Experimental Study: The Protection Mechanism Of The New Platelets Cryopreserving Solution

In these years,with the wild adoption of blood components therapy and extensive application of new technologies in medicine,high-dose blood transfusion and difficult complex surgeries largely increase.Meanwhile,the clinical applications of platelet amount also increase dramatically.Supplying fresh liquid of platelets in time has been far from meeting the clinical needs.The routine platelet storage methods of platelets used now(5 days,22℃) is unable to meet clinical needs because of the increased opportunities for bacterial contamination and the limit of shelf life of fresh platelet concentrates. Cryopreservation of platelets is a breakthrough in clinical transfusion.Platelet preserved in this way is more efficient in clotting with less side effects.It is safe,quick,and curative.It not only meets the increasing demand for clinical transfusion of platelets but also pioneers a new path for research on platelet preservation.Presently cryopreservation is one of the best methods to keep activeness of living cells and tissues.However there are still many issues unsolved,such as the overall preserving process,the select,infusion and remove of the cryoprotective agent. Cryoprotective agent itself is toxic to platelets.It will activate and cause damage to them. Platelets are easily activated,ephemeral,and instable.These characteristics also make platelet cryopreservation more difficult.Nowadays dimthyl sulfoxide(DMSO) with a concentration of 6%is normally used as cryopreservative agent for platelet concentrates.However,the amount and function of thawed platelets decrease tremendously;besides,DMSO shows its dose-dependently toxicity and produces stench in products of metabolization in human body.An ideal method is to reduce DMSO concentration as much as possible to deduce its side effect on the premise of guaranteed preserving effect.The new platelets cryopreserving solution mixes low-concentrated DMSO(2%) and the second-messenger effectors.By reversibly acting on distinctive activation pathway and therefore restraining ectogenetic activation of platelets,it stables in-cell liveliness of platelets,reduces damage in cryopreservation, lowers DMSO concentration,increases platelet activation,and improves preserving quality.In the previous experiments,we used the new platelets cryopreserving solution to cryopreserve fresh platelets under the temperature of -80℃.One week later we study effect of thawed platelet concentrates,and found the cryoprotective agent was the same effective as 6%DMSO by the function test in vitro and platelet count.On the basis of previous research,in order to explore the new platelets cryopreserving solution and obtain related biological and physical parameters in the cryopreservative procedure,our experiment tentatively explores its mechanism from the perspective of cryobiology and cryophysics.The research is composed of the following four parts:PartⅠ:Toxicity experiment of the new platelets cryopreserving solutionObjectiveTo investigate toxicity of the new platelets cryopreserving solution towards platelet concentrates.MethodsPlatelet concentrates were obtained by stepwise by the platelet rich plasma(PRP) method after fresh whole blood being collected in 1 h,and then were divided into four groups.Group 1,new platelets cryopreserving solution in 22℃;group2,new platelets cryopreserving solution in 4℃;group3,was infused with 6%DMSO in 22℃;group 4,6% DMSO in 4℃.Expression of CD62p on surface of platelet was checked in 10min,20min,30min,40min,50min,60min with FCM and fresh platelet concentrates as control.ResultsCD62p expression of every group does not increase in extension of time(P＞0.05). There was no difference in group 1,group 2 and fresh platelet concentrates(P＞0.05). Group 3 displayed the highest CD62p expression(P＜0.05) among all the groups.Group 4 displays a higher CD62p expression(P＜0.05) than group 1,group 2 and fresh platelet concentrates but lower than group 3(P＜0.05).ConclusionThe new platelets cryopreserving solution is not toxic to platelets.When platelets are infused with it and cyropreserved in an hour,no obvious effect towards activation of CD62p on surface of platelets has been exhibited.PartⅡ:Effect of cooling rates on cryopreservation of platelets ObjectiveExplore the best freezing procedure for the new platelets cryopreserving solution.MethodsWith new platelets cryopreserving solution,platelets were preserved in the low temperature refrigeratory immediately after its temperature drop to -80℃,by 1℃/min, 10℃/min,20℃/min,and non-programming cooling rates respectively.After freezing for a week,thaw it and test its function in vitro in with fresh platelets as control.ResultsWe calculated the amount of thawed platelets and found that the group of 20℃/min＜the group of non-programming cooling rate＜the group of 1℃/min＜the group of 10℃/min＜the control group of fresh platelets,among which the group of 10℃/min and the group of 20℃/min are more statistically meaningful(P＜0.05),and the comparison between the group of 1℃/min and the group of non-programming cooling rate is less statistically meaningful(p＞0.05).The maximum aggregation induced by adrenalin showed that the group of 20℃/min＜the group of 1℃/min＜the group of non-programming cooling rate＜the group of 10℃/min＜the control group of fresh platelets.When respectively comparing the group of 20℃/min,the group of 1℃/min,and the group of non-programming cooling,we found no obvious difference,but a much lower rate than the control group of fresh platelets(P＜0.05) and a higher one than the group of 20℃/min (P＜0.05).Comparing to fresh platelets,the groups of platelets which are recovered to room temperature after freezing show no obvious change in CD41 on platelets(p＞0.05). The CD42b expression on platelets is that the group of 20℃/min＜the group of non-programming cooling rate＜the group of 1℃/min＜the group of 10℃/min＜the control group of fresh platelets,among which the comparison between the control group of fresh platelets and the group of 10℃/min is less statistically meaningful(p＞0.05) and the expression is higher than the other three groups.The CD62p expression on platelets is that the control group of fresh platelets＜the group of 10℃/min＜the group of 1℃/min＜the group of non-programming cooling rate＜the group of 20℃/min.The comparison between the group of 10℃/min and the control group of fresh platelets is not statistically different(P＞0.05),while comparison with the other three groups is statistically meaningful(P＜0.05). ConclusionFunction of platelets cryopreservated by the new cryopreserving solution has the most similarity to fresh platelets.With the new platelets cryopreserving solution,the best cooling rate is 10℃/min.PartⅢ:Experimental study of thermal properties of the new eryopreserving solutionObjectiveThe relevant low-temperature physical parameters is accumulated when platelet is kept in frozen condition to provide guidance on the ideal method of cryopreservation of platelets.MethodExothermic curve is detected by the DSC repeatly during the process of the platelet temperature being fallen when using new and 6%DMSO cryoprotectant respectively.ResultThe fusion and phase temperture of platelet group without adding cryoprotectant,2% DMSO group,new platelet cryoproserving solution group and 6%DMSO group are distributed beteen-1.02℃and-0.41℃,-1.86℃and-2.99℃,-3.78℃and-4.95℃,-4.64℃and -5.72℃respectively.The freezble water content differents significantly(P＜0.01) among 2%DMSO group,new platelet cryoproserving solution group and 6%DMSO group. However,the free water content donot differents significantly(P＞0.01) between new platelet cryoproserving solution group and 6%DMSO group.ConclusionCryoprotectant can change the platelet low-temperature physical property,and the new platelet cryoproserving solution has better function in changing the platelet freezing and melting temperture and free water content.PartⅣ:Cryomicroscopic study on ice formation of the cryopreserved plateletObjectiveTo understand the ice inside and outside of cell growth deeply and find means to inhibit the growth of ice crystals.To observe IIF of platelet concentrates cryopreservated by the new cryopreserving solution.MethodThe platelet formation of ice crystals under new platelet cryoprotectant protection is observed by using cyromicroscopy.ResultThe platelet formation of ice crystals under new platelet cryoprotectant protection was observed by using cryomicroscopy.At the micro size,direct observation and intake in the ice changes during the temperature is up and down.Ice crystals form is subjected to the cryoprotectant type,concentration and cooling rate impact.The number of ice nucleus formation is smaller at the slower cooling rate,the shape is in formation of tree branch and less coarse,and intracellular ice formation is later.Under the hight cooling rates,there is a large number of ice nucleus,growth relatively narrow growth space.the initial ice embryos cannot form into the shape of tree branch,and become meticulous dendritic of ice finally. The intracellular ice formation is earlier.ConclusionThe number and shape of the concertrated platelet are subjected to the different cryoprotectant and different cooling rate.Rational cooling rate should be adopted to get better outcomes of cryopreservation.PartⅤ:Experimental research on how cryopreservated platelets affect systemic hematogenic immunological functionObjectiveTo investigate how cryopreserved platelets protected by the new cryopreserving solution influence systemic hematogenic immunological function by means of contemporary systemic immunology theory and methodology. MethodBy means of analytical methodology for immunofluorescence labeling flow cytometry, explore effect of freezing platelets acted by the new frosting protective on system hemal immunity at molecule level.Detect variability affected by expressions of concerning immune molecule on blood corpuscle(CD55,CD59,CD25).ResultThe expression of platelet CD55:the group of homomorphic platelets＞the group of homomorphic cryopreservated platelets(the new cyropreserving solution)＞the group of 6%DMSO＞the group of autologous platelets.The comparison result between the group of autologous platelets(16.58±5.41) and the group of homomorphic platelets(23.58±7.55) is statistically meaningful(P＜0.05).However the comparison result with the other two groups shows little difference and therefore is statistically unmeaningful.The expression of platelet CD59:The comparison result between the group of autologous platelets(18.76±4.33) and the group of homomorphic platelets(24.39±6.68) is statistically meaningful(P＜0.05).However the comparison result with the other two groups shows little difference and therefore is statistically unmeaningful.The group of homomorphic platelets＞the group of homomorphic cryopreservated platelets(the new cyropreserving solution)＞the group of 6%DMSO＞the group of autologous platelets＞the group of platelets-poor plasma The expression of erythrocyte CD55:The comparison result between the group of autologous platelets and platelets-poor plasma is statistically meaningful(P＜0.05).However the comparison result with the other groups shows little difference and therefore is statistically unmeaningful.The expression of erythrocyte CD59: The comparison result between the experimental groups and the group of 6%DMSO,the group of platelet-poor plasma is very statistically meaningful respectively(P＜0.05,P＜0.01).The expression of lymphocyte CD25:The comparison result between the group of autologous platelets(12.44±5.09) and the group of homomorphic platelets(17.56±5.63) is statistically meaningful(P＜0.05).However the comparison result with the other two groups shows little difference and therefore is statistically unmeaningful.ConclusionThis experiment confirmed that some of the red blood cells and platelets can enhance expression of immune molecules on the platelet surface.By contrast we found that the effect of fresh allogeneic red blood cell on immune response is weaker than cryopreservated platelets,but activation of fresh allogeneic platelets is stronger than the cryopreservated.The influence of the cryopreservated platelet on immune system is related to the cryopreserving solution,and the new cryopreserving solution is better than 6% DMSO by comparing the influence on immune system.