C37982: Point of care diagnostics

Dry eye is complex, often involving a combination of aqueous tear deficiency and evaporative stress that leads to inflammation and hyperosmolarity1 and defined by the DEWS study2 as ‘a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.…the core mechanisms of dry eye are driven by tear hyperosmolarity and tear film instability. Tear hyperosmolarity causes damage to the surface epithelium by activating a cascade of inflammatory events at the ocular surface and a release of inflammatory mediators into the tears. Epithelial damage involves cell death by apoptosis, a loss of goblet cells, and disturbance of mucin expression, leading to tear film instability.’

The challenge of diagnosing dry eye is finding an objective method to measure its severity. Traditional clinical tests for dry eye, including the Schirmer test, ocular surface staining, tear film break-up time (TFBUT), and meibomian gland grading, all provide useful diagnostic information. However, they suffer from variability. Add to this the common disconnect between symptoms, clinical signs, and diagnostic test results makes the diagnosis and treatment of dry eye challenging.2,3 New and emerging clinical tests for dry eye are increasingly specific and quantitative, moving us further toward that goal.3

POCT

Point of care diagnostics (or point of care testing, POCT) bypass the need for sophisticated laboratory systems by using new technologies to diagnose, determine and deliver management plans in a timely manner for a range of health concerns in the chair, or ‘point of care’. It has been estimated that diagnostics account for 2 per cent of the cost of healthcare yet can affect 60-70 per cent of treatment decisions. Common examples of POCT range from blood glucose screening and pregnancy testing to assessing factors associated with heart disease, stroke and malignancies.4,5,6

POCT is beginning to impact upon eye care with new and developing dry eye tests focusing on identifying specific biomarkers and taking precise, non-invasive measurements. While none of these has yet displaced traditional dry eye tests, the new tests add objectivity, reproducibility, and new insights into disease progression and treatment efficacy. Devices currently available include InflammaDry (Rapid Pathogen Screening, Inc/NicOx) and the TearLab Osmolarity System (TearLab Corporation) for dry eye evaluation and AdenoPlus (Rapid Pathogen Screening, Inc/NicOx) for adenovirus detection. We will now review these (and other) POCT systems and how they may integrate into a clinical setting. But first, a brief revision of tears.

Tear structure and function

The tear film is responsible for providing a smooth refractive surface for clear vision, maintaining the health of corneal and conjunctival epithelia, and acting as the first line of defence against microbial infections. Table 1 summarises the major components of human tears.

The aqueous layer of the tear film consists of water, electrolytes, peptide growth factors, immunoglobulins, cytokines, vitamins, antimicrobials, and hormones secreted by the lacrimal glands. Matrix metalloproteinases (MMPs) are proteolytic enzymes (produced by inflammatory/immune cells and the ocular surface when under stress).

Of the many types of proteins present in the tears (some estimates suggest as many as 80 to 100), four are present in large amounts; lysozyme, lactoferrin, lipocalin, and sIgA. All contribute to the primary defence system of the ocular surface through a mixture of non-specific immunity (lysozyme, lactoferrin) and the specific immunity of antibodies (secretory immunoglobulin A or sIgA).7

In aqueous-deficient dry eye syndrome, the concentration of lysozyme, lactoferrin, lipocalin and sIgA are reduced, compromising the integrity of the defence system. This may make the ocular surface more susceptible to infection in addition to contributing to the symptoms of dry eye.8,9 Interestingly, there exists a near linear relationship between lactoferrin and the tear-secreting function of the lacrimal gland10 and this allows lactoferrin to serve as an accurate biomarker for assessing aqueous production.11

Electrolytes present in the tear film include sodium, potassium, magnesium, calcium, chloride, bicarbonate, and phosphate ions. The electrolytes dictate the osmolarity of tears, acting as a buffer to maintain a constant pH and contributing to the maintenance of the epithelial integrity of the ocular surface. An increase in osmolarity of the aqueous layer is a global feature of dry eye syndrome, damaging the ocular surface directly and indirectly by triggering inflammation.12,13

Lipids derive from both the meibomian glands and as lipocalin-associated lipid, apparently delivered with the protein from the lacrimal gland. Lipid production promotes tear film stability by reducing evaporation from the open eye. Meibomian gland dropout is associated with the effects of ageing on meibomian gland secretions and anatomy.14

This brief overview of tear constituents forms the basis of interest currently shown in potential biomarkers of dry eye disease and which go some way to explain the often poor correlation between symptoms and conventional test results encountered in clinical practice.

Osmolarity biomarkers

Osmolarity is a measure of solid particles in a solution and is expressed in milliosmoles per litre (mOsmol/L). Hyperosmolarity implies an increased number of particles in the solution.15

Tear hyperosmolarity is well established as a defining characteristic of dry eye disease16 and represents an increased concentration of solutes due to reduced tear production and/or increased tear evaporation. Osmolarity testing has been declared the ‘gold standard’ of objective dry eye diagnosis, and the single best marker of disease severity.17,18

Osmolarity testing

Osmolarity instruments are used to test all kinds of organic matter including blood, serum, plasma, urine, bili-rubin, milk, cell culture media, and many others, including, of course, tears. The challenge of diagnosing dry eye through osmolarity has been to objectively measure it within a clinical setting.

Of the three methods used for measuring tear osmolarity, the TearLab Osmometer (TearLab) is used most commonly in a clinical setting and utilises the method of electrical impedance (Figures 1a and 1b). This method correlates well with the laboratory-based freezing point depression technique (considered the gold standard) and the vapour pressure technique, both of which are found more commonly in research.19

[CaptionComponent="2757"]

Electrical impedance (used in blood glucose monitors) is a technique whereby the ionic content of a fluid can be characterised by its electrical properties. Changes in the composition or concentration of the ions within the tear fluid will affect its electrical conductivity. This change correlates to its osmolarity measurement.

[CaptionComponent="2758"]

The TearLab osmometer consists of two hand-held analyser pens (each with disposable test cards or chips) and a docking station. The test card houses the so-called ‘lab on a chip’ at the tip of the analyser pen (Figure 2) and contains all the technology for the collection and analysis of the 50 nanolitre tear sample (smaller than the size of the full stop at the end of this sentence), providing a way of measuring tear osmolarity that is ‘almost’ independent of volume. This single-use microchip embedded with gold electrodes measures the electrical impedance of the tear fluid sample in a tiny channel in the chip.20,21

[CaptionComponent="2759"]

To use the instrument, the clinician inserts a disposable test card into the pen and places the tip of the handheld pen flat down onto the inferior lateral meniscus of the tear film (Figure 3). A very fine capillary channel is found on the underside of the microchip which automatically draws up the 50nL of tear fluid by passive capillary action. The pen will beep and a green light will switch off, indicating successful collection. The pen is then docked into the table-top reader and a calibration code (from the test card) is entered. The results are received within 10 seconds.19,20

[CaptionComponent="2760"]

Osmolarity is measured in milliosmoles per litre reading, and the higher the reading, the ‘drier’ the eye. Of the several suggested dry eye cut-off values in the literature, 308mOsms/L represents the cut-off for earlier, more mild, forms of the disease. Use of this value as a diagnostic has a high sensitivity (95 per cent) and specificity (88 per cent).16

Normal tear osmolarity is equivalent to normal blood osmolarity, which averages between 280mOsms/L and 300mOsms/L. Mild dry eye disease beginnings at 308mOsml/L, mild to moderate between 308mOsml/L to 328mOsml/L and severe as greater than 328mOsml/L.20

Variability, often considered the hallmark of the disease, of about 5mOsms/L between tests and between eyes or day to day is considered normal, with variability greater than 10mOsms/L considered a sign of dry eye.21,22

As osmolarity can be affected by external influences, precautions are needed to improve accuracy. As a general rule, no procedure that can alter the tear fluid should be undertaken within two hours prior to TearLab testing. These would include:

• InflammaDry
• Tonometry
• Tear break-up time
• Schirmer testing
• Slit-lamp examination
• Use of topical eye drops.

Interestingly, the manufacturer states that readings are not affected by contact lens use.21 Ongoing therapies, such as punctal plugs, topical eye drops, oral medication or nutritional supplementation, need to be factored into interpretation of the osmolarity score.

Tear osmolarity can now be considered a suitable test to incorporate into a clinical setting with good performance in dry eye diagnosis. It offers better sensitivity and specificity than other clinical tests alone, particularly in cases of moderate to severe dry eye. It has been suggested that TearLab results correlate with disease better than current clinical tests and could be effective as a single objective test for the discrimination of those with dry eye from those without the condition.19 Generally the osmolarity scores should be combined with slit-lamp examination of the anterior segment to aid in differentiating the type of dry eye and in tailoring management decisions (for example assessing meibomian gland involvement), or diagnosing non dry eye related ocular surface conditions (such as allergies, infection, and structural factors).3,15

Matrix metalloproteinases (MMP-9) biomarkers

It is known that inflammatory mechanisms are involved in the pathophysiology of dry eye disease and that changes in tear composition can destabilise the tear film and result in alterations to the ocular surface.23,24,25

Matrix metalloproteinases (MMPs) are proteolytic enzymes produced by stressed ocular surface and by inflammatory/immune cells. Increased activity of MMPs has been implicated in pathologic ocular surface changes.26 Among the MMPs, the non-specific inflammatory marker MMP-9 has been found to play an important role in wound healing and inflammation,23 and is primarily responsible for the alterations to the ocular surface that lead to a dysfunctional tear film.1

The expression of MMP-9 by the ocular surface epithelia in normal healthy eyes is low.27 In the normal tear film, the level of MMP-9 is between 3 and 40ng/mL; an MMP-9 level greater than 40ng/mL is indicative of non-specific ocular surface inflammation, correlating with the conventional diagnostic tests of symptom severity scores, fluorescein TBUT, corneal and conjunctival fluorescein staining scores.26

The changes in clinical findings can be attributed to the ability of MMP-9 to degrade epithelial tight junction and basement membrane proteins, leading to altered epithelial permeability and poor epithelial adherence.28 As MMP-9 is a non-specific marker of inflammation, a thorough patient history and assessment of clinical signs are necessary to confirm a diagnosis of dry eye, but other inflammatory conditions affecting the eye are generally not difficult to rule out. MMP-9 is elevated even in mild dry eye disease and may be a more sensitive diagnostic marker than traditional clinical signs alone.1,26

MMP testing

The lnflammaDry test (Figure 4) is based on the principle of lateral flow immunoassays using direct sampling micro-filtration technology and detects the presence of elevated MMP-9 in the tear fluid.4 While a positive result is not expressed in numerical units, the intensity of the positive result is generally thought to be proportional to the level of MMP-9 present. The brighter the stripe, the more MMP-9 activity is present.29,30 The test takes less than 30 seconds to perform and only about 10 minutes to produce results.31

[CaptionComponent="2761"]

The InflammaDry test consists of a sample collector (with sampling fleece), test cassette (containing gold labelled antibodies to MMP) and a buffer vial.30 The patient’s lower eyelid is lowered to expose the inferior palpebral conjunctiva. The sampling fleece is gently dabbed (not dragged) in multiple locations along the palpebral conjunctiva, releasing the lid after every two to three dabs to allow the patient to blink, until the sampling fleece glistens, indicating saturation. You then gently click the sample collector and test cassette together, ensuring the absorbent tip is uncovered. After immersing the absorbent tip of the test cassette into the buffer vial for 20 seconds, replace the protective cap onto the tip and lay the test flat on a horizontal surface for 10 minutes.

Once the background within the result window is white and 10 minutes have elapsed, the test may be accurately read.30 The solution in the vial is drawn up through the absorbent tip, picking up antibodies to MMP-9 (that have been labelled with nanoparticles of gold), and distributed throughout a piece of filter paper in the test cassette. This antigen-antibody complex is localised in the test window at an immobilised test line. The formation of a blue line at the control zone indicates the correct application and performance of the test, and must appear for the test to be valid. The presence of both a blue line in the control zone and a red line in the result zone indicates a positive result. An uneven or incomplete red line is due to an uneven distribution of tear fluid on the sampling fleece. If the red line is faint, incomplete over the width of the test strip, or uneven in colour, it must be interpreted as positive. The intensity of the positive result is generally thought to be proportional to the level of MMP-9 present: The brighter the stripe, the more MMP-9 activity is present.30,32 A positive result indicates the presence of MMP-9 =40ng/ml. The presence of only a blue line in the control zone indicates a negative result. A negative result is indicative of an MMP-9 level <40ng/ml. MMP-9 appears as a potentially useful biomarker for diagnosing, classifying, and monitoring dysfunctional tear syndrome.29

Testing tips

Accuracy can be improved by following certain steps:

• MMP-9 is a nonspecific indicator for the presence of inflammation. A positive test result should not be used as the sole basis for treatment or other management decisions
• If you are doing both an osmolarity test and InflammaDry on a patient, you should do the former test32
• Slit-lamp biomicroscopy is required to aid in the overall assessment of the ocular surface
• Since it is looking for an inflammatory protein produced throughout the lacrimal system, there is no risk of result variability problems associated with reflex tearing31
• InflammaDry should be performed prior to instilling ocular anaesthetic, topical dyes, or performing Schirmer testing
• A full history of ocular surgery or infection, allergic conjunctivitis, or other ocular surface diseases should be ascertained as they might be the cause of elevated levels of MMP-9
• Medication history is needed as systemic immunomodulators, topical or oral steroids, cyclosporine, tetracycline, and topical azithromycin, are known to inhibit metalloproteinase activity. Use of these medications may lead to false negative results
• The accuracy of the test is not affected by use of most topical medications. However, a minority of medications can affect the accuracy if administered within two hours of testing (Iquix, Quixin, Proparacaine and Trusopt)
• Patients with a history of contact lens use or recent ocular surgery have yet to be studied, so no data supports any claims for safety and efficacy in these populations.

Using the data

An objective, albeit non-specific, test like InflammaDry can add important diagnostic information with high sensitivity (85 per cent) and specificity (94 per cent).33 Dry eye is often under-diagnosed as a result of poor communication between the clinical assessment of dry eye severity between clinicians and patients. This often leads to a lack of effective treatment. A fairly common presentation of dry eye disease can involve symptoms with little improvement with artificial tears coupled with an unremarkable slit-lamp appearance.1 A positive InflammaDry result confirms the presence of ocular surface inflammation and should suggest the use of anti-inflammatory therapies.34 Corticosteroids inhibit inflammation and decrease production of MMP-9 by the corneal epithelium. Doxycycline preserves the tight junction network, preserving the corneal barrier function and leads to a reduction in the production and activity of MMP-9. The use of cyclosporine is well known in the US, and inhibits T-lymphocyte proliferation and decreases MMP-9 expression in the conjunctival epithelium.35

Because dry eye disease is a progressive disorder, the earlier that treatment is started, the better the response to therapy. If additional treatment such as punctal occlusion is required, identifying and treating patients with ocular inflammation beforehand prevents the potential accumulation of inflamed tissues that may exacerbate the ocular surface disease.

A negative InflammaDry test suggests a non-inflammatory cause of dry eye disease, in which case the clinician may consider artificial tears or punctal plugs as possible options.1

Other tests

Both TearlLab and InflammaDry test for different markers, so the clinician would not necessarily have to choose one over the other. Both offer high sensitivity, with InflammaDry offering better specificity. TearLab appears to be more helpful in determining the severity of dry eye disease, while InflammaDry helps formulate a treatment plan when it is positive.32

But what of other potential investigations currently available?

Lactoferrin and IgE biomarkers

Another new tool designed to allow quantification of key markers in the tear film is the TearScan MicroAssay System (Advanced Tear Diagnostics). Although not currently available in the UK, it measures markers relating to both dry eye and allergy.11 The instrument has a high sensitivity and quantifies both lactoferrin (83 per cent) and IgE (93 per cent) in the tears with high specificity (96 per cent for both tests)3,11

Lactoferrin as mentioned earlier is an iron-binding protein secreted by the lacrimal gland and exhibits anti-microbial qualities. It is produced in quantities roughly linear to other secretions produced by the lacrimal gland, including aqueous tears.3 Lactoferrin’s direct actions have little to do with dry eye disease, but this near-linear relationship between lactoferrin and the tear-secreting function of the lacrimal gland allows lactoferrin to serve as an accurate biomarker for assessing aqueous production.36 The concentration of lactoferrin has been shown to be significantly decreased in tears of dry eye.11

Tear IgE has long been established as the key immunologic mechanism in allergic conjunctivitis37 and the measurement of tear IgE concentrations can be used to confirm the condition.38 Total IgE in tear fluid increases with the severity of the allergic response,39 making the test useful not only for making a clinical diagnosis of allergic conjunctivitis, but also for the severity of the allergic presentation.

Allergy can mimic the signs and symptoms of dry eye disease.40 The evaluation of IgE and tear dynamics are important for the differential diagnosis of patients with suspected allergic conjunctivitis and dry eye.41 The TearScan system enables the testing for two biomarkers essential in the differential diagnosis of dry eye (evaporative versus aqueous deficient) and ocular allergy .The two conditions have different mechanisms of action and are managed differently, yet an allergic episode can aggravate a concurrent dry eye, worsening symptoms that had been tolerable.

The TearScan microassay test is not as portable as other POCT, requiring use of a slit lamp to collect 0.5 microlitres (500nL) of tears from the patient’s canthus via a micropipette. The sample is placed in a diluent and shaken to amplify the biomarker. This mix is then put in a small well in a disposable cassette and the cassette is introduced into the table-mounted microassay unit. The unit measures the amount of biomarker in the test sample via a reflectance photometer specifically designed to interpret concentration from small tear samples. The test time for lactoferrin is 90 seconds, while the IgE test takes five minutes.

The data obtained from TearScan system has the potential to reduce the ‘shotgun’ approach to dry eye therapies, allowing clinicians to address the actual cause, be it an aqueous production problem with or without an allergy component. The applications of the TearScan system go beyond the differential diagnosis of dry eye and ocular allergy. Knowing the presenting levels of both lactoferrin and IgE in potential contact lens wearers and refractive surgery patients may help guide treatment options and avoid unwanted outcomes.

Adenovirus biomarkers

Another example of lateral flow immunoassays using direct sampling microfiltration technology is the AdenoPlus test (Rapid Pathogen Screening, Inc/NicOx), capable of detecting the presence of adenovirus serotypes in the tear fluid. Adenovirus is the most common cause of acute viral conjunctivitis, with epidemic keratoconjunctivitis (EKC) (serotypes 8,19 and 37) being one of the most severe forms of conjunctivitis we face.42

EKC is highly contagious and can present with bilateral (asymmetric) inferior palpebral follicular conjunctivitis, with epithelial and stromal keratitis.5,43

Although duration can vary, EKC generally has an initial seven-day incubation period, followed by an active infection persisting for seven days, then an immune response and the presence of pseudomembrane and sub-epithelial infiltrates (the latter leading to decreased vision) for another seven days, concluding with seven additional days of morbidity.44,45

Initially, many signs and symptoms of viral conjunctivitis overlap those of bacterial conjunctivitis, and can be indistinguishable by empirical observation (some ophthalmologists have quoted detection accuracy rates among clinicians as ranging from 25-60 per cent), making diagnosis difficult. This can lead to inappropriate antibiotic treatment, yielding little therapeutic effect while at the same time increasing the probability of localised resistance. It is essential, therefore, to make a prompt diagnosis and begin treatment immediately to decrease the infectious potential of EKC as well as limit its associated complications.5,43,46

Traditional tests for EKC involved costly and time-consuming diagnostic lab testing.43,46 The AdenoPlus test (Figure 5) has been found to offer a high degree of sensitivity (88 per cent) and specificity (91 per cent) and can be used in a clinical setting.47

[CaptionComponent="2762"]

The test involves touching a sampling fleece to the lower bulbar conjunctiva (Figure 6), which allows it to absorb tears. The saturated fleece is snapped into a cartridge. A reagent is applied and absorbed into the system. After 10 minutes, the result will be available for review. A single line in the reading window indicates a negative result for adenovirus, whereas two lines support a positive adenovirus diagnosis.48

[CaptionComponent="2763"]

Since EKC is contagious yet self-limiting, management often includes patient education and other palliative treatment, such as cool compresses, artificial tears and vasoconstrictors. In instances of pseudomembrane and/or sub-epithelial infiltrates, topical corticosteroids have been advocated.49,50,51

Others

As dry eye has a significant evaporative component resulting from dysfunctional meibomian gland produced lipids, more attention is focused on the treatment of meibomian gland dysfunction to remedy evaporative dry eye. As a result of this, more sophisticated lipid diagnostic technology is also becoming available. Tear film interferometry has been developed to visualise and measure the tear film lipids as they spread over the ocular surface.

The LipiView Ocular Surface Interferometer (TearScience) is a table-top device that illuminates the tear film and records and measures the interference pattern of the reflected light.3 This ‘interferogram’ is captured and analysed by software included with the device, allowing lipid layer thickness to be determined. If the lipid layer is too thin or the tear film composition abnormal, then the associated LipiFlow Thermal Pulsation System treatment may be advised, provided the meibomian glands remain expressible.

The Oculus Keratograph (Oculus, UK distributor Birmingham Optical Group) offers qualitative and quantitative assessment of the tear film using non-invasive scanning software. The device has a tear film scan module that provides an objective assessment of tear break-up time, Placido ring-based corneal topography. It can also assess tear meniscus height. A meibography function is also available to monitor the status of the meibomian glands (a review of meibography will be published in Optician early next year).

Other additional tests include use of the OCT which, benefiting from less reflex tearing than other traditional tear film assessments, offers a non-invasive, highly accurate measurement of the tear film and its changes in response to various therapies. Confocal microscopy has been used to study peripheral corneal nerves, and may show promise in monitoring progression of dry eye.3

Conclusion

The increasing availability of POCT techniques offer greater objectivity to dry eye disease diagnosis and, when combined with traditional clinical tests for dry eye, add more detail to the diagnosis and management of dry eye.

Model answers

The correct answers are in bold text

1 Which of the following statements about lactoferrin is true?

A It is a lipid

B It is secreted by antibodies

C It is found in increased amounts with increased secretion by the lacrimal gland

D It is found in increased concentrations in aqueous deficiency states

2 Which of the following is indicated by an osmolarity reading of 320 mOsml/L?

A Hypo-osmolarity

B Normal

C Mild to moderate dry eye disease

D Severe dry eye disease

3 Which of the following best explains the term specificity with relation to diagnostic testing?

A The ability to detect a disease

B The ability to confirm a normal as not having a disease

C The ability to detect one disease from another

D The ability to distinguish one patient from another

4 A patient is found to have MMP-9 levels of 45 ng/ml. Which of the following is true?

A The patient is likely to have normal healthy eyes

B The patient has adenovirus infection

C The patient is likely to be symptomatic in the absence of any significant observable clinical signs

D The patient is likely to have a non-specific ocular surface inflammation

5 Which of the following drugs might lead to a falsely low MMP-9 reading?

A Prednisolone

B Digoxin

C Warfarin

D Atenolol

6 Which of the following is indicated by raised IgE levels?

A Viral conjunctivitis

B Bacterial conjunctivitis

C Allergic conjunctivitis

D Chlamydial conjunctivitis

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Dr Rohit Narayan runs a dedicated dry eye clinic in Nuneaton