RH1 (RhD) is highly immunogenic and may cause alloimmunization in RH1 negative recipients. Serological RH1 testing of blood donations is often not sensitive enough to detect very weak RHD variants. Therefore, all serological RH1 negative first-time donors in Switzerland are subject to mandatory molecular screening for the presence of RHD DNA sequences. Here, we present the results of two independent investigations undertaken to resolve discrepancies in RH1 pheno- and genotyping.

Genomic DNA from pools of up to 23 serological RH1 negative donor EDTA blood samples was extracted using the EZ1 DSP DNA Blood Kit (Qiagen) and analyzed using the RBC-FluoGene D-Screen kit (inno-train). Positive pools were resolved to individual donor samples and re-tested. Serological analyses were performed using column agglutination testing (DG Gel Rh Pheno+Kell, Grifols), an anti-D antibody panel with 9 monoclonal antibodies (D-Screen, Diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-D antibodies or anti-D (clone ESD1). DNA was initially analyzed by SSP-PCR for common RHD variants (RH-TYPE, BAG Health Care; RBC-Ready Gene CDE, inno-train). RHD DNA and cDNA were amplified and sequenced using published and in-house primers.

In both samples RHD exons 3, 5 and 10 were detected by PCR. However, red cells were phenotyped as RH:-1 using standard column agglutination and an additional anti-D panel. Adsorption-elution analysis showed a negative result in donor 1 and a very weak positive reaction was observed in donor 2. Commercial SSP-PCR kits did not identify known variant alleles. By exon and cDNA sequencing, a hemizygous deletion c.970del was detected in donor 1, causing a translation frame shift (p.His324Thrfs*35). In donor 2 a hemizygous substitution c.265C>T was found causing a premature stop codon (p.Gln89Ter).

We present two novel RHD variant alleles. To the best of our knowledge neither has been reported so far. According to our results individuals harboring RHD*970del would be considered RH:-1 both as donors and patients. In contrast, individuals harboring RHD*265T might weakly express a truncated version of RH1 posing a risk for alloimmunization in RH1 negative recipients. Therefore, this donor is considered as RH:-1 as patient, but RH:1 as donor, which emphasizes the importance of RHD molecular screening in serologically RH1 negative donors.

no conflict of interest