How does the D antigen affect Rhesus (Rh) typing?

Updated: Aug 01, 2018
  • Author: Victoria K Gonsorcik, DO; Chief Editor: Jun Teruya, MD, DSc, FCAP  more...
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The RhD protein has two clinically significant variations, "partial D"” and "weak D." Because the D antigen comprises multiple epitopes, red cells that lack components of the D antigens are often described as "partial D." Molecular studies have identified that many partial D phenotypes result from amino acid substitutions or a protein segment switch on the extracellular portion of the RhD protein. Individuals with partial D are usually typed as Rh-positive but may form anti-D when alloimmunized.

Rh antigen diagram. Image created by Jaye Parsley. Rh antigen diagram. Image created by Jaye Parsley.


Red cells that carry weak forms of D antigen are classified as "weak D," which usually results from amino acid substitutions within the internal portion or in the membrane-crossing portion of the RhD protein causing quantitative changes. An individual with weak D has a decreased amount of D antigens expressed on the red cell.

With current serologic testing, most individuals with weak D are typed as Rh-positive via direct agglutination testing using anti-D. However, demonstration of some individuals with weak D requires the use of antihuman globulin reagent after incubation with anti-D (indirect antiglobulin test [IAT]). Relative to partial D individuals, weak D individuals are less likely to form anti-D antibodies. It is important to understand that certain molecular types of weak D are capable of making anti-D. [5]

These two variations of the D antigen have caused considerable debate and discussions about how to interpret laboratory test results, how to manage these individuals in different clinical settings to select proper blood products for transfusion, and how to safely identify the characteristics of donors to avoid transfusion-related alloimmunization. According to the American Association of Blood Banks (AABB) Standards for Blood Banks and Transfusion Services, it is required to test donors for weak D, because transfusion of blood from weak D individuals carries the risk of alloimmunization in Rh-negative transfusion recipients. [6]

There is significant variation in terms of interpretation and reports of transfusion recipients with weak D. A 2017 review estimates 0.2%-1% of routine RhD blood typings result in a "serological weak D phenotype." [7] A 1999 survey of 3000 participating institutions showed that 50.7% would consider the patient "Rh(D)–positive," 20.9% would report the patient as "Rh(D)–positive, weak D–positive/variant," 20.1% would report the patient as "Rh(D)–negative, weak D–positive/variant," and 3.3% would report the patient as "Rh(D)–negative." [8] These same institutions also reported their actual transfusion policies; the survey showed that 43.5% would administer Rh(D)–negative blood, 42.4% would give Rh(D)–positive blood, and 10.2% of the institutions would provide Rh(D)–negative blood to a childbearing woman. [8] Therefore, there is no current consensus as to how to interpret weak D individuals.

A 2012 update to the 1999 survey again found a lack of consensus for interpretation of weak D individuals as well as a reduction in the number of transfusion services (from 58.2% to 19.8%) performing serologic weak D testing on patients as a strategy to manage those with weak D as Rh(D)-negative. [9] Although testing in general decreased, testing rose from 26.6% in 1999 to 33.7% in 2012 in specific patient populations: women of childbearing years, pregnant women, and others (newborns). [9]

The formation of Rh antibody usually requires an initial exposure, either through a transfusion or pregnancy. During the first exposure to foreign Rh antigen, the host’s immune system will not produce clinically significant amounts of antibodies that cause hemolysis. Rh antibody causing hemolysis is seen after the second and subsequent exposures.

Of the five most common clinically significant Rh red cell antigens, D is well known for causing the most severe immunogenic responses. Studies have indicated that Rh-negative individuals have an 80% chance of making an antibody to D upon exposure to 1 unit of D-positive red blood cells.

Two methods of Rh typing appear below. Use of both tube testing and gel testing as a two-method strategy for Rh typing appears to be a strong screening tool for identifying candidates for RHD genotyping. In a study that used the two-method strategy for Rh typing of patients with no historical blood type at a medical institution, 50 patients were identified, of whom genomic testing confirmed D variants in 49 (98% positive predictive value). [10]

Manual tube testing. Image created by Jay Parsley. Manual tube testing. Image created by Jay Parsley.
Column agglutination "gel card." Image created by Column agglutination "gel card." Image created by Jaye Parsley.

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