DNA content and nuclear size of megakaryocytes in thrombocythaemia

Total nuclear DNA content and nuclear size of megakaryocytes were studied in biopsies of the iliac bone marrow of individuals with normal or increased platelet counts. The DNA content was determined using Feulgen cytophotometry of bone marrow smears and the nuclear area by morphometric analysis of megakaryocytes of bone marrow sections. The mean DNA content and the mean nuclear area were both significantly larger in megakaryocytes of patients with thrombocytosis as a result of myeloproliferative disease than in patients with secondary thrombocytosis as well as in two control groups of individuals with normal platelets counts, one comprising healthy volunteers, the other with various non-haematological disorders. There was a statistically significant correlation between the DNA content and nuclear area of the megakaryocytes (r = 0.92) in the entire group of bone marrows studied.     

Pro-megakaryoblasts in bone marrow tissue from patients with primary (idiopathic) osteo-myelofibrosis (agnogenic myeloid metaplasia). An immunomorphometric study on trephine biopsies

An immunomorphometric study was performed on bone marrow biopsies from 40 patients with primary osteomyelofibrosis--OMF, (agnogenic myeloid metaplasia) by employment of a monoclonal antibody against glycoprotein IIIa (Y2/51) to determine the number of pro-megakaryoblasts. Specimens from 15 individuals without any hematological disorder served as controls. With reference to the pertinent literature on megakaryocyte precursors and following a pilot study on corresponding smears, in tissue sections pro-megakaryoblasts were characterized by a size of 42.1 +/- 2.6 microns 2 (diameter 7.5 +/- 0.3 microns). In comparison with controls, in OMF no relevant increase in the number of pro-megakaryoblasts per square and cubic millimeter bone marrow was evaluable. The relative frequency of these precursors was significantly reduced due to an increase in the total amount of conspicuously large and abnormal megakaryocytes. Statistical analysis failed to reveal any correlations between counts for pro-megakaryoblasts or the total number of Y2/51--positive megakaryocytic elements with the density of argyrophilic fibers (determined by morphometry) or the platelet values. Our findings imply that in OMF the marked increase in circulating progenitor cells of the megakaryocyte lineage may be generated by extramedullary, probably splenic hematopoiesis. Moreover, the evolution of medullary fibrosis is thought to be associated with the striking predominance of large atypical, possibly overaged and hyperpolyploid megakaryocytes and not with an increase in precursor cells.     

CD34/QBEND10 immunostaining in the bone marrow trephine biopsy: a study of CD34-positive mononuclear cells and megakaryocytes

CONTEXT: The immunohistochemical detection of CD34 protein using QBEND10 antibody in bone marrow trephine biopsies was shown recently to be a precise method for quantitation of blasts and a possibly useful approach in diagnosis and classification of myelodysplastic syndrome. OBJECTIVES: To evaluate CD34+ cells in bone marrow biopsies with various diagnoses and to assess how counts obtained using this method correlate with blast counts obtained by traditional morphologic evaluation of bone marrow smears. DESIGN: Bone marrow trephine biopsies from 108 adult patients were evaluated by immunohistochemistry using anti-CD34 antibody (QBEND10). CD34+ mononuclear cells were counted and compared with the blast counts in the bone marrow aspirate smears or imprints. CD34+ mononuclear cell clusters and CD34+ megakaryocytes were also recorded. The type of positivity (membranous vs cytoplasmic) and the percentage of CD34+ megakaryocytes were evaluated because the presence of CD34+ megakaryocytes was recently suggested to be present in myelodysplastic syndrome, but not in myeloproliferative disease or nonneoplastic bone marrow. RESULTS: Six of 24 biopsies with partial involvement by non-Hodgkin lymphoma and 5 of 60 biopsies with reactive changes had 5% to 10% CD34+ mononuclear cells and were associated with lymphocytosis and increased hematogones. The CD34+ mononuclear cell clusters were found only in myelodysplastic syndrome and myeloproliferative disease. The CD34+ megakaryocytes were present in all diagnostic groups. CONCLUSION: The number of CD34+ mononuclear cells was often slightly higher than the number of myeloid blasts in the bone marrow smears, probably due to increased hematogones. The presence and the number of CD34+ megakaryocytes do not appear to have diagnostic value, but this finding should be further investigated in relation to clinical parameters.     

Postmortem Diagnosis of Acute Megakaryocytic Leukemia Usefulness of lmmunohistochemistry and Tissue Hemogram

An autopsy case of acute megakaryocytic leukemia (AMKL) is presented. The bone marrow was hypercellular with proliferation of three lineages, especially megakaryocytes. Immunohistochemical examination revealed many platelet glycoprotein IIb/IIIa (GP IIb/IIIa)- positive blast cells in bone marrow. The proportion of the blasts was 26.4% by tissue hemogram. GP IIb/IIIa-positive blasts and megakaryoblasts were deposited massively in lymph nodes. lmmunohistochemistry against GP IIb/IIIa and tissue hemograms by paraffin section are needed to diagnose AMKL by postmortem examination, since the identification of ultra-structural platelet peroxidase in autopsy materials is difficult.     

Postmortem diagnosis of acute megakaryocytic leukemia. Usefulness of immunohistochemistry and tissue hemogram

An autopsy case of acute megakaryocytic leukemia (AMKL) is presented. The bone marrow was hypercellular with proliferation of three lineages, especially megakaryocytes. Immunohistochemical examination revealed many platelet glycoprotein IIb/IIIa (GP IIb/IIIa)-positive blast cells in bone marrow. The proportion of the blasts was 26.4% by tissue hemogram. GP IIb/IIIa-positive blasts and megakaryoblasts were deposited massively in lymph nodes. Immunohistochemistry against GP IIb/IIIa and tissue hemograms by paraffin section are needed to diagnose AMKL by postmortem examination, since the identification of ultrastructural platelet peroxidase in autopsy materials is difficult.     

Dysmegakaryopoiesis in myelodysplastic syndromes (MDS): an immunomorphometric study of bone marrow trephine biopsy specimens.

An immunohistochemical and morphometric analysis was performed on trephine biopsy specimens of the bone marrow in 40 patients (23 men and 17 women, mean age 62 years) with different subtypes of myelodysplastic syndromes (MDS) to determine dysmegakaryopoiesis, but particularly precursor cells--that is, pro- and megakaryoblasts. In 31 of the 40 patients the numbers of megakaryocytes were increased which was associated with a predominance of smaller cell forms (micromegakaryocytes). Compared with periodic acid Schiff, immunostaining with a formalin resistant monoclonal antibody against glycoprotein IIIa (Y2/51(CD61) showed a clinically important proportion of immature elements. These could be designated pro- and megakaryoblasts by taking morphometric measurements on smears and bone marrow sections. There was a relevant increase in the number of promegakaryoblasts in 32 patients, consistent with uncontrolled expansion of the precursor pool. Seventeen repeated bone marrow biopsy specimens taken after chemotherapy largely showed a decrease in the numbers of megakaryocytes including the precursor cell population. Moreover, morphometric evaluation disclosed that micromegakaryocytes in MDS differ significantly from those in chronic myeloid leukaemia (CML) due to distinctive nuclear features and a disturbed nuclear:cytoplasmic ratio. These changes generate a more pleomorphic or atypical appearance of this cell population in MDS, compared with micromegakaryocytes in CML. It is concluded that the disproportionate increase in megakaryocyte precursors and the grossly abnormal aspects of micromegakaryocytes in MDS are characteristics of the severe defect involving haematopoiesis in this disorder.     

The relationship between ploidy and morphological classification of human megakaryocytes]

The relation between polyploidy and morphological classification of human megakaryocytes was studied in bone marrow aspirates from five normal individuals. On a Wright-Giemsa stained smear, megakaryocytes were morphologically classified into four groups according to a modification of Feinendegen's classification which is considered to reflect megakaryocyte maturation. The DNA of the morphologically classified cell is measured by microcytofluorometry using DAPI (4',6-diamidino-2-phenylindole) staining after removing the Wright-Giemsa stain. Most of the normal megakaryocytes were classified into type III (mature megakaryocytes) and the maximum peak in population of the megakaryocyte ploidy was observed at 16N. In each individual, the ploidy showed a similar pattern regardless of the classification. These findings suggest that the development of ploidy depends on a factor different from the one that determined the megakaryocyte maturation of cytoplasm and the ploidy is determined at the level of a megakaryocyte precursor or the most juvenile megakaryocyte.     

Physiologic role of TPO in thrombopoiesis

Recombinant human thrombopoietin (rHuTPO) serves as a megakaryocyte colony-stimulating factor and predominantly acts on GPIIb/IIIa+ rat late megakaryocyte progenitor cells, colony forming units-megakaryocyte (CFU-MK). The GPIIb/IIIa+ fraction of CFU-MK differentiates into mature megakaryocytes and further into proplatelets in liquid culture containing rHuTPO. rHuTPO stimulates cultured megakaryocytes generated from rat GPIIb/IIIa+ CFU-MK to enhance proplatelet formation and to increase megakaryocyte size. rHuTPO also induces a big size of megakaryocyte colonies from human cord blood CD34+ cells. rHuTPO does not cause aggregation of platelets from normal mice and mice made thrombocytotic by consecutive administration of rHuTPO, but preincubation with rHuTPO enhances adenosine diphosphate-induced aggregation, suggesting that platelets induced by rHuTPO administration may have a normal function. Administration of rHuTPO to normal mice daily for five days causes a dose-dependent thrombocytosis. On the other hand, rHuTPO induces a significant decrease in hemoglobin concentration and does not affect white blood cell counts. rHuTPO increases the size and number of marrow megakaryocytes and the number of marrow CFU-MK, and also influences the development of other hematopoietic progenitor cells. The effects of rHuTPO on thrombocytopenia associated with myelosuppression were examined in animal models. Following treatment with mitomycin C, mice received daily injections of various doses of rHuTPO. rHuTPO reduced the severity of thrombocytopenia, accelerated the recovery of platelets and improved neutropenia. Similar therapeutic efficacy was observed in cynomolgus monkeys treated with nimustine. These results suggest the clinical usefulness of rHuTPO for the treatment of thrombocytopenia.     

Megakaryocytes in fine-needle aspiration biopsy of breast

An unusual case in which megakaryocytes were found in fine needle aspirate of a breast lesion is reported. The megakaryocytes in the smears were initially mistaken to be malignant cells. However, the cell block sections available later showed normal bone marrow particles, indicating that an inadvertent puncture of a rib was the reason for the presence of megakaryocytes in the smears.     

Analysis of megakaryocytes by flow cytometry

A flow cytofluorometric measurement of megakaryocyte ploidy has been adapted from Tomer's method. Briefly, bone marrow is aspirated through a medium containing theophyllin, prostaglandin-E1 and other antiaggregant agents. Megakaryocytes-enriched buffy coats are recovered. Megakaryocytes are then stained for GP IIIa coupled with FITC and for DNA (propidium iodide). A Becton Dickinson FACScan flow cytometer is used for measuring the ploidy distribution of GP IIIa positive cells. The method we developed presents several advantages. Firstly, the time required is greatly decreased in comparison with other studies. Secondly, the washing steps are limited in number allowing a diminution of the cell loss. Thirdly, the method ensures a better megakaryocyte preservation. Finally, selection of megakaryocytes by ploidy and expression of GP IIIa can be made easily because of the simultaneity of the two stainings as well as by the use of a precise gating on the flow cytometer. Based on these results, we conclude that the present method provides a better means for the isolation and analysis of human normal megakaryocytes. This technique has been applied to the analysis of megakaryocyte populations from patients with abnormal platelet counts. In chronic myeloid leukemia patients, analysis of ploidy distribution shows a shift toward the low ploidy while in patients with immune thrombocytopenic purpura, polycythemia vera and essential thrombocythemia the ploidy distributions are shifted toward the high ploidy.     

Detection of mRNA in megakaryocytes using in situ hybridization]

The usefulness of in situ hybridization for detecting mRNA encoding various kinds of proteins in megakaryocytic cell lines (K562, HEL and CMK) was examined. Using in situ hybridization, glycoprotein (GP) IIIa mRNA was found to be present in K562, HEL and CMK cells. Both GPIb and GPIIb mRNA were also present in HEL and CMK cells, while only HEL cells expressed platelet factor 4 mRNA. These findings were identical to those obtained with Northern blotting. It should be emphasized that in situ hybridization was useful to clearly define each cell expressing platelet specific protein mRNA. The expression of interleukin-6 (IL-6) and IL-6 receptor mRNA in mononuclear cells prepared from bone marrow aspirates was examined. In situ hybridization study demonstrated the presence of IL-6 receptor mRNA in the recognized megakaryocytes only, while IL-6 mRNA was found to be present in the megakaryocytes and a few mononuclear cells. These findings suggest that differentiation and proliferation of normal megakaryocytes might be controlled by an IL-6 autocrine loop, and detection of IL-6 receptor mRNA might be useful to identify megakaryocytes.     

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.     

Myelodysplastic syndromes: Immunohistochemical and morphometric evaluation of proliferative activity in erythropoiesis and endoreduplicative capacity of megakaryocytes

An immunohistochemical and morphometric analysis was performed on bone marrow trephine biopsies in 40 patients with primary myelodysplastic syndromes (MDS) to evaluate the proliferative activity in erythropoiesis and the endoreduplicative capacity of megakaryocytes. Control groups included normal bone marrow and marrow from cases presenting with pernicious anaemia. Double-immunostaining was applied with a monoclonal antibody (PC10) directed against proliferating cell nuclear antigen (PCNA), followed by antibodies against glycophorin C (Ret40f) or platelet glycoprotein IIIa (Y2/51-CD61) for the identification of the erythroid and megakaryocytic cell lineage. Comparison with normal bone marrow showed a reduction of erythropoiesis accompanied by an increase in atypical (micro-) megakaryocytes. Erythroid precursors displayed significant enhancement of PCNA-immunostaining. Megakaryocytes showed no increase in the relative frequency of PC10-positive cells (PCNA-labelling index). In pernicious anaemia, predominance of macrocytic-megaloblastoid erythropoiesis was associated with a striking increase in PCNA-labelling. Cell kinetic studies in this disorder revealed an abnormal arrest, particularly in S-phase which generates an over-expression of PCNA. Similar conditions were believed to be present in MDS with secondary folate deficiency. This mechanism explains the relatively high rate of positively-reacting pro- and erythroblasts which is not invariably accompanied by an increase in cell proliferation. Determination of megakaryocyte size and PCNA-staining capacity resulted in a significant increase in PC10-positive cells among micromegakaryocytes. Our findings on this cell lineage are in keeping with the assumption of a block in endoreduplicative activity at higher ploidy levels, associated with an apparently not-deregulated endomitosis in small-sized megakaryocytes of lower ploidy stages.Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG-Th 390/1-3)     

Osteoclasts contain macrophage and megakaryocyte antigens

The origin and mechanism of formation of the osteoclast remains controversial. Although it is known to be derived from a circulating mononuclear percursor, the identity of this cell is unknown. Using a panel of monoclonal antibodies raised against macrophage and other marrow-derived cells, we determined the immunocytochemical staining of human osteoclasts in both fetal bone metaphyseal imprints and frozen sections. Osteoclasts and marrow mononuclear cells were stained by three broad spectrum antimacrophage antibodies, EBM-11, Y182a and BM2. T310, an antibody which stains macrophages and T helper cells, and C17, an antimegakaryocyte antibody, also stained osteoclasts. EBM-11, Y182a and BM2 also stained megakaryocytes in bone imprints as well as normal bone marrow smears. The presence of macrophage-associated antigens in osteoclasts, megakaryocytes and bone marrow mononuclear cells indicates that they are phenotypically similar to macrophages.     

Cytogenetic clonality analysis of megakaryocytes in myelodysplastic syndrome by dual-color fluorescence in situ hybridization and confocal laser scanning microscopy.

In the myelodysplastic syndrome (MDS), cytogenetic abnormalities are often present and can be used as markers in studies for cell lineage involvement. Little is known of the involvement of the megakaryocytic lineage due to the variable ploidy of these cells. We applied dual-color fluorescence in situ hybridization (FISH) to routinely prepared bone marrow (BM) smears of cytogenetically normal patients and seven patients with MDS and monosomy 7 or trisomy 8. Probes specific for the centromeric regions of chromosomes 7 and 8 were detected with fluorescein isothiocyanate (FITC) and Texas Red, respectively. This enabled us to assess the ratio between the numbers of chromosomes 7 and 8 in the polyploid cells. We utilized confocal laser scanning microscopy to count the FITC and Texas Red FISH signals in the different focal layers of the megakaryocytes. Fifty-six megakaryocytes in six normal BM smears were analyzed, giving a mean ratio of 1.0, a standard deviation (SD) of 0.12, and a range of 0.8-1.33. This ratio was applied to evaluation of clonal involvement of individual megakaryocytes in the patients with MDS. In two patients with monosomy 7, the majority of the megakaryocytes were monosomic. In the five patients with trisomy 8, all or a majority of the analyzed megakaryocytes were trisomic. These results add direct evidence that in MDS megakaryocytes are involved in the malignant clone. Genes Chromosomes Cancer 25:332-338, 1999.     

A monoclonal antibody against rat platelets. I. Tissue distribution in vitro and in vivo.

In this study we describe a new monoclonal antibody (MoAb PL.1) against rat platelets. Immunohistology of various rat tissues showed staining of platelets, especially in the spleen, and staining of megakaryocytes in bone marrow and spleen red pulp. In the liver small platelet aggregates and endothelial cells were stained. After in-vivo administration of MoAb PL.1 an acute severe thrombocytopenia was observed. In general the distribution of the antibody and/or antibody-coated platelet aggregates showed the same pattern as after in-vitro incubation, i.e. staining of rat platelets and platelet aggregates in spleen red pulp, and staining of megakaryocytes in spleen and bone marrow. Platelet aggregates were observed in the liver and electron microscopy indicated that they were associated with Kupffer cells. Furthermore, liver endothelial cells were positively stained. Comparison of the molecular weight of the antigens recognized by this MoAb and by human anti-platelet MoAbs, as well as comparison of staining patterns of megakaryocytes indicated that MoAb PL.1 is probably directed to a GPIIb/IIIa complex analogue. Since MoAb PL.1 is of the non-complement-binding mouse IgG1 isotype, it can be used for studying clearance of platelet aggregates by Fc-receptors of the MPS. It also promises to be a useful tool in the study of platelet involvement in rats with experimental nephritis.     

Loss of membrane expression of E-cadherin in leukemic erythroblasts

CONTEXT: The special societal relationships existing between various cell types in bone marrow suggests that there may be a link between the adhesive characteristics of hematopoietic cells and their maturation. Egress of the developing hematopoietic cells is also a highly regulated process governed by adhesive interactions. In leukemia, immature blasts are not retained within the marrow, suggesting a breakdown of adhesive mechanisms. Recent reports suggest that E-cadherin, an epithelial adhesion molecule, is expressed on erythroid precursors and megakaryocytes, but not on other hematopoietic marrow elements. OBJECTIVE: To characterize the expression pattern of E-cadherin in normal and leukemic erythroid precursors by immunohistochemistry in paraffin-embedded tissue and bone marrow aspirate smears. METHODS: Five normal bone marrow specimens from rib resections, 15 trephine bone marrow biopsy specimens, and 6 bone marrow aspirate smears from the iliac crest of patients with no known leukemia were selected. Fourteen bone marrow biopsy specimens from patients with erythroleukemia were also studied. Immunoperoxidase staining of paraffin-embedded tissue and air-dried aspirate smears for E-cadherin (1:200 dilution, HECD-1 clone) was performed using the avidin-biotin peroxidase technique. RESULTS: In paraffin-embedded bone marrow biopsy and rib specimens and in air-dried bone marrow aspirate smears, strong membrane expression of E-cadherin was seen in the normal erythroid precursors in all cases. In contrast, no membrane expression of E-cadherin was present in any of the bone marrow biopsy specimens from patients with erythroleukemia. CONCLUSIONS: Immunohistochemical detection of membrane expression of E-cadherin may be a useful tool for identification of erythroid precursors. Cells of erythroleukemia lack membrane expression of E-cadherin, in contrast to their normal counterparts. Further studies are needed to define the potential role of E-cadherin in the maturation of erythroid precursors and to ascertain the significance of loss of membrane expression of E-cadherin in erythroleukemia.     

Megakaryocytes in steel mutant mice

Summary The megakaryocytes in the bone marrow of steel mutant mice (Sl/Sl^d) and their normal littermates (+/+) were studied by light and electron microscopy with special emphasis on their maturity and distribution in the hematopoietic cords. A higher percentage of megakaryocytes lying against the sinus wall, a higher percentage of the sinus perimeter covered by megakaryocytes and a higher percentage of large megakaryocytes were found in Sl/Sl^d mice than in +/+ mice. In addition, more large megakaryocytes as well as senile megakaryocytes were observed in the spleen of Sl/Sl^d mice than in that of +/+ mice. These observations suggest that more platelets are produced in Sl/Sl^d mice than in +/+ mice on the basis of per unit area of the marrow tissue. Heretofore, the fate of the senile megakaryocytes in the marrow was not known. However, in Sl/Sl^d mice senile megakaryocytes were often found entering the marrow sinuses from the hematopoietic cords. They were also seen in the lung and the spleen where degradation of senile megakaryocytes was observed. These observations suggest that senile megakaryocytes in Sl/Sl^d mice leave the marrow and are removed by the reticuloendothelial system outside the marrow.