Diagnostic Testing for Dry Eye
The Tear Film and Ocular Surface Society (TFOS) Dry Eye WorkShop (DEWS) defines dry eye as "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 definition implies that the diagnosis of dry eye is complex and requires different aspects of the tear film to be assessed using different diagnostic modalities.
Loss of homeostasis is difficult to measure directly, but usually, it is assessed through interpretation of a battery of diagnostic test results. Tear film instability is usually evaluated by a simple tear break-up time (BUT) test using a vital dye, fluorescein. Some of the newer imaging techniques offer non-invasive ways to measure BUT. Tear osmolarity can be directly measured using a point-of-care device in-office. Ocular surface inflammation can also be assessed utilizing a point-of-care device in-office to determine whether a non-specific marker, matrix metalloproteinase-9 (MMP-9), is positive in the tear film. Neurosensory abnormalities can be demonstrated examining corneal sensitivity simply using a whim of cotton or tissue or an esthesiometer and using in-vivo confocal microscopy to image corneal nerves. However, more traditional ways to diagnose dry eye include ocular surface staining using vital dyes and measuring tear volume using Schirmer's test. There are other diagnostic tests that can be utilized to evaluate the tear film lipid layer or meibomian gland structure. Also, an in-office finger-prick test is used to detect autoantibodies in patients who are suspected of an underlying systemic inflammatory/autoimmune disease such as Sjogren's syndrome.
Most commonly utilized diagnostic tests for dry eye and some of the newer tests and their technique were summarized below.
Tear Film Stability
- Fluorescein BUT: The tear break-up time test is easily and quickly performed via fluorescein dye instillation. After the fluorescein is instilled, the patient is asked to stare without blinking. The cornea is observed under the cobalt blue light and time is counted in seconds, from the time of the last blink until a pocket of cornea appears that is no longer covered with fluorescein stained tears. The longer the BUT, the more stable the tear film. A BUT > 8-10 seconds is usually considered normal. 
- Non-Invasive BUT (NIBUT): As fluorescein dye instillation may affect the stability of the tear film itself, non-invasive measurement of tear BUT using technological advancements has become popular recently. Tear film stability is measured automatically using a specific software through detection of the break-up on the image reflected from the ocular surface, either a grid pattern or Placido disk images. To assess the NIBUT, the software asses the different segments and distortion in the reflected mires. 
Fluorescein Break-Up Patterns
A recent study reported that the pattern of tear break-up may suggest which layer of the tear film is primarily affected, such as aqueous tear layer/secretory mucins or surface epithelial layer/membrane-associated mucins.  Based on the observed pattern, the treatment plan can be customized. 
Classification of the patterns, suggested mechanisms, and treatment recommendations:
- Area break – no, or limited, movement of fluorescein-stained tear film with instant BUT over large areas of the cornea. Severe surface staining is usually observed. This pattern may suggest severe aqueous deficiency and is best treated via punctal occlusion and preservative-free artificial tears use.
- Spot break – spot-like shape immediately after eye opening with instant BUT usually in the upper part of the cornea. This pattern suggests locally impaired wettability that is associated with deficiency of membrane-associated mucin, MUC16,  or contamination of glycocalyx with lipids. Diquafasol sodium (P2Y2 purigenic receptor agonist) eye drops are recommended which is currently not available in the United States. 
- Dimple break – irregular but vertical line–like shape in the central part of the cornea. Similar to the spot break pattern, the wettability of the cornea is affected.
- Line break – vertical line-like shape in the lower part of the cornea. This pattern is observed in patients with mild to moderate aqueous deficiency dry eye. BUT is usually between 2-3 seconds. Superficial punctate keratopathy is observed in the lower part of the cornea. The thin aqueous layer exposes the glycocalyx to cell debris and lipid which affects the wettability of the surface. Diquafosol sodium eye drops are recommended to treat patients who show this pattern. 
- Random break – irregular and indefinite shapes that occur in different shapes with each blink. BUT is usually more than 5 seconds. This pattern can be treated using a variety of choices, such as artificial tears, hyaluronic acid, diquafosol sodium, ophthalmic ointment. 
Ocular Surface Staining
Corneal staining is often part of a routine eye exam. The ocular surface is most commonly stained with fluorescein dye. The fluorescein is applied on a moistened sterile strip of paper to the inner lining of the lower eyelid or applied in an eye drop mixed with a topical anesthetic. The ophthalmic strips might be mildly irritating and the drop may briefly burn. When abnormal or missing epithelial cells are stained with fluorescein and observed under the cobalt blue light, they appear bright green. Bright green areas of the cornea may indicate dry eye, as well as other conditions. Other less commonly utilized stains are lissamine green and rose bengal. Lissamine green and rose bengal have similar staining characteristics for evaluating the conjunctiva. Lissamine green is less toxic and better tolerated than rose bengal.
Tear Volume Assessment
Schirmer's Test: A small paper strip with rulers printed along their length is placed over the temporal one-third of the lower lid margin inserting the folded end inside the inferior conjunctival fornices. The strips are removed after 5 minutes and the amount of tears produced in that time is measured by reading off of the length of wetting in millimeters. This test can be performed with or without anesthesia. Interpretations can vary but usually < 10mm of tear production in 5 minutes is suggestive of some form of dry eye. 
Tear Meniscus Assessment: Tear meniscus can be observed over the lower lid margin after instillation of fluorescein vital dye using Cobalt blue filter. Its height can be roughly estimated compared to the slit lamp beam height although this method is not easily reproducible.  Anterior segment OCT can be used to measure the tear meniscus height more objectively; however, the analysis of the image is dependent on the technology and/or software used and the operator who performed the test. 
The test is relatively painless and quick (seconds) and can be performed by an ophthalmic technician. Increased osmolarity indicates dry eye as suggested by TFOS DEWS II.  The tear lab osmometer is a nanoliter instrument that offers a relative expertise free method for tear osmolarity measurement which uses a lab on chip technology. The test card is mounted on the device and placed over the lower tear meniscus to get a reading. It requires less than 100 nanolitres of tears, hence useful in severely dry eyes. Normal osmolarity was defined as < 300 mOsm/L in both eyes and an inter-eye difference of < 8 mOsm/L. Values greater than 300 mOsm/kg are suggestive of dry eye. From 300 mOsm/L to 320 mOsm/L, is graded as mild; from 320 mOsm/L to 340 mOsm/L, is graded as moderate; and greater than 340 mOsm/L, is graded as severe. 
Matrix Metalloproteinase-9 Test
The matrix metalloproteinases (MMP) in tears are indicators of the loss of ocular surface barrier function and found to be elevated in tears of patients with dry eye.  InflammaDry® (Quidel Corporation, San Diego, CA) is a point-of-care test that measures MMP-9 levels in tears. It is performed prior to other tear testings, instillation of ocular anesthesia, or topical dyes. A tear sample is collected from the palpebral conjunctiva and combined with a test buffer. Color-coded results are read after 10 minutes. It is primarily a qualitative test, just like a pregnancy test, where the blue line is negative and inflammation is crossed out. If the values are above 40 µg/ml, then it is positive. It can tell if the underlying cause is inflammatory, thereby decisive in the commencement of anti-inflammatory treatment. It is important to be aware that the test can be falsely positive in certain settings, such as allergic conjunctivitis and infection. 
Lactoferrin is a protein produced by the acinar cells of the lacrimal gland and can be detected in tears.  Lactoferrin has antimicrobial and anti-inflammatory properties.  Lower concentrations of lactoferrin were demonstrated in patients with dry eye, which was associated with decreased aqueous tear production.    TearScan 270 MicroAssay System (Advance Tear Diagnostic, Birmingham, AL) is a point-of-care test unit that determines tear lactoferrin level through the collection of 0.5 microliter tears. 
The thickness of the lipid layer can be estimated based on surface reflection patterns and dynamics.  The first clinically available interferometer was the LipiView (TearScience, Morrisville, NC). This instrument measures the lipid layer thickness between blinks and gives a quantitative assessment in interferometric color units (ICU). The measurements are obtained through images of the tear film that are captured over 5 minutes. Blink dynamics are also assessed as a part of this system. Among the parameters, the partial blinks and the C factor can be taken most valued, among others. The accuracy of the interference pattern is measured as the C factor. The color scale resulting from the interference pattern is evaluated, which occurs at the boundary of the tear film. It ranges from 0 to 240 with a precision of 1 ICU.
The silhouette of the meibomian gland structure can be visualized using different techniques. The imaging of illuminated everted eyelids using black-and-white photography, infrared camera, or a near-infrared charge-coupled device (CCD) video camera allows morphology and structure of meibomian glands to be captured.   A quantitative analysis of the meibomian gland structure is possible using different scoring systems, such as the meiboscore, through software included in the imaging systems. 
The Sjo® Test
Early diagnosis of Sjögren’s syndrome (SS) in patients with clinically significant dry eye is relevant to prevent vision-threatening complications. The Sjo test is an in-office diagnostic blood test. A finger prick blood sample is collected to saturate a specimen filter paper and sent to the lab. The diagnostic testing panel includes four traditional SS antibodies (anti-SS-A/Ro, anti-SS-B/La, rheumatoid factor [RF], and antinuclear antibody [ANA]) along with three tissue-specific autoantibodies (anti-SP-1, anti-PSP, and anti-CA-6). The 2012 American College of Rheumatology (ACR) classification criteria for SS suggested that a positive anti-SSA and/or positive anti-SSB or a combination of RF and ANA at a titer ≥ 1: 320 are needed to fulfill the positive serology criterion. However, recently revised 2016 classification criteria included anti-SSA antibody as the only serological marker for SS.  It is important to note that these tissue-specific antibodies are not included in any classification criteria for SS; therefore, they do not have a diagnostic value. Some of these antibodies were associated with more severe dry eye regardless of SS diagnosis. Based on current knowledge, a positive novel autoantibody may suggest either early stages of SS or another form of an autoimmune disease. Their utility is still under investigation. Strong suspicion of SS, even in the absence of the traditional or novel antibodies, should lead to a rheumatology referral as negative serology is not exclusive and further testing and evaluation, such as a lip biopsy, may still reveal the diagnosis of SS.
- ↑ 1.0 1.1 1.2 1.3 1.4 Wolffsohn JS, Arita R, Chalmers R, Djalilian A, Dogru M, Dumbleton K, Gupta PK, Karpecki P, Lazreg S, Pult H, Sullivan BD, Tomlinson A, Tong L, Villani E, Yoon KC, Jones L, Craig JP. TFOS DEWS II Diagnostic Methodology report. Ocul Surf. 2017 Jul;15(3):539-574.
- ↑ Lemp MA, Hamill JR. Factors affecting tear film breakup in normal eyes. Arch Ophthalmol 1973;89:103e5.
- ↑ Abelson MBO, G 3rd W, Nally LA, Welch D, Krenzer K. Alternative reference values for tear film break up time in normal and dry eye populations. Adv Exp Med Biol 2002;506:1121e5.
- ↑ Goto T, Zheng X, Okamoto S, Ohashi Y. Tear film stability analysis system: introducing a new application for videokeratography. Cornea 2004;23:S65e70.
- ↑ Gumus K, Crockett CH, Rao K, Yeu E, Weikert MP, Shirayama M, et al. Noninvasive assessment of tear stability with the tear stability analysis system in tear dysfunction patients. Invest Ophthalmol Vis Sci 2011;52: 456e61.
- ↑ 6.0 6.1 Yokoi N, Georgiev GA, Kato H, Komuro A, Sonomura Y, Sotozono C, Tsubota K, Kinoshita S.Classification of Fluorescein Breakup Patterns: A Novel Method of Differential Diagnosis for Dry Eye. Am J Ophthalmol. 2017 Aug;180:72-85.
- ↑ 7.0 7.1 Yokoi N, Georgiev GA. Tear Film-Oriented Diagnosis and Tear Film-Oriented Therapy for Dry Eye Based on Tear Film Dynamics. Invest Ophthalmol Vis Sci. 2018 Nov 1;59(14):DES13-DES22.
- ↑ Argüeso P. Glycobiology of the ocular surface: mucins and lectins. Jpn J Ophthalmol. 2013;57(2):150–155.
- ↑ King-Smith PE, Reuter KS, Braun RJ, Nichols JJ, Nichols KK. Tear Film Breakup and Structure Studied by Simultaneous Video Recording of Fluorescence and Tear Film Lipid Layer Images. Invest Ophthalmol Vis Sci. 2013 Jul 22;54(7):4900-9.
- ↑ 10.0 10.1 Yokoi, N., Sonomura, Y., Kato, H. et al. Three percent diquafosol ophthalmic solution as an additional therapy to existing artificial tears with steroids for dry-eye patients with Sjögren’s syndrome. Eye 29, 1204–1212 (2015).
- ↑ Nichols KK, Mitchell GL, Zadnik K. The repeatability of clinical measurements of dry eye. Cornea 2004;23:272e85.
- ↑ Lemp MA, Bron AJ, Baudouin C, Benítez Del Castillo JM, Geffen D, Tauber J, Foulks GN, Pepose JS, Sullivan BD. Tear Osmolarity in the diagnosis and management of dry eye disease. Am J Ophthalmol. 2011 May;151(5):792-798.e1.
- ↑ TearLab. https://www.tearlab.com/ Accessed October 9, 2017.
- ↑ InflammaDry®. A Simple 4-Step Process to Aid in Dry Eye Diagnosis. https://www.quidel.com/immunoassays/inflammadry https://www.quidel.com/sites/default/files/product/documents/HT1024000EN01.pdf Accessed October 9, 2017.
- ↑ Gillette TE, Allansmith MR. Lactoferrin in Human Ocular Tissues. Am J Ophthalmol 1980;90:30-7.
- ↑ Flanagan JL, Willcox MD. Role of Lactoferrin in the Tear Film. Biochimie 2009;91:35-43.
- ↑ Danjo Y, Lee M, Horimoto K, et al. Ocular Surface Damage and Tear Lactoferrin in Dry Eye Syndrome. Acta Ophthalmol 1994;72:433-7.
- ↑ Li Y, Wei Z, Yu Z. The Relationship between Dry Eye and Lactoferrin Levels in Tears. Asian Biomed 2012;6:81-5.
- ↑ Da Dalt S, Moncada A, Priori R, et al. The Lactoferrin Tear Test in the Diagnosis of Sjogren's Syndrome. Eur J Ophthalmol 1996;6:284-6.
- ↑ 20.0 20.1 Chao C, Tong L.Tear Lactoferrin and Features of Ocular Allergy in Different Severities of Meibomian Gland Dysfunction. Optom Vis Sci. 2018 Oct;95(10):930-936.
- ↑ Sonobe H, Ogawa Y, Yamada K, Shimizu E, Uchino Y, Kamoi M, Saijo Y, Yamane M, Citterio D, Suzuki K, Tsubota K. A novel and innovative paper-based analytical device for assessing tear lactoferrin of dry eye patients. Ocul Surf. 2019 Jan;17(1):160-166.
- ↑ Craig JP, Tomlinson A. Importance of the lipid layer in human tear film stability and evaporation. Optom Vis Sci 1997;74:8e13.
- ↑ Finis D, Pischel N, Schrader S, Geerling G. Evaluation of lipid layer thickness measurement of the tear film as a diagnostic tool for Meibomian gland dysfunction. Cornea 2013;32:1549e53.
- ↑ Robin JB, Jester JV, Nobe J, Nicolaides N, Smith RE. In vivo transillumination biomicroscopy and photography of meibomian gland dysfunction. A clinical study. Ophthalmology 1985;92:1423e6.
- ↑ Mathers WD, Daley T, Verdick R. Video imaging of the meibomian gland. Arch Ophthalmol 1994;112:448e9.
- ↑ Srinivasan S, Menzies K, Sorbara L, Jones L. Infrared imaging of meibomian gland structure using a novel keratograph. Optom Vis Sci 2012;89:788e94.
- ↑ Nichols JJ, Berntsen DA, Mitchell GL, Nichols KK. An assessment of grading scales for meibography images. Cornea 2005;24:382e8.
- ↑ Liew M, Zhang M, Kim E, Akpek EK. Prevalence and predictors of Sjögren’s syndrome in a prospective cohort of patients with aqueous-deficient dry eye. British Journal of Ophthalmology. 2012;96(12):1498–1503.
- ↑ Shiboski SC, Shiboski CH, Criswell L, Baer A, Challacombe S, Lanfranchi H, Schiødt M, Umehara H, Vivino F, Zhao Y, Dong Y, Greenspan D, Heidenreich AM, Helin P, Kirkham B, Kitagawa K, Larkin G, Li M, Lietman T, Lindegaard J, McNamara N, Sack K, Shirlaw P, Sugai S, Vollenweider C, Whitcher J, Wu A, Zhang S, Zhang W, Greenspan J, Daniels T, Sjögren's International Collaborative Clinical Alliance (SICCA) Research Groups. American College of Rheumatology classification criteria for Sjögren's syndrome: a data-driven, expert consensus approach in the Sjögren's International Collaborative Clinical Alliance cohort. Arthritis Care Res (Hoboken). 2012 Apr; 64(4):475-87.
- ↑ Shiboski C. H., Shiboski S. C., Seror R., et al. 2016 American College of Rheumatology/European League Against Rheumatism classification criteria for primary Sjögren’s syndrome: a consensus and data-driven methodology involving three international patient cohorts. Annals of the Rheumatic Diseases. 2016;76(1):9–16.
- ↑ Suresh L, Malyavantham K, Shen L, Ambrus JL Jr. Investigation of novel autoantibodies in Sjogren's syndrome utilizing Sera from the Sjogren's international collaborative clinical alliance cohort. BMC Ophthalmol. 2015 Apr 10;15:38.
- ↑ Karakus S, Baer AN, Agrawal D, Gurakar M, Massof RW, Akpek EK. Utility of Novel Autoantibodies in the Diagnosis of Sjögren's Syndrome Among Patients With Dry Eye. Cornea. 2018 Apr;37(4):405-411.
- ↑ Karakus S, Baer AN, Akpek EK. Clinical Correlations of Novel Autoantibodies in Patients with Dry Eye. J Immunol Res. 2019 Jan 13;2019:7935451.
- ↑ Bunya VY, Massaro-Giordano M, Vivino FB, Maguire MG, Baer AN, Gonzales JA, Ying GS. Prevalence of Novel Candidate Sjögren Syndrome Autoantibodies in the Penn Sjögren's International Collaborative Clinical Alliance Cohort. Cornea. 2019 Dec;38(12):1500-1505.