The Pathologist's Perspective on Vitreous Opacities

ABIG PTAMLG SYMPOSIUM OPHTHALMOLOGY CAMBRIDGE 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).

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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, snowﬂake proteinaceous material degeneration, familial exudative vitreoretinopathy)

Inﬂammatory, non-infectious Pars planitis Cell aggregates of small T-lymphocytes, macrophages, glial cells, lytic cells Sarcoidosis Macrophages and PAS, Grocott, PCR to exclude multinucleate giant Warthin-Starry, mycobacterial infection cells, possibly forming Ziehl-Neelsen to granulomata, scattered exclude fungi and small lymphocytes mycobacteria

Vitiliginous HLA A29 in Small T-lymphocytes, chorioretinitis (i.e. 80–90% cases macrophages; scattered Birdshot) eosinophils Behc¸et’s syndrome HLA B5/BW51 Neutrophils, Bacterial and fungal in 450% lymphocytes, plasma stains to exclude patients cells, monocytes microorganisms

Crohn’s disease Sarcoid-like PAS, Grocott, granulomata with Warthin–Starry, admixed lymphocytes Ziehl–Neelsen to exclude fungi and mycobacteria

Juvenile Macrophages and CD68, S100P, xanthogranuloma multinucleate (Touton) CD1a to giant cells differentiate from Langerhan’s cell histiocytosis Fuchs’ cyclitis Non-specific chronic infiltrates with small lymphocytes, scattered macrophages Vogt–Koyanagi– Chronic granulomatous Harada syndrome inflammation with melanin-containing epithelioid cells and T-cells, scattered plasma cells Multiple sclerosis Non-specific chronic CD20 and CD3 to inflammatory cells exclude lymphoma

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Table 2 (Continued )

Disease Cause Morphology Relevant special Immunocytology Molecular biological stains analysis of vitreous

Inﬂammatory infectious Bacterial Endophthalmitisa See text Abudant neutrophils Gram stain Tuberculosis Mycobacterium Macrophages and Ziehl-Neelsen for PCR directed at tuberculosis multinucleate giant acid-fast bacilli mycobacteria cells with granulomata, scattered small lymphocytes Whipple’s disease Tropheryma Foamy macrophages PAS for Ab. against whipplei cytoplasmic causative agent inclusion bodies

Fungal Endophthalmitis Candida sp Numerous mixed PAS, Grocott, Aspergillus sp macrophages and Warthin-Starry, Cryptococcus neutrophilic Gomori to neoformans granulocytes; possibly demonstrate fungal Fusarium granulomatous; hyphae and spores ﬁbrin-platelet aggregates; necrotic debris

Viral Chorioretinitis Cytomegalovirus Non-specific PCR directed against Herpes simplex virus chronic infiltrates viruses Varicella zoster with small lymphocytes, scattered macrophages, possibly forming granulomata; background lytic cells Parasitic Toxoplasmosis T. gondii Chronic inflammatory PAS to demonstrate PCR directed against Toxocariasis T. canis cells with numerous parasites microorganisms Ocular cysticerosis T. solium eosinophils, lymphocytes, plasma cells & macrophages

Spirochaetal Syphilis Treponema Non-speciﬁc chronic Silver stains to PCR directed against Lyme disease pallidum inﬂammatory cells demonstrate microorganisms Borrelia spirochaetes burgdorferi

Inflammation, iatrogenic eg, ICCE, pan-retinal Non-specific chronic photocoagulation, inflammatory cells, cryotherapy RPE cells, retinal fragments, proteinaceous deposits

Degenerative Vitreous detachment Non-speciﬁc chronic inﬂammatory cells Pigment granules Melanocytic or Pigment granules, Perl’s to exclude/ haemorrhage dispersed or within detect haemosiderin vitreous strands

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Table 2 (Continued )

Disease Cause Morphology Relevant special Immunocytology Molecular biological stains analysis of vitreous

Traumatic Vitreous detachment Non-specific chronic (eg, traumatic avulsion of inflammatory cells vitreous base) Haemorrhage Clumps or strands of Perl’s for Only Only appropriate in erythrocytes in fresh haemosiderin appropriate in suspected underlying haemorrhage; deposition; bacterial cases of lymphoma (see below) dispersed ‘‘ghost’’ & fungal stains to suspected or viral infection cells with scattered exclude underlying underlying macrophages in older causative agents malignancy haemorrhage (see below). Sympathetic Chronic granulomatous ophthalmia inflammation with melanin-containing epithelioid cells and T-cells, scattered plasma cells: varying amount of eosinophils

Neoplastic Primary Retinoblastoma Small atypical ‘‘blue Synaptophysin cell’’ neoplasm cells positive Retinal lymphoma Atypical lymphocytes, B-cell antigens IgH-PCR IL10:IL6 Retinovitreal lymphoma macrophages, lytic cells (CD20, CD79a); (ELISA) Vitreal lymphoma IgH or IgL; Ki-67 Uveal melanoma Tumour cells MelanA, HMB-45 haemorrhage

Neoplastic Secondary Metastatic Clumps of varying- Pancytokeratin carcinoma sized atypical cells Cytokeratin subtypes Metastatic Atypical melanocytes Melan, HMB-45 cutaneous melanoma Secondary ocular Atypical myeloblasts Myeloperoxidase, involvement by with or without CD34, CD117 leukaemia maturation Secondary ocular Atypical lymphocytes B-cell antigens IgH-PCR involvement by (CD20, CD79a), lymphoma T-cell antigens (CD3), plasma cell marker (CD138, IgL) Idiopathic Asteroid hyalosis Calcium Round varying-sized Von Kossa pyrophophate- bodies with minimal spheres inﬂammatory reaction Synchysis scintillans Cholesterol Crystals with minimal crystals inﬂammatory reaction

Ab ¼ antibody; CD ¼ cluster of differentiation; ICCE ¼ intracapsular cataract extraction; IgH-PCR ¼ Immunoglobulin heavy chain polymerase chain reaction; PAS ¼ periodic acid Schiff; PCR ¼ polymerase chain reaction; RPE ¼ retinal pigment epithelium. aIncluding post-surgical endophthalmitis.

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Table 3 Useful stains in the assessment of vitreous biopsies

Conventional Histochemical Stains Detects Examples/comments

Morphological stains (eg, May Grunewald-Giemsa, Morphology modiﬁed Papanicolou)

Giemsa Bacteria Neisseria sp. Fungi Actinomyces sp. Parasites Toxoplasmosis Protozoa Leishmania tropica

Gram Twort Gram-positive bacteria (blue) Gram-negative bacteria (red)

Periodic Acid-Schiff (PAS) with or without diastase Bacteria Tropheryma whipplei digestion (d-PAS) Fungi Candida sp. Parasites T. gondii

Silver stains (eg, Gomori’s methenamine silver; Fungi Grocott)

Mucicarmine Fungi Encapsulated fungi eg, Cryptococcus neoformans

(Modiﬁed) Ziehl-Neelsen Mycobacteria M. tuberculosis Nocardia M. leprae

Warthin starry Bacteria Helicobacter sp. Fungi Spirochaetes Treponema pallidum

Perl’s prussian blue Haemosiderin (ferric iron) Congo red Amyloid Green birefringence Parasites Taenia echinococcus

Immunohistochemistry CD3 Pan T-cell marker CD20 B-cell marker Negative on precursor B-cells and plasma cells CD68 Pan-macrophage marker MelanA, HMB-45 Melanocyte markers Pancytokeratin Epithelial cells eg, MNF-116 Anti-Cytomegalovirus CMV Against early and late antigens allowing for detection in nucleus and cytoplasm Anti-Herpes simplex viruses I and II HSV I and II Anti-Varicella virus Varicella Anti-Trophyrema whipplei Whipple’s disease Polyclonal antibody

Cellular vitreous samples haemosiderin-laden macrophages and acellular eosinophilic material, in various proportions, depending On microscopy, cells in vitreous samples can be on the age of the haemorrhage. (Figure 2). Neoplastic categorized as: haemorrhagic, inflammatory cells or microorganisms within the haemorrhage may non-infectious, inflammatory infectious and reveal the underlying cause. neoplastic. Inflammatory non-infectious vitritis Haemorrhage Inflammatory non-infectious vitreous cells consist Vitreous specimens with haemorrhage predictably predominantly of small T-lymphocytes, which are contain varying amounts of erythrocytes, ‘ghost cells’ usually of the CD4 þ helper type.9 Varying proportions

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Figure 1 (a) Clinical photograph of asteroid hyalosis in a patient with uveal melanoma (Courtesy of Professor Bertil Damato); (b) Cytospin of a diagnostic vitrectomy in another patient showing the circular spheres seen in asteroid hyalosis (May Grunewald Giemsa); (c) Clinical photograph of a patient with primary amyloidosis of the vitreous; (d) Congo red stain of the centrifuged vitreous specimen demonstrating positive material, which was bi-refringent under polarized light; (e) Conjunctival squames, artiﬁcially displaced into the vitreous on sampling (HE); (f) Retained lens fragments in a vitreous biopsy with a mild foreign-body inﬂammatory reaction (HE).

of admixed macrophages, monocytes, plasma cells and vitritis’, despite the use of special stains, neutrophils may be present (Figure 2). Once malignant immunocytology and/or PCR for clonality. In such cases, cells and microorganisms have been excluded, the the clinical history and examination ﬁndings are diagnosis may amount only to ‘chronic non-speciﬁc especially important.

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Inflammatory, infectious vitritis should be taken into account. Tissue biopsies from the Abundant neutrophils suggest bacterial (suppurative) uvea may confirm this diagnosis, and may also exclude endophthalmitis, most commonly caused by organisms juvenile xanthogranulomatosis in younger patients. such as Streptococcus sp, Staphylococcus aureus, Exceptionally rarely, intranuclear and intracytoplasmic Staphylococcus epidermis, coagulase-negative viral inclusion bodies (eg, cytomegalovirus or Herpes staphylococcus, Neisseria sp, Bacillus cereus, Haemophilus simplex) may be demonstrated in association with influenzae, Propionibacterium acnes, Pseudomonoas necrotic cells in the vitreous sample. Such inclusion aeruginosa, Klebsiella pneumoniae and Escerichia coli.4 bodies are, however, more likely to be demonstrated in Microbiological cultures or molecular biological chorioretinal biopsies, using immunohistochemical or techniques are usually necessary for the exact immunofluorescence techniques. PCR analysis of ocular identification of the genus and determination of fluid samples may expedite the diagnosis and treatment antibiotic sensitivities,37 however, bacterial stains of of such conditions, which is important as the retinitis can vitreous specimens may identify the causative agent progress rapidly.11,15,16,42,43 should cultures be negative. It is important to note that Syphilis, once the main cause of vitreous opacities,1 neutrophilic infiltrates in the aqueous and/or vitreous accounts for only about 1% of all vitreous opacities but is can occur with non-bacterial conditions such as Behçet’s increasing in incidence, particularly in the HIV-positive disease. population,44 and should always be considered in the The presence of eosinophils in the vitreous suggests differential diagnosis of an inflammatory cellular conditions such as nematode-induced endophthalmitis vitreous infiltrate. (eg, Toxocara canis), sympathetic ophthalmia, Lyme disease, Eale’s disease, and, rarely, birdshot Neoplastic disease retinochoroidopathy or chronic eosinophilic myeloid Malignant neoplasms can simulate vitritis (‘masquerade leukaemia.38 Scattered eosinophils with non-specific syndrome’). They include primary ocular tumours such vitritis can occur in Toxoplasmic retinochoroiditis. In this as retinal lymphoma (also known as ‘primary intraocular condition, the bradyzoites and tachyzoites may be seen lymphoma’) and retinoblastoma, as well as diseases such occasionally in vitreous samples (Figure 2) but more as leukaemia, metastatic carcinomas and metastatic often in the outer layers of the retina in chorioretinal cutaneous melanomas.45–50. The latter are most often biopsies stained with periodic acid-Schiff (PAS). localized to the choroid, but can produce brown Additional molecular analyses of ocular fluids are often ‘cannonballs’ of tumour cell aggregates within the required for confirmation in this condition.39 vitreous. Ocular metastases arise most commonly from A predominance of macrophages in the vitreous lung and breast in men and women, respectively. Other sample may occur in Whipple’s disease, ocular primary sites include kidney and prostate. toxoplasmosis, and endophthalmitis due to The main ‘masquerader’, however, is retinal Mycobacterium avium, Histoplasmosis capsulatum, lymphoma, which is located primarily within the Pneumocystis carinii, Cryptococcus and Blastomyces. subretinal space. This often seeds into the vitreous In these conditions, fungal stains such as PAS and (ie, ‘retinovitreal lymphoma’) and may be located purely mucicarmine may reveal cytoplasmic inclusions or cysts within the vitreous (ie, ‘vitreal lymphoma’). of particular sizes and shape. (Figure 2) This allows for a Cytomorphological examination of vitreous infiltrates in morphological suggestion of the possible causative retinal lymphoma demonstrates medium-to-large cells microorganism, to be confirmed however, with with minimal cytoplasm, pleomorphic or round nuclei immunocytology, cultures and/or PCR. and prominent nucleoli (Figure 3). Necrotic material and Macrophages and multinucleated giant cells suggest a macrophages are commonly present. Immunocytology granulomatous process (Figure 2), caused by fungal and shows the neoplastic cells to express B-cell antigens mycobacterial infections. The most common fungi (CD79a, CD20, PAX-5) in the majority of cases. Many of causing fungal endophthalmitis include Candida sp, these cells stain positively with the MIB-1 antibody, Aspergillus fumigatus and flavus, as well as Cryptococcus which indicates a high growth fraction, that is, a high- neoformans (Figure 2) (Table 2).4,40 Acid-fast bacilli, grade of malignancy (Figure 3). The diagnosis of highlighted with Ziehl-Neelsen, are rarely observed in lymphoma is supported by demonstration of cellular aqueous and vitreous samples but may be found monoclonality, with monotypic expression of either a intracytoplasmically within macrophages or retinal light and/or heavy chain of the immunoglobulin gene pigment epithelial cells in tissue biopsies.41 (usually IgM). Should sufficient material be available for Granulomatous inflammation with negative staining for examination, investigation of the vitreous specimens for fungi, mycobacteria and other microorganisms suggests rearrangements of the immunoglobulin heavy chain gene sarcoidosis although the history and clinical findings using PCR provides further evidence of the neoplastic

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nature of the lymphocytic inﬁltrate in retinal also support the diagnosis of retinal lymphoma.52–54 If lymphoma.17,18,20,22,25,35,49,51 Biochemical analysis of the possible, retinal lymphoma should be distinguished from vitreous specimen for interleukin ratios (IL10 : IL6) may other types of intraocular lymphoma, namely primary

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uveal lymphoma and secondary (metastatic) intraocular gel may not contain any material of diagnostic relevance, lymphoma. The reader is referred to detailed reviews as in primary uveal lymphomas and some cases of retinal outlining the morphological and immunocytological lymphoma where the tumour cells are conﬁned to the characteristics of these rare entities.55,56 subretinal space with minimal vitreal involvement. Secondly, the patient may have been treated with steroids prior to vitrectomy, increasing the cell fragility. Thirdly, Non-diagnostic vitreous biopsy the size of the vitreous sample may be insufﬁcient, A ‘negative’ vitreous biopsy, which is particularly perhaps because of leakage from a poorly-sealed problematic in suspected cases of intraocular container during transport to the laboratory. Fourthly, the lymphoma,57 has several causes. First of all, the vitreous specimen may not be handled properly, for example, not

Figure 3 (a and b) Two vitrectomy specimens with varying degrees of cellularity demonstrating atypical lymphocytes with convoluted nuclei on a background of lytic cells and scavenging macrophages (May Grunewald Giemsa); (c) Retinovitreal lymphoma cells with clear membranous positivity for the B-cell antigen, CD20 (APAAP stain); (d) MIB-1 antibody directed against the Ki-67 antigen, showing a high growth fraction in retinovitreal lymphoma cells, indicating the high degree of malignancy (APAAP stain).

Figure 2 (a) Resolving vitreous haemorrhage with some scattered melanomacrophages (HE stain); (b) Perl’s stain showing haemosiderin in an older vitreous haemorrhage; (c) Non-specific inflammatory infiltrate in ‘chronic non-specific vitritis’, comprising macrophages, plasma cells, lymphocytes and occasional neutrophils (HE stain); (d) Suppurative bacterial endophthalmitis (HE) with evidence of Gram-positive organisms (inset); (e) Pars plana vitrectomy sample with macrophages and scattered bradyzoites (PAS stain); (f) Granulomatous vitritis (HE); the Ziehl-Neelsen stain was negative, and the clinical findings were suggestive of sarcoidosis; (g) Chorioretinal biopsy demonstrating a granulomatous inflammation with evidence of microorganisms, morphologically consistent with Histoplasma capsulatum (PAS stain); (h) Agar cell block of a vitrectomy sample showing fungal elements, consistent with Candida sp (PAS stain).

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being placed in the correct fixative or being left unfixed 6 Ljung BM, Char D, Miller TR, Deschenes J. Intraocular for an excessive time. Fifthly, the specimen may be lost lymphoma. Cytologic diagnosis and the role of during laboratory processing, perhaps as a result of a immunologic markers. Acta Cytol 1988; 32: 840–847. 7 Augsburger JJ. Invasive diagnostic techniques for uveitis technical error. Most of these problems can be avoided by and simulating conditions. Trans Am Ophthalmol Soc 1990; taking the precautions already mentioned in this article. 88: 89–104; discussion 104–107. Whenever a non-diagnostic biopsy occurs, it is especially 8 Wilson DJ, Braziel R, Rosenbaum JT. Intraocular lymphoma. important for the clinician and pathologist to confer Immunopathologic analysis of vitreous biopsy specimens. without delay, so that any errors can be avoided if the Arch Ophthalmol 1992; 110: 1455–1458. 9 Davis JL, Solomon D, Nussenblatt RB, Palestine AG, Chan investigation is repeated. CC. Immunocytochemical staining of vitreous cells. Indications, techniques, and results. Ophthalmology 1992; 99: 250–256. Conclusions 10 Obata H, Horiuchi H, Tsuru T. Clinical application of new membrane filter for cytopathological diagnosis in The diagnosis of vitreous opacities demands close ophthalmology. Jpn J Ophthalmol 1993; 37: 344–351. collaboration between the clinician, the pathologist and 11 Garweg J, Fenner T, Bohnke M, Schmitz H. An improved the microbiologist. This involves documentation of all technique for the diagnosis of viral retinitis from samples of relevant clinical information in the pathology request aqueous humor and vitreous. Graefes Arch Clin Exp Ophthalmol 1993; 231: 508–513. form, comprehensive and up-to-date guidelines and 12 Shields JA, Shields CL, Ehya H, Eagle Jr RC, De Potter P. protocols, timely discussions between the various Fine-needle aspiration biopsy of suspected intraocular specialists (for example, telephone communications just tumors. The 1992 Urwick Lecture. Ophthalmology 1993; 100: before a vitreous biopsy is performed), regular audits 1677–1684. and multidisciplinary meetings. The laboratory 13 Abe T, Tsuchida K, Tamai M. A comparative study of the polymerase chain reaction and local antibody production in investigation of vitreous specimens is demanding and acute retinal necrosis syndrome and cytomegalovirus depends not only on a pathologist with special expertise retinitis. Graefes Arch Clin Exp Ophthalmol 1996; 234: but also on the support of experienced technical staff 419–424. capable of using a wide range of investigations. 14 Verbraeken H. Diagnostic vitrectomy and chronic uveitis. Graefes Arch Clin Exp Ophthalmol 1996; 234(Suppl 1): S2–S7. 15 Doornenbal P, Seerp Baarsma G, Quint WG, Kijlstra A, Rothbarth PH, Niesters HG. 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