Microrheology and structural quantification of hypercoagulable clots

Abstract

Hypercoagulability is a pathology that remains difficult to explain today in most cases. It is likely due to a modification of the conditions of polymerization of the fibrin, the main clot component. Using passive microrheology, we measured the mechanical properties of clots and correlated them under the same conditions with structural information obtained with confocal microscopy. We tested our approach with known alterations: an excess of fibrinogen and of coagulation Factor VIII. We observed simultaneously a rigidification and densification of the fibrin network, showing the potential of microrheology for hypercoagulability diagnosis.

Fig. 1.

Experimental setup of the detection system in reflection. A laser at 1069 nm is focused through a microscope objective on a bead inserted in the clot. The cameras CCD 1 and CCD 2 are used for centering the bead on the laser focus and the quadrant photodiode (QPD) for measuring the Brownian motion of the bead. Lenses with focal lengths 300 and 400 nm form a telescope that expands the laser beam and conjugate CCD 2 with the back focal plane of the objective. Dotted and solid lines show two series of conjugate planes: the bead, CCD 1 and the QPD on one side, CCD 2 and the back focal plane of the objective on the other.

Fig. 2.

Step-by-step method for centering the bead on the laser focus. (a) Schematics of the laser interacting with the bead in the centered position. (b) Image of the bead on CCD 1 when the bead is 2 µm below the focal plane (Z0 + 2 µm), when the bead is in the focal plane (Z0) and 2 µm above the focal plane (Z0 - 2 µm). The image with a dark center first becomes smaller, then its center changes rapidly from dark to bright. (c) Interference pattern on CCD 2 for a centered bead in X and Y (left) and for a bead displaced 200 nm in the X direction (right). (d) Normalized voltage measured by the QPD as a function of the displacement in X and Y (in µm) applied to the bead by the piezo. The slopes on this plot give the calibration factors of the QPD signal in X and Y in µm^-1.

Fig. 3.

Determination of the viscoelastic moduli. (a) Principle of the moduli determination. Starting from the recorded bead positions, the PSDs in each direction were computed, and filtered to reduce the noise: subfigure (b) shows an example of a PSD graph in one direction after filtering. Then, the compliances were determined, followed by the moduli G’ (storage) and G’’ (loss) for each direction. Finally, we took the mean of the moduli in the two directions to obtain the moduli G’ and G’’: subfigure (c) shows an example of the moduli G’ (storage) and G’’ (loss) as a function of the angular frequency ω. Finally, to determine their values at 30 and 3000 rad/s, the moduli are fitted by a power-law around each of those two angular frequencies.

Fig. 4.

Three-dimensional (3D) quantification of a standard clot. All images shown here are the Z-projection of 10 optical sections for clarity, but the quantification is done on the full Z-stack of 50 optical sections, recorded with a confocal microscope. Subpictures below show a zoom on the same region of the main image at the different steps. A bead can be seen at the center of subpictures, appearing as a black disk as beads are non-fluorescent. Analysis was made using Fiji software up to the skeletonized image. (a) Raw micrograph of a fibrin network. (b) The fluorescence distribution is used to determine an optimal threshold, and thus a noisy binary image. (c) Filtered image. (d) The filtered image is skeletonized. A last step is performed to remove the artifacts (see Supp. Mat.) due to the skeletonization. Density of branching points, density of fibers, and length of the fibers are then determined. Scale bar: 25 µm

Fig. 5.

Height dependence of viscoelastic moduli and of structural parameters. (a) Storage modulus measured for multiple beads at different heights in 4 normal clots (one dot represents one bead); (b) Fiber density (number of fibers per unit of volume) and (c) fiber length measured in normal clots at different heights. In (b) and (c), one dot represents the average measurement and its standard deviation for 4 clots.

Fig. 6.

Mechano-structural properties of clots enriched in fibrinogen. (a) Storage modulus at 30 rad/s, (b) fiber density, and (c) fiber length, measured for control and clot enriched with 10% of fibrinogen. In (a), each point is the average value from three to five different beads in one given clot. In (b) and (c), each point is the measurement on a given clot. (*): p < 0.05, (**): p < 0.01.

Fig. 7.

Mechano-structural properties of clots enriched in Factor VIII at 400%. (a) Storage modulus at 30 rad/s, (b) fiber density, and (c) fiber length, measured for control and clot enriched with 10% of coagulation Factor VIII. In (a), each point is the average value from three to five different beads in one given clot. In (b) and (c), each point is the measurement on a given clot. (**): p < 0.01, (***): p < 0.001.