FDA, using input provided by Advanced Medical Technology Association (AdvaMed), has developed this document in response to numerous adverse event reports of falsely elevated results sent by manufacturers and users of troponin assays and many published articles related to this subject.
What is the Purpose and Scope of this Communication?
The purpose of this communication is to inform laboratorians about the possibility of falsely elevated troponin results when troponin is used as a cardiac marker, and to provide information as to how to identify and verify cases of suspected falsely elevated results. This communication may also be of interest to cardiologists, primary care physicians, and emergency care physicians. In this document, we use the term “false-positive” troponin test result to refer to cases when the troponin immunoassay result is positive in the absence of acute myocardial infarction (AMI) or any other clinical condition in which troponin markers may be elevated.
This document addresses issues related to false-positive troponin results only when troponin testing is used in the diagnosis of acute myocardial infarction and not when it is used to diagnose other diseases. The information applies to both cardiac troponin I and cardiac troponin T immunoassays since the issue of falsely elevated results applies to both assay systems.
Cardiac troponins are highly sensitive and specific biochemical markers of myocardial cell necrosis and are widely used for the diagnosis of acute myocardial infarction.   Troponin levels rise and fall with acute myocardial infarction and with other clinical syndromes associated with infiltrative/inflammatory causes of myocyte death.
Troponin testing is subject to analytical interfering factors and assay malfunctions; both may lead to a falsely elevated troponin result. In addition, there are clinical conditions in which the troponin level may rise in the absence of an acute myocardial infarction . Although the elevation of troponin in association with non-AMI is generally rare, it may mislead the clinician to suspect that the patient has an acute myocardial infarction when that is not the case.
Therefore, it is important that laboratorians, working in conjunction with physicians who order the test, are aware of falsely elevated results and non-AMI causes of elevated troponin results in order to assist physicians to properly utilize troponin results in patient management.
Can Results of Different Troponin Assay Systems Be Compared?
No. The results of different troponin assays are not generally comparable. A large variation in cardiac troponin I concentration, in terms of absolute value, may be observed for a given patient specimen with different analytic methods. Until recently, there was no accepted reference standard for troponins. However, a new Standard Reference Material for Human Cardiac Troponin Complex (SRM 2921) has been introduced by the National Institute of Standards and Technology (NIST) which should aid in the future standardization of troponin assays. The SRM 2921 is intended for use in evaluating the accuracy of clinical procedures for the determination of cardiac troponin I in human serum.
The analytical variability among troponin assays is due to the fact that different troponin assays have wide variations in lower detection limits, upper reference limits, diagnostic cut points, assay imprecision (coefficient of variation), and specimen matrices (i.e., serum versus plasma samples). The presence of a large number of manufacturers of troponin assays in the United States market makes standardization more difficult.
What Are the Analytical Interfering Factors That May Lead to Falsely Elevated Troponin Results?
- fibrin clots in serum as a result of incompletely clotted specimen, e.g. in patients with coagulopathy or on anticoagulant therapy
- heterophile antibodies, human anti-animal antibodies, rheumatoid factor  , and autoantibodies
- interference from other endogenous components in the blood such as bilirubin and hemoglobin
- immunocomplex formation
- microparticles in specimen
- high concentration of alkaline phosphatase
- analyzer malfunction
What Are Interfering Antibodies (Heterophile and Human Anti-Animal) and How Can Laboratorians Address This Issue?
Definition and Sources: Circulating heterophile antibodiesi and anti-animal antibodiesii have the potential to interfere with two-site (sandwich) or competitive immunoassays, such as troponin assays, by cross-linking the capture and label antibodies in the absence of specific analyte.   The estimated prevalence of interfering antibodies in the general population is up to 40% of normal serum samples.  
Most modern immunoassays contain nonspecific blocker immunoglobulins (which originate from the same species as the analyte-specific antibodies) in order to limit the effect of the interfering antibodies. However, in some instances the blocking proteins can not sufficiently neutralize the interfering antibodies. Thus, analytical errors may occur. In case of troponin assays, the presence of high levels of these antibodies may lead to falsely elevated values.
An individual may acquire these antibodies from a variety of sources including the use of mouse monoclonal antibodies in diagnostic imaging and cancer therapy; exposure to microbial antigens; exposure of veterinarians, farm workers, and food preparers to foreign proteins; the presence of domestic animals in the home; or autoimmune diseases which can give rise to autoantibodies such as rheumatoid factor.  
i Heterophile antibodies are antibodies produced against poorly defined antigens (frequently foreign proteins). The general term "heterophile antibodies" is sometimes used in the literature interchangeably to refer to heterophile antibodies, human anti-animal antibodies, rheumatoid factor, and autoantibodies.
iiHuman anti-animal antibodies are circulating human antibodies reactive with animal proteins. Circulating antibodies with specificities for a wide range of animal immunoglobulins have been reported such as mouse, rat, rabbit, and others.
- Laboratorians May Suspect or Recognize the Presence of Interfering Antibodies in a Specimen Tested for Troponin: Because of a broad range of interfering antibodies, there is not a specific test to verify or rule out their presence in an assay. Laboratorians should suspect the occurrence of interfering antibodies in a troponin assay when the test result:
- does not agree with the patient’s clinical information for an acute myocardial infarction (see items 6 & 7 for more information)
- may not be reproducible on the same or different assay system
- is not linear after serial dilutions
- Methods to Reduce or Remove Interfering Antibodies from Troponin Assays: After ruling out the possibility of technical errors and analyzer malfunction and after repeating the assay, the following methods may be applied for reducing or removing the effect of interfering antibodies from troponin assay results.
- Run the specimen on a different assay system
- Use commercially available heterophile blocking reagents
- Remove endogenous immunoglobulins by adding endogenous immunoglobulin-free serum samples to the specimen. For example, use normal mouse (animal) sera as blocking reagent
If a Troponin Test Result Does Not Match the Patient’s Clinical Picture for Acute Myocardial Infarction, What Should Laboratorians Consider Doing?
If the troponin test result does not match the patient’s clinical picture for acute myocardial infarction, it is possible that the test result is falsely elevated due to analytical interfering factors, and/or analyzer malfunction. The laboratory should investigate the presence of any potentially interfering factors and verify the test result by taking the following steps.
- Find out whether the patient is on anti-coagulant therapy or has coagulopathy.
- Check for bilirubin and/or hemoglobin interference.
- Visually examine the specimen tube for any particles or clots (fibrin interference). If present, centrifuge the specimen for a second time.
- Rerun the specimen after doing a serial dilution and look for interfering antibodies by examining the linearity of results.
- If sufficient amount of specimen is available, save the sample for further testing by the laboratory or the manufacturer.
- Rerun the same specimen on a different assay system, if available, to verify the results. If another assay system is not available, send out the specimen to another laboratory to be run on a different assay system.
- Examine the analyzer thoroughly to investigate the presence of any malfunction.
- Communicate to the treating physician the discrepancy between the test result and clinical information, and inform him/her of the possibility of a false-positive value.
- If the problem does not get resolved, report the issue to the manufacturer and FDA and send the specimen to the manufacturer for further testing and investigation.
If a Troponin Test Result Does Not Match the Patient’s Clinical Picture for Acute Myocardial Infarction, What Should Physicians Consider Doing?
In cases when a troponin test result does not match the patient’s clinical picture for acute myocardial infarction, it is important that the physician gather all the available information and reassess the patient. The physician may:
- consider the possibility that some other clinical condition may be causing an elevated troponin level in the absence of acute myocardial infarction.
- communicate with the laboratory about the test result and ask the laboratory to rule out technical errors, analytical interfering factors, and analyzer malfunction.
- consider repeating the blood draw and retesting.
- review the clinical presentation and consider additional diagnostic testing (e.g. reconsider the nature of chest pain, repeat ECG, etc.); bear in mind that the troponin test result is only one piece of the diagnostic puzzle.
Is It Important that Laboratorians Follow Manufacturers’ Recommended Instructions?
Yes. For troponin testing, it is critical that patient specimens be collected and processed according to the manufacturers’ recommendations included in the device product insert. Improper collection, handling, and preparation of specimens can impact the accuracy of results.
- Store unused collection tubes and blood specimens according to the manufacturers’ recommendations.
- Follow manufacturers’ instructions for using collection tubes with anticoagulants. Some may contain insufficient anticoagulant and lead to elevated or decreased results.
- Mix the content of tubes properly at the time of blood collection to prevent incomplete clot formation (serum) and platelet clumping or clotting (plasma).
- Process specimens according to the tube manufacturer’s recommendations. Different types of tubes may have different requirements.
- Use a refrigerated, horizontal centrifuge head for best results. Use the centrifuge settings recommended by the tube manufacturer.
- Inspect samples for clots, fibrin, particulate matter, and other debris prior to processing them on an analyzer. Cellular debris from grossly hemolyzed samples may elevate test results.
- Follow manufacturer’s recommendation for running proper quality control samples. At least one control should be run at the cutoff level. If the risk stratification and acute myocardial infarction cutoff are different, separate controls should be considered at those levels.
- Follow the manufacturers’ recommended calibration and/or maintenance schedules. Analyzer malfunction is one of the common assay interfering factors that leads to inaccurate results. Laboratories reporting troponin results should perform thorough and regular system maintenance to ensure peak performance of their analyzers and to reduce the possibility of inaccurate results.
What Are the Current Cardiology Practice Guidelines for Troponin Reference Intervals and Decision Limits?
Current practice guidelines issued by the American College of Cardiology (ACC), the National Academy of Clinical Biochemistry (NACB), and the European Society of Cardiology (ESC) make similar recommendations regarding reference intervals, decision limits, and precision. These recommendations include:   
- establishing an upper reference limit at the upper 99 th percentile of normal population
- using a single cutoff positioned at or slightly above the upper reference limit
- accomplishing a total imprecision (coefficient of variation) ≤10% at the decision limit (Currently, few commercially available assays can achieve this precision level .)
What Are Some Clinical Conditions Associated with Cardiac Troponin Elevations in the Absence of Acute Myocardial Infarction?
Myocardial cell damage (reversible or irreversible) and elevation of cardiac troponins can occur under conditions OTHER than acute myocardial infarction due to obstructive coronary artery disease. A short list of some of these clinical conditions includes but is not limited to:  
- myocardial cell trauma
- congestive heart failure
- left ventricular hypertrophy
- pulmonary embolism
- renal failure
- infiltrative disease with cardiac involvement
- acute neurological disease including cerebrovascular accidents and subarachnoid bleeds
- rhabdomyolysis with cardiac injury
Reporting to FDA
If there are any questions or concerns regarding the performance of troponin test method, contact the assay manufacturer. You should report all occurrences of unusual test performance to the manufacturer, and you are encouraged to also report them to FDA. To obtain more information about medical device reporting you can refer to the FDA’s Medical Device Reporting web site.
All reports should be sent to:
Food and Drug Administration
Center for Devices and Radiological Health
Medical Device Reporting
P.O. Box 3002
Rockville , MD 20847-3002
For any questions or concerns regarding the content of this communication contact:
Ms. Ruth Chesler
Food and Drug Administration
Center for Devices and Radiological Health
Office of In Vitro Diagnostic Device Evaluation and Safety
2098 Gaither Road , HFZ-440
Rockville , MD 20850
Phone: (240) 276-0686
Fax: (240) 276-0651
1 Heterophile antibodies are antibodies produced against poorly defined antigens (frequently foreign proteins). The general term "heterophile antibodies" is sometimes used in the literature interchangeably to refer to heterophile antibodies, human anti-animal antibodies, rheumatoid factor, and autoantibodies.
Human anti-animal antibodies are circulating human antibodies reactive with animal proteins. 9 Circulating antibodies with specificities for a wide range of animal immunoglobulins have been reported such as mouse, rat, rabbit, and others. 9
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