Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases

Abstract

Introduction: Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures.

Methods: In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals.

Results: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+) and CD8(+) T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction).

Conclusions: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

Figure 1. Analysis of SF MVs by EM and NTA.

(a). Electron micrographs of the 20,500 g pellets from SFs are shown. Scale bars denote 500 nm, original magnification was 30,000×. (b). NTA screenshot from an RA SF pellet, showing light scattering particles. The field of view is 120 µm by 80 µm. (c). Size histograms of particles in SFs obtained by NTA measurements. A small peak is visible in the MV size range indicated by an arrow (between 100 and 200 nm), however, most particles are below 100 nm in diameter.

Figure 2. Differential detergent lysis by FC.

Events are shown within the MV gate. Background fluorescence was determined using an isotype control antibodies. Values show percents of positive events. Counting beads are visible in the upper right corner of the dot plots. (a). CD3⁺ and CD8⁺ vesicles were detected by FC. Most positive events disappear after the addition of 0.1% Triton X-100. Remaining events were subtracted from the original event counts. (b). Detergent-resistant IgG and IgM staining in JIA SF samples suggested that these events were related to immune complexes rather than vesicular structures.

Figure 3. Immunophenotyping of SF MVs by FC.

MV count/µl is shown in the y axis. *p<0.05, **p<0.01.