Review Article

Ocular Cytology: Diagnostic Features and

Ongoing Practices
Nora M. V. Laver, MD

Ocular cytology specimens are small, with limited options for a repeat biopsy. Appropriate handling of these specimens and
triaging for ancillary testing can be taxing. In this article, the author reviews a selection of potentially challenging diagnoses
and current common practices and methods used in diagnosing ocular diseases by cytology. The majority of cytology speci-
mens submitted for evaluation of ocular diseases can be divided into 3 major categories: surface epithelial corneal and con-
junctival cytology samples, intraocular fluids from the anterior (aqueous fluid) or posterior (vitreous fluid) chambers of the
eye, and intraocular fine-needle aspiration specimens. The clinical findings, testing, and cytologic features of ocular surface
epithelial infections, inflammations and neoplasia are discussed; and challenges in processing and diagnosing intraocular
infections, chronic uveitis, and vitreoretinal lymphoma are reviewed. Novel molecular testing in the cytologic diagnosis and
classification of uveal melanoma also is explored. Cytology evaluation of corneal epithelial and stromal cells, anterior cham-
ber and vitreous samples, and fine-needle aspiration biopsies can provide detailed diagnostic findings to aid in the treatment
and follow-up of patients with ocular diseases. Cancer Cytopathol 2021;129:419-431. © 2020 American Cancer Society.

KEY WORDS: Acanthamoeba keratitis; corneal scrapings; dry-eye disease; impression cytology; intraocular lymphoma;
ocular cytology; ocular surface intraepithelial neoplasia; uveal melanoma; uveitis.

INTRODUCTION
Ocular cytology can be challenging to general cytopathologists. Ocular diseases are unique, and the methods
used for diagnosis are highly specialized. Adequate sampling and processing of ocular samples require under-
standing of clinical scenarios and the diagnostic methods commonly used. Ocular cytology specimens are
small, with limited options for a repeat biopsy, requiring meticulous allocation to the appropriate ancillary
tests. This article reviews a selection of potentially challenging diagnoses and current common practices and
methods used in diagnosing ocular diseases by cytology.
Common ocular cytology specimens submitted for evaluation can be divided into 3 major types, namely,
surface epithelial corneal and conjunctival cytology samples, intraocular fluids from the anterior (aqueous fluid)
or posterior (vitreous fluid) chambers of the eye, and intraocular fine-needle aspiration specimens (Table 1).

SURFACE OCULAR EPITHELIAL CYTOLOGY SAMPLES

Conjunctival and corneal samples are usually submitted as direct smears, brush cytology, impression cytology,
or small biopsies, depending on the clinical suspicion and differential diagnoses.1-3 The epithelial surface of the
cornea and conjunctiva may be scraped under local anesthesia with a small spatula or brush and the cellular
yield smeared onto tissue slides or placed into liquid-based cytology fixative.4 If impression cytology is used,
an absorbent filter paper is pressed onto the ocular surface to acquire surface epithelial cells. Direct smears may
be alcohol-fixed or air-dried; brush cytology samples may be prepared using liquid-based cytology, placing

Corresponding Author: Nora M. V. Laver, MD, Tufts Medical Center, 800 Washington Street, Suite 6700, Boston, MA 02111 (nlaver@tuftsmedicalcenter.org).

Departments of Ophthalmology, Pathology and Laboratory Medicine, Tufts Medical Center and Tufts University School of Medicine, Boston, Massachusetts
Received: September 16, 2020; Revised: September 29, 2020; Accepted: September 30, 2020

Published online November 2, 2020 in Wiley Online Library ­(wileyonlinelibrary.com)

DOI: 10.1002/cncy.22384, wileyonlinelibrary.com

Cancer Cytopathology  June 2021 419

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TABLE 1.  Common Ocular Cytopathology Specimens

Infections
Acanthamoeba
Bacteria
Fungus
Herpes virus (HSV-I and HSV-II)
Inflammations
Dry-eye disease
Ocular surface squamous neoplasia
Intraocular fluids: Anterior chamber and vitrectomy samples
Intraocular infections
Uveitis
Vitreoretinal lymphoma
Intraocular fine-needle aspiration biopsies
Uveal malignant melanoma
Abbreviation: HSV, herpes simplex virus.

the scraped cells in appropriate alcohol-based fixative.

Impression cytology filters may be placed directly into
fixative and, depending on the number of cells harvested,
cytology and further testing can be performed.4,5 More
details are provided below regarding current practices in
the cytologic diagnosis of corneal infections, dry-eye dis-
ease, and ocular surface intraepithelial neoplasia (Table 1).

Corneal Infections
Most ocular infections are diagnosed clinically and
confirmed by culture or by polymerase chain reaction
(PCR) analysis.6 Clinically challenging bacterial, viral,
fungal, and Acanthamoeba keratitis may require cor-
neal surface epithelial scrapings and ocular cytology
interpretation.
Acanthamoeba keratitis
Acanthamoeba keratitis remains an uncommon diagno-
sis seen most often in contact lens wearers; often, the
infection goes undiagnosed until later stages of disease.
Two of the 8 known species of Acanthamoeba, A. castel-
lani and A. polyphaga, are responsible for most infec-
tions; these free-living amoebas can be found in pools,
hot tubs, shower water, and contact lens solutions.7
Even when clinical findings suggest the diagnosis, con-
firmatory testing is needed to rule out other infectious
organisms. Early clinical signs may be mild and nonspe-
cific, such as cornea epithelial irregularities or epithelial
or anterior stroma infiltrates and pain out of propor-
tion to the findings. More typical clinical findings
include deep ring-shaped stromal infiltrates, corneal
perforation, scleritis, anterior uveitis, and hypopyon,
among others (Fig. 1A).7,8 Cultures for Acanthamoeba
Figure 1.  (A) An external corneal photograph is shown from a
male aged 18 years who was a contact lens wearer. The cornea
shows a stromal ring-shaped infiltrate, epithelial irregularities,
and conjunctivitis. (B) A confocal microscopic image reveals
corneal epithelial cells with highly reflective Acanthamoeba
cysts. Internal structures are visible in some of the organisms.
require time6 and may be negative, especially if a super-
ficial scraping is submitted but the infection is localized
in the deeper corneal stroma. PCR analysis can also be
used for the diagnosis but has limited utility because,
depending on sample quality, DNA compared with
culture may be reported as negative.9,10 In vivo confo-
cal laser microscopy is a useful and rapid, noninvasive
method used in the clinic that enables morphologic and
quantitative analysis of ocular surface microstructures,
capturing multiple 2-dimensional images at different
depths in a sample and enabling the reconstruction
of 3-dimensional structures within an object. This
technology allows the identification of characteristic
double-walled cysts or larger trophozoites in patients
who have corneas infected with Acanthamoeba species.

420 Cancer Cytopathology  June 2021

 Ocular Cytology: Diagnostic Features and Best Practices/Laver

Typical findings include hyperreflective cysts in clusters

or chains with a double layer, and sometimes these are
star-shaped (Fig. 1B). In early infections, trophozoites
can be seen in the epithelial layer; they are larger than
cysts and appear egg-shaped or pear-shaped.11,12

Cytologic findings
Cytology sample smears may be stained with Papanicolaou
(Pap), Diff-Quik, or hematoxylin and eosin (H&E) stains.
Depending on the availability of cells, additional stains may
be performed, including Grocott’s methenamine silver stain,
calcofluor white, acridine orange, and lactophenol-cotton
blue.3,13,14 Typical double-walled cysts of Acanthamoeba
species (Fig. 2A) can be observed. The cyst walls provide a
physical barrier that renders the amoeba resistant to treat-
ments. The double-walled cysts consist of an ectocyst and an
endocyst. Occasional single-walled structures, representing
single-walled exocysts, can be present. Electron microscopy
studies have shown that the exocyst station is accompanied
by partial enzymatic digestion of the endocyst, explaining
the single-wall appearance of the empty cysts.15 When such
cysts occur, cultures are not possible, and genomic DNA
for PCR detection would not be available.15 Acanthamoeba
trophozoites are found in corneal epithelial infections with
an environment more favorable for their survival (Fig. 2B). Figure 2.  (A) A corneal smear shows an Acanthamoeba cyst
Trophozoites are small, usually from 15 to 25 µm in length, (arrow) (Diff-Quik stain, original magnification ×60). (B)
Another corneal smear reveals an Acanthamoeba trophozoite
and amoeboid in shape. (arrow) surrounded by reactive epithelial cells (Diff-Quik stain,
original magnification ×40).
Differential diagnoses
The main differential diagnoses of Acanthamoeba keratitis
include infections caused by bacteria, fungi, and viruses. glaucoma may occur.7 Corneal scrapings for smears and
Bacterial keratitis is the most common type of infection cultures are performed to determine the causative organ-
seen in the clinic. Like Acanthamoeba infections, the ism in all ulcers that are either large, involve the middle to
most common risk factor for bacterial keratitis is contact deep stroma, are sight-threatening, are chronic in nature,
lens wear. Other risk factors include swimming, trauma, are atypical, or are unresponsive to treatment (Fig. 3B).
previous eye surgeries, and eye diseases. The most com- Confocal microscopy can be performed in challenging
mon etiologies of bacterial keratitis are Streptococcus, cases to rule out Acanthamoeba and reveals surface ulcera-
Pseudomonas, Enterobacteriaceae (including Klebsiella, tion, loss of stromal cells, numerous acute inflammatory
Enterobacter, Serratia, and Proteus), and Staphylococcus cells, and cellular debris, as seen in smears, but not the
species.7 Clinical signs of bacterial keratitis include con- characteristic findings of Acanthamoeba infection.16
junctival injection, focal white infiltrates, corneal thin- Fungal keratitis is much less common than bacterial
ning, stromal edema, endothelial inflammatory plaque, keratitis in the United States. Risk factors include trauma
Descemet membrane folds, mucopurulent discharge, an- with vegetable matter (thorn injury, wheat trauma in ag-
terior chamber reaction, and hypopyon (Fig. 3A). Eyelid ricultural workers, etc), immunosuppression (especially
edema may be present in some patients. In severe infections, associated with topical corticosteroids), and contact lens
additional findings like posterior synechiae, hyphema, and wear. Fungal keratitis may be caused by any of several

Cancer Cytopathology  June 2021 421

Review Article
Figure 3.  (A) An external corneal photograph is shown from a
woman aged 43 years with Propionibacterium acne keratitis.
The cornea shows white infiltrates, stromal edema, anterior
chamber hypopyon, and conjunctival injection. (B) Acute
inflammatory cells and debris are observed in a bacterial
keratitis smear (ThinPrep; periodic acid-Schiff stain, original
magnification ×40).
fungi, including Candida spp., Aspergillus spp., Fusarium
spp., Cladosporium spp., Curvularia, and in nonseptated
fungi, such as Rhizopus.7 Clinical findings include blurred
vision, redness, tearing, photophobia, pain, foreign body
sensation, and secretions. The classic appearance of fun-
gal keratitis is a gray-white, dry-appearing infiltrate with
feathery borders (Fig. 4A). However, these findings are
not always present; therefore, corneal cultures, smears, and
scrapings are often used to aid in the diagnosis. In vivo
confocal microscopy, depending on the fungus infection
and operator experience, often allows the visualization of
fungal-like hyphae throughout the cornea (Fig. 4B).17,18
Because fungal cultures can take up to 3 weeks to grow
and identify specific fungi, direct smears may be submit-
ted to cytology (Fig. 4C) to aid in the identification of
fungi and the appropriate modification of treatment plans.
422
Herpes simplex keratitis is the most common viral
infection of the cornea. It is a serious infection that may
present as repeated attacks triggered by stress, exposure
to sunlight, or any condition that lowers the immune
response. Risk factors involve direct contact with infected
secretions or lesions. Herpes simplex type 1 (HSV-I) and
HSV-II may cause blepharon-conjunctivitis, epithelial
keratitis, stromal keratitis (necrotizing or nonnecrotiz-
ing), iridocyclitis, and/or retinitis. The hallmark of HSV
keratitis is the presence of multiple, small, branching epi-
thelial dendrites on the surface of the cornea (Fig. 5A).7,19
A diagnosis of HSV is usually made clinically; however, a
definitive diagnosis can be made using conjunctival scrap-
ings, impression cytology specimens, and scrapings from
vesicular lesions on the skin tested by cytology, culture, or
PCR for the presence of HSV.19,20 A superficial corneal
smear can reveal multinucleated giant cells and intranu-
clear eosinophilic inclusion bodies (Fig. 5B). Serum anti-
body testing is typically of limited use.19,20
Inflammations of the Cornea and
Conjunctiva—Dry-Eye Disease
Dry-eye disease is a very common eye condition caused
by a chronic lack of lubrication and moisture of the ocular
surface. Epidemiological studies indicate that the preva-
lence of dry eye in the United States in patients aged >50
years is approximately 7% in women and 4% in men.21
Dry-eye disease may be due to meibomian gland dysfunc-
tion, failure of the lacrimal glands to produce enough
watery fluid to keep the eyes adequately moistened, and
other conditions, including contact lens wear, aging,
menopause, indoor environment, smoking, medications,
and eyelid problems, among others.21,22 The normal ocu-
lar surface of the cornea and conjunctiva is composed of
a stratified nonkeratinizing epithelium. Different num-
bers of goblet cells, which produce the mucous part of
the tear film, can be found in the conjunctiva (Fig. 6A).
Pathologic changes, such as squamous metaplasia of the
epithelial cells, keratinization, and reduction of goblet cell
density up to total goblet cell loss, occur in numerous
disorders, including dry eye, Sjogren syndrome, blepha-
roconjunctivitis, inflammatory keratoconjunctivitis, and
vitamin A deficiency.23 Multiple classification schemes
have been proposed to aid in the diagnosis of dry eye and
other inflammatory conditions.24-26 Conjunctival im-
pression cytology specimens may be graded into 3 groups
based on the appearance of the epithelial cells and the
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 Ocular Cytology: Diagnostic Features and Best Practices/Laver

Figure 4.  (A) An external corneal photograph is shown from a man aged 51 years with fungal keratitis. The cornea appears gray-
white and has feathery borders. (B) A confocal microscopic image of the corneal stroma reveals infiltration by Aspergillus spp. (C)
A cell block preparation shows the fungal spores and hyphae of Paecilomyces spp. (Grocott’s methenamine silver stain, original
magnification ×40).

numbers of goblet cells. A cytology specimen exhibiting
small, round epithelial cells with large nuclei and >500
goblet cells/mm2 is classified as grade 0, the presence of
100 to 500 goblet cells/mm2 is classified as grades 1 and
2; and the presence of large polygonal epithelial cells with
small nuclei and <100 goblet cells/mm2 is classified as
grade 3. All specimens classified as grade ≥2 are consid-
ered abnormal (Fig. 6B). The findings of grades 2 and 3
on the interpalpebral conjunctiva in the absence of in-
flammatory cells suggest a diagnosis of keratoconjunctivi-
tis sicca, whereas grades 2 and 3 on both the bulbar and
palpebral conjunctiva suggest an intrinsic ocular surface
disease, such as ocular cicatricial pemphigoid, Stevens-
Johnson syndrome, or severe chemical burns.25,26
Additional studies may be performed by impression
cytology, including quantitative detection of HLA-DR,
upregulation of ICAM-1 (CD54) in inflammatory condi-
tions, and quantitative PCR of decreased mucins MUC2,
MUC4, MUC5AC, and MUC7, which make up the tear
film in dry-eye disease.27 Cytology specimens can provide
detailed cytologic data in confirming a diagnosis of squa-
mous metaplasia in dry-eye disease and other inflamma-
tory conditions.
Corneal and Conjunctival Ocular Surface
Squamous Neoplasia
Ocular surface squamous neoplasia (OSSN) encompasses
a wide and varied spectrum of disease involving the ab-
normal growth of dysplastic squamous epithelial cells
on the surface of the eye. The etiology of OSSN appears
to be multifactorial and involves various environmental
factors in a susceptible host, including ultraviolet light
exposure, immunosuppression, HIV infection, human
papillomavirus infection, mutation or deletions of p53,
smoking, and exposure to petroleum products, among
other factors.28 The clinical presentation of OSSN varies,
making diagnosis challenging. Typically, patients present
with a gelatinous or plaque-like, interpalpebral, conjunc-
tival gray or white lesion. The great majority of lesions
occur at the limbus, in which the most actively mitotic

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Review Article
Figure 5. (A) An external corneal photograph is shown from
a woman aged 36 years with Herpes simplex type 1 (HSV-I)
keratitis. The inset reveals the typical dendritic branching seen
on slit-lamp examination with fluorescein dye. (B) Corneal
epithelial cells with nuclear enlargement are observed in
HSV keratitis (cell block preparation; H&E stain, original
magnification ×40). The inset shows immunohistochemistry
staining for HSV-I and HSV-II (cell block preparation; HSV I-II
immunohistochemistry, original magnification ×40).
cells reside. The lesion may be flat or elevated and may
be associated with feeder vessels (Fig. 7A).28,29 Optical
coherence tomography has been used as an in vivo diag-
nostic modality for the detection of OSSN. Distinctive
features, such as hyperreflectivity, thickened epithelium,
and abrupt transition from normal to abnormal tissue
seen on ultra–high-resolution optical coherence tomogra-
phy, differentiate it from other conjunctival lesions, such
as pterygia.30 Cytology smears, brushes, or impression
cytology may be used as noninvasive methods for con-
junctival and corneal sampling of suspected OSSN.27,28
The surface layers of normal conjunctival epithelium are
composed of stratified, cuboidal-to-columnar epithelium
with mucin-secreting goblet cells. They are usually readily
424
Figure 6.  (A) Normal conjunctival epithelial cells from a smear
are shown (H&E stain, original magnification ×20). The inset
reveals goblet cells (cell block preparation; H&E stain, original
magnification ×60). (B) Conjunctival epithelial cells from
woman aged 65 years who had dry eye are shown. Smears
reveal predominantly conjunctival epithelial cells with rare
goblet cells (<300 goblet cells/mm2) from the interpalpebral
conjunctiva, consistent with grade 2 dry-eye disease (Diff-Quik
stain, original magnification ×60).
removed using a brush or by impression cytology and are
often seen as large, cohesive sheets (Fig. 7B). The con-
junctival epithelium merges into the nonkeratinized strat-
ified squamous epithelium of the cornea at the limbus.
Because healthy corneal cells are tightly bound to each
other and to their basement membrane, only a few rag-
ged, intermediate squamous cells are usually released. In
contrast, neoplastic cells are readily obtained from both
areas of the ocular surface. Conjunctival and corneal squa-
mous neoplasms may be keratinizing or nonkeratinizing.
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 Ocular Cytology: Diagnostic Features and Best Practices/Laver

Figure 7.  (A) This is an external photograph from a man aged 56 years who had a papillary, gelatinous, vascularized limbal lesion
involving the cornea and conjunctiva. (B) Sheets of conjunctival and limbal epithelial cells are observed with reactive changes
(Diff-Quik stain, original magnification ×20). (C) This image shows an ocular surface squamous neoplasia (keratinizing dysplasia/
carcinoma in situ) arising from the limbus and involving the cornea and conjunctiva in a man aged 45 years (H&E stain, original
magnification ×20). The inset reveals cells with nuclear enlargement, increased nuclear-to-cytoplasmic ratios, prominent nucleoli,
and keratinization (H&E stain, original magnification ×40).

Three-fold nuclear enlargement, syncytial-like groupings,
increased nuclear-to-cytoplasmic ratios, indistinct cyto-
plasms, prominent nucleoli, and keratinization are fea-
tures present in ocular surface squamous intraepithelial
neoplasia and squamous cell carcinoma (Fig. 7C).1,3,31
INTRAOCULAR FLUIDS
The anterior chamber of the eye, located between the pos-
terior surface of the cornea and the anterior surface of
the iris, contains approximately 0.3 mL of aqueous fluid.
Anterior chamber aqueous fluid aspirations are usually
small samples (0.2-0.3 mL). The undiluted aqueous fluid
may be diluted with saline and sent for molecular diagno-
sis of infections, inflammation, and lymphoma. Alcohol-
fixed or air-dried smears or a cytospin may be prepared
from a few drops of the fluid and stained with Diff-Quik,
Pap, or H&E stains.1,3
Vitrectomy surgery is performed to remove some
or all the vitreous humor from the posterior chamber of
the eye. Vitrectomy specimens are submitted as diluted
or undiluted samples, depending on the clinical diagno-
sis. In diagnostic vitrectomies, an initial approximately
1 mL of undiluted vitreous sample is collected before
the beginning of an infusion. A diluted vitreous sample
is submitted in both diagnostic and therapeutic vitrec-
tomies, representing a sample containing the removed
vitreous plus an irrigation. When processing therapeutic
vitrectomy samples, the undiluted vitreous sample can be
used to prepare direct smears and the rest of the sample
triaged for additional testing, depending on the clinical
differential diagnoses. The diluted vitreous sample can be
used to prepare alcohol-fixed, air-dried smears or cyto-
spins from a few drops of the fluid and, if enough tissue
is available, can be used for liquid-based cytology and a

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Figure 8. (A) A liquid-based smear of a retinal fungal infection is shown from a woman aged 78 years who had a history of
diabetes mellitus (Papanicolaou stain, original magnification ×20). (B) Vitreous fluid with pigment-laden macrophages in retinal
toxoplasmosis is shown from a woman aged 35 years (Papanicolaou stain, original magnification ×20). The inset shows positive red
staining for toxoplasmosis in macrophages (immunohistochemistry for toxoplasmosis; original magnification ×40).

cell block. The samples can be stained with Pap and other
stains (Grocott’s methenamine silver stain, etc).1,3,32 The
most common differential diagnoses in both ocular an-
terior and posterior (vitreous) chamber cytology samples
include infections, inflammatory conditions, and lym-
phoma (Table 1).
findings include acute and chronic inflammatory cells
with groups of macrophages (Fig. 8B). However, typi-
cal cytopathic changes of specific infections are usually
absent. Therefore, the cytology findings coupled with
PCR or culture studies are needed to render useful clini-
cal diagnostic interpretations.33

INTRAOCULAR INFECTIONS Uveitis

Undiluted fresh-fluid cytology samples (0.5-1.0 mL), in
ice or refrigerated, can be submitted to rule out HSV,
Herpes zoster virus, Toxoplasmosis species, Tuberculosis
(TB complex), and Cytomegalovirus by PCR analysis. If
Toxocara canis infection is suspected, antibody titers can
be drawn.1,3,32 Bacterial, fungal, and viral cultures can be
performed, depending on the available fluid. If the sam-
ple is small, PCR is the best option to rule out infections.
Cytology smears prepared from these fluids can show
typical hyphae in a fungus infection (Fig. 8A); in other
infections (eg, viral, toxoplasma, tuberculosis), cytology
Uveitis refers to inflammation of the iris (anterior uvei-
tis), ciliary body (intermediate uveitis), choroid (poste-
rior uveitis), or all layers of the uvea (panuveitis). It may
be seen in association with autoimmune diseases or in
otherwise healthy individuals. Uveitis is not limited to
the uvea and may affect other ocular structures, includ-
ing the lens, retina, optic nerve, and vitreous, resulting
in reduced vision or blindness. People of all ages, but
especially those between ages 20 and 60 years, can be
affected.34,35 Uveitis can have a short, acute course or
may become chronic with recurrences. Patients present

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Figure 9. A liquid-based vitreous sample is shown from a
woman aged 71 years with chronic uveitis. The sample reveals
mixed chronic inflammation, including lymphocytes and
macrophages (Papanicolaou stain, original magnification ×60).
with ocular pain, eye redness, floaters, light sensitiv-
ity, or blurred vision. Laboratory testing is performed
to identify causes of uveitis, which include infectious
organisms, autoimmune diseases, and, in some cases,
lymphoma.
Undiluted fresh anterior chamber or vitreous sam-
ples can be used for PCR analysis of heavy-chain gene
rearrangement and T-cell receptor rearrangement. In ad-
dition, a cytokine assay for interleukin 6 (IL-6) and IL-10
can also be performed. An undiluted sample ≥0.5 mL
is needed for PCR analysis, and a volume of 2.0 mL is
needed for an interleukin assay. If an undiluted sample
is insufficient in volume, the sample may be diluted with
balanced salt solution to reach a volume from 2.5 to 3.0
mL to triage for the different studies needed.32 In chronic
uveitis, cytology smears from the anterior and posterior
chamber fluids show chronic inflammatory cells, includ-
ing lymphocytes, plasma cells, and macrophages (Fig. 9).
Increased levels of IL-6, IFN-γ, and TNF-α have been
observed in idiopathic uveitis, Behcet disease, and anky-
losing spondylitis. IL-6/IL-10 ratios >1.0 show high sen-
sitivity (93%) and specificity (100%) in the diagnosis of
vitreous lymphoma.36
Vitreoretinal Lymphoma
The vitreous can be the site of primary involvement in
cases of B-cell lymphoma, a rare but potentially fatal
eye condition for which early diagnosis is crucial to
Cancer Cytopathology  June 2021
ensure appropriate treatment and improvement of an
unfavorable prognosis. The great majority of cases are
CD20-positive B-cell lymphomas, but rare cases of
T-cell lymphomas have been reported. Up to 50% of
patients who present with ocular findings have concom-
itant involvement of the central nervous system.37 The
disease has an insidious course, most commonly pre-
senting as posterior uveitis, making an accurate clinical
diagnosis challenging. The cells are usually spilled over
from neoplastic infiltrates in the retina and subretinal
pigment epithelium. The main differential diagnosis
includes reactive lymphocytic infiltration of the uveal
tract (chronic uveitis), sarcoidosis, sympathetic uveitis,
and Vogt-Koyanagi-Harada disease. These conditions
are ruled out by clinical tests and diagnostic biopsy
samples.36,38 Undiluted, refrigerated anterior chamber
or vitreous fluids can be tested to rule out lymphoma
(sample volume, 0.5-1.0 mL). Flow cytometry may be
done on vitrectomy samples; however, it has limited ap-
plication because of scant cellularity. IL-6 and IL-10
levels can also be tested. IL-10 cutoff values of 65 pi-
cograms per milliliter (pg/mL) in the vitreous chamber
and 30 pg/mL in the anterior chamber are associated
with high sensitivity (93% and 78%, respectively) and
specificity (100% and 97%, respectively) in diagnosing
intraocular lymphoma.36 PCR analysis of heavy-chain
gene rearrangement and T-cell receptor rearrangement
can be performed, but the test depends on the available
DNA present in the sample. Recent studies have shown
the utility of testing for a hotspot mutation of the mye-
loid differentiation primary response gene 88 (MYD88)
in the anterior chamber fluids and vitreous samples.
This mutation is present in approximately 75% of pa-
tients with lymphoma by PCR analysis and is absent
in nonneoplastic proliferations. This technique is very
useful in the diagnosis of lymphoma in ocular cytol-
ogy fluids with low cellularity or with cell lysis. In this
mutation, there is a change in the amino acid leucine to
proline at position 265 (L265P).38 Clinically suspected
vitreoretinal lymphoma that exhibits cytology features
suspicious for lymphoproliferative disorder, coupled
with an abnormal flow cytometry result, a heavy-chain
gene immunoglobulin rearrangement, or an MYD88
L265P mutation, can be diagnosed with vitreoretinal
lymphoma and receive prompt chemotherapy treat-
ment.38 Cytology preparations reveal a mixed popula-
tion of cells that have atypical, large lymphoid cells with
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Review Article
Figure 10.  A liquid-based vitreous sample is shown from a man
aged 55 years who had new floaters and vision changes. The
smears reveal malignant, large lymphoid cells with convoluted
nuclear membranes, multiple conspicuous nucleoli, and
plasmacytoid scant cytoplasm (Papanicolaou stain, original
magnification ×60).
convoluted nuclear membranes, multiple conspicuous
nucleoli, and plasmacytoid-scant cytoplasms (Fig. 10).
An accompanying infiltrate of small, reactive T lym-
phocytes is usually present. If enough tissue is avail-
able for a cell block preparation, then monoclonality by
immunohistochemistry stains can be demonstrated.32
INTRAOCULAR FINE-NEEDLE
ASPIRATION BIOPSIES
Malignant uveal melanoma is the most common in-
traocular tumor of adults and has a strong propensity
for fatal metastasis.39,40 Patients with uveal melanoma
may present with complaints of visual loss, but many
are without symptoms and are discovered on routine
ocular examination. In eyes with clear media, the di-
agnosis of posterior uveal melanoma can be made by
indirect ophthalmoscopy. Ultrasonography is the most
accurate method of substantiating the diagnosis and de-
termining tumor size; serial fundus color photographs
and fluorescein angiography aid in determining the
greatest tumor dimension.40,41 Most malignant mela-
nomas are diagnosed clinically and treated with radia-
tion therapy; large tumors are treated by enucleation.
Tumor location, size, extraocular tumor extension,
presence of ocular/oculodermal melanocytosis, his-
topathologic features, cytogenetic abnormalities, and
tumor profiling are all used in determining the risk of
428
death from metastatic disease of choroidal melanocytic
lesions. Tumor thickness >2.0 mm, subretinal fluid,
ocular symptoms, orange pigment overlying the tumor,
margin of the tumor near the optic nerve disc, ultra-
sonographic hollowness, and absence of halo or drusen
are features used in the clinic to differentiate a nevus
from uveal melanoma.39,41 There are clinical settings in
which the clinical distinction between a melanoma and
a simulating lesion, including a choroidal nevus, reti-
nal hamartoma, retinal pigment epithelial adenoma,
or carcinoma, is uncertain, and a fine-needle biopsy
can be used in the diagnosis.1,42 The great majority of
cytology samples are submitted in cases of uveal mela-
noma to confirm the diagnosis, to perform molecular
studies, or to rule out uveal nevi and other potential le-
sions involving the uvea and retina (Table 1). Multiple
direct smears can be prepared by using both air-dried
and alcohol-fixed methods. The remainder of the spec-
imen can be submitted for a cytospin, liquid-based
cytology, and a cell block preparation, depending on
the volume submitted. Both SurePath and ThinPrep
liquid-based methods can be used. Slides can be used
for cytogenetics testing for loss of chromosome 3 (or
monosomy 3), gain on 6p, loss on 6q, and gain on
8q. Loss of an entire chromosome 3 (or monosomy 3),
which is an early event in tumorigenesis, is detected
in approximately 50% of tumors. The presence of
monosomy 3 may be determined by karyotyping using
fluorescence in situ hybridization on cultured cells or
by fluorescence in situ hybridization analysis of tissue
sections or cells obtained by fine-needle aspiration bi-
opsy. Long-term studies have demonstrated that almost
70% of patients who have monosomy 3 in the primary
tumor have died of metastases within 4 years after the
initial diagnosis, whereas tumors with normal chromo-
some 3 status rarely give rise to metastatic disease.43,44
Gene expression profiling can be done to classify ma-
lignant melanomas using undiluted fresh-tumor fine-
needle aspiration samples placed in a special fixative
provided by a collection kit (DecisionDx-UM; Castle
Biosciences Incorporated). The gene expression profile
test has been refined to a 15-gene assay performed on
a microfluidics PCR platform that allows accurate and
reproducible molecular classification from minute sam-
ples obtained from small-gauge needle biopsy.45,46 The
development of this new molecular classification of
Cancer Cytopathology  June 2021
 Ocular Cytology: Diagnostic Features and Best Practices/Laver

uveal melanoma allows patients at high risk of metasta-

sis to be identified early in the disease process. Class 1
tumors have a low risk, and class 2 tumors have a high
risk of metastasis. Class 1 tumors often show 6p and
8q gain; and class 2 tumors are strongly associated with
inactivating mutations of BRCA1-associated protein-1
(BAP1), located at 3p21.46 BAP1 mutations occur
almost exclusively in class 2 tumors and are strongly
associated with metastasis, a phenotype that presum-
ably arises later in tumor evolution. GNAQ (guanine
nucleotide-binding protein G subunit) encodes the α
subunit (Gαq), and GNA11 encodes the α subunit 11
(Gα11); both are proteins of importance in transmem-
brane signaling systems. Mutations in GNAQ/GNA11
were the first described as driver mutations in uveal
melanoma and found to upregulate the MAPK (mito-
gen-activated protein kinase) pathway. Approximately
85% of all uveal melanomas carry a mutation in either
GNAQ or GNA11. These mutations are mutually ex-
clusive and appear to represent early or initiating events
found in premalignant nevi and in uveal melanoma of
all stages; they have not been associated with the 2 dif-
ferent molecular classes of uveal melanoma nor have
they demonstrated prognostic value.47-50 In contrast,
mutations in SF3B1, located at chromosome 2 (splic-
ing factor 3b subunit 1), occur mostly in class 1 tu-
mors and are largely mutually exclusive with the BAP1 Figure 11.  (A) A direct smear from a fine-needle aspiration of a
mutations seen in class 2 tumors. Similarly, EIF1AX choroidal lesion in a man aged 44 years is shown. The smears
reveal a population of spindled and epithelioid melanoma cells
(eukaryotic translation initiation factor 1A, X-linked) (H&E stain, original magnification ×40). (B) A direct smear
mutations appear to occur primarily in class 1 tumors of a fine-needle aspiration choroidal lesion in a woman aged
61 years is shown. The smears reveal spindle and epithelioid
in a mutually exclusive fashion with SF3B1 and BAP1 melanoma cells with pigment deposits (H&E stain, original
mutations.48-50 magnification ×60).

The cytology of uveal melanoma reveals atypical

melanocytes observed in clusters and singly with spin-
dle cell and/or epithelioid cell features (Fig. 11A,B).
Epithelioid melanoma cells have abundant cytoplasm,
large vesicular nuclei, and prominent, centrally located
nucleoli.
The application of advanced molecular biology
and genetic techniques has revolutionized uveal ma-
lignant melanoma classification and clinical manage-
ment. These highly accurate clinical prognostic testing
techniques allow high-risk patients to be identified and
offered more intensive metastatic surveillance. Despite
successful local tumor control, up to 50% of patients
develop metastatic disease within 15 years of diagnosis;
currently, there is no effective form of chemotherapy or
immunotherapy for metastatic disease.39,40,42 However,
there are new therapeutic trials underway that may offer
more options to patients in the future.46,48,50
Conclusion
Ocular cytology plays a very important role in the diagno-
sis of ocular diseases, tumor classification, and the clinical
management and treatment of patients. The field of ocu-
lar cytology is in continuous evolution as new molecular
diagnostic tests are developed. To render useful cytology
diagnoses, general cytopathologists need to keep abreast
of advancements in clinical methods in the diagnosis of
ocular diseases.

Cancer Cytopathology  June 2021 429

Review Article
FUNDING SUPPORT
No specific funding was disclosed.
CONFLICT OF INTEREST DISCLOSURES
The author made no disclosures.
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