Methods of Testing

Testing can be performed using different methods or techniques depending on local laboratory resources. In all cases, the presence of visible RBC agglutination following the addition of patient plasma is interpreted as meaning that the plasma contained an antibody against an antigen present on the RBC membrane. However, the actual appearance of this agglutination differs significantly depending upon the testing method used.


This is the traditional method in blood bank compatibility testing. In its simplest form, RBCs are incubated with plasma, centrifuged and washed with normal saline to remove any unbound antibodies from the solution, and then tested for agglutination using antiglobulin reagent: this is known as saline indirect antiglobulin testing, or testing by SIAT. Reactions are graded by the size of the RBC agglutinin seen.

Check cells, or RBCs deliberately coated with antibody, must be added to any negative reactions by tube testing, so as to confirm that the antiglobulin reagent wasn’t accidentally neutralized by residual antibodies that were not removed during the wash phase. If the check cells do not agglutinate, the test result is not valid.

Although use of test tubes requires larger sample volumes and is not as sensitive or gel- or solid-phase based testing (described below), it is more amenable to test modifications such as:

  • Performance at different temperatures
  • Addition of various enhancing and inhibiting substances such as polyethylene glycol (PEG) and dithiothreitol (DTT)
  • Cycling of the same sample through a progression of different testing procedures.

Gel microcolumn

In this method, antiglobulin reagent is embedded within a column of dextran acrylamide gel, with a small volume of low ionic saline solution (LISS) layered on top. RBCs and plasma are incubated together in the LISS, which serves to potentiate binding of antibodies to RBC antigens. The RBCs are then driven through the gel by centrifugation, which effectively separates them from any unbound antibody still present in the LISS layer.

If the RBCs were opsonized with antibodies during the incubation phase, they agglutinate during passage through the gel and become trapped within it. Reactions are graded by how high in the column the agglutinins are trapped. If all RBCs are centrifuged to the bottom of the column, the reaction is negative. Because there is no washing phase, the use of check cells is not required to confirm negative reaction.

This testing method is referred to as gel-LISS-indirect antiglobulin testing (GLIAT) and is faster, more sensitive, less prone to operator error and needs smaller sample volumes than tube testing and can also be performed in batches using automated devices. However, it is more expensive and less flexible than tube testing.

Solid phase red cell adherence assay (SPRCA)

In this method, testing is performed with microwells that have been pre-coated with fragments of RBC membrane. These wells are incubated with patient plasma and LISS to facilitate binding of anti-RBC antibodies, and then washed to remove unbound antibody.

Special indicator cells (RBCs coated with anti-IgG) are then added to the microwell then centrifuged. If antibodies have bound to the microwell, the indicator cells will be captured across the surface of the microwell; a negative reaction is reflected by a large “button” of unbound indicator cells at the centre of the microwell.

As opposed to tube testing, the complete absence of an RBC button in solid phase testing actually indicates a strongly positive reaction. SPRCA shares many of the same advantages of gel over tube testing but, like gel, is also less amenable to more advanced testing techniques.

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