Software update enables improved RHCE/RHD genotyping on allele level for real-time PCR system

Individuals from African descent often carry variant RHCE and RHD alleles. To handle the high number of SNPs investigated for a detailed genotyping including variants in a routine setting a sophisticated software-based solution is indispensable. With this, the alloimmunization risk may be lowered, as the absence of high prevalence antigens in patients and the presence of low prevalence antigens in donors could be predicted with more accuracy.

The RBC-FluoGene CDE eXtend kit is a real-time PCR assay based on the TaqMan probe technique. It comprises 43 RHD and 30 RHCE SNPs, provided as pre-dropped and dried primer probe mixes in a 96-well PCR plate. Hand-on time is less than 3 minutes and real-time PCR is less than 1 hour. The automated result analysis with the FluoGene Software (FGSW) update version 1.8 has been improved for calling a heterozygous RHD result, calling a genotype for RHCE on an allele level in ISBT nomenclature and the prediction of the respective phenotypes. 15 DNA samples from African descent were genotyped with RBC-FluoGene CDE eXtend kit (lot R920823) and the result displays of FGSW versions 1.7 vs. 1.8 were compared.

The FGSW version 1.8 calls for all samples RHD and RHCE genotyping results in numerical ISBT nomenclature as listed in table 1 (RHD) and table 2 (RHCE). In the previous version 1.7 only the most likely heterozygous combinations of two variant alleles were displayed, but others gave no call. For results comprising a RHD*01 allele and a variant allele the FGSW version 1.8 also displays the possible combinations of two variant alleles, which are very unlikely but cannot be excluded. Table 2 shows the display of each positive specificity (C, Cw, c, E, e, plus SNPs) in FGSW 1.7 compared to the RHCE genotyping result in numerical ISBT nomenclature in FGSW 1.8. For 14 out of 15 samples the version 1.8 calls a clear RHCE genotyping result.

Complex blood group systems as RH require investigation of an increasing number of SNPs for a better characterization of variant alleles. To handle RHD and RHCE genotyping in a routine setting, a sophisticated software solution is essential. The update of the FGSW facilitates the routine blood group reporting with ISBT genotypes and predicted phenotypes.

Sabrina König is an employee of inno-train Diagnostik GmbH.