A Study On The Cryopreserved Platelet Preparation, Cryobiologic Parameters Determination And The Characteristics Of It's New Subpopulations

Platelet demands increase twenty percent per year with the development of medical tech in China. Because of the increase demand and the limited shelf life (five days when it stored at 22 degree C with gentle rotation), platelet shortages occur frequently, and the conflict will become severely in the future. In recent four years, platelets preservation and application tech research is hotspot and difficulty in international transfusion medicine area. The relative reports are rare in China. Many literatures indicated that cryopreservation is effective solution for platelets long storage. Presently frozen platelets are the only accepted alternative to conventional platelet concentrations even if cryopreservation is cumbersome and expensive. The platelets storage techs were hold back by the complex functions, small volume and susceptibility to be activated of platelets. This paper is one part of the item-Physical Characteristics and Laws of Platelets during the Cryopreservation Process at Low Temperature - the research plan presided by my tutor Prof. Liu. The factors affect the quality of platelets during the course of preparation, two cryobiology parameters of platelets and the new characteristics of new subpopulations in cryopreservated platelets were studied based on methods selected or set up in our laboratory. This paper was divided into five parts.The First Part, Flow cytometry assay research for molecules in thesurface of plateletsSection l Flow cytometry methodology research for detecting CD62preserve of preserved platelets stimulated with thrombinObjective 1) To establish and optimize a flow cytometry method which is used to test CD62p reserve incubated with thrombin; 2) Daily determinating the CD62p reserve of platelets stored at 22 癈 to assess its value in evaluation of preserved platelet quality. Methods 1) Statistic methods including factorial experiment was carried out to optimize the major conditions for sample management, and the feasible negative and positive control for FCM analysis of CD62p expression were check out. 2) Thirteen donors' platelets including ten PRP and three apheresis concentrations incubated in a flatbed shaker at 22? "C were prolonged to twelve days. The CD62p reserve were detected daily. The line corelation between CD62p reserve and in vivo residual life 歱an of 22 癈 stored platelet were analyzed. Results 1) Satisfying conditions for analysis the CD62p reserve by FCM were obtained. 2)The CD62p reserve and in vivo residual life 歱an of 22 "C stored platelet have a imitate positive linear correlation. The value of respective r range from 0.91 to 0.99, and that of the total r is 0.99癈onclusion 1) This FCM method is simple, sensitive, stable, and easy to repeat out. 2) It is valuable to monitor the quality of preserved platelet by detecting its CD62p reserve, and the potential value for new tech research in platelet preservation is predictable.Section 2 Flow Cytometry Combined Assay for Phosphatidylserine and CD62pExpressed by Preserved PlateletsHuman platelets have distinct characters when preserved by different methods. A efficient cytometric assay for different preserved platelets expression of CD62p and phosphatidylserine(PS) is in dire need. Efficient flow cvtometric assay for CD62p and PS expressed by preserved platelets was established and the major conditions were optimized. The platelets need not to be washed to wipe off plasma and can be labelled diredtly during the sample preparation. It is efficient for flow cytometric analysis when fresh platelet riched plasma(FPRP) was set as negative control, thrombin actived FPRP and liquid nitrogen treated FPRP were set as positive control respectivelv. Gly-Pro-Arg-Pro acetate salt (GPRP) was applied to prevent platelets aggregation and fibrin formation, stabilize platelets and minimize the artifical platelets activation. This is also the key to conquer difficulty of flow cytometric quantitive analysis when platelet, Ca2+ and plasma coexist. This flow cytometric method is specially suitable for the...