Methodology

A suspension of red blood cells from the D-Negative mother is mixed with reagent containing anti-D and is incubated for 5 minutes at room temperature. During incubation any minor population of D-positive cells are coated with anti-D. After incubation cells are washed to remove all unbound antibody and indicator cells are added. One antigen-binding site free to attach to an Rh antigen of the indicator cells. the red cell mixture is centrifuged and examined microscopically for mixed-field agglutination in the form of rosettes at low power. The indicator cells added after washing form rosettes around the individual cells leading to larger agglutinates for detection.