Serologically D-negative blood donors in Thailand: molecular variants and diagnostic strategy

Abstract

Background -Discriminating individuals with “Asian type DEL” from those who are “true D-negative” (D–) among serologically D– donors/patients in Asia would be very valuable, as clinical outcomes are different in these groups. Here we investigated the molecular basis of D-negativity in Thai blood donors, designing a specific strategy for this purpose.
Materials and methods - After routine testing, a total of 1,270 serologically D– blood donors originating from Central, Northeastern and South Thailand underwent analysis of the RHD gene by (i) quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF); (ii) direct sequencing of exon 9 to identify the c.1227G>A variant defining the Asian type DEL allele; and (iii) direct sequencing of the other exons.
Results - The most common observation was whole deletion of the gene (i.e. RHD*01N.01; allele frequency: 86.81%), followed by the Asian type DEL allele (RHD*01EL.01; 7.60%) and a D-negative hybrid allele (RHD*01N.03; 3.46%). Four novel alleles, including one with a 13.1 kilobase-deletion, were identified and characterized. All but one RHD*01EL.01 allele carriers (183/184) were C-positive (C+), suggesting that this latter subset may be screened specifically when investigating the c.1227G>A variant, which can be identified with 100% accuracy by a specific Tm-shift genotyping assay.
Discussion - On the basis of our extensive molecular findings, we have designed a dedicated diagnostic strategy based on Rh C antigen typing followed by a genotyping test. Implementation of this method in all or selected groups of serologically D– donors/patients will contribute to improve the management of transfusion and pregnancy in Thailand.