Determination of RhD variants in blood donors and patients is crucial for transfusion strategies in order to prevent RhD alloimmunisation. Molecular RHD testing can complement serological assessments, especially in case of massive blood transfusion or reduced antigen expression. The objective of this study was to validate an RHD typing method using the newly introduced LightCycler® PRO instrument. Our typing strategy includes real-time PCR for the most common aberrant RhD types in Upper Austria, followed by Sanger sequencing for samples tested negative in PCR.

Fifty previously characterized samples derived from proficiency panels, blood donors and patients were included in this study. These samples exhibited a variety of aberrant RhD types, with alterations found in each exon. Real-time PCRs targeting weak D type 1, weak D type 2, weak D type 3 and DVII as well as RHD exons 1-10 were carried out on a LightCycler® PRO platform using LightCycler® 480 SYBR Green Master I. Additionally, Sanger sequencing was conducted using a SeqStudio instrument.

Molecular typing confirmed all previously obtained results. Melting curve analysis using the LightCycler® PRO instrument was proven to be specific and precise. Besides, we confirmed a novel RHD allele with a c.885G>A alteration during the validation process. This point mutation results in an amino acid change from Methionine to Isoleucine at position 295, which correlates with the characteristics of weak partial type 11.

Our RHD typing method has proven to be specific and accurate. Notably, the LightCycler® PRO instrument provides a significant improvement in throughput compared to the LightCycler® 2.0 instrument. This testing approach seems suitable for RhD determination in clinical situations in which serological typing may be challenging or less effective.

There are no financial conflicts of interest to disclose.