We prospectively enrolled 243 consecutive diabetic patients at the Institute of endocrinology and metabolism between August 2011 and November 2012.
Exclusion criteria include use of medications or history of any other ocular or systemic disease that can affect tear production or quality, history of anterior segment surgery, Keratorefractive procedures (LASIK, LASEK, PRK) within one year prior to enrollment, trauma, contact lens wear, incomplete lid closure, entropion, ectropion, nasolachrymal drainage obstruction, punctual plugs placement, or cauterization; ocular allergy, glaucoma, pregnancy or lactation, and use of ocular medications or nutritional tear supplements.
All of the participants underwent a general physical examination and a thorough ophthalmologic exam. The visual acuity of both eyes was tested using Snellen’s chart. Both eyes were examined first using the board beam of the slit lamp to know the condition of the ocular surface and adenexa, observing the tear film meniscus, tear film, corneal changes, conjunctival changes, and eyelids. They were also clinically evaluated with direct and indirect ophthalmoscopy to know the status of retina. The study population underwent tear osmolarity test (standard test) before the Ocular Surface Disease Index (OSDI) questionnaire, Schirmer I test, Rose Bengal and fluorescein staining, and tear film break-up time test (TFBUT) (index tests); all were performed on the same day. All of the tests were performed according to the randomization table for one eye.
OSDI questionnaire was administrated to the participants by a research associate trained by an expert ophthalmologist to score the questions and to follow the ambiguous information. Schirmer I test, Rose Bengal, fluorescein staining, TFBUT were performed for all of the participants by an expert ophthalmologist in consecutive sessions. Tear osmolarity test was done by a single specialist. The investigators were blinded to the patients’ history and the obtained information.
To avoid diagnostic error, all of the examinations were performed in the same physical condition and in the morning to standardize the tests and to avoid possible diurnal variation. Assessments were made in a room controlled for enlightment (dim light), temperature, humidity, and airflow, to avoid ocular surface stress. The tests that need slit lamp were performed in a darkened room with the same slit lamp and by the same physician.
Tear osmolarity was measured using tear lab osmolarity system, (BON Co. Germany). The Tear lab instrument is based on a lab- on – a- chip technology working as both a collection device and an analytical system, in absence of any chemical reagent. This avoids the need for a capillary tube or absorbing acetate disc. The equipment consists of single use test cards containing microchannels to collect tear fluid, held by a pen designed to facilitate tear collection, and a portable reader unit which elaborates and displays the osmolarity results. A tear sample, approximately 50 nl, was collected from the inferior lateral tear meniscus of the ocular surface. To facilitate tear collection, patients were asked to position their head laterally for a few seconds before approaching the tip of the test card microchip: in this way, tears were driven laterally and collection made easier. Subjects had been requested not to wear makeup on their eyelids. Quality control procedures were applied at the beginning of each day of patient testing by using reusable electronic check cards (provided by the manufacture as a procedural quality control) to confirm the function and calibration of the TearLab osmolarity system. The 308 mOsm/L cutoff was used to diagnose DED [20]. At this diagnostic cutoff, osmolarity was found to have 88% specificity, 75% sensitivity in mild/moderate disease and 95% sensitivity in severe disease [20].The very rapid acquisition of tear samples by the TearLab would be less likely to be influenced by evaporation.
The OSDI questionnaire has a Likert design and assesses frequency of ocular subjective symptoms (soreness, blurred vision), difficulty with vision-related function (television, visual display unit, driving, reading) and discomfort due to environmental triggers (low humidity, high wind). The patients answer 12 questions, with higher scores representing greater disability [21].
Subjective symptoms of dry eye were graded on the basis of dry eye discomfort symptoms questionnaire (OSDI) [9]. The score range was from 0–12 (no disability), 13–22 (light dry eye), to 23–32 (moderate dry eye), and 33–100 (sever dry eye) [9].
Schirmer I test and TFBUT were carried out as outlined in the DEWS report [4].
The Schirmer test is an invasive and indirect method to measure change in volume of the tears in the tear reservoir. This test involves insertion of a wick into the lower conjunctival sac and measurement of the wetting length over a set period of time. The Schirmer test uses filter papers to assess tear production. There are two commonly used variations of the Schirmer test: Schirmer I measures total tear secretion (reflex and basal tears) and Schirmer II utilizes anesthetic to measure basal secretions, although this has not been validated [22-24].
The strip was folded at the notch and placed at the junction of the middle and lateral thirds of the lower eyelids and allowed there to stay in place for 5 minutes [25].
A value of less than 5 mm wetting in 5 minutes is considered abnormal, more than 10 mm per 5′ seconds as normal and 6-10 mm per 5′seconds as borderline [4]. Medial and lateral placements of the paper have been described, as well as having the patient looking up, but no method has been deemed more reliable [26].
TFBUT is the standard test for estimating tear film stability. The results are explained as seconds. Patients with break-up time of more than ten seconds was consider as normal, those with less than ten seconds was labeled as unstable tear film, 6 to 10 seconds as moderate dry eye disease, and ≤5 sec as sever dry eye disease [9].
The TFBUT was performed by applying a fluorescein strip after moistening it with a drop of sterile saline, to the lower tarsal conjunctiva without the use of topical anesthesia. The time lapse between the last blink to the appearance of the first random dry spot was taken as the tear film break up time [25].
Epithelial damage to the exposed surface of the eye can be demonstrated with vital and supra-vital stains. Fluorescein and Rose Bengal staining are the standard but invasive methods used to demonstrate ocular surface damage. This technique reveals surface damage on both the cornea and conjunctiva [27].
The eye is best viewed with anexciter (Wratten 47/47a; Edmund Optics, Barrington, NJ, USA) and barrier filter (Wratten 12/15) to assess staining on the cornea and conjunctiva.
Evaluation of staining is highly subjective, but the use of charts such as the Oxford grading scheme can help by enabling consistent recording of staining severity and is used to estimate surface damage in dry eye. The scheme has five panels, labeled A--E, with staining represented by punctuate dots that increase logarithmically between the panels [28]. The clinician compares the appearance of staining on the exposed interpalpebral conjunctiva and cornea with each panel and the closest match determines the grade. On a specially designed form, a grade between 0 and 3was given for staining on the cornea, based on the number of “dots” seen, which was to be added to a grade between 0 and 3 for the nasal and the same for the temporal conjunctiva. This gave a maximum possible total of 9 points. Three additional points were then allocated for fluorescein only if there was confluent staining (+1), staining in the papillary area (+1), or one or more filaments (+1), giving a maximum possible score of 12. This was performed for each eye. An abnormal score was considered to be 3 and above [21].
The study was approved by the local ethical committee of Tehran University of Medical Sciences and conducted in accordance with the ethical principles of the Declaration of Helsinki and all of the participants signed the written informed consent.
Statistical analysis
Statistical analysis was performed using Statistical Package for Social Sciences (SPSS, SPSS Inc., Chicago, IL, USA) version 18.0. Descriptive statistics were summarized as mean ± SD, or median and interquartile. Kolmogorov Smirnov was performed to assess normality for continuous variables.
An evaluation was made of the linear relationship between the tear osmolarity values and TFBUT, OSDI score, Schirmer I test and Rose Bengal and fluorescein staining. This was performed by using Spearman’s correlation coefficient (rho). Student’s t test and Mann–Whitney U test was applied for comparisons between two groups (significance p < 0.05).
Chi square test was used to compare discrete variables. Results were given with their 95% CIs.
Diagnostic accuracy tests was performed to analyze sensitivity, specificity, receiver operating characteristic (ROC) curves, positive likelihood ratio (LR+), and positive predictive values (PPV).