Serum thyroxine binding capacity-dependent bias in five free thyroxine immunoassays: assessment with serum dilution experiments and impact on diagnostic performance

Objectives: To assess the presence and magnitude of a serum thyroxine binding capacity (sBC)-dependent bias in five free thyroxine (FT 4) immunoassays, compared with equilibrium dialysis (ED). The exhibited bias is confronted with clinical results from previous studies to evaluate its impact on FT 4 determination in sera with various sBC. Design and methods: The sera of three pregnant women and three non thyroidal ill (NTI) patients were serially diluted in an inert buffer to progressively decrease the sBC. FT 4 values were measured in diluted and undiluted samples with the six assays. Results: As a function of increasing dilution performed on pregnancy sera, except for ED and Vitros ECi FT 4, the other four FT 4 assay results decreased to different degrees, in the following order: two-step GammaCoat RIA< Elecsys< ADVIA:Centaur< Amerlex-MAB RIA. In sera from NTI patients, the decrease was more marked and found at high dilution with the Vitros ECi assay. Data from previous studies showed that FT 4 measured with biased assays were decreased only in samples from very severely NTI patients with low sBC and that FT 4 results in pregnancy sera with high sBC were not significantly biased. Conclusions: The dilution test is a sensitive alarm to assess sBC-dependent bias in FT 4 assays. For all FT4 assays and particularly when a bias is observed, documentation should be sought on the diagnostic performance of the assay and supported by a detailed clinical study including samples with low sBC. Physicians should still be educated about the limitations of FT 4 assays.