From 2003–2006, framework V of the European commission funded a demonstration project, the objective of which was to illustrate the applicability of glass array based genotyping approaches to the determination of a large number of blood group polymorphisms. The Bloodgen consortium (Avent et al, 2007) comprises academic laboratories, blood banks and Progenika Biopharma SA, a Spanish biotech company that specialises in genotyping and personalised medicine. The project developed the commercially available product, BLOODchip, which is an oligonucleotide array with multiple probes corresponding to allelic pairs of blood-specific SNPs. PCR products corresponding to DNA sequences flanking each SNP are amplified by a multiplex amplifiable probe hybridisation (MAPH)-based multiplex reaction (Beiboer et al, 2005). The PCR products are fragmented, fluorescently labelled, and then hybridised to probes that are arranged on a glass array. Differential hybridisation to allelic probe pairs for each SNP is then detected using a conventional scanner, and then genotyping is completed by software devised to detect hetero or homozygosity for each SNP (Fig 2). Bloodchip includes ABO (33 haplotypes) RHD (91 haplotypes) RHCE (9 alleles), KEL (8 alleles), JK (4 alleles, including 2 JKnull) FY (4 alleles), MNS (9 haplotypes), DI, DO and CO. The major content of the array are the various RHD alleles that cause D-negative, partial, weak D and D-elute phenotypes. BLOODchip has recently been CE-marked for diagnostic purposes in the European Union (currently for RHCE, KEL, FY, JK, CO, MNS, DI and DO) with RHD in progress, and this clinical validation proved the superiority of this platform over conventional serology. Thousand samples, which included patient samples, newborns, weak D and A or B positive samples were analysed by BLOODchip and 116 different SNPs were analysed for each sample and phenotypes scored by the detection of each SNP and the inheritance of a combination of SNPs (for example in hybrid gene type partial D phenotypes). Only two errors in genotype compared to phenotype were observed, one RhC scoring and one Kpa/Kpb scoring. The mis-typed RhC sample was later resolved by an improved fragmentation protocol, although the reasons for the incorrect Kpa/Kpb typing are unclear. Two unknown combinations of SNPs were found in the ABO system, and six in the RH system and are currently being investigated by DNA sequence analysis. BLOODchip demonstrated effectively that a high throughput genotyping technology has superior accuracy over serology, for Rh CcEe typing 999/1000 were scored correctly by genotyping, whereas 995/1000 were concordant by serology. For the following blood groups there was 100% concordance between BLOODchip defined genotype and serological phenotype, (except 1/358 Kpa/Kpb typings). In contrast serological testing recorded the following concordances K/k (1000/1000); Kpa/Kpb (358/358); Jsa/Jsb (122/123); Jka/Jkb (596/597); FY serotypes (498/506), MN (425/445), Ss (479/483), CO (169/170). DI and DO types were confirmed by DNA sequence analysis due to the unavailability of serological reagents. At present the source of serological errors, which could either be technical or clerical, is unclear (see Table I).
Figure 2. Technical approach using one commercially available blood group genotyping platform – BLOODchip. Genomic DNA extracted from peripheral blood is (1) subjected to multiplex amplifiable probe hybridisation (MAPH) multiplex (MPX) polymerase chain reaction (PCR) where the PCR contains a mix of gene-specific primers (3 here shown for clarity), and primers which are tagged with MAPH sequences. The MAPH tagged primer sets are at a higher concentration in the MPX and ensures uniform amplification of all PCR products due to their common sequence. (2) The MPX PCR products are fragmented, labelled and made single stranded by denaturation. Fluorescent dyes are incorporated into the DNA. (3) The labelled DNA is hybridised to a BLOODchip array using an automated hybridisation station (for example Ventana discovery), and arrays are rinsed and scanned using a conventional scanner (4) Bespoke software analyses the data output (there are multiple probes per SNP, and positive and negative controls) and scores the spot as ‘good’ hybridisation (perfect match) or ‘poor’ (imperfect match). Zygosity for each SNP is devised using a patented algorithm. The software then interprets combinations of SNPs and converts this into a phenotype where these combinations are known. This is simple for KEL/DARC/GYPA(/B/E)/SLC4A1/ART$/AQP1 genotyping but complex for ABO and RH genotypes.
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