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 ...

Stanford University

This protocol describes an ex vivo model to quantify enzymatic effects on vitreous liquefaction using porcine eyes in a transwell-plate system via induced syneresis. It provides a standardized dissection process and performs critical steps for gel-liquid separation with high precision, minimal tissue loss, and scalability. The protocol can be applied to other studies investigating vitreous liquefaction or gelatinous tissue analysis and can also serve to study vitreous liquefaction in vivo as it may occur during aging or disease progression.

Protocol to quantify enzymatic effects on vitreous liquefaction in porcine eyes using a transwell-plate system.

New methods for basic research are finding their way into ophthalmology and expand the options for research, diagnostics and treatment. This article centers on the study of gene activity in cells and tissues by next-generation sequencing (NGS). Following a brief introduction to the basic principles of NGS, this article focuses on transcriptome analysis by RNA sequencing using examples from ophthalmology. The RNA sequencing provides a comprehensive and unbiased overview of gene activity in cells and tissues and thus forms an important foundation for generating new testable scientific hypotheses. It thus contributes to the in-depth characterization of pathological changes and supports the development of new diagnostic and therapeutic approaches.

[Next-generation sequencing in ophthalmology].

Purpose: To determine the prevalence of SARS-CoV-2 in tear samples and to investigate whether it correlates with ocular findings and patients’ prognosis in Brazil. Methods: Tears were collected using Schirmer strips (SS) and conjunctival swabs (CS) from patients hospitalized with laboratory-confirmed SARS-CoV-2 infection. Samples were analyzed using qRT-PCR. Demographic and clinical data, ocular symptoms, and Schirmer tests (ST) were collected from patients. Charlson Comorbidity Index (CCI) was used to rate comorbidities, and patients were monitored until hospital discharge or death. Results: There were 61 hospitalized patients, 33 of which were diagnosed with COVID-19. Within the confirmed COVID-19 patients, SARS-CoV-2 was detected in 18.2% (n = 6) of CS and 12.1% (n = 4) of SS samples. Subjective and objective parameters for dry eye syndrome (e.g., ST COVID-19: 8.3 ± 6.4mm, non-COVID-19: 8.9 ± 6.6mm, p > 0.05) were comparable between COVID-19 (n = 33) and non-COVID-19 patients (n = 28). Among the 16 COVID-19 patients exhibiting ocular symptoms, only tearing was reported significantly more frequently when tear samples were positive for SARS-CoV-2 (p < 0.05). Strikingly, patients whose tears tested positive for SARS-CoV-2 had significantly inferior CCI (pos.: 34.0 ± 31.8%, neg.: 67.6 ± 36.4%, p < 0.05) and higher mortality rates (pos.: 50.0%, neg.: 7.4%, p < 0.01). Conclusions: SARS-CoV-2 was detected with a prevalence of 18.2% on the ocular surface. Decreased CCI and increased mortality rate in the positive tear group suggests that viral detection may relate to prognosis and highlight the need of personal protective measures for healthcare professionals. Most of the patients, regardless of COVID-19 diagnosis, had low tear production and eye discomfort, possibly pointing to the need for artificial tear use during hospitalization.

Conjunctival Swabs Reveal Higher Detection Rate Compared to Schirmer Strips for SARS-CoV-2 RNA Detection in Tears of Hospitalized COVID-19 Patients.

Corneal transplantation is one of the most common forms of tissue transplantation worldwide. Donor corneal tissue used in transplantation is provided by eye banks, which store the tissue in culture medium after procurement. To date, the effects of cell culture on human corneal tissue have not been fully elucidated. Using the 3' RNA sequencing method for massive analysis of cDNA ends (MACE), we show that cultivation of corneal tissue leads to significant changes in a variety of molecular processes in human corneal tissue that go well beyond aspects of previously known culture effects. Functionally grouped network analysis revealed nine major groups of biological processes that were affected by corneal organ culture, among them keratinization, hypoxia, and angiogenesis, with genes from each group being affected by culture time. A cell type deconvolution analysis revealed significant modulations of the corneal immune cell profile in a time dependent manner. The results suggest that current culture conditions should be further refined and that prolonged cultivation may be detrimental. Recently, we showed that MACE enables transcriptional profiling of formalin-fixed and paraffin-embedded (FFPE) conjunctival tissue with high accuracy even after more than 10 years of storage. Here we demonstrate that MACE provides comparable results for native and FFPE corneal tissue, confirming that the technology is suitable for transcriptome analysis of a wide range of archived diseased corneal samples stored in histological archives. Finally, our data underscore the feasibility of bioinformatics cell-type enrichment analysis in bulk RNA-seq data to profile immune cell composition in fixed and archived corneal tissue samples, for which RNA-seq analysis of individual cells is often not possible.

Transcriptional Profiling Provides New Insights into Organ Culture-Induced Changes in Human Donor Corneas.

This study characterizes the transcriptional profile and the cellular tumor microenvironment of conjunctival extranodal marginal zone lymphoma (EMZL) and identifies prognostically relevant biomarkers. Ten formalin-fixed and paraffin-embedded conjunctival EMZL and eight healthy conjunctival specimens were analyzed by Massive Analysis of cDNA Ends (MACE) RNA sequencing. The 3417 upregulated genes in conjunctival EMZL were involved in processes such as B cell proliferation and Rac protein signaling, whereas the 1188 downregulated genes contributed most significantly to oxidative phosphorylation and UV protection. The tumor microenvironment, as determined by deconvolution analysis, was mainly composed of multiple B cell subtypes which reflects the tumor's B cell lineage. However, several T cell types, including T helper 2 cells and regulatory T cells, as well as innate immune cell types, such as anti-inflammatory macrophages and plasmacytoid dendritic cells, were also strongly enriched in conjunctival EMZL. A 13-biomarker prognostic panel, including  and , classified ocular and extraocular tumor recurrence, exceeded prognostic accuracy of Ann Arbor and American Joint Committee on Cancer (AJCC) staging, and demonstrated prognostic value for patient survival in 21 different cancer types in a database of 12,332 tumor patients. These findings may lead to new options of targeted therapy and may improve prognostic prediction for conjunctival EMZL.

Julian Wolf

Molecular Surgery Laboratory,
Department of Ophthalmology,
Byers Eye Institute,
Department of Ophthalmology, Byers Eye Institute

Stanford University

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses

Julian Wolf

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Molecular Surgery Laboratory,Department of Ophthalmology,Byers Eye Institute,Department of Ophthalmology, Byers Eye Institute

Stanford University

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses

Molecular Surgery Laboratory,
Department of Ophthalmology,
Byers Eye Institute,
Department of Ophthalmology, Byers Eye Institute