Introduction

The diagnosis of posterior segment infectious or inflammatory disease is based primarily on clinical examination. In the majority of cases, the slit-lamp biomicroscopic and ophthalmoscopic appearance of the vitreous, retina, and choroid is diagnostic of a specific disorder, especially when combined with the findings from ancillary tests such as fluorescein angiography, ocular echography, and radiologic and serologic tests.¹

However, not all patients have the characteristic clinical features or classic appearance of a specific disease, and the etiology of their disorder can be a diagnostic dilemma. The inability to make a specific diagnosis has been reported in up to 33% of patients who present with intraocular inflammation.² Moreover, some infectious and malignant processes are manifested first, or only, in the eye, in which case the diagnostic yield from systemic testing is low. This is often the situation in patients with atypical malignancy in the eye, chronic low-grade endophthalmitis after cataract surgery, intraocular inflammation of atypical viral, bacterial, or fungal origin, and some types of progressive retinitis, choroiditis, and pigment epitheliopathies. In these patients, particularly if they are immunocompromised or if one of the possible diagnoses is malignancy, the disease process may threaten not only vision but also life. Intraocular biopsy provides affected ocular tissue for culture, examination, and other tests to help with therapeutic decision-making.³ In most of these cases, therapeutic intervention will be either antimicrobial, antineoplastic, or anti-inflammatory.^4,5

A number of factors in recent years have brought about an increased demand for tissue diagnosis in posterior segment inflammatory disease. One of these is the rise in the number of individuals who have been iatrogenically immunosuppressed as a result of systemic disease, malignancy, or organ transplantation. In addition, the frequent use of intravitreal steroids has also led to the report of opportunistic ocular infections in immunocompetent individuals.⁶ The clinical presentation of ocular inflammatory disease may be variable and atypical in these immunocompromised patients; the possible etiologic agents are numerous and diverse, and serologic testing is frequently misleading. Another factor increasing the indication for intraocular biopsy is the technologic advance of modern cataract surgery. With phacoemulsification and in-the-bag intraocular lens placement, every year millions of patients are left with most of their capsular bags intact, forming small potential reservoirs in which, rarely, sequestered organisms of normally low-grade pathogenicity can multiply. Clinicians are confronted with a number of patients whose chronic, smoldering, postoperative endophthalmitis is due to organisms such as Candida parapsilosis or Propionibacterium acnes (Figs 124.1–124.5), which cannot be adequately treated until a tissue diagnosis is made. In addition, ongoing advances in laboratory testing continue to improve the diagnostic yield from biopsied material. For example, polymerase chain reaction (PCR) testing, a biochemical technique that allows for the detection of infinitesimal amounts of specific nucleic acids, now permits identification of genetic components of infectious organisms. Finally, advances in vitreoretinal surgical instrumentation and increasing use of 25- and 23-gauge (G) transconjunctival pars plana vitrectomy⁷ have allowed for possibly safer access and acquisition of ocular tissue for analyses. Table 124.1 lists common indications for ocular biopsy.

Fig. 124.1 After cataract surgery, inflamed eye of a patient with chronic indolent uveitis is caused by Candida parapsilosis.

Fig. 124.2 Candida parapsilosis organisms crowd this capsular bag, surrounded by an area of dense neutrophilic incursion.

Fig. 124.3 Characteristic posterior capsular plaque seen in a patient with chronic postcataract inflammation caused by Propionibacterium acnes.

(From Sawusch MR, Michels RG, Stark WJ, et al. Endophthalmitis due to Propionibacterium acnes sequestered between IOL optic and posterior capsule. Ophthalmic Surg 1989;20:90–2. By kind permission of Slack Inc.)

Fig. 124.4 Propionibacterium acnes organisms fill the capsular bag in this patient with smoldering endophthalmitis and retinal detachment more than 1 year after cataract surgery.

Fig. 124.5 High-power view of the Propionibacterium acnes organisms seen in Figure 124.4.

Table 124.1 Common indications for biopsy

The efficacy of ocular biopsy depends partly on the expertise of the surgeon who obtains the specimen and the laboratory personnel who analyze the specimen(s). Before embarking on surgery, harvesting and handling of the specimen should be carefully mapped out in advance. The pathologist should be familiar with ophthalmic disease processes, aware of the diagnoses being considered, and involved at the outset in planning how the specimen will be handled from the moment it is taken – a step that is critical to the success of the procedure.^4,8,9

The pathologist’s available investigative methods include, among others, light microscopy, electron microscopy, immunohistochemistry, and PCR. PCR can be used to identify unique DNA and RNA sequences specifically from infectious agents and, occasionally, from other disease processes, such as proliferating monoclonal B lymphocytes in ocular malignant lymphoma.^9–13 The laboratories responsible for culture and identification of bacterial, fungal, and viral disease should receive the specimen as soon as possible. It should be plated on a variety of specific media and maintained under the correct (aerobic, anaerobic, temperature) conditions. Plates may need to be specially treated or held for extra time to reveal growth from some of the more fastidious organisms.

Vitreous biopsy

With the evolution of modern vitrectomy techniques, including 23G, 25G, and 27G vitrectomy, removal of vitreous from the eye has become a common and relatively safe procedure.¹⁴ A number of different goals are achieved with diagnostic vitrectomy^5,15 In vitritis with a possible neoplastic, infectious, or inflammatory cause, diagnostic vitrectomy may provide a definitive diagnosis, thereby allowing appropriate therapy to be instituted.^5,8 In some cases of retinitis and choroiditis, a diagnosis can be made from vitreous sampling alone, particularly if marked vitritis is present. The vitrectomy also removes or debulks infectious material within the eye, reduces the intraocular load of inflammatory cells and associated biologically active material and debris, effectively removes material (e.g. retained lens material and blood) that may be part of the pathogenesis of the ocular disorder, allows clinicopathologic correlation in the active stages of disease for some conditions, and helps to define the pathophysiologic basis of many disorders.⁵ Cytopathologic diagnosis has proved valuable in reported series or case reports of patients with: endogenous and exogenous bacterial and fungal infections (Fig. 124.6); nematode endophthalmitis; inflammatory pseudotumor of the iris and ciliary body; pars planitis; phacoanaphylaxis; intraocular lymphoma; leukemia; metastatic melanoma; metastatic carcinoma; medulloepithelioma of the ciliary body; secondary glaucoma (including blood-induced, phacolytic, and melanomalytic glaucoma); epithelial ingrowth; proliferative vitreoretinopathy, and miscellaneous conditions with vitreous opacities (including chronic inflammation in patients with suspected lymphoma, acute retinal necrosis, birdshot chorioretinopathy, toxoplasmosis, Whipple’s disease, amyloidosis, and asteroid hyalosis).^4,5,16,17 Vitrectomy may also help to clear vitreous opacities, remove scaffolding on which postoperative cicatricial proliferation can grow, relieve traction, and allow for improved intraocular penetration of antibiotics.^5,15 Table 124.2 lists a sample of vitreous findings in major disease categories.

Fig. 124.6 This 61-year-old woman had a history of idiopathic uveitis, quiescent for years, and cataract surgery 10 years previously. She developed a red eye and decreased vision with active anterior segment inflammation, shown here with cellular and fibrinous anterior chamber deposits. A retinal detachment was noted on echography. Examination of smears and culture of the vitrectomy specimen from the time of detachment repair revealed Cryptococcus organisms.

Surgical technique

To obtain an undiluted specimen, a variety of vitrectomy techniques (with either 20-, 23-, 25-, or 27G instrumentation) may be used. If a large sample (>0.5 mL) of vitreous is needed, a standard three-port vitrectomy system should be utilized. A 10 mL Luer–Lok syringe is spliced via a three-way stopcock into the aspiration line (Fig. 124.7). Manual aspiration and specimen collection are carried out for retrieval of an undiluted specimen from the most intensely involved area (usually posteriorly, inferiorly, or over the area of retinitis or chorioretinitis).

Fig. 124.7 Technique of specimen collection in diagnostic vitrectomy. A 10 mL syringe is spliced into the vitrectomy aspiration tubing, and a choice undiluted specimen is obtained with manual aspiration.

Alternatively, a vitreous “trap” method may also be performed using two 18G needles, a male adaptor, and a sterile red-topped test tube.¹⁸ In the vitreous trap method, the male adaptor is attached to one of the needles, and the needles are attached to the ends of the aspiration tubing. The two needles are then inserted into the test tube. The diagnostic vitrectomy is performed and the pure vitreous sample is directed into the test tube.

After a sufficient amount of sample is obtained, the infusion is turned on and either fluid or air is infused before large choroidals form and the eye begins to collapse.¹⁴ The vitrector is withdrawn from the eye, and the vitreous specimen is aspirated into the syringe. Vitrectomy can then be completed. At completion of the surgery, the cassette with the dilute residual vitreous is also sent to the pathology laboratory, where the specimen is concentrated, using a micropore filter, or cytospin smears are made.^4,19

If a small sample (≤0.5 mL) of vitreous is needed, a 27G vitrectomy with a single incision may be utilized.^20,21 In this technique, the 27G vitrector is inserted perpendicular to the pars plana. Using a cut rate of 800 cuts/minute and an aspiration rate of 600 mmHg, a small amount of undiluted vitreous may be safely removed from the eye without the need of an additional infusion line or endoillumination port. The 27G scleral wound is also self-sealing and does not require sutures.