Corneal staining procedure

CORNEAL
STAINING
PROCEDURE
PRESENTED BY- Dr samarth mishra
Tuesday, August 2, 2016 1

USE OF VITAL STAINS IN PRACTICE
——Vital stains most commonly used for ocular use
◦ Sodium fluorescein
◦ Lissamine green
◦ Rose bengal
Use
Determine the fit of contact lenses
—Visualize tear film components
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

FLUORESCEIN STAIN

Fluorescein – Historical Perspective
• Baeyer(1871): First fluorescein dye was made.
• M Straub (1888) : First used fluorescein for vital staining
of the eye.
• Burk (1910): First used fluorescein to detect retinal
disease

Properties of fluorescein
• A yellow water-soluble dibasic dye
of xanthine series
• Orange red in powder and yellow in
solution.
• Chemical formula: C2H12O5Na
• Molecular weight: 376.27
• Solubility : 50% (in water at 15 C)

Optimum condition for observation of
fluorescein
• For dilute concentrations of fluorescein in an
aqueous solution
• Peak absorption:wavelength between 485 and 500 nm
• Peak emission: wavelength between 525 and 530nm
 The fluorescent light appears
yellow green in blue light.
 The flourescence increases with
greater concentration upto
0.001% and greater pH upto 8.

Important clinical characterstics
• Stains epithelial defects bright green
• Diffuses into intercellular space
• Will not stain devitalized
• Tear film appears yellow orange
• Can exhibit pseudoflare, Fischer Schweitzer mosaic
• Promotes growth of pseudomonas aeruginosa in
solution
• Will stain soft contact lens

Preparation for tropical ocular use

Available forms
• Can be applied to eye
• Topically in form of solution
• By Fluorescein impregnated
filter paper strips (developed
by kimura)
• Injectable form for IV use

A. Topical Indication
• Assessment of ocular surface integrity -
Detection of defects in corneal epithelium
• Fitting assessment of rigid contact lens.
• Applanation tonometry - Goldmann
tonometer/Perkins hand-held tonometry
• Seidel's test- Detection of site of perforation/bleb
• Lacrimal testing ( Tear flim breakup time
(TBUT), Jone dye test, Fluorescein dye
disappreance test(FDDT)

1.Assessment of ocular surface integrity
• Frequently used to detect lesions of ocular
surface owing to its high degree of ionization, it
neither penetrates the intact corneal epithelium
nor forms a firm bond with any vital tissue.
• Instillation of dye in cul-de-sac allows
determination of corneal & conjunctival lesions
such as abrasions ulcers& edema & aids in
detection of foreign bodies.
• Epithelial defect appears as vivid green
fluorescence

How does staining take place?
Any break in
epithelium
Penetration of
Fluorescein in
adjoining
bowman’s &
stromal layer
Dye makes
contact
with an
alkaline
interstitial
fluid
Fluid turns bright
green owing to its
PH indicator
properties &
depending to
extent of lesion

Staining of corneal infiltrate
Corneal abrasion Conjunctival lesion

• Each of the major grading systems employs a method of
dividing the cornea into zones and for evaluating one or
more staining variables in each zone.
• In the five-zone model,
• Zone 1 is a circle in the center of the cornea and
• the four equal segments of the ring surrounding this
central zone are Zones 2 through 5 (superior, temporal,
nasal and inferior zones).
• The observer makes an estimate of the zonal area
involvement and calculates the staining score based on
the number and/or type of staining variables.

• Efron The Efron system is based on the work of Nathan
Efron, DSc, MCOptom, FAAO, FCLSA, FBCLA, FIACLE
(currently based at the University of Queensland,
Australia) when he was at the European Centre for
Contact Lens Research, Department of Optometry and
Neuroscience, University of Manchester in the United
Kingdom.
• The Efron system uses one variable, the degree of
staining per zone based on an ordinal scale of 0 to 4:
0=no staining, 1=trace staining, 2=mild staining, 3=
moderate staining, 4=severe staining.
• The Efron system has been validated for clinical use with
an expected accuracy of �1.2 grading scale units, a
rather large range of error for a four-point scale.

CCLRU
• CCLRU The scale developed by the Cornea and Contact
Lens Research Unit (CCLRU), School of Optometry and
Vision Science, The University of New South Wales,
Sydney, Australia, calls for three variables per zone: type,
depth and extent of surface area staining.
Figure 1. Types of punctate fluorescein staining in CCLRU standards
superimposed on a photo from the CCLR group. Image copyright is owned by
the Centre for Contact Lens Research, School of Optometry, University of
Waterloo, Ontario, Canada. Used with permission.
It shows the CCLRU punctate staining types.
Type 0 means there is no staining.
Type 1 is micropunctate;
Type 2, macropunctate;
Type 3, coalescent macropunctate staining; and
Type 4 is a coalescent patch of 1mm or greater in size.

• The other two variables in the CCLRU system are
1. depth and
2. extent of staining.
• If there is no staining, the depth is graded as 0.
• Superficial epithelial involvement is Grade 1.
• presence of a stromal glow within 30 seconds is
Grade 2.
• immediate localized stromal glow is Grade 3, and
• immediate diffuse stromal glow is Grade 4.
• If there is no staining, the extent is graded as 0.
• From 1 to 15 percent of surface involvement is Grade
1,
• 16 to 30 percent surface involvement is Grade 2,
• 31 to 45 percent surface involvement is Grade 3 and
• 46 percent or greater surface involvement is Grade 4.

• The CCLRU scale uses the zone of greatest staining to
determine clinical significance.
• Under their criteria, corneal staining is clinically
significant when
• it is persistent,
• its type is greater than Type 2 (macropunctate) and/or
• its depth is greater than Grade 1 (superficial epithelial
involvement) and/or
• its extent is greater than Grade 1 (1 to 15 percent surface
involvement) in a given zone.
• Micropunctate staining is considered not clinically
significant by the CCLRU unless it involves more than
15 percent of the corneal surface.

CCLR/Global Staining Score

CCLR/Global Staining Score
• CCLR/Global Staining Score Lyndon Jones, PhD, FCOptom, FAAO, at the Centre
for Contact Lens Research (CCLR), School of Optometry, University of Waterloo,
Ontario, Canada, expanded on the CCLRU system to make it more sensitive by
changing the ordinal scale of 0 to 4 to an integer scale of 0 to 100.
• Under this system, the type of staining in each zone is graded on
• a 0 (none) to 100 (total) scale.
• The mean outcome measure, the Global Staining Score, is the product of the
type of staining in each of five zones (one central and four peripheral) times
the percentage area of the zone with the staining.
• Under this system, the scale ranges from 0 (none per zone) to 50,000 (total staining
in all 5 zones).
• Recently, the scoring was modified slightly in an attempt to normalize the scores to
represent a typical score for a given sector.
• The Global Staining Score is now divided by 5, for a maximum average sector
staining score of 10,000. For example, one corneal sector with micropunctate
staining (score 25) over 10 percent of its surface would have a total score of 250 (25
x 10). If all five sectors had the same score, the total score would be 1,250 (250 x 5),
but this would be normalized to 250. An average sector score of less than about
1,200 (or total Global Staining Score of 6,000) is considered clinically
insignificant.

Global staining scores are shown for each type of
punctate fluorescein staining (CCLR standards).

2. Seidel's test
Detection of site of perforation/bleb

• A major aid in fitting of RGP contact lenses is
vital staining of tear film
• Observation of Fluorescein stained tear film with
a cobalt filter of slit lamp allows determination of
the fit of lens
• Useful in assessing the integrity of cornea in CL
users as the dye can disclose areas where the
CL disrupts the corneal epithelium
3.Contact lens fitting and management

4.Applanation tonometry
• Important component in measuring IOP with Goldmann
applanation tonometer
• Requires the meniscus of tear fluid surrounding the
flattened corneal surface be sufficiently stained so that
apex of the wedge shaped meniscus is visible.
• Procedure
1.Anaesthetic & fluorescein instilled in conjuntival sac
2.With Cobalt blue filter,brightest illumination and prism
advanced until touches apex of cornea
3.A pattern of 2 semicircles one above ,other below the
horizontal midline

5.Evaluation for dry eye & lacrimal system
• Topically applied Fluorescein –used to evaluate integrity
of the precorneal tear film& patency of the lacrimal
drainage system
• Assessment of TBUT
• Evaluating the EPIPHORA
• Assessment of FDDT
• To distinguish between
Anatomical and functional
outflow problems-JONES DYE TEST

• PROCEDURE FOR JONES DYE TEST
• Instil one drop of fluorescein into the conjunctival sac (Figure
13).
• Put a cotton bud soaked in anaesthetic in the inferior meatus.
• If fluorescein is detected after five minutes, the system is
patent (positive Primary Jones Test).
• If no fluorescein is discovered, this is a negative Primary Jones
Test (Figure 14) and the functional obstruction could be
anywhere from the punctum to the Valve of Hasner.
• Next, wash the excess fluorescein from the conjunctival sac
and syringe. If fluorescein is detected, then this shows it had
entered the sac and constitutes a positive Secondary Jones
Test (Figure 15) and suggests a functional obstruction of the
nasolacrimal duct.
• If no dye is found on the cotton bud after syringing, this is
termed a negative Secondary Jones Test, because fluorescein
had not entered the sac and, thus, there is stenosis of the
puncta or canalicular system (Figure 16).
• If no saline appears in the nose, there is a complete obstruction
somewhere in the lacrimal drainage system.

Contamination of fluorescein
• Contamination of fluorescein eyedrops is a serious risk
-even greater than that encountered with the majority of
other eyedrops.
• As these individual drops are liable to become infected
with bacteria and, at the same time, are frequently used
on damaged tissue that is prone to infection, very great
care must be taken in their use.
• Pseudomonas aeruginosa – most dangerous
microorganism with which fluorescein eyedrops are
inclined to become invaded.
Sharma IP

Contd...
• Phenylmercuric acetate or nitrate in 0.002%
-Best bactericide for preserving fluorescein
drops, and this is effective against
Pseudomonas, given adequate contact time.
• However, the safest method is sterile single-
dose units or sterile fluorescein-impregnated
paper strips, both are readily available and to
be highly recommended.
Sharma IP

ROSE BENGAL
• Rose bengal is actually a derivative of fluorescein.
• stain dead or degenerated cells and mucous strands
• used for evaluation of ocular pathologies including herpetic
corneal epithelial dendrites, superficial punctate keratitis,
meibomian gland dysfunction, and dysplastic or squamous
metaplastic cells of conjunctival squamous neoplasms.
• it’s blocked from staining the ocular surface where molecules such as mucins,
albumin, or even an artificial tear compound such as carboxymethylcellulose are
present.
• Rose bengal have intrinsic cellular toxicity.
• Rose bengal has a dose-dependent, toxic effect on human
corneal epithelial cells that is further enhanced by light
exposure.
• patient discomfort, particularly stinging upon instillation, which can become severe,
is often a deterrent from using rose Bengal.

Lissamine green
• Lissamine green: It preferentially stains membrane-
damaged or devitalized cells, and, like rose bengal,
localization of the dye to the cell nucleus has been noted.
• lissamine is unique in this group of three in that it has not
been shown to stain healthy ocular surface cells.
Evaluation of lissamine green staining in corneal epithelial
cells revealed that it doesn’t stain healthy, proliferating
cells and has a minimal effect on cell viability.
• There is no stinging or discomfort such as that associated
with rose bengal.

Contact lens induced conjunctival
staining with lissamine green

LISSAMINE GREEN
• • Stains degenerate cells, dead cells, and mucous fibrils
in the same manner as rose Bengal
• • The nucleus is generally stained more intensely that the
cytoplasm
• • Suitable for vital staining of the cornea and conjunctiva
• • Lissamine green detects dead or degenerated
conjunctival cells

THANK YOU

Corneal staining procedure

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