Elution and adsorption (overview)

Principle

• Elution removes antibody bound to red cells so it can be identified; the recovered fluid is the eluate.
• Adsorption removes antibody from plasma using red cells carrying the target antigen, leaving other antibodies in the adsorbed plasma for testing.

Procedure

• Elution – heat (56 °C for about 10 min): best for IgM or ABO antibodies, e.g. ABO HDFN.
• Elution – acid (glycine-HCl at low pH, commercial kits): most common for IgG alloantibodies and warm autoantibodies.
• Elution – freeze–thaw (Lui method): ABO antibodies. Organic solvents (ether, chloroform, dichloromethane) are now rarely used due to toxicity.
• Before elution, wash cells thoroughly (about 4–6 times) and test the last wash in parallel; it must be negative to show that reactivity comes from bound antibody, not free plasma antibody.
• Autoadsorption: patient's own cells, only if not transfused within about 3 months. For warm autoantibodies the cells are first treated with ZZAP (DTT + papain or ficin) or heat to free antigen sites; for cold autoantibodies incubate at 4 °C.
• Allogeneic (differential) adsorption: when recently transfused, use group O cells of selected phenotypes (e.g. R1R1, R2R2, rr, each lacking certain common antigens) so alloantibodies can still be identified.
• Rabbit erythrocyte stroma removes cold autoantibodies such as anti-I and anti-IH, but may also remove some anti-B, anti-D and anti-E.

Results

• Eluate reacting with all panel cells suggests a warm autoantibody; a specific pattern suggests an alloantibody.
• Adsorbed plasma is tested with a panel to reveal alloantibodies hidden by the autoantibody.

Quality control and pitfalls

• Repeat adsorptions until the autoantibody is removed; test the adsorbed plasma against the adsorbing cells.
• Adsorption dilutes plasma slightly (especially with ZZAP), so weak antibodies may be missed.
• A positive last-wash control invalidates the eluate.

Clinical use

• DAT-positive patient after recent transfusion: identify an alloantibody causing a delayed haemolytic reaction.
• HDFN: identify the antibody on cord cells.
• Warm AIHA: eluate is usually panreactive; adsorption detects underlying alloantibodies before transfusion.
• Adsorption–elution confirms weak A or B subgroups (e.g. Ax, Ael).