SUN-144 - Assessment of Free Testosterone Measurement Methods: Findings from an Interlaboratory Comparison Study

Free testosterone (FT) is widely used in clinical diagnosis, management, and research of various conditions, such as hypogonadism and female hyperandrogenism. Accurate and reliable laboratory FT measurements are critical for the correct diagnosis and treatment of patients, and interpretation of research data. FT is currently measured in clinical practice using direct commercial immunoassays, assays using equilibrium dialysis (ED) or other techniques or estimated by calculating free testosterone (cFT) using different formulas based on the levels of total testosterone and sex hormone binding globulin (SHBG). There are concerns about the accuracy and comparability of current methods, that can limit the use of FT testing in patient care, public health, and research.
To address these concerns, CDC’s Clinical Standardization Programs (CDC CSP) conducted an interlaboratory comparison study to evaluate the analytical performance and inter-assay agreement of different FT measurement methods used in clinical laboratories. A total of 10 FT methods performed by 8 laboratories and assay manufacturers participated: six laboratory developed tests (LDTs) based on ED liquid chromatography tandem mass spectrometry, two commercial immunoassays (IAs), and two calculated FT methods based on the Vermeulen formula. Participants analyzed 43 single-donor adult male and female donor serum samples in duplicate on two days (N=4 per sample). FT concentrations of the samples ranged from 2.9 to 339 pg/mL. The reported results were compared against those obtained using CDC ED LC-MS/MS routine FT method. The study showed significant inconsistencies across methods. Immunoassays (IAs) exhibited notably lower measurements, with a negative mean bias ranging from -86.5% to -67.7%. While some ED LDTs demonstrated results comparable to CDC method (mean bias: -9.2% to 2.1%), other ED-based LDTs and cFT estimates showed substantial overestimation, with positive bias ranging from 46% to 136%. Large variability was observed between LDTs and IAs.
CDC CSP interlaboratory comparison of FT methods found large variability among participating methods and highlights the need for measurement standardization to improve the accuracy of FT measurements. The findings support the Endocrine Society’s recommendation that direct IAs should not be used in clinical practice. The study also allowed CDC CSP to identify factors contributing to assay variabilities such as use of various sample-to-buffer ratios during the ED step and application of dilution factors when calculating FT concentrations in the dialysate. These results emphasize the importance of addressing inconsistencies in FT measurement and provide valuable insights for advancing future efforts to standardize FT measurements.

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