Emahazion Tesfai, Badri Ahlam, Jalkesten Elisabeth, Flodström Mari, Ajne Gunilla, Uzunel Mehmet, Wikman Agneta
Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden (Emahazion, Badri, Jalkesten, and Wikman); Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institute, Huddinge, Sweden (Emahazion and Wikman).
Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden (Emahazion, Badri, Jalkesten, and Wikman).
Am J Obstet Gynecol MFM. 2025 Jul;7(7):101694. doi: 10.1016/j.ajogmf.2025.101694. Epub 2025 May 13.
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.
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.
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.
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.
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.
孕期红细胞和血小板同种免疫可能很严重，在整个孕期都受到密切监测。然而，胎儿的血型及其相关风险往往未知。在此，利用下一代测序技术测定母体血浆中的胎儿红细胞和血小板基因型，为监测同种免疫妊娠提供了一种替代传统方法的无创产前检测手段。
本研究旨在验证一种创新的下一代测序方法，用于检测母体血浆来源的游离DNA中的特定血型抗原，重点关注与免疫相关的关键抗原。通过分析不同妊娠阶段的游离循环胎儿DNA，该研究旨在实现对临床相关红细胞和血小板抗原的高精度鉴定。
在瑞典斯德哥尔摩县，连续收集了74名免疫和未免疫孕妇在妊娠13至27周时的全血样本。使用瑞典斯德哥尔摩Devyser AB公司的原型试剂盒对母体血浆进行下一代测序分析。该试剂盒可检测多种红细胞血型标志物、2种人类血小板抗原标志物（HPA-1和HPA-5）、XY染色体标志物以及来自不同染色体的12个插入和缺失，以识别和定量胎儿DNA。出生后，采用多种方法对脐带血样本的基因组DNA进行基因分型，并与孕期获得的无创产前检测结果进行比较。
共分析了74例妊娠的95份样本。使用插入缺失和Y染色体标志物，在74例中的72例（97.3%）成功鉴定出胎儿DNA。2例因缺乏信息性标志物未检测到胎儿DNA。无创产前检测结果与基因分型新生儿的结果100%一致。在22名免疫孕妇的胎儿中，6例抗原阴性，10例抗原阳性，5例具有下一代测序检测板未包含的抗原（3例抗Cw、1例抗M和1例抗Ge2），1例结果不确定。
我们的研究突出了使用下一代测序进行全面胎儿抗原筛查的可行性，为同种免疫妊娠的个性化管理方法铺平了道路。通过准确识别表达与母体抗体对应的抗原的胎儿和无风险胎儿，提高了针对性护理的精准度。
