Drug-induced agranulocytosis: evidence for the commitment of bone marrow haematopoiesis

Clinical and haematological features of 61 patients with drug-induced agranulocytosis (63 episodes) are presented. Multiple drug consumption was a common observation and complicated the attempt to incriminate a particular drug as being aetiologically involved. Bone marrow analysis shortly after the diagnosis revealed evidence for an impairment of proliferative granulopoiesis in the majority of cases. This observation was confirmed by in vitro culturing of granuloid precursor cells (CFU-c). Moreover, the data clearly demonstrated that drug-induced agranulocytosis may not be restricted to the granulocytic series. Thrombocytosis and reticulocytosis during the recovery phase are taken as an indication for the commitment of all haemopoietic cell lineages in agranulocytosis. These observations were in accordance with cytomorphological studies and in vitro culture data of erythroid precursor cells (CFU-e, BFU-e) of bone marrow aspirates taken in the initial phase of agranulocytosis. More than 25% of the patients showed a marked erythroid depression in the marrow.     

Agranulocytosis induced by propylthiouracil: evidence of a drug dependent antibody reacting with granulocytes, monocytes and haematopoietic progenitor cells

A patient with agranulocytosis and myeloid marrow hypoplasia following a second exposure to propylthiouracil (PTU) was studied for antibodies against mature blood cells and bone marrow precursor cells. During the acute phase of the agranulocytosis, significant growth inhibition of the myeloid committed progenitor cells (CFU-GM) was found following incubation with complement, indicating the presence of in-vivo cell bound cytotoxic antibodies. Using immunofluorescence and complement dependent cytotoxicity techniques it was demonstrated that acute phase and recovery phase sera contained circulating antibodies, reactive not only with differentiated granulocytes and monocytes but also with myeloid and erythroid (BFU-E/CFU-E) progenitor cells. Complement dependent lysis of the progenitor cells was facilitated by preincubation with PTU. These results indicate that the agranulocytosis was mediated by a PTU dependent antibody that affected both mature blood cells and bone precursor cells.     

The role of drugs and lymphocytes in granulocyte-macrophage colony formation in patients with drug induced agranulocytosis

The in vitro effects of the causative drugs and lymphocytes from the patients with agranulocytosis were tested against granulocyte-macrophage colony formation (CFU-C) of bone marrow cells from normal individuals and the patients in recovery stage. A semisolid culture system was used for CFU-C assay. The drug concentrations were adjusted to the therapeutic levels in sera, and the lymphocytes were obtained from the patient's peripheral blood. Three patients with agranulocytosis and one patient with pancytopenia caused by disopyramide, methimazole, sodium valproate, and Towasaal, respectively, were examined. Each of the four drugs except disopyramide suppressed the CFU-C of normal and patient's bone marrow cells in a dose-dependent manner. When the patient's bone marrow cells were cultured with respective drugs and their own lymphocytes or with the culture supernatant of the drug and lymphocytes, CFU-C suppressions was significantly augmented. Phenacetin, an agent of Towasaal, significantly suppressed CFU-C and also CFU-E. These results indicate that humoral factor(s) produced from patient's lymphocytes by reacting with the drugs may function as an immunological mechanism in the patients with drug-induced agranulocytosis.     

Procainamide-induced agranulocytosis with reversible myeloid sensitivity

A 56-year-old man developed procainamide-induced agranulocytosis. Bone marrow aspiration showed the absence of myeloid elements beyond the promyelocyte stage. Procainamide at therapeutic concentrations in vitro depressed bone marrow granulocyte-macrophage colonies (CFUc) while CFUc growth was normal in the absence of drug. Following the patient's recovery, CFUc growth was no longer suppressed by procainamide in vitro. The observations in this patient showing reversible sensitivity to drug contrast with those on other patients with drug-induced agranulocytosis that show persistent marrow injury or drug sensitivity despite clinical recovery.     

Erythroid colony studies on sickle cell anemia in hypoproliferative crisis

Bone marrow cells, peripheral blood lymphocytes, and sera from patients with sickle-cell anemia in hypoproliferative crisis were studied in the plasma clot culture system in the presence or absence of erythropoietin (Epo). Bone marrow cells from five patients demonstrated a marked ability to form erythroid colonies in the presence of Epo. These studies also suggested that bone marrow cells from some patients may have an increased sensitivity to Epo. The most outstanding observation in the present study was the marked erythroid colony inhibition by serum taken from one patient during crisis. Serum taken from the same patient two months after hypoproliferative crisis had no suppressive effect on erythroid colony formation. Lymphocytes taken from three patients in crisis had a stimulatory effect on erythroid colony formation when included in culture. The conclusion is that the defect of erythropoiesis in sickle-cell anemia during hypoproliferative crisis is not due to the absence of erythroid precursor cells or to the presence of suppressor lymphocytes, but may in some cases be associated with a circulating inhibitor of erythroid maturation.     

Serum myeloperoxidase and lactoferrin in neutropenia.

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.     

Serum Myeloperoxidase and Lactoferrin in Neutropenia

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more mature granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.     

Isobutyramide, an orally bioavailable butyrate analogue, stimulates fetal globin gene expression in vitro and in vivo

Summary. Clozapine, a novel antipsychotic drug that is particularly effective in treatment-resistant schizophrenia, causes severe agranulocytosis of unknown aetiology in approximately 0·8% of U.S. patients. We evaluated potential toxic mechanisms of drug-induced agranulocytosis. Clozapine, the two major metabolites N-desmethylclozapine and N-oxide clozapine, and five other clozapine derivatives were screened for toxicity to normal haemopoietic precursors. For all compounds except N-des-methylclozapine, toxicity to CFU-GM, BFU-E and CFU-GEMM occurred at concentrations at least 10 times the normal serum levels reported in unaffected patients. In contrast, the LD₅₀ for N-desmethylclozapine was 2·5 μg/ml for CFU-GM, 3·2 μg/ml for BFU-E, and 2·4 μg/ml for CFU-GEMM, only 3–6 times the normal serum concentration. Bone marrow from patients with acute clozapine-induced agranulocytosis was not more sensitive to clozapine or N-desmethylclozapine than bone marrow from normal donors. These studies suggest that N-desmethylclozapine, the major metabolite of clozapine, is itself toxic or is further metabolized to an unstable compound which is toxic to haemopoietic precursors of both myeloid and erythroid lineages.     

Mebhydrolin Induced Agranulocytosis

Abstract: Mebhydrolin induced agranulocytosis . G. A. R. Young, P. Forrest, S. F. Deveridge, R. B. Gates and P. C. Vincent, Aust. N.Z. J. Med., 1982,12, pp. 173–176.
Two cases of agranulocytosis are described, in which the antihistamine mebhydrolin (Fabahistin, Bayer) was taken shortly before the onset of agranulocytosis. In one case, agar culture techniques demonstrated that the patient's acute phase plasma in the presence of complement inhibited the growth of granulocyte/monocyte precursor cells (CFU–C) from her marrow. Both patients recovered; in one, lithium carbonate may have aided recovery.     

Granulopoiesis in Infantile Genetic Agranulocytosis In vitro Cloning of Marrow Cells in Agar Culture

A 3-year-old boy and a 2-year-old girl with infantile genetic agranulocytosis have been studied by in vitro cloning of bone marrow cells in agar culture. The patients display a normal concentration of colony forming cells and the morphological maturation is identical with that of control marrow cultured in vitro. The marrow cells of the patients show some degree of auto-stimulation indicating that endogenous production of colony stimulating factor is operating. As an inverse relationship is expected between the peripheral neutrophil count and the percentage of marrow colony forming cells in S-phase a high percentage was expected. On the contrary, we find that the percentage of colony forming cells in S-phase is extremely low indicating a genetic unresponsiveness of granulopoietic precursor cells to feed back regulation in infantile genetic agranulocytosis.     

Granulopoiesis in infantile genetic agranulocytosis. In vitro cloning of marrow cells in agar culture.

A 3-year-old boy and a 2-year-old girl with infantile genetic agranulocytosis have been studied by in vitro cloning of bone marrow cells in agar culture. The patients display a normal concentration of colony forming cells and the morphological maturation is identical with that of control marrow cultured in vitro. The marrow cells of the patients show some degree of auto-stimulation indicating that endogenous production of colony stimulating factor is operating. As an inverse relationship is expected between the peripheral neutrophil count and the percentage of marrow colony forming cells in S-phase a high percentage was expected. On the contrary, we find that the percentage of colony forming cells in S-phase is extremely low indicating a genetic unresponsiveness of granulopoietic precursor cells to feed back regulation in infantile genetic agranulocytosis.     

Erythropoiesis during an erythroblastic phase of chronic myeloproliferative disorder associated with monosomy 7

A chronic myeloproliferative disorder associated with monosomy 7 is described in a 3 1/2-year-old boy. His presenting features closely resembled those of juvenile chronic myeloid leukaemia (JCML). Cytogenetic study of bone marrow cells showed that all of the metaphases examined had chromosome 7 deletions. He developed an erythroblastic phase, characterized by anaemia, marked erythroid hyperplasia of bone marrow and the appearance of nucleated red blood cells in the peripheral blood. During the erythroblastic phase, blood transfusion resulted in a suppression of erythropoiesis as evidenced in both the peripheral blood and bone marrow. The in vitro culture studies showed that the erythroid precursor was dependent upon erythropoietin (Ep) for differentiation and proliferation during the erythroblastic phase. However, the Ep dose-response curve showed that a peak of erythroid colony formation occurred at a lower concentration than in the healthy controls. These findings suggest that although the erythroid precursor remains under the control of Ep, it has an increased sensitivity to Ep during the erythroblastic phase of monosomy 7.     

Chlorpropamide-induced pure white cell aplasia

We investigated the mechanism for isolated agranulocytosis and marrow pure white cell aplasia in an elderly man receiving 0.5 to 1.0 g per day of chlorpropamide (Chl) without other toxic drug exposure or overt systemic illness. Patient marrow revealed an absence of recognizable granulocytic precursors; megakaryocytes and erythroid precursors were normal. The WBC count was 1800/mm3 on admission with only 2% neutrophils; the absolute neutrophil count first exceeded 500/mm3 on the 17th day following cessation of Chl. A serum Chl level on admission was 100 micrograms/mL (acute phase, AP); no Chl was detected in serum (convalescent phase, CP) assessed on the 22nd hospital day. Antineutrophil antibodies were not detected, and T cell depletion failed to augment patient in vitro granulopoiesis. Patient AP serum produced potent complement-mediated inhibition (87% +/- 7%) of autologous granulocyte progenitors (CFU-GM) with minimal inhibition of erythroid (11% +/- 5%) or multipotent (5% +/- 4%) progenitor cells. Selective inhibition by patient AP serum of CFU-GM (74% +/- 11%) was also seen against two allogeneic marrows. Patient CP serum no longer inhibited (6% +/- 4%) autologous CFU-GM. Addition of Chl (5 to 120 micrograms/mL) to CP serum but not to control serum resulted in potent drug concentration-dependent complement-mediated inhibition of autologous and allogeneic CFU-GM. Inhibition of CFU-GM in the presence of Chl was no longer demonstrable following immunoabsorbent removal of IgG from patient serum. Patient serum in the presence of Chl had limited activity against morphologically recognizable marrow granulocytic precursors in a microimmunofluorescence assay. These results are most consistent with the development of Chl-dependent, selective antibody-mediated immune inhibition of granulopoiesis.     

Mechanism of aprindine induced agranulocytosis: direct toxicity on CFU-C and CFU-GEMM

The in vitro bone marrow growth of 2 patients with aprindine-induced agranulocytosis was studied. Granulocyte-macrophage committed stem cell (CFU-C) growth is inhibited by aprindine in a dose dependent manner. 50% inhibition of CFU-colony growth (TD50) was seen at 5.1 and 3.4 micrograms aprindine/ml medium respectively. The TD50 of control marrow CFU-C was 3.2 micrograms/ml. 100% inhibition was seen at 16 micrograms aprindine/ml, both in patients and controls. Pluripotential stem cell growth (CFU-GEMM) in control marrow was equally inhibited in a dose dependent manner by aprindine, though to a lesser extent (TD50: 9.1 micrograms/ml) and with relative sparing of pure megakaryocyte and erythroid colonies. Co-culturing of patients marrows with their respective acute phase serum did not inhibit CFU-C growth.     

Characterization of the hematopoietic defect in paroxysmal nocturnal hemoglobinuria

We studied the relationship between paroxysmal nocturnal hemoglobinuria (PNH) and bone marrow failure using in vitro hematopoietic colony culture assays. Most of 17 patients with PNH showed decreased colony formation, by erythroid burst-forming cells (BFU-E) and granulocyte-macrophage colony-forming cells (CFU-C) in methylcellulose, disproportionate to their degree of bone marrow biopsy cellularity. Only a minority of the hematopoietic progenitors were sensitive to complement-mediated lysis in vitro. In contrast, normoblasts from maturing erythroid bursts removed from culture and exposed to acidified serum were sensitive to complement-mediated lysis. The size of bursts and the sensitivity of their progeny correlated strongly, suggesting that the PNH defect was acquired in culture as a function of the generational age of erythroid precursor cells. In addition, BFU-E of PNH patients were very sensitive to 3H-thymidine suicide, in comparison with normal individuals and patients with other hemolytic anemias, indicating that a large proportion of primitive erythroid progenitors in PNH bone marrow were in cell cycle. All of these results imply that acquisition of the PNH defect during erythropoiesis may lead to intramedullary destruction of developing erythroid cells. The increased demand that results on the progenitor pool may lead to stem cell depletion and bone marrow failure.     

Effect of mast cell growth factor (c-kit ligand) on clonogenic leukemic precursor cells.

Mast cell growth factor (MGF), the ligand for the c-kit receptor, has been shown to be a hematopoietic growth factor that preferentially stimulates the proliferation of immature hematopoietic progenitor cells (HPC). We studied the effect of MGF on the in vitro growth of clonogenic leukemic precursor cells in the presence or absence of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or erythropoietin (EPO). Leukemic blood and bone marrow cells from patients with various types of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) in chronic phase, as well as bone marrow samples from patients with myelodysplastic syndromes (MDS) were studied. MGF as a single factor did not induce significant colony formation by clonogenic leukemic precursor cells. In the presence of IL-3 and/or GM-CSF, MGF weakly stimulated the colony formation by clonogenic precursor cells from patients with AML. In contrast, in the presence of IL-3 and/or GM-CSF, MGF strongly induced both size and number of leukemic colonies from patients with CML in chronic phase. Furthermore, in the presence of EPO, MGF strongly stimulated erythroid colony formation by CML precursor cells. Cytogenetic analysis of the colonies showed that all metaphases after 1 week of culture were derived from the leukemic clone. In patients with MDS, MGF strongly stimulated myeloid colony formation in the presence of IL-3 and/or GM-CSF (up to fourfold), and erythroid colony formation in the presence of EPO (up to eightfold). Not only the number, but also the size of the colonies increased. In the presence of MGF, the percentage of normal metaphases increased in three patients tested after 1 week of culture compared with the initial suspension, suggesting that the normal HPC were preferentially stimulated compared with the preleukemic precursor cells. In the absence of exogenous EPO and in the presence of 10% human AB serum, MGF in the presence of IL-3 and/or GM-CSF induced erythroid colony formation from normal bone marrow and patients with MDS or CML, illustrating that MGF greatly diminished the EPO requirement for erythroid differentiation. These results indicate that MGF may be a candidate as a hematopoietic growth factor to stimulate normal hematopoiesis in patients with acute myeloid leukemia, or with myelodysplastic syndromes.     

Agranulocytosis associated with "Mexican aspirin" (dipyrone): evidence for an autoimmune mechanism affecting multipotential hematopoietic progenitors

A case of acute, transient agranulocytosis and thrombocytopenia associated with ingestion of dipyrone is reported. This once widely used analgesic, which is now banned in the United States, was obtained by the patient as "aspirin" while traveling in Mexico. Studies of the effects of this patient's serum on purified CD34+ marrow cells, which were highly enriched for hematopoietic progenitors, showed not only a drug-dependent suppression of the in vitro growth of myeloid progenitors, as has been reported previously, but also a drug-dependent suppression of primitive multipotential progenitors (CFU-Mix) and erythroid progenitors (BFU-E). These findings indicate that autoimmune, antibody-hapten interactions which have been reported to occur in dipyrone- and aminopyrine-induced agranulocytosis are not restricted to the neutrophil lineage.     

Methimazole-induced agranulocytosis: growth inhibition of myeloid progenitor cells by the patient's serum

The mechanism for agranulocytosis induced by antithyroid drugs is not established. The few available studies have proposed an immune-mediated process against mature granulocytes. We investigated the effect of methimazole and propylthiouracil and serum from a patient with methimazole-induced agranulocytosis on marrow myeloid colony growth. In the presence of normal serum or patient's recovery serum, antithyroid drugs had no effect on the growth of CFU-GM colonies from normal or patient's marrow. However, the patient's serum obtained during agranulocytosis inhibited the in vitro myeloid colony growth from both autologous and allogeneic bone marrow. These results are compatible with an immune-mediated mechanism for methimazole-induced agranulocytosis rather than a direct toxic effect of the drug on abnormally sensitive cells.     

Expression of transferrin receptor 2 in normal and neoplastic hematopoietic cells.

Iron is essential for cell proliferation, heme synthesis, and a variety of cellular metabolic processes. In most cells, transferrin receptor-mediated endocytosis is a major pathway for cellular iron uptake. Recently, transferrin receptor 2 (TfR2), another receptor for transferrin, was cloned. High levels of expression of TfR2 messenger RNA (mRNA) occur in the liver, as well as in HepG2 (a hepatoma cell line) and K562 (an erythroid leukemia cell line). In this study, TfR2 mRNA expression was analyzed in hematological cell lines, normal erythroid cells at various stages of differentiation, and leukemia and preleukemia cells. High levels of TfR2 expression occurred in all of the erythroid cell lines that were examined. Erythroid-specific expression of TfR2 protein in bone marrow cells was confirmed by immunohistochemical staining. Expression of TfR2 mRNA was high in normal CD34(+) erythroid precursor cells, and levels decreased during erythroid differentiation in vitro. Levels of expression of TfR2-alpha mRNA were significantly higher in erythroleukemia (M6) marrow samples than in nonmalignant control marrow samples. In addition, relatively higher levels of TfR2-alpha mRNA expression occurred in some samples of myelodysplastic syndrome that had erythroid hyperplasia in bone marrow, acute myelogenous leukemia M1, M2, and chronic myelogenous leukemia. Expression profiles of normal members of the erythroid lineage suggest that TfR2-alpha may be a useful marker of early erythroid precursor cells. The clinical significance of TfR2-alpha expression in leukemia cells remains to be determined.     

Isolation and in vitro differentiation of human erythroid precursor cells.

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.