The test tube method, as suggested by the name, occurs in a glass tube that provides sufficient surface area for optimal antibody-antigen interaction and macroscopic interpretation. Test tube method is often performed in three phases:
- Immediate spin (IS)
Patient serum or plasma and reagent red cells are combined. Some facilities then centrifuge and observe for agglutination (immediate spin readings are optional for routine testing). Enhancement media such as albumin or LISS can be added. Note that enhancement media will be discussed in the upcoming pages.
- 37°C incubation
The tubes are incubated at 37°C for 15–60 minutes, depending on the enhancement media used. Each stage of the process, such as using enhancement media, centrifugation, or changing the temperature conditions, is aimed at enhancing the binding of antibodies to antigens. After the incubation period, the test tubes are centrifuged. After centrifugation, a red cell button appears at the bottom of the test tube. The scientist must carefully dislodge the red blood cell button by gentle agitation and either observe for hemolysis or agglutination. Note that 37°C readings are optional for routine testing.
- Indirect antiglobulin test or Antihuman globulin (AHG)
After incubation, the patient plasma and reagent red blood cell mixture is washed 3–4 times to remove any unbound antibodies. A wash consists of adding saline to the mixture to roughly 3/4 of the test tube, centrifuging the contents, and discarding the supernatant. Cell washing is a critical step in the tube method. Insufficient or improper washing may impede the antihuman globulin reagent from reacting with the target antigen and can lead to false-negative results. AHG reagent is then added, the tubes are centrifuged, and are observed for agglutination.
Check cells are added to all negative tubes (tubes with no agglutination). Check cells are manufactured reagent red blood cells coated with IgG. Check cells serve as a control to verify that washing was thorough and IgG was properly added in cases where no agglutination is observed. As a result, check cells should react positively with the AHG in the tube. If check cells are negative, it indicates that the test should be performed again.
This image illustrates a setup of test tubes, representing an antibody identification panel. Each tube contains one drop of manufactured reagent red blood cells mixed with two drops of the patient's serum.
