madman
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Abstract
The aim of this thesis was to evaluate and improve laboratory measurements in diagnosing thyroid disease. This thesis demonstrates the extensive scope of laboratory measurements in diagnosing thyroid disease and its tendency towards a more personalized diagnostic approach. In the first part, we provided better insight into the performance of the current free thyroxine (fT4) immunoassays in specific populations. In the second part, we described newly developed liquid chromatography tandem-mass spectrometry (LC-MS/MS) methods to measure fT4, total T4, total triiodothyronine (T3), and reverse T3 (rT3). The third part shed light on thyroid disease diagnostics and proposed and developed improvements in this diagnostic process. Clinicians are not always aware of possible drawbacks of laboratory measurements. However, this thesis shows it is worth diving into this important part, with a focus on thyroid disease diagnostics as it can have major clinical consequences. Collaboration between laboratory specialists and requesting medical specialists is thus vital for achieving optimal thyroid care.GENERAL INTRODUCTION
Thyroid hormone action
Thyroid hormones (THs) are essential for development, growth, and metabolism and almost all human cells are influenced by THs 1. THs are produced by the thyroid gland. The name ‘thyroid’ originates from the Greek word ‘thyreoeides’ meaning ‘shield-shaped’, and is also known as the butterfly shaped endocrine organ (Figure 1). The thyroid gland is located in the neck below the Adam’s apple and is controlled by the pituitary and the hypothalamus in the brain.The hypothalamus releases thyrotropin-releasing hormone (TRH), this stimulates the pituitary to release thyroid-stimulating hormone (TSH) which stimulates the thyroid gland to produce the prohormone thyroxine (T4) and to a smaller extent the bioactive hormone triiodothyronine(T3). T4 and T3 are released into the bloodstream and transported to the tissues mostly bound to binding proteins (99.95%), such as thyroxine-binding globulin (TBG; 75%), albumin (12%) and transthyretin (10%) 2. The small percentage of T4 and T3 that circulates freely, unbound to binding proteins, is called free T4 (fT4) and free T3 (fT3) respectively. Circulating TH inhibits the production of TRH and TSH in the hypothalamus and the pituitary gland, respectively, together called the hypothalamus-pituitary-thyroid (HPT) axis. THs need to enter the cells via TH transporters to exert their function 3-6. In the cell, the inactive T4 is converted to the active T3 by deiodinase (DIO) type 1 and 2 7. Subsequently, T3 binds to the thyroid hormone receptor (TR) to exerts its effect either via gene expression (canonical pathway) or without gene expression (noncanonical pathway) 8, 9.There are several metabolizing pathways to inactivate TH; the conversion of T4 into reverse T3 (rT3) and T3 into the inactive metabolite 3,3’-diiodothyronine (T2) by type 3 deiodinase (DIO3) is one of them. A schematic summary of TH action can be found in Figure 2.
Thyroid hormone measurements TH status can be assessed by measuring TSH and THs in blood. Currently, TSH is often measured first after which fT4 concentrations can be measured to further define thyroid status. TSH and fT4 concentrations are mostly measured using automated immunoassays, but this was not always the case. After the discovery of a thyroid stimulator named TSH 10, bioassays measuring TSH stimulated 131-I-T4 release were developed in the 1950s 11. At the same time, total T4 was estimated by measuring protein-bound iodine (PBI) by colorimetry 12, which was then considered the best routinely available test to determine thyroid function. In the 1960s, the radioimmunoassay (RIA) was developed to measure TSH, T4, and T3. However, the TSH RIA suffered from interferences and low specificity and could not be used as a diagnostic test. T4 and T3 measurements using RIA superseded PBI measurement at the start of 1970s. In the 1980s, TSH RIAs were greatly improved by the development of non-competitive immunometric assays using monoclonal antibodies and consequently proved useful in the diagnosis of thyroid diseases 13. The measurement of T4 and T3 was complicated by the large influence of binding proteins. This led to the ‘free hormone index’ where the total TH concentrations were mathematically corrected for their binding protein concentration. In the 1980s, direct methods to measure fT4 were developed to overcome the influence of binding proteins. The technique of equilibrium dialysis (ED) or ultrafiltration that separated free and bound T4 were combined with the measurement of this (f)T4 using RIA. Later, this last step was replaced by liquid chromatography tandem-mass spectrometry (LC-MS/MS) measurement and equilibrium dialysis followed by this LC-MS/MS is now considered as the gold standard 14. However, it was, and still is, a challenge to directly measure fT4 reliably, due to its concentration in the picomolar range and the subtle equilibrium between free and bound T4. The technique of equilibrium dialysis followed by RIA or LC-MS/MS is an expensive, time consuming, and labor intensive technique which makes it unsuitable to use as routine test in clinical laboratories. Instead, one-step analog competitive ‘estimate’ immunoassays were developed in the 1980s to determine fT4 and fT3. These assays limited the effect of TBG disturbances, but were highly influenced by albumin or other binding protein deviations. Subsequently, two-step competitive assays were developed to reduce the impact of binding proteins by incorporating a washing step.
Nowadays, nonisotopic two-site ‘sandwich’ automated immunoassays based on the immunometric assay developed in the 1980s are most often used to measure TSH and became increasingly sensitive 15. FT4 measurements are still performed by both one- and two-step approaches, with the addition that the currently used immunoassays are automated and nonisotopic. These immunoassays measure fT4 concentrations quickly and are user-friendly. Still, a disadvantage of automated immunoassays is their susceptibility to interference from other factors/substances, although mostly limited to specific situations, such as a complex serum matrix, interference with antibodies (anti-Streptavidin, anti-Ruthenium, heterophilic, autoantibodies) or interference with other substances (medication, biotin) 16. Measuring fT4 can be particularly challenging due to its low concentration and the subtle equilibrium between free and bound T4 as mentioned above. Immunoassays rely on a stable equilibrium to accurately measure fT4 concentrations. However, changes in T4 binding protein concentrations, such as those seen during pregnancy with increased TBG concentrations, can disturb this equilibrium and affect the results 17.Solutions to more reliably measure fT4 concentrations may lay in the establishment of alternative methods, such as LC-MS/MS preceded by equilibrium dialysis 14. Total THs, such as T4, T3, and rT3, are not routinely measured in clinical practice, but are indices of thyroid function as well and can be measured in specific situations. Again, interference with other substances is the largest contributor of inaccuracies in T4, T3, and rT3 measurement.
Thyroid diseases
Thyroid diseases are very common with a prevalence of 3.82% in Europe 22 and are mainly diagnosed based on TSH concentrations that are outside their RI and subsequent fT4 concentrations The most common thyroid diseases can be classified into (subclinical) hypothyroidism and (subclinical) hyperthyroidism. Overt hypothyroidism is characterized by increased TSH (above the upper reference limit) and decreased fT4 (below the lower reference limit) concentrations,while overt hyperthyroidism shows the exact opposite results. Subclinical hypo/hyperthyroidism show an increased and decreased TSH concentration, respectively, but with a fT4 concentration within the RI. Overt hypo- and hyperthyroidism necessitate treatment and also subclinical hypo and hyperthyroidism are regularly treated. It is important to realize that diagnosis of these thyroid diseases, and thus treatment, heavily depends on accurate TSH and fT4 measurements and RIs 23.
Hypothyroidism
The prevalence of hypothyroidism in Europe in the adult population is between 0.2 and 5.3% and subclinical hypothyroidism is even more common with a prevalence of 4-20% 24, 25. Hashimoto’s hypothyroidism is an autoimmune disease and the most common cause of hypothyroidism.Symptoms of hypothyroidism include amongst others fatigue, weight gain, constipation, and brain fog, which are rather non-specific. Cornerstone of treatment for auto-immune hypothyroidism is lifelong supplementation with levothyroxine (LT4). However, despite treatment, a large number of patients who are biochemically euthyroid do not feel recovered from their symptoms. Treatment with LT4 for subclinical hypothyroidism is often initiated, although European and US guidelines do not necessarily advise to treat this group in all cases 26, 27 and interestingly, symptomatic relief is often disappointing for subclinical hypothyroid patients treated with LT4 28. Currently, we rely on serum/plasma TSH and fT4 measurements to determine euthyroidism in LT4 treated patients.However, we do not have insight into cellular TH concentrations, which may be different from serum/plasma concentrations and may help in our understanding of yet unexplained (continuing) symptoms.
A subcategory of hypothyroidism involves its inborn form, congenital hypothyroidism(CH). CH can be divided into primary CH, the most prevalent form that results from an un(der)developed thyroid gland or a defect in TH synthesis, and central CH, which is caused by insufficient stimulation of an otherwise normal thyroid gland due to a defect at the level of the pituitary and/or hypothalamus 29. CH is treated with LT4 as well. CH is mainly detected using the newborn screening(NBS), which was implemented in the Netherlands in 1981. The Dutch NBS uses a T4-TSH-TBG algorithm to detect CH and these parameters are measured in dried blood spots taken in the first days after birth 30. The Dutch algorithm detects primary CH as well as central CH, but at the cost of a high percentage of false-positive referrals, accompanied with a positive predictive value (PPV) of only 21% 31. Most other countries use a TSH-based screening, that only aims to detect primary CH 32 and comes with a higher PPV and thus less false-positive referrals 33. Therefore, improving the PPV of the Dutch NBS for CH while maintaining the detection of primary and central CH is desired.
Hyperthyroidism
Graves’ Disease (GD) is an autoimmune disease and the most common cause of hyperthyroidism. Besides TSH and fT4 measurement, TSH-receptor antibodies (TRAb) can be measured in serum to better differentiate GD from other causes of hyperthyroidism. GD is most often treated with anti thyroid drugs. After discontinuation of this medication, GD relapses in approximately 50% of patients. A remaining challenge in GD treatment is the possibility to predict this risk of relapse.The ‘Graves recurrent event after therapy’+ (GREAT+) score was developed to provide a more personalized GD relapse risk 34, although validation and implementation are not yet performed.
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