R. Bruce Wilcox

Analog-Based Free Testosterone Test Results Linked to Total Testosterone Concentrations, Not Free Testosterone Concentrations

BACKGROUND Analog-based free testosterone test results, sex hormone binding globulin (SHBG) concentrations, and total testosterone concentrations are somehow related. This study used new experiments to clarify these relationships. METHODS An analog-based free testosterone immunoassay and a total testosterone immunoassay were applied to well-defined fractions of serum testosterone. First, they were applied to the 2 fractions (retentate and dialysate) of normal male serum obtained by equilibrium dialysis. Second, they were applied to covaried concentrations of SHBG and total testosterone. Third, they were applied to decreasing concentrations of SHBG and protein-bound testosterone, offset by increasing concentrations of protein-free testosterone, while total testosterone was held constant. RESULTS The analog-based free testosterone assay and the total testosterone assay detected and reported serum testosterone test results from serum retentate, whereas neither assay detected the free testosterone in serum dialysate. Test results reported by the analog-based free testosterone assay followed varied concentrations of SHBG and total testosterone. When total testosterone was held constant, however, analog-based free testosterone test results did not follow varied concentrations of serum proteins or of free testosterone. CONCLUSION An analog-based free testosterone immunoassay reported free testosterone test results that were related to total testosterone concentrations under varied experimental conditions. This alleged free testosterone assay did not detect serum free testosterone (the test results it reported were nonspecific) and should not be used for this purpose.

Counterpoint: Legitimate and Illegitimate Tests of Free-Analyte Assay Function: We Need to Identify the Factors That Influence Free-Analyte Assay Results

There have been, and continue to be, reports of discordant free thyroxine (T4)1 measurements when different free-T4 methods are applied to the same serum samples (1)(2)(3)(4). Our laboratory has attempted to identify some of the factors that contribute to these disparities. There are 4 separate components of serum T4: free T4, albumin-bound T4, transthyretin-bound T4, and thyroxine-binding globulin (TBG)-bound T4. Free T4 is the one component that moves across semipermeable membranes during equilibrium dialysis and ultrafiltration. As Midgley and Christofides point out, the gold standard free-T4 methods are designed to measure this component. We applied analog-type methods and an equilibrium-dialysis/immunoassay gold standard method to carefully prepared solutions in which we varied these 4 components of serum T4. This equilibrium-dialysis method agrees closely with a liquid chromatography–tandem mass spectrometry method applied to serum ultrafiltrates (4). We also applied this equilibrium dialysis/immunoassay and a group of 7 analog-type free-T4 assays to solutions of T4 that did not include T4-binding proteins (5); that is, the only form of T4 was free T4. The analog-type assays required 28- to 588-fold higher concentrations of free T4 than …

Assessing thyroid hormone status in a patient with thyroid disease and renal failure: from theory to practice.

A 35-year-old Asian male, treated for hyperthyroidism, systemic lupus erythematosis, and uremia presented with low serum total thyroxine (T4) and normal serum thyrotropin (TSH) levels. He had been receiving prednisone and methimazole for 15 weeks. Free T4 measured by direct equilibrium dialysis was in the hypothyroid range (0.3 ng/dL; normal, 0.8-2.7). Two possibilities were considered: (1) a weakly bound dialyzable inhibitor in uremic serum that interfered with this serum free T4 determination or (2) hypothyroidism with persistent TSH suppression because of prior hyperthyroidism. To determine whether a weakly bound inhibitor was involved, the patients serum was serially diluted using two diluents: (1) an ultrafiltrate of the patients serum, which would contain any unbound inhibitor, as well as free T4 and (2) an inert diluent. Free T4 measurements were similar with both, providing evidence against the presence of a dialyzable and ultrafilterable inhibitor. In conclusion, this patient was hypothyroid because of antithyroid drug administration, associated with prolonged central TSH suppression from preexisting hyperthyroidism. Discontinuation of methimazole resulted in normalization of serum total T4 and TSH values. Thus, paired, serial serum dilutions, using two different diluents, provided evidence for differentiation of appropriately low free T4 measurements (because of hypothyroidism), from spuriously low free T4 measurements (because of an interfering inhibitor).