Raman spectroscopy: a real-time tool for identifying microcalcifications during stereotactic breast core needle biopsies.

Microcalcifications are an early mammographic sign of breast cancer and a target for stereotactic breast needle biopsy. We present here a Raman spectroscopic tool for detecting microcalcifications in breast tissue based on their chemical composition. We collected ex vivo Raman spectra from 159 tissue sites in fresh stereotactic breast needle biopsies from 33 patients, including 54 normal sites, 75 lesions with microcalcifications and 30 lesions without microcalcifications. Application of our Raman technique resulted in a positive predictive value of 97% for detecting microcalcifications. This study shows that Raman spectroscopy has the potential to detect microcalcifications during stereotactic breast core biopsies and provide real-time feedback to radiologists, thus reducing non-diagnostic and false negative biopsies.

An evidence-based appraisal of the use of hyperbaric oxygen on flaps and grafts.

Hyperbaric oxygen has been advocated, both as an adjunctive or primary form of treatment, for a variety of disorders, including gas gangrene, osteoradionecrosis, and carbon monoxide poisoning. It has also been used to improve ischemic wounds before skin grafting and to support ischemic flaps. In this review, we analyze the available literature that investigates the use of hyperbaric oxygen for composite grafts, skin grafts, random flaps, distant flaps, and free flaps. An appraisal of the level of evidence for each of these uses of hyperbaric oxygen is offered. Although there are a significant amount of animal data supporting the application of hyperbaric oxygen for grafts and flaps, there is very little clinical information other than case reports and series to sustain its choice over other modalities of therapy. Multicenter prospective clinical studies are clearly needed comparing hyperbaric oxygen to other mechanical or pharmacologic interventions to improve wound healing for grafting or to support flap survival.

The use of biomedical sensors to monitor capsule formation around soft tissue implants.

Piezoelectric sensors have been shown to respond reproducibly to changes in tissue mechanical properties surrounding an implant over a 4-month period. The vibrational amplitude at a frequency corresponding to the radial resonance shows a statistically significant change over time. The initial period of inflammation is marked by a significant reduction in amplitude, which is indicative of an increase in viscous dissipation of the tissue. As collagen displaces the cellular response, the amplitude continues to decrease. Finally, as the tissue matures, the capsule becomes stiffer, and the viscous dissipation lessens. These results are consistent with qualitative assessments of explanted capsules. Strain gauges encased in a monolithic block of silicone exhibited a greater degree of variability, yet show similar trends over time. The strain increases in the initial 4-week period and remains relatively steady over the following 4 weeks. Beyond 8 weeks, the gauges begin to extrude from the animal or suffer a loss of electrical continuity. Steps are being taken to improve the strain sensor longevity in the animals.

Raman microspectroscopy of human coronary atherosclerosis: biochemical assessment of cellular and extracellular morphologic structures in situ.

BACKGROUND: We have previously shown that Raman spectroscopy can be used for chemical analysis of intact human coronary artery atherosclerotic lesions ex vivo without tissue homogenization or extraction. Here, we report the chemical analysis of individual cellular and extracellular components of atherosclerotic lesions in different stages of disease progression in situ using Raman microspectroscopy. METHODS: Thirty-five coronary artery samples were taken from 16 explanted transplant recipient hearts, and thin sections were prepared. Using a high-resolution confocal Raman microspectrometer system with an 830-nm laser light, high signal-to-noise Raman spectra were obtained from the following morphologic structures: internal and external elastic lamina, collagen fibers, fat, foam cells, smooth muscle cells, necrotic core, beta-carotene, cholesterol crystals, and calcium mineralizations. Their Raman spectra were modeled by using a linear combination of basis Raman spectra from the major biochemicals present in arterial tissue, including collagen, elastin, actin, myosin, tropomyosin, cholesterol monohydrate, cholesterol linoleate, phosphatidyl choline, triolein, calcium hydroxyapatite, calcium carbonate, and beta-carotene. RESULTS: The results show that the various morphologic structures have characteristic Raman spectra, which vary little from structure to structure and from artery to artery. The biochemical model described the spectrum of each morphologic structure quite well, indicating that the most essential biochemical components were included in the model. Furthermore, the biochemical composition of each structure, indicated by the fit contributions of the biochemical basis spectra of the morphologic structure spectrum, was very consistent. CONCLUSIONS: The Raman spectra of various morphologic structures in normal and atherosclerotic coronary artery may be used as basis spectra in a linear combination model to analyze the morphologic composition of atherosclerotic coronary artery lesions.

Diagnosis of human coronary atherosclerosis by morphology-based Raman spectroscopy.

BACKGROUND: Recent studies have shown that chemical composition and morphology, rather than anatomy (degree of stenosis), determine atherosclerotic plaque instability and predict disease progression. Current clinical diagnostic techniques provide accurate assessment of plaque anatomy, but have limited capability to assess plaque morphology in vivo. Here we describe a technique for a morphology-based diagnosis of atherosclerosis in the coronary arteries using Raman spectroscopy that can potentially be performed in vivo using optical fiber technology. METHODS: Raman tissue spectra were collected from normal and atherosclerotic coronary artery samples in different stages of disease progression (n=165) from explanted transplant recipient hearts (n=16). Raman spectra from the elastic laminae (EL), collagen fibers (CF), smooth muscle cells (SMC), adventitial adipocytes (AA) or fat cells, foam cells (FC), necrotic core (NC), cholesterol crystals (CC), beta-carotene containing crystals (beta-C), and calcium mineralizations (CM) were used as basis spectra in a linear least squares-minimization (LSM) model to calculate the contribution of these morphologic structures to the coronary artery tissue spectra. RESULTS: We developed a diagnostic algorithm that used the fit-contributions of the various morphologic structures to classify 97 coronary artery samples in an initial calibration data set as either nonatherosclerotic, calcified plaque, or noncalcified atheromatous plaque. The algorithm was subsequently tested prospectively in a second validation data set, and correctly classified 64 (94%) of 68 coronary artery samples. CONCLUSIONS: Raman spectroscopy provides information about the morphologic composition of intact human coronary artery without the need for excision and microscopic examination. In the future, it may be possible to use this technique to analyze the morphologic composition of atherosclerotic coronary artery lesions and assess plaque instability and disease progression in vivo.

Endoscopic detection of dysplasia in patients with Barrett's esophagus using light-scattering spectroscopy.

BACKGROUND & AIMS: We conducted a study to assess the potential of light-scattering spectroscopy (LSS), which can measure epithelial nuclear enlargement and crowding, for in situ detection of dysplasia in patients with Barrett's esophagus. METHODS: Consecutive patients with suspected Barrett's esophagus underwent endoscopy and systematic biopsy. Before biopsy, each site was sampled by LSS using a fiberoptic probe. Diffusely reflected white light was spectrally analyzed to obtain the size distribution of cell nuclei in the mucosal layer, from which the percentage of enlarged nuclei and the degree of crowding were determined. Dysplasia was assigned if more than 30% of the nuclei exceeded 10 microm and the histologic findings compared with those of 4 pathologists blinded to the light-scattering assessment. The data were then retrospectively analyzed to further explore the diagnostic potential of LSS. RESULTS: Seventy-six sites from 13 patients were sampled. All abnormal sites and a random sample of nondysplastic sites were reviewed by the pathologists. The average diagnoses were 4 sites from 4 different patients as high-grade dysplasia (HGD), 8 sites from 5 different patients as low-grade dysplasia (LGD), 12 as indefinite for dysplasia, and 52 as nondysplastic Barrett's. The sensitivity and specificity of LSS for detecting dysplasia (either LGD or HGD) were 90% and 90%, respectively, with all HGD and 87% of LGD sites correctly classified. Decision algorithms using both nuclear enlargement and crowding further improved diagnostic accuracy, and accurately classified samples into the 4 histologic categories. CONCLUSIONS: LSS can reliably detect LGD and HGD in patients with Barrett's esophagus.

Principles and pitfalls of diagnostic test development: implications for spectroscopic tissue diagnosis.

Diagnostic spectroscopy has the potential to supplant the time-honored "gold standard" of light microscopy and herald an era of in vivo tissue diagnosis. However, the lessons in disease diagnosis learned by pathologists over the years should not be forgotten. This discussion will focus on the basis principles and pitfalls of diagnostic test development, and how they apply to optical spectroscopy tissue diagnosis.

Prospects for in vivo Raman spectroscopy.

Raman spectroscopy is a potentially important clinical tool for real-time diagnosis of disease and in situ evaluation of living tissue. The purpose of this article is to review the biological and physical basis of Raman spectroscopy of tissue, to assess the current status of the field and to explore future directions. The principles of Raman spectroscopy and the molecular level information it provides are explained. An overview of the evolution of Raman spectroscopic techniques in biology and medicine, from early investigations using visible laser excitation to present-day technology based on near-infrared laser excitation and charge-coupled device array detection, is presented. State-of-the-art Raman spectrometer systems for research laboratory and clinical settings are described. Modern methods of multivariate spectral analysis for extracting diagnostic, chemical and morphological information are reviewed. Several in-depth applications are presented to illustrate the methods of collecting, processing and analysing data, as well as the range of medical applications under study. Finally, the issues to be addressed in implementing Raman spectroscopy in various clinical applications, as well as some long-term directions for future study, are discussed.

Diffuse reflectance spectroscopy of human adenomatous colon polyps in vivo.

Diffuse reflectance spectra were collected from adenomatous colon polyps (cancer precursors) and normal colonic mucosa of patients undergoing colonoscopy. We analyzed the data by using an analytical light diffusion model, which was tested and validated on a physical tissue model composed of polystyrene beads and hemoglobin. Four parameters were obtained: hemoglobin concentration, hemoglobin oxygen saturation, effective scatterer density, and effective scatterer size. Normal and adenomatous tissue sites exhibited differences in hemoglobin concentration and, on average, in effective scatterer size, which were in general agreement with other studies that employ standard methods. These results suggest that diffuse reflectance can be used to obtain tissue information about tissue structure and composition in vivo.

Histopathology of human coronary atherosclerosis by quantifying its chemical composition with Raman spectroscopy.

BACKGROUND: Lesion composition, rather than size or volume, determines whether an atherosclerotic plaque will progress, regress, or rupture, but current techniques cannot provide precise quantitative information about lesion composition. We have developed a technique to assess the pathological state of human coronary artery samples by quantifying their chemical composition with near-infrared Raman spectroscopy. METHODS AND RESULTS: Coronary artery samples (n=165) obtained from explanted recipient hearts were illuminated with 830-nm infrared light. Raman spectra were collected from the tissue and processed to quantify the relative weights of cholesterol, cholesterol esters, triglycerides and phospholipids, and calcium salts in the examined artery location. The artery locations were then classified by a pathologist and grouped as either nonatherosclerotic tissue, noncalcified plaque, or calcified plaque. Nonatherosclerotic tissue, which included normal artery and intimal fibroplasia, contained an average of approximately 4+/-3% cholesterol, whereas noncalcified plaques had approximately 26+/-10% and calcified plaques approximately 19+/-10% cholesterol in the noncalcified regions. The average relative weight of calcium salts was 1+/-2% in noncalcified plaques and 41+/-21% in calcified plaques. To make this quantitative chemical information clinically useful, we developed a diagnostic algorithm, based on a first set of 97 samples, that demonstrated a strong correlation of the relative weights of cholesterol and calcium salts with histological diagnoses of the same locations. This algorithm was then prospectively tested on a second set of 68 samples. The algorithm correctly classified 64 of these new samples, thus demonstrating the accuracy and robustness of the method. CONCLUSIONS: The pathological state of a given human coronary artery may be assessed by quantifying its chemical composition, which can be done rapidly with Raman spectroscopic techniques. When Raman spectra are obtained clinically via optical fibers, Raman spectroscopy may be useful in monitoring the progression and regression of atherosclerosis, predicting plaque rupture, and selecting proper therapeutic intervention.

Raman spectroscopy and fluorescence photon migration for breast cancer diagnosis and imaging.

We are developing optical methods based on near infrared Raman spectroscopy and fluorescence photon migration for diagnosis and localization of breast cancer. We demonstrate the ability of Raman spectroscopy to classify accurately normal, benign and malignant breast tissues, an important step in developing Raman spectroscopic needle probes as a tool for improving the accuracy of needle biopsy. We also show that photon migration imaging can be used to localize accurately small fluorescent objects imbedded in a thick turbid medium with realistic optical properties, thus demonstrating the potential of this technique for optical imaging.

Spectral pathology.

We are investigating the use of optical spectroscopy (fluorescence, reflectance, Raman scattering) for detecting precancerous lesions in the mucosal linings of hollow organs. We present a morphological model for extracting quantitative pathological information from fluorescence spectra, using colonic dysplasia as an example. The potential of this technique in providing histological information in real time without the need for tissue removal is discussed.

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.

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.

Biochemical analysis and mapping of atherosclerotic human artery using FT-IR microspectroscopy.

We report the application of FT-IR microspectroscopy for in situ spectroscopic characterization of molecular constituents of human atherosclerotic lesions. Since water content in tissue affects conformation-sensitive protein vibrational bands, tissue specimens were examined under moist conditions. In all measurements, vibrational bands from water were found to dominate the spectrum. By removing these water contributions, well resolved bands due to tissue components were readily observed. Utilizing the high sensitivity and good spatial resolution of IR microspectroscopy, spectra from a sample volume of 40 x 40 x 4 microns3 were collected using unstained cryostat sections mounted on a BaF2 flat in neutral isotonic saline. Microstructures were confirmed histologically by light microscopy in stained serial sections. In the spectrum of normal intima, major bands due to amide I (1656 cm-1), amide II (1556 cm-1), and CH bending (1457 cm-1) vibrations of the proteins collagen and elastin were observed. In the spectrum of the intima of noncalcified atherosclerotic plaque, major bands due to both proteins and lipids were observed. The lipid bands at 1734, 1468, 1171 and 1058 cm-1 were assigned to the C = O (ester) stretch, CH2 bend, C--O (ester) stretch and C--O stretch, respectively. At a more detailed level, bands specific to free cholesterol, and cholesterol esters were identified. A plot of the integrated intensity ratio of these bands to the protein amide II mode versus depth from the luminal surface confirmed a heterogeneous distribution of these constituents in the atheromatous core. In the spectra of calcified atherosclerotic plaque, bands were attributed to three types of biochemical microstructures: proteins (1657, 1555, 1243 cm-1), lipids (1735, 1466, 1170, 1085, 1055 cm-1) and calcium minerals such as hydroxyapatite (1094, 1040, 962 cm-1), and carbonated apatite (1463, 1412, 872 cm-1). The results demonstrate that IR microspectroscopy can be used for in situ characterization of molecular constituents in human unstained arterial sections. The molecular information obtained from these studies could be important in understanding the pathogenesis of atherosclerosis.

A characterization of the fluorescent properties of circulating human eosinophils.

This paper presents a characterization of the fluorescence properties of human eosinophils isolated from peripheral blood of normal donors over a wide range of excitation and emission wavelengths. Circulating eosinophils possess three fluorescence excitation emission maxima: one at 280 nm excitation, 330 nm emission, attributable to tryptophan fluorescence, and currently unassigned peaks at 360 nm excitation, 440 nm emission and 380 nm excitation, 415 nm emission. Fluorescence microscopy studies show that the fluorescence of eosinophils may be site dependent; specifically, when observed at 365 nm excitation, circulating eosinophil fluorescence appears blue-violet, while the fluorescence of tissue-dwelling eosinophils appears amber-gold. These results should be considered in developing an optical biopsy technique to identify eosinophils in human tissue.

Fine-needle aspiration cytologic study of myofibroblastoma of the breast. Immunohistochemical and ultrastructural findings.

Myofibroblastoma of the breast is a recently recognized benign stromal tumor arising from the breast mesenchyma. Myofibroblastomas are grossly circumscribed, unencapsulated tumors that are most commonly found in males. Histologically, myofibroblastomas comprise predominantly bipolar spindle cells arranged either haphazardly or in fascicles that traverse a collagenous background. Their ultrastructural and immunohistochemical profiles are consistent with myofibroblastic differentiation. Myofibroblastoma of the breast was discovered in a 64-year-old man. For the first time the fine-needle aspiration findings are reported, as are the histologic, immunohistochemical, and ultrastructural findings.

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.

476 nm excited laser-induced fluorescence spectroscopy of human coronary arteries: applications in cardiology.

We have shown that normal coronary arteries and noncalcified and calcified atherosclerotic plaque can be differentiated on the basis of the 476 nm excited fluorescence spectra, providing the basis of a spectroscopic guidance system for coronary artery laser angiosurgery. This discrimination is based on extraction of parameters from tissue fluorescence spectra, which are proportional to the tissue concentrations of structural proteins (collagen and elastin) and ceroid via a model of tissue fluorescence. We use these parameters to calculate the likelihood that an area of interest in a coronary artery is normal, noncalcified, or calcified plaque. This method of diagnosing atherosclerosis provides information about the histochemical composition of atherosclerotic lesions and is thus fundamentally different from the diagnostic methods currently used. It may ultimately have bearing on a number of pertinent clinical problems. We have discussed applications to studying initiating factors in formation and progression of plaque, healing after interventional treatments, and the likelihood of restenosis after PTCA.