Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next‐generation sequencing

Abstract

A next-generation sequencing-based method that can accurately define all known human platelet antigen polymorphisms was developed. With the ability to sequence up to 96 samples simultaneously, our HaloPlex design could be used for high-throughput human platelet antigen genotyping. This method is also applicable for investigating fetal neonatal alloimmune thrombocytopenia when rare or novel human platelet antigens are suspected.

The first reported case of neonatal alloimmune thrombocytopenia due to low‐frequency human platelet antigen‐6b antibodies in the United Kingdom

“…At this center, we genotype for HPA‐1, ‐2, ‐3, ‐4, ‐5, ‐6, ‐9, and ‐15 by Sanger Sequencing. Davey et al argue that NGS 22 would allow us to target all exons containing HPA genes, thus enabling the identification of LFHPA and novel HPAs, which could direct laboratory investigations toward a crossmatch sooner. Our current donor platelet panel for HPA antibody screening is limited to “common” high‐frequency HPA types.…”

Neonatal alloimmune thrombocytopenia due to a new alloantigen Bl(a) defined by an Asp458Gly substitution in GPIIIa

“…Indexed DNA libraries were prepared from 50 ng genomic DNA obtained from the infant and mother using a custom‐designed HaloPlex high‐sensitivity target‐enrichment assay (Agilent Technologies) following the manufacturer's instructions. All HPA coding exons plus 50 nucleotides of the respective 5′ and 3′ flanking intronic regions of the ITGB3 and ITGA2B genes were targeted …”

Current understanding and future perspectives for anti‐human platelet antigen‐15 antibodies in patients with alloimmune thrombocytopenia: History, laboratory testing, and clinical impact

“…A number of genotyping platforms have been developed for HPA‐15, including: sequence‐specific primer polymerase chain reaction (PCR‐SSP), 5 restriction fragment length pattern PCR (PCR‐RFLP), 5 real‐time PCR, 5 melting curve analysis, 35 sequence‐specific oligonucleotide (PCR‐SSO), 36 DNA sequencing‐based typing (PCR‐SBT), 37 matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS), 38 PCR‐single‐base extensions (PCR‐SBE), 39 DNA microarray, 40 next‐generation sequencing, 41,42 droplet digital PCR, 43 and high resolution melting PCR (PCR‐HRM) 44 (Table 1). While PCR‐SSP is the most popular method for HPA‐15 genotyping, current methods may be less time‐consuming, more cost‐effective, and may achieve high‐throughput 45 .…”