Nov 25, 2024

Public workspaceSinai SCENT TMC - Peripheral Blood Collection V.2

DOI
dx.doi.org/10.17504/protocols.io.ewov1q6q7gr2/v2
  • 1Icahn School of Medicine at Mount Sinai
  • Cellular Senescence Network (SenNet) Method Development Community
Sojin Kim
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DOI: dx.doi.org/10.17504/protocols.io.ewov1q6q7gr2/v2
Protocol Citation: Santos Bermejo, Sojin Kim, Patty J Lee 2024. Sinai SCENT TMC - Peripheral Blood Collection. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q6q7gr2/v2Version created by Sojin Kim
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 02, 2024
Last Modified: November 25, 2024
Protocol Integer ID: 112742
Abstract
This Standard Operating Procedure is to provide guidance toresearch team members who are involved in the retrieval, processing, and storing of peripheral blood samples obtained from research participants.

The research team shall use this SOP within the scope of an IRB-approved protocol.
Materials
Blood Collection 
  • 2x Yellow Top Tubes (PBMC) - Acid citric dextrose additive (ACD)
  • 1x Red Top Tube (Serum) – No additive
  • 3x Purple Top (Plasma) - Ethylenediaminetetraacetic Acid (EDTA)
  • 1x PaxGene Tube
  • Blood Collection Supplies: Gloves, Butterfly needle, vacutainer, tourniquet, gauze, alcohol pads, Band-Aids
  • Biohazard bag
  • Transport container (check with lab manager for appropriate transport container)

Processing
  • Microtubes (5x 0.2mL, 1-2x 1.5mL)
  • PBS
  • Histopaque – 1077 (Sigma, 10771)
  • 10% bleach solution (to discard used tips, tubes, etc.)
Scope
Scope
This document outlines the process of collecting, processing, and storing peripheral blood from research participants enrolled in SCENT U54. 
CRITERIA 1 - Collecting blood samples from colonoscopy and bronchoscopy
dx.doi.org/10.17504/protocols.io.bp2l628yrgqe/v3
dx.doi.org/10.17504/protocols.io.rm7vzjz4xlx1/v1

CRITERIA 2 - Collecting blood samples (banked, FAMRI)
Inclusion criteria: a) age 65 or older, b) persons who agreed to be contacted for the EDOPeCC study, c) persons who provide a smoking history and agree to be contacted for the current study.

Exclusion criteria: a) immunocompromised adults (HIV, malignancy diagnosis in past 5 years, systemic chemotherapy, end-stage renal failure), as these factors may influence the expression of TLRs; b) adults with other obstructive lung disease (history of asthma, cystic fibrosis, bronchiectasis, bronchiolitis obliterans, or vocal cord dysfunction); c) adults who cannot give informed consent due to language, cognitive, or other barriers; d) adults who cannot perform spirometry due to cognitive, health or other reasons.
Materials
Materials
Blood Collection 
  • 2x Yellow Top Tubes (PBMC) - Acid citric dextrose additive (ACD)
  • 1x Red Top Tube (Serum) – No additive
  • 3x Purple Top (Plasma) - Ethylenediaminetetraacetic Acid (EDTA)
  • 1x PaxGene Tube
  • Blood Collection Supplies: Gloves, Butterfly needle, vacutainer, tourniquet, gauze, alcohol pads, Band-Aids
  • Biohazard bag
  • Transport container (check with lab manager for appropriate transport container)
Processing
  • Microtubes (5x 0.2mL, 1-2x 1.5mL)
  • PBS (Phosphate Buffered Saline)
  • Histopaque – 1077 (Sigma, 10771)
  • 10% bleach solution (to discard used tips, tubes, etc.)
Procedures - Blood collection and Transport
Procedures - Blood collection and Transport
Sample retrieval:  
Peripheral blood shall be collected during visit 2, immediately before the bronchoscopy procedure.
Order of blood draw:  
Red → Yellow → Purple → PaxGene
Inversion times
Once blood has stopped flowing in the tube, remove and gently invert (Avoid vigorous shaking or mixing):
  • Red – No inversion necessary
  • Yellow – 8 times
  • Purple – 8 times
  • PaxGene – 8 times. Do not force blood into the tube, which can affect RNA quality.
Critical
Sample Transport:
Samples should be transported to the lab and processed 30-60 minutes after collection. Transport should occur at TemperatureRoom temperature

Procedures - Sample Processing
Procedures - Sample Processing
10m
10m
Serum (Red top) and Plasma (Purple top):

  1. Label all tubes with Study ID and ‘S’ for serum, and ‘P’ for plasma.
  2. Centrifuge tubes Centrifigation2200 rpm, 00:10:00 , Break #9, #9
  3. After spinning, aliquot supernatant to 5 x 0.2mL microtubes, and the remaining supernatant into 1.5mL tubes (label top of 1.5mL w/ volume (e.g. ‘300cc’)
  4. Store in Temperature-80 °C freezer
  5. Update sample inventory

10m
PaxGene:

  1. Store the PAXgene Blood RNA Tube upright TemperatureRoom temperature (18°C–25°C) for a minimum of 2 hours and a maximum of 72 hours before processing or transferring to the refrigerator (2–8°C) or freezer (−20°C).
  2. Update sample inventory
PBMC Isolation and Cryopreservation:

See PBMC isolation protocol in LRT (Lee Research Team) shared drive
Protocol references