Development of an ultrastructure preservation protocol for platelet and fibrin networks utilizing high pressure freezing and subsequent comparison of new and established protocols

Abstract

Abnormalities in the structure of fibrin fibers and blood platelets have been broadly studied and well correlated to functional abnormalities in the coagulation system due to disease, genetics or environmental factors. The importance of these and further ultrastructural investigations of fibrin fiber networks and platelets is therefore paramount in understanding iii the aetiology of haemorrhagic or thrombogenic tendencies and disorders. To study structural abnormalities and variations of fibrin fibers and platelets, electron microscopy is essential; unfortunately the use of electron microscopy necessitates several preparation steps to make a biological specimen stable enough to withstand the high vacuum environment of an electron microscope and also electron beam irradiation. The two most common procedures to accomplish this is chemical fixation and freeze fixation. Chemical fixation entails the chemical alteration of a specimen by means of the introduction of chemical bonds and cross-links that keep molecules and structures in place, followed by several rinsing and dehydration steps. Freeze fixation preserves biological specimens by the removal of thermal energy from the specimen at an extremely fast rate (> 105 In this study, methods to fixate fibrin fiber and platelet networks by freeze fixation was developed, optimised and subsequently compared to chemical fixation methods to ascertain the optimum preparation technique for transmission and scanning electron microscopy for ultrastructural studies of platelets and fibrin networks. K/s) allowing the water in a biological specimen to reach a super-cooled stabilized state (vitrification). The general consensus in the scientific community is that ultrastructural preservation by high pressure freeze fixation is superior to that of chemical fixation, although the facts are that different fixation methodologies have dissimilar chemical and physical interactions with different specimens and as a result different artefact introductions. Therefore the best possible specimen preparation method to ensure an accurate likeness of the fixated specimen to its in vivo condition needs to be ascertained and used. In this study, methods to fixate fibrin fiber and platelet networks by freeze fixation was developed, optimised and subsequently compared to chemical fixation methods to ascertain the optimum preparation technique for transmission and scanning electron microscopy for ultrastructural studies of platelets and fibrin networks. K/s) allowing the water in a biological specimen to reach a super-cooled stabilized state (vitrification). The general consensus in the scientific community is that ultrastructural preservation by high pressure freeze fixation is superior to that of chemical fixation, although the facts are that different fixation methodologies have dissimilar chemical and physical interactions with different specimens and as a result different artefact introductions. Therefore the best possible specimen preparation method to ensure an accurate likeness of the fixated specimen to its in vivo condition needs to be ascertained and used. Ultimately it was found that high pressure freezing coupled with freeze substitution is a superior method for fine structure preservation of fibrin fiber networks and platelets when utilizing transmission electron microscopy. Contrastingly for scanning electron microscopy ultrastructural studies it was found that chemical fixation is the more optimal method for the preparation of fibrin networks and platelets.