Underestimates of serum free thyroxine (T4) concentrations by free T4 immunoassays.

Abstract

Different free T4 (FT4) assays often give different FT4 measurements, and conflicting measurements have been striking in nonthyroidal illness. Because FT4 immunoassays depend upon serum protein-bound T4 (PBT4) dissociation to stabilize the FT4 concentration during assay perturbations, interassay differences in perturbations combined with variation in serum PBT4 concentrations could produce discordant FT4 measurements. This study examined the effects of PBT4 on FT4 measurements obtained by direct immunoassay methods. Standard solutions with constant FT4 levels and varying PBT4 concentrations were prepared and analyzed by direct equilibrium dialysis, two-step immunoextraction, one-step labeled T4 antibody, and one-step labeled T4 analog FT4 methods. Direct equilibrium dialysis results were independent of PBT4 concentrations and gave correct measurements of serum FT4 when the PBT4 concentration was above 8 nmol/L or 0.6 micrograms/dL, but were PBT4 dependent and underestimated serum FT4 at lower PBT4 concentrations. The other three methods were PBT4 dependent and variably underestimated serum FT4 at all levels of PBT4 up to 256 nmol/L (19.9 micrograms/dL), the highest level studied. Thus, PBT4-dependent underestimates of serum FT4 occurred with all four methods, whereas the measured FT4 level at each PBT4 concentration varied widely between methods. A serum PBT4 dependent bias causes discordant FT4 measurements and probably explains the observed underestimates of FT4 in nonthyroidal illness.

Circulating Thyroid Hormone Concentrations and Placental Thyroid Hormone Receptor Expression in Normal Human Pregnancy and Pregnancy Complicated by Intrauterine Growth Restriction (IUGR)

“…The relatively small sample volume obtained at cordocentesis determined that it was not feasible to employ a direct equilibrium dialysis method for measurement of free thyroid hormone concentrations in fetal serum. Underestimates of free thyoid hormones have been reported with related methods in several clinical situations, including nonthyroidal illness (31), a factor that should be taken into account when interpreting absolute concentrations from immunoassays reported here. Results for free T 4 obtained with this method of two-step, back-titration, immunoassay have been compared with equilibrium dialysis (8,30) and have been shown to correlate with free thyroid hormone concentrations measured using equilibrium dialysis.…”

Serum substances that interfere with thyroid hormone assays in patients with chronic renal failure

“…The addition of these organic acids to pooled serum from healthy subjects did not interfere with this assay. Serum protein bound T4 is reported to affect various FT4 immunoassays (Nelson ., 1994). Since the concen_trations of protein‐bound T4 and albumin may be lower in uraemic sera than in normal sera, the interference by organic acids with the assay in uraemic sera may be more prominent.…”

Thyroid Hormone Metabolism and Thyroid Diseases in Chronic Renal Failure

“…Immunoextraction or RIA methods estimate free T 4 by either a T 4 analog or two-step-back-titration with a solid phase T 4 antibody, without use of semipermeable membranes to separate free from bound hormone. Free T 4 values paralleled concentrations of protein-bound T 4 in these nondialysis methods (52). However, in patients with significant reductions of serum T 4 binding to serum carrier proteins, such as in nonthyroidal illnesses, all but the direct equilibrium dialysis method may provide spurious results (51).…”

“…Free T 4 index methods correct total T 4 values directly or indirectly for altered serum concentrations of TBG. Free T 4 immunoassays depend upon serum protein-bound T 4 dissociation to stabilize free T 4 concentrations during assay perturbations (52). By design, all free T 4 methods provide normal values for healthy euthyroid subjects with modestly increased or decreased TBG concentrations (low levels in hypothyroidism and high values in hyperthyroidism) in oth-* erwise well patients (51).…”