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

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

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

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)

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

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

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

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

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)

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

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

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

Speckled Red/Green Top & Blue/Black Top tubes Lab Manual p 17

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

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

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

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

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

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!

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

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.