Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Summary

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Abstract

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).

Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)'s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Introduction

Accurate and repeatable analysis of EVs and other nanometer-scaled particles presents numerous challenges across research and industry. Replication of EV research has been difficult, in part, due to the lack of uniformity in reporting necessary parameters associated with data collection. To address these deficiencies, the ISEV proposed industry guidelines as a minimal set of biochemical, biophysical, and functional standards for EV researchers and published them as a position statement, commonly referred to as MISEV2014¹. The accelerating pace of EV research required an updated guideline, and the "MISEV2018: a position statement of the ISEV" ex....

Protocol

1. General protocol guidelines

1. Maintain the microscope on an air table or at a minimum on a vibration-free table. Ensure that extraneous vibrations (e.g., foot tapping on the floor, touching the table, door closures, laboratory traffic) are kept to a minimum.
2. Set and maintain the temperature of the laser module at a constant temperature for all video recordings.
   NOTE: The temperature chosen was 25 °C because the nanoparticle size analyzer was calibrated at that temperature. Therefore, it is important that all users of the instrument know and use the calibrated temperature. Room temperature is not an acceptable setting becaus....

Results

Table 1 contains the results of the NTA videos for the liposome samples (18 filtered and 18 unfiltered) and a representative DPBS diluent. Comparisons across the two groups were completed regardless of the camera level or detection threshold in this paper. Filtered samples had a mean particle diameter of 108.5 nm, a particle mode of 86.2 nm, and a concentration of 7.4 × 10⁸ particles/mL. In contrast, unfiltered samples had a mean particle diameter of 159.1 nm, a particle mode of 105.7 nm, and .......

Discussion

There are several methods available to estimate the size and concentration of nanoparticles¹¹. These include ensemble methods that generate a size estimate from a population, including dynamic light scattering (DLS), centrifugal sedimentation, and single-particle level analysis-electron microscopy, NTA, atomic force microscopy, and tunable resistive pulse sensing. Of these, DLS and NTA are widely used, nondestructive size and concentration measurement methods, based on Brownian movement in an idea.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automatic Pipetter
Centrifuge Tubes, Conical, Nunc 15 mL	Thermo Sci.	339650
Kimwipes
Lens Cleaner
Lens Paper
NanoSight LM-10	Malvern Panalytical
NanoSight LM-14 Laser Module	Malvern Panalytical
Nanosight NTA Software Ver. 3.2	Malvern Panalytical
Paper Towels
Pipette Tips, 1-200 µL, Filtered, Sterile, Low Binding	BioExpress	P -3243-200X
Pipette Tips, 50-1,000 µL, Filtered, Sterile	BioExpress	P-3243-1250
Saline, Dulbecco's Phosphate Buffered (No Ca or Mg)	Gibco	14190-144
Standards, Latex Transfer- 100 nm (3 mL)	Malvern	NTA4088
Standards, Latex Transfer- 50 nm  (3 mL)	Malvern	NTA4087
Syringe Filter, 33 mm, .22 µm, MCE, Sterile	Fisher brand	09-720-004
Syringe, TB, 1 mL, slip tip	Becton Dickinson	309659
Waste fluid container