Download DIFFERENTIATING_THE_STAIN-_WHAT_DOES_IT_ALL_MEAN

4/4/12 DR. BLOOMENSTEIN’S
DISCLOSURE
•  Presenter is on speakers panel of Alcon, Allergan, Abbott,
Bausch & Lomb, Inspire, STAAR Surgical, Odyssey
DIFFERENTIATING THE STAIN:
WHAT DOES IT ALL MEAN?
•  President of the Optometric Council on Refractive
Technology (OCRT)
•  Presenter has NO financial interest in any products
mentioned
Marc R. Bloomenstein OD, FAAO
Scottsdale, Arizona
•  Except he does have stock in a certain coffee company...
REFRESHER: THE CORNEAL EPITHELIUM
•  The cornea is exposed to the
environment on its outer surface, and
is epithelial in nature
Mucin
Layer
Epithelium
•  The outermost part of the cornea is a
stratified squamous layer, the
corneal epithelium
Bowman’s
Membrane
•  The corneal epithelium is covered by
a layer of mucin
•  The corneal epithelium is continuous
at its margins with the conjunctiva of
the eyelids and the eyeball
Stroma
REFRESHER: THE CONJUNCTIVAL EPITHELIUM
•  The conjunctiva houses goblet cells responsible for mucus
production
•  Highly vascularized
•  Even in healthy individuals, mononuclear cells are resident in
the conjunctival stroma
Goblet Cells
Epithelium
Stroma
USE OF VITAL STAINS IN PRACTICE
USE OF VITAL STAINS IN PRACTICE (CONT’D)
—  Determine the fit of contact lenses
—  Visualize tear film components or lack of
—  Localization of corneal foreign bodies
—  Enhancement of palpebral conjunctival pathology
—  To detect ocular abnormalities, such as dry-eye, corneal
damage, and inflammatory conditions (ie, corneal infiltrates)
◦  Depending on the eye care practitioner, this may be
performed as part of an annual check-up or only when a
patient presents with a problem or both
—  Vital stains are often used to determine the live/dead
cell ratio in a cell population
—  Vital stains most commonly used for ocular use
◦  Sodium fluorescein
◦  Lissamine green
◦  Rose bengal
1 4/4/12 WHAT WE LEARNED IN SCHOOL: LISSAMINE
GREEN
WHAT WE LEARNED IN SCHOOL: ROSE
BENGAL
•  Stains degenerate cells, dead cells, and mucous fibrils in the
same manner as rose bengal
•  Derivative of sodium fluorescein (NaFL)
•  Believed to stain dead or degenerated cells and mucous
strands
•  The nucleus is generally stained more intensely that the
cytoplasm
•  Best used to examine the conjunctiva
•  Suitable for vital staining of the cornea and conjunctiva
Norn MS. Acta Ophthalmol (Copenh). 1973;51:483.
Norn MS. Acta Ophthalmol (Copenh). 1972;113(Suppl):20
FLUORESCEIN CORNEAL
STAINING
LISSAMINE GREEN STAINING
Exposure zone staining with
limbal sparing
Exposure zone staining with
limbal staining
Intense diffuse staining of
exposure zone, limbal
staining
•  Degree of severity increases from left to right
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Images from Dry Eye and Ocular Surface Disorders, 2004
ž 
Synthetic organic compound
ž 
Widely used as a fluorescent tracer
ž 
Self-quenching at high concentrations
ž 
Fluorescent intensity is highly pH dependent
ž 
How it interacts with cornea is largely unknown
Optimum viewing time is 3-5mns after application
ž 
Absorbs light with a maximum absorbance
wavelength (λ) of ≈490 nm, resulting in the emission
of light (or fluorescence) at a maximum absorbance
λ≈530 nm
ž 
Although visible to the naked eye, this emitted λ can
be enhanced when viewed through a cobalt filter of
a slit lamp biomicroscope
o  Visual intensity is enhanced when
•  Lissamine green detects dead or degenerated conjunctival cells
FLUORESCEIN MOLECULE
ž 
the signal is viewed with an
additional yellow filter, such as
the Kodak Wratten 12
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WHAT WE LEARNED IN SCHOOL:
FLUORESCEIN CORNEAL STAINING
•  Taught in 1st year that the fluorescence observed with NaFL
represents dead or damaged cells
•  The three potential mechanisms of corneal staining (CS) with NaFL
that are most commonly described are:1
•  Surface pooling
•  Uptake by cells
•  Ingress around cells
•  However, no one actually knows WHAT fluorescein is
binding to in or on cells!
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1. Morgan PB, Maldonado-Codina C. Cont Lens Ant erior Eye. 2009;32:48.
2 4/4/12 INFERIOR STAINING
PATIENT CCF
•  Lid laxity conditions such as ectropion
•  Lagophthalmos
•  Incomplete blink
•  MGD/blepharitis
•  Exposure keratopathy
•  Environmental contributors
•  Elevated computer screen
SUPERIOR STAINING
•  Superior Limbic
Keratoconjunctivitis
•  Limbal stem cell deficiency
3 4/4/12 LSCD CAUSES
•  Chemical burns
•  Chronic inflammation e.g. scleritis
•  Abuse of contact lens wear
NEW FINDINGS SUGGEST THIS MAY NOT
BE ENTIRELY ACCURATE…
HOW LONG DOES EPITHELIAL STAINING LAST
AND HOW DO WE GET RID OF IT?
—  NaFL enters healthy cells and result in transient “corneal staining” with an intact
epithelium
FL staining after impression cytology in
normal patient
3 hours after impression cytology later of
same eye
Bakkar M, et al. Optom Vis Sci. 2010; E-abstract 100959; Thinda S, et al. Br J Ophthalmol. 2010;94:406.
4 4/4/12 ENHANCING PALPEBRAL PATHOLOGY
INDENTIFYING SIGHT OF IMPACT – BB TO EYE
5 4/4/12 SOMETIMES YOU DON’T NEED FL TO TELL THE
SITE OF IMPACT…
DIFFERENTIAL STAINING –
RB VS FL
AGAIN USING RB TO DIFFERENTIATE STAINING
6 4/4/12 3 DAYS LATER – STEROID AND ANTIBIOTICS
WE ARE NOT DONE YET.
DO WE ALWAYS NEED TO DO A DIFFERENTIAL
STAIN?
CORNEAL STAINING
•  Thygesons
•  EKC
•  Herpetic
•  Zoster (pseudodendritic)
•  etc.
DRY EYE STAINING
42
43
7 4/4/12 44
45
46
47
48
49
8 4/4/12 EPITHELIAL BASEMENT DYSTROPHY (EBMD)
•  Abnormal corneal epithelial regeneration and maturation
•  Abnormal basement membrane
•  Very common dystrophy
•  Considered age related
•  Prevalence increases with age
•  Onset is around 40-70 y.o.
•  Late onset supports degeneration vs. dystrophy
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50
9 4/4/12 EBMD
PHOTO COURTESY TRACY SWARTZ OD, FAAO
RCE
64
65
10 4/4/12 2 DAYS LATER – LOOK AT STAINING PATTERN
RCE
66
FUCH’S DYSTROPHY
• 
Characterized
•  Corneal Guttata
•  Small refractile “drops” on
corneal endothelium
•  Affects the “pump” action of
the endothelium
•  Edema
•  Greater in the AM
•  Desiccates as day goes on
•  Long standing edema may
lead to corneal scarring
•  RCE’s common
FUCH’S DYSTROPHY
•  Symptoms vary with degree of guttata and compromise of the endothelial
tissue
•  Moderate guttata
•  May affect visual function
•  May induce mild-moderate edema
•  Halos around lights
•  Hazy vision > a.m.
•  Severe guttata
•  Vision decreases
•  Possible bullous develops
PHOTO COURTESY TRACY SWARTZ OD, FAAO
FUCH’S DYSTROPHY
CORNEAL DEGENERATIONS
PHOTO COURTESY TRACY SWARTZ OD, FAAO
11 4/4/12 CORNEAL DEGENERATIONS
DEGENERATIONS
•  Defined as a deterioration or change from a higher to a lower
form, especially change of tissue to a lower or less functionally
active
•  Arcus
•  Non-inherited
•  Amyloid
•  Unilateral or bilateral
•  Limbal girdle of Vogt
•  Asymmetric
•  Band keratopathy
•  Develop in later years
•  Salzman’s nodular degeneration
•  Variable progression
•  Spheroidal degeneration
•  Systemic disease can be associated
DEGENERATIONS
•  Coats white Ring
•  Hassal-Henle bodies
•  Crocodile shagreen
•  Senile furrow
•  Dellen
•  Pingueculae
•  Pterygium
ENHANCING CROCODILE SHAGREEN
DRY EYE EVALUATION
Lissamine Green
12 4/4/12 USING FL TO VISUALIZE TEAR FILM
SLITLAMP EXAMINATION
Fluorescein
Staining
Evaluation of
Tear Meniscus
INCOMPLETE BLINK/NOCTURNAL
LAGOPHTHALMOS
Rose Bengal/
Lissamine Green
Staining
Tear film break up viewed with fluorescein stain on a patient with dry eye
0 seconds
1 second
2 seconds
3 seconds
4 seconds
5 seconds
6 seconds
16 seconds
Tear film break up is indicated by the
dark areas that appear on the cornea.
13 4/4/12 POTENTIAL SEVERE CONSEQUENCES OF
UNTREATED DRY EYE DISEASE
Sterile Melting
BLEPHARITIS
Is the most common and
arguably the most
important diagnosis
presenting to the
ophthalmologist
Anterior
Inflammation
mainly
centered
around the
eyelash and
follicles
Blepharitis
Bacterial Keratitis
SIGNS AND SYMPTOMS
Morning
crusting
Recurrent
hordeola
Loss of
lashes
Conjunctival
hyperemia
Foreign
body
sensation
Inflammation
that involves
the meibomian
gland orifices
Collarretes
(scales that
encircle
lash)
Posterior
Staph
immune
disease
•  Phlyctenula
•  Pannus
•  Catarrhal
infiltrates
•  etc
Lid Wiper Epitheliopathy
•  Characterized by presence of damaged epithelial cells on
lid wiper portion of marginal conjunctiva
•  Fluorescein or rose bengal can be used to dye the lid wiper
•  Staining graded on scale of 0 to 3: 0 = no staining, 3 =
heavy staining
2
1
FLUORESCEIN TEAR CLEARANCE
•  Meant to differentiate the inflammatory dry eye
•  Reduced clearance leads to stasis, which allows
inflammatory mediators to remain in contact with
ocular tissue
• 
Tseng, Pflugfelder
1. Korb DR, Herman JP, Greiner JV, et. al: Lid Wiper epitheliopathy and dry eye symptoms. Eye & Contact Lens 31(1): 2-8, 2005.
2. Korb DR, Herman JP, Greiner JV, et. al: Lid Wiper epitheliopathy and dry eye symptoms in contact lens wearers. CLAO J 28: 211-216, 2002.
14 4/4/12 CONTACT LENS/SOLUTION STAINING GRIDS
PRESERVATIVES IN MPS ARE TAKEN UP AND THEN
RELEASED BY SOFT CONTACT LENSES
•  All preservatives are taken up by all
soft contact lenses during the
soak1-5
• 
Amount absorbed and rate of absorption
depends on lens material and preservative
•  After application, the lens releases
the preservative into the tear film2
• 
The release rate depends on preservative
and lens material combination2,4
•  The tears slowly dissipate the
preservatives
1. Data on File. CFR-6091/CFR-6047. Bausch & Lomb, Inc. 2008. 2. Powell CH, et al. Cont Lens Anterior Eye. 2010;33:9. 3. Sentell KB, Beaullieu E. Invest Ophthalmol Vis Sci.
2004;45:E-Abstract 1573. 4. Willcox MD, et al. Optom Vis Sci. 2010 Sep 2. [Epub ahead of print].
5. Dassanayake N, et al. Invest Ophthalmol Vis Sci. 2005;46:E-abstract 915.
15 4/4/12 THE MPS PRESERVATIVE PHMB DOES NOT
DAMAGE CORNEAL CELL MEMBRANES
MPS PRESERVATIVES AND TRANSIENT
HYPERFLUORESCENCE WITH NAFL
®  At the cornea, preservatives may interact with the cell
membrane
•  The commonly used preservative PHMB (ie, renu) binds
to mucin…
•  PHMB may be binding to other components of the ocular
surface…
®  In the presence of PHMB, the corneal cell membrane is
not affected
PHMB
Cell
Membrane
•  PHMB may be interacting with NaFL…
PHMB (polyhexamethylene biguanide) is a preservative in COMPLETE® Easy Rub®*, Boston Simplus® Multi-Action**, Biotrue™**,
Renu® family**, AQuify®†, MeniCare Plus, Sauflon All in One Lite, SOLO-care Aqua™‡
THE PRESERVATIVE PHMB HAS AN
EXTREMELY STRONG AFFINITY FOR NAFL
®  NaFL adheres to MPS PHMB on the eye
® 
NaFL has a strong affinity for PHMB, which is up to 50-times greater than that for
PQ-1
Fluorescein
FluoresceinPHMB Complex
PHMB
Fluorescein
Trademark of *AMO, **B+L, †Novartis, ‡CIBA Vision.
1. Bright FV, et al. Poster presented at: The 6th Biennial Scientific Symposium of the Contact Lens Association of Ophthalmologists Education & Research Foundation;
September 23-25, 2010; Las Vegas, Nevada, USA.
ALDOX CONCENTRATION AT PEAK RELEASE IS ~20X GREATER VS
PHMB PEAK RELEASE CONCENTRATION
•  Aldox concentration1 at peak
time is ~20x greater in
comparison to PHMB peak
concentration (ppm)1,2
0.5
•  Aldox concentration peaks:
within 30 min1
•  PHMB concentration peaks:
within 2 hours1,2
•  Every preservative is taken up
and released by lenses:
Cell
Membrane
Concentration (ug/uL)
Muya L, et al. Invest Ophthalmol Vis Sci. 2008;49:E-Abstract 4869; Bright FV, et al. Poster presented at: The 6th Biennial Scientific
Symposium of the Contact Lens Association of Ophthalmologists Education & Research Foundation; September 23-25, 2010; Las
Vegas, Nevada, USA.
PQ-1 (polyquaternium-1/POLYQUAD®) is a preservative in COMPLETE® RevitaLens OcuTec™*, Biotrue™**, OPTI-FREE®
Express®†, and OPTI-FREE® RepleniSH®†
PHMB RELEASE FROM SILICONE HYDROGEL LENS*
GREATEST BETWEEN INSERTION AND TWO HOURS
System
PHMB (B&L)
PHMB (Powell)
Aldox (Powell)
0.3
Peak Maxima (h)
0.74
0.71
0.24
0.2
0.1
0.0
0
•  Detection is based on:
Trademark of *AMO, **B+L, †Alcon.
Bright FV, et al. Poster presented at: The 6th Biennial Scientific Symposium of the Contact Lens Association of Ophthalmologists Education & Research Foundation; September
23-25, 2010; Las Vegas, Nevada, USA.
PHMB w/ Balafilcon A (B&L)
PHMB w/ Balafilcon A (Powell)
Aldox w/ Balafilcon A (Powell)
0.4
2
4
6
8
10
12
14
Time (h)
•  Time of observation
•  Fluorescent probe used
1. Powell CH, et al. Cont Lens Anterior Eye. 2010;33:9. 2. Data on File. Bausch & Lomb, Inc. 2010.
ASSESSING FLUORESCEIN:
PRESERVATIVE AGENT ASSOCIATION
0.014 Fluorescence greatest
+
≈2 hrs
0.01
PHMB release
0.008
0.006
0.004
0.002
0
0
2
4
6
8
10
12
14
Time (hrs)
Does not consider tear exchange rate which would reduce absolute values but not profile shape
*SiHy lens is PureVision (balafilcon A).
Data on File. Bausch & Lomb, Inc. 2010.
Steady-State
Fluorescence Anisotropy
PHMB Concentration (µg/µl)
0.012
0.04
Strong association
0.03
Modest association
0.02
0.01
0.00
Weak/No association
0
2000
4000
6000
8000
10000
Preservative Agent
16 4/4/12 THE BINDING OF PHMB AND NAFL RESULTS IN A
BENIGN, TRANSIENT FLUORESCENCE1
®  The signal intensity with PHMB solutions:1,2
­  Peaks at 2 hours
­  Dissipates within 6-8 hours indicating no ocular surface damaged
occurred
Insertion
2 hours
THIS PRESERVATIVE-ASSOCIATED TRANSIENT
HYPERFLUORESCENCE OR PATH IS DIFFERENT
THAN CLINICALLY RELEVANT STAINING
®  The science suggests this visual signal is not representative of
pathological or true corneal staining1-5
6 hours
Fluorescence at 2 hrs
Pathological Staining
The punctate appearance of the signal results from many PHMB molecules aggregating
and binding with NaFL3
1. Bright FV, et al. Poster presented at: The 6th Biennial Scientific Symposium of the Contact Lens Association of Ophthalmologists Education & Research Foundation;
September 23-25, 2010; Las Vegas, Nevada, USA. 2. Garofalo RJ, et al. Eye Contact Lens. 2005;31:166. 3. Alila S, et al. Langmuir. 2005;21:8106.
1. Garofalo RJ, et al. Eye Contact Lens. 2005;31:166. 2. Jones L, et al. Optom Vis Sci. 2002;79:753. 3. Ward KW. Optom Vis Sci. 2008;85:8. 4. Andrasko GJ, Ryen KA. Rev
Cornea Contact Lenses. March 2007:36. 5. Carnt N, et al. Optom Vis Sci. 2007;84:309.
PRESERVATIVE-ASSOCIATED TRANSIENT
HYPERFLUORESCENCE OR PATH IS DIFFERENT
THAN CORNEAL STAINING (PATHOLOGICAL)
Corneal Staining
PATH
• 
• 
• 
Etiology likely due to benign
preservative interactions1
Generally
Surface
asymptomatic2-4
phenomenon3,4
commonly)3-5
• 
Punctate (most
• 
Resolution - within a few hrs* 2,4,6
• 
Onset – within a few hrs* 2,6,7
• 
Not associated with future
complications4,8
• 
Etiology due to epithelial damage9
• 
Commonly symptomatic4,10
• 
Depth varies9
• 
Macropunctate to coalesced9
• 
Resolution – dependent on epithelial
turnover rate; commonly several
days11
• 
May or may not be associated with
future complications12,13
*Post-lens insertion.
1. Bright FV, et al. Poster presented at: The 6th Biennial Scientific Symposium of the Contact Lens Association of Ophthalmologists Education & Research Foundation;
September 23-25, 2010; Las Vegas, Nevada, USA. 2. Garofalo RJ, et al. Eye Contact Lens. 2005;31:166. 3. Jones L, et al. Optom Vis Sci. 2002;79:753. 4. Ward KW. Optom
Vis Sci. 2008;85:8. 5. Andrasko GJ, Ryen KA. Rev Cornea Contact Lenses. March 2007:36. 6. Kislan T. Optometry. 2008;79:Poster 69. 7. Bandamwar KL, et al. Cont Lens
Anterior Eye. 2010;33:199. 8. FDA Ophthalmic Devices Panel meeting, June 10, 2008. Available at: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfAdvisory/details. cfm?
mtg5699. 9. Snyder C. Clin Refract Optom. 2005;16:1. 10. Sweeney DF, et al. Cornea. 2003;22:435. 11. Korb DR, Korb JM. J Am Optom Assoc. 1970;41:233. 12. Barr JT, et al.
Cornea. 2006;25:16. 13. Sweeney DF, et al. Poster presented at: The 31st Annual Meeting of the British Contact Lens Association; May 29-June 1, 2008; Birmingham, UK.
“Superficial corneal staining as captured during
uneventful clinical visits…is not associated with
an eventual infiltrative response”1
– L. Szczotka-Flynn
EVEN IN EXTENDED WEAR – WHICH
CARRIES THE HIGHEST RISK FOR
COMPLICATIONS – ASYMPTOMATIC
“REAL” CORNEAL STAINING IS NOT
ASSOCIATED WITH CORNEAL
INFLAMMATION
“supported by Fleiszig et al…
who have shown in animal models
that the superficial epithelium can be
severely abraded in the presence of virulent strains of Pseudomonas
species with no increased susceptibility to infection.”
Image reproduced from Lee EJ, Evans DJ, Fleiszig SM. Invest Ophthalmol Vis Sci 2003;44:5220–7.
1. Szczotka-Flynn L, et al. Invest Ophthalmol Vis Sci. 2010 Jun 10. [Epub ahead of print]. 2. Evans DJ, Fleiszig SM. Invest
Ophthalmol Vis Sci 2003;44:5220.
17 4/4/12 SO WHAT DOES THIS MEAN?
CLINICAL LESSONS: VITAL STAINS
•  In the absence of symptoms, results from the use of vital
stains, in particular NaFL, may be misleading…especially in
MPS users
•  In the presence of acute signs and symptoms:
•  NaFL should be used to assess the cornea
•  Either lissamine green/rose bengal best to examine the
conjunctiva
•  For persistent symptoms, such as discomfort, scratchiness,
tearing, look to the conjunctiva with lissamine green/rose
bengal for dry eye!
•  Put vital stains in the context
•  In the absence of symptoms, results may be misleading as
PATH ≠ Pathology
•  In the presence of persistent low grade symptoms: Don’t forget
the conjunctiva! Use lissamine green or rose bengal in
diagnosis
•  When using any corneal stain: Don't paint the cornea, less is more
•  You can always add more, but too much dye can obstruct the
view of subtle staining patterns
THANK YOU
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