title: Super-resolved calibration-free flow cytometric characterization of platelets and cell-derived microparticles in platelet-rich plasma.
abstract: Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward-versus-side-scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light-scattering profiles and side scattering from MPs, respectively. The developed two-stage method allowed accurate separation of PLTs and MPs in platelet-rich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light-scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs-median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument-independent quantitative information on MPs, which can be used in studies of various factors affecting MP population.
authors: Institute of Chemical Kinetics and Combustion SB RAS, 630090, Novosibirsk, Russia.
authors: Anastasiya I
authors: AI
authors: Konokhova
authors: Institute of Chemical Kinetics and Combustion SB RAS, 630090, Novosibirsk, Russia.|Novosibirsk State University, 630090, Novosibirsk, Russia.
authors: Darya N
authors: DN
authors: Chernova
authors: Institute of Chemical Kinetics and Combustion SB RAS, 630090, Novosibirsk, Russia.|Novosibirsk State University, 630090, Novosibirsk, Russia.
authors: Alexander E
authors: AE
authors: Moskalensky
authors: Institute of Chemical Kinetics and Combustion SB RAS, 630090, Novosibirsk, Russia.|Novosibirsk State Medical University, 630091, Novosibirsk, Russia.
authors: Dmitry I
authors: DI
authors: Strokotov
authors: Institute of Chemical Kinetics and Combustion SB RAS, 630090, Novosibirsk, Russia.|Novosibirsk State University, 630090, Novosibirsk, Russia.
authors: Maxim A
authors: MA
authors: Yurkin
authors: Institute of Chemical Kinetics and Combustion SB RAS, 630090, Novosibirsk, Russia.|Novosibirsk State University, 630090, Novosibirsk, Russia.
authors: Andrei V
authors: AV
authors: Chernyshev
authors: Institute of Chemical Kinetics and Combustion SB RAS, 630090, Novosibirsk, Russia.|Novosibirsk State University, 630090, Novosibirsk, Russia.|Novosibirsk State Medical University, 630091, Novosibirsk, Russia.
authors: Valeri P
authors: VP
authors: Maltsev
mesh_terms: D001792:Blood Platelets / Q000502:physiology*; D002138:Calibration; D055252:Cell-Derived Microparticles / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008027:Light; D053657:Platelet-Rich Plasma / Q000166:cytology; D012542:Scattering, Radiation
keywords: flow cytometry; inverse light-scattering problem; light scattering; microparticles; platelets; refractive index; vesicles