Premature Corneal Fluorescein Stain Grading May Underestimate Dry Eye Disease Severity

Corneal ulcer
Corneal ulcer (green, just below pupil) on the eye of an 67-year-old woman with microbial keratitis. The yellow and green staining is caused by a fluorescein dye, which allows the ulcer to be seen. A corneal ulcer is an infected break in the surface of the eye that fails to heal and becomes inflamed. In this case the ulcer was caused by microbial keratitis, an infection of the cornea (the transparent membrane covering the front of the eye). Treatment depends on the infectious agent present; for example, bacterial ulcers are treated with antibiotics.
Lissamine green maximum grade of staining did not exhibit any significant differences in time between different dry eye disease subtypes.

Reaching the maximum grade of staining (Gmax) following corneal fluorescein staining varies between patients with non-Sjogren’s dry eye disease (nSS DED) and those with Sjogren’s dry eye disease (SS DED), and clinicians who assess eyes prior to them reaching Gmax may underestimate corneal staining severity, according to a study published in Ocular Surface. Lissamine green staining Gmax, however, was shorter and did not vary significantly between patients with nSS DED and SS DED.

Researchers assessed photographs of 100 study participants with nSS DED (n=68, mean age 61±13, 81% women) or SS DED (n=32, mean age 59±14 years, 88% women). They performed  both corneal fluorescein staining and conjunctival lissamine green staining on participants and took photographs every 30 seconds for at least 5 minutes. A team of 2 clinicians randomly ordered and graded photographs of 1 eye on a scale from 0 to 5 (severe staining) and calculated the average time to reach Gmax. 

Investigators found that the median time to corneal fluorescein Gmax was 2.6 minutes for patients with nSS DED and 3.8 minutes for patients with SS DED (P =.018). The median time to lissamine green staining Gmax for the nasal and temporal conjunctiva was 0.5 minutes for both individuals with nSS DED and SS DED. The SS DED group had significantly higher grades of corneal and conjunctival staining compared with the nSS DED group. 

Researchers state that ocular staining has been used clinically for over 100 years and an optimal time for observation and grading has still not been established, according to the report. They suggest that corneal fluorescein staining Gmax should be an important consideration in clinical care and clinical trials and caution that “early grading of corneal fluorescein staining may lead to under-grading, which could greatly impact the assessment of DED severity and treatment efficacy.”

Study limitations include possible differences in illumination between sites which may have affected the grade of staining, failure to use a red filter for a better view of lissamine green staining, and use of a large volume (5μl) of 2% fluorescein which may have caused concentration quenching and increased time to Gmax in those with low tear volume.


Begley CG, Caffery B, Nelson JD, Situ P. The effect of time on grading corneal fluorescein and conjunctival lissamine green staining. Ocul Surf. 2022;25:65-70. doi:10.1016/j.jtos.2022.05.003