Visual Confirmation of Residual Erythrocytes and Platelets in Human Plasma

Abstract

Human plasma stored in biobanks serves as a starting material for many biomarker studies. Despite being termed “platelet-free plasma”, plasma still contains erythrocytes and platelets at concentrations below the limit of detection of routine haematology analysers, approximately 10 6 mL -1 . The presence of these residual cells affects biomarker studies of extracellular vesicles (EVs). In previous work, we used flow cytometry to study residual cells in double-centrifuged plasma. However, because direct visualization of such low cell concentrations is technologically challenging, residual cells detected by flow cytometry in plasma have not yet been directly visualized. Our goal is to visualize residual erythrocytes and platelets in double-centrifuged plasma and confirm that the flow cytometry events we previously detected indeed correspond to these cell types. We developed a workflow combining cell sorting, flow cytometry, and super-resolution microscopy to enrich and image residual erythrocytes and platelets, and applied it to the plasma of a single donor. Flow cytometric analysis from the plasma of a healthy donor revealed concentrations of 4.5×10 5 mL -1 for erythrocytes and 3.05×10 5 mL -1 for platelets, consistent with previous reports. Sorting enriched these cells by 64-fold and 40-fold, respectively, enabling super-resolution microscopic imaging. Microscopy confirmed the presence of activated erythrocytes expressing CD235a and platelets expressing CD63. For the first time, we linked flow cytometry measurements of cells in double-centrifuged plasma to super-resolution microscopy images, thereby providing a visual confirmation of the presence of erythrocytes and platelets. The presence of these cells further underscores the need to account for them in EV-focused biomarker exploration.