Table 3.

The Relative Merits and Demerits of Various Methods of Measuring Free and Bioavailable Testosterone Levels

Method	Merits	Problems
Bioavailable Testosterone
Ammonium sulfate precipitation of SHBG-bound testosterone	• Correlates well with free testosterone obtained by equilibrium dialysis	• Technically difficult • Not easily automated • Few clinical laboratories measure it routinely • Conceptually measures non−SHBG-bound testosterone, which approximates but does not equal HSA-bound plus unbound testosterone
Concanavalin A method	• More selective and less variable than ammonium sulfate precipitation to precipitate SHBG	• Technically difficult • Not easily automated • Not used currently by clinical laboratories • Measures non−SHBG-bound testosterone, which approximates but does not equal HSA-bound plus unbound testosterone
Calculated bioavailable testosterone	• Based on law-of-mass-action theory or empirical equations • Simple to obtain	• Correlation between different algorithms is poor unless revalidated in a local laboratory • Dependent on correct estimation of the association constants for the binding of testosterone to SHBG (KT) and HSA (KHSA) • Results affected by the quality of total testosterone and SHBG and HSA measurements

Free Testosterone
Equilibrium dialysis	• The reference method against which other methods are compared	• Technically difficult; operations in which the dialysis is performed vary across laboratories, contributing to high interlaboratory variability • Not easily automated • Few hospital clinical laboratories perform this assay • Expensive • Relies on accuracy and precision of total testosterone
Ultracentrifugation	• Comparable to equilibrium dialysis	• Technically difficult • Not easily automated • Few clinical laboratories measure it routinely • Expensive • Relies on accuracy and precision of total testosterone
Free androgen index	• Represents the ratio of total testosterone/SHBG • Has been shown to correlate with free testosterone measurements • Simple to obtain	• Overly simplistic and inaccurate measure of free testosterone concentrations • Poor indicator of gonadal status • Dependent on accurate measurements of total testosterone and SHBG • Most experts do not favor its use
Analogue immunoassays	• Commercially available kits • High throughput and precision • Has been shown to correlate with free testosterone measurements	• Provides inaccurate estimates of free testosterone • Experts recommend against the use of direct analogue assays for measurement of free testosterone.
Salivary testosterone	• Simple to obtain	• May not be an accurate marker of circulating free testosterone concentrations • Affected by sample desiccation, contamination by food and blood
Calculated free testosterone	• Easy to use algorithms based on various models of testosterone binding to SHBG or empirical equations • Simple to obtain	• Dependent upon correct estimates of the association constants and stoichiometry for binding of testosterone to SHBG and HSA • Accuracy and precision affected by the accuracy and precision of the total testosterone and SHBG assays