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Available online at www.sciencedirect.com

www.elsevier.com/locate/semdp

Ocular cytopathology: A primer for the generalist Nora MV Laver, MDn Tufts University School of Medicine, Boston, Massachusetts

article info

abstra ct

Keywords:

The cytomorphological features of normal ocular structures compared to those found in

Eye

diagnostic samples from the anterior and posterior segments of the eye are discussed.

Fine-needle aspiration

& 2014 Elsevier Inc. All rights reserved.

Melanoma Retinoblastoma Vitrectomy Ocular surface cytology

Introduction

Normal eye histology

Ocular cytology specimens can be challenging for cytopathologists. Specimens are usually scanty as lesions in the eye are generally small compared to other sites in the body. Furthermore, the diseases in the eye are unique and require specialized expertise to interpret. Knowledge of the clinical aspects of the disease is often critical to the overall interpretation of the sample. Appropriate specimen handling is very important in yielding a definitive diagnosis. Ocular cytology is useful for the evaluation of inflammatory, neoplastic, degenerative, and congenital conditions of the ocular surface, anterior chamber, and posterior segment of the eye. The goal of ocular cytology diagnosis is to provide quality patient care with accurate clinico-pathological correlation. This article discusses the cytomorphological features of the normal ocular structures and contrasts these findings to those encountered in samples from the anterior and posterior segments of the eye. These include surface epithelial scrapings, anterior chamber aspirations, vitrectomy specimens, and intraocular fine-needle aspirations commonly encountered in clinical practice. A discussion of the best methods and ancillary tests currently used is included. Eyelid and ocular adnexal lesion fineneedle aspiration biopsies are not included in this article.

Accurate cytology interpretation of ocular specimens requires a fundamental knowledge of ocular histology. A general overview with emphasis on the areas relevant to cytology specimens is presented. The average adult eye measures about 25 mm horizontally, 23 mm vertically, and 21–26 mm anteroposteriorly. The lacrimal gland is located superolaterally in the orbit. The sclera, or outer coat of the eye seen anteriorly as the white of the eye, encircles the entirety of the eye except anteriorly, where it is continuous with the cornea (Fig. 1). The cornea is clear, permitting transmission and refraction of light into the eye (Fig. 2A). The conjunctiva overlies the sclera anteriorly; the bulbar conjunctiva covers the anterior surface of the eye, and the palpebral conjunctiva covers the posterior surface of the eyelids (Fig. 2B). Behind the cornea, a small space called the anterior chamber holds a small amount of watery fluid called the aqueous humor (Fig. 1). The uvea or vascularized layer of the eye is formed anteriorly by the iris and ciliary body (Fig. 2C) and posteriorly by the choroid. Posterior to the anterior chamber is the iris, with the pupil centrally located. Behind the pupil is the lens, which, like the cornea, is normally transparent and refracts light (Fig. 2D). The ciliary body is located posterior to the iris at the equator of the

n Correspondence to: Ocular Pathology Laboratory, Box 6700, New England Eye Center at Tufts Medical Center, 800 Washington St, Boston, MA 02111. E-mail addresses: [email protected], [email protected]

http://dx.doi.org/10.1053/j.semdp.2014.12.013 0740-2570/& 2014 Elsevier Inc. All rights reserved.

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surface of the cornea and conjunctiva may be scraped under local anesthesia with a small spatula or a brush, and the cellular yield is smeared onto tissue slides. Smears may be alcohol-fixed or air-dried for the diagnosis of inflammatory, infectious, and neoplastic disorders stained with Diff-Quik, Papanicolaou stain (PAP), H&E, or other stains. The cells may be prepared utilizing liquid-based cytology, placing the scraped cells in an appropriate alcohol-based fixative.

Vitrectomy samples

Fig. 1 – The eye: low-power view of normal ocular structures (  1). lens (Fig. 1). It is responsible for making the aqueous humor, holding the lens in place, and changing the shape of the lens by contracting and relaxing a small muscle to facilitate the eye's ability to focus between distant and close objects. The choroid is a highly vascular layer that lies between the sclera and the retina and is responsible for the blood supply to the outer parts of the retina (Fig. 2E and F). Throughout the uvea are many melanocytes that give these tissues their pigmentation, which can be quite variable from person to person. Retinal signals leave the eye through the optic nerve and are carried to the brain for higher-level perception of light and light patterns, known as vision. The retinal pigment epithelium (RPE) is a cuboidal pigmented layer found between the retinal photoreceptors and the choroid (Fig. 2F). The large cavity of the eye between the lens anteriorly and the retina posteriorly is filled with a viscous substance called the vitreous humor. The average volume in adults is 4 ml, and it is composed primarily of water (99%) and collagens types II and IX, glycosaminoglycans, soluble proteins, and glycoproteins.

Sampling and cytopreparatory techniques Ocular cytology specimens are most commonly submitted for evaluation as (1) corneal or conjunctival scrapings, (2) vitrectomy specimens, and (3) fine-needle aspiration (FNA) biopsies (from the anterior and posterior segments of the eye).1

Corneal and conjunctival cytology specimens Conjunctival and corneal scrapings are usually submitted as direct smears or utilizing liquid-based cytology. The epithelial

Vitrectomy is surgery performed to remove some or all of the vitreous humor from the eye. Vitrectomy specimens are submitted as diluted or undiluted specimens depending on the clinical suspicion and presumptive clinical diagnosis. Additionally, small retinal or choroidal FNA biopsies may be performed during vitrectomy procedures. Therapeutic and diagnostic vitrectomies differ: when a diagnostic vitrectomy is performed, the initial 1 ml of undiluted vitreous sample is collected prior to the beginning of infusion. A diluted vitreous sample is submitted in therapeutic and diagnostic vitrectomies; this sample has an irrigation solution added that is used during the removal of the vitreous. In processing therapeutic vitrectomy samples, the diluted vitreous sample can be used to prepare PAP and PAS-stained slides with liquid-based cytology methodology and a cell-block when enough tissue is present. The following protocols can be used in triaging diagnostic vitrectomy samples.2

Clinical suspicion of lymphoma The undiluted refrigerated vitreous sample is used to prepare 1–3 air-dried Diff-Quik stained smears. The remaining fluid can be used for polymerase chain reaction (PCR) analysis [heavy chain gene rearrangement (IgH) and T cell receptor (TCR)] and cytokine assay for interleukins 6 and 10 (IL-6/IL-10) if indicated. An undiluted sample of at least 0.5 ml is needed for PCR analysis, and a volume of 2 ml is needed for interleukin assay. If the undiluted sample is insufficient in volume, the undiluted sample may be diluted with a balanced salt solution to reach a volume of 2.5–3 ml, of which 2 ml can be used for IL-10 and IL-6 analysis (1 ml each in 2 separate syringes) and 0.5–1 ml for PCR analysis. The diluted vitreous sample is used to prepare a liquid-based cytology sample stained with PAP and other stains (GMS, etc.) A cell-block is prepared if enough tissue is available. Flow cytometry (FCM) is a diagnostic tool for lymphoma diagnosis in fluids with adequate cellularity. Most laboratories require a minimum cell count (10,000–40,000 per ml depending on laboratory methods) in the sample to obtain valid results. FCM can have very limited application in vitreous samples due to paucicellular samples. If performed on vitreous samples, FCM may require customized protocols, which may not be available in all laboratories.

Clinical suspicion of infection The undiluted refrigerated vitreous sample can be tested (0.5– 1 ml) for PCR analysis of Herpes simplex virus (HSV), Herpes zoster virus (HZV), Toxoplasmosis species, Tuberculosis (TB complex), and Cytomegalovirus (CMV); if suspected, Toxocara canis antibody titers can be drawn. The diluted vitreous sample

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Fig. 2 – (A) Normal anterior corneal histology showing corneal epithelial cells, Bowman's layer, and anterior stromal fibers (H&E,  20). (B) Normal bulbar conjunctiva histology from the plica semilunaris area (H&E stain,  10). (C) Normal histology of the iris, anterior chamber of the eye, angle region, and ciliary body (H&E,  2). (D) Histology of the anterior portion of the lens with a thick capsule and a single layer of lens epithelial cells and lens fibers (PAS,  60). (E) Histology of the normal retina, choroid, and sclera (H&E,  4). (F) Histology of the normal choroid showing larger blood vessels closer to the sclera (bottom of the picture) and smaller vessels and capillaries under the retina; pigmented melanocytic cells are normally present in a loose connective tissue matrix. The choroid is located under the retinal pigment epithelium (top of the picture), a layer of cuboidal pigmented cells (H&E,  40). can be used for preparation of cultures and liquid-based cytology-stained slides with PAP and other stains (GMS, AFB, and Giemsa). A cell-block is prepared if enough tissue is available.

on 6p, loss on 6q, and gain on 8q. Additional molecular tests comprising a gene expression profile can be done in order to classify MM as class 1 or class 2, utilizing undiluted fresh tumor FNAB sample (DecisionDx-UM, Castle Biosciences Inc.).

Fine-needle aspiration biopsies Fine-needle aspiration biopsies (FNAB) may be performed for the diagnosis of anterior chamber pathologies and for tumors involving the uvea, retina, orbit, and ocular adnexa. FNAB usually requires cytopathology assistance at the time of the procedure. Multiple direct smears are prepared, both air-dried and alcoholfixed and stained with Diff-Quik, PAP, and H&E stains. The remainder of the specimen can be submitted for cytospin, liquid-based cytology, and/or cell-block preparation if enough material is present. Both SurePath and ThinPrep liquid-based methods can be used to prepare a monolayer of representative cells and stained with PAP, PAS, or other histochemical and immunohistochemical stains. If there is clinical suspicion of uveal malignant melanoma (MM), multiple slides (2–3) can be prepared with a single drop of fluid placed on the center of the slide and air-dried. These slides can be used for cytogenetic testing, looking for loss of chromosome 3 (or monosomy 3), gain

Cornea and conjunctiva ocular surface cytology Normal histology and cytology The conjunctiva is a nonkeratinized stratified squamous epithelium with goblet cells (Fig. 2B). Most conjunctival swabs or scrapings are taken from the interior fornix of the conjunctiva and show clusters of single epithelial cells, cuboidal to columnar, with abundant cytoplasms, eccentric nuclei, and occasional nucleoli (Fig. 3A). Goblet cells show a clear vacuolated cytoplasm. In allergic conjunctivitis,3 a common inflammation of the conjunctiva due to a type I hypersensitivity reaction, cytological smears show large numbers of eosinophils admixed with other inflammatory cells (Fig. 3B). The corneal epithelium is composed of 5–6 layers of modified, stratified, nonkeratinizing squamous epithelium.

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Fig. 3 – (A) Conjunctival cells in chronic conjunctivitis showing reactive changes (H&E,  20). (B) Smear in a case of allergic conjunctivitis showing numerous eosinophils (H&E,  40). (C) Corneal epithelial cell smear in acute keratitis showing reactive changes (PAP,  20). (D) Surface epithelial cells with abnormal keratinization in a cell-block preparation (H&E,  20).

Basal cells are smaller with higher nuclear to cytoplasmic ratios compared to other cells in the corneal epithelium. There are 2–3 layers of epithelial cells with interdigitating cytoplasmic processes. The top 2 layers are flattened superficial epithelial cells with small round nuclei and inconspicuous nucleoli. Corneal epithelial cells are attached to a basement membrane and beneath lies Bowman's layer, a specialized layer of the corneal stroma that does not regenerate after injury (Fig. 2A). Surface epithelial smears from the normal cornea will demonstrate cohesive sheets of nonkeratinizing squamous epithelial cells (Fig. 3C). The presence of keratinized cells in smears from the cornea is abnormal (Fig. 3D).

Infections involving the ocular surface Chlamydia trachomatis infections involve the cornea and the conjunctiva, resulting in trachoma, a bacterial infection spread through direct contact. If left untreated, repeated trachoma infections can cause severe scarring and lead to blindness.4 The infection affects the most marginalized communities in the world, with almost 8 million people visually impaired. Cytologic smears from trachoma yield epithelial cells with multiple small (0.5 mm) cytoplasmic basophilic inclusions with halos as the characteristic finding in Giemsa-stained slides (Fig. 4A). Current methods of detection include chlamydial cultures of conjunctival cells, direct immunofluorescent (DFA) staining of conjunctival scrapings, enzyme-linked immunosorbent assay, and serum immunoglobulin IgG titers against Chlamydia species.5 Bacterial infections that cause acute conjunctivitis predominantly

show neutrophils and bacteria in smears. Conjunctival scrapings and cultures are frequently obtained in severe bacterial conjunctivitis or those cases when antibacterial therapy is ineffective. Bacteria most commonly responsible for bacterial keratitis are Streptococcus, Pseudomonas, Enterobacteriaceae (including Klebsiella, Enterobacter, Serratia, and Proteus), and Staphylococcus species.6 Fungal keratitis usually occurs secondary to corneal defects in the epithelium due to trauma (e.g., contact lens wear, foreign material, and prior corneal surgery). The organisms can penetrate an intact Descemet's membrane and gain access into the anterior chamber or the posterior segment. Fungal keratitis also has been described secondary to fungal endophthalmitis. In these cases, fungal organisms extend from the posterior segment through Descemet's membrane and into the corneal stroma. Another possibility is entry through corneo-scleral trabeculae into the cornea. Fungal corneal infections are not common in the U.S. but are fairly common in developing countries. Fungi that have been isolated from corneal infections include Moniliaceae (non-pigmented filamentary fungi, including Fusarium and Aspergillus species), Dematiaceae (pigmented filamentary fungi, including Curvularia and Lasiodiplodia species), and yeasts (including Candida species).7 Up to 20% of cases of fungal keratitis (particularly candidiasis) are complicated by bacterial co-infections. Viral infections8 are a common cause of painful inflammation of the cornea and the conjunctiva, and their identification may be of assistance in the clinical management of the disease. Specimens should be obtained for culture and smear if inflammation is severe, in chronic or recurrent infections, with atypical conjunctival reactions, and with failure to

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Fig. 4 – (A) Chlamydia. Conjunctival epithelial cells with multiple small cytoplasmic basophilic inclusions (Giemsa,  300). (Original image from the files of Moshe Lahav, M.D., New England Eye Center Ocular Pathology Collection.) (B) Corneal keratitis smear showing Acanthamoeba double-walled cysts (H&E,  300). (Courtesy of Nasreen A. Syed, M.D.) respond to treatment. Giemsa (Diff-Quik) staining of conjunctival scrapings may aid in characterizing the inflammatory response; mononuclear cells and lymphocytes are seen with viruses. Adenoviruses usually involve the conjunctiva and show multiple, small eosinophilic intranuclear inclusions in small conjunctival cells. If present, diagnostic findings are seen in few cells. Herpes Simplex and Zoster viruses involve both the cornea and the conjunctiva, and cells have typical eosinophilic, “ground-glass” intranuclear inclusions, multinucleation, and nuclear molding. Measles may involve the conjunctiva with multinucleated cells with multiple eosinophilic inclusions with sharp halos present. Corneal keratitis caused by Acanthamoeba species may occur mainly in contact lens wearers; corneal scrapings may be used for diagnosis. Trophozoites, ameboflagellate measuring 15–30 mm and the double-walled cysts of the parasite may be identified (Fig. 4B). Early diagnosis and aggressive therapy are essential to prevent visual loss.9 Less common infections seen in the US include tuberculosis, syphilis, and parasites.

Epithelial dysplasia and neoplasia Squamous intraepithelial neoplasia includes intraepithelial dysplastic changes to full-thickness epithelial neoplasia or carcinoma in situ arising in the limbus (corneal stem cells), with either or both conjunctival and corneal involvement. The spectrum of abnormalities ranges from mild to severe dysplasia or carcinoma in situ and invasive squamous cell carcinoma.10 Risk factors for the development of these lesions include ultraviolet sun exposure; HPV 6, 11, 16, and 18; advanced age; male gender; immunosuppression; therapeutic radiation; burns; and genetic conditions (xeroderma pigmentosa) among others.11 Mucoepidermoid and spindle cell carcinomas have also been described. A cytology sample shows small to moderately sized atypical epithelial cells to frankly malignant squamous epithelial cells with keratinization if the lesion is a keratinizing dysplasia or carcinoma (Fig. 5A and B). The differential diagnosis includes sebaceous carcinoma, which shows numerous mitotic figures, large cells with high nuclear to cytoplasmic ratios, and clear cytoplasm containing

intracellular fatty deposits that stain with Oil Red O or Sudan Black stains (Fig. 5C and D).

Anterior chamber cytology The anterior chamber of the eye, located between the posterior surface of the cornea and the anterior surface of the iris, contains approximately 0.3 ml of fluid. Anterior chamber fluid aspirations are usually small samples, and optimal handling is imperative. The procedure may be performed in the management of uveitis and to rule out infectious diseases. Anterior uveitis involves inflammation of the iris seen in association with autoimmune diseases or in otherwise healthy individuals.12 Cytologic smears show chronic inflammatory cells including lymphocytes, plasma cells, and macrophages. IL-6 measurements in these samples may be requested; levels of IL-6, IFN-γ, and TNF-α have been found to be increased in idiopathic uveitis, Behcet's disease, and ankylosing spondylitis. Lens-induced uveitis is due to an immune complex reaction to lens protein exposure in cases of disrupted lens capsule. Numerous neutrophils are found within the lens cortex surrounded by a ring of macrophages. Other diseases that may undergo anterior chamber aspiration biopsy include ghost-cell glaucoma, phacolytic glaucoma, epithelial downgrowth after anterior segment surgical procedures, juvenile xanthogranuloma, iris epithelial cysts or single cells, and suspected neoplasms.13 Ghost cell glaucoma develops secondary to a vitreous hemorrhage with disruption of the anterior hyaloid of the vitreous. Senescent red blood cells or “ghost” erythrocytes may be found in the anterior chamber. Ghost erythrocytes cannot drain through the angle trabecular meshwork, and their presence may lead to increased intraocular pressure, i.e., ghost cell glaucoma (Fig. 6A). Lens-induced glaucoma or phacolytic glaucoma occurs in advanced cataracts with leakage of lens cortex proteins into the anterior chamber through an intact lens capsule. These proteins may cause aqueous fluid drainage alterations through the trabecular meshwork and lead to open-angle glaucoma. In these samples, macrophages filled with liquefied lens cortical material are present (Fig. 6B).

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Fig. 5 – (A) Cornea. Atypical cells from a case of low-grade squamous intraepithelial neoplasia (mild dysplasia) (PAP,  20). (B) Atypical dyskeratotic corneal epithelial cells in a cell-block from a squamous cell carcinoma (H&E,  20). (C) Smear of sebaceous carcinoma involving the cornea and the conjunctiva (Oil Red O,  40). (D) Tissue section of sebaceous carcinoma with involvement of the conjunctival epithelium (H&E,  20).

Epithelial downgrowth develops secondary to a perforating wound to the cornea or the limbus. Squamous epithelial cells may grow into the wound and along the ocular surfaces lining the anterior chamber. This growth may result in blinding complications with angle-closure glaucoma and retinal detachment. Juvenile xanthogranuloma may present in children as iris nodules with spontaneous hemorrhages (hyphema) in the anterior chamber (Fig. 6C). Cytology samples of aqueous humor show histiocytes, occasional eosinophils, and rare Touton giant cells. Iris epithelial cysts, although uncommon, are usually paucicellular with pigmented iris cells and macrophages (Fig. 6D). Melanin granules in iris-pigmented epithelial cells are typically larger than those seen in MM, and are uniform in size and shape. In contrast, melanin granules within macrophages tend to vary in size and shape. Iris epithelial cells may also be found in response to ocular injury, inflammation, and certain types of glaucoma (pigmentary glaucoma). Tumor cells in the anterior chamber may be seen in retinoblastoma (Rb) with extensive vitreous and anterior chamber seeds. The diagnosis of Rb is based on clinical grounds, not on cytology examination. Most primary tumors of the iris and ciliary body are slowgrowing nevi or low-grade MM, and tumor cells seldom shed from these tumors into the anterior chamber. Rarely, however, metastasis may involve the iris and shed into the aqueous humor.

Cytology of the posterior segment of the eye Adequate cytologic and immunologic evaluation of vitrectomy specimens is important to increase the diagnostic yield in

suspected cases of inflammatory, infectious, and neoplastic diseases. A high diagnostic yield begins with safe and precise specimen acquisition and requires optimum handling practices for cytopathological processing. The number of tests that can be carried out on a sample depends on the quality and quantity of the specimen.2 Vitrectomy has become a common technique in performing vitreous biopsy and is an important step in obtaining retinal and choroidal biopsies. Vitrectomy is an invasive procedure with potential surgical complications, and the benefits of cytologic evaluation of a diagnostic vitrectomy must outweigh the risk of these complications. It is often performed consecutive to equivocal or negative ancillary systemic tests and as a final option.14 A standard 3-port pars plana vitrectomy technique can be used to obtain a sufficient sample of vitreous, ideally at least 1.5 ml. To obtain an undiluted specimen, a single incision can be used. A self-sealing sclerotomy is created and a vitrector is inserted into the pars plana of the ciliary body. A small amount of undiluted vitreous is removed from the eye. Optimal handling of this low-volume sample involves prioritizing ancillary tests (cytology, measurement of cytokine levels, immunohistochemistry, flow cytometry, and PCR analysis), and this largely depends on clinical suspicion and presumptive diagnosis. As the vitreous sample can be paucicellular, prompt transportation to the pathologist can avoid degeneration of diagnostic cells. In cases with a strong suspicion of an infectious etiology, it is important to send samples for microbiologic evaluation with preoperative consultation with the microbiology laboratory. In addition to preoperative communication with the ocular pathologist and the laboratories, retinal surgeons should obtain maximal yield of vitrectomy specimens by discontinuing systemic steroids before vitrectomy. The following diagnostic categories may be used to stratify vitrectomy

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Fig. 6 – (A) Ghost cell glaucoma. Senescent or “ghost” erythrocytes present in an anterior chamber FNAB (PAP,  20). (B) Lensinduced glaucoma. Macrophages ingesting lens fibers (H&E,  60). (C) Juvenile xanthogranuloma. Cell-block of an anterior chamber FNAB showing numerous macrophages (H&E,  40). (D) Histologic section through the root of the iris and ciliary body showing an iris cyst with pigmented macrophages (H&E,  4). cases: negative for malignancy (including vitreous hemorrhage, non-pathologic findings, inflammatory/infectious findings, and other findings), atypical, suspicious for malignancy, and positive for malignancy (Table).

Normal cytologic components of a vitrectomy sample are the vitreous cells or hyalocytes: small, slender columnar cells with eosinophilic cytoplasm, vitreous fibers, and macrophages

Table – Vitrectomy diagnostic categories.

Therapeutic vitrectomies Therapeutic vitrectomies are performed to excise visually significant vitreous opacity, as seen in cases of vitreous hemorrhage, cataract, inflammatory, or vitreous opacification, causing visual loss or precluding adequate view of the posterior segment. Lens-induced uveitis, endophthalmitis, control of inflammation, retinal detachment, epiretinal membrane excision, chronic hypotony, and sustained intra-vitral drug delivery are also indications of therapeutic vitrectomies. Although rare, an unsuspected malignancy might be diagnosed in a patient undergoing therapeutic vitrectomy. Cytologic evaluation of therapeutic vitrectomies provides a baseline diagnostic exposure to cytopathologists and cytotechnologists to help distinguish features in diagnostic vitrectomy samples.2

Intraocular cytology diagnoses Atypical Chronic uveitis Lymphoproliferative disorders Uveal nevus Retinopathy of prematurity Suspicious for malignancy Lymphoproliferative disorders Uveal malignant melanoma Retinal pigment epithelium, iris, and ciliary body tumors Metastases Malignant Retinoblastoma Uveal malignant melanoma Intraocular lymphoma/leukemia Metastases

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(Fig. 7A). Other components include fragments of peripheral retina (Fig. 7B), anterior uveal tissues, retinal pigment epithelial cells (Fig. 7C), subretinal membrane, lens fiber fragments, lens epithelial cells, lens capsule material (Fig. 7D), fibrovascular tissue fragment (tractional retinal detachment), and red blood cells. Conjunctival cells from the aspiration needle entry port can also be seen. In asteroid hyalosis, spherical acellular material is found in vitrectomy specimens measuring 10– 100 mm, when stained with PAS, Alcian blue, neutral fats, phospholipids, and calcium (Fig. 7E). These spheres reflect the light of the ophthalmoscope and appear to the examining ophthalmologist as stars in a galaxy, but do not interfere with patient's vision. They are birefringent under polarized light and may be removed incidentally in the treatment of other eye conditions (for example in diabetic retinopathy). Vitreous amyloidosis is found in a group of life-threatening multisystem disorders named familial amyloid polyneuropathies that lead to extracellular amyloid deposits. Multiple genetic mutations of the transthyretin protein have been described, but the first identified mutation Val30Met is the most common of more than 100 amyloidogenic point mutations. Less often, the precursor is mutant apolipoprotein A-1 or gelsolin. Vitreous amorphous material is found, positive with Congo red stain, and shows birefringence under polarized light (Fig. 7F). Systemic manifestations include vitreous opacities, perivascular infiltrates, peripheral neuropathy, and cardiomyopathy among others. Fragments of retina or individual retinal cells can be observed in vitrectomies from patients with retinal tears and retinal detachments. Retinal tears form from vitreous traction on the retina, secondary to proliferative vitreoretinopathy or ocular trauma. Retinal detachments occur when vitreous traction and fluid currents during eye movements lead to detachments between the retina and the RPE. In rhegmatogenous retinal detachment, the most common type of retinal detachment, a tear in the retina leads to fluid accumulation with a separation of the neurosensory retina from the underlying RPE, and cellular membranes may form on either anterior or posterior surfaces of the retina. These membranes are a proliferation of retinal pigment epithelial cells, glial cells, macrophages, fibroblasts, myofibroblasts, and hyalocytes. Idiopathic macular holes form as a result of degenerative changes in the vitreous, leading to full-thickness macular retinal defects with rounded tissue margins. Cytologic samples of macular holes are usually paucicellular. Persistent fetal vasculature (or persistent hyperplastic primary vitreous) is characterized by the persistence of variable components of the primary vitreous found in ocular development. In cases of clinically significant persistent fetal vasculature, a fibrovascular plaque is present behind the lens, seen in contiguity with a remnant of the hyaloid artery that may attach to the optic nerve head. The cytology shows vascular fragments and lens fragments.

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and non-infectious uveitis. Infectious causes include bacteria, fungi, viruses, and parasites.2,14 Endophthalmitis may be posttraumatic, post-surgical, endogenous, exogenous, or sterile. It can involve the vitreous (vitritis), the retina (retinitis and retinal vasculitis), or the choroid (choroiditis). Noninfectious forms of uveitis that may require a diagnostic vitrectomy include autoimmune uveitis, primary intraocular lymphoma, metastatic tumors including leukemic infiltration, and uveal MM. The presence of numerous neutrophils (Fig. 8A), necrotic debris, focal abscess formation, necrotic retinal tissue, bacteria, and fungi can be found in cases of acute endophthalmitis secondary to bacterial and fungal infections. Viral infections are usually paucicellular with monocytes, lymphocytes, and macrophages and can be diagnosed by PCR analyses. Viral cytopathic effects (cytomegaly, multinucleation, and nuclear inclusions with margination of nuclear chromatin) are rarely found on cytologic examination of vitrectomy specimens. Cytologic findings of eosinophils and hydatid cyst wall may be seen in parasitic infections. Ancillary studies helpful in infectious endophthalmitis include cultures, PAS and GMS staining for fungal infections, PCR analysis for toxoplasmosis, herpes viruses types I and II, cytomegalovirus infections, Treponema pallidum, and tuberculosis. Bacterial infections include staphylococcus, fusarium, and propionibacterium among others. In chronic uveitis, vitrectomy samples show benign lymphocytes and macrophages (Fig. 8B). In cases suspicious for lymphoproliferative disorders, atypical lymphocytes are present that may represent activated B-lymphocytes or lymphoma cells.12,14 Interleukin analysis ratios of IL-6 and IL-10 are helpful in distinguishing inflammatory versus lymphoproliferative disorders. A ratio greater than 1 is most likely indicative of an inflammatory diagnosis; a ratio of less than 1 favors a diagnosis of lymphoma. The vitreous can be the site of primary involvement in cases of B-cell non-Hodgkin lymphoma (NHL).15 T-cell lymphoma rarely involves the vitreous. The most common presentation is as a posterior uveitis. Lymphoid cells usually spill over from neoplastic infiltrates in the retina and sub-RPE. Up to 50% of patients presenting with ocular findings have concomitant involvement of the central nervous system. The cytologic diagnosis of intraocular NHL shows a mixed population of cells with atypical large lymphoid cells having convoluted nuclei, multiple conspicuous nucleoli, and plasmacytoid scant cytoplasms (Fig. 8C). An accompanying infiltrate of small reactive Tcells is usually present. FCM, IHC, IL-10 to IL-6 ratios, and PCR for the detection of IgH gene rearrangement and translocation all can be used to aid in a diagnosis of NHL. Small rounded cells of early mesenchymal origin simulating Rb can be found in documented cases of retinopathy of prematurity, the abnormal blood vessel development found in retinas of babies who are born before 30 weeks or weigh fewer than 3 pounds at birth (Fig. 8D).

Diagnostic vitrectomies Fine-needle aspiration biopsies Diagnostic vitrectomies are performed to aid in a particular diagnosis and in some cases are accompanied by intraocular biopsies. They are performed in the diagnosis of infectious

The FNAB technique is usually reserved to collect samples in suspected tumors. Although referred to as FNAB, the

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Fig. 7 – (A) Hyalocytes with vitreous fibers in the background, vitrectomy specimen (PAP,  4). (B) Fragment of peripheral retina with vessels, vitrectomy specimen (H&E,  20). (C) Retinal pigment epithelial cells, vitrectomy specimen (PAP,  10). (D) Fragment of lens capsule, lens epithelial cells and fibers, vitrectomy specimen (PAP,  10). (E) Spherical acellular material found in asteroid hyalosis, vitrectomy specimen (PAP,  10). (F) Amyloid with characteristic “smudged” appearance, vitrectomy specimen (PAP,  100).

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Fig. 8 – (A) Acute endophthalmitis. Numerous neutrophils from a diagnostic vitrectomy (PAP,  20). (B) Chronic vitritis containing macrophages and lymphocytes (PAP,  40). (C) B-cell NHL involving the vitreous. Large lymphocytes are singly dispersed in this smear (PAP,  40). (D) Retinopathy of prematurity. Crushed small blue cells simulate retinoblastoma. Cellblock (H&E,  40).

procedure is actually a fine-needle core aspiration rather than a biopsy. A needle is place in a single meridian to minimize visual morbidity; sampling in multiple planes is not usually performed due to the risk of retinal detachment or vitreous hemorrhage. The most common intraocular tumors in adults are metastases to the eye. Intraocular metastases may occur in the choroid, in the posterior pole of the eye, or in the iris stroma. The treatment of ocular metastasis is radiation, and life expectancy is short after intraocular metastasis is detected. The most common metastatic tumors are breast/ lung carcinomas for women and lung/gastrointestinal carcinomas for men (Fig. 9A). These tumors show similar cytologic features as found in samples from other body sites. Retinoblastoma (Rb), the most common intraocular tumor of childhood, is usually diagnosed without difficulty by ophthalmoscopic examination supplemented when necessary by ultrasonography.16 Many lesions may mimic Rb, and on rare occasions, FNAB may be requested when the diagnosis is uncertain. Rb cells are highly friable, and many ophthalmic oncologists are opposed to needle biopsy for fear of needle tract dissemination by the tumor. Rb cells are undifferentiated with rounded hyperchromic nuclei, coarse chromatin,

and scant cytoplasm (Fig. 9B). They frequently exist within in a necrotic background. Coat's disease is a condition that can mimic Rb clinically. Cytologic samples of Coat's disease yield cholesterol crystals and macrophages containing melanin from RPE cells (Fig. 9C). Other tumors that may enter in differential diagnosis are uveal MM, a rare malignancy in children. Toxocara canis infection may also simulate Rb clinically, and an FNAB from these eyes typically yields numerous eosinophils. Uveal MM is the most common intraocular tumor of adults. Most are diagnosed clinically and treated with radiation therapy; large tumors are treated by enucleation. FNAB may be applicable in settings where the clinical distinction between MM and a simulating lesion (choroidal nevus, retinal hamartoma, RPE adenoma, or carcinoma) is uncertain. The cytopathology of MM exhibits malignant cells in loose clusters and as single forms having spindle and/or epithelioid shapes (Fig. 9D). Epithelioid MM cells have abundant cytoplasm, large vesicular nuclei, and centrally located macronucleoli; some are binucleated. Cytogenetic testing of airdried smears can be performed to rule out the presence of monosomy 3 and or trisomy 8, allowing for stratification of

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Fig. 9 – (A) Lung adenocarcinoma metastatic to choroid and retina, vitrectomy specimen. Large cells with convoluted nuclear contours and visible nucleoli (PAP,  60). (B) Retinoblastoma. Primitive malignant small rounded cells with high N/C ratios, FNAB (Diff-Quik,  40). (C) Cell-block of Coatʼs disease showing vessels, fibrous and proteinaceous material, and macrophages, vitrectomy specimen (PAS,  10). (D) Uveal melanoma. Malignant pigmented spindle and epithelioid cells. Note binucleated cell at lower right. FNAB (H&E,  60). the tumor into type 1 or type 2 depending on the absence or presence of monosomy 3. The mortality of uveal MM averages nearly 50%; there is no effective chemotherapy or immunotherapy for metastatic uveal MM.17 Congenital simple RPE hamartomas, choroidal nevi, and RPE adenomas and adenocarcinomas, although uncommon, may be sampled by FNAB. Congenital simple hamartoma of the RPE appears ophthalmoscopically as a small localized, elevated black lesion usually located in the foveal region and on the surface of the retina.18 Lesions are o1 mm in diameter and composed of a proliferation of the RPE with benign cytologic features (Fig. 10A). Choroidal nevi are common melanocytic lesions of the posterior uvea, with a prevalence in the US ranging from 6% to 10% of the population.17 Although usually asymptomatic, choroidal nevi can be associated with symptoms that mimic choroidal MM and therefore may need to be sampled. The cytology of choroidal nevi shows spindle amelanotic or pigmented cells with bland oval or cigar-shaped nuclei, finely dispersed chromatin, and lacking nucleoli, nuclear folds or mitotic activity. Intranuclear cytoplasmic inclusions, balloon cell degeneration, or dendritic plump cells may be seen.

Adenoma and adenocarcinoma of the RPE appear as an oval-shaped, abruptly elevated, usually pigmented mass arising from the peripheral RPE. The affected eye often has vitreous cells due to inflammation. As it enlarges, adenoma of the RPE can assume a retinal blood supply with large, dilated tortuous vessels; intraretinal and subretinal exudation is commonly found adjacent to the tumor. Histopathologically, adenoma of the RPE usually shows cords of proliferating RPE cells separated by fibrous stroma. Tumor cells are large and polyhedral with eccentric round-to-oval nuclei and abundant granular cytoplasm. Adenocarcinoma of the RPE exhibits similar patterns, but the cells present show increased pleomorphic features18 (Fig. 10B).

Conclusion Ocular cytology can be challenging to cytopathologists not only in the interpretation of a specimen, but also in understanding the best sampling and preparatory methods and the implications of a specific diagnosis in the treatment of the condition. Cytologic evaluation of ocular fluids and tissues

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Fig. 10 – (A) Congenital simple hamartoma of retinal pigment epithelium with benign proliferating cells embedded in fibrous tissue deposits. FNAB cell-block (PAS,  10). (B) Retinal pigment adenocarcinoma with malignant cells, mitosis, and basement material present. FNAB cell-block (H&E,  10). plays an important role in the management of a variety of important ophthalmic conditions. The utility of cytologic evaluation in this highly specialized area is directly proportional to the level of communication between the cytopathologist and the surgeon and the cytopathologist's grasp of ocular cytology.

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