63 Acid Elution (ELU KITTM PLUS)

Principle

Acid elution is suitable for investigation of positive DAT’s in association with warm-reactive (IgG) auto- or alloantibodies. In conjunction with adsorption techniques, it can be used to separate mixtures of IgG antibodies against red cell antigens.

Specimen

Packed DAT-positive red cells.

Reagents

1. Wash solution concentrate; contains 1% sodium azide as an antimicrobial agent. Dilute the concentrate (1/10-1+9) with distilled or deionized water to prepare a working wash solution. Store at 1-10°C
2. Solution I; Acid eluting solution; a low pH glycine buffer containing a coloring agent (pH indicator)
3. Solution II; Base buffering solution; a Tris solution containing bovine albumin with 0.1% sodium azide as an antimicrobial agent.
4. Supernatant saline from final wash of the red cells to be tested.

Procedure

1. Place a little over 1 ml of packed red cells (in student labs we will use ALL the red cells provided) in a 12X75mm test tube.
2. Wash the packed cells with the working wash solution a minimum of four times to remove the unbound antibody. Be sure to reserve a small amount of the last wash to test for antibody activity. Label this tube “last wash”. This last wash should be negative if all the unbound antibody has been removed (quality control step).
3. Transfer the washed cells to a clean labeled test tube (12X75mm). To 1 ml (in student labs we will use ALL the washed cells) of washed packed red cells add 20 drops (~1 ml) of Solution I to elute the antibody. Mix very Centrifuge immediately for 45-60 seconds. Excess mixing or failure to centrifuge immediately may cause hemolysis which alters pH of eluate.
4. Transfer the supernatant fluid off these cells to a clean labeled test tube. Discard red cells.
5. Add 5 drops of Solution II to buffer the eluate. Mix well. Continue adding Solution II dropwise, mixing well after each drop until a distinct blue color appears. The blue color indicates a pH range of 6.5-7.5.
6. Centrifuge this eluate for 2-3 minutes to remove precipitate that forms after neutralization (there may be cellular debris). Even if you don’t visually “see” debris this tube needs to be centrifuged.
7. Transfer the supernatant eluate (even if it looks like there is no cellular debris) to a clean, labeled test tube (patient name and “eluate”) and test in parallel and the supernatant saline from the “last wash” saved on step 2 using the “Modified Antiglobulin Test” methodology provided below.

Modified Antiglobulin Test

1. Prepare properly labeled tubes for eluate panel (may only be enough time in student labs to run eluate screening cells) and “last wash” panel (or screening cells). Tubes are labeled with patient’s name, eluate and the cell number.
2. Add 1 drop of panel or screening cells to appropriately labeled tubes and prepare a “dry” red cell button (wash 1 time in cell washer).
3. Add 2-4 drops of eluate or “last wash” to the relevant “dry” cell buttons. Do not add potentiators.
4. Mix well and incubate at 37°C for 15 minutes
5. After incubation add 5-10 drops of working wash solution to each tube. Centrifuge and completely decant/blot resulting in a “dry” cell button
6. Add 2 drops of AHG, mix, spin for 20 seconds and read/grade. Record results immediately while holding tubes in hand.
7. Add CCs to all negative results.

Interpretation

1. Positive results: agglutination. To identify the antibody perform antibody panels using the eluate and perform exclusion/antibody identification procedure.
2. Negative results: no agglutination
3. Invalid test: failure of IgG sensitized cells to react when added to negative tests OR reserved “last wash” demonstrates positive results.