PB0619 - Clot Collapse in Scanning Electron Microscopy (SEM)

Background: Preparation of blood clots for SEM imaging and analysis is technically challenging. Dehydration and drying steps may result in shrunken clots, fibers that are collapsed upon each other, and fibers having a ribbon-like rather than a cylindrical appearance. Improper clot preparation may result in images that are a poor representation of the native clot structure.

Aims: Our aim was to evaluate current methods of clot preparation for SEM imaging and analysis and present a method that results in representative images of the overall 3D structure of native clots.

Methods: We compared hexamethyldisilazane (HMDS) and critical point drying preparation methods of blood clots. In vitro clots were made from fresh frozen plasma, purified fibrinogen and lyophilized fibrinogen. Resulting clots (> n=5 for each method/ sample) were imaged and compared qualitatively under SEM. Average fiber diameters were measured for each clot and preparation method.

Results: Standard HMDS drying methods resulted in clots that were more likely to be collapsed with larger average fiber diameters than those from clots dried using critical point drying with either ethanol or acetone.

Conclusion(s): Critical point drying to prepare clots for SEM imaging and analysis is more likely to produce clots that are un-collapsed with smaller fibers than HMDS drying. HMDS preparation can still result in representative clots, similar to those from critical point drying; however extra precautions, like structurally supportive clot containers, may be needed.