Novel Human Acute Ischemic Stroke Blood Clot Analogs for In Vitro Thrombectomy Testing.

BACKGROUND AND PURPOSE
Previous studies have successfully created blood clot analogs for in vitro endovascular device testing using animal blood of various species. Blood components vary greatly among species; therefore, creating clot analogs from human blood is likely a more accurate representation of thrombi formed in the human vasculature.


MATERIALS AND METHODS
Following approval from the Mayo Clinic institutional review board, human whole-blood and platelet donations were obtained from the blood transfusion service. Twelve clot analogs were created by combining different ratios of red blood cells + buffy coat, plasma, and platelets. Thrombin and calcium chloride were added to stimulate coagulation. Clot composition was assessed using histologic and immunohistochemical staining. To assess the similarities of mechanical properties to patient clots, 3 types of clot analogs (soft, elastic, and stiff) were selected for in vitro thrombectomy testing.


RESULTS
The range of histopathologic compositions produced is representative of clots removed during thrombectomy procedures. The red blood cell composition ranged from 8.9% to 91.4%, and fibrin composition ranged from 3.1% to 53.4%. Platelets (CD42b) and von Willebrand Factor ranged from 0.5% to 47.1% and 1.0% to 63.4%, respectively. The soft clots had the highest first-pass effect and successful revascularization rates followed by the elastic and stiff clots. Distal embolization events were observed when clot ingestion could not be achieved, requiring device pullback. The incidence rate of distal embolization was the highest for the stiff clots due to the weak clot/device integration.


CONCLUSIONS
Red blood cell-rich, fibrin-rich, and platelet-rich clot analogs that mimic clots retrieved from patients with acute ischemic stroke were created in vitro. Differing retrieval outcomes were confirmed using in vitro thrombectomy testing in a subset of clots.

In the treatment of acute ischemic stroke, the achievement of complete revascularization from a single mechanical thrombectomy attempt, termed First Pass Effect (FPE), is associated with significantly improved outcomes for patients 1,2 . Removing the clot in a fragmented manner increases the potential of embolization to new territories, a major contributing factor to poor neurological outcomes due to additional brain infarction [3][4][5] .
Despite the advancement in the second generation mechanical thrombectomy devices, the rates of FPE remain low, as low as 29% in the recently reported Aspiration versus Stent Retriever (ASTER) trial 6 .
Previous studies have demonstrated that a wide variety of occlusive clots can cause an LVO [7][8][9][10][11] and clot composition has been shown to have a significant impact on the success of mechanical thrombectomy procedures 7,12,13 . This suggests that in order to further advance the success rates of stroke intervention, we must turn our attention to clot composition and compare treatment strategies using in-vitro thrombectomy models of the cerebral vasculature. Previous studies have successfully created blood clot analogues for In-vitro testing using animal blood of various species which have significantly advanced our understanding of clot biomechanics and imaging characteristics [13][14][15][16][17][18][19] . However, blood components and blood groups vary amongst species 20 and thus, creating clot analogues with human blood is likely a more accurate representation of thrombi formed in the human vasculature.
The hypothesis of the study is that the diverse range of clots retrieved from AIS patients can be accurately replicated using human blood by mimicking the process by which clots form In-vivo. The rationale for this study is that, as the success of mechanical thrombectomy procedures is influenced by the composition of the clot, creating human clot F o r P e e r R e v i e w analogs that accurately represent the different phenotypes retrieved form patients and testing them in an in-vitro thrombectomy system will allow us to compare the performance of different thrombectomy devices and techniques. We will thereby be able to determine the optimum treatment approach for each clot phenotype, thereby optimizing the chances of achieving the desired First Pass TICI3 outcome in the clinical setting 1 . To assess the similarities of mechanical properties to patient clots, three types of clot analogues (soft, elastic, and stiff) were selected for in-vitro thrombectomy testing.

Human Clot Analogue Creation
This Study received Institutional Review Board approval from Mayo Clinic Rochester in accordance with the ethical standards of the Declaration of Helsinki. A total of 12 clot analogues types were created as per Table 1. These clots analogues were selected to be representative of the previously identified phenotypes of clots retrieved from AIS patients; including; RBC-Rich, Fibrin-Rich, and Platelet-Rich clots 21 .
A human whole blood donation and a human platelet donation from two separate donors were obtained from the Mayo Clinic Blood Transfusion Service. The whole blood was centrifuged at 1,200RPM for 20 minutes at 20°C to separate it into its constituents 22   1,485µ 1,485µ 30µ *3µL of Thrombin (1NIH/mL) and 300µL of 5% CaCl 2 were added to stimulate coagulation.

Patient Cohort
Clots were collected from 100 patients who underwent mechanical thrombectomy for the treatment of acute ischemic stroke at Mayo Clinic Rochester. Where more than one procedural pass was needed to retrieve the occlusive clot, all fragments of clot were combined for histological analysis. The inclusion criteria were; >18 years, having undergone mechanical thrombectomy treatment for acute ischemic stroke and with clot material available for analysis. A waiver of informed consent was granted for the purposed of collecting retrieved clot material from acute ischemic stroke patients for this study.

Histological Processing and Staining
Gross photos were taken of each clot and analogue before fixation overnight in 10% phosphate-buffered formalin. All clots and analogues were then processed using a standard tissue processing protocol and embedded in paraffin. The formalin-fixed paraffin-embedded (FFPE) material was cut into 3µm sections. The Martius Scarlett Blue stain is now regarded as the gold-standard for assessing clot composition as it identifies platelet-rich regions of thrombi in addition to RBCs, WBCs and Fibrin 21,23 . Two representative slides were stained with Martius Scarlett Blue (MSB) to identify the common clot constituents; RBCs, White Blood Cells, Fibrin, Platelets/other, Collagen and Calcification as described previously 21,23 . Clot phenotype for both the clinical samples and the clot analogs was defined based on the dominant component (%) in each clot as determined by the MSB histological staining.
Immunohistochemical staining for platelets (CD42b) and von Willebrand Factor (vWF) was performed on a Leica Bond RX autostainer. Antigen retrieval with Tris-EDTA was performed for platelet staining (anti-CD42b); no antigen retrieval was used for vWF staining. Primary Percentage area of positive IHC staining was calculated separately for CD42b and vWF 25 .

Thrombectomy Testing in a Benchtop Stroke Platform
The mechanical properties of clots vary based on their histological composition; clot analogues with an increasing volume of Platelets contract to a greater degree due to the force of platelet contraction, resulting in stiffer clot analogues 27 . Clots analogs that have a high RBC content will typically be softer, more friable clots and clots made from plasma only, will produce clot analogs with a network of thin Fibrin-stands. Three phenotypes of clot analogues with varying compositions of RBCs, Plasma and Platelets were selected to represent prominent phenotypes of clots retrieved from AIS patients during thrombectomy; Soft (1:10 RBC + buffy coat: Plasma Only), Elastic (1:5 RBC + buffy coat:platelets + plasma), and Stiff (1:10 RBC + buffy coat:platelets + plasma).
Thrombectomy testing were carried out on these clots inside a benchtop stroke platform as previously described 28,29 . Briefly, a cerebrovascular glass model, where the lumen resembles the intracranial internal carotid artery, the anterior cerebral artery, and the middle cerebral arteries, is connected to a customized flow system to deliver flow with physiologically representative flow rate and pressure. Clot analogues measuring 6mm in length were introduced into the flow system and embolized to the M1-M2 bifurcation.  Ingestion was defined as complete ingestion of the clot into the catheter, First Pass Effect was defined as complete removal of the clot from the target artery in the first procedural pass, Successful Recanalization (SR) was defined as the as complete removal of the clot from the target artery within 3 procedural passes and Distal Embolization was defined as the occurrence of visible fragments of clots being dislodged and migrating distally from the target vessel. The thrombectomy processes were recorded and the failure mechanism, including the presence of Distal Embolization, were confirmed following the procedure.

Statistical analysis
All statistical correlations were assessed and graphs were generated using GraphPad Prism 8.
MSB histological composition was reported as % of the total clot area, positive immunohistochemistry staining (CD42b & vWF) was reported as % of the total clot area. A Shapiro-Wilk test indicated that quantitative variables did not follow a standard normal distribution. The non-parametric Spearman rho correlation was used to assess the similarity between clot analogues and clinical samples.

Clot Analogue Appearance
The gross appearance of each of the human clot analogues after clot formation and also post fixation in 10% Neutral buffered formalin is show in Figure 1. Clots analogues that are rich in Red Blood cells clots (e.g. 1:10 RBC+Buffy Coat:Plasma Only) have a dark red color after creation and a black color post-fixation. Clots that contain high platelet content (e.g. 1:100 RBC+Buffy Coat:Platelets Only) have white platelet-rich regions that are visible both pre and post fixation. Clots that are Fibrin-rich but not platelet-rich have a light red color after creation and a brownish color post-fixation (e.g. 1:50 RBC+Buffy Coat:Plasma Only). The Platelet-rich clots are smaller in clot volume due to the effect of platelet stimulated contraction of the clots. The clot analogues produced have a similar gross appearance to clots extracted from patients during mechanical thrombectomy procedures for the treatment of AIS.

Histological Composition
The MSB stain was used to assess the histological composition of the clot analogues (Table 2) and of the clots retrieved from AIS patients ( Figure 2). Red Blood cell-rich, Fibrin-rich, and Platelet-rich clot analogues that mimic clots retrieved from acute ischemic stroke patients were created. The range of histopathological compositions of the clot analogues is similar to that of the clinical samples ( Figure 2). The addition of a large volume of Red Blood Cells leads to a RBC-Rich clot regardless of the whether platelets and/or plasma were also added ( Figure   2). The Red blood cell composition of the clot analogs ranged from 8.9% to 91.4% and the clots retrieved from the patients ranged from <1% to 85%, there was a significant positive correlation between the RBC composition of the analogs and the clinical samples (r s =.755, p=0.010*). The platelet composition of the clot analogs ranged from 5.4% to 83.7% whilst the clinical samples ranged from 3% to 88% (r s =.618, p=0.048*). Fibrin composition of the clot analogs ranged from 3% to 53% and from 3% to 77% in the clinical samples (r s =.136, p=0.694).        Figure 3.

Revascularization Results
The three types of clot analogues are associated with different revascularization outcome results ( For the stiff clots and using the DA technique, only 20% (1 out of 5) of the clots can be ingested. During device pull, 80% (4 out of 5) of the clots lost integration with the suction catheter due to the antegrade pressure gradient and resulted in failed revascularization. The integration of the clots to the stent retriever was stronger than the aspiration catheter alone, resulting in a lower distal embolization rate and higher revascularization rate (Table 3). In this study a range of novel In-vitro human clot analogues that mimic the gross appearance and histological composition of clots retrieved from acute ischemic stroke patients were created. The composition of the clot analogues was confirmed using the MSB histological stain for the main components and immunohistochemical staining for the identification of Platelets and vWF. Furthermore, a subset of clot analogues were tested in an in-vitro thrombectomy model and demonstrated that revascularization outcome is related to both the composition of the clot and the technique used to retrieve them. The results of this study are important because they 1) prove that human clot analogs that accurately replicate the histological composition of clots retrieved from patients can be created and 2) demonstrate that these clot analogs can be used in an in-vitro thrombectomy setup to compare the performance of different treatment approaches, potentially leading to a clinical benefit for the patients.
The inability of Second Generation thrombectomy aspiration and stentriever devices to dramatically improve the rates of FPE following endovascular treatment of acute ischemic stroke suggests the effect is not specifically device related 6  RBC-rich, Fibrin-rich, and Platelet-rich clot analogues that mimic clots retrieved from Acute Ischemic Stroke Patients were created In-vitro. Differing retrieval outcomes were confirmed using in-vitro thrombectomy testing in a subset of clots. The use of human clot analogs and accurate in-vitro thrombectomy systems could be a valuable training resource for physicians to optimize their chances of achieving complete revascularization for every clot phenotype.

Acknowledgements:
This work was supported by the National Institutes of Health grant number (R01 NS105853), the    2. Pease delete the last sentence of the Conclusion. The sentence, "These novel human clot analogues will help to advance the field of endovascular device testing and clot imaging research". has no clear meaning. If you wish, replace this with 1 or 2 sentences as to, 1) how this work advances end-vascular device testing, and, 2) how this work advances clot imaging research.
Response: As suggested, this sentence has been removed and replaced with the following: 'The use of human clot analogs and accurate in-vitro thrombectomy systems could be a valuable training resource for physicians to optimize their chances of achieving complete revascularization for every clot phenotype. Comments to the Author 1. Although a large scale verification with the human specimen might be necessary as there should be numerous types of blood clot composition. meticulous testing of blood clot analogues, along with mechanical thrombectomy shown in this article is useful.
2. The data is fully examined and convincing.
3. I believe an experimental study like this will help the physicians to use the right device in the right situation, which will be beneficial for the patients as well as cost-conscious.
Response: The authors thank Reviewer 2 for their compliments in relation to our manuscript.
Reviewer: 3 Comments to the Author The authors describe a study in which analogues of human trombi are made, in order to resemble characteristics of thrombi removed from patients with an acute ischemic stroke.
Different types (n=12) of thrombi are made, by changing the amount of RBC +buffy coat, platelets and plasma. These thrombi are thereafter compared to thrombi collected from 100 ischemic stroke patients.
Comparison is made based on gross appearance, as well as histological composition (by MSB stain and immunohistochemistry) in order to differentiate between RBC, WBC, fibrin, platelets, collagen and calcification.
In the second part of the study, they describe results regarding ingestion, first pass effect, and succesfull recanalization rate from three different artificially made clot types.
Overall, the article is well written and reads easily.
Response: The authors thank Reviewer 3 for their accurate summation of our manuscript.
However, there are some comments I would like to make. I have listed them below.
Several points stand out -Please clearly describe the aim and hypothesis of the study so that the article can be 'attached' to this hypothesis. It will improve the clarity of the study Response: The authors have added the following hypothesis to the Introduction of the paper: Introduction: - The rational of the study is not argued very clearly in my opinion. The authors suggest that because of clot fragmentation and failure to remove the clot, clot composition and treatment strategies should be compared using in vitro models.
In the next paragraph, they explain it more clearly. But try to formulate accurately.
Response: As suggested by the Reviewer, the authors have re-written parts of the introduction in an effort to better formulate the rationale of the study.
-A statement is made that blood components vary between species, and that therefore this study should be undertaken. However, no reference about the difference between species is given.
Response: The authors have added an appropriate reference on the variability of blood and blood groups between species to support this statement.
-Please formulate the research question/hypothesis of the study clearly in the end of the introduction. For me, it is not clear what you will be studying in the paper.
Response: The authors have formulated the hypothesis of the study and the rationale at the end of the introduction as suggested by the reviewer.

-
Please use references on why the authors choose for the methodology of human clot analogue creation.
Response: The authors have added a reference in relation to the separation of whole blood into its constituents. The method used to create the human clot analogues is part of the novelty of this study and therefore a reference was not added for the methodology. - The authors do not clearly specify and explain on what grounds the different clot analogues were chosen.
Response: The authors have added the rationale for the choice of clots analogues included in the study has been added to Paragraph 1 of the 'Human Clot Analogue Creation' subsection. Please specify how embolization events were recorded and scored. Were distal emboli measured in the collected fluid from the different 'vessels', or was only visual fragmentation used?
Response: The authors have edited the 'Thrombectomy Testing in a Benchtop Stroke Platform' subsection to confirm that only visual fragmentation was used; 'Distal Embolization was defined as the occurrence of visible fragments of clots being dislodged and migrating distally from the target vessel. The thrombectomy processes were recorded and the failure mechanism, including the presence of Distal Embolization, were confirmed following the procedure.'

-
The statistical methods are not sufficient for this article. Only one sentence is used on 'correlations', however, not one correlation is mentioned in the article. Also, statistical statements are made, but without explanation on what statistics are used. Please provide a power analysis, null hypothesis, p-value, exact statistical methods and change the results and discussion of the article accordingly.
Response: The statistical analysis section has been updated to include the exact statistical methods used and the results and discussion sections of the article have been changed accordingly.

-
On what criteria are the three different clot types based? Some of the fibrin-rich and plateletrich seem to have some overlap. Response: The reviewer is correct, the histological composition of one clot type had been omitted in error from Figure 2B in the original submission, although the composition was reported in Table 2.
The authors have updated Figure 2 to now include all 12 clots described. - The three dominant groups are not pre-defined in the methodology, and therefore seem somewhat artificial and overlap between fibrin-rich and platelet-rich is present.
Response: The three dominant clot phenotypes are now defined at the beginning of the 'Human Clot Analogue Creation' subsection in the Methodology. Additionally, as mentioned above, the criteria for determining the clot phenotype is now also stated.  Response: The non-parametric Spearman rho correlation data has been added to the manuscript as requested.

Discussion -
Please update your statistical methods before make statements that clot analogues are the same as clots from AIS patients. The same for revascularization outcomes Response: The statistical and revascularization outcome methods have been updated as recommended. - The second part of the discussion (previous studies have demonstrated-of clot composition) seems more something for in the introduction Response: The authors have removed paragraph 2 as suggested by the reviewer. In addition, the authors have move some information from paragraph 2 to paragraph 3.
-Adequate references are missing throughout the discussion (and manuscript) Response: The authors have updated references throughout the manuscript to support their statements.
-Suddenly, statements on ingestion rates are made, however, the criteria for these measurements are not described in the methodology. 'Clots were collected from 100 patients who underwent mechanical thrombectomy for the treatment of acute ischemic stroke at Mayo Clinic Rochester. Where more than one procedural pass was needed to retrieve the occlusive clot, all fragments of clot were combined for histological analysis.' 2) Statistical Analysis: Please provide a more robust description of the analytic approach used to demonstrate the similarities between the mechanical properties between patient clots and clot analogues, as well as a description of any other statistical analyses utilized.
Response: The authors have added a more robust description of the analytic approach used to demonstrate the similarities between the patient clots and clot analogues as requested by the statistical editor.  Figure 2 presents MSB quantification between patient clots and clot analogues, and the Results text concludes on the basis of the data in this figure that the histopathological compositions are 'similar' between the two sets of samples. How was 'similarity' defined and how were differences assessed between patient clots and clot analogues?
Response: The authors have assessed the similarity between patient clots and clot analogues using the non-parametric Spearman rho correlation.

Background and Purpose: Previous studies have successfully created blood clot analogues for
In-vitro endovascular device testing using animal blood of various species. Blood components vary greatly among species, therefore, creating clot analogues from human blood are likely a more accurate representation of thrombi formed in human vasculature.

Materials and Methods:
Following approval from Mayo Clinic Institutional Review Board, human whole blood and platelet donations were obtained from the Blood Transfusion service. 12 clot analogues were created by combining different ratios of RBC+Buffy coat, Plasma, and Platelets. Thrombin and CaCl 2 were added to stimulate coagulation. Clot composition was assessed using histological and Immunohistochemical staining. To assess the similarities of mechanical properties to patient clots, three types of clot analogues (soft, elastic, and stiff) were selected for in-vitro thrombectomy testing.

Results:
The range of histopathological compositions produced is representative of clots removed during thrombectomy procedures. The RBC composition ranged from 8.9%-91.4% and Fibrin composition ranged from 3.1%-53.4%. Platelet (CD42b) and von Willebrand Factor (vWF) ranged from 0.5%-47.1% and 1.0%-63.4%, respectively. The soft-clots had the highest first-pass-effect and successful revascularization rates followed by the elastic and stiff clots.
Distal embolization events were observed when clot ingestion cannot be achieved, requiring device-pullback. Incidence rate of distal embolization is the highest for the stiff clots due to the weak clot/device integration.
Conclusions: RBC-rich, Fibrin-rich, and Platelet-rich and mixed clot analogues that mimic clots retrieved from Acute Ischemic Stroke Patients were created In-vitro. Differing retrieval outcomes were confirmed using in-vitro thrombectomy testing in a subset of clots.  In the treatment of acute ischemic stroke, the achievement of complete revascularization from a single mechanical thrombectomy attempt, termed First Pass Effect (FPE), is associated with significantly improved outcomes for patients 1,2 . Removing the clot in a fragmented manner increases the potential of embolization to new territories, a major contributing factor to poor neurological outcomes due to additional brain infarction [3][4][5] .
Despite the advancement in the second generation mechanical thrombectomy devices, the rates of FPE remain low, as low as 29% in the recently reported Aspiration versus Stent Retriever (ASTER) trial 6 .
Previous studies have demonstrated that a wide variety of occlusive clots can cause an LVO 7-11 and cClot composition varies significantly in acute ischemic stroke patients and has been shown to have a significant impact on the success of mechanical thrombectomy procedures 7,12,13 . High rates of clot fragmentation and failure to remove the clot in one pass.
This suggests that in order to further advance the success rates of stroke intervention, we must turn our attention to clot composition and compare treatment strategies using in-vitro thrombectomy models of the cerebral vasculature.
Clot composition varies significantly in acute ischemic stroke patients and has been shown to have a significant impact on the success of mechanical thrombectomy procedures [7][8][9] . Previous studies have successfully created blood clot analogues for In-vitro testing using animal blood of various species which have significantly advanced our understanding of clot biomechanics and imaging characteristics [13][14][15][16][17][18][19] . However, blood components and blood groups vary greatly amongst species 20 and thus, creating clot analogues with human blood is likely a more accurate representation of thrombi formed in the human vasculature. We present a novel method of The hypothesis of the study is that the diverse range of clots retrieved from AIS patients can be accurately replicated using human blood by mimicking the process by which clots form In-vivo. The rationale for this study is that, as the success of mechanical thrombectomy procedures is influenced by the composition of the clot, creating human clot analogs that accurately represent the different phenotypes retrieved form patients and testing them in an in-vitro thrombectomy system analogues from human blood and platelets that mimic the process by which clots form In-vivo.will allow us to compare the performance of different thrombectomy devices and techniques. We will thereby be able to determine the optimum treatment approach for each clot phenotype, thereby The human clot analogues created can then be inserted in anatomical models of the human vasculature and retrieved using different endovascular strategies. The ability to make accurate clot analogues coupled with the availability of realistic human vasculature replicators will be an invaluable tool to help neurointerventional surgeons test clot analogues of different compositions in various clinical scenarios, thus ultimately moving towards optimizing the chances of achieving the desired First Pass TICI3 outcome in the clinical setting 1 . To assess the similarities of mechanical properties to patient clots, three types of clot analogues (soft, elastic, and stiff) were selected for in-vitro thrombectomy testing.

Human Clot Analogue Creation
This Study received Institutional Review Board approval from Mayo Clinic Rochester in accordance with the ethical standards of the Declaration of Helsinki. A total of 12 clot analogues types were created as per Table 1. These clots analogues were selected to be representative of the previously identified phenotypes of clots retrieved from AIS patients 17 ;  1,485µ 1,485µ 30µ *3µL of Thrombin (1NIH/mL) and 300µL of 5% CaCl 2 were added to stimulate coagulation.

Patient Cohort
Clots were collected from 100 patients who underwent mechanical thrombectomy for the treatment of acute ischemic stroke at Mayo Clinic Rochester. Where more than one procedural pass was needed to retrieve the occlusive clot, all fragments of clot were combined for histological analysis. The inclusion criteria were; >18 years, having undergone mechanical thrombectomy treatment for acute ischemic stroke and with clot material available for analysis. A waiver of informed consent was granted for the purposed of collecting retrieved clot material from acute ischemic stroke patients for this study.

Histological Processing and Staining
Gross photos were taken of each clot and analogue before fixation overnight in 10% phosphate-buffered formalin. All clots and analogues were then processed using a standard tissue processing protocol and embedded in paraffin. The formalin-fixed paraffin-embedded

Imaging and Quantification
Following staining, a representative slide of each stain was scanned at 20x magnification (Motic Easyscan Pro, Motic Digital Pathology). Histologic quantification was performed on the digital slide using Orbit Image Analysis Software (www.Orbit.bio) as  Percentage area of positive IHC staining was calculated separately for CD42b and vWF 25 .

Thrombectomy Testing in a Benchtop Stroke Platform
The mechanical properties of clots vary based on their histological composition; clot analogues with an increasing volume of Platelets contract to a greater degree due to the force of platelet contraction, resulting in stiffer clot analogues 27 . CThree types of clot analogues were selected to represent a variety of patient clots retrieved from thrombectomy: lots analogs that have a high RBC content will typically be softer, more friable clots and clots made from plasma only, will produce clot analogs with a network of thin Fibrin-stands. Three phenotypes of clot analogues with varying compositions of RBCs, Plasma and Platelets were selected to represent prominent phenotypes of clots retrieved from AIS patients during thrombectomy; soft Soft (1:10 RBC + buffy coat: Plasma OnlyRBC:plasma), Eelastic (1:5 RBC + buffy coat:platelets + plasma), and Sstiff (1:10 RBC + buffy coat:platelets + plasma).
Thrombectomy testing were carried out on these clots inside a benchtop stroke platform as previously described 28, 29 . Briefly, a cerebrovascular glass model, where the lumen resembles the intracranial internal carotid artery, the anterior cerebral artery, and the middle cerebral arteries, is connected to a customized flow system to deliver flow with physiologically representative flow rate and pressure. Clot analogues measuring 6mm in length were introduced into the flow system and embolizeembolized to the M1-M2 bifurcation.

Statistical analysis
All statistical correlations were assessed and graphs were generated using GraphPad Prism 8.
MSB histological composition was reported as % of the total clot area, positive immunohistochemistry staining (CD42b & vWF) was reported as % of the total clot area. A Shapiro-Wilk test indicated that quantitative variables did not follow a standard normal distribution. The non-parametric Spearman rho correlation was used to assess the similarity between clot analogues and clinical samples.

Histological Composition
The MSB stain was used to assess the histological composition of the clot analogues (Table 2) and of the clots retrieved from AIS patients (Figure 2). Red Blood cell-rich, Fibrin-rich, and Platelet-rich and mixed clot analogues that mimic clots retrieved from acute ischemic stroke patients were created. The range of histopathological compositions of the clot analogues is similar to that of the clinical samples ( Figure 2). The addition of a large volume of Red Blood Cells leads to a RBC-Rich clot regardless of the whether platelets and/or plasma were also added ( Figure 2). The Red blood cell composition of the clot analogs ranged from 8.9% to 91.4% and the clots retrieved from the patients ranged from <1% to 85%, there was a      Figure 3.

Revascularization Results
The three types of clot analogues are associated with different revascularization outcome results ( For the stiff clots and using the DA technique, only 20% (1 out of 5) of the clots can be ingested. During device pull, 80% (4 out of 5) of the clots lost integration with the suction catheter due to the antegrade pressure gradient and resulted in failed revascularization. The integration of the clots to the stent retriever was stronger than the aspiration catheter alone, resulting in a lower distal embolization rate and higher revascularization rate (Table 3). The inability of Second Generation thrombectomy aspiration and stentriever devices to dramatically improve the rates of FPE following endovascular treatment of acute ischemic stroke suggests the effect is not specifically device related 6   For the DA technique, FPE is associated with successful clot ingestion and depends on the clot mechanical properties. Of the 15 clots tested, FPE were achieved for 12 clots and 7 (or 58%) of them were due to successful ingestion. The ingestion rates are 80%, 40%, and 20% for the soft, elastic, and stiff clots. Clots with higher compositions of platelets and fibrin have higher stiffness and friction coefficient 17,40 , making them difficult to deform into the catheter tip and get ingested. For the 8 clots without successful ingestion, 6 (or 75%) of them presented distal embolization and resulted in repeated device passes or failed revascularization. Suction catheters that can generate large suction force to deform the clots and overcome the clot friction to ingest the clots could be beneficial 41 . However, the thrombectomy tests are carried out in a glass phantom and arterial response to suction was not captured. Under suction, the vessel could collapse due to the reduced intraluminal pressure and evacuation of fluid, which was hypothetically related to the more sever vessel injuries using the suction catheters than the stent retrievers 42 . The safety profile of the new-generation large bore suction catheters needs to be further validated.  This study has some limitations. First, the whole blood and platelet donations were not collected from the same patient and the blood phenotypes of each were not available.
Second, blood phenotype has been shown to impact coagulation and this method may need to be adjusted slightly for clot phenotype. Finally, the thrombectomy tests are carried out in a glass phantom and arterial response to suction was not captured. RBC-rich, Fibrin-rich, and Platelet-rich and mixed clot analogues that mimic clots retrieved from Acute Ischemic Stroke Patients were created In-vitro. Differing retrieval outcomes were confirmed using in-vitro thrombectomy testing in a subset of clots. These novel human clot analogues will help to advance the field of endovascular device testing and clot imaging research.