Abstract
MacroTSH still interferes with TSH assays. We present here a case report illustrating the difficulties that can arise in such conditions and attempt to discuss the steps involved in diagnosis.
Introduction
Diagnosis of thyroid disease is based on thyroid-stimulating hormone (TSH) measurement. Occasionally, this measurement may be erroneous due to analytical interference. Clinical biochemists and clinicians need to be aware of this in order to avoid repeated, costly, and time-consuming investigations, as well as unnecessary treatments. Clinicians should consider the possibility of interference and contact the laboratory to investigate the potential presence of this. Clinical biochemists must know how to use the tools available to identify analytical interference or turn to specialized laboratories.
Case presentation
A 30-year-old woman was referred to an endocrinologist due to abnormal thyroid function tests obtained in the context of constitutional thinness (TSH: 63.9 mUI/L (reference interval (RI): 0.27–4.2 mUI/L), free T4: 16 pmol/L (RI: 12.51–21.39 pmol/L), and free T3: 5.8 pmol/L (RI: 3.10–6.8 pmol/L); cobas analyser, Roche Diagnostics). Anti-thyroid peroxidase and anti-thyroglobulin antibodies were negative, and thyroid ultrasonography was normal. A short treatment with levothyroxine (initially 25 µg/day and then 50 µg/day) was initiated, and the TSH decreased to near-normal values (17 mUI/L), but the patient complained of hypertyroxinemic signs (weight loss, nervousness, sleep disorder, and thermophobia). The levothyroxine was then discontinued, and TSH increased to around 50 mUI/L (Fig. 1). Over the following 6 months, thyroid function tests were repeated several times, and the profile of subclinical hypothyroidism was confirmed. The patient was not symptomatic. Because of the discrepancy between thyroid function tests, the absence of symptoms, and the intolerance to treatment, an analytical interference test was prescribed, and the search of heterophilic antibodies was found negative. The patient was then referred to an endocrine unit to perform further exams. Although thyroid hormone concentrations were normal, genetic testing, thyrotropin-releasing hormone test, and pituitary MRI were performed and formally excluded thyroid hormone resistance or TSH-secreting pituitary adenoma. The patient was then followed up without any treatment. Two years later, she became pregnant, and levothyroxine was promptly reintroduced at 25 µg/day during the first trimester as elevated TSH (43.8 mUI/L) was associated with a low free T4 concentration (9 pmol/L (non-pregnant RI: 10–22 pmol/L)). The treatment was increased to 37.5 µg/day during the second and third trimesters, while TSH levels were 43 and 41 mUI/L, and free T4 were 11.2 and 11.7 pmol/L, respectively. The pregnancy went well, and the patient gave birth to a healthy baby. After pregnancy, levothyroxine was discontinued. Ten years after the first thyroid function test, TSH was still around 50 mUI/L with normal free thyroid hormone concentrations. Since the patient appeared clinically euthyroid, her new endocrinologist raised the question of a biological artifact and asked the laboratory to check for analytical interference of TSH measurement (1). Heterophilic antibodies that could interact with the TSH assay were still negative. The blood sample was then transferred to our laboratory for macroTSH determination research. TSH measurement was first performed using another reagent. The TSH value was 56.7 mUI/L using the Roche assay and only 2.03 mUI/L with the Abbott assay (i2000® analyser, Abbott Laboratories) on the same sample (Fig. 1). Then, TSH was measured after polyethylene glycol (PEG) precipitation. The post-PEG TSH recovery for this patient was 1.4% (pre-PEG: 56.7 mUI/L; post-PEG: 0.795 mIU/L). Finally, the suspected interference was investigated using gel filtration chromatography (GFC), the reference technique. This procedure found >95% of large molecular sized TSH (MW >150 kDa).
Evolution of TSH (A) and FT4 (B) concentrations during the follow-up of the patient. The hatched area corresponds to the TSH reference intervals and the dotted lines are the FT4 lower and upper reference intervals.
Citation: European Thyroid Journal 13, 3; 10.1530/ETJ-24-0013
Discussion
We confirm herein that macroTSH identification is still a challenging issue for clinicians. In this observation, it took 10 years to make the right diagnosis, and the patient underwent, during this long period of time, inappropriate explorations and treatments. This observation adds to the literature as long-term evolution has been rarely studied (2). In the present case, biological abnormalities remained unchanged over time. Additionally, if case reports have described the challenges of this diagnosis, the prevalence of analytical interference due to macroTSH is not known (3). MacroTSH is a compound that may lead to falsely elevated TSH, simulating the result of subclinical hypothyroidism. Clinicians should consider macroTSH when subclinical hypothyroidism treatment is not well tolerated, and when the patient gains no benefit from it. In the case presented here, the patient was treated twice. As a reminder, TSH was very elevated, but thyroid hormone concentrations were strictly normal; the patient had no symptoms of hypothyroidism and no signs of thyroiditis. The difficulties in establishing a diagnosis led to inappropriate medical explorations. Indeed, there was then no reason, in the present case, to look for thyroid hormone resistance or pituitary adenoma.
When suspected, several methods may be used to identify analytic interference. The easiest is to perform the measurement using a reagent from another manufacturer (1) (Fig. 2). In the case presented herein, there was a marked difference between the two reagents used, indicating a major analytic interference. It has been reported that the Abbott assay may be the least sensitive to macroTSH (4), whereas many published case reports describe interference with the Roche assay (5, 6, 7, 8, 9, 10). MacroTSH results most often from the binding of TSH molecule to anti-TSH autoantibodies and is known to precipitate in presence of PEG (2, 4, 10). However, care should be taken when interpreting results, as the 65% cutoff very commonly used for macroprolactin is not adapted for macroTSH. It is recommended to suspect macroTSH in samples where post-PEG TSH recovery is lower than 10–30% (2, 10, 11, 12, 13) since sera from patients with subclinical hypothyroidism (elevated TSH without macroTSH) have a percentage of recovery around 63% (2). The gold standard procedure to prove the presence of macroTSH (and more generally macrohormone) is GFC. It is not commonly used owing to a combined lack of equipment and technical skills; it is also of note that GFC should exclusively be used on samples with less than 10–30% of post-PEG TSH recovery (1, 4, 14). Analytical interference affects all platforms and in different ways;. for instance, the prolactin reagent from Roche Diagnostics is less sensitive to macroprolactin compared to the reagent from Abbott Laboratories (15, 16, 17, 18), which is the opposite of that found for macroTSH.
MacroTSH interference on immunoassays. TSH concentrations are measured with immunometric assays, meaning that two different antibodies bind the TSH. One antibody is linked to a magnetic bead, and the other to a tracer whose signal can be measured after a wash step to remove antibodies that are not bind. Some reagent (reagent A) use antibodies that recognize epitopes that are available when TSH is combined with IgG to form macroTSH. In such a case, TSH value is overestimated. When antibodies recognize epitopes that are not available in the presence of macroTSH (reagent B), then the reagent is not or less sensitive to macroTSH. Figure created in part with BioRender.
Citation: European Thyroid Journal 13, 3; 10.1530/ETJ-24-0013
As the macroTSH led to TSH analytical interference, it is important to remember that free thyroid hormone concentrations will be within the reference interval and are not impacted by macroTSH.
In some cases, this interference may cross the placenta and also impact the TSH determination of neonatal hypothyroidism screening (19, 20). In this case report, because of a low FT4 (9 pmol/L) during pregnancy, the patient was treated with thyroxine, fortunately without deleterious impact on the pregnancy. The child was born in good health. Moreover, it is also important to remember that free T4 during pregnancy has to be interpreted in relation to reference values adapted to pregnancy (21).
In conclusion, macroTSH may lead to misdiagnosis, unnecessary and expansive evaluation, and inappropriate treatment. There is no clear recommendation on when to screen for this interference. However, Hattori et al. suggest screening for macroTSH, especially in childbearing women before starting treatment for subclinical hypothyroidism (4). We also suggest that macroTSH should be considered in patients with isolated elevated TSH (>10 mUI/L) with no other signs of thyroid dysfunction, particularly in childbearing-age women before starting supplementation with levothyroxine (4, 5).
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this case report.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Statement of ethics
The study was approved by the Scientific and Ethics Committee of the Hospices Civils de Lyon. The patient provided written informed consent before publication.
Author contribution statement
VR drafted the manuscript and SM, PP, JA, and HL reviewed it critically.
Acknowledgements
We would like to thank Françoise Borson-Chazot and Philip Robinson for their help with manuscript preparation.
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