Multiphoton microscopy in the evaluation of human bladder biopsies

Abstract

Context: Multiphoton microscopy (MPM) is a nonlinear imaging approach, providing cellular and subcellular details from fresh (unprocessed) tissue by exciting intrinsic tissue emissions. With miniaturization and substantially decreased cost on the horizon, MPM is an emerging imaging technique with many potential clinical applications.

Objectives: To assess the imaging ability and diagnostic accuracy of MPM for human bladder biopsies.

Design: Seventy-seven fresh bladder biopsies were imaged by MPM and subsequently submitted for routine surgical pathology diagnosis. Twelve cases were excluded because of extensive cautery artifact that prohibited definitive diagnosis. Comparison was made between MPM imaging and gold standard sections for each specimen stained with hematoxylin-eosin.

Results: In 57 of 65 cases (88%), accurate MPM diagnoses (benign or neoplastic) were given based on the architecture and/or the cytologic grade. The sensitivity and specificity of MPM in our study were 90.4% and 76.9%, respectively. A positive (neoplastic) diagnosis on MPM had a high predictive value (94%), and negative (benign) diagnoses were sustained on histopathology in two-thirds of cases. Architecture (papillary versus flat) was correctly determined in 56 of 65 cases (86%), and cytologic grade (benign/low grade versus high grade) was assigned correctly in 38 of 56 cases (68%).

Conclusions: The MPM images alone provided sufficient detail to classify most lesions as either benign or neoplastic using the same basic diagnostic criteria as histopathology (architecture and cytologic grade). Future developments in MPM technology may provide urologists and pathologists with additional screening and diagnostic tools for early detection of bladder cancer. Additional applications of such emerging technologies warrant exploration.

Fig 1. A 2 × 2 contingency table comparing multiphoton microscopy diagnosis with the final histopathologic diagnosis

Abbreviations: PPV - positive predictive value; NPV - negative predictive value

Fig 2. Normal structures of bladder wall and flat intraurothelial lesions without atypia

Multiphoton microscopy (MPM) image (A) and corresponding H&E image (B) at low magnification showing clear demarcation between normal urothelium (a) and lamina propria (b). Lamina propria is composed of collagen bundles (red) and elastin fibers (green). MPM image (C) and corresponding H&E image (D) at high magnification showing multi-layered urothelium and superficial umbrella cells. Umbrella cells in each image are boxed and shown at higher digital zoom in the inset. MPM image (E) and corresponding H&E image (F) at low magnification showing von Brunn nests, some with cystic dilatation (cystitis cystica; arrow). (MPM magnifications: A=120X, C=240X, E=306X;H&E magnifications: B=40X, D=200X, F=200X).

Fig 3. Carcinoma in situ (CIS) and invasive urothelial carcinoma

Multiphoton microscopy (MPM) image (A) and the corresponding H&E image (B) at low magnification showing flat urothelium with loss of polarity (a) and underlying lamina propria (b). MPM image (C) and corresponding H&E image (D) at high magnification showing high grade cytologic features [marked pleomorphism and increased Nuclear:Cytoplasmic (N:C) ratio; arrow]. MPM image (E) and corresponding H&E image (F) at high magnification showing nests of invasive tumor cell (a; color-coded green) in collagen fibers of lamina propria (b; color-coded red). (MPM magnifications: A=48X, C=293X, E=480X; H&E magnifications: B=40X, D=200X, F=400X)

Fig 4. Papillary urothelial carcinoma, low grade, non-invasive

Multiphoton microscopy (MPM) image (A) and corresponding H&E image (B) at low magnification showing the papillary nature of the lesion (complex papillae with thin fibrovascular cores; arrow). MPM image (C) and corresponding H&E image (D) at high magnification showing thin fibrovascular cores (arrow). MPM image (E) and corresponding H&E image (F) at high magnification showing thickened urothelium lined by cells with minimal pleomorphism and relatively low Nuclear:Cytoplasmic (N:C) ratio. MPM magnifications: A=48X, C=240X, E=480X; H&E magnifications: B=40X, D=200X, F=400X)

Fig 5. Papillary urothelial carcinoma, high grade, non-invasive

Multiphoton microscopy (MPM) image (A) and corresponding H&E image (B) at low magnification showing the papillary nature of the lesion (complex, thickened papillae with thin fibrovascular cores; arrow). MPM image (C) and corresponding H&E image (D) at high magnification showing thickened fibrovascular cores and disorderly arrangement of cells. MPM image (E) and corresponding H&E image (F) at high magnification showing cells with marked pleomorphism, high Nuclear:Cytoplasmic (N:C) ratio, and hyalinized fibrovascular cores. MPM magnifications: A = 48X, C = 240X, E = 480X; H&E magnifications: B=40X, D=200X, F=400X).