Susceptibility of Cardiac Troponin Assays to Biotin Interference

Ithiel J. Frame, MD, PhD; Parag H. Joshi, MD, MHS; Caroline Mwangi, MT; Ian Gunsolus, PhD; James A. de Lemos, MD; Sandeep R. Das, MD, MPH; Ravi Sarode, MD; Jyoti Balani, MD; Fred S. Apple, PhD; Alagarraju Muthukumar, PhD

Disclosures

Am J Clin Pathol. 2019;151(5):486-493. 

In This Article

Abstract and Introduction

Abstract

Objectives: To investigate biotin interference on three cardiac troponin (cTn) assays and demonstrate a method to overcome biotin interference.

Methods: cTn levels were measured in (1) plasma from healthy volunteers on 10-mg daily biotin supplementation mixed with a plasma with known elevated troponin, (2) plasmas with known elevated cTn after mixing in reagent biotin to simulate supplementation, and (3) biotin-spiked plasma specimens pretreated with streptavidin-agarose beads.

Results: Daily biotin ingestion (10 mg) and studies simulating daily biotin use resulted in significant interference in the Gen5 cardiac troponin T (cTnT) assay; the contemporary Gen 4 cTnT and high-sensitivity cardiac troponin I (hs-cTnI) assays were unaffected. The biotin interference threshold was 31, 315, and more than 2,000 ng/mL for Gen5 cTnT, cTnT, and hs-cTnI assays, respectively. Streptavidin pretreatment blocked biotin interference in cTn assays.

Conclusions: Biotin interference is possible at plasma concentrations achievable by ingestion of over-the-counter supplements that may lead to delayed or missed diagnosis of myocardial injury with the Gen5 cTnT assay.

Introduction

Over-the-counter (OTC) biotin (vitamin B7) sales have increased due to perceived health and cosmetic benefits at doses of 5 to 10 mg/d, which are more than 150 times the Adequate Intake recommendation.[1,2] Individuals may be unaware they are taking biotin as it is sold under various commercial names.[3] Very high doses of biotin up to 300 mg/d are currently under investigation as a therapy for multiple sclerosis.[4,5]Plasma biotin can interfere in immunoassays that have a streptavidin-biotin interaction step.[1,6,7] As a result, several routine assays are susceptible to biotin interference, including cardiac troponin (cTn) assays.[1] Biotin interference can lead to either overestimation or underestimation in test results depending on the assay design.

Sandwich immunoassays are a common format for detecting proteins, such as cTn, relying on two antibodies of different epitopes: one capture antibody that has been biotinylated and one antibody coupled to a reporter molecule. The cTn-antibody complexes can then be bound to immobilized streptavidin, washed, and subjected to a detection reaction. For cTn assays relying on this format, excess biotin could bind the streptavidin, thereby blocking binding of the cTn-antibody complexes. Thus, biotin interference can potentially result in missed diagnosis of myocardial injury, including myocardial infraction (MI), due to underestimation.

Consequently, the US Food and Drug Administration (FDA) issued a safety warning on biotin interference in diagnostic tests.[3] A number of recent studies have demonstrated how biotin can affect laboratory testing, particularly thyroid function tests.[1,6,8–12] Accordingly, manufacturers advise a washout period of several hours to weeks prior to sample collection. For critical and time-sensitive tests such as cTn, the biomarkers of myocardial injury, a washout period is not feasible.[1,8,13]

The fifth-generation cardiac troponin T (cTnT) assay (Gen 5 Stat cTnT; Roche Diagnostics), marketed by Roche as a high-sensitivity (hs)–cTnT assay, is clinically available in the United States. The Gen 5 cTnT assay offers the advantage over many contemporary cTnT and cardiac troponin I (cTnI) assays for safely and rapidly ruling out acute MI within 3 hours of presentation.[14–17] This potential for faster triage is an attractive option for emergency departments (EDs). We note the distinction between the FDA-cleared Gen 5 cTnT assay vs the hs-cTnT assay Roche markets worldwide as high sensitivity for two reasons. First, the Gen 5 assay and the high-sensitivity assay have different assay characteristics as published in their package inserts to customers. Second, by International Federation of Clinical Chemistry (IFCC) definition, while both the Gen 5 and high-sensitivity assays are excellent assays for clinical use, neither meet the IFCC guideline criteria as a high-sensitivity assay, as neither assay is able to provide measurable concentrations above the limit of detection in 50% or more of women.[18]

The Gen 5 cTnT and contemporary Gen 4 cTnT assays are biotinylated assays, rendering them susceptible to biotin interference.[19] Given the critical importance of the Gen5 cTnT assay in rapidly assessing acute MI, we evaluated the biotin tolerance threshold of this assay and the potential impact of biotin interference in patients seeking treatment for chest pain at the ED.[20–23] Methods to overcome biotin interference using magnetic streptavidin microparticles have been described.[24,25] We also demonstrate a possible method to overcome biotin interference for cTn using streptavidin-agarose beads.

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