What are the phases of an antibody screening test procedure?

Updated: Jun 24, 2019
  • Author: Ashok Tholpady, MD, MSc; Chief Editor: Jun Teruya, MD, DSc, FCAP  more...
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The procedure is separated into 3 phases: immediate spin, 37°C, and AHG.

The purpose of the immediate spin is to detect "cold" antibodies, usually of the IgM class. A drop of RBC suspension from each set of the screening cells is placed into a centrifuge tube and mixed with 2 drops of the recipient's plasma. The tubes are then spun for 15 seconds at room temperature to facilitate antigen-antibody interaction. Resuspension of the pellet allows for observation of agglutination or hemolysis.

After immediate spin, the tubes are incubated at 37°C. To promote the detection of warm reactive antibodies, especially of the IgG class, additional enhancement techniques such as low ionic strength saline (LISS) and polyethylene glycol (PEG) are often used. LISS is usually added to reduce clustering by Na+ and Cl- ions and speed antigen-antibody attraction. With the addition of LISS, incubation times can be reduced from 30-60 minutes to 10 minutes. [1, 2] PEG, a water-soluble linear polymer, appears to accelerate antibody-RBC binding by steric exclusion of water molecules in the diluents and to promote antibody detection.

AHG (indirect antiglobulin test [IAT], indirect Coombs): The tubes are washed 3-4 times with saline to remove any unbound globulins, and AHG is added to each tube. AHG is an animal antibody that binds to the Fc portion of human immunoglobulin. The AHG detects bound RBC antibodies that do not produce direct agglutination (sensitizing antibody). The presence of agglutination with the addition of AHG indicates antibody binding to a specific red cell antigen.

The last 2 phases (37°C and AHG phases) are necessary to detect clinically significant IgG antibodies.

With column (gel) agglutination (see Methods), the procedure varies by the type of test to be carried out (typing or screening). Patient plasma and reagent RBC suspension are placed at the top of the tube and then centrifuged.

With the solid-phase red cell adherence assay (SPRCA), in the first step, a combination of patient plasma and LISS is added to the reagent RBC-coated plate and incubated for a predetermined time (usually 15-60 minutes) to allow binding between antibody and antigen, if present. Afterward, the plate is washed multiple times with phosphate-buffered saline to remove unbound proteins. In the final step, a suspension of indicator RBCs with attached anti-IgG antibodies are added to the well and centrifuged.

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