Abstract
Clinical performance of platelet products processed or stored under novel conditions is difficult to predict based on in vitro studies alone. Evaluation of such products involves determination of recovery and survival of radiolabeled platelets in human volunteers as a surrogate endpoint for platelet efficacy. Such human studies pose some risk to volunteers, are a financial burden on the sponsor, and stifle innovation in the development of platelet products. The development of an animal model for evaluating human platelets has been limited by rapid, immunemediated clearance of human cells. In the current studies, severe combined immunodeficient (SCID) mice were used to circumvent the need to block the reticuloendothelial system and prolong circulation of human cells. Human platelets were infused via tail vein into normal and SCID mice, and the recoveries and survival times compared. Mouse whole blood was collected at various time points post-infusion, and human platelets were detected by flow cytometry using an anti-human CD41 monoclonal antibody. Recovery was defined as percent human platelets in circulation relative to time zero, and survival time in circulation as the t1/2 of the human platelets. Recoveries and survival times were different between normal and SCID mice, with a maximal difference in recovery of 60.3% at 4 hours post-infusion (normal recovery, 11.1 ± 9.1%; SCID recovery, 71.4 ± 8.8%), and survival times of 1.4 ± 0.4 hours and 10.7 ± 2.3 hours in normal and SCID mice, respectively (N=3). Chemically treated and aged platelets were used to evaluate the ability of the model to detect differences in control and damaged platelets. Chemical damage was induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler which mimics the platelet storage lesion. Platelets were exposed to 10 μM CCCP in methanol, control platelets were exposed to an equal volume of methanol (N=3). CCCP treatment of platelets decreased agonist-induced aggregation (Control aggregation, 73.3 ± 6.8%; CCCP-treated platelet aggregation, 13.8 ± 5.3%). Recovery of control and CCCP-treated platelets were 31.5 ± 16.9% and 7.9 ± 5.1%, respectively, at 4-hours post-infusion. Survival times were 1.3 hours for control and 1.9 hours for CCCP-treated platelets. For storage studies, in vitro cell quality parameters were evaluated in three products, and each product was infused into 3 animals on Day 1 and 3 different animals on Day 7. In Day 7 platelets, in vitro platelet parameters were decreased compared to Day 1. Platelet counts decreased an average of 22.8% ± 2.2% between Day 1 and Day 7. pH decreased from 6.7 ± 0.1 at Day 1 to 5.8 ± 0.1 at Day 7. All platelet products had visible swirl on Day 1 and no swirl on Day 7. Platelets stored for 7 days showed decreased recovery over Day 1 platelets at 4 hours post-infusion (Day 1, 66.9 ± 12.8%; Day 7, 0.2 ± 0.08%).
The SCID mouse may be a useful model for evaluating the impact of new technologies (apheresis devices, anticoagulants, storage containers, pathogen inactivation systems) on the in vivo efficacy of human platelets. In two different models of platelet damage (chemical and storage induced damage), this model can distinguish between normal and damaged platelets.
Recovery of Infused Day 1 and Day 7 Human Platelets in SCID Mice Recovery of Infused Day 1 and Day 7 Human Platelets in SCID Mice
TARG1 protects against toxic DNA ADP-ribosylation