Background 
Hemolytic disease of the fetus and the newborn (HDFN) is caused by maternal alloantibodies directed against fetal red cell surface antigens that the mother herself lacks. In allo-immunized pregnant women, it is beneficial to determine whether her fetus has the corresponding antigen to tailor pregnancy management. In the Rh system, after anti-D, the most common cause of HDFN is anti-c followed by anti-E and anti-C although these two last ones only very occasionally cause severe HDFN. Prenatal diagnosis of fetal RHD genotype from maternal blood is done routinely in many countries in Europe but is still uncommon for RHCE. We report our Belgian 7-year clinical experience of non-invasive fetal blood group genotyping of RHCE C/c and E alleles to determine for Rh C/c, E fetal status in allo-immunized pregnant women.  
Objective 
Retrospective study to estimate the diagnostic performance of non invasive fetal RHCE C/c and E genotyping and its accuracy by comparing the results with the Rh C/c, E phenotype of the newborn. 
Methods 
Real-time polymerase chain reaction was performed for RHCE intron 2, RHCE exon 2 and RHCE exon 5 for the RhC, c and E allele respectively in alloimmunized pregnant women from 2010 up to 2017. The results were compared with available serology tests performed on cord red blood cells (RBC) after delivery.
Results 
A total of 86 fetal blood group genotyping tests were performed in 76 allo-immunized pregnant women.  We obtained retrospective informations about 55 fetal RHCE genotypes (C: n = 25; c: n = 12; E: n = 18) in 49 allo-immunized pregnant women (3 women are immunized against 2 antigens and 3 women had 2 pregnancy). The median gestational age was 20,4 weeks. Genotyping result was issued in 53 tests (96%): in two samples, there was a lack of reproducibility in the amplification of RHCE exon 5 (RhE) and RHCE intron 2 (RhC). Seven-teen samples were excluded for accuracy testing because Rh phenotype was not available (stillbirth/abortion (n=1), in utero-transfusion (n=2), positive antiglobulin reaction at birth (n=3) and Rh phenotype not tested (n=11).Genotyping was correctly determined with only 1 false negative in RhC test among those women for whom cord blood serology was available (n=37). This discrepancy concerns an allo-immunized anti-C woman whose newborn has got a negative direct antiglobulin test and no indices of hemolytic process. The accuracy of noninvasive fetal RH C/c, E blood group genotyping is 100% for Rhc and RhE and 94.7% for RhC. 
Conclusion
In spite of the small number of samples analyzed, we confirm that the noninvasive fetal RH C/c, E blood group genotyping in Rh C/c or E alloimmunized pregnant women is an accurate and performant method to determine whether a fetus is at risk of hemolytic disease and can be used in clinical diagnostic setting. In a secure approach, a negative result could be controlled from a second maternal plasma sample a few weeks later. Invasive procedures to determine the fetal Rh C/c or E status are no longer necessary.

Non-invasive fetal genotyping of RHCE C/c and E alleles in allo-immunized pregnant women: evaluation of a 7-year experience