Analytical Considerations in Nanoscale Flow Cytometry of Extracellular Vesicles to Achieve Data LinearityFunding H.S.L. is supported by a Movember/Prostate Cancer Canada Rising Star Award (#RS2012–008 and #RS2016–56) and received operating grant support by Prostate Cancer Fight Foundation and Ontario Institute for Cancer Research (SPS 0613–03). Y.K. is supported by an Ontario Graduate Scholarship Award. K.C.W. is funded by a CIHR Postdoctoral Fellowship (140880). L.K. and F.L.L. are supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada.
25 April 2018
11 July 2018
15 August 2018 (eFirst)
Background Platelet microparticles (PMPs) and their abundance in the blood are a prognostic biomarker in thrombotic disorders and cancer. Nanoscale flow cytometry (nFC) is ideal for high-throughput analysis of PMPs but these clinical assays have not been developed previously.
Objective This article demonstrates that nFC is a suitable technology to enumerate PMPs present in plasma samples in a clinical setting.
Materials and Methods nFC was performed using the Apogee A50-Micro instrument. Instrument settings and acquisition parameters were developed with the use of fluorescent beads and plasma samples. Sample preparation and handling was also optimized.
Results nFC allows for linear detection of particles between approximately 200 and 1,000 nm based on calibration beads and was dependent on dilution factor and flow rate. Linearity in event analysis as samples became more diluted was lost when events approximately 100 nm were gated while linearity was maintained despite dilution of sample in events larger than 200 nm in diameter. Higher flow rates lead to an under-estimation of events analysed per microlitre of analyte and this was more pronounced when plasma samples were not diluted more than 1/20×.
Conclusion nFC offers multi-parametric analysis of PMPs when optimal calibration of acquisition and sample processing settings is performed. Analysis of plasmas from metastatic prostate cancer patients and leukaemia patients revealed that PMP levels were larger than 100 nm and were equally abundant in patients that responded to or failed androgen deprivation therapy or between patients representing different stages of leukaemia.
J.G. and F.L. performed all experiments, designed all experiments and wrote the manuscript. Y.K., K.C.W., T.L., L.K. and F.L.L. performed experiments. C.M. and H.S.L. wrote the manuscript. N.E.K. and J.B. provided plasma samples and reviewed and edited the manuscript.
* Janice Gomes and Fabrice Lucien equally contributed to the study.
- 1 Boulanger CM, Amabile N, Tedgui A. Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease. Hypertension 2006; 48 (02) 180-186
- 2 Lynch SF, Ludlam CA. Plasma microparticles and vascular disorders. Br J Haematol 2007; 137 (01) 36-48
- 3 George FD. Microparticles in vascular diseases. Thromb Res 2008; 122 (Suppl. 01) S55-S59
- 4 Barteneva NS, Fasler-Kan E, Bernimoulin M. , et al. Circulating microparticles: square the circle. BMC Cell Biol 2013; 14: 23
- 5 Mause SF, Weber C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ Res 2010; 107 (09) 1047-1057
- 6 Jy W, Horstman LL, Jimenez JJ. , et al. Measuring circulating cell-derived microparticles. J Thromb Haemost 2004; 2 (10) 1842-1851
- 7 Hargett LA, Bauer NN. On the origin of microparticles: from “platelet dust” to mediators of intercellular communication. Pulm Circ 2013; 3 (02) 329-340
- 8 Wolf P. The nature and significance of platelet products in human plasma. Br J Haematol 1967; 13 (03) 269-288
- 9 Kornek M, Lynch M, Mehta SH. , et al. Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis. Gastroenterology 2012; 143 (02) 448-458
- 10 Burger D, Schock S, Thompson CS, Montezano AC, Hakim AM, Touyz RM. Microparticles: biomarkers and beyond. Clin Sci (Lond) 2013; 124 (07) 423-441
- 11 Xue S, Cai X, Li W, Zhang Z, Dong W, Hui G. Elevated plasma endothelial microparticles in Alzheimer's disease. Dement Geriatr Cogn Disord 2012; 34 (3-4): 174-180
- 12 Diamant M, Tushuizen ME, Sturk A, Nieuwland R. Cellular microparticles: new players in the field of vascular disease?. Eur J Clin Invest 2004; 34 (06) 392-401
- 13 Matzdorff A, Kemkes-Matthes B, Pralle H. Microparticles and reticulated platelets in Wiskott-Aldrich syndrome patients. Br J Haematol 2000; 109 (03) 673
- 14 Ando M, Iwata A, Ozeki Y, Tsuchiya K, Akiba T, Nihei H. Circulating platelet-derived microparticles with procoagulant activity may be a potential cause of thrombosis in uremic patients. Kidney Int 2002; 62 (05) 1757-1763
- 15 Italiano Jr JE, Mairuhu ATA, Flaumenhaft R. Clinical relevance of microparticles from platelets and megakaryocytes. Curr Opin Hematol 2010; 17 (06) 578-584
- 16 Burnouf T, Goubran HA, Chou M-L, Devos D, Radosevic M. Platelet microparticles: detection and assessment of their paradoxical functional roles in disease and regenerative medicine. Blood Rev 2014; 28 (04) 155-166
- 17 Tseng C-C, Wang C-C, Chang H-C. , et al. Levels of circulating microparticles in lung cancer patients and possible prognostic value. Dis Markers 2013; 35 (05) 301-310
- 18 Platt M, Willmott GR, Lee GU. Resistive pulse sensing of analyte-induced multicomponent rod aggregation using tunable pores. Small 2012; 8 (15) 2436-2444
- 19 Dragovic RA, Gardiner C, Brooks AS. , et al. Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine (Lond) 2011; 7 (06) 780-788
- 20 Lawrie AS, Albanyan A, Cardigan RA, Mackie IJ, Harrison P. Microparticle sizing by dynamic light scattering in fresh-frozen plasma. Vox Sang 2009; 96 (03) 206-212
- 21 Leong HS, Podor TJ, Manocha B, Lewis JD. Validation of flow cytometric detection of platelet microparticles and liposomes by atomic force microscopy. J Thromb Haemost 2011; 9 (12) 2466-2476
- 22 György B, Szabó TG, Turiák L. , et al. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases. PLoS One 2012; 7 (11) e49726
- 23 Abrams CS, Ellison N, Budzynski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 1990; 75 (01) 128-138
- 24 van der Pol E, van Gemert MJC, Sturk A, Nieuwland R, van Leeuwen TG. Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost 2012; 10 (05) 919-930
- 25 Biggs CN, Siddiqui KM, Al-Zahrani AA. , et al. Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry. Oncotarget 2016; 7 (08) 8839-8849
- 26 Kibria G, Ramos EK, Lee KE. , et al. A rapid, automated surface protein profiling of single circulating exosomes in human blood. Sci Rep 2016; 6: 36502
- 27 Chandler WL. Measurement of microvesicle levels in human blood using flow cytometry. Cytometry B Clin Cytom 2016; 90 (04) 326-336
- 28 Marcoux G, Duchez A-C, Cloutier N, Provost P, Nigrovic PA, Boilard E. Revealing the diversity of extracellular vesicles using high-dimensional flow cytometry analyses. Sci Rep 2016; 6: 35928
- 29 van der Pol E, Coumans FAW, Grootemaat AE. , et al. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost 2014; 12 (07) 1182-1192
- 30 Montoro-García S, Shantsila E, Orenes-Piñero E, Lozano ML, Lip GY. An innovative flow cytometric approach for small-size platelet microparticles: influence of calcium. Thromb Haemost 2012; 108 (02) 373-383
- 31 van der Pol E, de Rond L, Coumans FAW. , et al. Absolute sizing and label-free identification of extracellular vesicles by flow cytometry. Nanomedicine (Lond) 2018; 14 (03) 801-810
- 32 Robert S, Poncelet P, Lacroix R. , et al. Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?. J Thromb Haemost 2009; 7 (01) 190-197
- 33 Lacroix R, Judicone C, Poncelet P. , et al. Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol. J Thromb Haemost 2012; 10 (03) 437-446
- 34 Coumans FAW, Brisson AR, Buzas EI. , et al. Methodological guidelines to study extracellular vesicles. Circ Res 2017; 120 (10) 1632-1648
- 35 van der Pol E, Coumans FAW, Sturk A, Nieuwland R, van Leeuwen TG. Refractive index determination of nanoparticles in suspension using nanoparticle tracking analysis. Nano Lett 2014; 14 (11) 6195-6201
- 36 de Rond L, van der Pol E, Hau CM. , et al. Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry. Clin Chem 2018; 64 (04) 680-689
- 37 van der Pol E, Sturk A, van Leeuwen T, Nieuwland R, Coumans F. ; ISTH-SSC-VB Working group. Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation. J Thromb Haemost 2018; 16 (06) 1236-1245
- 38 Stoner SA, Duggan E, Condello D. , et al. High sensitivity flow cytometry of membrane vesicles. Cytometry A 2016; 89 (02) 196-206
- 39 Arraud N, Gounou C, Turpin D, Brisson AR. Fluorescence triggering: a general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry. Cytometry A 2016; 89 (02) 184-195
- 40 Leong HS, Podor TJ, Manocha B, Lewis JD. Validation of flow cytometric detection of platelet microparticles and liposomes by atomic force microscopy. J Thromb Haemost 2011; 9 (12) 2466-2476
- 41 Gray WD, Mitchell AJ, Searles CD. An accurate, precise method for general labeling of extracellular vesicles. MethodsX 2015; 2: 360-367
- 42 Bambace NM, Holmes CE. The platelet contribution to cancer progression. J Thromb Haemost 2011; 9 (02) 237-239
- 43 Ogura H, Kawasaki T, Tanaka H. , et al. Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. J Trauma 2001; 50 (05) 801-809
- 44 van Doormaal F, Kleinjan A, Berckmans RJ. , et al. Coagulation activation and microparticle-associated coagulant activity in cancer patients. An exploratory prospective study. Thromb Haemost 2012; 108 (01) 160-165
- 45 Helley D, Banu E, Bouziane A. , et al. Platelet microparticles: a potential predictive factor of survival in hormone-refractory prostate cancer patients treated with docetaxel-based chemotherapy. Eur Urol 2009; 56 (03) 479-484
- 46 Cointe S, Judicone C, Robert S. , et al. Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop. J Thromb Haemost 2017; 15 (01) 187-193
- 47 Andersen MN, Al-Karradi SNH, Kragstrup TW, Hokland M. Elimination of erroneous results in flow cytometry caused by antibody binding to FC receptors on human monocytes and macrophages. Cytometry A 2016; 89 (11) 1001-1009