Accuracy issues in free thyroxine testing methods.

The incidence of low neonatal free thyroxine (T(4)) assay results is methodology-dependent. Nonanalog free T(4) assay results represent free T(4) concentrations when free T(4) is the only form of T(4). Similar analog-based free T(4) assay results are produced by an extraordinary range of free T(4) concentrations, when free T(4) is the only form of T(4). Adding albumin or transthyretin to free T(4) concentrations greatly decreased free T(4) concentrations, as expected, but increased analog-based free T(4) assay results. By contrast, adding thyroxine-binding globulin decreased free T(4) concentrations and free T(4) assay results; but these free T(4) concentrations were not represented by assay results. There was no specificity for the free form of T(4) versus bound forms of T(4) in some free T(4) assay results. The protein that binds T(4) can have a major influence on some of the total T(4) assay results that may be used in free T(4) index methods.

Analog-based free testosterone test results linked to total testosterone concentrations, not free testosterone concentrations.

BACKGROUND: Analog-based free testosterone test results, sex hormone binding globulin (SHBG) concentrations, and total testosterone concentrations are somehow related. This study used new experiments to clarify these relationships. METHODS: An analog-based free testosterone immunoassay and a total testosterone immunoassay were applied to well-defined fractions of serum testosterone. First, they were applied to the 2 fractions (retentate and dialysate) of normal male serum obtained by equilibrium dialysis. Second, they were applied to covaried concentrations of SHBG and total testosterone. Third, they were applied to decreasing concentrations of SHBG and protein-bound testosterone, offset by increasing concentrations of protein-free testosterone, while total testosterone was held constant. RESULTS: The analog-based free testosterone assay and the total testosterone assay detected and reported serum testosterone test results from serum retentate, whereas neither assay detected the free testosterone in serum dialysate. Test results reported by the analog-based free testosterone assay followed varied concentrations of SHBG and total testosterone. When total testosterone was held constant, however, analog-based free testosterone test results did not follow varied concentrations of serum proteins or of free testosterone. CONCLUSION: An analog-based free testosterone immunoassay reported free testosterone test results that were related to total testosterone concentrations under varied experimental conditions. This alleged free testosterone assay did not detect serum free testosterone (the test results it reported were nonspecific) and should not be used for this purpose.

A direct free thyroxine (T4) immunoassay with the characteristics of a total T4 immunoassay.

BACKGROUND: Direct free thyroxine (T(4)) measurements have been linked to both T(4)-binding serum protein concentrations and protein-bound T(4) concentrations. Whether this is evidence of a relationship to total T(4) concentrations has not been reported. METHODS: We compared an analog-based direct free T(4) immunoassay and a total T(4) immunoassay. Each assay was applied to the fractions of serum T(4) obtained by ultrafiltration and equilibrium dialysis. Both were applied to serum-based solutions in which free T(4), T(4)-binding proteins, protein-bound T(4), and total T(4) were systematically varied, held constant, or excluded. RESULTS: Neither the free T(4) assay nor the total T(4) assay detected dialyzable or ultrafilterable serum T(4). Both assays detected and reported the T(4) retained with serum proteins. Both free and total T(4) results were related to the same total T(4) concentrations in the presence and absence of T(4)-binding proteins. Both results were similarly related to total T(4) concentrations when free T(4) was held constant while total T(4) was varied. Both were similarly related to a total T(4) concentration that was held constant while free T(4) progressively replaced protein-bound T(4). These free T(4) results, like total T(4) results, were unresponsive to a 500-fold variation in dialyzable T(4) concentrations. CONCLUSION: New experiments extend the characterization of a longstanding and incompletely characterized analog-based free T(4) immunoassay. These free T(4) measurements relate to total T(4) concentrations in the same way that total T(4) measurements do.

Quantifying spurious free T4 results attributable to thyroxine-binding proteins in serum dialysates and ultrafiltrates.

BACKGROUND: Direct equilibrium dialysis and direct ultrafiltration free thyroxine (T(4)) assays rely on semipermeable membranes to exclude T(4)-binding serum proteins from dialysates and ultrafiltrates. The presence of these proteins in dialysates or ultrafiltrates will yield spuriously high free T(4) values when free T(4) is quantified by RIA. METHODS: We used a nonanalog free T(4) RIA that detects and quantifies dialyzable and ultrafilterable serum free T(4) to detect T(4)-binding serum proteins. Two equilibrium dialysis devices and 3 ultrafiltration devices were used to illustrate this application. Displacements of [(125)I]T(4) from anti-T(4) by various concentrations of T(4)-depleted thyroxine-binding globulin, albumin, and serum total protein were compared to displacements by various concentrations of free T(4). RESULTS: Both dialysis devices excluded detectable T(4)-binding serum proteins from dialysates. Two of 3 ultrafiltration devices excluded detectable T(4)-binding serum proteins from ultrafiltrates. One did not, and its ultrafiltrate yielded spurious free T(4) values that correlated directly with serum protein concentrations. CONCLUSION: The presence or absence of T(4)-binding proteins in dialysates and ultrafiltrates and the spurious free T(4) values that these proteins cause can be documented using a nonanalog free T(4) RIA.

Underestimates and overestimates of total thyroxine concentrations caused by unwanted thyroxine-binding protein effects.

The first evidence of unwanted serum protein effects on analogue-based total thyroxine (T4) determinations came from a study that varied serum protein concentrations while total T4 concentrations were constant. The present study approached this issue by varying total T4 concentrations while protein concentrations were constant. Four analogue-based total T4 immunoassays were applied to solutions that contained either free T4 without binding protein, a T4-binding protein without T4, or protein-bound T4. When total T4 concentrations were 3-12 microg/dL, the assays reported total T4 determinations that ranged from none detected to 23 microg/dL. These T4 determinations reflected the protein to which T4 was bound, in addition to the level of T4. Total T4 was underrepresented when T4 was unbound, or thyroxine-binding globulin (TBG) bound. Total T4 was overrepresented when T4 was albumin-bound, or transthyretin-bound. There were substantial disparities among assays applied to the same total T4 solutions. These assays reported no detectable T4 when applied to T4-binding protein solutions without protein-bound T4. Nonetheless, T4-binding proteins contributed to the underestimates and overestimates of protein bound T4. Different forms of protein bound T4 were quantified differently, evidence that protein-T4 complexes persist during quantification. We attribute the unexpected total T4 values to a combination of incomplete protein-bound T4 release from T4-binding proteins during quantification, and variably inaccurate quantifications of the protein-bound T4 that remained.

Assessing thyroid hormone status in a patient with thyroid disease and renal failure: from theory to practice.

A 35-year-old Asian male, treated for hyperthyroidism, systemic lupus erythematosis, and uremia presented with low serum total thyroxine (T4) and normal serum thyrotropin (TSH) levels. He had been receiving prednisone and methimazole for 15 weeks. Free T4 measured by direct equilibrium dialysis was in the hypothyroid range (0.3 ng/dL; normal, 0.8-2.7). Two possibilities were considered: (1) a weakly bound dialyzable inhibitor in uremic serum that interfered with this serum free T4 determination or (2) hypothyroidism with persistent TSH suppression because of prior hyperthyroidism. To determine whether a weakly bound inhibitor was involved, the patient's serum was serially diluted using two diluents: (1) an ultrafiltrate of the patient's serum, which would contain any unbound inhibitor, as well as free T4 and (2) an inert diluent. Free T4 measurements were similar with both, providing evidence against the presence of a dialyzable and ultrafilterable inhibitor. In conclusion, this patient was hypothyroid because of antithyroid drug administration, associated with prolonged central TSH suppression from preexisting hyperthyroidism. Discontinuation of methimazole resulted in normalization of serum total T4 and TSH values. Thus, paired, serial serum dilutions, using two different diluents, provided evidence for differentiation of appropriately low free T4 measurements (because of hypothyroidism), from spuriously low free T4 measurements (because of an interfering inhibitor).

The nature of analogue-based free thyroxine estimates.

Clinical laboratories often use analogue-based immunoassays to estimate serum free thyroxine (FT(4)) concentrations. These assays yield FT(4) estimates that correlate closely with thyroxine (T(4)) binding protein concentrations. This correlation implies that either T(4) binding proteins or protein bound T(4) contribute to analogue-based FT(4) values. To study the contributions made by T(4) binding proteins to these FT(4) estimates further, four analogue-based FT(4) assays were applied to: (1) FT(4) solutions without T(4) binding proteins, (2) to T(4) binding protein solutions without T(4), and (3) to total T(4) solutions containing T(4) binding protein, FT(4), and protein-bound T(4). The FT(4) estimates obtained with these solutions ranged from 0.2-8.6 ng/dL, when FT(4) concentrations ranged from less than 0.2-12,000 ng/dL. In the FT(4) solutions, gravimetrically determined FT(4) concentrations were 500-12,000 ng/dL (0.5-12.0 microg/dL) without protein-bound T(4), and the FT(4) estimates obtained were 0.3-6.9 ng/dL. In the total T(4) solutions, dialyzable FT(4) concentrations were less than 0.2-59 ng/dL, retained T(4) concentrations were 499.8-11,441 ng/dL, and the analogue-based FT(4) estimates obtained were 0.2-8.6 ng/dL. Similar FT(4) estimates (0.2-8.6 ng/dL and 0.3-6.9 ng/dL) were obtained with similar concentrations of either protein-bound T(4) or FT(4). Similar test results were associated with similar total T(4) concentrations, not similar FT(4) concentrations. Protein-bound T(4) and T(4) binding protein contributed variably to test results. T(4) quantifications included large analytical losses that are unaccounted for. These assays passed tests of correlation with FT(4) concentrations, but they failed tests of specificity for FT(4) and accuracy in T(4) quantification.