Non-invasive diagnosis of fetal RHD status in an admixed population

TRUCCO BOGGIONE, CAROLINA; LUJÁN BRAJOVICH, MELINA ELIANA; DI MÓNACO, RENE; BIONDI, CLAUDIA; COTORRUELO, CARLOS

Copenhague

Encuentro; International Meeting on Cell-free DNA. Special focus: fetal RHD genotyping; 2013

Background: non-invasive fetal RHD genotyping allows an early risk assessment of Hemolytic Disease of the Newborn in pregnancies with anti-D alloantibodies. It has also the potential to avoid antenatal anti-D prophilaxis in D negative pregnant women carrying a RHD negative fetus. The genetic background of the Argentinean population is the result of admixture events between Europeans, Amerindians and Africans. Aim: to assess the feasibility of RHD genotyping of the fetus by PCR amplification of fetal DNA obtained from plasma samples of pregnant women of an admixed population. Materials and Methods: 126 D negative pregnant women were studied. Cell free fetal (cff) DNA was obtained from 800 µl of plasma, using the QIAamp® DNA Blood Mini Kit (Qiagen). RHD exon 10 (3 replicates) and exon 7 (2 replicates) were investigated using allele specific primers and TaqMan® probes by real time (rt) PCR. A SRY sequence was amplified in duplex with RHD exon 10. The D phenotype of the newborns was determined by cord blood serology. Results: fetal RHD status could de assigned in 123 samples. The 3 inconclusive results were due to silent RHDØ (n=2) and r?s (n=1) maternal alleles. A total of 84 (68.3%) fetuses were RHD positive and 39 (31.7%) were RHD negative. When no amplification was observed (RHD and SRY negative, n=15), the test was repeated in a new maternal sample. The same results were obtained and RHD negative fetuses assigned. Cord blood phenotyping showed 1 false positive result by cff DNA typing. Further analysis determined a RHDØ allele in this cord blood sample. No false negative results were found. The assay showed 97.4% specificity and 100% sensitivity. Conclusions: the test performance is acceptable. However, to improve specificity RHDØ detection will be incorporated in the rt-PCR strategy. To detect cases in which the test is not applicable, scanning for aberrant RHD alleles in maternal DNA obtained from leucocytes will be implemented before performing fetal genotyping.