F.A.W. Coumans

Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing

Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25‐fold in size, 300 000‐fold in concentration, 20 000‐fold in volume, and 10 000 000‐fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 104 to 1012 vesicles mL–1.

Single-step isolation of extracellular vesicles by size-exclusion chromatography

Background Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim To develop a single-step protocol to isolate vesicles from human body fluids. Methods Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles.

All circulating EpCAM+CK+CD45− objects predict overall survival in castration-resistant prostate cancer

BACKGROUND Presence of five or more circulating tumor cells (CTC) in patients with metastatic carcinomas is associated with poor survival. Although many objects positive for epithelial cell adhesion molecules and cytokeratin (EpCAM+CK+) are not counted as CTC, they may be an important predictor for survival. We evaluated the association between these objects and survival in patients with prostate cancer. PATIENTS AND METHODS Included in this follow-up study were 179 patients with castration-resistant prostate cancer. CellSearch was used to isolate EpCAM+ objects and to stain DNA, cytokeratin and CD45. All EpCAM+CK+ objects were subdivided into seven classes on the basis of predefined morphological appearance in 63 independent samples. Association of each class with survival was studied using Kaplan-Meier and Cox regression analyses. RESULTS Each EpCAM+CK+CD45- class showed a strong association with overall survival (P < 0.001). This included small tumor microparticles (S-TMP), which did not require a nucleus and thus are unable to metastasize. A higher number of objects in any class was associated with decreased survival. A good prediction model included large tumor cell fragments (L-TCF), age, hemoglobin and lactate dehydrogenase. Models with S-TMP or CTC instead of L-TCF performed similarly. CONCLUSION EpCAM+CK+CD45- that do not meet strict definitions for CTC are strong prognostic markers for survival.

Innovation in detection of microparticles and exosomes

Cell‐derived or extracellular vesicles, including microparticles and exosomes, are abundantly present in body fluids such as blood. Although such vesicles have gained strong clinical and scientific interest, their detection is difficult because many vesicles are extremely small with a diameter of less than 100 nm, and, moreover, these vesicles have a low refractive index and are heterogeneous in both size and composition. In this review, we focus on the relatively high throughput detection of vesicles in suspension by flow cytometry, resistive pulse sensing, and nanoparticle tracking analysis, and we will discuss their applicability and limitations. Finally, we discuss four methods that are not commercially available: Raman microspectroscopy, micro nuclear magnetic resonance, small‐angle X‐ray scattering (SAXS), and anomalous SAXS. These methods are currently being explored to study vesicles and are likely to offer novel information for future developments.

Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system.

Challenges in the enumeration and phenotyping of CTC

Purpose: Presence of circulating tumor cells (CTC) in metastatic carcinoma is associated with poor survival. Phenotyping and genotyping of CTC may permit “real-time” treatment decisions, provided CTCs are available for examination. Here, we investigate what is needed to detect CTC in all patients. Experimental Design: CTCs enumerated in 7.5 mL of blood together with survival from 836 patients with metastatic breast, colorectal, and prostate cancer were used to predict the CTC concentration in the 42% of these patients in whom no CTCs were found and to establish the relation of concentration of CTCs with survival. Influence of different CTC definitions were investigated by automated cell recognition and a flow cytometric assay without an enrichment or permeabilization step. Results: A log-logistic regression of the log of CTC yielded a good fit to the CTC frequency distribution. Extrapolation of the blood volume to 5 L predicted that 99% of patients had at least one CTC before therapy initiation. Survival of patients with EpCAM+, cytokeratin+, CD45− nucleated CTCs is reduced by 6.6 months for each 10-fold CTC increase. Using flow cytometry, the potential three-fold recovery improvement is not sufficient to detect CTC in all patients in 7.5 mL of blood. Conclusions: EpCAM+, cytokeratin+, CD45− nucleated CTCs are present in all patients with metastatic breast, prostate, and colorectal cancer and their frequency is proportional to survival. To serve as a liquid biopsy for the majority of patients, a substantial improvement of CTC yield is needed, which can only be achieved by a dramatic increase in sample volume. Clin Cancer Res; 18(20); 5711–8. ©2012 AACR.

Methodological guidelines to study extracellular vesicles

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

Refractive Index Determination of Nanoparticles in Suspension Using Nanoparticle Tracking Analysis

The refractive index (RI) dictates interaction between light and nanoparticles and therefore is important to health, environmental, and materials sciences. Using nanoparticle tracking analysis, we have determined the RI of heterogeneous particles <500 nm in suspension. We demonstrate feasibility of distinguishing silica and polystyrene beads based on their RI. The hitherto unknown RI of extracellular vesicles from human urine was determined at 1.37 (mean). This method enables differentiation of single nanoparticles based on their RI.

Reproducible extracellular vesicle size and concentration determination with tunable resistive pulse sensing

Introduction The size of extracellular vesicles (EVs) can be determined with a tunable resistive pulse sensor (TRPS). Because the sensing pore diameter varies from pore to pore, the minimum detectable diameter also varies. The aim of this study is to determine and improve the reproducibility of TRPS measurements. Methods Experiments were performed with the qNano system (Izon) using beads and a standard urine vesicle sample. With a combination of voltage and stretch that yields a high blockade height, we investigate whether the minimum detected diameter is more reproducible when we configure the instrument targeting (a) fixed stretch and voltage, or (b) fixed blockade height. Results Daily measurements with a fixed stretch and voltage (n=102) on a standard urine sample show a minimum detected vesicle diameter of 128±19 nm [mean±standard deviation; coefficient of variation (CV) 14.8%]. The vesicle concentration was 2.4·109±3.8·109 vesicles/mL (range 1.4·108–1.8·1010). When we compared setting a fixed stretch and voltage to setting a fixed blockade height on 3 different pores, we found a minimum detected vesicle diameter of 118 nm (CV 15.5%, stretch), and 123 nm (CV 4.5%, blockade height). The detected vesicle concentration was 3.2–8.2·108 vesicles/mL with fixed stretch and 6.4–7.8·108 vesicles/mL with fixed blockade height. Summary/conclusion Pore-to-pore variability is the cause of the variation in minimum detected size when setting a fixed stretch and voltage. The reproducibility of the minimum detectable diameter is much improved by setting a fixed blockade height.

Filtration parameters influencing circulating tumor cell enrichment from whole blood.

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·104∶102∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.