A previously type O, Rh-negative individual is now positive on the direct antiglobulin test (DAT). Which pattern would you most likely see in a sample tested for anti-D?

A previously type O, Rh-negative individual is now positive on the direct antiglobulin test (DAT). Which pattern would you most likely see in a sample tested for anti-D?

• A. Cells with anti-D: IS = 0; AHG = 1; Control: IS = 0; AHG = 1
• B. Cells with anti-D: IS = 0; AHG = 0; Control: IS = 0; AHG = 0
• C. Cells with anti-D: IS = 0; AHG = 0; Control: IS = 0; AHG = 1
• D. Cells with anti-D: IS = 0; AHG = 1; Control: IS = 0; AHG = 0

Answer: (A)

The key idea is that a positive direct antiglobulin test means some antibodies are already bound to red cells in the patient in vivo. When you test for anti-D in the serum (the indirect antiglobulin test), you expose patient serum to red cells bearing the D antigen and then use the antihuman globulin to see if that serum antibody can bind and bridge the cells. If the patient has been sensitized to the D antigen (as suggested by the DAT being positive) and anti-D antibodies are present, those antibodies can be in the serum as well as already bound to any D-positive cells added in the test. In this situation, during the incubation step there may be no visible agglutination (IS) because the binding is not yet crosslinked. But when AHG is added, it will crosslink any IgG anti-D antibodies that are either already coating cells in vivo or bound to the test cells, producing agglutination. Because the DAT positive state reflects antibodies on red cells, you can observe AHG-mediated agglutination in both the test cells and the control cells in this anti-D assay, with no reactivity in the saline (IS) phase. So the pattern you’d expect is no reaction at IS for either column, but AHG-phase agglutination in both the cells being tested and the control cells. This reflects the in vivo antibody coating indicated by the DAT and the presence of anti-D antibodies capable of bridging with AHG in the in vitro test.

The key idea is that a positive direct antiglobulin test means some antibodies are already bound to red cells in the patient in vivo. When you test for anti-D in the serum (the indirect antiglobulin test), you expose patient serum to red cells bearing the D antigen and then use the antihuman globulin to see if that serum antibody can bind and bridge the cells.

If the patient has been sensitized to the D antigen (as suggested by the DAT being positive) and anti-D antibodies are present, those antibodies can be in the serum as well as already bound to any D-positive cells added in the test. In this situation, during the incubation step there may be no visible agglutination (IS) because the binding is not yet crosslinked. But when AHG is added, it will crosslink any IgG anti-D antibodies that are either already coating cells in vivo or bound to the test cells, producing agglutination. Because the DAT positive state reflects antibodies on red cells, you can observe AHG-mediated agglutination in both the test cells and the control cells in this anti-D assay, with no reactivity in the saline (IS) phase.

So the pattern you’d expect is no reaction at IS for either column, but AHG-phase agglutination in both the cells being tested and the control cells. This reflects the in vivo antibody coating indicated by the DAT and the presence of anti-D antibodies capable of bridging with AHG in the in vitro test.