Fluorescein staining

During the application of the various guidelines for ophthalmic, contact lens, and intraocular products, ocular examination and biomicro scopic examination of rabbit eyes are completed with objective reproducible grading for conjunctival congestion, conjunctival swelling, conjunctival discharge, aqueous (humor) flare, iris involvement, severity and area of corneal opacity or cloudiness, pannus, and intensity of fluorescein staining [39,103]. Other available methods... 

In choosing a fluorescent tag, the most important factors to consider are good adsorption (high extinction coefficient), stable excitation without photobleaching, and efficient, high quantum yield of fluorescence. Some fluorophores, such as fluorescein, exhibit rapid fluorescent quenching which lowers the quantum yield over time. Up to 50 percent of the fluorescent intensity observed on a fluorescein-stained slide can be lost within 1 month in storage. AMCA and... 

Fluorescein Staining. Cup the lower lid of the eye to be tested and instill one drop of a 2% (in water) sodium fluorescein solution onto the surface of the cornea. After 15 s, thoroughly rinse the eye with physiological saline. Examine the eye, employing a hand-held long-wave ultraviolet illuminator in a darkened room. Comeal lesions, if present, appear as bright yellowish-green fluorescent areas. 

At least 1 h after fluorescein staining, the test substance is placed in one eye of each animal by gently pulling the lower lid away from the eyeball to form a cup (conjunctival cul-de-sac) into which the test material is dropped. The upper and lower lids are then gently held together for 1 s to prevent immediate loss of material. 

In 1969, 31 subjects had ocular Instillations of either 0.12 or 0.25X CS In water with 0.5X polyaorbate 20 or 0.05- 1.0Z CS In trioctyl phosphate In their right eyes. The subjects experienced intense ocular Irritation and lacrlmatlon. Acute conjunctival Injection lasted 1 h. No fluorescein staining of the cornea was seen under ultraviolet Illumination. In one subject, corneal staining, visualized with the slit lamp, resolved In 24 h.34 35... 

The characteristic color of a wet fluorescein stain under ultraviolet light is a bright yellow. Care must be exercised not to confuse this stain with many oils which also appear yellow under ultraviolet light. Most metals have a bluish cast under ultraviolet light. Plastic materials under ultraviolet light vary in color but have little or no tendency to appear yellow. 

Fig. 6.5. The use of a phycoerythrin (PE) isotype control to help in deciding where, in a dual-color plot, to draw the horizontal line between fluorescein-stained cells to be considered positive and those to be considered negative for the PE stain. Misplacing of the horizontal line will affect the number of CD19 cells determined to express the CD5 antigen in the stained sample. Data courtesy of Jane Calvert.

This test has been used since then many times each with slight variations. For instance, exposure of only one eye to the test substance and no exposure of the other eye or exposure of the other eye to the vehicle. To minimize discomfort to the animal, a local anesthetic is sometimes instilled before use of the test substance. The qualitative scoring system hasbeen extended using also measures of eye blinks or wipes. Examinations can also include magnifying glasses, slit lamp examination, fluorescein staining and photodocumentation. After observed toxicity, animals are sacrificed, the eyes removed and subjected to microscopic and histological examinations. 

The toxieity of both neat and vapor exposures of SM to the eye is highly predictable. After a time period of no symptoms, laerimation begins and quickly progresses to eonjunetivitis, pain, blepharospasm, and photophobia. Comeal edema follows rapidly as a result of the loss of the epithelium, whieh allows water to enter the stroma from the preeomeal tear film (Slatter, 1990). By 24 h after exposure, it is not unusual to see a 100 to 300% increase in comeal thiekness evident by a substantial increase in opacification. Fluorescein staining at 24 h will reveal ulcers that may eover a majority of the eomeal surface. By 48 h, evidence of healing is seen, and by 96 h, it is not imusual to have... 

Figure 1-1 Self-induced injury. Fluorescein staining of the inferior bulbar conjunctiva shows a typical epithelial defect caused by contact with an ointment tube tip. (From Solomon A. Inadvertent conjunctival trauma related to contact with drug container tips. Ophthalmology 2003 110 798.)...

A significant positive correlation has been observed between the levels of inflammatory cytokines in the conjunctival epithelium and the severity of ocular irritation symptoms and corneal fluorescein staining. The inflammatory cytokines and other inflammatory mediators also correlate positively to severity of conjimctival squamous metaplasia in Sjogren patients. These proin-flammatory cytokines also have been implicated in regulation of epithelial mucin expression, with several studies suggesting inflammation is central to the pathogenesis of meibomian gland dysfunction as well. 

Figure l6-3 Fluorescein photograph of conjunctival staining taken without barrier filter. (From Courtney RC, Lee JM. Predicting ocular intolerance of a contact lens solution by use of a filter system enhancing fluorescein staining detection. Int Contact Lens CUn 1982 9 302-310.)... 

The mechanism of fluorescein staining of ocular epithelia has been subject to some conjecture. In earlier work it was suggested that staining occurred due to accumulation in intraepithelial spaces rather than direct staining of the cells. However, it has become clear that fluorescein can directly stain diseased human corneal cells and rabbit epithelial cells. Moreover, the hyperfluorescence that probably represents micropunctate clinical staining is likely due to optimum dye concentration and fluorescence within the cell rather than simple pooling. Cellular hyperfluorescence occurred from both mechanical abrasion and chemically induced toxicity, conditions that presumably promote an intracellular concentration that allows definitive clinical visualization. An issue that has received some attention is whether repeated... 

Contact Lens Fitting and Management Fluorescein staining of the tear film is a major aid in the fitting of rigid gas-permeable contact lenses.After topical application of fluorescein to the eye the tear layer becomes visible, with a characteristic pattern of green fluorescence. Observation of the fluorescein-stained tear film with an ultraviolet light or the cobalt blue filter of the slit lamp allows determination of the fit of the lens. 

The practitioner should be cautious in interpreting apparent fluorescein staining in a contact lens wearer, as areas of indentation, which do not represent cellular damage, also demonstrate increased fluorescence. Indentation may result from the accumulation of bubbles (known as dimple veiling) or from compression by a lens edge or poorly finished junction, which often results in an arcuate pattern of fluorescein pooling. 

Figure l6-6 Cornea partially flattened by applanation tonometer. The apices of the fluorescein-stained wedges above and below the flattened area are too dilute to be visible. The 3.06-mm end point of applanation appears to have been reached but in reality consists of a smaller flattened area. (Modified and reprinted with permission from Moses RA. Fluorescein in applanation tonometry. Am J Ophthalmol 1960 49 1149-1155.)... 

Lakkis C, Brennan NA. Bulbar conjunctival fluorescein staining in hydrogel contact lens wearers. CLAO J 1996 22 189-193. 

Tabery HM. Dual appearance of fluorescein staining in vivo of diseased human comeal epithelium. A non-contact photomicrographic smdy. BrJ Ophthalmol 1992 76 43-44. 

Wilson G, Ren H, Laurent J. Comeal epithelial fluorescein staining. J Am OptomAssoc 1995 66 435-441. 

Fluorescein-stained fluid exits the nose this indicates a partial distal (ferther away from the punctum) obstruction. Occasionally, the obstruction is composed of a bolus of mucopurulent material that is dislodged by the force of the irrigation. In these cases the irrigation procedure itself is therapeutic, and often the epiphora disappears immediately. Other cases may require surgical intervention to maintain the patency of the lower lacrimal drainage system (dacryocystorhinostomy). 

Following severe exposure these symptoms progress for 24 h or longer, and are followed by spotty hemorrhagic discoloration of the iris and roughened lusterless surface of the corneal epithelium, which demonstrate punctuate fluorescein staining. The corneal epithelium may exfoliate. 

As a part of the Federal Hazardous Substances Act (FHSA), a modified Draize test was adopted (96-98) as the official method for evaluation of acute ocular irritancy (99). It is a pass/fail determination that remains in effect today. The test may use a small volume more consistent with the capacity of the inferior conjunctival sac (100), and incorporate biomicroscopic slit-lamp examination and/or fluorescein staining to assess ocular changes (71,97,98). While various in vitro tests have been proposed to replace this in vivo evaluation and a U.S. interagency committee has recommended certain in vitro tests to detect severe irritants and corrosives, none has yet been officially accepted or validated (101-104). 

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