Isolation and Quantification of Blood Apoptotic Bodies, a Non-invasive Tool to Evaluate Apoptosis in Patients with Ischemic Stroke and Neurodegenerative Diseases

BACKGROUND: Improper regulation of apoptosis has been postulated as one of the main factors that contributes to the etiology and/or progression of several prevalent diseases, including ischemic stroke and neurodegenerative pathologies. Consequently, in the last few years, there has been an ever-growing interest in the in vivo study of apoptosis. The clinical application of the tissue sampling and imaging approaches to analyze apoptosis in neurological diseases is, however, limited. Since apoptotic bodies are membrane vesicles that are released from fragmented apoptotic cells, it follows that the presence of these vesicles in the bloodstream is likely due to the apoptotic death of cells in tissues. We therefore propose to use circulating apoptotic bodies as biomarkers for measuring apoptotic death in patients with ischemic stroke and neurodegenerative diseases. RESULTS: Since there is no scientific literature establishing the most appropriate method for collecting and enumerating apoptotic bodies from human blood samples. Authors, here, describe a reproducible centrifugation-based method combined with flow cytometry analysis to isolate and quantify plasma apoptotic bodies of patients with ischemic stroke, multiple sclerosis, Parkinson’s disease and also in healthy controls. Electron microscopy, dynamic light scattering and proteomic characterization in combination with flow cytometry studies revealed that our isolation method achieves notable recovery rates of highly-purified intact apoptotic bodies. CONCLUSIONS: This easy, minimally time consuming and effective procedure for isolating and quantifying plasma apoptotic bodies could help physicians to implement the use of such vesicles as a non-invasive tool to monitor apoptosis in patients with cerebrovascular and neurodegenerative diseases for prognostic purposes and for monitoring disease activity.