The RHD(1227G>A) DEL-associated allele is the most prevalent DEL allele in Australian D– blood donors with C+ and/or E+ phenotypes
Corresponding Author
Stacy A. Scott
Research and Development, Australian Red Cross Blood Service, Brisbane, Australia
Address reprint requests to: Catherine A. Hyland, Research and Development Division, Australian Red Cross Blood Service, 44 Musk Avenue, Kelvin Grove, Brisbane, Qld 4079, Australia; e-mail: [email protected].Search for more papers by this authorLisa Nagl
Research and Development, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorLouise Tilley
Bristol Institute for Transfusion Sciences, Bristol, United Kingdom
Search for more papers by this authorYew-Wah Liew
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorJenny Condon
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorRobert Flower
Research and Development, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorCatherine A. Hyland
Research and Development, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorCorresponding Author
Stacy A. Scott
Research and Development, Australian Red Cross Blood Service, Brisbane, Australia
Address reprint requests to: Catherine A. Hyland, Research and Development Division, Australian Red Cross Blood Service, 44 Musk Avenue, Kelvin Grove, Brisbane, Qld 4079, Australia; e-mail: [email protected].Search for more papers by this authorLisa Nagl
Research and Development, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorLouise Tilley
Bristol Institute for Transfusion Sciences, Bristol, United Kingdom
Search for more papers by this authorYew-Wah Liew
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorJenny Condon
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorRobert Flower
Research and Development, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorCatherine A. Hyland
Research and Development, Australian Red Cross Blood Service, Brisbane, Australia
Search for more papers by this authorAbstract
Background
Red blood cells (RBCs) with D antigen levels only detected by anti-D adsorption-elution and an antiglobulin test express a DEL phenotype. For two DEL types, including RHD(1227G>A), immunization of D– recipients has been reported. This study's aim was to measure the prevalence of DEL-associated RHD alleles in a cohort of Australian D– donors to develop a model to estimate alloimmunization risk.
Study Design and Methods
D–, C+ and/or E+ blood donors were screened for RHD exons using quantitative polymerase chain reaction. Donors with RHD signals were DEL phenotyped with MCAD6 anti-D. RHD alleles were characterized via single-nucleotide polymorphism array or sequencing. Extended DEL phenotyping was performed with an anti-D panel.
Results
Among 2027 donors, 39 carried RHD alleles that have been previously reported to associate with either the DEL or the weak D phenotype. An additional five donors carried previously unreported RHD alleles and exhibited the DEL phenotype: RHD(IVS2-2delA), RHD(IVS1+5G>C), RHD(ex9:del/CE), and RHD(ex8:del/CE) represented twice. In total, DEL/weak D–associated RHD alleles were detected in 44 of 2027 donors or 2.17% (95% confidence interval, 1.54%-2.81%). The RHD(1227G>A) DEL allele was the most frequent (n = 16). The risk of transfusing D– females not more than 40 years of age with an RHD(1227G>A) DEL RBC unit (when managed as D–) is estimated to be one in 149,109 transfusions (range, 100,680-294,490).
Conclusion
DEL/weak D–associated RHD alleles were found in 2.17% of Australian D–, C+ and/or E+ blood donors. This differs from previous European reports in that the clinically significant RHD(1227G>A) DEL allele is the most prevalent.
Supporting Information
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| trf12701-sup-0001-si.pdf192.2 KB |
Fig. S1. qPCR reactions (singleplex and duplex) and thermal cycling conditions were initially tested using gDNA extracted from D– donors with ccee, Ccee and ccEe phenotypes, and RhD positive donors either hemizygous for RHD with DCcee phenotype or homozygous for RHD with DCcEe phenotype (n = 5). To allow RHD exon 4&10 and RHD exon 5&CCR5 duplexing, RHD exon 4 and 5 TAMRA hydrolysis probes were FAM-labelled (green channel) and RHD exon 10 and CCR5 TAMRA hydrolysis probes were VIC-labelled (yellow channel). a) Green channel analysis of singleplex reactions (RXN), “Exon 4” and “Exon 5” and duplex reactions “Exon 4/Exon 10” and “Exon 5/CCR5”. No Ct values for gDNA samples derived from D– RBC samples with ccee, Ccee and ccEe phenotypes are shown as fluorescence was not above threshold and/or did not exhibit a total change in fluorescence >10% relative to the largest change in any tube. b) Yellow channel analysis of singleplex reactions, “CCR5” and “Exon 10” and duplex reactions “CCR5/Exon 5” and “Exon 10/Exon 4.” Fig. S2. PCR/agarose gel analysis of RHD(ex8:del/CE) donors using primer set 5&6 and different annealing temperatures. Lane: 1) ladder; 2-7) annealing temperatures 50°C, 52°C, 54°C, 56°C, 58°C and 60°C, respectively. a) RHD(ex8:del/CE) donor #1. b) RHD(ex8:del/CE) donor #2. c) Overexposed gel for RHD(ex8:del/CE) donor #1. d) Overexposed gel for RHD(ex8:del/CE) donor #2. The arrow indicates a non-specific band approximately 400bp in size. |
| trf12701-sup-0002-table_s1.doc62.5 KB |
Table S1. The corresponding name, sequence, thermal cycling conditions, RHD position and amplicon size for primer set 1&2-7&8. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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