Multiplex Assay Genotypes 35 Erythrocyte Antigens

A multiplex polymerase chain reaction (PCR) assay has been designed for typing 35 red blood cell (RBC) antigens in six reaction mixes.

The high throughput assay is to be used to screen a significant number of donors with genotyping instead of serologically testing a variety of antigens, which is a costly and through the shortage of reagents, also limits the procedure.

Scientists at the Medical University of Vienna (Austria) validated the assay using 370 selected serologically typed samples. Subsequently 6,000 individuals were screened to identify high frequency antigen (HFA)-negative donors and to facilitate the search for compatible blood for alloimmunized patients.

They chose a conventional qualitative PCR and tested for 35 genotypes, carried out in six multiplex reaction mixes consisting of up to seven different amplification targets per mix. The PCR products are analyzed subsequently using agarose gel electrophoresis. The assay also detects genotypes related to 12 high frequency antigens (HFA). The high-incidence single-nucleotide polymorphisms (SNPs) are included in every PCR reaction and serve as internal controls. By combining two or three HFA-related alleles with alleles of average or low frequency in one reaction mix, the results on the gel are clearly separated by the lines or grid of the high-frequency bands and can easily be interpreted.

The screening revealed 55 donors with a rare blood type. These individuals were lacking one of the 12 high-incidence antigens tested. They identified nine donors that are Lutheran system 2 (LU2)-negative, five LU8-negative, five Kell system 2 (KEL2)-negative, one KEL4-negative, 24 YT (‘Cartwright’) 1 (YT1)-negative and 11 Colton 1 (CO1)-negative individuals. The results were always confirmed by serology. In all cases, there was concordance between serological testing and the PCR result. The screening of 6,000 donors revealed 57 new HFA-negative donors and the blood group database was extended by approximately 210,000 results.

The authors conclude that their method for blood donor red cell genotyping is a feasible alternative to other high-throughput donor RBC typing approaches. Despite some limitations, it can be deployed in most blood centers with reasonable investments. The use of this method could be a strategy to bridge the gap until higher resolutions techniques, such as microarray technology, are available at an affordable price. The study was published in the April 2012 issue of the journal Vox Sanguinis.

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Medical University of Vienna