A PBMC-based proof-of-principle framework for fit-for-purpose validation of flow cytometry assays in cell therapy development.
- 1Department of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Heidelberg, Germany.
- 2Department of Hematology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.
Abstract
Cellular immunotherapy offers personalized therapeutic benefits but faces challenges such as manufacturing consistency and regulatory compliance. Validated analytical methods based on product's mechanism of action are essential to guarantee product quality and patient safety. Despite regulatory emphasis on analytical validation, a structured framework applicable to broader cellular therapies remains undefined. This study evaluated a PBMC-based proof-of-principle framework for fit-for-purpose validation of flow cytometry-based analytical method to cellular therapy development. Key assay parameters were optimized, including antibody concentration, photomultiplier tube (PMT) voltage, compensation, and steric hindrance verification. Assay validation assessed specificity, sensitivity, linearity, precision, robustness, and carryover across different conditions. The validated method demonstrated high specificity (false-positive rates <1%), strong assay sensitivity (LLOQ within acceptable limits), robust linearity (R2 > 0.9), and reproducibility across intra-assay, inter-instrument, and inter-operator evaluations. Practical considerations, including sample stability, antibody cocktail preparation, and instrument carryover, were also addressed to enhance workflow efficiency. The proposed framework may support product-specific analytical method development; however, application to a defined cell therapy producet requires product-, matrix-, and analytical decision-specific qualification or validation.
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BibTeX
@article{han2026pbmcbased,
title = {{A PBMC-based proof-of-principle framework for fit-for-purpose validation of flow cytometry assays in cell therapy development.}},
author = {Huixiu Han and Anita Schmitt and Angela Hückelhoven-Krauss and Brigitte Neuber and Theresa Tretter and Mandy Hinkelbein and Ann-Kathrin Bauer and Carsten Müller-Tidow and Michael Schmitt and Lei Wang},
journal = {Frontiers in medical technology},
year = {2026},
month = oct,
doi = {10.3389/fmedt.2026.1905186},
url = {https://europepmc.org/article/MED/42952705},
note = {CC BY},
}
RIS
TY - JOUR
TI - A PBMC-based proof-of-principle framework for fit-for-purpose validation of flow cytometry assays in cell therapy development.
AU - Huixiu Han
AU - Anita Schmitt
AU - Angela Hückelhoven-Krauss
AU - Brigitte Neuber
AU - Theresa Tretter
AU - Mandy Hinkelbein
AU - Ann-Kathrin Bauer
AU - Carsten Müller-Tidow
AU - Michael Schmitt
AU - Lei Wang
PY - 2026
DA - 2026/10/14
JO - Frontiers in medical technology
DO - 10.3389/fmedt.2026.1905186
UR - https://europepmc.org/article/MED/42952705
AB - Cellular immunotherapy offers personalized therapeutic benefits but faces challenges such as manufacturing consistency and regulatory compliance. Validated analytical methods based on product's mechanism of action are essential to guarantee product quality and patient safety. Despite regulatory emphasis on analytical validation, a structured framework applicable to broader cellular therapies remains undefined. This study evaluated a PBMC-based proof-of-principle framework for fit-for-purpose validation of flow cytometry-based analytical method to cellular therapy development. Key assay parameters were optimized, including antibody concentration, photomultiplier tube (PMT) voltage, compensation, and steric hindrance verification. Assay validation assessed specificity, sensitivity, linearity, precision, robustness, and carryover across different conditions. The validated method demonstrated high specificity (false-positive rates <1%), strong assay sensitivity (LLOQ within acceptable limits), robust linearity (R2 > 0.9), and reproducibility across intra-assay, inter-instrument, and inter-operator evaluations. Practical considerations, including sample stability, antibody cocktail preparation, and instrument carryover, were also addressed to enhance workflow efficiency. The proposed framework may support product-specific analytical method development; however, application to a defined cell therapy producet requires product-, matrix-, and analytical decision-specific qualification or validation.
SN - 2673-3129
ER -