Supplementary Material for: Indocyanine green labeled platelets for survival and recovery studies

Introduction: Before being implemented in daily clinical routine, new production strategies for platelet concentrates (PCs) must be validated for their efficacy. Besides in vitro testing, the establishment of new methods requires the labeling of platelets for in vivo studies of platelets’ survival and recovery. Indocyanine green (ICG) is a Food and Drug Administration approved near-infrared fluorescent dye for diagnostic use in vivo, suitable for non-radioactive direct cell labeling of platelets. Methods: Platelets from PCs in storage solutions with different plasma concentrations were labeled with ICG up to concentrations of 200 µM. Whole blood was used as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. The impact of labeling processes was assessed by the quantification of CD62P expression and PAC-1 binding as platelet function markers. Platelet aggregation was analyzed by light transmission aggregometry. ICG labeling efficiency and stability of platelets was determined by flow cytometry. Results: Platelets from PCs could be successfully labeled with 10 µM ICG after 1 and 4 days of storage. The best labeling efficiency of 99.8% ± 0.1% (immediately after labeling) and 81% ± 6.2% (after 24 hours incubation with whole blood) was achieved by plasma replacement by 100% platelet additive solution for the labeling process. Since the washing process slightly impaired platelet function, ICG labeling itself did not affect platelets. Immediately after the ICG labeling process plasma was re-added resulting in a recovered platelet function. Conclusion: We developed a Good Manufacturing Practice compatible protocol for ICG fluorescent platelet labeling suitable for survival and recovery studies in vivo as a non-radioactive labeling alternative.