Investigation of megakaryocytes from normal and myeloproliferative bone marrow biopsies

Abstract

Megakaryocytes are large, distinctive cells found mainly in the bone marrow. They produce platelets in accordance with changes in peripheral demand. Regulatory mechanisms for the modulated production of platelets are ill-defined. The recent identification of thrombopoietin (TPO) as the primary regulator of megakaryocytopoiesis, and its receptor, c-Mpl, allows the possibility of further characterising megakaryocytes in normal haemopoiesis and chronic myeloproliferative disorders (CMPD). Detection of TPO and c-Mpl messenger ribonucleic acid (mRNA) sequences by in-situ hybridisation (ISH) and Northern blot analysis techniques using custom-designed probes was undertaken in this project. Optimisation of ISH proved unsuccessful in wax-embedded bone marrow trephine biopsy (BMT) sections and a possible signal was obtained in cytocentrifuge preparations of unfixed bone marrow cells. Confirmation that the probe molecules were capable of binding with the correct target sequence was achieved by Northern blot analysis. A band corresponding to 3746 nt, the molecular size of c-Mpl mRNA, was obtained from total RNA isolated from HEL cells. To aid further studies, megakaryocytes were enriched from aspirated bone marrow specimens following labelling for CD61 or CD42b and separation by magnetic activated cell sorting (MACS). In addition, a panel of antibodies reactive with megakaryocyte- and platelet-associated antigens was used to characterise megakaryocytes by immunohistochemistry in normal and CMPD paraffin-embedded BMT sections. CD42b-stained megakaryocytes from BMT sections from patients with ET were larger than those from patients with PRV but variation between individual patients in these groups renders this distinction unfeasible for use as a diagnostic tool. Anti-CD42b antibody stained megakaryocytes intensely and more reliably than other markers. The results of this study strongly suggest recommendation of CD42b as a megakaryocyte marker for routine diagnostic use.

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