An analysis of endothelial microparticles as a function of cell surface antibodies and centrifugation techniques.

Chronic vascular disease is partially characterized by the presence of lesions along the vascular endothelial wall. Current FDA-approved clinical techniques lack the ability to measure very early changes in endothelial cell health. When endothelial cells are damaged, they release endothelial microparticles (EMPs) into circulation. Thus, blood EMP concentration may represent a useful cardiovascular disease biomarker. Despite the potential value of EMPs, current flow cytometry techniques may not consistently distinguish EMPs from other small cell particles. The purpose of this study was to use imaging flow cytometry to modify existing methods of identifying EMPs based on cell-surface receptor expression and visual morphology. Platelet poor plasma (PPP) was isolated using four different techniques, each utilizing a two-step serial centrifugation process. The cell-surface markers used in this study were selected based on those that are commonly reported in the literature. PPP (100μL) was labeled with CD31, CD42a, CD45, CD51, CD66b, and CD144 for 30-min in dark on ice. Based on replicated experiments, EMPs were best identified by cell-surface CD144 expression relative to other commonly reported EMP markers (CD31 & CD51). It is important to note that contaminating LMPs, GMPs, and PMPs were thought to be removed in the preparation of PPP. However, upon analysis of prepared samples staining CD31 against CD51 revealed a double-positive population that was less than 1% EMPs. In contrast, when using CD144 to identify EMPs, ~87% of observed particles were free of contaminating microparticles. Using a counterstain of CD42a, this purity can be improved to over 99%. More research is needed to understand how our improved EMP measurement method can be used in experimental models measuring acute vascular responses or chronic vascular diseases.