Assay of Megakaryocytopoiesis in Thrombocytopenic Rats

S ummary . Rats were given exchange transfusions with different volumes of platelet-poor blood, injected 24 hr later with tritiated thymidine, and killed after another 24 or 36 hr. Autoradiograms of bone marrow smears were made and the labelling index of megakaryocytes was determined. The labelling index was proportional to the degree of thrombocytopenia induced by exchange transfusion. The results therefore demonstrate that the number of circulating platelets exerts a rather delicate control over megakaryocytopoiesis. In addition, the potential of this procedure for studying megakaryocytopoiesis, its regulation, and its abnormalities is indicated.     

Thrombopoiesis- and Megakaryocyte Colony-stimulating Factors in the Urine of Patients with Idiopathic Thrombocytopenic Purpura

The urinary extract from patients with severe, chronic idiopathic thrombocytopenic purpura (ITP) was capable of inducing significant thrombocytosis in rats in vivo and enhancing megakaryocyte colony formation in culture of mouse bone marrow cells. The apparent specific activity of megakaryocyte colony stimulating factor (MEG-CSF) in the ITP extract was approximately one-half of that of the urinary extract from patients with aplastic anaemia (AA). Daily injections of ITP extract did not cause an increase in Hb concentration, while rats receiving AA urinary extract revealed profound erythropoiesis 3 weeks later. In vitro assay of erythropoietin (EPO) failed to show significant EPO activity in the extract from patients with ITP. These findings excluded the possibility that the observed activities of thrombopoiesis-stimulating factor (TSF) and MEG-CSF in the ITP urinary extract are due to contaminating EPO. Urine of patients with severe ITP appears to be a good source of TSF and MEG-CSF.     

The Maturation Rate of Reticulocytes

An in vitro culture technic for the study of reticulocyte maturation wasdescribed. The method gave reproducible results and proved to be of valuein the comparative study of reticulocyte maturation in blood disorders. By thismethod it was shown that variations in the reticulocyte maturation in vitroparalleled similar variations present in vivo.

The maturation of reticulocytes from patients with different types ofanemia was investigated. In some anemias the in vitro maturation of reticulocytes was prolonged, not only because younger reticulocytes were present inthe blood, but also because the rate at which the reticulum substance disappeared was delayed. This was particularly evident in the anemia of chronicuremia, in Cooley’s anemia and in pernicious anemia in relapse. In only occasional cases of hereditary spherocytosis and of autoimmune hemolyticanemia was the rate of reticulocyte maturation found to be moderatelydelayed. In patients with iron deficiency anemia or bleeding anemia it wasalways normal.

From the above findings the following conclusions were derived:

1. The reticulocyte number in the circulating blood is the resultant of threevariables: (a) the rate of output of new reticulocytes from the bone marrow;(b) the stage of maturation at which reticulocytes are delivered into theperipheral circulation; (c) the rate of disappearance of the reticulum substance.

2. The number of reticulocytes in the circulating blood cannot be indiscriminately used as a precise index of red cell production in erythrokinetics.

3. There is good reason to believe that a defect in the rate at which thereticulocytes mature in the circulating blood is an index of a similar defectin the process of erythroblastic differentiation in the bone marrow.

     

Megakaryocyte Polyploidization in Myeloproliferative Disorders

Summary In 5 cases of polycythaemia vera and 2 cases with other myeloproliferative disorders accompanied by thrombocythaemia (megakaryocytic myelosis), the megakaryocytes were differentiated and studied by use of the combined application of cytophoto-metric determination of the DNA content and autoradiography with tritiated thymidine (³H-TdR) in vitro.A shift to the right of the megakaryocyte series, occurence of high polyploidy cells at 64c and a decrease of the³H-TdR-labeling indices were observed. The data suggest a disturbance of the rhythmical polyploidization of the megakaryocytes, consisting of an elevated proportion of rest cells at the different ploidy stages. The maturation capacity of megakaryocytes may be related more to the resting than to the DNA synthesizing cells.

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Zusammenfassung Die Polyploidisierung der Megakaryozyten wurde bei 5 Patienten mit Polycythaemia vera und bei 2 Patienten mit einem myeloproliferativen Syndrom, das mit einer starken Thrombozytose einherging (megakaryozytäre Myelose), mit Hilfe der Kombination von zytophotometrischer Bestimmung des DNA-Gehalts und Autoradiographie mit tritiiertem Thymidin (³H-TdR) in vitro untersucht.Dabei wurde eine Rechtsverschiebung der Megakaryozytopoese mit Zunahme des Anteils von Typ III-Megakaryozyten, das Auftreten von hochpolyploiden Megakaryozyten bei 64c und eine Abnahme der³H-TdR-Markierung beobachtet.Die Befunde sprechen für eine Störung der rhythmischen Polyploidisierung der Megakaryozyten, wobei die Abnahme des Anteils DNA-synthetisierender Zellen als Folge einer Zunahme von Ruhezellen auf den verschiedenen Polyploidiestufen angesehen wird. Die zu beobachtende starke Reifungskapazität der Megakaryozyten ist offenbar mehr an die Ruhephasen als an die DNA-Synthesephasen gebunden.

     

Megakaryocyte apoptosis in immune thrombocytopenia

The mechanisms of platelet underproduction in immune thrombocytopenia (ITP) remain unknown. While the number of megakaryocytes is normal or increased in ITP bone marrow, further studies of megakaryocyte integrity are needed. Megakaryocytes are responsible for the production of platelets in the bone marrow, and they are possible targets of immune-mediated injury in ITP. Since the biological process of megakaryocyte apoptosis impacts platelet production, we investigated megakaryocyte DNA fragmentation as a marker of apoptosis from ITP bone marrow biopsies. Archived bone marrow biopsy specimens from ITP patients, bone marrow specimens from controls with normal platelet counts, and bone marrow specimens from thrombocytopenic controls with myelodysplastic syndrome (MDS) were evaluated. Sections were stained with anti-CD61 for megakaryocyte enumeration, and terminal deoxynucleotidyl transferase dUTP nick-end labeling was used as an apoptotic indicator. In ITP patients, megakaryocyte apoptosis was reduced compared to nonthrombocytopenic controls. Megakaryocyte apoptosis was similarly reduced in thrombocytopenic patients with MDS. These results suggest a link between megakaryocyte apoptosis and platelet production.     

Changes in Megakaryocyte Development Following Thrombocytopenia

S ummary . Rats were made thrombocytopenic by total-body X-irradiation and changes in megakaryocyte development studied in shielded tibial bone marrow. The platelet count fell from the fourth day after irradiation to reach a minimum value of 19.5% of the initial count 7 days after irradiation. No changes in megakaryocyte development were observed 5 days after irradiation. Megakaryocyte size was increased at day 7, and the increment maintained until day 12. Megakaryocyte numbers were only significantly increased 12 days after irradiation, but differential counts and electron-microscope autoradiographs suggested increased influx at day 9. Changes in the labelling index of megakaryocytes suggested that some cells already present were undergoing one or more extra endoreduplications. No evidence was obtained for an accelerated megakaryocyte maturation rate or for a sudden release of platelets from mature megakaryocytes. Morphological changes, signifying increased synthesis of protein and demarcation membranes were apparent only in young cells 7 days after irradiation but were present in mature cells by day 12, demonstrating that the response to thrombocytopenia is confined to immature cells.     

Impaired Thromboxane Production by Newly Formed Platelets after Aspirin Administration to Thrombocytopenic Rats

The in vivo inhibitory effect of aspirin on platelet cyclo-oxygenase is irreversible and lasts for the entire platelet life-span. Reappearance of cyclo-oxygenase activity in blood after aspirin has been proposed as a measure of the formation of new platelets and as an indirect indicator of platelet survival. A delay of 24–72 h in recovery, however, has been observed and it has been suggested that aspirin might also inhibit megakaryocyte cyclo-oxygenase. To test this possibility, aspirin (100 mg/kg) or saline were administered i.p. to rats made thrombocytopenic 2 h later (platelet count less than 5% of basal value) by a specific antiplatelet antiserum. Malondialdehyde (MDA) and thromboxane B₂ (T×B₂) production by platelets was measured by spectrophotometry and radioimmunoassay respectively, during the period of platelet count restoration. By 24 h after thrombocytopenia was induced, platelet count was about 15% of basal values in control and aspirin-treated rats. However, while in controls MDA and T×B₂ production was restored to about 20% of basal values, in aspirin-treated rats less than 5% return of activity was detected. A marked difference between the two groups was still found 96 h after induction of thrombocytopenia, when platelet count restoration was similar. Since aspirin disappeared very rapidly from the circulation, the delay in recovery of cyclo-oxygenase activity supports the hypothesis of a megakaryocyte effect of this drug.     

Megakaryocyte maturation in long-term marrow culture.

Evidence has been sought for megakaryocyte maturation in long-term cultures of mouse bone marrow. Cultures up to 14 weeks of age were examined for the presence of megakaryocytes with processes, that is, resembling the morphological appearance seen in vivo prior to platelet liberation. Such cells were found floating just above the adherent stromal layer using low magnification phase contrast microscopy. It was rare to observe as many as 20 of these cells per 25-cm2 flask. At higher magnification, processes were seen to be attenuated with constrictions at intervals along their length. Time-lapse photography was used to follow the development and behavior of the processes. Direct evidence of rupture was very rare; generally the megakaryocytes retracted their processes within 48 h. Careful searching of cultures occasionally revealed the presence of several process fragments, and sometimes individual platelets were found. Ultrastructurally, the processes were seen to contain organelles that are usually associated with platelets. The observations applied to both Dexter and Whitlock-Witte cultures. It is concluded that maturation of megakaryocytes occurs in long-term marrow culture to the point where platelet release appears imminent. Final rupture is rare and may require shearing forces, which in vivo would be provided by blood flow.     

A Method for Evaluating the Hemostatic Effect of Various Agents in Thrombocytopenic Rats and Mice

A simple method for measuring the control of blood loss in thrombocytopenicrats and mice is described.

The administration of lyophilized platelets, brain phospholipid extractand soya bean extract failed to correct the blood loss in the thrombocytopenicanimal.

The results obtained with this method have failed to support the effectiveness of "platelet substitutes" in in vivo systems and reaffirm the requisites ofviability and recirculation for platelet transfusions.

Submitted on June 5, 1959 Accepted on August 5, 1959     

Megakaryocyte structure and function.

Recent advances in the understanding of megakaryocyte (MK) function largely have been made through the careful observation of the morphological and structural events underlying MK development. Ultrastructural localization of enzymatic activities has facilitated the specific recognition of their committed diploid precursors. Observation of the sequential features of endomitosis demonstrates that although similar to normal mitosis, cell division aborts at the anaphase stage. The ability of thrombopoietin to induce the full maturation MKs in vitro not only facilitates platelet release but has increased our knowledge of various subcellular aspects of the phenomenon and eventually will improve the in vivo detection of the site of platelet formation and shedding. Finally, the structural and functional consequences of MK molecular dysfunction leading to thrombocytopenia or myelofibrosis can now be investigated because of the development of transgenic animal models. This review aims to incorporate these new findings within the classical knowledge of MK structure related to its function.     

Megakaryocyte Polyploidization in Acute Leukaemia and Preleukaemia

S ummary . Megakaryocyte polyploidy was studied in six cases of preleukaemic acute leukaemia (PL), and in acute leukaemia (AL), five cases in the overt phase and three during complete remission. The megakaryocyte polyploidization was determined cytophotometrically and in some cases also by in vitro labelling with tritiated thymidine (³H-TdR). The results obtained in PL and untreated AL. demonstrate an impairment of the ³H-TdR labelling index and altered megakaryocyte polyploidy such as decrease of the maximum polyploidization capacity (five cases), evidence of diploid megakaryocytes (two cases) and of hyperploid megakaryocytes (six cases). These disturbances reflected the cytological abnormalities (microkaryo-cytes, polykaryocytes) observed in most of the cases studied.
In contrast, in cases studied during complete remission of AL. a normal labelling index and polyploidy composition was observed, suggesting that the polyploidization disturbances are reversible. A strict correlation of the polyploidization defect and the platelet production could not be established from the patients investigated.     

Megakaryocyte polyploidy and maturation in chronic granulocytic leukemia

To understand abnormal platelet production in chronic granulocytic leukemia, polyploidization and maturation of megakaryocytes in 10 patients were studied using a technique which allows sequential immunofluorescence identification by a monoclonal platelet antibody (C17), cytophotometric determination of the relative DNA content and cytological characterization of megakaryocytes in panoptically stained smears. Compared to normal conditions the proportion of diploid promegakaryocytes was not increased, suggesting an undisturbed influx of progenitor cells into the megakaryocytic cell compartment. Small tetraploid (4c) megakaryocytes undergo maturation without further polyploidization, the so-called microkaryocytes being mature rather than immature cells. Most of the megakaryocytes show rhythmical polyploidization only up to octoploid (8c) level, indicating the inability to produce high-polyploidy cells.