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Seminars in Fetal & Neonatal Medicine 16 (2011) 301e304

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Seminars in Fetal & Neonatal Medicine

journal homepage: www.elsevier .com/locate/s iny

Developmental hemostasis: Primary hemostasis and evaluation of plateletfunction in neonates

T. Strauss a,b,c, R. Sidlik-Muskatel a,b,c, G. Kenet b,c,*aNeonatology and Pediatric Departments of the Safra Children’s Hospital, Tel Hashomer, Israelb Sackler Medical School, Tel Aviv University, Tel Aviv, Israelc Thrombosis Unit, Sheba Medical Center, Tel Hashomer, Israel

Keywords:AggregationFlow cytometryNeonatesPlatelet functionPlatelets

* Corresponding author. Address: Thrombosis Unit,Sheba Medical Center, Tel Hashomer 52621, Israel. Tel3 5351806.

E-mail address: Gii.kenet@sheba.health.gov (G. Ke

1744-165X/$ e see front matter � 2011 Published bydoi:10.1016/j.siny.2011.07.001

s u m m a r y

Hemostasis is a dynamic process and physiologic concentrations of coagulation proteins graduallyincrease with gestational age. Nevertheless, the risk for bleeding in term neonates is counterbalanced bythe protective effects of physiologic deficiencies of the inhibitors of coagulation. Although laboratorydiagnosis of coagulation disorders in infants may be difficult to establish, due to the need to adapt allassays for small amounts of blood and the age-related interpretation required for test results e evalu-ation of infants with secondary hemostatic defects is quite feasible, whereas laboratory assessment ofprimary hemostasis in neonates remains a challenge. While platelet number and volume are similar inneonates as compared to adult values, neonatal platelets certainly exhibit hyporesponsiveness. Analysisof platelet function may include aggregation studies or flow cytometry assays, using fluorescence-stainedmonoclonal antibodies against platelet membranes and cellular antigens. Data on platelet function incorrelation with gestational age are scarce and the duration of platelet hyporeactivity and its clinicalsignificance have not yet been completely elucidated. Whole-blood-based platelet function assays haveshown in neonates as well as in premature infants progressive improvement of clot formation withgestational age. This article reviews platelet function, assessed by various techniques, and its develop-ment in the premature as well as healthy term neonate.

� 2011 Published by Elsevier Ltd.

1. Introduction

Hemostasis is a dynamic process, which evolves in utero. Thus,physiologic concentrations of coagulation proteins graduallyincrease during pregnancy and are lower in premature infants ascompared to full-term babies or healthy children.1e5 Recent studieshave provided reference ranges that delineate age-dependentfeatures of coagulation factors and proteins e these may vary withthe use of different reagents and analyzers.2e4

In the neonate, plasma concentrations of vitamin-K-dependentcoagulation factors (II, VII, IX, X) and contact factors (XI, XII, pre-kallikreine and high molecular weight kininogen) are about 50% ofadult values.2e4Thecapacityofnewborns togenerate thrombin is alsoreduced.6,7 Yet, the hemostatic system is balanced by the protectiveeffects of physiologic deficiencies of the inhibitors of coagulation, aswell as by the decreased fibrinolytic capacity in infants.5e9

National Hemophilia Center,.: þ972 3 5302120; fax: þ972

net).

Elsevier Ltd.

Although laboratory diagnosis of coagulation disorders ininfants may be difficult to establish, due to the need to adapt allcoagulation assays and analytic instruments for small amounts ofblood and the age-related interpretation required for test results,9

still it enables evaluation of infants with secondary hemostasisdefects. On the other hand, data for evaluation of primary hemo-static defects are lacking.

Since neonates exhibit high hematocrit, with presence of largenucleated red cells,10 as well as increased concentrations andenhanced function of von Willebrand factor (VWF) and VWF largemultimers,2,4,11 bleeding time, the test that measures primaryhemostasis, e.g. platelets and vessel wall interaction, is shorter inhealthy neonates as compared to adults.12

Whereas platelet number and volume are similar in neonates ascompared to adult values,13e15 neonatal platelets certainly exhibithyporesponsiveness.16e20

Analysis of platelet function may include aggregation studies orflow cytometry assays, using fluorescence-stained monoclonalantibodies against platelet membranes and cellular antigens. Thesetechniques are troublesome since the first requires relatively largevolume of blood and the second requires special expertise and thus

Table 1Platelet flow cytometry tests reported in neonates.a

Authors Publicationyear

Tests No. ofneonates

Rajasekharet al.18,19

1994, 1997 GPIIbeIIIa, GPIbeIX,P-selectin

30

Kuhne andImbach26

1996 GPIIbeIIIa, GPIbeIX,P-selectin

7

Pietruchaet al.30

2001 GPIbeIX, P-selectin 42

Keh et al.31 2001 GPIIbeIIIa 10Saving

et al.272002 Adhesion receptorsb 7

Hezardet al.21

2003 GPIIbeIIIa, GPIbeIX, P-selectin;PAC-1 and fibrinogen binding

36

Sitaruet al.32

2005 GPIIbeIIIa, GPIbeIX, P-selectin;PAC-1

20 term,37 pre-term

Wasiluket al.34

2008 P-selectin 16 preterm

a Original research studies only.b Fibronectin, laminin, thrombospondin.

T. Strauss et al. / Seminars in Fetal & Neonatal Medicine 16 (2011) 301e304302

cannot serve as a routine and real-time diagnostic technique. Dataon platelet function in correlation with gestational age are scarceand the duration of platelet hyporeactivity and its clinical signifi-cance have not yet been completely elucidated.21 Whole bloodplatelet function assays have demonstrated physiologicallyimproved platelet function that correlates with gestational age.22

This chapter will review platelet function, assessed by varioustechniques, and its development in the premature as well ashealthy term neonate. Rare platelet function disorders are notdiscussed.

2. Neonatal platelet physiology and morphology

Newborn infants have a smaller proportion of reticulatedplatelets as compared to adults. This observation may representa developmental phenomenon related to platelet maturation.23 Theplatelet volume of neonates is similar to adults yet slightly lowerplatelet size in fetal blood was reported.24,25 With regard to ultra-structure of platelets in neonates and premature infants: variationin electron lucencywas reported as well as less visiblemicrotubularstructures, lower numbers of pseudopodia, reduced glycogendeposits and significantly decreased quantities of alpha granules.26

Neonatal platelets exhibit lower expression of adhesion recep-tors.27 These changes may represent age-dependent functionalplatelet maturation processes.16

Platelet surface glycoproteins are already present in fetuses,25

but their expression and differences in response to agonistssuggest deficient platelet function.26e28

3. Platelet aggregation studies

Classical aggregation studies, despite their diagnostic poten-tial, are not routinely used for platelet function assessment inneonates and premature babies, due to the limitation alreadymentioned. Neonatal aggregation response to thrombin andarachidonic acid may be similar to adults whereas aggregation toepinephrine is markedly reduced due to decreased number ofalpha adrenergic receptors on platelets’ surfaces.1,26,28 Collagenhyporeactivity is also noted, despite presence of GPIaeIIa, andmay be explained by a signal transduction defect.26 Deficientthromboxane signaling and response has been documented inplatelets of premature infants.28 Neonatal platelets demonstratelower activation in response to thrombin, TRAP (thrombin acti-vation peptide) and nitric oxide.26e31 In a study of neonatalplatelets obtained from cord blood and peripheral blood of 20term and 37 preterm babies, applying flow cytometry to detectand quantify platelet membrane glycoprotein expression andbinding studies before and after TRAP stimulation, the authorsshowed that platelet reactivity increases with gestational age.32

Another study, of 125 healthy neonates, infants and older chil-dren, showed persistence of unexplained platelet hyporeactivitybeyond the neonatal period.21 Despite the fact that aggregationstudies are reliable and reference values for adults and childrenhave long been established, the large volume of blood requiredfor these assessments reduces their applicability for plateletfunction testing in term and preterm neonates.

4. Flow cytometry studies

Since platelet function is mediated by membrane glycoproteins,detection of surface glycoproteins via flow cytometry and mono-clonal antibodies that bind either to resting or activated plateletsprovides important insights into platelet function. Whole bloodflow cytometry is particularly advantageous for neonatal plateletfunction testing, because only very small blood volumes are

required for analyses.33 It has been shown that neonatal plateletreactivity increases with gestational age32,33 and platelets of verylow birth weight infants are maximally hyporeactive on day 3 or 4post delivery.33 Platelet dysfunction may, at least partially, stemfrom decreased appearance of adhesion receptors27 and low P-selectin expression, manifested by reduced surface CD 62P.34

Whereas some authors claim that platelet function may berestored after 10 days following delivery,33,35 hyporeactivitydetected by flow cytometry studies may persist beyond theneonatal period.21 Table 1 summarizes data of recent neonatalflow cytometry studies, demonstrating the variant surface glyco-proteins and receptors studied and the small number of infantstested. Among the disadvantages of flow cytometry studies, highcost of equipment and reagents, the required experienced labo-ratory staff and the need for prolonged sample processingdiminish the use of these tests as potential routine screening toolsfor neonatal platelet function. Since neonatal platelet physiology iscomplex, standardization of reference values and more studies arecertainly required.

5. Whole blood platelet function analysers: PFA-100, CPA

Assessment of platelet function via whole blood analyzers, suchas PFA-100 or the cone and plate(let) analyzer (CPA) is a relativelysimple and fast technique that measures both aggregation andadhesion under flow conditions, requiring minimal volumes ofblood samples.

ThePlatelet FunctionAnalyzer (PFA-100�) deviceaspiratesa smallsample of anticoagulated blood under constant vacuum froma sample reservoir through a narrow-diameter capillary anda microscopic aperture cut into a membrane. The membrane isimpregnated with the platelet agonists collagen and epinephrine orcollagen and adenosine 50-diphosphate. The presence of thesechemical stimuli, and the high shear rates generated under thestandardized flow conditions, result in platelet adhesion, activation,and aggregation, building a stable platelet plug at the aperture. Thetimerequired toobtain full occlusionof theaperture is reportedas theclosure time (CT). ThemeanCTs of healthychildrenare very similar tothose of healthy adults, but longer than those of healthy neonates.36

The Impact-R [Cone and Plate(let) Analyzer (CPA)] was designedin an attempt to test platelet adhesion and aggregation on a poly-styrene surface under close to physiological shear conditions.37e39

Platelet function tested in cord blood was shown to correlatewith gestational age,22 and further study of 195 full-term and 36preterm infants disclosed that pregnancy-induced hypertension or

Table 2Platelet function tests in neonatal whole blood (including cord blood).a

Authors Publicationyear

Device No. ofneonates

Carcao et al.36 1998 PFA-100 17Levy Shraga et al.22 2006 CPA 195 term,

36 pretermChan et al.42 2007 TEG 24 (<1 year)Strauss et al.41 2010 ROTEM 184 term,

47 preterm

PFA-100, platelet function analyzer; CPA, cone and platelet analyzer; TEG, throm-boelastogram; ROTEM, rotating thromboelastogram.

a Original research studies only.

T. Strauss et al. / Seminars in Fetal & Neonatal Medicine 16 (2011) 301e304 303

gestational diabetes of mothers may be associated with even moreimpaired platelet function of neonates. The clinical impact of thesefindings and their potential predictive value is yet to be studied.39

Importantly, PFA-100 and CPA tests are sensitive to samplestorage and time of testing; the results of the PFA-100 CTs and theCPA are affected by platelet counts and hematocrit levels.37e40

Recent studies of whole blood platelet function in neonates arepresented in Table 2 e despite the relatively larger numbers ofinfants tested, as compared to flow cytometry, no uniform inter-national protocol for neonatal platelet function assays has beenestablished so far.

6. Whole blood assays evaluating primary and secondaryhemostasis: TEG, ROTEM

One of the currently available methods for assessing clotformation by using whole blood and testing small volumes isapplication of thromboelastography. First described more than 50years ago, this method is now gaining popularity an a clinicalassessment tool due to its point-of-care capabilities with real-timeresults, its ability to analyze the initiation of clot formation, actualclot firmness and its fibrinolysis. The use of thromboelastogram(TEG) and rotating thromboelastogram (ROTEM) to evaluate clotformation in neonates has recently been reported,41,42 and refer-ence values for ROTEM clot formation parameters were estab-lished in cord blood of 184 full term and 47 preterm infants.Maximal clot firmness, a parameter affected by platelet numberand function, correlated with gestational age, yet the predictivevalue of clot formation tests in neonates deserves furtherattention.

7. Evaluation of platelet function in neonates conclusions

Developmental hemostasis is an important concept that hasspecific implications in the diagnosis of platelet disorders inneonates.28 The physiological causes responsible for the observedage–related changes in neonatal platelets remain to be elucidatedand are of tremendous biological significance. Whereas the ratio-nale for observed age-related changes in the hemostatic systemremains unknown, determination of risk:benefit ratios for thera-peutic manipulations of the hemostatic system in neonates reliesupon proper diagnosis. Age-appropriate, analyzer- and reagent-specific reference ranges should always be used.

As new assays are developed, these assays need to be validatedin neonates and their clinical utility may still be uncertain.

Conflict of interest statementNone declared.

Funding sourcesNone.

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