High-throughput genotyping of human platelet antigens using the 5'-nuclease assay and minor groove binder probe technology.
OBJECTIVE
Human platelet antigen (HPA) genotyping is a valuable tool for typing platelets to assist in the management of alloimmunized thrombocytopenic patients. We describe, for the first time, 5' nuclease assays (NA) to genotype HPA-5 and -15, and improved 5'-NA to genotype HPA-1, -2 and -3, by utilizing minor groove binder (MGB) and non-fluorescent quencher (NFQ) technology. Superior probe specificity and fluorescent performance is attained through MGB-NFQ probe modifications compared with previous 5'-NA designs.


METHODS
Primers and dye-labelled MGB-NFQ probes were designed and synthesized to detect the single nucleotide polymorphism responsible for each HPA-1, -2, -3, -5 and -15. One-hundred blood samples were tested for the combinations of HPA genotypes 1, 2, 3 and 5 by our traditional sequence-specific primer-polymerase chain reaction (SSP-PCR) method, and 41 blood samples were tested for HPA-15 by SSP-PCR at an external laboratory. These results were then compared with those obtained by using the new 5'-NA.


RESULTS
There was complete concordance of results for all samples tested by SSP-PCR and 5'-NA. The 5'-NA offers distinct advantages over non-fluorescent genotyping methods. DNA amplification and allele discrimination occurs in a single closed tube for each antigen with no post-PCR manipulation required. This minimizes the risk of cross-contamination and mislabelling of samples, as well as making the assay less time-consuming to perform. In comparison with other fluorescent assays, the 5'-NA has the highest sample throughput, resulting from the use of a 96-well platform, identical cycling conditions for all assays and the potential for automation.


CONCLUSIONS
Genotyping for HPA-1, -2, -3, -5, and -15 by the 5'-NA is suitable for routine analysis. The latest 5'-NA design, using MGB probe technology, ensures superior detection of all alleles and is the most versatile fluorescent assay, ideal for both urgent clinical samples and large-scale screening programs.
Higgins
M
M
Department of Haematology, Royal Perth Hospital, Perth, Western Australia. melinda.higgins@health.wa.gov.au
Hughes
A
A
Buzzacott
N
N
Lown
J
J
D016824:Antigens, Human Platelet / Q000235:genetics*; D003851:Deoxyribonucleases / Q000032:analysis*; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D013921:Thrombocytopenia / Q000276:immunology
D016824:Antigens, Human Platelet; D003851:Deoxyribonucleases