Diagnostic accuracy of cell-free DNA for the determination of fetal red blood cell antigen genotype: a systematic review and meta-analysis.

Abstract

This study aimed to evaluate the diagnostic accuracy of cell-free DNA in identifying fetal red blood cell antigen genotypes. A comprehensive systematic search was conducted across 3 databases between 2000 and 2024. We included cohort studies using cell-free DNA for fetal red blood cell 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. Reporting followed the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy and the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 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 approach. The analysis was conducted for all studies and then per each laboratory technique, including polymerase chain reaction, next-generation sequencing, and matrix-assisted laser desorption/ionization-time of flight. Subgroup analyses were performed for each antigen type, including RhD, RhC, Rhc, RhE, Kell, and Duffy (Fy^a) antigens. A total of 84 studies encompassing 77,187 antigen samples with neonatal genotype concordance were included. Seventy-six studies used polymerase chain reaction (75,692 antigen samples), 5 used next-generation sequencing (328 antigen samples), and 3 used matrix-assisted laser desorption/ionization-time of flight (1167 antigen samples). Combining all laboratory techniques, the sensitivity and specificity of cell-free DNA in identifying fetal red blood cell antigen were 99% (95% confidence interval, 99-100). The diagnostic accuracy by technique was as follows: (1) polymerase chain reaction: for RhD (74,786 samples), RhC (189 samples), Rhc (232 samples), RhE (276 samples), and Kell (209 samples) the pooled sensitivities were 99% (95% confidence interval, 99-100), 100% (95% confidence interval, 98-100), 99% (95% confidence interval, 96-100), 100% (95% confidence interval, 97-100), and 99% (95% confidence interval, 96-100), respectively, and the pooled specificities were 99% (95% confidence interval, 99-100), 100% (95% confidence interval, 98-100), 100% (95% confidence interval, 94-100), 100% (95% confidence interval, 99-100), and 100% (95% confidence interval, 98-100), respectively; (2) next-generation sequencing: for RhD (68 samples), RhC (42 samples), Rhc (52 samples), RhE (88 samples), and Kell (108 samples) the pooled sensitivities were 100% (95% confidence interval, 96-100), 100% (95% confidence interval, 92-100), 100% (95% confidence interval, 95-100), 100% (95% confidence interval, 95-100), and 100% (95% confidence interval, 90-100), respectively, and the pooled specificities were 100% (95% confidence interval, 91-100), 100% (95% confidence interval, 93-100), 100% (95% confidence interval, 83-100), 100% (95% confidence interval, 97-100), and 100% (95% confidence interval, 98-100), respectively; and (3) matrix-assisted laser desorption/ionization-time of flight: this method was used only for investigating RhD (1167 samples), with sensitivity of 99% and specificity of 98%. The Fy^a antigen (19 samples) was assessed using only the next-generation sequencing technique, with both sensitivity and specificity of 100%. Cell-free DNA can accurately detect 6 fetal blood group antigens involved in red cell alloimmunization that can lead to severe fetal anemia. Integrating cell-free DNA into clinical practice for detecting fetal red blood cell antigens, particularly for RhD-negative pregnancies, can streamline care, reduce unnecessary treatment and monitoring, and decrease patient anxiety. In addition, it can be performed earlier than amniocentesis.