Giovanni Cuda¹, Margherita Lentini¹, Luigia Gallo¹, Fortunata G. Lucia¹, Lorenzina Giaquinto Carinci¹, Serafina Mancuso¹, Rosa A. Biondi¹, Raffaella Sinopoli¹, Rita Casadonte¹, Pietro H. Guzzi², Mario Cannataro², Annalisa Mongiardo³, Claudio Iaconetti³, Angela Bochicchio³, Antonio Curcio³, Daniele Torella³, Pietroantonio Ricci⁴, Ciro Indolfi³, Francesco Costanzo¹

1Laboratory of Clinical Biochemistry and Molecular Biology, Fondazione, T. Campanella, University of Magna Graecia, Catanzaro, Italy, ²Laboratory of Bioinformatics, University of Magna Graecia, Catanzaro, Italy, ³Division of Cardiology, University of Magna Graecia, Catanzaro, Italy, ⁴Division of Forensic Medicine, University of Magna Graecia, Catanzaro, Italy

TABLE OF CONTENTS

1. Abstract

2. Introduction

3.Materials and methods

3.1. Patients population

3.2. Specimen collection and measurement of cardiac troponin T

3.3. Statistical analysis

4.Results

5.Discussion

6. References

1. ABSTRACT

This study was aimed at assessing the bias of high sensitive cardiac troponin T vs. the standard cardiac troponin T in a selected population with chest pain of presumed cardiac origin. Serum cTnT was determined in 132 patients and in 106 apparently healthy controls by both assays. The hs-cTnT outperformed the standard generation assay by: i) allowing a larger and earlier diagnosis of AMI (74.2 percent vs. 64.3 percent patients resulted positive at the final diagnosis of AMI when tested with the hs-cTnT or the std-cTnT assay, respectively); ii) showing a better time-dependent dynamics in patients with AMI due to a higher precision at low concentrations; iii) identifying, within the controls, 6 subjects in whom a further examination revealed the presence of chronic asymptomatic cardiac ischemia. The results underscore the excellent performance of the hs-cTnT assay in our population. The use of this test can thus be strongly recommended in subjects presenting to the emergency unit with chest pain of presumed ischemic origin in order to increase the probability of earlier diagnosis of AMI, especially in non-STEMI.

2. INTRODUCTION

Acute myocardial infarction (AMI) is a disease characterized by death of myocardial cells due to a prolonged ischemia that overwhelms the physiological mechanisms of cell repair. Ischemia may occur as a consequence of increased myocardial metabolic demand, decreased delivery of oxygen to the cardiac muscle, or both.According to the Joint ESC-ACCF-AHA-WHF Task Force for the Redefinition of myocardial infarction, AMI can be diagnosed when cardiac troponin (cTn) is present and detectable in the blood of an individual exhibiting signs and symptoms of myocardial infarction (1).Incidence of AMI is still very high: in Italy it is estimated that 130000 new cases of AMI occur each year; of those, about 33000 die and, in more than 18000 cases, the death occurs prior to reaching the hospital.

Rapid, reliable and sensitive diagnostic tools are therefore highly needed for management optimization of these critical patients. Electrocardiography is the most important and widely used approach to complement clinical assessment in the diagnosis of AMI (2), but is often insufficient due to the fact that electrocardiographic patterns suggestive of myocardial ischemia/necrosis can be detected in several non-AMI conditions (3, 4). Detection of the highly specific cTn T or I proteins in the serum of individuals with a clinical suspect of myocardial infarction has been shown to improve significantly the diagnosis of cardiac injury (3, 5-7), displaying a higher sensitivity than other biomarkers, such as creatin kinase MB and myoglobin (8, 9). In healthy subjects, serum cTn level is estimated to be within the range of 0.0001-0.0002 �g/L, due to a physiological loss of cardiomyocytes (45x10⁶/year in the left ventricle) (10, 11). Changes in the 20% range of concentration of serum cTn are generally considered significant to differentiate between acute and chronic cardiac accidents (12); however, several reports suggest that a higher increment may be required (13). An important limitation of standard cTn assays resides in their low sensitivity, leading to a delay in confirming the diagnosis of AMI, with many drawbacks in the clinical management of the affected patients.

Very recently, high sensitivity cTn diagnostic tests have been released by several companies, which allow to further increase the detection limit of this protein in the circulating blood at the 99^th percentile of an apparently healthy reference population with <10% coefficient of variation (CV), therefore complying with the requirements of the ESC-ACCF-AHA-WHF Task Force and the Study Group on Biomarkers in Cardiology of the Working Group on Acute Cardiac Care of the European Society of Cardiology (1, 14).

In the present study, we show the results of a comparative analysis of the 4^th generation cTnT assay (std-cTnT) with the high sensitivity cTnT (hs-cTnT) assay (Elecsys Troponin T hs, Roche Diagnostics), performed on 132 samples from patients referred to us from the Cardiology Unit, as well as from 106 randomly selected and apparently healthy individuals.

3. MATERIALS AND METHODS

3.1. Patients population

The study has been conducted in the Clinical Biochemistry Laboratory of the Magna Graecia University, School of Medicine, Catanzaro (Italy), fromMarch 2009 through February 2010. The study has been cleared by the Institution Ethics Review Board for human studies and patients have signed an informed consent. The patients enrolled in the study ranged from 29 to 88 years of age, and were admitted to the Emergency Unit (EU) because of an episodeof chest pain of presumed ischemic origin, lasting for at least 5 minutes, but less than 6 hours,within the previous 24 hours. Baseline characteristics are reported in Table 1. Patients were excluded from the studyin the presence of a documentedmyocardial infarction within the previous three weeks, thrombolytic therapyor angioplastywithin the previous 6 months. Unstable angina or non-Q-wave myocardial infarction were diagnosed on the basis of serial electrocardiograms and determinationsof creatine kinase or CK-MB. Blood sample collection was performed at the time of admission and after 6, 9 and 12 hours.

3.2. Specimen collection and measurement of cardiac troponin T

Serum specimens were collected in standard tubes or tubes containing separating gel and immediately subjected to analysis accordingly with the manufacturer's specifications. For the purposes of this study, the analysis was performed on the Cobas e411/e 601 analyzers (Roche Diagnostics) under the following conditions: std-cTnT with a detection limit of 0.01 �g/L, a 99^th-percentile cutoff point of less than 0.01 �g/L and a coefficient of variation of less than 10% at 0.035 �g/L; hs-cTnT with a detection limit of 0.002 �g/L, a 99^th-percentile cutoff point of less than 0.014 �g/L and a coefficient of variation of less than 10% at 0.013 �g/L. As recommended by the Study Group on Biomarkers in Cardiology of the Working Group on Acute Cardiac Care of the European Society of Cardiology, the decision limit for cardiac injury was established as the concentration of cTnT (either std-cTnT or hs-cTnT) that corresponds to the 99^th percentile limit of the reference distribution in a sex- and age-matched healthy reference population. Diagnosis of myocardial necrosis was made on the basis of a rising and falling cTnT pattern, with at least one value above the 99^th percentile. All samples were measured as a single determination.

3.3. Statistical analysis

Data analysis was done by the use of the Pearson chi-square test and the Fisher exact test. Nonparametric analysis was performed for comparison of assays. The criterion for significance was P< 0.05. Continuous variable are presented as means (+SD).

4. RESULTS

A total of 238 individuals were enrolled in the study: of these, 132 patients had been admitted to the EU, while the remaining 106 were apparently healthy individuals. The mean (+SD) age was 66,9+11,6 and 65,6+17,3 for patients and healthy controls, respectively; 68,9% man and 31,1% women in the patient's cohort, 64,7% man and 35,3% women in the control group. The presence of AMI was confirmed in 98 patients (74.2%), in 21 a diagnosis of unstable angina was made (15.9%); the remaining 13 patients (9.8%) were classified as affected by non coronary artery disease (non-CAD). At presentation, both std-cTnT and hs-cTnT assays showed more elevated troponin levels in patients who underwent AMI compared to individuals with unstable angina or non-CAD (Figure 1A). As expected, the diagnostic accuracy of hs-cTnT was very high at presentation; the high sensitivity assay outperformed the standard one detecting the presence of cTnT in the blood of AMI patients earlier and longer than the std-cTnT (P<0.05) (Figure 1B and 1C). The adjudicated final diagnosis of AMI significantly increased from 64.3% to 74.2% in patients tested with the std-cTnT vs. the hs-cTnT assay, respectively (P<0.02).

hs-cTnT and std-cTnT levels measured in the 132 patients and 106 apparently healthy controls are reported in Table 2. The performance of hs-cTnT was significantly higher with respect to the std-cTnT assay: in particular, with the hs-cTnT assay, concentrations of cTnT were at or above the limit of detection (0.002 �g/L) in 292 out of 294 determinations (99.3%) and at or above the 99^th percentile (0.014 �g/L) in 245 out of 294 determinations (83.3%) (P<0.05). On the other side, the std-cTnT assay gave the following results: 234 out of 294 determinations (79.6%) were at or above the limit of detection (0.01 �g/L), 221 out of 294 (75.1%) were at or above the 99^th percentile (P<0.02). In the 106 apparently healthy subjects enrolled in the study as controls, hs-cTnT was at or above the detection limit in 20 cases (18.9%) and at or above the 99^th percentile in 16 cases (9.4%), while the std-cTnT assay resulted at or above the detection limit in 4 cases (3.8%) and at or above the 99^th percentile in 6 cases (5.7%) (Table 3). The distribution of cTnT levels, as measured by the high and the standard sensitivity assay in patients is shown in Figure 2A. A fair degree of concordance between the two assays emerged from the overall measuring range, but a significant difference was observed at the very low end, both in the patient (Figure 2B) and in the control population (Figure 2C) (P<0.05), suggesting that, below 0,01 �g/L, determinations obtained with std-cTnT and hs-cTnT cannot be directly compared.

5. DISCUSSION

A correct risk assessment of patients referred to the EU because of acute chest pain is a daily challenge, even for expert physicians; this is due to the wide variety of symptoms, especially in individuals with non-ST elevation acute coronary syndrome, and to the low specificity of instrumental diagnostic tools (i.e. electrocardiography). The availability of reliable, sensitive and specific markers of cardiac necrosis is therefore highly desirable. The development during the past 10-15 years of cTn assays has represented a major advancement in the diagnosis of AMI. The detection of cTnT and I subunits in the circulating blood has significantly increased the percentage of AMI cases correctly identified; moreover, many reports clearly demonstrate that elevation of cTn levels can be correlated to the extent of the necrotic area and that its detection may substantially improve the early diagnosis of AMI, especially in patients with a recent onset of chest pain (15). According to the recent literature, the definition of increased levels of cTn can be made only when they exceed the 99^th percentile upper reference limit in a healthy reference population (CV<10%) (3, 16-18). To this end, more sensitive assays are needed. Very recently, several companies have released new, better performing cTn assays, which can be used for an earlier and more accurate identification of AMI patient, as well as for a better risk stratification.

In the present study we evaluated two different cTnT assays: the 4^th generation cTnT assay (std-cTnT) and the high sensitivity cTnT (hs-cTnT) assay (both from Roche Diagnostics). The diagnostic performance of the hs-cTnT was significantly higher than that of the std-cTnT assay. Hs-cTnT was, in fact, able to detect the presence of circulating cTnT earlier and longer than the standard assay in AMI patients. Moreover, hs-cTnT outperformed the std-cTnT assay in the correct identification of AMI vs. non-AMI patients (i.e. individuals affected by unstable angina and/or non coronary artery disease). Remarkably, with the hs-cTnT assay, 292 out of 294 determinations (99.3%) were above the limit of detection (0.002 �g/L) in the patient population, while only 234 out of 294 (79.6%) overcame the limit of detection (0,01 �g/L) in the same population when the standard cTnT assay was used. We further compared the analytical performance of the two assays by studying the distribution of cTnT levels and found that, even if hs-cTnT and std-cTnT show a reasonable concordance over a wide range of concentrations, the 4^th generation assay cannot provide reliable data below the 0.01 �g/L limit, leading to either an underestimation or a late diagnosis of AMI patients. Interestingly, the high sensitivity assay, applied to an apparently healthy cohort (106 individuals) in which clinical conditions potentially associated to non-ischemic myocardial damage, as well as non-cardiac cTnT-associated diseases were excluded, was able to identify six subjects in whom a further and accurate examination revealed the presence of chronic asymptomatic cardiac ischemia. This finding, which is still under investigation, appears of particular interest and highlights to the usefulness of the hs-cTnT test in a specific subset of individuals with silent cardiac ischemia.

All together, our results underscore the excellent performance of the hs-cTnT assay in the studied population compared to the 4^th generation assay. Even small elevations of cTnT, undetectable with the standard assay, have been reported to be associated to a worse outcome during follow up (higher incidence of cardiovascular death and heart failure), even in subjects with stable coronary artery disease (19). Thus, the availability of sensitive, specific and accurate tools is highly desirable. It must be considered that this is an observational, retrospective analysis of a relatively small cohort of patients admitted to the EU and therefore, as pointed out in similar studies, definitive conclusions cannot be drawn. However, we feel that our findings are sufficient to strongly encourage the use of high sensitive cardiac troponin assays in subjects presenting to the emergency department in order to increase the likelihood of improving early diagnosis of AMI, especially in non-ST elevation acute coronary syndrome.

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Abbreviations: cTn, cardiac troponin; hs-cTnT, high sensitive cardiac troponin T; std-cTnT, standard cardiac troponin T; AMI, acute myocardial infarction; UA, unstable angina; Not-CAD, not coronary artery disease; SD, standard deviation

Key Words: Cardiac troponin T, Acute myocardial infarction, Coronary artery disease, Early diagnosis, Biomarkers