Isolation of microvesicles from peripheral blood

A clinically relevant test with the corresponding equipment and laboratory material will be developed to determine microvesicles (sub-micron fragments of cells) from blood for diagnostics and treatment. The market consists of research and clinical laboratories worldwide.

Blood tests are a widely used method for diagnostics in human and veterinary medicine. In the proposed project, blood test with the corresponding equipment and laboratory material will be developed to determine microvesicles (MVs) from blood. Background: Microvesicles (MVs) are very small (sub-micron sized, on the average several hundred times less extensive than cells) membrane-enclosed fragments of cell interior which are created is the final stage of membrane budding. All cells can shed MVs and MVs are present in all body fluids. Being able to move with lymph and blood, they carry the contents deriving from the mother cell to the distal cells where they can become functional. As MVs are considered procoagulant, determination of the number and the content of MVs could be considered as a complementary method for determination of the instant risk for thromboembolic events. By an appropriate characterization, they can be used for determination of the presence of ocult cancer and early diagnostics of other diseases. Diagnostic procedures based on determination of MVs could in the future partly replace invasive biopsies. Development of diagnostic methods based on assessment of MVs would have extensive positive medical, ethical and economical consequences and imply widespread use in human and veterinary medicine. Further, specific MVs are potentially beneficial in treatment of some diseases. State of art: Methods for assessment of MVs from peripheral blood which are presently used in research, consist of centrifugation and washing of samples, labelling and counting of MVs by flow cytometry, however, the protocols are sensitive to the dynamics of the process and to many parameters (such as temperature) in a yet systematically undetermined manner. Preliminary results show the important effect of temperature, through activation of platelets and through the dynamics of sedimentation of cells during centrifugation. It is yet unclear how MVs interact with surfaces of laboratory material. Further, samples are presently handled manually which is a source of large noise in results. Presently, results are not accurate and repeatable enough to be used in clinical practice. Moreover, even in research, the number of samples treated under the same conditions is limited to relatively small numbers. The project: It is the scope of the proposed project to improve the method for isolation of MVs from blood and their characterization, and to develop the equipment and laboratory material for this purpose. We will thoroughly elaborate parameters of each step of the protocol. We will develop appropriate heat baths and laboratory material which will keep the temperature during manipulation at the required level and enable controlled manipulation of the temperature during the process. We will elaborate the effect of the centrifugation acceleration and speed and the corresponding temperature and speed regulation in a centrifuge and automate gathering of plasma from the tubes. We will study and optimize the surface of laboratory tubes and pipette tips. We will study different labelling techniques for assessment of MVs. It is our aim to develop a method and the corresponding equipment which would ensure adequate repeatability and accuracy for the use in clinical practice which seems to be possible only by advance in technology. To validate the method, clinical studies involving patients with different diseases, and healthy donors will be performed. There are six partners in the project (University of Ljubljana (UL), Domel, d.o.o. (TŽ), Abo Akademi University (AA), Medical University of Gdansk (MUG), University of Ghent (UG) and Zentech (ZT)). TŽ produces equipment for laboratory analyses, UL and AA have a history of successful collaboration in scientific research based on the equipment produced by TŽ while MUG, UG and ZT have recently begun a collaboration with UL regarding clinical and experimental studies. The research groups from UL leaded by prof. Kralj-Iglič and from AA leaded by doc. Hagerstrand are coauthors of around 30 scientific papers in distinguished international journals, mostly on membrane budding and vesiculation. The isolation of MVs in Ljubljana is being performed by using centrifuges produced by TŽ. Partners will collaborate in developing a clinically relevant method for assessment of MVs from blood. UL, AA and UG will determine parameters (temperature, dynamics of centrifugation and gathering samples) while TŽ will develop equipment and ZT will collaborate to determine the appropriate labels to optimize the method for assessment of MVs.
Project ID: 
6 260
Start date: 
Project Duration: 
Project costs: 
960 000.00€
Technological Area: 
Heart and blood circulation illnesses
Market Area: 
Diagnostic services

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