Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses

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 strategies^2,3,4. 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

1. Institutional review board approval
   1. Obtain the approvals from the local IRB before the start of the experiment and perform sample collection accordingly.
2. Study population
   1. Inclusion criteria: Include all patients (ages 0 - 99 years) that undergo intraocular surgery at the institution that will provide an adequate quantity of aqueous humor or vitreous liquid in excess of that required for appropriate diagnostic tests to evaluate the patient's condition and patients who wish to participate.
   2. Exclusion criteria: Exclude patients who decline to participate and pregnant women.

3. Informed consent
   1. Obtain written informed consent from each patient following the IRB approved protocol.
   2. Archive the signed consent in a secured database.
   3. Train involved personnel (surgeons, laboratory technicians, operating room (OR) staff, scientists) as described in this protocol.
   4. Set up a sample managing database. Use REDCap as a HIPAA-compliant web-based sample database that is designed to support data capture for research studies⁵.
      NOTE: This article describes the use of the web-based interface provided by REDCap to design forms, define fields, set up branching logic, and apply data validation rules without the need for extensive programming knowledge. Alternatively, other software, such as standard spreadsheet applications, may also be suitable.
   5. Ensure the availability of a cooling box, dry ice, syringe for sample collection, and cryovials (see Table of Materials). Use cryovials with barcodes that are permanently etched onto the vials. This eliminates the need to add patient identifiers on the vial and the possibility of losing a label under freezing conditions.
   6. Notify the surgeon on the case and the lab technician who will help with the sample collection in the OR at least 24 h prior to the scheduled surgery.

2. Acquisition of surgical specimens in the operating room

1. Mobile operating room lab interface (MORLI)
   1. Establish a MORLI in the operating room. MORLI includes a flat lab bench surface, a computer/tablet with a barcode scanner with access to the REDCap database, and a cooling box with dry ice (see Table of Materials).
      CAUTION: Dry ice is extremely cold. Always wear gloves when handling dry ice, and avoid touching it.
2. Preparation of sample collection in the OR
   1. Log in to the computer/tablet on MORLI and open the REDCap database.
   2. Check that informed consent has been signed by the patient and confirm that with the surgeon. Remind him/her that an undiluted sample is required.
   3. Wear gloves. Obtain the appropriate number of barcoded cryovials (0.5 mL for aqueous humor and 1.9 mL for vitreous samples) and place them where they are easily accessible.
3. Collection of aqueous humor liquid biopsies
   CAUTION: Consider human tissue samples as biohazardous material, which requires appropriate precautions such as a lab coat and gloves to assure the safety of the involved personnel.
   NOTE: The following steps should only be performed by a trained ophthalmic surgeon. Aqueous humor liquid biopsies can be obtained, for example, at the beginning of cataract surgery, one of the most frequent surgeries worldwide.
   NOTE: A sterile field is maintained as per standard of care protocols in the operating room. Preoperative procedures related to patient anesthesia follow standard of care steps for anterior chamber and vitreoretinal surgeries.
   1. Prepare and drape the eye for surgery and place a sterile lid speculum for optimal visualization of the sterile field.
   2. Use an operating microscope to perform an anterior chamber paracentesis perpendicular to the limbus using a 30-32 G needle connected to a 1 mL syringe. Use a cotton tip or small forceps to stabilize the eye during this procedure.
      NOTE: Ensure that the needle and the syringe are locked and that there is no pressure in the syringe (by moving the plunger). Ensure that the tip of the needle remains over the peripheral iris in the mid-anterior chamber to avoid damage to intraocular structures. In the case of cataract surgery, the needle to obtain the liquid biopsy can also enter the anterior chamber via one of the paracenteses that are created for the cataract surgery.
   3. Under direct visualization via the microscope, manually aspirate approximately 100 µL of undiluted aqueous humor using a 1 mL syringe. Move the syringe plunger with the surgeon's non-dominant hand or by a trained assistant without moving the needle.
      NOTE: Obtain less than 100 µL of aqueous humor in case the anterior chamber should collapse.
   4. Carefully remove the needle from the anterior chamber.
      NOTE: In a phakic eye, keep the needle over the iris to avoid touching the lens. Positive pressure on the globe can increase reflux. Releasing the cotton tip before the needle is withdrawn helps reduce reflux.
   5. Pull back the plunger and see how the air and the collected fluid are moving.
   6. Inject the syringe into the cryovial. The extra air clears the syringe's dead space.
   7. Use the barcode on the cryovial to scan the sample to the REDCap form on a computer in the operating room (more details in steps 3.1 to 3.9).
   8. Immediately transfer the cryovial to dry ice in the cooling box.
   9. Continue with the surgery scheduled for the patient (e.g., a cataract surgery as previously described⁷ ).
4. Collection of vitreous liquid biopsies
   NOTE: The following steps should only be performed by a trained vitreoretinal surgeon. Vitreous liquid biopsies can be obtained at the beginning of a vitrectomy⁸. Since the goal is to collect an undiluted vitreous sample, the vitrectomy cutter will not be primed with fluid¹.
   NOTE: A sterile field is maintained as per standard of care protocols in the operating room. Preoperative procedures related to patient anesthesia follow the standard of care steps for anterior chamber and vitreoretinal surgeries.
   1. Prepare and drape the eye for surgery and place a sterile lid speculum for optimal visualization of the sterile field.
   2. Create sclerotomies with a 23-, 25-, or 27-G trocar cannula, following the standard of care procedures. Insert the infusion cannula and visually confirm the appropriate placement in the vitreous cavity.
   3. In the vitreous cavity, activate the vitreous cutter without infusion to collect an undiluted vitreous sample. Manually aspirate 0.5 to 1.0 mL of vitreous using a syringe that is connected to the vitreous extrusion cannula¹.
   4. Remove the vitreous cutter from the eye and turn on the fluid infusion.
   5. Aspirate the remaining fluid within the tubing into the syringe.
   6. Disconnect the syringe.
   7. Process the sample as described for an aqueous humor specimen in section 2.3 (step 2.3.5 to step 2.3.9).

3. Processing of samples in the OR and adding samples to the database

1. Ask the lab technician to take the prepared cryovial (0.5 mL for aqueous humor and 1.9 mL for vitreous samples) and walk to the surgeon without touching any sterile OR equipment.
2. Ask the lab technician to open the cryovial.
3. Unload the syringe directly into the cryovial.
4. Ask the lab technician to immediately recap the cryovial.
5. Ask the lab technician to walk back to the MORLI and immediately transfer the sample onto dry ice in the cooling box (-80 ˚C). Close the lid of the box.
6. Open a new sample collection form. Enter the following information in the respective field of the form: case surgeon, location and date of collection, patient identifier number, and other basic information, such as age, sex, right or left eye, diagnosis, preoperative history (free text), information about the procedure (e.g., type of surgery), as well as information about the samples, such as number of samples collected, type of samples (aqueous humor, vitreous), and other details such as volumes. Add the tube barcode using the barcode scanner.
7. Click on Submit/Next.
8. Repeat steps 3.1 to 3.7 if any additional samples are collected.
9. When all samples are secured, click Save and submit on the REDCap sample collection form. Then log out of the database and the computer/tablet.

4. Transferring cryovials to storage

1. Transport the samples on dry ice in the cooling box from the OR to the lab and place it on a lab bench next to a lab computer.
2. Log in to REDCap on the lab computer using your login ID and password.
3. Wear gloves. Take one of the collected samples and scan the barcode of the cryovial into the database (more details in section 5). Immediately place the sample back on dry ice.
4. Obtain a second container filled with dry ice.
5. Obtain a rack for the cryovials from the -80 °C freezer. Place it in the second container on dry ice.
   NOTE: A 96-format rack will be needed for the 0.5 mL aqueous humor tubes and a 48-format rack for the 1.9 mL vitreous tubes.
6. Scan the barcode of the rack to the database (more details in section 5).
7. Transfer the sample to the rack.
8. Add the position of the vials in the rack to the database (more details in section 5).
9. Click Save and submit.
10. Transport the rack with the vials on dry ice to the fridge for storage at -80 °C. Add the rack to a specific position in the fridge using a coordinate system. This will later allow to retrieve samples for downstream analysis easily.

5. Sample storage form

1. Complete a storage form for each sample that is collected during the entry form phase. Click on the empty circle or the "+" under Sample Storage to create and open a new storage form.
2. Enter the date that this form has been completed under Record Archival Date.
3. Scan or type the tube barcode under Specimen Tube Barcode. Immediately place the sample back on dry ice.
4. Select whether a sample is transferred out or if the sample is going into internal biorepository storage.
5. Verify that written informed consent was obtained from the patient and select the box under Verify Consent Compliance and enter your name under Consent verified by.
6. Select a free and suitable location for the cryovial in the rack. Transfer the cryovial at this position into the rack (e.g., position A1). Keep the rack on dry ice.
7. In the Location phase, enter the following information: the location of the freezer under Freezer, the shelf number where the sample will be stored under Shelf, the box barcode under Box Barcode, the tube position in the box by row (Tube Position (Row)) and column (Tube Position (Column)).
   NOTE: Optionally, a box label can also be entered under Box Label, which may facilitate to find the box in the freezer.
8. Under the Usage section, enter the following information: the name of the project that the sample is used for (Project Name), the specimen volume in one of the following categories: full, partial, near-empty, or empty (Specimen Volume), and storage notes if applicable under Storage Notes.
   NOTE: The date, time and user that last accessed the form is auto populated to ensure a chain of custody that can be reviewed and audited as needed.
9. Confirm that the form is completed by clicking Complete under Complete?.
10. Click Save & Exit Form. This will bring you back to the patient overview.
11. For every tube that was collected, generate another sample collection form by clicking on the "+" under Sample Storage. Then repeat steps 5.1 to 5.10.
12. Click Save and Exit to complete the form and log out of the database and the computer/tablet.
13. Transfer the sample rack (on dry ice) to the fridge at the prespecified position.

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.

1. Identify samples of interest for the experiment by using the search function of the database. This will allow finding e.g., all aqueous humor samples from patients between 20 and 40 years with diabetic retinopathy.
2. Obtain the location of the cryovials of interest (freezer, shelf/rack, sample rack, coordinates within the rack). Write them down, print them, or have them available on a mobile computer/tablet to facilitate the finding of the samples in the freezer.
3. Mark the samples as used in the database.
4. Click Save and Exit to complete the form and log out of REDCap and the computer/tablet.

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 analyses^3,10 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 humans^2,3,4,14, and may help overcome the limitations of cell and animal disease models that do not fully recapitulate human disease^15,16. 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 individual^2,18,19.

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 qualities^9,20. 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 disease^18,21. 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 trials^4,16.

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.