Best Practices in Performing Flow Cytometry in A Regulated Environment: Feedback from Experience Within The European Bioanalysis Forum

Strate, Barry van der; Longdin, Robin; Geerlings, Marie; Bachmayer, Nora; Cavallin, Maria; Litwin, Virginia; Patel, Minesh; Passe-Coutrin, Wilfried; Schoelch, Corinna; Companjen, Arjen; Fjording, Marianne Scheel

doi:10.4155/bio-2017-0093

Cited by 41 publications

(29 citation statements)

References 39 publications

(75 reference statements)

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“…Currently, the only existing guidelines for validating flow cytometric assays are gathered from guidance documents and recommendations published in specific journals [9,10]. So far, several harmonisation and standardisation programs have been promoted and activated, e.g., by the Human Immune Phenotyping Consortium (HIPC) [11], the EuroFlow Consortium [3,12], and the ONE Study Consortium [13].…”

Section: Introductionmentioning

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

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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.

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Section: Introductionmentioning

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

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“…Control cells can be used as positive and procedural controls for flow cytometric applications such as phenotyping of leukemia and lymphoma, immune monitoring and drug discovery. Scientists have been exploring cell preservation techniques to achieve both consistency and performance for a few decades (68,76,77,83). This workshop covered all current products as well as laboratory-developed approaches.…”

Section: Discussionmentioning

confidence: 99%

“…FCM assays should be developed and validated in accordance with recommendations presented in seminal papers . These support best practices for performing FCM in a regulated environment and are cited in feedback from the experience of cytometrists within the European Bioanalysis Forum .…”

Section: Ws11: Flow Cytometry Application In Multi‐center Global Clinmentioning

confidence: 92%

Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2018 Conference Workshops

Czechowska¹,

Lannigan

Wang

et al. 2019

Cytometry Pt A

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“…Immune cells were gated according to size and granularity defined in the forward (FSC) and side light scatter (SSC) plot (see Additional file 1 : Figure S1, Supplemental Digital Content). Gating boundaries were set on the basis of control samples at the 24 h time point to minimize data variability (see van der Strate et al [ 15 ]). Cell populations were further characterized based on their live/dead appearance and CD45 expression pattern.…”

Section: Methodsmentioning

confidence: 99%

Experimental murine acute lung injury induces increase of pulmonary TIE2-expressing macrophages

et al. 2018

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BackgroundBreakdown of the alveolo-capillary wall is pathognomonic for Acute Lung Injury (ALI). Angiopoietins, vascular-specific growth factors, are linked to endothelial barrier dysfunction, and elevated Angiopoietin-2 (ANG2) levels are associated with poor outcome of ALI patients. Specialized immune cells, referred to as ‘TIE2-expressing monocytes and macrophages’ (TEM), were shown to specifically respond to ANG2 binding. However, their involvement in acute inflammatory processes is so far completely undescribed. Thus, our aim was to assess the dynamics of TEMs in a murine model of ALI.ResultsIntratracheal instillation of LPS induced a robust pulmonary pro-inflammatory response with endothelial barrier dysfunction and significantly enhanced ANG2 expression. The percentage number of TEMs, assessed by FACS analysis, was more than trebled compared to controls, with TEM count in lungs reaching more than 40% of all macrophages. Such distinct dynamic was absent in all other analyzed compartments (alveolar space, spleen, blood). Incubation of the monocytic cell line THP-1 with LPS or TNF-α resulted in a dose-dependent, significant upregulation of TIE2, suggesting that not recruitment from extra-pulmonary compartments but TIE2 upregulation in resident macrophages accounts for increased lung TEM frequencies.ConclusionsFor the first time, our data provide evidence that the activity of TEMs changes at sites of acute inflammation.Electronic supplementary materialThe online version of this article (10.1186/s12950-018-0188-5) contains supplementary material, which is available to authorized users.

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Best Practices in Performing Flow Cytometry in A Regulated Environment: Feedback from Experience Within The European Bioanalysis Forum

Cited by 41 publications

References 39 publications

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

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

Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2018 Conference Workshops

Experimental murine acute lung injury induces increase of pulmonary TIE2-expressing macrophages

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