Comparative Proteomic Study On Plasma Treated With Methylene Blue For Pathogen Inactivation And Fresh Frozen Plasma

Objectives:Plasma takes approximately 50% of blood component,and plasma infusion is an important part of clinical therapy.The main clinical application are methylene blue virus inactivated plasma and fresh frozen plasma.The comparative analysis on the above two kinds of plasma has been focused on plasma coagulation factor,plasma protein content and the corresponding clinical biochemical changes,but there are few studies on changes of specific proteins between the two kinds of plasma.In this study,comparative proteomics methods were used to identify the alterations of some specific differential proteins between fresh frozen plasma and plasma treated with methylene blue for pathogen inactivation,in order to provide the experimental and theoretical basis for clinicians to treat patients who need blood transfusion,to avoid irrational and ineffective infusions,especially for some particular cases.Methods:1.Prothrombin time and fibrinogen content after transfusion in 62 patients with different plasma infusions were analyzed statistically.2.Trichloroacetic acid(TCA)/ acetone precipitation method was used to remove high abundance albumin in plasma.3.Ammonium sulfate precipitation method was used to remove high abundance albumin in plasma.4.SDS-PAGE was used to examine the effectiveness of high abundance albumin removing in plasma by TCA/acetone precipitation or ammonium sulfate precipitation.5.Clean up kit was used to remove interfering substances in the protein before2 DE.6.The effectiveness of using clean up kit was determined by running SDS-PAGE and 2DE.7.Proteins of both virus inactivated plasma and fresh frozen plasma wereseparated and analyzed by 2D electrophoresis.8.Identification of differential proteins between the two kinds of plasma was analyzed by mass spectrometry and searching of database.9.Selected proteins with obvious differences were verified by Western blot and ELISA analysis.Result:1.The statistical analysis of the two groups of plasma transfusion revealed that both plasma are effective in clinical treatment,but the fresh frozen plasma group of patients showed better prothrombin time(PT)value than the virus inactivated plasma group with statistical significance(P<0.05).While the difference of plasma fibrinogen content was not statistically significant between groups(P = 0.072).2.TCA/acetone precipitation method was better than saturated ammonium sulfate precipitation method to remove high-abundance albumin.3.The effectiveness of using clean up kit to remove interference substances in plasma before 2D electrophoresis was not obvious.4.Pathogen inactivated fresh frozen plasma by using methylene blue method,showed some modifications of some special proteins in 2DE map.5.The modified proteins were as follows: Complement C1 r subcomponent,Inter-alpha-trypsin inhibitor heavy chain H4,Keratin,type II cytoskeletal 1,Hemopexin,Fibrinogen gamma chain,Transthyretin.6,Keratin 1(Keratin,type II cytoskeletal 1)was detected in 18 cases of fresh frozen plasma and virus inactivated plasma by Western Blot with no significant differences.7.Hemoglobin binding protein(Hemopexin)detected in 48 cases by ELISA showed higher concentrations in cases of fresh frozen plasma than that in plasma treated by methylene blue for virus inactivation.Conclusion:The prothrombin time(PT)in patients with fresh frozen plasma infusion is significantly better than that of patients with methylene blue treated plasma infusion.The results by comparative proteomics reveal that MB has made some significantmodifications in some plasma proteins,such as Complement C1 r subcomponent,Inter-alpha-trypsin,Inhibitor heavy chain H4,Keratin,type II cytoskeletal 1,Hemopexin,Fibrinogen gamma chain and Transthyretin.The effects of protein modifications on their functions,as well as on the profound influences of the MB treated plasma infusion in clinic,need to be further studied.