Carrie Brookner

Autofluorescence Microscopy of Fresh Cervical-Tissue Sections Reveals Alterations in Tissue Biochemistry with Dysplasia ¶

Abstract Fluorescence spectroscopy offers an effective, noninvasive approach to the detection of precancers in multiple organ sites. Clinical studies have demonstrated that fluorescence spectroscopy can provide highly sensitive, specific and cost-effective diagnosis of cervical precancers. However, the underlying biochemical mechanisms responsible for differences in the fluorescence spectra of normal and dysplastic tissue are not fully understood. We designed a study to assess the differences in autofluorescence of normal and dysplastic cervical tissue. Transverse, fresh tissue sections were prepared from colposcopically normal and abnormal biopsies in a 34-patient study. Autofluorescence images were acquired at 380 and 460 nm excitation. Results showed statistically significant increases in epithelial fluorescence intensity (arbitrary units) at 380 nm excitation in dysplastic tissue (106 ± 39) relative to normal tissue (85 ± 30). The fluorophore responsible for this increase is possibly reduced nicotinamide adenine dinucleotide. Stromal fluorescence intensities in the dysplastic samples decreased at both 380 nm (102 ± 34 [dysplasia] vs 151 ± 44 [normal]) and 460 nm excitation (93 ± 35 [dysplasia] vs 137 ± 49 [normal]), wavelengths at which collagen is excited. Decreased redox ratio (17–40% reduction) in dysplastic tissue sections, indicative of increased metabolic activity, was observed in one-third of the paired samples. These results provide valuable insight into the biological basis of the differences in fluorescence of normal and precancerous cervical tissue.

Autofluorescence Patterns in Short‐Term Cultures of Normal Cervical Tissue

Abstract Fluorescence spectroscopy has potential to improve cervical precancer detection. The relationship between tissue biochemistry and fluorescence is poorly understood. The goal of this study was to characterize normal cervical autofluorescence, using fresh tissue short-term tissue cultures and epithelial cell suspensions. Transverse, short-term tissue cultures were prepared from 31 cervical biopsies; autofluorescence images were obtained at 380 and 460 nm excitation. Fluorescence excitation–emission matrices were measured from normal, precancerous and cancerous cervical cell suspensions. Observed fluorescence patterns contrast those reported for frozen–thawed tissue, and were placed into groups with (1) bright epithelial and weak stromal fluorescence; (2) similar epithelial and stromal fluorescence; and (3) weak epithelial and bright stromal fluorescence. The average ages of women in the groups were 30.9, 38.0 and 49.2 years. Epithelial fluorescence intensity was similar in Groups 1 and 2, but weaker in Group 3. Stromal intensity was similar in Groups 2 and 3, but weaker in Group 1. The ratio of epithelial to stromal fluorescence intensity was significantly different for all groups. EEMs of cell suspensions showed peaks consistent with tryptophan, reduced form of nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide. Short-term tissue cultures represent a novel, biologically appropriate model to understand cervical autofluorescence. Our results suggest a biological basis for the increased fluorescence seen in older, postmenopausal women.

Screening for squamous intraepithelial lesions with fluorescence spectroscopy

OBJECTIVE To evaluate the accuracy of fluorescence spectroscopy in screening for squamous intraepithelial lesions (SILs) and to compare its performance with that of Papanicolaou smear screening, colposcopy, cervicoscopy, cervicography, and human papillomavirus (HPV) testing. DATA SOURCES Receiver operating characteristic (ROC) curve analysis was used to analyze performance by fluorescence spectroscopy (primary data) and other methods (secondary data). METHODS OF STUDY SELECTION In our search, 275 articles were identified in MEDLINE (1966-1996). Articles were included if the investigators had studied a population in whom low disease prevalence was expected; used either Papanicolaou smear screening and colposcopy or colposcopically directed biopsy as a standard against which the screening technique was measured, and included enough data for recalculation of reported sensitivities and specificities. TABULATION, INTEGRATION, AND RESULTS Receiver operating characteristic curves for fluorescence spectroscopy were calculated using a Bayesian algorithm, and ROC curves for the other screening methods were constructed using metaanalytic techniques. Areas under the ROC curves and Q points were calculated. Screening colposcopy had the highest area under the curve (0.95), followed by screening cervicography (0.90), HPV testing (0.88), cervicoscopy (0.85), fluorescence spectroscopy (0.76), and Papanicolaou smear screening (0.70). CONCLUSION In terms of screening for SILs, fluorescence spectroscopy performed better than the standard technique, Papanicolaou smear screening, and less well than screening colposcopy, cervicography, HPV testing, and cervicoscopy. The promise of this research technique warrants further investigation.

Fluorescence spectroscopy of the cervix: influence of acetic acid, cervical mucus, and vaginal medications.

Fluorescence spectroscopy has been shown to provide information useful in the detection of cervical dysplasia. The goal of this study was to determine if substances found on the cervix such as acetic acid, mucus, and vaginal medications can influence the fluorescence in the spectral region useful for discriminating normal cervical tissue from abnormal tissue.

Cervical Fluorescence of Normal Women

Cervical tissue fluorescence spectra have previously been measured in vivo in women with a recent abnormal Papanicolaou smear. Diagnostic algorithms have been developed to diagnose squamous intraepithelial lesions (SILs) based on these fluorescence emission spectra. However, algorithms have not been tested in women with no history of cervical neoplasia.

Effects of biographical variables on cervical fluorescence emission spectra.

Diagnostic algorithms can classify tissue samples as diseased or nondiseased based on fluorescence emission collected from the intact cervix. Such algorithms can distinguish high-grade squamous intraepithelial lesions from low-grade squamous intraepithelial lesions. An understanding of the effects of the values of biographical covariates, such as age, race, smoking, or menopausal status on the emission spectra for each patient could improve diagnostic efficiency. The analysis described was performed using data collected from two previously published clinical trials; one study measured spectra from 395 sites in 95 patients referred to a colposcopy clinic with abnormal Pap smears, and the second study measured spectra from 204 sites in 54 patients self-referred for screening and expected to have a normal Pap smear. For this analysis, data about age, race, menstrual cycle, and smoking were collected. The principal components from normalized data were compared. There are clear intensity differences observed with age and menopausal status; postmenopausal patients exhibit higher emission intensities. Differences associated with biographical variables need to be tested in larger studies, which stratify adequately for these variables. The addition of these biographical variables in the preprocessing of data could dramatically improve algorithm performance and applicability.

Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: Results from an animal model

Using the hamster cheek pouch carcinogenesis model, we explore which fluorescence excitation wavelengths are useful for the detection of neoplasia. 42 hamsters were treated with DMBA to induce carcinogenesis, and 20 control animals were treated only with mineral oil. Fluorescence excitation emission matrices were measured from the cheek pouches of the hamsters weekly. Results showed increased fluorescence near 350-370 nm and 410 nm excitation and decreased fluorescence near 450-470 nm excitation with neoplasia. The optimal diagnostic excitation wavelengths identified using this model - 350-370 nm excitation and 400-450 nm excitation - are similar to those identified for detection of human oral cavity neoplasia.

Safety Analysis: Relative Risks of Ultraviolet Exposure from Fluorescence Spectroscopy and Colposcopy Are Comparable*

Fluorescence spectroscopy is a promising tool for use in the diagnosis of disease in human tissue. However, few published reports have evaluated the safety of this technique, despite the fact that many spectroscopic systems use UV illumination. This study determined the relative risk associated with light exposure from spectroscopic systems compared with the traditional light sources that are used to illuminate tissue and direct biopsies. We compared spectroscopic detection systems for the cervix to the colposcope, a low‐power microscope routinely used to illuminate the cervix, which does not cause any known photochemical damage. We measured the average spectral irradiance (W/[cm2nm]) and the average tissue exposure time during a diagnostic colposcopy examination. To quantify the relative risks, we multiplied illumination spectra by several action spectra from the literature and compared the areas under the curves corresponding to each procedure. The risk associated with the average power colposcope served as our basis for comparison. We conclude that the risks of illumination using spectroscopic systems are lower than or comparable to those already encountered in routine diagnostic procedures such as colposcopy with an average power colposcope. Spectroscopic examination can be associated with a somewhat higher risk than a colposcopy with the lowest power colposcope or a shorter than average colposcopy. The analysis presented can be repeated to estimate the magnitude of risks associated with other spectroscopic diagnostic devices.

Biological basis of cervical tissue autofluorescence

The goal of this study was to characterize the biological basis of cervical autofluorescence. Fluorescence of transverse fresh tissue slices was imaged using a fluorescence microscope. Fluorescence spectra were measured from cervical epithelial cell suspensions.

In-vivo fluorescence EEM collection of normal and cancerous epithelial tissue in the hamster cheek pouch model

Fluorescence spectroscopy has been shown to be a viable technique for the diagnosis of epithelial dysplasia and neoplasia both in-vitro and in-vivo.1 Since excision and fixation change important characteristics of tissue such as vascularity and metabolic rate, it is desirable to obtain excitation-emission matrices (EEMs) in-vivo for more accurate assessment of the fluorescence properties of the tissue being probed. We have designed and constructed an instrument which allows for the collection of fluorescence emission spectra at 18 different excitation wavelengths in about 3 minutes, a time short enough to allow in-vivo probing. The system consists of a white light source which is filtered by a monochromator and delivered to the sample via an optic fiber probe. The same probe collects the fluorescence which is passed through long-pass filters into an imaging spectrograph and detected with a TE-cooled CCD camera. The entire system is under computer control.