Holographic imaging flow cytometry for radiation response assessment in lymphocytes

Radiotherapy (RT) is a crucial component of cancer treatment, with over 50% of patients undergoing ionizing radiation therapy. RT aims to deliver precise radiation doses to tumors while minimizing damage to surrounding healthy tissues. However, RT has yet to become fully personalized, as general dose prescriptions are still applied despite significant interindividual variability in response. A major challenge associated with RT is radiation-induced lymphopenia (RIL), a condition caused by the high radiosensitivity of lymphocytes (LCs). RIL, which affects 40-70% of RT patients, impairs the antitumor immune response and correlates with poor clinical outcomes. Recent advances in radiobiology leverage single-cell analysis techniques such as flow cytometry to study RT effects on LCs. For example, Fluorescence Imaging Flow Cytometry has been used to assess radiation responses, but its reliance on fluorescence staining introduces limitations such as photobleaching, phototoxicity, and high costs. Instead, Quantitative Phase Imaging (QPI) based on Digital Holography has emerged as a promising label-free imaging modality enabling non-invasive analysis of single-cell biophysical properties, such as refractive index and dry mass, which could be indicative of radiation-induced alterations. Here a novel application of Holographic Imaging Flow Cytometry (HIFC) is discussed to characterize LC responses to X-ray doses (2 Gy and 10 Gy) over multiple time points (0, 24, 48, 72 h). The combination of QPI with multiparametric statistical analysis and machine learning enables accurate classification of irradiated versus non-irradiated LCs. These findings highlight HIFC's potential for RT monitoring and biodosimetry, with implications for personalized treatment strategies and space radiation exposure assessments.