Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT05760326 |
| Other study ID # |
5153 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
September 2, 2022 |
| Est. completion date |
September 30, 2024 |
Study information
| Verified date |
February 2023 |
| Source |
Fondazione Policlinico Universitario Agostino Gemelli IRCCS |
| Contact |
Giovanni Frisullo, MD |
| Phone |
3476612284 |
| Email |
giovanni.frisullo[@]policlinicogemelli.it |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial 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 (3.7% 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 (H&E) will allow visualization of general thrombus structures and
identification of aggregates of fibrin/platelets (colored pink), red blood cells (red),
and nucleated cells (dark blue).
- Martius Scarlet Blue (MSB), selectively stains fibrin (dark pink/red), 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-IIb/IIIa+, CD61-GpIIIa),
- white blood cells (CD45+, common leukocyte antigen), or monocytes/macrophages
(CD14+, CD1a+, CD68+),
- T lymphocytes (CD3+, CD8/CD4+), or natural killers (CD16+, CD56+), or mobile
premature endothelial cells and blood progenitors (CD34+), or neutrophils (CD45+,
CD16+), or fibrinogen or von Willebrand factor.
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" (ESUS)3. 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 therapy7,8.
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 purposes9,10.
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.
Patient's 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 renal/liver function, bleeding history or
predisposition, labile international normalized ratio, drugs/alcohol concomitantly),
diagnosis of atrial fibrillation, Oxfordshire Community Stroke Project (OCSP) classification,
previous treatment with thrombolysis, ASPECTS/pc-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 and/or 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 0.5xAxBxC, 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 (3,7% 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 (H&E) will allow to visualize the general overall structures of the thrombus
and the identification of fibrin/platelet aggregates (colored in pink), RBC (red) and
nucleated cells (dark blue). The Martius Scarlet Blue (MSB) staining, instead, will
selectively stain fibrin (Dark pink/red), RBC (yellow) and collagen (Blue). RBC-rich
fibrin-poor material will appears red on H&E staining and yellow on MSB, while
RBC-poor/fibrin-rich areas will appear as light pink areas on H&E 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-rich/Platelets-poor, mixed and platelets-rich/RBC-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-IIb/IIIa+, CD61-GpIIIa), white blood cells
(CD45+, leukocyte common antigen), monocyte/macrophages (CD14+, CD1a+, CD68+), T lymphocytes
(CD3+, CD8/CD4+), 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,
W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,
https://imagej.nih.gov/ij/, 1997-2018.) will allow to apply thresholds for color and thus
selective quantification of specific colors/components, 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 Student's 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) v.20.
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 approval/favorable opinion by the Ethics Committee
(IRB/IEC). 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 patient's 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.
