Paediatric cardiac surgery is associated with high blood losses that may even surpass the patient's entire blood volume . Factors specific to children, to their diseases and to the procedure may play a role in these high blood losses . Up to approximately 6 months of age, neonates have reduced blood levels of factors II, V, VII, X, XI and XII, protein C, protein S, and antithrombin III [3,4]. The activated partial thromboplastin time is prolonged at birth and reaches adult levels at approximately 3 months of age. Fifty percent of neonates undergoing open-heart surgery with cardiopulmonary bypass (CPB) come to the operating room with significantly lower coagulation factor levels than expected for their age, a phenomenon that might be explained by delayed hepatic maturation secondary to poor organ perfusion .
Children with cardiac disease and body weight <10 kg have decreased platelet aggregation [5,6] while those with cyanotic heart disease may also have thrombocytopaenia proportional to the degree of their chronic hypoxaemia  and reduced blood levels of high-molecular weight multimers of von Willebrand factor . So far, risk factors clearly associated with increased blood losses in children undergoing CPB are age <1 month , body weight <8 kg , complex procedures (arterial switches for transposition of the great vessels, Fontan procedures, Glenn shunts and truncus arteriosus repairs) , length of CPB [11,12], resternotomy , and platelet count during CPB . Although present in a significant number of patients, the amplitude of fibrinolysis, as measured by thromboelastography, does not correlate with blood losses . Pronounced dilution of coagulation factors from the high prime pump volume/child blood volume ratio (up to 50% dilution)  and platelet activation have not been clearly related to blood losses in children undergoing CPB. In adults, CPB-induced platelet activation/dysfunction is considered to be the most important contributor to non-surgical postoperative blood losses . P-selectin (CD62), an adhesion molecule found in the α-granule membrane protein of platelets, becomes externalized on the platelet surface following platelet activation . Flow cytometry is a useful tool for the detection of changes in the expression of membrane glycoproteins induced by platelet activation in vitro and during platelet storage .
The aim of this study was to evaluate the effects of CPB on platelet activation of children undergoing open-heart surgery.
After parental informed consent, the following data from 18 consecutive children undergoing cardiac surgery with the use of CPB were prospectively collected: age, sex, body weight, length of surgery, CPB and aortic clamp times, intraoperative blood losses (based on sponges weights, suction bottle volumes and chest tubes drainage) and postoperative chest tube blood losses, preoperative and postoperative haemoglobin levels (immediately after surgery and at 20 h of the operative day), and platelets count before and after CPB. Anaesthesia was maintained with fentanyl, isoflurane (<1 minimum alveolar concentration (MAC)) and neuromuscular relaxants. Reconstituted blood (packed red blood cells and fresh frozen plasma) was added to the pump prime to maintain a haematocrit of 30%. The minimal body core temperature was lowered between 25°C and 32°C depending on the complexity of the planned surgery. Heparin was administered to maintain an activated clotting time ≥400 during CPB. After bypass, heparin was neutralized with protamine at 1 mg of protamine per 100 unit of heparin administered (first dose). No steroids were given. Administration of blood products was guided by haemoglobin values and laboratory coagulation tests, and followed our previously published protocol .
Haemoglobin, haematocrit and platelet counts were determined with a Technicon H-1 (Tarrytown, NY, USA). For all patients 10 mL of blood was collected via the central venous line immediately before and after CPB for measurement of CD62 by flow cytometry. Samples were anticoagulated with 3.8% sodium citrate at a 1:10 ratio. To 5 μL of whole blood, an equal volume of phycoerythrein-conjugated human monoclonal anti-CD62P antibodies (clone, AC1.2; Beckton Dickinson, San Jose, CA, USA) detecting human P-selectin antigens expressed on platelet surface, was added and the suspension was incubated in the dark for 20 min. Samples were then fixed by 1% paraformaldehyde (Sigma-Aldrich, St-Louis, MO, USA) and assayed with flow cytometry using an FACScan cytometer and CellQuest software (Beckton Dickinson, San Jose, CA, USA). The precision of this technique was ±2%.
Data were analysed with the JMP 5.0.1, Professional Edition 2002 (SAS Campus Drive, Cary, NC, USA) using analysis of variance (ANOVA), repeated measures ANOVA and the Wilcoxon rank sum test (pre-bypass values of CD62). P < 0.05 was considered statistically significant. Apart from age, all data were normally distributed. In view of the small number of patients tested, however, data are given as median (range) for tables and mean ± standard error of the mean (SEM) in the Figure.
Two patients were excluded from the analysis because they had received a prophylactic dose of aprotinin. Patient characteristics and surgical data are given in Table 1. Cyanotic patients, as compared to non-cyanotic, had significantly longer duration of surgery (P = 0.09), CPB (P = 0.04) and aortic clamp (P = 0.005). There was a significant variation in haemoglobin over time (P = 0.0005), but the change followed a similar pattern for the two study groups (P = 0.11, Table 2). CPB induced a significant decrease in platelet count (P < 0.0001), but the magnitude of the change did not differ between cyanotic and non-cyanotic children (P = 0.89, Table 3). The percentage of platelets with CD62 expression (percentage of activated platelets) was significantly higher in cyanotic children at the beginning of the surgery (P = 0.07). It increased significantly (P = 0.03) with CPB in all children but the change did not differ between the two groups (Table 3, Fig.). The mean change (6.7 ± 6.0% vs. 6.5 ± 8.7%) was similar for both types of cardiac defects. However, the small sample size does not allow detection of a difference <53% between the two groups in the magnitude of the change (α = 0.05, one-tailed; β = 0.2). Blood loss did not differ among the two study groups (Table 2).
CPB-induced platelet activation is a well-known phenomenon in adult cardiac surgery. Some authors have speculated that CPB would induce t-PA liberation with subsequent plasmin formation and that the newly generated plasmin would induce modifications of platelet receptors. Some of these modifications, e.g. the down regulation of glycoprotein Ib/IX complexes would be preventable by the prophylactic administration of aprotinin but not by α2-antiplasmin or tranexamic acid . The paediatric population has been less extensively studied for this aspect.
Evidence of activation of the coagulation system has been found even in the younger patients despite administration of anti-inflammatory drugs . Increased prothrombin activation fragments 1 and 2 reached their peak level immediately at or close to the end of CPB and remained elevated up to 6 h after the surgery. In the present study, there was a significant increase in the percentage of activated platelets after CPB (CD62 pre- vs. post-CPB) (Table 3). The exact contribution of these changes to bleeding was not evaluated in the present study.
Indications of preoperatively increased platelet activation (degranulation) as found in the present study (5% [range 3-8] for cyanotic patients vs. 1% [range 0-23] for acyanotic patients; P = 0.07; Table 2) has also been reported by others . This could be in agreement with one previous study where the authors reported evidence of disseminated intravascular coagulation in children with cyanotic heart disease, a finding that was not corroborated by subsequent studies [6,21,22].
The significant increase in platelet activation observed in the present study after CPB is an argument to reinforce the practice adopted by many clinicians [2,9] that, when all other haemostatic parameters have already been corrected, platelet concentrates should be administered to children with ongoing bleeding after CPB even if the platelet count is within normal limits (>75-100 × 103 mL).
In conclusion CPB induces significant platelet activation (degranulation) as measured by flow cytometry (CD62) in children undergoing open-heart operations.
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