John R. Kramer

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.

Serum Lp(a) level as a predictor of vein graft stenosis after coronary artery bypass surgery in patients.

Although the serum lipoprotein fraction Lp(a) has been associated with coronary artery atherosclerosis, its relationship to narrowing of saphenous vein grafts has not previously been elucidated. We therefore measured serum Lp(a) levels in 167 symptomatic patients undergoing cardiac catheterization who had had coronary artery bypass surgery 0.7 to 14.3 years earlier. Lp(a), total cholesterol, and total triglyceride levels were compared with the degree of saphenous vein graft stenosis to test for any association. Serum Lp(a) levels were significantly associated with the degree of stenosis of saphenous vein grafts (r = .24, p = .002). Mean Lp(a) levels (mg/dl) in the 135 patients with stenosis were almost double (32.0 +/- 32.7, mean +/- SD) those in the 32 patients with no graft stenosis (16.7 +/- 22.6; p = .002). Graft stenosis was not associated with previous myocardial infarction, hypertension, obesity, diabetes, or smoking. Serum cholesterol levels (mg/dl) were slightly higher in the stenosis group (251.3 +/- 69) than in the no-stenosis group (231.8 +/- 48.8), but the difference was of borderline significance (p = .06). A stepwise increase in mean Lp(a) was found in groups of patients with increasing vein graft stenosis. At a serum Lp(a) level of 31.6 mg/dl or above, 92% of the patients demonstrated vein graft stenosis. Thus, patients with elevated Lp(a) levels have an increased risk of developing saphenous vein graft stenosis after coronary bypass surgery.

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.

Clinical significance of isolated coronary bridges: Benign and frequent condition involving the left anterior descending artery

Review of 658 normal cineangiograms performed in the cardiac laboratory of the Cleveland Clinic during 1974 revealed myocardial bridging in 81 patients (12 % ). Maximal systolic compression was measured and compared to diastolic dimension above and below the obstruction by means of a programmable digital caliper. Systolic bridging involved the left anterior descending coronary artery in all cases and was mild in 26 patients, moderate in 55 patients, and severe in 11 patients. Seventy per cent of the patients were men and 30% were women. The majority had atypical symptoms although 15 patients were thought to have angina pectoris. Fiveyear survival rate was 97.5 % . No survivor had acute myocardial infarction during this follow-up period of 5 years. In the patient with normal coronary arteries and normal left ventricular function, myocardial bridging is benign. Myocardial bridging of coronary arteries was recognized and described by Black’ in 1796. If specifically sought at autopsy, such bridges are found in 5 % to 86% of hearts examined.2-4 These bridges also can be recognized arteriographically as a systolic compression of the vessel involved. A prevalence of 0.5% to 1.6% has been reported.5p6 It has been suggested that myocardial bridges, by reducing myocardial blood flow in systole, are responsible for cardiac ischemia,5*7 acute myocardial infarction (AMI),7-g ventricular fibrillation,‘O and sudden death.” Furthermore, it has been implied that myocardial bridging may have the same significant clinical morbidity and mortality as coronary atherosclerosis.12

Optical fiber probe for biomedical Raman spectroscopy

In vitro experiments have demonstrated the ability of Raman spectroscopy to diagnose a wide variety of diseases. Recent in vivo investigations performed with optical fiber probes were promising but generally limited to easily accessible organs, often requiring relatively long collection times. We have implemented an optical design strategy to utilize system throughput fully by characterizing the Raman distribution from tissue. This scheme optimizes collection efficiency, minimizes noise, and has resulted in small-diameter, highly efficient Raman probes that are capable of collecting high-quality data in 1 s. Performance has been tested through simulations and experiments with tissue models and several in vitro tissue types, demonstrating that this new design can advance Raman spectroscopy as a clinically practical technique.

Progression of coronary atherosclerosis.

Two hundred sixty-two patients with 50% or greater obstruction of at least one coronary artery on the initial study underwent recatheterization 2–182 months later and were evaluated for progressive arterial changes. Progression was considered present if (1) a change from less than total obstruction to total obstruction occurred in any vessel; (2) a change from 70% or less to 90% or more occurred in any vessel; (3) a change from 30% or less to 50% or more occurred in any vessel not initially obstructed by 50%; or (4) a 20% or more increase in obstruction was noted in any vessel already narrowed 50% or more. Of the 262 patients, 116 of 238 men (49%) and 12 of 24 women (50%) met the criteria for progression. Risk factors determined at the time of the initial catheterization in patients who met the criteria for progression were compared with risk factors in patients who did not. No significant difference could be found between the groups in relation to family history, blood pressure, diabetes, smoking habits, weight, cholesterol levels, triglyceride values, initial ECG and initial catheterization findings.The frequency of detecting progressive arterial changes tended to increase as the interval between studies increased (p < 0.001). The frequency of multiple vessel progression increased as the interval between studies increased. Progression was more frequent in patients younger than 50 years than it was in patients 50 years and older.

Determination of Human Coronary Artery Composition by Raman Spectroscopy

BACKGROUND We present a method for in situ chemical analysis of human coronary artery using near-infrared Raman spectroscopy. It is rapid and accurate and does not require tissue removal; small volumes, approximately 1 mm3, can be sampled. This methodology is likely to be useful as a tool for intravascular diagnosis of artery disease. METHODS AND RESULTS Human coronary artery segments were obtained from nine explanted recipient hearts within 1 hour of heart transplantation. Minces from one or more segments were obtained through grinding in a mortar and pestle containing liquid nitrogen. Artery segments and minces were excited with 830 nm near-infrared light, and Raman spectra were collected with a specially designed spectrometer. A model was developed to analyze the spectra and quantify the amounts of cholesterol, cholesterol esters, triglycerides and phospholipids, and calcium salts present. The model provided excellent fits to spectra from the artery segments, indicating its applicability to intact tissue. In addition, the minces were assayed chemically for lipid and calcium salt content, and the results were compared. The relative weights obtained using the Raman technique agreed with those of the standard assays within a few percentage points. CONCLUSIONS The chemical composition of coronary artery can be quantified accurately with Raman spectroscopy. This opens the possibility of using histochemical analysis to predict acute events such as plaque rupture, to follow the progression of disease, and to select appropriate therapeutic interventions.

Spectral diagnosis of atherosclerosis using an optical fiber laser catheter.

This communication demonstrates that fluorescence spectra of human aorta with good S/N ratios can be collected using an optical fiber laser catheter. The performance of this catheter is compared to a non-fiber optic collection system with an equivalent delivery/collection geometry. For a given sample, fluorescence lineshapes obtained using the two systems are identical; differences in peak fluorescence intensity are related to the different collection efficiencies of the two systems. It is shown that the fluorescence lineshape of arterial tissue depends on the delivery/collection geometry of the detection system, and that this is due to the interaction of absorption and fluorescence within the artery wall. This effect is investigated systematically using a specially designed collection system. Results are analyzed qualitatively using a simple, one-dimensional model of tissue fluorescence. With this analysis, we present design requirements for a collection system in which such geometric effects are eliminated, and show that our optical fiber laser catheter satisfies these requirements.

The dynamics of progression of coronary atherosclerosis studied in 168 medically treated patients who underwent coronary arteriography three times.

To study the dynamics of progression of coronary atherosclerosis we analyzed findings in 168 patients who underwent coronary arteriography three times without undergoing coronary surgery or percutaneous transluminal coronary angioplasty. Of 66 patients who had progression in interval 1 (from first to second coronary arteriogram), 32 also had progression in interval 2 (from second to third coronary arteriogram); of 102 patients who had no progression in interval 1, 37 had progression in interval 2. In only 9 of the 32 patients who had progression during both intervals was the same lesion involved, and in six of these patients other lesions were also involved. Progression correlated with the duration of the interval between catheterizations, an increase in symptoms, and the occurrence of myocardial infarction. Analysis of variance showed no significant differences in mean values for age, blood pressure, total serum cholesterol, and serum triglycerides in the four main progression groups. Progression of coronary atherosclerosis is a highly unpredictable process. It follows a nonlinear course, and information derived from sequential coronary arteriograms is of little value in predicting future progression.

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.