Biotin
Biotin based Immunoassays: Biotin is a water-soluble vitamin belonging to vitamin B family. It is also an essential component of streptavidin-biotin immobilising systems (SBIS) used for most immunoassays. This system exploits the interaction between the fungal protein streptavidin and biotin which is one of the strongest non-covalent interactions in nature. It remains undisturbed by multiple washing steps in assays.1 Furthermore, biotinylation does not alter the biological activity or immunologic specificity when bound to any test molecule. SBIS is widely used in many Food and Drug Administration approved immunoassay systems using fully automated platforms, including Access, DxI, and DxC (Beckman Coulter, California, US); the Elecsys, Cobas, and Modular platforms (Roche Diagnostics, Basel, Switzerland); the Isys platform (Immuno Diagnostic System, East Boldon, United Kingdom); the Ortho Vitros platform (Ortho Clinical Diagnostics, New Jersey, US); the Dimension Vista, Exl, Immulite platforms (Siemens Healthineers, Erlangen, Germany), Abbott Architect i2000® (Abbott Diagnostics, Illinois, United States), and Diasorin Liaison XL® (DiaSorin, Saluggia, Italy).2
Type of abnormality: The presence of excess biotin in test samples causes abnormalities in accordance with the type of immunoassay. Falsely low readings occur with sandwich immunoassays (glycoprotein hormones like TSH) and falsely high values in competitive immunoassays (e.g. triiodothyronine (T3), thyroxine (T4), steroid hormones and 25-hydroxy-vitamin D).2
  1. Mechanism of interference with sandwich immunoassays: When the assay has a “sandwich” design as employed for estimation of TSH, the test serum is incubated with biotinylated monoclonal TSH antibodies and radiolabelled monoclonal antibodies. Immune complex ”sandwiches” thus formed are captured by streptavidin-coated magnetic microparticles. Chemiluminescence produced by application of a voltage to these magnetic microparticles is directly proportional to the TSH levels in the test serum. The presence of excess biotin in the serum will saturate the streptavidin binding sites and reduce binding of these immune sandwiches to the solid phase (magnetic microparticles) resulting in low chemiluminescence and falsely low readings as illustrated in Figure 1.
  2. Mechanism of interference with competitive immunoassays: When a competitive immunoassay is utilised as in the FT4assay, the test serum is incubated with biotinylated T4 molecules and radiolabelled anti-T4 monoclonal antibodies. Biotinylated T4 molecules compete with the analyte (or T4) in the test serum for binding with the radiolabelled anti-T4 antibodies. The biotinylated T4 molecules are then immobilised in the streptavidin-coated microparticles. The chemiluminescence signal generated from the application of voltage to these magnetic microparticles is inversely proportional to the levels of FT4. The presence of excess biotin in the test serum will prevent the complexes of biotinylated T4-radiolabelled antibodies from binding to the magnetic microparticles. This would result in lower chemiluminescence, translating to falsely higher levels of T4 in the serum as depicted in Figure 2.
Biotin dose and assay interference: The recommended daily intake of biotin ranges from 30-70 mcg daily. While dietary biotin intake does not generate significant enough blood levels to interfere with in-vitro diagnostic tests, supraphysiologic levels achieved through the therapeutic use of biotin as supplements for skin and hair growth, multiple sclerosis and rare inherited metabolic disorders like biotinidase deficiency, propionic academia, thiamine responsive basal ganglia disease, holocarboxylase synthase deficiency and mitochondrial disorders, can cause problems with these tests.3 The minimal dose required and the degree, duration, and magnitude of this interference is variable and might be specific to the analyte being tested and the assay characteristics.4,5 The level of interference depends on the serum concentration attained rather than the dose of biotin consumed, which in turn is determined by the length of the washout period before testing.6,7 Grimsey et al. evaluated washout periods, required for assays with interference thresholds ranging from 10 to 100 ng/ml, at biotin dose regimens ranging from 1 mg once daily to 300 mg four times daily.7 For assays with an in-vitro interference threshold of more than 30 ng/ml, biotin doses of up to 5 mg twice daily or 10 mg once daily, an 8-hr washout period is sufficient to mitigate the risk. If assays have an in-vitro interference threshold of < 30 ng/ml, or in rare cases of biotin intake of more than 10 mg per day, sampling should be delayed for a more extended period (up to 73 h) after the last dose of biotin. Though the effect of biotin on FT3, FT4 and TSH estimation wanes in hours, anti-TSH receptor antibodies (TRAbs), which can also be falsely elevated due to this assay interference, may take up to 7 days to normalise.2,8–10
Clinical correlate: The TFT could falsely suggest a diagnosis of overt or subclinical thyrotoxicosis or thyroid hormone resistance, and falsely elevated TRAbs can further mislead the diagnosis. History of intake of OTC vitamin supplements containing biotin should be elicited. If an erroneous report is suspected, retesting should be done in serial dilutions (if using the same platform) or on another platform which does not utilise SBIS (Centaur FT4, Diasorin, Abbott).11,12Another option would be to repeat the test, after stopping biotin supplements for a duration which is determined by the dosage and analyte being assessed. Depletion protocols which involve pre-treatment of the test sample with substances (e.g. streptavidin-coated particles) that bind biotin can also be used.13