S F W M Libregts1, G J A Arkesteijn1, A Németh2, E N M Nolte-'t Hoen1, M H M Wauben1. 1. Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands. 2. Department of Genetics, Cell- and Immunobiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary.
Abstract
Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. SUMMARY: Background Extracellular vesicles (EVs) in plasma are increasingly being recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets with a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria, as it allows multiparameter analysis of individual EVs. However, analysis of plasma EVs is challenging, because of their size and heterogeneity, and the presence of other submicrometer-sized particles in plasma that could interfere with EV analysis. Objectives To explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labeled or differently labeled EVs and particles. Methods Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry with fluorescence threshold triggering. Results We found that light scatter detection of low-abundance or rare EV subsets during fluorescence threshold triggering was severely affected by particles of non-interest, owing to coincidence and swarming. Importantly, we show that interfering particles labeled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. Conclusions We demonstrate how particles of non-interest in plasma can impact on the light scatter and fluorescence detection of low-abundance EVs of interest during fluorescence-based flow cytometric analysis, and provide a means to prevent erroneous data interpretation.
Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. SUMMARY: Background Extracellular vesicles (EVs) in plasma are increasingly being recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets with a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria, as it allows multiparameter analysis of individual EVs. However, analysis of plasma EVs is challenging, because of their size and heterogeneity, and the presence of other submicrometer-sized particles in plasma that could interfere with EV analysis. Objectives To explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labeled or differently labeled EVs and particles. Methods Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry with fluorescence threshold triggering. Results We found that light scatter detection of low-abundance or rare EV subsets during fluorescence threshold triggering was severely affected by particles of non-interest, owing to coincidence and swarming. Importantly, we show that interfering particles labeled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. Conclusions We demonstrate how particles of non-interest in plasma can impact on the light scatter and fluorescence detection of low-abundance EVs of interest during fluorescence-based flow cytometric analysis, and provide a means to prevent erroneous data interpretation.
Authors: Kyle Fraser; Ala Jo; Jimmy Giedt; Claudio Vinegoni; Katherine S Yang; Pierepaolo Peruzzi; E Antonio Chiocca; Xandra O Breakefield; Hakho Lee; Ralph Weissleder Journal: Neuro Oncol Date: 2019-05-06 Impact factor: 12.300
Authors: Tom A P Driedonks; Sanne Mol; Sanne de Bruin; Anna-Linda Peters; Xiaogang Zhang; Marthe F S Lindenbergh; Boukje M Beuger; Anne-Marieke D van Stalborch; Thom Spaan; Esther C de Jong; Erhard van der Vries; Coert Margadant; Robin van Bruggen; Alexander P J Vlaar; Tom Groot Kormelink; Esther N M Nolte-'t Hoen Journal: J Extracell Vesicles Date: 2020-05-26
Authors: Clotilde Théry; Kenneth W Witwer; Elena Aikawa; Maria Jose Alcaraz; Johnathon D Anderson; Ramaroson Andriantsitohaina; Anna Antoniou; Tanina Arab; Fabienne Archer; Georgia K Atkin-Smith; D Craig Ayre; Jean-Marie Bach; Daniel Bachurski; Hossein Baharvand; Leonora Balaj; Shawn Baldacchino; Natalie N Bauer; Amy A Baxter; Mary Bebawy; Carla Beckham; Apolonija Bedina Zavec; Abderrahim Benmoussa; Anna C Berardi; Paolo Bergese; Ewa Bielska; Cherie Blenkiron; Sylwia Bobis-Wozowicz; Eric Boilard; Wilfrid Boireau; Antonella Bongiovanni; Francesc E Borràs; Steffi Bosch; Chantal M Boulanger; Xandra Breakefield; Andrew M Breglio; Meadhbh Á Brennan; David R Brigstock; Alain Brisson; Marike Ld Broekman; Jacqueline F Bromberg; Paulina Bryl-Górecka; Shilpa Buch; Amy H Buck; Dylan Burger; Sara Busatto; Dominik Buschmann; Benedetta Bussolati; Edit I Buzás; James Bryan Byrd; Giovanni Camussi; David Rf Carter; Sarah Caruso; Lawrence W Chamley; Yu-Ting Chang; Chihchen Chen; Shuai Chen; Lesley Cheng; Andrew R Chin; Aled Clayton; Stefano P Clerici; Alex Cocks; Emanuele Cocucci; Robert J Coffey; Anabela Cordeiro-da-Silva; Yvonne Couch; Frank Aw Coumans; Beth Coyle; Rossella Crescitelli; Miria Ferreira Criado; Crislyn D'Souza-Schorey; Saumya Das; Amrita Datta Chaudhuri; Paola de Candia; Eliezer F De Santana; Olivier De Wever; Hernando A Del Portillo; Tanguy Demaret; Sarah Deville; Andrew Devitt; Bert Dhondt; Dolores Di Vizio; Lothar C Dieterich; Vincenza Dolo; Ana Paula Dominguez Rubio; Massimo Dominici; Mauricio R Dourado; Tom Ap Driedonks; Filipe V Duarte; Heather M Duncan; Ramon M Eichenberger; Karin Ekström; Samir El Andaloussi; Celine Elie-Caille; Uta Erdbrügger; Juan M Falcón-Pérez; Farah Fatima; Jason E Fish; Miguel Flores-Bellver; András Försönits; Annie Frelet-Barrand; Fabia Fricke; Gregor Fuhrmann; Susanne Gabrielsson; Ana Gámez-Valero; Chris Gardiner; Kathrin Gärtner; Raphael Gaudin; Yong Song Gho; Bernd Giebel; Caroline Gilbert; Mario Gimona; Ilaria Giusti; Deborah Ci Goberdhan; André Görgens; Sharon M Gorski; David W Greening; Julia Christina Gross; Alice Gualerzi; Gopal N Gupta; Dakota Gustafson; Aase Handberg; Reka A Haraszti; Paul Harrison; Hargita Hegyesi; An Hendrix; Andrew F Hill; Fred H Hochberg; Karl F Hoffmann; Beth Holder; Harry Holthofer; Baharak Hosseinkhani; Guoku Hu; Yiyao Huang; Veronica Huber; Stuart Hunt; Ahmed Gamal-Eldin Ibrahim; Tsuneya Ikezu; Jameel M Inal; Mustafa Isin; Alena Ivanova; Hannah K Jackson; Soren Jacobsen; Steven M Jay; Muthuvel Jayachandran; Guido Jenster; Lanzhou Jiang; Suzanne M Johnson; Jennifer C Jones; Ambrose Jong; Tijana Jovanovic-Talisman; Stephanie Jung; Raghu Kalluri; Shin-Ichi Kano; Sukhbir Kaur; Yumi Kawamura; Evan T Keller; Delaram Khamari; Elena Khomyakova; Anastasia Khvorova; Peter Kierulf; Kwang Pyo Kim; Thomas Kislinger; Mikael Klingeborn; David J Klinke; Miroslaw Kornek; Maja M Kosanović; Árpád Ferenc Kovács; Eva-Maria Krämer-Albers; Susanne Krasemann; Mirja Krause; Igor V Kurochkin; Gina D Kusuma; Sören Kuypers; Saara Laitinen; Scott M Langevin; Lucia R Languino; Joanne Lannigan; Cecilia Lässer; Louise C Laurent; Gregory Lavieu; Elisa Lázaro-Ibáñez; Soazig Le Lay; Myung-Shin Lee; Yi Xin Fiona Lee; Debora S Lemos; Metka Lenassi; Aleksandra Leszczynska; Isaac Ts Li; Ke Liao; Sten F Libregts; Erzsebet Ligeti; Rebecca Lim; Sai Kiang Lim; Aija Linē; Karen Linnemannstöns; Alicia Llorente; Catherine A Lombard; Magdalena J Lorenowicz; Ákos M Lörincz; Jan Lötvall; Jason Lovett; Michelle C Lowry; Xavier Loyer; Quan Lu; Barbara Lukomska; Taral R Lunavat; Sybren Ln Maas; Harmeet Malhi; Antonio Marcilla; Jacopo Mariani; Javier Mariscal; Elena S Martens-Uzunova; Lorena Martin-Jaular; M Carmen Martinez; Vilma Regina Martins; Mathilde Mathieu; Suresh Mathivanan; Marco Maugeri; Lynda K McGinnis; Mark J McVey; David G Meckes; Katie L Meehan; Inge Mertens; Valentina R Minciacchi; Andreas Möller; Malene Møller Jørgensen; Aizea Morales-Kastresana; Jess Morhayim; François Mullier; Maurizio Muraca; Luca Musante; Veronika Mussack; Dillon C Muth; Kathryn H Myburgh; Tanbir Najrana; Muhammad Nawaz; Irina Nazarenko; Peter Nejsum; Christian Neri; Tommaso Neri; Rienk Nieuwland; Leonardo Nimrichter; John P Nolan; Esther Nm Nolte-'t Hoen; Nicole Noren Hooten; Lorraine O'Driscoll; Tina O'Grady; Ana O'Loghlen; Takahiro Ochiya; Martin Olivier; Alberto Ortiz; Luis A Ortiz; Xabier Osteikoetxea; Ole Østergaard; Matias Ostrowski; Jaesung Park; D Michiel Pegtel; Hector Peinado; Francesca Perut; Michael W Pfaffl; Donald G Phinney; Bartijn Ch Pieters; Ryan C Pink; David S Pisetsky; Elke Pogge von Strandmann; Iva Polakovicova; Ivan Kh Poon; Bonita H Powell; Ilaria Prada; Lynn Pulliam; Peter Quesenberry; Annalisa Radeghieri; Robert L Raffai; Stefania Raimondo; Janusz Rak; Marcel I Ramirez; Graça Raposo; Morsi S Rayyan; Neta Regev-Rudzki; Franz L Ricklefs; Paul D Robbins; David D Roberts; Silvia C Rodrigues; Eva Rohde; Sophie Rome; Kasper Ma Rouschop; Aurelia Rughetti; Ashley E Russell; Paula Saá; Susmita Sahoo; Edison Salas-Huenuleo; Catherine Sánchez; Julie A Saugstad; Meike J Saul; Raymond M Schiffelers; Raphael Schneider; Tine Hiorth Schøyen; Aaron Scott; Eriomina Shahaj; Shivani Sharma; Olga Shatnyeva; Faezeh Shekari; Ganesh Vilas Shelke; Ashok K Shetty; Kiyotaka Shiba; Pia R-M Siljander; Andreia M Silva; Agata Skowronek; Orman L Snyder; Rodrigo Pedro Soares; Barbara W Sódar; Carolina Soekmadji; Javier Sotillo; Philip D Stahl; Willem Stoorvogel; Shannon L Stott; Erwin F Strasser; Simon Swift; Hidetoshi Tahara; Muneesh Tewari; Kate Timms; Swasti Tiwari; Rochelle Tixeira; Mercedes Tkach; Wei Seong Toh; Richard Tomasini; Ana Claudia Torrecilhas; Juan Pablo Tosar; Vasilis Toxavidis; Lorena Urbanelli; Pieter Vader; Bas Wm van Balkom; Susanne G van der Grein; Jan Van Deun; Martijn Jc van Herwijnen; Kendall Van Keuren-Jensen; Guillaume van Niel; Martin E van Royen; Andre J van Wijnen; M Helena Vasconcelos; Ivan J Vechetti; Tiago D Veit; Laura J Vella; Émilie Velot; Frederik J Verweij; Beate Vestad; Jose L Viñas; Tamás Visnovitz; Krisztina V Vukman; Jessica Wahlgren; Dionysios C Watson; Marca Hm Wauben; Alissa Weaver; Jason P Webber; Viktoria Weber; Ann M Wehman; Daniel J Weiss; Joshua A Welsh; Sebastian Wendt; Asa M Wheelock; Zoltán Wiener; Leonie Witte; Joy Wolfram; Angeliki Xagorari; Patricia Xander; Jing Xu; Xiaomei Yan; María Yáñez-Mó; Hang Yin; Yuana Yuana; Valentina Zappulli; Jana Zarubova; Vytautas Žėkas; Jian-Ye Zhang; Zezhou Zhao; Lei Zheng; Alexander R Zheutlin; Antje M Zickler; Pascale Zimmermann; Angela M Zivkovic; Davide Zocco; Ewa K Zuba-Surma Journal: J Extracell Vesicles Date: 2018-11-23
Authors: Estefanía Lozano-Andrés; Sten F Libregts; Victor Toribio; Félix Royo; Sara Morales; Soraya López-Martín; Mar Valés-Gómez; Hugh T Reyburn; Juan Manuel Falcón-Pérez; Marca H Wauben; Manuel Soto; María Yáñez-Mó Journal: J Extracell Vesicles Date: 2019-03-04
Authors: A Rodallec; C Franco; S Robert; G Sicard; S Giacometti; B Lacarelle; F Bouquet; A Savina; R Lacroix; F Dignat-George; J Ciccolini; P Poncelet; R Fanciullino Journal: Sci Rep Date: 2020-03-05 Impact factor: 4.379