Clinical requirements for extracellular vesicle assays

Figures & data

Table 1. Technique comparison of clinical EV analysis.

Technique	Advantages	Limitations	References
Conventional Flow Cytometry	Widely available in clinical laboratories Clinical validated machines Possible to phenotype High throughput and automation Good reproducibility	Limited sensitivity Variability known to occur between different machines Requires expertise	[31,56]
High Sensitivity Flow Cytometry	Good resolution Possible to phenotype High throughput Good reproducibility	Expensive Requires expertise	[10,42,57,58]
Nanoparticle Tracking Analysis	High resolution Possible to phenotype Good reproducibility	Throughput Specificity – Designed for monodisperse populations	[10,18,59–62]
Dynamic Light Scattering	High resolution Good reproducibility	Throughput Specificity – Designed for monodisperse populations	[60,62–64]
Resistive Pulse Sensing	High resolution Good reproducibility	Throughput Specificity – Designed for monodisperse populations	[10,62,65]
Electron Microscopy	High resolution Possible to phenotype	Not widely available Expensive Throughput Poor Reproducibility	[11]

Table 2. Definitions of terms.

Term	Definition
Clinical Assay	Any test being used to generate results that will be used diagnostically and may influence clinical decisions about a patient.
Clinical Laboratory Accreditation	A process to ensure the quality of results generated meet ISO 15189 standards
User Required Specifications (URS)	The required performance of a clinical assay, as defined by the clinical users either within an institution or nationally/internationally by a consensus publication
Validation	Analysis of performance characteristics to ensure that the assay meets the URS
Verification	Confirmation of performance characteristics of a commercial kit to ensure that the assay meets the URS

Figure 1. Introduction of clinical assays.

Table 3. Performance characteristics for assay validation.

Performance characteristic	Definition
Trueness	Qualitative assessment of the closeness of the measured value to the true value
Precision	Measurement of how close a group of measurements are to each another
Clinical Sensitivity	Ability to identify all individuals with a condition
Clinical Specificity	Ability to identify all individuals without a condition
Linearity	Ability to detect an analyte in a linear fashion across the reportable range
Analytical Sensitivity	Lowest level that can be reliably detected
Analytical Specificity	Effect of interfering substances on the assay
Sample Stability	Acceptable conditions and time that a sample can be stored prior to analysis
Sample Diversity	Range of sample types that can be used on the assay
Uncertainty of Measurement	Range of the values that could reasonably be attributed to the measured quantity

Table 4. Challenges for clinical validation of EV diagnostics.

Challenges	Solutions
Lack of International Reference Preparation to determine Trueness	Setting up and use of EQA schemes
Overlap between patients and healthy controls	Setting an appropriate cut-off using ROC analysis
Interference from lipaemic, haemolytic and platelet contaminated samples	Applying strict, but achievable sample requirements for assays
Issues with sample stability	Determining appropriate collection and storage conditions that minimise EV generation and contaminating fractions of blood components
Issues with sample diversity	Stringent guidelines for sample acceptance criteria
Establishing uncertainty of measurement	Consideration of pre-analytical variables to ensure accurate reporting of EV levels

van der Pol E, Sturk A, van Leeuwen T, et al.. Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation. J Thromb Haemost. 2018;16:1236–1245.

 PubMed Web of Science ®Google Scholar

Erdbrugger U, Rudy CK, Etter ME, et al. Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry. Cytometry A. 2014;85:756–770.

 Web of Science ®Google Scholar

Maas SL, de Vrij J, van der Vlist EJ, et al. Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics. J Control Release. 2015;200:87–96.

 PubMed Web of Science ®Google Scholar

Robert S, Lacroix R, Poncelet P, et al. High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles–brief report. Arterioscler Thromb Vasc Biol. 2012;32:1054–1058.

 PubMed Web of Science ®Google Scholar

Chandler WL, Yeung W, Tait JF. A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer. J Thromb Haemost. 2011;9:1216–1224.

 PubMed Web of Science ®Google Scholar

Groot Kormelink T, Arkesteijn GJ, Nauwelaers FA, et al. Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry. Cytometry A. 2016;89:135–147.

 Web of Science ®Google Scholar

Ayers L, Kohler M, Harrison P, et al. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay. Thromb Res. 2011;127:370–377.

 PubMed Web of Science ®Google Scholar

Dragovic RA, Gardiner C, Brooks AS, et al. Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis. Nanomedicine. 2011;7:780–788.

 PubMed Web of Science ®Google Scholar

Nicolet AMF, van der Pol E, Yuana Y, et al. Inter-laboratory comparison on the size and stability of monodisperse and bimodal synthetic reference particles for standardization of extracellular vesicle measurements. Meas SciTechnol. 2016;27.

 Google Scholar

Gercel-Taylor C, Atay S, Tullis RH, et al. Nanoparticle analysis of circulating cell-derived vesicles in ovarian cancer patients. Anal Biochem. 2012;428:44–53.

 PubMed Web of Science ®Google Scholar

Almizraq RJ, Seghatchian J, Holovati JL, et al. Extracellular vesicle characteristics in stored red blood cell concentrates are influenced by the method of detection. Transfus Apher Sci. 2017;56:254–260.

 PubMed Web of Science ®Google Scholar

Coumans FA, van der Pol E, Boing AN, et al. Reproducible extracellular vesicle size and concentration determination with tunable resistive pulse sensing. J Extracell Vesicles. 2014;3:25922.

 PubMedGoogle Scholar

Chuo ST, Chien JC, Lai CP. Imaging extracellular vesicles: current and emerging methods. J Biomed Sci. 2018;25:91.

 PubMed Web of Science ®Google Scholar