Central Lab and CSO Samples

1. Central Lab and CSO Samples George VlamisLab Data Manager Jennifer NaroznyGlobal Biosamples Specialist

2. Types of Samples SHIP TO: COVANCE CENTRAL LAB SHIP TO: ROCHE CSO

3. NH20052 Covance Central Laboratory Procedures For Europe George VlamisLab Data Manager

4. Central Laboratory Topics • Site Responsibilities • Supplies/Kits/Requisition Forms • Specimen Collection Procedures • Packaging and Shipping • Laboratory Reports / Alerts • Site Support Services

5. Site Responsibilities • Centrifuge • Gauze and Alcohol Swabs • Tourniquets and Bandages • Non-automatically Defrosting Freezer (-20º C) • temperature monitored by site • storage capacity for samples until shipment • Urine Cups • Kits and Supplies Storage Capacity • to ensure quality specimens, store supplies between 4º C to 25º C (39º F to 77º F)

6. Start-up Supplies • Prior to start up shipment, Covance will telefax: • confirmation request of site address, contact names, telephone and fax numbers • dry ice provider and courier information • Visit-specific collection kits • Expired kits or kits/supplies in excess at end of study must be destroyed at site, do not return to Covance

7. Start-up Supplies • Shipping boxes and site specific shipping documents (air waybills, Commerical Invoices, address labels) supplied in envelopes designated by shipping condition • in some countries boxes/shipping documents are supplied by courier • Covance Central Laboratory Services Manual • available in English. Laminates will be made available in local languages • Bulk supplies • a standard bag of extra collection supplies • use if collection kit materials are damaged • laminated synopsis pages in appropriate language • training DVD (upon request)

8. Covance Central Laboratory Services Manual • Protocol-specific: • Laboratory Visit Test Schedule • laboratory requisition forms • reference ranges and flagging • Specimen collection and processing information • Packaging and shipping instructions • Laboratory certifications • renewals to be found at www.covance.com • Resupply form • Specimen Stabilities and Holiday Observations • Covance contact information • Section to file Covance communications Please reviewmanual prior tofirst patient visit

9. Covance Automated Kit Resupply Automated Kit Resupply • Review inventory in preparation of patient visits and for expired kits • Inform Covance of lost, damaged or expired kits • Contact Covance if enrollment is faster than expected to increase supply levels 1 4 2 3

10. Country Specific Numbers provided here on Form Covance Resupply Requests • Sites must request: • additional shipping supplies • boxes, airway bills, address labels and Commerical Invoices • damaged, lost or expired kit replacements • extra tubes • Resupply form in manual (make copies) • e-mail/fax completed form • call-in order via telephone • Please allow a minimum of 7 working days for the delivery of your resupply order

11. 1205 Creatinine Collection Kit Contents • Visit-specific requisition forms, collection suppliesand packaging materials • 2 extra bar coded labels • to label, if needed, replacement tubes and vials • write the testing name and patient identifier on label Do not interchange kits between investigators or other protocols ! Do not interchange tubes and/or requisitions between kits !

12. Accession Number & Barcode • Located on kit box, tubes, vials and laboratory requisition form • Unique accession number assigned to each kit • protocol, site number and visit name are encoded • One accession number per visit per patient • reference this number with all queries to Covance ACCESSION NUMBER EXPIRATION DATE Do not collect specimens in expired containers !

13. Laboratory Requisition Form • Complete with a black ballpoint pen • draw a single line through incorrect information entered, date and initial the correction • First page: • mandatory for reporting of laboratory results • patient demographic section • special Instructions • optional Testing • tick box to request testing • Subsequent page(s): • collection instructions • Ship white copies to Covance with samples • Retain pink copies for your records • Once visit is completed, please fax requisition form to: • Covance CLS Geneva +41 22 989 7576

14. Demographic Section Completion of demographics is mandatory for reporting of laboratory results Required to identify your patient At Week 1, Week 9, Week 17, Week 23 & Final Visit check the appropriate box to indicate visit Required to report age and gender-specific reference ranges Required to calculate specimen stability Required to assist in site communication

15. Unscheduled/Optional Visits • Unscheduled or Early Term • for patients who terminate before study completion • for follow-up testing or to redo analyses at any time during trial • in substitution of any kit, if no appropriate kit in stock. Simply cross through “Unscheduled” and write correct visit name example: Unscheduled Week 1 • Make sure you order all tests required at visit being performed • Immediately call Covance for resupply

16. Sample Collection, Labels and Process • All tubes are pre-labelled • test name(s) and Accession number • check expiration date on tubes • Write the patient identifiers (Patient CRF Number) on each label at the time the specimen is drawn • identifiers on tubes/vials, requistion form, and case report form must match 1205 1205 Incorrect patient identifiers could cause cancellation of testing

17. Specimen Types Returned To Covance • All samples shipped to Covance Frozen by batch every 3 months

18. Serum Collection:Intact Parathyroid Hormone • Fill Serum Separation tube until blood flow stops • Allow blood to clot for 30 minutes (tube standing upright) • Centrifuge at a minimum of 1500 x g for 15 minutes • Clot and serum must be well separated • Carefully pipette all the serum into labelled plastic vial • Discard collection tube • Freeze immediately at –20°C until shipment

19. Urine Collection:Creatinine & Total Protein • Have patient void into a clean collection cup • Transfer specimen into labelled transport tube • Do not fill above the 10 mL mark on tube • minimum of 3 mL urine is required to perform testing • Screw cap on tightly to avoid leakage • DO NOT SHAKE – preservative tablet is not intended to dissolve completely

20. General Shipping Preparation • BEFORE first patient visit, contact site specific MARKEN courier • provide exact location for pick-up • determine last call-in time and LAST PICK-UP TIME • Instructions outlined in laboratory manual • Plan to ship frozen shipments early in week (Mon-Thurs) • Specimens are delivered to Covance within 24-48 hours • Customs/local holidays should be considered prior to sample shipment • refer to Covance Central Laboratory Manual or www.covance.com/investigators/holidays for list of most dates • do not ship 2 days before a holiday • Complete all documentation; attach to shipping carton

21. Dry Ice • In Europe, primary provider is MARKEN unless notified otherwise at site level • IATA (International Air Transport Association) Dangerous Goods Training • Mandatory and site’s responsibility

22. Frozen Packaging • Shipping Box • pre-printed with the Class 9 and UN1845 label • place site address label on box • Packaging • insert tube(s) into Specimen Collection Bag • If including requisition place in outside pouch (bar code visible) • place Specimen Collection Bag(s) in Styrofoam container • completely surround with dry ice • consolidate shipments as much as possible • replace lid and close box securely

23. Shipping Documents:Air Waybill • Documents are pre-printed, specific to shipping condition (frozen), supplied by Covance or Courier • need to be attached to shipping box(Marken Place in plastic sleeve) • Marken, frozen shipper provided with documents and address labels • Required Information • number of boxes • total box weight (record in whole numbers) • frozen • Verify amount of dry ice (in kg) on airway bill and UN1845 label on shipping box. These must match! • Friday Shipments (and Thursday shipments if located 48 hours from Covance) must have air waybill marked for “Saturday Delivery” • date and signature • Retain a copy (Marken: Investigator Receipt) for site records

24. Shipping Documents:Commercial Invoice • Documents • are pre-printed and specific to shipping condition (frozen) • need to be attached to shipping box, (Marken place in plastic sleeve) • Marken, frozen shipper provided with Commerical Invoice • Required Information • number of shipping boxes • total box weight (record in whole numbers) • date and signature • Retain a copy for site records

25. Courier Information • Europe: • The courier is country and site specific • Courier name and exact contact numbers will be communicated to the site via fax

26. Courier Pick-up Information • If sample pick-up is missed, proceed as follows: • FROZEN samples should remain frozen at the appropriate temperature • Contact the Covance Call Center with any problems due to missed pick-ups (# in the manual) Do not hesitate to call Covance,if you need further advice

27. Reporting Delays • Reasons for laboratory report delays: • missing, incomplete or inconsistent data on requisition • additional data requested on the requisition form is missing • incorrect visit kit used • incorrect sample condition • Sample testing will be performed upon receipt • Covance will query site for data clarification • once resolved the laboratory report will be released • for PANIC alert values, Covance will immediately contact the investigator to ensure patient safety • the report will be held pending data clarification

28. Laboratory Reports • Automatically faxed to the site • Sites must have a reliable fax service • Investigator must date and sign/initial each report • Report must be filed at site • Do not send back to Covance

29. Site Support Services • Covance will call you: • to obtain missing or inconsistent patient demographic or administrative data • when incorrect visit kits are used • to resolve collection date or time discrepancies • to report abnormal values • Contact Covance for questions about: • collections instructions • resupply of kits or shipping supplies • reports • courier problems • change in investigator or patient information • when enrollment is complete

30. Geneva-CH: Belgium France Germany Italy Spain Tel 00 800 88 77 44 11 Fax 00 800 88 77 44 22 Greece Tel 00 800 41 12 871 141 Fax 00 800 41 12 871 140 Poland Tel 00 800 41 11 231 Fax 00 800 41 11 230 Site Support Services Contact Numbers If unable to get through, please call your telephone operatorand ask for a collect call to: Covance CLS in Geneva - Switzerland Telephone + 41 22 989 79 00 / Fax + 41 22 989 75 21 English: + 41 22 989 79 01 / French: +41 22 989 79 02 / German: +41 22 989 79 03 / Italian: +41 22 989 79 04 / Spanish: +41 22 989 79 05

31. Sampling Overview Jennifer NaroznyGlobal Biosamples Specialist

32. Samples Being Collected & Shipped to Roche CSO Mandatory for all patients • Anti-Erythropoietin Antibodies • NT-proBNP and Troponin T Optional samples (Consented patients ONLY) • Biomarker Sample Repository (BSR) – Blood • Biomarker Sample Repository (BSR) – Urine

33. Roche Will Supply: • Pre-labelled plain blood collection tubes and polypropylene serum storage tubes for Anti-Erythropoietin Antibody and NT-proBNP/Troponin T samples • Bulk blood collection tubes, urine cups and centrifuge tubes, polypropylene serum storage tubes, and pre-printed CSO labels for optional Biomarker Sample Repository (BSR) Blood and Urine samples • Needles and holders for blood collection • Back up kits and materials including spare/blank labels • Shipping materials including dry ice for shipment (must be requested on the Courier Booking Form)

34. Re-supply: • If re-supply is needed during the study, inform your monitor as soon as possible • Allow at least 2-3 weeks from the time the request is made at Roche for supplies to be sent to the site

35. Documentation Provided by Roche • Sample Collection, Handling and Logistics Manual • All appropriate Shipping Forms • Sample Processing, Storage and Shipping Log • Freezer Log • Sample Processing Laminates

36. Anti-Erythropoietin AntibodiesSampling Times & Associated Sample Numbers

37. Anti-Erythropoietin AntibodiesSample Collection and Handling Procedure • 5 mL blood draw • Centrifuge • Transfer SERUM to 2 storage tube • Storage at -20°C or lower • Complete Sample Processing, Storage and Shipping log and the eCRF

38. Why Two Serum Samples? • First sample analyzed using an ELISA • If sample positive, neutralizing antibody test performed immediately • Neutralizing antibody test performed in different country • Prevents problems caused by freeze / thaw

39. NT-proBNP and Troponin TSampling Times & Associated Sample Numbers

40. NT-proBNP and Troponin TSample Collection and Handling Procedure • 3 mL blood draw • Centrifuge • Transfer SERUM to storage tube • Storage at -20°C or lower • Complete Sample Processing, Storage and Shipping log and the eCRF

41. Unscheduled Anti-Erythropoietin Antibodies & NT-proBNP and Troponin T • Unscheduled samples are ONLY taken by request of Investigator or Roche • Send to the Roche Clinical Sample Operations (CSO) immediately for transfer to the Analytical Lab • Follow the same Sample Collection and Handling Procedure for the scheduled Anti-Erythropoietin Antibodies and NT-proBNP and Troponin T • Associated sample numbers • Unscheduled Anti-Erythropoietin Antibodies: 301 – 308 • Unscheduled NT-proBNP/Troponin T: 401 – 408

42. Biomarker Sample Repository (BSR) SamplesSampling Times & Associated Sample Numbers

43. BSR Policies • BSR is governed by policies which ensure the ethically, scientifically and socially responsible use of the resource • Plasma and Urine samples will be collected from consenting individuals participating in the BSR portion of NH20052 • definitive list TBD • analysis of hepcidin/prohepcidin • Samples and associated data are stored for 15 years • Patient can withdraw BSR consent any time during the 15 year storage period • Research restricted to drug efficacy or AE, indication and related disease • BSR does NOT look at inherited DNA

44. BSR Blood – Sample Collection and Handling Procedure Blood / Serum BSR Samples No.: 901-905

45. BSR - BloodSample Collection and Handling Procedure • Label all tubes according to instructions in the sample handling manual • Draw 4.9 mL blood into monovette tube provided • Centrifuge • Transfer SERUM supernatant into SERUM TRANSFER tube • Transfer SERUM from transfer tube into 2 storage tubes • Storage at -70°C / -80°C • Complete Sample Processing, Storage and Shipping log and the eCRF

46. BSR Urine – Sample Collection and Handling Procedure Urine BSR Samples No.: 906-910

47. BSR - UrineSample Collection and Handling Procedure • Label collection cup and tubes according to instructions in the sample handling manual • Collect urine into urine collection cup provided • Transfer URINE to the 15 mL centrifuge tube(s) and centrifuge • Aliquot total of 4 mL URINE SUPERNATANT into 4 storage tubes (1 mL each storage tube) • Storage at -70°C / -80°C • Complete Sample Processing, Storage and Shipping log and the eCRF

48. Sample Labeling Non-Barcoded Label: Barcoded Label: • Non-barcoded labels are used for blood collection and transfer tubes and urine collection containers and centrifuge tubes which can be discarded after sample processing is complete • Barcoded labels are used for the plasma and urine storage tubes

49. 00000000346857 Subject No. 123 Sample No. 302 NH20052 012345 Smith SERUM Sample Labeling –BSR Blood & Urine or Spare Blank Labels • Use of the CSO labels provided is mandatory • DO NOT place any adhesive tape over labels • Apply label to the tube in the orientation illustrated (right) with the colored portion at the bottom of the tube • Check that the sample number and timepoint correspond to the eCRF sample number and timepoint being collected • Sample number and patient number on spare blank labels must be written in indelible ink

50. BSR eCRF Completion • “Is the patient participating in the BSR project?” • Must be answered for all patients • If Yes is ticked, dates of consent and sampling must be recorded • BSR Withdrawal • while the study is ongoing, • site must complete eCRF with date of patient withdrawal • notify your monitor to obtain the BSR Patient Withdrawal form • if study complete • notify your monitor