Detection of extracellular vesicles: size does matter

Type: PhD thesis PhD thesis Edwin van der Pol
Author: Edwin van der Pol
Date: January 2015
University: University of Amsterdam
Location: Amsterdam, The Netherlands
ISBN: 978-9-46-295069-6
Doctoral advisors: Prof. Dr. A.G.J.M. van Leeuwen
Prof. Dr. A. Sturk
Co-supervisor: Dr. R. Nieuwland
Attachment: PhD Thesis Edwin van der Pol.pdf (5 MB)

Summary

Extracellular vesicles

The human body is made up of cells. Cells release small sacks filled with fluid, which are called “extracellular vesicles”. The diameter of extracellular vesicles (EV) typically ranges from 30 nm to 1 μm, the smallest being some 100-fold smaller than the smallest cells of the human body. Because cells release EV into their environment, our body fluids, such as blood, saliva, and urine, contain numerous EV.

Detection hampers clinical applications of EV

Cells release EV to remove waste, and to transport and deliver cargo, such as receptors and genetic information, to other cells. Since the size, concentration, cellular origin, and composition of EV in body fluids change during disease, EV have promising clinical applications, such as diagnosis of cancer and monitoring the efficacy of therapy. However, clinical applications of EV are not realized yet, because currently used detection techniques lack the sensitivity to detect the majority of EV.

Aim of this thesis

The aim of this thesis is to improve the detection of EV by (1) obtaining insights into physical properties of EV, and (2) gaining a profound understanding of techniques to detect EV.

Physical properties of EV

Detection is the act of perceiving “something”. To specify “something”, physically detectable properties of EV are defined in Chapter 2. Examples of these properties are size, concentration, density, morphology, biochemical composition, refractive index, zeta potential and deformability. This thesis focuses on the properties size, concentration and refractive index of EV, since these three properties play a key role in the optical detection of EV.

Gaining understanding of detection techniques

In Chapter 3, an overview of currently available and potentially applicable techniques to detect the size and concentration of EV is provided. The working principle of all techniques is briefly discussed, as well as their capabilities and limitations based on the underlying physical parameters of the technique. To compare the precision in determining the size of EV between the discussed techniques, a mathematical model is developed to calculate the expected size distribution for a reference EV population.

In Chapter 4, the most applicable techniques of Chapter 3 are selected for an experimental evaluation. For these techniques, the accuracy and precision in measuring the EV size and concentration are determined. Although each technique gives a different size distribution and concentration for the reference EV population, all techniques indicate that the concentration of EV decreases with increasing diameter. Consequently, the minimum detectable EV size of a technique affects the measured concentration. Differences between the minimum detectable EV size of techniques explain the 100,000,000-fold difference in the reported concentrations of EV in human blood plasma. The relationship between the concentration of EV and their diameter can be described by the power-law function.

EV detection by flow cytometry

Chapter 5 addresses EV detection by flow cytometry, which is the most widely used technique to study single EV. Due to their small size and high concentration, however, multiple EV are simultaneously illuminated by the laser beam of the flow cytometer, and therefore are counted as a single event signal. This phenomenon is christened “swarm detection”. In addition, the relationship between light scattering and the diameter of EV is modeled using Mie theory. This relationship is used to demonstrate that a currently widely applied standardization procedure for EV detection selects EV and cells with a diameter of 800-2400 nm instead of the envisioned 500-900 nm. Consequently, in many studies other particles than the envisioned EV were studied.

Refractive index of EV

A variable of Mie theory is the refractive index of EV, which determines how efficiently a EV scatters light. In Chapter 6, a method based on nanoparticle tracking analysis is developed to determine the size and refractive index of single EV and other nanoparticles. For urinary EV a mean refractive index of 1.37 at 405 nm was obtained, which is much lower than the frequently and often unintentionally assumed values between 1.45 and 1.63. The low refractive index of EV implies that EV scatter light less efficiently than calibration beads. Consequently, detecting scattering from EV demands a sensitive detector. The determined refractive index of EV can be used to relate scattering to diameter, which is useful for data interpretation and calibration.

EV detection by tunable resistive pulse sensing

Tubable resistive pulse sensing is a technique to measure the size and concentration of EV in suspension. In Chapter 7, a protocol is developed to determine and improve the reproducibility of tunable resistive pulse sensing.

Single-step isolation of EV

Because body fluids contain many particles other than EV, EV require isolation prior to detection. Isolation of EV particularly from plasma is challenging due to the presence of proteins and lipoproteins. In Chapter 8, a single-step protocol to isolate EV from human body fluids is developed. The protocol is based on size-exclusion chromatography and has excellent recovery and enrichment.

The future of EV-based diagnostics

In the future, EV will be included in reference tables, such as hematology reference tables, as their physical properties are expected to correlate with disease. Prerequisites to establish EV as clinical biomarkers are: (1) knowledge of physical properties of EV, (2) insight into capabilities and limitations of detection techniques, (3) availability of techniques with the capability of deriving the cellular origin and function of EV and with improved sensitivity compared to current state-of-art technology, and (4) standardization of measurements. Standardization is important for data comparison between laboratories. In Chapter 9, the applicability of EV detection by techniques that are beyond the current state-of-art is discussed. Chapter 10 enlightens the future of EV-based diagnostics.

This thesis provides solid insight into (1) the physical properties of EV and (2) the capabilities and limitations of current detection techniques. This knowledge is the onset to (3) the development of novel detection techniques and (4) improved standardization procedures, which are important steps towards EV-based diagnostics.