Info

"Modified from ref. 78.

"Modified from ref. 78.

future markers of myocardial necrosis

Accurate recognition of myocardial necrosis is important for achieving an accurate diagnosis but, more important, for assessing risk and directing appropriate therapy that improves clinical outcome. Most investigators agree that future efforts in diagnostics and therapeutics should focus on that phase of myocardial ischemia during which injury is reversible so that myocardial salvage can be maximized. Multimarker strategies using established and new biomarkers for risk stratification and clinical decision making have potential for improving the outcomes of patients with ACS (74).

In the context ofmyocardial necrosis, cTnl and cTnT measurements are fundamental for the diagnosis of MI by definition (6). Therefore, identifying biomarkers that will improve the tissue specificity and clinical performance possible with cardiac troponin for MI is a significant undertaking. Advances based on the detection ofspecific patterns ofdegrada-tion/modification of cardiac troponin that might improve the early detection of myocar-dial necrosis or provide information regarding the specific mechanism of myocardial injury would be extremely valuable. In addition, continued enhancements to the analytic performance of assays for cardiac troponin will enable reliable detection of necrosis at lower concentrations.

Two additional efforts may also improve the ability to detect and diagnose myocardial necrosis: proteomics and modeling decision aids. These areas are related, but decision aids will be broadened beyond biomarkers to combine objective clinical indicators such as classification of ECG findings, history, age, and gender.

Modeling Computer Aids

The rationale behind the use of multiple marker strategies is that each biomarker provides independent and additive information for the diagnosis of MI and risk assessment. Biomarkers are continuous variables, yet diagnostic interpretation generally utilizes cut points that force data into a binary scenario. One notion that adds value to modeling the combination of markers is that biomarkers that have not crossed the decision threshold for "positive" may contain useful information. In addition, modeling approaches must yield a single straightforward output, rather than several results that must be combined by the user.

One early approach to modeling involved a multimarker strategy in the context of diagnosing MI utilizing myoglobin, CK-MB, cTnI, and B-type natriuretic peptide (BNP) measurements to develop a multivariable index function (75). This strategy was piloted in a cohort that included 210 patients with noncardiac chest pain and 105 patients with MI; all samples were collected within 10 h of the onset of symptoms (76). Table 4 shows the diagnostic performance of each biomarker and that performance of the panel was substantially better than any ofthe biomarkers alone. However, there are numerous caveats with this study, including prevalence of disease, timing of samples, definition of MI, and quality of each component biomarker. Nevertheless, it is interesting that the data indicate promise for the concept of combining multiple markers and improving the ability to diagnose myocardial necrosis.

conclusion

Utilization of biomarkers for the assessment of myocardial necrosis has had a rich history that spans nearly half a century. The earliest biomarkers, such as AST and LD, have fallen out of use with the development of assays for CK-MB and particularly cardiac troponin. The current order of diagnostic value is cTnT or cTnl > CK-MB mass > CK-MB activity > total CK (6,39). Myoglobin must be considered a niche biomarker that may lack sensitivity for the diagnosis of MI in the future but appears to have value for risk stratification. The quality of assays for biomarkers varies substantially, so assay choice must be a collaboration between laboratory and clinical staff. The future will undoubtedly involve automated multimarker strategies that will aim not only at the diagnosis of MI, but also the probability of ACS. In addition, assessing risk of future events in addition to defining the diagnosis will be included in the new paradigm. Biomarkers are absolutely fundamental to the (re)definition and diagnosis of MI, but it must be noted that MI is a clinical diagnosis and that a setting of myocardial ischemia must be established.

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Analytical Issues for Clinical Use of Cardiac Troponin

Contents

Introduction

Release of Troponin After Myocardial Injury Quality Specifications for Analytic Assays AMI Cutoff Concentrations and Assay Imprecision for Troponin Cardiac Troponin Cutoff for Risk Stratification Pre- and Postanalytic Variables Conclusion References

Summary

Guidelines jointly developed by the European Society of Cardiology and the American College of Cardiology have established cardiac troponin (T or I) as the biomarker of choice for the diagnosis of acute coronary syndromes (ACSs) and risk stratification of patients who present with ischemic symptoms suggestive of ACS. Despite these international guidelines, a number of analytic issues have slowed the acceptance and implementation ofthis test worldwide. Regarding precision, analytic sensitivity, and specificity, the performance of commercial assays is variable. Moreover, there is a lack of assay standardization for troponin I results among commercial assays. The release of a standard reference material should initiate the process of assay harmonization. It is important that clinicians be familiar with the analytic performance (i.e., level of 10% coefficient of variation) and appropriate cut points (99th percentile and/or evidence-based decision limits) for all troponin assays used (both laboratory-based and point of care) at their institution. In time, it is hoped that all troponin assays will be standardized and exhibit similar performance to each other, as is the case for the majority of other clinical laboratory analytes today.

Key Words: Troponin; imprecision; interference; harmonization; false positive.

From: Contemporary Cardiology: Cardiovascular Biomarkers: Pathophysiology and Disease Management Edited by: David A. Morrow © Humana Press Inc., Totowa, NJ

introduction

Guidelines that were jointly developed by the European Society of Cardiology (ESC) and the American College ofCardiology (ACC) have established cardiac troponin T (cTnT) or cTnI as the biomarker of choice for the diagnosis of acute coronary syndromes (ACSs) and risk stratification of patients who present with ischemic symptoms suggestive of ACS

(I). Despite the international guidelines on the use of troponin by cardiologists, emergency department (ED) physicians, and clinical laboratory scientists (1-3), there are a number of analytic issues that have slowed the acceptance and implementation of this test worldwide. The performance of commercial assays is quite variable regarding precision, analytic sensitivity, and specificity. Furthermore, there is a lack of assay standardization for troponin I results among commercial assays. Recently, the National Institute of Standards and Technology (NIST) released a standard reference material that should initiate the process of assay harmonization. These problems in cardiac troponin have led to confusion as to the proper cutoff concentrations that should be used in routine clinical practice and in clinical trials. In addition, the reagent costs for cardiac troponin are higher than for other older markers such as creatine kinase (CK) and the CK-MB isoenzyme. In this chapter, these important issues are discussed with reference to their impact on the clinical interpretation of test results.

release of troponin after myocardial injury cTnT and cTnI are part of a complex of three regulatory proteins that includes troponin C. This ternary complex is bound to the thin filament of striated muscle and regulates the contraction of actin and myosin filaments. Following myocardial damage, the troponin T-I-C complex is gradually released into the blood, where it degrades into the binary IC complex and free troponin T, the predominant forms of cardiac troponin in the circulation (Fig. 1) (4). In addition, a small amount of free troponin T and I exists within the cytoplasm and appears within the initial hours after the onset of cardiac damage (5). In Fig. 2, peak A illustrates the biphasic release pattern of cardiac troponin from damaged myocytes. Troponin I can also exist in phosphorylated forms and in oxidized/reduced forms. The degree of phosphorylation affects cardiac myocyte contractility in normal and failing hearts (6). Once in the blood, the various troponin forms undergo further degradation into smaller molecular weight fragments by serum proteolytic enzymes (see next section) (7).

An ongoing controversy is whether or not troponin can be released following reversible ischemia. Cardiac markers that are large in molecular weight, such as CK-MB (84 kDa) and lactate dehydrogenase (135 kDa), are released into the blood only after irreversible injury. However, some investigators have questioned whether cTnT and cTnI, at 37 and 24 kDa, respectively, can be released during ischemia (8,9). Although these proteins are themselves too large to traverse across viable cell membranes, studies have shown that troponin can undergo in situ degradation into smaller fragments during prolonged periods of myocardial ischemia (10). Using denaturing Western blot analysis, troponin fragments appear in the blood of patients with acute coronary syndromes within the first hour after the onset of chest pain, well before detection by commercial troponin assays

(II). Others have suggested that the release of troponin following reversible injury is not likely (12). Although the debate is intellectually interesting, most agree that a definitive answer regarding this area of question will be difficult, if not impossible, to prove (13).

Fig. 1. The troponin T-I-C complex of the thin filament is released from damaged myocytes into various molecular forms. The T-I-C complex appears in the blood and degrades first into the I-C complex (the predominant form of cTnI in blood) and free TnT. The I-C complex further degrades into free troponin subunits and fragments of intact subunits (both immunoreactive and nonimmuno-reactive). A small proportion of troponin I and T and fragments is also found in the cytoplasm. Troponin I can exist in either oxidized or reduced forms, or up to two phosphorylations.

Fig. 1. The troponin T-I-C complex of the thin filament is released from damaged myocytes into various molecular forms. The T-I-C complex appears in the blood and degrades first into the I-C complex (the predominant form of cTnI in blood) and free TnT. The I-C complex further degrades into free troponin subunits and fragments of intact subunits (both immunoreactive and nonimmuno-reactive). A small proportion of troponin I and T and fragments is also found in the cytoplasm. Troponin I can exist in either oxidized or reduced forms, or up to two phosphorylations.

3 4 5 6 7 Days After Onset of AMI Fig. 2. Biphasic release pattern of cardiac troponin and relationship of cutoff concentrations. Peak A, myocardial infarction (MI); peak B, unstable angina. AMI, acute myocardial infarction. (Modified from ref. 3.)

Fig. 3. (A) Two-site ("sandwich") immunoassays. The capture antibody is immobilized to a solid surface (e.g., test tube wall). The detection antibody is labeled with a detecting enzyme, fluorophore, or chemiluminescent tag. (B) The presence of analytes (circles) enables capture. Unbound labeled antibody is removed. (C) The signal of the labeled antibody (left) is generated and measured. The concentration of the analyte is extrapolated from a calibration curve (right). (D) Mechanism for interferences owing to the presence of unusual antibodies. Heterophilic or human antimouse antibodies bind to both the capture and detection antibodies, producing an analytic signal in the absence of the analyte. (Modified from ref. 14).

Fig. 3. (A) Two-site ("sandwich") immunoassays. The capture antibody is immobilized to a solid surface (e.g., test tube wall). The detection antibody is labeled with a detecting enzyme, fluorophore, or chemiluminescent tag. (B) The presence of analytes (circles) enables capture. Unbound labeled antibody is removed. (C) The signal of the labeled antibody (left) is generated and measured. The concentration of the analyte is extrapolated from a calibration curve (right). (D) Mechanism for interferences owing to the presence of unusual antibodies. Heterophilic or human antimouse antibodies bind to both the capture and detection antibodies, producing an analytic signal in the absence of the analyte. (Modified from ref. 14).

quality specifications for analytic assays

All assays for cardiac troponin require the use of immunoassay techniques. Two-site "sandwich" immunoassays make use of a capture antibody to bind to the analyte of interest, and a labeled antibody that is used to determine the quantity that is bound to the capture antibody (Fig. 3A-C). The concentration is determined from a calibration curve (plot of the analytic signal vs concentration of calibrators). An immunoassay analyzer is used to measure these proteins. Point-of-care (POC) testing devices offer an alternative to a large analyzer/central laboratory testing approach (see Chapter 32). Because the analytic performance of assays for cardiac markers can have a major impact on how results are interpreted, the International Federation of Clinical Chemistry Committee on Standardization of Markers of Cardiac Damage (IFCC C-SMCD) developed quality specifications for cardiac troponin assays (15). Table 1 summarizes the major recommendations made by this committee.

Table 1

Quality Specification for Cardiac Troponin Assays

Antibody specificity: Antibodies in troponin assays should recognize the stable part of the molecule and should not be affected by complex formation or in vivo modifications. Calibration: The natural and native troponin ternary complexes should be used. Sample dilution: There should be no matrix effects for immunoassays, as determined by superimposable curves on dilution of high samples. Assay specificity: There should be no crossreactivity with heterophilic, rheumatoid factor, or human antianimal antibodies. Documentation of preanalytic factors: The types of blood collection tube and in vitro stability at different temperatures should be documented.

Adapted from ref. 36.

Epitope Specificity of Commercial Antibodies

The performance of immunoassays is greatly dependent on the reactivity of the antibodies used toward the epitopes of the targeted proteins. Proper selection of antibodies is particularly important for troponin assays, because blood from patients with ACS contains a variety of different forms of troponin (Fig. 1). Whereas some assays produced an equimolar response to these forms, others produced a higher signal to the binary and ternary troponin forms (16). Thus, a tight linear regression of troponin I results from human patients was not achieved with a comparison of early commercial troponin assays (e.g., r = 0.811 for Beckman Access vs Stratus) (17).

The primary sites of in situ and in vitro degradation for cTnT and cTnI are the C- and N-terminal sequences. These low-molecular-weight fragments are rapidly cleared from the circulation by glomerular filtration. As a consequence, assays that use antibodies directed toward the central stable portion of the molecule will exhibit greater relative increases in concentration over time and a longer duration of elevation (Fig. 4) (18). Antibodies directed toward the fragments will have a limited window of detectability. However, if troponin is released during reversible ischemia, development of assays directed to the N-and C-terminal fragments (the unstable parts of the molecule) may have added clinical utility.

Assay Standardization

A major issue for cTnl assays is the current lack of industry standardization among commercial assays. Although this is not a problem for troponin T assay, because only one manufacturer (Roche) has the intellectual property rights for use of this test, it is an issue for other cardiac markers such as myoglobin and CK-MB (19).

Clinical Implications of Lack of Standardization

Figure 5 illustrates the extent of the problem for cTnl regarding the lack of standardization. The data are from the College ofAmerican Pathologists 2004 Proficiency Survey (20). Although each of the commercial assays demonstrates linearity between individual concentrations and the zero point (demonstrating minimal offset bias), there is substantial proportional bias between assays, as recognized by the slope of each line against an arbitrarily selected predicate assay. Using results of assays that produce the lowest and highest cTnl results (Triage and AxSYM, respectively), the slopes differ by a factor of nearly 100 to 1, resulting in very different reported values for the same sample. For

Fig. 4. Effect of antibody selection on measurement of cardiac troponin. (Top) Epitopes to four antibodies to the intact primary amino acid sequence of cTnl are shown. After troponin is released from the blood, it undergoes degradation at the C-terminal and then N-terminal sequences. These smaller peptides are rapidly removed from the circulation. (Bottom) Dual-site troponin assay using various combinations of antibodies. Assay AB3-4 shows a rapid return to baseline levels, owing to removal ofthe C-terminal fragment; assay AB1-2 shows an intermediate return to baseline, owing to removal of the N-terminal fragment; and assay AB2-3 shows a prolonged return to baseline, because the central portion of the peptide is the most stable.

Fig. 4. Effect of antibody selection on measurement of cardiac troponin. (Top) Epitopes to four antibodies to the intact primary amino acid sequence of cTnl are shown. After troponin is released from the blood, it undergoes degradation at the C-terminal and then N-terminal sequences. These smaller peptides are rapidly removed from the circulation. (Bottom) Dual-site troponin assay using various combinations of antibodies. Assay AB3-4 shows a rapid return to baseline levels, owing to removal ofthe C-terminal fragment; assay AB1-2 shows an intermediate return to baseline, owing to removal of the N-terminal fragment; and assay AB2-3 shows a prolonged return to baseline, because the central portion of the peptide is the most stable.

example, for a sample reported as 1.0 ng/mL with the Dimension assay, results with other assays will vary from <0.2 with the Triage to >2.0 with the AxSYM. Table 2 provides the reasons for the lack of concordance between assays. The most influential reason is the fact that different manufacturers have used dif-ferent calibrator materials and value assignment of calibrator concentrations.

The lack of assay standardization poses a major problem when interpreting results generated from different assays, such as when a patient is transferred from another hospital whose laboratory uses a different cTnl assay. Confusion may also occur if the same hospital uses different testing platforms, such as a POC assay when the patient is in the ED followed by a central laboratory assay when the patient is admitted to the coronary care unit. For risk stratification of patients with ACS, differences in cTnl cutoff concentrations make it very difficult to determine the proper cutoff concentrations for one assay when the published data from clinical studies use a different cTnl assay.

Development of Reference Material for cTnI

To address the standardization issue, the American Association for Clinical Chemistry (AACC) established The Troponin I Standardization Committee (21). This committee obtained recombinant and heart-purified cTnI materials in the T-I-C and I-C complex forms, and free cTnI. Candidate reference materials (cRMs) were characterized for purity by liquid chromatography/mass spectrometry (LC/MS), and the cTnI concentrations were assigned by a combination of amino acid analysis and LC/MS. cRMs were evaluated to

Dade Dimension HM

Fig. 5. Lack of standardization for cTnl assays. Data are from the 2004 College of American Pathologists CAR-A Survey for Cardiac Markers (see ref. 20). Although each commercial assay (y-axis) demonstrates linearity for the two survey materials, they differ in the slope ("s") of the line relative to the Dimension HM assay (arbitrarily selected as the predicate x-axis). *First-generation assay, no longer available.

Table 2

Reasons for Lack of Concordance Among Cardiac Troponin Assays

Lack of standardization of the calibrating materials Differences in the specificity of the antibodies used in the assays

Variability in the various forms of troponin found in blood and the reactivity of antibodies to these forms

Differences in the analytic performance of assays with particular reference to analytic sensitivity and assay imprecision determine whether they had acceptably low matrix-associated variations ("commuta-bility") when diluted in human serum or a suitable diluent selected by the manufacturer, and whether they could be used as calibrators to produce identical results for different cTnl assays ("harmonization"). To select the best material, each participating manufacturer of troponin I assays was given cRMs and instructed to calibrate their analyzers using these materials, and then measure serum pools prepared by the committee containing varying concentrations of cTnl. Based on three round-robin cycles of testing, the AACC standardization committee determined that the CIT ternary complex was most commut-able. Using manufacturer-specified calibrations, results of the serum pools produced a variability of 90% among results of different assays. This variability was reduced by seven- to fivefold to 12% for these same assays when the cRM troponin CIT complex was used as a calibrator (22). The NIST has certified a Standard Reference Material based on the CIT complex (SRM #2921), and it is available at www.nist.gov. One manufacturer (Abbott) has released an improved cTnI assay that is calibrated to the new NIST standard. Although other manufacturers have no obligation to use this material, many are in the process of reformulating their assays. The availability of a standard reference material will not enable complete standardization among assays, because of the variability of epitopes targeted by antibodies used in the assays. However, results from individual patients using standardized assays will differ only by a small percentage, instead of orders of magnitude, as is the case today.

Assay Interferences

All antibody-based assays are subjected to interferences owing to the presence of unusual antibodies such as heterophiles and human antianimal antibodies. As shown in Fig. 3D, these antibodies recognize and bind to antitroponin antibodies, thereby mimicking the analyte itself. Several case reports have documented this problem (23,24). There has been increasing attention by manufacturers of troponin I assays to reformulate their assays, but the incidence of false positive results has not been eliminated (25). Although less commonly encountered, false-negative results can also occur (26).

Repeated testing showing the absence of a rise and fall characteristic of acute myocardial injury can be very helpful in suggesting the presence of an interfering substance. Reversible interference such as that from fibrin strands resulting from incomplete isolation of serum ought to resolve completely with repeat testing using appropriate sample handling (e.g., centrifugation). Testing for heterophile antibodies may be conducted by a laboratory when an interfering antibody is strongly suspected.

ami cutoff concentrations and assay imprecision for troponin

For the majority of clinical chemistry analytes, a reference range can be established, because the analyte is present in measurable concentrations in healthy individuals. The reference range is determined by measuring the analyte in a cohort of subjects who are free from the disease in question. Ideally, the healthy subjects should be matched to the targeted disease group regarding age, gender, race, and other factors. The reference range for two-tailed tests (i.e., clinical significance for both low and high results) is calculated by the mean ± 2 SDs if the distribution of results is parametric, or by the central 95% of results if the distribution is nonparametric. Because there is no significance for low cardiac marker results, the reference range can be computed as the upper 97.5% of healthy individuals using a one-tailed test.

In contrast to the majority of clinical chemistry analytes, cutoff concentrations for cardiac markers such as CK-MB and myoglobin were established on the basis of clinical criteria for the diagnosis of MI and not from a cohort of healthy individuals. Using receiver operating characteristic (ROC) curve analysis, which plots clinical sensitivity vs 1 - specificity, the optimum cutoff concentrations were determined at an analyte value that best discriminated between subjects with disease (i.e., AMI) and those without disease (Fig. 6). The latter category consisted of patients who presented to the ED with chest pain owing to a noncardiac etiology or who had cardiac disease that was not ruled out for AMI. Patients with unstable angina were considered to be in the non-AMI group, despite a minor increase in cardiac biomarker results. The cutoff concentrations were therefore higher than the 97.5% of a cardiac-healthy population.

The notion that biomarkers were only useful for diagnostic purposes changed with the demonstration that troponin was more sensitive than CK-MB for the detection of MI. Clinical trials showed that troponin could also be used for risk stratification of patients who were previously ruled out for AMI by the older biomarkers. The term minor myocardial damage was initially coined to refer to troponin concentrations that were between

Fig. 6. ROC curve analysis plotted from distribution of cardiac marker results in disease vs a healthy population. CAD, coronary artery disease.

cTnl concentration (ng/ml_)

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