Letter to the Editor

Ann Lab Med 2023; 43(5): 524-527

Published online September 1, 2023

Copyright © Korean Society for Laboratory Medicine.

RHD*DNT (RHD*38) Showing D-Positive Reactivity on Rhesus D Typing and Forming Anti-D Antibody

Gun-Hyuk Lee, M.D.1 , Hanah Kim, M.D., Ph.D.1 , Mina Hur, M.D., Ph.D.1 , Kyeong A So, M.D., Ph.D.2 , Dong Woo Shin, M.D.3 , Yun Ji Hong, M.D., Ph.D.3,4 , and Kyoung Un Park, M.D., Ph.D.3,4

1Department of Laboratory Medicine, Konkuk University School of Medicine, Seoul, Korea; 2Department of Obstetrics and Gynecology, Konkuk University School of Medicine, Seoul, Korea; 3Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Korea; 4Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea

Correspondence to: Kyoung Un Park, M.D., Ph.D.
Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul National University Bundang Hospital, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 13620, Korea
Tel: +82-31-787-7692, E-mail:

Mina Hur, M.D., Ph.D.
Department of Laboratory Medicine, Konkuk University School of Medicine, Konkuk University Medical Center, 120-1 Neungdong-ro, Gwangjin-gu, Seoul 05030, Korea
Tel: +82-2-2030-5581, E-mail:

Received: November 29, 2022; Revised: January 25, 2023; Accepted: February 17, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Dear Editor,

In the Rhesus (Rh) blood group system, D variant antigens are mainly due to a single-nucleotide variant or a RHD/RHCE hybrid gene and are classified into three groups: molecularly defined weak D phenotypes, molecularly defined partial D phenotypes, and DEL phenotypes [1]. Individuals having D variant antigens (except for Asian-type DEL and weak D types 1, 2, and 3) may have anti-D antibodies, which have clinical significance in transfusion or pregnancy [2]. Partial D may show variable serological reactivity. In some cases with positive reactivity rather than weak positive reactivity on RhD typing, it is difficult to detect partial D unless the individual has been exposed to D antigens, resulting in anti-D formation [3, 4]. Among various partial D alleles, RHD*DNT (RHD*38) shows a single missense variant in exon 3, namely c.455A>C (N152T) [5].

We report the first Korean case of RHD*DNT, which is the fourth case worldwide. This study was exempted from approval by the Institutional Review Board of the Konkuk University Medical Center (KUMC; approval number: 2022-09-051), Seoul, Korea, and the need for obtaining informed consent was waived. A 49-year-old Korean woman was admitted to the KUMC with adenomyosis. She had had two pregnancies (without Rh immunoglobulin [RhIG] administration) and a transfusion history (no information on the blood component). ABO and RhD typing using the Ortho BioVue Innova System (Ortho Clinical Diagnostics, Raritan, NJ, USA) revealed A and D 4+ reactivity, respectively. Antibody screening using the Ortho BioVue Innova System and antibody identification using 0.8% Resolve Panel A and Ficin-treated Panel C (Ortho Clinical Diagnostics, High Wycombe, UK) suggested the presence of anti-D. RhD typing using the tube method (Shinyang Diagnostics, Siheung, Korea) revealed 4+ reactivity. Rh phenotyping using an Ortho BioVue System RH/K II Cassette (Ortho Clinical Diagnostics) revealed the ccDEe phenotype.

Further evaluation was conducted in the Korean Rare Blood Program (KRBP) reference laboratory [6]. All anti-D reagents showed 4+ reactivity in both tube and column agglutination technology methods. Antibody screening using DiaCell I+II, Dia Positive (Bio-Rad, Glattbrugg, Switzerland) and antibody identification using ID-DiaPanel (Bio-Rad) with dithiothreitol-treated cells excluded anti-LW and confirmed anti-D. Real-time PCR and melting curve analysis revealed the presence of RHD, and PCR using sequence-specific primers (SSP) with BAGene RH-TYPE, Partial D-TYPE, and/or Weak D-TYPE SSP Kits (BAG Health Care GmbH, Lich, Germany) revealed partial D, suggesting RHD*DIIIc (RHD*03.03) or other alleles harboring N512T [6, 7]. Sanger sequencing of all 10 RHD exons and flanking regions of RHD confirmed RHD*DNT (method protocol available from the KRBP reference laboratory on request) [2].

The four RHD*DNT cases are summarized in Table 1 [8-10]. Case 1 was a pregnant German woman having anti-D without a history of RhIG administration; case 2 was an African American male donor with D+hrB–; case 3 was a Caucasian man referred for RHD zygosity testing; and case 4 is the present one. All four cases showed 4+ reactivity on all anti-D reagents tested, which made it difficult to identify D variant antigens. The two female cases (cases 1 and 4) were incidentally identified because they showed anti-D after pregnancy without RhIG administration, which hinted at partial D. All four cases were confirmed as RHD*DNT by Sanger sequencing of the RHD exons.

Table 1 . Summary of the four RHD*DNT cases reported worldwide to date

Basic and serological findingsCase 1 [ref. 8]Case 2 [ref. 8]Case 3 [ref. 8]Case 4 (present case)

RaceGermanAfrican AmericanCaucasianKorean
Age/sexNA/FNA/MNA/M49 yr/F
Rh phenotypeCcDeeNTNTccDEe
RhD typing methodCATCATCATTube & CAT

Anti-D reagent information and reactivity


BS226NA [7]IgM6.44+NTNTNT
BS232NA [7]IgM6.44+NTNTNT
D415 1E4ImmucorIgGNANTNTNT4+
LHM169/80DiaMed, QuotientIgG6.34+4+4+NT
LHM57/17DiaMed, QuotientIgG6.34+4+4+NT
LHM59/19DiaMed, QuotientIgG8.14+4+4+NT
LHM70/45DiaMed, QuotientIgG1.24+4+4+NT
LHM76/55DiaMed, QuotientIgG3.14+4+4+NT
LHM77/64DiaMed, QuotientIgG9.14+4+4+NT
LOR17-6C7NA [7]IgG4.14+NTNTNT
P3X212 11 F1DiagastIgM8.24+NTNTNT
P3X212 23 B10DiagastIgM9.14+NTNT4+

*Manufacturer information for the anti-D reagents: Immucor (Immucor, Inc., Norcross, GA, USA); Diagast (Diagast Laboratories, Lille, France); Quotient (Quotient Biodiagnostics, Newtown, PA, USA); DiaMed (DiaMed GmbH, Cressier sur Morat, Switzerland); Biotest (Biotest AG, Dreieich, Germany); Shinyang (Shinyang Diagnostics, Siheung, Korea); Ortho (Ortho Clinical Diagnostics, Raritan, NJ, USA); D antigen epitopes were divided into epD 1–16 [9]; if not available, they were divided into epD 1–9 [10].

Abbreviations: M, male; F, female; epD, D antigen epitope; NA, not available; NT, not tested; CAT, column agglutination technology.

The 20 RHD alleles harboring c.455A>C (N152T) are listed in Table 2 [3-5, 8]. The designation RHD*DNT was derived from the asparagine (N) to threonine (T) amino acid substitution, and N152T has been suggested to cause a high D antigen density (>20,000 antigens per red blood cell) by facilitating the integration of the RhD protein into the red blood cell membrane. Alleles harboring N152T may remain undetected (typed as D-positive) and anti-D immunization may occur, resulting in transfusion incompatibility and complications during pregnancy [8]. Individuals with partial D and anti-D were mostly females, and pregnancy was the most frequent cause of immunization; thus, women of childbearing age and girls would benefit from measures to prevent further anti-D immunization [8]. Although further research is required to define the underlying mechanism and to investigate the relationship between the increased antigen density and epitope change, it is assumed that the internally located N152T substitution (in the transmembrane zone) may alter extracellular epitopes. Among the 20 alleles harboring N152T, 11 alleles have been associated with anti-D formation [3, 4]; RHD alleles harboring N152T do not necessarily imply anti-D formation, and conversely, any alleles harboring N152T may have the potential to form anti-D. In RhD-positive individuals with anti-D formation, Sanger sequencing of the RHD exons can help clinically differentiate the RHD alleles harboring N152T. RHD*DNT could not have been identified without Sanger sequencing of the RHD exons; this implies that there may have been missed or unidentified RHD*DNT cases worldwide.

Table 2 . All 20 RHD alleles harboring c.455A>C (N152T)

AllelePhenotypeAnti-D formation
RHD*DIIIa (RHD*03.01)DIIIaReported [3]
RHD*DIIIc (RHD*03.03)DIIIcReported [3]
RHD*DIII.4 (RHD*03.04)DIII type 4Reported [3]
RHD*DII.04.02 (RHD*03.04.02)Not testedNot reported [5]
RHD*DIII.6 (RHD*03.06)DIII type 6Not reported [3]
RHD*DIII.07 (RHD*03.07)DIII type 7Not reported [5]
RHD*DIII.08 (RHD*03.08)DIII type 8Reported [3]
RHD*DIII.09 (RHD*03.09)DIII type 9Not reported [5]
RHD*DIVa (RHD*04.01)DIVaReported [3]
RHD*04.01.02DIVa-like (DIVa type 3)Not reported [5]
RHD*DVI.3 (RHD*06.03.01)DVI type 3Reported [3]
RHD*DVI.03.02 (RHD*06.03.02)DVI type 3.2Reported [3]
RHD*DVI.4 (RHD*06.04)DVI type 4Reported [3]
RHD*DOL4 (RHD*12.04)DOL4Reported [4]
RHD*DFR5 (RHD*17.05)DFR5Reported [3]
RHD*DNT (RHD*38)RHD*DNTReported [8, present case]
RHD*D-SPM (RHD*40)D-SPMNot reported [5]
RHD*DKK (RHD*45)DKKNot reported [5]
RHD*62DNT(V270G)Not reported [5]
RHD*03N.01D–, C+Not reported [5]

In conclusion, we reported the first Korean case of RHD*DNT, which is the fourth case worldwide. RHD*DNT showed D-positive reactivity on RhD typing, which made it difficult to identify rare D variant antigens. Our case underscores the importance of molecular characterization for the identification of rare D variant antigens.

Lee GH and Shin DW analyzed the data and wrote the draft; Hur M and Park KU conceived and designed the study, analyzed the data, and finalized the draft; Kim H, So KA, and Hong YJ collected the samples, analyzed the data, and participated in manuscript drafting. All authors have read and approved the final manuscript.

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