Mar 04, 2026
Dilute plasma biobanking protocol
- Taylor P. Johnson1,
- Ariel Saurí1,
- Daniel W. Sirkis1,
- Jennifer S. Yokoyama1,2,3
- 1Fein Memory and Aging Center, Department of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA;
- 2Global Brain Health Institute, University of California, San Francisco, San Francisco, CA, USA;
- 3Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, USA
- Taylor Johnson
Protocol Citation: Taylor P. Johnson, Ariel Saurí, Daniel W. Sirkis, Jennifer S. Yokoyama 2026. Dilute plasma biobanking protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8xq7rv2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 25, 2026
Last Modified: March 06, 2026
Protocol Integer ID: 244128
Keywords: Plasma, whole blood, PBMC, UCSF, Yokoyama, Biomarker, Protein, dilute plasma biobanking protocol, biobanking of dilute plasma, dilute plasma biobanking protocol this protocol presents method, dilute plasma available as supernatant, dilute plasma, accurate quantification of analyte, total volume whole blood, phlebotomy tube anticoagulant volume, peripheral blood mononuclear cell, generated dilute plasma, density differential of whole blood, analyte, 1x pbs volume for pbmc processing, human whole blood, proteomic assessment, cryopreservation tube, biobanking, pbmc isolation, ficoll layering for pbmc isolation, whole blood, dilution factor, pbmc processing, storage with additional centrifugation step, pbmc isolation protocol
Abstract
This protocol presents methods for separation and biobanking of dilute plasma from human whole blood, specifically in parallel with a peripheral blood mononuclear cell (PBMC) isolation by density gradient centrifugation. Whole blood is diluted with Ca²⁺- and Mg²⁺ -free 1x PBS prior to Ficoll layering for PBMC isolation to increase the density differential of whole blood relative to separation medium to promote effective layering. After centrifugation in accordance with PBMC isolation protocol, dilute plasma available as supernatant is aliquoted into cryopreservation tubes for long-term storage at -80°C. Generated dilute plasma can be clarified prior to storage with additional centrifugation steps at 4°C or performed upon thawing, depending on study goals. Accurate quantification of analytes for proteomic assessment may be achieved by correcting through a dilution factor, considering (i) phlebotomy tube anticoagulant volume, (ii) total volume whole blood drawn, and (iii) added 1x PBS volume for PBMC processing.
Materials
- 8.5 ml acid-citrate-dextrose (ACD) phlebotomy tube (whole blood drawn)
- Gasketed internally threaded cryovials
- 70% ethanol
- Kimwipe
- Serological controller
- Serological tips (5 ml)
- 1x PBS (-Ca²⁺, -Mg²⁺)
- Tissue culture biosafety cabinet
- Dry ice
- Ice bucket
Protocol materials
70% ethanol
1X PBS
Troubleshooting
Materials preparation
Prepare whole blood for density gradient centrifugation according to respective PBMC isolation protocol, diluted with 1X PBS to improve separation.
During density gradient centrifugation for PBMC isolation, prepare materials for plasma biobanking.
Label four cryovials according to study requirements in tissue culture hood.
Dry ice in ice bucket for immediate freezing.
After density gradient centrifugation, spray separated diluted whole blood and Ficoll tube into tissue culture hood with 70% ethanol . Take care to maintain the distinct, clean layers.
Dilute plasma collection
Using a5 mL pipette tip, remove 6 mL of dilute plasma from the top layer.
Pipette slowly to prevent disturbances below. Follow volume down from the top with pipette tip to maintain maximum distance from PBMC. Avoid air bubbles.
Dilute plasma biobanking
Aliquot 1.5 mL volumes into each of the four prepared, labeled, cryovials. Ensure gaskets are correctly seated and caps are secured before removing aliquoted cryovials from the tissue culture hood.
Place dilute plasma cryovials immediately on dry ice for freezing. Place vertically in dry ice to prevent freezing of dilute plasma onto caps. Keep on dry ice until frozen.
Proceed with PBMC isolation or other primary phlebotomy tube application.
Place dilute plasma aliquots at -80 °C for long-term storage.
Record all observations and storage locations in secure spreadsheet or LIMS database system.