Traceable Refractive Index Measurements of Liquids to Standardize Extracellular Vesicle Flow Cytometry

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Biophysics

Background:

• Extracellular vesicles (EVs) are crucial biomarkers found in body fluids.
• Flow cytometry is used to quantify EV concentrations, relying on light scattering.
• Accurate sizing and quantification depend on Mie theory, which requires accurate refractive index (RI) of the surrounding medium.

Purpose of the Study:

• To measure traceable refractive indices (RIs) of common suspension liquids used in EV analysis.
• To evaluate the impact of RI accuracy on EV size and concentration measurements.
• To provide recommendations for improving the accuracy of EV flow cytometry.

Main Methods:

• Traceable measurement of RIs for Dulbecco's phosphate buffered saline (DPBS) and diluted human blood plasma.
• Utilized wavelengths between 405 and 644 nm with an uncertainty of $1.4\times {10}^{-5}$.
• Compared EV concentrations derived from assumed versus measured RIs using Mie theory and flow cytometry.

Main Results:

• Traceable RIs for DPBS and plasma dilutions were accurately determined.
• Differences between assumed and measured RIs significantly altered optical diameter gates and reported EV concentrations.
• Using the currently assumed RI for DPBS in Rosetta Calibration resulted in 9% to 35% discrepancies in EV concentrations.

Conclusions:

• Accurate RI determination of suspension liquids is critical for reliable EV quantification.
• Current RI assumptions in EV flow cytometry calibration lead to significant measurement errors.
• Implementing traceably measured RIs in Mie theory calculations will enhance the accuracy of EV optical diameter and concentration measurements.