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    Poster Networking

    Fibrinolysis and Proteolysis

    Poster Networking Session

    PB1201 - Update on the proposed standardized method for scanning electron microscopy analysis of fibrin clots

    Tuesday, June 27, 2023
    18:30 – 19:30 ET
    Room: Exhibition
    Background: In the absence of an internationally accepted standardized method for determining fibrin fiber diameter from scanning electron microscopy (SEM) analysis, a large discrepancy in fiber diameter has been reported in the literature for healthy individuals. This precludes inter-laboratory comparison and prevents the establishment of normal and disease ranges, as are available for other cardiovascular disease risk factors.

    Aims: To develop a standardized protocol for determining fibrin fiber diameters from SEM analysis of plasma clots.

    Methods: We performed an extensive review of published protocols and synthesized a best practices protocol. This protocol describes, in detail, the steps for clot formation, washing, fixation, dehydration, drying (using both critical point and chemical drying), sputter coating and imaging. The protocol aims to preserve clot structure, and to maintain ionic strength and molality of blood through the fixation step. The protocol also aims to use materials that are widely available and has been tested by nine labs in six different countries. Human α-thrombin and fresh-frozen commercial pooled citrated plasma were provided to the participating labs.

    Results: Labs used the standardized protocol and their respective in-house protocols to prepare and process clots and determine fiber diameter values. These values will be compared and reported, as well as values obtained by centrally analyzing the diameter values using both manual measurement and an automated measurement tool (Diameter J, an Image J plug-in).

    Conclusion(s): The proposed standardized protocol should aid in diminishing discrepancies in fibrin fiber diameter determination via SEM analysis. This will facilitate interpretation of results, allow direct comparison of data between laboratories and aid in the development of ranges for healthy, pro-thrombotic and hemophilic individuals.