Beyond PBMCs: Polymer-Based Cell Mimics for Robust TBNK Immunophenotyping Assay Validation.

Flow cytometry-based TBNK immunophenotyping is widely used to assess immune status in research, clinical diagnostics, and cell therapy development. Biological control materials such as PBMCs often serve as physiologically relevant controls, but suffer from considerable variability due to donor differences, limited stability, and fluctuations in antigen expression levels between lots. These factors make it challenging to achieve consistent assay performance, especially in longitudinal or multi-site environments. TBNK Cell Mimic (synthetic controls in PhenoCyte product line, developed by Slingshot Biosciences; henceforth referred to as TBNK Cell Mimic in this manuscript) are polymer-based cell mimics engineered to provide defined scatter properties, controlled antigen density, and stable subset ratios. Their scatter profiles are designed to be biologically relevant and comparable to those of native leukocyte populations. In this study, we performed a comprehensive analytical validation of a TBNK immunophenotyping assay using these TBNK Cell Mimics as standardized reference controls. Validation parameters included repeatability, intermediate precision, accuracy, linearity, specificity, robustness, stability, and carryover. The TBNK Cell Mimic met all predefined acceptance criteria, demonstrating ≤ 5% CV for intra- and inter-assay precision and R2 values > 0.998 across the linearity range. Accelerated stability studies performed at 25°C and 37°C showed < 5% variation in population frequencies, supporting the material's suitability for extended quality monitoring. These results indicate that TBNK Cell Mimic provides a consistent and reproducible reference material that can support assay validation and routine performance assessment. While not a replacement for biological samples when evaluating donor-specific or viability-dependent biology, their stability and lot-to-lot consistency offer a practical tool for reducing technical variability and improving harmonization across instruments, operators, and testing sites.