- Email: [email protected]
Immunohistochemical Analysis of Debris Captured by Filter-Type Distal Embolic Protection Devices for Carotid Artery Stenting
ARTICLE IN PRESS
Immunohistochemical Analysis of Debris Captured by Filter-Type Distal Embolic Protection Devices for Carotid Artery Stenting Yukinao Kambayashi, MD,* Ichiro Yuki, MD,* Toshihiro Ishibashi, MD,* Ayako Ikemura, MD,* Takashi Umezawa, CT, PhD, (C.M.I.A.C.),† Masafumi Suzuki, MD,† Issei Kan, MD, PhD,* Hiroyuki Takao, MD, PhD,* and Yuichi Murayama, MD*
Background: Little is known about the micro-debris captured in filter-type distal embolic protection devices (EPD) used for carotid stenting (CAS). This study aimed to determine the histological and immunohistochemical characteristics of such debris by using a new liquid-based cytology (LBC) technique. Methods: Fifteen patients who underwent CAS using a filter-type distal EPD (FilterWire EZ; Boston Scientific, Marlborough, MA, USA) were included in the study. After gross inspection of each recovered filter device, micro-debris were collected using a new LBC technique (SurePath; TriPath Imaging, Inc., Burlington, NC). Histological and immunohistochemical analysis of the recovered debris was performed. The preand postoperative brain magnetic resonance imaging and neurological status of each patient were also reviewed. Results: No patient developed ipsilateral symptomatic stroke due to a thromboembolic event. All 15 patients (100%) had microscopically identifiable debris in the filters, whereas gross inspection detected visible debris only in 5 patients (33.3%). Histological analysis revealed various types of structural components in an advanced atheromatous plaque, including fragments of fibrous cap, calcified plaque, smooth muscle cells, and necrotic tissue fragment infiltrated with monocytes and macrophages. Conclusions: Filter-type EPDs may contribute to reducing the risk of CAS-related embolic events by capturing micro-debris even when gross inspection of the recovered filter shows no visible debris in the device. Key Words: Carotid artery stenting—embolic protection device—micro-debris—histological analysis. © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.
Introduction From the *Department of Neurosurgery, Jikei University School of Medicine, Japan; and †Department of Pathology, Jikei University School of Medicine, Japan. Received June 11, 2016; revision received October 2, 2016; accepted October 23, 2016. Address correspondence to Ichiro Yuki, MD, Department of Neurosurgery, Jikei University School of Medicine, 3-25-8 NishiShinbashi, Minato-ku, Tokyo 105-8461, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2016.10.025
Studies suggest that post-procedural ipsilateral stroke, most likely caused by a thromboembolic event during stent deployment, remains a serious complication of carotid artery stenting (CAS).1-4 Filter-type embolic protection devices (EPDs) are designed to reduce the risk of distal embolic event during CAS procedures. Several clinical studies have shown that the use of EPDs is associated with lower risk of embolic events.4-6 However, little is known about the micro-debris captured in these devices, partially because a layer of blood in the recovered filter often obscures the visibility of the miniscule contents.
Journal of Stroke and Cerebrovascular Diseases, Vol. ■■, No. ■■ (), 2016: pp ■■–■■
1
ARTICLE IN PRESS Y. KAMBAYASHI ET AL.
2
Liquid-based cytology (LBC) test is a cytology technique used in gynecology to detect cellular components in Pap smear samples, which are frequently replete with red blood cells.7 A new LBC test called “SurePath” (TriPath Imaging, Inc., Burlington, NC) is known to have a higher detection rate than conventional LBC tests, mainly owing to its unique density-gradient centrifugation process.8-10 In this study, micro-debris captured in filter-type EPDs used in CAS were recovered using SurePath and underwent thorough histological and immunohistochemical analysis. The clinical characteristic and image findings of each treated patient were also reviewed.
Methods The CAS Procedure From March 2013 to October 2014, 29 patients with carotid stenosis (4 symptomatic) underwent the CAS procedure. Among them, 15 patients who were treated using FilterWire EZ (FWEZ; Boston Scientific, Marlborough, MA) were selected for this study. All of the patients were started on dual antiplatelet therapy with the combination of 75 mg clopidogrel per day and 100 mg aspirin per day. If the patient was a poor or nonresponder to clopidogrel, the combination of 100 mg of aspirin and 200 mg of cilostazol per day was selected. Dual antiplatelet therapy was started at least 7 days before the procedure. All procedures were performed via femoral access under local anesthesia. Systemic heparinization was performed with the goal of maintaining the activating clotting time within 250-300 seconds during the procedure. A 6 Fr shuttle sheath was advanced via the femoral artery and placed at the treatment side of the common carotid artery. After the stenotic lesion was crossed with a FWEZ, the filter device was deployed at the distal segment of the cervical internal carotid artery. Predilatation was performed using an angioplasty balloon (Sterling; Boston Scientific, Natick, MA), and an appropriate sized Carotid WALLSTENT (Boston Scientific) was deployed over the FWEZ. Post-dilation was performed using the same angioplasty balloon. After confirming sufficient dilatation of stenotic lesion in the post-procedure angiogram, FWEZ was re-sheathed into a dedicated recovery sheath. The Institutional Review Board of the Jikei University School of Medicine approved the study protocol.
Evaluation of Post-Procedure Imaging and Clinical Findings For the pre- and post-procedure imaging analysis, all of the patients underwent brain magnetic resonance imaging (MRI) (1.5 T). The sequences included diffusionweighted image (DWI), fluid-attenuated inversion recovery,
susceptibility-weighted imaging, and magnetic resonance angiography. Any high-intensity signal detected in the post-procedure DWI was considered a thromboembolic complication and was recorded. Neurological examination was performed immediately after the procedure and daily during the hospitalization. A modified Rankin scale on the day of discharge was also recorded as a clinical outcome.
Debris Analysis Immediately after recovery of the FWEZ from the guiding catheter, the delivery wire near the device was cut with sterilized scissors and the filter was carefully submerged in the SurePath preservative fluid. Rinsing or flushing the filter with saline was avoided to prevent the loss of micro-debris. The preservative was composed of multiple alcohols (ethanol 21.7%, methanol 1.2%, and isopropanol 1.1%) for the osmotic hemolysis of the red blood cells and fixation and rinsing of the cellular components. Each sample was then mixed by vortexing, and the debris were separated from the supernatant by centrifugation. The supernatant was discarded, and for the next enrichment step, centrifugal sedimentation through 3 mL of a hemolytic fixative, CytoRich Red Preservative (TriPath Imaging, Inc.), was performed. After centrifugation, the pelleted micro-debris were re-suspended with 500 μmL of distilled water, mixed, and transferred to a PrepStain Settling Chamber (TriPath Imaging, Inc.) mounted on a SurePath PreCoat slide. The debris were then sedimented by gravity for 10 minutes. The settling chamber was used to deposit a thin layer of micro-debris concentrated within a circular area (φ13 mm) on the glass slides, which were coated with poly-L-lysine (beta-helix structure). After rinsing the slide using 95% ethanol, the sample was fixed with 100% ethanol. Each prepared slide glass was then stained by multiple staining, including hematoxylin and eosin (H&E), Masson trichrome, elastic Verhoeff-van Gieson (EVG), Papanicolaou, and Kossa. Immunohistochemical analysis was performed using alfa smooth muscle actin (αSMA) and CD68. The debris, presented within a 13-mm-diameter circle on the slide glass, were examined under a microscope by a trained neuropathologist and neurosurgeons. Because of the limited amount of recovered tissue sample, H&E staining was prioritized and the other types of staining, including immunohistochemical analysis, were selected based on the types of tissue fragment observed in the primary staining. For instance, if a sample showed preponderance of noncellular component, stains focusing on the extracellular matrix (e.g., Masson trichrome and EVG) were selected. For samples with rich cellular component, immunohistochemical analysis e.g. CD68 staining and αSMA staining were performed.
ARTICLE IN PRESS ANALYSIS OF DEBRIS CAPTURED BY EPD DURING CAS
For controls, blood samples collected from a healthy volunteer were used for the analysis. A total of 20 mL of whole blood was drawn via the right median cubital vein and mixed with .25 cc of heparin (250 units). The blood was then poured into the unused FWEZ device, which was placed in a SurePath preservative fluid. The filter was then processed in the procedure above. The association between the number of debris found on each sample and the postoperative DWI findings was investigated. The relationship between the debris number and the symptomatology of the carotid stenosis was also evaluated.
Results Post-Procedure Clinical and Imaging Findings The patients’ demographics and the summary of their clinical and imaging findings were shown in Table 1. Every CAS procedure was performed uneventfully, and no patient had any neurological deficit immediately after the procedure. Patient 8 had concomitant severe stenosis at the petrous segment of the ipsilateral internal carotid artery (tandem lesion). He showed unstable transient ischemic symptoms, including consciousness disturbance pre and post procedure, and underwent percutaneous transluminal angioplasty for the lesion a day after the CAS procedure. The symptoms completely resolved after percutaneous transluminal angioplasty, and the patient was discharged home without any neurological deficit (modified Rankin scale = 0). Post-procedure MRI was performed 1 day after the procedure for every patient except for patient 5, who had an implanted cardiac pacemaker and who instead underwent head computed tomography scan that revealed no interval change. Eight of 14 patients (57.1%) showed various degrees of small high-intensity lesion(s) on postoperative DWI. All lesions measured <5 mm in size, except for that of patient 9.
Debris Analysis Microscopic evaluation demonstrated micro-debris in all 15 filter samples (100%) after the LBC processing, whereas only 5 cases (33.3%) showed visible debris on gross inspection (Figs 1, 2). No micro-debris was detected in the control samples. In all samples, H&E staining showed a preponderance of micro-debris that had eosinophilic fibrous structure and size of 10-100 μm. The structures appeared to be mature fibrin fibers or a part of necrotic tissue frequently observed in atheromatous plaque (Fig 3). Occasionally, Kossa-positive deposits, the calcified component of plaque, were detected in the debris (Fig 4, A,B). In samples stained with αSMA, fragments of αSMApositive debris that were presumably derived from layers
3
of smooth muscle cells were also detected (Fig 4, C,D). In samples stained with Masson trichrome, fragments of collagenous fibrous tissue measuring 100 μm-1 mm with few cellular components were seen (Fig 5, A,B). For those processed with CD68 staining, debris fragments infiltrated with positively stained cells (macrophages and foam cells) were observed (Fig 5, C,D). Elastic fibers were not detected in EVG staining.
Association between the Number of Debris and Image or Clinical Findings The number of debris larger than 50 μm within a field (132.7 mm2) was manually counted, and the association between the debris number and the postoperative DWI findings was evaluated. Average number of the debris found on the evaluated field was 38.9 (range: 2-115, standard deviation: 42.6). The sample from patient number 1 showed the largest number of debris of 115; however, the DWI findings showed 1 high-intensity lesion of 2.5 mm in the MCA territory. On the other hand, patient number 8 showed only 2 debris, which was the lowest number in the group, and the DWI findings of the same patient showed 8 different lesions in different vascular territories, with each of them measuring approximately 3 mm. Because of the multifactorial nature of the debris sample, for example, the size, shape, and type of debris based on histological findings, a statistical analysis assessing the association between the histological and the image findings was avoided; however, the qualitative inspection of the 2 datasets indicated that there was no clear association observed in this particular patient group. Three patients (patient number 4, 8, and 9) were originally presented as having symptomatic carotid stenosis (Table 1). The numbers of debris discovered in these patients were 20, 2, and 12 per field, respectively. The debris numbers were relatively low compared with the samples from the asymptomatic group (mean: 50.7, range: 3-115, standard deviation: 46.3), although the sample size was too small to conclude the statistical significance.
Discussion Even after the development of a variety of EPDs, distal embolic events remain serious procedure-related complications of the CAS procedure.6 A filter-type EPD is one of the commonly used protection devices. It is especially preferred for the treatment of patients with poor collateral circulation via the circle of Willis because it does not require a temporary occlusion of blood flow during the procedure. The filter device used in this study, FWEZ, has a pore size of 80 μm, which is the smallest among the currently available filter-type EPDs.4 With the limited number of patients in this study, all filter devices recovered from the patients showed micro-debris (100%), and many of these micro-debris measured between 50 and 100 μm,
4
Table 1. Summary of the patient demographics and the clinical results Posttreatment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gender
Age
Symptomatic or not
Degree of stenosis (%)
Hypertension
Dyslipidemia
Diabetes
Symptomatic stroke
M M F F M M M M M M M M M M M
79 65 76 76 70 62 82 69 61 75 71 63 66 77 72
(−) (−) (−) (+) (−) (−) (−) (+) (+) (−) (−) (−) (−) (−) (−)
69 70 62 88 73 74 76 50 50 66 95 68 82 90 70
(+) (+) (+) (−) (−) (+) (−) (−) (+) (+) (+) (+) (−) (+) (+)
(+) (−) (+) (−) (−) (−) (−) (+) (+) (+) (+) (+) (−) (+) (+)
(−) (−) (−) (+) (+) (−) (−) (+) (−) (+) (+) (−) (−) (+) (+)
(−) (−) (−) (−) (−) (−) (−) (−)* (−) (−) (−) (−) (−) (−) (−)
DWI lesion 2.5 mm × 1 5 mm × 1 2 mm × 8 (−) N/A† 2 mm × 2 (−) 3 mm × 5 10 mm × 1 2 mm × 2 (−) 2 mm × 2 (−) (−) (−)
mRS at D/C
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Patient #
0 0 0 0 1 0 0 0 0 1 0 0 0 0 0
*The patient had concomitant severe stenosis at the petrous segment of the ipsilateral ICA (tandem lesion) and presented with unstable transient ischemic symptoms. The symptoms resolved after percutaneous transluminal angioplasty (PTA) for the intracranial lesion a day after the CAS procedure (mRS = 0). † The patient had an implanted cardiac pacemaker and underwent head computed tomography (CT) scan that revealed no interval change. Y. KAMBAYASHI ET AL.
ARTICLE IN PRESS ANALYSIS OF DEBRIS CAPTURED BY EPD DURING CAS
Figure 1. A recovered filter positive for recovered micro-debris by (A) gross inspection and (B) microscopic findings (Papanicolaou stainings, 100×). The arrow indicates the yellowish debris in the filter. (Color version of figure is available online.)
whereas gross inspection showed only 33.3% of filter devices recovered had visible debris. It is possible that filter devices can contribute to the prevention of embolic complications, even when gross inspection of the recovered filter device shows no debris inside. Giannakopoulos et al previously reported that the LBC technique could be used to recover micro-debris from filtertype EPDs.14 Their study used the ThinPrep system and they concluded that 30 out of 53 cases (56.6%) showed micro-debris in the filter. SurePath is a new LBC technique designed to improve the detection rate of conventionally used LBC, for example, ThinPrep platform. SurePath is reported to have more optimized cell preservation and reduced artifacts, which sometimes hinder microscopic interpretation.7,8,9,10 The present series has a
Figure 2. A recovered filter negative for recovered micro-debris by (A) gross inspection and (B) microscopic findings (Papanicolaou stainings, 100×). Note the micro-debris found in the microscopic observation even though the gross inspection showed no visible debris in the filter.
5
Figure 3. (A) A sample stained by hematoxylin and eosin showed abundant eosinophilic fibrous structures measuring 10-100 μm (40×) in size. (B) A magnified view of the same sample showed a few cellular components with inflammatory cell infiltration (arrowheads). The structures appeared to be the mature fibrin fibers or part of necrotic tissue frequently observed in atheromatous plaque.
100% detection rate of micro-debris. The improved detection rate may be associated with the improved LBC technology. Histological classification proposed by the American Heart Association defines the advanced (type IV) plaque as a sub-intimal accumulation consisting of extracellular lipid including cholesterol crystals and macrophage foam cells surrounding a lipid core. The intimal coverage of the plaque eventually becomes a collagenous tissue or fibrous cap. The lesion is then classified as type V.11-13 The histological and immunohistochemical analysis in this series reveals almost every typical structural component of advanced plaque (types IV and V), such as
Figure 4. (A) Kossa-positive deposits, or the calcified components of plaque, were also detected in the debris (von Kossa stain; 200×). (B) Magnified view of the same sample. The arrow indicates the calcified component in the debris. (C) A sample processed using alfa smooth muscle actin (αSMA) stain (400×) revealed a fragment of debris with layers of smooth muscle cells. (D) Magnified view of the same sample (C).
ARTICLE IN PRESS Y. KAMBAYASHI ET AL.
6
was not sufficient for the multiple slides and could not be tested with different stains. Further refinement on the method of dividing the micro-debris into several slides for the multiple staining is required.
Conclusions
Figure 5. (A) A sample processed using Masson trichrome stain (400×) revealed a fragment of collagen-rich debris (blue). (B) Magnified view of the same sample. (C) A sample processed with CD68 stain (400×) revealed micro-debris infiltrated with CD68-positive cells (macrophages). (D) Another sample processed with CD68 staining (400×) demonstrated a roundshaped macrophage with multiple small bright inclusions in the cytoplasm, suggesting that it might be a foam cell derived from the carotid plaque. (Color version of figure is available online.)
macrophages infiltrating old fibrin tissue and fragments of fibrin cap. These findings suggest that the CAS procedure may either induce the rupture of plaque or, in some cases, push the plaque contents out through erosion. To the best of our knowledge, this is the first study to report a detailed histological analysis, including immunohistochemistry, on captured micro-debris.14,15
Study Limitations With a limited case number in this study, concluding on the efficacy of filter-type device in preventing the development of symptomatic stroke will be premature. The comparative analysis between the debris number and the DWI findings showed no association between the 2 results. Potential explanations that account for this result include (1) occasional inappropriate apposition of the filter against the wall of the vessel, (2) potential involvement of the lipid component that was not detected in the histological evaluations, and (3) the thromboembolic events happened before or after the effective filter placement. Further investigation with a larger case number is warranted to evaluate the true efficacy of EPDs. Also, as described above, even though LBC is a useful method for detecting cellular component or fragments of extracellular matrix, the lipid component of plaque can be resolved during the preparation process owing to the use of alcohol-based reagents. Thus, determining whether the ruptured lipid core is caught in the filter device during the procedure may be difficult. Other staining methods must be performed separately to address this issue. Lastly, the amount of micro-debris recovered from each sample
Using a novel LBC method, micro-debris can be detected in every filter EPD used in the CAS procedures. Histological and immunohistochemical analysis on the recovered debris revealed that majority of the structural components are from advanced atheromatous plaque, including old fibrin fibers with infiltrated macrophages and leukocytes, and fragments of fibrin cap, indicating potential rupture of the plaque during the procedure. Filtertype EPDs may contribute to reducing the risk of CASrelated embolic events by capturing micro-debris even when gross inspection of the recovered filter show no visible debris in the device.
References 1. Yadav JS, Wholey MH, Kuntz RE, et al. Protected carotid-artery stenting versus endarterectomy in high-risk patients. N Engl J Med 2004;351:1493-1501. 2. Brott TG, Hobson RW 2nd, Howard G, et al. Stenting versus endarterectomy for treatment of carotid-artery stenosis. N Engl J Med 2010;363:11-23. 3. Silver FL, Mackey A, Clark WM, et al. Safety of stenting and endarterectomy by symptomatic status in the Carotid Revascularization Endarterectomy Versus Stenting Trial (CREST). Stroke 2011;42:675-680. 4. Sangiorgi G, Colombo A. Embolic protection devices. Heart 2003;89:990-992. 5. Iyer V, de Donato G, Deloose K, et al. The type of embolic protection does not influence the outcome in carotid artery stenting. J Vasc Surg 2007;46:251-256. 6. Mousa AY, Campbell JE, Aburahma AF, et al. Current update of cerebral embolic protection devices. J Vasc Surg 2012;56:1429-1437. 7. Bentz JS. Liquid-based cytology for cervical cancer screening. Expert Rev Mol Diagn 2005;5:857-871. 8. Kenyon S, Sweeney BJ, Happel J, et al. Comparison of BD Surepath and ThinPrep Pap systems in the processing of mucus-rich specimens. Cancer Cytopathol 2010;118:244249. 9. Fontaine D, Narine N, Naugler C. Unsatisfactory rates vary between cervical cytology samples prepared using ThinPrep and SurePath platforms: a review and metaanalysis. BMJ Open 2012;2:e000847. 10. Rosa M, Pragasam P, Saremian J, et al. The unsatisfactory ThinPrep(R) Pap test: analysis of technical aspects, most common causes, and recommendations for improvement. Diagn Cytopathol 2013;41:588-594. 11. Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 1995;92:1355-1374. 12. Nighoghossian N, Derex L, Douek P. The vulnerable carotid artery plaque: current imaging methods and new perspectives. Stroke 2005;36:2764-2772.
ARTICLE IN PRESS ANALYSIS OF DEBRIS CAPTURED BY EPD DURING CAS 13. Falk E, Nakano M, Bentzon JF, et al. Update on acute coronary syndromes: the pathologists’ view. Eur Heart J 2013;34:719-728. 14. Giannakopoulos TG, Moulakakis K, Sfyroeras GS, et al. Association between plaque echogenicity and embolic material captured in filter during protected carotid angioplasty and stenting. Eur J Vasc Endovasc Surg 2012;43:627-631.
7 15. van Laanen JH, Hendriks JM, Verhagen HJ, et al. Quantity, particle size, and histologic composition of embolic debris collected in a distal protection filter after carotid angioplasty and stenting: correlation with patient characteristics, timing of carotid artery stenting, and procedural details. J Thorac Cardiovasc Surg 2013;146:492495.
Immunohistochemical Analysis of Debris Captured by Filter-Type Distal Embolic Protection Devices for Carotid Artery Stenting Yukinao Kambayashi, MD,* Ichiro Yuki, MD,* Toshihiro Ishibashi, MD,* Ayako Ikemura, MD,* Takashi Umezawa, CT, PhD, (C.M.I.A.C.),† Masafumi Suzuki, MD,† Issei Kan, MD, PhD,* Hiroyuki Takao, MD, PhD,* and Yuichi Murayama, MD*
Background: Little is known about the micro-debris captured in filter-type distal embolic protection devices (EPD) used for carotid stenting (CAS). This study aimed to determine the histological and immunohistochemical characteristics of such debris by using a new liquid-based cytology (LBC) technique. Methods: Fifteen patients who underwent CAS using a filter-type distal EPD (FilterWire EZ; Boston Scientific, Marlborough, MA, USA) were included in the study. After gross inspection of each recovered filter device, micro-debris were collected using a new LBC technique (SurePath; TriPath Imaging, Inc., Burlington, NC). Histological and immunohistochemical analysis of the recovered debris was performed. The preand postoperative brain magnetic resonance imaging and neurological status of each patient were also reviewed. Results: No patient developed ipsilateral symptomatic stroke due to a thromboembolic event. All 15 patients (100%) had microscopically identifiable debris in the filters, whereas gross inspection detected visible debris only in 5 patients (33.3%). Histological analysis revealed various types of structural components in an advanced atheromatous plaque, including fragments of fibrous cap, calcified plaque, smooth muscle cells, and necrotic tissue fragment infiltrated with monocytes and macrophages. Conclusions: Filter-type EPDs may contribute to reducing the risk of CAS-related embolic events by capturing micro-debris even when gross inspection of the recovered filter shows no visible debris in the device. Key Words: Carotid artery stenting—embolic protection device—micro-debris—histological analysis. © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.
Introduction From the *Department of Neurosurgery, Jikei University School of Medicine, Japan; and †Department of Pathology, Jikei University School of Medicine, Japan. Received June 11, 2016; revision received October 2, 2016; accepted October 23, 2016. Address correspondence to Ichiro Yuki, MD, Department of Neurosurgery, Jikei University School of Medicine, 3-25-8 NishiShinbashi, Minato-ku, Tokyo 105-8461, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2016.10.025
Studies suggest that post-procedural ipsilateral stroke, most likely caused by a thromboembolic event during stent deployment, remains a serious complication of carotid artery stenting (CAS).1-4 Filter-type embolic protection devices (EPDs) are designed to reduce the risk of distal embolic event during CAS procedures. Several clinical studies have shown that the use of EPDs is associated with lower risk of embolic events.4-6 However, little is known about the micro-debris captured in these devices, partially because a layer of blood in the recovered filter often obscures the visibility of the miniscule contents.
Journal of Stroke and Cerebrovascular Diseases, Vol. ■■, No. ■■ (), 2016: pp ■■–■■
1
ARTICLE IN PRESS Y. KAMBAYASHI ET AL.
2
Liquid-based cytology (LBC) test is a cytology technique used in gynecology to detect cellular components in Pap smear samples, which are frequently replete with red blood cells.7 A new LBC test called “SurePath” (TriPath Imaging, Inc., Burlington, NC) is known to have a higher detection rate than conventional LBC tests, mainly owing to its unique density-gradient centrifugation process.8-10 In this study, micro-debris captured in filter-type EPDs used in CAS were recovered using SurePath and underwent thorough histological and immunohistochemical analysis. The clinical characteristic and image findings of each treated patient were also reviewed.
Methods The CAS Procedure From March 2013 to October 2014, 29 patients with carotid stenosis (4 symptomatic) underwent the CAS procedure. Among them, 15 patients who were treated using FilterWire EZ (FWEZ; Boston Scientific, Marlborough, MA) were selected for this study. All of the patients were started on dual antiplatelet therapy with the combination of 75 mg clopidogrel per day and 100 mg aspirin per day. If the patient was a poor or nonresponder to clopidogrel, the combination of 100 mg of aspirin and 200 mg of cilostazol per day was selected. Dual antiplatelet therapy was started at least 7 days before the procedure. All procedures were performed via femoral access under local anesthesia. Systemic heparinization was performed with the goal of maintaining the activating clotting time within 250-300 seconds during the procedure. A 6 Fr shuttle sheath was advanced via the femoral artery and placed at the treatment side of the common carotid artery. After the stenotic lesion was crossed with a FWEZ, the filter device was deployed at the distal segment of the cervical internal carotid artery. Predilatation was performed using an angioplasty balloon (Sterling; Boston Scientific, Natick, MA), and an appropriate sized Carotid WALLSTENT (Boston Scientific) was deployed over the FWEZ. Post-dilation was performed using the same angioplasty balloon. After confirming sufficient dilatation of stenotic lesion in the post-procedure angiogram, FWEZ was re-sheathed into a dedicated recovery sheath. The Institutional Review Board of the Jikei University School of Medicine approved the study protocol.
Evaluation of Post-Procedure Imaging and Clinical Findings For the pre- and post-procedure imaging analysis, all of the patients underwent brain magnetic resonance imaging (MRI) (1.5 T). The sequences included diffusionweighted image (DWI), fluid-attenuated inversion recovery,
susceptibility-weighted imaging, and magnetic resonance angiography. Any high-intensity signal detected in the post-procedure DWI was considered a thromboembolic complication and was recorded. Neurological examination was performed immediately after the procedure and daily during the hospitalization. A modified Rankin scale on the day of discharge was also recorded as a clinical outcome.
Debris Analysis Immediately after recovery of the FWEZ from the guiding catheter, the delivery wire near the device was cut with sterilized scissors and the filter was carefully submerged in the SurePath preservative fluid. Rinsing or flushing the filter with saline was avoided to prevent the loss of micro-debris. The preservative was composed of multiple alcohols (ethanol 21.7%, methanol 1.2%, and isopropanol 1.1%) for the osmotic hemolysis of the red blood cells and fixation and rinsing of the cellular components. Each sample was then mixed by vortexing, and the debris were separated from the supernatant by centrifugation. The supernatant was discarded, and for the next enrichment step, centrifugal sedimentation through 3 mL of a hemolytic fixative, CytoRich Red Preservative (TriPath Imaging, Inc.), was performed. After centrifugation, the pelleted micro-debris were re-suspended with 500 μmL of distilled water, mixed, and transferred to a PrepStain Settling Chamber (TriPath Imaging, Inc.) mounted on a SurePath PreCoat slide. The debris were then sedimented by gravity for 10 minutes. The settling chamber was used to deposit a thin layer of micro-debris concentrated within a circular area (φ13 mm) on the glass slides, which were coated with poly-L-lysine (beta-helix structure). After rinsing the slide using 95% ethanol, the sample was fixed with 100% ethanol. Each prepared slide glass was then stained by multiple staining, including hematoxylin and eosin (H&E), Masson trichrome, elastic Verhoeff-van Gieson (EVG), Papanicolaou, and Kossa. Immunohistochemical analysis was performed using alfa smooth muscle actin (αSMA) and CD68. The debris, presented within a 13-mm-diameter circle on the slide glass, were examined under a microscope by a trained neuropathologist and neurosurgeons. Because of the limited amount of recovered tissue sample, H&E staining was prioritized and the other types of staining, including immunohistochemical analysis, were selected based on the types of tissue fragment observed in the primary staining. For instance, if a sample showed preponderance of noncellular component, stains focusing on the extracellular matrix (e.g., Masson trichrome and EVG) were selected. For samples with rich cellular component, immunohistochemical analysis e.g. CD68 staining and αSMA staining were performed.
ARTICLE IN PRESS ANALYSIS OF DEBRIS CAPTURED BY EPD DURING CAS
For controls, blood samples collected from a healthy volunteer were used for the analysis. A total of 20 mL of whole blood was drawn via the right median cubital vein and mixed with .25 cc of heparin (250 units). The blood was then poured into the unused FWEZ device, which was placed in a SurePath preservative fluid. The filter was then processed in the procedure above. The association between the number of debris found on each sample and the postoperative DWI findings was investigated. The relationship between the debris number and the symptomatology of the carotid stenosis was also evaluated.
Results Post-Procedure Clinical and Imaging Findings The patients’ demographics and the summary of their clinical and imaging findings were shown in Table 1. Every CAS procedure was performed uneventfully, and no patient had any neurological deficit immediately after the procedure. Patient 8 had concomitant severe stenosis at the petrous segment of the ipsilateral internal carotid artery (tandem lesion). He showed unstable transient ischemic symptoms, including consciousness disturbance pre and post procedure, and underwent percutaneous transluminal angioplasty for the lesion a day after the CAS procedure. The symptoms completely resolved after percutaneous transluminal angioplasty, and the patient was discharged home without any neurological deficit (modified Rankin scale = 0). Post-procedure MRI was performed 1 day after the procedure for every patient except for patient 5, who had an implanted cardiac pacemaker and who instead underwent head computed tomography scan that revealed no interval change. Eight of 14 patients (57.1%) showed various degrees of small high-intensity lesion(s) on postoperative DWI. All lesions measured <5 mm in size, except for that of patient 9.
Debris Analysis Microscopic evaluation demonstrated micro-debris in all 15 filter samples (100%) after the LBC processing, whereas only 5 cases (33.3%) showed visible debris on gross inspection (Figs 1, 2). No micro-debris was detected in the control samples. In all samples, H&E staining showed a preponderance of micro-debris that had eosinophilic fibrous structure and size of 10-100 μm. The structures appeared to be mature fibrin fibers or a part of necrotic tissue frequently observed in atheromatous plaque (Fig 3). Occasionally, Kossa-positive deposits, the calcified component of plaque, were detected in the debris (Fig 4, A,B). In samples stained with αSMA, fragments of αSMApositive debris that were presumably derived from layers
3
of smooth muscle cells were also detected (Fig 4, C,D). In samples stained with Masson trichrome, fragments of collagenous fibrous tissue measuring 100 μm-1 mm with few cellular components were seen (Fig 5, A,B). For those processed with CD68 staining, debris fragments infiltrated with positively stained cells (macrophages and foam cells) were observed (Fig 5, C,D). Elastic fibers were not detected in EVG staining.
Association between the Number of Debris and Image or Clinical Findings The number of debris larger than 50 μm within a field (132.7 mm2) was manually counted, and the association between the debris number and the postoperative DWI findings was evaluated. Average number of the debris found on the evaluated field was 38.9 (range: 2-115, standard deviation: 42.6). The sample from patient number 1 showed the largest number of debris of 115; however, the DWI findings showed 1 high-intensity lesion of 2.5 mm in the MCA territory. On the other hand, patient number 8 showed only 2 debris, which was the lowest number in the group, and the DWI findings of the same patient showed 8 different lesions in different vascular territories, with each of them measuring approximately 3 mm. Because of the multifactorial nature of the debris sample, for example, the size, shape, and type of debris based on histological findings, a statistical analysis assessing the association between the histological and the image findings was avoided; however, the qualitative inspection of the 2 datasets indicated that there was no clear association observed in this particular patient group. Three patients (patient number 4, 8, and 9) were originally presented as having symptomatic carotid stenosis (Table 1). The numbers of debris discovered in these patients were 20, 2, and 12 per field, respectively. The debris numbers were relatively low compared with the samples from the asymptomatic group (mean: 50.7, range: 3-115, standard deviation: 46.3), although the sample size was too small to conclude the statistical significance.
Discussion Even after the development of a variety of EPDs, distal embolic events remain serious procedure-related complications of the CAS procedure.6 A filter-type EPD is one of the commonly used protection devices. It is especially preferred for the treatment of patients with poor collateral circulation via the circle of Willis because it does not require a temporary occlusion of blood flow during the procedure. The filter device used in this study, FWEZ, has a pore size of 80 μm, which is the smallest among the currently available filter-type EPDs.4 With the limited number of patients in this study, all filter devices recovered from the patients showed micro-debris (100%), and many of these micro-debris measured between 50 and 100 μm,
4
Table 1. Summary of the patient demographics and the clinical results Posttreatment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gender
Age
Symptomatic or not
Degree of stenosis (%)
Hypertension
Dyslipidemia
Diabetes
Symptomatic stroke
M M F F M M M M M M M M M M M
79 65 76 76 70 62 82 69 61 75 71 63 66 77 72
(−) (−) (−) (+) (−) (−) (−) (+) (+) (−) (−) (−) (−) (−) (−)
69 70 62 88 73 74 76 50 50 66 95 68 82 90 70
(+) (+) (+) (−) (−) (+) (−) (−) (+) (+) (+) (+) (−) (+) (+)
(+) (−) (+) (−) (−) (−) (−) (+) (+) (+) (+) (+) (−) (+) (+)
(−) (−) (−) (+) (+) (−) (−) (+) (−) (+) (+) (−) (−) (+) (+)
(−) (−) (−) (−) (−) (−) (−) (−)* (−) (−) (−) (−) (−) (−) (−)
DWI lesion 2.5 mm × 1 5 mm × 1 2 mm × 8 (−) N/A† 2 mm × 2 (−) 3 mm × 5 10 mm × 1 2 mm × 2 (−) 2 mm × 2 (−) (−) (−)
mRS at D/C
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Patient #
0 0 0 0 1 0 0 0 0 1 0 0 0 0 0
*The patient had concomitant severe stenosis at the petrous segment of the ipsilateral ICA (tandem lesion) and presented with unstable transient ischemic symptoms. The symptoms resolved after percutaneous transluminal angioplasty (PTA) for the intracranial lesion a day after the CAS procedure (mRS = 0). † The patient had an implanted cardiac pacemaker and underwent head computed tomography (CT) scan that revealed no interval change. Y. KAMBAYASHI ET AL.
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Figure 1. A recovered filter positive for recovered micro-debris by (A) gross inspection and (B) microscopic findings (Papanicolaou stainings, 100×). The arrow indicates the yellowish debris in the filter. (Color version of figure is available online.)
whereas gross inspection showed only 33.3% of filter devices recovered had visible debris. It is possible that filter devices can contribute to the prevention of embolic complications, even when gross inspection of the recovered filter device shows no debris inside. Giannakopoulos et al previously reported that the LBC technique could be used to recover micro-debris from filtertype EPDs.14 Their study used the ThinPrep system and they concluded that 30 out of 53 cases (56.6%) showed micro-debris in the filter. SurePath is a new LBC technique designed to improve the detection rate of conventionally used LBC, for example, ThinPrep platform. SurePath is reported to have more optimized cell preservation and reduced artifacts, which sometimes hinder microscopic interpretation.7,8,9,10 The present series has a
Figure 2. A recovered filter negative for recovered micro-debris by (A) gross inspection and (B) microscopic findings (Papanicolaou stainings, 100×). Note the micro-debris found in the microscopic observation even though the gross inspection showed no visible debris in the filter.
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Figure 3. (A) A sample stained by hematoxylin and eosin showed abundant eosinophilic fibrous structures measuring 10-100 μm (40×) in size. (B) A magnified view of the same sample showed a few cellular components with inflammatory cell infiltration (arrowheads). The structures appeared to be the mature fibrin fibers or part of necrotic tissue frequently observed in atheromatous plaque.
100% detection rate of micro-debris. The improved detection rate may be associated with the improved LBC technology. Histological classification proposed by the American Heart Association defines the advanced (type IV) plaque as a sub-intimal accumulation consisting of extracellular lipid including cholesterol crystals and macrophage foam cells surrounding a lipid core. The intimal coverage of the plaque eventually becomes a collagenous tissue or fibrous cap. The lesion is then classified as type V.11-13 The histological and immunohistochemical analysis in this series reveals almost every typical structural component of advanced plaque (types IV and V), such as
Figure 4. (A) Kossa-positive deposits, or the calcified components of plaque, were also detected in the debris (von Kossa stain; 200×). (B) Magnified view of the same sample. The arrow indicates the calcified component in the debris. (C) A sample processed using alfa smooth muscle actin (αSMA) stain (400×) revealed a fragment of debris with layers of smooth muscle cells. (D) Magnified view of the same sample (C).
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was not sufficient for the multiple slides and could not be tested with different stains. Further refinement on the method of dividing the micro-debris into several slides for the multiple staining is required.
Conclusions
Figure 5. (A) A sample processed using Masson trichrome stain (400×) revealed a fragment of collagen-rich debris (blue). (B) Magnified view of the same sample. (C) A sample processed with CD68 stain (400×) revealed micro-debris infiltrated with CD68-positive cells (macrophages). (D) Another sample processed with CD68 staining (400×) demonstrated a roundshaped macrophage with multiple small bright inclusions in the cytoplasm, suggesting that it might be a foam cell derived from the carotid plaque. (Color version of figure is available online.)
macrophages infiltrating old fibrin tissue and fragments of fibrin cap. These findings suggest that the CAS procedure may either induce the rupture of plaque or, in some cases, push the plaque contents out through erosion. To the best of our knowledge, this is the first study to report a detailed histological analysis, including immunohistochemistry, on captured micro-debris.14,15
Study Limitations With a limited case number in this study, concluding on the efficacy of filter-type device in preventing the development of symptomatic stroke will be premature. The comparative analysis between the debris number and the DWI findings showed no association between the 2 results. Potential explanations that account for this result include (1) occasional inappropriate apposition of the filter against the wall of the vessel, (2) potential involvement of the lipid component that was not detected in the histological evaluations, and (3) the thromboembolic events happened before or after the effective filter placement. Further investigation with a larger case number is warranted to evaluate the true efficacy of EPDs. Also, as described above, even though LBC is a useful method for detecting cellular component or fragments of extracellular matrix, the lipid component of plaque can be resolved during the preparation process owing to the use of alcohol-based reagents. Thus, determining whether the ruptured lipid core is caught in the filter device during the procedure may be difficult. Other staining methods must be performed separately to address this issue. Lastly, the amount of micro-debris recovered from each sample
Using a novel LBC method, micro-debris can be detected in every filter EPD used in the CAS procedures. Histological and immunohistochemical analysis on the recovered debris revealed that majority of the structural components are from advanced atheromatous plaque, including old fibrin fibers with infiltrated macrophages and leukocytes, and fragments of fibrin cap, indicating potential rupture of the plaque during the procedure. Filtertype EPDs may contribute to reducing the risk of CASrelated embolic events by capturing micro-debris even when gross inspection of the recovered filter show no visible debris in the device.
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ARTICLE IN PRESS ANALYSIS OF DEBRIS CAPTURED BY EPD DURING CAS 13. Falk E, Nakano M, Bentzon JF, et al. Update on acute coronary syndromes: the pathologists’ view. Eur Heart J 2013;34:719-728. 14. Giannakopoulos TG, Moulakakis K, Sfyroeras GS, et al. Association between plaque echogenicity and embolic material captured in filter during protected carotid angioplasty and stenting. Eur J Vasc Endovasc Surg 2012;43:627-631.
7 15. van Laanen JH, Hendriks JM, Verhagen HJ, et al. Quantity, particle size, and histologic composition of embolic debris collected in a distal protection filter after carotid angioplasty and stenting: correlation with patient characteristics, timing of carotid artery stenting, and procedural details. J Thorac Cardiovasc Surg 2013;146:492495.