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Editorials: Mauro Panteghini Selection of Antibodies and Epitopes for Cardiac Troponin Immunoassays: Should We Revise Our Evidence-Based Beliefs? Clin Chem 2005; 51: 803-804 [Full text] [PDF]

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Beliefs in cardiac troponin testing

Susann Eriksson, Kim Pettersson   (23 June 2005)

Beliefs in cardiac troponin testing 23 June 2005
Susann Eriksson, Research scientist Department of Biotechnology, University of Turku, Turku, Finland, Kim Pettersson Send letter to journal: Re: Beliefs in cardiac troponin testing susann.eriksson{at}utu.fi Susann Eriksson, et al.	To the Editor, In a recent editorial, Panteghini (1) asks himself whether there is a need to revise the evidence-based beliefs regarding the presently recommended design of troponin immunoassays as a consequence of our recent and previous reports (2-4) on the occurrence of circulating anti-troponin antibodies, which can significantly distort patient results when using existing cardiac troponin I (cTnI) assays. In response to some of the statements in the editorial we want to make some clarifying comments. It is evident from our studies (5) that autoantibodies interfere most severely with the measurement of cTnI when antibody combinations most frequently favored by commercial manufacturers are used. However, within the so-called stable mid-fragment area (amino acids 30 to 110), some epitope combinations are less prone to this interference than others. Nevertheless, we do maintain that the present IFCC recommendation for antibody selection (6) clearly becomes inadequate in individuals with high titers of anti-troponin autoantibodies. Panteghini (1) asserts us that troponin testing would never have become the cornerstone for diagnosis of myocardial infarction (MI) if there had been false-negative results in a large proportion of patients. This statement is a misrepresentation of what we are claiming. Despite many methodological difficulties, immunoassays for cardiac troponins have extensively been taken into clinical routine and replaced the other biochemical markers, especially CK-MB. This is mainly due to the fact that cardiac troponins are not normally found in the large majority of healthy subjects using state-of-the-art analytical techniques. However, it is important that one acknowledges that with the adoption of the new definition of MI (7), which was largely spurred by the troponin assays themselves, the number of patients classified as having an MI has increased by an estimated 20-30% (8, 9). In hindsight, these additional patients would have been false negatives in the pre-troponin era, i.e. missed by the evidenced-based belief of that time. Against this background it may be wise not to categorically rule out the possibility that even today with the existing generations of troponin tests, cases of myocardial injury can be overlooked. This is a sound approach for reasons of general scientific caution, but especially pertinent considering the extensive analytical difficulties and challenges in troponin assay methodologies that we have witnessed over the past ten to fifteen years. In fact, it has recently been demonstrated (10, 11) that troponin assays with high analytical sensitivity do not necessarily show equivalent performance in detecting minor cardiac injuries. These injuries, although not acutely fatal, are still important to detect, because patients showing even minor leakages of troponins represent a group with increased short- and long-term risks for future adverse cardiac events. Neither should we forget the frequently acknowledged circumstance that 1-2% of chest pain patients who are sent home have subsequent myocardial infarction and adverse outcomes (12). Panteghini (1) also finds it difficult to understand that disturbing autoantibodies does not necessarily give rise to false negatives, but only produces partial inhibition. It is important that this misconception be cleared out. Autoantibodies are characterized by their concentration (titer), their binding specificity (mono- or polyspecific), and their affinity (with variable on- and off-rate constants). Their inhibiting effect on the formation of the two-site immunoassay complex is dependent on the same qualities of the immunoreagents used. The net effect is not an all-or-none or irreversible situation, but a result of at least two competing reactions, which follow the law of mass action. Autoantibodies will have their highest inhibiting effect early during the release of troponins from the injured myocytes. As the troponin release continues the inhibiting effect will be gradually overcome. According to our results (4,13), one of the most noteworthy consequences of circumventing the inhibiting effect on cTnI assays by careful selection of assay design is an unhindered recognition of the small and/or early releases in chest pain patients. The troponins have generally been considered markers of high specificity but with a relatively slow release. By combining high analytical sensititivity with assay designs that circumvent the interference from autoantibodies, we are confident that the troponins will present markers as early as myoglobin but with a superior specificity. Regarding the doubts that Panteghini still harbors in his editorial (1) about the validity and importance of our findings, we do understand that changes of the official recommendations from regulatory bodies call for additional independent studies to determine the overall significance of troponin autoantibodies in differently designed, widely used immunoassays using large patient cohorts. Meanwhile, our priorities will continue with investigations of mechanisms for anti-troponin antibody formation, further characterization of the fine specificities of the autoantibodies and their effect on assays of cTnI as well as troponin T (14), and exploration of possible correlations to cardiac disease etiologies. We also believe that in close connection to this is the development of analytical tools with further improved detection limits. This will serve to promote the troponins towards their full clinical potential as cardiac markers. References 1. Panteghini M. Selection of antibodies and epitopes for cardiac troponin immunoassays: should we revise our evidence-based beliefs? Clin Chem 2005;51:803-4. 2. Eriksson S, Halenius H, Pulkki K, Hellman J, Pettersson K. Negative interference in cardiac troponin I immunoassays by circulating troponin autoantibodies. Clin Chem 2005;51:839-47. 3. Eriksson S, Junikka M, Laitinen P, Majamaa-Voltti K, Alfthan H, Pettersson K. Negative interference in cardiac troponin I immunoassays from a frequently occurring serum and plasma component. Clin Chem 2003;49:1095-104. 4. Eriksson S, Ilva T, Becker C, Lund J, Porela P, Pulkki K, et al. Comparison of cardiac troponin I immunoassays variably affected by circulating autoantibodies. Clin Chem 2005;51:848-55. 5. Eriksson S, Junikka M, Pettersson K. An interfering component in cardiac troponin I immunoassays - Its nature and inhibiting effect on the binding of antibodies against different epitopes. Clin Biochem 2004;37:472 -80. 6. Panteghini M, Gerhardt W, Apple FS, Dati F, Ravkilde J, Wu AH. Quality specifications for cardiac troponin assays. Clin Chem Lab Med 2001;39:175-9. 7. Alpert JS, Thygesen K, Antman E, Bassand JP. Myocardial infarction redefined - a consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. J Am Coll Cardiol 2000;36:959-69. 8. Koukkunen H, Penttilä K, Kemppainen A, Penttilä I, Halinen MO, Rantanen T, et al. Differences in the diagnosis of myocardial infarction by troponin T compared with clinical and epidemiologic criteria. Am J Cardiol 2001;88:727-31. 9. Kontos MC, Fritz LM, Anderson FP, Tatum JL, Ornato JP, Jesse RL. Impact of the troponin standard on the prevalence of acute myocardial infarction. Am Heart J 2003;146:446-52. 10. Venge P, Johnston N, Lagerqvist B, Wallentin L, Lindahl B. Clinical and analytical performance of the Liaison cardiac troponin I assay in unstable coronary artery disease, and the impact of age on the definition of reference limits. A FRISC-II substudy. Clin Chem 2003;49:880 -6. 11. Venge P, Lagerqvist B, Diderholm E, Lindahl B, Wallentin L. Clinical performance of three cardiac troponin assays in patients with unstable coronary artery disease (a FRISC II substudy). Am J Cardiol. 2002;89:1035-41. 12. Apple FS, Wu AH, Mair J, Ravkilde J, Panteghini M, Tate J, et al. Future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome. Clin Chem. 2005;51:810-24. 13. Ilva T, Eriksson S, Lund J, Porela P, Mustonen H, Pettersson K, et al. Improved early risk stratification and diagnosis of myocardial infarction, using a novel troponin I assay concept. Eur J Clin Invest 2005;35:112-6. 14. Eriksson S, Junikka M, Alfthan H, Pettersson K. Do troponin autoantibodies negatively influence measurement of troponin T? [Abstract]. Clin Chim Acta 2005;355 (Suppl 1):S95.