Editorial

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The impact of biotin interference on

laboratory testing and patient diagnosis in
clinical practice
Luca Giovanella*,1
1
Clinic for Nuclear Medicine & Competence Centre for Thyroid Diseases, Imaging Institute of Southern Switzerland/Ente
Ospedaliero Cantonale, Via Ospedale 6, 6500 Bellinzona, Switzerland
*Author for correspondence: Tel: +41 (0) 91 811 86 72; Luca.Giovanella@eoc.ch

First draft submitted: 31 January 2019; Accepted for publication: 1 May 2019; Published online:
26 July 2019

Keywords: biotin • clinical practice • laboratory errors • laboratory testing • immunoassay • interference

Biotin-streptavidin coupling has been utilized for decades by diagnostic immunoassay manufacturers. However,
concerns have been raised regarding the risk of biotin interference with biotin/streptavidin-based immunoassays
due to the use of high-dose biotin in cosmetic supplements, and treatments for multiple sclerosis [1–4], and some
inherited metabolic diseases (e.g., biotinidase, multiple carboxylase and holocarboxylase synthetase deficiencies) [5].
A recent study characterized the biotin pharmacokinetic profile in healthy participants following high-dose biotin
administration and provided guidance on washout periods to avoid false assay results from biotin interference [6].
Here, we discuss the prevalence of assay interferences, particularly biotin interference, consider its impact on
clinical practice, and suggest potential strategies to reduce this impact and ensure accurate patient diagnosis.

Incidence & types of laboratory errors

The total laboratory testing error rate is estimated to vary widely from 0.012 to 0.6% [7]. Most errors occur
in the preanalytical phase (e.g., inappropriate sample collection/handling; up to 68% of total errors), followed
by the postanalytical phase (e.g., erroneous data entry/reporting; up to 47% of total errors); analytical errors
(e.g., interference from endogenous/exogenous substances) constitute 7–13% of total errors, with errors caused by
interferences comprising a small proportion of these [8].
Immunoassays are susceptible to many interferences. Although it is difficult to estimate the overall frequency
with which interferences occur [7], hemolysis is thought to be the leading cause of unsuitable samples for analysis
(40–70% of cases) [9]. Importantly, the prevalence of clinically relevant interferences can be substantially lower than
the overall interference prevalence. For example, prevalence estimates for human antimouse antibodies range from
<1 to 80% [10], while the proportion of clinically relevant interferences from these antibodies is considerably lower
(0.03–0.05%) [11]. In our specialist clinic in Switzerland, we typically observe four to five cases of clinically relevant
interferences per 1000 patients each year. In the last 2 years, we have observed six clinically relevant interference
cases: two due to biotin interference in patients with multiple sclerosis who were taking high-dose biotin as part of
a clinical trial, and the others due to heterophilic antibodies.
Interferences should be suspected when the test result is not consistent with the wider clinical picture (e.g., a
patient with no thyroid dysfunction symptoms but abnormal thyroid function test results). Identifying interferences
can be more challenging in patients with an established diagnosis, such as cancer, where biomarkers are used to
evaluate disease progression or guide treatment. For example, thyroglobulin and calcitonin are used as early
indicators of disease relapse in patients with differentiated and medullary thyroid cancer, respectively. Similarly, in
prostate and testicular cancer, early identification of disease relapse is based on prostate-specific antigen and human
chorionic gonadotropin, respectively. The uncertainty caused by a potentially false test result can be particularly
challenging for these patients, so it is important that clinicians are aware of potential interferences and interpret
anomalous results correctly. Additional imaging and/or complementary tests should be performed and interference
should be considered before proceeding with invasive procedures/therapies.

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Editorial Giovanella

Some interferences are relatively easy for laboratories to identify using techniques such as dilution experiments.
Unfortunately, sophisticated imaging can be negative in some cases as the biomarkers are more sensitive; serial
biomarker evaluation may be informative in these cases, as a continuous rise is rarely due to interferences. It is also
useful to have a reference test to confirm the original results; for example, simultaneous procalcitonin measurement
can help exclude false-positive calcitonin results due to heterophilic antibody interference [12].

The impact of biotin interference on clinical practice

All biotin/streptavidin-based immunoassays are susceptible to biotin interference. Until recently, this risk was
considered relatively rare, as assay biotin interference thresholds are considerably higher than serum biotin concen-
trations in individuals taking no more than the recommended adequate biotin intake in adults of 30 μg daily [13].
Therefore, biotin concentrations required to pose a risk to immunoassay results are virtually impossible to achieve
from a biotin-rich diet alone. However, biotin interference is of increasing concern due to the marketing of high-
dose biotin supplementation (up to 10 mg in single-ingredient preparations) and trials of experimental multiple
sclerosis treatments containing very high biotin doses (up to 300 mg daily) [1–4].
Biotin interference can be particularly challenging with the use of thyroid hormone biomarkers as it is possible
to mimic Graves’ disease without having the condition [4,14,15]. The two biotin interference cases observed in our
clinic resulted in erroneous thyroid hormone results [14]. In both cases, the patients were referred to our center
with very high thyroid hormones and suppressed thyroid-stimulating hormone, mimicking hyperthyroidism, but
with no typical symptoms of thyroid overactivity and an unremarkable physical examination. Both patients were
being treated at a specialist neurologic center for multiple sclerosis and had received high-dose biotin as part of a
clinical trial, but the patients and their clinicians were unaware of the possibility of biotin interference, illustrating
that this risk is not well recognized. As with any interference, it is important that thyroid disorders indicated
by biomarker testing are confirmed by clinical examination and imaging (e.g., ultrasound scan of the thyroid
gland and, if indicated, thyroid scan/uptake test). Although this may involve the patient undergoing unnecessary
investigations, it is preferable to start inappropriate treatments. Of course, it may be possible to start treatment
based on a positive test result, without other confirmatory examinations, if the patient has clear symptoms of
hyperthyroidism (e.g., increased heartbeat, weight loss). However, in the absence of clear signs and symptoms,
clinicians should avoid starting treatment based on biomarker results alone.
A key question is the distinction between the risk of analytical interference versus the risk of clinically relevant
misclassification of patients. Physicians are primarily interested in detecting clinically relevant interferences. A
prevalence study of 1442 patients presenting to a US emergency department showed that 7.4% had biotin
≥10 ng/ml (lowest biotin interference threshold among Roche Diagnostics immunoassays) [16]. However, only
a few assays have biotin interference thresholds as low as 10 ng/ml; most have thresholds much higher than
this and the proportion of samples with biotin >30 ng/ml was only 0.5%. Therefore, very few of these biotin
concentrations would likely lead to clinical misclassification. The misclassification risk due to biotin interference
can vary considerably between assays, and a combination of high peak biotin concentrations and a sensitive assay
is needed for a clinically relevant interference to occur; for example, biotin interference thresholds for cardiac
troponin assays range from 2.5 to 10,000 ng/ml [17,18]. Although most Roche Diagnostics assays are minimally
affected at biotin concentrations of 15.6 and 31.3 ng/ml (simulating 5 and 10 mg biotin intake, respectively), some
assays exhibit greater sensitivity, including troponin T, thyroid-stimulating hormone and antithyroid antibodies [18].
Regarding troponin T, a recent study showed that elevated biotin (>20 ng/ml) is rare in US patients with suspected
acute coronary syndrome and the likelihood of false-negative acute myocardial infarction prediction due to biotin
interference with the Elecsys R
Troponin T Gen 5 assay was very low (0.026%), having a minimal effect on the assay’s
negative predictive value (93.4%) at 3 h [19]. It is important to note that the benefit of diagnostic immunoassay
testing largely outweighs the risk of potential misclassification of patients due to interferences, including biotin
interference, and laboratory error rates are considerably lower than those seen in overall clinical healthcare [20].

Strategies to reduce the risk of biotin interference

Laboratory staff and clinicians should be aware of the risk for biotin interference, paying particular attention to
results from certain patient groups (e.g., patients with multiple sclerosis) or results that do not fit the overall clinical
picture. A focus on increased reporting and education on biotin interference, as recommended by the US FDA [2],
will hopefully improve awareness and reduce the likelihood of errors occurring. There is also increasing emphasis
on education around biotin interference in the multiple sclerosis patient population, with participants in trials of

10.4155/ipk-2019-0001 Int. J. Pharmacokinet. (Epub ahead of print) future science group

 The impact of biotin interference on laboratory testing & patient diagnosis in clinical practice Editorial

high-dose biotin receiving a card detailing their involvement and the risk for assay interference. This approach has
been extended to wider patient groups not directly involved in these trials. However, the need to raise awareness
should be balanced against the fact that the risk of clinically relevant misclassification due to biotin interference is
low and the overall laboratory error rate has fallen in the past few decades [8]. If biotin interference is suspected,
clinicians should enquire about recent biotin consumption. It may be helpful to provide guidance to patients and
ask them to avoid taking certain medications or supplements before blood tests. Lastly, many biomarker and/or
disease algorithms involve serial testing, which may help to reduce the impact of interference with a single result.

Future perspective
The use of high-dose biotin in cosmetic supplements and multiple sclerosis treatments has raised concerns regarding
the risk of assay interference. However, recent research suggests the risk of clinically relevant misclassification due
to biotin interference may be low. Crucially, this issue reiterates that laboratory results should not be considered
in isolation. Instead, patient symptoms, clinical examination findings, laboratory results and imaging should be
evaluated in concert to gain a holistic clinical picture and avoid misdiagnosis.

Financial & competing interests disclosure

The author has served as a member of Scientific Advisory Boards for Eisai, Roche Diagnostics, and Sanofi Genzyme, has received
speaker fees from BRAHMS, Roche Diagnostics and Sanofi Genzyme, and received research grants from Roche Diagnostics. The
author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or
financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Third-party medical writing assistance, under the direction of the author, was provided by Thomas Burton, BMBS (Gardiner-
Caldwell Communications, Macclesfield, UK), and was funded by Roche Diagnostics International Ltd, Rotkreuz, Switzerland.

Open access
This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license,
visit http://creativecommons.org/licenses/by-nc-nd/4.0/

References
1. Peyro Saint Paul L, Debruyne D, Bernard D, Mock DM, Defer GL. Pharmacokinetics and pharmacodynamics of MD1003 (high-dose
biotin) in the treatment of progressive multiple sclerosis. Expert Opin. Drug Metab. Toxicol. 12(3), 327–344 (2016).
2. US Food and Drug Administration. The FDA warns that biotin may interfere with lab tests: FDA safety communication (28 November
2017). www.fda.gov/medicaldevices/saf ety/alertsandnotices/ucm586505.htm
3. Clerico A, Plebani M. Biotin interference on immunoassay methods: sporadic cases or hidden epidemic? Clin. Chem. Lab. Med. 55(6),
777–779 (2017).
4. Piketty ML, Polak M, Flechtner I, Gonzales-Briceño L, Souberbielle JC. False biochemical diagnosis of hyperthyroidism in
streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences. Clin. Chem. Lab. Med. 55(6), 780–788
(2017).
5. Wolf B. Biotinidase deficiency: ‘if you have to have an inherited metabolic disease, this is the one to have’. Genet. Med. 14(6), 565–575
(2012).
6. Grimsey P, Frey N, Bendig G et al. Population pharmacokinetics of exogenous biotin and the relationship between biotin plasma levels
and in vitro immunoassay interference. Int. J. Pharmacokinet. 2(4), 247–256 (2017).
7. Sturgeon CM, Viljoen A. Analytical error and interference in immunoassay: minimizing risk. Ann. Clin. Biochem. 48(Pt 5), 418–432
(2011).
8. Plebani M. The detection and prevention of errors in laboratory medicine. Ann. Clin. Biochem. 47(Pt 2), 101–110 (2010).
9. Lippi G, Blanckaert N, Bonini P et al. Hemolysis: an overview of the leading cause of unsuitable specimens in clinical laboratories. Clin.
Chem. Lab. Med. 46(6), 764–772 (2008).
10. Kricka LJ. Human anti-animal antibody interferences in immunological assays. Clin. Chem. 45(7), 942–956 (1999).
11. Tate J, Ward G. Interferences in immunoassay. Clin. Biochem. Rev. 25(2), 105–120 (2004).
12. Giovanella L, Giordani I, Imperiali M, Orlandi F, Trimboli P. Measuring procalcitonin to overcome heterophilic-antibody-induced
spurious hypercalcitoninemia. Clin. Chem. Lab. Med. 56(8), e191–e193 (2018).
13. Institute of Medicine. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, vitamin B6, folate, vitamin B12, pantothenic acid,
biotin, and choline.A Report of the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on
Folate, Other B Vitamins, and Choline. National Academy Press, Washington, DC, USA (1998). https://www.nap.edu/catalog/6015/die
tary-ref erence-intakes-f or thiamin-riboflavin-niacin-vitamin-b6-folate-vitamin-b12-pantothenic-acid-biotin-and-choline

future science group 10.4155/ipk-2019-0001

Editorial Giovanella

14. Giovanella L, Imperiali M, Kasapic D, Ceriani L, Trimboli P. Euthyroid Graves’disease with spurious hyperthyroidism: a diagnostic
challenge. Clin. Chem. Lab. Med. 57(5), e94–e96 (2019).
15. Koehler VF, Mann U, Nassour A, Mann WA. Fake news? Biotin interference in thyroid immunoassays. Clin. Chim. Acta 484, 320–322
(2018).
16. Katzman BM, Lueke AJ, Donato LJ, Jaffe AS, Baumann NA. Prevalence of biotin supplement usage in outpatients and plasma biotin
concentrations in patients presenting to the emergency department. Clin. Biochem. 60, 11–16 (2018).
17. Saenger AK, Jaffe AS, Body R et al. Cardiac troponin and natriuretic peptide analytical interferences from hemolysis and biotin:
educational aids from the IFCC Committee on Cardiac Biomarkers (IFCC C-CB). Clin. Chem. Lab. Med. 57(5), 633–640 (2018).
18. Trambas C, Lu Z, Yen T, Sikaris K. Characterization of the scope and magnitude of biotin interference in susceptible Roche Elecsys
competitive and sandwich immunoassays. Ann. Clin. Biochem. 55(2), 205–215 (2018).
19. Mumma B, Diercks D, Ziegler A, Dinkel-Keuthage C, Tran N. Quantifying the prevalence of elevated biotin in a cohort with suspected
acute coronary syndrome. Presented at: 70th Annual Scientific Meeting of the American Association for Clinical Chemistry, Chicago, USA,
29 July–2 August 2018, Abstract A105.
www.aacc.org/science-and-practice/annual-meeting-abstracts-archive/2018-annual-meeting-abstracts
20. Leappe LL. Striving for perfection. Clin. Chem. 48(11), 1871–1872 (2002).

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