A 69-year-old man with diabetes mellitus type II, hypertension, dyslipidemia, and prior ischemic strokes presented to the emergency department with complaints of balance difficulties and inability to stand unassisted of 2 weeks’ duration. The patient’s home medication regimen included atenolol, lisinopril, amlodipine, metformin, and glipizide. He is a retired chef and a former smoker (20 pack-years). He has 2 brothers, both of whom had myocardial infarctions in their 50s. The patient’s physical examination was remarkable for frequent premature contractions, left lower extremity weakness, and impaired coordination. His electrocardiogram revealed sinus rhythm with frequent premature ventricular contractions and diffuse nonspecific T-wave abnormalities.
Results of a comprehensive metabolic chemistry panel were within the reference intervals except for increases in glucose (158 mg/dL; reference interval, 74–99 mg/dL) and creatinine (1.5 mg/dL; reference interval, 0.7–1.3 mg/dL). The hemoglobin A1c value was 7.4% (reference interval, <6.0%). Cardiac troponin I (cTnI)1 concentrations were increased at 0.27, 0.22, and 0.25 μg/L (Abbott Architect assay; 99th percentile, <0.03 μg/L) over a span of approximately 8 h. The patient was admitted to the cardiology service on the basis of these abnormal results.
A transthoracic echocardiogram revealed a preserved left ventricular systolic function with evidence of impaired diastolic filling. A cardiac catheterization evaluation revealed an ulcerated plaque in the left anterior descending artery with >70% stenosis, which was treated with a bare-metal stent. The presenting symptom of balance difficulty failed to resolve after the coronary intervention, and the patient remained unable to stand unassisted. A neurologic evaluation included magnetic resonance imaging, which found no evidence of an acute stroke. He was discharged with a diagnosis of orthostatic hypotension.
Three months later, the patient presented to the emergency department with several days of balance difficulty. The initial cTnI value was increased at 0.10 μg/L, prompting admission to the cardiology service. Over the subsequent 12 h, cTnI values for 2 additional samples remained stable at 0.10 μg/L. Other laboratory tests included a comprehensive metabolic panel and a complete blood count, with results within reference intervals except for a hemoglobin concentration of 12.5 g/dL (reference interval, 14.0–18.0 g/dL) and a hematocrit of 35.5% (reference interval, 40.0%–52.0%). The patient’s symptoms were not consistent with a cardiac etiology, and no further cardiac evaluation was pursued. A neurologic evaluation again found no evidence of acute stroke, and his symptoms were believed to reflect a combination of orthostatic hypotension, pontine gliosis, and cerebellar atrophy. The unexplained abnormal troponin results prompted the attending cardiologist to contact the director of clinicalchemistry.
Cardiac troponins play a central role in diagnosis and risk stratification in acute coronary syndromes(1), but troponins are recognized as markers of cardiac myocyte injury, not of the etiology of injury. A wide range of clinical conditions has been associated with increased troponin values(1), most or all of which have been shown to entail cardiac injury. Thus, it is necessary to consider these conditions when investigating unexpected increases in cardiac troponins.
In the patient described above, the rise and fall in troponin concentrations that are characteristic of acute myocardial infarction was not seen on either admission. This finding pointed toward other etiologies for the increased troponin values(1). The clinical findings and other laboratory information excluded most if not all of these other causes of increased troponin (Table 1⇓ ).
laboratory investigation of increased ctni
Two important interferences reported to produce false-positive results in cardiac troponin immunoassays are fibrin clots and heterophile antibodies, such as human antimouse antibodies (HAMAs) or rheumatoid factor(2). No fibrin clots were seen in the patient’s sample and increased cTnI values were seen in multiple samples and upon their reanalysis, providing presumptive evidence that fibrin clots were not involved. This patient’s plasma was evaluated for HAMAs by measuring cTnI before and after treatment with a heterophile blocking tube from Scantibodies Laboratory. The plasma cTnI concentration remained unchanged, suggesting that HAMAs were not a source of analytical interference in the immunoassay.
investigation of possible immunoglobulin-ctni complex
We suspected the presence of a macrocomplex of cTnI with an immunoglobulin molecule in the patient’s plasma. We reasoned that such a complex would delay the clearance of cTnI from the circulation and thus produce persistently increased troponin concentrations(3). We therefore treated the patient’s plasma either with protein A bound to Sepharose to deplete the sample of IgG, along with any cTnI complexed with IgG, or with an equal volume of buffer. Protein A decreased the cTnI concentration from 0.10 μg/L to undetectable (<0.02 μg/L), whereas buffer produced only the decrease expected from dilution, to 0.06 μg/L (the between-day total imprecision of the assay at 0.04–0.06 μg/L is <10%). In samples from 5 other patients, the cTnI concentrations obtained for the protein A–treated sample matched those of the dilutional control. Unfortunately, no plasma from the index patient’s initial hospitalization was available for a similar analysis.
macrocomplexes of troponin: chemistry, prevalence, and clinical findings
Naturally occurring immunoglobulin–cardiac troponin complexes in plasma were first recognized only recently. In 1996, Bohner et al. suspected the presence of such a complex in a patient after elective coronary artery bypass graft surgery. The patient had undetectable cTnI despite increased concentrations of creatine kinase isoenzyme MB (CK-MB) and cTnT(4). The authors added increasing amounts of cTnI to the sample, up to 38.5 μg/L, and found that cTnI continued to be undetectable. The authors concluded that an antibody that binds cTnI was complexing cTnI, rendering it unable to bind to the antibodies in the immunoassay reagent.
In 2002, an immunoglobulin–cTnI complex was identified as the source of increased cTnI concentrations(3). The patient described in the report had increasing cTnI concentrations over several months without clinical evidence of myocardial infarction. Treatment of a sample with antihuman IgG antiserum removed the cTnI. The authors hypothesized that the complex extended the normally brief half-life of the cTnI and allowed accumulation of cTnI in the blood to measurable concentrations.
Recent reports indicate that IgGs that bind cardiac troponins are common in healthy blood donors. IgG autoantibodies against cTnI, cTnT, or both cTnI and cTnT were found in 12.7% (n = 750), 9.9% (n = 467), and 1.7% (n = 345) of the donors, respectively(5)(6)(7). In a study of patients with cardiomyopathy(8), antibodies to cTnT (or cTnI) were present in 1.7% (or 7.7%) of patients with dilated cardiomyopathy (n = 272) and in 0.5% (or 9.2%) of 185 patients with ischemic cardiomyopathy. The lower prevalence than in the healthy blood donors is thought to reflect a difference in the types of assays used to identify autoantibodies(6).
Pettersson et al.(9) showed that the presence of antibodies to cTnI slowed the apparent clearance of cTnI from the circulation (Fig. 1⇓ ). The plasma cTnI concentrations in the patient described here are indicated by the asterisks and dashed line added to the figure of Pettersson et al. (Fig. 1⇓ ). As can be seen, the rate of clearance of cTnI from the circulation of this patient is like that of the patients of Pettersson et al., who had anti-cTnI antibodies, and is much slower than that seen in the patients without antibodies to cTnI (shaded area).
Circulating immunoglobulins complexed with enzymes and other proteins that are measured in the diagnostic laboratory have been recognized for many years, back at least to the recognition of macroamylase(10). None of the other macrocomplexes are likely to have a more serious immediate clinical impact than an immunoglobulin–cardiac troponin complex (or macrotroponin). Even small increases in cardiac troponin in plasma or serum will be identified as abnormal by the sensitive cardiac troponin assays currently available (and even more sensitive assays are forthcoming). Such results carry the potential to lead to unnecessary interventions, as seen in this case. Results of increased cardiac troponin concentrations are expected with any cardiac troponin assay because the increased cardiac troponin concentration measured does not represent an interference, but rather an analytically correct result that is nonetheless misleading.
In light of the high prevalence of immunoglobulin–cardiac troponin complexes and the critical importance of cardiac troponin testing, it is necessary to be aware of the possibility of these antibodies in patients whose clinical presentations do not match their increased cardiac troponin concentrations. A second blood sample obtained a few hours later should provide clarification. With acute myocardial injury, cardiac troponin will continue to increase, whereas it will be stable with an immunoglobulin–cardiac troponin complex, as it was on both admissions of this patient. Another approach is parallel analysis of CK-MB. A rise and fall in the CK-MB concentration suggests an acute myocardial infarction, but the test is less diagnostically sensitive and specific than troponin assays.
One approach to identify an immunoglobulin–cardiac troponin complex is to measure cardiac troponin before and after treatment of the plasma or serum with protein A, which removes IgG and thus the complex containing the cardiac troponin and IgG. Heterophile blocking tubes, as were used in this case, provide a simple approach to exclude the possibility that protein A is removing an interfering HAMA. Other approaches for identifying the presence of macrocomplexes include electrophoresis and immunofixation(10).
We conclude that this patient’s cTnI most likely circulated as a complex with IgG. Recognition of such complexes is critical to avoid erroneous diagnosis of cardiac injury.
QUESTIONS TO CONSIDER
Describe common pathologic reasons for increased plasma concentrations of cardiac troponins in the absence of an acute coronary syndrome.
What analytical interferences increase measured cTnI concentrations?
How would you investigate the abnormal cTnI results in this patient?
POINTS TO REMEMBER
Plasma concentrations of cardiac troponins are increased in a large number of conditions that injure cardiac muscle (Table 1⇑ ).
Autoantibodies to cTnI or cTnT have been identified in roughly 10% of the healthy population and form complexes with troponin.
Antibodies to cardiac troponin can produce a result of either a decreased troponin concentration (by blocking an epitope recognized by a reagent antibody) or an increased troponin value that appears to reflect the presence of increased cardiac troponin concentration.
The apparent slow clearance of immunoglobulin–cardiac troponin complexes from the circulation leads to persistent increases in measured cardiac troponin, especially after cardiac injury.
Measurement of cTnT in a patient with an immunoglobulin-cTnI complex may or may not be useful, because patients with such complexes may also have macrocomplexes involving cTnT; measurement of CK-MB may thus be useful.
The presence of immunoglobulin–cardiac troponin complexes can be inferred from exclusion of a HAMA interference, combined with demonstration of cardiac troponin removal by protein A, protein G, or antihuman immunoglobulin antiserum.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
- © 2010 The American Association for Clinical Chemistry