The Nature of Analogue-Based Free Thyroxine Estimates

Abstract

Clinical laboratories often use analogue-based immunoassays to estimate serum free thyroxine (FT₄) concentrations. These assays yield FT₄ estimates that correlate closely with thyroxine (T₄) binding protein concentrations. This correlation implies that either T₄ binding proteins or protein bound T₄ contribute to analogue-based FT₄ values. To study the contributions made by T₄ binding proteins to these FT₄ estimates further, four analoguebased FT₄ assays were applied to: (1) FT₄ solutions without T₄ binding proteins, (2) to T₄ binding protein solutions without T₄, and (3) to total T₄ solutions containing T₄ binding protein, FT₄, and protein-bound T₄. The FT₄ estimates obtained with these solutions ranged from 0.2–8.6 ng/dL, when FT₄ concentrations ranged from less than 0.2–12,000 ng/dL. In the FT₄ solutions, gravimetrically determined FT₄ concentrations were 500–12,000 ng/dL (0.5–12.0 µg/dL) without protein-bound T₄, and the FT₄ estimates obtained were 0.3–6.9 ng/dL. In the total T₄ solutions, dialyzable FT₄ concentrations were less than 0.2–59 ng/dL*, retained T₄ concentrations were 499.8–11,441 ng/dL, and the analogue-based FT₄ estimates obtained were 0.2–8.6 ng/dL. Similar FT₄ estimates (0.2–8.6 ng/dL and 0.3–6.9 ng/dL) were obtained with similar concentrations of either protein-bound T₄ or FT₄. Similar test results were associated with similar total T₄ concentrations, not similar FT₄ concentrations. Proteinbound T₄ and T₄ binding protein contributed variably to test results. T₄ quantifications included large analytical losses that are unaccounted for. These assays passed tests of correlation with FT₄ concentrations, but they failed tests of specificity for FT₄ and accuracy in T₄ quantification.