Advances in the Noninvasive Diagnosis of Dry Eye Disease

: Dry eye disease (DED) is a multifactorial disease that represents one of the most common ophthalmologic conditions encountered in everyday clinical practice. Traditional diagnostic tests for DED, such as subjective questionnaires, tear ﬁlm break-up time and the Schirmer test, are often associated with poor reproducibility and reliability, which make the diagnosis, follow-up, and management of the disease challenging. New advances in imaging technologies enable objective and reproducible measurements of DED parameters, thus making the diagnosis a multimodal imaging-based process. The aim of this review is to summarize all the current and emerging diagnostic tools available for the diagnosis and monitoring of DED, such as non-invasive tear breakup time, thermography, anterior segment optical coherence tomography, meibography, interferometry, in vivo confocal microscopy, and optical quality assessment. Although there is not a gold standard imaging technique, new multi-imaging-integrated devices are precious instruments to help clinicians to better cope with the diagnostic complexity of DED.


Introduction
Dry eye disease (DED) is one of the most common ophthalmologic conditions encountered in everyday clinical practice [1]. The 2017 report by the Tear Film and Ocular Surface Society (TFOS) Dry Eye Workshop (DEWS II) published the renewed definition of DED, which was defined as an ocular surface disorder in which multiple pathological events, including tear film instability, hyperosmolarity, inflammation and neurosensory abnormalities, lead to the loss of the homeostasis of the entire system [2]. Common symptoms of DED include, among others, irritation, redness, foreign body sensation, blurry vision, tearing, and sensitivity to light. It is a chronic condition that also represents an important economic burden for both patients and society [3].
Performing a suitable clinical diagnosis of DED can be challenging, as its signs and symptoms are often poorly correlated. Combinations of subjective symptoms evaluated by questionnaires, slit lamp examination and (non-)invasive diagnostic tests have been used to obtain the diagnosis. The TFOS DEWS II Diagnostic Methodology report identified a diagnostic algorithm for a stepwise analysis that includes, after triaging questions, the evaluation of symptoms and homeostasis markers (noninvasive or fluorescein break-up time, tear osmolarity and ocular surface staining) [4]. Nonetheless, the report acknowledged that no single gold standard test has yet been established, and that there is a need for new, reliable diagnostic biomarkers.
In recent years, a great number of imaging techniques and devices for the examination of the ocular surface have been developed and placed on the market (Table 1). These devices offer the advantage of providing automated results of the examined tests, thus avoiding observer bias; moreover, since most of these examinations are noninvasive, they do not alter the results of subsequent tests, representing useful screening tools for discriminating healthy subjects from patients affected by or at risk for DED. Finally, combining different techniques in a comprehensive ocular surface workup may increase sensitivity and specificity to diagnose the disease and monitor its course after specific treatments [5][6][7].
In this review, we summarize the current research available about the development and use of novel noninvasive diagnostic techniques for the diagnosis and follow-up of DED.

Noninvasive Tear Break-Up Time
Pre-corneal film stability plays a critical role in the homeostasis of the ocular surface, and it is an important parameter to be considered for DED diagnosis [4,8]. In fact, the tear film is the first optical interface between the air and the ocular surface, and since there is a large difference in refractive index between the air and the tear, inhomogeneity in its structure has a major impact on the optical quality. In DED, there is a quantitative and/or qualitative tear deficiency that determines irregularities and/or the early break-up of the tear film [9,10]. In clinical practice, the most frequently employed test for the measurement of tear film stability is the fluorescein tear film breakup time (BUT), which is the interval time between the eyelid opening after a complete blink and the first break in the tear film. Since BUT requires the use of fluorescein, whose quantity and concentration can also affect the final measurement, noninvasive BUT (NIBUT) has become widely used in both clinical practice and research settings. DEWS II indicated that a cutoff value of NIBUT ≤ 10 s is an indicator for the diagnosis of DED, with a sensitivity of 82-84% and a specificity of 76-94% [4]. There are several commercially available NIBUT systems, based on topographic or videokeratographic methods, which analyze the inter-blink changes of reflected placido mires. A change in the edges of the mires reflects compromised tear film integrity. Bandlitz et al. demonstrated that there is agreement and repeatability of measure between subjective and objective devices [11], while Lee et al. demonstrated that the agreement between the results extrapolated from two different instruments (Tomey RT-7000 Auto Refractor-Keratometer and Oculus Keratograph) was poor [12]. The Keratograph 5 M (Oculus Optikgeräte GmbH, Wetzlar, Germany) allows the measure of quantitative values, such as the NIBUTf, which is the initial tear film breakup, whereas the NIBUTav represents the average of all tear film breakups occurring over the entire cornea. Both parameters have been reported to be correlated with Ocular Surface Disease Index (OSDI) score [13,14].

Light Scatter
Light scattering is a physical phenomenon in which light when hits a small object (a particle or a molecule) changes its direction. In human eyes, aberrations and light scattering are the main factors in the degradation of optical quality [15]. In DED, the loss of integrity of the tear film and the early exposure of the rough epithelium can lead to an increase in the ocular forward light scattering detected by C-Quant straylight meter (Oculus GmbH, Wetzlar, Germany). In DED eyes with superficial punctate keratopathy, there is also an increase in corneal backward light scattering from the anterior corneal part, detected by a Scheimpflug camera (Pentacam HR; Oculus GmbH), compared to normal eyes [16]. Tan et al. evaluated optical quality changes in DED by using the Optical Quality Analysis System (OQAS II; Visiometrics S.L., Tarrasa, Spain). They analyzed aberrations and intraocular scattering by using the Objective Scatter Index (OSI) [17] and found that this parameter was increased in patients with DED, and its rate of change was correlated with severity of DED [18]. Furthermore, Ge et al. demonstrated that several parameters (OSI, OSI standard deviation, ∆OSI, ∆OSI/time, blinking change, and blinking frequency) were correlated with BUT and staining score [19].

Aberrometer
Aberrometry uses wavefront sensing to analyze deviations in the wavefront exiting the eye from a reference wavefront. This technique is useful for measuring the complete refractive status, including irregular astigmatism, or any other optical irregularity [20].
Koh et al. demonstrated that BUT was associated with increased higher-order aberrations (HOAs) both in photopic and scotopic conditions [21]. Sequential measurement of HOAs demonstrated that normal eyes can be classified into three sub-groups as having stable, small-fluctuation, and sawtooth patterns, respectively. In the latter group, significant changes were found in the sequential post-blink changes in the coma-like and sphericallike aberrations and total HOAs [22]. In eyes with DED or irregularities in their refractive surfaces, dynamic wavefront analysis post-blink can detect an increase of the HOAs, and this is correlated with the OSDI score and BUT [23].
In patients with DED, superficial punctate keratopathy may aggravate both baseline HOAs and sequential post-blink changes in HOAs [24]. In eyes with a short BUT, a prolonged blink interval leads to increased HOAs, suggesting that suppressed blinking (e.g., for workers who operate video display terminals), can result in reduced optical quality [25].
Deschamps et al. demonstrated the impact of tear film-related aberration changes on activities of daily living in DED, specifically during a driving simulator test [26].
An interventional study investigated the effects of topical drops (sodium hyaluronate) on dynamic aberrometry in patients with DED and discovered no significant changes [27]. On the other hand, another study examining the effect of rebamipide 2% (a mucin secretagogue) demonstrated decreased HOAs after 4 weeks of treatment in DED patients with short BUT [28].

Anterior Segment Optical Coherence Tomography
Optical coherence tomography (OCT) is a noninvasive technique developed to obtain tomographic image reconstruction of a biological tissue with a longitudinal and lateral spatial resolution of a few micrometers by using low-coherence interferometry [29,30]. Anterior segment OCT (AS-OCT) can obtain images of important structures of the ocular surface [31] and is a useful tool in the diagnosis and follow-up of several diseases of the anterior segment of the eye. In DED, the measurement of parameters of the tear film by means of AS-OCT, such as pre-corneal tear film thickness (TFT), tear meniscus height (TMH), curvature (TMR), cross-sectional area (TMA) and tear meniscus depth (TMD), are widely used in routine clinical practice thanks to the noncontact nature of these examination and the rapid image acquisition by the instrument [32,33]. The average value of TFT measured by AS-OCT in healthy subjects is approximately 5 µm, while it is reduced in DED patients. This reduction is correlated with objective and subjective assessments, such as BUT and OSDI [34]. The value of TFT increases up to 24 h with the topical administration of chitosan-N-acetylcysteine, perfluorohexyl octane or low-dose hydrocortisone [35,36], but is less influenced by low-viscosity agents. TMH and TMA are two useful biomarkers of DED, as suggested by the DEWS II Report. Spectral Domain AS-OCT demonstrated a sensitivity of 80.5% and 86.1% and a specificity of 89.3% and 85.3% for TMH and TMA, respectively; however, the diagnostic ability of evaporative DED was low (<50%) [37]. In clinical practice, some confounding factors related to the environment or the ocular surface's anatomy (e.g., conjunctivochalasis, lid-parallel conjunctival folds, disorders of lid margin congruity) should be taken into account. AS-OCT is also useful for the 3D high-resolution of Meibomian glands (MG) [38]. In patients with Meibomian gland dysfunction (MGD), AS-OCT demonstrated a decreased MG length and width, which correlated with ocular discomfort symptoms and signs [39].

In Vivo Confocal Microscopy
In vivo confocal microscopy (IVCM) is a useful imaging technique for the in vivo microscopic observation of the corneal microstructure that enables clinicians to gain deep insight into the pathophysiology of ocular surface diseases [40]. Although it is a non/minimally invasive test, there are concerns about its routine use in clinical practice: (i) the examination requires direct contact with the ocular surface; (ii) a small field of view is obtained; (iii) only one z-plane is investigated; (iv) the eye tracker function is not feasible [41].
IVCM of the corneal epithelium demonstrated significant alterations in DED patients, presumably due to increased desquamation of the superficial cell layer caused by hyperosmolarity of the tear film associated with increased tear evaporation and elevated inflammatory mediators. Furthermore, IVCM allows the evaluation of immune and inflammatory cells, corneal nerves, keratocytes, and MG structures on a cellular level [42].
Several studies demonstrated a reduced density of superficial epithelial cells in Sjögren's and non-Sjögren's DED (respectively, PSDE and NSDE) [43]. Tuominen et al. reported a reduction in stromal thickness in patients with PSDE as well as abnormal keratocyte hyperreflectivity [44]. Benitez del Castillo et al. presented an increased density in anterior and posterior stromal cells in the PSDE group compared to the NSDE group, but without statistical significance [45].
In accordance with the recent evidence of neural dysregulation in DED, studies regarding corneal nerve parameters reported an increased tortuosity and reflectivity of the corneal sub-basal nerve plexus and an increased number of bead-like formations in both PSDE and NSDE compared to healthy controls [41,43]. The results obtained by Lin et al. demonstrated that there was also immune dysregulation: in fact, while in healthy subjects dendritic and leukocytes epithelial cells of the cornea decrease from the periphery towards to the center, in DED they show the opposite trend [46].
IVCM enables the visualization of the ultrastructure of MG [47][48][49]. Villani et al. demonstrated an increased acinar dilatation, a higher reflectivity (grades 1-4) of meibum, a decreased diameter of MG orifices and increased inhomogeneity of the acinar wall in patients with SS and MGD compared to controls [47]. Ibrahim et al. demonstrated a reduction in the density and diameters of the acinar units in MGD [49]. Randon et al., proposed a new classification of MGD based on IVCM findings (type 0 for normality, type 1 for meibum obstruction, type 2 for inflammation, and type 3 for fibrosis), and demonstrated a strong correlation between the IVCM score and the meibography scores [50]. In a crosssectional study, Zhao found that DED symptoms were negatively correlated with IVCM parameters of MG and positively correlated with conjunctival inflammatory cells [51].

Meibography
Meibography allows the observation of the morphological structure of the MG in a two-dimensional plane. The technique was introduced for the first time in 1977 by Tapie, who used transillumination from the palpebral skin with an illuminating probe to capture images of MGs. The approach was then refined by other methodologies that allow the visualization of MG on black-and-white film, on infrared film with a near-infrared CCD (charge-coupled device) camera, or with an infrared CCD camera [52]. Non-contact techniques based on an infrared filter and infrared CCD camera that made possible recording the transillumination image from the conjunctival side of the eyelid were developed in the last decade [53]. Non-contact meibography is based on the autofluorescence of a healthy meibum when illuminated with infrared light, which can be detected by an infrared chargecoupled device camera; thus, the glands appear as light areas against a darker background and any alterations in meibum or loss of acinar tissue appear as a dark area [54]. Recent technology has led to the development of several mobile, handheld, pen-shaped and multifunctionality (slit lamp-based, mobile and topography-equipped) systems with infrared light-emitting diodes fixed to infrared cameras that allow the capture of videos and images of MG and increase the feasibility of outpatient care assessment [55,56].
In clinical practice, the grading systems of MG structures can be used to document the presence, progression, and treatment response to MGD [57]. The most useful systems are the Meiboscore and the Meibograde; both feature a score based on the percentage of loss of the glandular area, although the latter offers a higher sensitivity and should be able to detect minor changes in MG [58]. Gulmez Sevim et al. demonstrated that OSDI score, BUT and lissamine green staining were significantly correlated with MGD grade and MG area loss [59]. Although functional and morphological changes in MG are often thought to be well correlated [60], meibography alone does not appear to be sufficient for reaching the diagnosis of MGD, but it should be considered in the context of other clinical parameters (e.g., BUT, lid margin examination) or other imaging techniques (e.g., IVCM).

Interferometry
The tear film lipid layer is chiefly composed of meibomian lipid, which is derived from the marginal reservoirs of the lids. The lipid layer plays a critical role in tear film stability for the maintenance of ocular surface health by preventing excessive evaporation of the aqueous layer. In obstructive or hyposecretory MGD, thinning of the lipid layer leads to excessive evaporation, causing evaporative-type DED [61]. When adequate light hits an oily layer, the result is the generation of an interferometric fringe. Furthermore, interference patterns are produced due to the phase difference between the light reflected from the lipid layer and the light that is reflected from the corneal epithelium [62,63]. Interferences by thin films display different colors, depending on the thickness of the film, from a dark color, caused by a thinner film area, to a brighter color, caused by a thicker film area. Imaging-based ocular surface interferometry is a noninvasive technique that allows the measurement of lipid layer thickness (LLT) on a nanometer (nm) scale.
The DR-1α camera allows a qualitative analysis of the lipid layer. Yokoi et al. proposed a grading system based on the first stable frames from the DR-1α camera [76].
Goto et al. produced a computer-synthesized color chart of a human tear lipid interference image for the conversion from tear interference color information to tear lipid layer film thickness data [77]. By using the DR-1α camera, it is possible to evaluate the kinetic spread and stability of the lipid layer. Goto et al. evaluated the speed and pattern of lipid spread after eye opening and the stability of the lipid film after spread, and found that in patients with lipid layer deficiency, the lipid spread was slow and resulted in a vertically streaking and non-uniform pattern [78]. Arita et al. demonstrated that the DR-1α interferometer can measure the TMH as reliably as AS-OCT and that the interferometric TMH correlated with Schirmer's score. [79]. DR-1α interferometry device can assist in the differential diagnosis of different subtypes of DED; indeed, Arita et al., by comparing three interferometric patterns (pearl-like, Jupiter-like, or crystal-like), found a direct correlation with BUT, NIBUT and Schirmer test value [80].
The LipiView II (LVII) interferometer can quantitatively measure the average LLT by analyzing the interferometric pattern of the tear film, a partial blink rate, and uses an infrared light source for imaging the MG. Lee Y. et al. showed that the average LLT value has a significantly positive correlation with age, OSDI, and Ocular Staining Score, and no correlation with the Schirmer test type I, BUT, or meiboscore [81]. By using LVII, Weng demonstrated that younger patients with DED experienced more severe subjective symptoms, more incomplete blinks, and a thinner LLT [82].

Thermography
The stability of the tear film is strictly linked to the integrity of the lipid layer that controls evaporation. There is currently no commercially available instrument dedicated to the detection of tear film evaporation, but thermography permits an indirect evaluation of the evaporation rate by measuring the temperature of the ocular surface in a noninvasive manner, using a thermographic camera operating within the infrared range [83][84][85]. In 1995, Morgan et al., measured ocular surface temperature in patients with DED and reported that the surface temperature was significantly higher than in normal eyes, while the temperature at the center of the cornea of DED eyes became lower than that in normal eyes after sustained eye opening [86]. In the last decade, advances in technology have allowed the measurement of the ocular surface temperature with increasing accuracy, resolution, and speed [87]. Su et al., showed a strong spatial and temporal correlation between BUT and tear film evaporation [88].
Kamao et al. reported that DED was associated with a greater decrease in the ocular surface temperature at 10 s after eye opening and suggested that measurements obtained over 10 s have sensitivity and specificity values of 0.83 and 0.80, respectively, for screening DED eyes [87].

Bulbar Redness Assessment
Bulbar redness (BR) is a non-specific ocular condition caused by the vasodilation of the conjunctival and/or anterior scleral blood vessels, which is itself caused by enhanced blood flow to and capillary permeability in the anterior ocular tissues in response to various stimuli [89]. Hyperemia is a feature of several ocular conditions: inflammation of the anterior segment of the eye [90], the adverse effects of glaucoma medications [91,92], contact lens wearing [93], allergic or infective conjunctivitis [94], and DED [95].
Since the first introduction by McMonnies and Chapman-Davies in 1987 of an imagebased bulbar hyperemia grading scale [89], several subjective rating scales have been introduced to improve inter-or intra-observer variability [96,97]. Scientific studies tried to overcome these limitations by developing novel models of computer-based photographanalysis techniques, which have now progressed to automated image analysis through the application of artificial intelligence (AI) [98]. Villumesen et al. developed the first computerassisted conjunctival hyperemia quantification system by using the pixel edge detection analysis of a 3 × 3 mm region of bulbar conjunctiva [99]. Willingham analyzed the mean relative redness (RR) and the blood vessel area ratio (VA) of digital images of the external eye by using a clinical photographic biomicroscope, an MVC video camera and computer imaging system, and custom software [100]. Rodriguez et al. developed an automated computer redness grading system (Ora, Inc., Andover, MA, USA, patent pending) for the evaluation of the location and prominence of fine horizontal conjunctival vessels in DED and demonstrated agreement with a group of investigators using the established clinical scale [101].
Wu et al. evaluated the clinical assessment of bulbar redness by using an Oculus Keratograph 5M Topographer (Oculus Optikgerate GmbH, Wetzlar, Germany) equipped with automated scanning and scoring software. They found a statistically significant correlation between the Oculus Index score and the scores determined using three subjective scales (the Institute for Eye Research scale, the Efron scale, and the Validated Bulbar Redness 10-picture grading scale) [96,102]. Moreover, they found that the Keratograph yielded higher intra-and inter-observer reproducibility, suggesting that the Keratograph could be a time-saving device [103].
Despite the introduction of novel incorporated instruments that make the clinical assessment of BR in DED quicker and easier, the majority of automatic grading systems are unable to differentiate bulbar conjunctival hyperemia from episcleral and scleral hyperemia; thus, there are many concerns over the use of BR as a biomarker [104].

Image Modality Based Computerized Detection Techniques
Through recent advances in AI and the rapid progression of analytic techniques, researchers are trying to overcome the main pitfalls of clinical testing, such as the long time required for acquisition and the need for skillful maneuvers [105]. The aim of the computerization of clinical tests is to help clinicians to increase accuracy and reduce the time taken to form a diagnosis.
In the last decade, several computerized DED detection techniques have been proposed, each based on different technologies. The first attempt to automatically detect DED was undertaken by Yedidya et al., who used a multi-step algorithm to evaluate BUT from an eye video that was captured using an Eyescan device [106]. Ramos et al. proposed an automatic methodology for characterizing tear film dynamics over the exposed corneal surface from the emergence of the first break-up in the tear film until the subsequent blink. To this end, the BUT measurement was computed, and the break-up areas were segmented in each video frame to analyze other break-up parameters, such as the size or spatial extension of the BUT [107]. Su et al. proposed an automatic method to detect the BUT area using a deep learning convolutional neural network (CNN) model, a hierarchical multilayered neural network that can learn visual patterns directly from image pixels [108,109]. The proposed CNN model detected break-up with an accuracy rate of 98% [110].
Two techniques based on the interference pattern of the lipid layer have been proposed. The first is a computer-aided system to support DED diagnosis based on tear film maps (CASDES), and the second is called iDEAS. The former system, which analyzes the images acquired by Tearscope, allows the recognition of dry area regions in agreement with the region annotated manually by the ophthalmologist [111]. The latter is a web-based system for DED assessment, in which, once the patient's interference pattern images are uploaded, a support vector machine classifier based on statistical learning theory allocates the images into five categories based on Guillon classification [112,113]. Acharya et al. developed a technique based on thermal infrared images. The result produced by this technique is binary; that is, input images of either normal eye or dry eye are converted into 1D data and then fed into different classifiers, such as k-Nearest Neighbor (KNN), decision tree, Probabilistic Neural Network (PNN), SVM and Naive Bayes (NB). The authors obtained an average sensitivity of 99.8%, specificity of 99.8% and classification accuracy of 99.8% using PNN and KNN classifier [114].

Pros and Cons of Noninvasive DED Imaging
The novel non-invasive diagnostic techniques for DED offer numerous advantages over conventional tests. The main advantage is related to their automated nature, which means that they do not require the clinician's judgment to determine a score. This is clinically relevant as subjective DED markers, such as fluorescein BUT and corneal staining, feature low inter-observer repeatability due to their lack of standardization [4]. Moreover, most of these novel diagnostic techniques do not require direct contact with the eye, and therefore have little/no effect on the volume and properties of the tear film. Thus, they can be used as screening tools (e.g., prior to ocular surgery) by trained medical personnel (not necessarily an ophthalmologist), before proceeding with more invasive ocular surface examinations in case of the detection of abnormal values. Furthermore, they provide clear and detailed reports summarizing all the results of the tests for each patient examination, which can be used for future reference and comparison (Figures 1 and 2). The daily report is useful for educating patients and encouraging treatment compliance: "A picture is truly worth a thousand words". All-in-one devices (one machine for more tasks) make it possible to reach the diagnosis of DED in less time compared to a traditional workup (e.g., 3 min are enough to perform a complete ocular surface workup with Idra), and represent a guide for imaging-based treatment: the deficient layer(s) of the tear film is/are identified and a targeted therapy can be prescribed. Similarly, monitoring patient's course over time is also made easier by using these devices and the trend of a single parameter (e.g., NIBUT) after a given therapy can be graphically reported and analyzed ( Figure 3).  Nevertheless, there are some drawbacks to these new diagnostic techniques that need to be mentioned. In particular, the cost may represent a major limitation for their widespread acceptance, and cost-effectiveness evaluations are still required to support their adoption on a large scale. Moreover, ophthalmological conditions other than DED may cause some phenomena that reduce the diagnostic accuracy, particularly if the image interpretations are not performed by an ophthalmologist and/or if the slit lamp examination is not performed. For instance, conjunctivochalasis may hamper the accurate measurement of TMH, leading to overestimation of the value (Figure 4), while conjunctival lesions, such as pterygium, may significantly alter bulbar redness by increasing its value in the affected area ( Figure 5). It should also be noted that novel tests cannot yet replace all the traditional DED metrics and slit lamp examination is still required to complete the diagnostic workup.  Finally, we would like to provide some recommendations for the improved use of noninvasive diagnostic tools. Firstly, as in traditional DED tests, in the noninvasive workup attention should be paid to the order in which diagnostic exams are performed. In fact, although these tests usually do not require contact with the ocular surface, lightinduced tearing should be considered, since it could influence the results of subsequent examinations. Secondly, in the case of significant differences between the two eyes of the same patient, results should be interpreted with caution, and multiple evaluations of both eyes are advisable.

Conclusions
DED represents a very frequent ocular disease worldwide and its prevalence is continuously growing [115]. Several diagnostic tools have recently been introduced in clinical practice to assist clinicians in the (early) diagnosis and monitoring of DED; furthermore, these devices allow the identification of the affected layer(s) of the tear film and represent a guide for imaging-based targeted treatment. The information generated provides objective results and pictures that are useful for helping patients' awareness about DED, thus improving treatment compliance. Soon, the advent of AI may further help clinicians in this complex diagnostic pathway. In this direction, further, larger-scale, clinical trials should provide more evidence on the role of these emerging diagnostic techniques in the DED diagnostic battery by establishing universal cut-off values for each exam.

Conflicts of Interest:
The authors declare no conflict of interest.