Experimental Study Of Protective Techniques Before Platelets Lyophilization

Objective The function and quality of platelets before freeze-drying was the key to lyophilize platelets successfully. The study was to establish serial retreating techniques of platelets freeze-drying, including conditions of trehalose loading and composing of loading solution. Aim was to load trehalose into platelets before lyophilizing without platelet-activation. Methods (1) Using 0.5M trichloroacetic acid to extract trehalose and deposit proteins from platelets loaded trehalose and sulfuric-anthrone reaction to determine trehalose, to set up the method for cellular trehalose determination. And conditions of loading trehalose including temperature, concentration, duration and the basis solution were studied. (2) PLT and MPV were detected by automatic hemanalysis; Expressions of CD62p and PAC-1 on platelet were investigated before and after induced by thrombin with Flow cytometry (FCMs). The maximal aggregation of platelets induced by thrombin, ADP, propyl gallate and ristocetin was determined with APACT-2. The platelet aggregating time was detected by SPAT. Function and status of platelets were observed using above-mentioned methods before and after trhalose loading with adenosine, prostaglandin E₁ (PGEi), L-Arginine, cilostazol, phyticacid and bivalirudin. Otherwise the six inhibitors and trehalose loading solution were analyzed using the orthogonal experimental design. Then the best compounding of trehalose loading was selected. Results (1) The method for cellular trehalose determination was established. The basic parameters of trehalose loading technique include 37℃, 4 hours, 45mM of trhalose and loading in plasma. (2) Loaded with trehalose, MPV had no change, but the expression of platelets CD62p and PAC-1 increased significantly. Trehalose loading affected platelets aggregation induced by ristocetin and ADP,...