An easy and reliable whole blood freezing method for flow cytometry immuno‐phenotyping and functional analyses

Abstract

Background: Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by 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.

The cytokinesis-block micronucleus assay for cryopreserved whole blood

“…The highest viability and functionality of PBMCs was also achieved when cryopreserved at low temperatures (liquid nitrogen) and thawed by the slow addition of prewarmed washing medium (Fowke et al 2000;Ramachandran et al 2012;Hønge et al 2017). Despite reduced absolute counts of leukocytes in thawed whole blood samples, flow cytometric analysis revealed that the frequency of cell subtypes recovered from A c c e p t e d M a n u s c r i p t cryopreserved whole blood samples were in agreement with those obtained from fresh whole blood samples (Langenskiöld et al 2018;Verschoor & Kohli 2018;Braudeau et al 2021). In addition, the frequency of T cell sub populations in thawed whole blood samples were very similar to those in thawed isolated PBMC samples (Alam et al 2012).…”

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2-specific T-cells

“…Additionally, further optimisations should be performed to improve the standardisation and flexibility of the proposed SARS-COV-2-specific whole blood assay including using tubes containing lyophilised reagents for cell stimulation, pre-mixed antibody cocktail for cell staining and automated gating for flow analyses. Another alternative to alleviate collection time and transport constrains and for limited-resource sites would be to directly cryopreserve whole blood samples using cryopreservative solution, such as CryoStor® CS10 solution [ 40 ], allowing to perform the cell stimulation and staining in batch at adequately equipped sites. Lastly, in this study, cell stimulation was performed using a pool of peptides covering the SARS-CoV-2 S, N and M proteins.…”

The frequency of interleukin-1β-producing monocytes is significantly associated with varicella-zoster responses of nursing home residents

“…In the previous and present study, the mean frequency of CD14 ++ CD16 + intermediate monocytes was 0·58 versus 1·5%, respectively, while the frequency of CD14 + CD16 + non‐classical monocytes was 0.55 versus 0.8%, respectively. Unfortunately, we are unable to evaluate the impact of cryopreservation on the observed cytokine production following treatment; however, a recent study shows that frequencies of IL‐1β + , IL‐6 + and TNF‐α + monocytes following LPS stimulation were similar in fresh and frozen blood [48].…”