Study On The Methodology And Mechanism Of Riboflavin Photochemical Bacteria Inactivation In Blood

Background The rapid development of transfusion is an important guarantee for the development of modern medicine. But everything has its two sides, blood transfusion also companied with risks, immunological risk and pathogen infection risk. In order to control the risk of infection, in recent years has developed a variety of technologies, such as blood donor screening, leukocyte filter, third and fourth-generation enzyme-linked immunosorbent assay (ELISA), nucleic acid detection technology (NAT). The application of these technologies has made the risk of transfusion-transmitted HBV infection decrease to less than1/106. However, the risk for bacterial infections has remained stable in general and is estimated to range from1:2000to1:3000. In some countries, the rate of bacterial contamination of platelets is up to15.6%, especially66.7%of those have multiple drug resistance. The initial amount of bacteria in contaminationed blood is usually10to100per bag, existing detection means can not achieve such a low detection limit, current detection means consume too much long time, and require professional technical staffs. Photochemical pathogen inactivation technique shows a strong advantage. Riboflavin itself is a human vitamins (vitamin B2) and the illumination product flavin is also a normal human metabolites. Secondly, previous studies in our laboratory have found riboflavin photochemical methord can effectively inactivated cells and some viruses suspended in plasma. Therefore, this study aimed to study riboflavin photochemical method for bacterial pathogen inactivation in blood.Objective Identify the most effective appliance and programs for bacteria inactivation, research the platelet function after handled by the pathogen inactivation system.Methods1. Irradiated the E. coli suspended in plasma with different wavelengths of UV-visible excitation light between300nm and550nm.2.Added different types of quenchers, mannitol, vitamin C, histidine and tryptophan to the inactivation system to identify the reaction type.3. Determined the optimal inactivation equipment and methods considering the inner filter effect.4. Treated the platelets with the established program and add type II quenchers (ROS quencher) to the system; Sade the treated platelets to3days or5days and examined the function of platelets.Results1.313nm and420nm excitation light could effectively inactivate E. coli suspended in plasma by2log/ml.2. When operated the research in cell culture plate, vitamin C enhanced the inactivation effect, other quenchers have no impact on the inactivation effect. All quencher had no impact on the inactivation effect when handled the plasma in PVC bag.3. In PVC bags, reducing the thickness of the liquid layer significantly improved Escherichia coli and Staphylococcus epidermidis inactivation effect to approximately31og/ml.4. Vitamin C and alpha lipoic acid were not conducive to platelet storage, glutathione (GSH) effectively preserved platelet function to5days.Conclusions1. Riboflavin combined with313nm excitation light can effectively inactivate E.coli and Staphylococcus about3log/ml. Specific inactivation conditions: radiation dose is5J/ml, irradiation time is30minutes, and irradiation temperature is22～27℃, irradiation equipment is PVC blood bags (d=0.26mm), vibration frequency is60r/min. Compared with similar foreign equipment can only deal with one person platelets, the experimental device we developed make it possible for large-scale platelets processing.2. In terms of the inactivation mechanism,1) discovered adding quencher has no effect on riboflavin photochemical bacterial inactivation;2) We discovered that thiol-containing protective agents can effectively protect the riboflavin photochemical function of treated platelets, especially adding4mM glutathione (GSH) can effevtively protect the function of platelets to5days;3) first discovered oxygen environment is not conducive to inactivate riboflavin photochemical method, base on that we cleared that the inactivation of bacteria in the blood depends on the non-oxygen type I reaction pathways;4) explicitly proof that inner filter effect is an important factor affecting the inactivation effect.