The REDOXS© Study REducing Deaths due to OXidative Stress

A RandomizEd Trial of ENtERal Glutamine to MinimIZE Thermal Injury

Laboratory Training

Study SponsorsDr. Paul Wischmeyer (US)Dr. Dominique Garrel (Canada)

Clinical Evaluation Research UnitDirector: Dr. Daren HeylandProject Leader: Rupinder DhaliwalProject Assistant: Maureen Dansereau

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

Lab Manual p 4

Blood Draws 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 Manual p 4

Materials Provided

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)

Lab Manual p 5, 6

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

Lab Manual p 5

Supplies for shipping specimens on dry ice

Cryovial/Tube Labels

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 Manual p 7

Lab Sample Log Labels

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

Lab Manual p 8

Blood Draw Preparation

BEFORE drawing blood:

1. Ensure all necessary materials and equipment are present (including a cup/container of ice bedside)

2. Fill out and affix labels to tubes

Lab Manual p 8

Blood Draw Order

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)

Lab Manual p 8

Green Top Tube (Sodium Heparin)Lab Manual p 9,

16

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

Process within 30 minutes of collection

Green Top Tube (Sodium Heparin)

Lab Manual p 16

Speckled Red/Green & Blue/Black Top tubesLab 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

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 TubeLab Manual p 18

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

Speckled Red/Green Top TubesLab Manual p 19

PAXGENE DNA TubeLab 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

Lab Sample LogLab 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!

Packaging & Shipping

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

Lab Manual p 15

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.