Megakaryocytes and sinus walls in primary osteomyelofibrosis: transendothelial migration as revealed by three-dimensional reconstruction of serial sections following sequential double-immunostaining

Using sequential double-immunostaining and a newly-developed three-dimensional (3D-) reconstruction technique on serially cut sections from bone marrow trephines, we studied the transmural passage of megakaryocytes through the sinus wall. Biopsies derived from patients with primary (idiopathic) osteomyelofibrosis were exposed to monoclonal antibody against type IV collagen to delineate the sinus walls and also the frequently thickened basement membrane. Staining with the primary antibody was followed by Y2/51 (CD61) to identify all elements of megakaryopoiesis. In most instances serial sectioning and 3D-reconstruction revealed an amoeboid shape of megakaryocytes and a tandem-like arrangement in close spatial contact with the abluminal surface of the sinus wall. Preceded by formation of cytoplasmic processes, straight penetration of entire megakaryocytes through gaps in the sinus walls into the lumen was seen. Where collagen deposits apparently presented a barrier, a mole-like tunnelling through the basement membrane material (type IV collagen) was recognizable. Our findings are in keeping with the assumption that megakaryocyte locomotion is an essential requirement for normal thrombocytogenesis.