Universal DNA Arrays for High-Throughput SNP Detection of Human Platelet Antigens (HPA)

DNA analysis of human platelet antigens is known to improve the diagnosis and treatment of neonatal alloimmune thrombocytopenia and minimize the risks of post transfusion purpura and refractoriness to random donor platelet therapy. The availability of reliable molecular platforms such as HPA eMAP assays analyzing HPA-1, -2, -3, -4, -5, -6, -7, -8, -9, -11, and -15, are routinely used and eliminate the need for reference sera or large platelet volumes. New generations of HPA assays (HPA eMAP-S Beadchip™ Kit) described here are developed by identification of specific allele in solution and detection of targets by a generic probe array called the Universal BeadChip™ by imaging, HPA-13 was also incorporated in the panel. These assays are easily automatable due to fewer protocol steps.

The BioArray Solutions HPA eMAP-S BeadChip Kit uses the novel Elongation Mediated Multiplexed Analysis of Polymorphisms in Solution (eMAP-S) technology (Lin, X., et al., 2010) to identify the presence or absence of the selected alleles associated with a given phenotype. We amplified 10 DNA fragments covering 12 allele variants which are associated with 12 platelet antigens by using multiplex PCR and HPA gene specific primer pairs. PCR was followed by multiplex allele specific primer extension (ASPE) and Beadchip detection by using the AIS-400 Array Imaging System. To evaluate the overall performance of HPA eMAP-S BeadChip™ Kits, a total 552 clinical samples with known HPA eMAP phenotype and/or serology were analyzed to determine polymorphisms associated with common human platelet antigen (HPA-1, -2, -3, -5, -15) and less common human platelet antigen (HPA-4, -6, -7, -8, -9, -11, -13). For internal study, 176 clinical samples were analyzed at BioArray Solutions (BAS). For the external study, 186 and 190 (total 376) donor samples were tested at two sites and analyzed by BASIS™ software. Phenotype results obtained from the HPA eMAP-S Beadchip™ Kit during the studies were compared to the data generated from the HPA eMAP BeadChip™ Kit and/or serology.

For internal and external studies, the concordance between the HPA eMAP-S Beadchip™ kit and HPA eMAP Beadchip™ kit was 99.99%. The discordant call (0.01%) on HPA-1b antigen was investigated. One HPA-1 discordant sample was sent out for sequencing and re-analyzed by using increased amount of DNA from DNA precipitation. The result from sequencing is consistent with the result from HPA eMAP Beadchip™ Kit. The HPA eMAP-S result from higher concentration DNA is also consistent with the result of sequencing. The discordance in the original HPA eMAP-S assay was attributed to less DNA input in the multiplex PCR, which will be corrected in the Package Insert of HPA eMAP-S Beadchip™ Kit.

HPA eMAP-S Beadchip™ Kit could be used reliably for the determination of human platelet antigens using DNA analysis.

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