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Phosphorylcholine Coating of Bypass Systems Used for Young Infants Does Not Attenuate the Inflammatory Response

Leiden University, Leyden, South Holland, Netherlands
The Annals of thoracic surgery (Impact Factor: 3.85). 05/2006; 81(4):1455-9. DOI: 10.1016/j.athoracsur.2005.11.058
Source: PubMed
ABSTRACT
Contact of blood with the artificial surfaces of the cardiopulmonary bypass (CPB) system is considered to be a main cause of complement activation. Improving the biocompatibility of the system by reduction of contact activation of blood elements and thereby producing less inflammatory response is evidently desired, especially for neonates and infants who are more susceptible to the deleterious effects of CPB. A phosphorylcholine coating, Phisio, is designed to mimic the natural interfaces of blood. The aim of this study is to compare the influence of a phosphorylcholine-coated CPB system versus an uncoated CPB system on complement activation and clinical outcomes.
In this prospective, randomized, blind, one-center study, 28 neonates and infants with a bodyweight between 3 and 6 kg who were undergoing cardiopulmonary bypass were divided in two groups, the phosphorylcholine group and the control group. Thirteen patients were assigned to the phosphorylcholine group and 15 patients to the control group. Patients with Down syndrome, prematurity, cyanosis, or reoperation were excluded. Complement factor C3b/c, human neutrophil elastase (HNE), interleukin-6, and C-reactive protein were measured before, during, and after CPB. Duration of intensive care stay, ventilation time, highest body temperature, and inotropic medication were the clinical variables.
No significant differences were found between the groups for complement factor C3b/c, HNE, interleukin-6, or C-reactive protein during and after CPB. No clinical differences were observed between the groups.
Phosphorylcholine coating does not attenuate the complement activation during CPB in neonates and infants.

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Available from: Nanning Anes, Feb 06, 2014
DOI: 10.1016/j.athoracsur.2005.11.058
2006;81:1455-1459 Ann Thorac Surg
Augueste Sturk and Robert A.E. Dion
Anjo M. Draaisma, Mark G. Hazekamp, Nanning Anes, Paul H. Schoof, C. Erik Hack,
Attenuate the Inflammatory Response
Phosphorylcholine Coating of Bypass Systems Used for Young Infants Does Not
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Phosphorylcholine Coating of Bypass Systems Used
for Young Infants Does Not Attenuate the
Inflammatory Response
Anjo M. Draaisma, BS, EKP, Mark G. Hazekamp, MD, PhD, Nanning Anes, EKP,
Paul H. Schoof, MD, PhD, C. Erik Hack, PhD, Augueste Sturk, MD, PhD, and
Robert A. E. Dion,
MD, PhD
Departments of Extra Corporeal Circulation and Cardiothoracic Surgery, Leiden University Medical Center, Leiden, Department
of Immunopathology, Sanquin Research at CLB, Amsterdam, and Internal Medicine, Academic Medical Center, Amsterdam, the
Netherlands
Background. Contact of blood with the artificial sur-
faces of the cardiopulmonary bypass (CPB) system is
considered to be a main cause of complement activa-
tion. Improving the biocompatibility of the system by
reduction of contact activation of blood elements and
thereby producing less inflammatory response is evi-
dently desired, especially for neonates and infants
who are more susceptible to the deleterious effects of
CPB. A phosphorylcholine coating, Phisio, is designed
to mimic the natural interfaces of blood. The aim of
this study is to compare the influence of a phosphoryl-
choline-coated CPB system versus an uncoated CPB
system on complement activation and clinical
outcomes.
Methods. In this prospective, randomized, blind, one-
center study, 28 neonates and infants with a bodyweight
between 3 and 6 kg who were undergoing cardiopulmo-
nary bypass were divided in two groups, the phospho-
rylcholine group and the control group. Thirteen patients
were assigned to the phosphorylcholine group and 15
patients to the control group. Patients with Down syn-
drome, prematurity, cyanosis, or reoperation were ex-
cluded. Complement factor C3b/c, human neutrophil
elastase (HNE), interleukin-6, and C-reactive protein
were measured before, during, and after CPB. Duration
of intensive care stay, ventilation time, highest body
temperature, and inotropic medication were the clinical
variables.
Results. No significant differences were found be-
tween the groups for complement factor C3b/c, HNE,
interleukin-6, or C-reactive protein during and after
CPB. No clinical differences were observed between
the groups.
Conclusions. Phosphorylcholine coating does not at-
tenuate the complement activation during CPB in neo-
nates and infants.
(Ann Thorac Surg 2006;81:1455–9)
© 2006 by The Society of Thoracic Surgeons
S
eghaye and colleagues [1] reported that the full-
term neonate shows significant complement acti-
vation and leukocytes stimulation when undergoing
cardiopulmonary bypass (CPB). Complement activa-
tion and leukocytes stimulation may result in postop-
erative organ dysfunction. Contact of blood with the
nonbiologic surfaces of the CPB system has been
designated as the main cause of complement activa-
tion. Improving the biocompatibility of CPB systems by
means of less contact activation of blood elements and
thereby less inflammatory response is evidently desir-
able [2].
A phosphorylcholine coating, Phisio (Dideco, Miran-
dola, Italy), is designed to mimic natural cellular sur-
faces and thereby to avoid recognition by the blood as
foreign material. The outer cell surface is composed of
predominantly phosphorylcholine polar groups that
largely contribute to the nonthrombogenic properties
exhibited by blood cells. Because of the hydrophilic
character of the polar head group, there is less protein
adsorption [3]. Furthermore, phosphorylcholine is a
regular component of the outer cell membrane of all
human cells [4].
In this prospective, randomized, blind, one-center
study, we aimed to compare the effects of phosphoryl-
choline coating versus noncoating of the CPB systems on
complement activation, its effect on leukocytes stimula-
tion, and clinical outcomes in neonates and infants with
a bodyweight between 3 and 6 kg. For this purpose, we
measured C3b/c, a stable complement activation prod-
uct, human neutrophil elastase (HNE), as a marker of
neutrophils degranulation [1], the cytokine interleukin-6
(IL-6), which is a good predictor of clinical outcome [5],
and C-reactive protein (CRP), an acute phase protein, as
a marker for inflammatory response. Clinical variables
were intensive care unit stay, ventilation time, the high-
est body temperature in the first 24 postoperative hours,
and use of inotropic medication.
Accepted for publication Nov 28, 2005.
Address correspondence to Dr Draaisma, Department of ECC, J4-Q-85,
Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, the
Netherlands; e-mail: a.m.draaisma@lumc.nl.
© 2006 by The Society of Thoracic Surgeons 0003-4975/06/$32.00
Published by Elsevier Inc doi:10.1016/j.athoracsur.2005.11.058
CARDIOVASCULAR
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Page 2
Material and Methods
Patients
Twenty-eight patients with a bodyweight between 3
and 6 kg who were undergoing surgical repair of their
congenital heart defects with the use of CPB and
moderate (28
o
C) hypothermia were included in this
prospective, randomized, blind study. The Medical and
Ethical Committee of our institution approved the
study in January 2002. Written informed consent from
parents or guardians was obtained for all patients.
Patients were randomly assigned into the phosphoryl-
choline-coated group (PC group; n 13) or into the
noncoated control group (NC group; n 15). Patients
with Down syndrome, other syndromes or chromo-
somal abnormalities, prematurity, cyanosis (defined as
oxygen saturation lower than 75%), use of circulatory
arrest, and cardiac reoperation were excluded from
this study. Postoperatively, inotropic support was used
if necessary. No steroids, aprotinin, and other medica-
tion that might affect the inflammatory response were
used throughout the study.
Anesthesia
All patients were premedicated with oral midazolam (0.5
mg/kg). Anesthesia was induced with sevoflurane and
continued with midazolam (0.10 mg · kg
-1
·h
-1
), sufen
-
tanil (0.04 ug · kg
-1
· min
-1
), and pancuronium (0.15
mg/kg).
Cardiopulmonary Bypass
Before aortic cannulation, heparin (300 IU/kg) was given,
and the activated clotting time was maintained above 480
seconds during CPB. For all patients, a Dideco Lilliput
D901 (Dideco, Mirandola, Italy) closed system was used,
with the difference of the phosphorylcholine coating. The
coating was from arterial canula to venous canula, from
“tip to tip.” A Stockert roller pump (Stockert Instrumente
GMBH, Munich, Germany) was used, inducing a nonpul-
satile flow. The priming solution of 270 mL consisted of
100 mL fresh frozen plasma, 50 mL 20%, w/v, human
albumin (Sanquin, Amsterdam, Netherlands), Mannitol
(0.5 g/kg), Ringers’ solution, and packed red blood cells if
necessary to maintain a hematocrit of 25%. The CPB flow
was maintained at 2.4 L · m
-2
· min
-1
at 37
o
C and 1.6
L·m
-2
· min
-1
at 25
o
C nasopharyngeal. The alpha-stat
method was used for blood gas management. For myo-
cardial protection, St Thomas cardioplegia solution was
given and repeated every 30 minutes. Ultrafiltration,
conventional or modified, was not used throughout the
study.
Samples
Samples of whole blood (2 mL) were collected in tubes
containing 10 mmol/L ethylenediamine tetraacetic acid,
10 mmol/L benzamidine, and 100 mg/mL soy bean tryp-
sin inhibitor, final concentrations. Sample moments were
after induction of anesthesia but before sternotomy
(baseline), 10 minutes after start of CPB, 5 minutes before
end of CPB, and 15 minutes and 6 hours after protamine
administration. Samples were centrifuged (3,000 rpm, 10
minutes) and stored at -70
o
C until analysis.
Laboratory Measurements
Plasma levels of activated C3 were measured with en-
zyme-linked immunosorbent assay (ELISA) in which
specific monoclonal antibody against a neoepitope on
activated C3 was used to catch the activation fragments,
and biotinlayed polyclonal sheep antibodies against C3
to detect bound complement fragments [6]. As the assay
does not discriminate between C3b, C3bi, or C3c, the
activation products detected in the assay are further
referred to as C3b/c. Results were expressed as nmol/L
C3b/c, referring to an in-house standard with known
levels of activation products. Elastase-1-antitrypsin
complexes were measured with an ELISA in which anti-
elastase antibodies were coated onto an ELISA plate, and
bound complexes detected with biotinylated monoclonal
antibody against complexed-1-antitrypsin. Purified hu-
man neutrophili elastase added to pooled plasma was
used as a standard [6]. Results were expressed as ng/mL
elastase (HNE). Interleukin 6 and CRP were measured
with sandwich-type ELISAs (CLB; Department Immune
Reagents, Amsterdam, Netherlands).
Collection of Clinical Data
Data on intensive care unit stay, ventilation time, highest
body temperature in the first 24 hours, and use of
inotropic medication were collected in a retrograde
fashion.
Statistical Analyses
Statistical analyses were performed using the statistical
computing package SPSS12.01 (SPSS, Chicago, Illinois).
Patients’ data were compared with unpaired t test. Data
corresponding to cytokines values were not normally
distributed. After logarithmic transformation of the raw
data, we used repeated measures analysis of variance
with the Greenhouse-Geisser correction to test for differ-
ences within the groups throughout the five time periods
and between the two groups.
Clinical data were also not normally distributed and
were analyzed with the Mann-Whitney U test. Raw data
are expressed as mean SD values. All p values less than
0.05 were considered statistically significant.
Results
There was no difference between the two groups in
patients’ age (p 0.80) bodyweight (p 0.97), and
diagnoses. The CPB time (p 0.60) and aortic cross-
clamp time (p 0.94) showed no differences between the
groups (Table 1). Four patients in the PC group and 3
patients in the NC group were preoperatively treated
with prostaglandin E
1
(PGE
1
).
No deaths or major complications occurred in either
group.
There was a significant rise in complement factor
C3b/c for both groups, but there was no significant
1456
DRAAISMA ET AL Ann Thorac Surg
COATING IN PEDIATRIC SURGERY 2006;81:1455–9
CARDIOVASCULAR
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difference between the groups. The C3b/c increased
rapidly in both groups after the start of CPB and
reached the highest point after the protamine admin-
istration. Six hours after CPB, complement factor C3b/c
values had almost returned to baseline in both groups
(Table 2). The HNE peaked at the end of the CPB
procedure. The values decreased rapidly and had al-
most returned to baseline after 6 hours. Also, for HNE,
there was a significant time difference in the groups,
but over time, both groups showed equal behavior
(Table 2). Interleukin-6 started to increase slightly
during CPB and rose rapidly after the CPB procedure
and protamine administration. After 6 hours, IL-6 was
still increased (Table 2). The mean baseline of CRP of
the PC group is higher (5.8 8.8 mg/L versus 2.0 4.1
mg/L) compared with that of the control group. When
the patients who received preoperative PGE
1
were left
out, no difference was observed anymore between the
groups at baseline point (0.9 1.5 mg/L versus 1.5
4.2 mg/L). The CRP level did not increase during the
CPB procedure but rapidly increased after the prota-
mine administration. No significant differences were
found between the groups (Fig 1A and B; Table 2). The
mean length of stay in the pediatric intensive care
unit was not different between the two groups. Also
ventilation time, body temperature, and inotropic dos-
age were not significantly different in both groups
(Table 3).
Comment
The present study was performed to compare the effects
of CPB on the inflammatory response and clinical out-
comes of a phosphorylcholine-coated CPB system versus
an uncoated CPB system in pediatric patients with a
bodyweight of 3 to 6 kg. We were not able to show any
differences between the two groups.
Yu and colleagues [3] showed an improved biocom-
patibility during an in vitro study with reduced protein
adsorption and complement activation. The results
during in vivo studies are controversial. De Somer and
colleagues [7] showed lower complement activation
and a better preservation of platelets in a study with 10
Table 1. Demographic and CPB Data
PC Group NC Group
p
ValueMean SD Mean SD
Number of patients 13 15
Demographic data
Age (days)
a
79.4 84.6 87.5 80.4 0.80
Body weight (kg)
a
4.2 0.98 4.2 0.96 0.97
Sex (M/F) 3/10 8/7
Diagnosis
AVSD 0 3
VSD 5 2
TAPVC 1 2
Truncus arteriosus 0 1
TGA 5 5
TOF 0 1
DORV 1 1
ASD 1 0
CPB data
CPB time (min) 117.5 48.6 108.6 38.0 0.60
Clamp time (min) 68.9 32.1 69.7 26.3 0.94
a
Values expressed as mean.
ASD atrial septal defect; AVSD Atrioventricular septum defect;
CPB cardiopulmonary bypass; DORV double outlet right ven-
tricle; min minutes; NC noncoated control group; PC
phosphorylcholine-coated group; p value Student t test; TAPVC
totally abnormal pulmonary connection; TGA transposition of
the great arteries; TOF tetralogy of Fallot; VSD ventricular
septal defect.
Table 2. Laboratory Measurement Results
Group t 1
p
Value t 2
p
Value t 3
p
Value t 4
p
Value t 5
p
Value
Complement
factor
C3b/c
(nmol/L)
(SD)
PC
NC
49.1 (36.28)
32.47 (12.12)
0.15 94.08 (42.10)
93.07 (43.63)
0.82 184.62 (57.94)
164.47 (64.70)
0.26 269.46 (126.02)
234.40 (98.32)
0.46 80.31 (52.79)
52.47 (32.40)
0.12
Human
neutrophil
elastase
(ng/mL)
(SD)
PC
NC
33.23 (23.80)
43.86 (79.03)
0.58 29.23 (12.95)
37.60 (54.95)
0.76 150.15 (91.46)
137.20 (109.50)
0.48 130.62 (69.42)
124.07 (101.82)
0.53 96.23 (70.59)
62.73 (25.87)
0.09
Interleukin-6
(pg/mL)
(SD)
PC
NC
5.91 (2.49)
5.42 (1.14)
0.56 6.07 (3.16)
6.16 (2.46)
0.77 15.85 (14.56)
25.67 (19.66)
0.16 32.95 (24.09)
40.29 (21.75)
0.36 247.84 (230.43)
231.71 (117.56)
0.48
C-Reactive
protein
(mg/L)
(SD)
PC
NC
0.95 (1.58)
1.54 (4.21)
0.22 1.04 (0.96)
1.31 (3.01)
0.25 0.74 (0.57)
1.34 (2.97)
0.36 0.81 (0.82)
1.38 (3.29)
0.26 6.25 (2.69)
6.93 (4.06)
0.78
Values expressed as mean.
PC phosphorylcholine-coated group; NC noncoated control group; t time.
1457Ann Thorac Surg DRAAISMA ET AL
2006;81:1455–9 COATING IN PEDIATRIC SURGERY
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patients [7]. Böning and coworkers [8] demonstrated
that phosphorylcholine coating or heparin coating of
the CPB system used for pediatric surgery causes the
same biologic effects. They also demonstrated that the
group with a larger prime volume shows higher values
of IL-6 and higher tumor necrosis factor- [8]. Horten
and coworkers [9] were not able to show any differ-
ences between a heparin-coated system and an un-
coated system with a study population of 200 patients.
Interestingly, Tárnok and colleagues [10] demon-
strated that children undergoing major cardiovascular
surgery without CPB show almost the same comple-
ment activation as the children undergoing major
cardiovascular surgery with the use of CPB. These
findings are also reported in adults [11, 12].
The inflammatory response after CPB is not only
initiated by the artificial surface of the CPB system,
although it is seen by many as being the main cause [13].
The response is also triggered by the gas-blood inter-
faces, ischemia-reperfusion injury, and other proinflam-
matory stimuli [10, 11]. The duration of the CPB proce-
dure and the condition of the patient play roles in the
extent of the inflammation after CPB [14]. It is also known
that suction and retransfusion of mediastinal shed blood
contribute to the inflammatory response [15].Inour
study, suction and retransfusion was performed equally
in both groups.
The pediatric population is a widely spread population
regarding to the differences in age, bodyweight, diag-
noses, and syndromes. To achieve a homogeneous group
of patients for this study, we used strong exclusion
criteria.
Radical oxygen species (ROS) activates nuclear fac-
tor-B (NF-B), which is an important protein in the
regulation of the acute phase response of inflammation.
Nuclear factor-B stimulates the production of, among
others, IL-1, IL-6, and tumor necrosis factor- [16]. Down
syndrome is a genetic disorder associated with ROS, and
patients with Down syndrome show a wide range of
defects regarding either specific or nonspecific immunity
[17, 18]. Body and blood values of cyanotic patients are
severely affected by the chronic hypoxia, and uncon-
trolled reoxygenation is associated with ROS [19]. Circu-
latory arrest with deep hypothermia gives a higher post-
operative leukocyte count, probably due to a higher
-adrenergic stimulation [20]. Complement levels corre-
late with gestational age, particularly in the late prenatal
period. Preterm infants have lower complement activity
and complement component levels than full-term infants
[21]. Also, neutrophil counts are lower in preterm infants
[22]. Patients indicated for reoperations were excluded
because of the expected longer surgery time.
In conclusion, phosphorylcholine coating does not
attenuate the complement activation during CPB, nor
does it have an influence on clinical outcomes. As
mentioned above, the inflammation after CPB is not
only triggered by the artificial surface but is dependent
upon many factors. Coating alone will not affect the
inflammation as desired. Other agents should be con-
sidered for this purpose, such as ultrafiltration during
or after CPB and the use of corticosteroids and pro-
tease inhibitors.
Fig 1. (A) Values of C-reactive protein (CRP), with patients who
used preoperative prostaglandin E
1
included for statistical analysis.
(Solid line with phosphorylcholine coating; dashed line no
coating.) (B) Values of C-reactive protein (CRP), with patients who
used preoperative prostaglandin E
1
excluded from statistical analy
-
sis. (S