This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.
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.
Molecular profiling of liquid biopsies from the human eye can capture locally enriched fluids containing molecules such as DNA, RNA, proteins, glycans, and metabolites from highly specialized ocular tissues. Liquid biopsies from the vitreous in the posterior chamber of the human eye proved to be a generally safe procedure1. They allow molecular characterization of ocular diseases in living humans and offer the potential to identify novel diagnostic and therapeutic strategies2,3,4. The aqueous humor in the anterior chamber of the eye has even higher surgical accessibility and could be obtained in large numbers, e.g., during cataract surgery, which is one of the most frequently performed surgeries. However, no standardized protocol for the collection, annotation, and biobanking of human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics, is available until now.
Here, we developed a protocol for the collection and biobanking of high-quality liquid biopsies for molecular analyses from patients undergoing eye surgery. A Mobile Operating Room Lab Interface (MORLI) enables a researcher to immediately snap-freeze the collected samples in barcoded cryovials on dry ice at -80 ˚C in the operating room (OR). This procedure ensures a high and consistent sample quality for downstream molecular analysis. In addition to excellent sample quality, accurate annotation of samples in a biobank is critical. Using a web-based HIPAA (Health Insurance Portability and Accountability Act)-compliant REDCap (research electronic data capture) database5, our workflow allows storage of detailed metadata for each sample, including age, sex, disease, disease stage, sample type, and unique features of the surgery. This will allow accurate future searching capacity, e.g., for samples from a specific disease or a particular group of patients. In addition, the exact location of each sample in the freezer is archived using a cartesian grid system, which enables efficient sample retrieval for downstream experiments. We show examples of DNA, protein, glycan, and metabolite analyses.
Our workflow represents a practical and effective connection between the OR and the research laboratory and provides a valuable foundation for translational research.
The protocol follows the guidelines of the Institutional Review Board for Human Subjects Research (IRB) at Stanford University, USA.
CAUTION: This protocol is a guide for qualified ophthalmic surgeons. In the context of intraocular malignancies, extraocular tumor seeding in the setting of aqueous humor or vitreous liquid biopsies cannot be ruled out. However, the risk of extraocular extension and orbital involvement is extremely low in transvitreal choroidal tumor biopsy, that is done in safety enhanced manner and with careful entry site consideration6. This protocol does not cover and may be contraindicated in cases of retinoblastoma or tumors with high metastatic risk.
1. Before sample collection
2. Acquisition of surgical specimens in the operating room
3. Processing of samples in the OR and adding samples to the database
4. Transferring cryovials to storage
5. Sample storage form
6. Retrieval of surgical specimens for downstream analysis
NOTE: Specimens are often archived for several years before they are analyzed. The barcoded cryovials and the searchable REDCap database system allow to find and locate each sample easily for downstream analysis.
The collected liquid biopsy specimens can be subjected to a variety of molecular analyses, including the analysis of DNA, proteins, glycans, and metabolites. It has been shown before that long-term storage over several years at -70 °C did not significantly affect the integrity of the proteomic profile9. The REDCap database enables simple and quick retrieval of samples. The database can be searched for samples from a specific group of patients, e.g., all patients with diabetic retinopathy. The database will then provide the barcodes of the tubes and the positions in storage. Until now, we have collected and archived more than 1,000 liquid biopsies. The database allowed us to quickly find the samples for downstream analyses3,10 and helped to perform the following experiments.
A 17-year-old female presented with retinal and optic nerve inflammation. She was immunocompromised, and there was a concern for infection. Aqueous humor was collected from her right eye and sent for DNA PCR analysis. The results were positive for Cytomegalovirus and negative for Herpes simplex virus and toxoplasmosis. These findings illustrate that aqueous humor liquid biopsies can help to distinguish infectious from non-infectious forms of intraocular inflammation, which is critical to select the appropriate therapy.
Liquid chromatography-mass spectrometry enables an unbiased and semi-quantitative analysis of the proteome. In a liquid biopsy from the vitreous of a patient undergoing vitrectomy, the technique was able to identify 484 unique proteins, including Complement C3 (C3), Opticin (OPTC), and Collagen Type II Alpha 1 (COL2A1) (Figure 1A).
Three vitreous liquid biopsies were analyzed using a glycoproteomics multiplex ELISA (see Table of Materials)11. The assay detected the glycosylation profiles of 500 human proteins, capturing a variety of biological pathways, such as metabolism, immune response, cell adhesion, and actin organization (Figure 1B).
A metabolomics screen using capillary electrophoresis coupled with Fourier transformed mass spectrometry12 (see Table of Materials) identified 292 different metabolites in three aqueous humor liquid biopsy samples. A pathway analysis (see Table of Materials)13 identified a variety of metabolic pathways, including amino acid metabolism, urea cycle and carnitine synthesis (Figure 1C).
Figure 1: Representative results. (A) Proteomics analysis of human vitreous humor using liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 484 unique proteins in a single liquid biopsy. Protein levels are shown and ranked based on spectral counts. Representative proteins are highlighted in blue. (B) A glycoproteomics multiplex ELISA detected glycosylation levels of 500 unique proteins in three vitreous liquid biopsies. A STRING protein interaction analysis identified clusters of protein interactions (clusters with at least 10 proteins are shown). The most significantly enriched pathway is shown for each cluster. (C) Metabolomics analysis using mass spectrometry identified 292 different metabolites in three aqueous humor liquid biopsies. Each point represents one sample. The height of the bar corresponds to the mean number of metabolites, the error bar represents the standard deviation. The right panel shows significantly enriched pathways. The number of detected metabolites (numerator) as well as the total number of metabolites in each pathway (denominator) are shown. Please click here to view a larger version of this figure.
Surgical specimens from patients allow direct molecular characterization of disease in living humans2,3,4,14, and may help overcome the limitations of cell and animal disease models that do not fully recapitulate human disease15,16. Molecular analysis of human tissue could improve the selection of new drug targets and may contribute to a higher success rate of clinical trials and drug approval17. In addition, this approach offers the potential for personalized medicine, as the obtained tissue retains the unique genomic, epigenomic, metabolomic, glycomic, and proteomic fingerprint of each individual2,18,19.
High and consistent sample quality is fundamental for all molecular analysis applications. Previous studies have shown that immediate freezing after sample retrieval and avoiding repeated freeze/thaw cycles are critical for high sample qualities9,20. Long-term storage over several years at -70 °C did not significantly affect the integrity of the proteomic profile9. A standardized protocol is an important foundation to reduce bias and improve the comparability of scientific data, especially when several people (surgeons, technicians, and others) or different institutions are involved in the sampling process. Apart from the sample quality, the annotation of samples is another important factor that requires standardization to allow the correlation of molecular findings with clinical data. Our protocol relies on three essential principles to accomplish this: 1) a standardized sampling procedure for aqueous humor and vitreous liquid biopsies by an ophthalmic surgeon, 2) the immediate processing and snap-freezing of samples in the OR by laboratory personnel, and 3) a metadata annotation of each sample in a web-based database that allows researchers to quickly find samples for later experiments.
In addition to vitreous specimens20, this workflow also establishes the standardized collection of aqueous humor liquid biopsies for molecular analysis. The aqueous humor is a highly accessible, complex fluid in the anterior chamber of the eye that does not only reflect ocular diseases of the anterior but also of the posterior segment of the eye, including retinal disease18,21. Along with the fact that a high number of aqueous humor samples could be collected e.g., during cataract surgery, one of the most frequently performed surgery worldwide, these features make it an interesting source for liquid biopsies from the human eye. The standardized metadata annotation of each sample established in this workflow could also allow the correlation of proteome data with prospective clinical follow-up data. This provides the exciting opportunity to identify new prognostic biomarkers which may help to estimate the prognosis for future patients.
However, molecular analysis of human surgical specimens also has important limitations. For example, complex experimental manipulations are often only possible in animal and cell models. A solution may be to compare the molecular profile of animal or cell models with that of human disease. This strategy can identify overlapping protein biomarkers and therapeutic targets that can be validated in animals or cell models to identify the most promising candidates that correlate with human disease and are likely to succeed in clinical trials4,16.
In conclusion, our workflow establishes a practical interface between the OR and the research laboratory that allows standardized and high-throughput collection, annotation, and storing of high-quality surgical specimens for molecular downstream analysis, providing a valuable foundation for future translational research.
The authors have nothing to disclose.
VBM is supported by NIH grants (R01EY031952, R01EY031360, R01EY030151, and P30EY026877), the Stanford Center for Optic Disc Drusen, and Research to Prevent Blindness, New York, USA. JW and DR are supported by the VitreoRetinal Surgery Foundation, USA. DR is supported by the DARE Fellowship, which is sponsored by the Lundbeck Foundation.
0.5ml Tri-coded Tube, 96-format, External Thread | Azenta Life Sciences, Burlington, MA 01803, USA | 68-0703-12 | used for aqueous humor samples |
1 mL syringe | surgical grade, whatever available in hospital | – | for aqueous humor biopsies |
1.9ml Tri-coded Tube, 48-format, External Thread | Azenta Life Sciences, Burlington, MA 01803, USA | 65-7643 | used for vitreous samples |
3 mL syringe | surgical grade, whatever available in hospital | – | for vitreous biopsies |
30-32-gauge needle | surgical grade, whatever available in hospital | – | for aqueous humor biopsies |
Capillary electrophoresis coupled with Fourier transformed mass spectrometry (CE-FTMS) | Human Metabolome Technologies, Inc., Tsuruoka, Japan | – | – |
Constellation vitrectomy system with 23-, 25-, or 27-gauge trocar cannula system | Alcon Laboratories Inc, Fort Worth, TX, USA | – | for vitreous biopsies |
Cooling box | Standard styrofoam box, whatever available in lab | – | – |
Dry ice | Whatever available in lab | – | – |
Handsfree Standard Range Scanner Kit with Shielded USB Cable | Zebra Symbol | DS9208-SR4NNU21Z | Barcode scanner |
Human Glycosylation Antibody Array L3 | RayBiotech, Peachtree Corners, GA, USA | GAH-GCM-L3 | – |
Mac mini | Apple Inc., Cupertino, CA 95014, USA | – | – |
MetaboAnalyst software | Pang et al., 2021, PMID: 34019663 | – | – |
Rack for 0.5ml tubes, 96-Format | Azenta Life Sciences, Burlington, MA 01803, USA | 66-51026 | for aqueous humor samples |
Rack for 1.9ml tubes, 48-Format | Azenta Life Sciences, Burlington, MA 01803, USA | 65-9451 | for vitreous samples |
REDCap browser-based sample database | REDCap Consortium, Vanderbilt University, https://www.project-redcap.org | – | – |