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
- 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.
- 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