As discussed previously, T4 is highly protein-bound (99.97%) and, when bound to proteins, is metabolically inactive. Historically, T4 was determined using competitive protein-binding methods, followed by immunoassay methods employing radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and enzyme immunoassay (EIA) principles. Despite high degrees of analytical specificity, such assays do not provide adequate clinical information to diagnose thyroid disease, as the protein binding effects of T4 must be considered.
More recently, methods for determining the free levels of T4 (FT4) have become common in clinical laboratories to directly determine the metabolically available level of circulating T4. Direct immunoassays for FT4 are far more sensitive than traditional total T4 assays, capable of measuring nanogram concentrations of the free and active hormone.
A suggested FT4 reference interval is shown, however assays from different manufacturers are known to vary widely; reference intervals must be established in each clinical laboratory.
For accurate assessment of thyroid function during pregnancy, measurement of total T4 is recommended over FT4, because the alterations in serum proteins may cause falsely low levels of FT4.