples). The intra-assay CVs for POC (L1, 15.3%; L2, 7.0%; L3, 4.7%) were higher than those for CLIA (L1, 5.89%; L2, 4.89%; L3, 3.44%). Based on the more rapid increase in serial serum progesterone concentrations in ovulating bitches and the greater imprecision of the POC, the clinical interpretations of serum progesterone measurements as they relate to canine breeding management should be made with caution.

Comparison of the Vcheck and IMMULITE 2000 methods for cortisol measurement in canine serum

The aim of this study was to compare canine cortisol results obtained by the Vcheck method with those obtained by the IMMULITE 2000 immunoassay, which had previously been validated for canine serum. The concentration of cortisol in 44 canine serum samples was determined concurrently by the Vcheck and IMMULITE methods, the latter as the reference method. Cortisol values were compared using Pearson’s correlation analysis and simple regression analysis. Agreement between the two methods was calculated with a Bland-Altman plot. According to regression analysis and the Bland-Altman plot, the two methods gave comparable results. The results of the Vcheck method were comparable to those of the IMMULITE 2000 reference method, so the Vcheck may be used as an alternative assay to evaluate serum cortisol concentration in dogs for the diagnosis of adrenal disease.

Identification of Protein Carbonyls (PCOs) in Canine Serum by Western Blot Technique and Preliminary Evaluation of PCO Concentration in Dogs With Systemic Inflammation

In people, serum Protein Carbonyls (PCOs) increase during oxidative stress (OS) due to oxidative damage to proteins. OS is often associated with inflammation and especially with sepsis, a condition hard to diagnose in veterinary medicine because reliable markers are lacking. The aim of this study was to assess whether PCOs in canine serum may be detected by antibody-based methods such as Western Blotting (WB), and to preliminarily investigate the possible utility of this marker in dogs with inflammation. A serum sample oxidized in vitro was used to set up the method; the coefficient of variation obtained by repeated analysis varied from 24 to 36%. In order to assess whether the technique may cover the range of PCOs concentration detectable in routine practice, PCOs were measured in 4 healthy dogs and in 15 with inflammatory diseases, in some cases potentially associated with sepsis, as suggested by the results of other inflammatory markers such as C-Reactive Protein (CRP) and the anti-oxidant enzyme Paraoxonase 1 (PON-1): the concentration of PCOs was low in dogs with normal PON-1 activity, moderately increased in the majority of dogs with low-normal PON-1 activity, and severely increased in dogs with very low PON-1 activity. In conclusion this study demonstrates that PCOs, may be detected in canine serum, using antibody-based techniques such as WB. The preliminary results in dogs with and without systemic inflammation encourage further studies on the possible role of PCOs as inflammatory markers.

Evaluation of an automated immunoturbidimetric assay for detecting canine C-reactive protein

C-reactive protein (CRP) is a major acute-phase protein, and it is produced by the liver in response to a pro-inflammatory stimulus. Given that human and canine CRP have a similar molecular structure, the assays used for human CRP detection have been used to measure CRP concentrations in dogs. We evaluated the use of a human CRP assay (Biotecnica CRP assay) and validated its application in dogs. We analyzed 91 canine serum samples with a fully automated analyzer. Our validation was based on the evaluation of imprecision, limits of linearity, limits of quantification, and an evaluation of interferences. The new assay was also compared with the Randox CRP assay, a validated assay for the measurement of CRP. Intra- and inter-assay repeatability were <8% and <11%, respectively. The tested assay proportionally measured canine CRP in an analytical range up to 60 mg/L; however, hemoglobin, triglycerides, and bilirubin interfered with the determination. Good agreement, with the presence of proportional systematic bias, was observed between Biotecnica and Randox assays. The Biotecnica CRP assay provides reliable measurement of CRP in canine serum, provided that samples are free of interferents.

Discovery of N-glycan Biomarkers for the Canine Osteoarthritis

Protein glycosylation is a post-translational modification that impacts on protein activity, stability, and interactions. It was sensitively altered by the cellular state and, therefore, is now used for a diagnostic or prognostic indicator of various human diseases such as cancer. To evaluate the clinical feasibility in the veterinary area, the N-glycan biomarkers were discovered from canine serum for the diagnosis of osteoarthritis (OA), which is one of the most common diseases of dogs. N-glycome was obtained from 20 μL of canine serum by the enzymatic cleavage followed by the purification and enrichment using solid-phase extraction. Independent compositions of 163 and 463 N-glycans were found from healthy control (n = 41) and osteoarthritis patients (n = 92), respectively. Initially, 31 of the potential biomarkers were screened by the p-values below 1.0 × 10−10 from ANOVA. Then, the area under the curve (AUC) and the intensity ratio between OA patient and healthy control (P/C ratio) were calculated. Considering the diagnostic efficacy, the AUC bigger than 0.9 and the P/C ratio larger than 3.0 were used to discover 16 N-glycans as diagnostic biomarkers. Particularly, five of the diagnostic biomarkers were AUC above 0.99 and three of N-glycans had AUC 1.0. The results suggest a clear possibility for N-glycan biomarkers to be used as a clinical tool in the veterinary medical area enabling to provide objective and non-invasive diagnostic information.

Validation of a commercial magnetic bead–based multiplex assay for 5 novel biomarkers of acute kidney injury in canine serum

Interest is growing in measurement of novel biomarkers for the diagnosis of acute kidney injury. Multiplex assays may provide a rapid and cost-effective way of measurement; however, sparse information is published regarding their use in dogs. We aimed to validate a commercial magnetic bead–based assay for 5 biomarkers: clusterin (Clus), cystatin C (CysC), kidney injury molecule 1 (KIM-1), monocyte chemoattractant protein 1 (MCP-1), and neutrophil gelatinase–associated lipocalin (NGAL). Intra- and inter-assay imprecision, linearity under dilution (LUD), spike recovery (S-R), and hemoglobin interference were evaluated using serum from healthy and diseased dogs. Additionally, the effect of sample type (serum vs. plasma) was investigated. All values for Clus and MCP-1 were outside the assay’s measurable range. Intra- and inter-assay precision were acceptable for NGAL (CVs 8.8% and 13.2%, respectively). Regression analysis of LUD and S-R indicated good linearity for CysC and NGAL. Hemolysis did not affect measurement of any biomarker. Measured concentrations of CysC ( p = 0.018) and NGAL ( p = 0.015) were significantly lower in sodium citrate plasma compared to serum. We conclude that this magnetic bead–based assay is precise and accurate for NGAL measurement in canine serum. Inappropriate standards for MCP-1 and Clus, and poor accuracy for KIM-1 measurement, suggest that this assay cannot reliably quantify those biomarkers in canine blood. Measurements of CysC in canine blood using this assay must be interpreted with caution given inter-assay imprecision.