Knowledge of fetal RhD status enables targeted administration of anti-D prophylaxis in RhD-negative pregnant women carrying RhD-positive fetuses, thereby preventing unnecessary health risks and resulting in cost savings in cases of RhD-negative fetuses.

The aim was to compare the new commercial RBC-FluoGene fetal RHD Q kit (inno-train) with currently used commercial and in-house kits. The study utilized EDTA blood samples collected during early gestational weeks with longer transport times to assess the new kit's capability to detect low amounts or partially degraded cell-free fetal DNA.

21 maternal peripheral blood samples collected between gestational weeks 12 and 26 and transported for a maximum of 5 days at room temperature were typed in parallel using the mentioned test systems. The blood samples were centrifuged at 1700 g for 10 minutes. The resulting plasma supernatant was then transferred to a 13mL tube and stored frozen at -80°C until cell-free DNA (cfDNA) extraction. The extraction of cfDNA was carried out from 2mL of plasma using the QIASymphony DSP Circulating DNA kit, following the manufacturer's protocol. The elution volume was 60µL. All test systems are real-time assays based on the TaqMan probe principle. With RBC-FluoGene fetal RHD Q, exon 5 and 7 of the RHD gene and an internal control are amplified in triplicates on QuantStudio 7 Pro Real-Time PCR System (ThermoFisher Scientific). The commercial kit and the in-house kit detect RHD exon 5, 7, 10, and an internal control in two mixes without replicates. With the RBC-FluoGene fetal RHD Q Kit, at least 3 out of the 6 replicates must be positive to classify the sample as RHD positive.

In all 21 samples, the RBC-FluoGene fetal RHD Q kit confirmed the results obtained with the commercial kit and in-house test method. 7 out of 21 samples were RHD negative and 14 samples were RHD positive. The mean Ct values were very similar across different tests. Samples with longer transport times showed slightly higher Ct values compared to samples with shorter transport times.

Results obtained with the new commercial kit are fully consistent with those of the current commercial test kit and in-house method for fetal RHD genotyping. Therefore, the RBC-FluoGene tetal RHD Q Kit can be utilized for fetal RHD detection in maternal plasma, even during early gestational weeks and prolonged sample transport times.

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