Evaluation of serum cardiac troponin I values in children less than 1 year of age

Objective: The objective is to verify the cardiac troponin I values in children less than 1 year of age without clinical cardiac dysfunction. Methods: The cardiac troponin I values were determined in 99 children less than 1 year of age, including term infants without diseases related to cardiac dysfunction using the specific kit Opus T Troponin I (cTn) (Dade Behring Inc. Newalk, DE 19714, USA). Results: All children have values of cardiac troponin I less than 0.1 ng/ml. Conclusion: We verified that the cardiac troponin I value is less than 0.1 ng/ml in children less than 1 year, including term infants without cardiac dysfunction, when analized by the kit Opus T Troponin I (cTn) test


INTRODUCTION
Discovered by Setsuro Ebashi [1], in 1963, the troponins constitute a complex of proteins which modulates the velocity and the strength of striated muscle contraction. The complex consists of three subunits: Troponin T (TnT), responsible for binding the complex to tropomyosin; Troponin C (TnC) is a calcium-binding protein and has a key role in muscle contraction; and Troponin I (TnI) inhibits muscle contraction [2].
TnI can be found in three isoforms with different protein structures determined by distinct and specific genes for each isoform [3]. Two isoforms are present in the skeletal muscle, one in fast-twich fibers and other in slow-twich fibers. The third isoform, present in the myocardium, é called Cardiac Troponin I (cTnI) [4].
The structural difference between TnI isoforms allowed that Cummins et al. [5] to develop the first radioimmunoassay capable of identify the release of cTnI in the acute myocardial infarction (AMI) and to publish their results in 1987.
The improvement of the method [6] and the great number of studies investigating its usefulness in the diagnosis of myocardial injury [7] have made it to become one of the major biochemical markers of AMI [8].
In pediatrics, researches are focused on the use of this marker in the diagnosis of myocardial injury related to surgical repair of congenital heart defect. These studies demonstrate the application of the method in different situations, since the diagnosis of events following the performance of operative procedures to its use as a prognostic marker [9][10][11][12].
Also, cTnI values have been presented as an important method to assist in the management of other pediatric diseases, such as in myocarditis [13], sepsis [14], and in Kawasaki disease [15], diseases in which cardiac dysfunction is a common event.
For an adequate use of biochemical markers in daily clinical practice, it is necessary to define which reference values to use [16]. Some studies have emerged with this purpose, achieving results in pediatrics similar to those of adult researches [17], however, others have presented increased cTnI values in children under one year of age [18,19].
The definition in relation to the reference values for cTnI in pediatrics and the importance of this diagnosis method, by its characteristics and applicability, has motivated this research.

METHODS
This research was undertaken after being approved by the Research and Ethics Committee of the participant institutions. This is a non-randomized, prospective observational clinical trial to refine new diagnosis approaches.
Between January 15 and February 13 2003, 99 children under the age of one year were included in the study after a written informed consent was signed by their legal authorized representatives. cTnI values were then measured.
The children enrolled in the study have met the following inclusion criteria: age under 1 year, including full term infants, no previous diagnosis of heart diseases, or any clinical condition somehow related to cardiac dysfunction, hemodynamic stability, determined through physical examination without using drug therapy or mechanical support.
The patients were divided into two groups according to their age group. Group I consisted of newborns up to 28 days of life. Group II consisted of infants between 29 days of life and one year. The newborns in group I have cTnI values collected at the moment of nursery routine examinations, and children in group II, at the moment of preoperative ward examinations and for child care followup; the exams were ordered by practitioners not involved in the study.
The sample collection, storage, and assay followed the manufactures guidelines and the pertinent literature.
Samples were collected using conventional peripheral venipuncture techniques, preferably in the right cubital region, using a disposable plastic syringe and needles with no anticoagulant solution.
After sample collection, 2 mL of blood was poured into a glass tube and set out for serum separation by means of centrifugation at 3000 rpm for 10 minutes. Serum was stored in a glass tube at +3ºC. Samples remained stored at an average of 24 hours not surpassing a 72-hour period storage until cTnI dosage.
A specific kit, Opus T Troponin I (cTn) test modules (Dade Behring Inc. -Newalk, DE 19714, USA) and the device Opus plus ® analyzer (Behring Diagnostics Inc. -Westwood, MA 02090, USA) were used to analyze the samples.
The assay is based on fluorogenic immunoassay principles linked to a double antibody sandwich enzyme (antibody-antigen-antibody), or linked to two sites with mouse monoclonal antitroponin I antibodies.
Analysis was performed automatically by the Opus plus analyzer.
Serum concentration is expressed in nanograms per milliliter (ng/mL).
Method assay range varies from 0.1 to 50.0 ng/mL. Assay analytic sensitivity is 0.1 ng/mL. All values below are referred by the analyzer as < 0.1 ng/mL. The reference value for normalization established by the laboratory is lower than 0.5 ng/mL.
The method used in our study, the Opus T Troponin I (cTn) test modules (Dade Behring Inc. -Newalk, DE 19714, USA) was chosen due to its commercial use and with which an important ability in daily usage has been developed; in addition, it was available at our institution.
The daily use of serum dosage of cTnI for AMI diagnosis in adult patients through this diagnostic method has yielded outcomes contrary to literature data regarding sensibility, specificity, and temporal course, turning this method into an important aid tool in the diagnosis and follow-up of these patients [20].
As the method analytical sensitivity was set at 0.1 ng/ mL, our result did not allow the performance of statistical processes. Likewise, with the analyzer Opus Plus assigning values <0.1 ng/mL, our result was rather a qualitative variable than a quantitative one, not showing variation.
Levin [26] defined Statistics as "…a set of techniques to reduce quantitative data (i.e., a series of number) into a more convenient and easily communicable number of smaller descriptive terms".
Taking into consideration the characterization of our result and the definition proposed by Levin, we believe to be justifiable not to apply a statistical outlining to our study, however, this does not interfere in the assessment regarding its applicability, once the distribution of all values below 0.1 ng/mL allows concluding that all patients presented normal values, regardless its variation within this interval.
After assessing all data presented, it becomes difficult to justify the findings of increased cTnI in normal children; on the contrary, we believe these data cause our study result to be expected, and they justify the incisive fashion as this result turned to be, which has been repeated in newly published studies [27,28].
Despite the variability in the results found among different methods, the use of this myocardial injury biochemical marker has been playing an important role in patients' diagnosis and follow-up. It is important that the interpretation of the results in daily practice be carried out relatively to local laboratory standards and comparatively to other methods.
New studies are needed to investigate several aspects related to the dosage of serum cTnI and its applicability.

CONCLUSION
We concluded that the reference value (0.1 ng/mL) for serum cardiac troponin I dosage, when performed through Opus T Troponin I (cTn) test modules (Dade Behring Inc.-Group I infants were sampled in their first day of life, except one who was sampled on the 13 th day of life. Male and female infants' mean age (group II) at the moment of sampling was 7.6 ± 2.8 months and 8.2 ± 1.9 months, respectively.
The dosage of cTnI was found to be below the assay analytic sensitivity level (0.1 ng/mL) in all children and it was registered by the analyzer (Opus plus) as < 0.1 ng/mL.

DISCUSSION
The results of our study are compatible with the results of other studies which have observed normal values of cTnI in pediatric patients without myocardial injury [17]. The distribution of all values found within the reference range for normality (<0.1 ng/mL) is not an unexpected fact, once the literature data relates a high specificity to the method [20] and the study population did not present any clinical data suggesting the existence of myocardial injury, which was established by the study inclusion criteria.
Among the studies, we have found two publications aiming at defining the reference values in a pediatric population. The authors considered a new and exciting datum to have as a result a large percentage of patients in the first year of life presenting increased cTnI values [18,19].
The abovementioned authors suggest that the increased serum levels of cTnI is related with the apoptosis of myocardial cells. However, it is difficult to establish this relationship because apoptosis is a physiological phenomenon playing an important role in the development of several organs, being suppressed at the beginning of extrauterine life, and despite of being named as cell death, it occurs usually in isolated cells and it is not followed by an inflammatory process. The cells undergo herniation of cell membrane, volume reduction, condensation of nuclear chromatin and, finally, they are phagocytosed by macrophages and surrounding cells [21][22][23].
An important consideration to be made when different study results are compared is the influence of the methodology used. There is a great variability of results between two methods [24]. This variability is related to a series of processes that will change the cTnI presentation form after its release. Only 10% of the released cTnI remains in its free form; the other 90% build up complexes, especially with Troponin C; moreover, cTnI can be submitted to other biochemical processes, causing it to be oxidized or reduced. These several changes interfere with the reaction of cTnI with the