Biotinylated Platelets: A Promising Labeling Technique?

Wonderen, Stefan F. van; Baarle, Floor L.F. van; Bruin, Sanne de; Peters, Anna; Korte, Dirk de; Bruggen, Robin van; Vlaar, Alexander P.J.

doi:10.1016/j.tmrv.2023.01.001

Cited by 5 publications

(6 citation statements)

References 37 publications

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“…One of the limitations of NHS-biotin is non-specific binding to plasma proteins which reduces labelling efficiency and necessitates multiple wash steps, increasing activation [2]. We found that inhibition of CAM-labelling by plasma was negligible (Figure 3a,b).…”

Section: Resultsmentioning

confidence: 91%

“…Development of alternative labels provides an opportunity to further optimize the BEST protocol. Although advances in closed system labelling have been made for biotin and indocyanine green, a fluorescent dye approved for clinical use, non-specific binding to plasma proteins necessitates washing steps and protein-free buffer to achieve efficient labelling [2,10]. Our results suggest the effect of plasma on calcein labelling is minimal enough for labelling without prior manipulation of the platelet unit.…”

Section: Discussionmentioning

confidence: 92%

“…The gold standard method to evaluate platelet kinetics uses radioisotopes ( 51 Cr and 111 In) to label platelets for infusion in healthy volunteers according to standardized and validated procedures published by the Biomedical Excellence for Safer Transfusion (BEST) Collaborative [1]. Although radiolabelling enables evaluation of recovery and survival (R&S) of platelets in circulation, the use of radioisotopes imposes significant expense and regulatory burden on clinical sites that can impede development and licensure of new products [2]. Radiolabelling further confines evaluation of therapeutic potential to healthy volunteer studies, requires extensive manipulation of platelets during labelling, results in low labelling efficiency for platelets (5%-15% with 51 Cr) and limits assessment of a complex cell population to a single parameter.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of a method to fluorescently label platelets for in‐human recovery and survival studies

Feldman,

Brown

2024

Vox Sanguinis

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Background and ObjectivesPlatelets 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 MethodsProtocols 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.ResultsLabelling 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.ConclusionThese data provide a rationale for further development of CAM and other fluorescent dyes as tools for measuring post‐transfusion kinetics of platelets.

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Section: Resultsmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

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Evaluation of a method to fluorescently label platelets for in‐human recovery and survival studies

Feldman,

Brown

2024

Vox Sanguinis

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“…The assessment of platelet function after transfusion has always been a theoretical advantage of biotinylation compared to radiolabeling. However, no other studies have thus far investigated this as thoroughly as we did 30,31 …”

Section: Discussionmentioning

confidence: 89%

Biotin labeling allows for post‐transfusion functional assessment of stored human platelets in mice

Bailey,

Bochenek,

Chauhan

et al. 2024

Transfusion

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BackgroundPlatelet radiolabeling with radioisotopes is currently used for human platelet recovery and survival studies. Biotinylation enables ex vivo post‐transfusion platelet function testing. Whether platelet biotinylation itself affects platelet function is controversial.Study Design and MethodsPlatelet concentrates from healthy humans were stored for 6 days. Samples were obtained at 1 or 2 and 6 days, and platelets were labeled following a radiolabeling protocol using saline instead of radioactive indium‐111 (sham radiolabeling [sham‐RL]). Alternatively, a newly developed biotinylation protocol, a washing protocol, or an unmanipulated control sample were used. Platelet function was assessed by flow cytometry after stimulation with platelet agonists and labeling of platelets with platelet activation markers. To test whether platelets can be activated after transfusion, labeled platelets were transfused into nonobese diabetic/severe combined immunodeficiency mice, and samples were obtained 1 h after transfusion.ResultsThe activation profile of biotinylated platelets was comparable to sham‐RL platelets before transfusion except for significantly less α‐degranulation and more phosphatidyl serine exposure on storage day 1/2. There was no significant difference between sham‐RL and biotinylated platelets on storage day 6. Sham‐RL and biotinylated platelets were significantly less activatable than washed and unmanipulated control platelets. After transfusion, the activation profile of biotinylated platelets was largely indistinguishable from unmanipulated ones.DiscussionThe decrease in activation level in biotinylated platelets we and others observed appears mainly due to the physical manipulation during the labeling process. In conclusion, biotinylated platelets allow for post‐transfusion function assessment, a major advantage over radiolabeling.

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“…However, exposure of patients or healthy volunteers to radioactive isotopes is no longer accepted in the European Union and strictly regulated [9]. Besides radiation exposure, radioactive cell labeling is limited due to impaired platelet function after labeling and the difficulty of producing radioactive isotopes in a Good Manufacturing Practice (GMP)-compliant manner [8,10]. Highly required alternative approaches have been developed such as biotinylation of platelets.…”

Section: Introductionmentioning

confidence: 99%

Indocyanine Green-Labeled Platelets for Survival and Recovery Studies

von Behren,

Wesche,

Greinacher

et al. 2023

Transfus Med Hemother

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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 μ<sc>m</sc>. 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 μ<sc>m</sc> 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.

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Biotinylated Platelets: A Promising Labeling Technique?

Cited by 5 publications

References 37 publications

Evaluation of a method to fluorescently label platelets for in‐human recovery and survival studies

Evaluation of a method to fluorescently label platelets for in‐human recovery and survival studies

Biotin labeling allows for post‐transfusion functional assessment of stored human platelets in mice

Indocyanine Green-Labeled Platelets for Survival and Recovery Studies

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