The pathologist’s approach to the vitreous...

The pathologist’s approach to the vitreous biopsy

Sarah E CouplandGeorge Holt Chair in Pathology

Dept. of Molecular & Clinical Cancer MedicineInstitute of Translational Medicine, University of Liverpool, UK

Normal Vitreous

•Composition: Hyaluronic acid, collagenous filaments

•Minimal cellular content: Hyalocytes, astrocytes and glial cells

Vitreous opacities

Congenital

Acquired

Remnants of hyaloid vasculatur system (e.g. cysts)

Persistent hyperplastic primary vitreous

Endogenous: colloid coagula, crystalline deposits

Exogenous: protein coagula, amyloid, cells

Duke Elder

Chapter V, p322

Vitreous opacities

Acquired

Inflammatory: Non-Infectious

Inflammatory: Infectious

Autoimmune

Degenerative

Traumatic

Neoplastic

Genetic

Vitreous opacities

Acquired



Autoimmune

Degenerative

Traumatic

Neoplastic

Pars planitis

Sarcoidosis

Behçets disease

Vogt-Koyanagi Harada

Juvenile xanthogranuloma

Genetic

Vitreous opacities

Acquired



Bacterial

Fungal

Viral

ParasiticAutoimmune

Degenerative

Traumatic

Neoplastic

Genetic

Spirochaetal

TBC

CMV

Fungal

Syphilis

Toxoplasmosis

Vitreous opacities

Acquired



Crohn’s disease

Multiple sclerosis

Autoimmune

Degenerative

Traumatic

Neoplastic

Genetic

Behçet’s disease

Vitreous opacities

Acquired



Amyloid

Autosomal dominant VR disorderse.g. Wagner-Stickler syndrome

Familial exudative vitreoretinopathy

Snowflake degeneration

Autoimmune

Degenerative

Traumatic

Neoplastic

Genetic

Vitreous opacities

Acquired



Degenerative

Traumatic

Neoplastic

Asteroid hyalosis

Synchysis scintillans

Haemorrhage

Vitreous detachment

Autoimmune

Genetic

Syneresis

Pigment granula

Vitreous opacities

Acquired



Post-operativee.g. Irvine Gass

Cellophane retina

Haemorrhage

Vitreous detachment

Degenerative

Traumatic

Neoplastic

Autoimmune

Genetic

Pigment granula

Vitreous opacities

Acquired



Autoimmune

Degenerative

Traumatic

Idiopathic

Neoplastic

Primary

Secondary

Retinoblastoma

Vitreoretinal lymphoma

Metastatic

cutaneous melanoma

Acute myeloid leukaemia

• To identify the nature of an infectious /inflammatory process in vitreous

• To establish the aetiological agent causing the vitritis

• To confirm or refute the clinical impression of an intraocular malignancy

• To classify, subtype and grade the intraocular neoplasm

Pathologist‘s role in vitreous samples

Vitreous/aqueous tap

Diagnostic vitrectomy

Chorioretinal biopsy

Subretinal aspirates

Ocular fluid sample types

Glutaraldehyde

EM

CD20

Cytolyt, PreserveCyt, HOPE (fluid samples)

•Conventional stains

•Immunohistochemistry

•FISH(Fluorescence in situ hybridisation)

•PCR(Polymerase chain reaction)

•MLPA(Multiplex ligation probe-dependent

amplification) • MSA

(Microsatellite analysis)•CGH array

(Comparative genomic hybridisation array)

•SNP array(Single nucleotide polymorphism array)

Formalin (tissue Bx)

IgH-PCR

GeneScan

FISH

Fresh

•Microbiological analysis• Biochemical analysis•Gene expression profiling

Immunofluorescence

ELISA

GEP

Intraocular biopsies

Pathologist‘s approach to vitreous samples

Inadequate sample

Adequate sample

Cytomorphology

Non-Cellular

Cellular

Acellular Vitreous Samples

Conjunctival squames Vitreous condensed fibrils

Retained lens material Asteroid hyalosis

Synchisis scintillans

Cholesterol Crystals

Cellular Vitreous Sample: Haemorrhage

Perl’s

Inflammatory non-infectious, macrophage-poor vitritis

CD3

PAS

Immunocytology

Cultures

(IgH-PCR)

Chronic non-specific vitritis

Inflammatory non-infectious, macrophage-rich vitritis

PAS stain - important to exclude cytoplasmic inclusions

DD: Whipple’s disease, Histoplasma capsulatum

CD68PAS

Giemsa

Inflammatory non-infectious, granulomatous vitritis

ZNPASWSGrocott

Juvenile XanthogranulomatosisSarcoidosis

Chorioretinal

biopsy

Vitreous

CulturesPCR

Inflammatory infectious, neutrophil-rich vitritis

Bacterial

HE HE

Grocott Gram

E. coliStaphylococcu aureusStreptococcus sp.Neisseria sp.P. acnes

e.g. Behçets disease

DD: Non-Bacterial

Inflammatory infectious, eosinophil-rich vitritis

HEPapanicolou

DD:

Parasitic-induced endophthalmitis

Toxocara canis

Birdshot chorioretinopathy

(Chronic myeloid eosinophilic leukaemia)Borrelia burgdorferi.

HE

Levaditi

Inflammatory infectious, granulomatous vitritis

Mycobacteria

Ziehl-Neelsen

Ziehl-Neelsen

Acid fast bacilli often found within macrophages and RPE

Inflammatory infectious, (non)granulomatous vitritis

Candida sp.

Candida sp.

PAS PAS

Aspergillus sp.

PAS

Inflammatory infectious viral vitritis/ chorioretinitis

Herpes simplex

virus

Immunofluorescence

Cytomegalovirus

CMV Ab

Inflammatory infectious parasitic vitritis

Toxoplasmosis

MGG

CD20 Ki-67MGG H&E

Barry RJ, et al. Br J Ophthalmol 2018

Vitreoretinal lymphoma

• Flow cytometry

• ELISA

• IgH-PCR

• MYD88 mutational analysis

IgH-PCR GeneScan

Bonzheim et al., Blood 2015

IL10:IL6

+/- IFN-Gamma

Vitreoretinal lymphoma: adjunctive diagnostics

• Flow cytometry

• ELISA

• IgH-PCR


IgH-PCR GeneScan

Bonzheim et al., Blood 2015

IL10:IL6

+/- IFN-Gamma

JAMA Ophthalmol. 2018;136(10):1098-1104


• Flow cytometry

• ELISA

• IgH-PCR

• NGS panelse.g. MYD88, CDKN2A and AKT1

IL10:IL6

+/- IFN-Gamma

IgH-PCR GeneScan

Cani AK et al., Oncotarget, 2016

Bonzheim I, Fend F, Coupland SE et al: European Association Haematopathology Meeting 2018

MYD88, CD79B, PIM1, TBL1XR1, CARD11, IRF4, BTG1/2, PRDM1, CREBBP, IGLL5, ETV6


IL10:IL6

+/- IFN-g

IgH-PCR GeneScan

• Flow cytometry

• ELISA

• IgH-PCR


• miRNAs

(miRs 19, 22, 92 and 155) Kakkassery V et al., 2016


Intraocular LymphomasPrimary

Secondary

Choroidal

Low grade B-cell ‘MALT’ Ly

Iridal

CD20High grade B-cell Ly

Choroidal infiltration: B-CLL

Retinal

Vitreoretinal

VitrealHigh grade B-cell Ly

Vitreous Infiltrates: Neoplastic other

Primary

Retinoblastoma

Secondary

Met. cutaneous melanoma

Acute myeloidleukaemia

Pathologist‘s findings in vitreous samples

Non-diagnostic !

Diagnostic

Cytomorphology

Non-Cellular

Cellular

Cytomorphology

Immunocytology

Fresh

Fixed (Cytolyt; HOPE fixation medium)

Culture medium

CytospinsAgar cell blockFilter techniques

Vitreous Sample: insufficient for diagnosis!

Possible Causes

•Normal vitreous•Sampling error•Small sample and/or scanty cells•Prior steroid therapy •Loss of material in transport•Loss of material in processing•Insufficient material for all tests•Transport delays (Fri -> Mon)•Wrong fixative

• Extensive DDx -> Essential = detailed history on request form

• Delicate specimens require proper and careful handling

• Close collaboration between clinicians, pathologists and microbiologists

• Experienced technical staff with up-to-date techniques

• Specialist expertise in assessing vitreous samples

Conclusions

“It used to be said that syphilis - particularly syphilitic chorioretinitis - was the mostcommon cause of exogenous opacities but, while they are numerous andpronounced in this disease, it by no means holds a monopoly”

(Duke Elder, page 360, Vol. XI)

Some photographs courtesy of:Heinrich HeimannBertil DamatoNikolaos BechrakisMatthias BeckerNarsing RaoPaty Chevez-Barios

AcknowledgmentsSome scanned photographs from:“Diseases of the Ocular Fundus” Kanski, Milewski, Damato, Tanner“Retina and Vitreous” Federman, Gouras, Schubert, Slusher & Vrabec“Clinical Ophthalmology”, Kanski“Ocular Pathology” Yanoff and Fine“Atlas of Clinical Ophthalmology”, Spalton, Hitchings and Hunter

The pathologist’s approach to the vitreous...

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