Quantifying the mechanical and histological properties of thrombus analog made from human blood for the creation of synthetic thrombus for thrombectomy device testing

Abstract

Background

Untreated ischemic stroke can lead to severe morbidity and death, and as such, there are numerous endovascular blood-clot removal (thrombectomy) devices approved for human use. Human thrombi types are highly variable and are typically classified in qualitative terms – ‘soft/red,’ ‘hard/white,’ or ‘aged/calcified.’ Quantifying human thrombus properties can accelerate the development of thrombus analogs for the study of thrombectomy outcomes, which are often inconsistent among treated patients.

Methods

‘Soft’human thrombi were created from blood samples ex vivo (ie, human blood clotted in sample vials) and tested for mechanical properties using a hybrid rheometer material testing system. Synthetic thrombus materials were also mechanically tested and compared with the ‘soft’ human blood clots.

Results

Mechanical testing quantified the shear modulus and dynamic (elastic) modulus of volunteer human thrombus samples. This data was used to formulate a synthetic blood clot made from a composite polymer hydrogel of polyacrylamide and alginate (PAAM-Alg). The PAAM-Alg interpenetrating network of covalently and ionically cross-linked polymers had tunable elastic and shear moduli properties and shape memory characteristics.

Conclusions

Due to its adjustable properties, PAAM-Alg can be modified to mimic various thrombi classifications. Future studies will include obtaining and quantitatively classifying patient thrombectomy samples and altering the PAAM-Alg to mimic the results for use with in vitro thrombectomy studies.

INTRODUCTION

In general terms, ‘soft/red’ thrombi have also been defined as red blood cell (RBC)-rich, whereas ‘hard/white’ are typically fibrin-rich.^1–3 Both RBC-rich and fibrin-rich thrombi can also contain regions of calcifications, also referred to as ‘aged’ thrombus. For this study, aged thrombus samples will be referred to as calcified RBC-rich or calcified fibrin-rich thrombus. Although fibrin dominance is not well defined in the literature, histological analysis of thrombus with 55% or greater fibrin content constitutes a fibrin-rich clot.⁴ Alternately, thrombus containing less than 50% fibrin content, or greater than 50% RBC content constitutes an RBC-rich clot.^{2 3} The volume fractions of RBCs and fibrin within a thrombus can be correlated to its mechanical properties, using techniques to assess engineered composite materials.⁴

Neurovascular thrombectomy procedures are becoming widespread. Clinical outcomes with these devices have varied greatly from patient to patient since the devices entered the market in 2012.^5–7 Variability in clinical outcome is often attributed to the location of the thrombus in the brain (accessibility), and the ability of the thrombectomy device to incorporate with and capture the thrombus to facilitate complete thrombus removal.⁶ All four general types, calcified and non-calcified forms of RBC-rich or fibrin-rich thrombi, are found in ischemic stroke patients that undergo thrombectomy. dgjnb^{2 8–10}

Prior studies have analyzed the histological composition of thrombi retrieved following endovascular thrombectomy procedures and attempted to correlate clot composition with various clinical and radiographic procedure-related variables.^{7 8} Niesten et al classified 22 thrombi, retrieved from acute stroke patients, as fresh, lytic, or organized, and calculated percentages of RBCs, platelets, and fibrin in these clots.⁹ Neisten et al found a correlation between the RBC component and thrombus attenuation on CT scan, suggesting that pre-procedure imaging may ultimately provide some data regarding clot composition. Marder et al analyzed 37 clots from patients who underwent gradient echo (GRE) imaging prior to endovascular therapy.¹ They then analyzed the proportion of RBCs, fibrin, platelets, and white blood cells, and sought correlations between clot compositions and stroke subtypes and susceptibility vessel signs on GRE imaging. They found that histologic composition differed between clots from patients with cardio embolism relative to those secondary to large artery atherosclerosis. They also found that a susceptibility vessel sign on GRE is strongly associated with a high proportion of RBCs and a low proportion of fibrin and platelets.

There is a disconnect in the literature with respect to thrombus composition, clinical findings, histological data, and the resulting mechanical properties. While increased fibrin content and calcification are not exclusive properties of an aged thrombus, literature does relate the age and calcification effects of thrombus to its resulting stiffness. A calcified RBC-rich thrombus has a stiffness (elastic modulus – E) approximately 10 times that of a RBC-rich only thrombus. A calcified fibrin-rich thrombus has a stiffness approximately six times greater than a calcified RBC-rich thrombus, and is there-fore 60x stiffer than an RBC-rich thrombus.⁵ These stiffness values do not consider the fibrin content of the thrombi. Shear modulus (G), an indicator of thrombus viscous (permanent deformation) and elastic (recoverable) mechanical properties, has not been extensively studied in the literature with respect to thrombus composition.

The aims of the research effort are threefold. First, to non-destructively test and quantify the mechanical properties (specifically elastic and shear moduli) of RBC-rich human blood clots, which can be readily-formed from human blood samples. Second, use the intact thrombus samples that were mechanically tested and prepare them for histological analysis for calculating percent fibrin content. Third, use this quantified data to create a fully synthetic thrombus material that mimics the human thrombus mechanical properties and has ‘tunable’ elastic and shear modulus properties.

MATERIALS AND METHODS

Blood samples were acquired from volunteers, following NAU’s Institutional Biosafety Committee (IBC) Protocol. Samples were taken from two healthy demographics: male (n=6) and female (n=6) participants 20–30 years’ old. All donors lacked hematology pathologies.

Human thrombus procedure

Six thrombus samples were tested from each demographic. All human thrombus analogs were created with the same procedure: whole blood was aseptically collected in sterile Z-serum collection tubes from the healthy donors and spun down for 10 minutes at 3000 rpm, per the standard hospital protocol, to elicit Z-serum platelet activation and thrombus sample formation. The resulting samples were classified qualitatively as RBC-rich only samples. To quantify the RBC-rich human clot, the samples were tested for mechanical properties. To minimize degradation of the human thrombus, the samples were tested for mechanical properties and then fixed for histology – all within 30 min of being removed from the sterile Z-serum vials.

Rheometer setup

To properly simulate the forces acting on an in vivo thrombus, the laboratory simulated a biologic environment while mechanically testing human and synthetic thrombus samples. A novel non-destructive testing procedure was performed with a TA Instruments Discovery HR-2 Rheometer (TA Instruments, New Castle, DE). The samples were tested for shear modulus and dynamic (elastic) modulus using a 4 mm and 8 mm diameter testing head and a temperature-controlled quick-exchange Peltier plate base. The 8 mm diameter head was used for the larger human thrombus samples retrieved from the Z-serum vials. The 4 mm diameter head was used for the smaller synthetic thrombus samples, which simulate the size of thrombus samples from thrombectomy patients (typically from vessels of 4–5 mm diameters).

150-grit adhesive sandpaper disks, cut to fit the geometry, were attached to the testing head and the Peltier plate, to reduce slippage of the sample during shear and dynamic modulus testing. An immersion ring was then attached to the Peltier plate and filled with approximately 1 cc of phosphate-buffered saline (PBS) to cover the Peltier plate surface and wet the thrombus sample without submerging the testing head. The Peltier plate was maintained at 37°C for all testing procedures.

A 4 mm or 8 mm biopsy punch was used to create cylindrical thrombus samples. The cylindrical samples were cut with a scalpel to a height of approximately 3 mm (±1 mm). Thickness does vary from sample to sample, however, studies by the research team found that thrombus samples between 2–4 mm thick, exhibited consistent shear and elastic modulus results. Alignment of the thrombus sample under the top plate was critical for obtaining consistent data and reducing testing error.

Shear modulus determination

The shear modulus test devised was a non-destructive rheometer test procedure. A constant normal force of 0.02 N±0.01 N was applied to the thrombus sample throughout the shear test procedures. The normal force ensured constant contact with the sample during head rotation on the sample, eliminating any relaxation or creep effects.

A 1% rotational shear strain (0.0628 radians) was applied and released from the sample across a linear frequency range from 0.1 to 10 rad/s (20 points per test range). This range was determined from literature sources and from experience with previous hydrogel sample testing.^{11 12} Most hydrogel-like materials begin to slip at strains higher than 1% and oscillation speeds greater than 10 rad/s.^{13 14} Data from the samples were compared at 6.28 rad/s (1 Hz), which simulates pulsatile physiological blood flow.

Elastic modulus determination

The elastic modulus test devised was a non-destructive rheometer test procedure. An initial normal force of 0.01 to 0.02 N was applied to the thrombus sample at the start of the elastic modulus test. A 0.5% axial shear strain (0.5% displacement of the original height of the sample) was applied and released from the sample across a frequency range from 0.1 to 10 rad/s (20 points per test range). This range was also determined from literature sources and previous hydrogel sample testing.^11–14 Data from the samples were also compared at 6.28 rad/s (1 Hz). The intact mechanically-tested thrombus samples were then prepared for histological analysis to determine fibrin content.

Histological analysis procedure

Male and female thrombus samples, that underwent non-destructive mechanical testing with the rheometer, were immediately moved from the PBS and fixed for 72 hours in 10% solution of formaldehyde at 20°C. The samples were embedded in paraffin wax blocks. Samples were then chilled and sectioned at 5 μm. Two sections were chosen from the beginning, middle, and end of each sample, one section stained with a standard Hematoxylin-and-Eosin stain process (H&E – highlights RBCs, platelets, and fibrin) and the other section stained with Martius Scarlet Blue (MSB – stains fresh fibrin bright red, aged fibrin blue, and RBCs yellow).^{2 15}

Male and female thrombus samples were histologically prepared with H&E and MSB stains, imaged at various micro-scope magnifications, and analyzed for fibrin content using Photoshop CS6 (Adobe Systems Inc., San Jose, CA) and ImageJ 1.50i (National Institutes of Health (NIH), Bethesda, MD).

Using Photoshop, random regions of the H&E and MSB images (taken at 10X magnification) were selected by an independent investigator. The regions selected were at least 1 mm² in area, to ensure a large distribution of RBCs and fibrin. ‘Color Range’ with a ‘Fuzziness’ between 70 and 150 was used to select the fibrin in the image. Photoshop then automatically selected all fibrin in the image with a similar color range. The expanded histogram window was selected, which listed the number of pixels that represented the selected fibrin. The total number of fibrin pixels (F_p) were recorded. Image processing was repeated on the original image regions, this time selecting the RBC content. The total number of RBC pixels (RBC_p) were recorded. Percent fibrin content was determined using equation 1:

$$\frac{F_p}{F_p+RB{C}_p}\times 100\%=\%\mathit{\text{ fibrin}}$$ (1)

The percent fibrin was verified by a second investigator using ImageJ. In ImageJ, a random region of the original H&E and MSB images was selected. ‘Color Threshold’ was used to adjust the image range of hue, saturation, and brightness. Red was used as the threshold color that resulted in the best contrast. The ‘filtered’ button was used to determine the selection of fibrin content. Once selected, the ‘Analyze-Measure’ option recorded the number of pixels of fibrin. This procedure was repeated to filter out and record the number of pixels of RBCs as well. Percent fibrin was re-calculated and compared with the Photoshop results (Equation 1).

Synthetic thrombus, procedure

Once human thrombus samples were quantitatively assessed, a synthetic thrombus material was formulated to exhibit similar shear and elastic modulus properties. Hydrogels and polymer constructs were investigated as synthetic thrombus candidates. Hydrogels typically have large viscous properties and minimal elasticity. This was verified with the testing of PVA and PVA-hydrogel composites. However, RBC-rich thrombus exhibit ‘soft’ and highly elastic properties.

Polyacrylamide and alginate (PAAM-Alg), when combined with a crosslinking agent, formed an interpenetrating network of covalent and ionic bonds that resulted in tunable elastic and shear moduli with shape memory characteristics.^{13 14}

The composition of acrylamide, alginate, and cross-linking agents was altered to tune the mechanical properties of the resulting PAAM-Alg gel.¹⁶ The gel creation procedure is described in detail in the Results section.

Various compositions were created by varying three factors: weight percentage of acrylamide to alginate; the weight percentage of the solvent; and the viscosity of the solvent (DI water plus liquid contrast agent). As such, each variation was tested independently to determine its effect on the resulting hydrogel.

In order to develop a narrow scope of the changes in elastic properties, the PAAM-Alg gel was created at 3 weight-percent levels: 47.1 wt%, 72 wt%, and 88.9 wt%, PAAM. At 47.1 wt% PAAM. In order to increase the elastic properties of the gel, the percentage of PAAM was increased incrementally to 88.9 wt%. In addition, the weight percentage of solvent (DI water) was varied to four levels: 85%, 87 wt%, 90 wt%, and 93 wt% DI water. Lastly, high molecular weight liquid contrast agents were added to the solvent volume (50:50 vol% water:contrast) to reduce polymer elasticity and improve thrombus visualization during in vitro thrombectomy studies conducted using fluoroscopic imaging.

The resulting gels were then tested for shear and elastic moduli.

RESULTS

The male and female human thrombus samples (n=6 of each) were tested for shear and elastic modulus and results were reported at 1 Hz. Among all thrombus samples, the average shear modulus at 1 Hz was 200 Pa and the elastic modulus was 12 800 Pa with no significant variation between male and female samples (P values >0.05 – table 1).

Table 1.

Summary of human thrombus elastic and shear modulus values at a physiologically-relevant pulsatile frequency of 1 Hz

Thrombus	Shear modulus		Elastic modulus
Sample	(Pa)	st. dev	(Pa)	st. dev
Female	212	±25.7	13 800	±3800
Male	189	±26.1	11 700	±3760
All	200	±27.4	±12 800	±3770
P values		0.16		0.36

Human thrombus samples were found to be extremely soft in shear modulus, but over 60x stiffer in elastic modulus.

Thrombus samples exhibited modulus levels typical of low fibrin (RBC-rich) thrombus samples.³ Fibrin content was verified with histology performed on the same samples that underwent non-destructive shear and elastic modulus testing.

Histology results

An example of a female thrombus histology (H&E and MSB sections) can be found in figure 1. Fibrin is evenly distributed throughout the middle of the thrombus, with regions of concentration around the edges. Image analyses of the samples resulted in a percent fibrin range from 4% to 12%, with the overall percentage being 7% fibrin for all human thrombi samples collected and prepared in the Z-serum vials.

Figure 1.

MSB and H&E stain of a serial-sliced female thrombus sample. MSB: top left (2.5X) and bottom left (10X). Platelets and fresh fibrin stain bright red, old fibrin stains blue, and RBC content stains a dull yellow. H&E: top right (2.5X) and bottom right (10X). Platelets and fibrin stain purple, whereas RBC content stains a bright pink. Photoshop and ImageJ analysis verified the average fibrin content of 7 vol%.

Synthetic thrombus results

Results show that variations in PAAM wt% and DI water wt% (with and without liquid contrast) can independently alter shear and elastic modulus results. Lower PAAM content (72 wt%) resulted in stiffness values (elastic modulus) higher than human thrombus. Higher PAAM content (88.9 wt%) resulted in a highly elastic gel that was statistically similar in elastic modulus to human thrombus (figure 2). Elastic modulus can be further adjusted by varying water content. 88.9 wt% PAAM samples exhibited a relatively small decrease in elastic modulus with decreased solvent (DI water) content (figure 2). 72 wt% PAAM samples exhibited large decreases in elastic modulus with increased solvent (50:50 water:contrast) content.

Figure 2.

Elastic modulus of various PAAM-Alg formulations (five bars on the left) reported at a physiologically-relevant pulsatile frequency of 1 Hz and compared with human thrombus (rightmost bar). Results show that higher polyacrylamide content reduces elastic modulus, but when coupled with increased solvent content can exhibit a trend toward higher elastic modulus.

The 72 wt% PAAM samples also exhibited large decreases in shear modulus with increased solvent (50:50 water:contrast) content. However, at 88.9% PAAM, the addition of water had an inverse effect on elastic modulus – increasing elastic modulus with decreasing DI water content (figure 3).

Figure 3.

Shear modulus of various PAAM-Alg formulations (five bars on the left) reported at a physiologically-relevant pulsatile frequency of 1 Hz and compared with human thrombus (rightmost bar). Results show that higher polyacrylamide content reduces shear modulus, and when coupled with increased solvent content and liquid contrast content can exhibit a trend toward lower shear modulus.

Resulting synthetic thrombus formulation

The subsequent procedure was followed to create the final version of the ‘soft’ synthetic thrombus material. To start, the DI H₂O was placed in a vacuum flask. While stirring at 600 rpm, the sodium alginic salt (Alginate) (Sigma St. Louis, MO) and acrylamide (Sigma St. Louis, MO) was added. Stirring speed was increased to 1000 rpm to compensate for the increased viscosity of the solution. After the contents stirred for an hour, or was fully dissolved, methylenebisacrylamide (MBAA) (Sigma St. Louis, MO) and ammonium persulfate (AP) (Sigma St. Louis, MO) were also stirred into the mixture for another 30 min. Once the components were dissolved entirely, the mixture was connected to a vacuum and degassed for up to 1.5 hours. Tetramethylethylenediamine (TEMED) (Sigma St. Louis, MO) was additionally added to a DI water and CaSO₄ suspension. Immediately after the TEMED, DI H₂O, and CaSO₄ suspension was made, it was poured into the fully degassed mixture within the vacuum flask. The gelling fluid was quickly stirred and aspirated into 1 mL disposable polycarbonate syringes to mould the gel into 4 mm diameter cylinders. The syringes were left under UV light (254 nm wavelength) for 1 hour to cure. Once cured, the gel was aspirated out of the syringe and cut into desired lengths.

The final comprehensive gel consisted of 89.9% acrylamide dissolved in DI water to create a 93% wt percent DI H₂O solvent solution.

DISCUSSION

Human thrombus samples were successfully created and exhibited non-homogeneous material properties – very soft in shear, but dramatically stiffer in elastic modulus. This unique result requires a novel synthetic polymer system to accurately mimic human thrombus properties. Results show that an acrylamide concentration between 72 wt% and 88.9 wt%, with increased DI water and liquid contrast content, can approach statistically similar modulus values to human thrombus samples.

The synthetic polymer composition of 89.9% acrylamide and 93% solution (100% DI H₂O) closely mimicked the human thrombi quantitatively tested in this study. This synthetic polymer was the best initial match for both elastic and shear moduli. The research team believes increasing water content further, along with the combination of liquid contrast agent, will result in properties that can be tuned to statistically match various classifications of human thrombus samples. The research team believes the factors for controlling the synthetic modulus properties have been identified and verified through comparison with the initial human thrombus samples.

This finding will be further tested once the research team begins collecting and categorizing patient thrombectomy samples from its partnership with Barrow Neurological Institute (BNI). At that time, a factorial analysis will be run to determine the statistical significance of PAAM wt%, solvent wt%, and liquid contrast vol% on the resulting synthetic modulus properties. Once the research team begins to acquire and quantitatively classify thrombectomy samples, a statistically optimized version of synthetic thrombus will be created for each identified category of human thrombus.

Preliminary results show that PAAM-Alg has highly tunable properties and can maintain its properties for months when stored in an airtight container. PAAM-Alg is potentially an ideal candidate for thrombus modeling. Thrombectomy device optimization using synthetic thrombus analogs could reduce the need for biological-based thrombus analogs which are difficult to handle, have a shortened shelf-life, and are classified as biohazards. PAAM-Alg polymers exhibit the material properties of human vascular tissue, including mechanical strength and hydrophilic lubricity. The use of these novel polymers could revolutionize vascular modeling and device development for vascular disease and stroke treatment.

Limitations

The preliminary RBC-rich thrombus analogs created for this study are relatively homogeneous, compared with human thrombectomy samples referenced in the literature. Also, the properties of the thrombus analogs created from our donor demographic (ages 20–30) likely differ from the properties of complex thrombectomy samples collected from the average stroke patient. However, the goal of this study is to develop a mechanical testing and histology process for classifying thrombus samples into categories that will improve the accuracy of modeling various human thrombus. First by developing a new line of adjustable synthetic polymers that can be correlated to RBC-rich only samples, and then adjusted to other classifications of human thrombi.

Future work

This work has provided a methodological foundation for future research. The investigators have attained Institutional Review Board (IRB) approval to collect thrombi from stroke patients in order to better understand the composition and mechanical properties of stroke-causing thrombi. Future studies intend to classify human thrombus beyond RBC-rich to include calcified RBC-rich, fibrin-rich, and calcified fibrin-rich thrombus. The ultimate goal: tune the synthetic thrombus to approximate the mechanical properties of each thrombus classification and validate long-term stability and shelf-life to simplify in vitro modeling of ischemic stroke for thrombectomy device development, testing, and training.

Data sharing statement

Additional data from this study includes shear and elastic modulus data for: (1) male and (2) female thrombus samples as well as (3) all synthetic thrombus formulations tested across a shear rate range of 1 – 10 rad/s. A detailed MSB staining procedure is also available. Additional MSB and H&E histology images are available, including images and detailed analysis techniques for calculating fibrin content using Photoshop and ImageJ software. The corresponding author and the authors affiliated with Northern Arizona University can access the data. Data can be obtained by contacting the corresponding author (TAB).