Ординатура / Офтальмология / Английские материалы / Clinical Ocular Pharmacology 5th edition_Bartlett, Jaanus_2008

Dyes for ophthalmic care came into use in the late 1800s, following Baeyer’s synthesis of sodium fluorescein in

1871 and Ehrlich’s use of it to study aqueous dynamics. Since then, several dyes have been used for ophthalmic diagnosis, including fluorescein sodium (e.g., to examine corneal damage, tear stability, intraocular pressure [IOP], and retinal vascular characteristics), fluorexon (a larger molecular weight fluorescein to facilitate examination in hydrogel contact lens wearers), rose bengal, and (more recently) lissamine green (for conjunctival staining). In addition, other dyes such as indocyanine green and methylene blue are developing acceptance in ocular vasculature observation and intraocular surgery, respectively.

FLUORESCEIN SODIUM

Fluorescein is probably one of the most widely used dyes for ophthalmic use. Several factors contribute to its utility, including its hydrophilicity, low toxicity, and excellent fluorescent properties in the visible spectrum, even in very dilute concentration. Early ocular applications were used in detection of corneal ulcers and aqueous flow, followed shortly thereafter by retinal diagnostic application.

Pharmacology

Fluorescein sodium, 3¢,6¢-dihydroxyspiro[isobenzofuran- 1(3H),9¢-[9H]xanthen], C20H10Na2O5, CAS number 518- 47-8, is a yellow acid dye of the xanthene series. Its molecular weight is 376 Da, and its solubility in water at 15° C is 50% (i.e., it is freely soluble). It is generally formulated as its sodium salt (Figure 16-1). When exposed to light, fluorescein maximally absorbs light at approximately 493 nm and emits (fluoresces) at approximately 520 nm.

Figure 16-2 illustrates the excitation and emission spectra of dilute fluorescein in phosphate buffer.

Because fluorescein is a weak acid, depending on the pH of the solution, it can exist in various ionic states. Below pH 2, the cationic form predominates, and a weak blue-green fluorescence occurs. Between pH 2 and

4 the cations dissociate to neutral molecules. At pH 7

negative ions prevail and are associated with a brilliant yellow-green fluorescence. The pH sensitivity has been used to noninvasively examine human stromal pH.

Several factors can alter the fluorescence of fluorescein in solution: its concentration, the pH of the solution, the presence of other substances, and the intensity and wavelength of the absorbed light. In the eye the thickness of the media being measured becomes important in quantitative measurements due to fluorescein self-absorption (or “quenching”). One clinical effect can be to obscure corneal staining if the tear concentration is too great. The intensity of fluorescence increases with increasing pH, reaching a plateau at approximately pH 8. Thus at physiologic pH the fluorescence is nearly maximum. Further increases in pH above 8 reduce the intensity of fluorescence.

Clinical Uses

Fluorescein may be applied topically to the eye in the form of a solution or by fluorescein-impregnated filter paper strips (Table 16-1). It is also available in injection form for intravenous use (Table 16-2).

Fluorescein in solution is highly susceptible to bacterial contamination, especially by Pseudomonas aeruginosa, which grows easily in the presence of fluorescein. Major methods of reducing the possibility of bacterial growth include sterile formulation and air-tight seal of solutions (e.g., injection fluorescein), use of effective

COONa

Figure 16-1 Molecular structure of fluorescein sodium.

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RELATIVE INTENSITY

WAVELENGTH IN NANOMETERS (nm)

Figure 16-2 Excitation and emission spectra of a 0.00005% solution of sodium fluorescein in KH2PO4–K2HPO4 buffer at pH 8. (Reprinted with permission from Romanchuk KG. Fluorescein. Physiochemical factors affecting its fluorescence. Surv Ophthalmol 1982;26:269–283.)

preservatives in fluorescein solution (e.g., Fluress®), and the development of sterile fluorescein impregnated strips.

Although both injection fluorescein and that used clinically from sterile strips are used once and discarded, fluo- rescein–anesthetic combination solutions are used repeatedly on sequential patients. Thus the problem of maintaining sterility, particularly when accidental contamination from patient contact can easily occur, becomes a major issue.

Many studies have examined the issue of microbial contamination of fluorescein–anesthetic solutions designed for clinical use. Although most have directly inoculated the liquid solution, it seems more useful to consider the evidence of contamination from products retrieved from clinical use and from direct contamination

Table 16-1

Fluorescein Preparations for Topical Ocular Use

Product (Manufacturer) Composition

Fluorescein sodium solutions

Fluorescein strips

Table 16-2

Fluorescein Preparations for Intravenous Use

of the bottle tip (if attached, as for some generic products) and dropper tips (for separate droppers), as would occur clinically.

Both dropper tip or bottle tip contamination was examined using Staphylococcus and Pseudomonas species, and Fluress® was found to prevent colonization after 1 minute but the generic fluorescein–anesthetic combinations all allowed growth up to 2 hours. It was suggested that the preservative used in Fluress®, chlorobutanol, worked more rapidly than the thimerosal used in the competing formulations and also that the benoxinate and weak boric acid in Fluress® may confer additional antibacterial properties.

Bottles of Fluress® sourced from the clinic, which might be expected to demonstrate contamination, were examined and found to be largely free of either

Staphylococcus or Pseudomonas species. Although it appears that resistance to bacterial contamination is quite good for Fluress®, the potential for viral contamination appears more serious.

The resistance to adenoviruses types 8 and 19, both common causes of epidemic keratoconjunctivitis, in Fluress® was studied and survival was found for 3 to 4 weeks for types 19 and 8, respectively. Extreme care should be taken when examining suspect patients. Conversely, resistance to contamination for Fluress® from herpes simplex virus type 1 was examined and found to be quite good. Overall, it appears that Fluress®, with its unique formulation, is generally the most effective of the combination fluorescein–anesthetic solutions for clinical use but that care must be taken when using generic versions.

Topical Ocular Applications

Assessment of Ocular Surface Integrity

Instillation of the dye in the cul-de-sac allows detection of corneal and conjunctival lesions, such as abrasions, ulcers, and edema, and aids in the detection of foreign bodies.When the cobalt blue filter of the slit lamp is used to excite the dye, the epithelial defect usually appears outlined in vivid green fluorescence.The dye turns green in the tear film, in spite of being introduced as a yellow-orange liquid, due to dilution with tear fluid.

Figure 16-3 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 Clin 1982;9:302–310.)

Although the use of the cobalt blue excitation illumination is adequate for some observations, the addition of a yellow barrier filter over the observation system of the slit lamp, Burton lamp, or camera greatly enhances visibility of the stained areas, especially on the conjunctiva. Kodak Wratten No. 12 or No. 15 photographic filters or Tiffen No. 2 photographic filters are relatively inexpensive and serve well in this capacity (Figures 16-3 and 16-4). The yellow barrier filter must be placed over the optics of the instrument, not in the path of the blue excitation light. It is highly recommended that the yellow barrier filter be used for staining assessment and for other tests such as fluorescein breakup time.

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

Figure 16-4 Fluorescein photograph of conjunctival staining taken with a Wratten No. 12 yellow barrier filter in place. (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 Clin 1982; 9:302–310.)

instillations of fluorescein might serve as a predictive test for corneal compromise.

Sequential instillations of fluorescein (up to six times, 5 minutes apart) may have value as a mildly provocative test of corneal integrity. Although only 19% of patients showed fluorescein staining after a single instillation of fluorescein, an additional 23% exhibited staining after repeated instillations. It was also noted that the severe degrees of staining appeared to be correlated with contact lens intolerance. However, it was demonstrated that the fluorescein itself was inducing the staining, apart from physicochemical formulation properties or preservatives. Additional work related to the predictive value of sequential staining, using nonpreserved and physiologically compatible formulations, may be warranted.

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.

However, it should be understood that many recently developed contact lens materials contain polymers that block the transmission of light in the ultraviolet region.

Therefore when an ultraviolet light source, such as a

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Figure 16-5 Contact lens fluorescein pattern in eye with keratoconus. Central dark area reflects the absence of fluorescein, indicating central contact lens bearing (touch). There is also bearing in the intermediate area, surrounded by peripheral clearance indicated by the pooling of fluorescein. (Courtesy A. Christopher Snyder, O.D.)

Burton lamp, is used, the fluorescein behind the lens may not be visible. Visualizing the fluorescein necessitates changing to a blue light source in the visible region.This may be accomplished by fitting the Burton lamp or other ultraviolet light with a white light source covered with a deep blue excitation filter, such as a Kodak Wratten No. 47, 47A, or 47B photographic filter. Areas where the lens makes corneal contact show minimal fluorescence or absence of the fluorescein dye (Figure 16-5).

In addition to its usefulness during contact lens fitting procedures, fluorescein is essential for assessing the integrity of the cornea in contact lens wearers. Common contact lens complications that stain with sodium fluorescein include those thought to be due to mechanical etiologies (e.g., foreign body abrasions, 3- to 9-o’clock staining, edge desiccation, vascularized limbal keratitis, superior epithelial arcuate lesions, conjunctival flaps, etc.) and those related to other causes such as solution incompatibilities (e.g., superficial punctate keratopathy) and lack of lens tear exchange (e.g., inferior “smile face” or arcuate staining).

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.

Lacrimal System Evaluation

Topical ocular fluorescein can be used to evaluate two key aspects of the lacrimal system.These are the stability of the precorneal tear film and the patency of the lacrimal drainage system.

Tear breakup time (TBUT) is used clinically as a diagnostic aid in dry eye syndromes and for testing the efficacy of therapeutic approaches. Assessment of TBUT, typically defined as the interval between the last complete blink and the development of the first randomly distributed dark spot in the tear film, is commonly used to estimate tear film stability. Fluorescein can be instilled into the eye with either a pipette or wetted fluorescein strip and observed with cobalt blue excitation and with or without a yellow barrier filter for observation.* Unfortunately, there is still no global standard as to how TBUT should be determined and no consensus as to appropriate cut-off values.

Historically, TBUTs of less than 10 seconds were thought to indicate an unstable tear film. However,TBUTs in normal asymptomatic Hong Kong and Singapore Chinese were found ranging from approximately 8 seconds to 6 seconds, which suggests that a cut-off value for instability less than perhaps 7 seconds may be sensible.

Fluorescein is also useful clinically in evaluating epiphora. Fluorescein testing for lacrimal obstruction usually involves instilling the dye into the conjunctival cul-de-sac and then observing for the presence of fluorescein in the nose. Appearance of the dye in the nose or posterior oropharynx indicates that the lacrimal drainage system of that eye is functional. Generally, a 2% fluorescein solution is used, and this test can be used in conjunction with other procedures for diagnosis of lacrimal obstruction (see Chapter 24).

Applanation Tonometry

The use of topical fluorescein is an important component in the measurement of IOP with the Goldmann applanation tonometer. The dye permits visualization of the applanated area, which is 3.06 mm2 for accurate IOP measurement.

Measurement of IOP with the Goldmann applanation tonometer requires the meniscus of tear fluid surrounding the flattened corneal surface to be sufficiently stained with fluorescein so that the apex of the wedge-shaped meniscus is visible. If the fluid apex is not visible, IOP will be underestimated due to inadequate applanation (Figure 16-6).

The mire visualization problem is likely the reason why IOP measurement without fluorescein has been discredited. For example, it was found that readings without fluorescein were lower by an average of 7.01 mm Hg (Table 16-3). The mean reading with Fluress was 18.03 mm Hg compared with 11.02 mm Hg with the anesthetic Ophthetic in the absence of fluorescein. By performing a regression analysis, it was further suggested that the difference in IOP readings with and without fluorescein becomes even greater as the pressure rises.

*NB:The author recommends the yellow filter for use in TBUT because it seems to provide more definitive TBUT end points.

Figure 16-6 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-mm2 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.)

Intravenous Applications

The introduction of fluorescein angiography in the early 1960s provided a useful method for studying various parameters of ocular function. Intravenous fluorescein is used extensively to delineate vascular abnormalities of the fundus and occasionally to evaluate anterior segment blood and aqueous flow.

Fluorescein Angiography

In the bloodstream fluorescein is excited by a wavelength of 465 nm and emits a wavelength of 525 nm. Circulating fluorescein binds to albumin and red blood cells. It is also metabolized to a weakly fluorescent conjugate, fluorescein monoglucuronide, which exhibits less plasma protein binding than fluorescein. The amount of binding

can affect the penetration of fluorescein through blood–ocular barriers. After injection of 5 ml of a 10% or 3 ml of a 25% fluorescein solution in the antecubital vein, the dye usually appears in the central retinal artery in 10 to 15 seconds. Both circulation time and integrity of the retina and choroid may be examined. Injection of the 25% concentration is well visualized and may cause fewer side effects.

Fluorescein angiography shows retinal blood vessels in high contrast. Nonvascularized pigmented retinal and subretinal lesions appear as dark areas against the green fluorescing background. The abnormal fluorescence of various retinal and choroidal lesions is explained by several mechanisms, including (1) some abnormalities in the retina, allowing for greater visibility of choroidal fluorescence; (2) neovascularization, producing enhanced fluorescence due to new vascular channels; and (3) pathologic processes resulting in enhanced capillary permeability, allowing for leakage of fluorescein into the lesion. These types of abnormalities may often be differentiated by time at onset of fluorescence.

Choroidal fluorescence appears early and usually precedes the arterial phase by about 1 second. Depending on the origin of the new vessels, neovascular fluorescence coincides with the arteriolar or venous phase of fluorescence. Enhanced capillary permeability (leakage) delays fluorescence, followed by a slow increase in fluorescence as the dye recirculates and stains the affected tissues.

Fluorescein angiography has proven helpful in the diagnosis of a variety of pathologic conditions of the fundus. Various macular lesions, central serous choroidopathy, diabetic retinopathy, and disciform macular degeneration show typical fluorescein patterns. Moreover, tumors such as malignant melanoma, those arising from metastasis, and hemangiomas of the choroid demonstrate fluorescence. Fluorescein angiography does not illustrate well the choroidal vasculature, which can be complemented by indocyanine green angiography (see below).

Chapter 31 discusses the clinical procedure and interpretation of fluorescein angiography.

Iris Angiography. Intravenous injection of fluorescein can be useful for visualization of iris tumors and vessel abnormalities such as rubeosis irides. After injection into

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the antecubital vein, the dye first appears in the radial vessels of the iris, which are demonstrated as linear spokes with slow leakage. The amount of iris pigmentation and the pattern of its distribution affect the amount of detail observed in a normal iris angiogram. Blue irides generally show the vessels in greater detail than do brown irides. An adapter mounted in front of a fundus camera lens has rendered possible more complete visualization of the vascular structure in heavily pigmented irides.

Aqueous Flow. Changes in the concentration of fluorescein in the anterior chamber after intravenous injection were measured as early as 1950. Using a slit lamp or objective fluorophotometer, the time course of the fluorescence in the circulating blood and the anterior chamber can be determined in humans. The rate of aqueous flow is approximately 1.5% to 2.0% of the volume of the anterior chamber per minute. Following the early work other methods were devised to measure aqueous turnover, and all have given comparable results. Anterior chamber fluorometry is also useful in monitoring inflammation after oral or injected fluorescein.

Vitreous Fluorophotometry

Vitreous fluorophotometry is a noninvasive quantitative method for measuring small amounts of fluorescein in various ocular compartments, including assessment of the blood–retinal barrier. Both slit-lamp–based and objective scanning fluorometers have been used to characterize the fluorescence of the vitreous in health and disease.

Because the normal blood–retinal barrier resists various substances, including fluorescein, the presence of fluorescein in the vitreous humor indicates a functional breakdown of this barrier. Although physiologic factors and instrument artifacts can influence vitreous fluorescence, this technique has been used to detect retinal vascular disease, especially in diabetes.The procedure has also been used to study the integrity of the blood–retinal barrier in various other diseases, including retinitis pigmentosa, optic neuritis, and essential hypertension.

Oral Fluorescein Angioscopy

Because the integrity of the normal ocular physiologic barriers to fluorescein depends less on dye administration velocity than on certain other parameters, such as retinal circulation time, fluorescein has also been administered by mouth to study posterior pole lesions. The oral procedure in adults usually involves administering 1 to 2 g of fluorescein powder or three vials of 10% injectable fluorescein mixed in a citrus drink over ice. In children, fruit juice containing 1 ml of a 10% fluorescein solution per 20 ml juice per 5 kg body weight has been used to determine macular leakage after removal of congenital cataracts. The dye begins to appear in the fundus in approximately 15 minutes, but maximal fluorescence is not obtained until 45 to 60 minutes after ingestion.

The oral route of administration yields adequate clinical angiograms in approximately 97% of cases and has the advantage that side effects are rare.

Adverse Reactions

Studies of humans undergoing fluorescein angiograms indicate an incidence range of adverse effects ranging from 1.1% to 10%. The most common mild adverse reaction is nausea, accompanied less frequently by vomiting. The nausea usually occurs 15 to 30 seconds after injection and subsides within several minutes. Moderate adverse reactions include fainting, localized reactions, and urticaria (hives), although no severe adverse reactions were reported. Interestingly, in patients with a history of adverse reaction to injected fluorescein, the incidence of adverse reactions becomes nearly 50%, suggesting that careful history and medical monitoring of these patients are imperative.A significant adverse effect that can occur with intravenous fluorescein injection includes pain at the site of injection,especially if the dye becomes extravasated. Patients should be advised that intravenous fluorescein temporarily discolors both skin and urine and can appear in breast milk for up to 76 hours after administration. Itching, discomfort, or nausea was found in 1.7% of 1,787 patients taking oral fluorescein for fundus angiography, approximately the same percentage as for injected fluorescein.

Adverse effects associated with topical fluorescein and anesthetic–fluorescein combinations are usually limited to transient irritation of the cornea or conjunctiva.

Contraindications

Because of the possibility of adverse reactions a family and personal history of allergies, and especially prior angiographic procedures, should be obtained from every patient undergoing fluorescein angiography. Appropriate emergency kits need to be available that range from a minimum of oxygen, cardiopulmonary resuscitation equipment, antihistamine, and smelling salts to a full crash cart.

Because topically administered fluorescein discolors soft contact lenses, the eye should be thoroughly irrigated with sterile saline until the tears show no discoloration or a less absorbent dye such as fluorexon (see below) should be used.

FLUOREXON

Because fluorescein sodium can penetrate into many hydrogel contact lenses, the lenses become discolored, which raises bacterial growth issues and renders the lenses cosmetically objectionable. In addition, the boundary between lens and tears becomes obscured, which precludes the use of fluorescein in soft contact lens fitting. Fluorexon, a molecule similar in fluorescent characteristics to that of fluorescein, is less readily absorbed by the soft lens material, which renders it useful in fitting and evaluating soft and hybrid design lenses.

Figure 16-7 Molecular structure of fluorexon.

Pharmacology

Fluorexon, N,N-bis((carboxymethyl)-amino)ethylfluores- cein tetrasodium salt (Chemical Abstract Registry no. 1461- 15-0), has a molecular weight of approximately 710 Da, about twice that of sodium fluorescein (Figure 16-7). It is a hydrophilic dye due to its multiple polar moieties. Compared with sodium fluorescein, fluorexon has a paler yellow-brown color. Its staining properties are similar to those of fluorescein, although the fluorescence is much less (due to a lower quantum yield) and thus it must be used at greater concentration.

Like sodium fluorescein, fluorexon is vulnerable to bacterial contamination, but it appears to support bacterial growth longer than does a comparable solution of fluorescein sodium. For clinical use, therefore, it is dispensed as single-dose sterile pipettes (see Table 16-1), a preserved solution with benoxinate (Flura-Safe™, Rose Stone Enterprises, Rancho Cucamonga, CA, USA), or recently as fluorexon-impregnated sterile strips.

Clinical Uses

Fluorexon can aid in the fitting of soft contact lenses and is particularly useful in evaluating hybrid designs, such as the SoftPerm lens (Ciba Vision, Duluth, GA, USA), which consists of a rigid gas-permeable center with a hydrogel surround. The use of fluorexon allows visualization of the tear film under the rigid portion of the lens without discoloring the hydrogel portion. Similarly, in a piggyback lens system, wherein a rigid lens is placed on a hydrogel lens for fitting special cases (e.g., advanced keratoconus), the use of fluorexon can be a valuable adjunct to the fitting process. It can be applied to the eye with the lens in place, but it is more effective when placed in the posterior bowl of the lens before insertion.

Recently, fluorexon was examined relative to TBUT in contact lens wearers and nonwearers and the stability time was found to be not statistically different. Moreover, a fluorexon–benoxinate combination (Flura-Safe™, Rose Stone Enterprises) was compared with a Fluress® analogue

(AccuFluoro, Altaire Pharmaceuticals Inc., Aquebogue, NY, USA) in Goldmann applanation tonometry in normal individuals.They found that IOP readings were comparable and that the Flura-Safe formulation induced greater comfort and less stinging and burning compared with the gold standard preparation. Flura Safe® may become the diagnostic aid of choice in practices with large soft lens populations.

Side Effects

Fluorexon stains the soft lens if it remains in contact with the lens for more than a few minutes. However, repeated rinsing with saline usually removes the dye from the lens. Occasional conjunctival injection may occur. Topical application to the eye of a fluorexon–benoxinate combination solution for tonometry has been suggested to produce less stinging and burning compared with a standard fluorescein–benoxinate solution. In clinical use fluorexon has proven nontoxic to ocular tissue.

Contraindications

Fluorexon is not recommended for use with highly hydrated soft lenses having a water content of 60% or higher. In such cases the lens can absorb significant amounts of dye, resulting in unwanted lens discoloration.

ROSE BENGAL

Widely used in the diagnosis of ocular surface disease, the understanding of the staining characteristics of rose bengal has evolved. Relatively recent evidence suggests that it is not a vital dye but one that may actually cause toxicity and cell death under certain circumstances.

Pharmacology

Rose bengal is the 4,5,6,7-tetra-chloro-2′,4′,5′,7′-tetraiodo derivative of fluorescein (Figure 16-8; CAS no. 632-69-9, MW 1017.6) and is a dye commonly used in ophthalmic diagnosis.Tissues stained with rose bengal display a vivid pink or magenta color when viewed with white light. It has been formulated as a 1% solution and in the form of sterile impregnated paper strips that require moistening with sterile saline or extraocular irrigation solution.When using a rose bengal–impregnated strip a variable volume of dye is delivered to the eye based on differing strip soak times (e.g., 15, 30, or 45 seconds). Moreover, the estimated volume applied to the eye using an in vitro eye model was approximately 17 mcl, possibly explaining the discomfort commonly reported with the strip application method.

CI

Figure 16-8 Molecular structure of rose bengal.

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Rose bengal is a photoreactive compound.With excitation light it generates singlet oxygen, which may be responsible for its ability to kill microorganisms such as bacteria and viruses.

Relatively recent studies demonstrated that cells do not need to be devitalized or necrotic to display rose bengal staining. In fact, rose bengal will stain numerous types of healthy cultured cells, including rabbit and human corneal epithelial cells, in a dose-dependent manner. These studies have confirmed earlier observations that the nucleus of the cell retains the dye. A toxic response to rose bengal has been observed. Cells exposed to the dye demonstrated instantaneous morphologic changes, loss of cellular motility, cell detachment, and cell death. Exposure to light further augmented this effect, indicating that photosensitivity may be an additional factor in the dye’s intrinsic toxicity on unprotected epithelial cells. However, this staining could be blocked by the addition of albumin and mucin to the culture medium. This strongly suggests that rose bengal staining results not from a lack of cell vitality, but rather from the lack of the protective preocular tear film. This theory appears consistent with the clinical disorders traditionally associated with rose bengal staining, such as dry eye wherein the mucous layer is compromised.

Clinical Uses

Traditionally, the most frequent use of rose bengal is in the differential diagnosis of dry eye syndromes. However, rose bengal also has other uses in clinical practice. Rose bengal is helpful in the evaluation of most types of corneal and conjunctival lesions, including abrasions, ulcerations, and foreign bodies, and conjunctival dysplasia or metaplasia.A clinical conundrum exists for the use of rose bengal in diagnosing corneal viral disease. Although it is helpful in differentiating herpes simplex from herpes zoster, it is toxic to herpes simplex virus type 1 and thus may prevent accurate identification if used before culturing.

The use of rose bengal in dry eye evaluation is by far the most common use of the dye. Use of liquid volumes of 1% has been reported in dry eye diagnosis ranging from 1 to 20 mcl. The use of 3.0 mcl of nonpreserved 1% rose bengal, instilled with a laboratory pipette, seems to be comfortable for a majority of dry eye subjects.

The use of rose bengal for dry eye diagnosis remains controversial, with numerous workers continuing to value the test and others championing lissamine green. Interestingly, the several worldwide criteria (i.e., the Japanese,American, and European) suggested for the diagnosis of Sjögren’s syndrome include use of rose bengal to assist the diagnosis (Figure 16-9). Although it could be argued that Sjögren’s syndrome is perhaps the most morbid dry eye condition and thus easily visualized using rode bengal, the working group endorsements are powerful statements. Greater comfort in dry eye patients using lissamine green was demonstrated compared with rose bengal, and the conjunctival staining was found to be

Figure 16-9 Rose bengal staining in patient with keratoconjunctivitis sicca (arrows). Note the typical triangular shape and location in the area of eyelid gap of the cornea and conjunctiva. (Courtesy Mark Williams, O.D.)

helpful in diagnosis. A new concept in dry eye diagnosis is the concept of rose bengal staining on the upper eyelid junctional epithelium, demonstrating greater upper lid staining in patients symptomatic for dryness compared with asymptomatic patients.

Contraindications

Because rose bengal also stains skin, clothing, and contact lenses, contact with these entities should be avoided. Wearers of soft contact lenses should perform a thorough irrigation of the ocular surface and fornices before resuming contact lens wear. Irrigation after dry eye evaluation may be helpful to some patients.

A dilemma exists with the use of rose bengal in the differential diagnosis of dendritic lesions of the cornea. Rose bengal is particularly useful in identifying epithelial herpetic corneal ulcers, by virtue of the characteristic staining of the edges of the dendritic lesion, whereas fluorescein stains the center. However, because of its potent antiviral activity, rose bengal used on a suspected herpetic ulcer may preclude a positive culture result, thus delaying the appropriate course of therapy. Therefore the severity of the corneal lesion and the importance of positive identification of the causative organism must be carefully considered in deciding whether to use rose bengal. A false-negative culture result can lead to inappropriate treatment.

LISSAMINE GREEN

Lissamine green is a vital stain that stains degenerate cells, dead cells, and mucus in much the same way as rose bengal. It is also widely used in the food industry as a colorant.

Pharmacology

Lissamine green has a chemical formula of C27H25N2NaO7S2, CAS number 3087-16-9, and a molecular

Figure 16-10 Molecular structure of lissamine green B.

weight of 576.6 Da. Figure 16-10 shows the molecular structure of lissamine green B.

Clinical Uses

Lissamine green 1% stains in a fashion identical to that of 1% rose bengal. It is currently available in sterile strips, which when wetted with saline solution probably deliver variable concentrations and volumes to the eye similar to that for rose bengal. It may be useful when a patient is known to be sensitive to rose bengal. Lissamine green stains membrane-damaged epithelial cells as well as corneal stroma in a manner similar to that of fluorescein and, like rose bengal, also binds to the nuclei of severely damaged cells.An antiviral effect in vitro was also reported with lissamine green B concentrations as low as 0.06%.

In contact lens wear it has become apparent that conjunctival staining in general is related to symptoms of irritation and that lissamine green in particular may be more specific compared with fluorescein for those with symptoms.

Side Effects

Instillation of lissamine green B into the conjunctival sac appears to cause no ocular irritation, and no other adverse effects have been reported. Clinical experience suggests the staining effect of lissamine green to be longer lasting than that of rose bengal.

INDOCYANINE GREEN

Although the possibility of using indocyanine green (ICG) to observe vasculature of the human choroid was first introduced in the early 1970s,not until years later did it gain widespread recognition as a clinical diagnostic tool. Modifications to the original technique and the development of commercial ICG angiographic instrumentation were primary factors leading to its emergence into clinical practice.

Pharmacology

ICG is a water-soluble tricarbocyanine dye, chemical formula C43H47N2NaO6S2 (molecular weight, 774.96 Da;

Figure 16-11 Molecular structure of indocyanine green.

CAS number 3599-32-4). It has a peak absorption in the near-infrared spectrum at 805 nm and maximal emission at 835 nm (Figure 16-11). This feature constitutes an important difference between ICG and fluorescein angiography. In the 800-nm region of ICG absorption, the pigment epithelium and choroid absorb only 21% to 38% of the light, as compared with 59% to 75% in the 500-nm region with fluorescein. Photography in the near-infrared region also enhances angiogram viewing in the presence of media opacities and subretinal exudation of fluid or blood. Moreover, unlike fluorescein, ICG is rapidly and completely bound to plasma proteins after intravenous injection in blood (especially albumin), so that it does not leak through the fenestrated capillaries of the choriocapillaris to obscure underlying details.

Clinical Uses

ICG’s primary use is as a fluorescent dye for retinal and choroidal angiography. Its low fluorescence property initially limited its use in angiography studies. Improvements in video technology, the introduction of appropriate excitation and barrier filters, and the development of the scanning laser ophthalmoscope with a modification to permit infrared recording ultimately allowed choroidal angiograms with high temporal and spatial resolution.

ICG videoangiography (ICGV) is useful in studying a variety of choroidal abnormalities, including congenital anomalies and ischemic, inflammatory, and degenerative disorders. It is used most frequently to identify and characterize choroidal neovascularization (CNV) in agerelated macular degeneration.The collaborative work of the Macular Photocoagulation Study Group has shown that laser photocoagulation is of value in treating CNV. However, fewer than one-half of patients with newly diagnosed agerelated macular degeneration are eligible for laser therapy on the basis of results of fluorescein angiography alone. Cases of ill-defined (or occult) CNV on the fluorescein angiogram are generally associated with poorer results with laser photocoagulation.The efficacy of angiography could be improved with the ability of the ICGV technique to locate and image more accurately the vessels targeted for photocoagulation.

ICG is available commercially from Akorn, Inc., as a twopart system; the dry dye powder (25 mg) is dissolved into a volume of aqueous diluent and should be used within 10 hours. Amounts up to 40 mg of dye dissolved in 2 ml

292 CHAPTER 16 Dyes

of diluent yield acceptable angiograms, depending on the imaging equipment used (package insert). Images can be obtained at 1- or 2-second intervals until the retinal and choroidal circulations are at maximum brightness and at increasing intervals over 30 to 40 minutes until fluorescence subsides. An ICG concentration of 50 mg/ml and injected 3.0 mg/kg followed by a 5.0-ml flush of sterile saline was found to be well tolerated.

ICGV studies are better able to visualize the choroid than is fluorescein angiography, and they allow imaging of rapid choroidal filling not captured by fluorescein angiography. Moreover, the ICG remains in the area of the CNV long after the dye has cleared from the surrounding retinal and choroidal circulation. Thus the ICGV technique appears to be particularly beneficial for visualizing poorly defined membranes, especially those with overlying hemorrhage and those near the edge of previously treated areas. Use of the infrared scanning laser ophthalmoscope can provide the high resolution required to render the ICGV technique even more successful.

Adverse Reactions

Intravenous ICG has proven essentially as safe as sodium fluorescein. Few toxic effects have occurred, but severe allergic reactions have been reported. In one study ICG was generally well tolerated and caused fewer reactions than did fluorescein. However, two patients developed hives, and one experienced transient nausea and vomiting. In another series it was found that the near-infrared illumination of the technique was more comfortable than that used in fluorescein angiography. Patients did not experience nausea or other adverse effects from ICG. Because ICG remains bound to proteins in the blood and is rapidly metabolized by the liver, discoloration of the urine, skin, or mucous membranes does not occur.

Contraindications

Because ICG contains a small amount of sodium iodide, it should not be used in patients with sensitivities to iodine or shellfish or in patients at high risk for anaphylactic reaction. The safety of this agent in pregnancy has not been established.

METHYLENE BLUE

Methylene blue, a vital stain (Urolene blue), has properties similar to those of rose bengal. It can stain both devitalized cells and mucus and corneal nerves. It is not a specific stain when applied to the eye because the blue areas may be either cells or mucus. Clinically, methylene blue is useful for staining the lacrimal sac before dacryocystorhinostomy and outlining glaucoma filtering blebs, and it may prove useful in gonioscopic laser sclerostomy. More recently it has been used in vitro (tissue extraction and absorbance at 660 nm) to examine the effects of

artificial tear preparations on corneal integrity in dry eye models.

Pharmacology

Methylene blue, 3,7-bis (dimethylamino)-phenazathion- ium chloride tetramethylthionine chloride (C16H18N3C1S; CAS no. 61-73-4) has a molecular weight of 373.91 Da. It is an aniline dye with an absorption peak of 660 nm.The dye is usually used as a 5% solution, and benzalkonium chloride may be added to the dye solution to enhance sterility. Methylene blue precipitates in alkaline solutions.

Clinical Uses

Vital staining of corneal nerves requires up to three instillations at 5-minute intervals.The bluish ocular discoloration may remain for 24 hours.

For staining of the lacrimal sac before surgery, the sac is irrigated with methylene blue. The dye should remain in the sac for several minutes. Before the beginning of surgery the dye should be washed out of the sac, because it can spill out on incision and stain the surrounding tissues.

Methylene blue can also be administered intracamerally to stain the crystalline lens capsule to aid in visualization during cataract surgery.

Adverse Reactions

When topically applied methylene blue can be fairly irritating to ocular tissue. A topical anesthetic may be used, because it enhances penetration of the drug at the same time as it relieves the discomfort.

Contraindications

Methylene blue is contraindicated in patients allergic to the dye.

SELECTED BIBLIOGRAPHY

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Berkow JW, Flower RW, Orth DH, Kelley JS. Fluorescein and indocyanine green angiography: technique and interpretation. Am Acad Ophthalmol 1997;5–6.

Bonnano J,Polse K.Measurement of in vivo human corneal stromal pH: open and closed eyes. Invest Ophthalmol Vis Sci 1987; 28:522–530.

Bright DC, Potter JW, Allen DC, Spruance RD. Goldmann applanation tonometry without fluorescein. Am J Optom Physiol Opt 1981;58:1120–1126.

Brubaker RF. Flow of aqueous humor in humans. Invest Ophthalmol Vis Sci 1991;32:3145–3166.

Brubaker RF, Maurice DM, McLaren JW. Fluorometry of the anterior segment. In: Masters BR, ed. Noninvasive diagnostic techniques in ophthalmology. New York: Springer-Verlag, 1990: 248–280.