Characterization and enumeration of platelet microvesicles in human platelet concentrates by using transmission electron microscopy including electron tomography

Abstract

Platelet microvesicles (PMV) carry receptors and contain genetic information. They are delivered from platelets by budding or by exocytosis of α-granules and are able to activate leukocytes and endothelial cells, resulting in inflammatory reactions. Therefore, the ultrastructural investigation and counting of PMV in platelet concentrates (PC) produced by apheresis or pooled buffy coats were investigated. High numbers of PMV in PC can occasionally provoke severe transfusion reactions in recipients suffering from thrombocytopenia caused by different diseases or therapeutic interventions. The ultrastructural investigation of PMV in PC, produced by different manufacturing methods, can significantly contribute to their quality evaluation. PMV was investigated and enumerated using transmission electron microscopy (TEM) on filmed grids by the negative contrasting method and a special photomontage option integrated with TEM. Image aspects of four areas of about 8,500 × 8,500 nm could be sifted through. Using reference gold particles with a known concentration, added to the sample of the PC preparation, and applied to the grid, the number of PMV/μl of the sample could be calculated. Using morphometry, their distribution in terms of area was determined. Visualization of single PMV in ultracentrifuged or alginate-embedded PC samples was enabled by using electron tomography (ET).