AACC Guidance Document on Biotin Interference in Laboratory Tests AUTHORS Danni Li, PhD, DABCC, FAACC Kara L. Lynch, PhD, DABCC, FAACC Department of Lab Medicine and Pathology Department of Laboratory Medicine University of Minnesota Medical Center University of California San Francisco Minneapolis, MN San Francisco, CA Angela Ferguson, PhD, DABCC, FAACC Patrick B. Kyle, PhD, DABCC, FAACC Department of Pathology and Laboratory Medicine Department of Pathology Children’s Mercy Kansas City University of Mississippi Medical Center Kansas City, MO Jackson, MS Mark A. Cervinski, PhD, DABCC, FAACC Department of Pathology and Laboratory Medicine The Geisel School of Medicine at Dartmouth Hanover, NH TABLE OF CONTENTS Background . 2 Content . 2 Mechanisms of Biotin Interference . 2 Biotin Pharmacokinetics . 2 Summary of Case Reports . 3 Summary of In Vivo Studies . 3 Summary of In Vitro (Spiking) Studies . 3 Recommendations from the FDA and In Vitro Diagnostic Test Manufacturers . 4 Summary . 5 References . .. 5 CITATION Li D, Ferguson A, Cervinski MA, Lynch KL, Kyle PB . AACC guidance document on biotin interference in laboratory tests . [Epub] J Appl Lab Med January 13, 2020, as doi:10 .1373/10 .1093/jalm/jfz010 . BACKGROUND assay readout, which increases the ability of the assay to detect Laboratory tests that use streptavidin–biotin binding mecha- lower quantities or concentrations of the analyte, decreases the nisms have the potential to be affected by high circulating bio- number of steps required for analyte measurement, and allows tin concentrations, which would produce positive and negative for more rapid measurement of biomolecules of interest (14). interference in biotinylated competitive and noncompetitive Normal circulating concentrations of biotin derived from the (sandwich) immunoassays, respectively. Consumption of high- diet and normal metabolism are too low to interfere with bioti- dose biotin supplements for cosmetic or health-related reasons nylated immunoassays. Biotin in over-the-counter multivitamins has drawn attention to biotin interference in clinical laboratory (doses up to 1 mg) has not been reported to cause immunoas- tests. Case reports and in vivo studies show that ingestion of sup- say interference. However, ingestion of high-dose biotin supple- - biotinylated immunoassays. centrations that can interfere with commonly used biotinylated plemental biotin can cause clinically significant errors in select immunoassays.ments (e.g., ≥5 mg)Evidence results of ininterference significantly has elevated been described blood con in CONTENT case reports, in vivo studies (with study participants), and in vi- This AACC Academy document is intended to provide guidance to tro studies (biotin addition to mimic high blood concentrations), laboratorians and clinicians for preventing, identifying, and dealing as described herein. In November 2017, the FDA released a Safety with biotin interference. In vivo and in vitro spiking studies have Communication warning the public that biotin supplementation demonstrated that biotin concentrations required to cause inter- may interfere with laboratory tests (15). The potential for bio- ference vary by test and by manufacturer. This document includes tin interference in some assays may be substantial, as one study discussion of biotin’s mechanisms for interference in immunoas- determined that approximately 7% of emergency room patients says, pharmacokinetics, and results of in vitro and in vivo studies exhibited circulating biotin concentrations >10 ng/mL (>40.9 and cites examples of assays known to be affected by high biotin nmol/L) (16). concentrations. This document also provides guidance recommen- dations intended to assist laboratories and clinicians in identifying MECHANISMS OF BIOTIN INTERFERENCE and addressing biotin interference in laboratory testing. The mechanisms of biotin interference with biotinylated im- munoassays can be explained in the context of how biotin and Biotin (vitamin B7) is a coenzyme involved in multiple metabolic streptavidin binding is used in biotinylated assays. Immunoas- processes, including carbohydrate metabolism, fatty acid synthe- says are generally categorized as either competitive or noncom- sis, amino acid catabolism, and gluconeogenesis (1). The Insti- petitive (sandwich). In general, streptavidin–biotin binding is tute of Medicine recommends a daily biotin intake of 30 µg (2). used during assay incubation to couple biotinylated antibodies Although the inclusion of biotin in over-the-counter multivita- in sandwich immunoassays, or biotinylated antigens in compet- mins is common, biotin supplementation is not usually necessary itive immunoassays, to streptavidin-coated magnetic surfaces (e.g., beads). When a biological specimen contains excess biotin, grains, eggs, and dairy products). High doses 35 of biotin (up to the biotin competes with the biotinylated antibodies or antigens 3333because times biotin the isrecommended found in numerous daily dose foods [i.e., (e.g., 100 meat, 000 µgfish, or nuts, 100 for binding to the streptavidin-coated magnetic surfaces, result- mg]) are available over-the-counter and have been used for a va- ing in reduced capture of the biotinylated antibodies or antigens. riety of medical conditions, including diabetes, lipid disorders, Excess biotin produces falsely low results in sandwich immuno- 65 assays because the assay signal is directly proportional to the neuropathy (3–5), as well as for hair, nail, and skin health (6, 7). analyte concentration. In competitive immunoassays, excess bio- Althoughbiotinidase no deficiency, clinical evidence carboxylase supports deficiencies, the use of and high-dose peripheral bio- tin causes falsely elevated results because the assay signal is in- tin for beauty concerns, ongoing clinical trials have highlighted the potential therapeutic role of daily 300 mg biotin in slowing of biotin interference have been described extensively in other the morbidity associated with progressive and relapsing multiple publicationsversely proportional (17–27). to Of the note, analyte excessive concentration. biotin in aSpecific blood sample details sclerosis (8–12). is not likely to interfere with immunoassays that do not utilize bi- Biotin is also an essential reagent in biochemical applications. otin and streptavidin binding in the assay design or that utilize a Its binding with avidin (e.g., streptavidin), one of the strongest streptavidin–biotin complex bound in reagents before incubation noncovalent bonds, has been exploited in biochemical studies with blood samples. since the 1970s (13). Many laboratory tests that have been ap- proved or cleared by the US Food and Drug Administration (FDA) BIOTIN PHARMACOKINETICS are immunoassays that utilize biotin and streptavidin binding in Normal circulating concentrations of biotin typically range from the assay design (i.e., biotinylated immunoassays). Their strong 0.1 to 0.8 ng/mL in individuals consuming the recommended dai- binding is particularly useful for amplifying and isolating the ly dose of 30 µg (28). Biotin is rapidly absorbed after ingestion AACC GUIDANCE DOCUMENT on Biotin Interference in Laboratory Tests Published in The Journal of Applied Laboratory Medicine, January 13, 2020 ∙ academic.oup.com/jalm 2 and exhibits peak plasma concentrations within 1 to 2 hours (29, mone [FSH]). Li et al. (18) demonstrated that, among 6 healthy 30). Oral doses of 10 mg, equivalent to some of the highest doses volunteers who took 10 mg of biotin per day for 7 days, biotin-as- in over-the-counter products, resulted in peak plasma concen- sociated interference was found in 9 of 23 biotinylated immuno- trations ranging from 55 to 140 ng/mL (225–573 nmol/L) (29), assays by 3 manufacturers (Roche, Ortho Clinical Diagnostics, whereas oral doses of 100 mg resulted in peak plasma concen- Siemens), including TSH, total T3, free T4, free T3, PTH, NT-proB- trations ranging from 375 to 450 ng/mL (1535–1842 nmol/L) NP, and 25-hydroxyvitamin D. The assays exhibited varying de- (11). The accumulation of biotin does occur, and sequential daily grees of biotin interference among manufacturers. dosing revealed circulating concentrations twice as high on Day Biotin is predominately eliminated in the urine, so the poten- 7 compared with the same time point on Day 1 (29). Data anal- tial for interference in urine immunoassay tests is also of concern. ysis revealed that steady-state concentrations are reached after To this end, Williams et al. (44) examined biotin interference in 3 days of constant biotin intake (29). Low concentrations of bi- 7 different urine human chorionic gonadotropin pregnancy tests otin are cleared quickly from the circulation of healthy individ- and demonstrated that one test failed to develop a control line uals, with an elimination half-life of approximately 2 hours (30), when tested with nonpregnant volunteers’ urine collected 2 to 4 whereas experiments with high doses indicate a half-life up to hours after 10 mg biotin ingestion. This result was likely caused 18.8 hours (11). A pharmacokinetics study of apparently healthy by interference with the process in which immobilized streptavi- individuals determined that blood concentrations can be expect- din binds to a biotinylated dye to display the control line. ed to drop below 20 ng/mL (81.9 nmol/L) in 1, 5.5, 20, 108, and 146 hours after oral biotin doses of 1, 5, 10, 100, and 300 mg, SUMMARY OF IN VITRO (SPIKING) STUDIES respectively (29). However, delayed clearance and/or higher cir-