Extramedullary haemopoiesis in fetal and adult human spleen: a quantitative immunohistological study

Haemopoietic cells were assessed in spleens from normal adults, adults with splenic extramedullary haemopoiesis due to chronic myeloproliferative disorders and fetuses of 17–21 weeks' gestation. A variety of antigens expressed by developing granulocytes and erythrocytes were demonstrated immunohistochemically. The relative proportions of early and late precursor cells of these two lineages were quantified. There was no significant haemopoiesis in normal adult spleen, while there was abundant (predominantly granulocytic) haemopoiesis in patients with chronic myeloproliferative disorders. Fetal spleens contained numerous late erythroid precursors but few early erythroid or granulocytic cells. The relative numbers of early and late haemopoietic cells in adult chronic myeloproliferative disorders and fetal spleens showed statistically significant differences. Our findings indicate that haemopoiesis in the spleens of adult patients with these disorders differs fundamentally from that occurring in fetal life. They support the view that the human spleen does not have a significant role in fetal haemopoiesis, but that it filters circulating nucleated erythroid precursors and is permissive of their terminal differentiation only. Our results also favour the view that adult splenic haemopoiesis originates by displacement of precursor cells from the bone marrow rather than by activation of stem cells which have lain dormant in the spleen since fetal life.     

Bone marrow mast cell morphologic features and hematopoietic dyspoiesis in systemic mast cell disease

Systemic mast cell disease (SMCD) cannot be distinguished from reactive mastocytosis (RM) by quantitation of mast cells in aspirate smears, and few studies have analyzed systematically the morphologic features of mast cells in SMCD vs RM. In addition, although SMCD is associated with myeloproliferative disorders/myelodysplastic syndromes (MPD/MDS), it is not known whether subtle signs of dysplasia or MPD can be found in SMCD, suggesting most cases are part of a dysplastic or myeloproliferative process. We compared 18 bone marrow specimens with SMCD with 10 bone marrow specimens from patients with RM. Mast cells in SMCD were more likely to show cytoplasmic hypogranularity, uneven granule distribution, and fusiform morphologic features. Eight cases of SMCD (44%) demonstrated MPD/MDS, and 9 cases (50%) showed subtle evidence of dyspoiesis, with megaloblastic change, nuclear budding of erythroid precursors, and/or atypical megakaryocytes. Mast cells in SMCD appear morphologically different from those in reactive proliferations. Dyspoietic features were present in most cases of SMCD, suggesting that SMCD is part of a spectrum of chronic myeloproliferative/myelodysplastic disorders.     

Histomorphological study of cellular interactions between stromal and haemopoietic stem cells in normal and leukaemic bone marrow

The interactions between haemopoietic and stromal elements are crucial for stem cell proliferation and differentiation. The bulk of this evidence is derived from experiments in rodents and in-vitro culture studies. We have studied the spatial relationships between the stromal and haemopoietic components and their cellular composition in histological sections of bone marrow (BM) from seven healthy fetuses, 10 normal adults and over 60 patients with acute myeloid leukaemia (AML) and chronic granulocytic leukaemia (CGL) at different stages of the disease. During the early developmental stage (16–18 weeks) fetal BM showed focal haemopoiesis with a characteristic spatial localization of haemopoiesis near bony trabeculae and around small blood vessels. In AML following the treatment-induced hypoplasia, large uniform unilocular fat cells arranged in groups designated ‘structured fat’ developed from scattered multilocular precursor fat cells. Early foci of haemopoietic regeneration were present almost exclusively in areas of structured fat. In the marrow of patients with CGL in blast transformation (BT) treated by intensive therapy and autografting with cryopreserved haemopoietic stem cells, the haemopoiesis was focal. Clusters of regenerating erythroid precursors or of megakaryocytes were seen in intimate contact with marrow sinusoids and granulopoietic precursors in intimate association with small blood vessels and also in close contact with the endosteal surface of bony trabeculae. We conclude that the endosteal cells, fat cells and the vascular endothedial cells comprise the critical non-haemopoietic stromal elements of human BM. The close associations observed between the regenerating haemopoietic cells and the stromal cells provide strong evidence in support of the existence of a permissive haemopoietic micro-environment in man and emphasize the structural and functional interrelationships that exists between bone, fat, the microvascular system and haemopoiesis in human bone marrow.     

Myelodysplastic syndromes: pathogenesis, functional abnormalities, and clinical implications.

The myelodysplastic syndromes represent a preleukaemic state in which a clonal abnormality of haemopoietic stem cell is characterised by a variety of phenotypic manifestations with varying degrees of ineffective haemopoiesis. This state probably develops as a sequence of events in which the earliest stages may be difficult to detect by conventional pathological techniques. The process is characterised by genetic changes leading to abnormal control of cell proliferation and differentiation. Expansion of an abnormal clone may be related to independence from normal growth factors, insensitivity to normal inhibitory factors, suppression of normal clonal growth, or changes in the immunological or nutritional condition of the host. The haematological picture is of peripheral blood cytopenias: a cellular bone marrow, and functional abnormalities of erythroid, myeloid, and megakaryocytic cells. In most cases marrow cells have an abnormal DNA content, often with disturbances of the cell cycle: an abnormal karyotype is common in premalignant clones. Growth abnormalities of erythroid or granulocyte-macrophage progenitors are common in marrow cultures, and lineage specific surface membrane markers indicate aberrations of differentiation. Progression of the disorder may occur through clonal expansion or through clonal evolution with a greater degree of malignancy. Current attempts to influence abnormal growth and differentiation have had only limited success. Clinical recognition of the syndrome depends on an acute awareness of the signs combined with the identification of clonal and functional abnormalities.     

Aplastic anaemia and the hypocellular myelodysplastic syndrome: histomorphological, diagnostic, and prognostic features.

In a retrospective study of 111 patients with aplastic anaemia iliac crest biopsies were evaluated for the presence of morphological features statistically related to the evolution of the disease. Prognostic variables for a transition to acute non-lymphatic leukaemia were: cellular atypias of the three haemopoietic lineages, as observed in the myelodysplastic syndrome, and especially "micromegakaryocytes"; high numbers or irregular distribution of megakaryocytes, or both; and (slight) marrow fibrosis. Clinical variables did not influence these prognostic correlations. Prognosis in relation to death from bone marrow failure without leukaemia might well have been influenced by a strong plasma cell reaction, but this correlation was weakened by clinical factors. On the basis of this study aplastic anaemia can thus be subdivided morphologically into two disease entities--namely, hypocellular myelodysplastic syndrome with a 23-82% risk of acute non-lymphatic leukaemia developing within three years, depending on how many variables associated with acute non-lymphatic leukaemia are present, and non-dysplastic myelohypoplasia.     

Thrombopoietin receptor (Mpl) expression by megakaryocytes in myeloproliferative disorders

The thrombopoietin receptor (Mpl) is involved in the pathogenesis of chronic myeloproliferative disorders (CMPD). In this study, we determined Mpl expression by bone marrow cells and megakaryocytes in CMPD by applying laser microdissection, real-time RT-PCR, and immunohistochemistry. Mpl mRNA expression was significantly increased up to 9-fold in total bone marrow cells (p < 0.001) and up to 4-fold in megakaryocytes in chronic myeloproliferative disorders (n = 73) compared to normal controls (n = 26, p = 0.01). Immunohistochemistry revealed heterogeneous Mpl expression by megakaryocytes in CMPD with a stronger accentuation in idiopathic myelofibrosis (IMF) in comparison to polycythaemia vera (PV) and essential thrombocythemia (ET). In addition to megakaryocytes, the erythropoietic lineage was prominently labelled by Mpl antiserum, with considerably stronger staining in polycythaemia vera. We conclude that, in CMPD, megakaryocytes and erythroid cells exhibit increased Mpl expression levels which may contribute to the sustained proliferation of both cell lineages in CMPD.     

Myelodysplastic syndromes: Immunohistochemical and morphometric evaluation of proliferative activity in erythropoiesis and endoreduplicative capacity of megakaryocytes

An immunohistochemical and morphometric analysis was performed on bone marrow trephine biopsies in 40 patients with primary myelodysplastic syndromes (MDS) to evaluate the proliferative activity in erythropoiesis and the endoreduplicative capacity of megakaryocytes. Control groups included normal bone marrow and marrow from cases presenting with pernicious anaemia. Double-immunostaining was applied with a monoclonal antibody (PC10) directed against proliferating cell nuclear antigen (PCNA), followed by antibodies against glycophorin C (Ret40f) or platelet glycoprotein IIIa (Y2/51-CD61) for the identification of the erythroid and megakaryocytic cell lineage. Comparison with normal bone marrow showed a reduction of erythropoiesis accompanied by an increase in atypical (micro-) megakaryocytes. Erythroid precursors displayed significant enhancement of PCNA-immunostaining. Megakaryocytes showed no increase in the relative frequency of PC10-positive cells (PCNA-labelling index). In pernicious anaemia, predominance of macrocytic-megaloblastoid erythropoiesis was associated with a striking increase in PCNA-labelling. Cell kinetic studies in this disorder revealed an abnormal arrest, particularly in S-phase which generates an over-expression of PCNA. Similar conditions were believed to be present in MDS with secondary folate deficiency. This mechanism explains the relatively high rate of positively-reacting pro- and erythroblasts which is not invariably accompanied by an increase in cell proliferation. Determination of megakaryocyte size and PCNA-staining capacity resulted in a significant increase in PC10-positive cells among micromegakaryocytes. Our findings on this cell lineage are in keeping with the assumption of a block in endoreduplicative activity at higher ploidy levels, associated with an apparently not-deregulated endomitosis in small-sized megakaryocytes of lower ploidy stages.Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG-Th 390/1-3)     

The effect of prolonged treatment with haemin on pluripotent haemopoietic progenitors of normal and hereditarily anaemic Belgrade laboratory (b/b) rats

The Belgrade laboratory (b/b) rat has an intracellular iron and haem deficiency-induced anaemia, due to a mutation affecting intracellular iron transport. Many disturbances in haemopoiesis of the b/b rat have been described, including impaired proliferation and a decreased number of pluripotent and committed progenitor cells in bone marrow as well as expanded granulocytic and erythroid progenitor cell populations in the spleen. In this study, the effects of prolonged treatment with haemin were evaluated on femoral and splenic pluripotent progenitors (spleen colony formingunits day 8, CFU-Sd8) in normal (+/+) and anaemic (b/b) rats. The treatment of b/b rats with haemin did not correct their severe anaemia. However, the abrogation of the proliferative block and an increased proliferative activity of the bone marrow CFU-Sd8 were found, demonstrating that the proliferative block of the b/b rat bone marrow CFU-Sd8 was induced by intracellular iron deficiency, rather than by severe hypoxia. Accordingly, the initially very low number of CFU-Sd8 per femur had increased. The results also revealed an enormously expanded population of splenic CFU-Sd8 in b/b rats, whereas after the haemin treatment a significant decrease in the number of these cells was found. The treatment of +/+ rats with haemin resulted in a significant decrease in the number of femoral CFU-Sd8, but the cycling status of these cells was not affected. In the spleen, the initially low number of CFU-Sd8 was modestly increased after the same treatment. These results imply that the effects of haemin treatment are different in a steady state than in a state of impaired medullar haemopoiesis. It is also evident that for both +/+ and b/b rats there exists an inverse number of CFU-Sd8 in the bone marrow and spleen.     

Enrichment of murine haemopoietic stem cells: diverging roads

The cellular elements of the peripheral blood must be constantly replenished by the process of haemopoiesis, since most blood cells have a limited life span of only days or weeks. Although the developmental lineages of haemopoietic differentiation have been depicted in textbooks for decades, the actual details of the early stages of haemopoiesis are relatively unknown due to the very low numbers of haemopoietic stem cells in bone marrow or spleen. Only by isolating these rare stem cells and developing in-vitro culture systems to maintain them can a complete understanding of the early stages of haemopoiesis be achieved. This approach has already been successfully applied to the study of the later stages of haemopoiesis. In this review, Gerald Spangrude examines several experimental approaches that have been used to enrich murine haemopoietic stem cells.     

DNA去甲基化对大鼠骨髓间充质干细胞增殖细胞核抗原的调节

背景：DNA去甲基化是一种重要的表观遗传修饰。 目的：观察DNA去甲基化对骨髓间充质干细胞增殖及增殖细胞核抗原蛋白表达的影响。 方法：全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞,免疫组化法鉴定骨髓间充质干细胞CD44和CD45蛋白的表达,MTT法检测5-杂氮胞苷对骨髓间充质干细胞增殖的影响,免疫组化法检测5-杂氮胞苷对骨髓间充质干细胞增殖细胞核抗原蛋白表达的影响。 结果与结论：①第3代骨髓间充质干细胞不表达或低表达CD45,高表达CD44。②与未加入5-杂氮胞苷组比较,5-杂氮胞苷干预48 h,6,12,24 μmol/L组显著促进细胞增殖活性(P < 0.05),无浓度依赖性。③与未加入5-杂氮胞苷组比较,5-杂氮胞苷干预48 h,6,12,24 μmol/L 组增殖细胞核抗原蛋白积分吸光度值显著增加 (P < 0.05),组间差异无显著性意义。结果显示在一定浓度范围内,5-杂氮胞苷发挥去甲基化作用,激活增殖细胞核抗原蛋白表达,从而促进骨髓间充质干细胞增殖。     

Proliferating cell nuclear antigen expression by erythroid precursors in normal bone marrow, in reactive lesions and in polycythaemia rubra vera

Using a sequential double-imunostaining technique, a morphometric analysis was performed on routinely processed bone marrow trephines from 20 patients with secondary (reactive) polycythaemia and 28 patients with polycythaemia rubra vera to determine the proliferation capacity of erythropoiesis. Monoclonal antibodies PC10—anti-proliferating cell nuclear antigen (PCNA)—and Ret40f—anti-glycophorin C—were employed. For comparison with the PCNA-labelling index, in a pilot study, Ki-67 was additionally used on frozen-section material. In comparison with normal bone marrow (15 patients) morphometric and statistical evaluation revealed a numerical increase in erythroid precursors (pro-, erythro- and normoblasts) in secondary polycythaemia and to a pronounced degree in polycythaemia rubra vera. In comparison with secondary polycythaemia and the control group, in polycythaemia rubra vera there was a significant enhancement of proliferation according to PCNA-staining reactivity in all haematopoietic cell elements and particularly in the erythroid series. Evaluation of PCNA v . Ki-67 immunostaining disclosed only a slight difference, which could be mainly attributed to various modalities of antigen expression during the cell cycle. Our findings are in keeping with in vitro studies on cultured erythroid progenitor cells and, in problematic cases, may present a valuable aid in differentiation between reactive lesions and polycythaemia rubra vera.     

A new case of congenital dyserythropoietic anaemia, type III: studies of the cell cycle distribution and ultrastructure of erythroblasts and of nucleic acid synthesis in marrow cells.

The clinical and laboratory findings in an asymptomatic 19-year-old Welshman with congenital dyserythropoietic anaemia (CDA) type III are described. The blood film showed macrocytosis and red cell fragmentation and there was biochemical evidence of intravascular haemolysis. The bone marrow showed erythroid hyperplasia, megaloblastic erythropoiesis and several giant multinucleate erythroblasts. Some mononucleate erythroblasts were large and had relative DNA contents of 4-8c and the bi- and multinucleate erythroblasts had total DNA contents of 2-16c. Some of the multinucleate erythroblasts displayed a variety of ultrastructural abnormalities, including marked differences in the appearances of the individual nuclei within the same cell. The marrow cells gave a normal deoxyuridine-suppressed value indicating that the megaloblastic changes were not caused by an impairment of the methylation of deoxyuridylate. The rates of incorporation of 14C-glycine and 14C-adenine into both the DNA and RNA of bone marrow cells were within the normal range. Furthermore, the average rate of elongation of newly-synthesised, 3H-thymidine-labelled daughter DNA strands, assessed by hydroxyapatite chromatography of alkali-denatured DNA was found to be normal. The results suggest that there is no impairment of DNA replication in the majority of the erythroblasts and that the abnormality of erythropoiesis resulted from disturbances during mitosis and the G2 phase.     

Intracellular hemoglobin-a specific marker for erythroid cells in paraffin sections. An immunoperoxidase study of normal, megaloblastic, and dysplastic erythropoiesis, including erythroleukemia and other myeloproliferative disorders.

Intracellular hemoglobin represents an excellent marker for specific characterization of normal, megaloblastic, or dysplastic erythroid cells in paraffin sections. Using an immunoperoxidase indirect sandwich technique for detection of intracellular hemoglobin, erythroid cells at all stages of maturation were readily identified in bone marrow biopsies (58 specimens total) with a) normal erythropoiesis, b) megalobastic erythropoiesis, and c) various myeloproliferative disorders, including erythroleukemia. In other tissues (6 spleens, 2 lymph nodes, 1 liver) with extramedullary hematopoiesis, erythroid cells were similarly defined on the basis of this immunohistochemical method. Initial fixation in Zenker's-acetic acid solution (employed for bone marrow biopsies), B5 solution, or formalin, appeared equally effective in preserving the antigenicity of intracellular human hemoglobin. This sensitive and specific immunoperoxidase technique for erythroid cell characterization is particularly applicable to tissues with abnormal erythropoiesis, in which precise cell identification generally presents a diagnostic problem.     

Phospho-STAT5 expression pattern with the MPL W515L mutation is similar to that seen in chronic myeloproliferative disorders with JAK2 V617F

Abnormal nuclear megakaryocytic staining for phospho-STAT5 (pSTAT5) correlates with JAK2 V617F mutational status in non-chronic myelogenous leukemia chronic myeloproliferative disorders. However, a proportion of wild-type JAK2 non-chronic myelogenous leukemia chronic myeloproliferative disorders cases also demonstrate this abnormal pSTAT5 expression pattern. We report a patient with a JAK2 V617F-negative myeloproliferative/myelodysplastic syndrome who had abnormal megakaryocytic pSTAT5 expression and a MPL W515L mutation. The patient was a 71-year-old man with anemia and thrombocythemia on laboratory examination. His peripheral blood smear demonstrated occasional dysplastic neutrophils. Bone marrow biopsy revealed hypercellular marrow with features consistent with myeloproliferative/myelodysplastic syndrome. Immunohistochemistry for pSTAT5 showed abnormal nuclear megakaryocyte positivity. Cytogenetic analysis revealed a normal karyotype, fluorescence in situ hybridization for BCR-ABL was negative, and JAK2 genotyping demonstrated wild-type JAK2. However, MPL genotyping showed a MPL W515L mutation. Abnormal nuclear megakaryocytic staining for pSTAT5 expression, previously associated with the JAK2 V617F mutation, is also associated with MPL W515L, likely reflecting activation of the JAK-STAT signaling pathway.     

Interleukin-3

Synopsis

Interleukin-3 is an early- and late-acting haemopoietic growth factor which promotes the proliferation and differentiation of multilineage and single lineage committed progenitor cells. It also has modulatory effects on mature cells, including basophils, monocytes and eosinophils. Interleukin-3 stimulates the proliferation of bone marrow and peripheral blood progenitor cells and increases bone marrow cellularity, causing a shift of haemopoiesis to the left, with a subsequent increase in the proportion of immature haemopoietic cells and in numbers of megakaryocytes and eosinophils. Interleukin-3 also potentiates granulocyte-macrophage colony-stimulating factor- (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) mediated mobilisation of peripheral blood progenitor cells from the bone marrow to the blood. Preliminary clinical trials show that recombinant human interleukin-3 (referred to as interleukin-3) reduces haematological toxicity in patients following standard- or high-dose chemotherapy with or without autologous bone marrow transplantation. The duration of neutropenia was reduced and importantly, platelet numbers were generally increased such that the duration of thrombocytopenia was also reduced in the majority of studies. There was a trend towards a lower requirement for platelet transfusions in treatment cycles that included interleukin-3 compared with those that did not, and the need to postpone further chemotherapy cycles due to prolonged haematological toxicity was reduced with interleukin-3. Sequential combination therapy with interleukin-3 followed by recombinant GM-CSF appears to further enhance haemopoietic recovery in these indications. Other potential applications of interleukin-3 include disease states with intrinsic bone marrow dysfunction, such as myelodysplasia and aplastic anaemia. Thus, the ability of interleukin-3 to reduce both neutropenia and thrombocytopenia in preliminary studies suggests that this cytokine will be useful, either alone or in sequential combination with other cytokines, for the treatment of haematological toxicity associated with chemotherapy, following bone marrow transplantation, and in the treatment of haemopoietic stem cell disorders.

Pharmacological Properties

Interleukin-3 (multipotential colony-stimulating factor) is a haemopoietic growth factor, secreted predominantly by activated T-helper lymphocytes. It stimulates the proliferation of early haemopoietic progenitor cells in the bone marrow, including colony-forming unit (CFU)-granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM), CFU-granulocyte-macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), CFU-eosinophil (CFU-Eo), CFU-megakaryocyte (CFU-Meg), and CFU-basophil (Bas) cells. It also stimulates the proliferation of peripheral blood progenitor cells in vitro. Bone marrow cellularity is increased by interleukin-3 in patients with normal haemopoiesis, bone marrow failure or myelodysplastic syndrome, and there is a shift of haemopoiesis to the left, increasing the proportion of immature haemopoietic progenitor cells and the numbers of eosinophils and megakaryocytes. The cytokine also affects cells at later stages of maturation including basophils, eosinophils and monocytes. It is unclear whether interleukin-3 can induce the mobilisation of peripheral blood progenitor cells when administered alone; however, it potentiates the effect of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) and recombinant granulocyte colony-stimulating factor (rG-CSF). In vitro studies indicate that interleukin-3 enhances the cytotoxic effects of cytarabine (ara-C) on acute myeloid leukaemia cells; however, its efficacy in humans in this setting remains to be determined. Data on the pharmacokinetic properties of recombinant human interleukin-3 are limited. Peak plasma concentrations are reached within 2 to 4 hours following subcutaneous administration of interleukin-3 and are dose-proportional. The plasma elimination half-life is 1.3 to 3.3 hours following subcutaneous administration and 0.4 to 1 hour following intravenous administration. The apparent systemic clearance of interleukin-3 is 0.3 L/h/kg, irrespective of the route of administration or dosage.

Clinical Potential

Recombinant human interleukin-3 (referred to as interleukin-3) has shown clinical efficacy in reducing haematological toxicity following standard- and high-dose chemotherapy treatment regimens in small phase I/II clinical trials reported to date. A daily dosage of 5 to 10 μg/kg, administered either subcutaneously or by continuous intravenous infusion, caused a significant increase in neutrophil and platelet counts after chemotherapy and reduced the duration of neutropenia and thrombocytopenia compared to that observed in control chemotherapy cycles in which interleukin-3 was not given. A reduction in the need to postpone further chemotherapy due to prolonged haematological toxicity was observed with interleukin-3 treatment, and the number of patients requiring platelet transfusions was also reduced. Sequential administration of interleukin-3 followed by rGM-CSF or rG-CSF may further reduce myelosuppression. Interleukin-3 also enhanced platelet and neutrophil recovery following autologous bone marrow transplantation, and in patients with bone marrow failure. Its relative benefits in reducing myelosuppression compared with those of other cytokines remain unclear. Preliminary findings indicate that sequential combination therapy with interleukin-3 and rGM-CSF may be beneficial compared with monotherapy with either agent in these indications. In addition, interleukin-3/rG-CSF-induced mobilisation of peripheral blood progenitor cells prior to apheresis and re-transplantation, or in vitro incubation of patients’ bone marrow cells with interleukin-3 and rGM-CSF or rG-CSF prior to transplantation, both appeared to enhance marrow recovery of platelet numbers. These findings require further investigation. Interleukin-3 may also offer some clinical benefit to patients with myelodysplastic syndromes in that their leucocyte counts are increased. However, it has shown only transient effects, if any, on platelet counts in these patients. Initial findings indicate that patients with aplastic anaemia may also derive some clinical benefit from treatment with interleukin-3. Further study of its efficacy relative to that of other agents in these indications is warranted.

Tolerability

There are few tolerability data available for interleukin-3. While adverse events have been reported frequently, they have been generally tolerable at dosages ≤ 10 μg/kg/day. Influenza-like symptoms (myalgia, arthralgia, fatigue), headache, and low grade fever have occurred most frequently. Severe headache occurring at dosages > 10 μg/kg/day was often a dose-limiting event. Other adverse events have included nausea, vomiting, skin rash, mild local erythema at the injection site, flushing, oedema, facial erythema, diarrhoea, rigors, malaise and dyspnoea; these were generally mild and resolved at the end of treatment. The tolerability profile of interleukin-3 appeared similar when the drug was administered prior to or following chemotherapy, and during combined sequential treatment with rGM-CSF or rG-CSF.

Dosage and Administration

In clinical trials, interleukin-3 has been administered once daily by subcutaneous injection or by continuous intravenous infusion. For the treatment of myelosuppression following chemotherapy, ≥ 5 μg/kg/day for 7 to 14 days appears to be an effective dosage, and 10 μg/kg/day has shown efficacy in patients following autologous bone marrow transplantation. A lower dosage of interleukin-3 2.5 μg/kg/day for 10 days followed by rGM-CSF therapy has also shown efficacy in the bone marrow transplantation setting. Interleukin-3 30 to 500 μg/m²/day for 15 days induced a haematological response in patients with bone marrow failure, and 250 to 500 μg/m²/day for 14 to 15 days induced a response in patients with myelodysplastic syndromes or aplastic anaemia.     

Immunohistochemical assessment of human bone marrow macrophages in hematologic disorders

Changes in bone marrow macrophages may be associated with abnormal hematopoiesis in various hematologic disorders. We immunohistochemically evaluated the density of macrophages in bone marrow trephine biopsies. In reactive erythroid hyperplasia (hemolytic anemia and megaloblastic anemia), the macrophages slightly increased in density, extending their cytoplasmic processes between hematopoietic cells. In erythroid hypoplasia (pure red cell aplasia), they became rounded and frequently had hemosiderin granules. There was no significant difference in the macrophage density in the hematopoietic area between erythroid hyperplasia and hypoplasia. The macrophages increased in density in myeloproliferative disorders (polycythemia vera, chronic myelogenous leukemia and primary thrombocythemia). In myelofibrosis, some macrophages became extremely elongated along the line of the fibroblastic cells. In contrast, in conditions in which myelopoietic activity is considerably impaired (aplastic anemia, acute leukemia and multiple myeloma), they significantly decreased in density. These results suggest that the morphologic change in bone marrow macrophages is associated with erythropoietic activity and that there is a correlation between macrophage density and myelopoietic activity.     

Immunohistochemical identification of adult and fetal haemopoiesis in the spleen in lymphoma, leukaemia and myeloproliferative disease

Antisera to adult and fetal haemoglobins were used in the immunoperoxidase method to identify haemopoietic cells in spleens from patients with lymphoproliferative and myeloproliferative disorders. Splenic extramedullary haemopoiesis in adults often contains a major fetal component. Spleens infiltrated by Hodgkin's lymphoma are invariably negative, but other lymphomas, leukaemias and myeloproliferative diseases are commonly associated with splenic fetal haemopoiesis. Immunoperoxidase staining for haemoglobins is a useful way of determining the haemopoietic origin of round cell infiltrates in tissue sections.     

Erythroid proliferations in myeloid neoplasms

Prominent erythroid proliferations (in which erythroid elements comprise ≥50% of total bone marrow cells) can be seen in various hematopoietic stem cell neoplasms. The myeloproliferative neoplasm polycythemia vera exhibits effective, overexuberant erythropoiesis resulting in an increased red blood cell mass; in contrast, most other diseases characterized by erythroid predominance exhibit ineffective hemopoiesis. The latter include acute erythroid leukemia (erythroid-myeloid and pure erythroid leukemia subtypes) as well as some cases of myelodysplastic syndromes, acute myeloid leukemia with myelodysplasia-related changes, and therapy-related myeloid neoplasms. Some nonneoplastic reactive conditions may also manifest a striking bone marrow erythroid predominance. In this article, we review the literature relevant to this group of diseases for a better understanding of their clinicopathologic features and surrounding controversies. We also examine the position of neoplastic erythroid proliferations in the current 2008 World Health Organization Classification of Myeloid Neoplasms and provide recommendations as to how to approach the differential diagnosis of this group of diseases.     

An autopsy case of myelodysplastic syndrome (MDS): diagnostic problems between MDS and the other haematopoietic disorders including acute myelofibrosis

We report an autopsy case of myelodysplastic syndrome (MDS) in a 35-year-old male, who presented with pancytopenia and bleedings. Bone marrow specimens disclosed myelofibrosis and hypercellular marrow with more than 60% atypical erythroblasts in the bone marrow cells. Type I or type II blasts were less than 10% of the peripheral blood and bone marrow cells during the clinical course. At autopsy, infiltration by myeloid and erythroid cells and megakaryocytes was noted in the liver, spleen and lymph nodes. According to the FAB classification, this case might be classified into refractory anemia with excess of blasts (RAEB) or RAEB in transformation. However, the remarkable neoplastic proliferation of three haematopoietic cell lines also indicates acute myeloproliferative disorder such as acute myelofibrosis or acute panmyelosis.     

Myelodysplastic/myeloproliferative disease with erythropoietic hyperplasia (erythroid preleukemia) and the unique translocation (8;9)(p23;p24): first description of a case

We report on a patient fulfilling the diagnostic criteria of unclassifiable myelodysplastic/myeloproliferative diseases with prominent erythropoietic hyperplasia/dysplasia (erythroid preleukemia) and the unique translocation (8;9)(p23;p24). The patient presented with B-symptoms, erythroblastemia, thrombopenia, marked eosinophilia, presence of myeloid precursors in the peripheral blood, and decreased erythropoietin level. Nodular peritrabecular polymorphous blasts, dysplastic megakaryocytes, and a diffuse argyrophilic fibrosis were detected in the trephine bone marrow biopsy. Immunohistochemically, the blasts stained positively for glycophorin C and hemoglobin A; the proliferation fraction was nearly 90% in the Ki-67 stain. Expression of the phosphorylated Janus kinase 2 was detected in almost all megakaryocytes and in isolated erythroblast islets, suggesting a probable activation of Janus kinase 2, the jak-2 gene being mapped on 9p24. Ten months after initial diagnosis, the disease progressed to frank acute erythroid leukemia. We report for the first time a myelodysplastic/myeloproliferative disease (erythroid preleukemia) accompanied by the specific chromosomal aberration t(8;9)(p23;p24), distinct histopathology, and clinical and laboratory symptoms, and progress to acute erythroid leukemia.