(c) STEP 3. Remove plasma from the 37C incubated cells. Wash the
cells 3 times, in a large volume of warm (37C) saline, using warmed centrifuge cups.
(d) STEP 4. Prepare a small volume of 2 percent to 5 percent
suspension of the RBCs in saline, using the washed, packed, RBCs from step 3. Use
these cells for ABO and other testing.
(e) STEP 5. Test the RBCs from step 4 with the serum from step 2, at
4 C. If cold agglutinin was removed in the initial incubation, tests will be negative.
(f) STEP 6. If antibody remains, add 1 volume of serum to 1 volume
of patient's washed, packed, cells from step 3, and place in an ice bath for 30 to 60
minutes. Shake frequently for maximum absorption.
(g) STEP 7. After cold centrifugation, remove the supernatant serum
as quickly as possible, and test with autologous RBCs from step 4. Test at 4C as in
step 5. The procedure should be repeated if cold antibody remains.
c. Absorption with Enzyme-Treated Cells.
(1) STEP 1. With one volume of washed, packed, autologous RBCs, add
one volume of enzyme solution. Any enzyme may be used (see the Special Methods
STEP 2. Mix and incubate at 37C for 15 minutes.
(3) STEP 3. Wash cells several times to remove enzyme solution. Be sure
to mix the cells well with each new volume of saline. This is best done by adding half a
tube of saline, mixing the cells by inversion to remove them all from the bottom, then
filling the tube with saline.
(4) STEP 4. To one volume of washed, packed, enzyme-treated cells, add
one volume of patient's serum.
(5) STEP 5. Mix and incubate in ice bath for 15 to 60 minutes; mix
frequently for maximum absorption. In some cases when the antibody is very potent, an
even shorter incubation will result in almost complete agglutination of the cells.
STEP 6. Centrifuge and harvest serum, as quickly as possible.
(7) STEP 7. Test patient's absorbed serum against autologous cells to
determine if cold autoagglutinin has been completely removed. If not, repeat absorption
with fresh enzyme-treated cells.