Megakaryocyte precursors (promegakaryoblasts and megakaryoblasts) in the normal human bone marrow. An immunohistochemical and morphometric study on routinely processed trephine biopsies

On routinely processed trephine biopsies of the normal human bone marrow derived from 15 patients, immunostaining with a monoclonal antibody against platelet glycoprotein IIIa (Y2/51) and morphometric measurements were performed for the determination of megakaryocyte precursor cells. Based on cell sizes and on comparison with (1) specimens stained by the periodic acid-Schiff reaction and (2) smears, the smallest elements clearly identifiable as belonging to the megakaryocyte series were classified as promegakaryoblasts. Promegakaryoblasts had a frequency of 1.7/sq mm and 140/cu mm of bone marrow and constituted about 8% of the total positively stained megakaryocytic elements; they were characterized by a size of 41.5 sq microns, a diameter of 7.7 microns, a high nuclear-cytoplasmic ratio (0.32) and a nearly circular outline of their nuclear and cellular perimeters.     

Primary (essential) thrombocythemia versus polycythemia vera rubra. A histomorphometric analysis of bone marrow features in trephine biopsies

A morphometric analysis of bone marrow biopsies was performed in 25 patients each with clinical diagnoses of primary (essential) thrombocythemia (PTH) and polycythemia vera rubra (P. vera) according to the rigid diagnostic criteria of the Polycythemia Vera Study Group to reveal significant differences in the histomorphologic features between these disorders. In comparison with control specimens of patients without any hematologic disease, megakaryocyte proliferation was most prominent in PTH, even exceeding that of P. vera with concomitant thrombocythemia (11 of 25 cases with a platelet count greater than 600 X 10(9)/L). Moreover, in P. vera there were wide ranges of megakaryocyte sizes, consisting of micro-megakaryocytes as well as giant forms with highly segmented nuclei (four nuclear lobes), which gave the cells a pleomorphic appearance. As compared with the normal bone marrow, the amount of neutrophilic granulopoiesis and erythropoiesis was not significantly increased in PTH, in contrast to P. vera. Similar results were obtainable regarding the density of reticulin (argyrophilic) fibers: a normal content was encountered in the control specimens and PTH, whereas P. vera displayed a minimal-to-slight increase. Finally, the bone marrow of P. vera was totally devoid of stainable iron while hemosiderin deposits were detected in about two-thirds of the patients without hematologic disorders and in PTH. The characteristic differences revealed by this morphometric study may lead to an improvement of the controversial histologic diagnosis in these disorders.     

Evaluation of the JAK2-V617F gene mutation in Turkish patients with essential thrombocythemia and polycythemia vera

An activating mutation of Janus kinase 2 (JAK2-V617F) was previously described in chronic myeloproliferative disorders (MPD). In previously published studies, the frequency of the JAK2-V617F mutation was determined to be 80-90 % for patients with polycythemia vera (PV) and 40-70 % for essential thrombocythemia (ET). In this study, we analyzed the relationship between the JAK2-V617F mutation and clinical-hematological parameters in Turkish patients with MPD and compared these findings with published studies from other geographic regions. A total of 148 patients were studied; of which, 70 were diagnosed with PV and 78 with ET. The mutation status of JAK2 was determined using a tetra-primer polymerase chain reaction. We found that 80 % of the PV group and 42 % of the ET group were positive for the JAK2-V617F mutation. When all patients were analyzed, the levels of white blood cells, hemoglobin and splenomegaly were significantly different in patients with the JAK2-V617F mutation (p < 0.05). To our knowledge, this study is the first to evaluate the relationship between MPD and JAK2-V617F in Turkish patients. The JAK2-V617F mutation is frequently detected in the Turkish patients with MPD, and especially in patients with PV. Hence, it would be useful to include JAK2 mutation screening in the initial evaluation of patients suspected to have MPD.     

RNA-dependent DNA polymerase from a cell line derived from the bone marrow of a patient with polycythemia vera.

A RNA-dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)-cellulose, and glycerol gradient centrifugation. The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) - oligo(dG) and its molecular size of about 70000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co-purified. Upon dodecylsulfate-polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68000 and 66000 and three minor bands were detected. The enzyme preferentially used poly(rA) - oligo(dT) as template-primer compared with poly(dA) - oligo(dT). It incorporated dGMP into polymer on poly(rC) - oligo(dG).     

Polycythemia vera: electron microscopy of the bone marrow in 10 non-treated patients. A thin section and freeze-fracture study.

Electron microscopy (thin sections and freeze-fracture replicas) was performed on the bone marrow of ten patients with Polycythemia vera prior to any treatment. In addition to a hyperplasia of all three cell lineages and the sinuses, atypias were observed in the maturing erythroblasts. These aberrations of normal development consisted mainly of deep invaginations of the nuclear envelope in proerythroblasts and conspicuous nuclear clefts in erythro- and normoblasts. In comparison with similar changes in dyserythropoietic and aplastic anemia as well as leukemia these alterations are discussed in connection with disturbances of DNA synthesis. Further atypias involved megakaryopoiesis which displayed microforms probably as an evidence for maturation arrest. These ultrastructural abnormalities and their morphological features of a neoplastic proliferation of all three cell lineages in Polycythemia vera are in good agreement with the new concept of a transformation of a pluripotent stem cell with clonal character.     

JAK2 mutation status, hemostatic risk factors and thrombophilic factors in essential thrombocythemia (ET) patients

The recently discovered JAK2 V617F point mutation, found in 50-60% of ET patients, has been reported to be associated with a higher risk of thrombotic events. In this study, we explored if JAK2 V617F mutation, or coexisting thrombophilic and hemostatic risk factors, contributed to these complications. We examined 32 patients with ET, and looked for pathogenetic JAK2 V617F mutation and prothrombotic genes mutations: factor V Leiden, prothrombin and MTHFR. We also evaluated plasma levels of fibrinogen, factors VIII and XII, AT, protein C, protein S and serum level of homocysteine. Urokinase concentration was assessed in patients' plasma as well as platelet lysates. There was no difference in the number of thrombotic complications between ET patients with and without JAK2 mutation. However, we found a number of thrombophilic and hemostatic risk factors that could contribute to thrombotic complications in ET patients.     

Development of early erythroid precursors (BFUe) from mouse bone marrow in a serum-free culture media. Effect of hemin

In culture medium containing albumin, iron saturated transferrin, phospholipids, cholesterol and erythropoietin, BFUe growth requires foetal Calf serum. Without serum the BFUe development is advantageously restored by the addition of hemin associated with spleen conditioned medium. In erythropoietin supplemented serum-free medium the growth of primitive erythroid precursor cells is closely dependent on growth factor supply found in spleen conditioned medium. Using this medium allows us to clearly distinguish between the respective effects of erythropoietin and of BPA on erythroid progenitor cells.     

Reptilian bone marrow. An ultrastructural study in the spanish lizard,Lacerta hispanica

The ultrastructure of hemopoietic bone marrow of the Spanish lizard, Lacerta hispanica, has been studied for the first time. The organ consists of a stroma formed by venous sinuses and reticular cells. Erythropoiesis takes place in the lumen of blood vessels, while granulopoiesis is extravascular. Pluripotent stem cells are structurally differentiated into erythrocytes and granulocytes. Two types of granulocytes, heterophils and acidophils, have been found, and a third granular cell type is tentatively identified as granular leukocyte. Remarkably, plasmacytopoiesis occurs in the bone marrow of Lacerta hispanica. The possible functional significance of these results is discussed with emphasis on their importance for the reptilian immune system.     

B lymphocyte precursors in human bone marrow: an analysis of normal individuals and patients with antibody-deficiency states.

Lymphoid cells containing cytoplasmic IgM but lacking stable surface IgM are believed to be the direct precursors of B lymphocytes. We have characterized these pre-B cells in the bone marrow of normal individuals and patients with a variety of immunoglobulin deficiencies or hematologic disorders by using immunofluorescence and autoradiography. Pre-B cells comprised 5.8 +/- 5.7% of lymphoid cells in normal bone marrow. Eleven patients with infantile X-linked agammaglobulinemia (X-LA) lacked B lymphocytes but had a normal frequency (3.8 +/- 3.6%) of bone marrow pre-B cells. A smaller proportion of marrow pre-B cells from patients with X-LA were engaged in spontaneous DNA synthesis than was found for normal controls. In individuals other than the group with X-LA, the number of circulating B cells was positively correlated with the frequency of marrow pre-B cells. These results indicate that patients with X-LA have a defect in maturation of pre-B cells, and suggest that some patients with acquired B lymphocyte deficiency may have lost the capacity to generate pre-B cells from stem cells.     

Brain-derived neurotrofic factor (BDNF) secretion of human mesenchymal stem cells isolated from bone marrow,endometrium and adipose tissue

The ability of mesenchymal stem cells (MSCs) to differentiate into neuronal lineage determines the potential of these cells as a substrate for a cell replacement therapy. In this paper we compare the neurogenic potential of the MSCs from different donors, isolated from the bone marrow (BMSC), subcutaneous adipose tissue (AD MSC) and menstrual blood (eMSC). It was established that the native eMCSs, BMSCs and AD MSCs express neuronal marker β-III-tubulin with a frequency of 90, 50 and 14%, respectively. Also we showed that the eMSCs have a high endogenous level of brain-derived neurotrophic factor (BDNF), whereas the BMSCs and the AD MSCs are characterized by low basal BDNF levels. An induction of neuronal differentiation in the studied MSCs using differentiation medium containing B27 and N2 supplements, 5-azacytidine, retinoic acid, IBMX and dbcAMP induced changes in the cells morphology, the increase of β-III-tubulin expression, and the appearance of neuronal markers GFAP, NF-H, NeuN and MAP2. During the differentiation the BDNF secretion was significantly enhanced in the BMSCs and decreased in the eMSCs cultures. However, no correlation between the basal and induced levels of the neuronal markers expression in the studied MSCs has been established.     

Pathological cell aggregates in bone marrow cultures from patients with various hematological diseases.

Non-clonal growth of macroscopic cell aggregates in methylcellulose cultures of abnormal marrow is described. They were seen in all patients with Graft-versus-Host Disease, graft rejection, and autoimmune disease presumably directed against hemopoietic cells, we found them in 35% of patients with primary hematological neoplasias and rarely in patients with solid tumors. They were never encountered in 80 healthy controls. The aggregates originated from small cell clumps which sedimented with the "buffy coat" in contrast to normal bone marrow particles. They contained tumor cells, grafted myeloid cells, or target cells of autoimmune disease in association with a widely varying amount of macrophages. Preliminary results suggest that the frequency of macrophages within the aggregates correlates inversely with the aggressiveness of the clinical condition. We propose that appearance of such aggregates in an indicator of immune activation; we expect that further quantitation of the phenomenon will reveal important clinical correlations and provide a model for the study of host defense to "foreign" cells.     

Ultrastructure of neutrophilic granulopoiesis in the bone marrow of patients with chronic myeloid leukemia (CML). A morphometric study with special emphasis on azurophil (primary) and specific (secondary) granules

In seven patients with chronic myeloid leukemia (CML) and ultrastructural and morphometric study was performed on neutrophilic granulopoiesis in bone marrow trephine biopsies. Bone marrow specimens from five patients without hematological abnormalities served as controls. In stable phases of CML, abnormalities of the maturing granulocytic lineage were most conspicuously expressed by an infrequently occurring nuclear disfiguration (blebs and disturbed bridging of segments). Morphometric evaluation included the numbers of azurphil (primary) and specific (secondary) granules, the cisternal length of the endoplasmic reticulum and the area of the mitochondrial profiles. These variables could be determined in early and late myeloblasts, promyelocytes, metamyelocytes, band cells and mature polymorphonuclear granulocytes. Statistical analysis with regard to control specimens demonstrated no significant differences in the total amount of neutrophil granules or of the other cell organelles.     

Recombinant interleukin-2 (rIL-2) after autologous bone marrow transplantation (BMT): a pilot study in 19 patients.

In vivo use of rIL-2 autologous BMT may be the means of reproducing a kind of "adoptive immunotherapy" from grafted cells after allogeneic BMT. This approach may enhance the spontaneous generation of cytotoxic T-cells and NK cells which are presumably involved in this immunotherapy. Potential risks of such an approach would be to increase the usual toxicity of rIL-2 and to jeopardize the hemopoietic reconstitution. To determine the feasibility of this approach we have treated 19 poor prognosis patients with a succession of autologous BMT followed 78 +/- 12 days later by a continuous infusion of rIL-2. Eighteen million international units (IU) per m2 per day of Proleukine (CETUS, Amsterdam, The Netherlands) were administrated over 6 or 12 days. No patient died of the procedure. Clinical toxicity related to rIL-2 was not increased. Hemopoietic toxicity, significant both for platelets and granulocytes, was transient. Immune stimulation was dramatic for lymphocytes and subpopulations (CD3+ and NK cells) and for cytolytic functions (NK and LAK activity). This trial establishes the feasibility of administration of high doses of rIL-2, 2 months after autologous BMT. In this setting a 6 day period of continuous infusion of 18 million per m2 per day of Proleukine appears to be a regularly tolerable dosage conducting to a major immune activation and invites further studies to determine the clinical impact of such an approach.     

Diagnostic differentiation of essential thrombocythaemia from thrombocythaemias associated with chronic idiopathic myelofibrosis by discriminate analysis of bone marrow features--a clinicopathological study on 272 patients

Until now diagnosis of essential thrombocythaemia (ET) is generally performed by following the criteria of the Polycythaemia Vera Study Group (PVSG) that only marginally regards morphological features. Bone marrow biopsies were studied from 272 patients with ET in strict accordance with the PVSG guidelines and also from 35 control patients with reactive thrombocytosis. To define morphological features of distinctive impact more accurately, we performed a stepwise discriminant analysis of 16 morphological parameters based on histochemical staining reactions and semiquantitative grading of standardized features. A clear-cut separation into three distinctive histological patterns was accomplished that showed in more than 96% a correct predicted classification. Variables of significant impact included fibre content, quantity and cytological abnormalities of megakaryopoiesis like bulbous (cloud-like) nuclei, degree of nuclear lobulation and presence of giant forms. These changes were not detectable in the control group. The different constellations of histopathological features could be assigned to true ET (98 patients) and false ET, i.e. 136 patients with prefibrotic and 38 patients with early fibrotic chronic idiopathic myelofibrosis (IMF) accompanied by thrombocythaemia. A re-evaluation of clinical findings was in keeping with this classification into three categories that exerted significant differences to develop myelofibrosis during observation time and also different survival patterns. Contrasting IMF true ET is characterized by a pronounced proliferation of the megakaryocyte lineage showing large to giant cells without maturation defects and no relevant increase in reticulin fibres. Discrimination between these entities is warranted, because of a significant difference in presenting haematological data, follow-up and life expectancy.     

Ability of plasma from patients with immune thrombocytopenic purpura (ITP) to promote the growth of megakaryocyte progenitors from normal bone marrow.

The ability of plasma from ITP patients (before and after splenectomy) to support the growth of megakaryocyte progenitors was compared with that from healthy subjects. Plasma Factor Index-Megakaryocyte PFI-Mk (ITP) which expressed resultant colony growth was significantly lower before splenectomy, but it normalized after splenectomy. (PFI-Mk) (ITP) did not relate neither to megakaryocyte nor to platelet counts. A positive correlation has been observed between megakaryocyte and platelet numbers in healthy subjects and in ITP patients after splenectomy, but not before splenectomy. The proportion of immature megakaryocytes was markedly higher in ITP marrow before splenectomy. This study indicates, that in ITP apart from antibodies directed to platelets and megakarocytes a low plasma stimulatory activity affected megakaryocytopoiesis.