Temperature Effects on Free-Thyroxine Measurements: Analytical and Clinical Consequences

Abstract

We compared the analytical and clinical performance of two free-thyroxine (FT4) assays--a solid-phase radioimmunoassay, Spectria, and a time-resolved fluoroimmunoassay, Delfia, both of them two-step methods--with the performance of a direct radioimmunoassay, Nichols, to measure FT4 concentration in equilibrium dialysate of undiluted serum. The three assays showed comparable analytical performance. We tested clinical utility in sera from 135 healthy subjects with and without thyroxine-binding abnormalities and in 61 patients with and without thyroidal illnesses. We found significant differences for FT4 measured by different assays in sera from the same euthyroid patients. To explain the differences, we studied the influence of temperature on performance and calibration. Most important was the neglected fact that the association constant for the binding of thyroxine to thyroxine-binding globulin decreases when the temperature rises from 20 to 37 degrees C, causing a doubling of FT4. The two-step assays, if performed at room temperature without a well-defined calibration, can give misleading FT4 concentrations. This is the case when sera from patients with thyroxine-binding abnormalities are measured against kit standards, made up in normal human sera. If an assay is to reflect the in vivo FT4 concentration at body temperature in all types of samples, it should be performed at body temperature. For practical reasons 37 degrees C is recommended, and reference values should be defined at 37 degrees C. The same might be valid for other free-hormone assays.

Temperature-responsive release of thyroxine and its environmental adaptation in Australians

“…Kd measurements were repeated more than three times in duplicate; data are means ± s.d. ( b ) The fluorophore Kd/Kd 37°C derived changes in free thyroxine (FT4) with temperature (shown in bold) are in consistent agreement with previous independently derived values with thyroxine and isolated plasma TBG [ 20 ] (non-bold), and the assay of free thyroxine in plasma at 21°C and 37°C, [ 19 , 20 ] ( figure 2 ). Calculations based on FT4 of 20 pM and TBG saturation of 20% at 37°C.…”

“…The use of the fluorophore–thyroxine Kd/Kd 37°C ratios to calculate the proportional changes that will occur to the binding affinity of thyroxine to TBG in plasma is validated in table 1 a and figure 2 by the agreement with values independently determined with thyroxine [ 18 ] and with the direct assays by others of free-thyroxine concentrations in the blood at room and body temperature [ 19 , 20 ]. The plot of Kd/Kd 37°C ratios versus temperature in figure 1 d and of consequent blood free-thyroxine concentrations ( table 1 b and figure 2 ) demonstrate how changes in the binding affinity of TBG provide an inherent adjustment of thyroxine levels to match metabolic needs.…”

“… Variation in free thyroxine with temperature from Kd/Kd 37°C ratios (hatched bars), 20 pM at 37°C rising to 25 pM at 39°C with wt-TBG, but dampened to 22 pM in the 191/283 TBG (Aus) variant (upper normal limit, dashed line). The open (non-hatched) bars show values from independent determinations by others using plasma-derived thyroxine [ 18 ] and direct assays of plasma-free thyroxine [ 19 , 20 ]. The comparative bars are based on a defined Kd of 80 pM, free thyroxine of 20 pM and a TBG saturation of 20%, at 37°C.…”

“…Other proteins that bind thyroxine in the plasma do so with a very much lower affinity than TBG. As Schreiber describes [ 5 ] and others show experimentally [ 19 , 20 ], if the concentration of free thyroxine is less than the Kd of TBG, as in plasma, the changes in free thyroxine can be directly derived from the changes in the Kd of TBG. This allows the calculation here of the concentration of free thyroxine [FT4] and the percentage saturation of TBG, Y, from the law of mass action: …”

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“…Perusal of the literature also showed a disconcerting variation in the quoted 'normal ranges' of FT4 and an inconsistency in defining the demarcations between euthyroid, hypo-, and hyperthyroid function. Enquiries to individual laboratories as to their normal ranges were met by evasive replies until obtaining an unpublicised referencecentre range (Thienpont, et al, 2010 (Christofides & Midgley, 2009;Jonklaas, Kahric-Janicic, et al, 2009;Ross & Benraad, 1992;van der Sluijs Veer, et al, 1992). Moves are apparently now underway with manufacturers and kitset suppliers to re-calibrate FT4 analyses at 37 o C by 2018.…”