Abstract
Introduction: Microparticles (MPs) are phospholipid-rich vesicles shed from a variety of eukaryotic cells. Several types of MPs exist: membrane vesicles, exosomes, exosome-like vesicles and apoptotic bodies and they can be released from the cells undergoing activation, apoptosis, and several stress induced activations. MPs express proteins in and on the surface, which are indications of which cell the MP originates from. The major cellular source of MPs has been shown to be platelets. MPs can in theory possess various functions depending on their cellular origin. The major known and described functions of MPs are the involvement in coagulation, inflammation and in the vascular system, but MPs have also been shown to act as intracellular mediators and have the potential to transfer part of their components to a target cell. The clearance of MPs from the circulation is poorly understood, but it has been proposed that MPs are mainly cleared by phagocytosis. MPs are present in both healthy individuals and in various pathologies. Elevated levels of MPs have been shown in numerous types of diseases, such as cardiovascular diseases, cancer, and infectious diseases. Despite increasing interest in MPs, the isolation, detection, and characterisation of MPs are hampered by both limitations in the available technology and the biological complexity of body fluids. The need for new methods for the characterisation of MPs is therefore crucial.
Aim: The aim of this project is to evaluate the potential of two novel detection methods for the characterisation of MPs, namely Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS).
Methods: NTA and DLS were evaluated by investigating the reproducibility and accuracy of measurements by the use of microbeads in sizes 50 nm, 100 nm, and 200 nm. Dilution of plasma samples were investigated to find suitable dilutions of plasma samples for measurements of MPs. Different pre-analytical parameters were investigated to evaluate the potential of NTA and DLS for usage in clinical experiments. The effect of freezing was investigated by comparing fresh samples with the same samples that have been frozen for 1, 7, 15, and 40 days at -80°C. The influence of centrifugation on the particle size distribution and the concentration of MPs were investigated by comparing three different centrifugation methods. Centrifugation A, a centrifugation at 3220g for 20 minutes at 20°C, Centrifugation B, a double-step centrifugation, in which blood samples were centrifuged twice at 2500g for 15 minutes at room temperature, and finally Centrifugation C, a two-step centrifugation, in which blood samples were first centrifuged at 2500g for 15 minutes at room temperature, and the resultant platelet-poor-plasma was then ultracentrifuged at 13000g for 2 minutes at room temperature. Finally, the potentials of NTA and DLS for measurement and differentiation of healthy plasma samples and pathological samples were investigated.
Results: Both NTA and DLS showed relative reproducibility and accuracy for measurement of microbeads and MPs in plasma samples. The most suitable dilution of plasma samples in buffer for measurement on NTA was found to be 2 µL of plasma sample in 1000 µL DPBS for both healthy and diseased plasma samples. Freezing did not affect the particle size distributions of measured MPs, but the concentration of MPs was found to be higher for the fresh sample when measuring the samples with NTA. The DLS results were inconclusive for the fresh-frozen experiment. Centrifugation showed to have little or no effect on the particle size distribution and concentration of MPs in plasma samples for measurements with both methods. The clinical potential of both NTA and DLS was proven when both methods were able to differentiate between healthy plasma samples and pathological plasma samples.
Conclusion: Both methods exhibit relative reproducibility and they can be used as complementary methods for the detection and characterisation of MPs in plasma.