Abstract：
OBJECTIVE: 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.
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.
CONCLUSIONS: These data provide a rationale for further development of CAM and other fluorescent dyes as tools for measuring post-transfusion kinetics of platelets.
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.
CONCLUSIONS: These data provide a rationale for further development of CAM and other fluorescent dyes as tools for measuring post-transfusion kinetics of platelets.
摘要：
目的：使用健康人受试者的恢复和存活测量来评估用于输血的血小板的体内质量。放射性标记是输血后追踪血小板的标准，但存在后勤和技术限制。这项研究调查了用钙黄绿素荧光染料家族标记血小板作为放射性同位素或生物素替代品的体外可行性。
方法:放射性标记的方案适用于钙黄绿素乙酰氧基甲酯(CAM)和生物素。通过流式细胞术分析标记的血小板并评估其活化和功能。我们测试了在不操作血小板的情况下标记和样品多重化的可行性。
结果:标记2μgCAM/1010血小板导致>99%的CAM+血小板。CAM标记的或生物素化的血小板与载体对照之间的活化或聚集没有显著差异,尽管在未经标记处理的血小板中%CD62P+显著较低。向标记为>95%的血小板的血小板储存袋中添加CAM。用不同染料标记的血小板群可以通过流式细胞术区分。
结论：这些数据为进一步开发CAM和其他荧光染料作为测量血小板输注后动力学的工具提供了理论基础。
方法:放射性标记的方案适用于钙黄绿素乙酰氧基甲酯(CAM)和生物素。通过流式细胞术分析标记的血小板并评估其活化和功能。我们测试了在不操作血小板的情况下标记和样品多重化的可行性。
结果:标记2μgCAM/1010血小板导致>99%的CAM+血小板。CAM标记的或生物素化的血小板与载体对照之间的活化或聚集没有显著差异,尽管在未经标记处理的血小板中%CD62P+显著较低。向标记为>95%的血小板的血小板储存袋中添加CAM。用不同染料标记的血小板群可以通过流式细胞术区分。
结论：这些数据为进一步开发CAM和其他荧光染料作为测量血小板输注后动力学的工具提供了理论基础。
