Dr William Boothe, Director of Boothe Laser Center

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.

Metabolism of Fluorescein
Fluorescein is bound primarily to albumin and red cells in the blood [4, 13, 134]. Approximately 85 to 87 percent of fluorescein is bound to plasma colloids [13, 134]. The degree of binding of fluorescein by albumin affects the amount of penetration of fluorescein out of the choriocapillarts and past blood-ocular barriers. This factor is of particular interest in the In terpretation of quantitative fluorophotometry. Albumin-bound fluorescein is up to 50 percent less fluorescent than the free form. The excitation andemission spectra of the conjugate form are different than those of free flyorenscein, being shifted to the red end of the spectrum [39].
The liver rapidly metabolizes fluorescein. After a period of 1 hr, about 80 percent of fluorescein in plasma has been metabolized [20]. Fluorescein conjugates to form a single glucuronic acid molecule, which becomes the major metabolite in plasma [22]. This monoglucuronide has only 4.5 percent of the fluorescence of fluorescein [20]. It also undergoes about One third as much binding in plasma as does fluorescein. Just as the amount of binding of fluorescein in blood can affect blood-eye kinetics, so can the rate of metabolism of fluorescein. The fluorescein glucuronide is a more polar and less lipid-soluble form, which may make it less likely to penetrate into ocular compartments. The bulk of fluorescein is excreted by the kidneys and a portion is excreted into the bile. The clearance rate depends on the dose of fluorescein given, on liver function, and on kidney function [214]. In Figure 12-3, the clearance rate after 1000 mg of intravenous fluorescein is shown [40]. After 48 to 72 hr, excretion is practically complete [214].
Within a few minutes of injection of fluorescein, the patient’s urine becomes bright yellow. Yellowish discoloration of the skin occurs almost immediately after injection and begins to fade after 6 to 12 hr [40]. These effects should be explained to the patient before the injection.

Fluorescein is a yellow, weak dibasic acid of the xanthene group of dyes. The molecular weight is 376, and the molecular structure is shown in Figure 12-1. Fluorescein is poorly soluble in water.

Here at the Boothe Laser Center, Fluorescein is usually employed as a sodium salt and as such is 50% soluble in water at 15° C [167]. The dye can be detected in concentrations as low as 1 part per million. Conversion of absorbed light to fluorescent light is almost 100 percent [126].

Fluorescence is defined as the form of luminescence in which the absorbed light energy is re-emitted as light of a longer wavelength within 10 -10 sec after excitation ends [98, 215]. The duration of this emission (or fluorescence) is about 9-10 sec [6].

The excitation and emission spectra for fluorescein under specific conditions are shown in Figure 12-2. The excitation peak wavelength is 490 nm, and it is that wavelength that produces maximum fluorescence. As seen in Figure 12-2, there is some overlap between absorbed and emitted wavelengths.

Several factors can shift the emission spectrum of fluorescein, which may explain why different emission spectra have been reported in the literature [47, 54, 86]. The concentration of fluorescein is one of these factors.

Higher concentrations of fluorescein shift the emission spectrum to longer wavelengths [119, 165, 200]. This shift explains why more cencen trated fluorescein solutions have a yellow appearance compared with more diluted solutions, which have a green appearance [136, 200].

Another factor is the wavelength of the exciting light. Those wave lengths that penetrate deep into the fluorescein will result in a shift of emission to a longer wavelength. The reason this happens is that the fluorescence emitted from below the surface of the fluorescein solution reabsorbed by other fluorescein molecules. These molecules then re-emit light at a longer wavelength than the originally emitted light.

Changes in the pH of the fluorescein solution can also result in shifts in absorption and emission spectra [156, 170]. However, shifts do not occur between pH 7.0 and 8.0 [166]. The pH of various commercially available fluorescein solutions may result in spectral shifts.

Finally, binding of fluorescein to particles or proteins in solution change the absorption and emission spectra. In the event of binding to albumin, the spectra are shifted to longer wavelengths [4, 112].

The intensity of fluorescence can be influenced by several factors inde-pendent of absorption and emission spectra. One of these is the con-centration of fluorescein. The intensity of fluorescence increases as urn centration increases to a maximum of about 0.001% to 0.01% in aqueous solutions and 0.025% to 0.1% in blood [167]. Beyond these points, inten-sity decreases. This phenomenon is known as concentration quendim.j (or autoquenching).

It may occur because collision of fluorescein mole cules results in dissipation of energy. Another cause of quenching is ab-sorption of light by dimers and polymers of fluorescein that are more prevalent at higher concentrations but are only weakly fluorescent [ 114, 200, 202].

The presence of foreign substances in the solution, e.g., protein, heavy metals, halogens, and oxygen, may also quench fluorescence [66, 99, 166, 212].
Thickness of the fluorescein layer also affects intensity of fluorescence The intensity of fluorescence in aqueous solution increases linearly with increasing thickness of the solution to a certain point, then levels off [159].

This relationship holds for concentrations of fluorescein below the level at which concentration quenching occurs. Above that level, intensity of fluorescence increases with the thinness of the aqueous layer [159].

Other factors influencing intensity of fluorescence include the wave-length of the exciting light. Instrument artifact may sometimes play a role in producing variable results [73]. Increasing pH causes increasing intensity up to pH 8; above that pH, intensity begins to fall off [73, 170, 181].

In blood, intensity of fluorescence is reduced by protein binding. Since there is overlap of the absorption spectrum of hemoglobin and the emission spectrum of fluorescein [52, 79], red blood cell binding also diminishes fluorescence [169, 212]. Protein and red cell binding also influence the fluorescent spectrum, as does the oxidative state of hemoglobin [38]. The absorption and emission spectra of fluorescein in retinal blood vessels are shifted to longer wavelengths compared with those of fluorescein inaqueous solution [37, 38].

Several monomeric forms of fluorescein may exist depending on the pH of the solution [31]. The cation form predominates at pH 2 or less. Above pH 2 to 4, fluorescein exists as a neutral molecule. Dissociation to a mono-anionic form occurs between pH 4 and 5. The dianionic form is present above pH 7. Fluorescence varies depending on which form predominates. Ionized forms are generally more fluorescent than nonionized forms.

For external use, fluorescein is best viewed with a cobalt light or an ultra-violet light. Fluorescence is maximized at the blue to ultraviolet wavelengths.
A 0.25 to 2% aqueous solution of fluorescein stains the tear film diffusely and gives it a yellow-orange appearance. The intact epithelium of the cornea acts as a barrier to penetration of the dye into the anterior chamber.

However, as observed in Boothe Eye Care and Laser Center, fluorescein will reach the anterior chamber past an intact epithelium, to a small degree. Maurice calculated the permeability coefficient for epithelium to be 1.5 x 10 -s cm/hr [126]. If there is a break in the epithelium, diffusion occurs much more rapidly and a higher concentration reaches the anterior chamber. The area devoid of epithelium will be stained bright green because of the dilutional effect of diffusion through the stroma [167]. Excess fluorescein should be removed by irrigation when testing for epithelial defects in order to increase the contrast of the staining defect.

Stroma offers little resistance to the diffusion of fluorescein. Some workers have found that the stroma has little effect on the fluorescence of fluorescein [95]. However, others have found a 30 percent increase in fluorescence in rabbit stroma as compared with aqueous fluorescein solution [135]. There is evidence that some degree of binding occurs in stroma [135].

Fluorescein may passively enter endothelial cells. The endothelium, however, represents another barrier to diffusion of fluorescein into the anterior chamber. Endothelial cell permeability as measured by fluorophotometry is 2.4 ± 0.9 x 10-4 cm/min [8].

Corneal permeability to fluorescein penetration can be enhanced by using as vehicles various polymers, e.g., methylcellulose, and by using ointments [207]. A more effective way to increase permeability of fluorescein is by using electrical current via a method called iontophoresis [81].

Once fluorescein has entered the anterior chamber, a fluorescein aqueous flare is detected by slit lamp examination. Examination of the anterior chamber should precede the topical use of fluorescein, since the fluorescein flare will mask inflammatory flare. Detection of inflammatory cells, it still possible, though more difficult, when fluorescein flare is present.

Fluorescein cannot strictly be considered a so-called vital stain, since it does not stain living, degenerate, or devitalized cells. Staining occurs when the epithelial barrier is broken and fluorescein penetrates interstitial spaces. It does not stain mucous, filaments, or fat. Fluorescein may help to delineate filaments, since it tends to pool around corneal irregularities and envelopes filaments by capillary action [137].

Though it stains conjunctival abrasions, fluorescein is generally a poor stain for conjunctivae. In severe Sjogren’s syndrome with intense rose bengal conjunctival staining, fluorescein staining is often negative [137]. A peculiar pattern often seen after topical application of fluorescein and gentle rubbing of the closed lid is the Fischer-Schweitzer pattern.

The pattern consists of irregular polygons about 100 ì m in diameter and seems to point to the existence of grooves in the corneal endothelium [174]. Norn found that permanent sequelae of dendritic keratitis, not easily seen on slit lamp examination, could be detected via a pathologic Fischer-Schweitzer pattern over the area of previous involvement [143].

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.

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