How do you check for assay interference in thyroid hormone measurements in a patient with a suspected thyroid disorder and potential interactions with medications such as biotin, heparin, or immunoglobulins?

How to Check for Assay Interference in Thyroid Hormone Measurements

Recognize When to Suspect Interference

Suspect assay interference whenever thyroid function test results are discordant with clinical presentation or when results are internally inconsistent (e.g., suppressed TSH with low free T4, or elevated TSH with elevated free T4). 1

Key clinical scenarios requiring high suspicion:

• Biochemical discrepancies: TSH and free T4 moving in the same direction rather than opposite directions 1
• Clinical-laboratory mismatch: Patient appears clinically euthyroid but labs suggest severe thyroid dysfunction 1
• Unexpected changes: Dramatic shifts in thyroid function tests without medication changes or clinical explanation 2
• Treatment resistance: Thyroid function tests fail to respond appropriately to adequate levothyroxine therapy 3

Identify the Most Common Interfering Substances

Biotin (Vitamin B7)

Biotin is the most common and clinically significant interference in modern thyroid assays, particularly affecting streptavidin-biotin immunoassays used by Roche and Siemens platforms. 4, 3

• Biotin causes falsely low TSH and falsely elevated free T4/T3 on Roche Cobas platforms, mimicking hyperthyroidism 3
• Biotin causes falsely low thyroglobulin on Siemens IMMULITE platforms, potentially masking thyroid cancer recurrence 3
• Interference occurs at doses as low as 10 mg/day (well above the 30 mcg RDA but commonly found in supplements) 3
• Maximum interference occurs 2-5 hours after biotin ingestion 3
• The FDA warns that biotin levels higher than recommended daily allowance may cause interference with cardiovascular and hormone tests 4

Heterophile Antibodies

These antibodies cause unpredictable interference patterns by cross-linking capture and signal antibodies in two-site immunoassays. 5

• Most commonly affect TSH measurements, causing falsely elevated or falsely low values 5
• Human anti-mouse antibodies (HAMA) are a specific subtype that interfere with assays using mouse monoclonal antibodies 5, 6
• Can occur in patients with no apparent exposure to mouse proteins 5

Thyroid Hormone Autoantibodies

• Cause falsely elevated total T4 and T3 measurements 5
• Can cause discordant free and total hormone measurements 1
• Present in both thyroid and non-thyroid autoimmune disorders 5

Macro-TSH

• TSH complexed with immunoglobulins, causing falsely elevated TSH with normal free T4 1
• Mimics subclinical hypothyroidism biochemically but patient is clinically euthyroid 1

Anti-Streptavidin and Anti-Ruthenium Antibodies

• Interfere with specific assay platforms using these labels 1
• Cause variable patterns of thyroid function test abnormalities 2

Systematic Detection Algorithm

When interference is suspected, follow this stepwise approach:

Step 1: Obtain Detailed Medication and Supplement History

• Ask specifically about biotin supplementation, including multivitamins, hair/nail supplements, and high-dose B-complex vitamins 2, 3
• Document timing of last biotin dose relative to blood draw 3
• Inquire about iodine-containing medications (amiodarone, contrast agents) 2
• Review all medications that affect thyroid function or hormone binding 2

Step 2: Repeat Testing After Biotin Washout

If biotin use is confirmed or suspected, discontinue biotin for 48-72 hours and repeat thyroid function tests. 3

• This is the simplest and most cost-effective first step 3
• If results normalize after biotin discontinuation, interference is confirmed 3
• For urgent clinical decisions, cannot wait for washout period 3

Step 3: Perform Assay Method Comparison

Measure thyroid hormones using a different analytical platform or methodology. 1

• Compare results from streptavidin-biotin platforms (Roche, Siemens) with non-biotin platforms (Abbott Architect) 3
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the gold standard as it is not subject to immunoassay interferences 3
• If results differ significantly between platforms, interference is likely 1
• Abbott Architect platform showed no biotin interference in comparative studies 3

Step 4: Serial Dilution Studies

Dilute the patient's serum sample and remeasure thyroid hormones. 1

• True hormone concentrations should decrease proportionally with dilution 1
• Non-linear dilution curves suggest interference 1
• Particularly useful for detecting heterophile antibodies and macro-TSH 1

Step 5: Polyethylene Glycol (PEG) Precipitation

Add PEG to precipitate immunoglobulins and immunoglobulin-bound hormones. 1

• Useful for detecting macro-TSH and thyroid hormone autoantibodies 1
• If TSH or hormone levels decrease significantly after PEG treatment, macro-complexes are present 1
• Requires specialized laboratory capability 1

Step 6: Blocking Reagent Studies

Use heterophile blocking reagents or animal serum to neutralize interfering antibodies. 1

• If results normalize after adding blocking reagents, heterophile antibodies are confirmed 1
• Most commercial assays now include blocking reagents, but breakthrough interference still occurs 5

Platform-Specific Interference Patterns

Roche Cobas Platform (Streptavidin-Biotin Based)

• Biotin causes falsely low TSH and falsely elevated free T4/T3 3
• Creates biochemical picture of hyperthyroidism 3
• Most vulnerable to biotin interference 3

Siemens IMMULITE Platform

• Biotin causes falsely low thyroglobulin 3
• Particularly problematic in thyroid cancer surveillance 3
• Also uses biotin-streptavidin technology 3

Abbott Architect Platform

• Not affected by biotin interference 3
• Preferred alternative when biotin interference suspected 3
• Uses different immunoassay technology 3

Clinical Impact and Consequences

More than 50% of documented thyroid interferences lead to misdiagnosis and inappropriate management. 1

Common clinical errors resulting from unrecognized interference:

• Unnecessary levothyroxine therapy initiated for falsely elevated TSH 1
• Inappropriate discontinuation of levothyroxine for falsely suppressed TSH 1
• Unnecessary thyroid scans and imaging 1
• Missed thyroid cancer recurrence due to falsely low thyroglobulin from biotin 3
• Inappropriate antithyroid drug therapy for falsely elevated thyroid hormones 1

Critical Pitfalls to Avoid

• Never rely on a single abnormal thyroid function test without clinical correlation 7
• Always ask about biotin supplementation before interpreting thyroid tests, as it is increasingly common in over-the-counter supplements 4, 3
• Do not assume all assay platforms are equally affected by the same interference 3
• Recognize that interference can occur even with "normal" supplement doses of biotin (10 mg/day) 3
• Do not dismiss discordant results as laboratory error without systematic investigation 1
• Remember that TSH levels can vary by 40-50% day-to-day due to physiological factors, which is different from assay interference 7, 8

When to Involve the Laboratory

Contact the laboratory immediately when:

• Results are internally inconsistent (TSH and free T4 moving in same direction) 1
• Clinical presentation contradicts laboratory findings 1
• Patient is taking biotin and urgent clinical decision needed 3
• Serial dilution or alternative platform testing is required 1

Strong interaction between clinician and laboratory is necessary to identify and resolve thyroid assay interferences. 1