Ignite Creation Date:
2024-05-06 @ 6:44 PM
Last Modification Date:
2024-10-26 @ 2:53 PM
Study NCT ID:
NCT05760326
Status:
RECRUITING
Last Update Posted:
2023-03-08
First Post:
2023-02-27
Brief Title:
Diagnostic and Prognostic Role of Clot Analysis in Stroke Patients
Sponsor:
Fondazione Policlinico Universitario Agostino Gemelli IRCCS
Organization:
Fondazione Policlinico Universitario Agostino Gemelli IRCCS
Study Overview
Official Title:
Diagnostic and Prognostic Role of Clot Analysis in Stroke Patients
Status:
RECRUITING
Status Verified Date:
2023-02
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
DICAS
Brief Summary:
BACKGROUND AND RATIONALE OF THE STUDY
The analysis of the composition of the clot constitutes a promising tool for investigating the possible pathogenetic mechanisms underlying ischemic stroke This analysis was made possible thanks to the numerous mechanical thrombectomy operations which have now become routine
Several studies have attempted to explore the possible relationship between the primary site of thrombus formation and clot composition reporting that cardioembolic stroke may have a higher percentage of platelet-rich areas than noncardioembolic thrombosis However the data are conflicting and do not seem to support an association between clot histology and etiology Furthermore thrombus composition appears to influence thrombolysis and the efficacy of thrombectomy For example fibrin-rich thrombi appear to reduce the effectiveness of thrombolytic treatment and require more steps with mechanical thrombectomy treatment
Primary ENDPOINT
Evaluate how clot composition relates to stroke etiology according to the TOAST classification
Secondary ENDPOINT
relationship between different clot components and the degree of thrombectomy recanalization as defined by treatment modified cerebral ischemia score mTICI
relationship between the different components of the clot and the number of steps required to achieve recanalization
relationship between the different clot components and outcome indicators NIHSS score and mRS score
TARGET POPULATION Patients with ischemic stroke with occlusion of large intracranial vessels will be included in the study if deemed suitable for mechanical thrombectomy therapy in accordance with national and international guidelines
INCLUSION CRITERIA
age 18 years
Patients diagnosed with large vessel occlusion stroke in the emergency room CT Angio-study undergoing mechanical thrombectomy procedure
Recovered thrombus available for analysis
EXCLUSION CRITERIA
Lack of written informed consent
MATERIALS AND METHODS The clot will be portioned Part of the sample will be fixed in a 10 formalin solution 37 formaldehyde part will be frozen in liquid nitrogen Within 24-48 hours of fixation formalin-fixed thrombi will be dehydrated by increasing the concentration of ethanol 70 -80 -95 100 and paraffin-embedded allowing good preservation of tissue morphology and easy long-term storage The included samples will be sectioned along the major axis of the thrombus in slices with a thickness between 4 and 5 µm
Base staining will be used to visualize RBC PLT and fibrin
Hematoxylin and Eosin HE will allow visualization of general thrombus structures and identification of aggregates of fibrinplatelets colored pink red blood cells red and nucleated cells dark blue
Martius Scarlet Blue MSB selectively stains fibrin dark pinkred red blood cells yellow and collagen blue
Mallory-Azan for collagen and phosphotungstic acid hematoxylin for fibrin
immunohistochemistry to detect the presence of
Platelets CD42-Gp-Ib CD41a-Gp-IIbIIIa CD61-GpIIIa
white blood cells CD45 common leukocyte antigen or monocytesmacrophages CD14 CD1a CD68
T lymphocytes CD3 CD8CD4 or natural killers CD16 CD56 or mobile premature endothelial cells and blood progenitors CD34 or neutrophils CD45 CD16 or fibrinogen or von Willebrand factor
The analysis of the composition of the clot constitutes a promising tool for investigating the possible pathogenetic mechanisms underlying ischemic stroke This analysis was made possible thanks to the numerous mechanical thrombectomy operations which have now become routine
Several studies have attempted to explore the possible relationship between the primary site of thrombus formation and clot composition reporting that cardioembolic stroke may have a higher percentage of platelet-rich areas than noncardioembolic thrombosis However the data are conflicting and do not seem to support an association between clot histology and etiology Furthermore thrombus composition appears to influence thrombolysis and the efficacy of thrombectomy For example fibrin-rich thrombi appear to reduce the effectiveness of thrombolytic treatment and require more steps with mechanical thrombectomy treatment
Primary ENDPOINT
Evaluate how clot composition relates to stroke etiology according to the TOAST classification
Secondary ENDPOINT
relationship between different clot components and the degree of thrombectomy recanalization as defined by treatment modified cerebral ischemia score mTICI
relationship between the different components of the clot and the number of steps required to achieve recanalization
relationship between the different clot components and outcome indicators NIHSS score and mRS score
TARGET POPULATION Patients with ischemic stroke with occlusion of large intracranial vessels will be included in the study if deemed suitable for mechanical thrombectomy therapy in accordance with national and international guidelines
INCLUSION CRITERIA
age 18 years
Patients diagnosed with large vessel occlusion stroke in the emergency room CT Angio-study undergoing mechanical thrombectomy procedure
Recovered thrombus available for analysis
EXCLUSION CRITERIA
Lack of written informed consent
MATERIALS AND METHODS The clot will be portioned Part of the sample will be fixed in a 10 formalin solution 37 formaldehyde part will be frozen in liquid nitrogen Within 24-48 hours of fixation formalin-fixed thrombi will be dehydrated by increasing the concentration of ethanol 70 -80 -95 100 and paraffin-embedded allowing good preservation of tissue morphology and easy long-term storage The included samples will be sectioned along the major axis of the thrombus in slices with a thickness between 4 and 5 µm
Base staining will be used to visualize RBC PLT and fibrin
Hematoxylin and Eosin HE will allow visualization of general thrombus structures and identification of aggregates of fibrinplatelets colored pink red blood cells red and nucleated cells dark blue
Martius Scarlet Blue MSB selectively stains fibrin dark pinkred red blood cells yellow and collagen blue
Mallory-Azan for collagen and phosphotungstic acid hematoxylin for fibrin
immunohistochemistry to detect the presence of
Platelets CD42-Gp-Ib CD41a-Gp-IIbIIIa CD61-GpIIIa
white blood cells CD45 common leukocyte antigen or monocytesmacrophages CD14 CD1a CD68
T lymphocytes CD3 CD8CD4 or natural killers CD16 CD56 or mobile premature endothelial cells and blood progenitors CD34 or neutrophils CD45 CD16 or fibrinogen or von Willebrand factor
Detailed Description:
2 BACKGROUND
Stroke is the second leading cause of death and the third cause of disability worldwide The majority of strokes are ischemic mainly caused by blood thrombi occluding a cerebral vessel1 According to the TOAST classification2 four cause of stroke due to large vessel occlusion LVO can be identified atherosclerosis cardioembolic other determined etiology like dissection or other undetermined causes Strokes without a defined etiology are now called embolic stroke of undetermined nature ESUS3 This definition implies the presence of an undefined embolic source as the cause of the stroke Not knowing the origin of the embolus the best medical therapy in these patients is actually matter of debate4-6 Research on this topic is focusing on finding a laboratory or radiologic marker capable of clearly identified the embolic source and guide in the choice of the best medical therapy78
Revascularization strategy such as thrombolysis and thrombectomy represent the gold standard therapy during stroke acute phase in particular for patients with large vessel occlusion LVO From 2015 a growing body of evidence showed the efficacy of endovascular procedures in stroke patients and furthermore allowed for the first time the opportunity to collect thrombus material for research purposes910
3 OBJECTIVES
The objective of this study is to evaluate how clot composition is related to stroke etiology according to the TOAST classification Furthermore we will also study the relationship between different clot components and thrombectomy grade of recanalization as defined by the modified treatment in cerebral ischemia score mTICI and the number of required passes to achieve recanalization
4 STUDY DESIGN Study population
Patients hospitalized in the - Catholic University of the Sacred Heart Policlinico Gemelli - Catholic University - Rome with a stroke due to LVO treated with mechanical thrombectomy will be presented the study
Patient will be enrolled upon their presentation to the emergency department The usual clinical practice will be followed
Considering the emergency setting and the impossibility for most patients to provide a valid and informed consent before e immediately after the procedures retrieved thrombi will be collected but not analyze An informed consent to the study will be collect in the following day Should the patient refuse the participation the thrombus will be discarded and the patient will be considered as a drop out from the study
Investigators plan to include 30 patients with acute ischemic stroke due to LVO treated with mechanical thrombectomy
Patients characteristics Ischemic stroke patients with LVO will be included in the study if considered suitable for mechanical thrombectomy therapy in accordance with national and international guidelines
Inclusion criteria
age 18 years
Patients with a diagnosis of large vessel occlusion stroke at the Angio-CT study performed in the ER undergoing a mechanical thrombectomy procedure
Thrombus retrieved available for analysis
Exclusion criteria
Deny informed consent
5 ENDPOINTS Primary endpoint
To evaluate the association between thrombus composition studied through histological and immunohistochemical coloration and stroke etiology according to the TOAST classification In particular TOAST voices for etiological category will be dichotomize between cardioembolic and not cardioembolic the latter including large vessel occlusion other determined etiologies and other undetermined etiologies
Secondary endpoints
To evaluate the association between thrombus composition and mechanical thrombectomy outcome
To evaluate the association between thrombus composition and number of passes needed to obtain recanalization
Correlation endpoints
To evaluate the correlation between thrombus composition and global outcome variable such as the National Institutes of Health Stroke Scale NIHSS at seven days and nineteen days after the event and the modified ranking scale mRS at three months
6 METHODS AND ASSESSMENT
Clinical variables and neuroimaging studies The following data will be collected medical history recording demographic data age sex potential vascular risk factors hypertension diabetes mellitus dyslipidemia smoking prior stroke transient ischemic attack TIA previous vascular diseases previous antiplatelet treatment congestive heart failure hypertension vascular disease abnormal renalliver function bleeding history or predisposition labile international normalized ratio drugsalcohol concomitantly diagnosis of atrial fibrillation Oxfordshire Community Stroke Project OCSP classification previous treatment with thrombolysis ASPECTSpc-ASPECTS based on TC scan performed 24 h after acute stroke andHemorrhagic trasformation subtype At three months follow-up the following data will be collected intracranial bleeding major and minor bleeding blood transfusion hospitalization deaths modified Rankin Scale mRS score adherence to treatment stroke recurrence and myocardial infarction Laboratory tests blood counts biochemistry and coagulation tests 12-lead ECG chest radiography carotid ultrasonography and Computed Tomography CT or Magnetic Resonance Imaging MRI will be performed at admission and during the hospitalization
To evaluate neurologic deficit the National Institute of Health Stroke Scale NIHSS will be performed at admission 24 48 72 h 7 days andor at discharge and at 3 months
Functional outcome will be evaluated at discharge and at 3 months by modified rankin scale mRS Stroke etiology will be classified according to TOAST criteria for ischemic stroke TOAST voices for etiological category will be dichotomize between cardioembolic and not cardioembolic the latter including large vessel occlusion other determined etiologies and other undetermined etiologies
To evaluate infarct volume and control hemorrhagic evolution a CT will be performed between 24 ours from treatment Infarct volume will be quantified in cubic centimeters cm3 and assessed according to the formula 05xAxBxC where A direction and C to the number of 10mm slices where infarct volume is present 20 All neuroimaging evaluations will be made by the a neuroradiologist blinded to clinical and laboratory data
Specimen fixation and sectioning Retrieved thrombi will be portioned Part of the specimen will be fixed in a solution of 10 formalin 37 formaldehyde part will be frozen in liquid azote Within 24-48 hours from fixation formalin-fixed thrombi will be dehydrated via increasing concentration of ethanol 70 -80 -95 100 and embedded in paraffin allowing a good preservation of tissue morphology and easy long-term storage Included samples will be sectioned along the major axis of the thrombus in slice with a 4 to 5 µm of thickness
Staining Base coloration will be employed to visualize RBC PLT and fibrin Hematoxylin and eosin staining HE will allow to visualize the general overall structures of the thrombus and the identification of fibrinplatelet aggregates colored in pink RBC red and nucleated cells dark blue The Martius Scarlet Blue MSB staining instead will selectively stain fibrin Dark pinkred RBC yellow and collagen Blue RBC-rich fibrin-poor material will appears red on HE staining and yellow on MSB while RBC-poorfibrin-rich areas will appear as light pink areas on HE staining and pink to red areas on MSB Since platelets are only present in large amount in RBC-poor fibrin-rich areas thrombi will be divided in RBC-richPlatelets-poor mixed and platelets-richRBC-poor Additional base coloration will include Mallory-Azan for collagen and phosphotungstic acid hematoxylin for fibrin Furthermore immunohistochemical staining will be employed to detect the presence of Platelets CD42-Gp-Ib CD41a-Gp-IIbIIIa CD61-GpIIIa white blood cells CD45 leukocyte common antigen monocytemacrophages CD14 CD1a CD68 T lymphocytes CD3 CD8CD4 Natural Killer cells CD16 CD56 premature endothelial and blood progenitor cells CD34 Neutrophils CD45 CD16 Fibrinogen and Von Willebrand factor
Analysis Standardized quantification algorithms to calculate thrombus composition will be employed Computational color-based segmentation analysis by the means of ImageJ Rasband WS ImageJ U S National Institutes of Health Bethesda Maryland USA httpsimagejnihgovij 1997-2018 will allow to apply thresholds for color and thus selective quantification of specific colorscomponents providing data in automated manner about the percentage of total thrombus areas
7 STATISTICAL ANALYSIS
In this study 28 consecutive patients fulfilling selection criteria will be included According to the analysis of Kim 2015 strong differences in mean percentages in clot composition between the two subgroups are reported therefore it is possible to make an hypothesis of an effect size equal to or greater than 1 using the Students t-test this sample size of 28 patients increased to 30 for possible technical difficulties included in one of the two groups determined by the TOAST classification will have a power of 80 to detect such differences at a significance level of 5 two-tailed Statistical analyses will be performed using the IBM-SPSS Statistical Package for Social Science SPSS v20 Kolmogorov-Smirnov test will be used to evaluate data distribution in case of deviation from normality U test di Mann-Whitney or Kruskal-Wallis test will be used for comparisons among two or more groups in case of respect of normality assumptions Students t-test or analysis of variance will be considered Association between dichotomomic items will be evaluated using the Fisher exact test or χ2 as appropriate Due to the explorative approach of this study no adjustment will be made for multiple testing
8 ETHICAL PRINCIPLES
This study will be conducted in accordance with the principles laid down by the 18th World Medical Assembly Helsinki 1964 and all applicable amendments laid down by the World Medical Assemblies and the ICH guidelines for good clinical practice GCP
9 INFORMED CONSENT
The Investigator should fully inform the patient of all pertinent aspects of the study including the written information giving approvalfavorable opinion by the Ethics Committee IRBIEC All participants should be informed to the fullest extent possible about the study in language and terms they are able to understand
Prior to thrombus analysis the written informed consent form should be signed name filled in and personally dated by the patient or by the patients legally acceptable representative and by the person who conducted the informed consent discussion A copy of the signed and dated written informed consent form will be provided to the patient
Stroke is the second leading cause of death and the third cause of disability worldwide The majority of strokes are ischemic mainly caused by blood thrombi occluding a cerebral vessel1 According to the TOAST classification2 four cause of stroke due to large vessel occlusion LVO can be identified atherosclerosis cardioembolic other determined etiology like dissection or other undetermined causes Strokes without a defined etiology are now called embolic stroke of undetermined nature ESUS3 This definition implies the presence of an undefined embolic source as the cause of the stroke Not knowing the origin of the embolus the best medical therapy in these patients is actually matter of debate4-6 Research on this topic is focusing on finding a laboratory or radiologic marker capable of clearly identified the embolic source and guide in the choice of the best medical therapy78
Revascularization strategy such as thrombolysis and thrombectomy represent the gold standard therapy during stroke acute phase in particular for patients with large vessel occlusion LVO From 2015 a growing body of evidence showed the efficacy of endovascular procedures in stroke patients and furthermore allowed for the first time the opportunity to collect thrombus material for research purposes910
3 OBJECTIVES
The objective of this study is to evaluate how clot composition is related to stroke etiology according to the TOAST classification Furthermore we will also study the relationship between different clot components and thrombectomy grade of recanalization as defined by the modified treatment in cerebral ischemia score mTICI and the number of required passes to achieve recanalization
4 STUDY DESIGN Study population
Patients hospitalized in the - Catholic University of the Sacred Heart Policlinico Gemelli - Catholic University - Rome with a stroke due to LVO treated with mechanical thrombectomy will be presented the study
Patient will be enrolled upon their presentation to the emergency department The usual clinical practice will be followed
Considering the emergency setting and the impossibility for most patients to provide a valid and informed consent before e immediately after the procedures retrieved thrombi will be collected but not analyze An informed consent to the study will be collect in the following day Should the patient refuse the participation the thrombus will be discarded and the patient will be considered as a drop out from the study
Investigators plan to include 30 patients with acute ischemic stroke due to LVO treated with mechanical thrombectomy
Patients characteristics Ischemic stroke patients with LVO will be included in the study if considered suitable for mechanical thrombectomy therapy in accordance with national and international guidelines
Inclusion criteria
age 18 years
Patients with a diagnosis of large vessel occlusion stroke at the Angio-CT study performed in the ER undergoing a mechanical thrombectomy procedure
Thrombus retrieved available for analysis
Exclusion criteria
Deny informed consent
5 ENDPOINTS Primary endpoint
To evaluate the association between thrombus composition studied through histological and immunohistochemical coloration and stroke etiology according to the TOAST classification In particular TOAST voices for etiological category will be dichotomize between cardioembolic and not cardioembolic the latter including large vessel occlusion other determined etiologies and other undetermined etiologies
Secondary endpoints
To evaluate the association between thrombus composition and mechanical thrombectomy outcome
To evaluate the association between thrombus composition and number of passes needed to obtain recanalization
Correlation endpoints
To evaluate the correlation between thrombus composition and global outcome variable such as the National Institutes of Health Stroke Scale NIHSS at seven days and nineteen days after the event and the modified ranking scale mRS at three months
6 METHODS AND ASSESSMENT
Clinical variables and neuroimaging studies The following data will be collected medical history recording demographic data age sex potential vascular risk factors hypertension diabetes mellitus dyslipidemia smoking prior stroke transient ischemic attack TIA previous vascular diseases previous antiplatelet treatment congestive heart failure hypertension vascular disease abnormal renalliver function bleeding history or predisposition labile international normalized ratio drugsalcohol concomitantly diagnosis of atrial fibrillation Oxfordshire Community Stroke Project OCSP classification previous treatment with thrombolysis ASPECTSpc-ASPECTS based on TC scan performed 24 h after acute stroke andHemorrhagic trasformation subtype At three months follow-up the following data will be collected intracranial bleeding major and minor bleeding blood transfusion hospitalization deaths modified Rankin Scale mRS score adherence to treatment stroke recurrence and myocardial infarction Laboratory tests blood counts biochemistry and coagulation tests 12-lead ECG chest radiography carotid ultrasonography and Computed Tomography CT or Magnetic Resonance Imaging MRI will be performed at admission and during the hospitalization
To evaluate neurologic deficit the National Institute of Health Stroke Scale NIHSS will be performed at admission 24 48 72 h 7 days andor at discharge and at 3 months
Functional outcome will be evaluated at discharge and at 3 months by modified rankin scale mRS Stroke etiology will be classified according to TOAST criteria for ischemic stroke TOAST voices for etiological category will be dichotomize between cardioembolic and not cardioembolic the latter including large vessel occlusion other determined etiologies and other undetermined etiologies
To evaluate infarct volume and control hemorrhagic evolution a CT will be performed between 24 ours from treatment Infarct volume will be quantified in cubic centimeters cm3 and assessed according to the formula 05xAxBxC where A direction and C to the number of 10mm slices where infarct volume is present 20 All neuroimaging evaluations will be made by the a neuroradiologist blinded to clinical and laboratory data
Specimen fixation and sectioning Retrieved thrombi will be portioned Part of the specimen will be fixed in a solution of 10 formalin 37 formaldehyde part will be frozen in liquid azote Within 24-48 hours from fixation formalin-fixed thrombi will be dehydrated via increasing concentration of ethanol 70 -80 -95 100 and embedded in paraffin allowing a good preservation of tissue morphology and easy long-term storage Included samples will be sectioned along the major axis of the thrombus in slice with a 4 to 5 µm of thickness
Staining Base coloration will be employed to visualize RBC PLT and fibrin Hematoxylin and eosin staining HE will allow to visualize the general overall structures of the thrombus and the identification of fibrinplatelet aggregates colored in pink RBC red and nucleated cells dark blue The Martius Scarlet Blue MSB staining instead will selectively stain fibrin Dark pinkred RBC yellow and collagen Blue RBC-rich fibrin-poor material will appears red on HE staining and yellow on MSB while RBC-poorfibrin-rich areas will appear as light pink areas on HE staining and pink to red areas on MSB Since platelets are only present in large amount in RBC-poor fibrin-rich areas thrombi will be divided in RBC-richPlatelets-poor mixed and platelets-richRBC-poor Additional base coloration will include Mallory-Azan for collagen and phosphotungstic acid hematoxylin for fibrin Furthermore immunohistochemical staining will be employed to detect the presence of Platelets CD42-Gp-Ib CD41a-Gp-IIbIIIa CD61-GpIIIa white blood cells CD45 leukocyte common antigen monocytemacrophages CD14 CD1a CD68 T lymphocytes CD3 CD8CD4 Natural Killer cells CD16 CD56 premature endothelial and blood progenitor cells CD34 Neutrophils CD45 CD16 Fibrinogen and Von Willebrand factor
Analysis Standardized quantification algorithms to calculate thrombus composition will be employed Computational color-based segmentation analysis by the means of ImageJ Rasband WS ImageJ U S National Institutes of Health Bethesda Maryland USA httpsimagejnihgovij 1997-2018 will allow to apply thresholds for color and thus selective quantification of specific colorscomponents providing data in automated manner about the percentage of total thrombus areas
7 STATISTICAL ANALYSIS
In this study 28 consecutive patients fulfilling selection criteria will be included According to the analysis of Kim 2015 strong differences in mean percentages in clot composition between the two subgroups are reported therefore it is possible to make an hypothesis of an effect size equal to or greater than 1 using the Students t-test this sample size of 28 patients increased to 30 for possible technical difficulties included in one of the two groups determined by the TOAST classification will have a power of 80 to detect such differences at a significance level of 5 two-tailed Statistical analyses will be performed using the IBM-SPSS Statistical Package for Social Science SPSS v20 Kolmogorov-Smirnov test will be used to evaluate data distribution in case of deviation from normality U test di Mann-Whitney or Kruskal-Wallis test will be used for comparisons among two or more groups in case of respect of normality assumptions Students t-test or analysis of variance will be considered Association between dichotomomic items will be evaluated using the Fisher exact test or χ2 as appropriate Due to the explorative approach of this study no adjustment will be made for multiple testing
8 ETHICAL PRINCIPLES
This study will be conducted in accordance with the principles laid down by the 18th World Medical Assembly Helsinki 1964 and all applicable amendments laid down by the World Medical Assemblies and the ICH guidelines for good clinical practice GCP
9 INFORMED CONSENT
The Investigator should fully inform the patient of all pertinent aspects of the study including the written information giving approvalfavorable opinion by the Ethics Committee IRBIEC All participants should be informed to the fullest extent possible about the study in language and terms they are able to understand
Prior to thrombus analysis the written informed consent form should be signed name filled in and personally dated by the patient or by the patients legally acceptable representative and by the person who conducted the informed consent discussion A copy of the signed and dated written informed consent form will be provided to the patient
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None