Molecular profiling of liquid biopsies from the human eye can capture locally enriched fluids containing thousands of different molecules from highly specialized ocular tissues. They allow molecular characterization of ocular diseases in living humans and of further potential to identify novel diagnostic and therapeutic strategies. Here we developed protocol for the standardized collection and biobanking of high quality aqueous humor and vitreous liquid biopsies during intraocular surgery.
Part of the protocol is a web-based data privacy compliant database to annotate each sample over its lifetime and the use of a coordinate system that allows to track the location of each barcoded specimen in storage, which enables efficient sample retrieval for downstream experiments such as protein, glycan, and metabolite analysis. Use an operating microscope to perform an anterior chamber paracentesis perpendicular to the limbus using a 30 to 32 gauge needle connected to a one ml syringe. A cotton tip can be used to stabilize the eye during this procedure.
Ensure that the tip of the needle remains over the peripheral iris in the mid anterior chamber to avoid damage to intraocular structures. Under direct visualization via the microscope, manually aspirate approximately a hundred microliters of undiluted aqueous humor using a one ml syringe. The needle is then carefully removed from the anterior chamber.
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 Releasing the cotton tip before the needle is withdrawn helps reduce reflux.
Pull back the plumber and see how the air and the collected fluid is moving. Inject the syringe into the cryo vial. The extra air clears the syringe dead space.
Here you can see the barcode which is permanently etched onto the vial. The barcode is then used to scan the sample to the REDCap form on a computer in the operating room. The cryo vial will immediately be transferred on dry ice in the cooling box.
Vitreous liquid biopsies can be obtained at the beginning of a vitrectomy by a trained vitreoretinal surgeon. Note that the vitrectomy cutter will not be primed with fluid. In the vitreous cavity, activate the vitreous cutter without infusion to collect an undiluted vitreous sample.
Manually aspirate 0.5 to one ml of vitreous using a syringe that is connected to the vitreous extrusion cannula. The sample is then processed as we demonstrated for an accurate humeral specimen. This video is a walkthrough of a completed sample collection form in REDCap in survey view.
Once a patient completes the e-consent with the assistance of a clinical research coordinator, this form is populated. It can be accessed on the same device that the patient was e-consented on, or it can be accessed using any other approved device. A copy of the e-consent that was completed can be accessed here for signatures to be verified.
The coordinator then fills out the other relevant sections including the type of consent and in this blue section, details on the case itself, such as the case surgeon, where it was performed, the date of the collection, and other relevant info. In this next green patient section, we have patient demographics, including a sample MRN, the patient's first and last name, their sex, date of birth with an auto calculation of the age, the laterality of the affected eye, and diagnostic category. This form features branching logic, so when a checkbox is selected, it affects the appearance of check boxes below it.
In this case, this is an anterior segment and retina case. So first, the diagnosis retina box is populated. The coordinator has selected that this is a macular hole and the anterior segment diagnosis box has also been populated.
In this case, looks like it's a cataract. A pre-op history form is also populated as a free text box. Here, information from the EMR can be copy and pasted or new information on pre-op history can be entered for future reference.
Next, in this red section, we have the procedure box. In this case, looks like a pars plana vitrectomy was selected along with the a phaco IOL. Anything not populated here in any of these check boxes can be entered in free text using these other boxes that are peppered throughout the form.
Relevant details about the case itself are also completed. Finally, in this gold section, we have a collection area. The name of the coordinator collecting the specimen along with the number of specimen tubes being used have been entered here.
Again, using branching logic, we're able to collect as many samples as needed for the case. In this case, two samples were collected, looks like a vitreous core and an anterior chamber fluid sample. The barcodes of each of these samples have been scanned in here and recorded into the form.
Finally, there's a sample collection notes section where the coordinator can type in relevant details including volumes and quality of the fluid drawn. In this final file upload section, any relevant photos, videos, or documentation can be uploaded into the form itself and linked. Finally, once everything is completed, the coordinator can click submit next and the form is saved with all relevant info stored, connecting the sample successfully with the patient.
Transport the samples on dry ice from the operating room to the lab. Log in to REDCap on the lab computer. Take one of the collected samples and scan the barcode into the database.
Now, a second container with dry ice and a rack for the cryo vials is needed. The barcode of the rack will also be scanned to the database and the samples are transferred to the rack. The position of the vials in the rack is added to the database and the entry is saved and closed.
The rack with the vials is then transported on dry ice to the fridge for storage at minus 80 degrees Celsius. The rack is added to a specific position in the fridge using a coordinate system. This will later allow to easily retrieve samples for downstream analysis.
This video is a walkthrough of the sample storage form. Here, we see the patient overview record. This green circle indicates the sample collection entry form has already been completed.
This empty circle indicates that a storage form must be completed. A storage form must be completed for each sample that is collected during the entry form phase. Clicking this circle will take us into the first sample storage form.
Here we can see that a PID has been generated and the MRN has been masked. That information is still accessible and can be accessed using the previous page by clicking the green circle in the entry form. Here a specimen snapshot will be generated once this page is completed.
Under collection notes, we see that information has been auto imported from the entry form. Next, under record archival date, we can enter the date that this form is being completed. Under specimen tube barcode, we scan or reenter the barcode of the tube that is being archived.
In real time, this specimen snapshot is being updated. In this section, we can select whether a sample is being transferred out. Here we enter that relevant information.
For the sake of this example, we're going to indicate that this is a sample going into internal biorepository storage. Under verification, a research coordinator can go back and verify that the e-consent or paper consent was completed successfully and within protocols. The name of the person completing this verification is entered and using this dropdown, the verifier matches the barcode here with what was just typed.
This ensures accuracy. Next, in this location phase, we see the freezer where the sample is going to be stored. We can indicate the shelf where the sample will be stored as well as record the box barcode, which can be scanned or typed in.
A box label can also be typed in here and the label can be placed on the box for further matching. Then the two position by row and column are both recorded. This ensures that every barcode is associated with a position within the box that is recorded for easy retrieval in the future.
Under this usage section, we can enter our project name or select one from a dropdown box. Record the specimen volume. And here, the date and time as well as the user that last accessed this form is auto-populated to ensure a chain of custody that can be reviewed and audited as needed.
The person archiving the form enters their name here and indicates any relevant notes here that may be consistent with what they wanna record within this entry form for storage. Finally, there's another section to upload a file, and once all this is completed, this form can be completed and submitted by clicking save and exit, which brings us back to the patient overview. For each additional form that must be completed, for every tube that must be stored, we can click this plus button to go back into the sample storage form and generate another record.
This overview provides an access into the entry form that was generated as well as any subsequent storage forms for this patient record. In this way, it's easily retrievable and accessible by anyone with backend access or other given permissions within a REDCap. The collected liquid biopsies can be subjected to a variety of molecular analysis including proteomics, glycomics, and metabolomics.
The REDCap database enables simple and quick retrieval of samples, for example, by searching for samples from patients with a specific disease. This is a case of a 17 year old female patient who was immunocompromised and who presented with retinal and optic nerve inflammation. Being concerned about an infection, an aqueous humor liquid biopsy was performed and sent for DNA PCR analysis.
The results were positive for CMV and negative for HSV and toxoplasmosis and were critical to distinguish infectious from non-infectious forms of intraocular inflammation and 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, opticin, and collagen type 2.
Three vitreous liquid biopsies were analyzed using a glycoproteomics multiplex analyzer. 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. We also analyzed the metabolomics profile of aqueous humor liquid biopsies using a special form of mass spectrometry.
We identified 292 different metabolites in three aqueous human liquid biopsies. A pathway analysis identified a variety of metabolic pathways including amino acid metabolism, uria cycle, and carnitine synthesis. In conclusion, our workflow established a practical interface between the operating room and the research laboratory that allows standardized and high throughput collection, annotation, and storing of high quality surgical specimens.
The samples can be used for molecular downstream analysis including proteomics, glycomics, and metabolomic studies. Our protocol provides a valuable foundation for future translational research.
