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. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Anatomy / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/33162 |
| Date | 28 January 2014 |
| Creators | Buys, A.V. (Antonia Vergina) |
| Contributors | Pretorius. E |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2013 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria |
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