Variable underestimates by serum free thyroxine (T4) immunoassays of free T4 concentrations in simple solutions

The immunoassays used to measure free T4 in serum frequently underestimate free T4 concentrations. These underestimates of serum free T4 are often greater than can be attributed to sample dilution. Unrecognized T4 sequestration by assay materials could cause such underestimates, but T4 sequestration during free T4 measurements has not been well studied. To study T4 sequestration, we used simple solutions containing gravimetrically determined quantities of T4 without T4 binding proteins. T4 sequestration was calculated as the differences between the actual quantities of free T4 required to obtain a specific free T4 measurement and the quantities that would, in theory, have been required, assuming zero T4 sequestration. Sequestration occurred in every serum free T4 assay studied, was often in the nanomolar range, and varied from 26-99+% of the actual amount of free T4 required to obtain free T4 measurements. We conclude that unrecognized T4 sequestration is a major confounding variable in free T4 assay performance, and that it explains the underestimates of serum free T4 concentrations by free T4 immunoassays which cannot be explained by sample dilution.