Emperipolesis of neutrophils by dysmorphic megakaryocytes

Neutrophil engulfment by megakaryocytes was observed within 20 to 30% of megakaryocytes from two children: one with metastatic rhabdomyosarcoma, the other with fever of unknown origin. Other cell types and neutrophil precursors were not observed within megakaryocytes. Only late megakaryocytes were involved in the process, and often these cells appeared vacuolated or degenerating at the light and electron microscope level. Ultrastructurally the engulfed neutrophils were intact and were within the open canalicular system of the megakaryocyte cytoplasm. No evidence of neutrophil granule exocytosis could be demonstrated in ultrastructural morphologic and peroxidase preparations; however, many neutrophils appeared to be endocytosing portions of the megakaryocyte cytoplasm. The phenomenon could not be transferred to normal marrow incubated with patient serum or plasma. Thus, our patients differ from previous observations of emperipolesis in: 1) the extreme frequency of the observation; 2) the selective involvement of neutrophils; and 3) the association of the anomaly with dysmorphic and/or disrupted megakaryocytes. These observations are consistent with a neutrophil response to altered and/or injured megakaryocytes.     

Marrow Cell Uptake by Megakaryocytes in Routine Bone Marrow Smears during Blood Loss

In studying smears of marrow aspirates, we have encountered the presence of normal appearing haemopoietic cells within megakaryocytes. We have then searched routine marrow smears from 125 patients seen in our service during 3 years. The presence of marrow cells (granulocytic, erythroid, and lymphoid cells) within megakaryocytes was seen in 16 cases of whom 9 had documented bleeding and 5 had carcinoma. 3 patients were suspected of bleeding but this was not documented. 56 % of patients with bleeding and 83 % of patients with carcinoma seen during this period displayed this phenomenon. A search for this phenomenon in routine marrow smears may reveal unsuspected blood loss.     

Mechanisms of thrombocytopenia in patients with lymphoproliferative diseases.

Different factors are involved in the development of thrombocytopenia in patients with lymphoproliferative disorders. Significant correlation was detected between the number of megakaryocytes in bone marrow and platelet count (r = 0.485, p = 0.002, n = 37) and significant difference between the number of megakaryocyte in patients with normal platelet count (> 200,000/microliters) and patients with marked thrombocytopenia (platelet count < 100,000/microliters). All patients in the latter group (n = 15) had a relatively low number of megakaryocytes. Low but significant reverse correlation was found between the level of platelet-associated IgG (PA-IgG) and platelet count (r = -0.249, p = 0.024, n = 82) and significant difference between the mean levels of PA-IgG in the groups of patients with platelet count > 200,000/microliters and < 100,000/microliters. PA-IgG were increased in 46% of patients in the total group and in 65% of patients with platelet count < 100,000/microliters. The correlation between platelet count and PA-IgG was about 2 times higher in splenectomized (r = -0.549, p = 0.005, n = 24) than nonsplenectomized patients. All splenectomized patients with platelet count < 100,000/microliters (n = 8) had a significant increase in PA-IgG. Serum antibodies were detected in only 7% of tested patients. This group was characterized by severe thrombocytopenia (in 6 of 10 patients--platelet count < 50,000/microliters) and a high incidence of haemorrhages (in 5 of 10 patients). Thus the depression of platelet production was suggested to be the basic cause of thrombocytopenia in lymphoproliferative disorders. Involvement of immune mechanisms was revealed in a large number of patients and correlated with a deeper and more complicated thrombocytopenia.     

Megakaryocyte Quantifications in Relation to Thrombokinetics in Primary Thrombocythaemia and Allied Diseases

Megakaryocyte morphology was studied quantitatively in primary thrombocythaemia (PT) and in chronic myelogenous leukaemia (CML). The relation of thrombokinetics to megakaryocyte quantifications was evaluated in PT and compared to previously obtained results in polycythaemia Vera (PV) and idiopathic thrombocytopenic purpura (ITP). Megakaryocyte area, number and volume per μl bone marrow were significantly higher in PT as compared to controls. The nuclear lobe number was significantly increased and the megakaryocytes were shifted towards more mature forms, suggesting a prolonged megakaryocyte generation time. In CML the megakaryocyte number and volume per μl bone marrow were also significantly above normal, but the megakaryocyte area, number of lobes and degree of megakaryocytic maturation were significantly below normal. Platelet production was in PT 6.2 times normal and proportional to the increase in megakaryocyte volume which was 6.8 times normal. In PV with major splenomegaly the mean platelet production rate was higher (9.5 times normal) although their peripheral platelet count was lower than in PT. This discrepancy is explained by the greatly enlarged splenic platelet pool in the PV patients. In ITP the mean platelet production rate was 2.2 to 3 times normal and was significantly lower than in PT and PV.     

The relation of megakaryocyte ploidy to platelet volume

To determine how alterations of megakaryocyte proliferation will affect platelet production, we measured mean platelet volume (MPV), platelet volume heterogeneity, platelet count, and mean megakaryocyte ploidy in 42 patients. In normal subjects, mean platelet volume and megakaryocyte ploidy were related inversely but nonlin-early to platelet count, whereas mean platelet volume and platelet volume heterogeneity were related directly. In patients with immune thrombocytopenic purpura (low platelet count, MPV above normal, and increased megakaryocyte ploidy), and in those with reactive thrombocytosis (high platelet count, low MPV and megakaryocyte ploidy), the relation of MPV to megakaryocyte ploidy, platelet volume heterogeneity, and platelet count resembled or extended the relations found in normal subjects. By contrast, in patients with aplastic anemia or megaloblastic anemia, or in patients who were undergoing chemotherapy for leukemia, heterogeneity was increased abnormally at any MPV, and both MPV and megakaryocyte ploidy were substantially lower, at any platelet volume, than in normals or the above other groups. The most common ploidy class was 8N in all patients, and the mean megakaryocyte ploidy correlated directly and linearly with mean platelet volume. The data show that bone marrow with megakaryocytes of higher ploidy produces platelets that are both larger and more heterogeneous.     

The Relation of Platelet Kinetics to Bone Marrow Megakaryocytes in Chronic Granulocytic Leukaemia

The relation of thrombokinetics to quantitative determinations of megakaryocytes (mgkc) in bone marrow sections was studied in 11 consecutive cases of untreated Ph¹-positive chronic granulocytic leukaemia (CGL). The results were compared with controls and with previously obtained data in polycythaemia vera (PV), primary thrombocythaemia (PT) and in idiopathic thrombocytopenic purpura (ITP). Platelet survival was significantly reduced in CGL. Platelet production was 5.8 x normal and the mgkc number and volume/μl bone marrow were significantly increased as compared to controls. The increase in mgkc volume was not in proportion to that of number due to a significant decrease of mgkc size. Platelet production was strongly related to mgkc number/mm² and to the mgkc volume/μl bone marrow. The platelet production rate in relation to a unit of mgkc volume/μl bone marrow was, however, greater in CGL than in controls, PV, PT and ITP. The chief reason for this is most probably the greater expansion of the total bone marrow mass in CGL.     

Circulating Micromegakaryocytes Signaling Blast Transformation of Chronic Myeloid Leukaemia

A patient with chronic myeloid leukaemia had numerous micromegakaryocytes in the peripheral blood and bone marrow appearing coincidentally with the onset of blast crisis. These cells were initially confused with lymphocytes because of their size, configuration and scanty cytoplasm. The true identification of these cells can be suspected by careful scrutiny of well prepared Wrights stained preparations and proven electronmicroscopically. Such marked dysplasia of the megakaryocyte series appears to be a poor prognostic sign in chronic myeloid leukaemia.     

The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis

Thrombopoietin (TPO), the ligand for the c-Mpl cytokine receptor, is a recently identified cytokine with potent effects on platelet production. The receptor-binding portion of c-Mpl ligand is encompassed in another molecule known as megakaryocyte growth and development factor, or MGDF. Although it is clear that the administration of TPO or MGDF to animals dramatically increases the platelet count, the specific stage(s) of thrombopoiesis during which these molecules are principally active have not been unambiguously determined. Pharmacology studies administering MGDF at doses ranging from 0.1 to 630 μg/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 μg/kg/d resulted in platelet counts 5-fold above normal. However, doses >60 μg/kg/d resulted in less-than-optimal platelet production. This phenomenon was investigated in vitro. Using an established culture system for the generation of human megakaryocytes and platelets, MGDF was shown to be optimally and equivalently active in the generation of mature megakaryocytes at concentrations from 10 to 1000 ng/ml. However, the cytokine was not required for proplatelet formation and in fact was inhibitory to that process in a dose-dependent manner. When MGDF was added to human megakaryocytes at concentrations of 200 ng/ml or greater, proplatelet formation was inhibited to 30% of control values. MGDF-mediated inhibition was specific, since the addition of the truncated form of the c-Mpl receptor reversed the inhibition in a dose-dependent manner. Other recombinant factors, interleukin-6, interleukin-11 and erythropoietin had no significant positive or negative effects in this human proplatelet assay. Together, these data suggest that although TPO and MGDF promote the full spectrum of megakaryocyte growth and development, they are not necessary for proplatelet formation, and may in part regulate platelet shedding by their absence.     

Is the histological classification of chronic granulocytic leukaemia justified from the clinical point of view?

By means of morphometric techniques, in 100 untreated Ph'-positive chronic granulocytic leukaemia (CGL) patients the main features from the initial bone marrow biopsy were analyzed, with particular attention being paid to morphological and quantitative study of megakaryocytes. The number of megakaryocytes per mm2 of marrow tissue showed a mean value of 25.3 (SD +/- 18.8), and was positively correlated with either platelet counts, blood percentage of basophils and blast cells, or spleen and liver size. Based on the number and morphological characteristics of megakaryocytes, patients were classified as having granulocytic CGL (67 cases) or the so-called chronic megakaryocytic-granulocytic myelosis (33 cases), but except for higher platelet counts and blood percentages of basophils and blast cells in the latter, no relevant clinical, evolutionary or prognostic differences were observed between the groups. Such results cast doubt on the validity of histological classification of CGL from the clinical point of view.