Recurrent Pregnancy Loss in a 33-Year-Old Woman

A 33-year-old pregnant woman of Asian-Indian descent. History of recurrent miscarriages (G4, P1, A2). The patient presents at 15 weeks gestation in her fourth pregnancy for antenatal care. Her first and third pregnancies ended with first trimester (less than 12 weeks gestational age) miscarriages of unknown cause. The second pregnancy was complicated by pregnancy-induced hypertension (PIH) and was successfully carried to term, with a living boy delivered by Cesarean section at 40 weeks gestation. The newborn boy was healthy, but was small (2300 gm) for gestational age. The patient had never been transfused blood products. The patient had a history of mild asthma and remote uneventful tonsillectomy. There was no significant family history. Physical examination was normal for gestational age. Initial Laboratory Findings: CBC, routine coagulation testing, type and screen, and thrombophilia workup were performed on the mother, at 15 weeks gestation (Table 1). Additional Laboratory Testing: Additional blood bank testing was performed on the mother (Tables 2, 3, and 4) and on family members (Table 5). What are the patient’s most striking initial clinical laboratory findings? What is the clinical significance in pregnancy of the abnormal thrombophilia test result? What do the additional blood bank investigations demonstrate? What is the nature of the red cell antibody? What is the clinical significance of the red cell antibody for the patient? What is this patient’s chance to get compatible red blood cell (RBC) units if blood transfusion is urgently required, and what are other possible alternatives if no compatible blood is accessible? Does the maternal alloantibody in this case have any effect on the newborn baby and why? 1. The most striking laboratory findings include the positive RBC antibody screening test and the presence of heterozygosity for Factor V Leiden mutation. 2. The patient was shown to be heterozygous for the Factor V Leiden mutation, 1691 G>A(R506Q), using DNA-based polymerase chain reaction (PCR) assay.1 The allele frequency Factor V Leiden mutation has been reported to be 4.4% in Europeans2 and 0.6% in the indigenous populations of Asia-Minor, which is the origin of the patient in this case study.3 The R506Q, arginine to glutamine mutation at position 506, makes the Factor V protein resistant to proteolytic cleavage by activated Protein C and is a thrombophilia risk factor. In the pregnant patient with Factor V Leiden, this thrombophilic tendency may be manifested as early, recurrent and late pregnancy loss, stillbirth, placental infarction, placental abruption, low birth weight and significantly higher risks of pre-eclampsia, although controversy exists as to the extent of the risk.1,4,5 Pregnancy outcome with Factor V Leiden mutation is heterogeneous, and a severely affected pregnancy may be followed by a less severely affected, or even a normal pregnancy, regardless of therapy. In this case, there is the possibility that the presence of heterozygosity for Factor V Leiden mutation was possibly related to her previous 2 miscarriages, low birth weight baby, and pregnancy-induced hypertension (PIH). Due to her increased risk for thrombosis, the patient received prophylaxis with low molecular weight heparin after delivery. 3. The patient’s serum reacted with all red cells tested, using an indirect antiglobulin test (IAT) with saline and polyethylene glycol (PEG), except her own autologous red cells, compatible with an antibody to a high prevalence antigen (Table 2). The extended antibody panel (Table 3) showed all cells reacting with the patient’s serum, except for cell 1, the Crawford red cells with Lu(a−b−) phenotype. The patient’s phenotype was Lu(a+b+) and therefore antibodies directed against Lua and Lub were excluded. It was also noted that reactions were significantly weaker with other Lu3- cells, and since the reactions were inhibited with papain-treated cells, a small panel of red cells was used to test by PEG-IAT. This panel included red cells negative for Inb and some other high prevalence antigens of the following phenotype: Kp(b−), Js(b−), Co(a−), Lu -3, -17, -18, Yt(a−), Ge -2, -3, -4 (Table 4). The patient was found to have anti-Inb antibody. The patient’s phenotype was In(a+b−), and her husband’s phenotype was In(a−b+) (Table 5). The anti-Inb in this patient with no previous history of transfusion was most likely formed following pregnancy. The antibody titre in the maternal serum was =4 throughout the pregnancy. Initial Laboratory Findings WBCs, white blood cells; MCV, mean cell volume; RBCs, red blood cells; INR, international normalized ratio; PTT, partial thromboplastin time; DAT, direct antiglobulin test. Initial Laboratory Findings WBCs, white blood cells; MCV, mean cell volume; RBCs, red blood cells; INR, international normalized ratio; PTT, partial thromboplastin time; DAT, direct antiglobulin test. 4. Anti-Inb is a rare red blood alloantibody directed against Inb, a high prevalence RBC antigen. Ina and Inb antigens are the 2 antigens making up the Indian blood group system.6 These antigens reside on the leukocyte homing receptor and cellular adhesion molecule, CD44, noted for its ability to bind hyaluronan, a component of the extracellular matrix.7 Ina/Inb polymorphism represents an amino acid difference at position 46. When the amino acid is proline, the protein expresses Ina, and when the amino acid is arginine, the protein expresses Inb.6 Anti-Inb antibody is of IgG type reacting by IAT and at 37°C but not with enzyme-pretreated cells. The expression of Inb is reduced on Lu(a−b−) RBCs which result from the effect of the dominant inhibition gene In(Lu).6,7 This explains the negative reaction in the IAT using the patient’s serum against Crawford red cells. Crawford is a blood banker who found her own red cells of the dominant type Lu(a−b−) phenotype. Her red cells kept in our institution as 1 of the rare red cells for antibody identification. Protein Ina is a low prevalence antigen, rare in the Caucasian population, occurring in ranges from 0.01% to 0.09% in the Indian subcontinent and up to 0.4% in Iranians and Arabs.8 5. Anti-Inb is a clinically significant alloantibody. Individuals with anti-Inb could develop a transfusion reaction if transfused with In(b+) RBCs. RBC survival studies have shown that anti-Inb causes premature destruction of transfused In(b+) RBCs.8 A case report of an immediate hemolytic transfusion reaction has been described after transfusion of In(b+) blood to an In(b−) patient.9 Patient Serum Antibody Panel Results Patient Serum Antibody Panel Results Patient Extended Serum Antibody Panel Results Crawford’s red cells, Lu(a−b−) with dominant inhibitor In(Lu). Patient Extended Serum Antibody Panel Results Crawford’s red cells, Lu(a−b−) with dominant inhibitor In(Lu). 6. Finding compatible blood for this patient is problematic, as the patient requires In(a+b−) phenotype blood for transfusion. An extensive search for compatible blood through Canadian and American rare donor registries was pursued, but the search disclosed that no In(a+b−) blood was available for transfusion. Testing family members for a similar phenotype is 1 possible alternative if no compatible allogeneic blood is accessible. Unfortunately in this case, there were no other family members available for testing. Therefore autologous blood donation was the only option available. Several studies have shown autologous donation during pregnancy to be safe for both the mother and the fetus.10,11 She was put on a full dose of oral iron therapy and was able to donate 3 red cell units during her pregnancy, at 17 weeks gestation, 20 weeks gestation, and 38 weeks gestation. The patient tolerated the 3 autologous blood donations, of 450 mL each, during her pregnancy remarkably well, maintaining her hemoglobin level, as shown in Figure 1. The first 2 units collected were kept frozen at the Canadian Blood Services (CBS), and the last unit collected was kept refrigerated until after delivery and subsequently frozen. The patient has been encouraged to participate in the Canadian National Rare Donor Program (NRDP) in the event that blood with this rare In(b−) phenotype is required for transfusion. The NRDP is operated by CBS and Hema-Quebec, the 2 blood collection agencies in Canada. The NRDP works in collaboration with other international rare blood donor programs such as the American Rare Donor Program and the WHO International Rare Donor Program. 7. The maternal anti-Inb has no effect on the fetus/newborn, and hemolytic disease of the newborn due to anti-Inb has not been reported, as it appears that anti-Inb is unable to cross the placenta.12 It is hypothesized that anti-Inb is absorbed by CD44 expressed by placental macrophages thus blocking its passage to the fetus.8,13 If a RBC transfusion was indicated in this newborn, it is recommended that the baby’s serum be used for the cross-match. The patient was followed in the high-risk obstetric clinic to ensure high-quality care and an improved pregnancy outcome. Her blood pressure remained normal and the fetal growth was adequate throughout the pregnancy, with the fetus growing along the 25th percentile. There was no significant change in the anti-Inb titre in the maternal serum during the pregnancy. The titres at 10, 26, and 35 weeks gestation were 4, 2, and 2 respectively. The patient donated 3 autologous red cell units during her pregnancy. At 38½ weeks gestation, the patient delivered a healthy baby girl by Cesarean section, small for gestational age (2500 g), but with no clinical manifestations of hemolytic disease of newborn (HDN). The newborn girl’s RBC phenotype was In(a+b+) (Table 5). The cord RBCs gave a negative direct antiglobulin test (DAT) and a negative reaction when the cord serum was tested against maternal cells and a mini panel of red cells. Therefore, no anti-Inb antibody was detected in the cord sample, compatible with the absence of any laboratory evidence of HDN. Neither the mother nor her baby required red cell transfusion. Postpartum the current pregnancy, the patient received low molecular weight heparin prophylaxis. Patient Serum Antibody Results to Red Cells Negative for Selected High Prevalence Antigens for patient’s results: +indicates patient’s serum reacts with red cells negative for this high prevalence antigen 0 indicates patient’s serum does not react with red cells negative for this high prevalence antigen Patient Serum Antibody Results to Red Cells Negative for Selected High Prevalence Antigens for patient’s results: +indicates patient’s serum reacts with red cells negative for this high prevalence antigen 0 indicates patient’s serum does not react with red cells negative for this high prevalence antigen Indian Blood Group Phenotype Testing on Family Members Indian Blood Group Phenotype Testing on Family Members Hemoglobin levels (pink line) and timing of autologous blood –donations (black arrows). Hemoglobin levels (pink line) and timing of autologous blood –donations (black arrows). In summary, in this patient with a history of recurrent fetal loss, 2 significant laboratory abnormalities were detected at 15 weeks gestation, namely heterozygosity for Factor V Leiden mutation, and the presence of antibody to a high-prevalence antigen, anti-Inb. The presence of heterozygosity for Factor V Leiden was managed by closely following the patient in the high-risk obstetric clinic and with postpartum low molecular weight heparin prophylaxis. The anti-Inb was managed by autologous donation during pregnancy, to ensure the availability of RBCs for transfusion for the mother, should the need have arisen. Our case study shows the careful laboratory investigation and management of a patient with a high-risk pregnancy can result in a successful delivery of a healthy, live infant and ensure the good health of the mother. The expertise of the Transfusion Medicine technologists at the Ottawa hospital is gratefully acknowledged. The authors would also like to acknowledge and thank Rachel Berger for her support providing rare blood samples that were collected with help from Dr. M N Crawford (1923–1997). pregnancy-induced hypertension polymerase chain reaction indirect antiglobulin test polyethylene glycol Canadian Blood Services Canadian National Rare Donor Program hemolytic disease of newborn direct antiglobulin test white blood cells mean cell volume red blood cells international normalized ratio partial thromboplastin time

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