aWomens’ Health Theme (NDOG & Dept of Engineering Science), Evaluation Of Methods For Measurement Of Cellular Microparticles & Exosomes In Human Disease Dragovic, R.Aa, Harrison Pb, Sheldon Hc, Harris Ac, Ratoi Ma, Dobson Pa and Sargent I.L.a aWomens’ Health Theme (NDOG & Dept of Engineering Science), bBlood Theme (Oxford Haemophilia & Thrombosis Centre), cCancer Theme (WIMM) Introduction Current Methods of Detection ELISA Circulating blood contains a variety of cell membrane microparticles (MPs) derived from many different cell types, including platelets, white cells, red cells and endothelial cells. Cellular particles are divided into two types: Microparticles (100nm – 1mM), released from cells during activation or apoptosis. Exosomes or nanoparticles (40nm – 100nm) which are formed from internalised endocytic vesicles that are subsequently secreted from the cell. MPs are involved in cell to cell interactions and cell signalling, possibly by transferring functional molecules between different cell types. MPs have been implicated in a large number of vascular pathologies where they have been found to be elevated, raising the possibility of their use as potential prognostic and diagnostic markers for numerous disease states. Their use as biomarkers for pre-eclampsia, haemostatic disorders and cancer is currently being investigated as part of the BRC programme. Ultra centrifugation of biological samples is used to pellet cellular particles and separate them from soluble proteins. Antibodies specific for the particles of interest are used to capture the particles on the ELISA plate. Disadvantages: Only one particle marker can be studied at one time and this method cannot discriminate between MPs and exosomes. C NP PE 25 50 75 *** ** STBM (ng/ml) Total placental MP levels measured in the plasma of non-pregnant (C), normal pregnant (NP) and pre-eclamptic (PE) women using a specific ELISA FLOW CYTOMETRY Analysis carried out on biological samples. Multiple markers can be used to simultaneously distinguish particles of different cellular origins. Disadvantages: Limit of sensitivity is ~200nm. Cannot be used to detect small MPs or exosomes ELECTRON MICROSCOPY Becton Dickinson LSR-II Flow Cytometer Ultracentrifugation of culture supernatants or plasma to pellet cellular particles allows visualisation by electron microscopy. Disadvantages: Subjective, not quantitative and only limited phenotyping possible. 660nm 190nm 400nm 660nm 190nm 400nm 200nm LSRII analysis of a Mixture of 190nm, 400nm and 660nm polystyrene particles 0.9% 2.2% 96.1% 0.8% Electron microscopy of exosomes from cultured U87 tumour cell line Pre-eclamptic plasma double labelled with Annexin V-APC and CD61-FITC

New Technology Conclusions NANOSIGHT DYNAMIC LIGHT SCATTERING 50nm 189nm 390nm 659nm NANOSIGHT 50nm polystyrene beads Mixture of 190nm, 400nm and 660nm polystyrene particles Nanoparticle Tracking Analysis (NTA) is a light scattering method for determining particle size and concentration of monodisperse and polydisperse samples. Accurately sizes particles based on analysis of their Brownian motion. Dynamic range of 20nm – 1mM. 100nm 300nm Mixture of 100nm and 300nm polystyrene beads Mixture of 100nm and 300nm polystyrene beads post filtration 0.2mM Filter 238nm Beckman Coulter N5 Submicron Particle Analyser DYNAMIC LIGHT SCATTERING Intensity Size [nm] Linear 30º 60º 90º DLS analysis of Placental (Syncytiotrophoblast) MPs DLS sizes particles through analysis of their Browninan motion. The N5 Submicron Particle Analyser has a dynamic range of 3nm – 5mM. Disadvantages: Samples must be of a very narrow size range. Unable to accurately size highly polydisperse samples. Placental (Syncytiotrophoblast) MPs Non Pregnant Plasma 84nm CPS DISC CENTRIFUGE Normal Pregnant Plasma 150nm PE Plasma NP Plasma C Plasma Resolves particles between 5nm – 75mM in the same sample by density gradient sedimentation. Disadvantages: time taken to analyse samples CPS disc centrifuge analysis of pre-eclamptic (PE), normal pregnant (NP) and non-pregnant (C) plasma. Pre-eclamptic Plasma 87nm Conclusions These methods are currently being evaluated as effective tools to measure MPs and exosomes in human disease. NTA is a new robust technique that accurately determines particle size and concentration. It has many advantages in resolving particle size distribution profiles which are not possible with other current methodologies.