Diagnostic imaging of cervical intraepithelial neoplasia based on hematoxylin and eosin fluorescence

Abstract

Background: Pathological classification of cervical intraepithelial neoplasia (CIN) is problematic as it relies on subjective criteria. We developed an imaging method that uses spectroscopy to assess the fluorescent intensity of cervical biopsies derived directly from hematoxylin and eosin (H&E) stained tissues.

Methods: Archived H&E slides were identified containing normal cervical tissue, CIN I, and CIN III cases, from a Community Hospital and an Academic Medical Center. Cases were obtained by consensus review of at least 2 senior pathologists. Images from H&E slides were captured first with bright field illumination and then with fluorescent illumination. We used a Zeiss Axio Observer Z1 microscope and an AxioVision 4.6.3-AP1 camera at excitation wavelength of 450-490 nm with emission captured at 515-565 nm. The 32-bit grayscale fluorescence images were used for image analysis.

Results: We reviewed 108 slides: 46 normal, 33 CIN I and 29 CIN III. Fluorescent intensity increased progressively in normal epithelial tissue as cells matured and advanced from the basal to superficial regions of the epithelium. In CIN I cases this change was less prominent as compared to normal. In high grade CIN lesions, there was a slight or no increase in fluorescent intensity. All groups examined were statistically different.

Conclusion: Presently, there are no markers to help in classification of CIN I-III lesions. Our imaging method may complement standard H&E pathological review and provide objective criteria to support the CIN diagnosis.

Fig. 1

Fluorescent representation of normal, CIN I and CIN III cervical tissue specimens. a, d and g represent H&E stained cervical tissue. b, e and h represent inherent fluorescence of H&E sections at an excitation wavelength of 488 nm. c, f and i represent a color-map image in which fluorescent intensity is represented as shades (blue > green > yellow > red; high to low intensity). a, b, c = normal cervical tissue. d, e, f = CIN I cervical tissue. g, h, i = CIN III cervical tissue. Notice the lack of intensity in epithelium as you move from normal (c) to CIN I (f) to CIN III (i)

Fig. 2

Quantifying average fluorescent values for normal, CIN I and CIN III cervical tissue. a Cervical epithelium divided into equidistant segments: segment 1 (bottom third), segment 2 (middle), and segment 3 (upper third). b Plotted fluorescent intensity profile of a line drawn from segment 1 to segment 3 on a normal sample. Segments are derived empirically by dividing the line distance into three parts. c Average fluorescent intensity for each segment derived from Normal, CIN I, and CIN III. Normal tissue is significantly higher in fluorescence intensity in all three segments when compared to CIN I and CIN III (p < 0.05). No significant differences were evident between CIN I and CIN III. Results are generated by averaging greater than 20 segmented lines for each sample. Total samples in each histological category are Normal = 13, CIN I = 18, CIN III = 12

Fig. 3

Linear regression analysis of segmental fluorescence intensity. a Cervical epithelium divided into equidistant segments: segment 1 (bottom third), segment 2 (middle), and segment 3 (upper third). b Fluorescence intensity profile of the line plotted in (a). Bisecting lines represent the 89th percentile of each segment. c The highest fluorescence intensity values (top 10 %) of each segment from (b) are averaged, plotted in (c) and analyzed with linear regression. Total samples in each histological category are Normal = 13, CIN I = 18, CIN III = 12

Fig. 4

Normalizing linear regression to lower segment. a and b Fluorescent intensity averages in lower segments are set to 1 and all other data points are normalized to this value. Statistical analysis of the linear regression performed on normalized slopes indicates all histological types (N, CIN I, CIN III) are significantly different from each other (p < 0.05). Total samples in each histological category are Normal = 13, CIN I = 18, CIN III = 12. c Pseudo-colored intensity of fluorescent images from normal, CIN I, and CIN III epithelium that closely represent the average fluorescent intensity signature for each histological type

Fig. 5

Validation of unique signatures. a An analysis of normal, CIN I and CIN III samples (N = 34, CIN III = 16, CIN III = 18) collected from a different institution (Weill Cornell Medical College) indicated that at a threshold of 10 % the slopes for normal and CIN I samples are not significantly different from the samples analyzed from SIUH. b When all samples from both institutions are combined, slopes from each histological type are significantly different from each other indicating a unique signature for normal, CIN I, and CIN III