Platelet Cryopreservation Using A Combination Of Trehalose And Phosphate As Cryoprotectants

Objective: The aim of this study was to observe the protective function of trehalose and phosphate at -80℃on human apheresis platelets, and to realize the long-term(3 months) protective function of this platelet storage solution.Methods: Human apheresis platelets were treated with trehalose plus phosphate, 6% DMSO and 2% DMSO plus TS, then placed in -80℃refrigerator. After frozen for 1 week, 1 month and 3 months, samples were thawed and analyzed. Measurements included platelet count, hypotonic shock response, ADP induced aggregation, apoptosis and ultrastructure of platelets.Results: (1) The apoptosis rate of platelets before and after loading trehalose were (1.3±0.5)% and (1.9±1.1)% (P>0.05), respectively. (2) The apoptosis rate of platelets treated with trehalose(without phostphate) and trehalose plus phosphate were (11.6±3.9)% and (6.4±0.9)% (P<0.05), respectively. (3) The platelet count of platelets treated with trehalose plus phosphate for 1 week,1 month and 3 months were (905±52)×106, (920±50)×106 and (880±55)×106, respectively.There were no statistical difference among the platelet count of 6% DMSO group, 2% DMSO plus TS group and trehalose plus phosphate group (P>0.05). (4) The results of HSR of platelets treated with trehalose plus phosphate for 1 week,1 month and 3 months were (53.5±8.0)%, (51.3±7.8)% and (50.1±8.8)%, respectively. The results of HSR of trehalose plus phosphate group were significantly higher than 6% DMSO group and 2% DMSO plus TS group(P<0.05). (5) The results of PAG of platelets treated with trehalose plus phosphate for 1 week,1 month and 3 months were (34.3±3.0)%, (32.7±3.5)% and (31.3±3.0)%, respectively. The results of PAG of trehalose plus phosphate group were significantly higher than the other two groups(P<0.05). (6) The apoptosis rate of platelets treated with trehalose plus phosphate for 1 week, 1 month and 3 months were (2.8±0.9)%, (6.0±2.2)% and (6.4±0.9)%, respectively. The apoptosis rate of trehalose plus phosphate group were significantly lower than the other two groups (P<0.05). (7) Platelets treated with trehalose plus phosphate retained intact ultrastructure.Conclusion: The loading process of trehalose has no effect on apoptosis of platelets. The efficacy of trehalose plus phosphate in suppressing apoptosis of platelets during cryopreservation is superior to that of only trehalose. Compared to 6% DMSO and 2% DMSO plus TS, trehalose plus phosphate can retain the same platelet count, superior hypotonic shock response and platelet aggregatory reaction to ADP, and lower apoptosis rate of platelets. Platelets cryopreserved with trehalose plus phosphate can retain intact ultrastructure.