Microassay system tests for two tear film biomarkers

Since the late 1980s, the ophthalmic-scientific community has widely recognized that certain ocular surface disorders could be accurately diagnosed and graded by analyzing certain biomarkers in the tear film. Units for measuring tear film osmolarity have been commercially available for several years, as has a diagnostic test for adenovirus. The TearScan MicroAssay System (Advanced Tear Diagnostics [ATD]) is a new in-office diagnostic laboratory-testing platform for tear lactoferrin and IgE.

Dr. BowlingFor years, doctors have had to rely on patient symptoms and subjective evaluation of clinical signs to diagnose dry eye. With this system, we now have more objective means of determining whether or not a patient has dry eye or ocular allergy. Moreover, the device has significant applications outside of dry eye.

Tear biomarkers

The TearScan system measures the amount of lactoferrin and IgE in the patient’s tear film. Lactoferrin, lysozyme, and tear lipocalin are the main tear proteins of lacrimal gland origin. The concentrations of these proteins have been shown to remain constant in both non-stimulated and stimulated tear samples.¹ Lactoferrin, an iron-binding protein secreted directly by the acinar cells of the lacrimal gland, is an important component of the non-specific host defense mechanism of the external eye² and exhibits anti-bacterial, anti-viral, anti-fungal, anti-parasitic³ and anti-inflammatory⁴ biologic properties. Lactoferrin is produced in quantities roughly linear to other biologics produced by the lacrimal gland, including aqueous tears.

Figure 1. Disposable microcassetteWhile lactoferrin’s direct actions have little to do with dry eye disease, this near-linear relationship between lactoferrin and the tear-secreting function of the lacrimal gland⁵ allows lactoferrin to serve as an accurate biomarker for assessing aqueous production. The concentration of lactoferrin has been shown to be significantly decreased in tears of dry eye not associated with Sjögren’s syndrome, as well as in those dry eye patients with Sjögren’s or Stephens-Johnson syndrome.⁶ Tear lactoferrin has shown a high specificity (95%) and good sensitivity (72%) when combined with qualitative tear tests (Schirmer’s, vital dye staining) in the diagnosis of dry eye.⁷ In a study of 156 dry eye patients experiencing minimal ocular irritation, statistical analysis showed the Schirmer test in conjunction with lactoferrin assay provided the best balance between high test sensitivity and false positive rates.⁸

Figure 2. TearScan Microassay unitsThe ocular allergic response results from exposure of the conjunctiva to an environmental allergen and binding with specific IgE on the conjunctival mast cells triggering a cascade of inflammatory mediators.⁹ Tear IgE has long been established as the key immunologic mechanism in allergic conjunctivitis¹⁰ and the measurement of tear IgE concentrations can be used to confirm the condition.¹¹ Because total IgE in tear fluid increases with the severity of the allergic response,¹² determining the total IgE concentration is useful not only for making a clinical diagnosis of allergic conjunctivitis, but also for the severity of the allergic presentation.

In dry eye disease, the presence of an allergen often mimics the signs and symptoms of dry eye disease,¹³ so it would be clinically useful to assess the presence of an allergen during the initial dry eye workup to confirm, or rule out, its presence. The evaluation of antigen-specific IgE and tear dynamics are important for the differential diagnosis of patients with allergic conjunctivitis and dry eye.¹⁴ With the TearScan system, we have the ability to test for two biomarkers essential in the differential (evaporative vs. aqueous deficient) diagnosis of dry eye and ocular allergy . This capability is clinically important because 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 test

The microassay tests begin by collecting 0.5 microliter 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 (see Figure 1), and the cassette is introduced into the microassay unit, a small device that can easily sit on counter space in your office (Figure 2). The microassay unit measures the amount of biomarker in the test sample via a reflectance photometer specifically designed to interpret concentration from small tear samples.

Normal and abnormal levels of both lactoferrin and IgE according to ATD’s studies are shown in the following tables.

The test time for lactoferrin is approximately 90 seconds, while the test time for IgE is approximately 5 minutes. ATD reports a 83% sensitivity for the lactoferrin test and a 93% sensitivity for the IgE microassay. The specificity for both tests exceeds 96%. Each test has its own separate CPT code:

Lactoferrin           83520 (immunoassay for analyte other than an infectious agent)

IgE                      82785 (Immunoglobulin E, quantative, total)

Each microassay is reimbursable through Medicare and private insurers. The cost for the disposable cassettes is $10 per eye per test. The American Medical Association recommends using the “-59” modifier for the second eye to indicate a “distinct procedural service.” The unit output can go to a paired or wireless printer, and can push the data to a server- or cloud-based EMR. The test unit requires a license from Clinical Laboratory Improvement Amendments (CLIA), and this license number must be shown on all claims for reimbursement.

What is a CLIA license or waiver?

The Centers for Medicare and Medicaid Services (CMS) regulates all laboratory testing-except research-performed on humans in the U.S. through CLIA.¹⁵ Any person or facility that performs laboratory tests on human specimens for the purpose of diagnosis or treatment is required by law to have a CLIA certificate. As defined by CLIA, waived tests are categorized as “simple laboratory examinations and procedures that have an insignificant risk of an erroneous result.”¹⁶ The categorization of commercially marketed in vitro diagnostic tests under CLIA is the responsibility of the Food and Drug Administration (FDA). This categorization includes the process of assigning commercially marketed in vitro diagnostic test systems into one of three CLIA regulatory categories based on their potential for risk to public health:¹⁶

• Class I: Waived tests (i.e., adenovirus)

• Class II: Tests of moderate complexity

• Class III: Tests of high complexity

The ATD microassay tests of are considered Class II tests and, as such, require all persons involved in the taking, processing, or reading of lab samples to be properly licensed. Upon receipt of the proper license, each laboratory will be issued a CLIA number that must accompany any and all reimbursement requests. The regulatory burden associated with applying for, receiving, and maintaining a CLIA Class I (waived) or Class II license is not overwhelming. In fact, it is pretty simple and straightforward. “ATD will assist with the CLIA application process,” says Jeffry Busby, global sales officer for ADT.

While CLIA regulations cover specific educational, training, and experience requirements needed for proper CLIA licensure by the federal government, states have varying requirements. With few exceptions, optometrists and ophthalmologists meet the regulatory requirements to serve as lab director.¹⁷ “While currently the device requires a Class II CLIA license, ADT is actively pursuing a CLIA waiver for all tests,” said to Marcus Smith, chief executive officer of ADT.

Other clinical utility

The applications of the ATD system go far beyond the differential diagnosis of dry eye and ocular allergy. In a small, unpublished pilot study of 32 LASIK patients, by Bill Rafferty, OD, and Alan Carlson, MD, at Duke University, and Terry O’Brien, MD, of Bascom Palmer, the researchers found that preoperative lactoferrin levels may be associated with post-operative LASIK results. All the patients with low lactoferrin levels pre-operatively had a post-operative refractive correction of -0.25 D to -1.50 D. Only 19% of the patients with normal pre-operative lactoferrin levels had post-operative refractions outside the -0.25 to +0.25 range, while 80% of the patients with high pre-operative lactoferrin levels had post-LASIK hyperopic refractions of +0.50 D or greater. The researchers concluded “lactoferrin serves as an excellent marker for pre-existing conditions that influence the post-LASIK healing response. Pre-LASIK lactoferrin levels are a statistically significant predictor of post-LASIK spherical refractions.” The authors added: “Lactoferrin should be considered as one tool in assessing the pre-surgical corneal health of all LASIK candidates.”¹⁸

The level of IgE is increased in the eyes of some silicone-hydrogel wearers during an acute event of contact lens-related papillary conjunctivitis.¹⁹ Similarly, lactoferrin levels are decreased in patients with giant papillary conjunctivitis²⁰ and vernal conjunctivitis.²¹ 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.

“I’m excited about the science behind this technology,” says Young Choi, MD, a group practitioner in Homewood, AL. “I’ve been using the device for several months now, and the great thing about this test is that it’s accurate. The device helps me fine-tune the diagnosis in my dry eye patients.” Dr. Choi sees the benefits in surgical cases as well. “I perform the lactoferrin microassay on all my potential LASIK patients,” he adds. “It is now part of my LASIK pre-op protocol.”

The steadily growing use of in-office ocular diagnostic laboratory testing will, over time, become so widespread it will be considered vital to the practice of primary ocular care. This shift is inevitable as eyecare professionals better understand the significant clinical and commercial benefits of ocular lab testing. This device likely will gain a place in our ocular surface disease diagnostic regimen.ODT

References

1. Fullard RJ, Tucker DL. Changes in human tear protein levels with progressively increasing stimulus. Invest Ophthalmol Vis Sci 1991;32:2290-2301.

2. Ballow M, Donshik PC, Rapaz P, et al. Tear lactoferrin levels in patients with external inflammatory ocular disease. Invest Ophthalmol Vis Sci 1987;28:543-545.

3. Jenssen H, Hancock RE. Antimicrobial properties of lactoferrin. Biochimie 2009; 91(1):19-29.

4. Conneely OM. Anti-inflammatory properties of lactoferrin. J Am Coll Nutr 2001; 20 (5 Suppl): 389S-395S.

5. Danjo Y, Lee M, Horimoto K, et al. Ocular surface damage and tear lactoferrin in dry eye syndrome. Acta Ophthalmol (Copenh) 1994;72(4):433-437.

6. Ohashi Y, Ishida R, Kojima T, et al. Abnormal protein profiles in tears with dry eye syndrome. Am J Ophthalmol 2003;136(2):291-299.

7. DaDalt S, Moncada A, Priori R, et al. The lactoferrin tear test in the diagnosis of Sjogren’s syndrome. Eur J Ophthalmol 1996;6(3):284-286.

8. Goren MB, Goren SB. Diagnostic tests in patients with symptoms of ketatoconjunctivits sicca. Am J Ophthalmol 1988;106 (5):570-574.

9. Leonardi A, Motterle L, Bortlotti M. Allergy and the eye. Clin Exp Immunol 2008;153(S1): 17S-21S.

10. Friedlaender MH. Ocular allergy. Curr Opin Asthma Clin Immunol 2011;11:477-482.

11. Martinez R, Acera A, Soria J, et al. Allergic mediators in tears from children with seasonal and perennial allergic conjunctivitis. Arch Soc Esp Ofthalmol 2011;86(6):187-192.

12. Mimura T, Usui T, Yamagami S, et al. Relation between total tear IgE and severity of acute seasonal allergic conjunctivitis. Curr Eye Res 2012;37(10):864-870.

13. Bowling EL. Is it dry eye, allergy, or both? Rev Cornea Cont Lens. September 2012. http://www.revoptom.com/continuing_education/tabviewtest/lessonid/108499/. Accessed March 16, 2013.

14. Fujishima H, Toda I, Shimazaki J, et al. Allergic conjunctivitis and dry eye. Br J Ophthalmol 1996;80:994-997.

15. CLIA waiver. Available at: www.nm-pharmacy.com/Pharmacy_Resources/CLIA_Waiver_information/HowtogetCLIA-Waiver.ppt. Accessed March 16, 2013.

16. CMS. Clinical Laboratory Improvement Amendments (CLIA). Available at: www.cms.gov/Regulations-and-guidance/Legislation/CLIA/downloads/howtoobtaincertificateofwaiver.pdf. Accessed March 16, 2013.

17. Advanced Tear Diagnostics Web site. Available at : www.teardiagnostics.com/resources/technical-manuals/CLIA-regulations/. Accessed March 16, 2013.

18. Rafferty, Carlson, Cox, O’Brien, Myrowitz. Correlation of LASIK refractive outcomes with pre-surgical tear lactoferrin. Unpublished manuscript.

19. Zhao Z, Fu H, Skotnitsky CC, et al. IgE antibody on worn highly oxygen-permeable silicone hydrogel contact lenses from patients with contact lens induced papillary conjunctivitis (CLPC). Eye Contact Lens 2008;34(2):117-121.

20. Velasco-Cabrera MJ, Garcia-Sanchez J, Bermudez-Rodriguez FJ. Lactoferrin in tears in contact lens wearers. CLAO J 1997;23(2):127-129.

21. Rapacz P, Tedesco J, Donshik PC, et al. Tear lysozyme and lactoferrin levels in giant papillary conjunctivitis and vernal conjunctivitis. CLAO J 1988;14(4):207-209.

Take-Home Message

The TearScan MicroAssay System (Advanced Tear Diagnostics) enables a clinician to diagnose dry eye or ocular allergy. This in-office, diagnostic platform tests for tear lactoferrin and IgE could also be used for screening patients in advance of refractive surgery or contact lens fits.