AMERICAN JOURNAL OF PERINATOLOGY/VOLUME 9, NUMBER 5/6
Sept/Nov 1992
NEONATAL ALLOIMMUNE THROMBOCYTOPENIA AFTER MATERNAL IMMUNIZATION WITH PATERNAL MONONUCLEAR CELLS: SUCCESSFUL TREATMENT WITH INTRAVENOUS GAMMA GLOBULIN S.A. Pearlman, M.D., R.S. Meek, M.D., F.S. Cowchock, M.D., J.B. Smith, M.D.,J. McFarland, M.D., and R.H. Aster, M.D.
We report a case of neonatal alloimmune thrombocytopenia and intracranial hemorrhage in an infant whose mother received immunizations of paternal mononuclear cells. This therapy is designed to prevent unexplained first trimester miscarriages. No previous cases of platelet autoimmunization associated with maternal immunization with paternal mononuclear cells has been reported. Treatment with antenatal maternal infusions of intravenous gamma globulin (IVGG) did not preventfetal thrombocytopenia, but IVGG may become the treatment of choice for postnatal, antibody-mediated thrombocytopenia of the newborn.
Innovative work by Mowbray and others1 suggested that women who have repeated unexplained first trimester miscarriages may be able to achieve a successful pregnancy following immunization by paternal or donor mononuclear cell preparations. Although no formal reports of maternal or fetal complications related to this therapy have been reported, some authors have expressed concerns about its use.2"5 Initial concerns about the risks associated with this treatment have focused on the potential alloimmunization to red blood cell antigens and the possible exposure to blood-borne viruses. We are reporting a case of neonatal alloimmune thrombocytopenia (NAT) and intracranial hemorrhage in an infant whose mother received immunizations of paternal mononuclear cells. The mononuclear cell preparations contain 5 to 10% of platelets originally present in the blood unit. Stored sera from this pregnancy were used to demonstrate that immunization to P1A1 antigen had presumably occurred in a previous pregnancy, with a marked rise in maternal antibody titer probably related to the immunization procedure. The platelet content in this case may have been sufficient to boost levels of antiplatelet antibody already present. The infant's thrombocytopenia was successfully treated with intravenous gamma globulin (IVGG).
CASE REPORT
The mother was a 28-year-old Caucasian who had had three first trimester miscarriages. Evaluation revealed no known cause for repeated miscarriages. She had no history of systemic autoimmune disorders, thrombocytopenia, or transfusion. She had hyperthyroidism treated with propylthiouracil until 28 weeks' gestation. Her antinuclear antibody titer was 1:100 and her antithyroid microsomal antibody titer was 1:40. The parents underwent immunologic evaluation for possible immunization therapy using paternal mononuclear cells for treatment of unexplained recurrent miscarriages. Based on negative tests for maternal serum antipaternal lymphocytotoxic antibodies or blocking antibodies in mixed maternal-paternal lymphocyte reactivity, the patient was immunized at five postmenstrual weeks with paternal cells prepared according to a protocol that we have previously described.6 In the course of their evaluation maternal HLA type was noted to be A2, 10;B7,8(BW6; DR2,3(DRW52) and paternal HLA type was A11,28;B39,8 (BW6; Dr4,6 (DRW52). The mononuclear cells were prepared by centrifugation of 1 U of paternal blood to recover the "buffy coat"
Department of Pediatrics, Medical Center of Delaware, Newark, Delaware; Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania; and Blood Center Southeastern Wisconsin Supported in part by Grant HL13629 from the National Heart, Lung and Blood Institute, Bethesda, Maryland Reprint requests: Dr. Pearlman, Division of Neonatology, Department of Pediatrics, Medical Center of Delaware, P.O. Box 6001, Newark, DE 19718 448
Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
ABSTRACT
NAT AFTER MATERNAL IMMUNIZATION/Pearlman, et al.
ing three daily doses of 1 gm/kg of body weight, the IVGG, the platelet count rose and remained elevated, requiring no further treatment. A head computed tomography scan performed on day 1 revealed a large porencephalic cyst and an enlarged right lateral ventricle. Treatment included placement of a venticuloperitoneal shunt and phenobarbital for seizure activity, which began on day 3 of life. Neurologic follow-up revealed evidence of cortical visual impairment and earlyonset spasticity. Table 1 summarizes the results of maternal and paternal serologic studies. Samples were obtained before and 6 weeks after immunization and postpartum. The father was P1A1 homozygous and the mother was P1A2 homozygous (P1A1 negative). Both parents were positive for the platelet-specific alloantigen BakA. Maternal serum levels of antibody to P1A1 antigen were measured by using antigen capture enzyme-linked (ACE) immunosorbent assay.7 Before immunization with the paternal lymphocyte preparation, the mother's serum contained anti-PlA1 antibody detectable in dilutions up to 1:256. Six weeks following immunization, during the first trimester of pregnancy, the titer rose significantly and was detectable in dilutions of 1:1024 or greater (a threefold rise in the titer). This antibody was also measured by the 51Cr release assay.8 51Cr-labeled platelets were used as targets for antibody in the presence of complement. This test was only slightly positive for anti-PlA1 when the preimmunization serum was used undiluted. The postimmunization serum was strongly positive for anti-PlA1 at a dilution of 1:16. After delivery of this child, the mother had a fifth pregnancy without immunization treatment, which ended in miscarriage of a 9-week nonviable embryo. Before her sixth pregnancy with the same father, she was immunized with mononuclear cells from a P1A2 homozygous donor. No rise in her anti-PlA1 titer of 1:4 (51Cr release assay) was demonstrated after immunization. She conceived in the same menstrual cycle and antepartum prophylaxis for fetal thrombocytopenia in this pregnancy included monthly
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followed by density gradient separation of mononuclear cells and hypotonic lysis to remove remaining red cells. The isolated cells were resuspended in 5 ml of Ringer's lactate solution and 2.5 ml were given intravenously and the remaining cells were injected into two subcutaneous and two intradermal forearm sites. Platelet counts from similar preparations averaged 2.9 x 106/mm3 for an average injection of 1.5 x 1010 platelets. These amounts are equivalent to 5 to 10% of the platelets from the original blood units. A fetal ultrasound study performed at 11 weeks of gestation was unremarkable. The pregnancy continued uneventfully until 37 weeks' gestation when a fetal ultrasound showed evidence of macrocephaly, dilation of the lateral ventricles, and severe oligohydramnios. Because of documented fetal distress (low biophysical profile), a 2.3 kg male was delivered by cesarean section. Apgar scores were 8 and 9 at 1 and 5 minutes, respectively. Physical examination of the newborn revealed a height and weight at the 10th percentile and a head circumference greater than the 90th percentile (38 cm). The anterior fontanelle was bulging and the cranial sutures splayed. Generalized petechiae were noted and there was a large hematoma at the vitamin K injection site. There was no hepatosplenomegaly and initial neurologic examination was normal. Laboratory data showed a hemoglobin of 16.5 mg/dl, white blood cell count of 27,600/mm3, and a platelet count of 1000/mm3. The blood smear showed marked polychromasia and the absence of platelets. The infant's prothrombin time was 11.0 and the partial thromboplastin time was 30.0. The infant's blood type was A positive, direct and indirect Coombs tests were negative. Maternal complete blood count, platelet count and peripheral smear were normal. Blood cultures, urine culture, and cytomegalovirus (CMV), toxoplasmosis, CMV and rubella titers performed at birth were subsequently negative. Figure 1 shows the course of the thrombocytopenia. Treatment consisted of three maternally derived platelet transfusions followed by IVGG (Sandoglobulin). Follow-
500
400
300
si 200
100 Figure 1. Patient's platelet count versus age in days. T: platelet transfusion; TW: washed platelet transfusion; IVGG: intravenous gamma globulin given at dose of 1 gm/kg/day.
5
10
15 AGE IN DAYS
20
25
30 449
AMERICAN JOURNAL OF PERINATOLOGY/VOLUME 9, NUMBER 5/6 Table 1.
Mother Father
Sept/Nov 1992
Serologic Studies*
HLA Typing
Platelet Typing
A2, 10; B7, 8 (BW6); Dr2, 3 (DW52) All, 28, B39, 8 (BW6); Dr4, 6, (DW52)
P1 A 2 homozygous (P1 A1 negative) P1 A 1 homozygous
Anti-PTA1 Antibody (Antigen Capture)
Anti-P1A1 Antibody (5'Cr Release)
1:256 before 1:1024 after
Weak (+) undil before (+) at 1:16 after
intravenous infusions of 1 gm/kg IVGG from 12 postmenstrual weeks. Cordocentesis was performed at 21 postmenstrual weeks (1 week after maternal IVGG infusion) and revealed a fetal platelet count of 7000/mm3. Oral dexamethasone (4 mg daily) was added, and IVGG infusion frequency increased to weekly. However, when the patient returned the next week for treatment, the fetus was nonviable, secondary to exsanguination. DISCUSSION NAT may cause serious bleeding problems in affected newborns.9 NAT is diagnosed in about 1 of 3000 to 5000 pregnancies.1011 The mechanism is similar to that of Rh sensitization. Maternal-fetal incompatibility for the P1A1 antigen is the most common cause. Approximately 98% of the European and North American population have the P1A1 antigen12 and sensitization to P1A1 accounts for about 50% of the cases of NAT.11 Other antigen systems, such as BakA, Bra, and Pena may also be involved.13-17 sensitization usually occurs due to previous pregnancy or exposure to blood products. The prevalence of NAT-affected infants is lower than that expected from the incidence of antigen incompatibility in the population. The reason that some PlA1-negative women develop antibodies while others do not remains unclear. However, an association between maternal HLA type B8, DR3, and the development of anti-PlA1 alloimmunization has been noted.12-18 Although infants at risk for NAT are frequently delivered by cesarean section to protect them from the trauma of vaginal delivery,18 cases of intrauterine intracranial hemorrhage have been reported.19-20 Once delivered, these infants can be treated with maternal platelets that lack the target antigen. In our patient, two transfusions of maternal platelets temporarily increased the platelet count in this affected infant, but the third transfusion of washed maternal platelets was not effective. Other modalities of neonatal treatment for NAT include exchange transfusion and corticosteroids.9-20. IVGG is now being used to treat many forms of immune thrombocytopenia. IVGG has been used in childhood idiopathic thrombocytopenic purpura,21-23 platelet alloimmunization from frequent transfusions,24-25 and neonatal passive immune thrombocytopenia associated with maternal idiopathic thrombocytopenic purpura.26 There have been previous reports on the successful treatment of NAT with IVGG.27-39 Antenatal treatment of pregnant women with a prior affected pregnancy with infusions of IVGG given with or without dexamethasone was reported by Bussel and coworkers30 to prevent recurrent intracranial hemorrhage. However, in our patient's second affected pregnancy, maternal IVGG infusion the week before cordocentesis had no apparent effect on the fetal platelet count. Nicolini 450 and others31 reported a similar case in which the fetal
platelet count was apparently unchanged by maternal IVGG infusion, even though fetal hypergammaglobulinemia was achieved. We note that neither our patient, nor the case reported by Nicolini et al, were given dexamethasone in addition to the IVGG infusions. Previous studies have not demonstrated a striking increase in the antibody titers as measured by the ACE assay in at-risk pregnancies that have been followed serially.729 Other antibody tests, such as the 51Cr release assay32 may increase in strength throughout pregnancy, but this occurs in the minority of cases. Therefore, we speculate that the paternal mononuclear cell immunization was related to the boost in titer seen during the first successful pregnancy. In summary, we believe that this is the first case report of platelet alloimmunization associated with maternal immunization with paternal mononuclear cells. Although this patient was sensitized to her husband's platelet antigens through previous miscarriages, our investigation demonstrated a dramatic rise in anti-PlA1 titer following paternal mononuclear cell immunization. Most centers offering this treatment use screening tests to identify those at risk for Rh and Kell sensitization and give Rh-negative women prophylactic treatment with rhesus immune globulin after immunization. Either careful screening and Rh prophylaxis or more complete removal of red cells from the lymphocyte preparations may account for the observation that red cell alloimmunization attributable only to the immunization procedure has not been reported. Because a significant number of paternal platelets are still present after mononuclear cell preparation, platelet alloimmunization is a possible consequence of treatment for those at risk. The rise in our patient's platelet antibody titer after immunization was probably caused by recent antigen exposure as a consequence of her early pregnancy or immunization with mononuclear cells from the PlA1-positive paternal donor. The rise in antibody titer was probably not a nonspecific response to the immunization procedure or to pregnancy because no such increase was observed after immunization of this woman with a similar preparation of cells from a PlA1-negative donor in a subsequent pregnancy. Women at risk for platelet alloimmunization might be identified through the parental HLA typing often performed as part of immunologic evaluation of recurrent miscarriages. Those whose HLA type is known to be B8, DR3 (DRW52) could be offered platelet antigen typing, because they may be at highest risk for platelet alloimmunization. Furthermore, this case demonstrates that operative delivery of an infant with known or suspected NAT does not spare the infant from significant morbidity. Although IVGG was effective for postnatal treatment of the first affected infant's thrombocytopenia, antenatal maternal IVGG infusion did not prevent fetal thrombocytopenia in this woman's second affected pregnancy. IVGG may become the treatment of choice for postnatally diagnosed antibody-medicated thrombocytopenia of the newborn.
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"Serologic testing performed on parents. Serum tested postnatally included samples saved from prior to immunization procedure.
NAT AFTER MATERNAL IMMUNIZATION/Pearlman, et al.
1. Mowbray JF, Liddel H, Underwood J, Gibbings C, Reginald PW, Beard RW: Controlled trial of treatment of recurrent spontaneous abortion by immunization with paternal cells. Lancet 1:941,943, 1985 2. Scott JR: Immunologic aspects of recurrent spontaneous abortion. Fertil Steril 38:301-302, 1982 3. Hill JA: Blood transfusions for recurrent abortion: Is the treatment worse than the disease? Fertil Steril 46:151— 152, 1986 4. Unander AM, Lindholm A, Olding LB: Reply. Fertil Steril 38:152-153,1986 5. Redman CWG: Immune factors and recurrent abortion: A review. Am J Reprod Immunol 4:179-191, 1983 6. Smith JB, Cowchock FS: Immunologic studies in recurrent spontaneous abortion: Affects of immunization of women with paternal mononuclear cells on lymphocytotoxic and MLR blocking antibodies and correlation with HLA sharing and pregnancy outcome. J Reprod Immunol 14:99-113, 1988 7. McFarland JG, Frenzke M, Aster RH: Testing of maternal sera in pregnancies at risk for neonatal alloimmune thrombocytopenia. Transfusion 29:128-133, 1989 8. Cimo P, Pisciotta AV, Desai RG, Aster RH: Detection of drug-dependent antibodies by the 51Cr platelet lysis test. AmJ Hematol 2:65, 1977 9. Oski FA, Naiman JL: Hematologic Problems in the Newborn. Philadelphia: W.B. Saunders, 1982, pp 190-196 10. Shulman NR, Marder VJ, Hiller MC, Collier EM: Platelet and leukocyte isoantigens and their antibodies: Serologic physiologic and clinical studies. Prog Hematol 6: 222,1964 11. Mclntosh S, O'Brien RT, Schwartz AD, Pearson HA: Neonatal isoimmune purpura: Response to platelet infusions. J Pediatr 82:1020-1027, 1973 12. deWaal LP, van Dalen CM, Engelfriet CP, von dem Borne AEGK: Alloimmunization against the platelet-specific ZW antigen, resulting in neonatal alloimmune thrombocytopenia or posttransfusion purpura, is associated with supertypic Dre52 antigen including Dr3 and Drw6. Hum Immunol 17:45-53, 1986 13. Miller DT, Etzel RA, McFarland JG, et al: Prolonged neonatal alloimmune thrombocytopenic purpura associated with anti-Bak-a: Two cases in siblings. AmJ Perinatol 4: 55-58, 1987, and erratum 4:177-178, 1987 14. von dem Borne AEGK, van Leeuwen EF, von Riesz LE, van Boxtel CJ, Egelfriet CP: Neonatal alloimmune thrombocytopenia: Detection and characterization of the responsible antibodies by the platelet immunofluorescence test. Blood 57:649-656, 1981 15. von de Borne AEGK, von Reisz E, Verheugt FWA, et al: Baka, a new platelet-specific antigen involved in neonatal alloimmune thrombocytopenia. Vox Sang 39:113, 120, 1980 16. Freidman JM, Aster RH: Neonatal alloimmune thrombocytopenic purpura and congenital porencephaly in two siblings associated with a "new" maternal antiplatelet antibody. Blood 65:1412, 1985
17. 18. 19. 20. 21.
22.
23.
24.
25. 26. 27. 28. 29. 30. 31.
32.
Keifel V, Santoso S, Mueller-Eckhardt C: A new plateletspecific antigen Br-a. Vox Sang 54:101-106, 1988 Sitarz AL, Driscoll JM, Wolf JA: Management of isoimmune neonatal thrombocytopenia. AmJ Obstet Gynecol 124: 39, 1974 Zalneraitis EL, Young R, Krishnamoorthy KS: Intracranial hemorrhage in-utero as a complication of iso-immune thrombocytopenia. J Pediatr 95:611-614, 1979 Herman JH, Jumberlic MI, Ancona RJ, Kickler TS: Inutero cerebral hemorrhage in alloimmune thrombocytopenia. Am J Pediatr Hematol Oncol 8:312-317, 1986 Bussel JB, Goldman A, Imbach P, et al: Treatment of acute idiopathic thrombocytopenia of childhood with intravenous infusions of gamma globulin. J Pediatr 106:886— 890,1985 Imbach P, Barandun S, Hirt A, et al: Intravenous immunoglobulin for idiopathic thrombocytopenic purpura in childhood. Am J Pediatr Hematol Oncol 6:171-174, 1984 Imbach P, Berchtold W, Hirt A, et al: Intravenous immunoglobulin vs. oral corticosteroids in acute immune thrombocytopenic purpura in childhood. Lancet 464—468, 1985 Kekoma KR, Elfenbein G, Gardner R, et al: Improved response of patients refractory to random platelet transfusions by intravenous gamma globulin. Am J Med 199— 203,1984 Becton DL, Kinney TR, Chafee S, et al: High dose intravenous immunoglobulin for platelet alloimmunization. Pediatrics 74:1120-1123, 1984 Chirico G, Duse M, Ugazio A, et al: High dose intravenous gamma globulin therapy for passive immune thrombocytopenia in the neonate. J Pediatr 103:654-655, 1983 Sidiropoulos D, Straume B: The treatment of neonatal isoimmune thrombocytopenia with intravenous immunoglobulin. Blut 48:383-386, 1984 Massey GV, McWilliams NB, Mueller DG, et al: Intravenous immunoglobulin the treatment of neonatal isoimmune thrombocytopenia. J Pediatr 111:133-135, 1987 Derycke M, Dreyfus M, Ropert JC, et al: Intravenous immunoglobulin for neonatal isoimmune thrombocytopenia. Arch Dis Child 60:667-669, 1985 Bussel JB, Berkowitz RL, McFarland JG, et al: Antenatal Treatment of Neonatal Alloimmune Thrombocytopenia. N EnglJ Med 319:1374-1378, 1988 Nicolini V, Fannirandorn Y, Gonzalez P, et al: Continuing controversy in alloimmune thrombocytopenia: Fetal hyperimmunoglobulinemia fails to prevent thrombocytopenia. AmJ Obstet Gynecol 163:1144-1147, 1990 Mueller-Eckhardt C, Grubert A, Weisheit M, et al: 348 cases of suspected neonatal alloimmune thrombocytopenia. Lancet 1:363-366, 1989
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REFERENCES
The authors wish to thank Drs. E. Mamberg, T. Chronister, P. Huang, and E. McReynolds for their contributions to the management of this patient. We are grateful to Dr. Michael E. Norman for his review of the manuscript and to B. Simmons for her secretarial assistance.
451
Sept/Nov 1992
NEONATAL ALLOIMMUNE THROMBOCYTOPENIA AFTER MATERNAL IMMUNIZATION WITH PATERNAL MONONUCLEAR CELLS: SUCCESSFUL TREATMENT WITH INTRAVENOUS GAMMA GLOBULIN S.A. Pearlman, M.D., R.S. Meek, M.D., F.S. Cowchock, M.D., J.B. Smith, M.D.,J. McFarland, M.D., and R.H. Aster, M.D.
We report a case of neonatal alloimmune thrombocytopenia and intracranial hemorrhage in an infant whose mother received immunizations of paternal mononuclear cells. This therapy is designed to prevent unexplained first trimester miscarriages. No previous cases of platelet autoimmunization associated with maternal immunization with paternal mononuclear cells has been reported. Treatment with antenatal maternal infusions of intravenous gamma globulin (IVGG) did not preventfetal thrombocytopenia, but IVGG may become the treatment of choice for postnatal, antibody-mediated thrombocytopenia of the newborn.
Innovative work by Mowbray and others1 suggested that women who have repeated unexplained first trimester miscarriages may be able to achieve a successful pregnancy following immunization by paternal or donor mononuclear cell preparations. Although no formal reports of maternal or fetal complications related to this therapy have been reported, some authors have expressed concerns about its use.2"5 Initial concerns about the risks associated with this treatment have focused on the potential alloimmunization to red blood cell antigens and the possible exposure to blood-borne viruses. We are reporting a case of neonatal alloimmune thrombocytopenia (NAT) and intracranial hemorrhage in an infant whose mother received immunizations of paternal mononuclear cells. The mononuclear cell preparations contain 5 to 10% of platelets originally present in the blood unit. Stored sera from this pregnancy were used to demonstrate that immunization to P1A1 antigen had presumably occurred in a previous pregnancy, with a marked rise in maternal antibody titer probably related to the immunization procedure. The platelet content in this case may have been sufficient to boost levels of antiplatelet antibody already present. The infant's thrombocytopenia was successfully treated with intravenous gamma globulin (IVGG).
CASE REPORT
The mother was a 28-year-old Caucasian who had had three first trimester miscarriages. Evaluation revealed no known cause for repeated miscarriages. She had no history of systemic autoimmune disorders, thrombocytopenia, or transfusion. She had hyperthyroidism treated with propylthiouracil until 28 weeks' gestation. Her antinuclear antibody titer was 1:100 and her antithyroid microsomal antibody titer was 1:40. The parents underwent immunologic evaluation for possible immunization therapy using paternal mononuclear cells for treatment of unexplained recurrent miscarriages. Based on negative tests for maternal serum antipaternal lymphocytotoxic antibodies or blocking antibodies in mixed maternal-paternal lymphocyte reactivity, the patient was immunized at five postmenstrual weeks with paternal cells prepared according to a protocol that we have previously described.6 In the course of their evaluation maternal HLA type was noted to be A2, 10;B7,8(BW6; DR2,3(DRW52) and paternal HLA type was A11,28;B39,8 (BW6; Dr4,6 (DRW52). The mononuclear cells were prepared by centrifugation of 1 U of paternal blood to recover the "buffy coat"
Department of Pediatrics, Medical Center of Delaware, Newark, Delaware; Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania; and Blood Center Southeastern Wisconsin Supported in part by Grant HL13629 from the National Heart, Lung and Blood Institute, Bethesda, Maryland Reprint requests: Dr. Pearlman, Division of Neonatology, Department of Pediatrics, Medical Center of Delaware, P.O. Box 6001, Newark, DE 19718 448
Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
ABSTRACT
NAT AFTER MATERNAL IMMUNIZATION/Pearlman, et al.
ing three daily doses of 1 gm/kg of body weight, the IVGG, the platelet count rose and remained elevated, requiring no further treatment. A head computed tomography scan performed on day 1 revealed a large porencephalic cyst and an enlarged right lateral ventricle. Treatment included placement of a venticuloperitoneal shunt and phenobarbital for seizure activity, which began on day 3 of life. Neurologic follow-up revealed evidence of cortical visual impairment and earlyonset spasticity. Table 1 summarizes the results of maternal and paternal serologic studies. Samples were obtained before and 6 weeks after immunization and postpartum. The father was P1A1 homozygous and the mother was P1A2 homozygous (P1A1 negative). Both parents were positive for the platelet-specific alloantigen BakA. Maternal serum levels of antibody to P1A1 antigen were measured by using antigen capture enzyme-linked (ACE) immunosorbent assay.7 Before immunization with the paternal lymphocyte preparation, the mother's serum contained anti-PlA1 antibody detectable in dilutions up to 1:256. Six weeks following immunization, during the first trimester of pregnancy, the titer rose significantly and was detectable in dilutions of 1:1024 or greater (a threefold rise in the titer). This antibody was also measured by the 51Cr release assay.8 51Cr-labeled platelets were used as targets for antibody in the presence of complement. This test was only slightly positive for anti-PlA1 when the preimmunization serum was used undiluted. The postimmunization serum was strongly positive for anti-PlA1 at a dilution of 1:16. After delivery of this child, the mother had a fifth pregnancy without immunization treatment, which ended in miscarriage of a 9-week nonviable embryo. Before her sixth pregnancy with the same father, she was immunized with mononuclear cells from a P1A2 homozygous donor. No rise in her anti-PlA1 titer of 1:4 (51Cr release assay) was demonstrated after immunization. She conceived in the same menstrual cycle and antepartum prophylaxis for fetal thrombocytopenia in this pregnancy included monthly
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
followed by density gradient separation of mononuclear cells and hypotonic lysis to remove remaining red cells. The isolated cells were resuspended in 5 ml of Ringer's lactate solution and 2.5 ml were given intravenously and the remaining cells were injected into two subcutaneous and two intradermal forearm sites. Platelet counts from similar preparations averaged 2.9 x 106/mm3 for an average injection of 1.5 x 1010 platelets. These amounts are equivalent to 5 to 10% of the platelets from the original blood units. A fetal ultrasound study performed at 11 weeks of gestation was unremarkable. The pregnancy continued uneventfully until 37 weeks' gestation when a fetal ultrasound showed evidence of macrocephaly, dilation of the lateral ventricles, and severe oligohydramnios. Because of documented fetal distress (low biophysical profile), a 2.3 kg male was delivered by cesarean section. Apgar scores were 8 and 9 at 1 and 5 minutes, respectively. Physical examination of the newborn revealed a height and weight at the 10th percentile and a head circumference greater than the 90th percentile (38 cm). The anterior fontanelle was bulging and the cranial sutures splayed. Generalized petechiae were noted and there was a large hematoma at the vitamin K injection site. There was no hepatosplenomegaly and initial neurologic examination was normal. Laboratory data showed a hemoglobin of 16.5 mg/dl, white blood cell count of 27,600/mm3, and a platelet count of 1000/mm3. The blood smear showed marked polychromasia and the absence of platelets. The infant's prothrombin time was 11.0 and the partial thromboplastin time was 30.0. The infant's blood type was A positive, direct and indirect Coombs tests were negative. Maternal complete blood count, platelet count and peripheral smear were normal. Blood cultures, urine culture, and cytomegalovirus (CMV), toxoplasmosis, CMV and rubella titers performed at birth were subsequently negative. Figure 1 shows the course of the thrombocytopenia. Treatment consisted of three maternally derived platelet transfusions followed by IVGG (Sandoglobulin). Follow-
500
400
300
si 200
100 Figure 1. Patient's platelet count versus age in days. T: platelet transfusion; TW: washed platelet transfusion; IVGG: intravenous gamma globulin given at dose of 1 gm/kg/day.
5
10
15 AGE IN DAYS
20
25
30 449
AMERICAN JOURNAL OF PERINATOLOGY/VOLUME 9, NUMBER 5/6 Table 1.
Mother Father
Sept/Nov 1992
Serologic Studies*
HLA Typing
Platelet Typing
A2, 10; B7, 8 (BW6); Dr2, 3 (DW52) All, 28, B39, 8 (BW6); Dr4, 6, (DW52)
P1 A 2 homozygous (P1 A1 negative) P1 A 1 homozygous
Anti-PTA1 Antibody (Antigen Capture)
Anti-P1A1 Antibody (5'Cr Release)
1:256 before 1:1024 after
Weak (+) undil before (+) at 1:16 after
intravenous infusions of 1 gm/kg IVGG from 12 postmenstrual weeks. Cordocentesis was performed at 21 postmenstrual weeks (1 week after maternal IVGG infusion) and revealed a fetal platelet count of 7000/mm3. Oral dexamethasone (4 mg daily) was added, and IVGG infusion frequency increased to weekly. However, when the patient returned the next week for treatment, the fetus was nonviable, secondary to exsanguination. DISCUSSION NAT may cause serious bleeding problems in affected newborns.9 NAT is diagnosed in about 1 of 3000 to 5000 pregnancies.1011 The mechanism is similar to that of Rh sensitization. Maternal-fetal incompatibility for the P1A1 antigen is the most common cause. Approximately 98% of the European and North American population have the P1A1 antigen12 and sensitization to P1A1 accounts for about 50% of the cases of NAT.11 Other antigen systems, such as BakA, Bra, and Pena may also be involved.13-17 sensitization usually occurs due to previous pregnancy or exposure to blood products. The prevalence of NAT-affected infants is lower than that expected from the incidence of antigen incompatibility in the population. The reason that some PlA1-negative women develop antibodies while others do not remains unclear. However, an association between maternal HLA type B8, DR3, and the development of anti-PlA1 alloimmunization has been noted.12-18 Although infants at risk for NAT are frequently delivered by cesarean section to protect them from the trauma of vaginal delivery,18 cases of intrauterine intracranial hemorrhage have been reported.19-20 Once delivered, these infants can be treated with maternal platelets that lack the target antigen. In our patient, two transfusions of maternal platelets temporarily increased the platelet count in this affected infant, but the third transfusion of washed maternal platelets was not effective. Other modalities of neonatal treatment for NAT include exchange transfusion and corticosteroids.9-20. IVGG is now being used to treat many forms of immune thrombocytopenia. IVGG has been used in childhood idiopathic thrombocytopenic purpura,21-23 platelet alloimmunization from frequent transfusions,24-25 and neonatal passive immune thrombocytopenia associated with maternal idiopathic thrombocytopenic purpura.26 There have been previous reports on the successful treatment of NAT with IVGG.27-39 Antenatal treatment of pregnant women with a prior affected pregnancy with infusions of IVGG given with or without dexamethasone was reported by Bussel and coworkers30 to prevent recurrent intracranial hemorrhage. However, in our patient's second affected pregnancy, maternal IVGG infusion the week before cordocentesis had no apparent effect on the fetal platelet count. Nicolini 450 and others31 reported a similar case in which the fetal
platelet count was apparently unchanged by maternal IVGG infusion, even though fetal hypergammaglobulinemia was achieved. We note that neither our patient, nor the case reported by Nicolini et al, were given dexamethasone in addition to the IVGG infusions. Previous studies have not demonstrated a striking increase in the antibody titers as measured by the ACE assay in at-risk pregnancies that have been followed serially.729 Other antibody tests, such as the 51Cr release assay32 may increase in strength throughout pregnancy, but this occurs in the minority of cases. Therefore, we speculate that the paternal mononuclear cell immunization was related to the boost in titer seen during the first successful pregnancy. In summary, we believe that this is the first case report of platelet alloimmunization associated with maternal immunization with paternal mononuclear cells. Although this patient was sensitized to her husband's platelet antigens through previous miscarriages, our investigation demonstrated a dramatic rise in anti-PlA1 titer following paternal mononuclear cell immunization. Most centers offering this treatment use screening tests to identify those at risk for Rh and Kell sensitization and give Rh-negative women prophylactic treatment with rhesus immune globulin after immunization. Either careful screening and Rh prophylaxis or more complete removal of red cells from the lymphocyte preparations may account for the observation that red cell alloimmunization attributable only to the immunization procedure has not been reported. Because a significant number of paternal platelets are still present after mononuclear cell preparation, platelet alloimmunization is a possible consequence of treatment for those at risk. The rise in our patient's platelet antibody titer after immunization was probably caused by recent antigen exposure as a consequence of her early pregnancy or immunization with mononuclear cells from the PlA1-positive paternal donor. The rise in antibody titer was probably not a nonspecific response to the immunization procedure or to pregnancy because no such increase was observed after immunization of this woman with a similar preparation of cells from a PlA1-negative donor in a subsequent pregnancy. Women at risk for platelet alloimmunization might be identified through the parental HLA typing often performed as part of immunologic evaluation of recurrent miscarriages. Those whose HLA type is known to be B8, DR3 (DRW52) could be offered platelet antigen typing, because they may be at highest risk for platelet alloimmunization. Furthermore, this case demonstrates that operative delivery of an infant with known or suspected NAT does not spare the infant from significant morbidity. Although IVGG was effective for postnatal treatment of the first affected infant's thrombocytopenia, antenatal maternal IVGG infusion did not prevent fetal thrombocytopenia in this woman's second affected pregnancy. IVGG may become the treatment of choice for postnatally diagnosed antibody-medicated thrombocytopenia of the newborn.
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"Serologic testing performed on parents. Serum tested postnatally included samples saved from prior to immunization procedure.
NAT AFTER MATERNAL IMMUNIZATION/Pearlman, et al.
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REFERENCES
The authors wish to thank Drs. E. Mamberg, T. Chronister, P. Huang, and E. McReynolds for their contributions to the management of this patient. We are grateful to Dr. Michael E. Norman for his review of the manuscript and to B. Simmons for her secretarial assistance.
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