Fetal RHD detection from maternal plasma – comparison of two assays developed according to the "Workshop report on extraction of fetal DNA from maternal plasma"

The non-invasive method of fetal RHD genotyping from maternal plasma aids in the management of RhD alloimmunization potentially leading to hemolytic disease of the newborn (HDN). The clinical implications of fetal RHD genotyping are significant. For RhD-negative mothers carrying RhD-positive fetuses, knowledge of the fetal RhD status allows for timely interventions to prevent RhD alloimmunization. These interventions may include administration of Rh immunoglobulin (RhIg) to suppress the maternal immune response, thus reducing the risk of HDN in subsequent pregnancies. Conversely, RhD-negative fetuses can be identified early, sparing unnecessary RhIg administration and associated costs.

The aim was to compare the new commercial RBC-FluoGene fetal RHD Q kit (inno-train) with currently used inhouse kits. RBC-FluoGene fetal RHD Q detects cell-free fetal DNA in maternal plasma and detects the presence of exons 5 and 7 of the RHD gene in triplicates, identical to the currently used inhouse test system.

35 plasma samples from different weeks of gestation and after max 5 days of transport have been typed in parallel with the mentioned test systems. DNA isolation was done using Qiamp Blood kit mini. All test systems are real-time assays based on the TaqMan probe principle. For amplification, the real-time devices FluoQube® and StepOne Plus were used. With RBC-FluoGene fetal RHD Q, at least 3 of the 6 replicates have to be positive to call the sample RHD positive. The currently used technique requires 5 of 10 replicates to be positive to call the sample RHD positive.

32 out of 35 samples showed concordant results. 19 samples were typed with both systems as RHD positive; 9 samples were typed with both systems as RHD negative; 3 samples showed very early Ct values which was caused by a maternal partial RHD gene (RHD*01W.3, *08N.01; *06.02). 3 discrepancies were observed. Due to lack of further sample material, these could not be investigated further, unfortunately.

Both test systems showed reliable results and are suitable for detection of fetal RHD from maternal plasma. In conclusion, fetal RHD genotyping from maternal plasma represents a remarkable advancement in prenatal diagnostics, offering a safe, accurate, and non-invasive means of assessing the fetal RhD status. The new RBC-FluoGene fetal RHD Q kit is suitable for fetal RHD detection in maternal plasma.

The authors Sabine Scholz, Birgit Omengo, and Julia Kreuchauff are employees of inno-train Diagnostik GmbH.