The nature of analogue-based free thyroxine estimates

Clinical laboratories often use analogue-based immunoassays to estimate serum free thyroxine (FT4) concentrations. These assays yield FT4 estimates that correlate closely with thyroxine (T-4) binding protein concentrations. This correlation implies that either T-4 binding proteins or protein bound T-4 contribute to analogue-based FT4 values. To study the contributions made by T-4 binding proteins to these FT4 estimates further, four analogue-based FT4 assays were applied to: (1) FT4 solutions without T-4 binding proteins, (2) to T-4 binding protein solutions without T-4, and (3) to total T-4 solutions containing T-4 binding protein, FT4, and protein-bound T-4. 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 mug/dL) without protein-bound T-4, 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 T-4 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 T-4 or FT4. Similar test results were associated with similar total T-4 concentrations, not similar FT4 concentrations. Protein-bound T-4 and T-4 binding protein contributed variably to test results. T-4 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 T-4 quantification.