The procedure for elution is outlined below:
- Centrifuge the specimen and remove the plasma using a pipette. Do not pour off the plasma. The plasma can be saved for further testing as deemed appropriate.
- Place 1 mL (approximately 20 drops) of the patient's packed red blood cells into a clean glass test tube.
- Wash the packed red blood cells once with saline and remove the supernatant with a pipette. Discard supernatant.
- Wash the packed red blood cells with working wash solution (prepared per manufacturer's instructions) an additional 4 times inverting each time to mix well and remove the supernatant by pipette. Reserve a small aliquot of the last wash to serve as a control in a separate clean glass tube.
- Place approximately 20 drops of the washed red blood cells into a clean glass tube and add an equal amount of eluting solution. Mix immediately by gently inverting 4 times. Centrifuge immediately for 30–45 seconds at 3400 rpm. Delays in this step can cause hemolysis.
- Transfer the supernatant into a clean glass test tube with a pipette. Discard the red blood cells.
- Add buffering solution until the solution turns blue (approximately 1:1 volume ratio). Mix well and centrifuge to remove any debris.
- Remove and transfer the supernatant with a pipette to a clean glass test tube.
The eluate is now ready for panel testing to see if there are any underlying alloantibodies. The last wash is used as a control to make sure the antibody has been removed from a bound state on the red blood cells and that the red cells were adequately washed. If the antibody is present in the last wash, the elution should be repeated after a thorough washing of the red blood cells. A warm autoantibody will normally show panagglutination in the eluate panel testing.
