In a negative indirect antiglobulin test, after the addition of IgG-sensitized red blood cells, which statement is NOT necessarily true?

• A. Antihuman globulin reagent was added
• B. Adequate washing of cells was performed
• C. Antiglobulin serum has not reacted with the original red blood cells being tested
• D. Patient serum was added to the test tube

Answer: (D)

The indirect antiglobulin test detects antibodies in a patient’s serum by allowing them to bind to antigens on reagent red blood cells, then using anti-human globulin (AHG) to detect any IgG that has attached to those cells after thorough washing. In a negative result, there’s no agglutination after the AHG step, meaning no detectable IgG is bound to the test cells.

Antihuman globulin reagent must be added to reveal any IgG on the red cells, so that step is essential for interpretation. Adequate washing is crucial to remove unbound antibodies that could otherwise give a false impression of binding when AHG is added. The antiglobulin serum acts on IgG that is already bound to the red cells, not on unbound or original cells, so it should not react with cells that have no IgG attached. Given these principles, the statement about patient serum being added to the test tube is not guaranteed in every variation of the setup, so it’s the one that is not necessarily true.