Methodology for isolation, identification and characterization of microvesicles in peripheral blood

Rationale: Analyses of circulating cell membrane-derived microvesicles (MV) have come under scrutiny as potential diagnostic and prognostic biomarkers of disease. However, methods to isolate, label and quantify MV have been neither systematized nor validated. Objective: To determine how pre-analytical, analytical and post-analytical factors affect plasma MV counts, markers for cell of origin and expression of procoagulant surface phosphatidylserine. Methods and results: Peripheral venous blood samples were collected from healthy volunteers and patients with cardiovascular disease and/or diabetes. Effects of blood sample collection, anticoagulant and sample processing to platelet free plasma (PFP), and MV isolation, staining and storage (freeze-thaw) and cytometer design were evaluated with replicate samples from these populations. The key finding is that use of citrate or EDTA anticoagulants decreases or eliminates microvesicles from plasma by inducing adhesion of the microvesicles to platelets or other formed elements. Protease inhibitor anticoagulants, including heparin, preserve MV counts. A centrifugation protocol was developed in which recovery of isolated MV was high with resolution down to the equivalent light scatter of 0.2 μm latex beads. Each procedure was systematically evaluated for its impact on the MV counts and characteristics. Conclusion: This study provides a systematic methodology for MV isolation, identification and quantification, essential for development of MV as diagnostic and prognostic biomarkers of disease.