Quantitative Stain-Free Conjunctival Collagen Imaging in Cicatrizing Conjunctivitis Using Second Harmonic Generation-Two Photon Excitation Technology.

Purpose: We aim to study the structure of collagen in cicatrizing conjunctivitis (CC), a disease with significant morbidity, to find sensitive and quantitative measures of severity of scarring as current progression of conjunctival scarring is reliant only on clinical assessment. Methods: We used two-photon excitation and second harmonic generation to scan conjunctival tissues from patients with CC from Stevens-Johnson syndrome and its relation to disease severity by correlation with a validated clinical severity assessment tool. Collagen morphometry in region of interest was analyzed with FibroIndex software (HistoIndex Pte Ltd.) in conjunctival biopsies. Results: Eighteen patients (seven CC and 11 controls) were included. Mean age was 60.7 ± 14.4 years old, with no difference between groups (P = 0.89) Compared to controls, diseased group has significantly smaller collagen area ratio (CAR) (P < 0.01), collagen fiber number (CFN)/mm2 (P < 0.01) and larger collagen fiber density (P = 0.03) In diseased groups, CAR correlated with inflammation (R = -0.55, P = 0.011), scarring (R = 0.61, P = 0.0034), morbidity (R = 0.46, P = 0.035) and overall composite score (R = 0.52, P = 0.017). In all groups, CFN/mm2 negatively correlated with inflammation (R = -0.50, P < 0.01), scarring (R = -0.25, P = 0.07) morbidity (R = -0.37, P < 0.01), and overall composite score (R = -0.33, P = 0.02). Conclusions: We have further characterized the defining features in CC. CAR has significant correlation to all scores in diseased groups and, hence, may be a reliable marker for diagnosis. CFN/mm2, as the only parameter with significant negative correlation with all scores, can potentially be a predictor for severity.