Review

Impression cytology of the ocular surface: a review

Margarita Calonge*, Yolanda Diebold, Victoria Saez, Amalia Enrquez de Salamanca,Carmen Garca-Vazquez, Rosa M. Corrales, Jose M. Herreras

Instituto Universitario de Oftalmobiologa Aplicada (IOBA), University of Valladolid, Ramon y Cajal, 7, E-47005 Valladolid, Spain

Received 5 September 2003; accepted 15 September 2003

Abstract

Purpose. To historically review the technique of impression cytology as a minimally invasive diagnostic tool for ocular surface pathology.

Methods. A comprehensive review of published literature cited in PubMed since the first description of impression cytology in 1977 up to

date has been undertaken.

Results. A wide range of processing methods have been adapted to the technique of impression cytology (from conjunctiva, cornea or

limbus): regular light microscopy with different stainings, transmission and scanning electron microscopy, immunofluorescence,

immunocytochemistry, polymerase chain reaction analysis, immunoblotting analyses, or flow cytometry. At present, it is widely used as a

non-invasive alternative to the full-thickness biopsy for the obtention of epithelial cells from the ocular surface.

Conclusions. Impression cytology represents a non- or minimally invasive biopsy of the ocular surface epithelium with no side effects or

contraindications. It has demonstrated to be a useful diagnostic aid for a wide variety of processes involving the ocular surface. In addition,

and mainly during the last decade, its use as a research tool has experienced an enormous growth and has greatly contributed to the

understanding of ocular surface pathology.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: blepharitis; conjunctiva; cornea; dry eye; epithelium; goblet cells; impression cytology; keratoconjunctivitis sicca; mucins; ocular surface; tear film

1. Introduction

Impression cytology (IC) is the technique of collection of

the most superficial layers of the ocular surface by applying

different collecting devices (usually filter papers) so that

cells adherent to that surface are subsequently removed

from the tissue and further processed for a diversity of

techniques. It represents therefore a non- or minimally

invasive biopsy of, usually, the conjunctiva, although it can

be applied to the cornea and the limbal area.

IC was first introduced in 1977 simultaneously by

investigators from Moorfields Eye Hospital in London

(UK) (Thatcher et al., 1977) and from David Maurices

group at the Stanford University School of Medicine in

California (USA) (Egbert et al., 1977).

Thatcher et al. designed this technique with a plastic

impression disc in order to study the cytologic response of

the conjunctiva in various disorders of the ocular surface

and as an alternative to other methods of collecting

conjunctival cells already described at that time, such as

the scraping technique, the cotton swab technique, or the

pipette technique to collect tears. These authors concluded

that IC permitted the study of the cytologic response more

easily and rapidly than the other techniques, overcoming

their drawbacks and being comfortable to the patient (even

with no local anesthesia) (Thatcher et al., 1977).

It was, however, David Maurices group (Egbert et al.,

1977) who, simultaneously, designed the technique of IC as

it is currently used. These authors obtained imprints of the

surface of the bulbar and palpebral conjunctiva with an

absorbent filter, as they were interested in removing the

secretions of the goblet cells. They immediately noticed that

not just goblet cell secretions but also sheets of epithelial

cells (both goblet and non-goblet) were consistently

removed with cellulose filters. After reducing the processing

time up to 1015 min (staining was periodic acid Schiffs,

PAS, and hematoxylin), they showed results in normal

specimens and, additionally, they compared IC findings

0014-4835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

DOI:10.1016/j.exer.2003.09.009

Experimental Eye Research 78 (2004) 457472

www.elsevier.com/locate/yexer

* Corresponding author. Dr Margarita Calonge, Instituto Universitario de

Oftalmobiologa Aplicada (IOBA), University of Valladolid, Ramon y

Cajal, 7, E-47005 Valladolid, Spain.

E-mail address: calonge@ioba.med.uva.es (M. Calonge).

(sections of the membrane were also shown) with excisional

biopsies from adjacent areas in human cadavers and rabbits.

These authors showed that goblet cells and their secretory

granules were easily removed, having different densities in

different parts of the conjunctiva and perfectly matching

previous results with whole mounts of the conjunctiva. In

addition, they proved that up to five layers of epithelial cells

(sometimes the basal layer remained) could be pulled off.

The fact that not just goblet cell imprints but also sheets of

epithelial goblet cells were collected led to the wide use of

IC as a simple biopsy. But some years later, David

Maurice still tried to only remove goblet cell secretions free

from epithelial cells and goblet cells in order to study the

individual behaviour of goblet cells (Hareuveni and

Maurice, 1994).

From this initial description, the opportunity to study

goblet and non-goblet epithelial cells from the ocular

surface in a simple, rapid and atraumatic manner expanded

dramatically. Some years later, IC became the standard

technique to study squamous metaplasia and goblet cell loss

in ocular surface diseases such as dry eye syndrome,

cicatrizing conjunctivitis, chemical injuries, vitamin A

deficiency, and several other disorders of the ocular surface

as well as the effect of diverse medications. The finding of

goblet cells in corneal IC samples became the standard way

to assure the diagnosis of limbal deficiency, and it is still

very much used (Puangsricharem and Tseng, 1997). In the

following years, the conventional IC technique was first

adapted for transmission and scanning electron microscopy

for the study of several ocular surface disorders (Kruse et al.,

1986a,b), for the diagnosis of mucopolysaccharidoses

(Maskin and Bode, 1986), and also for the detection of

microorganism invasion of the ocular surface (Florakis et al.,

1988).

IC is at present in constant expansion, due to its

unquestionable advantages (Dart, 1997): (1) it is an

invaluable source of intact and well preserved epithelial

cells from the ocular surface from both normal subjects and

any kind of ocular surface pathology; (2) it is a nonsurgical,

minimally invasive, easy to perform, quick, and cheap

technique, that can always be done on an outpatient basis;

(3) only topical anesthesia is required, causing no

discomfort to the patient and no side effects or contra-

indications have been ever noticed; it can be applied to

children; (4) repeated IC sampling in the same patient along

time is an excellent way to demonstrate changes due to a

certain event (e.g.) contact lens wear, to monitor the

progress of a disease (i.e. dry eye syndrome, alkali burns,

etc.), or to follow the effect of a therapeutic intervention; (5)

IC provides a flat mount of an area as big as the surface of

the filter, avoiding the problems of conjunctival smears,

scrapings or brush cytology, which may destroy much of the

cell morphology and do not allow one to see cells the way

they are placed together in vivo, maintaining cellcell

contacts; (6) IC samples can be processed by a wide range of

techniques, from any kind of microscopy to polymerase

chain reaction (PCR), immunoblotting analyses or flow

cytometry.

Based on all these advantages, IC has become the

technique of choice for sampling ocular surface epithelium,

being in constant expansion as a very useful research tool in

both basic and clinical aspects.

2. Literature search

A medline (PubMedNCBI http://ncbi.nlm.nih.gov/)

search has been conducted matching the term impression

cytology with either eye or ocular. Additional more

limited searches were also conducted to seek several aspects

(i.e. electron microscopy and IC), which brought additional

reports. First references were found in 1977 (Thatcher et al.,

1977; Egbert et al., 1977), with a slight increase until the

end of the 1980s, from where the numbers of papers using

IC has constantly increased up to present level. The

approximate number of total publications found were over

450. All publications in English and Spanish were consulted

in their entire length as well as the English abstracts

available for those in other languages.

3. Literature review

Since the publication of the first reports on IC in 1977

(Egbert et al., 1977; Thatcher et al., 1977), many

modifications have been introduced, regarding aspects like

IC sampling, processing methods or the different appli-

cations. All these aspects are summarized below.

3.1. Techniques of cell collection

3.1.1. Materials used for cell collection

Regarding the materials used to collect cells from the

ocular surface, paper filters of different kind have been most

often used, with a few exceptions. One of these exceptions is

the first description of the technique by Thatcher et al. using

a plastic (polystyrene) applanation cytometer that was

pressed onto the conjunctiva and then snapped off and

processed (Thatcher et al., 1977). Some other authors have

used plastic materials such as plastic discs (Thermanox)

(Hershenfeld et al., 1981) or glass slides (Zaidman and

Billingsley, 1998).

Egbert et al. first reported good results using filters

composed of mixed esters of cellulose with submicroscopic

pores (MF-Millipore, type VS), after experiencing bad

results with cellophane tape, photographic film, and various

synthetic filters (Duralon, Polyvic, Mitex) (Egbert et al.,

1977). Later, and since the report by Nelson et al. using the

same cellulose acetate filters with a pore size of 0025 mm(Nelson et al., 1983), most authors agree to use paper filters

with pore sizes ranging between 0025 and 045 mm. Poresize is not irrelevant, as it affects the consistency of cell

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472458

collection (the larger the pore size, the better the collection)

and the resolution of the details under the microscope (small

pore size preserves details better) (Vadrevu and Fullard,

1994), and it has actually been defined that a medium pore

size of 022 mm renders the best results (Martinez et al.,1995).

In addition to cellulose acetate filters, other materials

have been used, such as nitrocellulose, Biopore membranes

or polyether sulfone filters. Biopore membranes (Millicell-

CM 04 mm, Millipore) have been preferentially used for

immunohistochemistry, either unmounted (Pflugfelder et al.,

1990) or mounted (Thiel et al., 1997). These membranes

have also been used for ELISA (Garcher et al., 1998) and for

the study of neoplasms of the ocular surface (Tole et al.,

2001).

Filters are usually cut in different shapes and sizes for

orientation purposes during processing and then applied to

the conjunctiva with forceps. They are pressed onto the

ocular surface, usually for 35 sec, originally with the aid

of a solid rod for 35 sec (Egbert et al., 1977; Adams et al.,

1988; Saini et al., 1990), and later with similar devices

(Nelson et al., 1983; Donisi et al., 2003). Alternatively, the

lateral aspect or the blunt end of forceps used to manipulate

filters can be used (Tseng, 1985). Nelson, in an attempt to

standardize the technique, introduced the use of an

ophthalmodynamometer so that the same amount of

pressure (40 g) was always applied to the filter (Nelson,

1988; Krenzer and Freddo, 1997). It was later demonstrated

that a pressure of 60 g gave better results than either 80 or

40 g (Martinez et al., 1995).

No anesthesia was first used in the method (plastic disk)

described by Thatcher et al. (Thatcher et al., 1977). Egbert

et al. described a frequent pricking sensation when epithelial

cells were pulled off with an absorbent filter and no

anesthesia (Egbert et al., 1977). At present, topical

anesthesia is consistently used, mainly if more adherent

filter papers are applied because the removal of cells can

cause discomfort without anesthesia but is absolutely

painless when a drop of topical anesthesia is instilled.

Although some authors initially suggested that topical

anesthetics might cause artifacts (Thatcher et al., 1977), it

was alternatively suggested from the beginning (Egbert

et al., 1977) that some anesthetics (05% proparacaine)

would not alter the morphologic appearance of cells (Nelson

et al., 1983). However, the influence of topical anesthesia on

the results of IC has not been evaluated.

IC has also been taken from buccal mucosa in cases of

Sjogrens syndrome and compared with labial salivary gland

biopsies, with a rate of agreement of 97%; pathological

changes were found in 94% of the conjunctival IC specimens

and 76% of buccal specimens (Aguilar et al., 1991).

Finally, Adams first described a similar method to that of

the IC to study the morphologic features of human

conjunctival mucus (Adams, 1979). In normal conditions,

more than 100 granules per mm2, sheets, and strands are

visible (Tseng et al., 1987; Herreras et al., 1992).

3.2. Processing methods

3.2.1. Light microscopy

Although over the last decade many techniques have

utilised IC samples, still light microscopy is the most used.

After the specimens have been fixed, different stain can be

used. Early reports used PAS to stain goblet cells and their

secretions and hematoxylin as counterstain to stain

epithelial cells (Egbert et al., 1977; Adams, 1979; Nelson,

1988; Adams et al., 1988). Both PAS and Papanicolau have

also been used together (Saini et al., 1990). Tseng modified

the conventional hematoxylin counterstain by the PAS and

Gills modified Papanicolaus stain, claiming to better

interpret the epithelial changes of squamous metaplasia

such as the metachronic changes of the cytoplasm and the

distinct nuclear patterns (Tseng, 1985). This staining

protocol has been and is still widely used. Alcian blue

staining has sometimes been used instead of PAS (Maskin

and Bode, 1986; Chowdhury et al., 1996). Other stain used

are a modified Wrights stain (Diff-Quik) (Hershenfeld et al.,

1981), the May-Grundwald and Giemsa stain (Midena et al.,

1991), carbol fuchsin (Chowdhury et al., 1996), or the PAS-

Giemsa (Figs. 13) (Saez et al., 2001).

Some authors initially described good results when cells

removed with filters were transferred onto a glass slide to be

viewed by light microscopy (Luzeau et al., 1988). Although

IC with transfer has not become popular for light

microscopy, it is routinely used for immunocytochemistry,

as it will be detailed later.

In order to evaluate IC specimens under the light

microscope, several features are universally evaluated: (1)

the quality of epithelial cells, i.e. the degree of squamous

metaplasia for which the cell size and shape, the

nuclear:cytoplasmic (N/C) ratio and the epithelial cell area

need to be determined; (2) the density, shape and PAS

Fig. 1. Conjunctival impression cytology specimen from a patient

diagnosed of mild blepharitis. Although this sample can be considered

normal, there is, however, a high density of goblet cell secretions (PAS

positive dots); in addition a sheet of PAS positive material can be observed,

somehow obscuring the view of the normal epithelial cells (Polyether

sulfone filters and PAS-Giemsa staining, 40).

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472 459

intensity of goblet cells present; (3) the presence non-

epithelial cells, i.e. inflammatory cells, microorganisms, etc.

It is important to understand that IC removes 13

layers (although sometimes even the basal layer) and it is

not therefore comparable to flat mounts or sections of the

ocular surface. For this reason, goblet cell densities

found with IC cannot be compared with goblet cell

densities harvested with biopsies. However, comparisons

can be made among IC specimens provided the way of

obtaining samples have been similar (Nelson, 1988).

Several grading systems have been published. Although

recognizing the usefulness of most of them, only the most

general ones are outlined below. Table 1 also summarizes

the most important characteristics of these grading systems.

3.2.1.1. Nelsons classification. In 1983, Nelson developed a

four-grade system (Nelson et al., 1983) that was improved

some years later (Nelson, 1988). It is still a widely used

classification system to evaluate the morphology of con-

junctival ocular surface and the degree of squamous

metaplasia by using a specific criteria based on the

appearance of the epithelial cells (morphological changes

in the nucleus, N/C ratio, and metachromatic changes in the

cytoplasm) and the density of the goblet cells and

subsequently assigning a grade (03) to the ocular surface,

where grades 0 and 1 are considered normal, whereas grades

2 and 3 are abnormal (Table 1). Based on this classification,

and attributing a grade to the interpalpebral bulbar and the

inferior palpebral conjunctiva, Nelson stated that squamous

metaplasia confined to the bulbar regions suggested

keratoconjunctivitis sicca or extrinsic disease, but if this

finding was present in both conjunctivas, the suspected

process should be what he called intrinsic ocular surface

diseases (cicatrizing conjunctivitis, alkali burns) (Nelson,

1988).

Interestingly, the cell size and N/C ratio have been

recently quantified by planimetry, correlating well with

Nelsons grading system (Blades and Doughty, 2000;

Doughty et al., 2000).

3.2.1.2. Adams classification. Also in 1988, Adams et al.

defined a simple scoring system of four grades based on

goblet and non-goblet epithelial cell morphology, also

considering the presence or absence of inflammatory cells.

The changes occurring from grade 0 to grade 3 were the

features representing squamous metaplasia (Table 1). Using

this grading system and repeating IC over the course of a

disease, authors were able to monitor the progression of a

disease or the changes occurring during treatment. They

found highly abnormal IC samples in severely damaged

eyes, returning to normal as the disease improved. The

sensitivity of the goblet cell population to disease was also

remarkable, its decrease being an early sign of squamous

metaplasia and a non-specific indicator of ocular surface

disease (Adams et al., 1988).

3.2.1.3. Tsengs classification. In 1985, Tseng proposed a

modification of the conventional IC technique so that

squamous metaplasia changes could be progressively

defined from normal (stage 0) to advanced keratinization

(stage 5). The first pathologic finding, meaning the

transition from a secretory to a nonsecretory epithelium is

recorded as early (stage 1) and total (stage 2) loss of goblet

cells. Stages 35 mean progression from early to moderate

and advanced keratinization (Table 1). Upon this author,

this grading system allows one to make the diagnosis of

squamous metaplasia more accurately than with previous

systems (Tseng, 1985). This grading system is one of the

most commonly used at present.

Fig. 2. This conjunctival impression cytology belongs to a patient

diagnosed of aqueous-deficient dry eye, in addition to meibomian gland

disease (evaporative dry eye). Goblet cells are absent. The cytoplasm of

epithelial cells is larger and the nucleo:cytoplasm ratio is between 1:4 and

1:6. It would correspond to a grade 3 of Adams et al.s classification, to a

grade 23 of Nelsons grading system and to a stage 4 in Tsengs

classification (Polyether sulfone filters and PAS-Giemsa staining, 40).

Fig. 3. This conjunctival impression cytology from a contact lens wearer

with mild dry eye shows typical snake-like chromatin arrangements in all

cell nuclei (Polyether sulfone filters and PAS-Giemsa staining, 40).

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472460

3.2.2. Electron microscopy

The very first description of IC filters processed for

electron microscopy, both transmission and scanning,

occurred nine years after the initial description of IC and

its processing for light microscopy. Kruse and colleagues

studied the ultrastructural characteristics of some ocular

surface diseases such as Sjogren syndrome (Kruse et al.,

1986a,b). The first description in the English literature came

from Maskin and Bode, who were able to make comparative

studies of excisional biopsies and impression specimens of

mucopolysaccharidosis cases (Maskin and Bode, 1986).

These authors established the potential of electron

microscopy-processed IC samples to study the subcellular,

cellular and intercellular morphology in ocular surface

disorders.

Some years later, electron microscopy of IC samples

allowed the demonstration of viral particles (retrovirus) in

the conjunctiva of AIDS patients suffering from cytomega-

lovirus retinitis (Pastor et al., 1991). Additionally, IC

samples processed for scanning and transmission

microscopy from long-term lens contact wearers, allowed

one to study the fine structure of snake-like chromatin

changes, attributing them to mechanical stress (Knop and

Reale, 1994). Moreover, the ultrastructure of squamous

metaplasia changes (Meller, 1996) and the fine structure of

chromatin alterations in dry eye patients (Meller, 1999)

have been well defined in IC specimens processed for

electron microscopy. Finally, immunoelectron microscopy

was used to study the binding of a monoclonal antibody

(H185, which recognizes carbohydrate epitopes on mucin

molecules) to conjunctival cell obtained by IC with

nitrocellulose filter papers (Danjo et al., 1998).

3.2.3. Immunocytochemistry

The technique of IC was successfully adapted for

immunocytochemistry in 1990 by Pflugfelder et al., who

referred to a 1989 ARVO presentation by Iwata and Burris.

They used Millicell-CM transparent Biopore membranes,

which were directly incubated with primary monoclonal

antibody, then with the fluorescein-conjugated secondary

Table 1

Main grading systems for conjunctival impression cytology specimens processed for light microscopy

Goblet cells Non-goblet epithelial cells

Size/form Density PAS

staining

Size/form Cyto staining N size N/C ratio Other

Tseng,

1985

Stage 0: normal

conj epithelium

NS Moderate NS Uniform Blue to green 1:1

Stage 1: early

GC loss, no K

NS Decreased NS Mild enlargement Blue to green 1:21:3

Stage 2: total

GC loss, no K

NS Absent NS Moderate enlargement

flattened (squamoid)

Blue-green to

mild pinkish

1:4

Stage 3: early

and mild K

NS Absent NS Markedly squamoid Pinkish 1:6 Visible KF mild

pyknotic N

Stage 4:

moderate K

NS Absent NS Markedly squamoid

large

Pinkish 1:8 Densely packed KF,

pyknotic N

Keratohyaline gr

Stage 5:

advanced K

NS Absent NS Shrunken cytoplasm NS May

be absent

NS Densely packed IF,

pyknotic N

Nelson,

1988

Grade 0 Plump,

oval

Abundant Intense Small, round Eosinophilic Large 1:2 Basophilic N

Grade 1 Plump,

oval

Decreased intense Slightly larger

more polygonal

Eosinophilic Smaller 1:3 NS

Grade 2 Smaller,

poorly

defined

border

Markedly

decreased

Less

intense

Larger polygonal Variable Small 1:41:5 Occasionally MN

Grade 3 NS Very few NS Large polygonal Basophilic Small .1:6 Pyknotic

Adams

et al., 1988

Grade 0 NS Abundant Intense Normal NS Normal 1:2 Good cell sheet

Grade 1 NS Slightly

decreased

Intense Larger NS NS 1:3 Good cell sheet

Grade 2 NS Decreased Reduced Larger NS NS NS Reduced cell sheet

Grade 3 NS Very

decreased

Pale Large, irregular NS Small NS Poor cell sheet

Conj, conjunctiva; Cyto, cytoplasm; GC, goblet cells; gr, granules; K, keratinization; KF, keratin filaments; MN, multinucleated; N, nucleus; NS, not

specified; PAS, periodic acid Schiffs staining.

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472 461

antibody and finally placed on a gelatin-coated slide

(Pflugfelder et al., 1990). Immunofluorescence on Biopore

membranes has been used and it is still used by the same

research group (Jones et al., 1994; Liu et al., 2000; Solomon

et al., 2001). Biopore membranes, but this time mounted,

have also been used for immunoperoxidase (taking 4 hr) and

immunofluorescence (taking 1 hr) to demonstrate the

presence of viral antigens in corneal or conjunctival IC

samples, with the advantage of the many cells that IC

provided for diagnosis (Thiel et al., 1997).

Baudouin et al. first performed immunofluorescence by

transferring cells from a filter paper to gelatin-coated glass

slides. They initially used cellulose acetate filters (Baudouin

et al., 1992, 1994, 1997a) and then polyether sulfone filters

in an attempt to increase the yield of cell removal (Baudouin

et al., 1997b). Recently, immunofluorescence has been

successfully done with Whatman nitrocellulose filters. The

adherent nitrocellulose strips were place on slides air dried

and washed repeatedly with 100% methanol until nitrocel-

lulose was totally dissolved (Avunduk et al., 2000, 2001).

Very recently, immunofluorescence has been tried with

polyether sulfone filters, without transferring (Baudouin

et al., 2003). We have been working with this technique by

processing two conjunctival IC samples from the same

patient and eye taken at the same moment from adjacent

areas, one being transferred to a coated glass slide and the

other being stained on the filter. In order to view the stained

cells, a confocal microscope is mandatory so that focus can

be moved in order to avoid the fluorescence coming from

the filter itself. This immunostaining made directly on the

filter paper seems to be promising because it avoids losing

cells during the transference step, while the quality seems to

remain unaltered (Fig. 4).

In 1993 was the first report of processing of IC with

immunohistochemistry other than with fluorescence. Cor-

neal IC specimens taken with nitrocellulose filters were

successfully stained by the peroxidase-antiperoxidase

method (Nakagawa et al., 1993). IC on cellulose acetate

filters has also been successfully processed for immunohis-

tochemical staining using a sensitive immunoenzymatic

alkaline phosphatase-monoclonal/anti-alkaline phosphatase

complex procedure (Leonardi et al., 2000). Recently,

immunoperoxidase staining has been performed directly

on Biopore membranes with corneal cells harvested by IC

(Donisi et al., 2003).

3.2.4. Polymerase chain reaction

IC samples have also been processed for RT-PCR

analysis. Jones et al. reported the expression of a panel of

inflammatory cytokines and ICAM-1 by RT-PCR in

epithelial cells of Sjogrens syndrome patients (Jones et al.,

1994). This study was later expanded and many more

cytokines have been found by PCR in IC samples

(Pflugfelder et al., 1999). Recently, corneal and conjunctival

IC samples processed for RT-PCR have served to

demonstrate the expression of the natural antibacterial

peptide b defensin in the ocular surface (Lehmann et al.,2000). In addition, cells harvested from the ocular surface

by IC are being used to investigate DNA polymorphisms

using PCR to examine the survival of donor human limbal

stem cells (Williams et al., 1995; Henderson et al., 2001a,b).

Conjunctival IC has also been used to demonstrate the

presence of a panel of ocular mucin genes in the normal

ocular surface by either RT-PCR (Inatomi et al., 1995;

Corrales et al., 2003a,b) or real time PCR (Argueso et al.,

2002; Corrales et al., 2003c). These approaches were also

used in several conditions such as contact lens wearing

(Corrales et al., 2003d) or dry eye (Argueso et al., 2002;

Calonge et al., 2003). In addition, real time PCR-processed

IC samples have been adequate to study the expression of

antioxidant enzyme genes in the human conjunctiva in

normal conditions (Herreras et al., 2003) and in contact lens

wearers (Galarreta et al., 2003). In general, nitrocellulose or

polyether sulfone filters have been used for PCR analyses.

Fig. 4. Conjunctival impression cytology specimens from the same normal

donor immunostained with antibody anti-muscarinic type 2 receptor.

Epithelial cells were harvested with polyether sulfone paper filters, indirect

imunofluorescence was performed and specimens were viewed with a

confocal microscope. The immunostaining procedure was made in two

different settings: (a) after transferring cells from the filter paper to a poly-

L-lysine-coated slide (note that many cells have been lost during the

transference); or (b) directly on the filter paper. Positive green

immunostaining can be seen around the red counterstained nuclei with

propidium iodide ( 63).

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472462

3.2.5. Flow cytometry

The application of this technique to IC samples was first

reported in 1997 by Baudouin et al., who performed HLA-

DR and CD23 analysis in IC samples processed for flow

cytometry from normal and inflamed tissue by using

polyether sulfone filters; findings with this technique

correlated significantly with findings with immunocytology

(Baudouin et al., 1997a,b). The development of flow

cytometry for the analysis of IC specimens has provided a

very sensitive, rapid, and objective tool to investigate ocular

surface pathology and monitor the effects of drug therapy

and it is very much used at present (Baudouin et al., 1997b;

Pisella et al., 2000; Brignole et al., 2000, 2001).

3.2.6. Immunoblot analysis

Krenzer et al. first developed a technique to extract

proteins by SDS-PAGE from IC samples (nitrocellulose

filters) that were further processed, in addition to immuno-

cytochemistry and immunofluorescence, for gel electro-

phoresis and subsequent western blot analysis (Krenzer and

Freddo, 1997). These authors defined the normal pattern of

expression of cytokeratins in the bulbar conjunctiva, giving

the tools to further research cytokeratin patterns in ocular

surface diseases. Additionally, western blot analyses,

correlating with immunofluorescent staining, have shown

the increased expression of the type 1 growth factor

receptors in the conjunctival IC samples of KCS patients

(Liu et al., 2000).

3.2.7. Other techniques

Immunoenzymofluorometry (CA 19-9 ELISA) has been

successfully applied to study mucus changes in conjunctival

specimens collected with Biopore membranes (Garcher

et al., 1998).

Pflugfelder et al. also used conjunctival IC lysates from

normals and Sjogrens patients to measure IL-6 protein

concentration with a commercial ELISA kit (Pflugfelder

et al., 1999).

Recently, Shen et al. have successfully adapted an acid

esterase staining technique to IC specimens and the use of

objective microspectrophotometry to measure staining

intensity (Shen et al., 2002).

3.3. Applications of impression cytology

3.3.1. Description of the normal ocular surface

IC has been used to describe the ocular surface in normal

conditions, often, as part of the required normal controls of

pathologic samples. However, some authors have studied

different aspects of the normal ocular surface only in normal

conditions. For instance, the variation in total cell counts

recovered by IC over a period of nine waking hours has been

performed, finding higher cell counts on waking, decreasing

thereafter, which was attributed to the neutrophils existing

upon waking (Hirji et al., 1984). Some years later, the IC

technique was used to describe the normal pattern of

conjunctival and corneal epithelium and their topographic

variations. IC samples were taken from 24 different

locations in the conjunctiva and from four places in the

cornea. Interestingly, an increase in N/C ratio was described

as specimens approached the limbal area, where it was

greater than 1:3 (Rivas et al., 1991).

In 1992, and using Nelsons classification (Nelson,

1988), normal conjunctiva of 30 healthy donors was

shown to be grade 0 in 26 subjects and grade 1 in four

individuals (Gadkari et al., 1992), which basically agrees

with studies from Adar et al., demonstrating 90% of grade 0

and 10% of grade 1 in 50 eyes of 25 healthy controls (Adar

et al., 1997) or from Ozkan et al., with 100% of grade 0

(from upper bulbar) or 85% grade 0 and 15% grade 1 (from

temporal bulbar) in 20 normal subjects (Ozkan et al., 1997).

Surprisingly, IC has also demonstrated variations in the

number of goblet cells according to gender, with women

having less goblet cells and lowest numbers around the time

of ovulation, while contraceptive intake increased these

numbers, suggesting a reproductive hormonal influence on

conjunctival goblet cell density (Connor et al., 1999).

Regarding correlations with age, no correlation between the

number of goblet cells harvested from IC and normal

volunteers with age was found, whereas a negative

correlation existed between N/C ratio and age (Paschides

et al., 1991). An interesting study demonstrated that

repetition of sampling in the same area (at days 04, 8

and 12) produced a localized decrease in goblet cell density

and an increase in N/C ratio, that recovered after 4 days

(Rolando et al., 1994).

Regarding snake-like chromatin, which was supposed to

be present only in pathologic conditions, Bjerrum described

it in 39% of normal elderly subjects (mean age was 64

years) (Bjerrum, 1995).

Another interesting finding was the demonstration that

human conjunctival epithelial cells from normals (as well as

that from Sjogrens syndrome patients) harbor latent

EpsteinBarr virus infection (Jones et al., 1994).

Immunofluorescence and immunohistochemistry studies

in normal controls have also shown that conjunctival

dendriform cells (mainly Langerhans cells) from normal

or inflammatory specimens had a similar immunopheno-

type, although the percentage of such cells was 15% in

normals and up to 13% in inflamed conjunctivas (Baudouin

et al., 1997a).

IC samples processed for immunoblotting techniques

have allowed one to define the pattern of cytokeratin

expression in the normal human conjunctiva as constituted

by cytokeratins characteristic of nonkeratinized, stratified

epithelia, in addition to other more typical of a simple

differentiation pattern, a glandular differentiation pattern or

both (Krenzer and Freddo, 1997). These approaches should

be further expanded to pathological conditions.

Recently, Liu et al. have described the expression in

human ocular surface epithelia of the receptor tyrosine

kinases, epidermal growth factor receptor (preferentially

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472 463

located in the basal epithelium), ErbB2, and ErbB3 (mainly

expressed by superficial epithelia) by immunofluorescent

staining of samples from conjunctiva, limbus and cornea

(Liu et al., 2000, 2001).

Our group has recently defined the range of transcripts of

conjunctival mucin genes present in normal healthy donors

(MUC1, MUC2, MUC4, MUC5AC, MUC7, MUC13,

MUC15, MUC16, MUC17) (Corrales et al., 2003a) and

have additionally defined threshold levels of normality for

MUC1, MUC2, MUC4, MUC5AC, and MUC7 (Corrales

et al., 2003c) in conjunctival IC samples.

3.3.2. Diagnostic aid in dry eye syndrome

Squamous metaplasia is a very typical phenomenon in

dry eye states although its origin is ubiquitous and it occurs

in multiple ocular surface disorders: cicatrizing conjuncti-

vitis, some chronic non-cicatrizing conjunctivitis (superior

limbic keratoconjunctivitis), vitamin A deficiency, dyspla-

sia/neoplasia of the ocular surface, long-term contact lens

wear, etc. and of course in dry eye disorders. Squamous

metaplasia is a continuous, dynamic process of abnormal

epithelial differentiation and it means the pathologic

transition from a nonkeratinized, stratified (secretory or

nonsecretory) epithelium (such as conjunctival or corneal)

to a nonsecretory keratinized epithelium. During squamous

metaplasia of the conjunctival epithelium, there is a

continuous spectrum of changes, with an early decrease

and eventual loss of goblet cells and progressive morpho-

logical changes of non-goblet epithelial cells such as

increased stratification and keratinization. Round blue

cells with N/C ratios of 1:1 transform into more elongated

and flattened (squamoid) cells with metachromatic changes

of the cytoplasm (pinkish color), and N/C ratios increasing

up to 1:8 and becoming pyknotic. As noted, these changes

occur in a variety of ocular surface disorders, mainly in dry

eye-related conditions (Tseng, 1985; Adams et al., 1988;

Nelson, 1988; Pflugfelder et al., 1990; Meller et al., 1996;

Murube and Rivas, 2003).

IC has been shown to be extremely helpful in the diagnosis

of various types of dry eye states as they are usually

accompanied by progressive squamous metaplasia of the

ocular surface and goblet cell decrease as the disease gets

more severe. In aqueous-deficient dry eye, it has been

demonstrated that the inferior palpebral conjunctiva is much

less affected than the exposed bulbar interpalpebral, provided

that no other factors are present, such as blepharitis or drug

toxicity.(Nelson and Wright, 1984; Tseng, 1985; Rolando

et al., 1990; Rivas et al., 1993). Some authors claimed that IC

and rose Bengal staining are the most specific and sensitive

methods for the diagnosis of primary Sjogrens syndrome

(Rivas et al., 1993).

Marner was the first author to describe the snake-like

appearance of chromatin in nuclei of epithelial cells from

dry eye patients (Fig. 3), and this finding was correlated with

severity of disease (Marner, 1980). Later on, many other

authors reported on this finding (Meller, 1999) and it is at

present clear that it is not exclusive to dry eye conditions

(Knop and Reale, 1994).

IC has also made an important contribution to the

understanding of dry eye as an immune-based inflammatory

condition (Stern et al., 1998). With IC and immunofluor-

escence, Pflugfelder et al. showed that between 60 and 80%

of the inflammatory cells seen in the inferior fornix of

Sjogrens syndrome patients were T lymphocytes (CD3

positive), correlating with findings in excisional biopsies by

immunoperoxidase staining (Pflugfelder et al., 1990). Later,

conjunctival epithelial cells from dry eyes harvested by IC

have been shown to overexpress inflammatory markers such

as HLA-DR, ICAM-1, the low affinity receptor for IgE

CD23, CD40-CD40L, or Fas and APO27 levels by

immunocytochemistry and flow cytometry (Baudouin

et al., 1992, 1997b; Bourcier et al., 2000; Pisella et al.,

2000; Brignole et al., 2000, 2001; Solomon et al., 2001).

Additionally, IC samples from Sjogrens patients have been

shown to express higher levels of ICAM-1 and many

proinflammatory cytokines in the conjunctival epithelium,

analysed by immunofluorescence, RT-PCR and ELISA

(Jones et al., 1994; Pflugfelder et al., 1999; Solomon et al.,

2001). These authors also demonstrated with IC samples

processed by RT-PCR that EpsteinBarr virus latent or lytic

infection of the conjunctiva is not an etiologic factor in the

ocular surface pathology of primary Sjogrens syndrome

(Jones et al., 1994).

Danjo et al. have defined the pattern of mucin binding to

an antibody recognizing carbohydrate epitopes by immuno-

cytochemistry and immunoelectron microscopy of IC

specimens. The pattern found in dry eye specimens,

described as starry sky, (lack of apical binding but

increased binding to goblet cells) clearly differed from the

one seen in normal conditions (mosaic pattern) (Danjo et al.,

1998). The levels of ocular mucin genes are at present being

characterized. So far, the gel-forming mucin gene

MUC5AC has been found to be decreased in Sjogren

syndrome patients IC samples (Argueso et al., 2002). In

addition, our group has reported a decrease in the number of

transcripts for several mucin genes (MUC1, MUC2, MUC4

and MUC5AC) in conjunctival epithelium from dry eye

patients harvested by IC and processed for real time PCR

(Calonge et al., 2003).

3.3.3. Aid in the diagnosis of chronic conjunctivitis

The demonstration of eosinophils in conjunctival IC

from allergic rhinoconjunctivitis patients is useful, but about

15% of cases do not show eosinophils (Sapci et al., 1999).

The real value of IC to demonstrate typical cells from

allergies remains to be investigated. Although vernal

patients show altered cell junctions, chromatin changes

and higher number of goblet cells (Aragona et al., 1996),

these findings do not help in the diagnosis of this disease.

Studies conducted by Baudouins group have greatly

contributed to the understanding of the etiology of chronic

inflammation in the ocular surface other than dry eye.

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472464

Special processing of IC samples has allowed the study of

the expression of several inflammatory markers in epithelial

cells, showing a higher expression of HLA-DR and the IgE

low affinity receptor CD23, a known inflammation marker

(Baudouin et al., 1997b), and CD40-CD40L (Bourcier et al.,

2000) in chronic allergic disease. Another study using

immunofluorescence on IC samples showed the presence of

CD4, CD8, CD23, and CD45RA (perinuclear staining) in

vernal keratoconjunctivitis (Avunduk et al., 2000). Inter-

estingly, the density of dendriform conjunctival cells was

higher in IC specimens from chronic inflammatory disorders

of the ocular surface than in normals, although the

immunophenotypic characterization was similar (Baudouin

et al., 1997a).

Lastly, decreased goblet cell numbers and squamous

metaplasia has been found in atopic dermatitis patients,

which was not correlated with duration of disease but rather

with the number of recurrences (Dogru et al., 1998).

IC has also been used as an aid in the diagnosis of

cicatrizing conjunctivitis. Initially, Nelson demonstrated the

percentage decrease in goblet cell numbers in these

conditions (Nelson and Wright, 1984). In these cicatrizing

conjunctivities such as ocular cicatricial pemphigoid,

Stevens-Johnsons syndrome or severe chemical injuries,

and in contrast with dry eye, squamous metaplasia and loss

of goblet cells affect the bulbar or palpebral conjunctiva

equally and abundant inflammatory cells can be seen (Ohji

et al., 1987; Nelson, 1988; Sanz et al., 2001). It is, however,

mandatory to understand that the diagnosis of immune-

mediated cicatrizing conjunctivitis, especially ocular cica-

tricial pemphigoid, needs a confirmatory excisional biopsy,

where immune deposition needs to be demonstrated at the

basement membrane zone, as these disease will need

systemic immunosuppression for long-term control (Foster,

1986).

IC has been tried in infectious cicatrizing conjunctivitis,

such as trachoma, where keratinization and conjunctival

scarring develops, IC has demonstrated a marked reduction

of goblet cells (Blodi et al., 1988).

3.3.4. To monitor the impact of contact lenses

on the ocular surface

IC has been widely used to study the impact of contact

lens wearing on the ocular surface. Initially, IC was used to

demonstrate significantly higher cell counts (neutrophils

and lymphocytes) 5 hr after insertion of soft contact lenses

compared with hard contact lenses or control subjects (Hirji

et al., 1985).

Later, Knop and Brewitt described striking conjunctival

changes by IC in contact lens wearers after 6 months of

wearing. Squamous metaplasia and nuclear alterations

(especially snake-like changes of chromatin) (Fig. 3), with

relatively low goblet cell density started a few weeks after

initiating contact lens wear and increased with time,

although wearers were asymptomatic and the conjunctiva

remained clinically normal (Knop and Brewitt, 1992).

Another study also showed epithelial alterations (squamous

metaplasia and goblet cell decreases) notably different from

normals, being more frequent and more severe in sympto-

matic than in asymptomatic wearers (Adar et al., 1997).

An interesting study showed that goblet cell numbers in

the inferior bulbar conjunctiva evaluated in the same

healthy individuals before and monthly up to 6 months

after initiating daily wear contact lens use actually increased

in 88% of them after 5 months, speculating that this could be

an adaptive response to the mechanical irritation caused by

the contact lens (Connor et al., 1994). These authors later

demonstrated that this response was almost absent when

contact lenses were used on a 2-week disposable schedule

(Connor et al., 1997).

The above mentioned alterations in goblet cell numbers

supports the concept that mucins are altered in contact

lens wearers, with some other studies further supporting

this (Pisella et al., 2001). To further analyse the alteration

of the mucin layer in contact lens wearers, our group

studied the expression of different mucin genes by real

time PCR in conjunctival IC specimens, showing that

hydrogel contact lens wearing for 12 months decreased

the expression of MUC2 and MUC7, and increased the

expression of MUC5AC (the most specific goblet cell

mucin gene), MUC16, and MUC17 mucin genes (Corrales

et al., 2003d). This needs to be further investigated.

A sign described as typical of contact lens wearers is the

presence of snakes (Knop and Reale, 1994; Adar et al.,

1997). The fine structure of snake-like chromatin changes at

the electron microscope was defined by Knop and Reale,

hypothesizing that a mechanical stimulus could be respon-

sible for the alteration of the nuclear and cytoplasmic

skeleton, producing their fragmentation and therefore the

appearance of snakes (Knop and Reale, 1994).

HLA-DR and CD23 expression has been reported to be

greater in contact lens wearers compared to normals and

even greater in those contact lens wearers who had dry eye

syndrome (Albietz, 2001). Another study also found,

increased expression of HLA-DR and ICAM-1 by flow

cytometry in IC samples of contact lens wearers compared

to normals (Pisella et al., 2001), suggesting the possibility

that contact lens use could cause inflammation of the

conjunctiva. An additional preliminary study by our group

showed that the capacity of conjunctiva to fight inflam-

mation may be impaired in contact lens wearers. We

evaluated the alteration of antioxidant enzyme genes in the

human conjunctival epithelium of hydrogel contact lens

wearers after 12 months (Galarreta et al., 2003) compared to

that of normals (Herreras et al., 2003), showing a decreased

expression of three (CuZn SOD, GSS, and GSR) of the

genes studied, while two more (MnSOD and ecSOD)

remained unchanged.

3.3.5. To diagnose vitamin A deficiency

Patients at risk of vitamin A deficiency in developed

countries include those with hepatic dysfunction and

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472 465

malasorption states, whose symptoms may go unrecognized

or unreported. In developing countries, the problem of

vitamin A deficiency and its consequences is a real public

health issue and it is mainly due to malnutrition and persistent

diarrhea, especially in small children. Subclinical vitamin A

deficiency puts children at risk of to severe and fatal

infections. In the eye, it produces keratomalacia, the major

cause of preventable childhood blindness in developing

countries. The use of conjunctival IC as a subclinical

indicator of vitamin A deficiency (squamous metaplasia

and goblet cell loss) is very widespread, although its real

usefulness is subjected to controversy. It has been concluded

in general that although it may not be a reliable indicator of a

certain individuals vitamin A status, it can accurately

characterize the risk of vitamin A deficiency in communities,

serving as a cheap and easy to perform screening method and

aiding in the recommendations of which community needs to

be given vitamin A supplements (Underwood, 1994).

3.3.6. To aid in the diagnosis of limbal deficiency

and its management

IC on the cornea has been used from the first limbal

transplantations to demonstrate restoration of the corneal

phenotype and regression of goblet cells (Kenyon and Tseng,

1989). It has also been demonstrated that IC applied in the

limbal area can be used to diagnose and monitor corneal

disease with limbal dysfunction, by showing goblet cells in

the cornea (Chen and Tseng, 1991) as sign of conjunctiva-

lization and predicting those patients who will be poor

candidates for keratoplasty (Puangsricharern and Tseng,

1997). This group also used IC to demonstrate that the

success of conjunctival surface reconstruction with large

patches of amniotic membrane correlated well with recovery

of the conjunctival epithelial phenotype; these patients failed

to show a corneal epithelial phenotype, even in avascular

corneas, proving that the concept of conjunctival transdiffer-

entiation seems not to occur in vivo and also indicating that

additional limbal stem cell transplantation is needed for

corneal reconstruction (Prabhasawat and Tseng, 1997).

More recently, it has been suggested that the positive

staining against cytokeratins 19 and 3 on corneal IC samples

is a simple and practical method to investigate limbal stem

cell deficiency (Donisi et al., 2003). In this way, IC on the

cornea has become an important tool to select those

keratoplasty patients who would additionally benefit from

a limbal stem cell transplantation.

Lastly, the identification of the origin of corneal cells by

DNA fingerprinting is becoming a very interesting tech-

nique to trace the survival of donor human limbal stem cells

(Williams et al., 1995; Henderson et al., 2001a) which has

obvious implications in corneal limbal grafting (Henderson

et al., 2001b,c).

3.3.7. Detection of microorganisms

There are several reports on the successful isolation of

organisms from the ocular surface. Cultures performed with

IC samples taken from conjunctiva and from elevated

corneal epithelial lines were able to grow Acanthamoeba

organisms in two patients (Florakis et al., 1988). Immuno-

fluorescent staining of corneal cells obtained after pressing

the cornea with a glass slide confirmed rabies as the etiology

of idiopathic acute encephalitis in a young girl (Zaidman

and Billingsley, 1998).

Using electron microscopy-processed IC samples, par-

ticles consistent with retrovirus were isolated from the

conjunctiva of acquired immunodeficiency syndrome

patients (Pastor et al., 1991).

IC has also being tried in the diagnosis of herpes simplex

virus (HSV) keratitis. HSV antigens were demonstrated in

30 of 32 patients with HSV keratitis using immunocyto-

chemistry of IC taken from the corneal lesions (Nakagawa

et al., 1993). Recently, collection of IC on a sterile glass

slide with further processing for immunocytochemistry

staining has been described as a simple, rapid (25 hr) and

inexpensive technique for the diagnosis of HSV keratitis

offering positivities in 80% of cases, against virus isolation

in 333% (Athmanathan et al., 2001).

3.3.8. In the evaluation of ocular surface neoplasia (OSSN)

The use of IC has also been evaluated for the specific

study of OSSN. The published positivity of OSSN with IC is

between 77 and 80% of cases confirmed histologically, and

either cellulose acetate or Biopore membranes have been

successfully used (Nolan et al., 1994; Tole et al., 2001). The

difficulty in interpretation of these IC specimens caused by

the paucity of published criteria can be overcome with the

recent publication by Nolan et al. where they describe in

detail the cytomorphology of OSSN based on a high number

of cases (Nolan, et al., 2001). IC has also been used to study

the effects of topical mitomycn C in the treatment of OSSN,

demonstrating that it produces cell death by apoptosis and

necrosis and that changes induced in the ocular surface may

persist for at least 8 months (McKelvie and Daniell, 2001).

There are additional reports on the use of IC for other

related purposes. Interestingly, in 73% of patients with

pigmented lesions, IC predicted the histological diagnosis of

melanocytic tumors by detecting atypical melanocytes

(Paridaens et al., 1992). Lastly, IC immunostained with

cytokeratin antibodies and HMB-45 was useful to differen-

tiate conjunctival seborrheic keratosis masquerading as

malignant melanoma (Tseng et al., 1999).

3.3.9. As a technique to monitor tolerance and efficacy

of therapeutic interventions

IC has been routinely used to prove efficacy of diverse

therapeutic options for dry eye, specially that of artificial

tears. Some authors failed to show reversal of squamous

metaplasia and goblet cell reduction with artificial tear

substitution (Nelson and Farris, 1988), autologous serum

treatment (Tananuvat et al., 2001) or topical retinoid therapy

(Soong et al., 1988). Soluble ocular inserts from porcine

scleral collagen for the treatment of dry eye improved clinical

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472466

signs and reduced the frequency of artificial tear adminis-

tration, but did not reduce the rose Bengal staining or IC

results (Shaker et al., 1989). Later, unpreserved carbox-

ymethylcellulose artificial tears (Grene, 1992) and hyalur-

onate-containing artificial tears (Aragona et al., 2002),

however, produced improvement in the IC grades of KCS

patients. Oral antioxidant therapy for marginal dry eye also

showed improvement in IC samples (Blades et al., 2001).

The same discrepancy is found in the evaluation of the

response to punctal plug occlusion. For instance, IC

abnormalities were shown to persist for 6 weeks after

punctal plug, even though patients had improved clinically

(Willis et al., 1987). Recently, however, improvement in IC

findings has been reported 6 weeks and one year after

punctal occlusion in dry eye patients (Dursun, et al., 2003),

6 months after silicone canalicular plugs were inserted in

trachomatous dry eye (Guzey et al., 2001) or after being

inserted for superior limbic keratoconjunctivitis (Yang et al.,

1997). In this same disease, thermocauterization of the

superior bulbar conjunctiva demonstrated to restore the loss

of goblet cells harvested by IC (Udell et al., 1986).

The number of PAS positive cells (mainly goblet cells) in

IC specimens can be used to judge the efficacy of treatments

that intend to increase its numbers, such as gefarnate

(Toshida et al., 2002) or P2Y2 agonists (Fujihara et al.,

2002). Care needs to be taken though, as sometimes many

PAS positive cells (supposedly those non-goblet epithelial

cells belonging to the secondary secretory system) can be

seen in IC specimens and sometimes a sheet of PAS positive

material can be observed.

Other therapeutic options have used IC findings to

demonstrate their benefits. For instance, treatment with

botulin toxin A-induced protective ptosis in cases of

indolent corneal disease showed recovery of the normal

conjunctival morphology (Kirkness et al., 1988). This

therapy, however, was not effective in improving cytologi-

cal changes in IC samples of dry eye patients who also

suffered from blepharospasm (Horwath-Winter et al., 2003).

IC has also been used to demonstrate the adverse effects

of long-term use of topical medications on the conjunctiva.

Antiglaucoma medications have been shown to induce

conjunctival metaplasia associated with the number of

medications used (Brandt et al., 1991). Another prospective

study showed no significant epithelial cell damage but

significant and progressive goblet cell decrease in patients

after 13 months of topical treatment with one preserved

antiglaucoma medication compared to baseline conditions

(same patients before initiating treatment) (Herreras et al.,

1992). These effects can be attributable to the medications

themselves, the preservatives and/or the duration of topical

treatment. Later, Baudouin et al. showed abnormal

expression of inflammatory markers (by immunofluores-

cence on IC samples) in conjunctival cells in the absence of

clinical inflammation in patients receiving preserved

(especially with benzalkonium chloride) antiglaucoma

drops chronically, which correlated with failure in filtering

glaucoma surgery (Baudouin et al., 1994). One study using

IC samples taken from the conjunctival surface after

filtering surgery showed long-term damage of the con-

junctival epithelium overlying filtering blebs, especially in

those patients treated with mitomicyn C (Kim, 1997). This

study, however, was not prospective and therefore changes

could have been already present before surgery due to

antiglaucoma medications. A posterior study showed

persistent HLA-DR overexpression on conjunctival cells

measured in IC by flow cytometry still 6 months after

glaucoma surgery, indicating the increased ability of

epithelial cells to induce inflammation and possibly

subsequent fibrosis (Ihan and Cvenkel, 2000).

The fact that preserved medications alter the ocular

surface is at present beyond any doubt and IC has greatly

contributed to its demonstration (Herreras et al., 1992;

Baudouin et al., 1994; Albietz and Bruce, 2001; Cvenkel

and Ohan, 2002).

Immunocytochemistry-processed IC samples can also be

used to evaluate the efficacy of certain treatments. For

instance, markers such as CD4, CD8, CD23 or CD45RA

have been determined on conjunctival IC samples by

immunofluorescence, in vernal keratoconjunctiviis speci-

mens in order to demonstrate the efficacy of treatment with

lodoxamide or cromoglycate (Avunduk et al., 2000) or with

topical cyclosporin A (Avunduk et al., 2001). Patients

treated with either drug had significantly lower CD4 and

CD23-positive cells, whereas the other two markers were

not affected. Recently, IC from patients having a con-

junctival provocation test had increased expression of

ICAM-1 as determined by immunohistochemical staining,

that significantly decrease after treatment with ketorolac

tromethamine (Leonardi et al., 2000).

But it is probably flow cytometry is one of the best

techniques to process IC specimens in order to objectively

and reliably prove the pharmacological effect of a certain

drug. For instance, the treatment of dry eye with topical

cyclosporin A significantly reduced HLA-DR and CD40

expression as well as the percentage of Fas-positive cells,

while APO27 expression was significantly increased in

conjunctival IC samples of dry eye patients processed for

flow cytometry (Brignole et al., 2001).

Finally, the use of IC to evaluate the success of diverse

transplantations of the ocular surface has also been already

commented. In addition, IC and the restoration of the

normal conjunctival phenotype have also been used to

evaluate the adequate healing of the conjunctival surface

after pterygium surgery with different treatment modalities,

showing that even though the healing is delayed with the use

of mitomycin C and promoted with autografting, the

conjunctival phenotype is still abnormal even one year

after surgery (Tseng at al., 2001).

3.3.10. Other applications

Some other interesting studies have described changes

primarily consisting of squamous metaplasia in diverse

M. Calonge et al. / Experimental Eye Research 78 (2004) 457472 467

ocular surface conditions. For instance, one study showed

that IC was the most discriminative technique for the

diagnosis of dry eye in diabetic patients, as 86% of the 92

diabetic patients examined showed pathologic conjunctival

epithelium (Tsengs stage IIIV) (Seifart and Strempel,

1994). Diabetic patients have also been shown to have signs

of conjunctival squamous metaplasia (Goebbels, 2000), and

goblet cell loss in IC samples which were correlated with

peripheral neuropathy, poor diabetic control, and decreased

corneal sensitivity (Dogru et al., 2001). In another disease

that can affect the ocular surface, thyroid associated eye

disease, IC has shown grades 2 or 3 of squamous metaplasia,

according to Nelsons grading system, in 32% of samples

from upper bulbar conjunctiva and in 82% of the temporal

bulbar area (Ozkan et al., 1997). Interestingly, IC has served

to objectively demonstrate that the workers suffering from

the so-called Sick Building Syndrome, who often complain

about dry eye-related complaints, have alterations in their

ocular surface, as IC specimens showed significant altera-

tions in specimen cellularity, cell-to-cell contact, N/C ratio,

chromatin pattern, goblet cell distribution, and keratinization

(Fenga et al., 2001).

Patients with chronic renal failure had their ocular

surface assessed by IC, showing higher grades of squamous

metaplasia that, however, did not correlate with the calcium

deposits that these patients often show (Dursun et al., 2000).

Squamous metaplasia and increased goblet cell density

have also been noted throughout the bulbar conjunctiva of

patients with pterygium, although the most advanced were

over the pterygium surface (Chan et al., 2002). Also

keratoconus patients have shown loss of goblet cells and

squamous metaplasia on conjunctival IC sample, which

seems to be related with the extent of keratoconus

progression (Dogru et al., 2003). IC samples of these

patients also served to demonstrate higher levels of

lysosomal enzyme (Shen et al., 2002).

Some other diseases that apparently would not affect the

ocular surface have, however, showed squamous metaplasia

changes. For example, the conjunctival surface of seven

anorexia nerviosa patients was prospectively studied and IC

demonstrated moderate to severe squamous metaplasia in 5

of the 7 patients studied, with rare loss of goblet cells, which

was not related to vitamin A deficiency (Gilbert et al.,

1990). A reduced number of goblet cells with some degree

of squamous metaplasia was also found in the bulbar

conjunctiva of Downs syndrome patients, which was

thought to be due to altered metabolism of some elements,

i.e. vitamin A (Filippello et al., 1997). Psoriatic patients

have also been studied by IC, showing more frequently

pathologic grades of squamous metaplasia than normal

controls, in addition to more frequent snake-like chromatin

changes (Karabulut et al., 1999).

Likewise, radiation has been shown to have a deleterious

effect on the ocular surface, even if not directly applied to

the eye. A very interesting recent study has used IC to

demonstrate impressive surface alterations (squamous

metaplasia, intraepithelial lymphocyte infiltration) in radi-

ology technicians exposed to diagnostic doses of radiation,

suggesting that routine examination by IC can be beneficial

in detecting early cytological radiation-induced and dry eye

changes in these workers (Gurdal et al., 2002). Striking

epithelial changes have been described (including abnormal

mitoses, nuclear fragmentation and atypia, snake-like

chromatin) in 10 patients after they were irradiated for

paranasal sinus tumors and dose related (changes were not

evident until 25 Gy were reached, and they were more

severe with 3035 Gy) (Midena et al., 1991).

IC has also been used as an impression debridement

technique, effectively removing degenerated cells, inflam-

matory cells and organisms from corneal ulcers, as well as

filaments of filamentary Keratitis (Arora and Singhvi, 1994).

Similarly, IC on the cornea of rabbits has been used to

remove some epithelial layers and subsequently create an

epithelial defect that facilitated the adhesion of pseudomona

aeruginosa (Klotz et al., 1989).

4. Summary

In summary IC is an extraordinarily useful technique. It

has become a routine technique to evaluate squamous

metaplasia and goblet cell changes in any ocular surface

disease, specially in those dry eye-related disorders, being

useful for diagnostic purposes and to follow the course of

therapeutic interventions. In addition, and especially during

the last decade, it has become the technique of choice to

obtain samples from the ocular surface epithelium, which

has greatly contributed to increase our knowledge on its

biology and its contribution to ocular surface pathology.

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Ophthalmologi