Journal of Perinatology (2013) 33,79–82 r 2013 Nature America, Inc. All rights reserved. 0743-8346/13 www.nature.com/jp PERINATAL/NEONATAL CASE PRESENTATION Massive intracranial hemorrhage caused by neonatal alloimmune thrombocytopenia associated with anti-group A antibody

S Kato1, T Sugiura1, H Ueda1, K Ito1, H Kakita1, I Kato1, K Kawabata2, H Ohto2 and H Togari1 1Department of Pediatrics and Neonatology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan and 2Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University School of Medicine, Fukushima, Japan

hemorrhage (ICH), which occurs in approximately 10–20% of Neonatal alloimmune thrombocytopenia (NAIT) is a rare but clinically affected newborns.2 It is well documented that antibodies to human important etiology of intracranial hemorrhage. There have been no reported platelet antigen (HPA) -1a and HPA-4 are the most important cases of intracranial hemorrhage caused by anti-group A or anti-group B cause of NAIT in Caucasians and Asians, respectively. NAIT may antibodies. A Japanese boy weighing 1550 g was born at 37 weeks. He rarely be caused by other antigens, and Curtis et al.3 reported blood suffered from refractory thrombocytopenia and developed severe intracranial group B antibodies as a causative antigen, but little is known about hemorrhage on his second day. Despite repeated platelet, red-cell and fresh- the pathogenesis of NAIT by ABO antigens. We report a case of frozen-plasma transfusions, he died at day 10 of life. Serological studies and severe intracranial hemorrhage associated with NAIT from an genotyping of the patient and his parents were performed. There were no anti-group A isoantibody. incompatible genotypes of platelet antigens between the patient and the mother. Serological studies revealed that the mother had extremely high-titer anti-group A immunoglobulin G2 (4096-fold) that reacted Case strongly with the father’s platelets. The reaction against the father’s platelets A Japanese boy was delivered at 37 weeks of gestation to a 36-year- disappeared when her serum was adsorbed with group A red blood cells. old gravida2 para1 by emergency cesarian section because of non- Maternal anti-group A antibody was associated with NAIT and severe reassuring fetal status. He did not have any relatives who were bilateral intracranial hemorrhage. known to have suffered NAIT previously. The outcome of her Journal of Perinatology (2013) 33, 79–82; doi:10.1038/jp.2011.204 first pregnancy had been spontaneous abortion. Our patient’s Keywords: intracranial hemorrhage; neonatal alloimmune thrombo- measurements at birth were: weight 1550 g (third percentile), cytopenia; intrauterine growth restriction; anti-group A antibody; length 44 cm (hemoglobin was 13.6 g dlÀ1, platelet count by maternal immunoglobulin (Ig) G alloantibodies directed 4 À1 against fetal platelet antigens. In a Japanese population study, was 2.1 Â 10 ml and gastrointestinal bleeding continued. He platelet antibodies were detected in 0.91% of maternal samples and was pale with generalized petechiae, but head ultrasound was thrombocytopenia was observed in 18 of 24 630 (0.07%) neonatal normal. He was transfused with fresh-frozen plasma and platelets. samples.1 The most serious complication of NAIT is intracranial Toxoplasma, rubella, cytomegalovirus and herpes simplex virus serologies were negative. On his first day, his hemoglobin dropped Correspondence: Dr T Sugiura, Department of Pediatrics and Neonatology, Graduate School to 6.4 g dlÀ1 without ICH and he was transfused with group-A of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, packed red cells. On his second day of life, though his platelet Aichi, 467-8601, Japan. 4 À1 E-mail: [email protected] count had increased to 12.4 Â 10 ml , his hemoglobin dropped À1 Received 14 October 2011; revised 7 December 2011; accepted 8 December 2011 to 5.4 g dl and his systolic blood pressure deteriorated despite Severe neonatal alloimmune thrombocytopenia S Kato et al 80

Figure 1 Axial and coronal sections of head computed tomography (CT) of the patient at nineth day of life. The CT showed old subdural and parenchymal hemorrhage around the left temporal lobe and new multiple parenchymal hemorrhages from the frontal to the parietal lobe. transfusion. Ultrasound revealed new ICH, and head computed Hiroshima, Japan) and Genosearch-HLA/A, /B, /C kit, Ver.2 tomography showed a severe subdural hematoma with parenchymal (Medical and Biological Laboratories, Nagoya, Japan), respectively. hemorrhage causing midline shift of the brain stem. Then he was transferred to our tertiary-referral NICU. He was in compensated hemorrhagic shock with continued pallor and generalized Results petechiae, a hemoglobin count of 6.7 g dlÀ1 and a platelet count of The anti-platelet-MPHA-Screen revealed that the patient and 4.5 Â 104 mlÀ1. His total serum bilirubin was 7.0 mg dlÀ1,and his mother had no anti-HPA or anti-HLA antibodies. The parents during his stay in our NICU, he never showed icterus. On his nineth and the case had no HPA or HLA genotype incompatibility day of life, he again developed anemia and thrombocytopenia, (Table 1). However, the mother’s serum sample contained an which was refractory despite continuous transfusion. His head extremely high level of anti-group A antibody (4096-fold) that computed tomography revealed new parenchymal hemorrhage reacted strongly with the father’s platelets. The reaction completely (Figure 1), and he died at tenth day of life. disappeared when the mother’s serum was absorbed with standard Informed consent for postmortem computed tomography, group A red blood cells but not group O red blood cells. Flow autopsy and presentation was obtained from the parents. Autopsy cytometry analysis showed that maternal serum contained showed encephalomalacia, hepatomegaly and splenomegaly. a high level of IgG2 antibody, with a sample to negative control Microscopic examination revealed liver cell necrosis and ratio of 8.3. cholestasis, but there was no hemophagocytosis.

Discussion Methods ICH is the most lethal complication in NAIT-affected newborns. Platelet-reactive antibodies of the patient and mother were A case series reported that the incidence of ICH in babies with NAIT characterized by measuring specificity and titer using mixed is about 14%.6 Wilke et al.7 reported a premature newborn that passive hemagglutination (MPHA)4 and were detected with the suffered from anti-HPA-1a antibody-derived NAIT and showed anti-PLT-MPHA-Screen kit (Beckman Coulter, Tokyo, Japan). massive ICH. Their case underwent operation on the third day of Crossmatching between maternal serum and paternal platelets life for a massive left parenchymal lesion. Our case also had a was performed using the Magnetic-MPHA method.5 subdural and parenchymal hemorrhage and a midline shift of his The IgG subclass isotype was determined by flow cytometry brain stem already at admission to our NICU. After discussion with (Cytomics FC500, Beckman Coulter, Brea, CA, USA) with the pediatric neurosurgeon and the patient’s parents, we concluded commercial murine anti-human monoclonal IgG1, IgG2, IgG3 that his massive ICH with midline shift of the brain stem was and IgG4 antibodies (The Binding Site Group Limited, inoperable. Birmingham, England). Intrauterine fetal demise and intrauterine growth restriction Their HPA and human leukocyte antigen (HLA) were genotyped are other well-known complications of NAIT.8 The most frequent by WAKFlow HPA typing kit Ver.5 (Wakunaga Pharmaceutical, antigen causing NAIT is different among ethnic groups; the HPA-4

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Table 1 Serological studies and genotyping of the patient and his parents

Serological testing Mother Patient Anti-HPA Ab Negative Negative Anti-HLA Ab Negative Negative Anti-group A Ab Positive (4096-fold; normal range 4–64-fold) S/N ratio Subclass IgG1 1.4 IgG2 8.3 Not available IgG3 1.0 IgG4 1.0 Anti-group B Ab Positive (512-fold)

Cross matching between the mother’s serum and the father’s platelet Before absorption Positive (512-fold) After absorption with RBC of group A Negative After absorption with RBC of group O Positive (512-fold)

Genotyping of HPA antigens HPA-1 HPA-2 HPA-3 HPA-4 HPA-5 HPA-6 Naka

Father a/a a/a a/b a/a a/a a/a + Mother a/a a/a a/b a/a a/a a/a + Patient a/a a/a a/b a/a a/a a/a +

Genotyping of HLA antigens HLA-A HLA-B HLA-C

Father A*0201 A*0206 B*3501 B*4403 C*0401 C*1403 Mother A*1101 A*3101 B*1527 B*4501 C*0102 C*0401 Patient A*0206 A*3101 B*1527 B*3501 C*0401 F

Abbreviations: Ab, antibody; HLA, human leukocyte antigen; HPA, human platelet antigen; Ig, immunoglobulin; S/N ratio, sample/negative control fluorescent intensity ratio. S/N>2.0 considered positive reaction. system is the most frequent cause of NAIT in Asians. There are a Secondary intracranial hemorrhage might have occurred in this few reports of NAIT caused by ABO antigens.3 Curtis et al.3 reported case because random-donor-transfused platelets might have been that high-titer blood group antibodies acquired from the mother destroyed by the anti-group A antibody in the patient’s circulation. can cause thrombocytopenia in high-expresser infants. They also Although washed, irradiated maternal platelets or antigen-negative reported that there is a population with platelet expression of group donor platelets are recommended in NAIT, random-donor platelets A antigen that is about 10 to 20 000 times higher than the general are recommended for patients, such as ours who bleed suddenly population.9 Ogasawara et al.10 showed that platelets from a high before any evidence of alloimmunization.11 expresser of blood group B were rapidly destroyed upon being The patient’s parents were counseled that NAIT could recur transfused to a group O patient, supporting the importance of in a subsequent pregnancy. As NAIT tends to be more severe in high-expressor status. subsequent pregnancies, antenatal management is critical. In our case, the direct cross match showed that maternal anti- Although the optimal strategy for prevention and treatment of group A IgG reacted with paternal group A antigen presented on NAIT is controversial,12 maternal intravenous immunoglobulin paternal platelets. Although we could not examine the patient’s and oral glucocorticoid therapy should be considered. own antigen-presenting levels on platelets because of his death, examination of his parents strongly suggested that high-titer anti- Conflict of interest group A antibody from his mother destroyed his platelets, which The authors declare no conflict of interest. expressed the group A antigen inherited from his father. Although we could not genotype for rare antigens or screen for rare antibodies, due to lack of sample availability, our data shows Acknowledgments that the chance that this case of NAIT was caused by such rare We thank Drs Norihisa Koyama, Sumio Fukuda, Keiko Mouri and Patricia phenotypes is very small. Bachiller for their helpful advice.

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References 7 Wilke M, Muller-Hansen I, Wernet D, Wernet D, Na¨gele T, Poets CF. Severe bilateral intracranial haemorrhage due to alloimmune thrombozytopenia in a premature 1 Ohto H, Miura S, Ariga H, Ishii T, Fujimori K, Morita S. The natural history of infant. Arch Dis Child Fetal Neonatal Ed 2009; 94: F427–F428. maternal immunization against foetal platelet alloantigens. Transfus Med 2004; 14: 8 Althaus J, Weir EG, Askin F, Kickler TS, Blakemore K. Chronic villitis in untreated 399–408. neonatal alloimmune thrombocytopenia: an etiology for severe early intrauterine 2 Bussel JB, Zabusky MR, Berkowitz RL, McFarland JG. Fetal alloimmune growth restriction and the effect of intravenous immunoglobulin therapy. Am J Obstet thrombocytopenia. N Engl J Med 1997; 337: 22–26. 3 Curtis BR, Fick A, Lochowicz AJ, McFarland JG, Ball RH, Peterson J et al. Neonatal Gynecol 2005; 193: 1100–1104. alloimmune thrombocytopenia associated with maternal-fetal incompatibility for 9 Curtis BR, Edwards JT, Hessner MJ, Klein JP, Aster RH. Blood group A and B antigens blood group B. Transfusion 2008; 48: 358–364. are strongly expressed on platelets of some individuals. Blood 2000; 96: 1574–1581. 4 Shibata Y, Juji T, Tohyama H, Sakamoto H, Ozawa N, Kano K. Mixed passive 10 Ogasawara K, Ueki J, Takenaka M, Furihata K. Study on the expression of ABH antigens hemagglutination with soluble platelet antigens. Int Arch Allergy Appl Immunol on platelets. Blood 1993; 82: 993–999. 1984; 74: 93–96. 11 Kiefel V, Bassler D, Kroll H, Paes B, Giers G, Ditomasso J et al. Antigen-positive platelet 5 Ohgama J, Yabe R, Tamai T, Nakamura M, Mazda T. A new solid-phase assay system transfusion in neonatal alloimmune thrombocytopenia (NAIT). Blood 2006; 107: using magnetic force on blood group serology. Transfus Med 1996; 6: 351–359. 3761–3763. 6 Ghevaert C, Campbell K, Walton J, Smith GA, Allen D, Williamson LM et al. 12 Rayment R, Brunskill SJ, Soothill PW, Roberts DJ, Bussel JB, Murphy MF. Antenatal Management and outcome of 200 cases of fetomaternal alloimmune thrombocyto- interventions for fetomaternal alloimmune thrombocytopenia. Cochrane Database penia. Transfusion 2007; 47: 901–910. Syst Rev 2011; 11: CD004226.

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