Methods

A total of 97 unselected consecutive patients with ischemic-type chest pain presenting at the emergency department of the HELIOS Heart Center Wuppertal (Wuppertal, Germany) were enrolled in this prospective study. The exclusion criteria were the inability or unwillingness to give informed consent, age <18 years, and interhospital transfer. Three patients were excluded from the study because of improper sample collection or timing that made it impossible to establish or rule out the final diagnosis of AMI; thus, 94 patients were included in the analysis. The clinical parameters assessed included history of cardiac events, Goldman risk score (reflecting clinical risk of myocardial infarction), type and duration of symptoms, arteriosclerotic risk factors, and presence of renal impairment with an estimated glomerular filtration rate of <60 ml/min. At admission to the hospital, the cTnT and H-FABP serum levels were measured from the initial blood samples, and a second cTnT measurement was performed ≥12 hours after admission. All samples were centrifuged and frozen at −70°C; H-FABP is known to show no significant changes in the serum concentration after 12 months of storage. Serum creatinine was also measured in the initial sample to assess chronic renal failure as a source of variation in the biomarker levels. The serum cTnT levels were measured using the Elecsys troponin T immunoassay (Roche Diagnostics, Mannheim, Germany). The upper reference limit (ninety-ninth percentile) is 0.01 μg/L and the lowest concentration with a coefficient of variation of ≤10% was 0.03 μg/L. H-FABP was measured by quantitative enzyme-linked immunosorbent assay (Hycult Bio/Technology Human H-FABP enzyme-linked immunosorbent assay test kit HK402, HyCult Biotechnology BV, Eindhoven, The Netherlands). The detection limit of the test was 250 pg/ml, and the detection range was 100 to 25,000 pg/ml (manufacturer’s data).

To determine the diagnostic test characteristics of H-FABP, we preclinically tested serum drawn from 51 patients with AMI at admission (STEMI, age 60 ± 13 years, 70% men) retrospectively. These patients with anterior (n = 18, maximum creatinine kinase serum level 960 ± 1,037 U/L), inferior (n = 24, maximum creatinine kinase 615 ± 391 U/L), and posterolateral (n = 7, maximum creatinine kinase 541 ± 226 U/L) STEMI were admitted 291 ± 207 minutes after the onset of symptoms. Blood samples from 80 patients without angiographic evidence of coronary artery disease and 80 patients with chronic stable coronary artery disease but no AMI (elective patients admitted for diagnostic coronary angiography) served as the controls. The median H-FABP serum concentration of normal patients without coronary artery disease was 5.4 ng/ml (95% CI 5.1 to 5.6), with an upper reference limit of 7.1 ng/ml (90% CI 6.8 to 7.4). The median H-FABP concentrations did not differ significantly from those for the patients with coronary artery disease (5.6 ng/ml, 95% CI 5.5 to 5.8). Receiver operating characteristics analysis found the optimal test accuracy for differentiating between patients with myocardial infarction (STEMI) and normal patients to be a cutoff value of 7.3 ng/ml (sensitivity 83%, specificity 97%). The upper limit of normal for H-FABP was therefore defined as 7.3 ng/ml for the clinical trial. Receiver operating characteristics analysis also revealed that the test characteristics of H-FABP (area under the receiver operating characteristics curve [AUC]) were not inferior to cTnT in all patients with STEMI and significantly better than cTnT in patients with STEMI presenting <4 hours after symptom onset (AUC 0.98 vs 0.86). On the basis of the retrospective control group analysis (STEMI vs controls without AMI, AUC for cTnT and H-FABP), the sample size estimation revealed a required sample size of 93 patients for the comparison of the 2 AUCs (derived from the same cases). To show that the discriminating power of the H-FABP and cTnT assays, performed on the same cases, was significantly different, we aimed at a sample size of 100 (assumptions: α level = 0.05, β level = 0.10, rank correlation coefficient = 0.4).

In the clinical study, we blindly assessed the initial 12-lead electrocardiograms of all patients. ST elevations ≥1 and ≥2 mm (at the J point) were noted. ST depression ≥0.5 mm at 80 ms after the J point and T inversion of ≥1.0 mm at the nadir were classified. Q waves were recorded if ≥0.03 s and ≥25% of the following R. The presence of left bundle branch block was noted separately. AMI was diagnosed when either the cTnT serum levels at admission or at 12 hours were >0.03 ng/ml, irrespective of the presence of ischemic features on the electrocardiogram in the absence of any other cause for the chest pain. In the absence of cTnT levels at 12 hours, a typical increase and decrease in creatinine kinase levels of more than twice the upper level of normal at 24 hours also confirmed the final diagnosis of AMI (type 1 or 2). When this definition was met, we then further classified cases as STEMI and non-STEMI according to the electrocardiographic features. STEMI was diagnosed when ST elevation was found in 2 contiguous leads of >1 mV in leads I to III, aVL, aVF, V ₅ to V ₆ , and ≥2 mV in V ₁ to V ₃ . Classification of non-STEMI was by exclusion of STEMI. Unstable angina pectoris was diagnosed when the history and/or electrocardiographic changes were consistent with an acute coronary syndrome but cTnT negative at 12 hours and/or no typical increase and decrease in creatinine kinase levels at 24 hours. The history parameters included previous myocardial infarction, percutaneous coronary intervention, or coronary artery bypass grafting. The electrocardiographic parameters included significant ischemic changes on the admission electrocardiogram (ST depression ≥0.5 mV, T inversion ≥1 mV) or evidence of coronary artery disease during the index hospital stay (positive coronary angiographic findings, positive stress test). Uninterpretable electrocardiographic tracings (pacemaker, left bundle branch block) did not contribute to the diagnosis of unstable angina but required a positive history of coronary artery disease and/or evidence of coronary artery disease with additional testing. Nonischemic chest pain was diagnosed by the exclusion of AMI and unstable angina.

The baseline patient characteristics were assessed using the Mann-Whitney U test for continuous variables and using the chi-square test for categorical variables. Two-tailed p values <0.05 were taken as significant. We compared the biomarker levels between independent groups using the Mann-Whitney U test. The median values and 95% confidence intervals (95% CI) of median were given for continuous variables that were not distributed normally. Receiver operating characteristics analysis was performed to analyze the performance of H-FABP as an early indicator of AMI. The AUC (AUC ± SE) was calculated as a quantitative measurement for test performance, taking into account sensitivity and specificity. Significant differences from random (AUC = 0.50) and differences between the AUCs of H-FABP and cTnT (repeated measurements) and between independent samples (STEMI and non-STEMI) were tested. The sensitivities and specificities were compared using McNemar’s test. Data analysis was performed using a standard statistical software package (MedCalc, version 10.0, MedCalc Software, Mariakerke, Belgium). We evaluated decision making in the context of acute chest pain using binary recursive partitioning. Decision tree analysis was performed using commercial decision tree software (DTREG, version 4.5, evaluation, Phillip H. Sherrod; available from: www.dtreg.com ). Tree building was performed by splitting each node into 2 child nodes aimed at minimizing node impurity and misclassification. Splitting was evaluated using the entropy algorithm by finding the best fit for each possible split for each predictor variable. The tree building stopping criterion was controlled by setting maximum splitting levels to 10 and a minimum node size for a split to 10. Tree pruning was performed using V-fold cross validation to find the optimal tree size. We used 10 V-fold cross-validation trees to minimize classification error size (cross-validated error, cross validation cost).

The present study complied with the Declaration of Helsinki, and the local ethics committee approved it. All patients gave informed consent at study enrollment.

Results

Patients with a final diagnosis of AMI did not differ significantly from those without AMI with respect to mean age, gender, median symptom duration, or presence of cardiac history ( Table 1 ). A total of 49 patients (52%) presented within 4 hours of symptom onset. They did not differ in terms of age, gender, history, clinical risk score, or electrocardiographic features from those patients presenting >4 hours after symptom onset. However, early presenters were less likely to have a positive initial cTnT test, with a greater rate of normal findings on the initial electrocardiograms and a lower rate of ST depression/T inversion found on the electrocardiograms than were late presenters ( Table 2 ). Acute management characteristics are listed in Table 3 . Figure 1 compares the median H-FABP serum concentrations in different patient subgroups, along with controls. In particular, patients with STEMI displayed significantly greater median H-FABP values than did patients without AMI, in controls and prospective patients alike. However, we found no significant difference in the H-FABP concentrations between patients with non-STEMI and those with unstable angina pectoris. The H-FABP serum levels in patients presenting at the emergency department with acute chest pain but no AMI were still significantly greater than those in the controls without AMI.

Median serum concentrations of H-FABP in patients with chest pain and controls. Boxes represent 25% and 75% percentiles, whiskers represent 10% and 90% percentiles. *p <0.05 (Mann-Whitney U test). CAD = coronary artery disease; MI = myocardial infarction; NICP = nonischemic chest pain; NSTEMI = non–ST-elevation MI; UA = unstable angina pectoris.

The cutoff values established from the preclinical analysis were used to dichotomize the biomarker concentrations. The test characteristics of H-FABP and initial cTnT serum levels are listed in Table 4 . The sensitivity of cTnT at presentation was lowest when the symptom duration was <4 hours and increased to 100% when the symptom duration was >4 hours. The sensitivity of H-FABP for AMI was superior to cTnT for patients admitted within 4 hours of symptom onset. The sensitivity of H-FABP was greatest at 2 to 4 hours after symptom onset and decreased after >8 hours. In contrast, cTnT maintained a sensitivity of 100% at later points ( Figure 2 ). Combining cTnT and H-FABP (either elevated) provided a significant improvement in sensitivity for patients presenting within the first 4 hours after symptom onset. This improvement in sensitivity for AMI could be maintained at later points. The specificity of H-FABP, however, was significantly lower than cTnT in all subgroups and for all periods. Taking into account the sensitivities and specificities, the AUC was calculated for the initial cTnT and H-FABP level as a quantitative parameter of test performance ( Table 5 ). In the total patient population, the AUC was significantly higher for initial cTnT than for H-FABP. However, when testing for patients presenting within 4 hours after symptom onset, no significant difference was found in test performance between the 2 biomarkers. When testing for acute STEMI, the AUC was slightly higher for H-FABP than for to the initial cTnT, without statistical significance. However, in those patients with STEMI who presented early (<4 hours), the AUC for H-FABP was significantly higher than for the initial cTnT. The same relation between the biomarker test performance in the first 4 hours held true for controls (STEMI). In contrast, when testing for acute non-STEMI, the AUC was markedly lower for H-FABP than for cTnT and did not prove significantly different from random (p = NS compared with an AUC of 0.50; Figure 3 ).

Table 4

Test characteristics of initial cTnT, H-FABP or either for diagnosis of acute myocardial infarction (AMI) stratified by symptom duration

Variable	Initial cTnT Positive	H-FABP Positive	Either cTnT or H-FABP Positive
All patients (n = 94) ⁎
Sensitivity	74% (66–74%)	71% (57–83%)	97% (86–99%) † ‡ §
Specificity	100% (96–100%)	65% (58–71%) † ¶	65% (60–66%) † §
Negative predictive value	89% (85–89%)	82% (73–89%)	98% (90–100%)
Positive predictive value	100% (88–100%)	50% (40–58%)	58% (51–59%)
Misclassification rate	9% (9–14%)	33% (25–42%)	25% (23–32%)
False-negative rate	26% (26–34%)	29% (17–43%)	3% (1–14%)
False-positive rate	0% (0–4%)	35% (11–42%)	35% (34–40%)
Number needed to diagnose	1.4 (1.4–1.6)	2.8 (1.9–6.8)	1.6 (1.5–2.2)
Symptom to admission <4 hours (n = 49) ∥
Sensitivity	42% (28–43%)	86% (64–96%) † ¶	93% (73–99%) † §
Specificity	100% (94–100%)	66% (57–70%) † ¶	66% (58–68%) † §
Negative predictive value	81% (77–81%)	92% (80–98%)	96% (84–99%)
Positive predictive value	100% (65–100%)	50% (38–56%)	52% (41–55%)
Misclassification rate	16% (16–25%)	29% (23–41%)	27% (23–38%)
False-negative rate	58% (57–72%)	14% (4–36%)	7% (1–27%)
False-positive rate	0% (0–6%)	34% (30–43%)	34% (32–42%)
Number needed to diagnose	2.3 (2.3–4.6)	1.9 (1.5–4.7)	1.7 (1.5–3.3)
Symptom to admission >4 hours (n = 45) #
Sensitivity	100% (90–100%)	59% (40–75%) † ¶	100% (85–100%) † ‡
Specificity	100% (94–100%)	64% (53–74%) † ¶	64% (55–64%) † §
Negative predictive value	100% (94–100%)	72% (59–83%)	100% (86–100%)
Positive predictive value	100% (90–100%)	50% (34–64%)	63% (54–63%)
Misclassification rate	0% (0–8%)	38% (26–52%)	22% (22–33%)
False-negative rate	0% (0–10%)	41% (25–60%)	0% (0–15%)
False-positive rate	0% (0–6%)	36% (26–47%)	36% (36–45%)
Number needed to diagnose	1.0 (1.0–1.2)	4.3 (2.0–15.0)	1.6 (1.6–2.5)

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