Anti-D alloimmunization by Asia type DEL red blood cell units in a D-negative Thai patient.

BACKGROUND: DEL phenotype is a rare Rh variant that cannot be detected by routine serological typing, and DEL individuals are thus typed D-negative (D-). Anti-D alloimmunization has been reported in "true" D- patients receiving DEL red blood cells (RBCs). CASE PRESENTATION: A 17-year-old, D- Thai male patient suffering from immunodeficiency syndrome with negative antibody screening received RBC units from 17 serological D- donors over a period of seven months due to acute respiratory failure with anemia. Before the 12th transfusion, anti-D production was detected. He was later transfused with RBCs from six other apparent D- donors. In order to elucidate anti-D production, all 17 blood donors were investigated by replicative serological testing and molecular analysis to identify potential RHD gene variants. All donors were confirmed D- by routine method, but as many as 12/17 were positive by adsorption-elution testing. Molecular analysis showed that five donors, including four whose blood was transfused before anti-D production occurred, carry the Asia type DEL allele, and are thus predicted to express a DEL phenotype. These data clearly suggest that 1/ the alloimmunized D- patient was exposed to D antigen, 2/ our adsorption-elution test is currently defective to identify DEL RBCs, and 3/ molecular analysis is highly valuable for Asia type DEL allele screening. CONCLUSION: For the first time in Thailand, we report anti-D alloimmunization in a serological D- patient transfused by Asia type DEL RBC units. This work definitely supports the implementation of a dedicated policy for DEL blood management including molecular testing.

Nation-wide investigation of RHD variants in Thai blood donors: Impact for molecular diagnostics.

BACKGROUND: Knowledge of the molecular determinants driving antigen expression is critical to design, optimize, and implement a genotyping approach on a population-specific basis. Although RHD gene variability has been extensively reported in Caucasians, Africans, and East-Asians, it remains to be explored in Southeast Asia. Thus the molecular basis of non-D+ blood donors was investigated in Thailand. STUDY DESIGN AND METHODS: First, 1176 blood samples exhibiting an inconclusive or negative result by automated serological testing were collected in the 12 Regional Blood Centres of the Thai Red Cross located throughout Thailand. Second, the RHD gene was analyzed in all samples by 1) quantitative multiplex PCR of short fluorescent fragments, and 2) direct sequencing, when necessary, for identifying structural variants and single nucleotide variants, respectively. RESULTS: Additional serological typing yielded 51 and 1125 samples with weak/partial D and D-negative (D-) phenotype, respectively. In the first subset, partial RHD*06.03 was the most common variant allele (allele frequency: 18.6%). In the second subset, the whole deletion of the gene is largely the most frequent (allele frequency: 84.9%), followed by the Asian DEL allele found in 15.6% of the samples. Eight novel alleles with various mutational mechanisms were identified. CONCLUSION: We report, for the first time at the national level, the molecular basis of weak/partial D and serologically D- phenotypes in Thai blood donors. The design and implementation of a dedicated diagnostic strategy in blood donors and patients are the very next steps for optimizing the management and supply of RBC units in Thailand.

Comprehensive Molecular Analysis of Serologically D-Negative and Weak/Partial D Phenotype in Thai Blood Donors.

BACKGROUND: Molecular genetics of the Rh system has been extensively studied in Caucasians, Black Africans, East Asians, and Indians more recently. In this work, we sought to investigate the molecular basis of variant D expression in the Thai population, which remains unknown. MATERIALS AND METHODS: Blood samples from 450 Thai donors showing the variant D phenotype were collected. The RHD gene was analyzed by quantitative multiplex polymerase chain reaction of short fluorescent fragments and/or Sanger sequencing. RESULTS: The most frequent alleles in 200 D-negative and 121 DEL samples were the whole RHD gene deletion and the Asian DEL alleles, respectively. In 129 weak/partial D samples, 36 variant alleles were identified, including eight novel alleles. RHD*06.03, which is common in variant D samples from South China, is the most prevalent variant allele, followed by the recently reported Indian RHD*01W.150 allele. DISCUSSION: For the first time, a comprehensive overview of the nature and distribution of variant RHD alleles in Thailand is reported. It is a milestone to pave the way towards improvement of the current screening strategy to identify DEL donors accurately. The next step will be the design and implementation of a simple molecular test for screening the most frequent alleles, specifically in this population.

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

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.