The frequency of megakaryocytes in autopsy sections

1. Megakaryocytes can be demonstrated with great frequency in the viscera.

2. Megakaryocytes were present in the lungs in all of 50 autopsies studied.

3. They were next most frequently found in the spleen.

4. The simultaneous occurrence in the various organs, in the absence of extra-medullary hematopoiesis, is roughly related to the concentration in the lungs.

5. Under normal conditions megakaryocytes in small numbers circulate in theblood stream.

     

Announcement

Book Reviews in This Articles:
Methods for Studying Platelets and Megakaryocytes. Edited by R. W. Colman and J. B. Smith.
Venous Thrombosis and Pulmonary Embolism: Diagnostic Methods. Edited by Jack Hirsch.     

A product of their environment: do megakaryocytes rely on extracellular cues for proplatelet formation?

Megakaryocytes have long been observed to form abundant filamentous extensions called proplatelets. A strong body of evidence strongly suggests these proplatelets are the mechanism by which platelets are released into the vasculature. Despite the recent advances in understanding proplatelet architecture, surprisingly little attention has been paid to identifying the ways in which the bone marrow environment regulates proplatelet formation. This review summarises this field and how these findings suggest a spatial and temporal regulation to ensure that platelets are produced in the correct location.     

A product of their environment: Do megakaryocytes rely on extracellular cues for proplatelet formation?

Megakaryocytes have long been observed to form abundant filamentous extensions called proplatelets. A strong body of evidence strongly suggests these proplatelets are the mechanism by which platelets are released into the vasculature. Despite the recent advances in understanding proplatelet architecture, surprisingly little attention has been paid to identifying the ways in which the bone marrow environment regulates proplatelet formation. This review summarises this field and how these findings suggest a spatial and temporal regulation to ensure that platelets are produced in the correct location.     

Culture in vitro of isolated guinea pig megakaryocytes: recovery, survival, morphologic changes, and maturation

Isolated guinea pig megakaryocytes were maintained in liquid cultures for up to 4 days. Megakaryocytes were incubated in siliconized glass vials in Dulbecco's Modified Eagle Medium with 5%-10% guinea pig serum and 2.3% bovine serum albumin. Cultured megakaryocytes did not adhere to glass vials and were almost entirely recovered by aspiration. No reproduction or cell division of megakaryocytes occurred. A small decline in viability occurred promptly on placing the freshly isolated cells in culture medium and could be attributed to reexposure to calcium. On incubation there was little further cell death. Up to 2 days in culture the megakaryocytes remained morphologically intact and appeared similar to megakaryocytes in situ. Megakaryocytes matured in culture with a loss of cytoplasmic basophilia, an increase in granule content, and progressive changes in nuclear configuration. The most mature megakaryocytes developed pseudopod formation but large-scale platelet liberation was not seen. The ability to culture megakaryocytes in vitro will allow more extensive biochemical and physiologic studies of this cell than previously possible.     

Isolation of human megakaryocytes by immunomagnetic beads

A simple method was developed to purify human megakaryocytes to homogeneity from normal bone marrow aspirates. An initial separation of marrow between 1.020 and 1.050 g/ml. Percoll density cut was used to enrich megakaryocytes. After washing, the cells were suspended with immunomagnetic beads which were coated with sheep anti-mouse IgG antibody and treated with anti-human glycoprotein (GP) IIb/IIIa monoclonal antibody, or the cells were treated with human platelet GP IIb/IIIa monoclonal antibody and suspended with the immunomagnetic beads which were coated with sheep anti-mouse IgG antibody. Megakaryocytes were selectively separated using a magnet. All of the isolated cells were morphologically recognizable megakaryocytes. 1.5-3.1 x 10(4) megakaryocytes were obtained from 1.7-4.5 x 10(8) bone marrow nuleated cells. These cells were all positive in immunoenzymatic staining for GP IIb/IIIa. Megakaryocytes obtained by this method responded to recombinant human GM-CSF (rhGM-CSF) showing an increased 3H-thymidine (3H-dT) incorporation. These data show that this method is useful for obtaining pure megakaryocyte populations which can be submitted to comprehensive biological studies.     

Regulation of megakaryopoiesis in long-term murine bone marrow cultures

Megakaryocytes and their precursor cells were sustained in mouse bone marrow suspension cultures for over 4-6 wk. Megakaryocyte precursor cells were detected by their capacity to form colonies of megakaryocytes in semisolid agar cultures. Colony formation was dependent on the presence of medium conditioned by a myelomonocytic leukemic cell line (WEHI-3CM). Megakaryocytes from the liquid and semisolid cultures were identified by cytoplasmic acetylcholine esterase and by ultrastructural analysis. The suspension medium from the bone marrow liquid cultures which sustained megakaryopoiesis was not directly acitive in stimulating megakaryocyte colony formation in the semisolid agar cultures, but potentiated the number of colonies detected when WEHI-3CM was present. Bone marrow-conditioned medium increased the sensitivity of megakaryocyte progenitor cells to the stimulus in WEHI-3CM. Addition of the activities present in the two sources produced a quantitative assay for the detection of mouse megakaryocyte progenitor cells. These studies showed: (1) that no inductive regulator of in vitro clones of megakaryocytes was present in the supernatants from the long-term marrow cultures and, (2) that at least two factors were necessary for the induction of megakaryocyte progenitors to proliferate and differentiate in semisolid cultures in vitro.     

Abnormalities of Megakaryocytes in W/Wv Mice

Megakaryocytopoiesis was evaluated ingenetically anemic mice of the W/W^v genotype and was found to be abnormal.Concentration and size of blood plateletswere normal. Megakaryocytes were decreased in number in tibial marrow andspleen, and the size of mature megakaryocytes was increased.

Submitted on April 9, 1973 Revised on May 31, 1973 Accepted on June 12, 1973     

Platelet heterogeneity and coronary artery thrombosis

Platelets are produced from megakaryocytes. Under normal physiological conditions changes in platelet volume and density are not secondary to ageing but are present at thrombopoiesis. Prolonged increased platelet destruction rate leads to the production of large platelets from large, high ploidy megakaryocytes. In vivo and ex vivo studies show such platelets have more haemostatic potential. Platelets are larger and denser in acute myocardial infarction, where bleeding time is shortened and bleeding time aspirin sensitivity is increased. Thromboxane A(2) formation is increased in unstable angina pectoris and in acute myocardial infarction. Megakaryocytes are enlarged in acute myocardial infarction and in sudden unexpected cardiac death. Megakaryocyte and platelet changes may precede coronary artery thrombosis.     

"Pawn Ball Megakaryocytes": From the Marvellous Medici and Dear Old Saint Nick to the Unsanctified Marrow of Myelodysplasia

The worlds of biology and medicine in general, and the discipline of hematology in particular, enjoy a rich lexicon full of fascinating etymologies. The term "Pawn Ball Megakaryocytes" has been used to describe a peculiar type of abnormal cell that can be found in bone marrow samples taken from some patients with the myelodysplastic syndrome (MDS). The three-ball pawnbroker's symbol that these megakaryocytes resemble is ancient and may have derived from the insignia of the Medici family or the symbol of Saint Nicholas of Myra. The murky history of the symbol and its significance for myelodysplasia are reviewed.     

"Pawn Ball Megakaryocytes": from the marvellous medici and dear Old Saint Nick to the unsanctified marrow of myelodysplasia

The worlds of biology and medicine in general, and the discipline of hematology in particular, enjoy a rich lexicon full of fascinating etymologies. The term "Pawn Ball Megakaryocytes" has been used to describe a peculiar type of abnormal cell that can be found in bone marrow samples taken from some patients with the myelodysplastic syndrome (MDS). The three-ball pawnbroker's symbol that these megakaryocytes resemble is ancient and may have derived from the insignia of the Medici family or the symbol of Saint Nicholas of Myra. The murky history of the symbol and its significance for myelodysplasia are reviewed.     

Platelets and megakaryocytes: protocols and perspectives volume 4

As our understanding of platelet and megakaryocyte function extends and deepens, access to clearly articulated and accurate experimental protocols that underpin new and innovative approaches is essential for the scientific community. This article reviews the contents of the most recent volume in the series “Platelets and Megakaryocytes, Advanced Protocols and Perspectives”, a valuable and essential addition to the laboratory resources of all platelet and megakaryocyte researchers.     

Aberrant quantity and localization of Aurora-B/AIM-1 and survivin during megakaryocyte polyploidization and the consequences of Aurora-B/AIM-1-deregulated expression

Megakaryocytes skip late anaphase and cytokinesis during endomitosis. We found normal expression and localization of a fundamental regulator of mitosis, Aurora-B/AIM-1, during prophase in polyploidizing mouse bone marrow megakaryocytes. At late anaphase, however, Aurora-B/AIM-1 is absent or mislocalized. Megakaryocytes treated with a proteasome inhibitor display Aurora-B/AIM-1 properly expressed and localized to the midzone, suggesting that protein degradation contributes to this atypical appearance. In contrast, survivin, an Aurora-B/AIM-1 coregulator of mitosis, is not detected at any stage of the endomitotic cell cycle, and in most megakaryocytes proteasome inhibition does not rescue this phenotype. To further explore the importance of reduced Aurora-B/AIM-1 for polyploidization, it was overexpressed in megakaryocytes of transgenic mice. The phenotype includes increased transgenic mRNA, but not protein, in polyploidy megakaryocytes, further suggesting that Aurora-B/AIM-1 is regulated at the protein level. Aurora-B/AIM-1 protein is, however, elevated in diploid transgenic megakaryocytes. Transgenic mice also exhibit enhanced numbers of megakaryocytes with increased proliferative potential, and some mice exhibit mild decreases in ploidy level. Hence, the molecular programming involved in endomitosis is characterized by the mislocalization or absence of at least 2 critical mitotic regulators, Aurora-B/AIM-1 and survivin. Future studies will examine the impact of survivin restoration on mouse megakaryocyte polyploidization.     

Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets.

There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.     

Flow cytometric analysis of the megakaryocyte DNA distribution in whole and partially purified human and rabbit bone marrow

Megakaryocytes are polyploid cells that constitute less than 0.1% of the nucleated cells in the bone marrow of most mammals including man. The DNA content of megakaryocytes has previously been measured by microdensitometry. Flow cytometry has also been used in the analysis of bone marrow megakaryocytes, but the general prerequisite for prior enrichment has made this technology less attractive. We describe here a modification to the mode of analysis of megakaryocyte DNA content that can be applied to whole, partially purified and elutriated human and rabbit bone marrow. The electronic masking of low-ploidy cells makes it possible to visualize the DNA content of minority, high-ploidy populations of megakaryocytes within a few minutes. In addition, this rapid technique can be combined with monoclonal antibody analysis of bone marrow cells to aid megakaryocyte identification.     

In vitro culture of murine megakaryocytes from fetal liver-derived hematopoietic stem cells

Megakaryocytes (MKs) are specialized precursor cells committed to producing and proliferating platelets. In a cytoskeletal-driven process, mature MKs generate platelets by releasing thin cytoplasmic extensions, named proplatelets, into the sinusoids. Due to knowledge gaps in this process and mounting clinical demand for non-donor-based platelet sources, investigators are successfully developing artificial culture systems to recreate the environment of platelet biogenesis. Nevertheless, drawbacks in current methods entail elaborate procedures for stem cell enrichment, extensive growth periods, low MK yield, and poor proplatelet production. We propose a simple, robust method of primary MK culture that utilizes fetal livers from pregnant mice. Our technique reduces expansion time to 4 days, and generates ~15,000–20,000 MKs per liver. Approximately, 20–50% of these MKs produce structurally dense, high-quality proplatelets. In this review, we outline our method of MK culture and isolation.     

Growth of mouse megakaryocyte colonies in vitro.

Mouse bone marrow and spleen cells formed pure or mixed colonies of up to 80 megakaryocytes in agar cultures after stimulation by medium conditioned by activated mouse lymphoid cells. Megakaryocytes were identified on the basis of their morphology, polyploid mitoses and DNA content, and high cytoplasmic content of acetylcholinesterase. Megakaryocyte colony-forming cells were relatively small with a peak sedimentation velocity of 4.2 mm/hr. Spleen, lymph node, and thymus cells produced the factor stimulating megakaryocyte proliferation after culture in medium containing 2-mercaptoethanol, with or without added mitogens or allogeneic spleen cells. Peak activity in conditioning medium was associated with the small lymphocyte fractions in mouse spleen.     

Pulmonary microvascular cytology. A new diagnostic application of the pulmonary artery catheter

When pulmonary disorders involve primarily the microvasculature, definitive diagnosis is difficult and, in some cases, is not possible until autopsy. In patients with amniotic fluid embolism, fat embolism, and lymphangitic carcinomatosis, terminal pulmonary arterioles and capillaries contain abundant diagnostic material. We hypothesized that withdrawal of blood from a pulmonary artery catheter, particularly in the wedge position, should recover diagnostic cells and debris in patients with these disorders. We describe the technique of pulmonary microvascular cytology and show examples of the recovery of fetal squames in amniotic fluid embolism, fat globules in fat embolism, and malignant cells in lymphangitic carcinomatosis. Megakaryocytes, normal inhabitants of the pulmonary capillary bed, are readily seen in wedged blood and confirm the microvascular origin of a blood sample.