A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials

Pitoiset, Fabien; Barbié, Michèle; Monneret, Guillaume; Braudeau, Cécile; Pochard, Pierre; Pellegrin, Isabelle; Trauet, Jacques; Labalette, Myriam; Klatzmann, David; Rosenzwajg, Michèlle

doi:10.1002/cyto.b.21622

Cited by 39 publications

(26 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Later on, the role of these cells in sepsis‐induced innate and adaptive alterations was showed . Thanks to the improvement in flow cytometry phenotyping such as the addition of CD127 staining and standardization of intracellular Foxp3 staining for its use in clinic , large prospective multicenter clinical studies have evaluated this parameter in ICU patients . It was showed that increased percentage of Treg in critically ill patients is associated with increased risk of subsequent nosocomial infections and that this parameter could be part of an immunopanel to stratify patients suitable for immuno‐intervention in a precision medicine approach .…”

Section: Landmarks Of Flow Cytometry In Sepsismentioning

confidence: 99%

“…In addition, the development of lyophilized pre‐formulated antibody panels also represents a major improvement for FCM standardization in RCTs. As an example, FOXP3‐lyophilized tubes are currently used for regulatory T cells determination in an RCT evaluating low‐dose IL‐2 treatment for type 1 diabetes (DIABIL‐2 study, NCT02411253). Furthermore, new technical developments (low cost compact portable flow cytometer, bedside flow cytometry or chip‐based flow cytometry) are now being proposed that will facilitate the use of Flow at the bedside in ICU (see above in mHLA‐DR paragraph ).…”

Section: Current Multicenter Studies and Clinical Trialsmentioning

confidence: 99%

See 1 more Smart Citation

How Clinical Flow Cytometry Rebooted Sepsis Immunology

et al. 2019

Self Cite

View full text Add to dashboard Cite

On May 2017, the World Health Organization (WHO) recognized sepsis as a global health priority by adopting a resolution to improve the prevention, diagnosis, and management of this deadly disease. While it has long been known that sepsis deeply perturbs immune homeostasis by inducing a tremendous systemic inflammatory response, pivotal observations based on clinical flow cytometry indicate that sepsis indeed initiates a more complex immune response that varies over time, with the concomitant occurrence of both pro-and anti-inflammatory mechanisms. As a resultant, some septic patients enter a stage of protracted immunosuppression. This paved the way for immunostimulation approaches in sepsis. Clinical flow cytometry permitted this evolution by drawing a new picture of pathophysiology and reshaping immune trajectories in patients. Additional information from cytometry by time of flight mass cytometry and other highdimensional flow cytometry platform should rapidly enrich our understanding of this complex disease. This review reports on landmarks of clinical flow cytometry in sepsis and how this single-cell analysis technique permitted to breach the wall of decades of unfruitful anti-inflammatory-based clinical trials in sepsis.On May 2017, the World Health Organization (WHO) recognized sepsis as a global health priority by adopting a resolution to improve the prevention, diagnosis, and management of this deadly disease (1). Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection (2). Septic shock is the most severe form of sepsis in which hypotension persists despite adequate volume resuscitation thus requiring the use of vasopressors. According to recent definitions, in response to an infectious trigger, the unbalanced host (immune) response is at the center of sepsis pathophysiology (2).Sepsis represents a major healthcare problem worldwide. As an example, in the US, sepsis is more frequent than myocardial infarction, breast, or colon cancers (3-6). Recent modeling of available data determined the number of cases of sepsis in the entire world to over 20-25 million corresponding to >6 million deaths per annum (7,8). Over the years, 28-day mortality has remained high, ranging from 20% for sepsis to over 40% for septic shock despite improvement in patients' care (fluid resuscitation, source control, antimicrobial therapy). Whereas few therapeutic options evaluated in phase II clinical trials have demonstrated the potential efficacy of immunostimulation in sepsis, to date, no therapeutic intervention targeting host response has specifically been approved and sepsis remains the leading cause of mortality in intensive care units (ICU)(9,10). In addition, sepsis incidence has dramatically increased over the past decade and is expected to further augment. This rising

show abstract

Section: Landmarks Of Flow Cytometry In Sepsismentioning

confidence: 99%

Section: Current Multicenter Studies and Clinical Trialsmentioning

confidence: 99%

How Clinical Flow Cytometry Rebooted Sepsis Immunology

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…Similar dried antibody mixtures have already proven to yield high-data reproducibility and reliability in large-scale projects such as the ONE study [13], the PreciseADS study [34], and others [35][36][37]. These reagents contain exactly the same amount of dried antibodies precoated in individual tubes for direct labelling of cells from the same batch and are very stable overtime, offering the advantage of speeding up and simplifying the labelling procedure, reducing the number of technical steps, and avoiding the maximum of biases.…”

mentioning

confidence: 99%

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring

Macchia

Sorsa²,

Ruspantini³

et al. 2020

Journal of Immunology Research

View full text Add to dashboard Cite

Background. Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intraand interoperator variabilities in a multicentre project. Methods. The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results. Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of crosscentre variability for naïve/memory subset frequencies particularly in the whole blood setting. Conclusion. Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

show abstract

“…In our studies, we have used whole peripheral blood as the starting sample which has been the routine over the years in this cytometry laboratory, instead of isolated mononuclear cells via Ficoll discontinuous gradient centrifugation (Böyum, ). Two recent reports actually favor the whole blood approach, as it saves time and is equally reliable as the staining of isolated mononuclear cells (Jimenez Vera et al, ; Pitoiset et al, ). While our report is centered on utilizing a two‐laser, four‐fluorescent labels plus forward scattering‐side scattering approach for gating on lymphocytes and then on CD4‐T cells, it is possible to utilize instruments with more lasers and thus more fluorescent labels.…”

Section: Discussionmentioning

confidence: 99%

A modified flow cytometry method for objective estimation of human CD4⁺ regulatory T cells (CD4⁺ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling

Petsiou

Paschou

Vartholomatos

et al. 2019

Cytometry Part B Clinical

View full text Add to dashboard Cite

Background: Several methods exist for flow-cytometric estimation of human peripheral blood CD4 + T regulatory cells (CD4 + Tregs). Methods:We report our experience with the estimation of human CD4 + Tregs via three different characterizations using flow cytometry (CD25 high FoxP3 + , CD25 high CD127 low/− FoxP3 + , and CD4 + CD25 high/int CD45ROFoxP3 + ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4 + T cell fractions obtained by the third method were differentially colored to ascertain the Abbreviations: CD45RA-RO, CD45RA or CD45RO; eTregs, effector Tregs (CD4 + CD25 high CD45RA − , or CD4 + CD25 high CD45RO + ), or active Tregs (aTregs); IPEX, immune deficiencypolyendocrinopathy-enteropathy-X-linked; iTregs, induced Tregs; mAb, monoclonal antibody; MFI, mean fluorescence intensity; nTregs, naïve Tregs (CD4 + CD25 int CD45RA + , or CD4 + CD25 int CD45RO − )

show abstract

A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials

Abstract: Our results highlight the reliability of this high-standard protocol that could become a reference method for the monitoring of Tregs in clinical trials. © 2018 International Clinical Cytometry Society.

Cited by 39 publications

References 18 publications

How Clinical Flow Cytometry Rebooted Sepsis Immunology

How Clinical Flow Cytometry Rebooted Sepsis Immunology

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring

A modified flow cytometry method for objective estimation of human CD4⁺ regulatory T cells (CD4⁺ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling

Contact Info

Product

Resources

About

A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials

Abstract: Our results highlight the reliability of this high-standard protocol that could become a reference method for the monitoring of Tregs in clinical trials. © 2018 International Clinical Cytometry Society.

Cited by 39 publications

References 18 publications

How Clinical Flow Cytometry Rebooted Sepsis Immunology

How Clinical Flow Cytometry Rebooted Sepsis Immunology

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring

A modified flow cytometry method for objective estimation of human CD4+ regulatory T cells (CD4+ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling

Contact Info

Product

Resources

About

A modified flow cytometry method for objective estimation of human CD4⁺ regulatory T cells (CD4⁺ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling