Abstract
A 19-year-old male presented with mild fatigue and chronic constipation and was found to have an elevated thyroid-stimulating hormone (TSH) level of 85 mIU/L, normal free thyroxine, and normal total triiodothyronine. This presentation was concerning for Macro-TSH, which is rare and may suggest hypothyroidism. This patient’s diagnosis of Macro-TSH was confirmed by TSH antibody testing and gel filtration chromatography. We present this case to highlight the need to further evaluate TSH results when thyroid function tests are incompatible with the clinical presentation. Assessment for TSH antibodies, a commercially available test, or gold-standard gel filtration chromatography testing, which is not commercially available, can be used to diagnose Macro-TSH.
Keywords
Introduction
Macro-thyroid-stimulating hormone (TSH) is a rare and therefore under-recognized cause of elevated TSH when measurements of free thyroxine (T4) and total triiodothyronine (T3) are normal.1,2 The binding of plasma proteins, particularly antibodies, to TSH results in the formation of macromolecular complexes (Macro-TSH) with very low biological activity.1,3 Macro-TSH can present a diagnostic challenge due to the detection of the TSH within the Macro-TSH complex being recognized by TSH immunoassays (IAs), resulting in a falsely elevated TSH level.3-5 As a consequence, misdiagnosis as subclinical hypothyroidism or other causes of euthyroid hyperthyrotropinemia may lead to the patient receiving unnecessary treatment.3,5,6
We present a case with Macro-TSH to guide the diagnostic approach and awareness of the condition.
Case Presentation
A 19-year-old male undergoing a routine workup with his primary care physician was found to have a serum TSH of 85 mIU/L (reference interval [RI] 0.36-4.0 mIU/L) measured by IA, with normal free T4 and total T3, and no symptoms suggestive of hypothyroidism.
The patient was referred for endocrine evaluation and had suspended the biotin supplement several weeks before the Endocrinology Clinic visit. He denied hypothyroid symptoms except for mild fatigue and chronic constipation. He was clinically euthyroid with an unremarkable physical exam. A normal thyroid exam revealed no visible or palpable thyroid enlargement, nodules, tenderness, or abnormal sounds; the gland was soft, smooth, and moved with swallowing. The patient’s mother has hypothyroidism, and both parents have a history of hypercholesterolemia. There is no family history of thyroid cancer.
The following test results were obtained: TSH 94.7 mIU/L (RI 0.40-4.50), free T4 1.36 ng/dL (RI 0.8-1.8), total T3 93 ng/dL (RI 76-181), negative thyroid peroxidase (TPO) antibody, and negative thyroglobulin (Tg) antibodies (Table 1). Additional testing revealed a serum-free T4 level by direct dialysis of 1.3 ng/dL (RI 0.9-2.2), alpha subunit of 0.2 ng/mL (RI 0.1-0.5), prolactin of 11.5 ng/mL, cortisol of 7.5 µg/dL, and ACTH of 13 pg/mL. Sequencing of the thyroid hormone receptor beta gene for known variants was negative. Human anti-mouse immunoglobulin G antibody (HAMA) testing identified no interference (Table 1).
Results of Endocrinology Laboratory Workup.
Abbreviations: ACTH, adrenocorticotropic hormone; HAMA, human anti-mouse immunoglobulin G antibody; T3, triiodothyronine; T4, thyroxine; THRβ, thyroid hormone receptor beta; TPO, thyroid peroxidase; TSH, thyroid-stimulating hormone.
Detailed Laboratory Investigations
The possibility of Macro-TSH triggered the submission of samples to determine whether antibodies binding to TSH were present. Antibodies binding to TSH were detected by demonstrating the presence of autoantibodies capable of immunoprecipitating radiolabeled TSH, which were absent from a sample of normal control serum. This positive TSH antibody test (Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, USA) suggested that the patient’s elevated TSH was due to antibodies bound to TSH. Gel filtration chromatography demonstrated that a large proportion of the circulating TSH was contained within large molecular- weight complexes, confirming the diagnosis of Macro-TSH (Figure 1). These biochemical findings were discussed with the patient and shared with his primary care provider, preventing the initiation of unnecessary treatment.
Gel filtration chromatography showed that the TSH peak of the control sample (blue line) eluted at the 13th fraction, corresponding to free TSH, whereas the patient’s TSH peak (orange line) eluted at the 10th fraction, overlapping with the reference Macro-TSH (green line). These results confirm that the patient’s markedly elevated serum TSH is due to the presence of Macro-TSH. TSH, thyroid-stimulating hormone.
Discussion
Macro-TSH, large aggregations of antibodies or other plasma proteins complexed with monomeric TSH, is uncommon, with an estimated prevalence of 0.6% to 1.6% of the adult population,1,2 and in 0.43% of 939 umbilical cord samples of neonates. 7 Whether Macro-TSH is likely to persist for a patient’s lifetime or represents a transient phenomenon remains unknown. 8 Case reports describe persistently elevated TSH levels in patients who are clinically euthyroid with normal free T4 and total T3 levels.3,6 These patients are frequently discovered after unnecessary treatment attempts, where they remain asymptomatic with stable free active hormones.1,6 Clinicians should suspect Macro-TSH in patients who have persistently elevated TSH despite normal free T4 and T3 levels, are clinically euthyroid, and have no goiter, pituitary abnormalities, or positive thyroid antibodies, especially when TSH values do not respond appropriately to treatment.4,9 Early recognition and diagnosis of Macro-TSH are important to prevent unnecessary interventions, such as inappropriate thyroid hormone therapy.
However, many other more common conditions can result in elevated TSH with normal total T3 and free T4 levels (Table 2), including subclinical hypothyroidism, malabsorption of T4 therapy, intermittent/poor adherence to T4 therapy, nonthyroidal illness recovery phase, resistance to thyroid hormone, human anti-mouse antibodies, TSH-secreting adenomas, and certain medications such as amiodarone. 9 Most patients with subclinical hypothyroidism have chronic autoimmune (Hashimoto’s) thyroiditis with anti-TPO or anti-Tg antibodies, 10 and have much lower elevations of TSH. Hyperprolactinemia and Cushing’s syndrome have also been associated with subclinical hypothyroidism.11,12
Evaluation of Elevated TSH With Normal FT4 and T3: Differential Diagnosis and Diagnostic Approach.
Abbreviations: CH, congenital hypothyroidism; HAMA, human anti-mouse IgG antibody; IgG, immunoglobulin G; IL-2, interleukin-2; Pax8, paired box 8; RTSH, resistance to thyrotropin; T3, triiodothyronine; T4, thyroxine; Tg, thyroglobulin; TKIs, tyrosine kinase inhibitors; TPO, thyroid peroxidase; TSHR, thyroid-stimulating hormone receptor.
Our patient was negative for both anti-TPO and anti-Tg antibodies, ruling out Hashimoto’s thyroiditis, and had normal levels of prolactin, adrenocorticotropic hormone, and cortisol levels (Table 1), which ruled out hyperprolactinemia and Cushing’s syndrome. Testing free T4 by dialysis removes potentially interfering proteins, but our patient’s T4 level remained within the normal range after dialyzing the sample. An alpha subunit and a beta subunit form the mature TSH, and an elevated level of free alpha subunit in the serum may suggest a possible TSH-secreting adenoma. 13 In the patient’s case, the alpha subunit level was within the reference range.
Most cases of Macro-TSH are caused by immunoglobulin G (IgG) complexed with TSH, although cases with IgA have been reported.14,15 Commercially available IAs are the methods of choice for thyroid function testing due to rapid turnaround time, high specificity and sensitivity, and complete automation. 2 Still, there are currently no commercial TSH IA platforms that do not cross-react with Macro-TSH.14,15 Reports detailing the use of various commercial TSH IAs to examine whether different assays yield different results have shown that these assays are similarly affected by Macro-TSH.3,14,15 TSH assays are also vulnerable to interference from heterophile antibodies and HAMA, which can bind to the antibodies used in IAs and cause false-positive or false-negative results.2,16
TSH antibody testing is commercially available and may assist in identifying cases in which anti-TSH immunoglobulins form complexes with TSH to form Macro-TSH, as in the present case. Still, the TSH antibody test (Quest Diagnostics Nichols Institute) has not been previously highlighted in previous reports. Other, more time-intensive methods, such as polyethylene glycol precipitation, pretreatment with a heterophilic antibody tube, affinity chromatography on protein A, and size fractionation chromatography, are also effective in identifying antibody interferences.1,5,17 Size fractionation chromatography is the gold-standard method for confirming Macro-TSH, and it should be done to confirm positive results from any of the other tests.17,18 Unfortunately, size fractionation chromatography is not commercially available.
In patients whose clinical pictures do not explain the cause of abnormal thyroid function tests, clinicians should have a low threshold to evaluate for substances that interfere with TSH testing. Macro-TSH and macroprolactin form Ig-bound, high-molecular-weight complexes that interfere with IAs.3,17,19 The aggregations of Ig-bound TSH are inactive and unlikely to harm the patient directly. Macroprolactin is more common, while Macro-TSH is rarer but important in thyroid testing. Less frequent macro forms of FSH, LH, and GH have been reported, and similar macro phenomena occur with other proteins, such as macroamylase or Macro-AST, representing a recognized diagnostic challenge.20,21
However, they jeopardize the integrity of the diagnostic process by masquerading as more common diagnoses, such subclinical as hypothyroidism, and, therefore, have the potential to lead to overtreatment. 22 There are already growing concerns regarding the overtreatment of subclinical hypothyroidism, both in specialty and primary care settings. 23 Therefore, reporting cases of Macro-TSH and relevant diagnostic approaches remains important.
Conclusion
Early suspicion and diagnosis of Macro-TSH are important for preventing unnecessary treatment. This report highlights the option of ordering a commercially available test to detect TSH antibody in a patient with macro-TSH. The results of this test provided useful clues for the management of this patient and were corroborated by size-exclusion chromatography, which is available only through specialized research laboratories.
Materials and Methods
Radio Labeling of Purified Human TSH
Preparations of purified human TSH (Calbiochem; MilliporeSigma, St Louis, MO, USA) were radiolabeled using 125I and separated from free iodine by size-exclusion chromatography. The resulting 125I-labeled TSH (“stock tracer”) was stored at −15 °C to −35 °C or at 2 °C to 8 °C when diluted (“working tracer”) until use. In this assay, the “working tracer” denotes the tracer reagent diluted to the final working concentration used for analysis.
Samples representing “positive controls” were constructed by spiking mouse anti-TSH antibodies (Fitzgerald Industries) into human serum containing no detectable anti-TSH antibodies. “Negative controls” were aliquots of normal human serum (Golden West Biologicals, Temecula, CA, USA) or pooled serum from normal healthy volunteers that tested negative for the presence of anti-TSH antibodies. Goat anti-human gamma globulin (“second antibody”) was from Golden West Biologicals.
TSH Antibody Detection Method
Aliquots of control serum or patient serum samples were added to glass tubes, followed by the addition of assay buffer, followed by the addition of working tracer to all tubes. Samples were mixed and incubated at room temperature for 18 to 24 hours. Following incubation, the second antibody was added to the tubes. Tubes were mixed and incubated at a refrigerated temperature for 60 minutes. Following incubation, 4% polyethylene glycol solution was added. Tubes were mixed and incubated at room temperature for 20 minutes. After incubation, samples were centrifuged at 1912 RCF under refrigerated temperature for 30 minutes. After centrifugation, the supernatant was decanted, and the pellets remaining in the tubes were counted in a gamma counter.
Radioactivity (counts per minute [CPM]) associated with four separate normal reference samples were used to generate the “normal reference value” (“normal assay specific cutoff”) for the assay. The assay “clinical cutoff” is fixed at 30% above the mean value established for the “normal assay specific cutoff.” Samples with CPMs below the clinical cutoff are reported as “Negative”; those exceeding this threshold are reported as “Positive.” Assays of the positive control displayed 2.5% coefficient of variation (CV) intra-assay precision, and a 12.4% CV inter-assay precision. Assays of the negative control provided a 6.0% CV intra-assay precision, and a 10.5% CV inter-assay precision.
Column Chromatography
High molecular mass TSH immunoreactivity was confirmed using gel filtration chromatography as previously described (Figure 1). The analysis was performed on serum from the patient, a control with normal TSH, and a reference Macro-TSH sample. A total of 200 µL of diluted serum (diluted 1:1 with elution buffer) was applied to a 23 × 3 cm Sephadex G-100 column, which was equilibrated with 50 mM Tris-HCl buffer containing 150 mM NaCl, pH 7.2. Fractions were collected dropwise (10 drops/tube) using an ISCO Retriever II fraction collector (SpectraLab Scientific, Inc, Markham, ON, Canada), and TSH concentrations were measured with the Immulite 1000 IA system (Siemens Healthineers, Erlangen, Marla, Germany). 5
Footnotes
Author Note
Prior Presentation of Abstract Statement: The abstract was presented at the Endo Society Meeting in June 2023, Chicago, Illinois.
ORCID iDs
Ethical Considerations
Our institution does not require ethical approval for reporting an individual case.
Consent for Publication
Written informed consent was not required by our institution for publication of anonymized clinical information.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Declaration of Conflicting Interests
The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: One of the authors serves as an editor for this journal. To avoid any potential conflict of interest, this author was not involved in the review process or editorial decision-making for this case report.