Biotin Interference on Diagnostic Testing

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Biotin, also known as vitamin B7 is a coenzyme that is involved in carbon dioxide transfer in carboxylase reactions. The USDA recommended dietary reference intake for biotin is 30 ug per day which should mostly come from food. The last few years biotin has been marketed heavily as a beauty supplement. It is used in hair, skin, and nail supplements, and is not FDA regulated and is sold as over-the-counter. Biotin can be found in B-complex vitamins, multivitamins, prenatal vitamins, vitamin H, and vitamin B7 supplements. The only FDA recommended use for biotin is in patients with secondary progressive multiple sclerosis who receive mega-doses of up to 300 mg per day. Even in such large doses biotin is considered nontoxic and has very little adverse effects.

The issue is that serum or plasma biotin may potentially interfere with any assay that uses biotin-streptavidin binding. Biotin is a small molecule that attaches covalently to a variety of targets with minimal effect on their biological activity. The biotin binding makes the target an easy capture because it forms a strong bond with avidin, streptavidin, and NeutrAvidin proteins who have an exceptionally high affinity for biotin. Biotin-streptavidin detection is a favorite among many immunoassays across many manufacturers including Roche, Ortho, Beckman, Siemens, and Dimension.

The direction of interference depends on the design of the assay. Some results may be falsely elevated, and some may be falsely decreased. The sandwich and competitive assays are among the most commonly impacted. Interference can occur with hormone tests such as parathyroid hormone (PTH), thyroid stimulating hormone (TSH), T4, T3, and even troponin tests.

Sandwich assays involve two antibodies that form a sandwich with the analyte being tested to be measured. The first antibody is labeled with a signal that can be quantified and the other antibody to the target is labeled with biotin. When the biotin:antibody complex binds to streptavidin-coated beads, the labeled antibody then binds creating a sandwich. The resulting complex is then measured. The more complexes that are created, the stronger the signal, i.e the more target analyte there is. Excess free biotin interferes by binding to the streptavidin-coated beads, leaving fewer binding opportunities for the antibodies. Antibody complexes that have successfully bound the analyze get washed away and are then undetected, resulting in falsely low results.

Competitive assays consist of an antibody to the analyte that is labeled with biotin. The analyte must compete for antibody binding sites with a reagent that is a supplied version of itself with a label for detection. If no analyte is present, the reagent occupies all the antibody binding sites and the complex is captured by streptavidin, and a strong signal is emitted. If analyte is present, that occupies antibody binding sites that outcompete the labeled reagent. When analyte is present, there is less detection and less signal measured. It is an inverse relationship. When analyte is not present, there is a strong signal detected, when analyte is present, there is a weak signal detected. Free biotin sticks to the streptavidin, leaving fewer antibody binding sites for the analyte:antibody or reagent:antibody complex. The complexes get washed away and causes weakening of the signal. This may give the impression that analyte is present, even in its absence.

This is an ongoing issue and the FDA advises the healthcare community; patients and physicians both to disclose any supplements that are being taken that contain biotin. Physicians should advise laboratory if interference from biotin is a possibility. Practice should be implemented to counsel patients to abstain from oral biotin 2-3 days before blood tests. Biotin has a rapid half-life of 2 hours, but patients taking mega-doses (>30 mg) have demonstrated interference on laboratory tests for up to 24 hours.

Physicians should educate patients to increase awareness of biotin interference. Adverse health effects can occur if test results are falsely skewed in any direction.

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Cardiac Markers

Approximately every 42 seconds an American will suffer from a myocardial infarction. A MI occurs in a hypoxic state and sections of the heart are unable to get the oxygen it needs. According to the CDC about 610,000 die of heart disease in the United States every year, an occurrence of about 1 in every 4 deaths. Heart disease is the leading cause of death in both men and women.

Also about 47% of sudden cardiac deaths occur outside a hospital environment suggesting that many people with acute heart disease either don’t recognize the early signs or they don’t act on them.

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There are cardiac markers that can give a physician a better picture of what is going on. These are routinely measured in a clinical laboratory and are almost all of the time STAT tests. The quick turn-around-time of these tests is imperative, because the sooner a patient with a heart condition gets treated, the better their prognosis.

The Troponin test is the most sensitive and specific test for myocardial damage. Troponin is released during and MI from the cytosolic pool of the myocytes. Its release is prolonged by the degradation of the actin and myosin filaments. Isoforms of protein, T, and I are specific markers for myocardium. After myocardial injury, troponin is released in 2-4 hours and peaks after 12 hours. It persists for up to 7 days after MI.

The Creatine Kinase-MB test is relatively specific when skeletal muscle damage is not present. Creatine kinase is an enzyme that is present in various tissues and cell types. It catalyzes the conversion of creatine to phosphocreatine utilizing adenosine triphosphate (ATP). The phosphocreatine serves as an energy reservoir in tissues that consumes ATP, especially the skeletal muscle and the brain. When mitochondrial creatine kinase is involved in the formation of phosphocreatine from mitochondrial ATP, cytosolic CK regenerates ATP from ADP and phosphocreatine kinase. In this instance, CK acts as an ATP regenerator. Clinically creatine kinase levels are assayed as a marker for damage of the CK-rich tissues in pathological states of myocardial infarction, rhabdomyolysis, muscle dystrophy, autoimmune diseases, and acute kidney injuries. There are two subunits of the cytosolic CK enzymes; Brain type (B) or Muscle type (M). The two subunits create three different isoenzymes CK-MM, CK-BB, and CK-MB. The different isoenzymes are present in different levels in various tissues. In skeletal tissue CK-MM is predominantly expressed, and in myocardial tissue, CK-MM and CK-MB is measured. Therefore measuring CK-MB levels is a good diagnostic test for heart damage from myocardial infarctions. CK-MB peaks about 10-24 hours after the attack and normalizes within 2-3 days.

Lactate dehydrogenase (LDH) was talked about in one of my previous posts and it can aid in the diagnosis of MI. Although it has been most recently replaced by the more specific and sensitive troponin test. LDH catalyzes the conversion of pyruvate to lactate. LDH-1 is found in the myocardium and LDH-2 is found in the serum. Normally LDH-2 is the predominate isoenzyme, but in cases of MI, LDH-1 is the predominate isoenzyme assayed and found. LDH takes about 72 hours to peak and normalizes within 10-14 days.

Myoglobin is an iron and oxygen binding molecule found in the muscle tissue. Myoglobin is a cytoplasmic protein that only harbors one heme group, although in contrast it has a much higher affinity for oxygen than does hemoglobin because its primary role is to store oxygen, where hemoglobins function is to transport oxygen. It contains a porphyrin ring with a proximal histidine group attached to the iron in its center. Myoglobin is only found in the bloodstream after muscle injury such as rhabdomyolysis. Myoglobin is a sensitive marker for muscle injury, making it a potential marker for MIs. However it lacks specificity and should be taken into account with other clinical findings to make a diagnosis. Myoglobin peaks the earliest of all other cardiac markers, that is within two hours, but it also falls quickly, usually before troponin or CK-MB.

Pro-brain natriuretic peptide is used as marker for acute congestive heart failure. The BNP is a hormone secreted by the cardiomyocytes in the ventricles of the heart in response to stretching caused by increased ventricular blood volume. The actions of BNP cause a decrease in systemic vascular resistance and venous pressure which causes a drop in blood pressure and causes after load. This causes a decrease in cardiac output.

Cardiac markers should be used to add to a clinical diagnosis, they should not be solely used to diagnose a patient with MI or CHF.

-Caleb