Several methods are used to evaluate binding proteins and to estimate the free T₄ concentration. A commonly used test is the T₃ uptake (T₃U) test.10 In this procedure, a patient’s serum sample is placed in a test tube to which is added a tracer amount of ¹²⁵I-T₃ and a resin or other solid phase matrix. During incubation, binding sites on the resin and on the patient’s serum binding proteins compete for the ¹²⁵I-T₃ until equilibrium is achieved. When the serum has an increased number of unoccupied binding sites, a greater percentage of the ¹²⁵I-T₃ binds to these serum proteins. When the resin is separated and counted for radioactivity, the decreased percentage of ¹²⁵I-T₃ resin uptake indicates an increased number of unoccupied TBG-binding sites. In contrast, when the patient’s TBG-binding sites are nearly saturated, the T₃U is elevated. The free thyroxine index (FT₄I) is calculated by multiplying the serum total T₄ by the percentage of resin uptake.

Several modifications have evolved in FT₄I determinations. To permit comparisons of varying methods, a thyroid hormone–binding ratio (THBR) can be derived by dividing the patient’s T₃U by the mean of a reference population. Multiplying the THBR by the total T₄ gives the FT₄I. A second change has been the development of nonisotopic automated methods. Rather than using T₃, T₄ is labeled (enzyme or fluorescein). This assay method is called a “T-uptake”.20