Experimental Studies On The Virus Inactivation In Platelet Suspension By Using Short-wave Ultraviolet Light C

ObjectiveThis topic aims to establish a method that can inactivate Sindbis and Pseudorabies virus in platelet suspension by using short wave ultraviolet light C to improve the safety of.platelet suspension under the premise of ensuring the quality of platelets.Methods1.Establish a method of virus inactivation(1)Preparation of platelet-rich plasma:Platelet-rich plasma were prepared in buffy-coat from whole blood,collected from healthy donors in 400ml bags containing CPDA.In the operation,blood need to be mixed slowly in order to prevent blood clots and hemolysis.When the blood collection finished,whole blood units were putted into a centrifuge with big content in the 22 ?.After centrifugation(4000rpm,10min),the buffy coat was separated from the plasma and red blood cells using the hemapheresis.The platelet-rich plasma was further separated from the mixture of red blood cells and white blood cells by a further centrifugation(800rpm,10min),followed by extraction using a splint.Platelet-rich plasma were stored at 22±2? with gentle vibration.(2)Virus inactivation in platelet suspension:Platelet additive solution(SSP+ salt solution)should be prepared before the experiment.Firstly,PRP was suspended in additive soltion SSP+ after centrifugation(3000rpm,20min)discarding plasma.Secondly,1/10 volume model virus(Pseudorabies or Sindbis virus)and riboflavin sodium phosphate were added into SSP+ solution to yield a working concentration of 200 ?M.Thirdly,20ml platelet-SSP+ solution with Pseudorabies or Sindbis virus were transferred into a UVC-permeable 14×5cm illumination bag resulting in a suspension depth of 3mm.Treatment of platelets with UVC is performed 0,15s,30s,lmin and 2min.Every time after the irradiation,0.5ml samples were collected for viral titer detection.(3)Virus assay:Pseudorabies virus and Sindbis virus were grown and assayed in Vero cells.Viral titers were determined by endpoint titration in microtiter plate assays(1:10 serial dilutions,eight parallel samples per dilution).The plates were incubated at 37? in humidified atmosphere containing 5%CO2.After an appropriate incubation period the cell lawns were inspected microscopically for virus-induced changes in morphology(cytopathic effect,syncytia formation).Titers were calculated and expressed as log of tissue culture infectious doses(logTCID5a).2.Measurement of platelet function(1)PLT:platelet counts were detected by blood cell analyzer after UVC treatment.(2)Concentration of six growth factors:VEGF,PDGF-BB,TGF-?,IGF,bFGF,EGF contents of activated PRP were tested by ELISA KIT.(3)Cell proliferation assay:HUVEC cells and CCK-8 reagent were chosen to run the experiment about cell proliferation.HUVEC cells were inoculated into 96-well plates with a concentration of 5×104/ml,after being adherent for drug delivery.Every well need 100?l sample(1:320 dilution with 1640 culture medium containing 0.4%FBS,three parallel samples)according to the result of the growth factor contents.Only 1640 culture medium were added into negative well.After the cultivation of 24h,48h,72h,add 10 u 1 CCK-8 reagent for 2h incubation in humidified atmosphere containing 5%CO2 at 37 ?.Absorbance values would be measured under the main wavelength of 570nm and the dual wavelength of 630nm.Cell proliferation could be evaluated on the comparison of absorbance values.(4)Ultrastructural observation of platelet:PRP were washed by PBS buffer twice after centrifugation(8000g,2min),Ultrathin slices were prepared after the platelets immobilized by 3%glutaraldehyde at 4?.Morphology and ultra structure of platelets were observed by transmission electron microscope.Results1 1.The Vero cells were in good condition with MEM medium which is containing 10%fetal bovine serum.After 4h,some of the cells had cell attachment,and some extended its pseudopodia.After 24h,Vero cells were all becomingadherent cells which were border and clear in long spindle shape.2-3 days later,cells were grown fast and well,finally,they were fused into a dense layer like the paving stones,When the Pseudorabies and Sindbis virus infected Vero cells,some cytopathic effects may be observed through the microscope,such as cell swelling,stronger refraction,syncytium and denudation.2.Virus in platelets can be efficiently reduced by the UVC virus inactivator.If the level of Pseudorabies and Sindbis virus were going to be reduced over 4 log grades,4.32mw/cm2 light and 30s irradiation time were needed.The levels of Pseudorabies virus decreased more than 5.38 log and Sindbis virus decreased more than 4.59 log by irradiation for 1 min.At the same irradiation dose,there was no significant difference among the groups with different contents of riboflavin sodium phosphate.3.In the evaluation of platelets function,PLT were markedly reduced after UVC treament(P<0.01);at the same irradiation dose,riboflavin group was slowly reduced than no riboflavin sodium phosphate group in platelet counts,and the difference was statistically significant(P<0.05).And The results of growth factors showed that other five growth factors in treatment groups,except the factor TGF-?,were significantly lower than that in control group(P<0.05)after irradiation for 30 seconds by UVC.The content of TGF-?was higher after the treatment(P<0.05),compared with the control group.In the cell proliferation test,all samples could promote the HUVEC cells proliferation.PRP from the control group had the strongest effect on cell proliferation on 24h,P<0.05.PRP treated with only UVC had better effect on HUVEC cell proliferation on 48h and 72h.Some morphologic changes such as pseudopodium,platelet cells swollen,contents release could be observed in the two treatment group under electron microscopy.ConclusionUVC treatment can inactivate Pseudorabies and Sindbis virus in platelets suspension effectively;there were more than 4 log reduction within a short time.This reduction meets the national demand for virus inactivation of blood pruducts.And riboflavin sodium phosphate had no synergism to combine with ultraviolet light C,but it could protect platelets from injury of UVC slowly.On the other hand,UVC irradiation may lead to some damage on platelets,such as the number of platelets reduced,structure damaged.It is particularly urgent to solve the problem in the next step.