Thomas Wagner

By Josef Neumüller, Adolf Ellinger and Thomas Wagner

Platelet concentrates are produced in order to treat bleeding disorders. They can be provided by apheresis machines or by pooling of buffy coats from four blood donations. During their manufacturing and storage, morphological alterations of platelets occur which can be demonstrated by transmission electron microscopy. Alterations range from slight and reversible changes, such as formation of small cell protrusions and swelling of the surface-connected open canalicular system, to severe structural changes, where platelets undergo transitions from discoid to ameboid shapes as a consequence of platelet activation. These alterations end in delivery of the contents of platelet granules as well as platelet involution caused by apoptosis and necrosis processes denoted as the platelet release reaction. Hereby, the involvement of the network of the open canalicular system, helping to deliver the contents of platelet granules into the surrounding milieu via pores, is distinctly shown by electron tomography. As a consequence of platelet activation, a delivery of differently sized microparticles takes place which is thought to play an important role in the adverse reactions in some recipients of platelet concentrates. In this article, the formation and delivery of platelet microparticles is illustrated by electron tomography. Above all, the ultrastructural features of platelets and megakaryocytes are discussed in the context of the molecular characteristics of the plasma membrane and organelles including the different granules and the expression of receptors engaged in signaling during platelet activation. Starting from the knowledge of the ultrastructure of resting and activated platelets, a score classification is presented, allowing the evaluation of different activation stages in a reproducible manner. Examples of evaluations of platelet concentrates using electron microscopy are briefly reviewed. In the last part, experiments showing the interaction of platelets with bacteria are presented. Using the tracer ruthenium red, for staining of the plasma membrane and the open canalicular system of platelets as well as the bacterial wall, the ability of platelets to adhere and sequestrate bacteria by formation of small aggregates, but also to incorporate them into compartments of the open canalicular system which are separated from the surrounding milieu, was shown. In conclusion, electron microscopy is an appropriate tool for the investigation of the quality of platelet concentrates. It can efficiently support results on the functional state of platelets obtained by other methods such as flow cytometry and aggregometry.

Part of the book: The Transmission Electron Microscope

By Josef Neumüller, Sylvia Emanuela Neumüller-Guber, Johannes Huber, Adolf Ellinger and Thomas Wagner

Dendritic cells (DCs) are antigen-presenting cells, which are mediated by MHC-class II molecules reacting with T-helper cells, eliciting a broad spectrum of immune reactions at cellular and humoral levels depending on their subtypes. DCs are also able to cross-present peptides from intracellular proteins as well as from intracellular pathogens via MHC-class I molecules by inducing MHC-class I–restricted cytotoxic T cells, which are also able to destroy cells undergoing malignant transformation. DCs originate from CD34+ hematopoietic stem cells but can also develop from monocytes. The local or systemic milieu of cytokines and steroid hormones significantly influences the generation of particular DC subtypes such as the classical myeloid DCs such as cDC1 and cDC2 as well as the plasmacytoid DCs. These subtypes are able to induce specific Th1- and Th17-dependent, Th2-dependent, or regulatory immune responses, respectively. Immature DCs take up extracellular pathogens that are presented by MHC molecules that are upregulated during maturation. Immature and mature DCs can be characterized by morphological and biochemical features that are outlined in this article. In addition, DCs are under control of sexual hormones. Estrogen receptor ligands are potent modulators of hemopoiesis and immune function in health and disease, influencing key cytokines promoting the maturation of DCs. DC differentiation is mainly regulated by binding of estradiol to ERα. Estrogen promotes the differentiation of immature DC subsets derived from bone marrow precursors or from myeloid progenitors. In contrast to estrogen, progesterone inhibits DC maturation, causing a decreased immunity in pregnancy or in postmenopausal women, where elevated levels of progesterone result in the production of Th2 cytokines. The influence of estrogen and progesterone on DC maturation has been demonstrated in own in vitro experiments using fluorescence microscopy and cell sorting and, above all, by visualization using SEM and TEM. At the end of this article, pits and falls concerning the treatment of malignancies with living DC vaccines are discussed.

Part of the book: Modern Electron Microscopy in Physical and Life Sciences

By Josef Neumüller, Christof Jungbauer and Thomas Wagner

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).

Part of the book: Electron Microscopes, Spectroscopy and Their Applications [Working title]