Characterization of Platelets Using P-Selectin Targeted Antibodies and Flow Cytometry

Open Access
Author:
Blankemeyer, Catherine Eileen
Area of Honors:
Biomedical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Keefe Manning, Thesis Supervisor
  • Justin Brown, Honors Advisor
Keywords:
  • Flow Cytometry
  • Cardiovascular
  • blood
  • platelet
  • platelet activation
  • Heart
Abstract:
Cardiovascular disease in its many forms is the leading cause of death for men and women in the United States, and its prevalence has led to an increased need for mechanical devices to restore or improve heart function. Inferior vena cava filters (IVCs), left ventricular assist devices (LVADs), and mechanical heart valves are among the most common type of cardiovascular devices, and although they improve heart function, they also introduce a greater risk of thrombosis in patients. Thrombosis is a result of increased platelet activation in the blood surrounding the device, and can lead to stroke, heart attack, or pulmonary embolism. Increased platelet activation surrounding devices is caused by mechanical stresses induced by changes in geometry or flow pattern. A prediction of thrombosis can be determined by measuring changes in activated platelet levels. The objective of this study is to quantify platelet activation levels using flow cytometry and fluorophore-conjugated P-Selectin specific antibodies. Two main protocols are compared to determine whether platelet rich plasma (PRP) or whole blood is more appropriate for platelet activation quantification. The effects of centrifugation speed on platelet activation are quantified, and it is determined that the whole blood protocol is more appropriate because it eliminates the need for centrifugation. Experimental conditions such as needle size, fixation with paraformaldehyde, and data post-processing are varied and compared to optimize the protocol. The resulting protocol is one that produces accurate platelet activation results that are repeatable and reliable. The protocol development process proves the importance of considering each experimental factor and provides opportunity for future improvement and applications.