Fetal Rhesus D genotyping on amniocytes in alloimmunised pregnancies using fluorescence duplex polymerase chain reaction

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Abstract
1. To establish the reliability of fetal amniocyte Rhesus D (RhD) genotyping using fluorescence duplex polymerase chain reaction (PCR) and 2. to assess the potential clinical impact on management of alloimmunised pregnancies. Multicentre observational study. Four departments of obstetrics and gynaecology in Germany. Fourty-four amniotic fluid samples were obtained by amniocentesis from a retrospective group of 27 RhD alloimmunised pregnancies and 15 samples from 14 women treated prospectively. Two RhD gene specific fragments (exon 7 and 10) were amplified using two separate fluorescence duplex PCR assays, and laser detected in an automated DNA sequence analyser. Amplification of the Rh gene sequences was successful in all samples. PCR at the two RhD gene regions resulted in complete concordance. Genotyping correctly predicted the RhD status of all fetuses serotyped (n = 41). After intrauterine transfusions, PCR identified the RhD type of two fetuses more accurately than serotyping. Earlier knowledge of a negative RhD status would have rendered unnecessary 12 amniocenteses in four fetuses of the retrospective study group, and prevented further invasive testing in one fetus treated prospectively. In the latter group, women with a positive fetal RhD genotype underwent intensive prenatal care including serial invasive monitoring and intrauterine treatment. Fetal RhD genotyping of amniocytes is a reliable technique with the potential to improve routine management of alloimmunised pregnant women.