Blood System Portfolio: Rhesus System

The Rhesus blood system is arguably the second most important blood system behind the ABO system. There are 50 defined blood group antigens, among which the five antigens; D, C, E, c, e are the most significant. Individuals who are Rh positive possess the D antigen and those who are Rh negative lack the D antigen. Antibodies to Rh antigens play a major role in hemolytic transfusion reactions and cause significant risk for hemolytic disease of the fetus and newborn (HDFN).


The gene locus for the Rh system antigens is located on chromosome 1. There are two genes that are closely related. RHD is a 417 amino acid sequence membrane protein that encodes for the D antigen. RHCE codes for a different membrane protein that carries the C/c and E/e antigens. A third gene, RHAG, located on chromosome 6 is associated with the expression of RHD and RHCE membrane proteins. RHAG NEEDS to be expressed for RHD and RHCE to be expressed. The Rh antigens are membrane bound non-glycosylated proteins (meaning that there is no carbohydrate attached) involved with membrane transport of cations. An individual who is C instead of c has a difference found in amino acid position 103, where C has a serine and c has a proline. An individual who has E antigen possesses a proline at amino acid position 226, and an individual who has the e antigen has an alanine at amino acid position 226.


The Rhesus blood system was discovered in 1937 by Karl Landsteiner and Alexander S. Wiener who named it the “Rhesus factor” because they believed it resembled an antigen found on rhesus monkey red cells. It was soon after that it was discovered that the human factor (Rh) is not at all similar to antigens found on the red cells of the rhesus monkey, although it stands today as a misnomer. Today in the United States 85% of the population are Rh positive and 15% are Rh negative. 70% of the population has the C antigen, 30% have the E antigen, 80% have the c antigen, and 98% have the e antigen. The Rh system currently has two sets of nomenclatures, one which was discovered by Ronald Fisher and R.R. Race, and the other by Alexander Wiener. Both systems are based on alternate theories which have both been since proved partially correct. The Fisher-Race system operates on the theory that separate genes control the product of each corresponding antigen. The Wiener system is based on the theory that there was a single gene on a single locus on each chromosome that gave rise to multiple antigens. Testing today shows that there are two genes that control the Rh system. The first one; RHD gene which produces a single antigen (D) and immune anti-D, and the RHCE gene which synthesizes the C, c, E, e antigens and corresponding antibodies.

Rh Testing

Some individuals can have a weak expression of the D antigen. They are Rh positive, but it is difficult to detect the presence of the antigen on the red cells. They require more sensitive methods of detection using anti-human globulin which is a poly specific CD3-IgG antibody reagent. It enhances the antigen-antibody complex formed so that agglutination is detected. Its important to detect weak D is cross-matching the donor and recipient blood samples especially when the recipient has anti-D in the serum. There are a few mechanisms for weak D expression. There can be a genetic weak D where a genetic variation of the D antigen is inherited. A partial D where the structure of the D antigen is made up of antigenic subparts where different D epitopes are missing or genetically altered.

Rh null is when there is absence of the RHAG gene. If individuals do not have a functioning RHAG gene there is no expression of genes RHD and RHCE and the corresponding antigens do not get expressed on the red cells. Red cell abnormalities have been observed with the phenotype including hemolytic anemia, decreased cell survival, stomatocytosis, spherocytosis, and altered activity of other blood group systems, most notably the MNS blood system.

Rh antibodies are IgG and are not detected at room temperature and need incubation at 37 degrees C. and the addition of a protein enhancement such as albumin or LISS to make detection more reliable. Anti-D is the most important antibody that can be formed. It takes just one exposure as the D antigen is extremely immunogenic. This typically happens through transfusion of antigen positive blood to an antigen negative recipient or through pregnancy and birth where there is maternal and fetal blood exchange where the mother gets sensitized. This is the basis of HDFN.

Important reminders regarding transfusion practice for the Rh system; Rh negative individuals should never receive Rh positive donor units. Rh positive individuals can receive Rh positive, but can in emergencies receive Rh negative. If there are Rh antibodies present, transfuse blood units that lack the Rh antigens to those antibodies. Sometimes its appropriate to phenotype and genotype a recipient or a donor. To do that the five different specific antisera is used to test for the five antigens that can be expressed. The purpose of Rh phenotypic and genotyping is to identify unexpected Rh antibodies, estimate the risk of HDFN in women, and in some cases can be used to exclude the male in paternity testing.