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Clinical Practice

JOHN E. SUTPHIN, MD, SECTION EDITOR

Impression Cytology: Recent Advances and


Applications in Dry Eye Disease
ELI LOPIN, MD, TATIANA DEVENEY, BA, AND PENNY A. ASBELL, MD, FACS, MBA

ABSTRACT Impression cytology (IC) allows cells to be


harvested from the ocular surface noninvasively. Supercial
layers of the epithelium are removed by application of cellulose
acetate lters or Biopore membranes, and the cells can be
subsequently analyzed by various methods, depending on the
objective of the investigation or pathology involved. IC techniques are easily learned, can be performed in an outpatient
setting, and cause virtually no discomfort to the patient. IC
facilitates the diagnosis of ocular surface disorders, including, among others, keratoconjunctivitis sicca, ocular surface
squamous neoplasia, and ocular surface infections. During the
past decade, IC has been used increasingly to assist in diagnosis of ocular surface disease, improve our understanding
of the pathophysiology of ocular surface disease, and provide
biomarkers to be used as outcome measures in clinical trials.
Dry eye disease is one area in which IC has contributed to
signicant advances.
KEY WORDS clinical trials, dry eye disease, ow
cytometry, goblet cell density, impression cytology,
keratoconjunctivitis sicca, squamous metaplasia

Accepted for publication February 2009.


From the Department of Ophthalmology, Mount Sinai School of Medicine,
New York, NY, USA.
Supported in part by NEI EY17626 and the Martin and Toni Sosnoff
Fund.
The authors have no commercial or proprietary interest in any product or
concept discussed in this article.
Single copy reprint requests to: Penny A. Asbell, MD, FACS, MBA (address
below).
Corresponding author: Penny A. Asbell, MD, FACS, MBA, Professor of
Ophthalmology, Director of Cornea and Refractive Services, Department
of Ophthalmology, Mount Sinai School of Medicine, One Gustave L. Levy
Place, New York, New York 10029. Tel: 212-241-7977. Fax: 212-241-4550.
E-mail: penny.asbell@mssm.edu.

2009 Ethis Communications, Inc. The Ocular Surface ISSN: 15420124. Lopin E, Deveney T, Asbell PA. Impression cytology: recent
advances and applications in dry eye disease. 2009;7(2):93-110.

I. INTRODUCTION
n 1954,Larmande and Tismit in France reported
using impression cytology (IC) to diagnose ocular
surface squamous neoplasia.1 References to IC in
the English-language literature did not appear until 1977.
At that time, Thatcher et al described an improvement in
ocular surface cell harvesting using a plastic disc, which was
considerably more comfortable for patients than techniques
that relied on scraping or swabbing with cotton.2 Also in
1977, Egbert et al documented the use of IC with absorbent
lter paper to harvest cells in essentially the same manner
that is still used today.3
Three review articles on impression cytology of the ocular surface have been published in recent years. McKelvie
(2003) addressed the technical aspects of IC, especially in
reference to the user-friendly Biopore membrane device. She
additionally addressed the application of IC in diagnosing
ocular surface squamous neoplasia.4 Calonge et al (2004)
provided a historical review of IC technique, showing it to
be a useful diagnostic aid for a wide variety of processes
involving the ocular surface, while being minimally invasive.5 Singh et al (2005) also underscored the ability of IC
to diagnose a wide range of ocular surface disorders and
also stressed the importance of cell harvesting technique, as
the number of cells obtained varies considerably depending
on the methodology used.6
Although this review article incorporates relevant data
from the three earlier reviews, its main purpose is to describe recent advances in knowledge obtained through the
use of IC. It specically addresses the use of IC as applied
to dry eye disease.
A PubMed search was conducted using the terms impression cytology and dry eye and impression cytology and keratoconjunctivitis sicca. Of the articles retrieved by this method,
we reviewed all publications in English, and we reviewed the
English abstracts of non-English publications. We included
articles that described developments in IC harvesting or processing techniques in addition to the vast number of articles
devoted to dry eye disease in the context of IC use. Emphasis
was placed on articles published since the review by Calonge
et al,5 but we included earlier articles that provided a more
comprehensive understanding of both dry eye disease and IC.

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IMPRESSION CYTOLOGY / Lopin, et al

OUTLINE
I. Introduction
II. Impression cytology techniques
A. Cell harvesting
B. Cell processing
C. Flow cytometry and immunocytochemistry
III. Application of IC to investigation of dry eye disease
A. Investigating the pathology of dry eye
1. Evaluation of inflammation
a. Cytokines/chemokines
b. Defensins
c. Inflammatory markers
1) HLA-DR
2) CD40
d. Summary of data on inflammation
2. Oxidative reactions
3. Snake-like chromatin
4. Ocular mucins
5. Growth factor receptors
6. Microvilli
B. Monitoring of clinical trials
1. Cyclosporine
2. Autologous serum
3. Umbilical cord serum
4. Artificial tears
a. Preservatives
b. Sodium hyaluronate
c. Carboxymethylcellulose (CMC)
5. Antioxidants
6. Essential fatty acids
7. Nerve growth factor
8. Steroids
9. Vitamin A
10. Botulinum toxin
11. Non-pharmacological treatments
C. Characterization of animal models
D. Association of dry eye disease with other conditions
IV. Summary and conclusion

II. IMPRESSION CYTOLOGY TECHNIQUES


A. Cell Harvesting
Impression cytology, as described by Egbert et al in 1977,
relied on the use of cellulose acetate lters to harvest ocular
surface cells.3 This method provided signicant advantages
over the then more prevalent techniques of conjunctival
smears or excisional biopsies. Morphological detail of the
cell was far more difcult to preserve with smears, and only
a limited number of cells were extractable with excisional
biopsy. In contrast, impression cytology was able to preserve
morphology, and it allowed a much broader sampling of
the ocular surface.7
However, IC still had some signicant limitations. Until
the mid-1990s, cell surface antigens could not be analyzed
94

by IC because the cellulose acetate lters were incompatible


with the lter preparation materials; thus, the technique
could not be used to detect antibody-antigen interactions or
other cell surface markers. Moreover, the cellulose acetate
lters had to be placed in specialized sample containers for
transport to the laboratory for testing. This rather inefcient
requirement dissuaded many ophthalmologists from utilizing IC as a conventional diagnostic tool in the outpatient
setting or as part of a clinical trial.7
By the late 1990s, the use of Biopore membranes for
IC circumvented many of the limitations of cellulose acetate lters. In the clinicians ofce, topical anesthetic eye
drops were applied to achieve a localized anesthetic effect,
allowing the small Biopore membrane device (approximately inch in diameter [Millicell-CM 0.4 PICM 012550,
Millipore Corp., Bedford, MA]) to be gently pressed on the
ocular surface for 3-5 seconds for a rather efcient cellular
yield (Figure 1).7,8
Brush cytology is an alternative method for harvesting
ocular surface cells. Yagmur et al described a disposable
brush similar to the brushes used to obtain cervical smears.9
Anesthetic is applied prior to careful scraping of the ocular
surface. Yagmur et al compared brush to impression cytology and found in a study of 63 patients that brush cytology
was superior with respect to quantity and quality of cells
harvested, ease of staining techniques, and cost. Ersz et
al, in their studies of ocular surface squamous neoplasia,
reported that brush cytology better preserved cellular morphology, the smears were easy to prepare, and slides maintained good quality even after several years.10 In contrast,
IC slides took longer to prepare and were more prone to
develop artifacts that obscured the cytologic view.
B. Cell Processing
Since the advent of IC, many processing methods have
been used to analyze the harvested ocular surface cells
(Table 1). By far the most prevalent method remains light
microscopy, with which epithelial and goblet cells can be
easily visualized through hematoxylin and periodic acid

Figure 1. Impression cytology sampling using the Biopore membrane


device. (Reprinted with permission from McKelvie PA, Daniell M, McNab
A, et al. Squamous cell carcinoma of the conjunctiva: a series of 26
cases. Br J Ophthalmol 2002;86:168-73.)

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IMPRESSION CYTOLOGY / Lopin, et al

Table 1. Techniques for analyzing impression cytology specimens


IC analysis technique

Advantages

Light microscopy

Easy slide preparation.


Epithelial/goblet cells well visualized enabling use as good screening tool.

Electron microscopy

Permits visualization of cell ultra-structures; important for disease types such as MPS.

Immunohistochemistry

Can detect sub-clinical inflammation of ocular surface.

Flow cytometry

Can also detect sub-clinical inflammation, but methodology is standardized and not userdependent.

RT-PCR/PCR

Allows examination of survival of donor human limbal stem cells in recipient with limbal cell
deficiency.
Permits identification of ocular surface genes such as anti-bacterial peptide defensin genes
and antioxidant enzyme genes.

Schiff (PAS) staining, respectively. To assess the progression and severity of ocular surface disease based on light
microscopy ndings, various grading systems have been
employed, including systems developed by Tseng in 1985,11
Nelson in 1988,12 and Adams et al in 1988 (Table 2).13 All
three grading systems utilize criteria to identify degree of
pathology, such as change in cell morphology (squamous
metaplasia), reduction in number or activity of goblet cells,
and the presence or lack of inammatory cells. These criteria
are nonspecic and can apply to a broad spectrum of ocular
surface pathology. However, light microscopy functions

extremely well as a screening tool, thus allowing further


workup to be implemented as needed.
Electron microscopys (EM) exponentially more powerful ability to magnify allows visualization of cell ultrastructures.14 This is especially signicant for diseases such as
mucopolysaccharidoses (MPS), which are best diagnosed
and evaluated at the subcellular level. Pastor et al used EM
to demonstrate that HIV viral particles were actively budding from the plasma membrane of conjunctival cells from
several AIDS patients with cytomegalovirus retinitis, which
implied that these particles were residing in the conjuncti-

Table 2. Goblet and non-goblet epithelial cell characteristics of three main grading systems11-13
Classication

Goblet cells

Non-goblet epithelial cells

N:C ratio

Grade 0

Moderate density

Uniform size/form

1:1

Grade 1

Decreased density

Mild enlargement

1:21:3

Grade 2

Absent

Moderate enlargement,
flattened (squamoid)

1:4

Grade 3

Absent

Markedly squamoid

1:6

Grade 4

Absent

Markedly squamoid, large

1:8

Grade 5

Absent

Shrunken cytoplasm

Grade 0

Plump/oval, abundant

Small, round

1:2

Grade 1

Plump/oval, decreased number

Slightly larger, more polygonal

1:3

Grade 2

Smaller, poorly defined border,


markedly decreased number

Larger, polygonal

1:41:5

Grade 3

Very few

Large, polygonal

> 1:6

Grade 0

Abundant

Normal

1:2

Grade 1

Slightly decreased number

Larger

1:3

Grade 2

Decreased number

Larger

NS

Grade 3

Very decreased number

Large, irregular

NS

Tseng11

Nucleus may be absent

Nelson12

Adams13

N:C = Nucleus:cytoplasm.

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IMPRESSION CYTOLOGY / Lopin, et al

val epithelium.15 This raised the question of how the virus


gained access to the conjunctiva, and thus carries broader
implications for the pathophysiology of HIV.
C. Flow Cytometry and Immunocytochemistry
Although ow cytometry has been used for several
decades, especially in the elds of hematology and immunology, it was rst applied to analysis of conjunctival
IC specimens by Baudouin et al in 1997.16 Until that time,
immunocytological staining was the most sensitive method
to detect subclinical inammation. While light microscopy
could detect absence of goblet cells and metaplastic cellular
changes, immunouorescent staining could demonstrate
the presence of HLA-DR and CD23, two inammatory
markers. The very same IC specimen could provide information on cell type and immune markers.17 However,
immunocytochemistry had signicant limitations. Biopore
membranes were difcult to use for cell collection and immunochemistry. More importantly, appreciation of both
percentage of positive cells and intensity of immunostaining was largely observer-dependent. Flow cytometry, on
the other hand, was a more objective and standardized
technique that could be performed in different laboratories
with little concern that results would be subject to operator-dependent interpretation.18
The ow cytometry apparatus consists of a uidics
system chamber that allows suspended particles in solution to pass through a progressively narrowing chamber
that enables cells to form single le formations (Figure 2).
Each cell passes through at least one beam of light, which
causes the light to scatter. A lens called a forward scatter
detector is able to determine the size of the cell, while a
side-scattered detector can assess intracellular density. Additionally, cellular surface antibodies can be labeled with
a uorescent dye, and as those cells pass through the light
beam, an excitation light of a specic wave length or color
is emitted and detected. Histograms can plot the number of
cells expressing this wavelength, revealing the percentage
of cells that contain the antibody in question. This allows
for a very sensitive, rapid, and objective tool to investigate
ocular surface pathology.19
The use of reverse transcriptase polymerase chain reaction (RT-PCR) involves the isolation of mRNA from IC
specimens. Complementary DNA (cDNA) is then made
from the mRNA specimen, using deoxyribonucleotide
monomers and reverse transcriptase. Standard PCR is
subsequently initiated, allowing exponential amplication
of DNA polymers. Jones et al used this method to identify
inammatory cytokines that contribute to ocular surface
changes associated with primary Sjogren syndrome. 20
Daya et al used PCR and IC to investigate the DNA genotype of the limbal epithelium belonging to the recipients
eyes following ex vivo expanded stem cell allograft transplantation.21 They found that nine months post-allograft
transplant, there was a complete absence of donor DNA,
which has broad implications for post-transplant therapy.
IC samples that are then processed by RT-PCR can reveal
96

Figure 2. The flow cytometry apparatus consists of a fluidics system


chamber that allows suspended particles in solution to pass through
a progressively narrowing chamber that enables cells to form single
file formations.

information about specic gene activation and can be used


to identify cell source, as in post-surgical manipulation of
the ocular surface.
III. APPLICATION OF IC TO STUDY OF
DRY EYE DISEASE
IC has been useful in the investigation of many aspects
of dry eye disease, including 1) pathophysiology of dry eye
disease, 2) monitoring of clinical trials to evaluate efcacy
of various dry eye disease treatments, 3) characterization
of animal models of dry eye disease, and 4) associating dry
eye disease with other systemic conditions.
A. Investigating the Pathology of Dry Eye Disease

1. Evaluation of Inammation
Although the exact cause of dry eye disease is not
known, increasing evidence suggests that inammation of
the ocular surface is a signicant component and leads to
the observed signs and symptoms of dry eye disease. The
DEWS report highlights some of the recent research relating
to inammation in dry eye disease.22
a. Cytokines/Chemokines
Using IC samples combined with different analysis
methodologies has been helpful in analyzing different
immune biomarkers related to dry eye disease. To evaluate cytokines and chemokines, researchers have used IC
samples combined with immunohistochemical and immunouorescent staining RT-PCR to determine cytokine
gene transcription, and ow cytometry.
To evaluate the role of cytokines and chemokines in
dry eye disease, IC samples have been analyzed by various
techniques for markers of inammation. Yoon et al utilized
immunohistochemical staining of IC samples incubated with
anti-IL-6 and TNF- antibodies from patients with dry eye
disease (with and without Sjogren syndrome) to demonstrate
level and expression of pro-inammatory cytokines IL6 and

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IMPRESSION CYTOLOGY / Lopin, et al

TNF-. IL-6 staining was strongest in samples from patients


with Sjogren syndrome, less in patients with non-Sjogren
dry eye disease, and was barely detected in normal control
samples. IL-6 levels in tears from dry eye patients signicantly
correlated with conjunctival goblet cell density, as measured
by IC. TNF- was not detected in any of the groups.23
Cejkova et al also used immunohistochemical staining to
demonstrate increased expression of inammatory cytokines
IL-1beta, IL-6, IL-8 and TNF- in association with increased
dry eye severity.24
Solomon et al, using immunouorescent staining of IC
samples from patients with aqueous tear-decient Sjogren
syndrome compared to normal controls, demonstrated
increased expression of IL-1alpha, mature IL-1beta, and
IL-1Ra, the last being a natural antagonist for IL-1. This
suggests that the balance of IL-1 and its antagonist may have
a role in controlling inammation of the ocular surface.25
RT-PCR applied to IC samples is a newer technique to
determine mRNA expression and evaluate the pathway
of cytokine production. It has been used to demonstrate
increased levels of inammatory cytokines IL-6, IL-8, IL1alpha, transforming growth factor beta1 (TGF-beta1),
and TNF- associated with increasing dry eye severity.26
Narayanan et al used RT-PCR to analyze IC samples from 10
subjects (5 with moderate dry eye and 5 healthy controls).
However, they found no difference in cytokine expression
between the moderate dry eye group and the control group.
Tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL) was constitutively expressed, whereas IL-1beta,
IL6 and GRO-beta were not present and intracellular adhesion molecule 1 (ICAM-1) was only weakly expressed.27
Flow cytometry is another, newer technique that has
been extensively used to analyze IC samples objectively.
Flow cytometry analysis of IC samples from 17 patients
with aqueous-decient keratoconjunctivitis sicca (KCS)
demonstrated a high percentage of cells expressing the
chemokine receptor CCR5 as compared to normal controls. Interestingly, chemokine receptor CCR4 expression
was not elevated. These results support the idea that KCS
is a Th1-mediated disease, as CCR5 is associated with Th1
immune pathways, whereas CCR4 is associated with Th2
immune pathways.28
Two-color ow cytometry is a newer modication to the
ow cytometry technique that uses double-immunostaining to analyze two cell surface markers on the same cell
simultaneously. Two-color ow cytometry revealed CCR5
expression by both CD45-positive cells (bone-marrow derived) and a larger group of CD45-negative (resident) cells
in KCS patients.29 Two-color ow cytometry analysis of IC
was also used by Gulati et al to further support this nding.
They also demonstrated an increased percentage of CCR5
chemokine receptor-positive resident conjunctival epithelial
cells (as opposed to bone marrow-derived CD45-positive
cells) in dry eye patients versus normal controls.30
These research efforts utilizing IC have suggested
through several studies that IL-6 is an important cytokine
in dry eye disease. Other work has pointed to an important

role of IL-1 and its antagonist in controlling inammation. RT-PCR has demonstrated increased transcription of
cytokines in dry eye disease, although some researchers
have not demonstrated such changes. IC combined with
ow cytometry has led to increased understanding of the
pathophysiology of inammation, suggesting that dry eye
disease is a Th1- mediated disease and that it primarily involves the response of resident conjunctival epithelial cells,
as opposed to inammatory cells coming from elsewhere
in the body.

b. Defensins
Human beta-defensins (hBD) are antimicrobial peptides,
but they also have an immune modulator role, inuencing
cytokine production and chemotaxis, for example. Some
defensins, such as hBD-2, are inducible by pro-inammatory cytokines.31 Using RT-PCR analysis of IC samples,
Narayanan et al showed that hBD-2 mRNA was expressed
only in conjunctival epithelial cells from patients with
moderate dry eye disease and that its up-regulation could
be induced by pro-inammatory cytokines (IL-1beta or
TNF-) in cultured conjunctival cells. They hypothesized
that this up-regulation compensates for the compromised
ocular surface in dry eye disease, providing additional defenses, but may also contribute to ocular surface damage.31
Further work by the same group utilizing IC samples from
dry eye disease patients did not demonstrate upregulation
of IL-1beta, suggesting that this cytokine is not responsible
for the up-regulation of hBD-2 in patients with moderate
dry eye disease. This work suggests that other cytokine
signaling pathways may be involved in the up-regulation
of hBD-2.27
Use of RT-PCR analysis of IC samples to explore defensins has demonstrated that in some inammatory processes,
there is reduced expression of specic defensins. Specically,
the beta-defensin DEFB-109 gene associated with antimicrobial peptides (AMPs) was reduced in all samples from
patients with ocular surface inammation and infection.
The least reduction was found in patients with dry eyes as
compared to normal controls.32
Use of IC to study defensins has demonstrated that
in dry eye disease up- and down-regulation of immune
modulators occurs.
c. Inammatory Markers
1) HLA-DR
Flow cytometry has expanded the opportunities to discover more information from IC samples and has provided
an objective metric to evaluate ocular surface changes.17 For
dry eye disease, evaluation of HLA-DR has been specically
explored using ow cytometry to gain a more complete
picture of the ocular surface inammation that is associated
with the disease.
Major histocompatibility complex (MHC) class II
molecule HLA-DR is a glycoprotein that is normally expressed on immune cells, such as B lymphocytes, but whose
expression has also been described on some nonimmune

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IMPRESSION CYTOLOGY / Lopin, et al

epithelial cells.17 Initially, abnormal HLA-DR expression in


samples from dry eye patients was demonstrated through
immunohistochemistry staining, but ow cytometry analysis of IC has allowed for a more objective metric on the
number of HLA-DR-positive cells and expression of HLADR. Baudouin et al conrmed the immunouorescence
ndings, using ow cytometry, and found an increase in
percent positive HLA-DR cells in patients with dry eye disease (although the range varied from 20% to 98% of cells)
compared to normal controls, in which fewer than 10% of
cells were HLA-DR- positive. They also found a positive
correlation between HLA-DR immunolabeling of IC samples
and ow cytometry results, conrming the validity of ow
cytometry results in comparison with more traditional immunhistochemistry.16
Baseline data from the cyclosporine trial (Section B.1,
below) showed that the percentage of positive HLA-DRpositive cells and HLA-DR expression in patients with Sjogren syndrome dry eye was higher than that in non-Sjogren
syndrome dry eye patients, although both groups had a signicantly higher percentage compared to normal controls.33
More recently, increased expression of HLA-DR, as
measured by ow cytometry analysis of IC samples, was
positively associated with diagnostic tests for dry eye, conrming the usefulness of HLA-DR expression as a measurement for monitoring changes in the inammatory state of
the ocular surface in dry eye disease.34 These results have
been conrmed in other studies as well.28,30,35

2) CD40
CD40 is a cell surface receptor whose up-regulation
has been observed in a variety of inammatory conditions.
This receptor belongs to the tumor necrosis factor receptor
superfamily. Using ow cytometry analysis of IC samples
from KCS, Bourcier et al demonstrated increased CD40
expression compared to normal controls and showed that
this was positively correlated with HLA-DR expression.35
d. Summary of Data on Inammation
Using IC in conjunction with a variety of analytical
techniques, researchers have been able to demonstrate
inammation associated with dry eye disease, providing
not only more insight into the mechanism and pathogenesis of dry eye disease, but providing the rationale and
inspiration for new anti-inammatory treatments for dry
eye disease.
2. Oxidative Reactions
IC sampling has been used to elucidate the mechanism
of action that results in ocular surface abnormalities associated with dry eye disease. Studies of reactive oxygen species
suggest that they may be an important factor contributing to
inammation and ocular surface damage and provide the rationale for the use of antioxidants to treat dry eye disease.36
Cejkova et al analyzed xanthine oxidoreductase/xanthine oxidasean enzymatic system responsible for the
generation of reactive oxygen speciesby histochemistry
98

and immunohistochemistry of IC samples from patients


with Sjogren syndrome. Activity and expression of these
enzymes was higher in patients with Sjogren syndrome
than in normal controls, suggesting that the reactive oxygen
species that are generated by this enzyme system contribute
to the damaging oxidative reactions associated with autoimmune diseases.36
In another study by Cejkova et al, immunohistochemistry processing of IC samples from dry eye Sjogren syndrome
patients demonstrated that increased endothelial nitric
oxide synthase (NOS3) and inducible nitric oxide synthase
(NOS2) were highly expressed in patients with dry eye;
expression increased with the degree of dry eye severity
and the immunodetection of pro-inammatory cytokines
(IL-1beta, IL6, IL8, TNF-). The authors suggest that NOS
expression may be involved in dry eye injury through the
formation of peroxynitrite (also found to be present in the
conjunctival epithelium of dry eye patients), an oxidizing
and nitrating agent that can be produced through the action
of the xanthine oxidoreductase enzyme.24
Since oxidative reactions may be important in generation of ocular surface damage in dry eye disease, the
presence, or decreased expression, of antioxidant enzymes
has also been evaluated. (See section B.5 for a discussion
of antioxidant treatment for dry eye disease.) Immunohistochemistry analysis of IC samples demonstrated that the
expression of antioxidant enzymes (superoxide dismutase,
glutathione peroxidase, catalase) was much less in patients
with dry eye disease versus normal controls in correlation
with increasing dry eye severity (although expression of
superoxide dismutase was reduced below normal only in
severe dry eye cases). This adds credence to the hypothesis that control and balance of oxidative reactions on the
ocular surface may be an important contributor to ocular
surface disease specically associated with dry eye disease
and Sjogren syndrome.37

3. Snake-like Chromatin
Snake-like chromatin (SLC) is an unusual arrangement
of chromatin found in the cell nucleus (Figure 3).38 It is
associated with certain pathological conditions, such as inammation. The presence of SLC in conjunctival cells from
patients with dry eye disease has been extensively noted,
most recently by Jirosova et al.39 In another paper, Jirsova
et al used light microscopy of IC samples to demonstrate
the presence of micronuclei associated with SLC-positive
cells and conrmed the correlation between increased
SLC-positive cell numbers, decreased goblet cell density,
and impaired clinical dry eye parameters.40
4. Ocular Mucins
Mucins are glycoproteins and are a major constituent
of the mucous layer that is adjacent to the surface cells of
the ocular surface epithelium. The mucous layer of the tear
lm is generated by both goblet cells and apical cells of the
cornea and conjunctiva.41 IC has aided us in understanding
the complexity of the role of mucins on the ocular surface.

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IMPRESSION CYTOLOGY / Lopin, et al

Figure 3. Snake-like chromatin: nucleus with a moderate degree of


central chromatin on TEM. This micrograph displays the alteration of
the fibrous lamina, the detachment of the condensed chromatin from
the nuclear periphery, and its strands (arrowheads) confluent centrally
into the snake. Note the multitude of cytoplasmic filaments, indicating
the squamous metaplastic nature of the cell. (Reprinted with permission from Knop E, Reale E. Fine structure and significance of snake-like
chromatin in conjunctival epithelial cells. Invest Ophthalmol Vis Sci
1994;35:711-9.)

Although initial work concentrated on the density of goblet


cells, recent ndings have suggested that goblet cell density
is not associated with tear lm break-up time (TFBUT).42
Other studies utilizing IC samples have shown that multiple
mucins may be important for increasing tear lm stability,
including MUC1 and MUC16, and that alteration of mucin
distribution may be associated with dry eye disease.41,43,44
Utilizing IC samples to evaluate glycosylation has shown
no signicant difference between dry eye and normal subjects.45 Yet another approach has been to use IC samples for
microarray analysis of expression of multiple genes and, at
the same time, to determine alterations in gene expression
in dry eye disease.46
It has been generally accepted that a decrease in TFBUT
is associated with the common nding of decreased goblet
cell density in patients with dry eye disease. However, a
recent study of Chinese patients with low noninvasive
tear break-up time (NITBUT) and TFBUT demonstrated
that there was no correlation with goblet cell density, as
measured by IC sampling. This nding suggests that other
non-goblet cell associated mucins may be important for
tear lm stability.42
MUC1 was evaluated by Hayashi et al, who used the
monocolonal antibody KL-6, which recognizes the ocular
surface epithelial cell membrane associated MUC1 mucin,
to evaluate IC samples from patients with dry eye disease
and normal controls. The expression of the KL-6 epitope of
MUC1 in corneal and conjunctival cells in mild and moderate dry eye disease was elevated, perhaps in an attempt to
relieve the goblet cell loss associated with dry eye disease,
but, interestingly, it was down-regulated in the conjunctiva
of patients with severe dry eye.43
MUC16 was evaluated by Caffery et al. They used real

time quantitative PCR (qPCR) of IC samples from patients


with Sjogren syndrome and KCS to demonstrate increased
concentration of MUC16 mRNA, a mucin expressed by
ocular surface epithelial cells, in Sjogren syndrome patients
compared to KCS patients and normal controls. Western
blotting analysis of protein samples collected by IC for membrane-bound MUC16 revealed no signicant differences
between samples from Sjogren syndrome, KCS, and normal
controls. The authors suggested that increased MUC16 production by conjunctival cells in Sjogren syndrome patients
might be the result of a compensatory mechanism to help
maintain ocular surface integrity.44
Mucin distribution patterns have also been evaluated
in IC samples. IC samples were analyzed by immunouorescent and immunoelectron microscopy for presence and
localization of the human specic monoclonal antibody
H185, which recognizes the O-linked carbohydrate epitope
on mucins. This analysis revealed signicantly different
patterns of binding associated with dry eye disease (starry
sky) and normal eyes (mosaic). H185 binding in the
starry sky pattern was also closely associated with the
severity of dry eye disease, as assessed by the rose bengal
staining score, suggesting that changes in mucin distribution are associated with dry eye disease. The authors also
suggest that variations in the nature of goblet-cell mucin
require more than one measurement technique, and that
traditional staining (PAS, for example) of IC samples may
not be sufcient to reveal all goblet cells.41
A negative nding involved study of glycosylation in
dry eye versus normal subjects. UDP-GalNAc:polypeptide
N-acetyl-galactosamnyltrasferase (ppGaNTase) family
members initiate mucin type O-glycosylation, and it is this
glycosylation process that provides mucins with viscoelastic
properties that are required for their role in maintaining a
healthy ocular surface. Imbert et al analyzed IC samples
from patients with aqueous-decient dry eye and normal
controls via quantitative mRNA analysis by real-time PCR
for 17 human ppGaNTase isoforms. Although multiple
isoforms were expressed, no signicant difference between
conjunctival epithelial cells from dry eye and normal groups
was found.45
An innovative use of IC samples was to apply microarray
analysis to determine gene expression in different patient
samples. Glycoconjugates are responsible for the control of
a variety of events on the mucosal surface and are encoded
by glycogenes. IC samples from patients with non-Sjogren
dry eye underwent RNA extraction, but in a new processing
technique, the RNA was further processed for microarray
analysis, a technique that allows analysis of many genes at
once. The microarray revealed signicant differences between dry eye and normal conjunctival epithelial cells; 46
of 424 genes were found to be signicantly reduced in dry
eye patients, including Notch1, 2 and 3 receptors, Notch
ligands Jagged1 and Delta1, and four members of the Wnt
signaling pathway, suggesting that these pathways may play
a role in the mechanism of dry eye disease development
and progression.46

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5. Growth Factor Receptors


Epidermal growth factor receptors have been shown to
play a role in wound healing. They may function similarly
on the ocular surface to maintain ocular surface integrity,
although altered expression of these receptors may contribute to abnormal ocular surface pathology. Epidermal growth
factor ErbB2, ErbB3, and ErbB4 are members of the type 1
growth factor receptor family and are expressed on corneal
and conjunctival epithelial cells.47 In 22 patients with KCS,
IC samples were processed by immunouorescent staining
for type 1 growth factor receptors using mouse monoclonal
antibodies. IC samples were also processed for Western blotting. KCS samples showed stronger staining for ErbB2 and
ErbB3 compared to normal controls, and Western blotting
conrmed the staining results. IC results positively correlated with clinical severity of KCS as measured by degree
of corneal uorescein staining and conjunctival lissamine
green staining score.47
6. Microvilli
Altered epithelial microvilli are thought to be part of
the process by which tear lm abnormalities develop, since
they play a role in tear lm stability. Cennamo et al, using
scanning electron microscopy (SEM) of IC samples from
patients with mild, moderate, and severe tear lm abnormalities compared with controls, described a reduction in
microvilli in all severities of tear lm abnormalities that
correlated with increasing severity of dry eye disease.48 This
study was the rst to analyze microvilli associated with
tear lm abnormalities using the noninvasive IC technique
rather than an invasive biopsy. The results suggest that SEM
can detect reduction in microvilli before more traditional
epithelial damage could be detected by light microscopy
and could be a new tool in classifying different stages of
tear lm abnormalities.48
B. Monitoring of Clinical Trials to Evaluate Efcacy
of Treatments for Dry Eye Disease
IC is an invaluable tool for analyzing the efcacy of
therapeutic treatments for dry eye disease. Most studies

using IC in connection with clinical trials have relied on


histology and immunohhistochemistry with light microscopy to analyze the results, using one of three scoring
systems to grade results. However, evaluation of IC by light
microscopy is open to observer bias and is time-consuming, as it requires an experienced reader. With the advent
of ow cytometry in conjunction with IC, it is possible to
provide an objective metric. Table 3 illustrates use of IC in
a multi-center clinical trial of a new treatment for dry eye
disease, cyclosporine, to document the change in ocular
inammation associated with treatment.

1. Cyclosporine
A body of evidence supports a role of inammation in dry
eye disease, and cyclosporine A (CsA), a T cell inhibitor, has
been developed for topical administration as a treatment for
the disease. A large, randomized, multi-center, clinical trial to
evaluate the efcacy of CsA employed ow cytometric analysis of IC samples as an objective tool to monitor the effect
of the drug on ocular surface cells. One hundred sixty-nine
dry eye disease patients from 28 centers in four European
countries were assigned to three treatment groups0.05%
CsA, 0.1% CsA, or vehicle (control)and monitored over
12 months (Table 3). IC samples were analyzed for immune markers HLA-DR and CD-40, and apoptotic marker
APO2.7. The percentage of HLA-DR positive cells and
level of HLA-DR expression were reduced in both groups
receiving CsA, whereas patients receiving vehicle showed
no signicant changes. CD40 and CD40 ligand signicantly
decreased in both CsA groups, as well. APO2.7 expression was more variable and increased in patients receiving
CsA. The objective results from IC analysis demonstrated
that CsA signicantly reduced expression of inammatory
markers on epithelial cells, and suggest that ow cytometry analysis of IC is an objective technique for describing
changes on the ocular surface in response to treatment.49,50
Cyclosporine treatment has also been compared with
other, more traditional treatments, such as articial tears
and topical sodium hyaluronate, with IC samples used to
assess any difference between treatments.

Table 3. The use of IC as an outcome measure in a dry eye clinical trial (topical CsA)
% HLA-DR-Positive Conjunctival Cells
Baseline
(meanSD)

3 months

6 months

12 months

0.05% CsA
(p/baseline)

61.67%29.54%
(p<0.0001)

39.03%31.36%
(p<0.001)

39.45%33.06%
(p<0.001)

26.55%27.33%

0.1% CsA
(p/baseline)

57.53%31.73%
(p=0.004)

41.73%33.57%
(p<0.001)

38.59%32.95%
(p<0.02)

37.84%27.80%

5.09%
(NS)

6.68%
(NS)

36.08%*
(p=0.02)

Group

Control
(p/baseline)

NS=nonsignificant. Compiled from Brignole et al, 200149 and Galatoire et al, 2003.50
*Patients initially randomized to receive the control vehicle starting at 6 months, received 0.1% CsA and were then evaluated at 12 months,
after 6 months of treatment with CsA.

100

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Utilizing IC combined with light microscopy and immmunostaining, one study evaluated six dry eye patients
who were treated with unpreserved artificial tears for
1 month, followed by topical 0.05% cyclosporine for 3
months. IC analysis after articial tear treatment showed
no change in goblet cell density in contrast to IC analysis
after 3 months of cyclosporine, which demonstrated a signicant increase in goblet cell density and of the number
of TGF-beta 2 positive goblet cells.51
Another study, which used light microscopy analysis of
IC samples to determine goblet cell density, showed that
both sodium hyaluronate and cyclosporine increased goblet
cell density. Thirty-six patients with dry eye were treated
with topical chondroitin sulfate with sodium hyaluronate
(CS-HA) in one eye and CsA in the other for 6-8 weeks.
Goblet cell density was signicantly higher in CsA-treated
eyes, although the authors suggest that administration of
both treatments simultaneously might result in a synergistic
response and even greater improvement in ocular surface
parameters.52

2. Autologous Serum
Articial tears made with autologous serum have long
been thought to be benecial in treating dry eye disease,
but until recently no controlled studies had been done. It
is believed that vitamins such as vitamin A, growth factors
such as TGF-beta and EGF, and other components of tears
that are critical to the maintenance of a healthy ocular surface are present in autologous serum, but not in articial
tears, which may contribute to their efcacy. The absence of
preservatives in autologous serum, such as benzalkonium
chloride (BAC), which is known to exacerbate dry eye
symptoms, may also explain the effectiveness of autologous
serum.53-55
Tananuvat et al performed a prospective, randomized,
placebo-controlled, single-masked 2-month study in 12
patients with dry eye disease to determine the safety and
efcacy of tears containing the patientsown serum.54 One
eye received the serum (20% solution) and the contralateral
eye received normal saline as a control. IC samples before
and after treatment began were stained with Papanicolau
stain and classied according to the Tseng classication.
Improvement in signs, including IC score, and symptoms
was observed in serum-treated eyes, although improvement
was nonsignicant because control groups also improved,
suggesting a large placebo effect. The authors state the need
for additional larger studies.54
In a later study by Noble et al, 50% autologous serum
drops were evaluated against more traditional treatments
in a prospective, randomized, partially masked, crossover
trial.56 Sixteen dry eye patients (11 with KCS/Sjogren syndrome and 5 with a variety of other ocular surface disorders)
were randomized to receive autologous serum for 3 months
followed by conventional treatment for 3 months, or conventional treatment for 3 months followed by autologous
serum for 3 months. IC scores in eyes treated with serum
showed signicant improvement, and subjective comfort

scores also were signicantly improved. The effect of serum


was conrmed by the crossover design, as the improvement
was lost when conventional therapy replaced serum.56
Platelet-rich plasma (PRP) is rich in growth factors, as
is autologous serum, but has platelets, which are reported
to speed wound healing and might help ocular surface
regeneration and decrease inammation that is associated
with dry eye disease. Alio et al performed a prospective,
nonrandomized, observational pilot study with 18 dry eye
disease patients treated with topical autologous PRP for 1
month. They found a statistically signicant increase in
goblet cell density on the superior bulbar conjunctiva as
assessed by IC.57

3. Umbilical Cord Serum


Like autologous serum, umbilical cord serum contains essential growth factors and components found in
tears. However, Yoon et al found signicantly higher EGF
and TGF- concentrations in umbilical serum than in
autologous serum. Additionally, umbilical serum has the
advantage of not requiring repeated blood collection from
patients. In a 2-month, uncontrolled and unmasked study
of 20% umbilical cord serum eye drops, 31 dry eye patients
had IC samples collected before and after 2 months of treatment, stained with PAS, and graded for goblet cell density
and degree of squamous metaplasia. Both conjunctival
squamous metaplasia and goblet cell density grades signicantly improved with umbilical cord serum treatment,
although the authors noted a need for larger, randomized
and controlled studies.55
Yoon et al compared autologous serum with umbilical
cord serum for the treatment of dry eye disease in a prospective, case-controlled study of 48 patients with severe dry eye
disease (Sjogren and non-Sjogren syndrome).53 Twenty-one
patients were treated with 20% autologous serum eye drops,
and 27 patients were treated with 20% umbilical cord serum
eye drops for 2 months. IC samples were stained with PAS
and analyzed using the grading scheme of Nelson (graded
on a scale from 0 to 3 with 3 being the worst, representing a higher degree of squamous metaplasia, abnormal
epithelial cell morphology and absence of goblet cells).12
The squamous metaplasia grade and goblet cell density
improved in non-Sjogren and Sjogren patients treated with
both autologous serum and umbilical cord serum at 1 and 2
months after beginning treatment. Interestingly, in Sjogren
syndrome patients, goblet cell density at 2 months was
higher with umbilical cord treatment than with autologous
serum treatment. Although both treatments improved signs
and symptoms of dry eye disease, umbilical cord serum was
more effective at decreasing symptoms.53,55
4. Articial Tears
a. Preservatives
Abietz and Bruce compared articial tears containing preservatives and preservative-free tears in 134 dry
eye subjects who were classied according to treatment:
untreated (57), nonpreserved topical treatment (30),

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IMPRESSION CYTOLOGY / Lopin, et al

preserved topical treatment with drops containing BAC,


chlorbutanol (CHB), or both.49 Twenty-one non-dry eye
subjects served as controls. IC samples were analyzed using
monoclonal antibodies with immunocytochemistry for expression of inammatory markers HLA-DR and CD23. The
nucleocytoplasmic ratio and goblet cell density were also
assessed using the Nelson grading scheme. The preserved
treatment group had signicantly lower goblet cell density
and higher HLA-DR and CD23 expression compared to
the nonpreserved treatment group, suggesting that dry eye
disease-associated inammation is worsened by preservative agents. However, there was no signicant difference
between the nonpreserved group and the untreated group,
suggesting that use of nonpreserved articial tears is not
sufcient for reducing inammation alone.58 The effects of
preservatives are still being explored. Most recently, a rabbit dry eye model was induced by topical administration
of 0.1% BAC, a common ophthalmic preservative, and IC
revealed dry eye-like conjunctival changes.59

b. Sodium Hyaluronate
Sodium hyaluronate (a biopolymer) drops have been
extensively studied as an alternative to other tear substitutes
for dry eye, because they may promote corneal wound healing, control inammation, and increase tear lm stability,
as well as having viscoelastic properties that help reduce
friction on the ocular surface.60 In a multi-center, randomized, double-blind study evaluating the long-term effect
of sodium hyaluronate-containing eye drops in patients
with dry eye disease, 44 patients with medium-to-severe
dry eye were evaluated and randomly treated with either
preservative-free sodium hyaluronate or preservative-free
saline. IC was the primary efcacy variable in this study. At
3 months, IC scores, based on the grading scheme designed
by Nelson, were signicantly lower in patients treated with
sodium hyaluronate compared to those treated with saline.
The authors suggest this is the direct result of the treatment, as the IC grade in the placebo group did not change
over the course of the study, whereas the treatment group
statistically improved from baseline.60
Sodium hyaluronate was further evaluated in an openlabel study designed to compare two different commercially
available solutions that varied in osmolarity. Forty patients
with Sjogren syndrome were randomly assigned to either
unpreserved 0.4% hypotonic sodium hyaluronate drops or
unpreserved 0.4% isotonic sodium hyaluronate drops for 3
months. IC score, based on evaluation of seven parameters,
including goblet cell distribution and presence of inammatory cells, and global symptom score were the primary
efcacy variables in this study. Both groups showed improvement in IC score, but the hypotonic group showed
a faster and more pronounced improvement in IC score,
suggesting that the hypotonic formulation may be the more
effective treatment. Some hypothesize that hypotonic solutions may help to correct the tear hyperosmolarity that is
associated with damage to the ocular surface and dry eye
disease.61
102

c. Carboxymethylcellulose (CMC)
Carboxymethylcellulose has long been investigated for
its therapeutic efcacy.62 An early study used Nelsons IC
score to demonstrate improvement in KCS patients treated
with a CMC-based tear.63 In a more recent randomized
study of LASIK patients by Lenton and Albietz, ten patients
treated with nonpreserved CMC articial tears during and
after LASIK surgery showed greater goblet cell density 1
month post-LASIK compared to a group that received salt
solution.64
IC sometimes demonstrates that there is no signicant
improvement in the ocular surface with a presumed effective treatment, and IC results may be in contrast to other
evaluations of dry eye disease, such as symptom scores. In
a prospective, randomized, masked-observer, single-center study, 19 patients with mild or moderate dry eye were
treated with a 0.5% isotonic CMC or salt solution. Although
subjective symptoms improved with CMC treatment, no
difference between the groups was observed with IC; degree
of conjunctival metaplasia was similar in the two groups
before and after treatment, and Nelsons IC score did not
correlate with improvement in subjective symptoms, as
measured by a custom questionnaire.62
Combining ow cytometry with IC samples can provide
a more objective endpoint for the effects of new treatment.
In a prospective, randomized, masked-observer trial comparing sodium hyaluronate drops with CMC in the treatment of dry eye syndrome with supercial keratitis, ow
cytometry was used to analyze IC samples for: HLA-DR (an
inammatory marker), Apo2.7 (an apoptosis-related marker), MUC5AC (a soluble mucin secreted by goblet cells),
and CD44 (hyaluronic acid receptor). CD44 expression was
signicantly decreased in sodium hyaluronate-treated eyes
compared to CMC-treated eyes, and there was a tendency
for reduction of the number of HLA-DR-positive cells in
both treatment groups, although this was not statistically
signicant.65
5. Antioxidants
Reactive oxygen species and oxidative reactions have
been associated with dry eye disease and ocular surface
injury and stress.66 Therefore, antioxidant therapy has
emerged as being potentially able to reverse or balance
these harmful reactions on the ocular surface by protecting
epithelial tissue from attack.67
Blades et al studied 40 patients with marginal dry eye in
a prospective, randomized, double-blind, placebo-controlled
trial with cross-over. Patients received no treatment for 30
days, placebo for 30 days, and oral antioxidant supplements
for 30 days in random order. IC samples were stained with
PAS and hematoxylin and evaluated by light microscopy
for number of goblet cells and appearance and degree
of squamous metaplasia. A signicant improvement in
goblet cell density and squamous metaplasia was observed
following antioxidant treatment, and increased tear lm
stability correlated with change in goblet cell density.
Interestingly, there was no signicant carry-over effect,

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IMPRESSION CYTOLOGY / Lopin, et al

and positive changes during antioxidant therapy did not


persist following cessation of treatment.66
Iodide is an oxygen free radical scavenger and can
therefore function as an antioxidant. In a prospective study,
16 patients were randomized to receive iodide iotophoresis
(a method by which an electrical current drives iodide across
tissue barriers, facilitating its penetration into tissues), and
12 were randomized to receive iodide application without
current for 10 days in a mock iontophoresis procedure. IC
samples taken before treatment and at 1 week, 1 month,
and 3 months post-treatment were evaluated by light
microscopy for epithelial cell changes and goblet cell
density. Although subjective symptoms and some clinical
signs improved in the iontophoresis group (with current),
no signicant change in IC ndings was observed between
the two groups.67

6. Essential Fatty Acids


Oral axseed oil, which is rich in essential fatty acids,
has been demonstrated to reduce inammation in systemic
autoimmune diseases. In a clinical trial of 38 dry eye patients with rheumatoid arthritis or lupus, patients were
randomized into three groups for 180 days: 1 g/day axseed
oil, 2 g/day axseed oil, or placebo. Ocular surface inammation was evaluated and quantied by conjunctival IC
before and after the study. In the two treatment groups, IC
demonstrated reduced ocular surface inammation.68
7. Nerve Growth Factor
Nerve growth factor (NGF) has been demonstrated
to promote corneal healing through recovery of corneal
sensory nerves in vitro and recovery of a healthy corneal
epithelium in vivo. NGF receptors have been found on
conjunctival and corneal epithelial cells and keratocytes.69,70
In an open study of three dogs, whose third eyelid lacrimal
gland had been excised to induce a human-like dry eye
disease state, NGF ointment was administered for 1 month
to 1 eye, while the contra-lateral eye served as a control.
IC samples revealed mucous laments and a signicant
increase in goblet cell density in NGF-treated eyes.69
Other research suggests that NGF is produced during
inammation by a variety of immune cells, and increased
NGF production has been observed in association with
inammatory diseases, such as asthma.70 In contrast to
the dog study noted above, a study in patients with dry
eye disease suggested that a decrease in NGF staining of
IC samples correlated with an improvement in the Nelson
score of the IC samples. Lee et al performed a prospective,
double-masked, randomized, comparative clinical trial in
41 patients with non-Sjogren syndrome dry eye. Patients
received a 0.1% anti-inammatory prednisolone solution
in one eye and 0.1% hyaluronic acid in the other eye for
28 days. Pre-treatment IC samples showed elevated NGF
immunostaining in dry eye patients compared to normal
controls. Post-treatment, the IC grading score (Nelsons
classication) and NGF staining (and tear concentration)
were signicantly lowered in the prednisolone group, while

the hyaluronic acid group had no signicant change in


these parameters, suggesting that NGF may play a role in
inammation associated with dry eye disease.70

8. Steroids
Because inammation is believed to play a role in the
pathophysiology of dry eye disease, treatment with antiinammatory topical corticosteroids has been investigated.
As noted above, the patients in the study of Lee et al demonstrated improvement (as measured by IC) with 0.1%
prednisolone treatment.70
In a prospective study of 53 primary or secondary Sjogren syndrome patients, a topical nonpreserved 1% methylprednisolone solution was administered in a pulse therapy
design, so that over the course of the trial, patients were
tapered off the medication. After treatment, a signicant
increase in the number of PAS-positive goblet cells was observed in conjunctival IC samples. The authors suggest that
steroid pulse therapy may be a safe and effective long-term
treatment for dry eye associated with Sjogren syndrome.71
In a single-masked, randomized, prospective clinical
trial, 32 dry eye patients with or without Sjogren syndrome
were randomly assigned to three groups: preservative-free
topical articial tear substitute (ATS); ATS plus nonsteroidal
anti-inammatory drops (NSAID); or ATS plus corticosteroid drops. IC samples were stained with PAS for analysis
of goblet cells and with monoclonal antibodies for HLADR and Apo2.7. Patients receiving ATS plus corticosteroid
drops had signicantly lower numbers of HLA-DR positive
cells, higher numbers of PAS-positive goblet cells, and lower
symptom severity scores compared to the other groups and
compared to their own baseline values. The authors suggest
that topical corticosteroids are effective treatment for dry
eye disease, producing a reduction in inammatory markers
and improvement in symptoms.72
9. Vitamin A
Because vitamin A deciency is associated with dry eye
disease, topical treatment with vitamin A and its derivatives
was studied over a decade ago as a potential treatment for
dry eye disease. An early study looked at the effects of topical tretinoin ointment in patients with severe dry eye disorders, including six patients with KCS, and found reduced
IC score (representing reduced squamous metaplasia) after
treatment.73 Schilling et al used IC to study the effect of
tretinoin treatment in 6 patients with KCS patients and 19
patients with mucin deciency. IC evaluation demonstrated
an improvement in squamous metaplasia in the mucin-decient group only.74 IC has also been used to demonstrate
goblet cell recovery in rabbit dry eye models treated with a
retinoic acid analog, CBS-211.75
10. Botulinum Toxin
Blepharospasm has been associated with dry eye disease.
Treatment with botulinum toxin injected into the lids (an
established blepharospasm treatment) has been reported to
be an effective treatment for some dry eye patients.76 Ocular

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IMPRESSION CYTOLOGY / Lopin, et al

surface changes were assessed in 16 blepharospasm patients


with dry eye disease who had had botulinum toxin A injected into the lids. IC samples obtained before treatment
and at 1 and 3 months post-treatment were analyzed by light
microscopy for parameters of the IC score, including epithelial cell morphology, squamous metaplasia, and presence of
snake-like chromatin. Before injection, reduced goblet cell
density was observed in 15 of the 16 patients. No signicant
changes in IC score were observed at 1 and 3 months postbotulinum toxin A injection, although TBUT increased.
However, rose bengal staining was slightly increased, and
Schirmer test results were signicantly worse.77

11. Non-Pharmacological Treatments


IC has been used to monitor the efcacy of non-pharmacological interventions for dry eye disease, such as insertion
of punctal plugs or surgery.
IC showed improvement in squamous metaplasia
in trachomatous dry eye patients randomized to receive
canalicular silicone plugs78 and in patients with severe
dry eye, who received Smart Plug (Medennium) lacrimal
plugs.79 Dursun et al evaluated the ocular surface changes
in aqueous-decient dry eye patients who received silicone
punctal plugs, and IC samples graded according Nelsons
classication showed signicant improvement after punctal
occulsion.80
In a retrospective study, patients with moderate-tosevere dry eye (with and without confounding diagnoses
(eg, rheumatoid arthritis or graft-versus-host disease) had
punctum or proximal canalicular stenoses after spontaneous
loss of their silicone punctal plug. No signicant changes
in IC score were observed after the procedure, although
subjective symptoms, Schirmer test, and staining improved
signicantly.81
A continuous articial tear reservoir to provide roundthe-clock tears to patients suffering from severe dry eye disease has been explored. Murube et al implanted an articial
tear pump-reservoir under the subcutaneous tissue of the
abdomen in six patients with severe dry eye disease without
other surgical options. Corneal and conjunctival IC samples
were taken pre- and post-operatively and showed improvement post-operatively, as did other clinical tests.82
In a prospective study of 24 patients with dermatochalasis and dry eye symptoms who underwent blepharoplasty,
light microscopy of IC samples revealed improvement in
epithelial cell morphology in some patients, but the majority
(70%) had no change during the 3 months after the operation. Inammation (represented by presence of polymorphonuclear leukocytes in IC samples) was reduced in some
patients, but the reason for this was unclear.83
C. Characterization of Animal Models
Although most studies that employ IC use human
samples, the technique has been helpful in evaluating new
animal models of dry eye disease, particularly in rabbits.
Analysis of IC samples from animals employs the same
methods used for human samples. IC sampling has been
104

an effective tool to determine whether or not an animal


model is appropriate for evaluating human dry eye disease,
and determination of changes in goblet cell density and cell
morphology have been most commonly used to evaluate
changes in the animal ocular surface. As in patients with
dry eyes, IC has also been used as an endpoint to evaluate
new treatment for ocular surface disease.
Xiong et al established a rabbit dry eye model using
topical administration of 0.1% BAC twice daily for 14 days
in one eye, while the other eye served as a contralateral
control. IC samples revealed a signicantly decreased goblet
cell density in the BAC-treated eyes (conrmed by immunouorescent stain of MUC5AC on conjunctival cryosections) at day 7 and 14, and evidence of increased squamous
metaplasia, consistent with ndings found in human dry
eye, making this possibly a viable dry eye model.59
A model developed by Toshida et al involved loss of
preganglionic parasympathetic neural control by surgical
removal of the greater supercial petrosal nerve. IC samples
taken from rabbits before and after surgery revealed lower
goblet cell density and abnormal appearance in denervated
animals.84
Animal models have also allowed for improvement in
IC technique. In a rabbit dry eye model induced by intramuscular atropine injection, a modied IC technique was
used. In an effort to increase cell numbers, after being placed
on the rabbit eye, the lter paper was immersed in distilled
water overnight, dried, and then stained. Cell pickup was
greatly increased by drying it overnight, and in the animal
group injected with atropine, a decrease in goblet cells was
observed, consistent with human dry eye disease.85
IC has also been used in animal models to evaluate the
effect of various dry eye interventions. For example, a P2Y2
receptor agonist (a mucin stimulator) and a synthetic retinoic acid analog were evaluated in rabbits using IC.75,86 In
another study, IC documented increased goblet cell density
with NGF treatment in a dog model of dry eye.69
Finally, IC has been used to evaluate the link between
dry eye disease and other diseases in animal models. In
a rat model of hypothyroidism, corneal IC samples were
evaluated for squamous metaplasia by light microscopy. The
samples showed abnormal corneal epithelial alteration in
the hypothyroid rats, suggesting that there is a link between
hypothyroidism and dry eye disease.87
D. Association of Dry Eye Disease With Other
Conditions
IC has been utilized to investigate the ocular surface in
a variety of disorders associated with dry eye disease (Table
4). Some ndings of interest are summarized below.
The most common IC findings were conjunctival
squamous metaplasia89,93,95-97,100,103,104,106,110112,114,116,117,119121 and decreased goblet cell density (Figure 4).89,92,97,102104,106,109-112,114-116,118,126 Other ndings included MUC5AC
staining,89,104,113 decreased density of dendritic cells,105
snake-like chromatin, 117,119 and increased HLA-DR
expression.91,98,105,111

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IMPRESSION CYTOLOGY / Lopin, et al

Table 4.

Impression cytology findings associated with various conditions


Impression cytology ndings

Condition/Reference #

# pts

Allergic rhinoconjunctivitis88

27
21
39
68

Atopic keratoconjunctivitis89
Chemotherapy90
Chronic conjunctivitis91
Chronic hepatitis
Jacobi et al92
Huang et al93
Collagen-vascular disease94
Congenital aniridia
Jastaneiah et al95
Rivas et al96
Cystic fibrosis
Mrugacz et al97
Mrugacz98
Dermatochalasis84

20
18

x
x

20
25
24

Graft vs host disease


Fei et al103

20

Wang et al104
HIV/AIDS105

Lipoprotein A elevation108

Ocular rosacea111
Oral carbamazepine Rx112
Polycystic ovary (PCO)
syndrome113
Pre-post-cataract surgery114
Pre/post LASIK surgery115

x
Abnormal IC findings correlated with clinical
signs of DED
Stem cell deficiency; increased goblet cells.
Metaplasia improved by limbal cell transplant.
x
x
Morphology unchanged by blepharaoplasty, but
PMLs reduced.
Tseng grade pathology.
Abnormal conjunctival epithelium.

Epithelial keratization, T cell infiltration.

15
30

1
906

x
x

13
1
20

x
x

37

x
x

x
x

x
x

x
x
x
x

Radiation (proton beam


for conjunctival melanoma)120
Renal disease/kidney transplant
Aktas et a1121
Strempel et al122

10

38
53

Sarcoidosis & dry eye disease123

56

Sjogren vs non-Sjogren
autoimmunity 124
Smoking125

91

SqM = Squamous metaplasia.

Other IC ndings
Eosinophilia, mononuclear cells.
Most severe in adults with childhood onset.
Conjunctival mucositis.

Possibly due to altered metabolism, such as


vitamin A deficiency.

11
24
50
70
15

Premature infants116
Psoriasis117
Pterygium118
Radiation (occupational)119

HLA-DR

27
50
20

Mucus deficiency syndrome109


Multinodular goiter110

MUC5AC

92
86
100
15

Decreased
GCD

71
19
60

Diabetes mellitus
Seifart & Strempel99
Goebbels100
Jin et al101
Down syndrome102

Hypovitaminosis A
Qureshi et al106
Farbos et al107

SqM

60

Inflammatory infiltrates.
Decreased dendritic cells, increased apoptotic
marker Apo2-7.
Enlarged keratinized squamous cells.
31% abnormal IC tests; inconsistent with
clinical results.
IC pathologic in 82% of patients.
GCD increased after mucosal grafting.
Metaplasia and GC count worse after thyroidectomy; suggests relationship between abnormal
ocular findings and post-op subclinical hypothyroidism.
Increase in ICAM-1.
PCOS, but not PCO, showed increase in GCD.
Decreased GCD and development of SqM
post-op.
Decreased GCD post-op.
Compared to full-term.
Snake-like chromatin, neutrophil clumping.
GCD increased after surgery.
Snake-like chromatin, lymphocytic infiltration,
hypertrophy.

Conjunctival calcification.
Moderate to severe morphological changes in
50% conjunctivas.
IC did not differentiate between sarcoidosis and
dry eye disease; similar findings.
Ocular surface disease differed among various
autoimmune diseases.
No difference in GCD between smokers and
non-smokers.

GCD = goblet cell density.

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105

IMPRESSION CYTOLOGY / Lopin, et al

Because IC is noninvasive and objective, its


routine use has been recommended to predict and
monitor dry eye disease
in premature babies, 116
patients with diabetes,99101 certain dermatologic
conditions,111,117 polycystic ovary, 113 and kidney
disease.121,122 It has also
been recommended as an
alternative to the more
invasive nasal smear evaluation in patients with allergic rhinoconjunctivitis,
as IC findings correlated
well with those obtained
from nasal smears.88 Cystic
fibrosis affects all secretory epithelia, including the
eye, and regular evaluation
with IC is recommended
to monitor inflammatory
processes.97,98 IC revealed
squamous metaplasia and
Figure 4. Photomicrographs of impression cytology specimens in patients with dry eye syndrome. (a) Normal
cytological picture with round-shaped epithelial cells, dense staining round nuclei, and abundant goblet cells.
intraepithelial lympho(b) Distinct squamous metaplasia of the epithelial cells (high nucleocytoplasmic ratio) with double nuclei
cytic inltration in all of
(arrowheads) and absence of goblet cells. (c) Distinct squamous metaplasia of the epithelial cells (high nucleo15 radiology technicians
cytoplasmic ratio) and different degrees of keratinization. (d) Distinct squamous metaplasia of the epithelial
cells (high nucleocytoplasmic ratio) and different degrees of keratinization. All nuclei pathologically altered:
examined, and routine
double nuclei, snake-like chromatin, and nuclear fragmentations (arrowheads). Stained with PAS- and Gillsophthalmic evaluation of
modified Papanicolau stain, microscopic magnification 40. (Reprinted with permission from Haller-Schober
radiology technicians was
EM, Schwantzer G, Berghold A, et al. Evaluating an impression cytology grading system (IC score) in patients
with dry eye syndrome. Eye 2006;20:927-33.)
recommended.119
IC has demonstrated
ocular surface damage asIV. SUMMARY AND CONCLUSION
sociated with cancer chemotherapy,90 proton beam radiation
IC is a minimally invasive technique that allows analysis
for conjunctival melanoma,120 and treatment with interferon
of human disease in a clinical setting. It can be used for
and ribavirin93 and oral carbamazepine (anticonvulsant).112
It has also been useful in demonstrating ocular surface imdiagnostic purposes, for understanding the mechanism that
has led to the disease, and for evaluating the efcacy of treatprovement after limbal transplantation in aniridia,96 after
ment. Research utilizing IC over the past several years has
pterygium surgery,118 after nasal mucosal grafting in severe
mucus deciency syndrome,109 and after cyclosporine treatled to an increased understanding of the pathophysiology
ment of chronic graft-vs-host-disease.104
of dry eye disease and helped conrm that ocular surface
IC revealed that squamous cell metaplasia and decreased
inammation is clearly associated with the clinical signs of
goblet cell density were both more pronounced in adult
the disease. Research utilizing IC has also begun to elucidate
patients with childhood onset of atopic keratoconjunctivitis
the mechanism of action of inammation, including work
than in children or in adults with adult-onset disease; these
that suggests dry eye disease is a Th1-mediated process
ndings suggest that prolonged inammation may be a facand that local surface cells (such as conjunctival epithelial
tor in the progression of ocular surface disease.89
cells), as opposed to systemic cells, play a key role in ocular
IC has demonstrated decreased goblet cells 1 month
inammation. Information from IC analysis has also proafter LASIK with use of a suction ring,115 and squamous
vided a rationale to consider new treatments.
metaplasia and low goblet cell density 3 months after cataIC has been effectively used in clinical trials of new treatract surgery.114
ments for ocular surface disease. In addition to providing
In other studies, IC showed no difference in goblet cell
an outcome measure to determine treatment efcacy and to
density between smokers and non-smokers,125 and it did
analyze the mechanism of action of any observed changes,
not differentiate between sarcoidosis and dry eye disease,
it has been effectively used to compare different treatments,
both showing signs of ocular surface disease.123
such as different formulations of a drug.
106

THE OCULAR SURFACE / APRIL 2009, VOL. 7, NO. 2 / www.theocularsurface.com

IMPRESSION CYTOLOGY / Lopin, et al

Often, IC results correlate with other signs and symptoms of dry eye disease, such as corneal staining and symptom scores. However, in some cases, IC results are incongruous with reported changes in symptoms, demonstrating no
improvement, although symptom scores appear to improve.
IC sampling has been used in animal models of ocular
disease, especially to conrm that ocular surface disease is
present and typical of human dry eye disease. IC sampling
has been done in rabbit, dog, and rat models, and has also
been used as an endpoint to evaluate new treatments utilizing these animal models.
Evaluation of IC to determine changes in the ocular surface has revealed that many conditions, systemic and local,
are, in fact, associated with changes to the ocular surface
that are typical of dry eye disease. All of this suggests that
IC may be a useful technique for evaluating ocular surface
changes in a variety of conditions.
A major change in the use of IC samples has been the incorporation of modern laboratory techniques, in particular,
ow cytometry, as a method to analyze the samples. Early
work with IC relied largely on light microscopy analysis of
histology and on immunohistochemistry. It was based on
grading schemes that were highly dependent on observer
technique and required an experienced observer to perform.
Conjunctival biopsies provided larger samples sizes, but
are clearly more invasive than IC. Although many studies
still employ these techniques, newer laboratory techniques
greatly expand the utility of IC. Flow cytometry, for example, provides objective metrics, and one sample can be
analyzed for multiple markers. Combining ow cytometry
with IC samples has led to new opportunities to provide
objective metrics on ndings on the ocular surface, such
as the number of HLA-DR-positive cells, other receptors,
and apoptotic factors. Flow cytometry, however, is a laboratory-based technique, and it requires specialized equipment
and an experienced researcher to process the samples and
analyze the results. Other new ways to use IC sampling
include RT-PCR to quantify mRNA levels and microarray
analysis to determine which genes are expressed.
IC has become an established tool to evaluate ocular surface changes and will likely continue to provide expanded
information that is useful for diagnosing and treating our
patients.
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