Evaluation Of Methods For Measurement Of Cellular Microparticles & Exosomes In Human Disease Introduction •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 – 1M), 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. Dragovic, R.A a , Harrison P b , Sheldon H c , Harris A c , Ratoi M a , Dobson P a and Sargent I.L. a a Womens’ Health Theme (NDOG & Dept of Engineering Science), b Blood Theme (Oxford Haemophilia & Thrombosis Centre), c Cancer Theme (WIMM) ELECTRON MICROSCOPY Current Methods of Detection 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 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 0 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 Electron microscopy of exosomes from cultured U87 tumour cell line Ultracentrifugation of culture supernatants or plasma to pellet cellular particles allows visualisation by electron microscopy. Disadvantages: Subjective, not quantitative and only limited phenotyping possible. LSRII analysis of a Mixture of 190nm, 400nm and 660nm polystyrene particles 660nm 190nm 400nm 660nm 190nm 400nm Pre-eclamptic plasma double labelled with Annexin V-APC and CD61-FITC 0.9% 2.2% 96.1% 0.8% 200nm Becton Dickinson LSR-II Flow Cytometer