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Updated: Jan 1, 2026
In Vivo Confocal Microscopy in the Diagnosis and Management of Dry Eye: A Focus on Imaging Protocols and Interpretation
Published on: November 11, 2025
Assessment of Corneal Fluorescein Staining in Different Dry Eye Subtypes Using Digital Image Analysis
Marc Pellegrini1, Federic Bernabei1, Fabian Moscardelli1
1Ophthalmology Unit, S.Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy.
Purpose:
To describe a new objective technique of digital image analysis for the quantification and the morphological characterization of corneal staining in the setting of dry eye disease (DED), and to apply it to distinguish Sjögren syndrome (SS) from ocular graft versus-host disease (oGVHD).
Methods:
Slit-lamp photographs of corneal staining obtained from 40 patients with DED (20 with SS and 20 with oGVHD; mean age 60.7 ± 12.3 years) were evaluated. Images were subjectively graded using Oxford and National Eye Institute (NEI) scales, the staining pattern was classified as micropunctate, macropunctate, coalescent, or patch. The corneal staining index (CSI) was calculated automatically using the software ImageJ 1.51s. Particles analysis was used to calculate mean area, circularity, and roundness of staining spots.
Results:
CSI was significantly correlated with Oxford and NEI scales (respectively Rs = 0.823 and Rs = 0.773; both P < 0.001), and showed a good interobserver reliability (intraclass correlation coefficient [ICC] = 0.988 [95% confidence interval [CI]: 0.978-0.994]). The mean area of staining spots calculated with particles analysis was significantly correlated with the subjective classification of the staining pattern (Rs = 0.550, P < 0.001). The circularity and roundness of staining spots were significantly higher in oGVHD patients compared with SS (respectively, 0.51 ± 0.11 vs. 0.44 ± 0.10, P = 0.040; 0.61 ± 0.03 vs. 0.59 ± 0.02, P = 0.004). Sensitivity and specificity to distinguish oGVHD from SS were respectively 65.0% and 60% for circularity and 80.0% and 70.0% for roundness.
Conclusions:
The new algorithm showed good reliability and was well correlated with the traditional subjective grading scales. Particles analysis for the objective assessment of the staining pattern may help to differentiate patients with oGVHD from those with SS.
Translational Relevance:
The digital image analysis technique may be a reliable alternative to evaluate corneal staining objectively in the clinic and in clinical trials.
