Summary Objectives This study aimed to investigate single‐nucleotide variants (SNVs) associated with P1 expression among Thai blood donors and develop a genotyping method using multiplex polymerase chain reaction (PCR) to predict P1 blood group status. Background The α1,4‐galactosyltransferase (A4GALT), also called Gb3/CD77 synthase or P1/Pk synthase enzyme, is encoded by the A4GALT gene and catalyses the transfer of galactose from uridine diphosphate‐galactose to lactosylceramide, creating the Pk antigen (Gb3). The same enzyme synthesises the P1 antigen by adding terminal galactose to paragloboside. The A4GALT transcripts are elevated in P1, and different SNVs in transcription factor‐binding regions of A4GALT correlate with P1 and P2 phenotypes. Material and Methods A total of 218 blood samples from Thai blood donors at the Thammasat University Hospital were tested for the P1 antigen using the conventional tube technique. Genomic DNA was extracted, and non‐coding regions of A4GALT were sequenced and analysed. A multiplex PCR assay was developed and validated to identify P1‐associated SNVs and was subsequently tested on 1022 Thai DNA samples of unknown P1 antigen status. Results In the tested cohort (n = 218), P1 and P2 phenotypes were found in 24.77% and 75.23% of donors, respectively. Moreover, three SNVs—rs8138197 (C/T), rs2143918 (T/G) and rs5751348 (G/T)—correlated 100% with both phenotypes. Finally, findings agreed with serological phenotyping and DNA sequencing results, confirming their validity for predicting P1 antigen positivity. Conclusions This study confirmed that three SNVs also correlated with P1/P2 phenotypes among Thais, as expected. A multiplex PCR found that SNVs rs2143918 (T) and rs5751348 (G) predicted blood group P1 and is an accurate, reproducible, cost‐effective and less time‐consuming alternative to traditional methods.

<b><i>Background:</i></b> High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. <b><i>Methods:</i></b> Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of <i>KEL</i>*<i>01</i> and <i>KEL</i>*<i>02</i>, <i>JK</i>*<i>01</i> and <i>JK</i>*<i>02</i>, <i>FY</i>*<i>01</i>, <i>FY</i>*<i>02</i>, and <i>FY</i>*<i>02 N.01</i>, <i>DI</i>*<i>01</i> and <i>DI</i>*<i>02</i>, <i>GYPB</i>*<i>03</i> and <i>GYPB</i>*<i>04</i>, <i>RHCE</i>*<i>E</i> and <i>RHCE</i>*<i>e,</i> and <i>DO</i>*<i>01</i> and <i>DO</i>*<i>02.</i> Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. <b><i>Results:</i></b> The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. <b><i>Conclusions:</i></b> We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

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