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A simple method to detect biotin interference on immunoassays

We read with interest the letter to the editor by Batista et al. [1] on the interference of biotin in immunoassays. Biotin is available over the counter usually at strengths of 5 mg or less per tablet taken as a health supplement for skin problems and hair loss. However, biotin in much higher doses is an established lifelong treatment for patients with inherited defects of biotin metabolism (e.g. biotinidase, holocarboxylase synthase deficiency). Very large doses of biotin are also used to treat certain neurological disorders (e.g. biotin responsive basal ganglia disease) and have recently been reported to improve disability in some patients with progressive multiple sclerosis [2]. To confirm interference caused by biotin, Batista et al. [1] collected blood specimens before and after the termination of biotin ingestion. While this approach is well suited for patients taking unusually large doses of biotin as a health supplement, it requires a second sample and may not be medically justified for those with underlying biotin-treatable illnesses. We report here a simple method for clinical laboratories to identify assay interference caused by biotin consumption using streptavidin-coated microparticles, a readily available reagent in many immunoassays. An endocrinologist prescribed and ingested 100 mg (1.3 mg/kg) of biotin compounded by a local pharmacy [Optimus Health, Auckland; a good manufacturing practice (GMP) registered company] at 8 A.M. in the morning. Thyroid and reproductive function tests requested on heparin plasma samples were taken prior to ingestion (0 h) and serially at 1, 2, 3, 4, 6, 8 and 24 h after ingestion. Written consent was obtained from the individual whose laboratory results were published. Residual streptavidin-coated microparticles (0.72 mg/ mL) used in Roche immunoassays were pooled from approximately 10 used reagent cartridges destined for disposal. Three milliliters of a suspension of streptavidincoated microparticles were centrifuged at 1000 rpm (relative centrifugal force; 180 g) for 5 min. The supernatant was removed, 500 μL of plasma was added, vortexed and the mixture left to stand at room temperature for 1 h. The mixture was centrifuged at 3000 rpm (1940 g) for 5 min and 450 μL of the supernatant placed into a new tube, taking care not to disturb the microparticle pellet. Immunoassays (Roche Diagnostics) of plasma with and without streptavidin-coated microparticle incubation were performed on a Roche Cobas e602 module. Baseline thyroid function tests for free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) were 5.1 pmol/L (reference interval 3.9–6.8), 16 pmol/L (12–22) and 2.0 mIU/L (0.27–4.2; Figure 1). For all assays, maximal interference was observed 1 h following the ingestion of biotin with FT4 falsely reported as greater than the reporting limit of the assay >100 pmol/L, FT3 increased to 13 pmol/L and TSH reduced to 0.02 mU/L. Although TSH was not undetectably low, this pattern of interference could not be reliably distinguished from clinically significant hyperthyroidism, especially of recent onset. Baseline reproductive function tests prior to the consumption of biotin for total testosterone, estradiol, progesterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were: 17 nmol/L (male reference interval 6.7–26), 64 pmol/L (0–200 pmol/L), 1.2 nmol/L ( < 1 nmol/L), 1.9 IU/L (2–9), 3.1 IU/L (2–12; Figure 2). After biotin ingestion testosterone, estradiol and progesterone were markedly increased to 47 nmol/L, 550 pmol/mL, 47 nmol/L, respectively; while LH and FSH decreased to 0.3 IU/L and 0.2 IU/L. The pattern of interference produced *Corresponding author: Campbell V. Kyle, Department of Chemical Pathology, Labplus, Auckland City Hospital, Auckland, New Zealand, Phone: + 649 367 4949, ext 22045, Fax: + 649 375 4301, E-mail: Campbellk@adhb.govt.nz; and Department of Biochemistry, Labtests, Auckland, New Zealand Leo Lam: Department of Chemical Pathology, Labplus, Auckland City Hospital, Auckland, New Zealand