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© Borgis - New Medicine 1/2003, s. 12-15
Leszek Bęc1, Maciej A. Karolczak1, Barbara Motylewicz1, Ewa Rogala2
Von Willebrand factor plasma levels depend on rate of hypothermia in paediatric cardiopulmonary bypass
1 2nd Department of Cardiac and General Pediatric Surgery, University Medical School of Warsaw
Head of Department: Prof. Maciej A. Karolczak
2 Department of Laboratory Diagnostics and Immunology, National Institute of Tuberculosis and Lung Diseases, Warsaw
Background. The use of a cardiopulmonary bypass (CPB) is associated with a risk of endothelial damage. Von Willebrand factor (vWf) is a marker of necroses endothelial cells. High levels of vWf increase the of postoperative complications and morality. Our objective was to find any correlation between cardiopulmonary bypass and plasma levels of vWf I paediatric patients who had been operated on for congenital heart defects.
Method. 20 patients with simple congenital heart defects were operated on using a cardiopulmonary bypass. Arterial blood was sampled after induction of anesthesia, 5 minutes after commencing CPB, and at 1 hour and 3 hours after surgery finished. The plasma levels of von Willebrand factor were measured and compared to selected clinical findings? What? How? Boes thin mean known clinical data! It so, what?
Results. Von Willebrand factor plasma levels were significantly elevated in all patients 1 hour and 3 hours after surgery. We found no correlation between vWf levels and type of defect, CPB duration, aortic cross clamp ing, or reperfusion time. However, we were able to demonstrate that the observed elevation of vWF levels was almost 4-fold higher in patients cooled down to lower temperatures (15 pts; mean rectal value 27.64ą0.7°C) than in 5 patients (mean rectal temp. 30.74ą1.56°C) where only slight elevation was found. There were no differences in He postoperative course of these patients. All patients were discharged from hospital 10ą3 days after operation (median 9 days).
Conclusion. Paediatric hypothermic cardiopulmonary bypass is associated with a significant elevation of von Willebrand factor plasma levels. It seems that the augmentation of vWf values could be independently associated with the rate of hypothermia.
Systemic inflammatory response is one of the most senious complications after cardiopulmonary bypass (CPB). Many factors including proinflammatory cytokines activation, presence of endotoxin, and activated leukocytes may lead to higher endothelial permeability, with loss of fluid into the third space. Aggregation of platelets and their adhesion to the endothelial surface may cause microemboli. The process is closely related to endothelial cell damage as well as to necrosis. The plasma levels of von Willebrand factor (vWf) released from Weibel-Palade bodies of necrosed endothelium correlate with postoperative morbidity and mortality. This prospective study was designed to find any correlation between cardiopulmonary bypass and plasma levels of vWf in paediatric patients operated on for congenital heart defects.
Materials and method
20 patients aged from 2 months to 15 years (mean value 45.85ą54.28 months) were operated on for congenital heart defects with the use of cardiopulmonary bypass in 2nd Department of Cardiac and General Pediatric Surgery, University Medical School of Warsaw, Poland between January 31st, 2000 and April 2nd, 2001. Fifteen patients were female and five were male. The spectrum of defects involved atrial septal defect type secundum/partial anomalous pulmonary vein drainage (ASD2/PAPVD; n=7), ventricular septal deffect (VSD; n=12), and anomalous origin of coronary artery from pulmonary artery (Bland-White-Gerland syndrome, BWG; n=1). The characteristics of patients are summarized in table 1.

Table 1. Characteristics of patients.
Sex (M/F)5/15 
Mean age (mo.), range45.85 ? 54,28(2-182)
Mean weight (kg), range13.74 ? 10,33(3.3-42)
CPB duration (min.)66 ? 26,5 
Aortic cross-clamping time (min.)31.4 ? 19,9 
Mean temperature (rectal, °C)29.89 ? 2,19 
The extracorporeal circulation was performed using membrane oxygenators (Dideco, Mirandola, Italy). The circuit was primed with Ringer acetate (100-500 mL), 20% Mannitol (1 mg/kg), 5% Albumin (100-200 mL), 20% Albumin (100 mL), 8.4% sodium bicarbonate (20-40mL) and heparin (1 mg/kg). Fresh (max. 72 hr old) blood was used for all patients.
Anaesthesia was induced by sodium thiopental and Pipecuronium bromide and maintained with Fentanyl citrate, Izofluran and Pipecuronium bromide. Heparin (Heparinum natricum, Polfa, Warsaw, Poland) was given at a dose of 3 mg/kg after an activated clotting time of 480 seconds was achieved the cardiopulmonary bypass was commenced. The aorta was clamped and antegrade cold crystalloid cardioplegic solution (St. Thomas cardioplegic solution) at a dose of 20 ml/kg was administered. Venticular septal defects were patched with PTFE (Gore-texŽ), ASD2 were closed with direct sutures or a pericardial patch, and PAPVD with pericardial patch. The Hamilton procedure (modification of Takeuchi repair) was applied in Bland-White-Gerland syndrome. Protamine sulfate was applied to neutralize heparin. All procedures were performed by one surgeon (M.A.K.) employing bicaval cannulation technique under moderate (27-32°C) hypothermia. The perfusion rate started at 2.8 l/min/m2 and was reduced 1.0 l/min/m2 at 27°C.
Four samples (3 ml each) of blood were collected from the arterial line of each patient. A baseline sample was obtained immediately after induction of anesthesia (sample 1). Sampling was also performed 5 minutes after commencing cardiopulmonary bypass (sample 2), and at 1 hour (sample 3) and 3 hours after skin closure (sample4). The samples were then centrifuged at 2000g and plasma stored at – 70°C until immunoassay. vWf plasma level was measured using a commercially – available enzyme immunoassay kit (ELISA – von Willebrand Activity Kit, Shield Co., Germany) according to the manufacturer´s instructions. All patients´ parents gave informed written consent for participation in this study.
Statistical analysis
Data are presented as the arithmetic meanąstandard deviation. All values of vWf plasma levels were examined by repeated measures. Changes in vWf plasma levels as well as clinical findings were compared with the use of the unpaired Student´s – t-test. A value of P less or equal to 0.05 was considered statistically significant.
Twenty patients with mean body weight 13.74ą ą10.33kg underwent total correction of congenital heart defects with the use of a cardiopulmonary bypass. There was no early (< 30 day) mortality. All patients were discharged from hospital 10ą3 days after operation. The mean operation time (i.e. from skin incision to skin closure) was 150 ą109 min (median 135 min). The mean cardiopulmonary bypass time and aortic cross clamp ing time were 66ą26.5 min (median 59 min) and 31.4ą20 min (median 28 min), respectively. Comparing various types of defects (group of patients with ASD2/PAPVD vs. group of patients with VSD) there were no statistically significant differences in body weight, age type of operation, CPB and aortic cross clamping times.
vWf plasma levels were significantly diminished after the onset of cardiopulmonary bypass in all patients. They were, however, significantly elevated in 15 patients one hour and/or three hours after finishing cardiopulmonary bypass (Fig. 1).

Fig. 1. Elevated plasma levels of vWf in 15 patients.
In the remaining 5 patients there was only a slight elevation of vWf plasma levels 1 hour after surgery (with no statistical significance), unchanged after 3 hours (Fig. 2).

Fig. 2. No significant changes in vWf plasma levels in remaining 5patients 1 hour and 3 hours after surgery.
There was no correlation between a lack of vWf plasma level elevation and type of defect, cardio-pulmonary bypass, aortic cross clamp ing time, or reperfusion times. The only significant difference (p=0.017) between patients with raised and unchanged (constant) levels of vWf factor was the rate of hypothermia (27.64ą0.7°C vs. 30.74ą1.56°C, respectively) applied during CPB.
We found no differences in monitored postoperative clinical findings and parameters. The rate of mechanical ventilatory support, total blood loss (substernaldrainge), and diuresis rate did not differ between these two groups of patients. The postoperative data are shown in table 2.

Table 2. The intraoperative data and post-op. findings.
 OverallvWf elevation groupvWf constant groupStatistical significance
CPB time (min.)66.15 ? 26.5667.2 ? 20.4363 ? 43.3N.S.
Mean AoCC Time (min.)31.4 ? 19.931.2 ? 14.1732 ? 34.33N.S.
Hypothermia (°C)29.89 ? 2.1927.64 ? 0.730.74 ? 1.56p = 0.017
Total substernal drainage (ml/kg)16.94 ? 11.7817 ? 13.2716.75 ? 6.46N.S.
Mechanical ventilation (h)29.65 ? 36.3533.94 ? 40.9116.8 ? 11.94N.S.
CPB – cardiopulmonary bypass; AoCC – aortic cross clamp. Data are given as mean ąstandard deviation. P< 0.05 was considered statistically significant
vWf is a glycoprotein synthesized in platelets or endothelial cells. Stored as the polymeric molecules in thrombocyte alpha-granules and the Weibel-Palade bodies of endothienal cells, it can be released in response to various factors such as thrombin or vasopressin (1). Weibel-Palade bodies, also known as dense granules, contain vWf and P-selectin, adhesion molecule. When activated, they move into the cell membrane and relese their contents (2, 3, 4).
This special situation takes place during a cardiopulmonary bypass. Activation of proinflammatory cytokines (i.e. interleukin-1, 6, 8, tumour necrosis factor-alpha) in the presence of endotoxin leads to synthesis of adhesion molecules. When present on various cells (endothelium, platelets, leukocytes), these molecules are responsible for leukocyte adhesion to the vascular wall. The first part of this process is known as rolling. This unstable reaction is closey related to a subgroup of selectins (including P-selectin) and is associated with marginalization of neutrophils in the vascular lumen. The second phase, depending on another group of adhesion molecules (superimmunoglobulins and integrins), is solid and leads to leukocyte migration through the vascular wall. Activated leukocytes release proteolitic enzymes and oxygen-free radical which damage the surrounding tissue (3, 5-7). This situation, multipled by the aggregation of plateles (vWf molecules bind to platelet receptor GPIb, thus permitting platelets to adhere to the sub-endothelial layer of the vascular wall), and reperfusion after CPB can cause serious endothelial cell damage and/or necrosis (1, 8-13). The plasma levels of vWf released from Weibel-Palade bodies of necrosed endothelium correlate with postoperative morbidity and mortality. vWf levels can therefore be used as a marker of endothelial damage (1). High vWf levels in acute myocardial infraction are associated with the development of other necrotic fields of the myocardium and augment the risk of deasth (3). It has been shown in vitro trials that vWf is released only from necrotic endothelial cells. There is no doubt that CPB – related release of vWf predominantly endothelium – derived. It has been confirmed (by blocking platelet action) that thrombocytes release only ≈15% of vWf found in plasma(1).
In our study demonstrated a significant elevation of vWf in the majority of patients 1 hour and 3 hours after surgery. This in agreement with previous studies. As has previously been reported, vWf plasma level elevation is present immediately after aortic declamping or within a few hours after a cardiopulmonary bypass. These levels still remain high 24 hours after surgery or even 6 days after CPB (1, 2, 3, 14).
The group subjected to trial was quite homogenous. We decided to measure vWf plasma levels in simple congenital heart defects. Neither the intraoperative technique nor CPB or aortic cross clamp ing times differed significantly between patients. We observed, however, that in 5 patients there was no vWf plasma level elevation within 3 hours after surgery. We did not manage to find any differences between this group and the remaining 15 patients, except for the rate of hypothermia used. Patients with stable vWf levels were cooled to 30.74ą1.56°C, whereas those with elevated vWf level were cooled to 27.64ą0.7°C. This suggests that the lower temperatures used during CPB have a more damaging effect on endothelial cells. It also raises the question is hypothermia really a benefit?
In our study we used mild (27-32°C in rectum) hypothermia, as patients were operated on for simple congenital defects. The use of whole body cooling is a confirmed method of preventing neurological and ischaemic disturbances after open heart surgery. Hypothermia allows us to minimize or even stop (TCA – total circulatory arrest) blood flow in vital organs. It is course safe when employed with full control of both cooling and rewarming (15). Low temperatures have an influence on the minimization of cytokine, super-immunoglobulins and elastase plasma levels (16,17). Hypothermic CPB is associated with a lower neurophil count in peripheral blood. Others give priority to normothermic cardiopulmonary bypass as it does not elongate CPB time and does not aggravate systemic vascular resistance (18). They have also proved that a lower neurophil count is only time-limited, and returns to previous levels after rewarming (18). We believe hypothermia is very useful in a paediatric cardio-pulmonary bypass. Nevertheless, the results of our study suggest greater endothelial damage after hypothermic CPB. Probably this grade of damage has no influence on the postoperative course in patients. Mechanical ventilatory support, diuresis rate and total blood loss (substernal drainage) did not differ between patients with elevated and those with stable vWf plasma levels. Comparison of simple heart defects with operations on complex defects (with hypothermia or not) would be helpful in determining if cooling is really endothelium-destructive.
This study has some limitations. The group of patients was relatively small, with various congenital heart defects. The group with constant vWf plasma levels contained only 5 patients and it was very difficult to compare unsteady groups of patients. The statistical significance of p=0.017 was however very clear and we do hope other studies will confirm our results.
The paediatric hypothermic cardiopulmonary bypass causes elevation of von Willebrand factor plasma levels. It seems that the augmentation of vWf values may be independently associated with the rate of hypothermia. Further studies are needed to confirm this hypothesis.
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New Medicine 1/2003
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