What methods are used to measure free thyroxine in euthyroid sick syndrome?

Updated: Aug 28, 2020
  • Author: Serhat Aytug, MD; Chief Editor: Romesh Khardori, MD, PhD, FACP  more...
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An ongoing controversy concerns true free T4 levels in NTI. Various studies use different techniques to measure free T4 in NTI, but all methods have been challenged. Using these methods, free T4 has been found to be within the reference range, low, and high. The results of free T4 assays in NTI are method dependent and may be influenced by many variables.

Several methods can be used to measure free T4 directly, including equilibrium dialysis, a 2-step immunoextraction technique, a 1-step (analog) method, FTI (T3 resin-binding ratio), and ultrafiltration. Equilibrium dialysis usually is the reference method. In equilibrium dialysis, a small amount of radioactive tracer T4 and the unknown sample are placed in a dialysis membrane, which limits the diffusion of bound T4. The proportion of the hormone that is dialyzable (ie, free) is determined. The dialyzable hormone–to–total hormone ratio is used, with the concentration of T4 determined in a standard assay and then used to calculate the concentration of free T4.

A second type of assay is the 2-step radioimmunoassay (RIA). The patient's serum is equilibrated with a solid phase antibody to T4. The unoccupied antibody binding sites are quantified in a second step in which labeled hormone is added to the solid phase system. The 2-step assay appears to have the best correlation with equilibrium dialysis results.

The 1-step (analog) assay uses an analog, usually an alanine substitution for T4. The analog does not bind to proteins in the serum but does compete for binding with antibody to T4. The analog also binds to albumin, which has a low affinity but high capacity; therefore, if albumin concentration changes, then free T4 measurements change (ie, if albumin increases, free T4 decreases and vice versa). Such changes can produce spurious results. This technique is not used widely.

An FTI is calculated by multiplying the total T4 concentration by the T3 uptake (T3U). The T3U is an indirect estimate free T4 fraction, which is obtained by adding labeled T3 to serum and estimating how much of it remains free for binding to a secondary binder (eg, charcoal, talc, ion-exchange resin, anti-T3 antibody, immobilized albumin) added to the serum. In this way, the FTI reflects the actual free T4 concentration, although this appears to be less accurate in cases of very low or high TBG concentrations. The use of FTI had poor reliability in patients with NTI; both artificially low and high FTI values were encountered frequently. This discrepancy in reported results probably is attributable to differences in patient selection (eg, the severity of illness and drugs used that interfere with serum T4 binding). These findings seriously limit the usefulness of the FTI tests in patients with NTI.

The ultrafiltration method is a research assay in which ultrafiltrates of undiluted serum are used to measure free T4. In a study of 504 patients by Docter et al, free T4 was elevated in 54% of the patients with mild-to-severe NTI, according to measurements using equilibrium dialysis, and free T4 was elevated in about 24% of patients in the most severely ill group, as illustrated in the diagram below. [12] Another study by Melmed et al demonstrated that free T4 was reduced in ICU patients as measured by 6 different methods, including equilibrium dialysis. [13] Free T4 was found to be uniformly reduced as measured by all methods, but patients with liver disease and chronic renal failure exhibited more variable results. This study demonstrated that, overall, patients with NTI who have serum total T4 levels within the reference range typically do not have reduced free T4 by most assay methods.

See the image below.

Euthyroid sick syndrome. Relationship between seru Euthyroid sick syndrome. Relationship between serum thyroid hormone concentrations and severity of nonthyroidal illness (NTI). Abbreviations: reverse triiodothyronine (rT3), thyroid-stimulating hormone (TSH), free thyroxine (FT4), thyroxine (T4), triiodothyronine (T3).

In an extensive comparison of 5 measurement methods, free T4 was extremely low in patients with NTI who had a serum level of total T4 less than 3 mcg/dL. Results obtained using ultrafiltration also are variable. Thus, although extensively studied, the question remains whether free T4 in patients with NTI actually is low, within the reference range, or even high.

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