ABSTRACT
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 (NIR) 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 (WB) 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 were 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 h incubation with WB) 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.
We developed a simple, reproducible method according to Good Manufacturing Practice guidelines for labeling of platelets from platelet concentrates (PCs) with indocyanine green (ICG) as a non-radioactive alternative. PCs are medicinal drugs that are transfused to prevent or treat bleeding. They consist of the blood cells' platelets which are responsible for clotting processes in the body. Manufacturing procedures of PCs are continuously refined, and for in vivo testing, these platelets have to be labeled to track and to distinguish them from proband's or patient's own cells. Radioactive labeling, for a long time the gold standard for cell labeling, is no longer accepted. ICG is a fluorescent dye approved by the drug authorities and already used for diagnostic purposes in humans. We used ICG to label platelets from PCs. With our method, we achieved a labeling efficiency of 99.8%. We used whole blood (WB) as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. After the addition of ICG-labeled platelets to WB, the labeling efficiency decreased to 81% after 24 h. However, we were still able to distinguish the ICG-labeled platelets from the WB platelets. We could also show that platelet function was not impaired by the labeling processes. The good tolerance of ICG indicates a short path to clinical application in healthy volunteers and investigations of novel PC-manufacturing procedures. As a read-out system, flow cytometry systems equipped with NIR lasers and filters could offer the possibility of rapid visualization, cell tracking, re-isolation, and ex vivo studies.