Preceding METVES project

Background

The costs of health care are increasing rapidly throughout Europe due to ageing of the population. One of the instruments to reduce health care costs is early diagnosis of disease, which improves the efficacy of medical treatment. Making a medical decision requires reliable information. A major part of this information comes from the routine analysis of body fluids such as blood and urine. These fluids contain cells and biomarkers that reflect health and disease. To gain access to this information, clinicians request the collection of blood, urine and other body fluid samples, which are then analysed in hospital laboratories.

Need

Cell-derived extracellular vesicles (EVs) have gained a strong clinical interest. We use the term EVs as a collective term for all of the different types of vesicles (microparticles, exosomes, etc.) present in human body fluids. EVs are released from cells and are present in all body fluids. As EVs in patients differ from EVs in healthy subjects, EVs can be regarded as novel and non-invasive biomarkers. Moreover, EVs affect progression of diseases such as cancer and cardiovascular disease by promoting coagulation, inflammation and angiogensis. The need will be illustrated by three examples.

Firstly, ovarian cancer, called the "silent killer", is almost invariably detected when the tumour is at an incurable stage. Blood samples from ovarian cancer patients contain EVs originating from tumour cells, and the relevance of these ovarian-derived EVs as biomarkers is currently under investigation.

Secondly, thrombosis, the second cause of death of all hospitalised cancer patients, is thought to be caused by EVs released from tumour cells into the blood. In 2012, a multi-centre trial will start (4,000 patients) to identify high-risk thrombosis cancer patients using a EV-based assay developed in the Amsterdam University Medical Centers.

Thirdly, preeclampsia is the most frequent disease of pregnant women and threatens the life of both the mother and the unborn child. Preeclampsia is now thought to be caused by the uncontrolled release of vesicles from placental cells into the blood of the mother. Because there are no biomarkers available to detect preeclampsia at an early stage, studies are now ongoing to evaluate the usefulness of placental-derived EVs in mother blood as an early and non-invasive biomarker.

Detection of EVs is a challenge due to their extremely small size (average diameter less than 100 nm) and heterogeneity. Even state-of-the-art detection techniques for EVs detect only 1-2% of all EVs present, and thus provide insufficient and incomplete information. Due to detection problems, data from EV research is qualitative rather than quantitative, and cannot be compared between laboratories. Obviously, biomarkers have clinical relevance only when measurements are reliable and comparable.

Objectives

The aim of the preceding METVES project was to develop reliable, comparable and quantitative analysis of EVs in biological fluids. Methods were developed for the standardized collection and handling of human body fluids for the isolation of EVs. Methods, available in metrological institutes, were explored to measure the size and size distribution, chemical composition, morphology and concentration of EVs. Synthetic and biological reference materials were tested as standards for EV measurements. Selected reference materials were analysed by all project partners and data was compared between them.

Impact

The preceding METVES project has defined the state-of-the-art for EV measurements. In METVES, procedures were developed for the collection and handling of EVs from biological fluids. The size distribution of EVs was measured using metrological and clinical instruments. Because EVs are polydisperse and have a complex composition, and because suitable reference materials and methods were lacking, traceable size measurements proved unfeasible with both primary and clinical methods.

However, METVES revealed that flow cytometry has clinical potential, because flow cytometers can identify cell-specific EVs at a rate of thousands per second. Therefore, an inter-laboratory comparison study was initiated to measure cell-specific EVs within the same size range. In this study, commercial synthetic EV reference materials were characterised by metrological instruments and used to standardise EV size measurements by 46 flow cytometers. The results were ground-breaking: although two out of three flow cytometers were sufficiently sensitive to detect EVs, flow rates deviated two-fold from the set flow rate, and local preparation of EV samples lead to undesired inter-laboratory variability. In sum, the preceding METVES project provided the onset to METVES II.

At this website, you can find an overview of the articles, posters, presentations, reports, and videos published by the preceding METVES project.