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a Modified from ref. 39.

a Modified from ref. 39.

Fig. 5. Structure of troponin complex and contraction of striated muscle.

are still useful for timing the occurrence of MI, detecting reinfarction, and sizing the extent of infarction (35).

Cardiac Troponin

The Troponin Complex

The troponin complex has three protein subunits and is located on the thin filament of striated muscle (Fig. 5). The three subunits are troponin T, a binding protein that attaches the troponin complex to tropomyosin; troponin I, which modulates the interaction of actin and myosin by acting as an inhibitor of actomyosin adenosine triphosphatase activity; and troponin C, the calcium-binding subunit of the troponin complex. After stimulation for contraction, excess calcium enters into cells and binds to troponin C. This binding causes a change in the conformation of tropomyosin, exposing the binding site on actin and actuating cross bridging; the result is muscle contraction. Troponin C has an identical amino acid sequence in both skeletal and cardiac tissues and, thus, has no potential as a cardiac-specific marker. However, troponin T and troponin I have different isoforms in cardiac and skeletal muscle, encoded by separate genes, and, consequently, have different amino acid sequences (9). The respective cardiac isoforms of cTnT and cTnI allow production of antibodies that exclusively recognize these myocardial-specific proteins. To date, cTnT and cTnI release has not been attributed to a tissue source other than myocardium, and except for rare analytical false positives, detection of cTnT or cTnI in the blood is indicative of heart injury (7).

Release After Necrosis cTnT and cTnI are structural biomarkers of cardiac necrosis whose kinetics require several hours after the onset of acute ischemia before they can be detected (Fig. 4). For this reason, cTnT and cTnI are not considered early biomarkers of necrosis. High diagnos tic sensitivity and specificity require specimen collection at patient presentation, 6-9 h later, and at 12-24 h if clinical suspicion is high and earlier results are negative (6). The increase in concentration of cTnl and cTnT after necrosis is prolonged compared with that of other biomarkers of necrosis (Fig. 4); this time course enables the diagnosis of MI many days after the acute event. The exact pattern of cTnT and cTnl release varies among individuals and is unpredictable; therefore, cTnT and cTnl may be less useful for assessing reocclusion or for infarct sizing compared to CK-MB mass (36).

Circulating Forms

Although the nature of the troponin complex released after myocardial injury is not fully characterized, several reports suggest that a large portion of troponin I enters into the circulation first as TnC-cTnT-cTnI and cTnI-TnC complexes, with only a small portion of troponin I circulating in a free form (43,44). Studies have examined degradation of the cardiac troponins both inside the cell and in circulation (43,44). The release forms of cTnl and cTnT after degradation in circulation are incompletely understood at present but may have clinical importance. Currently, release of cTnT and cTnl and detection using commercially available assays is a surrogate for myocardial cell death in the setting of acute cardiac ischemia (6).

The International Federation of Clinical Chemistry (IFCC) Committee for Standardization of Cardiac Laboratory Markers developed specifications for the cardiac troponin assays (45).

Redefinition of MI: Evolution With cTnT and cTnI

The evolution of the definition of MI based on the availability of cardiac troponin is discussed in detail in Chapter 3. Initial regulatory clearance of cTnT and cTnI assays by the US Food and Drug Administration was based in large part on showing that their clinical performance was equivalent to CK-MB mass. This demonstration would have been straightforward had CK-MB mass and cTnT and cTnI indeed provided identical diagnostic information. However, the superior signal/noise biology for cardiac troponin yielded results that were diagnostically "positive" for cardiac troponin, but "negative" for CK-MB mass for many patients with ACS (39). A body of literature evolved for cardiac troponin over the past 5-10 yr, allowing meta-analysis (41,42) that unequivocally demonstrated that "positive" troponin results identify high-risk ACS patients, independent of the CK-MB results. In large part, these data motivated the redefinition of MI based on cardiac troponin as the preferred biomarker of cardiac necrosis (6).

Clinical Cutoffs

Selecting the appropriate decision point or cutoff that should be used for designating a positive cTnT or cTnI is a topic of controversy. Issues related to selection of decision limits are also discussed in other chapters. Several studies have found that even subtle increases in cTnT or cTnI are associated with increased risk of adverse cardiac events (46,47) and, therefore, a low cardiac troponin should be considered indicative of cardiac necrosis. The ESC/ACC consensus group recommended that the 99th percentile of a reference control population (i.e., normal value) should be utilized as the diagnostic cutoff (6). This cutoff is a much lower value than had been specified previously based on utilization of CK-MB as the predicate biomarker for MI diagnosis. The ESC/ACC group also specified that the imprecision of assays should have a coefficient of variation (CV) of10% at the 99th percentile decision limit. This analytical goal was extremely ambitious and, in fact, impractical for implementation, because only a few cardiac troponin assays could meet it (48,49). As a stopgap measure, some have suggested that the 10% CV of assays be utilized as the decision point, at least until assays are improved (50). Others have defined what may be considered an evidence-based approach in which a value conferring high risk is utilized as the decision point, regardless of the CV at that decision point (47). For epidemiological purposes, the AHA Council for Epidemiology and Prevention defines a positive biomarker as being at least one value above the 99th percentile of the distribution in healthy populations or the level at which a 10% CV can be demonstrated for a particular laboratory (39). This definition is contingent on at least two measurements at least 6 h apart (39). The values at the 99th percentile and the level of 10% CV for the majority of available assays for troponin are provided in Chapter 2, Fig. 9. It is critical that laboratory medicine and clinical staff collaborate on the choice and implementation of cardiac troponin assays, as well as on the cut point utilized. This issue is vital because of the heterogeneous quality of cardiac troponin assays, as was demonstrated by significant outcome differences among troponin assays even when the same apparent cutoff was utilized (51).

Harmonization (i.e., ensuring that different assays will yield the same cTnl results in patient specimens) has not yet been achieved. Although the same assay applied repeatedly to the same sample will yield similar results, cTnl results for the same patient but measured with different assays may vary by more than 30-fold (10). This phenomenon is the result of manufacturers using antibodies that target different epitopes of cTnl as well as using different calibrators. The situation for cTnl may be resolved, in part, through ongoing efforts to define a reference material and harmonize cTnl methods (52,53).

Myoglobin

Function and Release

Myoglobin is a heme protein that is abundant in the cytoplasm of cardiac and skeletal muscle cells; its function is to transport intracellular oxygen (9). The tissue/plasma ratio of myoglobin is very high and, thus, when necrosis of these tissues occurs, blood levels of myoglobin increase rapidly (Fig. 4). Myoglobin is generally accepted as the earliest appearing biomarker that is routinely available for assessment of patients with ACS. Myoglobin is released only upon necrosis, as evidenced by overall poor performance as a biomarker ofacute cardiac ischemia (54). The earliest methods for measuring myoglobin were based on RIA (55). These assays had good performance but were untenable for routine use, because they used radioisotopes and required several hours for incubation. In the early 1990s, two-site immunoassays became available; with these assays, good performance and rapid turnaround times in a range of10-15 min are possible, and these assays now predominate in laboratories (56).

The amino acid sequence for myoglobin is the same for both cardiac and skeletal muscle, so elevations are not exclusive for injury to either tissue. Myoglobin is cleared by the kidneys and, therefore, renal insufficiency causes elevated blood levels in the absence of acute tissue injury. For these reasons, myoglobin measurements are viewed as having low diagnostic specificity for myocardial injury. Myoglobin assays are in the process of being standardized and harmonized by a committee of the IFCC (57).

Clinical Use for Diagnosis of MI

There is divergence as to whether myoglobin measurements are useful for the evaluation of patients with suspected ACS. Meta-analysis has shown that the clinical sensitivity of myoglobin is approx 90% with serial sampling and, somewhat surprisingly, about the same for diagnostic specificity in the emergency medicine population (54). Nonetheless, suboptimal specificity is considered to limit the use of myoglobin as a biomarker to rule in MI. The diagnostic sensitivity of the biomarker, however, has led to recommendations by the National Academy for Clinical Biochemistry and the ESC/ACC that the biomarker is useful as an early marker (6,35). Perhaps the most useful application of myoglobin is to rule out myocardial necrosis with a negative predictive value approx 96% (54). Owing to myoglobin's kinetics, patients presenting relatively late after their acute event (e.g., >30 h) may have falsely negative values within the normal reference interval (Fig. 4). Therefore, cardiac troponin and perhaps CK-MB should always be assessed in combination with myoglobin.

In an attempt to improve sensitivity for the detection of MI, investigators have focused on the brisk increase in myoglobin after cardiac injury. These efforts have given rise to strategies in which differences between measurements at presentation and 60 to 120 min later are compared as an early indicator of MI (54,55). Most of these strategies use a doubling in myoglobin values over the early hours as the diagnostic criterion (58,59). Evidence shows that this use of myoglobin is open to question, however. A recent study comparing the "delta" strategy for myoglobin with a similar approach using CK-MB mass indicates that the CK-MB strategy is more sensitive for the diagnosis of MI (60).

Although utilization of myoglobin may add little as a diagnostic tool, several studies have suggested that elevated levels of myoglobin are significantly and independently associated with adverse outcomes (61,62).

Quantitative vs Qualitative Biomarker Testing

Although the results of cardiac biomarker measurements are continuous values, the clinical assessment of myocardial necrosis typically is based on specific cut points. This dichotomization of cardiac biomarker results provides a context in which qualitative testing (i.e., positive vs negative) is clearly feasible, particularly for evaluation ofthe earliest specimen(s) as long as the appropriate cut point is used. In fact, a strategy for qualitative reporting of cardiac troponin may be most appropriate because quantitative assays may vary by up to 30-fold (10). Furthermore, qualitative testing (positive/negative) may help avoid discord between point-of-care testing and quantitative testing in the main laboratory, particularly when different technologies are used in these locations. Qualitative testing is less feasible for biomarkers that have different cut points that vary with race, gender, and/or age.

The key to successful qualitative testing is that the true biomarker concentration representing a positive result must be in harmony with the positive cut point for the qualitative assays used at an institution. When using both quantitative and qualitative assays, the cutoffs must be clinically evaluated to ensure that there is no discord in results between the methods. Discord will result in false-positive and false-negative results, leading to confusion (and understandable consternation) among clinical staff.

Quantitative assays are necessary for monitoring the release (rise) and clearance (fall) of cardiac markers. Applications other than the diagnosis of MI, such as risk stratification, reperfusion monitoring, and assessment of prognosis, also require continuous data.

Serial Sampling

To optimize performance, biomarkers of myocardial necrosis require serial sampling when the initial results are negative (6,63); the largest impact is on diagnostic sensitivity.

Serial sampling is necessary because in most cases myocardial-specific necrosis biomarkers, most notably cardiac troponin and CK-MB, require a few hours before their natural kinetics show a detectable response to clinical events (Fig. 4). To the extent possible, placing cardiac biomarkers in the temporal context of clinical signs and symptoms is critically important. Serial sampling at presentation, 6-9 h later, and after 12 h is recommended if the earlier results are negative and clinical suspicion remains high (6).

necrosis biomarkers still in development Heart-Type Fatty Acid-Binding Protein

Fatty acid-binding proteins (FABPs) are low-molecular-mass proteins (~14,500 Daltons) that are abundant in the cytoplasm of striated muscle cells (64,65). FABPs specifically and reversibly bind long-chain fatty acids and appear to function as the principle vehicle for cytosolic transport of long-chain unesterified fatty acids. Myocardium and skeletal muscle contain the same isoform of FABP, termed heart-type FABP (H-FABP), but the content ofthis protein in skeletal muscle is only 10-30% of that found in cardiac muscle. The level of H-FABP in healthy donors is relatively low (2-6 ^g/L) (66), and the bio-marker has a very good tissue/plasma ratio.

Like myoglobin, H-FABP is released from the heart soon after the onset of infarction, and it has been proposed as an early marker for the diagnosis of MI (64,65,67). Plasma concentrations of H-FABP increase within 3 h after MI and return to the normal range within 12-24 h in individuals without renal impairment (68). Differing relative amounts of H-FABP and myoglobin in myocardial vs skeletal muscle tissue led to the notion that the ratio of myoglobin/H-FABP may confer myocardial specificity. In one study, the ratio of myoglobin/H-FABP was found to be different in plasma from patients with myocardial injury (at 4.5) compared with skeletal muscle damage, which was associated with very high ratio values, in the range of20-70 (68). However, strategies to improve detection of myocardial injury by calculating ratios of myoglobin and H-FABP have not yielded a clear advantage over the measurement ofH-FABP alone (69). Moreover, the tissue specificity ofH-FABP is questionable in patients with renal failure or skeletal muscle injury. Although a relatively small number of clinical trials have been conducted to date, H-FABP appears to have similar or better performance than myoglobin.

Carbonic Anhydrase (III)

Carbonic anhydrase (III) (CAIII) is a 28-kDa cytosolic protein located almost exclusively in type I (slow-switch) skeletal muscle. Preliminary experiments showed that myoglobin and CAIII are released from skeletal muscle in a 3:1 fixed ratio during cell injury (70). Because CAIII is not present in myocardium, combining serum CAIII and myoglobin measurements has been proposed to improve the specificity of myoglobin as an early diagnostic marker for MI (71,72). Both serum myoglobin and CAIII have been shown to increase in healthy subjects following vigorous exercise and in patients with neuromuscular disease. By contrast, patients with acute MI showed markedly elevated myoglobin with no concomitant elevation in CAIII. Several studies have confirmed that myoglobin and CAIII are released in a fixed ratio following exercise, showed no significant difference in the ratio for trauma patients, and demonstrated a significant elevation in ratio in the setting of MI (73). These data suggest that the myoglobin/CAIII ratio may be a useful diagnostic indicator of MI (70). There is enthusiasm by some manufacturers to combine myoglobin and CAIII, but no commercial product is yet available.

Table 4

Relative Performance of Various Markers and Combined Model for Diagnosis of mia

Receiver operating Sensitivity at 92.5% Specificity at 92.5%

Analyte characteristic area specificity (%) sensitivity (%)

Table 4

Relative Performance of Various Markers and Combined Model for Diagnosis of mia

Receiver operating Sensitivity at 92.5% Specificity at 92.5%

Analyte characteristic area specificity (%) sensitivity (%)

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