Lab

Practical Blood Bank

Adsorption Definition: The removal of antibody from serum

by combining a serum sample with appropriate RBCs under optimal conditions.

After the antibody attaches to the membrane-bound antigens and the serum and cells are separated, the specific antibody remains attached to the red cells.

It may be possible to harvest the bound antibody by elution.

Anti-e

Anti-eAnti-e

Anti-SAnti-e

Anti-eAnti-e

Anti-eAnti-S

Mix serum with antibody with red cells with corresponding antigen. Incubate at appropriate temperature. Anti-e antibodies have been adsorbed out of the serum and onto the RBCs.

Anti-S

Anti-S

Adsorption techniques are useful in such situations as:

1. Separating multiple antibodies present in a single serum.2. Removing autoantibody activity to permit detection of

coexisting alloantibodies.3. Removing unwanted antibody (often anti-A and/or anti-B)

from serum that contains an antibody suitable for reagent use.

4. Confirming the presence of specific antigens on red cells through their ability to remove antibody of corresponding specificity from previously characterized serum.

5. Confirming the specificity of an antibody by showing that it can be adsorbed only to red cells of a particular blood group phenotype.

Adsorption: Separating Multiple Antibodies Patient red cell phenotype is very helpful:

Phenotyping the patient can help in choosing a red cell phenotype that will give the most information.

If None of the antibodies have been identified then a weak reacting cell can be used assuming that only one antibody may be reacting with it.

Adsorption: Separating Multiple Antibodies If One or More of the antibodies have been

identified, then red cells lacking those antigens are usually chosen so that only one antibody is removed.

Large volumes of red cells are required so Reagent vials are insufficient. Donor blood or staff members end up being the most convenient.

Adsorption: Positive DAT Auto adsorption is simply mixing patient

serum with patient red cells to remove the auto antibody remaining in the serum under optimal conditions. If it is a warm auto antibody then adsorb at warm temps, if it is a cold auto antibody then adsorb at cold temps.

Retest serum to determine degree of removal of auto antibody. May require second adsorption.

Test panel with adsorbed serum to check for presence of allo antibody that was masked by the auto antibody.

Specimen Serum or plasma containing antibody to be

adsorbed.

Reagents Red cells (eg, autologous or allogeneic) that carry

the antigen corresponding to the antibody specificity to be adsorbed.

Procedure1. Wash the selected red cells at least three times with

saline.

2. After the last wash, centrifuge the red cells at 800 to 1000 × g for at least 5 minutes and remove as much of the supernatant saline as possible. Additional saline may be removed by touching the red cell mass with a narrow piece of filter paper.

3. Mix appropriate volumes of the packed red cells and serum and incubate at the desired temperature for 30 to 60 minutes.

4. Mix the serum/cell mixture periodically throughout the incubation phase.

5. Centrifuge the red cells at 800 to 1000 × g for 5 minutes to pack cells tightly. Centrifuge at the incubation temperature, if possible, to avoid dissociation of antibody from the red cell membranes.

6. Transfer the supernatant fluid, which is the adsorbed serum, to a clean test tube. If an eluate is to be prepared, save the red cells.

7. Test an aliquot of the adsorbed serum, preferably against an additional aliquot of the cells used for adsorption, to see if all antibody has been removed.

Interpretation If reactivity remains, the antibody has not been

completely removed. No reactivity signifies that antibody has been completely adsorbed.

Notes1. Adsorption is more effective if the area of contact

between the red cells and serum is large; use of a large bore test tube (13 mm or larger) is recommended.

2. Multiple adsorptions may be necessary to completely remove an antibody, but each successive adsorption increases the likelihood that the serum will be diluted and unadsorbed antibodies weakened.

3. Repeat adsorptions should use a fresh aliquot of cells and not the cells from the prior adsorption.

4. Enzyme pretreatment of adsorbing cells can be performed to increase antibody uptake for enzyme-resistant antigens.