Structure, mechanical properties, and modeling of cyclically compressed pulmonary emboli

Abstract

Pulmonary embolism occurs when blood flow to a part of the lungs is blocked by a venous thrombus that has traveled from the lower limbs. Little is known about the mechanical behavior of emboli under compressive forces from the surrounding musculature and blood pressure. We measured the stress-strain responses of human pulmonary emboli under cyclic compression, and showed that emboli exhibit a hysteretic stress-strain curve. The fibrin fibers and red blood cells (RBCs) are damaged during the compression process, causing irreversible changes in the structure of the emboli. We showed using electron and confocal microscopy that bundling of fibrin fibers occurs due to compression, and damage is accumulated as more cycles are applied. The stress-strain curves depend on embolus structure, such that variations in composition give quantitatively different responses. Emboli with a high fibrin component demonstrate higher normal stress compared to emboli that have a high RBC component. We compared the compression response of emboli to that of whole blood clots containing various volume fractions of RBCs, and found that RBCs rupture at a certain critical stress. We describe the hysteretic response characteristic of foams, using a model of phase transitions in which the compressed foam is segregated into coexisting rarefied and densified phases whose fractions change during compression. Our model takes account of the rupture of RBCs in the compressed emboli and stresses due to fluid flow through their small pores. Our results can help in classifying emboli as rich in fibrin or rich in red blood cells, and can help in understanding what responses to expect when stresses are applied to thrombi in vivo.

Keywords: Cyclic compression; Hysteresis; Phase transition; Pulmonary emboli.

Figure 1:. Experimental set-up to obtain rheological data for compression experiments.

(A) Schematic illustration of compression of a clot formed between the rheometer plates. (B) Schematic illustration of compression of an embolus placed between the rheometer plates. Rheometer plates are shown in black. The dark gray shapes represent the clot in (A) and the embolus in (B), while the light gray shape represents the liquid expelled from the clot and embolus during the compression cycle. The clot and embolus were compressed as the upper rheometer plate moved down, squeezing liquid out of the clot and embolus. Dashed line shows changes in area after the clot and embolus were compressed. Arrows indicate liquid expelled from the clot and embolus.

Figure 2:. Fitting of stress-strain response curves

for embolus #1 to compression and decompression for the regions 0≤ϵ≤0.5, region 0.5≤ϵ≤0.8, and region 0.8≤ϵ≤0.92. The circles represent data points from the experiments, the solid blue lines represent the fully densified phase.

Figure 3:. Stress-strain curves of whole blood clots with different RBC and fibrin concentrations.

(A) clot made from whole blood with 40% RBCs, 2.5 mg/ml fibrinogen, 160,000 platelets/μl; (B) clot made from whole blood with 70% RBCs, 2.5 mg/ml fibrinogen, 160,000 platelets/μl; (C) clot made from whole blood with 26% RBCs, 2.5 mg/ml fibrinogen, 160,000 platelets/μl; (D) clot madeM from whole blood with fibrinogen concentration of 8 mg/ml, 40% RBCs, 160,000 platelets/μl.

Figure 4:. Structural changes in a whole blood clot at a compressive strain of 0.65 observed by confocal and scanning electron microscopy.

Panels A – C, Confocal microscope images, magnification bar 30 µm. Panels D – F, Scanning electron microscope images, magnification bar for (D, E) images 10 µm, for (E) 5 µm. (A) The optical section from top of compressed clot where force was applied; (B) The optical section from bottom of compressed clot, part which was lying on the stationary plate; (C) 3D reconstruction of optical sections of compressed clot; Arrows indicate broken RBCs; arrowheads indicate polyhedrocytes.

Figure 5:. Representative scanning electron microscope images of emboli before compression.

Panel (A) represent overview of pulmonary embolus at low magnification, magnification bar = 25 µm. Panel (B – E) show details at higher magnification, magnification bar = 15 µm. (B and D) Fibrin structure is primarily composed of fiber fibers, some of which are bent (1) and fibrin bundles (2). (C) Mostly deformed RBCs (4) with fibrin fibers (1) and fibrin bundles (2); some white blood cells were also present (6). (D) Dense contracted fibrin structures with deformed RBCs (4) trapped in the fibrin mesh. (E) Tightly packed polyhedrocytes (5).

Figure 6:. Representative scanning electron microscope images of emboli after compression.

Panel (A) represents overview of pulmonary embolus at low magnification, magnification bar = 25 µm. Panel (B – E) showing more detail, magnification bar = 15 µm. (A, B and D) Fibrin structures, mostly composed of fibrin bundles (white arrows) and deformed RBCs, trapped in fibrin (black arrows). (C) Tightly packed polyhedrocytes, which are oriented perpendicular to the compressive force (black arrowheads), big white arrow shows the direction of orientation. (E) Mostly broken RBCs (white arrowheads) with fiber ends (white narrow arrowheads).

Figure 7:. Heterogeneity of pulmonary emboli. Stress-strain curves and scanning electron microscope images.

Panels (A – D) scanning electron micrographs of emboli. (A, C) embolus which has high content of fibrin structures (black arrows) with some RBCs trapped in fibrin (white arrowheads). Panels (B, D), embolus that has a high content of densely packed RBCs (white arrowheads) and fibrin component mostly comprised of thin fibers (black arrows). Large white arrows show the area where fibrin was the primary component. Panels (A, B), magnification bar 25 µm; (C, D), magnification bar 15 µm. Panel (E), Maximum of normal stress at compressive strain 0≤ϵ≤0.5; 0.5≤ϵ≤0.8 and 0.8≤ϵ≤0.92 for all emboli. The white circles represent data points from embolus #2; white squares from embolus # 1; white triangles pointing up from embolus #3a; white triangles pointing down from embolus #3b; white diamonds from embolus #3c. Panel (F) Quantitative analyses of fibrin and RBC components identified in embolus #1 and #3c. Quantitative differences were compared statistically by Students t test. P values for differences between fibrin component and RBCs components are indicated by **, and *; **P=0.0004, *P=0.0001.