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The Pathologist's Perspective on Vitreous Opacities

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The Pathologist's Perspective on Vitreous Opacities Eye (2008) 22, 1318–1329 & 2008 Macmillan Publishers Limited All rights reserved 0950-222X/08 $32.00 www.nature.com/eye CAMBRIDGE OPHTHALMOLOGY SYMPOSIUM The pathologist’s SE Coupland perspective on vitreous opacities Abstract proper handling and expert application of a wide range of specialized techniques. Background Vitreous opacities are diverse in Eye (2008) 22, 1318–1329; doi:10.1038/eye.2008.31; nature. Many underlying diseases are sight- published online 14 March 2008 threatening and several are also lethal. This review presents the pathologist’s perspective Keywords: vitritis; vitreous inflammation; of vitreous opacities, correlates laboratory vitreous infiltrates; ocular lymphoma; retinal findings with the underlying disease and lymphoma; vitreoretinal lymphoma recommends safe methods for handling specimens. An aetiological classification of vitreous opacities is also proposed. Methods A gentle fixative such as Cytolyt or Introduction HOPE-fixation is required, unless delivery of the vitreous biopsy specimen to the laboratory The vitreous body is a transparent, extracellular can be guaranteed within two hours. Cells and gel, with a complicated structural framework of other material are precipitated onto slides or collagen, soluble proteins, hyaluronic acid, and into cell blocks by centrifugation. Light a water content of 99%. The few cells that are microscopy with the May-Grunewald Giemsa normally present in the vitreous gel are located stain is enhanced, as necessary, by the use of predominantly in the cortex and consist of special stains, such as Congo red for amyloid, hyalocytes, astrocytes, and glial cells. Vitreous Perl’s for iron, Periodic Acid-Schiff for abnormalities include opacification, microorganisms, and several others. liquefaction, and shrinkage.1 Immunocytological methods enable cell The terminology of visible vitreous typing, using labels such as CD3 for T-cells in abnormalities is confusing. The term ‘vitreous reactive inflammation; CD20 for B-cells in opacities’ refers to visible structures in the retinal lymphoma; CD34 and myeloperoxidase vitreous gel (as opposed to opacities within the for myeloid leukaemic cells. The polymerase Department of Pathology, vitreous cavity but outside the gel, exemplified University of Liverpool, chain reaction enhances the identification of by subhyaloid haemorrhage). ‘Vitreous Liverpool, England organisms in endophthalmitis and of infiltrates’ imply transport of extraneous immunoglobulin rearrangements in material through membranes in the retina. Correspondence: lymphoma. ‘Vitreous deposits’ would include such SE Coupland, Results Acquired vitreous opacities can be infiltrates as well as substances ‘precipitating’ Department of Cellular and classified according to their aetiology as: Molecular Pathology, from within the vitreous itself and becoming University of Liverpool, genetic; inflammatory non-infectious; visible (eg, synchisis scintillans). ‘Floaters’ Royal Liverpool Hospital, inflammatory infectious; inflammatory usually refer to visual phenomena experienced Duncan Building, Daulby iatrogenic; degenerative, traumatic; neoplastic by the patient. Street, Liverpool L69 3GA, and idiopathic. Non-diagnostic vitreous The differential diagnosis of vitreous UK. biopsies, unfortunately, still do occur with the Tel: þ 44 151 706 5885; opacities can be difficult, because there are Fax: þ 44 151 706 5883. main causes of failure including small sample many types of vitreous opacity, several having E-mail: s.e.coupland@ size; sampling error; inadequate fixation; and numerous causes. Biopsy can play an important liverpool.ac.uk leakage from container during transport. role, but requires proper handling of specimens Conclusions Vitreous biopsy can profoundly and application of a wide range of Received: 21 January 2008 influence the outcome in patients with histopathological and molecular biological Accepted in revised form: vitreous opacities. Success depends on close 21 January 2008 techniques. Published online: collaboration between clinicians, pathologists The aims of this article are to classify the 14 March 2008 and microbiologists. Vitreous samples require various types of vitreous opacity, and to provide Perspective on vitreous opacities SE Coupland 1319 an overview of vitreous biopsy, from the pathologist’s Table 1 Traditional subdivision of vitreous opacities 1 perspective. Class Subtype Examples Congenital Remnants of the Classification of vitreous opacities hyaloid vasculature Endogenous (A) Coagula of colloid (i) Asteroid bodies Traditionally, vitreous opacities were divided basis of gel (ii) synchisis scintillans aetiologically into two main classes: congenital and (B) Crystalline deposits acquired, with the latter class being further subdivided Exogenous (A) Protein (i) Exudative cells into two subclasses, endogenous and exogenous.1 As coagulaFthe plasmoid (ii) Blood seen in Table 1, there were additional subdivisions of the vitreous (iii) Tissue cells: (B) Amyloid epithelial, histiocytic, opacities within each of these subclasses. (C) Cells glial With advances in diagnostic and laboratory (iv) Tumour cells techniques, our understanding of the aetiology of (v) Pigment: melanotic acquired vitreous opacities has improved, making it & haematogenous necessary for this classification to be revised. Acquired causes of vitreous opacity can be categorized as genetic, inflammatory non-infectious, inflammatory infectious, inflammatory iatrogenic, degenerative, traumatic, neoplastic and idiopathic. These are summarized in worth discussing the case with the investigating Table 2, with corresponding non-exhaustive lists of laboratory before the specimen is collected, so that the diseases exemplifying each of the subdivisions. correct fixative and container are used and to make any special arrangements for transport. Various techniques have been described in the litera- Sample handling and processing ture for the preparation of vitreous and aqueous speci- Since the underlying pathological lesions resulting in mens for cytomorphological evaluation.4–7,9,10,20,21,26,30–35 vitreous opacities may be located in differing ocular These include: (1) vitreous ‘filtration’; (2) a celloidin bag tissues, such as the uvea or the retina, with possible technique; (3) cytospin and (4) cell block preparation. We involvement of the anterior chamber, the histopathologist are most familiar with the latter two methods, whereby may receive a variety of specimens, including aqueous the vitreous is spun at 500 r.p.m. for 5 min, concentrating tap, vitreous tap, diagnostic vitrectomy specimen, uveal the cells either onto glass slides or into an agar block or biopsy or subretinal aspirate. In all cases, careful paraffin. Vitreous and aqueous samples may be consideration should be given to the handling and accompanied by specimens obtained from tissues such as examination of the samples, allowing for the application retina and choroid.20,36 These solid specimens are usually of cytological, immunocytological, molecular biological fixed in buffered formalin, and processed in paraffin and microbiological analyses, to optimize the diagnostic using standard procedures. yield.2–26 Depending on the morphological findings of the Whether or not to fix a vitreous or aqueous sample vitreous samples, various additional special stains or depends on the clinical question and the time required immunocytological markers may be necessary, as for transport to the diagnostic laboratory. If it is possible summarized in Table 3. to deliver the vitreous sample within 1 h to the investigating laboratory, no fixative is required. If, however, longer delays are anticipated, for example, if the sample is being assessed at a remote laboratory, the Examination of acellular vitreous samples specimen should be placed in culture medium (eg, bovine serum albumin) or a mild cytofixative, such as Acellular vitreous samples contain any of the following: Herpes–glutamic acid buffer-mediated Organic solvent condensed vitreous strands; iridescent particles such as Protection Effect (HOPE) fixation27 or Cytolyt (Cytyc) for asteroid hyalosis (calcium soaps) and synchisis subsequent ThinPrep slide preparation.28 The latter two scintillans (cholesterol); amyloid deposits; squames, fixatives are preferable to formalin and glutaraldehyde consisting of conjunctival cells artefactually displaced fixation, because they provide superior preservation of during the vitrectomy procedure; retained lens fragments cytomorphology, immunoreactivity, and DNA extraction following cataract removal and pigment dust (Figure 1). for clonality assessment using the polymerase chain Depending on the initial findings, special stains may reaction (PCR). Several authors have advised against include congo red for amyloid, Prussian blue (or Pearl’s) alcohol fixation of vitreous samples.26,29 It is, therefore, stain for iron, etc. (Table 3). Eye Perspective on vitreous opacities SE Coupland 1320 Table 2 Classification of acquired vitreous opacities with associated causes, cytomorphological changes and recommended immunocytological and molecular biological analyses Disease Cause Morphology Relevant special Immunocytology Molecular biological stains analysis of vitreous Genetic Primary non-familial Transthyretin Amorphous, dense Congo red: material Not required amyloidosis gene mutation pink globular is metachromic, excrescences dichroic and bi- refringent Autosomal Macrophages with dominantly inherited cholesterol crystals, vitreoretinal disorders scattered small (eg, Wagner–Stickler lymphocytes, syndrome,
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