The controversy about controls for fetal blood group genotyping by cell-free fetal DNA in maternal plasma

Fetal blood group genotyping using cell-free fetal DNA from maternal plasma is routinely performed in alloimmunized women and has been introduced for targeted antenatal anti-D prophylaxis. The necessity to control for extraction of fetal DNA in these tests is questioned by many. This review describes the various types of controls for preventing false-negative results and discusses their value. Polymorphic markers like short tandem repeats, insertion/deletion polymorphisms or single nucleotide polymorphisms can be used as a fetal DNA control in pregnancies in which a Y-chromosome marker is not applicable, but workload is considerable and more than 99% coverage is only reached on platforms allowing high level of multiplexing. Recently, the universal fetal marker RASSF1A has been introduced, enabling the demonstration of fetal DNA after methylation-sensitive digestion of maternal DNA. The analysis of recently published noninvasive fetal blood group genotyping studies showed that false-negative results were only encountered in studies lacking a control for the presence of fetal DNA, albeit at a low frequency of 0.1-0.2%. Because of the potentially severe consequences of false-negative results in alloimmunized women, a blood group antigen-negative result should in these cases only be issued if fetal DNA is demonstrated. However, the low frequency of false-negative results makes it acceptable to perform screening studies without a fetal marker