Updated: Jan 22, 2019
  • Author: Ashok Tholpady, MD, MSc; Chief Editor: Jun Teruya, MD, DSc, FCAP  more...
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When blood products are ordered to be administered to a patient who requires transfusion, a series of tests are performed to decrease the risk of an immune-mediated hemolytic reaction caused by incompatible blood.

Hemolytic transfusion reactions occur when the recipient's immune system encounters antigens from donor blood. Antibodies may form in response to these antigens, resulting in destruction of donor red blood cells (RBCs), with sequelae leading to clinical manifestations of fever, hypotension, rigors, acute respiratory failure, and acute renal failure. [1] (Antibody Screening is discussed in a separate article.)

The type and screen are the first pretransfusion compatibility tests performed, and they are used to identify the patient's ABO group and Rh type as well as to detect expected and unexpected antibodies in the patient's serum, respectively. [2]

The crossmatch is the final step of pretransfusion testing as a routine procedure. A portion of donor blood is combined with patient plasma or serum and is checked for agglutination, which would signify incompatible blood. This important step, also known as major crossmatch, serves as the last guard to ensure a safe transfusion. [1]


Clinical Indications/Applications

The crossmatch is routinely used as the final step of pretransfusion compatibility testing. It serves two purposes: (1) to serve as a final check of ABO compatibility between donor red blood cells (RBCs) and patient plasma or serum and (2) to detect clinically significant antibodies that may have been missed by the antibody screening test.


Test Performance

Serologic crossmatch

To perform the serologic crossmatch, two samples must be collected, as follows:

  • Donor red blood cells (RBCs) from a "segment of tubing originally attached to the blood unit container" [3]

  • The patient's plasma or serum

An immediate spin phase is performed with donor RBCs prepared as a 2%-5% suspension in normal saline or ethylenediaminetetraacetic acid (EDTA) saline and the patient's serum.

Both samples are mixed together and centrifuged at room temperature to visualize hemolysis or agglutination, which would signify a positive test result and the presence of an additional antibody. Crossmatch-incompatible blood is rarely encountered, because donor blood units are selected after an effective antibody screen. When only the type and screen are combined with the immediate spin phase, this is known as an abbreviated crossmatch [4] and it is 99.9% effective in preventing an incompatible transfusion. [5]

The benefits of the abbreviated crossmatch include decreased cost and quicker blood availability. Further testing is unnecessary if no antibodies are identified with the immediate spin phase. However, if agglutination occurs during the immediate spin phase, one of the following situations may have occurred:

  • ABO-incompatible donor RBCs

  • Polyagglutinable donor RBCs

  • An A2 or A2 B recipient who has serum anti-A1 antibodies

  • Cold autoantibodies

  • Passively acquired anti-A or anti-B

  • An antibody reactive at room temperature (anti-M)

When the immediate spin phase is positive for agglutination or hemolysis, further testing is required. The donor unit is deemed incompatible for transfusion, and the antibody responsible for the reaction should be identified. The next phase of testing consists of the antiglobulin crossmatch, which may be performed via column agglutination, solid-phase systems, or tube. The antiglobulin crossmatch is the major component of a full serologic crossmatch.

Gel testing. Agglutination is graded on a scale fr Gel testing. Agglutination is graded on a scale from 0 to 4+. A: 4+ reaction = red blood cell agglutinates (RBCAs) remain at the top of the gel. B: 3+ reaction = RBCAs remain in the top half of the column. C: 2+ reaction = RBCAs are scattered throughout the column. D: 1+ reaction = RBCAs are primarily in the lower half of column. E: 0 = no agglutination and red blood cells pass all the way to the bottom.

In an antiglobulin crossmatch performed using the column agglutination system, the donor RBCs, suspended in a hypotonic buffered saline solution, are mixed with the recipient’s serum or plasma to allow antigen-antibody interaction in the upper chamber of the microtube. The antibody in the recipient’s serum or plasma is detected when the sensitized donor RBCs react with the anti–immunoglobulin G (IgG) in the microtube during centrifugation. Agglutination or hemolysis constitutes incompatibility between the donor RBC unit and the recipient’s serum. As shown in the image above (gel testing), the migration of donor RBCs through the tube is graded between 0 (representing no agglutination) to 4+ (the maximum amount of agglutination).

Before performing tube testing, it is advantageous to wash and resuspend donor RBCs in 2%-5% saline to remove small fibrin clots and some cold agglutinins. The ratio of patient plasma or serum to donor RBCs is also important, as too many donor RBCs could result in a false-negative result if there are not enough antibodies binding to the RBCs to cause a reaction.

The crossmatch incompatibility detected in antiglobulin crossmatch can be associated with the following conditions:

  • Presence of an alloantibody or alloantibodies to RBC-specific antigen(s) on the donor unit(s) that has/have not been identified, especially for antibodies that demonstrate dosage and donor RBCs from heterozygotes or donor RBCs carrying a low-incidence antigen
  • Presence of passively acquired anti-A and anti-B antibody

  • Presence of cold or warm-reactive autoantibody

  • Presence of antibody to an ingredient in the enhancement media or enhancement-dependent autoantibody

  • Positive direct antiglobulin test result of donor RBCs

Electronic or computer crossmatch

In the computer crossmatch, there is no mixing of the donor RBCs and patient serum. Instead, a computer verifies the ABO/Rh compatibility. [4, 6] This procedure may be used in lieu of an immediate-spin phase and antiglobulin crossmatch if certain criteria are met, as follows:

  • The patient's plasma or serum must not have current clinically significant antibodies or a history of clinically significant antibodies found via antibody detection test.

  • The patient's ABO blood group must be typed twice.

  • The computer system must be US Food and Drug Administration (FDA) approved and validated on site. It must contain the unit number, component name, ABO group/Rh type and the recipient's ABO group/Rh type, and antibody screen results.

  • The system must be able to alert the user to ABO/Rh–incompatible blood and must not issue the donor unit until its ABO/Rh is verified.



The following are limitations to crossmatching [6] :

  • Does not prevent delayed transfusion hemolytic reactions with transfusion of compatible blood if an antibody titer is not high enough to detect

  • Does not detect Rh incompatibility: Plasma from an Rh-negative recipient may not react with Rh-positive donor RBCs in the absence of anti-D antibodies

  • Does not detect minor ABO incompatibility (eg, group A donor blood given to group AB recipient)

  • Does not gurantee normal survival of the transfused red blood cells



The tests performed during the crossmatch vary depending on the results of the antibody screening test and patient history. A full serologic crossmatch is required in patients with a history of clinically significant antibodies found with previous testing or found in the current blood sample. The crossmatch may be performed with either computer or serology.

See also the Methods section in Antibody Screening.