A practical and effective strategy in East Asia to prevent anti‐D alloimmunization in patients by C/c phenotyping of serologic RhD‐negative blood donors

Abstract Serologic RhD‐negative red cells can cause anti‐D alloimmunization if they carry the Asian‐type DEL or other DEL variants. RHD genotyping is a viable countermeasure if available, but inexpensive alternatives are worthy of consideration. RhD‐negative blood donors in Japan were studied by anti‐D adsorption‐elution and RHD genotyping. We collated published case reports of RhD‐negative red cell transfusions associated with inexplicable anti‐D immunization. Of 2754 serologic RhD‐negative donors, 378 were genotyped D/d. Anti‐D adsorption‐elution revealed 63.5% (240 of 378) to be DEL, of whom 96.7% (232 of 240) had the 1227G > A variant, diagnostic for the Asian‐type DEL. All 240 donors also carried at least one C antigen; none had a cc phenotype. The chance of transfusing DEL red cells to genuinely RhD‐negative Asian patients (based on a three‐unit transfusion) ranges from 16.7% in Korea to 69.4% in Taiwan, versus 0.6% in Germany. Among 22 RhD‐negative recipients of serologic RhD‐negative red cells, who produced new or increased anti‐D antibody titers, all 17 from East Asia were transfused with red cells with a C‐positive phenotype or known to be Asian‐type DEL or both. Serologic RhD‐negative East Asians with a cc phenotype can be red cell donors for RhD‐negative recipients, especially those of childbearing potential.

D antigen sites and can only be detected by specialized serology, including adsorption-elution of anti-D [2]. This serologic confirmation of the DEL phenotype is labor intensive.
DEL is often transfused to RhD-negative recipients, because DEL donors are typed as RhD-negative, as their D antigens evade detection by routine serology. Among such recipients, some experience a primary alloimmunization and develop a new anti-D or secondary alloimmunization and increase their anti-D titer [3,4]. RhD-negative patients are rare in East Asia, but cultural imperatives call for practical measures to ensure their equitable transfusion care.
Progress in genotyping enabled the molecular characterization of red cell antigens. Access to economical genotyping may become universal soon. Based on Japanese donor data, literature review (including follow-up with authors), and biostatics, we propose in the meantime to do extended Rh phenotyping of all RhD-negative blood donors. Blood donors who are negative for the C antigen provide a reliable source of red cells that are negative for the Asian-type DEL and are safe for transfusion to truly D-negative, RHD gene negative, recipients in East Asia.

Donor samples
A total of 2754 serologic RhD-negative blood samples donated between April and December, 2019, were investigated after initial typing with anti-D reagent (clones TH28 and MS26, Wako, Tokyo, Japan) and automated blood grouping (PK7300 instrument, Beckman Coulter, Tokyo, Japan) at the Tohoku Block Blood Center of the Japanese Red Cross, which serves six prefectures of northeast Japan. No two samples were from the same donor.
The Ethics Committee of the Japanese Red Cross Society approved our study protocol (#2019-038), in accordance with institutional policies, national law, and the World Medical Association Declaration of Helsinki.

Serology
Antiglobulin tests were done by a standard tube method with anti-D

RHD gene analysis
Genomic DNA was extracted from whole blood (QIAamp: Qiagen, Tokyo, Japan). The RHD genotype was analyzed by polymerase chain reaction (PCR), with sequence-specific primers essentially as described previously [5]. Using PCR-sequence-specific primers, the RHD deletion (RHD*01N.01) of the hybrid Rhesus box and the c.1227G > A mutation of the RHD*01EL.01 were determined. Multiplex PCR was used to amplify all exons 1 through 10 of the RHD gene, with exon 8 as a positive control as it has no nucleotide difference between the RHD and RHCE genes. When alleles were encountered in DEL samples that could not be explained by our screening methods, RHD exons 1-10 were sequenced as previously described [5].

Likelihood of transfusing DEL red cells to genuinely RhD-negative patients
We calculated the chance of DEL RBC transfusion as follows: where X is the fraction of truly RhD-negative (lacking the RHD gene) among serologic RhD-negative red cell units, and N is the number of red cell units transfused from different blood donors.

Relationship between DEL and the C/c phenotypes
Of the 240 DEL in our cohort, 232 (97%) had the 1227G > A variant, diagnostic for the Asian-type DEL, whereas eight had other RHD alleles (Table 2). Among these eight, all of which were RhC-antigen positive, one was found to have a new allele: c.1228-1G > A at intron 9.

Likelihood calculation for transfusing DEL red cells to genuinely RhD-negative patients
Genuinely RhD-negative patients, who do not harbor an RHD gene, occur in East Asia with frequencies ranging between 0.19% among Chi-nese in Hong Kong, 0.23% of both Koreans and Chinese in Taiwan, and 0.46% of Japanese [6][7][8][9]. Based on these data, the chance of transfusing DEL red cells to genuinely RhD-negative recipients, assuming a 3-unit exposure, ranges between 16.7% in Korea and 69.4% in Taiwan (Table 3). This likelihood was substantially higher than the 0.6% calculated for Germany, representative of Caucasian populations [10].   (Table 4). Among these 17 recipients, five were presumed to have experienced a primary, and 12 a secondary alloimmunization (Table 4).
We propose to perform extended Rh phenotyping on the few blood donors who are serologic RhD-negative on initial routine testing, and then exclusively supply red cell units with a cc phenotype to RhDnegative patients when indicated, as shown in Figure 2. As approximately 80% of serologic RhD-negative East Asian blood donors (78% of Japanese and 82% of Chinese [7]) also carry the cc phenotype, this practical and effective strategy would not significantly alter the supply and demand calculus of RhD-negative red cells for RhD-negative patients in East Asia. It will increase patient safely and reliably prevent anti-D alloimmunizations by blood transfusion.
In Japan, we have identified only eight patients with primary or  Request of RhD-negative and Rhcc RBCs for RhD-negative women of child bearing potential F I G U R E 2 Proposed work-flow for testing routine serologic Rh testing. The RhD phenotyping for patients (left side) follows current practice in Japan, but implements a special request of C-negative blood units for persons of childbearing potential. The RhD phenotyping for donors (right side) requires additional serologic testing for the C and c antigens. The results will be registered in the donor database and used to fill requests for C-negative red cell units that are expected to be DEL negative (see Figure 1) low immunogenicity of DEL can be explained, at least in part by the extremely low number of <30 D-antigen sites per DEL red cell [26], compared to approximately 10,000->33,000 per normal D-positive [27] and 300 -5000 for weak D red cells [28]. neonates, but a DEL fetus, more common than routinely detected, could cause an anti-D sensitizing event. As a precautionary countermeasure, currently available C/c phenotyping, a standard practice in many European countries, would easily and inexpensively mitigate potentially adverse outcomes.
Our aggregated data of Asian-type DEL red cell transfusions (Table 4) document only two of 17 recipients who developed posttransfusion hemolytic transfusion reactions. Based on this clinical data, we propose to prioritize red cell units of cc phenotype, because they are scarce, for transfusion to recipients of childbearing potential, rather than to any RhD-negative patient.
In conclusion, while the technology and economy of genotyping progresses, it is practical, economical, and effective in East Asia to implement C/c phenotyping of all serologic RhD-negative blood donors, and select those who are C-negative (cc phenotype) for red cell transfusion to RhD-negative patients, predominantly those of child-bearing potential. This approach may be useful in other population as well, either for routine care or in the context of a crisis in which genotyping is unavailable.