Characterising Red Cell-Derived Vesicles in Sickle Cell Disease and Investigating Potential to Induce Tolerance to Human Red Cell Antigens

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Patients with sickle cell disease (SCD) receive regular blood transfusions, which can lead to alloimmunisation due to exposure to different red blood cell (RBC) antigens. The spleen is frequently damaged in these patients, resulting in higher numbers of red cell-derived particles (RCDP) and autophagic vesicles (AV). Most RCDP, produced through membrane budding, are right-side out and expose the external domains of RBC proteins, whereas AV are inside-out, exposing cytoplasmic domains. Since SCD patients have higher numbers of RCDP and AV, we aimed to characterise these particles and compare with healthy donor plasma and with storage vesicles (SV) from outdated blood. We also investigated the use of these particles to induce tolerance to RBC antigens, in a murine model. RCDP and AV from SCD and healthy plasma were isolated using immunomagnetic separation. Results using imaging flow cytometry (IS) showed that RBC markers like glycophorins A and C, band 3, glucose transporter1 and phosphatidylserine, were detected in all particles, confirming they originated from RBC. RCDP were more prevalent and generally larger than AV but both particle types were more abundant in SCD than healthy plasma. IS was more sensitive for small particle detection than flow cytometry. The latter detected large ghost membranes. All particle types were detected by transmission electron microscopy (TEM) and immunogold staining against extracellular or cytoplasmic GPA domains further confirmed their RBC origin. TEM, IS and dynamic light scattering showed that RCDP were larger than AV. A model system was developed to investigate tolerance induction in vivo and indicated that both SV and ghosts could be used to prime host animals. In conclusion, small RCDP and AV from SCD and healthy plasma have been characterised for first time, using IS. It may be possible to use particles from blood cells induce tolerance to RBC antigens and thereby reduce alloimmunisation.
Date of Award26 Nov 2020
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorAllison Blair (Supervisor) & David J Anstee (Supervisor)

Keywords

  • sickle cell disease
  • vesicles
  • microparticles
  • imaging flow cytometry
  • macrovesices
  • transfusion
  • alloimmunisation
  • storage blood

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