Assessment of a dimethyl sulfoxide-stabilized frozen canine platelet concentrate.

OBJECTIVE: To assess platelet count, mean platelet volume (MPV), metabolic characteristics, and platelet function in a dimethyl sulfoxide (DMSO)-stabilized canine frozen platelet concentrate (PC). SAMPLE POPULATION: 11 units of a commercial frozen PC in 6% DMSO and fresh platelet-rich plasma from 6 healthy control dogs. PROCEDURES: PCs were thawed, and the following data were collected: thaw time, platelet count, MPV, pH, PCO2, and PO2 and HCO3-, glucose, and lactate content. Phosphatidylserine translocation was determined by use of flow cytometry. Fresh platelet-rich plasma from healthy dogs served as a source of control platelets for flow cytometric analysis. RESULTS: At thaw, the platelet count in the frozen PC ranged from 243,000 to 742,000 platelets/microL. Median platelet count of paired samples was 680,000 platelets/microL and decreased significantly to 509,000 platelets/microL at 2 hours after thaw. Median MPV at thaw was 11.15 femtoliters and was stable after 2 hours. Compared with fresh platelets, frozen PC had increased amounts of phosphatidylserine in the outer leaflet of the platelet membrane in the resting (ie, not treated with thrombin) state (19% vs 99%, respectively) and alterations in cellular morphology, all of which were consistent with platelet activation. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this in vitro study indicated that there was a decrease in platelet quantity and function as well as an increase in platelet activation during the freeze-and-thaw process in DMSO-stabilized canine frozen PC. In vivo effects on PC remain to be determined.

Assessment of platelet growth factors in supernatants from rehydrated freeze-dried equine platelets and their effects on fibroblasts in vitro.

OBJECTIVE: To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. ANIMALS: 6 clinically normal adult horses. PROCEDURES: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor beta1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS). RESULTS: Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor-stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS. CONCLUSIONS AND CLINICAL RELEVANCE: The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.

In situ assessment of erythrocyte membrane properties during cold storage.

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.