The indirect antiglobulin test (IAT) is used to detect the presence of serum antibodies directed to erythrocyte blood group antigens, there must be between 100 and 200 IgG or C3 molecules on the cell to obtain a positive reaction.
The presence of these antibodies is identified by reacting (in vitro) the test serum with suitably selected red blood cells. The reaction is revealed by using a Coombs serum. If the indirect Coombs’ is positive, titration of the antibody and differentiation between IgG or IgM antibodies must always be carried out. It is also necessary to identify the erythrocyte blood group antigen towards which the antibody is directed. For the titration of the antibody a scalar dilution to doubling the serum in physiological is prepared.
A IAT is then prepared from each dilution, using a poly specific antiglobulin serum. The titer of the antibody is indicated by the highest dilution at which agglutination of the red cells that have given a positive reaction to the IAT still occurs.
To proceed with the identification of the specificity of an antibody, special RBC panels are used. If maternal-foetal incompatibility due to ABO incompatibility is studied, a panel consisting of 4 red blood cells of groups A1, A2, B and O is used. a panel consisting of group O polyantigenic red cells characterized by different phenotypes is used. Figure 1 shows screening for Indirect Antiglobulin Test using a three cells panel.
Fig1. Screening for Indirect antiglobulin test using a three cells panel. The figure above shows a screening search for anti-erythrocyte antibodies carried out using a panel of red blood cells test polyantigenic group O. This screening is positive for cells 1 and 2. Antibody specificity identification performed using a more extensive panel is reported in Fig.2
This screening is positive for cells 1 and 2. An antibody identification should be performed using an extended panel of fifteen group O polyantigenic red cells.
Figure 2 describes characterization of an alloantibody mixing using an extended identification panel untreated and treated with enzymes and Fig. 3 antibody titration.
Fig2. Identification of allantibodies using an extended identification panel with and without enzyme treatment. This is the same sample already screened, as shown in Fig. 1. For identification we tested the sample against a panel of polyantigenic group O red blood cells not treated with enzymes, the test was performed in Liss-Combs. Some negative test cells 6, 11 and 12 were observed; such as some positive tests with different score: ++++ for columns 4, 5, and 13; and +++− for 1, 2, 3, 7, 8, 10, 14, and 15. after this series of tests, based on the results obtained we hypothesized the presence of an antibody mixture to solve which we decided to use a different panel and a different reaction method, as shown in the second series of images. We then tested the sample against the same panel of polyantigenic group O test red cells, but enzyme-treated, the test was performed in Liss. As can be seen, the negativities for cells 6, 11 and 12 remain as well as the positivity ++++ for columns 4,5 and 13. The positivity for cells 1, 2, 3, 7, 8, 10, 14 and 15 disappear; a sign that the antigens to which the antibody was directed was destroyed by the enzymatic treatment of the red blood cells. The combined evaluation of the results of the two tests made it possible to resolve the mixture by identifying an anti-E (specificity that remains even in the panel treated with enzymes), and an anti-M anti body whose reactivity is eliminated by the enzymatic treatment
Fig3. Alloantibody titration. To carry out the titration of an antibody, serial factor two dilutions are prepared which are tested against a cell that has been tested reactive to IAT using the most appropriate phase. The dilution of the last positive column indicates the reciprocal of the antibody titer. Results: Titer 1/32
After reporting the reactivity pattern on the appropriate sheet, it is compared with the results reported on the accompanying sheet of the panel (identigram) (Fig. 4), excluding the antigens based on the negative results. It is good practice, after presumptively identifying an alloantibody directed towards a specific erythrocyte antigen, to demonstrate its absence on the patient’s red cells.
Fig4. Identigram. The figure shows the data sheet showing the antigenic composition of the panel of polyantigenic red blood cells of group O used for the identification of the antibody mixture shown in Fig.2. Each row corresponds to the typing of each of the 15 cells that make up the panel and for each column the positivity or negativity for each of the antigens is reported
