Non-invasive fetal platelet and red cell blood group genotyping with the use of targeted massively parallel sequencing of maternal plasma cell-free DNA

In pregnant women with a history feto-maternal incompatibility (fetal and neonatal alloimmune thrombocytopenia (FNAIT) or hemolytic disease of the newborn (HDN)), fetal human platelet antigen (HPA) or blood group genotyping is required to determine whether the fetus is at risk and whether prenatal interventions are required.

Published methods for non-invasive genotyping of fetal blood groups using maternal plasma cell-free DNA do not provide internal controls for exclusion of false-negative results.

Cell-free DNA was isolated from plasma of 3 RhD-negative pregnant women and 6 pregnant women with a history of FNAIT due to anti-HPA-1a (4 cases), anti-HPA-5b or anti-HPA-15a. The gestational age at the time of blood sampling was 24 weeks (median; range 15 – 32). A primer panel was designed to target sequences flanking single-nucleotide polymorphisms (SNPs)/exonic regions of ITGB3 (HPA-1), ITGA2B (HPA-3), ITGA2 (HPA-5), CD109 (HPA-15), RHD, RHCE, KEL, DARC, SLC14A1, GYPA, GYPB, and SRY. These regions and 14 anonymous SNPs were massively parallel sequenced by semiconductor technology. The implicated non-maternal fetal blood groups (RHD, HPA-1a, HPA-5b, HPA-15a) were correctly identified in all cases. The counting of non-maternal sequences of Y chromosomal regions and autosomal SNPs allowed quantification of fetal load and served as internal control for the presence of fetal DNA. The fractional fetal DNA concentration was 9.43% (mean; range 4.4%-27.14%).

We propose this method for reliable non-invasive detection of fetal blood group polymorphisms that are frequently involved in FNAIT and HDN.