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Evaluation of a method to fluorescently label platelets for in-human recovery and survival studies
Tamar P Feldman1, Bethany L Brown1
1American Red Cross, Holland Lab for the Biomedical Sciences, Rockville, Maryland, USA.
Background And Objectives:
Platelets for transfusion are evaluated for in vivo quality using recovery and survival measurements in healthy human subjects. Radiolabelling is the standard for tracing platelets post-transfusion but imposes logistical and technical limitations. This study investigates the in vitro feasibility of labelling platelets with the calcein family of fluorescent dyes as an alternative to radioisotopes or biotin.
Materials And Methods:
Protocols for radiolabelling were adapted for use with calcein acetoxymethyl ester (CAM) and biotin. Labelled platelets were analysed by flow cytometry and evaluated for activation and function. We tested feasibility for labelling without manipulation of platelets and for multiplexing of samples.
Results:
Labelling at 2 μg CAM/1010 platelets resulted in >99% of CAM+ platelets. There was no significant difference in activation or aggregation between CAM-labelled or biotinylated platelets and vehicle controls although %CD62P+ was significantly lower in platelets that were not processed for labelling. Addition of CAM to the platelet storage bag labelled >95% of platelets. Platelet populations labelled with different dyes could be distinguished by flow cytometry.
Conclusion:
These data provide a rationale for further development of CAM and other fluorescent dyes as tools for measuring post-transfusion kinetics of platelets.
