Ocular Pathology Basics

Christopher M Reilly, DVM, MAS, DACVP Basic Science Course June 5‐6, 2018 NC State University

1 Outline • General tips – Be nice to your pathologist – What the heck am I looking at? • Specific lesions not covered elsewhere • Stains

2 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

3 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

4 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

5 Example – cells v matrix v pigment

6 Mechanism Overviews/Examples

• Intracellular accumulations • Extracellular accumulations • Necrosis v Apoptosis • Tissue Degenerations • Inflammation • Neoplasia • Aging • Special stains*

7 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)

8 Corneal Epithelial Edema

9 Hemosiderin

Hematoidin

10 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 artifactual corneal clefting • When severe, fibers are wispy, irregular, and variably stained

11 Normal Canine Cornea

12 Corneal Edema

13 Mineralization

• Dystrophic – Due to cell death, normal calcium • Metastatic – Hypercalcemia, normal tissue • Mineral is basophilic in tissue section – Often shatters/fragments with sectioning

14 Stromal/BM mineralization

“Band Keratopathy”

15 Von Kossa Stain

16 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

17 Necrosis ‐ retina

More viable

Necrotic

18 Necrosis ‐ cornea • Can’t occur to extracellular matrix (ECM) • CAN occur in stromal tissue

19 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

20 Apoptotic keratinocytes

www.studyblue.com

21 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

22 Corpora Nigra

23 Atrophic Corpora Nigra

24 Optic nerve atrophy

25 Phthisis bulbi

• The end stage of severe ocular dz • Must be differentiated from microphthalmia • Criteria: – Shrinkage – Atrophy – Disorganization

26 Phthisis bulbi

27 Phthisis, Cat

28 Other: Solar Elastosis Apoptotic squamous cell (sunburn cell)

29 Inflammation

• Can affect any or all of the eye • Classified by: – Location – Chronicity – Cell type(s) – Etiologies

30 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

31 Types

• Neutrophilic – Acute, innate – Tissue destruction, necrosis • keratomalacia – Cavities/chambers – Surfaces

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

32 Fibrinosuppurative exudate

33 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)

34 Lymph/plasma cell/Mott cell

35 Eosinophilic

• Acute or chronic • Allergy, foreign body, parasites • Immune/idiopathic – Eosinophilic keratitis • Grossly characteristic – Granular on corneal surface • Luna’s stain can highlight

36 Luna’s Stain, Eos

37 Granulomatous

• Variably strict definitions – True granulomas – Granulomatous inflammation – sheets • Plump, activated, interdigitated – Histiocytic infiltrates • Idiopathic/Immune mediated histiocytoses – Common, confusing, poorly understood

38 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

39 Granuloma

40 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

41 Corneal Fibrosis

42 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?

43 Corneal endothelial dystrophy

Flattened Endothelium with irregular matrix

44 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

45 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

46 Asteroid Hyalosis, dog

Vitreous

Lens

47 Ciliary Hyalinization

48 Systemic Disease

• Metabolic – Diabetes mellitus (cataract, uveitis) – Hypertension (retinal hemorrhage, etc) • Neoplasia – Metastasis • Infection – FIP, systemic mycoses, West Nile Virus

49 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

50 Common Special Stains PAS-positive vascular deposits in diabetic vasculopathy • 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

51 Neurnal Ceroid Lipofuscinosis

H&E

RGCs

52 Neuronal Ceroid Lipofuscinosis PAS

RGCs

53 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.

54 B&B Tissue Gram Stain

55 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

56 Von Kossa Stain

57 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=AOv Vaw1wmD0gXZIKNvGiR7mQon35&ust=1528207143078519

58 Optic nerve, Trichrome

59 Common Special Stains III

• Luna’s Stain – eosinophil granules – Red/brown • Giemsa/Toluidine blue – metachromatic – Mast cells (purple) – Bacteria (pink)

60 Luna’s Stain, Eos

61 Giemsa

62 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)

63 Mucicarmine

64 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

65 Special Stains VI

• Luxol Fast Blue – Myelin, blue • Bodian’s – Axons, black • Combos: – LFB/HE, LFB/Bodians, LFB/PAS

66 Luxol Fast Blue ‐ Myelin