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A R andomiz E d Trial of EN t ER al G lutamine to Minim IZE Thermal Injury Laboratory Training. Lab Manual p 4. Blood Sample Overview. All patients will have blood samples taken unless they refuse consent - PowerPoint PPT Presentation
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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.