In indirect antiglobulin testing, when is the antihuman globulin reagent added?

In indirect antiglobulin testing, when is the antihuman globulin reagent added?

• A. After washing of cells
• B. Before incubation with patient serum
• C. During incubation with serum
• D. After mixing with albumin

Answer: (A)

In this test, you’re detecting antibodies that have actually attached to red cells, so the detection step must come after any unbound antibodies are removed. After the patient serum has been incubated with donor red cells to allow IgG antibodies to bind, the cells are thoroughly washed. This wash removes free antibodies from the serum. Only red cells with antibodies bound to their surface remain. The antihuman globulin reagent is then added; it binds to the Fc portion of those surface-bound IgG antibodies and cross-links them, producing visible agglutination if antibodies are present. Adding AHG before washing (or during incubation) could detect free antibodies or cause nonspecific reactions, leading to false results. Therefore, the antihuman globulin reagent is added after washing the cells.

In this test, you’re detecting antibodies that have actually attached to red cells, so the detection step must come after any unbound antibodies are removed. After the patient serum has been incubated with donor red cells to allow IgG antibodies to bind, the cells are thoroughly washed. This wash removes free antibodies from the serum. Only red cells with antibodies bound to their surface remain. The antihuman globulin reagent is then added; it binds to the Fc portion of those surface-bound IgG antibodies and cross-links them, producing visible agglutination if antibodies are present. Adding AHG before washing (or during incubation) could detect free antibodies or cause nonspecific reactions, leading to false results. Therefore, the antihuman globulin reagent is added after washing the cells.