Sex-specific 99th percentiles derived from the AACC Universal Sample Bank for the Roche Gen 5 cTnT assay: Comorbidities and statistical methods influence derivation of reference limits

Objectives Our purpose was to determine a) overall and sex-specific 99th percentile upper reference limits (URL) and b) influences of statistical methods and comorbidities on the URLs. Methods Heparin plasma from 838 normal subjects (423 men, 415 women) were obtained from the AACC (Universal Sample Bank). The cobas e602 measured cTnT (Roche Gen 5 assay); limit of detection (LoD), 3 ng/L. Hemoglobin A1c (URL 6.5%), NT-proBNP (URL 125 ng/L) and eGFR (60 mL/min/1.73m²) were measured, along with identification of statin use, to better define normality. 99th percentile URLs were determined by the non-parametric (NP), Harrell-Davis Estimator (HDE) and Robust (R) methods. Results 355 men and 339 women remained after exclusions. Overall < 50% of subjects had measureable concentrations ≥ LoD: 45.6% no exclusion, 43.5% after exclusion; compared to men: 68.1% no exclusion, 65.1% post exclusion; women: 22.7% no exclusion, 20.9% post exclusion. The statistical method used influenced URLs as follows: pre/post exclusion overall, NP 16/16 ng/L, HDE 17/17 ng/L, R not available; men NP 18/16 ng/L, HDE 21/19 ng/L, R 16/11 ng/L; women NP 13/10 ng/L, HDE 14/14 ng/L, R not available. Conclusions We demonstrated that a) the Gen 5 cTnT assay does not meet the IFCC guideline for high-sensitivity assays, b) surrogate biomarkers significantly lowers the URLs and c) statistical methods used impact URLs. Our data suggest lower sex-specific cTnT 99th percentiles than reported in the FDA approved package insert. We emphasize the importance of detailing the criteria used to include and exclude subjects for defining a healthy population and the statistical method used to calculate 99th percentiles and identify outliers.