Underestimates and Overestimates of Total Thyroxine Concentrations Caused by Unwanted Thyroxine-Binding Protein Effects

Abstract

The first evidence of unwanted serum protein effects on analogue-based total thyroxine (T₄) determinations came from a study that varied serum protein concentrations while total T₄ concentrations were constant. The present study approached this issue by varying total T₄ concentrations while protein concentrations were constant. Four analogue-based total T₄ immunoassays were applied to solutions that contained either free T₄ without binding protein, a T₄-binding protein without T₄, or protein-bound T₄. When total T₄ concentrations were 3–12 µg/dL, the assays reported total T₄ determinations that ranged from none detected to 23 µg/dL. These T₄ determinations reflected the protein to which T₄ was bound, in addition to the level of T₄. Total T₄ was underrepresented when T₄ was unbound, or thyroxine-binding globulin (TBG) bound. Total T₄ was overrepresented when T₄ was albumin-bound, or transthyretin-bound. There were substantial disparities among assays applied to the same total T₄ solutions. These assays reported no detectable T₄ when applied to T₄-binding protein solutions without protein-bound T₄. Nonetheless, T₄-binding proteins contributed to the underestimates and overestimates of protein bound T₄. Different forms of protein bound T₄ were quantified differently, evidence that protein–T₄ complexes persist during quantification. We attribute the unexpected total T₄ values to a combination of incomplete protein-bound T₄ release from T₄-binding proteins during quantification, and variably inaccurate quantifications of the protein-bound T₄ that remained.