Impression Cytology of The Ocular Surface - A Review

Experimental Eye Research 78 (2004) 457472
www.elsevier.com/locate/yexer
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
* 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).
0014-4835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
DOI:10.1016/j.exer.2003.09.009
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 10 15 min (staining was periodic acid Schiffs,
PAS, and hematoxylin), they showed results in normal
specimens and, additionally, they compared IC findings
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M. Calonge et al. / Experimental Eye Research 78 (2004) 457472
(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 contraindications 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 cell cell
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 applications. 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. Pore
size is not irrelevant, as it affects the consistency of cell
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 (MillicellCM 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 3 5 sec, originally with the aid
of a solid rod for 3 5 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).
459
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 PASGiemsa (Figs. 1 3) (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).
460
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 2 3 of Nelsons grading system and to a stage 4 in Tsengs
classification (Polyether sulfone filters and PAS-Giemsa staining, 40).
intensity of goblet cells present; (3) the presence nonepithelial cells, i.e. inflammatory cells, microorganisms, etc.
It is important to understand that IC removes 1 3
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.
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).
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 conjunctival 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 (0 3) 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 3 5 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.
461
Table 1
Main grading systems for conjunctival impression cytology specimens processed for light microscopy
Goblet cells
Tseng,
1985
Nelson,
1988
Size/form
Density
PAS
staining
Size/form
Cyto staining
NS
Moderate
NS
Uniform
Blue to green
1:1
NS
Decreased
NS
Mild enlargement
Blue to green
1:21:3
NS
Absent
NS
Absent
NS
Blue-green to
mild pinkish
Pinkish
1:4
NS
Moderate enlargement
flattened (squamoid)
Markedly squamoid
1:6
NS
Absent
NS
Markedly squamoid
large
Pinkish
1:8
NS
Absent
NS
Shrunken cytoplasm
NS
Abundant
Intense
Small, round
Decreased
intense
Markedly
decreased
Less
intense
Slightly larger
more polygonal
Larger polygonal
Grade 3
Grade 0
Plump,
oval
Plump,
oval
Smaller,
poorly
defined
border
NS
NS
Very few
Abundant
NS
Intense
Grade 1
NS
Grade 2
Grade 3
NS
NS
Slightly
decreased
Decreased
Very
decreased
Stage 0: normal
conj epithelium
Stage 1: early
GC loss, no K
Stage 2: total
GC loss, no K
Stage 3: early
and mild K
Stage 4:
moderate K
Stage 5:
advanced K
Grade 0
Grade 1
Grade 2
Adams
et al., 1988
Non-goblet epithelial cells

N size
N/C ratio
Other
Eosinophilic
May
be absent
Large
1:2
Visible KF mild
pyknotic N
Densely packed KF,
pyknotic N
Keratohyaline gr
Densely packed IF,
pyknotic N
Basophilic N
Eosinophilic
Smaller
1:3
NS
Variable
Small
1:41:5
Occasionally MN
Large polygonal
Normal
Basophilic
NS
Small
Normal
.1:6
1:2
Pyknotic
Good cell sheet
Intense
Larger
NS
NS
1:3
Good cell sheet
Reduced
Pale
Larger
Large, irregular
NS
NS
NS
Small
NS
NS
Reduced cell sheet

Poor cell sheet
NS
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.
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 cytomegalovirus 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
462
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 nitrocellulose 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. Corneal 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 immunohistochemical 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
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 polyL -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).
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.
3.2.5. Flow cytometry

The application of this technique to IC samples was first
reported in 1997 by Baudouin et al., who performed HLADR 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 immunocytochemistry and immunofluorescence, for gel electrophoresis 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
463
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
zkan et al., with 100% of grade 0
et al., 1997) or from O
(from upper bulbar) or 85% grade 0 and 15% grade 1 (from
zkan et al., 1997).
temporal bulbar) in 20 normal subjects (O
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 0 4, 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
Epstein Barr 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 immunophenotype, although the percentage of such cells was 1 5% 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
464
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 conjunctivitis, some chronic non-cicatrizing conjunctivitis (superior
limbic keratoconjunctivitis), vitamin A deficiency, dysplasia/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 morphological 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 immunofluorescence, 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 Epstein Barr 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 immunocytochemistry 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.
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). Interestingly, 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 immunemediated cicatrizing conjunctivitis, especially ocular cicatricial 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).
465
Another study also showed epithelial alterations (squamous

metaplasia and goblet cell decreases) notably different from
normals, being more frequent and more severe in symptomatic 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 responsible 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 inflammation 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 (Cu Zn 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
466
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 conjunctivalization 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 transdifferentiation 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 technique 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). Immunofluorescent 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, particles 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 immunocytochemistry 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 (2 5 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 differentiate 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
signs and reduced the frequency of artificial tear administration, but did not reduce the rose Bengal staining or IC
results (Shaker et al., 1989). Later, unpreserved carboxymethylcellulose artificial tears (Grene, 1992) and hyaluronate-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 cytological 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 1 3 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 immunofluorescence 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
467
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 conjunctival 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 specimens 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 conjunctival 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
468
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 III V) (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
zkan et al., 1997). Interestingly, IC has served
bulbar area (O
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 alterations 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 radiology 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 30 35 Gy) (Midena et al., 1991).
IC has also been used as an impression debridement
technique, effectively removing degenerated cells, inflammatory 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.
References
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Ophthalmol. 97, 730734.
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Impression Cytology of The Ocular Surface - A Review

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Experimental Eye Research 78 (2004) 457472

Impression cytology of the ocular surface: a review

the conjunctiva in various disorders of the ocular surface

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

(sections of the membrane were also shown) with excisional

chain reaction (PCR), immunoblotting analyses or flow

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

3.2. Processing methods

Fig. 1. Conjunctival impression cytology specimen from a patient

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

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

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

3.2.1.1. Nelsons classification. In 1983, Nelson developed a

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

Non-goblet epithelial cells

Good cell sheet

Reduced cell sheet

3.2.2. Electron microscopy

microscopy from long-term lens contact wearers, allowed

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

antibody and finally placed on a gelatin-coated slide

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

peptide b defensin in the ocular surface (Lehmann et al.,

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

3.2.5. Flow cytometry

conjunctival and corneal epithelium and their topographic

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

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

present clear that it is not exclusive to dry eye conditions

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

Special processing of IC samples has allowed the study of

Another study also showed epithelial alterations (squamous

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

malasorption states, whose symptoms may go unrecognized

IC samples taken from conjunctiva and from elevated

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

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

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

ocular surface conditions. For instance, one study showed

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

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

alterations in operating room workers. A single-institution pilot study.

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

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

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