b. Preparation of Cephalothin-Treated Cells.
STEP 1. Group O cells (preferably fresh) are washed three times in
(2) STEP 2. To one ml of packed, washed cells are added 400 mg
cephalothin dissolved in 10 ml of pH 9.5-10.0 buffered saline (for example, one part pH
9.5-10.0 barbital buffer, plus nine parts normal saline).
STEP 3. Incubate at 37C for two hours with gentle mixing.
STEP 4. Wash cells three times in saline.
c. Tests on Patient's Serum and Eluate.
(1) STEP 1. If the results of a direct antiglobulin test are positive, an eluate
should be prepared by a standard method (for example, heat or ether).
(2) STEP 2. The eluate and the patient's serum should be tested against
normal group O cells and the same cells treated with penicillin. Usually, serial dilutions
of the patient's serum are tested.
(3) STEP 3. Two volumes of eluate or serum dilutions should be incubated
with 1 volume of a 2 to 5 percent suspension of penicillin-treated and untreated group
O cells in saline.
(4) STEP 4. Incubate at room temperature for 15 minutes. Centrifuge and
inspect for agglutination.
(5) STEP 5. Move tubes to 37C for 30 minutes. Centrifuge and inspect for
STEP6. Wash cells 4 times in saline.
(7) STEP 7. Add antiglobulin serum to button of washed cells. Centrifuge.
Inspect for agglutination.
d. lgM Penicillin Antibodies. lgM penIcillin antibodies will agglutinate
saline-suspended penicillin-treated cells, but not the same cells untreated. lgG
penIcillin antibodies will react by the Indirect antigiobulun test against penicillin-treated
cells, but not against the same cells untreated.
e. Anitiglobulin Tests. If indirect antiglobulin tests are used to detect
cephalothin antibodies, it must be remembered that cephalothln-treated RBCs can
adsorb proteins non-immunologically. Therefore, all normal serums will give positive
results in indirect antiglobulin tests if incubated with cephalothin-treated cells for a long