Patients with Asian-type DEL can safely be transfused with RhD-positive blood

Abstract

Red blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; none of the recipients (0/42; 95% confidence interval, 0-8.40) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4045 serologic D- pregnant women throughout China; alloanti-D was found only in individuals with true D- (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

Graphical abstract

Figure 1.

Patient enrollment in the clinical trial (NCT03727230). The flow diagram documents the inclusion and exclusion criteria applied and the alloanti-D immunization studied in a cohort of patients with Asian-type DEL receiving D⁺ RBC transfusions.

Figure 2.

Detection of alloanti-D in the pregnant women with true D^– and Asian-type DEL phenotypes. (A) Flow diagram of enrollment of the pregnant women with serologic D^– phenotype. (B) The numbers and percentages of pregnant women, according to the gestational time (G1 to ≥G5), were identified with alloanti-D in the true D^– and Asian-type DEL groups. (C) The RHD genotypes of 127 patients who developed alloanti-D. The percentages of RHD gene variants, including deletion and 2 nonfunctional alleles accounting for the true D^– phenotype rather than Asian-type DEL, were shown. The gestations with D⁺ fetus were not prospectively confirmed because >95% of Chinese women with true D^– or women with Asian-type DEL carry a D⁺ fetus.

Figure 3.

Analysis of RHD transcripts from individuals with Asian-type DEL. (A) The schematic diagram of full-length RHD transcript and the primer positions for Sanger sequencing and Nanopore sequencing analyses. Specifically, the primers for fragment amplification of RHD transcripts and Sanger sequencing located on exon 8 to 9 and 3’-UTRs to cover the c.1227A mutation of RHD and the primers for whole coding region amplification of RHD transcripts and Nanopore sequencing located on 5’-UTRs and 3’-UTRs. (B) Schematic representation of RHD transcripts identified in individuals with Asian-type DEL. The RT-PCR products of full-length RHD coding region, which were amplified using cDNA from the cultured erythroblast of individuals with Asian-type DEL, were analyzed using Nanopore sequencing. The top 7 major types of RHD transcripts identified in individuals with Asian-type DEL are presented. The structural schematic diagram and frequency of RHD transcripts are indicated in the right panel. Different types of transcripts are indicated by various colors. (C) Barplot showing the frequencies for major types of RHD transcripts in each individual with Asian-type DEL (n = 8) or D⁺ control sample. The color codes for different types of transcripts are identical to those in panel B. The red bar indicates the full-length transcripts, which was identified in all individuals with Asian-type DEL with lower abundance (mean, 0.18%) but higher abundance in D⁺ control (56.6%). The gray bar indicates other types of transcripts. In the D⁺ control, 3 major transcripts including the full-length RHD transcript in the red bar, the truncated transcripts with exon 7 deletion (19.0% of total, shown in the gray bar accompanied by several other low-frequency transcripts), or exons 7/8/9 deletion (9.0%) in the orange bar, were identified as shown in one previous study. (D) Recognition of RhD antigenic epitopes using anti-D mAbs in vitro. The top 7 types of RHD transcripts identified in Asian-type DEL erythroblasts were cloned into an expression vector and cotransfected into HEK 293T cells with wt RHAG construct. RhD antigen on the transfected cell surface was measured compared with unstained cell negative control and wt RHD-positive control using flow cytometry with 7 different anti-D mAbs.