Visualising interaction of monocytes with red blood cells (RBS) sensitised with "clinically significant' antibodies

Abstract Background Anti-RBC antibodies in plasma are regarded as clinically significant when they promote clearance of transfused RBC by monocytes/macrophages. To date, processes involved in the attachment or phagocytosis of sensitised RBC by monocytes have been investigated using light or fluorescent microscopy. We used scanning electron microscopy (SEM) to facilitate a closer examination of the interaction between monocytes and RBCs sensitised with antibodies of specificities classified as clinically significant. Methods Peripheral blood mononuclear cells (PBMC) were seeded onto glass coverslips, and non-adhered cells were removed after 1hr. Antigen-positive RBCs were opsonised with clinically significant antibodies (anti-A, anti-B, anti-D and anti-K) before addition to adhered monocytes. Anti-M was used as an example of an antibody regarded as non-clinically significant. Coverslips were then prepared for SEM which included glutaraldehyde fixation followed by post-fixation with osmium tetroxide. Cells were further dehydrated with ethanol and hexamethyldisilazane before coated with gold nanoparticles Results We observed the interaction between monocytes and sensitised RBCs using SEM. Clinically significant antibodies induced monocyte activation, evidenced by multiple morphological changes including cell elongation, extension of membrane ruffles and the projection of pseudopodia to capture sensitised RBCs. Conclusions Detailed characterisation of interaction between monocytes and sensitised RBCs provides insight into the processes associated with RBC removal. The model provides a basis for in-depth analysis of the mechanisms associated with antibody-induced RBC destruction in vivo.