Analytical Considerations in the Evaluation of Thyroid Function

Last Literature Review: December 2020 Last Update:

Medical Experts



Kayode A. Balogun, PhD
Kayode A. Balogun, PhD
Clinical Chemistry Fellow, Pathology, University of Utah School of Medicine

Thyroid function tests are used in the initial evaluation of thyroid disease. Thyroid function immunoassays, including many thyroid-stimulating hormone (TSH, or thyrotropin), thyroxine (T4), and triiodothyronine (T3) tests, are subject to a number of sources of interference that may impact test results. Sources of interference include antibodies (eg, antiruthenium antibodies), biotin supplements, and other substances. Identification of interference is important to avoid inappropriate treatment and management.

Quick Answers for Clinicians

Do thyroid function test results vary depending on the methodology used?

Due to a lack of standardization, the results of thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) tests are subject to variation based on the method, assay platform, and protocol used. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with equilibrium dialysis or ultrafiltration is considered the gold standard for measuring free thyroid hormones (free T3 and free T4), but modern immunoassay results often correlate well with this technique and are more readily available and cost-effective. Due to method-related bias, however, method-specific reference intervals are recommended. It is important to note that immunoassays may be subject to a variety of interferences (see the Common Sources of Interference with Thyroid Function Tests section).

How do biotin supplements affect thyroid hormone tests?

Many commonly used thyroid function immunoassays make use of biotin-streptavidin technology.  High biotin intake, such as from biotin supplements, may interfere with these assays and lead to inaccurate test results.    Whether biotin interferes and what type of interference is expected depends on the design of the assay and will vary by laboratory. For more information, see the ARUP Laboratories Biotin Interference page and Common Sources of Interference with Thyroid Function Tests section.

What sources of interference might cause misleading thyroid function test results?

In addition to biotin, thyroid function immunoassays are subject to interference from macro thyroid-stimulating hormone (macro-TSH), antistreptavidin antibodies, antiruthenium antibodies, thyroid hormone autoantibodies, and heterophilic antibodies, among other sources of interference. Such interference should be suspected when results from a thyroid function test do not match previous results from the same test, other test results, or clinical presentation.  The appropriate approach to the detection and correction of interference depends on the type of interference suspected. For more information, see the Common Sources of Interference with Thyroid Function Tests section.

Common Sources of Interference With Thyroid Function Tests

Thyroid function testing often relies on immunoassays and is therefore susceptible to a number of sources of interference. Six major causes of interference, including biotin, macro thyroid-stimulating hormone (macro-TSH), antistreptavidin antibodies, antiruthenium antibodies, thyroid hormone autoantibodies, and heterophilic antibodies, may have a particularly large impact. Additional sources of interference include thyroid hormone transport protein variants, TSH variants, and paraprotein. 

Communication between clinicians, laboratorians, and assay manufacturers is required to avoid the potential negative consequences of interference. Interference should be suspected when test values do not match previous test values, other laboratory test parameters, or clinical presentation.  Interference is commonly detected by repeating the assay using a different method, performing dilutions, adding a blocking agent, or removing interfering antibodies.  


Normal dietary biotin intake does not typically interfere with biotin-streptavidin-based immunoassays for TSH, T3, and T4. However, high biotin intake, such as from dietary supplements for hair, skin, and nails, may lead to falsely low TSH or falsely high T3 or T4 test results.    Multiple factors may impact the extent of interference, including the specific immunoassay platform used, the type of assay (sandwich or competitive), the volume of the sample, and the protocol used.  

The recommended method for identifying suspected biotin interference is to ask the patient about biotin supplementation.  Patients should consult with their healthcare providers, and, if medically indicated, stop taking high-dose biotin supplements and repeat testing after 2 or more days.  If the patient does not report taking supplements, but suspicion remains, testing for biotin interference may be useful.  For more information, see the ARUP Laboratories Biotin Interference page.

Macro Thyroid-Stimulating Hormone

Macro-TSH is a large form of TSH that results from the formation of complexes with serum proteins, including immunoglobulin. This form of TSH is not considered biologically active. Macro-TSH is detected by all current immunoassays, but because it cannot be routinely distinguished from active TSH, it can lead to falsely elevated TSH test results. Macro-TSH elevation should be suspected in asymptomatic individuals with markedly elevated TSH concentrations in isolation.  Communication with the testing laboratory may be useful to determine possible testing strategies for macro-TSH.

Antistreptavidin Antibodies

Antistreptavidin antibodies may interfere with immunoassays that make use of biotin-streptavidin technology, which may lead to falsely elevated T3 and T4 and falsely low TSH test results. Antistreptavidin antibodies may also interfere with anti-TSH receptor tests, which may lead to an erroneous diagnosis of Graves disease. 

Antiruthenium Antibodies

Ruthenium may be used as a label in some TSH, T3, and T4 immunoassays. Antibodies against ruthenium may lead to low or elevated concentrations in any of these tests.  The possibility of interference will depend on the testing method and assay design used. Consultation with the testing laboratory may be useful to determine if antiruthenium antibodies are a potential source of interference.

Thyroid Hormone Autoantibodies

Autoantibodies against T3 and T4 may interfere with thyroid hormone immunoassays and lead to falsely elevated test results. These autoantibodies are most prevalent in patients with autoimmune conditions. Screening for thyroid hormone autoantibodies in patients with autoimmune conditions is recommended if interference is suspected. 

Heterophilic Antibodies

Antibodies that bind to components used in thyroid function immunoassays (these are referred to as heterophilic antibodies and include human antianimal antibodies) may lead to incorrect test results. Falsely elevated results are particularly likely.    In addition to affecting tests in adult patients, heterophilic antibodies may cross the placenta and interfere with tests in newborns.  Repeat testing with the inclusion of heterophilic blocking reagent may be useful. The testing laboratory should be contacted if heterophilic antibody interference is suspected.

ARUP Laboratory Tests

Recommended Immunoassays
Other Assays (Not Recommended for Routine Screening or Evaluation)