Troponin I (cTnI) fragments immunoreactivity and the development of high-sensitivity diagnostic assays for the associated diagnosis of acute myocardial infarction

update article Troponin I (cTnI) fragments immunoreactivity and the development of high-sensitivity diagnostic assays for the associated diagnosis of acute myocardial infarction Imunorreatividade de fragmentos da troponina I (cTnI) e desenvolvimento de ensaios de alta sensibilidade para o diagnóstico associado do infarto agudo do miocárdio Raíssa M. Teixeira1, 2; Ricardo David Couto1 1. Universidade Federal da Bahia (UFBA), Bahia, Brazil. 2. Faculdade Osvaldo Cruz, São Paulo, Brazil. abstract Among the cardiovascular diseases (CVD), acute myocardial infarction (AMI) is currently considered the most common cause of death and disability worldwide. Several laboratory tests have been developed for the detection of cardiac injury, including troponins that are considered the gold standard marker (surrogate biomarker) of myocardial injury. The high specificity of troponin for cardiomyocyte necrosis is related to a single unique peptide sequence present in troponin at the cardiac muscle. As a result, studies are currently focused on the development of troponin (hs-cTnI) determination tests with high diagnostic sensitivity value. These diagnostic tests aim to detect increasingly lower serum concentrations of cTnI biomarkers, from the detection of peptide fragments that are released after structural biochemical changes. This article discusses the differences between troponin fragments immunoreactivity to the development of cTnI determination tests, such as the high-sensitivity tests, which arise with the proposal of guaranteeing greater efficiency in the AMI associated diagnosis. Key words: acute coronary syndrome; troponin I; immunodominant epitopes. Introduction Cardiovascular diseases (CVD) are still the most common cause of death worldwide. Out of the 57 million deaths in 2008, 17.3 million (30%) occurred due to CVD(1). Among these, acute myocardial infarction (AMI) is the main cause of death and disability, and coronary atherosclerosis is one of its main causes. AMI may be a minor life event or a chronic illness; it may not even be detected, but it may be one of the main critical events, leading to sudden death or severe hemodynamic impairment. AMI may be the first manifestation of coronary artery disease, or may happen, repeatedly, in patients with the established disease(2). Nowadays, serum concentrations of tissue damage markers are assessed, such as cardiac enzymes and isoenzymes, which are essential for diagnosis or exclusion of myocardial lesion. Several laboratory tests have been developed for detection of cardiac injury, including troponins. Troponins are represented as a protein complex composed of three units that regulates the binding between actin and myosin, filaments that interact to produce muscle contraction. While the complex troponin, troponin T (TnT), troponin I (TnI), and troponin C (TnC) is found in both the skeletal and the cardiac muscle, the high specificity of troponin for necrosis of cardiomyocytes is due to a unique peptide sequence present in the troponin originated in the cardiac muscle (cTn), which is not found in the troponin of the skeletal muscle (sTn)(3). Therefore, this update article approaches aspects related to the presence of structural biochemical changes present in fragments of cardiac troponins I (cTnI), as well as the importance of these changes in these immunoreactive fragments for the development of new highly sensitive diagnostic tests to assure greater efficiency and performance in the AMI associated diagnosis. First submission on 07/05/17; last submission on 12/08/17; accepted for publication on 13/08/17; published on 20/10/17 305 10.5935/1676-2444.20170048 J Bras Patol Med Lab, v. 53, n. 5, p. 305-308, October 2017 Troponin I (cTnI) fragments immunoreactivity and the development of high-sensitivity diagnostic assays for the associated diagnosis of acute myocardial infarction Structural changes and immunoreactivity of cTn fragments Diagnostic efficiency: cTnI detection limits Although troponin (cTn) is specific for lesions of the cardiac muscle, we need to be aware of situations that can alter immunoreactivity, and, consequently, modify its structure. In those cases, both TnT and TnI are released in different molecular forms. For instance, TnT is mainly found as intact TnT:I:C complex, free TnT, and smaller immunoreactive fragments. TnI is mainly released as intact TnI:I:C and I: C complex. The binary form TIc:C seems to be the predominant molecule, besides having forms deriving from proteolysis (degraded) and other biochemical changes, such as phosphorylation, oxidation, and reduction(4). Thus, a lot of effort has gone into developing monoclonal antibodies to be used in assays of different manufacturers for the troponins that are comparable with each other(5). We can cite several instruments with their automated assays that are used in the diagnostic support of AMI, such as Architect – Abbott Diagnostics, Centaur – Siemens, Access – Beckman Coulter, Immulite – Siemens Dimension series – Siemens, and Vitros – Ortho Clinical Diagnostics, all of them used for determination of TnI; and Cobas/Modular – Roche Diagnostics, the only one for TnT. There are also nonautomated tests; rapid tests, such as i-STAT – Abbott Diagnostics; Triage Cardiac Panel – Alere; Reader – Roche Diagnostics and Radiometer AQT90 for TnI(5). The thorough investigation of new target epitopes is decisive for overall performance of the diagnostic tests currently most used in the detection of cTnI. With the identification of those epitopes, new tests gained specificity, so, in a short time assays are expected to follow the guidance of the European Society of Cardiology (ESC) and the American College of Cardiology (ACC): detection limit for TnI assays must be very low among healthy individuals, that is, low number of false-positive results, considering the 99th percentile for the definition of AMI(9). The diagnostic industry is focused on reduction of analytical imprecision and improvement of sensitivity to meet the recommendations necessary for manufacturing of diagnostic kits with highest coefficient of variation (CV) of 10% at the 99th percentile(10). Alternatively, the use of a lower serum concentration of measurable troponin has been suggested when CV is lower than 10% (CV < 10%)(11). However, most analytical methods for diagnostic support used in emergency hospitals do not satisfy the rule established by the guidelines, what characterizes lack of uniformity among the used assays(12). Still as an aggravating factor, although less precise assays of cardiac troponin do not generate relevant false-positive AMI diagnoses, even when CV is 20% at the 99th percentile, they are considered acceptable(7, 13). According to Bates et al. (2010)(14), after AMI, the circulating fragments of cTnI are present in the serum in the complexes TIc:C and IC, and the fragments cTnT present themselves combined as TIc:C, or exclusively in their free form, free cTnT. Th (...truncated)