REDOXS

1

A RandomizEd Trial of ENtERal Glutamine to
MinimIZE Thermal Injury Laboratory Training
Study Sponsors Dr. Paul Wischmeyer (US) Dr. Dominique Garrel (Canada) Clinical Evaluation Research Unit Director Dr. Daren Heyland Project Leader Rupinder Dhaliwal Project Assistant Maureen Dansereau
2
Blood Sample Overview
Lab Manual p 4
All patients will have blood samples taken unless
they refuse consent For DNA studies, lymphocyte
function studies, inflammatory cytokines heat
shock proteins
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Blood Draws
Lab Manual p 4

• Blood Draws
• Draw 1 (4 days post start of study intervention)
  48 hrs
• Draw 2 (7days post start of study intervention)
  48 hrs
• Draw 3 (14 days post start of study intervention)
  48 hrs
• Draw 4 (21 days post start of study intervention)
  48 hrs
• If a draw can not be obtained due to a weekend,
  draw on the closest working day
• Draw from a central line
• If no central line, draw with routine blood work
• DO NOT draw blood if no central line
• no scheduled blood draw

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Materials Provided
Lab Manual p 5, 6

• Lab Kits (4 kits per patient) each containing
  
• 2 Heparin CPT tubes
• 1 Citrate CPT tube
• 1 Sodium Heparin tube
• 1 Paxgene DNA tube (Kit 1 only)
• 30 Cryovials
• 40 Labels
• For expired tube replacement contact Elizabeth
  Luzier (Lab Manual p 7)

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Materials Needed
Lab Manual p 5

• Centrifuge variable speed
• Pipette calibrated for 50 to 500 microlitre
  pipetting
• - 80 C (-112 F) Freezer
• Phosphate Buffered Saline (PBS)
• Dimethyl Sulfoxide (DMSO)
• 0.9 Normal Saline Solution
• 15 ml conical tubes
• Timer
• Ice
• Gloves
• Biohazardous waste disposal system

Supplies for shipping specimens on dry ice
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Cryovial/Tube Labels
Lab Manual p 7

• Each kit will contain enough labels for each
  cryovial/tube
• Add one label to each cryovial/tube used
• Label includes an ID as follows

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Lab Sample Log Labels
Lab Manual p 8

• Add to labels
• enrollment number
• date and time of the blood draw

8
Blood Draw Preparation
Lab Manual p 8

• BEFORE drawing blood
• Ensure all necessary materials and equipment are
  present (including a cup/container of ice
  bedside)
• Fill out and affix labels to tubes

9
Blood Draw Order
Lab Manual p 8

• Draw blood in the following order 
• (1) 2 x Red/green speckled top tubes
• (2) 1 x Blue/black speckled top tube
• (3) 1 x Green top tube
• (4) 1 x PAXGENE tube (1st draw only)
• Invert all tubes 8-10 times
• Place Green Top tube (sodium heparin) only on ice
  immediately and keep it on ice until further
  processing (do not exceed 30 minutes)

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Green Top Tube (Sodium Heparin)
Lab Manual p 9, 16
Process within 30 minutes of collection

• Step 1
• Remove green top tube from ice and place in
  centrifuge
• Centrifuge at 1000 RCF (g) for 5 minutes
• If red cells are re-suspended in plasma
  when removing tube from
• centrifuge, repeat centrifuge process
  above.
• Step 2
• Pipette plasma in 500µL (0.5 mL) aliquots
  into 4 green topped
• cryovials
• Step 3
• Place cryovials in - 80 C freezer
• Store at - 80 C (-112 F) until shipment
  to central lab

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Lab Manual p 16
Green Top Tube (Sodium Heparin)
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Speckled Red/Green Blue/Black Top tubes
Lab Manual p 10, 17
Keep at room temperature and process within 2
hours

• Centrifuge for 30 minutes at room temperature at
  1800 RCF
• Pipette off the plasma without disturbing the
  cell layer
• Aliquot 500µL of plasma from the Blue/Black top
  tube into each of the 8 labeled (ACD) orange
  cryovials
• Aliquot 500µL of plasma from Red/Green top tubes
  into each of the 16 labeled red cryovials
• Freeze at 20 C (-4 F) for 1 day
• Store at -80 C until shipping

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Speckled Red/Green Top Blue/Black Top tubes
Lab Manual p 17
14
Speckled Blue/Black Top
Lab Manual p 11, 18
Using the already processed Blue/Black top tube

• Gently re-suspend the cells above the gel barrier
• Transfer into 2 labeled (ACD PBMC) orange
  cryovials
• Estimate volume and add DMSO to a final
  concentration of 10 (see table)
• Cap cryovials and mix gently by inverting several
  times
• Freeze at 20 C (-4 F) for 1 day
• Store at 80 C (-112 F) until shipping

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Speckled Blue/Black Top Tube
Lab Manual p 18
16
Speckled Red/Green Top
Lab Manual p 12, 19
Using the already processed Red/Green top tubes

• Pull out the white cell layer from both Red Green
  Top tubes
• Add to one new 15 ml centrifuge tube
• Add PBS to the conical tube to bring total volume
  to 15 ml
• Cap tube and mix by inverting several times
• Centrifuge for 15 minutes at 600 RCF
• Aspirate and discard supernatant without
  disturbing cell pellet
• Add PBS to the conical tube to bring total volume
  to 10 ml
• Cap tube and mix by inverting several times
• Centrifuge for 15 minutes at 600 RCF
• Aspirate and discard supernatant without
  disturbing cell pellet
• Freeze cell pellet at -80º C (-112 F) until
  shipping

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Speckled Red/Green Top Tubes
Lab Manual p 19
18
PAXGENE DNA Tube
Lab Manual p 12, 20

• Place tube upright in wire rack
• Freeze at - 20º C (-4º F) for 24 hrs NOTE Do
  not freeze in styrofoam,
• tube will crack
• After 24 hours, transfer to -80º C
• (-112 F) freezer
• Maintain at -80ºC (-112 F) until
• shipping

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Lab Sample Log
Lab Manual p 13, 21

• Complete One log per patient, add
• Affix cryovial labels with Patient ID to Lab
  Sample
• Log in space provided
• Date blood samples taken for each draw
• each cryovial/tube processed per day by colour
  
• Total samples processed per day

Date sample taken on label date recorded on
eCRF worksheet!
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Packaging Shipping
Lab Manual p 15

• Each patient place following in smaller
  individual blood draw boxes (total of 4 blood
  draw boxes per patient)
• Cryovials
• DNA tube
• PBMC 15mL centrifuge tube
• Place smaller boxes securely in a larger shipping
  box
• Add dry ice
• Add completed Lab Sample Logs (one log per
  patient)
• Ship via FedEx within 8 weeks of collection to
  Colorado (Mon-Wed)
• Indicate Bill Recipient on the FedEx shipping
  label
• Check local courier services for
  schedule

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Packaging Shipping

• Important Considerations
• Please be sure to inquire about your local
  service schedules and the latest time of day that
  you can call to arrange for a pick-up
• Local courier service (pickup and delivery) may
  be limited prior to, during and following
  observed holidays. It is imperative that you
  check local service schedules in advance of the
  holiday.
• Specimens should be shipped from Mondays through
  Wednesdays only.