Diagnostic Accuracy of Cell-Free DNA for the Determination of Fetal Red Blood Cell Antigen Genotype: A Systematic Review and Meta-Analysis.

OBJECTIVE To evaluate the diagnostic accuracy of cfDNA in identifying fetal RBC antigen genotypes. DATA SOURCES A comprehensive systematic search was conducted across three databases between 2000 and 2024. STUDY ELIGIBILITY CRITERIA We included cohort studies utilizing cfDNA for fetal RBC antigen detection in pregnancies at risk of or with red cell alloimmunization. Two reviewers independently assessed the studies for inclusion, resolving any disagreements through discussion. STUDY APPRAISAL AND SYNTHESIS METHODS Reporting followed the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Two authors independently extracted data and assessed the risk of bias using the quality assessment of diagnostic accuracy studies (QUADAS-2) tool. Pooled sensitivity and specificity were determined using a hierarchical summary receiver operating characteristic (HSROC) approach. The analysis was conducted for all studies and then per each laboratory technique, including polymerase chain reaction (PCR), next-generation sequencing (NGS), and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). Subgroup analyses were performed for each antigen type, including RhD, RhC, Rhc, RhE, Kell, and Duffy (Fya) antigens. RESULTS A total of 84 studies encompassing 77,187 antigen samples with neonatal genotype concordance were included. Seventy-six studies used PCR (75,692 antigen samples), five used NGS (328 antigen samples), and three used MALDI-TOF (1,167 antigen samples). Combining all lab techniques, the sensitivity and specificity of cfDNA in identifying fetal RBC antigen were 99% (95% CI 99-100). The diagnostic accuracy using i)PCR technique for the following antigens in order RhD (74,786 samples), RhC (189 samples), Rhc (232 samples), RhE (276 samples), and Kell (209 samples) were, the pooled sensitivity is 99% (95% CI 99,100), 100% (95% CI 98-100), 99% (95% CI 96-100), 100% (95% CI 97-100), and 99% (95% CI 96-100), respectively and the pooled specificity is 99% (95% CI 99-100), 100% (95% CI 98-100), 100% (95% CI 94-100), 100% (95% CI 99-100), 100% (95% CI 98-100), respectively, ii) NGS technique for the following antigens in order RhD (68 samples), RhC (42 samples), Rhc (52 samples), RhE (88 samples), and Kell (108 samples) were, the pooled sensitivity is 100% (95% CI 96-100), 100% (95% CI 92-100), 100% (95% CI 95-100), 100% (95% CI 95-100), and 100% (95% CI 90-100), respectively and the pooled specificity is 100% (95% CI 91-100), 100% (95% CI 93-100), 100% (895% CI 83-100), 100% (95% CI 97-100), and 100% (95% CI 98-100), respectively, and iii) MALDI-TOF technique studies only investigated RhD (1,167 samples) with sensitivity of 99% and specificity of 98%. Fya antigen (19 samples) was only available in the NGS technique (sensitivity and specificity of 100%). CONCLUSION CfDNA can accurately detect six fetal blood group antigens involved in red cell alloimmunization that can lead to severe fetal anemia. Integrating cfDNA into clinical practice for detecting fetal RBC antigens, particularly for RhD-negative pregnancies, can streamline care, reduce unnecessary treatment and monitoring, and decrease patient anxiety. Additionally, it can be performed earlier than amniocentesis.