Fluorescence and reflectance for atherosclerotic plaque characterization

Fluorescence and reflectance spectroscopy can provide valuable data regarding the diagnosis of atherosclerosis and vulnerable lesion characterization. Vulnerable plaques are responsible for clinical events with high morbidity and mortality, such as myocardial infarctions and strokes. Coronary artery disease is responsible for at least 450,000 deaths per year in the United States.[1] Atherosclerosis involving the carotid and femoral arteries is also a major cause of stroke and peripheral vascular disease.

Background experience
Fluorescence spectroscopy and the absorption-free intrinsic fluorescence spectroscopy (IFS) are analytical methods with a high signal to noise ratio and very low collection times, which offer the possibility of investigating a broad panel of proteins, lipoproteins and proteoglycans with importance in atherosclerosis. Our group has done pioneering work of fluorescence microscopy in atherosclerosis, with the purpose of characterizing chemical constituents such as ceroid and tryptophan, configuring the spectral features of these elements and identifying the histological structures that manifest similar fluorescent characteristics.[2-4] Taking this approach further, our group demonstrated the possibility of diagnosing with high accuracy calcified and non-calcified atherosclerotic arterial segments, both in coronary arteries and aorta.[5, 6]

With the creation and successive improvement of the fast emission-excitation (FastEEM) instrument equipped with a fiber optic probe,[7] our group became able to acquire and analyze fluorescence and reflectance data in clinical trials.[8-10] Combining fluorescence and reflectance allowed us to disentangle fluorescence spectra and extract IFS data non-influenced by absorption effects.[11] This is particularly important in atherosclerosis, where the blood absorption severely distorts the fluorescence spectra.

Diffuse reflectance spectroscopy (DRS) offers information on tissue scatterers and absorbers. DRS spectra are collected with the FastEEM instrument simultaneously with the fluorescence data. An important absorber in the atherosclerotic plaques is beta-carotene,[12] known to be present in lipid-rich structures of the plaque, such as the foam cells.[13] The study of this chemical component in the atherosclerotic plaque was pursued by our group as an adjunctive means for better spectroscopic characterization of the atherosclerotic plaque. By combining IFS at 480 nm excitation wavelength and DRS we were able to separate normal from atherosclerotic coronary arteries with sensitivity 95% and specificity 91%.[14]

Current work. An increasing emphasis has been placed by our group on identifying the constituent structures of the vulnerable plaques. Foam cells are the active cellular elements of the atherosclerotic plaques. Most important are especially those situated in the superficial layers of the plaque and coined by us superficial foam cells (SFC).

Figure 1	Figure 2

In an in-vitro study of 132 coronary specimens investigated with the FastEEM instrument, we used DRS and IFS at 480 nm excitation wavelength to identify those segments harboring SFC in a superficial region of interest (ROI) with the depth of 200 µm. To fit the data we used DRS spectra of beta-carotene and oxy-hemoglobin (Figure 1), and IFS morphological basis spectra extracted from specimens with SFC, and layers of fibrous cap and necrotic core respectively (Figure 2). We obtained fits of excellent quality as illustrated in Figure 3. Contribution coefficients to DRS and IFS at 480 nm excitation wavelength were subsequently used to build an algorithm for the identification of SFC specimens (Figure 4). The accuracy of identifying specimens with SFC area greater than 40%, 10% and 0% of the ROI was 98%, 93% and 87% respectively.

Figure 3	Figure 4

Foam cells are critical elements in atherosclerotic lesions and their detection is key to the diagnosis of plaques prone to erosion and rupture.[15] Spectroscopic means for identifying other important histological and chemical structures in these two types of lesions are currently under scrutiny.

Recent Publications

1. Heart Disease and Stroke Statistics - 2003 Update. 2002, American Heart Association: Dallas, TX.
2. Verbunt, R.J., et al., "Characterization of ultraviolet laser-induced autofluorescence of ceroid deposits and other structures in atherosclerotic plaques as a potential diagnostic for laser angiosurgery." Am Heart J. 1992, 123(1): p. 208-16.
3. Fitzmaurice, M., et al., "Argon ion laser-excited autofluorescence in normal and atherosclerotic aorta and coronary arteries: morphologic studies." Am Heart J. 1989, 118(5 Pt 1): p. 1028-38.
4. Baraga, J.J., et al., "Characterization of the fluorescent morphological structures in human arterial wall using ultraviolet-excited microspectrofluorimetry." Atherosclerosis 1991, 88(1): p. 1-14.
5. Richards-Kortum, R., et al., "A one-layer model of laser-induced fluorescence for diagnosis of disease in human tissue: applications to atherosclerosis." IEEE Trans Biomed Eng 1989, 36(12): p. 1222-32.
6. Richards-Kortum, R., et al., "476 nm excited laser-induced fluorescence spectroscopy of human coronary arteries: applications in cardiology." Am Heart J 1991, 122(4 Pt 1): p. 1141-50.
7. Zangaro, R.A., et al., "Rapid multiexcitation fluorescence spectroscopy system for in vivo tissue diagnosis." Applied Optics 1996, 35: p. 5211- 5219.
8. Georgakoudi, I., et al., "Fluorescence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett's esophagus." Gastroenterology2001, 120(7): p. 1620-9.
9. Georgakoudi, I., et al., "Trimodal spectroscopy for the detection and characterization of cervical precancers in vivo." Am J Obstet Gynecol 2002, 186(3): p. 374-82.
10. Müller, M.G., et al., "Spectroscopic detection and evaluation of morphologic and biochemical changes in early human oral carcinoma." Cancer 2003, 97(7): p. 1681-92.
11. Müller, M.G., et al., "Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption." Applied Optics 2001, 40: p. 4633-4646.
12. Ye, B. and G.S. Abela, "Beta-carotene enhances plaque detection by fluorescence attenuation in an atherosclerotic rabbit model." Lasers Surg Med 1993, 13(4): p. 393-404.
13. Carpenter, K.L., et al., "The carotenoids beta-carotene, canthaxanthin and zeaxanthin inhibit macrophage-mediated LDL oxidation." FEBS Lett. 1997, 401(2-3): p. 262-6.
14. Angheloiu, G.O., et al. "Diagnosing Coronary Atherosclerosis Using Intrinsic Fluorescence and Reflectance." in American College of Cardiology Annual Scientific Session. 2002. Atlanta, GA.
15. Virmani, R., et al., "Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions." Arterioscler Thromb Vasc Biol 2000, 20(5): p. 1262-75.