This study was performed at Pediatric Intensive Care Unit (PICU), Pediatric Department, Minoufia University Hospital, during the period from Jan 2018 to June 2020. The minimum sample size was calculated based on a previous study aimed to assess whether the relationship of serum copeptin at admission with clinical outcomes. Yoshikawa, et al. (2019) concluded that copeptin was a useful marker for predicting outcomes in patients with HF. Based on his findings, the minimal required sample size was 62 patients with HF assuming a significance level of 95% and statistical power of 80% with the assumption of the copeptin discrimination for the long-term clinical outcome (composite of all causes deaths and readmission for heart failure) was 70%. In total, 76 of 538 pediatric patients admitted with acute HF with different etiologies were selected randomly using the simple random generator. All children aged from 1 month to 16 years, admitted with a diagnosis of HF either of congenital or acquired etiologies were eligible for inclusion. Children with any chronic condition other than cardiac diseases, malignancy, metabolic syndrome, central nervous system (CNS) disorders, diabetes insipidus and diabetes mellitus were excluded. HF was diagnosed both clinically by signs of HF as tachypnea, tachycardia, and enlarged tender liver, and radiologically, by chest X ray showing cardiomegaly, plethoric lungs. Then assessing cardiac function and diagnosis of etiology by echocardiography. A total of 65 normal children matched in age and sex were included to identify the normal level of copeptin. The study was approved by the Ethical Committee of the Faculty of Medicine, Minoufia University. Informed written consent was signed by all caregivers of included children.
All children were subjected to detailed history taking, thorough clinical examination to determine the primary cause of HF either congenital or acquired causes, clinical signs of HF, ROSS classification of heart failure(Ross 2012), PRISM (Pollack, et al. 1988) score at PICU admission, inotropic score, vital signs were recorded appropriately, anthropometric measures. Routine investigations were performed as complete blood count (CBC), renal functions (blood urea, serum creatinine), liver enzymes (alanine transaminase ALT, aspartate transaminase AST), C reactive protein (CRP), blood electrolytes (serum sodium, potassium and calcium), and cardiac troponin.
Echocardiography:
Two pediatric cardiologists blindly performed cardiac examination using Philips’s ultrasound machine with 2-3 MHZ and 8MHZ probes. Two-dimensional, M mode and Doppler were used for diagnosis of type of congenital heart defect and other causes. Left ventricular systolic functions was measured by acquiring parasternal long axis view in 2D, and M mode was used to measure LVEDD and LVESD, EF% &FS% were measured using Teichon method. If there was any disagreement in diagnosis, consultant opinion was sought.
Plasma level of copeptin
Two ml of venous blood were withdrawn under aseptic condition and allowed to clot for 30 minutes before centrifugation for 15 minutes. Serum was separated into separate tubes and frozen at -20 to -80 degrees Celsius till the assay was performed after all samples were collected from all patients and controls. The kit assay Human Copeptin level in the sample, use Purified Human Copeptin antibody to coat microtiter plate add Copeptin to wells, Combined Copeptin antibody which with enzyme-labeled, become antibody — antigen — enzyme—antibody complex, after washing Completely, Add substrate, the substrate becomes blue color At Horse-radish peroxidase( HRP) enzyme—catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Copeptin in the samples is then determined by comparing the O.D. of the samples to the standard curve. Based on Copeptin level patients with heart diseases were divided into quartiles.
Patients follow up
Patients were managed with follow up of disease course, occurrence of other morbidities as sepsis (systemic inflammatory reaction (SIRS) in the presence of suspected or proven infection), multi-organ dysfunctions (MODS), need for mechanical ventilation (MV) and its duration, duration of PICU stay, and mortality.
Study outcome
The primary outcome of this study was to evaluate the level of plasma copeptin in children with HF of different etiologies. Secondly, we correlated different clinical, echocardiographic and hemodynamic characteristics with plasma copeptin levels. Finally, we assessed the role of plasma copeptin in predicting HF outcomes.
Statistical analysis
Statistical analysis Continuous variables were presented as mean ± standard deviation (SD) or median ± inter-quartile range (IQR) and were compared with Student's t-test or by Mann-Whitney U test (if not normally distributed). Categorical variables were expressed as rate or proportion and compared by the Chi-square test or the Fisher's exact test. Kendall’s coefficient of rank correlation
tau-sub-b was used to test the association between ordinal and continuous variables(Khamis 2008). Copeptin level was expressed as median with inter-quartile range (IQR) [25th–75th percentile] and the non-parametric Wilcoxon rank-sum test was adopted for group comparisons.