Hemolytic disease of the newborn (HDN) affects the fetus or neonate and results from the transplacental passage of maternal alloantibodies directed against fetal red cell antigens inherited from the father. Over 90% of all cases of clinically significant HDN affect rhesus D (RhD)-positive infants born to RhD-negative mothers. When fetomaternal hemorrhage occurs antenatally or at delivery, maternal B lymphocyte clones that recognize the RhD antigen are established. In a subsequent pregnancy, if stimulated by the RhD antigen on fetal red cells, they proliferate and produce immunoglobulin (Ig) G antibodies that cross the placenta and destroy any RhD-positive red cell, resulting in fetal anemia. The incidence of HDN has dropped dramatically (1-6/1000 live births) since 1968, when effective prophylaxis by anti-D Ig injection to the mother become available. In the United States, anti-D Ig is systematically administrated at 28 weeks' gestation and at delivery, or if an identifiable risk event occurs to RhD-negative mothers. Administration of blood derivative is not devoid of risk, and every effort should be made to improve techniques that reduce the number of injections. In 1991, the RhD locus was localized to the short arm of chromosome 1. The elucidation of the molecular basis of the various blood group system alloantigens allowed the development of polymerase chain reaction (PCR)-based assay for blood group typing. These assays can be performed with fetal DNA obtained from amniocentesis or chorionic villus sampling. In 1998, Lo et al. and Faas et al. discovered that fetal DNA was present in maternal blood and that fetal RhD status could be determined by a noninvasive method.
For clinical purposes, fetal RhD genotyping using PCR is a significant advancement. In case of a heterozygous father, fetal RhD genotype determination early in pregnancy is useful in the management of RhD-negative sensitized women. Amniocentesis is now accepted as the primary modality that is used to test fetal blood type. Chorionic villous sampling can also be used, but it should be discouraged in patients who wish to continue the pregnancy if the fetus is found to be RhD-positive. Disruption of the chorionic villi during the procedure can result in fetomaternal hemorrhage and an anamnestic response in maternal titer, thereby worsening the fetal disease. This phenomenon can also occur during amnio-centesis, and all attempts should be made to avoid transplacental passage. Although the quantity and quality of DNA obtained from fetal sampling are less than those of leukocytes, many groups showed concordant results with serology, although the importance of testing more than one region of the gene was established. Moreover, these sampling procedures are invasive, resulting in an increase risk of fetal loss (2%). Fetal RhD genotyping is also useful in RhD-negative pregnant women at risk for RhD immunization [American College of Obstetrics and Gynecology (ACOG) practice bulletin, 1999] to adapt prophylactic anti-D Ig infusion, to avoid unnecessary administration in case of an RhD-nega-tive fetus.
Many groups have worked on a reliable noninvasive approach to determine fetal RhD genotype. Recovery of fetal cells from maternal blood or cervical mucus has been reported, but techniques require isolation and enrichment of fetal cells, and questions regarding sensitivity and cost-effectiveness are still unresolved for routine use.[6,7] An RNA-based assay on fetal erythroblasts isolated from maternal blood was also evaluated, but results were not conclusive. Moreover, the persistence of fetal cells from previous pregnancies renders this approach susceptible to false-positive results. Despite the fact that many studies have shown that it is theoretically possible to isolate fetal cellular DNA from maternal blood, none of the techniques used meets the accuracy needed for clinical usage.
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