The RE-ENERGIZE Study A RandomizEd trial of ENtERal Glutamine to minimize thermal injury Lab Study Manual Human Research Protection Office (HRPO) Log Number A-15774.0 HRPO Proposal Number 09155001 Clinical trials.gov ID #NCT00985205 November 12, 2010 1
Lab Manual Table of Contents Study Contacts...3 Blood Sample Overview...4 Blood Draw Visits...4 Materials Provided...5 Equipment and Supplies Needed...5 Tube Kit Contents... 6-8 Blood Draw Instructions...8 Blood Processing Instructions... 9-13 Green Top... 9-10 Speckled Red/Green Top & Speckled Blue/Black Top...11 Speckled Blue/Black Top...12 Speckled Red/Green Top...13 PAXGENE DNA Tube...13 Sample Log and Data Entry...14 Packaging & Shipping...15 Appendix...16 A Green Top Tube...17 B Red/Green Speckled Top Tube & Blue/Black Speckled Top Tube...18 C PAXGENE DNA Tube...19 D Lab Sample Log...20 November 12, 2010 2
Study Contacts RE-ENERGIZE Sponsor Contacts Paul Wischmeyer, MD University of Colorado Denver Department of Anesthesiology 12700 East 19 th Avenue RC2, Room 7119 Aurora, CO 80045 Phone: 720.848.6745 Fax: 303.724.2936 Paul.Wischmeyer@ucdenver.edu Dominique Garrel, MD Université de Montreal Department of Nutrition 2405, ch. de la Côte Ste-Catherine Pavillon Lilian-de -Stewart Room 1280-5 Montréal, QC H3T 1A8 Phone: 514.343.6111 ext.1739 Hospital: 514.890.8100 Cell: 514.926.4848 dominique.garrel@umontreal.ca Clinical Evaluation Research Unit Contacts Rupinder Dhaliwal, RD Project Leader Phone: 613.549.6666 ext. 3830 Cell: 613.484.3830 dhaliwar@kgh.kari.net Maureen Dansereau Project Assistant Phone: 613.549.6666 ext. 6866 danserem@kgh.kari.net Clinical Evaluation Research Unit Angada 4 Kingston General Hospital 76 Stuart Street Kingston ON K7L 2V7 Central Laboratory Contacts Kelly Queensland Wischmeyer Translational PharmacoNutrition Laboratory University of Colorado Denver Dept. of Anesthesiology 12700 E. 19th Ave. RC2 Room P15 7490D Aurora, CO 80045 Tel: 303-724-2945 Kelly.Queensland@ucdenver.edu LPS Testing Contacts Lisa Hanson, Manager Immune Surveillance Laboratory Room W250 The Research Institute at Nationwide Children's Hospital 700 Children's Drive Columbus, OH, USA 43205 Tel: 614-355-2942 Lisa.Hanson@NationwideChildrens.org November 12, 2010 3
Blood Sample Overview All patients enrolled in the RE-ENERGIZE study will have blood samples taken unless they refuse consent. Blood samples will be used for DNA studies, lymphocyte function studies as well as inflammatory cytokines and heat shock proteins measurements. These studies will be designed to help understand how glutamine works, to measure the body s response to the study intervention and also to look at different markers in the blood of critical illness. Blood samples will be obtained on days 4, 7, 14 and 21 post start of the study intervention. We choose not to sample blood on day 1 because the analysis of our previous studies showed great variations in relation with fluid administration and water retention. The rationale for sampling at day 4 is based on the observation that the peak for inflammatory response occurs at that time. The 3 other samples are needed to assess the effects of glutamine on the time-course of inflammation, immunosuppression and the production of heat shock proteins. Since surgery has a significant and short lived impact on the parameters to be studied, the blood sampling will be made at least 12h post surgery. Therefore, the 4 sampling times will be identified as: acute phase (around day 4), day 7, 14 and 21 (post start of the study intervention). Blood Draw Visits Each RE-ENERGIZE patient enrolled is scheduled to have blood draws done on the following study days: 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 Blood samples are to be drawn from the central line, wherever possible. If there is no central line, then blood should be drawn at the same time as routine blood work to prevent unnecessary blood draws. If there is no central line and there is no routine blood work scheduled for 4, 7, 14 or 21 days post start of study intervention (or the next day if weekend), do not draw blood. Blood samples must NOT be drawn within the period of 12 hrs after surgery or a blood transfusion. It is OK to draw blood before surgery/transfusion. If a blood draw visit day falls on a weekend, and it is not possible to obtain and process the samples on the weekend, the sample should be drawn on the closest working day. For example, blood draws due on Saturdays can be drawn on Fridays. Blood draws due on Sundays can be drawn on Mondays. November 12, 2010 4
Materials Provided The following laboratory materials will be provided to all sites participating in the RE-ENERGIZE Study prior to the start of enrollment (contents per blood draw): Vacutainers o 2 heparin CPT tubes o 1 citrate CPT tube o 1 sodium heparin tube 1 Paxgene DNA tube (first blood draw only) LPS (Lipopolysaccharide) Stimulation media tubes (store at 4 Celsius) 32 Cryovials (16 red, 10 Orange, 6 green) Labels for the tubes, cryovials and Case report Forms Equipment and Supplies Needed All sites participating in the RE-ENERGIZE Study are responsible for ensuring that they have the following equipment/materials for the processing and storage of blood samples: Centrifuge (variable speed) capable of spinning at or above 1800 RCF (RCF = 0.00001118 x rotor radius x RPM2) (g) with a temperature range from 4 C - 25 C (RT) Centrifuge capable of accommodating 8mL CPT tubes, 6mL trace element free tubes, and 15mL conical tubes. 15mL conical tubes Phosphate Buffered Saline (PBS) 0.9% normal saline solution Dimethyl Sulfoxide (DMSO) Gloves Pipettes (capable of pipetting a range of 50 microliters to 500 microliters) Incubator (37 degree Celsius) Ice Timer -80 Freezer A safe way to dispose of human blood (as per standard procedures at site) Dry ice and Dry ice labels for shipping Biohazard bags/boxes for shipping November 12, 2010 5
Tube Kit Contents Each patient will have 1 white box containing 4 tube kit bags. Each tube kit is labelled according to the blood draw visit, Draw # 1 # 4 (4, 7, 14 and 21 days post start of study intervention). The tube kit contents are as follows: Draw 1 (4 days post start of intervention) 2 x red/green speckled top Heparin CPT tubes 8 ml each 1 x blue/black speckled top Citrate CPT tube 8 ml 1 x green top Sodium Heparin tube 4 ml 1 x PAXGENE DNA tube 8.5 ml 32 Cryovials: o 16 x red o 10 x orange o 6 x green Cryovial/tube labels Draw 2 (7 days post start of intervention) 2 x red/green speckled top Heparin CPT tubes 8 ml each 1 x blue/black speckled top Citrate CPT tube 8 ml 1 x green top Sodium Heparin tube 4 ml 32 Cryovials: o 16 x red o 10 x orange o 6 x green Cryovial/tube labels Draw 3 (14 days post start of intervention) 2 x red/green speckled top Heparin CPT tubes 8 ml each 1 x blue/black speckled top Citrate CPT tube 8 ml 1 x green top Sodium Heparin tube 4 ml 32 Cryovials: o 16 x red o 10 x orange o 6 x green Cryovial/tube labels Draw 4 (21 days post start of intervention) 2 x red/green speckled top Heparin CPT tubes 8 ml each 1 x blue/black speckled top Citrate CPT tube 8 ml 1 x green top Sodium Heparin tube 4 ml 32 Cryovials: o 16 x red o 10 x orange o 6 x green Cryovial/tube labels November 12, 2010 6
If you identify that tubes within your kits are expired, please contact Elizabeth Luzier to obtain replacement tubes. Elizabeth Luzier Wischmeyer Translational PharmacoNutrition Laboratory University of Colorado Denver Tel:(303) 724-3597 Email: Elizabeth.Luzier@ucdenver.edu Cryovial/Tube Labels Each kit will contain enough labels for each cryovial/tube. The person processing the samples will need to add one label to each cryovial/tube that will be used for blood sampling. Each label includes an ID that corresponds to the day of blood draw and site number (4 digits). The last 3 digits refer to the patient enrollment number (or randomization number) and these are to be filled in by the person at the site drawing the blood. Example: For patient #1 from site # 1012, the ID# on the label would look like this: Site Number (4 digit) Draw 1 Draw 2 Draw 3 Draw 4 1-1 0 1 2-1 - _ 2-1 0 1 2-1 - _ 3-1 0 1 2-1 - _ 4-1 0 1 2-1 - _ Patient enrollment number (3 digits). To be filled in by site as follows: Enrollment # 1= 001 Enrollment # 2= 002, etc Blood draw # CERU assigned # for randomized patient (always will be 1) November 12, 2010 7
Case Report Form (CRF) Labels In addition, ONE corresponding label for EACH day of blood draw will be provided for the Case Report Form. This label is to be attached to the Case Report Form titled Study Blood Work (page 25 of the Case Report Forms). Example: For patient #13 from site # 1012, for blood draw # 2 (7 days post start of study intervention), the Case Report Form label would look like this: Blood draw # Site Number (4 digit) Patient enrollment # 2-1 0 1 2 1-0 1 3 Date blood drawn (DD/MMM/YYYY) Time blood drawn (hh:mm) The person processing the samples must add the enrollment number and the date and time of the blood draw to the CRF Label and attach it to the row for the appropriate Draw # on the Study Blood Draw Case Report Form (refer to instructions under Sample Log and Data Entry) Blood Draw Instructions Using an existing central line, 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 (Hold vertically below patient s arm during collection)- 1 st draw only Invert all tubes 8-10 times. After tube inversion, place Green Top tube (sodium heparin) only on ice immediately and keep it on ice until further processing. NOTE: Prior to drawing blood, fill out and affix labels provided to blood draw tubes listed above. Ensure that you have a cup/container of ice with you when you draw the blood. November 12, 2010 8
Blood Processing Instructions While blood is spinning in centrifuge (refer to tube-specific instructions below), place provided labels on cryovials: 16 x red cryovials 16 x Heparin plasma labels 10 x orange cryovials 8 x ACD plasma labels 2 x ACD PBMC labels 6 x green cryovials 4 x LPS plasma labels 2 x LPS stimulation labels 10 Additional labels 4 x Vacutainer tube labels 1 x Paxgene DNA label (1 st kit only) 1 x Heparin PBMC label (to put on the 15mL conical tube) 1 x Case Report Form label 3 x Extra labels ACD = Acid Citrate Dextrose LPS = Lipopolysaccharide PBMC = Peripheral Blood Mononuclear cells Green top tube (Sodium Heparin tube, See appendix A) 1 x Green top tube Tube should be processed starting no later than 30min post collection Be sure the incubator s internal temperature is at 37 C LPS stimulation will be done in duplicate remove 2 microfuge tubes containing stimulation media from the refrigerator Remove the green top tube from ice and invert it gently a few times to ensure even mixing of the blood, remove lid and pipette 50µL of blood into each microfuge tube containing the stimulation media DO NOT DISCARD GREEN TOP TUBE AFTER BLOOD REMOVAL o Mix well (vortex or multiple inversions) o Place microfuge tubes into incubator for 4 HOURS EXACTLY! Spin remaining blood in green top tube at 1000 RCF (g) for 5 minutes. o Carefully remove plasma (top layer) in aliquots of 500µL and place into 4 green topped cryovials labeled plasma (6 empty cryovials provided, but 2 are used later in the procedure) o Store at -80 C until shipment November 12, 2010 9
After the 4 HOUR INCUBATION o Remove the microfuge tubes (containing the stimulation/blood mixture) from incubator o Centrifuge the microfuge tubes at 1000 RCF (g) for 5 minutes at 4 C if microcentrifuge has a temperature control o Remove microfuge tubes carefully not to disturb the supernatant (pinkish fluid) or pellet (red) at the bottom of the tube (if contents are accidently mixed repeat centrifugation) o o Pipette the supernatant off of each stimulation tube and place into the 2 green topped cryovials labeled LPS stimulation. BE SURE NOT TO PIPETTE ANY OF THE PELLET IN WITH THE SUPERNATANT Once supernatant has been removed the pellet can be discarded in biohazard waste Store supernatant at -80 C until shipment November 12, 2010 10
Speckled Red/Green top & Blue/Black tube (CPT tubes, see Appendix B) 2 x Red/Green speckled top tubes 1 x Blue/Black speckled top tube Tubes should be processed as soon as possible following collection. o Tubes can be kept at room temperature for a maximum of 2 hours Centrifuge at room temperature for 30 minutes at 1800 RCF o RCF = 0.00001118 x rotor radius x RPM2 Mononuclear cells (lymphocytes and monocytes) & platelets are in whitish layer just under the plasma layer (see Figure 1). Figure 1. CPT tube plasma & PBMC separation Remove the plasma from each tube without disturbing the cell layer and aliquot into the appropriate colored cryovials (see below). Discard any extra plasma. o Aliquot 0.5mL (500μL) of plasma from the Blue/Black top tube into each of the 8 labeled (ACD) orange cryovials. o Aliquot 0.5mL (500μL) of plasma from the 2 Red/Green top tubes into each of the 16 labelled red cryovials. See Figure 2 below. Figure 2. Cryovial showing 0.5 ml plasma November 12, 2010 11
Speckled Blue/Black Top Tube ONLY Using the already processed Blue/Black speckled top tube: Gently resuspend the cells above the gel barrier and transfer to 2 labelled (ACD PBMC) orange cryovials. Estimate the volume (should be ~ 0.8-1.5mL) and gently add the preserving solution (DMSO) to a final concentration of 10% (see table). Cap and mix gently by inverting tube 5 times. Freeze at -20ºC for approximately one day, then at -80ºC prior to shipment to the Central lab. Table 1: DMSO Table Volume of Volume of DMSO to add cell/plasma mixture (µl) (µl) 800 88 900 99 1000 111 1100 122 1200 133 1300 144 1400 155 1500 166 1600 177 1700 188 November 12, 2010 12
Speckled Red/Green Top Tube ONLY Using the already processed Red/Green speckled top tube: Pull out white cell layer from both tubes & add to the same new 15 ml centrifuge tube (Affix Heparin PBMC label provided.) o This is the second layer in the vacutainer (i.e. the lymphocyte and monocyte band) and, it will be turbid and sitting above the plug (see figure 1) o Do not be concerned if contaminated with a small amount of plasma above it Add PBS (Phosphate-buffered Saline) to bring volume of collected cells in the 15mL conical centrifuge tube to a total of 15mL Cap tube, mix by inverting tube 5 times Centrifuge for 15 minutes at 600 RCF o RCF = 0.00001118 x rotor radius x RPM2 o ONLY if a pellet does not form, increase the RCF to 1300 Aspirate and discard as much supernatant as possible without disturbing cell pellet Re-suspend cell pellet by tapping tube with finger Add PBS to bring volume to 10mL Cap tube, mix cells by inverting 5 times. Centrifuge for 15 minutes at 600 RCF Aspirate and discard as much supernatant as possible without disturbing cell pellet Freeze cell pellet at -80ºC until shipping PAXGENE DNA Tube (see Appendix C) 1 x PAXGENE DNA Tube Freeze PAXGENE DNA tube upright in wire rack at -20ºC for 24 hours (do NOT freeze in Styrofoam, tube will crack) Transfer tubes to -80ºC freezer until shipping November 12, 2010 13
Lab Sample Log and Data Entry Complete the Lab Sample Log (see Appendix D) for the patient (one log per patient) and list the following: Patient ID# (from the cryovial label) i.e. 1-1012-1-001 Date blood samples taken for each draw Number of each cryovial/tube processed per day by color Total number of samples processed per day Ensure that the date the sample was taken recorded on the Sample Log is the same as that on the CRF label. Affix the completed CRF label that corresponds to the day of blood draw and then add the date to the Case Report Form as shown below. November 12, 2010 14
Packaging & Shipping Cryovials, DNA tube, and PBMC 15mL centrifuge tube must be placed in individual blood draw boxes (total of 4 blood draw boxes per patient) and then these smaller boxes must be securely packed in a larger shipping box. Please check by gently shaking the box after packaging. If the containers are loose, repack the box by filling the empty spaces with paper. Mail processed study specimens on dry ice along with the completed Lab Sample Logs (one log per patient, see Appendix D) to the Wischmeyer TPN Lab within 8 weeks of collection at: Wischmeyer Translational PharmacoNutrition Laboratory UCD Dept. of Anesthesiology 12700 E. 19th Ave. RC2 Room P15 7490D Aurora, CO 80045 The FedEx account number to be used to ship samples to the Wischmeyer TPN Lab will be provided. 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. Timely pick-ups will ensure samples are shipped within stability for testing. Important Considerations 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. Frozen samples should NOT be shipped on the day before an observed holiday. Observed Holidays 2010: Christmas Day - Saturday, December 25 th Boxing Day - Sunday, December 26th 2011 2012 2013 New Year s Day Saturday, January 1 st Sunday, January 1 st Tuesday, January 1 st Good Friday Friday, April 22 nd Friday, April 6th Friday, March 29th Easter Monday Monday, April 25 th Monday, April 9 th Monday, April 1st Labour Day Monday, September 5 th Monday, September 3rd Monday, September 2nd Christmas Day Sunday, December 25 th Tuesday, December 25 th Wednesday, December 25 th Boxing Day Monday, December 26th Wednesday, December 26th Thursday, December 26 th Check with your courier regarding other holidays not listed above. November 12, 2010 15
Appendix A: Green Top Tube B: Red/Green Speckled Top Tube & Blue/Black Speckled Top Tube C: PAXGENE DNA Tube D: Lab Sample Log November 12, 2010 16
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Appendix B - Red/Green Speckled Top Tube & Blue/Black Speckled Top Tube Speckled Red/Green Top Speckled Blue/Black Top Red/Green Top Tubes X2 Blue/Black Top Tube Centrifuge at room temperature for 30 mins @ 1800 RCF Remove the plasma from each tube (without disturbing the cell layer) Red/Green From both tubes, aliquot 0.5mL (500μL) of plasma into each of the 16 labelled red cryovials Aliquot 0.5mL (500μL) of plasma into the appropriate coloured cryovials Blue/Black From 1tube, aliquot 0.5mL (500μL) of plasma into 8 labelled (ACD) orange cryovials Red/Green top tube Pull out the white cell layer from both tubes & add to the same new 15mL centrifuge tube (Affix Heparin PBMC label provided.) Add PBS to bring to total volume 15mL. Cap tube & invert 5 times. Centrifuge for 15 mins @ 600 RCF. Blue/Black top tube Gently resuspend the cells above the gel Transfer to 2 labelled (ACD PBMC) orange cryovials Aspirate the supernatant. Resuspend the cell pellet by tapping tube with finger. Add PBS to bring volume to 10 ml. Cap tube & invert 5 times. Centrifuge X 15 mins at 600 RCF. Aspirate as much supernatant as possible without disturbing cell layer. Freeze cell pellet at -80ºC. Estimate the volume remaining in the tube (0.8-1.5 ml), gently add DMSO (see table) to a final concentration of 10%. See Table. Cap & invert gently 5 times. Freeze at -20ºC for 1 day, then -80ºC November 12, 2010 18
Appendix C Paxgene DNA Tube Appendix C - PAXGENE DNA Tube DNA Freeze PAXGENE DNA tube upright in wire rack at -20ºC for 24 hours (Do NOT freeze in Styrofoam, tube will crack) Transfer tube to a -80ºC freezer Send to central lab within 8 weeks of drawing samples November 12, 2010 19
Appendix D November 12, 2010 20