Application of high-sensitivity flow cytometry in combination with low-voltage scanning electron microscopy for characterization of nanosized objects during platelet concentrate storage

Федоров, А. В.; Кондратов, К. А.; Kishenko, V. V.; Mikhailovskii, Vladimir; Kudryavtsev, I. V.; Belyakova, M; Sidorkevich, Sergey; Вавилова, Т. В.; Kostareva, Anna; Сироткина, О. В.; Головкин, А. С.

doi:10.1080/09537104.2019.1599337

Cited by 20 publications

(11 citation statements)

References 44 publications

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“…Instrument calibration setup was performed using the Cytometry Sub-Micron Particle Size Reference Kit, Molecular probes by Life Technologies), and Megamix-Plus FSC and Megamix-Plus SSC (Biocytex, Marseille, France) containing FITC-labeled reference beads of various diameters. All control samples, detergent effectiveness treatments, and possible false-positive or false-negative controls, were performed in accordance with the previously published requirements [ 7 , 11 ] and as previously described by our group [ 10 , 12 ]. Stained sample dilution was performed in accordance of prevention of coincidence effects, as previously described [ 10 , 12 , 13 ].…”

Section: Methodsmentioning

confidence: 99%

The Significance of Phenotyping and Quantification of Plasma Extracellular Vesicles Levels Using High-Sensitivity Flow Cytometry during COVID-19 Treatment

Kudryavtsev

Kalinina

Bezrukikh

et al. 2021

Viruses

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New investigation results point to the potential participation of extracellular vesicles (EVs) in the pathogenesis of coronavirus infection, its progression, and mechanisms of the therapy effectiveness. This dictates the necessity to transfer scientific testing technologies to medical practice. Here, we demonstrated the method of phenotyping and quantitative analysis of plasma EVs based on differential centrifugation, immunostaining, and high-sensitivity multicolor flow cytometry. We used EV markers that were potentially associated with SARS-CoV-2 dissemination via vesicles and cell-origination markers, characterizing objects from different cell types that could influence clinical manifestation of COVID-19. Plasma levels of CD235a+ and CD14+ EVs in patients with moderate infection were significantly increased while CD8+ and CD19+ EVs were decreased comparing with HD. Patients with severe infection had lower levels of CD4+, CD19+, and CD146+ EVs than HD. These findings demonstrate that EV concentrations in COVID-19 are severity related. Moreover, the three-point dynamic assessment demonstrated significant loss of CD63+ and CD147+ plasma EVs. The used method can be a convenient tool for vital infection pathogenesis investigation and for COVID-19 diagnostics.

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Section: Methodsmentioning

confidence: 99%

The Significance of Phenotyping and Quantification of Plasma Extracellular Vesicles Levels Using High-Sensitivity Flow Cytometry during COVID-19 Treatment

Kudryavtsev

Kalinina

Bezrukikh

et al. 2021

Viruses

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“…This research aimed to use a SEM for the characterisation of objects in platelet concentrate storage, which required the use of highly sensitive equipment exceeding the capabilities of conventional optical microscopy to detect objects with dimensions ranging from 25 to 700 nm. Conventional SEMs require samples to be coated with a layer of conductive metal with a thickness comparable to that of small extracellular vesicles, however low-voltage SEM approaches used by researchers obviated the need for this layer, enabling the examination of objects smaller than single cells [ 48 , 49 ].…”

Section: Microscopic Imaging Technologymentioning

confidence: 99%

A review of microscopic cell imaging and neural network recognition for synergistic cyanobacteria identification and enumeration

et al. 2022

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Real-time cyanobacteria/algal monitoring is a valuable tool for early detection of harmful algal blooms, water treatment efficacy evaluation, and assists tailored water quality risk assessments by considering taxonomy and cell counts. This review evaluates and proposes a synergistic approach using neural network image recognition and microscopic imaging devices by first evaluating published literature for both imaging microscopes and image recognition. Quantitative phase imaging was considered the most promising of the investigated imaging techniques due to the provision of enhanced information relative to alternatives. This information provides significant value to image recognition neural networks, such as the convolutional neural networks discussed within this review. Considering published literature, a cyanobacteria monitoring system and corresponding image processing workflow using in situ sample collection buoys and on-shore sample processing was proposed. This system can be implemented using commercially available equipment to facilitate accurate, real-time water quality monitoring. Graphical abstract

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“…Analysis of EVs was performed using Cytoflex S (Beckman Coulter, USA) flow cytometer equipped with 405 nm (violet), 488 nm (blue), 638 nm (red) and 561 nm (yellow-green) lasers. The flow cytometer instrument setup was performed as described previously [31,32]. The use of a side scatter at the shorter wavelength of 405 nm remarkably lowered the size-specific sensitivity limit for polystyrene beads compared to that of the 488 nm light scatter which signal was attenuated by a neutral density filter in the CytoFlex S.…”

Section: High-sensitivity Flow Cytometrymentioning

confidence: 99%

Heterogeneity of the nucleic acid repertoire of plasma extracellular vesicles demonstrated using high‐sensitivity fluorescence‐activated sorting

Кондратов

Nikitin

Федоров

et al. 2020

J of Extracellular Vesicle

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The aim of this study was to investigate cell source-dependent nucleic acids repertoire of diverse subpopulations of plasma extracellular vesicles (EVs). Blood plasma from nine healthy volunteers was used for the analysis. Samples of EVs were obtained by differential centrifugation of plasma. The application of high-sensitivity fluorescence-activated vesicles sorting (hs-FAVS) using fluorophore-conjugated anti-CD41-FITC (Fluorescein isothiocyanate) and anti-CD235a-PE antibodies allowed the isolation of three subpopulations of EVs, namely CD41+ CD235a-, CD41-CD235a+ and CD41-CD235a dim. The high purity (>97%) of the sorted subpopulations was verified by highsensitivity flow cytometry. Presence of nanosized objects in sorted samples was confirmed by combination of low-voltage scanning electron microscopy and dynamic light scattering. The amount of material in sorted samples was enough to perform Quantitative polymerase chain reaction (qPCR)-based nucleic acid quantification. The most prominent differences in the nucleic acid repertoire were noted between CD41+ CD235-vs. CD41-CD235a+ vesicles: the former contained significantly (p = 0.004) higher amount of mitochondrial DNA, and platelet enriched miR-21-5p (4-fold), miR-223-3p (38-fold) and miR-199a-3p (187-fold), but lower amount of erythrocyte enriched miR-451a (90-fold). CD41-CD235a+ and CD41-CD235a dim vesicles differed in levels of miR-451a (p = 0.016) and miR-21-5p (p = 0.031). Nuclear DNA was below the limit of detection in all EV subpopulations. The hs-FCM-based determination of the number of sorted EVs allowed the calculation of per single-event miRNA concentrations. It was demonstrated that the most abundant marker in CD41+ CD235a-subpopulation was miR-223-3p, reaching 38.2 molecules per event. In the CD41-CD235+ subpopulation, the most abundant marker was miR-451a, reaching 24.7 molecules per event. Taken together, our findings indicate that erythrocyte-and platelet-derived EVs carry different repertoires of nucleic acids, which were similar to the composition of their cellular sources.

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Application of high-sensitivity flow cytometry in combination with low-voltage scanning electron microscopy for characterization of nanosized objects during platelet concentrate storage

Cited by 20 publications

References 44 publications

The Significance of Phenotyping and Quantification of Plasma Extracellular Vesicles Levels Using High-Sensitivity Flow Cytometry during COVID-19 Treatment

The Significance of Phenotyping and Quantification of Plasma Extracellular Vesicles Levels Using High-Sensitivity Flow Cytometry during COVID-19 Treatment

A review of microscopic cell imaging and neural network recognition for synergistic cyanobacteria identification and enumeration

Heterogeneity of the nucleic acid repertoire of plasma extracellular vesicles demonstrated using high‐sensitivity fluorescence‐activated sorting

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