Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry

Abstract

The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform their use as biomarkers for disease.

Fig 1. NanoFCM allows identification of beads and liposomes down to 100 nm.

Separation of a mixture containing 200 and 500 nm latex beads by LSRII (A), and NanoView (B) instruments show more distinct separation with the NanoView Instrument. The NanoView is capable of separating a mixture of 100–500 nm beads into distinct populations (C) and can detect 100 nm liposomes (D). The gating strategy for these experiments to determine instrument and background noise are described in the methods section.

Fig 2. EVs detected in plasma from a healthy donor.

Plasma from 5 mL of blood was collected, centrifuged to remove cellular debris (see methods), and imaged following a series of dilutions in PBS (A) to test for ‘swarming’. An EV population (based on positioning of 100 nm liposomes and size distribution of plasma samples) from the plasma of a healthy donor was sorted (gate R2; B) and imaged using atomic force microscopy (C). The size distribution of sorted EVs was analyzed by qNano and is represented in D. The gating strategy for these experiments is detailed in the methods section.

Fig 3. The number and size distribution of plasma EVs isolated from healthy donors varies during the day.

Plasma from 6 healthy donors was isolated and cell-free plasma was diluted 1:500 in PBS and imaged using nanoFCM. Plots from all donors recorded at 7:30 AM, 2:30 PM, and 7:30 PM are shown.