Experience with a triple exon RHD-NIPT and detection of maternal and fetal RH variants

Background

Since mid-2021, RhD-negative pregnant women in Germany have the option of fetal RHD typing using a non-invasive cell-free fetal DNA (cffDNA) prenatal test (RHD-NIPT). The investigation is covered by the statutory health insurance. The molecular prerequisite for the RHD-NIPT is homozygosity for the complete deletion of the RHD gene (RHD*01N.01). We present our overall experience with the RHD-NIPT as well as in the presence of non-deletional maternal RHD alleles or fetal RH variants.

Methods

The RHD NIPT was performed using the FetoGnost® Kit RHD (Ingenetix), which detects exons 5, 7 and 10 of the RHD gene. Indications of maternal RHD sequences were low CT values or a small CT difference (<4) between the RHD-specific reaction and the internal positive control (δIPC). When maternal RHD gene sequences were suspected, Rh blood group antigens were determined serologically (ID-Card System, BioRad), followed by maternal RHD genotyping (ERY Q® Weak D, BAG Diagnostics). Samples yielding negative results with the latter assay were forwarded to specialized laboratories for further molecular genetic testing. Due to the relatively small amount of cffDNA, samples with possible fetal RH gene variants could not be further investigated.

Results

Between July 2022 and April 2023, we detected maternal RHD sequences in 7 out of 870 (approx. 0.8%) samples of pregnant women, who were serologically RhD-negative by routine recipient testing. Further genotyping revealed RHD*08N.01 (RHD*Ψ) in four cases as well as RHD*11, RHD*15 and RHD*03N.01 ((C)de^S) once each. The observed patterns of exon amplification corresponded to the detected maternal RHD genotypes. With regard to the fetal RHD status we clearly detected 537 positive (approx. 62.4%) and 323 negative (approx. 37.6%) fetal RHD genotypes. Furthermore, a variant at the fetal RH locus was suspected in three cases (aprox. 0.3%). One of them with a suspected fetal RHD*08N.01 was subsequently serologically typed RhD-negative after birth.

Conclusion

If serological RhD negativity is not due to RHD*01N.01 homozygosity, the RHD NIPT may be confounded by the presence of maternal sequences. The same applies to other RHD variants that are considered RhD-negative in the recipient setting. In these cases, the maternal genotype can be further identified. Upon suspicion of fetal RHD variants, a definite prediction of the serological status is not possible and rhesus prophylaxis needs to be administered to the pregnant woman.

Offenlegung Interessenkonflikt:

The authors have no conflicts of interest to disclose.