Rh Typing

Updated: Aug 01, 2018
  • Author: Victoria K Gonsorcik, DO; Chief Editor: Jun Teruya, MD, DSc, FCAP  more...
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After the ABO system, the Rh (Rhesus) blood group system is regarded as the second most important blood group system, as some of the severe hemolytic transfusion reactions and most hemolytic disease of the fetus and newborn (HDFN) cases are associated with antibodies to the Rh group antigens.

The Rh system consists of over 50 red cell antigens. There are five main Rh red cell antigens—D, C, c, E, and e—that involve most clinically significant transfusion complications. These five are the focus of this article.

Two separate genes for the Rh system are found on chromosome 1. One gene, RHD, encodes for the D antigen. Individuals with the D antigen present on their red blood cells are labeled as "Rh (D)–positive." Those who do not have the D antigen are labeled as "Rh (D)–negative."

The frequency of "Rh-negative" individuals varies among different ethnic groups, largely because of the different molecular mechanisms that cause the absent expression of the D antigen. For example, 15% of white individuals who are Rh-negative have this finding mainly caused by the absence of the RHD gene. Approximately 6% of blacks and less than 1% of Asians are Rh-negative. Mechanisms of Rh-negativity in these ethnic groups may include gene deletion, gene rearrangement, gene duplication, and gene mutation. It is important to note that a precise typing of the RH genotype can be achieved with the advancement of molecular testing techniques. [1, 2]

A second gene, RHCE, encodes for a combination of CE or ce antigens together. RHD and RHCE are highly homologous to each other. In essence, RHCE is the original gene, and RHD is a duplication of RHCE. The RHD and RHCE genes encode proteins such that each crosses the red cell membrane 12 times, with 6 protein loops on the exterior of the cell membrane.


It is important to note that the Rh blood group system has more than one accepted nomenclature. Most of this review uses the Fisher-Race nomenclature, which designates the antigens with letters DCE in either upper or lower case. The Weiner system uses the letters R, r; subscript y (y); z; the numbers 1, 2, 0; and symbols for prime (', "). These two nomenclatures are listed in the tables below.

Table 1. Rh Haplotype Frequencies (Open Table in a new window)

Fisher-Race Common Rh Red Cell Antigens

Weiner Common Rh Red Cell Antigens

White, %

Black, %

Asian American, %


































< 0.01

< 0.01



< 0.01

< 0.01

< 0.01

Adapted from Westhoff C. ABO, H, and Lewis blood groups and structurally related antigens and methods. In: Roback J, Coombs MR, Grossman B, Hillyer C, eds. Technical Manual. 16th ed. Bethesda, MD: American Association of Blood Banks (AABB); 2008. [3]

"d" = an individual who does not express the D antigen.

Table 2. Rh Phenotype Frequencies (Open Table in a new window)




Weiner System

White, %

Black, %

Asian, %



R1R1 (R1r')





R2R2 (R2r")





R1r (R1R0;R0r')





R2r (R2R0;R0r")





R0r (R0R0)





RzRz (Rzry)





R1Rz (Rzr'R1ry)





R2Rz (Rzr";R2ry)





R1R2 (R1r";R2r'Rzr;R0Rz;R0ry)































r'r" (ryr)





r'ry; r"ry;ryry




Adapted from Reid ME, Lomas-Francis C. The Blood Group Antigen Facts Book. 2nd ed. San Diego, CA: Academic Press; 2003. [4]

Partial D and weak D

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.

Clinical Indications/Applications


There is specific terminology involved in testing for Rh groups.

A "type and screen" includes the following tests:

  • ABO

  • Rh(D)

  • Antibody detection/identification

A "type and cross" includes the following tests:

  • ABO

  • Rh(D)

  • Antibody detection/identification

  • Compatible match with a donor unit

Transfusion recipient testing

Transfusion of a recipient requires a type and cross to be completed before blood is issued to prevent Rh incompatibility.

Transfusion donor testing

A type and screen is one of the required tests for blood donation.

Recipient organ/hematopoietic stem cell testing

The recipient has a type and screen performed to assess Rh compatibility between the recipient and potential organ donors. Hematopoietic stem cell testing is the only instance when Rh type does not have to be identical. Donor hematopoietic progenitor stem cells will engraft into the recipient's chemotherapy-induced "empty" or "wiped out" marrow and replace the recipient's blood ABO type and Rh type if the engraftment is successful.

Donor organ/hematopoietic stem cell testing

A type and screen is one of the required tests for organ/hematopoietic marrow donation to assess donor compatibility for an intended recipient.

Hemolytic disease of the fetus and newborn assessment and Rh testing

Evaluation of hemolytic disease of the fetus and newborn (HDFN) that is associated with Rh incompatibility involves multiple steps. HDFN occurs when a fetus has antigens on his or her red cells that the mother's immune system does not recognize as her own (usually an Rh-negative woman carrying an Rh-positive fetus). A small percentage of fetal blood may come into direct contact with the maternal blood circulation through fetal maternal hemorrhage (eg, amniocentesis, trauma, miscarriage/abortion, or placental abnormalities).

Certain antigens on fetal blood cells, like those of the Rh system, are more fully developed and are present in greater numbers than others. This allows the mother's immune system to easily recognize them and produce antibodies that may cause hemolysis. Anti-Rh antibodies are mainly immunoglobulin (Ig) G. The mother's IgG antibodies, because of their smaller structures, are able to cross the placenta and attack fetal red blood cells carrying the Rh antigens, causing fetal hemolysis. Once the mother's Rh antibody is identified, titers and a score are used to assess the amount of antibody present in the mother's plasma and to evaluate its strength/avidity. Clinicians then use this information to predict the risk of fetal hemolysis.

Titers and scores are performed in a blood bank reference laboratory. A sample of the mother's blood is centrifuged to separate her plasma (which contains the antibody) from her red blood cells. The plasma is then serially diluted and combined with reagent Rh-antigen–positive red cells. Agglutination or lack of agglutination is visually assessed. A score is then determined based on the strength of agglutination associated with each dilution.

In order to prevent HDFN arising from an Rh-negative mother in the first place, several steps can be taken. In the United States, when fetal Rh(D) status is unknown, Rh-negative pregnant women are given a form of Rh(D) immunoglobulin (ie, RhoGam or WinRho) at approximately 28 weeks’ gestation. Additional doses are administered after any procedures and at the time of delivery. Clinicians can also use amniocentesis samples to verify fetal Rh(D) status to predict current or future risk for HDFN due to Rh(D) incompatibility.

A less-invasive molecular genotyping procedure has been tested and used in portions of Europe. Throughout pregnancy, particularly during the second and third trimesters, fetal DNA is present in the pregnant woman's plasma. A blood sample from the pregnant woman is taken and separated via centrifugation. If the fetus is Rh-positive, the fetal RH DNA can be amplified and detected by polymerase chain reaction (PCR). The genotyping results assist in determining the fetal Rh(D) status. If the fetus is deemed Rh-negative, the pregnant woman may not need to receive the Rh(D) immunoglobulin to prevent antibody formation.

In a review of the literature from European centers that implemented large-scale nationwide noninvastive fetal RHD typing in the second trimester for targeted Rh(D) immunoglobulin administration, investigators found that depending on patients' ethnic backgrounds and the medical institution, fetal RHD typing using a duplex real-time PCR can safely and cost-effectively guide the administration of Rh(D) immunoglobulin to prevent D-alloimmunization during pregnancy. [11]  They calculated the unnecessary administration of 40% of antenatal Rh(D) immunoglobulin could be avoided, and cord blood serology could be omitted.

This molecular testing has undergone review in the United States. If implemented, there will not only be a decrease of more invasive techniques but also a decrease of unnecessary exposure to Rh(D) immunoglobulin. [9]


Test Performance and Limitations

Test performance

Rh(D) typing is usually performed at the time of ABO typing and antibody screening. A blood sample is placed in a centrifuge, which separates the red blood cells from the plasma. The individual's red cells are diluted to make a working suspension (2%-5%) in saline. The red cell suspensions are then mixed with anti-D reagent.

The test is used to measure visual agglutination or lack of agglutination. Agglutination refers to the clumping of cells in the presence of antibody. Unlike ABO typing, a "reverse" test is not performed, because a person should not have preformed antibodies to the D antigen, unless they have previously been alloimmunized to the D antigen through transfusion or pregnancy. [3]

Traditionally, blood typing used serologic testing, based on hemagglutination reactions against specific antisera; more recently, molecular typing, based on detecting the polymorphisms and mutations controlling the expression of blood group antigens, has been used as an alternative or supplemental typing method. [2]

Test limitations

A limitation to Rh typing is the amount of sample that is required to perform ABO and Rh typing using the standard tube testing, column agglutination method, or solid phase test systems: approximately 1 mL of blood.


Test Interpretation

Factors that can interfere with a type and screen are discussed below.

A history of Rh immunoglobulin (RhoGam/WinRho/Rhophylac) administration can cause interference with a type and screen. Rh immunoglobulin is used mainly in obstetrics to prevent hemolytic disease of the fetus and newborn (HDFN). In theory, the medication is composed of anti-Rh (D) immunoglobulin (Ig) G antibodies that will coat small amounts of exposed fetal red blood cells, which should prevent the mother's immune system from producing anti-D. In subsequent months, up to 6 months after receiving Rh immunoglobulin, the mother's plasma shows the presence of anti-D, which is interpreted as passively acquired anti-D secondary to Rh-immunoglobulin use; thus, there is no clinical concern for hemolysis. The mother still receives Rh-negative blood products if transfusion is indicated.

Another scenario that causes interference with a type and screen is that of an Rh-negative individual who received Rh-positive red cells because of a low inventory of Rh-negative blood; this person will show newly formed anti-D alloantibody. This is a true alloantibody situation and is mainly reserved for Rh-negative males or Rh-negative females beyond childbearing age. The patient should subsequently receive Rh-negative blood products to avoid hemolysis.

False-positive results with Rh typing may result from a positive direct antiglobulin test (DAT) result (coating of red cells with alloantibodies), rouleaux formation (can be seen with patients with multiple myeloma), or contamination, or reagents. False-positive results may also be seen when high-protein Rh reagents that contain 20% protein or other high molecular weight additives are used for typing. Therefore, a proper Rh control reagent should be tested simultaneously before the Rh typing results can be correctly interpreted. [3]

Finally, false-negative results with Rh typing may result from the use of incorrectly diluted red cell suspensions, reagent deterioration, failure to follow the manufacturer's directions, or inappropriate techniques. [3]



Manual agglutination testing was the main methodology used for ABO/Rh typing for many years. However, during the 1980s, automated testing methodology was introduced, including column agglutination and solid phase test systems. The following testing descriptions are adapted from Methods, section 2, of Westhoff C. ABO, H, and Lewis blood groups and structurally related antigens and methods. In: Roback J, Coombs MR, Grossman B, Hillyer C, eds. Technical Manual. 16th ed. Bethesda, MD: American Association of Blood Banks (AABB); 2008. [3]

Manual tube testing

An individual's red cells (in a 2%-5% saline suspension) are mixed in a test tube with anti-D reagent. The specimen is mixed gently. After centrifugation, the pelleted cell button is gently resuspended and examined for agglutination. Agglutination indicates a positive reaction. The reaction is graded on a scale of 0 to 4+. See the following image.

Manual tube testing. Image created by Jay Parsley. Manual tube testing. Image created by Jay Parsley.

Column agglutination ("gel," "gel card")

An individual's red cells are initially mixed in a small gel-filled tube that contains reagent anti-D. The specimen is centrifuged and assessed for agglutination. Agglutination seen at the top indicates a strong positive reaction; agglutination seen at the bottom signifies no reaction. The reaction is graded on a scale of 0 to 4+. See the image below.

Column agglutination "gel card." Image created by Column agglutination "gel card." Image created by Jaye Parsley.

Solid phase test systems (microplate test)

Commercial anti-D reagent is embedded in the bottom of micoplate wells. An individual's red cells (in a 2%-5% saline suspension) are mixed together with an enhancing reagent and centrifuged. The plates are then assessed. If there is just one tightly packed red cell pellet at the bottom of the well, there is no reaction. If there is a covering of red cells throughout the bottom of the well, this would indicate a positive reaction. The reaction is graded on a scale of 0 to 4+.

See table 3 below and the images above for further description of conventional agglutination grading.

Table 3. Standard Agglutination Grading (Open Table in a new window)

Marsh Score

Conventional Grading

Description of Grading



Complete agglutination



Strong reaction: 2-3 clumps



Strong reaction: several clumps



Many clumps



Scattered agglutination



Weak granularity



Even cell suspension