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Influence of multiple stenoses on thrombosis formation: an in vitro study

Influence of multiple stenoses on thrombosis formation: an in vitro study Multiple lesions in the same vessel is one of the most common situations found in patients suffering from cardiovas‑ cular diseases, this complicates not only the assessment of the severity of each one but also their treatment. To date, the effect of multiple stenoses on different parameters has been simulated by numerical studies. Few others have implemented in vitro platforms for their investigation. However, visualization of thrombosis formation in this kind of lesion is still needed. This in vitro study monitors the formation of thrombus inside microchannels having one, two, −1 and three stenoses. Whole blood was perfused through each channel at high shear rates (> 12,000 s ), generating thrombosis. Flow changes across each lesion as well as the final percentage of aggregations were monitored. Thus, the location where total occlusion could be produced was found to be the first stenosis for all the cases. Less flow reaching the second and third stenoses was observed which demonstrates that aggregations were growing at the first one. This was verified by measuring the percentage of aggregations at the end of the test. Keywords: Thrombosis, Multiple stenoses, Platelets, Whole blood, Microfluidics, In vitro model, Lab ‑ on‑a‑ chip Introduction Plaques in arteries can be different in their geometry, Cardiovascular diseases (CVDs) are the leading cause of size, and length. They can also, be present as single and death worldwide, it has been reported that it takes even multiple (or serial) lesions. Compared to single stenosis, more lives every year than all types of cancer and chronic fluid dynamics in serial stenoses is complex, therefore an lower respiratory disease together [1]. Some of the con- accurate diagnosis and treatment are still a medical chal- sequences of CVDs resulting in death are coronary heart lenge [3]. Keeley et al. [4] found that 69% of patients with disease, strokes, and heart attacks. These are mainly ST-segment elevation myocardial infarction had multiple caused by blood clots in the arteries blocking the normal complex stenoses, of which 26% had two stenoses and supply of blood which is known as thrombosis. Throm - 17% had three stenoses. According to the results of Gold- bosis develops when an atherosclerotic plaque within the stein et al. [5] patients with serial stenoses have a higher artery suffers an injury allowing the interaction between risk of recurrent ischemia as well as higher mortality fats and cholesterol inside the plaque with blood. Thus, compared to patients with single stenosis. leading to the growth of a thrombus composed of red High wall shear rates play an important role in coro- blood cells, platelets, and fibrin. This process is known as nary thrombosis development [6]. They are responsible thrombogenesis [2]. for starting the mechanism for platelets’ aggregation [7]. An in  vivo study supported that platelet aggregations develop not only by soluble agonists but also by shear stress gradients [8]. Wall shear rate is the result of the *Correspondence: wtpark@seoultech.ac.kr wall shear stress divided by the viscosity of the fluid [9]. Convergence Institute of Biomedical Engineering and Biomaterials, Seoul Numerical studies have simulated the flow dynamics National University of Science and Technology, Seoul, South Korea present in serial stenoses, demonstrating that wall shear Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Flores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 2 of 6 stress increases as the percentage of stenosis increases lesions. As the purpose was to find the effect of the [10]. However, the presence of multiple stenoses has number of stenoses, all of them were designed the more impact on flow impairment than single stenoses same. The geometry of the stenosed channels was con - with the same degree [11, 12]. centric. The diameter of the channel was 600 μm while While assessing multiple stenoses, one of the most the diameters in the stenotic parts were 90 μm. The dis - important parameters is pressure drop. The pressure tance between lesions was set as 5 mm (Fig. 1). gradient is used mainly when diagnostic techniques A silicon wafer was patterned by photolithography such as fractional flow reserve (FFR) are measured. FFR and master molds were created by soft lithography. index helps to evaluate the lesion severity and to decide Chips were made of PDMS 10:1, using the thermal air on treatment [13]. Pressure drop has been found to vary expansion method developed in our laboratory [16]. and depend on the number of stenoses in a vessel [10, This method allows the fabrication of self-aligned ellip - 14]. Also, a larger pressure gradient is produced when the tical channels, as the intention is to mimic the shape of distance between proximal and distal stenoses increases human arteries. [15]. It has been found that post-stenotic sections are Collagen type I from rat tail (1 mg/ml, Sigma-Aldrich, prone to develop particle aggregations due to recircula- Korea) was used for creating a prothrombotic coating tion zones and flow separation [14], which could lead to inside the channel. Channels were incubated overnight thrombosis development. at room temperature and rinsed with PBS 1X before Through these studies, prothrombotic sites can be esti - experiments [17]. mated, however, in vitro experiments proving these pre- dictions in thrombosis formation are still lacking. This study demonstrates the effect that the number of sten - Experiment oses has on thrombogenesis, the location of thrombus Whole blood was drawn from one volunteer and col- formation, and how multiple stenoses increase the risk lected in a bag containing CPDA-1 anticoagulant; of thrombosis and total occlusion. These results contrib - before use, it was recalcified with the proper amount of ute to a broader understanding of how thrombosis might CaCl 0.25  M. Blood was perfused through the chan- develop in multiple stenoses. Moreover, thrombus for- nels using a flow rate of 3 ml/h controlled by a syringe mation monitoring and final aggregations measurement pump, connected by tubing from the syringe to the allow the determination of the lesion prone to occlusion. inlet of the channel. Blood flow and thrombus forma - tion were observed by an optical microscope (DMSZ7, Fabrication Sunny Optical, China). Pressure drop was monitored A microfluidic chip with channels having one (a), by two pressure sensors (ABP series, Honeywell, US) two (b), and three stenoses (c) was designed. A clini- placed at the inlet and outlet. The setup and chip used cal severe narrowing of 85% was chosen for all of the for the experiment are shown in Fig. 2. Fig. 1 Microfluidic chip designed for the experiment. Channels with one (a), two (b), and three (c) lesions. Zoomed part shows the stenosis geometry and sizes F lores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 3 of 6 Fig. 2 A Setup for monitoring thrombosis formation. B PDMS microfluidic chip Analysis when clear areas appear, the area indicates the exist- To determine how much a second and/or third stenosis ence of a thrombus [18]. However, during our control is affected by thrombosis, each lesion was observed sepa - experiments, three different phenomena were observed rately. Video recordings were used for analysis. Frames that might be the reason explaining these clear areas. at each time of interest were obtained, and images were As is shown in Fig. 3, one of the causes of the color dif- processed using ImageJ software. A mask was used for ference is the formation of aggregations, they appear defining the area to be analyzed and the region of interest like clots with light color, as they are mainly composed was cropped. Then, clear areas were determined by color of platelets. Also, when aggregations appear, the flow is thresholding using the HSB (hue-saturation-brightness) deviated from its normal path, creating a light-colored method. The size of the whole region of interest and clear string. The third reason is the phenomenon known as areas were obtained, and percentages were calculated. “cell-free layer” (CFL). When the blood flows through For determining the flow reaching each stenosis, the microvessels, red blood cells tend to gather in the images during perfusion were used and clear area per- middle of the flow leaving a layer of plasma without centages were calculated for the first 600  s. Normally, Fig. 3 Three different reasons for clear areas in blood flow. a Formation of thrombus, b flow changes due to thrombus formation, c cell‑free layer Flores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 4 of 6 cells on the edges. This cell-free layer shows a different color from the usual blood color. As the appearance of clear areas could be ambigu- ous, another measurement was taken for verifying the risk of occlusion of each stenosis. Images were taken at the end of the experiment when blood was not flowing anymore, and they were also considered in the analysis. The percentage of clear areas at each lesion was calcu - lated and they were declared to be aggregations. As flow was already stopped two of the possible reasons for clear areas were discarded: flow changes and cell-free layer. Results and discussion Clear areas percentage was measured in the channel with only one stenosis. Through this analysis, changes at the stenotic part during blood flow were observed, such as Fig. 5 Clear area percentages trend at second (green) and third (blue) stenoses compared to first stenosis (red) behavior aggregations, and lysis or embolism. Figure  4 shows the percentage of clear areas at some points during 600  s of perfusion where these changes were evident. For confirming the results found during perfusion, the When blood started flowing, an increment in the clear aggregation percentage was measured at the end of the area was observed, which was due to aggregations form- experiment, these results are shown in Fig.  6. Aggrega- ing inside the channel. The clear area was then reduced tion percentages were analyzed at each stenosis in the meaning that the clot was dissolved and therefore, blood three channels and the highest percentage was found at occupied more space in the channel. Reaggregation was the first stenosis in all the cases. u Th s, demonstrating also observed at 600 s. that the first stenosis was the cause of the changes occur - Same observation and analysis were made for sec- ring in the second and the  third one. Even if the second ond and third stenoses. Contrary to what happened and the  third stenosis also showed a considerable per- in the first one, at these lesions, aggregations were not centage of aggregations, the first one showed the most. observed. Instead, a constant increment in clear area Results obtained through this study, give us an insight percentages was noticed (Fig.  5). Meaning that blood into the development of thrombosis in vessels with mul- flow reaching these stenoses was decreasing. Aggrega - tiple stenoses. Thrombosis formation at high shear rates tions being formed in the first stenosis could explain was confirmed. Aggregation and embolism or lysis were these changes. Thrombus growth at the first lesion causes observed at the first stenosis during blood perfusion, higher resistance to flow; therefore, less flow reaches the which demonstrates thrombus instability, however, total second and third stenosis. occlusion was not reached. It was observed that in a case where all the stenoses have the same severity, the responsible for total occlu- sion would be the first one. This could be explained because even if shear rates are almost the same at each lesion, the first lesion is the first one having contact with blood, therefore, the thrombosis mechanism starts there. Thrombus in second and third stenoses was not observed, however, less flow was found to reach them. Aggregations in a lower percentage were also found in these lesions. The behavior of severe stenosis of 85% was confirmed through this study, however, some limitations were also encountered. First, our design shows an ideal case where all the stenoses have the same degree of sever- ity, and all of them have been reached by the collagen product of a rupture somewhere. Besides collagen Fig. 4 Flow changes during blood perfusion in the channel with one coating not being localized, soluble collagen was used stenosis F lores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 5 of 6 Fig. 6 A Percentage of aggregations at each lesion in channels with one, two, and three stenoses. B Picture of each stenosis, where aggregations can be observed in a lighter color for these experiments, which contains less von Wille- endothelial cells to provide the biochemical effect and brand factor protein, which is necessary for arterial make it more similar to a living artery. thrombosis development [19]. This might be the reason why total occlusion was not reached and aggregation Conclusion percentages were not too significant. Also, a limited Thrombosis development in multiple stenoses was number of samples was used, therefore, findings need investigated using a microfluidic in  vitro model. The to be verified further. In terms of analysis, clear areas formation of thrombus at severe stenosis (85% narrow- were defined by the author based on the observations. ing) and high shear rates was confirmed. Flow reduc - However, normally the flow should be recovered after tion through the following stenoses was observed. Total the divergent. One explanation for this could be the occlusion was not reached due to unexpected lysis. At separation of blood components, as it happens with the the beginning, it was expected that embolus coming cell-free layer (CFL). Clear areas might be plasma, as it from the first stenosis could generate occlusion in the has been found that geometry variations of microfluid - following stenoses, but this was not observed. Clots ics enhance CFL. This has been used for blood plasma grew in all the stenoses; however, the highest aggrega- separation [20]. Regarding the monitoring, an optical tion percentage was found in the first stenosis. Based microscope was utilized, so the resultant image is 2D, on our results, the first stenosis is the most prone to which does not allow for observing the volume of the total occlusion, thus, it should be treated earlier. aggregations, thus, the area was measured. Monitoring was made separately for all the lesions; this could be Abbreviations improved later by implementing simultaneous moni- CVDs: Cardiovascular diseases; FFR: Fractional flow reserve; HSB: Hue ‑satura‑ toring for having a wider observation of thrombus and tion‑brightness; CFL: Cell‑free layer. blood flow behavior through all the stenoses. Acknowledgements This study demonstrated one of several conditions Not applicable. commonly found in patients with this disease. A simi- Author contributions lar fabrication method, experiment setup, and perhaps WTP supervised the findings of this work and reviewed the manuscript. CJS, evaluation technique could be applied to more complex HBFM, HDH, JHK, and PHJ fabricated the device. JHC, WTP, CJS, HBFM, and coronary artery lesions in the future. For example, ath- HDH designed the scope and performed the experiments. HBFM drafted the manuscript. WTP read and approved the final manuscript. erosclerotic plaques within bifurcation lesions, which is one of the most difficult lesions to diagnose and Funding treat. Furthermore, the device could be also lined with This work was supported by the Basic Research Program under the National Research Foundation of Korea (NFR 2020R1F1A1074995). Blood samples were Flores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 6 of 6 collected under Seoul National University of Science and Technology IRB consensus document. 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In: Proceedings of the IEEE international conference on micro electro mechanical systems Author details (MEMS). Institute of Electrical and Electronics Engineers Inc., New York, pp Convergence Institute of Biomedical Engineering and Biomaterials, Seoul 348–350 National University of Science and Technology, Seoul, South Korea. Depar t‑ 17. Li M, Hotaling NA, Ku DN, Forest CR (2014) Microfluidic thrombosis under ment of Mechanical and Automotive Engineering, Seoul National University multiple shear rates and antiplatelet therapy doses. PLoS ONE 9:e82493. of Science and Technology, Seoul, South Korea. Samsung Medical Center, https:// doi. org/ 10. 1371/ journ al. pone. 00824 93 Seoul, South Korea. 18. van Rooij BJM, Závodszky G, Hoekstra AG, Ku DN (2021) Haemody‑ namic flow conditions at the initiation of high‑shear platelet aggrega‑ Received: 17 August 2022 Accepted: 3 November 2022 tion: a combined in vitro and cellular in silico study. Interface Focus 11:20190126. https:// doi. org/ 10. 1098/ rsfs. 2019. 0126 19. Bernardo A, Bergeron AL, Sun CW, Guchhait P, Cruz MA, Lo JA, Fong JD, Pez LJJ‑F Von Willebrand D (2004) Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen‑induced References platelet aggregation. J Thromb Haemost 2:660–669. https:// doi. org/ 10. 1. American Heart Association (2021) Heart disease and stroke statistics 1111/j. 1538‑ 7836. 2004. 00661.x update fact sheet at‑a‑ glance; 2021. Circulation 626‑44 20. Faivre M, Abkarian M, Bickraj K, Stone HA (2006) Geometrical focusing 2. Chang JC (2018) Thrombogenesis and thrombotic disorders based on of cells in a microfluidic device: an approach to separate blood plasma. “two‑path unifying theory of hemostasis”: philosophical, physiological, Biorheology 43:147–159 and phenotypical interpretation. Blood Coagul Fibrinol 29:585–595. https:// doi. org/ 10. 1097/ MBC. 00000 00000 000769 Publisher’s Note 3. Kumbhani DJ, Bhatt DL (2016) Fractional flow reserve in serial coronary Springer Nature remains neutral with regard to jurisdictional claims in pub‑ artery stenoses. JAMA Cardiol 1:359–360. https:// doi. org/ 10. 1001/ jamac lished maps and institutional affiliations. ardio. 2016. 0219 4. Keeley EC, Mehran R, Brener SJ, Witzenbichler B, Guagliumi G, Dudek D, Kornowski R, Dressler O, Fahy M, Xu K, Grines CL, Stone GW (2014) Impact of multiple complex plaques on short‑ and long‑term clinical outcomes in patients presenting with ST‑segment elevation myocardial infarction (from the Harmonizing Outcomes with Revascularization and Stents in Acute Myocardial Infarction [HORIZONS‑A]. Am J Card 113:1621–1627. https:// doi. org/ 10. 1016/j. amjca rd. 2014. 02. 016 5. Goldstein J, Demetriou D, Grines C, Pica M, Mazen S, O’Neill W (2000) Multiple complex coronary plaques in patients with acute myocardial infarction. N Engl J Med 343:915–922. https:// doi. org/ 10. 1056/ NEJM2 00009 28343 1303 6. 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Influence of multiple stenoses on thrombosis formation: an in vitro study

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Abstract

Multiple lesions in the same vessel is one of the most common situations found in patients suffering from cardiovas‑ cular diseases, this complicates not only the assessment of the severity of each one but also their treatment. To date, the effect of multiple stenoses on different parameters has been simulated by numerical studies. Few others have implemented in vitro platforms for their investigation. However, visualization of thrombosis formation in this kind of lesion is still needed. This in vitro study monitors the formation of thrombus inside microchannels having one, two, −1 and three stenoses. Whole blood was perfused through each channel at high shear rates (> 12,000 s ), generating thrombosis. Flow changes across each lesion as well as the final percentage of aggregations were monitored. Thus, the location where total occlusion could be produced was found to be the first stenosis for all the cases. Less flow reaching the second and third stenoses was observed which demonstrates that aggregations were growing at the first one. This was verified by measuring the percentage of aggregations at the end of the test. Keywords: Thrombosis, Multiple stenoses, Platelets, Whole blood, Microfluidics, In vitro model, Lab ‑ on‑a‑ chip Introduction Plaques in arteries can be different in their geometry, Cardiovascular diseases (CVDs) are the leading cause of size, and length. They can also, be present as single and death worldwide, it has been reported that it takes even multiple (or serial) lesions. Compared to single stenosis, more lives every year than all types of cancer and chronic fluid dynamics in serial stenoses is complex, therefore an lower respiratory disease together [1]. Some of the con- accurate diagnosis and treatment are still a medical chal- sequences of CVDs resulting in death are coronary heart lenge [3]. Keeley et al. [4] found that 69% of patients with disease, strokes, and heart attacks. These are mainly ST-segment elevation myocardial infarction had multiple caused by blood clots in the arteries blocking the normal complex stenoses, of which 26% had two stenoses and supply of blood which is known as thrombosis. Throm - 17% had three stenoses. According to the results of Gold- bosis develops when an atherosclerotic plaque within the stein et al. [5] patients with serial stenoses have a higher artery suffers an injury allowing the interaction between risk of recurrent ischemia as well as higher mortality fats and cholesterol inside the plaque with blood. Thus, compared to patients with single stenosis. leading to the growth of a thrombus composed of red High wall shear rates play an important role in coro- blood cells, platelets, and fibrin. This process is known as nary thrombosis development [6]. They are responsible thrombogenesis [2]. for starting the mechanism for platelets’ aggregation [7]. An in  vivo study supported that platelet aggregations develop not only by soluble agonists but also by shear stress gradients [8]. Wall shear rate is the result of the *Correspondence: wtpark@seoultech.ac.kr wall shear stress divided by the viscosity of the fluid [9]. Convergence Institute of Biomedical Engineering and Biomaterials, Seoul Numerical studies have simulated the flow dynamics National University of Science and Technology, Seoul, South Korea present in serial stenoses, demonstrating that wall shear Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Flores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 2 of 6 stress increases as the percentage of stenosis increases lesions. As the purpose was to find the effect of the [10]. However, the presence of multiple stenoses has number of stenoses, all of them were designed the more impact on flow impairment than single stenoses same. The geometry of the stenosed channels was con - with the same degree [11, 12]. centric. The diameter of the channel was 600 μm while While assessing multiple stenoses, one of the most the diameters in the stenotic parts were 90 μm. The dis - important parameters is pressure drop. The pressure tance between lesions was set as 5 mm (Fig. 1). gradient is used mainly when diagnostic techniques A silicon wafer was patterned by photolithography such as fractional flow reserve (FFR) are measured. FFR and master molds were created by soft lithography. index helps to evaluate the lesion severity and to decide Chips were made of PDMS 10:1, using the thermal air on treatment [13]. Pressure drop has been found to vary expansion method developed in our laboratory [16]. and depend on the number of stenoses in a vessel [10, This method allows the fabrication of self-aligned ellip - 14]. Also, a larger pressure gradient is produced when the tical channels, as the intention is to mimic the shape of distance between proximal and distal stenoses increases human arteries. [15]. It has been found that post-stenotic sections are Collagen type I from rat tail (1 mg/ml, Sigma-Aldrich, prone to develop particle aggregations due to recircula- Korea) was used for creating a prothrombotic coating tion zones and flow separation [14], which could lead to inside the channel. Channels were incubated overnight thrombosis development. at room temperature and rinsed with PBS 1X before Through these studies, prothrombotic sites can be esti - experiments [17]. mated, however, in vitro experiments proving these pre- dictions in thrombosis formation are still lacking. This study demonstrates the effect that the number of sten - Experiment oses has on thrombogenesis, the location of thrombus Whole blood was drawn from one volunteer and col- formation, and how multiple stenoses increase the risk lected in a bag containing CPDA-1 anticoagulant; of thrombosis and total occlusion. These results contrib - before use, it was recalcified with the proper amount of ute to a broader understanding of how thrombosis might CaCl 0.25  M. Blood was perfused through the chan- develop in multiple stenoses. Moreover, thrombus for- nels using a flow rate of 3 ml/h controlled by a syringe mation monitoring and final aggregations measurement pump, connected by tubing from the syringe to the allow the determination of the lesion prone to occlusion. inlet of the channel. Blood flow and thrombus forma - tion were observed by an optical microscope (DMSZ7, Fabrication Sunny Optical, China). Pressure drop was monitored A microfluidic chip with channels having one (a), by two pressure sensors (ABP series, Honeywell, US) two (b), and three stenoses (c) was designed. A clini- placed at the inlet and outlet. The setup and chip used cal severe narrowing of 85% was chosen for all of the for the experiment are shown in Fig. 2. Fig. 1 Microfluidic chip designed for the experiment. Channels with one (a), two (b), and three (c) lesions. Zoomed part shows the stenosis geometry and sizes F lores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 3 of 6 Fig. 2 A Setup for monitoring thrombosis formation. B PDMS microfluidic chip Analysis when clear areas appear, the area indicates the exist- To determine how much a second and/or third stenosis ence of a thrombus [18]. However, during our control is affected by thrombosis, each lesion was observed sepa - experiments, three different phenomena were observed rately. Video recordings were used for analysis. Frames that might be the reason explaining these clear areas. at each time of interest were obtained, and images were As is shown in Fig. 3, one of the causes of the color dif- processed using ImageJ software. A mask was used for ference is the formation of aggregations, they appear defining the area to be analyzed and the region of interest like clots with light color, as they are mainly composed was cropped. Then, clear areas were determined by color of platelets. Also, when aggregations appear, the flow is thresholding using the HSB (hue-saturation-brightness) deviated from its normal path, creating a light-colored method. The size of the whole region of interest and clear string. The third reason is the phenomenon known as areas were obtained, and percentages were calculated. “cell-free layer” (CFL). When the blood flows through For determining the flow reaching each stenosis, the microvessels, red blood cells tend to gather in the images during perfusion were used and clear area per- middle of the flow leaving a layer of plasma without centages were calculated for the first 600  s. Normally, Fig. 3 Three different reasons for clear areas in blood flow. a Formation of thrombus, b flow changes due to thrombus formation, c cell‑free layer Flores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 4 of 6 cells on the edges. This cell-free layer shows a different color from the usual blood color. As the appearance of clear areas could be ambigu- ous, another measurement was taken for verifying the risk of occlusion of each stenosis. Images were taken at the end of the experiment when blood was not flowing anymore, and they were also considered in the analysis. The percentage of clear areas at each lesion was calcu - lated and they were declared to be aggregations. As flow was already stopped two of the possible reasons for clear areas were discarded: flow changes and cell-free layer. Results and discussion Clear areas percentage was measured in the channel with only one stenosis. Through this analysis, changes at the stenotic part during blood flow were observed, such as Fig. 5 Clear area percentages trend at second (green) and third (blue) stenoses compared to first stenosis (red) behavior aggregations, and lysis or embolism. Figure  4 shows the percentage of clear areas at some points during 600  s of perfusion where these changes were evident. For confirming the results found during perfusion, the When blood started flowing, an increment in the clear aggregation percentage was measured at the end of the area was observed, which was due to aggregations form- experiment, these results are shown in Fig.  6. Aggrega- ing inside the channel. The clear area was then reduced tion percentages were analyzed at each stenosis in the meaning that the clot was dissolved and therefore, blood three channels and the highest percentage was found at occupied more space in the channel. Reaggregation was the first stenosis in all the cases. u Th s, demonstrating also observed at 600 s. that the first stenosis was the cause of the changes occur - Same observation and analysis were made for sec- ring in the second and the  third one. Even if the second ond and third stenoses. Contrary to what happened and the  third stenosis also showed a considerable per- in the first one, at these lesions, aggregations were not centage of aggregations, the first one showed the most. observed. Instead, a constant increment in clear area Results obtained through this study, give us an insight percentages was noticed (Fig.  5). Meaning that blood into the development of thrombosis in vessels with mul- flow reaching these stenoses was decreasing. Aggrega - tiple stenoses. Thrombosis formation at high shear rates tions being formed in the first stenosis could explain was confirmed. Aggregation and embolism or lysis were these changes. Thrombus growth at the first lesion causes observed at the first stenosis during blood perfusion, higher resistance to flow; therefore, less flow reaches the which demonstrates thrombus instability, however, total second and third stenosis. occlusion was not reached. It was observed that in a case where all the stenoses have the same severity, the responsible for total occlu- sion would be the first one. This could be explained because even if shear rates are almost the same at each lesion, the first lesion is the first one having contact with blood, therefore, the thrombosis mechanism starts there. Thrombus in second and third stenoses was not observed, however, less flow was found to reach them. Aggregations in a lower percentage were also found in these lesions. The behavior of severe stenosis of 85% was confirmed through this study, however, some limitations were also encountered. First, our design shows an ideal case where all the stenoses have the same degree of sever- ity, and all of them have been reached by the collagen product of a rupture somewhere. Besides collagen Fig. 4 Flow changes during blood perfusion in the channel with one coating not being localized, soluble collagen was used stenosis F lores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 5 of 6 Fig. 6 A Percentage of aggregations at each lesion in channels with one, two, and three stenoses. B Picture of each stenosis, where aggregations can be observed in a lighter color for these experiments, which contains less von Wille- endothelial cells to provide the biochemical effect and brand factor protein, which is necessary for arterial make it more similar to a living artery. thrombosis development [19]. This might be the reason why total occlusion was not reached and aggregation Conclusion percentages were not too significant. Also, a limited Thrombosis development in multiple stenoses was number of samples was used, therefore, findings need investigated using a microfluidic in  vitro model. The to be verified further. In terms of analysis, clear areas formation of thrombus at severe stenosis (85% narrow- were defined by the author based on the observations. ing) and high shear rates was confirmed. Flow reduc - However, normally the flow should be recovered after tion through the following stenoses was observed. Total the divergent. One explanation for this could be the occlusion was not reached due to unexpected lysis. At separation of blood components, as it happens with the the beginning, it was expected that embolus coming cell-free layer (CFL). Clear areas might be plasma, as it from the first stenosis could generate occlusion in the has been found that geometry variations of microfluid - following stenoses, but this was not observed. Clots ics enhance CFL. This has been used for blood plasma grew in all the stenoses; however, the highest aggrega- separation [20]. Regarding the monitoring, an optical tion percentage was found in the first stenosis. Based microscope was utilized, so the resultant image is 2D, on our results, the first stenosis is the most prone to which does not allow for observing the volume of the total occlusion, thus, it should be treated earlier. aggregations, thus, the area was measured. Monitoring was made separately for all the lesions; this could be Abbreviations improved later by implementing simultaneous moni- CVDs: Cardiovascular diseases; FFR: Fractional flow reserve; HSB: Hue ‑satura‑ toring for having a wider observation of thrombus and tion‑brightness; CFL: Cell‑free layer. blood flow behavior through all the stenoses. Acknowledgements This study demonstrated one of several conditions Not applicable. commonly found in patients with this disease. A simi- Author contributions lar fabrication method, experiment setup, and perhaps WTP supervised the findings of this work and reviewed the manuscript. CJS, evaluation technique could be applied to more complex HBFM, HDH, JHK, and PHJ fabricated the device. JHC, WTP, CJS, HBFM, and coronary artery lesions in the future. For example, ath- HDH designed the scope and performed the experiments. HBFM drafted the manuscript. WTP read and approved the final manuscript. erosclerotic plaques within bifurcation lesions, which is one of the most difficult lesions to diagnose and Funding treat. Furthermore, the device could be also lined with This work was supported by the Basic Research Program under the National Research Foundation of Korea (NFR 2020R1F1A1074995). Blood samples were Flores Marcial et al. Micro and Nano Systems Letters (2022) 10:18 Page 6 of 6 collected under Seoul National University of Science and Technology IRB consensus document. 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Journal

Micro and Nano Systems Letters – Springer Journals

Published: Nov 19, 2022

Keywords: Thrombosis; Multiple stenoses; Platelets; Whole blood; Microfluidics; In vitro model; Lab-on-a-chip

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