Free Thyroid Hormone Measurements Research Articles

Approach to the Patient With Raised Thyroid Hormones and Nonsuppressed TSH.

Measurement of free thyroid hormones (THs) and thyrotropin (TSH) using automated immunoassays is central to the diagnosis of thyroid dysfunction. Using illustrative cases, we describe a diagnostic approach to discordant thyroid function tests, focusing on entities causing elevated free thyroxine and/or free triiodothyronine measurements with nonsuppressed TSH levels. Different types of analytical interference (eg, abnormal thyroid hormone binding proteins, antibodies to iodothyronines or TSH, heterophile antibodies, biotin) or disorders (eg, resistance to thyroid hormone β or α, monocarboxylate transporter 8 or selenoprotein deficiency, TSH-secreting pituitary tumor) that can cause this biochemical pattern will be considered. We show that a structured approach, combining clinical assessment with additional laboratory investigations to exclude assay artifact, followed by genetic testing or specialized imaging, can establish a correct diagnosis, potentially preventing unnecessary investigation or inappropriate therapy.

Analysis of free, unbound thyroid hormones by liquid chromatography-tandem mass spectrometry: A mini-review of the medical rationale and analytical methods.

Measurement of hormones is important for the diagnosis and management of endocrine diseases. The thyroid hormones thyroxine (T4) and triiodothyronine (T3) are among the most commonly measured hormones in clinical laboratories, and it is the concentration of free (not bound to proteins) thyroid hormones that is clinically most relevant. Free thyroid hormones are commonly measured using automated immunoassays, however, these are known to produce erroneous results due to interferences for some patients. Measurement of free thyroid hormones using equilibrium dialysis or ultrafiltration combined with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is considered a more accurate and robust method for free thyroid hormone analysis and overcomes many of the limitations of immunoassays. However, LC-MS/MS-based methods are often considered too technically difficult and not amendable to high throughput by clinical chemists and are not offered by many clinical laboratories. This mini-review aims to make it easier for clinical laboratories to implement LC-MS/MS-based measurement of free thyroid hormones. It describes the medical rationale for measuring free thyroid hormones, the benefits of LC-MS/MS-based methods with respect to interferences affecting immunoassay-based methods and physical separation methods. This mini-review highlights important parameters for ultrafiltration and equilibrium dialysis to obtain physiologically relevant free thyroid hormone concentrations and focuses on methods and devices used in clinical chemistry.

Clinical Consequences of Variable Results in the Measurement of Free Thyroid Hormones: Unusual Presentation of a Family with a Novel Variant in the THRB Gene Causing Resistance to Thyroid Hormone Syndrome.

Clinical Consequences of Variable Results in the Measurement of Free Thyroid Hormones: Unusual Presentation of a Family with a Novel Variant in the <i>THRB</i> Gene Causing Resistance to Thyroid Hormone Syndrome.

The Two Faces of Janus: Why Thyrotropin as a Cardiovascular Risk Factor May Be an Ambiguous Target.

Elevated concentrations of free thyroid hormones are established cardiovascular risk factors, but the association of thyrotropin (TSH) levels to hard endpoints is less clear. This may, at least in part, ensue from the fact that TSH secretion depends not only on the supply with thyroid hormones but on multiple confounders including genetic traits, medication and allostatic load. Especially psychosocial stress is a still underappreciated factor that is able to adjust the set point of thyroid function. In order to improve our understanding of thyroid allostasis, we undertook a systematic meta-analysis of published studies on thyroid function in post-traumatic stress disorder (PTSD). Studies were identified via MEDLINE/PubMed search and available references, and eligible were reports that included TSH or free thyroid hormone measurements in subjects with and without PTSD. Additionally, we re-analyzed data from the NHANES 2007/2008 cohort for a potential correlation of allostatic load and thyroid homeostasis. The available evidence from 13 included studies and 3386 euthyroid subjects supports a strong association of both PTSD and allostatic load to markers of thyroid function. Therefore, psychosocial stress may contribute to cardiovascular risk via an increased set point of thyroid homeostasis, so that TSH concentrations may be increased for reasons other than subclinical hypothyroidism. This provides a strong perspective for a previously understudied psychoendocrine axis, and future studies should address this connection by incorporating indices of allostatic load, peripheral thyroid hormones and calculated parameters of thyroid homeostasis.

Lapland extreme 900 km: Biochemical responses during training, competition and restoring period. A case study

This review discusses the state-of-the-art measurement of free and total thyroid hormones in clinical laboratories. We highlight some of the limitations of currently used immunoassays and critically discuss physical separation methods for the measurement of free thyroid hormone. Physical separation methods, such as equilibrium dialysis or ultrafiltration, followed by tandem mass spectrometry for the measurement of free thyroid hormones offer many advantages, which we feel, can deepen our understanding of thyroid hormone metabolism and improve patient diagnosis and care. Problems with direct analogue immunoassay methods for FT4/FT3 as well as immunoassay methods for total T3 at low T3 concentrations and during pregnancy are highlighted. Improved diagnosis and patient management can be achieved utilizing tandem mass spectrometry for these measurements.

Homeostatic Control of the Thyroid-Pituitary Axis: Perspectives for Diagnosis and Treatment.

The long-held concept of a proportional negative feedback control between the thyroid and pituitary glands requires reconsideration in the light of more recent studies. Homeostatic equilibria depend on dynamic inter-relationships between thyroid hormones and pituitary thyrotropin (TSH). They display a high degree of individuality, thyroid-state-related hierarchy, and adaptive conditionality. Molecular mechanisms involve multiple feedback loops on several levels of organization, different time scales, and varying conditions of their optimum operation, including a proposed feedforward motif. This supports the concept of a dampened response and multistep regulation, making the interactions between TSH, FT4, and FT3 situational and mathematically more complex. As a homeostatically integrated parameter, TSH becomes neither normatively fixed nor a precise marker of euthyroidism. This is exemplified by the therapeutic situation with l-thyroxine (l-T4) where TSH levels defined for optimum health may not apply equivalently during treatment. In particular, an FT3–FT4 dissociation, discernible FT3–TSH disjoint, and conversion inefficiency have been recognized in l-T4-treated athyreotic patients. In addition to regulating T4 production, TSH appears to play an essential role in maintaining T3 homeostasis by directly controlling deiodinase activity. While still allowing for tissue-specific variation, this questions the currently assumed independence of the local T3 supply. Rather it integrates peripheral and central elements into an overarching control system. On l-T4 treatment, altered equilibria have been shown to give rise to lower circulating FT3 concentrations in the presence of normal serum TSH. While data on T3 in tissues are largely lacking in humans, rodent models suggest that the disequilibria may reflect widespread T3 deficiencies at the tissue level in various organs. As a consequence, the use of TSH, valuable though it is in many situations, should be scaled back to a supporting role that is more representative of its conditional interplay with peripheral thyroid hormones. This reopens the debate on the measurement of free thyroid hormones and encourages the identification of suitable biomarkers. Homeostatic principles conjoin all thyroid parameters into an adaptive context, demanding a more flexible interpretation in the accurate diagnosis and treatment of thyroid dysfunction.

A pilot study: Subclinical hypothyroidism and free thyroid hormone measurement by immunoassay and mass spectrometry

BackgroundThe diagnosis of subclinical hypothyroidism is defined as the presence of an elevated thyroid stimulating hormone (TSH) with a normal free thyroxine (FT4) level. The commonly used direct analogue immunoassays for the measurement of FT4 have been shown to have poor performance at the upper and lower limits of the FT4 reference interval. PurposeThe purpose of this pilot study was to investigate the percentage of individuals classified as having subclinical hypothyroidism with a standard immunoassay, that actually have low free thyroid hormone levels by mass spectrometry measurements. DesignOutpatient samples with elevated TSH values and normal FT4 concentrations as per standard immunoassay methods were collected. FT4 and free triiodothyronine (FT3) analyses were performed on these samples using liquid chromatography–tandem mass spectrometry (LC–MS/MS). ResultsSixty five percent (n=26) of patients (n=40) had (LC–MS/MS) FT4 or FT3 or both FT4 and FT3 values below mass spectrometry reference limits. ConclusionsOur findings indicate that the direct analogue immunoassay method for FT4 measurement results in a significant proportion of patients being misclassified as having subclinical hypothyroidism.

Biotin interference on TSH and free thyroid hormone measurement

Biotin interference on TSH and free thyroid hormone measurement

Quantification of free serum cortisol based on equilibrium dialysis and isotope dilution-liquid chromatography–tandem mass spectrometry

Objectives Only unbound serum cortisol is bioactive and protein binding of cortisol is highly variable. Thus, the quantification of free serum cortisol (FSC) is of superior biological relevance compared to total serum cortisol quantification. Consequently, the development of automated routine tests for FSC for endocrine testing is desirable — along the lines of free thyroid hormone measurement. Since the availability of a reliable and matrix-independent method is an important tool for this goal, we have developed a highly standardised mass spectrometric FSC method. Design and methods We used equilibrium dialysis (ED) to obtain a protein-free fraction from serum samples. The cortisol content of the dialysate was quantified using isotope-dilution two dimensional liquid chromatography (LCxLC–MS/MS). Results Comprehensive evaluation characterised the method as reliable and robust; using commercially available dialysis cells, convenient handling was realised. Conclusions The method described in this article can be suggested for the implementation of a reference measurement system for FSC.

Standardization activities in the field of thyroid function tests: a status report

Laboratory testing is an essential tool for diagnosis and management of thyroid diseases. However, the current status of standardization hampers the interchangeability of results. To improve this situation, the Working Group for Standardization of Thyroid Function Tests was established. Method comparisons were organized for measurement of human thyroid stimulating hormone (TSH), and free and total thyroid hormone in serum from apparently healthy donors. The aim was to assess the status of standardization and the quality of the performance of current routine assays. A second objective was to investigate the effect of mathematical recalibration of the results using their relationship to the overall mean (TSH) or the reference measurement procedure values (other thyroid hormones). The need for standardization was shown to be highest for free thyroid hormone and total triiodothyronine measurements, while the majority of TSH and total thyroxine assays agreed within 10% of the reference. Most assays showed good performance. However, some could benefit from improved precision, consistency of calibration, or within- and between-run stability. Recalibration eliminated assay-specific bias. Thus, the residual spread was due to within-method effects. Not withstanding, sample-related effects remained. These studies confirmed the feasibility of standardization based on method comparison with native sera, but highlighted the need to resolve issues, such as sample-related effects. In view of the fact that in this phase the project worked with samples from individuals with euthyroid status, the next method comparison shall place emphasis on challenging the performance of the assays with clinical samples and expanding the covered measurement range.

Inaccuracies in Free Thyroid Hormone Measurement by Ultrafiltration and Tandem Mass Spectrometry

We have read with interest recent reports of the measurement of free thyroxine and free triiodothyronine by ultrafiltration and tandem mass spectrometry (1)(2)(3)(4). There is, however, a fundamental flaw in these studies that we wish to bring to general attention. Ultrafiltration of serum was routinely carried out close to room temperature (25 °C)(1 …

Correlations of Free Thyroid Hormones Measured by Tandem Mass Spectrometry and Immunoassay with Thyroid-Stimulating Hormone across 4 Patient Populations

Accurate measurement of free thyroid hormones is important for managing thyroid disorders. Ultrafiltration liquid chromatography tandem mass spectrometry (LC-MS/MS) can reliably measure the concentrations of small molecules, including thyroid hormones. Our study was designed to compare free thyroid hormone measurements performed with immunoassay and LC-MS/MS. We studied the performance of LC-MS/MS in 4 different populations comprising pediatric patients, euthyroid adults, and healthy nonpregnant and pregnant women. The samples obtained from each population numbered 38, 200, 28, and 128, respectively. Free thyroxine, free triiodothyronine, and thyroid-stimulating hormone (TSH) concentrations were documented. LC-MS/MS measurement of free thyroid hormones provided better correlation with log-transformed serum TSH in each population and also the populations combined. The correlations between free thyroxine measured by LC-MS/MS and log TSH in the pediatric outpatients and healthy adults were -0.90 and -0.77, respectively. The correlations for immunoassay were -0.82 and -0.48. The correlations between free triiodothyronine measured by LC-MS/MS and TSH for both pediatric and healthy adult populations were -0.72 and -0.68, respectively. Free thyroid hormone concentrations measured by LC-MS/MS correlate to a greater degree with log TSH values compared to concentrations measured by immunoassay. This correlation was maintained across the patient populations we studied and may reflect the accuracy and specificity of LC-MS/MS. The superior ability of LC-MS/MS to enable documentation of the well-known thyroid hormone-TSH relationship supports the use of this measurement technique in a variety of clinical situations.

A Major Step Forward in the Routine Measurement of Serum Free Thyroid Hormones

In the field of free thyroid hormone testing, methods based on equilibrium dialysis (ED) or ultrafiltration (UF) of serum, before direct measurement of the hormone concentration in the dialysate or ultrafiltrate, are accepted to be gold standards(1). These measurement procedures, however, physically isolate the free from the protein-bound hormone fractions and have been considered too technically demanding, inconvenient, and relatively expensive for routine clinical laboratory use(2)(3). Typically, such methods are better suited for use in reference laboratories(4). In this issue of Clinical Chemistry , Yue et al. report on the application of an ED isotope dilution–liquid chromatography/tandem mass spectrometric (ID–LC-MS/MS) procedure developed in-house …

Circulating thyroid hormone concentrations and placental thyroid hormone receptor expression in normal human pregnancy and pregnancy complicated by intrauterine growth restriction (IUGR).

Thyroid hormones are critical to growth and development of the human fetus. Abnormal placental development, a major cause of intrauterine growth restriction (IUGR), is associated with a high perinatal mortality and morbidity. Thyroid status has been postulated to play a role in the pathogenesis of such morbidity. In the present study, we have investigated fetal thyroid function and placental expression of thyroid hormone receptor (TR) alpha and beta variants during normal human pregnancy and in pregnancy associated with IUGR. Measurement of free thyroid hormones and TSH concentrations revealed significant rises in free T4 and free T3 between the second and third trimesters of normal pregnancy. Serum concentrations of free T4 and free T3 were lower in fetuses affected by IUGR, although serum TSH levels were not significantly different. Immunocytochemistry demonstrated the presence of TR alpha1, alpha2, and beta1 proteins within the nuclei of trophoblast and stromal placental cells. Immunostaining for these TR variants increased with increasing gestation in normal placenta. Comparison of IUGR placental samples with normal samples revealed greater immunostaining for TR alpha1, alpha2, and beta1 variants in IUGR. Examination of pretranslational expression of TR alpha1, alpha2, beta1, and beta2 variants by semiquantitative RT-PCR revealed increasing expression of TR alpha1, alpha2, and beta2 messenger RNAs with increasing gestation in normal pregnancy, which "mirrored" post-translational expression. However, and in contrast, there were no significant differences in expression of TR messenger RNAs in normal and IUGR placenta. The present findings of reduction in serum free thyroid hormones and increased expression of TR alpha and beta proteins in association with IUGR highlight the potential importance of thyroid status in influencing long-term fetal outcome in this condition.

Thyroid hormone-free albumin: Charcoal treatment or resin treatment

Free thyroid hormone measurement by means of immunoassay kits is greatly influenced by the altered serum albumin and free fatty acid (FFA) levels. In the evaluation of these kits, it is therefore essential to study the interferences due to these factors by adding FFA or thyroid hormone-free human serum albumin (HSA) to the assay mixture, but little attention has been paid to the selection of albumin. In the present study, FFA content in various preparations of thyroid hormone-free HSA was compared. Charcoal-treated HSA was free from both thyroid hormone and FFA, whereas anion exchange resin-treated HSA was only free from thyroid hormone. Commercially available "FFA-free HSA" was also free from thyroid hormone. Our results suggest that attention must be paid to the nature of albumin when studying the interference by albumin in free thyroid hormone measurement and that commercially available "FFA-free HSA" is a ready-to-use thyroid hormone-free HSA when HSA free from both FFA and thyroid hormone is desired.

Autoimmunity against Thyroid Hormones

The presence of thyroid hormone autoantibodies (THAA) is a common phenomenon. More than 270 cases have been reported by the end of 1993 involving not only thyroidal but also nonthyroidal disorders. Clinically, THAA in a patient's serum produces variation in thyroid hormone metabolism and, in particular, may interfere with the radioimmunoassay (RIA) results of total or free thyroid hormone measurements, which can cause unusually high or low values of the hormones depending on the B/F separation method used. This in vitro interference can give clinicians confusing information about the patient's thyroid state. As a result, the patient may receive inappropriate treatment from physicians who are unaware of this disorder. The presence of THAA has been reported not only in humans but also in dogs, chickens, and rats. In this review article, clinical features of THAA and the mechanism of autoantibody production are discussed.

Thyroid hormone autoantibodies and their implications for free thyroid hormone measurement.

Thyroid hormone autoantibodies (THAA) disrupt the equilibrium between thyroid hormones and their binding proteins. This may lead to spurious estimations of free thyroxine (FT4) and triiodothyronine (FT3) by radioimmunoassay (RIA). In the present study we highlight the importance of THAA by examining the frequency of THAA in consecutive sera sent to a routine district hospital laboratory. Over a period of six months, sera were collected from 200 consecutive hypothyroid, 200 hyperthyroid and seven patients whose clinical and biochemical thyroid status were contradictory. A further 200 patients with non-thyroid autoimmune conditions, 20 patients with insulin autoantibodies and 100 healthy blood transfusion donors were studied. In all sera, both effects of antigen removal on THAA detection and where THAA were found, the effect of their removal on FT4, were examined. The frequencies of THAA amongst hypothyroid, hyperthyroid and non-thyroid autoimmune conditions were 7%, 1.5% and 7.5% respectively, whilst no THAA were found in insulin autoantibody positive patients and 100 blood transfusion donors. However, THAA frequency was highest in those patients whose biochemical thyroid status was widely inappropriate to clinical state (5/7 = 64%). Sera stripped of thyroid hormones prior to THAA detection had significantly higher antibody activity than unstripped sera (p = 0.0027 and p = 0.0123 for T3 and T4 binding respectively). Free thyroxine levels measured by the Amerlex-M RIA kit after antibody removal fell in all 21 THAA positive sera tested. The correlation coefficient between antibody activity in serum with percentage fall in FT4 was 0.79 (Spearman's Rank Correlation Test).(ABSTRACT TRUNCATED AT 250 WORDS)

The free thyroid hormone hypothesis and measurement of free hormones

Journal Article The free thyroid hormone hypothesis and measurement of free hormones Get access J E Midgley J E Midgley Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 39, Issue 6, 1 June 1993, Pages 1342–1344, https://doi.org/10.1093/clinchem/39.6.1342 Published: 01 June 1993

In Reply: The Free Thyroid Hormone Hypothesis and Measurement of Free Hormones

In Reply: The Free Thyroid Hormone Hypothesis and Measurement of Free Hormones

Guidelines for Thyroid Testing

New assay methods for the measurement of human thyroid-stimulating hormone (TSH) and improved assays for the measurement of free thyroid hormones have led to changes in the use of available tests for diagnosis and management of thyroid disease. In this article, I review the normal physiologic and pathophysiologic features of the thyroid system and their relationship to the interpretation of thyroid tests. An analysis of the cost-effectiveness of various tests is beyond the scope of this presentation. The use of thyroid tests, however, is based on the assumption that they will be ordered to facilitate decisions concerning diagnosis or management, not as part of a multiphasic screening of large numbers of asymptomatic individuals, except in specific populations in which such screening is either mandatory or effective.