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TFOS DEWS III Diagnostic Methodology

James S. Wolffsohn , José Benı́tez-Del-Castillo ,

Denise Loya-Garcia , Takenori Inomata , Geetha Iyar ,
Lingyi Liang , Heiko Pult , Alfonso L. Sabater ,
Christopher E. Starr , Jelle Vehof , Michael TM Wang , Wei Chen ,
Jennifer P Craig , Murat Dogru , Victor L Perez Quinones ,
Fiona Stapleton , David A Sullivan , Lyndon Jones , TFOS
collaborator group

PII: S0002-9394(25)00275-2
DOI: https://doi.org/10.1016/j.ajo.2025.05.033
Reference: AJOPHT 13391

To appear in: American Journal of Ophthalmology

Received date: May 15, 2025

Accepted date: May 23, 2025

Please cite this article as: James S. Wolffsohn , José Benı́tez-Del-Castillo , Denise Loya-Garcia ,
Takenori Inomata , Geetha Iyar , Lingyi Liang , Heiko Pult , Alfonso L. Sabater ,
Christopher E. Starr , Jelle Vehof , Michael TM Wang , Wei Chen , Jennifer P Craig ,
Murat Dogru , Victor L Perez Quinones , Fiona Stapleton , David A Sullivan , Lyndon Jones , TFOS
collaborator group, TFOS DEWS III Diagnostic Methodology, American Journal of Ophthalmology
(2025), doi: https://doi.org/10.1016/j.ajo.2025.05.033

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 TFOS DEWS III Diagnostic Methodology
James S. Wolffsohn,1 José Benítez-Del-Castillo,2 Denise Loya-Garcia,3,4 Takenori Inomata,5
Geetha Iyar,6 Lingyi Liang,7 Heiko Pult,8,9 Alfonso L. Sabater,10 Christopher E. Starr,11 Jelle
Vehof, 12,13 Michael T. M. Wang,14 Wei Chen,15 Jennifer P. Craig,14 Murat Dogru,16 Victor L.
Perez Quinones,10 Fiona Stapleton,17 David A. Sullivan,18 Lyndon Jones.19 + TFOS
collaborator group
1) Optometry and Vision Science, College of Health and Life Sciences, Aston University, Birmingham, UK
2) University Complutense, Hospital Clinico San Carlos, Clinica Rementeria, Madrid, Spain
3) Instituto de Oftalmología Fundación de Asistencia Privada Conde de Valenciana, I.A.P, Mexico City,
Mexico.
4) Tecnologico de Monterrey, School of Medicine and Health Sciences, Monterrey, Mexico
5) Department of Ophthalmology, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo,
Japan
6) CJ Shah Cornea Services/Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Medical
Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India
7) State key laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University,
Guangzhou, China
8) College of Biomedical & Life Sciences, Optometry & Vision Sciences, Cardiff University, UK
9) Dr. Heiko Pult – Optometry and Vision Research, Weinheim, Germany
10) Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA
11) Weill Cornell Medicine, New York-Presbyterian Hospital, New York, NY
12) Department of Ophthalmology and Epidemiology, University of Groningen, Groningen, the Netherlands
13) Department of Ophthalmology, Vestfold Hospital Trust, Tønsberg, Norway
14) Department of Ophthalmology, New Zealand National Eye Centre, The University of Auckland,
Auckland, New Zealand
15) Eye Hospital of Wenzhou Medical University, National Eye Clinic Research Center, Peoples Republic of
China.
16) Ichikawa General Hospital Tokyo Dental College Dept of Ophthalmology Ichikawa, Chiba, Japan
17) School of Optometry and Vision Science, UNSW Sydney, NSW, Australia
18) Tear Film & Ocular Surface Society, Boston, USA
19) Centre for Ocular Research & Education (CORE), School of Optometry and Vision Science, University
of Waterloo, Waterloo, ON, Canada

TFOS Collaborator Group

Reiko Arita, Itoh Clinic, University of Tokyo, Japan ritoh@za2.so-net.ne.jp
Carlos Belmonte, Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Avda
Santiago Ramón y Cajal s/n, Alicante, Spain carlos.belmonte@umh.es
Robin L Chalmers, Clinical Trial Consultant, Atlanta, Georgia, USA
chalmers2097@gmail.com
Anat Galor, Miami Veterans Affairs Medical Center Ophthalmology, Bascom Palmer Eye
Institute, University of Miami Miller School of Medicine, Florida, USA
AGalor@med.miami.edu
Arkasubhra Ghosh, Molecular Signalling and Gene Therapy, GROW laboratory,
Narayana Nethralaya Foundation (nnf.org.in), Bangalore, India
Arkasubhra@narayananethralaya.com
Marc Labetoulle, Ophthalmology Department, Bicêtre Hôpital, APHP, Paris-Saclay
niversity, Kremlin-Bicêtre, France; Quinze-Vingts Hospital, Paris, France
marc.labetoulle@aphp.fr
Kelly K. Nichols, School of Optometry, The University of Alabama at Birmingham,
Alabama, USA nicholsk@uab.edu
Andrew D. Pucker, Eminent Ophthalmic Services, Milledgeville, Georgia, USA
apucker@uab.edu
Eduardo M. Rocha, Dept of Ophthalmology, Otorhinolaryngology and Head & Neck
Surgery Ribeirao Preto Medical School, University of Sao Paulo, Brazil
emrocha@fmrp.usp.br

1
 Benjamin Sullivan, Bausch & Lomb, Bridgewater, New Jersey, USA; Lµbris BioPharma,
Florida, USA bdsulliv@me.com
Piera Versura, Alma Mater Studiorum University of Bologna Cornea-Ocular surface Lab
and translation research in Ophthalmology IRCCS Azienda Ospedaliero-Universitaria di
Bologna, Bologna Italy piera.versura@aosp.bo.it
Mark D P Willcox, School of Optometry and Vision Science, UNSW, Sydney, Australia
mdpwillcox@gmail.com

Corresponding author: James S. Wolffsohn, Vision Sciences, Aston University, Aston

Triangle, Birmingham, B4 7ET, United Kingdom. J.s.w.wolffsohn@aston.ac.uk
ABSTRACT
A standard approach to the diagnosis of dry eye disease across eye care practitioners is
critical to reassuring the patient, providing consistency between practitioners and informing
governments as to the true prevalence and resulting healthcare needs. The Tear Film &
Ocular Surface Society (TFOS) Dry Eye Workshop (DEWS) III has reviewed the evidence-
base since their previous reports published in 2017 and revised the definition to “Dry eye is a
multifactorial, symptomatic disease characterized by a loss of homeostasis of the tear film
and/or ocular surface, in which tear film instability and hyperosmolarity, ocular surface
inflammation and damage, and neurosensory abnormalities are etiological factors.” Key
features from the definition include that dry eye disease is multifactorial, is a disease and not
a syndrome and is always symptomatic. Differential diagnosis and ocular examination
guidance is given along with the risk factors that should be discussed with the patient. The
recommended screening questionnaire is the OSDI-6 with a cut-off score ≥4. A positive
result together with a non-invasive breakup time <10s or alternatively tear film
hyperosmolarity (≥308mOsm/L in higher eye or an interocular difference >8mOsm/L) gives a
diagnosis of dry eye. In addition, the ocular surface should be stained and positive
symptomology together with >5 corneal fluorescein and/or >9 conjunctival lissamine green
punctate spots and/or lid margin lissamine green staining of ≥2mm length & ≥25 %width also
gives a diagnosis of dry eye. Subclassification was separated into tear film (lipid, aqueous
and mucin/glycocalyx) and ocular surface and adnexa (anatomical misalignment, blink/lid
closure, lid margin, neural dysfunction, ocular surface cell damage/disruption and primary
inflammation/oxidative stress) components, with appropriate clinical tests and cut-offs
provided to identify these etiological drivers in an individual, to inform appropriate
management and therapy.

Keyword: dry eye disease (DED); definition; diagnosis; differential diagnosis; subtypes;
subclassification; blepharitis, meibomian gland dysfunction (MGD)

2
Table of Contents
1 Report Aims ............................................................................................ 7

2 Definitions .............................................................................................. 8
2.1 Dry eye disease (DED) .......................................................................................................... 8

2.1.1 Multifactorial .................................................................................................................... 8

2.1.2 Disease, not a syndrome ................................................................................................ 8

2.1.3 Dry eye is a subset of ocular surface disease and can co-exist with other ocular
surface disease .............................................................................................................................. 8

2.1.3.1 Signs vs symptoms ......................................................................................................... 9

2.1.4 Ocular symptoms include vision ...................................................................................... 9

2.1.5 Pathophysiology elements ............................................................................................ 10

3 History and Symptoms ........................................................................ 10

3.1 Differential Diagnosis ......................................................................................................... 10

3.1.1 Eyelid-related disorders ................................................................................................ 12

3.1.2 Conjunctival and corneal abnormalities ........................................................................ 12

3.1.2.1 Conjunctival-related disorders ...................................................................................... 12

3.1.2.1.1 Allergic conjunctivitis ............................................................................................. 12
3.1.2.1.2 Viral conjunctivitis .................................................................................................. 13
3.1.2.1.3 Bacterial conjunctivitis ........................................................................................... 13
3.1.2.1.4 Cicatrizing conjunctivitis ........................................................................................ 14
3.1.2.1.5 Conjunctivochalasis................................................................................................ 14
3.1.2.1.6 Pinguecula and pterygium ..................................................................................... 14
3.1.2.1.7 Conjunctival concretions........................................................................................ 14
3.1.2.2 Corneal-related disorders.............................................................................................. 14
3.1.2.2.1 Neurotrophic keratitis............................................................................................ 14
3.1.2.2.2 Corneal dystrophies and degenerations ................................................................ 15
3.1.2.2.3 Bullous keratopathy ............................................................................................... 15
3.1.2.2.4 Infectious keratitis ................................................................................................. 15
3.1.2.2.5 Thygeson’s superficial punctate keratitis .............................................................. 15
3.1.3 Other mixed and miscellaneous ocular surface disorders ............................................ 15

3.1.3.1 Limbal stem cell deficiency ....................................................................................... 15

3.1.3.2 Episcleritis ................................................................................................................. 16
3.1.3.3 Mucus fishing syndrome ........................................................................................... 16

3
 3.1.3.4 Ocular neuropathic pain ............................................................................................ 16
3.1.4 Non-ocular surface disease disorders ................................................................................. 16

3.1.5 Summary ............................................................................................................................. 16

3.2 Risk factors .......................................................................................................................... 17

3.3 Symptomology .................................................................................................................... 19

3.3.1 Routine questions based on variability of symptoms .................................................... 19

3.3.2 Standardised questionnaires ......................................................................................... 19

3.3.3 Discordance between signs and symptoms .................................................................. 20

3.3.4 Paediatric considerations .............................................................................................. 23

3.4 Ocular Examination ............................................................................................................ 23

3.4.1 Diagnostic Homeostasis Test Battery ........................................................................... 23

3.4.1.1 What is a diagnosis ................................................................................................... 23

3.4.1.2 Need for standardisation ........................................................................................... 23
3.4.1.3 Other approaches since TFOS DEWS II .................................................................. 23
3.4.1.4 Use of sensitivity and specificity to select diagnostic tests ....................................... 24
3.4.1.5 Tests to establish a loss of homeostasis of the tear film .............................................. 24
3.4.1.6 Tests to establish a loss of ocular surface homeostasis ........................................... 25
3.4.1.7 Practical diagnostic criteria considerations ............................................................... 26
3.4.2 Advanced screening ...................................................................................................... 28

3.4.3 Severity rating ............................................................................................................... 29

3.5 Subclassification to identify DED etiological drivers ...................................................... 30

3.5.1 Purpose of a DED subclassification .............................................................................. 30

3.5.2 Tear Film Deficiencies ................................................................................................... 30

3.5.2.1 Lipid component ........................................................................................................ 31

3.5.2.1.1 Interferometry ................................................................................................... 31
3.5.2.1.2 Lipid turnover .................................................................................................... 31
3.5.2.1.3 Evaporimetry ..................................................................................................... 31
3.5.2.1.4 Thermography ................................................................................................... 32
3.5.2.1.5 Meibum expressibility and quality (meibometry) ............................................. 32
3.5.2.2 Aqueous .................................................................................................................... 32
3.5.2.2.1. Meniscometry or tear meniscus assessment....................................................... 32
3.5.2.2.2 Phenol red thread test....................................................................................... 33
3.5.2.2.3 Schirmer test ..................................................................................................... 33
3.5.2.2.4 Strip meniscometry ........................................................................................... 33

4
 3.5.2.2.5 Tear clearance ................................................................................................... 33
3.5.2.2.6 Lacrimal gland patency ...................................................................................... 34
3.5.2.2.7 Tear proteins and other components ............................................................... 34
3.5.2.3 Mucin / glycocalyx ..................................................................................................... 34
3.5.2.3.1 Mucins ............................................................................................................... 34
3.5.2.3.2 Rose bengal and lissamine green (see section 3.4.1.7) ..................................... 34
3.5.2.3.3 Conjunctival impression cytology ...................................................................... 35
3.5.2.3.4 Ferning test ........................................................................................................ 35
3.5.3 Eyelid anomalies ........................................................................................................... 35

3.5.3.1 Blink and lid closure anomalies ................................................................................. 35

3.5.3.2 Lid margin health ....................................................................................................... 36
3.5.3.2.1 Anterior blepharitis ........................................................................................... 36
3.5.3.2.1.1 Demodex associated blepharitis ..................................................................... 37
3.5.3.2.2 Meibomian gland dysfunction (MGD) ............................................................... 37
3.5.3.2.3 Ocular rosacea ................................................................................................... 39
3.5.4 Ocular Surface Abnormalities ....................................................................................... 40

3.5.4.1 Anatomical misalignment .......................................................................................... 40

3.5.4.2 Neural dysfunction..................................................................................................... 40
3.5.4.3 Ocular surface cellular damage / disruption .............................................................. 43
3.5.4.4 Primary inflammation / oxidative stress..................................................................... 48
3.5.4.4.1 Imaging-based diagnostic tests ......................................................................... 48
3.5.4.4.1.1 Ocular conjunctival redness ....................................................................... 48
3.5.4.4.1.2 In vivo confocal imaging ............................................................................. 49
3.5.4.4.2 Tear Biomarker diagnostic tests ........................................................................ 49
3.5.4.4.2.1 Matrix metalloproteinases ......................................................................... 49
3.5.4.4.2.2 Cytokines and chemokines......................................................................... 49
3.5.4.4.2.3 Neurotrophic Factors and Neuropeptides ................................................. 50
3.5.4.4.2.4 Ocular surface immune markers ................................................................ 50
3.5.4.4.2.5 Inflammasome markers ............................................................................. 50
3.5.4.4.2.6 MicroRNAs.................................................................................................. 50
3.5.4.4.2.7 Oxidative stress markers ............................................................................ 51
3.5.4.4.2.8 Serum markers ........................................................................................... 51
3.5.5 Systemic diseases leading to dry eye ........................................................................... 53

3.5.5.1 Autoimmune conditions ............................................................................................. 53

3.5.5.2 Hormonal imbalance ................................................................................................. 53

5
 3.5.5.3 Metabolic disease...................................................................................................... 53
3.5.5.4 Exposure ................................................................................................................... 54
3.6 Tests for monitoring treatment .......................................................................................... 54

4 Patients with only symptoms or ocular surface signs ..................... 54

4.1 Ocular surface disease in the absence of symptoms ..................................................... 55

4.1.1 Diagnosing Ocular Neurosensory Abnormalities .......................................................... 55

4.1.2 Diagnosing Neurotrophic Keratopathy .......................................................................... 55

4.2 Symptoms in the absence of ocular surface disease ..................................................... 57

4.2.1 Diagnosing corneal neuropathic pain ............................................................................ 57

5 Future advances................................................................................... 57
5.1 Artificial intelligence ........................................................................................................... 57

5.2 Tear biomarker testing of tears ......................................................................................... 58

5.3 Sustainability ....................................................................................................................... 58

5.4 Need for experience-informed approach to un/under-researched areas ...................... 58

6 Summary............................................................................................... 59
6.1 Workflow and enhanced link to individualised management ......................................... 59

6.2 Patient communication ....................................................................................................... 61

6.3 Key diagnostic methodological changes from TFOS DEWS II ....................................... 61

6 References ............................................................................................ 62

6
Abbreviations:
DED Dry eye disease
DEWS Dry eye workshop
IL Interleukin
LIPCOF Lid-parallel conjunctival folds
MGD Meibomian gland dysfunction
MMP Matrix metalloproteinase(s)
OCT Optical coherence tomography imaging
OR Odds ratio
OSDI Ocular Surface Disease Index
TFOS Tear Film & Ocular Surface Society

1 Report Aims
The Tear Film & Ocular Surface Society (TFOS) second dry eye workshop (DEWS II)
Diagnostic Methodology report 1 provided practical, clinical diagnostic recommendations for
dry eye disease (DED), based on the evidence available for tests with diagnostic potential to
align with the revised definition of DED.2 It also provided a clear rationale for the framework
which informed the number and characteristics of the selected tests. Questions to inform a
differential diagnosis were proposed. The need for subclassification post-diagnosis was
emphasised, to inform management approaches. The purpose of the TFOS DEWS III
Diagnostic Methodology report was to:
 Revisit the current definition 2 to ensure it aligns with current understanding of
DED
o Provide a rationale for the components of the definition
o Highlight considerations when a patient only has symptoms or signs
associated with DED
o Define associated conditions
 Draw on risk / associated factors for DED (from the TFOS DEWS III Digest
report) and masquerading diseases to guide appropriate history and symptom-
taking
 Identify any updates required to the 2017 diagnosis of DED, reiterating the
rationale for change and perceived challenges with regard to the available
evidence
 Propose a new etiological driver-based approach to subclassification, identifying
the tests that indicate the driver involved in an individual's dry eye, which can
then be linked to management approaches by the TFOS DEWS III Management
and Therapy report
 Discuss possible future directions that could help to inform further advances in
DED diagnosis and subclassification

7
2 Definitions
2.1 Dry eye disease (DED)
In 2017, after careful consideration of the terminology including diction, word order,
emphasis, and accepted meaning, DED was defined by a multidisciplinary and transnational
committee as “a multifactorial disease of the ocular surface characterized by a loss of
homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film
instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory
abnormalities play etiological roles.”. This was ratified by the full TFOS DEWS II authorship
consisting of 150 clinical and basic science research experts from around the world, who
utilized an evidence-based approach and a process of open communication, dialogue and
transparency to consolidate the understanding of DED 3.

It was the consensus of TFOS DEWS III that the definition did not require radical change
based on our updated understanding of the disease pathology and the tear film 4, but noted
the intrinsic role of the ocular surface tissues as well as the tear film in homeostasis leading
to repositioning of this aspect in the revised definition to read “Dry eye is a multifactorial,
symptomatic disease characterized by a loss of homeostasis of the tear film and/or ocular
surface, in which tear film instability and hyperosmolarity, ocular surface inflammation and
damage, and neurosensory abnormalities are etiological factors.” Key aspects of the
definition include:

2.1.1 Multifactorial
There are many aspects of our lifestyle that can impact the ocular surface including digital
environments 5, environmental conditions 6, nutrition 7, use of cosmetics 8, elective
medication and procedures 9, contact lenses 10, societal factors 11 and general lifestyle 12. All
of these, therefore, need to be considered in the management of the disease and
appropriate treatment or therapy is likely to include more than one approach or treatment. In
addition, genetic factors have been found to play a role in DED. 13-15

2.1.2 Disease, not a syndrome

When a combination of symptoms and physical findings are common to a group of patients,
but the direct cause is not yet understood, it is referred to as a syndrome (from the Greek
roots meaning ‘running together’) 16. However, once causative agents or processes have
been identified that have a moderately high degree of certainty, then it is appropriate to use
the term ‘disease’ rather than a ‘syndrome’.16 Dry eye was once considered a syndrome due
to insufficient understanding of its etiology. However, advances in medical knowledge have
revealed its clearly identifiable diagnostic features 1 and disease progression 4 and response
to specific treatments 17. These insights support the current recognition of dry eye as a
disease entity rather than a syndrome 2. Defining dry eye as a disease is an important issue
for patients (as this can affect reimbursement for clinical care and treatment as well as their
understanding of their symptoms that are impacting their quality of life), and for eye care
practitioners, since in some countries, certain professions are not permitted to treat
‘disease’. The TFOS DEWS III consensus is that all eye care practitioners play an important
role in managing patients with DED within the limits of their clinical competency, by providing
evidence-based advice on lifestyle factors 18 and over-the counter treatments, as a
minimum.

2.1.3 Dry eye is a subset of ocular surface disease and can co-exist with other ocular
surface disease
DED is defined as a symptomatic disease and thus, by definition, must always be
accompanied by ocular symptoms. Recognition of DED as a symptomatic disease is not
new; the National Eye Institute/Industry Workshop in 1995 defined dry eye as “a disorder of
the tear film … associated with symptoms of ocular discomfort” 19. The first TFOS DEWS
report identified dry eye as a “disease of the tears and ocular surface that results in
symptoms of discomfort …” 20 and TFOS DEWS II as a “disease of the ocular surface

8
characterised by a loss of homeostasis of the tear film and accompanied by ocular
symptoms ….” 2. Hence, ocular surface signs in the absence of symptoms reflects the
presence of ocular surface disease (Figure 1), but does not signify the existence of DED,
specifically.

2.1.3.1 Signs vs symptoms

It is acknowledged that there are pathological conditions that result in symptoms without
clinically significant signs (see Section 4.2), that present with signs but without symptoms
(see Section 4.1), or that exhibit both dryness symptoms and signs but are not DED as they
have a different pathophysiology (see Section 3.1). Such conditions can co-exist with dry
eye disease; for example a patient with severe symptoms, but only mild signs, may have
neuropathic pain in conjunction with mild DED. This is important to recognise from a
management perspective as it may indicate the need for a multimodal approach 17.

Figure 1: Venn diagram illustrating that dry eye disease requires the presence of both
signs and symptoms, and exclusion of differential diagnoses. Dry eye disease can co-exist
with other forms of ocular surface disease and symptomatic conditions.

2.1.4 Ocular symptoms include vision

Vision and vision-specific tasks can be significantly affected by DED due to destabilisation of
the tear film disrupting the smooth air-tear interface between blinks or damage to the ocular
surface affecting corneal transparency 21-25. An unstable tear film results in light scatter and
visual fluctuations between blinks 26-28. Sensitivity to light and “spots in vision” are more
commonly reported accompanying DED than glaucoma and/or cataract 29. Hence, while
vision is not specifically mentioned in the definition of DED (as there are many associated
symptoms), visual disturbance is acknowledged to be encompassed within the terminology
of “ocular symptoms”. As also previously noted by TFOS DEWS II, the term “symptoms” is

9
considered to cover a wide range of possible patient-reported sensations associated with
DED, which can include discomfort and / or visual disturbance 3.

2.1.5 Pathophysiology elements

The component of the definition describing the disease pathology (see TFOS DEWS III
Digest Pathophysiology section 4) needs to exclude other conditions with both signs and
symptoms, but with different pathophysiology and therefore appropriate management
therapies. Clinically, these can be excluded through a process of differential diagnosis (See
Section 3.1).

3 History and Symptoms

3.1 Differential Diagnosis

Both symptoms and signs of DED are heterogeneous. Therefore, the differential diagnosis of
DED is extensive and encompasses most ocular surface disease (Figure 2). Several of the
ocular surface changes caused by conditions identified as differential diagnoses of DED may
increase the risk of DED, and DED may sometimes exacerbate other ocular surface
diseases with similar symptoms (see section 3.2). Thus, many of the differential diagnoses
are often comorbid with DED. There are also non-ocular surface disease diagnoses that may
mimic dry eye symptoms, that are particularly important to consider when no ocular surface
disease signs are present. Table X gives an overview of differential diagnoses of DED and
their key differential features. The following sections focus on the most common differential
diagnoses of DED.

10
Figure 2: The key differential diagnoses of dry eye disease (DED) grouped by anatomy, and main differential features compared to DED.

11
3.1.1 Eyelid-related disorders
While patients with anterior blepharitis or MGD do not always have DED, as these conditions
can be asymptomatic, they are commonly a driver of DED (see section 3.5.3.2) Diseases
affecting the eyelids, such as chalazion, hordeolum, entropion, ectropion,
trichiasis/distichiasis, floppy eyelid syndrome, blepharospasm, ocular rosacea, Bell's palsy
and canaliculitis may result in symptoms similar to DED including watery eyes as a
neuroregulated response to dry or irritated ocular surface 30, and so a thorough eyelid
examination should be performed in every patient suspected of having DED 31.

3.1.2 Conjunctival and corneal abnormalities

To distinguish between DED and other etiologies, a thorough patient history is vital; this is
especially important in patients with a history of contact lens wear 32, use of multiple eye
drops, or exposure to toxic chemicals 33, 34. Observation of the ocular surface provides
further useful information. For example, a "curl" pattern of fluorescein staining (which may
indicate epithelial stress for example due to medication toxicity) 33 or corneal
conjunctivalization (which may be indicative of limbal stem cell deficiency) 32, 35. Fluorescein
staining in the superior cornea may point to superior limbic keratoconjunctivitis 36, floppy
eyelid syndrome 37 or can result from contact lens wear issues 38-40. Non-responsiveness to
standard DED therapies warrants consideration of concurrent systemic conditions such as
mucus membrane pemphigoid (ocular cicatricial pemphigoid) or Stevens-Johnson syndrome
41, 42
. Even though DED can co-exist in these conditions, early diagnosis is vital as advanced
therapies including systemic immunomodulatory therapy are often required.
Superficial punctate keratitis can commonly be observed in a variety of corneal disorders. If
it is primarily the superior cornea that is affected, possible causes include vernal
keratoconjunctivitis, superior limbic keratoconjunctivitis, trachoma, poorly fitting contact
lenses, floppy eyelid syndrome, trichiasis or distichiasis 38. If it is primarily inferior in location,
it might, more likely be attributable to ocular rosacea, atopic keratoconjunctivitis, allergic
keratitis, blepharitis, exposure keratopathy, lower eyelid margin lesions, topical medication
toxicity, entropion or ectropion, trichiasis or distichiasis 38. If the superficial punctate keratitis
is primarily interpalpebral, it may be contact lens-related (chemical toxicity, tight lens
syndrome, overwear syndrome), or due to exposure to ultraviolet light, neurotrophic
keratopathy or DED 38.

Filamentary keratitis is a persistent corneal condition, recognizable by the presence of fine

strands (from mucin bound degenerated epithelial cells) and mucus which adhere to the
corneal surface 43-45. It may arise secondary to DED, causing discomfort symptoms and
photophobia; blepharospasm and increased blinking are also common 46.

3.1.2.1 Conjunctival-related disorders

3.1.2.1.1 Allergic conjunctivitis
The signs and symptoms of dry eye overlap with those of allergic conjunctivitis, and the
conditions can coexist 47, 48. In a study in of 689 randomly sampled patients from an
ambulatory optometric practice in California, United States, 57% of those reporting itching
had clinically significant dryness and 45% of those with dry eye reported itching 49.
Immunoglobulin E (IgE) antibodies against seasonal or perennial allergens are commonly
evaluated by a blood test 50 and diagnostic tests are available to detect the presence of IgE
in the tear film. Additionally, typical ocular signs of allergy, such as eyelid edema, and
conjunctival papillae as well as conjunctival chemosis, help differentiate allergic conjunctivitis
from DED 51, 52. A strong family history of allergy, atopic dermatitis, and/or the presence of
asthma is common in those patients 53. Atopic and vernal keratoconjunctivitis, chronic,
bilateral, inflammatory and visually threatening forms of the disease also have signs and
symptoms similar to DED and can serve as a trigger for DED. Signs of inflammation can be
found in the cornea, conjunctiva, and eyelids. Typically, symptoms are photophobia, burning,
tearing, itching, mucous discharge, and hyperemia and papillary hypertrophy of the eyelids.

12
Some of the most common signs found in both atopic keratoconjunctivitis and DED
(although generally less severely in DED) include superficial punctate keratitis, conjunctival
injection or hyperemia, anterior blepharitis, meibomian gland dysfunction (MGD) and tear
instability 39, 54-57. Atopic keratoconjunctivitis should be considered if there are signs such as
conjunctivitis (possibly with scarring) and periorbital eczema 58, corneal neovascularization,
symblepharon, keratoconus, and sometimes anterior polar cataracts 59, 60. Again, a family
history of allergy, atopic dermatitis (occurring in 95%), asthma (occurring in 87%) and
periorbital eczema are common 39, 61, 62.

3.1.2.1.2 Viral conjunctivitis

While viruses cause approximately 80% of cases of acute infectious conjunctivitis in adults,
they may be responsible for only around 20% of pediatric cases 63, 64. Even though watery
discharge is typical of viral conjunctivitis this may also be seen in about 25% of bacterial
cases 63, 64. Most viral conjunctivitis involves the highly contagious adenovirus (65–90%),65
which has an incubation period of 4 to 10 days before being clinically observable 66 and may
have associated pharyngoconjunctival fever and epidemic keratoconjunctivitis. Other causes
of viral conjunctivitis include herpes viruses, picornaviruses, and several systemic viral
infections 39, 64. Unilateral herpetic keratitis can affect the tear film of both eyes 67, 68. Even
though viral conjunctivitis shares a number of findings with DED such as tearing, burning,
redness, irritation, photophobia and blurred vision, the following signs and symptoms may
help to differentiate a viral etiology 39, 69:
 Acute onset of signs and symptoms
 Redness and irritation initially in one eye, which often spreads to the other eye within
a few days.
 Recent upper respiratory tract infection or close contact with someone with a red eye.
 Crusting around the eyes in the morning.
 Examination findings are watery, mucoid discharge and red, edematous eyelids.
 Preauricular lymphadenopathy (swelling of the lymph nodes in front of the ears which
drain lymph fluid from the area around the eyes, cheeks and surrounding scalp).

Epstein-Barr virus infects more than 90% of the adult population 70. Even though the
infection of ocular structures with the Epstein-Barr virus results most commonly in transient
follicular conjunctivitis 71, it can also present with signs and symptoms similar to DED, as well
as with keratitis, uveitis, choroiditis, retinitis, ocular glandular syndrome, papillitis and
ophthalmoplegia 72. Several systemic viruses, including measles, rubella (German measles),
mumps, and influenza, are also frequently associated with conjunctivitis 69.

3.1.2.1.3 Bacterial conjunctivitis

Bacteria as a causative agent of conjunctivitis occurs more often in children than in adults
(70% vs 20% of cases) 63 73. Purulent conjunctival discharge and morning eyelash crusting
may suggest a bacterial involvement, but this does not rule out a viral cause 63. Patients
with bacterial conjunctivitis may complain of similar symptoms as DED, such as burning,
stinging, irritation, foreign body sensation and photophobia. In contrast to dry eye, there is
typically significant conjunctival hyperemia (often more than with viral conjunctivitis or DED)
and discharge (typically moist and mucopurulent). Affected patients often complain about
matting or clumping of the eyelashes, mostly in the morning. Bacterial conjunctivitis can
occur in one or both eyes and systemic findings may be present, especially in children, such
as purulent rhinorrhoea and respiratory infection, fever and malaise 74. Chlamydial
conjunctivitis should be considered in sexually active persons who present with a chronic
follicular conjunctivitis, that is more prominent in the lower palpebral conjunctiva, and with
mucopurulent discharge 75.

13
3.1.2.1.4 Cicatrizing conjunctivitis
Cicatrizing conjunctivitis is characterized by inflammation and scarring of the conjunctiva.
Cicatrization can range from mild and subtle, with only subconjunctival fibrosis, often seen
as fine white lines at the palpebral conjunctiva, to extensive, with fornix shortening, multiple
symblepharon and ankyloblepharon. It can induce ocular dryness and can lead to gross
distortion of the anatomy of the eyelid and ocular surface, including entropion, trichiasis,
limbal stem cell deficiency and keratinization of the ocular surface. Causes of cicatrizing
conjunctivitis are many and include autoimmune disease such as ocular pemphigoid,
epidermolysis bullosa, sarcoidosis, systemic sclerosis, Sjögren disease and lichen planus,
bacterial and viral conjunctivitis such as trachoma and adenoviral conjunctivitis, thermal and
chemical burns, graft versus host disease, Stevens-Johnson syndrome, ocular rosacea and
atopic keratoconjunctivitis. Conjunctival biopsy is important in confirming a diagnosis and to
identify the cause. 76-78

3.1.2.1.5 Conjunctivochalasis
Conjunctivochalasis is characterized by loose, redundant, non-oedematous folds in the
conjunctiva, the more mild expression of which is lid-parallel conjunctival folds (LIPCOF). It
is most often seen in the inferior bulbar conjunctiva, overlying the inferior lid margin. It is
often asymptomatic, but can lead to tear film instability and symptoms consistent with DED,
including irritation, dryness, foreign body sensation, mucus discharge, and tearing. The
etiology of conjunctivochalasis is still unclear, but it is more common with increasing age, in
patients with DED, and contact lens wearers. Symptoms may be increased during downward
gaze or with vigorous blinking 79, 80.

3.1.2.1.6 Pinguecula and pterygium

A pinguecula is a benign degeneration of the conjunctiva, that presents as a gray-white or
yellowish lesion on the bulbar interpalpebral conjunctiva, adjacent to the limbus, which may
cause localized disruption of the tear film due to a change in lid-globe juxtaposition. Wind,
dust, and UV exposure are factors associated with pinguecula and pterygium development.
Pinguecula are usually asymptomatic, but may present with mild foreign body sensation or
itch 81. A pterygium is a growth of epithelial and fibrovascular tissue from the conjunctiva
migrating over the corneal limbus in a wing-shaped way. Pterygia have similar associated
factors as pingueculae, and a pterygium is often preceded by, or is comorbid with, a
pinguecula. Irregular astigmatism with a pterygium can lead to visual symptoms, and
irritation may be present. Inflammation of pterygia and pingueculae is also possible, leading
to conjunctival hyperemia and edema, and increased chance of irritation 82, 83.

3.1.2.1.7 Conjunctival concretions

Conjunctival concretions are benign discrete yellow-white deposits, most commonly found in
the palpebral conjunctiva or fornices. Most are idiopathic and the result of degenerative
changes with ageing, but they may be secondary to conjunctival inflammation, such as that
associated with allergic keratoconjunctivitis, trachoma and DED. They are normally
asymptomatic, but when they erode through the conjunctiva they may irritate the cornea and
bulbar conjunctiva 84.

3.1.2.2 Corneal-related disorders

3.1.2.2.1 Neurotrophic keratitis

Neurotrophic keratitis is characterized by a decrease in corneal sensitivity and stems from
dysfunction in the ophthalmic division of the trigeminal nerve, which can be triggered by

14
conditions such as diabetes mellitus, ocular herpes simplex/zoster, non-ocular surface
neoplasia or ophthalmic surgery. This dysfunction ultimately leads to a decrease in aqueous
tear production 39.

3.1.2.2.2 Corneal dystrophies and degenerations

Corneal dystrophies and degenerations present with similar symptoms as DED. They can
also lead to recurrent corneal erosions. The more common cases seen in clinical practice
are Salzmann’s nodular degeneration, epithelial basement membrane dystrophy, and Fuchs
endothelial corneal dystrophy. Salzmann’s nodular degeneration is a non-inflammatory
degeneration of the anterior cornea with blue-white-grayish sub-epithelial nodules of the
cornea, usually mid-peripheral. It can lead to symptoms of decreased visual acuity and
irritation, pain, foreign body sensation, and blepharospasm. 85, 86 epithelial basement
membrane dystrophy, previously known as map-dot-fingerprint dystrophy affects around
42% of the general population and is characterized by abnormal epithelial turnover,
maturation and production of basement membrane. Typical signs are gray patches,
microcysts and fine lines in the corneal epithelium, and typical symptoms are blurred vision
and pain. 87, 88 Fuchs’ endothelial cornea dystrophy is a hereditary, progressive disease of
the posterior cornea, with progressive decline of corneal endothelial cells, and the formation
of extracellular matrix excrescences in Descemet’s membrane. This can eventually lead to
corneal edema, bullous keratopathy (see Section 3.1.2.2.3), loss of vision, photophobia,
epiphora, and pain. 89

3.1.2.2.3 Bullous keratopathy

Bullous keratopathy forms small vesicles or bullae in the cornea due to endothelial
dysfunction. These blister-like formations can rupture painfully and disrupt vision. Treatment
options may include 5% sodium chloride or other hyperosmotic eye drops to reduce swelling,
amniotic membranes, bandage contact lenses for comfort, antiglaucoma medications (when
associated with glaucoma) to decrease fluid flow into the cornea and corneal transplants to
replace damaged tissue 90.

3.1.2.2.4 Infectious keratitis

Infection of the cornea can be microbial (bacteria, fungal or parasitics), or viral (herpes
simplex or zoster), and is a sight-threatening condition, that always needs to be ruled out in
patients with ocular surface symptoms. Contact lens wear, topical steroid use, ocular
trauma, previous keratitis and previous ocular surgery are important risk factors. Blurred
vision, bulbar hyperaemia, pain, photophobia, tearing and discharge, and an acute,
unilateral clinical picture should particularly raise suspicion. 91.
3.1.2.2.5 Thygeson’s superficial punctate keratitis
Thygeson’s superficial punctate keratitis is a chronic and recurrent corneal epitheliopathy
with episodes of foreign body sensation, tearing, photophobia and visual symptoms. Clinical
signs are multiple, slightly elevated, round or oval, white-gray intraepithelial corneal lesions,
usually with little or no conjunctival involvement. Etiology and optimal treatment is unknown
as yet, but mild topical corticosteroids and immunomodulatory agents are often effective in
causing regression of the keratitis. 92

3.1.3 Other mixed and miscellaneous ocular surface disorders

3.1.3.1 Limbal stem cell deficiency
Limbal stem cell deficiency is characterized by loss or deficiency of the limbal epithelial stem
cells, that are essential for the maintenance of the corneal surface and its physiological
functioning. Diagnosis is primarily clinical, with varying signs depending on the stage, such
as a stippled staining pattern of the epithelium, late fluorescein staining, loss of corneal

15
transparency, conjunctivalization of the cornea and superficial corneal vascularization.
Symptoms include ocular redness, discomfort, pain, tearing, photophobia and decreased
vision, the latter occurring particularly when the visual axis is involved. 93

3.1.3.2 Episcleritis
Episcleritis is a usually benign and self-limiting disease of the episcleral tissue that presents
as superficial bulbar redness, most commonly in the interpalpebral area. In the majority of
patients, the inflammation is limited to a single sector, but may involve the entire episclera,
and it may present with a semi-mobile nodule. Symptoms are often not present, but may
include irritation and tenderness. 94

3.1.3.3 Mucus fishing syndrome

Mucus fishing syndrome is caused by repetitive eye-rubbing and self-extraction of mucus
discharge (for example that arises as a result of DED or allergic conjunctivitis) out of the eye,
leading to chronic inflammation of the ocular surface, and even more mucus production,
sparking a vicious cycle. An important clinical sign is epithelial damage (as seen by
lissamine green staining) of the nasal and temporal conjunctiva.95, 96

3.1.3.4 Ocular neuropathic pain

Ocular neuropathic pain, or corneal neuropathic pain, is characterized by increased
perception of pain in response to stimuli that are normally not painful. It is usually a
diagnosis of exclusion, when symptoms outweigh clinical signs. Questionnaires that
potentially could be used for the diagnosis of ocular neuropathic pain have been identified,
although no gold standard questionnaire has been established. 97 Neuropathic pain can
result from injury or disease of peripheral corneal nerves, but may also have a central
nervous system origin. The use of an esthesiometer may help in determining hypersensitivity
of the cornea 98. A proparacaine challenge test may help distinguish between neuropathic
pain of peripheral or central origin 99. Reported symptoms may be very similar to those of
DED, albeit often very severe, and treatment is often difficult and encompasses both regular
ocular surface treatments including anti-inflammatory options, and systemic analgesics,
tricyclic antidepressants, anticonvulsants (for example gabapentin and pregabalin), low dose
naltrexone, and electrical neurostimulation to reduce pain. 100-102.

3.1.4 Non-ocular surface disease disorders

Finally, the early stage of several non-ocular surface disease disorders of the eye, orbit and
surrounding tissues may mimic symptoms of DED. These should be considered particularly
when no ocular surface signs are visible. Ocular disorders include refractive error (such as
latent hypermetropia), digital eye strain, intermittent angle closure, anterior scleritis and
uveitis. Orbital disorders include Tolosa-Hunt-syndrome, thyroid eye disease (which may
ultimately lead to severe ocular surface disease secondary to chronic exposure), and carotid
cavernous fistula and disorders of the nasolacrimal duct that include dacryocystitis and
nasolacrimal duct obstruction. In addition, sinusitis and headache disorders may also
present with referred pain around the eye. 103.

3.1.5 Summary
Many conditions can mimic symptoms and/or signs of DED. Failing to investigate possible
comorbidities may lead to delayed diagnoses of source conditions and may impact the
optimal treatment. To aid in the differential diagnoses, specific questions are listed below.
For patients in whom the differential diagnostic history and symptoms suggest it may not be
DED, a detailed anterior eye examination using a slit-lamp biomicroscope is warranted 39,
including assessment of:

16
 • Eyelashes for blepharitis, trichiasis, distichiasis, milphosis, and/or poliosis
• Eyelids, including palpebral conjunctiva for irregularities or the presence of
follicles, papillae or swelling, and eyelashes for crusting or cylindrical dandruff,
and meibomian gland orifices for blockage, pouting or presence of Demodex tails
• Ocular surface for conjunctivochalasis, pinguecula, pterygium and any post
treatment/surgery signs of corneal disruption (see section 3.5.4.1)
• Bulbar conjunctiva for redness and/or swelling
• Cornea, including staining, for signs of ulceration, marginal keratitis, erosion,
lesions and possible trauma
• Anterior chamber for the presence of cells or keratic precipitates indicating
intraocular inflammation.
Medications which can cause DED and the use of cosmetics should be considered (as
reviewed by the TFOS Lifestyle reports 8, 9 as well as risk / associated factors (as reviewed in
the TFOS DEWS III Digest 4) including general health conditions.

Key clinical differential diagnosis questions, alongside asking about a patient’s general
health and medication, are:
 Do you feel eye pain rather than discomfort?
o DED commonly presents with symptoms of discomfort, light sensitivity or
blurred vision, rather than pain, especially in mild to moderate dry eye. If pain
is present, investigate for signs of trauma / erosion / infection / ulceration /
acute glaucoma and consider administering a pain questionnaire (see section
3.1.2.2).
 Do you have any facial flushing/redness, mouth dryness or enlarged salivary glands?
o Trigger for rosacea, sarcoidosis or S gren disease investigation.
 When did your symptoms start and can you recall any triggering event?
o DED is a chronic condition. If there is increased dryness when waking up or
dryness symptoms at night, consider incomplete lid closure, for example.
o If the onset was sudden or linked with an event, examine the eyes for trauma
/ infection / ulceration.
o If the symptoms are linked with contact lens use, refer to their contact lens
practitioner to consider alternative approaches that might improve symptoms
(such as changing lens material, fit or modality) 104
 Is your vision affected and if so, does it improve on blinking?
o A reduction in vision which does not improve with blinking, especially with
sudden onset, requires an urgent complete ophthalmic examination to rule
out conditions such as vascular occlusions.
 Are the symptoms or any redness much worse in one eye than the other?
o DED is commonly bilateral. If signs and/or symptoms are significantly greater
in one eye, investigate for signs of trauma / infection / ulceration.
 Do the eyes itch, are they swollen or crusty, or is there any discharge?
o Ocular itching is more likely associated with allergies/history of atopy, while a
mucopurulent discharge is associated with ocular infection. Reported itching,
specifically along the lash line, warrants assessment for Demodex related
anterior blepharitis.105

3.2 Risk factors

Identifying risk / associated factors may help in finding causes and pathophysiological
mechanisms of DED, in determining subtypes, in setting a differential diagnosis, in
identifying comorbidities, and in explaining possible discordance between symptoms and
signs (see section 3.3.3). In addition, it may help in patient education, and many associated
factors may be modifiable, offering the potential to improve symptoms and signs and help
prevent/slow progression of disease. In clinical practice, administering a questionnaire to the
patient (such as via paper or a digital application) for completion before the consultation is

17
recommended to save time and to ensure that all commonly associated factors have been
addressed.

DED is a multifactorial and heterogeneous disease, and over 200 associated factors have
been proposed in the literature. Not all these factors necessarily play a causative role, and
some associated factors are linked more strongly with symptoms, while others are linked
with signs. Figure 3 shows consistent and probable associated factors of DED as described
in the TFOS DEWS III Digest Epidemiology section.4 The following section briefly highlights
the most important risk / associated factors by category. Irrespective of a possible causal
mechanism, all associated factors described may be useful in elucidating the etiology of
DED and seeking to identify the possible disease driver(s) in an individual. Table 1 presents
frequently described risk factors that have inconclusive evidence (either directly conflicting
information in peer-reviewed publications, or inconclusive information but with some basis
for a biological rationale).
Figure 3: Consistent and probable associated factors of DED described in the TFOS
DEWS III Digest Epidemiology section 4.

18
Table 1: Frequently described risk factors that have inconclusive evidence (either directly
conflicting information in peer-reviewed publications, or inconclusive information but with
some biological rationale):

 Smoking 106-108
 Caffeine 7, 109-112
 Alcohol 7, 113, 114
 Water intake 7, 115, 116
 Food restriction 7
 Mediterranean diet (possible positive effect) 117-121
 Menopause 122, 123
 Air pollution from particulate matter of < 10µm 6
 Oral contraceptives 124-126

3.3 Symptomology
3.3.1 Routine questions based on variability of symptoms
Patients with DED often report sensations of grittiness and burning alongside dryness, while
contact lens wearers frequently complain of dryness, along with sensations of scratchiness
and watery eyes, driving likelihood of failure for long term successful wear 127-129. Individuals
diagnosed with DED present with variable symptom severity, ranging from mild to severe,
throughout the day.39 Dryness symptoms are typically worse upon waking than later in the
morning and tend to increase towards the end of the day in contact lens wearers 130-133.
Ocular allergies, exposure to air conditioning, and climatic factors that change between
seasons may be associated with seasonal variations in symptoms and signs. However, a
cross-sectional, retrospective cohort study in two Japanese clinics found that none of the
symptoms examined (dryness, irritation, pain, fatigue, blurring and photophobia) showed
significant seasonal variation 134. This was confirmed by another study, which failed to show
seasonal or weather-related variation in the severity of presenting signs or symptoms of
DED over a period of 3 years in 652 people in Oslo, Norway 135. Hence seasonality of DED
symptoms, doesn’t seem to warrant specific questioning beyond the differential diagnosis if
the patient reports the eyes generally itch. However, itching along the lash line may indicate
a Demodex blepharitis issue 105

3.3.2 Standardised questionnaires

Around 25 questionnaires can be identified from a literature search using terms "dry eye"
and "questionnaire" as well as by research articles cited in the identified publications. Each
questionnaire warrants evaluation to assess its clinical relevance, known population validity,
concurrent validity, internal consistency and reproducibility 136, 137. Additionally, uni-
dimensional evaluation validity should be confirmed using Rasch analysis and it should be
confirmed whether “health-related quality of life” has been assessed.

Questionnaires such as the Ocular Surface Disease Index (OSDI), Impact of Dry Eye in
Everyday Life (IDEEL), Dry Eye-Related Quality-Of-Life Score (DEQS), University of North
Carolina Dry Eye Management Scale (UNC DEMS) and the 25-Item National Eye Institute
Visual Function Questionnaire (NEI VFQ-25) are validated to a greater or lesser extent in
regard to their utility in assessing the impact of DED on health-related quality of life 137.
These questionnaires are not interchangeable, underscoring the necessity of recommending
a single, comprehensive, standardized tool in establishing robust diagnostic criteria 137. A
diagnostic questionnaire should have a low response burden on the patient, be simple to
score for the clinician and contribute to the assessment of severity and monitoring treatment
efficacy. TFOS DEWS II recommended the use of either the OSDI or the 5-item Dry Eye
Questionnaire 138. However, subsequent comparisons of the results from these
questionnaires have shown poor comparability. 139-141

19
The OSDI-6 was introduced in 2018 to reduce the response burden on patients.
Researchers conducted a study that included Rasch analysis, to determine if the
effectiveness of the 12 item OSDI questionnaire, could be maintained with a shortened
version (Figure 4). The questionnaire was further adapted such that questions would reflect
a recall period of one month versus only one week in the original OSDI. The resulting
abbreviated version, OSDI-6, consisting of six questions, was found consistently to be more
repeatable than either the full OSDI and DEQ-5 142. Moreover, the scoring method that
involves simply summing the item scores and using a diagnostic cutoff of ≥4 to indicate
symptoms of DED, allows for a quicker evaluation by clinicians 142. It is possible that this
abbreviated format might also be more suitable for children, who are known to better tolerate
questionnaires that require less time and assistance to complete. 143 Similarly to the original
OSDI, the OSDI-6 results can be indexed against severity, as normal (0-3 points), mild-to-
moderate DED (4-8 points), severe DED (>8)144. A Chinese translation of the OSDI-6 found
it to be repeatable, valid and psychometrically responsive to DED in a Chinese population
145
. The OSDI-6 thus yields comparable outcomes to the full OSDI, requires less time to
complete, and maintains similar variability and improved sensitivity to treatment effects 146.
Consequently, the OSDI-6 merits consideration as a suitable screening questionnaire for
DED diagnostic purposes while other questionnaires may offer supplementary aid for risk
factor identification and treatment selection, after confirming the initial diagnosis.

Constantly Mostly Often Sometimes Never

Have you experienced any of the following during a typical day within the last month?
1. Eyes that are sensitive to light? 4 3 2 1 0
2. *Vision blurring between blinks, 4 3 2 1 0
with your refractive correction*?
Symptoms and visual disturbance subscale 
Have problems with your eyes limited you in performing any of the following during a typical
day within the last month?
3. Driving or being driven at night? 4 3 2 1 0
4. Watching TV, or a similar task? 4 3 2 1 0
Visual function / tasks subscale 
Have your eyes felt uncomfortable in any of the following situations during a typical day within
the last month?
5. Windy conditions? 4 3 2 1 0
6. Places or areas with low humidity? 4 3 2 1 0
Environmental subscale 

Figure 4: Questions of the OSDI-6 146, 147. The diagnostic cut-off is a summed score of ≥4.

3.3.3 Discordance between signs and symptoms

It is well established that there is a poor correlation between symptoms and signs in DED. A
systematic review including 33 population-based and cohort studies, with a total of 175
individual sign-symptoms associations, found the vast majority of correlations to range
between +0.4 and –0.4 148. This means that dry eye signs generally explain only up to 15%
of the variation in symptoms within a population.
There are numerous methodological, statistical, but also physiological reasons why the
correlation between symptoms and signs in studies is low.
 Firstly, measurement error is a common problem for many of the DED tests, and the
order in which tests are performed or the instillation of fluorescein, for example, can
impact test findings 39. Outcomes are often subjectively scored, not only symptoms,
but also purportedly objective signs such as fluorescein break-up time, staining of the
ocular surface, and MGD.

20
  Secondly, DED is recognised to be a dynamic disease, in which symptoms can
fluctuate over time, and where test outcomes may be affected by environmental
factors, time of the day, and recent artificial tear use 39. Furthermore, symptoms are
often scored as an average over a defined period (such as ‘during the last week’ in
the OSDI), leading to differences in time period over which signs are measured and
symptoms are scored.
 Thirdly, DED is multifactorial, with potentially more than one etiology being identified
in clinical practice 47, 149. Studies that include different subgroups of DED in one
analysis risk a dramatic fall in correlation values, such as when patients with
neuropathic pain are included in studies focusing on more objective DED subtypes
150
. Indeed, when certain subgroups of patients with DED have been analysed
independently, higher correlations between symptoms and signs have been found 148.
 Fourth, neurosensory abnormalities and inflammation of the ocular surface, which are
considered two core mechanisms of dry eye, are not (directly) measured in common
clinical dry eye tests. With time, changes in nerve status may effect changes in
reported symptomology in DED. For example, a small study of patients with Sjögren
disease found that those with advanced corneal staining counterintuitively reported
fewer symptoms than patients with more mild corneal staining 151. Increasing age can
also lead to a reduction in corneal sensitivity 152, 153. A study in the United States of
America found correlations between a number of tear film measures and symptoms
scores on the 5-item Dry Eye Questionnaire and OSDI to have coefficients all lower
than r=0.18, while symptoms were much more strongly correlated with non-ocular
pain, depression and post-traumatic stress disorder scores 154. A large single-centre
study in the Netherlands also found poor correlation between common dry eye signs
and OSDI symptom scores (all r<0.30), and found correlations to be significantly
lower in women than in men, indicating sex differences in symptom reporting in DED
150
. A wide variety of factors, including depression, stress, age, sex and gender can
affect symptomology scores.
 Fifth, other comorbid ocular surface disease (that are often associated with DED) may
also lead to symptoms (see section 3.1), risking obscuration of true correlations
between dry eye signs and symptoms in analyses.
 Sixth, signs of DED may not necessarily lead to symptoms. For example, MGD is a
common finding in asymptomatic persons 155. Seventh, treatment may alter the
correlation between symptoms and signs, with certain treatments influencing one
variable more than another 156. Frequently, clinical trials show a positive effect of a
DED treatment on symptoms only, but not signs, or vice versa 157-162. In addition,
some longer duration trials have found signs to improve at a later stage than
symptoms (for example with artificial tears), or vice versa (with cyclosporine)163, 164.
 Finally, despite the longitudinal nature of these clinical trials, there remains a lack of
natural history studies that attempt to correlate dry eye signs and symptoms within the
individual patient rather than between patients; there is therefore a need for within
patient correlation studies across different time points, where it might be expected
that relationships between signs and symptoms may be stronger.

In recent years, several studies have explored predictors of discordance between symptoms
and signs in DED. In a study in the Netherlands, 648 patients were ranked based on a
composite dry eye signs’ severity score, and also ranked according to symptoms using the
OSDI score. Next, a risk factor association analysis was performed with the difference
between these two rank scores being the dependent variable. Factors associated with a
finding of symptoms exceeding signs were chronic pain syndromes, a history of atopic
diseases, allergies, use of antihistamines, depression, the use of antidepressants, and
osteoarthritis. Predictors of fewer symptoms than signs were increased age, Sjögren disease
and graft-versus-host disease 165. A similar study in the United States of America with 326
patients replicated many of these factors, finding associations between increasing age and

21
fewer reported symptoms, while mental health and chronic pain disorders associated with
more symptoms. In addition, the study found quantitative sensory testing scores that indicate
hyperalgesia to be associated with more symptoms than signs 166. A recent Taiwanese study
with 1229 patients using a similar approach found female sex, and a history of cataract,
pterygium and conjunctivochalasis surgery to be associated more with symptoms than signs,
and people of older age and those using artificial tears to be relatively less symptomatic than
their signs might suggest 167.

Other studies looking at discordance between signs and symptoms in DED have found
corneal microneuroma-like structures and increased corneal dendritic cell density 168, but
also decreased corneal nerve density 169, and tear and conjunctival cytokines 168, 170 in
patients where symptoms outweighed signs. Table 2 lists all the factors across studies that
were associated with symptoms outweighing signs, and conversely, those with significantly
fewer symptoms than signs. These factors may help in understanding outcomes in clinical
practice, aid in patient education (such as in explaining about central sensitisation
mechanisms or altered nerve status after surgery) and offer clues for differential diagnoses
and comorbid disorders (for example atopy and allergic conjunctivitis) in patients with DED
exhibiting discordance between signs and symptoms. The findings also emphasise the need
for clinicians not to rely solely on symptom-reporting in older patients, or those with Sjögren
disease or graft-versus-host disease, as symptoms may be understated in relation to the
severity of effects on the ocular surface.

Predictors of more symptoms than signs Predictors of fewer symptoms than signs

Demographics

Female sex 150, 167 Older age 165, 167, 171

Black race 166
Pathophysiological Factors
Tear and conjunctival cytokines Decreased corneal nerve density 169
(IL-10, IL-2, IL-6, IL-17a, tumor
necrosis factor alpha) 168, 170
Increased corneal dendritic cell density 168
Hyperalgesia as demonstrated with
quantitative sensory testing 166
Non-ocular pain intensity 166
Comorbidity Factors
Post traumatic stress disorder 166 Sjögren disease 165, 172
165
Allergy Graft-versus-host disease 165
Atopic disorders 165 Benign prostatic hyperplasia 166
165
Osteoarthritis Hypertension 166
165, 166
Depression
Anxiety 166
Chronic pain syndromes 165, 166, 173-175
Pharmaceuticals
Use of analgesics 166 Use of artificial tears 167
166
Use of anxiolytics
Use of antidepressants 165, 166
Use of antihistamines 165
Lifestyle Choices
Current smoking 166

22
 Surgery
167
Cataract surgery
Pterygium surgery 167
Conjunctivochalasis surgery 167

Table 2: Factors associated with a discordance between symptoms and signs in dry eye
disease. A higher number of studies showing an association is reflected by placement of the
factor higher on the list.

3.3.4 Paediatric considerations

While DED is common in children 176 (see TFOS DEWS III Digest epidemiology section),4
diagnostic tests have largely been validated only in adults. One study examined the
quantification of dry eye symptoms in children (6 to 15 years of age) using standardized
questionnaires, identifying the completion time was ≤2 min for each individual questionnaire
and while younger participants took longer to complete and required more assistance,
especially with longer visual analog scales, repeatability was noted to remain high across the
age range 177.

3.4 Ocular Examination

3.4.1 Diagnostic Homeostasis Test Battery

3.4.1.1 What is a diagnosis

As previously described, the ability to receive a diagnosis is critical for patients to
acknowledge their symptoms and / or signs as real and that they have the attention of health
care practitioners, as well as being required for healthcare insurance coverage, where
available. For practitioners, diagnostic criteria inform which evidence-based clinical practice
guidelines to follow and provide confidence in making patient diagnoses that are consistent
with their peers. A standardised diagnosis is essential for industry and researchers to be
able to target and validate the efficacy and safety of new products and to obtain regulatory
approvals. Appropriate health resource allocation requires robust epidemiological and
economic data that are based on consistent diagnoses. Hence diagnosis of a disease must
be characterized by standardised, universally-adopted criteria, based on widely available
and inexpensive tests with validated cut-off values. There are advantages in having a simple
screening element that can rapidly identify those individuals who would benefit from further
testing by a health care practitioner.

3.4.1.2 Need for standardisation

The definition of DED (see Section 2.1) dictates that symptoms must be present, and a loss
of homeostasis of the tear film and / or ocular surface must be established. Identifying that
the expected symptoms are present in a standardized way requires a validated
questionnaire as discussed in Section 3.3.2. While TFOS DEWS II recommended two
possible questionnaires that could be used, these questionnaires have since been shown to
not be equivalent, risking variability in diagnosis depending on the instrument chosen, so it is
appropriate to select a single questionnaire to be used in this setting. Based on evidence
published since TFOS DEWS II, this has been identified as the OSDI-6 with a summed cut-
off score of ≥4 to be positive for dry eye symptoms (see section 3.3.2).

3.4.1.3 Other approaches since TFOS DEWS II

The Asian Dry Eye Society 178, 179 supported by the Japanese Dry Eye Society 180 suggested
that DED should be diagnosed by confirming the presence of ocular symptoms (using any
one of four possible questionnaires) along with identification of an unstable tear film
(assessed with fluorescein tear breakup time of <5s). This approach shows similarities to

23
TFOS DEWS II, but lacks the standardisation that can be offered by a single questionnaire
as proposed by TFOS DEWS III. Furthermore, changing the chemical composition and
volume of the tear film by instilling fluorescein prior to assessment of its stability is not ideal.
The Korean Dry Eye Society defined dry eye as “a disease of the ocular surface
characterized by tear film abnormalities and ocular symptoms” without referring to the
pathophysiology, or specifying the means by which to diagnose ocular symptoms (no
questionnaire proposed). The diagnostic criteria included an unstable tear film in the form of
fluorescein breakup time <7s (a test which is invasive in itself, and recommended without
scientific justification for the cut-off), with a Schirmer test (<10 mm) and ocular surface
staining considered “ad unctive criteria” 181.

It has been suggested that diagnostic certainty (in the form of sensitivity and specificity) can
be increased by requiring multiple discriminatory tests to be positive 182; leading to the
recommendation that both corneal AND conjunctival staining needed to be present to match
a Bayesian-informed global prevalence of DED 183. However, the reported global prevalence
used had been generated from existing studies that relied on these various diagnostic
criteria which introduces bias into the approach. Further, using the sensitivity and specificity
of tests from multiple studies is also susceptible to bias 184 as previously identified in the
TFOS DEWS II report 1. There is increasing chance of misdiagnosing or under-diagnosing a
disease if standardised diagnostic criteria are not used because it can require excessive
time, consumables costs or equipment which is expensive or has limited availability. Corneal
staining often occurs in later and in more severe stages of DED, so while requiring its
presence will increase diagnostic specificity, it will exclude appropriate treatment for many
individuals whom clinicians recognise to have a marked loss of quality of life as a result of
their symptomatic ocular surface disease and would currently be identified as having DED
185
.
Attempts have been made to use dry eye tests to differentiate diseases that affect the ocular
surface such as S gren disease, graft-versus-host-disease, Graves’ orbitopathy, facial
nerve palsy, non-proliferative diabetes mellitus and glaucoma treated with preserved topical
drops, with mixed results 186-188.

3.4.1.4 Use of sensitivity and specificity to select diagnostic tests

Without there being a recognised gold standard, and due to the well-established low
correlation between signs and symptoms in DED 189-191, the level of certainty in derived
sensitivity and specificity values of new DED ‘diagnostic’ tests is low. Sensitivity and
specificity depend on the inclusion and exclusion criteria for the ‘healthy’ and ‘disease’
groups, increasing with the severity of signs or symptoms required to be allocated to the
DED group (spectrum bias). Individuals who do not meet the criteria of either group prevents
generalisability of the results across the broad population (sampling bias). Selection bias
occurs when the patient groups are selected with test(s) with a similar focus to the test being
evaluated 192. Parallel testing (requiring multiple tests to be positive) will increase the
sensitivity and specificity of differentiating a selected ‘healthy’ and ‘disease’ group, but this
approach is just as susceptible to the bias in group selection. Detailed discussion and
examples of these issues are reported in the TFOS DEWS II Diagnostic Methodologies
report 1.
3.4.1.5 Tests to establish a loss of homeostasis of the tear film
While the diagnostic or screening potential of tests such as thermography 193-195,
interferometry 196, lipid layer pattern/thickness 197 and tear evaporation rate 198 have been
evaluated, they are not widely used in clinical or research settings. Artificial intelligence has
been used to show that multiple factors including age, ocular surface staining and symptoms
are the most important predictors of an unstable tear film, followed by meibomian gland
drop-out and expressibility, blink frequency, osmolarity and meibum quality 199, but it would
not be practical to assess all of these in making a diagnosis of DED.
Tear film stability, as usually assessed by the tear film breakup time, is defined as the
measured time interval between a blink and the appearance of the first discontinuity in the

24
tear film 200, 201. While it is commonly assessed by instilling fluorescein, illuminating the ocular
surface with blue light and observing the fluorescence through a yellow filter 202, the
application of fluorescein itself reduces the stability of the tear film and increases its volume
so the tear film generally breaks up much more quickly 203-205 and the measurement may not
be an accurate reflection of the natural tear film status 206-208. Fluorescein breakup time is
also limited as a measure of tear film stability due to the requirement for subjective
assessment by the observer and, while an attempt has been reported to automate this
measurement 209, the required equipment is not available in clinical settings. Instead of the
time taken for the first break in the tear film to be detected, a metric to describe overall
disruption in tear film surface quality (assessed from a placido disc reflection) has been used
to assess tear film instability 210-212, but this has not been widely adopted. Non-invasive
objective assessment, usually with a Placido disc reflected from the tear film surface,
determines the time to the first detected break as well as mapping the locations of the tear
film disruption and the velocity of the destabilised area. Clinically insignificant differences
occur between the objective measurements by many automated devices to measure non-
invasive breakup 213, 214, but not all. 215-217 The detection algorithm results can be adjusted in
the validation stage of new instruments (such as adjusting the threshold of discontinuity in
the mires) to benchmark more closely to existing devices as there is no ‘gold standard’.
Controlling environmental factors such as temperature, humidity and air circulation during
and immediately before the measurement is important, as well as instructing the patient to
blink naturally several times and then to cease blinking until instructed to blink again. 218. The
Ocular Protection Index is the ratio of the tear film breakup time divided by the blink interval,
but as with other signs of a loss of homeostasis of the tear film and/or ocular surface, levels
deemed ‘pathological’ are poorly associated with patient reported DED symptoms 219.

Increased osmolarity of the tear film occurs within the pathophysiology of dry eye 4 with
estimated localized levels up of up to around 900 mOsm/kg predicted across the ocular
surface at points of tear film destabilisation 220. Point-of-care devices that measure osmolality
are currently limited to sampling from the tear meniscus, as the volume of tears is too small
to sample from the ocular surface. Osmolarity has also been reported to differ according to
the location along the lid margin from which it is sampled 221. While some studies have
advocated for its diagnostic role 222, 223, other studies suggest current instrumentation yields
results that are not repeatable from a single in vivo measurement 224 and values vary with
the device used 225. Tear meniscus osmolarity may account for <5% of the variability in other
tear film and ocular surface homeostasis signs 226 and is therefore unreliable in identifying
individuals where DED is diagnosed 227, or the DED severity is determined 228 with other dry
eye metrics. The daily variation of osmolarity has been suggested to be a better marker of
DED 229, but this is impractical for diagnosic purposes in a clinical setting. Tear osmolarity
was able to predict poor surgical outcomes and patient dissatisfaction in a cataract surgery
population with mild ocular surface disease, again suggesting it provides additional
information to tear stability and ocular surface damage 230 and hence is an important metric
in diagnosing DED 4.The inclusion of osmolarity amongst the possible signs of a loss of
homeostasis of the tear film in the TFOS DEWS II diagnostic algorithm made less than 5%
difference in the prevalence of DED 231, 232. Similarly to osmolarity, tear composition alters
where there is loss of homeostasis of the tear film, but not in a measurably consistent
manner, and current point-of-care tests for other tear film biomarkers are of limited value
(see Section 3.5.4.4.2) 233.

3.4.1.6 Tests to establish a loss of ocular surface homeostasis

Loss of homeostasis of the ocular surface is most widely established by topically applying
ophthalmic dyes such as fluorescein and lissamine green, and the resulting staining of the
tissues is considered to be a diagnostic sign of DED 234-236. Staining is one of the clinical
signs most strongly associated with dryness symptoms in moderate-to-severe DED 150, 237.
Punctate staining, while not pathognomonic of DED, is commonly associated with
desiccation stress, particularly when present in the inferior quadrant of the cornea 238, 239.

25
Fluorescein staining occurs due to a loss of corneal epithelial cell integrity such as a
disruption in superficial cell tight junctions or defective glycocalyx 238, 240. Lissamine green
has largely replaced the use of rose bengal as it is less toxic to the ocular surface 241,
staining epithelial cells if the cell membrane is damaged, irrespective of the presence of
mucin 238, 242-244. Staining of the eyelid margin conjunctiva that wipes the ocular surface
during blinking (termed lid wiper epitheliopathy) possibly due to mechanical stress resulting
from insufficient lubrication 245 is common in patients with DED and associated with poor lipid
spread, poor tear film stability, abnormal lid anatomy and blink speed 246, 247. It is also an
earlier diagnostic sign than corneal and conjunctival staining in the natural history of DED
pathophysiology 248-250. A clinically detectable poor seal between the eyelids has also been
identified as factor associated with symptoms of ocular discomfort 251.
The diagnostic potential of point-of-care inflammation testing (MMP-9) has been reviewed,
but while inflammation was common in severe DED, it was not sensitive to more mild to
moderate DED which is more common in the general population.252 Several studies have
investigated the potential of epithelial thickness as a diagnostic test for DED; while the
central epithelial thickness has largely to be found to be similar in DED compared to health
controls, the superior epithelium is generally thinner, especially in more severe disease,253,
254
but this finding is not consistent.255, 256
Squamous metaplasia and goblet cell density of the conjunctiva can be assessed using
impression cytology and, as goblet cell density reduces in patients with dry eye, it has been
suggested as a DED diagnostic technique 257, 258. Impression cytology removes cells from
the three most superficial layers of the epithelium, typically by applying cellulose acetate
filters or biopore membranes; the cells can then be analyzed by techniques such as
microscopy, flow cytometry, immunoblotting analysis, immunocytochemistry and polymerase
chain reaction to meet the aims of the investigation 259. It is a useful alternative to a biopsy,
but the changes observed are not pathognomonic of DED 260, 261.

3.4.1.7 Practical diagnostic criteria considerations

Sections 3.4.1.5.and 3.4.1.6 highlight that the key tests to assess the homeostatic status of
the tear film remain unchanged from those derived in TFOS DEWS II. The key homeostatic
markers are thus non-invasive tear film breakup time, osmolarity and ocular surface staining.
The sequence of diagnostic assessment can affect the results as restricting blinking and
bright lights can stimulate reflex tearing. It is therefore recommended that tear film
assessment tests are carefully ordered, from least to most invasive 262.
The OSDI-6 is a short questionnaire, ideal for screening and is recommended to be
conducted as the first component within routine eye examinations to identify those patients
who would benefit from a fuller diagnostic evaluation to determine the likely drivers of
disease.
As identified in section 3.4.1, it is critical that diagnosis follows a standard protocol. While
combining more tests can improve sensitivity, this may be at the expense of clinical utility.

TFOS DEWS II recommended that one of three signs of a loss of homeostasis needs to be
present:
 non-invasive breakup time (first break) <10s: as highlighted in Section 3.4.1.5, this
test establishes a loss of homeostasis of the tear film There are now a range of
affordable instruments available to the practitioner to avoid the adverse impacts of
fluorescein dye on the robustness of the test result as is well documented (see
Section 3.4.1.5). Where there is no access to such a device to allow non-invasive
measurement of breakup time, fluorescein can be applied, but the volume instilled
should be minimised and a cut-off of <5s applied as a positive sign of instability. 204,
205

• osmolarity ≥308 mOsm/L in either eye or interocular difference > 8 mOsm/L (cut-offs
established with the TearLab device only). This test serves as a marker of loss of
homeostasis for both the tear film and ocular surface (see Section 3.4.1.5).
Consideration of the inter-eye difference alone was found to be valid in one study 263

26
 but not in another (at least in relating to dryness symptoms) 264 and interocular
osmolarity was found to have modest, but superior discriminative ability than
absolute osmolarity (higher value of the two eyes) 265.
 ocular surface staining of >5 corneal or >9 conjunctival punctate spots or lid margin
staining of ≥2 mm length and ≥25 % width following the instillation of both fluorescein
and lissamine green dyes 238, 266. It is more common to observe corneal and
conjunctival staining in severe DED (see section 3.5.4.3)
Corneal observation: The corneal surface, following the installation of fluorescein
drops or application of a moistened fluorescein strip, should be observed using a
blue light of 495 nm, as this is the peak excitation wavelength for fluorescein (rather
than the 450 nm ‘cobalt blue’ peak of light filters historically used in slit-lamp
biomicroscopes) 202. An observation filter with a band pass at 500 nm limits visibility
to the wavelength of the excited fluorescence molecules (emittance around 515 nm)
while excluding those from the applied blue light 202. Consensus on the ideal time
after instillation for assessment is 1-4 minutes 238, 262, 267.

Conjunctival and lid margin observation: Lissamine green staining is critical for observation
of conjunctival and lid margin staining assessment. A lissamine green strip (note, not all
brands are equivalent)268, 269 should be moistened with sterile saline with the whole drop
applied to the eye after having been placed on the strip for at least 5s to allow the dye to be
eluted for maximal concentration 269, 270. The staining should be observed between 1 and 5
min post-instillation of lissamine green,269-271 potentially through a red filter to aid
visualization 241, 272. Everting the eyelids multiple times should be avoided as this can stress
the tissues and affect the degree of staining observed, whereas exposure time seems to be
less impactful on the outcomes.273 Assessment of lid wiper staining generally involves
subjective estimation of the length and sagittal depth of staining, but may be optimised using
semi-objective imaging techniques 274.

Grading: It should be noted that if using a grading scheme such as the Oxford scale,
applying half unit increments improves sensitivity and repeatability, while summing regional
grading doesn't give a comparable score to global ocular surface staining and increases
variability 275. For diagnosis, counting punctate spots should result in higher consistency.
Despite claims that objective assessment of corneal staining can be used as a highly
successful automated dry eye diagnostic system 276, a review of objective techniques
suggest this still has limited reliability 275. While there are challenges to grading staining of
the lid margin 277, a diagnostic criterion of 2mm or more length over at least 25% of the
sagittal lid width still seems appropriate.

A study found that a substantial proportion of patients ‘diagnosed’ with DED by a consultant
ophthalmologist without following any set criteria, reported symptoms and exhibited
hyperosmolarity, but no other obvious signs of DED; however, tear film stability was
assessed invasively and staining of only the corneal (not bulbar or lid margin conjunctiva)
was included 191.
In a UK population study, among the TFOS DEWS II diagnostic signs for DED, conjunctival
staining (with lissamine green dye) occurred in most people diagnosed with DED, followed
by reduced non-invasive tear breakup time, lower or upper lid wiper epitheliopathy staining,
corneal staining and signs of tear hyperosmolarity. 231 The prevalence of DED was notably
consistent if any one of the three markers indicating a loss of homeostasis was omitted from
the TFOS DEWS II diagnostic algorithm, indicating its robustness.231 In a larger study 232, it
was found that evaluating just one of the three TFOS DEWS II homeostatic signs resulted in
between 12.3% and 36.2% of patients who would otherwise have met the DED diagnostic
criteria, not being assigned this diagnosis; hence at least two signs need to be assessed,
even although only one needs to be positive. While comprehensive ocular surface staining
evaluation in combination with symptoms had the highest sensitivity (87.7%) of the three
markers, the sensitivity dropped to 44.6% if corneal staining only was evaluated; hence

27
conjunctival and lid margin staining assessment (with lissamine green) is critical to
diagnosing DED 182, 232. Omitting either non-invasive tear breakup time or osmolarity each
dropped the sensitivity by less than 5%. The prevalence of DED within the population was
substantially reduced if diagnosis required symptoms plus two of the three signs to be
positive (by between 43.7% to 61.2%) and by 65.9% if all three signs indicating a loss of tear
film homeostasis were required 232. The outcomes of this analysis did not change
significantly across differing severities of DED symptoms, confirming the robustness of the
DED diagnostic approach (Figure 5).

Figure 5: TFOS DEWS III diagnostic algorithm

3.4.2 Advanced screening

Diagnosing and monitoring DED often relies on specialized equipment (such as a slit-lamp
biomicroscope) and dyes, which are not readily available in non-eye care setting. A non-
invasive and simplified test for DED could enable earlier diagnosis and intervention to help
prevent disease onset or exacerbations 278. Additionally, improved accessibility to DED
screening could raise public awareness and encourage high-risk or undiagnosed individuals
to seek attention by an eye care practitioner.
Blinking is a natural process that refreshes tear film, removes ocular surface debris, and
maintains vision quality 279. Altered blinking physiology is a common feature in DED and is
implicated in its pathogenesis 280-282. As such, DED-associated blinking patterns could
potentially serve as a non-invasive biomarker. Blink rate, interblink interval and maximum
blink interval (defined as the length of time that the participants can comfortably keep the
eye open before blinking), have been reported to be useful in distinguishing between healthy
participants and patients with DED 283-288.

Maximum blink interval (cut-off 12.4 seconds) has demonstrated a sensitivity of 82.5% and
specificity of 51.0% for a DED diagnosis based on OSDI symptoms and fluorescein tear
breakup time) 289. A smartphone application has been tested 278, 287, 290, 291, showing
comparable results with conventional paper-based OSDI and subjectively observed
maximum blink interval 292, 293. The app-based maximum blink interval had an area under the
curve of 0.649, with a cut-off of 10.5 seconds on a digital device with an iOS, but a much
shorter 7.0 seconds on an Android operating system, (compared to 12.4 seconds
subjectively assessed) for a symptoms (perhaps due to differences in refresh rate and
software algorithms) and fluorescein breakup time DED based diagnosis 294. Similarly,
interblink interval, with a cut-off of 3.1 seconds, has shown an approximately 80% sensitivity
and 70% specificity for a DED diagnosis based on general symptoms and corneal staining
283
. Furthermore, patients with DED are reported to have higher blinking rates 295, with a

28
greater proportion of incomplete blinks 296. By analyzing such blinking characteristics,
clinicians may be able to gain valuable insights into a patient’s ocular surface status.

As maximum blink interval is dependent on an individual’s tolerance to eye pain, an

alternative approach is asking an individual to report when their eyes become uncomfortable
after a blink while keeping their eyes open, which demonstrated a sensitivity of 66%,
specificity of 88%, and an area under the curve of 0.77 (cut-off 10 seconds) compared to a
full TFOS DEWS II diagnosis of DED, improving to 71%, 90%, and 0.81 respectively when
considered in combination with the OSDI score 288.

3.4.3 Severity rating

The severity of a disease is rated for a diverse range of purposes, but primarily for
evaluation and communication 297:
 to predict prognosis
 to characterize the impact of the disease on the person’s well-being at a given point
in time
 to establish the basis for treatment decisions
 to evaluate disease activity and monitor response to treatment
Some classifications are based on pathological or physiological status, while others have
used impairments or specific symptoms (such as pain) and still others have characterized
severity based on exercise tolerance or functional status 297. Symptoms and outcomes tend
to be used in systems that are designed to reflect the patient experience. Pathological or
physiological measures have been incorporated in systems used to predict prognosis and
both tend to be used to guide treatment, or measure response to treatment 297. Some
severity classification systems also take into account the presence of other conditions,
diseases, demographics or behaviors in their staging, if these are considered risk factors for
a poorer prognosis.297

In other eye diseases, severity ratings vary from those based on the risk of advanced
disease progression, for example in age-related macular degeneration 298, to those variably
combining signs and symptoms which aligns with clinician consensus, for example in
keratoconus 299-301. The former approach requires a large sample of natural history data 302
and the latter, a reasonably high association between subjective and objective clinical
metrics 303, neither of which are currently available for DED.
Severity grading in DED has been based on factors such as inflammatory cytokine
concentrations 304, corneal fluorescein staining scores 305 and doctor (clinical judgement)
rating 306. The ODISSEY group proposed a severity rating based on the expert opinion of 10
ophthalmologists 307 where the DED diagnosis relied on poor tear stability alone, and severe
dry eye was considered to be those with an OSDI score ≥33 and corneal fluorescein staining
grade ≥3 on the Oxford scale, or with additional criteria if there was less staining. An
objective composite index of disease severity has been proposed using an independent
component analysis approach 308, however key non-invasive tests of the tear film and ocular
surface were missing such as non-invasive tear breakup time, interferometry, tear meniscus
height and lid wiper epitheliopathy and the cut-off for each test’s severity grade was based
on an “expert panel” of five; the amount of independent information provided by each test
(eigenvalue) was used as its weighting for each component’s contribution to a composite
score, which was based on the sum of the squared measures, divided by the square-root of
the sum of the weighting coefficients. A survey approach involving 37 corneal specialists in a
hospital setting (non-representative of dry eye practitioners) of unknown location identified
clinical tests and cut-offs they felt represented a diagnosis and reflected severity (mild,
moderate, severe and very severe) of DED 309; seven tests were identified and overall
severity was based on an algorithm of combining the scores with equal weighting (identified
as a limitation) which was based on clinician-ratings (not defined) of 50 patients. A recent
study suggested observing corneal cell morphology and density with in vivo confocal
microscopy in areas which stain with fluorescein and lissamine green dyes may be a reliable

29
basis for clinical grading of DED severity, but the cohort of 24 participants was classified into
severity grades based on the Chinese Cornea Society criteria which also involved staining,
as well as fluorescein tear breakup time, so the observation was not surprising.310

A recent review of how to “best diagnose severity levels of DED” erroneously described
‘asymptomatic DED’, only mentioned non-invasive tear breakup time as a form of
interferometry and concluded, without proposing a clinical algorithm, that the evaluation of
severity of the condition has often been difficult.311 While the original TFOS DEWS report
proposed a severity matrix 312 based on a prior Delphi panel of 17 DED specialists,313 this
was not adopted by TFOS DEWS II (2017) due to the limited association between the
characteristics included and the lack of evidence for the tests included to inform their
weighting in a composite algorithm. Likewise, the Asian Dry Eye Society did not propose a
severity algorithm for DED 179. However, the Korean Dry Eye Society proposed a severity
matrix, but without scientific justification for the proposed tests, and the severity levels
provided 181.The revised American Academy of Ophthalmology Preferred Practice Pattern
guidance 314 mentions the role of DED severity in informing management, but without
guidance on how to rate severity. In a recent survey in the UK, patients with DED rated
symptom frequency and severity along with tear film stability as the most desired aspects of
their DED to improve with treatment, although other factors such as ocular surface, corneal
nerve and tear gland damage followed by tear volume and constituents were rated only
slightly less important.315

3.5 Subclassification to identify DED etiological drivers

3.5.1 Purpose of a DED subclassification
Diagnostic subcategories (for diseases and most syndromes) are simply concepts. Their
purpose is to segregate multifactorial aspects to allow a better characterization of patient
outcomes and to guide decision-making regarding treatment 316. TFOS DEWS and DEWS II
confirmed the importance of subclassifying DED into aqueous deficient or evaporative forms,
or a combination of the two 2, 312. However, several studies have confirmed that at least two-
thirds of those with DED exhibit the evaporative form 317-321 which is recognised to have a
number of etiologies including lid-related and ocular surface-related,322 which, without
distinction, limits the ability to target treatment to the appropriate etiology. The selection of
tests used to differentiate evaporative from aqueous deficient forms of DED varies between
studies and a Delphi panel approach has attempted to establish agreement 323. In addition, it
has been noted that between 18 % and 29 % have no obvious signs of either a reduction in
tear volume or disruption to meibomian gland structure and function, suggesting the need to
acknowledge other subtypes of the disease 191, 319-321, 324. DED is accepted as a multifactorial
disease 2, 20 so addressing the different mechanisms leading to an individual’s DED could
impact treatment outcomes. Disease heterogeneity will reflect differences in the underlying
pathophysiology, genetic risk and environmental contributors of affected individuals 325.
Clinical tests that identify the possible drivers of an individual’s disease (which are not
mutually exclusive), in turn, inform the appropriate treatment approach(es) 326. The Asian Dry
Eye Society 179 proposed four targets for therapy (lipid, aqueous and mucin layers along with
ocular surface inflammation), but with a target of the epithelium consisting of membrane-
associated mucins and epithelial (goblet) cells. Expanding on this approach, the following
section outlines those clinical tests that inform the clinician about the contribution to DED
from:

3.5.2 Tear Film Deficiencies

The latest understanding on the tear film is reported in the TFOS DEWS III Digest.4. It has
been proposed that differences observed in the fluorescein tear breakup patterns can inform
the clinician about which tear film layer has been disrupted. Area break is thought to be
associated with severe aqueous-deficient DED; spot, dimple, and line breaks with rapid
expansion are associated with decreased wettability DED, with a line break thought to be
associated with mild-to-moderate aqueous-deficient DED; and a random break appearance

30
is associated with increased evaporation DED 327-329. However, to date, there has been only
limited published evidence to support these hypotheses 329.

3.5.2.1 Lipid component

3.5.2.1.1 Interferometry
Tear interferometry allows the tear film lipid layer thickness to be estimated, noninvasively
330, 331
. Due to nature of the thin lipid layer overlying a body of aqueous with different
refractive index, reflections from the air-lipid and the lipid-aqueous interfaces create
interference patterns, which can be analyzed quantitatively or semi‑quantitatively 332. Lipid
layer interference pattern grades correlate with corneal staining and tear film breakup time
333
. Lipid layer thickness should be increased in hypersecretory MGD and decreased in
obstructive meibomian MGD, but a direct association between the thickness of the lipid layer
and the rate of tear evaporation has not been proven 334, 335. There are various non‑invasive
diagnostic devices for assessing the tear film lipid layer 336-342. Some devices attach to a slit-
lamp biomicroscope base while others are stand-alone instruments. Most require subjective
grading of the lipid pattern 343, which equates the pattern observed to an estimated thickness
337
. A dynamic lipid layer interference patterns test has been proposed, reporting the optimal
number of blinks to observe a significant change in lipid pattern as being up to five forced
followed by 10 natural blinks at 2s intervals; in patients with DED, the number of blinks
required to change the lipid pattern (2.4 3.1 blinks) was statistically lower than in healthy
subjects (18.1 5.9 blinks)344. The LipiView interferometer has a sensitivity of around 68%
and specificity of 64% if a cut‑off value of 75 nm is used for MGD diagnosis 332, 345; the
coefficient of variability for inter‑observer repeatability was 13 nm and the intra‑observer
repeatability 16 nm in healthy individuals, with values less than 60 nm considered
pathological 346. In a study of 221 participants, optimal diagnostic cut-offs for DED based on
the TFOS DEWS II criteria were <72nm with the LipiView and a grade of ≤3 sub ectively,
based on interferometric patterns (modified Guillon scale).197 Lipid layer thickness values
obtained with the LipiView instrument have been reported to correlate well with meibomian
gland loss 347. Another technique, using a spectrophotometer, claims an ability to directly
image tear muco-aqueous and lipid layer thicknesses in vivo with nanometer axial resolution
348, 349
.

3.5.2.1.2 Lipid turnover

The turnover of the lipids of the tears assessed by fluorophotometry 0.9 ± 0.4 % / min) is
slower than the aqueous turnover (10.3 ± 3.7 % / min).350 Contrast-enhanced optical
coherence tomography (OCT) imaging has also been used to evaluate the clearance of
lipids in human tears 351. A system that combines simultaneous OCT and thickness-
dependent fringes interferometry can be used for in vivo assessment, simultaneously
imaging both the lipid layer thickness and overall tear film thickness 352, 353. The analysis of
the OCT’s en face maps of the tear lipid layer provides complementary information to
interferometry 354. The tear film imager uses spectral interference to allow real-time
evaluation of the rate of lipid thickness change and discontinuations over a large field of view
at nanometer axial resolution 330. The mucoaqueous thickness correlates with the Schirmer
score and lipid or fluorescein tear breakup time 349.

3.5.2.1.3 Evaporimetry
A meta-analysis of studies measuring evaporation found raised levels in patients with DED,
particularly evaporative DED (normal, 13.57 6.52 x 10-7 g/cm2/s; aqueous deficient dry eye,
17.91 10.49 x 10-7 g/cm2/s; evaporative dry eye, 25.34 13.08 x 10-7 g/cm2/s)355. Different
instruments have been used for assessing evaporation, although only one is currently
commercially available 356-359. Tear evaporation has been shown to be reduced by eyelid
warming therapy 360. Confounding factors are the sampling response rate to blinking,
perspiration within the sampling area, palpebral aperture, variation in blink speeds and
patterns, and in the level of chamber ventilation, and differences in resistance to evaporation
caused by humid, and still air 361, 362.

31
3.5.2.1.4 Thermography
Thermography uses a specialized camera to detect infrared radiation emitted from the ocular
surface, mapping changes in the ocular surface temperature that are presumed to be
caused by tear fluid evaporation 363. The technique seems repeatable 364. Thermal cooling of
the ocular surface is a predictor of soft contact lens induced dryness symptoms 365. Ocular
surface temperature decreases more rapidly following a blink in individuals with adequate
tear volume, but unstable tear films.366-368 The temperature differential between the central
cornea and limbus is higher in DED than in normal,369 with evaporative DED associated with
higher ocular surface temperature than aqueous deficient DED and patients with MGD
having higher ocular surface temperature than those with healthy eyes.367, 370

3.5.2.1.5 Meibum expressibility and quality (meibometry)

Meibomian gland functionality is assessed by testing the expressibility of meibum and the
quality of expressed meibum 332, 371. Meibum quality is typically graded as 0 (clear fluid), 1
(cloudy fluid), 2 (cloudy particulate) or 3 (toothpaste‑like). The expressibility of meibum from
the meibomian glands of the upper and lower eyelids is graded after 10-15 seconds of
applying pressure as 0 (all glands expressible), 1 (3 to 4 glands expressible), 2 (1 to 2
glands expressible) or 3 (no glands expressible). Meibomian gland expressor devices have
been developed as a means of standardizing the pressure of ‘diagnostic’ expression, which
aims to be equivalent to that of a natural blink 371. Meibum quality and expressibility are
correlated with gland loss and lipid layer thickness 372-375.
Meibometry is a technique applied to assess lipid volume 376, involving sampling lipid from
the lower lid margin with a loop of translucent plastic tape. The tape is air-dried for 3 min to
allow evaporation of any contaminating tear fluid and the optical density of the oil retained on
the tape is assessed using a diode laser. A correlation has been reported between lower lid
meibometry readings and expressibility of meibomian lipid from the central upper lid, as well
as a reduction in volume in patients with MGD and an improvement after meibomian gland
orifice probing 377.
A test strip made of hemp oil-absorbing material, designed specifically to absorb tear lipid,
has been developed. The folded end is placed in the conjunctival sac of lower eyelid and the
length of infiltration (over 1 minute seemed optimal) measured and recorded, similar to the
Schirmer test 378.

Tear lipidomics are reviewed in Section 3.5.4.4.2.

3.5.2.2 Aqueous
As the aqueous component represents the majority of the tear film volume, techniques
assessing tear volume are often used to quantify this tear component.
3.5.2.2.1. Meniscometry or tear meniscus assessment
Meniscometry involves non-invasive biometry of the lower eyelid tear meniscus, usually in
the form of a central height in primary gaze. Subjective methods of tear meniscus height
measurement, such as estimating the meniscus height relative to a height-adjusted slit-lamp
beam scale, has shown poor inter-visit reproducibility 379. Slit-image photography has also
been used to quantify tear meniscus height, radius, width, as well as cross-sectional area
and radius of meniscus curvature 376. The TFOS DEWS II Diagnostic Subcommittee
proposed tear meniscus height assessment as a differential factor for the subclassification of
DED, describing a cut-off value of 0.20 mm or lower as an indicator of aqueous-deficient
DED, which has subsequently been confirmed 39, 320, 380. Tear meniscus height should be
measured directly below the pupil midline (±1mm) as it is affected by varying lid
morphologies more peripherally.381 The timing of the assessment after a blink should be
controlled, with 1.0 to 2.5s after two blinks found to be most robust; a single measure of tear
meniscus height is sufficient, using either with infrared or visible white light (although these
are not interchangeable).382 Alternative meniscometry systems have been developed in

32
research settings, projecting a target to dynamically visualise the tear meniscus curvature,
without the need for fluorescein instillation 379, 383, 384.

OCT allows the cross-sectional area of the tear prism or even the volume along the lower lid
to be quantified 385, 386. Spectral-domain OCT has enabled higher resolution, greater imaging
depth and faster acquisition (facilitating three-dimensional volume imaging) compared to
time-domain OCT, improving image quality and measurement repeatability 330, 387-389. The
high repeatability allows changes in tear meniscus morphology after fluid instillation to be
tracked, to determine tear clearance rate 390. Meniscus measurements are instrument-
dependent 391 and can be distorted by anatomical features such as lid parallel conjunctival
folds, conjunctivochalasis, or other disruption to the shape of the lid margin or ocular surface
392
. Furthermore, analysis of the image may be complex, time-consuming and operator-
dependent 393.

3.5.2.2.2 Phenol red thread test

The slight alkalinity of the tear film (between pH 7 and 8)39 allows the length of a thin cotton
thread, with the folded end hooked over the temporal end of the eyelid, moistened by tear
absorption over a 15 second period to be observed from a color change of yellow to red.
Compared to the Schirmer test (see Section 3.5.2.2.3), the small profile and limited amount
of pH indicator in the thread is expected to limit the chance of triggering substantial reflex
tearing and as a result, intersession repeatability is good.394 While the phenol red test is
thought to indirectly measure the tear volume, it is only weakly correlated with other
established methods such as fluorophotometry or tear meniscus height.394, 395 In addition, it
is weakly correlated with dry eye symptoms396 Reported agreement with the Schirmer test is
variable between studies.397, 398 An arbitrary aqueous deficient DED cut-off value of 20 mm
has been adopted in clinical practice 399 and values of <9mm in 15 seconds suggest more
severe cases of aqueous deficient DED400. Due to issues with accessibility of a
commercialized product, techniques to develop an equivalent test have been described 401.

3.5.2.2.3 Schirmer test

The Schirmer test is an invasive test of tear volume, that involves assessing the length of a
(Whatman 41) filter paper strip that becomes saturated by tears, 5 minutes after hooking the
end of the strip, folded at the notch, over the lower lid margin, within the temporal one-third
332
. Technique variations, such as the use of anaesthetic which aims to differentiate basal
from reflex tearing 402, indicate poor repeatability, sensitivity and specificity 1. Using the
wetting of the Schirmer strip over the final 4 minutes out of the 5, seems to be more robust
than other time intervals including assessment over the full 5 minutes, but not surprisingly,
accounts for <3% of the variance in fluorescein breakup time, a key homeostatic marker for
all DED subtypes, or meibomian gland secretion, a recognised marker of evaporative
DED.403

3.5.2.2.4 Strip meniscometry

Strip meniscometry involves placing a strip with a 0.4mm diameter central duct into the lower
lid tear meniscus for 5 seconds 404-406. A cut-off of 2.5 mm has been adopted, with the
results correlating with other tear film assessments and the values are moderately
repeatable 407, 408.The combination of strip meniscometry and fluorescein breakup time have
been proposed as a more sensitive approach for assessing DED than either test alone.386

3.5.2.2.5 Tear clearance

Measuring the fluorescence of instilled quantities of fluorescein across the ocular surface
(termed fluorophotometry) can be used to quantify tear turnover, reported as the decrease
(in percent) per minute 409. Alternative approaches to measuring tear flow include conducting
a Schirmer test 5 minutes after instilling fluorescein with anesthetic, with both the length of
wetting and the intensity of strip staining compared to a standard color plate after 5 minutes
recorded 410. Due to the various factors that can affect Schirmer test results, the tear function

33
index, which is the value obtained by dividing the value of the Schirmer test by the tear
clearance rate, is a measure that has been used by a limited number of authors to assess
patients with DED 411-413. Other methods of tear clearance assessment include anterior
segment OCT, which has been used to evaluate the early phase of tear clearance 414.
Lacrimal scintigraphy (tracking a small amount of radioactive material instilled into the
conjunctival cul-de-sac) has also been used to measure tear clearance 415, 416.

3.5.2.2.6 Lacrimal gland patency

A clinical test for examining the patency of the palpebral lobe of the lacrimal gland has been
described, which involves having the patient look inferonasally while the upper eyelid is
retracted in the superotemporal direction. Dry 2% fluorescein ophthalmic strips are applied
onto the exposed palpebral lobe multiple times over 20s. This allows the number and
location of ductules per lobe as well as the tear flow rate to be assessed 417. Alternatively the
patency of the lacrimal gland can be assessed by stimulating the ocular surface with a pure
CO2 gas jet at 200 ml·min-1 for 3s, delivered 5 mm from the cornea and measuring the
increase in reflex tearing volume with a Schirmer strip 418.

3.5.2.2.7 Tear proteins and other components

The presence of the antibacterial and anti-inflammatory lacrimal gland proteins, lipocalin,
lactoferrin, and lysozyme, which possess anti-inflammatory and anti-bacterial properties 419
can be assessed as an indirect measure of lacrimal gland function. Tear protein
concentration has generally been found to decrease with age 420. Although lactoferrin has
been proposed as a biomarker of DED 421-423, low tear lactoferrin levels are also found in
giant papillary conjunctivitis, vernal keratoconjunctivitis, and chronic meibomitis associated
with acne rosacea 424-426. Tear film urea levels are linearly related to Schirmer’s test values;
for diagnosing DED, a cutoff of 37.2 mg/dL has been reported to provide a sensitivity of
96% and a specificity of 76% 427.Tear fluid proteomics are reviewed in section 3.5.4.4.2.7.

3.5.2.3 Mucin / glycocalyx

3.5.2.3.1 Mucins
Human conjunctival goblet cells synthesize and secrete the largest type of gel-forming, non-
surface-bound mucin in the eye, MUC5AC, which acts to protect and lubricate the ocular
surface, mitigating friction during blinking 4, 428. Patients with DED typically show reduced
concentrations of soluble MUC5AC in the tear film 429. Together with lipids, a concomitant
increase in MUC5AC protein expression in tears in infants may contribute to their greater
tear film stability 430. Goblet cell count and tear MUC5AC protein are decreased in Graves´
ophthalmopathy, thought to be possibly due to ocular surface inflammation secondary to
ocular surface exposure 431.

Immunohistochemistry and immunoelectron microscopy have been used to examine binding

of the HI85 antibody, which recognizes carbohydrate epitopes on the MUC16 mucin on the
surfaces of apical conjunctival cells 432, 433.

Other membrane-associated mucins, MUC1, MUC4, as well as MUC16 (glycocalyx) and gel-
forming mucin MUC5AC, have been studied using different techniques 244, 434, 435. In other
research large decreases in sialic acid (almost 7-fold) 436 and increases in galectin-3 (proxies
of the glycocalyx/mucin) 437 have been observed in the tear film and shown to strongly
correlate with clinically assessed disease severity 438.

3.5.2.3.2 Rose bengal and lissamine green (see section 3.4.1.7)

Rose bengal has been shown to stain ocular surface epithelial cells that are unprotected by
the mucin rich glycocalyx 439, but it has been shown to suppress human corneal epithelial
cell viability in vitro, indicating toxicity 240.. Lissamine green is less toxic to the ocular surface
and has largely replaced the use of rose bengal in clinical care and research. Lissamine

34
green is a vital dye that stains epithelial cells only if the cell membrane or intracell junctions
are damaged, irrespective of the presence of mucin 39, 440.

3.5.2.3.3 Conjunctival impression cytology

After the instillation of topical anesthetic, a filter strip is pressed against the bulbar
conjunctival surface, 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 light microscope 261. The Nelson classification system is used most frequently
to grade the density, morphology, cytoplasmic staining affinity and nucleus/cytoplasm ratio of
conjunctival epithelial and goblet cells across the ocular surface 39. There is variation in
goblet cell distribution across the conjunctival surface, with the lower forniceal conjunctiva
goblet cell density higher than in the bulbar conjunctiva 441. Recently, moxifloxacin-based
fluorescence microscopy has emerged as a novel technique that enables efficient, non-
invasive and in vivo animal imaging of goblet cells 442. Confocal imaging, on the other hand,
can be used to assess corneal cell morphology 443, goblet cell density 444 and conjunctival
squamous metaplasia (from nucleocytoplasmic ratios) 445 in vivo in humans.

3.5.2.3.4 Ferning test

Whole tears collected by one of many possible techniques 446 are transferred immediately to
a small plastic centrifuge tube (0.5 ml or less); a sample (1–2 μl) is then pipetted onto a
clean glass microscope slide and allowed to dry for 7–10 minutes under normal room
temperature (20–26°C) and room humidity (up to 50 %) 447. The slide then can be observed
under a light or digital microscope with various magnifications 446. Depending on the tear film
composition, a variety of ferning patterns can be observed. The classification is typically:
type I: uniform large arborization; type II: ferning abundant but of smaller size; type III:
partially present incomplete ferning; and type IV: no ferning 448. Healthy tear samples
typically produce full dense ferning patterns (types I and II), while the ferning pattern are
often fragmented or absent in patients with DED 446.
The exact nature of what determines the ferning pattern is still not fully understood 1, 449,
though a causal link has been proposed between tear ferning pattern and the ocular mucins
449
. Hyperosmolarity affects the appearance of ferning patterns 446 as does introducing
electrolytes into the tear film 450. Tear ferning patterns have been found to be repeatable 451.
A correlation has been found between the ferning test grade, non-invasive breakup time 452
and low Schirmer test values, but no correlation has been reported between ferning pattern
and various tear protein levels. 453

3.5.3 Eyelid anomalies

3.5.3.1 Blink and lid closure anomalies

Blinking is a complex neuromuscular process that plays a vital role in maintaining ocular
surface homeostasis and proper functioning of the tear film. Specifically, it facilitates the
even distribution of tears, mucin, and lipids, essential for lubrication, protection from eye
irritation, and removal of debris and foreign bodies 289, 454-456. Blinking is primarily controlled
by the orbicularis oculi muscle, innervated by the facial nerve (cranial nerve VII) 457. The
levator palpebrae superioris muscle, innervated by the oculomotor nerve (cranial nerve III),
and Müller’s muscle, innervated by sympathetic fibres, are also contributory 458. Sensory
input from the cornea and conjunctiva, relayed via the trigeminal nerve (cranial nerve V),
modulates the blink reflex 459.
Blinking can be categorized into three types 460. The first is spontaneous blinking, which is
the unconscious and coordinated closure of both upper eyelids occurring briefly and
symmetrically without any evident stimulus. The second is reflex blinking, which is triggered
by trigeminal, visual, and acoustic stimuli. Lastly, voluntary blinking is defined as the closure
of eyelids consciously initiated by the individual. The normal rate for spontaneous blinking
ranges from 10-15 blinks per minute 295, 461. This rate can be influenced by multiple factors,

35
such as age, cognitive load, social activity, neurological and psychiatric diseases, fatigue,
eye injury, medication, contact lens wear and dryness 295, 460.
Abnormalities in blinking patterns have been implicated in DED development 279, 282, 289. Blink
rates, interblink interval and maximum blink interval can differentiate patients who are
considered healthy from those with DED 285, 291, likely due to the changes in ocular surface
exposure and failure to restore tear film structure between blinks. Patients with DED often
exhibit increased blink rates 295 relative to normal. However, reduced blink rates have also
been observed both in individuals with and without DED, particularly during activities
requiring prolonged visual attention (e.g., screen use), leading to insufficient tear film
distribution and increased tear evaporation rates 462. Similarly, studies have shown that
patients with DED had shorter mean and maximum interblink intervals compared to healthy
controls 283, 289, 463. Notably, people with DED also demonstrated higher rates of incomplete
blinking 296, which in itself is associated with inadequate expression of meibomian gland
secretions into the lipid layer on the ocular surface, exacerbating tear film instability 464-466.
Given the multifactorial nature of DED, a comprehensive evaluation of blinking physiology is
essential for understanding its pathophysiology and employing effective treatment strategies.
Clinical monitoring is usually subjective and surreptitious to avoid a change in blink pattern
from the patient 467, but objective image analysis is becoming more widely available.
Improving blink quality and frequency through behavioral modifications and therapeutic
interventions can significantly benefit ocular surface health and patient comfort. Poor lid seal
is also linked to symptomology in DED and can be detected by placing a pen torch or
transilluminator against the relaxed, closed, outer upper eyelids of semi-reclined patients
and observing visible light emanating from the lid area between the lashes 251.

3.5.3.2 Lid margin health

3.5.3.2.1 Anterior blepharitis
Anterior blepharitis has been defined as “an inflammation of the lid margin anterior to the
gray line and concentrated around the lashes” which may be accompanied by squamous
debris or cylindrical dandruff around the lashes (Figure 6), and inflammation may spill onto
the posterior lid margin”47, 468, 469. The term ‘blepharitis’ is often used by clinicians to describe
anterior blepharitis, with posterior cases referred to by the more specific etiology, such as
meibomian gland dysfunction. The pathophysiology of anterior blepharitis is multi-staged and
relates to microbial changes that can culminate in inflammation: periocular bacteria build a
defensive structure known as a biofilm, which predisposes to forming on the eyelid margin
due to its moisture, nutrients and warmth. Biofilms are composed of a polysaccharide/protein
matrix, that adhere strongly to surfaces due to proteins like adhesin produced by bacteria
such as Staphylococcus epidermidis and Staphylococcus aureus. Biofilms allow bacteria to
evade the impacts of desiccation and host defense mechanisms, facilitating gene activation
and inflammatory virulence factors 470. Dry eye symptoms 471 and signs,472 as well as contact
lens discomfort,473, 474 are reduced on treatment anterior blepharitis,475 implicating anterior
blepharitis as one of the key triggers of the multifactorial disease.
Common clinical manifestations of anterior blepharitis include the presence of squamous
debris or cylindrical dandruff around the base of the lashes, accompanied by vascular
changes in the lid skin 468, 476 Anterior blepharitis is associated with ocular rosacea,
seborrhea and hypersensitivity caused by staphylococcal toxins, infectious processes
(bacterial or viral) or infestation by phthiriasis or Demodex, or combinations of these triggers
477
.
The most frequently identified species are Staphylococcus epidermidis (in about one third of
cases), followed by Pseudomonas (approximately 20%), Staphylococcus aureus,
Propionibacterium, Corynebacteria, and Moraxella 478, 479. Immunologic mechanisms have
been documented, with enhanced cell-mediated immunity to Staphylococcus aureus
detected in 40% of patients with chronic blepharitis. Seborrheic blepharitis, in contrast,
presents with greasy deposits and is commonly associated with seborrheic dermatitis of the
eyebrows and scalp 476, 480.

36
3.5.3.2.1.1 Demodex associated blepharitis
Anterior blepharitis can also result from the activity of Demodex folliculorum or brevis,
parasites identified in 14% to 89% of the population (especially in older patients) 105, 481-483.
There are two types identified in the human eyelids: Demodex folliculorum and Demodex
brevis 484. Demodex folliculorum is typically found in the eyelash follicles of the eyelids and
observed with high magnification slit-lamp microscopy 485.
Characteristic features are apparent at the base of the lashes, and these can be present in
asymptomatic individuals. Demodex mite presence is associated with changes in the
anterior lid margin, such as increased scale intensity and cylindrical dandruff or sleeves.
Cylindrical dandruff are considered pathognomonic for the presence of Demodex mites 485,
486
. Demodex can be detected by examining epilated eyelashes under white light microscopy
at high magnification 487, 488 or on a slit-lamp at high magnification. Manipulating an eyelash
with cylindrical dandruff around its axis in a circular motion 489, 490 or by applying lateral
traction 491 using fine-tipped metal forceps, can reveal mite tails at the insertion point of the
lashes. Detection of Demodex using in vivo confocal microscopy has also been described,
but this technique is cumbersome, time-consuming and costly, limiting its diagnostic utility
491, 492
.

Figure 6: Anterior blepharitis.

3.5.3.2.2 Meibomian gland dysfunction (MGD)

MGD, a major contributing factor to DED, is described as “a chronic, diffuse abnormality of
the meibomian glands, commonly characterized by terminal duct obstruction and/or
qualitative/quantitative changes in the glandular secretion” in the TFOS International Report
on Meibomian Gland Dysfunction Definition and Classification report published in 2011. 468.
The diagnosis of MGD is primarily clinically-based, focusing on the detection of signs
indicative of altered meibomian gland secretions, lid margin changes, and meibomian gland
dropout 372. The severity of MGD is classified based on subjective symptoms, abnormal
signs at the orifices (plugging, pouting and capping), vascularization or reddening of the

37
eyelid margin, anterior or posterior displacement of the mucocutaneous junction, eyelid
margin irregularity and rounding, gland drop-out observed by meibography (where imaging
both upper and lower lids is important 493), corneal and tear film abnormalities (such as
superficial punctate keratitis) and the quality of the expressed meibum 494-496. Various clinical
grading criteria for gland appearance have been proposed 495, 497-499. Additionally, the term
"plus disease" has been used to refer to co-existing or accompanying disorders of the ocular
surface and/or eyelids 331. Another scoring system combines subjective symptoms, slit-lamp
microscopy findings, and tear test results to classify MGD severity into stages 1 to 4 and
describes the relationship between meibomian gland dysfunction severity and serum lipids
500
. An alternative scoring system integrates subjective symptoms, in vivo confocal
microscopy findings, slit-lamp microscopy, meibography, tear film breakup time and corneal
staining scores, classifying MGD severity into three stages 494, 500. Yet another scoring
system elected to combine scores for secretion appearance and the digital pressure needed
to express the meibum 501.

The number of lower eyelid meibomian glands yielding liquid secretion has been assessed
as a metric of MGD 371. Assessing meibum expressibility, color and quantity is crucial 502,
either as the highest grade from eight expressed glands (total score range: 0 to 3) or
summing all eight gland expressibility scores (total score range: 0 to 24) 467, 468.
Anatomically, there are more meibomian glands in the upper eyelid, with a median count of
31, compared to a median count of 26 in the lower eyelid 469. The glands in the upper eyelid
are notably longer and slimmer than those in the lower eyelid. Studies on lower and upper
eyelids separately highlight secretion differences between the nasal and temporal sides.
The lower eyelid reportedly has higher secretion rates and poorer meibum quality than the
upper eyelid 467. Additionally, nasal glands appear more active, with activity diminishing
toward the temporal margin 503. Upper eyelid meibograpy has been proposed to be an
integral component of comprehensive meibomian gland evaluation.493

Non-invasive infrared or transillumination photography of meibomian glands has advanced

the assessment of two-dimensional gland silhouettes 498, 504. Meibography enables direct
visualization of meibomian gland morphology, highlighting partial or total non-visible
meibomian tissue as dropout or loss 372, 505. This can be assessed through subjective
grading 468, 506-508, or semi-quantitative or quantitative analyses 509. Asymptomatic individuals
have been found to have meibomian gland loss <16.9% for the upper and <28.7% for the
lower eyelid 498.Various gland morphological characteristics have been identified, including
truncation, dilation and tortuosity 510, 511, but none in isolation seems to be a good predictor
of DED 512.

Tortuosity of the meibomian glands in the upper eyelid has been observed to correlate
negatively with tear film stability, while tortuosity in the lower eyelid correlated with dry eye
symptoms 508. However, its variability makes it less reliable as a standalone diagnostic
parameter 503, 508 and it is only weakly correlated with meibomian gland expression 372.
Meibomian gland thickness increases with overall loss, potentially as a compensatory
response, although this does not improve expressibility 372, 503. On the other hand, distorted
and thinned glands appear to be transitional phases before dropout 468, 469, 502. However,
meibomian gland length has been identified as the key morphological metric for function in
terms of expressibility. 372.

OCT has been used to image meibomian glands and may be more sensitive than traditional
meibography techniques 513. In vivo confocal microscopy can observe meibomian gland
orifice tissues at a cellular level, enabling evaluation of acinar density, acinar diameter,
enlarged meibomian gland orifice and conjunctival inflammatory cell density 514; it can also
allow assessment of glandular atrophy and peri-glandular fibrosis. However, the equipment
is expensive, requires a learning curve to obtain good images, and the technique requires
contact with the epithelium, posing disadvantages with respect to invasiveness and

38
prolonged examination burden. The technique is unable to permit visualization of the glands
themselves in the human eyelid margin due to light attenuation at that tissue depth; the
structures imaged are rete ridges located at the dermal–epidermal junction, with alterations
believed to indicate a shift of the mucocutaneous junction 515. Clinical use remains limited 331,
494, 514, 516
. Lipid quantification at the eyelid margins and biochemical analysis of gland
secretions for lipid components and markers are still being researched (see Section
3.5.4.4.2).494

The inner border of the eyelid margin plays a crucial role in helping maintain ocular surface
integrity by ensuring even spread of the thin tear film with each blink. Normally, the eyelid
margin features a convex inner border with a keratinized epidermis, which ends abruptly
behind the posterior margin of the meibomian orifices. This is followed by the
mucocutaneous junction, creating a transition zone between the wet, non-keratinized
conjunctival tissue of the ocular surface and the dry, keratinized tissue of the eyelid margin
517
. This area, known as the lid wiper, comes into contact with the ocular surface to distribute
the tear film and any morphological changes at this site may be associated with tear film
instability and early signs of DED 266, 518. The lid wiper extends from the mucocutaneous
junction to the sub tarsal fold sagitally and from the medial punctum to the lateral canthus
horizontally. Posterior migration of the mucocutaneous junction leads to lid-margin
keratinization 519. Mechanical factors between the eyelid and the ocular surface, contribute to
diseases perceived to be friction-related (in the form of sheer forces), including superior
limbic keratoconjunctivitis, lid wiper epitheliopathy and conjunctivochalasis 520. Damage to
the epithelium of the marginal conjunctiva at the lid wiper zone is a clinical sign indicative of
DED 271 and its staining and grading is covered in Section 3.5.4.3.

Factors such as aging, inflammation, hormonal imbalance, bacterial growth, eye drops, and
oral medications can induce hyperkeratinization of the meibomian gland ductal epithelium,
altering meibum transparency and viscosity 494. These changes are hypothesized to lead to
gland obstruction and reduced secretion.
Keratinization of the lid margin can also result from long-term rigid contact lens use or
severe systemic conditions such as Stevens-Johnson syndrome/toxic epidermal necrolysis.
These conditions lead to loss of the mucocutaneous junction barrier, epidermalization,
whitish keratin deposits over the lid wiper zone, dyskeratosis, T-cell and neutrophil infiltration
and altered local microbiome.(Muntz et al., 2020, Singh et al., 2021)521, 522. Keratinization can
also occur with androgen insensitivity 523 and androgen deficiency 47, 123, Staphylococcus
aureus over-colonization 524, estradiol increases of cornulin 525, stearoyl-CoA desaturase-1
deficiency and related upregulation of ceramides due to altered fatty acid metabolism 526,
hyperlipidemia 527 and isotretinoin 528.

Figure 7: A) blocked meibomian glands; B) ‘toothpaste’ like meibum; C) upper

lid meibography of an eye with meibomian gland truncation.

3.5.3.2.3 Ocular rosacea

Acne rosacea has long been recognized as an inflammatory disease resulting from a
complex interaction of abnormalities of the innate and adaptive immune system,

39
accompanied by mast cell dysfunction and / or neurovascular compromise. However, the
exact mechanisms and roles of these different components of the pathophysiology remain
incompletely elucidated 529. Acne rosacea is currently diagnosed 530 based on the presence
of at least one ‘diagnostic phenotype’ (centro-facial erythema with periodic intensification or
phymatous changes) or at least two ‘ma or phenotypes’ (papules and pustules, flushing,
telangiectasia, or ocular rosacea); however, ocular rosacea is not well defined, with a list of
features proposed from blepharitis and conjunctival injection as indicative of mild to
moderate disease, to punctate keratitis, infiltrates, vascularization and scleritis indicating
moderate to severe disease 530. A recent review identified ten typical ocular signs and nine
diagnostic steps for recognising ocular rosacea, but many of these overlap with other
posterior blepharitic conditions, with the main differentiating features being concurrent signs
of rosacea on the skin, recurrent hordeola/chalazia, corneal vascularization, corneal
infiltrates/ulcers and anterior uveitis 531.

3.5.4 Ocular Surface Abnormalities

3.5.4.1 Anatomical misalignment
Ultraviolet radiation and other chronic environmental exposures can cause changes in the
corneal and conjunctival cells, leading to disruption of the smooth ocular surface, for
example pterygia,532, 533 pinguecula,534. LIPCOF (see section 3.1.4.6) 392, 535 and
conjunctivochalasis 535. Any such raised structures can affect the flow of the tear film, the
position/function of the glands and the conformity between the eyelids and the ocular
surface, reducing tear film stability and altering tear volume distribution 536.

LIPCOF are undulations in the inferior bulbar conjunctiva, parallel to the margin of the lower
lid. LIPCOF may be observed as the initial signs of conjunctivochalasis (possibly having the
same aetiology),537 but exhibit a smaller cross-sectional area 392, 538, do not occur centrally
539-541
and have no apparent relationship with age.539 LIPCOF have a moderately high
predictive ability for differentiating symptomatic eyes with poor tear stability 540, 542, 543, but not
in all studies 544. Independent groups have shown that OCT can clearly resolve LIPCOF
morphology, such as LIPCOF area, that correlates well with subjective grading 545, 546.
While other signs are associated with DED, such as ocular/conjunctival redness, epithelial
thickness 547, corneal nerve damage, inflammatory cell migration into the cornea, a loss of
corneal sensitivity, changes to the meibomian glands (morphology and expressibility) and
blinking, there is not strong evidence for the role of each in the overall diagnosis of DED.
These other tests are covered with respect to their role in determining the etiological drivers
of DED (section 3.5).

3.5.4.2 Neural dysfunction

Corneal neuropathic pain is complex with a pragmatic and systemised approach needed for
management 548. Abnormal corneal sensitivity has been associated with signs and
symptoms in individuals with ocular surface disease 549, 550. The corneal nerves serve both
sensing and nutritional functions. The sensing function is not only linked to the blink reflex,
but also to tear secretion by the lacrimal gland. Corneal sensitivity is a measure of corneal
nerve function and an indicator of the integrity of the protective mechanisms of the ocular
surface 551 . Morphological and anatomical features can be directly observed by in vivo
confocal imaging and objectively graded using software 552 553 , whereas function is
assessed with the aid of a contact or non-contact corneal esthesiometer to assess corneal
sensitivity. Lid margin sensitivity determined by non-contact esthesiometry has been
demonstrated to be strongly correlated to corneal sensitivity; 550 lid margin sensitivity
thresholds exhibited marginally higher predictive performance than corneal sensitivity for
clinical signs of DED, as defined by the TFOS DEWS II criteria, and were significantly
correlated with non-invasive tear film breakup time, corneal, conjunctival and lid wiper
staining.550 Liquid jet esthesiometers have also been developed and corneal sensitivity to
cold found to be related to digital eye strain.554 The corneal sub-basal nerves may be
evaluated in detail using non-invasive in vivo confocal microscopy 555 . Corneal sub-basal

40
nerve plexus density and length tend to decrease and the tortuosity increase, whereas the
loss of corneal endothelium is accelerated in patients with DED, indicating damaged nerve
fibres (Figure 8)556-558. Moreover, the damage to the nerves in DED may prevent the nervous
system from exerting its immunomodulatory role, leading to changes in corneal sensitivity
559
. Although many studies have shown that the number and density of the sub-basal nerves
in patients with DED decreases significantly and strongly correlates with the decrease in
corneal sensitivity 560-563, some studies show no relationship, perhaps due to variations in
DED subtypes and severity of disease being examined 564. Some studies have also found
that different clinical presentations of DED show corresponding corneal sensitivity
changes.565

Figure 8: Corneal nerves observed by in vivo confocal microscopy (IVCM).

(A) central corneal and (B) inferior whorl nerves in a health eye.
(C) central corneal and (D) inferior whorl nerves in an eye with dry eye disease.

Severe neuropathic corneal pain, as an abnormality of corneal sensitivity with extreme

effects, has attracted significant attention from researchers aiming to more fully understand
its underlying nerve abnormalities (see TFOS DEWS III Digest pain and sensation section).4
The decrease in corneal nerve density in neuropathic corneal pain is consistent with other
types of DED. However, the relationship between microneuromas and nerve beading (Figure
9) with corneal nerve pain is still unclear, and further studies are required to confirm 566, 567.

41
 A B

Figure 9： (A) microneuromas in dry eye. (B) nerve beading in dry eye

Study Cohort Design Results

Kaltenieceet al. N=35 with cross-sectional  high intra and inter-observer
2017 555 obesity reproducibility for all corneal
nerve parameters
 6 images sufficient for analysis
Tepelus et al. N=24 DED, cross-sectional  density of nerve fibres
2017 557 N=44 Sjögren decreased, primarily in Sjögren
N=10 control disease
 nerve tortuosity and reflectivity
decrease in all DED
 density of dendritic cells higher
in DED groups
 nerve and cell changes
correlated with symptomology
Kheirkhah et al. N=20 DED retrospective  endothelial cell density and
2017 558 N=13 control longitudinal nerves lower in DED
 loss is greater than literature
reported normal ageing
Levy et al. 2017 N=30 Sjögren prospective, non-  6 months topical cyclosporin A
559
[N=15 control] randomized 0.05% increased corneal sub-
treatment study basal nerve density (only)
 decreased dendritic cell
numbers, more so in those with
more severe baseline symptoms
and staining
 corneal sensitivity increased
Cardigos et al. N=116 with non- cross-sectional  corneal sub-basal nerve plexus
2019 556 Sjögren DED density and length lower, and
N=20 control tortuosity higher in DED
 corneal sub-basal nerve plexus
strongly associated with
Schirmer test score and tear
breakup time
Ross et al. 2020 N=14 with severe cross-sectional  sub-basal nerve density reduced

42
566
ocular pain compared with controls
>1year, 4 with  more activated keratocytes and
neuropathic pain spindle, lateral and stump
microneuromas in stroma in
those with neuropathic pain
Moein et al. N=30 DED retrospective,  similar lower nerve density and
2020 567 N=25 case-controlled higher dendritic cell numbers in
neuropathic pain DED and neuropathic groups
N=16 controls  no difference in nerve beading
 microneuromas present only in
neuropathic pain group
Maity et al. N=28 Sjögren Cross-sectional  similar dendritic cell density
2024 568 disease  nerve fibre length, density and
N=25 meibomian branching lower with Sjögren
gland disease
dysfunction  tear osmolarity weakly
negatively correlated with
corneal nerve parameters
Table 3: Confocal studies of corneal nerve abnormalities in DED.

Systemic diseases that are associated with DED can cause corneal hypoesthesia such as in
patients with diabetes 569-571. In addition to nerve reduction, a significant reduction in nerve
beading frequency has also been reported, possibly due to reduced metabolomic activity in
patients with diabetes 572-574 . Corneal nerve changes have also been observed in patients
with Graves’ ophthalmopathy (often associated with DED), perhaps due to nerve
degeneration 575 . However, there appears to be no research on corneal sensitivity related to
Graves’ ophthalmopathy, and its correlation with structural alterations in nerves.

3.5.4.3 Ocular surface cellular damage / disruption

Punctate staining of the cornea and bulbar conjunctiva observed following the application of
dyes, such as sodium fluorescein (Figure 10), rose bengal, and lissamine green, is a key
diagnostic marker of numerous anterior segment conditions, including DED 238. The
distribution of punctate staining may provide an indication of potential aetiology 238, 576, and
DED is traditionally thought to be predominantly associated with interpalpebral or inferior
staining 238. In recent years, there has been growing interest in the utility of eyelid marginal
conjunctival staining or lid wiper epitheliopathy in the diagnosis of DED 1, 577.

To facilitate standardised recording of the severity of ocular surface staining and lid wiper
epitheliopathy (Section 3.5.4.3), several grading systems have been devised, and the most
commonly used are summarised in Table 4 578. Corneal and conjunctival staining grading
systems include the van Bijsterveld system 579, the National Eye Institute (NEI) Industry
Workshop guidelines 19, the Collaborative Longitudinal Evaluation of Keratoconus (CLEK)
schema 580, the Oxford scheme 581, the Lexitas grading system 582, the area–density
combination index 583, and the Sjögren's International Collaborative Clinical Alliance (SICCA)
ocular staining score 584. A recent study modified the Oxford scheme to incorporate half-unit
increments for the assessment of corneal staining and reported improved sensitivity and
repeatability 275. The corneal and conjunctival staining component of the global consensus
TFOS DEWS II diagnostic criteria was based on the SICCA grading system 1. Lid wiper
epitheliopathy is most commonly assessed relative to Korb’s grading system, which
combines the horizontal length of staining in mm, as well as the sagittal width relative to the
eyelid margin 266. The eyelid margin staining component of the global consensus TFOS
DEWS II diagnostic criteria was based on this grading system.1

43
Key diagnostic accuracy studies evaluating the discriminative performance of ocular surface
staining and lid wiper epitheliopathy in the detection of DED are summarised in Table 5.
Overall, lid wiper epitheliopathy (C-statistic range, 0.69 to 0.80) demonstrated superior
discriminative ability relative to corneal (C-statistic range, 0.52 to 0.57) and conjunctival
staining (C-statistic range, 0.51 to 0.63) 222, 248, 250, 540, 585, which would support the
incorporation of all three staining parameters in the routine diagnostic workup of DED 1. The
reasons underlying the greater diagnostic performance of lid wiper epitheliopathy are not
completely understood. Previous studies have reported that corneal and conjunctival
staining more commonly present in patients with severe DED 248, 250 and demonstrate poorer
correlation with other dry eye signs and symptoms in mild-to-moderate disease 248, 250.
Moreover, a number of epidemiological and diagnostic studies have suggested that lid wiper
epitheliopathy might be an earlier clinical sign than corneal and conjunctival staining in the
natural history of DED 249, 586 and it remains uncertain whether the greater exposure to
shearing and viscosity-induced hydrodynamic forces during the blink cycle might predispose
the lid wiper region to earlier damage 537.

Table 4: Commonly used grading systems for ocular surface staining and lid wiper
epitheliopathy.

Grading system Details

van Bijsterveld Corneal staining scoring:

staining score 579 1: sparsely scattered spots
2: densely scattered spots
3: confluent spots

Conjunctival staining scoring:

Divided into nasal and temporal zones.
1: few separated spots
2: many separated spots
3: confluent spots

NEI staining score 19 Corneal staining scoring:

Divided into five sectors (central, superior, inferior, nasal, and
temporal), each scored from 0 to 3.

Conjunctival staining scoring:

Divided into superior paralimbal, inferior paralimbal, and
peripheral area, both nasally and temporally, each scored 0 to
3.

CLEK staining score Cornea staining scoring:

580
Divided into five sectors (central, superior, inferior, nasal, and
temporal), each scored 0 to 4 in 0.5 increments.

Conjunctival scoring:
Divided into four sectors (superior, inferior, nasal, and
temporal), each scored 0 to 4 in 0.5 increments.

Intra-class correlation coefficient 587:

Fluorescein: 0.76
Rose bengal: 0.40

Oxford staining score Corneal staining scoring:

44
581
Whole corneal area scored from 0 to 5 dependent on the
intensity of punctate staining displayed pictorially, with the
intensity of dots increasing on a logarithmic scale between
grades.

Conjunctival staining scoring:

Whole conjunctival area scored from 0 to 5 dependent on the
intensity of punctate staining displayed pictorially, with the
intensity of dots increasing on a logarithmic scale between
grades.

Korb grade 266 Lid wiper epitheliopathy horizontal length grading:

0: <2 mm
1: 2 to 4 mm
2: 5 to 9 mm
3: ≥10mm

Lid wiper epitheliopathy sagittal width grading:

0: <25% of the lid wiper
1: 25% to <50% of the lid wiper
2: 50% to <75% of the lid wiper
3: ≥75% of the lid wiper

Area–density Corneal staining area scoring:

combination index 583 A0: no punctate staining
A1: >⅓
A2: ⅓ to ⅔
A3: >⅔

Corneal staining density scoring:

D0: no punctate staining
D1: sparse
D2: moderate
D3: high with lesion overlap

SICCA staining score Corneal fluorescein staining scoring:

584
0: 0 dots
1: 1 to 5 dots
2: 6 to 30 dots
3: >30 dots
Extra points for confluent patches, staining within the pupil or
filaments

Conjunctival lissamine green staining scoring:

Divided into nasal and temporal zones
0: 0 to 9 dots
1: 10 to 32 dots
2: 33 to 100 dots
3: >100 dots

Intra-class correlation coefficient: 0.90 to 0.91 588

Table 5: Key diagnostic accuracy studies assessing the discriminatory performance of
ocular surface staining and lid wiper epitheliopathy in detecting dry eye disease.

45
Study Methods Outcomes

Lemp et Sample size: 314 Corneal staining:

al. 2011 C-statistic: 0.77
222
Index tests: Sensitivity: 54%
Corneal staining (NEI score) Specificity: 89%
Conjunctival staining (NEI score)
Conjunctival staining:
Reference standard: C-statistic: 0.88
Composite disease severity index, derived Sensitivity: 60%
from the TFOS DEWS severity scale (clinical Specificity: 91%
signs only).

Incorporation bias:
Ocular surface staining formed part of both
index testing and reference standard.

Pult et Sample size: 47 Corneal staining:

al. 540 C-statistic: 0.52
Index tests: Sensitivity: not reported
Corneal staining with fluorescein (CCLRU Specificity: not reported
scale)
Conjunctival staining with lissamine green Conjunctival staining:
(CCLRU scale) C-statistic: 0.51
Lid wiper epitheliopathy with lissamine green Sensitivity: not reported
(Korb grading) Specificity: not reported

Reference standard: Lid wiper epitheliopathy:

OSDI score (clinical symptoms only). C-statistic: 0.75
Sensitivity: 48%
Incorporation bias: Specificity: 96%
None.

Wang Sample size: 552 Corneal fluorescein

et al. staining:
2019 248 Index tests: C-statistic: 0.56
Corneal fluorescein staining (Oxford score) Sensitivity: 25%
Conjunctival lissamine green staining (Oxford Specificity: 86%
score)
Superior lid wiper epitheliopathy (lissamine Conjunctival lissamine
green) (Korb grading) green staining:
Inferior lid wiper epitheliopathy (lissamine C-statistic: 0.52
green) (Korb grading) Sensitivity: 11%
Specificity: 94%
Reference standard:
TFOS DEWS II criteria (excluding staining Superior lid wiper
parameters) epitheliopathy:
C-statistic: 0.71
Incorporation bias: Sensitivity: 65%
None. Specificity: 73%

46
 Inferior lid wiper
epitheliopathy:
C-statistic: 0.69
Sensitivity: 72%
Specificity: 66%

Wang Sample size: 2,066 Corneal fluorescein

et al. staining:
2024 250 Index texts: C-statistic: 0.57
Corneal fluorescein staining (SICCA score) Sensitivity: 38%
Conjunctival lissamine green staining (SICCA Specificity: 76%
score)
Superior lissamine green lid wiper Conjunctival lissamine
epitheliopathy (Korb grading) green staining:
Inferior lissamine green lid wiper C-statistic: 0.63
epitheliopathy (Korb grading) Sensitivity: 58%
Specificity: 64%
Reference standard:
TFOS DEWS II criteria (excluding staining Superior lid wiper
parameters) epitheliopathy:
C-statistic: 0.72
Incorporation bias: Sensitivity: 72%
None. Specificity: 66%

Inferior lid wiper

epitheliopathy:
C-statistic: 0.71
Sensitivity: 77%
Specificity: 60%

Yeniad Sample size: 86 Fluorescein lid wiper

et al. epitheliopathy:
2010 585 Index texts: C-statistic: 0.80
Fluorescein lid wiper epitheliopathy (Korb Sensitivity: 44%
grading) Specificity: 93%
Rose bengal lid wiper epitheliopathy (Korb
grading) Rose bengal lid wiper
Lissamine green lid wiper epitheliopathy epitheliopathy:
(Korb grading) C-statistic: 0.78
Sensitivity: 43%
Reference standard: Specificity: 90%
OSDI score (clinical symptoms only).
Lissamine green lid
Incorporation bias: wiper epitheliopathy:
None. C-statistic: 0.76
Sensitivity: 39%
Specificity: 90%

47
Figure 10: A) Fluorescein corneal staining illuminated with a blue light; B) Lissamine
green bulbar conjunctival staining under white light; C) Lissamine green lower lid wiper
staining under white light
3.5.4.4 Primary inflammation / oxidative stress
Inflammation plays an etiological role in the pathophysiology of DED 149. Assessing the
presence and intensity of inflammation is essential to determine the severity of the disease
589
, the risk of progression 590 and to inform its management. Inflammation at the ocular
surface can be both a cause and a consequence of DED 591. Ocular surface inflammation
can occur due to ocular surface damage 592, however autoimmune diseases are intrinsically
significant contributors to DED 593. In systemic immune-mediated conditions such as S gren
disease, lymphocyte infiltration in the lacrimal gland can result in damage and fibrosis 594, 595
, resulting in reduced tear secretion and elevated inflammatory cytokine levels in tears 596.
Therefore, the assessment of inflammation in DED frequently includes both local and
systemic investigations to help elucidate the source of inflammation. As an example, it has
been reported that MMP-9–positive patients respond more favorably to topical cyclosporine
than MMP-9–negative patients 597, and the use of topical anti-inflammatory therapy has been
associated with a reduction in HLA-DR 598 and MMP-9 tear levels 599, 600. Treatments
targeting novel inflammatory pathways in DED are continuously being explored and
developed 601, 602, although many to date have failed. Current diagnostic inflammatory tests
have some limitations 603, 604. While image-based comparative scales and noncomparative
methods based purely on clinical observation remain valid assessment tools for use in the
clinic, objective conjunctival redness quantification is substantially more sensitive and
reliable than subjective grading 605, 606 and can be performed using a smartphone.607
Moreover, enhancing the user-friendliness of confocal microscopy by developing a non-
contact imaging technique with adequate resolution or providing a wider field of imaging
would probably facilitate its adoption. In terms of molecular-based diagnostic tests, the lack
of standardized methods for tear fluid collection and biomarker quantification 608, 609, as well
as the absence of normal reference values 610, are key aspects that may limit the accuracy,
reproducibility and overall implementation of these techniques. The cost and technical
complexity of some of these diagnostic tests may also be an important limitation to their
implementation. However, improving clinical outcomes, avoiding unnecessary therapies, and
accelerating patients’ recovery not only stands to benefit patients but also to save costs.

3.5.4.4.1 Imaging-based diagnostic tests

3.5.4.4.1.1 Ocular conjunctival redness
Conjunctival ocular hyperemia occurs with dilation of the microvasculature arising from a
multitude of etiologies 606, 611, 612. The vasodilatation of conjunctival microvessels plays a
critical role in the efferent component of the immune system, providing both soluble
mediators and cellular elements to the site of inflammation 613. Accurate assessment of the
underlying causes of conjunctival hyperemia is key in differentiating systemic causes from a
localized inflammatory response 614. Both descriptive 615 and reference image-based 616-618
subjective grading scales can facilitate the detection and monitoring of changes in the
conjunctival microvasculature during follow-up, supporting decision-making in modifying the
treatment plan. Multiple studies have shown that these scales have limited inter- and intra-
observer repeatability 605, 619, which prompted the development of computer-based

48
photograph-analysis techniques to allow objective grading of conjunctival redness allowing
higher precision and repeatability 605, 606, 620-627.
3.5.4.4.1.2 In vivo confocal imaging
In vivo confocal imaging can aid in identifying characteristic structural changes in the cornea,
conjunctiva, meibomian glands and lacrimal gland in patients with DED 330, 628. Conflicting
findings in corneal and conjunctival epithelial cell changes in DED studies may result from
DED disrupting cell renewal, but simultaneously promoting cell repair at the same time,
affecting the apoptosis-proliferation balance 629.
Previous studies have reported characteristic changes in the corneal stroma of patients with
DED such as a significant increase in anterior corneal stromal keratocyte density 630 and
abnormal stromal hyper-reflectivity indicating increased activity 631. An increase in dendritic
cell density has been reported in patients with DED 630, 632, 633. Mature and immature dendritic
cells have been found in the corneal stroma of these patients 634. Interestingly, increased
dendritic cell density has been correlated with severe symptoms 557 as well as with aqueous
deficient DED due to immune disease 635. Dendritic cell and activated keratocyte density, as
well as reduced corneal sub-basal nerve fibre length have shown an indirect association with
inflammation on the ocular surface, through a significant reduction following treatment with
topical corticosteroids 636, 637. Similarly, long-term therapy with topical cyclosporine for more
than 6 months has shown a positive impact on corneal epithelial, stromal, dendritic, and
nerve confocal imaging parameters 559, 638 (see TFOS DEWS III Digest Pathophysiology
section).4

3.5.4.4.2 Tear Biomarker diagnostic tests

3.5.4.4.2.1 Matrix metalloproteinases
Matrix metalloproteinases (MMPs) are a family of enzymes that are core to several ocular
and systemic inflammatory processes 639, 640. MMPs are generated by connective tissues
and pro-inflammatory cells 641, and can be detected in tears of patients with DED 642, 643. In
DED, corneal epithelial damage can result in a local inflammatory reaction that leads to
increased secretion of MMPs 591. MMP-1, -3, -9, -10, and -13 are the MMPs most notably
elevated at the corneal surface, splitting epithelial basement membrane components and
tight junction proteins (such as ZO-1 and occludin) that maintain corneal epithelial barrier
function 643, 644. Studies have reported a significant correlation between MMP-9 degree of
positivity and ocular surface fluorescein staining 228, 597. However, tear volume has an impact
on the assay indicator, and therefore, a MMP-9 test degree of positivity may not correlate as
strongly with MMP-9 tear concentration in cases of either low tear volume or reflex tearing 645
3.5.4.4.2.2 Cytokines and chemokines
Measurement of cytokines and chemokines may enable differentiation of ocular surface
inflammation caused through the innate immune response, and adaptive immune response
(see TFOS DEWS III Digest Pathophysiology section).4 Further, within the adaptive immune
response, these markers may separate into Th1, Th2, Th17, and Treg-mediated responses
646
. IFN-γ, the dominant cytokine associated with the Th1 response, has been associated
with goblet cell loss and squamous epithelial hyperplasia. Consequently, some clinical
studies demonstrated correlation between higher IFN-γ and tear deficiency, though other
studies found contradictory results 647-649, possibly due to inherent differences in assay
methods, or population characteristics. IFN-γ may be specifically associated with an
increase in osmolarity 650. Th17-mediated ocular inflammation may be induced through IL-17
signaling. IL-17 activates MMP-9 which contributes to damage to the corneal epithelial
barrier 646, 651. A significant correlation between both corneal and conjunctival staining scores
and presence of Th cells has been reported, although Th subtypes such as Th1 and Th17,
were not detectable at high enough levels for establishing correlations with tear film stability
and volume in patients with DED 652; in addition, no correlation was found between DED and
the detection of IL-1β, Il-6, IL-8, IL-10, IL-17A, IFNγ, and tumor necrosis factor alpha in tears
653
, although cytokine upregulation has been detected in patients with Sjögren disease 654.
However higher MMP levels are found in patients with DED 655, likely from episodic flares 656
and MMPs have been found to correlate with osmolarity and tear volume, more strongly than

49
with tear stability and symptoms 657. The variable nature of cytokine levels and DED
corresponds with the heterogeneous nature of ocular surface inflammation 658-660, changes
over time 661 and the location of sampling 662. This confirms that inflammation is more often
downstream (a consequence) rather than intrinsic (a driver) in DED (see TFOS DEWS III
Digest Pathophysiology section).4
3.5.4.4.2.3 Neurotrophic Factors and Neuropeptides
Neuropeptides and neurotrophic factors have a role in mediating sensory information and in
regulating aspects of neuronal function and cell survival. 649.
Serotonin, which is a peripheral nerve sensitizer, is found at a higher concentration in tears
of patients with DED than in those of normal eyes and correlates with symptoms 663.
Serotonin is activated by inflammation and sensitizes peripheral nerves, perhaps playing a
role in the development of corneal hypersensitization in DED 664. Increased nerve growth
factor has a protective role in DED, improving the integrity of the epithelial cell layer and tear
secretion 665, 666. Lacrimal gland dysfunction has been associated with decreased calcitonin
gene-related peptide 665, 666. Substance P has been found to be raised in tears of DED
patients after refractive surgery 667 and nerve growth factor has also been found to be raised
in tear fluid of patients with neuropathic pain 668. Hence these neurotrophic factors and
neuropeptides appear to regulate tear aqueous production such that deficiencies in an
individual with DED may indicate a strategy for improving tear secretion.
3.5.4.4.2.4 Ocular surface immune markers
Major histocompatibility complex based markers have long been identified as a risk factor for
DED, particularly with Sjögren disease 669, 670. Antigen presentation through the major
histocompatibility process seems to play an intermediary role in T-cell activation and the
cytokine-based inflammatory cascade 646. While both major histocompatibility complex class
I and class II have been connected to inflammation in DED, HLA-DR, a member of the class
II family, has been more thoroughly investigated 671, 672. A gene expression analysis of
mRNA transcripts observed from conjunctival impression cytology sampling discovered a
correlation between HLA-DR, CD40, and IFN. This connection further links inflammatory
DED with T cell activation 671. Conjunctival impression cytology samples collected on a
heterogenous group of patients with DED revealed that the percentage total cells expressing
HLA-DR was positively correlated with conjunctival and corneal staining scores 672 and
weakly with tear volume 673; although HLA-DR percentage analysis was not a sensitive
diagnostic marker for DED in itself, it may represent a means of helping identifying specific
dry eye subtypes based on the lymphocytic response responsible for the ocular surface
inflammation in a particular patient, and guiding therapeutic decisions. Neutrophils,
macrophages, mast cells, T-cells and dendritic cells have been found to increase in DED
across several studies, particularly in more severe levels of dry eye found in autoimmune
disease such as Sjögren disease and graft versus-host disease. 674,
3.5.4.4.2.5 Inflammasome markers
The inflammasomes are innate immune system sensors that induce an inflammatory form of
cell death, known as pyroptosis, in response to harmful stimuli such as pathogens or
oxidative stress, among others 675, 676 (see TFOS DEWS III Digest Pathophysiology section).4
Reactive oxygen species are involved in the pathogenesis of DED 677, and have been
suggested as a priming signal for inflammasome activation 602. NOD-like receptor protein-3
(NLRP3) inflammasome, a key driver in the innate immune system, has a role in DED
pathogenesis 677, 678, is upregulated in the tear film of people with Sjögren disease 678 and is
activated by hyperosmolarity 677, 679. Tear levels of caspase-1, a molecule involved in the
inflammasome cascade, and various clinical signs of ocular surface damage in patients with
DED and patients using topical glaucoma medications have been found to be correlated 680.
Moreover, tear levels of Gasdermin-D, a pyroptosis executor, are also elevated in patients
with DED 681
3.5.4.4.2.6 MicroRNAs
MicroRNAs are non-coding RNAs that serve as significant regulators in a variety of
molecular pathways 682 (see TFOS DEWS III Digest pain and sensation section).4 Several

50
studies have identified tear microRNAs as potential biomarkers for ocular diseases, including
Sjögren disease 683 and DED 684-686, among others 608, 687. Nine tear microRNAs (miR-127-5p,
miR-1273h-3p, miR-1288-5p, miR-130b-5p, miR-139-3p, miR-1910-5p, miR-203b-5p, miR-
22-5p, and miR-4632-3p) associated with inflammation have been found to be upregulated
in the tears of patients with DED.
3.5.4.4.2.7 Oxidative stress markers
Oxidative stress, an imbalance of free radicals and antioxidants that leads to cell damage,
may play a role in the pathogenesis of DED 688. Proteomic analysis of tears from patients
with DED shows an upregulation of proteins associated with oxidative stress injury 689. It is
well established that oxidative damage triggers an inflammatory response, resulting in ocular
surface dysfunction 690-692. Moreover, oxidative stress may cause the progression of DED by
exacerbating inflammation by triggering the vicious circle of DED. Oxidative stress
biomarkers, which indicate the degree of oxidative stress, have been found elevated in the
tears and conjunctiva of patients with DED 693. The detection of oxidative stress biomarkers
through tear film or conjunctival impression cytology samples may be undertaken to evaluate
DED status, monitor the efficacy of drugs or evaluate disease progression. Oxidative
markers such as lactoferrin (tears), peroxiredoxin 2 (tears), SOD (tears), CAT (tears), and
GSH-Px (tears) are downregulated in DED 689, 694. In contrast, markers such as S100A8
(tears), S100A9 (tears), reactive oxygen species (conjunctiva), LPO (conjunctiva), 4-HNE
(conjunctiva), MDA (conjunctiva) and HEL (tears) are upregulated in these patients 692, 695-698.
A correlation between ocular surface oxidative stress markers and topical treatments has
been described by several authors 696, 699, 700, indicating the utility of these markers in
monitoring response to anti-oxidant therapies.
3.5.4.4.2.8 Serum markers
DED is associated with chronic inflammatory systemic conditions including, collagen
vascular diseases 701, rheumatoid arthritis 702, or S gren disease 703, among others 704.
Acute phase reactants such as erythrocyte sedimentation rate and C-reactive protein
indicate active systemic inflammation 705. However, previous studies found that levels of
these reactants do not correlate with ocular surface symptoms or tear parameters in DED 701,
706
, although serum inflammatory markers PM-Scl100 and Sm were associated with more
severe DED symptoms, while inflammatory markers U2SnRNP A', Ro52, La, DNA, and
Ro60 were associated with more severe ocular surface disease signs 707, 708. Other serum
inflammatory-related markers such as antinuclear antibody and IL-2 receptor(sIL-2R), or
anti-double-strand DNA antibody, have been associated with DED in primary S gren
disease 709, 710 or systemic lupus erythematosus 701. Similarly, serum levels of IL-17, a
proinflammatory cytokine 711, are significantly increased in patients with DED and high
fluorescein staining scores 712. S100A8/A9 and granulysin have been found to be higher in
more severe Stevens-Johnson Syndrome 713. In addition, in a large population-based study,
decreased serum androgens were found to be highly associated with DED diagnosis and
symptoms 714.

51
Table 6: Subclassification of DED etiological drivers recommended tests and cut-offs [where available]
Standard testing Advanced testing
Interferometry – grade ≤ 3 (non-amorphous or colored
197, 320, 343
pattern) or <72nm on LipiView
Lipid
Meibum expressibility/quality – meibum not clear or limited
332, 371, 715
expressibility .
39, 320, 380 404-406
Tear Film Meniscometry - tear meniscus height ≤0.20mm . Strip meniscometry - 2.5mm wetting length .
Aqueous
Deficiencies Tear proteins and other chemical components testing
Rose bengal or lissamine green staining - >9 punctate Immunohistochemistry and immunoelectron microscopy of
581
spots tear film
Mucin / glycocalyx
Impression cytology – goblet cell density and epithelial cell
morphology
463
Partial blinking observation - >40% occurrence
Blink / lid closure
Lagophthalmos / inadequate lid seal - observed
Anterior blepharitis observation
320,
MGD Meibography – gland length <75%
372, 509
Eyelid
715
Anomalies Gland plugging - observed
Lid margin 715
Telangiectasia - observed
Gland expressibility
Keratinization Slit-lamp biomicroscopy
Ocular rosacea Slit-lamp biomicroscopy
Anatomical misalignment Slit-lamp biomicroscopy Corneal topography
Puff or physical sensation - corneal and lid margin In vivo confocal microscopy – normative values available
550 717
Neural dysfunction sensitivity thresholds ≥0.8 mbar although instruments for nerve length, branch and density metrics
716
are not comparable
Ocular 250, 581
Corneal fluorescein staining - >5 punctate spots
Surface
Ocular surface cellular Conjunctival lissamine green staining - >9 punctate spots
Abnormalities 250, 581
damage / disruption
250
Lid wiper staining - >2mm length and 25% width
Primary inflammation / Bulbar conjunctival hyperaemia – >1.5 Efron scale or In vivo confocal microscopy
604
oxidative stress >0.95 objective JENVIS Tear film and ocular surface molecular testing
Systemic Check for systemic conditions
Drivers

52
3.5.5 Systemic diseases leading to dry eye
Many systemic diseases significantly contribute to DED through inflammation, autoimmunity,
metabolic dysregulation and ocular surface exposure. Interdisciplinary collaboration in the
management of DED patients with underlying systemic conditions is important, such as
coordinating care with rheumatologists, endocrinologists, or other relevant specialists to
optimize both ocular and systemic outcomes.

3.5.5.1 Autoimmune conditions

Systemic inflammatory diseases such as rheumatoid arthritis, systemic lupus
erythematosus, Stevens-Johnson syndrome and Sjögren disease are closely associated
with DED. Chronic inflammation leads to the infiltration of immune cells in the lacrimal gland,
reducing tear production and altering tear film composition 718. Key inflammatory cytokines
include IL-1, tumor necrosis factor-alpha and MMPs 719. Patients with ankylosing spondylitis
treated with tumor necrosis factor inhibitors showed improvement in clinical and laboratory
disease parameters, tear production, DED severity, and impression cytology scores,
suggesting tumor necrosis factor inhibitors may restore lacrimal gland acinar cells affected
by proinflammatory cytokines 720. In rheumatoid arthritis, the activation of the NF-κB pathway
and overexpression of tumor necrosis factor alpha and IL-6 lead to systemic and ocular
inflammation 721. The full potential of tumor necrosis factor alpha inhibitors to reduce ocular
surface inflammation and improve tear production in rheumatoid arthritis remains uncertain
722, 723
.

Autoimmune diseases, particularly Sjögren disease, are closely associated with DED.
Sjögren disease involves lymphocytic infiltration of exocrine glands, affecting the production
of aqueous tears and saliva, respectively, and leading to dry eye and dry mouth. Detection of
antibodies, such as anti-Ro/SSA and anti-La/SSB, support its autoimmune nature 724. Gene
analysis of conjunctiva imprints revealed 53 differentially expressed genes in Sjögren
disease compared to healthy controls, that indicated immune activation in patients with
Sjögren disease 725. Higher percentages of antigen-presenting cells and mature dendritic
cells in the conjunctiva are associated with more severe keratoconjunctivitis sicca in Sjögren
disease, which may contribute to goblet cell loss.726
.
Multidisciplinary treatment for both the underlying systemic disease as well as managing the
residual ocular symptoms of DED is generally appropriate. In rheumatoid arthritis, for
example, disease-modifying antirheumatic drugs such as methotrexate and biologics like
tumor necrosis factor inhibitors help control systemic inflammation and improve ocular
symptoms.721

3.5.5.2 Hormonal imbalance

Hormonal changes can trigger DED by affecting tear production and quality, especially in
women, who experience hormonal fluctuations throughout their lives 727. Hormonal
imbalance and its management in patients with DED is covered in the TFOS DEWS III
Digest.4

3.5.5.3 Metabolic disease

Metabolic diseases such as diabetes mellitus significantly impact the ocular surface and can
lead to DED 4, 7. Hyperglycemia and advanced glycation end-products contribute to
microvascular damage and neuropathy, affecting the lacrimal glands and corneal nerves 728.
Patients with diabetes often exhibit reduced tear secretion, increased tear osmolarity, and
altered corneal sensitivity 729. Antioxidant therapy, along with strict glycemic control, can
mitigate the effects of diabetes on the body 730 and the ocular surface 731, 732. The impact of
medications and procedures related to managing metabolic disease on the ocular surface
are covered in the TFOS Lifestyle reports.7, 9

53
3.5.5.4 Exposure
A multitude of diseases as well as trauma can cause intermittent (for example nocturnal) or
constant lagophthalmos (such as facial nerve palsy), leading to exposure of the ocular
surface and DED 733. A number of these conditions and the impact of medications and
procedures related to ocular exposure on the ocular surface are covered in the TFOS
Lifestyle reports 9, 12.

3.6 Tests for monitoring treatment

In monitoring treatment effects over time, it is important to consider indicators that may lag
(such as corneal staining 249), those that can change more rapidly (such as symptomology)
and the treatment’s mechanism of action 734. It is therefore recommended that practices
adopt a standardised, reproducible and repeatable DED protocol that involves validated
questionnaires, diagnostic tests and clinical exam that remains consistent from visit to visit.
While every real-life patient encounter will undoubtedly not be as clear cut as this
hypothetical example, clinicians are encouraged to make diagnostic and treatment decisions
based on scientific evidence and on tracking subjective and objective data over time, and
with the aid of clinical judgment, experience and acumen as outlined in this report.

As the number of testing options increases, the volume of clinical data that is required to be
processed at each patient visit also increases and can be, at times, overwhelming. This
becomes increasingly challenging as physician time becomes progressively constrained and
face-to-face consultation times are shortened. With handwritten paper records, processing
diagnostic data trends over long periods of time encompassing multiple office visits was
challenging, time consuming and rarely done thoroughly. However, in the current electronic
medical record era, the ability to seamlessly and instantaneously process, summarize, plot
and chart large volumes of disparate diagnostic data over long periods of time is much
easier to facilitate. DED/ocular surface disease-specific electronic medical record platforms
are now available and enhance the ability to track and monitor DED symptoms, objective
tests, exam findings and treatment outcomes 735 as well as real-world registries which also
provide the facility to monitor patient progress and benchmark against peers 736. Additionally,
with the ubiquity of smartphones and the burgeoning emergence of smart glasses and
wearable health monitors/sensors, there is unprecedented potential for gathering and
analyzing real-time continuous data in-between office visits data 292, 737-739. Machine learning
and artificial intelligence (see section 5.1), when integrated into electronic medical record
platforms and smart devices, with access to process reliable data from disparate physicians,
practices and geographic locations, may possibly provide novel insights into DED that are as
yet unknown, facilitating more accurate diagnoses, better treatments and more strategic
clinical trial designs 740. With the assistance of machine learning and artificial intelligence,
historically-challenging diagnoses such as neurotrophic keratopathy and neuropathic corneal
pain will likely be made earlier, with the potential to improve patient outcomes and reduce
late-stage sequelae.

4 Patients with only symptoms or ocular surface signs

There are numerous explanations highlighted in section 3.3.3 for the, often vexing, clinical
scenario of discordant signs and symptoms. Significant corneal staining in a pain-free
patient, colloquially referred to as ‘stain without pain’, may indicate neurotrophic keratopathy.
741
Conversely, when the clinical signs of ocular surface dysfunction are mild or subtle in the
absence of patient-reported symptoms it might indicate an early, preclinical or situational
DED 155. In the extreme, a clinical scenario involving significant symptomology in the
absence of, or out of proportion to, clinical signs, colloquially referred to as ‘pain without
stain’ might be indicative of neuropathic corneal pain 175.

54
4.1 Ocular surface disease in the absence of symptoms
It is recommended to treat any significant ocular surface disease prior to a patient
undergoing any type of eye surgery (such as laser vision correction or cataract removal),
initiating contact lens wear, starting any high risk topical or systemic medications and/or any
other intervention known to cause or exacerbate DED (see TFOS DEWS III Digest
Iatrogenic section).4 The surgical informed consent process should include a discussion of
the risk of worsening signs and/or symptoms of DED, including visual symptoms, which may
require intensive long-term treatment to control. The importance of identifying, offering
education about and treating early preclinical DED/ocular surface disease is particularly
critical in the setting of refractive, cataract or laser vision correction surgery where patient
expectations tend to be particularly high.742

There is a high prevalence of ocular surface disease in presurgical cataract patients, many
of whom have few or no reported symptoms 743, 744. It is speculated that the stark disconnect
between signs and symptoms in this older population is due to a combination of generational
stoicism, inherent bias to focus on the perceived ‘bigger’ problem (for example their poor
vision due to cataract) and/or age-related reductions in corneal nerve density and sensation
152, 153, 165, 167, 171, 745
. In the younger patient population, contact lens intolerance from DED is a
common reason for seeking laser vision correction surgery,746 but can also place the patient
at higher risk of postsurgical complications if not identified, discussed and managed
preoperatively.

Many studies have shown that DED, especially when significant corneal staining is present,
can adversely affect the accuracy of preoperative measurements (such as keratometry,
topography, optical pachymetry and aberrometry) potentially leading to post-surgical
refractive error misses, poor and/or fluctuating visual quality (especially when multifocal or
extended-depth of focus intraocular lenses are implanted) and overall lower patient
satisfaction 28, 747-751. Due to the high prevalence of ocular surface disease in this patient
population, an algorithm has been designed by the American Society of Cataract and
Refractive Society to identify and treat visually significant ocular surface disease
preoperatively 752.

4.1.1 Diagnosing Ocular Neurosensory Abnormalities

A medical history questionnaire or intake form should include often missed non-ophthalmic
risk factors for neurotrophic keratopathy (diabetes, herpes, Parkinson’s, multiple sclerosis,
prior brain surgery such as acoustic neuroma, cerebrovascular accidents and congenital
dysautonomia) and for neuropathic corneal pain (such as small fibre peripheral
neuropathies, fibromyalgia, migraine, irritable bowel, anxiety, depression and post-traumatic
stress disorder) 753. Clinical suspicion for neurosensory abnormalities should be raised in any
patient with marked discordance of signs and symptoms, risk factors and/or positive answers
to targeted triaging questions. Corneal surgical incisions, laser corneal ablation, suction prior
to flap creation by microkeratome or femtosecond laser, phototoxicity from the operating
microscope, benzalkonium chloride-preserved postoperative drops amongst other factors
can exacerbate preexisting or induce new corneal neurosensory abnormalities, potentially
leading to visually significant post-operative ocular surface disease 9, 33, 107, 754-761.

4.1.2 Diagnosing Neurotrophic Keratopathy

Neurotrophic keratopathy is considered a rare disease, but is likely underdiagnosed 762. It is
caused by a unilateral or bilateral abnormality of the trigeminal nerve, resulting in decreased
or absent corneal sensation, and leading to diffuse corneal punctate epithelial defects
(Mackie stage 1), persistent epithelial defects with characteristic smooth rolled edges (stage
2), and stromal melting with the potential for corneal perforation (stage 3) 763, 764. A more
nuanced and detailed 6-stage grading system has recently been proposed by a Neurotrophic
Keratopathy Study Group to better refine the stages of progression and to allow practitioners

55
to identify NK at earlier stages 741. Because early stages of neurotrophic keratopathy can
involve corneal epitheliopathy with staining, increased mucous viscosity and decreased tear
film stability, it is often misdiagnosed and treated as moderate-to-severe DED. The pattern of
corneal staining in neurotrophic keratopathy is often diffuse involving the entire cornea in
contrast to the inferior or interpalpebral staining typically seen in DED 551, 765. Patients with
neurotrophic keratopathy will also typically have lower blink rates and poorer blink quality 762
whereas patients with DED and poor blink quality, tend to blink more frequently 766. Patients
with neurotrophic keratopathy typically don’t self-report symptoms of pain or discomfort,
inconsistent with the level of corneal staining, but they may complain about reduced visual
acuity, quality, stability and performance. This clinical scenario of ‘stain without pain’ should
alert clinicians to suspect the possibility of NK as early as possible, prior to the patient failing
long-term DED treatment, and, ideally well before potentially blinding stromal breakdown
occurs in later stages.

Reduced or absent corneal sensitivity is suggestive of neurotrophic keratopathy and

therefore corneal sensation should be assessed as soon as there is a clinical suspicion and
prior to instillation of anaesthetic drops. Various methods are available for assessing corneal
sensitivity. The Cochet-Bonnet esthesiometer is quantitative and makes contact with the
ocular surface while the gas esthesiometers are quantitative, non-contact, and able to
assess chemical, thermal and/or mechanical corneal sensitivity 716, 767-770. Both are
commonly used in research and in specialty referral centres but historically have been costly
and impractical for most general eye care practitioners. Cheaper and simpler methods
involving contact with the eye’s surface that are commonly used in practice include a wisp
from a cotton-tip applicator, a corner of a disposable facial tissue and/or unwaxed and
unflavored dental floss and can provide a quick qualitative assessment of corneal sensation
in a primary eye care setting 741. A patient with normal sensation will blink and report
discomfort when the central cornea is touched, a patient with a hypoesthetic cornea may
blink but not report sensation and one with an anaesthetic cornea typically will not register a
blink or any sensation to the stimulus 741. A modified-Delphi expert panel on neurotrophic
keratopathy strongly recommended corneal sensitivity testing for persistent epithelial defects
after 14 days, new painless epithelial defects, a history of herpetic eye disease or
procedures that may injure the trigeminal nerve, and pain in an eye with multiple concurrent
risk factors for neurotrophic keratopathy such as poorly controlled diabetes and either
reduced blink rate or a history of corneal surgery 771. As more modalities for non-contact,
reproducible, quantifiable and inexpensive esthesiometry become available,769, 772-774 corneal
sensation can be performed earlier in the diagnostic subtyping process and ideally be
incorporated into office-based routine DED/ocular surface disease protocols (see section
6.1).

Once clinical suspicion for neurotrophic keratopathy and reduced corneal sensation are
identified, further investigation with corneal in vivo confocal microscopy can be diagnostically
confirmatory (see sections 3.5.3.6.1.2 and 3.5.3.4). Studies have consistently demonstrated
significant alterations in the corneal nerves, epithelial cells and corneal stroma in patients
with neurotrophic keratopathy 775-778. Of note, corneal sensation may remain relatively normal
despite significant reductions (of 50 to 80%) in sub-basal nerve density accompanied by
morphological changes such as increased tortuosity and beading 779, and may be clinically
detectable only when the nerve density drops below 1000 µm nerve length /frame 780, 781.
Epithelial abnormalities include enlarged and irregular cell shapes, decreased cell density
and squamous metaplasia correlating with disease severity 780, 781. Severe disorganization,
altered keratocyte morphology and presence of hyperreflective cells can be observed in
stage 3 along with increased dendritiform cell density, particularly in the central cornea,
suggesting a possible inflammatory component 782. Substantial reductions in sub-basal
nerve density are commonly seen in patients with neurotrophic keratopathy secondary to
herpetic eye disease 777, 778, 781. Interestingly, patients with unilateral herpes simplex and
zoster ophthalmicus may exhibit contralateral reductions in sub-basal nerve density 67, 780.

56
4.2 Symptoms in the absence of ocular surface disease
Unlike nociceptive pain that involves the triggering of nociceptors from local tissue damage,
neuropathic pain, which can be peripheral or central in origin, is caused by an abnormality in
the somatosensory nervous system 783. Diagnosing neuropathic corneal pain is primarily
clinical and exclusionary and based heavily on clinical history, risk factors and the
discordance in terms of the level of symptoms in the absence of corresponding clinical signs,
colloquially termed ‘pain without stain’ 784. While DED is commonly the initial diagnosis and
treatment target for many of these patients, neuropathic pain does not respond to
conventional treatments in the same way, and artificial tears do not provide the same
temporary symptom relief 785. DED and neuropathic corneal pain can coexist, requiring
treatment for both. Indeed, DED may have been the trigger, leading to neuropathic corneal
pain, often in patients with comorbid chronic pain (such as migraine and fibromyalgia),
psychiatric and/or mental health disorders 175, 786, 787. Patients with S gren disease also have
a higher risk of chronic ocular pain with neuropathic features 788. Other risk factors and
associated comorbidities of neuropathic ocular pain include DED, diabetes, sarcoidosis,
small fibre neuropathies, herpetic eye disease, prior eye surgery, infection, trauma, contact
lens wear and radiation keratopathy, and many of these overlap with risk factors for
neuropathic keratopathy 789. An association with long-COVID has also been identified 790, 791.
To confound the inherent diagnostic challenges even further, a subset of patients with stage
1 neurotrophic keratopathy and concomitant neuropathic corneal pain has also been
reported 792. If neuropathic corneal pain is suspected on the basis of clinical history,
symptoms and a lack of signs on examination, a pain-specific validated questionnaire such
as the Ocular Pain Assessment Survey or Neuropathic Pain Symptom Inventory, which has
been modified for the eye, can be utilized to score symptoms 793, 794.
4.2.1 Diagnosing corneal neuropathic pain
If the patient’s eyes are painful despite lubricating drops and the pain is incited by light, wind
or other triggers, neuropathic corneal pain should be suspected. In such a situation, a
proparacaine challenge test can be performed to aid in both diagnosis and differentiation
between peripheral and central etiologies 789, 795, 796. If the pain is completely ameliorated by
an anaesthetic drop the patient likely suffers from peripheral or nociceptive corneal pain
whereas, if the pain persists unchanged afterwards, then a central neuropathic pain
mechanism is likely; if only partial relief is achieved then a mixed mechanism of peripheral
and central neuropathic corneal pain is likely 795. Differentiating peripheral from central
neuropathic corneal pain is important as the treatment strategies differ significantly between
the two 797, 798. While corneal aesthesiometry is a critical test for neurotrophic keratitis it is
less useful in the workup of neuropathic corneal pain as studies have revealed both higher
and lower sensitivities in these patients and overall poor diagnostic correlation 152, 796, 799, 800
although more objective, non-contact techniques offer promise in identifying neuropathic
pain 769. Reduced density of sub-basal nerves is a finding common to both neurotrophic
keratitis and neuropathic corneal pain, but the increased presence of sub-basal and stromal
microneuromas, detectable by confocal microscopy, is more common in neuropathic corneal
pain 168, 169, 566, 567, 801. Further studies are needed to establish whether confocal microscopy
can be used reliably for differentiating the etiological drivers in a patient with DED.

5 Future advances

5.1 Artificial intelligence

Artificial Intelligence, a term coined by emeritus Stanford Professor John McCarthy in 1955,
was defined by him as “the science and engineering of making intelligent machines.” A wide
range of definitions have now been proposed, making it difficult to assess claims on its use.
It has been suggested that it is already widely used in DED clinical tests and research 802,
but it could be argued whether an algorithm to detect a change in pixel contrast or an ‘edge’

57
of a placido ring is truly “intelligence”. Machine learning thorough training has been used to
try and predict video frames that a specialist identified as showing breakup, with a sensitivity
of 78% and specificity of 86% 803. There is the potential that machine learning could make a
clinical test used in the diagnosis of dry eye more objective, but as such a diagnosis would
require a gold standard disease group against which to compare the individual, and a
consensus of which tests are most suitable to identify this group based on available
evidence, along with practical considerations such as cost and equipment availability, as
artificial intelligence cannot in itself drive development or change in the diagnostic algorithm
for dry eye. It certainly can aid in image analysis however, and particularly structural
segmentation such as in meibography 804-809. Rating of corneal fluorescein staining, on the
other hand, remains more challenging 810. Potential barriers to widespread adoption of
artificial intelligence include cost, accessibility, regulatory and ethical considerations, training
requirements and integration with existing diagnostic protocols 811.

5.2 Tear biomarker testing of tears

MMPs are one of many classes of proteases secreted into the tears in DED. Since MMPs
can destroy tight junctions in the ocular surface epithelium, increased levels of MMPs
reflects loss of ocular surface barrier function, 812-814. MMPs are produced as inactive
proenzymes and can be cleaved to become active enzymes 815. It is therefore important for
future MMP tests to detect enzyme activity levels and not simply total tear protein levels.
MMP-9 is detected more commonly in severe DED and has been proposed as a potential
means of monitoring the success of DED management; patients with a positive MMP-9 test
showed a greater benefit from topical cyclosporine than those who were MMP-9-negative 597.
A silicon nanowire-based field-effect transistor MMP-9 tear film biosensor was found to have
a sensitivity of 87% and specificity of 90% for DED 816. More advanced point-of-care tear
proteomic test kits for identifying DED subtype drivers are needed along with independent
validation / replication studies.

Lymphotoxin-alpha (LT-α), a member of the tumor necrosis factor superfamily, is expressed

by T cells, B cells, and natural killer cells, playing a crucial role in immune system
development and function 817-819; this includes the formation of lymphoid organs,
maintenance of lymphatic microenvironments, host defense, and modulation of
inflammation. Despite its established association with inflammation, emerging research has
identified a negative correlation between LT-α levels in the blood and fatigue symptoms
(often linked to proinflammatory processes) in patients with primary Sjögren disease 820, 821.
This suggests that LT-α's role in inflammation might be more complex than previously
thought. Levels of multiple tear protein markers (TNF-α, IL-10, IL-1β, IL-1Ra, IL-17A, and IL-
12/23 p40) were elevated in patients with DED with high LT-α (>700 pg/mL) compared to
those with lower LT-α (≤700 pg/mL), indicating possible differences in pathogenesis 822.

5.3 Sustainability
To date, there has been a significant paucity of literature examining the sustainability
implications of diagnostic testing and practices for DED 823. Future research is required to
characterise the potential sustainability implications, environmental effects, and carbon
footprint of the production, use, and disposal of different types of diagnostic instruments,
dyes, consumables, treatments and packaging.

5.4 Need for experience-informed approach to un/under-researched areas

Where research on best practice is either limited, conflicting or logistically or ethically difficult
to obtain, a group process using collective intelligence may help 824. This can for example be
applied to achieve consensus on the best clinical criteria for diagnosis or to initiate treatment
825
. The Delphi technique is a systematic process designed to establish consensus in a
group of experts. A series of questionnaires is distributed, and controlled feedback with
group statistical responses is given each time, until answers are converged and a predefined
criterion is reached to bring the process to a close. Important aspects, in order to obtain valid

58
outcomes, include systematic identification of the problem area, the selection of panel
members based on objective and predefined criteria, anonymity of panelists and responses,
controlled feedback, and stability of results including a priori defined closing criteria 824.

A Delphi approach has been used to define ocular surface disease activity and damage
indices 826. Several Delphi or other group process approaches have been conducted in the
past two decades to establish a best practice on a diagnostic aspect of DED and blepharitis.
The ODISSEY European Consensus group defined a two-step scoring algorithm for
diagnosing DED, but only at a severe level. Symptom-based assessment and corneal
fluorescein staining were considered to be the two most important criteria. In case of
discordance between these two tests, identification of additional criteria was
recommended.827 Separately, the DIDACTIC study used a Delphi approach to categorize
signs and symptoms to identify DED pathophysiology. A total of 19 items were deemed
indicative of evaporative dry eye, and 12 items of aqueous deficient DED.323. Using nominal
group and Delphi techniques, a group of Italian ophthalmologists reached consensus on
criteria for classification of DED. Three types were classified: a transient and reversible form,
a recurrent form, and a chronic form, each with its own clinical characteristics 828. Recent
Delphi panels on Demodex-associated blepharitis have achieved consensus on it being
chronic and recurrent 486, with the presence of cylindrical dandruff at the base of the
eyelashes, visible Demodex mites, lid margin telangiectasia, and a previous history of
anterior blepharitis not responding to treatment, being proposed as the most indicative
independent signs 485. Cylindrical dandruff is considered pathognomonic for Demodex
blepharitis, with the suggestion that patients with >10 collarettes should be treated even in
the absence of symptoms and that treatment efficacy can be tracked by the extent of
cylindrical dandruff resolution 829.

Examples of areas in the field of diagnostic methodology of DED that lack scientific evidence
and could benefit from a future Delphi approach include:
 how should DED severity be graded
 is site-specific itch useful in differentiating dry eye from allergy
 what cut-offs should be used for etiological drivers without current evidence-based
diagnostic thresholds
 the best sequence for instilling fluorescein and lissamine green to assess ocular
surface staining

6 Summary
A standardised approach is critical to providing robust epidemiological information on DED in
future.4 It is critical that all practitioners and researchers adopt the same approach for the
field to move forward for the benefit of patients.
6.1 Workflow and enhanced link to individualised management
In those patients that identify dryness type symptoms, the OSDI-6 screening questionnaire
should be used to quantify these (see Section 3.3.2; Figure 4) and a score of ≥4 used as a
prompt for further investigation. Risk factors should be explored (see Section 3.2; Figure 3;
Table 1) and a differential diagnosis conducted (see Section 3.1; Figure 2). If the practitioner
lacks the expertise and access to instrumentation to facilitate a detailed examination of the
eye, the triaging questions (see Section 3.1.5) will help to identify patients for whom referral
is appropriate. Diagnosis of DED requires evaluation of ocular surface staining (including the
cornea, conjunctiva and lid margin) along with tear film instability and/or hyperosmolarity
(Figure 5), and the criteria met by demonstrating a positive score in at least one of these
three indices of tear film and ocular surface homeostasis. In those diagnosed with DED, it is
important to identify the etiological driver(s) of the individual’s disease (see Section 3.5;
Table 6) to inform the most appropriate management and therapy option(s) (as described in
the TFOS DEWS III Management and Therapy report;17 Figure 11)

59
Figure 11: Classification of dry eye disease

An example of a test sequence to diagnose and identify the drivers (subtypes) of dry eye
disease based on key tests / observations is presented in Table 7. Tests should be ordered
from least to most invasive, to best maintain the integrity of the assessments and minimise
impact on subsequent test results. Slight variations in test order may be expected where
tests applied differ, but, in any case, the same test order should be utilised consistently on
each patient at any one site, to enhance consistency in diagnosis and subclassification.
Diagnosis and assessment for subtype drivers may be conducted in a single visit (if time
allows) or may be separated into two visits to include a rapid diagnosis as part of routine
testing, with follow up for subtype driver analysis at a separate visit. Test order will be
adjusted accordingly, always from least to most invasive.

Table 7: An example of a test sequence (from least to most invasive) to diagnose

(second column) and identify the drivers (subtypes – third column) of dry eye disease based on key
tests / observations in the same appointment.

Sequence Diagnosis Subtype Drivers

1 Symptoms Screening - OSDI-6
2 Blink / lid closure - rate / completeness
/ lid seal
3 Aqueous – tear meniscus height (using
infrared illumination)
4 Non-invasive tear breakup time
5 Anatomical lid/globe misalignment –

60
 features such as pterygia
6 Inflammation – redness
7 Lipid – interferometry
8 Osmolarity
9 Lid margin – eyelashes, lid margin,
diagnostic expression
[10] [Fluorescein tear breakup time]
11 Ocular surface staining Ocular surface damage- corneal,
conjunctival and lid wiper staining
Mucin – conjunctival staining
Lid margin – keratinisation staining
12 Lid margin – meibography
13 Neural dysfunction - corneal nerves /
sensation (if contact methods are
used)

6.2 Patient communication

When communicating with a patient experiencing dry eye, active, two-way communication
should be prioritized by openly discussing their symptoms, addressing concerns, educating
them on lifestyle modifications to manage the condition, and empowering them to actively
participate in their treatment plan. Key aspects include: explaining the chronic nature of
DED, setting realistic expectations, discussing environmental triggers, promoting proper eye
hygiene practices and recommending appropriate artificial tears or other treatments based
on their individual needs and as indicated by their individual identified etiological drivers.
Patients are usually highly engaged in their desired outcomes315 and if DED is suspected, a
follow up appointment is likely to be needed to perform a differential diagnosis, conduct the
diagnostic algorithm, identify the etiological drivers (with the potential for a technician to
conduct these measurements) and to discuss these with the patient and ideally to show
them the images capture, to make shared decisions on appropriate management and
therapy.17

6.3 Key diagnostic methodological changes from TFOS DEWS II

The revised definition: “Dry eye is a multifactorial, symptomatic disease characterized by a
loss of homeostasis of the tear film and/or ocular surface, in which tear film instability and
hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities
are etiological factors.” Key features of DED from the definition that have been
reemphasised include that DED is multifactorial, a disease not a syndrome and always
symptomatic.

The recommended screening questionnaire is the OSDI-6 with a cut-off of a score ≥4 (Figure
4). Other questionnaires can be used as desired to gain further understanding of the
environmental risk factors, but the standardised diagnostic questionnaire is necessary to
achieve diagnostic consistency for all patients.

Key clinical differential diagnosis questions, alongside asking about a patient’s general
health and medication, are:
 Do you feel eye pain rather than discomfort?
 Do you have any facial flushing/redness, mouth dryness or enlarged salivary glands?
 When did your symptoms start and can you recall any triggering event?
 Is your vision affected and if so, does it improve on blinking?
 Are the symptoms or any redness much worse in one eye than the other?
 Do the eyes itch, are swollen, crusty or have given off any discharge?

61
A detailed examination of the ocular surface is recommended where the responses to these
questions suggest possible presence of eye conditions that might masquerade as DED, with
a guide to the ocular examination presented in Figure 2.

Known DED risk / associated factors (Figure 3) have been updated based on the scientific
evidence4 and factors commonly assumed to be associated with, or even cause, DED for
which the evidence is equivocal, are reported (Table 1).

The diagnostic algorithm for DED has been refined (Figure 5) and in mitigating the
established variability between questionnaires, only one (the OSDI-6) is recommended for
the diagnostic algorithm. The impact on the diagnosis of each individual signs of a loss of
homeostasis of the tear film and ocular surface recommended in TFOS DEWS II has been
examined demonstrating that the lack of non-invasive breakup time or osmolarity only has a
minor effect. Hence the revised, TFOS DEWS III approach has been shown to be robust232
and further simplifies the procedure for application in clinical practice.

In aligning treatment strategies that possess different mechanisms of action with the multiple
established drivers of dry eye disease, it has become clear that a greater number of distinct
subtypes than simply aqueous and evaporative need to be acknowledged to ensure optimal
patient care. This report has compiled the evidence on a more detailed subclassification of
the disease based on the etiological drivers, so that these can be identified for an individual
patient for the purpose of informing appropriate management and therapy. Cut-offs for the
identified clinical tests have been provided where available (Table 6). Many of these clinical
tests are already part of a standard clinical DED routine (Table 7), with the TFOS DEWS III
approach offering structure and links to management and therapy (Figure 11).17

Acknowledgments
Sarp Orgul and Arthur Chan are acknowledged for their support in specific sections of the
report.

Funding
The TFOS DEWS III effort was supported by unrestricted donations from Alcon, Bausch +
Lomb, Azura, AbbVie, CooperVision, Dompé, Espansione Group, Harrow, Laboratoire Théa,
SIFI, SINQI, Tarsus, Topcon and Trukera.

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Conflict of Interest

The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.

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