Staining Patterns in Dry Eye Syndrome: Rose Bengal Versus Trypan Blue Rosane S. Castro (1) Lívia M. D. Freire (1), Renato Ambrosio Jr(2) 1-Ophthalmology Department UNICAMP-SP 2-Renato Ambrosio Eye Institute, Rio de Janeiro-RJ

Introduction ► Dry eye syndrome (DES) is a somewhat common disorder found in ophthalmic daily practice, it can be associated with many local and/or systemic disorders, and sometimes is a diagnostic challenge because of the lack of association between symptoms and clinical findings. ► There is no described gold standard diagnostic test that could reliably diagnose and stage the disease in every case. ► A number of different testings are commonly performed to help determining an accurate diagnosis of DES. These include subjective (as questionnaires) and objective ones, like tear breakup time (tBUT), ocular surface dye staining [fluorescein, rose bengal (RB), or lissamine green (LG)], Schirmer test, and conjunctival impression cytology.

Introduction ► Since its first report in ophthalmic practice, RB is one of the most used stains, examiner friendly, but providing a great patient discomfort. It is able to bind to epithelial cells that are uncoated by certain proteins (mainly mucin) and presents high cell toxicity. ► The Trypan Blue is reported to be used in ophthalmology since Although it is known to stain dead cells as well as cells that had been injuried, this vital dyer is not used much in the clinical practice for staining of the ocular surface. The Trypan Blue is considered to be toxic in certain manner for epithelial cells, however it is more torelable than the Rose Bengal on the patient´s eyes

Purpose ► In the present study, we evaluated and compared staining properties and patient’s tolerance for both RB and TB in DES and assessed the correlation of disease severity with staining patterns.

MATERIALS AND METHODS ► This was a prospective, randomized, comparative, crossover-fashioned study. The study protocol was approved by the State University of Campinas (UNICAMP) Review Board and followed the Declaration of Helsinki. ► Patients were randomly selected from the external eye disease ambulatory, Department of Ophthalmology of UNICAMP. ► Patients with any other ocular surface disorders, previous ocular surgery, contact lens wearers, and those who were using eyedrops other than lubricants were excluded. ► A validated questionnaire, the Ocular Surface Disease Index (OSDI, ranging from 0 to 100 points), reviewed elsewhere, was used to classify disease severity. ► Two percent fluorescein drops were used to measure tBUT. Then, both 1% RB and 1% TB drops were applied. Patients were randomly divided in 2 groups, regarding dye instillation order (group 1: RB first; group 2: TB first). The second dye was applied 1 hour apart (or after the first drop was washed out, whichever occurred last). An anesthetic drop preceded both.

MATERIALS AND METHODS ► A staining score (van Bijsterveld scale, ranging from 0 to 9) was used to evaluate the results, which were obtained by the same examiner. Staining scores for both RB and TB were compared between each other in both groups. The scores were also correlated to disease severity (measured by the OSDI score). ► Patient comfort to the colorant drop was evaluated by an objective questionnaire. After receiving the first drop, the patient was asked the question: ‘‘Did this drop feel uncomfortable?’’ and the patient answered either ‘‘yes’’ or ‘‘no.’’ When the second drop was applied, the question was asked again ► Clinical data were compared using a paired t test (for age, OSDI score, and tBUT), Fisher exact test (for patient sex, etiology, and comfort questionnaire), and Pearson r correlation test (for RB vs TB staining scores and for disease severity vs staining scores).

Van Bijsterveld Right Eye(RE) Left Eye(LE) Right Eye(RE) Left Eye(LE) ► 1 –temporal conjuctiva ► 2 - cornea ► 3 –nasal conjuctiva ► Punctuation ► 0 point ► 1 point ► 2 points ► 3 points ► Total= (1+2+3)RE+(1+2+3)LE 2

Results Group 1 Group 2 p Sex Female Male33 Etiology evaporative32 non SS Primary SS Secondary SS Age 52 ± ± OSDI 24 ± ± BUT 4 ± ± Table 1 - Patient Demographics and Clinical Features SS= Sjögren Syndrome OSDI= ocular surface desease index BUT= Breakup timeGroupEyesDye ± mean ± SD p Group 1 14TB 5.3 ± RB 6.2 ± 1.9 Group 2 14TB 6.2 ± 2.0 <0.001 RB 5.9 ± 1.9 Table 2 – Staining Scores TB – Trypan blue BR – Bengal rose

DISCUSSION ► Vital staining of the ocular surface remains a very useful, easy, and inexpensive diagnostic test in patients with dry eye. ► Among available stains, fluorescein and RB are still the most commonly used. Recent studies with RB warned for its toxicity, including decreasing the chance to recover herpes viruses in human cell cultures.It is also known to provide a great patient discomfort. ► As an alternative, TB staining has been used as a dye in cataract surgery. It seems to be a less toxic stain, providing less stinging upon instillation. They are easily observed under the slit-lamp examination, seen as blue (TB) or pink (RB) punctate or confluent staining of the ocular surface. ► Our main goal was to compare both stains in both eyes of the same patients in a crossover fashion. This criterion was used to try to eliminate any bias regarding staining properties (would the first drop interfere in the second drop score?). Besides this, patients were examined in only 1 visit, so that any alteration in patients’ ocular surface would not compromise the results in a second visit.

DISCUSSION ► As reported in other studies, we found a poor correlation between patient symptoms (assessed by the OSDI score) and signs (measured by the staining scores). One observation to be made is that currently there are no validated OSDI translations to Portuguese language, although Allergan, Inc, (Irvine, CA) provides it in a translated form in Brazil. Also, all patients included in this study were receiving treatment (with artificial teardrops) for their disease, which could possibly alter the results (both for the questionnaire and staining scores). ► In conclusion, both TB and RB showed similar staining patterns. RB was found to provide greater patient discomfort. There was no correlation between disease severity (addressed by the ocular surface disease index questionnaire) and staining patterns (measured by the van Bijsterveld scale).