An easy and reliable whole blood freezing method for flow cytometry immuno-phenotyping and functional analyses.
flow cytometry
immune monitoring
immunology
peripheral blood
Journal
Cytometry. Part B, Clinical cytometry
ISSN: 1552-4957
Titre abrégé: Cytometry B Clin Cytom
Pays: United States
ID NLM: 101235690
Informations de publication
Date de publication:
11 2021
11 2021
Historique:
revised:
01
12
2020
received:
12
08
2020
accepted:
26
01
2021
pubmed:
6
2
2021
medline:
17
3
2022
entrez:
5
2
2021
Statut:
ppublish
Résumé
Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Furthermore, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent to which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods. In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC. Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells, and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa). In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.
Sections du résumé
BACKGROUND
Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Furthermore, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent to which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods.
METHODS
In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.
RESULTS
Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells, and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa).
CONCLUSIONS
In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.
Identifiants
pubmed: 33544978
doi: 10.1002/cyto.b.21994
doi:
Types de publication
Journal Article
Multicenter Study
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
652-665Subventions
Organisme : "Investissements d'Avenir" program through the "Agence Nationale de la Recherche"
ID : ANR11-LABX-0016-01
Informations de copyright
© 2021 International Clinical Cytometry Society.
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