Study On The Inactivation Of Human Peripheral Blood Lymphocytes Using Riboflavin Photochemical Treatment

Background Transfusion is a public health measures which can both mobilize social resources and protect people’s health. It is also an important supporting condition for modern medicine. Along with the progress of social civilization and the development of clinical medical technology, its significance will highlight. However, transfusion exists the risk of adverse reaction. Therefore, how to insure clinical effect of transfusion and prevent its risk always be the hot topic in the medical profession and the public health administrative department. Blood transfusion risk including infectious blood transfusion risk and immunological blood transfusion adverse reactions. Viruses, bacteria and parasites can infect recipients through transfusion, which constitute the infectious risk of transfusion. Red blood cells, platelets, white blood cells and plasma proteins, which contain different immunogen, can lead to different immunity transfusion adverse reactions. The transfusion associated with graft versus host disease (TA-GVHD) caused by the allogeneic lymphocytes is the most serious consequences of immunity transfusion risk. Blood pathogens inactivated is the key technology to eliminate infectious transfusion risk. Riboflavin photochemical treatment (PRT) is a kind of blood pathogens inactivated technology. It is known that the riboflavin can combine with nucleic acid, and under the action of light it can lead to nucleic acid’s fracture and prevent its copy. Similarly, it can also inactivate lymphocytes in blood. The aim of this study was to discuss the feasibility and appropriate method of using PRT to inactivate lymphocytes in blood.Objective Research the effect and mechanism of PRT to human peripheral blood lymphocytes, which is under the irradiation of visible light in420nm.Methods1) Lymphocytes separated from whole blood were suspended in autologous plasma. For experimental groups, riboflavin was mixed with the cell suspensions in a final concentration of0μM,50μM,100μM and200μM. The mixture was transferred into P VC bags, and then exposed to420nm visible light at a final dose of10J/ml,20J/ml,30J/ml and40J/ml. The control group didn’t receive any processing. Both lymphocytes aforementioned were tested for the proliferative ability and the level of several cytokines after the stimulation of phytagglutinin (PHA). Eventually, determine the optimum conditions of RPT. on the inhibition of lymphocyte.2) AS for experimental group, lymphocytes were treated by PRT at the best conditions above. Another part of the lymphocytes were exposed to y-irradiation at25Gy, which were as for irradiation group. The control group didn’t have any processing. All treated and untreated samples were tested for cell morphology, Proliferation inhibition rate, the secretion of cytokines, cell apoptosis and DN A damage to analyze the inactivation effects and mechanisms of RPT on lymphocytes.Results1) RPT under the excitation of Visible can inactivate lymphocytes. And with the increase of riboflavin concentration and light dose, the inactivated effect was increasing. In the condition of riboflavin concentration at100μM and the light dose at40J/ml, the inactivated effect was the best, one.2) Further analysis shows that the number of experimental group cell was significantly lower than the control group and the irradiation group, and most of the cells were died, and under PHA stimulation, the experimental cell proliferation colony was significantly less than other two groups.After RPT, the proliferation inhibition rate of lymphocyte was82.8%stimulated by PHA, which was significantly higher than irradiation group. The amount of TNF-α、IFN-γ、IL-1β and IL-10cytokine secretions of experimental group were significantly lower than those of control group. And there was no difference in IL-6and IL-8cytokine secretions of these two groups. The results showed a significantly reduction of the number of CD3+CD4+T cell and the value of CD4+/CD8+.3) Both of experimental group and irradiation group were detected Phosphatidylserine exposure, DNA ladder and Cleaved PARP and DNA damage. This indicates that riboflavin photochemical treated induces lymphocyte apoptosis, and its targets for the nucleic acid of lymphocyte.Conclusions1) Riboflavin photochemical treatment with visible light can inactivate lymphocytes’ proliferation and secretion of cytokines. its inactivated effect is better than that of irradiation.2) The riboflavin photochemical treatment can also induce lymphocytes apoptosis and damage their nuclear acid. The research suggested that PRT is expected to prevent TA-GVHD and transfusion transmitted diseases at the same time.