Systemic inaccuracies, proportional to the concentrations of serum proteins and the thyroxine (T4) they carry, have been reported in direct free T4 immunoassays. However, analytical recoveries of free T4 have not been carefully examined in most current methods, and they have not previously been examined across the pathophysiological range of serum T4 binding. In the present study we investigated ranges of serum T4 binding using free and total T4 measurements from 1359 individuals. Carefully characterized, gravimetrically calibrated, serum-based free T4 test solutions were then prepared with a constant normal free T4 concentration (12 ng/L) and varied serum T4 binding (approximately 300:1 to 24,000:1, ng protein bound T4: ng free T4). These standardized test solutions were analyzed using five T4 analog based free T4 methods. Analytical recoveries were calculated as ratios of actual free T4 measurements to the target value, and expressed as a percent of the target. Analytical recoveries were directly proportional to the extent of serum T4 binding and ranged 2% to 155%, 25% to 131%, 53% to 106%, 37% to 93%, and 37% to 73%, lowest to highest, in different methods. These systemic inaccuracies will confound interpretations of free T4 test results in clinical conditions with altered T4 binding. Future investigations into free T4 status must examine the analytical recovery of the free T4 method(s) used, as they relate to the extent of serum T4 binding in the clinical condition(s) studied.

Progressive dilution of normal sera causes little change in free T4 concentrations. Similar dilution of sera containing drug inhibitors of T4 binding to serum proteins causes a progressive fall in free T4. The low T4 syndrome of nonthyroidal illness is thought to be associated with a circulating inhibitor(s) of T4 binding. It would be expected, therefore, that dilution of sera from the low T4 syndrome might also result in a fall in free T4 concentrations. We compared the effect of progressive serum dilution on free T4 concentrations in low T4 syndrome sera to those in normal, hyperthyroid, pregnancy, TBG-deficient and salicylate-containing sera. A tracer dialysis method proved inappropriate for studying dilution effects. Using a new dialysate RIA method to measure free T4, we found a progressive fall in free T4 concentrations in sera from patients with the low T4 syndrome similar to that in serum containing salicylate, but not to that in normal sera. The magnitude of the fall varied widely among individual patients. Free T4 methods which use a diluted serum sample will underestimate free T4 concentrations in the low T4 syndrome.

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.

A B S T R A C T A radioimmunoassay has been developed for the measurement of 3,5-diiodo-L-tyrosine (DIT)

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