Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

Estefanía Lozano-Andrés; Tina Van Den Broeck; Lili Wang; Majid Mehrpouyan; Ye Tian; Xiaomei Yan; Ger J A Arkesteijn; Marca H M Wauben

doi:10.1016/j.nano.2023.102720

Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

Estefanía Lozano-Andrés^*
, Tina Van Den Broeck
, Lili Wang
, Majid Mehrpouyan
, Ye Tian
, Xiaomei Yan
, Ger J A Arkesteijn
, Marca H M Wauben

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

Original language	English
Article number	102720
Number of pages	7
Journal	Nanomedicine: Nanotechnology, Biology, and Medicine
Volume	56
Early online date	23 Nov 2023
DOIs	https://doi.org/10.1016/j.nano.2023.102720
Publication status	Published - Feb 2024

Bibliographical note

Publisher Copyright:
© 2023 The Authors

Funding

This research is supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie (grant agreement No 722148 ) and by the National Natural Science Foundation of China ( 21934004 and 21627811 ). E. L. A. is supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 722148.

Funders	Funder number
H2020 Marie Skłodowska-Curie Actions	722148
National Natural Science Foundation of China	21627811, 21934004
Horizon 2020

Keywords

Extracellular vesicles
Flow cytometry
Fluorescence calibration
Nanoparticles
Standardization

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10.1016/j.nano.2023.102720Licence: CC BY

1-s2.0-S1549963423000710-mainFinal published version, 7.47 MBLicence: CC BY

Cite this

Lozano-Andrés, E., Van Den Broeck, T., Wang, L., Mehrpouyan, M., Tian, Y., Yan, X., Arkesteijn, G. J. A., & Wauben, M. H. M. (2024). Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry. Nanomedicine: Nanotechnology, Biology, and Medicine, 56, Article 102720. https://doi.org/10.1016/j.nano.2023.102720

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title = "Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry",

abstract = "Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.",

keywords = "Extracellular vesicles, Flow cytometry, Fluorescence calibration, Nanoparticles, Standardization",

author = "Estefan{\'i}a Lozano-Andr{\'e}s and \{Van Den Broeck\}, Tina and Lili Wang and Majid Mehrpouyan and Ye Tian and Xiaomei Yan and Arkesteijn, \{Ger J A\} and Wauben, \{Marca H M\}",

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doi = "10.1016/j.nano.2023.102720",

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Lozano-Andrés, E, Van Den Broeck, T, Wang, L, Mehrpouyan, M, Tian, Y, Yan, X, Arkesteijn, GJA & Wauben, MHM 2024, 'Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry', Nanomedicine: Nanotechnology, Biology, and Medicine, vol. 56, 102720. https://doi.org/10.1016/j.nano.2023.102720

Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry. / Lozano-Andrés, Estefanía; Van Den Broeck, Tina; Wang, Lili et al.
In: Nanomedicine: Nanotechnology, Biology, and Medicine, Vol. 56, 102720, 02.2024.

Research output: Contribution to journal › Article › Academic › peer-review

TY - JOUR

T1 - Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

AU - Lozano-Andrés, Estefanía

AU - Van Den Broeck, Tina

AU - Wang, Lili

AU - Mehrpouyan, Majid

AU - Tian, Ye

AU - Yan, Xiaomei

AU - Arkesteijn, Ger J A

AU - Wauben, Marca H M

PY - 2024/2

Y1 - 2024/2

N2 - Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

AB - Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

KW - Extracellular vesicles

KW - Flow cytometry

KW - Fluorescence calibration

KW - Nanoparticles

KW - Standardization

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DO - 10.1016/j.nano.2023.102720

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SN - 1549-9634

VL - 56

JO - Nanomedicine: Nanotechnology, Biology, and Medicine

JF - Nanomedicine: Nanotechnology, Biology, and Medicine

M1 - 102720

ER -

Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

Abstract

Bibliographical note

Funding

Keywords

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