Thrombus Profiling Assay: A Microfluidics-Based Platform for Comprehensively Characterizing Biomechanical Thrombogenesis

Thrombosis is a main cause of cardiovascular diseases, responsible for millions of deaths worldwide every year¹. Currently, no bioassay is available in standard clinical settings for evaluating risks of thrombosis. Among the commercialized laboratory and point-of-care hematological function assays, conventional coagulation assays and aggregometry have been proven unreliable in predicting thrombosis or major adverse cardiovascular events^2,3,4. Global thrombosis test⁵, PFA-100/200⁶, and global coagulation assays^7,8,9,10,11 also have limited data supporting their performance.

Based on the current understanding, the process of thrombogenesis is mainly contributed to by three mechanisms. Besides the two conventionally acknowledged mechanisms, namely, biochemical platelet aggregation and coagulation, a third mechanism that is under-studied and often under-estimated is shear-driven platelet aggregation, which was also termed as "biomechanical platelet aggregation"^12,13. In biomechanical platelet aggregation, high shear stress and shear gradient serve as the main drive for platelet crosslinking via GPIbα-von Willebrand factor (VWF), integrin α_IIbβ₃-VWF, and integrin α_IIbβ₃-fibrinogen interactions. In arterial thrombosis, biomechanical platelet aggregation likely serves as the most essential mechanism, considering that it is greatly reinforced by high shear flow caused by arterial stenosis. Therefore, thrombogenesis driven by biomechanical platelet aggregation was termed 'biomechanical thrombogenesis'^12,14.

In previous works, a common method for experimentally observing biomechanical platelet aggregation is the microfluidic stenosis assay, wherein a site of severe stenosis is embedded into a straight channel. When blood is perfused over the channel under a physiological wall shear stress, pathologically high shear stress is generated around the stenotic site, which drives the accumulation of platelets to form a thrombus. However, previous works only utilized a single (for platelets, reflecting the thrombus size)^{15,16,17,18,19} or at most two (one for platelets and one for another biomarker) readout^13,20, which are thus unable to achieve comprehensive characterization of the thrombus.

A thrombus profiling assay was recently developed, which incorporates multi-color fluorescence imaging in the microfluidic stenosis assay, achieving real-time tracking of 7 biomarkers (platelets, fibrinogen level, von Willebrand factor level, P-selectin expression level, phosphatidylserine exposure level, extended integrin α_IIbβ₃ expression level, fully active integrin α_IIbβ₃ expression level) in a thrombus, which sets the basis for comprehensively characterizing biomechanical thrombogenesis²¹. In this work, detailed protocols are provided on the preparation and performance of the thrombus profiling assay as well as the related data analysis. The hardware required for the assay includes an inverted multi-color fluorescence microscope and a microfluidic system. The assay uses a relatively small amount of human whole blood (less than 2 mL), has high cost-effectiveness (~$12 per sample), and derives results within 30 min. The assay can accurately detect the multi-dimensional prothrombotic abnormalities of individuals and evaluate the effects of anti-thrombotic agents in changing the size, composition, and platelet activation status of the thrombus, endorsing its wide application for both research and clinical purposes in the future²¹. It is noteworthy that the assay must use freshly collected heparinized blood. Storing the blood at 4 °C or for over 6 h or using anticoagulants other than heparin will either prevent thrombus from forming or render inaccurate results.

This protocol follows the guidelines of and has been approved by the Human Research Ethics Committee of The University of Texas Medical Branch. The experimental hardware setup consists of a microfluidic device, perfusion components (connectors, tubing, syringe, and syringe pump), and an inverted microscope with optical components enabling bright-field and multi-color fluorescence imaging. A multi-LED light house and a multi-pass filter cube are used in the microscope to split 4 fluorescent channels with minimal mutual bleed-through: excitation: 391/32, 479/33, 554/24, 638/31, and emission: 435/30, 519/25, 594/32, 695/58. Wear personal protective equipment, including gloves, eye protection, and a lab jacket, for all experimental procedures. The reagents and the equipment used are listed in the Table of Materials.

1. Microfluidic device preparation

1. Design a master mold to contain rectangular channels (200 µm wide × 50 µm high) that incorporate a site of 80% luminal narrowing. Each channel has dedicated inlets and outlets for tubing connections (Figure 1; see Supplementary File 1 for the original design file).
2. Based on the design, use a silicon wafer to fabricate an SU-8 photoresist master mold via standard photolithography. Tape the mold at the bottom of a 15-cm Petri dish (Figure 2A).
3. Prepare PDMS mixture by mixing the prepolymer base and curing agent in the silicone elastomer kit at a 10:1 weight ratio. Pour the mixture onto the master mold (Figure 2B).
4. Place the plate containing the PDMS mixture in a vacuum desiccator to degas and eliminate entrapped air bubbles.
   NOTE: Maintain the plate under vacuum for a minimum of 2 h until bubbles are completely removed.
5. Thermally cure the PDMS mixture by incubating it at 75 °C for 2 h.
6. Carefully cut out the cured PDMS from the master mold (Figure 2C,D) and section it into individual chip units.
7. Create the inlet and outlet by punching holes at the designated locations using a probe needle (Figure 2E).
   NOTE: Use a compressed air duster to remove any residual debris from the punched holes. To minimize surface contamination, clean the PDMS devices using adhesive sealing tape prior to bonding.
8. In a chemical hood, spray 75% ethanol on a task wipe, and use the wetted task wipe to wipe and clean glass slides. Leave the glass slides to dry.
9. In a chemical hood, treat the glass slides and PDMS chips with a high-frequency generator for 30 s and 20 s, respectively, and then align each chip with a glass slide. Gently press the chip onto the glass slide surface so that it can be bound.
   NOTE: This step needs to be done in a chemical hood because the high-frequency generator produces ozone during usage, which is harmful to health.
10. Thermally bond the devices by incubating them at 150 °C for 15 min. After cooling down, the devices will be ready to use (Figure 2F).
11. Store the devices at room temperature under dust-free conditions.
12. Use a pincer to remove the plastic part of the probe needles, and bend the metal tubes to 90°. These bent tubes will be used as connectors for the microfluidic devices.

2. Fluorescent sensor preparation

NOTE: The experiment uses a total of 7 fluorescent sensors: SZ22-FITC, fibrinogen-Alexa Fluor 405, 2.2.9-Alexa Fluor 555, AK4-Alexa Fluor 647, Annexin V-Pacific Blue, MBC 370.2-Alexa Fluor 555, PAC-1-Alexa Fluor 647. Among them, fibrinogen, 2.2.9, and MBC 370.2 are only commercially available in the unconjugated form and need to be fluorescently conjugated in the lab.

1. To conjugate fibrinogen with Alexa Fluor 405:
   1. Make a fresh 0.1 M sodium bicarbonate buffer, pH 8.5.
   2. Dilute fibrinogen stock to 1 mg/mL in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4).
   3. Add 1/10 volume of sodium bicarbonate buffer to the fibrinogen solution.
   4. Mix 1 mL fibrinogen with 1 mg Alexa Fluor 405 NHS-ester and incubate for 1 h at room temperature on a rotator. Protect from light.
   5. Remove the storage buffer from a purification column by centrifuging the column at 1,100 × g for 2 min, and discard the flow-through. Load the reaction solution onto the column, mount the column onto a compatible collection vial, and centrifuge at 1,100 × g for 5 min to harvest the fibrinogen-Alexa Fluor 405.
   6. Use a spectrophotometer to measure the absorbance at 280 nm (A₂₈₀; protein signal) and 405 nm (A₄₀₅; dye signal) wavelength. Calculate protein concentration and F/P ratio (fluorescence: protein molar ratio).
      NOTE: The F/P ratio should be in the range of 8-10.
   7. Store fibrinogen-Alexa Fluor 405 at 4 °C in the dark.
2. To conjugate 2.2.9 and MBC 370.2 antibodies with Alexa Fluor 555:
   1. Make a fresh 0.1 M sodium bicarbonate buffer, pH 8.5.
   2. Dilute antibody stock to 1 mg/mL in amine-free buffer.
   3. Add 1/10 volume of sodium bicarbonate buffer to the antibody solution.
   4. Mix 100 µL antibody with 1 vial (100 µg) of Alexa Fluor 555 NHS-ester and incubate for 1 h at room temperature on a rotator. Protect from light.
   5. Remove the storage buffer from a purification column by centrifuging the column at 1,100 × g for 2 min, and discard the flow-through. Load the reaction solution onto the column, mount the column onto a compatible collection vial, and centrifuge at 1,100 × g for 5 min to harvest the 2.2.9- or MBC 370.2-Alexa Fluor 555.
   6. Use a spectrophotometer to measure the absorbance at 280 nm (A₂₈₀; protein signal) and 555 nm (A₅₅₅; dye signal) wavelength. Calculate antibody concentration and F/P ratio (fluorescence: antibody molar ratio).
      NOTE: The F/P ratio should be in the range of 1-1.5.
   7. Store 2.2.9- and MBC 370.2-Alexa Fluor 555 in 4 °C in the dark.
      NOTE: Avoid over-labeling - high F/P ratio may impair biological function.

3. Blood collection from human subjects

NOTE: This procedure must be conducted by qualified medical personnel (e.g., licensed nurses, certified phlebotomists). Also, obtain written informed consent from the individuals participating in the study.

1. Prepare the syringe by aspirating 500 µL of Tyrode buffer (12 mM NaHCO₃, 10 mM Hepes, 137 mM NaCl, 2.7 mM KCl, 5.5 mM D-Glucose, 0.5% bovine serum albumin, pH 7.4) containing 0.32 U/mL heparin per 10 mL of blood to be collected.
2. Collect blood via venipuncture. Use an empty syringe to collect the first 2 mL of blood to discard. Then, change to the heparin-containing syringe to collect the blood sample. Pull the syringe slowly to minimize platelet activation.
3. Transfer the collected blood into a 15-mL centrifuge tube and keep it under 37 °C.
   NOTE: Blood samples should be used for experiments within 6 h of collection.

4. Thrombus profiling assay

NOTE: It is recommended to perform all procedures involving blood handling in a biosafety cabinet whenever possible to avoid blood spills on the experimentalist. If this is not possible, then use a benchtop splash shield.

1. Dilute VWF monomer to 2 µg/mL in PBS. Pre-coat the microfluidic devices with VWF monomer and incubate for 1 h at room temperature.
2. Incubate the blood sample with fluorescent sensor Set 1 or 2 for 10 min at room temperature:
   1. Set 1: SZ22-FITC (0.5 µg/mL), fibrinogen-Alexa Fluor 405 (60 µg/mL), 2.2.9-Alexa Fluor 555 (1 µg/mL), AK4-Alexa Fluor 647 (1 µg/mL).
   2. Set 2: SZ22-FITC (0.5 µg/mL), Annexin V-Pacific Blue (1 µg/mL), MBC 370.2-Alexa Fluor 555 (1 µg/mL), PAC-1-Alexa Fluor 647 (1 µg/mL).
      NOTE: Because two sensor sets need to be used separately, each blood sample needs to be tested at least twice (once with sensor Set 1 and once with sensor Set 2) to acquire a complete data set.
3. Connect a microfluidic device with inlet and outlet connectors and tubing. Connect the other end of the inlet connector with a tube containing PBS, and the other end of the outlet connector with a syringe. Mount the syringe onto a syringe pump. Mount the microfluidic device onto an inverted microscope, and manually maneuver the stage to find the site of stenosis under the microscope (Figure 3A).
4. Fill the microfluidic channel and tubing with PBS using the syringe pump at a flow rate of 0.5 mL/min. Ensure that no air bubbles are present around the site of stenosis.
5. Connect the inlet tubing with the blood sample, and perfuse the blood sample through the microfluidic channel using the syringe pump at a flow rate of 0.018 mL/min (Figure 3A).
6. Set the exposure time and gain of each fluorescence channel. Perform real-time multi-color fluorescence imaging by alternating among the 4 channels, exciting one kind of fluorophore at a time. Meanwhile, find the focus plane of the thrombus, and record signals at the site of stenosis, typically spanning 15-30 min (Figure 4).
   NOTE: The exposure time of each channel needs to be adjusted so that the fluorescent signal is easily distinguishable while avoiding saturation (Figure 4). In the present system, healthy subjects' blood samples typically render the following signal intensity range within the formed thrombus: SZ22-FITC, 70-110; fibrinogen-Alexa Fluor 405, 60-100; 2.2.9-Alexa Fluor 555, 70-110; AK4-Alexa Fluor 647, 45-75; Annexin V-Pacific Blue, 35-65; MBC 370.2-Alexa Fluor 555, 45-85; PAC-1-Alexa Fluor 647, 10-30. Notably, however, a small proportion of healthy subjects would render a non-typical readout. Therefore, it is recommended to test at least 5 healthy subject's blood samples to make sure that the exposure time for each channel is selected properly.

5. Data analysis

1. Open the data file using ImageJ 1.53 (Fiji, National Institutes of Health).
   NOTE: Each file should contain a total of 4 videos from 4 different channels. The following procedure is generally applicable to any time point that the user is interested in analyzing.
2. Use the SZ22-FITC channel to identify the contour of the thrombus, and use 'Polygon selections' to roughly select the area of the thrombus (Figure 5A,B).
3. For each channel, use Image -> Adjust -> Threshold to eliminate the background (Figure 5C), and then use Analyze -> Measure to measure the area and average intensity of the signal within the thrombus.
   NOTE: SZ22-FITC stains platelets and thus reflects thrombus size. In Set 1, fibrinogen-Alexa Fluor 405 reflects fibrinogen enrichment level, 2.2.9-Alexa Fluor 555 reflects von Willebrand factor (VWF) enrichment level, and AK4-Alexa Fluor 647 reflects P-selection expression. In Set 2, Annexin V-Pacific Blue reflects phosphatidylserine (PS) exposure, MBC 370.2-Alexa Fluor 555 reflects integrin α_IIbβ₃ activation to the extended conformation (E⁺ α_IIbβ₃), and PAC-1-Alexa Fluor 647 reflects integrin α_IIbβ₃ full activation (Act. α_IIbβ₃)²¹.

The thrombus profiling assay perfuses blood through a stenotic channel to allow shear-driven thrombus formation, and performs real-time multi-color fluorescence imaging to collect multi-dimensional information on the formed thrombus. By completing experiments using both Set 1 and Set 2 sensors, one should be able to characterize the thrombus in aspects including size, enrichment of crosslinking proteins (von Willebrand factor, fibrinogen), as well as platelet activation level (reflected by P-selection expression, PS exposure, and integrin αIIbβ3 activation)21.

Data collected from each experiment can be thoroughly analyzed to render 'signal vs. time' curves (c.f., Figure 6). However, this is time-consuming. Alternatively, one can pick a single time point, and therefore, one image per channel, for data analysis. For instance, 7.5 min after the onset of thrombus development typically corresponds to the time when healthy blood's thrombus starts to become relatively stable, and thus can be selected as a representative time point for data analysis21. If only one time point is used for data analysis, then the following procedure can be used to derive a 7-dimensional thrombus profile that comprehensively represents the blood sample's biomechanical prothrombotic tendency. Firstly, the area and average intensity of the fluorescent signal from each channel are multiplied to derive the total signal intensity, rendering one total signal intensity value from each of the 4 channels in sensor Set 1 and each of the 4 channels in sensor Set 2 (Figure 7A). Then, the total signal intensities of fibrinogen (from fibrinogen-Alexa Fluor 405), VWF (from 2.2.9-Alexa Fluor 555) and P-selectin (from AK4-Alexa Fluor 647) will be divided by that of platelets (from SZ22-FITC) in sensor Set 1 to acquired normalized fibrinogen, VWF and P-selectin signal intensity, and the total signal intensities of PS (from Annexin V-Pacific Blue), E+ αIIbβ3 (from MBC 370.2-Alexa Fluor 555) and Act. αIIbβ3 (from PAC-1-Alexa Fluor 647) will be divided by that of platelets (from SZ22-FITC) in sensor Set 2 to acquire normalized PS, E+ αIIbβ3, and Act. αIIbβ3 signal intensity, respectively. This normalization eliminates the impact of the thrombus size on the signal intensity of other readouts. Lastly, because signals from platelets are measured in both sensor Sets 1 and 2 (by SZ22-FITC), the two total signal intensities of platelets will be averaged to acquire the thrombus size. Eventually, a 7-dimension thrombus profile in the format of [thrombus size, normalized fibrinogen level, normalized VWF level, normalized P-selectin level, normalized PS level, normalized E+ αIIbβ3 level, normalized Act. αIIbβ3 level] can be derived (Figure 7B). Confirmed by previous quality control tests, the assay generates highly repeatable results21. Therefore, for each blood sample, one complete test (composed of two experimental runs, with Sensor Set 1 and Set 2, respectively) suffices to generate the thrombus profile. However, to increase the robustness of the thrombus profile, one can perform more replicates of the test and average the acquired data values.

To succinctly summarize the effects of a certain factor (e.g., pathological condition or drug) on biomechanical thrombogenesis, an 'effect barcode' can be used21. One effect barcode is composed of 7 digits, each corresponding to a dimension of the thrombus profile. A positive, neutral, or negative effect of a factor is numerically expressed as "+", "0", or "−", respectively. For example, a previous work compared the thrombus profiles of young hypertension patients with healthy young subjects, where hypertension was concluded to carry an 'effect barcode' of [+ + 0 0 0 + +], indicating a positive effect on the thrombus size, fibrinogen level, as well as E+ αIIbβ3 and Act. αIIbβ3 levels, but no effect on the VWF, P-selectin, and PS levels21.

Figure 1: Microfluidic chip design. (A) Design of the chip's mold. The mold contains 8 replicates of the microfluidic chip's pattern, and each pattern contains 10 separate microfluidic channels. The adjacent channels are designed to have different lengths to better spatially separate their inlets and outlets. Each channel contains an area filled with an array of small pillars (shown in yellow) upstream of the stenotic site, which is used to trap debris in the blood to avoid channel clogging. (B) Zoom-in of the stenotic site with all dimensions marked. Scale bar: 1 cm. Please click here to view a larger version of this figure.

Figure 2: Procedure for making the microfluidic devices. (A) A master mold taped to the bottom of a 15-cm Petri dish. Hardened PDMS can be seen at the periphery of the mold, which is the leftover of the device carve-out from previous preparations. (B) Pouring the PDMS mixture to cover the patterns on the master mold. (C,D) Using a knife to cut out the PDMS at the center of the master mold containing the microfluidic channel patterns. (E) An individual PDMS chip with inlet and outlet holes punched. (F) A ready-to-use microfluidic device with a quarter coin placed adjacent. Please click here to view a larger version of this figure.

Figure 3: Thrombus profiling assay assembly. (A) A single-channel setup. The inlet of a microfluidic device is connected to a blood sample, and the outlet to a syringe, both via tubing. The syringe is mounted on a syringe pump. The microfluidic device is mounted on the stage of an inverted fluorescence microscope. The stage is manually maneuvered to find the site of stenosis within the microfluidic channel. During the experiment, the syringe pump pulls the syringe to perfuse the blood through the channel within the microfluidic device. (B) A multi-channel setup designed for monitoring multiple channels simultaneously. A multi-channel syringe rack is utilized to pull 10 syringes at the same time. A programmable motorized stage on the microscope is pre-set to automatically move back and forth between the 10 channels over time, which allows alternate capturing of images of the growing thrombi within all 10 channels. Please click here to view a larger version of this figure.

Figure 4: Representative snapshots of thrombi formed in a healthy subject's blood. Dashed lines indicate the geometric contour of the PDMS channel. Sensor Set 1 (A) and Set 2 (B) each render images of 4 fluorescence channels. Names of the molecular sensors and their corresponding thrombus biomarkers are annotated in the figure. Scale bar: 40 µm. Please click here to view a larger version of this figure.

Figure 5: A typical process of data analysis on one snapshot of the thrombus. Set 1 SZ22-FITC channel is used as an example. However, for all other channels, the process is the same. (A) The initial image as shown in ImageJ. (B) 'Polygon selections' function is used to roughly select the area of the thrombus. (C) 'Image -> Adjust -> Threshold' function is used to eliminate the background area, following which 'Analyze -> Measure' function will be used to measure the area and average intensity of the signal within the thrombus. Please click here to view a larger version of this figure.

Figure 6: A representative 'signal vs. time' curve of the Set 1 SZ22-FITC channel from an experimental run of a healthy subject's blood sample. Please click here to view a larger version of this figure.

Figure 7: Illustration of the composition of the post-analysis data. (A) For each time point of the video, data analysis will render a total signal intensity (represented by a rectangular block with the corresponding name) for each of the 4 targeted biomarkers in Sensor Set 1 (colored in orange) and each of the 4 targeted biomarkers in Sensor Set 2 (colored in blue). (B) Further data analysis is performed to render the 7-dimensional thrombus profile, in which: ① thrombus size is calculated by averaging the two total signal intensities of platelets acquired from sensor Sets 1 and 2; ②-⑦ normalized fibrinogen, VWF, P-selectin, PS, E+ αIIbβ3, and Act. αIIbβ3 levels are calculated by respectively normalizing the total signal intensities of these biomarkers by that of platelets derived from the same sensor set. Please click here to view a larger version of this figure.

Supplementary File 1: Design file of the microfluidic device master mold. Please click here to download this File.

By combining the microfluidic stenosis assay with multi-color fluorescence imaging, the thrombus profiling assay provides a convenient and powerful approach to study biomechanical thrombogenesis and platelet mechanobiology. Meanwhile, the assay is useful in a wide range of applications. For instance, it can be used to screen anti-thrombotic agents that specifically inhibit biomechanical platelet aggregation, where the molecular target and mechanism of action can be deduced from the effect barcode²¹. It can also be used to study the prothrombotic tendency in specific populations to identify and characterize the effect of risk factors for cardiovascular diseases, and to gauge the prothrombotic tendency of individuals. We believe that the thrombus profiling assay is promising for clinical translation to evaluate the prothrombotic tendency of individuals and to help physicians develop personalized anti-thrombotic regimens for patients.

The thrombus profiling assay is versatile. The molecular sensors can be freely changed or removed to meet customized needs. For instance, if the experimentalist is only interested in the thrombus size and the E⁺ α_IIbβ₃ level, a single experimental run with blood containing SZ22-FITC and MBC 370.2-Alexa Fluor 555 shall be performed. If the experimentalist is interested in testing other biomarkers not included in the sensor Sets 1 and 2 listed here, corresponding molecular sensors can be added to replace those in the current setup, as long as the tagged fluorescent dye is compatible with the channel's optical setting. Moreover, as shown in a previous work²¹, a low-cost single-readout simplified version of the assay is also available, where the blood is solely stained with DiOC₆(3), a dye that is much cheaper than antibodies and can render strong signals at a very low concentration. This simplified assay is especially useful for preliminary tests, e.g., drug screening, where only thrombus size needs to be acquired.

The assay may run into several issues that can cause the failure of the experiment. If the blood does not perfuse through the microfluidic device smoothly, then it is likely that the inlet and/or outlet has a leakage. In that case, use a smaller-sized probe needle to punch the inlet and outlet holes, and larger-sized probe needles to make the connectors when making microfluidics chips. Occasional observation of debris is acceptable. However, if debris is frequently (>20% of all runs) observed to flow through and clog the channel, more caution should be taken to clean the PDMS chips and the glass before bonding. When filling the microfluidic channel and tubing with PBS, it is possible that the downstream corner of the stenotic site can have a residual air bubble remaining that cannot be flushed away. This bubble, even a small one, can cause detrimental issues because it can grow larger over time when running the blood sample. An effective way to eliminate it is to reverse the flow direction several times during PBS flushing. Lastly, if the microscope light path is not carefully designed, crosstalk among different fluorescence channels can occur. One can either revise the light path design to circumvent it or perform post-processing subtraction to eliminate the impact of the crosstalk on the data. On a separate note, due to the overlapping of excitation and emission spectra of the different dyes, Förster resonance energy transfer (FRET) will inevitably occur. Although with careful light path design, this should have a trivial impact on the quantification of the signal intensities, single-label control experiments are strongly recommended for confirmation.

It should also be pointed out that the thrombus profiling assay has certain limitations. Firstly, the microfluidic channel is not endothelialized and thus does not consider the contribution of endothelial cells to thrombosis. Secondly, by adding no soluble agonist and by using anticoagulant, the assay intentionally focuses on biomechanical platelet aggregation while minimizing biochemical platelet aggregation and coagulation, and thus is unable to assess the potency of blood samples in the latter two aspects. Thirdly, the Reynolds number in microfluidic systems is much lower than in large human arteries due to differences in lumen size, and thus the blood perfusion in this system cannot generate turbulence, which can potentially exist in stenotic human arteries and contribute to thrombus formation^22,23. Thus, it is important to note that the main purpose of the thrombus profiling assay is to evaluate the general prothrombotic tendency of blood samples in a biomechanical setting, but not to perfectly recapitulate the in vivo process of thrombosis. Lastly, photobleaching can affect the accuracy of intensity measurements when analyzing thrombi over time to generate 'signal vs. time' curves, which can be alleviated by lowering the frequency of data acquisition.

The assay has the potential to accommodate higher-throughput experimentation. Specifically, the 10 channels on the device can be run simultaneously by using a syringe pump mounted with a multi-channel syringe rack, and a microscope with a programmable motorized stage can be used to automatically alternate the image acquisition position back-and-forth between the 10 different channels (Figure 3B). With the use of a multi-camera array microscope²⁴and by re-designing the microfluidic chip, even more channels can be monitored concurrently in real time.