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

Extracellular vesicles (EVs) are potential disease biomarkers released by cells into body fluids. Flow cytometers measure EV concentrations within their size detection range. These size ranges can be quantified by relating the arbitrary units of measured light scattering signals to the optical diameter of EVs with Mie theory. However, Mie theory requires input parameters, including the refractive index (RI) of the suspension liquid surrounding the measured particles, which is not accurately known. Here we measure traceable RIs of suspension liquids, including Dulbecco's phosphate buffered saline (DPBS) and human blood plasma dilutions with an uncertainty of $1.4\times {10}^{-5}$ for relevant wavelengths between 405 and 644 nm. We show that differences between traceably measured and currently used RIs for suspension liquids lead to differences in applied optical diameter gates and reported EV concentrations. The currently assumed RI of DPBS in Rosetta Calibration leads to a difference of 9% to 35% between reported and actual EV concentrations in diluted plasma, depending on the lower detection limit of the flow cytometer. A traceably measured RI of the suspension liquid should be used to calibrate light scattering signals with Mie theory to improve accuracy of optical diameter and concentration measurements by EV flow cytometry.