Genotyping RBCs

The Use of Genotyping in Transfusion Medicine

Required testing for red blood cell (RBC) transfusion includes serologic ABO and RhD typing, as well as screening for alloantibodies to “minor” (non-ABO and non-RhD) RBC antigens.

ABO and RhD typing can now be accomplished quickly and accurately using monoclonal antibody reagents.

To confirm the ABO type, all donors and recipients are tested for the presence of isoagglutinins directed against A and B antigens, as plasma from all immunologically normal individuals without an A or B antigen should contain such antibodies.

If a recipient’s RBCs type as A, the plasma from that recipient will have anti-B antibodies.

Anti-D is neither expected nor naturally occurring in D-negative individuals.

A screen for alloantibodies to other blood group antigens is done to avoid the mostly delayed hemolytic transfusion reactions that can be caused by such alloantibodies.

Blood donor centers and hospital-based laboratories use high-throughput automated testing instruments to ascertain ABO and Rh type, as well as to perform antibody screens designed to detect alloantibodies.

One to two percent of all patients who receive transfusions develop antibodies to RBC antigens.

In patients receiving chronic transfusion, the frequency of alloimmunization is higher, affecting ≈20 percent to 30 percent of recipients.

Most patients who develop alloantibodies do so before the 15th transfusion.

For patients with hematologic malignancies, the incidence of RBC alloimmunization is estimated at nine to 15 % ,despite the immunosuppressive effects of the chemotherapy.

Once RBC antibodies have been induced, 20-25 percent of patients form additional antibodies after subsequent transfusions and become multiply alloimmunized.

To detect alloantibodies to minor blood group antigens, most laboratories rely on the indirect Coombs’ test.

In the indirect Coombs’ method patient antibodies bound to erythrocytes of the test panel are detected by agglutination mediated by Coombs’ serum, a rabbit antiserum against human IgG and complement C3.

A specific antibody against human IgG can be used rather than Coomb’s serum to mediate the RBC crosslinking.

The successful identification of the target of the alloantibody is reagent dependent, and identification of the target antigen of a single alloantibody is relatively uncomplicated.

When multiple alloantibodies are present, a transfusion laboratory needs a supply of often rare reagent RBCs with unusual phenotypes to be able to characterize fully and accurately the spectrum of alloantibodies present.

Once the presence or history of alloimmunization has been documented, the availability of RBC units for transfusion may be severely restricted by compatibility issues, and the time necessary to identify compatible units may be prolonged.

The majority of banked blood has only been tested for the presence of ABO and RhD antigens.

When specific antigen-negative units are needed, the transfusion service must test donor units for the presence of the relevant antigens that must be avoided.

Large transfusion services, and some blood donor centers, genotype selected samples.

The single most common context outside hemoglobinopathies in which patients are genotyped is hematologic malignancies.

As many as 26 erythrocyte blood group antigen systems have been characterized at the molecular level.

Most minor blood group antigens are polymorphisms due to exchange of one amino acid, arising from a single nucleotide substitution in the encoding gene.

The MN polymorphism involves exchange of two nonadjacent amino acids.

For carbohydrate antigens, such as ABO and Lewis, the genetic mechanism of polymorphism resides in the alteration of genes encoding glycosyltransferases involved in synthesis of the antigenic oligosaccharides.

Some variant phenotypes are caused by more complex genetic changes, including intra- and intergenic exchanges, inversions, insertions, and deletions.

The RH system alone has 51 characterized antigens.

In addition, the two RH genes (RHD and RHCE) each have a large number of alleles, with more than 200 alleles recognized in RHD.

The incidence of developing antibodies per unit transfused is diminished 10-fold when selected donors phenotype matching are used for transfusion of patients with sickle cell disease.

For sickle cell disease patients, who frequently undergo transfusion and are frequently alloimmunized, genotypic data can clarify their alloimmune status and facilitate provision of compatible RBC units.

Individuals with autoimmune hemolytic anemia have an autoantibody that coats the patient’s own RBCs and typically reacts with all cell tested, interfering with patient blood group antigen phenotyping, identification of alloantibodies, and crossmatching.

Transfusion of genotypically matched blood is a safer practice than the use of traditional serological methods in patients with an IgG autoantibody.

Genotyping in the setting of recent transfusion can accurately determine the recipient’s blood group antigen genotype without concerns about contamination from donor-derived DNA.

Routine serologic methods for the prediction of risk for hemolytic disease of the newborn consist of performing a serologic screen for unexpected alloantibodies in the mother’s serum, and then titering any antibodies found.

Determination of the fetus’ genotype allows alloimmunized mothers whose fetuses cannot express the target antigen to receive routine prenatal management, without concern for HDN.

Genotyping of a father can define zygosity (e.g., of RhD), which can also determine the likelihood that the fetus has inherited a particular allele.

Delayed hemolytic transfusion reactions rarely cause more than transient clinical problems.

Delayed hemolytic transfusion reactions more common in patients with frequent transfusion requirements, limited capacity to produce RBCs, or chronic hemolysis.

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