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Test Methods

One of three methods is used to detect and identify antibodies:
  • Tube
  • Gel
  • Solid phase
The Tube method consists of three phases:
Immediate spin (IS)
Patient serum or plasma and reagent red cells are combined, then centrifuged, and observed for agglutination. Enhancement media such as albumin, LISS or PEG, can be added.
37° C incubation
The tubes are incubated at 37°C for 15-60 minutes, depending on the enhancement media used. After the incubation period, the tubes are centrifuged and observed for agglutination. The cells are washed 3-4 times to remove any unbound antibody. This is an important step in the tube method. Improper washing may lead to false-negative results.
Indirect antiglobulin test or Anti-human globulin (AHG)
AHG is then added, the tubes are centrifuged, and are observed for agglutination. Check cells are added to all negative tubes. Check cells are cells coated with IgG and should react positively with the AHG in the tube. If check cells are negative, the procedure was not performed correctly and should be repeated.
Gel cards are used in the Gel method. The cards have microtubules filled with a dextran acrylamide gel containing anti-IgG. The gel acts as a filter for agglutinates. Patient serum and reagent red cells are added to the microtubules. The card is incubated at 37° C for 15 minutes and then centrifuged for 10 minutes. Washing and check cells are not required. If no agglutinates are present, the red cells form a pellet at the bottom of the gel, indicating that no clinically significant antibodies were detected. Agglutination indicates an antibody was detected. If large agglutinates are present, the red cells form a layer at the top of the gel. Small agglutinates are dispersed throughout the bottom of the gel. A gel card is depicted below.
In the Solid phase method, RBC antigens coat microtiter wells. Patient serum or plasma is added to each well along with a low ionic strength solution (LISS). The wells are incubated at 37°C, and washing is then required. Instead of AHG, indicator cells (cells coated with anti-IgG) are added. If sensitization occurred, indicator cells react with the antibody coating the well and form a diffuse pattern. This is a positive reaction indicating an antibody is present. If no sensitization occurred, indicator cells form a pellet at the bottom of the well. This is a negative reaction indicating no clinically significant antibodies are present. The image below illustrates negative and positive reactions.
Increased sensitivity in detection of antibodies is seen when using the tube method with PEG or the gel method (especially for mixed field reactions). Solid phase testing has a better sensitivity than just using the test tube method with LISS.