Underestimates and overestimates of total thyroxine concentrations caused by unwanted thyroxine-binding protein effects

The first evidence of unwanted serum protein effects on analogue-based total thyroxine (T-4) determinations came from a study that varied serum protein concentrations while total T-4 concentrations were constant. The present study approached this issue by varying total T-4 concentrations while protein concentrations were constant. Four analogue-based total T-4 immunoassays were applied to solutions that contained either free T-4 without binding protein, a T-4-binding protein without T-4, or protein-bound T-4. When total T-4 concentrations were 3-12 mug/dL, the assays reported total T-4 determinations that ranged from none detected to 23 mug/dL. These T-4 determinations reflected the protein to which T-4 was bound, in addition to the level of T-4. Total T-4 was underrepresented when T-4 was unbound, or thyroxine-binding globulin (TBG) bound. Total T-4 was overrepresented when T4 was albumin-bound, or transthyretin-bound. There were substantial disparities among assays applied to the same total T-4 solutions. These assays reported no detectable T-4 when applied to T-4-binding protein solutions without protein-bound T-4. Nonetheless, T-4-binding proteins contributed to the underestimates and overestimates of protein bound T-4. Different forms of protein bound T-4 were quantified differently, evidence that protein-T-4 complexes persist during quantification. We attribute the unexpected total T-4 values to a combination of incomplete protein-bound T-4 release from T-4-binding proteins during quantification, and variably inaccurate quantifications of the protein-bound T-4 that remained.