Real-world performance of a clinical droplet digital polymerase chain reaction assay for non-invasive foetal blood group and platelet antigen genotyping of alloimmunized pregnant women with antibodies directed against RhD, RhE, Rhc, RhC, K1, HPA-1a or HPA-5b: A 1-year experience.

Background and objectives: To test the performance of a new droplet digital polymerase chain reaction (ddPCR) non-invasive foetal blood group and platelet antigen genotyping assay in the setting of a Dutch reference laboratory for foetal blood group and platelet antigen genotyping. Our population comprised 229 consecutive alloimmunized pregnant women who presented between April 2022 and March 2023 with 250 requests for non-invasive foetal RHD, RHE, RHc, RHC, K1, HPA-1a or HPA-5b blood group and platelet antigen genotyping.

Materials and methods: Samples were genotyped for blood group and platelet antigen alleles along with methylated RASSF1a (mRASSF1a) and sex-determining region of Y (SRY) and DYS14 as positive foetal controls. Negative blood group and platelet antigen results were issued only when foetal controls were positive; otherwise, such samples were classified as inconclusive.

Results: The assay achieved a success rate of 98.4% (246 of 250) because one case was lost to follow-up, one case was solved with quantitative polymerase chain reaction (qPCR) and one case precluded foetal typing due to RHD variant mothers. Only 10 cases needed a second sample and one case a third for a valid final result. We identified 116 maternal-foetal blood group and platelet antigen incompatibilities.

Conclusion: Clinical non-invasive foetal blood group and platelet antigen typing of alloimmunized pregnant women via ddPCR is successful and represents an improvement over qPCR because of the addition of a foetal control and because ddPCR circumvents potential interference from maternal cell-free DNA (cfDNA) background for foetal HPA-1 and K1.