65 “General” Warm-Reactive Autoantibody Adsorption Procedure

Principle

Adsorption of plasma removes antibodies from the plasma by incubating with cells that have the corresponding antigen. Adsorptions are performed for a variety of reasons. Warm-reactive autoantibodies in serum/plasma may mask the presence of clinically significant alloantibodies. Adsorption of the serum/plasma with autologous red cells can remove autoantibody from the serum, permitting detection of underlying alloantibodies. Some potent cold-reactive autoantibodies may also mask an alloantibody.

Specimen

Serum containing antibody to be adsorbed.

Reagents

1. Red cells that carry the antigen corresponding to the antibody specificity to be adsorbed (may be autologous cells).
2. 1% cysteine-activated papain or 1% ficin
3. Phosphate buffered saline, pH 7.3
4. 0.2 M DTT

Procedure

1. Prepare ZZAP reagent by mixing 0.5mL of 1% cysteine-activated papain with 2.5 mL of 0.2 M DTT and 2 mL of PBS. Alternatively use 1 mL of 1% ficin, 2.5 mL of 0.2 M DTT and 1.5 mL of PBS.
2. Add 2 mL of ZZAP reagent to each of two tubes containing 1 mL of the respective packed red cells. There is no need to wash the cells before treatment.
3. Mix and incubate at 37oC for 30 minutes with periodic mixing.
4. Wash the red cells 3 times in saline, centrifuge the last wash at least 5 minutes and remove as much supernatant as possible.
5. Add equal volumes of the ZZAP–treated red cells and serum, mix and incubate at 37oC for approximately 30 to 45 minutes.
6. Centrifuge the red cells after incubation and carefully remove the serum.
7. If the original serum reactivity was only 1+ proceed to step 8; otherwise repeat steps 5 and 6 once more using the once-adsorbed patient’s serum and a second aliquot of the ZZAP-treated cells.
8. Transfer the supernatant fluid, which is the adsorbed serum, to a clean test tube.
9. Test the adsorbed serum against group O reagent red cells. If reactivity persists, repeat steps 5 and 6.

Interpretation

1. If reactivity remains, the antibody has not been completely removed.
2. If there is no reactivity this signifies the antibody has been completely adsorbed.

Notes

1. ZZAP treatment destroys all Kell system antigens and all other antigens are destroyed by proteases (eg, M, N, Fya and Fyb as well as the antigens of the LW, Cartwright, Dombrock and Knops systems). If the autoantibody is suspected to have specificity to a high incidence antigen in any of these blood groups an alternative procedure is to perform autoadsorption with untreated cells or with cells treated only with 1% ficin or 1% cysteine-activated papain.
2. Adsorption is more effective if the area of contact between the red cells and serum is large therefore use of a large bore test tube is recommended. (13 mm or larger)
3. Multiple adsorptions may be necessary to completely remove an antibody.
4. Repeat adsorptions should use a fresh aliquot of cells and not the cells from the prior adsorption.