Current Eye Research, 2014; 39(7): 673–679 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2013.865757

Basal values, intra-day and inter-day variations in tear film osmolarity and tear fluorescein clearance Noelia Garcı´a1, Marisa Teso´n1,2, Amalia Enrı´quez-de-Salamanca1,2, Laura Mena1, Amelia Sacrista´n1, Itziar Ferna´ndez1,2, Margarita Calonge1,2 and Marı´a J. Gonza´lez-Garcı´a1,2 1

Ocular Surface Group, Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain and 2CIBER de Biomateriales y Nanomedicina (CIBER-BBN), Valladolid, Spain

ABSTRACT Purpose: The aim of this study was to determine the normal inter-day and intra-day variations in tear film osmolarity and the tear fluorescein clearance test (T-FCT) in healthy subjects. Methods: Tear samples from 24 young, healthy adults were collected from 11:00 AM to 1:00 PM (midday) and 5:00 PM to 7:00 PM (evening) on three non-consecutive days. Tear osmolarity measurement and the T-FCT were performed to assess the basal values and inter-day and intra-day variations of the test results. A freezing point depression osmometer was used to analyze the tear osmolarity, and the T-FCT was performed using a fluorophotometer. Results: The mean osmolarity value was 270  4.4 mOsm/l and the mean T-FCT result was 2.97  0.17 fluorescence arbitrary units. The inter-day or intra-day tear osmolarity values did not differ significantly. The T-FCT results varied significantly during the day, with significantly (p = 0.0004) higher results in the evening; no significant differences were found in the inter-day analysis. Conclusions: Tear osmolarity was unaffected by intra-day variations; however, the T-FCT showed an inter-day variation, which indicated that the time of day when the test is performed must be considered when it is used to evaluate the diagnosis of dry eye disease, disease progression or therapeutic effectiveness. Keywords: Basal values, dry eye disease, ocular surface, osmolarity, tear fluorescein clearance test

rhythms,8 which may be related to changes in the ocular surface symptoms throughout the day; in fact, an evening worsening of visual function and symptoms have been described in patients with DED.8 Due to the unsatisfactory results provided by the currently available diagnostic tests, some new tests have been developed recently, e.g. measurement of the tear meniscus with optical coherence tomography,9 and others have been improved to allow easier use in the clinic; for example, tear osmolarity measurements, which had been primarily a laboratory test, is more accessible clinically for assessing dry eye due to recent technologic advances.10,11 The more common techniques used to measure tear osmolarity

INTRODUCTION Dry eye disease (DED) is a highly prevalent1 ocular surface disease that has multifactorial origins, leading to symptoms of discomfort, visual disturbance and tear film instability. DED is accompanied by an increased tear osmolarity and chronic ocular surface inflammation.2 Despite the number of clinical tests available for diagnosing DED, there is no gold standard test. Most tests are not standardized in the manner in which they are performed, and the tests results are not well correlated with the reported symptoms of DED.3–7 In addition, some tests seem to be altered by circadian

Received 29 April 2013; revised 13 October 2013; accepted 8 November 2013; published online 8 January 2014 Correspondence: Marı´a J. Gonza´lez-Garcı´a, PhD, Ocular Surface Group-IOBA, University of Valladolid, Valladolid 47011, Spain. Tel: þ34 983 184756. Fax: þ34 983 423235. E-mail: [email protected]

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674 N. Garcı´a et al. include the freezing point depression method12 and the vapor-pressure method.13 The most recently introduced nanoliter osmometer uses the electrical impedance of tears to measure the osmolarity.14–16 In 1941, Von Bahr17 first suggested an association between increased tear osmolarity and decreased tear secretion. In 1952, Balik first proposed that tear film hyperosmolarity was a pathogenic feature of DED.18 The recent definition of DED established hyperosmolarity as a pathogenic mechanism in dry eye.2 Measurement of tear secretion is important to differentiate evaporative from aqueous-deficient DED. Some tests measure this aspect of the lacrimal functional unit, and the one used most often is Schirmer’s test. However, this test has shown wide intra-subject, day-to-day and visit-to-visit variations.19 In contrast, measurement of tear turnover is a good indicator of tear secretion and tear dynamics with a greater predictive value for ocular symptoms.20,21 A number of different techniques have been reported for measuring tear clearance or turnover. Fluorophotometry, described by Mishima et al.22 in 1966, was the first technique for measuring the tear elimination coefficient based on the disappearance of sodium fluorescein dye, instilled on the ocular surface, from the tear film. Modified slit-lamp fluorophotometry was used in early studies, and the development of commercial instruments helped standardize the measurements;23,24 however, its use in clinical practice is uncommon. The tear fluorescein clearance test (T-FCT) emerged as a clinical solution to costly, time-consuming tests and the requirements of fluorophotometry.25 In 1995, Xu and Tsubota26 first described the T-FCT and reported a significant correlation with the basal tear turnover rate and tear flow obtained by fluorophotometry. Other authors20,21,26,27 have reported modifications of the T-FCT and reported its high sensitivity and specificity independent of the technique used,21–30 suggesting that the T-FCT has high potential for DED diagnosis. The aim of the current study was to determine the basal values of tear osmolarity with a freezing point depression osmometer and the T-FCT results in healthy subjects and if the tests results are affected by the time at which the measurements are obtained. Analysis of tear physiologic variations, such as intraday changes and inter-day variations is necessary to understand the outcomes of clinical tests.

METHODS Study Design The study followed the tenets of the Declaration of Helsinki; the institutional review board of Institute of Applied Ophthalmobiology and the ethics committee

of the University of Valladolid approved the study protocol. Written consent was obtained from all subjects after explanation of the protocol. This prospective study included 24 healthy volunteers, for whom tear osmolarity and the T-FCT were performed on three different non-consecutive days and at two different times during the same day. The criteria used for selecting subjects were age between 18 and 30 years, no systemic and ocular medication use, no previous history of systemic or ophthalmic diseases that affect tear production, no ophthalmic surgeries within the last 3 months and no contact lens use. To rule out ocular surface disease, subjects could not have ocular symptoms (ocular surface disease index [OSDI] score 12 points)31,32 and results had to be within normal limits in at least two of the following DED tests: tear film break-up time (TBUT) 7 s or longer,33 negative fluorescein corneal staining,34 phenol red thread test 20 s or longer,35 and a Schirmer’s test result without topical anesthesia of 5 mm or longer in 5 min.36 Once enrolled, each subject completed six examinations over three non-consecutive days that were performed between 11:00 AM and 1:00 PM (midday) and 5:00 PM and 7:00 PM (evening), with 2- to 5-day intervals between days on which tears were collected. During these visits, tear osmolarity was always performed first and the T-TFC was always performed 5 min after (to allow tear film normalization). The tests were performed in an examination room in which the temperature (mean  SEM temperature, 19.31  0.25  C; range, 18–21  C) and humidity (mean humidity, 31.79  1.37%; range, 23–38%) were measured at all visits. These measurements corresponded to the average humidity in Valladolid, Spain37 and a comfortable indoor temperature.38 Subjects were required to be awake for at least 2 h before each visit and they were instructed to not use artificial tears within 2 h before the evaluation. One randomly selected eye of each subject was included in the study.

Ophthalmic Evaluation The OSDI questionnaire31 was used to assess the presence or absence of symptoms of DED; the test questions are scored on a scale of 0–100. Tear stability was evaluated by measuring the TBUT; fluorescein strips (Fluorets, Chauvin, Aubenas, France) previously wetted with 0.9% sodium chloride were applied to the inferior fornix. After three blinks, the time lapse between fluorescein instillation and the appearance of the first dry spot was measured; the mean of three measurements was recorded.33 The corneal integrity was evaluated by assessing the fluorescein staining, and was scored on a scale of 0–5, with 0 indicating no corneal staining, according to the Oxford Scheme.34 Current Eye Research

Osmolarity and Tear Fluorescein Clearance Test Variability Tear production was measured using Schirmer’s test without topical anesthesia; one sterile strip (Schirmer Tear Test Strips, 5 mm  35 mm; Alcon Laboratories, Inc., Fort Worth, TX) was placed in the lateral canthus of the inferior lid margin of both eyes,36 and the subjects were asked to keep their eyes closed during the test. After 5 min, the length of the wet strip was measured in millimeters. Tear production also was measured using the phenol red thread test (Zone Quick Test, Menicon Co., Ltd., Nagoya, Japan), in which the thread was placed over the lateral canthus of the eye and the wetted portion was measured after 15 s.35

Tear Osmolarity Measurement An unstimulated 2-ml tear sample was collected with a 2 ml in a capillary tube (Drummond, Broomal, PA) from the lateral canthus of the eye, with extreme care not to touch the conjunctiva to avoid tear reflex collection. Subjects were allowed to blink normally during the sample obtaining. If any significant reflex tearing during the collection were observed, the sample would have to be discarded and 5 min were allowed for tear normalization before obtaining the sample again. When the 2-ml capillary tube was filled, samples were transferred immediately to an Eppendorf tube with a pipetboy (Integra Biosciences AG, Zizers, Switzerland), closed, sealed with Parafilm and stored at 20  C until analysis. The automatic pipetboy ensures that the 2-ml sample is completely transferred to the Eppendorf tube. The day of the analysis samples were kept (still closed and sealed) at room temperature for 5 min and centrifuged before dilution to 1/10 with 18 ml of ultrapure water. The sample was mixed with a vortex and centrifuged again to have the entire sample at the bottom of the Eppendorf tube where osmolarity was tested. The osmolarity was measured using the Fiske 210 osmometer (Advanced Instruments, Inc., Norwood, MA), following the standard instrument procedures (http:// www.tecil.com.). Tear osmolarity is typically expressed as mOsm/L, i.e. the concentration of active particles per liter of tears, but is also expressed as the active particle per kilograms of solvent (Osm/kg) and referred to as osmolality. However, both methods of expression are often considered equivalent for clinical purposes39 and in the current study we refer to them as osmolarity.

T-FCT We used the technique of tear collection to measure the clearance of fluorescein instilled into the tear film !

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with a sensitive commercially available spectrofluorophotometer.20 Five microliters of 2% sodium fluorescein (Colircusı´ Fluorescein 2%. Alco´n Cusı´, Barcelona, Spain) was instilled using a pipette into the selected eye to minimally interfere with the tear film.40,41 The subjects were instructed to blink several times to distribute the dye throughout the tear film; after 15 min, a 1-ml tear sample was collected with a 1 ml glass capillary tube as described previously, transferred to an Eppendorf tube, sealed and stored at 4  C protected from light until fluorescence analysis, which was performed within 24 h after sample collection. For its analysis, tear samples were diluted to 1/1000 with 0.9% sodium chloride using a SpectraMax M5 spectrophotometer (Molecular Devices, Berkshire, Wokingham, UK) with excitation and emission wavelengths set at 490 and 515 nm, respectively. All samples were analyzed in duplicate. Fluorescence at a higher dilution (1/10.000) also was measured to rule out a fluorescence quenching effect.

Statistical Analysis A random-effects analysis of variance model was used to evaluate the possible impact of days and times of the day on the test results. The sample size was checked in advance, and the number of subjects needed to obtain statistical significance was calculated. The data are expressed as the mean  SEM. p50.05 was considered statistically significant. The log of the tear fluorescein concentration was used for statistical analysis rather than the fluorescein concentration to assume normal distribution of data. A licensed statistician (coauthor I.F.) performed all data analyses. The SAS (version 9.2) for Windows software with PROC MIXED and SPSS (v15.0) for Windows software (SPSS Inc., Chicago, IL) were used.

RESULTS Clinical Tests Twenty-four healthy subjects (7 men, 17 women; mean age, 21.6  0.7 years; range, 18–30 years) were recruited. The results of diagnostic testing performed at the inclusion visit were: OSDI, 5  0.9; TBUT, 12  1.1 s; Schirmer’s test, 19  2.2 mm and phenol red threat test, 24  1.1 mm. All diagnostics tests were within normal limits for 15 of the 24 subjects; one value was out of the normal range for eight subjects and two were out of the normal range for one subject. Twenty-one subjects had no fluorescein corneal

676 N. Garcı´a et al. staining; three had Grade 1 corneal fluorescein staining, which in the absence of any other test that altered these values was considered valid for a healthy ocular surface. In fact, normal subjects can have small amounts of corneal or conjunctival staining.42,43 Twenty-three subjects had an OSDI score 512; one subject had a score of 19, with the remaining tests were within the criteria for inclusion. Four subjects had TBUTs below the normal limit. The Schirmer’s test results were within normal limits for all subjects; however, four subjects had phenol red thread test results that were below normal.

Osmolarity Tear samples for osmolarity measurement were obtained in 51 min and none had to be discarded because of reflex tearing. The mean osmolarity value from the six visits was 270  4.4 mOsm/l. The results of each visit are shown in Figure 1. There were no significant differences between different days and the times of day.

T-FCT Tear samples for T-TCT measurements were obtained in 51 min and none had to be discarded because of reflex tearing. The mean T-FCT value from the six visits was 2.97  0.17 fluorescence arbitrary units. The results of each visit are shown in Figure 2. There were no significant differences between different days. However, the T-FTC values were significantly (p = 0.0004) higher in the evening compared to midday on the first and third days; on the second day, the evening T-FCT values also were higher than the midday values, but the difference did not reach significance (p = 0.1834).

FIGURE 1 Tear film osmolarity on three different days and two different time points each day. There are no significant differences between different days and the time of the day.

DISCUSSION Although many clinical tests are available for diagnosing DED, there is no single test used universally for diagnosis of patients complaining of dry eye. One reason is that most tests show poor reproducibility and are poorly correlated with symptoms.3–7 In the current study, we selected healthy subjects without symptoms or signs of DED to obtain basal values and evaluate inter-day and intra-day variations in tear osmolarity values and T-FCT results. The ages of the subjects were between 18 and 30 years to avoid age-related changes in tear film as described in older populations.44 Some authors have reported that tear volume remains constant with increasing age,45 but tear turnover is slower in older persons compared with younger persons.20 Controversy exists in the literature regarding gender and age related differences in tear osmolarity,44,46 while some authors46 have not found changes related to age in the whole group, they found significantly lower values of osmolarity in females under 41 years. We used two timeframes, i.e. midday (11:00 AM– 1:00 PM) and evening (05:00–07:00 PM), which represent the morning and the afternoon during office hours in Spain. For the osmolarity analysis, the most common technique used is the freezing point depression osmometry of tear samples.12,47 The osmometer used in the current study is commercially available. However, due to the amount of sample this osmometer needs to take a measurement (20 ml), we diluted the tear samples. We choose 2 ml to assure that we could collect the same amount of tears for all subjects.

FIGURE 2 The tear fluorescein clearance test on different days and at different times of measurement. There are no significant differences between days in the time intervals; a significant (*p50.05) difference is seen between the 11:00 AM–1:00 PM and the 5:00 PM–7:00 PM measurements. FU, fluorescein units. Current Eye Research

Osmolarity and Tear Fluorescein Clearance Test Variability We found a mean tear osmolarity value of 270  4.4 mOsm/l, which was lower than those reported previously. Tomlinson et al.12 conducted a meta-analysis of data published between 1978 and 2005, and reported a mean tear osmolarity value of 302.2  9.7 mOsm/l for healthy subjects. However, recent studies have reported lower mean osmolarity values in normal subjects. Versura et al.16 reported a mean of 296.5  9.8 mOsm/l, Keech et al.48 of 288  4 mOsm/l and Fortes49 of 293  9 mOsm/l. One reason for the current results could be that we collected tear samples with a capillary tube, which may induce a hypersecretory reflex or artifactual variations, and indeed, increased reflex tear production results in decreased osmolarity.50 However, to minimize this effect highly trained personnel obtained the samples.51 An alternative explanation was the need to dilute the tear samples because of the osmometer used in the current study. This problem now seems to have resolved due to the availability of osmometers for which samples in nanoliters are needed and dilution is not required. Another factor that also could have affected our results was that the samples were frozen at 20  C until processing. Nelson and Wright52 reported that storing samples at 4  C increased the osmolarity by 1.3 mOsm/kg; while Stahl et al. (Invest Ophthalmol Vis Sci 2009;50:ARVO E-Abstract 2611) did not found differences in osmolarity when the samples were frozen; however, no decrease in tear osmolarity due to freezing of the tear samples has been reported. Another factor to consider is participant age and sex, the mean age in the current study was 21.6  0.7 years and 17 women were included in the 24 subjects. Craig and Tomlinson46 found significant correlation between age and osmolarity for females with a lower tear osmolarity in younger females. In the current study, no significant differences in tear osmolarity were found between days or in the results from measurements at two different times of the day, which agreed with several studies (Dalton et al. Optom Vis Sci 2005;82:E-Abstract 055070).53,54 Li et al.54 recently reported diurnal variations in tear osmolarity and found that osmolarity was unaffected by the time of day in normal subjects. Intra-day variations are seen in tear film dynamics using other tests, e.g. variations in the upper and lower tear menisci have been described.55,56 Measurements of collected tear samples have shown that certain properties vary intra-daily, including pH levels57,58 and inflammatory mediators (Enriquez-De-Salamanca et al. Invest Ophthalmol Vis Sci 2010;51:ARVO E-Abstract 4147).59 However, tear osmolarity does not look like to be affected by these variations. !

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Regarding the T-FCT, Xu and Tsubota26 were the first to measure the rate of tear clearance using a modified Schirmer’s test, and they simultaneously evaluated the volume of discharge by measuring the length of the cloth strip section and the tear clearance, and compared color intensity of this section with a standardized scale. This test, despite its simplicity, shows a significant correlation with the basal tear turnover rate and tear flow obtained using more sophisticated techniques such as fluorophotometry.21–25 However, there are disadvantages in that variability, sensitivity and specificity because the paper touches the eyelashes, which elicits reflex tearing that cannot be suppressed by topical anesthesia.60 Collecting tear samples with a capillary tube, besides the fact that small amounts of tears are necessary to analyze fluorescein concentration in the sample, eludes this problem. To measure the tear fluorescein concentration, we used a commercial plate spectrofluorometer (SpectraMax M5) based on a study by Alfonso et al.,20 who reported a mean value of 1.89  0.70 log fluorescence units at 15 min in normal subjects,20 which was slightly below the current mean of 2.97  0.17. Differences between the age groups studied or the measurement technique could explain this discrepancy. In the current study, no significant differences were found in the T-FCT results between days; however, we found that the evening tear fluorescein concentration was higher than the midday value, which suggested decreased tear turnover over the course of the day. This result agreed with that of Webber et al.61 who using fluorophotometry and reported that the morning tear turnover rate was higher than the afternoon rate in healthy subjects, which suggested a circadian rhythm in tear flow, and which also was suggested for aqueous tear evaporation,62 tear meniscus height,54 and tear film stability,63 tests that might be related to the T-FCT results. Also, it has been found a daily variation of visual function, keratitis, conjunctival hyperemia and symptoms in DED subjects.8 The present study has some limitations as the number of subjects included or the criteria used for selecting subjects. Although they were focused to include young healthy subjects without ocular surface disease, there were some subjects that could be classified as normal or mild DED.2 However, it has been described that normal subject can have small amounts of corneal staining42,43 or any altered tests; thus we cannot assure our subject were entirely normal. The current study was performed in young, healthy subjects to evaluate the effect of inter-day measurements or circadian rhythms in two DED diagnostic tests, eliminating factors such as age or presence of ocular surface disease. However, to better understand the clinical implications of these results,

678 N. Garcı´a et al. these effects should be studied in different groups of subjects, such as those with DED, patients who have undergone corneal refractive surgery, and those who wear contact lenses. In conclusion, the current study showed that tear osmolarity measured by freezing point depression osmometry and the T-FCT results measured by spectrophotometry showed no inter-day variations, which supports the test reliability, however T-FCT is affected by circadian rhythms and should be considered when interpreting the test data.

DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. Grant by the Regional Government of Castilla y Leo´n Ref: GR217. Presented in part at Optometry, Contact Lenses and Ophthalmic Optics International Congress, Madrid, Spain in 2010 and at the American Academy of Optometry, Boston, MA, USA, October 2011. Commercial relationships: N. Garcı´a (N), M. Teso´n (N), A. Enrı´quez-de-Salamanca (N), L. Mena (N), A. Sacrista´n (N), I. Ferna´ndez (N), Marga Calonge (C), Maria J. Gonza´lez-Garcı´a (N).

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Basal values, intra-day and inter-day variations in tear film osmolarity and tear fluorescein clearance.

The aim of this study was to determine the normal inter-day and intra-day variations in tear film osmolarity and the tear fluorescein clearance test (...
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