Supplementary Material for: Single-tube multiplex PCR-SSP for the detection of RHD variant alleles commonly found in serologically D– phenotype individuals in a Thai population

Background: The molecular basis of the RHD gene found in serologically D– phenotype individuals differs with race/ethnicity. Therefore, we aimed to develop a single-tube multiplex PCR-sequence specific primer (multiplex PCR-SSP) to detect RHD variant alleles commonly found in serologically D– phenotype individuals in a Thai population. Study design and methods: In total, 205 blood samples with a serologically D– phenotype were tested using a single-tube multiplex PCR-SSP targeted RHD exons 1, 4, 7, 10, and c.1227G>A in RHD exon 9 in combination with a hybrid Rhesus box, and results were confirmed by direct DNA sequencing. Results: In a single-tube multiplex PCR-SSP, three patterns of amplified RHD exons were observed: total deletion of the RHD gene, Asian-type DEL, and RHD-CE-D hybrid. The allele frequencies of RHD*01N.01, RHD*01EL.01, and RHD-CE-D hybrid were 83.4%, 12.9%, and 2%, respectively. All of the Asian-type DEL samples present the RHCE*C/E allele (predicted RhCE phenotype: C/E+). Conclusion: This study successfully established a simple and reliable molecular diagnostic platform for analyzing RHD variant alleles commonly found in serologically D– phenotype individuals in a Thai population. This technique could enable broader RHD*01EL.01 (Asian-type DEL) analyses in high-prevalence areas such as Thailand and other countries in East and Southeast Asia, serving as an example for blood bank routine settings.