Systemic Synuclein Sampling Study. Biospecimen Collection, Processing, and Shipment Manual

Transcription

1 S4 Biospecimen Collection, Processing, and Shipment Manual 25 February

2 Table of Contents 1.0 Biorepository Information Biorepository Contacts 1.2 Hours of Operation 1.3 Holiday Schedules and Observations 2.0 Research and Clinical Laboratory Collection Schedule Sample Collection Volumes and Quantities 2.2 Protocol Schedule for Sample Collection 3.0 Specimen Collection Kits and Supplies Kits Required at Each Visit 3.2 Specimen Collection Kit Contents 3.3 Initial Supply 3.4 Automatic Kit and Label Distribution 4.0 Equipment Required at Clinical Sites Training Videos Collection and Processing Procedures for Biofluid and Skin Biopsy Visit Order of Blood Draws 6.2 Labeling Samples 6.3 Filling Aliquot Tubes (Serum, Plasma, and CSF) 6.4 PAXgene RNA 6.5 Plasma 6.6 Whole Blood 6.7 DNA 6.8 Blood for Research Clinical Labs 6.9 Serum Determination 25 February

3 7.0 Saliva Collection and Processing Procedures Required Equipment 7.2 Preparation of PIC 7.3 Saliva Collection at the Day of Visit 8.0 Cerebrospinal Fluid Collection Lumbar Puncture Supplies 8.2 Setting Up the LP 8.3 Maintaining the Sterile Field 8.4 Tips for Clinicians Performing Lumbar Puncture 8.5 Detailed Lumbar Puncture Procedures 9.0 Tissue Collection and Processing Procedures Skin Biopsy 9.2 Colon Biopsy 9.3 Submandibular Biopsy 10.0 Packaging and Shipping Instructions Sample Quality Checks and Feedback to Sites Data Queries and Reconciliation Appendices Appendix A: Rate of Centrifugation Worksheet Appendix B: Sample Record Summary and Shipment Notification Form Appendix C: Detailed Shipping Instructions Appendix D: Blood for DNA Processing Diagram Appendix E: PAXgene RNA Processing Diagram Appendix F: Plasma EDTA Processing Diagram Appendix G: Whole Blood EDTA Processing Diagram Appendix H: Serum Processing Diagram 25 February

4 Appendix I: Appendix J: CSF Processing Diagram Aliquot Tube Label Diagram Appendix K: Low Fat Diet Menu Suggestions Appendix L: S4 Sample Submission Non-Conformance Report Appendix M: Preparation of Protease Inhibitor Cocktail (PIC) Appendix N: Saliva Collection Diagram Appendix O: Skin Biopsy Processing Diagram Appendix P: Colon Biopsy Processing Diagram Appendix Q: Submandibular Biopsy Processing Diagram Appendix R: Blood for Research Clinical Labs Processing Diagram 25 February

5 1.0 Biorepository Information 1.1 Biorepository Contacts General Study Contact Information Phone: International Phone: (00+1) Fax: International Fax: (00+1) Tatiana Foroud, PhD, Core Leader Phone: International Phone: (00+1) Danielle Smith, BS, Project Manager Phone: International Phone: (00+1) Madeline Potter, BA, Clinical Research Specialist Phone: International Phone: (00+1) Sample Shipment Mailing Address S4 Biorepository Indiana University School of Medicine Walther Hall R3 C W. Walnut Street Indianapolis, IN February

6 1.2 Hours of Operation Indiana University business hours are from 8 AM to 5 PM Eastern Time, Monday through Friday. 1.3 Holiday Schedules Please note that courier services may observe a different set of holidays. Please be sure to verify shipping dates with your courier prior to any holiday. Weekend/holiday delivery must be arranged in advance with the biorepository. Individual collection site questions should be directed toward the respective repositories. Frozen samples must be shipped Monday Wednesday only. Tissue and ambient samples (DNA and research clinical labs) must be shipped Monday Thursday only. Tissue samples may be shipped on Fridays ONLY IF prior arrangements have been made with IU. See Section 1.4 for details. Table 1. Holiday Observations Holiday New Year s Day Martin Luther King, Jr. Day Memorial Day Independence Day (observed on Friday if the holiday falls on a Saturday, and observed on Monday if it falls on a Sunday) Labor Day Thanksgiving Day and following Friday Christmas Day (observed on Friday if the holiday falls on a Saturday, and observed on Monday if it falls on a Sunday) Please note that between December 24 th and January 2 nd, Indiana University will be open Monday through Friday for essential operations ONLY and will re-open for normal operations on January 2nd. If at all possible, biological specimens for submission to Indiana University should NOT be collected and shipped to Indiana University between December 24 th and January 2 nd. Should it be necessary to ship samples to Indiana University during this period, please contact the Indiana University staff before December 24th by ing s4bio@iu.edu so that they can arrange to have staff available to process incoming samples. If biofluid samples are collected during this holiday period and cannot be shipped, please store them at -70 or -80 degrees Celsius and ship them on dry ice to Indiana University AFTER January February

7 1.4 Friday Biopsy Collections In order for the S4 Biorepository at IU to accept biopsy samples collected on Friday for overnight delivery to IU on Saturday, the site must: Obtain prior approval from IU before sending samples for Saturday delivery Notify IU of a Friday collection at least one (1) week in advance Confirm with IU on Friday that the visit did occur as scheduled Send the Sample Record Summary and Shipment Notification Form to s4bio@iu.edu (preferred) or fax to (317) on Friday, with tracking information Be mindful of holidays and confirm with IU that staff will be available to receive the samples 2.0 Research and Clinical Laboratory Collection Schedule The following samples will be collected according to the visit schedule noted below. Please note that visits after screening may occur in any order. Screening Visit o Blood for standard clinical safety analysis (analyzed by local lab at clinical site) Biofluid Collection and Skin Biopsy Visit (all samples shipped to Indiana University for further processing and storage) o Blood for research clinical labs including a CBC with differential and platelets as well as a reticulocyte count o Serum, plasma, and whole blood suitable for proteomic, metabolomic, and other analyte studies o Whole blood for DNA sequencing/genomic analysis o RNA for transcriptomic analysis o Saliva for proteomic, metabolomic, and other analyte studies o Cerebrospinal fluid for proteomic, metabolomic, and other analyte studies o Skin punch biopsies for histology and immunolabeling 25 February

8 Colonic Biopsy Visit o Submucosal biopsies from the sigmoid colon for histology and immunolabeling Submandibular Gland Biopsy Visit o Needle core biopsies of the submandibular gland for histology and immunolabeling If a sample is not obtained at a particular visit, this should be recorded on the appropriate data form and a reason should be provided. 2.1 Sample Collection Volumes and Quantities Sample Type Amount Whole blood for Clinical Safety Analysis 8 ml Whole Blood for Research Clinical Labs 4 ml Whole Blood for DNA 6 ml Whole Blood for RNA 10 ml Whole Blood for Plasma 10 ml Whole Blood for Serum 10 ml Whole Blood for Storage 6 ml Saliva 5 ml Cerebrospinal Fluid ml Skin Punch Biopsy 4 Colon Biopsy 8 Submandibular Gland Needle Core Biopsies 5 25 February

9 2.2 Protocol Schedules for Biospecimen Sample Collection Study Visit Screening Table 2: S4 Biospecimen Collection Biorepository Samples Collected none BSB (Biofluid Collection and Skin Biopsy Visit) CB (Colonic Biopsy Visit) SGB (Submandibular Gland Biopsy Visit) CSF; blood for research clinical labs, whole blood, plasma, serum, DNA, and RNA; saliva; skin biopsies Colon biopsies Submandibular biopsies 25 February

10 3.0 Specimen Collection Kits and Supplies Clinical Lab Collection Blood will be collected at the screening visit for local safety labs. Supplies for this will not be provided by IU. Research Clinical Lab and Biospecimen Collection Kits and Supplies For all visits after the screening visit, research specimen collection kits will be provided to sites by Indiana University. Kits will include most of the materials needed for saliva, blood, CSF, and tissue biopsy collection. Kits will include tube labels, which will be pre-printed with study information and the type of sample being drawn. It is important that you check to be sure that all tubes are properly labeled during processing and at the time of shipment. The kits will also include shipping labels and packaging materials necessary for sending samples back to the S4 Biorepository. The list of items that each site will need to provide is shown on the next page. Please review this list prior to scheduling subjects. Please note that most items are typical items found already at sites; however, there are a few items that you will not be likely to have on hand and will need to order. Vendor and product numbers are provided for the fixatives and protease inhibitor cocktail as it is a requirement that all sites use the same products to ensure standardization of processing procedures across sites. NO SUBSTITUTIONS. NOTE: Your institution may require additional training and certification to ensure proper handling and disposal of formalin fixative. Please refer to your institution s guidelines to ensure you are in compliance. 25 February

11 Table 3: Items to be Provided by Each Site Visit(s) Item(s) Needed Visit(s) Item(s) Needed BSB Dry Ice BSB Crushed Ice BSB Alcohol Prep Pads BSB Gauze Pads BSB Bandages BSB Butterfly Needles BSB Tourniquets BSB Tube Racks (2 ml to 10 ml) All Gloves BSB Sharps Bin and Lid BSB Pipettes and Pipette Tips BSB, SGB Lidocaine BSB Serological Pipette Filler BSB 10 ml Serological Pipettes BSB Permanent Markers BSB Plastic or glass containers to hold wet ice All 10% Formalin (Fisher ) BSB Protease Inhibitor Cocktail (PIC; Sigma-Aldrich P8340-1ML) All Fixative disposal bin BSB, CB Saline solution in spray bottle Screening Blood collection tubes for safety labs BSB Steri-strips BSB ddh 2 O for PIC dilution 3.1 Kits Required at Each Visit Study Visit Kit(s) Required Screening BSB (Biofluid Collection and Skin Biopsy) CB (Colonic Biopsy) SGB (Submandibular Gland Biopsy) NA* Sites will need to provide materials for Chemi-20 panel, CBC, PT/PTT, and serum pregnancy test if applicable 1. Research Clinical Labs 2. Blood for DNA + Biomic Blood + CSF + Saliva + Skin Biopsy Kits Colon Biopsy Kit Submandibular Biopsy Kit *Blood will be collected at the screening visit for local safety labs. Supplies for this will not be provided by IU. 25 February

12 3.2 Specimen Collection Kit Contents Specimen collection kits contain the items listed below under each kit type. Each kit provides the necessary supplies to collect samples for all study visits from one subject. S4 kit components have been carefully selected to suit the needs of this project. Do not replace or supplement any of the tubes or kit components provided by Indiana University with your own supplies unless you have received approval from The Michael J. Fox Foundation for Parkinson s Research (MJFF)/Indiana University to do so. Note that supplemental kits will be provided to sites should you require additional supplies from those contained in the visit specific kits. 25 February

13 3.2.1 Biofluid Collection and Skin Biopsy Visit Kit Figure 1: BSB Kit Components 25 February

14 Table 4: Biofluid Collection and Skin Biopsy Visit Kit Component List Quantity Kit Component 1 Vacutainer Purple-top EDTA tube (10 ml) 1 Vacutainer Red-top serum tube (10 ml) 2 Vacutainer Purple-top EDTA tube (6 ml) 4 Vacutainer - PAXgene tubes (2.5 ml) 1 Vacutainer Purple-top EDTA tube (4 ml) 27 Cryogenic vials (2 ml) 3 red cap, 4 purple cap, 20 clear cap 2 Cryoboxes 3 Transfer pipets 4 Warning label packets 4 Shipping instruction sheets 2 FedEx Overpacks 4 FedEx return Airbills 1 Shipping container for dry ice shipments 1 Shipping container for cold pack shipments 2 Shipping containers and kits for ambient shipments 2 Tyvek envelope/biohazard bag combos 2 Biohazard bags ml absorbent sheets ml absorbent sheets 1 6-tube bubble pouch 2 Cold packs REFRIGERATE AT 4⁰C UPON RECEIPT 1 Tissue specimen storage container 5 Screw-top centrifuge tubes (15 ml) 1 Screw-top centrifuge tube (50 ml) 1 Lumbar puncture tray (Lidocaine) 2 Skin biopsy punch tools with plunger 2 Gauze sponges 1 Sterile scissors 1 Sterile forceps 1 Coverlet adhesive dressing 1 Sterile drape 1 Gelfoam sterile compressed sponge 2 Vaseline ointment packets 2 Transparent film dressings 3 Alcohol prep pads 1 Tissue cassette (peach) 1 Tissue cassette (white) 4 Sponges (2 per cassette) 25 February

15 3.2.2 Colonic Biopsy Visit Kit Figure 2: CB Visit Kit Components Table 5: Colonic Biopsy Visit Kit List Quantity Kit Component Quantity Kit Component 1 Radial jaw sterile forceps with needle 1 Shipping container for cold pack shipments 1 Sterile forceps 2 Cold packs REFRIGERATE AT 4⁰C UPON RECEIPT 2 Tissue specimen storage containers 2 Biohazard bags 4 Tissue cassettes (tan) 1 Warning label packet 8 Sponges (2 per cassette) 1 FedEx return Airbill ml absorbent sheets 1 Shipping instruction sheet 25 February

16 3.2.3 Submandibular Gland Biopsy Visit Kit Figure 3: SGB Visit Kit Components Table 6: Submandibular Gland Biopsy Visit Kit List Quantity 1 Disposable core biopsy instrument 1 Sterile forceps 1 Tissue specimen storage container 2 Tissue cassettes (pink) 4 Sponges (2 per cassette) ml absorbent sheet Type C Kit Component 1 Shipping container for cold pack shipments 2 Cold packs REFRIGERATE AT 4⁰C UPON RECEIPT 1 Biohazard bag 1 Warning label packet 1 FedEx return Airbill 1 Shipping instruction sheet 25 February

17 3.2.4 Supplemental Kit Components Quantity Supplemental Kit Component ml absorbent sheets ml absorbent sheets 2 6-tube bubble pouches 2 Cryoboxes 20 Cryogenic vials (2 ml) with red caps 20 Cryogenic vials (2 ml) with purple caps 40 Cryogenic vials (2 ml) with clear caps 5 FedEx return Airbills 5 FedEx Overpacks 2 Needles introducer 2 Needles Sprotte spinal 15 Screw-top centrifuge tubes (15 ml) 2 Screw-top centrifuge tubes (50 ml) 2 Tyvek envelope/biohazard bag combos 10 Biohazard bags 4 Vacutainer - PAXgene tubes (2.5 ml) 2 Vacutainer Purple-top EDTA tubes (10 ml) 4 Vacutainer Purple-top EDTA tubes (6 ml) 2 Vacutainer Red-top serum tubes (10 ml) 2 Vacutainer Purple-top EDTA tubes (4 ml) 5 Transfer pipets 5 Warning label packets 5 Tissue specimen storage containers 16 Sponges (2 per cassette) 5 Sterile forceps 2 Skin biopsy punch tools with plunger 2 Gauze sponges 1 Sterile scissors 5 Coverlet adhesive dressings 2 Sterile drapes 2 Gelfoam sterile compressed sponges PROLENE suture packs 5 Vaseline ointment packets 5 Transparent film dressings 10 Alcohol prep pads 1 Needle driver 2 Radial jaw sterile forceps with needle 2 Disposable core biopsy instruments 2 Tissue cassettes (peach) 2 Tissue cassettes (white) 2 Tissue cassettes (tan) 2 Tissue cassettes (pink) 25 February

18 3.2.5 Extra Supplies Quantity Kit Component Quantity Kit Component ml absorbent sheets 5 Tyvek envelope/biohazard bag combos ml absorbent sheets 5 Cold Packs REFRIGERATE AT 4⁰C UPON RECEIPT ml absorbent sheets 5 Vacutainer - PAXgene tubes (2.5 ml) ml absorbent sheets 5 Vacutainer Purple-top EDTA tubes (10 ml) 5 6-tube bubble pouches 5 Vacutainer Purple-top EDTA tubes (6 ml) 5 Cryoboxes 5 Vacutainer Red-top serum tubes (10 ml) 10 Cryogenic vials (2 ml) with red caps 5 Vacutainer Purple-top EDTA tubes (4 ml) 20 Cryogenic vials (2 ml) with red caps 2 Skin biopsy punch tools with plunger 10 Cryogenic vials (2 ml) with purple caps 5 Sterile forceps 10 Cryogenic vials (2 ml) with clear caps 2 Gauze sponges 20 Cryogenic vials (2 ml) with clear caps 1 Sterile scissors 5 FedEx return Airbills 5 Coverlet adhesive dressings 5 FedEx Overpacks 2 Sterile drapes 2 Lumbar puncture trays (Lidocaine) 2 Gelfoam sterile compressed sponges 2 Needles introducer PROLENE suture packs 2 Needles Sprotte spinal 10 Vaseline ointment packets 10 Screw-top centrifuge tubes (15 ml) 10 Transparent film dressings 5 Screw-top centrifuge tubes (50 ml) 10 Alcohol prep pads 10 Screw-top centrifuge tubes (15 ml) 1 Needle driver 5 Screw-top centrifuge tubes (50 ml) 1 Radial jaw sterile forceps with needle 1 Shipping container for ambient shipments 5 Tissue specimen storage containers 2 Shipping containers for ambient shipment 8 Tissue cassettes (2 of each color) 1 Shipping container for cold pack shipments 1 Disposable core biopsy instruments 2 Shipping containers for cold pack shipments 16 Sponges (2 per cassette) 5 Biohazard bags 5 Transfer pipets 10 Biohazard bags 5 Warning label packets 25 February

19 3.3 Initial Supply Each site will be initially supplied with the following items, depending on storage space available: 1-2 BSB (Biofluid Collection and Skin Biopsy) Kit(s) 1-2 CB (Colon Biopsy) Kit(s) 1-2 SGB (Submandibular Gland Biopsy) Kit(s) 1 Supplemental Kit Subsequent kits and supplies should be ordered from Indiana University, when needed. 3.4 Indiana University Automatic Kit and Label Distribution Each site will be responsible for entering the necessary information into the study database to register the subject. Once this step is complete, an automatic will be generated to the Biorepository team that will trigger the development of kits and labels for that subject. IU will prepare all kits and labels for ALL visits for that subject. Kits and labels will be sent to the site within five business days. All tubes and containers used in specimen collection, processing, and aliquoting must be labeled using the provided labels (see diagram below). Please refer to Appendix J and section 6.2 for instructions on correct sample labeling. Figure 4: S4 Study Labels 25 February

20 Kits, labels, and supplemental kits or supplies can be ordered using Indiana University s online kit ordering module ( Refer to the kit schedule diagram (section 3.1) to verify which kits are needed for a particular visit. In the electronic kit ordering module, select site number and name from the list provided. This will automatically fill with the site coordinator s contact and shipping information. Verify that this information is correct (and modify it if necessary) and select the kit types and/or labels needed. Provide the quantity of kits needed, as well as the S4 IDs for the subjects for whom the kits will be used. Click Submit to send your request. Kits and labels will be sent to the site within five business days. Site coordinators should only need to use the kit request module to replace a study kit and/or labels, to order additional supplies, or to order a new supplemental kit. 25 February

21 4.0 Equipment Required at Clinical Sites In order to process samples consistently across all sites and ensure the highest quality sample possible, sites must have access to the following equipment: 4 C Refrigerated and Room Temperature Centrifuge -80 C Freezer -20 C Freezer 4 C Refrigerator Serological pipettes and pipette filler p1000 Micropipette and tips ***Important Note*** In order to ensure the highest quality samples are collected, processed, and stored, it is essential to follow the specific collection, processing, and shipment procedures detailed in the following pages. Please read the following instructions first before collecting any specimens. Have all your supplies and equipment out and prepared prior to the start of specimen collection. 25 February

22 5.0. Training Videos The following training videos are available to assist you with the S4 specimen processing, aliquoting, and shipping processes. The videos are available at Table 7: Video List Plasma Processing and Aliquoting Serum Processing and Aliquoting CSF Processing and Aliquoting RNA Processing Saliva Collection and Processing Ambient Shipping Cold Pack Shipping Frozen Shipping Use of Micropipettes Cassette Handling and Biopsy Devices 6.0 Collection and Processing Procedures for Biofluid and Skin Biopsy Visit Kit components for the collection of blood for research clinical labs, blood for DNA, blood for RNA, biomic blood, CSF, saliva, and skin biopsy specimens will be needed for this visit. 6.1 Labeling Samples *Refer to Appendix J for a supplemental diagram of labeling samples In order to ensure the label adheres properly and remains on the tube: Place labels on ALL collection and aliquot tubes BEFORE sample collection, sample processing, or freezing. This should help to ensure the label properly adheres to the tube before exposure to moisture or different temperatures. Place label horizontally on the tube (wrapped around sideways if the tube is upright) and just below the ridges of the aliquot tubes (see labeling diagram below). There is enough space on the aliquot tube for the label to be placed without overlapping the ridges. Take a moment to ensure the label is completely adhered to each tube. It may be helpful to roll the tube between your fingers after applying the label. Figure 5: Aliquot Tube Labeling 25 February

23 6.2 Filling Aliquot Tubes (Serum, Plasma, CSF, and Saliva) To assist in the preparation and aliquoting of samples, colored caps are used for the aliquot tubes. The chart below summarizes the correspondence between cap color and type of aliquot. Table 8: Figure 6: Aliquot Vial Cap Color by Sample Type Cap Color Sample Type Purple Plasma Purple CSF for local lab Red Serum Clear CSF for repository Clear Saliva Proper Filling of Aliquot Vials In order to ensure that the S4 Biorepository receives a sufficient amount of the sample for processing and storage and to avoid cracking of the tubes prior to shipment, each aliquot tube should ideally be filled to 1.5 ml (see Figure 6, above) after processing is completed (refer to detailed processing instructions per sample type for average yield per sample below). A 1.5 ml aliquot will reach the bottom of the ridged section of the cryovial, as shown. Over-filled tubes may burst once placed in the freezer, resulting in a loss of that sample. If there is biologic material remaining that will not fill a subsequent aliquot tube to 1.5 ml, that remaining amount should still be kept and sent in a partially filled aliquot tube. All material should be shipped to the S4 Biorepository. Aliquot the recommended maximum volume into as many aliquot tubes as will allow after processing the biospecimen sample. Fill as many tubes as possible with 1.5 ml of sample. For example, if 3.7 ml of sample is obtained, you should fill 2 cryovial tubes each with 1.5 ml, and one additional cryovial tube with the remaining 0.7 ml sample volume (see Figure 6, above). 25 February

24 6.3 Order of Blood Draws Blood samples should be collected in the morning between 8 am 10 am, preferably fasted. If fasting is not feasible, the low fat diet should be followed (see Appendix K). Record time of last meal (and whether low fat diet followed, if applicable) on the Blood Sampling Data Form (available from the Clinical Trials Statistical and Data Management Center at the University of Iowa). Tubes should be filled in the following order: 1. 4 x 2.5 ml PAXgene 2. 1 x 10 ml Plasma EDTA Purple Top 3. 1 x 6 ml Whole Blood EDTA Purple Top 4. 1 x 6 ml Blood for DNA EDTA Purple Top 5. 1 x 4 ml Blood for Research Clinical Labs EDTA Purple Top 6. 1 x 10 ml Serum Determination Red Top 25 February

25 6.4 PAXgene RNA See training video for blood draw for RNA Processing: 1. CRITICAL STEP: Store PAXgene Blood RNA Tubes at room temperature, 64 F - 77 F (18 C to 25 C), before use. 2. CRITICAL STEP: The PAXgene Blood RNA Tubes should be the first tubes drawn in the phlebotomy procedure (before Research Clinical lab, plasma, etc.). 3. Place RNA label on the PAXgene RNA tubes prior to blood draw (per Section 5.2). If possible, please ensure that the tubes are labeled in numerical order by barcode number. 4. Using a blood collection set and a holder, collect blood into the first of the four PAXgene Blood RNA Tubes using your institution s recommended procedure for standard venipuncture technique. The following techniques shall be used to prevent possible backflow: a. Place donor s arm in a downward position. b. Hold tube in a vertical position, below the donor s arm during blood collection. c. Release tourniquet as soon as blood starts to flow into tube. d. Make sure tube additives do not touch stopper or end of the needle during venipuncture. 5. Allow at least 10 seconds for a complete blood draw to take place in each tube. Ensure that the blood has stopped flowing into the tube before removing the tube from the holder. The PAXgene Blood RNA Tube with its vacuum is designed to draw 2.5 ml of blood into the tube. Record time of draw in the RNA section of the Blood Sampling Data Form. 25 February

26 6. CRITICAL STEP: Immediately after blood collection, gently invert/mix (180 degree turns) the PAXgene Blood RNA Tube 8-10 times. 7. REPEAT STEPS 4 TO 6 for the remaining three PAXgene Blood RNA Tubes to be collected. 8. CRITICAL STEP: Incubate the PAXgene Blood RNA Tubes UPRIGHT at room temperature, 64 F - 77 F (18 C to 25 C), for 24 hours. Record time and date of draw in the RNA section of the Blood Sampling Data Form. If blood is drawn on a Friday and you are unable to return on Saturday to place tubes in the freezer, transfer the tubes as late as possible before leaving on Friday. Samples must sit at room temperature for a minimum of 2 hours. 9. After 24 hours at room temperature, place the four PAXgene tubes UPRIGHT into a WIRE or PLASTIC type test tube rack (DO NOT use a solid Styrofoam test tube holder) and transfer into a -80 C (minus eighty) freezer. Keep the four PAXgene Blood RNA Tubes at -80 C until you ship on dry ice. If after the samples have been sitting at room temperature for 24 hours and they cannot be immediately transferred to a -80⁰C freezer, prepare a sufficient amount of dry ice for immediate freezing. Complete remainder the RNA section of the Blood Sampling Data Form. Samples should be shipped within two weeks of collection, following the instructions in Appendix C. Figure 7: RNA Incubation 25 February

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28 6.5 Plasma See training video for blood draw for Plasma Processing and Aliquoting: 1. CRITICAL STEP: Store empty Plasma Separation Tube at room temperature, 64 F - 77 F (18 C to 25 C), before use. 2. Place a pre-printed PLASMA label on the 10 ml EDTA lavender top tube prior to blood draw (per Section 5.2). 3. Using a blood collection set and a holder, collect blood into the 10 ml EDTA lavender top tube using your institution s recommended procedure for standard venipuncture technique. The following techniques shall be used to prevent possible backflow: a. Place donor s arm in a downward position. b. Hold tube in a vertical position, below the donor s arm during blood collection. c. Release tourniquet as soon as blood starts to flow into tube. d. Make sure tube additives do not touch stopper or end of the needle during venipuncture. 4. Allow at least 10 seconds for a complete blood draw to take place in each tube. Ensure that the blood has stopped flowing into the tube before removing the tube from the holder. The tube with its vacuum is designed to draw 10 ml of blood into the tube. 5. CRITICAL STEP: Immediately after blood collection, gently invert/mix (180 degree turns) the EDTA tube 8-10 times. 25 February

29 6. Within 30 minutes of plasma collection, centrifuge balanced tube at 4 C for 15 minutes at 1500 x g. It is critical that the tube be centrifuged at the appropriate speed to ensure proper plasma separation. For assistance, see Appendix A. Equivalent rpm for spin at 1500 x g = While centrifuging, record the time of centrifuge start in the Plasma section of the Blood Sampling Data Form. 7. Place pre-printed PLASMA labels on the 15 ml centrifuge and 2 ml aliquot tubes. If possible, please ensure that the aliquot tubes are labeled in numerical order by barcode number. Remove the plasma, being careful not to agitate the packed blood cells at the bottom of the lavender top tube, by tilting the tube and placing the pipette tip along the lower side of the tube wall without touching the pellet so that plasma is not contaminated by pellet material (see below). Using a disposable graduated transfer pipette, transfer plasma into the 15 ml centrifuge tube at room temperature and mix gently by inverting 3-4 times. Figure 8: Plasma Aliquoting NOTE: When pipetting plasma from the plasma tube into the 15 ml centrifuge tube, be very careful to pipet the plasma top layer only, leaving the buffy coat and the red blood cell layers untouched. 8. Pipette at least 1.5 ml of plasma from the 15 ml centrifuge tube into each labeled 2 ml aliquot tube. The EDTA tube should yield, on average, 4.5 ml of blood plasma for a total of 2-3 aliquot tubes per subject. Seal each aliquot tube with a purple cap. 9. Discard the used EDTA and 15 ml centrifuge tubes according to site guidelines for disposing of biomedical waste. 25 February

30 10. Within 60 minutes of plasma collection, freeze and store samples at -80 C. Samples should be frozen and stored UPRIGHT. A cryobox is provided for this purpose. If samples cannot be immediately transferred to a -80⁰C freezer, prepare a sufficient amount of dry ice for immediate freezing. Complete the remainder of the Plasma section of the Blood Sampling Data Form and ensure timely entry of data into the study database. Samples should be shipped within two weeks of collection, following the instructions in Appendix C. 25 February

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32 6.6 Whole Blood for Storage 1. CRITICAL STEP: Store empty Whole Blood EDTA tube at room temperature, 64 F - 77 F (18 C to 25 C), before use. 2. Place a pre-printed WBLD label on the 6 ml EDTA purple top tube prior to blood draw (per section 5.2). 3. Using a blood collection set and a holder, collect whole blood into the 6 ml EDTA purple top whole blood tube using your institution s recommended procedure for standard venipuncture technique. The following techniques shall be used to prevent possible backflow: a. Place donor's arm in a downward position. b. Hold tube in a vertical position, below the donor s arm during blood collection. c. Release tourniquet as soon as blood starts to flow into tube. d. Make sure tube additives do not touch stopper or end of the needle during venipuncture. 4. Invert the tube gently 3 times. 5. Transfer the tube immediately to a -80 C Freezer. If samples cannot be immediately transferred to a -80⁰C freezer, prepare a sufficient amount of dry ice for immediate freezing. Complete the Whole Blood section of the Blood Sampling Data Form and ensure timely entry of data into the study database. The sample should be frozen and stored UPRIGHT in a WIRE or PLASTIC type test tube rack (DO NOT use a Styrofoam test tube holder). Samples should be shipped within two weeks of collection, following the instructions in Appendix C. Figure 9: Whole Blood Draw for Storage 25 February

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34 6.7 Blood for DNA Blood samples for DNA must be received at the S4 Biorepository within 5 days of collection. Samples not received within 5 days of collection must be redrawn at the site. Please do not ship DNA samples on Fridays. 1. CRITICAL STEP: Store empty Blood for DNA EDTA tube at room temperature, 64 F - 77 F (18 C to 25 C), before use. 2. Place pre-printed DNA label on the 6 ml EDTA purple top tube prior to blood draw (per Section 5.2). 3. Using a blood collection set and a holder, collect blood into the 6 ml EDTA purple top tube using your institution s recommended procedure for standard venipuncture technique. The following techniques shall be used to prevent possible backflow: a. Place donor s arm in a downward position. b. Hold tube in a vertical position, below the donor s arm during blood collection. c. Release tourniquet as soon as blood starts to flow into tube. d. Make sure tube additives do not touch stopper or end of the needle during venipuncture. 4. Invert the tube gently 3 times. 5. Seal the EDTA tube in the ambient shipment kit. 6. Ship the sample back to the S4 Biorepository at room temperature according to kit instructions. If sample cannot be shipped the same day as collected, hold 25 February

35 at room temperature until shipping can be managed. Sample must be received at the S4 Biorepository within 5 days of being collected. Additional instructions are found in Appendix C. This sample may be shipped with the Research Clinical Lab sample as long as shipping guidelines for both sample types are followed. 8. Complete the DNA section of the Blood Sampling Data Form and ensure timely data entry of data into the study database. 25 February

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37 6.8 Blood for Research Clinical Labs Blood for research clinical labs will be collected at the BSB visit and sent to Indiana University. It is critical that this blood sample is obtained on the same day as the biologics samples (see section 6.3) as the results from this clinical lab informs the analysis of the biologics. Blood samples for Research Clinical Labs must be shipped on the day of collection and received at the S4 Biorepository the day after collection. PLEASE NOTE: Blood samples for Research Clinical Labs must be collected Monday Thursday ONLY! 1. CRITICAL STEP: Store empty Research Clinical Lab EDTA purple top tube at room temperature, 64 F - 77 F (18 C to 25 C), before use. 2. Place pre-printed label on the 4 ml EDTA purple top tube prior to blood draw (per Section 5.2). 3. Using a blood collection set and a holder, collect blood into the 4 ml EDTA purple top tube using your institution s recommended procedure for standard venipuncture technique. The following techniques shall be used to prevent possible backflow: a. Place donor s arm in a downward position. b. Hold tube in a vertical position, below the donor s arm during blood collection. c. Release tourniquet as soon as blood starts to flow into tube. d. Make sure tube additives do not touch stopper or end of the needle during venipuncture. 25 February

38 4. Invert the tube gently 3 times. 5. Seal the EDTA tube in the ambient shipment kit. 6. CRITICAL STEP: Ship the sample back to the S4 Biorepository at room temperature according to Appendix C on the same day of collection. This sample may be shipped with the DNA sample as long as shipping guidelines for both sample types are followed. 25 February

39 6.9 Serum Determination See training video for blood draw for Serum Processing and Aliquoting: 1. CRITICAL STEP: Store empty Serum Determination Tube at room temperature 64 F 77 F (18 C to 25 C), before use. 2. Place a pre-printed SERUM label on the 10 ml Serum red top tube prior to blood draw (per Section 5.2) 3. Using a blood collection set and a holder, collect blood into the serum determination red top tube using your institution s recommended procedure for standard venipuncture technique. The following techniques shall be used to prevent possible backflow: a. Place donor s arm in a downward position b. Hold tube in a vertical position, below the donor s arm during blood collection c. Release tourniquet as soon as blood starts to flow into tube. d. Make sure tube additives do not touch stopper or end of the needle during venipuncture. 4. Allow at least 10 seconds for a complete blood draw to take place in each tube. Ensure that the blood has stopped flowing into the tube before removing the tube from the holder. The tube with its vacuum is designed to draw 10 ml of blood into the tube. Record the time of draw in the Serum section of the Blood Sampling Data Form. 5. CRITICAL STEP: Immediately after blood collection, gently invert/mix (180 degree turns) the serum determination tube 8-10 times. 6. CRITICAL STEP: Allow blood to clot at room temperature for at least February

40 minutes. 7. Within 60 minutes of serum collection (after at least 15 minutes of clotting at room temperature), centrifuge balanced tube at 4 C for 15 minutes at 1500 x g. It is critical that the tubes be centrifuged at the appropriate speed to ensure proper serum separation. For assistance, see Appendix A. Equivalent rpm for spin at 1500 x g = While centrifuging, record the time of centrifuge start in the Serum section of the Blood Sampling Data Form. 8. Place pre-printed SERUM labels on the 15 ml centrifuge and 2 ml aliquot tubes. If possible, please ensure that the aliquot tubes are labeled in numerical order by barcode number. Remove the serum, being careful not to disturb the clot at the bottom of the tube by tilting the tube and placing the disposable graduated pipette tip along the lower side of the tube wall without touching the pellet. Using a disposable graduated transfer pipette, transfer all blood serum (top layer) into the 15 ml centrifuge tube at room temperature and mix gently by inverting 3-4 times. Figure 10: Serum Aliquoting NOTE: When pipetting serum from the serum tube into the 15 ml centrifuge tube, be very careful to pipet the serum top layer only, leaving the red blood cell layer untouched. 9. Pipette at least 1.5 ml of serum from the 15 ml centrifuge tube into each labeled 2 ml aliquot tube. The serum tube should yield, on average, 4.5 ml of serum, for a total of 2-3 aliquot tubes per subject. Seal each aliquot tube with a red cap. 10. Discard the used red-top and 15 ml centrifuge tubes according to site guidelines for disposing of biomedical waste. 11. Within 60 minutes of serum collection, freeze and store samples at 25 February

41 -80 C. If samples cannot be immediately transferred to a -80⁰C freezer, prepare a sufficient amount of dry ice for immediate freezing. Samples should be frozen and stored UPRIGHT. A cryobox is provided for this purpose. Complete the remainder of the Serum section of the Blood Sampling Data Form and ensure timely entry of data into the study database. Samples should be shipped within two weeks of collection, following the instructions in Appendix C. 25 February

42 25 February

43 7.0 Saliva Collection and Processing Procedures See training video for Saliva Collection and Processing: Saliva should be collected in the morning between 8 am 10 am, preferably fasted. Subjects should refrain from eating, drinking, or using oral hygiene products for at least 1 hour prior to collection. If fasting is not feasible, the low fat diet should be followed (see Appendix K). Record the number of hours since last meal and use of oral hygiene products on the Saliva Sampling CRF. Note that sites will purchase 100X Protease Inhibitor Cocktail (PIC; Sigma-Aldrich P8304-1ML) directly from the vendor and should store the PIC at -20⁰C. Sites will be responsible for ordering more PIC as needed. It should be noted that the cocktail has an expiration date of one year. 7.1 Required equipment ⁰C freezer for protease inhibitor cocktail (PIC) storage 2. Serological pipette filler ml serological pipette 4. p1000 micropipette and tips (NOTE: Sites are responsible for regular calibration of micropipettes according to manufacturer recommendations.) 7.2 Preparation of PIC (Appendix M) Sites are expected to provide ddh 2 O (sterile water) needed to prepare 100X PIC as a 10X reagent to add to saliva samples to prevent digestive enzymes in saliva from breaking down candidate biomarker proteins. Aliquoting of the 100X PIC should be done prior to the subject visit to minimize work during the subject visit. Aliquot the 100X PIC as follows: 1. Thaw 100X PIC at room temperature prior to aliquoting (avoid freeze thaw cycles). 2. Using permanent marker, label 10 x 2 ml centrifuge tubes with 100X PIC and date. 3. Aliquot 100 µl of 100X PIC into each labeled 2 ml centrifuge tube. 4. Store 100X PIC aliquots at -20⁰C and minimize exposure to light. 25 February

44 7.3 Saliva collection at the day of visit (Appendix N) On the day of the subject visit, remove a single aliquot of 100X PIC and thaw at room temperature. Add 900 µl sterile ddh 2 O to 100 µl of 100X PIC. (This will dilute the solution from 1:100 to 1:10). Invert 8-10 times to mix and keep it at room temperature until saliva sample processing. 1. Label (write subject ID) on a 50 ml conical tube. Place pre-printed labels on ALL 15 ml conical and 2 ml micro centrifuge tubes (CLEAR cap) and place on ice. If possible, please ensure that the aliquot tubes are labeled in numerical order by barcode number. 2. Place the labeled 50 ml conical tube in ice in a plastic or glass container. 3. Prepare subject for collection a. Instruct the subject to thoroughly rinse their mouth with water for 1 minute. b. Escort the subject to an empty, quiet room and let them rest for 5 minutes. c. Do not use induction techniques such as sugar. Instruct the subject not to spit saliva into the 50 ml conical collection tube, but rather have them lean forward and hold their head down over the collection tube. The saliva will slowly accumulate in their mouth, and will come out of the mouth by itself. Subject may swallow during collection while maintaining a leaning forward position. d. Record answers to each relevant item on the Saliva Sampling Data Form as you progress through the collection and processing procedures. e. Collect saliva for 20 minutes or until 5 ml is obtained, whichever comes first. 4. Saliva sample processing a. Using a serological pipette, transfer saliva to a 15 ml conical tube and note total volume collected on the Saliva Sampling CRF. b. Add the appropriate amount of 10X PIC using the table shown on page 42 and document this on the Saliva Sampling CRF: 25 February

45 Table 9: Volume of 10X PIC to add Based on Saliva Volume Obtained Volume of saliva Volume of 10X PIC to add transferred to 15 ml conical 0.5 ml 100 µl (0.1 ml) minimum volume possible with p ml 100 µl (0.1 ml) 1.5 ml 150 µl (0.15 ml) 2.0 ml 200 µl (0.2 ml) 5.0 ml 500 µl (0.5 ml) c. Mix the sample by vortexing or vigorously shaking/inverting the sample and PIC for seconds. This step is variable as the consistency of the saliva will vary among subjects. The more viscous the sample, the longer the sample will need to be mixed to create a homogenous sample. Within 30 minutes of collection, centrifuge samples at 2,000 g for 15 minutes at 4⁰C. It is critical that the tubes be centrifuged at the appropriate speed and time to remove cellular debris. Equivalent rpm for spin at 2000 x g = While centrifuging, record the time of centrifuge start on the Saliva Sampling Data Form. d. CRITICAL STEP: Place the labeled 2 ml centrifuge tubes on ice. e. Using a micropipette, transfer 1.5 ml of centrifuged saliva into each labeled, pre-cooled aliquot tube. Take caution not to disturb the white pellet at the bottom of the tube by tilting the tube and placing the pipette tip along the lower side of the tube wall without touching the pellet, so that saliva is not contaminated by pellet material. This will yield, on average, up to 4 aliquot tubes per subject. f. Freeze samples immediately following processing by transferring to a -80⁰C freezer. If samples cannot be immediately transferred to a -80⁰C freezer, prepare a sufficient amount of dry ice for immediate freezing. Store all samples in a -80⁰C freezer until you ship on dry ice. Samples should be frozen and stored UPRIGHT. A cryobox is provided for this purpose. Samples should be shipped within two weeks of collection, following the instructions in Appendix C. 25 February

46 8.0 Cerebrospinal Fluid Collection CSF should be collected in the morning between 8 am 10 am, preferably fasted. If fasting is not feasible, the low fat diet should be followed (see Appendix K). Record time of last meal (and whether low fat diet followed, if applicable) on the Lumbar Puncture Data Form. 8.1 Lumbar Puncture Supplies Table 11: Components of Lumbar Puncture Tray 25 February

47 The lumbar puncture tray contains the items listed in the table below, which will be used to perform lumbar puncture. Check the dates of expiration: these reflect the expiration date of the lidocaine, if included. Supplies for collection and shipment of CSF are sent to sites in a separate kit from Indiana University Lumbar Puncture Tray Components Quantity Lumbar Puncture Tray Kit Component 1 Pencil point spinal needle, 24g x Introducer needle, 1 mm x 30 mm 1 Hypodermic needle, 22G x Plastic syringe, (3 ml, luer lock) with 25G x 5/8 needle attached 4 Polypropylene syringe (6 ml, luer lock) 1 Needle stick pad 1 Adhesive bandage 1 Drape, fenestrated, 2 tabs, paper, 18 x 26 2 Towel, 13.5 x 18 6 Gauze pad, 2 x 2 3 Sponge stick applicator 1 Lidocaine 1%, 5 ml (US SITES ONLY) 1 Povidone-Iodine Topical Solution, 0.75 oz 25 February

48 8.2 Setting Up the LP 1. On an overbed table, remove the contents of the LP kit from the outer plastic packaging, leaving the contents wrapped in their sterile drape. Leave everything wrapped until the person performing the LP is seated and begins examining the subject. 2. Feel the outside of the LP kit (still wrapped) to determine which end contains the spongy swabs. Turn this end toward the person performing the LP and begin unwrapping the kit. 3. Touch only the outside of the paper wrapper. When you grab an edge to unfold it, touch only the folded under portions of the outside of the wrapper. Also, don t let the outside of the wrapper touch any part of the inside. If you touch any part of the paper wrapper, or if any non-sterile object or outside of the wrapper touches any part of the inside of the wrapper, discard the kit and start over. If you are in doubt as to whether something touched the inside of the paper wrapper, throw the kit away and start over. 8.3 Maintaining the sterile field Keep in mind that there may be other staff in the room during an LP, and a big part of assisting with the LP is keeping the field sterile keeping people away from it, and reminding them to be careful around it. If anyone touches the inside of the paper wrapper or any part of the contents of the kit, throw away the kit away and start over. If you are in doubt as to whether someone touched the kit, throw it away and start over. Also, you are the monitor for whether the person performing the LP has broken sterility, usually by touching something not sterile with a sterile gloved hand. Feel free to speak-up and inform people if need be. Be assertive. 8.4 Tips for Clinicians Performing Lumbar Puncture *Optimizing patient comfort and minimizing the risk of adverse events. 1. Talk the patient through the procedure so that there are no surprises. 2. If there are difficulties obtaining spinal fluid and the subject is willing, an LP may be performed using fluoroscopy or ultrasound on the same or another day. 3. Use of a Sprotte 24g atraumatic spinal needle and careful technique are optimal for reducing post-lp headache risk. A pencil point spinal needle such as Spinocan 22g or 24g may also be used. 25 February

49 4. Use adequate local anesthesia. Use the 25g 1/2" needle and inject lidocaine to raise a skin wheal. Then, inject lidocaine using the pattern of a square first the center, and then to all 4 corners. If the subject is thin, do not insert the deep infiltration needle OR the spinal introducer all the way. Use only about 2/3 of their length (to prevent entering the subarachnoid space with anything other than the 24g pencil point spinal needle). 5. Encouraging fluid intake immediately after LP is helpful. 6. Be sure to give post-lp care instructions verbally to the subject as found in the Operations Manual. 7. A follow-up call will be placed by the study coordinator 7 days (±2 days) after the procedure to assess for adverse events. 25 February

50 8.5 Detailed Lumbar Puncture Procedure CSF is processed at Room Temperature [64 0 F F (18 C to 25 C)]. Also, a portion of the CSF must be sent to your clinical lab and analyzed within 4 hours of collection. See training video for CSF Processing and Aliquoting: 1. Place a pre-printed CSF label on the 15 ml centrifuge tubes used for processing. Place pre-printed CSF labels on the 2 ml cryovial aliquot tubes (per section 5.2). Prepare at least 9 aliquot tubes based on the collection of total ml of CSF. If possible, please ensure that the aliquot tubes are labeled in numerical order by barcode number. 2. Place aliquot tubes on wet ice prior to the procedure so they are pre-cooled. 3. Perform lumbar puncture using the atraumatic technique. 4. Collect CSF into syringes (if a noticeably blood tap, discard the first 1-2 ml). After the LP has begun and fluid is being collected, take the first 1-2 ml of CSF from the first syringe and place in the CSF labs cryovial (PURPLE TOP), and send it to the local lab for routine diagnostic tests (protein level). Do not freeze this sample. Send at room temperature to local clinical lab for basic CSF analysis. NOTE: Sample must be analyzed within 4 hours of collection. 1. Cell count (erythrocytes first) 2. Total protein 5. Collect the remaining CSF, up to ml total, and transfer to 15 ml conical polypropylene tubes at room temperature. Mix gently by inverting 3-4 times. Record the time of draw (once collection is complete) on the Lumbar Puncture data form. 25 February

51 6. Within 15 minutes of collection, spin the remaining CSF sample down at 2000 x g for 10 minutes at room temperature, 64 F 77 F (18 C to 25 C). For assistance, see Appendix A. Equivalent rpm for spin at 2000 x g = While centrifuging, record the time of centrifuge start on the Laboratory Procedures data form. 7. Pipette (micropipette preferred) at least 1.5 ml of supernatant directly into pre-cooled polypropylene CSF collection aliquot tubes. This will yield, on average, 9-12 aliquot tubes per subject. (Use more aliquot tubes if needed- do not discard any CSF). Seal each aliquot tube with a clear cap. 8. Within 60 minutes of CSF collection, freeze aliquots immediately on dry ice and then store at -80 C or ship on dry ice in a shipping container. Samples should be frozen and stored UPRIGHT. A cryobox is provided for this purpose. Complete the remainder of the Laboratory Procedures data form and ensure timely entry of data into the study database. 25 February

52 25 February

53 9.0 Tissue Collection and Processing Procedures * See training video for Cassette Handling and Biopsy Devices: Skin biopsy samples must be shipped on the day of collection and received at the S4 Biorepository the day after collection. 9.1 Skin Biopsy using the punch biopsy tool The BSB kit contains the items listed in the table below, which will be used to perform the skin punch biopsy procedure. Check the dates of expiration. Additional supplies for collection and shipment are sent to sites in a separate kit from Indiana University. Place cold packs in refrigerator upon arrival to chill them. Note that sutures and needle drivers will be provided in each site s supplemental kit and should be on hand and ready in case they are necessary for this procedure Punch biopsy kit components Quantity Kit Component 1 Sterile drape 1 Tweezers 2 Gauze Pads 3 Alcohol prep pads 1 Scissors 2 Skin biopsy punch tools with plunger 1 Gelfoam sterile compressed sponge 2 Vaseline ointment packets 1 Coverlet adhesive dressing 2 Transparent film dressings 1 Tissue specimen storage container 1 Tissue cassette (peach) 1 Tissue cassette (white) 4 Sponges 2 Cold packs 25 February

54 9.1.2 Setting Up the Kit 1. On an overbed table, remove the contents of the kit from the outer packaging, leaving all sterile contents wrapped in their packaging. Leave everything wrapped until the person performing the biopsy is seated and begins examining the subject. 2. Open the sterile kit components, touching only the outside of the wrapper. Don t let the outside of the wrapper touch any part of the inside. If you touch any part of the paper wrapper, or if any non-sterile object or outside of the wrapper touches any part of the inside of the wrapper, discard the component and start over. If you are in doubt as to whether something touched the inside of the paper wrapper, throw the component away and start over Skin Sample Collection Skin Sample Collection Sites There will be a total of 4 skin biopsies obtained: 2 from the cervical paravertebral region and 2 from the thigh. The cervical paravertebral biopsies should be taken at approximately the C8 level, vertically aligned (see figure below). The second biopsy should be 3-4 cm above or below the previous one. The thigh biopsies will be taken from one thigh (right or left) approximately 15 cm above the patella. The second biopsy should be at approximately 3cm to the right or left of the previous one (see figure below). For each of the biopsy locations (cervical paravertebral region and thigh) there will be 2 biopsies. All will be processed in formalin (for immunohistochemistry staining). 25 February

55 Figure 12: Skin Biopsy Sampling Locations Precollection Steps for Specialist Preparation of Patient 1. Prepare patient for procedure per institution guidelines. 2. Before the biopsy is collected, the volunteer will be screened and complete the informed consent for the skin biopsy procedure. The doctor will explain the study, and the volunteer will have an opportunity to ask questions. Once this discussion is complete, the volunteer is ready for the biopsy procedure Precollection Steps for Coordinator Preparation of Containers and Cassettes 1. Prior to the procedure, label the tissue collection container with the Patient ID label. 2. Formalin containers will be pre-labeled with a Formalin Biohazard label. Label this tissue collection container with a Tissue label. 25 February

56 3. Fill this container 2/3 full of 10% formalin. 4. Record the barcode label from the peach and white cassettes on the Skin Biopsy CRF and Sample Record Summary and Shipment Notification Form. The peach cassette will be used for the paravertebral biopsies; the white cassette will be used for the thigh biopsies. Figure 13: Skin Biopsy Cassette Barcodes 5. Place the blue sponges on the top and bottom sections of the white and peach cassettes. 25 February

57 Figure 14: Preparation of Skin Biopsy Cassettes Biopsy Collection Procedure 1. Ensure that the biopsy site has been properly sterilized with alcohol wipes. A punch biopsy is a clean procedure, not a sterile procedure, and therefore, sterile gloves and gown are not required. Wearing safety glasses is recommended. 2. Anesthetize the area by injecting the Lidocaine solution (Lidocaine HCL 1% 1:100,000) just under the epidermis (subepidermally) using 3cc syringe just prior to the biopsy. The injection should continue until a bleb or small bubble forms under the skin (approximately 3mm in diameter). The injection will burn slightly (much like a bee sting) due to a ph difference between the skin and the solution. Injecting slowly decreases the burning sensation. The burning will subside quickly, and the site will become numb. It is acceptable to massage the area. 25 February

58 3. After the Lidocaine injection, the area anesthetized may be marked using a pen if helpful to the individual completing the biopsy. The area to be biopsied should be checked to ensure the skin is properly anesthetized. This can be tested by gently pressing the needle to the area. If the patient experiences neither pain nor sharp sensation, the area is ready to be biopsied. Experiencing a pressure sensation is normal but there should be no pain. If the area requires more anesthesia, another injection of Lidocaine solution is made with a new syringe. 4. Using a sterile 3mm skin punch, place the punch perpendicular to the skin, in the paravertebral C8 region, within 3 mm of the midline. Apply constant downward pressure while twirling the punch tool between thumb and index finger, rotating clockwise and counterclockwise until the blade has pierced the epidermis of the skin and the metal part of the punch tool is buried (there will be a give once the punch reached the subcutaneous fat). Once the tool has reached the lowest point, lift the tool straight up. 5. Depress the plunger to remove the specimen. Forceps may be needed to remove the specimen. If the specimen remains connected at the level of the subcutaneous fat, it may be necessary to cut at base of specimen to remove it. Do not try to tear a specimen that remains connected as it may damage the specimen. Tissue should be rinsed with a saline solution if it does not release. The specimen should be placed directly into a peach, sponge-lined cassette as instructed. Using a punch with a plunger should help to ensure that the epidermis is not crushed or damaged by during the process. 6. Collect a second biopsy 3-4 cm above or below the original collection site on the same side of the midline. Place the two biopsies into a peach, sponge-lined cassette. Tissue should be rinsed with a saline solution if it does not release. 7. Collect two punch skin biopsies from the thigh region, at least 3 cm laterally apart from each other, 15 cm above the patella, and place into a white, sponge-lined cassette. Tissue should be rinsed with a saline solution if it does not release. 25 February

59 8. CRITICAL STEP: Close cassettes securely by bringing the lid down onto the bottom and snapping it. 9. To restore hemostasis, hold pressure with gauze for ~30 seconds. Wipe any excess blood with a sterile 2x2 gauze to expose the site. Pack biopsy site with GelFoam. Apply the Vaseline ointment to the bandage and cover biopsy site. This can be reinforced with gauze and tape if necessary. If the biopsy site is oozing, apply a pressure bandage by applying Vaseline to small gauze and then apply Tegaderm. Other closure options include using a steri-strip and transparent film dressing closure system. In most cases, suturing a wound will not be necessary. Placing a suture can be considered if the wound base is still oozing after packing with GelFoam. To place a suture, grip the needle using the forceps approximately ½ to 1/3 of the distance between the suture attachment and the tip of the needle. Place the needle point perpendicular to the skin surface 2mm away from the wound edge, then turn the wrist to exit the skin on the opposite side of the wound again 2mm from the wound edge. To tie the suture, hold the needle holder parallel to the axis of the wound and at the center of the wound. Wrap the free end of the suture twice around the holder, then grasp the free end and pull through, tightening the knot. Repeat with just looping around needle holder once for repeat knots. Tie 3 knots (See Figure 15 below). Figure 15. Tying the suture knot. 25 February

60 10. The study coordinator will be responsible for completing the processing of the tissue once collected using the procedures described in detail below. 11. Be sure to give post care instructions verbally to the subject as found in the Operations Manual. A follow-up call will be placed by the study coordinator 7 days (±2 days) after the procedure to assess for adverse events Tissue Processing of Skin Biopsies 1. Two punch skin biopsies will be collected from either the right or left side of the Paravertebral C8 region within 3 mm of the midline. 2. These two paravertebral biopsies should be placed on the bottom section of a peach cassette lined with sponges. 3. Two punch skin biopsies will be collected from the Thigh region (at least 3 cm laterally apart from each other and 15 cm above the patella). 4. These two thigh biopsies should be placed on the bottom section of a white cassette lined with sponges. 5. Submerge both the peach and white cassettes into the container marked Formalin. 6. CRITICAL STEP: Be sure that the container lid clicks twice and that the arrows are aligned, indicating that the container is properly sealed. 7. Record time placed in Formalin on the Skin Biopsy CRF and Sample Record Summary and Notification Form. 8. Place container in a refrigerator until shipment. 9. CRITICAL STEP: Ship the samples back to the S4 Biorepository at 4 C according to Appendix C on the same day of collection. 10. Place a follow-up call to the subject 7 days (±2 days) after the procedure to assess for adverse events. 25 February

61 25 February

62 9.2. Colon Biopsy Visit The kit contains the items listed in the table below, which will be used to perform biopsy procedure. Check the dates of expiration. Supplies for collection and shipment are sent to sites in a separate kit from Indiana University. Place cold packs in refrigerator upon arrival to chill them. Colon biopsy samples must be shipped on the day of collection and received at the S4 Biorepository the day after collection Colon biopsy kit components Quantity Kit Component 1 Radial jaw sterile forceps with needle 1 Sterile forceps 2 Tissue specimen storage containers 4 Tissue cassettes (tan) 8 Sponges 2 Cold packs Setting Up the Kit On an overbed table, remove the contents of the kit from the outer plastic packaging, leaving the contents wrapped in their sterile drape. Leave everything wrapped until the person performing the biopsy is ready to begin examining the subject Colon Biopsy Collection Precollection Steps for Specialist - Preparation of Patient Before the colon biopsy procedure, the subject will have undergone informed consent and screening procedures, including evaluation of coagulation status and a review of medications that may lead to an increased bleeding time. The specialist completing the procedure will review the procedure and potential risks prior and address any questions the subject may have prior to performing the procedure. Specific discussion points are listed on the next page: 25 February

63 Thank the volunteer for participating in the study. Review that the procedure will include 8 biopsies of colon tissue obtained from the sigmoid colon using a flexible sigmoidoscope. The flexible sigmoidoscope (a lighted instrument with which one can view the lower intestine) will be inserted about 20 cm (about 8 inches) into the rectum. Explain that the biopsy procedure will take approximately 15 minutes. The subject should not experience pain when the biopsies are taken. Review the risks: minor discomfort, bleeding from the rectum, and perforation (tearing) of the intestinal wall. These procedures are performed routinely in clinical care and carry a risk of bleeding less than 1 in 100 persons and perforation or tearing in less than 1 in 50,000 persons Precollection Steps for Coordinator Preparation of Containers and Cassettes 1. Prior to the procedure, label the two tissue collection containers with the Patient ID labels. 2. Formalin containers will be pre-labeled with a Formalin Biohazard label. Label both tissue collection containers with a Tissue label. 3. Fill the containers 2/3 full of 10% formalin. 25 February

64 4. Record the barcode labels from the tan cassettes on the Colon Biopsy CRF and Sample Record Summary and Shipment Notification Form. Figure 16: Colon Biopsy Cassette Barcodes 5. Place the blue sponges on the top and bottom sections of the tan cassettes. Figure 17: Preparation of Colon Biopsy Cassettes 25 February

65 Colon Biopsy Collection Procedure Prior to the procedure, please be sure that all supplies are available (review supply list). Once the procedure has been reviewed and questions from the volunteer have been addressed, begin the biopsy procedure. The biopsy procedure will proceed as follows: 1. Position the subject on the procedure table per the preference of the gastroenterologist. 2. Apply a small amount of jelly to the rectum. 3. Insert 2 cotton swabs into the rectum (rotate 3 times). Remove and discard. 4. Insert the esophagogastroduodenoscopy (EGD) scope cm. 5. Using the radial jaws, take double tissue bites, until up to 8 tissue biopsies are obtained within the same 5 cm region (20-25 cm above the rectum). 6. Place two biopsies into each of the four tan, sponge-lined cassettes. If the tissues does not release, rinse with a saline solution until it releases. Spread out biopsies by gently spraying with sterile saline from a plastic squeeze bottle and/or using sterile forceps to lay out the tissue. 7. CRITICAL STEP: Close cassettes securely by bringing the lid down onto the bottom and snapping it. 8. The study coordinator will be responsible for completing the processing of the tissue once collected using the procedures described in detail below. 9. Be sure to give post care instructions verbally to the subject found in the Operations Manual. The patient is instructed to watch for bleeding or discomfort and to call with questions or concerns. 25 February

66 10. A follow-up call will be placed by the study coordinator 7 days (±2 days) after the procedure to assess for adverse events Sample Procedure Note The subject was prepped in a standard fashion following usual institutional procedures for a flexible sigmoidoscopy. The sigmoidoscope (list type) was inserted into the rectum to approximately cm. Eight biopsies were obtained from this region. The subject tolerated the procedure well and no complications occurred. No significant blood loss occurred. Procedure Code: Research test for mucosal survey and sample collection. Procedure performed by (insert name) Tissue Processing of Colon Biopsies 1. Two biopsies will be placed on the bottom section of each of the four tan cassettes. 2. Submerge the two cassettes into one container. Record time placed in Formalin on the Colon Biopsy CRF and Sample Record Summary and Shipment Notification Form. 11. Submerge the other two cassettes into the remaining container. Record the time placed in Formalin on the Colon Biopsy CRF and Sample Record Summary and Shipment Notification Form. 12. CRITICAL STEP: Be sure that the container lids click twice and that the arrows are aligned, indicating that the containers are properly sealed. 13. Place containers in a refrigerator until shipment. 14. CRITICAL STEP: Ship the samples back to the S4 Biorepository at 4 C according to Appendix C on the same day of collection. 15. Place a follow-up call to the subject 7 days (±2 days) after the procedure to assess for adverse events. 25 February

67 25 February

68 9.3 Submandibular Biopsy Visit The kit contains the items in the table below, which will be used to perform biopsy procedure. Check the dates of expiration. Supplies for collection and shipment are sent to sites in a separate kit from Indiana University. Place cold packs in refrigerator upon arrival to chill them. Submandibular gland biopsy samples must be shipped on the day of collection and received at the S4 Biorepository the day after collection Needle core biopsy kit components Quantity Kit Component 1 Disposable core biopsy instrument 1 Sterile forceps 1 Tissue specimen storage container 2 Tissue cassette (pink) 4 Sponges 2 Cold packs Setting Up the Kit 1. On an overbed table, remove the contents of the kit from the outer plastic packaging, leaving the contents wrapped in their sterile drape. Leave everything wrapped until the person performing the biopsy is seated and begins examining the subject. 2. Open the sterile kit touching only the outside of the wrapper. Don t let the outside of the wrapper touch any part of the inside. If you touch any part of the paper wrapper or if any non-sterile object or outside of the wrapper touches any part of the inside of the wrapper, discard the kit and start over. If you are in doubt as to whether something touched the inside of the paper wrapper, throw the kit away and start over. 25 February

69 9.3.3 Precollection Steps for Specialist Preparation of Patient Before the submandibular gland biopsy procedure, the subject will have undergone informed consent and screening procedures, including evaluation of coagulation status and a review of medications that may lead to an increased bleeding time. The specialist completing the procedure will review the procedure and potential risks prior and address any questions the subject may have prior to performing the procedure. Specific discussion points are listed below: Thank the volunteer for participating in the study. Review that the procedure will include 5 separate passes of the biopsy apparatus on the same side. Explain that the biopsy apparatus makes a loud pop ; demonstrate with the apparatus in the air if appropriate. The subject may experience a minimal amount of pain. Lidocaine will be given prior to biopsy to minimize discomfort. Most people do not complain of pain after the procedure. Review the risks: most common side effect is bleeding and bruising. Because the location of the facial vein can be difficult to identify, the biopsy may hit that vein and cause some bleeding during the procedure. Bleeding, should it occur, will be easily controlled by applying pressure. Bruising can last for a week, but there will not be any permanent or visible scars. Other possible side effects include infection, though no infections have been reported using this technique Precollection Steps for Coordinator Preparation of Containers and Cassettes 1. Prior to the procedure, label the tissue collection container with the Patient ID label. 2. A formalin container will be pre-labeled with a Formalin Biohazard label. Label the tissue collection container with a Tissue label. 25 February

70 3. Fill the container 2/3 full of 10% formalin. 3. Record the barcode label from the pink cassettes on the Submandibular Gland Biopsy CRF and Sample Record Summary and Shipment Notification Form. Figure 18: Submandibular Gland Biopsy Cassette Barcodes 4. Place the blue sponges on the top and bottom section of the pink cassettes. Figure 19: Preparation of Submandibular Gland Biopsy Cassettes 25 February

71 9.3.5 Submandibular Gland Biopsy Collection Procedure Once the procedure has been reviewed and questions from the volunteer have been addressed you ready to begin the biopsy procedure. Prior to the procedure, please be sure that all supplies are available (review supply list). The biopsy procedure will proceed as follows: 1. Palpate the submandibular glands bilaterally. 2. Mark the preferred gland with a pen. 3. Clean skin with an alcohol wipe. 4. Inject the skin over the submandibular gland with 1% lidocaine with 1:100,000 epi. 5. Use the needle biopsy injector to take 5 biopsies of the submandibular gland. 6. Have your assistant hold pressure over the area until hemostasis is achieved. 7. After each biopsy, your assistant will remove the tissue from the apparatus using a needle. The needle should be opened to 180 degrees to release the tissue and the open side of the needle should be placed against the sponge inside the cassette. Hold until the specimen adheres to the sponge. 8. Place two biopsies into a pink, sponge-lined cassette and place the remaining three biopsies into a second pink, sponge-lined cassette. 9. CRITICAL STEP: Close cassettes securely by bringing the lid down onto the bottom and snapping it. 10. The study coordinator will be responsible for completing the processing of the tissue once it is removed from the biopsy apparatus using the procedures described in detail below. 25 February

72 11. An assistant holds pressure on the biopsy site until hemostasis is achieved. 12. Antibacterial ointment in placed on the biopsy site. 13. A band aid is placed over the biopsy site. 14. The subject should be instructed to watch for increasing erythema, pain, swelling, or breathing and to call with questions or concerns. 15. A follow-up call will be placed by the study coordinator 7 days (±2 days) after the procedure to assess for adverse events Sample language for procedure note The skin over the left submandibular gland was injected with 1% lidocaine with 1:100,000 epinephrine. After anesthesia of the area was confirmed, a 16-gauge needle biopsy injector was used to take five biopsies of the submandibular gland. There was minimal bleeding. Pressure was held over the area until hemostasis was achieved. The patient tolerated the procedure well Tissue Processing of Submandibular Gland Biopsies 3. Two biopsies will be placed on the bottom section of one pink cassette and the remaining three biopsies on the bottom section of the second pink cassette. 4. Submerge the two cassettes into one container. Record the time placed in Formalin on the Submandibular Gland Biopsy CRF and Sample Record Summary and Shipment Notification Form. 5. CRITICAL STEP: Be sure that the container lid clicks twice and that the arrows are aligned, indicating that the container is properly sealed. 6. Place container in a refrigerator until shipment. 7. CRITICAL STEP: Ship the samples back to the S4 Biorepository at 4⁰C according to Appendix C on the same day of collection. 8. Place a follow-up call to the subject 7 days (±2 days) after the procedure to assess for adverse events. 25 February

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74 10.0 Packaging and Shipping Instructions Please refer to Appendix C for detailed shipping instructions regarding: S4 Ambient Shipping Instructions S4 Frozen Shipping Instructions S4 Tissue Sample Shipping Instructions ***Important Notes*** Include only one subject visit set of samples per frozen shipping carton in order to have room for a sufficient amount of dry ice to keep samples frozen up to 24 hours unless otherwise noted by Indiana University. Research Clinical Lab samples must be shipped on the day of collection for overnight delivery to IU. These samples should not be collected on Fridays. DNA samples must be shipped within FIVE DAYS of collection. Frozen samples must be shipped within TWO WEEKS of collection. Ship all frozen samples Monday through Wednesday ONLY! Ambient blood for DNA or Research Clinical Labs may be shipped on Thursday. BE AWARE OF HOLIDAYS!! Ship all tissue samples Monday through Thursday. Shipment of tissue samples on Fridays will only be permitted under prior arrangement with IU. Tissue Samples must be shipped same day after completion of sample processing! BE AWARE OF HOLIDAYS!! Remember to complete the Sample Record Summary and Shipment Notification Form (Appendix B). Include a completed copy of Appendix B in your shipment AND notify Indiana University IN ADVANCE to confirm the shipment. 25 February

75 11.0 Sample Quality Checks and Feedback to Sites In addition to tracking and reconciliation of samples, the condition and amount of samples received is tracked by Indiana University for each sample type received. Sites are responsible to ensure the requested amounts of each fluid and tissue type are collected to the best of their ability and that frozen samples are packed well with sufficient amounts of dry ice to avoid thawing in the shipment process. Indiana University will complete a Non-Conformance Report (Appendix L) should there be any issues with a shipment and will provide this feedback to the site. Issues of concern that may impact collection, processing, or future analyses of the samples will be addressed by the S4 Steering Committee and communicated to site personnel, including any applicable specialists, as soon as possible via an ed report indicating the issue(s) found with the samples Data Queries and Reconciliation The CRFs related to laboratory procedures must be completed within 14 days, according to the study protocol, since they capture information related to the details of the sample collection and processing. These forms include information that will be used to reconcile sample collection and receipt, as well as information essential to future analyses. Indiana University will be collaborating with the University of Iowa to reconcile information captured in the study database compared to samples received and logged in at Indiana University. Information that appears incorrect in the study database will be queried and additional discrepancies that may be unrelated to data entry will be resolved with sites in a separate follow-up communication. Data queries or discrepancies with samples shipped versus received at Indiana University may result from: Missing samples at Indiana University Incorrect samples collected and shipped to Indiana University Damaged or incorrectly prepared samples Unlabeled samples, samples labeled with incomplete information, or mislabeled samples Discrepant information documented on the Sample Record Summary and Shipment Notification Form and logged in at Indiana University compared to information entered into the study database. 25 February

76 APPENDIX A Rate of Centrifugation Worksheet Please complete and return this form by fax or , and alert the S4 Biorepository Project Manager if you have any questions regarding sample processing. The correct RPM will be sent back to you. Make note of this in your S4 Biologics Manual. Submitter Information Name: Site #: Submitter Centrifuge information Please answer the following questions about your centrifuge. Centrifuge Type: Fixed Angle Rotor Swing Bucket Rotor Radius of Rotation (mm): Determine the centrifuge s radius of rotation (in mm) by measuring distance from the center of the centrifuge spindle to the bottom of the device when inserted into the rotor (if measuring a swing bucket rotor, measure to the middle of the bucket). Calculating RPM from G-force: RRRRRR = RRRRRR 11, rr RCF = relative centrifugal force (G-force) RPM = rotational speed (revolutions per minute) R = centrifugal radius in mm = distance from the center of the turning axis to the bottom of the centrifuge Comments: Please send this form to Holly Riss at holly-riss@uiowa.edu or

77 APPENDIX B S4 Sample Record Summary and Shipment Notification Form Please complete this form electronically (i.e., typed). All fields are required. Site #: Site Name: Principal Investigator: Coordinator: Telephone: Instructions: Ship frozen samples Monday Wednesday ONLY. Ship all other samples Monday Thursday ONLY unless you have made prior arrangements with Indiana University (IU) to ship a biopsy sample on a Friday. This form must be completed for all biorepository sample shipments. Notify IU ( preferred) prior to shipment using the contact information below. Place a copy of the completed form in the shipment box and file a copy in the site study binder. The site will be contacted if any issues with the samples/form are noted upon receipt. Subject ID # Gender Specimen Type (ex. RNA, Colon Biopsy) Visit Type (BSB, CB, SGB) # of Tubes Completed by Submitter/Site List Subject ID that corresponds to pre-printed labels List only one Specimen Type per row Tube Volume (if less than standard) Biopsy Site Cassette Color Cassette ID # # of Biopsies Date of Draw/ Biopsy Time placed in fixative Completed by Biorepository Nonconformance report? Notation of problems Courier (check one): FedEx Other (specify): Date shipped: Tracking #: IMPORTANT! BEFORE SHIPPING, (PREFERRED) OR FAX A COPY OF THE COMPLETED FORM TO: Indiana University S4Bio@iu.edu Fax: Ph:

78 APPENDIX C S4 DNA and Research Clinical Lab Sample Shipping Instructions AMBIENT BLOOD SAMPLE PACKAGING AND SHIPMENT TO INDIANA UNIVERSITY Blood Samples Shipped Ambient: DNA Purple Top 6ml EDTA Blood Tube Research Clinical Labs 4ml EDTA Blood Tube IMPORTANT! AMBIENT SAMPLES MAY BE SHIPPED MONDAY-THURSDAY ONLY! DNA must be shipped within five (5) days of collection. Research clinical labs MUST be shipped on the day of collections. These samples may be sent together in the sample package--up to four ambient sample tubes may be shipped in a single package. 1. Contact FedEx to confirm service is available and schedule package to be picked up. 2. Notify Indiana University of shipment by ing S4Bio@iu.edu (preferred) or faxing ( ) a copy of the completed Sample Record Summary and Shipment Notification Form (Appendix B). 3. Insert up to four purple top EDTA tubes in the tube sleeve. Place the sleeve into the canister and close securely. Wrap the canister in the enclosed bubble wrap and place into the cardboard box. 4. Apply the UN3373 label to the outside of the cardboard box. 5. Place the cardboard box and the completed Sample Record Summary and Shipment Notification Form in the FedEx Clinical Pak and seal according to the instructions on the envelope. 6. Complete the From portion of the provided FedEx air waybill by filling in your name, address, and phone number. FedEx is likely to reject or return your shipment without this information. 7. Apply the completed FedEx air waybill to the outside of package and arrange for FedEx pick up. 8. Ship the sample(s) to Indiana University, following the guidelines in the box above. Hold at room temperature until the time of pick up.

79 APPENDIX C S4 Biopsy Shipping Instructions COLD PACK BIOPSY SAMPLE PACKAGING AND SHIPMENT TO INDIANA UNIVERSITY Biopsy Samples Shipped on Cold Packs: Skin biopsy samples 2 peach and 2 white tissue cassettes Colon biopsy samples 4 tan tissue cassettes Submandibular biopsy samples 2 pink tissue cassettes IMPORTANT! BIOPSY SAMPLES MAY BE SHIPPED MONDAY-THURSDAY ONLY UNLESS PRIOR ARRANGEMENTS HAVE BEEN MADE WITH IU! 1. Contact FedEx to confirm service is available and schedule package to be picked up. 2. Notify Indiana University of shipment by ing S4Bio@iu.edu (preferred) or faxing ( ) a copy of the completed Sample Record Summary and Shipment Notification Form (Appendix B). 3. Insert a large absorbent sheet into a biohazard bag (both provided) and place one tissue container, with cassettes inside, into the bag and seal it. Repeat as necessary for all tissue containers. 4. Insert two cold packs into the ambient shipping container provided. Insert the specimen container(s) into the shipping container. If necessary, add paper toweling or other material as padding. 5. Replace the lid on the Styrofoam carton, place the completed Sample Record Summary and Shipment Notification Form on top of the carton, and close and seal the outer cardboard shipping carton with packing tape. 6. Complete the From portion of the provided FedEx air waybill by filling in your name, address, and phone number. FedEx is likely to reject or return your shipment without this information. 7. Apply all provided warning labels and the completed FedEx air waybill to the outside of the package, taking care not to overlap labels. Arrange for FedEx pickup. 8. Hold packaged samples in a 4 C refrigerator until the time of FedEx pickup. 9. Ship the samples to Indiana University on the day of collection.

80 APPENDIX C S4 Frozen Sample Shipping Instructions FROZEN SAMPLE PACKAGING/SHIPMENT TO INDIANA UNIVERSITY Samples Shipped on Dry Ice: Frozen whole blood in 6 ml plastic EDTA tubes Frozen whole blood in PAXgene tubes Frozen plasma in 2ml polypropylene tubes Frozen serum in 2ml polypropylene tubes Frozen CSF in 2ml polypropylene tubes Frozen saliva in 2ml polypropylene tubes IMPORTANT! FROZEN SAMPLES MAY BE SHIPPED MONDAY-WEDNESDAY ONLY! Only ONE set of samples may be shipped in a single package. 1. Contact FedEx to confirm service is available and schedule package to be picked up. 2. Notify Indiana University of shipment by ing S4Bio@iu.edu (preferred) or faxing ( ) a copy of the completed Sample Record Summary and Shipment Notification Form (Appendix B). 3. Place all frozen 2ml aliquots in the provided cardboard cryoboxes. Label the outside of the cryoboxes with the subject ID and visit number. 4. Place the cryoboxes into a clear plastic biohazard bag with a small absorbent sheet and seal according to the instructions on the bag. Insert this into the white Tyvek biohazard envelope and seal according to the instructions on the envelope. 5. Insert frozen EDTA and PAXgene tubes into the bubble wrap pouch provided. In the event that the tubes cracked or broke during previous shipments, one is advised to package the bubble wrapped tubes with additional padding. 6. Place bubble-wrapped tubes into the 2 nd clear plastic biohazard bag with a small absorbent sheet and seal according to the instructions on the bag. Insert this into the 2 nd white Tyvek biohazard envelope and seal according to the instructions on the envelope.

81 APPENDIX C S4 Frozen Shipping Instructions Domestic 7. Place both envelopes upright and side-by-side in the provided Styrofoam-lined shipping carton, as shown below: 8. Fill the remaining space in the shipping carton with approximately 10 lbs of dry ice, ensuring ice surrounds both envelopes and reaches the top of the carton, as shown below: 9. Replace the lid on the Styrofoam carton, place the completed Sample Record Summary and Shipment Notification Form on top of the carton, and close and seal the outer cardboard shipping carton with packing tape. IMPORTANT! Complete the required fields on the FedEx air waybill and Class 9 Dry Ice label, or FedEx may reject or return your package.

82 10. Complete the FedEx air waybill with the following information: a. Section 1, From : fill in your name, address, and phone number b. Section 6, Special Handling and Delivery Signature Options : under Does this shipment contain dangerous goods? check the boxes for Yes, Shipper s Declaration not required and Dry Ice. Enter the number of packages (1) x the net weight of the dry ice in kg. 11. Complete the Class 9 UN 1845 Dry Ice label (black and white diamond) with the following information: a. Your name and return address b. Net weight of dry ice in kg c. Consignee name and address: S4 Biorepository, IU School of Medicine, Walther Hall R3-C158, 980 W. Walnut St., Indianapolis, IN d. Do not cover any part of this label with other stickers, including pre-printed address labels. 12. Apply all provided warning labels and the completed FedEx air waybill to the outside of the package, taking care not to overlap labels. 13. Hold packaged samples in a -80 C freezer until the time of FedEx pickup.

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88 APPENDIX I

89 APPENDIX J

90 APPENDIX K Low-Fat Diet Menu Suggestions Due to the interference of lipid content in blood specimens collected for biomarker evaluation in the S4 study, it is strongly advised that samples be collected after an 8 hour fast (no food or drink except fluids such as water, tea, black coffee). If fasting is not achievable, a subject should be on a low-fat diet for at least 8 hours prior to blood collection. Below is a list of suggested sample menus that could be consumed prior to blood collection. These lists are not all inclusive and sites should use their best judgment in this process. Sample Breakfast Items: Dry whole wheat toast Fruit salad no dressing Clear tea or coffee no milk or cream Fruit or vegetable juice Dry cereal without nuts/no granola; no milk Clear tea or coffee no milk or cream Fruit or vegetable juice Plain oatmeal or other cooked whole grain cereal topped with fresh or dried fruit no butter, milk, or cream Clear tea or coffee no milk or cream Fruit or vegetable juice Dry whole wheat toast Poached egg white or egg substitute Clear tea or coffee no milk or cream Fruit or vegetable juice Sample Lunch Items: Turkey breast sandwich on whole wheat bread Lettuce, Tomato, and Mustard Clear beverage Flavored gelatin Plain pasta with plain marinara sauce no butter or cheese Side of steamed vegetables or green salad Clear beverage Flavored gelatin Steamed chicken breast lean, without skin Side of steamed vegetables or green salad Clear beverage Flavored gelatin Large tossed green salad, assorted vegetables no dressing or cheese Clear beverage Flavored gelatin Cucumber sandwich on whole wheat bread Lettuce, tomatoes, shredded carrots, onions, etc. Clear beverage Flavored gelatin Clear broth with vegetables and pasta Fruit salad no dressing Clear beverage Flavored gelatin

91 Foods to avoid prior to blood collection: Avoid: All fats and nuts such as: Butter Cream Bacon fat Lard All oils All margarine All nuts Peanut butter Coconut Whole seeds such as pumpkin and sunflower Avoid: All milk and dairy products such as: All whole milk products All cheese All products containing cheese Cheese spreads such as cream cheese Sour cream All ice cream Milk chocolate APPENDIX K Low-Fat Diet Menu Suggestions Avoid: High fat prepared foods and foods naturally high in fat: All red meats or meats containing fat such as pork Fatty meats such as: o Luncheon meats o Organ meats o Bacon Fatty fish such as: o Salmon o Mackerel Salad dressing and mayonnaise Buttered, au gratin, creamed, or fried vegetables Fried foods Fried snacks such as: o Chips o Crackers o French fries Gravies and sauces Baked goods and frosting

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93 APPENDIX M Saliva Collection - PIC Preparation (prior to scheduled visit) Upon receipt 100X PIC 10 Aliquots 100X PIC (100 µl ea) At day of visit 100X PIC (thaw at RT) At day of visit Dilute to 10X PIC (1 ml after dilution) Dilute to 10X PIC 1: Store 1-ml 100X PIC according to manufacturer recommendation Thaw at room temp on bench top when aliquoting 2: Aliquot 100 µl into each 1.5 ml microcentrifuge tube Label 100x PIC with permanent marker on the tube and date 3: Store PIC aliquots at -20 o C 4: At day of visit, remove one 100X PIC aliquot from -20 o C Thaw at room temperature 5: Dilute to 10X by adding 900 µl sterile ddh 2 O. Invert few times to mix. Keep at room temp Ready to use

94 APPENDIX N Saliva Collection and Aliquot Transfer saliva (using a serological pipette of micropipetter) to a 15 ml conical tube Add appropriate amount of 10X PIC Mix vigorously for sec + 1: Write subject ID on the 50 ml conical tube Place the labeled tube on ice 2. Have prepared 10X PIC ready Volume of saliva transferred to a 15 ml tube Volume of 10X PIC to add to sample 1.0 ml 100 µl (0.1 ml) 1.5 ml 150 µl (0.15 ml) 2.0 ml 200 µl (0.2 ml) 5.0 ml 500 µl (0.5 ml) 10.0 ml Entire 1 ml of 10X PIC 3: Within 30 minutes of collection, centrifuge samples at 4 0 C, 2000 x g for 15 minutes 4: Label the 2 ml microcentrifuge tubes with pre-printed label Saliva. Place the labeled tubes on ice 5. Aliquot 0.5 ml of centrifuged saliva to each 2 ml microcentrifuge tube. Store saliva aliquots frozen at C until shipment.

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