Cardiac troponin and natriuretic peptide analytical interferences from hemolysis and biotin: educational aids from the IFCC Committee on Cardiac Biomarkers (IFCC C-CB)

Cardiac troponin I and T (cTnI, cTnT) and the natriuretic peptides (NP; B-type natriuretic peptide, BNP; N Terminal-proBNP; NT-proBNP) are the primary cardiac biomarkers utilized in the diagnosis of myocardial injury and infarction (MI) and heart failure (HF), respectively. As with any clinical laboratory test, there are exogenous and endogenous factors that adversely interfere with the analytical performance of the cTn and NP assays, potentially resulting in inappropriate clinical interpretation of the results if the interferences are not identified. Analytical interferences are particularly concerning when dealing with cardiac biomarker assays, which are utilized to make time sensitive critical clinical decisions. Two interferences recently brought to the forefront as patient safety issues include hemolysis (hemoglobin) and biotin (vitamin B7, vitamin H, coenzyme R). The International Federation for Clinical Chemistry Committee on Cardiac Biomarkers (IFCC-CB) obtained input from a majority of cTn and NP assay manufacturers to collate information related to high-sensitivity (hs)-cTnI, hs-cTnT, contemporary, and POC cTn assays (Table 1) [1], and NP assays (Table 2) [2] interferences due to hemolysis and biotin. The information contained in these tables was designed as educational tools to aid laboratory professionals and clinicians in troubleshooting cardiac biomarker analytical results that are discordant with the clinical situation.

Hemolysis is one of the major causes of pre-analytical errors, reportedly accounting for 40%–70% of all specimen rejections [3]. Furthermore, a substantial volume of hemolyzed samples occur from specimens collected in the emergency department and from indwelling catheters in many intensive care units [4]. The accuracy of cTn results is of significant importance because it is heavily relied upon for making appropriate and rapid patient care decisions. If hemolysis thresholds are exceeded, the specimen needs to be recollected, resulting in delays in patient care and an increased risk of iatrogenic injury, infection, and adverse clinical management in the absence of objective information. Hemolysis is a known confounder of hs-cTn and cTn assays, causing false positive or false negative results; either situation may hinder interpretation of single or serial values [5]. Detection of hemolyzed samples occurs either manually (visual, qualitative assessment) or through automated detection (quantitative or semi-quantitative assessment) using indices on the clinical chemistry platform. The latter approach is supported as a benchmark of good laboratory practice due to the improved reliability, accuracy and standardized approach to reporting results within a laboratory when using automated mechanisms to assess hemolysis. For cTn assays with a low threshold for hemolysis (>100 mg/dL, i.e. >1 g/L) the reported rate of incorrectly released results is as high as 76% [6]. Not all immunoassay platforms or point-of-care devices have the ability to routinely perform automated hemolysis detection, presenting potential patient safety issues for reporting accurate cTn and NP results due to the subjective nature of visual detection of hemoglobin. Moreover, hemolysis will be missed if whole blood is used as the matrix for measurements.

Biotin interference is a relatively new challenge to laboratories and highlighted by the Food and Drug Administration (FDA) warning statement to clinical laboratories (https://www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm586641.htm). Investigation of potential interferences from biotin in immunoassays is similar to methods utilized for decades in clinical laboratories to probe analytical interferences. Biotin is a water-soluble vitamin with a half-life ranging from 8 to 16 h, depending on renal function [7]. Adequate intake is defined at 0.03 mg/day, although consumption has expanded and retail sales of over-the-counter “mega” doses (2.5–10 mg) of biotin have increased significantly due to marketing efforts claiming healthier and stronger hair, skin and nails. Furthermore, individuals are often unaware that the supplements they are ingesting even contain biotin. There are ongoing randomized clinical trials in the US and Europe to evaluate biotin doses of 300 mg/day in patients with multiple sclerosis and other inflammatory diseases, resulting in circulating serum biotin concentrations between 170 and 700 μg/L [8]. Immunoassays comprised of biotin labeled antibodies or biotin-streptavidin labeled complexes are particularly susceptible to interferences for a wide array of clinical tests.

Data obtained from manufacturers regarding the analytical specificity and interference for the cTn and NP assays/platforms are presented in Tables 1 and 2, respectively. Interference thresholds were defined as the greatest concentration for either hemoglobin or biotin that did not compromise accuracy of the cTn or NP analytical results. When this threshold was exceeded results were classified as either falsely high or low, allowing laboratory professionals to ascertain the performance of their specific assay/platform in the scenario of gross hemolysis or potentially excessive endogenous biotin intake. Manufacturers defined their acceptance criteria when evaluating and validating interference thresholds. The “End User Assessment of Hemolysis” column in Tables 1 and 2 was designed to aid clinical laboratory personnel performing cardiac biomarker testing. If laboratory personnel must visually assess for hemolysis before reporting or releasing cTn results the assay was designated as “Qualitative”. If the instrument automatically assesses for hemolysis to allow erroneous results to be suppressed and alerting the laboratorian the threshold was exceeded, the assay was designated as “Quantitative”.

Biotin interference data in the tables state whether a biotinylated antibody is incorporated and/or if biotin is used in the assay configuration; it is notable that those assays with either characteristic are more susceptible to interference from endogenous biotin use. If high dose biotin supplementation is known or suspected due to results that do not correlate with the patient’s clinical condition, one possible mitigation strategy could involve analysis with another assay that is not susceptible to biotin interference. However, this is not always a practical solution and may be problematic due to the lack of standardization of cTn and NP assays. Other proposed strategies include adsorption of excess biotin using streptavidin-coated microparticles [8, 9], although this requires additional validation within the laboratory before implementation.

Cardiac biomarker assays, like a majority of clinical laboratory assays, are susceptible to endogenous and exogenous interferences to some extent, which may yield analytically incorrect results. There is particular concern about the effect of interferences with hs-cTn and NP assays, as these are widely used clinically in urgent care settings to guide critical clinical decisions but there is often less time to carefully consider potential analytical issues in this situation. For cTn assays, the analytical sensitivity and imprecision at the 99th percentile are of utmost importance and the consequences of false negative or false positive results at or near the 99th percentile due to hemolysis and/or biotin consumption have been highlighted in recent publications [10]. While diagnosis of acute MI, ischemia or heart failure should always be taken in conjunction with the clinical context of the patient, heightened awareness of these analytical issues and solutions should be implemented to avoid adverse events.