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Ocular Pathology Basics

Christopher M Reilly, DVM, MAS, DACVP

Basic Science Course

June 5‐6, 2018

NC State University

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Outline

• General tips

–Be nice to your pathologist

–What the heck am I looking at?

• Specific lesions not covered elsewhere

• Stains

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Be nice to your pathologist

• Help them help you

–History, when known

– Specific instructions when needed

–Description/diagrams for focal lesions

• Package specimens appropriately

• Don’t put big things in cassettes

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What the heck am I looking at?

• You gotta know normal: – www.youtube.com/watch?v=5n4nfMFb‐BU ‐ overview

– www.youtube.com/watch?v=bkGVB2CMXnY ‐ fibrous tunic

– www.youtube.com/watch?v=SI‐kfQae49o ‐ anterior uvea

– www.youtube.com/watch?v=r7cpMQqFqNc ‐ choroid/tapetum

– www.youtube.com/watch?v=bwMEEfFq3eU ‐ retina and optic nerve

– www.youtube.com/watch?v=lnLKaD675tU ‐ glaucoma

– www.youtube.com/watch?v=osYVARsMbUo ‐ some bird stuff

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The basics:

• Pink = protein = eosinophilic

– Cytoplasm/matrix/granules

• Blue = nuclei = basophilic

• Purple = cytoplasm/matrix = amphophilic

• Fat/Water = clear (washes out)

– except early/moderate corneal edema

• PIgments = their natural color

– Melanin, hemosiderin, hematoidin

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Example – cells v matrix v pigment

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Mechanism Overviews/Examples

• Intracellular accumulations

• Extracellular accumulations

• Necrosis v Apoptosis

• Tissue Degenerations

• Inflammation

• Neoplasia

• Aging

• Special stains*

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Intracellular accumulation 

• Water – Acute cellular swelling

• Other stuff

– Lipid – e.g. lipid corneal dystrophy

• Also may be extracellular

–Hemosiderin – e.g. chronic hemorrhage

– Lipofuscin – e.g. age, storage disease (e.g. neuronal lipofuscinosis) 

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Corneal Epithelial Edema

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Hemosiderin

Hematoidin

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Extracellular Accumulations

• Edema

• Generally clear space

– Uvea, retina, orbital tissues, dermis/lids

• Cornea ‐ unique appearance of edema

• Overhydration of GAGs between collagen fibers = “washed out” appearance of expected artifactualcorneal clefting

• When severe, fibers are wispy, irregular, and variably stained

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Normal Canine Cornea

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Corneal Edema

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Mineralization

• Dystrophic

–Due to cell death, normal calcium

• Metastatic

–Hypercalcemia, normal tissue

• Mineral is basophilic in tissue section

–Often shatters/fragments with sectioning

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Stromal/BM mineralization

“Band Keratopathy”

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Von Kossa Stain

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Necrosis

• Cellular swelling, then loss–Usually demarcated groups of cells

• Hypereosinophilia ‐ denatured protein–Nuclear changes – karyorrhexis

• Eventual loss of nuclear detail

– Secondary inflammation

• Connective tissue relatively spared

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Necrosis ‐ retina

More viable

Necrotic

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Necrosis ‐ cornea 

• Can’t occur to extracellular matrix (ECM)

• CAN occur in stromal tissue

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Apoptosis

• Programmed cell death

– Usually individual cells

• Can also be part of development

– Anterior segment, lymphoid development

• Intrinsic or extrinsic signals

• Classically: shrunken cells with uniform nuclear fragmentation 

• No overt inflammation

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Apoptotic keratinocytes

www.studyblue.com

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Tissue Degeneration

• Atrophy – loss of tissue bulk

– Senile iris atrophy

–Optic nerve atrophy – often with gliosis

• Proliferation can accompany atrophy

–Phthisis bulbi – widespread along with disorganization

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Corpora Nigra

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Atrophic Corpora Nigra

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Optic nerve atrophy

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Phthisis bulbi

• The end stage of severe ocular dz

• Must be differentiated from microphthalmia

• Criteria:

– Shrinkage

–Atrophy

–Disorganization

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Phthisis bulbi

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Phthisis, Cat

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Other: Solar ElastosisApoptotic squamous cell (sunburn cell)

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Inflammation 

• Can affect any or all of the eye

• Classified by:

– Location

–Chronicity

–Cell type(s)

– Etiologies

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Inflammation 

• Includes both the fluid (edema, flare) and cellular (infiltrate, cell) events

–Corneal edema can be non‐inflammatory (endothelial disease)

– Fluid dysregulation, however, may lead to inflammatory changes

• Most eye diseases have some inflammatory or immune component

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Types

• Neutrophilic–Acute, innate

– Tissue destruction, necrosis• keratomalacia

–Cavities/chambers

– Surfaces

https://bcrc.bio.umass.edu/courses/fall2011/biol/biol523/content/neutrophil

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Fibrinosuppurative exudate

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Lymphocytic plasmacytic

• Chronic, adaptive

– At least a couple days

– Tissue response (e.g. uvea)

• Often perivascular, sometimes nodular

• Etiologically nonspecific

–Proportion can help (plasma cells in FIP)

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Lymph/plasma cell/Mott cell

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Eosinophilic

• Acute or chronic

• Allergy, foreign body, parasites

• Immune/idiopathic 

– Eosinophilic keratitis

• Grossly characteristic

– Granular on corneal surface

• Luna’s stain can highlight

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Luna’s Stain, Eos

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Granulomatous

• Variably strict definitions

– True granulomas

– Granulomatous inflammation – sheets

• Plump, activated, interdigitated

– Histiocytic infiltrates

• Idiopathic/Immune mediated histiocytoses

– Common, confusing, poorly understood

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Granulomatous

• Search for etiology

– Fungal – stains: GMS, PAS, BCG IHC

–Mycobacterial ‐ Fite’s, Ziehl‐Neelsen AF, BCG

– Foreign body ‐ polarized light for plants, plastic, suture, hairs, cotton

• Wrong diagnosis = wrong treatment

– Steroids v antimicrobials

• May need fresh tissue for culture

– Think before you fix

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Granuloma

*

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Fibrosis

• Common end result of inflammation

• Indicates chronicity

• Corneal fibrosis/scarring

• Uvea is resistant

–But chambers and surfaces prone

• Pre‐iridal, cyclitic, retrocorneal, vitreal, epiretinal

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Corneal Fibrosis

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Dystrophy

• Inherited*, non‐inflammatory, bilateral lesions

–Corneal opacities in vet med

• Endothelial dystrophy

–Better characterized in humans – Boston Terriers, Dachshunds, Chihuahuas

• Often secondary – true dystrophy?

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Corneal endothelial dystrophy

DM

Flattened Endothelium with irregular matrix

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Neoplasia

• Round cell: leukocytes (melanoma?)

– Sheets

– No real pattern, architecture

• Epithelial – polygonal, cuboidal, columnar

–Nests, cords, tubules, acini, “islands”

• Mesenchymal – spindle cells, mostly

–More vague patterns, streams, whorls, etc

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Aging

• Variably significant, can be subtle

–Nuclear sclerosis, senile cataract

– Thickening of DM and lens capsule

–Hyaline material in ciliary body

–Cystic degeneration of ocualr

–Asteroid hyalosis

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Asteroid Hyalosis, dog

Lens

Vitreous

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Ciliary Hyalinization

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Systemic Disease

• Metabolic

–Diabetes mellitus (cataract, uveitis)

–Hypertension (retinal hemorrhage, etc)

• Neoplasia–Metastasis

• Infection – FIP, systemic mycoses, West Nile Virus

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Common Special Stains I

• Periodic Acid‐Schiff

– Starch (glycogen, glycoproteins, fungi)

–Magenta

• Alcian Blue (Alb)

–Mucopolysaccharides, GAGs

– Bright blue• i.e. vitreous

• Along with PAS

– Deep Blue ‐ Cartilage

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Common Special Stains

• Periodic Acid‐Schiff ‐ PAS

– Carbohydrates (BM, fungus, cellular debris, mucus, Lipofuscin)

– Magenta

– Often done w/ Alcian Blue

• Grocott’s Methenamine Silver ‐ GMS

– Similar to PAS, can stain differently

– Black – can be confusing in pigmented eyes

– Light green counterstain

PAS-positive vascular deposits in diabetic vasculopathy

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Neurnal Ceroid Lipofuscinosis

H&E

RGCs

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Neuronal Ceroid Lipofuscinosis

PAS

RGCs

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Common Special Stains II

• Tissue Gram stain 

– Typically Brown & Brenn (B&B)

• Gram +, Gram – (often weak)

• Yellow background

• Other techniques better for some

–Brown and Hopps for Klebsiella spp.

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B&B Tissue Gram Stain

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Common Special Stains III

• Von Kossa

– Phosphate, black

• Prussian Blue (or Gomori’s, Perl’s)

– Iron (hemosiderin)

– Ferrugination – iron in blood vessels adjacent to dead neurons (can look like mineral)

• Masson’s Trichrome

–Muscle = red; Collagen = Blue; Cytoplasm = Pink; Nuclei = Blue/black 

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Von Kossa Stain

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Hemosiderin/Prussian Blue

https://www.google.com/url?sa=i&source=images&cd=&ved=2ahUKEwjL4Ye8lbrbAhXCiVQKHfdPD14Qjhx6BAgBEAM&url=http%3A%2F%2Fslideplayer.com%2Fslide%2F8027407%2F&psig=AOvVaw1wmD0gXZIKNvGiR7mQon35&ust=1528207143078519

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Optic nerve, Trichrome

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Common Special Stains III

• Luna’s Stain – eosinophil granules

–Red/brown

• Giemsa/Toluidine blue – metachromatic

–Mast cells (purple)

–Bacteria (pink)

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Luna’s Stain, Eos

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Giemsa

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Common Special Stains IV

• Fontana Masson – Melanin

– Black in a VERY pale background

• Mucicarmine – Mucus

– Pink, can highlight Cryptococcus

• Acid Fast (Ziehl‐Neelson, Fite’s)

–Mycobacteria, other Acid Fast

– Red w blue background

– Fite’s for atypical AFB (e.g. M. leprae)

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Mucicarmine

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Special Stains V

• Oil Red O, Sudan Black– Lipid, can’t be on processed tissue

–Can do fixed, unprocessed

• Phosphatongstic acid‐hematoxylin

– Fibrin, black

• Vierhoff‐van Gieson– Elastin, black

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Special Stains VI

• Luxol Fast Blue

–Myelin, blue

• Bodian’s

–Axons, black

• Combos:

– LFB/HE, LFB/Bodians, LFB/PAS

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Luxol Fast Blue ‐ Myelin