impression cytology - recent advances and applications in dry eye disease

THE OCULAR SURFACE / APRIL 2009, VOL. 7, NO. 2 / www.theocularsurface.com 93

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].

THE OCULAR SURFACE / APRIL 2009, VOL. 7, NO. 2 / www.theocularsurface.com94

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.)


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.



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.



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



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.



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.)



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


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),



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,



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



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



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.



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.)



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



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



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

impression cytology - recent advances and applications in dry eye disease

Documents

ocular surface cell

use of ic

ocular surface infections

application of ic

ability of ic

technical aspects of

ic techniques

ocular surface