Antiglobulin Test

The antiglobulin (AHG) test was invented by Coombs, Race and Mourant in 1945 and is sometimes called the Coombs test after its main inventor. It is the single most important test we have for detection of red cell antibodies.

Principle of the AHG Test

Because antibodies are gamma globulins, an antibody to gamma globulin can form bridges between red cells sensitized with antibody and cause them to agglutinate (Figure 3-1). Since most incomplete antibodies are IgG, polyspecific AHG serum contains anti-IgG. Because some IgG and IgM antibodies also cause C3 to attach to red cells, polyspecific AHG serum also contains anti-C3, which can cause C3-coated red cells to agglutinate. The AHG serum that is routinely used by most labs for pretransfusion IATs is monospecific anti-IgG. When doing DATs to detect in vivo sensitization with IgG or C3, blood banks must use polyspecific AHG serum containing anti-IgG, anti-C3b, and anti-C3d.

Preparation and Source of AHG Serum

Indirect Antiglobulin Test (IAT, IDC, IAGT)

Today most labs use monospecific anti-IgG for IAT testing. This test is meant to detect in vitro sensitization, but will also detect in vivo sensitization if the red cells used already are sensitized with IgG. The method is as follows:

  1. 2 or 4 drops of serum (preferably 4) are incubated at 37°C for 15-60 minutes with 1 drop of 3 - 5% red cells. If the serum contains an antibody specific for an antigen on the red cells, the antibody will sensitize the cells (but not agglutinate them, if the antibody is IgG).
  2. Following incubation, the test is read macroscopically only for agglutination and hemolysis. This is the saline 37°C part of the AHG test. Note: the test could be read microscopically here, but it usually is not.
  3. After reading the saline 37°C part of the test, the red cells are washed well 3 - 4 times to remove all unbound protein. The washing stage is crucial, because if any unbound protein (IgG) remains, it could neutralize the AHG serum and cause a false negative. The washes are also decanted well, because residual saline will decrease the efficiency of protein removal. Also, residual saline after the last wash will dilute the AHG serum and can cause a false negative.
  4. After the last wash has been thoroughly decanted, 2 drops of AHG serum are added and the test is centrifuged and read macroscopically for agglutination only (no serum remains to show hemolysis). If negative, the test is read microscopically also, and if still negative, IgG sensitized cells are added. (See later under QC).

Uses of the Indirect Antiglobulin Test

Quality Control of the IAT

Direct Antiglobulin Test (DAT, DCT, DAGT)

The DAT detects only in vivo sensitization by IgG or C3. Unlike the IAT, no serum is added to the cells, and no incubation is required. The method consists of washing the cells well 3 - 4 times, adding polyspecific AHG serum, centrifuging,and reading macroscopically for agglutination, and microscopically (if negative). If still negative, IgG sensitized cells are added. The applications of the DAT include investigation of the following clinical conditions:

  1. HDN (maternal IgG antibody sensitizing the infant's cells)
  2. AIHA (IgG autoantibody or C3 sensitizing the patient's cells)
  3. drug-related hemolytic anemias or positive DATs (IgG autoantibody, IgG drug antibody, or C3 sensitizing the patient's cells)
  4. hemolytic transfusion reactions (IgG antibody or C3 sensitizing the donor cells that are in the patient)
Antiglobulin Test