Sigrid Beiboer, 1Tinka Wieringa-Jelsma, 2Petra Maaskant-van Wijk, 1Ellen van der Schoot, 1Dirk Roos, 3Johan den Dunnen, 1Masja de Haas
1Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands, 2Sanquin Blood Bank South West Region, Wytemaweg 10, 3015 CN Rotterdam, The Netherlands, 3Leiden Genome Technology Centre, LUMC, Wassenaarseweg 72, 2300 RA Leiden, The Netherlands
In the Netherlands, about 500,000 people volunteer to donate blood. Each year, 60,000 new donors come forward. For transfusion, blood of all these donors is currently serologically typed for only a few of the about 60 relevant blood group systems. Due to high costs and absence of test reagents only a subset of these donors is tested for more systems. Incomplete typing can lead to transfusion reactions. Therefore, it is our aim to develop a high-throughput technique to genotype by DNA microarray the whole donor cohort for 60 blood group systems. This means that 60,000 donors a year will have to be typed after two different donations, that is 138,000 genotypes each week.
The molecular basis for most blood group systems is known. Most blood group antigens are bi-allelic and are the result of a single nucleotide polymorphism (SNP). These SNPs are used for genotyping. After DNA isolation, gene fragments containing the SNP are amplified by PCR. To this end, a multiplex PCR has been developed to both amplify and fluorescently label gene fragments of 18 blood group systems in one reaction tube. The PCR products are then heat-denatured and hybridised to the DNA array without further purification. On each glass slide 12 arrays are present, containing spots of short (17-29 nt) allele-specific oligonucleotides. For each blood group 20 different oligonucleotides are spotted: 10 for each allele, sense and antisense. The allele-specific oligo hybridisation method (ASO) is used to discriminate between the two blood group alleles. To evaluate the reproducibility, different individuals performed hybridizations of two blinded panels, with two different batches of slides. No discrepancies and a good reproducibility were obtained after genotyping 94 different samples for
HPA-1, -2, -3, -4, -5 and –15. These results show that the microarray will provide a reliable and fast procedure, which can be further improved to obtain the necessary throughput. The availability of a completely genotyped donor cohort will facilitate the selection of correct donor blood and improve the safety of blood transfusion.