Introduction

The vitreous body is a transparent, extracellular gel, with a complicated structural framework of collagen, soluble proteins, hyaluronic acid, and a water content of 99%. The few cells that are normally present in the vitreous gel are located predominantly in the cortex and consist of hyalocytes, astrocytes, and glial cells. Vitreous abnormalities include opacification, liquefaction, and shrinkage.1

The terminology of visible vitreous abnormalities is confusing. The term ‘vitreous opacities’ refers to visible structures in the vitreous gel (as opposed to opacities within the vitreous cavity but outside the gel, exemplified by subhyaloid haemorrhage). ‘Vitreous infiltrates’ imply transport of extraneous material through membranes in the retina. ‘Vitreous deposits’ would include such infiltrates as well as substances ‘precipitating’ from within the vitreous itself and becoming visible (eg, synchisis scintillans). ‘Floaters’ usually refer to visual phenomena experienced by the patient.

The differential diagnosis of vitreous opacities can be difficult, because there are many types of vitreous opacity, several having numerous causes. Biopsy can play an important role, but requires proper handling of specimens and application of a wide range of histopathological and molecular biological techniques.

The aims of this article are to classify the various types of vitreous opacity, and to provide an overview of vitreous biopsy, from the pathologist's perspective.

Classification of vitreous opacities

Traditionally, vitreous opacities were divided aetiologically into two main classes: congenital and acquired, with the latter class being further subdivided into two subclasses, endogenous and exogenous.1 As seen in Table 1, there were additional subdivisions of the opacities within each of these subclasses.

Table 1 Traditional subdivision of vitreous opacities 1

With advances in diagnostic and laboratory techniques, our understanding of the aetiology of acquired vitreous opacities has improved, making it 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 Table 2, with corresponding non-exhaustive lists of diseases exemplifying each of the subdivisions.

Table 2 Classification of acquired vitreous opacities with associated causes, cytomorphological changes and recommended immunocytological and molecular biological analyses

Sample handling and processing

Since the underlying pathological lesions resulting in vitreous opacities may be located in differing ocular tissues, such as the uvea or the retina, with possible involvement of the anterior chamber, the histopathologist may receive a variety of specimens, including aqueous tap, vitreous tap, diagnostic vitrectomy specimen, uveal biopsy or subretinal aspirate. In all cases, careful consideration should be given to the handling and examination of the samples, allowing for the application of cytological, immunocytological, molecular biological and microbiological analyses, to optimize the diagnostic yield.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26

Whether or not to fix a vitreous or aqueous sample depends on the clinical question and the time required for transport to the diagnostic laboratory. If it is possible 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 specimen should be placed in culture medium (eg, bovine serum albumin) or a mild cytofixative, such as Herpes–glutamic acid buffer-mediated Organic solvent Protection Effect (HOPE) fixation27 or Cytolyt (Cytyc) for subsequent ThinPrep slide preparation.28 The latter two fixatives are preferable to formalin and glutaraldehyde fixation, because they provide superior preservation of cytomorphology, immunoreactivity, and DNA extraction for clonality assessment using the polymerase chain reaction (PCR). Several authors have advised against alcohol fixation of vitreous samples.26, 29 It is, therefore, worth discussing the case with the investigating laboratory before the specimen is collected, so that the correct fixative and container are used and to make any special arrangements for transport.

Various techniques have been described in the literature for the preparation of vitreous and aqueous specimens for cytomorphological evaluation.4, 5, 6, 7, 9, 10, 20, 21, 26, 30, 31, 32, 33, 34, 35 These include: (1) vitreous ‘filtration’; (2) a celloidin bag technique; (3) cytospin and (4) cell block preparation. We are most familiar with the latter two methods, whereby the vitreous is spun at 500 r.p.m. for 5 min, concentrating the cells either onto glass slides or into an agar block or paraffin. Vitreous and aqueous samples may be accompanied by specimens obtained from tissues such as retina and choroid.20, 36 These solid specimens are usually fixed in buffered formalin, and processed in paraffin using standard procedures.

Depending on the morphological findings of the vitreous samples, various additional special stains or immunocytological markers may be necessary, as summarized in Table 3.

Table 3 Useful stains in the assessment of vitreous biopsies

Examination of acellular vitreous samples

Acellular vitreous samples contain any of the following: condensed vitreous strands; iridescent particles such as asteroid hyalosis (calcium soaps) and synchisis scintillans (cholesterol); amyloid deposits; squames, consisting of conjunctival cells artefactually displaced during the vitrectomy procedure; retained lens fragments following cataract removal and pigment dust (Figure 1). Depending on the initial findings, special stains may include congo red for amyloid, Prussian blue (or Pearl's) stain for iron, etc. (Table 3).

Figure 1
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).

Cellular vitreous samples

On microscopy, cells in vitreous samples can be categorized as: haemorrhagic, inflammatory non-infectious, inflammatory infectious and neoplastic.

Haemorrhage

Vitreous specimens with haemorrhage predictably contain varying amounts of erythrocytes, ‘ghost cells’ haemosiderin-laden macrophages and acellular eosinophilic material, in various proportions, depending on the age of the haemorrhage. (Figure 2). Neoplastic cells or microorganisms within the haemorrhage may reveal the underlying cause.

Figure 2
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).

Inflammatory non-infectious vitritis

Inflammatory non-infectious vitreous cells consist predominantly of small T-lymphocytes, which are usually of the CD4+ helper type.9 Varying proportions of admixed macrophages, monocytes, plasma cells and neutrophils may be present (Figure 2). Once malignant cells and microorganisms have been excluded, the diagnosis may amount only to ‘chronic non-specific vitritis’, despite the use of special stains, immunocytology and/or PCR for clonality. In such cases, the clinical history and examination findings are especially important.

Inflammatory, infectious vitritis

Abundant neutrophils suggest bacterial (suppurative) endophthalmitis, most commonly caused by organisms such as Streptococcus sp, Staphylococcus aureus, Staphylococcus epidermis, coagulase-negative staphylococcus, Neisseria sp, Bacillus cereus, Haemophilus influenzae, Propionibacterium acnes, Pseudomonoas aeruginosa, Klebsiella pneumoniae and Escerichia coli.4 Microbiological cultures or molecular biological techniques are usually necessary for the exact identification of the genus and determination of antibiotic sensitivities,37 however, bacterial stains of vitreous specimens may identify the causative agent should cultures be negative. It is important to note that neutrophilic infiltrates in the aqueous and/or vitreous can occur with non-bacterial conditions such as Behçet's disease.

The presence of eosinophils in the vitreous suggests conditions such as nematode-induced endophthalmitis (eg, Toxocara canis), sympathetic ophthalmia, Lyme disease, Eale's disease, and, rarely, birdshot retinochoroidopathy or chronic eosinophilic myeloid leukaemia.38 Scattered eosinophils with non-specific vitritis can occur in Toxoplasmic retinochoroiditis. In this condition, the bradyzoites and tachyzoites may be seen occasionally in vitreous samples (Figure 2) but more often in the outer layers of the retina in chorioretinal biopsies stained with periodic acid-Schiff (PAS). Additional molecular analyses of ocular fluids are often required for confirmation in this condition.39

A predominance of macrophages in the vitreous sample may occur in Whipple's disease, ocular toxoplasmosis, and endophthalmitis due to Mycobacterium avium, Histoplasmosis capsulatum, Pneumocystis carinii, Cryptococcus and Blastomyces. In these conditions, fungal stains such as PAS and mucicarmine may reveal cytoplasmic inclusions or cysts of particular sizes and shape. (Figure 2) This allows for a morphological suggestion of the possible causative microorganism, to be confirmed however, with immunocytology, cultures and/or PCR.

Macrophages and multinucleated giant cells suggest a granulomatous process (Figure 2), caused by fungal and mycobacterial infections. The most common fungi causing fungal endophthalmitis include Candida sp, Aspergillus fumigatus and flavus, as well as Cryptococcus neoformans (Figure 2) (Table 2).4, 40 Acid-fast bacilli, highlighted with Ziehl-Neelsen, are rarely observed in aqueous and vitreous samples but may be found intracytoplasmically within macrophages or retinal pigment epithelial cells in tissue biopsies.41 Granulomatous inflammation with negative staining for fungi, mycobacteria and other microorganisms suggests sarcoidosis although the history and clinical findings should be taken into account. Tissue biopsies from the uvea may confirm this diagnosis, and may also exclude juvenile xanthogranulomatosis in younger patients.

Exceptionally rarely, intranuclear and intracytoplasmic viral inclusion bodies (eg, cytomegalovirus or Herpes simplex) may be demonstrated in association with necrotic cells in the vitreous sample. Such inclusion bodies are, however, more likely to be demonstrated in chorioretinal biopsies, using immunohistochemical or immunofluorescence techniques. PCR analysis of ocular fluid samples may expedite the diagnosis and treatment of such conditions, which is important as the retinitis can progress rapidly.11, 15, 16, 42, 43

Syphilis, once the main cause of vitreous opacities,1 accounts for only about 1% of all vitreous opacities but is increasing in incidence, particularly in the HIV-positive population,44 and should always be considered in the differential diagnosis of an inflammatory cellular vitreous infiltrate.

Neoplastic disease

Malignant neoplasms can simulate vitritis (‘masquerade syndrome’). They include primary ocular tumours such as retinal lymphoma (also known as ‘primary intraocular lymphoma’) and retinoblastoma, as well as diseases such as leukaemia, metastatic carcinomas and metastatic cutaneous melanomas.45, 46, 47, 48, 49, 50. The latter are most often localized to the choroid, but can produce brown ‘cannonballs’ of tumour cell aggregates within the vitreous. Ocular metastases arise most commonly from lung and breast in men and women, respectively. Other primary sites include kidney and prostate.

The main ‘masquerader’, however, is retinal lymphoma, which is located primarily within the subretinal space. This often seeds into the vitreous (ie, ‘retinovitreal lymphoma’) and may be located purely within the vitreous (ie, ‘vitreal lymphoma’). Cytomorphological examination of vitreous infiltrates in retinal lymphoma demonstrates medium-to-large cells with minimal cytoplasm, pleomorphic or round nuclei and prominent nucleoli (Figure 3). Necrotic material and macrophages are commonly present. Immunocytology shows the neoplastic cells to express B-cell antigens (CD79a, CD20, PAX-5) in the majority of cases. Many of these cells stain positively with the MIB-1 antibody, which indicates a high growth fraction, that is, a high-grade of malignancy (Figure 3). The diagnosis of lymphoma is supported by demonstration of cellular monoclonality, with monotypic expression of either a light and/or heavy chain of the immunoglobulin gene (usually IgM). Should sufficient material be available for examination, investigation of the vitreous specimens for rearrangements of the immunoglobulin heavy chain gene using PCR provides further evidence of the neoplastic nature of the lymphocytic infiltrate in retinal lymphoma.17, 18, 20, 22, 25, 35, 49, 51 Biochemical analysis of the vitreous specimen for interleukin ratios (IL10 : IL6) may also support the diagnosis of retinal lymphoma.52, 53, 54 If possible, retinal lymphoma should be distinguished from other types of intraocular lymphoma, namely primary uveal lymphoma and secondary (metastatic) intraocular lymphoma. The reader is referred to detailed reviews outlining the morphological and immunocytological characteristics of these rare entities.55, 56

Figure 3
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).

Non-diagnostic vitreous biopsy

A ‘negative’ vitreous biopsy, which is particularly problematic in suspected cases of intraocular lymphoma,57 has several causes. First of all, the vitreous gel may not contain any material of diagnostic relevance, as in primary uveal lymphomas and some cases of retinal lymphoma where the tumour cells are confined to the subretinal space with minimal vitreal involvement. Secondly, the patient may have been treated with steroids prior to vitrectomy, increasing the cell fragility. Thirdly, the size of the vitreous sample may be insufficient, perhaps because of leakage from a poorly-sealed container during transport to the laboratory. Fourthly, the specimen may not be handled properly, for example, not being placed in the correct fixative or being left unfixed for an excessive time. Fifthly, the specimen may be lost during laboratory processing, perhaps as a result of a technical error. Most of these problems can be avoided by taking the precautions already mentioned in this article. Whenever a non-diagnostic biopsy occurs, it is especially important for the clinician and pathologist to confer without delay, so that any errors can be avoided if the investigation is repeated.

Conclusions

The diagnosis of vitreous opacities demands close collaboration between the clinician, the pathologist and the microbiologist. This involves documentation of all relevant clinical information in the pathology request form, comprehensive and up-to-date guidelines and protocols, timely discussions between the various specialists (for example, telephone communications just before a vitreous biopsy is performed), regular audits and multidisciplinary meetings. The laboratory investigation of vitreous specimens is demanding and depends not only on a pathologist with special expertise but also on the support of experienced technical staff capable of using a wide range of investigations.