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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 tech-niques 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, includ-ing, among others, keratoconjunctivitis
sicca, ocular surface squamous neoplasia, and ocular surface
infections. During the past decade, IC has been used increasingly
to assist in diag-nosis 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
I. INTRODUCTIONn 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 ocu-lar
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 inva-sive.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 de-scribe 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 impres-sion
cytology and dry eye and impression cytology and
kerato-conjunctivitis 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
pro-cessing 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.
Impression Cytology: Recent Advances and Applications in Dry Eye
Disease
ELI LOPIN, MD, TATIANA DEVENEY, BA, AND PENNY A. ASBELL, MD,
FACS, MBA
Clinical PracticeJOHN E. SUTPHIN, MD, SECTION EDITOR
2009 Ethis Communications, Inc. The Ocular Surface ISSN:
1542-0124. Lopin E, Deveney T, Asbell PA. Impression cytology:
recent advances and applications in dry eye disease.
2009;7(2):93-110.
I
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: [email protected].
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II. IMPRESSION CYTOLOGY TECHNIQUESA. 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
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 utiliz-ing 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 ac-etate lters.
In the clinicians ofce, topical anesthetic eye drops were applied
to achieve a localized anesthetic effect, allowing the small
Biopore membrane device (approxi-mately 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 cytol-ogy 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
mor-phology, the smears were easy to prepare, and slides
main-tained 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
OUTLINE
I. Introduction II. Impression cytology techniques
A. Cell harvestingB. Cell processingC. Flow cytometry and
immunocytochemistry
III. Application of IC to investigation of dry eye diseaseA.
Investigating the pathology of dry eye
1. Evaluation of inflammationa. Cytokines/chemokinesb.
Defensinsc. Inflammatory markers
1) HLA-DR2) CD40
d. Summary of data on inflammation2. Oxidative reactions3.
Snake-like chromatin4. Ocular mucins5. Growth factor receptors6.
Microvilli
B. Monitoring of clinical trials1. Cyclosporine2. Autologous
serum3. Umbilical cord serum 4. Artificial tears
a. Preservativesb. Sodium hyaluronate c. Carboxymethylcellulose
(CMC)
5. Antioxidants6. Essential fatty acids7. Nerve growth factor8.
Steroids9. Vitamin A
10. Botulinum toxin 11. Non-pharmacological treatments
C. Characterization of animal modelsD. Association of dry eye
disease with other conditions
IV. Summary and conclusion
IMPRESSION CYTOLOGY / Lopin, et al
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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Schiff (PAS) staining, respectively. To assess the progres-sion
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 power-ful ability
to magnify allows visualization of cell ultrastruc-tures.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 bud-ding 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 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 user-dependent.
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.
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
Tseng11
Grade 0 Moderate density Uniform size/form 1:1
Grade 1 Decreased density Mild enlargement 1:21:3
Grade 2 Absent Moderate enlargement, 1:4 flattened
(squamoid)
Grade 3 Absent Markedly squamoid 1:6
Grade 4 Absent Markedly squamoid, large 1:8
Grade 5 Absent Shrunken cytoplasm Nucleus may be absent
Nelson12
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, Larger, polygonal 1:41:5
markedly decreased number
Grade 3 Very few Large, polygonal > 1:6
Adams13
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
N:C = Nucleus:cytoplasm.
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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 ImmunocytochemistryAlthough ow cytometry
has been used for several
decades, especially in the elds of hematology and im-munology,
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 in-formation on
cell type and immune markers.17 However, immunocytochemistry had
signicant limitations. Biopore membranes were difcult to use for
cell collection and im-munochemistry. More importantly,
appreciation of both percentage of positive cells and intensity of
immunostain-ing 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 opera-tor-dependent
interpretation.18
The ow cytometry apparatus consists of a uidics system chamber
that allows suspended particles in solu-tion 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.
Ad-ditionally, 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 re-action
(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 geno-type of the limbal epithelium belonging to
the recipients eyes following ex vivo expanded stem cell allograft
trans-plantation.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
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 Disease1. 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 evalu-ate cytokines and
chemokines, researchers have used IC samples combined with
immunohistochemical and im-munouorescent 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
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.
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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, IL-1alpha, 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 adhe-sion 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
con-trols. 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-immunostain-ing 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 de-rived) 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 inamma-tion.
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
in-volves 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-inamma-tory 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 de-fenses, 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 defen-sins has
demonstrated that in some inammatory processes, there is reduced
expression of specic defensins. Specically, the beta-defensin
DEFB-109 gene associated with antimi-crobial 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 Markers1) HLA-DR
Flow cytometry has expanded the opportunities to dis-cover 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 ex-pressed on immune cells, such
as B lymphocytes, but whose expression has also been described on
some nonimmune
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epithelial cells.17 Initially, abnormal HLA-DR expression in
samples from dry eye patients was demonstrated through
immunohistochemistry staining, but ow cytometry analy-sis of IC has
allowed for a more objective metric on the number of
HLA-DR-positive cells and expression of HLA-DR. 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 dis-ease (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 im-munhistochemistry.16
Baseline data from the cyclosporine trial (Section B.1, below)
showed that the percentage of positive HLA-DR-positive cells and
HLA-DR expression in patients with Sjo-gren syndrome dry eye was
higher than that in non-Sjogren syndrome dry eye patients, although
both groups had a sig-nicantly 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, con-rming the usefulness of HLA-DR
expression as a measure-ment 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) CD40CD40 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 InammationUsing 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 pathogen-esis of dry eye disease, but
providing the rationale and inspiration for new anti-inammatory
treatments for dry eye disease.
2. Oxidative ReactionsIC sampling has been used to elucidate the
mechanism
of action that results in ocular surface abnormalities
associ-ated 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 ra-tionale
for the use of antioxidants to treat dry eye disease.36
Cejkova et al analyzed xanthine oxidoreductase/xan-thine
oxidasean enzymatic system responsible for the generation of
reactive oxygen speciesby histochemistry
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 auto-immune diseases.36
In another study by Cejkova et al, immunohistochemis-try
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 gen-eration 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.) Immunohis-tochemistry 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 hypoth-esis 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 ChromatinSnake-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
in-ammation. 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 MucinsMucins 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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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 sub-jects.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 moder-ate 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 mem-brane-bound MUC16 revealed no signicant differences
between samples from Sjogren syndrome, KCS, and normal controls.
The authors suggested that increased MUC16 pro-duction 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 immunouo-rescent 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 distribu-tion 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 be-tween 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
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 permis-sion 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.)
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5. Growth Factor ReceptorsEpidermal 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 contrib-ute 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 blot-ting.
KCS samples showed stronger staining for ErbB2 and ErbB3 compared
to normal controls, and Western blotting conrmed the staining
results. IC results positively corre-lated with clinical severity
of KCS as measured by degree of corneal uorescein staining and
conjunctival lissamine green staining score.47
6. MicrovilliAltered 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 abnor-malities 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 micros-copy 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-consum-ing, 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. CyclosporineA 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 analy-sis 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 im-mune 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 expres-sion 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 cytom-etry 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 Group (meanSD) 3
months 6 months 12 months
0.05% CsA 61.67%29.54% 39.03%31.36% 39.45%33.06%
26.55%27.33%(p/baseline) (p
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Utilizing IC combined with light microscopy and
im-mmunostaining, 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
sig-nicant 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 sur-face 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 syn-drome 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 con-ventional 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 SerumLike autologous serum, umbilical cord
serum con-
tains 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
treat-ment, stained with PAS, and graded for goblet cell density
and degree of squamous metaplasia. Both conjunctival squamous
metaplasia and goblet cell density grades sig-nicantly 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 prospec-tive,
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,
represent-ing 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 Tearsa. Preservatives
Abietz and Bruce compared articial tears contain-ing
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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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 ex-pression 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 preserva-tive 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
rab-bit 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 HyaluronateSodium hyaluronate (a biopolymer) drops
have been
extensively studied as an alternative to other tear substitutes
for dry eye, because they may promote corneal wound heal-ing,
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, random-ized, 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 treat-ment, 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 open-label 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
inam-matory cells, and global symptom score were the primary efcacy
variables in this study. Both groups showed im-provement 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 solu-tions may help to correct the tear hyperosmolarity
that is associated with damage to the ocular surface and dry eye
disease.61
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 effec-tive 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-cen-ter 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 com-paring sodium
hyaluronate drops with CMC in the treat-ment 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 mark-er), 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. AntioxidantsReactive 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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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 AcidsOral 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 pa-tients
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 inam-mation 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 FactorNerve 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. SteroidsBecause inammation is believed to play a role in
the
pathophysiology of dry eye disease, treatment with
anti-inammatory topical corticosteroids has been investigated. As
noted above, the patients in the study of Lee et al dem-onstrated
improvement (as measured by IC) with 0.1% prednisolone
treatment.70
In a prospective study of 53 primary or secondary Sjo-gren
syndrome patients, a topical nonpreserved 1% methyl-prednisolone
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 ob-served 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 corticoste-roid drops. IC samples were stained
with PAS for analysis of goblet cells and with monoclonal
antibodies for HLA-DR 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 ABecause 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 topi-cal tretinoin
ointment in patients with severe dry eye disor-ders, 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-de-cient 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 ToxinBlepharospasm 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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surface changes were assessed in 16 blepharospasm patients with
dry eye disease who had had botulinum toxin A in-jected 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 epithe-lial 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
post-botulinum toxin A injection, although TBUT increased. However,
rose bengal staining was slightly increased, and Schirmer test
results were signicantly worse.77
11. Non-Pharmacological TreatmentsIC has been used to monitor
the efcacy of non-pharma-
cological 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-to-severe 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 round-the-clock
tears to patients suffering from severe dry eye dis-ease 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 improve-ment post-operatively, as did other clinical
tests.82
In a prospective study of 24 patients with dermatocha-lasis 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 opera-tion. Inammation (represented by presence of
polymor-phonuclear leukocytes in IC samples) was reduced in some
patients, but the reason for this was unclear.83
C. Characterization of Animal ModelsAlthough 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
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 immu-nouorescent stain of MUC5AC on
conjunctival cryosec-tions) 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 intra-muscular 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 reti-noic 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 ConditionsIC 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
metaplasia89,93,95-97,100,103,104,106,110112,114,116,117,119-121
and decreased goblet cell density (Figure
4).89,92,97,102-104,106,109-112,114-116,118,126 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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Table 4. Impression cytology findings associated with various
conditions
Impression cytology ndings
Condition/Reference # # pts SqM DecreasedGCD MUC5AC HLA-DR Other
IC ndings
Allergic rhinoconjunctivitis88 27 Eosinophilia, mononuclear
cells.
Atopic keratoconjunctivitis89 21 x x x Most severe in adults
with childhood onset.
Chemotherapy90 39 Conjunctival mucositis.
Chronic conjunctivitis91 68 x
Chronic hepatitis Jacobi et al92 Huang et al93
7119 x
x
Collagen-vascular disease94 60 Abnormal IC findings correlated
with clinical signs of DED
Congenital aniridia Jastaneiah et al95 Rivas et al96
2018
xx
Stem cell deficiency; increased goblet cells.Metaplasia improved
by limbal cell transplant.
Cystic fibrosis Mrugacz et al97 Mrugacz98
2025
x xx
Dermatochalasis84 24 Morphology unchanged by blepharaoplasty,
but PMLs reduced.
Diabetes mellitus Seifart & Strempel99 Goebbels100 Jin et
al101
9286100
xTseng grade pathology.Abnormal conjunctival epithelium.
Down syndrome102 15 x Possibly due to altered metabolism, such
as vitamin A deficiency.
Graft vs host disease Fei et al103 Wang et al104
20
15
x
x
x
x x
Epithelial keratization, T cell infiltration.
Inflammatory infiltrates.HIV/AIDS105 30 x Decreased dendritic
cells, increased apoptotic
marker Apo2-7.Hypovitaminosis A Qureshi et al106 Farbos et
al107
1906
x x Enlarged keratinized squamous cells.31% abnormal IC tests;
inconsistent with clinical results.
Lipoprotein A elevation108 27 IC pathologic in 82% of
patients.
Mucus deficiency syndrome109 50 x GCD increased after mucosal
grafting.
Multinodular goiter110 20 x x Metaplasia and GC count worse
after thyroidec-tomy; suggests relationship between abnormal ocular
findings and post-op subclinical hypo-thyroidism.
Ocular rosacea111 13 x x x Increase in ICAM-1.
Oral carbamazepine Rx112 1 x x
Polycystic ovary (PCO) syndrome113
20 x PCOS, but not PCO, showed increase in GCD.
Pre-post-cataract surgery114 37 x x Decreased GCD and
development of SqM post-op.
Pre/post LASIK surgery115 11 x Decreased GCD post-op.
Premature infants116 24 x x Compared to full-term.
Psoriasis117 50 x Snake-like chromatin, neutrophil clumping.
Pterygium118 70 x GCD increased after surgery.
Radiation (occupational)119 15 x Snake-like chromatin,
lymphocytic infiltration, hypertrophy.
Radiation (proton beamfor conjunctival melanoma)120
10 x
Renal disease/kidney transplant Aktas et a1121 Strempel et
al122
3853
x Conjunctival calcification. Moderate to severe morphological
changes in 50% conjunctivas.
Sarcoidosis & dry eye disease123 56 IC did not differentiate
between sarcoidosis and dry eye disease; similar findings.
Sjogren vs non-Sjogrenautoimmunity 124
91 Ocular surface disease differed among various autoimmune
diseases.
Smoking125 60 No difference in GCD between smokers and
non-smokers.
SqM = Squamous metaplasia. GCD = goblet cell density.
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Because IC is nonin-vasive and objective, its routine use has
been rec-ommended to predict and monitor dry eye disease in
premature babies,116 patients with diabetes,99-101 certain
dermatologic conditions,111,117 polycys-tic ovary,113 and kidney
disease.121,122 It has also been recommended as an alternative to
the more invasive nasal smear evalu-ation in patients with
al-lergic rhinoconjunctivitis, as IC findings correlated well with
those obtained from nasal smears.88 Cystic fibrosis affects all
secre-tory epithelia, including the eye, and regular evaluation
with IC is recommended to monitor inflammatory processes.97,98 IC
revealed squamous metaplasia and intraepithelial lympho-cytic
inltration in all of 15 radiology technicians examined, and routine
ophthalmic evaluation of radiology technicians was
recommended.119
IC has demonstrated ocular surface damage as-sociated with
cancer chemotherapy,90 proton beam radiation for conjunctival
melanoma,120 and treatment with interferon and ribavirin93 and oral
carbamazepine (anticonvulsant).112 It has also been useful in
demonstrating ocular surface im-provement after limbal
transplantation in aniridia,96 after pterygium surgery,118 after
nasal mucosal grafting in severe mucus deciency syndrome,109 and
after cyclosporine treat-ment of chronic
graft-vs-host-disease.104
IC revealed that squamous cell metaplasia and decreased goblet
cell density were both more pronounced in adult patients with
childhood onset of atopic keratoconjunctivitis than in children or
in adults with adult-onset disease; these ndings suggest that
prolonged inammation may be a fac-tor in the progression of ocular
surface disease.89
IC has demonstrated decreased goblet cells 1 month after LASIK
with use of a suction ring,115 and squamous metaplasia and low
goblet cell density 3 months after cata-ract surgery.114
In other studies, IC showed no difference in goblet cell density
between smokers and non-smokers,125 and it did not differentiate
between sarcoidosis and dry eye disease, both showing signs of
ocular surface disease.123
IV. SUMMARY AND CONCLUSIONIC is a minimally invasive technique
that allows analysis
of human disease in a clinical setting. It can be used for
diagnostic purposes, for understanding the mechanism that has led
to the disease, and for evaluating the efcacy of treat-ment.
Research utilizing IC over the past several years has led to an
increased understanding of the pathophysiology of dry eye disease
and helped conrm that ocular surface inammation is clearly
associated with the clinical signs of the disease. Research
utilizing IC has also begun to elucidate the mechanism of action of
inammation, including work that suggests dry eye disease is a
Th1-mediated process and that local surface cells (such as
conjunctival epithelial cells), as opposed to systemic cells, play
a key role in ocular inammation. Information from IC analysis has
also pro-vided a rationale to consider new treatments.
IC has been effectively used in clinical trials of new
treat-ments for ocular surface disease. In addition to providing an
outcome measure to determine treatment efcacy and to analyze the
mechanism of action of any observed changes, it has been
effectively used to compare different treatments, such as different
formulations of a drug.
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. (b) Distinct squamous metaplasia of the
epithelial cells (high nucleocytoplasmic ratio) with double nuclei
(arrowheads) and absence of goblet cells. (c) Distinct squamous
metaplasia of the epithelial cells (high nucleo-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: double nuclei, snake-like chromatin, and nuclear
fragmentations (arrowheads). Stained with PAS- and Gills-modified
Papanicolau stain, microscopic magnification 40. (Reprinted with
permission from Haller-Schober 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.)
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Often, IC results correlate with other signs and symp-toms of
dry eye disease, such as corneal staining and symp-tom scores.
However, in some cases, IC results are incongru-ous 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 utiliz-ing these animal models.
Evaluation of IC to determine changes in the ocular sur-face 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
in-corporation 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 ex-ample, 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
labora-tory-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 sur-face
changes and will likely continue to provide expanded information
that is useful for diagnosing and treating our patients.
REFERENCES 1. Larmande A, Tismit E. [Importance of cytodiagnosis
in ophthal-
mology: preliminary report of 8 cases of tumors of the
sclero-cor-neal limbus.] Bull Soc Ophtalmol Fr 1954;19:415-9. In
French
2. Thatcher RW, Darougar S, Jones BR. Conjunctival impression
cytology. Arch Ophthalmol 1977;95:678-81
3. Egbert PR, Lauber S, Maurice DM. A simple conjunctival
biopsy. Am J Ophthalmol 1977;84:798-801
4. McKelvie P. Ocular surface impression cytology. Adv Anat
Pathol 2003;10:328-37
5. Calonge M, Diebold Y, Sez V, et al. Impression cytology of
the ocular surface: a review. Exp Eye Res 2004;78:457-72
6. Singh R , Joseph A ,Umapathy T, et al. Impression cytology of
the
ocular surface. Br J Ophthalmol 2005;89:1655-9 7. Dart J.
Impression cytology of the ocular surfaceresearch tool
or routine clinical investigation? Br J Opthalmol 1997;81:984-88
8. McKelvie PA, Daniell M, McNab A, et al. Squamous cell carci-
noma of the conjunctiva: a series of 26 cases. Br J Ophthalmol
2002;86:168-73
9. Yamur M, Ersz C, Ersz TR, Varinli S. Brush technique in
ocular surface cytology. Diagn Cytopathol 1997;17:88-91
10. Ersz C, Yamur M, Ersz TR, Yalaz M. Preoperative brush and
impression cytology in ocular surface squamous neoplasms. Acta
Cytol 2003;47:13-5
11. Tseng SC. Staging of conjunctival squamous metaplasia by
impres-sion cytology. Ophthalmology 1985;92:728-33
12. Nelson JD. Impression cytology. Cornea 1988;7:71-81 13.
Adams GG, Dilly PN, Kirkness CM. Monitoring ocular disease by
impression cytology. Eye 1988;2(Pt5):506-16 14. Maskin SL, Bod
DD. Electron microscopy of impression-acquired
conjunctival epithelial cells. Ophthalmology 1986;93:1518-2 15.
Pastor SA, Shuster AR, Miller MM, Lam KW. Use of impression
cytology to demonstrate a retrovirus in AIDS patients with
cyto-megalovirus retinitis. Cornea 1991;10:511-5
16. Baudouin C, Brignole F, Becquet F, et al. Flow cytometry in
impres-sion cytology specimens. A new method for evaluation of
conjunc-tival inammation. Invest Ophthalmol Vis Sci
1997;38:1458-64
17. Baudouin C, Haouat N, Brignole F, et al. Immunopathological
nd-ings in conjunctival cells using immunouorescence staining of
impression cytology specimens. Br J Ophthalmol 1992;76:545-9
18. Brignole-Baudouin F, Ott AC, Warnet JM, Baudouin C. Flow
cytometry in conjunctival impression cytology: a new tool for
exploring ocular surface pathologies. Exp Eye Res
2004;78:473-81
19. Givan A. Flow cytometry: rst principles, 2002, ed 2 20.
Jones DT, Monroy D, Ji Z, et al. Sjogrens syndrome: cytokine
and Epstein-Barr viral gene expression within the conjunctival
epithelium. Invest Ophthalmol Vis Sci 1994;35:3493-504
21. Daya SM, Watson A, Sharpe JR, et al. Outcomes and DNA
analysis of ex vivo expanded stem cell allograft for ocular surface
recon-struction. Ophthalmology 2005;112:470-7
22. (No authors listed). Research in dry eye: report of the
Research Subcommittee of the International Dry Eye WorkShop 2007).
Ocul Surf 2007;5:179-93
23. Yoon KC, Jeong IY, Park YG, Yang SY. Interleukin-6 and tumor
necrosis factor-alpha levels in tears of patients with dry eye
syn-drome. Cornea 2007;26:431-7
24. Cejkova J, Ardan T, Zimonova Z, et al. Nitric oxide synthase
induction and cytotoxic nitrogen-related oxidant formation in
conjunctival epithelium of dry eye (Sjgrens syndrome). Nitric Oxide
2007;17:10-17
25. Solomon A, Dursun D, Liu Z, et al. Pro- and anti-inammatory
forms of interleukin-1 in the tear uid and conjunctiva of patients
with dry-eye disease. Invest Ophthalmol Vis Sci 2001;42:2283-92
26. Pugfelder SC, Jones D, Ji Z, et al. Altered cytokine balance
in the tear uid and conjunctiva of patients with Sjogren syndrome
keratoconjunctivitis sicca. Curr Eye Res 1999;19:201-21
27. Narayanan S, Miller W, McDermott A. Conjunctival cytokine
expression in symptomatic moderate dry eye subjects. Invest
Ophthalmol Vis Sci 2006;47:2445-50
28. Baudouin C, Liang H, Bremond-Gignac D, et al. CCR4 and
CCR5
IMPRESSION CYTOLOGY / Lopin, et al
-
THE OCULAR SURFACE / APRIL 2009, VOL. 7, NO. 2 /
www.theocularsurface.com108
expression in conjunctival specimens are differential markers of
TH1/TH2 in ocular surface disorders. J Allergy Clin Immunol
2005;116:614-9
29. Trinh L, Baudouin-Brignole F, Raphael M, et al. Th1 and Th2
responses on the ocular surface in uveitis identied by CCR4 and
CCR5 conjunctiva expression. Am J Ophthalmol 2007;144:580-5
30. Gulati A, Sacchetti M, Bonini S, Dana R. Chemokine receptor
CCR5 expression in conjunctival epithelium of patients with dry eye
syndrome. Arch Ophthalmol 2006;124:710-6
31. Narayanan S, Miller W, McDermott A. Expression of human
beta-defensins in conjunctival epithelium: relevance to dry eye
disease. Invest Ophthalmol Vis Sci 2003;44:3795-801
32. Abedin A, Mohammed I, Hopkinson A, Dua H. A novel
antimicro-bial peptide on the ocular surface shows decreased
expression in in-ammation and infection. Invest Ophthalmol Vis Sci
2008;49:28-33
33. Brignole F, Pisella P-J, Goldschild M, et al. Flow
cytometric analysis of inammatory markers in conjunctival
epithelial cells of patients with dry eyes. Invest Ophthalmol Vis
Sci 2000;41:1356-63
34. Mrugacz M, Zywalewska N. [HLA-DR antigen expression on
conjunctival epithelial cells in patients with dry eye.] Klin Oczna
2005;107:278-9. In Polish
35. Bourcier T, De Saint-Jean M, Brignole F, et al. Expression
of CD40 and CD40 ligand in the human conjuntival epithelium. Invest
Ophthalmol Vis Sci 2000;41:120-6
36. Cejkova J, Ardan T, Jirsova K, et al. The role of
conjunctival epi-thelial cell xanthine oxioreductase/xanthine
oxidase in oxidative reactions of the ocular surface of dry eye
patients with Sjogren syndrome. Histol Histopathol
2008;22:997-1003
37. Cejkova J, Ardan T, Simonova C, et al. Decreased expression
of antioxidant enzymes in the conjunctival epithelium of dry eye
(Sjogren syndrome) and its possible contribution to the
devel-opment of ocular surface oxidative injuries. Histol
Histopathol 2008;23:1477-83
38. Knop E, Reale E. Fine structure and signicance of snake-like
chromatin in conjunctival epithelial cells. Invest Ophthalmol Vis
Sci 1994;35:711-9
39. Jirsova K, Juklova K, Vesela V, Filipec M. Morphological and
im-muncytochemical characterization of snake-like chromatin cells.
Histol Histopathol 2006;21:355-60
40. Jirsova K, Juklova K, Alfakih A, Filipec M. Presence of
snake-like chromatin in epithelial cells of keratoconjunctivitis
sicca followed by a large number of micronuclei. Acta Cytol
2007;51:541-6
41. Danjo Y, Watanabe H, Tisdale A, et al. Alteration of mucin
in hu-man conjunctival epithelia in dry eye. Invest Ophthalmol Vis
Sci 1998;39:2602-9
42. Yeo A, Carkeet A, Carney L, Yap M. Relationship between
gob-let cell density and tear function tests. Ophthalmic Physiol
Opt 2003;23:87-94
43. Hayashi Y, Kao W, Kohno N, et al. Expression patterns of
sialylated epitope recognized by KL-6 monoclonal antibody in ocular
surface epithelium of normal and dry eye patients. Invest
Ophthalmol Vis Sci 2004;45:2212-7
44. Caffery B, Joyce E, Heynen ML, et al. MUC16 expression in
Sjogren syndrome, KCS, and control subjects. Mol Vis
2008;14:1547-55
45. Imbert Y, Jumblatt M, Foulks G, et al. Expression in human
ocular surface tissues of the GalNAc-transferases that initiate
mucin-type O-glycosylation. Cornea 2006;25:1193-9
46. Mantelli F, Schaffer L, Dana R, et al. Glycogene expression
in conjunctiva of patients with dry eye:Downregulation of notch
signaling. Invest Ophthalmol Vis Sci 2008 (epub ahead of print)
47. Liu Z, Carvajal M, Carraway CC, et al. Increased of the type
1 growth factor receptor family in the conjunctival epithelium of
patients with keratoconjunctivitis sicca. Am J Ophthalmol
2000;129:472-80
48. Cennamo GL, Prete A, Forte R, et al. Impression cytology
with scanning electron microscopy: a new method in the study of
conjunctival microvilli. Eye 2008;22:138-43
49. Brignole F, Pisella PJ, De Saint Jean M, et al. Flow
cytometric analysis of inammatory markers in KCS: 6-month treatment
with topical cycosporin A. Invest Ophthalmol Vis Sci
2001;42:90-5
50. Galatoire O, Baudouin C, Pisella PJ, et al. [Flow cytometry
in impression cytology during keratoconjuntivitis sicca: effects of
topical cyclosporine A on HLA-DR expression.] J Fr Ophthalmol
2003;26:337-43. In French
51. Pugfelder SC, De Paiva CS, Villarreal AL, Stern ME. Effects
of sequential articial tear and cyclosporine emulsion therapy on
conjunctival goblet cell density and transforming growth
factor-beta2 production. Cornea 2008;27:64-9
52. Moon JW, Lee HJ, Shin KC, et al. Short term effects of
topical cyclosporine and viscoelastic on the ocular surfaces in
patients with dry eye. Korean J Ophthalmol 2007;21:189-94
53. Yoon KC, Heo H, Im SK, et al. Comparison of autologous serum
and umbilical cord serum eye drops for dry eye syndrome. Am J
Ophthalmol 2007;144:86-92
54. Tananuvat N, Daniell M, Sullivan LJ, et al. Controlled study
of the use of autologous serum in dry eye patients. Cornea
2001;20:802-6
55. Yoon KC, Im SK, Park YG, et al. Application of umbilical
cord serum eyedrops for the treatment of dry eye syndrome. Cornea
2006;25:268-72
56. Noble BA, Loh RS, MacLennan S, et al. Comparison of
autologous serum eye drops with conventional therapy in a
randomised con-trolled crossover trial for ocular surface disease.
Br J Ophthalmol 2004;88:647-52
57. Alio JL, Colecha JR, Pastor S, et al. Symtomatic dry eye
treat-ment with autologous platelet rich-plasma. Ophthalmic Res
2007;39:124-9
58. Albietz J, Bruce A. The conjunctival epithelium in dry eye
subtypes: effect of preserved and non-preserved topical treatments.
Curr Eye Res 2001;22:8-18
59. Xiong C, Chen D, Liu J, et al. A rabbit dry eye model
induced by topical medication of a preservative benzalkonium
chloride. Invest Ophthalmol Vis Sci 2008;49:1850-6
60. Aragona P, Papa V, Micali A, et al. Long term treatment with
sodium hyaluronate-containing artical tears reduces ocular surface
dam-age in patients with dry eye. Br J Ophthalmol 2002;86:181-4
61. Aragona P, Stefano G, Ferreri F, et al. Sodium hyaluronate
eye drops of different osmolarity for the treatment of dry eye in
Sjogren syndrome patients. Br J Ophthalmol 2002;86:879-84
62. Bruix A, Adan A, Casaroli-Marano RP. [Efcacy of sodium
car-boxymethylcellulose in the treatment of dry eye syndrome.] Arch
Soc Esp Opftalmol 2006;81:85-92. In Spanish
63. Grene RB, Lankston P, Mordaunt J, et al. Unpreserved
carboxym-etylcellulose artical tears evaluated in patients with
keratocon-junctivitis sicca. Cornea 1992;11:294-301
IMPRESSION CYTOLOGY / Lopin, et al
-
THE OCULAR SURFACE / APRIL 2009, VOL. 7, NO. 2 /
www.theocularsurface.com 109
64. Lenton LM, Albietz JM. Effect of carmellose-based articial
tears on the ocular surface in eyes after laser in situ
keratomileusis. J Refract Surg 1999;15:S227-S231
65. Brignole F, Pisella PJ, Dupas B, et al. Efcacy and safety of
0.18% sodium hyaluronate in patients with moderate dry eye syndrome
and superficial keratitis. Graefes Arch Clin Exp Ophthalmol
2005;243:531-8
66. Blades KJ, Patel S, Aidoo KE. Oral antioxidant therapy for
marginal dry eye. Eur J Clin Nutr 2001;55:589-97
67. Horwath-Winter J, Schmut O, Haller-Schober E-M, et al.
Iodide iontophoresis as a treatment for dry eye syndrome. Br J
Ophthalmol 2005;89:40-4
68. Pinheiro MN, dos Santos PM, dos Santos RC, et al. Oral
axseed oil (Linum usitatissimum) in the treatment for dry-eye
Sjogren syn-drome patients. Arq Bras Oftalmol 2007;70:649-55. In
Portuguese
69. Coassin M, Lambiase A, Costa N, et al. Efcacy of topical
nerve growth factor treatment in dogs affected by dry eye. Graefes
Arch Clin Exp Ophthalmol 2005; 243:151-5
70. Lee H, Ryu I, Seo K, et al. Topical 0.1% prednisolone lowers
nerve growth factor expression in keratoconjunctivitis sicca
patients. Ophthalmology 2006;113:198-205
71. Hong S, Kim T, Chung S, et al. Recurrence after topical
nonpre-served methylprednisolone therapy for keratoconjunctivitis
sicca in Sjogren syndrome. J Ocul Pharmacol Ther 2007;23:78-82
72. Avunduk AM, Avunduk MC, Varnell ED, Kaufman HE. The
comparison of efcacies of topical corticosteroids and nonsteroidal
anti-inammatory drops on dry eye patients: a clinical and
im-munocytochemical study. Am J Ophthalmol 2003;126:593-602
73. Tseng SC. Topical tretinoin treatment for severe dry-eye
disorders. J Am Acad Dermatol 1986;15:860-6
74. Schilling H, Koch JM, Waubke TN, Frank B.[ Treatment of the
dry eye with vitamin A acidan impression cytology controlled
study.] Fortschr Ophthalmol 1989;86:530-4. In German
75. Driot JY, Bonne C. Benecial effects of a retinoic acid
analog, CBS-211 A, on an experimental model of keratoconjunctivitis
sicca. Invest Ophthalmol Vis Sci 1992;33:190-5
76. Spiera H, Asbell PA, Simpson DM. Botulinum toxin increases
tearing in patients with Sjogren syndrome: a preliminary report. J
Rheumatol 1997;24:1842-3
77. Horwath-Winter J, Bergloeff J, Floegel I, et al. Botulinum
toxin A treatment in patients suffering from blepharospasm and dry
eye. Br J Ophthalmol 2003;87:54-6
78. Guzey M, Ozardali I, Kilic A, et al. The treatment of severe
trachomatous dry eye with canalicular silicone plugs. Eye
2001;15:297-303
79. Li XM, Zhang J, Wang W. [Application of lacrimal plug for
dry eye patients.] Zhonghua Yan Ke Za Shi 2005;41:1098-1102. In
Chinese
80. Dursun D, Ertan A, Bilezikci B, et al. Ocular surface
changes in keratoconjunctivitis sicca with silicone punctum plug
occlusion. Curr Eye Res 2003;26:263-9
81. Boldin I, Klein A, Haller-Schober EM, Horwath-Winter J.
Long-term follow up of punctal and proximal canalicular stenoses
after silicone punctal plug treatment in dry eye patients. Am J
Ophthal-mol 2008;146:968-72
82. Murube J, Murube E, ChenZhuo L, Rivas L. Subcutaneous
ab-dominal artical tears pump-reservoir for severe dry eye. Orbit
2003;22:29-40
83. Floegel I, Horwath-Winter J, Muellner K, Haller-Schober EM.
A conservative blepharoplasty may be a means of alleviating dry eye
symptoms. Acta Ophthalmol Scand 2003;81:230-2
84. Toshida H, Nguyen D, Beuerman R, et al. Evaluation of novel
dry eye model: preganglionic parasympathetic denervation in rabbit.
Invest Ophthalmol Vis Sci 2007;48:4468-75
85. Altinors D, Bozbeyoglu S, Karabay G, Akova YA.. Evaluation
of ocular surface changes in a ra