Ceftriaxone-Induced Granulopenia Related to a Peculiar Mechanism of Granulopoiesis Inhibition DAVID REY, M.D., THIERRY MARTIN, M.D., ANNE ALBERT, M.D., JEAN-LOUIS PASQUALI, M.D., Ph.D. .wJSbOUfg, France

B eta-la&m antibiotics are sometimes responsible for severe neutropenia in treated patients. The

mechanisms of this potentially serious side effect seem heterogeneous, since the beta-la&am-induced neutro- penias have been reported to be related either to an immune-mediated process affecting peripheral leuko- cytes [l-3] or to direct toxicity of the drugs to bone- marrow precursors. In particular, recent in vitro data showed a correlation between the inhibitory capacity of beta-lactam antibiotics on granulocyte precursors and the doses inducing neutropenia in vivo [4]. We present a case report that emphasizes a higher degree of complexity of the mechanism underlying a beta- lactam (ceftriaxone)-induced granulopenia. This com- plexity can be related to two different events: one is humoral (a serum factor), the other is cellular and located at the level of the bone-marrow granulocyte precursors. Both of these drug-driven related events were reversible.

CASE REPORT The patient was an BO-year-old woman (body

weight: 69 kg) who was admitted to the hospital be- cause of fever, cough, and weight loss. The clinical examination and the chest radiograph showed pneu- monia of the lingula. Transtracheal needle aspiration isolated Pseudomonas stutzeri that was sensitive to cephalosporins. The patient was initially treated with cefotaxime (3 g/day) and netilmicin for six days. Be- cause repeat radiologic evaluation showed the forma- tion of a lung abscess, the treatment was changed to ceftriaxone plus netilmicin, then to ceftriaxone alone (2 g/day). On the fourth day of this regimen, the serum ceftriaxone peak level was 88 pg/mL and the trough level was 16 pg/mL (high-performance liquid chroma- tography). Two weeks after this change, severe neutro- penia occurred (Figure 1). The platelet, lymphocyte, and erythrocyte counts were not modified, as well as the serum creatine and the liver function tests A bone-marrow aspirate was analyzed at the onset of leukopenia and showed a hypercellular granulopoiesis with arrest at the promyelocytic and myelocytic stages. Ceftriaxone was discontinued, and the granulo- cyte count progressively returned to normal values. As shown in Figure 1, two other bone-marrow aspirates (A and B) were obtained 18 and 58 days, respectively, after the onset of leukopenia, both after the recovery of normal leukocyte counts. The cells of these two aspirates were cultured in soft agar using the colony assay technique to measure the colony-forming unite of the granulocyte-monocyte lineage (CFU-GM) in the presence of human placental-conditioned medium [5,6]. Table I shows the results of the different assays

that were developed to document the effects of the patient’s serum samples, which were drawn during leukopenia (serum no 1 sample) and after recovery (se- rum no2 sample), on her own granulocyte precursors. The patient’s serum no1 sample strongly inhibited early (seven days) and late (12 days) colony formation of the 18-day bone-marrow cells A. Normal serum Al3 samples (control) did not affect CFU-GM formation from the patient’s two bone-marrow aspirates A and B, whether or not ceftriaxone was added during the in vitro assay. Because of the limited number of bone- marrow cells A, we were unable to test different con- centrations of ceftriaxone. The concentration of 35 pg/ mL was chosen because it was within the range of the patient’s serum levels of the drug.

COMMENTS The mechanism of the present beta-lactam-induced

neutropenia seems to differ from previously reported cases of drug-induced neutropenia. The in vitro inhibi- tion of the patient’s CFU-GM colony formation (1) did not necessitate the presence of the inducing drug cef- triaxone, (2) occurred in the presence of a transient serum factor, and (3) was only documented in early precursors of the patient’s bone marrow cells.

Among the beta lactams, the cephalosporins are known to be responsible for in vitro inhibition of colo- ny formation in a dose-dependent manner [4,7]. How- ever, as shown in Table I, ceftriaxone (35 pg/mL) re- duced to 50% the cluster formation from the patient’s bone-marrow cells without affecting colony formation. Furthermore, the patient’s serum no1 sample was drawn five days after the drug was discontinued and did not contain any detectable ceftriaxone. We cannot formally exclude the possibility that some potential degradation products of the drug were still present in the serum no1 sample, although it seems unlikely that such products could be present five days after the drug was discontinued. Nevertheless, this serum sample had an important inhibitory effect on the patient’s colony formation in.vitro (bone-marrow A).

The existence of a pathogenic transient serum fac- tor was previously reported during quinidine-induced neutropenia in which the patient’s serum inhibited the in vitro growth of allogeneic granulocytic colonies only in the presence of the responsible drug [8,9]. The for- mation of drug-antibody complexes could explain this type of neutropenia as it was also suggested during amidopyrine-induced agranylocytosis [lo]. On the other hand, unknown degradation products of cef- triaxone could also represent this transient serum fac- tor, providing that they have a very long half-life, which does not seem to be the case [ll].

Finally, the autologous bone-marrow cells were only transiently sensitive to the patient’s serum no1 sam- ple. During the acute phase of leukopenia, the bone- marrow analysis showed typical maturation arrest of the granulocyte lineage. The possible explanations be- hind these granulocytic growth and differentiation in-

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CEFT’RIAXONE-INDUCEDGRANULOPENIA / REY ET AL

Ceftriaxone b.marrow A b.marrowB

serum1 serum 2 I

I 10

I 20

// I 0

30 72 Days Figure 1. Evolution of the patient’sgran- ulocyte counts.

TABLE I

CFU-GM Formation of the Patient’s Bone-Marrow Cells*

BoneMarrow A Bone-Marrow B (J 18) (J 58)

Early CFU-GM Late CFU.GM Early CFU-GM Late CFU-GM

Serum AB 172 colonies 51 colonies 174 colonies 52 colonies (controls) 39 clusters 48 clusters

Serum Al3 163 colonies 40 colonies ND* + ceftriaxone 35 pg/mL 18 clusters Serum no lf 1 colony 0 colony 214 colonies 60 colonies

J5 4 microclusters 45 clusters Serum no 2 219 colonies 72 colonies ND

J18 37 clusters

ND = not done l The pabent’s sera were frozen at -80” until use. The CFU-GM formations were tested at seven (early) and 12 (late) days of incubation (37°C. 5% carbon dioxide, and 100% humidity). r Serum no 1 did not contain any detectable ceftrraxone as judged by high-pressure liquid chromatography analysis.

hibitions could be related in oivo to the disease itself, to the drug itself, or to drug-induced events. It is unlikely that the infectious disease was responsible for the leu- kopenia since the patient was recovering from the lung abscess when the leukocyte count dropped. The drug alone was probably not responsible for the growth and maturation arrests of the granulocyte lineage since its in vitro addition to the patient’s cells plus control se- rum AB did not inhibit CFU-GM formation. Thus, it is tempting to suggest that ceftriaxone induced in vivo (1) transient modification of granulocyte precursors and (2) the appearance of a serum factor (immuno- globulin?) that was targeted to this modification.

The in vivo consequence of these events (granulo- penia) would have been reproduced in vitro by the colony assay using the early serum no1 sample and the early bone-marrow cells A.

It is difficult to estimate the frequency of this postu- lated mechanism of drug-induced neutropenia, since it would necessitate testing in vitro the patient’s own sensitive granulocyte precursors with an early autolo- gous serum sample and serum samples from patients not having ceftriaxone-induced neutropenia as a com- plementary control. These difficulties could account for eventual negative results as, for example, when

CFU formation is assayed too late, and could make impossible the identification of the serum factor if early precursor cells (target cells) are no longer avail- able, as occurred in our case.

REFERENCES 1. Weitzman SA. Stossel TP. Desmond M: Drug-induced immunological neutro- penia. Lancet 1978; II: 1068. 2. Rouveix B. Lassoved K. Vittecoq D. Regnier 8: Neutropenia due to r3 lactamine antibodies. Br Med J 1983; 287: 1832-1834. 3. Murphy MF. Metcalfe P, Grint PCA. et at Cephalosporin-induced immune neu- tropenia. Br J Haematol 1985; 59: 9-14. 4. Neftel KA, Hauser SP. Muller MR: fnhibition of granulopoiesis in vivoand in vitro by @actam antibiotics. J Infect Dis 1985: 152: 90-98. 5. Pike BL, Robinson WA: Human bone marrow colony growth in agar gel. J Cell Physiol 1970: 76: 77-84. 6. Burgess AW. Wilson EM. Metcalf D: Stimulation by human placental conditioned medium of hemopoietic colony formation by human bone marrow cells. Blood 1977; 49: 573-585. 7. Neftel KA. Hubscher U: Effects of ,%lactam antibiotics on proliferatingeukaryotic cells. Antimicrob Agents Chemother 1987; 31: 1657-1.661. 8. Eisner EV. Carr RM, MacKinney AA: Quinidine induced agranulocytosis. JAMA 1977; 238: 884-886. 9. Kelton JG. Huang AT, Mold N, Logue G. Rosse WF: The use of in vitro techniques to study drug-induced pancytopenia. N Engl J Med 1979; 301: 621624. 10. Barrett AJ. Weller E, Rozengurt N, Longhurst P. Humble JG: Amidopyrine agranulocytosis: drug inhibition of granulocyte colonies in the presence of patients serum. Br Med J 1976: 2: 850-851. 11. Pate1 IH. Kaplan SA: Pharmacokinetic profile of ceftriaxone in man. Am J Med 1984; 19 (suppl): 17-25.

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