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Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility

Sofie Van Dorpe (UGent) , Lien Lippens (UGent) , Robin Boiy (UGent) , Cláudio Pinheiro (UGent) , Glenn Vergauwen (UGent) , Pekka Rappu, Ilkka Miinalainen, Philippe Tummers (UGent) , Hannelore Denys (UGent) , Olivier De Wever (UGent) , et al.
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Abstract
Background Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers.Results Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine.Conclusions In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.
Keywords
Extracellular vesicles, Separation, Density gradient centrifugation, Blood, Urine, Automation, Proteomics, PROTEOME, EXOSOMES, BLOOD

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MLA
Van Dorpe, Sofie, et al. “Integrating Automated Liquid Handling in the Separation Workflow of Extracellular Vesicles Enhances Specificity and Reproducibility.” JOURNAL OF NANOBIOTECHNOLOGY, vol. 21, no. 1, BMC, 2023, doi:10.1186/s12951-023-01917-z.
APA
Van Dorpe, S., Lippens, L., Boiy, R., Pinheiro, C., Vergauwen, G., Rappu, P., … Hendrix, A. (2023). Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility. JOURNAL OF NANOBIOTECHNOLOGY, 21(1). https://doi.org/10.1186/s12951-023-01917-z
Chicago author-date
Van Dorpe, Sofie, Lien Lippens, Robin Boiy, Cláudio Pinheiro, Glenn Vergauwen, Pekka Rappu, Ilkka Miinalainen, et al. 2023. “Integrating Automated Liquid Handling in the Separation Workflow of Extracellular Vesicles Enhances Specificity and Reproducibility.” JOURNAL OF NANOBIOTECHNOLOGY 21 (1). https://doi.org/10.1186/s12951-023-01917-z.
Chicago author-date (all authors)
Van Dorpe, Sofie, Lien Lippens, Robin Boiy, Cláudio Pinheiro, Glenn Vergauwen, Pekka Rappu, Ilkka Miinalainen, Philippe Tummers, Hannelore Denys, Olivier De Wever, and An Hendrix. 2023. “Integrating Automated Liquid Handling in the Separation Workflow of Extracellular Vesicles Enhances Specificity and Reproducibility.” JOURNAL OF NANOBIOTECHNOLOGY 21 (1). doi:10.1186/s12951-023-01917-z.
Vancouver
1.
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, et al. Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility. JOURNAL OF NANOBIOTECHNOLOGY. 2023;21(1).
IEEE
[1]
S. Van Dorpe et al., “Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility,” JOURNAL OF NANOBIOTECHNOLOGY, vol. 21, no. 1, 2023.
@article{01HB68ZZ6A72QQXKXAXNCNCRVZ,
  abstract     = {{Background Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers.Results Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine.Conclusions In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.}},
  articleno    = {{157}},
  author       = {{Van Dorpe, Sofie and Lippens, Lien and Boiy, Robin and Pinheiro, Cláudio and Vergauwen, Glenn and  Rappu, Pekka and  Miinalainen, Ilkka and Tummers, Philippe and Denys, Hannelore and De Wever, Olivier and Hendrix, An}},
  issn         = {{1477-3155}},
  journal      = {{JOURNAL OF NANOBIOTECHNOLOGY}},
  keywords     = {{Extracellular vesicles,Separation,Density gradient centrifugation,Blood,Urine,Automation,Proteomics,PROTEOME,EXOSOMES,BLOOD}},
  language     = {{eng}},
  number       = {{1}},
  pages        = {{16}},
  publisher    = {{BMC}},
  title        = {{Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility}},
  url          = {{http://doi.org/10.1186/s12951-023-01917-z}},
  volume       = {{21}},
  year         = {{2023}},
}

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