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 one time with saline and remove supernatant with a pipette. Discard supernatant.
- Wash the packed red blood cells with working wash solution (prepared per manufacturers 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 20 drops (approximately) of the washed red blood cells into a clean glass tube and add an equal amount of drops 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 more thorough washing of the red blood cells. A warm autoantibody will normally show panagglutination in the eluate panel testing.
