This prospective study was performed at the outpatient clinic of the Second Affiliated Hospital of Zhejiang University School of Medicine in September of 2019. All subjects were divided as DE groups and non-dry eye (ND) groups. The criteria of DE diagnosis was OSDI higher than 13 points and fBUT shorter than 10 seconds and non-dry eye diagnosis was OSDI was not higher than 13 points or fBUT were not shorter than 10 seconds. The exclusion criteria were subjects who were under 18 years old and pregnancy, with eye allergies, conjunctival inflammation, corneal ulcer, eyelid inflammation, palsy, valgus and other ocular lesions, history of wearing contact lenses within 30 days, history of intraocular operation within 6 months, severe blepharitis, or severe systemic disease. This study followed the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine(NO. 2019-307). All individuals who agreed to enter this study provided written informed consent after explanation of the nature and possible consequences of the study.
2. Dry eye symptom questionnaires
2.1. The ocular surface disease index(OSDI)
The ocular surface disease index (OSDI) was a validated dry eye questionnaire that can measure the severity of DE, symptoms, functional problems, and environmental triggers queried for the past week 15. Each answer of OSDI in 12 questions was graded on a scale of 0-4. A total OSDI scores ranged from 0-100.
2.2. The standard patient evaluation of eye dryness (SPEED) questionnaire
The standard patient evaluation of eye dryness (SPEED) questionnaire was administered to grade the level of dry eye symptomology 16. The assessment standard of the SPEED questionnaire is derived by summing the scores from the frequency and severity parts of the questionnaire of 3 months. The value for each part of the frequency and severity questions in the SPEED questionnaire were obtained by summing the 0–4 scores of each of the 8 items and the total SPEED scores ranged from 0–28.
3. Dry eye symptom examinations
3.1. Schirmer test I
Schirmer test (ST) was performed once without topical anesthesia. In each test, both eyes were tested at the same time. The filter paper (Showa Yakuhin Kako Co, Ltd, Tokyo, Japan) was folded and placed between the lower eyelid and the globe at the junction between the middle and lateral thirds of the eyelid. The participants were asked to close their eyes, as since ST with close eyes may be more reliable in DE 17. The test lasted 5 minutes and the length of wetted paper was directly read off the scale on the paper. Wetting was measured in eyes at 1, 2, 3, 4, and 5 minutes. The participants were excluded from the study if any of the scores exceeded 30 mm (the whole paper was wetted) because of inability to give an exact measurement of the amount of wetting. Results were interpreted as follows: ≤5 mm as severe dry eye, ≤10 mm as borderline dry eye and >10 mm as normal tear secretion 18. We observed multiple indicators of Schirmer test as following:
1.ST1: the value of normal Schirmer test.
2.2-minute ST: the value of first 2 minutes of Schirmer test.
3.rear 3- minute ST: the value that five minute's value subtracting first two minutes of ST
4.rear 4-minute ST: the value that five minute's value subtracting first minute of ST
3.2. tear film breakup time(BUT)
Tear film instability was assessed using BUT measurements, which were obtained using the following method. TBUT test include fluorescein tear film break-up time (fBUT) and noninvasive tear film break up time (NIBUT). fBUT: Fluorescein was added to the tear film for both eyes and patients were asked to blink several times to ensure that it was uniformly distributed across their eyes. The time from the last blink of the eye to the first dry spot on the tear film was measured. Three consecutive measurements were made and recorded. The average values of three measurements were recorded. NIBUT: NIBUT were measured by Keratograph 5M (Oculus Optikgerate GmbH, Wetzlar, Germany). The examinee should place the lower jaw on the jaw rest and keep both eyes open. After successful focusing, examinee should blink 2 times and look at the red point in the center, keeping the eye open as much as possible until the next blink. Then the device can automatically detect the tear film distribution map and displays the measured value. The results of the BUT measurements were categorized as moderate (5–10s) or severe (<5s) 19-20.
3.3. Meibomian gland grading (MG grading)
The quality of the Meibomian gland dropout was briefly assessed using a Meibomian Gland Evaluator (TearScience, Morrisville, NC, United States). A total of 15 Meibomian glands (in the nasal, middle, and temporal parts of the lower tarsus, respectively) were selected. The quality of Meibomian dropout was graded as follows: 0, clear fluid; 1, thick and cloudy dropout; 2, inspissated and congealed dropout with the consistency of toothpaste; 3, none present. The total scores ranged from 0–45.
3.4. Corneal fluorescein staining score(CFS)
Corneal fluorescein staining score assessment was used to stain the patients’ corneas in this study. The cornea was divided equally into five quadrants of and the score for each quadrant was recorded after fluorescein staining, as follows: 0, no punctate staining; 1, less than 30 stained points; 2, more than 30 stained points but no fusion; 3, entirely stained with fusion. A total score of five part was given as 0–15.
All statistical analyses were performed using SPSS software (version 24, IBM, Armonk, NY, United States) for Mac. The chi-square test and Pearson’s rank-order correlation were used to identify the correlation between ST, BUT, OSDI, SPEED, MG grading and CFS. An independent sample t-test was used to compare the parameters of the ST indicators. One- way ANOVA test was uesd to compare the values of indicators in two groups. Figures were created by SPSS and GraphPad Prism, version 8 (San Diego, CA). Descriptive statistics were summarized as mean ± standard deviation (SD).A p-value < 0.05 was considered statistically significant.