1. REDOXS Training: Introduction & Site Investigator
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
November 2008

2. Lab Manual p 4
Blood Sample Overview
All patients will have blood samples taken unless they refuse consent
For DNA studies, lymphocyte function studies, inflammatory cytokines & heat shock proteins

3. Blood Draws 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
Remember:
Do NOT draw blood within a period of 12 hrs after surgery or blood transfusion. OK to draw before surgery

4. Lab Kits (4 kits per patient) each containing: 2 Heparin CPT tubes
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)

5. Materials Needed Centrifuge: variable speed
Lab Manual p 5
Materials Needed
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

6. 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

7. Lab Sample Log Labels Add to labels: enrollment number
Lab Manual p 8
Add to labels:
enrollment number
date and time of the blood draw

8. Blood Draw Preparation
Lab Manual p 8
Blood Draw Preparation
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 Draw blood in the following 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)

10. 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

11. Green Top Tube (Sodium Heparin)
Lab Manual p 16
Green Top Tube (Sodium Heparin)

12. Speckled Red/Green & Blue/Black Top tubes
REDOXS Training: Introduction & Site Investigator
Lab Manual p 10, 17
Speckled Red/Green & Blue/Black Top tubes
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
November 2008

13. 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

15. Speckled Blue/Black Top Tube
Lab Manual p 18
Speckled Blue/Black Top Tube

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
Freeze cell pellet at -80º C (-112° F) until shipping

17. Speckled Red/Green Top Tubes
Lab Manual p 19
Speckled Red/Green Top Tubes

18. PAXGENE DNA Tube Place tube upright in wire rack
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

19. Date sample taken on label = date recorded on eCRF worksheet!
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!

20. Check local courier services for schedule
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

21. Important Considerations
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.