Genotyping of HPA‐1 to ‐7 and ‐15 in the Thai population using multiplex PCR

Abstract

Background: Different polymerase chain reaction (PCR) techniques for human platelet antigens (HPA) genotyping have been implemented, in order to diagnose the clinical syndromes of patients with thrombocytopaenia and provide effective HPA‐matched platelet donors. Objectives: The aim of this study is to develop an in‐house multiplex PCR for HPA‐1 to ‐7 and ‐15 genotyping in the Thai population. Methods: One hundred DNA samples of known HPA genotyping by the PCR with sequence‐specific primers (PCR‐SSP), as previously described, were tested with the multiplex PCR. Additionally, 300 DNA samples of group O donors were tested for HPA‐1 to ‐7 and ‐15 genotyping using multiplex PCR. Results: The comparison of HPA‐1 to ‐7 and ‐15 genotype results between multiplex PCR and PCR‐SSP technique was in 100% concordance. Interestingly, HPA‐2b2b genotype was found in two samples; however, other low‐incidence genotypes such as HPA‐1b1b, HPA‐5b5b, HPA‐6b6b and HPA‐7b7b were not found in this study. Moreover, 30 samples were randomly tested twice for HPA genotyping using the multiplex PCR and demonstrated reproducible results. Conclusions: This study shows that the in‐house multiplex PCR is simple, cost‐effective and suitable for HPA genotyping for routine laboratories in other developing countries. Nevertheless, a large‐scale evaluation of this technique through multicentre analysis is suggested.

Red Cell Genotyping by Multiplex PCR Identifies Antigen-Matched Blood Units for Transfusion-Dependent Thai Patients

“…Nevertheless, individual testing for each antigen may not be suitable to find antigen-negative blood units. The implementation of multiplex-PCR is beneficial for transfusion-dependent patients for the following reasons: i) The results can be obtained even with a low concentration of DNA (20 ng/μl) within 2 h. ii) Multiplex PCR uses fewer tubes and less reagents, and the cost is approximately Eur 4.00/test, which is 50% less than PCR-SSP, and similar to the application of multiplex PCR to detect other genetic polymorphisms [30,31]. iii) Its simplicity and reproducibility is proper for routine testing.…”

Genetic polymorphisms of human platelet antigens in the Asian population: implications in the establishment of platelet donor registries

“…). Among East Asians, the HPA‐1b allele is extremely rare accounting for <0·5% in terms of allele frequency, which is in marked contrast to HPA‐1b allele frequency of 14–19% among those of European ancestry . Conversely, HPA‐4b and HPA‐6b are nearly non‐existent among Europeans and are seen nearly exclusively among East Asians.…”

Comparison of a Simple-Probe Real-Time PCR and Multiplex PCR Techniques for HPA-1 to HPA-6 and HPA-15 Genotyping

“…Five hundred blood group O DNA samples consisted of 300 samples from our previous study and an additional 200 samples were from the Thai National Stem Cell Donor Registry (TSCDR) of the National Blood Centre, Thai Red Cross Society. Informed consent was obtained from each subject.…”

“…Primers for multiplex PCR were identical to those previously described . All primers and hybridization probes for a simple‐probe real‐time PCR technique were commercially provided by Tib Molbiol (Berlin, Germany).…”

“…Several techniques have been established for HPA genotyping, such as polymerase chain reaction (PCR) with sequence-specific primer (PCR-SSP) (3,4), PCR with restriction fragment length polymorphism (PCR-RFLP) (5), real-time PCR (6)(7)(8)(9)(10)(11)(12), and DNA sequencing based typing (13)(14)(15). Although the PCR-SSP is widely used for HPA genotyping because of its simplicity and cost effectiveness, a previous study demonstrated that multiplex PCR could provide greater convenience at half the cost of PCR-SSP (16). The disadvantages of PCR-SSP and multiplex PCR include being labor intensive and being unsuitable for high-throughput HPA genotyping (13).…”