Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in vivo.

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.

Using Electronic Health Record Data to Determine the Safety of Aqueous Humor Liquid Biopsies for Molecular Analyses.

Purpose: Knowing the surgical safety of anterior chamber liquid biopsies will support the increased use of proteomics and other molecular analyses to better understand disease mechanisms and therapeutic responses in patients and clinical trials. Manual review of operative notes from different surgeons and procedures in electronic health records (EHRs) is cumbersome, but free-text software tools could facilitate efficient searches. Design: Retrospective case series. Participants: A total of 1418 aqueous humor liquid biopsies from patients undergoing intraocular surgery. Methods: Free-text EHR searches were performed using the Stanford Research Repository cohort discovery tool to identify complications associated with anterior chamber paracentesis and subsequent endophthalmitis. Complications of the surgery unrelated to the biopsy were not reviewed. Main Outcome Measures: Biopsy-associated intraoperative complications and endophthalmitis. Results: A total of 1418 aqueous humor liquid biopsies were performed by 17 experienced surgeons. EHR free-text searches were 100% error-free for surgical complications, >99% for endophthalmitis (<1% false positive), and >93.6% for anesthesia type, requiring manual review for only a limited number of cases. More than 85% of cases were performed under local anesthesia without ocular muscle akinesia. Although the most common indication was cataract (50.1%), other diagnoses included glaucoma, diabetic retinopathy, uveitis, age-related macular degeneration, endophthalmitis, retinitis pigmentosa, and uveal melanoma. A 50- to 100-µL sample was collected in all cases using either a 30-gauge needle or a blunt cannula via a paracentesis. The median follow-up was >7 months. There was only one minor complication (0.07%) identified: a case of a small tear in Descemet membrane without long-term sequelae. No other complications occurred, including other corneal injuries, lens or iris trauma, hyphema, or suprachoroidal hemorrhage. There was no case of postoperative endophthalmitis. Conclusions: Anterior chamber liquid biopsy during intraocular surgery is a safe procedure and may be considered for large-scale collection of aqueous humor samples for molecular analyses. Free-text EHR searches are an efficient approach to reviewing intraoperative procedures. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses.

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the research laboratory. Here, we developed a protocol for acquiring high-quality liquid biopsies for molecular analyses from the aqueous humor and the vitreous from patients undergoing eye surgery. In this workflow, a Mobile Operating Room Lab Interface (MORLI) cart equipped with a computer, a barcode scanner, and lab instruments, including onboard cold storage, is used to obtain and archive human biological samples. A web-based data privacy-compliant database enables annotating each sample over its lifetime, and a cartesian coordinate system allows tracking each barcoded specimen in storage, enabling quick and accurate retrieval of samples for downstream analyses. Molecular characterization of human tissue samples not only serves as a diagnostic tool (e.g., to distinguish between infectious endophthalmitis and other non-infectious intraocular inflammation) but also represents an important component of translational research, allowing the identification of new drug targets, development of new diagnostic tools, and personalized therapeutics.

Immunoprofiling of Nonarteritic Anterior Ischemic Optic Neuropathy.

Purpose: Nonarteritic anterior ischemic optic neuropathy (NAION) is a common acute optic neuropathy in those older than 50 years. There is no blood diagnostic test or efficient treatment for NAION. We investigated the suitability of blood inflammatory proteins as biomarkers and therapeutic targets of NAION. Methods: We conducted an exploratory, cross-sectional case-control study including 18 patients with NAION (n = 5 acute, and n = 13 chronic) and 9 controls. NAION was confirmed by clinical examination and optical coherence tomography. Subjects underwent peripheral blood collection; plasma was isolated within 2 hours and analyzed using a 76-plex array of cytokines, chemokines, and growth factors. Results: In acute NAION, there was increased peripapillary retinal thickness on optical coherence tomography consistent with optic disc edema. Plasma profiling revealed dramatic changes in inflammatory proteins in NAION. Statistical analysis generated a list of 20 top-ranked molecules in NAION, with 15% overlap in acute and chronic NAION. Principal component analysis, hierarchical clustering, and Spearman correlation generally segregated controls, acute and chronic NAION, with some overlap. Longitudinal data from one patient demonstrated an evolving inflammatory pattern from acute to chronic NAION. In acute NAION, Eotaxin-3, MCP-2, TPO, and TRAIL were the top biomarker candidates. In chronic NAION, IL-1α and CXCL10 emerged as the strongest therapeutic targets. Conclusions: Post-NAION inflammation occurs in both acute and chronic NAION. Statistical analysis of plasma profile changes generated a list of 20 potential biomarker and therapeutic targets of NAION. Translational Relevance: We identified blood molecular targets to improve NAION diagnosis and treatment.

CustomVue laser in situ keratomileusis treatment after previous keratorefractive surgery.

PURPOSE: To evaluate the efficacy, predictability, and safety of Visx CustomVue wavefront-guided enhancement after previous keratorefractive surgery. SETTING: Stanford University Eye Laser Center, Stanford, California, USA. METHODS: A retrospective analysis was used to evaluate wavefront-guided enhancement in a preliminary set of 120 eyes of 102 patients. All eyes had previous keratorefractive surgery (photorefractive keratoplasty [PRK] in 1 eye, laser in situ keratomileusis [LASIK] in 119 eyes); the prekeratorefractive surgery spherical equivalent (SE) refraction ranged from -1.25 diopters (D) to -7.00 D. Primary outcome variables including uncorrected visual acuity (UCVA), manifest refraction, and complications were evaluated at 1 and 3 months. RESULTS: At 1 month, the mean pre-enhancement SE was reduced from -0.91 D +/- 0.40 (SD) (range -2.375 to -0.125 D) to -0.13 +/- 0.33 D (range -1.25 to 0.75 D) with 91% of eyes within +/-0.5 D of emmetropia and 100% within +/-1.0 D. All eyes showed equal or improved UCVA (range 20/15 to 20/30) with 20/20 or better in 84 of 91 eyes. At 3 months, the mean was -0.20 +/- 0.32 D (range -0.75 to 0.75 D) with 100% of eyes within +/-0.75 D of emmetropia. All eyes showed equal or improved UCVA (range 20/15 to 20/30) with 20/20 or better in 74 of 84 eyes. Higher-order wavefront aberration analysis showed that the mean root-mean-square error was reduced from 0.39 +/- 0.14 microm (range 0.16 to 0.86 microm) to 0.34 +/- 0.12 microm (range 0.12 to 0.78 microm). Coma was reduced from 0.22 +/- 0.13 microm (range 0.02 to 0.71 microm) to 0.16 +/- 0.11 microm (range 0.01 to 0.62 microm), and trefoil was reduced from 0.16 +/- 0.09 microm (range 0.01 to 0.62 microm) to 0.11 +/- 0.07 microm (range 0.01 to 0.27 microm). Spherical aberration was unchanged from 0.14 +/- 0.14 microm (range -0.18 to 0.59 microm) to 0.14 +/- 0.14 microm (range -0.16 to 0.5 microm). CONCLUSION: Preliminary data show that Visx CustomVue wavefront-guided enhancement after keratorefractive surgery is an effective, predictable, and safe procedure.