Rh Blood Group System

Fisher and Race¹² postulated that the Rh blood group system antigens were inherited as a gene complex or haplotype that codes for three closely linked sets of alleles. The D gene is inherited at one locus, C or c genes are inherited at the second locus, and E or e genes are inherited at the third locus. Each parent contributes one haplotype or set of Rh genes. Fig. 6.1 illustrates this concept. Each gene expresses an antigen that is given the same letter as the gene. When referring to the gene, the letter is italicized. For example, the gene that produces the C antigen is C. Each red cell antigen can be recognized by testing with a specific antibody. The original theory assumed the d allele was present when the D allele was absent. According to the Fisher-Race theory, the order of the genes on the chromosome is DCE; however, it is often written alphabetically as CDE.

In contrast to the Fisher-Race theory, Wiener¹³ postulated that alleles at one gene locus were responsible for expression of the Rh blood group system antigens on red cells. Each parent contributes one RH gene. The inherited form of the gene may be identical (homozygous) to or different (heterozygous) from each parent. According to Wiener, eight alleles exist at the RH gene locus: R⁰, R¹, R², R^z, r, r′, r″, and r^y. The gene encodes a structure on the red cell called an agglutinogen, identified by its parts or factors. These factors are identified with the same antisera that agglutinate the D, C, c, E, and e antigens mentioned earlier in the Fisher-Race nomenclature. The difference between the Wiener and Fisher-Race theories is the inheritance of the Rh blood group system on a single gene locus rather than three separate genes. The antigen complex or agglutinogen comprises factors that are identifiable as separate antigens (Table 6.2). For example, in Wiener terminology, the R¹ gene codes for the Rh₁ agglutinogen, which is made up of factors Rh₀, rh′, and hr″ that correspond to D, C, and e antigens, respectively. The r gene codes for the rh agglutinogen, made up of factors hr′ and hr″ that correspond to c and e antigens, respectively. The longhand factor notations of Rh₀, rh′, hr′, rh″, and hr″ that correspond to D, C, c, E, and e antigens, respectively, are outdated and rarely used.

Both the Wiener and the Fisher-Race terminologies are based on genetic concepts. The Rosenfield system was developed to communicate phenotypic information more suited for computerized data entry; it does not address genetic information.¹⁴ In this system, each antigen is given a number that corresponds to the order of its assignment in the Rh blood group system. A red cell’s phenotype is expressed with Rh followed by a colon and the numbers corresponding to the tested antigens. If a red cell sample is negative for the antigen tested, a minus sign is written before the number. For example, red cells that tested D+, C+, E−, c+, e+ would be written as Rh:1,2,−3,4,5. Table 6.1 compares Fisher-Race and numeric terminology.

Most red cells can be phenotyped for the D antigen directly with anti-D commercial reagents. Although the antibody to D antigen is typically of the IgG class, reagent manufacturers have developed monoclonal anti-D antibodies for concurrent use with anti-A and anti-B testing. When the D antigen is weakly expressed on the red cell, its detection may require the indirect antiglobulin test (IAT) (Fig. 6.6). Red cells that are positive for D only by the IAT are referred to as weak D. Recently the serologic weak D definition has been modified to reactivity of RBCs with anti-D reagent giving no or weak (≤2+) reactivity in initial testing with moderate or strong reactivity with antihuman globulin (AHG). Table 6.6 shows the interpretation of this test, which must always include a control. The Rh or D control is a reagent made by manufacturers that consists of all additives except the D antibody. It is used to determine whether agglutination by anti-D at immediate-spin is false positive, which could be due to the reagent additives, such as albumin. The Rh or D control tested at the antiglobulin phase determines whether patient cells are already coated with IgG antibodies before testing. Reagent manufacturers specify the use of controls in their package inserts, and it is important to become familiar with these guidelines. Chapter 3 discusses Rh reagents in detail. If the control is positive, additional serologic techniques may be required.

Weaker expression of the D antigen can be found when the Ce (r′) gene is inherited in trans to the RHD gene (Fig. 6.7). Genes inherited in trans are inherited on opposite chromosomes. The Ce (r′) gene paired with a CDe (R¹) or a cDe (R⁰) gene weakens the expression of the D antigen. Weak D antigen due to position effect is usually detected with monoclonal anti-D reagents because of the increased sensitivity of monoclonal reagents. The occurrence of the Ce (r′) gene is less than 2%.

Although rare, some individuals who are positive for the D antigen can make an alloantibody that appears to be anti-D after exposure to D-positive red cells. Investigation of this phenomenon revealed that some D-positive cells could be missing parts of the D antigen complex. When these individuals are exposed to the “whole D” antigen, they can make an antibody to the part they are missing. In the past, the partial D phenotype was termed D variant or D mosaic. Lomas et al.²⁰ established nine partial D phenotypes, which are classified by their parts or epitopes. The nine-epitope model was later expanded to accommodate new reaction patterns with different monoclonal anti-D reagents.²¹ Red cells of most partial D phenotypes react as strongly with monoclonal antibody anti-D reagents as red cells composed of the complete D antigen. For this reason, partial D phenotypes are infrequently detected. A partial D phenotype should be suspected if a D-positive individual makes an antibody that reacts with D-positive cells but is nonreactive with his or her own cells.¹⁹ In addition, the partial D phenotype should be suspected if two different manufacturers’ monoclonal anti-D reagents are used and the interpretation as D-positive or D-negative does not agree. In this circumstance, the clone used to manufacture the reagent may vary in its ability to detect all the epitopes or parts of the D antigen. The types of weak D are summarized in Table 6.7.

The AABB Standards requires testing for weak D on donor red cells that do not directly agglutinate with anti-D reagents using a method designed to detect weak expression of D.²² There is no requirement for a test that uses an IAT.¹⁹ Weak D–positive units are labeled D-positive and should be transfused only to D-positive recipients. A D control or an autocontrol is an important part of the weak D test because it verifies that a positive result is not due to red cells already coated with antibodies. If red cells are coated with IgG antibodies before testing with anti-D at the antiglobulin phase, the test is invalid and additional procedures are required to determine the D antigen status of the donor. Scan the QR code for more information.

The G antigen is located on red cells that possess C or D antigens. Red cells that are negative for both the D and the C antigen are negative for the G antigen. Because an antibody to G reacts with red cells that are either D-positive or C-positive, the specificity appears to be anti-D and anti-C. Antibodies to G appear as an anti-D plus an anti-C; however, the specificities cannot be separated. In other words, anti-G antibodies mimic the reactions observed with anti-D and anti-C antibodies. In some cases, a D-negative person may receive D−, C+ red cells and appear to produce anti-C as well as anti-D. The antibody produced in this case was most likely anti-G. Distinguishing anti-G, anti-D, and anti-C antibodies requires adsorption and elution procedures.¹⁸ Extensive testing to identify anti-G is not usually necessary. Individuals making anti-G or what appears to be anti-D or anti-C should receive red cells that are negative for both D and C antigens. Rare cells exist that are negative for D and positive for G (r^G). G is not a compound antigen; G is present when D or C is inherited. Scan the QR code for more information on G antigen.

Rare Rh phenotypes demonstrate no reactions when the red cells are tested with anti-E, anti-e, anti-C, or anti-c. Genetic material was deleted or rendered nonfunctional at the RHCE site. Red cells that lack C/c or E/e antigens may demonstrate stronger D antigen activity (see Fig. 6.5). Individuals who have the “D-deletion” phenotype may produce an antibody that reacts as a single specificity (anti-Rh17) or separable specificities such as anti-e and anti-C. An individual who produces anti-Rh17 would require D-deleted RBC units if transfusions are necessary. The D-deletion phenotype is written as -D- or D—.

The Rh_null phenotype appears to have no Rh antigens and can be produced from two distinct genetic mechanisms. Cells that type as Rh_null have membrane abnormalities that shorten their survival and cause hemolytic anemia of varying severity.²³ Antibodies produced by immunized individuals who lack all Rh antigens may be directed to “total-Rh” (Rh29) or to an individual Rh-antigen specificity. If an anti-Rh29 is detected, Rh_null cells are needed for transfusion. Donations from siblings, autologous donations, and donations from the rare donor registry could be potential sources of compatible RBC units.