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Putting vital stains in context

Clin Exp Optom 2013; 96: 400–421 DOI:10.1111/j.1444-0938.2012.00802.x

Nathan Efron BScOptom PhD DScFAAOInstitute of Health and Biomedical Innovation,and School of Optometry and Vision Science,Queensland University of Technology, Kelvin Grove,Queensland, AustraliaE-mail: [email protected]

While vital staining remains a cornerstone in the diagnosis of ocular disease and contactlens complications, there are many misconceptions regarding the properties of com-monly used dyes by eye-care practitioners and what is and what is not corneal stainingafter instillation of sodium fluorescein. Similarly, the proper use and diagnostic utility ofrose Bengal and lissamine green B, the other two ophthalmic dyes commonly used forassessing ocular complications, have similarly remained unclear. Due to the limitationsof vital stains for definitive diagnosis, concomitant signs and symptoms in addition to acomplete patient history are required. Over the past decade, there have been manyreports of a type of corneal staining—often referred to as solution-induced cornealstaining (SICS)—that is observed with the use of multipurpose solutions in combinationwith soft lenses, more specifically silicone hydrogel lenses. Some authors believe thatSICS is a sign of lens/solution incompatibility; however, new research shows that SICSmay be neither a measure of lens/solution biocompatibility nor ‘true’ corneal staining,as that observed in pathological situations. A large component of SICS may be a benignphenomenon, known as preservative-associated transient hyperfluorescence (PATH).There is a lack of correlated signs and/or symptoms with SICS/PATH. Several proper-ties of SICS/PATH, such as appearance and duration, differentiate it from pathologicalcorneal staining. This paper reviews the properties of vital stains, their use and limita-tions in assessment of the ocular surface, the aetiology of corneal staining, characteris-tics of SICS/PATH that differentiate it from pathological corneal staining and what theSICS/PATH phenomenon means for contact lens-wearing patients.

Submitted: 9 June 2011Revised: 17 June 2012Accepted for publication: 19 June 2012

Key words: cornea, fluorescein, preservative-associated transient hyperfluorescence, solution-induced corneal staining, vital stains

The use of vital stains to detect ocularsurface abnormalities has been a mainstayfor eye-care professionals for nearly half acentury.1 Since their first use,2–4 much hasbeen learned regarding their physicalproperties and what the observed ocularsurface staining means with respect toocular health. With the advent of contact

lenses, ocular surface defects, disordersand diseases have increased in magni-tude.5,6 Differential diagnosis of ocularsurface disorders in contact lens wearers isoften difficult due to the overlappingsymptomatology and signs among differ-ent contact lens complications, as wellas non-contact lens-related disorders.7–11

The use of the three most commonlyused vital dyes, rose Bengal (RB),lissamine green B (LG), and sodiumfluorescein (NaFl),12 which havevarious properties and preferred uses(Table 1),1,3,13–35 can impart a wealth ofinformation regarding the health of theocular surface and aid in differential

C L I N I C A L A N D E X P E R I M E N T A L

OPTOMETRY

Clinical and Experimental Optometry 96.4 July 2013 © 2012 The Author

400 Clinical and Experimental Optometry © 2012 Optometrists Association Australia

diagnosis of ocular complications(Table 2).1,36,37 Due to ease of use andfamiliarity of eye-care professionals withvital dyes, there may have been an over-reliance on them for assessment of ocularhealth and a definitive cause of complica-

tions. Their value as a reliable diagnostictool is hampered due to several factors,especially our incomplete knowledge ofwhat these dyes do at a cellular andmolecular level. This has become appar-ent over the past decade due to the vast

research in dry eye syndromes (DES)38

and widespread use of silicone hydrogel(SiHy) lenses.39,40

The seminal study of Jones, McDougalland Sorbara39 in 2002 showed thathigh levels of asymptomatic corneal

Fluorescein Rose bengal Lissamine green B

At ocular pH (predominantly) Dianion (two negative charges);large population of monoanionswill also exist23

Dianion (two negative charges)24 Anion (single negative charge);large population of dianions willalso exist19

pKa a1 = 2.50; a2 = 3.81; a3 = 6.1023 a1 < 0; a2 = 1.89; a3 = 3.9324 a1 = 1.31; a2 = 7.66; a3 = 11.7219

Spectra* Abs (nm): lmax = 474–500[blue to cyan]23,28,30,75,80

Em (nm): lmax = 510–550[green]28,29,75

Reflect/transmit = Orange75

Abs (nm): lmax = 543–567,507–522†

[green to yellowish green]24,31,34,75

Em (nm): lmax = 565–570[yellowish green to orange]31–33,75

Reflect/transmit = Magenta75†

Abs (nm): lmax = 624–635[red]25,72,73,75

Reflect/transmit = Bluish green75

Illumination (Filter) Cobalt blue (Yellow Wratten #12)20 White light (None)1 White light (Red Wratten #24)84,161

Preferred use Cornea;13,26 conjunctiva (only withboth absorption and emissionfilters)27,144

Conjunctiva13,26; cornea (HSK, DE,other disorders with mucindeficiency)13,26,27

Conjunctiva;13,26 may be of valueassessing the cornea with a redfilter161

Test conductance NaFI-impregnated filter stripmoistened with a drop of sterilesaline (excess shaken off); assess1 to 4 minutes after applicationand several blinks27,84

RB-impregnated filter stripmoistened with a drop of sterilesaline (excess shaken off) or 25 mlof a 1% solution; assessed after afew minutes; perform towards endof ocular surface exam38,152

LG-impregnated filter stripmoistened with a drop of sterilesaline (excess shaken off) or 10 mlof 1% solution; assessed 1 to 4minutes after application84,161

Stains Healthy cells15,17,22,50,83

Damaged cells14,50

Dead cells17

Intracellular spaces14,17,21

Healthy cells18,21,35

Dead cells3,17

Damaged cells3,21

Mucous strands3

Damaged cells3,21

Dead cells3

Mucous strands3

Correlation with other stains Little correlation with RB andLG1,13,21

Little correlation with NaFI1,13,21

High correlation with LG3,13,16,84,95Little correlation with NaFI13

High correlation with RB3,13,16,84,95

pKa: the pH value at which equal concentrations of a molecule that differ by a single charge exist (X X X X- -+ ← →⎯⎯ ← →⎯⎯ ← →⎯⎯pKa pKa pKa1 2 3 2 )lmax: peak/dominant wavelength(s), Abs: absorption, DE: dry eye, Em: emission, HSK: herpes keratitis, LG: lissamine green, NaFI: sodium fluorescein,RB: rose bengal.* Absorption, emission and reflection are highly sensitive to environment; emission and reflection spectra are also highly dependent upon lightsource/excitation wavelength.79

† RB has a second lesser peak at 507–522 nm; the two peaks are responsible for the magenta, which falls in the non-spectral purples (a combinationof blue and red).24,31

Table 1. Characteristics of the most commonly used vital stains for ocular surface visualisation

Putting vital stains in context Efron

© 2012 The Author Clinical and Experimental Optometry 96.4 July 2013

Clinical and Experimental Optometry © 2012 Optometrists Association Australia 401

Observation Staining Signs Symptoms

MechanicalForeign body(RGP>SCL)1,11

NaFl—zig-zag track1 Tearing121 Sudden stinging; pain/discomfort while trappedunder lens/eyelid;121

asymptomatic to milddiscomfort once foreignbody is removed123

Abrasion NaFl—Deep or coalescedepithelial defect122

Tearing, blepharospasm, ciliaryinjection (severity of signsbased on extent of cornealinjury)121

Pain (often after lensremoval), FBS, reducedvisual acuity,photophobia122,123

Superior epithelialarcuate lesion (SEAL)1

(SiHy>non-SiHySCL or RGP)11

NaFl—superior arcuate;parallel to limbus7

None; some may experiencehyperaemia7

Most often asymptomaticduring lens wear/FBS afterlens removal; some mayexperience burning,irritation, lens awareness7

Lens binding/tight lenssyndrome1,11

(RGP/EW high waterhydrogel lenses)11

NaFl—Arc correspondingto lens edge or diffusestain in excessive bearing;1

RB/LG—possiblecircumlimbal103,124

Corneal oedema, conjunctivalhyperaemia, poor lensmovement, limbal indentation7,11

Irritation, visual defects,constant pain/discomfort,photophobia or may beasymptomatic123

ExposureKeratoconjunctivitissicca/dry eye syndrome(non-Sjögren’s) (causedor exacerbated bycontact lens wear)122

NaFl—punctate epithelialkeratopathy ininterpalpebral region;7,104

RB/LG—conjunctiva stainsmore intensely than cornea;cornea may also stain inmore severe cases37,127

Negligible tear meniscus at thelower lid, reduced tear break-uptime (TBUT)104

Dryness, burning/stinging,‘sandy’ or ‘gritty’ FBS,itchiness, excess tearing(epiphora), contact lensintolerance104–106

3/9 o’clock staining(RGP)1,108

NaFl—at the 3 and 9o’clock positions;1,108

conjunctiva adjacent tocornea at 3/9 o’clock (mostcommonly with highergrade corneal staining andsymptomatic patients)91

None to moderate bulbarhyperaemia adjacent to cornealstaining91,109

Asymptomatic (especiallywith low grade cornealstaining), discomfort, FBS,tearing, burning, reducedwearing times, andphotophobia91,126

Incomplete blink/lagophthalmos(inferior epithelialarcuate lesion/‘smilestain’)1

(SCL)11

NaFl—inferior arcuatepunctate band;1

RB—limited to inferonasalcornea and conjunctiva;37

LG—conjunctiva possiblewhen symptomatic110

None; though low blink rate,increased contact lensdeposition, inferior limbalhyperaemia, lid wiperepitheliopathy may beobserved110,125

Asymptomatic or milddiscomfort, irritation,dryness, or contact lensintolerance110,125

Metabolic and toxicHypoxia [non-SiHySCL/PMMA RGP]11

NaFl—central and diffuse111 Depending on severity: epithelial/stromal oedema and/or cell deathand desquamation, conjunctivalhyperaemia: chronic hypoxiacauses microcysts andneovascularisation111

Depending upon severity:temporary blurred vision,discomfort, photophobia orcan be asymptomatic111

Table 2. Pathological corneal staining: appearance, signs and symptoms. Lens type typically associated with complication in bracketsin first column. Images courtesy of Gary Foulks (keratitis sicca), Lyndon Jones (foreign body, lagophthalmos), the Association ofOptometric Contact Lens Educators (lens binding), the Digital Reference of Ophthalmology at the Edward S Harkness Eye Institute(bacterial keratitis) and the Bausch & Lomb Image Library (abrasion, SEAL, hypoxia, chemical keratoconjunctivitis, CLPU, CLSD,HSV and protozoan keratitis). Reproduction of the 3/9 o’clock image with permission from Morgan and colleagues.108





Chemicalkeratoconjunctivitis/conjunctivitismedicamentosa(SCL>>RGP)11

NaFl—Diffuse punctatestaining of cornea and/orconjunctiva;112

RB/LG—conjunctiva moreprominent than with NaFI112

Hyperaemia, eyelid oedema,corneal oedema, chemosis,pseudodendrites, follicles,scattered and/or disciform stromalinfiltrates, corneal epithelialerosions, SLK7,112

Irritation, ocular pain,stinging and burning (uponlens insertion in acutetoxicity), photophobia,blurred vision7,112

lnflammatoryContact lens-inducedperipheral ulcer(CLPU)

NaFl—overlying smallepithelial defect stains113,114

Mild to moderate conjunctivalinjection near round, focalperipheral infiltrate �2 mm, focalexcavation, watery discharge;anterior chamber reaction, ifpresent, is very mild113,114

Moderate to severe pain/discomfort, photophobia,FBS (all milder than inmicrobial keratitis);asymptomatic in somecases113,114

Contact lens-inducedacute red eye(CLARE) (EW)113

NaFl—when present isusually punctate,superficial, scattered113,114

Unilateral severe conjunctival andcircumlimbal hyperaemia, bothdiffuse and focal subepithelialinfiltrates most often in cornealperiphery, white and hazyoedematous cornea, anterioruveitis is common113,114

Acute onset of symptomsoften awakening patientsfrom sleep with irritation tomoderate/severe pain, FBS,burning, extremephotophobia113,114

Infiltrative keratitis(IK)(SCL)114

NaFl—overlay infiltratesor SPK, if present7,103

Mild to moderate redness, cornealoedema, usually single, peripheral/mid-peripheral small, round, hazy,grey-white, cloudy, or amorphouscorneal infiltrates that may besubepithelial or anterior stromal,occasional watery discharge113,114

Mild to moderate symptoms(occasionallyasymptomatic) includingphotophobia, blurred vision,irritation113,114

Contact lens-inducedsuperior limbickeratoconjunctivitis(CL-SLK)(DW?)103

NaFl—fine upper cornealpunctate staining, uppertarsal, bulbar, and limbalconjunctiva115

Mild to moderate superior tarsalpapillary hypertrophy (withmoderate reaction), superiorbulbar conjunctival hyperaemia,superior limbal hypertrophy,micropannus of superior cornealepithelium7,115

Mild to moderatediscomfort, dryness,burning, itching, blurredvision, contact lensintolerance7,114

Corneal limbalstem cell deficiency(CLSD)

NaFl—atypical stainingof the conjunctivalisedepithelium on corneaadjacent to limbus116

Poor epithelialisation of thecorneal surface, recurrent erosion,chronic stromal inflammation,corneal neovascularisation,conjunctivalisation of the cornea116

Reduced vision,photophobia, tearing,blepharospasm, andrecurrent episodes of pain,contact lens intolerance116

InfectiousBacterial keratitis(SCL)11

NaFl—ulceration stainsintensely117

Epithelial ulcer, dense, suppurativestromal inflammation withindistinct edges, stromal oedemaand tissue loss, anterior chamberreaction (hypopyon possible),Desçemet’s membrane folds,upper lid oedema, posteriorsynechiae, focal or diffuse cornealinflammation, conjunctivalhyperaemia, mucopurulentdischarge, endothelialinflammatory plaque7,117

Moderate to severe FBS,ocular pain, photophobia,tearing, decreased vision7,117

Table 2. Continued





Viral—herpes simplexvirus keratitis (HSK)(Not associated withcontact lenses)

NaFl—early: mild finepunctate; late: branchingpatterns stain brightly;RB/LG—early: brightfine punctate; late: stainepithelial cells surroundingthe ulcer, ‘terminalend-bulbs’118

Unilateral, punctate keratitis,may progress to single ormultiple branching dendriticulcers, may present also asstromal keratitis, endothelitis,uveitis and retinitis118

Tearing, FBS, photophobia,ocular pain and blurring ofvision118

Viral—epidemickeratoconjunctivitis(EKC)(Not typicallyassociated withcontact lenses)

NaFl—diffuse fine,superficial keratitis duringfirst week and focal,elevated, punctate epitheliallesions develop by Day 6 to13118

Acute onset of unilateral,followed by bilateral papillaryand follicular reaction,bilateral preauricularlymphadenopathy, centralcorneal erosions during firstor second week after onset,coalescence of lesions toform coarse spots ofsubepithelial infiltrates,small-rounded subepithelialopacities may bepersistent118

FBS, photophobia,conjunctival hyperaemia,eyelids stuck togetherupon waking, blepharitis,serofibrinous discharge;ocular symptomscommonly preceded bysystemic symptoms118

Fungal NaFl—may overlayinfiltrates or SPK7,114

Mild to moderatehyperaemia, discharge,fine or coarse, granular,grey-white epithelial/stromalinfiltrates, typically irregularfeathery-edged, white ring inthe cornea, satellite lesionsprimary focus of theinfection; in advance cases:suppurative stromal keratitis,conjunctival hyperaemia,anterior chamberinflammation, hypopyon,iritis, endothelial plaque orpossible cornealperforation7,114

Mild to moderate FBS,decreased vision, ocularpain, photophobia7,114

Protozoan—Acanthamoebakeratitis

NaFl—Overlying epithelialdefect7

Usually unilateral,conjunctivitis or conjunctivalhyperaemia, cornealulceration, lid oedema,characteristic corneal ringstromal infiltrate, anterioruveitis of fluctuating severity,increased intraocularpressure, hypopyon; scleritismay be found in advancedcases7

FBS, severe ocular pain,photophobia, blurred vision;pain often disproportionateto signs in early course ofthe disease7

DW: daily wear, EW: extended wear, FBS: foreign body sensation, LG: lissamine green B, NaFl: sodium fluorescein, PMMA: poly(methyl methacrylate),RB: rose Bengal, RGP: rigid gas-permeable contact lenses, SCL: soft contact lenses, SiHy: silicone hydrogel contact lenses, SLK: superior limbickeratoconjunctivitis, SPK: superficial punctate keratitis.

Table 2. Continued




hyperfluorescencea or ‘corneal staining’can be observed after instillation of NaFl,when subjects used polyhexamethylenebiguanide (PHMB)-based contact lensmultipurpose solutions (MPS). There hasbeen much controversy regarding theimplications and cause of this phenom-enon, which is often referred to as solution-induced corneal staining (SICS). Jones,McDougall and Sorbara39 stated thatsome subjects showed ‘a level of stainingconsistent with a classical solution-basedtoxicity reaction’,39 igniting widespreadresponse. Eye-care professionals generallyconsider corneal staining after instillationof NaFl to indicate corneal compromise.2,41

The implication of this statement is thatMPS must be damaging the corneal epithe-lium in some manner, even in the absenceof any other signs or symptoms.42 Since thatstudy,39 much research has shown that SICScan be largely explained by a benign, non-pathological phenomenon referred to as‘preservative-associated transient hyper-fluorescence’ or PATH.15,22,40,42–71 Addition-ally, recent studies have begun to questionwhat vital dyes—especially NaFl—are orare not staining.

The purposes of this review are to: givean overview of the structure, dynamics andproperties of the molecules that make upvital stains; discuss the benefits and draw-backs of vital stains and the means of meas-uring it; review the prevalence of ocularvital staining and consider the reasonsfor the inconsistencies observed in theliterature, and detail how SICS/PATHb

may differ from pathological and othernon-pathological situations commonly

observed in contact lens wearers based onNaFl corneal staining/hyperfluorescencepattern and concomitant signs and/orsymptoms.

VITAL STAINS: PROPERTIES ANDMECHANISMS OF ACTION

Understanding what the observedstaining/hyperfluorescence with vital dyesmay indicate is essential to understandingtheir properties and our perception ofthem. Briefly, stains (dyes) contain mol-ecules that absorb specific wavelengths (l)of light within the visible range of the elec-tromagnetic spectra (l = 400 to 700 nm).Similar to other dyes, fluorescein (FL, thefree non-salt form), RB and LG eachabsorb a range of wavelengths thatencompasses a dominant wavelength(s) orpeak (lmax), where the most energy isabsorbed (FL: lmax = 474 to 500 nm[spectra: blue to cyan; total range400 to 530 nm]; RB: lmax = 543 to 567,507 to 522 nm [spectra: green to yellow-ish green; total range 480 to 590 nm]and LG: lmax = 624 to 635 nm[spectra: red; total range 550 to 690 nm])(Table 1).23–25,28,30–33,72,73

When dyes are illuminated with whitelight, our eyes detect the wavelengthsabsorbed by the dye and respond to thelack of a complete range of wavelengths byseeing the non-absorbed wavelengths ascoloured light.74 The colour observed isreferred to as the complementary colour(wavelengths) of that which was removed,typically of the dominant wavelength(s)(Table 1).74,75 This is not fluorescence butrather reflection or transmission of light.Due to their absorption peaks, duringa slitlamp examination with white light,RB appears magenta (Figure 1) and LGappears bluish green (Figure 2a).75,76

When visualising LG using a red filter,which transmits wavelengths that LGabsorbs, it appears black on the conjunc-tiva (Figure 2b).27,76

Depending upon the molecular struc-ture of the dye, absorbed energy isdissipated through heat, bond breakage(photobleaching), a photon of light (fluo-rescence) or energy transfer to a nearby

a Based on recent findings by Bakkar andcolleagues22 and the group at the JulesStein Eye Institute,13,37 hyperfluorescence/hyperfluorescent is defined as the bright greensignal observed after instillation of fluorescein(either free or as a disodium salt) on the cornea(for example, corneal staining) in vivo or ex vivoand cell-based assays in vitro. Fluorescence/fluorescent is defined as the background glowseen on the cornea in vivo or ex vivo or in cell-based assays in vitro and the emitted lightobserved in non-cell-based assays using any fluo-rescent molecule either free or as a label/probe.

b Throughout this paper, the associated term‘SICS/PATH’ will be used, as these phenomenaare largely synonymous (that is, SICS can belargely attributed to PATH).

Figure 1. Rose Bengal staining of a dendritic ulcer in a case ofHerpes simplex virus keratitis. Image courtesy of Peter FahmyMD. Available at: http://xn–jenlgekbenhavn-3ib7zia.dk/herpes-hornhindebetaendelse-herpes-simplex-keratitis/.




entity (for example, quenching).77,78

While all dyes absorb various wavelengthsof light, not all are fluorescent.79 Thoseshowing fluorescence usually containseveral ring structures (aromatic) or con-jugated double bonds (that is, alternatingsingle and double bonds between atoms)in a rigid configuration, which reducesthe loss of absorbed energy by motion(heat).77,78 The dye molecules can onlymaintain the excited energy state (certainelectrons are raised to a higher energystate by absorbed light) for a very shorttime as it must return to the lowest, moststable energy state.79 As fluorescent mol-ecules use little of the energy for molecu-lar motion, they emit the excess energy asa photon.77 Fluorescent molecules absorbshorter wavelengths of light (moreenergy) and emit longer wavelengths(less energy) (Table 1).77 For example, thepeak absorption of FL is between 474 and500 nm (blue to green blue range)23,28,30,80

and the peak emission is between 510and 550 nm,28,29 which is within the greenrange that characterises the non-saltfluorescein.75,81

The use of NaFl to assess the health ofthe cornea dates back as early as 1882;2

however, its full potential was not realiseduntil the introduction of the modern slit-lamp biomicroscope82 with a cobalt filterto visualise the cornea in greater detail.20

The use of a blue filter is essential as thenarrow band of fluorescent emission has

low contrast against the wide range ofwavelengths contained in white light thatmakes up the background illumination.48

The potential cellular mechanisms in-volved in corneal surface hyperfluores-cence most commonly described aresurface pooling,14 uptake by dead17 ordamaged cells,14,83 and ingress aroundcells where there is a loss of epithelial celljunction integrity17 (Table 1). Nonethe-less, several studies have shown NaFlcorneal staining/hyperfluorescence to bea normal physiological phenomenon dueto desquamation, where the intensityand duration vary based on the mitoticrate49 and/or entry of FL into healthycells.15,17,22,50,83

Most studies do not report whethermicropunctate or macropunctate hyper-fluorescent spots on the cornea after instil-lation of NaFl represent a single epithelialcell or a group of epithelial cells. Based onthe physics of human vision and the prop-erties of FL, a single hyperfluorescentcorneal epithelial cell would be easilyobservable via slitlamp biomicroscopy atthe most commonly used settings (10¥ or16¥ magnification84). While the unaidedhuman eye can resolve objects of approxi-mately 100 mm85 (approximately the widthof a human hair86), objects of muchsmaller size (down to about 10 mm [10¥]or 6.25 mm [16¥]) should be visible undera slitlamp using the appropriate filters.Under these conditions, single corneal

epithelial cells should be discernible asthey range from 8 to 52 mm.87,88 Further-more, FL is extremely luminous and emitsthe most light within the 510 to 550 nmrange, which is very close to the wave-lengths where the human eye showsmaximal sensitivity (555 nm).81 Based onseveral calculations, this assumption isconfirmed by a recent study,15 whereresearchers demonstrated via membranecytology that the hyperfluorescent micro-punctate spots visualised on the cornea viaslitlamp were localised to a single cellwithin the epithelium (Figure 3) in everycase. Specifically, the cells distributed tothe first three cell layers, mainly superfi-cial and wing cells that range in size from30 to 50 mm,88 are the cells responsiblefor micropunctate spots (Figure 3).15

Although this study was performed inpatients with ocular complications,15

based on this information in conjunctionwith the previously mentioned studies inhealthy eyes and cells,17,22,50,83 micropunc-tate spots most likely represent a singlecell or a few cells and macropunctatespots represent a group of contiguouscells.

While NaFl can be used to visualise theconjunctiva, using both blue and yellowfilters,20 its rapid stromal diffusion makesit difficult to discriminate between stainedcells and intercellular staining, making itless ideal for conjunctival staining.17,89

Studies assessing the relationship betweencorneal staining/hyperfluorescence andconjunctival staining with NaFl haveshown that there is either no correlationor a low correlation with questionableclinical relevance between the two loca-tions in contact lens wearers.51,90 Oneinteresting exception is 3/9 o’clock stain-ing in rigid gas-permeable lens wearers,91

which seems specific to this aetiology, asadditional findings in these subjects areinconsistent with other types of cornealstaining/hyperfluorescence.92,93

The use of RB to visualise the ocularsurface was first described in 1919.4 RoseBengal stains live as well as dead cells andcan be blocked by mucin, suggesting thatnormally, the absence of RB staining is dueto the protective function of the tearfilm.18,94 While RB is derived from FL, they

Figure 2. Lissamine green staining of the bulbar conjunctiva under low white lightillumination without a barrier filter (A) and with a red barrier filter (Wratten #25equivalent) (B). Reproduced with permission from McDonnell.76




differ as FL lacks intrinsic toxicity, photo-sensitising capacity, and the ability to beblocked by some tear components and sub-cellular localisation.17,18 While it has been along-held belief that NaFl corneal stainingis not blocked by mucin,17 new research hascalled this into question.50 It has often beennoted that little correlation is observedbetween NaFl staining/hyperfluorescenceand RB/LG staining.1,13,17

In 1973, Norn3 described the use of LGfor staining the conjunctiva and cornea. Itis generally believed that similar to RB, LGstains damaged or dead cells and mucousfibrils (Table 1) but with less toxicityand stinging than RB.3,95,96 Additionally,LG does not stain healthy corneal cells21

but only those with membrane damage,

whereas RB stains healthy cells and nega-tively affects their vitality.3,96 Irrespective ofthese differences between RB and LG,there is a high correlation between theirstaining pattern in patients with dry eyesyndromes.16 The lack of toxicity has madeLG the preferred dye to assess the con-junctiva in practice.13

While vital stains are essential tools inthe armamentarium of eye-care profes-sionals, there are several limitations of allthree stains. The first and possibly themost important is that we do not trulyknow:1. what each dye binds to14,21,36

2. why molecules with very similarproperties show different staining pat-terns13,14,17,73,80,89,94 and

3. why those more dissimilar have thesame staining pattern.3,16,19,21,95

This lack of specificity highlights the factthat these dyes are not molecular probes.It is only when they are attached to a tar-geting system (that is, an antibody), whichis directed to specific proteins or cellularlocations, that conclusions can be madewith regard to specificity.97,98

Additional drawbacks of all threestains are their propensity for photo-bleaching.99–101 This is especially true ofFL, which loses approximately 70 per centof its fluorescent intensity within 60seconds.100

While RB has acid dissociation con-stants (the pH at which there is a changein the charge of the molecule) well below

Figure 3. Impression cytology of a punctate spot. A: Clinical photograph of limbal area exhibiting a hyperfluorescent spot, encircled.Linear limbal fluorescence provided fiducials. B: Disappearance of the punctate spot after impression cytology. C: After repeatinstillation of fluorescein, a hyperfluorescent outline of the membrane remains. D: Enlargement of the punctate spot encircled in (A)and the corresponding spot on the impression membrane, viewed with epifluorescence (E). F: The hyperfluorescent spot localised toa cell (circle) after rapid, air-dried staining of the membrane, (G) which can be visualised at high-magnification to confirm that thehyperfluorescence was observed from a single cell (staining of sample with Diff-Quik). Original magnification: (A–D) 10¥; (E,F) 100¥;(G) 400¥. Reproduced with permission from Mokhtarzadeh, Casey and Glasgow.15




the pH range found in the eye,24 FL andLG have dissociation constants of approxi-mately 6.10 and 7.66, respectively, makingtheir absorption and/or emission spectraextremely sensitive to ocular pH.19,23,73,102

Another undesirable property of FLis that fluorescent intensity is highlyconcentration-dependent due to self-quenching.102 Therefore, a narrow rangeof concentrations exists where the absorp-tion and fluorescence is proportional.

Based on the most contemporary infor-mation, there is much that still needs to beelucidated regarding the interactions ofvital stains and the cornea and conjunctivabefore it can definitively be said whatNaFl, RB and LG staining or hyperfluores-cence of the ocular surface actually meanswith regard to pathophysiology. Further,vital dyes are extremely sensitive to theirenvironment, which makes it difficultto compare ocular surface staining/hyperfluorescence in a clinical setting witha high degree of accuracy. Therefore, theuse of these dyes may best be reserved forgross examination of the ocular surfacerather than to gather molecular insights.

CORNEAL AND CONJUNCTIVALSTAINING

While FL, RB and LG are not molecularprobes, they are excellent tools forhighlighting gross structural changesin pathological conditions. There aresix types of clinically important cornealstaining in contact lens wearers: mechani-cal, exposure, metabolic, toxic, inflamma-tory and infectious (Table 2).1,7,91,103–118

These pathological conditions in con-tact lens wearers where corneal stai-ning may occur can be differentiatedfrom:1. each other2. pathological situations in non-contact

lens wearers that may mimic thosefound in contact lens wearers and

3. hyperfluorescence in non-patho-logical situations (Tables 2 and3).1,39,42,43,48,119–124

This differential diagnosis is based on thepattern and/or location of cornealstaining/hyperfluorescence, the presenceof concomitant signs and/or symptomsand the patient history.1,7

In situations of corneal abrasion, thepattern often provides the necessary cluesto indicate the aetiology of the staining(Table 2).1,7,103,120,121 These conditionstend to be unilateral and are accompaniedby mild-to-moderate signs and symptoms,although in the case of superior epithelialarcuate lesions (SEALs),7 patients maybe asymptomatic without any additionalsigns.

Exposure keratopathy may be character-ised by its staining pattern, bilateralpresentation and symptoms.1,91,104–110,125

Staining located in the nasal-temporalregions (3/9 o’clock)91 can be easily dif-ferentiated from other conditions, as wellas keratoconjunctivitis sicca (KCS) andlagophthalmos simply by corneal stainingpattern (Table 2).1,37 Further differentia-tion of KCS and lagophthalmos from eachother and other pathological conditions,which may present with similar signsand/or symptoms, may be achieved withuse of RB or LG. In lagophthalmos, RBstaining is limited to the inferonasalcorneal and conjunctival epithelium witha clear line between stained and unstained


Dimple veil NaFl—pooling rather thanstaining, usually with RGPlenses48,119

None48,119 Asymptomatic119 ormild temporary loss ofvisual acuity48

Mucin balls NaFl—pooling rather thanstaining48

None48 Asymptomatic48

Physiologic staining NaFl—superficial punctatestaining of grade 2 or less1

None1 Asymptomatic1

SICS/PATH NaFl—superficial punctate,commonly central sparing39,42

None39 Asymptomatic39,43

Table 3. Non-pathological corneal hyperfluorescence: appearance, signs, and symptoms. Images courtesy of theBausch & Lomb Image Library (dimple veil) and the CCLRU (SICS/PATH). Image (mucin balls) reproduced withpermission from Morgan and Maldonado-Codina.48




tissue.37,110 In KCS early or mild cases aredetected more easily with RB/LG thanwith NaFl and the conjunctiva usually isstained more intensely than the cornea.In mild KCS, nasal conjunctival RB/LGstaining is commonly observed. As theseverity of the condition progresses,RB/LG staining is observed on the nasaland temporal conjunctiva (moderateKCS) and then finally on the nasal andtemporal conjunctiva and cornea (severeKCS).37,104,126

Metabolic or toxic staining will presentbilaterally with central corneal staining. Adetailed history and concomitant signs,including LG conjunctival staining andsymptoms will aid in diagnosis.7,111,112 WhileRB conjunctival staining may aid in thediagnosis of toxic staining, this is not rec-ommended as these patients are alreadyexperiencing substantial discomfort.7,112

With inflammatory and infectious condi-tions, only contact lens-induced superiorlimbic keratoconjunctivitis (CL-SLK) maybe differentiated based solely on the stain-ing pattern,7,115 whereas others requirecareful consideration of additional signs,symptoms and patient history.7,113,114,116–118

Similar to KCS, RB staining is highly diag-nostic for Herpes simplex virus keratitis

(HSK).37 Conditions such as epidemickeratoconjunctivitis (EKC) and Thyge-son’s superficial punctate keratopathy(SPK)127 may be confused with con-tact lens-associated infiltrative keratitis(CLAIK)114,128 based on pattern andlocation of infiltrates.9,111,114,128,129 Cornealstaining is highly apparent in EKC duringthe second week of the outbreak118 butNaFl corneal staining is variable, oftenmild or absent, during the first week ofEKC infection,118 in CLAIK114,130 and inThygeson’s SPK,131 so a detailed history andfull examination are essential. These threeconditions can be differentiated by patienthistory, since only CLAIK is associatedwith contact lens wear,114,128,132 EKC is char-acterised by acute onset unilaterally fol-lowed by bilateral involvement and contactwith an individual with red eyes or an upperrespiratory infection, usually themselves oranother family member114,133,134 and Thyge-son’s SPK has a chronic, remittent course.9

There are several instances of non-pathological hyperfluorescence, includ-ing mucin balls, dimple-veil and SICS/PATH,48,119 with distinct characteristichyperfluorescent patterns.39,48,119 Addi-tionally, many persons have low-gradephysiological corneal staining/hyper-

fluorescence, typically Grade 1 or 2 thatis considered a normal phenomenon(Table 3).1,41,135,136 All of these situationstend to be asymptomatic without addi-tional signs,39,43 with resolution of hyper-fluorescence typically after several hours,and at most overnight in the case of phy-siological corneal staining/hyperfluore-scence, without any intervention.1,5,43,137

INCIDENCE OF OCULAR SURFACESTAINING IN NON-MULTIPURPOSESOLUTION USERS

In the literature, the incidence of cornealstaining in normal non-contact lenswearers ranged from six to 79 per cent(Table 4).41,135,136,138–140 Similarly, success-ful contact lens wearers display micro-punctate corneal staining in up to 66 percent of individuals (Table 5).55,90,141–143

One report from 2002 found that even insuccessful contact lens wearers, eight percent had moderate-to-severe corneal stain-ing (cumulative staining score of 3 ormore with at least one quadrant score of 2or more on a 0–4 scale).55 In agreementwith these results, a recent study by thesame group found 54 per cent of contactlens-wearing patients had corneal staining

Number of Observations Prevalence (%) Age (years)‡ NaFI Concentration Method of Instillation Reference

84 subjects 6 11–13 NR NR Soni 1996138

411 eyes 17 Adults 0.125% Drop (0.01 mL) Norn 197041

100 subjects 19–42† NR 2% (1–7 times) Drop (volume NR) Korb 1979107

21 subjects 48 18–36 2% (7 times) Drop (volume NR) Josephson 1992139

300 subjects 58 <35 2% Drop (volume NR) Korb 1970141

49 eyes 73 Adults 1% Drop (0.01 mL) Norn 197041

16 subjects (620observations)

78 29–44(mean 34 years)

0.6 mg§ Ful-Glo strip wetted with2 drops of unpreservedsterile saline

Schwallie 1997135

102 subjects 79 18 to 50(median 22 years)

1.0 mg§ Fluoret wetted with 1 dropof unpreserved saline.

Dundas 2001136

NR: not reported.† Lower number after first instillation and highest number following sequential instillations.‡ Mean or median age included if reported.§ As reported in Bron, Evans and Smith.27

Table 4. Prevalence of superficial punctate corneal staining in normal, asymptomatic, non-contact lens wearers




present, but the average overall sum andnumber of patients with at least moderatestaining (3 or more out of 4) were greaterwith an average overall summed cornealstaining grading score of 1.96 � 3.40 andwith 26 per cent of patients showing atleast moderate staining.52

Recently, Szczotka-Flynn and col-leagues40 reported that in the Longitudi-nal Analysis of Silicone Hydrogel (LASH)Contact Lens Study, where subjects worelenses for up to 30 days of continuouswear (CW) (no MPS were used), 80.5 percent of subjects had at least one episode ofmild corneal staining (Cornea andContact Lens Research Unit [CCLRU]scale) and 38.1 per cent had at least oneepisode of moderate corneal staining.Much of the corneal staining was asympto-matic and observed during regularlyscheduled visits.

Few reports exist in the literatureregarding the prevalence of NaFl con-junctival hyperfluorescence/staining inhealthy non-contact lens and contact lenswearers and of those found, no two studiescontained the same population, used thesame lens material, modality or type orused the same grading system.90,144–146 Inthe three studies that used cobalt blue illu-mination and a yellow emission filter, NaFlconjunctival hyperfluorescence/stainingwas observed in 33 to 98 per cent of sub-jects (both contact lens and non-contactlens wearers), with clinically significantgrades (Grade 2 or more) in 12 per cent ofnon-contact lens wearers and four to 37per cent of contact lens wearers.90,144,146

While these data imply that NaFl conjunc-tival hyperfluorescence/staining probablyoccurs in a large proportion of contactlens wearers and may even be quite high,there are not enough similar studies toestimate the prevalence and level of con-junctival hyperfluorescence/staining to beexpected in healthy contact lens wearers.

Similar to NaFl, studies showed con-junctival LG staining is increased incontact lens wearers compared withhealthy non-contact lens wearers, irrespec-tive of ocular region.145,147–149 A recentstudy by Maissa, Guillon and Wong148

found that 51 per cent of healthy non-contact lens wearers showed LG staining

compared with 73 per cent of contactlens wearers (p < 0.001). There are toofew studies in contact lens wearers andof those available, inconsistent use ofgrading systems does not allow for an esti-mation of a true rate of LG staining or thelevel of severity likely to be observed inhealthy non-contact lens and contact lenswearers.

Rose Bengal ocular surface staining inhealthy non-contact lens wearers was lessthan or equal to 0.5 on scales that range0–9 or 0–12, irrespective of location.150–152

The one study in soft contact lens wearersfound that staining increased from 0.25 �

0.44 in healthy non-lens wearers to 2.0 �

1.2 points in soft-lens wearers, using thevan Bijsterveld (0–9) scale.150 Rates inhealthy contact lens wearers are notknown.

VITAL STAINING ANDDISCREPANCIES IN THELITERATURE

There are several reasons for the largeranges and discrepancies in the percent-age of patients and degree of vital ocularsurface staining, especially NaFl cornealstaining/hyperfluorescence, reported inthe literature. With regard to prevalence,outstanding factors in both non-contactlens wearers and lens wearers are theage of the population being studied, asthose with younger subjects have fewer‘stainers’ (Tables 4 and 5)41,55,90,135,136,138–143

and the method of instillation and theconcentration of NaFl being applied(Table 4).41,107,135,136,139,140

With regard to severity, the scale used tograde corneal staining/hyperfluorescencemay influence results, as investigators usedthe Efron, Cornea and Contact LensResearch Unit or the modified CCLRU/global grading systems (Table 5).55,90,141–143

The potential impact of using differentgrading scales on outcomes is supportedby two studies that compared variousgrading systems and found the gradingestimates to be significantly different (p =0.0001).153,154 For example, the artist-rendered systems (that is, Efron scale), ingeneral, showed lower severity of grades

with better grading reliability than thephotographic systems (that is, CCLRU).153

The CCLRU grading system incorpo-rates scales for grading the type, extentand depth of corneal staining and studieshave shown a significant positive correla-tion (Spearman’s rho [r] = 0.49–0.54; p =0.001) between these measures.142,145 Fur-thermore, whether corneal staining typeand extent were graded separately or com-bined, a similar reliability relationship hasbeen observed.155 These studies suggestthat additional information, in most situa-tions, may not be obtained by using morethan one measure of corneal staining.Interestingly, studies have shown that theextent of SICS/PATH hyperfluorescencecan range from one to 74 per cent of thecorneal surface,42 while almost all corneasshowed punctate (mostly micropunctate[type up to Grade 1]) hyperfluorescentspots, which were all located in the super-ficial epithelium (that is, at a depth of upto Grade 1).39,42–44 These findings suggestthat along with measuring area, depth andtype should be considered for accurateassessment of SICS/PATH.

Additionally, the increments of thegeneral grading scales were best describedby a quadratic rather than a linear func-tion and the printed grading scales weremore sensitive for grading features of lowseverity, but grades were not comparablebetween grading scales.156 For generalcorneal staining/hyperfluorescence grad-ing scales, the linearity for using edgedetection was r2 = 0.49 and r2 = 0.51, forthe Efron and CCLRU scales, respec-tively.156 In contrast, a scale developedspecifically for 3/9 o’clock staining hada linearity value of r2 = 0.99.156,157 Theseresults suggest that grading scales forspecific conditions may improve thesensitivity and specificity of gradingstaining/hyperfluorescence. Differencesof corneal staining incidence andseverity in studies may also be due to themethod of notation in patient records,as this may impact on optometricpractices.158

Another source of discrepancy is therepeatability and consistency of gradingvital staining. In a study by Nichols,Mitchell and Zadnik,159 the repeatability




of NaFl corneal staining overall was fair,with all but one region having moderaterepeatability and NaFl conjunctival stain-ing having poor to fair repeatabilityin patients with dry eye syndrome. Addi-tionally, percentage agreement betweenvisits showed low to moderate consistency(59 to 83 per cent). Similarly, RB stainingshowed little agreement between observ-ers except in the temporal region ofboth the cornea and conjunctiva, wheremoderate agreement was found.159 Incontrast, a separate study found mod-erate to substantial levels of intra- andinter-observer reliability of NaFl andRB corneal staining using a differentgrading scale.38 While intra-observer reli-ability when assessing conjunctival stain-ing with NaFl and RB was similar to thatwhen assessing the cornea, there was onlymodest inter-observer reliability whenassessing conjunctival staining.38

Conjunctival staining with LG has sub-stantial within-grader reliability160 but onlymoderate between-grader reliability.160,161

Disease severity influences grading reli-ability of the conjunctiva with LG, sincegrading was less reliable when more sig-nificant staining was present.160 Lissaminegreen corneal staining scores were also sig-nificantly less reliable than those for theconjunctiva, although use of the red filterwas valuable for evaluating LG cornealstaining.161 Additionally, the use of 10 mlLG showed the highest reliability andthis rose with greater experience of theobserver.161

PRESERVATIVE-ASSOCIATEDTRANSIENT HYPERFLUORESCENCEAS A PARTIAL OR COMPLETEEXPLANATION FORSOLUTION-INDUCED CORNEALSTAINING

A recent study by Nichols and Sinnott52

found several factors were relatedto increased corneal staining/hyper-fluorescence, including increased dailywearing times (p = 0.0006), lower income(p = 0.0008), LG conjunctival staining (p =0.002), contact lens deposition (p =0.007), increased tear meniscus height (p= 0.007) and decreased hydrogel nominal

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water content (p = 0.02). The wearing ofSiHy lenses was protective against cornealstaining/hyperfluorescence (p = 0.001).Notably, neither contact lens care solutionbrands nor preservatives, such as PHMBfound in some lens care solutions,were associated with corneal staining/hyperfluorescence. Additionally, over-night wear, a well-known risk factor forinfiltrative events, was not associated withcorneal staining/hyperfluorescence (p =0.27). A similar study published in 200255

evaluating only successful hydrogel lenswearers (defined as minimum wear eighthours per day, five days per week for morethan a month), also found that neitheroverall (all grades; p = 0.165) normoderate-to-severe (p = 0.567) cornealstaining/hyperfluorescence were associ-ated with lens care systems.

Based on the findings of this reviewin conjunction with a large body ofdata,1,37,39,42–45,48,58–63,91,104,109,110,112,115,117,151,162–

172 there are several important differencesbetween NaFl corneal staining in patho-logical situations and those observed insubjects with SICS, suggesting that thisform of staining can be largely explainedby PATH. Furthermore, it appears thatSICS/PATH largely represents a benignartifact, rather than the pathologicalcorneal staining seen during contact lenscomplications.

DURATION OF SICS/PATH

Healing, defined as absence of cornealstaining/hyperfluorescence after topicaladministration of NaFl,173 in clinicallysignificant ocular complications canrange from approximately one to sevendays, depending upon depth and sizeof lesion.162,173 In conditions such ascontact lens-induced peripheral infil-trates, corneal staining persists over dayseven with significant improvement ofsymptoms.165,174

SICS/PATH typically peaks between 30minutes and four hours after lens insertionand is reduced or absent after six to eighthours, depending on the preservative inthe MPS.43,59,62 Solutions containing thepreservative PHMB show peak SICS/PATH response one to four hours after

lens insertion, depending upon the studyand solution,43,44 which is substantiallyreduced to low levels or absent after six toeight hours.43,62,172 Solutions containingthe preservatives polyquaternium-1 (PQ-1)and Aldox show substantially higher levelsof SICS/PATH at 30 minutes, when com-pared with levels observed at two hourspost-lens insertion.59,175

Studies have suggested that the differ-ences in the timing of peak SICS/PATHbetween solutions are a result of differ-

ences in peak release of preservativesfrom soft lenses (Figure 4).64,65,176 All pre-servatives are adsorbed by all soft contactlens materials during the soak.64,65,176,177

The amount adsorbed and rate ofadsorption depends on lens material, pre-servative and MPS formulation.64,65,176,177

Research suggests that after insertion, thelens releases the preservative into thetear film.64,65,176,177 Similar to adsorption,the release rate depends on the pre-servative and lens material combination

Etafilcon AAlphafilcon ABalafilcon ASenofilcon ALotrafilcon AEtafilcon A release curveAlphafilcon A release curveBalafilcon A release curveSenofilcon A release curveLotrafilcon A release curve

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Figure 4. A: Time course of preservative release from soft contact lenses and thecorresponding degree of solution-induced corneal staining/preservative-associatedtransient hyperfluorescence (SICS/PATH) observed at specified timepoints. The tableat top displays percentage of cornea with SICS/PATH in subjects wearing lenses soakedin the Aldox/PQ-1-based multipurpose solution (MPS) assessed at 30 minutes post-lensinsertion (ETA, BA and SA)59 and at 120 minutes post-lens insertion (AA, BA, ETA, SAand LA).178,226 SICS/PATH was not assessed at 30 minutes post-lens insertion with AAand LA lenses. Graph: Bars = concentration of preservative released into artificial tearfluid (ATF) from soft contact lenses at the noted timepoints; Lines: continuous pre-servative release after insertion (polynomic regression analysis). A: Aldox release wasmeasured at 30, 60 and 120 minutes after transfer to ATF (graph).64




(Figures 4A and 4B).64,65,176 In contrast toadsorption, the release rate shows agreater dependence upon the preservativethan on lens material.64 At 30 minutes,when release of Aldox from lenses wasgreatest (Figure 4A), 16 to 33 per centof the cornea showed SICS/PATH(Figure 4A). By two hours, when Aldoxrelease from lenses was lowest, only one toseven per cent of the cornea showedSICS/PATH (Figure 4A).42,178 In contrast,PHMB release is high from one to fourhours and low by eight hours (Figure 4B),with peak release at approximately twohours (Figure 4B). The levels of SICS/PATH observed with PHMB-based solu-tions correspond to the level of pre-servative released at two hours, wherelenses that showed the highest release con-centrations (alphafilcon A and balafilconA) also showed the highest levels of SICS/PATH (15 and 17 per cent of cornea,respectively). Similarly, those lenses thatreleased lower concentrations of PHMB(etafilcon A, lotrafilcon A and senofilconA) also showed the lowest levels of SICS/PATH (one to four per cent; Figure 4B).The ‘transient’ nature of SICS/PATH firstdescribed by Garofalo and colleagues43

to a certain extent may be attributed tothe release of preservative from lensesrather than ocular surface damage, whichwould persist until the defect has healed.

AETIOLOGY OF SICS/PATH

Recently presented25 and published47

research in conjunction with previouslypublished studies64,65 that analysed theuptake and release of preservatives fromsoft contact lenses may explain how SICScan be largely attributed to PATH.

At the corneal surface, preservativesmay interact with the cell membrane, aselegantly demonstrated in experimentsusing a liposome model,47,179–181 which is awidely accepted in vitro model of cellmembranes.182 These studies showedthat PHMB can reversibly bind to cellmembrane phospholipids (a majorcorneal cell membrane component183)without affecting the integrity and stabilityof the fragile membrane model at thehighest concentrations measured (100

mmol), which is up to 100 times that foundin marketed MPS.47,181,184

The most commonly used preservativesin MPS are PHMB, PQ-1 and Aldox,

which are positively charged (cationic)molecules.185 These cations are attractedto negatively charged molecules, such asFL,25 which carries two negative charges

PH

MB

Rel

ease

(μg

/lens

)

Minutes

0 60 120 240 360 480-0.05

0

0.1

0.05

0.15

0.3

0.25

0.2

0.35

0.4

Lens Material

Alphafilcon A

Balafilcon A

Etafilcon A

Lotrafilcon A

Senofilcon A

Extent of PATH

2 hours (120 min)

17%

15%

1%

1%

4%

Figure 4. B: Polyhexamethylene biguanide (PHMB) release was measured at 60, 120,240 and 360 minutes after transfer to artificial tear fluid (ATF) (graph) [Note: themanuscript that reported the raw data, from which the graph was generated, did notcontain the 30 minute data for release of PHMB from lenses]. Prior to incubation inATF, each lens was soaked in at least 100 mL of MPS (either PHMB-based CompleteEasy Rub or Aldox/PQ-1-based Opti-Free RepleniSH) for three days. Post-soak, eachlens was placed into 2.0 mL of mucin-containing ATF without proteins and lipids. At thespecified times, preservative release concentration was assessed via reverse phase highpower liquid chromatography and UV detection at 200 nm for Aldox and UV spectros-copy at 236 nm, with baseline correction at 280 nm, for PHMB.64 SICS/PATH wasmeasured at the specified times after insertion of the presoaked lens.59,226 Lenses wereremoved and hyperfluorescence was assessed after instillation of fluorescein. Slitlampexamination was performed at 10¥ magnification with the use of cobalt blue and yellowWratten #12 filters. The hyperfluorescent area (zero to 100 per cent in 10 per centincrements) was graded in each of five corneal regions (central, superior, inferior, nasaland temporal). The area of hyperfluorescence that covered each region was estimated,summed and averaged.42




(FL=) at ocular pH (Table 1). Fluoresceininteracts with all three preservatives to dif-ferent degrees.25 Studies show that FL hasan extremely strong affinity for PHMB andcan be up to 50 times greater than that forPQ-1 (Figure 5A).47 While the affinity ofFL for Aldox cannot be truly measured bythe highly sensitive technique used byBright and colleagues to measure thestrength of binding of PHMB and PQ-1(due to differences in its chemicalproperties/structure from PHMB andPQ-1, which act similarly), mathematicalmodels suggest the affinity of FL for Aldoxis similar to PHMB and greater thanPQ-1. Further, several negatively chargedmolecules (that is, FL=) can bind toeach preservative polymer (Figure 5B).57

This ionic interaction between FL= andpreservatives, such as PHMB, which revers-ibly binds to phospholipids47,179,180,184

may in part explain the short-lived hyper-fluorescence observed in contact lenswearers who use MPS. In addition to thereversible nature of PHMB binding to

the cell membrane, the instantaneousconcentration of PHMB due to the releasekinetics from the contact lens explainsboth the transient nature as well as thetime of peak hyperfluorescence observedwith different preservatives. As pH,solvents and surfactants influence ionicinteraction, such as those between PHMBand FL=, and the fluorescent propertiesof FL itself,28–30 the different com-ponents of MPS may account for the vari-ance in the extent of SICS/PATHobserved between different MPS formula-tions, even in those with the samepreservative.42

A paper presented at the 2010 Ameri-can Academy of Optometry annual meet-ing,22 showed that when NaFl is added tocells in culture, all cells readily took up FL;however, a sub-population of cells (five to15 per cent of the total) stained moreintensely (mirroring the distribution ofstaining on the cornea). Exposure to aPHMB-based MPS caused a significantincrease in the proportion of hyperfluo-

rescent cells; yet, no correlation betweenpropidium iodide staining (a marker ofdead cells) and hyperfluorescence wereobserved (p < 0.0001).22

Healthy cells may take up FL by normalphysiological processes, such as a non-specific endocytosis called pinocytosis(‘cell drinking’)186 and/or transportthrough a monocarboxylic acid cotrans-porter (MCT) present in mucosal epithe-lial cells,66,67 including those of thecornea.187 The interaction of PHMB withboth FL and corneal cell membrane com-ponents may increase the concentrationof FL at the cell surface that can thenenter the cell through either mechanism(or some other mechanism not yet eluci-dated). Additionally, PHMB can increasethe number of FL molecules with theappropriate charge (anions at physi-ological pH) to enter cells throughMCTs.66,67,188

A preliminary study has also shown thatPHMB can interact with mucins,68 whichare highly negatively charged.189 Aggre-gated PHMB–FL complexes binding tomucin is also a plausible explanation forthe transient nature of SICS/PATH, asredistribution and turnover of the mucinlayer are dynamic.50,190

The benign nature of the aetiologyof SICS/PATH is further supported bythe safe use of PHMB over the past20 years.60,191,192 Additionally, most in vitrostudies show PHMB-based solutions haveminimal toxicity,193–197 including thosethat used the most sensitive assays.194,195,197

Furthermore, a large proportion ofstudies has shown PHMB-based solutionsare less cytotoxic than those containingPQ-1/Aldox,193–198 which show a lowerintensity of SICS/PATH.

The aetiology of corneal staining withNaFl in pathological conditions based onthis review of the literature may be due toepithelial damage and the pattern of thecorneal staining is dependent upon theunderlying cause (Table 2).1,7

APPEARANCE AND DEPTH OFSICS/PATH

Another difference between SICS/PATHand pathological staining (regardless of

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0.9

1.0

1.1

1.2

1.3

1.4

Preservative (µM)

Extremely strong binding affinity

On/

Off

Rat

e

A B

PHMB: Kd = 0.09102 ± 0.05349PQ-1: Kd = 4.687 ± 1.794

No binding affinity

Modest affinity

PQ-1

PHMB

Figure 5. Multipurpose solution preservatives and fluorescein (FL) binding. A: Bindingkinetics of FL to polyhexamethylene biguanide (PHMB) and PQ-1. Solid lines (green,PQ-1; blue, PHMB) represent the best fits to a single site binding model (1:1 stoichi-ometry).25 Lines representative of extremely strong (black), modest (orange) and no(red) binding affinity are included for reference. B: Schematic of several molecules offluorescein binding to a single molecule of PHMB. PHMB is a polymer containingbetween two and 40 hexamethylene biguanide repeats (mean 11), each of which con-tains a positively charged amine group (NH2

+)57 that is able to interact with negativelycharged FL molecules. Images courtesy of Dr Frank Bright (A) and Bausch & Lomb (B).Equilibrium binding of dianionic FL (1 mmol) to increasing concentrations of PHMB(Mw = 2500 g/mol) and PQ-1 (Mw = 8700 g/mol) was assessed at 37 °C in 20 mmol pH7.4 TRIS buffer with 100 mmol KCl.




the cause) is appearance. Where patho-logical corneal staining can range frommicropunctate to coalescent,1 SICS/PATH is punctate (most commonly)and generally annular with centralsparing.39,42,62

This pattern may be explained byseveral theories. The ability of PHMB toself-aggregate or ‘clump’56,185 may accountfor the punctate pattern of SICS/PATHseen at higher levels with PHMB-basedMPS than PQ-1/Aldox-based solutions.Alternately, through passive electricalcell communication, FL readily diffusesthrough the cytoplasm and across celljunctions between adjacent healthy epi-thelial cells.199 Both the mucin layer andcells in the peripheral cornea are signifi-cantly impacted by contact lenses. Thechange/disruption of the mucin layer bycontact lenses may increase access of FL toepithelial cells. Additionally, in activelydividing cells, located predominantly inthe peripheral cornea,200 both pinocyto-sis201 and paracellular communication202

are reduced. Normal physiological proc-esses in conjunction with changes at thecorneal periphery, may explain why thepunctate annular pattern is observed withthe SICS/PATH phenomenon.

The depth of FL penetration into thecorneal epithelium following a pathologicevent varies depending upon the insultand its severity. This can range fromsuperficial epithelial involvement (forexample, Thygeson’s SPK),9 to immediatediffuse stromal glow (for example,contact lens-induced peripheral ulcers[CLPU]). In contrast, SICS/PATH is asurface phenomenon,39,44 involving pri-marily the superficial epithelium, which isconsistent with the proposed aetiology ofSICS/PATH discussed above.

SIGNS AND SYMPTOMS OFSICS/PATH

Corneal staining in pathological condi-tions is commonly symptomatic (Table 2);however, some may be asymptomatic butare often accompanied by additional signsor have a characteristic NaFl corneal stain-ing pattern. One example is corneal des-

iccation staining (3/9 o’clock) in rigid gas-permeable (RGP) lens wearers. In a studyby van der Worp and colleagues,91 3/9o’clock staining was not correlated withVisual Analog Scale comfort scores. Yet, ina subset of subjects with greater than orequal to Grade 1 staining, symptomaticsubjects showed a higher level of stainingthan those that were asymptomatic.Nevertheless, even asymptomatic subjectsshowed the distinct 3/9 o’clock cornealstaining pattern.91,203

While RB and LG staining of the con-junctiva are better predictors of dry eyesyndromes than NaFl,204,205 they are stillpoor predictors of disease severity over-all.16,206 One study showed NaFl conjuncti-val staining was increased in symptomaticcontact lens wearers but only clinically sig-nificant LG conjunctival staining (Grade 2or greater) was a discriminating factor. Asubstantial proportion of symptomaticsubjects showed none to slight LG con-junctival staining. These data furthersupport the limitations of vital stains as adiagnostic tool in isolation.

Subjects with SICS/PATH are generallyasymptomatic. The majority of the scien-tific literature shows little or no associa-tion or correlation of SICS/PATHwith discomfort39,43,62,65,166,170,172,207 or dry-ness.39,43 Of the few publications thatreported an association of comfort ordryness with SICS/PATH, a close review oftheir methods revealed a small samplesize208 and poor methodology and datareporting.42 This makes one question thevalidity of their findings considering theplethora of literature that found no suchassociation. Further, it is well establishedthat comfort is poorest in soft contact lenswearers in the evening,209 when SICS/PATH has been absent for several hours,43

further supporting a disconnect betweensymptoms such as comfort and SICS/PATH. Signs such as conjunctival hyperae-mia39,166,170 and papillae170 also show noassociation with SICS/PATH. Althoughlonger and larger studies may add someclarity around the presence or absenceof an association of SICS/PATH andsymptoms, the clear consensus at thepresent time is that no such associationexists.39,43,58,61,62,65,69,166,167,169,170,172,175,210–214

Only five studies have measured LGconjunctival staining specifically duringuse of lens care solutions. In three studies,there were no significant differences inthe level of LG staining between solutionsthat show high levels of SICS/PATH at twohours (that is, PHMB-based) and thosethat show low levels of SICS/PATH at thesame two-hour assessment (that is, PQ-1/Aldox- and hydrogen peroxide-basedsolutions).175,210,215 Two studies found sta-tistically significant differences in the levelof LG conjunctival staining betweenPHMB- and PQ-1/Aldox-based solu-tions,166,211 although the clinical signifi-cance is unclear. One study showed levelsof staining for PHMB and PQ-1/Aldoxsolutions were very low (less than Grade 1on a zero to 4 scale)211 and the differencebetween solutions (grade less than 0.25)were far below the levels for clinical sig-nificance (Grade 3 or greater per theauthors211 and clinically significant differ-ences defined as Grade 1 or more153 on azero to 4 scale).211 When clinically signifi-cant staining was present, there was nodifference between those using PHMB-and PQ-1/Aldox-based solutions.211 Inthe second study, a clinically and statisti-cally significant difference between themedian levels of LG conjunctival stainingwith PHMB- and PQ-1/Aldox-based solu-tions was observed (p < 0.001). Similar tothe previous study that showed a differ-ence, the levels of LG conjunctival stainingwere lower (up to six out of 16; summedzero to 4 scale for four zones) than whatwould be considered clinically significant(eight or more based on a clinical sig-nificance of Grade 2 or more in fourzones204).216

SUBSEQUENT COMPLICATIONS OFSICS/PATH

Corneal staining may or may not be asso-ciated with subsequent complicationsdepending upon whether asymptomaticor symptomatic and the aetiology of thestaining.163,217 In keratoconus, even whensymptoms are not severe, marked centralcorneal staining is predictive of scarring.163

Conversely, some studies have shownthat corneal staining is not predictive




of subsequent complications in somepathological situations, such as the dry eyesyndromes.71,168,211,218,219

Even in extended wear, which carriesthe highest risk for complications, asymp-tomatic corneal staining is not associatedwith corneal inflammation. In the LASHStudy, over half (53.3 per cent) of the sub-jects had repeated episodes of at least mildcorneal staining and a substantial propor-tion (11.3 per cent) had repeated episodesof moderate or severe corneal staining.There were no associations between anystaining variables (type, overall severity orchronicity) and the development of acorneal infiltrative event (CIE) (p > 0.2).40

While corneal staining in some patho-logical conditions has been associatedwith subsequent events, no evidence hasshown SICS/PATH to be predictive ofany subsequent complications, includinginflammatory events.70,220,221 A re-analysisof the data from the single study that dem-onstrated an association of SICS/PATHwith inflammation published in 2007 byCarnt and colleagues222 was performed bythe authors to assess whether MPS use wasa confounding factor in the original analy-sis. In the initial publication, Carnt andcolleagues222 reported that subjects withSICS/PATH were three to six times morelikely to have had asymptomatic CIEs. Thisimplies a possible causative effect. In there-analysis, when the risk of asymptomaticCIEs was assessed in MPS users versushydrogen peroxide users, odds of anasymptomatic CIE in MPS users was 10.3times as great as peroxide users (p =0.025). No causative effect exists afterremoving those eyes with SICS/PATHfrom the analysis; the odds of experienc-ing an event and the statistical significancewere highly similar (OR = 10.3; p =0.028).70,220

Additionally, in the two studies thatmeasured ‘hallmarks’ of inflammation,results were similar between eyes wearinga lens soaked in MPS containing eitherPHMB or PQ-1/Aldox.61,223 In one study,SICS/PATH and the levels of 27 inflam-matory markers were measured at two andsix hours post-lens insertion. A statisticallysignificant difference in the extent ofSICS/PATH was found between the group

wearing lenses soaked in PHMB-basedMPS versus those wearing PQ-1/AldoxMPS-soaked lenses (p < 0.01).61 In con-trast, no statistically significant differenceswere observed between the PHMB and thePQ-1/Aldox group for levels of inflamma-tory markers or a treatment-time interac-tion (p > 0.05).61 A second study measuredthe density of immune cells, dendritic cellsand non-dendritic inflammatory cells, incontact lens-naive subjects during dailywear via in vivo confocal microscopy(IVCM) at baseline and one week post-contact lens wear.223 Vital dye assessmentsof the cornea, limbus and conjunctivawere also performed. The density of theseinflammatory cells was not significantlydifferent between the PHMB- and PQ-1/Aldox-based MPS in any regions of thecornea and no correlation was foundbetween clinical hyperfluorescence/staining and the IVCM parameters.223 Asmay be expected in pathological condi-tions, like meibomian gland dysfunction,inflammatory cell density measured byIVCM was correlated with NaFl (r = 0.524;p < 0.0001) and RB (r = 0.479; p < 0.005)ocular surface staining.224 Together, thesestudies make a strong argument thatSICS/PATH is not a causative factor inCIEs.

As no correlation between SICS/PATHand adverse events, such as keratitis, hasbeen demonstrated, the OphthalmicDevices Panel recommended that theUnited States Food and Drug Administra-tion should not require a two-hourcorneal staining assessment in an applica-tion for marketing approval of lenses andlens care solutions nor in any lens or lenscare guidance.225 To date, no regulatorybody has added the two-hour cornealstaining assessment for marketingapproval or labelling.

CONCLUSIONS

Our ever increasing knowledge regardingthe fundamental science underlying theinterplay of contact lenses, lens care solu-tions and the ocular surface enables theinformed eye-care practitioner to makebetter decisions and to achieve the bestoutcome for the patient.

There are certain situations duringcontact lens wear that can be accuratelydiagnosed based on corneal staining withNaFl alone or in conjunction with ocularsurface staining with LG or RB. Nonethe-less, in many instances, the use of NaFl, RBand LG for diagnosing contact lens com-plications needs to be viewed within thecontext of other signs and symptoms foraccurate diagnosis due to the limitationsof these dyes.

There are several characteristics thatdifferentiate SICS/PATH versus thecorneal staining observed in pathologicalconditions. First, the intensity of SICS/PATH peaks in about 30 minutes post-lensinsertion in users of PQ-1/Aldox-basedsolutions and one to four hours post-lensinsertion in users of PHMB-based solu-tions. In contrast, NaFl corneal staining ispresent in pathological conditions untilthe defect has been repaired. Additionally,the appearance of corneal hyperfluores-cence observed in SICS/PATH is mostcommonly superficial, micropunctate andannular, while pathological corneal stain-ing can be located in any region of thecornea, range from micropunctate to acoalescent patch and from superficialinvolvement to diffuse stromal glow.Moreover, corneal staining in pathologicalconditions is accompanied by additionalsigns, symptoms and/or has a distinctpattern that differentiates it from non-pathological corneal hyperfluorescencesuch as mucin balls, dimple veil, SICS/PATH and physiological cell turnover.Further, SICS/PATH is neither associatedwith, nor predictive of, subsequent com-plications, where pathological cornealstaining may or may not be.

Recent research regarding the interac-tions of FL, preservatives, contact lensesand corneal epithelial cells may explainthe disconnection between what was beingobserved under the slitlamp and the lackof signs and symptoms in contact lenswearers with high levels of SICS. Based onour continually evolving understandingregarding the aetiology of SICS, I feel thatthis phenomenon can be largelyexplained by PATH. Thus, SICS is mostlikely benign and may not indicate in-compatibility between the contact lens




and lens care solution or indicate ocularsurface damage.

Notwithstanding limitations in the clini-cal application of vital staining for exami-nation of the ocular surface, it is still anessential tool to evaluate the health ofthe cornea and conjunctiva. With ourimproved understanding of the propertiesand interactions of the physicochemicaland biological systems involved, eye-care practitioners should place what isobserved with vital stains in context ofother equally important (and some moreimportant) factors, such as additionalsigns, symptoms and lens/solutioncombinations.

ACKNOWLEDGEMENTS

The author receives funds as a consultantfor Bausch & Lomb. Editorial and scien-tific support (writing assistance, assem-bling tables and figures, grammaticalediting, fact-checking and referencing)were provided by BioScience Communica-tions, New York, NY, USA. BioScienceCommunications received unrestrictedfunding support from Bausch & LombVision Care, Rochester, USA. The authorwould also like to thank Dr Frank Brightfor his helpful feedback and use of hisfigures. Dr Bright has received an unre-stricted research grant from Bausch &Lomb and receives funds as a consultant/advisor.

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