The Experience of RHD Genotyping in D-negative Blood Donors

Background: There have been some domestic and overseas cases of anti-D alloimmunization caused by the transfusion of serologically D-negative blood. However, it is difficult to distinguish between true D-negative and DEL variants using conventional serologic typing. Therefore, we established the RHD genotyping algorithm for the detection of DEL variants and applied this algorithm to serologic D negative donors who voluntarily consented to testing. Methods: From September 2016 to December 2020, 216 RhD negative donors who were C ＋ and/or E+ in previous serologic typing were recruited. The screening test was PCR amplification of the RHD exons 4, 7, 10, and a promotor. Based on the results of PCR screening, true D-negative samples and RHD variants (including DEL) were discriminated. When the result was a RHD variant, exon 9 was sequenced to identify the nucleotide changes. Full sequencing was performed if no mutations were detected at exon 9. Results: Among the 216 participants, 39 cases with the C − E − c ＋ e ＋ phenotypes that did not meet the recruitment criteria were excluded from data analysis. Among the remaining 177 samples, 68 cases (38.4%) were RHD total deletions, 35 cases (19.8%) were RHD -CE-D hybrids, and 74 cases (41.8%) were RHD variants. Among the cases of RHD variants, 73 cases (98.6%) had c.1227G ＞ A substitutions and were confirmed as Asian-type DEL. Conclusion: Seventy-four cases of serologic D negative donors were reclassified as RHD variants by RHD genotyping. This is believed to have contributed to the improvement of transfusion safety by lowering the risk of anti-D alloimmunization in D-negative patients. (Korean J Blood Transfus 2021;32:91-101)