Laboratory testing in hyperthyroidism

Introduction

The clinical diagnosis of hypo- or hyperthyroidism is difficult[1][2][3]. Clinical symptoms and signs are often non-specific, and there is incomplete correlation between structural and functional thyroid gland changes. Laboratory testing is therefore indispensible in establishing the diagnosis of thyrotoxicosis. Similar considerations apply to treatment monitoring.

Laboratory testing also plays a crucial role in establishing the most likely cause for a patient’s hyperthyroidism.

Finally, during pregnancy, when isotopic scanning is relatively contraindicated and ultrasound is more difficult to interpret, laboratory testing becomes even more important.

Diagnosis of thyrotoxicosis

Thyroid hormone production is tightly regulated. Pituitary thyrotropin (TSH) secretion stimulates thyroidal synthesis and release of thyroxine (T4) and triiodothyronine (T3). The resultant increase in circulating thyroid hormone levels down-regulates TSH production, closing the feedback loop.

Over- or underproduction of thyroid hormone can be due to abnormal function of either the thyroid gland itself (primary hyper- or hypothyroidism) or due to pituitary/hypothalamic disease (secondary/tertiary hyper- or hypothyroidism). Because of their reciprocal relationship, paired measurements of serum TSH and T4/T3 concentrations should allow unequivocal assessment of all states of thyroid function (Figure 1). In practice, TSH measurement alone is usually sufficient for the initial diagnosis of hyperthyroidism, because secondary thyroid dysfunction is rare. TSH is preferred over T3/T4, because the pituitary response to changes in peripheral thyroid hormone concentrations is exponential: a 2-fold change in free T4 (FT4) results in a 30-60-fold reciprocal change in TSH (Figure 2). Consequently, TSH is the most sensitive indicator of thyroid function status in most situations[4]. Exceptions include pituitary disease, rapidly changing T4/T3 levels (e.g. during treatment of hyperthyroidism), and transient changes in free thyroid hormone concentrations due to severe illness or drug treatments that displace thyroid hormones from binding proteins. In these situations, measurement of FT4 and total T3 (TT3) might be required in addition to TSH measurement (Figure 3).

For optimal diagnostic performance, a TSH assay needs to have a limit of quantification (functional sensitivity: lowest analyte concentration with an interassay coefficient of variation of <20%) of ≤0.02 mIU/L[4]. Virtually all modern TSH assays achieve this goal. However, even after >20 years of standardization, TSH assays from different manufacturers continue to show significant systematic biases to each other, with differences between the highest and lowest biased assays of about 40%[5]. Therefore, if serial TSH measurements are to be performed on a patient, the same assay should be used throughout.

Treatment monitoring and detection of recurrence

During treatment of hyperthyroidism there can be quite rapid changes in serum thyroid hormone levels. Since pituitary TSH production can be completely suppressed in long-standing or severe hyperthyroidism[6], there may be a lag in the pituitary TSH response to such treatment-related changes. Serum T4/T3 should therefore be monitored much more frequently than TSH during treatment, particularly when therapy is first initiated. However, based on biological half-life, there is little point in checking T4 (half-life: 5-7 days) more often than weekly. Since T3 is mostly generated in the periphery from T4, a minimum testing interval of a week seems also reasonable for T3, despite its shorter half-life of ~1 day. In most situations, however, testing intervals will be longer than weekly; the two most popular treatments, radioiodine and antithyroid drugs, require usually at least 2-4 weeks before they reduce thyroid hormone levels significantly.

Two questions much debated are, whether T4 or T3 should be measured and whether it is preferable to monitor total- or free T4 or T3 levels.

Theoretically, measurement of free hormones is less subject to confounding by the concentrations of thyroid hormone binding proteins (thyroxine binding globulin, transthyretin, and albumin). Binding protein concentrations can fluctuate in response to changing thyroid hormone levels, and as a consequence of other influences (e.g. sex hormones, drugs, and non-thyroidal illness).

Unfortunately, the theoretical advantages of free hormone assays are not fully realized in practice, because most FT4 and FT3 assays are analog immunoassays. Such assays use analogs of T4 or T3, which are designed to have similar binding affinity as T4 or T3, respectively, to the antibodies used in the assays, while at the same time demonstrating much less avid binding than natural thyroid hormones to the thyroid hormone binding proteins[7]. In some commercial assays, such analogs do not perform quite as desired, and what is measured is something in-between a total and a free hormone measurement[89]. Furthermore, even analog immunoassays that display satisfactory performance in healthy individuals, might fail to give accurate results at the extremes of concentrations of thyroid hormones- or thyroid hormone binding proteins, which might be observed in thyrotoxicosis[89]. An argument can therefore be made to use total thyroid hormone measurements instead, or to consider using free thyroid hormone measurements that are performed by a reference methodology, such as dialysis.

Another issue with thyroid hormone immunoassays is that they are all competitive immunoassays. Exogenous labeled thyroid hormone (or analog) competes with the thyroid hormone in patient samples for a limited concentration of assay antibodies. This design results in a limited dynamic measurement range of only 1-1.5 orders of magnitude of thyroid hormone concentrations. Unfortunately, the concentration span of thyroid hormones observed in some thyrotoxic patients during treatment may exceed 2 orders of magnitude, reaching from severe thyrotoxic levels to severe hypothyroid levels. Depending on the design choices an assay manufacturer has made, good accuracy and precision can only be achieved at either the high- or at the low end of this range, but not at both. I would recommended that clinicians treating thyrotoxic patients, request information from their laboratory that allows them to estimate the part of the measurement range that will provide reliable results.

The second decision is whether to monitor T4 or T3. The high thyroid hormone turnover in most forms of thyrotoxicosis might reduce the proportion of fully iodinated tyrosine residues that are formed on thyroglobulin (Tg). Consequently, the Tg in the thyroid glands of hyperthyroid patients might contain more T3 than in euthyroid patients[10]. Furthermore, T3 has a much shorter half-life than T4, making T3 potentially a more sensitive indicator of short-term changes in thyroid gland activity than T4. Many experts therefore favor measurement of T3 over T4 measurement during monitoring of hyperthyroid patients[11]. However, pure T3-toxicosis is rare[12][13][14][15] and there are no compelling studies that suggest that the potential advantages of T3 measurements impact clinical outcomes significantly. At the same time, serum total T3 and FT3 concentrations are always considerably lower than total T4 and FT4 concentrations, respectively, resulting in greater analytical challenges. In particular, most, if not all, FT3 assays are of somewhat dubious analytical reliability. A further disadvantage is that there is very little agreement between results obtained by different analog FT3 immunoassays (Figure 4), absolutely mandating that the same FT3 assay must always be used for serial measurements in a given patient.

Determining the cause of hyperthyroidism

Once a diagnosis of hyperthyroidism has been reached, optimal patient management requires its cause to be established.

Primary autoimmune thyrotoxicosis (Graves’ disease) is the most common cause of hyperthyroidism, followed closely, at least in many countries other than USA, Canada or Japan, by thyroid autonomy (unifocal or multifocal) and subacute thyroiditis (including the painless variant). While the relative proportion of these top three depends in part on environmental factors, in particular the nutritional iodine supply status of a population, they account collectively for >95% of cases of hyperthyroidism. The remainder is made up by genetic disorders, nutritional or iatrogenic thyroid hormone intoxication, secondary hyperthyroidism, and a few other exotic causes.

If a patient presents with a relatively short history (a few weeks or months) of rapidly worsening symptoms, has a large, smooth goiter with an audible bruit, and has clear extrathyroidal stigmata of Graves’ disease (exophthalmos, pretibial myxedema or acropachy), the differential diagnosis is easy; likewise, for the patient with large solitary nodule that has grown over several years, accompanied by gradually increasing symptoms (thyroid autonomy). However, many a patient’s presentation may not be as typical as in the above scenarios. In addition, during the initial thyrotoxic phase of their disease, patients with subacute thyroiditis, particular of the painless variety, can be clinically indistinguishable from those patients with Graves’ disease without extrathyroidal manifestations. Finally, with both toxic nodular goiter and Graves’ disease being fairly common, there are bound to be patients who suffer from both diseases[16].

It is therefore often necessary to perform additional testing to establish the cause of a patient’s hyperthyroidism. In the USA, an isotopic thyroid uptake (99mTc, 123I or 131I) with or without scan is predominately used for this purpose and is recommended in the American Thyroid Association (ATA) hyperthyroidism management guidelines[11]. By contrast, Europeans are relying increasingly on Ultrasound (US), including color Doppler measurement of blood flow[17]. However, there are pitfalls to both methods[18]. For example, amongst many other causes, iodine deficiency can mimic the increased uptake and blood flow of Graves’ disease on isotope scanning and US, respectively. Furthermore, these imaging procedures are not as standardized or reproducible as laboratory tests. US quality in particular is highly operator-dependent, and for either type of testing, there is diagnostic overlap between the different forms of thyrotoxicosis[19]. In many cases, both isotope uptake/scan and US with Doppler will also require a separate patient visit, often to another facility. Isotope uptake/scan and US can also be quite costly; in a 200 mile radius around my home town of Rochester Minnesota, the total charges for thyroid uptake range from US-$ 139 to US-$ 509 and from US-$ 265 to US-$ 1,813 for scan plus uptake, while neck US is charged at rates of US-$ 96 to US-$ 866 (http://www.mymedicalcosts.com/Home.aspx; accessed 7/6/2011).

For all these reasons, there has been increased interest in using laboratory testing to discriminate between the different causes of hyperthyroidism. This approach is based on distinguishing Graves’ disease from other causes of thyrotoxicosis by detecting the TSH receptor autoantibodies (TSHR-AB), which are responsible for the hyperthyroidism in Graves’ disease (Figure 3). Two types of assays are available for this purpose:

1. Conventional autoantibody assays, which detect autoantibodies in patient sera that are capable of binding to the human TSHR. These are configured as competitive assays – TSHR-ABs in patient serum compete with either TSH or standardized TSHR-AB preparations for binding to intact or partial TSHRs.
2. Functional assays, which measure the ability of patient serum, compared to healthy reference serum, to stimulate growth, iodine uptake, or cAMP production of animal or human tissue isolates/cells that express the TSHR, either naturally (derived from mature or embryonal thyroid tissue/cells), or after transfection (most commonly Chinese Hamster Ovary [CHO] cells).

These methods were originally developed during the late 1960s and early 1970s[20][21]. Many variations of TSHR-AB assays are now offered, and there is a bewildering range of test names and performance characteristics (Table). However, all assays fall into these two assay-type categories[22][23][24].

Both conventional autoantibody assays and functional assays have improved greatly during the last 30-40 years[25]. Analytical turnaround times are now well under 2 days, in some instances (automated conventional TSHR-AB assay), less than 2 hours. The most recent iterations of conventional- and functional assays show clinical sensitivity and specificity of >90% for the diagnosis of Graves’ disease versus other causes of hyperthyroidism[24][26][27][28][29][30][31]. In fact, these assays are now so accurate and reliable that some investigators are wondering whether so called autoantibody-negative Graves’ disease actually exists[25][32].

In children, these assays have historically shown lesser detection rates of Graves’ disease, around 60%[33], but modern assays now show comparable sensitivity and specificity to what is observed in adults [34][35][36].

As to differences between conventional autoantibody assays and functional assays, comparisons of their dose-response curves after application of serial dilutions of international TSHR-AB standard material suggests that functional assays might be more sensitive for diagnosing subtle disease, at the expense of accuracy at very high TSHR-AB concentrations (Figure 5). Functional assays also provide information about the biological effect of TSHR-ABs, while conventional antibody assays can only report the presence or absence of TSHR-AB and their concentrations, but can not determine, whether these TSHR-AB are thyroid stimulating or blocking. In practice, however, this is not a big issue, because a patient’s thyroid function status will be known by the time TSHR-AB testing is performed.

Lack of assay standardization has been a problem in the past[11][37]. This situation has improved with the availability of international standard material and the use of monoclonal human TSHR-ABs instead of TSH as competitor and as calibration material in TSHR-AB assays[38][39]. For conventional TSHR-AB assays, this development has resulted in very close correlations between assays from different manufacturers. The table summarizes the main characteristics of TSHR-AB assays.

Diagnosis of 1st trimester presentation of Graves’ disease

The prevalence of Graves’ disease during pregnancy is about 0.1-0.4%[40]. A proportion of these women will have pre-existing, untreated, or partially treated Graves’ disease, while others might present for the 1st time during the 1st trimester. In this latter group, it can be difficult to distinguish between Graves’ disease and transient 1st trimester thyrotoxicosis induced by human chorionic gonadotropin (hCG)[40][41], a condition with comparable prevalence to 1st trimester Graves’ disease, which occurs in the context of hyperemesis gravidarum[40][41]. It is associated with very high hCG levels and with the presence of certain isoforms of hCG[42]. Presumably, the disease mechanism is hCG cross-talk on the TSHR. Since hyperemesis gravidarum remits between weeks 12 and 14, coincident with a gradual decline in hCG and a hCG isoform switch, only supportive therapy is required. By contrast, Graves’ disease requires ongoing close supervision of mother and fetus, and anti-thyroid drug treatment, if it does not remit spontaneously during the 2nd trimester (as is often the case, due to pregnancy-associated immune response down-regulation).

TSHR-AB measurements are indispensible to differentiate between these two conditions, because isotope scanning is contraindicated, and because the much increased maternal blood volume and cardiac output make thyroid US more difficult to interpret. The same diagnostic cut-offs as in non-pregnant women can be used (Table).

Risk of fetal/neonatal thyrotoxicosis in women with current or prior Graves’ disease

TSHR-ABs can cross through the placenta to the fetus. Thyroid hormone production and basic hypothalamic/pituitary-thyroid feedback are established by embryonal day 70[43]. From this moment onwards, maternal stimulating TSHR-AB can cause fetal or neonatal hyperthyroidism[44][45]. Therefore, whenever a woman presents with Graves’ disease during pregnancy, both maternal and fetal thyroid function need to be considered.

Women who have had thyroid-ablative treatment prior to pregnancy – radioiodine or surgery – represent a particular challenge. A proportion of these women will continue to have circulating TSHR-AB, because ablative treatment does not cure the underlying autoimmune disorder. In fact, TSHR-AB levels tend to rise substantially during the first 6 months following radioiodine treatment[46]. The TSHR-AB status of all pregnant women with a past history of Graves’ disease should therefore be determined during the 1st trimester. A result within the non-pregnant population reference range makes fetal/neonatal thyrotoxicosis exceedingly unlikely[47][48][49][50][51][52]. Such patients usually require no further testing. By contrast, patients with TSHR-AB levels above the non-pregnant reference range require further investigations or follow-up. The risk for fetal/neonatal thyrotoxicosis increases in parallel with the TSHR-AB concentrations and modest elevations – less than 2.5 times the upper limit of the non-pregnant reference range – are rarely associated with fetal/neonatal disease. Such patients may only require retesting sometime during the 2nd trimester. The cut-off of 2.5 times the non-pregnant cut-off translates into conventional TSHR-AB levels of ~40% binding inhibition or ~4IU/L (~4 mIU/mL) and a functional assay index ~2.5-3 (~250-300%). Patients with higher TSHR-AB levels should have repeated TSHR-AB measurements during pregnancy[47][48][49][50][51][52]. Given the apparent half-life of TSHR-AB of 8-20 days, repeat measurements every 2-4 weeks are appropriate[53][54]. These patients should also be linked into high risk obstetric care.

Either conventional TSHR-AB assays or functional assays can be used. Since many patients with Graves’ disease have blocking TSHR-AB in addition to stimulating TSHR-AB[55][56][57], functional assays may offer somewhat greater diagnostic specificity. During the 2nd and 3rd trimester there also appears to be a switch from stimulating TSHR-AB to blocking TSHR-AB, with no major change in total TSHR-AB concentrations[57]. A combination of conventional TSHR-AB testing and functional testing might give indication of such a change, with conventional TSHR-AB assays showing little change, while functional TSHR-AB assays show declining values.

Finally, measurement of maternal TSHR-AB in women with previous Graves’ disease has additional benefits for mother and child: (i) high levels in the 1st trimester indicate a high risk of a flare-up of Graves’ disease after delivery for women, who still have residual thyroid tissue[58], and (ii) a high level in the 3rd trimester can be associated with maternal transmission of TSHR-AB to infants during breastfeeding, with resultant thyrotoxicosis in the baby, even if it was not affected at birth[59].

Other laboratory testing in hyperthyroidism

When the common causes of thyrotoxicosis have been excluded, a variety of laboratory tests can assist in the diagnosis of the rarer causes. Detailed discussion of this topic is beyond the scope of this review, but genetic testing (thyroid hormone resistance, activating TSRH mutations, McCune-Albright syndrome, etc.), pituitary hormone measurements (TSH secreting pituitary tumor), and various other test might be used.

In addition, some of the testing discussed in this review might have other, not as yet mainstream, applications. For examples, there is an increasing body of literature that suggests that TSHR-AB measurement might be useful for determining the risk of relapse in patients on anti-thyroid drug treatment[60][61][62][63][64][65]. Likewise, worsening of Graves’ ophthalmopathy might be predicted by TSHR-AB levels[66][67][68]. Over time, we can expect the clinical usefulness of such testing to be defined more precisely.

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