A Dilution Method to Mitigate Biotin Interference in Cardiac Troponin T Testing

Abstract

Background: Oral biotin supplementation is known to interfere with biotin-streptavidin-based immunoassays, including Roche's fifth-generation cardiac troponin T (cTnT) assay, which plays a critical role in the diagnosis of myocardial infarction (MI). The utility of dilution, a quick and easy method to detect and remove interferences, has not been published for biotin interference. Methods: Concentrations of cTnT were measured in pooled serum from clinical samples. Serum samples were supplemented with biotin to known concentrations, then cTnT concentrations were remeasured to assess for biotin interference. Samples were then diluted to assess for effective removal of biotin interference. Results: At cTnT values near the critical reporting range for our institution (100 ng/L) we observed significant interference in measured values with added biotin concentrations above 50 ng/mL. In specimens without added biotin, autodilution at a 1:10 ratio yielded a mean 157% capture of measured cTnT, precluding the use of autodilution for detecting and mitigating biotin interference. A 1:10 dilution with serum containing 20-30 ng/L cTnT yielded a mean capture of 107%, which was suitable for detecting underlying biotin interference in supplemented samples. Conclusions: Biotin interference, at supraphysiologic concentrations, may create an artifactual reduction in measured cTnT to levels that could lead to delayed detection of an MI. Dilution with serum of known cTnT concentration of 20-30 ng/L is a fast and effective method to mitigate the analytical consequences of biotin interference. IMPACT STATEMENT Quantitative measurements of serum cardiac biomarkers are critical components in the timely diagnosis and management of suspected myocardial infarction (MI). The fifth-generation cTnT test provides an opportunity for earlier and more sensitive detection of MI events. Oral biotin supplementation can interfere with the test in a manner that may lead to missed or delayed MI diagnosis and subsequent patient morbidity. Here we demonstrate the efficacy of a simple method (dilution with serum with 20-30 ng/L cTnT) to detect and mitigate the effect of biotin interference.

False positive result of human chorionic gonadotropin caused by human anti-mouse antibodies

“…the interference of biotin (used as a drug or dietary supplement) in kits based on the fixation of the immune complex to microparticles through binding of streptavidin to biotin ( 11 - 13 );…”

“…• the presence of complexes of the determined antigens with immunoglobulins (i.e., with autoantibodies), such as in the case of macroenzymes, macrotroponin, macroprolactin, or macro-human chorionic gonadotropin (hCG) (1-7); • the presence of cross-reactive substances with an antibody against the antigen to be determined, which is a component of a reagent, such as digoxin-like immunoreactive substances (8-10); • the interference of biotin (used as a drug or dietary supplement) in kits based on the fixation of the immune complex to microparticles through binding of streptavidin to biotin (11)(12)(13);…”

Development and Evaluation of an Anti-Biotin Interference Method in Biotin-Streptavidin Immunoassays

“…There have been several studies that sought to detect and counter biotin interference using different approaches, such as the detection of biotin concentrations in serum samples [ 2 ]; inquisition of patients regarding consumption of biotin supplements [ 10 ]; manual incubation of the serum sample with streptomycin-coated magnetic particles outside the instrument, following which the incubated supernatant is separated and used as a sample for detection [ 11 , 12 ]; sample dilution [ 13 ]; and reagent improvement by adding biotin antibody to reagent [ 14 , 15 ]. Biotin concentration detection using these methods is limited because techniques such as enzyme-linked immunosorbent assays (ELISA) and high-performance liquid chromatography (HPLC), which require additional reagents or equipment, are not widely available; investigating patients’ biotin supplementation history is time-consuming and laborious, and the reliability is poor; and it takes a long time to adsorb and neutralize biotin through streptavidin magnetic particles outside the instrument before detection, and this process is complicated and liable to large human error.…”