To our knowledge, this is the first exploratory study in our Institute that suggests that the determination of these laboratory biomarkers could help in the evaluation of pediatric patients who are candidates for cardiac surgery with CPB, where contact activation of the blood cells with artificial surfaces, air, surgical trauma etc., trigger an inflammatory process with cell activation and endothelial dysfunction [30, 31]. The bioactive peptide ET-1 has been shown to mediate vasoconstriction of the systemic circulation and influence myocardial contractility [15]. The results of the present study demonstrated that ET-1 levels were significantly elevated in the post-operative period (24H) and were significantly lowest in the pre-operative period (48H) in patients undergoing CPB. These results are consistent with some previously published studies in which elevated ET-1 levels were detected during the perioperative period of cardiac operations in children [32, 33].
We measured higher baseline values of ET-1 than did Komai et al [32] and Xia et al [33]; this discrepancy cannot be completely explained by variation in specificity of the monoclonal antibodies used in each study. Despite the variations that ET-1 concentrations detected in the plasma of healthy subjects by different researchers may be between 0.1 and 48 fmol/mL (0.25-120 pg/mL) [32], it may reflect patient differences given to age or ethnic origin. The mean age of the patients in Komai’s study was 1.6 years, the patients in Xia's study were older, and our patients had an intermediary age, so it is possible that ET-1 levels increase in patients with congenital heart defects at different ages. In our study, the concentrations of ET-1 in pediatric patients after CPB were much higher than the documented plasma levels, which can be explained by the use of aprotinin for inactivity of kallikreins on the plasma samples, that limits the peptide detection. The elevated number of CEC in preoperative patients provides evidence that in CPB there is a pronounced endothelial injury and damage [34, 35]. We also assume that the detection of elevated numbers of CEC may be the most direct marker of endothelial activation or injury and, perhaps, may enable the quantification of the inflammatory response in conjunction with CPB. Since these cells are found very rarely in healthy people's blood; the increased number may reflect the degree of endothelial activation or damage, and even represent a prognostic indicator in patients developing an overwhelming inflammatory response after a period of CPB [35, 36].
Previous studies have used different protocols for the measurement of CEC and CEPC. Up to now, several assays enabling the detection of CEC and CEPC have been described and others are likely to be published in the near future. In addition to establishing the true value of CEC and CEPC enumeration, general consensus on the best way to enumerate these cells is now definitely required [37, 38].
Using this protocol, two populations were detectable by FC: [CD34+CD146+VEGFR2+CD133-] and [CD34+CD146+VEGFR2+CD133+], which have been reported, ranging typically from 0.1% to 6.0% of blood mononuclear cells from CEC and 0.01–0.20% of blood mononuclear cells for CPC [39].
CEC were easily quantified in this pediatric population, and counts in the 15 pediatric control subjects were close to those described in adults by the consensus network [38]. Besides, CEC counts in peripheral blood were similar in control subjects and patients with reversible CBP and were consistent with normal values defined by consensus (<10 CEC/mL), similar to the number found in normal subjects, as defined with the consensus protocol [38].
The CEC%, CEC number or CEPC% and CEPC number were not statistically significant and apparently did not correlate with the patient´s course. However, our report had few patients, and a type II error is possible, and it may be that higher number of patients would have shown a clinical and statistical significance, especially in infants, where the changes were more important. Sun et al [22], reported than the CEPC were higher in infants than in older children; they included children from a month to 3 years old. In our study, one advantage was that some children were from 0 months to 108 months, so this is the first observational study to report this CHD age range. At the first analysis, both groups had the same behavior, among these, differences were not found before and after of surgery. However, the age was the main doubtful variable. When the infants were analyzed separately, the most important differences were showed before and after surgery. According to Sun et al [22], this is the group where the value of endothelial cells would be representative and have more clinical significance, thus CEPC would play an important role in maintaining endothelial function and vascular repair; its higher count and percentage in infants would represent a prognostic factor in this age group [22].
We had the disadvantage that just two samples were collected from the patients (24 hours before surgery and 48hrs after of surgery). However, 24 hours before surgery has been considered a good parameter of reference of basal level of ET-1 and CEC and CEPC [22]. Schmid et al, [25], have also reported that most changes would be seen after 48 of the surgery. Finally, we intended to correlate the Aristotle Score [40] as a mortality risk, with the CPB and Aortic clamping times, and also the number of CEC and CEPC in patients who died, noting that these last biomarkers were higher in comparison with the median group of children with CHD. Thus, were correlated with the Aristotle Score of these patients, suggesting the presence of hypoxic damage secondary to CHD, leading these to be useful as an optimal biomarker for mortality in children with complex CHD, and furthermore, as aid in adult patients with coronary disease. In addition, our study's main weakness is thought to be the sample size, for it reduced to possibilities to find differences between groups. To confirm these data, prospective clinical trials need to be performed.
Limitations of the Study
The main limitation of the present pilot study was the number of patients analyzed, since its grouping process into age categories, shortened the groups even more. And although, in the methods (statistical analysis) we comment: The power of the sample size, especially for CPB and CPE, was higher than 80% when the comparisons were between at least 15 children, and the proportion between cases and controls was 1: 3; but if the ratio was 1: 1 with at least 5 cases the power was 60.9% and with 10 children the power was greater than 80% heterogeneity in the type of surgery. However, despite the difficulties given by the selection of the population and the performance of all these tests, it is important to highlight the heterogeneity of all the patients in the study with CPB in terms of the cardiac surgery performed. In general, the patients underwent a correction of septal ventricular defects, tetralogy of Fallot, transposition of the great arteries, and atrial septal defects. These different surgeries involve different surgical insults, different CPB times and, therefore, different activations of systemic inflammatory responses. Table 1 shows that the CPB time was reached an average of 107 minutes, but in the case of a broader range, from 37 to 168 minutes, different activations of the inflammatory system. If the sample size were larger, we consider that it would be important to analyze these data according to CPB time. Control group. The control group is different from the CPB group, in terms of age, and especially in terms of the different surgeries. However, due to the ethical implications in this pilot study, we were able to include only a group of older children in relation to the group of cases, with the idea stress that triggered the inflammatory response to surgical stimulation would be reflected. Therefore. Our results do not show considerable differences between the CPB and control groups or between the age groups, at least in this specific scenario after surgical procedures.