Lupus (2014) 23, 986–993 http://lup.sagepub.com
PAPER
Antiphospholipid antibodies in neonates with stroke—a unique entity or variant of antiphospholipid syndrome? Y Berkun1, MJ Simchen2,6, T Strauss3,5,6, S Menashcu4,6, S Padeh1,6 and G Kenet5,6 1
Departments of Pediatrics; 2Obstetrics and Gynecology; 3Neonatology; 4Pediatric Neurology; 5National Hemophilia Center and Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Israel and 6Sackler Medical School, Tel Aviv University, Tel Aviv, Israel
Objective: Antiphospholipid syndrome (APS) may present with thrombosis and persistently elevated titers of antiphospholipid antibodies (aPL) in the neonatal period. Our aim was to investigate the course and impact of elevated titers of aPL in a cohort of infants presenting with either perinatal arterial ischemic stroke (PAS) or cerebral sinus vein thrombosis (CSVT) during the perinatal period. Study design: Sixty-two infants with clinically and radiologically confirmed PAS or CSVT presenting in the neonatal period underwent thrombophilia workup that included Factor V Leiden (FVL), PII20210A mutation, MTHFR 677T polymorphism, protein C, protein S, aPL namely either circulating lupus anticoagulant (CLA), anticardiolipin antibodies (aCL) or anti-b2-glycoprotein-1 (b2GP1). Mothers also underwent thrombophilia workup. Results: Twelve infants with persistently elevated aPL were prospectively followed. Infants with positive aPL showed no concordance with presence of maternal aPL. All children were followed for a median of 3.5 years (range: nine months to 19 years) with repeated aPL testing every three to six months. Anticoagulant therapy initiation and therapy duration varied at the physician’s discretion. In 10/12 cases aPL decreased to normal range within 2.5 years; one female with complex thrombophilia risk factors required indefinite prolonged anticoagulation. None of the infants showed recurrent thrombosis or any other APS manifestations, despite lack of prolonged anticoagulation. Conclusions: The presence of aPL may be important in the pathogenesis of cerebral thrombosis in neonates. Nevertheless, the nature of thrombophilia interactions in this period and their therapeutic impact warrants further investigation. Lupus (2014) 23, 986–993. Key words: Anticardiolipin antibodies; antiphospholipid syndrome; pregnancy; thrombosis; perinatal arterial stroke; PAS
Introduction Ischemic perinatal stroke was defined in 2006 at an international perinatal stroke workshop (the National Institute of Child Health and Human Development-National Institute of Neurological Disorders and Stroke (NICHD-NINDS) perinatal stroke workshop) as ‘‘a group of heterogeneous conditions in which there is focal disruption of cerebral blood flow secondary to arterial or cerebral Correspondence to: Gili Kenet, National Hemophilia Center, Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, 52621, Israel. Email: [email protected] YB current affiliation: Department of Pediatrics, Hadassah-Hebrew University Medical Center, Mount Scopus, Israel YB and MJS contributed equally to the study and should be regarded as joint first authors on this manuscript. Received 17 November 2013; accepted 25 March 2014
venous thrombosis or embolization, between 20 weeks of fetal life through the 28th postnatal day, confirmed by neuroimaging or neuropathologic studies.’’1 Newborns with cerebral infarction may present either acutely during the neonatal period with neurologic symptoms such as seizures,2,3 or may be clinically asymptomatic until signs of motor impairment or seizures appear. In the latter case, a delayed diagnosis of presumed perinatal arterial ischemic stroke (PAS) is made according to neuroradiologic criteria.3,4 Perinatal stroke may lead to future cerebral palsy (CP) and other neurologic disabilities, including epilepsy and cognitive impairment.5–8 The prevalence of PAS is approximately one in 4000–5000 live births,2,9,10 while cerebral sinus vein thrombosis (CSVT) occurrence, although much rarer (0.7 per 100000), also prevails among neonates.11
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10.1177/0961203314531842
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The pathophysiology of perinatal stroke is complex and multifactorial. Risk factors may relate to both fetal and neonatal disorders as well as maternal and placental conditions. The role of genetic and acquired thrombophilia in the pathogenesis of PAS is quite controversial and not completely understood,12–15 yet the presence of antiphospholipid antibodies (aPL) in neonates has been reported as increasing the risk of PAS occurrence. Maternal aPL may also play a role in the pathogenesis of PAS.14 Antiphospholipid syndrome (APS) is a multisystem autoimmune disease, characterized by arterial and venous thrombosis, recurrent fetal loss, and persistent circulating aPL, such as circulating lupus anticoagulant (CLA), anticardiolipin (aCL) and anti-b2 glycoprotein 1 (b2GP1) antibodies.16,17 A diagnosis of the syndrome can be either primary or secondary to other autoimmune diseases, with 9% to 14% in children with systemic lupus erythematosus (SLE).18,19 APS plays an important role in the pathogenesis of pediatric thromboembolism (TE) and stroke.15,20,21 Pediatric APS is very rare. The preliminary classification criteria for definite APS require the presence of one of the laboratory criteria and one of the clinical criteria in order to comply with the diagnosis.16,17 Because of its rarity, pediatric APS has been addressed by few studies only.22,23 In a previous pediatric APS cohort study we reported a special subgroup of infants presenting with perinatal thrombosis, mostly PAS.24 In the current study we aimed to assess the potential influence of persistent aPL on the clinical expression and outcome of infants diagnosed with perinatal stroke.
Patients and methods Study group Children between the newborn period and 18 years of age diagnosed with cerebral infarction between April 1996 and October 2010 were entered into the Israeli Pediatric Stroke registry, an ongoing prospective registry of pediatric stroke patients, consecutively referred to our tertiary center for thrombophilia workup. PAS was diagnosed by demonstrating either onset of symptoms during the perinatal period and/or late motor impairment, accompanied by magnetic resonance imaging (MRI) or computed tomography (CT) findings of cerebral vascular ischemic lesions or venous occlusion.
PAS definition was applied by experienced neuroradiologists, referring to porencephalic lesions, periventricular cysts, volume loss and brain atrophy as signs of non-acute strokes. Children with symptoms beyond the perinatal period without radiographic confirmation of non-acute, presumed perinatal stroke were excluded. Among 168 pediatric stroke registry patients, 62 children were diagnosed with perinatal stroke. Follow-up and outcome data were obtained from medical records and assessment during visits at ambulatory clinics. Children with seizures, hemiparesis, or other neurological abnormalities requiring speech or motor therapy or prolonged neuro-developmental follow-up were defined as neurologically impaired, whereas children with no such abnormalities were considered neurologically intact. Children in the study group were consecutively referred for laboratory evaluation of thrombophilic risk factors. All thrombophilia testing was performed in the same tertiary referral center laboratory, using standard assays. Thrombophilia data were available on 49/ 62 infants with PAS, and elevated aPL (see definition below) were detected in a sub-cohort of 12/49 infants tested. These infants were prospectively followed with repeated antibody testing every three to six months. Coagulation tests for analysis of thrombophilia Blood samples were obtained on the first referral visit. Blood samples were collected into 3.8% trisodium citrate anti-coagulant in a 9:1 ratio (blood:citrate). Citrated blood was centrifuged within 30 minutes of blood sampling at 2000 g (20 minutes) and plasma aliquots were stored at –35 C until analysis. Prothrombin, partial thromboplastin (PTT), and thrombin times as well as fibrinogen assays were performed using standard techniques. Protein C and antithrombin (AT) activities were measured by chromogenic assays (Baxter Dade, Bonnstrasse, Switzerland). Free protein S antigen was measured by enzyme-linked immunosorbent assay (ELISA) (Gradipore ELISA test kit, Riverside Corporate Park, Australia). Repeatedly low-for-age values established the diagnosis of a coagulation inhibitor deficiency. CLA was determined using two coagulation-based tests: a PTTLA test (Diagnostica, Stago, Ansiers, France) and the dilute Russell’s viper venom test (DRVVT) time expressing the ratio of assay times using a poorphospholipids-containing reagent (LA Screen, Gradipore, Riverside Corporate Park, Australia) as compared with a rich-phospholipid-containing reagent (LA Confirm, Gradipore, Riverside Lupus
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Corporate Park, Australia). Prolonged ratios (above 1.5 or 1.7 for CLA-RVVT and CLA-PTT, respectively) were repeated for confirmation at least 12 weeks following the initial tests. aCL were measured using the Autoenzyme aCL Kit (Cambridge Life Sciences, Cambridge, UK). aCL were considered positive if medium-to-high titers (>20 GPL or MPL units (immunoglobulin (Ig)G phospholipid units or IgM phospholipid units)) were present on two or more occasions at least 12 weeks apart. b2GP1 aCL antibodies were measured using a standard kit (Asco Diagnostica, Germany). Medium (15–20 U/ml) to higher values were repeated at least twice in order to confirm the diagnosis. Genomic DNA was extracted from ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood samples using standard methods. Factor V Leiden (FVL) was detected by polymerase chain reaction (PCR) amplification of a 267 bp fragment and MNII digestion. The C677T polymorphism in the methylene-tetrahydrofolate reductase (MTHFR) gene was identified using the Hinfl cleavage of a 198 bp PCR-amplified product. For identification of the G20210A substitution in the factor II gene (FIIG20210A), a modification of the method used by Poort et al. was performed, as previously described.20
Follow-up Prospective follow-up visits and repeated aPL tests were documented every three to six months in the pediatric ambulatory coagulation clinic. Anticoagulant care was individually tailored at the physician’s discretion.
Ethics The study was approved by the institutional ethics board in concordance with the Declaration of Helsinki. All guardians gave their consent for anonymous data collection of their children’s clinical data as well as thrombophilia workup result.
Statistics The statistical analyses performed included descriptive and Box-plot graphs to describe the distribution of the blood tests’ results during the follow-up
period (six months to 36 months). The antibodies’ tests results during the follow-up period were compared by using Related Samples Freidman’s TwoWay Analysis of Variance by Rank. Analyses were performed using SPSS software, version 19 (SPSS Inc, Chicago, IL, USA). A value of p < 0.05 was considered to be statistically significant.
Results The cohort consisted of 12 Caucasian infants (11 Jews, one of Arab origin) of whom six (50%) were males. Neonatal APS was diagnosed due to PAS (n ¼ 11) or CSVT (n ¼ 1) and persistently elevated aPL. Family history of TEs disclosed two grandfathers with early cardiovascular events (myocardial infarct and stroke at ages 40 and 45 years, respectively) and one brother with pediatric stroke (Table 1). Three mothers had a history of previous obstetric complications: intrauterine fetal death (IUFD) in two cases, and two pregnancy losses in one case. Elevated aPL was discovered in two of 12 mothers tested. One mother presented with persistently elevated CLA and was previously diagnosed with APS due to stroke (at the age of 22 years), and subsequent fetal demise; the second mother was asymptomatic with high levels of anti-b2GP1 IgG antibodies that persisted one year following delivery of the child with PAS. Pregnancies of our PAS cohort were overall normal, apart from the mother with APS who was treated with low molecular weight heparin (LMWH) and aspirin (case #10, Table 1). Eleven pregnancies were singletons, and one was a twin pregnancy, of which a single twin only was affected with PAS. Most infants were born at term, and two were premature deliveries at 26 and 31 weeks (cases #1 and #8, respectively). There were seven spontaneous vaginal deliveries, one vacuum extraction (case #3, Table 1) and four cesarean sections (including cases #1 and #8—both for prematurity, and #4 and #7, see Table 1). All infants were appropriate for gestational age. Perinatal complications (respiratory distress syndrome, sepsis, asphyxia and perinatal thrombosis other than stroke) were observed in the two preterm infants only. Apgar score at one and five minutes were 9 and 10, respectively, in 10 infants, 8 and 9 in one, and one preterm infant had Apgar score of 3 and 6. Mean age of stroke diagnosis was 5.8 8.1 months, range two days to 30 months (Table 1). The most common initial manifestations included seizures (n ¼ 6, diagnosed within two to three days
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Table 1 Demographic and clinical data of infants with PAS
Gender
Maternal obstetric history
1 2
Female Male
Normal Alloimmune TCP
3
Female
Normal
4 5
Male Male
6
Patient #
Family risk factor
Age (months) at diagnosis
Presenting symptom
Imaging
Mother MTHFR677T Mother persistent antibGP1 Grandmother: early stroke
0.1 0.1
Seizure Seizure
Parieto-occipital stroke, IVH Left MCA stroke
0.03
Seizure, full fontanel
Normal Repeated pregnancy losses
NA Mother PS deficiency, grandfather early MI
3.0 7.0
Hemiparesis Hypertonia
Female
Normal
NA
4.0
Dystonic hemiplegia
7
Female
Normal
30.0
Epilepsy, hemiparesis
8
Male
IUFD, umbilical artery clot
9 10
Female Male
Normal IUFD, LMWH
Mother: FVL, father: FIIG20210A Mother persistent FVIII high, brother with stroke NA Mother APS (stroke)
Frontoparietal bleeding, suspected stroke, edema, herniation Left ventriculomegaly Ventriculomegaly, infarct of basal ganglia and left parietal infarct Dilated ventricle, left cerebral hypoplasia, corpus thin, right MCA occlusion Left MCA stroke
11 12
Female Male
Normal Normal
NA NA
8.0
4.0 12.0 0.1 0.1
Perinatal asphyxia, ICH, seizure, hemiplegia
Left parietotemporal stroke
Hemiparesis Hypertonia, developmental delay Seizure Lethargy, seizure
Left MCA stroke Dilated cysterna magnum Infarct right MCA Cerebral sinus vein thrombosis
APS: antiphospholipid syndrome; FVL: Factor V Leiden; ICH: intracranial hemorrhage; IUFD: intrauterine fetal death; IVH: intraventricular hemorrhage; LMWH: low molecular weight heparin therapy; MCA: median cerebral artery; MI: myocardial infarction; NA: not applicable; PAS: perinatal arterial ischemic stroke; PS: protein S; TCP: thrombocytopenia.
following delivery) or hemiparesis/hypertonia (n ¼ 6, diagnosed between three and 30 months of age). The imaging studies (CT or MRI) at presentation revealed median cerebral artery (MCA) stroke (n ¼ 8), intracranial hemorrhage (ICH) (n ¼ 2) brain cysts or atrophy (n ¼ 2) and cerebral sagital sinus thrombosis (n ¼ 1) (Table 1). No hematological, cardiac, skin or other than stroke thrombotic manifestations of APS at presentation were observed. Notably, seven of 12 infants with PAS had delayed diagnosis of presumed perinatal stroke, according to imaging studies. The antibodies detected in our cohort included: elevated anti-b2GP1 IgG (n ¼ 6), IgM (n ¼ 1), aCL IgG (n ¼ 5) IgM (n ¼ 3), LA (n ¼ 6), combined antibodies six of 12, aPL concomitant to another thrombophilia risk factor (three of 12, including MTHFR homozygous 677T (cases #1 and #9, Table 1) or combined FVL-FIIG20210A heterozygosity in one case, #7, see Table1). The type of antibodies in mothers with aPL (n ¼ 2) was concordant with that of their offsprings’. The patients were followed for a median time of 3.5 years (range: one to 19 years; mean: 5.5 7.9 years) with clinic visits of at least every six months.
At the end of the study period, a total of nine children (75%) had some degree of neurological impairments: hemiplegia (n ¼ 1), hemiparesis (n ¼ 2), cerebral palsy (n ¼ 2), dystonia (n ¼ 1), developmental delay (n ¼ 3). Our patients suffered neither recurrent thrombosis, nor autoimmune disease or additional APS manifestations. In 10/12 cases aPL levels decreased to normal range within a median of 2.5 years (range: one year to three years). aPL persisted in two cases only (one with borderline CLA followed until age 9.5 years, and another with high levels of aCL IgG þ IgM until age 29). Figures 1 and 2 show the levels of aCL and b2GP1 antibodies (IgG þ IgM) as tested in our cohort and their variation with age. Similar decline to normal values was noticed for CLA (data not shown). Notably, following the diagnosis of perinatal cerebral event, 11/12 patients, diagnosed with PAS, received no anticoagulant therapy, whereas the single infant out of the five diagnosed in the perinatal period who suffered from perinatal CSVT was treated with LMWH until the age of 6 months. Prolonged anticoagulant therapy was administered to one patient only following the delayed Lupus
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(a)
β2GP1 IgG36mnt
(b)
* mnt – months of age
Figure 1 Progression of b2 glycoprotein 1 antibodies during follow-up in study cohort infants. (a). b2GP1 IgG. (b). b2GP1 IgM. b2GP1: b2-glycoprotein 1; Ig: immunoglobulin; mnt: months of age.
diagnosis of perinatal stroke along with presence of complex thrombophilia (aPL as well as FVL heterozygosity and presence of FIIG20210A polymorphism). This patient, followed for 19 years, underwent successful pregnancy without any complications while receiving LMWH and aspirin therapy and delivered a healthy baby girl, negatively tested for aPL.
Discussion In our study we describe a large cohort of neonates who presented with cerebral TEs and persistent aPL, fulfilling the current criteria for primary APS. The important finding of our prospective cohort follow-up is the absence of recurrent
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(a)
(b)
Figure 2 Progression of anticardiolipin (aCL) antibodies during follow-up in study cohort infants. (a). aCL IgG. (b). aCL IgM. aCL: anticardiolipin; Ig: immunoglobulin; mnt: months of age.
thrombosis or any other APS manifestations in these neonates, despite lack of prolonged anticoagulation, along with gradual disappearance of aPL. Perinatal timing of acquired brain injury including stroke introduces multiple, often competing factors in brain maturation and development that will ultimately determine outcome. These include neuronal maturation and organization, myelination,
pruning, and synaptogenesis. The relative impact of each of these processes on recovery varies depending on fundamental aspects of the injury, including timing and location. Data on the clinical manifestations and the outcome of neonatal APS are very limited.23 In a case series25 of 16 infants with perinatal thrombosis born to mothers with APS, most thrombotic Lupus
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events were arterial (13/16), and among them strokes (eight of 16) prevailed. Persistent aPL was described in two neonates with PAS only. Our cases are different since index cases were infants with PAS and persistent aPL, rather than mothers with APS. The revised APS criteria include a combination of clinical (thrombosis or recurrent abortions) and laboratory (persistent aPL) manifestations.16 These criteria may not be valid for the pediatric population for several reasons. Pediatric patients are less prone to vascular thrombosis because of the rarity of acquired risk factors (e.g. atherosclerosis, pregnancy and use of oral contraceptives), whereas the criteria of obstetric morbidity are not applicable in children. Nevertheless, the same diagnostic criteria are used for classification of pediatric APS without validation in children and neonates.22–26 Although the clinical and laboratory manifestations of our cohort are consistent with the diagnostic criteria of APS, their disease behaved differently. APS is a multi-system disorder. Various hematological, gastrointestinal, cardiac, central nervous system (CNS), skin and renal manifestations have been reported in adult APS patients. However, perinatal cerebral event was the only presenting symptom in our patients. Furthermore, no thrombotic events or any other APS-related manifestations evolved during follow-up, despite lack of long-term anticoagulant therapy. Nonetheless, among adult APS patients, many years may intervene between episodes/events. Whereas among patients with APS females prevail, since risk may be altered by sex hormones, in our neonatal cohort no gender preference was noted. This discrepancy may be explained either by male predominance reported in children with stroke,27 or by the different nature of the disease in this age group. Several environmental factors were suggested as possible triggers for APS, including trauma, invasive procedures, vaccinations, and drugs.26,28 Since elevated levels of aPL antibodies are frequent in the pediatric population26 while thrombosis is quite rare, we assume that an additional trigger is probably required. The stress of delivery may induce de novo aPL formation in neonates. Prematurity, indwelling central lines, dehydration, sepsis and coexistent hereditary thrombophilia may act as a ‘‘second hit’’ mechanism on top of the aPL. Developmental hemostatic alterations typical for the neonatal period such as physiologically impaired coagulation inhibition and fibrinolysis
may considerably increase the risk for perinatal thrombosis.29 Epidemiologic pediatric studies show that from 1 year to puberty pediatric patients are less prone to thrombosis because of developmental reductions in prothrombotic coagulation factors. Prior to age 1 year these factors are in a prothrombotic balance. Indeed, in our study group two infants were premature and suffered from perinatal asphyxia, respiratory distress syndrome (RDS) and sepsis, a term infant with CSVT also suffered from sepsis, and three additional infants had co-morbid hereditary thrombophilic risk factors. All these may have contributed to the pathogenesis of cerebral thrombosis. Recently, several studies have shown that aPL is a risk factor for pediatric thrombotic cerebral events.13,15,24,21 In an Israeli perinatal stroke cohort, persistently elevated aPL prevailed among patients (23.4% vs 5.4% in controls) as well as their mothers.14 In pediatric APS patients, we previously reported a very high rate of CLA (96%), whereas the rate of other aPL was comparable to adult APS studies.26 In our patients with neonatal cerebral events, antibody distribution was quite equal among LA, aCL and anti-b2GP1 antibodies. Notably, anti-b2GP1 IgG increased when repeatedly tested in children younger than 1 year (Figure 2(a)). This phenomenon could be attributed to presence of maternal antibodies as well as frequent infections and recurrent immunizations applied to the infants. Furthermore, long-term follow-up yielded decline of aPL level to normal by age of 3 years in most (10/12) patients. The disappearance of aPL, which may be acquired during or after delivery (because of immunological triggers, stress or infection or transfer of maternal antibodies), is a unique finding for neonatal APS. There are several case reports describing neonatal thrombosis associated with transplacentally acquired antibodies of IgG type.25 The concordance of antibodies detected in both mother-infant pairs may indicate transplacental passage of pathogenic antibodies. In our cohort maternal aPL was detected in two cases only. In one case both mother and son had elevated anti-b2GP1 IgG whereas in the second pair the mother had persistently elevated CLA and aCL-IgG and the child demonstrated abnormal CLA only. Interestingly, within the Israeli PAS cohort of mother-infant pairs, aPL discordance was noted between mothers and their offspring.14 These findings indicate that maternal antibodies are not the major pathogenic factor for neonatal aPL. In summary, although patients in our PAS cohort have diagnostic criteria of APS, their disease
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behaves differently, is transitory and does not recur, unlike patients with neonatal SLE. Thus, perinatal stroke in children with aPL deserves special consideration and may not require anticoagulant therapy unless other risk factors prevail. The contribution of aPL to the multifactorial nature of PAS and perinatal CSVT warrants further investigation. Notably, these patients do fulfill criteria for APS and thus their risk may change as they age. Further studies should address this issue.
Funding This research received no specific grant from any funding agency in the public, commercial, or notfor-profit sectors.
Conflict of interest statement The authors have no conflicts of interest to declare.
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10 Laugesaar R, Kolk A, Tomberg T, et al. Acutely and retrospectively diagnosed perinatal stroke: A population-based study. Stroke 2007; 38: 2234–2240. 11 De Veber G, Andrew M, Adams C, et al. Cerebral sinovenous thrombosis in children. N Engl J Med 2001; 345: 417–423. 12 Gunther G, Junker R, Strater R, et al. Symptomatic ischemic stroke in full-term neonates: Role of acquired and genetic prothrombotic risk factors. Stroke 2000; 31: 2437–2441. 13 Golomb MR. The contribution of prothrombotic disorders to periand neonatal ischemic stroke. Semin Thromb Hemost 2003; 29: 415–424. 14 Simchen MJ, Goldstein G, Lubetsky A, Strauss Z, Schiff E, Kenet G. Factor V Leiden and antiphospholipid antibodies in either mothers or infants increase the risk for perinatal arterial ischemic stroke. Stroke 2009; 40: 65–70. 15 Kenet G, Lu¨tkhoff LK, Albisetti M, et al. Impact of thrombophilia on risk of arterial ischemic stroke or cerebral sinovenous thrombosis in neonates and children: A systematic review and meta-analysis of observational studies. Circulation 2010; 121: 1838–1847. 16 Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006; 4: 295–306. 17 Giannakopoulos B, Krilis SA. The pathogenesis of the antiphospholipid syndrome. N Engl J Med 2013; 368: 1033–1044. 18 Levy DM, Massicotte MP, Harvey E, Hebert D, Silverman ED. Thromboembolism in paediatric lupus patients. Lupus 2003; 12: 741–746. 19 Campos LM, Kiss MH, D’Amico EA, Silva CA. Antiphospholipid antibodies and antiphospholipid syndrome in 57 children and adolescents with systemic lupus erythematosus. Lupus 2003; 12: 820–826. 20 Kenet G, Sadetzki S, Murad H, et al. Factor V Leiden and antiphospholipid antibodies are significant risk factors for ischemic stroke in children. Stroke 2000; 31: 1283–1288. 21 Kenet G, Aronis S, Berkun Y, et al. Impact of persistent antiphospholipid antibodies on risk of incident symptomatic thromboembolism in children: A systematic review and meta-analysis. Semin Thromb Hemost 2011; 37: 802–809. 22 Avcin T, Cimaz R, Meroni PL. Recent advances in antiphospholipid antibodies and antiphospholipid syndromes in pediatric populations. Lupus 2002; 11: 4–10. 23 Avcin T, Cimaz R, Rozman B, Ped-APS Registry Collaborative Group. The Ped-APS Registry: The antiphospholipid syndrome in childhood. Lupus 2009; 18: 894–899. 24 Berkun Y, Padeh S, Barash J, et al. Antiphospholipid syndrome and recurrent thrombosis in children. Arthritis Rheum 2006; 55: 850–855. 25 Boffa MC, Lachassinne E. Infant perinatal thrombosis and antiphospholipid antibodies: A review. Lupus 2007; 16: 634–641. 26 Berkun Y, Kenet G. Pediatric antiphospholipid syndrome. Isr Med Assoc J 2008; 10: 45–47. 27 Normann S, de Veber G, Fobker M, et al. Role of endogenous testosterone concentration in pediatric stroke. Ann Neurol 2009; 66: 754–761. 28 Zinger H, Sherer Y, Goddard G, et al. Common infectious agents prevalence in antiphospholipid syndrome. Lupus 2009; 18: 1149–1153. 29 Andrew M. Developmental hemostasis: Relevance to thromboembolic complications in pediatric patients. Thromb Haemost 1995; 74: 415–425.
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PAPER
Antiphospholipid antibodies in neonates with stroke—a unique entity or variant of antiphospholipid syndrome? Y Berkun1, MJ Simchen2,6, T Strauss3,5,6, S Menashcu4,6, S Padeh1,6 and G Kenet5,6 1
Departments of Pediatrics; 2Obstetrics and Gynecology; 3Neonatology; 4Pediatric Neurology; 5National Hemophilia Center and Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Israel and 6Sackler Medical School, Tel Aviv University, Tel Aviv, Israel
Objective: Antiphospholipid syndrome (APS) may present with thrombosis and persistently elevated titers of antiphospholipid antibodies (aPL) in the neonatal period. Our aim was to investigate the course and impact of elevated titers of aPL in a cohort of infants presenting with either perinatal arterial ischemic stroke (PAS) or cerebral sinus vein thrombosis (CSVT) during the perinatal period. Study design: Sixty-two infants with clinically and radiologically confirmed PAS or CSVT presenting in the neonatal period underwent thrombophilia workup that included Factor V Leiden (FVL), PII20210A mutation, MTHFR 677T polymorphism, protein C, protein S, aPL namely either circulating lupus anticoagulant (CLA), anticardiolipin antibodies (aCL) or anti-b2-glycoprotein-1 (b2GP1). Mothers also underwent thrombophilia workup. Results: Twelve infants with persistently elevated aPL were prospectively followed. Infants with positive aPL showed no concordance with presence of maternal aPL. All children were followed for a median of 3.5 years (range: nine months to 19 years) with repeated aPL testing every three to six months. Anticoagulant therapy initiation and therapy duration varied at the physician’s discretion. In 10/12 cases aPL decreased to normal range within 2.5 years; one female with complex thrombophilia risk factors required indefinite prolonged anticoagulation. None of the infants showed recurrent thrombosis or any other APS manifestations, despite lack of prolonged anticoagulation. Conclusions: The presence of aPL may be important in the pathogenesis of cerebral thrombosis in neonates. Nevertheless, the nature of thrombophilia interactions in this period and their therapeutic impact warrants further investigation. Lupus (2014) 23, 986–993. Key words: Anticardiolipin antibodies; antiphospholipid syndrome; pregnancy; thrombosis; perinatal arterial stroke; PAS
Introduction Ischemic perinatal stroke was defined in 2006 at an international perinatal stroke workshop (the National Institute of Child Health and Human Development-National Institute of Neurological Disorders and Stroke (NICHD-NINDS) perinatal stroke workshop) as ‘‘a group of heterogeneous conditions in which there is focal disruption of cerebral blood flow secondary to arterial or cerebral Correspondence to: Gili Kenet, National Hemophilia Center, Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, 52621, Israel. Email: [email protected] YB current affiliation: Department of Pediatrics, Hadassah-Hebrew University Medical Center, Mount Scopus, Israel YB and MJS contributed equally to the study and should be regarded as joint first authors on this manuscript. Received 17 November 2013; accepted 25 March 2014
venous thrombosis or embolization, between 20 weeks of fetal life through the 28th postnatal day, confirmed by neuroimaging or neuropathologic studies.’’1 Newborns with cerebral infarction may present either acutely during the neonatal period with neurologic symptoms such as seizures,2,3 or may be clinically asymptomatic until signs of motor impairment or seizures appear. In the latter case, a delayed diagnosis of presumed perinatal arterial ischemic stroke (PAS) is made according to neuroradiologic criteria.3,4 Perinatal stroke may lead to future cerebral palsy (CP) and other neurologic disabilities, including epilepsy and cognitive impairment.5–8 The prevalence of PAS is approximately one in 4000–5000 live births,2,9,10 while cerebral sinus vein thrombosis (CSVT) occurrence, although much rarer (0.7 per 100000), also prevails among neonates.11
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The pathophysiology of perinatal stroke is complex and multifactorial. Risk factors may relate to both fetal and neonatal disorders as well as maternal and placental conditions. The role of genetic and acquired thrombophilia in the pathogenesis of PAS is quite controversial and not completely understood,12–15 yet the presence of antiphospholipid antibodies (aPL) in neonates has been reported as increasing the risk of PAS occurrence. Maternal aPL may also play a role in the pathogenesis of PAS.14 Antiphospholipid syndrome (APS) is a multisystem autoimmune disease, characterized by arterial and venous thrombosis, recurrent fetal loss, and persistent circulating aPL, such as circulating lupus anticoagulant (CLA), anticardiolipin (aCL) and anti-b2 glycoprotein 1 (b2GP1) antibodies.16,17 A diagnosis of the syndrome can be either primary or secondary to other autoimmune diseases, with 9% to 14% in children with systemic lupus erythematosus (SLE).18,19 APS plays an important role in the pathogenesis of pediatric thromboembolism (TE) and stroke.15,20,21 Pediatric APS is very rare. The preliminary classification criteria for definite APS require the presence of one of the laboratory criteria and one of the clinical criteria in order to comply with the diagnosis.16,17 Because of its rarity, pediatric APS has been addressed by few studies only.22,23 In a previous pediatric APS cohort study we reported a special subgroup of infants presenting with perinatal thrombosis, mostly PAS.24 In the current study we aimed to assess the potential influence of persistent aPL on the clinical expression and outcome of infants diagnosed with perinatal stroke.
Patients and methods Study group Children between the newborn period and 18 years of age diagnosed with cerebral infarction between April 1996 and October 2010 were entered into the Israeli Pediatric Stroke registry, an ongoing prospective registry of pediatric stroke patients, consecutively referred to our tertiary center for thrombophilia workup. PAS was diagnosed by demonstrating either onset of symptoms during the perinatal period and/or late motor impairment, accompanied by magnetic resonance imaging (MRI) or computed tomography (CT) findings of cerebral vascular ischemic lesions or venous occlusion.
PAS definition was applied by experienced neuroradiologists, referring to porencephalic lesions, periventricular cysts, volume loss and brain atrophy as signs of non-acute strokes. Children with symptoms beyond the perinatal period without radiographic confirmation of non-acute, presumed perinatal stroke were excluded. Among 168 pediatric stroke registry patients, 62 children were diagnosed with perinatal stroke. Follow-up and outcome data were obtained from medical records and assessment during visits at ambulatory clinics. Children with seizures, hemiparesis, or other neurological abnormalities requiring speech or motor therapy or prolonged neuro-developmental follow-up were defined as neurologically impaired, whereas children with no such abnormalities were considered neurologically intact. Children in the study group were consecutively referred for laboratory evaluation of thrombophilic risk factors. All thrombophilia testing was performed in the same tertiary referral center laboratory, using standard assays. Thrombophilia data were available on 49/ 62 infants with PAS, and elevated aPL (see definition below) were detected in a sub-cohort of 12/49 infants tested. These infants were prospectively followed with repeated antibody testing every three to six months. Coagulation tests for analysis of thrombophilia Blood samples were obtained on the first referral visit. Blood samples were collected into 3.8% trisodium citrate anti-coagulant in a 9:1 ratio (blood:citrate). Citrated blood was centrifuged within 30 minutes of blood sampling at 2000 g (20 minutes) and plasma aliquots were stored at –35 C until analysis. Prothrombin, partial thromboplastin (PTT), and thrombin times as well as fibrinogen assays were performed using standard techniques. Protein C and antithrombin (AT) activities were measured by chromogenic assays (Baxter Dade, Bonnstrasse, Switzerland). Free protein S antigen was measured by enzyme-linked immunosorbent assay (ELISA) (Gradipore ELISA test kit, Riverside Corporate Park, Australia). Repeatedly low-for-age values established the diagnosis of a coagulation inhibitor deficiency. CLA was determined using two coagulation-based tests: a PTTLA test (Diagnostica, Stago, Ansiers, France) and the dilute Russell’s viper venom test (DRVVT) time expressing the ratio of assay times using a poorphospholipids-containing reagent (LA Screen, Gradipore, Riverside Corporate Park, Australia) as compared with a rich-phospholipid-containing reagent (LA Confirm, Gradipore, Riverside Lupus
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Corporate Park, Australia). Prolonged ratios (above 1.5 or 1.7 for CLA-RVVT and CLA-PTT, respectively) were repeated for confirmation at least 12 weeks following the initial tests. aCL were measured using the Autoenzyme aCL Kit (Cambridge Life Sciences, Cambridge, UK). aCL were considered positive if medium-to-high titers (>20 GPL or MPL units (immunoglobulin (Ig)G phospholipid units or IgM phospholipid units)) were present on two or more occasions at least 12 weeks apart. b2GP1 aCL antibodies were measured using a standard kit (Asco Diagnostica, Germany). Medium (15–20 U/ml) to higher values were repeated at least twice in order to confirm the diagnosis. Genomic DNA was extracted from ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood samples using standard methods. Factor V Leiden (FVL) was detected by polymerase chain reaction (PCR) amplification of a 267 bp fragment and MNII digestion. The C677T polymorphism in the methylene-tetrahydrofolate reductase (MTHFR) gene was identified using the Hinfl cleavage of a 198 bp PCR-amplified product. For identification of the G20210A substitution in the factor II gene (FIIG20210A), a modification of the method used by Poort et al. was performed, as previously described.20
Follow-up Prospective follow-up visits and repeated aPL tests were documented every three to six months in the pediatric ambulatory coagulation clinic. Anticoagulant care was individually tailored at the physician’s discretion.
Ethics The study was approved by the institutional ethics board in concordance with the Declaration of Helsinki. All guardians gave their consent for anonymous data collection of their children’s clinical data as well as thrombophilia workup result.
Statistics The statistical analyses performed included descriptive and Box-plot graphs to describe the distribution of the blood tests’ results during the follow-up
period (six months to 36 months). The antibodies’ tests results during the follow-up period were compared by using Related Samples Freidman’s TwoWay Analysis of Variance by Rank. Analyses were performed using SPSS software, version 19 (SPSS Inc, Chicago, IL, USA). A value of p < 0.05 was considered to be statistically significant.
Results The cohort consisted of 12 Caucasian infants (11 Jews, one of Arab origin) of whom six (50%) were males. Neonatal APS was diagnosed due to PAS (n ¼ 11) or CSVT (n ¼ 1) and persistently elevated aPL. Family history of TEs disclosed two grandfathers with early cardiovascular events (myocardial infarct and stroke at ages 40 and 45 years, respectively) and one brother with pediatric stroke (Table 1). Three mothers had a history of previous obstetric complications: intrauterine fetal death (IUFD) in two cases, and two pregnancy losses in one case. Elevated aPL was discovered in two of 12 mothers tested. One mother presented with persistently elevated CLA and was previously diagnosed with APS due to stroke (at the age of 22 years), and subsequent fetal demise; the second mother was asymptomatic with high levels of anti-b2GP1 IgG antibodies that persisted one year following delivery of the child with PAS. Pregnancies of our PAS cohort were overall normal, apart from the mother with APS who was treated with low molecular weight heparin (LMWH) and aspirin (case #10, Table 1). Eleven pregnancies were singletons, and one was a twin pregnancy, of which a single twin only was affected with PAS. Most infants were born at term, and two were premature deliveries at 26 and 31 weeks (cases #1 and #8, respectively). There were seven spontaneous vaginal deliveries, one vacuum extraction (case #3, Table 1) and four cesarean sections (including cases #1 and #8—both for prematurity, and #4 and #7, see Table 1). All infants were appropriate for gestational age. Perinatal complications (respiratory distress syndrome, sepsis, asphyxia and perinatal thrombosis other than stroke) were observed in the two preterm infants only. Apgar score at one and five minutes were 9 and 10, respectively, in 10 infants, 8 and 9 in one, and one preterm infant had Apgar score of 3 and 6. Mean age of stroke diagnosis was 5.8 8.1 months, range two days to 30 months (Table 1). The most common initial manifestations included seizures (n ¼ 6, diagnosed within two to three days
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Table 1 Demographic and clinical data of infants with PAS
Gender
Maternal obstetric history
1 2
Female Male
Normal Alloimmune TCP
3
Female
Normal
4 5
Male Male
6
Patient #
Family risk factor
Age (months) at diagnosis
Presenting symptom
Imaging
Mother MTHFR677T Mother persistent antibGP1 Grandmother: early stroke
0.1 0.1
Seizure Seizure
Parieto-occipital stroke, IVH Left MCA stroke
0.03
Seizure, full fontanel
Normal Repeated pregnancy losses
NA Mother PS deficiency, grandfather early MI
3.0 7.0
Hemiparesis Hypertonia
Female
Normal
NA
4.0
Dystonic hemiplegia
7
Female
Normal
30.0
Epilepsy, hemiparesis
8
Male
IUFD, umbilical artery clot
9 10
Female Male
Normal IUFD, LMWH
Mother: FVL, father: FIIG20210A Mother persistent FVIII high, brother with stroke NA Mother APS (stroke)
Frontoparietal bleeding, suspected stroke, edema, herniation Left ventriculomegaly Ventriculomegaly, infarct of basal ganglia and left parietal infarct Dilated ventricle, left cerebral hypoplasia, corpus thin, right MCA occlusion Left MCA stroke
11 12
Female Male
Normal Normal
NA NA
8.0
4.0 12.0 0.1 0.1
Perinatal asphyxia, ICH, seizure, hemiplegia
Left parietotemporal stroke
Hemiparesis Hypertonia, developmental delay Seizure Lethargy, seizure
Left MCA stroke Dilated cysterna magnum Infarct right MCA Cerebral sinus vein thrombosis
APS: antiphospholipid syndrome; FVL: Factor V Leiden; ICH: intracranial hemorrhage; IUFD: intrauterine fetal death; IVH: intraventricular hemorrhage; LMWH: low molecular weight heparin therapy; MCA: median cerebral artery; MI: myocardial infarction; NA: not applicable; PAS: perinatal arterial ischemic stroke; PS: protein S; TCP: thrombocytopenia.
following delivery) or hemiparesis/hypertonia (n ¼ 6, diagnosed between three and 30 months of age). The imaging studies (CT or MRI) at presentation revealed median cerebral artery (MCA) stroke (n ¼ 8), intracranial hemorrhage (ICH) (n ¼ 2) brain cysts or atrophy (n ¼ 2) and cerebral sagital sinus thrombosis (n ¼ 1) (Table 1). No hematological, cardiac, skin or other than stroke thrombotic manifestations of APS at presentation were observed. Notably, seven of 12 infants with PAS had delayed diagnosis of presumed perinatal stroke, according to imaging studies. The antibodies detected in our cohort included: elevated anti-b2GP1 IgG (n ¼ 6), IgM (n ¼ 1), aCL IgG (n ¼ 5) IgM (n ¼ 3), LA (n ¼ 6), combined antibodies six of 12, aPL concomitant to another thrombophilia risk factor (three of 12, including MTHFR homozygous 677T (cases #1 and #9, Table 1) or combined FVL-FIIG20210A heterozygosity in one case, #7, see Table1). The type of antibodies in mothers with aPL (n ¼ 2) was concordant with that of their offsprings’. The patients were followed for a median time of 3.5 years (range: one to 19 years; mean: 5.5 7.9 years) with clinic visits of at least every six months.
At the end of the study period, a total of nine children (75%) had some degree of neurological impairments: hemiplegia (n ¼ 1), hemiparesis (n ¼ 2), cerebral palsy (n ¼ 2), dystonia (n ¼ 1), developmental delay (n ¼ 3). Our patients suffered neither recurrent thrombosis, nor autoimmune disease or additional APS manifestations. In 10/12 cases aPL levels decreased to normal range within a median of 2.5 years (range: one year to three years). aPL persisted in two cases only (one with borderline CLA followed until age 9.5 years, and another with high levels of aCL IgG þ IgM until age 29). Figures 1 and 2 show the levels of aCL and b2GP1 antibodies (IgG þ IgM) as tested in our cohort and their variation with age. Similar decline to normal values was noticed for CLA (data not shown). Notably, following the diagnosis of perinatal cerebral event, 11/12 patients, diagnosed with PAS, received no anticoagulant therapy, whereas the single infant out of the five diagnosed in the perinatal period who suffered from perinatal CSVT was treated with LMWH until the age of 6 months. Prolonged anticoagulant therapy was administered to one patient only following the delayed Lupus
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(a)
β2GP1 IgG36mnt
(b)
* mnt – months of age
Figure 1 Progression of b2 glycoprotein 1 antibodies during follow-up in study cohort infants. (a). b2GP1 IgG. (b). b2GP1 IgM. b2GP1: b2-glycoprotein 1; Ig: immunoglobulin; mnt: months of age.
diagnosis of perinatal stroke along with presence of complex thrombophilia (aPL as well as FVL heterozygosity and presence of FIIG20210A polymorphism). This patient, followed for 19 years, underwent successful pregnancy without any complications while receiving LMWH and aspirin therapy and delivered a healthy baby girl, negatively tested for aPL.
Discussion In our study we describe a large cohort of neonates who presented with cerebral TEs and persistent aPL, fulfilling the current criteria for primary APS. The important finding of our prospective cohort follow-up is the absence of recurrent
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(a)
(b)
Figure 2 Progression of anticardiolipin (aCL) antibodies during follow-up in study cohort infants. (a). aCL IgG. (b). aCL IgM. aCL: anticardiolipin; Ig: immunoglobulin; mnt: months of age.
thrombosis or any other APS manifestations in these neonates, despite lack of prolonged anticoagulation, along with gradual disappearance of aPL. Perinatal timing of acquired brain injury including stroke introduces multiple, often competing factors in brain maturation and development that will ultimately determine outcome. These include neuronal maturation and organization, myelination,
pruning, and synaptogenesis. The relative impact of each of these processes on recovery varies depending on fundamental aspects of the injury, including timing and location. Data on the clinical manifestations and the outcome of neonatal APS are very limited.23 In a case series25 of 16 infants with perinatal thrombosis born to mothers with APS, most thrombotic Lupus
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events were arterial (13/16), and among them strokes (eight of 16) prevailed. Persistent aPL was described in two neonates with PAS only. Our cases are different since index cases were infants with PAS and persistent aPL, rather than mothers with APS. The revised APS criteria include a combination of clinical (thrombosis or recurrent abortions) and laboratory (persistent aPL) manifestations.16 These criteria may not be valid for the pediatric population for several reasons. Pediatric patients are less prone to vascular thrombosis because of the rarity of acquired risk factors (e.g. atherosclerosis, pregnancy and use of oral contraceptives), whereas the criteria of obstetric morbidity are not applicable in children. Nevertheless, the same diagnostic criteria are used for classification of pediatric APS without validation in children and neonates.22–26 Although the clinical and laboratory manifestations of our cohort are consistent with the diagnostic criteria of APS, their disease behaved differently. APS is a multi-system disorder. Various hematological, gastrointestinal, cardiac, central nervous system (CNS), skin and renal manifestations have been reported in adult APS patients. However, perinatal cerebral event was the only presenting symptom in our patients. Furthermore, no thrombotic events or any other APS-related manifestations evolved during follow-up, despite lack of long-term anticoagulant therapy. Nonetheless, among adult APS patients, many years may intervene between episodes/events. Whereas among patients with APS females prevail, since risk may be altered by sex hormones, in our neonatal cohort no gender preference was noted. This discrepancy may be explained either by male predominance reported in children with stroke,27 or by the different nature of the disease in this age group. Several environmental factors were suggested as possible triggers for APS, including trauma, invasive procedures, vaccinations, and drugs.26,28 Since elevated levels of aPL antibodies are frequent in the pediatric population26 while thrombosis is quite rare, we assume that an additional trigger is probably required. The stress of delivery may induce de novo aPL formation in neonates. Prematurity, indwelling central lines, dehydration, sepsis and coexistent hereditary thrombophilia may act as a ‘‘second hit’’ mechanism on top of the aPL. Developmental hemostatic alterations typical for the neonatal period such as physiologically impaired coagulation inhibition and fibrinolysis
may considerably increase the risk for perinatal thrombosis.29 Epidemiologic pediatric studies show that from 1 year to puberty pediatric patients are less prone to thrombosis because of developmental reductions in prothrombotic coagulation factors. Prior to age 1 year these factors are in a prothrombotic balance. Indeed, in our study group two infants were premature and suffered from perinatal asphyxia, respiratory distress syndrome (RDS) and sepsis, a term infant with CSVT also suffered from sepsis, and three additional infants had co-morbid hereditary thrombophilic risk factors. All these may have contributed to the pathogenesis of cerebral thrombosis. Recently, several studies have shown that aPL is a risk factor for pediatric thrombotic cerebral events.13,15,24,21 In an Israeli perinatal stroke cohort, persistently elevated aPL prevailed among patients (23.4% vs 5.4% in controls) as well as their mothers.14 In pediatric APS patients, we previously reported a very high rate of CLA (96%), whereas the rate of other aPL was comparable to adult APS studies.26 In our patients with neonatal cerebral events, antibody distribution was quite equal among LA, aCL and anti-b2GP1 antibodies. Notably, anti-b2GP1 IgG increased when repeatedly tested in children younger than 1 year (Figure 2(a)). This phenomenon could be attributed to presence of maternal antibodies as well as frequent infections and recurrent immunizations applied to the infants. Furthermore, long-term follow-up yielded decline of aPL level to normal by age of 3 years in most (10/12) patients. The disappearance of aPL, which may be acquired during or after delivery (because of immunological triggers, stress or infection or transfer of maternal antibodies), is a unique finding for neonatal APS. There are several case reports describing neonatal thrombosis associated with transplacentally acquired antibodies of IgG type.25 The concordance of antibodies detected in both mother-infant pairs may indicate transplacental passage of pathogenic antibodies. In our cohort maternal aPL was detected in two cases only. In one case both mother and son had elevated anti-b2GP1 IgG whereas in the second pair the mother had persistently elevated CLA and aCL-IgG and the child demonstrated abnormal CLA only. Interestingly, within the Israeli PAS cohort of mother-infant pairs, aPL discordance was noted between mothers and their offspring.14 These findings indicate that maternal antibodies are not the major pathogenic factor for neonatal aPL. In summary, although patients in our PAS cohort have diagnostic criteria of APS, their disease
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behaves differently, is transitory and does not recur, unlike patients with neonatal SLE. Thus, perinatal stroke in children with aPL deserves special consideration and may not require anticoagulant therapy unless other risk factors prevail. The contribution of aPL to the multifactorial nature of PAS and perinatal CSVT warrants further investigation. Notably, these patients do fulfill criteria for APS and thus their risk may change as they age. Further studies should address this issue.
Funding This research received no specific grant from any funding agency in the public, commercial, or notfor-profit sectors.
Conflict of interest statement The authors have no conflicts of interest to declare.
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