The RE-ENERGIZE Study. A RandomizEd trial of ENtERal Glutamine to minimize thermal injury. Lab Study Manual

Transcription

1 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 HRPO Proposal Number Clinical trials.gov ID #NCT August 17,

2 Lab Manual Table of Contents Study Contacts...3 Blood Sample Overview...4 Blood Draw Visits...4 Materials Provided...5 Equipment and Supplies Needed...5 Legend of Abbreviated Terms and Symbols...5 Tube Kit Contents Blood Draw Instructions...8 Blood Processing Instructions Green Top...9 Speckled Red/Green Top & Speckled Blue/Black Top...10 Speckled Blue/Black Top...11 Speckled Red/Green Top...12 PAXGENE DNA Tube...12 Sample Log and Data Entry...13 Packaging & Shipping...14 Appendices...15 A Green Top Tube...16 B Red/Green Speckled Top Tubes & Blue/Black Speckled Top Tube...17 C Blue/Black Speckled Top Tube ONLY...18 D Red/Green Speckled Top Tubes ONLY...19 E PAXGENE DNA Tube...20 F Lab Sample Log...21 August 17,

3 Study Contacts RE-ENERGIZE Sponsor Contacts Paul Wischmeyer, MD University of Colorado Denver Department of Anesthesiology East 19 th Avenue RC2, Room 7119 Aurora, CO Phone: Fax: Dominique Garrel, MD Université de Montreal Department of Nutrition 2405, ch. de la Côte Ste-Catherine Pavillon Lilian-de -Stewart Room Montréal, QC H3T 1A8 Phone: ext.1739 Hospital: Cell: Clinical Evaluation Research Unit Contacts Rupinder Dhaliwal, RD Project Leader Phone: ext Cell: Maureen Dansereau Project Assistant Phone: ext 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 E. 19th Ave. RC2 Room P D Aurora, CO Tel: 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 Tel: Lisa.Hanson@NationwideChildrens.org August 17,

4 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. August 17,

5 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) 30 Cryovials (16 red, 10 Orange, 4 green) Labels for the tubes, cryovials and Lab Sample Log 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 (g) (RCF = x rotor radius x RPM 2 ) Centrifuge capable of accommodating 8mL CPT tubes, 6mL trace element free tubes, and 15mL conical tubes. 15mL conical tubes Phosphate Buffered Saline (PBS) Dimethyl Sulfoxide (DMSO) Gloves Pipettes capable of pipetting a range of 50 microlitres to 500 microlitres (0.05 ml to 0.5 ml)) 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 Legend of Abbreviated Terms and Symbols RCF = Relative Centrifugal Force: also known as (g) force RPM = Rotations per Minute PBS = Phosphate Buffered Saline DMSO = DiMethyl SulfOxide ml = milliliter µl = microlitre Blood Draw Tubes ACD = Acid Citrate Dextrose PBMC = Peripheral Blood Mononuclear Cells CPT = Cell Preparation Tube August 17,

6 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 30 Cryovials: o 16 x red o 10 x orange o 4 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 30 Cryovials: o 16 x red o 10 x orange o 4 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 30 Cryovials: o 16 x red o 10 x orange o 4 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 30 Cryovials: o 16 x red o 10 x orange o 4 x green Cryovial/tube labels August 17,

7 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) 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 _ _ _ _ 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) August 17,

8 Lab Sample Log Labels In addition, ONE corresponding label for EACH day of blood draw will be provided for the Lab Sample Log. This label is to be attached to the Lab Sample Log (Appedix F) in the space indicated. Example: For patient #13 from site # 1012, for blood draw # 2 (7 days post start of study intervention), the Lab Sample Log label would look like this: Blood draw # Site Number (4 digit) Patient enrollment # 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 Label and attach it to the appropriate Draw # on the Lab Sample Log (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 (invert tube 8 10 times and then place on ice until processing and process within 30 minutes of blood draw) The following tube is only collected as part of the first study lab draw: (4) 1 x PAXGENE tube (Hold vertically below patient s arm during collection)- 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. August 17,

9 Blood Processing Instructions While blood is spinning in centrifuge (refer to tube-specific instructions below), place provided labels on cryovials: 16 x red topped cryovials 16 x Heparin plasma labels 10 x orange topped cryovials 8 x ACD plasma labels 2 x ACD PBMC labels 4 x green topped cryovials 4 x Plasma 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 Lab Sample Log label 3 x Extra labels ACD = Acid Citrate Dextrose 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 30 minutes post collection Remove Green Top tube from ice and place in centrifuge. Spin blood at 1000 RCF (g) for 5 minutes. o Once tube has stopped spinning, gently remove tube from centrifuge, being careful not to resuspend the red blood cells in the plasma. If the layers become remixed, repeat centrifuge process above. o Carefully pipette plasma (top layer) out of tube in aliquots of 500µL (0.5mL) and place into 4 green topped cryovials labelled plasma. o Place all 4 green topped cryovials, containing 500µL (0.5mL) of plasma each, in an -80 C freezer. o Store at -80 C until shipment August 17,

10 Speckled Red/Green Top & Blue/Black Top Tubes (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 (g) 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 Cell Layer Remove the plasma from each tube without disturbing the cell layer and aliquot into the appropriate coloured 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 labelled (ACD) orange topped cryovials. o Aliquot 0.5mL (500μL) of plasma from the 2 Red/Green top tubes into each of the 16 labelled red topped cryovials. See Figure 2 below. Figure 2. Cryovial showing 0.5 ml plasma Freeze the 8 orange topped and 16 red topped cryovials at -20ºC for approximately 24 hours. Then, transfer all 24 cryovials to -80ºC freezer and store until shipment to the Central Lab. August 17,

11 Blue/Black Speckled Top Tube ONLY (See Appendix C) Using the already processed Blue/Black speckled top tube: Gently resuspend the cells above the gel barrier in the blue/black top tube and transfer equal amounts (approximately) into 2 labelled (ACD PBMC) orange topped cryovials. Note: Once you have transferred the cell layer into the two cryovials you may dispose of the blue/black top tube in a biohazardous waste container (per your local requirements). Estimate the volume in each cryovial (should be ~ mL) and gently add the preserving solution Dimethyl Sulfoxide (DMSO) to the cryovials to a final concentration of 10% (see table below). Cap the cryovials and mix gently by inverting 5 times. Freeze at -20ºC for approximately 24 hours. Then, transfer to -80ºC freezer and store until shipment to the Central lab. Table 1: DMSO (Dimethyl Sulfoxide) Table Volume of Volume of DMSO to add cell/plasma mixture µl ml µl ml August 17,

12 Red/Green Speckled Top Tubes ONLY (see Appendix D) Using the already processed Red/Green speckled top tubes: Pull out white cell layer from both tubes & add to the same new 15 ml conical centrifuge tube o The white cell layer 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 o Affix Heparin PBMC label provided to the 15mL tube now containing the white cell layer from both red/green speckled top tubes. 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 (g) o ONLY if a pellet does not form, increase the RCF (g) 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 (g) Aspirate and discard as much supernatant as possible without disturbing cell pellet Freeze cell pellet at -80ºC until shipping PAXGENE DNA Tube (see Appendix E) 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 August 17,

13 Lab Sample Log and Data Entry Complete the Lab Sample Log (see Appendix F) for the patient (one log per patient) and list the following: Date blood samples taken for each draw Number of each cryovial/tube processed per day by color Total number of samples processed per day Affix the completed label that corresponds to the day of blood draw to the Lab Sample Log (Appendix F) where indicated. Make sure you have added the 3 digit patient enrolment number to the labels. Ensure that the specimen ID and date recorded on the data collection worksheets is the same as that on the Lab Sample Log label. August 17,

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 E. 19th Ave. RC2 Room P D Aurora, CO 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 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. August 17,

15 Appendices A: Green Top Tube B: Red/Green Speckled Top Tubes & Blue/Black Speckled Top Tube C: Blue/Black Speckled Topped Tube ONLY D: Red/Green Speckled Topped Tubes ONLY E: PAXGENE DNA Tube F: Lab Sample Log August 17,

16 August 17,

17 August 17,

18 August 17,

19 August 17,

20 August 17,

21 Appendix F: Lab Sample Log August 17,