17:45 - 19:15 Improving the Quality of Diagnosis and Prognosis (IV)
We used near-infrared (NIR) Raman spectroscopy to assess the chemicalcomposition and pathological state of atherosclerosis in perfused intacthuman coronary artery tissue.
Human coronary artery samples of 5-6 cm length were obtained duringautopsy, and mounted in an in vitro set-up and perfused with anisotonic salt solution at ~80 mm Hg. Near infrared laser light (830 nm;50 mW) was delivered to the tissue through the central fiber of an opticalfiber probe (Visionex, GA USA) that was inserted transluminally into theartery sample. The probe has a central fiber of 400 µm core-diameter,guiding laser light to the tissue, and is surrounded by seven collectionfibers of 300 µm core-diameter. To suppress Raman scattered lightfrom the quartz fibers the excitation and collection fibers were coatedwith dielectric filters at the probe tip. The collected Raman light waslaunched into an imaging spectrometer equipped with a CCD-camera, optimizedfor the NIR region of the spectrum. The spectral information was storedon computer disk. High quality tissue Raman signals were collected in 10-60s.
Figure 1. Raman spectrum from human coronary arteryrecorded with an intravascular optical fiber probe (dotted line) modeledwith the set of spectra from individual components (line) to quantify thechemical composition of the artery wall. The curve under the spectrum showsthe difference of the spectrum and the fit.
The collected spectra were linearly modeled with Raman spectra of thechemical components comprising the artery. These components were free cholesterol,cholesterol esters, calcium salts, triglycerides and phospholipids, twodelipidized artery segments and b -carotene.The linear superpositions of these chemical components modeled the measuredcoronary artery spectra well, judged by the residual of the fit that wasobtained by subtracting the model fit from the artery spectrum. An exampleof the modeling routine result is shown in Figure 1. A diagnostic algorithm,based on the chemical fitting parameters, allows classification of theexamined tissue into one of three pathological classes (non-atherosclerotictissue, non-calcified plaque, or calcified plaque), according to classicalclassification schemes.
From these results we conclude that intravascular optical fiber Ramanspectroscopy can provide in situ histopathology, which may be usedto study vascular disease in the patient in vivo, and for predictingplaque rupture and evaluating of therapeutic effects.
In recent years, Raman spectroscopy has been used to study static anddynamic properties of biologically important molecules in solution, insingle living cells, in cell cultures, and finally in tissues. Raman signalsacquired from biological tissues display fingerprint features which canbe related to the specific molecular structure of the constituents.
Raman spectroscopy has been investigated for detection of cancers ofthe breast, brain, colon, bladder, uterus, ovary and cervix in vitro.However, the development of fiber optic probes for remote delivery andcollection of Raman spectra in vivo has proven to be more challenging.Typically, the optical elements and optical fibers used for light deliveryin a remote sensing spectroscopic system are made of silica. Silica hasan intense fluorescence and Raman signal which can interfere with the detectionof the weak tissue signal.
Recent developments in the attempt to develop innovative fiber opticprobes that have the potential to detect tissue Raman signals in vivowhile minimizing the effect of silica Raman will be reviewed. One suchdesign will be described in the context of its application for cervicalprecancer detection. Other possible instrument considerations will be discussed.Finally, Raman spectra obtained from intact issues will be comprehensivelyreviewed and discussed in terms of the molecular and microscopic literatureto develop a framework for analyzing Raman signals to yield informationabout the molecular changes that occur with cancers and precancers.
Pathological conditions involve changes in molecular composition oftissue, both as a cause and as a consequence of disease. Raman spectroscopyenables (in vivo) detection of these changes in a non/minimal-invasive,non-destructive manner. Such clinical applications require rapid, invivo signal collection � preferably in the order of seconds � and onlinedata analysis, using databases of reference spectra. The data that canbe obtained from the Raman spectra could then guide further clinical action.
Realisation of the Raman-potential requires advances in several areas,such as:
Presently our lab is pursuing Raman spectroscopy applications in thefields of skin research, atherosclerosis, detection of (pre-)malignanttissue, and transplant liver characterisation. In collaboration with industryin vivo instruments are being developed.
During the workshop some of the latest results of these projects willbe presented, which will provide an insight into current potentials andlimitations.
Particularly during operative neurosurgical intervention and post insultmonitoring, real time knowledge of neuronal biochemical and structuralchange has important clinical implications.
The escalating efficacy of Raman technology may well provide importantinsights in such occasion. Potential intracellular markers of impairedneuronal function either secondary to trauma or ischemia include increasedconcentration of water, decreased concentration of Oxygen, decreased concentrationglucose, increased concentration of lactate, increased concentration oflactic acid, increased concentration of sodium, increased concentrationof Cloride and decreased concentration of potassium.
In ischemic brain disease the metabolites detected in the brain arelactate and lactic acid, increased extracellular potassium, increased water,lipids Cho, Pcr-Cr, where feasible concentrations of metabolic and structuralmarkers are going to be discussed.
Additional markers useful in differentiation of normal neuronal tissuefrom glial brain tumors would be myioinositol, increased amount of Lipids,increased amount of Choline, decreased amount of N-Acetyl-Aspartate.
The diagnostic implications of such variances will be discussed in regardsto emphasizing Raman technology to maximize clinical outcome among patientswith neurosurgical problems.
It has been proposed that Raman spectroscopy may be used to diagnosecancer. Previously, alternative techniques such as fluorescence spectroscopyhave been successfully used in vivo to diagnose lung and gastrointestinalcancers. Fluorescence point spectra may be collected in less than a secondand fluorescence imaging is possible due to the high fluorescence intensityobserved in tissue. Unfortunately, the diagnostic specificity of this techniqueis limited by the lack of features in tissue spectra. Raman spectroscopyprovides detailed information about the biomolecular composition of tissuewhich may be used to distinguish between normal and diseased tissue witha higher specificity than fluorescence spectroscopy. Early detection oftissue transformation may also be possible.
A portable fiber optic-based In vivo Raman system (IVRS) hasbeen built that is suitable for clinical use. It has a high collectionefficiency and a high detection sensitivity and spectra may be collectedin vivo in less than 1 min. Recent work has been done to improvesystem performance through the development of specialized fiber optic probes.
Future work involves the testing of the IVRS with animal models andhuman clinical trials. During the animal studies, spectral sorting algorithmswill be developed for tissue diagnosis. The diagnostic sensitivity andspecificity of the Raman system will also be determined for different diseases.System performance will be continuously monitored to ensure that optimalsystem performance is achieved prior to clinical trials. Human in vivotrials will involve three different anatomical sites: (1) skin, (2) brain,and (3) the gastrointestinal tract. These represent a diverse samplingof clinical conditions in which the IVRS will be evaluated (non invasive,inter-operative and endoscopic examination, respectively). In this talk,the proposed clinical trials will be discussed. Special emphasis will beplaced on the limitations of the IVRS which are relevant for each anatomicalsite.