Next-generation sequencing-based approach for fetal DNA quantification and blood antigen detection in alloimmunized pregnancies

Abstract

Background: Erythrocyte and platelet alloimmunization during pregnancy can be severe and are closely monitored throughout pregnancy. However, the blood type of the fetus and the associated risk are often unknown. Here, fetal erythrocyte and platelet genotype from maternal plasma were determined using next-generation sequencing, providing noninvasive prenatal testing as an alternative to traditional methods for monitoring alloimmunized pregnancies.

Objective: This study aimed to validate an innovative next-generation sequencing method for the detection of specific blood antigens in maternal plasma-derived cell-free DNA, focusing on key antigens associated with immunizations. By analyzing free-circulating fetal DNA across various gestational stages, the study seeks to achieve high-precision identification of clinically relevant erythrocyte and platelet antigens.

Study design: Maternal whole blood samples were consecutively collected from 74 immunized and nonimmunized pregnant women at 13 to 27 weeks of gestation in Stockholm County, Sweden. Next-generation sequencing analysis was performed on maternal plasma using a prototype kit from Devyser AB (Stockholm, Sweden). The kit detects several erythrocyte blood group markers, 2 human platelet antigen markers (HPA-1 and HPA-5), XY chromosome markers, and 12 insertions and deletions from different chromosomes to identify and quantify fetal DNA. After birth, genomic DNA from umbilical cord blood samples were genotyped with various methods and compared with noninvasive prenatal testing results obtained during pregnancy.

Results: A total of 95 samples from 74 pregnancies were analyzed. Fetal DNA was successfully identified in 72 of 74 cases (97.3%) using insertion and deletion and Y chromosome markers. In 2 cases, fetal DNA could not be detected because of the absence of informative markers. The noninvasive prenatal testing results showed 100% concordance with the genotyped newborns. Among the fetuses of 22 immunized women, 6 were antigen negative, 10 were antigen positive, 5 had antigens not included in the next-generation sequencing panel (3 anti-Cw, 1 anti-M, and 1 anti-Ge2), and 1 had an inconclusive result.

Conclusion: Our study highlights the feasibility of using next-generation sequencing for comprehensive fetal antigen screening, paving the way for a personalized approach to managing alloimmunized pregnancies. By accurately identifying fetuses expressing antigens corresponding to maternal antibodies and those not at risk, it enhances the precision of targeted care.

Keywords: alloimmunization; blood group; fetal fraction; next-generation sequencing; noninvasive prenatal testing.