64 WAIHA Elutions using Warm Autoantibody Removal Medium (W.A.R.M.)

Principle

The red cells from a patient with a positive DAT and free plasma autoantibody are treated with W.A.R.M. This treatment removes autoantibody, thereby reducing the strength of the positive DAT and freeing antigen sites for adsorption. At the same time, the enzyme pre-modifies the red cells. The red cells, thus prepared are combined with autologous serum and the mixture is incubated at 37oC. The warm-reactive autoantibody binds to the antigen present on the patient’s red cells. This antigen-antibody interaction is enhanced by the enzyme treatment of the cells. Following incubation, the mixture is centrifuged and adsorbed serum is harvested and tested. By comparing the results of W.A.R.M.-adsorbed serum with those obtained with the un-adsorbed serum, it is usually possible to confirm the presence of the warm-reactive autoantibody, and detect or identify any additional alloantibody(ies) that may be present.

Specimen 

1. Specimens can be collected with or without anticoagulant. If using EDTA the testing should be performed within 48 hours.
2. A control specimen will be used having Rh antigen and Kell antigen to insure the W.A.R.M. enhancement and denaturation has occurred.

Reagent

1. W.A.R.M. is a specially formulated preparation of dithiothreitol (DTT) and cysteine-activated papain in a phosphate buffer. It has been filtered and lyophilized. This product is reconstituted with DH2O.
2. Isotonic saline
3. Serum or plasma to be adsorbed

Procedure

1. Label the tubes to be used in the procedure according to the tube labeling SOP
2. Reconstitute a vial of W.A.R.M. by adding 5 ml of deionized water. Mix thoroughly.
3. Add 1 ml of packed patient red cells to the tube. It is not necessary to wash patient’s red cells prior to W.A.R.M. treatment.
4. Add 2 mls of reconstituted W.A.R.M. to the tube.
5. Mix well and incubate at 37+/- 2oC for approximately 30 minutes.
6. Wash the W.A.R.M.-treated cells with saline a minimum of three times. Remove as much saline as possible after each wash.
7. Add an equal volume of patient serum or plasma to the W.A.R.M.-treated, packed red cells.
8. Mix well and incubate for 30-60 minutes at 37+/- 2oC. Note: Due to the fragility of certain red cells, some hemolysis may be observed upon treatment. The treatment of such red cells should be limited to thirty minutes.
9. Centrifuge for approximately 2 minutes or until red cells are well packed.
10. Harvest the adsorbed serum (plasma) using a Pasteur pipette.
11. Test an aliquot of the adsorbed specimen at 37+/- 2oC and at the antiglobulin phase to determine if all warm-reactive autoantibody has been removed. If results indicate that adsorption was insufficient repeat step 1-10 using a new aliquot of patient red cells. If adsorption is complete, the adsorbed specimen is ready for use in antibody detection and identification.

Interpretation

1. Complete adsorption of warm-reactive autoantibody has been achieved when the adsorbed serum no longer reacts where previously detected with the patient’s own W.A.R.M.-treated red cells (auto using adsorbed serum and W.A.R.M.-treated red cells) and reagent red cells that lack the antigens toward which additional alloantibodies are directed.
2. Incomplete adsorption will be demonstrated if agglutination is detected in either of the situations listed above.

Notes

1. Complete adsorption of warm-reactive autoantibodies may not always be possible, however in some instances, only partial adsorption of the autoantibody may be sufficient to reveal underlying alloantibody (ies) that might also be present.
2. W.A.R.M. may be deemed sufficiently active if it can be shown that treated red cells show enhancement of Rh antigen and denaturation of a Kell system antigen. It is recommended that a red cell of this phenotype be treated along with the patient to demonstrate enhancement and denaturation. (control)
3. Store W.A.R.M. at 1-10oC. Do Not Freeze. Do not use beyond five days after reconstitution. Some particulate matter and slight turbidity may be noted upon reconstitution.
4. If the autoantibody is in the Kell system Since Kell system antigens are destroyed by DTT autoantibodies of the Kell system can’t be adsorbed.
5. Some autoantibodies are directed against enzyme sensitive antigens (M or Xga) Since W.A.R.M. destroys these antigens, autoadsorption using these treated cells will not remove the corresponding autoantibody.
6. Do not use the W.A.R.M.-treated red cells for antigen typing of the patient since commercial antisera may not be specific when tested this way.
7. Crossmatches performed on a W.A.R.M. autoadsorbed serum can’t be considered “compatible” since the autoantibodies removed by this procedure are warm reactive antibodies and would be clinically significant.
8. The potential exists for adsorption of significant alloantibodies onto transfused red cells. This procedure should not be used on samples from recently transfused patients.