dyes and stains

• Diagnostic agents

  • No therapeutic effects as they are not given to treat a condition
  • Non-irritable and non-toxic
  • Compatible with other agents used alongside other things such as anaesthetics
  • Reversible – not stuck with dyed eye
  • No loss of vision
  • Selectively stains cells/structures – helps to see what is damaged. Not helpful if all the cells are affected
• Dyes

  Briefly adds colour to the ocular fluids or surface tissue. Doesn't penetrate deep within the eye. E.g. fluorescein sodium
• Stain

  Lasts several hours. E.g. Rose Bengal
• Ocular structures

  • 1 - limbus
  • 2 – bulba conjunctiva
  • 3 – conjunctival fornix
  • 4 – palpebral conjunctiva
  • 5 – punctum
  • 6 – edge of eyelid
• Goblet cells

  • Maximum distribution in fornices
  • Form the mucous layer of tears
• Accessory glands of Krouse & Wolfring

  • Located in the fonices
• What are the main functions of the conjunctiva?

  • Protection
  • Provides mucous layer of tear film
  • Supplies cornea with oxygen when eyes are closed
• Conjunctival epithelium

  • Made up of about 7 layers of single cells
  • Most superficial layer is the epithelium
  • If these cells are damaged, they take about 24hours to regenerate
  • The anterior surface is continuous with the conj
  • The posterior surface is firmly attached to the basement membrane
  • The stroma is the main layer and is composed of collagen fibrils
  • These maintain the strength of the cornea and are transparent
  • Most superficial layer is the epithelium
  • Descemet's membrane attaches the endothelium to the stroma
• Describing and recording abnormalities

  • Colour
  • Elevated o flat
  • Size
  • Location
  • Depth
  • Size and location can be described in relation to adjacent structures
• Describing eyes with dyes/stains

  • Size
  • Location
  • Depth
  • Size and location can be described in relation to adjacent structures
• Dry eye diagnosis

  Diagnosed by the number of spots on the conjunctiva and the cornea
• Simple descriptions of staining patterns

  May lack accuracy and the reliability of the observer may be questionable
• Van Bijsterveld

  • 0 to 3 points for each of nasal conjunctiva, cornea and temporal conjunctiva
  • > 3.5 points on scale of 9 = dry eye
• Popular anterior eye health grading scales

  • Photographic scales: CCLRU, Brian Holden Vision Institute (BHVI)
  • Artist rendered: Efron (a bit more uniform)
• National Eye Institute/Industry Workshop guidelines (1995)

  • 5 areas each scored 0-3
  • Total out of 15 for cornea
  • Total out of 18 for conjunctiva
• Oxford scale (1984, revised 2003)

  • Number of dots across cornea and conjunctiva O-V pictorial scale
  • Log scale
  • Used with any stain
• SICCA-Ocular staining score (2010)

  • Grade cornea with fluorescein and conjunctiva with lissamine green. 0-3 scale
  • Dependent on dot number with extra points for specific features
  • Total score out of 12 per eye
• Inserting dye/stains using strips

  1. Wash your hands

  2. Check expiry date

  3. Remove strip from packet

  4. Use saline to wet the strip without contaminating the saline bottle

  5. Get patient to look up

  6. Hold lower lid down

  7. Touch flat part of strip onto inferior temporal conjunctiva

  8. Release lids and ask patient to blink
• Rose Bengal

  • Stain
  • Category: xanthene dye
  • Brownish-red powder, soluble in water producing a solution (usually 1%)
  • Absorption and staining not pH dependant
  • Absorption at 550mn with smaller peak 510mn
  • Little fluorescence i.e. illumination does not make much difference, can view with white light
  • Fluorescein derivative but different staining characteristics
  • Crosses cell membranes of dead or degenerated cells
  • Stains mucous threads
  • Close eyes for 30s after insertion
  • Discomfort initially, goes within a few minutes
• Rose Bengal preparations and availability

  • Not manufactured in UK
  • Minims Rose Bengal discontinued 2008
  • Licensed suppliers of overseas products. E.g. CE – rose Bengal strips, Omni rose, rosets
  • Contain 1.3 or 1.5mg of rose Bengal
• Indications and use of Rose Bengal

  • Rarely used in UK currently
  • Dry eye: Staining of conjunctiva is a better indication of dry eye than Shirmer test
  • Grading: Van Bijsterveld often favoured for Rose Bengal
  • Contact lens pressure area: Intolerance to contact lenses, may need to change lens
  • Dendritic keratitis: Stains ulcer nicely, stains only ulcer
• Adverse effects of Rose Bengal

  • May substantially reduce fluorescein fluorescence (NaFl) – use NaFl 1st
  • Irritation of ocular surface, especially in dry eye patients
  • Anti-viral effects which limit its use in suspected viral ulcers if planning to culture for viral agents
  • May damage corneal epithelial cells
  • Stains normal proliferating cells
• Lissamine Green

  • Proper name is Green S or Light Green SF
  • Dark (olive) green colour
  • Washes off quickly, doesn't appear to stain nuclei
  • Non-fluorescent, strong absorption at 620-640 nm (red)
  • Illuminated with white light – see the reflected (non-absorbed) colours = blueish-green
  • Red filter enhances visualization by transmitting wavelengths that lissamine green absorbs
  • Stains similar cells to Rose Bengal: Lipid-like structures like mucous strands, Dead/dying epithelial cells, Leading edges of viral ulcer
  • Inserting too little will result in no staining
  • Clinics – impregnated strips, minimal/no discomfort as dilute
  • Research – solutions so that can control concentration (1% ideal, 2-3% can cause irritation)
• What are some extra steps inserting Lissamine Green

  1. Use saline to wet the strip without contaminating the saline bottle. Ensure a whole drop (or two) stays on the strip for more than five seconds

  2. Touch flat part of strip onto inferior temporal conjunctiva. A larger volume is needed compared with fluorescein

  3. If combined assessment with fluorescent: both strips can be wet and applied at the same time, if done separately apply fluorescein first, consider using two strips/insertions with lissamine green with one fluorescein
• Lissamine Green preparations and availability


  • Most strips have ~1.5mg dye
  • Some CE marked for use as a 'diagnostic agent when superficial corneal or conjunctival change is suspected'
• Indications and use of Lissamine Green

  • Corneal epithelial cells – limited use as poor visibility in font of iris, shows only damaged epithelial cells (not missing cells), only visible on severe dry eye patients, fluorescein is better for this
  • Conjunctival epithelial cells – good for this, best choice of dye/stain for this
  • Pressure for contact lens around limbus (peri-limbal contact lens staining)
  • Dry eye disease: List number of corneal and conjunctival spots
  • Lid-wiper epitheliopathy: Stained with any of the vital stains but lissamine green is preferred
• Lissamine green: adverse reactions

  • No major adverse reactions reported
  • Vs Rose Bengal, LG is: Better tolerated, Doesn't contaminate if px suspect viral infection, Doesn't stain normal proliferating cells, Doesn't damage corneal epithelial cells
• Fluorescein Sodium

  • Orange-red coloured xanthine dye
  • Topical stain and injected during fluorescein angiography
  • It colours the tear film
  • Absorbs blue light (485-500nm) at neutral pH
  • Absorbed energy excites fluorescein molecules
  • Emits longer wavelengths of lower energy (peak at 525-530nm)
  • Blue filter on the slit lamp limits the illumination to those absorbed by the fluorescein, eliminating glare from other wavelengths
  • Can use Wratten filter 45A over illumination
  • Emits longer wavelengths of lower energy at neutral pH (peak emission 525-530nm)
  • Gives yellow-green appearance
  • Best viewed with yellow filter: Wratten 15
  • Available as card mounted or slit lamp mounts
  • Converts yellow fluorescence on blue background to green, yellow on green background
  • Normal eye: Colours tear film, Normal corneal epithelium impermeable to tear film as lipid membranes are a barrier to water-soluble polar substances
  • Damaged eye: Epithelium damaged – tear film penetrates tissue (cornea/conjunctiva), pH difference causes green colour in area of desquamation, NaFl pools in the defects or spaces to show as highly fluorescent, Diffuses sideways when in deeper layers
• Fluorescein sodium: preparations and availability

  • Strips are preparations of choice in practice
  • Strips not currently manufactured in UK
  • Single use fluorescein impregnated strips 1mg dye
  • Fluorexon is a high molecular weight fluorescein 0.5mg dye
  • P Fluorescein Sodium: 1 and 2% available, Preservative free single dose unit
  • PoM Minims Lidocaine and Fluorescein: Lidocaine hydrochlor 4%, fluorescein sodium 0.25%, Preservative-free single use eye drops
• Fluorescein Sodium: Indications and use

  • Store in a cool dry place, avoid contamination of strips, don't want to pass infection into eye
  • Before use: Check px not allergic to NaFl
  • Integrity of cornea and conjunctiva: Damage from foreign bodies, Dryness, Recurrent corneal erosions, Corneal ulcers, Determining depth of defect, Full thickness break in cornea
  • Contact lens fitting
  • Applanation tonometry
  • Tear flow assessment and nasolacrimal duct patency
  • Fluorescein tear break up time
• Fluorescein sodium: indications and use

  • Contact lens fitting of rigid lenses: Rigid gas permeable (RGP)
  • Orthokeratology
  • Applanation tonometry
  • Tear flow and nasolacrimal duct patency
  • Fluorescein tear break up time
• Fluorescein Sodium: contraindications

  Soft contact lenses
• Fluorescein Sodium: adverse reactions

  • Very rare
  • Allergic reaction: Acute-onset inflammation/redness/oedema of the eyelids and periocular skin, Anaphylactic reaction, Skin rash