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. 2025 Jun 19;23(1):686.
doi: 10.1186/s12967-025-06716-8.

Transitioning a multiethnic donor pool from serologic D-negative to molecularly RHD-negative at a hospital-based blood donor service

Willy A Flegel  1 , Kshitij Srivastava  2 , Lorraine G Caruccio  2 , Pirmin Schmid  2 , David A Stiles  2 , Marina U Bueno  2 , Nadine R Dowling  2 , Traci D Paige  2 , Sita Shrestha  2
Affiliations

Transitioning a multiethnic donor pool from serologic D-negative to molecularly RHD-negative at a hospital-based blood donor service

Willy A Flegel et al. J Transl Med. .

Abstract

Background: Some individuals carry a very low expression of the D-antigen, called a Del phenotype. Red cell units from such blood donors with DEL alleles are RhD protein-positive, despite being routinely labelled D-negative. Molecular typing offers a more sensitive method to identify Del individuals by detecting the presence of the RHD gene. Pools of 20 or more donor samples are routinely screened for the RHD gene in some, mostly European, donor populations.

Methods: A modular real-time PCR assay targeting RHD intron 4, exon 5, and exon 7 was developed for individual testing. We screened for the RHD gene among all blood donors who typed D-negative in routine serology.

Results: Over 15 years, 2254 D-negative donors were individually tested for the RHD gene. With a sensitivity of detecting 5 RHD positive gDNA copies per reaction, 42 donors tested positive (1.9%). Among them, 34 carried the common RHDΨ allele (80.9%), while 7 harbored 5 known RHD alleles, and 1 a novel RHD deletion. We inadvertently detected 2 other donors with DVI, establishing a population frequency of 1 in 731 for the U.S.

Conclusions: A modular approach for RHD screening is suitable for blood donors when sample pooling is not feasible among multiethnic donor populations. We transitioned donors since 2009 from serologic D-negative to molecularly RHD-negative status at the NIH Clinical Center. Molecular RHD screening of serologic D-negative donors is an effective way to identify individuals harboring DEL alleles that can cause alloimmunization in transfusion recipients.

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Conflict of interest statement

Declarations. Competing interests: The authors declare no competing interests relevant to this article. 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 (WAF); the Swiss Armed Forces or the Swiss Government (PS).

Figures

Fig. 1
Fig. 1
All serologic D-negative donors and the RHD gene-positive donors over 15 years. A total of 2254 serologic D-negative donors (□) were screened between November 2009 and October 2024. Among them, 42 donors tested positive for the RHD gene (■). Inadvertently, 2 additional donors with a DVI phenotype were found (not shown)
Fig. 2
Fig. 2
RHD intron 4 real-time PCR assay. A RHD intron 4 real-time PCR melting curves have a single peak at 83.2 °C for a double-stranded PCR amplicon. The horizontal line represents the instrument’s default setting for the threshold of positive results. B Real-time standard curve of RHD intron 4 displaying the relationship between Cq and RHD-positive chromosome copies. The circles represent RhD-positive genomic DNA standards along the curve. The 2 standard points at Cq = 45 are outside of the linear dynamic range of the assay
See this image and copyright information in PMC

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