Asian-type DEL among D-negative patients and donors in Malaysia can ease the demand for D-negative red cells and RhIG Open Access

In East and Southeast Asia, D-negative red cell units and donors are rare (<0.5%).^1,2 The Del phenotype is a rare variant of the D antigen in the Rh blood-group system.³ Del red cells carry an extremely low number of D antigens on their surface, undetectable by any routine serologic methods and thus typed as D-negative.⁴ Currently, there are 50 DEL alleles known as molecular causes of the Del phenotype.⁵ 

Among the DEL alleles, 1 variant, originally described as RHD(K409K),⁶ is particularly prevalent among serologic D-negative East Asian populations.⁵ This variant, now referred to as the “Asian-type DEL”⁷ (RHD∗DEL1 or RHD∗01EL.01), is clinically relevant in China⁸ and other East Asian countries.⁹ Asian-type DEL individuals express a complete repertoire of D-epitopes¹⁰ and can safely receive D-positive blood transfusions without the risk of developing an anti-D.^8,11 Meanwhile, transfusion of Asian-type DEL red cells to D-negative patients may cause anti-D alloimmunization, subsequently predisposing transfusion recipients to the risk of hemolytic transfusion reaction and pregnant woman to the risk of hemolytic disease of the fetus and newborn.¹² 

Studies from East Asian countries typically report prevalences of 17%⁵ for the Asian-type DEL among D-negative individuals, ranging from 10% to 30%. In Myanmar, the prevalence of any Del phenotype was 15.8%, most possibly caused by Asian-type DEL.¹³ There are no reports on DEL from Vietnam, Laos, and Indonesia. In Southeast Asia, the prevalence of Asian-type DEL among D-negative individuals has been reported as 15.6% in Thailand¹⁴ and 33% among Malaysian Chinese.¹⁵ 

Data on the prevalence of Asian-type DEL among the Malay population in Malaysia are lacking. The Malay ethnicity refers to an Austronesian ethnoreligious group native to parts of Southeast Asia and representing 52.3% of the 34.1 million population in Malaysia (Figure 1).

We determined the prevalence of D-negative and Asian-type DEL among blood donors and patients on the Malaysian peninsula. We discuss how identifying Asian-type DEL among D-negative patients in all Southeast Asia can improve the practice of rare blood inventory and enhance transfusion safety.

Clinical records at Hospital Sultanah Nur Zahirah (HSNZ), Terengganu, Malaysia from January 2020 to July 2024 were reviewed using the Blood Bank Information System. We collated the Rh phenotypes and self-identified ethnicity of all D-negative blood donors. Blood samples were collected from all nonrepeat D-negative blood donors between March and July 2024. During the same 5-month period, samples were also collected from all D-negative inpatients at HSNZ, along with their self-identified ethnicity. The study was approved by the Medical Research & Ethics Committee, Ministry of Health Malaysia (NMRR ID-24-01803-9UD).

Hemagglutination tests for D antigen were performed with licensed reagents using the microplate method in blood donors (Immucor, Norcross, GA) and gel matrix in patients (Bio-Rad Laboratories, Hercules, CA) at HSNZ. An indirect antiglobulin test was conducted for all D-negative blood donors. All samples were tested for C, c, E, e phenotyping using the tube method, with licensed reagents (CSL Behring, Melbourne, Australia). EDTA-anticoagulated whole blood samples are shipped to the National Institutes of Health (NIH).

Genomic DNA was extracted from the buffy coat (Qiagen EZ1 DNA blood kit on the BioRobot EZ1; Qiagen, Valencia, CA) at NIH. All serologic D-negative samples were screened for the presence of RHD intron 4, exon 5, and exon 7 by real-time polymerase chain reaction (PCR). If all 3 tests were negative, we tested for RHD exon 1 and exon 10 to identify potential RHD-CE-D hybrids by PCR with gel electrophoresis. Positive samples underwent further testing with RHD∗DEL1 specific real-time PCR. We used published primers (supplemental Table 1). RHD genotyping (RHD BeadChip, Immucor BioArray Solutions, Warren, NJ) was performed to identify variant RHD alleles, with ambiguous cases further resolved by Sanger sequencing.¹⁷ Zygosity of the RHD gene was determined with the previously published restriction fragment length polymorphism assay.¹⁷ 

Hardy-Weinberg equilibrium was assessed using the χ2 goodness of fit test.¹⁸ 95% confidence intervals (CIs) for allele frequencies were calculated using the Poisson distribution.

Among 33 829 distinct blood donors studied, only 179 were D-negative (0.53%) with a prevalence of 0.49% in Malays, 0.69% in Chinese, and 4.97% in Indians, (Table 1) confirming data from previous studies.^15,19-21 Our study highlights the rarity of D-negative individuals in Malaysia, which presents practical challenges for D-negative transfusion support. The most prevalent D-negative phenotype was ccee, representing 68.16% (122/179), and the D-negative phenotypes expressing a C antigen, such as Ccee, CCee, and CcEe, accounted for 29.05% (52/179).

We detected 5 distinct RHD alleles that caused the serologic D-negative phenotype among 32 D-negative donors and 40 patients (Table 2), using published methods and primers (supplemental Table 1). The majority of samples (n = 52) exhibited the RHD gene deletion (RHD∗01N.01), whereas the remaining samples (n = 20) carried 1 of the 4 additional RHD alleles (Table 2). The unbiased estimate of Asian-type DEL (RHD∗01EL.01) among 32 donors was 28% and among 40 patients was 18% (Table 2; supplemental Table 2). All Asian-type DEL cases in this study were associated with the presence of the C antigen, consistent with known results.¹ 

The distribution of RHD alleles by ethnicity among blood donors and patients reflected the demographic composition of the study population, with Malays being the majority (supplemental Table 3). The prevalence of Asian-type DEL among 28 Malay donors was 25.0% (95% CI, 11.73-49.13) and among 33 Malay patients it was 21.21% (95% CI, 9.95-41.68). Data are, however, still lacking for Vietnam and Indonesia, where the prevalence may be similar: Vietnam with 100 million inhabitants is surrounded by countries with documented high prevalences of Asian-type DEL, and the 280 million population of Indonesia has language, ethnic, and genetic similarities with the Malay population. Therefore, our data are highly relevant, as it addresses the large population of 700 million in Southeast Asia, where Malaysia is centrally located.

Based on overwhelming evidence from East Asia since 2006, we support that patients and pregnant women in Malaysia expressing Asian-type DEL should be managed as D-positive^7,8 and excluded from Rh immunoglobulin (RhIG) administration.²² In Malaysia, blood-group genotyping is conducted at the National Blood Centre in Kuala Lumpur (Figure 1), primarily for transfusion-dependent thalassemia patients and select complex cases. However, with existing infrastructure and expertise, large-scale genotyping of blood donors and pregnant women is feasible. We propose revising the country’s transfusion practices to align with current evidence, starting with national-level discussions among policymakers, health care providers, and transfusion medicine experts. These discussions may eventually result in establishing updated guidelines and standards to improve the safe and effective management of Asian-type DEL patients, particularly in transfusion and pregnancy settings. Similar unbiased random surveys have prompted practice changes in the United States²³ and internationally²⁴ for the Rh blood-group system.

In the interim, a case-by-case approach to management may be considered for patient groups with a lower risk of immunization by D-antigen exposure. For example, transfusing D-positive red cells to Asian-type DEL patients who are women beyond child-bearing potential and males beyond 50 years may be considered as an acceptable strategy, as these groups are at lower risk of complications related to anti-D alloimmunization.

To document patient safety, rigorous follow-up protocols can be established. Patients receiving D-positive transfusions or those managed without RhIG prophylaxis should be monitored for the development of anti-D at 3- and 6-months after exposure. Such monitoring would identify unexpected alloimmunization, if any, with only 1 case reported²⁵ since the Del phenotype was recognized 40 years ago.⁴ 

By implementing these interim measures and emphasizing personalized medical decisions, immediate patient needs can be addressed while gathering data to inform evidence-based national guidelines and standards in Malaysia and other countries of this most populous region. Our approach represents a pragmatic clinical decision based on the current evidence, ensuring the safe management of Asian-type DEL patients while facilitating a smooth transition to updated practices.

In Malaysia, the D-antigen status in pregnant women is determined by serologic testing, with no routine genotyping. The current practice is to administer RhIG to all D-negative pregnant women. This includes 2 antenatal doses (typically at 28 and 34 weeks of gestation) and 1 postnatal dose, provided the newborn is confirmed to be D-positive. Avoiding unnecessary RhIG injections for pregnancies with Asian-type DEL would not only simplify clinical management but also ease RhIG supply. This is especially relevant in regions like Terengganu, with a fertility rate of 2.9.²⁶ In 2023, among 26 261 pregnant women in Terengganu, an estimated 140 were D-negative, of whom 35 were Asian-type DEL (25%).²⁷ Nationwide estimates suggest 607 Asian-type DEL cases every year among 2427 D-negative pregnancies from a total of 457 954 pregnancies. For all East and Southeast Asia, the applicable figures are 70 to 100 times larger. These findings highlight the potential economic benefits of omitting routine RhIG administration for Asian-type DEL pregnancies without compromising patient safety.

Our study contributes to the growing understanding of D-negative phenotypes and their molecular underpinnings in Southeast Asian populations in general, and the Malay ethnic group in particular. Incorporating molecular testing into routine blood donor and patient screening exemplifies the principles of precision medicine, where individualized genetic information can guide more accurate and safer transfusion practices.

Identification of specific RHD variants, such as the Asian-type DEL, enables more efficient utilization of D-negative blood by potentially preventing ∼1600 unnecessary D-negative transfusions and ∼1800 RhIG administrations, annually. This approach supports improved management of limited blood-product resources, particularly in regions such as East and Southeast Asia, where health care resources are often constrained.

The authors thank Marina Ursula Bueno, Nur Athirah Shukuri, and the staff of Transfusion Medicine Department at HSNZ for sample coordination and serologic testing; and Nuri Cha, and the staff of Transfusion Service and HLA Laboratories at National Institutes of Health for technical assistance.

This work was supported by the intramural research program (project ZIC CL002128) of the National Institutes of Health (NIH) Clinical Center (W.A.F.). The fellowship placement at NIH Clinical Center (KKM510-4/5/1/16715(9) and NIHCA2496840) was supported by the Ministry of Health Malaysia (M.M.K.).

The contributions of the NIH authors are considered Works of the United States Government. The findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.

Contribution: M.M.K. and W.A.F. designed the study approach and developed the rationales; M.M.K. and K.S. designed and performed the molecular experiments, retrieved literature, and wrote the draft; and all authors analyzed the molecular data, discussed the data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interest.

Correspondence: Willy Albert Flegel, Laboratory Services Section, Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, Bethesda, MD 20892; email: waf@nih.gov.