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2009 Ethis Communications, Inc. The Ocular Surface ISSN: 15420124. Lopin E, Deveney T, Asbell PA. Impression cytology: recent
advances and applications in dry eye disease. 2009;7(2):93-110.
I. INTRODUCTION
n 1954,Larmande and Tismit in France reported
using impression cytology (IC) to diagnose ocular
surface squamous neoplasia.1 References to IC in
the English-language literature did not appear until 1977.
At that time, Thatcher et al described an improvement in
ocular surface cell harvesting using a plastic disc, which was
considerably more comfortable for patients than techniques
that relied on scraping or swabbing with cotton.2 Also in
1977, Egbert et al documented the use of IC with absorbent
lter paper to harvest cells in essentially the same manner
that is still used today.3
Three review articles on impression cytology of the ocular surface have been published in recent years. McKelvie
(2003) addressed the technical aspects of IC, especially in
reference to the user-friendly Biopore membrane device. She
additionally addressed the application of IC in diagnosing
ocular surface squamous neoplasia.4 Calonge et al (2004)
provided a historical review of IC technique, showing it to
be a useful diagnostic aid for a wide variety of processes
involving the ocular surface, while being minimally invasive.5 Singh et al (2005) also underscored the ability of IC
to diagnose a wide range of ocular surface disorders and
also stressed the importance of cell harvesting technique, as
the number of cells obtained varies considerably depending
on the methodology used.6
Although this review article incorporates relevant data
from the three earlier reviews, its main purpose is to describe recent advances in knowledge obtained through the
use of IC. It specically addresses the use of IC as applied
to dry eye disease.
A PubMed search was conducted using the terms impression cytology and dry eye and impression cytology and keratoconjunctivitis sicca. Of the articles retrieved by this method,
we reviewed all publications in English, and we reviewed the
English abstracts of non-English publications. We included
articles that described developments in IC harvesting or processing techniques in addition to the vast number of articles
devoted to dry eye disease in the context of IC use. Emphasis
was placed on articles published since the review by Calonge
et al,5 but we included earlier articles that provided a more
comprehensive understanding of both dry eye disease and IC.
93
OUTLINE
I. Introduction
II. Impression cytology techniques
A. Cell harvesting
B. Cell processing
C. Flow cytometry and immunocytochemistry
III. Application of IC to investigation of dry eye disease
A. Investigating the pathology of dry eye
1. Evaluation of inflammation
a. Cytokines/chemokines
b. Defensins
c. Inflammatory markers
1) HLA-DR
2) CD40
d. Summary of data on inflammation
2. Oxidative reactions
3. Snake-like chromatin
4. Ocular mucins
5. Growth factor receptors
6. Microvilli
B. Monitoring of clinical trials
1. Cyclosporine
2. Autologous serum
3. Umbilical cord serum
4. Artificial tears
a. Preservatives
b. Sodium hyaluronate
c. Carboxymethylcellulose (CMC)
5. Antioxidants
6. Essential fatty acids
7. Nerve growth factor
8. Steroids
9. Vitamin A
10. Botulinum toxin
11. Non-pharmacological treatments
C. Characterization of animal models
D. Association of dry eye disease with other conditions
IV. Summary and conclusion
Advantages
Light microscopy
Electron microscopy
Permits visualization of cell ultra-structures; important for disease types such as MPS.
Immunohistochemistry
Flow cytometry
Can also detect sub-clinical inflammation, but methodology is standardized and not userdependent.
RT-PCR/PCR
Allows examination of survival of donor human limbal stem cells in recipient with limbal cell
deficiency.
Permits identification of ocular surface genes such as anti-bacterial peptide defensin genes
and antioxidant enzyme genes.
Schiff (PAS) staining, respectively. To assess the progression and severity of ocular surface disease based on light
microscopy ndings, various grading systems have been
employed, including systems developed by Tseng in 1985,11
Nelson in 1988,12 and Adams et al in 1988 (Table 2).13 All
three grading systems utilize criteria to identify degree of
pathology, such as change in cell morphology (squamous
metaplasia), reduction in number or activity of goblet cells,
and the presence or lack of inammatory cells. These criteria
are nonspecic and can apply to a broad spectrum of ocular
surface pathology. However, light microscopy functions
Table 2. Goblet and non-goblet epithelial cell characteristics of three main grading systems11-13
Classication
Goblet cells
N:C ratio
Grade 0
Moderate density
Uniform size/form
1:1
Grade 1
Decreased density
Mild enlargement
1:21:3
Grade 2
Absent
Moderate enlargement,
flattened (squamoid)
1:4
Grade 3
Absent
Markedly squamoid
1:6
Grade 4
Absent
1:8
Grade 5
Absent
Shrunken cytoplasm
Grade 0
Plump/oval, abundant
Small, round
1:2
Grade 1
1:3
Grade 2
Larger, polygonal
1:41:5
Grade 3
Very few
Large, polygonal
> 1:6
Grade 0
Abundant
Normal
1:2
Grade 1
Larger
1:3
Grade 2
Decreased number
Larger
NS
Grade 3
Large, irregular
NS
Tseng11
Nelson12
Adams13
N:C = Nucleus:cytoplasm.
95
1. Evaluation of Inammation
Although the exact cause of dry eye disease is not
known, increasing evidence suggests that inammation of
the ocular surface is a signicant component and leads to
the observed signs and symptoms of dry eye disease. The
DEWS report highlights some of the recent research relating
to inammation in dry eye disease.22
a. Cytokines/Chemokines
Using IC samples combined with different analysis
methodologies has been helpful in analyzing different
immune biomarkers related to dry eye disease. To evaluate cytokines and chemokines, researchers have used IC
samples combined with immunohistochemical and immunouorescent staining RT-PCR to determine cytokine
gene transcription, and ow cytometry.
To evaluate the role of cytokines and chemokines in
dry eye disease, IC samples have been analyzed by various
techniques for markers of inammation. Yoon et al utilized
immunohistochemical staining of IC samples incubated with
anti-IL-6 and TNF- antibodies from patients with dry eye
disease (with and without Sjogren syndrome) to demonstrate
level and expression of pro-inammatory cytokines IL6 and
role of IL-1 and its antagonist in controlling inammation. RT-PCR has demonstrated increased transcription of
cytokines in dry eye disease, although some researchers
have not demonstrated such changes. IC combined with
ow cytometry has led to increased understanding of the
pathophysiology of inammation, suggesting that dry eye
disease is a Th1- mediated disease and that it primarily involves the response of resident conjunctival epithelial cells,
as opposed to inammatory cells coming from elsewhere
in the body.
b. Defensins
Human beta-defensins (hBD) are antimicrobial peptides,
but they also have an immune modulator role, inuencing
cytokine production and chemotaxis, for example. Some
defensins, such as hBD-2, are inducible by pro-inammatory cytokines.31 Using RT-PCR analysis of IC samples,
Narayanan et al showed that hBD-2 mRNA was expressed
only in conjunctival epithelial cells from patients with
moderate dry eye disease and that its up-regulation could
be induced by pro-inammatory cytokines (IL-1beta or
TNF-) in cultured conjunctival cells. They hypothesized
that this up-regulation compensates for the compromised
ocular surface in dry eye disease, providing additional defenses, but may also contribute to ocular surface damage.31
Further work by the same group utilizing IC samples from
dry eye disease patients did not demonstrate upregulation
of IL-1beta, suggesting that this cytokine is not responsible
for the up-regulation of hBD-2 in patients with moderate
dry eye disease. This work suggests that other cytokine
signaling pathways may be involved in the up-regulation
of hBD-2.27
Use of RT-PCR analysis of IC samples to explore defensins has demonstrated that in some inammatory processes,
there is reduced expression of specic defensins. Specically,
the beta-defensin DEFB-109 gene associated with antimicrobial peptides (AMPs) was reduced in all samples from
patients with ocular surface inammation and infection.
The least reduction was found in patients with dry eyes as
compared to normal controls.32
Use of IC to study defensins has demonstrated that
in dry eye disease up- and down-regulation of immune
modulators occurs.
c. Inammatory Markers
1) HLA-DR
Flow cytometry has expanded the opportunities to discover more information from IC samples and has provided
an objective metric to evaluate ocular surface changes.17 For
dry eye disease, evaluation of HLA-DR has been specically
explored using ow cytometry to gain a more complete
picture of the ocular surface inammation that is associated
with the disease.
Major histocompatibility complex (MHC) class II
molecule HLA-DR is a glycoprotein that is normally expressed on immune cells, such as B lymphocytes, but whose
expression has also been described on some nonimmune
97
2) CD40
CD40 is a cell surface receptor whose up-regulation
has been observed in a variety of inammatory conditions.
This receptor belongs to the tumor necrosis factor receptor
superfamily. Using ow cytometry analysis of IC samples
from KCS, Bourcier et al demonstrated increased CD40
expression compared to normal controls and showed that
this was positively correlated with HLA-DR expression.35
d. Summary of Data on Inammation
Using IC in conjunction with a variety of analytical
techniques, researchers have been able to demonstrate
inammation associated with dry eye disease, providing
not only more insight into the mechanism and pathogenesis of dry eye disease, but providing the rationale and
inspiration for new anti-inammatory treatments for dry
eye disease.
2. Oxidative Reactions
IC sampling has been used to elucidate the mechanism
of action that results in ocular surface abnormalities associated with dry eye disease. Studies of reactive oxygen species
suggest that they may be an important factor contributing to
inammation and ocular surface damage and provide the rationale for the use of antioxidants to treat dry eye disease.36
Cejkova et al analyzed xanthine oxidoreductase/xanthine oxidasean enzymatic system responsible for the
generation of reactive oxygen speciesby histochemistry
98
3. Snake-like Chromatin
Snake-like chromatin (SLC) is an unusual arrangement
of chromatin found in the cell nucleus (Figure 3).38 It is
associated with certain pathological conditions, such as inammation. The presence of SLC in conjunctival cells from
patients with dry eye disease has been extensively noted,
most recently by Jirosova et al.39 In another paper, Jirsova
et al used light microscopy of IC samples to demonstrate
the presence of micronuclei associated with SLC-positive
cells and conrmed the correlation between increased
SLC-positive cell numbers, decreased goblet cell density,
and impaired clinical dry eye parameters.40
4. Ocular Mucins
Mucins are glycoproteins and are a major constituent
of the mucous layer that is adjacent to the surface cells of
the ocular surface epithelium. The mucous layer of the tear
lm is generated by both goblet cells and apical cells of the
cornea and conjunctiva.41 IC has aided us in understanding
the complexity of the role of mucins on the ocular surface.
99
1. Cyclosporine
A body of evidence supports a role of inammation in dry
eye disease, and cyclosporine A (CsA), a T cell inhibitor, has
been developed for topical administration as a treatment for
the disease. A large, randomized, multi-center, clinical trial to
evaluate the efcacy of CsA employed ow cytometric analysis of IC samples as an objective tool to monitor the effect
of the drug on ocular surface cells. One hundred sixty-nine
dry eye disease patients from 28 centers in four European
countries were assigned to three treatment groups0.05%
CsA, 0.1% CsA, or vehicle (control)and monitored over
12 months (Table 3). IC samples were analyzed for immune markers HLA-DR and CD-40, and apoptotic marker
APO2.7. The percentage of HLA-DR positive cells and
level of HLA-DR expression were reduced in both groups
receiving CsA, whereas patients receiving vehicle showed
no signicant changes. CD40 and CD40 ligand signicantly
decreased in both CsA groups, as well. APO2.7 expression was more variable and increased in patients receiving
CsA. The objective results from IC analysis demonstrated
that CsA signicantly reduced expression of inammatory
markers on epithelial cells, and suggest that ow cytometry analysis of IC is an objective technique for describing
changes on the ocular surface in response to treatment.49,50
Cyclosporine treatment has also been compared with
other, more traditional treatments, such as articial tears
and topical sodium hyaluronate, with IC samples used to
assess any difference between treatments.
Table 3. The use of IC as an outcome measure in a dry eye clinical trial (topical CsA)
% HLA-DR-Positive Conjunctival Cells
Baseline
(meanSD)
3 months
6 months
12 months
0.05% CsA
(p/baseline)
61.67%29.54%
(p<0.0001)
39.03%31.36%
(p<0.001)
39.45%33.06%
(p<0.001)
26.55%27.33%
0.1% CsA
(p/baseline)
57.53%31.73%
(p=0.004)
41.73%33.57%
(p<0.001)
38.59%32.95%
(p<0.02)
37.84%27.80%
5.09%
(NS)
6.68%
(NS)
36.08%*
(p=0.02)
Group
Control
(p/baseline)
NS=nonsignificant. Compiled from Brignole et al, 200149 and Galatoire et al, 2003.50
*Patients initially randomized to receive the control vehicle starting at 6 months, received 0.1% CsA and were then evaluated at 12 months,
after 6 months of treatment with CsA.
100
Utilizing IC combined with light microscopy and immmunostaining, one study evaluated six dry eye patients
who were treated with unpreserved artificial tears for
1 month, followed by topical 0.05% cyclosporine for 3
months. IC analysis after articial tear treatment showed
no change in goblet cell density in contrast to IC analysis
after 3 months of cyclosporine, which demonstrated a signicant increase in goblet cell density and of the number
of TGF-beta 2 positive goblet cells.51
Another study, which used light microscopy analysis of
IC samples to determine goblet cell density, showed that
both sodium hyaluronate and cyclosporine increased goblet
cell density. Thirty-six patients with dry eye were treated
with topical chondroitin sulfate with sodium hyaluronate
(CS-HA) in one eye and CsA in the other for 6-8 weeks.
Goblet cell density was signicantly higher in CsA-treated
eyes, although the authors suggest that administration of
both treatments simultaneously might result in a synergistic
response and even greater improvement in ocular surface
parameters.52
2. Autologous Serum
Articial tears made with autologous serum have long
been thought to be benecial in treating dry eye disease,
but until recently no controlled studies had been done. It
is believed that vitamins such as vitamin A, growth factors
such as TGF-beta and EGF, and other components of tears
that are critical to the maintenance of a healthy ocular surface are present in autologous serum, but not in articial
tears, which may contribute to their efcacy. The absence of
preservatives in autologous serum, such as benzalkonium
chloride (BAC), which is known to exacerbate dry eye
symptoms, may also explain the effectiveness of autologous
serum.53-55
Tananuvat et al performed a prospective, randomized,
placebo-controlled, single-masked 2-month study in 12
patients with dry eye disease to determine the safety and
efcacy of tears containing the patientsown serum.54 One
eye received the serum (20% solution) and the contralateral
eye received normal saline as a control. IC samples before
and after treatment began were stained with Papanicolau
stain and classied according to the Tseng classication.
Improvement in signs, including IC score, and symptoms
was observed in serum-treated eyes, although improvement
was nonsignicant because control groups also improved,
suggesting a large placebo effect. The authors state the need
for additional larger studies.54
In a later study by Noble et al, 50% autologous serum
drops were evaluated against more traditional treatments
in a prospective, randomized, partially masked, crossover
trial.56 Sixteen dry eye patients (11 with KCS/Sjogren syndrome and 5 with a variety of other ocular surface disorders)
were randomized to receive autologous serum for 3 months
followed by conventional treatment for 3 months, or conventional treatment for 3 months followed by autologous
serum for 3 months. IC scores in eyes treated with serum
showed signicant improvement, and subjective comfort
101
b. Sodium Hyaluronate
Sodium hyaluronate (a biopolymer) drops have been
extensively studied as an alternative to other tear substitutes
for dry eye, because they may promote corneal wound healing, control inammation, and increase tear lm stability,
as well as having viscoelastic properties that help reduce
friction on the ocular surface.60 In a multi-center, randomized, double-blind study evaluating the long-term effect
of sodium hyaluronate-containing eye drops in patients
with dry eye disease, 44 patients with medium-to-severe
dry eye were evaluated and randomly treated with either
preservative-free sodium hyaluronate or preservative-free
saline. IC was the primary efcacy variable in this study. At
3 months, IC scores, based on the grading scheme designed
by Nelson, were signicantly lower in patients treated with
sodium hyaluronate compared to those treated with saline.
The authors suggest this is the direct result of the treatment, as the IC grade in the placebo group did not change
over the course of the study, whereas the treatment group
statistically improved from baseline.60
Sodium hyaluronate was further evaluated in an openlabel study designed to compare two different commercially
available solutions that varied in osmolarity. Forty patients
with Sjogren syndrome were randomly assigned to either
unpreserved 0.4% hypotonic sodium hyaluronate drops or
unpreserved 0.4% isotonic sodium hyaluronate drops for 3
months. IC score, based on evaluation of seven parameters,
including goblet cell distribution and presence of inammatory cells, and global symptom score were the primary
efcacy variables in this study. Both groups showed improvement in IC score, but the hypotonic group showed
a faster and more pronounced improvement in IC score,
suggesting that the hypotonic formulation may be the more
effective treatment. Some hypothesize that hypotonic solutions may help to correct the tear hyperosmolarity that is
associated with damage to the ocular surface and dry eye
disease.61
102
c. Carboxymethylcellulose (CMC)
Carboxymethylcellulose has long been investigated for
its therapeutic efcacy.62 An early study used Nelsons IC
score to demonstrate improvement in KCS patients treated
with a CMC-based tear.63 In a more recent randomized
study of LASIK patients by Lenton and Albietz, ten patients
treated with nonpreserved CMC articial tears during and
after LASIK surgery showed greater goblet cell density 1
month post-LASIK compared to a group that received salt
solution.64
IC sometimes demonstrates that there is no signicant
improvement in the ocular surface with a presumed effective treatment, and IC results may be in contrast to other
evaluations of dry eye disease, such as symptom scores. In
a prospective, randomized, masked-observer, single-center study, 19 patients with mild or moderate dry eye were
treated with a 0.5% isotonic CMC or salt solution. Although
subjective symptoms improved with CMC treatment, no
difference between the groups was observed with IC; degree
of conjunctival metaplasia was similar in the two groups
before and after treatment, and Nelsons IC score did not
correlate with improvement in subjective symptoms, as
measured by a custom questionnaire.62
Combining ow cytometry with IC samples can provide
a more objective endpoint for the effects of new treatment.
In a prospective, randomized, masked-observer trial comparing sodium hyaluronate drops with CMC in the treatment of dry eye syndrome with supercial keratitis, ow
cytometry was used to analyze IC samples for: HLA-DR (an
inammatory marker), Apo2.7 (an apoptosis-related marker), MUC5AC (a soluble mucin secreted by goblet cells),
and CD44 (hyaluronic acid receptor). CD44 expression was
signicantly decreased in sodium hyaluronate-treated eyes
compared to CMC-treated eyes, and there was a tendency
for reduction of the number of HLA-DR-positive cells in
both treatment groups, although this was not statistically
signicant.65
5. Antioxidants
Reactive oxygen species and oxidative reactions have
been associated with dry eye disease and ocular surface
injury and stress.66 Therefore, antioxidant therapy has
emerged as being potentially able to reverse or balance
these harmful reactions on the ocular surface by protecting
epithelial tissue from attack.67
Blades et al studied 40 patients with marginal dry eye in
a prospective, randomized, double-blind, placebo-controlled
trial with cross-over. Patients received no treatment for 30
days, placebo for 30 days, and oral antioxidant supplements
for 30 days in random order. IC samples were stained with
PAS and hematoxylin and evaluated by light microscopy
for number of goblet cells and appearance and degree
of squamous metaplasia. A signicant improvement in
goblet cell density and squamous metaplasia was observed
following antioxidant treatment, and increased tear lm
stability correlated with change in goblet cell density.
Interestingly, there was no signicant carry-over effect,
8. Steroids
Because inammation is believed to play a role in the
pathophysiology of dry eye disease, treatment with antiinammatory topical corticosteroids has been investigated.
As noted above, the patients in the study of Lee et al demonstrated improvement (as measured by IC) with 0.1%
prednisolone treatment.70
In a prospective study of 53 primary or secondary Sjogren syndrome patients, a topical nonpreserved 1% methylprednisolone solution was administered in a pulse therapy
design, so that over the course of the trial, patients were
tapered off the medication. After treatment, a signicant
increase in the number of PAS-positive goblet cells was observed in conjunctival IC samples. The authors suggest that
steroid pulse therapy may be a safe and effective long-term
treatment for dry eye associated with Sjogren syndrome.71
In a single-masked, randomized, prospective clinical
trial, 32 dry eye patients with or without Sjogren syndrome
were randomly assigned to three groups: preservative-free
topical articial tear substitute (ATS); ATS plus nonsteroidal
anti-inammatory drops (NSAID); or ATS plus corticosteroid drops. IC samples were stained with PAS for analysis
of goblet cells and with monoclonal antibodies for HLADR and Apo2.7. Patients receiving ATS plus corticosteroid
drops had signicantly lower numbers of HLA-DR positive
cells, higher numbers of PAS-positive goblet cells, and lower
symptom severity scores compared to the other groups and
compared to their own baseline values. The authors suggest
that topical corticosteroids are effective treatment for dry
eye disease, producing a reduction in inammatory markers
and improvement in symptoms.72
9. Vitamin A
Because vitamin A deciency is associated with dry eye
disease, topical treatment with vitamin A and its derivatives
was studied over a decade ago as a potential treatment for
dry eye disease. An early study looked at the effects of topical tretinoin ointment in patients with severe dry eye disorders, including six patients with KCS, and found reduced
IC score (representing reduced squamous metaplasia) after
treatment.73 Schilling et al used IC to study the effect of
tretinoin treatment in 6 patients with KCS patients and 19
patients with mucin deciency. IC evaluation demonstrated
an improvement in squamous metaplasia in the mucin-decient group only.74 IC has also been used to demonstrate
goblet cell recovery in rabbit dry eye models treated with a
retinoic acid analog, CBS-211.75
10. Botulinum Toxin
Blepharospasm has been associated with dry eye disease.
Treatment with botulinum toxin injected into the lids (an
established blepharospasm treatment) has been reported to
be an effective treatment for some dry eye patients.76 Ocular
103
Table 4.
Condition/Reference #
# pts
Allergic rhinoconjunctivitis88
27
21
39
68
Atopic keratoconjunctivitis89
Chemotherapy90
Chronic conjunctivitis91
Chronic hepatitis
Jacobi et al92
Huang et al93
Collagen-vascular disease94
Congenital aniridia
Jastaneiah et al95
Rivas et al96
Cystic fibrosis
Mrugacz et al97
Mrugacz98
Dermatochalasis84
20
18
x
x
20
25
24
20
Wang et al104
HIV/AIDS105
Lipoprotein A elevation108
Ocular rosacea111
Oral carbamazepine Rx112
Polycystic ovary (PCO)
syndrome113
Pre-post-cataract surgery114
Pre/post LASIK surgery115
x
Abnormal IC findings correlated with clinical
signs of DED
Stem cell deficiency; increased goblet cells.
Metaplasia improved by limbal cell transplant.
x
x
Morphology unchanged by blepharaoplasty, but
PMLs reduced.
Tseng grade pathology.
Abnormal conjunctival epithelium.
15
30
1
906
x
x
13
1
20
x
x
37
x
x
x
x
x
x
x
x
x
x
10
38
53
56
Sjogren vs non-Sjogren
autoimmunity 124
Smoking125
91
Other IC ndings
Eosinophilia, mononuclear cells.
Most severe in adults with childhood onset.
Conjunctival mucositis.
11
24
50
70
15
Premature infants116
Psoriasis117
Pterygium118
Radiation (occupational)119
HLA-DR
27
50
20
MUC5AC
92
86
100
15
Decreased
GCD
71
19
60
Diabetes mellitus
Seifart & Strempel99
Goebbels100
Jin et al101
Down syndrome102
Hypovitaminosis A
Qureshi et al106
Farbos et al107
SqM
60
Inflammatory infiltrates.
Decreased dendritic cells, increased apoptotic
marker Apo2-7.
Enlarged keratinized squamous cells.
31% abnormal IC tests; inconsistent with
clinical results.
IC pathologic in 82% of patients.
GCD increased after mucosal grafting.
Metaplasia and GC count worse after thyroidectomy; suggests relationship between abnormal
ocular findings and post-op subclinical hypothyroidism.
Increase in ICAM-1.
PCOS, but not PCO, showed increase in GCD.
Decreased GCD and development of SqM
post-op.
Decreased GCD post-op.
Compared to full-term.
Snake-like chromatin, neutrophil clumping.
GCD increased after surgery.
Snake-like chromatin, lymphocytic infiltration,
hypertrophy.
Conjunctival calcification.
Moderate to severe morphological changes in
50% conjunctivas.
IC did not differentiate between sarcoidosis and
dry eye disease; similar findings.
Ocular surface disease differed among various
autoimmune diseases.
No difference in GCD between smokers and
non-smokers.
105
Often, IC results correlate with other signs and symptoms of dry eye disease, such as corneal staining and symptom scores. However, in some cases, IC results are incongruous with reported changes in symptoms, demonstrating no
improvement, although symptom scores appear to improve.
IC sampling has been used in animal models of ocular
disease, especially to conrm that ocular surface disease is
present and typical of human dry eye disease. IC sampling
has been done in rabbit, dog, and rat models, and has also
been used as an endpoint to evaluate new treatments utilizing these animal models.
Evaluation of IC to determine changes in the ocular surface has revealed that many conditions, systemic and local,
are, in fact, associated with changes to the ocular surface
that are typical of dry eye disease. All of this suggests that
IC may be a useful technique for evaluating ocular surface
changes in a variety of conditions.
A major change in the use of IC samples has been the incorporation of modern laboratory techniques, in particular,
ow cytometry, as a method to analyze the samples. Early
work with IC relied largely on light microscopy analysis of
histology and on immunohistochemistry. It was based on
grading schemes that were highly dependent on observer
technique and required an experienced observer to perform.
Conjunctival biopsies provided larger samples sizes, but
are clearly more invasive than IC. Although many studies
still employ these techniques, newer laboratory techniques
greatly expand the utility of IC. Flow cytometry, for example, provides objective metrics, and one sample can be
analyzed for multiple markers. Combining ow cytometry
with IC samples has led to new opportunities to provide
objective metrics on ndings on the ocular surface, such
as the number of HLA-DR-positive cells, other receptors,
and apoptotic factors. Flow cytometry, however, is a laboratory-based technique, and it requires specialized equipment
and an experienced researcher to process the samples and
analyze the results. Other new ways to use IC sampling
include RT-PCR to quantify mRNA levels and microarray
analysis to determine which genes are expressed.
IC has become an established tool to evaluate ocular surface changes and will likely continue to provide expanded
information that is useful for diagnosing and treating our
patients.
REFERENCES
1. Larmande A, Tismit E. [Importance of cytodiagnosis in ophthalmology: preliminary report of 8 cases of tumors of the sclero-corneal limbus.] Bull Soc Ophtalmol Fr 1954;19:415-9. In French
2. Thatcher RW, Darougar S, Jones BR. Conjunctival impression
cytology. Arch Ophthalmol 1977;95:678-81
3. Egbert PR, Lauber S, Maurice DM. A simple conjunctival biopsy.
Am J Ophthalmol 1977;84:798-801
4. McKelvie P. Ocular surface impression cytology. Adv Anat Pathol
2003;10:328-37
5. Calonge M, Diebold Y, Sez V, et al. Impression cytology of the
ocular surface: a review. Exp Eye Res 2004;78:457-72
6. Singh R , Joseph A ,Umapathy T, et al. Impression cytology of the
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