A PBMC-based proof-of-principle framework for fit-for-purpose validation of flow cytometry assays in cell therapy development
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Updated: Oct 31, 2026
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Published on: July 10, 2026
Huixiu Han1,2, Anita Schmitt1, Angela Hückelhoven-Krauss1
1Department of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Heidelberg, Germany.
Introduction:
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.
Methods:
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.
Results:
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.
Discussion:
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.
