Biotin Interference on Diagnostic Testing

biotin-b7

Biotin, also known as vitamin B7 is a coenzyme that is involved in carbon dioxide transfer in carboxylase reactions. The USDA recommended dietary reference intake for biotin is 30 ug per day which should mostly come from food. The last few years biotin has been marketed heavily as a beauty supplement. It is used in hair, skin, and nail supplements, and is not FDA regulated and is sold as over-the-counter. Biotin can be found in B-complex vitamins, multivitamins, prenatal vitamins, vitamin H, and vitamin B7 supplements. The only FDA recommended use for biotin is in patients with secondary progressive multiple sclerosis who receive mega-doses of up to 300 mg per day. Even in such large doses biotin is considered nontoxic and has very little adverse effects.

The issue is that serum or plasma biotin may potentially interfere with any assay that uses biotin-streptavidin binding. Biotin is a small molecule that attaches covalently to a variety of targets with minimal effect on their biological activity. The biotin binding makes the target an easy capture because it forms a strong bond with avidin, streptavidin, and NeutrAvidin proteins who have an exceptionally high affinity for biotin. Biotin-streptavidin detection is a favorite among many immunoassays across many manufacturers including Roche, Ortho, Beckman, Siemens, and Dimension.

The direction of interference depends on the design of the assay. Some results may be falsely elevated, and some may be falsely decreased. The sandwich and competitive assays are among the most commonly impacted. Interference can occur with hormone tests such as parathyroid hormone (PTH), thyroid stimulating hormone (TSH), T4, T3, and even troponin tests.

Sandwich assays involve two antibodies that form a sandwich with the analyte being tested to be measured. The first antibody is labeled with a signal that can be quantified and the other antibody to the target is labeled with biotin. When the biotin:antibody complex binds to streptavidin-coated beads, the labeled antibody then binds creating a sandwich. The resulting complex is then measured. The more complexes that are created, the stronger the signal, i.e the more target analyte there is. Excess free biotin interferes by binding to the streptavidin-coated beads, leaving fewer binding opportunities for the antibodies. Antibody complexes that have successfully bound the analyze get washed away and are then undetected, resulting in falsely low results.

Competitive assays consist of an antibody to the analyte that is labeled with biotin. The analyte must compete for antibody binding sites with a reagent that is a supplied version of itself with a label for detection. If no analyte is present, the reagent occupies all the antibody binding sites and the complex is captured by streptavidin, and a strong signal is emitted. If analyte is present, that occupies antibody binding sites that outcompete the labeled reagent. When analyte is present, there is less detection and less signal measured. It is an inverse relationship. When analyte is not present, there is a strong signal detected, when analyte is present, there is a weak signal detected. Free biotin sticks to the streptavidin, leaving fewer antibody binding sites for the analyte:antibody or reagent:antibody complex. The complexes get washed away and causes weakening of the signal. This may give the impression that analyte is present, even in its absence.

This is an ongoing issue and the FDA advises the healthcare community; patients and physicians both to disclose any supplements that are being taken that contain biotin. Physicians should advise laboratory if interference from biotin is a possibility. Practice should be implemented to counsel patients to abstain from oral biotin 2-3 days before blood tests. Biotin has a rapid half-life of 2 hours, but patients taking mega-doses (>30 mg) have demonstrated interference on laboratory tests for up to 24 hours.

Physicians should educate patients to increase awareness of biotin interference. Adverse health effects can occur if test results are falsely skewed in any direction.

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