Introduction

The initial evaluation of chest pain and possible acute coronary syndrome (ACS) in the emergency department (ED) includes laboratory testing to identify non-ST elevation myocardial infarction (NSTEMI). The specific serum biomarkers for ACS have evolved over time, and troponin is the primary biomarker currently used in most ED settings.

The troponin complex is made up of three subunits involved in myocardial sarcomeric contraction. Cardiac isoforms—cardiac troponin T (cTnT) and cardiac troponin I (cTnI)—are subunits exclusively expressed in myocardial tissue. Elevated serum levels are therefore markers of myocardial injury. But non-ACS elevations in troponin may result from a wide range of conditions, including sepsis, renal disease, and pulmonary embolism. Analytic issues can produce false-positive results in healthy subjects [1]. The conventional cTnT has been reported to be 72 % sensitive and 95 % specific. High-sensitivity troponin assays increase sensitivity to 94 % with a decrease in specificity to 73 % [2]. cTnI shows similar tradeoffs with conventional and high-sensitivity assays [3]. As a diagnostic test, troponin is effective and widely utilized, but many have explored its limitations [4–6]. These limitations highlight the need for alternative biomarkers to improve the accuracy of ACS diagnosis.

While reviews of alternative diagnostic cardiac biomarkers have previously been published, we focus on the strongest evidence from the past three years. The search for a better biomarker has been challenging. To illustrate this, Lin’s 2012 review of 58 studies that evaluated 37 novel biomarkers found clinical value for only five biomarkers: matrix metalloproteinase-9, copeptin, ischemia-modified albumin, heart-type fatty acid binding protein, and B-type natriuretic peptide [7•]. They represent discrete aspects in the pathogenesis of ACS, such as plaque instability to myocardial ischemia and necrosis. The latest evidence on each of these alternative biomarkers is explored here.

We have excluded from our review B-type Natriuretic Peptide (BNP) and its N-terminal fragment, N-terminal Pro-B-type Natriuretic Peptide (NT-pro-BNP), which are secreted in response to overloaded cardiac ventricles. Emerging evidence suggests that BNP or NT-pro-BNP is a potential prognostic tool rather than a diagnostic tool [8–14]. As a diagnostic tool, they only modestly predict severity of underlying coronary disease and ischemia [7•, 15–17]. There seems to be little new evidence for supporting BNP or NT-pro-BNP in ACS diagnosis.

Biomarkers

Matrix Metalloproteinases

Metalloproteinases (MMP) hydrolyze components of the extracellular matrix and possibly identify ACS and plaque instability. There are 23 recognized MMPs [18]. MMP-9 levels correlate with Framingham risk score, and elevations may identify risk for future myocardial infarction [19]. MMP-2 and -9 have been evaluated in the diagnosis of STEMI [20, 21], or ACS more generally [22]. It is theorized that “increased expression of MMP-2 and MMP-9 metalloproteinases and their tissue inhibitor (TIMP-2) is responsible for disturbed equilibrium of the metalloproteinase/tissue inhibitors system and as a consequence, for destabilization of atherosclerotic plaque and occurrence of the acute coronary syndrome” [22]. Small studies have found possible utility of MMP-9 in ACS diagnosis [23, 24]. However, countervailing evidence was provided by a study finding no elevation in blood mononuclear cells’ expression of MMP-9 on admission in acute myocardial infarction (AMI) [25].

Pregnancy-associated plasma protein A (PAPP-A), another metalloproteinase, may have a diagnostic role [26], including potential utility in unstable angina not detected by troponin elevation [27]. In fact, PAPP-A has figured more prominently in recent literature than the matrix metalloproteinases. Yet PAPP-A is only 90 % sensitive [28], and a five-study meta-analysis encompassing 2050 patients found it to be inferior to troponin [29]. In the only study from that meta-analysis to explore supplementing troponin with PAPP-A, there were only marginal non-significant improvements in diagnostic accuracy [29, 30]. Currently, there is an inadequate support for any of the metalloproteinases in ACS diagnosis for routine clinical practice.

Copeptin

Copeptin, the C-terminus of the vasopressin prohormone, is a surrogate of vasopressin release and indicates neurohormonal stress activation and vasoactive response. In a recent small study, copeptin blood levels mirror cTnT in AMI [31]. But the area under the curve (AUC) in ROC analysis of copeptin in NSTEMI was only 0.71. A cutoff of 10.25 pmol/l showed a sensitivity of 88.8 % and specificity of 69.8 % [31]. Other investigators found equally poor diagnostic characteristics when copeptin was used alone [32, 33•, 34].

A recent meta-analysis of 9244 patients from 14 studies found that patients with AMI and an initially negative troponin had a higher copeptin level than those without AMI (22.8 vs. 8.3 pmol/l) [35••]. Rather than being used alone, copeptin may complement troponin in early chest pain, identifying ACS missed by troponin. Piyanuttapull states that “the additional use of copeptin to cTnT allows for a rapid triage of chest pain patients to an early diagnosis of non-ST elevation myocardial infarction” [31]. In one study of 1927 patients in which NSTEMI had a 6 % rule-in rate, elevated copeptin identified approximately half of NSTEMI patients with initially negative cTnI (10 of 19 patients) [36••]. Furthermore, in 12 studies on 6988 chest pain patients without STEMI, adding copeptin to troponin had a sensitivity of 95 % (compared to 87 % sensitivity for troponin alone for AMI). However, this was at the expense of considerable loss of specificity (57 % for troponin plus copeptin, compared to 84 % for troponin) [37]. Charpentier et al. added copeptin to troponin in 641 ED patients which increased the sensitivity from 55.3 to 90.4 % in their cohort and improved the negative predictive value (NPV) from 92.8 to 97.6 % [38]. The AUC of the combination of copeptin and cTnI was significantly higher than for cTnI alone on presentation (0.89 vs. 0.77) [38], and similar results have been noted in diabetic patients [34]. Others report a NPV of 98 % in patients with known coronary disease presenting within 6 h of symptom onset [39]. A large meta-analysis seems to reinforce these results [35••].

While it is better with copeptin, one must still ask if the combined NPV is high enough to be applied in our current medico-legal environment. Charpentier et al. contend that “the sensitivity of this combination even using a conventional troponin assay remains insufficient to safely rule out NSTEMI at the time of presentation” [38]. A NPV around 98 % [38, 39] is certainly high but still not perfect. Moreover, the lost specificity and perhaps only modest increase in identified cases cannot be ignored. The large CHOPIN trial (copeptin helps in the early detection of patients with acute myocardial infarction) encompassed 16-sites and 1927 patients in EDs with less than six hours of chest discomfort onset and without electrocardiographic evidence of ST segment elevation. Combined normal initial cTnI and copeptin was seen in 58 % of patients, with a NPV of 99.2 %. But almost one-third (503/1646) of patients with normal troponin had an increased copeptin, and just ten of these patients ruled in as NSTEMI [36••]. A persistent copeptin elevation on serial testing may identify those with actual infarction [40]; but even with a negative copeptin and troponin, almost as many patients (9/1143) ruled in, so copeptin barely identified half of the unrecognized NSTEMI [36••].

In the COPED–MIRRO trial—a multicenter, prospective, observational, longitudinal cohort study of 1018 patients—copeptin plus troponin on arrival did not fully rule out AMI without necessitating additional evaluation [32]. In the Randomized Assessment of Treatment using Panel Assay of Cardiac Markers (RATPAC) trial, a randomized controlled trial of point-of-care cardiac markers in the ED that included 2243 subjects, copeptin did not add significantly to troponin in suspected-AMI [10]. Thus larger trials have cast doubt on the utility of copeptin in routine clinical practice.

High-sensitivity assays may provide better results. When high-sensitivity (hs) cTnT was used in combination with copeptin, the summary sensitivity and specificity were 98–50 % [37]. With multiple hs-cTnI assays in comparison, the sensitivity combined with copeptin may be slightly less–ranging from 89.4 to 97.8 %—with modest increases in specificity. The best reported AUC for hs-cTnI and copeptin was 0.94 [41]. In discussing their study of 1170 patients which showed a trend toward improved diagnostic accuracy using hs-cTnT combined with copeptin, Potoki et al. stated “copeptin provides independent prognostic information, largely by overcoming the challenging interpretation of mild increases in hs-cTnT” [42]. However, others have argued that copeptin does not enhance the diagnosis of NSTEMI over hs-cTnT alone [10, 34, 43, 44, 45•] and that the sensitivity of this combination is still not high enough to definitively exclude AMI [46].

The addition of ultrasensitive (us) copeptin has also been studied. The “ultrasensitive copeptin in addition to medium sensitivity cardiac troponin for the early diagnosis of non-ST-elevation Acute Coronary Syndromes” (COPACS) trial included 190 subjects enrolled within the first 6 h of onset of non-traumatic chest pain who did not have ST elevation on EKG. If validated, this might support an expedited cardiac rule out in the ED, and do so with a numerically higher sensitivity (100 vs 89.7 %, although not reaching statistical significance in this study). Unfortunately, the sensitivity increase is at the expense of specificity—only 74.2 % for the combination. The single-sample strategy of combined us-copeptin and cTnI is statistically non-inferior to serial cTnI for NSTEMI, although the AUC was actually numerically decreased from 0.939 to 0.871 [47]. Using high-sensitivity troponin assays instead of conventional cTnI, the AUC in one recent study improved to 0.93 for hs-cTnT plus us-copeptin, compared to 0.89 for hs-cTnT alone. In this case, sensitivity improved from 76 to 96 % and NPV from 95 to 98.9 % [48].

Regardless of which assays are used, a high NPV for a combination of troponin and copeptin would be advantageous for ruling out ACS. The NPV approaches 100 % in some studies [47, 49–51], but other investigators have found that even high-sensitivity assays for both troponin and copeptin have yielded NPVs similar to standard troponin and copeptin alone [36••, 37, 39]. To summarize, based on the literature that currently exists, there is inadequate evidence to support the routine use of copeptin—either alone or combined with troponin—as a diagnostic tool for the evaluation of AMI.

Ischemia-Modified Albumin

Ischemia changes the structure of serum albumin into ischemia-modified albumin (IMA). This modified protein is measured by its decreased binding of cations [52]. Since troponin will not rise in reversible myocardial ischemia without myocardial necrosis, IMA might be sensitive for parts of the ACS spectrum missed by troponin, notably angina.

A specific weakness of troponin is its poor sensitivity early in the course of symptom onset and clinical progression. Alternative biomarkers might rise earlier and address this weakness. Liebetrau et al. used a surgical treatment for hypertrophic cardiomyopathy as a model for AMI. IMA concentrations increased significantly 30 min after induction of this model of AMI versus baseline, with values of 26.0 U/ml (Interquartile range [IQR] 21.8–38.6 U/ml) versus 15.6 U/ml (IQR 10.1–24.7 U/ml), and then decreased after 75 min [53].

IMA was examined in a small cohort of ED patients in a third-world population in India presenting with suspected ACS within 6 h of onset of symptoms [54]. In the group determined to be non-ischemic, the mean IMA value was 56.38 ± 23.89 U/ml. In unstable angina, IMA levels were 89.00 ± 7.76 U/ml, and in AMI levels reached 87.50 ± 9.62 U/ml. Using a cutoff of ≤80 U/ml, IMA was normal in 21 of 24 patients without ischemia, and elevated in all but 2 of 25 patients with unstable angina and 6 of 40 patients with AMI. This yielded a sensitivity of 92 % and specificity of 87 %, with NPV of 94 % [54].

In another recent study where the sensitivity of IMA was only 88 %, this was improved to 96 % when used in combination with cTnI [55]. In 2014, Bhakthavatsala Reddy et al. found in a study of 89 patients that in 16 patients an early diagnosis could be made when compared with cTnT [54]. However, the results of other studies have been less favorable [26, 56–58]. Here again, conflicting findings make it difficult to recommend adoption of this marker for clinical practice.

Heart-Type Fatty Acid Binding Protein

Heart-type Fatty Acid Binding Protein (hFABP) is a small cytoplasmic protein released from cardiac myocytes following cardiac injury. As with IMA, levels of hFABP significantly increase 15 min after induction of a surgical model of “AMI” to reach a level of 9.0 ng/mL (IQR 7.0–15.4 ng/ml) compared to a baseline of 4.6 ng/mL (IQR 3.4–7.1 ng/ml). hFABP showed a continuous increase until the 8th hour with a decline afterward [53]. As previously noted in the discussion of IMA, the generalizability of this model to ED evaluation of undifferentiated chest pain is unclear. But with its promising biokinetics, hFABP may prove to have better sensitivity in the early presentation of chest pain [59].

Despite this promise, hFABP has not yet been shown to improve the diagnosis of an ED patient with chest pain. Freund and colleagues’ recent data found that hFABP does not provide useful additional information to cTnI for ruling out AMI with a NPV of 96 % versus a NPV of 95 % for cTnI alone [60]. Others have also failed to find any improvements in sensitivity, specificity, PPV, and NPV for the diagnosis ACS by adding hFABP [61]. In contrast, in the previously described RATPAC study, the combination of hFABP and troponin actually increased diagnostic sensitivity [10]. Further, adding copeptin to this combination of cTnI and hFABP has been shown in one study to provide a net NPV of 95.8 % and AUC of 0.88 [62]. But it is noteworthy that the NPV is not significantly different from the NPV for cTnI alone by Freund and colleagues [60]. Jacobs et al. still argue that the combination of cTnI, hFABP, and copeptin actually was superior to cTnI alone. These authors point to the first three hours—when troponin’s limitations are most apparent—and when the NPV of 92.9 % for the cTnI–hFABP–copeptin combination was clearly superior to the NPV of 84.6 % for cTnI alone [62].

But if most others find that hFABP does not add to the diagnostic sensitivity to standard troponin, it also generally fails to add to the high-sensitivity troponin based on emerging evidence. In 2013, Reiter and colleagues demonstrated this in a large 1247-patient multicenter study [45]. Smaller studies also revealed that the diagnostic characteristics did not significantly improve by addition of hFABP [63–67], but adding hFABP to hs-cTnT did increase the sensitivity from 83 to 96.8 % and the NPV 95.6–98.9 % for NSTEMI, enhancing the rule-out capability at the expense of significant loss of specificity [63]. Only the small study by Gami and colleagues found the combination could reach a NPV of 100 % while maintaining a specificity of 88.89 % [68]. Others have not reproduced such promising diagnostic characteristics. Thus, despite favorable kinetics, hFABP does not reliably live up to its promise of adding to the sensitivity of the troponins currently used—or at least not without compromising specificity.

The Patient Selection Challenge

The most recent research suggests that cardiac biomarkers—both troponin assays and novel biomarkers—do not in themselves provide clarity in ED evaluation of chest pain. The “holy grail” of perfect sensitivity without sacrificing specificity and ideal biokinetics has not yet been found.

The answer may not be a better assay alone but the selection of appropriate patients using risk stratification tools, algorithms, and risk score models to improve diagnostic accuracy. In cases of STEMI, MMP-9 level correlated with Global Registry of Acute Coronary Event (GRACE) risk score, although individually each demonstrated relatively poor diagnostic characteristics [69]. At a minimum, the correlation between the biomarker and the risk score suggests they may be measuring the same thing, although this relationship does not determine whether the use of GRACE risk scoring in conjunction with MMP-9 would add any diagnostic value, or whether this would extend to NSTEMI as well.

A 2014 study of 537 ED patients found that adding copeptin to cTnI improved the NPV for ruling out AMI. But it did so in low-to-intermediate risk patients specifically. When applied to patients with a GRACE risk score below 140 (11.3 % rate of AMI in this group), copeptin plus high-sensitivity troponin had an AUC of 0.925 and NPV of 98.6 %. At a lower cutoff below 108 (4.4 % AMI rate), the AUC was 0.935 and the NPV for AMI reached 100 % (95 % CI 97.5–100.0) [70]. Thus a single troponin and copeptin measurement might safely rule out AMI in a low-to-intermediate risk population [71, 72]. The Thrombolysis in Myocardial Infarction (TIMI) score has also been proposed as a risk-stratifying option when using copeptin [46].

The Manchester Acute Coronary Syndromes (MACS) decision rule was formulated for suspected cardiac chest pain in the ED [73]. Among its eight variables were high-sensitivity cTnT and hFABP. These biomarkers were used in conjunction with a series of electrocardiographic and clinical findings, in effect incorporating a clinical assessment. The decision rule categorizes patients into four different risk assessment categories. The AUC for major cardiac events was 0.95 in the derivation study [73, 74]. With the validation, the MACS rule had a sensitivity of 100.0 % (95 % CI 95.4–100.0 %) and NPV 100.0 % (97.1–100.0 %) for AMI [74]. Perhaps there is some promise for hFABP in the context of the MACS, with its incorporation of clinical features. But Freund and colleagues looked at hFABP in a low-risk group, based on empirical clinician judgment. While the NPV was strong, this held true for troponin alone as well. Even in the low-risk group, there was no significant improvement using hFABP [60].

Risk stratification may theoretically enhance the ability to rule out ACS. It would seem that the most promising applications for the novel biomarkers might not be in simply replacing or complementing troponin. Rather, the novel biomarkers may be the most useful to enhance the diagnostic capabilities of risk stratification tools and troponin together. Nevertheless, more robust evidence is needed, including determining which biomarkers and which risk stratification tools are most effective.

Conclusion

The diagnosis of acute chest pain in the ED setting is a common clinical challenge requiring substantial health care resource utilization. To improve the effectiveness and efficiency of care, significant efforts have been made to identify novel biomarkers that will aid in the diagnosis of AMI.

This article reviews literature on non-troponin cardiac biomarkers from the past three years, specifically those that were previously identified as having early promise [7•]. However, despite an array of options being explored, no clear solution exists, and it is possible that ACS may be too complex to be reduced to a laboratory test alone. While hope still exists for identifying a breakthrough diagnostic biomarker strategy, definitive answers and consensus remain elusive. The novel biomarkers cannot yet be endorsed for standard use in routine clinical practice.

The contradictions and limitations in the recent literature reviewed here on potential replacements or supplements to the troponin assay suggest that the search for the “perfect” biomarker, or combination of biomarkers, may be futile. A few studies have incorporated clinical risk stratification with some promise, but this evidence is preliminary at best. Further research on existing biomarkers as well as the possible development of new biomarkers—will be needed before any significant advancements in the evaluation of patients presenting with symptoms of AMI in the ED can be made.