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/1.73m(2)) 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.