Official Journal of the British Blood Transfusion Society
Transfusion Medicine
| SHORT COMMUNICATION
Perinatal management of neonatal alloimmune thrombocytopenia associated with anti-group A antibody H. Ueda,1 T. Sugiura,1 K. Katano,2 M. Matsuhashi,3 S. Kato,1 K. Ito,1 R. Nagasaki,1 T. Kato,1 N. H. Tsuno3 & S. Saitoh1 Department of Pediatrics and Neonatology, 2 Department of Obstetrics and Gynecology, Graduate School of Medical Sciences Nagoya City University, Nagoya, Japan, and 3 Department of Transfusion Medicine, The University of Tokyo, Tokyo, Japan
1
Received 9 July 2014; accepted for publication 9 February 2015
SUMMARY Objective: To prevent neonatal alloimmune thrombocytopenia due to anti-group A antibody perinatal management was performed. Background: We previously reported a case of severe intracranial haemorrhage associated with neonatal alloimmune thrombocytopenia due to anti-group A isoantibody. Material/Methods: A 40-year-old Japanese woman, gravida 4 para 1, was pregnant with her second baby. The previous sibling developed severe thrombocytopenia and died 10 days after birth due to intracranial haemorrhage. He was diagnosed with neonatal alloimmune thrombocytopenia; the causative antibody was found to be the anti-group A antibody. Prednisone was started at 7 weeks’ gestational age. Intravenous immunoglobulin 1 g kg−1 week−1 was started at 29 weeks’ gestational age and continued to delivery. Serological studies and genotyping were performed. Results: The second boy was delivered at 33 weeks’ gestational age by caesarean section. He was discharged without intracranial haemorrhage or thrombocytopenia. The anti-group A antibody titre in the maternal serum was 2048–4096 (normal range: 4–64). The anti-group A antibody titre in the newborn’s serum was 4. Cross-matching between the maternal serum and the paternal platelets was positive. Conclusion: Owing to the history of neonatal alloimmune thrombocytopenia causing intracranial haemorrhage and death of the previous sibling, strict follow-up of the subsequent pregnancy was conducted. Key words: anti-group A antibody, immunoglobulin, neonatal alloimmune thrombocytopenia, perinatal management.
Correspondence: Tokio Sugiura, MD, PhD, Department of Pediatrics and Neonatology, Graduate School of Medical Sciences Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81 52 853 8246; fax: +81 52 842 3449; e-mail: [email protected]
First published online 6 March 2015 doi: 10.1111/tme.12178
Neonatal alloimmune thrombocytopenia (NAIT) is a rare but clinically important aetiology of thrombocytopenia of the fetus/neonate caused by maternal immunoglobulin (Ig) G alloantibodies directed against fetal platelet antigens. In a Japanese population study, platelet antibodies were detected in 0·91% of maternal samples, and thrombocytopenia was observed in 18 of 24 630 (0·07%) neonatal samples (Ohto et al., 2004). It is well documented that antibodies to human platelet antigen (HPA)-1a and -4 are the most important cause of NAIT in Caucasians and Asians, respectively. NAIT may rarely be caused by other antigens expressed on the platelet surface, including blood group ABO antigens. Previously, blood group B antibodies have been reported as the causative factor (Curtis et al., 2008). We previously reported a case of severe intracranial haemorrhage (ICH) associated with NAIT due to anti-group A isoantibody (Kato et al., 2013). In the case of a sibling with ICH, the risk of severity of NAIT in the subsequent pregnancy is reported to be higher (Porcelijn et al., 2008). Here, we report a pregnancy subsequent to a case of severe NAIT with ICH, and describe the perinatal management for preventing NAIT due to anti-group A antibody.
MATERIALS AND METHODS Patient A 40-year-old Japanese woman, gravida 4 para 1, was pregnant with her second baby. Her first baby was a male delivered at 37 weeks’ gestation with a birth weight of 1550 g (third percentile). He developed anaemia and severe thrombocytopenia, which was refractory to repeated platelet transfusions. He died on the tenth day of life because of severe ICH associated with severe thrombocytopenia. He was diagnosed with NAIT, and the causative antibody was found to be the anti-group A antibody (Kato et al., 2013). The blood group types of the family were as follows: father, group A, Rh D(+); mother, group O, Rh D(+); first boy, group A, Rh D(+). The outcome of the mother’s first and third pregnancies had been spontaneous abortion. She was referred to our hospital for the prevention of NAIT in the subsequent pregnancy. This study was approved by the Ethics Committee of Nagoya City University.
© 2015 British Blood Transfusion Society
Prevent neonatal alloimmune thrombocytopenia 43
Antenatal treatment Prednisone p.o. 25 mg day−1 was started at 7 weeks’ gestational age. After written informed consent was obtained from the patient, intravenous immunoglobulin (IVIG) 1 g kg−1 week−1 was started at 29 weeks’ gestational age and continued to delivery.
Serological studies and genotyping Detection of anti-HPA and anti-human leukocyte antigen (HLA) antibodies was performed using the anti-PLT-MPHAScreen kit (Beckman Coulter, Tokyo, Japan) and WAKFlow HLA Ab Class I & II (MR) using Luminex method (Wakunaga Pharmaceutical, Hiroshima, Japan), respectively. The genotyping of ABO blood group type, HPA and HLA was performed by the Genosearch ABO kit (Medical and Biological Laboratories, Nagoya, Japan), the WAKFlow HPA typing kit (Wakunaga Pharmaceutical) and the WAKFlow HLA typing kit (Wakunaga Pharmaceutical), respectively. Cross-matching between maternal serum and paternal or the newborn’s platelets was performed using the monoclonal antibody immobilisation of platelet antigens (MAIPA) method as previously described (Matsuhashi et al., 2010). The following mAbs were used in the MAIPA assay, anti-CD31: Gi18, kindly provided by Dr S. Santoso (The Immunohematology Research Department at the Institute for Clinical Immunology and Transfusion Medicine, Giessen, Germany); anti-GPIIb/IIIa: P2 (Acris Antibodies, Herford, Germany); anti-GPIa/IIa: Gi9 (Acris Antibodies); anti-Ib/IX: AK2 (MERCK, Darmstadt, Germany), anti-CD109: W7C5 (MBL, Nagoya, Japan), and anti-HLA class I: W6/32 (abcam, Cambridge UK). The A antigen level on paternal platelets was checked by flow-cytometry.
RESULTS Clinical course The second boy was delivered at 33 weeks’ gestational age by elective caesarean section with a birth weight of 1876 g, suggestive of appropriate for gestational age. The blood type was confirmed to be A, Rh D(+). Blood cell counts were within normal values, with haemoglobin of 17·1 g dL−1 , and platelet count of 276 000 μL−1 . Transient tachypnea was treated only with oxygen therapy, without need for intubation. Head ultrasound and magnetic resonance imaging revealed no ICH. The infant was discharged at 1 month in good general condition, without anaemia or thrombocytopenia.
Serological studies and genotyping Serological studies and the genotyping of the newborn and the parents are shown in Tables 1–4. The ABO genotype of the father and the newborn were A102/A104 and O01/A102, respectively. The titre of the anti-group A antibody in the maternal serum was
© 2015 British Blood Transfusion Society
2048–4096 (normal range: 4–64), when tested against the paternal or the newborn’s red blood cells (RBC), as well as against commercially available RBC (A1 RBC; Bio-Rad, Tokyo, Japan). The titre of anti-group A antibody in the newborn’s serum was 4 (normal range: 4–64). The direct Coombs test using the newborn’s RBC was negative, but the elution test of the newborn revealed 1+ for anti-group A antibody. Cross-matching between the maternal serum and the paternal platelets was positive for anti-CD31 (PECAM-1). However, cross-matching between the maternal serum and the newborn’s platelets at 6 months was negative for anti-CD31 (PECAM-1). The flow-cytometric analysis revealed that the father was not a high expressor for A antigen on platelets.
DISCUSSION Owing to the history of severe NAIT causing ICH and death of the previous sibling, strict follow-up of the subsequent pregnancy was conducted. The previous sibling developed severe thrombocytopenia and anaemia after birth, and died 10 days after due to extensive ICH. Maternal platelet counts were normal, and neither anti-HLA nor anti-HPA antibodies were detected in her serum. However, cross-matching between the maternal serum and paternal platelets was positive, which was confirmed to be dependent on the presence of high-titre anti-A antibody in the maternal serum. Therefore, this case was reported as NAIT caused by anti-A antibody (Kato et al., 2013). Unfortunately, the previous sibling was lost; cross-matching between his platelets and the maternal serum was not possible because it was not feasible to collect blood at the time of birth. Thereafter, the mother experienced a spontaneous abortion, and later became pregnant with the infant described in this report. It has been observed that the severity of NAIT in a subsequent gestation tends to be higher (Porcelijn et al., 2008). There are recommendations in the literature for the use of IVIG for the prevention of severe NAIT, especially its associated complication ICH, in high-risk pregnancies (Espinoza et al., 2013). Although algorithms exist for the prevention or treatment of problems in subsequent siblings of an infant with severe NAIT due to anti-HPA-1 incompatibility (the most common and severe cause of NAIT in Caucasians), it is unknown if an algorithm can be generalised to NAIT caused by other antibodies. Because HPA-1 incompatibility is rare in Japan, no algorithms for the prevention or treatment of NAIT presently exist in Japan. Following the international algorithms, the administration of IVIG has recently become mainstream in Japan and steroids can be added according to the risks in the individual case (Berkowitz et al., 2006). IVIG is usually administered weekly at 1–2 g kg−1 week−1 until delivery. Also, reports exist on intrauterine fetal blood sampling for evaluating the effectiveness of treatment, and the use of intrauterine platelet transfusions (Birchall et al., 2003); however, due to the risk of emergency delivery or fetal death, these procedures are indicated only after 32 weeks’ gestation.
Transfusion Medicine, 2015, 25, 42–46
44 H. Ueda et al. Table 1. Genotyping of ABO, HPA and HLA antigen.
Father Mother Second infant
Type 1
Type 2
A102 O 01 O 01
A104 O 02 A102
HPA type
Father Mother Second infant
Father Mother Second infant
1
2
3
4
5
6
7
15
21
Naka
a/a a/a a/a
a/a a/a a/a
a/b a/b b/b
a/a a/a a/a
a/a a/a a/a
a/a a/a a/a
a/a a/a a/a
a/b b/b b/b
a/a a/a a/a
+ + +
A* 02:01 A* 11:01 A* 02:01
A* 02:06 A* 31:01 A* 11:01
B* 35:01 B* 15:27 B* 15:27
B* 44:03 B* 54:01 B* 44:03
Cw* 04:01 Cw* 01:02 Cw* 04:01
Cw* 14:03 Cw* 04:01 Cw* 14:03
A new platelet specific antigen Nak (a) are usually represented as “Naka ”.
Table 2. The titre of anti-group A antibody.
Table 4. Cross-matching between mother’s serum and father’s or second infant’ platelets by the MAIPA.
Anti-group A Ab
Mother’s serum
Second infant’s serum (at birth)
Father’s RBC Second infant’s RBC A1 RBC
2048–4096 fold 512–2048 fold 2048–4096 fold
N.T. Negative Four-fold
N.T., not tested.
Table 3. The detection of anti-HPA and anti-HLA antibodies. Anti-HPA antibodies were detected by MPHA and MAIPA. Anti-HLA antibodies were detected by the Luminex method. Mother’s serum Anti-HPA Ab Anti-HLA Ab (Class I) Anti-HLA Ab (Class II)
Negative Negative Negative
MPHA, mixed passive hemagglutination.
In Japan, the administration of IVIG in pregnant women at high risk for NAIT is not covered by the Japanese Health Insurance System, and because of the high cost of IVIG in Japan, it is rarely available to most pregnant women for treatment of fetomaternal alloimmune thrombocytopenia. In the present case, considering the severity of NAIT, together with ICH, in the previous sibling, we started prednisolone administration at 7 weeks’ gestation, and IVIG administration at 29 weeks, continuing until delivery. Also, considering that this was a high-risk pregnancy, the baby was delivered by elective caesarean section at 33 weeks’ gestation. For evaluation of the treatment efficacy, intrauterine fetal sampling was considered, but because this was a high-risk procedure and the parents did not give informed consent, it was not carried out.
Transfusion Medicine, 2015, 25, 42–46
Anti-CD31 Anti-GPIIb/IIIa Anti-GPIa/IIa Anti-GPIb/IX Anti-CD109 Anti-HLA Class I
Father’s platelets
Second infant’s platelets
Positive Negative Negative Negative Negative Negative
Negative Negative Negative Negative Negative Negative
Ab, antibody.
There is no consensus on the most appropriate mode of delivery. Porcelijn et al. (2008) recommended that in the case of a previous sibling with NAIT, IVIG administration should be started at 28–32 weeks’ gestation, and caesarean section should be performed at 28–32 weeks in cases where ICH has been observed. In the present case, the treatment strategy was discussed with the parents. Considering the financial aspect, IVIG administration was started at 29 weeks’ gestation, and elective caesarean section was performed at 33 weeks. Because there is no coverage by the Japanese Health Insurance System for the prevention of NAIT, IVIG administration was given for 4 weeks, which was charged to the parents. Also, long-term administration of IVIG starting from early gestation would be a burden to the parents; thus the administration of steroids, of which the cost is less onerous, was indicated in the early phase of gestation. Although anti-A antibody titres in the maternal serum persisted at high levels (2049–4094) throughout the gestation, the infant was born with low levels of anti-A antibody in his serum. Also, no evidence of thrombocytopenia or anaemia was found, suggestive of the effectiveness of steroid therapy plus IVIG administration.
© 2015 British Blood Transfusion Society
Prevent neonatal alloimmune thrombocytopenia 45 Although the mechanism to prevent NAIT in high-risk pregnancies remains to be elucidated, the following speculations can be made: (i) IVIG helps reduce the titre or the activity of anti-HPA antibodies; (ii) IVIG blocks the Fc receptor of the placenta, inhibiting the placental transfer of anti-HPA antibodies (IgG); and/or (iii) IVIG binds to the Fc receptor of fetal macrophages, inhibiting the binding of anti-HPA antibodies bound to the surface of platelets (Espinoza et al., 2013). The same mechanism as for anti-HPA antibody may occur with anti-A antibody. Presently, the adverse effects of long-term IVIG administration to the mother or the fetus are not known. In this case, no adverse events were observed in either the mother or the newborn, but long-term follow-up is required. CD31 captured from the infant’s platelets was not positive when tested with maternal serum, as a surrogate for detection of maternal serum anti-A. The reason why the cross-match between maternal serum and the newborn’s platelets was negative is intriguing. We suspected that the ABO blood type of the newborn was A2, and performed the DNA sequencing of the father and the second infant. The results, however, revealed A1, not A2. It is well-known that PECAM-1 also carry blood group A and B antigens (Curtis et al., 2000), and some individuals are high expressers. Thus, we used the monoclonal to CD31 as the capture antibody in MAIPA to confirm the reactivity of maternal serum with A-antigen. Monoclonals to GPIIbIIIa, GPIbIX, GPIaIIa, CD109 and HLA-I were also used, but resulted negative. The only positive reaction in the MAIPA was for the detection of antibodies against PECAM-1, which was consistent with reactivity with ABO antigens. Because NAIT may occur at the first pregnancy, there are reports of antenatal screening (Kjeldsen-Kragh et al., 2007). HPA-1a is the causative antibody in approximately 85% of NAIT cases in Caucasians; therefore, screening of the maternal serum for the presence of anti-HPA-1a antibody is conducted (Kjeldsen-Kragh et al., 2007). On the other hand, in Japan, more than half of the NAIT cases are due to anti-HPA-4b antibody, and the most severe cases are due to anti-HPA-3 antibody. As we have previously reported, the IgG type anti-A or anti-B antibody may also be causative of NAIT, leading to ICH (Curtis et al., 2008
REFERENCES Berkowitz, R.L., Kolb, E.A., McFarland, J.G., Wissert, M., Primani, A., Lesser, M. & Bussel, J.B. (2006) Parallel randomized trials of risk-based therapy for fetal alloimmune thrombocytopenia. Obstetrics and Gynecology, 107, 91–96. Birchall, J.E., Murphy, M.F., Kaplan, C., Kroll, H. & European Fetomaternal Alloimmune Thrombocytopenia Study Group (2003) European collaborative study of the antenatal management of feto-maternal alloimmune thrombocytopenia. British Journal of Haematology, 122, 275–288.
© 2015 British Blood Transfusion Society
and Kato et al., 2013). Thus, prediction of the risk of NAIT and preventive measures are necessary to avoid this life-threatening condition. The implementation of preventive strategies, as well as of screening programmes, is urgently needed in Japan. In summary, we have reported a case of severe NAIT with ICH in the previous sibling due to anti-A antibody, and a subsequent pregnancy in which the infant was born without any signs or symptoms of thrombocytopenia or anaemia after a strict follow-up regimen with steroids and IVIG. The limitations of this study are that the platelets from the first newborn were not available for compatibility testing with maternal serum, and the compatibility of the second newborn could be tested only after 6 months. Because the father was homozygous for blood group type A, but with two different haplotypes, it is possible that the first and the second newborns had inherited different haplotypes. It is reported that there exist high expressers of ABO antigens on platelets (Curtis et al., 2008), and it is possible that platelets with high ABO expression are destroyed by anti-A or anti-B antibodies preferentially to red blood cells. In such a case, it is possible that the present newborn would not have developed NAIT, and that IVIG administration was an overtreatment. However, considering the severe NAIT of the previous sibling, and the parental concerns about the subsequent pregnancy, we are confident that the treatment strategy was acceptable. Because reports on NAIT due to anti-A antibody are limited in the literature, there is a need to accumulate more cases in order to develop an overview of the condition and propose preventive and therapeutic strategies in the future.
ACKNOWLEDGMENTS We thank N. Koyama and H. Ohto for their helpful advice to perinatal management. H. U., T. S., K. K., S. K., K. I., R. N. and T. K. followed the patients. N. T. and M. M. performed the study. H. U. and T. S. wrote the paper. N. T. and S. S. supervised and contributed to the writing of the paper.
Curtis, B.R., Edwards, J.T., Hessner, M.J., Klein, J.P. & Aster, R.H. (2000) Blood group A and B antigens are strongly expressed on platelets of some individuals. Blood, 96, 1574–1581. Curtis, B.R., Fick, A., Lochowicz, A.J., McFarland, J.G., Ball, R.H., Peterson, J. & Aster, R.H. (2008) Neonatal alloimmune thrombocytopenia associated with maternal-fetal incompatibility for blood group B. Transfusion, 48, 358–364. Espinoza, J.P., Caradeux, J., Norwitz, E.R. & Illanes, S.E. (2013) Fetal and neonatal
alloimmune thrombocytopenia. Reviews in Obstetrics & Gynecology, 6, e15–e21. Kato, S., Sugiura, T., Ueda, H., Kakita, H., Kato, I., Kawabata, K., Ohto, H. & Togari, H. (2013) Massive intracranial hemorrhage caused by neonatal alloimmune thrombocytopenia associated with anti-group A antibody. Journal of Perinatology, 33, 79–82. Kjeldsen-Kragh, J., Killie, M.K., Golebiowska, E. et al. (2007) A screening and intervention program aimed to reduce mortality and serious morbidity associated with severe neonatal alloimmune thrombocytopenia. Blood, 110, 833–839.
Transfusion Medicine, 2015, 25, 42–46
46 H. Ueda et al. Matsuhashi, M., Tsuno, N.H., Kawabata, M. et al. (2010) The first case of alloantibody against human platelet antigen-15b in Japan: possible alloimmunization by a hydatidiform mole. Transfusion, 50, 1126–1130.
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Ohto, H., Miura, S., Ariga, H., Ishii, T., Fujimori, K. & Morita, S. (2004) The natural history of maternal immunization against foetal platelet alloantigens. Transfusion Medicine, 14, 399–408.
Porcelijn, L., Van den Akker, E.S. & Oepkes, D. (2008) Fetal thrombocytopenia. Seminars in Fetal and Neonatal Medicine, 13, 223–230.
© 2015 British Blood Transfusion Society
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Transfusion Medicine
| SHORT COMMUNICATION
Perinatal management of neonatal alloimmune thrombocytopenia associated with anti-group A antibody H. Ueda,1 T. Sugiura,1 K. Katano,2 M. Matsuhashi,3 S. Kato,1 K. Ito,1 R. Nagasaki,1 T. Kato,1 N. H. Tsuno3 & S. Saitoh1 Department of Pediatrics and Neonatology, 2 Department of Obstetrics and Gynecology, Graduate School of Medical Sciences Nagoya City University, Nagoya, Japan, and 3 Department of Transfusion Medicine, The University of Tokyo, Tokyo, Japan
1
Received 9 July 2014; accepted for publication 9 February 2015
SUMMARY Objective: To prevent neonatal alloimmune thrombocytopenia due to anti-group A antibody perinatal management was performed. Background: We previously reported a case of severe intracranial haemorrhage associated with neonatal alloimmune thrombocytopenia due to anti-group A isoantibody. Material/Methods: A 40-year-old Japanese woman, gravida 4 para 1, was pregnant with her second baby. The previous sibling developed severe thrombocytopenia and died 10 days after birth due to intracranial haemorrhage. He was diagnosed with neonatal alloimmune thrombocytopenia; the causative antibody was found to be the anti-group A antibody. Prednisone was started at 7 weeks’ gestational age. Intravenous immunoglobulin 1 g kg−1 week−1 was started at 29 weeks’ gestational age and continued to delivery. Serological studies and genotyping were performed. Results: The second boy was delivered at 33 weeks’ gestational age by caesarean section. He was discharged without intracranial haemorrhage or thrombocytopenia. The anti-group A antibody titre in the maternal serum was 2048–4096 (normal range: 4–64). The anti-group A antibody titre in the newborn’s serum was 4. Cross-matching between the maternal serum and the paternal platelets was positive. Conclusion: Owing to the history of neonatal alloimmune thrombocytopenia causing intracranial haemorrhage and death of the previous sibling, strict follow-up of the subsequent pregnancy was conducted. Key words: anti-group A antibody, immunoglobulin, neonatal alloimmune thrombocytopenia, perinatal management.
Correspondence: Tokio Sugiura, MD, PhD, Department of Pediatrics and Neonatology, Graduate School of Medical Sciences Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81 52 853 8246; fax: +81 52 842 3449; e-mail: [email protected]
First published online 6 March 2015 doi: 10.1111/tme.12178
Neonatal alloimmune thrombocytopenia (NAIT) is a rare but clinically important aetiology of thrombocytopenia of the fetus/neonate caused by maternal immunoglobulin (Ig) G alloantibodies directed against fetal platelet antigens. In a Japanese population study, platelet antibodies were detected in 0·91% of maternal samples, and thrombocytopenia was observed in 18 of 24 630 (0·07%) neonatal samples (Ohto et al., 2004). It is well documented that antibodies to human platelet antigen (HPA)-1a and -4 are the most important cause of NAIT in Caucasians and Asians, respectively. NAIT may rarely be caused by other antigens expressed on the platelet surface, including blood group ABO antigens. Previously, blood group B antibodies have been reported as the causative factor (Curtis et al., 2008). We previously reported a case of severe intracranial haemorrhage (ICH) associated with NAIT due to anti-group A isoantibody (Kato et al., 2013). In the case of a sibling with ICH, the risk of severity of NAIT in the subsequent pregnancy is reported to be higher (Porcelijn et al., 2008). Here, we report a pregnancy subsequent to a case of severe NAIT with ICH, and describe the perinatal management for preventing NAIT due to anti-group A antibody.
MATERIALS AND METHODS Patient A 40-year-old Japanese woman, gravida 4 para 1, was pregnant with her second baby. Her first baby was a male delivered at 37 weeks’ gestation with a birth weight of 1550 g (third percentile). He developed anaemia and severe thrombocytopenia, which was refractory to repeated platelet transfusions. He died on the tenth day of life because of severe ICH associated with severe thrombocytopenia. He was diagnosed with NAIT, and the causative antibody was found to be the anti-group A antibody (Kato et al., 2013). The blood group types of the family were as follows: father, group A, Rh D(+); mother, group O, Rh D(+); first boy, group A, Rh D(+). The outcome of the mother’s first and third pregnancies had been spontaneous abortion. She was referred to our hospital for the prevention of NAIT in the subsequent pregnancy. This study was approved by the Ethics Committee of Nagoya City University.
© 2015 British Blood Transfusion Society
Prevent neonatal alloimmune thrombocytopenia 43
Antenatal treatment Prednisone p.o. 25 mg day−1 was started at 7 weeks’ gestational age. After written informed consent was obtained from the patient, intravenous immunoglobulin (IVIG) 1 g kg−1 week−1 was started at 29 weeks’ gestational age and continued to delivery.
Serological studies and genotyping Detection of anti-HPA and anti-human leukocyte antigen (HLA) antibodies was performed using the anti-PLT-MPHAScreen kit (Beckman Coulter, Tokyo, Japan) and WAKFlow HLA Ab Class I & II (MR) using Luminex method (Wakunaga Pharmaceutical, Hiroshima, Japan), respectively. The genotyping of ABO blood group type, HPA and HLA was performed by the Genosearch ABO kit (Medical and Biological Laboratories, Nagoya, Japan), the WAKFlow HPA typing kit (Wakunaga Pharmaceutical) and the WAKFlow HLA typing kit (Wakunaga Pharmaceutical), respectively. Cross-matching between maternal serum and paternal or the newborn’s platelets was performed using the monoclonal antibody immobilisation of platelet antigens (MAIPA) method as previously described (Matsuhashi et al., 2010). The following mAbs were used in the MAIPA assay, anti-CD31: Gi18, kindly provided by Dr S. Santoso (The Immunohematology Research Department at the Institute for Clinical Immunology and Transfusion Medicine, Giessen, Germany); anti-GPIIb/IIIa: P2 (Acris Antibodies, Herford, Germany); anti-GPIa/IIa: Gi9 (Acris Antibodies); anti-Ib/IX: AK2 (MERCK, Darmstadt, Germany), anti-CD109: W7C5 (MBL, Nagoya, Japan), and anti-HLA class I: W6/32 (abcam, Cambridge UK). The A antigen level on paternal platelets was checked by flow-cytometry.
RESULTS Clinical course The second boy was delivered at 33 weeks’ gestational age by elective caesarean section with a birth weight of 1876 g, suggestive of appropriate for gestational age. The blood type was confirmed to be A, Rh D(+). Blood cell counts were within normal values, with haemoglobin of 17·1 g dL−1 , and platelet count of 276 000 μL−1 . Transient tachypnea was treated only with oxygen therapy, without need for intubation. Head ultrasound and magnetic resonance imaging revealed no ICH. The infant was discharged at 1 month in good general condition, without anaemia or thrombocytopenia.
Serological studies and genotyping Serological studies and the genotyping of the newborn and the parents are shown in Tables 1–4. The ABO genotype of the father and the newborn were A102/A104 and O01/A102, respectively. The titre of the anti-group A antibody in the maternal serum was
© 2015 British Blood Transfusion Society
2048–4096 (normal range: 4–64), when tested against the paternal or the newborn’s red blood cells (RBC), as well as against commercially available RBC (A1 RBC; Bio-Rad, Tokyo, Japan). The titre of anti-group A antibody in the newborn’s serum was 4 (normal range: 4–64). The direct Coombs test using the newborn’s RBC was negative, but the elution test of the newborn revealed 1+ for anti-group A antibody. Cross-matching between the maternal serum and the paternal platelets was positive for anti-CD31 (PECAM-1). However, cross-matching between the maternal serum and the newborn’s platelets at 6 months was negative for anti-CD31 (PECAM-1). The flow-cytometric analysis revealed that the father was not a high expressor for A antigen on platelets.
DISCUSSION Owing to the history of severe NAIT causing ICH and death of the previous sibling, strict follow-up of the subsequent pregnancy was conducted. The previous sibling developed severe thrombocytopenia and anaemia after birth, and died 10 days after due to extensive ICH. Maternal platelet counts were normal, and neither anti-HLA nor anti-HPA antibodies were detected in her serum. However, cross-matching between the maternal serum and paternal platelets was positive, which was confirmed to be dependent on the presence of high-titre anti-A antibody in the maternal serum. Therefore, this case was reported as NAIT caused by anti-A antibody (Kato et al., 2013). Unfortunately, the previous sibling was lost; cross-matching between his platelets and the maternal serum was not possible because it was not feasible to collect blood at the time of birth. Thereafter, the mother experienced a spontaneous abortion, and later became pregnant with the infant described in this report. It has been observed that the severity of NAIT in a subsequent gestation tends to be higher (Porcelijn et al., 2008). There are recommendations in the literature for the use of IVIG for the prevention of severe NAIT, especially its associated complication ICH, in high-risk pregnancies (Espinoza et al., 2013). Although algorithms exist for the prevention or treatment of problems in subsequent siblings of an infant with severe NAIT due to anti-HPA-1 incompatibility (the most common and severe cause of NAIT in Caucasians), it is unknown if an algorithm can be generalised to NAIT caused by other antibodies. Because HPA-1 incompatibility is rare in Japan, no algorithms for the prevention or treatment of NAIT presently exist in Japan. Following the international algorithms, the administration of IVIG has recently become mainstream in Japan and steroids can be added according to the risks in the individual case (Berkowitz et al., 2006). IVIG is usually administered weekly at 1–2 g kg−1 week−1 until delivery. Also, reports exist on intrauterine fetal blood sampling for evaluating the effectiveness of treatment, and the use of intrauterine platelet transfusions (Birchall et al., 2003); however, due to the risk of emergency delivery or fetal death, these procedures are indicated only after 32 weeks’ gestation.
Transfusion Medicine, 2015, 25, 42–46
44 H. Ueda et al. Table 1. Genotyping of ABO, HPA and HLA antigen.
Father Mother Second infant
Type 1
Type 2
A102 O 01 O 01
A104 O 02 A102
HPA type
Father Mother Second infant
Father Mother Second infant
1
2
3
4
5
6
7
15
21
Naka
a/a a/a a/a
a/a a/a a/a
a/b a/b b/b
a/a a/a a/a
a/a a/a a/a
a/a a/a a/a
a/a a/a a/a
a/b b/b b/b
a/a a/a a/a
+ + +
A* 02:01 A* 11:01 A* 02:01
A* 02:06 A* 31:01 A* 11:01
B* 35:01 B* 15:27 B* 15:27
B* 44:03 B* 54:01 B* 44:03
Cw* 04:01 Cw* 01:02 Cw* 04:01
Cw* 14:03 Cw* 04:01 Cw* 14:03
A new platelet specific antigen Nak (a) are usually represented as “Naka ”.
Table 2. The titre of anti-group A antibody.
Table 4. Cross-matching between mother’s serum and father’s or second infant’ platelets by the MAIPA.
Anti-group A Ab
Mother’s serum
Second infant’s serum (at birth)
Father’s RBC Second infant’s RBC A1 RBC
2048–4096 fold 512–2048 fold 2048–4096 fold
N.T. Negative Four-fold
N.T., not tested.
Table 3. The detection of anti-HPA and anti-HLA antibodies. Anti-HPA antibodies were detected by MPHA and MAIPA. Anti-HLA antibodies were detected by the Luminex method. Mother’s serum Anti-HPA Ab Anti-HLA Ab (Class I) Anti-HLA Ab (Class II)
Negative Negative Negative
MPHA, mixed passive hemagglutination.
In Japan, the administration of IVIG in pregnant women at high risk for NAIT is not covered by the Japanese Health Insurance System, and because of the high cost of IVIG in Japan, it is rarely available to most pregnant women for treatment of fetomaternal alloimmune thrombocytopenia. In the present case, considering the severity of NAIT, together with ICH, in the previous sibling, we started prednisolone administration at 7 weeks’ gestation, and IVIG administration at 29 weeks, continuing until delivery. Also, considering that this was a high-risk pregnancy, the baby was delivered by elective caesarean section at 33 weeks’ gestation. For evaluation of the treatment efficacy, intrauterine fetal sampling was considered, but because this was a high-risk procedure and the parents did not give informed consent, it was not carried out.
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Anti-CD31 Anti-GPIIb/IIIa Anti-GPIa/IIa Anti-GPIb/IX Anti-CD109 Anti-HLA Class I
Father’s platelets
Second infant’s platelets
Positive Negative Negative Negative Negative Negative
Negative Negative Negative Negative Negative Negative
Ab, antibody.
There is no consensus on the most appropriate mode of delivery. Porcelijn et al. (2008) recommended that in the case of a previous sibling with NAIT, IVIG administration should be started at 28–32 weeks’ gestation, and caesarean section should be performed at 28–32 weeks in cases where ICH has been observed. In the present case, the treatment strategy was discussed with the parents. Considering the financial aspect, IVIG administration was started at 29 weeks’ gestation, and elective caesarean section was performed at 33 weeks. Because there is no coverage by the Japanese Health Insurance System for the prevention of NAIT, IVIG administration was given for 4 weeks, which was charged to the parents. Also, long-term administration of IVIG starting from early gestation would be a burden to the parents; thus the administration of steroids, of which the cost is less onerous, was indicated in the early phase of gestation. Although anti-A antibody titres in the maternal serum persisted at high levels (2049–4094) throughout the gestation, the infant was born with low levels of anti-A antibody in his serum. Also, no evidence of thrombocytopenia or anaemia was found, suggestive of the effectiveness of steroid therapy plus IVIG administration.
© 2015 British Blood Transfusion Society
Prevent neonatal alloimmune thrombocytopenia 45 Although the mechanism to prevent NAIT in high-risk pregnancies remains to be elucidated, the following speculations can be made: (i) IVIG helps reduce the titre or the activity of anti-HPA antibodies; (ii) IVIG blocks the Fc receptor of the placenta, inhibiting the placental transfer of anti-HPA antibodies (IgG); and/or (iii) IVIG binds to the Fc receptor of fetal macrophages, inhibiting the binding of anti-HPA antibodies bound to the surface of platelets (Espinoza et al., 2013). The same mechanism as for anti-HPA antibody may occur with anti-A antibody. Presently, the adverse effects of long-term IVIG administration to the mother or the fetus are not known. In this case, no adverse events were observed in either the mother or the newborn, but long-term follow-up is required. CD31 captured from the infant’s platelets was not positive when tested with maternal serum, as a surrogate for detection of maternal serum anti-A. The reason why the cross-match between maternal serum and the newborn’s platelets was negative is intriguing. We suspected that the ABO blood type of the newborn was A2, and performed the DNA sequencing of the father and the second infant. The results, however, revealed A1, not A2. It is well-known that PECAM-1 also carry blood group A and B antigens (Curtis et al., 2000), and some individuals are high expressers. Thus, we used the monoclonal to CD31 as the capture antibody in MAIPA to confirm the reactivity of maternal serum with A-antigen. Monoclonals to GPIIbIIIa, GPIbIX, GPIaIIa, CD109 and HLA-I were also used, but resulted negative. The only positive reaction in the MAIPA was for the detection of antibodies against PECAM-1, which was consistent with reactivity with ABO antigens. Because NAIT may occur at the first pregnancy, there are reports of antenatal screening (Kjeldsen-Kragh et al., 2007). HPA-1a is the causative antibody in approximately 85% of NAIT cases in Caucasians; therefore, screening of the maternal serum for the presence of anti-HPA-1a antibody is conducted (Kjeldsen-Kragh et al., 2007). On the other hand, in Japan, more than half of the NAIT cases are due to anti-HPA-4b antibody, and the most severe cases are due to anti-HPA-3 antibody. As we have previously reported, the IgG type anti-A or anti-B antibody may also be causative of NAIT, leading to ICH (Curtis et al., 2008
REFERENCES Berkowitz, R.L., Kolb, E.A., McFarland, J.G., Wissert, M., Primani, A., Lesser, M. & Bussel, J.B. (2006) Parallel randomized trials of risk-based therapy for fetal alloimmune thrombocytopenia. Obstetrics and Gynecology, 107, 91–96. Birchall, J.E., Murphy, M.F., Kaplan, C., Kroll, H. & European Fetomaternal Alloimmune Thrombocytopenia Study Group (2003) European collaborative study of the antenatal management of feto-maternal alloimmune thrombocytopenia. British Journal of Haematology, 122, 275–288.
© 2015 British Blood Transfusion Society
and Kato et al., 2013). Thus, prediction of the risk of NAIT and preventive measures are necessary to avoid this life-threatening condition. The implementation of preventive strategies, as well as of screening programmes, is urgently needed in Japan. In summary, we have reported a case of severe NAIT with ICH in the previous sibling due to anti-A antibody, and a subsequent pregnancy in which the infant was born without any signs or symptoms of thrombocytopenia or anaemia after a strict follow-up regimen with steroids and IVIG. The limitations of this study are that the platelets from the first newborn were not available for compatibility testing with maternal serum, and the compatibility of the second newborn could be tested only after 6 months. Because the father was homozygous for blood group type A, but with two different haplotypes, it is possible that the first and the second newborns had inherited different haplotypes. It is reported that there exist high expressers of ABO antigens on platelets (Curtis et al., 2008), and it is possible that platelets with high ABO expression are destroyed by anti-A or anti-B antibodies preferentially to red blood cells. In such a case, it is possible that the present newborn would not have developed NAIT, and that IVIG administration was an overtreatment. However, considering the severe NAIT of the previous sibling, and the parental concerns about the subsequent pregnancy, we are confident that the treatment strategy was acceptable. Because reports on NAIT due to anti-A antibody are limited in the literature, there is a need to accumulate more cases in order to develop an overview of the condition and propose preventive and therapeutic strategies in the future.
ACKNOWLEDGMENTS We thank N. Koyama and H. Ohto for their helpful advice to perinatal management. H. U., T. S., K. K., S. K., K. I., R. N. and T. K. followed the patients. N. T. and M. M. performed the study. H. U. and T. S. wrote the paper. N. T. and S. S. supervised and contributed to the writing of the paper.
Curtis, B.R., Edwards, J.T., Hessner, M.J., Klein, J.P. & Aster, R.H. (2000) Blood group A and B antigens are strongly expressed on platelets of some individuals. Blood, 96, 1574–1581. Curtis, B.R., Fick, A., Lochowicz, A.J., McFarland, J.G., Ball, R.H., Peterson, J. & Aster, R.H. (2008) Neonatal alloimmune thrombocytopenia associated with maternal-fetal incompatibility for blood group B. Transfusion, 48, 358–364. Espinoza, J.P., Caradeux, J., Norwitz, E.R. & Illanes, S.E. (2013) Fetal and neonatal
alloimmune thrombocytopenia. Reviews in Obstetrics & Gynecology, 6, e15–e21. Kato, S., Sugiura, T., Ueda, H., Kakita, H., Kato, I., Kawabata, K., Ohto, H. & Togari, H. (2013) Massive intracranial hemorrhage caused by neonatal alloimmune thrombocytopenia associated with anti-group A antibody. Journal of Perinatology, 33, 79–82. Kjeldsen-Kragh, J., Killie, M.K., Golebiowska, E. et al. (2007) A screening and intervention program aimed to reduce mortality and serious morbidity associated with severe neonatal alloimmune thrombocytopenia. Blood, 110, 833–839.
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46 H. Ueda et al. Matsuhashi, M., Tsuno, N.H., Kawabata, M. et al. (2010) The first case of alloantibody against human platelet antigen-15b in Japan: possible alloimmunization by a hydatidiform mole. Transfusion, 50, 1126–1130.
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Ohto, H., Miura, S., Ariga, H., Ishii, T., Fujimori, K. & Morita, S. (2004) The natural history of maternal immunization against foetal platelet alloantigens. Transfusion Medicine, 14, 399–408.
Porcelijn, L., Van den Akker, E.S. & Oepkes, D. (2008) Fetal thrombocytopenia. Seminars in Fetal and Neonatal Medicine, 13, 223–230.
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