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. 2025 Jun 23;23(5):409–417. doi: 10.2450/BloodTransfus.1053

Molecular characterization of variant D antigen expression among 56,445 blood donors in East India

Suvro Sankha Datta 1, Kshitij Srivastava 2, Mercy Rophina 3, Vinod Scaria 3, Yew-Wah Liew 4, Jenny Morrison 4, Glenda Maree Millard 4, Willy Albert Flegel 2,✉
PMCID: PMC12425644  PMID: 40681466

Abstract

Background

Transfusion of red cells from RhD-positive donors to recipients lacking all or some epitopes of the D antigen can lead to the development of anti-D, potentially resulting in hemolytic transfusion reactions. Over 500 RHD alleles affect the qualitative or quantitative expression of the D antigen. There are huge differences in their prevalence among populations. Despite the distinct genetic diversity across multiple sub-populations in India, the prevalence of RHD alleles in the East Indian region has remained unknown.

Material and methods

Standard hemagglutination tests were performed, along with molecular techniques to determine the nucleotide sequence of the RHD gene.

Results

Over 3 years, 56,445 blood donors were tested at the Tata Medical Center, Kolkata by serology. We found 23 samples with D-discrepant test results in immediate spin anti-D technique (0.04%), all of them were C+c+. Variant RHD alleles were identified in 15 samples (15/23, 65.2%). The Indian-type weak D (RHD*01W.150) was the most common variant (30.4%, No.=7), occurring in 1 in 8,063 blood donors in East India versus 1 in 729 blood donors in South India (p<0.001). Other identified variants included RHD*06.03.01 (No.=3), RHD*17.05 (No.=2), RHD*01.EL.37 (No.=2), and RHD*01W.96 (No.=1). The remaining 8 samples (8/23, 34.8%) carried the reference RHD allele (RHD*01.01).

Discussion

Our findings reveal a lower prevalence of Indian-type weak D in East India than South India, indicating region-specific genetic diversity. The high incidence of wild-type RHD allele with D-discrepancy in East India suggests a potentially novel molecular mechanism. While RHD*06.03.01 and RHD*17.05 have, the 3 others identified RHD alleles have not been associated with anti-D alloimmunization. We recommend D-negative transfusion for individuals with D-variants that have documented alloimmunization, and advocate to investigate the clinical significance of other D-variant alleles prevalent in East India, such as the Indian-type weak D.

Keywords: D-variant, weak D, partial D, serologic typing, red cell genotyping, Indian-type weak D

INTRODUCTION

The Rh blood group system is the most complex, immunogenic, and polymorphic system among human blood groups. Variations in the RHD gene can affect the quantitative or qualitative expression of the D antigen, leading to D-variant phenotypes such as weak D, partial D, and Del1. Individuals with D-variant phenotypes often show discrepant, weak, or inconclusive serologic D antigen test results. Accurate D antigen typing in pre-transfusion testing reduces the risk of anti-D related hemolytic transfusion reaction (HTR) and hemolytic disease of the fetus and newborn (HDFN)2. Previous studies have shown that routine serologic testing with monoclonal anti-D reagents may not always accurately detect D-variant phenotypes3,4. Limitations of serologic testing can be overcome by analyzing individuals using DNA-based methods5.

The distribution of D-variant phenotypes varies considerably across different populations, including White6,7, African8, and East Asian groups9,10. Many institutions worldwide have established systematic RHD screening to detect variant RHD alleles in serologic D-discrepant individuals11. The prevalence of serologic weak D (~0.2%) and partial D phenotypes (~0.15%) has been reported for the western region of India12. Only a limited number of studies13 have molecularly screened RHD in individuals with a D-variant phenotype within India’s genetically diverse 1.4 billion population14. A major finding identified the weak D type 150 allele (RHD*01W.150), which accounted for the majority (58.3%) of weak D samples in South India15–18.

East India19 represents more than 270 million people (Figure 1)20. No data have been published on the molecular mechanisms, frequencies and characteristics of D-variants for this large population. We aimed to determine the prevalence and molecular basis of variant D antigen expression in East India.

Figure 1.

Figure 1

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Map of India divided into 6 regions

The Eastern region (red) is bounded by the Bay of Bengal to the south-east and Bangladesh to the east. East India comprises the states of Bihar, Jharkhand, Odisha, West Bengal, and the union territory of Andaman & Nicobar Islands. Map created using https://www.mapchart.net/ under the Creative Commons Attribution-ShareAlike 4.0 international license (CC BY-SA 4.0).

MATERIAL AND METHODS

Blood samples

EDTA-anticoagulated whole blood samples were collected between 1st January 2021 and 30th September 2024. The blood donors came from different districts of West Bengal and adjacent states in East India, except Andaman & Nicobar Islands. The study focused on the urban population of East India, who may be considered relatively homogeneous. Tribal ethnic groups in the region, who may have different blood group prevalences, were not among our blood donors21,22. Whole blood donations were collected by the Department of Transfusion Medicine at Tata Medical Center. Informed written consent for blood group typing, as part of their blood donation, was obtained from each donor in accordance with Government of India regulations. The whole blood donations comprised volumes of 350 mL or 450 mL, depending on the donor’s body weight, with a minimum 3-month donation interval. An ethical committee review was waived for this study, as it follows the routine institutional policy established at Tata Medical Center, Kolkata, since 2020.

Immunohematology

D antigen typing was performed using the column agglutination technique (CAT), and the results were verified using the conventional tube technique (CTT). We used 3 anti-D reagents: (i) Bioclone (IgM clone LDM3 + IgG clone ESD1 [Ortho Clinical Diagnostics, Raritan, NJ, USA]), (ii) DiaClon (IgM clone TH-28 + IgG clone MS-26 [Bio-Rad, DiaMed, Cressier, Switzerland]), and (iii) Eryclone (IgM clones P3x61 + NaTH119 [Tulip Diagnostics, Goa, India]). Samples were tested for the DVI phenotype using an oligoclonal reagent (ID card DiaClon ABO/D [Bio-Rad, Hercules, CA, USA]) of which 1 anti-D agglutinates the clinically relevant D category VI (DVI) cells (IgM clone ESD-1M) while the other does not (IgM clone LHM59/20), a procedure introduced in 199523. Samples that were negative or weakly reactive (≥2+) upon initial immediate spin testing were further tested in the indirect antiglobulin test (IAT) using a monoclonal IgG reagent [ID-DiaClon Anti-D (IgG clone ESD1); Bio-Rad]. Direct antiglobulin test (DAT) was performed for all samples with discrepant immediate spin results.

Agglutination strengths were noted ranging from “w” indicating weak reactivity to 4+ indicating very strong reactivity24. An agglutination strength of 0 indicated a complete lack of reactivity. Samples with weak or initially undetectable D antigen during serologic typing despite normal reactions in the indirect antiglobulin test (IAT) were referred for RHD genotyping in 3 institutions: Council of Scientific and Industrial Research, Institute of Genomics and Integrative Biology, India (15 samples); NIH Clinical Center, USA (4 samples); and Lifeblood, Australia (4 samples).

RhD-negative and D-discrepant samples were also tested for other Rh antigens (C, E, c, and e) by CAT using commercially available monoclonal (IgM) antisera (Bioclone, Ortho Clinical Diagnostics).

Molecular investigation in India

Genomic DNA was extracted from EDTA-anticoagulated whole blood (QIAamp DNA blood mini kit [Qiagen, Hilden, Germany]) from 15 donors and quantified (NanoDrop 1000 [Thermo Fisher Scientific, Wilmington, DE, USA]). Samples were tested on a sequence specific primer-PCR (SSP-PCR) based commercial kit targeting RHD exons 1, 5, and 10 (Inno-Train Diagnostik, Kronberg, Germany). Samples were screened for the weak D type 150 allele (RHD*01W.150), as previously described15.

All samples were also tested by next-generation sequencing (NGS) specifically targeting RHD at an average coverage of 100X. Library preparation, indexing, and target enrichment for sequencing were carried out using enrichment with a custom targeted blood group probe panel recognized by the International Society of Blood Transfusion (ISBT)25. Paired-end (2×87 cycle) single-plex sequencing was performed using NGS (MiSeq platform [Illumina, San Diego, CA, USA]). The raw sequence reads were assessed for quality (FastQC v0.11.9), aligned onto GRCh38 human reference genome (DRAGEN v3.4 Bio-IT platform, Illumina), and variants systematically annotated (ANNOVAR v. 2019 Oct 24). Computational methodologies were employed for calculating the copy number variation (CNV) ratio, following established protocols26–28.

Molecular investigation in US

Samples from 4 donors underwent molecular testing to identify RHD alleles using a commercial genotyping kit (BioArray RHD BeadChip, Immucor, Norcross, GA, USA), published methods15,29, or nucleotide sequencing30.

Molecular investigation in Australia

Genomic DNA from 4 donors was extracted, amplified, and sequenced by NGS (MiSeq platform, Illumina). DNA enrichment was done using a custom-designed panel, which enables comprehensive genotyping for 44 of the recognized 47 blood group systems (systems not covered: ER, ATP11C and MAL) and transcription factors KLF1 and GATA1 as per the ISBT25. The DNA sequence reads were aligned onto GRCh37 human reference genome to identify variants relative to the reference sequence (NG_007494.1). Copy number variation analysis was used to call RHD zygosity31,32.

Nomenclature

The RHD*01W.150 or RHD*weak D type 150 allele, as labelled by ISBT25, has been variably referred to in the literature before as RHD Exon 3 duplication15, Indian weak D15, the Indian-specific variant RHD (327_487-4164dup)17 and the Indian specific weak D type 15018. We propose designating a trivial name for this allele and its associated phenotype as “Indian-type weak D”13.

RESULTS

A comprehensive screening of blood donors from East India was carried out over a 3-year period. A total of 56,445 whole blood donors were tested. All donors were unpaid, non-directed volunteers, aged between 18 and 65 years. Among these 56,445 donors, 2,421 were confirmed as D-negative (4.3%), while 23 (0.04%) showed discrepant D-typing with weak reactions during the immediate spin test (Table I). Only 2 other samples (not shown), found to be DAT positive, were excluded from further analysis33.

Table I.

Serologic screening of blood donors for East India over a 3-year period

Characteristic Whole blood donors
Serologic D-typing Total (No.)
D-positive D-negative D-discrepant1
No. % No. % No. %
Sex
Male 44,844 83.04 1,985 81.99 21 91.30 46,850
Female 9,157 16.96 436 18.01 2 8.70 9,595
Total 54,001 95.67 2,421 4.29 23 0.04 56,445
Rh Phenotype
ccee Not tested 2,227 91.99 0 0 n.a.
Ccee Not tested 171 7.06 23 100 n.a.
ccEe Not tested 18 0.74 0 0 n.a.
CCee Not tested 5 0.21 0 0 n.a.
Total 2,421 100 23 100
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1

Positive IAT but negative DAT reaction. Two samples were found to be DAT positive and excluded from the analysis33.

n.a.: not available.

Blood donors with RHD alleles

Among the 23 donors with D-discrepant results, 7 harbored the RHD*01W.150 allele (30.4%), which is typical in individuals of Indian ancestry with a weak D phenotype (Table II). Among the remaining 16 donors, 8 carried 4 known RHD alleles, while the other 8 carried the reference RHD allele (RHD*01.01, Table II). No novel RHD allele was identified in the study.

Table II.

Serologic reactivity and RHD alleles identified in 23 individuals with discrepant D antigen typing

Sample Rh phenotype Serologic typing1 Red cell genotyping assays Test site ISBT allele
Immediate spin IAT5 DVI assay6 Zygosity SSP-PCR7 Indian-type weak D8 RHD BeadChip NGS
DiaClon2 Bioclone3 Eryclone4
1 CcDee w 0 mf 3+ Negative Hemizygous Not tested Positive Not tested c.327_487-4164dup Australia RHD*01W.150
2 CcDee 1+ 0 2+ 3+ Negative Hemizygous Not tested Positive Not tested c.327_487-4164dup Australia RHD*01W.150
3 CcDee 0 0 w 3+ Negative Hemizygous +/+/+ Positive Not tested c.327_487-4164dup India RHD*01W.150
4 CcDee 1+ w w 3+ Negative Hemizygous +/+/+ Positive Not tested c.327_487-4164dup India RHD*01W.150
5 CcDee mf w 1+ 3+ Negative Hemizygous Not tested Positive RHD-positive Not tested USA RHD*01W.150
6 CcDee mf 2+ 1+ 3+ Negative Hemizygous Not tested Positive RHD-positive Not tested USA RHD*01W.150
7 CcDee 0 0 0 3+ Negative Hemizygous +/+/+ Positive Not tested c.327_487-4164dup India RHD*01W.150
8 CcDee 1+ w 1+ 4+ Negative Hemizygous +/+/+ Negative Not tested c.731C>T India RHD*01W.96
9 CcDee 2+ 2+ 2+ 4+ Negative Hemizygous +/+/+ Negative Not tested c.1154-31T>C India RHD*01.EL.37
10 CcDee 2+ 2+ 2+ 4+ Negative Hemizygous +/+/+ Negative Not tested c.1154-31T>C India RHD*01.EL.37
11 CcDee 1+ w 2+ 3+ Negative Hemizygous Not tested Negative Not tested Multiple9 Australia RHD*17.05
12 CcDee 1+ w 2+ 3+ Negative Hemizygous Not tested Negative Not tested Multiple9 Australia RHD*17.05
13 CcDee 0 0 0 3+ 4+ Hemizygous Not tested Negative DVI Not tested USA RHD*06.03.01
14 CcDee 0 0 0 4+ 3+ Hemizygous Not tested Negative DVI Not tested USA RHD*06.03.01
15 CcDee 0 0 0 3+ 4+ Hemizygous +/−/+ Negative Not tested Multiple10 India RHD*06.03.01
16 CcDee 1+ w 1+ 3+ Negative Hemizygous +/+/+ Negative Not tested No variant India RHD*01.01
17 CcDee w 1+ 1+ 3+ Negative Hemizygous +/+/+ Negative Not tested No variant India RHD*01.01
18 CcDee 1+ 1+ 1+ 3+ Negative Hemizygous +/+/+ Negative Not tested No variant India RHD*01.01
19 CcDee 1+ w 1+ 4+ Negative Hemizygous +/+/+ Negative Not tested No variant India RHD*01.01
20 CcDee 1+ w 1+ 3+ Negative Hemizygous +/+/+ Negative Not tested No variant India RHD*01.01
21 CcDee w 1+ 1+ 3+ Negative Hemizygous +/+/+ Negative Not tested No variant India RHD*01.01
22 CcDee 1+ w 1+ 4+ Negative Hemizygous +/+/+ Negative Not tested No variant India RHD*01.01
23 CcDee w 1+ 1+ 3+ Negative Hemizygous +/+/+ Negative Not tested No variant India RHD*01.01
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1

mf: mixed field, w: weak reactivity;

2

DiaClon: IgM clone TH-28 + IgG clone MS-26;

3

Bioclone: IgM clone LDM3 + IgG clone ESD1;

4

Eryclone: IgM clones P3x61 + NaTH119;

5

ID-DiaClon (IgG clone ESD1); Indirect antiglobulin test (IAT): saline method with polyclonal anti-human globulin in tubes;

6

ID card DiaClon ABO/D (IgM clone ESD-1M); can agglutinate DVI cells in immediate spin;

7

SSP-PCR for RHD exon 1, exon 5 and exon 10;

8

Genotyping assay to detect RHD exon 3 duplication causing weak D type 15015;

9

c.361T>A, c.380T>C, c.383A>G, c.455A>C, c.505A>C, c.509T>G, c.514A>T, c.544T>A, c.577G>A, c.594A>T, c.602C>G;

10

c.505A>C, c.509T>G, c.514A>T, c.544T>A, c.577G>A, c.594A>T, c.602C>G, c.667T>G, c.676G>C, c.697G>C, c.712G>A, c.733G>C, c.744C>T, c.787G>A, c.800A>T.

Donors with reference RHD allele (RHD*01.01)

The reference RHD allele (RHD*01.01) was identified in 8 donors with a weak D phenotype (Table II), all carrying the allele in hemizygous form. The assay for the Indian-type weak D was negative. Even using NGS, we could not detect any known or novel nucleotide variations in RHD for these 8 donors.

DISCUSSION

Errors in blood group identification can cause improper red cell unit labeling. Serologic methods sometimes miss variations, and red cell genotyping offers a more accurate and sensitive approach. Routine molecular RHD screening for donors with serologic D-negative or D-discrepant results has been recommended and implemented in many institutions worldwide34–40. The D antigen was routinely tested for 56,445 whole blood donors in East India. We molecularly characterized RHD alleles in the 23 donors with D-discrepant results.

We delineated 5 RHD alleles as the molecular basis of variant D antigen expression in 15 individuals (Table II, Samples 1 to 15). All 5 alleles were known before. The prevalence of RHD variant alleles among blood donors in East India, all linked to the C antigen41, was lower in our cohort of East India (0.03%) compared to North (0.89%)16 and South India (0.14 to 0.18%)17,18.

In our cohort, we identified a DEL allele (RHD*01EL.37) with the causative variant RHD: c.1154-31T>C (rs28669938), initially reported in 2012 with variable adsorption/elution test results39. Another DEL allele, RHD*01EL.32, associated with the RHD: c.149-29G>C (rs2301153) substitution, has also been documented in the Indian population28. Computational and functional splicing assays have ruled out any impact of rs2301153 (intron 1)42 or rs28669938 (intron 8)43–45 on exon skipping. Given the global variant nucleotide frequencies of 7%46 for rs2301153 and 20%46 for rs28669938, along with our observation of rs28669938 in 2 weak D samples, these substitutions appear to be neutral, commonly present in various RHD alleles, and not associated with any distinct RhD phenotype42. In conclusion, no proper DEL allele has been found so far in the large Indian population, in striking difference to East and Southeast Asia9,47.

Fichou et al.15 originally described an allele encoding a weak D phenotype, common in India, which was designated as RHD*01W.150 and RHD*weak D type 150 by the ISBT. The RHD: c.327_487-4164dup variant, with its duplication located within RHD intron 3, is the common cause of the weak D phenotype representing a clinically relevant RHD allele in the Indian population13,15. Observations in previous reports included 8 cases of Indian-type weak D in 9,27917 and 28 cases in 16,974 blood donors18. Therefore, the Indian-type weak D occurs in approximately 1 in 729 blood donors in South India, compared to 1 in 8,063 in our East Indian cohort (p<0.001, Fisher’s exact test, two-tailed). The observed difference in the prevalence of Indian-type weak D may be associated with underlying ethnic and genetic differences between North and South Indian populations. North Indians share genetic similarity with Middle Easterners, Central Asians, and Europeans, whereas South Indians are not closely related to any group outside India48.

Our study identified 3 additional RHD alleles, including RHD*DVI.3 (RHD*06.03.01) in 3 donors, RHD*DFR5 (RHD*17.05) in 2 donors, RHD*weak D type 96 (RHD*01W.96) in 1 donor, all previously reported in the Indian population15. These RHD alleles are rare and vary in clinical significance. D category VI (DVI) is the most clinically significant partial D, associated with HTR and HDFN29. In contrast, RHD*DFR5, an RHD-RHCE(3-4)-RHD hybrid, and RHD*weak D type 96 have not been linked to anti-D alloimmunization.

A large number of serologic D-discrepant donors in our cohort (8/23, 34.8%) carried the wild-type RHD allele (RHD*01.01, Table II). This finding is consistent with previous reports of the wild-type RHD among donors with a weak D phenotype in the Indian population (10 to 18%)15–18, as well as similar observations in other populations (3 to 16%)7,49–54. The higher incidence of weak or variant D individuals with the wild-type RHD allele in our East Indian cohort suggests the potential presence of an unknown region-specific molecular variant, possibly located in the untranslated regions of RHD. It has been proposed that variants located in the non-coding regions of RHD, such as the 5′- and 3′-untranslated regions and introns, may influence gene expression and contribute to the weak D phenotype. Recent studies have identified variants in the GATA1 motifs within the RHD promoter55,56 that reduce RHD transcription. We could not evaluate the RHD promoter region in our sequencing approach and further research will be needed to discern the molecular basis in donors with seemingly wild-type RHD allele and D-discrepant serology.

The Indian-type weak D allele was initially suggested to be managed as D-negative using the same transfusion and Rh immunoglobulin (RhIG) guidelines that were applied to partial D variants15. However, the prevalence ranges from 1 in 729 to 1 in 8,063 in India and the accumulated data lacks any evidence for anti-D immunization to date15,17,18. Hence, we recommend to consider RhD-positive transfusion for individuals who harbor the Indian-type weak D allele, particularly when older than 50 years, including women. A similar pragmatic, yet cautious, strategy has proven effective in managing Asian-type DEL individuals57, even before a randomized controlled trial58. Ongoing collection and publication of case reports detailing clinical outcomes would help to support and refine this evolving transfusion practice. An observation of anti-D formation in Indian-type weak D, if any, could include the HLA type. For example, the HLA-DRB1*15:0159–61 and HLA-DQB1*02:0162 alleles have been associated with anti-D immunization63, and such HLA*DRB1*15:01-DQB1*02:01 haplotype is, with approximately 2.5%, more prevalent in India than anywhere else64.

Our study underscores the importance of region-specific molecular RHD screening to accurately identify individuals harboring variant RHD alleles that could permit alloimmunization in transfusion recipients. Characterizing RHD alleles is also crucial for guiding decisions regarding RhIG administration in pregnant women.

CONCLUSIONS

Molecular testing is essential for accurately determining the D antigen status of donors whose serologic typing results may be misleading or inconclusive. Given the diversity of RHD alleles observed in this study and previous studies from India, testing practices for donors with D-variant phenotypes can be tailored to specific communities. This approach will not only inform transfusion policy decisions but also have positive ethical and economic impacts, contributing to the creation of a regional donor database.

ACKNOWLEDGMENT

The Authors would like to thank Shikha Malhotra and her team at QuidelOrtho, India for the logistic support.

Footnotes

AUTHORSHIP CONTRIBUTION: WAF and SSD designed the study. WAF, SSD and KS interpreted the data. SSD performed the serologic testing. MR, VS, KS, YWL, JM, and GMM performed the nucleotide sequencing. SSD and KS wrote manuscript drafts. WAF wrote the final manuscript. All Authors edited and approved the manuscript.

The Authors declare no conflicts of interest.

STATEMENT OF DISCLAIMER: The views, information or content, and conclusions presented do not necessarily represent the official position or policy of, nor should any official endorsement be inferred on the part of, the Clinical Center, the National Institutes of Health, or the Department of Health and Human Services Funding.

FUNDING: This work was supported by the Intramural Research Program (projects ZIC CL002128 and RASCL727301) of the NIH Clinical Center at the National Institutes of Health.

DATA SHARING

The datasets from the 3 testing sites will be made available on reasonable request by the respective principle investigators of the sites.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets from the 3 testing sites will be made available on reasonable request by the respective principle investigators of the sites.

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