The Study Of Deep Hypothermia Of Viruses Inactivated Platelets

ObjectiveUVB inactivation device developed in our laboratory was made to explore the inactivation effects of riboflavin combined with UVB on the two indicator viruses,Sindbis virus(SINV)and pseudorabies virus(PRV),in platelet suspension.Effects including the effective inactivated conditions and the changes in platelet quality was evaluated to ensure the functional quality of platelets and also improve the safety of platelets.The platelets treated with riboflavin/UVB mentioned above were frozen to study their functional and quality change,which was designed to prolong the shelf-life of platelet while improving the safety of platelets.MethodsOur laboratory cooperated with LANGPRO company had designed a UVB inactivation device based on the principle of riboflavin combined with ultraviolet(riboflavin/UVB)inactivation.In this research,we aimed to study about:1)Explore the effective inactivation conditions of the inactivation device against PRV and SINV;2)Whether the screened effective conditions will affect the quality of platelets;3)Whether cryopreservation the platelets treated with riboflavin/UVB under the condition above will affect their quality;I.Screening conditions for effective inactivation of viruses in plateletsPRV,one of the indicated virus hepatitis B(HBV),and SINV,one of the indicated virus of hepatitis C(HCV)were chosen as tool viruses.1/10 volume of tool viruses were added into the Preparation of platelet concentrates(PCs),called the platelet suspensions with viruses.Referring Mirasol system(based on the riboflavin/UVA-UVB develop commercial systems)and other conditions inactivate riboflavin photochemical techniques,in this study,riboflavin solutions with different working concentrations(0,50,150,300mmol/L)were added to the platelet virus suspension,and then transferred to a disposable blood dialysis treatment container,and the platelet virus suspension was irradiated(0min,5min,10min,15min).Before and after irradiation at each time point,0.5 mL samples were taken for virus titer detection,and the microcytopathic method was used to detect the titer before and after virus inactivation,and the virus inactivation heat map was drawn.II.Evaluation in vitro of changes in platelet quality,function,metabolic parameters,etc.The platelet concentrate after virus inactivation is aseptically transferred to a special platelet storage bag,and stored in a shaker at 20 ℃-24 ℃.The day of virus inactivation treatment is defined as day 0.Take out 1mL sample on the first day for scanning electron microscopic observation,and perform other tests on the first,third,fifth,and seventh day.The main contents of the test are:detect the change of platelet count by the automatic blood routine analyzer;detect the mean platelet volume(MPV),hypotonic shock response capacity(HSR)and changes in platelet phenotype,including activation marker CD62p,apoptosis marker Annexin V analyzes the changes in platelet quality;the thromboelastogram test(TEG)is used to detect the changes in platelet contraction and hemostasis;finally,the platelet glucose and lactic acid are detected by an automatic biochemical analyzer to analyze the changes in its metabolic function.III.Cryogenic treatment of platelets after inactivation of the above virusesAccording to the preparation method of frozen platelets in the literature,5%dimethyl sulfoxide(DMSO)was added to the platelets and quickly transferred to-80 ℃ for storage.IV.In vitro evaluation of changes in platelet quality,function,and metabolic parameters after cryopreservationBefore evaluating changes in parameters such as the functional quality of platelets preserved at cryogenic temperatures,the platelets should be removed from the-80 ℃refrigerator and placed in a constant temperature water bath at 37℃for 10 minutes.The evaluation in vitro parameters are consistent with the above parameters,and the parameter detection is carried out on day 1,3,5,and 7(scanning electron microscope observation on day 1).ResultsWhen the working concentration of riboflavin was 150umol/L and the irradiation dose was 1.5J/cm2,according to the TCID50,PRV reduced the titer by 4.17±0.12logs,and SINV reduced by 4.25±0.201ogs,both are greater than 41ogs,in line with the "Guidelines for the Technical Methods and Verification of Blood Products Removal/Inactivation of Viruses" in 2002(this principle requires that the titer of the virus after inactivation reduced by at least 4 logs).Therefore,we selected the riboflavin of 150 μ mol/L and the radiation dose of 1.5J/cm2 as the best effective conditions.In scanning electron microscopy,fresh platelets had regular ultrastructure,with fewer pseudopods sticking out,which indicating that most of the platelets were in a resting state;the platelets after virus inactivation have irregular ultrastructure,with relatively multiple pseudopods stretched out,the platelets were obviously activated.After inactivating,the platelet volume increasing,hypotonic shock response(HSR)capacity decreasing,and the positive rate of Annexin V-FITC(apoptosis marker)and CD62P(activation marker)increasing;In the functional test,MA value of the TEG test was not significantly different from fresh platelets;in the energy metabolism test,the platelet glucose content after virus inactivation decreased significantly,the lactic acid content increased significantly,and the pH decreased significantly(P<0.05).Deep cryopreservation caused certain changes in platelet quality and function.In scanning electron microscopy,it could be observed that the ultrastructure of the platelets preserved irregular at deep low temperature,with more pseudopods sticking out,indicating that the platelets were in activated state;MPV increasing,HSR capacity decreasing,and the positive rate of Annexin VFITC and CD62P increasing,in the functional index test,the MA value in the TEG test was not significantly different from that of the conventionally platelets;in the energy metabolism test,it could be found that the glucose content of the platelets stored at deep low temperature increases significantly,and the lactic acid content Significantly decreased significantly(P<0.05).ConclusionIn summary,when added 150 μmol/L riboflavin as a photosensitizer,combining with 1.5J/cm2 UVB irradiation,the titers of PRV and SINV could be reduced effectively;inactivating and cryopreserving had an effect on platelet morphology and quality.The deep cryopreservation process had a significantly better impact on the platelet morphology and quality than inactivation,but its function and metabolism index change is not obvious.In summary,there is a certain feasibility for deep cryopreservation of riboflavin/UVB-inactivated platelets.