Protocol TN01 Pathway to Prevention Henry Rodriguez, M.D.

Transcription

1 Protocol TN01 Pathway to Prevention Henry Rodriguez, M.D. Laboratory Manual of Operations Version 2.0 LABORATORY MANUAL OF OPERATIONS

2 TN01 Pathway to Prevention Study Laboratory Manual of Operations -Table of Contents- 1. INTRODUCTION Document Description Role of the TrialNet Coordinating Center TNCC Laboratory Contacts Participating Laboratories SPECIMEN COLLECTION AND PROCESSING INSTRUCTIONS Important Reminders Specimen Labeling Specimen Shipping Biochemical Autoantibody (BAA) and Islet-Cell Autoantibody (ICA) Core Laboratory Assays Serum Autoantibodies (Screening, Annual Rescreening, & Monitoring) Serum Autoantibodies (Confirmatory & Tiebreaker)... 8 Figure 1. Autoantibody Processing Guidelines Beta-Cell Function Core Laboratory Assays Whole Blood HbA1c Plasma Oral Glucose Tolerance Test (OGTT) HLA/DNA Extraction Core Laboratory Assays Whole Blood HLA (DNA Storage Sample) Mechanistic Specimen Collections Whole Blood PBMC/Plasma (North American sites only) Whole Blood RNA Serum Mechanistic Serum Mechanistic Specimen Collection Volumes PACKAGING AND SHIPPING GUIDELINES Routine Ambient Shipment...16 Figure 2. Ambient Shipment Packaging Instructions (excluding Na Heparin Tubes) Ambient Shipment of Na Heparin Tubes...17 Figure 3. Ambient Shipment Packaging Instructions Na Heparin Tubes Refrigerated Shipment...18 Figure 4. Refrigerated Shipment Packaging Instructions Frozen Shipment...19 Figure 5. Frozen Shipment Packaging Instructions LABORATORY RESULT REPORTING Clinical Alerts Laboratory Result Turnaround Times (TAT) Turnaround Time Exceptions Priority Specimen Processing Guidelines Instructions for Marking a Specimen as Priority ONLINE SPECIMEN MANAGEMENT Overview Timing of Electronic and Physical Specimen Shipment Specimen Holds SPECIMEN HANDLING TIPS Temperature Changes Causes of Hemolysis in Evacuated Tubes...23 Page 2 of 26

3 TN01 Pathway to Prevention Study Laboratory Manual of Operations 7. SPECIMEN QUALITY CONTROL (QC) PROGRAM Overview of Participation SPECIMEN DESTRUCTION Overview Specimen Destruction Requests ONLINE LABORATORY REFERENCES Current Manuals and SOPs Current Training Materials TNCC Laboratory Contacts Actions Current Lab Certifications and Reference Ranges Current Tools Archive SUPPLIES Page 3 of 26

4 TN01 Pathway to Prevention Study Laboratory Manual of Operations 1. INTRODUCTION 1.1 Document Description This Manual of Operations has been created to provide details concerning specimen handling for the TN01 Pathway to Prevention Study, including: collection, processing, storage, and shipping of laboratory specimens, laboratory testing and result reporting timelines, clinical alert criteria. Guideline: A Laboratory Manual of Operations (MOO) will be provided for each TrialNet protocol. Principles: The Laboratory MOO is a cooperative work between each participating laboratory, the TNCC, and the Laboratory Monitoring Subcommittee (LMS) to document proper and standard specimen handling procedures. 1.2 Role of the TrialNet Coordinating Center The TrialNet Coordinating Center (TNCC) was established to support the data management and data analysis for the network. The TNCC Laboratory Operations Group coordinates all aspects of specimen handling, result reporting, inventory management, and facilitation of laboratory-related subcommittees within TrialNet. The TNCC Laboratory Clinical Research Administrator (CRA) serves as the primary liaison between clinical sites and core laboratories TNCC Laboratory Contacts TrialNet Coordinating Center (TNCC) University of South Florida Pediatrics Epidemiology Center Tampa, FL TNCC Laboratory Contacts Primary Contact: Cristin Linton, MS Secondary Contact: Archana Sharma Ph.: (813) Fax: (813) Cristin.Linton@epi.usf.edu Ph.: (813) Fax: (813) Archana.Sharma@epi.usf.edu 1.3 Participating Laboratories Note: Sites should contact the TNCC for all specimen and result inquiries. If there is a shipping emergency and the TNCC is not available to assist, sites may contact the individual labs. Core Screening / Islet Cell Autoantibody (ICA) Laboratory Shipping Address: Specimens: TrialNet Core Screening Laboratory (UFDRL) University of Florida 4800 SW 35th Drive Gainesville Florida, ICA testing, autoantibody sample aliquoting & storage Principal Investigator: William Winter, MD Main Contact: David Pittman Main Phone: (352) x FedEx online system: pittman@pathology.ufl.edu LAB: Screening ICA Core Page 4 of 26

5 TN01 Pathway to Prevention Study Laboratory Manual of Operations Biochemical Autoantibody (BAA) Core Laboratory Principal Investigator: Liping Yu, MD Sites ship BAA samples to the ICA Core Laboratory; ICA Lab then ships an aliquot of serum to the BAA Lab. Specimens: GAD65A, IA-2A/ICA512A, miaa, ZnT8A Beta-Cell Function Core Laboratory Shipping Address: Specimen Processing Northwest Lipid Research Laboratories University of Washington 401 Queen Anne Ave. North Seattle, WA Principal Investigator: Jerry Palmer, MD Main Contact: Jessica Harting Main Phone: (206) FedEx online system: LAB: Beta-Cell/Biochemistry Core Specimens: HbA1c, C-peptide, Glucose, Insulin HLA/DNA Extraction Core Laboratory Shipping Address: Attention: HLA/DNA Lab Barbara Davis Center 1775 Aurora Ct, UC Denver, AMC M C Aurora, CO Principal Investigator: Liping Yu, MD Main Contact: Taylor Armstrong Main Phone: (303) FedEx online system: LAB: HLA/DNA Extraction Core Specimens: HLA typing, DNA extraction & storage PBMC Core Laboratory Shipping Address: Specimens: Rutgers University Cell Repository 604 Allison Road, Room C11 Piscataway, NJ PBMC & plasma extraction from whole blood Principal Investigator: Jay Tischfield, PhD Main Contact: David Sokolowski Main Phone: (732) FedEx online system: LAB: PBMC Core RNA Processing Core Laboratory Shipping Address: Specimens: The TrialNet RNA Lab Jinfiniti Biosciences Center of Innovation for Life Sciences Medical College of Georgia th Street, CA 2105 Augusta, GA RNA extraction from whole blood Principal Investigator: Jin-Xiong She, MD Main Contact: Haito Liu Main Phone: (706) FedEx online system: LAB: The TrialNet RNA Lab NIDDK Biosample Repository Shipping Address: NIDDK Biosample Repository Fisher BioServices Century Blvd, Bldg 6, Suite 400 Germantown, MD Main Contact: Heather Higgins Main Phone: (240) FedEx online system: NIDDK Repository Specimens: Specimen storage, including PBMCs, plasma, serum, RNA, and DNA Page 5 of 26

6 2. TN01 Pathway to Prevention Study Laboratory Manual of Operations SPECIMEN COLLECTION AND PROCESSING INSTRUCTIONS This section provides detailed specimen handling instructions. Refer to Appendix A for specimen handling flow charts. Note: N.A. refers to North American sites; Int l refers to International sites. 2.1 Important Reminders Specimen Labeling Label collection tubes and cryovials before use. o Specimens should never be collected or processed in unlabeled tubes. Always include participant ID, FTL, and draw date. o Samples without proper labeling will be placed on hold to verify identification. o Be sure to use the 6-digit Participant ID, not the Local ID. Site may write participant identifiers directly onto the tube/vial or use a sticker label. Always use an alcohol-proof pen. Do not cover barcodes. When affixing a barcode label to a vial, apply the label vertically as pictured below. If the barcode is applied incorrectly, the lab is unable to scan the specimen upon arrival. This delays accessioning and creates the opportunity for data entry errors Specimen Shipping Ship sample electronically in online Specimen Management System (SMS) on the same day as physical shipment. If expedited processing is required, select the Priority checkbox in the Specimen Collection Form prior to shipping. o Marking samples as priority does not decrease result reporting timeline unless indicated in Laboratory Result Turnaround Time (TAT) Table. However, the lab will make every effort to expedite. Frozen shipments: o Use 2 freezer boxes for cryovials < 4.0 ml in size o Only use 3 freezer boxes for cryovials 4.0 ml in size o Samples from multiple subjects may be placed in the same freezer box, but should be arranged by participant. Critical for efficient logging of tolerance test samples! Page 6 of 26

7 2.2 TN01 Pathway to Prevention Study Laboratory Manual of Operations Biochemical Autoantibody (BAA) and Islet-Cell Autoantibody (ICA) Core Laboratory Assays Serum Autoantibodies (Screening, Annual Rescreening, & Monitoring) Parameter Assay Specifics Barcode Qty Collection Tube: 2.5 ml red top SST gel tube T N/A 1 Shipping Tube: 1.8 ml red top cryovial Etched 1 Storage: Maximum Stability: Shipping: Laboratory: Ship Days: Extended Storage Guidelines: Frozen at -20 C Indefinite Dry Ice Core Screening / Islet-Cell Autoantibody Laboratory (University of Florida) Monday Wednesday Refer to Figure 1 Collection and Processing Instructions 1. Label one 2.5 ml red top SST gel tube with a participant identifier. 2. Write the participant ID, FTL, and draw date on one 1.8 ml red top etched cryovial. 3. Scan barcode into the online Specimen Collection Form. 4. Draw 2.5 ml of blood into the collection tube. 5. Gently invert tube 5 times and place upright in a tube rack. Allow blood to clot for minutes at room temperature (65-75º F; 20-25º C). 6. Centrifuge at maximum speed for 15 minutes. 7. Transfer serum into the labeled 1.8 ml cryovial. Screw top on tightly. 8. Place specimen upright in a 2 partitioned freezer storage box. 9. Freeze at -20 C. Page 7 of 26

8 TN01 Pathway to Prevention Study Laboratory Manual of Operations Serum Autoantibodies (Confirmatory & Tiebreaker) Parameter Assay Specifics Barcode Type Qty Collection Tube: 8.5 ml tiger top SST gel tube N/A 1 Shipping Tube: 8 ml polypropylene shipment tube Etched 2 Storage: Maximum Stability: Shipping: Laboratory: Ship Days: Extended Storage Guidelines: Frozen at -20 C Indefinite Dry Ice Core Screening / Islet-Cell Autoantibody Laboratory (University of Florida) Monday Wednesday Refer to Figure 1 Collection and Processing Instructions 1. Label one 8.5 ml tiger top SST gel tube with a participant identifier. 2. Write the participant ID, FTL, and draw date on two 8 ml etched polypropylene shipment tubes. 3. Scan barcodes into the online Specimen Collection Form. 4. Draw 8.5 ml of blood into the collection tube. 5. Gently invert tube 5 times and place upright in a tube rack. Allow blood to clot for minutes at room temperature (65-75º F; 20-25º C). 6. Centrifuge at maximum speed for 15 minutes. 7. Transfer serum into the labeled 8 ml polypropylene shipment tubes. Screw tops on tightly. 8. Place specimen upright in a 3 partitioned freezer storage box. 9. Freeze at -20 C. Page 8 of 26

9 H O U R S TN01 Pathway to Prevention Study Laboratory Manual of Operations Figure 1. Autoantibody Processing Guidelines } Room Temperature }Begin to Centrifuge for 15 minutes } Separate Optimal & Freeze } W/O Freezer Room Temperature Begin to Centrifuge for 15 minutes } Separate & Refrigerate (SST tubes need not be separated prior to refrigeration) Maintain Refrigeration & Freeze within 24 hours W/O Centrifuge } Room Temperature Maintain Refrigeration (Centrifuge & separate within 8 hours) Maintain Refrigeration & Freeze within 24 hours 24 Summary: 1. Autoantibody samples must be clotted at room temperature for 30 minutes. 2. Storage Time: a. < 8 hours: Store at 4 C (refrigerator or cold packs) b. 8 to < 24 hours: Centrifuge, separate, and refrigerate at 4 C (refrigerator or cold packs) c. 24 hours: Centrifuge, separate, and freeze at -20 C. 3. Once frozen, samples can be stored indefinitely. Page 9 of 26

10 TN01 Pathway to Prevention Study Laboratory Manual of Operations 2.3 Beta-Cell Function Core Laboratory Assays Whole Blood HbA1c Parameter Assay Specifics Barcode Type Qty Collection Tube: N.A.: 1.2 ml lavender top K 3 EDTA tube Int l: 2.0 ml lavender top K 3 EDTA tube Sticker Label 1 Shipping Tube: Shipped in collection tube Sticker Label 1 Storage: Maximum Stability: Shipping: Laboratory: Ship Days: Extended Storage Guidelines: *N.A.: Sarstedt S-monovette tube **Int l: standard vacutainer N.A.: Refrigerated at 4 C Int l: Must be frozen at -70 C Refrigerated: 6 days Frozen: Indefinite at -70 C Refrigerated: Pre-chilled cold packs Frozen: Dry Ice Beta-Cell Function Core Laboratory (Northwest Lipids Research Lab) Collection and Processing Instructions Monday Thursday If blood must be held longer than 5-6 days, freeze whole blood at -70 C immediately after collection. N.A.* 1. Affix a preprinted TN barcode label onto one lavender top K 3 EDTA tube. Write the participant ID, FTL, and draw date on the tube. a. N.A.: Use 1.2 ml tube. b. Int l: Use 2.0 ml tube. 2. Scan barcode into the online Specimen Collection Form. 3. Draw 1.2 ml of blood into the collection tube. 4. Immediately invert tube gently 6-8 times to mix, avoiding jarring or shaking. DO NOT CENTRIFUGE. 5. Place tube upright until ready for shipping. a. N.A.: Store on ice or in the refrigerator (4 C). b. Int l: Freeze at -70 C. 6. Ship as whole blood. or Int l** Page 10 of 26

11 TN01 Pathway to Prevention Study Laboratory Manual of Operations Plasma Oral Glucose Tolerance Test (OGTT) Parameter Assay Specifics Barcode Type Qty Glucose: 1.2 ml gray top K Oxalate/Na Fluoride tube N/A 6 Collection Tube: C-Peptide: 1.2 ml lavender top K 3 EDTA tube N/A 6 Insulin: 1.2 ml green top Li Heparin tube N/A 3 Glucose: 1.8 ml gray top cryovial Etched 6 Shipping Tube: C-Peptide: 1.8 ml lavender top cryovial Etched 6 Insulin: 1.8 ml green top cryovial Etched 3 Storage: Frozen at -70 C (preferred) or -20 C Maximum Stability: 60 days Shipping: Dry ice Laboratory: Beta-Cell Function Core Laboratory (Northwest Lipids Research Lab) Ship Days: Monday Thursday OGTTs use Sarstedt S-monovette tubes Collection and Processing Instructions 1. Label each collection tube with specimen type, time point, and a participant identifier. a. Glucose (GLU) = 6 time points (-10, 0, 30, 60, 90 and 120 minutes) b. C-peptide (CPEP) = 6 time points (-10, 0, 30, 60, 90 and 120 minutes) c. Insulin (INS) = 6 time points, 3 collections* (-10, 0 and 120 minutes) *Results for 30, 60 and 90 min are analyzed using the c-peptide collections for these time points. 2. Write the participant ID, FTL, specimen type (GLU, CPEP or INS), time point, and draw date on each 1.8 ml etched cryovial. 3. Ensure that the appropriate color code (Glucose = Gray, C-Peptide = Lavender, Insulin = Green) is snapped into the lid of each cryovial. 4. Scan each barcode into the appropriate online Specimen Collection Form. Be sure to match sample type and time point on the collection form with the labeling on the vial. 5. Draw 1.2 ml of blood into the pre-labeled collection tubes at the specified time points. 6. Immediately invert tubes gently 6-8 times to mix, avoiding jarring or shaking. Place tubes upright on ice or in the refrigerator. 7. Centrifuge each sample at maximum speed for 15 minutes in a chilled (4 C) centrifuge within 1 hour of collection. 8. Transfer plasma into the labeled 1.8 ml cryovials. Screw tops on tightly. 9. Place specimens upright in a 2 partitioned freezer storage box. 10. Freeze at -70 C (preferred) or -20 C. Page 11 of 26

12 2.4 TN01 Pathway to Prevention Study Laboratory Manual of Operations HLA/DNA Extraction Core Laboratory Assays Whole Blood HLA (DNA Storage Sample) Parameter Assay Specifics Barcode Type Qty Collection Tube: 6 ml lavender top K 2 EDTA tube Sticker Label 1 Shipping Tube: Shipped in collection tube Sticker Label 1 Storage: Maximum Stability: Shipping: Laboratory: Ship Days: Extended Storage Guidelines: N.A.: Room Temperature Int l: Frozen at -70 C (preferred) or -20 C Ambient: 24 hours Frozen: 1 month Collection and Processing Instructions Ambient: No temperature-stabilization required Frozen: Dry Ice HLA/DNA Extraction Core Laboratory (Barbara Davis Center) Monday Thursday The preferred method of storage beyond 24 hours is freezing whole blood at -70 C (preferred) or -20 C. Frozen blood is stable for up to one month but should be shipped as soon as possible. 1. Affix a preprinted TN barcode label onto one 6 ml lavender top K 2 EDTA tube; apply vertically. Write the participant ID, FTL, and draw date on the. 2. Scan barcode into the online Specimen Collection Form. 3. Draw 6 ml of blood into the collection tube. 4. Immediately invert tube gently 8-10 times to mix. DO NOT CENTRIFUGE. 5. Keep sample upright until ready for shipping. a. N.A.: Store at room temperature. b. Int l: Freeze at -70 C (preferred) or -20 C. 6. Ship as whole blood. Page 12 of 26

13 2.5 TN01 Pathway to Prevention Study Laboratory Manual of Operations Mechanistic Specimen Collections Whole Blood PBMC/Plasma (North American sites only) Parameter Assay Specifics Barcode Type Qty Collection Tube: 10 ml green top Na Heparin tube Sticker Label 1-6* Shipping Tube: Shipped in collection tube Sticker Label 1-6* Storage: Maximum Stability: Shipping: Room Temperature 24 hours (whole blood) Ambient Laboratory: PBMC Core Laboratory (Rutgers University) Monday Friday Ship Days: (Sat/Sun shipping via WC at site expense) Whole blood must be processed within 24 hours; cell viability decreases Extended Storage with storage time. If samples cannot be shipped on day of draw, the Guidelines: collection should be missed. *Refer to Appendix B for visit-specific collection volumes per subject age and weight. Collection and Processing Instructions 1. Affix a preprinted TN barcode label onto each 10 ml green top Na Heparin tube; apply vertically. Write the participant ID, FTL, and draw date on each tube. 2. Scan barcodes into the online Specimen Collection Form. 3. Draw 10 ml of blood into each collection tube. The number of tubes collected depends on the protocol and/or the weight of the subject at enrollment. Please refer to the collection schedule in Appendix B to determine the collection volume required at each visit. 4. Immediately invert tubes gently 8-10 times to mix, avoiding jarring or shaking. DO NOT CENTRIFUGE. 5. Prepare the tubes for shipping immediately; keep at room temperature. Ship as whole blood. 6. SAMPLES MUST BE SHIPPED ON THE DAY OF COLLECTION. If samples cannot be shipped on the day of draw, the collection should be missed. Page 13 of 26

14 TN01 Pathway to Prevention Study Laboratory Manual of Operations Whole Blood RNA As of November 5, 2013, RNA will only be collected from subjects currently enrolled in Semi-Annual Monitoring. RNA collections will be discontinued on: All new subjects enrolled on or after November 5, 2013 Existing and new autoantibody-negative controls. Parameter Assay Specifics Barcode Type Qty Collection Tube: 3 ml blue top Tempus tube Sticker Label 1-2* Shipping Tube: Shipped in collection tube Sticker Label 1-2* Storage: Maximum Stability: Shipping: Laboratory: Ship Days: Frozen at -70 C Indefinite Dry Ice RNA Processing Core Laboratory (The TN RNA Lab, Jinfiniti Biosciences, Augusta, GA) Monday Wednesday *Refer to Appendix B for visit-specific collection volumes per subject age and weight. Collection and Processing Instructions 7. Affix a preprinted TN barcode label onto each 3 ml blue top Tempus tube; apply vertically. Write the participant ID, FTL, and draw date on the tube. 8. Scan barcode(s) into the online Specimen Collection Form. 9. Draw 3 ml of blood into each collection tube. 10. Immediately shake the tube extremely vigorously for a full 15 seconds. The shaking should be vigorous enough to cause some foaming of the sample. It is important to shake the tube hard enough to break up any small clots that have begun to form during drawing and to break the cells themselves to allow the buffer to protect the RNA inside. 11. Let sample sit at room temperature for 1-2 hours before freezing. 12. Freeze at -70 C. 13. Batch-ship samples quarterly, or monthly if freezer space onsite is limited. Page 14 of 26

15 TN01 Pathway to Prevention Study Laboratory Manual of Operations Serum Mechanistic Serum Parameter Assay Specifics Barcode Type Qty Collection Tube: 5 ml gold top SST gel tube with clotting activator N/A 1 Shipping Tube: 1.8 ml cryovial Etched 3 Storage: Frozen at -70 C Maximum Stability: Indefinite Shipping: Laboratory: Ship Days: Dry Ice NIDDK Biosample Repository (Fisher BioServices, Germantown, MD) Monday Wednesday Collection and Processing Instructions 1. Label one 5 ml SST gel tube with a participant identifier. 2. Write the participant ID, FTL, and draw date on three 1.8 ml etched cryovials. 3. Scan barcodes into the online Specimen Collection Form. 4. Draw 5 ml of blood into the collection tube. 5. Gently invert tube 5 times and place upright in a tube rack. Allow blood to clot for 30 minutes at room temperature (65-75º F; C). 6. Centrifuge sample at maximum speed for 15 minutes. 7. Transfer serum into the pre-labeled 1.8 ml etched cryovials. 8. Place specimens upright in a 2 partitioned freezer storage box. 9. Freeze at -70 C. 10. Batch-ship samples quarterly, or monthly if freezer space onsite is limited Mechanistic Specimen Collection Volumes Mechanistic specimen collection volumes vary according to participant age and weight to prevent exceeding blood volume limits. All samples may not be collected for younger subjects. Refer to Appendix B for visit-specific mechanistic collection schedules. Page 15 of 26

16 3. TN01 Pathway to Prevention Study Laboratory Manual of Operations PACKAGING AND SHIPPING GUIDELINES 3.1 Routine Ambient Shipment The following instructions detail the method for packaging and shipping of routine* ambient laboratory specimens (Figure 2). *Note: Applicable to all ambient shipments except those containing green top Na Heparin tubes. See section 3.2 for packaging of Na Heparin tubes. 1. Place the collection tube(s) into a Styrofoam tube holder(s) with an absorbent pad. Tape the holder securely closed. 2. Place each Styrofoam tube holder into a cardboard sleeve and then into a biohazard zip lock bag. 3. Use the online Specimen Management System (SMS) to prepare the shipment manifest and to notify the laboratory of the shipment. 4. Place the biohazard zip lock bag with specimens and a printed copy of the shipment manifest into an ambient tube shipper (5 3/8 x 4 5/8 x 1 5/8 box). 5. Place the ambient shipper into a FedEx Clinical Pak. 6. Affix the following label to the outside of the Clinical Pak (if not already pre-printed on the Pak): a. Diamond UN 3373 Biological Specimen Category B OR Exempt Human Specimen Label.* 7. Prepare and print a pre-paid FedEx airbill to ship specimens Priority Overnight to the appropriate TrialNet laboratory. *Each shipping site is responsible for the proper classification of biological specimens according to IATA Dangerous Goods Regulations (DGR). Figure 2. Ambient Shipment Packaging Instructions (excluding Na Heparin Tubes) Absorbent pad Collection tube + absorbent pad Styrofoam tube holder cardboard sleeve (not pictured) biohazard zip lock specimen bag + printed copy of manifest ambient tube shipper (5 3/8 x 4 5/8 x 1 5/8 box) FedEx UN3373 Clinical Pak Page 16 of 26

17 3.2 TN01 Pathway to Prevention Study Laboratory Manual of Operations Ambient Shipment of Na Heparin Tubes In the TN01 Pathway to Prevention Study, green top Na Heparin tubes are used to collect Whole Blood PBMC/Plasma samples. Due to the fragility of the peripheral mononuclear blood cells (PBMCs), additional temperature stability measures are required. The following instructions detail the method for packaging and shipping Na Heparin tubes (Figure 3). 1. Gel packs are to be left at room temperature (65-75 ºF; ºC) at least overnight prior to use. 2. Place the collection tube(s) into a bubble wrap sleeve(s). 3. Place the bubble wrap sleeve into a biohazard zip lock bag with an absorbent pad (place the pad near the pouch openings) and seal. 4. Use the online Specimen Management System (SMS) to prepare the shipment manifest and to notify the laboratory of the shipment. 5. Place the biohazard zip lock bag and a printed copy of the shipment manifest inside a Tyvek outer envelope and seal. 6. Place one gel pack flat on the bottom of the Styrofoam shipping container. 7. Place the sealed Tyvek envelope on top of the gel pack. 8. Place the second gel pack flat on top of the Tyvek envelope. 9. Securely seal the outer cardboard box of the Styrofoam shipper. 10. Affix the following label to the outside of the shipping box: a. Diamond UN 3373 Biological Specimen Category B OR Exempt Human Specimen Label.* 11. Prepare and print a pre-paid FedEx airbill to ship specimen(s) Priority Overnight to the appropriate TrialNet laboratory. *Each shipping site is responsible for the proper classification of biological specimens according to IATA Dangerous Goods Regulations (DGR). Figure 3. Ambient Shipment Packaging Instructions Na Heparin Tubes Bubble wrap sleeve Absorbent pad Tyvek envelope Gel packs Na Heparin tubes bubble wrap sleeve + absorbent pad biohazard zip lock specimen bag + printed copy of manifest Tyvek outer envelope + 2 gel packs insulated shipping box. Page 17 of 26

18 3.3 TN01 Pathway to Prevention Study Laboratory Manual of Operations Refrigerated Shipment The following instructions detail the method for packaging and shipping refrigerated laboratory specimens (Figure 4). 1. Cold packs are to be stored in the refrigerator (4 C) at least overnight prior to use. Do not freeze cold packs. 2. Place the collection tube(s) into a Styrofoam tube holder(s) with an absorbent pad. The Styrofoam tube holders provided by TrialNet hold 3 or 5 tubes. 3. Place each Styrofoam tube holder into a cardboard sleeve and then into a biohazard zip lock bag. 4. Use the online Specimen Management System (SMS) to prepare the shipment manifest and to notify the laboratory of the shipment. 5. Place the biohazard zip lock bag with two pre-chilled cold packs into a Styrofoam shipping container. 6. Place a printed copy of the shipment manifest in the outer cardboard box and tape securely closed. (Ensure manifest is not placed directly under tape line so it will not tear when the box is opened.) 7. Affix the following label to the outside of the box: a. Diamond UN 3373 Biological Specimen Category B OR Exempt Human Specimen Label.* 8. Prepare and print a pre-paid FedEx airbill to ship specimens Priority Overnight to the appropriate TrialNet Laboratory. *Each shipping site is responsible for the proper classification of biological specimens according to IATA Dangerous Goods Regulations (DGR). International sites: Use World Courier. Figure 4. Refrigerated Shipment Packaging Instructions Absorbent pad Cold packs Collection tube + absorbent pad Styrofoam tube holder cardboard sleeve (not pictured) biohazard zip lock specimen bag + 2 chilled cold packs + printed copy of manifest (placed outside cooler) insulated shipper Page 18 of 26

19 3.4 TN01 Pathway to Prevention Study Laboratory Manual of Operations Frozen Shipment The following instructions detail the method for packaging and shipping frozen laboratory specimens (Figure 5). 1. Place the 2 or 3 partitioned freezer storage box into a biohazard zip lock bag with an absorbent pad. a. Use 2 freezer storage box when shipping small cryovials (<4.0 ml). b. Use 3 freezer storage box only when shipping larger tubes and vials ( 4.0 ml). 2. Use the online Specimen Management System (SMS) to generate a shipment manifest and to notify the laboratory of the shipment. 3. Place the biohazard zip lock bag into a large Styrofoam shipping container filled to capacity with dry ice (at least 5 lbs or 3 kg). 4. Place a printed copy of the shipment manifest in the outer cardboard box and tape securely closed. (Ensure manifest is not placed directly under tape line so it will not tear when the box is opened.) 5. Affix the following labels to the outside of the box: a. Black Diamond UN1845 Dry Ice Label b. Diamond UN 3373 Biological Specimen Category B OR Exempt Human Specimen Label* (placed on same side as Dry Ice Label). 6. Prepare and print a pre-paid FedEx airbill to ship specimens Priority Overnight to the appropriate laboratory. *Each shipping site is responsible for the proper classification of biological specimens according to IATA Dangerous Goods Regulations (DGR). International sites: Use World Courier. 1.8 ml cryovials shipped in larger storage boxes fall out during transit, increasing accessioning time and the potential for vial breakage. Figure 5. Frozen Shipment Packaging Instructions Fiberboard freezer storage box Absorbent pad Fill with dry ice Freezer storage box (with vials inside) + absorbent pad biohazard zip lock specimen bag + dry ice + printed copy of manifest (placed outside cooler) insulated shipper Page 19 of 26

20 4. TN01 Pathway to Prevention Study Laboratory Manual of Operations LABORATORY RESULT REPORTING 4.1 Clinical Alerts A Clinical Alert is activated when specified laboratory results are outside their respective acceptable reference ranges. In the TN01 Pathway to Prevention Study, clinical alerts are activated if: 1. Plasma Glucose test results meet either of the following criteria: o Fasting Plasma Glucose 126 AND/OR o 2-hour Plasma Glucose HbA1c results meet the following criteria: o HbA1c test results 6.0% o HbA1c test results that have increased by 0.5% or more from the last visit*. *Last test must have been completed within 2 calendar years. When a clinical alert is activated, the laboratory will notify the clinical site and the TNCC immediately of the results via Laboratory Result Turnaround Times (TAT) Laboratory tests that are used to determine eligibility for participation in the TN01 Pathway to Prevention Study, treatment course, continued eligibility for follow-up, and monitor safety, etc. have posted turnaround times for result reporting. Laboratory result turnaround time (TAT) reflects the following factors: assay frequency, weekday sample arrival, processing time, assay preparation time, and assay run time. Improperly-packaged and disorganized shipments may increase accessioning time and delay result reporting. If special circumstances require additional sample testing, results may not be generated within the expected TAT. The TNCC laboratory operations team routinely monitors result reporting timelines and actively follows up on the status of delayed results. For specific assay turnaround times, refer to the TN Laboratory Result Reporting Turnaround Time Table. Samples not listed in the table do not have established TATs. The Turnaround Time table is located in the Tools portlet on the Specimen Processing References page of the TN members website (see section 9.6). TAT Calendars for assays specific to the TN01 Pathway to Prevention are included in Appendix C Turnaround Time Exceptions Turnaround times are agreed upon by the laboratory and the TrialNet Laboratory Monitoring Subcommittee (LMS). Requests for results within an assay s posted turnaround time cannot be accommodated unless related to eligibility for a TN Prevention Study or a safety concern. In those cases, the TNCC and laboratory will make every effort to expedite result reporting, if possible. However, participants should be scheduled to accommodate posted TATs. Page 20 of 26

21 4.3 TN01 Pathway to Prevention Study Laboratory Manual of Operations Priority Specimen Processing Guidelines Specimens may be marked as Priority at the discretion of the site. TrialNet laboratories will make every effort to process these specimens as soon as possible upon receipt in an effort to expedite result reporting. All specimen types can be marked as Priority. However, the result reporting turnaround time (TAT) is only reduced for specimens that have an expedited processing timeline indicated in the table. The Clinical Site Investigator must determine the standard for qualifying specimens with a Priority status. For example, a specimen(s) may be designated as Priority when a participant is screened near the end of the window of eligibility or results are needed for clinical confirmation Instructions for Marking a Specimen as Priority Identify a priority specimen in the specimen collection form by selecting the priority check box corresponding to that sample. Specimens indicated as Priority in the online Specimen Management System (SMS), appear in BOLD font on the printed shipment manifest. As an added, optional measure, the site may place red tape around the shipment tube/cryovial below the barcode label as per the figure below. Be sure not to cover up the sample barcode or participant identification information. Please note that this is an optional step and entirely up to the site. The primary method to mark specimens as priority is through the collection form; specimens with red tape that have not been marked as priority in the specimen collection form may not be treated as priority samples. For tolerance tests, only one vial needs to be marked with red tape. For consistency, please mark the glucose -10 minute sample. (If this specimen was not collected, the site may mark any specimen collected at the earliest time point). TNXXXXX OPTIONAL: Place Red Tape around the tube under the etched barcode or barcode label. Page 21 of 26

22 TN01 Pathway to Prevention Study Laboratory Manual of Operations Prioritization must be indicated at the time of specimen shipment. Requests for prioritization after shipment cannot be accomodated unless related to eligibility or a safety concern. 5. ONLINE SPECIMEN MANAGEMENT 5.1 Overview Specimen collection information is recorded in electronic case report forms, called online Specimen Collection Forms, on the TrialNet members website. Samples are shipped and tracked using the online Specimen Management System (SMS). For further detail on how to electronically collect, ship and track specimens, please reference the TN Specimen Management System MOO. 5.2 Timing of Electronic and Physical Specimen Shipment Specimens should be shipped electronically on the same day as physical shipment. The electronic shipment log generated by the system lets the lab know when to expect the specimen. If a specimen is not received as expected, a Specimen Not Received notification will be sent to the person at the site who shipped the sample, prompting them to check the site for the missing sample. 5.3 Specimen Holds Specimens received at the lab will be placed on hold for the following reasons: 1. Missing identifiers on tube 2. Mismatched information between tube and electronic shiplog 3. No electronic shiplog. When a lab places a specimen on hold, the TNCC is notified. The Protocol CRA will contact the site and ask them to consult their source documentation to confirm specimen identity. If the sample has not yet been shipped through the Specimen Management System (SMS), the site will be requested to ship the specimen immediately. If there is a correction that needs to be made, the TNCC will work with the site to update the system. Until specimen identity is confirmed and the specimen has been shipped through the SMS, the lab will not process the sample. This causes a delay in turnaround time. For some samples, the lab may be able to begin testing immediately due to the time-sensitive nature of the sample itself. However, results will continue to be held until a specimen resolution is provided to the lab. 6. SPECIMEN HANDLING TIPS 6.1 Temperature Changes When storing biological specimens, the temperature of the samples should always move in one direction from warmest to coldest. Stabilize specimen temperatures so that they do not warm to room temperature after they have been refrigerated or thaw after they have been frozen. Specimens may be stored at temperatures colder than specified. For example, if the specified temperature is -20 C, storage in a -70 C freezer is permitted. The samples must not be stored at temperatures warmer than -20 C. Page 22 of 26

23 TN01 Pathway to Prevention Study Laboratory Manual of Operations 6.2 Causes of Hemolysis in Evacuated Tubes 1. An improper choice in the venipuncture site, such as drawing from a distal site to the antecubital region of the arm rather than drawing from an antecubital site, has been shown to result in more hemolysis. 2. Prolonged tourniquet time causes the interstitial fluid to leak into the tissue and cause hemolysis. 3. Cleansing the venipuncture site with alcohol and not allowing the site to dry may cause hemolysis. 4. An improper venipuncture, indicated by a slow blood flow, may indicate occlusion due to the lumen of the needle being too close to the inner wall of the vein, causing hemolysis. 5. The use of a small-bore needle, resulting in a large vacuum force applied to the blood, may cause shear stress on the red blood cells, causing them to rupture. Detailed information on hemolysis can be found in the document How to Avoid Hemolysis, provided by the Beta-Cell/Biochemistry Laboratory. This guide is posted on the Specimen Processing page of the TN members website (see section 9.6). 7. SPECIMEN QUALITY CONTROL (QC) PROGRAM 7.1 Overview of Participation The Specimen QC Program, also known as the Split Duplicate Program, was designed to conduct a quality control assessment of the external and internal factors that may affect the reproducibility of an assay. All TrialNet Clinical Centers are required to participate in this program. Clinical Centers may also choose to train their affiliates in QC collection, but it is not mandatory for affiliates to participate. Please refer to the TN Laboratory QC MOO for complete instructions on how to collect, label, and ship QC specimens. 8. SPECIMEN DESTRUCTION 8.1 Overview Per the TN01 Pathway to Prevention protocol and model consent form, participants must consent to the storage of samples while TrialNet is ongoing in order to participate in the study. Participants have the option to allow storage of samples in the NIDDK Biosample Repository after TrialNet is over. If a participant withdraws consent for storage after TN is over, a Specimen Destruction Request is submitted to the TNCC by the site. At the conclusion of TrialNet, the TNCC will ensure these specimens are destroyed prior to transfer to the NIDDK Repository. A Specimen Destruction Request may also be submitted at the discretion of the site. For example, a site may request destruction if the identity of a sample cannot be confirmed. In this case, the TNCC notifies the applicable laboratory of the site s request and ensures it has been successfully completed. When confirmation of destruction is submitted by the laboratory to the TNCC, a notification is sent to the requesting site. 8.2 Specimen Destruction Requests The Sample Destruction Request form can be found on the TrialNet website on the Specimen Processing References page (see section 9.1). Page 23 of 26

24 TN01 Pathway to Prevention Study Laboratory Manual of Operations 9. ONLINE LABORATORY REFERENCES The Specimen Processing References page is designed to provide general laboratory references applicable to all TrialNet studies. Protocol-specific laboratory manuals, are posted on the TN01 Pathway to Prevention Study homepage. From the members website homepage, navigate to the Specimen Processing References page from the Laboratory References for Sites portlet. 9.1 Current Manuals and SOPs From the Specimen Processing References homepage, the latest laboratory SOPs and general laboratory manuals are available in the Current Manuals and SOPs portlet. 1. Electronic Specimen Management a. TN Specimen Management System User Manual 2. QC/Split Duplicate Program a. TN Laboratory QC Manual b. TN Laboratory QC Program Shipping Guide 3. Specimen Destruction a. Destruction of Sample SOP (includes Specimen Destruction Form) 9.2 Current Training Materials The Current Training Materials portlet includes general TNCC trainings on specimen handling, both physical and electronic. 1. General Training Materials 2. Specimen Processing 3. Electronic Specimen Management 4. QC/Split Duplicate Program 9.3 TNCC Laboratory Contacts From the Specimen Processing References homepage, all TNCC laboratory operations and supply system contacts are available in the TNCC Laboratory Contacts portlet. 9.4 Actions The Actions portlet includes access to result display and specimen browsing functions, as well as a link to the TN Fisher BioServices Supply Ordering System (SOS). 1. Specimen Data a. View Test Results b. Browse Specimens c. Browse Shipments Page 24 of 26

25 TN01 Pathway to Prevention Study Laboratory Manual of Operations d. Browse QC Collection Lists 2. Supplies a. Order Supplies (link to TN SOS website) 9.5 Current Lab Certifications and Reference Ranges The Current Lab Certifications and Reference Ranges portlet contains the most current versions of all TN core laboratory certifications and result reporting ranges, including: 1. Laboratory Certifications a. TN BAA Lab CLIA b. TN Beta/Biochem Lab CAP c. TN Beta/Biochem Lab CLIA d. TN ICA Lab CAP e. TN ICA Lab CLIA f. TN Viral Clinical Lab CAP g. TN Viral Clinical Lab CLIA h. TN Viral Research Lab CLIA 2. Result Reference Ranges a. TN Core Laboratory Reference Ranges 9.6 Current Tools Under the Current Tools header are the most current versions of general laboratory materials to aid sites in proper specimen handling procedures, including: 1. Specimen Processing References a. How to Avoid Hemolysis b. Sarstedt S-Monovette Tube Instructions c. Tips for Optimizing RNA Quantity and Yield 2. Blood Volume Guidelines a. TN Memo Blood Volume Guidelines in TrialNet 3. Laboratory Shipping Schedules a. TN Laboratory Holiday Closure Schedule (for current year) 4. Result Turnaround Times a. TN Laboratory Result TAT Table 5. Barcode Scanners a. TN Barcode Scanner Reference Guide b. TN Calibrating Scanner Document c. Request Barcode Scanner (link to 6. Shipping a. TN Online FedEx System Training b. WC Customs Invoice Templates (for international sites; form provided for each applicable laboratory) 9.7 Archive In the Archive portlet, previous laboratory SOPs, certifications, and reference ranges are posted for the site s reference. Page 25 of 26

26 10.SUPPLIES TN01 Pathway to Prevention Study Laboratory Manual of Operations All supplies referenced in the specimen collection, processing, and shipping instructions can be ordered through the online Fisher BioServices TrialNet Supply Ordering System (SOS). For more information on how to navigate the SOS and place an order, please refer to the section on supplies in the main protocol MOO. Page 26 of 26