Lecture 8 - The Rhesus System (Part 2) PDF

SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 5 Lecture 8- The Rhesus System (Part 2) 5.5 D Antigen The D antigen, found exclusively on RBCs, is very immunogenic as evidenced by the high rate of hemolytic disease of the fetus and newborn (HDFN) due to anti...

SCIE 2040 – Transfusion Medicine 1 Textbook Chapter 5 Lecture 8- The Rhesus System (Part 2) 5.5 D Antigen The D antigen, found exclusively on RBCs, is very immunogenic as evidenced by the high rate of hemolytic disease of the fetus and newborn (HDFN) due to anti-D that was observed before Rh immune globulin prophylaxis became available. Rh(D)-negative recipients should receive red blood cells containing products that lack the D antigen to avoid alloimmunization. Rh(D)-positive individuals may receive either Rh(D)-positive or Rh(D)- negative blood products containing red blood cells. The D antigen does not express dosage in the traditional sense, however the number of D antigen sites on each red blood cell varies among phenotypes: Among those commonly encountered, the R2R2 phenotype possesses the most D antigen sites, with 16,000 – 33,000 sites per red blood cell. The Rh phenotype demonstrating the greatest overall number of D sites is caused by a partial deletion. The rare D– – (“D dash dash”) cell has about 100,000–200,000 D antigen sites. These individuals have undergone a CE deletion, and thus express no CcEe antigens. 5.6 Weakened Expression of D Most Rh(D)-positive red blood cells give easily readable, macroscopic reactions when tested with anti-D. However, some individuals’ cells may appear to be Rh(D)- negative with some anti-D reagents and positive with others. Others type as Rh(D) negative unless the Rh typing is taken through the antiglobulin phase of testing. There are several mechanisms by which D antigen expression could be reduced or altered so that it is not detected by all anti-D reagents. 5.6.1 C in Trans Position to D The RHC gene inherited in the trans position (on the opposite chromosome) to the RHD gene has a suppressive effect on D antigen expression. For example, the genotype R0r’ (Dce/dCe). The D antigen produced is functionally normal but decreased in number. These individuals do not produce anti-D. RHC gene in the cis position (on the same chromosome) as the RHD gene has no suppressive affect. 5.6.2 Weak D Formerly known as Du, weak D results from the inheritance of a mutated RHD gene that codes for a reduced number of D antigens expressed. Weak D is found more commonly among people of African ethnicity. The most common form of weak D identified in people of European ethnicity is known as weak D, type 1. ML2401 Course Notes Chapter 5: Rh System (Part 2) Fall 2021 Weak D is generally a quantitative deficiency of the D antigen, and some amount of normal D antigen is still expressed. Most types of weak D will still type as Rh(D)+ using sensitive modern reagents, and most are unlikely to develop an anti-D. 5.6.3 Partial D Most partial D (formerly known as D-mosaic) phenotypes are due to an exchange of genetic information between RHD and RHCE genes. This results in a qualitative alteration of the D antigen where it lacks one or more epitopes found on normal D antigens. The most common partial-D type in people of European ethnicity is partial D, type VI. The partial D phenotype types as Rh(D) positive or negative with modern reagents depends on the clone(s) used in the reagent. Partial D individuals may produce anti- D against an epitope that they lack. This antibody will react with most other D- positive red blood cells but not their own. 5.6.4 Weak D Test Weak D testing involves Rh(D) typing taken beyond immediate spin through to an antiglobulin test phase using anti-D reagents that have an IgG component. Weak D testing must be performed on all blood donors appearing Rh(D)-negative: If a donor types as D+ routinely or by weak D test, the unit is labeled as Rh(D) positive. Only when the routine D typing and weak D tests are negative is the unit given the Rh(D)-negative designation. This is necessary as weak D or partial- D donor red cells are still potentially capable of immunizing a Rh(D)-negative recipient. Weak D test is not routinely performed on patients as, in general, a weak or partial- D transfusion recipient should be considered Rh(D)-negative. Some of these individuals can develop an anti-D (especially partial-D individuals) so these patients are transfused Rh(D)-negative RBCs to avoid possible alloimmunization. Antisera used in routine patient typing is intentionally of a limited specificity to avoid typing a weak/partial D patient as D-positive. Patients that demonstrate a weak or negative reaction on initial Rh testing undergo confirmatory re-typing using a broadly reactive anti-D reagent. Prenatal patients and women of child- bearing potential with a suspected variant D expression based on this testing may undergo Rh genotyping to distinguish weak D from partial D. However, weak D testing is still performed on Rh(D)-negative appearing neonates who are born to an Rh(D)-negative mother to determine alloimmunization risk. Weak D testing may also be used to resolve discrepancies between current and previous results. 2 ML2401 Course Notes Chapter 5: Rh System (Part 2) Fall 2021 5.7 Rh Antisera 5.7.1 Clonal Antisera Historically, reagent antisera, including anti-D, were made from the plasma of people who had formed the antibody of interest. However, clonal antisera has replaced human source antisera in most cases. A human plasma cell that produces anti-D may be combined with a mouse cancer cell to form a hybridoma producing monoclonal anti-D, specific for a singular epitope of the D antigen. Reagent anti-D is usually a blend of monoclonal antibodies so that the reagent can detect more than one epitope of the D antigen. This ensures that more partial-D phenotypes are identified, which is especially important when typing donors. Clonal reagent anti-D may be IgG or IgM or may be a blend of both. 5.7.2 High- and Low-Protein Human Antisera In the past anti-D and other Rh reagents made from human plasma were divided into high-protein and low-protein formulations. High-protein anti-D reagents were historically formulated with IgG anti-D. To allow agglutination without the need for an antiglobulin phase, 22% albumin (along with other macromolecules) were added to the reagent. This would help the red blood cells come close enough together for the IgG anti-D to span the distance between them and cause agglutination. However, cells already coated with IgG (i.e. if the patient has a positive DAT) would also agglutinate. Therefore, it was important to use a control to identify potential false-positive reactions. The control contains everything in reagent antisera except the anti-D. The Rh type is only valid if the control gives a negative reaction. Low-protein formulations, often called “saline” antisera, use either: an IgG anti-D that had been chemically modified to permit a wider span of the IgG, or an IgM anti-D This allows the anti-D to cause visible agglutination of Rh(D)-positive cells without the need for antiglobulin or high concentrations of protein. Procedures involving low-protein medium, including modern monoclonal blend reagents, often do not explicitly require a parallel control as spontaneous agglutination in these mediums is unlikely. However, a positive control can still be included, and must be assessed for a patient exhibiting a positive reaction against all reagents tested (for example, a patient appearing AB, D+) or if taking the Rh typing to AHG phase in a weak D test. 3 ML2401 Course Notes Chapter 5: Rh System (Part 2) Fall 2021 5.8 Other Cell Antigens 5.8.1 C/c and E/e The C, c and E, e antigens are co-dominate alleles, thus if the gene is present for the antigen it will be expressed on the red blood cells. The C/c antigens arise from a single amino acid difference at position 103, whereas the E/e antigens are different at position 226: These antigens express dosage and are less immunogenic than the D antigen. The immunogenicity of the common Rh antigens in order of ability to stimulate an antibody response is: (most immunogenic) D>c>E>C>e (least immunogenic) 5.8.2 G The G antigen is almost always found on red blood cells that express the D antigen or the C antigen or both. Red cells that lack both D and C antigens are almost always G-. Anti-G will look like a combination of anti-D and anti-C. In some cases, further investigation is needed to distinguish between the two possibilities. 5.8.3 Cw Initially thought to be an allele of the C/c locus, it was later demonstrated that Cw may be present with or without the C and c antigens. Cw is antithetical to the high- incidence antigen MAR. The Cw antigen is found in about 2% of people of European ethnicity and is very rare in people of African ethnicity. Anti-Cw may develop in Cw- patients after exposure, through pregnancy or transfusion, to red blood cells carrying the antigen. 5.8.4 f (ce) When both c and e antigens are present on the red blood cell and the respective genes are in the cis position the f antigen is expressed. Therefore, red blood cells of the Dce/dCE genotype will have the f antigen, while red blood cells of the DcE/dCe will be f negative, even though both cells have a similar Rh phenotype. 5.8.5 Ce When C and e are found in the cis position the Ce (formerly rhi) antigen is also present. Thus, an anti-Ce will tend to react against red cells from an individual that has a R1 (DCe) or r’ (dCe) haplotype. 4 ML2401 Course Notes Chapter 5: Rh System (Part 2) Fall 2021 5.9 Rh Antibodies Most Rh antibodies are of the immunoglobulin class IgG. They may be detected after incubation at 37° C, but many may not react until the antiglobulin phase of testing. Reactivity of Rh antibodies is enhanced when incubated with enzyme- treated red blood cells. 5.9.1 Alloantibodies Rh antibodies are red blood cell stimulated; the patient must be exposed to the antigen before the antibody will be formed. Exposure to red blood cells can occur during pregnancy or through transfusion of blood products that contain red blood cells. Many Rh antigens are very immunogenic, with the Rh(D) antigen being the most immunogenic. Even exposure to amounts of Rh(D)-positive blood less than 1 mL may stimulate anti-D formation in an Rh(D)-negative patient. Once Rh antibodies are formed they tend to remain in circulation for long periods of time and are detected by routine antibody screening methods. These antibodies do not bind complement but cause extravascular hemolysis when the IgG-coated red blood cells are removed from circulation. Individuals who develop one Rh antibody are more prone to develop other Rh system antibodies. For example, R1R1 (DCe/DCe) individuals who develop and anti- E will often also develop and anti-c. 5.9.2 Autoantibodies Sometimes developing autoantibodies have apparent Rh specificity. Unlike patients with alloantibodies, many patients with an autoantibody show no difference in red blood cell survival whether antigen-positive or antigen-negative cells are transfused. 5