Sung K. Chang

Reflectance spectroscopy for in vivo detection of cervical precancer

Optical technologies, in particular fluorescence spectroscopy, have shown the potential to provide improved detection methods for cervical neoplasia that are sensitive and cost effective through accurate, objective, instantaneous point-of-care diagnostic tools. The specific goals of this study were to analyze reflectance spectra of normal and neoplastic cervical tissue in vivo and to evaluate the data for use in diagnostic algorithm development. Spectroscopic measurements were obtained at four distinct source-detector separations from 324 sites in 161 patients. As the source-detector separation increases, greater tissue depth is probed. The average spectra of each diagnostic class differed at all source-detector separations, with the greatest differences occurring at the smallest source-detector separations. Algorithms, based on principal-component analysis and Mahalanobis distance classification, were developed and evaluated for all combinations of source-detector separations relative to the gold standard of colposcopically directed biopsy. The diagnostic combination of squamous normal versus high-grade squamous intraepithelial lesions gave good discrimination with a sensitivity of 72% and a specificity of 81%; discrimination of columnar normal versus high-grade squamous intraepithelial lesions also was good, with sensitivity of 72% and specificity of 83%. Thus, reflectance spectroscopy appears promising for in vivo detection of cervical precancer. Strategies that combine fluorescence and reflectance spectroscopy may enhance the discrimination capabilities.

Analytical model to describe fluorescence spectra of normal and preneoplastic epithelial tissue: comparison with Monte Carlo simulations and clinical measurements

Fluorescence spectroscopy has shown promise for the detection of precancerous changes in vivo. The epithelial and stromal layers of tissue have very different optical properties; the albedo is relatively low in the epithelium and approaches one in the stroma. As precancer develops, the optical properties of the epithelium and stroma are altered in markedly different ways: epithelial scattering and fluorescence increase, and stromal scattering and fluorescence decrease. We present an analytical model of the fluorescence spectrum of a two-layer medium such as epithelial tissue. Our hypothesis is that accounting for the two different tissue layers will provide increased diagnostic information when used to analyze tissue fluorescence spectra measured in vivo. The Beer-Lambert law is used to describe light propagation in the epithelial layer, while light propagation in the highly scattering stromal layer is described with diffusion theory. Predictions of the analytical model are compared to results from Monte Carlo simulations of light propagation under a range of optical properties reported for normal and precancerous epithelial tissue. In all cases, the mean square error between the Monte Carlo simulations and the analytical model are within 15%. Finally, model predictions are compared to fluorescence spectra of normal and precancerous cervical tissue measured in vivo; the lineshape of fluorescence agrees well in both cases, and the decrease in fluorescence intensity from normal to precancerous tissue is correctly predicted to within 5%. Future work will explore the use of this model to extract information about changes in epithelial and stromal optical properties from clinical measurements and the diagnostic value of these parameters.

Reflectance spectroscopy for diagnosis of epithelial precancer: model-based analysis of fiber-optic probe designs to resolve spectral information from epithelium and stroma

Reflectance spectroscopy is a promising technology for detection of epithelial precancer. Fiber-optic probes that selectively collect scattered light from both the epithelium and the underlying stroma are likely to improve diagnostic performance of in vivo reflectance spectroscopy by revealing diagnostic features unique to each layer. We present Monte Carlo models with which to evaluate fiber-optic probe geometries with respect to sampling depth and depth resolution. We propose a probe design that utilizes half-ball lens coupled source and detector fibers to isolate epithelial scattering from stromal scattering and hence to resolve spectral information from the two layers. The probe is extremely compact and can provide easy access to different organ sites.

Optimal excitation wavelengths for discrimination of cervical neoplasia

Fluorescence spectroscopy has shown promise for the in vivo, real-time detection of cervical neoplasia. However, selection of excitation wavelength has in the past been based on in vitro studies and the availability of light sources. The goal of this study was to determine optimal excitation wavelengths for in vivo detection of cervical neoplasia. Fluorescence excitation-emission matrices (EEMs) were measured in vivo from 351 sites in 146 patients. Data were analyzed in pairs of diagnostic classes to determine which combination of excitation wavelengths yields classification algorithms with the greatest sensitivity and specificity. We find that 330-340-, 350-380-, and 400-450-nm excitation yield the best performance. The sensitivity and specificity for discrimination of squamous normal tissue and high-grade squamous intraepithelial lesion (HGSIL) were 71 % and 77 % on cross validation using three excitation wavelengths. These results are comparable with those found in earlier in vivo studies; however, in this study we find that the proportion of samples which are HGSIL influences performance. Furthermore stratification of samples within low-grade squamous intraepithelial lesion and HGSIL also appears to influence diagnostic performance. Future diagnostic studies should be carried out at these excitation wavelengths in larger groups so that data can be stratified by diagnostic subcategory, age and menopausal status. Similarly, large studies should be done in screening populations.

Ball lens coupled fiber-optic probe for depth-resolved spectroscopy of epithelial tissue.

A ball lens coupled fiber-optic probe design is described for depth-resolved measurements of the fluorescence and reflectance properties of epithelial tissue. A reflectance target, fluorescence targets, and a two-layer tissue phantom consisting of fluorescent microspheres suspended in collagen are used to characterize the performance of the probe. Localization of the signal to within 300 microm of the probe tip is observed by use of reflectance and fluorescence targets in air. Differential enhancement of the fluorescence signal from the top layer of the two-layer tissue phantom is observed.

Fluorescence spectroscopy for cervical precancer detection: Is there variance across the menstrual cycle?

This study assesses one possible cause of inter-patient variation in fluorescence spectroscopy of the cervix: the menstrual cycle. Ten patients with no history of an abnormal Pap smear were seen daily throughout 30 consecutive days of their cycle. Fluorescence excitation-emission matrices were measured from three cervical sites on each patient. Principal component analysis was used to determine which spectral regions varied with the day of the cycle. Classification was performed to assess the influence of menstrual cycle on precancer diagnosis. Variations in the principal component scores and the redox ratio values show that the fluorescence emission spectra at 340-380 nm excitation appear to correlate with the cell metabolism of the cervical epithelium throughout the menstrual cycle; these changes do not affect diagnostic classification. The menstrual cycle affects intra-patient variation but does not appear to cause a significant level of inter-patient variation. It does not need to be controlled for in optical detection strategies based on fluorescence spectroscopy.

Detecting the Signal of the Menstrual Cycle in Fluorescence Spectroscopy of the Cervix

Fluorescence spectroscopy of the cervix has been shown to be an effective noninvasive diagnostic tool for cervical intraepithelial neoplasia (precancer). To assess the effect of the menstrual cycle on fluorescence spectroscopy, daily measurements were made on ten subjects for the length of their cycle. These measurements were analyzed to determine if there was a statistically significant signal associated with the menstrual cycle. A signal was found for emission wavelengths between 425 and 445 nm inclusive—near the main hemoglobin absorption band, the Soret band, at 420 nm. We suspect that the slight displacement of the Soret band is due to the nearby dominant NAD(P)H peak, which increases the signal-to-noise ratio and affects statistical significance. The signal consists of a reduction in fluorescence intensity for the first few days of the cycle. This analysis indicates that hemoglobin absorption is the main menstrual-cycle effect on the use of fluorescence spectroscopy on the cervix. The effect is confined to a small set of excitation/emission wavelengths and to approximately the first 8 days of the cycle. This suggests that any problems from the menstrual cycle can be avoided with a simple requirement that the device not be used during the period of menstrual bleeding.

Combination of fluorescence and reflectance spectroscopy for in vivo detection of cervical pre-cancers

The goal of this study is to identify fluorescence excitation wavelengths and reflectance source-detector separations that are significant for diagnosis of cervical pre-cancers. Measurements from fluorescence and reflectance spectroscopy were searched exhaustively using Mahalanobis distance based classification. Fluorescence excitation wavelengths at 340 nm, 350 nm and 410 nm and reflectance source-detector separation of 250 /spl mu/m appear significant for discriminating a pair of diagnostic classes.