(P-CB-14) Feasibility of Fluorescent Labeling for in Vivo Evaluation of Emerging Platelet Products

Background/Case Studies: Historical acceptance criteria for platelet in vivo recovery and survival are well-suited for products intended for prophylactic transfusion but offer limited utility when evaluating products for acute bleeds. Current methods to trace platelets in vivo are based on radioisotope labeling which precludes study of platelet function and characterization of subpopulations in circulation. We asked whether methods for radiolabeling could be adapted to label cold stored platelets (CSPs) and platelets stored in whole blood (WB) with a fluorescent dye to enable analysis by flow cytometry.

Study

Design/Methods: Apheresis platelets and concurrent plasma (n=3) and whole blood processed with a platelet-sparing filter (n=4) were stored at 2-6°C within eight hours of collection. Units were sampled prior to cold storage and on days 7 and 14 for labeling with a fluorescent viability dye, calcein acetoxymethyl ester (CAM). Apheresis platelets were labeled by either 1) direct addition of CAM or 2) centrifugation, resuspension in protein-free buffer, and addition of CAM. After incubation, platelets were centrifuged and resuspended in platelet-poor plasma (PPP). CAM was directly added to WB. WB samples for flow cytometry were taken immediately after labeling, and platelet-rich plasma and PPP were prepared for light transmission aggregometry (LTA). Samples were analyzed for quality of labeling (%CAM⁺, MFI, robust CV) and activation (%CD62P⁺). On days 0 and 14, LTA was performed using a dual agonist of collagen (10 µg/mL) and ADP (10 µM). Statistical testing was conducted by repeated measures one-way or two-way ANOVA. Significant results (p < 0.05) were analyzed between days or treatments by the Holm-Sidak test for multiple comparisons (p_adj < 0.05).

Results/Findings: There was no significant effect of labeling method on the %CAM⁺ or robust CV, but labeling CSPs by method 1 produced a lower CAM MFI (p=0.0041, Figure). Although >95% of platelets were CAM⁺ after labeling by either method, cold storage resulted in reduced CAM signal of 5-10% of the population. This was also observed in labeled WB. The additional centrifugation in method 2 increased platelet activation when platelets were labeled prior to cold storage (method 1: 18±11%, method 2: 30±16%, p<sub>adj=0.0440). Labeling in WB did not increase activation except on day 0 (no label: 19±5%, CAM: 23±5%, p<sub>adj=0.0008) but caused visible hemolysis. Platelets labeled in WB had reduced aggregation (p=0.0186), but aggregation of CSPs was unaffected.

Conclusions: These data support feasibility of fluorescent labeling for in vivo evaluation of CSPs and platelets in WB and suggest that the benefit of reduced manipulation enabled by CAM labeling could outweigh the cost of a mild reduction in fluorescent signal in refrigerated products. Subtle effects of cold storage on quality of labeling highlight the need for methods that can interrogate a heterogeneous product post-transfusion.