Rapid genotyping of blood group antigens by multiplex polymerase chain reaction and DNA microarray hybridization.
BACKGROUND
In the Netherlands, 500,000 blood donors are active. Blood of all donors is currently typed serologically for ABO, the Rh phenotype, and K. Only a subset of donors is typed twice for a larger set of red cell (RBC) and/or platelet (PLT) antigens. To increase the direct availability of typed RBCs and PLTs, a high-throughput technique is being developed to genotype the whole donor cohort for all clinically relevant RBC and PLT antigens.


METHODS
A multiplex polymerase chain reaction was developed to both amplify and fluorescently label 19 gene fragments of RBC and PLT antigens in one reaction. To test the setup of the genotyping method by microarray, a pilot study with human PLT antigen (HPA)-typed donor samples was performed. On each slide, 12 arrays are present containing 20 probes per PLT antigen system (28 for HPA-3). The allele-specific oligohybridization method was used to discriminate between two different alleles.


RESULTS
Two blinded panels encompassing 94 donors were genotyped for HPA-1 through -5 and -15; no discrepancies were found compared to their serologic typing (HPA-1, -2, -3, -4, and -5) and genotyping (HPA-15; TaqMan, Applied Biosystems).


CONCLUSIONS
This study shows that the HPA microarray provides a reliable and fast genotyping procedure. With further development an automated throughput for complete typing of large donor cohorts can be obtained.
Beiboer
Sigrid H W
SH
Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. s.beiboer@sanquin.nl
Wieringa-Jelsma
Tinka
T
Maaskant-Van Wijk
Petra A
PA
van der Schoot
C Ellen
CE
van Zwieten
Rob
R
Roos
Dirk
D
den Dunnen
Johan T
JT
de Haas
Masja
M
D016824:Antigens, Human Platelet / Q000235:genetics; D000094902:Blood Banking / Q000379:methods; D001789:Blood Group Antigens / Q000235:genetics*; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D004912:Erythrocytes; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results
D016824:Antigens, Human Platelet; D001789:Blood Group Antigens