Development and validation of the IFN-γ FlowCytoScreen for cytokine-based potency assessment of advanced therapy medicinal products
4 affiliations
- 1Immuno-Gene Therapy Factory, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”
- 2Miltenyi Biotec s.r.l.
- 3Unit of Biostatistics and Clinical Trials, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”
- 4Advanced Cellular Therapies and Rare Tumors Unit, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”
Abstract
Introduction: Robust quality control methods are required to ensure the safety, efficacy and consistency of advanced therapy medicinal products. Potency assays play a critical role in evaluating their biological activity. While ELISpot remains the reference method for single-cell cytokine analysis, it has several limitations such as a lack of standardisation and limited capacity for multiparametric characterisation. This study aimed to develop and validate a flow cytometry-based potency assay for the assessment of single-cell IFN-γ secretion.
Methods: The commercially available Miltenyi Biotec IFN-γ Secretion Assay - Detection Kit, based on flow cytometry, was modified to assess the potency of dendritic cell-based vaccines. To ensure its reliability, our assay was rigorously evaluated for specificity, linearity, accuracy, robustness and precision. Comparative analysis with the ELISpot assay was also performed to assess agreement between the two methods.
Results: Method development resulted in reduced sample requirements and optimisation of cytokine secretion conditions to generate a response to antigen-presenting cell stimulus. Validation showed that the specificity, linearity, accuracy, robustness and precision consistently met the acceptance criteria. Comparative analysis with ELISpot demonstrated high correlation and agreement, with no evidence of systematic or proportional error.
Discussion: The developed assay provides a reliable, scalable and efficient flow cytometry-based approach for cytokine secretion analysis and represents an alternative method to ELISpot. Its advantages in sensitivity, efficiency and cost effectiveness make it a valuable tool for advanced therapy quality control and immune monitoring in clinical research. The flow cytometry further enhances its potential by enabling multiparametric immune profiling, supporting comprehensive characterisation of cellular products in both preclinical and clinical applications.
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BibTeX
@article{carloni2026development,
title = {{Development and validation of the IFN-γ FlowCytoScreen for cytokine-based potency assessment of advanced therapy medicinal products}},
author = {Silvia Carloni and Claudia Piccinini and Chiara Arienti and Dario Armando Leone and Elena Pancisi and Francesca Fanini and Sara Pignatta and Valentina Soldati and Monica Stefanelli and Anna Maria Granato and Flavia Foca and Jenny Bulgarelli and Laura Ridolfi and Massimiliano Petrini},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1959178},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1959178/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Development and validation of the IFN-γ FlowCytoScreen for cytokine-based potency assessment of advanced therapy medicinal products
AU - Silvia Carloni
AU - Claudia Piccinini
AU - Chiara Arienti
AU - Dario Armando Leone
AU - Elena Pancisi
AU - Francesca Fanini
AU - Sara Pignatta
AU - Valentina Soldati
AU - Monica Stefanelli
AU - Anna Maria Granato
AU - Flavia Foca
AU - Jenny Bulgarelli
AU - Laura Ridolfi
AU - Massimiliano Petrini
PY - 2026
DA - 2026/10/20
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1959178
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1959178/full
AB - Introduction: Robust quality control methods are required to ensure the safety, efficacy and consistency of advanced therapy medicinal products. Potency assays play a critical role in evaluating their biological activity. While ELISpot remains the reference method for single-cell cytokine analysis, it has several limitations such as a lack of standardisation and limited capacity for multiparametric characterisation. This study aimed to develop and validate a flow cytometry-based potency assay for the assessment of single-cell IFN-γ secretion. Methods: The commercially available Miltenyi Biotec IFN-γ Secretion Assay - Detection Kit, based on flow cytometry, was modified to assess the potency of dendritic cell-based vaccines. To ensure its reliability, our assay was rigorously evaluated for specificity, linearity, accuracy, robustness and precision. Comparative analysis with the ELISpot assay was also performed to assess agreement between the two methods. Results: Method development resulted in reduced sample requirements and optimisation of cytokine secretion conditions to generate a response to antigen-presenting cell stimulus. Validation showed that the specificity, linearity, accuracy, robustness and precision consistently met the acceptance criteria. Comparative analysis with ELISpot demonstrated high correlation and agreement, with no evidence of systematic or proportional error. Discussion: The developed assay provides a reliable, scalable and efficient flow cytometry-based approach for cytokine secretion analysis and represents an alternative method to ELISpot. Its advantages in sensitivity, efficiency and cost effectiveness make it a valuable tool for advanced therapy quality control and immune monitoring in clinical research. The flow cytometry further enhances its potential by enabling multiparametric immune profiling, supporting comprehensive characterisation of cellular products in both preclinical and clinical applications.
SN - 1664-3224
ER -