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Licensed Unlicensed Requires Authentication Published by De Gruyter January 27, 2022

Delays during PBMC isolation have a moderate effect on yield, but severly compromise cell viability

Tanja Golke, Patrick Mucher, Patricia Schmidt, Astrid Radakovics, Manuela Repl, Philipp Hofer, Thomas Perkmann, Martina Fondi, Klaus G. Schmetterer and Helmuth Haslacher ORCID logo

Abstract

Objectives

Peripheral blood mononuclear cells (PBMCs) are a versatile material for clinical routine as well as for research projects. However, their isolation via density gradient centrifugation is still time-consuming. When samples are taken beyond usual laboratory handling times, it may sometimes be necessary to pause the isolation process. Our aim was to evaluate the impact of delays up to 48 h after the density gradient centrifugation on PBMC yield, purity and viability.

Methods

PBMCs were isolated from samples of 20 donors, either with BD Vacutainer CPT tubes (CPT) or with the standard Ficoll method. Isolation was paused after initial density gradient centrifugation for 0, 24, or 48 h. PBMC yield (% output/input), purity (% PBMCs/total cells) and viability (% Annexin V-/propidium iodide-) were compared.

Results

The yield did not change significantly over time when CPT were used (55%/52%/47%), but did after isolation with the standard method (62%/40%[p<0.0001]/53%[p<0.01]). Purity was marginally affected if CPT were used (95%/93%[p=n.s./92%[p<0.05] vs. 97% for all time points with standard method). Whereas viable PBMCs decreased steadily for CPT isolates (62%/51%[p<0.001]/36%[p<0.0001]), after standard Ficoll gradient isolation, cell apoptosis was more pronounced already after 24 h delay, and viability did not further decrease after 48 h (64%/44%[p<0.0001]/40%[p<0.0001]).

Conclusions

In conclusion, our findings suggest that while post-centrifugation delays ≥24 h might have only a minor effect on cell yield and purity, their impact on cell viability is substantial, even when CPT are used.


Corresponding author: Priv. Doz. Mag. DDr. Helmuth Haslacher, BSc, BA, Department of Laboratory Medicine, Medical University of Vienna, Waehringer Guertel 18-20 1090 Vienna, Austria, Phone: +43 1 40400 53190, Fax: +43 1 40495 15547, E-mail:
Tanja Golke, Patrick Mucher, Klaus G. Schmetterer and Helmuth Haslacher contributed equally to this work.

Acknowledgments

We thank all participants for their valuable donations. The MedUni Wien Biobank is part of the Austrian biobanking consortium BBMRI. at.

  1. Research funding: None declared.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study (ethics committee of the Medical University of Vienna, No. 404/2012).

  5. Ethical approval: The Ethics Committee of the Medical University of Vienna reviewed and approved this study (No. 1022/2021)

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Received: 2022-01-02
Accepted: 2022-01-19
Published Online: 2022-01-27
Published in Print: 2022-04-26

© 2022 Walter de Gruyter GmbH, Berlin/Boston

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