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From the Cornea and Contact Lens Research Unit, School of Optometry and the Cooperative Research Centre for Eye Research and Technology (CRCERT), University of New South Wales, Sydney, Australia.
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Abstract |
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METHODS. Fluorophotometry and a nonpenetrating tracer (70-kDa FITC-dextran) were used with a single extended-wear soft contact lens (EWSCL) material on 11 subjects to characterize tear-exchange kinetics. Twenty to 30 measurements were obtained over a 30-minute period to allow accurate modeling and estimation of the several tear-exchange parameters. Calculated values included tear-replenishment rate (TRR), elimination rate (ER), and the time for 95% of the signal to be eliminated (T95). Major experiments were (1) comparison of ER under controlled and physiological conditions, (2) comparison of right and left eyes, (3) repeatability of ER and T95 on five occasions, and (4) comparison of a marketed lens (oxygen permeability [Dk] 28 x 10-9 [cm/sec][ml O2/ml mm Hg]) to a prototype silicone hydrogel lens (Dk 140 x 10-9 [cm/sec][ml O2/ml mm Hg]).
RESULTS. Tracer elimination behind a hydrogel contact lens (CL), up to 30 minutes after insertion, was optimally described by double-exponential kinetics. Physiological ER (5–30 minutes after CL insertion) was optimally described by single-exponential kinetics. Overall, physiological ER was 8.8% ± 3.8% per minute, and T95 was 31.0 ± 16.1 minutes (n = 76 and 72 determinations, respectively). Differences between right and left eyes in ER and T95 were not significant at the 0.05 level. No difference in ER or T95 was found between habitual and controlled blinking. Mean TRR was 0.67% ± 0.26% per blink (n = 11 determinations). No differences were shown between ER or T95 measurements over time. A prototype highly oxygen-permeable silicone hydrogel lens showed higher ER than did a marketed hydrogel lens (P < 0.01).
CONCLUSIONS. Estimates of postlens tear exchange using a slit lamp fluorophotometer are similar to previously reported rates using similar fluorophotometric techniques. Fluorescent decay behind a hydrogel lens is most precisely described using a double-exponential curve equation and tear exchange may be described using ER, TRR and T95, although the T95 may be the least reliable of these measures. The technique appears capable of discriminating between lens types.
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Introduction |
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Tear exchange appears to be important in preventing the accumulation of debris, cellular material, and metabolites behind a soft lens.5 Mertz and Holden6 presented the first direct evidence that the postlens debris obtained from a patient with an inflammatory reaction from an extended-wear lens was composed of mucus, epithelial cells, and neutrophils. They speculated that the presence of the debris might instigate an inflammatory reaction that becomes acute if the debris remains in situ for a sustained period.6 More recently, the increased risk of corneal infection associated with overnight lens wear has again been reported,7 confirming that there has been no reduction in the risk associated with hydrogel lenses over the past 10 years.8 9 Limited tear exchange has been cited as a key factor in the pathogenesis of CL-related infection.7
Recent in vitro and ex vivo data studies of bacterial virulence suggest that postlens tear stagnation, perhaps related to inadequate tear exchange, may predispose the cornea to Pseudomonas aeruginosa infection.10 11 Cytotoxic strains of P. aeruginosa cause epithelial cell disruption and death with prolonged cell contact. Other strains of P. aeruginosa cause epithelial cell invasion after 3 hours of contact. It is conceivable that impaired tear exchange delays the removal of infected corneal epithelial cells and provides the basis for bacterial invasion of corneal tissue.
In summary, tear exchange may have relevance to successful flexible lens wear in the absence of hypoxia, although direct evidence of causal relationships to infection and inflammation has not been found. Before examination of these relationships, accurate and reliable techniques for measuring tear exchange are required.
There are few complete reports of postlens tear exchange, particularly in relation to current hydrogel materials.2 3 12 Sorensen et al.13 examined tear flow in the presence of a hydrogel lens, but did not characterize the kinetics of tracer elimination from beneath the material. In addition, tear exchange repeatability and reproducibility remain largely undocumented, and several methodological questions persist.
In two early reports, a fluorescent tracer (Fluorexon; molecular weight 710; Dioptic Laboratories, Markham, Ontario, Canada) was used to estimate tear exchange.2 3 In subsequent studies, the tracer size was increased to 4.4 kDa (FITC-dextran).12 Fluorexon is absorbed into a hydrogel lens within 10 minutes,14 which likely occurs during the 8- to 12-minute measurement time of the experiments.2 Moreover, Fluorexon probably penetrated the cornea, because much larger fluorescent compounds such as FITC-dextrans have demonstrated rabbit scleral penetration in vitro.15 16 Tissue penetration overestimates the elimination rate (ER) by as much as 25% compared with a nonpenetrating dye.17
Another consideration may be the manner in which the decay data are collected. Polse et al.,2 Wagner et al.,3 and McNamara et al.12 used a controlled blink rate to calculate the tear-replenishment rate (TRR, denoted as percentage of volume exchange per blink) based on concepts put forth by Cucklanz and Hill in 1969.18 19 Although the rate used, 15 blinks per minute, is physiological, it is not clear whether controlling the blink rate affects tear exchange under typical wearing conditions under which wearers blink at will.
With the renewed interest in accurately quantifying postlens tear exchange, the purpose of this study was to characterize the behavior of a nonpenetrating fluorescent tracer behind the lens. The investigation reported herein included an examination of decay kinetics and the development of useful models, comparison of tear mixing under controlled and noncontrolled blinking conditions, and a comparison of tear exchange using an optimized approach between a current hydrogel lens and a prototype highly oxygen-permeable silicone hydrogel lens.
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Methods |
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max 488 nm,
full width at half maximum [FWHM] 10 nm; Omega Optics, Brattleboro,
VT). The emitted light was transmitted through the standard instrument
optics onto a side-on photomultiplier tube (PMT) mounted on the camera
port (PMT housing, model 70680; PMT, model 77349; Oriel Corp.,
Stratford, CT). The emitted light passed through a 10-mm diameter black
metal mask (to reduce stray light) and an emission filter (530RDF40,
max 530 nm, FWHM 30 nm, Omega Optics). The PMT output was directed
to an amplifier (model 7070, Oriel Corp.) and the final signal fed to a
standard strip chart recorder (Fig. 1)
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Fluorescent intensities were measured as deflection distances from the hard copy of the chart recorder output. These distances were converted into arbitrary fluorescence units, such that all intensities were standardized to the 10-8 amplifier setting. Intrinsic or autofluorescence intensities were subtracted from all postinstillation values before normalization. Normalization of fluorescent intensity data was performed to obviate variation due to tracer concentration and the anatomic variation between subjects. Peak fluorescent intensities were obtained in all cases immediately after lens insertion. Commercial graphing software (Kaleidagraph, ver. 3.01; Synergy Software, Reading, PA) was used to analyze the 20 to 30 data points available for each decay curve.
Tracer
Although several potential fluorescent tracers may be acceptable
for tear-exchange investigation, FITC-dextran was selected because its
spectral characteristics are similar to sodium fluorescein. Key
attributes of the tracer are its stability, lack of protein affinity,
and safety.20
21
22
In addition, this tracer was known to
have no scleral or corneal penetration.16
23
24
FITC-dextrans have been widely used in research on the circulatory system and have been shown to be stable in vitro and in vivo.20 Plasma disappearance curves suggest little or no protein binding, because FITC-dextran leaves plasma at rates close to those of similar-size unlabeled dextran20 and inulin.21 Further evidence that there was no protein binding was found in an investigation of labeled and unlabeled dextran permeation across the blood–lymph barrier, wherein no difference in transport rate was observed.21 FITC-dextrans appear to be safe for in vivo use, because direct injection of 25% concentration in microlymphography studies have shown no adverse reactions.22
An FITC-dextran of molecular weight 70,000 (Sigma Chemical Co., St. Louis, MO) at 0.1% wt/vol concentration was used as the tracer in these studies. The tracer was dissolved into borate-buffered saline at pH 7.0 and sterile filtered before use. Microbiologic and toxicological data were obtained for these preparations before human use. These assays demonstrated no bacterial growth and no cytotoxicity. Viscosity of the 0.1% wt/vol FITC-dextran formulation was compared with that of the buffered saline alone and was found to be identical (i.e., the viscosity was approximately 1.0 mPa/sec at a shear rate of 19.2 seconds and temperature of 25°C).
Subject Selection
Eleven nonhabitual CL wearers were selected to participate in
the study. Subjects had no preexisting ocular disease, reported no eye
symptoms, and were using no topical or systemic medication. All
subjects had normal tear film by slit lamp microscopy and had a
noninvasive tear break-up time of at least 15 seconds. Central corneal
curvature and corneal topography were measured in all subjects, and
only those with normal topographic maps and less than 0.75 D of
corneal astigmatism were included. The subject demographic data
are shown in Table 1
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Study Design
The sample size required was estimated based on pilot study
data. A minimum sample size of 11 subjects was established based on
estimates of type I error 
= 0.05, for a power of 80%, with
five repeated measures, assuming a null hypothesis that there was no
difference between days. Eleven subjects were recruited and
measurements were taken on six occasions. The eye initially measured
was randomly selected, and five repeatability measurements were
performed at the same time of day to mitigate potential effects of
diurnal variation.25
Bilateral measurements were made on
two occasions: once for the comparison of right and left eye data and
on a second occasion for the comparison of controlled blinking with
physiological blinking rate. On a final occasion, subjects wore a
prototype lens of extended-wear design.
Procedures
Baseline intrinsic corneal fluorescence measurements were
determined for the right and left eyes. For the methods
characterization, all subjects were fitted with disposable etafilcon A
lenses (Vistakon; Johnson & Johnson, Jacksonville, FL) of power -3.00
D, back optic zone radius of 8.80 mm, diameter of 14.0 mm, and material
oxygen permeability of 28 x 10-9
(cm/sec)(ml O2/ml mm Hg). A prototype lotrafilcon
silicone hydrogel lens (CibaVision, Duluth, GA), of power -1.00 D,
back optic zone radius of 8.8 0 mm, diameter of 14.00 mm, and material
oxygen permeability of 140 x 10-9
(cm/sec)(ml O2/ml mm Hg), was fitted for the
final series of measurements. Two microliters of the fluorescent tracer
was applied to the back surface of the CL before insertion. The lens
was applied directly to the corneal apex with minimal manipulation, and
the central corneal fluorescence was measured at the following
intervals: immediately after insertion, every 30 seconds until 5
minutes, every minute until 10 minutes, and every 2 minutes until 30
minutes after insertion. The fluorescence intensity was measured for a
minimum of 3 seconds at each interval. The lens was removed after 30
minutes, and the central corneal fluorescence intensity was remeasured
after buffered saline irrigation to verify the absence of corneal
penetration. Subjects were asked to report their subjective comfort
with the CL for each experiment, using a 0 to 100 scale, where 0 was
"intolerable" and 100 was "could not be felt." Habitual blink
rates with lenses were estimated on a separate occasion at which
subjects naïve to the purpose of the experiment were examined
by slit lamp microscope under low illumination and low magnification.
The number of complete blinks only was recorded.
Data Modeling
Preliminary evaluation suggested that a custom exponential curve
fit using either single- or double-exponential equations would most
accurately describe the data. An approach using the philosophy of
fewest parameters with physical significance was adopted. Curve fits
were evaluated on the basis of maximizing the
R2 and 
2
statistics. Curve fits were performed for both 0- to 30-minute and 5-
to 30-minute data sets for each subject and each repeat. The equations
used were as follows.
Single exponential:
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is the lifetime of the decaying species in
minutes. The reciprocal of is the fractional loss per minute
(k) or the ER.
Double exponential:
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1 is the shorter lifetime related
to the rapid-elimination constant (k1 = 1/1);
(1 - f) is the slower decaying fraction;
1/2 is the longer lifetime related to the slower, more physiological
ER constant (k2 = 1/2). The general curve fit option in the graph software (Kaleidagraph; Synergy Software) uses the Levenberg-Marquardt fitting algorithm to minimize the sum of the squared errors of prediction. It allows weighting input, allowable error specification (set at 0.1%) and input of partial differentials to guide the program through parameter space for greater accuracy.
Parameters of the approximate order of magnitude were input to guide
the program through the iterative process. Decisions were made
concerning the accuracy of each of the curve fits by examination of the
individual parameter errors and the 2
(goodness of fit) and R2 (coefficient
of determination values). Initial guesses were altered or the equation
type (single to double) was altered until no further improvement in fit
could be attained.
Based on the best-fit approach, the ER (percentage per
minute), the T95 (time for 95% of the
dye to be eliminated) and the TRR per blink were calculated
for each data set as appropriate: (1) ER per minute was
estimated as 1/1 or 1/2 for single and double-exponential curve
fits, respectively. (2) T95 was
estimated from the curve-fitting coefficients derived from the 0- to
30-minute post–lens-insertion data set. The time taken for 95% of the
dye to be eliminated—that is, for a fluorescent intensity of 0.05 to
be reached from the normalized data, was estimated from the derived
curve equation. (3) TRR is the fractional volume exchanged
per blink and was estimated using the curve-fitting coefficients
derived from the 5- to 30-minute post–lens-insertion data set. The
curve equation was used to derive the fluorescence intensity at 5
minutes (Co) and at 30 minutes
(Cn) after insertion, and TRR
was calculated as follows, where n is the number of
blinks18
:
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The exponential signal decay obtained with a nonpenetrating tracer instilled into the base curve of the CL is illustrated in Figure 2 . The decay followed first-order kinetics, similar to that reported for normal tear flow.13 26 In the majority of determinations, the fluorescent signal decayed rapidly, probably due to reflex tearing after lens insertion, and required rapid measurement during the initial 5 minutes after insertion to capture the decay kinetics. Data collection speed with this system was limited to measurements every 30 to 45 seconds, which appears adequate, given the accuracy of the regression fits to these data.
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Laterality Effects
Both eyes of all subjects were measured similarly on a single day
to assess potential laterality effects. The first eye to be tested was
randomly selected. Table 3
shows the mean ER and T95
values for each eye. Differences between eyes were not significant for
either variable. Mean ERs (n = 11) were 9.1% ±
3.6% per minute in the right eye (T95
29.9 ± 11.5 minutes) and 8.3% ± 3.6% per minute
(T95 36.2 ± 19.1 minutes) in the
left eye.
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Comparison of Lens Designs
The 5- to 30-minute ER was used as the optimized
measure to compare the prototype extended-wear soft contact lens
(EWSCL) design with the marketed lens. Significantly higher tear
exchange was found for the prototype lotrafilcon design (14.2% ±
3.9%/min, n = 7) compared with the etafilcon A design
(8.8% ± 3.8%/min, n = 11; P < 0.01).
Slightly but not significantly greater primary gaze movement was measured with the prototype EWSCL (0.3 ± 0.2 mm) than with the etafilcon A lens (0.2 ± 0.1 mm, P > 0.05). Lens comfort was slightly but not significantly poorer with the prototype EWSCL than the etafilcon A lens (mean comfort score, 75 vs. 83, P > 0.05).
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Discussion |
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These decay kinetics have not previously been presented for hydrogel
lenses, although similar decay behavior has been reported for tear flow
with both fluorescent26
27
28
and 
-emitting
tracers.17
Accurate curve fitting based on the complete
kinetics can have a significant impact on parameters such as the
T95 calculated from the data. For
example, the data in Figure 2
were best described by the
double-exponential fit, giving a T95
of 27.3 minutes. If the same data were fitted with a single-exponential
curve (Y =
0.870e-t/4.8139;
2 = 0.17, R = 0.96), the
T95 would be 13.7
minutes—approximately a 50% decrease.
The mean ER in this study was 8.8% ± 3.6% per minute for 76 determinations, which is around half of the normal, non-CL physiological ER of 16% to 18% per minute.13 24 26 29 Both ER (percentage per minute) and TRR (percentage volume exchange per blink) data are similar to those in studies reported previously, using fluorophotometry to estimate postlens tear exchange, in which single exponential equations were used in curve fitting.2 Mean TRR in the present study was 0.62% ± 0.26% per blink, which is comparable to a previous report of 0.5% per blink (95% confidence interval [CI], 0.39–0.62),30 using an identical lens.
The prototype silicone hydrogel lens demonstrated an ER of approximately 14%/minute, compared with the marketed etafilcon design with an ER of approximately 9%. Mechanical characteristics of the lens material have been recognized to influence tear exchange behind hydrogel lenses.31 The modulus of rigidity, defined as the measurement of the resistance to deformation of a material under compression, of the lotrafilcon lens has been measured at 1.2 MPa,32 compared with 0.26 MPa for etafilcon A.31 The modulus of elasticity, defined as the measurement of the resistance to deformation of a material under tension (stretch) may be up to 20 times higher in silicone hydrogel materials than in conventional hydrogels.31 Both increased material modulus and elasticity are likely to result in higher blink-induced lens movement. However, in the present study, mean lens movement for the lotrafilcon lenses was 0.3 mm, and for the etafilcon A lenses, 0.2 mm; and differences in lens movement between designs in this group were not significant. In a recent study,33 tear exchange was shown to be related to the degree of lens movement on blink and for each 1-mm increase in lens movement, TRR increased 1.05% per blink.
Apart from lens movement, lens comfort is also likely to affect tear exchange.33 Slightly increased lens movement and reduced comfort associated with the lotrafilcon lens are unlikely to have been responsible for differences in ER of the magnitude described in the present study. We hypothesize that material characteristics play a major role in tear ERs in the open-eye environment.
In this study tear ERs were similar for the right and left eyes, and there were no differences in repeated measures of ERs within this subject group. The variances for repeated measurements were similar on each occasion for ER and TRR. Within-subject variations due to measurement variations and physiological fluctuations were low, despite the inherent variability in tear turnover, even in the absence of CL wear.17 As expected, the main variability in the current measurements was attributed to the between-subjects variability. Similarly, large individual variations in physiological tear turnover have been documented.25 The coefficient of repeatability data will assist in gauging meaningful differences between population groups, by using this technique. These data suggest that the ER technique offers reasonable discriminatory ability and the reproducibility of this technique, defined as interlaboratory agreement using similar techniques,34 is acceptable.
The sample size required for statistical validity was estimated from
the present data. The presumed experiment is a randomized crossover
comparison of two hydrogel lenses. The sample size was estimated based
on the ER parameter, a two-sided paired t-test
( = 0.05, power = 80%) using the pooled SD of the
differences in ER between the first and second repeatability
determinations (5.72). The results are plotted in Figure 5
and suggest that a sample size of 31 or 19 subjects is required for a
clinically significant change in ER of 3% or 4% per
minute, respectively. ER in this study showed moderate
repeatability; however, this sample-size analysis demonstrates that the
technique is sufficiently precise to determine differences in tear
ER between groups of subjects wearing different types of
lenses.
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ER is also affected by penetration of tracer into the CL and ocular tissue. Ocular penetration of sodium fluorescein artificially elevated ER by approximately 25% compared with a large-molecular-weight nonpenetrating fluorescent tracer.17 In the present study, no elevated corneal fluorescence was measured, suggesting that no penetration of the 70-kDa FITC-dextran occurred, thus obviating this potential source of error.
Lens-related factors, such as design; fitting relationship between the lens, cornea, and lids; postlens tear volume; lens diameter; and material modulus and elasticity are likely to influence tear exchange. However, the present study was undertaken to validate a method of measurement rather than to assess the impact of different lens and fitting relationship parameters. The effect of the lens-cornea–bearing relationship, based on the difference in radius between the lens base curve and the central keratometry, was shown not to alter TRR in a small group of subjects.3
Controlling the blink rate in this study did not modify the ER compared with the habitual blink rate; however, only one rate was tested in this study. The habitual blink rate reported here (19 ± 7 per minute) is similar to that previously reported during soft CL wear36 (20 ± 1 per minute). One previous study37 demonstrated a marginal increase in corneal oxygenation (0.7%) when blink rate increased from 20 to 60 times per minute. This increase was attributed to increased tear exchange.
In addition to fluorophotometry, other techniques and tracers have been used to estimate tear ERs. Gamma scintigraphy, which uses a gamma emitter such as technetium-99m and a gamma camera to record radioactivity, has also been used to monitor post–hydrogel-lens tear elimination.13 This method is limited by the size of the gamma emitter, the lack of mobility of the gamma camera, and the restriction to two-dimensional observation. Another issue for scintigraphy is sensitivity. Approximately a 1-log-unit range of detection is afforded by scintigraphy over the observation period.38 In contrast, fluorescein techniques typically demonstrate a 2- to 3-log range of signal detection.39
A further approach to the measurement of tear ER uses calibrated microspheres in a range of sizes from 6 to 40 µm in diameter applied to the back surface of a hydrogel lens. The rate of exchange of microspheres is estimated by imaging residual particles over time.40 A linear reduction in small (6-µm) particles over time was demonstrated. This technique may predict the rate of postlens debris removal.
The precision of fluorophotometric techniques may be limited by the long duration of measurement, the small area sampled, and the large depth of focus, depending on the instrumentation. The slit lamp technique described herein allows short measurements (2- to 3-second duration) to be taken at any interval over a wide area of the central precorneal tear film. The alignment technique is repeatable, allowing precise repositioning to the same ocular site, and the depth of focus is small, which minimizes the detection of extraneous fluorescence. The use of large-molecular-weight tracers eliminates confounding fluorescence due to lens or corneal penetration.
An additional source of confounding fluorescence may be tracer expelled from the postlens tear film and moving into the prelens tear film. Tear flow has been shown to be reduced in the presence of a hydrogel lens in one study in which the tracer was applied to the prelens tear film.13 This phenomenon may underestimate the measured ERs. The magnitude of this effect has not been quantified. However, if both the pre- and postlens fluorescence were being measured simultaneously in the present study, we would expect it to have a consistent effect on the measured ER with similar design lenses. Thus, on a comparative basis, differences in lens behavior can be characterized, as this study has demonstrated.
In summary, this study has shown that this slit lamp fluorophotometric technique for measuring postlens tear exchange is feasible and convenient and that the results are comparable to those in previous studies. Postlens tracer decay was most precisely described using a double-exponential curve equation in the majority of data sets. The ER may be described using ER (percentage per minute), TRR (percentage per blink), or T95 (time for 95% of the signal to be eliminated). Regression fits involving early time points after lens insertion may be imprecise, and thus ER or TRR may be more precise estimates of ER than T95. Within ER data, the major variation in the measurements is due to the between-subject variability. No differences were found between the right and left eyes of subjects and a habitual blink rate did not modify ER. Based on these data, it will be possible to design experiments to establish the role of lens–cornea–lid interactions and lens design and material properties in ER. This will allow rational strategies to be developed to maximize postlens tear exchange.
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Acknowledgements |
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Footnotes |
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Submitted for publication April 2, 2001; revised July 2, 2001; accepted August 1, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Fiona Stapleton, CRCERT, University of New South Wales, Level 4, North Wing, RMB, Sydney, New South Wales 2052, Australia. f.stapleton{at}cclru.unsw.edu.au
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