Pretransfusion Compatibility Testing


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Subject: Pretransfusion Compatibility Testing

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Definition and Use

  • Demonstration of RBC antigen–antibody reactions is the foundation for pretransfusion compatibility testing and the key to immunohematology. Agglutination is the end point for most of these tests (including performing the blood type, the antibody screen, and the crossmatch). Increased genetic knowledge has added a new approach to red cell antigen typing using DNA sequence determination, but these tests are currently not being used for routine blood bank testing, and the tests described in this chapter are based on the classical agglutination methodology.

  • Three major requirements must be satisfied for safe RBC transfusions:

    • The RBCs to be transfused must be ABO compatible

    • RhD-positive RBCs should not be given to RhD-negative patients

    • Transfused RBCs should lack blood group antigens to which the patient has preexisting clinically significant antibodies

  • To achieve these objectives, pretransfusion compatibility testing begins with the type and screen, where the recipient's ABO group and Rh (D) typing is determined. Antibodies to the ABO antigens are naturally occurring and are used to determine a person's ABO blood group. Next, the patient's serum is screened for the presence of clinically significant (non-ABO) antibodies directed against other blood group antigens. If an antibody is detected by the antibody screen, an antibody identification panel must be performed to identify the antibody.

  • Safe transfusion can be ensured for most recipients by the correct ABO and Rh typing of patients and donors.

ABO Group Determination

  • The forward and reverse ABO blood grouping of all patients must be determined prior to the transfusion of blood products using commercial reagents and the patient's red cells and serum (or plasma).

  • The forward ABO blood grouping is determined by checking the patient's (or donor's) red cells for the presence of A and B antigens using commercial anti-A and anti-B reagent antibodies.

  • The reverse ABO grouping is determined by checking the patient's (or donor's) serum for the presence of anti-A and anti-B antibodies using commercial reagent red cells.

  • The strength of the agglutination during testing is usually graded.

  • A group A individual has anti-B but not anti-A antibodies. A group B individual has anti-A but not anti-B antibodies. A group AB individual has neither anti-A nor anti-B antibodies. The serum of group O persons contains both anti-A and anti-B antibodies.

Rh Typing

  • The Rh (D) type of all patients should be determined prior to transfusion or if the patient is pregnant in order to prevent immunization to the D antigen and production of the anti-D alloantibody. Anti-D is a clinically significant antibody that can cause hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN).

  • The Rh typing is performed by testing the RBCs for the presence of D antigen using anti-D reagent antibodies and checking for agglutination.

  • Some patients may not show clear agglutination after centrifugation with anti-D but still have the D antigen. This is known as weak D and requires the addition of AHG for identification of the D antigen. These weak D patients are still considered Rh positive.

  • Weak D testing is not required for patients but is required for donors.

  • In practice, the terms Rh positive and Rh negative, respectively, refer to the presence or absence of the D antigen and routine pretransfusion tests only include testing for the D antigen. However, in addition to the D antigen, there are many other antigens in the Rh blood group system. When alloantibodies are present, it may be necessary to type patients and/or donors for these (other) Rh antigens, most commonly C, E, c, e.

  • Antibodies to Rh antigens are immune stimulated in most cases, mostly following pregnancy or transfusion.

Antibody Screening

  • Antibody screening is used to detect the presence of unexpected alloantibodies in the recipient's serum, directed against non-ABO blood group antigens (e.g., Kell, Duffy, and Kidd). This is accomplished by using commercially available screening RBCs. Generally, an IAT is performed using the patient's serum and two or three group O RBCs with known but varied blood group antigens.

  • If agglutination is detected in the antibody screen, the antibody must be identified and antigen-negative RBCs must be selected for transfusion.


  • The crossmatch assay involves testing the patient's serum with the donor RBC taken from a segment attached to the selected blood unit. Unless there is a very urgent need for blood, crossmatching is mandatory. The method used must be able to demonstrate incompatibility to ABO and other clinically significant RBC antibodies.

  • If the recipient/patient has a negative antibody screen, an abbreviated (immediate spin) crossmatch is adequate. However, if the patient has a positive antibody screen or there is a previous history of alloimmunization, antigen-negative donor units must be selected and a full Coombs crossmatch must be performed (by incubating the patient's plasma and the donor red cells at body temperature and then adding AHG).

  • An immediate spin crossmatch is one additional means of ensuring that the patient receives ABO compatible red cells as ABO antibodies will cause agglutination without incubation at body temperature and addition of AHG.

  • In order to detect many of the clinically significant non-ABO antibodies, a Coombs crossmatch is necessary as agglutination will not be seen without incubation at body temperature and addition of AHG.


  • Acquired antigens, such as “acquired B” antigens in group A individuals.

  • Forward and reverse typing discrepancies. When they occur, the cause must be immediately investigated. The most common cause is a patient who is a subgroup of A and who has formed anti-A1 antibodies (80% of group A patients are subgroup A1). Most of the remainder are A2 and may develop anti-A1 antibodies.

  • Warm and cold autoantibodies can interfere with pretransfusion testing.

  • Pretransfusion testing may be complicated in recently transfused and marrow transplant patients.