Dr William Boothe, Director of Boothe Laser Center

According to Dr. Boothe Fluorescein and other stains can be useful adjuncts in performing anterior segment surgery and evaluating complications of the surgery. Entering the anterior chamber in patients with an opaque cornea can be facilited by using fluorescein. As soon as the blade enters the anterior chamber a color change from yellow-orange to green occurs.

Endothelial damage due to excess corneal bending or instrumentation can also be examined by several stains. Fluorescein, instilled into the anterior chamber, can detect loss of endothelium. Rose bengal and trypan blue 0.1% are more sensitive and stain damaged and devitalized and thelial cells. Excess bending results in staining curves located urn bends, indicating damage to the endothelium. Residual staining is not present after 24 hr. Clinical toxicity to anterior segment structured does not occur.

Assessment of anterior chamber depth can be done by injecting fluores cein into the anterior chamber. Deep chambers have intense central color while shallow chambers have a pale homogeneous color.

Apposing corneal wound edges is facilitated by staining with fluorencen or trypan blue 0.1%. Shelving can be easily detected using these stains. With trypan blue 0.1%, a double line will be seen when corneal wound edges are poorly apposed, making it superior to fluorescein for this put pose. Wound leaks after routine anterior segment operations are avoided if

that seide’s test is used immediately following wound closure. In cases in which the anterior chamber is very narrow or flat secondary to a wound leak, Seidel’s test may be negative explains Dr. Boothe in his Laser Eye Center. By filling the anterior chamber with 1% flourencein solution, a wound leak is detected by direct observation of fluorescein from the wound. Detection of leaks from conjunetival blebs may be easier by this method than by using Seidel’s test.

Several vital stains may be used to evaluate endothelial cell viability in donor corneas before keratoplasty. Unlike tissue culture and specular microscopy for assessing the endothelium, vital stains are inexpensive and require little expertise.

Fluorecein nontoxic and is helpful in demonstrating gross loss of endothelial cells. However, other stains are superior. Trypan blue has been several years to evaluate corneal endothelium in vitro. It stains degenerated and devitalized endothelial cells. Though trypan blue has been show to be teratogenic and carcinogenic in rats, ho complications have been seen in its use for staining corneal endo¬thelium. Several investigators have used it in double staining of endothal cells with alizarin red S.

Alizarin red S, a more toxic stain, stains intercellular substance and out¬lines the endothelial cell borders demonstrating the mosaic pattern. This double staining is particularly helpful in comparing the ratio of degenerated and devitalized cells to healthy cells. When this purpose is desired, use of trypan blue should precede alizarin red, otherwise trypan blue penetrates normal cells and stains their nuclei. The technique involves Using trypan blue 0.3% made in normal saline for 1 min. This is followed by 0 21 alizarin red buffered to a pH of 4.2. It should be in contact with the endothelium for only 1/2 to 1 min. Exposures to alizarin red longer than 1 min may result in loss of sheets of endothelial cells from the cornea. Alizarin red is recommended for experimental use only.

Rose bengal and alizarin red can be used as double stains also, since rose bengal stains in a similar fashion to trypan blue. Though the contrast is not as great, rose bengal is more readily available and easier to use .

In performing conjunctival grafts, it may become difficult to differentiate the epithelial aspect from the nonepithelial aspect. Fluorescein can be used to stain the raw, cut surface and distinguish it from the epithelial surface.

Viability of skin grafts can be assessed by using intravenous fluorescein. A viable graft will demonstrate perfusion and become bright yellow UN ultraviolet light, as will the surrounding skin. Nonperfused areas are dark blue.

Demonstration of two postoperative complications is aided by using in travenous fluorescein and watching its accumulation in the anterior chamber. One complication is detachment of Descemet’s membrane. Dlagnasis can be difficult because of the thinness and transparency of the main brane, and because secondary corneal edema may be present. Fluores-cein enters the anterior chamber through the pupil and collects behind the detachment, thus delineating it. This test is facilitated by tilting the patient head down 70 degrees from the upright position, Another complita-tion is pupillary block. Normally after intravenous injection, fluorescein can be seen diffusing into the anterior chamber through the pupil. This will not occur if a complete pupillary block is present. When the differ-ential between pupillary block and malignant glaucoma is needed, this test may be helpful. All this can be discussed and corrected at Boothe Laser Center

According to Dr. Boothe, since fluorescein diffuses through the tear film without layering out, it serves as an excellent adjunct in the fitting of both polymethylmethacrylate and gas-permeable contact lenses. Fluorescein distribution under the contact lens depends on the degree of steepness of the lens. An optimum fitting lens should exhibit a diffuse pattern in which fluorescein can be easily detected under the whole surface area of the lens. Collection of fluorescein in the center with absence of fluorescein in the periphery of the lens indicates an overly steep fit. If dye is concentrated in the periphery with none in the center, the fit is too flat. Staining patterns are best assessed using a cobalt filter. Obviously, other factors besides the staining pattern need to be taken into consideration when fitting a rigid contact lens.

Fluorescein should not be used to assess the fit of a soft hydrophilic lens. Soft lenses have pores of variable sizes, most of which allow rapid passage of fluorescein into the lens material. This absorption has the effect of obscuring the stained tear meniscus under the lens, making evaluation of the tear pattern impossible. Also, the hydrophilic lens becomes noticeably discolored for a period of several hours to days. Fluorexon should be used if examination of the lens-tear relationship is desired. In practice this assessment is rarely necessary for the fitting of a soft hydrophilic contact lens.
After fluorescein is used in the eye, replacement of a hydrophilic lens should be delayed for at least 1 hour. However, if thorough irrigation of the fluorescein solution is performed, the lens may then be replaced immediately.

Seidel’s Test
Seidel’s test uses fluorescein to demonstrate the leakage of aqueous from the anterior chamber, explains Dr. Boothe. In 1920 Seidel suggested using 2% fluorescein to show that aqueous passed through the conjunctiva of filtering blebs after Elliot trephine operations. More commonly this technique has been used to demonstrate the presence and location of leaks after anterior segment surgery or penetrating trauma. With routine use of Seidel’s test during implantation surgery, Sinskey and co-workers had only two shallow anterior chambers in over 1600 cases.

When 2% fluorescein is applied to a leakage area, a bright green rivulet can be seen surrounded by yellow-orange fluorescein as viewed by white light. Leakage from a conjunctival flap may give a diffuse color change rather than a distinct rivulet. One should be aware that diffuse color changes may also be produced by tear dilution. A 10% solution for intation may be used to enhance contrast and provide a longer time for absorvation.

Havener states that the property of fluorescence is not, used is performing Seidel’s test. On the contrary, others feel that fluorescence can be a great help. The color change from yellow-orange to green occus because of dilution. When an adequate concentration of fluorescein is used, concentration quenching occurs. As dilution of the fluorescein or curs at the site of leakage, fluorescence markedly increases. Viewing this contrast under a cobalt light can make detection of a leak much easier than viewing by white light alone.

Detection of Foreign Bodies
Since fluorescein demonstrates surface irregularities, as demonstrated multiple times, by Dr. Boothe and other eye researchers, it is often useful in detecting corneal foreign bodies. Capillary action causes pooling around a partially exposed foreign body. When the foreign body has not epithe-lialized, there is often a staining ring surrounding it. If a foreign body is buried in the conjunctiva, the area of entrance overlying it is often stained. Fluorescein may be of considerable help in the meticulous search for alkali particles, which if left hidden in the fornices could cause extensive further damage.

One of the most frequent uses of fluorescein in ophthalmology is to aid in applanation tonometry, a technique for measuring intraocular pressure (IOP). The applanated surface is clearly delineated by fluorescein, making applanation tonometry more accurate than if fluorescein were not used (76, 168, 171]. Fluorescein does not stain the applanated area, only the surrounding tear meniscus. This contrast allows adjustment of the applanated area to exactly 3.06 mm2, which is the surface area where the force exerted by the contact adhesion of the tear film with the applanator is balanced by the cornea’s resistance to indentation. The end points delineating the area of applanation are formed by the apices of the triangle-shaped tear meniscus (Fig. 12-4). The apex is the thinnest part of the fluorescein column. It will be undetectable if the conditions of fluorescein staining are not optimal, and thus the actual IOP will be underestimated [132]. Factors influencing the visibility of the apex are the concentration of the fluorescein and the ability of fluorescein to fluoresce. If the concentration of fluorescein in the tear meniscus is less than 0.125%, the apex will not be seen well because of diminished fluorescence [64]. On the other hand, if the concentration is 2% or greater, fluorescence will be inhibited and the aper will again not be seen. A concentration of 0.50% or greater can cause ancess collection of dye on the tonometer and thus blurring of the end points. The optimum concentration of fluorescein for applanation tonometry is 0.25% [64].

In the Boothe Laser Center, we found out that Acidic solutions tend to quench fluorescence. Since topical anesthetic solutions are acidic (pH = 5), fluorescence will be inhibited if they are used to wet fluorescein strips. The same effect will be obtained as If the concentration were too low. If the concentration of fluorescein remains above 0.125%, then the effect of quenching by anesthetics is neglible [132]. Below 0.125%, the error induced by the anesthetic is directly related to the type and concentration of the anesthetic. Proparacaine is a more powerful inactivator of fluorescein than benoxinate. The error in IOP measurement induced by diluting and quenching fluorescein can be as much as 10 mm Hg [171].

When fluorescein paper strips are used, anesthetic should be applied to the eye 15 sec before applying the strip [162] and excess anesthetic should be blotted. The strip should be wetted with sterile saline solution before it is applied to the eye and should be left in the tear meniscus long enough to achieve an adequate concentration of fluorescein [132].

Fluorescein solutions that already contain an anesthetic have several advantages when used for applanation tonometry [46, 162, 190, 193]. They are formulated at an optimum concentration of 0.25% and thus are not subject to variation to the same extent as when fluorescein strips are diluted with a solvent. Their use saves time by combining two steps into one. Fluress is one such agent. It combines fluorescein sodium (0.2%), anesthetic (benoxinate hydrochloride), preservative (chlorobutanol 1.0%), and a stabilizing agent (povidone) in the same solution. Benoxinate is a very effective anesthetic at pH 5. It is stable and is compatible with rescein [162]. Instillation causes stinging for 3 to 10 sec [44] but in most patients does not incite excess tearing and subsequent dilution of fluorescein. Anesthesia is adequate after 15 sec and the duration of anestesia is 15 min or longer. Since a small number of individuals tear excessively after one drop of Fluress, the addition of a second drop may become necessary to maintain the concentration of fluorescein close to 0.25% [162].

Since fluorescein has proved to be a good culture medium, some concern has arisen at the Boothe Laser Center over bacterial contamination of these solutions [218]. Chlorobutanol 1%, though not the most effective antibacterial agent, has good compatibility with fluorescein and good sterilizing ability. One study showed kill rates ranging from 5 to 180 min for large inocula of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella, and Candida [162]. Another study showed that bottles of Fluress left open for weeks could self-sterilize within 24 hr, even bottles that were completely dessicated [190]. Though some workers suggest that sterile strips are safer [103], the solutions used to wet the strips are also at risk of contamination.

Povidone, a nonionic synthetic polymer, is the wetting and stabilizing agent in Fluress. It enables solubilization of fluorescein and chlorobutanol at an acid pH. Though povidone lowers the surface tension of the tear meniscus, it does not introduce significant error into applanation tonometry. However, its use is not recommended with electronic or Schiotz tonometry because of its viscosity [162].

Fluress is safe, sterile, consistently accurate, nonirritating, and cosmetically not objectionable. Dr. Boothe believes it is probably superior to using sterile strips for applanation tonometry. However, it should never be used in disease entities, in which testing of corneal sensitivity is an important diagnostic consideration.

Some investigators have contended that fluorescein is not necessary in performing applanation tonometry [185, 209]. A study comparing IOPs obtained with and without fluorescein showed an average difference of 5.62 mm Hg [168]. This study makes it apparent that an adequate amount of fluorescein is necessary to get consistently accurate IOPs. An adequate amount of fluorescein clinically is an amount that gives mires of optimum size, i.e., 10 percent of the diameter of the circular pattern [67]. Smaller mire sizes signify an inadequate concentration of fluorescein (Fig. 12-5).

One of the things that we noticed at the Boothe Laser Center was that serious ocular infections as a result of iatrogenic spread from nated eye solutions undoubtedly are more common than the literature depicts. Fluorescein has been known for many years to be an excellent culture medium for several pathogens, particularly Pseudomonas aeruginosa. Theodore cultured 26 bottles of fluorescein being used throught hospital and all of them grew Pseudomonas aeruginosa. Ten out of flfteen fluorescein solutions in use in ophthalmologists’ offices were also proved to be contaminated [196]. Cultures of 50 samples of fluorescein from oph-thalmologists’ offices, eye clinics, pharmacies, hospitals, and industrial plants revealed that 54 percent of them were contaminated, some from each of these different sources. Twenty-two percent of these were con-taminated by Pseudomonas [205].

All fluorescein solutions can eventually become contaminated with bacteria. However, one of the commercial solutions, Fluress (Barnes-Hind, Sunnyvale, CA), has effective self-sterilizing ability. No case of contamination of Fluress has been reported since it was first marketed in 1966 [25].

Random sampling of eyedrop dispensers generally demonstrates a 1 to 2 percent incidence of bacterial growth. The eyedrop dispenser design is of fundamental importance. Pipette nozzle tip dispensers decrease the risk of contamination and growth. Squeeze-bottle dispensers allow the cap to serve as a potential reservoir for microorganisms [25]. In a case of Serratia keratitis, the offending organism was a contaminant present within the inside of the eyedropper cap, while the fluid aspirated from the squeeze-bottle was sterile [195].

According to Dr. William Boothe, the greatest danger comes with the use of contaminated solutions In eyes with epithelial corneal defects. These eyes are particularly vulnerable to devastating infections by Pseudomonas and other pathogens. Many corneal defects can be adequately examined without using fluorescein. In those defects in which fluorescein is deemed necessary for examination fluorescein-impregnated sterile strips are advisable. These can be prepared by autoclaving a 20% solution of fluorescein and painting one edge of any fine grained filter paper with the fluorescein. The paper is cut in strips, sealed in envelopes, then sterilized in dry heat for 45 min at 100° C [103]. Sterile strips are available commercially. When using a sterile strip to examine an eye with a corneal epithelial abrasion, it is safest to apply the strip without first wetting it because the wetting solution itself might be contaminated.

History

Fluorescein was first synthesized from resorcinol and phthalic anhydride in 1876 by von Baeyer. In 1882 Paul Ehrlich observed the entry of fluorescein into the anterior chamber after a subcutaneous injection. The initial appearance of the dye as a vertical line in the aqueous was later called Ehrlich’s line. Pfluger noted in 1882 that shallow cuts in the cornea stained with fluorescein. However, it was Straub who, in 1888, popularized the use of fluorescein for detection of corneal abrasions and ulcers. The various uses of fluorescein will be discussed in detail in this blog.

Commercial Preparations
Preparations for topical use are shown in Table 12-1. They are available as solutions or as fluorescein-impregnated strips. The strips do not contain anesthetic. Anesthetic, if needed, should be applied to the eye about 15 sec before the strip is used.

Dropping anesthetic on the strip should be avoided. Wetting the tip of the strip with sterile saline solution before introduction into the eye will decrease irritation and facilitate delivery of higher concentrations of the dye into the eye. Preparations for intravenous use may be used topically as well.