Changes in Conjunctiva Morphology Using Impression Cytology in Patients with Hashimotos Thyroiditis without Thyroid-Associated Ophthalmopathy
Mustafa ALTAY1, Tuğba ŞAHİN2, Zennure YILDIZ2, Gülçin ŞİMŞEK3, Mehmet ÇITIRIK4, İhsan ATEŞ5, Murat DAĞDEVİREN1, Muharrem BİTİREN3
1Department of Endocrinology and Metabolism, University of Health Sciences, Keçiören Health Administration and Research Center, ANKARA, TURKEY
2Department of Ophthalmology, University of Health Sciences, Keçiören Health Administration and Research Center, ANKARA, TURKEY
3Department of Pathology, University of Health Sciences, Keçiören Health Administration and Research Center, ANKARA, TURKEY
4Department of Ophthalmology, University of Health Sciences, Ulucanlar Health Administration and Research Center, ANKARA, TURKEY
5Department of Internal Medicine, University of Health Sciences, Ankara Numune Health Administration and Research Center, ANKARA, TURKEY
Keywords: Anti-TPO, Conjunctival impression cytology, Hashimotos thyroiditis, Inflammation
To compare the conjunctival morphological changes in patients with Hashimotos thyroiditis (HT) without thyroid-associated
ophthalmopathy (TAO) and controls using impression cytology technique.
Material and Method: We included 25 HT patients and 33 healthy controls who did not have TAO findings or dry eye. For both groups, thyroid
stimulating hormone (TSH), free T4(FT4), and anti-thyroid peroxidase (anti-TPO) were measured. Thyroid ultrasonography was performed,
together with all routine eye check-ups and the Schirmers test. Also, conjunctival impression cytology (CIC) test was performed to analyze the
Results: When the CIC of HT patients was observed; 12% had grade 0, 40% had grade 1, 28% had grade 2 and 20% had grade 3. While patients
with squamous metaplasia made up 48% of the HT group, this was observed at 6.1% in the control group (p < 0,001). In the regression model
formulated, it was observed that obesity (OR=7.500; p=0.017) and anti TPO levels (OR=1.370, p=0,007) were independent stipulations for the
Conclusion: Conjunctival squamous metaplasia was more frequently seen in HT than controls and serum Anti-TPO level and obesity were
detected as independent predictors of the worsening at the conjunctival impression cytology.
Hashimotos thyroiditis (HT) is one of the most common
human autoimmune diseases leading to significant
morbidity in women. Also HT is the most common cause
of hypothyroidism in iodine-sufficient regions in the world
. Up to 2% of the general population is affected by HT
and it is more common in elderly women, and ten times
more frequently observed in women compared to men
. Similar to possibly all autoimmune disorders, the
noxious interaction between external (environmental and
endogenous) and internal (genetic) factors is required to
trigger HT. HT results from loss of immune tolerance and
reactivity to thyroid auto-antigens: thyroid peroxidase,
thyroglobulin and thyroid stimulating hormone receptor
(TSHR). This causes the gland to be infiltrated by T cells
and B cells producing antibodies specific for clinical
manifestations. Additionally, T cells included in HT induce apoptosis in thyroid follicular cells, which ultimately leads
to the destruction of the gland 3
. A significant proportion
of patients are asymptomatic. When anti-thyroid
peroxidase (anti-TPO) and/or anti tyhroglobulin (anti-
TG) was positive in the serum in line with the presence of
parenchymal heterogeneity on thyroid ultrasonography,
Hashimotos thyroiditis was diagnosed.
Some changes and signs named as thyroid-associated
ophthalmopathy (TAO) have been described in HT, as in
Graves disease. These are dry eye, upper eyelid retraction,
and eye muscle dysfunction as reduced eye movements in
upgaze and proptosis 4,5. Only a few cases and studies
about HT-associated eye diseases have been reported
in the literature (4,5). In previous studies, ocular surface
inflammation (Grade 2-3 changes of impression cytology)
could be the single present clinical sign in Graves
disease before the onset of overt findings of TAO 6,7.
Unfortunately, there is no report about how conjunctival
structure is affected in HT. We therefore wondered if we
could detect these changes before the development of
ophthalmopathy in Hashimotos thyroiditis. We therefore
compared the conjunctival morphological changes in
patients with HT without TAO and controls using the
impression cytology technique. In addition, we also look
into the possible association of conjunctival morphological
changes with thyroid hormones and thyroid antibodies.
First of all, approval was asked for from the hospital ethical
commission before the start of the study. The work was
conducted according to the principles of the Helsinki
Declaration, and a written consent concerning the study
was confirmed from each of the participants.
Twenty five patients in total with no known disorder other
than Hashimotos thyroiditis and 33 healthy individuals
which were all over 18 years of age were enrolled in the
study. Those with an eye disorder correlated to the thyroid
were excluded based on their clinical activity scores (CAS)
8 and those with any eye disease such as dry eye or
active infection, taking systemic-topical drugs for thyroid
disease or for some reason, using contact lenses, and with
a previous eye operation or drug use were excluded from
the study. Serum thyroid stimulating hormone (TSH),
free thyroxine (fT4) and free triiodothyronine (fT3) levels
in addition to anti-thyroid peroxidase (anti-TPO) levels
were studied from the blood samples taken at 08:00-09:00
in the morning following a 10-hour fasting period. This
process was followed by thyroid ultrasonography (USG).
Ultrasonography was used to support the diagnosis of HT.
Ultrasonography by itself is not sensitive for the diagnosis
of HT. When anti-TPO and/or anti-TG was positive
in the serum in line with the presence of parenchymal
heterogeneity on thyroid ultrasonography, Hashimotos
thyroiditis was diagnosed.
We excluded the cases who reported topical cyclosporine,
NSAID (locally/systemically) and steroid (locally/
systemically) use within the previous 6 months. Other
exclusion reasons were contact lens wear, previous
punctal plug, active ocular infection, use of other topical
treatments, recurrent herpes keratitis within the last 6
months, blepharitis, ocular surgery or trauma within the
last 6 months, punctate epithelial erosions of the cornea,
other ocular surface diseases and non-keratoconjunctivitis
sicca inflammation including atopic keratoconjunctivitis.
No patient had a history of Stevens-Johnson syndrome; a thermal, chemical or radiation injury, or any other ocular
surgery that would cause an ocular surface problem. Patients
who had a history of systemic disease other than HT were
excluded. Patients with HT had no signs and symptoms of
dry eye. The controls had no ocular problems, no previous
ocular surgery, and no major systemic disease that affects
the ocular tissues. None of the patients or controls had a
history of alcohol use or smoking. None of the patients
with HT or those in the control group had a history of
using diuretics, antihistamines, vitamins, anti-depressants,
and any medications with anticholinergic properties.
Following the routine eye examinations, Schirmers test was
conducted with a standardized kit consisting of a strip of
filter paper 5×30-mm2 in size that was placed on the lower
lid margin in a temporal position. The patient looks up and
blinks normally for 5 minutes, when the strip is removed
and the wetted papers length is measured. A value of < 5
mm was considered abnormal and patients with abnormal
results were excluded from the study.
Conjunctiva of the topically anaesthetized eye was subjected
to impression cytology. Nelson graded conjunctival
impression cytology specimens (grades 03) according
to the number of goblet cells and the appearance of the
epithelial cells 9. This grading score was used in the
present study. Small disks of cellulose acetate filter paper
(MFS, Advantec MFS, Pleasanton, USA, pore size 0.2μm)
were divided into pieces approximately 4mm×5mm
in size, placed on the superior temporal interpalpebral
conjunctiva at a 5 mm distance to the limbus, softly
pressed for 5 seconds, and then lifted. After placing in a
fixative solution, the relevant specimens were stained
with Papanicolaus modification of Gills technique.
A pathologist who was masked to the history of each
specimen examined the specimens with light microscopy.
Employing Nelsons method, the examination recorded the
appearance of conjunctival epithelial cells and goblet cells
(if any) 9,10. All slides were examined by two observers,
similarly masked. All specimens were graded according to
the following four-levels. Grade 0: the epithelial cells are
small and round with eosinophilic-staining cytoplasm.
The nucleis nucleocytoplasmic ratio is 1:2, which is large.
The goblet cells are ample, fleshy and oval with strongly
Periodic Acid Schiff (PAS)-positive cytoplasm (Figure
1). Grade I: slightly larger epithelial cells than those in
grade 0 and more polygonal, with eosinophilic-staining
cytoplasm. Smaller nuclei with a nucleocytoplasmic ratio
of 1:3 are evident. Fewer number of goblet cells appear;
nevertheless, their plump, oval shape with strongly PASpositive
cytoplasm are still maintained (Figure 2). Grade II: the epithelial cells are larger than those in grade I
and polygonal, occasionally multinucleated, with an
eosinophilic-staining cytoplasm. Their nucleocytoplasmic
ratio is 1:4 to 1:5. The goblet cells are significantly fewer
in number and are minor, insufficiently defined and less
strongly PAS-positive (Figure 3). Grade III: the epithelial
cells are larger than those in grade II and polygonal with
a basophilic-staining cytoplasm. The nuclei are small,
pycnotic, or completely absent in many cells. The goblet
cells are completely non-existent (Figure 4).
Click Here to Zoom
|Figure 1: Grading system of impression cytology. Grade 0: small,
ground epithelial cells have a prominent nucleus (PAS stain;
Click Here to Zoom
|Figure 2: Grading system of impression cytology. Grade 1: the
epithelial cells are slightly larger. The nuclei are smaller. Goblet
cells are decreased minimally (PAS stain; x200).
Click Here to Zoom
|Figure 3: Grading system of impression cytology. Grade 2: larger
and polygonal epithelial cells (N/C ratio 1:4-1:5). Goblet cells are
smaller and markedly decreased (PAS stain; x200).
Click Here to Zoom
|Figure 4: Grading system of impression cytology. Grade 3: goblet
cells have disappeared (N/C ratio 1:6) (PAS stain; x400).
Statistical evaluation was done with the Statistical Package
for Social Sciences for Windows 20 (IBM SPSS Inc., Chicago,
IL). Normal distribution of data was evaluated with the
Kolmogorov-Smirnov test. Average&standard deviation
was calculated from the numerical variables showing
normal distribution, and those not showing deviation
have been represented as median (min-max). Categorical
variables have been shown in numbers and percentages.
Differentiation between HT and the control group has been cross-referenced as necessary with the Mann-Whitney
U test, Students t-test, Hashimotos thyroid and Fishers
exact chi-square test. The differentiation between grade
groups was tested with the ANOVA test, Kruskal-Wallis
H and Monte Carlo chi-square with simulation. Possible
independent predictors over grade 2-3 risk were tested with
stepwise logistic regression analysis. p < 0.05 was accepted
as statistically significant in all comparisons.
In the Nelson system, grade 0-1 was accepted as normal
and grade 2 and 3 is accepted as abnormal (squamous
metaplasia) 4,7. While statistical analyses were being
done, the grades which were normal (grade 0 and 1) and
those which were abnormal (grade 2 and 3) were therefore
separated into two groups and analyses were performed.
The research population was formed from a total of 58
participants, 25 of which were in the HT group, and 33
were the healthy volunteers. Demographic and clinical data
pertaining to both groups have been depicted in Table I
Among HT patients, 12% (n=3) were found to have Grade
0, 40% (n=10) had Grade 1, 28% (n=7) had Grade 2, and 20% (n=5) had Grade 3. HT patients were observed to have
an increase as compared to normal patients as the grade
level increased (p < 0.001). Abnormal CIC findings were
found at a rate of 48% in the HT group while this figure was
6.1% in the control group (p < 0.001).
The obesity rate in Grade 3 cases was higher than in the
other grades (p = 0.003). In addition, in Grade 3 cases, the
median TSH (1.7 μIU/mL vs. 2.8 μIU/mL vs. 2.1 μIU/mL
vs. 12.1 μIU/mL; p = 0.003) was higher as compared to
other TSH grades while the median sT4 (1 ng/dL vs. 1 ng/
dL vs. 1 ng/dL vs. 0.7 ng/dL; p = 0.041) was lower. The anti-
TPO level was higher in Grade 3 cases as opposed to Grade
1-2 cases, while the anti-TPO level showed uniformity
in Grade 1-2 cases (Figure 5). All the Grade 3 cases were
found to be formed of HT patients (Table II).
Click Here to Zoom
|Figure 5: Anti TPO levels by CIC grade (Anti TPO unit: IU/mL)
Anti TPO: Anti thyroid peroxidase, CIC: Conjunctival impression
Click Here to Zoom
|Table II: Demographic and clinical features of all participant according to Grading system.
The average BMI level in Grade 2-3 cases was higher than
in Grade 0-1 cases (25.5 ± 3.7 kg/m2 vs. 28.6 ± 4.5 kg/
m2; p = 0.015). Also, while 42.9% of grade 2-3 cases were
obese, 9.1% of grade 0-1 cases were obese (p = 0,020). The
Hashimotos thyroiditis rate in Grade 2-3 cases was much
higher compared to grade 0-1 cases (29.5% vs. 85.7%; p < 0.001). The median anti-TPO rate was higher in Grade 2-1
cases compared to the other grades (14 IU/mL vs. 57.8 IU/
mL; p = 0.001). Other demographic and clinical findings
did not yield a significant difference (Table III).
Click Here to Zoom
|Table III: Demographic and clinical features of all study populations according to Grade 0-1 / 2-3.
Identification of independent predictors that may be
effective on Grade 2-3 was done with the regression model
formed from variables with a p value below 0.25 as found
in Table III. Accordingly, it was identified that obesity (OR
= 7.500; p = 0.017) and anti-TPO level (OR = 1.370, p =
0.007) is an independent predictor for Grade 2-3 (Table
In the present study we found that conjunctival squamous
metaplasia was more frequently seen in HT than controls
and serum Anti-TPO level and BMI were detected as the
independent predictors of the worsening of conjunctival
impression cytology findings.
Impression cytology refers to the application of a cellulose
acetate filter to the ocular surface to remove the ocular
surface epitheliums superficial layers. The removed cells
can thus be subjected to immune-histological, histological,
or molecular analysis. It is easy to perform and noninvasive,
and provides reliable information regarding the
area sampled with minimal discomfort to the patient.
Therefore, it is a fundamental method in understanding
ocular surface disorders. Impression cytology applications
can be used for the aetiological diagnosis of various ocular surface disorders, documenting sequential changes in the
conjunctival and corneal surface over time, monitoring
the treatment effects and staging conjunctival squamous
metaplasia, and as a useful tool for analyzing ocular surface
disease with immunostaining and DNA analysis 10.
Normal conjunctiva has a non-keratinized stratified
epithelium. This layer is necessary to keep the surface
smooth. Modification of the stratified non-keratinized
secretory epithelium to non-secretory keratinized
epithelium is called squamous metaplasia 10,11. It has been reported that squamous metaplasia is seen in many
eye diseases that go with inflammation (dry eye, atopic
conjunctivitis) and systemic diseases 10,11. Inflammation
and loss of vascularization with regards to scar tissue have
been proposed as the causes of squamous metaplasia 10.
We previously reported that increased duration of chronic
renal failure increases the risk of metaplasia depending on
the degree of inflammation 12. The mechanism of the
loss of goblet cells due to vascularization and inflammation
According to recent studies, thyroid hormone deficiency
predisposes to structural changes of the ocular surface and
dry eye 13. It has been demonstrated that the lacrimal
gland is a target organ for thyroid hormone and expresses
thyroid hormone receptor β-1 (Thrb). In rats, it has
been proven that chronically reduced thyroid hormone
levels modulate the expression of Thrb in the lacrimal
gland and can therefore cause dry eye 13. To the best
of our knowledge, there are only a few studies that have
investigated the association between thyroid diseases and
CIC. First, Özkan et al. reported in 1997 a much higher
rate (82.3%) of Grade 2 and 3 conjunctival changes in TAO
(11). They investigated the association between TAO and
CIC. Only two components of TAO listed as increased
interpalpebral distance and proptosis had a weak relation
with squamous metaplasia, and inflammation was the
primary reason for these changes. Thereafter similar ocular
surface changes were found in several studies on Graves
disease. For example, İsmailova et al. detected worsening
conjunctival structure in TAO by using impression
cytology, vital staining and incisional biopsy methods
6,7,14,15. They state that vital staining may indicate
absence of transmembrane and secretory mucin that
protects the superficial epithelial cells. They speculated that
this glycoprotein deficiency (clinically identified by vital
staining) may cause or worsen secondary damage of both
the superficial epithelial cells and the deeper epithelial cells
15. In another study, Gürdal et al. found an increased rate
(75.7%) of Grade 2 and 3 CIC changes in Graves disease.
In subgroup analysis, Grade 2-3 changes were similar in
Graves disease with-or-without TAO 6. They evaluated
TAO clinically in GD patients and 24 of 42 eyes (57%) were
diagnosed with TAO. Eighteen eyes did not have a clinical
diagnosis of TAO but had the signs of dry eye and higher
grades of squamous metaplasia similar to the eyes with
TAO. These patients signified ocular surface inflammation
before the classic signs of the disease developed. The
authors emphasized that inflammation was the main factor
for these changes. Even so, before the onset of overt findings
of TAO, ocular surface inflammation (Grade 2-3 changes
of CIC) can be the single clinical sign in Graves disease
6,7. Inflammation is reported to be T cell mediated and
pro-inflammatory agents like IL-1, MMP-9 and TNF-alpha
are also associated with it 6,7,14. Similarly Gupta et al.
reported dry eye in occult TAO, which was diagnosed by
orbital echography in the dry eye patients with no classic
clinical findings of TAO such as exophthalmos and lid
retraction 16. Interestingly enough, we spotted squamous
metaplasia changes in half of the HT patients without
TAO or dry eye findings or any eye symptoms. This has not previously been reported in the literature. To top it off,
the increase of TSH in patients with Grade 3 changes has
been proven to be related to the drop in fT4. Perhaps most
importantly, the CIC levels of HT patients whose anti TPO
levels were high became worse. In the above mentioned
studies, Grade 2-3 metaplasia is reported to be usually seen
with dry eyes, and inflammation is held responsible for
both of these conditions. However, we have seen that CIC
grade was higher in HT patients despite the fact that none
of our patients had dry eyes. The CIC Grade became worse
as autoantibodies (Anti-TPO) increased according to the
results of our study. This suggested that autoantibodies
could be held responsible for the development of
conjunctival metaplasia in HT as we know that TSHR, a
thyroid autoantibody, plays an important role in Graves
ophthalmopathy 17. The well-known association between
high levels of thyroid autoantibodies and inflammation also
contributes to this conclusion 18. Similarly, we found that
Grade 2-3 changes were significantly higher in obese people
and obesity was determined as an independent predictor
for grade worsening which made us think that fat tissue
gain had a role in the pathogenesis. We know that release of
pro-inflammatory cytokines due to the increase of fat tissue
and also of immune-modulatory factors due to insulin
resistance are seen in obesity and that these molecules
may cause inflammation and squamous metaplasia in
conjunctiva as they do in other tissues and organs 19.
However there is no study providing information about the
The higher rate of Grade 2-3 changes in patients compared
to the control group with even dry eye patients were
excluded. This suggests that the triggering factors of eye
inflammation in HT might be different from Graves
ophthalmopathy. We think further research is definitely
needed on this topic.
There were some limitations in our study. Firstly, the
number of patients is relatively small. Secondly, other
thyroid autoantibodies (i.e. anti-TG, TSHR) and cytokines
(such as IL-1 and TNF-α) that can possibly play a role in
conjunctival squamous metaplasia pathogenesis were not
In conclusion, this is the first study to show that the
conjunctiva is affected in HT distinct from TAO and
dry eye. Anti-TPO levels and obesity were possible
triggering factors in this process. We need early detection
of conjunctival changes before the development of
ophthalmopathy in patients with Hashimotos thyroiditis
because early detection may prevent ophthalmopathy
complications such as blindness.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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