Technical Note: Bimodal Negative Interference of Biotin in Roche IL-6 Immunoassay.

OBJECTIVE: Interleukin -6 (IL-6) is an important diagnostic test in COVID-19 patients to determine whether to initiate tocilizumab therapy or mechanical ventilation. We investigated potential interference of biotin in Roche IL-6 assay which utilizes biotinylated antibody. METHODS: We prepared three serum pools from left-over specimens which showed IL-6 values over 40 pg/mL. Then aliquots of each serum pool were further supplemented with various amounts of biotin expected in patients taking biotin supplement and then IL-6 values were measured again using Roche IL-6 assay on the Cobas e411 analyzer. RESULTS: We observed negative interference of biotin in IL-6 assay but interference was bimodal as maximum negative interference was observed with 100 ng/mL biotin but not with 1000 ng/mL. However, no interference was observed in the presence of 25 ng/mL biotin. CONCLUSIONS: Biotin showed negative interference with IL-6 assay.

The prevalence of elevated biotin in patient cohorts presenting for routine endocrinology, sepsis, and infectious disease testing.

Elevated blood biotin levels may interfere with some biotin-streptavidin immunoassays, used in clinical laboratories to aid diagnosis. The objective of this study was to determine the prevalence of elevated blood biotin levels in three at risk patient cohorts, where misclassification of disease status would have a high clinical impact. This retrospective, single-center study screened residual, de-identified plasma samples (N = 700) from adult patients undergoing routine thyroid stimulating hormone (TSH) (n = 500), procalcitonin (PCT) (n = 100), or human immunodeficiency virus (HIV) (n = 100) testing using the Elecsys® BRAHMS PCT (Roche Diagnostics), Access TSH (3rd IS) (Beckman Coulter Inc), and ARCHITECT HIV Ag/Ab Combo (Abbott Laboratories) immunoassays, respectively, for elevated levels of biotin (quantified by gas chromatography-time of flight mass spectrometry). Patients taking biotin supplements were included and dosages recorded from medical records. In the overall study cohort, blood biotin levels ranged 0.1-21.3 ng/mL; 44.3% (310/700) of samples were < 1 ng/mL, 54.7% (383/700) were 1-<10 ng/mL, and 1% (7/700) were ≥ 10 ng/mL. The sub-cohorts had similar ranges of biotin levels: 0.5-21.3 ng/mL (TSH), 0.1-12.1 ng/mL (PCT), and 0.3-7.3 ng/mL (HIV). In the 44 patients (6.3% of overall study cohort) who were documented as taking biotin supplements (range of doses, 2.5-10 mg/day), blood biotin levels ranged 0.9-21.3 ng/mL; 2.3% (1/44) of samples were < 1 ng/mL, 86.4% (38/44) were 1-<10 ng/mL, and 11.4% (5/44) were ≥ 10 ng/mL. Most patients who reported taking biotin supplements had blood biotin levels ≥ 1 ng/mL and the highest blood biotin level detected was 21.3 ng/mL.

Serum biotin interference: A troublemaker in hormone immunoassays.

OBJECTIVES: Biotin therapy can affect the results of many immunoassay procedures. The present study investigates biotin's interference on 25-hydroxy vitamin D (25-OHD), parathyroidhormone (PTH) and thyroid-stimulating hormone (TSH) tests using four different assay systems and biotin neutralization. DESIGN AND METHODS: Enrolled in the study were 50 children diagnosed with biotinidase deficiency (BTD) undergoing treatment with biotin (5-20 mg/day) who were subjected to a series of analyses involving 25-OHD (Roche Diagnostics assays, Beckman Coulter assays, HPLC, LC/MS-MS), TSH, PTH (Roche Diagnostics assays, Beckman Coulter assays) and biotin (LC/MS-MS), before and after biotin neutralization with Streptavidin-coated magnetic particles (SMP). RESULTS: The median biotin concentration was found to be 175.2 [94.0-307.1] µg/L. There was no significant difference in the 25-OHD results before and after neutralization with the Beckman Coulter, HPLC and LC-MS/MS assays. In contrast, the median 25-OHD level was seen to decrease from 90.2 [35.9-105.3] ng/mL to 29.1 [22.6-37.6] ng/mL after neutralization with the Roche assay (p < 0.0001). While there was no statistically significant difference in the values recorded before and after neutralization in PTH analysis using Beckman assay, the median PTH levels increased from 7.8 [1.6-21.6] pg/mL to 28.2 [22.5-41.9] pg/mL after neutralization with the Roche assay (p < 0.0001). The cut-off values at which serum biotin interfered in the Roche assay PTH test, with 25-OHD levels determined as 51.4 µg/L and 62.9 µg/L, respectively. A significant increase was detected in the TSH levels analyzed with a Roche assay after neutralization (from 2.36 [1.85-3.00] mIU/L to 2.74 [1.93-3.70] mIU/L, p < 0.0001). CONCLUSIONS: The PTH, 25-OHD and TSH results were found to be affected by high biotin concentrations in Roche assays, leading to a risk of misdiagnosis, although SMP neutralization can suppress any such interference efficiently.

The biotin interference within interference suppressed immunoassays.

BACKGROUND: Reports of false laboratory findings due to a biotin supplementation have raised concerns about the safety of immunoassays. According to current research, biotin is known to cause interference in immunoassays. Since up to 70% of medical decisions are based on laboratory results and the significantly increased intake of biotin supplements in the recent years, the reliability of immunoassays is essential. METHODS: To evaluate this reliability two experiments were conducted. In the first experiment 59 interference suppressed immunoassays of the manufacturer Roche Diagnostics were examined regarding their sensitivity to a biotin interference. In the second experiment the pharmacokinetic of biotin was examined by supplementing volunteers with biotin. RESULTS: A combination of the results of both experiments suggests that a biotin interference in laboratory findings is probable. Contrary to the current state of research on sandwich immunoassays, falsely elevated test results occur more frequently than falsely low results. CONCLUSION: The interference suppressed immunoassays have shown in the experiment that they are susceptible to a biotin interference. Therefore, laboratory institutions, medical staff and patients must be aware of the possibility of a biotin interference. As a result, Roche Diagnostics may consider reviewing the interference suppression and their indications of the tests.

Effects of magnesium biotinate supplementation on serum insulin, glucose and lipid parameters along with liver protein levels of lipid metabolism in rats.

The objective of this study was to investigate the effects of a novel form of biotin (magnesium biotinate) on serum glucose, lipid profile, and hepatic lipid metabolism-related protein levels in rats. Forty-two rats were divided into six groups and fed a standard diet-based egg white powdered diet supplemented with either d-biotin at 0.01, 1, or 100 mg/kg BW or magnesium biotinate at 0.01, 1, or 100 mg/kg BW for 35 days. Neither form of biotin influenced (p > 0.05) serum glucose or insulin concentrations. Serum total cholesterol and triglyceride decreased with biotin from both sources (p < 0.05). Concentrations were lower with magnesium biotinate when comparing the 1 mg/kg dose (p < 0.05). Serum, liver, and brain biotin and liver cyclic guanosine monophosphate (cGMP) concentrations were greater when rats were treated with magnesium biotinate versus d-biotin, particularly when comparing the 1 and 100 mg/kg dose groups (p < 0.05). Both biotin forms decreased the liver SREBP-1c and FAS and increased AMPK-α1, ACC-1, ACC-2, PCC, and MCC levels (p < 0.05). The magnitudes of responses were more emphasized with magnesium biotinate. Magnesium biotinate, compared with a commercial d-biotin, is more effective in reducing serum lipid concentrations and regulating protein levels of lipid metabolism-related biomarkers.

Effect of Biotin on Cardiac Troponin I and High Sensitivity Cardiac Troponin I Assays on Vista 1500 and ADVIA Centaur Analyzer.

OBJECTIVE: Biotin interferes with biotinylated antibody based immunoassays. We investigated effect of biotin on conventional troponin I and two high sensitivity troponin I assays, all manufactured by Siemens. MATERIALS AND METHODS: One high sensitivity troponin I assay (TNIH-1) was run using ADVIA Centaur analyzer. The second high sensitivity troponin I assay (TNIH-2) as well as conventional troponin I assay (CTNI) were run using Dimension Vista 1500 analyzer. We analyzed 25 specimens using CTNI, TNIH-1 and TNIH-2 assays for comparison of these assays. Moreover, serum pools prepared from additional specimens containing various amounts of troponin I were further supplemented with biotin to achieve biotin concentrations between 50 and 1000 ng/mL followed by reanalysis using CTNI, TNIH-1 and TNIH-2 assays. RESULTS: Although both high sensitivity troponin I assays correlated well, there was a significant positive bias with TNIH-2. We observed no significant negative biotin interference at a level up to 250 ng/mL. Highest observed negative bias was 29.7%. CONCLUSIONS: All three troponin I assays were free from biotin interferences up to a biotin concentration of 250 ng/mL.

Prevalence of Detectable Biotin in Five US Emergency Department Patient Cohorts.

BACKGROUND: The objective of this study was to estimate the prevalence of biotin supplementation in United States emergency department patients using a multi-site, geographically distributed sampling model. METHODS: Biotin was measured using an Abbott ARCHITECT Biotin research use only assay in 7118 emergency department patient serum or plasma samples from five US medical centers. Samples with biotin ≥10 ng/mL underwent additional LC-MS/MS confirmatory testing for biotin and its primary metabolites. The overall and site-specific prevalence of detectable biotin was determined using the screening assay while biotin speciation (i.e., prevalence of detectable metabolites) was determined using LC-MS/MS. RESULTS: Of 7118 samples screened, 291 (4.1%) had biotin ≥10 ng/mL and were considered positive. Across five medical centers, the fraction of positive samples ranged from 2.0% to 5.4%. The maximum biotin concentration observed was 355 ng/mL. Of the 285 positive screens that underwent additional LC-MS/MS testing, 89 (31%) showed detectable biotin, bisnorbiotin, and/or biotin sulfoxide. Biotin, bisnorbiotin, and biotinsulfoxide were detected in 82/89 (92.1%), 61/89 (68.5%), and 18/89 (20.2%) samples, respectively; biotin was detected in the absence of either metabolite in 18/89 (20.2%) samples. CONCLUSIONS: Using a screening assay, 4.1% of emergency department patient samples were found to be potentially susceptible to interference from biotin. Confirmatory testing showed detectable biotin and/or biotin metabolites in 31% of positive screens (1.3% overall). The prevalence of biotin ≥10 ng/mL varied 2-3-fold across US emergency department patient cohorts. Biotin metabolites were observed in 80% of samples confirmed to have detectable biotin species by LC-MS/MS, suggesting that rigorous assessments of assay susceptibility to biotin interference, often performed using in vitro studies, should consider the potential role of biotin metabolites present in vivo.

Quantification of biotin in plasma samples by column switching liquid chromatography - tandem mass spectrometry.

Biotin (or Vitamin B7) is a vitamin where deficiency can be caused by inadequate intake. Biotin deficiency is rare, as most people get enough biotin from diet, since many foods contain biotin. In addition to biotin from food, intestinal bacteria can synthesize biotin, which can then be absorbed by the body. Supplementation with biotin has been advocated, mainly due to proposed beneficial effects on skin, nail and hair growth. There is no evidence that high biotin intakes are toxic, but a high intake may interfere with diagnostic assays that use biotin-streptavidin technology. These tests are commonly used to measure plasma concentrations of a wide range of hormones. Erroneous results may lead to misdiagnosis of various endocrine disorders. Supplementation with high-dose biotin has been used experimental for the treatment of diseases (e.g. multiple sclerosis) and high doses are used to obtain effect on nail and hair growth. On this background a demand for tests to determine if there is a risk of obtaining false test results when using biotin-streptavidin based tests have appeared. In this paper we present a method based on column switching liquid chromatography tandem mass spectrometry for the quantification of biotin in plasma and serum and explore the effects of biotin on an immunoassay based on biotin strept(avidin) chemistry.

Biotin immunoassay interference: A UK-based prevalence study.

BACKGROUND: Biotin interference in biotin-streptavidin-based immunoassays is increasingly reported due to individuals taking biotin-containing supplements and patients prescribed biotin. The reported prevalence of serum biotin above the lowest threshold (≥10 µg/L) for interference in Roche Diagnostics immunoassay tests is 0.8% in Australia and 7.4% in the USA. There are, however, no such data in UK populations. In a service evaluation, we therefore studied the prevalence of biotin interference in routine serum samples received in our laboratory. METHODS: Biotin was measured in 524 anonymized surplus serum samples in which at least one immunoassay test had been requested. RESULTS: The median (95% confidence intervals) for serum biotin was 0.27 µg/L (0.07-0.93 µg/L). Serum biotin was <10 µg/L in all samples, <5 µg/L in 522 (99.6%) and <1 µg/L in 513 (98.1%) samples. In four samples, serum biotin was ≥2.5 µg/L (0.8%). CONCLUSIONS: These data indicate that the probability of biotin immunoassay interference in our patient population is extremely low, with the exception of assays reporting the lowest interference thresholds (e.g. Ortho Troponin I assay [threshold ≥2.5 µg/L]).

Urinary 3-hydroxyisovaleryl carnitine excretion, protein energy malnutrition and risk of all-cause mortality in kidney transplant recipients: Results from the TransplantLines cohort studies.

BACKGROUND: Leucine is an essential amino acid and a potent stimulator of muscle protein synthesis. Since muscle wasting is a major risk factor for mortality in kidney transplant recipients (KTR), dietary leucine intake might be linked to long-term mortality. Urinary 3-hydroxyisovaleryl carnitine (3-HIC) excretion, a functional marker of marginal biotin deficiency, may also serve as a marker for dietary leucine intake. OBJECTIVE: In this study we aimed to investigate the cross-sectional determinants of urinary 3-HIC excretion and to prospectively investigate the association of urinary 3-HIC excretion with all-cause mortality in KTR. DESIGN: Urinary 3-HIC excretion and plasma biotin were measured in a longitudinal cohort of 694 stable KTR. Cross-sectional and prospective analyses were performed using ordinary least squares linear regression analyses and Cox regression analyses, respectively. RESULTS: In KTR (57% male, 53 ± 13 years, estimated glomerular filtration rate 45 ± 19 mL/min/1.73 m2), urinary 3-HIC excretion (0.80 [0.57-1.16] µmol/24 h) was significantly associated with plasma biotin (std. ß = -0.17; P < 0.001). Subsequent adjustment for potential covariates revealed urinary creatinine excretion (std. ß = 0.24; P < 0.001) and urinary urea excretion (std. ß = 0.53; P < 0.001) as the primary determinant of urinary 3-HIC excretion. Whereas plasma biotin explained only 1% of the variance in urinary 3-HIC excretion, urinary urea excretion explained >45%. During median follow-up for 5.4 [4.8-6.1] years, 150 (22%) patients died. Log2-transformed urinary 3-HIC excretion was inversely associated with all-cause mortality (HR: 0.52 [0.43-0.63]; P < 0.001). This association was independent of potential confounders. CONCLUSIONS: Urinary 3-HIC excretion more strongly serves as a marker of leucine intake than of biotin status. A higher urinary 3-HIC excretion is associated with a lower risk of all-cause mortality. Future studies are warranted to explore the underlying mechanism. TRIAL REGISTRATION ID: NCT02811835. TRIAL REGISTRATION URL: https://clinicaltrials.gov/ct2/show/NCT02811835.

Hair Loss After Sleeve Gastrectomy and Effect of Biotin Supplements.

Aim: In this study, we aimed to determine the incidence of hair loss in patients who underwent laparoscopic sleeve gastrectomy (LSG), and to observe whether use of Biotin has an impact on hair loss. Methods: This study included 156 female patients who underwent LSG for obesity and completed a 1-year follow-up. All patients with vitamin deficiency were screened in the pre- and postoperative period. Hair loss was defined as the subjective perception of the women of losing a higher amount of hair when compared with normal situation. Results: Hair loss was observed in 72% of the patients after LSG (n = 112). Seventy-nine percent of the patients reported hair loss between the third and fourth-month interval, and continued for an average of 5.5 ± 2.6 months. Permanent alopecia was not observed in any of the patients. Patients who experienced hair loss and Biotin deficiency after LSG were prescribed 1000 mcg/day of Biotin for 3 months. Of these 22 patients; only 5 (23%) patients reported a remarkable decline in hair loss. In addition, 29 patients were found to take 1000 mcg/day of Biotin for average 2.5 months after onset of hair loss by their own initiative, despite optimal blood Biotin levels. Eleven (38%) patients reported a remarkable decline in hair loss. The effect of biotin use on hair loss in patients with and without biotin deficiency was compared. There was no significant difference (P = .2). Conclusion: Temporary hair loss after LSG is common. It was found that biotin supplementation used to prevent hair loss does provide low efficacy.

Clinical risk assessment of biotin interference with a high-sensitivity cardiac troponin T assay.

Objectives Biotin >20.0 ng/mL (81.8 nmol/L) can reduce Elecsys® Troponin T Gen 5 (TnT Gen 5; Roche Diagnostics) assay recovery, potentially leading to false-negative results in patients with suspected acute myocardial infarction (AMI). We aimed to determine the prevalence of elevated biotin and AMI misclassification risk from biotin interference with the TnT Gen 5 assay. Methods Biotin was measured using an Elecsys assay in two cohorts: (i) 797 0-h and 646 3-h samples from 850 US emergency department patients with suspected acute coronary syndrome (ACS); (ii) 2023 random samples from a US laboratory network, in which biotin distributions were extrapolated for higher values using pharmacokinetic modeling. Biotin >20.0 ng/mL (81.8 nmol/L) prevalence and biotin 99th percentile values were calculated. AMI misclassification risk due to biotin interference with the TnT Gen 5 assay was modeled using different assay cutoffs and test timepoints. Results ACS cohort: 1/797 (0.13%) 0-h and 1/646 (0.15%) 3-h samples had biotin >20.0 ng/mL (81.8 nmol/L); 99th percentile biotin was 2.62 ng/mL (10.7 nmol/L; 0-h) and 2.38 ng/mL (9.74 nmol/L; 3-h). Using conservative assumptions, the likelihood of false-negative AMI prediction due to biotin interference was 0.026% (0-h result; 19 ng/L TnT Gen 5 assay cutoff). US laboratory cohort: 15/2023 (0.74%) samples had biotin >20.0 ng/mL (81.8 nmol/L); 99th percentile biotin was 16.6 ng/mL (68.0 nmol/L). Misclassification risk due to biotin interference (19 ng/L TnT Gen 5 assay cutoff) was 0.025% (0-h), 0.0064% (1-h), 0.00048% (3-h), and <0.00001% (6-h). Conclusions Biotin interference has minimal impact on the TnT Gen 5 assay's clinical utility, and the likelihood of false-negative AMI prediction is extremely low.

Serum biotin level during pregnancy is associated with fetal growth and preterm delivery.

Background : Biotin is a water-soluble vitamin that plays various biological roles through histone modification, such as immune functions and fetal growth. Mammalian maternal biotin deficiency during gestation induces fetal growth restriction. Preterm infants are known to be marginal biotin deficiency. However, studies on the biotin status of pregnant women under various conditions are lacking. Method : This was a retrospective case control study to analyze serum biotin concentration during pregnancy and cord blood in normal pregnancy, preterm delivery and small-for-gestational-age (SGA). Results : Twenty pregnant women with normal term delivery, 35 with preterm delivery, 24 with SGA, and 10 non-pregnant adult women were enrolled. Serum biotin concentrations of pregnant women remained low from first to third trimester. The levels of serum biotin in cord blood showed a significant positive correlation with gestational age, and that of pregnant women showed a weak positive correlation with gestational age. The maternal serum biotin levels during second and third trimester of SGA group were significantly lower than those of normal term delivery. Conclusion : This study suggests that maternal biotin deficiency during pregnancy might be the risk of preterm labor or fetal growth restriction. Further studies are required to clarify the roles of biotin in perinatal medicine. J. Med. Invest. 67 : 170-173, February, 2020.

Prevalence of detectable biotin in The Netherlands in relation to risk on immunoassay interference.

Despite the increasing awareness about biotin interference with immunoassays, so far, only two studies have quantified the prevalence of elevated biotin in patient populations. In a US study, over 7% had biotin concentrations exceeding 10 ng/mL, whereas in an Australian study only 0.8% of ED samples contained biotin exceeding 10 ng/mL. At present, representative data for the European population are lacking. In this study, we investigated biotin prevalence in The Netherlands in a representative cohort of routine laboratory requests in our laboratory using an LC-MS/MS assay for quantification of biotin in human plasma. In our study, we found 0.2% of samples exceeding 10 ng/mL of biotin, a finding more or less in line with the Australian data. Even though the biotin prevalence appears to be low, with concomitant low to moderate biotin concentrations, it is by no means a rare phenomenon. Laboratories like ours are likely to experience biotin positive samples on a daily basis with variable impact on patient care depending on the analytical bias from the immunoassay platform used. Our simple and robust LC-MS/MS assay for quantification of biotin in human samples may contribute to better understanding of the systemic concentrations seen after moderate- and high-dose biotin supplementation and the extent of immunoassay interference.

Assessment of Risk for Interference by Circulating Biotin in Samples Received for High Sensitivity Troponin-T, Thyrotropin, and for Prostate Specific Antigen Testing by Immunoassays.

BACKGROUND: Biotin interference in streptavidin-based immunoassays is known and may lead to erroneous results and thus to diagnostic error. The recent increase in reports of biotin interference in immunoassay-based testing has been attributed to increased intake of biotin supplements by the public and to the high dose biotin therapy in patients with neurological and inherited disorders. Circulating biotin levels greater than 20 ng/mL are reported to exhibit interference in high sensitivity troponin T (hs-TnT), thyroid stimulating hormone (TSH), and in prostate specific antigen (PSA) among other assays when using our Cobas® 6000 immunoassay analyzer (Roche Diagnostics, IN, USA). This study aims to examine the risk for biotin interference among our patient population. METHODS: Serum and plasma leftover samples from 183 different patients were collected following completion of hs-TnT (53 samples), TSH (45 samples), and PSA (85 samples) testing. Aliquots were stored frozen at -20°C until analysis. Biotin concentrations in these samples were measured using an ELISA (ALPCO, Salem, NH, USA) according to the manufacture's protocol. Samples with biotin levels of 20 ng/mL or greater were considered as high-risk samples (HRS) for biotin interference. RESULTS: The overall concentrations of biotin in our patients' samples ranged from 0.02 ng/mL to 11.38 ng/mL (median 0.42 ng/mL). The median and (range) biotin concentrations in hs-TnT, TSH, and PSA samples were 0.27 ng/mL (0.02 - 6.86 ng/mL), 0.39 ng/mL (0.08 - 11.38 ng/mL), and 0.47 ng/mL (0.09 - 7.73 ng/mL), respectively. Although there was no significant difference between biotin levels in samples for TSH or PSA measurement (p = 0.85), biotin in samples for PSA and for hs-TnT and in samples for TSH and hs-TnT were significantly different (p = 0.049 and 0.089), respectively. None of the samples had biotin levels greater than or equal to 20 ng/mL. CONCLUSIONS: Using representative samples with requests for hs-TnT, TSH, and PSA testing, where reliable performance for the selected assays at their lowest measurement range is required for clinical intervention, among our study population the risk was considered minimal as their circulating biotin levels were less than 20 ng/mL. However, educating clinicians and laboratory users regarding the potential of biotin interference is always recommended.

Mimicking the human environment in mice reveals that inhibiting biotin biosynthesis is effective against antibiotic-resistant pathogens.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets1. In Mycobacteria tuberculosis and Francisella tularensis, biotin biosynthesis is a key fitness determinant during infection2-5, making it a high-priority target. However, biotin biosynthesis has been overlooked for priority pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. This can be attributed to the lack of attenuation observed for biotin biosynthesis genes during transposon mutagenesis studies in mouse infection models6-9. Previous studies did not consider the 40-fold higher concentration of biotin in mouse plasma compared to human plasma. Here, we leveraged the unique affinity of streptavidin to develop a mouse infection model with human levels of biotin. Our model suggests that biotin biosynthesis is essential during infection with A. baumannii, K. pneumoniae and P. aeruginosa. Encouragingly, we establish the capacity of our model to uncover in vivo activity for the biotin biosynthesis inhibitor MAC13772. Our model addresses the disconnect in biotin levels between humans and mice, and explains the failure of potent biotin biosynthesis inhibitors in standard mouse infection models.