Prenatal Fetal DNA Testing for Predicting HDFN, FNAIT, and RhIG Candidacy
Authors: van der Schoot C. Ellen , Thurik Florentine , Scheffer Peter G. , Abbink Frithjofna , P.B. Catharina van der Ploeg , Veldhuisen Barbera , de Haas Masja 
Published: October 2019

Abstract
Red cell blood group antigen and platelet antigen incompatibility between a pregnant women and her fetus can result in maternal alloimmunisation and, consequently, hemolytic disease of the fetus and newborn (HDFN) and fetal neonatal alloimmune thrombocytopenia (FNAIT), respectively. For a long time, immunization against D-antigen has been the major cause of HDFN, but postnatal immune-prophylaxis and more recently antenatal immunoprophylaxis, has successfully decreased its incidence. The majority of severe FNAIT cases are caused by antibodies against HPA-1a, and a comparable immunoprophylaxis program is presently investigated. Postnatal immunoprophylaxis is traditionally given based on the fetal blood group determined on cord blood, since only 60 percent of the newborns of a D-negative mother are D-positive. But as long as the fetal blood group is not known, antenatal prophylaxis is administered to all D-negative women. Also in alloimmunised pregnant women, knowledge of the fetal antigen status is beneficial to tailor pregnancy management. In case of red cell alloantibodies, the antibody titers have to be followed during pregnancy and the fetus is careful monitored to recognize fetuses needing intrauterine blood transfusions. In many countries, pregnant women with anti-HPA-1a alloantibodies are treated with intravenous IgG during the last trimester of pregnancy to prevent intracranial hemorrhages. Traditionally, fetal blood group genotyping has been performed through amniocentesis. This invasive procedure carries a small risk of miscarriage (especially in FNAIT cases) and could potentially enhance maternal sensitization. The discovery of cell-free fetal DNA in the blood of pregnant women in 1997 presented a noninvasive, and thus safe, method to determine the fetal blood group genotype. Cell-free fetal DNA is released from trophoblastic cells undergoing apoptosis. Within the maternal circulation, cell-free fetal DNA is present among an overwhelming background of maternal cell-free DNA, predominately of hematopoietic origin. It can be detected at as early as five weeks of gestation, and gradually increases during pregnancy, from 10 genome equivalents (GE) per ml to around 300 GE late in pregnancy, although quantities vary between pregnancies. After birth, cell-free fetal DNA is cleared from the maternal circulation within several hours, with an observed half-life of 16 minutes. Since 2000, many laboratories have developed assays that are sensitive and specific enough to genotype the fetus reliably using cell-free DNA isolated from maternal plasma. This assay is relatively simple for RHD, because in Caucasians D-negativity is caused by the complete absence of the RHD gene. However, due to the high frequency of variant RHD genes, especially in African blacks and Asians, the interpretation of the assay can be difficult. Most other blood groups are caused by SNPs, and the design of those assays is, therefore, technically more challenging. However, nowadays for most clinical blood group antigens (D, c, C, E, e, K, HPA-1a), reliable, non-invasive genotyping assays are offered worldwide on a routine basis, and invasive procedures for fetal blood group typing have become obsolete. In several European countries, large-scale feasibility studies have been performed to investigate whether non-invasive fetal RHD genotyping could be safely applied to restrict antenatal immunoprophylaxis to those D-negative women carrying D-positive fetuses. This would prevent the unnecessarily exposure of 40 percent of D-negative women to the small but non-negligible risk of infection with a blood-borne disease, as anti-D is still produced from plasma of hyperimmunised donors. Furthermore, worldwide supplies of RhD immunoglobulin are limited. Based on the promising outcomes of these studies, the Danish and Dutch government decided in 2010 and 2011, respectively, to implement fetal RHD typing. With our fully automated assay we encountered only eight false negative results in more than 25,000 tested pregnancies (0.03%, 95% CI: 0.01-0.05%). In the Netherlands, postnatal immunoprophylaxis is also now given based on the PCR result in week 27 of pregnancy, and no routine cord blood serology is performed anymore. Overall, this program is cost-effective in the Dutch setting. In several other European countries (e.g., Sweden, UK, France) studies are ongoing on the implementation of fetal RHD genotyping to guide prophylaxis.

Disclosures:
No relevant conflicts of interest to declare.