(6) STEP 6. Test the absorbed serum for complete absorption, using the
method of optimum reactivity, against a freshly prepared suspension of the RBCs used
for absorption. If the absorbed serum still reacts with the cells, absorption has not been
complete, and should be repeated with a fresh aliquot of washed, packed celIs.
STEP 7. If an eluate is to be made, save the cells with the absorbed
a. Background. Elution is removal of antibody that has been absorbed onto
RBC surfaces. The absorption may have been "in vivo" (autoimmune) or "in vitro."
Elution may be undertaken for a number of reasons:
(1) To demonstrate and identify the antibody of an infant's cells in hemolytic
disease of the newborn. If the mother's serum is unavailable, the eluate may be used
for crossmatching for exchange transfusion.
(2) To demonstrate and identify the antibody producing the positive
antiglobulin test in acquired hemolytic anemias, or in suspected transfusion reactions.
(3) To produce small amounts of useful single-antibody preparations after a
mixture of antibodies has been separated by absorption onto cells of the appropriate
(4) To free the cells from adsorbed antibody (usually autoantibody,
cold-active) so they are suitable for further testing, or for auto-absorbing the serum.
(5) To demonstrate that cells have adsorbed an antibody; and therefore,
possess an antigen, despite the failure of the antibody to agglutinate the cells. (This is
usually done with weak antigens of the ABO system, since antiglobulin-testing provides
this kind of information in the Rh and most other systems.)
b. Preparation of Cells. In all elution methods, the most critical detail is the
complete removal of unabsorbed antibody surrounding the RBCs. The cells must be
washed at least three times; in some cases, as many as 12 times. The procedure for
cell-washing is the same for all methods. To test for complete removal of unabsorbed
antibodies after the third washing and centrifugation, remove all the supernatant fluid
except for the 0.5 ml directly over the RBC mass. Remove this fluid and test it with
RBCs known to react with the "unwanted" antibody. If the test is negative, proceed with
the preparation of the eluate. The eluate usually contains hemoglobin, which does not
interfere with testing. Because complement is not