Beate Kirchharz (Ratingen / DE), Tanja Klinkenberg (Ratingen / DE), Nicole Baatz (Ratingen / DE), Sarah Petermann (Bad Kreuznach / DE), Brigitte Flesch (Bad Kreuznach / DE; Hagen / DE), Karin Scheulen (Ratingen / DE), Sabine Simpkins (Ratingen / DE), Katharina Siepmann (Ratingen / DE), Michaela Steffen (Ratingen / DE), Melanie Stump (Ratingen / DE), Claudia Wigger (Ratingen / DE), Eva-Maria Zayc-Schmidt (Hagen / DE), Bogdan Puscasu (Ratingen / DE; Hagen / DE), Birgit Fleiter (Ratingen / DE)
The occurrence of phenotypes in RhCE differs according to ethnic origin. People with partial phenotypes can immunize against the wild type. Some RhCE variants can cause cross-reactions with monoclonal Anti-D. The detection of RhCE variants and the correct D typing are important for people with these phenotypes.
In these two cases blood group typing was performed by serological standard tests. Due to conspicuous serological results further serological and molecular tests were added.
Standard blood typing was carried out using monoclonal reagents in gel and tube test (BioRad, DiaMed GmbH, Cressier, Switzerland and CE-Immundiagnostika GmbH, Neckargemünd, Germany). Genotyping was performed using PCR-SSP (RBC-Ready Gene CDE/RHCE variants, Inno-Train, Kronberg, Germany). The results were compared with the data from the ISBT antigen table. Sanger sequencing on an ABI Prism 310 (Applied Biosystems, Weiterstadt) was used to clarify the findings.
Both samples were positive in the typing for C, c and e and negative for E. C was weakened and negative with one clone (both cases, gel technique). Additional typing in the tube technique (case two) showed very weakened C and e, c was tested positive, E negative. Using PCR-SSP we detected: RHCE*c.48G>C, c.514T>A, c.544A>T, c.577A>G, c.594T>A, c.602G>C matching RHCE*02.10 (encodes Partial C, Partial e) and c.733C>G. RHCE*c.733G was consistent with RHCE*01.20 (encodes Partial c, Partial e) assumed that the SNP c.733G was on the other allele. In the first case the genotype was confirmed by Sanger sequencing (RHCE*02.10.01, *01.20.01). The second case has not yet been sequenced.
Both cases were positive for RhD (confirmed by SSP-PCR as both variants can lead to false D-typing).
Reliable serological diagnostics can be the basis for recognizing variants in the Rh blood group system. In the second case, the detailed serological findings show that both alleles encode variants. Findings of the PCR-SSP can be classified even in complex cases, taking serology into account. Sanger sequencing based on allele specific amplicons can help to resolve ambiguities, and to ensure reliable classification.
We have no conflicts of interest to disclose.