Specific Features of Interactions between Megakaryocytic and Granulocytic Hematopoiesis Lineages and Myelofibrosis during the Acute Phase of Chronic Myeloid Leukemia, Chronic Lymphocytic Leukemia, and Multiple Myeloma.

The specific features of interactions between megakaryocytic and granulocytic hematopoiesis lineages and myelofibrosis were studied in patients with active phase (before treatment) of chronic myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. In patients with chronic myeloid leukemia, a direct correlation was found between the severity of both early and advanced myelofibrosis and the number of megakaryocytes in the bone marrow irrespectively of the type of the bone marrow tumor. The parameters of granulocytic hematopoiesis lineage were higher in myelofibrosis. In patients with chronic lymphocytic leukemia and multiple myeloma, negative correlations between the severity of early and advanced myelofibrosis and the number of megakaryocytes in the bone marrow and platelets in the peripheral blood were found. In chronic lymphocytic leukemia, negative correlations between the severity of early and advanced myelofibrosis and the number of neutrophils in the bone marrow and peripheral blood were detected. In patients with multiple myeloma, negative correlations between the severity of advanced myelofibrosis and number of neutrophils in the bone marrow and peripheral blood were detected.

A megakaryocyte morphological classification-based predictive model for steroid sensitivity in primary immune thrombocytopenia.

The first-line therapy for primary immune thrombocytopenia (ITP) is steroids, but about one-third of patients do not respond to steroids. Recent studies have shown megakaryocyte (MK) growth and development abnormalities and poorly compensated thrombopoiesis. Here, we attempted to determine the impact of MK morphological classification on steroid response. We enrolled 170 adult patients with primary ITP and divided them into steroid-sensitive ITP (109/170) and non-steroid-sensitive ITP (61/170) groups. In the univariate logistic model, female, reduced thrombocytogenic MK count (TMC), increased granular MK count to total MK count ratio (GMC/TM ratio), and elevated naked nucleus MK count to TM count ratio were significantly associated with steroid-sensitive ITP. In the multivariate logistic model, sex, reduced TMC, and increased GMC/TM ratio were independent predictors of steroid-sensitive ITP diagnosis. Based on the regression parameters, we established a predictive index with weighted risk score of 1 assigned each to sex, TMC, and GMC/TM ratio. A predictive index ≥2 points had the best area under the curve value (0.63) with 47.7% sensitivity and 78.7% specificity for predicting steroid sensitivity. These findings may help guide early treatment strategies in ITP.

CD45 expression discriminates waves of embryonic megakaryocytes in the mouse.

Embryonic megakaryopoiesis starts in the yolk sac on gestational day 7.5 as part of the primitive wave of hematopoiesis, and it continues in the fetal liver when this organ is colonized by hematopoietic progenitors between day 9.5 and 10.5, as the definitive hematopoiesis wave. We characterized the precise phenotype of embryo megakaryocytes in the liver at gestational day 11.5, identifying them as CD41++CD45-CD9++CD61+MPL+CD42c+ tetraploid cells that express megakaryocyte-specific transcripts and display differential traits when compared to those present in the yolk sac at the same age. In contrast to megakaryocytes from adult bone marrow, embryo megakaryocytes are CD45- until day 13.5 of gestation, as are both the megakaryocyte progenitors and megakaryocyte/erythroid-committed progenitors. At gestational day 11.5, liver and yolk sac also contain CD41+CD45+ and CD41+CD45- cells. These populations, and that of CD41++CD45-CD42c+ cells, isolated from liver, differentiate in culture into CD41++CD45-CD42c+ proplatelet-bearing megakaryocytes. Also present at this time are CD41-CD45++CD11b+ cells, which produce low numbers of CD41++CD45-CD42c+ megakaryocytes in vitro, as do fetal liver cells expressing the macrophage-specific Csf receptor-1 (Csf1r/CD115) from MaFIA transgenic mice, which give rise poorly to CD41++CD45-CD42c+ embryo megakaryocytes both in vivo and in vitro In contrast, around 30% of adult megakaryocytes (CD41++CD45++CD9++CD42c+) from C57BL/6 and MaFIA mice express CD115. We propose that differential pathways operating in the mouse embryo liver at gestational day 11.5 beget CD41++CD45-CD42c+ embryo megakaryocytes that can be produced from CD41+CD45- or from CD41+CD45+ cells, at difference from those from bone marrow.

An automated image analysis methodology for classifying megakaryocytes in chronic myeloproliferative disorders.

This work describes an automatic method for discrimination in microphotographs between normal and pathological human megakaryocytes and between two kinds of disorders of these cells. A segmentation procedure has been developed, mainly based on mathematical morphology and wavelet transform, to isolate the cells. The features of each megakaryocyte (e.g. area, perimeter and tortuosity of the cell and its nucleus, and shape complexity via elliptic Fourier transform) are used by a regression tree procedure applied twice: the first time to find the set of normal megakaryocytes and the second to distinguish between the pathologies. The output of our classifier has been compared to the interpretation provided by the pathologists and the results show that 98.4% and 97.1% of normal and pathological cells, respectively, have testified an excellent classification. This study proposes a useful aid in supporting the specialist in the classification of megakaryocyte disorders.

Normal human bone marrow CD34(+)CD133(+) cells contain primitive cells able to produce different categories of colony-forming unit megakaryocytes in vitro.

OBJECTIVE: To evaluate the megakaryocyte potential of normal bone marrow (NBM) CD34(+)CD133(+) cells, a subset offering a possible alternative for clinical CD34 immunoselection, we evaluated their colony-forming unit megakaryocyte (CFU-Mk) content and their ability to produce clonogenic Mk progenitors in comparison with the CD133(-) subset. MATERIALS AND METHODS: Sorted NBM CD34(+)CD133(+) and CD34(+)CD133(-) subsets were evaluated for Mk clonogenic capacity before and after in vitro proliferation in serum-free liquid culture containing kit ligand, Flt3 ligand, thrombopoietin, interleukin-3, and interleukin-6. The segregation of CFU-Mk according to the expression of CD34, CD133, and CD41 was compared between fresh BM cells and expanded cells. RESULTS: Although the fresh NBM CD133(-)CD34(+) subset included two thirds CFU-Mk, only the CD133(+) subset contained primitive cells able to produce all categories of CFU-Mk in vitro. Immunophenotyping confirmed that CD41 antigen is nonspecific for Mk lineage and showed that the usual CD34(+)CD41(+) subset does not specifically define a CFU-Mk population. The segregation of CFU-Mk before and after expansion according to CD34, CD41, or CD133 was modified in relation with down-regulation of CD34 and CD133 antigens and up-regulation of CD41 antigen. CONCLUSIONS: The NBM CD133(+) subset contains primitive cells able to generate CFU-Mk, a subset probably relevant to platelet recovery after infusion. The alteration of antigen expression during in vitro proliferation calls for caution in the identification of the different categories of Mk subsets produced and in the assessment of their predictivity for in vivo platelet production.

A practical quick staining method using hydrochloric acid-fast metachromatic dye for megakaryocytes.

A specific stain using violet polymethine dye (VPM stain) for megakaryocytes was first developed by Kass (1995). We have modified this method for practical use in bone marrow specimens. The modified VPM stain labels megakaryocytes very well, while other marrow cells are poorly colorized. This staining procedure was more stable, and its color intensity was finer and clearer than the original. Using this stain, morphologic classification of megakaryocytes in bone marrow specimens from 11 normal and 8 myelodysplastic syndrome (MDS) patients was performed. Many megakaryocytes observed in MDS patients were juvenile compared with normal subjects according to their morphology. Blasts from acute megakaryoblastic leukemia (M7) and from a megakaryoblastic cell line (Mo7e) were also clearly stained with our method. This staining method is practical and very useful for rapid identification of megakaryocyte distribution and morphology.

Peripheral blood megakaryocytes in experimental hemorrhagic shock.

The number and morphological types of megakaryocytes (MK) in the rat inferior cava vein blood were evaluated in untreated hemorrhagic shock lasting 60 min and in 6 to 48 hours after treatment with a standard polyelectrolyte solution (PES). The rats were bled through carotid artery. MK were isolated using the 5 microns filters. The results were compared with those found in the control animals not subjected to surgical manipulation and subjected to sham operation (cervical incision only, and cervical incision + carotid artery cannulation). The most considerable increase in the circulating MK occurred in 12 hours after the PES treatment. The slight increase in the number of MK was also observed in rats with carotid artery cannulation without hemorrhage. Increase in the MK number was accompanied by a shift in their morphological types.

[Circulating megakaryocytes in blood during experimental acute hemorrhagic shock in rats]. / Megakariocyty krazace we krwi w doswiadczalnym wstrzasie krwotocznym u szczurów.

The circulating megakaryocytes (MK) in the central venous blood of rats during haemorrhagic shock have been studied. Nucleopore polycarbonate membranes with a pore size of 5 microns were used to isolate circulating MK. Different morphologic types of MK were analysed. The results showed an increased number of MK migrating from the bone marrow to blood and confirm that circulation MK are a normal physiologic component of blood. The number of the rise during haemorrhagic shock, especially large MK and MK with scant cytoplasm ("naked nuclei" MK).

Alterations in megakaryocyte and platelet compartments following in vivo IL-1 beta administration to normal mice.

IL-1 has been shown to stimulate the release of granulocyte-macrophage CSF, granulocyte-CSF, and macrophage-CSF from "accessory cell populations" in vitro, and it stimulates the appearance of colony-stimulating activity in the sera of mice in vivo. This cytokine has also been proposed to act on primitive hematopoietic progenitor cells to stimulate expression of receptors for the CSF. We sought to determine whether IL-1 beta could influence platelet and/or megakaryocytes and their progenitor cells following in vivo administration to normal mice. Our results demonstrated that, although administration of IL-1 beta clearly expands the pool of megakaryocyte-CFU and acetylcholinesterase-positive megakaryocytic cells (primarily in the spleen), it causes a transient and dose-dependent reduction of circulating platelets. The associated thrombocytopenia can be abolished by splenectomy before IL-1 beta administration, and is not temporally associated with the development of splenomegaly.

Characterization of guinea pig megakaryocyte subpopulations at different phases of maturation prepared with a Celsep separation system.

We introduce a new method for preparing subpopulations of guinea pig megakaryocytes (MK). MK, partially purified by a density gradient, were separated according to size by sedimentation, starting as a monolayer, in an albumin gradient at unit gravity. Twenty-two fractions were collected. Cells were cytocentrifuged, ploidy was assessed by microdensitometry, and small MK were identified with anti-von Willebrand factor (vWF) immunoglobulin. Immaturity was assessed by uptake of 3H thymidine and synthesis of proteoglycans from 35S sulfate. About 88% of cells in fractions 2 through 18 were MK, of which 90% were viable. Fractions containing the largest cells were composed of 98% stage III and IV MK; fractions with the smallest cells contained up to 80% stage I and II MK. Six MK classes were isolated: immature cells, both stage I and II cells, at either the 8N, 16N or 32N ploidy class; mature cells, both stage III and IV cells, at either the 8N, 16N or 32N ploidy class. The fractions were pooled into three groups: (a) 8% of MK in group 1, fractions 2 through 11, were immature, and group 1 was composed of 92% of 16N and 32N mature classes; (b) 29% of MK in group 2, fractions 12 through 15, were immature, and group 2 was composed of 52% 16N mature, 24% 16N immature, and 13% 8N mature classes; 67% of MK in group 3, fractions 16 through 18, were immature, and group 3 contained 51% 8N immature, 14% 16N immature, and 18% mature 16N classes. The mean protein content of the three groups was 1.251, 0.624, and 0.284 mg/10(6) MK, respectively. Nine percent of cells in group 3 but no cells in group 1 took up large amounts of 3H thymidine. The synthesis of high-molecular-weight (high-mol-wt) proteoglycans in group 3 and synthesis of lower mol wt proteoglycans in groups 1 and 2 provided further evidence for differences in MK maturity. Thus, the method can isolate MK subpopulations that are viable and can be used to investigate the biochemical characteristics of MK at different phases of maturation.

Comparative effects of thrombopoietic-stimulatory factor and spleen cell-conditioned medium on megakaryocytopoiesis in a short-term bone marrow liquid culture system.

The effect of partially purified thrombopoietic stimulatory factor (TSF) on megakaryocytopoiesis was studied using the soft-gel colony-forming assay and a short-term marrow liquid culture system (STLC) and compared to the effects of megakaryocyte colony-stimulating activity present in pokeweed mitogen-stimulated spleen cell-conditioned medium (PWCM). Nonadherent cells from STLC were sampled daily for acetylcholinesterase-positive cells and megakaryocyte progenitor cells (CFU-M). CFU-M were assayed in the soft-gel colony-forming system using PWCM as a source of colony-stimulating activity. Proliferative capacity of CFU-M obtained from liquid culture was determined from megakaryocyte colony size (number of megakaryocytes per colony) following plating of cells in a secondary colony-forming assay. Megakaryocytes were grouped into four maturation classes and megakaryocyte diameter was determined on acetylcholinesterase-stained cytocentrifuged cells using an eye-piece micrometer. TSF produced no CFU-M-derived colonies in the soft-gel colony-forming assay. Addition of TSF to STLC had no effect on the total number of CFU-M, megakaryocyte colony size, or total number of megakaryocytes compared to unstimulated STLC. However, on days 4-9 there was a significant increase in megakaryocyte diameter and the proportion of mature (stage III, IV) megakaryocytes obtained from TSF containing STLC compared to unstimulated STLC. In contrast, 5 days after addition of PWCM to STLC a sixfold increase in the total number of CFU-M per flask and a threefold increase in megakaryocytes was observed compared to unstimulated STLC. However, megakaryocyte colony size and megakaryocyte size were significantly reduced and a greater number of immature (stage I, II) megakaryocytes were present in STLC containing PWCM compared to unstimulated STLC. These results indicate that TSF accelerates the maturation of megakaryocytes in vitro and that a factor or factors present in spleen cell-conditioned medium, in addition to influencing megakaryocyte progenitor cell proliferation, also affect(s) megakaryocyte size.

New insights into the regulation of human megakaryocytopoiesis.

It is apparent that multiple cellular stages and biologic processes can be identified during megakaryocytopoiesis that are potentially subject to control by hematopoietic growth factors and marrow accessory cell populations. Two classes of megakaryocyte progenitor cells, the colony forming unit-megakaryocyte (CFU-MK) and the burst forming unit-megakaryocyte (BFU-MK), have now been detected in normal human bone marrow cells. The BFU-MK by virtue of the greater cellular content of its resultant colonies and the delayed time of appearance of these colonies appears to be a more primitive progenitor cell with a greater proliferative potential than the CFU-MK. A number of hematopoietic growth factors including megakaryocyte colony stimulating factor, (MK-CSF), recombinant erythropoietin (EPO) and granulocyte macrophage colony stimulating factor (GM-CSF) are each capable of increasing cloning efficiency of human megakaryocyte progenitor cells. It is presently unknown whether these factors act directly on the CFU-MK or whether they stimulate marrow accessory cells to elaborate growth factors that influence CFU-MK proliferation. In order to answer this question, the effect of these growth factors on the cloning efficiency of a human megakaryocytic cell line, EST-IU, was examined. Each of these factors was capable of increasing leukemia cell colony formation. One can conclude from these studies that MK-CSF, EPO, and GM-CSF act directly on cells of the megakaryocytic lineage. The physiologic significance of the lineage nonspecific effects of EPO and GM-CSF on megakaryocytopoiesis is yet to be determined. On the basis of these observations, a model of human megakaryocytopoiesis was suggested. Several factors appear able to influence multiple steps in megakaryocytic development, whereas others influence only specific stages or cellular events occurring during megakaryocytopoiesis.

Fluorescence-activated sorting of individual cells onto poly-L-lysine-coated slide areas.

Cells sorted by a fluorescence-activated cell sorter are collected onto small areas of a glass slide. These collection areas have been coated with poly-L-lysine to attach the cells firmly to the glass surface. This simple procedure proved to be suitable to sort single cells and small cell populations with preservation of cytomorphology and viability without modifying the cell sorter. Additional studies on sorted cells may be performed, as shown by peroxidase-anti-peroxidase analysis of cellular antigens and by mRNA in situ hybridization.

Morphological studies on the forming processes and patterns of the platelet demarcation membrane system in the megakaryocytic series of embryonic rat livers.

Using injections of horseradish peroxidase (HRP) and the osmium-tannic acid method, megakaryocytic cells in the livers of rat embryos at 12-16 days of gestation were examined for the purpose of classification of the stages of formation of the platelet demarcation membrane. Megakaryoblasts were classified into the following three types according to the formation patterns of the demarcation membrane. The P-type megakaryoblasts showed plate-like membrane invaginations in large localized areas at early stages. The invaginating membrane developed toward the periphery of the nucleus. The L-type megakaryoblasts showed localized labyrinthine membrane invaginations but no definite direction in its development. The T-type megakaryoblasts had tubular invaginations at multiple sites on the plasma membrane. The P- and L-type cells were observed at 12 and 13 days of gestation. The T-type cells were found after the 14th day. In all the types of megakaryoblasts the membrane invagination occurred in the areas making contact with hepatocytes. It was agreed that the cells of the megakaryocytic series in which the demarcation membrane developed contrary to the basic pattern were ordinary promegakaryocytes. The megakaryocytes forming networks of the demarcation membrane dividing into platelet areas were small in cell size. Examination of the patterns of formation of the demarcation membrane proved useful for classifying the megakaryocytic series at each stage of maturation.