Cross-Reactivity of Cefotetan and Ceftriaxone Antibodies, Associated With Hemolytic Anemia, With Other Cephalosporins and Penicillin

Coagulation and Transfusion Medicine / CROSS-REACTIVITY OF CEFOTETAN AND CEFTRIAXONE ANTIBODIES Cross-Reactivity of Cefotetan and Ceftriaxone Antibodies, Associated With Hemolytic Anemia, With Other Cephalosporins and Penicillin Patricia A. Arndt, MS, MT(ASCP)SBB, and George Garratty, PhD, FRCPath Key Words: Cross-reactivity; Drug-induced immune hemolytic anemia; Cefotetan antibody; Ceftriaxone antibody; Antibiotics Abstract Most drug-induced immune hemolytic anemias since the late 1980s have been caused by the secondand third-generation cephalosporins, cefotetan and ceftriaxone, respectively. Cross-reactivity of cefotetan and ceftriaxone antibodies with other cephalosporins or penicillin has been studied only minimally. We tested 7 serum samples previously identified to contain cefotetan antibodies and one serum sample previously identified to contain ceftriaxone antibodies against 9 other cephalosporins, penicillin, and 7-aminocephalosporanic acid in the presence of RBCs and also used hapten inhibition to indicate cross-reactivity. Serum samples containing cefotetan antibodies showed some cross-reactivity with cephalothin and cefoxitin (and to a much lesser extent with penicillin and ceftazidime). The ceftriaxone antibodies showed very weak cross-reactivity with cefotaxime, cefamandole, and cefoperazone. There was very little cross-reactivity between cefotetan antibodies and the drugs tested in the present study. We have no data to determine whether the in vitro data relate to in vivo reactivity. 256 Am J Clin Pathol 2002;118:256-262 Drug-induced immune hemolytic anemia (IHA) occurs rarely, with an estimated incidence of about 1 case per million of the population.1 In the 1970s, 67% of drug-induced IHA was due to methyldopa and 23% to penicillin.2 Since the late 1980s, 88% of drug-induced IHA that our laboratory has studied has been due to the second- and third-generation cephalosporins, cefotetan (75%) and ceftriaxone (13%).3,4 ❚Table 1❚ shows that first-generation cephalosporins rarely caused IHA. The clinical and serologic manifestations in these cases were similar to penicillin-induced IHA.2 In contrast, 3 second-generation cephalosporins and 4 thirdgeneration cephalosporins have been associated with numerous cases of IHA (Table 1). In some cases, severe intravascular hemolysis occurred, which was supported by serologic findings that indicated an “immune complex” mechanism (the patient’s serum reacts with untreated RBCs in the presence of drug) rather than the “drug adsorption” mechanism (the patient’s serum reacts with drug-coated RBCs). Fatal hemolysis occurred in 1 (33%) of 3 cases due to cefoxitin, 11 (17%) of 64 cases due to cefotetan, and 9 (47%) of 19 cases due to ceftriaxone. The extent of cross-reactivity of cefotetan and ceftriaxone antibodies with RBCs in the presence of other cephalosporins and penicillin is poorly documented. In a few reports, the patient’s serum samples containing cefotetan (n = 9) or ceftriaxone (n = 3) antibodies were tested by the drug adsorption or immune complex methods against 1,7,10,24 2,8,12,25,26 3,27 4,28 or 518 different antibiotics. We present results of testing 7 cefotetan antibody samples and 1 ceftriaxone antibody sample against 9 other cephalosporins, penicillin, and 7-aminocephalosporanic acid, the basis for semisynthetic cephalosporins. © American Society for Clinical Pathology Coagulation and Transfusion Medicine / ORIGINAL ARTICLE ❚Table 1❚ Cephalosporins Associated With Immune Hemolytic Anemia* No. of Cases Mechanism References First-generation Cephalothin Cefazolin Cephalexin Second-generation Cefamandole Cefoxitin Cefotetan 5 1 1 DC DC DC Garratty3 Garratty3 Manoharan and Kot5 1 3 64 DC DC, IC DC, IC, AA Garratty3 Garratty,3 Arndt et al4 Garratty,3 Arndt et al,4 Badon et al,6 Johnson et al,7 Stroncek et al,8 Naylor et al,9 Moes and MacPherson,10 Marques et al,11 Ray et al,12 Chai et al13 Third-generation Cefotaxime Ceftriaxone 2 19 DC, IC IC Ceftazidime Ceftizoxime 2 4 DC, IC IC, DC Garratty3 Garratty,3 Arndt et al,4 Longo et al,14 Maraspin et al,15 Meyer et al,16 Viner et al,17 Seltsam and Salama,18 Falezza et al19 Garratty,3 Fueger et al20 Shammo et al,21 Endoh et al,22 Calhoun et al23 AA, autoantibody (serum reacts with RBCs without addition of drug); DC, serum reacts with drug-coated RBCs; IC, “immune complex” (serum reacts in presence of drug + uncoated RBCs). * Serum samples from patients with immune hemolytic anemia due to the first-generation cephalosporins and cefamandole were not tested by the immune complex method. Materials and Methods Antibodies Antibodies studied were 7 cefotetan antibodies previously shown4,29 to react with cefotetan-treated RBCs and untreated RBCs in the presence of cefotetan and 1 ceftriaxone antibody previously shown24 to react with untreated RBCs in the presence of ceftriaxone. The source of complement was a pool of serum samples from healthy individuals that were separated, pooled, and frozen at –70°C on the day the serum samples were obtained. Drugs The following drugs were studied: cefotetan disodium (Cefotan, Zeneca Pharmaceuticals, Wilmington, DE), ceftriaxone sodium (Rocephin, Roche Laboratories, Nutley, NJ), cephalothin sodium (Keflin, Eli Lilly, Indianapolis, IN), cefazolin sodium (Kefzol, Eli Lilly), cefamandole nafate (Mandol, Eli Lilly Italia, Florence, Italy), cefoxitin sodium (Mefoxin, Merck, West Point, PA), cefotaxime sodium (Claforan, Hoechst-Roussel, Somerville, NJ), cefoperazone sodium (Cefobid, Roerig, Pfizer, New York, NY), penicillin G potassium (Pfizerpen, Roerig, Pfizer), ceftazidime (Fortaz, Glaxo Wellcome, Research Triangle Park, NC), and cefepime hydrochloride (Maxipime, Bristol Myers Squibb, Princeton, NJ). The 7-aminocephalosporanic acid was obtained from Sigma Chemical (St Louis, MO). Antibody Detection Using Drug-Treated RBCs Penicillin-treated and cephalothin-treated RBCs were prepared by previously described methods.2,30,31 Briefly, 40mg/mL solutions of penicillin G and the cephalosporins were © American Society for Clinical Pathology prepared in a 0.1-mol/L concentration of sodium barbital (Sigma) buffer, pH 9.8, or phosphate-buffered saline (PBS), pH 7.3, respectively. Group O RBCs were added, incubated for 1 to 2 hours at room temperature (penicillin) or 37°C (cephalosporins), washed, and suspended to 3% to 5% (vol/vol) in PBS for use. Ceftriaxone has never been shown to bond to RBCs by the aforementioned methods. 4 A previously described method using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma) was used31,32 to try to bond ceftriaxone to RBCs. Briefly, 3 mL of ceftriaxone (6.7 mg/mL PBS), 0.1 mL 50% washed type O RBCs, and 1 mL freshly prepared carbodiimide (50 mg/mL in PBS) were incubated for 50 minutes at 4°C and then poured into 4°C PBS + 2% Na4EDTA (Sigma). Treated RBCs were washed 3 times and resuspended to 3% to 5% (vol/vol) for use. Six serum or plasma samples containing cefotetan (historic a (...truncated)