Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 273–277
REVIEW ARTICLE
The mystifying nomenclature of cardiac troponin immunoassays
GIUSEPPE LIPPI Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy Abstract The laboratory assessment of cardiospecific troponins(s) represents the cornerstone for the diagnosis of acute coronary syndrome (ACS). Although troponin immunoassays are classified according to either analytical imprecision or percentage of measurable values in a presumably healthy population, it is rather clear that the nomenclature of commercial methods according to these systems of classification carries several drawbacks. The leading problems in classification according to imprecision are represented by the arbitrarity of optimal imprecision threshold, the uncertain correspondence between analytical performance and clinical outcomes and the improper use of terms, which has also been magnified by the lack of specific focus on this topic by regulating bodies such as the US Food and Drug Administration (FDA) and the European Union. Additional issues emerging from classification according to percentage of measurable values include the characterization of healthy population, the variation of values according to age, gender and race, as well as the influence of comorbidities. Considering that what really matters from a clinical standpoint is the clinical performance of the assay rather than the claimed analytical characteristics, it seems reasonable at this point in time to introduce a paradigm shift and gradually abandon the former analytical classification in favour of a different approach, preferable based on clinical outcomes. Key Words: Myocardial infarction, troponin, immunoassays, high-sensitivity, nomenclature
Cardiac troponin immunoassays in the diagnostics of acute coronary syndrome It is now well established that laboratory assessment of cardiac troponin(s), either troponin I (TnI) or troponin T (TnT), represents the cornerstone for diagnosis, management and prognostication of acute coronary syndrome (ACS), especially for patients with non-ST elevation myocardial infarction (NSTEMI) and unstable angina, wherein biomarker elevation in the context of a suggestive clinical scenario represents a virtually exclusive diagnostic feature [1,2]. The clinical use of cardiac troponin(s) immunoassays has now celebrated its 13th anniversary, considering that the first endorsement has been credited in the year 2000, when the European Society of Cardiology (ESC) and the American College of Cardiology (ACC) released a joint document for the redefinition of myocardial infarction (MI), supplying a clear indication that a diagnosis of an acute, evolving or recent MI is satisfied by a
typical rise (or gradual fall) of cardiospecific troponin(s), with at least one among ischemic symptoms, development of pathologic Q waves on the ECG, ECG changes indicative of ischemia (ST segment elevation or depression), or coronary artery intervention [3]. Since then, more than a decade has passed, MI has been further and slightly redefined in other guidelines [4–6] and – especially – cardiospecific troponin(s) immunoassays have been subjected to remarkable technical developments and analytical improvements culminated in the introduction of more (analytically) sensitive methods, conventionally defined as high-sensitivity (HS) immunoassays [7]. The basic assumption underlying the clinical use of a given cardiospecific troponin test is that its optimal imprecision, as defined by the use of the coefficient of variation [CV] at the 99th percentile of the upper reference limit (URL) of a presumably healthy population, should be equal or lower than 10% [3–6]. Around this standpoint, the various
Correspondence: Giuseppe Lippi, U.O. Diagnostica Ematochimica, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci, 14, 43126 - Parma, Italy. Tel: ⫹ 39 0521 703050. Fax: ⫹ 39 0521 703791. E-mail: [email protected], [email protected] (Received 25 June 2013 ; accepted 25 January 2014 ) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2014.888590
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immunoassays have been conventionally classified as ‘guidelines acceptable’ (when the CV at the 99th percentile URL is ⱕ 10%), ‘clinically usable’ (when the CV at the 99th percentile URL is comprised between 10 and 20%) or ‘not acceptable’ (when the CV at the 99th percentile URL is ⬎ 20%) [8]. Another widespread classification is that based on the percentage of measurable values below the 99th percentile URL of a presumably healthy population, so that ‘thirdgeneration-HS’ immunoassays are defined as those providing more than 95% of measurable values, ‘second-generation-HS’ are those providing 75–95% of measurable values, ‘first-generation-HS’ are those providing 50–75% of measurable values, and ‘contemporary-sensitive’ are those providing ⬍ 50% of measurable values [8]. It is now undeniable, however, that the nomenclature of the current cardiospecific troponin immunoassays according to these two systems of classification carries several drawbacks and mystifications that should be clearly acknowledged by both laboratory professionals and clinicians (Table I).
Classification according to analytical imprecision When assessing the optimal analytical imprecision of cardiospecific troponin(s) immunoassay, the metrics itself is a problem. Despite a growing consensus that a minimum change of ⬎ 20% in follow-up testing is required for diagnosing acute myocardial necrosis [6], the 10% CV imprecision at 99th percentile URL that has been originally advocated in 2000 by the ESC/ACC recommendations [3], and further reiterated in the following documents [4–6], does not appear to have a strong theoretical support, while it more seems a reasonable compromise between the analytical characteristics of laboratory immunoassays (in general), the pathophysiology of myocardial ischemic injury and the kinetics of troponin(s) release in blood of patients with ACS. By definition, the value of imprecision is thus strongly dependent upon the 99th percentile URL troponin concentration, and this may induce some manufacturers to artificially inflate the 99th percentile URL value to obtain much favourable imprecision data owing to the good relationship existing between the Table I. Mystifying nomenclature of cardiac troponin(s) immunoassay. Problems in classification according to analytical imprecision ✓ Arbitrarity of optimal imprecision threshold. ✓ Correspondence between analytical performance and clinical outcomes. ✓ Improper use of terms. Problems in classification according to percentage of measurable values ✓ Characterization of healthy population. ✓ Variation of values according to age, gender [and race]. ✓ Lack of standardization of troponin I immunoassays. ✓ Influence of comorbidities.
concentration of an analyte and the relative imprecision of the method within its range of linearity (i.e. the higher the concentration, the better the imprecision). This approach seems also a sort of vicious circle, wherein the 99th percentile limit is calculated on a presumably healthy population, and then the assay is classified according to the same principle, i.e. the percentage of healthy individuals with troponin values below such limit. We would all agree that this looks like ‘a dog chasing its own tail ’. It is also noteworthy that the clinical characterization of methods according to specific imprecision thresholds may be questionable. Venge and Lindahl recently showed that a ‘not acceptable’ immunoassay was equivalent or even superior in clinical terms (i.e. outcome prediction) to other methods classified as ‘clinically acceptable’ from an analytical perspective [9]. It is hence clear that troponin immunoassays should not be (only) classified on their analytical characteristics, but rather on the clinical performance, both in diagnostic and prognostic terms. Another actual issue is the improper use – often iniquitous – of terms in the diagnostic market. It is a matter of fact that some commercial immunoassays are labelled with odd definitions such as ‘ultra-sensitive’, ‘modified-sensitive’ or even ‘extrasensitive’, whereas other manufacturers add the term ‘HS’ to the name of the immunoassay despite the fact that the analytical characteristics only meet those of a ‘contemporary-sensitive’ method [10]. Although these practices are rather understandably driven by economic issues, i.e. for allowing participation of manufacturer(s) to tenders where HS methods (or similar) are specifically requested in the terms of contract, it is almost impossible to appreciate which is the real analytical significance of prefixes like ‘ultra’, ‘modified’ or ‘extra’, nor which is their reflection of clinical performance. Indeed, this is a further source of mystification and even dispute between laboratory professionals and clinicians, due to the widespread perception that the claims of manufacturers are not always verified once the tender has been adjudicated (e.g. ‘the term HS on the brand is itself a guarantee’), so that the clinical implementation of these methods will not ultimately meet the original expectations of cardiologists and emergency physicians. It is also noteworthy that the use of analytical specifications for labelling immunoassays is not without precedence (e.g. methods for measuring thyroid stimulating hormone or prostate specific antigen are currently classified according to their functional sensitivity), and regulating bodies such as the US Food and Drug Administration (FDA) and the European Union (for the European Conformity Mark) have been instrumental in labelling different generations of analytical methods. Unfortunately, these agencies have been silent regarding the nomenclature of troponin methods, thus causing uncontrolled marketing of troponin immunoassays.
Nomenclature of troponin immunoassays Classification according to percentage of measurable values The definition of reference ranges and diagnostic thresholds for a given laboratory parameter is always a challenging enterprise. According to the joint recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and the Clinical and Laboratory Standards Institute (CLSI) [11], the reference range is described as variation of measurements or values in a population of presumably healthy individuals, which should conventionally include at least 120 subjects. Here, at least three major drawbacks emerge. The appropriate definition of ‘healthy individual’ in the context of ACS diagnostics is anything but simple, since it can not be limited to the lack of suggestive preclinical and clinical information collected by means of short medical examinations and/or questionnaires, as currently advocated [11,12]. The lack of significant history of cardiovascular disease, risk factors or signs and symptoms of heart disease is technically inadequate to define that a person has no burden of cardiovascular disease, especially if one considers that the concentration of cardiac troponin(s) is increased in patients with stable coronary artery disease, especially when these biomarkers are measured using HS immunoassays [13]. It is hence clear that the most suitable approach to establish whether a reference population is really (‘cardiovascularly’) healthy, is to exclude the presence of atherosclerotic lesions by means of coronary angiogram or other invasive techniques, which is by far impractical, expensive and even unethical in this context. The substantial variation of troponin(s) values in the general – presumably healthy – population is another important issue that has prepotently emerged using more analytically sensitive techniques. More specifically, the troponin values of a presumably healthy population significantly increase with ageing, and are also higher in the male than in the female gender. After a healthy population has been partitioning according to age and gender, Giannoni et al. found that the concentration of HS TnT was less than 2 ng/L in healthy females aged less than 20 years, but approximated 30 ng/L in healthy males aged more then 71 years, thus exhibiting a more than 10-fold increase [14]. Normann et al. also recently showed that increased values of HS TnT are more frequent in the elderly for a higher prevalence of non-ACS conditions, and this may substantially impair the diagnostic performance of this biomarker for diagnosing NSTEMI [15]. The question as to whether specific cut-offs of cardiospecific troponin should be developed according to decades of age and gender appears hence pleonastic, but practically challenging, economically and
275
technically unaffordable. The existence of a genetic heterogeneity of cardiospecific TnT between European Americans and Blacks poses a further challenge, since partitioning of reference ranges and diagnostic thresholds according to ethnic origin may also be advisable [16]. Another important issue is the current lack of standardization of TnI immunoassays. As recently emphasized by Fred Apple, the various commercial methods for measuring TnI use a cocktail of antibodies that recognize different epitope regions in the sequence of the protein (the latest IFCC list of troponin assays is reported in reference [10]). Moreover, some protein moieties are more susceptible than others to physiological or pathological modifications, and exhibit heterogeneous sensitivity to a variety of interfering substances including rheumatoid factor, heterophile antibodies and autoantibodies, which thus cause a rather unpredictable impairment of immunoreactivity [17]. It is hence inevitable that standardization of TnI immunoassays will be much more challenging that determining and optimizing a primary or secondary reference material and is not likely to occur very soon [17]. The influence of comorbidities is a fourth important caveat in classifying troponins immunoassays according to the rate of measurable values in the general population. It is now clear that baseline troponin values may be increased by a kaleidoscope of acute and chronic disorders, including acute heart failure, myocarditis, cancer, liver and renal impairment, sepsis, acute pulmonary embolism or chronic pulmonary arterial hypertension [6,13], acute localized infections [18], as well as mild head injury [19], among others. It appears hence clear that the use of the conventional diagnostic cut-off in a patient with non-cardiac disorders and suspected ACS may be ambiguous, leading to a notably high rate of false positive results and – potentially – to unnecessary and expensive invasive testing. Even the use of the delta approach (i.e. observation of a significant rise of the biomarker during serial measurements) requires cautions in these patients, because turnover and clearance of cardiospecific troponins is impaired as a consequence of renal and liver disease, for example [14,20]. A potential solution to this problem is represented by the identification of universally agreed criteria for selection of reference population(s), that should be effective to harmonize the practices for identification of diagnostic cut-offs, and which should steadily define the overall number of subjects, the age and gender distribution, the principles used for assessing the ‘cardiovascular health status’ (e.g. physical examination, echocardiography, diagnostic imaging), the absence (or presence) of other comorbidities, along with the preferred statistical approach.
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Conclusions Due to coexistence of various ‘generations’ of commercial troponin immunoassays, their nomenclature is a major challenge for clinical laboratory professionals and users of troponin assays, a confusion that has been substantially fuelled by manufacturers by applying labels without substantiation to their assay in the hope of gaining market advantage to their assay over competitors. From a clinical standpoint, what really matters for diagnosis and prognostication of patients with suspected ACS is the clinical performance of the assay, rather than its claimed analytical characteristics. In the daily practice of emergency room and cardiology departments, it does not really make a difference whether an assay is ‘clinically usable’ or ‘not acceptable’, provided that the latter still enables us to achieve the desired (optimal) diagnostic performance. Accordingly, recent data strongly supports the evidence that the introduction of HS cardiac troponin assays leads to a modest to null increase in the overall incidence of MI [21,22]. At this point in time it appears thereby pleonastic to maintain an open debate around the use of one term rather than another for classifying the different methods, while it seems much more reasonable to abandon the former analytical classification in favour of a different approach, funded on well-defined clinical outcomes such as diagnostic efficiency using fixed cut-offs or serial sampling, as well as the predictive value of the individual test for identifying patients at risk of death or with a substantial likelihood of developing cardiac and extra-cardiac complications after an ACS, also including unstable angina [23]. Although this seems the ideal goal at this point in time, it is however far from being practically achieved, because head-to-head comparison of different troponin assays requires testing of identical patient samples with each single method and analytical platform. This is indeed a challenging enterprise, which would require active participation of manufacturers, something that can be hardly achieved especially for those companies that commercialize ‘guideline acceptable’ assays and would be thereby scarcely attracted by comparing their methods with those currently classified as ‘clinically usable’ or even ‘not acceptable’. It is also noteworthy that a gold standard, not relying on troponin values, should be established for defining clinical outcomes. In this perspective, modern imaging such as cardiac magnetic resonance imaging (MRI) might be a viable approach in this context [24]. Nevertheless, until such an ideal study can be concluded, characterization based on analytical attributes remains a necessary evil. The current crisis that plagues the economy of the in vitro diagnostic industry in several countries is another aspect that should be considered. In a
world of limited resources, where laboratories are increasingly invited to participate to large tenders for laboratory equipment in order to save money, the choice of one troponin immunoassay over another may become challenging, wherein the acquisition of specific methods and/or analytical platforms for only testing cardiac biomarkers is no longer sustainable for many national healthcare systems [25]. Declaration of interest: The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper.
References [1] Lippi G, Franchini M, Cervellin G. Diagnosis and management of ischemic heart disease. Semin Thromb Hemost 2013;39:202–13. [2] Casagranda I, Cavazza M, Clerico A, Galvani M, Ottani F, Zaninotto M, Biasucci LM, Cervellin G, Lenzi T, Lippi G, Plebani M, Tubaro M. Proposal for the use in emergency departments of cardiac troponins measured with the latest generation methods in patients with suspected acute coronary syndrome without persistent ST-segment elevation. Clin Chem Lab Med 2013;51:1727–37. [3] 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. [4] Thygesen K, Alpert JS, White HD; Joint ESC/ACCF/AHA/ WHF Task Force for the Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. Eur Heart J 2007;28:2525–38. [5] Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD; Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction. Third universal definition of myocardial infarction. Circulation 2012;126:2020–35. [6] Thygesen K, Mair J, Mueller C, Huber K, Weber M, Plebani M, Hasin Y, Biasucci LM, Giannitsis E, Lindahl B, Koenig W, Tubaro M, Collinson P, Katus H, Galvani M, Venge P, Alpert JS, Hamm C, Jaffe AS; Study Group on Biomarkers in Cardiology of the ESC Working Group on Acute Cardiac Care. How to use high-sensitivity cardiac troponins in acute cardiac care. Eur Heart J 2012;33: 2252–7. [7] Lippi G, Montagnana M, Aloe R, Cervellin G. Highly sensitive troponin immunoassays: navigating between the scylla and charybdis. Adv Clin Chem 2012;58:1–29. [8] Apple FS. A new season for cardiac troponin assays: it’s time to keep a scorecard. Clin Chem 2009;55:1303–6. [9] Venge P, Lindahl B. Cardiac troponin assay classification by both clinical and analytical performance characteristics: a study on outcome prediction. Clin Chem 2013;59:976–81. [10] Apple FS, Ler R, Murakami MM. Determination of 19 cardiac troponin I and T assay 99th percentile values from a common presumably healthy population. Clin Chem 2012;58:1574–81. [11] Siest G, Henny J, Gräsbeck R, Wilding P, Petitclerc C, Queraltó JM, Petersen PH. The theory of reference values: an unfinished symphony. Clin Chem Lab Med 2013;51: 47–64. [12] Plebani M, Lippi G. Personalized [laboratory] medicine: a bridge to the future. Clin Chem Lab Med 2013;51: 703–6.
Nomenclature of troponin immunoassays [13] Lippi G, Cervellin G. Identification of troponin determinants for improving its diagnostic performance in the emergency department. J Emerg Med 2012;43:e487–8. [14] Giannoni A, Giovannini S, Clerico A. Measurement of circulating concentrations of cardiac troponin I and T in healthy subjects: a tool for monitoring myocardial tissue renewal? Clin Chem Lab Med 2009;47:1167–77. [15] Normann J, Mueller M, Biener M, Vafaie M, Katus HA, Giannitsis E. Effect of older age on diagnostic and prognostic performance of high-sensitivity troponin T in patients presenting to an emergency department. Am Heart J 2012;164:698–705. [16] Yu B, Barbalic M, Brautbar A, Nambi V, Hoogeveen RC, Tang W, Mosley TH, Rotter JI, deFilippi CR, O’Donnell CJ, Kathiresan S, Rice K, Heckbert SR, Ballantyne CM, Psaty BM, Boerwinkle E; CARDIoGRAM Consortium. Association of genome-wide variation with highly sensitive cardiac troponin-T levels in European Americans and blacks: a meta-analysis from atherosclerosis risk in communities and cardiovascular health studies. Circ Cardiovasc Genet 2013;6:82–8. [17] Apple FS. Counterpoint: standardization of cardiac troponin I assays will not occur in my lifetime. Clin Chem 2012; 58:169–71. [18] Lippi G, Margapoti R, Aloe R, Cervellin G. Highly-sensitive troponin I in patients admitted to the emergency room with acute infections. Eur J Intern Med 2013;24:e57–8. [19] Lippi G, Dipalo M, Carbucicchio A, Aloe R, Benatti M, Cervellin G. The concentration of highly-sensitive troponin
[20]
[21]
[22]
[23]
[24]
[25]
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I is increased in patients with brain injury after mild head trauma. Int J Cardiol 2013;168:1617–8. Lippi G, Tessitore N, Montagnana M, Salvagno GL, Lupo A, Guidi GC. Influence of sampling time and ultrafiltration coefficient of the dialysis membrane on cardiac troponin I and T. Arch Pathol Lab Med 2008; 132:72–6. Reichlin T, Twerenbold R, Reiter M, Steuer S, Bassetti S, Balmelli C, Winkler K, Kurz S, Stelzig C, Freese M, Drexler B, Haaf P, Zellweger C, Osswald S, Mueller C. Introduction of high-sensitivity troponin assays: impact on myocardial infarction incidence and prognosis. Am J Med 2012;125:1205–13. Lippi G, Cervellin G. Assay characteristics and diagnostic improvement from contemporary to high-sensitivity troponin I immunoassays. Am J Med 2013;126:e9–e10. Reichlin T, Twerenbold R, Maushart C, Reiter M, Moehring B, Schaub N, Balmelli C, Rubini Gimenez M, Hoeller R, Sakarikos K, Drexler B, Haaf P, Osswald S, Mueller C. Risk stratification in patients with unstable angina using absolute serial changes of 3 high-sensitive troponin assays. Am Heart J 2013;165:371–8. Chan W, Ellims AH, Duffy SJ, Kaye DM, Taylor AJ. Principles, current status and clinical implications of ischaemic heart disease assessment by cardiac magnetic resonance imaging. Intern Med J 2012;42:7–17. Lippi G, Cervellin G. Choosing troponin immunoassays in a world of limited resources. J Am Coll Cardiol 2013;62:647–8.
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REVIEW ARTICLE
The mystifying nomenclature of cardiac troponin immunoassays
GIUSEPPE LIPPI Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy Abstract The laboratory assessment of cardiospecific troponins(s) represents the cornerstone for the diagnosis of acute coronary syndrome (ACS). Although troponin immunoassays are classified according to either analytical imprecision or percentage of measurable values in a presumably healthy population, it is rather clear that the nomenclature of commercial methods according to these systems of classification carries several drawbacks. The leading problems in classification according to imprecision are represented by the arbitrarity of optimal imprecision threshold, the uncertain correspondence between analytical performance and clinical outcomes and the improper use of terms, which has also been magnified by the lack of specific focus on this topic by regulating bodies such as the US Food and Drug Administration (FDA) and the European Union. Additional issues emerging from classification according to percentage of measurable values include the characterization of healthy population, the variation of values according to age, gender and race, as well as the influence of comorbidities. Considering that what really matters from a clinical standpoint is the clinical performance of the assay rather than the claimed analytical characteristics, it seems reasonable at this point in time to introduce a paradigm shift and gradually abandon the former analytical classification in favour of a different approach, preferable based on clinical outcomes. Key Words: Myocardial infarction, troponin, immunoassays, high-sensitivity, nomenclature
Cardiac troponin immunoassays in the diagnostics of acute coronary syndrome It is now well established that laboratory assessment of cardiac troponin(s), either troponin I (TnI) or troponin T (TnT), represents the cornerstone for diagnosis, management and prognostication of acute coronary syndrome (ACS), especially for patients with non-ST elevation myocardial infarction (NSTEMI) and unstable angina, wherein biomarker elevation in the context of a suggestive clinical scenario represents a virtually exclusive diagnostic feature [1,2]. The clinical use of cardiac troponin(s) immunoassays has now celebrated its 13th anniversary, considering that the first endorsement has been credited in the year 2000, when the European Society of Cardiology (ESC) and the American College of Cardiology (ACC) released a joint document for the redefinition of myocardial infarction (MI), supplying a clear indication that a diagnosis of an acute, evolving or recent MI is satisfied by a
typical rise (or gradual fall) of cardiospecific troponin(s), with at least one among ischemic symptoms, development of pathologic Q waves on the ECG, ECG changes indicative of ischemia (ST segment elevation or depression), or coronary artery intervention [3]. Since then, more than a decade has passed, MI has been further and slightly redefined in other guidelines [4–6] and – especially – cardiospecific troponin(s) immunoassays have been subjected to remarkable technical developments and analytical improvements culminated in the introduction of more (analytically) sensitive methods, conventionally defined as high-sensitivity (HS) immunoassays [7]. The basic assumption underlying the clinical use of a given cardiospecific troponin test is that its optimal imprecision, as defined by the use of the coefficient of variation [CV] at the 99th percentile of the upper reference limit (URL) of a presumably healthy population, should be equal or lower than 10% [3–6]. Around this standpoint, the various
Correspondence: Giuseppe Lippi, U.O. Diagnostica Ematochimica, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci, 14, 43126 - Parma, Italy. Tel: ⫹ 39 0521 703050. Fax: ⫹ 39 0521 703791. E-mail: [email protected], [email protected] (Received 25 June 2013 ; accepted 25 January 2014 ) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2014.888590
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G. Lippi
immunoassays have been conventionally classified as ‘guidelines acceptable’ (when the CV at the 99th percentile URL is ⱕ 10%), ‘clinically usable’ (when the CV at the 99th percentile URL is comprised between 10 and 20%) or ‘not acceptable’ (when the CV at the 99th percentile URL is ⬎ 20%) [8]. Another widespread classification is that based on the percentage of measurable values below the 99th percentile URL of a presumably healthy population, so that ‘thirdgeneration-HS’ immunoassays are defined as those providing more than 95% of measurable values, ‘second-generation-HS’ are those providing 75–95% of measurable values, ‘first-generation-HS’ are those providing 50–75% of measurable values, and ‘contemporary-sensitive’ are those providing ⬍ 50% of measurable values [8]. It is now undeniable, however, that the nomenclature of the current cardiospecific troponin immunoassays according to these two systems of classification carries several drawbacks and mystifications that should be clearly acknowledged by both laboratory professionals and clinicians (Table I).
Classification according to analytical imprecision When assessing the optimal analytical imprecision of cardiospecific troponin(s) immunoassay, the metrics itself is a problem. Despite a growing consensus that a minimum change of ⬎ 20% in follow-up testing is required for diagnosing acute myocardial necrosis [6], the 10% CV imprecision at 99th percentile URL that has been originally advocated in 2000 by the ESC/ACC recommendations [3], and further reiterated in the following documents [4–6], does not appear to have a strong theoretical support, while it more seems a reasonable compromise between the analytical characteristics of laboratory immunoassays (in general), the pathophysiology of myocardial ischemic injury and the kinetics of troponin(s) release in blood of patients with ACS. By definition, the value of imprecision is thus strongly dependent upon the 99th percentile URL troponin concentration, and this may induce some manufacturers to artificially inflate the 99th percentile URL value to obtain much favourable imprecision data owing to the good relationship existing between the Table I. Mystifying nomenclature of cardiac troponin(s) immunoassay. Problems in classification according to analytical imprecision ✓ Arbitrarity of optimal imprecision threshold. ✓ Correspondence between analytical performance and clinical outcomes. ✓ Improper use of terms. Problems in classification according to percentage of measurable values ✓ Characterization of healthy population. ✓ Variation of values according to age, gender [and race]. ✓ Lack of standardization of troponin I immunoassays. ✓ Influence of comorbidities.
concentration of an analyte and the relative imprecision of the method within its range of linearity (i.e. the higher the concentration, the better the imprecision). This approach seems also a sort of vicious circle, wherein the 99th percentile limit is calculated on a presumably healthy population, and then the assay is classified according to the same principle, i.e. the percentage of healthy individuals with troponin values below such limit. We would all agree that this looks like ‘a dog chasing its own tail ’. It is also noteworthy that the clinical characterization of methods according to specific imprecision thresholds may be questionable. Venge and Lindahl recently showed that a ‘not acceptable’ immunoassay was equivalent or even superior in clinical terms (i.e. outcome prediction) to other methods classified as ‘clinically acceptable’ from an analytical perspective [9]. It is hence clear that troponin immunoassays should not be (only) classified on their analytical characteristics, but rather on the clinical performance, both in diagnostic and prognostic terms. Another actual issue is the improper use – often iniquitous – of terms in the diagnostic market. It is a matter of fact that some commercial immunoassays are labelled with odd definitions such as ‘ultra-sensitive’, ‘modified-sensitive’ or even ‘extrasensitive’, whereas other manufacturers add the term ‘HS’ to the name of the immunoassay despite the fact that the analytical characteristics only meet those of a ‘contemporary-sensitive’ method [10]. Although these practices are rather understandably driven by economic issues, i.e. for allowing participation of manufacturer(s) to tenders where HS methods (or similar) are specifically requested in the terms of contract, it is almost impossible to appreciate which is the real analytical significance of prefixes like ‘ultra’, ‘modified’ or ‘extra’, nor which is their reflection of clinical performance. Indeed, this is a further source of mystification and even dispute between laboratory professionals and clinicians, due to the widespread perception that the claims of manufacturers are not always verified once the tender has been adjudicated (e.g. ‘the term HS on the brand is itself a guarantee’), so that the clinical implementation of these methods will not ultimately meet the original expectations of cardiologists and emergency physicians. It is also noteworthy that the use of analytical specifications for labelling immunoassays is not without precedence (e.g. methods for measuring thyroid stimulating hormone or prostate specific antigen are currently classified according to their functional sensitivity), and regulating bodies such as the US Food and Drug Administration (FDA) and the European Union (for the European Conformity Mark) have been instrumental in labelling different generations of analytical methods. Unfortunately, these agencies have been silent regarding the nomenclature of troponin methods, thus causing uncontrolled marketing of troponin immunoassays.
Nomenclature of troponin immunoassays Classification according to percentage of measurable values The definition of reference ranges and diagnostic thresholds for a given laboratory parameter is always a challenging enterprise. According to the joint recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and the Clinical and Laboratory Standards Institute (CLSI) [11], the reference range is described as variation of measurements or values in a population of presumably healthy individuals, which should conventionally include at least 120 subjects. Here, at least three major drawbacks emerge. The appropriate definition of ‘healthy individual’ in the context of ACS diagnostics is anything but simple, since it can not be limited to the lack of suggestive preclinical and clinical information collected by means of short medical examinations and/or questionnaires, as currently advocated [11,12]. The lack of significant history of cardiovascular disease, risk factors or signs and symptoms of heart disease is technically inadequate to define that a person has no burden of cardiovascular disease, especially if one considers that the concentration of cardiac troponin(s) is increased in patients with stable coronary artery disease, especially when these biomarkers are measured using HS immunoassays [13]. It is hence clear that the most suitable approach to establish whether a reference population is really (‘cardiovascularly’) healthy, is to exclude the presence of atherosclerotic lesions by means of coronary angiogram or other invasive techniques, which is by far impractical, expensive and even unethical in this context. The substantial variation of troponin(s) values in the general – presumably healthy – population is another important issue that has prepotently emerged using more analytically sensitive techniques. More specifically, the troponin values of a presumably healthy population significantly increase with ageing, and are also higher in the male than in the female gender. After a healthy population has been partitioning according to age and gender, Giannoni et al. found that the concentration of HS TnT was less than 2 ng/L in healthy females aged less than 20 years, but approximated 30 ng/L in healthy males aged more then 71 years, thus exhibiting a more than 10-fold increase [14]. Normann et al. also recently showed that increased values of HS TnT are more frequent in the elderly for a higher prevalence of non-ACS conditions, and this may substantially impair the diagnostic performance of this biomarker for diagnosing NSTEMI [15]. The question as to whether specific cut-offs of cardiospecific troponin should be developed according to decades of age and gender appears hence pleonastic, but practically challenging, economically and
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technically unaffordable. The existence of a genetic heterogeneity of cardiospecific TnT between European Americans and Blacks poses a further challenge, since partitioning of reference ranges and diagnostic thresholds according to ethnic origin may also be advisable [16]. Another important issue is the current lack of standardization of TnI immunoassays. As recently emphasized by Fred Apple, the various commercial methods for measuring TnI use a cocktail of antibodies that recognize different epitope regions in the sequence of the protein (the latest IFCC list of troponin assays is reported in reference [10]). Moreover, some protein moieties are more susceptible than others to physiological or pathological modifications, and exhibit heterogeneous sensitivity to a variety of interfering substances including rheumatoid factor, heterophile antibodies and autoantibodies, which thus cause a rather unpredictable impairment of immunoreactivity [17]. It is hence inevitable that standardization of TnI immunoassays will be much more challenging that determining and optimizing a primary or secondary reference material and is not likely to occur very soon [17]. The influence of comorbidities is a fourth important caveat in classifying troponins immunoassays according to the rate of measurable values in the general population. It is now clear that baseline troponin values may be increased by a kaleidoscope of acute and chronic disorders, including acute heart failure, myocarditis, cancer, liver and renal impairment, sepsis, acute pulmonary embolism or chronic pulmonary arterial hypertension [6,13], acute localized infections [18], as well as mild head injury [19], among others. It appears hence clear that the use of the conventional diagnostic cut-off in a patient with non-cardiac disorders and suspected ACS may be ambiguous, leading to a notably high rate of false positive results and – potentially – to unnecessary and expensive invasive testing. Even the use of the delta approach (i.e. observation of a significant rise of the biomarker during serial measurements) requires cautions in these patients, because turnover and clearance of cardiospecific troponins is impaired as a consequence of renal and liver disease, for example [14,20]. A potential solution to this problem is represented by the identification of universally agreed criteria for selection of reference population(s), that should be effective to harmonize the practices for identification of diagnostic cut-offs, and which should steadily define the overall number of subjects, the age and gender distribution, the principles used for assessing the ‘cardiovascular health status’ (e.g. physical examination, echocardiography, diagnostic imaging), the absence (or presence) of other comorbidities, along with the preferred statistical approach.
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Conclusions Due to coexistence of various ‘generations’ of commercial troponin immunoassays, their nomenclature is a major challenge for clinical laboratory professionals and users of troponin assays, a confusion that has been substantially fuelled by manufacturers by applying labels without substantiation to their assay in the hope of gaining market advantage to their assay over competitors. From a clinical standpoint, what really matters for diagnosis and prognostication of patients with suspected ACS is the clinical performance of the assay, rather than its claimed analytical characteristics. In the daily practice of emergency room and cardiology departments, it does not really make a difference whether an assay is ‘clinically usable’ or ‘not acceptable’, provided that the latter still enables us to achieve the desired (optimal) diagnostic performance. Accordingly, recent data strongly supports the evidence that the introduction of HS cardiac troponin assays leads to a modest to null increase in the overall incidence of MI [21,22]. At this point in time it appears thereby pleonastic to maintain an open debate around the use of one term rather than another for classifying the different methods, while it seems much more reasonable to abandon the former analytical classification in favour of a different approach, funded on well-defined clinical outcomes such as diagnostic efficiency using fixed cut-offs or serial sampling, as well as the predictive value of the individual test for identifying patients at risk of death or with a substantial likelihood of developing cardiac and extra-cardiac complications after an ACS, also including unstable angina [23]. Although this seems the ideal goal at this point in time, it is however far from being practically achieved, because head-to-head comparison of different troponin assays requires testing of identical patient samples with each single method and analytical platform. This is indeed a challenging enterprise, which would require active participation of manufacturers, something that can be hardly achieved especially for those companies that commercialize ‘guideline acceptable’ assays and would be thereby scarcely attracted by comparing their methods with those currently classified as ‘clinically usable’ or even ‘not acceptable’. It is also noteworthy that a gold standard, not relying on troponin values, should be established for defining clinical outcomes. In this perspective, modern imaging such as cardiac magnetic resonance imaging (MRI) might be a viable approach in this context [24]. Nevertheless, until such an ideal study can be concluded, characterization based on analytical attributes remains a necessary evil. The current crisis that plagues the economy of the in vitro diagnostic industry in several countries is another aspect that should be considered. In a
world of limited resources, where laboratories are increasingly invited to participate to large tenders for laboratory equipment in order to save money, the choice of one troponin immunoassay over another may become challenging, wherein the acquisition of specific methods and/or analytical platforms for only testing cardiac biomarkers is no longer sustainable for many national healthcare systems [25]. Declaration of interest: The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper.
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