Rapid Point-of-Care Inflammatory Cytokine Monitoring during Normothermic Liver Perfusion via a Multiplexed Paper-Based Vertical Flow Assay

The advent of normothermic machine perfusion (NMP) has substantially enhanced liver transplantation outcomes by enabling physiologic preservation and functional assessment of donor grafts. However, despite the well-established role of donor-derived inflammation influencing graft viability and post-transplant outcomes, current viability assessment metrics, such as lactate clearance, are insufficiently sensitive or specific to capture the underlying immunological status of the organ. Conventional immunoassays, while analytically robust, are limited by prolonged processing times that preclude rapid clinical decision-making and fail to reflect the multifactorial nature of immune-mediated graft injury. To address this critical gap, we developed a paper-based, multiplexed cytokine vertical flow assay (xcVFA) capable of detecting interleukin-6, tumor necrosis factor-α, and interleukin-8 directly from NMP perfusate within 30 min, enabling rapid and actionable inflammatory profiling during organ preservation. The xcVFA miniaturizes conventional sandwich ELISAs and integrates a hand-held smartphone reader for image capture, where signal readouts feed into three independent neural network classifiers, each trained to categorize respective cytokine levels. Lastly, an integrated decision tree translates cytokine profiles into overall inflammation status. The xcVFA achieved limits of detection of ∼2 ng/mL, with each classifier achieving macro-averaged F1 scores of >0.9 and one-vs-rest AUCs ≥ 0.9. In a blinded validation test, the integrated decision tree inflammation classifier achieved 90.9% overall accuracy, correctly distinguishing noninflamed grafts from a graft discarded due to inflammation. In summary, these results show that the xcVFA enables rapid, decentralized inflammatory profiling during NMP and may augment current viability criteria by incorporating inflammatory biomarkers into clinical decision-making. This point-of-care device has the potential to improve liver graft selection, optimize organ utilization, and reduce post-transplant complications.