Comment on “Diagnostic Accuracy of Cell-free DNA for the Determination of Fetal Red Blood Cell Antigen Genotype: A Systematic Review and Meta-analysis”

Hemolytic disease of the fetus and newborn (HDFN) occurs when maternal alloantibodies target fetal red blood cell antigens and is associated with neonatal morbidity and mortality. While anti-D prophylaxis to prevent maternal sensitization has nearly eliminated RhD-related cases, alloimmunization to other antigen variants remains unaddressed. Accurate and early determination of fetal antigen status helps guide decisions surrounding surveillance, management, and resource utilization. Previously, this has required invasive testing with amniocentesis, which has procedural risks and is limited to pregnancies past 15 weeks. Advances in cell-free DNA (cfDNA) analysis now allow for noninvasive fetal antigen genotyping from maternal plasma as early as 10 weeks’ gestation. This study assesses the diagnostic accuracy of cfDNA for detecting fetal red blood cell antigen status across different antigens and laboratory methods. This systematic review and meta-analysis conducted in 2024 employed a comprehensive literature search of MEDLINE, Embase, and the Cochrane Library to identify studies evaluating cfDNA for fetal red blood cell antigen genotype. Eligible studies included pregnant individuals undergoing cfDNA testing for fetal antigen status, with neonatal genotype or serology as the reference standard. Both observational and interventional studies were included, while case reports and reviews were excluded. Data were independently extracted and evaluated for diagnostic accuracy by comparing cfDNA results with confirmed neonatal antigen status. The included studies used several laboratory techniques, including quantitative, multiplex, and digital PCR, next-generation sequencing, and MALDI-TOF. Overall test performance was summarized by calculating pooled sensitivity, specificity, and diagnostic odds ratios. Study quality and bias risk were assessed using QUADAS-2. After the search and review, a total of 84 studies with a cumulative 77,187 fetal antigen assessments met the inclusion criteria. Most studies were prospective and predominantly evaluated RhD-negative pregnancies, although some included alloimmunized cohorts and additional antigens such as RhC, Rhc, RhE, Kell, and Fy a . PCR was the most commonly used method, with fewer studies using next-generation sequencing or MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight). Across all laboratory techniques, cfDNA demonstrated pooled sensitivity and specificity of approximately 99% (95% CI: 99-100). For PCR, the sensitivities for RhD, C, c, E, and Kell were 99% to 100%; for next-generation sequencing, the sensitivities for RhD, C, c, E and Kell were all 100%; and for MALDI-TOF, the sensitivity for RhD was 99%. The Fy a antigen was only assessed with NGS, and sensitivity was 100%. Study quality was found to be high, with low risk of bias and minimal variability in diagnostic performance between studies. The results support cfDNA testing as a highly accurate and reliable method for determining fetal red blood cell antigen status in alloimmunized and RhD-negative pregnancies, offering a safe and effective alternative to invasive procedures. In addition, cfDNA can be assessed earlier in pregnancy, thereby improving risk stratification, reducing unnecessary monitoring, and allowing for more conservative use of anti-D immunoglobulin, which is particularly relevant given current supply shortages. Strengths of this study include the large pooled sample size, high overall study quality, and consistent diagnostic performance across laboratory methods and populations, while limitations include variability in study design, differences in laboratory techniques, and limited validation for less common antigens. These findings support broader implementation of cfDNA testing to improve the management and safety of pregnancies at risk for HDFN. (Summarized from Mustafa HJ, Najjariasl P, Aghajani F. Diagnostic accuracy of cell- free DNA for the determination of fetal red blood cell antigen genotype: a systematic review and meta-analysis. Am J Obstet Gynecol . 2025;233:428–445.e16. doi: 10.1016/j.ajog.2025.05.004)