Lyophilization Of Human Platelets And Study Of Its Hematostatic Function

ObjectiveThis topic aims to freeze-dry platelets and realize platelets storing under room temperature,paving the way for the platelets hemostatics.MethodsIn the study,trehalose was used as a stabilizer to protect the platelets during freezing and drying conditions;the numbers and function of lyophilized platelets preserved under different temperature were evaluated;freeze-dried platelets were investigated as an hemostatic materials in experimental non-compressible hemorrhage,using a rat liver and femoral vein injury model.In the initial studies we used orthogonal design to optimize the platelets pretreatment formulation before freeze-drying.Three factors,that might affect platelet lyophilized effect,with three levels,are selected to arrange experiment according to the orthogonal design table L9(34).These factors are trehalose concentration,reversible platelet activation inhibitors and protein protective agent.Platelet numerical recovery(recovery),D-value of MPV and relative platelet aggregation rate(aggregation)were evaluation indexs.Platelets in nine groups were pretreated with their own pretreatment formulation,then they were pre-frozen and freeze-dried.Rehydrated freeze-dried platelets(RFDF)were tested after keeping under room temperature for one day(1d).Three bags of platelets were pretreated by the optimal combination,then freeze-dried.The FDP were compared with the FDP of orthogonal test group,with the recovery,D-value of MPV and aggregation as evaluation index,to verify the optimal combination.The ultrastructure of RFDP was observed under transmission electron microscope.At the same time,platelet were treated two other pretreatment formulation and freeze-dried,with platelet recovery and D-value of MPV as evaluation indexs.Next,the most suitable temperature for freeze-dried platelets(FDP)preservation were studied.FDPs,those processed with the best pretreatment formulation,were stored under-20?,4?,and room temperature separately.Recovery,D-value of MPV,aggregation and PDGF-BB,VEGF,TGF-beta content of RFDF were estimated when the samples were saved for Od,10d,30d,90d,180d.Finally,FDP,that were stored at room temperature for 90d,were investigated as an hemostatic materials in experimental non-compressible hemorrhage,using a rat liver and femoral vein injury model.Rats were assigned randomly to received either the FDP powder or Yunnan Baiyao(YNBY)powder.Standardized,severe liver and femoral vein injuries were induced.After the modeling,powder were applied and press initiated immediately.Bleeding time and blood loss were quantified.ResultsThe screening results of platelet lyophilized pretreatment formulation displayed that:In all groups,Recovery of RFDF fluctuated between(75.4±3.7)%and(87.7±2.4)%;D-value of MPV drifted from(1.9±0.4)fl to(3.0±0.3)fl:Aggregation floated between(29.0±4.2)%and(81.1±9.6)%.The best formulation is A3B1C2 based on orthogonal design range analysis and variance analysis results.That is to say,50 mmol/L trehalose,1?g/ml PGE1 and 30%protein protective agent a are the best combination.The verify experiment results show that,recovery of FDP treated with optimal formula was(91.0±4.0)%;D-value of MPV was(2.0±0.3)fl,aggregation rate was(80.8±6.3)%.Scanning electron microscopy studies showed the absence of pseudopodia on the membrane surface of the rehydrated platelets;however,the structure of FDP was similar to fresh platelets,the cell membrane was complete and intracellular granules structure remains.In addition,rehydrated cells were mildly swelling compared to control cells.The most suitable temperature for FDP study showed:FDPs were preserved under different temperature for Od,10d,30d,90d and 180d.RFDP recovery was no significant difference between the three groups(P>0.05);RFDP aggregation rate was no significant difference between each other(P>0.05);the content of VEGF was no significant difference between each other(P>0.05);and TGF-beta content was no significant difference between each other(P>0.05).MPV of FDPs under the same retention time was no significant difference between each other(P>0.05),while significant different from fresh platelets(P<0.05);the content of PDGF-BB under the same retention time was no significant difference between each other(P>0.05),and PDGF-BB of FDPs saved for Od were no significant different from fresh platelets(P>0.05),while saved for 10d,30d,90d and 180d significant different from control platelets(P<0.05).FDP hemostatic effect in rats demonstrated that post-treatment bleeding time was reduced(P<0.05)in the FDP group{(1.63±0.26)min/(1.94±0.27)min }and YNBY group {(2.08±0.50)min/(2.67±0.64)min } compared with the blank group{(4.49±0.59)min/(5.42±1.09)min };however the bleeding times treated with FDP or YNBY were no significant difference(P>0.05).The blood loss was reduced(P<0.05)in the FDP group{(0.293±0.131)g/(0.598±0.146)g} and YNBY group {(0.312±0.166)g/(0.636±0.235)g } compared with the blank group{(0.755±0.182)g/(1.125±0.239)g };however the blood losses treated with FDP or YNBY were no significant difference(P>0.05).ConclusionIn summary,trehalose concentration and reversible platelet activation inhibitors in pretreatment solution have an observably impact on platelet lyophilization.This study demonstrated that FDP pretreated by optimal formula with 50 mmol/L trehalose,1?g/ml PGE1 and 30%protein protective agent a have been better preserved on the quantity,function and morphology than the othergroups.FDP stored at room temperature are similar with those stored under 4?and-20?.The FDP are stable for at least 180d in the freeze-dried state at room temperature.FDP powder,the same as YNBY,can reduce hemorrhage after severe liver and femoral vein injury in rats.