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

Anton Fedorov, Kirill Kondratov, Vasilii Kishenko, Vladimir Mikhailovskii, Igor Kudryavtsev, Margarita Belyakova, Sergey Sidorkevich, Tatyana Vavilova, Anna Kostareva, Olga Sirotkina, Alexey Golovkin

Результат исследований: Научные публикации в периодических изданияхстатья

Выдержка

Platelet concentrates are used in clinic for therapy and prophylaxis of conditions associated with platelet deficiency or malfunction. The characteristics of platelet concentrates gradually change during pretransfusion storage, affecting their clinical effectiveness and the risk of adverse transfusion reactions. The presence of platelet-derived membrane vesicles is an important characteristic of platelet concentrates. Due to their functionality, changes in the number and molecular compositions of platelet-derived vesicles have major effects on the clinical properties of platelet preparations. The existence of different subpopulations of membrane vesicles requires analytical methods capable of providing information at the individual vesicle level. Such methods include flow cytometry and electron microscopy. However, conventional flow cytometry has certain limitations, since the diameters of many platelet-derived membrane vesicles are smaller than its detection limit. The use of classical scanning electron microscopy is also limited due to the requirement for coating with a layer of conductive material, which impedes the detection of small extracellular vesicles. Here, a combination of high-sensitivity flow cytometry and low-voltage scanning electron microscopy was used to increase sensitivity and resolution in the detection of nanosized objects present in platelet concentrates during storage. Apheresis platelet concentrates from eight healthy adult donors were investigated on days 2 and 7 of storage. Fractions of nanosized objects were obtained by differential centrifugation. Fluorophore-conjugated antibodies were used to detect marker-positive vesicles derived from platelets (CD41), red blood cells (CD235a), leukocytes (CD45), and endothelial cells (VEGFR2). Near-spherical objects with diameters ranging from 25 to 700 nm were observed by low-voltage scanning electron microscopy in platelet concentrates and its fractions. On day 7 of storage, objects with diameters of less than 100 nm were attached to and clustered near the terminal ends of pseudopod-like projections. High-sensitivity flow cytometry showed that during storage numbers of CD41(pos) vesicles elevated more than fivefold and numbers of marker-negative nanosized objects, which did not carry any of the investigated cell type-specific markers elevated more than twofold. Major changes in both CD41(pos) vesicles and marker-negative nanosized objects abundances were observed for objects with diameters around 100 nm bead equivalents. Overall, these results emphasized the importance of application of high-sensitivity methods for monitoring the characteristics of cell-derived nanosized objects during platelet concentrate storage.

Язык оригиналаанглийский
ЖурналPlatelets
DOI
СостояниеОпубликовано - 1 янв 2019

Отпечаток

Electron Scanning Microscopy
Flow Cytometry
Blood Platelets
Membranes
Blood Component Removal
Pseudopodia
Centrifugation
Limit of Detection
Electron Microscopy
Leukocytes
Endothelial Cells
Erythrocytes
Tissue Donors

Предметные области Scopus

  • Гематология

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Fedorov, Anton ; Kondratov, Kirill ; Kishenko, Vasilii ; Mikhailovskii, Vladimir ; Kudryavtsev, Igor ; Belyakova, Margarita ; Sidorkevich, Sergey ; Vavilova, Tatyana ; Kostareva, Anna ; Sirotkina, Olga ; Golovkin, Alexey. / Application of high-sensitivity flow cytometry in combination with low-voltage scanning electron microscopy for characterization of nanosized objects during platelet concentrate storage. В: Platelets. 2019.
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abstract = "Platelet concentrates are used in clinic for therapy and prophylaxis of conditions associated with platelet deficiency or malfunction. The characteristics of platelet concentrates gradually change during pretransfusion storage, affecting their clinical effectiveness and the risk of adverse transfusion reactions. The presence of platelet-derived membrane vesicles is an important characteristic of platelet concentrates. Due to their functionality, changes in the number and molecular compositions of platelet-derived vesicles have major effects on the clinical properties of platelet preparations. The existence of different subpopulations of membrane vesicles requires analytical methods capable of providing information at the individual vesicle level. Such methods include flow cytometry and electron microscopy. However, conventional flow cytometry has certain limitations, since the diameters of many platelet-derived membrane vesicles are smaller than its detection limit. The use of classical scanning electron microscopy is also limited due to the requirement for coating with a layer of conductive material, which impedes the detection of small extracellular vesicles. Here, a combination of high-sensitivity flow cytometry and low-voltage scanning electron microscopy was used to increase sensitivity and resolution in the detection of nanosized objects present in platelet concentrates during storage. Apheresis platelet concentrates from eight healthy adult donors were investigated on days 2 and 7 of storage. Fractions of nanosized objects were obtained by differential centrifugation. Fluorophore-conjugated antibodies were used to detect marker-positive vesicles derived from platelets (CD41), red blood cells (CD235a), leukocytes (CD45), and endothelial cells (VEGFR2). Near-spherical objects with diameters ranging from 25 to 700 nm were observed by low-voltage scanning electron microscopy in platelet concentrates and its fractions. On day 7 of storage, objects with diameters of less than 100 nm were attached to and clustered near the terminal ends of pseudopod-like projections. High-sensitivity flow cytometry showed that during storage numbers of CD41(pos) vesicles elevated more than fivefold and numbers of marker-negative nanosized objects, which did not carry any of the investigated cell type-specific markers elevated more than twofold. Major changes in both CD41(pos) vesicles and marker-negative nanosized objects abundances were observed for objects with diameters around 100 nm bead equivalents. Overall, these results emphasized the importance of application of high-sensitivity methods for monitoring the characteristics of cell-derived nanosized objects during platelet concentrate storage.",
keywords = "High-sensitivity flow cytometry, low-voltage scanning electron microscopy, membrane vesicles, nanosized objects, storage of platelet concentrate",
author = "Anton Fedorov and Kirill Kondratov and Vasilii Kishenko and Vladimir Mikhailovskii and Igor Kudryavtsev and Margarita Belyakova and Sergey Sidorkevich and Tatyana Vavilova and Anna Kostareva and Olga Sirotkina and Alexey Golovkin",
year = "2019",
month = "1",
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journal = "Platelets",
issn = "0953-7104",
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Fedorov, A, Kondratov, K, Kishenko, V, Mikhailovskii, V, Kudryavtsev, I, Belyakova, M, Sidorkevich, S, Vavilova, T, Kostareva, A, Sirotkina, O & Golovkin, A 2019, 'Application of high-sensitivity flow cytometry in combination with low-voltage scanning electron microscopy for characterization of nanosized objects during platelet concentrate storage', Platelets. https://doi.org/10.1080/09537104.2019.1599337

Application of high-sensitivity flow cytometry in combination with low-voltage scanning electron microscopy for characterization of nanosized objects during platelet concentrate storage. / Fedorov, Anton; Kondratov, Kirill; Kishenko, Vasilii; Mikhailovskii, Vladimir; Kudryavtsev, Igor; Belyakova, Margarita; Sidorkevich, Sergey; Vavilova, Tatyana; Kostareva, Anna; Sirotkina, Olga; Golovkin, Alexey.

В: Platelets, 01.01.2019.

Результат исследований: Научные публикации в периодических изданияхстатья

TY - JOUR

T1 - Application of high-sensitivity flow cytometry in combination with low-voltage scanning electron microscopy for characterization of nanosized objects during platelet concentrate storage

AU - Fedorov, Anton

AU - Kondratov, Kirill

AU - Kishenko, Vasilii

AU - Mikhailovskii, Vladimir

AU - Kudryavtsev, Igor

AU - Belyakova, Margarita

AU - Sidorkevich, Sergey

AU - Vavilova, Tatyana

AU - Kostareva, Anna

AU - Sirotkina, Olga

AU - Golovkin, Alexey

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Platelet concentrates are used in clinic for therapy and prophylaxis of conditions associated with platelet deficiency or malfunction. The characteristics of platelet concentrates gradually change during pretransfusion storage, affecting their clinical effectiveness and the risk of adverse transfusion reactions. The presence of platelet-derived membrane vesicles is an important characteristic of platelet concentrates. Due to their functionality, changes in the number and molecular compositions of platelet-derived vesicles have major effects on the clinical properties of platelet preparations. The existence of different subpopulations of membrane vesicles requires analytical methods capable of providing information at the individual vesicle level. Such methods include flow cytometry and electron microscopy. However, conventional flow cytometry has certain limitations, since the diameters of many platelet-derived membrane vesicles are smaller than its detection limit. The use of classical scanning electron microscopy is also limited due to the requirement for coating with a layer of conductive material, which impedes the detection of small extracellular vesicles. Here, a combination of high-sensitivity flow cytometry and low-voltage scanning electron microscopy was used to increase sensitivity and resolution in the detection of nanosized objects present in platelet concentrates during storage. Apheresis platelet concentrates from eight healthy adult donors were investigated on days 2 and 7 of storage. Fractions of nanosized objects were obtained by differential centrifugation. Fluorophore-conjugated antibodies were used to detect marker-positive vesicles derived from platelets (CD41), red blood cells (CD235a), leukocytes (CD45), and endothelial cells (VEGFR2). Near-spherical objects with diameters ranging from 25 to 700 nm were observed by low-voltage scanning electron microscopy in platelet concentrates and its fractions. On day 7 of storage, objects with diameters of less than 100 nm were attached to and clustered near the terminal ends of pseudopod-like projections. High-sensitivity flow cytometry showed that during storage numbers of CD41(pos) vesicles elevated more than fivefold and numbers of marker-negative nanosized objects, which did not carry any of the investigated cell type-specific markers elevated more than twofold. Major changes in both CD41(pos) vesicles and marker-negative nanosized objects abundances were observed for objects with diameters around 100 nm bead equivalents. Overall, these results emphasized the importance of application of high-sensitivity methods for monitoring the characteristics of cell-derived nanosized objects during platelet concentrate storage.

AB - Platelet concentrates are used in clinic for therapy and prophylaxis of conditions associated with platelet deficiency or malfunction. The characteristics of platelet concentrates gradually change during pretransfusion storage, affecting their clinical effectiveness and the risk of adverse transfusion reactions. The presence of platelet-derived membrane vesicles is an important characteristic of platelet concentrates. Due to their functionality, changes in the number and molecular compositions of platelet-derived vesicles have major effects on the clinical properties of platelet preparations. The existence of different subpopulations of membrane vesicles requires analytical methods capable of providing information at the individual vesicle level. Such methods include flow cytometry and electron microscopy. However, conventional flow cytometry has certain limitations, since the diameters of many platelet-derived membrane vesicles are smaller than its detection limit. The use of classical scanning electron microscopy is also limited due to the requirement for coating with a layer of conductive material, which impedes the detection of small extracellular vesicles. Here, a combination of high-sensitivity flow cytometry and low-voltage scanning electron microscopy was used to increase sensitivity and resolution in the detection of nanosized objects present in platelet concentrates during storage. Apheresis platelet concentrates from eight healthy adult donors were investigated on days 2 and 7 of storage. Fractions of nanosized objects were obtained by differential centrifugation. Fluorophore-conjugated antibodies were used to detect marker-positive vesicles derived from platelets (CD41), red blood cells (CD235a), leukocytes (CD45), and endothelial cells (VEGFR2). Near-spherical objects with diameters ranging from 25 to 700 nm were observed by low-voltage scanning electron microscopy in platelet concentrates and its fractions. On day 7 of storage, objects with diameters of less than 100 nm were attached to and clustered near the terminal ends of pseudopod-like projections. High-sensitivity flow cytometry showed that during storage numbers of CD41(pos) vesicles elevated more than fivefold and numbers of marker-negative nanosized objects, which did not carry any of the investigated cell type-specific markers elevated more than twofold. Major changes in both CD41(pos) vesicles and marker-negative nanosized objects abundances were observed for objects with diameters around 100 nm bead equivalents. Overall, these results emphasized the importance of application of high-sensitivity methods for monitoring the characteristics of cell-derived nanosized objects during platelet concentrate storage.

KW - High-sensitivity flow cytometry

KW - low-voltage scanning electron microscopy

KW - membrane vesicles

KW - nanosized objects

KW - storage of platelet concentrate

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U2 - 10.1080/09537104.2019.1599337

DO - 10.1080/09537104.2019.1599337

M3 - Article

AN - SCOPUS:85064479543

JO - Platelets

JF - Platelets

SN - 0953-7104

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