Megakaryocyte ploidy in thrombocytosis: improved microdensitometric measurements with a new image analysis system

Megakaryocyte (MK) nuclear deoxyribonucleic acid (DNA) content was measured on a new image analysis system. Feulgen-stained bone marrow aspirate smears were analysed from 9 patients with thrombocytosis. Average optical density (OD) of stained nuclei was evaluated by pixel discrimination over 128 grey levels. Projected nuclear area was delineated manually. The product of these two parameters gave an index of nuclear DNA content. Neutrophils were used as 2N reference cells. Reproducibility of OD, nuclear area and their product was excellent for individual cells (CV less than 2.0% for MKs, less than 4.0% for neutrophils). The average CV for DNA content of 18 groups of 10 neutrophils was 5.5% (range 3.0-9.7%). A significant linear regression existed between MK nuclear area and DNA content (p much less than 0.001) for 627 MKs in essential thrombocythaemia (ET) and 346 MKs in reactive thrombocytosis (RT). Compared with RT, more 8N and 64N MKs were seen in ET (p less than 0.05). 128N MKs were unique to ET. Image analysis of MK ploidy may assist the clinical discrimination between primary and secondary thrombocytosis.     

Flow cytometric analysis of megakaryocytes from patients with abnormal platelet counts

Megakaryocytes (MKs) from 40 patients with quantitative platelet disorders and 19 normal volunteers were analyzed by flow cytometry for size, fine cell internal structure and granularity, membrane expression of the glycoprotein (GP) IIb/IIIa complex, and for ploidy distribution. Analysis was performed on unfractionated minimally manipulated marrows obtained from routine bone marrow aspirates. MKs were labeled with a fluorescent lineage-specific monoclonal antibody to the GPIIb/IIIa complex followed by DNA staining with propidium iodide. Eight hundred to 3,000 MKs were analyzed in each sample. The modal ploidy distribution in normals was 16N, comprising about half of the megakaryocytic population, with 22.6% of the cells less than or equal to 8N and 22.0% greater than or equal to 32N. Twelve thrombocytopenic patients with decreased marrow MKs on biopsy (mean platelet count [MPC] 44,600/microliters) showed an increase in low ploidy cells with 53.2% less than or equal to 8N (P less than .01); cell size was reduced in three patients when compared to normal cells of identical ploidy (P less than .05). Eight thrombocytopenic patients with enhanced platelet destruction (with normal or increased MKs on biopsy and shortened platelet survival; MPC 41,400/microliters) showed an increased proportion of high ploidy cells greater than or equal to 32N to 39.2% (P less than .01). Increased cell size and granularity were found in four of these patients (P less than .05). Six patients with thrombocytopenia secondary to multiple mechanisms affecting both platelet production and destruction (MPC 66,700/microliters) showed no shift in ploidy. Four patients with primary thrombocytosis (two with thrombocythemia and two with polycythemia vera; MPC 822,500/microliters) showed a marked shift toward high ploidy cells with 42.3% greater than or equal to 32N and 7.6% greater than or equal to 64N cells (P less than .01). The shift was accompanied by a marked increase in cell size and granularity in the patients with thrombocythemia. Ten patients with thrombocytosis secondary to chronic blood loss, malignant or inflammatory disorders (MPC 714,000/microliters), showed variable distributions with four patients exhibiting a shift in ploidy to the right similar to that found in the patients with increased platelet destruction. Based upon the present data, flow cytometric ploidy distribution may be diagnostically useful in thrombocytopenic patients by discriminating between disorders of platelet production and destruction. (ABSTRACT TRUNCATED AT 400 WORDS)     

Uncontrolled thrombocytosis in chronic myeloproliferative disorders

S ummary . A retrospective study was performed to examine the natural course of uncontrolled thrombocytosis associated with chronic myeloproliferative disorders. Thirty-eight patients with polycythaemia rubra vera (PV), myelofibrosis/myeloid metaplasia (MM), chronic myelogenous leukaemia (CML) or essential thrombocythaemia (ET) had platelet counts greater than 1000 × 10⁹/1 and were followed closely for a total of 246 patient years. Eleven of the patients experienced haemorrhagic episodes. Bleeding was twice as frequent in patients over 59 years old as in those younger and no bleeding occurred in those less than 51 years of age. There was no correlation between frequency of bleeding and extent of thrombocytosis. Bleeding events occurred concurrently with use of anti-inflammatory agents in 32% of episodes. The gastrointestinal tract was the most frequent site. Documented thrombotic events occurred in three patients, two of whom had PV with haematocrits greater than 53%. This study suggests that the thrombocytosis of myeloproliferative processes may pose a less serious threat than originally thought and that aggressive lowering of the platelet count may not be indicated in all cases.     

Heterogeneity of in vitro growth pattern of megakaryocyte progenitors (CFU-M) in myeloproliferative disorders

In groups of 26 patients with myeloproliferative disorders (MPD), 8 with chronic myelogenous leukaemia (CML); 8 with polycythaemia vera (PV); 10 with essential thrombocythaemia (ET); and 6 patients with reactive thrombocytosis (RT), we studied the growth characteristics of bone marrow CFU-M in agar culture. The bone marrows from all the patients with MPD formed so called endogenous CFU-M colonies, in the absence of PHA-LCM, that increased in a dose-dependent manner with the addition of increasing concentrations of normal human AB-citrated plasma (NH-ABCP), while the bone marrows from all the patients with RT and from healthy controls formed few or no endogenous CFU-M colonies. In MPD, the endogenous CFU-M growth was enhanced by normal T cells in a dose-dependent fashion, and was decreased with the depletion of T cells from the marrow cells. These results suggest that the formation of endogenous CFU-M colonies is caused by hypersensitivity of CFU-M in MPD to NH-ABCP, which may contain a small amount of Meg-CSF, and/or by in vitro T cell stimulation. Among MPD, the endogenous CFU-M growth in ET was significantly lower than that of other MPD patients; however, the total number of ET CFU-M grown in the presence of PHA-LCM was the highest. These data show that the bone marrow CFU-M in MPD are heterogeneous with respect to in vitro growth pattern or sensitivity to exogenous Meg-CSF.     

Platelets in Myeloproliferative Disorders

Certain platelet functions were evaluated in 24 patients with secondary polycythaemia (SP) and in a large number of patients suffering from myeloproliferative disorders (MD'S): 89 patients with chronic myeloid leukaemia (CML) at different stages of development, 58 with polycythaemia vera (PV), 23 with essential thrombocythaemia (ET), and 25 with agnogenic myeloid metaplasia (AMM). Bleeding time, epinephrine-induced platelet aggregation and adhesiveness agree with those generally reported in the literature; they are independent of thrombocytosis, the haemoglobin level and the leucocyte count. Macrothrombocytosis, evaluated by an electronic method, was only found in CML, mainly during acute blast crisis. An increased percentage of light platelets was a constant feature in all groups except in the SP and in 20 % of the PV. The most severe abnormalities were observed in AMM and CML in the acute stage; in the chronic phase of CML there is no correlation between the severity of platelet abnormalities and the survival of the patients.     

Platelet density in essential thrombocythemia and polycythemia vera

This study aims to compare platelet density in myeloproliferative disorders (essential thrombocythemia (ET) and polycythemia vera (PV)) with platelet density of healthy subjects. Platelet density peaks were determined using a specially designed apparatus for scanning light transmission variations along test tubes containing density-separated platelets. Eighteen patients with myeloproliferative disorders (nine ET and nine PV) were compared with a control group consisting of 12 healthy volunteers. Compared with healthy volunteers, patients with myeloproliferative disorders had significantly lower platelet peak density ( P< 0.001). It is concluded that determination of peak platelet density may be a useful tool for excluding ET and PV. A high platelet density peak makes a clonal disorder less likely and a low density peak would confirm the suspicion.     

Platelet density in essential thrombocythemia and polycythemia vera

This study aims to compare platelet density in myeloproliferative disorders (essential thrombocythemia (ET) and polycythemia vera (PV)) with platelet density of healthy subjects. Platelet density peaks were determined using a specially designed apparatus for scanning light transmission variations along test tubes containing density-separated platelets. Eighteen patients with myeloproliferative disorders (nine ET and nine PV) were compared with a control group consisting of 12 healthy volunteers. Compared with healthy volunteers, patients with myeloproliferative disorders had significantly lower platelet peak density ( P< 0.001). It is concluded that determination of peak platelet density may be a useful tool for excluding ET and PV. A high platelet density peak makes a clonal disorder less likely and a low density peak would confirm the suspicion.     

Leukocyte Function in Chronic Myeloproliferative Disorders

ABSTRACT: The myeloproliferative disorders (MPD) are clonal diseases that originate from a transformed stem cell and involve all myeloid lineage. The affected cells have both proliferative and functional impairment. Therefore, we evaluated and compared neutrophil function in 31 patients with polycythemia vera (PV), idiopathic myelofibrosis (MF), chronic myeloid leukemia (CML), and essential thrombocytosis (ET). Neutrophil chemotaxis, random migration, bactericidal activity and superoxide anion release in these patients were simultaneously compared to those of 31 healthy controls. In this study, chemotactic activity was significantly impaired in patients with PV and CML as compared to controls (M±SE: 42 ± 6 vs. 69± 5 cells/field; p<0.005 and 47±7 vs. 68± 5; p<0.05, respectively). The assessment of the bactericidal activity of neutrophils showed no impairment in most of the patients. In the CML group, the serum had a very strong “lytic” effect on bacteria, possibly due to the high levels of serum lysozyme (22 ± 2 ug/ml). The superoxide anion release was found to be normal in most of the patients. Nevertheless, in 25% of PV patients the superoxide production was impaired (less than 60% of the simultaneous controls). In ET most patients had normal neutrophil function. Regarding the effect of treatment, neutrophil chemotactic activity was found to be significantly reduced in the hydrea-treated patients, as compared to the non-treated patients (p<0.001) or healthy controls (<0.0001). We conclude that disturbances in neutrophil function are present in patients with various MPDs, except ET. This probably reflects abnormal maturation of ancessors of the damaged stem cells. Nevertheless, we should keep in mind that therapy itself could affect neutrophil functions. This matter should be studied more extensively. Although infections are not common in MPD disorders, they occasionally occur. It is possible that impairment in the phagocytic function contribute to the development of infections in patients with myeloproliferative disorders.     

Chronic myeloproliferative neoplasms and subsequent cancer risk: a Danish population-based cohort study

Patients with chronic myeloproliferative neoplasms, including essential thrombocythemia (ET), polycythemia vera (PV), and chronic myeloid leukemia (CML), are at increased risk of new hematologic malignancies, but their risk of nonhematologic malignancies remains unknown. In the present study, we assessed the risk of both types of malignancies after an ET, PV, or CML diagnosis. We linked 2 population-based nationwide registries, the Danish National Registry of Patients, covering all Danish hospitals and the Danish Cancer Registry, and assessed subsequent cancer risk in a cohort of all 7229 patients diagnosed with a chronic myeloproliferative neoplasm during 1977-2008. We compared the incidence of subsequent cancer in this cohort with that expected on the basis of cancer incidence in the general population (standardized incidence ratio). Overall, ET, PV, and CML patients were at increased risk of developing both new hematologic and nonhematologic cancers. The standardized incidence ratio for developing a nonhematologic cancer was 1.2 (95% confidence interval [95% CI]): 1.0-1.4) for patients with ET, 1.4 (95% CI: 1.3-1.5) for patients with PV, and 1.6 (95% CI: 1.3-2.0) for patients with CML. We conclude that patients with chronic myeloproliferative neoplasms are at increased risk of developing a new malignant disease.     

Effects of recombinant interferons on human megakaryocyte growth

Interferons (IFNs) have been shown to suppress the proliferation of human pluripotent and single-lineage hematopoietic progenitor cells. Treatments with IFNs have reduced platelet counts in patients with myeloproliferative disorders (MPD) but have not altered platelet counts in patients with healthy marrows. We assessed recombinant alpha and gamma IFN (rIFN-alpha and rIFN-gamma) preparations for their effect on the growth of marrow megakaryocytes (MKs) from normal donors and from patients with MPD. In addition, the interactions of recombinant interleukin 3 (rIL-3) recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) with the suppressive effects of rIFNs were examined. The addition of rIFN-alpha to liquid cultures resulted in a dose-dependent inhibition of normal marrow and MPD marrow MK growth. Inhibition with rIFN-gamma was only observed in normal marrows at 2500 U/ml and 12,500 U/ml; and rIFN-gamma unexpectedly stimulated MK growth in some culture conditions. Thirty units per milliliter rIL-3 overcame the inhibitory effect of rIFN-alpha on MK growth, but rGM-CSF or 3 U/ml rIL-3 did not. These studies with rIFN-alpha demonstrate that MPD marrow MKs and their precursor cells are no more sensitive to rIFN-alpha than are normal marrow MKs. Clinically, rIFN-gamma would be expected to be less effective than rIFN-alpha at controlling thrombocytosis in patients with MPD.     

Multimeric composition of plasma von Willebrand factor in chronic myeloproliferative disorders

Summary In chronic myeloproliferative disorders (CMPD) thrombohaemorrhagic complications occur occasionally in association with thrombocytosis. We studied the multimeric composition of plasma von Willebrand factor (vWf) in 15 patients with polycythaemia vera (PV), 12 with essential thrombocythaemia (ET) and eight with primary myelofibrosis (PMF). The relative content of large (multimer band ≥ 11) multimers calculated by densitometer scan following SDS-agarose gel electrophoresis was 18.5 ± 4.4% (mean±SD) in normal controls, 8.3 ± 7.9% in PV, 8.1 ± 4.6% in ET and 19.6 ± 6.7% in PMF. The patients with PV and ET but not PMF had a significantly lower percentage of large multimers than normal controls (P < 0.001). The relative content of large multimers was negatively correlated with WBC and platelet count (P < 0.02 each) in PV. It was negatively correlated with platelet count (P < 0.005) and was positively correlated with a ratio of ristocetin cofactor/vWf antigen (RCof/vWf: Ag)(P < 0.01) in ET. These results indicate that acquired defects of vWf are quite common in PV and ET but not in PMF. In addition, some CMPD patients with high platelet counts completely lacked large multimers. The negative correlation of the relative content of large multimers with platelet count suggests that large multimers may be preferentially consumed during thrombocytosis or degraded by protease(s) from increased blood cells.     

Spontaneous megakaryocytic colony formation does not discriminate between essential thrombocythemia and polycythemia vera

Laboratory detection of spontaneous growth of colony-forming unit-megacaryocytes (CFU-MK), allowing us to distinguish essential thrombocythemia (ET) from reactive thrombocytosis, is therefore useful for the diagnostic of this myeloproliferative disorder. Whether CFU-MK assays allow us to discriminate at least partly between ET and other myeloproliferative disorders such as polycythemia vera (PV) remains, however, to be established. To gain insights about this point, we have performed CFU-MK cultures from bone marrow cells of patients diagnosed with ET (n = 42) or PV (n = 50) using a standardized collagen-based serum-free method. Spontaneous growth of CFU-MK was similarly detected in both 40/42 patients with ET and 47/50 patients with PV. These data suggest clearly that the CFU-MK assay is useful to detect not only ET, but also PV, but fails to discriminate, even partly, between these two myeloproliferative disorders.     

Hypersensitivity of circulating progenitor cells to megakaryocyte growth and development factor (PEG-rHu MGDF) in essential thrombocythemia

Hematopoietic progenitor cells in 2 myeloproliferative disorders, juvenile chronic myelomonocytic leukemia and polycythemia vera, are known to be hypersensitive to cytokines that control normal progenitor cell proliferation, differentiation, and survival in their respective granulocyte/macrophage and erythroid lineages. Because thrombopoietin controls these functions in the normal megakaryocytic lineage, we asked the question: Are megakaryocytic progenitor cells in the myeloproliferative disorder essential thrombocythemia (ET) hypersensitive to thrombopoietin? Peripheral blood mononuclear cells from patients with ET, or secondary (reactive) thrombocytosis (2 degrees T), or healthy volunteers were grown in strictly serum-free agarose culture containing interleukin 3 (IL-3) and all-trans-retinoic acid, with various concentrations of PEG-rHu megakaryocyte growth and development factor (MGDF). The concentration of cytokine at half-maximum colony number served as a measure of progenitor cell sensitivity. Hypersensitivity to PEG-rHu MGDF was found in circulating progenitors from 18 of 20 (90%) informative patients with presumptive diagnosis ET, 1 of 8 (12.5%) 2 degrees T patients, and none of the 22 healthy volunteers. Median MGDF sensitivity ratio in ET patients was approximately 53 times greater than in the controls. This hypersensitivity, which was also directed to rHu thrombopoietin, was highly specific with respect to cytokine, disease, and cell lineage. We propose that, despite their single pluripotential cell origin, the different clinicopathologic phenotypes in different chronic myeloproliferative disorders are determined by lineage-restricted hypersensitivities of hematopoietic progenitor cells to endogenous cytokines. This work emphasizes the importance of stringent serum-free conditions for revealing true sensitivities to cytokines. The findings also offer a basis for evolving a positive test for ET, a diagnosis now made essentially by exclusion.     

Serum interleukin (IL)-1, IL-2, sIL-2Ra, IL-6 and thrombopoietin levels in patients with chronic myeloproliferative diseases

A number of growth factors are involved in clonal haematopoietic expansion and their clinical significance in patients with chronic myeloproliferative diseases requires further evaluation. Using enzyme-linked immunosorbent assays, we analysed serum levels of interleukin (IL)-1a, IL-1b, IL-2, IL-6, the soluble IL-2 receptor alpha (sIL-2Ra), and thrombopoietin (TPO), in 25 individuals with myelofibrosis with myeloid metaplasia (MMM), 40 with essential thrombocythaemia (ET), eight with polycythaemia vera (PV), 10 patients with chronic myeloid leukaemia (CML) and 27 normal controls. These were correlated with clinicopathological characteristics including overall survival, and histopathological bone marrow features, including angiogenesis. The serum derived from patients with MMM, ET, PV and CML contained significantly higher IL-2 and sIL-2Ra than healthy subjects, while IL-6 levels were higher only in MMM and CML than controls. IL-2, sIL-2Ra and IL-6 levels were raised during the transformation phase of CML, during progression of MMM to AML, and ET and PV to myelofibrosis (P < 0.001). There was a positive correlation between IL-2, sIL-2Ra, IL-6 and angiogenesis in bone marrow samples. Cytokines may be useful markers for predicting clinical evolution, reflecting increased angiogenesis. This requires further evaluation to guide diagnostic and therapeutic options.     

Megakaryocyte cultures in the chronic phase and in the blast crisis of chronic myeloid leukaemia: studies on the differentiation of the megakaryocyte progenitors and on the maturation of megakaryocytes in vitro

Megakaryocyte (MK) colony formation has been studied in the chronic phase and in the blast crisis of chronic myeloid leukaemia (CML). Blood cells were grown in plasma clot for 13 d. MKs were subsequently identified by immunofluorescent techniques using two monoclonal antiplatelet antibodies (AN51 and J15). The maturation process was studied by ultrastructural methods. A marked increase in the number of circulating CFU-MK was observed in all the 10 cases studied prior to chemotherapy (70-fold increase per ml of blood). No significant modification in the regulation of MK colony formation as compared to that of normal subjects was observed. The predominant abnormality in maturation in culture was the occurrence of many hypoploid MKs (microMKs). However, the cytoplasmic maturation of the MKs was identical to that of normal subjects with occasional platelet shedding. Since microMKs predominated in some patients, scoring of MK colonies in CML necessitated immunofluorescent labelling to permit identification of MKs. During the blast crisis, MK colony formation occurred in four out of five patients with an extremely high plating efficiency in the case of promegakaryoblastic transformation. In contrast, MK colonies could not be grown from blood samples of patients with acute leukaemia, including two cases of promegakaryoblastic leukaemia. Maturation of MKs in blast crisis was identical to that of the chronic phase. Furthermore, after short periods of culture in liquid medium, circulating promegakaryoblasts from spontaneously to become large MKs exhibiting demarcation membranes and α-granules, while those from two cases of megakaryoblastic leukaemias did not mature. In consequence, the blast crisis of CML exhibits a different culture pattern from acute leukaemia. These results suggest, therefore, that the acute transformation of CML cannot be simply explained by clonal changes and that environmental and regulatory factors could be also involved.     

Platelet c-mpl expression is dysregulated in patients with essential thrombocythaemia but this is not of diagnostic value

Essential thrombocythaemia (ET) can be difficult to discriminate from an occult case of reactive thrombocytosis (RT). Since thrombopoietin (TPO) is the primary regulator of thrombopoiesis, we have investigated whether levels of TPO and/or its receptor, c-mpl, are of value in the differential diagnosis of ET. Plasma TPO levels in patients with ET, RT and other myeloproliferative disorders (MPDs) did not differ significantly from normal controls. However, surface c-mpl expression was significantly reduced in platelets from 18 ET patients, 0-65.5% of controls (P < 0.001). Immunoblots on five of these and five additional patients were consistent with absent or reduced c-mpl protein levels. The surface c-mpl expression results were significantly different from those in eight RT patients (21. 3-95.5%, P = 0.0015), but there was considerable overlap between the two groups and a reduced level was not restricted to ET. Furthermore, c-mpl expression in ET patients was not different from eight patients with other MPDs (0-87.6%, P = 0.06), nor could it differentiate between ET patients with monoclonal and polyclonal haemopoiesis. Although a low or absent c-mpl level is suggestive of a primary rather than a secondary thrombocytosis, it is insufficiently discriminatory to be used as a diagnostic marker for ET.     

Bone marrow stromal cells in myeloproliferative disorders

The number of bone marrow-derived fibroblastoid colony-forming cells (CFU-F) and the production of colony-stimulating activity (CSA) by bone marrow stromal cells were studied in 71 patients with myeloproliferative disorders (MPD). The numbers of CFU-F in chronic-phase chronic myelogenous leukemia (CML), polycythemia vera (PV) and essential thrombocythemia (ET) were not different from those in normal subjects. However, the number of CFU-F in acute-phase CML was markedly decreased. Bone marrow adipocyte colony-forming capacity (adipo-CFC), which was previously shown to reflect both the number of preadipocytes and the stromal cell function in vivo, was increased in patients with chronic-phase CML, PV and ET, but was absent in acute-phase CML patients. The production of CSA by marrow stromal cells of MPD patients, however, was not different from that of normal subjects. These results suggest that the characteristics of marrow stromal and its precursor cells of chronic-phase MPD patients were not different from those of normal subjects, however, they became changed in acute-phase CML patients.