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.