A harmonised protocol for generating human extracorporeal photopheresates for research

Abstract Extracorporeal photopheresis (ECP) is an established second-line therapy for T cell-mediated immune diseases such as graft-versus-host disease and organ transplant rejection. The ECP procedure induces apoptosis in autologous leucocytes using 8-methoxypsoralen (8-MOP) and UVA irradiation, which are then reinfused into patients leading to immunomodulatory effects. To ensure the comparability and relevance of investigations into these immunomodulatory actions, we developed and validated an ECP protocol for research purposes that closely reproduces the apoptotic profile of clinical ECP products from the CELLEX system. We optimised key parameters, including 8-MOP concentration, UVA dose and sample preparation, such that apoptosis rates matched clinical ECP products. An 8-MOP concentration of 400 ng/ml and UVA dose of 2 J/cm 2 resulted in consistent T cell apoptosis rates (84.7 ± 2.1% at 48 h). Analysis by flow cytometry confirmed similar kinetics of early and late apoptosis in research-grade and clinical photopheresates. Our research-grade protocol, which was validated by an independent laboratory, establishes a reliable and therapeutically relevant model for harmonised mechanistic and translational ECP research.