SURVEY OF OPHTHALMOLOGY VOLUME 57
NUMBER 4
JULY–AUGUST 2012

MAJOR REVIEW

Evaluation of Dry Eye

Samantha McGinnigle, BSc, Shehzad A. Naroo, MSc, PhD, and Frank Eperjesi, MBA, PhD

School of Life and Health Sciences, Aston University, Birmingham, United Kingdom

Abstract. Dry eye is a common yet complex condition. Intrinsic and extrinsic factors can cause
dysfunction of the lids, lacrimal glands, meibomian glands, ocular surface cells, or neural network. These
problems would ultimately be expressed at the tear film--ocular surface interface. The manifestations of
these problems are experienced as symptoms such as grittiness, discomfort, burning sensation,
hyperemia, and secondary epiphora in some cases. Accurate investigation of dry eye is crucial to correct
management of the condition. Techniques can be classed according to their investigation of tear
production, tear stability, and surface damage (including histological tests). The application, validity,
reliability, compatibility, protocols, and indications for these are important. The use of a diagnostic
algorithm may lead to more accurate diagnosis and management. The lack of correlation between signs
and symptoms seems to favor tear film osmolarity, an objective biomarker, as the best current clue to
correct diagnosis. (Surv Ophthalmol 57:293--316, 2012. Ó 2012 Elsevier Inc. All rights reserved.)

Key words. dry eye
tear quality
ocular surface
tear secretion
tear break up

osmolarity
evaporation
lipid layer
staining

I. Introduction dysfunctional tear syndrome, but redefined dry eye

Dry eye is a common, complex condition that can
reduce ocular comfort and visual performance. The
impact on quality-of-life has been rated as similar to
the effect of moderate angina226 and, in more severe
cases, dialysis and severe angina.33 In 2006 a panel
of dry eye experts used the Delphi approach to
establish diagnosis and treatment guidelines for dry
eye. This consensus method had been used success-
fully to standardize diagnosis and treatment in
cardiovascular disease.193 Four levels of disease
severity were outlined as well as recommendations
for patients with lid margin disease and abnormal
tear distribution. These guidelines primarily focused
on patient signs and symptoms and were accompa-
nied by the suggestion that the terminology
dysfunctional tear syndrome could replace the term
‘‘dry eye disease.’’17 The Dry Eye Workshop (DEWS)
added to the criteria and made additional treatment
recommendations. They did not adopt the term
as ‘‘a multifactorial disease of the tears and ocular
surface that results in symptoms of discomfort,
visual disturbance, and tear film instability with
potential damage to the ocular surface, accompa-
nied by increased osmolarity of the tear film and
inflammation of the ocular surface.’’154 The key
additions to their 1995 definition were the inclusion
of symptoms of visual disturbance, osmolarity, and
inflammation.149,154
The importance of inflammation in the patho-
genesis of dry eye had been established and
confirmed by the presence of immune-based in-
flammation in Sjögren and non-Sjögren dry eye250
and marked improvement in signs and symptoms
following use of anti-inflammatory therapies such as
cyclosporine, corticosteroids, tetracyclines, and
autologous serum.206 In 2009 a Canadian consensus
panel decided there was little difference between
grades 1 and 2 of the severity scale and combined

293
Ó 2012 by Elsevier Inc. 0039-6257/$ - see front matter
All rights reserved. doi:10.1016/j.survophthal.2011.11.003
294 Surv Ophthalmol 57 (4) July--August 2012
them to produce a simplified system to grade dry eye
as mild, moderate, or severe.110 A similar conclusion
was drawn in a prospective, multi-site clinical study,
where a combination of clinical measures were
converted to a ‘score’ for dry eye severity. Seven
clinical indicators were measured and the resulting
data showed ‘‘insufficient resolution to separate
mild and moderate patients into two groups,’’
leading to a simplified classification system of
normal, mild/moderate, and severe.253
The categorization of dry eye is complicated by
considerable variation in the disease’s symptoms
and signs and the often multiple causes. The
condition can involve the tear film, lids, main and
accessory lacrimal glands, meibomian glands, and
a wide spectrum of ocular surface cells including
epithelial, goblet, inflammatory, and immune
cells.112,146 The two main classes of dry eye
identified by the DEWS report were aqueous deficient
dry eye and evaporative dry eye. In aqueous deficient
dry eye, there are insufficient tears secreted by the
lacrimal glands, and this encompasses Sjögren
syndrome (an autoimmune disease) and non-
Sjögren. The term keratoconjunctivitis sicca has
been used historically instead of non-Sjögren
syndrome, although it is now recognized as a more
general term for any form of dry eye.154 The
common feature of these conditions is lacrimal
gland dysfunction. In evaporative dry eye there are
intrinsic and extrinsic causes. Intrinsic causes are
structural (e.g., abnormalities of the lids) or
functional (e.g., meibomian gland dysfunction).
Extrinsic causes include ocular surface irregulari-
ties, allergies, and contact lens wear.154 Smoking
causes deterioration in the lipid layer of the
precorneal tear film leading to dry eye symptoms
such as grittiness and burning sensations.7 The rate
of evaporation also increases with a larger palpebral
aperture (such as in up gaze), a longer blink
interval, increased air flow, increased temperature,
or reduced humidity.290
Tear hyperosmolarity, tear film instability, and
inflammation are all mechanisms for dry eye. Tear
hyperosmolarity may occur as a result of increased
evaporation or reduced aqueous secretion. The
increase in concentration of proteins and electrolytes
causes a reduction in tear volume that initially
irritates the ocular surface, but goes on to cause
inflammation and subsequent damage in evaporative
dry eye, as thinning of the lipid layer allows increased
evaporation.28,74 Tear film instability detected by
reduced tear film break-up time has also been linked
with the increased rate of local evaporation from the
tear film surface.127,129 Inflammation as a result of
auto-immune disease or even ageing can act as an
inciting event for dry eye.206
MCGINNIGLE ET AL
Epidemiological studies of dry eye in the United
Kingdom indicated a prevalence of 10% in people
under the age of 60, not including those who wear
contact lenses. The number of symptomatic patients
consulting an optometrist over this age is consider-
ably higher, with a larger proportion of women
being affected, especially post menopause.6 In the
United States prevalence of dry eye has been
reported as being between 0.6% and 57% depend-
ing on the classification.257 The Beaver Dam Study
found an incidence of 21.6% in a population
ranging from 43--86 years in age, increasing with
female sex and with age in both sexes.177 Johnson
and Murphy112 give a more generalized estimation
of 10--20% prevalence in the adult population,
based on a number of large studies.
In summary, aqueous deficient dry eye and
evaporative dry eye may co-exist and have features
in common, including increased tear film osmolarity
and reduced stability. Various assessment techniques
need to be carried out in order to be able to manage
the patient appropriately. We summarize the main
features of clinical and research techniques used to
assess dry eye and the indications for their use.
II. Subjective Evaluation
A. HISTORY AND SYMPTOMS
Examination of a patient with dry eyes invariably
starts with history and symptoms; there is often a lack
of correlation between the severity of the symptoms
and ocular signs of dry eye, however.16,99,116,190,280
Symptoms of dry eye have been found to be quite
severe, even with relatively mild surface changes, yet
paradoxically when the severity of dry eye reaches
a certain level, symptoms decrease as a result of loss of
corneal sensitivity.3,16,23,116,240,293 Reduced ocular
surface sensitivity has been documented as a normal
age-related change222 and as a consequence of
contact lens wear.207 Symptoms assessed by investiga-
tors have included dryness, grittiness, soreness,
redness, photophobia, and ocular fatigue, although
this is not an exhaustive list of patient’s complaints
with this condition. The variability of reported
symptoms can be simplified by a defined list of
questions to make comparisons between visits and
between patients more straightforward.
B. VALIDATED QUESTIONNAIRES
Validated questionnaires are available to ensure
consistency in recording symptomatic information.
They consist of a series of questions with numerical
values attributed to the answers, allowing the
symptoms to be scored. This also means that
patients can be monitored by comparing subsequent
EVALUATION OF DRY EYE
scores. The most widely used of these are the
McMonnies Dry Eye Index and The Ocular Surface
Disease Index (OSDI), the latter being deemed the
more reliable.191,225 The McMonnies survey is
a screening test using dichotomous yes/no re-
sponses and considers epidemiological risk factors,
frequency of symptoms, and sensitivity to environ-
mental triggers.161 The OSDI has a Likert design
and assesses frequency of ocular symptoms (sore-
ness, blurred vision), difficulty with vision-related
function (television, visual display unit, driving,
reading) and discomfort due to environmental
triggers (low humidity, high wind). The patient
answers 12 questions, with higher scores represent-
ing greater disability. The questionnaire has un-
dergone psychometric testing and has been
accepted by the US Food and Drug Administration
as suitable for use in clinical trials.225 Limitations of
the OSDI include variation of difficulty between
categories of questions, no linear relationship of the
results to symptom severity, and analysis issues from
the use of ordinal ranking. The final percentage
score may also be artificially high when difficult
questions are not answered or deemed not applica-
ble by the patient.113 Neither of these question-
naires indicate symptom severity, but The
International Workshop on Meibomian Gland Dys-
function: Report of the Diagnosis Subcommitee
suggested that the OSDI may be useful for monitor-
ing disease progression, despite the difference in
symptoms in meibomian gland dysfunction,259
based on a recent validation of the survey.166 The
Symptom Assessment in Dry Eye questionnaire
quantifies frequency and severity of symptoms and
demonstrated good repeatability within a few
days.224 A basic Likert design questionnaire asking
two symptom questions (1) ‘‘How often do your eyes
feel dry (not wet enough)?’’ and (2) ‘‘How often do
your eyes feel irritated?’’—with a final question
asking if the subject had ever been diagnosed with
dry eye syndrome before—was sensitive and repeat-
able (sensitivity, 77%, and specificity, 83%, when
Schirmer ! 10 mm/tear break-up time is less than
10 sec) in a large cohort and may prove useful in
a busy clinic or for large epidemiologic studies.95 A
more complete comparative listing of dry eye
questionnaires is available in the report of the
Epidemiology Subcommittee of the International
Dry Eye Workshop 2007.241
III. Objective Evaluation
A scientific roundtable on dry eye ranked tear
break up time (93%), corneal staining (85%), tear
film assessment (76%), conjunctival staining (74%),
295
and the Schirmer test (54%) as the most commonly
used diagnostic tests for initial assessment of dry
eye.230 A previous retrospective report by Nichols of
447 patients with dry eye in a clinic-based sample
found symptom assessment (82.8%), fluorescein
staining (55.5%), and tear break-up time (40.7%)
to be the most frequently used tests in cases coded
as a dry eye diagnosis.192 A wide variety of tests have
been used to evaluate dry eye, some more complex
than others and with varying sensitivity and speci-
ficity. A note of caution is warranted regarding
interpretation of sensitivity and specificity figures,
which may reflect spectrum bias, particularly in
smaller studies.154 This refers to recruitment of
moderate to severe diseased states that are more
easily distinguished from patients with a normal eye
state, and therefore the figures cannot be applied to
a generalized dry eye population, which will contain
many more mild cases.
The techniques are categorized depending on
which particular aspect of the condition is being
investigated.
A. TEAR PRODUCTION
1. Schirmer and Phenol Red Thread Test
(Hamano Threads)
The Schirmer test and phenol red thread test are
invasive methods to measure change in volume of
the tears in the tear reservoir. Both involve insertion
of a wick into the lower conjunctival sac and
measurement of the wetting length over a set period
of time. The Schirmer test uses filter papers to assess
tear production. There are two commonly used
variations of the Schirmer test: Schirmer I measures
total tear secretion (reflex and basal tears) and
Schirmer ll utilizes anesthetic to measure basal
secretions, although this has not been vali-
dated.46,252,273 A value of less than 5 mm wetting
in 5 minutes is considered abnormal for both tests,30
and Schirmer II values inversely correlate with
age.285 There is also a version of the Schirmer I test
using nasal stimulation for use in severe dry eye.268
Serin et al suggest that performing the test on
a closed eye gives more reliable and repeatable
results, although their test population did not have
dry eyes.230 Medial and lateral placements of the
paper have been described, as well as having the
patient looking up, but no method has been
deemed more reliable.155 Confounding factors in-
clude discomfort, difficulty of the 5-minute duration
when assessing children, controversy over what is
actually being measured, measuring inconsistencies
with uneven wetting edge margins, poor repeatabil-
ity, and damage to the surface of the eye.43 A study
evaluating the utility of a number of clinical tests
296 Surv Ophthalmol 57 (4) July--August 2012
found that the Schirmer test was particularly poor at
discriminating between mild to moderate cases of
dry eye and showed variable results, but that it is
a marker for the aqueous deficient subtype of dry
eye.253 A 1-minute (with anesthesia)14 and 2-
minute120 Schirmer test have been suggested, with
cut-offs of 6 mm and 10 mm (99% confidence
interval), respectively.
The phenol red thread is less invasive than the
Schirmer test (although more difficult to perform)
and has been described as an index of tear
volume.258 The thread is inserted for 15 seconds
and the pH of the tears changes the thread from red
to yellow to facilitate measurement. In normal eyes,
values for the test done in closed eyes were found to
be slightly less than open eyes.61 A value of less than
10 mm indicates dry eye.98 A study leaving the
thread in place for 120 seconds differentiated
between aqueous deficient and evaporative dry eye
using a cut-off of 20 mm (sensitivity, 86%; specificity,
83%).199 No correlation has been found between
phenol red thread test and tear meniscus radius in
dry eye, whereas the Schirmer I test showed
significant correlation when both tests were per-
formed for one minute.298
2. Research Techniques to Measure Tear
Thickness
Non-invasive methods to measure the total tear
film thickness are currently investigative because of
the cost and complexity of the instrumentation. A
normal tear thickness measurement of 3 mm was
found by King-Smith et al using reflectance spectra
from the cornea.126 Wang et al found similar results
using optical coherence tomography (OCT) (low
coherence interferometry);284 Hosaka et al, how-
ever, found normal values of 6.0  2.4 mm and dry
eye values of 2.0  1.5mm using an interference thin-
film thickness measurement device.105 Recent eval-
uation of indirect measurement by Azartash et al has
led to the development of Fluctuation Analysis by
Spatial Image Correlation using a laser and camera
system to measure interference patterns from the
tear film, then applying a mathematical model to
calculate the tear film thickness. They found
a statistically significant difference between the tear
film thickness of patients with normal eyes and
those with dry eye and suggested a cut-off value of #
2.75 mm for dry eye, with a sensitivity of 86% and
specificity of 94%.10
3. Tear Clearance/Tear Turnover Rate
Delayed tear clearance contributes to chronic
ocular inflammation that sensitizes the nerves, caus-
ing irritation.65,205 Tear clearance can be measured in
MCGINNIGLE ET AL
a number of different ways. One of the earlier
methods was visual comparison of fluorescein col-
lected on Schirmer strips to photographic stan-
dards,205 but reflex tearing was an issue, even with
the use of anesthetic.201 Alfonso et al analyzed samples
collected from the lower meniscus 15 minutes after
the instillation of 5 ml of 2% fluorescein, measuring
fluorescence using a fluorescence multiplate reader
(Cytofluor II; Perseptive Biosystems, Framingham,
MA, USA). Their findings suggested that delayed tear
clearance (independent of levels of aqueous tear
production) correlated with irritation as a symptom.4
This method was deemed impractical for routine
clinical use,156 and a standardized visual scale was
proposed. The color of the lateral inferior tear
meniscus was matched to a standardized scale with
a score ranging from 0--6. Scores greater than 3
indicated delayed fluorescein clearance. Research
using this technique to investigate the effect of
punctual occlusion in normals found reduced tear
clearance initially, followed by improvement in both
the occluded and nonoccluded eye, suggesting the
stimulation of mechanisms to regulate the ocular
surface. They also suggested that this could be why
individuals with ocular irritation find symptomatic
relief after the insertion of punctual plugs.294
Gamma scintigraphy and fluorophotometry use
the electromagnetic spectrum to monitor a tracer
molecule in the tear film. A radioisotope is required
for scintigraphy, raising issues of safety and expense,
so fluorophotometry is the favored technique using
fluorescein as the tracer. In addition, fluoropho-
tometry has been found to be more sensitive201 and
is regarded as the gold standard in estimating tear
flow or volume.160,201 A study in which 2 ml of
fluorescein was applied to the ocular surface and
then assessed with the Fluorotron Master fluoropho-
tometer (Coherent Radiation, Palo Alto, CA, USA) at
intervals for 30 minutes found patients experiencing
dry eye had a reduced tear turnover rate.246 Nelson
found the tear turnover to be 42% lower in patients
with keratoconjunctivitis sicca than in patients with
normal eyes, implying that dry eye increases the risk
for toxic effects of topically applied substances.181
Tomlinson et al conducted a meta-analysis and
found aqueous deficient dry eye patients showed
a 60% reduction in tear turnover rate, and
evaporative dry eye patients showed a reduction of
30%. They concluded that tear turnover rate was
a useful way to determine dry eye and its subtypes,
with cut-offs of O12.9 ml/min (sensitivity, 74.9%;
specificity, 73.6%) for dry eye versus patients with
normal eyes; O15.1 ml/min (sensitivity, 80.2%;
specificity, 58.7%) for patients with normal eyes
versus those with evaporative dry eye, and O9.6 ml/
min (sensitivity, 69.5%; specificity, 96.8%) for those
EVALUATION OF DRY EYE
with aqueous deficient dry eye versus those with
evaporative dry eye.260 This is still, however, an
expensive, laboratory-based technique with special
expertise required.
The Tear Function Index291 is used to measure
tear secretion (using the Schirmer test with anes-
thetic291), divided by drainage (measured by the
fluorescein clearance test292) to obtain a value that
discriminates between patients with normal eyes and
those with dry eyes. This combined test showed
improved discrimination over the individual tests,
with 78.9% sensitivity and 91.8% specificity for values
below 34 in Sjögren syndrome.292 The test became
less accurate with higher values, however, possibly
due to evaporation.158 McCann et al used a commer-
cially available modified filter paper strip impreg-
nated with a predetermined amount of fluorescein in
an extended version of the Schirmer test to measure
tear flow and turnover rates. The length of wetting in
3 minutes with a closed eye was measured, and the
intensity was compared with a pre-determined
standard to calculate the tear clearance rate. Their
findings for the Tear Function Index (Liverpool
modification) strip were 83% sensitive in discrimi-
nating between dry and normal eyes, but specificity
was poor at 40%. The investigators suggested the
results were comparable to those found using
fluorophotomety, but the limited number of grades
reduces the utility of the index, particularly in
borderline cases.160 The simplicity of the test and
potential improvements in the grading scheme could
make this technique useful in the clinical setting.
4. Evaporimetry
Tsubota proposed that tear volume is governed by
tear production, drainage, and evaporation, the latter
of which he measured by evaporimetry (capture of
fluid loss from the ocular surface).268,269 Tsubota felt
this method could be helpful in sub-classifying dry
eye268,269 and possibly determining patients with an
unstable tear film due to meibomian gland dysfunc-
tion82 (measuring tear evaporation rates with con-
trolled levels of humidity using tight-fitting goggles
and an eyecup). Tomlinson et al carried out meta-
analyses of studies measuring evaporation and found
raised levels in dry eye, particularly evaporative dry
eye (normal, 13.57  6.52  10ˉ7 g/cm2; aqueous
deficient dry eye, 17.91  10.49  10ˉ7 g/cm2;
evaporative dry eye, 25.34   10ˉ7 g/cm2).260 Korb
found that higher humidity increased the thickness
of the lipid layer.139 Evaporimetry has been used to
evaluate treatment for evaporative dry eye, including
thermotherapy83 and the use of lipid eye drops.84
Confounding factors are the sampling response
rate to blinking, perspiration, palpebral aperture,
297
variation in blink speeds and patterns and in un-
ventilated chambers, and resistance to evaporation
caused by humid, still air.82,129,268
5. Meniscometry
The tear meniscus is the concave reservoir of
tears at the inferior and superior lid margins from
which the pre-ocular tear film is formed after
a blink.256 Measurement of tear meniscus is con-
sidered to indicate total tear volume.115,297 Factors
influencing tear meniscus dimensions include tear
secretion,297 location of the punctum,285 lacrimal
drainage,34 lid length,285 eyelid tension,232 and
palpebral aperture.59,232 Confounding factors
for measurements of the tear meniscus include
conjunctivochalasis210 and debris at the cornea-
lower eye lid junction.306 Tear meniscus dimen-
sions including height,19,59,60,135,196,210,232,285,306
196,232,297
radius, depth,135,210,306, area,135,210,232,285,306
19,285,297
volume, and curvature297 have been measured
using a slit lamp, graticule, and photography;223 video
keratography;115,272 pachymetry115 video meniscome-
try using specular reflection;196,297 tear interference
(Tearscope; Keeler Ophthalmic Instruments, Windsor,
UK);272 time domain OCT (high resolution images
using infra-red light);16,19,107,135,232,285 and Fourier
domain optical coherence tomography.210,306
Many studies focused on the lower tear meniscus
as measurement of the upper meniscus proved
difficult because of the mobility of the upper lid and
shadowing from the upper lashes.115 Fluorescein
improved visualization of the meniscus, but, al-
though improving reproducibility, may have given
higher values as a result of the addition of fluid and
reflex tearing.306 The inherent natural variability of
the tear meninscus led to the suggestion that
repeated measures are required to get any meaning-
ful clinical information.115 Research into the simi-
larity of the upper and lower menisci has produced
conflicting results. Wang and Shen found the two
menisci to be similar, as measured by OCT,232,283 but
Johnson and Murphy found the superior meniscus
height to be 20% less than the inferior meniscus
height measured using a video slit lamp.114 Studies
have agreed that tear meniscus dimensions are
reduced with age and dry eye, although there was no
consensus in which specific dimension was superior
in the assessment of dry eye. The significant amount
of overlap in values between patients with normal
eyes and those with dry eye has also meant that
a range of referent values from a height of 0.1mm60
to 0.35mm157 (using fluorescein) have been
suggested as the cut-off for dry eye.
Earlier studies using OCT tended to underesti-
mate meniscus dimensions because of poor image
298 Surv Ophthalmol 57 (4) July--August 2012
resolution and artificial image coloration.115 Later
OCT studies have been more repeatable,19 particu-
larly those using Fourier domain OCT.210,306 Shen
suggested lower tear meniscus height and radius
were the best indicators of dry eye and gave a cut-off
height of 1.64 mm (sensitivity, 0.92; specificity, 0.9)
and radius of 1.82 mm (sensitivity, 0.92; specificity,
0.87).232 The use of a slit lamp and graticule/
micrometer is an inexpensive and useful way to
assess lower meniscus height, and therefore volume,
although using a cross section may lead to over-
estimation.115 Measurements by meniscometry or
pachymetry are really only suitable for research
because of the specialized nature of the equipment,
and OCT also requires expensive equipment and
may be confined to research. Fourier domain OCT
is capable of detailed and rapid measurements,
removing the error factor of eye movement and
providing improved qualitative information, such as
discontinuity and striae in the border of dry eye
menisci.210 Comparisons of Asian and white pop-
ulations may have led to erroneous results as a result
of the difference in lid tension and palpebral
aperture between races; measurement of tear
meniscus area using Fourier domain OCT and
‘‘removing the interference factor of interpalpebral
aperture’’210 may eradicate this problem in future
research. Yokoi et al have also suggested wider
applications of meniscometry in the assessment of
lacrimal plugs, punctal occlusion, general tear
clearance, tear supplement kinetics, and lacrimal
secretogogues.297,301
B. TEAR STABILITY
To prevent the damage of underlying tissues, the
tear film must be redistributed and reformed with
every blink,274 and, in a normal eye, this happens
approximately 17 times per minute at rest.18 Blink
rate has been shown to vary within and between
subjects and vary with tasks, decreasing with increased
concentration.62,100,227 Dry eye subjects have been
shown to exhibit increased blink rates, attributed to
reduced tear film stability.100,180 A tear film break-up
time of less than 10 seconds indicates instability,151
and the Report of the Diagnosis Subcommittee of
The International Workshop on Meibomian Gland
Dysfunction suggests that the role of lipids in the
stability of the tear film warrants investigation of
‘‘meibomian gland function and expressibility’’ in all
cases of low tear break-up times.259
The tear film can be assessed using invasive and
non-invasive techniques. The most commonly used
invasive technique uses a slit lamp with a cobalt blue
light source to view the tear film after the instillation
of fluorescein. The first appearance of a dark spot
MCGINNIGLE ET AL
marks the tear break-up time; fluorescein has
a destabilising effect on the tear film, however, and
can actually reduce the measured value.164,200 For
this reason, the trend in recent years, particularly in
research, has been towards non-invasive techniques
to allow tear film evaluation in the natural state.301
The non-invasive tear break-up time is measured
by observing standard keratometer mires45 or a
grid pattern projected onto 70--80% of the corneal
surface.101,301 This can be achieved by using
a xeroscope (modified bowl perimeter),165 modified
keratometer mires with a HIR-CAL grid,48,101 or
a hand-held keratoscope with a Loveridge grid.101
These instruments all project a white grid on a black
background. The Keeler Tearscope and Keeler Tear-
scope Plus (Keeler Ophthalmic Instruments) with
a corneal topography grid attachment46,94,187 project
a black grid on a white background. Guillon and
Guillon suggest that the black background instru-
ments measure tear thinning and the white back-
ground instruments measure tear breakup.93 This is
why some refer to tear thinning time or pre-rupture
phase time, which occurs just before tear break-up
and may account for the differing results found in
various studies. Another area of discrepancy is the
endpoint criterion, which varies from defocus of
mire image101 to the first discontinuity of the grid.165
These studies gave results of 18.6  11.0 seconds and
47.9 seconds, respectively.
The grid image size can also affect results as, in
some patients, the tear film breaks up more quickly
in the periphery.42 Thus, if the area of cornea
covered by the grid differs, so will the apparent tear
break-up time. Dry eye can cause the surface of the
eye to become irregular and therefore disrupt an
already unstable tear film more rapidly.131 There-
fore, despite the concerns about reproducibility and
variability, assessment of the tear film is considered
to be an important diagnostic test for dry eye, as tear
dysfunction is the common link between all forms of
dry eye.220 Cho et al found that if the fluorescein
tear break-up was repeated several times, the first
measurement was significantly different than the
second; the second was usually very similar to the
third, however. This suggests that if two measure-
ments are taken, the second should be used and
regarded as being representative of the patient’s
tear break-up. This has not been verified by non-
invasive techniques.44
1. Interferometry
The superficial lipid layer can be observed and
measured by interferometry, but a straightforward
relationship between the thickness of the lipid layer
and the rate of tear evaporation has not be proven.
EVALUATION OF DRY EYE
The composition and structure of this layer, rather
than its overall thickness, seems to determine the rate
of tear thinning.128,148 The lipid layer can be observed
by looking at the images produced by specular
reflection using the Tearscope Plus (Keeler Ophthal-
mic Instruments) and the DR-1 interferometer (Kowa
Co Ltd, Tokyo, Japan). One study has suggested that
the DR-1 interferometer is the superior of the two as it
eliminates the background iris color giving a clearer
image.81 The images can be graded using the Guillon-
Keeler Tear Film Grading System for the Tearscope
and the Yokoi severity grading system for the
DR-1.11,302 The Guillon-Keeler system describes
patterns that relate to lipid layer thicknesses: open
meshwork (13--50 nm), closed meshwork (13--50
nm), wave/flow (50--70 nm), amorphous (80--90
nm), color fringes (90--180 nm), and globular
(O200 nm). This scheme was upgraded to 10 levels
with 0 representing no lipid and 9 representing a lipid
layer thickness predominantly found in young in-
fants.108 The Yokoi scheme has five grades: grade 1,
grayish and uniform; grade 2, grayish and non-
uniform; grade 3, few colors but non-uniform; grade
4, very colorful but non-uniform; and grade 5,
exposed cornea from absence of lipid layer. Normal
eyes are grades 1 and 2, whereas dry eye tends to be
higher, although some overlap occurs.301 Meibomian
gland dysfunction can be differentiated from normal
eyes by a change from horizontal to vertical patterns
at grades 1 and 2 and a darker interference color with
no fringing.85 This is not definitive, however, as later
research found aqueous deficient dry eye could also
produce this pattern.88 Goto and Tseng measured the
interference patterns before and after punctual
occlusion and found the thickness of the lipid layer
and the volume of aqueous were interconnected, as it
took longer for the lipid layer to stabilize in aqueous
deficient eyes.86 They also assessed the lipid spread
time and pattern and the stability of the lipid layer
in lipid deficient dry eye and found distinctive
differences between those with dry eyes and normals,
which they suggested could be utilized in evaluating
treatment.85
Videokeratography uses distortion of a ring pat-
tern image on the corneal surface to analyse the
surface regularity index and the surface asymmetry
index. These indices effectively describe the smooth-
ing properties of the tear film, and studies have
shown that reduced tear stability in dry eye can
adversely affect image quality.173,271 High-speed
videokeratoscopy allows continuous acquisition of
corneal topography maps demonstrating the
dynamic nature of the tear film in between blinks,
previously depicted as static, constant and uni-
form.184 Kojima et al developed this idea and created
new software to detect more subtle changes in
299
distortion and two new aspects termed tear stability
regularity and asymmetry indices were analyzed.
They concluded that these new indices were partic-
ularly useful in detecting mild dry eye.136 This Tear
Analysis Stability System has recently been improved
further to correct for unstable fixation, and in
addition the brightness difference of data points
has been used to calculate the tear break-up. These
changes improved sensitivity to 82%, with a specificity
of 60%, for a 5-second cut off, improving to 82.2%
and 88%, respectively, for a 3-second cut-off. There
was increased variability as the severity of dysfunc-
tional tear syndrome worsened.96 In a 2008 survey,
videokeratoscopy was chosen by most of the expert
panel as their technique of choice for initial
evaluation a patient with Lasik-related dry eye.242
2. Aberrometry
Wavefront sensing using a Hartmann-Shack aberro-
meter (Topcon Corp, Tokyo, Japan) evaluates the
optical quality of the eye. This can give an indication
of visual disturbances created by higher order
aberrations, such as those created by tear film
instability and break-up. Changes in tear volume
and dynamics induce changes in higher order
aberrations, which appear as characteristic patterns.
Post blink, the sequential changes in these patterns
were classified in normal eyes as ‘‘stable’’, ‘‘saw tooth’’
and ‘‘other’’ by Koh et al.132 In evaporative dry eye
patients, the pattern changed to ‘‘saw tooth’’, with
a marked upward curve that increased after blink-
ing.133 In patients with an excessive tear volume and
epiphora after punctual plug insertion, there was
a ‘‘reverse saw tooth’’ pattern.134 It has been
suggested that the time course of increased aberra-
tions is accelerated in patients with an abnormal tear
film.171,172 Low tear volumes in severe dry eye may
cause increased, but stable, aberrations. This may be
to the result of the irregular, damaged epithelium in
the optic zone in the absence of dynamic tear film
changes.131 The instillation of artificial tears was
shown to reduce aberrations in dry eye when the
measurement was taken after several sec-
onds,172,174,175 although a study measuring 2 seconds
after a blink found an initial increase in aberrations
because of the increase in tear volume and the
disturbance to the tear film.216 Extended use of
artificial tears altered the short term effects of a drop
of artificial tears, restoring aberrations to levels
found in normal eyes. It was suggested that the
mechanism for this may have been a change in the
tear film structure because of a decrease in the
immune-mediated response.216 Aberrometry is a use-
ful tool for non-invasive assessment of the ocular
surface and optical performance of the eye. A recent
300 Surv Ophthalmol 57 (4) July--August 2012 MCGINNIGLE ET AL

conference suggested it could be utilized for detect- distance of the objective lens.169 Mathers and Daley
ing and monitoring dry eye and also to measure used a non-contact, tandem-scanning confocal
efficacy of treatments.55 microscope demonstrating real-time images to
observe the tear film at magnification of approxi-
3. Functional Visual Acuity mately 150. Debris was discovered in the central
corneal region of tear film in dry and normal eyes.
The ocular surface needs to be smooth, with
They concluded that the minimal alteration of tear
a stable tear film, to facilitate clear vision.117 Patients
film function and excellent focusing characteristics
with dry eye have ocular surface irregularity caused
made this a valuable tool for detailed imaging of
by tear film instability and are more likely to
tear film properties.159
complain about blurred vision when driving, read-
Wakamatsu et al used confocal scanning laser
ing, and using a computer.87 Functional visual acuity
microscopy for the quantitative assessment of the
(FVA) was originally defined as the monocular
conjunctival inflammation and epithelial cell densi-
recognition acuity at a specific time point.109 Later
ties, as well as evaluation of conjunctival morphologic
studies used a range of values, as well as the visual
alterations, such as microcysts in patients with
maintenance ratio (the ratio between the FVA and
Sjögren (SSDE) and non-Sjögren (NSSDE) dry eye
the baseline visual acuity) in an effort to reflect
disease. Their findings of conjunctival inflammatory
everyday vision more accurately.117,118 All indices
cells were 433  435.8 cells/mm2 in SSDE, 134.8 
evaluate real-time decay in visual function during
124.2 cells/mm2 in NSSDE, and 10  17.9 cells/mm2
the blink interval; statistical analysis of results in
in healthy control eyes. They have suggested that this
studies where the base visual acuities differ is only
technique could be used for non-invasive impression
possible using the visual maintenance ratio, how-
cytology in dry eye evaluation.282 Higher goblet cell
ever.118 FVA was assessed by Ishida et al using
densities were detected in samples using confocal
a measurement system (SSC-350; Nidek, Gamagori,
microscopy (332  137 cells/mm2) versus impression
Japan) where Landolt optotypes were presented on
cytology (200  141mm2) in SSDE by Hong et al.104
a monitor, starting at a size corresponding to the
Despite the difference, the results did correlate, but
patient’s best corrected visual acuity. The optotypes
more accurate representation of ocular surface
decreased in size when the patient gave a correct
changes may be possible using confocal microscopy.
answer about the orientation using a joystick, or
Zhang et al assessed subjects with ‘‘mild self-reported
increased in size if the patient answered incorrectly
office dry eye.’’ They found decreased cell density in
or gave no response. Topical anesthesia was instilled
superficial, intermediate, and basal epithelial layers
and the patients were instructed not to blink for 30
in mild, moderate, and severe cases of dry eye. They
seconds. Measurements were taken at 10-, 20-, and
suggested this could be a consequence of increased
30-second intervals, and FVA was reduced in dry eyes
osmolarity, inflammatory mediators, or insufficiency
versus controls.109 This technique was also used to
of tear components. Sub-basal nerve rupture and
evaluate punctual plug insertion in ‘‘short break-up
tortuosity were also present in moderate and severe
time dry eye’’ where FVA improved in patients
cases.304 Confounding factors such as cost and
without epiphora.119
maintenance issues may limit the use of laser
FVA has also been compared with wavefront
scanning confocal microscope in a clinical setting.
aberrations in dry eye and normal eyes. Greater
Adjustment all of the parameters to obtain a good
instability was found in the dry eye group, although
image has been described as ‘‘extremely difficult,’’
no correlation was found between higher order
particularly for the novice user, and therefore this can
aberrations and FVA in either group. The authors
only be regarded as a research tool.308
suggested factors in addition to higher order
aberration—including pupil size, light scattering,
and visual processing—may also affect FVA. Dry eye 5. Tear Osmolarity
individuals with a higher blink rate were found to
The role of tear hyperosmolarity in dry eye is
have more stable FVA, suggesting increased blinking
undisputed.70,261,278 As the osmolarity of the tear
may be a compensatory mechanism to improve
film increases, it starts a cascade of inflammatory
vision.286
changes resulting in damage to the epithelial
surface of the eye. Pflugfelder et al identified
4. Confocal Microscopy ‘‘increased concentrations of inflammatory cyto-
A confocal microscope focuses the light source kines in the tear fluid’’ and found a positive
and objective lens on to the same small area of correlation between the severity of dry eye symptoms
interest, the focal volume of which is defined by the with this marker and the severity of staining and
numerical aperture, magnification, and working conjunctival squamous metaplasia.204 In addition to
EVALUATION OF DRY EYE
this, surface wettability and tear film stability are
further reduced by loss of the glycocalyx and goblet
cell dropout, as found by Rivas et al in Sjögren
syndrome.217 The DEWS report identified expense,
the requirement of specialist technical support, and
the lack of a rapid and simple commercial device as
reasons why the measurement of osmolarity has
been mainly confined to research projects.30
Tear film osmolarity can be measured in three ways:
the gold standard is freezing point depression
(FPD),69,78 requiring samples as small as 0.1 ml;77
Other methods are vapor pressure167,202 and electrical
conductivity or impedance.195 These two techniques
require minimum tear samples of 0.8 ml.202 FPD
technically measures osmolality, which is the concen-
tration of solutes per unit weight. Osmolality is
a colligative property and therefore independent of
temperature and not subject to interpolation.103 The
difference between normal, mild, moderate or severe
values are so small that precision is critical. Attempts
to obtain sufficient quantities of tears in severe dry eye
may induce reflex tearing, contaminating the sample
and giving inaccurate lower osmolarity read-
ings.77,182,202 The variation of osmolarity across the
tear film and the potential for increased osmolarity in
the lower meniscus (the most common sampling
location) may also lead to inaccuracy.77
An increase in tear osmolarity is common to all
types of dry eye. Farris reviewed a number of studies
and found the measurement of tear osmolarity to
provide a sensitivity of 90% and a specificity of 95%.
He suggest that osmolarity should become the
standard test for the investigation of dry eye
syndrome.70 This has been confirmed by a recent
study finding ‘‘tear osmolarity to be the single best
marker of disease severity across normal, mild/
moderate and severe categories (p. 6129).’’253 This
argument is tempered slightly by Khanal et al who
advised caution in regarding osmolarity as the
definitive test. They concluded that although tear
osmolarity seemed a reliable test for dry eye, it was
‘‘ineffective in differentiating between aqueous de-
ficient dry eye and evaporative dry eye (p. 1239).’’121
The TearLab system (Tearlab Inc., San Diego, CA,
USA) determines osmolarity through the electrical
impedance of tear samples, a method that may be
suitable for use in a clinical setting.278 The
advantage of this technique is the 0.05-ml sample
size and the fact that it takes only 30 seconds to
obtain values for both eyes. The ‘‘lab-on-a-chip’’ is
said to mitigate the risk of evaporation using
microfluidics (fluid is pumped into tiny micro-
channels in the disposable tip), which means only
very small volumes of tears are required. Osmolarity
values greater than 308 mOsms/l are a sensitive
indicator of mild dry eye disease, although Lemp
301
et al emphasized that this should be seen as ‘‘a
guidepost along the gradient of severity,’’ due to the
intermittent nature of compensatory factors. There-
fore the higher value in either eye should be used
(variability between eyes is a hallmark of dry eye
disease), and the result should be considered in
conjunction with tests performed during a full
clinical examination, particularly when the signs
and symptoms do not match. It was suggested that in
moderate to severe cases of dry eye, a more sensitive
detection was achieved at 312 mOsms/l (sensitivity,
73%; specificity, 92%).150
The Tear Osmometer (Advanced Instruments Inc,
Norwood, MA, USA) calculates osmolarity using
FPD. Evaporation is minimized by the microcapil-
lary used to obtain the sample, although this fluid
does have to be transferred to a fabricated tip to go
into the instrument for analysis and requires
samples of 0.5 ml to obtain reliable results, so may
not be suitable for use in severe dry eye.296 Recent
studies used this method to show the correlation
between increased osmolarity and dry eye disease
severity,253,254 although it was stated that ‘‘tear
osmolarity cannot be used as the sole indicator of
dry eye disease (p.4560).’’254
6. Meibomian Gland Evaluation
Meibomian gland dysfunction is the most common
cause of evaporative dry eye causing problems with
ocular comfort, visual function, and, in more severe
cases, cosmesis. Red-rimmed, notched lids may
become permanently disfigured.152 A Spanish study
found a strong association between meibomian gland
dysfunction and the signs and symptoms of dry eye.
They found a prevalence of 11% for dry eye and
30.5% for meibomian gland dysfunction, which was
also present in 45.8% of the subjects with dry eye.279
Meibomian gland dysfunction denotes functional
abnormalities of the meibomian glands, whereas
meibomianitis or meibomitis indicates the presence
of inflammation. Anterior blepharitis is normally
associated with bacteria, particularly Staphylococcus,
and posterior blepharitis results from inflammatory
conditions of the posterior lid margin. The use of the
terms low delivery (hyposecretory) and high delivery
(hypersecretory) are used to further categorize
meibomian gland dysfunction,188 although there is
no universal classification.72 A recent study in the
United States reported a frequency of blepharitis of
37% in ophthalmological practice and 47% in
optometric practice.152 Meibomian glands are mod-
ified sebaceous glands numbering 25--40 along the
upper lid margin and 20--30 along the lower lid
margin.27 They secrete lipids, facilitated by the
contractile forces of blinking.41,137 This proposed
302 Surv Ophthalmol 57 (4) July--August 2012
mechanism may explain why many meibomian
blockages occur after sleep and why lipid accumula-
tion is common upon waking.137
Meibomian glands can be assessed visually using
a slit lamp. Grade 0 indicates all glands clear of
blockages; grade 1 indicates a few capped glands;
grade 2 indicates several blocked glands and
secretions appearing thick; grade 3 indicates half
the glands are blocked; and grade 4 indicates more
than half the glands are blocked, combined with
viscous secretions/tears.27 The most common form
of meibomian gland dysfunction may start with
minimal signs (non-obvious obstructive meibomian
gland dysfunction), requiring clinical evaluation of
meibomian gland expressibility.20 The International
Workshop on Meibomian Gland Dysfunction rec-
ommends that gland expression should be included
routinely in asymptomatic patients.188 There have
been several techniques described: digital
force;46,233,234 sterile cotton buds;73 or an instru-
ment such as a glass rod, speculum, or custom made
device;73,138,194 but this lack of standardization
makes comparisons of results difficult. Two similar
semiquantative grading scales (0--3) have been
proposed, with 0 representing clear, easily expressed
fluid (normal). In one scheme grade 1 represents
cloudy meibum expressed with mild pressure; grade
2 represents cloudy meibum expressed with more
than moderate pressure; and grade 3 represents
meibum that cannot be expressed with even strong
pressure.233,234 The other scale describes the secre-
tions, but not the pressure required. Grade 1
represents expression of yellowish-white oily fluid,
grade 2 represents thick cheesy material, and grade
3 represents no expression possible.247
Meiboscopy involves the transillumination of the
eyelid using a white light. Robin et al used cool
distal light from a Finoff transilluminator to assess
morphology and density of the meibomian glands as
well as quantifying any drop out.219 Yokoi et al have
recently improved on this technique by inventing
a new probe connected to a digital recorder.
Patients did not report any discomfort.300 A study
comparing obstructive and seborrhoeic meibomian
gland dysfunction everted the lids and used a slit
lamp with an infrared device and camera. The lids
were graded by a scheme from 0 (no loss of
meibomian glands) to 3 (loss of two-thirds of the
total area of meibomian glands). They found
shortening of the meibomian glands in obstructive
meibomian gland dysfunction and changes in
meibomian glands with age.8 This technique was
described as ’’useful, quick and patient-friendly,’’
suggesting its potential in a clinical setting.
Meibography is also a method of quantification of
meibomian gland drop-out. It is considered
MCGINNIGLE ET AL
superior as the photodocumentation enables
masked evaluation and therefore increased objec-
tivity in clinical trials.259 Nichols carried out a study
on the reliability of two methods of grading using
this technique;186 there is still no consensus on
grading scales, however.259
Meibometry involves the use of a plastic tape to
blot the central lower lid margin. The lipid blot
changes the optical density and can be measured in
a photometer. On the basis of this measurement,
Chew et al. estimated 300 ml of meibomian lipid are
present in the marginal reservoir of a normal adult
man.40,41 Versura et al used a laser meibometer to
estimate the lipid uptake by the tape and found this
technique be highly accurate and reliable in
distinguishing patients with meibomian gland
dysfunction.277
C. OCULAR SURFACE
1. Corneal and Conjunctival Staining
Corneal and conjunctival staining is an invasive
procedure which enables the assessment of ocular
surface damage by instilling a dye such as sodium
fluorescein, rose bengal, or lissamine green. Fluo-
rescein is tolerated well by patients, and single
applications do not sting. The dye suspended in the
tear film can obscure the staining pattern, but the
pattern must be read immediately or stromal
diffusion may cause inaccurate recording.29 The
amount of dye instilled should be minimized,
particularly in dry eyes, as fluorescein is not
fluorescent at concentrations above 2%. This is
called ‘‘quenching,’’ and the dye remains dark in
appearance. Self-absorption can also decrease fluo-
rescence, as the occlusive effect of the outer layers of
solution prevents the illumination of the deeper
layer.162 Other factors which may affect the result
are the pH (fluorescence increases up to pH 8 and
then decreases), the presence of preservative or
other substances, and the wavelength of the
absorbed light. The eye is best viewed with an
exciter (Wratten 47/47a; Edmund Optics, Barring-
ton, NJ, USA) and barrier filter (Wratten 12/15) to
assess staining on the cornea and conjunctiva.29
Rose bengal requires a threshold concentration to
achieve staining and is thought to stain areas of the
superficial epithelium which are not protected by
mucin because of a discontinuous tear film.71
Historically it has been the most commonly used
test in the assessment of dry eye (usually in
combination with a Schirmer test) and as a measure
of treatment success.58 It does not stain the tear
film, so any epithelial staining shows up well,
especially with a green filter. It does sting on
application, however, and topical anesthetic needs
EVALUATION OF DRY EYE
to be applied before instillation. It is phototoxic (re-
activated by sunlight post instillation, causing pain)
and stains the skin quite markedly.29
Lissamine green also requires a threshold con-
centration and, although it is accepted that it stains
the eye in a similar manner to rose bengal,58 there is
controversy over what is actually stained. Some claim
it stains epithelial cells that are not protected by
mucin or protein,125 whereas others suggest it
actually stains degenerated cells and mucus.58 It is
tolerated similarly to fluorescein by most patients
and is less toxic than rose bengal and is used
increasingly in place of rose bengal for these
reasons,. A repeat instillation of 10--20 ml is required,
and the staining is best viewed after 2 minutes. Korb
suggests the use of 2% fluorescein in combination
with 1% lissamine green for ‘‘optimal corneal and
conjunctival staining.’’141
Evaluation of staining is highly subjective, but the
use of charts such as the Oxford grading scheme can
help by enabling consistent recording of staining
severity. The scheme has five panels, labelled A--E,
with staining represented by punctuate dots that
increase logarithmically between the panels.29 The
clinician compares the appearance of staining on
the exposed interpalpebral conjunctiva and cornea
with each panel, and the closest match determines
the grade. Other grading schemes with scoring
systems are Van Bijsterveld,273 which has a narrower
range of scoring, and CLEK,13 which assesses several
zones over the cornea, including the visual axis
(which enables comparison with visual function).
The repeatability of staining tests has been found to
be poor,189 and they lack discriminatory power in
mild to moderate cases of dry eye.253
The Sjögren Syndrome International Registry
modified the Oxford grading scheme29 to enable
concurrent grading of the cornea and bulbar
conjunctiva using a combination of one drop of
0.5% fluorescein (for corneal staining) and one drop
of 1% lissamine green (for conjunctival staining). On
a specially designed form, a grade between 0 and 3
was given for staining on the cornea, based on the
number of ‘‘dots’’ seen, which was to be added to
a grade between 0 and 3 for the nasal and the same
for the temporal conjunctiva. This gave a maximum
possible total of 9 points. Three additional points
were then allocated for fluorescein only if there was
confluent staining (þ1), staining in the papillary area
(þ1), or one or more filaments (þ1), giving
a maximum possible score of 12. This was performed
for each eye. An abnormal score was considered to be
3. This study identified two forms of keratoconjunc-
tivitis sicca with potentially different causes and
showed a strong association between the ocular
staining score and other phenotypic characteristics
303
of Sjögren syndrome. It was concluded that this
would be a suitable test ‘‘for diagnosing the ocular
component of Sjögren syndrome in future classifica-
tion criteria.’’287
2. Fluorophotometry
Fluorophotometry has also been used as ‘‘a
sensitive measure of epithelial integrity.’’181 To
detect changes in fluorescence emitted from the
ocular surface following the instillation of fluores-
cein, measurements are taken 10 minutes after
washing out the dye with saline, then again every 10
minutes up to an hour. The fluorescein uptake at
the center of the cornea is assessed, and patients
with dry eye demonstrate an increased corneal
permeability and a slower rate of fluorescein
elimination compared to patients with normal eyes.
Residual fluorescein in the tear film can give a false
reading with this technique.68,181 Other potential
sources of error are the biphasic decay, with the first
5 minutes representing reflex rather than basal
secretions; fluorescence affected by variable tear
thickness layers rather than just concentration of
fluorescein (forced blinking increases the tear layer
thickness and weak blinking decreases it); and the
inherent autofluorescence of the cornea.181
3. Lid Wiper Epitheliopathy Evaluation
Lid wiper epitheliopathy (LWE) can often be
found in patients who have dry eye symptoms, but
no signs.295 This condition was first described by
Korb et al, who described the lid wiper as ‘‘that
portion of the marginal conjunctiva of the upper
eyelid that wipes the ocular surface during blink-
ing.’’142 The epithelium in this area is stratified
squamous epithelium, and if the lid is everted and
stained with lissamine green or rose bengal, Marx’s
line is visible along the length of the lid (approx 0.1
mm wide106). Jones et al interpreted this area as
a lubrication region which undergoes higher
stresses and suffers damage as a consequence and
therefore stains.A The staining in this area is often
more severe in dry eye patients.32,142 The diagnosis
of LWE involves sequential staining with a mixture
of 2% fluorescein and 1% lissamine green. The lid is
then everted and the fluorescein is graded from 0--3
depending on the linear area and severity of the
staining, followed by the same grading system for
lissamine green. The highest final score is taken to
be the LWE severity grade. The classification is no
LWE, grade 1 LWE (mild, 0.25--1.0), grade 2
LWE (moderate, 1.25--2.0), or grade 3 LWE (severe,
2.25--3.0).140
304 Surv Ophthalmol 57 (4) July--August 2012
4. Lid Parallel Conjunctival Folds
Lid parallel conjunctival folds (LIPCOF) may be
observed in the temporal and nasal areas bordering
the posterior lid margin in primary gaze. There is
controversy with regard to their predictive ability for
dry eye symptoms.102,168,208 Conjunctivochalasis has
been implicated in some dry eye cases,54,163,299 and
it is thought that LIPCOF may represent the early
stages of this condition.209 There are various
grading scales available: Schiffman et al proposed
0 to indicate no folds; 1 to indicate a single fold less
than tear prism height; 2 to indicate multiple folds
up to the tear prism height; and 3 to indicate
multiple folds, higher than tear prism height.225
Pult et al suggested an optimized grading scale
where 0 indicated no conjunctival folds; 1 indicated
one permanent and clear parallel fold; 2 indicated
two permanent and clear parallel folds (normally !
0.2 mm); 3 indicated more than two permanent
folds (normally O 0.2 mm).209 OCT has recently
been used in a study to grade LIPCOF, correlating
well with slit lamp evaluation. This technique may
allow a more detailed and objective method of
assessment of this condition in the future.275
D. LABORATORY TESTS
During normal tear production, the surface
environment is maintained by the secretion of
aqueous, mucin, and lipid components and is
regulated by the neural reflex arcs and anti-
inflammatory substances including immunoglobu-
lins, antimicrobial proteins and growth factors. Any
inciting stimulus disrupting the homeostasis of this
environment initiates an inflammatory cascade. The
following techniques are used to measure specific
molecules in the generation or immune expression
of dry eye disease and assess the resulting cellular
changes.
1. Tear Ferning Test
When mucus air dries on a microscope slide,
a characteristic arborization, or ferning, occurs. The
test requires a 1-ml drop of tears from the lower
meniscus taken with a micropipette and then
dropped onto a microscope slide and allowed to
dry at 20  3C for 10 minutes. The diagnosis of dry
eye is made according to the pattern made by the
crystals. The classifications are as follows: type 1 5
uniform large arborisation; type 2 5 ferning
abundant but of smaller size; type 3 5 partially
present incomplete ferning; and type 4 5 no
ferning. Dry eyes result in either type 3 or 4.221
Evans et al found a poor correlation between tear
ferning and tear film stability or their validated
symptoms questionnaires. They concluded that this
MCGINNIGLE ET AL
technique demonstrated ‘‘limited sensitivity and
specificity for the prediction of ocular surface
comfort in both contact lens and non-contact lens
wearers (p.199).’’67
2. Lacrimal Gland Function Test
Lactoferrin, lysozyme, and lipocalin are proteins
produced in the acini of the lacrimal glands.50,79
Lactoferrin makes up approximately 25% by weight of
the total tear proteins (approximately 2.2 mg/ml).123
There has been research suggesting that ocular
surface epithelial cells also produce lactoferrin in
the conjunctival and, to a lesser extent, corneal
epithelial tissue.145 More recently, the meibomian
gland has been investigated as a source of lactofer-
rin.265 The actions of lactoferrin are as a modulator to
the inflammatory response,15 an antimicrobial with
a bacteriostatic action competing with bacteria for
free iron,38 and an anti-oxidant, protecting mucosal
surfaces from oxidative damage.124 It can also bind
with the lipopolysaccharide from Gram negative
bacteria, increasing the membrane permeability—
thus causing osmotic shock and, in the right
concentration, inhibiting bacterial biofilm formation
on contact lenses.238,288,289 There is also evidence to
suggest it has a role in UV protection.235
Lactoferrin concentration in the tears can be
measured by commercial plates such as the Lacto-
plate test, using tear samples taken with filter paper
discs or by enzyme-linked immunosorbent assay
(ELISA). Farris et al found that lysozyme and
lactoferrin concentrations in basal and reflex tears
were variable, but the results of either could
correctly identify whether a patient had keratocon-
junctivitis sicca. Their findings overall were a re-
duction in lysozyme and lactoferrin concentrations
in dry eyes.69 Ohashi et al197 found mean levels of
2.05  1.12 mg/ml in normal eyes and 0.13  0.22
mg/ml in Sjögren syndrome, confirming findings in
a previous study by Da Dalt who had found a high
sensitivity (95%) and specificity (72%) using this
test.49
The LactoCard (Touch Scientific Inc., Raleigh,
NC, USA) is a solid-phase ELISA requiring 2 ml of
tears colorimetrically measured by a spectrometer.
The test takes approximately 15 minutes. Results
lower than 0.90 mg/dl are considered abnormal.70
Lipocalins are proteins with lipid-binding, anti-
microbial, and scavenging properties.76,80,214 The
lipocalin--lipid bond is felt to be key to maintaining
the stability of the tear film by enabling a rapid
formation of a homogenous lipid layer on the
surface of the tear film to limit evaporation.24,263
Caffery et al found reduced lipocalin levels in
SSDE and NSSDE compared with normal eyes
EVALUATION OF DRY EYE
using electrophoresis and the Western blot. All
three groups showed a difference in their bio-
chemical distribution of total tear proteins, which
could prove useful in distinguishing different
forms of aqueous-deficient dry eye.37 Versura et al
investigated tear proteins in early evaporative dry
eye using electrophoresis, immunoblotting, and
enzymatic digestion, followed by liquid chroma-
tography and mass spectrometry. The ratio of the
different forms of lipocalins was found to be
altered in comparison to normals. They hypothe-
sized that the tear lipid distribution change was
caused by decreased expression of affinity lipid-
proteins, including lactoferrin, even with early
inflammatory changes. These changes may repre-
sent dysfunction of the lacrimal gland, undetect-
able by other methods.276
Epidermal growth factor helps to maintain the
ocular surface and control corneal wound healing.
In a study using 1 ml of tears sampled using a glass
microcapillary pipette followed by sensitive bead
immunosorbent assay, reduced levels were found in
Sjögren syndrome than controls; however, increased
levels were found in meibomian gland dysfunction
with delayed tear clearance than controls. Previous
research showed the role of epidermal growth factor
in epithelial hypertrophy, increased epithelial cell
division,90 and corneal vascularization,185 and the
authors suggest that epidermal growth factor could
be the causative factor in meibomian gland orifice
metaplasia and subepithelial fibrosis in these
patients.212
3. Tear Protein Analysis
Evidence for the role of inflammation in dry eye
was initially focussed on T-cell involvement, specif-
ically the effector T-cell responses.250,307 This was
supported by the effective treatment of dry eye with
cyclosporine, a T-cell immunosuppressant.144,251
Stern et al, using conjunctival biopsy specimens,
found a common cellular basis of inflammation in
Sjögren and non-Sjögren dry eye that was pre-
dominantly CD4þ and CD8þ T-cells.250 The focus
has since shifted to the function of regulatory T
cells, which control the effector T-cells. Research on
autoimmune diseases such as rheumatoid arthritis
and multiple sclerosis has shown a connection
between interleukin or Th17 and inflamma-
tion.64,249,303 Investigators induced dry eye in mice
and found that dysfunctional regulatory T-cells
from the dry-eye mice were less effective at
suppressing the action of pathogenic effector T-
cells, particularly Th17, than those in normal
mice.39 A combination of real-time polymerised
chain reaction and flow cytometry was used. A
305
combination of immunohistochemistry, which ex-
ploits the specific binding features of antibodies
and antigens to target proteins, and Luminex
immunobead assay was used by a different group
who also suggested that the presence of both Th1
and Th17 were required for full expression of dry
eye.52 The authors suggest that this association
could be a new target for dry eye therapy.
There is a correlation between tear cytokine levels
and the severity of symptoms and ocular surface signs
in all forms of dry eye,147 and it has also been
demonstrated that different tear proteins are present
in dry eye disease with and without meibomian gland
disease.66,147 The technique used in these studies was
multiplex bead analysis using a Luminex IS-100
(Luminex Corp, Austin TX, USA) and a 10-ml tear
sample. This system enables simultaneous analysis of
up to 100 analytes and is based on the principles of
flow cytometry. In mild cases of dry eye without
staining and with normal tear production levels, an
increase in inflammatory cells could still be found.
This indicates that inflammatory mediators in the
tears are perhaps a better indicator of dry eye disease
than the measure of tear production or staining. Grus
et al obtained samples from dry eye subjects using
Schirmer strips and used ProteinChip Array (Cipher-
gen Biosystems, Inc., Fremont, CA, USA) technology,
which performs automated analysis of the proteins
and peptides.92 They found large quantities of
calgranulin A, a calcium binding protein thought to
play a significant role in fighting inflammation.179
Tong et al used absolute quantification based
proteomics combined with two-dimensional nano-
liquid chromatography and nano-electrospray mass
spectrometry to analyse tear proteins in various
grades of meibomian gland dysfunction (0--3 based
on slit lamp findings). They proposed that calgranu-
lin A and B were related to disease severity and
specific symptoms of ‘‘redness and transient blur-
ring.’’ In addition they correlated calgranulin A to
symptoms of ‘‘grittiness’’ and Lipocalin-1 with ‘‘heavi-
ness of the eyelids and tearing.’’262 Tear proteomics
gives detailed analysis of the structure and function of
proteins in microvolume samples, even at low
concentrations.228 Almost 500 proteins have been
identified in tear fluid using these methods, and the
characterization of these proteins could be an
important step towards the determination of new
biomarkers for dry eye disease.53
Matrix metalloproteinases (MMPs) are proteolytic
enzymes produced by the ocular surface, glandular
epithelial cells, and immune cells under stress. The
absence of inflammation in normal eyes leads to
a low expression of MMP-9, but in inflamed and dry
eyes, the expression is high and associated with
a disruption of the corneal epithelial barrier
306 Surv Ophthalmol 57 (4) July--August 2012
function.203,243 MMP-9 has also been found at
higher levels in eyes with conjunctivochalasis,1
meibomian gland disease, and Sjögren syndrome.245
4. Tear Lipid Analysis
The meibomian gland secretes a complex mixture
comprising families of non-polar lipids, polar lipids,
and proteins,35 although Tiffany estimated that
although over one hundred major compounds have
been identified, there could be thousands yet un-
discovered.255,256 Fatty acids and fatty alcohols are
surfactants that stabilize the interface between the
aqueous and the lipid layer,35 but they can easily
collapse to form droplets because of their thermody-
namic instability, causing disruption to the layer and
increased evaporation.236 Waxes and sterol (choles-
terol) esters are hydrophobic. Glycerol can bond with
different molecules, rendering some partially water
soluble and others hydrophobic,35 and it has been
shown that bacteria secrete lipases that hydrolyse
ester bonds in glycerides and phospholipids. This can
alter the lipid ratio, and increased levels of such
bacteria have been found in patients with chronic
blepharitis, and altered lipid ratios have been found
in patients with meibomianitis.38,57,237
Historically, lipid analysis was performed using
techniques based on hydrolysis and derivatization,
including thin-layer chromatography, nuclear mag-
netic resonance spectroscopy, infrared and Raman
spectroscopy, gas chromatography, and gas chroma-
tography mass spectrometry.35,91 The specifics of
these techniques are beyond the scope of this
review; an extensive review by Butovich identified
confounding factors in studies utilising these
techniques, however, including difficulty in obtain-
ing uncontaminated samples in sufficient quantity
to analyze; low sensitivity and resolution of the tests;
and degradation of the sample from prolonged
exposure times, sustained high temperatures, and
prolonged analysis times.35 Recent advances in
technology and instrumentation has seen huge
progress in the field of lipidomics,36 which involves
the identification and quantification of lipid molec-
ular species and their interactions with other
molecules. Analysis of minute quantities of meibum
and tear film lipids is now possible by combining
technologies and mathematical procedures such as
principal component analysis (PCA) that can also be
applied to the measurements of chemical data. PCA
reduces the dimensionality of complex data sets in
order to identify new, meaningful underlying
variables.
Borchman et al applied PCA to the infrared
spectroscopy results of 1-mg meibum samples ex-
pressed from patients with normal eyes and patients
MCGINNIGLE ET AL
with meibomian gland dysfunction. The samples
were collected using a platinum spatula after
individually compressing all four eyelids between
cotton tipped applicators. The main difference
between normal eyes and those with meibomian
gland disease was the increase in protein and
compositional differences leading to increased
viscosity in the diseased samples.21 Further research
revealed that changes in the amount of lipid
saturation governing lipid--lipid strength could
cause a change in the conformation of lipids in
individuals with meibomian gland disease.22 In-
frared and visible absorbance spectroscopy were
used to quantify skin and meibum lipids taken from
patients with normal eyes and patients with meibo-
mian gland disease. It was concluded that the
quantity of lid margin lipid was similar between
the cohorts, so was not contributing to tear film
instability, and that forehead sebum was not a bio-
marker for meibomian gland disease.9 Meibomian
fatty acids collected with sterile Schirmer strips and
analyzed using gas chromatography and mass
spectrometry were found to differ in meibomian
gland dysfunction when compared to aqueous
deficient dry eyes and normals.111 The significant
increase in branched chain fatty acids was reduced
by treatment with minocycline, leading to the
suggestion that fatty acids, particularly iso-C20,
could be a useful biomarker for meibomian gland
dysfunction.248
E. HISTOLOGICAL TESTS
Histology provides information about the ocular
surface, but the limitations of such techniques
include the variable and partial nature of the
sampling procedure and the relevance of informa-
tion gained, given the dynamic nature of the tear
film. These tests do enable the mucin content of the
tear film to be evaluated, however. Mucins are large
glycoproteins and are classified as transmembrane
or secretory, with secretory mucins further sub-
categorized as gel-forming or soluble according to
their ability to form polymers.112 The transmem-
brane mucins are anchored to the corneal epithelial
cells and form the glycocalyx.56 They make the
surface wettable and effectively fill in gaps where
there is epithelial desquamation or degeneration231
and are an effective protective barrier against
infectious agents.47 There is controversy over
whether the secretory mucins lower the surface
tension of the whole tear film or just the mucin
phase.112 Goblet cells in the conjunctiva secrete
both soluble and gel-forming mucins, and it has
been suggested that the density of these cells could
be a marker for dry eye disease.130,143 Conjunctival
EVALUATION OF DRY EYE
goblet cell density in a normal eye has been
measured as 8.84  4.66/mm in a normal eye and
2.11  0.78/mm in an eye with keratitis sicca.211
1. Conjunctival Impression Cytology
This technique is a rapid, minimally invasive, and
relatively painless method of harvesting conjunctival
epithelial, goblet, and inflammatory cells from the
bulbar mucosa. The most superficial epithelial cells
of the conjunctiva have reached their final differ-
entiation when they are harvested; therefore,
abnormalities in this process can be analyzed.26
Filter papers, 13 mm in diameter, are used, and the
most common are cellulose acetate, nitrocellulose,
Biopore (Millipore, Billerica, MA, USA) or Supore
(Gelman sciences, Ann Arbor, MI, USA), which is
polyethersulfone. Pores range from 0.025--0.45 mm,
but most are 0.20 mm.26 After the instillation of
topical anesthetic, a filter strip is pressed against the
conjunctiva, usually upper, for 5--10 seconds using
forceps. The samples are then fixed using 95%
ethanol, stained with periodic acid-Schiff reagent
and fixed on a slide to be viewed with a micro-
scope.239 Rivas et al were one of many groups to
demonstrate how the inferior palpebral conjunctiva
was much less affected than the exposed bulbar
conjunctiva in aqueous deficient dry eye and
claimed that impression cytology combined with
rose bengal staining was the most sensitive and
specific test for primary Sjögren syndrome.218
Impression cytology can show a diverse sample of
cells, depending on the staining agents used, but with
reference to dry eye, it has classically been used to
calculate goblet cell density and staging of squamous
metaplasia.213 Changes of this type have been seen in
other ocular surface disorders but are primarily
associated with dry eye.178 Squamous metaplasia
describes various changes of the epithelium accom-
panied by a decrease and eventual loss of goblet cells.
Morphological changes such as stratification and
keratinization of non-goblet cells (e.g. lymphocytes)
are also progressive. Various subjective grading
schemes considering the number, size, shape and
nucleus: cytoplasm ratio have been used. Rivas and
Tseng described six grades, and Nelson and Adams
described four.2,178,183,266 The absence of a scale on
the representative images63 and inability to grade
non-typical combinations have led to new schemes
being proposed.63,97
Flow cytometry usually involves the analysis of cells
released from filter papers into buffered solutions,
harvested as described in classic cytometry. Samples
can also be collected using brush cytology. The cells
are centrifuged in phosphate-buffered saline be-
fore analysis using immunofluorescent procedures.
307
Indirect methods of flow cytometry amplify weakly
expressed antigens; direct methods give more rapid
results, however.26 Solomon et al measured expres-
sion of cytokines in dry eye and found positive
immunofluorescent staining for cytokines IL-1a,
mature IL-1b, and IL-1Ra in a significantly greater
percentage of conjunctival cytology specimens from
eyes with Sjögren syndrome, than in those from
normal eyes (p ! 0.01 for IL-1a, p ! 0.009 for
mature IL-1b, and p ! 0.05 for IL-1Ra).245 In-
tercellular adhesion molecule causes T-cell homing
as well as acting as a ligand, leading to lymphocytic
infiltration.170 The expression of this and other
inflammatory markers such as human leucocyte
antigen (HLA-DR), which also acts as a T-cell ligand,
has been demonstrated using flow cytometry analysis
of tear samples from dry eyes.25
New methods of flow cytometry include the use of
different mediums in an attempt to preserve cell
numbers. Barabino et al placed right eye samples in
phosphate-buffered saline containing 0.05% para-
formaldehyde and left eye samples in cell culture
medium containing 10% fetal calf serum. After
phenotypic analysis, the cells collected in fetal calf
serum were stained for the expression of various
inflammation markers and analyzed by flow cytom-
etry. The calf serum samples contained a statistically
increased number of cells when compared with
parafomaldehyde samples. In the dry eye group
there was a significant difference in the CD4/CD8
ratio compared with normal eyes, and almost
double the number of CD14þ cells (monocytes/
macrophages). HLA-DR expression was also in-
creased in CK19þ conjunctival epithelial cells of
dry eye patients.12 Reinoso et al carried out a study
on meibomian gland dysfunction--related evapora-
tive dry eye subjects. After a 2-month treatment
regime of lid hygiene, artificial tears, and a short
course of steroids, 87% of subjects improved.
Increased CD8 cells, reduced CD4 cells and a re-
duced CD4/CD8 ratio were found, compared with
normal eyes. The authors suggested CD4 and CD8
as possible biomarkers for the disease.215 These
studies illustrate how immune cells isolated from the
superficial layer of the conjunctiva may influence
the pathogenesis of dry eye. With new methods to
preserve impression cytology samples, flow cytom-
etry analysis of epithelial and immune cells of the
conjunctiva may be enhanced and new biomarkers
identified.
2. Conjunctival Brush Cytology
This technique again requires topical anesthesia,
utilising a soft brush to obtain not only superficial
cells as in the previous technique, but also basal
308 Surv Ophthalmol 57 (4) July--August 2012
cells. The sample can then be assessed for the
presence of squamous metaplasia, inflammatory
cells, and the expression of surface markers on the
ocular surface epithelium.267 This is often combined
with flow cytology, which gives a highly sensitive and
specific analysis of epithelial cell markers, inflam-
matory cells, and goblet cells.75 Wakamatsu et al
found mean inflammatory densities of 320 cells/
mm2 and 856 cells/mm2 in normal eyes and those
with atopic keratoconjunctivitis, respectively. The
polymorph and dendritic cell densities in the
conjunctiva and cornea were also significantly high-
er in patients with atopic keratoconjunctivitis, the
normal values being estimated as 190 polymorph
cells/mm2, versus 920 cells/mm2 in atopic kerato-
conjunctivitis, and 10 dendritic cells/mm2, versus
þ50 cells/mm2 in atopic keratoconjunctivitis. This
correlated strongly with confocal scanning laser
microscopy.281
IV. Discussion
Dry eye is one of the most prevalent eye diseases
causing discomfort, deterioration in visual quality,
and increased risk of infection.89 Problems setting
diagnostic criteria remain, because of the wide range
of methods used in diagnosis, which creates difficulty
with comparative measurement of efficacy of treat-
ment.122,153,176,229,270 There is variation in the way
the tests are conducted and in the interpretation of
the results. The International Workshop on Meibo-
mian Gland Dysfunction259 presents a useful table
listing the diagnostic efficacy of tests for evaporative
and aqueous deficient dry eye, giving the sensitivity
and specificity of tests distinguishing normal eyes
from dry eyes in general and from aqueous deficient
and evaporative dry eye specifically. In addition,
there is some information differentiating evaporative
from aqueous deficient dry eye; in the absence of
a standard reference test and the possibility of
spectrum bias, however, careful consideration should
be given to the relevance of an individual test to
a particular patient. Paradoxically, some putative new
and improved methods have been shown to offer
poor accuracy and reproducibility.
The overlap in normal and dry eye values for many
tests is also a confounding factor. There is as yet no
gold standard test to diagnose dry eye disease, or even
a battery of tests that are universally accepted. The
clinical picture of dry eye disease changes through its
course; therefore, different tests and diagnostic
criteria may be appropriate at different stages.31
Bron described how boundaries between dry eye
classifications are not clear-cut and that hybrid forms
can evolve. Aqueous deficient dry eye is a high
osmolarity, low volume disorder. A decrease in
MCGINNIGLE ET AL
lacrimal secretions through the loss of the corneal
reflex leads to a decrease in tear volume, which thins
the aqueous and retards the spreading of the lipid
layer. This effectively adds a functional evaporative
component. Evaporative dry eye is a high osmolarity,
high volume disorder. Compensatory lacrimal flow is
lost due to inflammatory damage as the disease
progresses and aqueous production is reduced,
resulting in an aqueous deficient component.31
Testing across the range of etiologies, presenta-
tions, and severities in dry eye patients has been
addressed by Sullivan et al., whose method involved
the use of a composite index, where individual tests
including osmolarity and a symptom score were
combined and equally weighted to determine
severity. This approach was thought to insulate
against measurement noise when assessing disease
severity and allowed the role of each test to be
assessed at different levels of severity.253 Khanal et al
suggested the use of discriminant function analysis,
where several tests are combined in a diagnostic
algorithm that can be constantly updated and
improved as more data become available.122 They
suggested that tear physiology tests were repeatable
and reliable, unlike the currently used diagnostic
tests, but they excluded patients with blepharitis or
meibomian gland dysfunction, despite the high
prevalence of these conditions. Tear turnover rate
(TTR), evaporation, and osmolarity were the best
combination (86% overall accuracy, 100% sensitivity,
100% negative predictive values), but TTR is
relatively invasive as it uses fluorescein and evapo-
ration is really more of a research technique. King-
Smith did cite evaporation as a major cause of tear
film break-up, however, and suggested that huge
increases in osmolarity were possible through this
mechanism.129 The link between osmolarity and the
production of cytokines has been proven,198,305 and
the increase in concentration of cells such as Th17
and MMP-9 may be sensitive and specific for the
presence of early dry eye. Differentiation between
aqueous deficient and evaporative dry eye may also
be possible by identification and quantification of
specific proteins such as lipocalin, which has been
shown to stimulate reflex tearing in evaporative dry
eye via a compensatory mechanism.262,276 The
correlation of decreased tear clearance with in-
creased interleukin and MMP-95,244 further supports
the use of clinical measures of tear clearance/
turnover as a useful clinical measure of dry eye.
In terms of future treatment of dry eye, a stratified
clinical approach may soon be possible with the
selection of suitable biomarkers. This requires the
addition of a clinical biomarker-assessment step to
the traditional history and symptoms, clinical
evaluation, and laboratory investigations. This could
EVALUATION OF DRY EYE
possibly lead to the end of empirical prescribing and
the advent of tailored therapy, with increased
efficacy and reduced toxicity. This approach is
already being developed in the treatment of cancer,
although mainly in patients who have not
responded to the usual treatment strategies.51,264
V. Conclusion
Ideally a test needs to be accurate (sensitive and
specific to changes from normal values), objective,
and give reproducible results, preferably in a non-
invasive manner. The purpose of the test governs its
suitability, whether it is for use in clinical practice or
clinical trials. Research projects may utilize different
methods as sophisticated equipment is more readily
available; there is a need to establish reliable clinical
tests to measure the same parameters, however.
Where possible, the use of digital imaging will
enable external and therefore impartial grading in
trials and more consistent evaluation of dry eye in
clinics. In a clinical setting it will also free up
valuable consulting time as experienced technicians
will be able to complete the required investigations
so the clinician can focus on history, symptoms, and
treatment. The molecular composition of the tear
film is gradually being deciphered, as advances in
proteomics and lipidomics as well as improved
methods of mathematical analysis of results give
insight into specific differences between diseased
and non-diseased states. This may lead to the
discovery of specific molecules suitable for use as
biomarkers to identify dry eye and differentiate
between subtypes. The implementation of ‘‘lab-on-a-
chip’’ technology has enabled rapid, in-depth
assessment of minute quantities of tear fluid in
a clinical setting. As small and portable osmolarity
devices become affordable, this method could
become more commonly used and possibly become
the new standard. Improved software could also see
anterior segment OCT used in the diagnosis and
monitoring of some dry eye conditions, particularly
as lenses to adapt retinal OCT for anterior use are
becoming available. The substitution of lissamine
green for rose bengal may also mean conditions
such as lid wiper epitheliopathy are diagnosed as
part of a routine examination. Improved grading
scales with meibomian gland evaluation and the tear
function index (Liverpool modification) strip for
measuring tear clearance may enable more rapid,
accurate assessment of patients, enabling differenti-
ation between aqueous deficient and evaporative dry
eye conditions. Increased awareness of lid-parallel
conjunctival folds and an optimized grading system
may further knowledge with regard to the role of
309
this condition. Improvements in standardization for
all techniques will also help researchers, as the
number of validated methods of investigation
increases.
VI. Method of Literature Search
A search of pertinent articles using the PubMed
database (National Library of Medicine) and the Web
of Science database was performed to identify all
relevant articles published between 1990 and March
2011. Terms and words used for the search included
dry eye, tear quality, ocular surface, tear production, tear
components, meibomian gland dysfunction, osmolarity and
imaging. Articles from all languages were considered,
providing the English abstracts were included in
non-English articles. Copies of the entire articles
were obtained. Bibliographies of the retrieved
articles were manually searched using the same
guidelines. The articles were reviewed and informa-
tion relating to evaluation of dry eye were incorpo-
rated in the article. We included articles relating to
techniques using recognized and obtainable equip-
ment. Studies using researchers’ own experimental
designs of instrumentation or other instrumentation
that is not available for commercial use or research
purposes were excluded.
VII. Disclosure
The authors reported no proprietary or commer-
cial interest in any product mentioned or concept
discussed in this article.
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s.a.naroo@aston.ac.uk.

Outline
I. Introduction C. Ocular Surface
II. Subjective Evaluation
1. Corneal and conjunctival staining
A. History and symptoms 2. Fluorophotometry
B. Validated questionnaires 3. Lid wiper epitheliopathy evaluation
4. Lid parallel conjunctival folds
III. Objective evaluation
A. Tear production
D. Laboratory Tests
1. Schirmer and Phenol Red Thread Test
1. Tear ferning test
(Hamano Threads)
2. Lacrimal gland function test
2. Research techniques to measure tear
3. Tear protein analysis
thickness
4. Tear lipid analysis
3. Tear clearance/tear turnover rate
4. Evaporimetry
5. Meniscometry E. Histological tests
1. Conjunctival impression cytology
B. Tear Stability 2. Conjunctival brush cytology
1. Interferometry
IV. Discussion
2. Aberrometry
V. Conclusion
3. Functional visual acuity
VI. Method of literature search
4. Confocal microscopy
VII. Disclosure
5. Tear osmolarity
6. Meibomian gland evaluation