VARIABLE UNDERESTIMATES BY SERUM-FREE THYROXINE (T-4) IMMUNOASSAYS OF FREE T-4 CONCENTRATIONS IN SIMPLE SOLUTIONS

The immunoassays used to measure free T-4 in serum frequently underestimate free T-4 concentrations. These underestimates of serum free T-4 are Often greater than can be attributed to sample dilution. Unrecognized T-4 sequestration by assay materials could cause such underestimates, but T-4 sequestration during free T-4 measurements has not been well studied. To study T-4 sequestration, we used simple solutions containing gravimetrically determined quantities of T-4 without T-4 binding proteins. T-4 sequestration was calculated as the differences between the actual quantities of free T-4 required to obtain a specific free T-4 measurement and the quantities that would, in theory, have been required, assuming zero T-4 sequestration. Sequestration occurred in every serum flee T-4 assay studied, was often in the nanomolar range, and varied from 26-99+% of the actual amount of free T-4 required to obtain free T-4 measurements. We conclude that unrecognized T-4 sequestration is a major confounding variable in free T-4 assay performance, and that it explains the underestimates of serum free Ta concentrations by free T-4 immunoassays which cannot be explained by sample dilution.