Dissertation committee:
Πανουλής Κωνσταντίνος, Καθηγητής, Ιατρική Σχολή, ΕΚΠA
Ιακωβίδου Νικολέττα, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Πολίτου Μαριάννα, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Ξάνθος Θεόδωρος, Καθηγητής, Σχολή Επιστημών Υγείας, Πανεπιστήμιο Δυτικής Αττικής
Βαλσάμη Σερένα, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Μπούτσικου Θεοδώρα, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Ηλιοδρομίτη Ζωή, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Summary:
INTRODUCTION: The function of human hemostasis is based on the delicate balance
between stopping bleeding and avoiding clotting. It consists of 3 main stages: primary
hemostasis, secondary hemostasis and fibrinolysis. Primary hemostasis relies on
vasoconstriction and platelet thrombus formation. Platelets, as key players, participate
through a series of interrelated processes that include adhesion to the injured
endothelium, activation (changes in shape and degranulation), and aggregation into
the initial unstable thrombus. These processes are based on the connection of
macromolecules such as von Willebrand factor (VWF) and collagen with glycoproteins
on platelets’ surface, which lead, through various signaling pathways, to their activation
and degranulation. The microenvironment in combination with shear stress in vessels
determine the connections that will take part in platelet aggregation.
The study of platelet function and primary hemostasis has been based in recent
years on a series of tests based on platelet aggregation, adhesion with high shear
stress conditions (e.g., PFA-100), changes of viscoelastic nature, flow cytometry and
study of degranulation. Each one has distinct advantages and disadvantages. During
last decades, an attempt to apply point-of-care tests (POCTs) has been made, which
aims to their widespread use in clinical practice.
The PFA-100 method is a rapid, simple, and reproducible test to assess
primary hemostasis. It is based on the adhesion, activation, and aggregation of
platelets in conditions of high shear stress, representing, in vitro, the processes of
primary hemostasis. The evaluation of platelet function is based on the time required
to close the hole of a membrane with platelet aggregates. This time is expressed as
closure time (CT). Depending on the cartridge, the membrane is covered with either
collagen and epinephrine (COL/EPI) or collagen and ADP (COL/ADP). The results are
two CTs, the value and combination of which point to various disorders of primary
hemostasis.
Neonatal hemostatic balance is an evolving process that differs from the one
in adults. Regarding primary hemostasis, in vitro platelet hyporeactivity is
compensated, in healthy term neonates, effectively by other mechanisms such as high
VWF levels, high hematocrit, and should be considered part of a carefully balanced neonatal hemostatic system, rather than a developmental defect. Regarding
secondary hemostasis and fibrinolysis, it seems that the reduced levels of coagulation
factors are compensated by the reduced levels of natural inhibitors and the reduced
fibrinolytic capacity. These adaptive changes are known by the term "Developmental
Hemostasis". Whether and how prematurity and various maternal and neonatal
diseases disrupt this sensitive system remains a field of interest and research.
Intrauterine growth restriction (IUGR) is defined as fetal growth rate that is less
than normal for the growth potential of a particular infant based on the race and sex of
the fetus. It is important to distinguish the term “IUGR” from the term “SGA”, which
refers to infants with a birth weight less than the 10th percentile for that gestational
age. IUGR neonates represent 25% of births worldwide, i.e. approximately 30 million
newborns per year. Causes of IUGR include maternal, placental or fetal factors and
neonates are divided into two major categories, the symmetric and the asymmetric
type. IUGR is associated with both immediate postnatal complications and serious
long-term effects. The hematological profile of IUGR neonates is characterized by
increased hematocrit, decreased leukocyte and platelet count, but their hemostatic
profile has not been studied extensively and in depth.
In recent years, the study of the impact of IUGR on neonatal hemostasis seems
to be gaining interest. Thrombocytopenia is a common feature of IUGR neonates
through a variety of proposed pathophysiological mechanisms. The studies concerning
the platelet function of IUGR neonates are few and do not reach clear conclusions,
with some showing hyperreactivity and others hypofunction of platelets in this group of
neonates.
OBJECTIVE: The present study initially aimed to determine the reference range of
CTs in umbilical cord blood samples from healthy appropriate for gestational age
(AGA) preterm and term neonates. The main objective was to determine the CTs in
umbilical cord blood samples of preterm and full-term IUGR neonates, to compare
them with the CTs of AGA neonates and to correlate them with several perinatal
parameters.
SUBJECTS AND METHODS: The population of this study consists of 192 newborns,
who were born at Aretaieio University Hospital between January 2017 and December
2018. In the group of 118 healthy AGA newborns (control), 104 full-term neonates (with
gestational age greater than 37 weeks and normal birth weight for gestational age) and 14 preterm infants (with gestational age of less than 37 weeks and normal birth weight
for gestational age) were included. The group of 74 IUGR neonates consists of 48 fullterm neonates (gestational age greater than 37 weeks, who met criteria for IUGR) and
26 preterm neonates (gestational age less than 37 weeks, who met criteria for IUGR).
A detailed family, obstetric, perinatal and neonatal history was obtained for all
participants of the study. In each pregnancy, the cause of IUGR was recorded. Strict
exclusion criteria were applied to both groups (AGA and IUGR). All blood samples
were obtained from the umbilical vein of a double-clamped umbilical cord by trained
personnel and analyzed, without additional processing, via the PFA-100® - Platelet
Function Analyzer (DADE BEHRING) within 4 hours of blood collection. For each blood
sample, two CTs were recorded in seconds, COL/EPI CT and COL/ADP CT.
RESULTS:
AGA neonates: Our laboratory’s reference values, in cord blood samples, are for term
AGA neonates COL/EPI CT = 76s - 164s and COL/ADP CT= 59s - 85s. For preterm
AGA neonates are COL/EPI CT = 101s - 178s and COL/ADP CT = 64s - 86s. COL/EPI
CT is prolonged in preterm AGA neonates (149s) compared to term AGA neonates
(111s) (p 0.0236). This finding is consistent with the greater platelet hyporeactivity that
accompanies prematurity. There is a negative correlation between COL/ADP CT and
gestational age (Spearman’s r = -0.184, p= 0.0475) and COL/ADP CT and platelet
count (Spearman’s r = -0.188, p= 0.0437).
IUGR neonates: Our study shows that IUGR neonates present prolonged COL/EPI
CTs (132s) compared to AGA neonates (112.5s) (p=0.04), while the corresponding
difference is not found for COLs /ADP CTs. COL/ADP CT is shorter in IUGR neonates
delivered via vaginal delivery (p=0.007), in IUGR neonates whose mothers received
ampicillin peripartum (p=0.005) and prolonged in IUGR neonates whose mothers
received epidural anesthesia (p=0.032). Both COL/EPI CT and COL/ADP CT do not
appear to differ according to the cause of IUGR. COL/EPI CT is prolonged in preterm
IUGR neonates (137s) compared to full-term IUGR neonates (126s) (p=0.001), while
no corresponding difference is observed for COL/ADP CT.
CONCLUSIONS: COL/EPI CT is prolonged in both preterm AGA neonates and
preterm IUGR neonates, reflecting the platelet dysfunction that accompanies
prematurity. As for IUGR neonates, the hyporeactivity of platelets seems to be
expressed through the prolonged COL/EPI CTs. The increased hematocrit could act as a compensatory mechanism explaining the absence of serious bleeding
manifestations. In the group of IUGR neonates, apart from prematurity, we should
consider other perinatal parameters in the evaluation of closure times. The PFA-100
method offers the advantage of a quick and simple initial assessment of primary
hemostasis and is a useful tool in clinical practice. Umbilical cord blood samples could
have a prognostic character among high-risk neonates who require increased
surveillance. Our study could help to spread the use of the PFA-100 method in clinical
neonatology by contributing to an optimum assessment of hemostasis in IUGR
neonates.
