review

Neonatal haemostasis and the management of neonatal thrombosis Andrew Will Department of Paediatric Haematology, Royal Manchester Children’s Hospital, Manchester, UK

Summary Two detailed reviews of the management of neonatal thrombosis were published in 2012; one was an up-dated version of guidance first issued in 2004 and the other was a comprehensive review. Both of these publications gave very similar advice regarding the practical aspects of the indications, dosage and management of antithrombotic therapy. The authors stated that the evidence supporting most of their recommendations for anti-thrombotic therapy in neonates remained weak and so the therapy for a neonate with a thrombosis has to be based on an individualized assessment of estimated risk versus potential benefit. The aim of this present review is to give the treating physician an outline of the unique physiology of neonatal coagulation and how this affects the monitoring, dosing and even the choice of therapeutic strategy for the management of thrombosis in the neonate. Keywords: neonatal thrombosis, thrombophilia, heparin, thrombolysis, recombinant tissue plasminogen activator. Over the last two decades developments in the care of the newborn baby have made it possible for neonatal intensive care units (NICU) to treat newborns delivered as early as 22–23 weeks’ gestation. Many of the advances that have made the treatment of ever smaller and sicker neonates a reality rely on the use of central venous and arterial access. With 15% of all babies admitted to NICU and 50% of those weighing ≤1000 g requiring umbilical venous catheters (UVC) (Veldman et al, 2008) as well as the increasing requirement for umbilical arterial catheters (UAC) and other arterial access, it is not surprising that the incidence of both neonatal venous and arterial thrombosis is increasing. Catheter-related thrombosis accounts for the majority of all thrombotic events in the neonatal period. In a study involving nearly 4734 neonates, 34 (0
7%) had clinical thrombosis (van Elteren et al, 2011), equivalent to 6
8 per 1000 NICU

Correspondence: Dr Andrew Will, Department of Paediatric Haematology, Royal Manchester Children’s Hospital, Oxford Road, Manchester M13 9WL, UK. E-mail: [email protected]

ª 2015 John Wiley & Sons Ltd, British Journal of Haematology

admissions, which was almost three times the number found in the prospective Canadian study published 16 years earlier, of 2
4 per 1000 NICU admissions (Schmidt & Andrew, 1995), and more than the 5
1 per 100 000 live births found in a 2 year German survey in 1997 (Nowak-Gottl et al, 1997). Despite the increasing incidence, neonatal thrombosis remains uncommon but can no longer be considered a rare phenomenon. In the absence of information from large controlled clinical trials, the management of neonatal thrombosis remains largely the subjective choice of the treating team. Nevertheless, the work of the international registries and the many experts working in this field have led to an improved understanding about how to manage the different types of neonatal thromboses and how to use the available antithrombotic drugs more effectively and more safely. The increased in incidence of thrombosis makes the need for large national or international trials more urgent. However the design of these trials will be challenging. There are so many variables that need to be considered; maternal factors, such as pre-eclampsia or diabetes, peri-natal factors, such as asphyxia, traumatic delivery, the effect of different gestational ages on the haemostatic system, the different sites and extent of thrombosis, the differing general condition of the neonates, including the presence or not of haemorrhage, sepsis, dehydration or other medical conditions. There have been many successful trials assessing different approaches to the management of thrombosis in adults but these cannot be simply modified for application in neonatal thrombosis. To a significant extent this is due to marked differences between normal adult and normal neonatal haemostatic physiology (Andrew et al, 1987, 1988; Monagle et al, 2006).

Developmental haemostasis The neonatal haemostatic system differs significantly from that of older children and adults. All aspects of the coagulation system are affected with low levels of pro-coagulants except for Factors V and VIII and von Willebrand factor (VWF); the latter two being elevated throughout the neonatal period. The natural anticoagulants are reduced with the notable exception of alpha-2-macroglobulin, which is increased. The activity of the plasmin/plasminogen system is

doi: 10.1111/bjh.13301

Review also relatively reduced. Notably, the vitamin K-dependent proteins are all reduced, as is anti-thrombin (Andrew et al, 1987, 1988; Monagle et al, 2006). These differences have practical implications for both the dose requirements and laboratory monitoring of anticoagulant and antithrombotic drugs. The reasons for the qualitative and quantitative differences in the developing fetal and neonatal haemostatic system are unclear but probably relate to factors unconnected with blood coagulation. Monagle et al (2006), who first hypothesized that the anti-inflammatory, anti-viral and especially the anti-angiogenic properties of antithrombin III (ATIII) could potentially be detrimental to the developing fetus and neonate, has recently published further animal studies that support this hypothesis; in the neonatal anti-coagulant system the relatively low levels of ATIII are compensated for by supra-adult plasma concentrations of alpha-2-macroglobulin thus permitting unrestricted angiogenesis at a time of intense growth whilst maintaining an effective natural anticoagulant pathway (Monagle et al, 2006; Monagle & Massicotte, 2011). Furthermore, low intra-uterine levels of vitamin K may also be beneficial to the developing embryo. Reduction of the synthesis of the vitamin K-dependent enzyme, Osteocalcin, a promoter of cartilage mineralization, may prevent premature maturation of fetal cartilage though not at levels low enough to produce punctuate dyschondroplasia (Booth, 1997). Similarly, the reduced in-utero production of other, less well understood vitamin K-dependent proteins, such as growth arrest-specific protein 6, which has roles in the control of cell adhesion, cell proliferation and protection against apoptosis, may also be important to normal fetal development (Manfioletti et al, 1993).

adhesive activity due to the presence of large VWF multimers (Chalmers, 2004) and this probably explains why, despite reduced platelet function, the bleeding time in neonates is shorter than in adults and older children (Andrew et al, 1990a). Theoretically at least, the infusion of fully active adult platelets into a neonate might then be potentially thrombogenic. Although not usually considered to be a clinical problem, a recent in-vitro study using blood from full term neonates did demonstrate a reduction in platelet function analyser (PFA)-100 closure times when adult platelets were mixed with newborn plasma (Ferrer-Martin et al, 2011). It would seem plausible that this effect might be even pronounced in pre-term neonates, who are the predominant recipients of platelet transfusions.

The coagulation proteins Under normal physiological conditions, blood remains free flowing. This due to the balanced, dynamic equilibrium of the coagulation proteins that can be considered to act as three interacting systems; the intrinsic and extrinsic prothrombotic coagulation system, which is counteracted by the natural anticoagulant proteins and then any thrombus that does form is broken down by the plasmin/plasminogen pathway. In the neonate there are significant differences in all three of these systems compared to older children and adults. The safe and effective management of neonatal thrombosis has to take the unique features of developmental haemostasis into consideration rather than merely trying to extrapolate from studies in older children and adults.

The pro-coagulants Platelets The human haemostatic mechanism depends on complex, interactive systems of checks and balances between the endothelial cells of the blood vessel wall, the circulating platelets and the haemostatic proteins (Chalmers, 2004). The interaction between the endothelium and platelets is, to a large extent, dependent on the total number of platelets, their function and the activity of VWF. By 22 weeks gestation, the well fetus has a platelet count within the adult normal range at 247  59 9 109/l, which remains at similar levels until term (Forestier et al, 1991) but then rises during the first 9 week of life up to 750 9 109/l (Wiedmeier et al, 2009). However neonates may be less able than adults to respond to platelet stress because, unlike adults who can react by both increasing megakaryocyte ploidy and megakaryocyte numbers, neonates are unable to increase megakaryocyte ploidy and can only respond by increasing the number of megakaryocytes in the marrow (Sola-Visner et al, 2007). Platelet function is also reduced in the neonatal population (Israels et al, 2003; Sitaru et al, 2005). In the healthy neonate the relative platelet hypofunction is compensated for by a high haematocrit and increased levels of VWF with increased 2

The development of the neonatal coagulation system has been well documented (Andrew et al, 1987, 1988; Monagle et al, 2006) and there are clear differences in the level of most coagulation factors compared to adults, with the notable exception of high levels of factor (F) VIII and VWF and VWF function. FV is low on day 1 of life but reaches the adult normal range within days. Overall, the levels of the other pro-coagulants are approximately 50% of normal adult values. These differences are even more marked in pre-term infants but, in the pre-term infant, haemostatic development is accelerated and by 6 months of post-natal life the preterm neonates’ factor assay results are equivalent to those seen in term neonates and are nearly equivalent to adult levels (Andrew et al, 1988).

The natural inhibitor system With the notable exception of a2-macroglobulin, which is approximately 20% higher than normal adult levels in both healthy term and 30-to 36-week premature neonates, all other measured natural inhibitors are significantly reduced during the neonatal period (Andrew et al, 1987, 1988; ª 2015 John Wiley & Sons Ltd, British Journal of Haematology

Review Monagle et al, 2006). In the first few days of life, term infants have mean ATIII levels that are about 30% of the normal adult values and protein C (PC) and Protein S (PS) results that are more than 50% reduced. By the end of the neonatal period for term babies, mean ATIII was approaching the adult range, PS was in the normal adult range whereas PC remained 30% below; the 95% centile ranges were significantly wider in the infants (Andrew et al, 1987, 1988; Monagle et al, 2006). In premature infants (Andrew et al, 1988) ATIII, PC and PS levels were even more reduced at birth compared to term infants, with lower 95% centile values down to 0
14, 12
0 and 14
0 u/ml respectively; equivalent normal adult lower 5th centiles in the same study being ATIII 78
0 u/ml, PC 64
0 u/ml and PS 60
0 u/ml. At day 30 of life the mean ATIII and PS results had risen but only to just over 50% of the adult normal and PC only to 40% of adult normal. There are no studies on infants born before 30 weeks gestation but it is likely that premature infants below 30 weeks gestation have even lower plasma concentrations of ATIII, PC and PS. These findings have major implications for neonates, irrespective of gestational age. The reduction in ATIII is probably the major cause of the relative resistance to heparin anticoagulants in newborns and neonates require relatively higher doses of heparins to reach accepted therapeutic ranges. Furthermore the diagnosis of thrombophilia at the time of presentation becomes problematical, particularly in premature neonates, not just due to the potential consumption of thrombophilic proteins by the thrombotic process itself but because the lower end of the normal ranges is so low as to make discrimination between heterozygote deficiencies and normal very difficult (Greenway et al, 2004). DNA-based diagnoses of thrombophilic disorders (TP) are unaffected by gestational age.

The plasminogen/plasmin system On day one of life, the term neonate has plasminogen levels that are approximately 60% of the normal adult range, tissue plasminogen activator (TPA) is over twice that of a normal adult, a2-antiplasmin (a2AP) is moderately reduced and plasminogen activator inhibitor (PAI) is almost double the adult values (Andrew et al, 1990b; Riverdiau-Moalic et al, 1996; Williams, 2009). In the 30- to 36-week premature infant group plasminogen and a2AP are approximately 10% lower than the term infants with no significant differences in TPA or PAI. By the fifth day of life in both premature and term infants TPA and PAI have already reached adult levels and in the term infants so has a2AP, which remains low in preterm infants. In both groups plasminogen, the major fibrinolytic protein (Chalmers, 2004) remains well below adult values throughout the neonatal period. In the presence of active thrombosis this effect is amplified by the slow rate of plasminogen generation seen in newborn infants (Corrigan et al, 1989). ª 2015 John Wiley & Sons Ltd, British Journal of Haematology

The reduction in plasminogen significantly interferes with the efficacy of thrombolytic therapy throughout the neonatal period (Andrew et al, 1992). Despite this, the levels of D-Dimers, which indicate the extent of active fibrinolysis, are markedly increased in the newborn reflecting activation of the coagulation system during childbirth (Hudson et al, 1990). In their cohort of normal term babies, Monagle et al (2006) found that, in the first 3 d of life, D-Dimers were almost eight times higher than in normal adults. D-Dimer assays cannot be used effectively to diagnose thrombosis during the neonatal period. Nothing is known about how the mode of delivery or prematurity affects the plasminogen/plasmin system proteins. Although the neonatal haemostatic system has both quantitative and qualitative differences, the system seems balanced overall in the well neonate (Andrew et al, 1987, 1988; Monagle et al, 2006) and the healthy neonate has an effective haemostatic system. Nevertheless, using the cascade model of haemostasis it is difficult to appreciate why this is the case. The apparent contradiction of normal neonates having as an effective haemostatic mechanism as older children and adults but with prolongation of the prothrombin time (PT) and the activated partial thromboplastin time (APTT) is probably best explained using the cell-based model of haemostasis (CBMH) (Hoffman & Monroe, 2001), which considers the cohesive interaction between endothelial cells and platelets and the coagulation proteins. In CBMH the primary initiator of coagulation is the release of tissue factor (TF) from TF-bearing cells, usually endothelial cells. TF is released from the cytosol to the surface of damaged endothelial cells and binds to FVII to form a TF/activated FVII (FVIIa) complex, which in turn activates factors IX and X. Activated FX (FXa) activates FV, resulting in a production of a small amount of thrombin. The initial activation of FX by TF/FVIIa is rapidly inhibited by TFPI and ATIII, except on the cell surface, thus localizing the initiation phase to the surface of the damaged endothelial cells. In the amplification phase, platelets adhere to the damaged endothelium and come into contact with the small amounts of thrombin produced on the surface of the TFbearing cells. This partially activates the platelets and the coagulation process moves away from the damaged endothelial cells onto the platelets. VWF-bound FVIII (VWF/FVIII) binds onto the partially activated platelets and VWF is cleaved, leaving FVIIIa on the platelet surface, increasing platelet activation further. In the final propagation phase ‘tenase’ complexes (FVIIa/FIXa) form on the platelet surface and activate ‘prothombinase’ complexes (FXa/FVa), which, in turn, promote the burst of thrombin required for fibrin clot formation. In the neonate TF levels are high compared to adult levels whilst both TFPI and ATIII are present in reduced quantities (Tay et al, 2003). The high levels of TF probably compensate for the relatively low levels of pro-coagulants and, as discussed above, the relative hypo-activity of neonatal 3

Review platelets is counteracted by increased levels of FVIII and VWF. If assessed using appropriate laboratory conditions, neonatal thrombin generation is equivalent to approximately 90% of that in adults; certainly adequate for haemostatic clot formation (Cvirn et al, 2003). Thus, in neonates, the CBMH can explain how, despite the presence inherently of low levels of coagulation factors that would predict inefficient thrombin production by the cascade coagulation theory, increased levels of TF promote formation of effective fibrin clots. In the CBMH, undamaged endothelial cells are considered to be actively anti-thrombotic. One mechanism is the wellunderstood inactivation of thrombin by binding to thrombomodulin on the surface of undamaged cells. However healthy endothelial cells are also able to accelerate the degradation of activated coagulation factors by enhancing the anti-thrombotic effects of ATIII and TFPI. In neonates the reduced levels of ATIII and TFPI diminish the effectiveness of this second mechanism that enables healthy endothelial cells to contain thrombosis and thus may encourage clot propagation. This might partly account for the increased incidence of thrombosis seen in sick neonates.

Thrombophilic disorders and neonatal thrombosis The potential for TP to provoke neonatal thrombosis has been the subject of much debate and differing opinion. As most neonatal thrombosis has a multi-factorial aetiology, it might well be that sick neonates, particularly in the first few days after delivery, with high haematocrit, active sepsis, hypoxic lung disease and central venous or arterial lines, may well have a greater propensity to develop a thrombosis if there is also an underlying primary pro-thrombotic disorder present. The relevant literature is confusing and often does not clearly define important variables, such as the gestational age, the postnatal age when testing was performed and the presence of any relevant family history of thrombosis and there is often no accurate delineation of the presence of other non-TP pro-thrombotic factors. As discussed above, the gestational age and timing of testing related to this is critical to the interpretation of plasma anti-coagulants. Some experts suggest extensive testing of neonates with thrombosis with panels of investigations that can be applied in a stepwise protocol (Nowak-Gottl et al, 2003/2004). Others advocate limiting investigation of neonates to those with clinically significant thromboses, particularly if spontaneous, unanticipated or extensive, or if there is a positive family history of neonatal purpura fulminans (Williams et al, 2002; Seth, 2009). All are agreed that the presence of ischaemic skin lesions or apparently unprovoked extensive thrombosis should immediately instigate investigation for homozygous or bi-heterozygous PC or PS deficiency. Complete absence of PC and the much rarer PS requires urgent emergency 4

treatment with replacement of the missing proteins with fresh frozen plasma or PC concentrates if available and anticoagulation with heparin followed by maintenance with a vitamin K antagonist and prophylactic PC concentrates for PC deficiency (Price et al, 2011). In most cases the levels of PC are almost undetectable in the affected neonates, making the diagnosis obvious despite the sometimes insubstantial plasma concentrations at the lower limit of the neonatal normal range. In cases where there is no family history it may be helpful to confirm that both biological parents are heterozygotes (Williams et al, 2002). Accurate diagnosis of other co-existent TP is only important if its presence would be of prognostic significance, alter the choice or duration of anti-thrombotic therapy, predict recurrence in later life or give families information as to the potential of recurrence in future pregnancies. There is some evidence that this may be the case. Much of this evidence comes from research into neonatal stroke. In a single centre study that followed up 24 infants with perinatal cerebral infarction, 8/11 (73%) of infants with a poor outcome with hemiplegia and global developmental delay had the F5 R506Q (FV Leiden) mutation and/or a raised FVIII whereas only 1/13 (8%) with a good outcome had a TP, suggesting a significant role of pro-thrombotic risk factors, notably F5 R506Q, in the long term outcome of neonatal cerebral infarction (Mercuri et al, 2001). In a 2003 study of recurrence rate of thrombosis in 215 consecutive neonates with arterial stroke with a median follow-up of only 3
5 years, seven children (3
3%) had a second thrombotic event (Kurnik et al, 2003). Initially pro-thrombotic risk factors had been found in 127 (59
1%) of the neonates at presentation. In the recurrent thrombosis group 5 (71
4%) had evidence of a TP. In five of the seven infants with recurrent thrombosis this was triggered by a second episode of ill health, perhaps suggesting that thromboprophylaxis be considered in survivors of neonatal arterial ischaemic stroke with underlying persistent TP to cover operative procedures or periods of significant immobility in later childhood. In a recent comprehensive review of neonatal renal vein thrombosis (RVT), the commonest site of non-catheterrelated neonatal thrombosis, 79/149 (53%) infants tested had at least one underlying TP (Lau et al, 2007). One study reported on the recurrence rate of thrombosis during the follow-up of 59 newborns with RVT (Kosch et al, 2004); at presentation 40/59 (67
8%) had at least one TP compared to only 14/118 (11
9%) of controls. In a limited follow-up with a median of 4 years, 4/59 (6
8%) had a second thrombosis; all four of these had one or more TP. In three of these patients recurrence was during puberty, a time when there is a second, though less pronounced peak of thrombosis that is also associated with a reduction in thrombolytic activity (Siegbahn & Ruusuvaara, 1988). Two of the 4 children with recurrent thrombosis in the study reported by Kosch et al (2004) were from a sub-group of 12 with more than one TP. In an earlier study the same group had followed up 301 ª 2015 John Wiley & Sons Ltd, British Journal of Haematology

Review children with thromboses, of whom 100 were neonates (Nowak-Gottl et al, 2001). With a median follow-up of 7 years (range 6 months–15 years), 31/176 with a single TP recurred, 30/62 with two or more TP recurred and only 3/64 of the group with none. The aetiological role of TP in neonatal thrombosis remains uncertain. There is an excess of TP found in neonates with clinical thrombosis, even in neonatal portal vein thrombosis and neonatal aortic thrombosis where the vast majority of thrombotic events are secondary to the use of UVC and UAC, respectively (Nagel et al, 2010; Williams & Chan, 2011). It may be that the presence of an inherited thrombophilia merely tips the balance in favour of thrombosis in sick neonates with multiple other risk factors at a stage of haematological development that puts them at particular risk. However, late recurrence may be important and although the evidence to confirm this may take many years for registry data to define the extent of the risk, even now neonates with TP, particularly with two or more TP, may need to be considered for thromboprophylaxis at times of increased pro-thrombotic risk in later childhood and adolescence. International co-operation with clear agreement on the range of tests to perform and their timing would greatly assist with the accuracy and analysis of these essential data.

Clinical management of neonatal thrombosis Laboratory factors Accurate laboratory analysis of blood samples is critical to the diagnosis and therapeutic monitoring of neonatal thrombosis. In no other age group is attention to pre-analytical factors more important. The practical difficulties of blood sampling have been well documented (Williams et al, 2002; Chalmers, 2004; Seth, 2009). Laboratory analytical factors are also important and this even extends to the choice of analyser. There are two basic types of coagulometer; those that detect clot formation mechanically by reduction of movement in a metal ball as a clot forms, and those that rely on optical changes in the absorbance of laser light passing through the clot as it forms. Optical systems are affected by the presence of bilirubin and lipids, both of which are particularly common in neonatal plasma. Irrespective of any preference that there might be for one or other type of analyser at other times of life, laboratories serving NICUs may need to consider the need for access to a mechanical system for neonatal samples (Monagle et al, 2006). The pre-analytical and analytical variables coupled with the variation in even simple coagulation screening test results that are dependent on gestational age and the difficulty of obtaining adequate numbers of ‘normal’ neonatal samples, make it almost impossible to develop in-house normal ranges. So quoted laboratory normal values for neonates depend to a large extent on published historical data based ª 2015 John Wiley & Sons Ltd, British Journal of Haematology

on a relatively small number of individuals. The interpretation of neonatal coagulation results is not an exact science.

The therapeutic options The American College of Chest Physicians has recently up-dated its guidance on the management of thrombosis in neonates and children (Monagle et al, 2012). This extensive document provides detailed recommendations for the management of thrombosis with particular attention to neonates and it provides a comprehensive reference for the options available for the management of neonatal thrombosis, including detailed descriptions of the drugs available and dosage regimens for these. The problem for the treating neonatologist is when to treat and how to decide on which option to use. This may be best done as a multi-disciplinary approach involving a paediatric haematologist with advice from radiology, nephrology, cardiology hepatology and vascular surgery as appropriate. In the case of peripheral hospital NICUs, this may require transfer of the sick neonate with a clinically significant thrombosis to a unit in a tertiary children’s hospital to ensure that all the diagnostic and clinical expertise are available on site. In contrast to thrombosis during childhood when venous thromboembolism (VTE) predominates, the distribution of thrombosis during the neonatal period is approximately 50% arterial and 50% venous with up to 90% of VTE being catheter-related (Greenway et al, 2004). Catheter removal is an important part of the management of catheter-related thrombosis. However, even where this is possible, it is suggested that 3–5 d of heparin should precede removal (Monagle et al, 2012) though this advice is not based on clinical trial data (Saxenhouse, 2012). Where lines need to be retained, therapeutic anticoagulation should continue for 6–12 weeks followed by monitoring and heparin prophylaxis until the line can be removed (Monagle et al, 2012). Detection of a thrombus at any site should instigate an immediate assessment for the presence of reversible, co-existent factors, such as polycythaemia, dehydration, hypoxia and sepsis, either alone or in combination. Although a thrombotic event may present with obvious signs relating its location, the presence of an occult thrombosis should be considered where there is prolonged sepsis or there is persisting thrombocytopenia with no obvious other cause (van Elteren et al, 2011). There are three options in the management of neonatal thrombosis; anticoagulation with unfractionated (UFH) or low molecular weight heparin (LMWH), thrombolysis with recombinant TPA (rTPA) or vascular microsurgery. Microsurgery is only applied infrequently and the relevant expertise is not always available, even in tertiary centres. However vascular surgery may have a role to play in the management of thrombosis in a major artery where anticoagulation or thrombolysis are contraindicated. Vascular surgery is included in the schemata developed by Nagel et al (2010) for 5

Review the management of neonatal aortic thrombosis and in a single-centre study of the management of major artery thrombosis, 2/10 infants in whom thrombolysis was contraindicated had vascular microsurgery as first line treatment; both had a good outcome (Ade-Ajayi et al, 2008). There seems little doubt from the literature that thrombolysis is more effective in achieving complete or partial clot lysis than anticoagulation with heparin, which is, in turn, more effective than non-interventional policy (Nowak-Gottl et al, 1997; Manco-Johnson et al, 2002). This view was challenged in a recent small, single centre study in which there was no difference in clot lysis between heparin and a ‘wait and see’ policy (van Elteren et al, 2011). However both groups had what could be considered as low-risk thromboses that might not have warranted interventional anticoagulation anyway. There will have been a tendency to report only positive findings and that may have made active antithrombotic therapy appear more effective than it actually is but, generally, heparins are usually the first option with thrombolysis reserved for neonates with clinically significant thromboses that are life-, organ- or limb-threatening (Williams et al, 2002; Monagle et al, 2012; Saxenhouse, 2012). The balance of risk, mainly in the form of serious haemorrhagic side effects, versus potential benefit is gradually changing as more clinical experience is gained with the use of heparin and rTPA and as more neonates are being exposed to anti-thrombotic therapy at increasingly lower gestational age. It will be important that the various National and International registries continue to monitor and report on the situation. In a proportion of neonates there will be significant contraindications to the use of any antithrombotic therapy. In these it is still important to monitor the thrombus for extension or the development of organ or limb damage, which, if it develops, might well trigger escalation to antithrombotic therapy. Anti-coagulation with heparins. Anticoagulation with heparin can be either with the unfractionated form (UFH) often followed by LMWH or with LMWH alone. Although UFH has been increasingly replaced by LMWH, UFH still has a role to play, particularly at the time of presentation of thrombosis if the infant is clinically unstable. Because of its short half-life it may be useful initially in case there is unanticipated haemorrhage or there is doubt as to whether the child may need to undergo an invasive procedure. UFH has both marked anti-thrombin and anti-Xa activity; the former being responsible for the increase in APTT that is traditionally used to monitor UFH’s anticoagulant effect. APTT monitoring in neonates is problematical because of a raised baseline and a non-linear dose effect on the APTT seen in neonates (Manco-Johnson, 2006). Furthermore, using the APTT to assess the efficacy of UFH requires frequent blood sampling and that often creates practical difficulties in an ill newborn. Ideally anti-Xa activity would be a 6

better assessment of UFH with an aim to achieve anti-Xa activity of between 0
3–0
7 u/ml (Manco-Johnson, 2006; Monagle et al, 2012; Saxenhouse, 2012). This is a lower range than for LMWH because of UFH’s combined effect on thrombin and FXa. At the present time the APTT remains the most common method of monitoring UFH; MancoJohnson (2006) suggests aiming for two to three times the child’s baseline APTT as long as this is within the age-appropriate normal range. Just as with LMWH, care has to be taken with the laboratory anti-Xa assay because there is a tendency to under-estimate the amount of heparin present due to the low levels of neonatal ATIII. This can be addressed by correcting the low ATIII concentration in the assay system or adjusting the initial results using available nomograms (Williams et al, 2002). Low molecular weight heparin offers advantages over UFH that are particularly important in neonates. In particular it is administered sub-cutaneously and, because of its more predictable pharmacokinetics, LMWH requires less frequent monitoring. However, as with UFH, higher doses are required in neonates because of reduced ATIII levels. This is the cause of heparin resistance seen during the neonatal period. The aim is to achieve a 4-h post-dose anti-Xa concentration of between 0
5–1
0 u/ml (Manco-Johnson, 2006; Monagle et al, 2012; Saxenhouse, 2012). Premature neonates require even higher doses than term babies (Monagle et al, 2012). In the light of evidence that prompt attainment of therapeutic antiXa levels may be important to the final outcome (Lulic-Botica et al, 2012; Piersigilli et al, 2013), therapy should be instigated at the age-appropriate dose. However it is well known that neonatal anti-Xa assays are prone to fluctuating results, with only 27% being within the desired therapeutic range in one study and yet, despite this, efficacy appears to be unaffected (Lulic-Botica et al, 2012). Heparin resistance can be addressed by further increases in dose or, if this strategy fails, by replacing ATIII by infusions of fresh frozen plasma; one paper described five neonates who received ATIII concentrate with good effect (Manco-Johnson, 2006). The main side effect of both UFH and LMWH is bleeding. This is probably less with LMWH compared to UFH (Greenway et al, 2004; Saxenhouse, 2012) but it remains the main barrier to the use of heparin anticoagulants in the management of thrombosis in neonates. Although there is a clear relationship between anti-Xa results and efficacy, bleeding usually occurs in the absence of supra-therapeutic anti-Xa levels (Lulic-Botica et al, 2012). UFH should not be given if there is a known allergy to UFH or a history of heparininduced thrombocytopenia (HIT), which although rare, has been described in the neonatal period (Spadone, 1996). LMWH should also not be used if there is a history of allergy to LMWH or previous HIT and should be used with caution within 24 h of an invasive procedure and avoided if possible in the presence of renal failure, which, because LMWH are renally excreted, may lead to accumulation of anti-Xa activity (Seth, 2009). ª 2015 John Wiley & Sons Ltd, British Journal of Haematology

Review Thrombolysis. Recombinant tissue plasminogen activator has superseded both streptokinase (SK) and urokinase (UK), though recombinant urokinase (rUK) is under clinical assessment (Williams, 2009). rTPA is a very powerful thrombolytic but its potential to cause haemorrhage limits its use to neonates with extensive thrombosis or thrombosis that places life, organ function or a limb in danger (Williams et al, 2002; Greenway et al, 2004; Monagle et al, 2012). To reduce the perceived risk of administering rTPA to newborns, a set of contra-indications were developed that are still in use today; these include major surgery or significant bleeding within 10 d, severe asphyxia within 7 d, an invasive procedure within 3 d, a seizure within 2 d, gestational age of 1
0 g/l (Manco-Johnson, 2006). It is clear from the more recent literature that with increasing experience in the use of rTPA, these contraindications are no longer considered to be absolute. Recombinant tissue plasminogen activator is most often administered systemically but it can by also be locally infused directly into the affected blood vessel (Manco-Johnson, 2006; Williams, 2009). rTPA is also very effective when instilled at low dose to unblock central lines. Many different regimens have been described for systemic rTPA. Dosage advice varies from relatively high dose infusions of 0
1–0
6 mg/kg/h for arterial and for some cases of extensive venous thrombosis, usually administered over 6 h to low dose rTPA at 0
01– 0
06 mg/kg/h for up to 48 h for venous thrombosis (Manco-Johnson, 2006; Williams, 2009; Monagle et al, 2012). Theoretically prolonged, low-dose regimens might allow for a longer duration of contact of rTPA with the thrombus and because rTPA preferentially acts on fibrin-bound plasminogen, i.e., acting on the clot surface, this may increase its effectiveness. Because the neonate has low levels of plasminogen, many rTPA protocols include supplementation of plasminogen using infusions of fresh frozen plasma at 10 ml kg/d. Because rTPA does not immediately affect clot propagation, heparin is sometimes used concomitantly, usually at a low dose of 10 lg/kg/h (Manco-Johnson, 2006; Williams, 2009). There is no simple laboratory test that can be used to monitor rTPA therapy. That can only be achieved through radiological follow-up of the thrombus or if there is obvious clinical improvement. Nevertheless, before starting rTPA therapy it is important to ensure that the platelet count is >100 9 109/l and the fibrinogen concentration is >1
0 g/l. It may also be helpful to measure pre-treatment D-Dimers, a simple laboratory test of fibrinolytic activity. D-Dimer assays are usually significantly but unpredictably raised in most neonates and they are also elevated following fibrinolysis induced by rTPA. This can be potentially used to assess the effectiveness of rTPA thrombolysis. Confirmation of effective thrombolysis can be inferred if the post-infusion D-Dimers are elevated compared

ª 2015 John Wiley & Sons Ltd, British Journal of Haematology

to the pre-infusion levels. Where this occurs fibrinolysis has been successfully achieved and increasing the rTPA dose would not be indicated but if the D-dimers remain at or below the pre-treatment level then dose escalation might result in more effective thrombolysis (Manco-Johnson, 2006). Once a thrombus has been lysed, further therapy with rTPA is not indicated and the infant changed to therapeutic doses of UFH or LMWH. The major complication of thrombolytic therapy is bleeding and in the neonate the main concern is intracerebral haemorrhage (ICH). Most paediatric studies and reviews are unsatisfactory in that they include children of all ages, not just neonates, and do not fully detail the different dosage regimens used. Most were published in the 1990’s. A 1997 review of ICH in children that covered the previous 30 years included 83 term and 86 premature infants (Zenz et al, 1997). ICH occurred in 1/83 term neonates and in 11/86 preterm babies, 10 of the 11 preterm infants had been treated in the first week of life. However the incidence of ICH in all neonates increases with reducing gestational age and, as noted by Greenway et al (2004), some of the premature neonates included in Zenz’s review had taken part in a randomized controlled trial and the incidence of ICH (15%) was the same in both the treated and control group. Nowak-Gottl et al (1997) noted minor haemorrhage in 10% and major bleeding in 4% of 182 neonates given thrombolytic therapy. A more recent study (Albisetti, 2006) found higher rates of bleeding in a group of 413 children following thrombolysis with minor bleeding in 22% and major haemorrhagic complications in 15%. Intracranial bleeding remains the major concern in the neonatal period and so, prior to starting treatment with rTPA, brain imaging is mandatory.

Concluding remarks The unique features of neonatal haemostasis make the diagnosis, monitoring and management of thrombosis more challenging than at any other time of life. Currently, the evidence base remains insufficient to give clear guidance to the treating paediatrician as to how to assess the balance between risks and benefits of the available treatment options. In time, the various national and international registries should have clearer data on the effectiveness of more aggressive initial therapy and, importantly, on the long-term outcome of neonates treated with antithrombotic therapy. However, it is not just the registries that need to be pro-active. Groups with an interest in specific sub-types of neonatal thrombosis need to collaborate to produce agreed treatment protocols that can be widely implemented and the outcome data collected. Two recent publications have produced management protocols with treatment based on the severity of neonatal thrombosis in neonatal aortic (Nagel et al, 2010) and portal vein thrombosis (Williams & Chan, 2011), suggesting that this may become possible in the relatively near future.

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Review

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