Optimizing the methodology for the assessment of bulbar conjunctival lissamine green staining

SIGNIFICANCE: When using lissamine green for bulbar conjunctival evaluation, the most staining was observed with two applications of the same strip in the same eye, wetted with a drop of saline. The first application was 5 seconds after wetting the strip, and the second was 1 minute later, followed by immediate viewing. This suggests that bulbar staining should be viewed before the lid wiper region. PURPOSE: This study aimed to optimize the assessment of lissamine green staining of the bulbar conjunctiva by investigating the application technique and subsequent observation period. METHODS: Twenty-two participants with dry eye (as defined by the Tear Film and Ocular Surface Society Dry Eye Workshop II) were recruited to trial five application methods in randomized order: (1) application immediately after wetting a single strip, (2) application 5 seconds after wetting a single strip, (3) application 10 seconds after wetting a single strip, (4) consecutive single application of sodium fluorescein followed by lissamine green 5 seconds after wetting, and (5) two applications using the same lissamine green strip 5 seconds after wetting, 1 minute apart. Slit-lamp photography of the conjunctiva was performed immediately following application and at 30, 60, 90, and 300 seconds after application. Three experienced (masked) practitioners independently quantified the visible punctate spots and graded the staining intensity within the images in a random order. RESULTSValues for punctate spot count (F = 6.29, p<0.0001) and lissamine green staining intensity (F = 6.29, p<0.0001) varied significantly between the different application methods. Using two applications of the same lissamine green strip, 1 minute apart, in the same eye resulted in the greatest values for both punctate spot count and lissamine green staining intensity. Lissamine green staining decreased with time for both spot count (F = 18.87, p<0.0001) and lissamine green staining intensity (F = 11.33, p<0.0001), with the most staining found immediately after application followed by a gradual decline. There was no evidence of any interaction effect between time and application method for either measure (p>0.05 for both). CONCLUSIONS: The optimal approach for assessing bulbar conjunctival lissamine green staining involves two applications of a whole infused drop resting on the same strip for 5 seconds, applied 1 minute apart. The ocular surface should be viewed immediately after application to maximize the conjunctival staining observed.