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Year : 2021  |  Volume : 8  |  Issue : 4  |  Page : 410-414

A retrospective study on the prevalence of weak D antigen (Du) in a blood bank in a tertiary care hospital in Maharashtra, India

Department of Transfusion Medicine, MGM Medical College and Hospital, Navi Mumbai, Maharashtra, India

Date of Submission20-Oct-2021
Date of Acceptance26-Nov-2021
Date of Web Publication22-Dec-2021

Correspondence Address:
Dr. Abhiniti Rahul Srivastava
Department of Transfusion Medicine, MGM Medical College and Hospital, Kamothe, Navi Mumbai 410209, Maharashtra.
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mgmj.mgmj_77_21

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Background: One of the most immunogenic blood groups known to humans is the Rhesus (Rh) blood group. The Rh blood group with variable expression of D antigen is one of the complex systems in immunohematology. Sometimes, the variable expression of RhD antigen leads to the presence of weaker forms that are not detected by routine laboratory procedures. Aims: The study was conducted to determine the frequency of RhD negativity and weak D antigens. Settings and Design: This retrospective study was conducted in the Department of Immunohematology and Transfusion Medicine at a tertiary care center hospital for three years from June 2017 to June 2020. Materials and Methods: All the serial Rh grouping tests were conducted by the gel technique or by the tube method with two Anti-D reagents. Negative D antigen samples were further tested for weak D antigen by Coombs Test. Statistical analysis used the Statistical Package for Social Sciences (SPSS). Results: Out of a total of 17262 samples, 15400 (89.2%) tested Rh-positive, and 1866 (10.8%) tested Rh-negative. Weak D was positive in 52(0.027%) samples out of the RhD negative samples. Conclusion: The frequency of weak D antigen is low, but the strong immunogenicity of RhD antigen calls for the need for appropriate testing. This is of concern in RhD negative pregnant females, as it can cause alloimmunization if accidentally weak D antigen-positive blood is transfused.

Keywords: Alloimmunization, D antigen, Du, Rh blood group, Weak D

How to cite this article:
Srivastava AR, Dhote SW, Singh I. A retrospective study on the prevalence of weak D antigen (Du) in a blood bank in a tertiary care hospital in Maharashtra, India. MGM J Med Sci 2021;8:410-4

How to cite this URL:
Srivastava AR, Dhote SW, Singh I. A retrospective study on the prevalence of weak D antigen (Du) in a blood bank in a tertiary care hospital in Maharashtra, India. MGM J Med Sci [serial online] 2021 [cited 2022 Dec 7];8:410-4. Available from: http://www.mgmjms.com/text.asp?2021/8/4/410/333329

  Introduction Top

The presence or absence of the Rh antigen determines the Rh blood grouping, which is separated into two categories: Rh Positive and Rh Negative. Levine and Stetson defined Rh antigen for the first time in 1939. After Landsteiner discovered ABO blood types in 1901, this became a breakthrough in immunohematology.[1]

There are more than 50 antigens in the Rh system, with D being the most important one, expressed by the RhD Protein. Clinical problems are believed to be caused by five antigens: D, C, c, E, and e.[2] There are two homologous genes found at the RH locus. D Polypeptide is encoded by the RHD gene. C or c with E or e is coded for by the RHCE gene. These are the Rh proteins D and Cc/Ee.[3]

Rh expression is controlled by two genes, RHD and RHCE. These can be found on 1p34-p36 of chromosome one. RhCE-specific amino acids in RhD or RhD-specific residues in RhCE can be coded by RHCE, which are responsible for the generation of novel antigens in the Rh blood group system.[4]

The global incidence of the Rh-negative blood group is estimated to be between 3% and 25%. Du, one of the weaker D variations, is characterized as a quantitative variation, with just a few typical D antigens per red cell but all epitopes present. The incidence of weak D, which requires detection by Antihuman Globulin, varies from 0.2% to 1%.[5],[6]

When Rh-positive RBC samples are tested for the D antigen, Anti-D reagents show significant positive results. Some patients have D antigen-positive RBCs, which may be detected only with an Indirect Antiglobulin Test. Initially, the researchers believed they had identified a novel antigen. Individuals did not produce Anti-Du; instead, they generated Anti-D, proving that this was not a novel antigen.[7]

The use of molecular biology has aided in the demonstration of blood group systems’ genetic foundation. There are 51 antigens in the Rh system and more than 200 alleles for the RHD gene.[8]

All people with altered D antigen were known as having weak D for many years. A mechanism that results in weakened expression of D antigen can be described as a position effect or gene interaction effect. The allele carrying RHD is trans to the allele carrying C. Weak D, Partial D, and Del are other phenotypes that these altered D antigens can present with. It has been shown that 1% to 2% of individuals with European ancestry possess some altered form of D antigen. Finally, to further complicate matters, some rare individuals possess D epitopes on their RhCE protein.[7]

The present study aimed at evaluating the prevalence of weak D. The objectives were to find the distribution of weak D antigen in the total study samples, in the specific blood groups of the total samples, and in males and females.

  Materials and methods Top


The retrospective observational study was conducted at the Department of Immunohematology and Transfusion Medicine (Blood Center) of a tertiary care hospital, for a period of three years, that is, from June 2017 to June 2020.

The approval to conduct the study was obtained by the Institutional Ethics Committee.

Materials required

  1. All the blood units were collected over three years.

  2. All the samples were received from the central lab for blood group confirmation.

Method of data collection

Records used:

  1. Donor register

  2. Blood Grouping Register

  3. Blood Group Confirmation Request Forms from Central Laboratory

All the forms for blood grouping and registers were evaluated.

  1. The donor Detail Registers were analyzed to determine the sex of the donors and the blood group at camp that were maintained while registering the donors.

  2. The blood group confirmation forms that were received from the Central Laboratory were analyzed in detail to determine the sex of the patient, the blood group, and the final result.

  3. The blood grouping register was evaluated for the Du results.

Inclusion criteria

All the blood grouping done in the Department, during a period of three years from June 2017 to June 2020, was included in the study.

Exclusion criteria

The blood group confirmation request of samples received from outside the hospital was not included.

A total of 17,262 samples were analyzed. For all samples at the time of grouping, Rh typing was done by the immediate spin tube technique. The testing was done by two different companies’ Anti-Rh reagents by adding two drops of Anti-D serum (IgM, IgG+IgG) and one drop of 3–5% washed red cell suspension in a clean glass test tube. They were slightly shaken and mixed. The tubes were then centrifuged at 1,000 rpm for 1 min. The tubes were gently resuspended and then observed for agglutination. If the test result was recorded as positive (3+/4+), then a grade was assigned to it. If the test result was doubtful (1+/2+) or negative, a confirmation of weak D Antigen was done. A tube was labeled as Du; two drops of Anti-D (IgG) and one drop of 3–5% washed red cell suspension were added. Then, they were mixed and incubated at 37°C for 45 min. They were then washed three to four times with normal saline. After the final wash, the saline was decanted and two drops of antiglobulin serum were added. After this, the contents of the test tube were mixed, and the tube was centrifuged at 1,000 rpm for 1 min. The cell button was then gently resuspended and examined for agglutination. All negative results were confirmed under the microscope.

  Results Top

In this study, 17262 samples were analyzed retrospectively, of which 52 were found to be weak D antigen-positive. The distribution of RhD Antigen was conducted in the study. Approximately 89.1% were Rh-positive whereas 10.5% were Rh-negative [Table 1]. Blood group A was found to have the maximum number of weak D antigen samples [Table 2]. The prevalence of weak D antigens was found to be more in females [Table 3] and [Table 4].
Table 1: Distribution of RhD antigen in total study samples

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Table 2: Distribution of RhD antigen in the specific blood groups of the total samples

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Table 3: Distribution of RhD antigen in males and females in the total sample percentage

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Table 4: Distribution of RhD antigen in males and females gender wise percentage

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Statistical analysis

SPSS and Microsoft Excel were used to generate the results. The first table was not subjected to any tests, because it contains the frequency of Weak D Antigen. Weak RhD Antigen was shown to have no statistically significant relationship with particular blood groups (P > 0.05). Another finding was that the distribution of Weak D Antigen was significantly higher in females (%) as compared with males (%) (P < 0.01).

  Discussion Top

After Landsteiner discovered ABO blood types in 1901, the discovery of Rh antigen became a breakthrough in immunohematology.[1] Following the comments of the contradictory categorization of the Rh system, Stratton described a Weak D Antigen in 1946.[2]

In certain individuals, D Antigen expression on red blood cells is diminished, resulting in Weak D, a form of D phenotype seen in human beings. The usual procedures (spin tube method) utilized at the blood center are unable to identify this Antigen. As a result, numerous ways are employed to illustrate this weakly expressed Antigen. The most popular approaches are extended incubation and the use of the Antihuman Globulin Indirect Coombs Test. Antibody responses can be elicited in responder Rh-Negative people after exposure to 0.1 ml of Rh (D) Antigen-Positive cells. Only red blood cells have Rh antigens. The beginning of Rh Antibody synthesis is irreversible, and circulating Antibodies may persist for years.[8]

Weakened expression of normal D Antigen, that is, less than normal D Antigens per red cell, is what weaker forms of D are characterized as. “Du” was its previous name. This is an inherent trait. Weak D in blood donors is classified as D-Positive, whereas weak D in receivers is classified as Rh-Negative. Immuno-prophylaxis is required for mothers with Du-Positive fetuses.[9] For individuals who are prone to receive transfusions frequently, British standards require that transfusions are Rh DCE and K phenotypically matched and then transfused. This is because the transfusion of Rh and Kell-matched blood helps in reducing alloimmunization risk among nonchronically transfused patients.[10]

In their study, Desai et al.[11] observed that of 84,697 units of donated blood, 4,541 (5.36%) were Rh-negative and 38 (0.8% of Rh-negative donors) were Weak D Positive. A total of 3506 Rh-negative donors were tested for Weak D by using the Conventional Tube Method, and 34 donors (0.9%) were found to be Weak D Positive. Weak D testing was performed on 1,035 Rh-negative donors by using gel cards, and only four (0.3%) were found to be Weak D Positive. It was determined that this was not statistically significant (P = 0.08).

In 2011, Lamba et al.[12] evaluated 5,267 blood samples, which was 5,138 in 2012 and 2,638 in 2013. Between 2011 and 2013, a total of 13,043 blood samples were evaluated. There were 12,196 RhD positive people (95.51%) and 847 RhD negative people (6.49%). Out of the Rh-negative samples, eight (0.06%) of them were found to be Weak D or Du Positive. There were 12196 RhD positive people (95.51%) and 847 RhD negative people (6.49%). When these negative samples were examined for being Weak D or Du Positive, eight (0.06%) were found to be Weak D or Du Positive. Pallab et al.[13] observed that three of the 172 RhD negative blood donors over four years were Weak D Antigen Positive.

During the period January 2012 to August 2014, Deepthi Krishna et al.[14] analyzed a total of 46,654 blood samples (22,326 donors and 24,328 patients). RhD positives comprised 43,771 (93.82%) of the total, whereas RhD negatives comprised 2,883 (6.18%). Weak D Positive people accounted for 1.04% of RhD negatives and 0.06% of total people (16 donors and 14 patients). Some alleles are known to be restricted to cultural groups, whereas others are distributed widely. Some alleles are known to be restricted to cultural groups, whereas others are distributed widely.[15]

For determining the various configurations in the genotypic organization of the Rh blood group system, numerous innovative tests based on Sequence-Specific Primers (SSP)–Polymerase Chain Reaction (PCR) are now being employed. Exploring the molecular basis of a Weak D-Rh Del, an Rh-Negative Phenotype is produced by deletion of Exon 9 in the RhD gene. The genetic lesion of Weak D is investigated by using fragment length polymorphism (PCR-RFLP) and direct sequencing techniques.

Weak D is nearly always tested since it is well known that it might produce Alloimmunization in recipients. Individuals with the Du phenotype are regarded as D Positive while donating blood since D Antigen is known to be highly immunogenic. Patients with the Weak D phenotype are designated as D Negative and should get D Negative blood products. Sensitization may occur as a result of the passing of Weak D cells from the fetus to the pregnant mother. As a precaution, women who have a Weak D fetus should get Rh Immune-Prophylaxis to avoid Rh Alloimmunization.

To our knowledge, there have been no investigations on the distribution of ABO and D antigens in diverse ethnic groups.[9]

In our study, out of a total of 17,262 samples, 15,400 (89.2%) tested Rh Positive and 1,866 (10.8%) tested Rh-negative. Weak D was Positive in 52 (0.027%) samples out of the RhD negative samples. To minimize misdiagnosis and transfusion-related problems, extensive and precise national transfusion guidelines serve to regulate the methodology for D Antigen testing for donors and recipients.[16]

  Conclusion Top

Many patients who are transfused chronically in conditions such as Thalassemia, Sickle Cell Anemia, or Chronic Renal Failure require Weak D Antigen testing and detection. If these RhD negative people test positive for Weak D Antigen, they should be given Rh-negative blood from the same ABO group. This decreases the risk of alloantibody formation against the Weak D antigen that could be missed on routine methods of blood grouping. They are recognized as Rh-Positive donors. Weak D Antigen testing should be done in a highly controlled lab setting since erroneous Positive results can lead to unintentional transfusion of RhD positive blood to RhD negative patients, which can have devastating immunological and medical repercussions. The necessity of accurate Rh testing and its ramifications in transfusion medicine may be demonstrated by using several instances from the literature.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

Ethical consideration

Approval was obtained by the Institutional Ethical Committee of MGM Medical College and Hospital, Navi Mumbai, India vide their letter no. N-EC/2020 /09/85 dated 05th October 2020.

  References Top

Kumar H, Mishra DK, Sarkar RS, Jaiprakash M. Difficulties in immunohematology: The Weak D Antigen. Med J Armed Forces Indi 2005;61:348-50.  Back to cited text no. 1
Brar RK, Shaiji PS, Sehgal S. Testing for weak D antigen: Spectrum and its applied role in rhesus-negative transfusions in Andaman and Nicobar islands. Ci Ji Yi Xue Za Zhi 2020;32:167-70.  Back to cited text no. 2
Ansart-Pirenne H. Identification strategy of RHCE gene variants at the national blood group reference laboratory: Impact on transfusion safety. Transfus Clin Biol 2006;13:13-8.  Back to cited text no. 3
Rizzo C, Castiglia L, Arena E, Gangi S, Mazzola G, Caruso C, et al. Weak D and partial D: Our experience in daily activity. Blood Transfus 2012;10:235-6.  Back to cited text no. 4
Lin IL, Shih MC, Hsieh MH, Liu TC, Chang SE, Lin CL, et al. Molecular basis of weak D in Taiwanese. Ann Hematol 2003;82:617-20.  Back to cited text no. 5
Urbaniak SJ, Robertson AE. A successful program of immunizing Rh-negative male volunteers for anti-D production using frozen/thawed blood. Transfusion 1981;21:64-9.  Back to cited text no. 6
Suwe G, Wiler M. The Rh blood group system. In: Denise M editor. Harmening. Modern Blood Banking and Transfusion Practices. 7th ed. Philadelphia, PA: FA Dewis editors; 2019. p. 149-72.  Back to cited text no. 7
Flegel WA. Molecular genetics and clinical applications for Rh. Transfus Apher Sci 2011;44:81-91.  Back to cited text no. 8
Kotwal U, Raina TR, Sidhu M, Dogra M. Distribution of ABO &amp; Rh (D) blood groups among blood donors of Jammu region with respect to various ethnic groups. J Med Thesis 2014;1:3-6.  Back to cited text no. 9
Kumpel B. Are weak D RBCS really immunogenic? Transfusion 2006;46:1061-2; discussion 1062-6.  Back to cited text no. 10
Desai HM, Amonkar GP, Chandekar S, Valvi N, Puranik G. The feeble antigen “weak D”: Prevalence in a tertiary care hospital blood bank in Mumbai. J Lab Physicians 2017;9:148.  Back to cited text no. 11
Lamba HS, Kaur K, Kaur K, Vij AS. Prevalence of weak D (Du) in blood donors in a referral teaching hospital. Int J Med Dent Sci 2017;6:1509-12.  Back to cited text no. 12
Introduction I. Retrospective study on prevalence of Weak D Antigen (D u) in blood donors in a tertiary care hospital of Tripura. North-East 2019;18:38-40.  Back to cited text no. 13
Deepthi KG, Sreedhar BKV, Reji A, Jothbai DS. A study on Rh incompatibility and frequency of Weak D among blood donors and patients at a tertiary care referral teaching hospital in Tirupati, Andhra Pradesh. J Clin Sci Res 2015;4:281-4.  Back to cited text no. 14
Denomme GA, Wagner FF, Fernandes BJ, Li W, Flegel WA. Partial D, weak D types, and novel RHD alleles among 33,864 multiethnic patients: Implications for anti-D alloimmunization and prevention. Transfusion 2005;45:1554-60.  Back to cited text no. 15
Pahuja S, Pujani M, Sethi N, Kushwaha S, Jain M, Kumari R. Frequency of variant D in Delhi, India. Asian J Transfus Sci 2014;8:142-3.  Back to cited text no. 16
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