(P-CB-13) Evaluation of a Calcein-Based Fluorescent Label for Tracking in Vivo Recovery and Survival of Human Platelets in a SCID Mouse Model of Transfusion

Background/Case Studies: Background/Case Studies: Evaluation of novel and modified platelet (PLT) products includes their comparison to fresh autologous PLTs for in vivo recovery and survival in circulation of healthy human volunteers. Such studies currently use radioactive labels for the PLTs, but the radioactive isotopes are being phased out of production and alternate labels for PLTs are urgently needed for development of new PLT products. We used fluorescent calcein acetoxymethyl ester (CAM) to label human PLTs (hPLTs) and compared the in vivo recovery and survival of labeled and non-labeled hPLTs stored for 1-5 days at room temperature (RT) in a validated animal model (SCID mouse; Transfusion. 2020;60(10):2379-2388).

Study

Design/Methods: Study

Design/Methods: Single apheresis hPLT-units in 100% plasma, were stored for 1 or 5-days at RT with agitation (N=3 donations). On days 1 or 5, hPLTs were prepared using the BEST method and labeled with CAM. Labeled hPLTs and control non-labeled hPLTs were infused (5x10⁸ PLTs IV) into SCID mice (n=5-6 animals /condition). Murine blood samples were collected over time to generate survival and clearance curves of the hPLTs in circulation using flow cytometry analysis. Non-labeled hPLTs were detected ex vivo by an APC labeled anti-human monoclonal antibody (CD41-APC).

Results/Findings: Results/Findings: Mice tolerated CAM labeled hPLTs for 2 hours post infusion with no evidence of discomfort or adverse events. 1-day old control hPLTs had a higher recovery (62.1%, (95%CI:53.1-76.1)) than CAM hPLTs (39.9%, (95%CI:31.5-54.5)) and both had similar clearance curves. 5-day old control hPLTs (44.3%, (95%CI:37.2-53.0)) also had a higher recovery than 5-day CAM PLTs (34.3%, (95%CI:27.0-45.3)), but the CAM hPLTs had a faster clearance. When 5-day old non-labeled hPLTs were infused together with 5-day old CAM hPLTS into the same mouse there was no difference in recoveries which were 36% (95%CI:28.5-52.5) and 36.5% (95%CI:25.9-55.0), respectively and no difference in clearance rates (Figure A).

Conclusions:

Conclusion: CAM labeled hPLTs are well tolerated by mice but have lower in vivo recovery when infused alone compared to non-labeled hPLTs injected alone. When mixed 1:1 and infused together, non-labeled and CAM labeled PLTs have identical recovery and survivals. CAM dye may have local effects that reduce the initial recovery in circulation, but this can be overcome by co-injecting labeled and non-labeled PLTs together. Even with moderate mouse-to-mouse variability, CAM fluorescent dye appears to be suitable for tracking hPLTs in vivo.