Free T4 Results Research Articles

Evaluating thyroid function in the clinical laboratory

Background: Thyroid disorders are common in clinical medicine and laboratory confirmation is essential, especially in cases where overt signs and symptoms are absent. The measurement of thyroid-stimulating hormone (TSH) is recommended as the best indicator of thyroid function and is utilized along with thyroxine (T4) and in some circumstances triiodothyronine (T3). Aim: The aim is to evaluate the relationship between TSH and free T4 (FT4), and TSH and FT3. Methods: Thyroid function tests (TFTs) – TSH, FT4, and FT3 – performed in the Chemical pathology laboratory from February to December 2015 were retrieved from the laboratory information management system. Results were classified based on TSH results into suppressed, mildly suppressed, normal, mildly elevated and elevated, and correlated with FT3 and FT4 results. Results were also categorized as overt hyperthyroidism, subclinical hyperthyroidism, normal, subclinical hypothyroidism, overt hypothyroidism, and “others” and compared based on age and gender. Results: FT4 and FT3 correlate best with TSH at suppressed and elevated levels. Mean TSH was significantly higher in males than in females only in those with normal FT4 and TSH. TSH was significantly higher in the 41–60 age group, with females in this group having significantly higher levels as compared to males. Conclusions: There is an inverse correlation between FT4 and TSH, and FT3 and TSH, especially significant at suppressed and elevated levels of TSH. Highest TSH levels were observed in women in the 41–60 age cohort.

Influência da função tireoidiana nos níveis séricos de CPK

To determine serum CPK variation based on TSH e free T4 (FT4), and to assess serum CPK in pathological states of the thyroid (hyperthyroidism and hypothyroidism), in relation to the euthyroidism. We evaluated retrospectively 6,230 laboratory results of TSH and CPK from 2007 to 2011. From these, 3,369 had free T4 results. We evaluated the correlation between CPK and TSH and the pathological states of the thyroid. The correlation between TSH and CPK was positive (r = 0.065), while that between CPK and FT4 was negative (r = -0.091, p < 0.05). From the total of results analyzed, 586 (9.4%) were measures of hyperthyroidism, with a median (range) of CPK of 98 U/L (27 to 1,113), and 556 (8.9%) were of hypothyroidism, with CPK of 114 U/L (25-4,182). A positive correlation was found between serum CPK and TSH, and a negative correlation between CPK and FT4. CPK was lower in the group with hyperthyroidism, and greater in that with hypothyroidism.

Familial Dysalbuminemic Hyperthyroxinemia: A Persistent Diagnostic Challenge

Familial dysalbuminemic hyperthyroxinemia (FDH)1 is a well-characterized condition associated with increased circulating total thyroxine (T4) concentrations and normal physiological thyroid function. It is caused by mutations in the ALB (albumin) gene that increase the affinity of albumin for T4 by approximately 60-fold. When measured by a technique that minimally disturbs the equilibria between T4 and its serum binding proteins, such as equilibrium or symmetric dialysis (SyD) performed in a near-physiological medium, the free T4 (FT4) value is characteristically within the reference interval. Assays that rely on the competition of a T4 analog with unbound T4 in the sample can give spuriously high results in FDH patients, because albumin binding of the T4 analog is enhanced by the FDH mutation. “Two step” methods, in which the T4 analog never comes into contact with serum albumin owing to a wash step immediately after capture, avoid this problem. Such assay methods are expected to give FT4 results within the reference interval in FDH patients, but this expectation has been questioned (1)(2). Thyroid-function tests, including 1- and 2-step methodologies, were examined in 4 affected individuals from different families who had their FDH diagnoses proved genetically …

A Direct Free Thyroxine (T4) Immunoassay with the Characteristics of a Total T4 Immunoassay

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. 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. 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. 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

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. 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). 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. 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.

The measurement of free thyroxine by isotope dilution tandem mass spectrometry

Background Most clinical chemistry laboratories measure free thyroxine (FT4) by an analogue (direct) method. Nevertheless, the validity of analogue FT4 immunoassays has been questioned and patient's results using this approach frequently do not fit in with the clinical presentation. Because of these problems we routinely send out all direct free T4's that are < 2.5th percentile and many that are > 97.5th percentile for measurement by equilibrium dialysis, the gold standard method. In approximately half of these cases, the FT4 by equilibrium dialysis was normal. We developed a rapid, reliable free T4 method employing isotope dilution tandem mass spectrometry and compared our results with both the analogue (direct) free T4 and equilibrium dialysis procedures. Methods An API-4000 tandem mass spectrometer (Sciex, Toronto, Canada) equipped with TurboIonSpray and Agilent HPLC system was used employing isotope dilution with deuterium labeled internal standard (IS = l-thyroxine-d 2). Serum was filtered through the Centrifree YM-30 ultrafiltration device by centrifugation, IS added to the ultrafiltrate, and 400 μL injected onto a C-18 column. After washing, the switch valve is activated and a methanol gradient allows for elution of both T4 and the IS into the LC/MS/MS system. Quantitation was by MRM analysis in the negative mode. Equilibrium dialysis was performed by the Nichols method and analogue free T4 results were obtained on the Dade Dimension RxL. Results The within-day and between-day CV's were < 7.1% at all concentrations tested. The results correlated well with equilibrium dialysis (Eq Dial = 0.971 MS + 0.041, n = 68, Syx = 1.381, r = 0.954). A poor correlation was found with the analogue (direct) free T4 method (IA = 0.326 MS + 6.27, n = 154, Syx = 1.96, r = 0.459). Conclusions Samples can be processed in batches of 30. The free T4 tandem MS method has a rapid turn-around-time vs the equilibrium dialysis procedure, with a chromatographic run time of 8 min per sample.

Hypothyroidism.

Hypothyroidism.

FREE THYROID HORMONE MEASUREMENT: A Critical Appraisal

The main purpose of free T4 and free T3 assays is to distinguish reliably between thyrotoxicosis, hypothyroidism, and the euthyroid state, an objective that cannot be attained with assays of total T4 and T3 because of hereditary and acquired variations in the concentrations of binding proteins. Effective correction for changes in the serum concentration of TBG can be achieved with numerous types of free hormone estimate, but other changes in binding are not well accommodated. Despite remarkable methodologic ingenuity, no current method reflects the free T4 concentration in undiluted serum under in vivo conditions. Equilibrium dialysis, widely considered the reference method for free T4 measurement, is also subject to error, either preanalytic, owing to generation of NEFA in the sample leading to an overestimate of free T4, or analytic with underestimation of the effect of competitors to increase free T4. Current approaches to free T4 measurement are vulnerable to several method-dependent artifacts: abnormal albumin binding of T4 or of the assay tracer, the inhibition of T4 binding to TBG by medications, and the effects of critical illness, especially in heparin-treated patients, pregnancy, and the abnormalities in sick premature infants. Because of systematic variation between methods (i.e., whether a technique is albumin dependent or prone to incubation or dilution artifacts), it is essential to consider methodologic details in evaluating free T4 estimates in these situations and whenever estimates of free T4 are clinically discordant. False-positive abnormalities are more frequent than false-negative results. When free T4 results are correlated with the serum TSH concentration with attention to the assumptions that define this relationship, the majority of false-positive results can be readily identified. If a free T4 anomaly remains unexplained on repeat sampling, it is appropriate to use an alternative free T4 method that depends on a different assay principle and to correlate the result with an authentic total T4 measurement.

Meaning of serum free-thyroxin values in nonthyroidal illnesses: seven methods compared.

Serum free thyroxin (FT4) was determined in 40 patients with various nonthyroidal illnesses. We studied seven methods: (1) a free thyroxin index calculated from total T4 and triiodothyronine resin uptake; (2) a free T4 index determined by enzyme inhibitor assays (Abbott's "Tetrazyme" and "Thyrozyme"); (3) a free T4 index calculated from total T4 and thyroxin-binding globulin; (4) free T4 by equilibrium dialysis; (5) Amersham's free T4 RIA; (6) Clinical Assays' one-step free T4 RIA; and (7) Clinical Assays' two-step free T4 RIA. Approximately half of the free T4 results were in the euthyroid range and the other half in the hypothyroid range by methods 1, 2, 5, and 6. Results for free T4 by methods 3 and 7 were similar to those by equilibrium dialysis (method 4), the percentages of patients with results in the euthyroid range being 68%, 65%, and 76%, respectively.

Free thyroxin by radioimmunoassay: evaluation of a new direct method involving a radiolabeled thyroxin analog.

We present here the first performance evaluation of a new direct method for free thyroxin (T4) in serum by radioimmunoassay, with use of coated tubes and a radiolabeled T4 analog (Diagnostic Products Corp.). The assay is precise and robust: within-run imprecision (CV), 3.1-6.6%; between-run imprecision, 4.0-7.9%; no demonstrable variation between technologists irrespective of experience with the method. No outliers were observed when we compared the free T4 results with serum total T4. Reference values are reported for a total of 1243 euthyroid subjects; there was no significant age effect on serum free T4 in women 26 to 72 years old. The biological variation was about +/- 35% of the mean (2 SD). Free T4 results are the same for serum and plasma. The assay performs well in hypothyroidism and hyperthyroidism, and distinguishes individuals with thyroid disease from normal individuals. Free T4 values in women taking oral contraceptives are normal. Depressed results were often observed in acute nonthyroidal illness and continuing pregnancy. These results were directly comparable with those of another commercial direct radiolabeled-T4 analog kit for free T4.