3.3  Fluorophotometry Applications

The fluorophotometer has been used to determine tear flow rates (Occhipiniti et al. 1988), normal corneal epithelial permeability in the human (de Kruijf et al. 1987; Schalnus and Ohrloff 1990) and rabbit (McCarey and al Reaves 1995), corneal endothelial permeability (Mishima and Maurice 1971; Ota et al. 1974), aqueous humor turnover rate (Jones and Maurice 1966), blood aqueous barrier permeability (van Best et al. 1987), crystalline lens transmission (van Best et al. 1985), and blood–retinal barrier (Cunha-Vaz et al. 1975; Grimes et al. 1982). The fluorophotometer provides a tool to access ocular toxicity as an alteration in normal physiological parameters. The effects have been measured for drugs altering basal tear turnover (Kuppens et al. 1992, 1994), contact lenses on corneal epithelial permeability (Boets et al. 1988), contact lenses on endothelial permeability (Ramselaar et al. 1988; Gobbels and Spitznas 1992; and drug effect on the permeability of the blood–retinal barrier (Cunha-Vaz et al. 1975; Tsuboti and Pedersen 1987). Noninvasive ocular fluorophotometry can provide a sensitive indicator of the ocular toxicity effects of a drug, preservative, surgical procedure, etc. on the normal physiology parameters of epithelial permeability, McCarey and Reaves 1997), endothelial permeability, aqueous turnover rate, etc.

3.3.1  Tear Turnover Rate (%/min)

The fluorophotometer can be used to measure the basal or reflex tear turnover rate noninvasively. Kuppens et al. (1992) used 1 mL of a 2% solution of sodium fluorescein and scanned the eye for 30 min at 1.5 min intervals to measure the basal tear turnover rate. An accurate measure can only be performed by carefully avoiding the reflex tear response. In my laboratory, I used either 1 mL of a 0.2% sodium fluorescein or 10 mL of a 0.2% sodium fluorescein. The additional volume to the tear will not overwhelm the natural tear and cause the extra volume to produce erroneous data for the first 7 min. The tear/cornea peak fluorescence at 2-min intervals between 5 and 30 min after the tear application is plotted on a linear scale relative to post-fluorescein application (Fig. 3.7). An exponential curve is fit to the data to calculate the curve coefficients (intercept a0, slope a1, correlation coefficient r).

Tear turnover = min% =100(1 −ea1 ).

After 30 min, the fluorescein concentration is so low that six to eight drops of BSS will clear the tear film of applied fluorescein. The added fluorescein will be cleared after an additional 20 min; at which time the eye can be evaluated for tear turnover again.

Fig. 3.7  Following a single topical application to the conjunctiva of 1 mL of 0.2% sodium fluorescein,­ the human subject had tear/cornea fluorophotometer scans between 6 and 29 min postapplication. The exponential decrease in fluorescein resulted in a 9.3%/min tear turnover

3.3.2  Corneal Epithelial Cell Layer Permeability Methodologies

There are three basic techniques to quantify the fluorescein uptake in the cornea for epithelial cell layer permeability determination.

1. Eye bath technique to measure epithelial permeability: A 20 mL eye bath of 1% sodium fluorescein is maintained against the eye for 3 min. After the fluorescein exposure, the eye receives five 30 s rinses with a balanced salt solution. During the next hour four consecutive fluorophotometric scans are performed every 10 min (de Kruijf et al. 1987; Boets et al. 1988; Ramselaar et al. 1988). The epithelial cell layer permeability coefficient can be calculated.

2. Single drop technique to measure fluorescein uptake in the cornea: A 20 mL drop of 2% sodium fluorescein is instilled onto the cul-de-sac. Forty-five minutes later the fluorescein concentration is measured in the cornea. This measurement is referred to as the F45 (Berkowitz et al. 1981; Gobbels et al. 1989; Gobbles and Spitznas 1989; Chang and Hu 1993). The F45 provides relative epithelial cell layer permeability.

3. Single drop technique to measure epithelial permeability: A 1.0 mL drop of 10% sodium fluorescein is instilled onto the cul-de-sac. A tear sample is measured to determine the tear-diluted fluorescein concentration. Four minutes later fluorophotometric scans are performed every 90 s for 15 min, then at 45 min after fluorescein application (Gobbels and Spitznas 1992). A modification of this technique was published by Joshi et al. (1996). They used a 2.0 mL drop of 0.75% sodium fluorescein instilled onto the cul-de-sac and requested the patient to blink vigorously. The fluorescence of the tear film was followed for 20 min until it became comparable

to that in the cornea. Scans were repeated as rapidly as possible for 8 min, then every 2 min for 20 min. The fluorescein was flushed from the cul-de-sac for 1 min. Two more scans were performed to determine corneal stromal fluorescein concentration. The epithelial cell layer permeability coefficient can be calculated.

3.3.3  Eye Bath Technique

de Kruijf et al. (1987) were the first to report the use of an eyebath to deliver a known concentration of fluorescein to the cornea of human subjects. The epithelial permeability is estimated by dividing the resultant increase in corneal fluorescein by the product of the bath concentration and bathing time. Although the bath technique ­provides an easy method to estimate human in vivo epithelial permeability, its clinical use is limited because the method of fluorescein application is difficult for many ­subjects to tolerate and may lead to fluorescein staining of the skin. Despite the subject acceptance limitation, the technique is still being used (Tognetto et al. 2001).

Noninvasive fluorophotometry bath technique is easily performed with the ­unanesthetized rabbits with n = 6 per experimental group. The stock sodium fluorescein solution (376.3 mol. wt.) is 0.075% (750,000 ng/mL) dissolved in BSS. Store the fluorescein solution at 4°C and protected from light. Check daily the concentration of the corneal sodium fluorescein bathing­solution by diluting the stock solution 1:1,600 to yield 469 ng/mL. Corneal autofluorescence should be determined from the average of three fluorescence values on either side of the peak value from the anterior segment scan of the eye. The values will be 3–5 ng/mL for the rabbit. Bathe the cornea in 0.075% sodium fluorescein in vivo by the following technique. Place the unanesthetized rabbit on paper towels. Position yourself behind the rabbit while holding its lower eyelid open in a cup-like position. Fill the cul-de-sac with 0.075% sodium fluorescein at 33°C for 3 min. Add fresh sodium fluorescein as needed to keep the cornea covered. Make sure the eyelids are pulled away from the cornea to permit good bathing of the corneal epithelium. After the 3-min sodium fluorescein exposure, wick off the sodium fluorescein with a tissue. Flush away remaining fluorescein with 40 mL of 33°C BSS applied with a disposable pipette and bulb. Examine the eye with a slit lamp and blue filtered light. The normal cornea should not demonstrate general stromal uptake of sodium fluorescein. Note any staining pattern and location on the data form. Measure the corneal sodium fluorescein concentration with the Fluorotron Master Anterior Segment Adaptor. Measure and save three to six repeated readings immediately after BSS rinse. Measure the corneal thickness with an ultrasonic pachymeter. Print the data files from the Fluorotron. Select the peak corneal sodium fluorescein concentration for each repeated scan. Epithelial permeability ­analysis uses the following formula:

P = CT (FO −FC ) ,

FBt

CT = corneal thickness

FO = stromal fluorescence

Fig. 3.8  The in vivo noninvasive corneal epithelial cell layer permeability to carboxyfluorescein was determined in the rabbit eye (McCarey and al Reaves 1995)

FC = autofluorescence FB = fluorescein bath t = bath duration

FO and FC are adjusted for spatial resolution of the focal diamond scanning the thin cornea

The normal rabbit corneal epithelial cell layer permeability to carboxyfluorescein was measured by McCarey and al Reaves (1995). A skewed bell-shaped histogram­ (Fig. 3.8) has epithelial cell layer permeability to carboxyfluorescein of 0.0646 ± 0.00700 nm/s.

3.3.4  Single Drop Technique to Measure Epithelial Permeability

There have been many strategies to assess corneal epithelial permeability by applying a single drop of fluorescein to the ocular surface and estimating the amount of fluorescein remaining in the cornea at a specific time after application. Although simple to perform, it is difficult to know the concentration of fluorescein bathing the epithelium. The tear turnover continuously reduces the concentration of fluorescein available for penetration. Variations in eyelid blink rate (tear mixing) and variations in tear production amplify the test accuracy. In dry eye patients with abnormal tear factors the accuracy of the F45 style test (relative fluorescein uptake at 45 min) is even further stressed.

Yet, recent reports in the literature have used the technique to measure corneal epithelial­ barrier function with aging (Chang and Hu 1993). Yokoi et al. (1998) applied 3 mL of 0.5% sodium fluorescein into the lower cul-de-sac with a 20 mL BSS Plus rinse 30 min later (F30 relative fluorescein uptake at 30 min). The corneal fluorescein uptake was determined from ten fluorophotometer scans. The control and treatment groups were compared by the corneal fluorescein uptake in nanogram per milliliter. The authors were able to use the technique to reveal subclinical ocular surface epithelial­cell layer problems in atopic dermatitis with blepharoconjunctivitis. The F30 and F45 do not provide an epithelial permeability coefficient.

Gobbles and Spitznas (1989) attempted to improve the single drop technique by accounting for individual tear turnover rates and changing concentration gradients. They estimated permeability by dividing the increase in stromal fluorescein from baseline autofluorescence levels 45 min after instillation by the tear film fluorescein concentration integrated at the examination time of 45 min. The investigators failed to consider the fluorescein trapped in the cul-de-sac which can be a depot for the dye.

Joshi et al. (1996) improved on Gobbels technique by adding a saline rinse 20 min after instillation of the initial fluorescein drop. Even with this modification, the technique yielded human corneal epithelial values six times greater than the eye bath technique. This would indicate that an unknown methodological problem may exist. The basic single drop technique described by Joshi et al. cannot be used with confidence until certain issues are resolved. It is important to highlight the observations from the Joshi et al. (1996) publication. High concentrations of fluorescein were required to achieve a sufficient penetration into the cornea, and this led to an error in estimating the tear film concentration of fluorescein which may be beyond the linear range of the fluorophotometer. The Fluorotron Master (OcuMetrics, Mountain View, CA) is only linear for 1–2,000 ng/mL before quenching reduces the reliability. Figure 3.9 is reproduced from Joshi et al. (1996) to demonstrate the effects of fluorescein quenching of a 2 mL drop of 0.5 and 1.5% fluorescein. The explanation for the difference in the two curves in Fig. 3.8 is fluorescein quenching at the higher fluorescein concentration.

In Fig. 3.10, the points on the straight line correspond to the fluorescein levels to be expected in the tear if 2 mL drops of different concentrations were instilled and the fluorescein was diluted three times in the tear film by already present tear. Thus, a 1.5% drop should give rise to an initial fluorescein level corresponding to point A, but because of absorption and more serious quenching; its value is depressed to A¢. Similarly, if a drop of 0.5% fluorescein is applied, a fluorescein concentration at B should be achieved, but the fluorophotometer will read the fluorescein concentration at B¢. The dashed line is the relationship for the absorption of light by fluorescein calculated in the algorithm used by the Joshi et al. technique.

The data suggest that the maximum eye drop concentration that gives reliable results is approximately 0.75% fluorescein. The scans should be taken at the same time of day to avoid potential diurnal variations (Webber et al. 1987). Joshi et al. (1996) preferred a 2 mL drop of 0.75% fluorescein. They found no correlation between tear turnover and permeability (Table 3.1), which suggests that no systemic error is introduced by the proposed algorithms.

If only permeability values are needed, then the time can be considerably reduced by washing out the dye after a few minutes, and there is little reduction in the corneal penetration since the tear fluorescein concentrations are low after 10 min (Joshi et al. 1996). A practical constraint between concentration of the fluorescein drop and fluorophotometer quenching is that the permeability of the epithelium in a normal person can be so low that, to obtain a measurable increase in corneal fluorescein over the natural background, the fluorescein ­concentration in the tears must be increased to a level at which its value may be underestimated by the fluorophotometer reading.

Polse and associates (McNamara et al. 1997, 1998; Lin et al. 2002) further modified the Joshi et al. (1996) technique with a 2.0 mL drop of 0.35% sodium fluorescein to minimize quenching, while maintaining sufficient concentration gradient to provide enough stromal uptake of fluorescein for permeability calculations. Within 45 s, fluorophotometric scans are performed to determine initial tear fluorescein concentration. Scans are performed at 2-min intervals for 20 min to determine tear turnover dilution of the tear fluorescein concentration. As the newly excreated tear dilutes the applied fluorescein, the plot of fluorescein ­concentration vs. time will decrease. The area under the curve is the amount of fluorescein exposed to the cornea per duration in the tear film, i.e., equivalent to the eye bath concentration of fluorescein. Next, the eye received three 1-min ­gentle BSS rinses (the volume of BSS used is not defined). Finally, four consecutive fluorophotometric scans are performed for the next 10 min to determine stroma uptake of fluorescein. The tear film fluorescein concentration prior to rinsing is defined by the area under the curve produced by the multiple scans during the 20-min period after instillation and an assumed tear film thickness of 8 mm. With this technique there is a 95% chance that a second measurement from a human subject could be as much as 2.88 times higher or 0.35 times lower than the first reading. This substantial variability between repeated measurements indicates that the single drop technique is unreliable for monitoring individual patient changes in corneal epithelial permeability. However, McNamara et al. feel with careful sample size planning that the technique can be used in population-based research to compare differences in treatment effects between groups of subjects (Fig. 3.11). Sixty subjects (one treatment and one control eye/subject) would be necessary to reject the null hypothesis of no difference between treatment and control with a probability of 0.90 given that the treatment in

Fig. 3.11  Sample size estimates and the accompanying 95% pointwise confidence intervals for a paired eye comparison where a treatment is randomly assigned to one eye of each subject, whereas the fellow eye serves as control. The vertical axis represents the number of subjects necessary to detect various average percentage changes on permeability with a power of 0.9 and type I error of 0.05 using a two-sided z-test, assuming negligible between subject variation on treatment (McNamara et al. 1997)

fact indicates a 25% difference in corneal epithelial permeability on the average compared with the control intervention.

In our laboratory, we have found good results with Maurice’s higher fluorescein concentration and McNamara’s more aggressive saline rinse. The selection of fluorescein concentration between 0.35 and 0.75% is dependent upon subject age and amount of reflex tearing. Reflex tearing will dilute the applied fluorescein to zero concentration before 20 min of data can be obtained. Older individuals will have low basal tear productions resulting in minimal dilutions of the applied fluorescein. For these subjects 0.35% fluorescein will perform better. Conduct the following steps:

(a)Perform all steps alternating the OD and OS, i.e., “in parallel.”

(b)The individual scan of the tear/cornea fluorescence is defined as the area under the curve of peak value ±16 values vs. time in minutes.

(c)Scan each eye three times with the fluorophotometer. Average the area under the curve for three scans of each eye to obtain the autofluorescence values.

(d)Instill one drop every 30 s of the assigned eye drop (20 doses) in the OD and OS eye. Wait 2 min while carefully removing excess tear fluid.

(e)Instill 2.0 mL of 0.75% NaFl in the OD and OS eyes. Instruct subjects to blink vigorously, then scan the eye with the fluorophotometer within 45 s of the application to determine initial tear dilution of the applied fluorescein concentration.

(f)Scan the OD and OS cornea every 2 min for 20 min to determine tear turnover rate from the slope of the exponentially fit line.

(g)Plot the individual scan areas vs. time and determine the area under the resulting curve. This value will represent the fluorescein concentration per duration in the tear film of an estimated 8 mm thickness.

(h)Rinse the OD and OS eyes with a gentle stream of warmed BSS for 1 min. Repeat two additional times for a total rinse time of 3 min with each rinse the entire ocular surface including the superior and inferior cul-de-sac.

(i)Scan the OD and OS eyes four times with the fluorophotometer.

(j)Subtract the average OD and OS eyes autofluorescence from the area under the curves for the four readings. The resulting values will represent the stroma uptake of fluorescein.

(k)The values determined in sections (g) and (j), as well as an assumed tear film thickness of 8 mm, are used to calculate epithelial cell layer permeability (nm/s).

(l)Issues: The initial tear fluorescein concentration must not be too much greater than 2,000 ng/mL in order to avoid quenching and inaccurate concentration readings. Therefore, the problem of the initial drop of fluorescein being washed away by reflex tearing before sufficient amount can penetrate the cornea cannot be solved by increasing the concentration of the applied fluorescein.

(m)Thoughts: If the initial fluorescein concentration is too high for accurate readings, then could the values be estimated by reverse estimates from the “readable” range of values?

(n)Our laboratory has created a Microsoft Excel worksheet to process the data.

The fluorophotometry technique to measure epithelial cell layer permeability must include the tear fluorescein dilution caused by the reflex tearing (Nelson 1995). This can best be achieved with the Joshi et al. technique (Nelson 1995; Joshi et al. 1996; Paugh et al. 1998). They used a 2.0 mL drop of 0.75% sodium fluorescein instilled onto the cul-de-sac and requested the patient to blink vigorously. The fluorescence of the tear film was followed for 20 min until it became comparable to that in the cornea. Scans were repeated as rapidly as possible for 8 min, then every 2 min for 20 min. The fluorescein was flushed from the cul-de-sac for 1 min. Two more scans were performed to determine corneal stromal fluorescein concentration. The algorithm determines epithelial permeability (Pdc, nm/s). The final algorithm determines epithelial permeability, Pdc, from (3.1).

where Pdc is epithelial permeability, qd is tear film thickness, ac is fluorescein within the cornea, integration of ad is the fluorescein within the tear film, and dt is the time duration of the integration.

Paugh et al. (1998) performed an exaggerated exposure protocol with preserved (0.01% benzalkonium chloride and 0.03% EDTA) and nonpreserved artificial tearlubricating solutions in human subjects with ocular pathology. The solution application protocol was to perform a 5-min application of one-drop six times at intervals of 1 min, or a multiple-day application of one drop eight times/day for 3 or 7 days. In each protocol, the corneal epithelial permeability was determined by fluoropho-