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.

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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.

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.

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.