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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 4, July/August 2016
AFRICA
223
stenosis, 32 had isolated pulmonary stenosis and 24 were
healthy. Children with aortic and pulmonary stenosis associated
with other congenital heart diseases were excluded. Children
with hypothyroidism, renal or liver disease, malignancy or
autoimmune disease were also excluded.
A standardised screening questionnaire was used to evaluate
each patient’s bleeding symptoms. Written informed consent was
obtained from the parents of each patient, and the local ethics
committee approved the study. Drugs and foods that could
affect the coagulation tests were stopped one week before the
evaluation. All the children in the study underwent a detailed
physical examination and echocardiographic evaluation.
Transthoracic echocardiography was performed with a Vivid
7 (GE Vingmed, Horten, Norway) echocardiograph. M-modes
of two-dimensional images were obtained from the parasternal
long-axis views. Interventricular septal wall thickness, left
ventricular posterior wall thickness and left ventricular internal
diameters were measured in all the children. Cardiac chamber
sizes and left ventricular systolic and diastolic function were
assessed in accordance with the guidelines of the American
Society of Echocardiography.
6
The mean and peak transvalvular
pressure gradients were calculated using the modified Bernoulli
equation.
Patients with aortic stenosis were classified according to peak
pressure gradient as insignificant (
<
25 mmHg), mild (25–50
mmHg), moderate (50–74 mmHg) or severe (
>
75 mmHg).
7
Patients with pulmonary stenosis were classified according to
peak gradient as mild (
<
36 mmHg), moderate (36–64 mmHg)
and severe (
>
64 mmHg).
8,9
The following blood collection and laboratory assays were
performed: whole blood count, blood group, factor VIII level,
prothrombin time (PT), activated partial thromboplastin time
(aPTT), von Willebrand factor antigen (VWF:Ag), ristocetin
co-factor (VWF:RCo), bleeding time using a platelet-function
analyser (PFA-100; Dade Behring, Marburg, Germany) and
platelet aggregation using a lumi-aggregometer.
The PFA-100 is a high-shear system for
in vitro
testing of
platelet function that simulates primary haemostasis after injury
to a small vessel by determining the closure time of adenosine
diphosphate (ADP) cartridges. It is a highly sensitive way to
screen patients for von Willebrand factor defect.
10,11
Plasma VWF:Ag levels were evaluated by immunoturbido-
metry. VWF:Ag and VWF:RCo levels were standardised accord-
ing to the blood group of each patient. Platelet aggregation
tests were studied in 14 patients in whom the PFA-100 ADP
collagen closure time was prolonged by low levels of VWF:Ag
and VWF:RCo.
A diagnosis of AVWS was established by the following:
(1) acquired history of bleeding; (2) low values of VWF:RCo
and collagen-binding capacity (VWF:CB); and (3) VWF:RCo/
VWF:Ag and VWF:CB/VWF:Ag ratios less than 0.7 in cases of
borderline or normal values of VWF:RCo and VWF:CB, and
no curves in the platelet-aggregation test with ristocetin (RIPA).
2
The gold standard for the detection of structural abnormalities
of VWF is multimer analysis using electrophoretic separation
and immunostaining. However this labour-intensive and
time-consuming assay is not available in many laboratatories.
Interpretation of the von Willebrand profile analysis is
summarised in Table 1.
Statistical analysis
SPSS software version 13.0 (SPSS Inc, Chicago, IL) was used for
analysis. All results were expressed as mean
±
SD. Assessment
of significance between the groups was evaluated with the
chi-squared and Mann–Whitney
U
-test and one-way ANOVA.
Correlations between variables were assessed with Pearson’s
rank-correlation test. A
p
-value
<
0.05 was considered significant.
Results
The study group consisted of 28 patients with aortic stenosis
(23 males, five females), 32 patients with pulmonary stenosis
(18 males, 14 females), and 24 healthy children (14 males, 10
females). The mean ages were 8.09
±
3.73 years in the aortic
stenosis group, 5.72
±
3.79 years in the pulmonary stenosis
group, and 9.12
±
4.70 years in the control group.
A history of bleeding was positive in five patients with aortic
stenosis, three with pulmonary stenosis and in one healthy
child (Table 2). The mean follow-up time in patients with aortic
stenosis was 4.19
±
1.91 years, and 3.28
±
1.98 years in patients
with pulmonary stenosis (Table 3).
Echocardiographic findings: the distribution of patients
grouped by degree of stenosis is provided in Table 4.
Haematological parameters: although the PT was in the
normal range in patients with aortic and pulmonary stenosis,
their PT values were found to be significantly shorter than that
of the control group (
p
=
0.03 and 0.006, respectively). There
was no significant difference between the PTs of the aortic and
pulmonary stenosis groups (
p
=
0.52). The aPTT of the patients
Table 1. Abbreviations and interpretations in the study
Test name
Abbreviation Interpretation
Von Willebrand
factor antigen
VWF:Ag
Measurement of the quantity of VWF
monomers but no information given
about its functional ability
Ristocetin co-factor
assay
VWF:Rco
Measurement of the ability of VWF
to agglutinate formalin-fixed platelets
in presence of ristocetin
Ristocetin-to-VWF
antigen ratio
VWF:Rco/
VWF:Ag
Parameter of the capacity of available
VWF to bind platelets
Collagen-binding
capacity
VWF:CB Measurement of the ability of high
molecular weight VWF multimers to
bind to sub-endothelial collagen
Collagen-binding
capacity-to-VWF
antigen ratio
VWF:CB/
VWF:Ag
Measurement of the biological capac-
ity of available VWF for binding to
collagen.
Platelet-functional
analyser (PFA-100)
closure time (collagen
and epinephrine) or
(collagen and ADP)
PFA-100 CEPI
or PFA-100
CADP
Screening test for primary haemosta-
sis. Evaluates platelet disorders and
functions.
Ristocetin-induced
platelet aggregation
RIPA
Measurement of the ability of various
agonists to platelets to aggravate
in
vitro
activation and platelet-to-platelet
activation
Table 2. Episodes of bleeding of study group
Bleeding type
Patients (n) Diagnosis
Bleeding after circumcision
2
1 AS, 1 PS
Epistaxis
3
1 healthy, 2 AS
Bleeding after minor trauma
2
1 AS, 1 PS
Bleeding after dental extraction
1
AS
Postoperative bleeding
1
PS
AS: aortic stenosis, PS: pulmonary stenosis.