(P-CB-18) Toward a Fluorescent Label for In Vivo Evaluation of Platelet Products

Measurement of in vivo recovery at 24 hours (h) and survival is a key step in licensing platelet products for transfusion. The current use of radioisotope labeling to detect infused platelets in samples of peripheral whole blood (WB) is limited by regulatory considerations, cost, and supply chain disruptions. Here, we build on a published method for labeling platelets with a fluorescent dye, calcein acetoxymethyl ester (CAM), by investigating in vitro stability of labeled platelets, feasibility of labeling cold-stored platelets (CSPs), and detection of labeled cells in WB samples.

Study

Design/Methods:

A high-yield apheresis platelet unit and concurrent plasma were provided by a single donor. Platelets were prepared for labeling by centrifugation and resuspension in ACD-A/saline. To evaluate stability, platelets were labeled in triplicate with 2 µg CAM or an equivalent volume of vehicle for 15 minutes at room temperature (RT). Labeled platelets were then resuspended in concurrent plasma with PAS and stored at RT in transfer packs with agitation. Samples were taken at 0, 24, 48, and 72h post-labeling for analysis by flow cytometry. To evaluate feasibility of labeling 24-h delayed CSPs, the mother high-yield platelet unit was split into six low-dose units for storage at RT or 2-6°C in triplicate. Samples for labeling were taken 1-, 3-, and 6-days post-collection. Packed red cells were also tested for uptake of CAM to investigate feasibility of directly labeling WB to trace both platelets and red cells. To develop the gating syntax for post-transfusion samples, WB was stained with antibodies against CD61 and CD235a. Five million events were collected per donor (n=2) to estimate the rate of false positive events in the CAM⁺ gate.

Results/Findings:

CAM-labeling was stable for 72h at RT. Although the median fluorescence intensity (MFI) decreased over time, there was no significant difference in the percentage of CAM⁺ platelets at 0 (98.7 ±0.5%) and 72h (98.2 ±0.7%). Storage at 2-6°C up to 5 days did not affect uptake of the CAM label compared to storage at RT (day 6; MFI, p<sub>adj = 0.6; %CAM⁺, p<sub>adj = 0.7). We also found that red cells stored for one week at 2-6°C could also be labeled with CAM but produced a lower MFI than labeled platelets. Using WB samples without labeled platelets, our gating syntax (Figure A) produced a false positive rate of 1 in 2.1 million collected events with a 99% confidence interval.

Conclusions:

These data demonstrate that CAM labeling is stable for the minimum duration necessary to calculate recovery and survival though further preclinical experiments are needed to determine if labeling can be maintained over the 7–10-day lifespan of a platelet in vivo. Given increased usage of CSPs and WB, feasibility of labeling unconventional platelet products supports further development of CAM as a tool for in vivo studies.