Antenatal detection of the fetal RHD status for guiding immunoprophylaxis

TRUCCO BOGGIONE, CAROLINA; LUJÁN BRAJOVICH, MELINA ELIANA; MATTALONI, STELLA MARIS; GARCIA BORRAS, SILVIA; BIONDI, CLAUDIA; COTORRUELO, CARLOS

Los cocos, Córdoba

Congreso; LXI Reunión Anual de la Sociedad Argentina de Inmunología; 2013

Sociedad Argentina de Inmunología

Routine antenatal immunoprophylaxis is given to all D negative pregnant women to prevent alloimmunization against the D antigen. Considering that the anti-D IgG is a hemoderivative in short supply, non-invasive fetal RHD genotyping has the potential to reduce the unnecessary administration of this treatment to women carrying a RHD negative fetus. The aim of this work was to develop a RHD genotyping strategy using fetal DNA obtained from maternal plasma. A total of 173 D negative pregnant women were studied. Cell free (cf) DNA was obtained from 800 µl of plasma by adsorption/elution in silica gel membranes. The isolated DNA is supposed to be 95% of maternal and 5% of fetal origin. 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 to confirm the presence of fetal DNA. The D phenotype of the newborns was determined by cord blood serology. Fetal RHD status could be assigned in 171 samples. The 2 inconclusive results were due to silent RHDΨ maternal alleles. A total of 116 (67,84%) fetuses were RHD positive and 55 (32,16%) were RHD negative. When no amplification was observed (RHD and SRY negative, n=15), the presence of fetal DNA could not be confirmed and the test was repeated in a new maternal sample. The same results were obtained and RHD negative fetuses assigned. Cord blood phenotyping showed 3 false positive results by cf DNA typing due to silent alleles (RHDΨ n=2, RHD-CE-Ds n=1) inherited from the father, as determined by further studies. No false negative results were found. The test is suitable for guiding antenatal anti-D administration. The detection of null alleles, associated with African ancestry, will be incorporated in the rt-PCR strategy to improve specificity. To detect cases in which the test is not applicable, scanning for aberrant RHD alleles in maternal DNA will be implemented before fetal genotyping.