This document outlines the process of collecting, processing, and storing peripheral blood from research participants enrolled in SCENT U54. 

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.)

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.

Sample Transport:

Samples should be transported to the lab and processed 30-60 minutes after collection. Transport should occur at Room temperature

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 2200rpm
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 -80°C freezer
5. Update sample inventory

PaxGene:

1. Store the PAXgene Blood RNA Tube upright Room 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