Beauty fad’s ugly downside: test interference

Anne Paxton

September 2016—It’s the kind of health promotion advice one might pick up casually over lunch with friends, in a quick Google search, or during a visit to the hairdresser. Take megadoses of an over-the-counter vitamin called biotin—a common supplement in multivitamin compounds—and watch your skin improve and your hair and nails thicken and gleam. In recent years, online social networks and health-related websites have begun to teem with ads claiming that people have seen a transformation since they jumped on the biotin bandwagon.

The trend has meant that biotin megadoses up to hundreds of times the recommended daily intake of 30 µg per day have become a wellness routine for many. For clinical laboratories, for clinicians, and for patients getting diagnostic tests, however, this phenomenon is happening without regard for an alarming fact: taking unusually large doses of biotin can interfere with a broad range of immunoassay test results. In an unexpected and dismaying coincidence, it turns out, the design of many immunoassays, including thyroid tests, cardiac markers, and others, relies on biotin to capture antibodies.

In fact, “a significant percentage of immunoassays rely on biotin-streptavidin binding as part of the assay architecture,” says Ed Reineks, MD, PhD, staff pathologist at the Cleveland Clinic, which conducts millions of immunoassays per year on automated systems. “It’s a common mechanism used to capture antibodies. So this biotin interference could affect virtually every area with any immunoassay testing.”

Potentially hundreds of assays could be affected by high-dose biotin—and not just because of high hopes for skin, hair, and nails. “There are certain areas like rheumatology and multiple sclerosis where high-dose biotin is thought to be helpful by certain clinicians,” says André Valcour, PhD, director of the Esoteric Immunoassay, Allergy, Coagulation, Toxicology, and Biological Monitoring departments at LabCorp’s Center for Esoteric Testing, Burlington, NC.
The possibility that supplements might interfere with assay results, of course, is familiar. “There are so many over-the-counter formulations that are new and novel, so many different tests we measure. And the regulatory process for over-the-counter supplements is such that we don’t know their purity. We don’t know their contents or whether other substances are in there,” Dr. Valcour points out. “So it’s always possible, and should be considered by clinicians, that a patient taking over-the-counter supplements or drugs might produce anomalous results in a lab test.”

However, the use of high-dose biotin supplements, either over the counter or by prescription, is fairly new. “I believe it’s less than a couple of years old,” Dr. Valcour says. And biotin is in a category of its own. “I’m not aware of any other situation where a component of an assay design is being taken as a supplement,” he says. “Biotin is actually attached to one of the proteins of the assay or to the surface of the substrate. It’s part of the design and it’s chemically bound to a component of the assay. I don’t know of anything else that’s quite like that.”

At meetings in which he has participated and asked about biotin use, Dr. Valcour has been surprised at how many of the participants say they take biotin—usually based on the belief that biotin’s role as a key contributor to keratin will help improve hair and nails. “This included people on both sides of the table and a large portion of people in the room. So utilization is higher than one might expect. And they didn’t know anything about the potential effect of biotin megadoses on lab testing.”

Package inserts from Roche Diagnostics, Abbott Diagnostics, and Siemens Healthcare Diagnostics all contain warnings about limitations of their tests due to possible interferences, Dr. Reineks notes. “The diagnostics manufacturers are required, when they seek Food and Drug Administration approval, to examine common interferences for tests, and the most routine are things that can go wrong with the specimen, such as hemolysis,” Dr. Reineks says. “But then certain tests have additional interferences they have to consider.”

Biotin is one such potential interference. Patients who are taking high doses of biotin should not have their blood drawn for laboratory testing for a certain period after they take their biotin. However, Dr. Valcour says, what typically would happen is a clinician might get unexpected test results for a patient, then call the lab to find out if something was wrong with the test. “If it’s determined that the patient was on a high dose of the vitamin, they usually ask the patient to skip one day.”

Biotin, Dr. Reineks notes, has a very short half-life in most patients, according to the literature—under two hours—and it is usually 99 percent cleared from the body within four to five half-lives. “That’s one mitigating factor in this. The other is that the recommended intake for biotin is something on the scale of 30 µg per day, under expert guidelines for daily supplements.” When he learned of biotin interference a few years back, he looked at what kind of doses people are taking. “And the Roche package inserts I reviewed warn about patients taking 5 mg a day, which is more than 150 times the recommended dose.” So he considered an interference to be an unlikely problem.

“What I’ve learned since then, from reading and informal ground level research, is that the manufacturers of these supplements set the pills at 10,000 µg, more than 300 times the recommended dose. The pills are far in excess of what the recommended supplement level is,” Dr. Reineks says. And despite almost no one having a biotin deficiency, an extremely rare condition, usually associated with a genetic abnormality, “there are a lot more people taking biotin than I would have guessed.”

Dr. Valcour had a similar experience when he investigated. “When the problem first came to my attention in January 2016 with an Endocrine News article, I immediately went to the Web, and saw many, many sources of biotin advertised at megadoses and many claims of its clinical utility as an over-the-counter supplement. So it was clearly something that may have an impact on the results of immunoassays performed in a laboratory.”

Dr. Valcour, who oversees testing in LabCorp’s special coagulation laboratory, says that there is precedent for medication adversely affecting the accuracy of laboratory testing.

“Clinicians sometimes order clotting-based laboratory tests on patients who are treated with anticoagulants. In these cases, test results do not reflect the patient’s underlying condition, but rather the impaired clotting caused by the medication. Like with biotin, an exogenous compound taken by the patient can result in clinically erroneous results being reported.” What is new about the biotin situation, he says, is that, in many cases, the patients are taking the drug without informing their clinicians. “However, the common feature to both situations is the need for clinicians to be aware of their patient’s treatment regimen, both prescribed and unprescribed, and the potential impact of the treatment on testing results.”

With biotin, it’s not always the case that an obvious interference exists, Dr. Valcour notes. “You may have a patient on a very high dose of biotin that can cause results to be significantly abnormal, alerting the doctor there is something funny going on. In these cases, the results can suggest an extreme pathology that is inconsistent with the patient presentation. What we don’t understand is the effect of residual biotin in the patient’s blood several hours after they take their dose. This may produce inaccurate results that are clinically plausible. This is the more insidious situation,” he says.

In Dr. Valcour’s experience, “Many clinicians are unaware of the effect of biotin on laboratory results.” Labs are beginning to address the issue, he says. “I know our lab has been aware for several months, we’ve engaged with the diagnostics manufacturers, and are formulating an approach for dealing with it.” He has experienced three cases in which clinicians contacted him, and he discovered through investigation that biotin was causing anomalous results. “That’s helped me become more aware of it too.”

The diagnoses that could be affected by potential
biotin interference run the gamut, Dr. Reineks says. “We use the biotin-streptavidin technique in many immunoassays. That’s how we measure cardiac markers such as troponin, natriuretic peptides, some iron studies. That’s how we do hormone studies. In addition to thyroid, we have testosterone, estradiol, and hCG—it could affect all of those.”

A New Zealand study published this year (Elston MS, et al. J Clin Endocrinol Metab. Epub ahead of print June 30, 2016. doi:10.1210/jc.2016-1971) demonstrated that these possibilities are real. In the study, a patient who had markedly abnormal thyroid function tests that did not match the clinical context was given a factitious Graves’ disease diagnosis due to biotin immunoassay interference. The researchers found that once the patient’s biotin ingestion was halted, her thyroid function tests normalized far more rapidly than possible given the half-life of thyroxine. Significantly, multiple other analytes also tested abnormal in the presence of biotin.

In a letter to the editor published Aug. 18 in the New England Journal of Medicine (Kummer S, et al. 2016;375[7]:704–706), physicians from Heinrich Heine University Hospital in Duesseldorf, Germany, report on six children receiving high-dose biotin treatment in the context of inherited metabolic diseases for whom laboratory results were suggestive of Graves’ disease. The children had excessively elevated levels of free thyroxine and total triiodothyronine, low levels of thyrotropin, and elevated levels of anti-thyrotropin receptor antibodies. Only one child had symptoms attributable to hyperthyroidism, and scans of the thyroid were unremarkable in all examined patients. After biotin was discontinued, thyrotropin and thyroid hormone levels were normalized 24 to 28 hours later; levels of anti-thyrotropin receptor antibodies took up to seven days to normalize. “Although manufacturers are aware of this potential problem,” the authors write, “this source of error is usually not referenced to the clinician in laboratory reports.”

At the Cleveland Clinic, Dr. Reineks, who is medical director for automated chemistry at the clinic’s main campus, is not being flooded with calls about laboratory results, and he cautions that the kind of widespread impact that might result from biotin interference does not seem to be happening yet. “I’d be getting a lot more complaints. The number of calls I get about potential interference are pretty consistent—maybe one or two calls a week—but the denominator is very large, because we run so many tests. I would say biotin is on the radar as a possible explanation, but there are a lot of other interfering substances, or causes for inconsistent immunoassay results, that are probably higher on the list of suspects.”

For example, he says, a number of people have autoimmune diseases and are making antibodies that interfere with tests, and a number of therapeutic antibodies are emerging too. “There are basically in-vitro–produced medications that are antibodies and people get those as treatments for various things like arthritis or lymphoma. Some prescribed drugs can also cause false-positives, but they are usually very specific to a particular individual test.”

Biotin is unusual, Dr. Reineks emphasizes. “Something that applies to so many tests that can cause problems—there are not that many substances that would fall into that category. Biotin would have a very unique position in that regard.”

At least two types of common assays use the biotin-streptavadin formulation: competitive assays, which are typically used for low-molecular-weight targets such as thyroid hormone T4, and sandwich assays, used for bigger molecules such as thyroid-stimulating hormone.

“For an example of a competitive assay,” Dr. Reineks says, “we put in some kind of label on a therapeutic drug that’s in the reagent. When it’s bound to the antibody, it’s not giving off a signal, but when I put in a patient sample and it has the same unlabeled drug, the patient’s drug is going to compete with the labeled drug. The drug that’s labeled can come off the antibody and the patient drug can bind to it, so the two are competing.”

Competitive assays, because of this mechanism, tend to produce elevated values just because of the way they use biotin-streptavadin in the assay, while sandwich assays can cause falsely low values. “So it depends on the assay type and the analyte,” Dr. Valcour says. “For the same analyte, one assay might produce a falsely high value while a different assay might produce a falsely low value.”

“You can’t make assumptions about the extent of the effect, though. It could be influenced by factors such as incubation period or buffers. For some assays, there will be a large effect, while for other assays there will be a small effect even for the same principle of the assay—sandwich or competitive. The bottom line is we don’t know a lot about this because the studies haven’t been done.”

Diagnostics manufacturers confirm that they are conducting studies on biotin interference now. “But those studies will be complicated, because not only do the actual blood levels of biotin make a difference, but also the rate at which the biotin is cleared,” Dr. Valcour points out. In certain patients—those with renal function issues, for example—clearance may take longer. “So there’s just an awful lot we don’t know about the specific effect on a given assay by a given manufacturer and the specific effect of a dose on a given patient.”

Lack of clinician awareness of biotin’s potential to interfere with laboratory test results is an important aggravating factor in that interference. A professor of clinical medicine who presented at the recent International Thyroid Congress, according to the January 2016 article in Endocrine News, noted that most endocrinologists don’t know about this problem.

“The Endocrine Society has done quite a bit of work to educate endocrinologists on the issue of biotin interference,” Dr. Valcour says. “But quite frankly, most thyroid testing is done in primary care and not by endocrinologists, and internal medicine doctors may not know about it. What’s more, testing in other disciplines like infectious disease and gastroenterology might be affected as well, but different doctors may not tie an anomalous result to a particular dose of biotin taken by a patient.”

The FDA takes a virtually hands-off approach when it comes to regulating supplements, Dr. Reineks notes. “If the supplement manufacturers don’t make medical claims, but only wellness or health claims, I think supplements can fly under the radar unless they are saying they actually cure or treat something.” One result is that patients who are ingesting supplements may not even know that biotin, which can be marketed as vitamin H, coenzyme R, or vitamin B7, is what they are taking. So even if asked directly whether they are taking biotin, patients may answer incorrectly.

That is one of the questions manufacturers are studying as they work out how to deal with biotin interference.