Study On Transfusion-associated Pathogen Inactivation And Detection Technology

Background Transfusion is one of the important medical sources in medical therapy and an indispensable method in modern medicine. But there are some risks of transfusion complications. Transfusion risks primarily consist of immunological transfusion reactions and tranfusion-transmitted infectious diseases. Particularly, HBV/HCV/HIV and other viruses can cause severe infectious diseases. How to reduce and even overcome these risks has become the focus of medical research. With the social progress and development of blood testing technology, the prevalance rate of transfusion-related infectious diseases has decreased greatly, however because of the "window period" of pathogen testing and other non-tested blood transmitted viruses such as WNV, CMV, HTLV, EB etc, the donor screening and blood testing can not completely block viral infection. The fundamental measure to prevent viral infectious diseases is virus inactivation of blood and blood components. Recently, several related technique have been applied to inactivate the viruses in blood products, such as methylene blue (MB) photochemical treatment,8-methoxypsoralen+UVA and riboflavin (RB) photochemical treatment, etc. Among these technique, MB treatment has already been extended to be applied for clinic blood supply in our country. RB photochemical treatment has also been authorized to inactivate the pathogene in plasma and platelet by EU. Some reports even indicated that MB treatment can be used in clinic to treat the HBV patients'peripheral blood to decrease the titer of virus and cure the patients. But its mechanisms are unknown. Nowaday, the main studies focused on the effectiveness and the mechanisms of photochemical virus inactivation methods. However, there are fewer reports on the immuno function regulated by the inactivated virus. Objectives In this study, MB and RB photochemical virus inactivation methods has been used to treat HBV. and then, PBMCs collected from blood donors were incubated with the inactivated virus to analyze the changes of viral immunogenicity and the effect on the cytokine secretion by mononuclear cells. Methods The supernatant of HepG2.2.15 cell culture medium which contained HBV particles was collected and purified. Then the supernatant was treated by MB+ visible light and RB+visible light for inactivation respectively, and then incubated with PBMCs from 6 healthy donors. The supernatant was collected at Oh,24h and 72h. ELISA was performed to test HBsAg and HBeAg, and quantitative real-Time PCR assay was performed to test the up or down-regulated of the mRNA level of co-stimulatory molecules and cell adhesion molecules. ELISA was also performed to detect the secretion of cytokines of PBMCs. Results The concentrations of HBsAg were not changed in both of the two kinds of photochemical treatment groups (P>0.05). So the antigenicities of HBV were not affected by the treatment. After MB photochemical treatment, the concentration of HBeAg was not significantly changed (P>0.05). Compared with the control, MB treatment could increase the secretion of TNF-a to 14±10 folds, and IFN-γto 12±6 folds, but the secretion of IL-2, IL-4, IL-10 were not significantly changed. And the mRNA expression level of costimulatory molecules and cell adhesion molecules had also been up-regulated in MB treatment group (P<0.05). After RB photochemical inactivation, however, the concentration of HBeAg was decreased significantly when compared with the control (P<0.05). The secretions of TNF-a, IFN-y, IL-2, IL-4, and IL-10 were not changed, and the expression level of costimulatory molecules and cell adhesion molecules also were not changed in RB treatment group. It showed that the immunogenicity of HBV was disappeared after RB treatment. These results also suggested that the mechanisms of two viral inactivation methods were different. Conclusion The mechanisms of MB photochemical virual inactivation treatment and RB photochemical virual inactivation treatment were different. The antigenicity and immunogenicity of HBV were not affected in MB photochemical treatment, however, although the antigenicity of HBV was not changed, the immunogenicity was lost when perform RB photochemical treatment.