Biotin recommended intake of 30 μg/day is easily obtained from a balanced diet, and real biotin deficiency seems to be a very rare condition in the modern world. Biotin is present in some popular dietary supplements, alone or as a component in multi-vitamin formulas. It is advertised as a remedy for losing hair and for the fragility of hair and nails (< 20 mg/d) [4].

Automated immunoassays used to evaluate thyroid function are vulnerable to different types of interference that can affect clinical decisions [2]. However, in recent years, the widespread use of streptavidin–biotin technology, and the common use of biotin-containing supplements, has led to numerous reports of erroneous results of hormone- and non-hormone parameters measured by immunoassays [5,6,7,8]. In the vast majority of the studies previously reported, the amount of biotin ingested was in the range of moderate to high doses, and biotin (> 10 mg/day) interference resulted in either falsely high or low values.

Interestingly enough, interference has not been limited to thyroid tests and has the potential to affect a wide range of analytes, such as thyreoglobulin (Tg), luteinizing hormone (LH), follicle stimulating hormone(FSH) e.t.c [2, 6]. Namely, Biotin can inhibit immune complex separation, leading to analytical errors in some patient’s labwork; this may be encountered in biotin supplementation or in the presence of anti-streptavidin antibody [2]. In these cases, the interference may induce both false positive and false negative results, and simulate a seemingly coherent hormonal profile [5, 7]. This bidirectional behavior with opposing effects may mimic the biochemical diagnosis of hyperthyroidism with falsely elevated concentrations of FT4, free T3 (FT3), anti-TSH receptor Ab, and falsely lowered TSH concentration [6, 7]. Conversely, several endocrine disorders, including primary hypothyroidism, may be misdiagnosed because of biotin distortion of the assays decreasing the elevated TSH levels, primarily in patients with chronic kidney disease or biotinidase deficiency [2, 8,9,10]. The reformulation of immunoassays for biotin-resistant methods is in progress, but biotin-susceptible methods remain in widespread use throughout the world [11].

A topic remaining incompletely understood is the duration of the washout period needed in subjects consuming BCS, for the accurate evaluation of either TSH or FT4 levels. Based on available literature data the washout periods in several studies ranged from a few hours to 8 days [12, 13]. Given the variability in these results, it has been impossible to provide a consensus recommendation on that issue [8, 14]. The impact of biotin supplements on thyroid hormone levels can differ depending on the amount and length of time biotin has been taken [12, 13]. Therefore we focused on patients where BCS was received from at least 10 days to two months. One the other hand, once biotin interference is suspected to last from several hours to several days, we consider that the reassessment of thyroid hormones 15 days after supplements withdrawal is probably a sufficient wash out period to provide us with solid data.

The main goal of the present clinical study was to investigate whether various biotin supplements alter thyroid hormone profiles in hypothyroid patients and to what extent that “biochemical fault” might lead clinicians to unnecessary changes in the amount of prescribed thyroxine dose to their patients. We investigated potential alterations in the measurement of TSH and FT4 seen in real world practice, since these two parameters are used commonly as part of the assessment of patients with hypothyroidism both in endocrine, but also in non-specialty clinics, such as Internal Medicine and General Practice.

Studies have tested various doses of biotin, which were given to volunteers and demonstrated that the extent of analytical error is less remarkable as far as endocrine assays are concerned. The dose applied was approximately 100 times higher than the required daily dose (30–100 µg), and corresponded well to the amount of biotin found in commercial dietary supplements [15].

Using a moderately high intake such as 10 mg daily [15], it may lead to inaccurate results. The major determinants of such errors for susceptible immunoassays are the biotin dose; time elapsed since last intake, and kidney function [14,15,16,17]. Various factors may contribute to the artifactually falsely high or low results, including the degree of blood biotin elevation based in terms of the amount of biotin ingested, the time interval from biotin ingestion to blood specimen collection, the biotin interference threshold, and the patient's own relative biotin metabolism.

Although supra-nutritional amounts of biotin are often taken over the counter for hair, skin and nail benefits in the thousands of micrograms many multivitamin supplements contain biotin in smaller amounts, ranging from 20 μg to 300 μg and this probably explains the lack of any effect in thyroid hormone levels, observed in some studies. Recently, the high-dose biotin (100 mg to 300 mg/day, which is 10,000 times the standard dietary reference intake) has been accepted as a valid treatment strategy in patients with progressive multiple sclerosis [18], potentially threatening an overflow of abnormal TFTs in treated patients. Therefore, cautious interpretation of measured thyroid parameters should be exerted in such circumstances, or a biotin insensitive assay should be used.

The discrepancy between a clinical exam which is not indicative of thyroid dysfunction and markedly abnormal thyroid function tests should lead to a search for biotin intake, which can interfere with thyroid function tests. Additionally, most patients fail to mention taking this supplement to their physician, because it is not seen as a “real medication.” Both clinicians and patient awareness of this issue are necessary in high doses of biotin intake supplements. However, in daily practice, the impact of most multivitams (containing lower than 300 μg of biotin) seems to be irrelevant in terms of clinical decision and no further reassessment is needed.

We understand that the present study has two major limitations: First is the lack of biotin concentrations measurement in our cohort. Previous studies however have evaluated the biotin concentration in patient samples from different countries. A study quantifying biotin in plasma samples from emergency department patients in the USA showed that 7.4% of samples had a biotin concentration at or above 10 ng/mL (considering a low threshold, above which interference will occur for the most sensitive immunoassays) [19]. In another study from Australia, 0.8% (4/490) of subjects sampled presented with a biotin concentration above 10 ng/mL, whereas in the Netherlands, this percentage was estimated to be 0.2% [20, 21]. Secondly, the absence of patients receiving larger doses of biotin that could lead to major alterations in TFTs as recently mentioned [22]. The biotin interference threshold depends on the test and the platform used [23]. It is conceivable that the interference can become progressively more significant with higher biotin levels or less significant with biotin levels below the threshold. Since the process of biotin measurement is cumbersome and costly, we aimed straight to the clinical impact of supplement ingestion in daily clinical practice. For the same reasons, our protocol is mimicking the “real world” tactic where only TSH and/or FT4 are used to assess thyroid status in patients with chronic hypothyroidism receiving usual over the counter multivitamins.

Alterations observed in our study in both ΤSH and FT4 levels, (although reached statistical significance) were modest and cannot be solely attributed to analytical interference of biotin with the applied assays. Of note, in the only two patients where clinical decision might be misleaded, TSH was decreased after supplement withdrawal, opposite than expected. Differences between functional sensitivities can be found among laboratories using the same automated system even in the same laboratory. These discrepancies can be attributed to the way sera pools are prepared, the periodicity of measuring, and even to differences among automated systems that are theoretically the same [24]; whether recommended calibration prosses is performed regularly remains also questionable. Among the important criteria that influence desirable analytical performance is the biological variation of a given parameter [25]. Seasonal variations in thyroid hormones levels have been documented previously, mainly assigned to alterations in the central sensitivity to thyroid hormones (increased in summer and decreased in winter) [26]. Hence, the statistical difference in this study should not be interpreted uncritically as clinical significance. Unfortunately, a knowledge gap amongst physicians regarding the risks and benefits of biotin supplementation still exists. A recent study reported that almost half of physicians did not advise biotin cessation prior to laboratory testing, implying knowledge of biotin interference may not translate into change in practice [27].