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Contains fulltext : 167810.pdf (Publisher’s version ) (Open Access) To guide anti-D prophylaxis, Dutch D- pregnant women are offered a quantitative fetal-RHD-genotyping assay to determine the RHD status of their fetus. This allowed us to determine the frequency of different maternal RHD variants in 37 782 serologically D- pregnant women. A variant allele is present in at least 0.96% of Dutch D- pregnant women The D- serology could be confirmed after further serological testing in only 54% of these women, which emphasizes the potential relevance of genotyping of blood donors. 43 different RHD variant alleles were detected, including 15 novel alleles (11 null-, 2 partial D- and 2 DEL-alleles). Of those novel null alleles, one allele contained a single missense mutation (RHD*443C>G) and one allele had a single amino acid deletion (RHD*424_426del). The D- phenotype was confirmed by transduction of human D- erythroblasts, consolidating that, for the first time, a single amino acid change or deletion causes the D- phenotype. Transduction also confirmed the phenotypes for the two new variant DEL-alleles (RHD*721A>C and RHD*884T>C) and the novel partial RHD*492C>A allele. Notably, in three additional cases the DEL phenotype was observed but sequencing of the coding sequence, flanking introns and promoter region revealed an apparently wild-type RHD allele without mutations.

Haemolytic Disease of the Fetus and Newborn (HDFN) is caused by maternal alloimmunization against red blood cell antigens. In severe cases, HDFN may lead to fetal anaemia with a risk for fetal death and to severe forms of neonatal hyperbilirubinaemia with a risk for kernicterus. Most severe cases are caused by anti-D, despite the introduction of antental and postnatal anti-D immunoglobulin prophylaxis. In general, red blood cell antibody screening programmes are aimed to detect maternal alloimmunization early in pregnancy to facilitate the identification of high-risk cases to timely start antenatal and postnatal treatment. In this review, an overview of the clinical relevance of red cell alloantibodies in relation to occurrence of HDFN and recent views on prevention, screening and treatment options of HDFN are provided.

Background & Aims Wilson disease is a genetic copper storage disorder that causes hepatic and neurologic symptoms. Chelating agents (D-penicillamine, trientine) are used as first-line therapies for symptomatic patients, but there are few data from large cohorts. We assessed the safety of D-penicillamine and trientine therapy and outcomes of patients with Wilson disease. Methods We performed a retrospective analysis of data on 380 patients with Wilson disease from tertiary care centers in Germany and Austria, and 25 additional patients from the EUROWILSON registry. Chelator-based treatment regimens were analyzed for their effect on neurologic and hepatic symptoms and for adverse events that led to discontinuation of therapy (Kaplan–Meier estimation; data were collected for a mean of 13.3 y after therapy began). Results Changes in medication were common, resulting in analysis of 471 chelator monotherapies (326 patients receiving D-penicillamine and 141 receiving trientine). Nine of 326 patients treated with D-penicillamine and 3 of 141 patients given trientine underwent liver transplantation. Adverse events leading to discontinuation of treatment were more frequent among those receiving D-penicillamine than trientine ( P = .039). Forty-eight months after therapy, hepatic deterioration was reported in only 4 of 333 patients treated initially with a chelating agent. Hepatic improvements were observed in more than 90%, and neurologic improvements were observed in more than 55%, of therapy-naive patients, and values did not differ significantly between treatments. However, neurologic deterioration was observed less frequently in patients given D-penicillamine first (6 of 295) than those given trientine first (4 of 38; P = .018). Conclusions Chelating agents are effective therapies for most patients with Wilson disease; D-penicillamine and trientine produce comparable outcomes, although D-penicillamine had a higher rate of adverse events. Few patients receiving chelation therapy had neurologic deterioration, which occurred more frequently in patients who received trientine.

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.