Jefferson Medical College
Professor of Medicine
Thomas Jefferson University Hospital
Cardiac troponins (cTn) are highly sensitive and specific biochemical markers for detecting myonecrosis. The two that are most commonly used to distinguish between myocardial infarction (MI) and unstable angina in patients presenting with ischemic discomfort are cTnI and cTnT. Abnormal cTn levels are occasionally found in non-MI patients, including those who have tachycardia, myocarditis, or other conditions that can result in myonecrosis. Heterophile antibodies, such as rheumatoid factor, also have been found to interfere with cTn immunoassays, leading to positive readings despite a lack of cardiac pathology. While there is extensive evidence in the laboratory literature to suggest that cTn levels can be falsely elevated, little has been published on this topic in the clinical literature.
Cardiac troponins (cTn) are the most sensitive and specific biochemical markers for detecting myonecrosis.1 Over the past 10 years, the role of cTn biomarkers in diagnosing acute coronary syndromes (ACS) has grown, and the threshold values used to determine a positive result have decreased. According to the American College of Cardiology (ACC)/American Heart Association guidelines, elevated cTn levels in patients with ischemic discomfort is diagnostic of an acute myocardial infarction (MI).2
The Joint European Society of Cardiology/ACC Committee for the Redefinition of Myocardial Infarction has recommended that an increased concentration of cTn be defined as a measurement exceeding the 99th percentile of cTn concentrations observed in a healthy reference group.1 Because this is a very low threshold, the total imprecision (CV) at this level should be less than 10% to minimize false-positive results.1 This means that the measured cTn values from one serum sample, when evaluated on at least three occasions, cannot vary by more than 10%. If they do, assay imprecision should be suspected. This information is critical to understanding the current debate regarding cTn testing, because most of the 20 different assays on the market are not precise at the threshold value suggested for determining a positive result (99th percentile in a healthy reference group). Many false-positive results occur at the lower ranges of analytic sensitivity, where it is most difficult to separate clinically relevant myocardial cell damage from spurious cTnI elevation.3
A 44-year-old white woman was transferred to our institution from an outside hospital due to an elevated cTnI level and previous abdominal pain. On the day of her admission to the outside hospital, she experienced sharp, right-sided epigastric pain that radiated down her right arm. There were no associated symptoms and the pain remitted after about 20 minutes. When it recurred approximately 1 hour later, it was localized to her right upper quadrant and did not radiate to her right arm, but it was stronger compared with the first episode. The pain did not change with breathing and was not accompanied by a cough. She also experienced nausea but no vomiting, chest pain, shortness of breath, palpitations, or diaphoresis.
At the outside hospital, the patient was found to be anemic and to have increased cTn levels. She noted having heavy menstrual cycles over the past few months, which would last between 12 and 18 days. The patient's medical history was significant for a previous episode of syncope that had been worked up at an outside facility, where elevated cTn levels were identified. She had a negative stress test on that occasion and did not receive cardiac catheterization.
On presenting to our institution, physical examination revealed a heart rate of 58 beats/min, a respiratory rate of 16 breaths/min, and a blood pressure of 102/60 mm Hg. Cardiac examination noted a regular heartbeat with no murmurs, rubs, or gallops discernable on auscultation. The first heart sound was loudest at the apex and was accentuated when the patient was in the left lateral decubitus position. The second heart sound was loudest at the base and increased when the patient leaned forward. Her lungs were clear to auscultation bilaterally. Abdominal examination revealed a nondistended abdomen, normal bowel sounds, mild tenderness to deep palpation in the right upper quadrant, no rebound or guarding, and a negative Murphy's sign. The patient's extremities revealed no edema. Her pedal pulses were 1+ bilaterally and her radial pulses were 2+ bilaterally.
Laboratory examinations revealed a hemoglobin of 8.5 g/dL (normal, 12.0-15.0 g/dL), hematocrit of 26.2% (normal, 35.0%?45.0%), and a normal platelet count. The patient's cTnI levels from the outside hospital were 1.38, 1.32, and 1.46 ng/mL. At our institution, her cTnI was 1.48 ng/mL; the normal range for cTnI at both institutions is between 0.05 and 0.50 ng/mL. Before being transferred to our institution, her creatine kinase (CK) and CK-MB levels were checked three times and found to be within normal limits.
The patient's electrocardiogram (ECG) demonstrated normal sinus rhythm, a ventricular rate of 68 beats/ min, and no acute ST-segment changes (Figure). She underwent cardiac catheterization, which revealed no angiographic evidence of occlusive coronary artery disease. She had a normal left ventricular function, with an estimated ejection fraction (EF) of 0.55. A transthoracic echocardiogram showed an EF of 0.65 ± 0.05 with mild aortic regurgitation and minimal tricuspid regurgitation.
The patient was deemed stable and was discharged from the hospital. Because of the persistent elevations in cTnI, further work-up was undertaken. Eight plasma samples were collected: four from our patient and four from a patient with a confirmed MI. Two samples from our patient and two samples from the MI patient were treated with a heterophile blocking reagent (HBR). All eight samples were assayed for cTnI at Thomas Jefferson Hospital's clinical chemistry laboratory using the AccuTn1 assay. The HBR-treated samples from our patient showed an average decrease of 63% in cTnI compared with the untreated samples, whereas the HBR-treated samples from the patient with a confirmed MI had the same cTnI levels as the untreated samples.
We also had our patient's blood tested at two outside laboratories, using different assays (Axsym, Dade Dimension) to measure cTnI. On both of these assays, her cTnI was normal. These results confirm that our patient's positive troponin level was caused by interference from heterophile antibodies in her blood.
In a study that included eight different troponin assays, only one demonstrated a CV of less than 10% when calculated at the 99th percentile of the healthy reference group.4 In addition, two troponin assays had a 1.2- to 2.5-fold higher 99th percentile for males versus females and two had a 1.1- to 2.8-fold higher 99th percentile for blacks versus whites. There was also a 13-fold variance between the lowest measured 99th percentile and the highest.4 Age-related differences in troponin concentrations were identified in a study that demonstrated significantly higher cTn1 concentrations in healthy individuals who were older than 60 years.5
Another study of 14 different troponin assays found that no assay was able to achieve the 10% CV recommendation at the 99th percentile reference limit defined by the manufacturer.1 Only cTn concentrations that met the goal of 10% CV as an MI cutoff provided a false-positive rate below 1% in patients suspected of having ACS.6 Because different troponin concentrations were observed among healthy reference populations based on age, race, and sex, reliable cutoff points to determine a positive result have yet to be identified.
Abnormal levels of serum cTn can be found in non-MI patients and have been reported after prolonged strenuous exercise and in individuals with pulmonary embolism, pericarditis, myocarditis, coronary vasospasm, sepsis, congestive heart failure, supraventricular tachycardia with hemodynamic compromise, and renal insufficiency.3 In the only clinical study of elevated troponin in non-MI patients, the authors retrospectively identified 217 patients from their catheterization database with elevated troponin levels and normal coronary arteries.7 Tachycardia (35%) and myocarditis (23%) were identified as the most common causes of elevated troponin. Other physiologic causes included congestive heart failure, gastrointestinal bleeding, severe aortic stenosis, pericarditis, sepsis, septic shock, and left anterior descending artery bridging. Although these physiologic entities all cause myonecrosis and subsequent troponin elevations, they were not the focus of the study. In addition, the cause of cTn elevation could not be identified in 10% of study patients.
An elevated cTn in the absence of any pathology leading to myonecrosis generally results from analytical interference with the troponin assay. This occurs when an unintended analyte causes a positive result in the absence of the intended analyte. Troponin immunoassays often use two-site (sandwich) or competitive reactions, which contain two antibodies (capture and label antibodies) at two sites for the measured analyte. The capture antibody initially binds to any troponin in the sample. The label antibody is added after a wash phase and binds to the captured troponin, providing a quantifiable signal.
Heterophile antibodies are sometimes present in serum samples. These multispecific antibodies are produced against poorly defined antigens and are generally weak, but they can crosslink the capture and label antibodies, resulting in a false-positive troponin test.8 Heterophile antibodies may be acquired from a variety of sources, including mouse monoclonal antibodies used for therapeutic and imaging purposes; blood transfusions; vaccinations against infectious diseases; exposure to microbial antigens; animal husbandry; domestic pets; transfer of dietary antigens across the gut wall in celiac disease; and autoimmune diseases that may regsult in autoantibodies, such as rheumatoid factor (RF).8
Although the prevalence of heterophile antibodies is unknown, interference from these antibodies in troponin assays has been reported in the literature.7-11 A 1986 study of 188 patients found that 40% had heterophile antibodies that caused interference in two-site immunoassays.8 Larger investigational studies of newer generation troponin assays also report analytical interference. Studies from 2002, 2004, and 2006 evaluated serum samples from healthy blood donors and found heterophile antibody interference in 4 of 989 cases, 4 of 150 cases, and 6 of 480 cases, respectively.12-14
The effect of RF on troponin assays has been studied.8,15,16 Fifteen of 100 serum samples taken from a population of outpatients who had an RF greater than 100 and were not under evaluation for cardiac disease demonstrated cTn elevations consistent with an MI using one troponin assay but showed normal levels using a different assay.15 Another study found that 7 of 12 patients with RF and no indication of an MI had positive troponin levels.16 Although using polyclonal antisera has been shown to eliminate RF interference in one popular troponin assay,8 a 2006 study of troponin levels in patients with RF found that 11.5% had increased troponin levels attributable to RF even after pretreatment with heterophile blocking agents, and half of the false-positive results could not be corrected.8 False-positive troponin results can also result from fibrin clots, hemolysis interference, immunocomplex formation interference, and instrument malfunction; however, these factors have not been reported to cause the same level of interference observed with het-erophile antibodies.
In patients with an MI or other well-known causes of myonecrosis, cTn is detectable in the blood from 4 to 6 hours after myocardial injury, peaks from 12 to 16 hours after the injury, and decreases over the next 4 to 6 days. When an elevated troponin level remains constant over the course of repeated testing, analytical interference should be suspected. The lack of a pattern of increase or decrease suggests a constant level of analyte. This pattern would not be seen in patients suffering from an MI or other myonecrotic conditions because cTn levels fluctuate in these patients.
A positive troponin should be investigated when a patient's presentation and the clinical findings are not consistent with cardiac ischemia or an MI. The findings that increase the probability of an MI and its associated likelihood ratios (LRs) include new ST-segment elevation (LR 5.7-53.9) and/or a new Q wave (LR 5.3-24.8) on an ECG; chest pain radiating to both arms (LR 7.1); presence of a third heart sound (LR 3.2); and hypotension (LR 3.1). The findings that decrease the probability of an MI include a normal ECG (LR 0.1-0.3), pleuritic chest pain (LR 0.2), chest pain reproduced by palpations (LR 0.2-0.4), sharp or stabbing chest pain (LR 0.3), and positional chest pain (LR 0.3).17
When analytical interference is suspected, clinicians should consider ordering a stress test and confirmatory enzyme assays, including CK, CK-MB, or both. If the cTn elevation is related to analytical interference with the assay, the CK and CK-MB values should be within normal limits and the stress test should be negative for ischemia. In such cases, the clinician should notify the laboratory so that heterophile blocking assays can be performed in accordance with the manufacturers' protocol. If the troponin value decreases after heterophile blocking antibodies are added to the reagent, then the presence of these antibodies are causing the troponin elevation.8
We do not recommend ordering an RF test because the result has no impact on the patient's work-up. A positive RF level in a patient with suspected false-positive cTn is not enough to rule out an MI. Furthermore, if RF is truly causing the troponin elevation, its interference should be eliminated with the use of an HBR.
A false-positive troponin should be suspected when the cTn elevation does not fit the rise and fall pattern classically observed in cases of acute myonecrosis, especially if the patient's clinical presentation does not suggest an ACS. If a false-positive cTn is suspected, we recommend that the cTn assay be repeated with the techniques used to determine whether there is heterophile antibody interference.
(Answers at end of references list)
Answers: 1. B; 2. C; 3. C; 4. A; 5. B