Evaluation of Canine Platelet Cryopreservation Methods
ACVIM 2008
E.H. Appleman1; B.S. Sachais2; R. Patel1; K.J. Drobatz1; R.P. Groman1; D. Kennedy1; P. O'Donnell1; C. Bryan1; M.B. Callan1
University of Pennsylvania, 1School of Veterinary Medicine and 2School of Medicine
Philadelphia, PA, USA

Platelet cryopreservation allows long-term storage and immediate availability of platelet products for transfusion. The aims of this study were: 1) to compare two methods of canine platelet cryopreservation, the standard rapid freeze (-80°C) in 6% DMSO (DMSO) vs. 2% DMSO plus Thrombosol (Thrombosol--a mixture of second-messenger effectors that may lessen platelet freezing damage); and 2) to determine if either cryopreservation method produces a platelet product with acceptable function and survival as compared to fresh platelets.

Platelet concentrates (PC), containing ~ 3 × 1011 platelets in ~ 220 mls plasma, were collected via apheresis from 10 healthy research dogs. Each PC was split into 3 units: fresh and cryopreserved in DMSO and Thrombosol. In vitro platelet evaluation included optical aggregometry (agonists and final concentration: γ-thrombin 100 nM, convulxin 10 nM), baseline and post-thrombin stimulated P-selectin expression, and platelet morphology via phase microscopy. In vivo platelet survival was determined by administration of biotinylated platelets to 30 healthy research dogs which had not been previously transfused or pregnant; no dog received more than one platelet transfusion. Cryopreserved units were evaluated 1-10 weeks post-freezing.

Both γ-thrombin- and convulxin-induced platelet aggregation (median amplitude, % increase light transmittance) were markedly diminished in DMSO (10% and 9%, respectively) and Thrombosol (0% and 12%, respectively) cryopreserved platelets in comparison to fresh platelets (64% and 80%, respectively) (P = 0.001 for all cryopreserved vs. fresh). Baseline P-selectin expression was < 1% for fresh and frozen platelets. To determine if platelets could be activated, P-selectin expression was determined post-thrombin stimulation. There was no difference in thrombin-induced P-selectin expression between the cryopreserved platelets (mean, DMSO 20.9%, Thrombosol 16.5%), but both exhibited significantly less activation than fresh platelets (mean, 43.8%) (P < 0.0001). The percentage of discoid and spherical platelets was not different between the 3 platelet groups. Fresh biotinylated platelets survived 7-9 days in all 10 recipients, with a mean half-life of 3.8 days. In the DMSO and Thrombosol groups, 4 of 10 dogs in each group had > 1% circulating biotinylated platelets on day 7, but the mean half-life was 1.7 days and 2.5 days, respectively, both significantly less than fresh platelets but not different from each other.

Thrombosol did not provide any appreciable benefit for maintaining in vitro function or prolonging in vivo survival of cryopreserved platelets in comparison to DMSO. Cryopreserved platelets can be activated, as demonstrated by thrombin-induced P-selectin expression, and survive in the circulation long enough to potentially be of benefit in the management of life-threatening hemorrhage in severely thrombocytopenic or thrombopathic patients. Further studies are needed to assess in vivo function of cryopreserved platelets.

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Elizabeth Appleman


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