Benjamin Beppler (Presenter)
TriCore Reference Laboratories
Bio: Ben Beppler has worked at TriCore Reference Laboratories for 3 years, where he currently serves as a Development Scientist specializing in separation sciences. His assay development responsibilities include hormone, vitamin, therapeutic drugs, and drugs of abuse assays.
Authorship: Benjamin Beppler, Teal Clocksin, Michael Crossey
TriCore Reference Laboratories, Albuquerque, NM
Most clinical laboratories opt to calculate free testosterone based on direct measurements of total testosterone and appropriate binding proteins. The reliability of this calculation, however, is complicated by a variety of factors. In this talk, we demonstrate the effect of choosing different calculations, total testosterone methods, and Sex Hormone Binding Globulin (SHGB) assays, as well as the importance to compare calculated results against a reference method using the most appropriate regression analysis. Options regarding choosing appropriate reference ranges will also be discussed.
Free testosterone (unbound, freely circulating testosterone) or bioavailable testosterone (free and weakly bound testosterone) is often a more clinically useful measurement than total testosterone due do the inability of testosterone tightly bound to Sex Hormone Binding Globulin (SHBG) to perform a variety of biological functions[1-3]. Direct measurement of free testosterone is either tedious and impractical (dialysis or ultrafiltration) or unreliable (RIA) for clinical laboratories. Therefore, most clinical laboratories opt to calculate free testosterone based on direct measurements of total testosterone and appropriate binding proteins (SHBG and Albumin). This calculation, however, is complicated by the choice of equation, the value of association constants used in the equation, and the quality of the measurement methods of total testosterone, SHBG, and Albumin. In this study, we have investigated 3 independent total testosterone methods (LC-MS/MS and IA), 2 independent SHBG methods (IA), incorporation of measured versus average albumin concentrations, 3 different published calculation methods, and a comparison of calculated free testosterone to a direct measurement via tracer equilibrium dialysis LC-MS/MS for over 150 specimens. Our analysis has led to the following conclusions: (1) The CV% of calculated free testosterone is significant lower than that of the ED-LC-MS/MS, especially at low concentrations (5-7% vs 13-20% below 3 pg/mL), provided that the CV% of total testosterone is sufficiently low; (2) The choice of SHBG assay can introduce a significant (>30%) bias in the calculated free testosterone result; (3) The use of a LC-MS/MS total testosterone method is critically important for low testosterone samples (< 50 ng/dL); (4) Comparison to a reference free testosterone method (e.g. ED-LC-MS/MS) is necessary even if the accuracy of the total testosterone method is verified, and an offset may be necessary to align with the comparison method; and (5) The choice of regression analysis has a significant effect on the comparison of methods, and an inappropriate choice can lead to falsely confirmed agreement.
References & Acknowledgements:
 Bhasin S, Cunningham GR, Hayes FJ, et al. Testosterone therapy in adult men with androgen deficiency syndromes: An Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2010; 95:2536-2559.
 Bremer, AA. Polycystic Ovary Syndrome in the Pediatric Population. Metab Syndr Relat Disord. 2010 Oct; 8(5): 375–394.
 Miller KK. Androgen Deficiency in Women. J Clin
Endocrinol Metab. 2001; 86:2395-2401.
 De Ronde W, Van der Schouw YT, Pols HAP, Gooren LJG, Muller M, Grobbee D, De Jong FH. Calculation of Bioavailable and Free Testosterone in Men: A Comparison of 5 Published Algorithms. Clin Chem 2006; 52:1777-1784.
IP Royalty: no
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