Aug 22, 2023

Public workspaceCAMbank: cfDNA BCT Field Processing v1

DOI
dx.doi.org/10.17504/protocols.io.6qpvr33yovmk/v1
  • 1Weill Cornell Medicine;
  • 2BioAstra
Eliah G Overbey
Open access
DOI: dx.doi.org/10.17504/protocols.io.6qpvr33yovmk/v1
Protocol Citation: Eliah G Overbey, Krista A Ryon, JangKeun Kim, Christopher E Mason 2023. CAMbank: cfDNA BCT Field Processing v1. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr33yovmk/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: August 22, 2023
Last Modified: June 27, 2024
Protocol Integer ID: 86810
Keywords: CAMbank, astronaut, SOMA, cfDNA BCT, Streck, plasma
Funders Acknowledgement:
WorldQuant Foundation
NASA
Grant ID: NNX14AH50G
NASA
Grant ID: NNX17AB26G
NASA
Grant ID: 80NSSC22K0254
NASA
Grant ID: NNH18ZTT001N-FG2
NASA
Grant ID: 80NSSC22K0254
NASA
Grant ID: NNX16AO69A
NASA
Grant ID: 80NSSC21K0316
Abstract
Field processing of cfDNA BCTs for the Cornell Aerospace Medicine Biobank (CAMbank).

Instructions for preserving: plasma and RBC Pellets.
Materials
Tube Type: Streck Cell-Free DNA BCT® RUO (Streck: #230470)
Perform Venipuncture
Perform Venipuncture
5m
5m
After venipuncture, invert the tubes gently 8 to 10 times to fully mix tube anticoagulant with blood sample.

Store the tube upright at room temperature until centrifugation.

Note: The tube stabilizes cell-free DNA for up to 14 days at 6 °C to 37 °C and CTCs for up to 7 days at 15 °C to 30 °C.
5m
Centrifuge Settings: Plasma Separation
Centrifuge Settings: Plasma Separation
25m
25m
Note: A swing bucket centrifuge is required.

Set centrifuge:
  • acceleration: 9
  • deceleration: 9
  • temperature: RT
  • duration: 20 minutes
  • speed: 300xg
3m
Place the cfDNA BCTs in the centrifuge.
Place a protective cover over the swing buckets in case of tube leakage.
Start the centrifuge.
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity.
22m
Separate Plasma
Separate Plasma
7m
7m
Carefully remove cfDNA BCTs from the centrifuge and inspect for separation of red blood vs plasma layers.

Transfer cfDNA BCTs to a sanitized laminar flow hood.
2m
Remove the upper plasma layer and transfer to a new 15mL conical tube.
Cap the conical tubes and return to the centrifuge.
5m
Centrifuge Settings: Plasma Purification
Centrifuge Settings: Plasma Purification
15m
15m
Note: A swing bucket centrifuge is required.

Set centrifuge:
  • acceleration: 9
  • deceleration: 9
  • temperature: RT
  • duration: 10 minutes
  • speed: 5000xg
3m
Place the conical tubes in the centrifuge.
Place a protective cover over the swing buckets in case of tube leakage.
Start the centrifuge.
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity.
12m
Freeze Red Blood Cell Pellets
Freeze Red Blood Cell Pellets
5m
5m
Recap and freeze the cfDNA tubes to preserve DNA in the red blood cell pellet
5m
Aliquot Plasma
Aliquot Plasma
17m
17m
Carefully remove conical tubes from centrifuge.
Transfer conical tubes to a sanitized laminar flow hood.
2m
Using a P1000, carefully aliquot plasma into 2D barcoded tubes at 500µL each.

Return 2D tubes with plasma to the rack and place in the -80C freezer.
15m