The Egyptian Journal of Medical Human Genetics (2011) 12, 127–133 Ain Shams University The Egyptian Journal of Medical Human Genetics www.ejmhg.eg.net www.sciencedirect.com ORIGINAL ARTICLE Correlation between the RhD genotyping and RhD serotyping in isoimmunized pregnancies Sahar M. Nour El Din a,*, Ahmed R.M. ARamy b, Mohamed S. Ali b a Medical Genetics Center, Ain-Shams University, Cairo, Egypt b Gynecological and Obstetric Department, Faculty of medicine, Ain Shams University, Cairo, Egypt Received 2 January 2011; accepted 3 April 2011 Available online 5 July 2011 KEYWORDS Abstract Alloimmunisation was one of the most important causes of perinatal mortality and mor- Maternal alloimmunization; bidity by the middle of the last century. The objective of the present study was to investigate the Rhesus; presence of the RHD gene in fetal cells (amniocytes) obtained from amniotic fluid by genotyping RHD; to compare it with the RhD serotyping. Also to correlate the presence of RhD gene with the neo- Isoimmunization; natal outcome. This work was carried out at Maternity hospital and Medical Genetics center, while Hydrops fetalis; PCR testing was done at the Medical Research center, Faculty of Medicine, Ain Shams University Fetus; in the period from 2008 to 2010. The present study included recruiting of 20 RhD negative (sensi- Rh negative tized to the RhD antigen) pregnant mothers. The entire study group was subjected to complete general, obstetric and a detailed obstetric ultrasonographic examination. Rh typing and indirect Coomb’s test were also done. Amniocentesis was performed with a 20-gauge needle under contin- uous ultrasound guidance. RhD serotyping of the fetuses showed that, 14 fetuses (70%) were positive and six fetuses (30%) were negative. While using RhD gentyping 13 cases (65%) were positive and seven cases (35%) were negative (P value = 0.002). Among fetuses positive for RhD genotyping six fetuses (46%) had received postnatal treatment, while among fetuses negative for RhD genotyping, neither of them had received postnatal treatment (P value = 0.032), which is statistically significant. From the present study we can conclude that, the identification of an * Corresponding author. Address: 82 (B) El-Sewasry District, Naser City, Cairo 11331, Egypt. Tel./fax: +20 2 24109228. E-mail addresses: [email protected] (S.M.N. El Din), [email protected] (A.R.M. ARamy), mohamed_ali906@ yahoo.com (M.S. Ali). 1110-8630 Ó 2011 Ain Shams University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Ain Shams University. doi:10.1016/j.ejmhg.2011.06.005 Production and hosting by Elsevier 128 SM.N. El Din et al. antigen-negative fetus on the basis of the blood group genotype provides significant advantages in managing the pregnancy at risk for HDFN. Ó 2011 Ain Shams University. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction data in 2003 reported the incidence of Rh sensitization to be approximately 6.8 per 1000 live births [11]. Alloimmunisation was one of the most important causes of The prevalence of the RhD-negative blood type is depen- perinatal mortality and morbidity by the middle of the last dent on ethnicity with whites having the highest prevalence, century. It was affecting 1–2% of all pregnancies and approx- while Asians and American Indians having the lowest. Of imately 50% of Rhesus (Rh) immunized pregnancies were lost the existing Rh antigen, the D antigen is the most immuno- [1]. This was reduced by100-fold in the last decades using pro- genic. Approximately 10% of pregnancies in white women phylactic immunoglobulin and fetal therapy procedures, par- are Rh incompatible [12]. However, because the risk of ticularly an intrauterine intravascular transfusion (IUIVT) alloimmunization in a susceptible RhD-negative woman is [2]. Today the incidence of hemolytic disease of the newborn significantly affected by several factors, less than 20% of secondary to Rh disease is approximately one to six cases RhD-incompatible pregnancies actually lead to maternal per 1000 pregnancies [3]. Despite the development and imple- alloimmunization. These factors include the volume of fetoma- mentation of Rh(D) immune globulin prophylaxis, maternal ternal hemorrhage, the degree of maternal immune response, Rh alloimmunization is still a cause of erythroblastosis fetalis concurrent ABO incompatibility, and fetal homozygosity ver- and hemolytic disease of the newborn. However, with appro- sus heterozygosity for the D antigen [13]. priate monitoring and intervention, hemolytic disease of the Two genes have been identified on the short arm of chro- fetus and newborn can be treated successfully in almost all mosome 1 that encode the major Rh antigen groups (c/C, D, cases. The outcome of alloimmunized pregnancies is generally e/E): RHD and RHCE, with differential protein production very good, with no long-term sequelae in offspring [4]. in the latter is likely to be due to alternative splicing. Inheri- Maternal alloimmunization, also known as isoimmuniza- tance of these so-called Rh locus genes is closely linked, thus tion, occurs when a woman’s immune system is sensitized to allowing estimations of paternal heterozygosity at the Rh D lo- foreign erythrocyte surface antigens, stimulating the produc- cus to be made using gene frequency tables that incorporate tion of immunoglobulin G (IgG) antibodies [5]. Antenatal data on paternal ethnicity, blood type, and number of previous determination of fetal blood group is important in pregnancies RhD-positive infants [14]. with a significant risk of hemolytic anemia due to maternal Causes of maternal alloimmunization include: Blood trans- alloimmunization. Evaluation for the presence of maternal fusion, fetomaternal hemorrhage (antepartum, intrapartum), anti-D antibody should be undertaken at the first prenatal visit abortion (therapeutic, spontaneous), ectopic pregnancy, pla- [6]. First-time sensitized pregnancies are followed with serial cental abruption, abdominal trauma, obstetric procedures maternal titers and, when necessary, serial amniocenteses is (amniocentesis, chorionic villus sampling (CVS), percutaneous indicated to detect fetal bilirubin. The bilirubin level is quanti- umbilical blood sampling, manual removal of the placenta) fied by spectrophotometry and expressed as the change in [15]. optical density at a wavelength of 450 nm (DOD450); the IgG antibody-mediated hemolysis of fetal erythrocytes, DOD450 values are then plotted on a chart devised by Liley known as hemolytic disease of the fetus and newborn (HDFN), to estimate the severity of anemia [7]. To reduce the risk of varies in severity and can have a variety of manifestations [16]. Rh alloimmunization in a subsequent pregnancy, RhD-nega- The degree of anemia can range from mild to severe with tive women are given anti-RhD antibodies after miscarriage, associated hyperbilirubinemia and jaundice. In severe cases, the birth of an RhD-positive baby, or any obstetrical proce- hemolysis may lead to extramedullary hematopoiesis and retic- dure that may cause fetomaternal hemorrhage [8]. uloendothelial clearance of fetal erythrocytes. This may result Among the more than 50 different antigens capable of caus- in hepatosplenomegaly; decreased liver function; and ensuing ing maternal alloimmunization and fetal hemolytic disease, the hypoproteinemia, ascites, and anasarca. When accompanied Rhesus (Rh) blood group system is the most common. The Rh by high-output cardiac failure and pericardial effusion, this blood-group antigens are carried by a series of at least three condition is known as hydrops fetalis. Without intervention, homologous but distinct membrane-associated proteins10 [9]. this syndrome is often fatal. Intensive neonatal care, including Two of these proteins have immunologically distinguishable emergent exchange transfusion [17]. isoforms designated C, c and E, e. Of note, The principal Assessment of fetal RhD status is critical in determining protein, D, has no immunologically detectable isoform d. whether a pregnancy in an alloimmunized woman is at risk Therefore, the presence or the absence of the RhD gene in for the development of hemolytic disease of the fetus and new- the genome determines the genetic basis of the polymorphisms born. Once a fetus is found to be at risk (i.e., RhD positive), associated with Rh positivity and Rh negativity. the goals of managing the alloimmunized pregnancy are The D antigen of the Rh blood group system (RhD) causes twofold. First is the detection of fetal anemia prior to the most cases of severe hemolytic disease [10]. The incidence of fe- occurrence of fetal compromise. After detection, the goal is tuses at risk for anemia due to maternal alloimmunization to to minimize fetal morbidity and mortality by correcting this red cell antigens has decreased dramatically since the institution anemia until fetal lung maturity and delivery can be achieved. of routine anti-D immune globulin (RhoGAM) prophylaxis for Because of the potential need for invasive diagnostic and ther- Rh-negative women in the 1960s. A review of birth certificate apeutic procedures, pregnancies complicated by erythrocyte Correlation between the RhD genotyping and RhD serotyping in isoimmunized pregnancies 129 alloimmunization should be managed by maternal-fetal medi- The samples were transferred to the Medical Research cen- cine specialists [18]. ter (MRC) within half an hour. In fact, Rhesus antibodies when managed by close monitor- In 16 cases the procedure