Development of a 32-color spectral flow cytometry panel for comprehensive immunophenotyping of PBMCs from cancer patients receiving immunotherapy and radiotherapy

Background: High-dimensional immune profiling is essential for understanding how radiotherapy (RT) and immunotherapy (IO) reshape systemic immunity. Spectral flow cytometry offers major advantages over traditional flow by enabling >30 markers to be measured simultaneously in a single tube, which is critical when PBMC samples are limited and multiple panels are not feasible. This approach also improves detection of rare or functionally important immune subsets that cannot be reliably captured with smaller conventional panels. Using Sony ID7000, we developed a 32-color spectral flow cytometry panel to profile PBMCs from cancer patients enrolled in an ongoing RT/IO clinical trial. Methods: A 32-color panel was designed to resolve major immune populations (T cells, B cells, NK cells, monocytes, dendritic cells) and key functional markers of differentiation (CD45RA, CCR7, CD27), activation (CD69, HLA-DR, CD38), proliferation (Ki-67), and exhaustion (PD-1, TIGIT, TIM-3, CTLA-4). Panel development incorporated fluorochrome similarity assessments and spectral signature analyses from the five-laser, 147-detector Sony ID7000. Single-color controls, FMOs, and fully stained references were used for spectral unmixing. Validation included both unstimulated PBMCs and anti-CD3/CD28-stimulated samples to assess activation and exhaustion profiles. Data was analyzed with Sony Spectral Analysis Software and FlowJo. Results: The panel reliably identified all major PBMC lineages and resolved fine T-cell differentiation states, including naïve, memory, regulatory, and exhausted subsets. Myeloid subsets and dendritic cell populations were consistently distinguished with minimal spectral spreading. Stimulation assays produced expected functional changes-such as increased activation/proliferation marker expression-confirming panel sensitivity and functional applicability. Spectral unmixing, gating strategies, and marker resolution were highly reproducible across acquisition days and sample batches. Conclusion: We developed and validated a robust 32-color spectral flow cytometry panel capable of comprehensive immunophenotyping of PBMCs from cancer patients. The spectral platform provides substantial advantages for limited clinical samples, enabling deep single-panel profiling and improved detection of rare populations. This panel is now being implemented in an active clinical trial (UCDCC#272) involving combined radiotherapy and immunotherapy, supporting immune monitoring and biomarker discovery in translational RT/IO research.