Model-based analysis of clinical fluorescence spectroscopy for in vivo detection of cervical intraepithelial dysplasia.

We present a mathematical model to calculate the relative concentration of light scatterers, light absorbers, and fluorophores in the epithelium and stroma. This mathematical description is iteratively fit to the fluorescence spectra measured in vivo, yielding relative concentrations of each molecule. The mathematical model is applied to a total of 493 fluorescence measurements of normal and dysplastic cervical tissue acquired in vivo from 292 patients. The estimated parameters are compared with histopathologic diagnosis to evaluate their diagnostic potential. The mathematical model is validated using fluorescence spectra simulated with known sets of optical parameters. Subsequent application of the mathematical model to in vivo fluorescence measurements from cervical tissue yields fits that accurately describe measured data. The optical parameters estimated from 493 fluorescence measurements show an increase in epithelial flavin adenine dinucleotide (FAD) fluorescence, a decrease in epithelial keratin fluorescence, an increase in epithelial light scattering, a decrease in stromal collagen fluorescence, and an increase in stromal hemoglobin light absorption in dysplastic tissue compared to normal tissue. These changes likely reflect an increase in the metabolic activity and loss of differentiation of epithelial dysplastic cells, and stromal angiogenesis associated with dysplasia. The model presented here provides a tool to analyze clinical fluorescence spectra yielding quantitative information about molecular changes related to dysplastic transformation.

Calibration standards for multicenter clinical trials of fluorescence spectroscopy for in vivo diagnosis.

In the context of clinical trials, calibration protocols for optical instruments that ensure measurement accuracy and the ability to carry out meaningful comparisons of data acquired from multiple instruments are required. A series of calibration standards and procedures are presented to assess technical feasibility of optical devices for cervical precancer detection. Measurements of positive and negative standards, and tissue are made with two generations of research grade spectrometers. Calibration accuracy, ability of standards to correct and account for changes in experimental conditions, and device components are analyzed. The relative frequency of measured calibration standards is investigated retrospectively using statistical analysis of trends in instrument performance. Fluorescence measurements of standards and tissue made with completely different spectrometers show good agreement in intensity and lineshape. Frequency of wavelength calibration standards is increased to every 2 h to compensate for thermal drifts in grating mount. Variations in illumination energy detected between standards and patient measurements require probe redesign to allow for simultaneous acquisition of illumination power with every patient measurement. The use of frequent and well-characterized standards enables meaningful comparison of data from multiple devices and unambiguous interpretation of experiments among the biomedical optics community.

Diffuse reflectance patterns in cervical spectroscopy.

OBJECTIVES: Our laboratory seeks to develop minimally invasive cost-effective methods to improve screening and detection of curable precursors to cervical cancer. Previously, we have presented pilot studies that assess the diagnostic power of auto-fluorescence and diffuse reflectance spectroscopy. In the present study, we evaluate diffuse reflectance spectra from a comprehensive 850 patient clinical trial to determine its ability to discriminate normal tissue from several grades of abnormal cervical tissue. METHODS: Diffuse reflectance spectra at four source detector separations measured from 549 cervical sites were available for analysis. Three classifiers were implemented: one used spectral data directly as input, a second used simple spectral features such as peak position and intensity, and one used principal component analysis for feature selection. Algorithms were developed and evaluated using leave-one-out cross-validation to classify normal and precancerous cervical tissue. The percentage of samples correctly classified was used to evaluate and compare the performance of the algorithms, as compared to histology. RESULTS: Diffuse reflectance spectra of cervical precancer showed consistent differences from that of normal tissue at all source detector separations; reflectance intensity of precancer was lower than that of normal tissue on average. Normal cervical tissue spectra show more intensity variation between patients than other tissue grades. Reflectance spectra acquired from the closest source detector separations consistently demonstrated the most relevant information for tissue classification. Two persistent spectral patterns demonstrated that the contribution of hemoglobin absorption and the wavelength-dependent spectral slope contained relevant information for classification. CONCLUSIONS: Spectral patterns in diffuse reflectance spectra can be used for the discrimination of normal cervical tissue from low grade and high grade squamous intraepithelial lesions.