Removal of surgically induced fibrous arterial plaques by argon ion laser angiosurgery using a multifiber delivery system. An experimental study in the dog.

Removal of intravascular atherosclerotic obstructions by laser irradiation has gained the attention of many investigators, but has proven to be considerably more difficult to accomplish than initially envisioned. We tested, in an animal model, an argon ion laser delivery system that permits control of (1) laser power, (2) exposure time, and (3) laser beam spot size. The study was conducted on surgically, induced focal fibrous plaques in the carotid arteries of nine dogs. Plaque removal, vessel patency, and healing were evaluated angiographically and by light and electron microscopy at intervals up to 60 days after treatment. Results showed that intravascular obstructions could be removed, healing occurred, and vessels remained patent for up to 60 days.

Quantitative analysis of inflammatory cells in aortic atherosclerosis of young adults.

Cellular analysis of aortic atherosclerotic lesions has been pursued extensively in recent years, although most of these investigations have involved the detection of inflammatory cells in chronically diseased tissue or artificially induced atherosclerosis in an animal model. Few studies have attempted to quantify accurately, using computer analysis systems, the degree of cellular infiltration in a statistically significant number of samples, in tissue from young adults. In this study, segments of human aortae were collected at autopsy from 29 individuals ranging in age from 15 to 35 years. The tissue was embedded in paraffin and stained using routine histological and automated immunohistochemical staining techniques. The sections were evaluated using advanced image analysis techniques to investigate the differences in cellular composition and cell activation between the dorsal and ventral aspects of the human aorta and to correlate these findings to the age of the subjects. These regions have been previously shown to have a high (dorsal) and low (ventral) probability of developing sudanophilic lesions. Our data demonstrated that statistically different cell populations exist in the dorsal and ventral regions of each vessel. The dorsal aspect (i.e., high-probability region) had a greater number of HAM56(+) (36.9% increase,p = 0.0002) and HLA-DRα(+) cells (44.2% increase,p = 0.0035) than did the ventral surface (i.e., low-probability region), although there were no significant differences in the number of CD43(+) lymphocytes. When grouped according to age, results showed significant increases in the dorsal region when considering HAM56(+) and HLA-DRα(+) cells (p = 0033 and 0.046, respectively). Morphologically, a greater number of foam cell aggregates were found to occur in the dorsal region of the vessel than in the ventral portion. Our results indicate that the microarchitecture and cellular composition of the dorsal and ventral aorta are significantly different, with these variations becoming more marked with age.

Frequency content of normal and diabetic plantar pressure profiles: implications for the selection of transducer sizes.

How small do pressure transducers need to be in order to faithfully measure the plantar pressure profiles (PPPs) under normal and diabetic feet? In this study, pressures were collected from five diabetic and six non-diabetic subjects using a commercial measurement system with 25 mm2 transducers. Discrete Fourier Transform techniques were then used to determine (i) the spatial frequency content of diabetic and non-diabetic PPPs, and (ii) the effects of quadrupling the transducer area (from 5 mm x 5 mm to 10 mm x 10 mm). When the data were filtered to represent the effects of using 10 mm x 10 mm transducers, it was found that the ensuing reductions in peak pressure in the toe region (50 kPa) were significantly greater than in all other regions of the foot (p < 0.05). There was a significant correlation between pressure underestimations and measured peak pressures in the metatarsal regions. Based on data collected with 25 mm2 transducers it was concluded that transducer sizes greater than 6.36 mm x 6.18 mm (medio-lateral and antero-posterior directions) would result in sub-optimal sampling of PPPs.

Morphological model of human colon tissue fluorescence.

Fluorescence spectroscopy of tissue is a promising technique for early detection of precancerous changes in the human body. Investigation of the microscopic origin of the clinically observed tissue fluorescence can provide valuable information about the tissue's histology. The objective of this study was the development of a morphological model of colon tissue fluorescence which connects the clinically observed spectra with their underlying microscopic origins. Clinical colon tissue fluorescence which connects the clinically observed spectra with their underlying microscopic origins. Clinical colon tissue fluorescence spectra were modeled by measuring the intrinsic fluorescence properties of colon tissue on a microscopic level and by simulating light propagation in tissue using the Monte-Carlo method. The computed spectra were in good agreement with the clinical spectra acquired during colonoscopy, and exhibited the characteristic spectral features of the in vivo collected spectra. Our analysis quantitated these spectral features in terms of the intrinsic fluorescence properties of tissue and its general histological characteristics. The fluorescence intensity difference between normal and adenoma observed in vivo was found to be due to the increased hemoglobin absorption, the reduced mucosal fluorescence intensity, and the absence of submucosal fluorescence in adenomatous polyps. The increased red fluorescence in adenoma was found to be associated with the dysplastic crypt cell fluorescence.

Comparison of texture analysis methods for the characterization of coronary plaques in intravascular ultrasound images.

Intravascular Ultrasound (IVUS) is a diagnostic imaging technique that provides tomographic visualization of coronary arteries. The aim of this study was to evaluate five texture analysis techniques and determine their ability to distinguish between plaque lesions of different composition. Using histological correlation, regions of calcified, fibrous, and necrotic core plaque were chosen from 27 coronary plaques. First-order statistics, Haralick's method, Laws' texture energy method, the neighborhood gray-tone difference matrix method, and texture spectrum features were examined using discriminant analysis. Self-validation indicated that Haralick's method yielded the most accurate results, with resubstitution and cross-validation error rates of 0.00 and 14.76%, respectively. Further optimization gave error rates of 6.67%, using only two discriminating features, IDM and entropy.

Three-dimensional segmentation of luminal and adventitial borders in serial intravascular ultrasound images.

Intravascular ultrasound (IVUS) provides exact anatomy of arteries, allowing accurate quantitative analysis. Automated segmentation of IVUS images is a prerequisite for routine quantitative analyses. We present a new three-dimensional (3D) segmentation technique, called active surface segmentation, which detects luminal and adventitial borders in IVUS pullback examinations of coronary arteries. The technique was validated against expert tracings by computing correlation coefficients (range 0.83-0.97) and William's index values (range 0.37-0.66). The technique was statistically accurate, robust to image artifacts, and capable of segmenting a large number of images rapidly. Active surface segmentation enabled geometrically accurate 3D reconstruction and visualization of coronary arteries and volumetric measurements.

The use of lasers in vascular and cardiac surgery. Clinical review.

Use of lasers is routine in medical fields such as ophthalmology and dermatology, but in spite of intensive research in recent years, its role in cardiac and vascular surgery still remains to be determined. Laser energy can vaporize atherosclerotic plaques in human arteries obtained at necropsy. Fiberoptic catheters have been constructed to deliver the laser energy to atheromas at a distance from the arteriotomy. The healing of the arterial wall after laser treatment is rapid and results in complete reendothelialization. Lasers have been used in patients undergoing coronary surgery as well as peripheral vascular reconstructions. In some cases, the percutaneous approach has also been used. In these clinical series, the laser treatment was in almost all cases followed by either transluminal balloon angioplasty or by-pass operation. Complications after laser treatment include vessel perforation, reocclusions, thrombosis, or aneurysm formation. The development of more advanced fiberoptic catheters and better understanding of dosimetric parameters are promising features to avoid these complications as well as development of methods for steering of the laser catheter itself and means for "real time" diagnosis (e.g. angioscopes, spectroscopic diagnosis). The future use of lasers in cardiac and vascular surgery seems promising, especially as an alternative to balloon angioplasty.

Automated morphometry of coronary arteries with digital image analysis of intravascular ultrasound.

We designed and tested digital image processing strategies to perform fully automated segmentation of luminal and medial-adventitial boundaries in intravascular ultrasound images of human coronary arteries. Automated segmentation is an essential tool for advanced techniques of clinical visualization and quantitative measurement. Vascular compliance measurements and three-dimensional reconstructions are demonstrated as examples of such applications. Digital image processing was performed in three phases: (1) preprocessing, including a polar transform, local contrast enhancement, and speckle noise filtering; (2) segmentation, involving radial scanning, region growing, or cost-function minimization techniques; and (3) postprocessing, involving dropout filtering and outline smoothing. Cross-sectional areas were compared with manual tracings from experienced operators and showed good agreement. The algorithm bias ranged from -0.34 to 1.18 mm2; interclass and intraclass correlation coefficients ranged from 0.83 to 0.94. The designed techniques currently allow fully automated segmentation without operator interaction of the luminal and, if present, medial-adventitial boundary.

Preliminary evaluation of the prototype stereoscopic endoscope: precise three-dimensional measurement system.

A prototype stereoscopic endoscope (incorporating two charged-coupled devices), developed for the accurate three-dimensional measurement of gastrointestinal tract lesions, was initially evaluated with two-dimensional target grids and in vitro measurement of 15 objects of known size (marbles, cubes, and rectangular prisms) placed in a plastic model of the sigmoid colon. Images of the objects were captured and stored in a computer. Stereoscopic measurements were compared with results from the standard (open biopsy forceps) method by a blinded endoscopist. The volume measured with the stereoscope did not differ significantly from the true volume, whereas the volume obtained with the open biopsy forceps method differed significantly from the actual volume, consistently underestimating the actual size. The aberration ratios (percentage deviation between the measured and true volume, expressed as mean +/- SD) obtained with the stereoscopic endoscope were superior (9.2% +/- 9.5%) to those obtained with the open biopsy forceps method (-34.0% +/- 26.8%). These preliminary in vitro results with the stereoscope show considerable promise for the simple and precise three-dimensional measurement of gastrointestinal lesions and warrant human clinical trials.

Automated identification of stained cells in tissue sections using digital image analysis.

OBJECTIVE: To develop a novel automated image analysis system to differentiate immunohistochemically stained cells from background. STUDY DESIGN: Cell segmentation was performed by applying global thresholding algorithms to find an approximate threshold at which cells could be separated from background followed by a novel refinement algorithm to erode edge pixels of the region. To separate overlapping cells, a new decomposition method was developed that uses both semantic knowledge and high-level relational information. Both the cell segmentation and separation methods were evaluated on images of stained tissue sections and the manually outlined cell areas and numbers compared to the computed. RESULTS: Macrophage areas computed at the first stage by Otsu's algorithm did not differ significantly (P = .07) from those traced manually, while the areas computed by Kittler's and Kurita's algorithms did not agree (P < .01). Both Otsu's and Kurita's algorithms performed well when combined with edge pixel erosion. Kittler's algorithm proved unsuccessful even with edge erosion. Comparison of the computed and manually determined cell numbers showed a significant correlation, and regression analysis resulted in the unity curve. CONCLUSION: A combination of global thresholding and a novel edge erosion technique allowed identification of immunohistochemically stained macrophages; the computed cell areas agreed with the manual results.

Quantitative comparison of immunohistochemical staining intensity in tissues fixed in formalin and Histochoice.

Formaldehyde fixatives have traditionally been used to preserve tissues as they impart excellent morphological preservation. Formaldehyde fixes tissue by cross linking, a process which can reduce the antigenicity of tissue and weakens the intensity of immunohistochemical stains. Preliminary studies have shown that Histochoice tissue fixative offers equal or greater staining intensity than neutral buffered formalin (NBF). This study compares these fixatives quantitatively and presents the results in unambiguous statistical terms. Tissue samples were collected, bisected, and fixed in NBF or Histochoice. The sections were stained with a total of 21 antibodies, and color images were collected. The hue, saturation, and value were determined for each positive pixel and an ANOVA performed. Small differences in hue were noted in 8 of 21 cases. Histochoice-fixed tissue gave a greater mean saturation than NBF with 57.1% of the antibodies tested. No significant difference in the saturation was detected in 28.6% of the cases; NBF gave higher mean saturation levels with only 14.3% of the antibodies. Histochoice-fixed tissue was found to give lower values in 66.7% of cases than those prepared with NBF, indicating darker staining. These results show that Histochoice produces staining intensity that is comparable, and in many cases superior, to formalin.

Three-dimensional reconstruction of the coronary artery wall by image fusion of intravascular ultrasound and bi-plane angiography.

BACKGROUND: Intravascular ultrasound (IVUS) is becoming increasingly accepted for assessing coronary anatomy. However, its utility in visualizing and quantifying coronary morphology has been limited by its 2D tomographic nature. This study presents a 3D reconstruction technique that accurately preserves 3D geometric information. METHODS AND RESULTS: Images obtained from manual IVUS pullbacks and continuous bi-plane angiography were fused, using angiography to reconstruct the transducer trajectory and aid in solving for the correct rotational orientation. A novel 3D active surface method automatically identified the luminal and medial-adventitial borders which, when superimposed on the transducer trajectory, could be surface-rendered for visualization and morphometry. Segmentation agreed well with manual assessment, and 3D luminal shape matched that of angiography when projected to 2D. CONCLUSIONS: We conclude that this method provides an accurate reconstruction of the vessel's anatomy, which accounts for the true curvature of the vessel.

Laser-induced fluorescence microscopy of normal colon and dysplasia in colonic adenomas: implications for spectroscopic diagnosis.

OBJECTIVES: To determine what structures fluoresce and to what extent in normal colon and colonic adenomas to fully exploit laser-induced fluorescence spectroscopy as a tool for the diagnosis of dysplasia at endoscopy. METHODS: Unstained frozen sections of normal colon and colonic adenomas were studied by fluorescence microscopy under 351-364-nm argon ion laser excitation. Tissue fluorescence was observed and compared to morphology in serial sections stained with hematoxylin and eosin (H&E), Movat pentachrome, mucicarmine, and oil red O. RESULTS: In normal colon, fluorescence correlated morphologically with connective tissue fibers (principally collagen) in all layers of the bowel wall and with cytoplasmic granules within eosinophils present between the crypts in the lamina propria of the mucosa. Fluorescence of absorptive cells in normal crypts was very faint, and Goblet cells did not fluoresce. However, marked fluorescence was observed in the cytoplasms of dysplastic epithelial cells in the crypts of colonic adenomas. Fewer fluorescent connective tissue fibers were present in the lamina propria of colonic adenomas resulting in decreased fluorescence intensity as compared to that of normal colon. Fluorescent eosinophil granules were present in larger numbers in adenomas as compared with normal colon. CONCLUSION: Laser-induced fluorescence in normal colon and colonic adenomas correlates with morphology. Previous reported differences in laser-induced fluorescence emission spectra of normal colon and colonic adenomas obtained in vitro and in vivo may be due to differences in the cytoplasmic fluorescence between the dysplastic epithelium in colonic adenomas and normal colonic epithelium. Laser-induced fluorescence spectroscopy may be useful in studying other forms of epithelial dysplasia such as that which occurs in ulcerative colitis.

Effects of varying argon ion laser intensity and exposure time on the ablation of atherosclerotic plaque.

Using continuous wave (CW) argon ion laser light, a total of 253 laser exposures of varying power (1.5, 3, 5, 8 or 10 W) and duration (20-1,333 ms) were delivered to four segments of human atheromatous aorta obtained at autopsy. Exposure conditions were controlled by using an optically shielded laser catheter that provided a 500 micron spot of light of known power. Two thresholds for consistently reproducible ablation could be defined-an intensity threshold at 25.5 W/mm2 and a fluence threshold at 3.2 J/mm2. Above threshold, a fluence of 5.1 J/mm2 was found to produce the most efficient ablation, ie, removed the greatest volume (mm3) per energy delivered (J) compared to other fluence levels employed (p less than 0.0001). Between aortic segments, however, considerable variability in efficiency (mm3/J) was observed, possibly owing to different optical properties and/or plaque composition. Low-intensity laser radiation produced inconsistent ablation and extensive coagulation effects to surrounding tissue. When a fluence of 5.1 J/mm2 was constructed with a high-intensity laser beam and a short exposure time, consistent and efficient tissue removal resulted without histologic evidence of coagulation necrosis.

Characterization of ultraviolet laser-induced autofluorescence of ceroid deposits and other structures in atherosclerotic plaques as a potential diagnostic for laser angiosurgery.

Unstained frozen sections of normal and atherosclerotic human aorta and coronary artery were examined using histochemical and fluorescence microscopic techniques to identify the structures responsible for autofluorescence under 351 to 364 nm laser excitation. These structures included elastin and collagen in normal and atherosclerotic specimens, calcium deposits in calcified plaques, and granular or ring-shaped deposits histochemically identified as ceroid found in both calcified and non-calcified plaques. Qualitatively, both the color and intensity of ceroid autofluorescence differed greatly from that of elastin or collagen. The emission spectra of elastin, collagen, and ceroid were examined by microscopic spectrofluorimetry, and were found to differ significantly as well. When compared with spectra of elastin and collagen, spectra of ceroid were broader, shifted to the red, and were somewhat resistant to bleaching. We conclude that detection of laser-induced ceroid autofluorescence may aid in identifying plaques for laser ablation.

Quantification of intramural calcification in coronary intravascular ultrasound images with automated image analysis.

BACKGROUND: Recent studies have documented the utility of intravascular ultrasonography in quantifying coronary morphologic characteristics and determining an appropriate intervention. Unfortunately, its potential for quantifying lesion calcification is limited by subjective evaluation and manual tracing. The aim of this study was to develop an objective automated method for quantifying calcification in intracoronary images with digital image analysis. METHODS: Images of human coronary arteries acquired with a 30 MHz intracoronary ultrasound catheter were evaluated with digital image analysis and compared with manual tracings. Calcifications were automatically identified as highly echogenic regions detected by global thresholding within sectors of acoustic shadowing defined as regions devoid of texture. RESULTS: The mean percentage agreement, sensitivity, and specificity of detecting calcification in 1-degree sectors of calcified vessels were 82%, 73%, and 87%, respectively. Similar results were obtained in noncalcified images. CONCLUSION: The accuracy of this automated technique was comparable to interoperator and intraoperator variability in manually tracing calcification.

A model for thermal ablation of biological tissue using laser radiation.

We present a theory of thermal laser ablation based on the heat equation and on an energy balance equation derived from it. Ablation is assumed to be brought about by the heating and evaporation of tissue water. The model is three-dimensional, and scattering and the water-steam phase transition are explicitly taken into account. The model predicts threshold parameters and a steady-state ablation velocity in terms of the optical and thermal properties of the tissue and the laser beam intensity and spot diameter.

Gastrointestinal tissue diagnosis by laser-induced fluorescence spectroscopy at endoscopy.

An endoscope-compatible, optical fiber system has been developed which can be used to obtain laser-induced fluorescence spectra of mucosal abnormalities during endoscopy in real time. The results of our previous in vitro studies have suggested that laser-induced fluorescence tissue spectra are sufficiently unique that they can be used to accurately diagnose mucosal abnormalities in some systems. To test this hypothesis in vivo, laser-induced fluorescence spectra were obtained during colonoscopy from 31 colonic adenomas, 4 hyperplastic polyps, and 32 examples of normal mucosa in 20 patients. The resulting spectra could be used to correctly differentiate adenomas from normal colonic mucosa and hyperplastic polyps in 97% of the specimens studied with the resulting sensitivity, specificity, and positive predictive value of 100%, 97%, and 94%, respectively. These results, although preliminary in nature, suggest that laser-induced fluorescence spectra can be used in the recognition and differential diagnosis of mucosal abnormalities at endoscopy.

Detection of dysplasia at colonoscopy using laser-induced fluorescence: a blinded study.

BACKGROUND: Laser-induced fluorescence spectroscopy has the potential to detect colonic dysplasia in vivo. However, previous studies have limited their analyses to multivariate regression techniques and unblinded retrospective evaluation. The purpose of this study was to develop a probability-based algorithm to detect colonic dysplasia using laser-induced fluorescence spectroscopy and to evaluate it in a blinded manner. METHODS: Fluorescence spectra were collected from normal mucosa and colonic polyps during colonoscopy using 370 nm excitation. Tissue was classified as normal, hyperplastic, or adenomatous by histologic examination. Preliminary data was used to devise an algorithm to differentiate tissue type based on probability distributions of the fluorescence intensity at 460 nm and the ratio of the intensity at 680 nm to that at 600 nm. The algorithm was then tested in a blinded fashion. RESULTS: The algorithm correctly determined the tissue type in 88% of cases, equal to the agreement of independent pathologists. Sensitivity, specificity, and positive predictive value for the detection of dysplasia was 90%, 95%, and 90%, respectively. CONCLUSIONS: Dysplasia was detected in vivo using fluorescence spectroscopy and a probability-based algorithm. This method may form the basis for a new surveillance technique for patients with increased risk for dysplastic transformation.