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Monitoring VEGF levels with low-volume sampling in major vision-threatening diseases: age-related macular degeneration and diabetic retinopathy Min-Yen Hsu,ab Shih-Jen Chen,c Kuan-Hung Chen,a Yu-Chien Hung,b Hin-Yeung Tsaid and Chao-Min Cheng*a The purpose of this article is to demonstrate the capacity of paper-based ELISA (P-ELISA) to monitor VEGF in patients requiring treatment for vision-threatening diseases. The most commonly encountered visionthreatening diseases are age-related macular degeneration (AMD) and diabetic retinopathy (DR), both of which may require short-term or life-long anti-VEGF injection treatment therapy. Accurate measurement of VEGF concentration in aqueous humor can provide significant and timely information to diagnose the disease state. Adequate and precise therapy may consequently be provided. At odds with conventional diagnostic approaches is the fact that a maximum of only 200 microliters of aqueous humor can be safely removed from the eye for testing. Fortunately, new diagnostic platforms, such as P-ELISA, require only

Received 7th September 2014, Accepted 17th April 2015 DOI: 10.1039/c4lc01052c

minute volumes, i.e., approximately 2 microliters per test “well” and approximately 40 microliters total to quantify VEGF levels, and the testing process takes less than an hour. Thus, point-of-care (POC) diagnostics, such as P-ELISA, should be examined and improved upon as needed in order to develop an efficient tool for outpatient clinics and others to obtain semi-quantitative results that might facilitate accurate dos-

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ing of anti-VEGF treatment and delay or prevent the progression of AMD and DR.

I. Introduction Critical role of VEGF in the pathogenesis of two major visionthreatening diseases: age-related macular degeneration and diabetic retinopathy The loss of vision can significantly decrease the quality of life and results in a considerable healthcare burden in terms of both cost and effort required to provide appropriate care. The impact of blindness on individuals, families, and society is substantial, yet may be alleviated with adequate care, which starts with adequate diagnostics.1 In developed countries, the prevalence of age-related macular degeneration (AMD) and diabetic retinopathy (DR) has gradually increased.2,3 Both diseases are the leading causes of blindness. AMD accounts for 45% of low-vision patients in the United States, and every year 200 000 Americans lose significant vision due to AMD and require rehabilitation assistance.4,5 DR with complications

a

Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan. E-mail: [email protected] b Department of Ophthalmology, Taichung Veterans General Hospital, Taichung 407, Taiwan c Department of Ophthalmology, Taipei Veterans General Hospital, Taipei 112, Taiwan d Department of Ophthalmology, Taichung Tzu Chi Hospital, Taichung 427, Taiwan

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such as macular edema and hemorrhage accounts for 12% of all new cases of blindness annually in the USA.6 Both diseases are characterized by new vessel formation and fluid leakage at the posterior part of the eyeball, the retina or choroid, secondary to ischemia by degeneration (AMD) or ischemia (DR with vascular hypoperfusion). It has been shown that VEGF plays a critical role in the pathogenesis of both AMD and DR.7 Elevated VEGF levels within the eyeball lead to neovascularization or retinal edema.7,8 Several studies have also shown that the sample fluid from the anterior part of the eyeball, the aqueous humor, can be used to determine the VEGF level, which is proportionally correlated with the severity of these two diseases at the posterior part.9,10 For a decade, intravitreal injection of anti-vascular endothelial growth factor (VEGF) antibodies (bevacizumab, Avastin or ranibizumab, Lucentis) to neutralize the intraocular VEGF of diseased eyes has been commonly used to treat wet AMD, DR and retinal vein occlusion (RVO) with favorable results.11 These antibodies were injected at 4- to 8-week intervals for life and whether to treat or not depended on the functional and anatomical responses. It is estimated that 817 000 intravitreal injections of anti-VEGF antibodies were performed in the United States in 2008.12 Many research efforts have been undertaken to elucidate the pharmacodynamics of intravitreal injection of anti-VEGF monoclonal antibodies, in order to

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determine the optimal injection duration and to evaluate other therapeutic modalities.13–16 However, treatment response varied so therapeutic plans were highly individualized and implemented according to the response in each case. Approximately one third of AMD patients responded well after 3 loading injections and the improved response lasted for 12 months with quarterly injections, while one third required frequent dosing (monthly or bimonthly) and the other one third did not respond at all even with frequent injections.17 The reasons for this variation are unknown. Decisions for retreatment were based on the presence of edema in clinical ocular imaging studies, a symptom that appears before changes in visual acuity occur (Fig. 1). Patients were monitored via the use of ocular imaging and post-treatment visual acuity testing and follow-up monitoring and treatments were subsequently, and repeatedly, scheduled. Physicians were required to perform a number of imaging studies and use the results and treatment history to determine the timing of subsequent visits and treatment approaches.

do not provide any quantifying information regarding retinal function. In Fig. 1, schematic graphs illustrate the progression sequence of AMD and DR. It has been proven that VEGF levels within the eye increase before possible detection of structural changes brought about by FAG/OCT.18 The sequence of progression is as follows: (1) initiation of biochemical changes (e.g., VEGF elevation) (Fig. 1a); (2) retinal structural changes (e.g., edema, neovascularization detected by optical imaging modalities (FAG/OCT)) (Fig. 1b); (3) deterioration of organ function (e.g., impaired visual acuity) (Fig. 1c). As retinal structural changes occur, extensive damage to photoreceptors within the retina progresses, eventually resulting in the loss of retinal function. A lag of approximately 16 days exists between VEGF elevation and initiation of structural changes (Fig. 1a and b). Without prompt treatment to lower VEGF levels within the eye, a progressive cascade ensues that will result in irreversible visual acuity damage.15 Early detection of the rise of VEGF levels while the structure remains unaffected would provide for timely retreatment that may prevent vision loss.

Available optical imaging modalities for evaluation of treatment response: fluorescence angiography (FAG) and optical coherence tomography (OCT)

Available biochemical tools for VEGF evaluation: ELISA and Luminex bead-based ELISA

Optical imaging modalities such as fluorescence angiography (FAG) and optical coherence tomography (OCT) provide physicians with information related to structural changes (i.e., normal or abnormal structural changes, leakage or edema), but

Conventional plate ELISA requires large volumes (100 microliters) and a long incubation time (>4 hours in total) to quantify VEGF concentration. Luminex bead-based ELISA requires smaller volumes than conventional plate ELISA (50 microliters) and 3.5 hours to produce results. Both approaches

Fig. 1 Schematic graphs describing the relationship between VEGF concentration/retinal structural change/vision impairment and time before/ after intravitreal injections of the anti-VEGF antibody. The Y coordinates represent VEGF levels (red line), structural changes (green line) and vision impairment (purple line). The X coordinates represent time. (a) The VEGF level can be detected in less than previous scheduled intervals by paperbased ELISA (P-ELISA) or other POC diagnostics that use tiny sampling volumes and the elevated VEGF level can be determined at that point. Thus, intravitreal injection of anti-VEGF antibodies can be performed earlier than presumed by optical imaging modalities. (b) Optical imaging modalities: FAG and OCT are examinations of structural changes in the retina. FAG and OCT findings are determinable only after approximately 16 days following initial VEGF elevation. In this manner, progression of the disease cannot be determined before retinal structural damage has begun. POC biochemical diagnostics (e.g., P-ELISA, Luminex or others) can detect VEGF elevation before retinal structural changes start. (c) Vision impairment occurs after retinal structural changes begin.

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require a series of incubation and washing steps, and neither is well suited for VEGF analysis in point-of-care (POC) or outpatient clinical settings.

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II. New innovations in microliterscaled sample diagnostics The current standard for relevant disease biomarker examination requires highly invasive procedures such as vitreous humor sampling. It has been demonstrated that, at most, 200 μL of aqueous humor (usually 50–100 μL per tapping) can be collected from the anterior chamber of the eye before the threat of anterior chamber collapse ensues. Thus, dilution methods for aqueous sampling, commonly performed by most centralized laboratories, are widely used for ELISA (Fig. 2a).19 An ELISA plate requires a minimum sample volume of 100 microliters and the process can only be carried out twice for (undiluted) aqueous humor analysis (maximum 200 microliters of drawn sample). However, a previous study showed that ELISA with serial dilution resulted in less sensitivity than that with single dilution.20 Theoretically then, a method that can be carried out without dilution should be more sensitive than that with dilution techniques. The sensitivity of conventional plate ELISA or Luminex multi-beads for aqueous humor VEGF detection was approximately 4–5 pg mL−1.18 So, new aqueous humor-based diagnostic tools for monitoring VEGF levels that require small sample volumes are needed. The ability to use a biomarker would make the treatment plan safer and more accurate (prompt, earlier treatment and avoidance of overdose).21 The optimal treatment schedule for intravitreal injection of anti-VEGF antibodies is, in fact, difficult to determine individually due to the lack of laboratory biomarkers in ophthalmology.18,22

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Paper-based ELISA (P-ELISA) Paper has previously been used as an economical, convenient, and user-friendly platform for examination in ophthalmology, e.g., Schirmer's test, the strip test for the diagnosis of Sjogren's syndrome and tear film insufficiency.23 Recently, microfluidic paper-based analytical devices (μPADs) have emerged as an alternative for use in general healthcare.24–26 One such design, known as P-ELISA, employs a slightly different approach to paper-based diagnostic devices by using paper substrate microzone plates as an alternative to plastic microtiter plates.27 P-ELISA has been used to successfully quantify HIV loading and detect autoantibodies with 2 μLsamples in patients with bullous pemphigoid.28 In ophthalmology, the minute available volume of aqueous humor makes traditional diagnostic tools for cytokine quantification difficult to use, but P-ELISA can overcome this limitation because it requires very small sample volumes. To measure VEGF in minute aqueous humor samples, P-ELISA uses a flow-through assay based on the conjugation of the horseradish peroxidase ligand on bevacizumab (Avastin) as the primary antibody.29 This allows a colorimetric reaction without a secondary antibody (Fig. 2b), and thus the use of HRP-conjugated Avastin as a colorimetric antibody also reduces the cost of manufacturing antibodies (about 400 USD per 3 mL). The P-ELISA protocol used here was a slightly modified version of that used in our previous studies.29 The protocol described herein comprises multiple steps, requiring 40 minutes to complete. First, 2 μL of PBS was placed onto the test zone to rinse the plate. Then, 2 μL of aqueous humor was placed onto the paper-based zone. To block spare space, 1% BSA was placed onto the paper-based zone. 5 μL of HRP– Avastin (0.8 mg mL−1), which has strong affinity to VEGF, was then placed onto the test zone for 10 minutes. 2.5 μL of streptavidin was added onto the paper-based test zones to

Fig. 2 Comparison of conventional ELISA and P-ELISA for VEGF detection. (a) In conventional ELISA, dilution of the sample from aqueous humor, usually for ELISA, reduces sensitivity. (b) Illustration of the step-by-step protocol that permits bevacizumab-conjugated HRP to bind specifically to VEGF, simultaneously serving as a colorimetric indicator. This was recorded via serial cell phone camera and a desktop scanner.

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enhance the signal until the test zones dried. After carrying out the washing step again with washing buffer, the HRP substrates, TMB and H2O2 at a ratio of 1 : 1, were then placed onto the plate to form the resulting blue product. Colorimetric results were recorded every minute using a portable scanner (Fig. 2b). Serial captured images were used in the analysis, and semi-quantification of VEGF levels was performed after fitting to the calibration curve. The limit of detection for P-ELISA was 33.7 fg mL−1 (compared with those for conventional ELISA, which was 5 pg mL−1, and the Luminex xMAP system, which was 4 pg mL−1).30 The sensitivity of this new bioassay was much higher than that of conventional methods. With this platform, VEGF can be used as a feasible biomarker with measurable characteristics that indicate activity of AMD, PDR, and RVO. The cost of diagnosis using this technique is considerably lower than that which can be achieved by conventional methods (typically dozens of USD per test).

Further POC diagnostic devices that could provide ophthalmologists with instant results to aid in re-treatment determination We particularly note that a primary care physician (ophthalmologist, GP) is not likely to be a trained laboratory practitioner. Thus, POC diagnostics, such as P-ELISA, may offer a suitable and simplified technique that could provide GPs with semi-quantitative diagnostic results.26,31 Such POC diagnostic devices may be further developed for use in outpatient clinics for rapid results. Immediate re-treatment may be

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performed if a POC diagnostic device indicated, for instance, significantly elevated VEGF levels while patients were still in an outpatient clinical setting.

III. Importance of surveillance for VEGF detection In Fig. 3a, the schematic graphs show how VEGF levels fell and rose following intravitreal injection of the anti-VEGF antibody. P-ELISA provides a POC diagnostic tool to guide physicians administering anti-VEGF antibody injection therapy. Note that a rise in VEGF levels is detectable (here, as low as ~20 pg mL−1, Fig. 3a) earlier than the occurrence of structural changes noted by optical imaging modalities, such as OCT or fluorescein angiography (Fig. 3b), which may present misleading diagnosis especially in terms of edema and VEGF levels. This supports the notion of using biomarkers to guide earlier intervention via intravitreal injection of anti-VEGF antibodies and the idea that individualized medicine can be achieved with POC diagnostics. Further, current FAG examination protocols require intravenous fluorescein injection, which may induce a systemic adverse reaction and, in rare instances, may even cause death.32,33 POC diagnostics avoids this risk. Clearly, precise measurement of VEGF levels can lead to the avoidance of recurrent vision loss in patients (Fig. 3a).15,16 An adverse reaction following intravitreal injection of antiVEGF antibodies may develop when standardized biomarkers are not used.34 The systemic safety of Avastin (bevacizumab) and Lucentis (ranibizumab) for AMD has not been established

Fig. 3 A follow-up sequence of one real patient with wet age-related macular degeneration (AMD). (a) Here, P-ELISA functions as a POC diagnostic tool to detect VEGF concentration at different time points. A supernormal aqueous VEGF level was detected and re-treatment was performed (asterisk), thus preventing further occurrence of macular edema and vision loss. (b) OCT showed a significant decrease in macular edema following anti-VEGF medication. However, the follow-up showed no obvious structural change (double asterisk). This supports the notion that structural changes occur later than VEGF elevation and can result in undesirable vision loss.

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P-ELISA as an easy-to-use tool for surveillance of ocular VEGF levels

Fig. 4 The mean aqueous VEGF level decreased markedly when using P-ELISA for monitoring. The patient is an 80-year-old man with AMD. P-ELISA showed that the patient's VEGF level was 93.762 pg mL−1 (intensity = 22.301). A retinal image showed haemorrhage around the right ocular macular area (left column). One week after intravitreal injection of the anti-VEGF antibody, P-ELISA indicated that the VEGF level decreased to 20.740 pg mL−1 (intensity = 17.883). The retinal image showed remission of haemorrhage around the right permacular area (arrow, right column).

precisely.35 P-ELISA, Luminex or other POC diagnostic tools can also help build safety profiles after injection. In addition to therapeutic advantages provided by this approach, the use of P-ELISA for VEGF measurement would deliver considerable cost savings compared to frequent OCT and intravitreal injection, and the procedural duration of P-ELISA is much shorter than that of conventional ELISA (i.e., 44 versus 213 minutes) (Fig. 2). Because only microliters of a sample are required for P-ELISA to quantify VEGF concentration, aqueous humor analysis can be repeated and more sensitive results can be achieved.

We initiated a follow-up study in order to monitor VEGF levels in patients with AMD, PDR, or RVO, i.e., to evaluate the therapeutic effects of an anti-VEGF antibody, ranibizumab (Lucentis), using our P-ELISA approach before and after treatment. We obtained permission from the Institutional Review Board of Taichung Veterans General Hospital (IRB number: CF14120) to use P-ELISA as an evaluative tool. According to recent research, monthly intravitreal injection of Lucentis was highly beneficial in the treatment of wet-type AMD and PDR with macular edema.22,36 After obtaining consent from a patient, we collected a sample of aqueous humor for analysis. Before intravitreal injection with the anti-VEGF antibody, P-ELISA showed a VEGF level of 93.762 pg mL−1 (Fig. 4a) and the retinal image showed massive hemorrhage around the macular area. One week after injection, P-ELISA showed that the VEGF level dropped to as low as 20.74 pg mL−1 and the retinal image showed remission of hemorrhage around the macular area (Fig. 4b). Our P-ELISA platform provides healthcare personnel with a promising diagnostic tool to monitor the disease state by tracking the levels of a specific biomarker (i.e., VEGF) in ocular diseases, especially in outpatient clinical settings (Fig. 5). Existing POC diagnostics or other quantifying methods for VEGF concentration detection are currently inadequate (Fig. 5a). In conventional ELISA for VEGF concentration detection, patients must return home, await results, and schedule a follow-up visit. Such delays hasten disease

Fig. 5 Schematic illustrations of current scenarios and application of POC diagnostics (e.g. P-ELISA) in ocular VEGF detection. (a) Current scenario: no VEGF detection approach is applied and optical imaging modalities can only be applied for response evaluation. (b) Conventional ELISA can be applied but patients must leave the clinic and await VEGF concentration results. Retreatment on re-visit can be performed 4–7 days later. (c) Patients would benefit from POC diagnostics and immediate treatment (if needed).

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progression and increase the likelihood of vision loss (Fig. 5b). By using P-ELISA or other potential POC devices, patients can get earlier, more productive treatment (Fig. 5c). The technique outlined in this manuscript is inexpensive, rapid and easy-to-use, and requires a low test sample volume, a feature that is especially useful when the sample volume is extremely limited. Optimal scheduling of therapies using intravitreal injection of anti-VEGF antibodies can be achieved with a diagnostic tool that provides rapid, accurate results regarding relevant biomarker levels. The disadvantages of our novel method include the fact that it is an invasive procedure (aqueous sampling), and it includes risks of traumatic cataract, in phakia patients, and possible infection. A less invasive instrument for aqueous sampling would be ideal. Our study indicates an improved approach for the codevelopment of drug therapy (for specific disease/biomarkers such as VEGF in our study) and a diagnostic tool (in order to evaluate the drug function or performance; ranibizumab in this study) using an accurate, rapid, inexpensive, and easy-tohandle technique that only requires low sample volumes.

Conclusion To summarize, we have developed an HRP-conjugated Avastin approach for detecting substrates to determine the aqueous VEGF level in patients with AMD, PDR, and RVO. Only 40 μL (2 μL and 20 tests per sample) was required for determination of the VEGF level. P-ELISA is an inexpensive, easy-to-handle technique that offers ultra-high sensitivity and is capable of detecting VEGF levels in low volumes of aqueous humor. It potentially solves the problem of low aqueous humor sample volume for cytokine detection. By employing POC diagnostics in an outpatient clinical setting, dosing and treatment regimens for those in need may be provided. Future innovations in microliter-related bio-engineering may promote the wide application of POC diagnostics in the field of ophthalmology.

Acknowledgements We would like to thank the National Science Council of Taiwan (NSC 101-2628-E-007-011-MY3 for C.-M. Cheng) and Taichung Veterans General Hospital (TCVGH-1046901B for M.-Y. Hsu) for financially supporting this research. We also thank Dr. Yu-Ping Wang for providing the excellent illustrations in the figures.

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Monitoring VEGF levels with low-volume sampling in major vision-threatening diseases: age-related macular degeneration and diabetic retinopathy.

The purpose of this article is to demonstrate the capacity of paper-based ELISA (P-ELISA) to monitor VEGF in patients requiring treatment for vision-t...
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