Proteomic Biomarker Identification in Age-Related Macular Degeneration, Diabetic Retinopathy and Retinal Detachment

Identification And Validation Of Biomarkers In Ophthalmological Diseases (Age-Related Macular Degeneration, Diabetic Retinopathy And Retinal Detachment) Through Clinical Proteomic Approaches

This prospective interventional translational study aims to identify and validate protein biomarkers associated with major ophthalmological diseases, including age-related macular degeneration, diabetic retinopathy (DR), and retinal detachment (RD). A total of approximately 260 participants (cases and controls) will be enrolled at a single center.

Biological samples, including peripheral blood, tears, aqueous humor, vitreous humor, and subretinal fluid, will be collected during routine clinical and surgical procedures. Advanced clinical proteomics approaches will be applied to characterize molecular signatures associated with disease onset, progression, and response to treatment.

The study seeks to improve the understanding of disease pathophysiology and support the development of novel diagnostic and prognostic biomarkers in ophthalmology.

This is a single-center, prospective interventional translational study designed to identify and validate proteomic biomarkers in patients affected by age-related macular degeneration, diabetic retinopathy, and retinal detachment, compared with control subjects without retinal disease.

Study Population

Approximately 260 subjects will be enrolled, including:

180 patients (100 age-related macular degeneration, 50 diabetic retinopathy, 30 retinal detachment) 80 controls (cataract surgery or macular surgery patients without retinal disease). Participants will be recruited among patients routinely treated at IRCCS Fondazione G.B. Bietti.

Study Design and Procedures

All participants will undergo a baseline visit including:

• Medical and ophthalmological history comprehensive eye examination (visual acuity, tonometry, biomicroscopy, ophthalmoscopy)
• Non-invasive imaging (e.g., Optical Coherence Tomography, Optical Coherence Tomography Angiography)
• Collection of tears and peripheral blood samples

Patients undergoing therapeutic procedures will also participate in a second visit (within 1-30 days), during which additional biological samples will be collected during routine clinical care, including:

• Aqueous humor (via paracentesis)
• Vitreous humor
• Subretinal fluid (in retinal detachment surgery) Post-injection vitreous reflux (if applicable) All sampling procedures are performed as part of standard clinical or surgical practice and do not introduce additional risk to the patient.

Proteomic Analysis Collected biological matrices (ocular fluids and blood-derived components such as PBMCs and extracellular vesicles) will be analyzed using advanced clinical proteomics techniques. These include high-resolution mass spectrometry (Orbitrap-based platforms) combined with multiplex quantitative approaches (e.g., Tandem Mass Tags).

The study will follow a two-phase approach:

Biomarker discovery phase, aimed at identifying candidate protein biomarkers Validation phase, aimed at confirming their diagnostic and biological relevance Additional targeted analyses (e.g., ELISA) and functional studies (e.g., in vitro cellular models) may be performed to further characterize identified molecular pathways.

Objectives The primary objective is the identification and validation of protein biomarkers associated with age-related macular degeneration, diabetic retinopathy, and retinal detachment.

Secondary objectives include:

Characterization of molecular mechanisms underlying disease pathogenesis Identification of potential therapeutic targets Investigation of post-translational protein modifications (e.g., phosphoproteomics, ubiquitin-related signatures) Evaluation of surgical variables (e.g., vitreous fragmentation) in biomarker identification

Statistical Analysis Data will be analyzed using appropriate parametric and non-parametric statistical methods. Associations between clinical and molecular variables will be explored, with statistical significance defined as p < 0.05.

Ethical and Data Management Considerations The study will be conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. All participants will provide written informed consent prior to enrollment. Data will be pseudonymized and handled in compliance with applicable data protection regulations (General Data Protection Regulatio)

Inclusion Criteria:

• Age ≥ 18 years
• Ability to understand the study procedures and provide written informed consent
• Willingness and ability to comply with study procedures and visits

For case subjects:

• Patients with age-related macular degeneration (intermediate or advanced, including geographic atrophy or neovascular AMD)
• Patients with diabetic retinopathy (non-proliferative or proliferative, with or without diabetic macular edema)
• Patients undergoing surgery for primary or recurrent retinal detachment

For control subjects:

• Patients scheduled for cataract surgery with no documented retinal diseases (e.g., AMD, diabetic retinopathy, retinal vascular occlusions, or retinal epithelial disorders)
• Outpatients with no documented retinal diseases
• For retinal detachment subgroup: patients undergoing macular surgery (e.g., macular pucker or macular hole) or cataract surgery with attached retina

Exclusion Criteria:

• Age < 18 years
• Presence of infectious ocular diseases
• Alterations in the electrophoretic profile of gamma globulins