Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;7(11):e49726.
doi: 10.1371/journal.pone.0049726. Epub 2012 Nov 20.

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

Bence György  1 , Tamás G Szabó, Lilla Turiák, Matthew Wright, Petra Herczeg, Zsigmond Lédeczi, Agnes Kittel, Anna Polgár, Kálmán Tóth, Beáta Dérfalvi, Gergő Zelenák, István Böröcz, Bob Carr, György Nagy, Károly Vékey, Steffen Gay, András Falus, Edit I Buzás
Affiliations

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

Bence György et al. PLoS One. 2012.

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: Bob Carr is the inventor of NanoSight technology and a co-founder of the NanoSight Ltd. company. Matthew Wright is an employee of NanoSight Ltd. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. The authors declare no other competing interests.

Figures

Figure 1
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
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
Figure 3. Immunophenotyping of SF MVs by FC.
MV count/µl is shown in the y axis. *p<0.05, **p<0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gyorgy B, Szabo TG, Pasztoi M, Pal Z, Misjak P, et al. (2011) Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 68: 2667–2688. - PMC - PubMed
    1. Gyorgy B, Modos K, Pallinger E, Paloczi K, Pasztoi M, et al. (2011) Detection and isolation of cell-derived microparticles are compromised by protein complexes due to shared biophysical parameters. Blood. 117: e39–48. - PubMed
    1. Distler JH, Jungel A, Huber LC, Seemayer CA, Reich CF, 3rd, et al (2005) The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles. Proc Natl Acad Sci U S A 102: 2892–2897. - PMC - PubMed
    1. Artoni A, Merati G, Padovan L, Scalambrino E, Chantarangkul V, et al. (2012) Residual platelets are the main determinants of microparticles count in frozen-thawed plasma. Thromb Res. 130: 561–2. - PubMed
    1. Suštar V, Bedina-Zavec A, Stukelj R, Frank M, Bobojević G, et al. (2011) Nanoparticles isolated from blood: a reflection of vesiculability of blood cells during the isolation process. Int J Nanomedicine 6: 2737–48. - PMC - PubMed

Publication types

MeSH terms

Grants and funding

This work was supported by the Semmelweis Foundation, grants OTKA K 73247,NK 84043 and K 77537, Kerpel-Fronius Ödön Fellowship, Baross Gábor (REG-KM-09-1-2009-0010) and FP7-PEOPLE-2011-ITN - PITN-GA-2011-289033 “DYNANO”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
-