Cardiac troponin T reference interval of healthy Nigerian children aged 5–17 years

Anthony Chibueze Nlemadim; Henry Chima Okpara; Maxwell Udo Anah; Martin Madu Meremikwu

doi:10.4103/njc.njc_39_20

ORIGINAL ARTICLE

Year : 2021 | Volume : 18 | Issue : 1 | Page : 22-27

Cardiac troponin T reference interval of healthy Nigerian children aged 5–17 years

Anthony Chibueze Nlemadim¹, Henry Chima Okpara², Maxwell Udo Anah¹, Martin Madu Meremikwu¹
¹ Department of Paediatrics, University of Calabar Teaching Hospital, Calabar, Cross-River State, Nigeria
² Department of Chemical Pathology, Nnamdi Azikiwe University, Anambra State, Nigeria

Date of Submission	19-Oct-2020
Date of Decision	30-Aug-2021
Date of Acceptance	07-Jan-2022
Date of Web Publication	11-Aug-2022

Correspondence Address:
Dr. Anthony Chibueze Nlemadim
Department of Paediatrics, University of Calabar Teaching Hospital, PMB 1278, Calabar, Cross-River State
Nigeria

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/njc.njc_39_20

Abstract

Background: Cardiac troponin T (cTnT), a biomarker for myocardial injury, reference values is unknown in Nigeria. The establishment and use of pediatric cTnT normative values may be better than the use of adult-derived cTnT values.
Aim: The aim of this study is to determine cTnT reference interval among healthy Nigerian children.
Methods: One hundred and thirty-two healthy children aged 5–17 years were consecutively recruited over a 9-month period after meeting the inclusion criteria. Blood was taken and the sera analyzed for cTnT by the electrochemiluminescence immunoassay method using high-sensitive assay. Data were analyzed using RefVal software and SPSS version 23.0.
Results: The male: female ratio was 1.03:1 with median (interquartile range) serum cTnT of 5.1 (5.0–5.1) ng/L. The reference interval has a lower reference limit (2.5^th percentile) of 5.0 (5.0–5.0) ng/L and upper reference limit (97.5^th percentile) of 6.8 (5.7–7.2) ng/L. The 99^th percentile of cTnT was 7.2 ng/L. Serum cTnT had weak association with age (r_s[130] = 0.19, P = 0.033) but no significant relationship with body mass index-for-age-and-sex, sex and social class.
Conclusion: This cTnT reference interval can be beneficial in low-middle-income settings who lack indigenous reference intervals. We recommend its use for the identification of myocardial injury among children instead of adult intervals to prevent under-treatment. Stakeholders are encouraged to make cTnT affordable to the less privileged.

Keywords: Cardiac troponin T, children, low-middle-income, reference interval

How to cite this article:
Nlemadim AC, Okpara HC, Anah MU, Meremikwu MM. Cardiac troponin T reference interval of healthy Nigerian children aged 5–17 years. Nig J Cardiol 2021;18:22-7

How to cite this URL:
Nlemadim AC, Okpara HC, Anah MU, Meremikwu MM. Cardiac troponin T reference interval of healthy Nigerian children aged 5–17 years. Nig J Cardiol [serial online] 2021 [cited 2023 May 29];18:22-7. Available from: https://www.nigjcardiol.org/text.asp?2021/18/1/22/353686

Introduction

Troponin T is one of the regulatory proteins in the interaction between actin and myosin during muscular (cardiac and skeletal) contraction.^[1] It forms a complex with troponins C and I which are located on the thin actin filament of the muscles.^[1] Similar amino acid sequences are seen in troponin C of cardiac and skeletal muscles, unlike that of troponins I and T which are unique to the myocardium.^[1] Hence, the elevation of cardiac troponins T (cTnT) and I in the serum specifically implies loss from the cardiomyocytes (i.e., myocardial injury) following myocardial proteolysis which may be as a result of ischemia, infection, hypoxia, and malnutrition among others.^[2],[3] cTnT has gained attention globally because of its standardization by a globally acclaimed assay kit manufacturer, unlike cTnI.^[4] Hence, there may be difficulty in comparability and reproducibility of results using cTnI unlike cTnT.

Risk factors for cardiovascular diseases have been tracked from childhood into adulthood.^[5],[6] This means that there may be increasing requirements for the measurement of cTnT serum levels in at-risk patients, especially sickle cell anemic children and asphyxiated neonates who have been shown to develop myocardial injury as a disease complication.^[7],[8] In these categories of patients, myocardial injury can lead to myocardial necrosis and death. Therefore, there is a need for establishment of pediatric cTnT reference interval which is, to the best of our knowledge, currently not available.

Reference interval and clinical decision limit (i.e., 99^th percentile cutoff point) of cTnT used in pediatrics were obtained from studies in the adult population.^[9] This can cause inappropriate interpretation of result in pediatric clinical decision-making with attendant misdiagnosis, under-treatment, or delayed treatment. Hence, the development of indigenous cTnT reference interval cannot be overstated. This study aimed to establish cTnT reference interval among healthy young children and adolescents in a low-middle income country using the Clinical and Laboratory Standards Institute (CLSI) guideline.

Methods

Subjects

Healthy children who met the inclusion criteria, after obtaining ethical clearance from the Health Research Ethical Committee of University of Calabar Teaching Hospital (UCTH) and written informed consent from their parents, were enrolled. The recruitment was consecutively done when they attended the Children's General Out-patient Clinic of UCTH, Calabar, in the Niger Delta region of Nigeria for routine medical examination. The CLSI guideline states that the minimum sample size for reference interval determination is 120 reference individuals.^[10] Thorough examination was done for each participant including body mass index (BMI)-for-age-and-sex

calculation and categorization.^[11],[12] This took place from August 2017 to April 2018. Social classification was according to Oyedeji.^[13]

Inclusion criterion

Participants, 5–17 years of age, with hemoglobin-genotype AA or AS.

Exclusion criteria

This included clinical features suggestive of renal, cardiac, and muscular diseases; receiving drugs such as digoxin, frusemide, captopril, or chemotherapy; current or history of respiratory tract infection in the previous 2 weeks including upper respiratory tract infection; physical findings that can elevate serum cTnT level: malnutrition, fever, hypertension, anemia, jaundice, heart failure, or shock;^[2],[3] and low glomerular filtration rate-for-age-and-sex.

Sample collection and preservation

Four milliliters (4 ml) of blood sample were collected. Three milliliters were put into plain tubes, held in ice packs, and carried to the Chemical Pathology Laboratory of UCTH. Clotted samples were centrifuged at 3000 revolutions per minute for 5 min and the sera separated. Within 2 h of blood collection, fraction of sera were analyzed for creatinine using the modified Jaffe's kinetic method while the remaining 1 ml of blood were put into EDTA tubes and analyzed for hemoglobin genotype using citrate agar electrophoresis method. Participants with normal creatinine, normal GFR, and genotype AA or AS were enrolled. The sera were stored in a refrigerator at −20°C for a maximum of 2 weeks before batch analysis for serum cTnT.

Serum cardiac troponin T analysis

This was done by the electrochemiluminescence immunoassay method using Elecsys^® 2010 Analyzer. The analyzer and the high sensitive troponin T assay test kit with catalog number 05092744190 were manufactured by Roche Diagnostics/Hitachi High Tech. Corp. The assay has limit of detection of 5 ng/L, ≤10% coefficient of variation at 13 ng/L, and 99^th percentile among healthy adult controls of 14 ng/L.^[9]

Statistical analysis

Reference limits of serum cTnT values were calculated using nonparametric method of RefVal version 4.11 (July 2006) software.^[14] Outlier management was done using the software's Dixon's algorithm. Statistical Package for the Social Sciences 136 (SPSS) version 23 software (https://www.ibm.com/analytics/spss-statisticssoftware) was used for other data analyses. Proportions were expressed in percentages. Categorical variables were compared using the Chi-square test and Fisher's exact test. Continuous variables were not normally distributed with dissimilar histograms; hence comparison was with Mann–Whitney U-test. Spearman rank correlation was used to determine the relationship between serum cTnT and continuous variables. Kruskal–Wallis H-test was used to compare three groups. Statistical significance was set at P < 0.05 at 95% confidence interval.

Results

General data

One hundred and thirty-two participants with median (interquartile range) age of 10 (7–13) years participated in the study. The age groups 5–10 years and 11–17 years had median ages of 7 and 14 years, respectively. Male: female ratio was 1.03:1. The median (interquartile range) of the BMI-for-age-and-sex and serum cTnT were 15.9 (14.7–17.8) kg/m² and 5.1 (5.0–5.1) ng/L, respectively. BMI-for-age-and-sex were all normal (−2 to + 1 standard deviation [SD]).^[12] The majority of the participants had genotype AA and were of high social class, although their relationship with the age group was not statistically significant [Table 1].

Table 1: General characteristics of the participants

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Reference values

The serum cTnT values showed positively skewed distribution [Figure 1]. The percentile values are as shown in [Table 2].

Figure 1: Distribution of serum cardiac troponin T

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Table 2: Serum cardiac troponin T percentile values

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Serum cardiac troponin T and other variables

Serum cTnT levels had significant weak positive correlation with age (r_s(130) = 0.19, P = 0.033) but no correlation with BMI-for-age-and-sex (r_s(130) = 0.00, P = 0.996). However, the relationship between serum cTnT, sex, social class group, and genotype were not statistically significant [Table 3].

Table 3: Relationship between serum cardiac troponin T, sex, social class, and genotype

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Discussion

This study shows that the reference interval of serum cTnT values is 5.0–6.8 ng/L with upper 95% confidence limit of 5.7–7.2 ng/L. In addition, the 99^th percentile value obtained was 7.2 ng/L.

Reference interval is a common decision-making landmark for interpretation of laboratory test results in clinical practice.^[10] It refers to a range of values that are bordered by two values, the lower reference limit and upper reference limit (URL), usually obtained from well-selected healthy reference individuals. In practice, reference interval for a given analyte is usually estimated based on the central 95% values (between 2.5^th and 97.5^th percentiles) from the reference population.^[10] For clinically important cardiac biomarkers including cTnT, diagnostic cutoff values (clinical decision limits) which defines arbitrary risk level, are also determined in addition to reference intervals for clinical decision-making.^[15],[16] Pediatric cTnT reference interval should be able to detect children with cTnT concentrations above the URL.^[15] These children may require follow-up and/or treatment because they are at risk of myocardial injury which is cTnT >99^th percentile of an URL.^[2]

Serum cTnT level >99^th percentile (7.2 ng/L) obtained in this study may be associated with absolute risk of myocardial damage among children 5–17 years of age. cTnT is usually detected in the plasma due to ongoing loss from the cardiomyocytes.^[17] The proposed mechanisms of these normal losses of cTnT are normal myocyte cell turnover, formation, and release of membranous blebs, cellular release of troponin degradation products without cell death among others.^[18] Although we were not able to find any previous study on reference interval of cTnT in postneonatal children as at the time of writing, a similar study by Bailey et al.^[19] reported postneonatal URL of cTnI to be 9.0 ng/L with 95% confidence limits of 6.0-17.0 ng/L. This finding is similar to that obtained in this study because cTnT and cTnI are specific to the myocardium and have similar release kinetics.^[20] Furthermore, neonatal troponin values are higher than the 99^th percentile (14.2 ng/L) for adults and they decline to a nadir by 3 months of age to adulthood.^[19] The reason for this early life high value can be traced to the cTnT 95^th percentile value (50 ng/L) in the 1^st day of life which is due to the stress of birth.^[21] This implies that the reference values obtained in this study may be the nadir for cTnT which obviously is less than that of adults.

Due to the lack of pediatric cTnT reference values, adult values are hitherto being used for diagnosis in children. A multicenter study showed that the upper 95% confidence limit of healthy adult serum cTnT values was 12.7–24.9 ng/L with 99^th percentile of 14.2 ng/L.^[9] These values are higher than those obtained in this study may be because adults have larger body size and muscle mass, including cardiac muscle. Children are not little men and women; they have phases of growth and development with varying cardiac sizes and functions.^[22] This is the reason pediatric cTnT reference values are needed because using adult reference values for children may be misleading and may not detect children with myocardial injury early enough for intervention.

The interpreted BMI-for-age-and-sex were all normal (−2 to + 1 SD) for each participant.^[12] However, adolescents had significantly higher BMI-for-age-and-sex than the younger children. Serum levels of cTnT increased with older ages of the children. This may be due to varying cardiac sizes during the phases of childhood growth and it is expected that with increase in age, there is increase in growth, body size, and weight. de Simone et al.^[23] showed a linear relationship with weight and left ventricular mass implying that as the heart increases during growth, there may be more normal loss of cTnT from the cardiac cells. Therefore, it can be said that as weight increased, the cardiac mass with serum cTnT also increased because increase in BMI functionally implies increase in weight or reduction in height.^[11] This contrasts the finding by Bailey et al.^[19] who showed no influence of age and sex on postneonatal serum cTnI levels on 1482 healthy children. However, in this research, males and females had similar serum cTnT levels while the difference between the cTnT levels in adolescents and younger children was not significant. This presupposes that partitioning (subgrouping) of pediatric cTnT reference interval based on age, sex, or ethnicity may not be necessary.

The majority of the participants were mainly of the high social class group who routinely assess and can afford health care. Nevertheless, there was no significant difference in serum cTnT levels among the social classes.

Similarly, levels of cTnT were not different among genotypes AA and AS. This is noteworthy because AS genotype is also known as sickle cell trait. It has hemoglobin S and is usually asymptomatic. Serum cTnT levels elevated above a particular cutoff point have been demonstrated among children with sickle cell anemia (genotype SS) by Adegoke et al.^[7] A similar study also showed that very low number of children with sickle cell anemia during a steady-state of health had both elevated cTnT and ischemic electrocardiographic changes.^[24] This may not be obtainable in the participants of this study because they lack factors such as sickled erythrocyte-induced coronary microvascular obstruction. Most of the known factors that cause rise in serum cTnT were excluded from these participants.

Conclusion

The generated reference interval may be of benefit, especially in low-and middle-income settings similar to ours for screening and follow-up of children at risk of myocardial damage. It can also serve as a prognostic tool for dilated cardiomyopathy, infective endocarditis, and asphyxia-induced neonatal encephalopathy.^{[25],[26],[27]}

During the course of this study, the cost of cTnT was 5100 Naira (US$ 12) which the less privileged may not afford. However, with high index of suspicion, serum cTnT estimation is requested by cardiologists, and many patients afford the investigation. Efforts should be made to encourage stakeholders in the health sector, for example, health insurance policymakers and nongovernmental organizations, to assist the indigent child in cardiac evaluations including cTnT measurements.

Whereas it was beyond the scope of this work to establish clinical decision limit which is lacking in pediatrics, serum cTnT values >99^th percentile obtained in this study may be taken as increased risk for myocardial injury in children until there is a study with larger population.

Limitations of the study

The performance of electrocardiography could have helped in identifying healthy children with asymptomatic cardiac ischemia-induced cTnT elevation. Although rare, the presence of myocardial infarction in these children would have been ruled out affirmatively by echocardiography, using the World Heart Federation criteria.^[2]

Lines of future research

Multicenter analysis for clinical decision limit of myocardial infarction among adolescents: the 99^th percentile cutoff point
Influence of age, sex, and ethnicity on pediatric cTnT reference values.

Acknowledgments

We are very grateful to the parents of these children for allowing their wards to take part in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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