the pathologist’s approach to the vitreous...
TRANSCRIPT
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
Inflammatory: Non-Infectious
Inflammatory: Infectious
Autoimmune
Degenerative
Traumatic
Neoplastic
Pars planitis
Sarcoidosis
Behçets disease
Vogt-Koyanagi Harada
Juvenile xanthogranuloma
Genetic
Vitreous opacities
Acquired
Inflammatory: Non-Infectious
Inflammatory: Infectious
Bacterial
Fungal
Viral
ParasiticAutoimmune
Degenerative
Traumatic
Neoplastic
Genetic
Spirochaetal
TBC
CMV
Fungal
Syphilis
Toxoplasmosis
Vitreous opacities
Acquired
Inflammatory: Non-Infectious
Inflammatory: Infectious
Crohn’s disease
Multiple sclerosis
Autoimmune
Degenerative
Traumatic
Neoplastic
Genetic
Behçet’s disease
Vitreous opacities
Acquired
Inflammatory: Non-Infectious
Inflammatory: Infectious
Amyloid
Autosomal dominant VR disorderse.g. Wagner-Stickler syndrome
Familial exudative vitreoretinopathy
Snowflake degeneration
Autoimmune
Degenerative
Traumatic
Neoplastic
Genetic
Vitreous opacities
Acquired
Inflammatory: Non-Infectious
Inflammatory: Infectious
Degenerative
Traumatic
Neoplastic
Asteroid hyalosis
Synchysis scintillans
Haemorrhage
Vitreous detachment
Autoimmune
Genetic
Syneresis
Pigment granula
Vitreous opacities
Acquired
Inflammatory: Non-Infectious
Inflammatory: Infectious
Post-operativee.g. Irvine Gass
Cellophane retina
Haemorrhage
Vitreous detachment
Degenerative
Traumatic
Neoplastic
Autoimmune
Genetic
Pigment granula
Vitreous opacities
Acquired
Inflammatory: Non-Infectious
Inflammatory: Infectious
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
• MYD88 mutational analysis
IgH-PCR GeneScan
Bonzheim et al., Blood 2015
IL10:IL6
+/- IFN-Gamma
JAMA Ophthalmol. 2018;136(10):1098-1104
Vitreoretinal lymphoma: adjunctive diagnostics
• 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
Vitreoretinal lymphoma: adjunctive diagnostics
IL10:IL6
+/- IFN-g
IgH-PCR GeneScan
• Flow cytometry
• ELISA
• IgH-PCR
• MYD88 mutational analysis
• miRNAs
(miRs 19, 22, 92 and 155) Kakkassery V et al., 2016
Vitreoretinal lymphoma: adjunctive diagnostics
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