Standardization of scanning electron microscopy analysis of fibrin fiber diameter measurement: communication from the ISTH SSC subcommittee on FXIII and fibrinogen.

backgroundThe microscopic characteristics of the fibrin clot structure, such as fibrin fiber length and diameter, network fiber density, and pore size, can be correlated with mechanical properties, permeability, susceptibility to lysis, and diseases. Several of these properties are commonly assessed using scanning electron microscopy (SEM); however, there are currently no standardized protocols on clot preparation for fiber diameter determination using SEM. This has led to a large discrepancy in values reported for healthy individuals and precludes interlaboratory comparisons.

objectivesTo develop a standardized protocol for the preparation of blood plasma clots for SEM analysis and fibrin fiber diameter determination.

methodsA standardized sample preparation protocol using widely available materials and matching fibrinogen concentration and molality of normal human blood was established. This protocol, in addition to in-house protocols, was reproduced and tested by 8 laboratories across the world. Testing also included evaluations of sample air drying with hexamethyldisilazane versus critical point drying, fiber diameter measurements on the surface and inside a fractured clot, and manual versus automated measurement of fiber diameter.

resultsReported fiber diameter values were in good agreement across laboratories, with an average median diameter of 110 ± 13 nm, coefficient of variation = 12% (hexamethyldisilazane drying) and 99 ± 7 nm, coefficient of variation = 7% (critical point drying). There were only small, practically insignificant differences between the diameters of surface fibers and internal fibers. Manual and automated measurements showed reasonable agreement.

conclusionThe standardized protocol is recommended as a reference point for preparing plasma clots from the blood of healthy individuals for SEM.