Emperipolesis--a peculiar feature of megakaryocytes as evaluated in chronic myeloproliferative diseases by morphometry and ultrastructure

A combined morphometric and ultrastructural study was performed on so called emperipolesis or internal wandering of myeloid cells in the cytoplasm of large mature megakaryocytes. Measurements were made on material from a total of 115 patients comprising a normal control group and 5 groups with subtypes of chronic myeloproliferative diseases, including primary (essential, idiopathic) thrombocythemia (PTH). A significant increase in this peculiar phenomenon was noted in myeloproliferative disorders and especially in PTH where the frequency of emperipolesis showed a positive correlation with the number of anuclear cytoplasmic fragments sectioned, with the circular deviation of the shapes of megakaryocytes and with the extent to which the peripheral thrombocyte count was elevated. Electron microscopy in selected cases displayed the integrity of the plasma membranes of engulfed hematopoietic cells within the dilated cavities of the megakaryocytic demarcation membrane system (DMS) and no evidence of phagocytosis. Moreover there was a close relationship between engulfed myeloid cells and the presumptive sites of platelet shedding which had their openings from the cisternal lumina of the DMS. Our results demonstrate that emperipolesis of hematopoietic cells within megakaryocytes should not be regarded as a special nosological feature, but as an indication of enforced thrombocytogenetic activity which is expressed particularly in PTH.     

Abnormalities of platelet aggregation in chronic myeloproliferative disorders

A large variety of platelet dysfunctions has been described in chronic myeloproliferative disorders. These abnormalities may be due to deficiency of platelet granules, arahidonic acid metabolism defects or platelet membrane glycoproteins abnormalities. In this study we intend to detect the incidence of platelet function defects in 76 patients with various types of chronic myeloproliferative disorders. The platelet activity was studied in vitro by measuring platelet aggregation in response to ADP, epinephrine, collagen, arachidonic acid and ristocetin. These results were subsequently correlated with bleeding time and clinical aspects (bleeding or thrombosis). We found complex changes in platelet response with all agonists, in varied proportions. These abnormalities include absent, decreased or abnormal platelet aggregation response. In a few cases we found a markedly decreased, almost absent platelet response to all agonists while in some patients a normal platelet aggregation was noted. The correlation between these results and template bleeding time, thrombotic or hemorrhagic events and the type of diseases was difficult to establish and sometimes conflictual. Despite this fact, we consider that investigating platelet aggregation may be useful not only for the assesment of the hemostatic balance in chronic myeloproliferative disorders but also for a better insight into cell abnormalities occuring in these pathologic conditions.     

Planimetric analysis of megakaryocytes in the four main groups of chronic myeloproliferative disorders.

Planimetry of megakaryocytes (MK) was performed in bone marrow biopsies (BMBs) from patients with chronic myeloproliferative disorders (CMPD) to substantiate cytomorphologic differences in this cell lineage between the four main groups of CMPD. The biopsy specimens were classified histologically prior to morphometry, according to the Hannover Classification of CMPD. Five histological groups were investigated, evaluating between 21 and 30 biopsies in each group. The five groups were as follows: (1) Chronic myelocytic leukemia (CML) of common type (CML.CT), (2) CML with megakaryocytic increase (CML.MI), (3) polycythemia vera (P. vera), (4) primary thrombocythemia (PTH), and (5) chronic megakaryocytic-granulocytic myelosis (CMGM). The results of five variables, i.e. the cellular and nuclear size, the cellular and nuclear form factor, and nuclear segmentation, were determined in at least 50 MK per BMB. The results reveal significant differences in MK nuclear and cellular size, as well as in nuclear segmentation between CML and the three other groups in that the nuclear and cellular size of the MK in CML are smaller than in P. vera, PTH, and CMGM. Moreover, the degree of nuclear segmentation or lobulation differs significantly between the three disorders characterized by large MK. Discriminant analysis permits 78-100% reliability of reclassification by morphometry compared with the histologic classification. A reduced reliability of the morphometric classification to around 80% was found between P. vera and PTH, as well as between P. vera and CMGM. In the design of this study, morphometry of MK lends added weight to the subjective classification of these disorders.     

Cytochemical studies in megakaryocytes in hematologic diseases]

Bone marrow smears of 48 patients consisting of 12 normal cases, 36 patients with different haematological diseases-among them 9 cases of idiopathic thrombopenia, 4 cases of polycythaemia, and 9 cases of Hodgkin's disease - were examined cytochemically. Acid phosphatase, unspecific esterases, naphthol-AS-D-chloroacetate esterase, peroxydase, and leucin-aminopeptidase were represented; in addition the PAS reaction, fastgreen staining at pH 1.1, methyl-green pyronin staining and the lipid representation with Sudan black B were carried out. Besides those responses known from literature the different behaviour of acid megacaryocyte phosphatase in different haematological diseases must be particularly emphasized from all reactions.     

Monoclonal gammopathy in chronic myeloproliferative disorders

The incidence of monoclonal gammopathy in 61 patients with chronic myeloproliferative disorders (CMPD) was studied. The distribution of patients among the CMPD subgroups was: chronic myelocytic leukemia, 24 patients; myelofibrosis, 11; polycythemia vera, 15; essential thrombocythemia, 7; unclassified MPD, 4 patients. Monoclonal gammopathy was found in 5 patients (8.2%). Two of these patients (1 IgA/k and 1 IgM/k) had myelofibrosis and 3 (2 IgG/k and 1 IgG/lambda) polycythemia vera. The presence of monoclonal gammopathy indicates an involvement of the lymphoplasmatic system in CMPD.     

Myelofibrosis in chronic myeloproliferative disorders--dynamics and clinical impact

In chronic myeloproliferative disorders, presenting or evolving myelofibrosis (MF) is associated with significant morbidity and mortality. A systematically conducted evaluation of previous studies and data from our own material reveals a strikingly expressed heterogeneity of findings. Assessment of MF should be performed by a recently established semiquantitative scoring system regarding quantity and quality (reticulin versus collagen). It is important to differentiate between a fiber increase in bone marrow specimens and the clinical diagnosis that is explicitly based on extramedullary hematopoiesis (myeloid metaplasia). For this reason, prodromal stages of (reticulin) fibrosis are overlooked by the clinicians. Up to 30% of patients with chronic myelogenous leukemia show a minimal to advanced MF that is significantly associated not only with corresponding clinical parameters but more importantly with prognosis. In polycythemia vera about 20% of patients may display some degree of reticulin fibrosis at diagnosis, depending on stage of the disease. Transformation into (collagen) MF after more than 10 years is accompanied by clinical signs of myeloid metaplasia (spent phase). Essential thrombocythemia (ET) is characterised by the absence of increased reticulin at onset and an insignificant progression into MF, provided diagnosis is performed by the WHO criteria. Discrimination of prefibrotic and early stages of chronic idiopathic myelofibrosis (CIMF) from ET is relevant, especially concerning the rate and time usually required for the development of MF with myeloid metaplasia (full-blown CIMF). In conclusion, more elaborate evaluations including standardized grading of MF is warranted by regarding bone marrow biopsy specimens in association with clinical parameters including follow-up examinations.     

The use of the anti-factor VIII method on trephine biopsies of the bone marrow for the identification of immature and atypical megakaryocytes in myeloproliferative diseases and allied disorders. A morphometric study

A morphometric analysis was performed on trephine biopsies of the bone marrow to identify atypical megakaryocyte proliferation following PAS staining and the immunohistological demonstration of factor VIII. This study includes nine patients with a megakaryoblastic crisis in chronic myeloid leukemia (CML), four with acute megakaryoblastic leukemia (AM) and three with myeloid dysplasia later evolving into overt acute leukemia. Comparison and statistical evaluation of the PAS reaction with anti-factor VIII staining reveals that the latter technique not only facilitates the recognition of immature and abnormal megakaryocytes, but leads to a significantly increased count for all megakaryocytic elements in the bone marrow. Thus our retrospective investigation of routinely processed and paraffin-embedded trephine biopsies shows that the diagnosis of a megakaryoblastic crisis in CML as well as AM may be easily established with the aid of the anti-factor VIII method. In all cases of megakaryoblastic proliferation in CML and AM, the appearance of blasts was associated with moderate to pronounced myelofibrosis which could be also determined by morphometry.     

Expression of the surface membrane receptors of neutrophils in myeloproliferative diseases

For 147 patients with myeloproliferative diseases, a study was made of the expression of Fc-receptors to immunoglobulins IgC, IgA, and that of FcH-receptor and receptors to C3-components of the complement in peripheral blood neutrophils. The data obtained show the lower level of neutrophils having membrane receptors in patients with acute myeloblastic leukemia and chronic granulocytic leukemia at the stage of blast transformation. A decrease in expression of membrane receptors of neutrophils was shown in patients with chronic granulocytic leukemia and benign subleukemic myelosis. The finding of a higher level of neutrophils having surface receptors revealed in patients with real polycythemia is close to the data obtained in the study of expression of membrane receptors in patients with chronic myeloproliferative diseases and healthy persons.     

CD34+ stem cells in chronic myeloproliferative disorders

Contrasting the wealth of information that is available about various biological and therapeutic aspects of human CD34+ stem cells, little data exist concerning their quantity and dynamics as well as their mutual relationships with other hematopoietic constituents in the bone marrow of patients with chronic myeloproliferative disorders. In comparison with a control group frequency of progenitors is significantly increased in chronic myeloid leukemia (CML). Following different therapeutic modalities their quantity reflects therapeutic efficacy (responder and non-responder patients) and therefore exerts a predictive value regarding acceleration and blastic crisis. The significant correlations between fiber content and number of these precursors elucidates the complex interactions between stroma and progenitor cell differentiation and maturation. Following allogeneic bone marrow transplantation there is a rapid recovery of the CD34+ stem cell population in the first month. A higher number of these cells is related with graft size, an earlier independence for platelet transfusion and a more extended regeneration of erythro- and megakaryopoiesis. The slight increase in reticulin fibers in these patients may be associated with the complex and so far ill-defined pathomechanism of homing (adherence to the fibrous matrix). In idiopathic myelofibrosis (IMF) an increased number of CD34+ stem cells is found predominantly in the early (prefibrotic or mild fibrotic) hypercellular stages and probably indicates a higher proliferative activity of the precursor cell pool. According to sequential biopsies most patients with early IMF that later evolved into an overt fibrosclerotic stage usually display a reduction of progenitor cells during the development of myelofibrosis. The unequal distribution of CD34+ stem cells in the bone marrow versus spleen in IMF (advanced fibrosclerotic stage) is in support of the currently discussed hypothesis of splenic filtration and concentration of precursor cells as an essential feature of myeloid metaplasia. Regarding prognosis in CML a higher amount of CD34+ stem cells is significantly associated with an unfavorable survival and thus confirms the assumed implication of an accelerated phase of disease at onset. On the other hand, in polycythemia vera (PV) and IMF a low number of progenitors is probably due to a decreased proliferation rate (reduced hematopoietic turnover index) and therefore reflects a reduction in the regenerative capacity of hematopoiesis. For this reason, a presumptive defect in the recovery of normal and clonally transformed stem cells is speculated to add to the worsening of prognosis by causing the well-known bone marrow insufficiency in terminal stage PV and IMF.     

Abnormalities of the erythrocyte membrane in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by excessive growth of myeloid cells and their progenitors. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A (Con-A) agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3. The anion transport activities of erythrocytes from patients with CML and normal donors were comparable. In CML erythrocytes, significant reduction in the number of ankyrin-binding sites, present in the cytoplasmic domain of band 3, may lead to partial loss of cytoskeletal anchorage to the bilayer and account for their increased Con-A agglutinability and heat sensitivity and may lead to their premature removal from the circulation.     

Nuclear magnetic resonance investigation of erythrocyte membranes in chronic myeloproliferative disorders

The temperature dependence of the apparent water diffusional exchange through erythrocyte membranes in cases of policitemia vera, chronic granulocytic leukemia and primary myelofibrosis was measured by using a nuclear magnetic resonance method in the presence of Mn2+. The thermal transition shifted to lower temperatures in all cases, regardless of the stage of the disease, suggesting a structural alteration of the membrane. The shift of transition indirectly suggests a lower penetration of the erythrocytes by Mn2+. The water exchange time at 37 degrees C also increased, mainly in the blast crisis; it seems to have a prognostic value of some clinical interest. No simple correlation of the water exchange and the following clinical investigations was observed: the white count, the percentage of promyelocites and myeloblasts, the sedimentation rate of blood, the osmotic fragility of erythrocytes, the total concentration of proteins, albumin and immunoglobulins, respectively, in plasma.