Analytical validation and establishment of reference intervals for a ‘high-sensitivity’ cardiac troponin-T assay in horses

Abstract

BackgroundCardiac troponin-I assays have been validated in horses.’High-sensitivity’ cardiac troponin assays are now the standard in human cardiology. Objective: Appropriately validate the’high-sensitivity’ cardiac Troponin-T (hscTnT) assay for clinical use in horses, establish reference intervals, determine the biological variation, and demonstrate assay utility in selected clinical cases. Methods: Analytical validation of the Roche hscTnT assay included within- and between-run precision, linear dose response, limit of quantitation (LoQ), stability, and comparison with cTn-I (iSTAT). Reference intervals and biological variation were determined using adult, healthy, Non-Competition Horses (N = 125) and Racing-Thoroughbreds (N = 178). HscTnT levels were measured in two horses with cardiac pathology.ResultsThe hscTnT demonstrates acceptable within-run (L1 = 6.5 ng/L, CV 14.9 %, L2 = 10.1 ng/L, CV 8.7 %, L3 = 15.3 ng/L, CV 5.4 %) and between-run precision (L1 = 12.2 ng/L, CV 8.4 %, L2 = 57.0 ng/L, CV 8.4 %, L3 = 256.0 ng/L, CV 9.0 %). The assay was linear from 3 to 391 ng/L. The LoQ was validated at 3 ng/L. Samples demonstrated insignificant decay over freeze-thaw cycle. Comparison with cTnI assay showed excellent correlation (range: 8.0–3535.0 ng/L, R2 = 0.9996). Reference intervals: The upper 95th and 99th percentile of the hscTnT population distribution were 6.8 and 16.2 ng/L in Non-Competition Horses, and 14.0 and 23.2 ng/L in Racing-Thoroughbreds. Between-breed, diurnal effect, and between-day variation was below LoQ. Two clinical cases with presumed cardiac pathology had hscTnT levels of 220.9 ng/L and 5723.0 ng/L.ConclusionsThis benchmark study is the first to comply with CLSI guidelines, thus further establishing the performance characteristics of the hscTnT assay, and reference intervals in healthy horses. Two clinical cases demonstrated further the clinical utility of the assay.

24‐Hour Kinetics of Cardiac Troponin‐T Using a “High‐Sensitivity” Assay in Thoroughbred Chuckwagon Racing Geldings after Race and Associated Clinical Sampling Guidelines

“…Thirty‐seven apparently healthy, actively competing Thoroughbred Chuckwagon racing geldings (ages 5–16 years; median, 9 years) were included in the study. Another horse (1/38) had cTnT plasma concentrations that exceeded the 99th percentile URL previously published for the hscTnT assay at all but one time‐point, and was removed from the study population before data analysis.…”

“…In addition, all horses entered to race passed a veterinary lameness examination before competing. Any horse with baseline plasma cTnT concentrations that exceeded the 99th percentile URL using the hscTnT assay was excluded from data analysis because pre‐existing myocardial damage could not be definitively ruled out.…”

“…Specimens were immediately centrifuged at 2000 × g for 10 minutes, separated, frozen within 90 minutes, and stored at −80°C until batch analyzed. The hscTnT assay,b a fifth‐generation electrochemiluminescence immunoassay, was used on the Cobas‐e601 Analyzerc for analysis of plasma samples, complying with manufacturer's instructions and Calgary Laboratory Services (CLS) internal quality controls systems, and recently validated for use on equine plasma . Degree of hemolysis in all samples was assessed using the serum indices instrument application on the Roche Cobas‐e601 platformc and no specimen reached the hemolysis threshold for exclusion.…”

“…They are central to the diagnoses of acute myocardial infarctions (AMI in humans), but increases in cTn also are apparent in conditions that result in cardiac stress in the absence of obstructive coronary disease . Meeting the stringent recommendations of the Clinical and Laboratory Standards Institute (CLSI), the National Academy of Clinical Biochemistry (NACB), and the American Society of Veterinary Clinical Pathology (ASVCP), the “high‐sensitivity” cTnT (hscTnT) assay recently has been validated for use in horses with total imprecision ≤10% at the 99th percentile and measurable cTn analyte concentrations obtained in >50% of the populations studied . Upper reference limits (URL) for the 95th and 99th population distribution percentiles have been defined for apparently healthy horses and Thoroughbred Chuckwagon racing geldings and plasma concentrations have been described in 2 populations of horses with cardiac damage .…”

“…Meeting the stringent recommendations of the Clinical and Laboratory Standards Institute (CLSI), the National Academy of Clinical Biochemistry (NACB), and the American Society of Veterinary Clinical Pathology (ASVCP), the “high‐sensitivity” cTnT (hscTnT) assay recently has been validated for use in horses with total imprecision ≤10% at the 99th percentile and measurable cTn analyte concentrations obtained in >50% of the populations studied . Upper reference limits (URL) for the 95th and 99th population distribution percentiles have been defined for apparently healthy horses and Thoroughbred Chuckwagon racing geldings and plasma concentrations have been described in 2 populations of horses with cardiac damage . In numerous studies of healthy humans, in laboratory rodents, in sled‐dogs, as well as a few studies of horses, increases in cTn concentrations have been identified after exercise .…”