Moran CORE

Stains and Processing

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Tissue Processing

• Fixation in most cases is 10% neutral buffered formalin
• For electron microscopy, fix tissue in glutaraldehyde
• Fix in ethyl alcohol for cytologic preparations
• For immunofluorescence, fix with Michel medium
• Frozen sections and special stains (Oil Red O) require fresh tissue
  • Can keep tissue fresh for 24 hours if pack with ice
  • Place fresh tissue on saline-soaked gauze, not in dry gauze or container of saline
• Embedding process removes water and replaces with paraffin
  • Dissolves IOLs made of PMMA (polymethylmethacrylate), silicone, and haptics made of polypropylene
  • Does not dissolve sutures
  • Overnight process
• 4-6 μm sections of paraffin-embedded tissue are cut, affixed to a glass slide, and stained with various tissue dyes

Hematoxylin and Eosin (H&E)

 

H&E stain of cornea

• Used in routine histopathologic staining
• Blue: hematoxylin stains nucleus blue
• Red: eosin stains cytoplasm red/pink
• Does not stain lipidor mucin

Periodic Acid-Schiff (PAS)

 

PAS stain of corneal epithelium, Bowman’s membrane, and stroma

• Used to stain basement membranes (Descemet’s membrane, lens capsule, epithelial basement membrane)
• Magenta: glycogen and proteoglycans

Alcian Blue

 

Alcian Blue stain in Macular corneal stromal dystrophy

• Used to stain mucopolysaccharides
  • Useful in recognizing Macular corneal stromal dystrophy and cavernous optic atrophy of Schnabel
• Blue: acid mucopolysaccharide

Colloidal Iron

• Stain for mucopolysaccharides
  • Helps with recognition of Macular corneal stromal dystrophy and cavernous optic atrophy of Schnabel
• Blue: acid mucopolysaccharides
• Red/purple: collagen

Alizarin Red

 

Calcification on explanted IOL

• Used in band keratopathy and IOL calcification
• Red: calcium

Von Kossa

• Used to identify calcium, so useful in band keratopathy and IOL calcification
• Less specific for calcium than Alizarin Red
• Black: calcium phosphate salts

Congo Red

 

Congo red stain in Lattice corneal stromal dystrophy

 

Apple-green birefringence of corneal tissue

• Used to stain amyloid
  • Diagnoses amyloid deposits in all ocular tissues
  • Also useful in Lattice corneal stromal dystrophy
• Orange: amyloid
• Shows apple-green birefringence under polarized light

Crystal Violet

• Less commonly used to stain amyloid and used to recognize Lattice corneal stromal dystrophy
• Purple/violet: amyloid

Thioflavin T

• Stains for amyloid and used to recognize Lattice corneal stromal dystrophy in immunocytochemistry
• Fluorescent yellow: amyloid

Masson’s Trichrome

 

Masson’s Trichrome stain in Granular corneal stromal dystrophy

• Tri-color stain used to distinguish cells from connective tissue
  • Also used to diagnose Granular corneal stromal dystrophy
• Blue: collagen
• Dark red/purple: nuclei
• Red: muscle and cytoplasm

Gram Stain

 

 

Gram stain showing gram-positive bacteria

• Used if question of bacterial infection
• Specifically stains the thick peptidoglycan cell walls of certain types of bacteria
• Blue: Gram-positive bacteria
• Red: Gram-negative bacteria
• Yellow: corneal stroma

Ziehl-Neelsen

• Used to identify atypical mycobacteria
• AKA Acid-fast stain
• Red: acid-fast organisms

Gomori Methenamine Silver (GMS)

 

GMS stain showing yeast

• Primarily used to identify fungal elements
• Black: fungal elements
• Green/blue: background

Gridley

 

Gridley stain showing Acanthamoeba cysts in the cornea

• Primarily used in the diagnosis of acanthamoeba
• Gray/silver: acanthamoeba cysts
• Green: background/stroma

Prussian Blue

 

Prussian blue stain of cornea

• Used to stain for iron in tissue, such as in hemosiderosis bulbio
• Iron lines are left wherever tears pool and at elevations (keratoconus, pterygium)
• Blue: iron

Verhoeff-van Gieson

• Stains for elastin, which can be helpful in identifying the elastic layer of temporal artery
• Black: elastic fibers

Oil Red O

 

Oil Red O stain showing arcus senilis of the cornea

• Stains for lipid in frozen sections of fresh tissue
  • Helps diagnose sebaceous carcinoma versus other epithelial-derived malignancies
• Red: lipid

Sudan Black

• Also stains for lipid in frozen sections of fresh tissue
  • Helps diagnose sebaceous carcinoma versus other epithelial-derived malignancies
• Used less often than Oil Red O
• Black: lipid

Immunohistochemistry (IHC)

 

S-100 immunohistochemistry stain identifying melanocytes

• How IHC works
  • Cells present specific antigens
  • Primary antibody binds cell antigen
  • Secondary antibody binds to primary antibody
  • Chromophore on secondary antibody gives color signal seen on microscopy
• Common antigens and cell types in IHC
  • Cytokeratins: cells of epithelial origin (adenoma, carcinoma)
  • Desmin, myoglobin, actin: smooth and skeletal muscle (leiomyoma, rhabdomyosarcoma)
  • S-100: neuroectoderm (schwannoma, neurofibroma, melanoma)
  • HMB-45, Melan A: melanocytes (nevus, melanoma)
  • Chromogranin, synaptophysin: neuroendocrine cells (metastatic carcinoid, small cell carcinoma)
  • Leukocyte common antigen (CD45): hematopoietic cells (leukemia, lymphoma)
  • CD antigens: white blood cell subtypes
  • Her2/Neu, c-Kit: metastatic breast carcinoma prognosis and treatment

Electron Microscopy

 

TEM of Iris Pigment Epithelium Leiomyoma

• Uses an electron beam rather than a beam of light (photons)
• Electrons have a smaller wavelength, so can achieve a higher resolution
• Specimen must be dehydrated and embedded in resin
• Fix tissue with glutaraldehyde instead of formalin
• Stain with heavy metal (lead, uranium, tungsten) to scatter electrons and create contrast in different structures
• Needs high-vacuum environment to allow electron beam to be straight
• Generates black-and-white images
  • Black: electron dense
• Transmission electron microscopy (TEM)
  • Creates cross-section image of specimen
  • Requires very thin sections that are 100 nm thick
• Scanning electron microscopy (SEM)
  • Creates image of the specimen’s surface
  • Electrons do not pass through specimen, so thin sections not needed
  • Lower voltage electron beam than TEM
  • Can use low-vacuum environment
  • Coat with gold to prevent charging the specimen from the electron beam and increase the signal-to-noise ratio
• Lengthy specimen preparation and very expensive equipment, therefore very rarely used in ocular pathology

Summary of Common Stains in Ocular Pathology

Stains	Colors	Examples
H&E	Blue: nucleus Red/pink: cytoplasm	Routine tissue stain
PAS	Magenta: basement membrane glycogen and proteoglycans	Descemet membrane
Alcian Blue	Blue: acid mucopolysaccharides	Macular corneal stromal dystrophy
Colloidal Iron	Blue: acid mucopolysaccharides Red/purple: collagen	Macular corneal stromal dystrophy
Alizarin Red	Red: calcium	Band keratopathy
Von Kossa	Black: calcium phosphate	Band keratopathy
Congo Red	Orange with red-green birefringence: amyloid	Lattice corneal stromal dystrophy
Crystal Violet	Purple/violet: amyloid	Lattice corneal stromal dystrophy
Thioflavin T	Fluorescent yellow: amyloid	Lattice corneal stromal dystrophy
Masson’s Trichrome	Blue: collagen Dark red/purple: nuclei Red: cytoplasm, muscle	Granular corneal stromal dystrophy
Gram Stain	Blue: Gram-positive bacteria Red: Gram-negative bacteria Yellow: corneal stroma	Bacterial infections
Ziehl-Neelsen	Red: acid-fast organisms Blue: background	Tuberculosis
GMS	Black: fungal elements Blue-green: background	Fungal infections
Gridley	Gray-silver: acanthamoeba cysts Green: background	Acanthamoeba infections
Prussian Blue	Blue: iron	Hemosiderosis bulbi
Verhoeff-van Gieson	Black: elastic fibers	Temporal artery biopsy
Oil Red-O	Red: lipid	Sebaceous carcinoma
Sudan Black	Black: lipid	Sebaceous carcinoma

References

Harper RA. Basic Ophthalmology. Amer Academy of Ophthalmology; 2010.

Yanoff M, Fine BS. Ocular Pathology, A Text and Atlas. Lippincott Williams & Wilkins; 1989.