Catheterization and Cardiovascular Interventions 85:1141–1149 (2015)
Comparison of Scanning Electron Microscopy and Optical Coherence Tomography for Imaging of Coronary Bifurcation Stents Guilherme V. Silva,1 MD, Amir Gahremanpour,1 MD, Guilherme F. Attizzani,2 MD, Yi Zeng,1 MD, Wei Wang,2 PhD, Hirosada Yamamoto,2 MD, Tomoaki Kanaya,2 MD, Marian K. Rippy,3 DVM, PhD, Hiram G. Bezerra,2 MD, PhD, Marco A. Costa,2 MD, PhD, and Emerson Perin,1* MD, PhD Background: Optical coherence tomography (OCT) is a new intracoronary imaging modality that has excellent resolution and image quality and has been used to image neointimal coverage after stent implantation. OCT has been compared to histologic, intravascular ultrasound, and scanning electron microscopy (SEM) studies. However, OCT has not been compared with SEM for imaging stent coverage over side branches. Objective: The aim of this study was to compare OCT with SEM in imaging neointimal coverage over stent struts bridging coronary side-branch ostia. Methods: Using a balloon-overstretch in-stent restenosis model, we deployed 38 everolimus-eluting stents across coronary bifurcations in nine pigs. We performed OCT immediately after stenting and 4 weeks later; SEM was performed after euthanizing the pigs. OCT images of each stent were compared to the corresponding SEM image. Results: We analyzed OCT frames (n 5 111) for strut-level neointimal coverage and compared them to corresponding SEM images. The concordance correlation coefficient was 0.809 (95%CI; 0.734–0.864) and 0.951 (95%CI; 0.930–0.966) for covered and uncovered struts, respectively. Conclusions: In a non-atherosclerotic pig model, we showed strong agreement between OCT and SEM in imaging coverage of stent struts bridging side-branch ostia. VC 2015 Wiley Periodicals, Inc. Key words: neointima; coronary restenosis; stent
INTRODUCTION
Drug-eluting stents (DES) have been widely used because they markedly reduce in-stent restenosis (ISR) [1]. However, late stent thrombosis remains a major clinical concern because studies have raised the issue of the lack of endothelialization over DES, even 5 years after implantation [2]. We have previously shown that DES are associated with increased inflammation 1
Texas Heart Institute, Houston, Texas Harrington Heart and Vascular Institute, Case Medical Center, Cleveland, Ohio 3 Rippy Pathology Solutions, St. Paul, Minnesota. 2
Conflict of interest: Drs. Silva and Perin serve as consultants for St. Jude, Inc.; Dr. Bezerra receives consulting fees and honoraria from St. Jude, Inc.; Dr. Attizzani receives consulting fees from St. Jude, Inc.; Dr. Costa receives consulting fees from St. Jude, Inc. and Boston Scientific.
C 2015 Wiley Periodicals, Inc. V
and delayed neointimal coverage [3]. In addition, longer stent length has been associated with higher rates of in-stent thrombosis, which could be associated with lack of coverage over side branches [4, 5]. Optical coherence tomography (OCT) has been validated as a new intracoronary imaging modality that has better axial and lateral resolution than intravascular ultrasound [6]. Because of its excellent resolution and image quality, OCT is well suited for examining Grant sponsor: United States Department of Education through the Texas Higher Education Coordinating Board using, at least in part, American Recovery and Reinvestment Act funds... *Correspondence to: Emerson C. Perin, MD, PhD, 6624 Fannin, Suite 2220, Houston, TX 77030. E-mail: [email protected] Received 10 October 2013; Revision accepted 14 July 2014 DOI: 10.1002/ccd.25612 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com)
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neointimal coverage after stent implantation. In fact, studies in pigs have shown that OCT imaging results of stent coverage were comparable to those obtained by histologic and electron microscopy studies [7, 8]. However, there are no data comparing OCT results with electron microscopy in assessing stent coverage over side branches. Thus, in this study, we examined the use of a commercially available frequency-domain OCT system [6] in the imaging of coronary side branch ostia after stent placement in a pig model of balloon overstretch ISR, and we compared OCT results with those obtained from scanning electron microscopy (SEM) images of stent struts. MATERIALS AND METHODS
Domestic Yorkshire swine were obtained from The University of Texas MD Anderson Cancer Center’s Department of Veterinary Sciences (Bastrop, Texas) 5– 7 days before initiation of the study. All pigs received humane care in compliance with the Principals of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). The Institutional Animal Care and Use Committee at the Texas Heart Institute at St. Luke’s Episcopal Hospital approved this study. Study Design
Balloon-overstretch injury was induced in the coronary artery, and stents were implanted as described below. OCT imaging was performed immediately after stent placement (baseline) and 4 weeks after stent placement (follow-up). The pigs were humanely euthanized after the 4-week OCT imaging, and low-vacuum SEM imaging of the coronary arteries was used to assess neointimal coverage of the stent struts. Animal Model The femoral artery was accessed percutaneously, and a 6F sheath was inserted. A 6F AR1 guiding catheter was selectively engaged in the left anterior descending (LAD) and left circumflex (LCx) arteries. A 0.014-in coronary guide wire was positioned in the distal target artery. Before stent implantation, OCT imaging was used to determine the appropriate balloon size for the balloonoverstretch injury. A rapid-exchange balloon was inflated either at the proximal LAD artery (including the bifurcation of the first and second diagonal branches) or at the proximal LCx (including first and second obtuse marginal [OM] branches). In the LAD/diagonal and LCx/OM bifurcations, either a single or multiple everolimus stents (15 2.75, 3.5 10, or 3.5 18 mm2; Promus Boston Scien-
tific, Natick, MA) were deployed across the referred side branches (diagonal or OM), and side branches were then labeled as single layer/non-overlapping or more than one layer/overlapping, respectively. Final angiograms were obtained before and after guide-wire removal for followup analyses. OCT Imaging
OCT images were acquired after intracoronary administration of 50–200 mg of nitroglycerin through conventional guiding-catheters with a commercially available system (C7-XR, OCT Imaging System, St. Jude Medical, St. Paul, MN), which delivers axial resolution to 15 mm and lateral resolution to 25 mm. Because of its faster pullback speed compared with the previous time-domain OCT system, frequency-domain OCT does not require proximal balloon occlusion during image acquisition [7]. After coronary angiography, the 2.7 French OCT catheter (Dragonfly, St. Jude Medical) was advanced over a conventional angioplasty guidewire (0.014 in.) into the desired vessel and across the target location under fluoroscopic guidance. The images were calibrated by automated adjustment of the Z-offset, and the entire length of the region of interest was scanned using an integrated automated pullback device at 10 mm/sec. During image acquisition, coronary blood flow was replaced by continuous flushing of contrast media with a power injector (4 ml/sec, pressure limit: 300 psi) to create a virtually blood-free environment. All images were acquired at 100 frames/ sec and displayed with a color look-up table; the images were digitally archived. If the quality of the initial pullback was inadequate, pullbacks were performed until an optimal sequence was achieved. OCT Image Analysis All images were digitally stored and were evaluated offline by a core laboratory (Imaging Core Laboratory, Harrington Heart and Vascular Institute, University Hospitals Case Medical Center, Cleveland, OH). The images were then analyzed with the use of proprietary software (Offline Review Software, version C.0.2; St Jude Medical). After being examined by two experienced OCT analysts, the images were reviewed by a third reader. Any discrepancy between the analysts was resolved on the basis of consensus. All cross-sectional images (i.e., frames) were initially screened for quality; images were excluded from analysis if any portion of the image was out of the screen or if the image had poor quality because of residual blood, a sew-up artifact, or reverberation [6]. A strut was considered suitable for analysis only if it had (1) a well-defined, bright “blooming” appearance and (2) a characteristic
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd. Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
OCT Imaging of Side Branch Strut Coverage
shadow. Qualitative image evaluations and quantitative assessments of the bifurcation segments were performed in every frame (0.1-mm intervals) [9]. In stented segments with multiple overlapping sites, overlapping segments were labeled in numerical order distally to proximally. The sharp contrast between the lumen and the vessel wall in OCT images allowed fully automated delineation of the lumen contour. The inner and outer contours of each strut reflection (blooming) were delineated semi-automatically. The center of the luminal surface of the strut blooming was determined for each strut, and its distance to the lumen contour was calculated automatically to determine whether malapposition was present in baseline and follow-up cases and to determine strut-level intimal thickness (SIT) in follow-up cases. Struts covered by tissue had positive SIT values, whereas uncovered struts or malapposed struts had negative SIT values. Data were stored in an integrated database system, which corrects for the strut thickness of different stent types once the study is completed and the data are locked; this approach allows for blinding of the readers. Strut malapposition was determined when the negative value of SIT was higher than the strut thickness plus the polymer thickness, according to the specifications of the stent manufacturer with the addition of a compensation factor of 20 mm to correct for strut blooming [10]. The blooming compensation factor was determined on the basis of the analysis of 2,250 struts. Highly reproducible OCT measurements for strut apposition and coverage using the methodology described here have been previously reported [10–13] Qualitative imaging assessment was performed in every frame to determine whether abnormal intra-stent tissue was present. We defined abnormal intra-stent tissue as any mass that protruded beyond the stent struts into the lumen and that had an irregular surface and a sharp intensity gap between mass and neointimal tissue [14]. The methodology used for bifurcation quantitative assessment was based on our previous report [15]. Briefly, bifurcations that had a side branch larger than 1.5 mm in diameter upon baseline angiography were selected for analysis. For this study, bifurcations jailed by overlapping stents were also included. Bifurcation segments were identified and labeled according to the opening (start) and closing of the side branch (end), as identified by cross-section assessment. The diameter of the side branch ostium was measured in every frame (0.1-mm interval). Ostium area was obtained by multiplying mean ostium diameter with the length of the bifurcation, while ostium length was obtained by multiplying the frame interval (i.e., 0.1 mm) by the number of frames in which the ostium was present. To evaluate axial tissue distribution, cross-sectional OCT
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images were divided into three regions: the stent-struts bridging the side branch ostium (OS), the half circumference of the vessel opposite the ostium (OO), and the vessel wall adjacent to the ostium (AO) (Fig. 1). Struts located in AO and OO regions were evaluated as previously described [15], but the coverage in OS struts (i.e., floating) was qualitatively evaluated and defined as follows: uncovered, covered (only in the main vessel luminal surface), or proliferative (which required the strut to be fully encapsulated by tissue or tissue connecting adjacent struts). Tissue Preparation and SEM Imaging
The stented coronary arteries from all pigs were imaged. At the time of explant, the hearts were perfusion rinsed with saline and perfusion fixed with 2% Karnovsky’s fixative with subsequent immersion fixation in 2% Karnovsky’s until the time of processing. The vessels containing stents were then removed and marked with a stainless steel staple (proximal end), sutured (to mark anatomical positioning), photographed, and radiographed. Vessels were then bisected (parallel with the long axis) and processed for SEM, which included dehydration in a graded series of ethanol solutions. Specimens were examined by SEM (JSM-6460LV). Images were taken of the entire length of the vessel, and additional images were obtained of the stent struts bridging the side branch ostia. Stent struts were categorized as “covered” if the entire luminal surface of the struts was covered by neointima. SEM images of the stent struts bridging side branches were taken at various magnifications and always included a 50 image, which was used as the standard magnification upon which a calibration was made in the ImagePro software using both the calibrated micrometer included by the SEM software in the captured image; this allowed us to calibrate the two programs. Comparison of OCT and SEM Images of Side Branch Ostia
OCT frames that corresponded to SEM images were selected for comparative analysis. Each SEM image of a bifurcation was carefully matched to the OCT image by identifying the involved side branches and the number of stent layers bridging each side branch ostium. The locations of the OCT frames (distal to proximal) were selected using the pullback speed and frame number to correspond to the in vivo image within the stent. Then, each OCT image of OS struts and the corresponding SEM image (stent struts bridging side branch ostium) were classified as covered or uncovered for both methods.
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd. Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
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Fig. 1. Longitudinal and cross-sectional (FD-OCT) images of a bifurcation region at baseline and 4-week follow-up. (A) Longitudinal image at baseline of a stented segment. The bifurcation region is highlighted in yellow. (B) A crosssectional image of the bifurcation shown in (A). The red dashed line with the double arrows represents the diameter of the side branch ostium. The two blue dashed lines indicate the angle of the side branch and help to define the AO and OS regions. The yellow dashed line divides the side branch into two equal parts and passes through the center of the main vessel. The perpendicular line (white dashed line) that also passes though the center of the vessel divides the OO region from the others. (C) Longitudinal image of the same
stented region shown in (A) at the 4-week follow-up. The yellow line shows the reduction in the bifurcation length as a result of the vascular response to the stent implantation. (D) Cross-sectional image of (C) showing the significant reduction in the diameter of the side branch ostium, as well as in its angle. The white dashed boxes show covered, well-apposed AO struts that in the baseline image were either malapposed AO struts or OS struts. AO 5 vessel wall adjacent to the ostium; OS 5 stent-struts bridging the ostium; OO 5 the half circumference of the vessel opposite the ostium. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Statistical Analyses All statistical analyses were performed using SAS software (version 9.2; SAS Institute, Cary, NC); statistical significance was assessed at the 0.05 level. Continuous variables are expressed as mean 6 SD, and categorical variables are expressed as number (proportion). Agreement of covered and uncovered struts numbers for OCT and SEM was evaluated with concordance correlation coefficient (CCC) statistics. For bifurcation level analysis (AO vs. OO, baseline vs. follow-up), continuous variables were compared using a generalized estimating equations model with
exchangeable correlation structure to account for the clustering of values within each subject. RESULTS
We placed a total of 38 stents across LAD/diagonal and LCx/OM bifurcations; 11 side branches were bridged by a single stent and 17 by overlapping (OLP) stents. The entire stent length was imaged by SEM; however, OCT strut level analysis (a total of 4,056 struts) was confined to bifurcation segments only. OCT showed that the mean number of struts covering ostia
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd. Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
OCT Imaging of Side Branch Strut Coverage
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Fig. 2. Demonstration of concordance between OCT and SEM for imaging stent strut coverage over side branch ostium. The left side of the image shows complete coverage. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was 11.64 6 18.29 (range, 0–88). Overall, 80.44% 6 20.44% of struts were uncovered. The coverage percentage was 26.02% 6 24.84% for OLP stents (n ¼ 15) and 7.46% 6 9.52% for non-OLP stents (n ¼ 8) (P ¼ 0.053 for the comparison). OCT Versus SEM on Tissue Coverage of Struts Bridging Side Branch Ostia
The OCT and SEM images of OS struts were classified as either covered or uncovered (no proliferative pattern was identified), and corresponding images were compared (Fig. 2). There was strong, significant agreement between OCT and SEM in the evaluation of OS strut coverage (Table I). SEM of Strut Coverage in Bifurcation Versus Non-bifurcation Stent Segments
In this experimental pig balloon overstretch injury model, SEM showed complete coverage of the nonostial stent struts over the 4-week period, but coverage
was mostly incomplete for struts over the ostium of side branches. Strut coverage as imaged by SEM varied from 0% to 100% over the side branches (Fig. 3); the mean coverage was significantly less than the strut coverage of non-ostial areas (63.6% 6 6.3% vs. 100% 6 0.0%; P < 0.0001). Serial OCT Imaging: Dynamic Anatomic Changes of Coronary Bifurcations Serial OCT imaging of the side branch ostia. We measured the length of the bifurcations and the areas of 28 side branches. When baseline and follow-up OCT images were compared, we observed that tissue ingrowth from the adjacent ostial border resulted in significant shrinkage of the side branch ostium (Table II).
Serial OCT imaging of stent strut malapposition adjacent to the side branch ostia (AO) or opposite to the side branch ostia (OO). When NIT over OO and AO struts was assessed and compared, the
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd. Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
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TABLE I. Agreement of SEM and OCT in Imaging Ostium Strut Coverage Concordance correlation coefficient
Covered struts Uncovered struts
SEM
OCT
CCC
95% CI Lower Limit
95% CI Upper Limit
95 182
88 197
0.809 0.951
0.734 0.930
0.864 0.966
Abbreviations: SEM: scanning electron microscopy, OCT: optical coherence tomography, CCC: concordance correlation coefficient.
Fig. 3. (A) Scanning electron microscopy showing strut coverage in the left circumflex artery. A longitudinal cross-sectional image of a left circumflex artery segment with a stent containing one side branch ostium showing one uncovered strut and several covered struts. (B) Scanning electron microscopy showing strut coverage in the left anterior descending artery. A longitudinal cross-sectional image of a left anterior descending artery segment with a stent showing complete strut coverage.
percentage of malapposed stent struts was comparable in AO and OO areas (26.33% 6 20.81% vs. 20.79% 6 21.87%; P ¼ 0.150) at baseline. We found that in both areas the percentage of malapposed stent struts significantly decreased over time; however, there were significantly fewer malapposed stents in OO areas compared to AO areas. Strut-level NIT was significantly more pronounced in OO regions compared to AO regions (0.25 6 0.14 mm vs. 0.20 6 0.11 mm, respectively, P ¼ 0.003) (Tables III and IV).
DISCUSSION
Stent strut coverage in general and at bifurcations has been studied using OCT and light and electron microscopy [16–19]. To our knowledge, this is the first study comparing SEM with frequency-domain OCT in imaging bifurcation stents. The serial baseline to follow-up OCT imaging provided the opportunity to examine the temporal evolution of the in vivo response of the coronary side branches bridged by DES stent struts. Our findings indicate that (1) OCT and SEM show excellent agreement in imaging the neointimal
TABLE II.
FD-OCT Measures Bifurcation Level
Bifurcation length (mm) Ostium length (mm) Ostium area (mm2)
Baseline
Follow-up
Difference
P-value
1.15 6 0.52
0.95 6 0.60
0.20 6 0.71
0.146
1.55 6 0.42
1.06 6 0.38
0.48 6 0.40
Comparison of Scanning Electron Microscopy and Optical Coherence Tomography for Imaging of Coronary Bifurcation Stents Guilherme V. Silva,1 MD, Amir Gahremanpour,1 MD, Guilherme F. Attizzani,2 MD, Yi Zeng,1 MD, Wei Wang,2 PhD, Hirosada Yamamoto,2 MD, Tomoaki Kanaya,2 MD, Marian K. Rippy,3 DVM, PhD, Hiram G. Bezerra,2 MD, PhD, Marco A. Costa,2 MD, PhD, and Emerson Perin,1* MD, PhD Background: Optical coherence tomography (OCT) is a new intracoronary imaging modality that has excellent resolution and image quality and has been used to image neointimal coverage after stent implantation. OCT has been compared to histologic, intravascular ultrasound, and scanning electron microscopy (SEM) studies. However, OCT has not been compared with SEM for imaging stent coverage over side branches. Objective: The aim of this study was to compare OCT with SEM in imaging neointimal coverage over stent struts bridging coronary side-branch ostia. Methods: Using a balloon-overstretch in-stent restenosis model, we deployed 38 everolimus-eluting stents across coronary bifurcations in nine pigs. We performed OCT immediately after stenting and 4 weeks later; SEM was performed after euthanizing the pigs. OCT images of each stent were compared to the corresponding SEM image. Results: We analyzed OCT frames (n 5 111) for strut-level neointimal coverage and compared them to corresponding SEM images. The concordance correlation coefficient was 0.809 (95%CI; 0.734–0.864) and 0.951 (95%CI; 0.930–0.966) for covered and uncovered struts, respectively. Conclusions: In a non-atherosclerotic pig model, we showed strong agreement between OCT and SEM in imaging coverage of stent struts bridging side-branch ostia. VC 2015 Wiley Periodicals, Inc. Key words: neointima; coronary restenosis; stent
INTRODUCTION
Drug-eluting stents (DES) have been widely used because they markedly reduce in-stent restenosis (ISR) [1]. However, late stent thrombosis remains a major clinical concern because studies have raised the issue of the lack of endothelialization over DES, even 5 years after implantation [2]. We have previously shown that DES are associated with increased inflammation 1
Texas Heart Institute, Houston, Texas Harrington Heart and Vascular Institute, Case Medical Center, Cleveland, Ohio 3 Rippy Pathology Solutions, St. Paul, Minnesota. 2
Conflict of interest: Drs. Silva and Perin serve as consultants for St. Jude, Inc.; Dr. Bezerra receives consulting fees and honoraria from St. Jude, Inc.; Dr. Attizzani receives consulting fees from St. Jude, Inc.; Dr. Costa receives consulting fees from St. Jude, Inc. and Boston Scientific.
C 2015 Wiley Periodicals, Inc. V
and delayed neointimal coverage [3]. In addition, longer stent length has been associated with higher rates of in-stent thrombosis, which could be associated with lack of coverage over side branches [4, 5]. Optical coherence tomography (OCT) has been validated as a new intracoronary imaging modality that has better axial and lateral resolution than intravascular ultrasound [6]. Because of its excellent resolution and image quality, OCT is well suited for examining Grant sponsor: United States Department of Education through the Texas Higher Education Coordinating Board using, at least in part, American Recovery and Reinvestment Act funds... *Correspondence to: Emerson C. Perin, MD, PhD, 6624 Fannin, Suite 2220, Houston, TX 77030. E-mail: [email protected] Received 10 October 2013; Revision accepted 14 July 2014 DOI: 10.1002/ccd.25612 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com)
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neointimal coverage after stent implantation. In fact, studies in pigs have shown that OCT imaging results of stent coverage were comparable to those obtained by histologic and electron microscopy studies [7, 8]. However, there are no data comparing OCT results with electron microscopy in assessing stent coverage over side branches. Thus, in this study, we examined the use of a commercially available frequency-domain OCT system [6] in the imaging of coronary side branch ostia after stent placement in a pig model of balloon overstretch ISR, and we compared OCT results with those obtained from scanning electron microscopy (SEM) images of stent struts. MATERIALS AND METHODS
Domestic Yorkshire swine were obtained from The University of Texas MD Anderson Cancer Center’s Department of Veterinary Sciences (Bastrop, Texas) 5– 7 days before initiation of the study. All pigs received humane care in compliance with the Principals of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). The Institutional Animal Care and Use Committee at the Texas Heart Institute at St. Luke’s Episcopal Hospital approved this study. Study Design
Balloon-overstretch injury was induced in the coronary artery, and stents were implanted as described below. OCT imaging was performed immediately after stent placement (baseline) and 4 weeks after stent placement (follow-up). The pigs were humanely euthanized after the 4-week OCT imaging, and low-vacuum SEM imaging of the coronary arteries was used to assess neointimal coverage of the stent struts. Animal Model The femoral artery was accessed percutaneously, and a 6F sheath was inserted. A 6F AR1 guiding catheter was selectively engaged in the left anterior descending (LAD) and left circumflex (LCx) arteries. A 0.014-in coronary guide wire was positioned in the distal target artery. Before stent implantation, OCT imaging was used to determine the appropriate balloon size for the balloonoverstretch injury. A rapid-exchange balloon was inflated either at the proximal LAD artery (including the bifurcation of the first and second diagonal branches) or at the proximal LCx (including first and second obtuse marginal [OM] branches). In the LAD/diagonal and LCx/OM bifurcations, either a single or multiple everolimus stents (15 2.75, 3.5 10, or 3.5 18 mm2; Promus Boston Scien-
tific, Natick, MA) were deployed across the referred side branches (diagonal or OM), and side branches were then labeled as single layer/non-overlapping or more than one layer/overlapping, respectively. Final angiograms were obtained before and after guide-wire removal for followup analyses. OCT Imaging
OCT images were acquired after intracoronary administration of 50–200 mg of nitroglycerin through conventional guiding-catheters with a commercially available system (C7-XR, OCT Imaging System, St. Jude Medical, St. Paul, MN), which delivers axial resolution to 15 mm and lateral resolution to 25 mm. Because of its faster pullback speed compared with the previous time-domain OCT system, frequency-domain OCT does not require proximal balloon occlusion during image acquisition [7]. After coronary angiography, the 2.7 French OCT catheter (Dragonfly, St. Jude Medical) was advanced over a conventional angioplasty guidewire (0.014 in.) into the desired vessel and across the target location under fluoroscopic guidance. The images were calibrated by automated adjustment of the Z-offset, and the entire length of the region of interest was scanned using an integrated automated pullback device at 10 mm/sec. During image acquisition, coronary blood flow was replaced by continuous flushing of contrast media with a power injector (4 ml/sec, pressure limit: 300 psi) to create a virtually blood-free environment. All images were acquired at 100 frames/ sec and displayed with a color look-up table; the images were digitally archived. If the quality of the initial pullback was inadequate, pullbacks were performed until an optimal sequence was achieved. OCT Image Analysis All images were digitally stored and were evaluated offline by a core laboratory (Imaging Core Laboratory, Harrington Heart and Vascular Institute, University Hospitals Case Medical Center, Cleveland, OH). The images were then analyzed with the use of proprietary software (Offline Review Software, version C.0.2; St Jude Medical). After being examined by two experienced OCT analysts, the images were reviewed by a third reader. Any discrepancy between the analysts was resolved on the basis of consensus. All cross-sectional images (i.e., frames) were initially screened for quality; images were excluded from analysis if any portion of the image was out of the screen or if the image had poor quality because of residual blood, a sew-up artifact, or reverberation [6]. A strut was considered suitable for analysis only if it had (1) a well-defined, bright “blooming” appearance and (2) a characteristic
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd. Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
OCT Imaging of Side Branch Strut Coverage
shadow. Qualitative image evaluations and quantitative assessments of the bifurcation segments were performed in every frame (0.1-mm intervals) [9]. In stented segments with multiple overlapping sites, overlapping segments were labeled in numerical order distally to proximally. The sharp contrast between the lumen and the vessel wall in OCT images allowed fully automated delineation of the lumen contour. The inner and outer contours of each strut reflection (blooming) were delineated semi-automatically. The center of the luminal surface of the strut blooming was determined for each strut, and its distance to the lumen contour was calculated automatically to determine whether malapposition was present in baseline and follow-up cases and to determine strut-level intimal thickness (SIT) in follow-up cases. Struts covered by tissue had positive SIT values, whereas uncovered struts or malapposed struts had negative SIT values. Data were stored in an integrated database system, which corrects for the strut thickness of different stent types once the study is completed and the data are locked; this approach allows for blinding of the readers. Strut malapposition was determined when the negative value of SIT was higher than the strut thickness plus the polymer thickness, according to the specifications of the stent manufacturer with the addition of a compensation factor of 20 mm to correct for strut blooming [10]. The blooming compensation factor was determined on the basis of the analysis of 2,250 struts. Highly reproducible OCT measurements for strut apposition and coverage using the methodology described here have been previously reported [10–13] Qualitative imaging assessment was performed in every frame to determine whether abnormal intra-stent tissue was present. We defined abnormal intra-stent tissue as any mass that protruded beyond the stent struts into the lumen and that had an irregular surface and a sharp intensity gap between mass and neointimal tissue [14]. The methodology used for bifurcation quantitative assessment was based on our previous report [15]. Briefly, bifurcations that had a side branch larger than 1.5 mm in diameter upon baseline angiography were selected for analysis. For this study, bifurcations jailed by overlapping stents were also included. Bifurcation segments were identified and labeled according to the opening (start) and closing of the side branch (end), as identified by cross-section assessment. The diameter of the side branch ostium was measured in every frame (0.1-mm interval). Ostium area was obtained by multiplying mean ostium diameter with the length of the bifurcation, while ostium length was obtained by multiplying the frame interval (i.e., 0.1 mm) by the number of frames in which the ostium was present. To evaluate axial tissue distribution, cross-sectional OCT
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images were divided into three regions: the stent-struts bridging the side branch ostium (OS), the half circumference of the vessel opposite the ostium (OO), and the vessel wall adjacent to the ostium (AO) (Fig. 1). Struts located in AO and OO regions were evaluated as previously described [15], but the coverage in OS struts (i.e., floating) was qualitatively evaluated and defined as follows: uncovered, covered (only in the main vessel luminal surface), or proliferative (which required the strut to be fully encapsulated by tissue or tissue connecting adjacent struts). Tissue Preparation and SEM Imaging
The stented coronary arteries from all pigs were imaged. At the time of explant, the hearts were perfusion rinsed with saline and perfusion fixed with 2% Karnovsky’s fixative with subsequent immersion fixation in 2% Karnovsky’s until the time of processing. The vessels containing stents were then removed and marked with a stainless steel staple (proximal end), sutured (to mark anatomical positioning), photographed, and radiographed. Vessels were then bisected (parallel with the long axis) and processed for SEM, which included dehydration in a graded series of ethanol solutions. Specimens were examined by SEM (JSM-6460LV). Images were taken of the entire length of the vessel, and additional images were obtained of the stent struts bridging the side branch ostia. Stent struts were categorized as “covered” if the entire luminal surface of the struts was covered by neointima. SEM images of the stent struts bridging side branches were taken at various magnifications and always included a 50 image, which was used as the standard magnification upon which a calibration was made in the ImagePro software using both the calibrated micrometer included by the SEM software in the captured image; this allowed us to calibrate the two programs. Comparison of OCT and SEM Images of Side Branch Ostia
OCT frames that corresponded to SEM images were selected for comparative analysis. Each SEM image of a bifurcation was carefully matched to the OCT image by identifying the involved side branches and the number of stent layers bridging each side branch ostium. The locations of the OCT frames (distal to proximal) were selected using the pullback speed and frame number to correspond to the in vivo image within the stent. Then, each OCT image of OS struts and the corresponding SEM image (stent struts bridging side branch ostium) were classified as covered or uncovered for both methods.
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd. Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
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Fig. 1. Longitudinal and cross-sectional (FD-OCT) images of a bifurcation region at baseline and 4-week follow-up. (A) Longitudinal image at baseline of a stented segment. The bifurcation region is highlighted in yellow. (B) A crosssectional image of the bifurcation shown in (A). The red dashed line with the double arrows represents the diameter of the side branch ostium. The two blue dashed lines indicate the angle of the side branch and help to define the AO and OS regions. The yellow dashed line divides the side branch into two equal parts and passes through the center of the main vessel. The perpendicular line (white dashed line) that also passes though the center of the vessel divides the OO region from the others. (C) Longitudinal image of the same
stented region shown in (A) at the 4-week follow-up. The yellow line shows the reduction in the bifurcation length as a result of the vascular response to the stent implantation. (D) Cross-sectional image of (C) showing the significant reduction in the diameter of the side branch ostium, as well as in its angle. The white dashed boxes show covered, well-apposed AO struts that in the baseline image were either malapposed AO struts or OS struts. AO 5 vessel wall adjacent to the ostium; OS 5 stent-struts bridging the ostium; OO 5 the half circumference of the vessel opposite the ostium. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Statistical Analyses All statistical analyses were performed using SAS software (version 9.2; SAS Institute, Cary, NC); statistical significance was assessed at the 0.05 level. Continuous variables are expressed as mean 6 SD, and categorical variables are expressed as number (proportion). Agreement of covered and uncovered struts numbers for OCT and SEM was evaluated with concordance correlation coefficient (CCC) statistics. For bifurcation level analysis (AO vs. OO, baseline vs. follow-up), continuous variables were compared using a generalized estimating equations model with
exchangeable correlation structure to account for the clustering of values within each subject. RESULTS
We placed a total of 38 stents across LAD/diagonal and LCx/OM bifurcations; 11 side branches were bridged by a single stent and 17 by overlapping (OLP) stents. The entire stent length was imaged by SEM; however, OCT strut level analysis (a total of 4,056 struts) was confined to bifurcation segments only. OCT showed that the mean number of struts covering ostia
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd. Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
OCT Imaging of Side Branch Strut Coverage
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Fig. 2. Demonstration of concordance between OCT and SEM for imaging stent strut coverage over side branch ostium. The left side of the image shows complete coverage. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was 11.64 6 18.29 (range, 0–88). Overall, 80.44% 6 20.44% of struts were uncovered. The coverage percentage was 26.02% 6 24.84% for OLP stents (n ¼ 15) and 7.46% 6 9.52% for non-OLP stents (n ¼ 8) (P ¼ 0.053 for the comparison). OCT Versus SEM on Tissue Coverage of Struts Bridging Side Branch Ostia
The OCT and SEM images of OS struts were classified as either covered or uncovered (no proliferative pattern was identified), and corresponding images were compared (Fig. 2). There was strong, significant agreement between OCT and SEM in the evaluation of OS strut coverage (Table I). SEM of Strut Coverage in Bifurcation Versus Non-bifurcation Stent Segments
In this experimental pig balloon overstretch injury model, SEM showed complete coverage of the nonostial stent struts over the 4-week period, but coverage
was mostly incomplete for struts over the ostium of side branches. Strut coverage as imaged by SEM varied from 0% to 100% over the side branches (Fig. 3); the mean coverage was significantly less than the strut coverage of non-ostial areas (63.6% 6 6.3% vs. 100% 6 0.0%; P < 0.0001). Serial OCT Imaging: Dynamic Anatomic Changes of Coronary Bifurcations Serial OCT imaging of the side branch ostia. We measured the length of the bifurcations and the areas of 28 side branches. When baseline and follow-up OCT images were compared, we observed that tissue ingrowth from the adjacent ostial border resulted in significant shrinkage of the side branch ostium (Table II).
Serial OCT imaging of stent strut malapposition adjacent to the side branch ostia (AO) or opposite to the side branch ostia (OO). When NIT over OO and AO struts was assessed and compared, the
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd. Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
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TABLE I. Agreement of SEM and OCT in Imaging Ostium Strut Coverage Concordance correlation coefficient
Covered struts Uncovered struts
SEM
OCT
CCC
95% CI Lower Limit
95% CI Upper Limit
95 182
88 197
0.809 0.951
0.734 0.930
0.864 0.966
Abbreviations: SEM: scanning electron microscopy, OCT: optical coherence tomography, CCC: concordance correlation coefficient.
Fig. 3. (A) Scanning electron microscopy showing strut coverage in the left circumflex artery. A longitudinal cross-sectional image of a left circumflex artery segment with a stent containing one side branch ostium showing one uncovered strut and several covered struts. (B) Scanning electron microscopy showing strut coverage in the left anterior descending artery. A longitudinal cross-sectional image of a left anterior descending artery segment with a stent showing complete strut coverage.
percentage of malapposed stent struts was comparable in AO and OO areas (26.33% 6 20.81% vs. 20.79% 6 21.87%; P ¼ 0.150) at baseline. We found that in both areas the percentage of malapposed stent struts significantly decreased over time; however, there were significantly fewer malapposed stents in OO areas compared to AO areas. Strut-level NIT was significantly more pronounced in OO regions compared to AO regions (0.25 6 0.14 mm vs. 0.20 6 0.11 mm, respectively, P ¼ 0.003) (Tables III and IV).
DISCUSSION
Stent strut coverage in general and at bifurcations has been studied using OCT and light and electron microscopy [16–19]. To our knowledge, this is the first study comparing SEM with frequency-domain OCT in imaging bifurcation stents. The serial baseline to follow-up OCT imaging provided the opportunity to examine the temporal evolution of the in vivo response of the coronary side branches bridged by DES stent struts. Our findings indicate that (1) OCT and SEM show excellent agreement in imaging the neointimal
TABLE II.
FD-OCT Measures Bifurcation Level
Bifurcation length (mm) Ostium length (mm) Ostium area (mm2)
Baseline
Follow-up
Difference
P-value
1.15 6 0.52
0.95 6 0.60
0.20 6 0.71
0.146
1.55 6 0.42
1.06 6 0.38
0.48 6 0.40
