b. Monospecific antiglobulin serums are useful when performing the direct
antlglobulin test as the immune-complex group usually presents with complement only
on their RBCs, in contrast to the penicillin and methyldopa group, where, only lgG
usually is present. The cells from a patient with positive results in a direct antigiobulin
test as a result of cephalothln can react with some or all antiglobulin serums (for
example, anti-lgG, -IgA, -lgM, -C3, -C4, -CC, -B globulins, and anti-albumin).
c. The patient's serum should be screened by the usual blood bank procedures.
If antibody is detected, specificity tests should be performed and the previously
mentioned facts concerning specificity-testing in warm autoimmune hemolytic anemia
taken into account. If the patient's serum does not react against normal untreated
RBCs, the serum should be tested against ABO-compatible RBCs in the presence of
any drugs the patient has been receiving. A saturated solution of the drug should be
prepared. Two useful reference books on properties, such as solubility of drugs, are the
Physicians' Desk Reference, and the Merck Index. It is preferable to use several
dilutions of this saturated solution, progressing to an approximate physiologic dose (for
example, the average dose of drug circulating per milliliter of blood). Enzyme-treated
and untreated normal RBCs should be tested. It is also wise to add fresh complement
(for example, fresh compatible normal serum), to the system. The tests should be
inspected for agglutination, hemolysis, and sensitization to antiglobulin sera.
d. An essential part of the investigation is to prepare an eluate from the patient's
RBCs. This is particularly important in the penicillin group where, a definite diagnosis
can be reached only by proving that the eluate will react with penicillin-treated cells, and
yet is negative with the same cells untreated. It should be remembered that the
immune complex group is often associated with red cells sensitized with complement
components alone; thus, the eluates are often negative even when the drug is added to
2-11. DETECTION OF ANTIBODIES TO PENICILLIN AND CEPHALOTHIN
a. Preparation of Penicillin-Treated Cells.
STEP 1. Group 0 cell, (preferably fresh) are washed three times in
(2) STEP 2. To one ml of packed washed cells are added 1 X 106 units
(approximately 600 mg) of K-benzyl penicillin G dissolved in I5 ml of 0.1 M barbital
buffer, pH 9.5 to 10.0.
STEP 3. Incubate for 1 hour at room temperature with gentle mixing.
(4) STEP 4. Wash cells three times in saline. Slight lysis may occur during
incubation, and a small "clot" may form in the RBCs, which can be removed with
applicator sticks before washing cells. Once prepared, the cells may be kept in ACD, at
4C for up to 1 week; however, they do deteriorate slowly during this time.