Neutropenia induced in a patient by treatment with ritodrine: in vitro evaluation of myeloid progenitor colony-forming cells

Ritodrine hydrochloride, a β₂ symphathomimetic agent, has been widely used for tocolysis for many years. Although ritodrine can be used relatively safely if pulse rate, respiration, blood pressure, serum glucose and potassium are monitored, the drug has been associated with some undesirable maternal complications, such as pulmonary oedema [1], myocardial ischaemia [2] and neutropenia [3–5]. The pathophysiological mechanisms of drug actions leading to neutropenia are poorly understood. We report a case of ritodrine-induced neutropenia at 34 weeks' gestation, in which we studied the effect of ritodrine on the formation of erythroid burst-forming units (BFU-E) and granulocyte macrophage–colony-forming units (CFU-GM) from the mononuclear fraction of her bone marrow.

A 31-year-old Japanese woman, gravida 2, para 1, was referred to our hospital at 30 weeks and 5 days of gestation for preterm labour. She had no history of infection, premature rupture of membrane, fever, neutropenia or recent exposure to any drugs or toxins except a continuous intravenous infusion of ritodrine at 200 µg min⁻¹ since the previous day. On admission, the white blood cell (WBC) count was 7790 mm⁻³ and the neutrophil count 6638 mm⁻³. She was started on MgSO₄ at 1 g min⁻¹ intravenously.

The WBC count decreased to 3580 mm⁻³ at 32 weeks' gestation and to 2390 mm⁻³ at 33 weeks' gestation. At 34 weeks and 2 days of gestation, the WBC count was 790 mm⁻³ and the neutrophil count 16 mm⁻³. Haemoglobin remained 10–11 g dl⁻¹, and the platelet count also stayed in the normal range. Ritodrine was discontinued and she entered labour. On the same day, she delivered a male infant weighing 2202 g. The neonate had a normal WBC count. After delivery, granulocyte–colony-stimulating factor (Sankyo Pharm. Co., Tokyo, Japan) was administered for 4 days (100 µg daily). The WBC counts rose to 2210 mm⁻³, 5190 mm⁻³ and 8470 mm⁻³ on the second, third and seventh postpartum days, respectively. Blood and urine cultures were negative. She was discharged on the day 8 postpartum without any complications.

We obtained bone marrow samples at 34 weeks and 2 days of gestation. Bone marrow aspiration showed decreased myelopoiesis, especially of mature neutrophils (promyelocytes 5.8%, myelocytes 2.2%, metamyelocytes 1.0%, stab cells 0.8%, segmented cells 0.4%), without myelodysplastic change, but normal erythropoiesis, megakaryoctyopoiesis, and normal stroma. We also studied the effects of ritodrine on the growth of colonies from the mononuclear fraction of the bone marrow. Mononuclear cells were isolated from the bone marrow by density centrifugation and incubated in soft agar cultures. The colony assays of BFU-E and CFU-GM were performed as previously described [6]. Ritodrine hydrochloride (Kissei Pharm. Co., Ltd, Matsumoto, Japan) was added to the mononuclear cell suspension in final concentrations of 300 and 3000 ng ml⁻¹, and cells were incubated for 10 days, at which point colonies were counted. Colonies with >40 cells were scored using an inverted microscope. The effect of serum from the patient at 34 weeks and 2 days of gestation on the growth of colonies was also studied in the same manner.

Ritodrine inhibited the in vitro growth of BFU-E and CFU-GM when added to a concentration of 3000 ng ml⁻¹ (Figure 1). However, at a ritodrine dose of 300 ng ml⁻¹ there were no significant differences between control and ritodrine-containing medium in BFU-E and CFU-GM assays. Note that 300 ng ml⁻¹ ritodrine exceeded the therapeutic blood level in preterm labour patients under ritodrine treatment (infusion rates of 0.06–4.0 µg kg⁻¹ min⁻¹ yielded steady-state ritodrine serum concentrations of 5–168 ng ml⁻¹[7]). In addition, serum from the patient did not inhibit the growth of BFU-E and CFU-GM.

Figure 1.

Effect of ritodrine on the growth of colonies from the mononuclear fraction of the bone marrow. Controls were incubated with 40% fetal bovine serum, 10 ng ml⁻¹ of interleukin (IL)-3 and 10 ng ml⁻¹ of granulocyte macrophage–colony-stimulating factor in the granulocyte macrophage–colony-forming unit colony assay, and 40% fetal bovine serum, 10 ng ml⁻¹ of IL-3 and 2 U ml⁻¹ of erythropoietin in the erythroid burst-forming unit colony assay. Results are expressed as mean value ± SD of three separate experiments. *Statistically significant differences (P < 0.05) between treatment and controls

Drug-induced neutropenia may be the result of nondose-related immune-mediated toxic reactions and/or dose-related bone marrow suppression [8]. The immune-mediated reaction causes a precipitous decline in granulocytes a short time after ingestion of a small amount of the drug [8]. In ritodrine-induced neutropenia, bone marrow examination showed an absence of mature WBCs, suggesting that ritodrine-induced neutropenia is related to reduced production of granulocytes [4] or an impaired WBC maturation process [5]. In the present case, there was a decrease in the number of mature neutrophils in the bone marrow aspirate. Interestingly, in vitro colony assays showed that ritodrine did not inhibit the growth of colonies of either BFU-E or CFU-GM at therapeutic blood levels. It has been reported that ritodrine did not inhibit in vitro colony formation of CFU-BM obtained from healthy nonpregnant women [9]. These results suggest that ritodrine has no effect on precursor cell proliferation, but on their maturation process. Although further study is necessary to clarify the exact mechanisms by which ritodrine induces neutropenia, this complication needs to be considered in cases where long-term as well as high-dose administration of ritodrine is contemplated.