Detection of UV Induced Damage to Human Platelets by an In Vivo Animal Model.

Abstract

Evaluation of novel platelet products for transfusion currently involves determination of recovery and survival of radiolabelled platelets in human volunteers since in vitro studies are not predictive of platelet clinical performance and a practical animal model has not been universally accepted. We previously demonstrated that severe combined immunodeficient (SCID) mice could be used as a model to identify severely damaged human platelets (Blood 106 (11), p537a, 2005). To further characterize the sensitivity of this model we used exposure of human platelets to UV light to induce moderate damage, and quantitated this lesion by in vitro tests and in vivo recovery in the SCID mouse model. This type of damage is clinically relevant since UV radiation of blood-platelet concentrates is used to prevent the development of post-transfusion alloimmunization and has been used as part of pathogen reduction systems to inactivate bacteria and viruses. Apheresis platelet products, stored for 1 or 7 days, were exposed to UV light (280–315 nm) for either 20 or 40 min (intensity: 750 μW/cm2). In vitro testing included platelet count, pH, CD62P expression, mitochondrial membrane potential and collagen-induced (2 mg/ml) aggregation. The mitochondrial membrane potential was measured by flow cytometry (FL2-to-FL-1 ratio of platelets loaded with JC-1 (final: 2μM) showed a ratio 6.8 ± 1.5 for the 1 day old platelets and 3.25 ± 0.75 for the 7 day old platelets. Additional flow cytometric testing showed an increase of p-selectin (ant-human CD62P, clone AK-4) expression for 1 day old platelets (31.4 ± 3.5%) and 7 day old platelets (54.6 ± 6.6) after 20 min of UV light. Tables 1 and 2 show in vitro and in vivo results as percent of control (except for pH and JC-1).Table 11 Day Old PlateletsUV ExposurePlt CountpHJC-1 RatioCollagen Aggregationin vivo Recovery20 min76.27.46.5 ± 1.15101.683.8 ± 13.840 min42.27.23.5 ± 0.466.156.8 ± 12Table 27 Day Old PlateletsUV ExposurePlt countpHJC-1 RatioCollagen Aggregationin vivo Recovery20 min78.17.22.85 ± 0.95116.231.8 ± 6.240 min66.17.1nd5.52.27 ± 0.31For in vivo recovery, approximately 1x10 9 platelets (UV-treated or control) were injected into the tail vein of SCID mice (n=4 per each condition) and serial blood samples were collected. Human platelets were detected in mouse whole blood by flow cytometry using an anti-human GPIIbIIIa mAb (clone P2). Recovery was defined as percent of human platelets in mouse circulation five minutes post infusion. Comparison of recovery between control and UV treatments was done at 4 hours post infusion. UV light treatments, 20 and 40 minutes, reduced the in vivo recovery to 83.8 ± 13.8% and 56.8 ± 12% of control, respectively, for 1 day old platelets and to 31.8 ± 6.2% and 2.27 ± 0.31% of control, respectively, for 7 day old platelets. Our data suggest that the SCID mouse model can identify a relatively subtle UV-induced platelet lesion and that it may be more sensitive than in vitro tests. With further validation, this animal model could be useful for evaluation of the impact of new technologies on the in vivo efficacy of human platelets. The views of the authors represent a scientific opinion and should not be construed as FDA policy.