Cefotetan

* Cefotetan disodium-induced hemolytic anemia has been reported previously, and some of these cases have been severe or fatal. We describe a case of severe hemolytic anemia that occurred in an 80-year-old woman who received cefotetan prophylactically after surgery for a small bowel obstruction. Eight days after the first dose of cefotetan, the patient developed a severe Coomb test-positive hemolytic anemia. Using flow cytometry, we demonstrated cefotetan-specific antibodies in her posttreatment serum, which were detectable at a serum dilution up to 1:10 000. The patient received corticosteroid therapy and blood transfusions, with improvement of her hematologic parameters, but died 54 days after admission for respiratory failure. To our knowledge, this is the first use of flow cytometry for the detection of cefotetan-induced red blood cell antibodies. This technique offers a sensitive, rapid, objective method for detecting drug-induced antibodies.

(Arch Pathol Lab Med. 2000;124:1344-1346)

Cefotetan disodium is a popular second-generation cephalosporin antimicrobial agent active against gram-positive and gram-negative aerobes and anaerobic bacteria. Its broad spectrum of activity makes cefotetan a common choice for antimicrobial use after surgical and obstetrical procedures.1 Although only 10 cases of cefotetan-induced hemolytic anemia have been published,1-6 many additional cases have been reported in abstract form. It is noteworthy that in the published cases moderate-to-severe hemolytic anemia with fatalities have been reported.1-5 In this report, we describe a case of severe hemolytic anemia caused by cefotetan. We also describe a method to identify antibodies specific to cefotetan based on flow cytometry.

REPORT OF A CASE

The patient was an 80-year-old white woman who presented with symptoms of acute small bowel obstruction. Her medical history was unremarkable, and she had not received blood transfusions in the recent past. The patient underwent a partial small bowel resection, receiving 2 g of cefotetan disodium on postoperative day (POD) 1 and 1 g of cefotetan disodium on POD 3 and POD 4. In addition, she received 2 g of cefazolin sodium on POD 4 and POD 7. Postoperatively, the patients course was remarkable for tachycardia, fever, and progressive respiratory distress, requiring intubation. Her hemoglobin level declined, reaching a nadir of 57 g/L on POD 12. At that time, the patient became jaundiced, without evidence of splenomegaly. A hematology consult was obtained to evaluate the possibility of hemolytic anemia.

Results of the patients direct antiglobulin test (DAT) were strongly positive using broad-spectrum reagents and anti-immunoglobulin G (IgG) and C3 antibodies. Results of a DAT performed on an admission blood sample were negative. Examination of the peripheral blood smear revealed microspherocytes, polychromasia, and a few nucleated red blood cells. Other pertinent laboratory results included the following values: conjugated bilirubin, 214 (mu)mol/L; unconjugated bilirubin, 29 (mu)mol / L; serum creatinine, 71 (mu)mol / L; plasma free hemoglobin, 546 mg/L; lactate dehydrogenase, 5881 U/L; and reticulocyte count, 3.3%.

The patient was treated with steroids and received 6 U of packed red blood cells. Her hematologic parameters improved, and her bilirubin levels returned to normal. However, the patient's serum creatinine level increased to a peak of 442 (mu)mol/L and remained elevated throughout her hospital course. During the next several weeks, the patient's respiratory status deteriorated, and she died of respiratory failure 54 days after admission.

MATERIALS AND METHODS

Direct Antiglobulin Test

Following standard techniques, DATs were performed with commercial polyclonal antiglobulin reagents (IgG, Ortho, Raritan, NJ; broad spectrum, BioPool, Werchester, Pa).7 In addition, DATs were performed on the admission blood sample and 2 postdrug samples.

Antibody Detection by Flow Cytometry

The red blood cells were coated using a modification of the techniques described by Gallagher et a13 and Garratty et al.4 Cefotetan disodium (Stuart Pharmaceuticals, Wilmington, Del) and cefazolin sodium (SmithKline Beecham Pharmaceuticals, Philadelphia, Pa) were adjusted to a concentration of I mg / mL using phosphate-buffered saline (pH 7.9). One milliliter of this solution was incubated with an equal volume of blood group O, Rh-negative screening cells for 60 minutes at 37 deg C. The coated and uncoated red blood cells were washed 3 times in phosphate-buffered saline. Two drops of the patients undiluted predrug and postdrug serum samples were mixed with one drop of a 5% saline suspension of drug-coated and uncoated red blood cells and incubated for 30 minutes at 37 deg C. The cells were then observed for agglutination. The positive control consisted of check cells coated with anti-D. The negative controls consisted of drug-coated and uncoated red blood cells incubated with serum from an AB-negative individual. Phycoerythrin-labeled goat anti-human F(ab) antibodies (Biosource International, Camarillo, Calif) were added to each sample, incubated for 30 minutes, and washed 3 times in saline. Flow cytometric analysis was performed using a Coulter EPICS XL flow cytometer (Coulter Electronics, Hialeah, Fla). Ten thousand events were acquired by gating on forward scatter and right angle side scatter, collecting 40000 events. A single parameter histogram was displayed with fluorescence intensity on the x-axis and a 4-decade, logarithmic scale. Results were recorded as the percentage of positive cells above a threshold defined by the upper limit of fluorescence observed with the negative control.

RESULTS

The results of DATs performed on a pretransfusion blood sample drawn on POD 8 were positive using broadspectrum antiglobulin reagents IgG (3+) and C3 (strong microscopic +). The results of a DAT performed on the admission blood sample were negative. The patients antibody screen was negative using both admission and pretransfusion samples. An eluate performed on the red blood cells from a pretransfusion sample was nonreactive with panel red blood cells.

The results of flow cytometry using red blood cells coated with cefotetan and uncoated red blood cells are shown in the Figure and are summarized in Table 1. Antibody binding to cefotetan-treated red blood cells was observed using the pretransfusion, postdrug sample drawn on POD 8 but not observed with the admission sample. No antibody binding to cefazolin-treated red blood cells or untreated cells was observed (Table 1). Dilutions of the patients serum from 1:10 to 1:1000 000 were tested by flow cytometry using cefotetan-coated red blood cells (Table 2). Antibody binding to coated cells was detected at dilutions as high as 1:10 000.

COMMENT

Only 10 previous case reports of immune hemolytic anemia secondary to cefotetan therapy have been published, but many additional cases have been reported in abstract form.1-6 It is likely that other cases have gone unrecognized or unreported. Positive results of DATs have been described in 1% to 2% of patients receiving cefotetan,1 although hemolytic anemia associated with this drug appears to be rare.

The patient described in this case report had a clinical course similar to those described previously.2,5 She received a brief course of cefotetan prophylactically following abdominal surgery, then developed severe immune hemolytic anemia requiring blood transfusions 8 days later. In previous reports, most patients with cefotetan-induced hemolytic anemia have required blood transfusions, and 2 fatalities have been described.4,5

We used a flow cytometric technique to evaluate this patient's serum and demonstrated that a serum drawn at the time when hemolysis was evident reacted with cefotetan-treated red blood cells but not with normal red blood cells or red blood cells coated with cefazolin, a firstgeneration cephalosporin. This antibody was detectable at a titer of 1:10 000 using flow cytometry. A serum sample drawn at the time of admission failed to react with cefotetan-treated red blood cells. Furthermore, all the patients serum samples were nonreactive when tested against untreated red blood cells. Flow cytometry has been used previously to detect red blood cell autoantibodies and alloantibodies,8 but this is the first report, to our knowledge, of its use for the detection of drug-induced antibodies. Although the antiglobulin test is sensitive and specific, flow cytometry offers a straightforward technique that is rapid, quantitative, objective, and sensitive. Furthermore, flow cytometry lends itself well to the detection of immunoglobulin classes and subclasses, as well as complement components, and to investigations of cross-reactivity between antibodies against closely related drugs, such as cefotetan and cefazolin.

The mechanism of immune hemolysis observed in this patient involved the so-called drug absorption or haptenspecific mechanism, in common with most of the previous case reports.2-6 Eluates from the patient's red blood cells and her serum samples failed to react with red blood cells in the absence of cefotetan. This antibody appears to be directed against a structural moiety unique to cefotetan, since her serum failed to react with cefazolin-treated red blood cells. Alternatively, cefotetan may impart a unique conformational change to the red blood cell surface as proposed by Garratty in a review article of drug-induced hemolytic anemia.9

Although, the drug absorption mechanism has been implicated in most previous reports of cefotetan-induced hemolytic anemia, a few examples of immune complex-mediated hemolysis2-5 and even autoantibody formation have been described.4,6 We found no evidence of autoantibody formation in this case using either the standard DAT or flow cytometry.

In summary, we have described a case of severe cefotetan-induced hemolytic anemia that we evaluated by flow cytometry. A thorough investigation to evaluate the possibility of drug-induced hemolytic anemia is warranted in severely anemic patients receiving second- and third-generation cephalosporins.

References

1I Wagner BKJ, Heaton AL, Flink JR. Cefotetan disodium-induced hemolytic anemia. Ann Pharmacother. 1992;26:199-200.

2. Ehmann CW. Cephalosporin-induced hemolysis: a case report and review of the literature. Am J Hematol. 1992;40:121-125.

3. Gallagher NI, Schergen AK, Sokol-Anderson ML, Sheahan EJ, Chaplin H. Severe immune-mediated hemolytic anemia secondary to treatment with cefotetan. Transfusion. 1992;32:266-269.

4. Garratty G, Nance S, Lloyd M, Domen R. Fatal immune hemolytic anemia due to cefotetan. Transfusion. 1992;32:269-271.

5. Peano MG, Menardi G, Quaranta L, Abello P, Landra M, Fenoglio S. A rapidly fatal case of immune haemolytic anaemia due to cefotetan. Vox Sang. 1994;66:84-85.

6. Chenoweth CE, Judd WJ, Steiner AE, Kauffman CA. Cefotetan-induced immune hemolytic anemia. Clin Infect Dis. 1992;15:863-865.

7. Vengelen-Tyler V, ed. Technical Manual of the American Association of Blood Banks. 12th ed. Bethesda, Md: American Association of Blood Banks; 1996:669-670.

8. Garratty G, Arndt P. Applications of flow cytometry to transfusion science. Transfusion. 1995;35:157-178.

9. Garratty G. Immune hemolytic anemia and/or positive direct antiglobulin tests caused by drugs. Immunohematology. 1994; 10:41-50.

Accepted for publication January 11, 2000.

From the Department of Pathology, Fletcher Allen Health Care, Burlington, Vt.

Financial support provided by the Department of Pathology at Fletcher Allen Health Care/University of Vermont College of Medicine.

Reprints: Gregory S. Moes, MD, Department of Pathology, Fletcher Allen Health Care, 111 Colchester Ave, Burlington, VT 05401.

Copyright College of American Pathologists Sep 2000
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