Journal of Veterinary Cardiology (2014)
-, -e-
www.elsevier.com/locate/jvc
Analytical validation and clinical evaluation of a commercially available high-sensitivity immunoassay for the measurement of troponin I in humans for use in dogs Randolph L. Winter, DVM a,*, Ashley B. Saunders, DVM a, Sonya G. Gordon, DVM, DVSc a, Matthew W. Miller, DVM, MS a, Katharine T. Sykes, DVM b, Jan S. Suchodolski, Dr. med. vet., ¨rg M. Steiner, Dr. med. vet., PhD c PhD c, Jo a
Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA b North Houston Veterinary Specialists, Spring, TX, USA c Gastrointestinal Laboratory, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA Received 26 January 2014; received in revised form 11 March 2014; accepted 12 March 2014
KEYWORDS Arrhythmias; Biomarker; Cardiac; Congenital; Valve disease
Abstract Objective: To analytically validate a commercially available highsensitivity immunoassay for measurement of cardiac troponin I (cTnI) in humans for use in dogs and to evaluate serum cTnI concentrations in healthy dogs and 3 well-defined groups of dogs with common cardiac diseases. Animals: Canine serum samples were used for validation. 85 client-owned dogs including 24 healthy controls, 20 with myxomatous mitral valve disease, 19 with congenital heart disease, and 22 with arrhythmias. Methods: Four serum samples were used to analytically validate the ADVIA Centaur TnI-Ultra assay by assessing intra-assay variability, inter-assay variability, spiking recovery, and dilutional parallelism. Dogs were grouped based on examination, echocardiography, and additional testing as clinically indicated, and serum cTnI concentrations were compared.
* Corresponding author. E-mail address: [email protected] (R.L. Winter). 1760-2734/$ - see front matter ª 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jvc.2014.03.002
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
2
R.L. Winter et al. Results: Analysis of the serum samples used for validation revealed an intra-assay coefficient of variation between 3.6% and 5.7%, and an inter-assay coefficient of variation between 2.4% and 5.9%. Observed to expected ratios for spiking recovery were 97.9 8.6% (mean, SD). Observed to expected ratios for dilutional parallelism were 73.0 11.5% (mean, SD). Dogs with cardiac disease had significantly higher serum cTnI concentrations (P < 0.005) than healthy dogs. Conclusions: The ADVIA Centaur TnI-Ultra’s low limit of detection allows measurement of serum cTnI in the majority of dogs even with no or mild cardiac disease. Dilution of samples for measurement of values above the upper limit of detection is not reliable and therefore not recommended. Serum cTnI concentrations are significantly higher in dogs with cardiac disease compared to healthy dogs. ª 2014 Elsevier B.V. All rights reserved.
Abbreviations ACVIM American College of Veterinary Internal Medicine cTnI cardiac troponin I CV coefficient of variation hs-cTnI high-sensitivity cardiac troponin I MMVD myxomatous mitral valve disease
Introduction Cardiac troponin I (cTnI) is a cardiac-specific protein involved in cardiomyocyte contraction and relaxation.1 More specifically, it is one of 3 subunits of the cardiac troponin complex that plays a role in excitationecontraction coupling of the cardiomyocytes. Troponin I is also present in skeletal muscle, however, the cardiac-specific isoform of troponin I has a different amino acid sequence from its skeletal counterpart and both isoforms thus do not cross-immunoreact.1 Cardiac troponin I is normally bound to the actin filament and present in low concentration in the cytosol, but cardiomyocyte damage allows cTnI release into the extracellular space.1 Therefore, circulating concentrations of cTnI provide information about cardiac-specific injury. Canine and human cTnI share significant amino acid homology, thus cTnI assays designed for human patients can be used in canine patients with proper analytical validation.2 Circulating cTnI concentration has been used for the detection of both acute and chronic myocardial damage in multiple species, and is often utilized as adjunctive information to other diagnostic tests, such as thoracic radiography, electrocardiography, and echocardiography in case management.1e9 The concentration of circulating cTnI has been shown to be proportional to the degree of cardiomyocyte damage.1 Many different commercial assays are available,
both standard sensitivity cTnI assays and highsensitivity cTnI (hs-cTnI) assays. The main distinguishing characteristic of hs-cTnI assays is a lower level of cTnI detection compared to standard sensitivity cTnI assays which make them capable of detecting lower concentrations of circulating cTnI, potentially providing better sensitivity for detecting myocardial damage.10,11 In human medicine, cTnI assays can identify increases in serum cTnI in patients with acute diseases such as myocardial infarction,10,11 but also in chronic heart disease,12,13 congenital heart disease,14,15 and patients with clinically significant arrhythmias.16e18 In veterinary medicine, serum cTnI concentrations have been reported using standard sensitivity cTnI assays in dogs with myxomatous mitral valve disease (MMVD), dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and congenital heart disease.6,7,19e22 With standard cTnI assays, the lower limit of detection can be too high, limiting the ability to detect increases in cTnI and thereby limiting the assay’s clinical utility.7,21,23,24 The ADVIA Centaur TnI-Ultrad is a commercially available high-sensitivity 3-site assay for cTnI analysis that utilizes direct chemiluminometry with detectable cTnI serum concentrations reported by the manufacturer ranging from 0.006 to 50.0 ng/mL. This cTnI immunoassay has been previously validated for use in humans and cattle.25 The objectives of this study were to analytically validate the assay for use in dogs and to evaluate the clinical use of this assay in a population of healthy dogs and stratified in 3 welldefined groups of dogs with common cardiac diseases.
d ADVIA Centaur TnI-Ultra, Siemens Medical Solutions Diagnostics, New York, NY, USA.
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
Validation of a high-sensitivity cTnI assay in dogs
Animals, materials and methods Assay Cardiac troponin I analysis was performed with the ADVIA Centaur TnI-Ultra immunoassay, a highsensitivity 3-site assay using direct chemiluminometry. One polyclonal goat antibody directed against antitroponin I and labeled with acridium ester and 2 biotinylated mouse monoclonal antibodies directed against antitroponin I are used for cTnI detection. These capture antibodies that recognize amino acid sequences 41e49 and 87e91 in the central region of the cTnI molecule.10,25 The solid phase reagent is made of magnetic latex particles that have been conjugated with streptavidin. The biotinylated antibodies bind the cTnI in the serum, and the biotin is then captured by the streptavidin-labeled magnetic particles. A direct relationship exists between the cTnI amount in the sample and the amount of relative light emitted from the chemiluminescent reaction that ensues.10,25 The working range for cTnI is reported by the manufacturer as 0.006e50.0 ng/mL. The amount of serum required for the assay is 100 mL. The manufacturer’s specifications for this assay allow the use of either plasma (heparinized or EDTA) or serum. The data reported herein are for serum samples. Blood samples were collected from dogs presented to the Texas A&M University Veterinary Medical Teaching Hospital from October 2011 to December 2012. Blood was collected from the external jugular vein, and samples were placed into commercially available additive free glass tubes and allowed to rest for 15 min at room temperature to allow for complete blood clot formation. Serum was obtained via centrifugation and stored in 500e1000 mL aliquots in polypropylene tubes. Samples were immediately frozen and stored at 80 C until analysis. Frozen samples were allowed to slowly thaw at room temperature immediately prior to batch analysis. Assay linearity was assessed using dilutional parallelism (i.e. assessing assay performance by diluting samples with subsequent cTnI measurement) of serial twofold dilutions from 1:2 to 1:8.26 Assay accuracy was evaluated by spiking different sera with other sera of known concentrations of cTnI. The percentages of observed to expected values for serial dilution and spiking recovery were calculated as [observed value (ng/mL)/expected value (ng/mL)] 100.26 Precision was evaluated by the evaluation of selected serum samples 10 times within the same assay over a single day (i.e. within 1 assay plate) and calculating the intra-
3 assay coefficients of variation (CV).26 Reproducibility was determined by the evaluation of selected serum samples in 10 consecutive assay runs over 10 days (i.e. across multiple assay plates) and calculating inter-assay CV.26 Both the intraassay and inter-assay CV were calculated by % CV ¼ [SD/mean] 100.
Dogs Serum from 10 client-owned dogs was submitted for cTnI analysis for the purpose of using some of these dogs’ samples for analytical assay validation. Ten mL blood samples were obtained from each dog, and the serum cTnI values were evaluated in these dogs. Samples from 4 of the dogs were chosen to be used for the analytical assay validation. These 4 samples were chosen specifically because they were found to have different serum cTnI concentrations that covered a range of cTnI values. Serum samples were obtained from an additional 85 client-owned dogs that were presented for evaluation and assigned to 1 of 4 groups. Group 1 (G1-healthy) consisted of dogs that were considered apparently healthy with no abnormalities based on history, physical examination, echocardiogram, and laboratory analysis consisting of a complete blood-cell count and a serum biochemical analysis. The remaining 3 groups of dogs had cardiac disease and clinical evaluation consisted of a history, physical examination, and clinically indicated diagnostic tests. Dogs assigned to group 2 (G2-MMVD) had MMVD.27 Dogs assigned to group 3 (G3-congenital) had congenital heart disease. Dogs assigned to group 4 (G4-arrhythmia) had clinically relevant arrhythmias based on the presence of clinical signs and abnormalities on electrocardiography and 24h ambulatory electrocardiography. The dogs in G4-arrhythmia had clinical signs directly caused by the arrhythmia. This study was reviewed and approved by the Institutional Animal Care and Use Committee at Texas A&M University, College Station, TX.
Statistical analysis The D’Agostino and Pearson omnibus test was used to test each data set for age, weight, and troponin concentration for normality using a commercially available software package.e Data sets that failed normality testing were expressed as medians and e GraphPad Prism (v.6.0), GraphPad Software, San Diego, CA, USA.
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
4
R.L. Winter et al. Table 1 Precision and reproducibility of the ADVIA Centaur TnI-Ultra immunoassay for concentrations of cardiac troponin I in canine serum.
Intra-assay variability (n ¼ 10) Inter-assay variability (n ¼ 10)
Serum sample
Mean (ng/mL)
SD (ng/mL)
CV (%)
1 2 3 4 1 2 3 4
0.066 0.736 2.955 4.432 0.068 0.736 2.882 4.296
0.004 0.027 0.128 0.217 0.003 0.018 0.171 0.146
5.7 3.6 4.3 4.9 4.7 2.4 5.9 3.4
ranges, otherwise data were reported as mean SD. A KruskaleWallis one-way analysis of variance test was used to assess all data sets that failed normality testing for differences between groups. A Dunn’s multiple comparison test was used to test for differences between each group. A P-value < 0.05 was considered significant for all statistical analyses. Serum samples with cTnI concentrations below the lower limit of detection were assigned a value of 0.006 ng/mL.
Results Samples used for analytical assay validation had measured cTnI concentrations of 0.066, 0.768, 2.826, and 4.117 ng/mL. The 4 serum samples had intra-assay CV that ranged between 3.6 and 5.7%
and inter-assay CV that ranged between 2.4 and 5.9% (Table 1). Observed to expected ratios for serial dilutions for the 4 samples were 72.7 11.5% (mean, SD) with a range of 60.4e94.3% at dilutions of 1 in 2, 1 in 4, and 1 in 8 (Table 2). Observed to expected ratios for spiking recovery were 97.9 8.6% with a range of 87.0e109.6% for spiking each of the 4 serum samples with each of the other 3 serum samples (Table 3). The samples from 85 client-owned dogs were evaluated and group characteristics are reported in Table 4. Serum cTnI concentrations were detectable in 79/85 dogs. Values were below the lower detection limit in 5 dogs from G1-healthy and 1 dog from G2-MMVD. Breeds in G1-healthy consisted of mixed (n ¼ 4), labrador retriever (n ¼ 3), border collie (n ¼ 2), catahoula hog dog (n ¼ 2), bull mastiff (n ¼ 2), and all other breeds were represented by 1 dog each. Breeds in G2MMVD consisted of Cavalier King Charles Spaniel (n ¼ 14), and 1 each of mixed, border collie, dalmatian, Australian shepherd, and miniature schnauzer. Stage based on the American College of Veterinary Internal Medicine (ACVIM) guidelines included B1 (n ¼ 7), B2 (n ¼ 7), and C (n ¼ 5). Dogs with MMVD ACVIM Stage C were considered clinically stable based on the absence of active pulmonary edema. One dog in G2-MMVD with the highest cTnI value of 5.393 ng/mL was diagnosed as MMVD ACVIM Stage C with mild pulmonary hypertension and multiform ventricular tachycardia that required anti-arrhythmic therapy. Based on the presence of a clinically important
Table 2 Results for dilutional parallelism of the ADVIA Centaur TnI-Ultra immunoassay for measurement of cardiac troponin I in canine serum. Assay linearity was assessed at 2-fold dilutions ranging from 1:2 to 1:8. Serum sample 1
2
3
4
Dilution
Observed cTnI concentration (ng/mL)
Expected cTnI concentration (ng/mL)
O/E ratio (%)
Undiluted 1:2 1:4 1:8 Undiluted 1:2 1:4 1:8 Undiluted 1:2 1:4 1:8 Undiluted 1:2 1:4 1:8
0.066 0.028 0.010 0.006 0.768 0.268 0.116 0.059 2.826 1.229 0.505 0.246 4.117 1.942 0.820 0.373
e 0.033 0.017 0.008 e 0.384 0.192 0.096 e 1.413 0.707 0.353 e 2.059 1.029 0.515
e 84.8 60.6 60.6 e 69.8 60.4 61.5 e 87.0 71.5 69.6 e 94.3 79.7 72.5
cTnI, cardiac troponin I. O/E, observed-to-expected ratio.
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
Validation of a high-sensitivity cTnI assay in dogs
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Table 3 Results of spiking recovery for the ADVIA Centaur TnI-Ultra immunoassay for canine serum. Each sample was spiked into each of the other samples. Serum sample combination 1
2
3
4
Observed cTnI concentration (ng/mL)
Expected cTnI concentration (ng/mL)
O/E ratio (%)
0.386 1.443 2.069 0.386 1.853 2.214 1.443 1.853 3.330 2.069 2.214 3.330
0.407 1.353 2.208 0.407 1.690 2.545 1.353 1.690 3.491 2.208 2.545 3.491
95.0 107.0 93.7 95.0 109.6 87.0 107.0 109.6 95.4 93.7 87.0 95.4
þ2 þ3 þ4 þ1 þ3 þ4 þ1 þ2 þ4 þ1 þ2 þ3
cTnI, cardiac troponin I. O/E, observed-to-expected ratio.
arrhythmia of unknown etiology complicating the diagnosis of MMVD ACVIM Stage C, this dog was not included in the statistical analysis. Breeds in G3congenital consisted of boxer (n ¼ 3), maltese (n ¼ 2), Rhodesian ridgeback (n ¼ 2), and all other breeds were represented by 1 dog each. Diagnoses of dogs in G3-congenital included moderate to severe pulmonic stenosis (n ¼ 10), severe subaortic stenosis (n ¼ 3), left-to-right shunting patent ductus arteriosus (n ¼ 3), tricuspid valve dysplasia with concurrent atrial fibrillation (n ¼ 2), and a restrictive left-to-right shunting ventricular septal defect (n ¼ 1). Breeds in G4-arrhythmia consisted of boxer (n ¼ 5), labrador retriever (n ¼ 5), Doberman pinscher (n ¼ 3), collie (n ¼ 2), and all other breeds were represented by 1 dog each. Dogs in G4-arrhythmia had ventricular tachyarrhythmias (n ¼ 15), supraventricular tachycardia (n ¼ 5), and sick sinus syndrome (n ¼ 1). Dogs in G1-healthy and G3-congenital were both significantly (P < 0.05) younger than those in both G2-MMVD and G4-arrhythmia. There was no significant difference in age between dogs in G1healthy and G3-congenital, or between dogs in
G2-MMVD and G4-arrhythmia. Dogs in G2-MMVD weighed significantly (P < 0.05) less than those of G1-healthy and G4-arrhythmia, and those of G3congenital weighed significantly less than those of G4-arrhythmia. There was no significant difference in weight between dogs of G2-MMVD and those of G3-congenital, between those of G1-healthy and those of both G3-congenital and G4-arrhythmia. Dogs with cardiac disease (G2-MMVD, G3congenital, G4-arrhythmia) had significantly higher serum cTnI concentrations (P < 0.005) than healthy dogs (G1-healthy) (Fig. 1). There was no significant difference in serum cTnI concentrations between dogs of any of the 3 groups with cardiac disease (G2-MMVD, G3-congenital, or G4arrhythmia).
Discussion Our results indicate that the ADVIA Centaur TnIUltra immunoassay, a commercially available hs-cTnI assay for use in humans, can be used to accurately and reliably measure serum cTnI in dogs at lower detection limits. The majority of dogs
Table 4 Sex, age, weight, and serum cardiac troponin I concentrations (cTnI) for 84 client-owned dogs classified as healthy (G1-healthy) or having myxomatous mitral valve disease (G2-MMVD), congenital heart disease (G3congenital), or clinically relevant arrhythmias (G4-arrhythmia). Data for age, weight, and cTnI is expressed as median (range). M ¼ male; F ¼ female. G1ehealthy G2-MMVD G3-congenital G4-arrhythmia
N
M/F
Age (years)
Weight (kg)
cTnI (ng/mL)
24 19 19 22
9/15 7/12 8/11 9/13
2.6 (0.8e8.2) 9.6 (4.0e11.9) 5.3 (0.3e8.5) 8.7 (0.9e12.3)
27.1 (5.1e42.5) 8.7 (4.8e22.6) 19.0 (1.6e54.8) 33.2 (12.3e54.2)
0.017 (0.006e0.128) 0.085 (0.006e0.414) 0.088 (0.035e1.405) 0.247 (0.020e25.211)
cTnI, cardiac troponin I.
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
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Figure 1 Serum cardiac troponin I (cTnI) concentrations in healthy dogs (G1), dogs with myxomatous mitral valve disease (G2), dogs with congenital cardiac disease (G3), and dogs with clinically relevant arrhythmias (G4). There was no significant difference in serum cTnI concentrations between dogs in any of the 3 groups with cardiac disease (G2, G3, or G4). Dogs with cardiac disease had significantly higher serum cTnI concentrations than healthy dogs (G1). Lines depict the median for each group. P values < 0.05 were considered significant. *denotes a significant difference compared to the 3 other groups.
with and without cardiac disease had measurable concentrations of cTnI using this assay. Five healthy dogs in G1-healthy and 1 dog with cardiac disease in G2-MMVD (MMVD e ACVIM Stage B1) had cTnI measurements below the lower limit of detection of this assay. Dogs with cardiac disease had significantly higher concentrations of cTnI than dogs without cardiac disease. Validation for this assay consisted of using 4 canine serum samples with different concentrations of cTnI to assess linearity, spiking recovery, intra-assay CV, and inter-assay CV. Intra-assay CV and inter-assay CV were each
-, -e-
www.elsevier.com/locate/jvc
Analytical validation and clinical evaluation of a commercially available high-sensitivity immunoassay for the measurement of troponin I in humans for use in dogs Randolph L. Winter, DVM a,*, Ashley B. Saunders, DVM a, Sonya G. Gordon, DVM, DVSc a, Matthew W. Miller, DVM, MS a, Katharine T. Sykes, DVM b, Jan S. Suchodolski, Dr. med. vet., ¨rg M. Steiner, Dr. med. vet., PhD c PhD c, Jo a
Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA b North Houston Veterinary Specialists, Spring, TX, USA c Gastrointestinal Laboratory, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA Received 26 January 2014; received in revised form 11 March 2014; accepted 12 March 2014
KEYWORDS Arrhythmias; Biomarker; Cardiac; Congenital; Valve disease
Abstract Objective: To analytically validate a commercially available highsensitivity immunoassay for measurement of cardiac troponin I (cTnI) in humans for use in dogs and to evaluate serum cTnI concentrations in healthy dogs and 3 well-defined groups of dogs with common cardiac diseases. Animals: Canine serum samples were used for validation. 85 client-owned dogs including 24 healthy controls, 20 with myxomatous mitral valve disease, 19 with congenital heart disease, and 22 with arrhythmias. Methods: Four serum samples were used to analytically validate the ADVIA Centaur TnI-Ultra assay by assessing intra-assay variability, inter-assay variability, spiking recovery, and dilutional parallelism. Dogs were grouped based on examination, echocardiography, and additional testing as clinically indicated, and serum cTnI concentrations were compared.
* Corresponding author. E-mail address: [email protected] (R.L. Winter). 1760-2734/$ - see front matter ª 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jvc.2014.03.002
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
2
R.L. Winter et al. Results: Analysis of the serum samples used for validation revealed an intra-assay coefficient of variation between 3.6% and 5.7%, and an inter-assay coefficient of variation between 2.4% and 5.9%. Observed to expected ratios for spiking recovery were 97.9 8.6% (mean, SD). Observed to expected ratios for dilutional parallelism were 73.0 11.5% (mean, SD). Dogs with cardiac disease had significantly higher serum cTnI concentrations (P < 0.005) than healthy dogs. Conclusions: The ADVIA Centaur TnI-Ultra’s low limit of detection allows measurement of serum cTnI in the majority of dogs even with no or mild cardiac disease. Dilution of samples for measurement of values above the upper limit of detection is not reliable and therefore not recommended. Serum cTnI concentrations are significantly higher in dogs with cardiac disease compared to healthy dogs. ª 2014 Elsevier B.V. All rights reserved.
Abbreviations ACVIM American College of Veterinary Internal Medicine cTnI cardiac troponin I CV coefficient of variation hs-cTnI high-sensitivity cardiac troponin I MMVD myxomatous mitral valve disease
Introduction Cardiac troponin I (cTnI) is a cardiac-specific protein involved in cardiomyocyte contraction and relaxation.1 More specifically, it is one of 3 subunits of the cardiac troponin complex that plays a role in excitationecontraction coupling of the cardiomyocytes. Troponin I is also present in skeletal muscle, however, the cardiac-specific isoform of troponin I has a different amino acid sequence from its skeletal counterpart and both isoforms thus do not cross-immunoreact.1 Cardiac troponin I is normally bound to the actin filament and present in low concentration in the cytosol, but cardiomyocyte damage allows cTnI release into the extracellular space.1 Therefore, circulating concentrations of cTnI provide information about cardiac-specific injury. Canine and human cTnI share significant amino acid homology, thus cTnI assays designed for human patients can be used in canine patients with proper analytical validation.2 Circulating cTnI concentration has been used for the detection of both acute and chronic myocardial damage in multiple species, and is often utilized as adjunctive information to other diagnostic tests, such as thoracic radiography, electrocardiography, and echocardiography in case management.1e9 The concentration of circulating cTnI has been shown to be proportional to the degree of cardiomyocyte damage.1 Many different commercial assays are available,
both standard sensitivity cTnI assays and highsensitivity cTnI (hs-cTnI) assays. The main distinguishing characteristic of hs-cTnI assays is a lower level of cTnI detection compared to standard sensitivity cTnI assays which make them capable of detecting lower concentrations of circulating cTnI, potentially providing better sensitivity for detecting myocardial damage.10,11 In human medicine, cTnI assays can identify increases in serum cTnI in patients with acute diseases such as myocardial infarction,10,11 but also in chronic heart disease,12,13 congenital heart disease,14,15 and patients with clinically significant arrhythmias.16e18 In veterinary medicine, serum cTnI concentrations have been reported using standard sensitivity cTnI assays in dogs with myxomatous mitral valve disease (MMVD), dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and congenital heart disease.6,7,19e22 With standard cTnI assays, the lower limit of detection can be too high, limiting the ability to detect increases in cTnI and thereby limiting the assay’s clinical utility.7,21,23,24 The ADVIA Centaur TnI-Ultrad is a commercially available high-sensitivity 3-site assay for cTnI analysis that utilizes direct chemiluminometry with detectable cTnI serum concentrations reported by the manufacturer ranging from 0.006 to 50.0 ng/mL. This cTnI immunoassay has been previously validated for use in humans and cattle.25 The objectives of this study were to analytically validate the assay for use in dogs and to evaluate the clinical use of this assay in a population of healthy dogs and stratified in 3 welldefined groups of dogs with common cardiac diseases.
d ADVIA Centaur TnI-Ultra, Siemens Medical Solutions Diagnostics, New York, NY, USA.
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
Validation of a high-sensitivity cTnI assay in dogs
Animals, materials and methods Assay Cardiac troponin I analysis was performed with the ADVIA Centaur TnI-Ultra immunoassay, a highsensitivity 3-site assay using direct chemiluminometry. One polyclonal goat antibody directed against antitroponin I and labeled with acridium ester and 2 biotinylated mouse monoclonal antibodies directed against antitroponin I are used for cTnI detection. These capture antibodies that recognize amino acid sequences 41e49 and 87e91 in the central region of the cTnI molecule.10,25 The solid phase reagent is made of magnetic latex particles that have been conjugated with streptavidin. The biotinylated antibodies bind the cTnI in the serum, and the biotin is then captured by the streptavidin-labeled magnetic particles. A direct relationship exists between the cTnI amount in the sample and the amount of relative light emitted from the chemiluminescent reaction that ensues.10,25 The working range for cTnI is reported by the manufacturer as 0.006e50.0 ng/mL. The amount of serum required for the assay is 100 mL. The manufacturer’s specifications for this assay allow the use of either plasma (heparinized or EDTA) or serum. The data reported herein are for serum samples. Blood samples were collected from dogs presented to the Texas A&M University Veterinary Medical Teaching Hospital from October 2011 to December 2012. Blood was collected from the external jugular vein, and samples were placed into commercially available additive free glass tubes and allowed to rest for 15 min at room temperature to allow for complete blood clot formation. Serum was obtained via centrifugation and stored in 500e1000 mL aliquots in polypropylene tubes. Samples were immediately frozen and stored at 80 C until analysis. Frozen samples were allowed to slowly thaw at room temperature immediately prior to batch analysis. Assay linearity was assessed using dilutional parallelism (i.e. assessing assay performance by diluting samples with subsequent cTnI measurement) of serial twofold dilutions from 1:2 to 1:8.26 Assay accuracy was evaluated by spiking different sera with other sera of known concentrations of cTnI. The percentages of observed to expected values for serial dilution and spiking recovery were calculated as [observed value (ng/mL)/expected value (ng/mL)] 100.26 Precision was evaluated by the evaluation of selected serum samples 10 times within the same assay over a single day (i.e. within 1 assay plate) and calculating the intra-
3 assay coefficients of variation (CV).26 Reproducibility was determined by the evaluation of selected serum samples in 10 consecutive assay runs over 10 days (i.e. across multiple assay plates) and calculating inter-assay CV.26 Both the intraassay and inter-assay CV were calculated by % CV ¼ [SD/mean] 100.
Dogs Serum from 10 client-owned dogs was submitted for cTnI analysis for the purpose of using some of these dogs’ samples for analytical assay validation. Ten mL blood samples were obtained from each dog, and the serum cTnI values were evaluated in these dogs. Samples from 4 of the dogs were chosen to be used for the analytical assay validation. These 4 samples were chosen specifically because they were found to have different serum cTnI concentrations that covered a range of cTnI values. Serum samples were obtained from an additional 85 client-owned dogs that were presented for evaluation and assigned to 1 of 4 groups. Group 1 (G1-healthy) consisted of dogs that were considered apparently healthy with no abnormalities based on history, physical examination, echocardiogram, and laboratory analysis consisting of a complete blood-cell count and a serum biochemical analysis. The remaining 3 groups of dogs had cardiac disease and clinical evaluation consisted of a history, physical examination, and clinically indicated diagnostic tests. Dogs assigned to group 2 (G2-MMVD) had MMVD.27 Dogs assigned to group 3 (G3-congenital) had congenital heart disease. Dogs assigned to group 4 (G4-arrhythmia) had clinically relevant arrhythmias based on the presence of clinical signs and abnormalities on electrocardiography and 24h ambulatory electrocardiography. The dogs in G4-arrhythmia had clinical signs directly caused by the arrhythmia. This study was reviewed and approved by the Institutional Animal Care and Use Committee at Texas A&M University, College Station, TX.
Statistical analysis The D’Agostino and Pearson omnibus test was used to test each data set for age, weight, and troponin concentration for normality using a commercially available software package.e Data sets that failed normality testing were expressed as medians and e GraphPad Prism (v.6.0), GraphPad Software, San Diego, CA, USA.
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
4
R.L. Winter et al. Table 1 Precision and reproducibility of the ADVIA Centaur TnI-Ultra immunoassay for concentrations of cardiac troponin I in canine serum.
Intra-assay variability (n ¼ 10) Inter-assay variability (n ¼ 10)
Serum sample
Mean (ng/mL)
SD (ng/mL)
CV (%)
1 2 3 4 1 2 3 4
0.066 0.736 2.955 4.432 0.068 0.736 2.882 4.296
0.004 0.027 0.128 0.217 0.003 0.018 0.171 0.146
5.7 3.6 4.3 4.9 4.7 2.4 5.9 3.4
ranges, otherwise data were reported as mean SD. A KruskaleWallis one-way analysis of variance test was used to assess all data sets that failed normality testing for differences between groups. A Dunn’s multiple comparison test was used to test for differences between each group. A P-value < 0.05 was considered significant for all statistical analyses. Serum samples with cTnI concentrations below the lower limit of detection were assigned a value of 0.006 ng/mL.
Results Samples used for analytical assay validation had measured cTnI concentrations of 0.066, 0.768, 2.826, and 4.117 ng/mL. The 4 serum samples had intra-assay CV that ranged between 3.6 and 5.7%
and inter-assay CV that ranged between 2.4 and 5.9% (Table 1). Observed to expected ratios for serial dilutions for the 4 samples were 72.7 11.5% (mean, SD) with a range of 60.4e94.3% at dilutions of 1 in 2, 1 in 4, and 1 in 8 (Table 2). Observed to expected ratios for spiking recovery were 97.9 8.6% with a range of 87.0e109.6% for spiking each of the 4 serum samples with each of the other 3 serum samples (Table 3). The samples from 85 client-owned dogs were evaluated and group characteristics are reported in Table 4. Serum cTnI concentrations were detectable in 79/85 dogs. Values were below the lower detection limit in 5 dogs from G1-healthy and 1 dog from G2-MMVD. Breeds in G1-healthy consisted of mixed (n ¼ 4), labrador retriever (n ¼ 3), border collie (n ¼ 2), catahoula hog dog (n ¼ 2), bull mastiff (n ¼ 2), and all other breeds were represented by 1 dog each. Breeds in G2MMVD consisted of Cavalier King Charles Spaniel (n ¼ 14), and 1 each of mixed, border collie, dalmatian, Australian shepherd, and miniature schnauzer. Stage based on the American College of Veterinary Internal Medicine (ACVIM) guidelines included B1 (n ¼ 7), B2 (n ¼ 7), and C (n ¼ 5). Dogs with MMVD ACVIM Stage C were considered clinically stable based on the absence of active pulmonary edema. One dog in G2-MMVD with the highest cTnI value of 5.393 ng/mL was diagnosed as MMVD ACVIM Stage C with mild pulmonary hypertension and multiform ventricular tachycardia that required anti-arrhythmic therapy. Based on the presence of a clinically important
Table 2 Results for dilutional parallelism of the ADVIA Centaur TnI-Ultra immunoassay for measurement of cardiac troponin I in canine serum. Assay linearity was assessed at 2-fold dilutions ranging from 1:2 to 1:8. Serum sample 1
2
3
4
Dilution
Observed cTnI concentration (ng/mL)
Expected cTnI concentration (ng/mL)
O/E ratio (%)
Undiluted 1:2 1:4 1:8 Undiluted 1:2 1:4 1:8 Undiluted 1:2 1:4 1:8 Undiluted 1:2 1:4 1:8
0.066 0.028 0.010 0.006 0.768 0.268 0.116 0.059 2.826 1.229 0.505 0.246 4.117 1.942 0.820 0.373
e 0.033 0.017 0.008 e 0.384 0.192 0.096 e 1.413 0.707 0.353 e 2.059 1.029 0.515
e 84.8 60.6 60.6 e 69.8 60.4 61.5 e 87.0 71.5 69.6 e 94.3 79.7 72.5
cTnI, cardiac troponin I. O/E, observed-to-expected ratio.
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
Validation of a high-sensitivity cTnI assay in dogs
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Table 3 Results of spiking recovery for the ADVIA Centaur TnI-Ultra immunoassay for canine serum. Each sample was spiked into each of the other samples. Serum sample combination 1
2
3
4
Observed cTnI concentration (ng/mL)
Expected cTnI concentration (ng/mL)
O/E ratio (%)
0.386 1.443 2.069 0.386 1.853 2.214 1.443 1.853 3.330 2.069 2.214 3.330
0.407 1.353 2.208 0.407 1.690 2.545 1.353 1.690 3.491 2.208 2.545 3.491
95.0 107.0 93.7 95.0 109.6 87.0 107.0 109.6 95.4 93.7 87.0 95.4
þ2 þ3 þ4 þ1 þ3 þ4 þ1 þ2 þ4 þ1 þ2 þ3
cTnI, cardiac troponin I. O/E, observed-to-expected ratio.
arrhythmia of unknown etiology complicating the diagnosis of MMVD ACVIM Stage C, this dog was not included in the statistical analysis. Breeds in G3congenital consisted of boxer (n ¼ 3), maltese (n ¼ 2), Rhodesian ridgeback (n ¼ 2), and all other breeds were represented by 1 dog each. Diagnoses of dogs in G3-congenital included moderate to severe pulmonic stenosis (n ¼ 10), severe subaortic stenosis (n ¼ 3), left-to-right shunting patent ductus arteriosus (n ¼ 3), tricuspid valve dysplasia with concurrent atrial fibrillation (n ¼ 2), and a restrictive left-to-right shunting ventricular septal defect (n ¼ 1). Breeds in G4-arrhythmia consisted of boxer (n ¼ 5), labrador retriever (n ¼ 5), Doberman pinscher (n ¼ 3), collie (n ¼ 2), and all other breeds were represented by 1 dog each. Dogs in G4-arrhythmia had ventricular tachyarrhythmias (n ¼ 15), supraventricular tachycardia (n ¼ 5), and sick sinus syndrome (n ¼ 1). Dogs in G1-healthy and G3-congenital were both significantly (P < 0.05) younger than those in both G2-MMVD and G4-arrhythmia. There was no significant difference in age between dogs in G1healthy and G3-congenital, or between dogs in
G2-MMVD and G4-arrhythmia. Dogs in G2-MMVD weighed significantly (P < 0.05) less than those of G1-healthy and G4-arrhythmia, and those of G3congenital weighed significantly less than those of G4-arrhythmia. There was no significant difference in weight between dogs of G2-MMVD and those of G3-congenital, between those of G1-healthy and those of both G3-congenital and G4-arrhythmia. Dogs with cardiac disease (G2-MMVD, G3congenital, G4-arrhythmia) had significantly higher serum cTnI concentrations (P < 0.005) than healthy dogs (G1-healthy) (Fig. 1). There was no significant difference in serum cTnI concentrations between dogs of any of the 3 groups with cardiac disease (G2-MMVD, G3-congenital, or G4arrhythmia).
Discussion Our results indicate that the ADVIA Centaur TnIUltra immunoassay, a commercially available hs-cTnI assay for use in humans, can be used to accurately and reliably measure serum cTnI in dogs at lower detection limits. The majority of dogs
Table 4 Sex, age, weight, and serum cardiac troponin I concentrations (cTnI) for 84 client-owned dogs classified as healthy (G1-healthy) or having myxomatous mitral valve disease (G2-MMVD), congenital heart disease (G3congenital), or clinically relevant arrhythmias (G4-arrhythmia). Data for age, weight, and cTnI is expressed as median (range). M ¼ male; F ¼ female. G1ehealthy G2-MMVD G3-congenital G4-arrhythmia
N
M/F
Age (years)
Weight (kg)
cTnI (ng/mL)
24 19 19 22
9/15 7/12 8/11 9/13
2.6 (0.8e8.2) 9.6 (4.0e11.9) 5.3 (0.3e8.5) 8.7 (0.9e12.3)
27.1 (5.1e42.5) 8.7 (4.8e22.6) 19.0 (1.6e54.8) 33.2 (12.3e54.2)
0.017 (0.006e0.128) 0.085 (0.006e0.414) 0.088 (0.035e1.405) 0.247 (0.020e25.211)
cTnI, cardiac troponin I.
Please cite this article in press as: Winter RL, et al., Analytical validation and clinical evaluation of a commercially available highsensitivity immunoassay for the measurement of troponin I in humans for use in dogs, Journal of Veterinary Cardiology (2014), http://dx.doi.org/10.1016/j.jvc.2014.03.002
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Figure 1 Serum cardiac troponin I (cTnI) concentrations in healthy dogs (G1), dogs with myxomatous mitral valve disease (G2), dogs with congenital cardiac disease (G3), and dogs with clinically relevant arrhythmias (G4). There was no significant difference in serum cTnI concentrations between dogs in any of the 3 groups with cardiac disease (G2, G3, or G4). Dogs with cardiac disease had significantly higher serum cTnI concentrations than healthy dogs (G1). Lines depict the median for each group. P values < 0.05 were considered significant. *denotes a significant difference compared to the 3 other groups.
with and without cardiac disease had measurable concentrations of cTnI using this assay. Five healthy dogs in G1-healthy and 1 dog with cardiac disease in G2-MMVD (MMVD e ACVIM Stage B1) had cTnI measurements below the lower limit of detection of this assay. Dogs with cardiac disease had significantly higher concentrations of cTnI than dogs without cardiac disease. Validation for this assay consisted of using 4 canine serum samples with different concentrations of cTnI to assess linearity, spiking recovery, intra-assay CV, and inter-assay CV. Intra-assay CV and inter-assay CV were each
