Sources in Time JUAN MURUBE, MD, PHD,
EDITOR
Fluorescein: The Most Commonly Used Surfocular Vital Stain JUAN MURUBE, MD, PHD
he microscope was invented about four centuries ago,1,2 and when the laboratory microscope was first used to observe anatomical parts of the body, it was without stains. The first natural dye for microscopic observation appears to have been carmin, used by Hill to study the structure of wood fiber.3 In the XIX century, indigo, aniline, and other stains began to be used to dye microscopic specimens and histologic sections. Because these stains, and later others, were used to study the human body in vivo (blood, urine, ocular surface, etc), they were termed “vital stains.” The stain used most commonly today in clinical ophthalmology is fluorescein.
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I. INVENTION OF FLUORESCEIN Fluorescein is a synthetic organic powder with a yellow-orange-red color. It was synthesized in 1871 by the Berlin chemist Adolf von Baeyer (1835-1917 [Figure 1]), who created a yellowish fluorescent intense colorant, which he named resorcinphthalein. It could permeate vascular and other body barriers, was nonanaphylactic with little toxicity, and could be applied to textile cloths as well as to human and animal tissues. Baeyer founded the Bayer pharmaceutical company and patented resorcinphthalein with the name of fluorescein. In 1905, he received the Juan Murube, MD, PhD is Professor of Ophthalmology, University of Acalà, Madrid, Spain. © 2013 Elsevier Inc. All rights reserved.
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Nobel Prize in Chemistry for his various discoveries in organic chemistry, including fluorescein, which was not yet very well known. Fluorescein can be prepared from phthalic anhydride and resorcinol in the presence of zinc chloride (Figure 2A). The disodium salt form of fluorescein is known as uranine, but in ophthalmic and medical terminology, it is referred to simply as fluorescein (Figure 2B). Earlier biocolorants of the ocular surface, such as carmin or indigo, were not introduced in standard ophthalmological practice, although some oculists began to use them in late XIX and early XX century. II. INTRODUCTION OF FLUORESCEIN IN OCULAR SURFACE EXAMINATION The medical examination of the external eye became increasingly precise over the XIX century. Doctors began using a positive lens of about þ10 or þ20 Diopters, and illuminated the eye with the ambient light from a window or with a lateral oblique light (wax candle, oil or petroleum lamp, incandescent lamp) focused on the eye. This was the usual technique in the XIX century, and in the XX century, it was still described in many handbooks of ophthalmology, some of which did not mention any other procedures.4 Czapski introduced the binocular biomicroscope for eye examination in 1896. In 1910, Gullstrand created the slit lamp, which he introduced in 1911.5 That year, he received the Nobel Prize in “Physiology or Medicine” for
his “work on the dioptrics of the eye,” including the slit lamp. Henker combined the Czapski biomicroscope with Gullstrand’s slit lamp.6 Diffusion of new inventions was slower then than it is now, and two decades later, the combination instrument was still little used. Similarly, the newly patented and manufactured fluorescein was slow to achieve practical use. In about 1880, Richard Ulrich injected fluorescein in the subcutaneous and periocular tissues to study the permeability of the cornea and the lens capsule, aqueous humor dynamics, and the causes of glaucoma and papilledema.7 Soon after that, Pflüger8 and Straub9 described the use of sodium fluorescein to detect breaks in the continuity of the corneal epithelium (abrasions and ulcers) in rabbits or humans. Icard then used it in “legal medicine” to study the dead or apparently dead, examining pupil color after intravenous injection of fluorescein (Icard’s test).10 In 1905, Angelucci subcutaneously injected 3.5 ml of a 20% fluorescein solution to study the origin of the aqueous humor,11 and in 1913, Strauss injected it in the body to study renal function in urine elimination.12 However, fluorescein did not surpass other vital stains until blueviolet-ultraviolet light (referred to below simply as “violet light”) was incorporated into the illumination system of the binocular slit lamp. This light gives fluorescein a luminous and brilliant yellow-orange color that contrasts with the non-illuminated areas. Over the third to fifth decades
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SOURCES IN TIME / Murube determined the tear film breakup time (TFBUT) without a slit lamp, fluorescein, or violet light illumination, and Sjögren did not report the use of fluorescein in his early publications, preferrring rose bengal.15 With the incorporation of violet light illumination, the use of fluorescein staining of the lacrimal pool and ocular surface spread quickly.
Figure 1. Adolf von Baeyer, Inventor of fluoresein (1835-1917).
of the XX century, the illumination system of slit lamps was gradually complemented with a cobalt glass filter (which has a large transmittance of blue-violet-ultraviolet wave frequencies). By the second half of the
century, violet light illumination was present in almost all slit lamps, and fluorescein became the most widely used surfocular vital stain. The process had been a slow one. Marx13 (1921) and Go Ing Hoen14 (1926) had
Figure 2. A. Preparation of fluorescein from phthalic anhydride and resorcinol in the presence of zinc chloride. B: Fluorescein sodium C20H10Na2O5, calculated on the anhydrous basis.
A. Instillation and Observation Fluorescein is aqueous-soluble but not liposoluble, so it does not dye the lipid layer of the tear film or the lipid secretion of the lid rims and caruncula.16 The fluorescence of fluorescein varies according to its concentration. In high concentrations over 1%, it is not very fluorescent, but in a diluted concentration of 0.1%, it is very fluorescent, reaching its maximal fluorescence in a concentration of 0.08g/l. The fluorescence diminishes with additional dilution.17 The most commonly used fluorescein solutions have a 1% concentration of sodium fluorescein. It does not allow optimal observation of the ocular surface immediately after instillation, but improves some seconds or minutes later, when diluted by the secreted tears and blinking. The solution is usually instilled on the exposed surface of the open eye, or in the lower conjunctival fornix, or in another selected specific place. The ocular surface is then examined at the ophthalmological biomicroscope under illumination with white light or with violet light produced by a blue exciter source (eg, “cobalt blue light”). The drop size of a usual fluorescein flask is about 20-30 ml, and when a smaller drop is needed for specific types of examination, a micropipette is used. Ousler et al noted that to detect ocular surface damage, it is sufficient to instill 5 ml of fluorescein in the lacrimal pool and to use violet illumination (with barrier filters such as Wratten #12 or Tiffen #11) and 16x magnification.18 The time between the instillation of the fluorescein and the slit lamp examination to optimize the observation varies with the amount
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SOURCES IN TIME / Murube of fluorescein, its concentration, the tear volume, and the tear turn-over rate. Depending on these factors, the optimal time may be from seconds to several minutes. Fluorescein is used in ophthalmology for many pleiotropic functions, as described below. In addition to its use in observing various ocular surface pathologies (Figures 3 and 4) and assessing tear properties and dynamics in humans, fluorescein has been useful in the measurement of intraocular pressure with Goldmann applanation tonometry (Figure 5), evaluation of the tear layer under rigid gas permeable contact lenses, study of aqueous humor in experimental animals,19 permeability of the vascular system of the chorioretina,20 penetration in the anterior chamber of liquids of various tonicities,21 etc. B. Staining of the Damaged Cornea In 1882, Pflüger was the first author to describe fluorescein vital staining of the cornea and conjunctiva.8 In 1891, Fromm and Groenouw reported the use of sodium fluorescein to stain damaged corneal epithelium, observing that the fluorescein penetrated the cornea when the epithelium was damaged but without actual cell loss.22 In 1905, Morax instilled a drop of saturated solution of fluorescein in the upper part of the cornea to identify corneal ulcers; following blinking, fluorescein was not visible on the corneal surface except where there was a loss of epithelium, seen as a red stain with greenish reflexes. At that time, neither the slit lamp nor cobalt filter violet illumination existed.23 In 1921, Brückner et al reported instilling a drop of fluorescein solution on the ocular surface to observe damaged corneas, although they usually employed the method commonly used at that time. That is, while the patient faced a paned window, they observed how regular or irregular the reflected grid appeared on the corneal surface. As fluorescein, the binocular microscope, and violet illumination became 146
Figure 3. Corneal ulcer seen with use of fluorescein. (Courtesy of Dr. Pino Cidad. Madrid.)
more widely used, it was realized that a healthy epithelium is practically impermeable to fluorescein, while corneal and conjunctival areas that lacked epithelial cells were stained. Later, it was realized that when the junctions between the cells are altered and when the fluorescein remains on the surface for some time, a small amount of fluorescein can go through these junctions and under the epithelium.18,24-26 Recently, it has been discovered that damage to the perilimbal stem cells surrounding the cornea facilitates the conjunctivalization of the corneal surface. The most evident manifestations of corneal conjunctivalization are the presence of mucin-secretory goblet cells in the corneal epithelium and metabolic alterations of the subjacent corneal stroma induced by the migrated conjunctival epithelium. The
intercellular permeability of the conjunctival epithelium to fluorescein is greater than that of the corneal epithelium,27 and therefore conjunctivalized corneas stain with fluorescein more than normal corneas. Margo noted that the cornea of aniridic patients is easily invaded by fibrovascular pannus,28 and Tseng et al associated the limbal insufficiency of congenital aniridia with its corneal opacification and vascularization.29,30 Fluorescein is used to examine many types of ocular surface lesions, including keratitis, mechanical wounds, chemical abrasion, keratosis, hypovitaminosis A, conjunctival invasion of cornea, etc.31 C. Staining of the Damaged Conjunctiva Fluorescein staining of the damaged conjunctiva yields information about
Figure 4. Keratopathia punctata seen with fluorescein. (Courtesy of Dr. E. Mateos. Madrid.)
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Figure 5. Intraocular tonometry with Goldmann applanation tonometer. Right image: A truncated cone is applied on the eye after instillation of fluorescein and with violet illumination. Second image: The size of the flattened ocular surface seen by the examiner through the cone with the stained meniscus manifests the intraocular pressure. (Courtesy of Dr. Pino Cidad. Madrid.)
different types of lesions and the tears retained in abnormal folds.32 The pleats of the normal conjunctiva are referred to as vertical and horizontal. The more or less vertical conjunctival pleats are in the medial and lateral exposed conjunctival trigoni, produced by the horizontal movements of the eyes. With fluorescein, these vertical pleats appear as many short, approximately vertical lines that occupy almost the whole conjunctival trigoni. They are less abundant in the medial trigonus,26 probably because these pleats are to some extent substituted by the caruncular and the semilunar pleats. With aging, some of these short wrinkles join vertically and form longer vertical folds. The horizontal conjunctival pleats in the upper and lower conjunctiva facilitate ductions in up- and down-gaze. They are present in the upper and lower conjunctival fornices from youth, but they are not in the exposed conjunctiva. The surface and pleats of the conjunctiva may be increased (conjunctivochalasis) or retracted. Conjunctivochalasis is an excessive redundancy and laxity of the conjunctiva. The compression of the lid on the conjunctiva displaces the redundant conjunctiva to the interpalpebral space and causes the formation of pleated lip-like folds, especially visible over the rim of the lower lid. Conjunctivochalasis was first reported by Middlemore in 1835,33 then by Ferradas in 187934 and Ehrlich in 1908.35 Conjunctivochalasis disrupts
the lacrimal meniscus of the lower lid, and hinders the spreading of tear over the cornea after blinking. It is usually diagnosed without fluorescein, but the fluorescein makes it more evident. Conjunctivochalasis is easily corrected with the new technique of surgical Z-plasty and YV-plasty.36,37 Retraction of the conjunctiva can be produced by various disorders, including chemical and thermal burns, synechiae, cicatricial pemphigoid, Stevens-Johnson syndrome, Lyell syndrome, Reiter syndrome, and many other pathologies. Most of these diseases cause a general retraction of the conjunctiva, including the horizontal and vertical pleats. The instillation of fluorescein and eversion of the lids allows evaluation of the progression or stabilization of the disease, but it is rather inexact, because these pleats also depend on the eversion applied to the lid and on the position of the eye. A better test for evaluating conjunctival retraction is to apply a drop of fluorescein solution on the eye and to measure the degree of ocular abduction necessary to make the folds disappear between the conjuntiva and plica semilunaris, and between the plica semilunaris and the caruncula (Figure 6).26,38 Successive examinations –days, months or years later– can be performed to measure the retraction of the conjunctiva and evaluate its progression or stability. The three furrows of the lacus lacrimalis in the medial part of the exposed ocular surface (episcleral conjunctiva/plica semilunaris, plica
semilunaris/caruncula, and caruncula/ medial canthus) can be better observed when a drop of fluorescein is instilled on the ocular surface. Observation depends on the quantity of fluorescein fluid instilled and on the position of the eye. Usually this test is done under topìcal anesthesia in successive examinations with slit lamp and violet light illumination–always with the same quantity and concentration of fluorescein. In the primary position of gaze, the first and second sulci of the healthy eye retains fluorescein across their whole length. When the eye is abducted, the superior halves of the sulci flatten out and disappear earlier than the lower ones. With greater abduction, the fluorescein disappears along the whole length of the sulci. The examination determines the degrees of ocular abduction at which the fluorescein line disappears in the upper and lower half of the sulci. Repetition of this test can determine whether or not the retraction of the ocular surface tissues is evolving. López-García et al have found a correlation between reduced lacunar furrows and shortened TFBUT.39 IV. ESTABLISHED CRITERIA FOR OCULAR SURFACE STAINING Fluorescecein staining is specifically cited in most of the criteria for diagnosis of dry eye syndromes. The National Eye Institute/Industry Workshop guidelines include fluorescein for clinical trials in dry eye as an indicator of the corneal epithelial integrity and for the TFBUT, and lissamine
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Figure 6. Sulci of the medial angle of the exposed conjunctiva in a normal eye, and evaluation with fluorescein with abduction of the eye. A. With eye looking straight ahead, the first (conjunctiva-plica semilunaris) and second (plica semilunaris-caruncula) sulci retain the fluorescein solution. B. When abducting 15 , the upper part of the sulci becomes flat, and the fluorescein disappears. C. When abducting 30 , the fluorescein disappears in almost all the exposed first and second sulci.26,38
green and rose bengal as indicators of conjunctival surface integrity.40 The Pisa Criteria (Workshop on Diagnostic Criteria for Sjögren’s Syndrome. Pisa, Italy, 1988) achieve a high degree of sensitivity and specificity with a small number of tests. The system divides the tests into office tests and laboratory tests. Office tests include a volumetric test for secretion (eg, Schirmer or Schirmer-Jones test, and a staining test using fluorescein or rose bengal. The laboratory tests measure the content of lysozyme, lactoferrin, and IgA, and the osmolarity.41 The diagnostic methodology recommended in the DEWS Report includes the use of fluorescein with yellow barrier filter (“violet” light) to measure TFBUT and corneal staining. Lissamine green is proposed to grade conjunctival staining.42 Various criteria and methods have been established for diagnosing 148
Sjögren syndrome. The first Japanese criteria (Government Japanese Criteria 1977-1986) for keratoconjunctivitis sicca include surfocular staining with rose bengal or fluorescein, although they considered the two stains to be different.43,44 The second and third Japanese Criteria (1997,1999) also accepted one or both stains. The San Diego Criteria (1986, 1999) accept surfocular staining with fluorescein or with rose bengal, because both stain the areas of devitalized surfocular tissues.45,46 The American-European Consensus Criteria (2002) allow surfocular staining with rose bengal to be substituted for other stains, such as fluorescein or lissamine green.47 The Oxford Scheme grades corneal and conjunctival staining for clinical trials. A series of panels, labeled A-E, illustrates staining patterns in order of increasing
severity. The use of fluorescein and lissamine green are recommended for staining.48 To meet the criteria for all the methods, the staining must have been performed specifically for the purpose of grading of the ocular surface, and cannot be the inadvertent or incidental staining produced by the Goldmann applanation tonometry or another circumstance.49 AUTHOR’S NOTE The use of fluorescein in lacrimal evaluation will be discussed in the October 2013 issue of this journal. REFERENCES 1. Kircher A. Ars Magna Lucis et Umbrae. Roma, Hermanni Scheus, 1646. Latin 2. van Leeuwenhoek A, 1674. Cited by Schierbeek A. Measuring the invisible world: The life and works of Antoni van Leeuwenhoek. London, Abelard-Schuman Publishing, 1959
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20. Novotny HR, Alvis DL. A method of photographic fluorescence in circulating blood in the human retina. Circulation 1961;24:82-6 21. Maurice D. The tonicity of an eye drop and its dilution by tears. Exp Eye Res 1971;11:30-3 22. Fromm, Groenouw. [About the diagnostic applications of the fluorescein stain in eye diseases. Arch Augenheilkd 1891;22: 247-257. German 23. Morax V. [Examination of patient and ocular semiology], in Lagrange F, Valude E. [French encyclopedia of ophthalmology]. Paris, Doin, Vol IV, 1905. p 352. French 24. Norn MS. Micropunctate fluorescein vital staining of the cornea. Acta Ophthalmol (Copenh) 1970;48:108-18 25. MacDonald EA, Maurice DM. Loss of fluorescein across the conjunctiva. Exp Eye Res 1991;53:427-30 26. Murube J, Chen Zhuo. Femto-biomicroscopic examination of dry eye. In: Murube J, ed. Ojo seco-dry eye. Madrid, Tecnimedia, 1997. pp123-34, 138-39 27. Huang AJW, Tseng SCG, Kenyon KR. Paracellular permeability of corneal and conjunctival epithelia. Invest Ophthalmol Vis Sci 1989;30:684-89 28. Margo CE. Congenital aniridia: A histopathologic study of the anterior segment in children. J Pediatr Ophthalmol Strabismus 1983;20:192-98 29. Tseng SCG. Application of corneal impression cytology to study conjunctival transdifferentiation defect. In: Orsoni JG, ed. Ophthalmic cytology. Proceedings of the International Symposium on Ophthalmic Cytology, 1987. Parma, Centro Grafico Edit, Univ di Parma, 1988. pp. 65-76 30. Tseng SCG, Chen JJY, Huang AJW, et al. Classification of conjunctival surgeries for corneal diseases based on stem cell concept. Ophthalmol Clin North Am 1990;3:595-610 31. Sommer A, Emran N, Tamba T. Vitamin A responsive punctate keratopathy in xerophthalmia. Am J Ophthalmol 1979;87:330-33 32. MacDonald EA, Maurice DM. The kinetics of tear fluid under the lower lid. Exp Eye Res 1991;53:421-25 33. Middlemore R. Chapter II: Chronic diseases and various affections of the conjunctiva and of the subconjunctival cellular membrane. A treatise on the diseases of the eye and its appendages. Vol. II. London. 1835 34. Ferradas J. [Clinical lessons on eye diseases]. Madrid, Impr, Telloo, 1879. Pp 35. Elschnig A. [Contribution to the etiology and therapy of chronic conjunctivitis]. Deuts Med Wochenschr 1908;34:1133-135. German
36. Murube E, Morum M, Murube J, Arnalich F. Conjunctivochalasis. A century of history. Its correction with Z-plasty and with YV-plasty. Studium Ophthalmologicum 2004;22:125-128 37. Murube J. Conjunctivochalasis. Its surgical treatment. Saudi J Ophthalmol 2005;19: 71-72 38. Murube J, ChenZhuo L, Murube E, et al. Measuring the lacunar sulci as a new indicator of shrinkage of the ocular surface. Eur J Ophthalmol 2001;11:227-32 39. López García S, García-Lozano I, MartínezGarchitorena J. Lacunar folds study in dry eye diagnosis. Arch Soc Esp Oftalmol 2003;78:21-8 40. Lemp MA. Report of the National Eye Institute/Industry Workshop on Clinical Trials in Dry Eye. CLAO J 1995;21:221-32 41. Murube J, Cortés Rodrigo MD. Eye parameters for the diagnosis of xerophthalmos. Criteria of Pisa (Italy). Clin Exp Rheumatol 1989;7:145-50 42. (No authors listed). Methodologies to diagnose and monitor dry eye disease: Report of the Diagnostic Methodology Subcommittee of the International Dry Eye Workshop (2007). Ocul Surf 2007;5:108-23 43. Homma M, Tojo T Akizuki M, Yamagata H. Criteria for Sjögren’s syndrome in Japan. Scand J Rheumatol 1986;61(Suppl):26-7 44. Ohfuji T. [Summary of the studies on Sjögren’s disease in 1977]. in Annual Report of the Ministry of Health and Welfare: Sjögren’s Disease Research Committee. Tokyo. Ministry of Health and Welfare. Japan, 1977-8:3-6. Japanese 45. Fox RI, Robinson CA, Curd JC, et al. Sjögren’s syndrome: proposed criteria for classification. Arthritis Rheum 1986;29: 577-85 46. Fox RI, Tornwall J, Michelson P. Current issues in the diagnosis and treatment of Sjögren’s syndrome. Curr Opin Rheumatol 1999;11:364-71 47. Vitali C, Bombardieri S, Johnsson R, et al. Classification criteria for Sjögren’s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002;61: 554-58 48. Bron AJ, Evans VE, Smith JA. Grading of corneal and conjunctival staining in the context of other dry eye tests. Cornea 2003;22:640-50 49. Zhivov A, Stachs O, Kraak R, et al. In vivo confocal microscopy of the ocular surface. Ocul Surf 2006;4:81-93
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