Spuriously High Free Thyroxine Values in Familial Dysalbuminemic Hyperthyroxinemia

To the Editor: Cartwright et al. recently showed that current commercially available free thyroxine (FT4)1 methods yield spuriously high results in individuals with familial dysalbuminemic hyperthyroxinemia (FDH) (1). In particular, variants of the “one-step labeled analog” technique have been notorious in this respect, whereas the 2-step approach, in which the sample is washed away before the antibody-bound T4 is brought into contact with the labeled T4 analog, is thought to eliminate the problem of tracer binding to albumin and especially albumin in FDH patients. Some 2-step assays, however, also yield spuriously high values in individuals with this condition; thus, interference is not simply a problem of 1-step assays. Inhibitors of T4 and T4 analog binding to the albumin included in the incubation buffer have been suggested to play a role for both the 1- and 2-step designs. In addition, the report of Cartwright et al. referred to similarly increased results obtained by symmetric dialysis when the incubation buffer of the Beckman Coulter Access 2-step assay (known to give falsely increased results for FDH samples) was used. We present evidence for the most probable candidate substance that causes the spurious results, at least for this particular assay platform. Symmetric dialysis with 4 different media and 2 different analyzers were used in the measurement of the FT4 concentration, and fT4% in a pool of serum samples from healthy individuals and serum samples from 3 patients with proven FDH. The total T4 concentration was measured by Vitros ECi. When available, reference intervals (±2 SDs) are given. HEPES, a buffer with physiological concentrations of the most relevant mineral ions in serum [Van Uytfanghe et al (4)]. Cl− = 0, a 0.15-mol/L phosphate buffer, pH 7.4. 2 mol/L Cl−, a 0.05-mol/L phosphate buffer with 2 mol/L NaCl. Cl−>>, the proprietary incubation buffer used in the Access 2-step FT4 assay. The Vitros analyzer medium (Cl− = 0) is a chloride-free medium consisting of 0.15 mol/L phosphate with other minor additions; the medium used for the Access assay (Cl−>>) is Beckman Coulter's proprietary formulation. ND, not determined. Symmetric dialysis with 4 different media and 2 different analyzers were used in the measurement of the FT4 concentration, and fT4% in a pool of serum samples from healthy individuals and serum samples from 3 patients with proven FDH. The total T4 concentration was measured by Vitros ECi. When available, reference intervals (±2 SDs) are given. HEPES, a buffer with physiological concentrations of the most relevant mineral ions in serum [Van Uytfanghe et al (4)]. Cl− = 0, a 0.15-mol/L phosphate buffer, pH 7.4. 2 mol/L Cl−, a 0.05-mol/L phosphate buffer with 2 mol/L NaCl. Cl−>>, the proprietary incubation buffer used in the Access 2-step FT4 assay. The Vitros analyzer medium (Cl− = 0) is a chloride-free medium consisting of 0.15 mol/L phosphate with other minor additions; the medium used for the Access assay (Cl−>>) is Beckman Coulter's proprietary formulation. ND, not determined. The importance of the correct electrolyte composition of the medium used in assessing FT4 has been stressed repeatedly for the last 4 decades (2–4), and the chloride concentration appears to be particularly important. As an illustration, the FT4 percentage (i.e., the FT4/total T4 ratio expressed as a percentage) in a 20-g/L solution of human serum albumin as measured by symmetric dialysis is 0.48% in HEPES buffer at a physiological chloride concentration of 100 mmol/L, 0.24% in a 0.15 mol/L phosphate buffer without chloride, and 0.75% in a 0.05 mol/L phosphate containing 2 mol/L NaCL. Thus, chloride appears to be an inhibitor of T4 binding to albumin. We used symmetric dialysis to measure the free T4 percentage in 3 serum samples from patients with proven FDH and a serum pool from healthy individuals in 4 different media with different chloride contents (see Table 1). One of these media was the incubation buffer for the Access assay. We compared results obtained with both the Access and Vitros ECi (Ortho Clinical Diagnostics) analyzers. In the latter assay, we measured FT4 with a 1-step, labeled-antibody format that uses an isotonic but chloride-free medium. We expressed our free T4 results as a percentage of the total T4 concentration, as well as the FT4 concentration, because the former provides a clearer view of the effect of the various media on T4 binding. Table 1 shows that the omission of chloride has only a slight effect on the sample of pooled serum (control) with typical albumin concentrations, but it has a profound effect on the FDH samples. Conversely, increasing the chloride concentration has a moderate effect on the control serum sample but strongly increases the percentage of free T4 in FDH sera up to the range seen with the control serum. Use of the Access buffer gave comparably high results. These results are readily understood when one considers the fact that a much larger proportion of the total T4 is bound to albumin in FDH, so that measurements that are affected predominantly by albumin binding are altered to a larger extent in FDH serum samples than in control sera. The measurements obtained by symmetric dialysis with chloride-free medium paralleled those of the Vitros analyzer, and the symmetric dialysis measurements in high-chloride medium paralleled those of the Access instrument, given that the latter generates lower values than other assays, likely because of calibration effects. In summary, the absence of chloride amplifies the differences in percent free T4 between normalbuminemic and FDH sera, whereas use of high chloride concentrations and the buffer from the 2-step assay reduces the differences to practically nil. For the corresponding free T4 concentrations in FDH sera, this result implies a typically euthyroid FT4 value in a physiological medium, a slightly decreased FT4 value in chloride-free medium, and a strongly increased FT4 concentration in a high-chloride medium. Although we found a chloride concentration of 2.5 mol/L in the incubation medium for the 2-step assay, we are not absolutely certain that this agent causes the spurious values. Given that the manufacturer is reluctant to disclose the composition of the buffer, it is possible that the effect is caused by a different inhibitor of T4 binding to albumin. For the same reason, we cannot rule out interference in the chloride assay. Therefore, we recommend that all manufacturers disclose the composition of their FT4 incubation buffers so that investigators can perform simulation studies with symmetric or equilibrium dialysis to investigate potential sources of interference. free thyroxine familial dysalbuminemic hyperthyroxinemia.