Coombs Test - Procedure, Solid-phase test, Microcolum, Antiglobulin gel test

Anti-human reagents in Coombs Test

For the Coombs Test, there are three types of reagents that may be used:

• monospecific reagents to detect bound immunoglobulin G (IgG)

• monospecific reagents to detect bound complement (C3)

• polyspecific reagents that can simultaneously detect IgG and/or C3

For the step of screening RBCs, the most commonly used reagents used are polyspecific reagents that can simultaneously detect IgG and/or C3. If the result is positive, the RBC sample is then subsequently tested with monospecific reagents, either bound immunoglobulin G or bound complement. This finalizes the identification of a substance that is bound to the RBC surface.

Procedure for Coombs Test

The Coombs Test can be done by several methods – Tube test, Microcolumn test, and solid phase, polyethylene glycol (PEG).

Microcolumn method/Antiglobulin gel test

The Microcolumn method is also known as the antiglobulin gel test. It uses a dextran acrylamide column impregnated with an anti-human globulin reagent (anti-IgG) which traps antibody-bound RBCs.

Some major advantages of the microcolumn method include longevity of the result (can be reported up to 24 hours after its run) and a small volume of sample is enough to run the test.

Procedure

1. Take a gel microtube containing anti-IgG

2. Add 25 μl of test serum to the microtube

3. Add 50 μl of LISS (low ionic strength solution) suspended RBC at 0.8% concentration to the top of the column in a reaction chamber.

   * LISS is a potentiator that assists IgG antibodies to bridge and form visual agglutination.

4. Incubate for 15 minutes at 37ºC.

5. Centrifuge the sample for 10 minutes at 70 x g

   * during centrifugation in the reaction chamber, cells are pulled from the chamber into the column with unbound RBCs passing through the pores of the column.

   * This results in the formation of a button at the bottom of the column while sensitized RBCs are trapped in the gel.

Result, Interpretation

The negative reaction is indicated by the formation of a pellet at the bottom of the microtube with no agglutination.

Positive reaction (4+) is represented by a band of RBCs at the top of the column and graded from 0 to 4+.

In grade 1+, erythrocytes agglutinate at the lower half of the gel column.

In grade 2+, erythrocytes are dispersed throughout the microtube.

In grade 3+, erythrocytes are displayed in the upper half of the gel column.

In grade 4+, a solid band of RBCs is formed at the top of the microtube chamber.

Solid-phase test (SPT)

Solid phase test (SPT), also known as solid-phase anti-globulin test, is a machine-readable form of indirect anit-human globulin test (IAHG) or Indirect Coombs Test. This was developed as other immuno-hematological methods for antibody measurement, identification or cell typing are not suitable for compatibility testing.

This method uses a microtiter plate coated with agents Immucor, and Protein A – depending upon the manufacturers.

Procedure

1. Take a microtiter plate coated with agents (Immucor, Protein A).

2. Add the sample blood to the microtiter plate.

   * The agents bind the RBC sample to the microtiter plate

3. Add anti-IgG–coated indicator cells to the microtiter plate.

4. Centrifuge the microtiter plate

Result, Interpretation

A positive reaction is indicated by the presence of a carpet of RBCs over the well while a negative result is interpreted by the formation of a button.

Grading is done from 0 to 4+ scale.

Advantages

The Gel and solid phase methods have several advantages over tube test methods.

• Gel and solid phase methods are automatable

• are technically easier than the tube method

• higher sensitivity than tube testing