Staining Patterns in Dry Eye Staining Patterns in Dry Eye Syndrome: Rose Bengal Versus Syndrome: Rose Bengal Versus

Trypan BlueTrypan Blue

Rosane S. Castro (1) Lívia M. D. Freire (1), Renato Rosane S. Castro (1) Lívia M. D. Freire (1), Renato Ambrosio Jr(2)Ambrosio Jr(2)

1-Ophthalmology Department UNICAMP-SP1-Ophthalmology Department UNICAMP-SP2-Renato Ambrosio Eye Institute, Rio de Janeiro-RJ2-Renato Ambrosio Eye Institute, Rio de Janeiro-RJ rosane@med-odonto.com.brrosane@med-odonto.com.br

IntroductionIntroduction

► Dry eye syndrome (DES) is a somewhat common disorder found in Dry eye syndrome (DES) is a somewhat common disorder found in ophthalmic daily practice, it can be associated with many local and/or ophthalmic daily practice, it can be associated with many local and/or systemic disorders, and sometimes is a diagnostic challenge because of the systemic disorders, and sometimes is a diagnostic challenge because of the lack of association between symptoms and clinical findings. lack of association between symptoms and clinical findings.

► There is no described gold standard diagnostic test that could reliably There is no described gold standard diagnostic test that could reliably diagnose and stage the disease in every case.diagnose and stage the disease in every case.

► A number of different testings are commonly performed to help determining A number of different testings are commonly performed to help determining an accurate diagnosis of DES. These include subjective (as questionnaires) an accurate diagnosis of DES. These include subjective (as questionnaires) and objective ones, like tear breakup time (tBUT), ocular surface dye staining and objective ones, like tear breakup time (tBUT), ocular surface dye staining [fluorescein, rose bengal (RB), or lissamine green (LG)], Schirmer test, and [fluorescein, rose bengal (RB), or lissamine green (LG)], Schirmer test, and

conjunctival impression cytology.conjunctival impression cytology.

IntroductionIntroduction

► Since its first report in ophthalmic practice, RB is one of the Since its first report in ophthalmic practice, RB is one of the most used stains, examiner friendly, but providing a great patient most used stains, examiner friendly, but providing a great patient discomfort. It is able to bind to epithelial cells that are uncoated discomfort. It is able to bind to epithelial cells that are uncoated by certain proteins (mainly mucin) and presents high cell by certain proteins (mainly mucin) and presents high cell toxicity.toxicity.

► The Trypan Blue is reported to be used in ophthalmology since The Trypan Blue is reported to be used in ophthalmology since 1967. Although it is known to stain dead cells as well as cells 1967. 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 that had been injuried, this vital dyer is not used much in the clinical practice for staining of the ocular surface. The Trypan clinical practice for staining of the ocular surface. The Trypan Blue is considered to be toxic in certain manner for epithelial Blue is considered to be toxic in certain manner for epithelial cells, however it is more torelable than the Rose Bengal on the cells, however it is more torelable than the Rose Bengal on the

patient´s eyespatient´s eyes

PurposePurpose

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

MATERIALS AND METHODSMATERIALS AND METHODS

► This was a prospective, randomized, comparative, crossover-fashioned study. This was a prospective, randomized, comparative, crossover-fashioned study. The study protocol was approved by the State University of Campinas The study protocol was approved by the State University of Campinas (UNICAMP) Review Board and followed the Declaration of Helsinki.(UNICAMP) Review Board and followed the Declaration of Helsinki.

► Patients were randomly selected from the external eye disease ambulatory, Patients were randomly selected from the external eye disease ambulatory, Department of Ophthalmology of UNICAMP. Department of Ophthalmology of UNICAMP.

► Patients with any other ocular surface disorders, previous ocular surgery, Patients with any other ocular surface disorders, previous ocular surgery, contact lens wearers, and those who were using eyedrops other than lubricants contact lens wearers, and those who were using eyedrops other than lubricants were excluded.were excluded.

► A validated questionnaire, the Ocular Surface Disease Index (OSDI, ranging A validated questionnaire, the Ocular Surface Disease Index (OSDI, ranging from 0 to 100 points), reviewed elsewhere, was used to classify disease from 0 to 100 points), reviewed elsewhere, was used to classify disease severity. severity.

► Two percent fluorescein drops were used to measure tBUT. Then, both 1% 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 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). 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 The second dye was applied 1 hour apart (or after the first drop was washed out, whichever occurred last). An anesthetic drop preceded both.out, whichever occurred last). An anesthetic drop preceded both.

MATERIALS AND METHODSMATERIALS AND METHODS

► A staining score (van Bijsterveld scale, ranging from 0 to 9) was A staining score (van Bijsterveld scale, ranging from 0 to 9) was used to evaluate the results, which were obtained by the same used to evaluate the results, which were obtained by the same examiner. Staining scores for both RB and TB were compared examiner. Staining scores for both RB and TB were compared between each other in both groups. The scores were also between each other in both groups. The scores were also correlated to disease severity (measured by the OSDI score).correlated to disease severity (measured by the OSDI score).

► Patient comfort to the colorant drop was evaluated by an Patient comfort to the colorant drop was evaluated by an objective questionnaire. After receiving the first drop, the patient objective questionnaire. After receiving the first drop, the patient was asked the question: ‘‘Did this drop feel uncomfortable?’’ was asked the question: ‘‘Did this drop feel uncomfortable?’’ and the patient answered either ‘‘yes’’ or ‘‘no.’’ When the and the patient answered either ‘‘yes’’ or ‘‘no.’’ When the second drop was applied, the question was asked againsecond drop was applied, the question was asked again

► Clinical data were compared using a paired t test (for age, OSDI Clinical data were compared using a paired t test (for age, OSDI score, and tBUT), Fisher exact test (for patient sex, etiology, and score, and tBUT), Fisher exact test (for patient sex, etiology, and comfort questionnaire), and Pearson r correlation test (for RB vs comfort questionnaire), and Pearson r correlation test (for RB vs TB staining scores and for disease severity vs staining scores).TB staining scores and for disease severity vs staining scores).

Van BijsterveldVan Bijsterveld

Right Eye(RE) Left Eye(LE)Right Eye(RE) Left Eye(LE)

1 2 3 3 2 1 1 2 3 3 2 1 ► 1 –temporal conjuctiva1 –temporal conjuctiva► 2 - cornea2 - cornea► 3 –nasal conjuctiva3 –nasal conjuctiva► PunctuationPunctuation► 0 point 0 point ► 1 point1 point► 2 points2 points► 3 points3 points► Total= Total= (1+2+3)RE+(1+2+3)LE(1+2+3)RE+(1+2+3)LE 22

ResultsResultsGroup 1Group 1 Group 2Group 2 pp

SexSex

FemaleFemale 1111 1111 ----

MaleMale 33 33 ----

EtiologyEtiology

evaporativeevaporative 33 22 ----

non SSnon SS 11 33 ----

Primary SS Primary SS 33 00 ----

Secondary SS Secondary SS 77 99 ----

AgeAge 52 ± 1452 ± 14 56 ± 6.856 ± 6.8 0.390.39

OSDIOSDI 24 ± 9.724 ± 9.7 20 ± 10.620 ± 10.6 0.370.37

BUTBUT 4 ± 1.84 ± 1.8 3.7 ± 1.73.7 ± 1.7 0.750.75

Table 1 - Patient Demographics and Clinical

Features

SS= Sjögren Syndrome

OSDI= ocular surface desease index

BUT= Breakup time

GroupGroup EyesEyes DyeDye mean ± ± SD

pp

Group 1Group 1 1414 TBTB 5.3 ± 5.3 ± 1.61.6

0.0090.009

RBRB 6.2 ± 6.2 ± 1.91.9

Group 2Group 2 1414 TBTB 6.2 ± 6.2 ± 2.02.0

<0.00<0.0011

RBRB 5.9 ± 5.9 ± 1.91.9

Table 2 – Staining Scores

TB – Trypan blue

BR – Bengal rose

DISCUSSIONDISCUSSION► Vital staining of the ocular surface remains a very useful, easy, and Vital staining of the ocular surface remains a very useful, easy, and

inexpensive diagnostic test in patients with dry eye.inexpensive diagnostic test in patients with dry eye.► Among available stains, fluorescein and RB are still the most commonly used. Among available stains, fluorescein and RB are still the most commonly used.

Recent studies with RB warned for its toxicity, including decreasing the 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 chance to recover herpes viruses in human cell cultures.It is also known to provide a great patient discomfort.provide a great patient discomfort.

► As an alternative, TB staining has been used as a dye in cataract surgery. It 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 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 are easily observed under the slit-lamp examination, seen as blue (TB) or pink (RB) punctate or confluent staining of the ocular surface.(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 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 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 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 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 alteration in patients’ ocular surface would not compromise the results in a second visit. second visit.

DISCUSSIONDISCUSSION

► As reported in other studies, we found a poor correlation between As reported in other studies, we found a poor correlation between patient symptoms (assessed by the OSDI score) and signs patient symptoms (assessed by the OSDI score) and signs (measured by the staining scores). One observation to be made is (measured by the staining scores). One observation to be made is that currently there are no validated OSDI translations to that currently there are no validated OSDI translations to Portuguese language, although Allergan, Inc, (Irvine, CA) Portuguese language, although Allergan, Inc, (Irvine, CA) provides it in a translated form in Brazil. Also, all patients provides it in a translated form in Brazil. Also, all patients included in this study were receiving treatment (with artificial included in this study were receiving treatment (with artificial teardrops) for their disease, which could possibly alter the results teardrops) for their disease, which could possibly alter the results (both for the questionnaire and staining scores). (both for the questionnaire and staining scores).

► In conclusion, both TB and RB showed similar staining patterns. In conclusion, both TB and RB showed similar staining patterns. RB was found to provide greater patient discomfort. There was RB was found to provide greater patient discomfort. There was no correlation between disease severity (addressed by the ocular no correlation between disease severity (addressed by the ocular surface disease index questionnaire) and staining patterns surface disease index questionnaire) and staining patterns (measured by the van Bijsterveld scale).(measured by the van Bijsterveld scale).