1040-5488/12/8906-0868/0 VOL. 89, NO. 6, PP.

868874 OPTOMETRY AND VISION SCIENCE Copyright 2012 American Academy of Optometry

ORIGINAL ARTICLE

Corneal Staining and Cell Shedding during the Development of Solution-Induced Corneal Staining
Doerte Luensmann*, Amir Moezzi, Rachael Claire Peterson, Craig Woods, and Desmond Fonn

ABSTRACT Purpose. This non-dispensing cross-over study was conducted to determine if lenses presoaked in Opti-Free RepleniSH (OFR) or ReNu MultiPlus (RMP) cause solution-induced corneal staining (SICS) and subsequent cell sloughing before the typical 2 h in vivo examination point. Methods. Study lenses (PureVision) were worn bilaterally by 13 participants for periods of 15, 30, 60, and 120 min using two different contralateral care regimen pairings. The lens worn on the test eye was soaked overnight in either OFR or RMP and the control eye in Clear Care (CC). After lens removal, corneal staining was rated on a scale of 0 (negligible) to 100 (severe) for four peripheral quadrants and the central region, and the differential global staining score was calculated by subtracting baseline staining scores. Following the staining assessment, corneal cells were collected from the ocular surface using a non-contact irrigation system to determine ocular cell shedding rates. Results. Differential global staining score with OFR was greater than CC with the differences being statistically significant at 30 and 60 min (p 0.01). Maximum staining with RMP was significantly greater than OFR and peaked after 60 and 120 min of lens wear (p 0.01). On average, 710 470 ocular cells were collected after lens wear, with similar shedding seen independent of solution or lens wear duration (p 0.05). Conclusions. SICS occurred earlier but to a significantly lower degree when PureVision lenses were presoaked in OFR compared with RMP, while lenses presoaked in CC did not cause SICS. Ocular surface cell shedding after lens removal was not impacted by lens wear durations of 2 h. (Optom Vis Sci 2012;89:868874) Key Words: solution-induced corneal staining, SICS, cell shedding, silicone hydrogel lenses, contact lens cleaning solution

orneal staining can occur for different reasons, including ocular dryness,1 mechanical surface erosions,2 and contact lens wear.35 Corneal staining is best observed using a biomicroscope equipped with a cobalt blue excitation filter and a yellow barrier filter to enhance contrast following topical installation of sodium fluorescein.6,7 The occurrence of corneal staining after contact lens wear can be related to lens type and fit8 or may be caused by certain soft lens material and care regimen combinations.9 Solution-induced corneal staining (SICS) has been described as diffuse punctate staining
*PhD, Dipl. Ing.(AO) MSc, BOptom PhD, MCOptom MOptom, FAAO Centre for Contact Lens Research, School of Optometry, University of Waterloo, Waterloo, Ontario, Canada.

(extent grade 1 and above) in at least four of the five regions (central, superior, inferior, nasal, and temporal) of the cornea.3 Previous studies have shown that SICS can be induced by a number of lens/solution combinations9 and typically dissipates over time.10 The staining response appears to be most apparent 2 h after lens insertion, with little evidence being detected after 6 h.10 For PureVision (PV) lenses, SICS was observed with all lens care systems, including RMP, Opti-Free RepleniSH (OFR) and Clear Care (CC) though, the last two with diminishing degrees.9,11 This difference in staining response is in agreement with another study that found higher levels of corneal staining with long-term users of polyhexamethylene biguanide-based solutions compared with polyquaternium-1-based products.12 Data from an in vitro study, however, demonstrated a similar reduction in cell viability on PV lenses after being exposed to either ReNu fresh or Opti-Free Express. The study further demonstrated that both solutions caused a

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FIGURE 1.
The OSCCA. (A) Head rest, (B) saline jet recessed inside funnel, (C) collection tube, (D) peristaltic pump, and (E) saline supply/water bath.

TABLE 1. Lens care regimens used for overnight soaking before lens wear
ReNu MultiPlus Manufacturer Preservative Bausch and Lomb 0.0001% PHMB Clear Care CIBA VISION Canada. Hydrogen peroxide 3% Opti-Free RepleniSH

Alcon Laboratories 0.001% polyquaternium-1 (Polyquad); 0.0005% MAPD (Aldox) Buffer Boric acid Phosphate Boric acid Other components Sodium borate; hydroxyalkylphosphonate Pluronic 17R4 (non-ionic) Citrate (citric acid); poloxamine (hydranate) 0.1% EDTA; poloxamine (Tetronic 1304); non-anoyl (Tetronic 1107) ethylenediaminetriacetic acid (C-9 ED3A) EDTA, ethylenediaminetetraacetic acid; MAPD, myristamidopropyl dimethylamine; PHMB, polyhexamethylene biguanide.

significantly lower cell viability compared with the phosphatebuffered saline (PBS) control solution.13 Powell et al.14 investigated in vitro release kinetics of multipurpose solution including OFR and RMP using PV lenses. They suggested that SICS with OFR is likely to occur over a very short period of time (i.e., within 1 h of lens insertion), which could be shorter than that observed with RMP. In other words, SICS may appear and dissipate so quickly that it is not observed at the 2 h time point. While in vivo observations of SICS demonstrate changes on the ocular surface, they cannot describe the response on a cellular level. To better understand cell responses on the ocular surface, the ocular surface cell collection apparatus (OSCCA; Fig. 1) has been developed at the Centre for Contact Lens Research, which is based on the concept introduced by Fullard and Wilson.15 With each eyewash, an average of 364 230 primarily loosely bound and shed cells have been collected from the corneal surface.16 The objective of this study was to determine whether lenses presoaked in OFR or RMP with CC as the control induce SICS

and subsequent cell sloughing before the 2 h in situ examination point. The null hypotheses (H0) are as follows: SICS occurs at a similar time point independent of the type of solution (1) and cell shedding is independent of time and solution (2).

METHODS
In this non-dispensing study, PV (Bausch & Lomb, Rochester, NY) lenses were worn bilaterally over four independent periods of time, with a cross-over of two different contralaterally controlled lens care system pairings. Participants were randomly assigned to start the study in either arm A or B. In arm A, participants wore one lens soaked overnight in OFR and one lens soaked overnight in CC. In arm B, the same participants wore one lens soaked overnight in RMP and one soaked overnight in CC. All lens care regimens used in this study are listed in Table 1. Each visit was scheduled at a different, randomly assigned duration of lens wear: 15, 30, 60, or 120 min. Visits were separated by wash-out periods of at least 2 days, during which no lenses were worn. Each partic-

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ipant was typically scheduled within a 2 h time window (midday) to control for diurnal variations. After completing all four visits within one arm (A or B), participants crossed-over and repeated the four visits for the second arm (A or B). Ethics clearance was obtained through the Office of Research Ethics at the University of Waterloo and each participant signed the informed consent letter before entering in the study. All participants were treated in accordance with the tenets of the Declaration of Helsinki.

The cell content of each well was manually counted by differentiating between nucleated epithelial cells (N) and cells that have a similar cell plasma appearance but are missing the nucleus (ghost cellsN). Examples for both cell types are seen in Fig. 2.

Data Analysis
Data analyses were conducted using Statistica 9 (StatSoft Inc. Tulsa, OK). A significance level of p 0.05 was used for all analyses. Data are presented in tables as mean standard deviation and in figures as the mean with 95% confidence intervals represented as error bars, unless otherwise noted. Repeated measures analysis of variance was used for the main outcome variables (GSS and cell counts) and the effects of solution and time were examined. Tukeys honestly significant difference post hoc tests were used to determine the significance of pairwise differences.

Participants and Study Visits

Seventeen healthy participants with previous experience of contact lens wear were recruited, excluding those using systemic or topical medications, or with any ocular pathology. During the screening visit, the investigator assessed ocular health, determined participant eligibility, and fitted PV lenses according to the manufacturers guidelines to ensure adequate lens movement and centration. This was followed by a 2-day wash-out period during which no lenses were worn. At each study visit, corneal staining was graded with fluorescein using a slitlamp biomicroscope. Corneal staining was rated on a scale of 0 (negligible) to 100 (severe) for each peripheral quadrant (nasal, temporal, superior, and inferior) and the central region. Corneal staining scores were calculated as the product of severity and percent of coverage. A global staining score (GSS) was calculated as the mean of these five quadrants (0 10,000). The differential GSS GSS is finally calculated by deducting the baseline GSS at the screening visit from the GSS observed after lens removal.17 Contact lenses, presoaked in different care systems, were worn for the designated duration of time, after which they were both removed directly from the cornea by the investigator wearing nitrile gloves and stored in 1 mL of PBS (pH 7.4). Immediately after removal, corneal staining was assessed using fluorescein. This was done by an investigator, who was masked to the presoaked lens condition. Following the staining assessment, non-invasive corneal cell collection was performed using the OSCCA as described in a previous study.16

RESULTS
Thirteen participants with a mean age of 35.8 14.9 years (median 27 years, ranging from 19 to 61 years) completed the study. None of the participants experienced an ocular adverse event during the study. Four participants were discontinued for reasons that were not study related. PV lenses presoaked in RMP showed significantly higher staining scores after 60 and 120 min of lens wear compared with the CC control solution (p 0.001) with similar staining levels seen at shorter wearing periods of 15 and 30 min (Fig. 3). For the PV/RMP combination, there was no significant difference in staining between 15 and 30 min (p 0.80); however, levels were higher after 60 min (p 0.001) with no further increase after 120 min of lens wear (p 0.70). Fig. 4 shows a typical staining appearance after 60 min of lens wear, with the lens being soaked overnight with RMP.

Cell Processing
Following the eye wash, the OSCCA was used for contact lens cytology (CLCy) to wash off the remaining cells from the worn contact lens. The lens was mounted on a precleaned spherical glass tube and thoroughly rinsed on one side with 5 mL of warm (35C) PBS, while gently being moved with a pair of silicone tipped tweezers. It was then flipped inside out and rinsed likewise. Eye wash and CLCy samples were immediately spun down in the tubes for 10 min using a centrifuge (Hettich, Edmonton, AB, Canada) at 400 relative centrifugal force. The content of each tube was reduced to 1 mL by aspirating off the supernatant. Both samples (eye wash and CLCy) were pooled (per eye), transferred into a 24 flat bottom well plate (Falcon, Mississauga, ON, Canada) and stained with Hoechst 33,342 (Invitrogen, Eugene, OR). Cells were examined using an Axiovert 40 CFL fluorescent microscope (Carl Zeiss, ON, Canada) equipped with a 4,6-diamidino-2-phenylindole filter.

FIGURE 2.
Typical fluorescent image of nucleated (N) ( 3 1) and ghost cells (N) ( 3 2) stained with Hoechst.

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FIGURE 3.
GSS after contralateral lens wear for 15, 30, 60, and 120 min (mean 95% confidence intervals [CI]) (p 0.001). Before lens wear, PureVision lenses were soaked in RMP or CC.

FIGURE 5.
GSS after contralateral lens wear for 15, 30, 60, and 120 min (mean 95% confidence intervals [CI]) (p 0.04). Before lens wear, PureVision lenses were soaked in OFR or CC.

FIGURE 4.
Typical example of SICS after 60 min of lens wear. PureVision lenses were soaked in ReNu MultiPlus before lens wear.

FIGURE 6.
Typical example of SICS after 60 min of lens wear. PureVision lenses were soaked in Opti-Free RepleniSH before lens wear.

Lenses presoaked in OFR showed statistically significantly higher levels of corneal staining compared with lenses presoaked in CC after 30 and 60 min of lens wear (p 0.01). No significant difference was seen between these two care regimens when lenses were worn for either 15 or 120 min (p 0.05) (Fig. 5). Comparisons of the different time points for the test eye alone (OFR soaked lenses) showed similar staining levels after 15, 30, and 60 min of lens wear (p 0.95) with a slight but non-significant trend showing less staining after lenses have been worn for 120 min (p 0.07). Fig. 6 shows a typical staining appearance after 60 min of lens wear, with the lens being soaked overnight with OFR. Comparisons between both test solutions showed that RMP induced significantly greater GSS than OFR at both 60 and 120 min (p 0.001 for both) but not at 15 or 30 min (p 0.95 for both). There were no differences in GSS between the control eyes wearing the CC presoaked lens (p 0.20). The ocular cell shedding results from pooled eyewash CLCy cell counts are listed in Table 2. The total number of N cells was

typically 1.3 to 1.8 times higher than the N cells; however, no significant effect of solution or time on total cell count or cell type (N or N) was found (p 0.05 for all). About 700 cells were found per irrigation, with no significant difference between each solution and its CC control at any time point (Figs. 7 and 8, Table 2). Furthermore, cell shedding rates were similar for RMP and OFR at all time points (p 0.05 for all). Correlation analysis did not suggest a relationship between the occurrence of corneal staining and cell shedding rates for the 15, 30, 60, or 120 min time points.

DISCUSSION
A number of publications have discussed the phenomenon of corneal staining in the past, raising new questions surrounding the clinical relevance of what corneal staining is and whether fluorescein is primarily pooling between the cell junctions or stains the cell plasma.6,18 In a recent review article, Morgan pointed out that

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TABLE 2. Total number of nucleated (N) and ghost (N) cells after lens wear (mean SD)
Time Nucleated cells (N ) 15 min 30 min 60 min 120 minutes Ghost cells (N) 15 min 30 min 60 min 120 min Total cells (N N) 15 min 30 min 60 min 120 min

OFR 268 356 210 184 277 184 298 245 487 256 327 177 453 321 481 431 756 417 537 252 731 310 779 619

CC 191 106 274 222 338 417 191 109 548 606 426 328 422 372 431 229 739 625 668 415 760 644 622 240

RMP 270 247 179 130 275 172 424 273 319 134 336 208 479 274 433 288 616 281 548 229 767 346 873 468

CC 308 278 271 454 373 454 288 183 450 368 808 1389 605 583 371 265 759 498 734 681 974 901 665 315

CC, Clear Care; OFR, Opti-Free RepleniSH; RMP, ReNu MultiPlus.

FIGURE 7.
Total epithelial cell count after contralateral lens wear for 15, 30, 60, and 120 min (mean 95% confidence intervals [CI]) (p 0.77). Before lens wear, PureVision lenses were soaked in RMP or CC.

FIGURE 8.
Total epithelial cell count after contralateral lens wear for 15, 30, 60, and 120 min (mean 95% confidence intervals [CI]) (p 0.18). Before lens wear, PureVision lenses were soaked in OFR or CC.

the interpretation of corneal staining is mainly based on assumption, extrapolation, and clinical intuition, rather than solid evidence, and asserted that there is a need for more fundamental research to better understand this phenomenon.6 While contact lens staining in general appears to be most severe in the inferior region of the cornea,4 SICS typically affects larger areas of this surface showing a wider spread of micropunctate staining in the superficial epithelial region (see Fig. 4).3 The results of this study confirm a prediction from a previous report that SICS may occur with PV lenses presoaked in OFR before the 2 h in vivo examination point.14 A staining response could be seen as early as 15 min after lens insertion, reaching peak levels at 30 to 60 min after insertion. Although the GSS associated with OFR at these peak levels were statistically significantly greater than that associated with CC, the differences were small and can be considered clinically irrelevant. In contrast to OFR

and consistent with previous findings with PV lenses presoaked in RMP, SICS with RMP started to peak at 60 min and this level of staining was only slightly less at 120 min, which is again consistent with recent studies.19 H0 (1) has been rejected. The GSS associated with RMP was statistically greater than that found with CC or OFR at both of the two last time points (p 0.01). Inter-subject differences in GSS with RMP indicate that more than two thirds of the participants reached peak GSS at 60 min and the rest had the peak response at 120 min with RMP. Despite significant differences in GSS between OFR and RMP at 60 and 120 min, no differences were found between the two study controls. This lack of sympathetic effect validates the feasibility of the contralateral study design for the observation of SICS; however it remains unclear whether a sympathetic effect may have occurred in the cell shedding response.

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There was no effect of time, solution, or interaction between time and solution for corneal epithelial cell shedding in this study; we have failed to reject H0 (2). An earlier study conducted at the Centre for Contact Lens Research confirmed that epithelial cell shedding with RMP peaked after 4 h of lens wear, which is 2 h after the peak of observed SICS and staining levels further decreased from the 2 h to the 4 h time point.20 The goal of this study was to determine the initial staining and cell shedding response after as little as 15 min of lens wear, at which time the concentration of the respective care regimens in the lens/eye is highest. However, despite the relatively high solution concentration, 15 min of exposure may not have been long enough for the cells to show a noticeable change, and while corneal staining and cell shedding response was investigated only immediately after lens removal, it remains unclear whether changes may have occurred at a later time after lens removal. Furthermore, no change in cell shedding was found for the two multipurpose solutions or the CC control after a maximum of 2 h of lens wear. Whether or not cell shedding will increase with OFRpresoaked lenses when they are worn for more than 2 h as seen with RMP20 cannot be suggested from these data. Further studies are necessary to answer this question. Cell shedding rates, however, were higher after contact lens wear, compared with a previous study that collected ocular surface cells from non-lens wearers (about 700 vs. 364).16 This higher shedding rate could be caused by stronger shear forces during lens wear and/or corneal cells that may be trapped under the lens and only released after the lens was removed from the eye. The viability of the shed cells was not further analyzed in this study; the appearance of ghost cells, however, is suggesting that these cells are in a certain stage of apoptosis or at a later state of terminal differentiation as described by Estil and Wilson.21 (A ghost cell has further been defined as a dead cell, in which the outline remains visible, but whose nucleus and cytoplasmic structures are not stainable.22) Another limitation that needs to be considered when evaluating the results of this study relates to the purity of the cell sample: Although the saline irrigation was directed to the central cornea, only about 75% of the collected cell samples are corneal epithelial cells as determined by positive K3 expression with AE5 in a previous study.16 Presumably, conjunctival cells that are floating in the tear film and potentially facial skin cells are the remaining 25% non-corneal epithelial cells, which may impact the sensitivity of the data.16 While SICS is often asymptomatic to the patient,23 a recent retrospective analysis has found a small but statistically significant reduction in subjective lens comfort and vision for patients who experienced SICS.24 The clinical implication of SICS has further been investigated by Carnt et al.,25 who demonstrated that patients who exhibited SICS were three times more likely to develop lowgrade corneal infiltrates. In general, it is therefore desirable that contact lens wear does not induce temporary or permanent changes on the ocular surface and more research is needed to understand and prevent lens wear-related complications.

pared with lens presoaked in RMP. Ocular surface cell shedding following lens removal was independent of the solution used for lens soaking over the time period of this study.

ACKNOWLEDGMENTS
We have no commercial or financial interest in the products used in this study. This study was supported by CIBA VISION. Received September 30, 2011; accepted February 10, 2012.

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CONCLUSIONS
SICS with PV lenses presoaked in OFR appeared to peak sooner, to a much lesser degree and resolved more quickly com-

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Ocular Response to Solution-Induced Corneal StainingLuensmann et al. disinfected with a polyaminopropyl biguanide-preserved care regimen. Optom Vis Sci 2002;79:75361. 24. Diec J, Evans VE, Tilia D, Naduvilath T, Holden BA, Lazon de la Jara P. Comparison of ocular comfort, vision, and SICS during silicone hydrogel contact lens daily wear. Eye Contact Lens 2012;38: 26. 25. Carnt N, Jalbert I, Stretton S, Naduvilath T, Papas E. Solution toxicity in soft contact lens daily wear is associated with corneal inflammation. Optom Vis Sci 2007;84:30915.

18. Ward KW. Superficial punctate fluorescein staining of the ocular surface. Optom Vis Sci 2008;85:816. 19. Bandamwar KL, Garrett Q, Cheung D, Huang J, Lee L, Ng C, Papas EB. Onset time course of solution induced corneal staining. Cont Lens Anterior Eye 2010;33:199201. 20. Peterson RC, Gorbet M, Woods CA, Fonn D. The transient nature of solution induced corneal staining. Optom Vis Sci 2009;87:EAbstract 90816. 21. Estil S, Primo EJ, Wilson G. Apoptosis in shed human corneal cells. Invest Ophthalmol Vis Sci 2000;41:33604. 22. Editors of the American Heritage Dictionaries. Compact American Medical Dictionary. A Concise and Up-to-date Guide to Medical Terms. Boston: Houghton Mifflin Harcourt; 1998. 23. Jones L, MacDougall N, Sorbara LG. Asymptomatic corneal staining associated with the use of balafilcon silicone-hydrogel contact lenses

Doerte Luensmann Centre for Contact Lens Research, University of Waterloo 200 University Avenue West Waterloo, ON, Canada N2l 3G1 e-mail: dluensma@uwaterloo.ca

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