Endothelial Progenitor Cell (EPC) Core Laboratory Instructions Version 5.0 March 29, 2008

1. Endothelial Progenitor Cell (EPC)Core Laboratory InstructionsVersion 5.0 March 29, 2008 For questions regarding collection, processing, storage, or shipping of EPC core lab samples, contact Dr. Tom Povsic (919) 681-XXXX (M–F, 8–5 ET) (919) 970-xxxx (pager)

2. Equipment and Supplies • REVEAL will supply  8 mL cell processing tubes (CPTs)  Cryovials for plasma storage  3 cc syringes  Cryovials for EPCs  16 gauge needles  Freezer-safe labels  PBS (phosphate buffered saline) 50 mL conical tubes  RPMI-FCS DMSO solution  Shipping containers • (Ten 15 mL conical tubes with  FedEx supplies 10 mL per tube. Freeze at -20 C in non-frost-free freezer or at -70 C ) • Site will supply •  Centrifuge  UN3373 Diagnostic Specimen labels •  Timer  Shipping box •  -80 or -70 C Freezer  Dry ice (to locate providers in your area, go to www.dryiceinfo.com)

3. Collecting the Sample • Obtain venous blood in an 8 mL cell preparation tube (CPT). A minimum of 5 mL blood is required • Gently invert tube 8–10 times • Maintain at room temperature • Process within 2 hours (sooner is better)

4. Processing the Sample If this is the baseline sample, remove RPMI-FCS DMSO solution (freezing buffer) from the freezer to thaw. At room temperature it will thaw in 20–30 min. Store remainder of DSMO solution in refrigerator until all three samples are processed.

5. Processing the Sample Full CPT tubes before spin

6. Processing the Sample • Separate the plasma • Invert CPT 8–10 times before centrifuging at 1500g for 30 minutes

7. Processing the Sample—After Spin Plasma Buffy Coat Polyester Gel Red Cells

8. Processing the Sample—After Spin Plasma Buffy Coat Polyester Gel Red Cells

9. 3 cc syringe and 16g needle Processing the Sample

10. Processing the Sample • Remove the plasma • Carefully remove ~ ½ of the plasma (top yellowish layer) using a 3 cc syringe with 16g needle. • Do not disturb the buffy coat layer. • Polyester gel ~ ¼ inch

11. Processing the Sample • Remove the plasma • If consent signed for plasma storage, divide plasma evenly into cryo vials (no more than 1.5 mL per vial). Label each vial. • If no consent for plasma storage, discard the plasma according to your institutional guidelines for disposal of blood and body fluid.

12. Processing the Sample Plasma Buffy Coat Polyester Gel Red Cells

13. Processing the Sample • Plasma in cryovial • Do not add more than 1.5 mL per vial • Place label on each vial

14. Processing the Sample EPC Washing • Measure ~ 40 mL of PBS into a 50 mL conical tube

15. Processing the Sample • Remove the buffy coat • Use the same needle/syringe to remove the buffy coat layer and remaining plasma. Place the needle in the middle of the buffy coat layer, being careful not to touch the polyester gel layer. • Carefully aspirate the buffy coat and remaining plasma. • Discard the CPT and red blood cells

16. Processing the Sample • CPT tube after removal of buffy coat • Discard according to your institutional guidelines for disposal of blood and body fluid

17. Processing the Sample EPC Washing • Inject the buffy coat into the 50 mL conical tube containing the PBS • Cap the tube and invert 2 times

18. Processing the Sample • 50 mL conical tube with cells and wash buffer

19. Processing the Sample EPC Washing • Balance centrifuge with another conical tube with equal volume of water • Label tubes • Centrifuge 50 mLconical tube at 500g for 10 minutes

20. Processing the Sample EPC Washing • The EPCs will be at the bottom of the tube

21. Processing the Sample

22. Processing the Sample EPC Washing • Carefully pour off (decant) and discard the supernatant • Do not disrupt the cells!

23. Processing the Sample • Cells at the bottom of the conical tube

24. Processing the Sample EPC Freezing • Using a clean 3 cc syringe with a 16g needle, add ~ 2 mL of thawed RPMI-FCS DMSO freezing solution to the 50 mL conical tube with the EPCs. • Store remaining RPMI-FCS DMSO solution in a refrigerator and use with next sample for this patient. Discard remainder after all samples collected.

25. Processing the Sample • This is what the thawed RPMI-FCS DMSO (freezing buffer) tubes should look like

26. Processing the Sample EPC Freezing • Resuspend EPCs by aspirating and injecting the solution 4–5 times

27. Processing the Sample • Cells are now suspended in buffer

28. Processing the Sample EPC Freezing • Aspirate the solution into the syringe. Some froth will remain in the bottom of the tube

29. Processing the Sample Label and freeze • Immediately transfer EPCs into cryovial(s). Do not add more than 1.5 mL of EPCs per vial. • Label vial(s) and place in a -80 (or -70) freezer or on dry ice.

30. Storing the Samples • Store cryo vials in a -70° or -80° freezer until all 3 specimens for the patient are obtained. If a freezer is not available, place the vial(s) on dry ice and immediately ship to the EPC Core Lab.

31. Shipping the Samples • Ship only on Monday–Thursday using the provided FedEx airbills

32. Shipping the Samples • Follow all IATA requirements for shipping of clinical specimens

33. Shipment Notification • Log on to the REVEAL Website to notify the EPC Lab of the shipment • From the Home page, select “Reveal Shipments”

34. Shipment Notification • Select your site folder. You will only have access to shipping information for your site • Select the Shipments folder within your site folder

35. Shipment Notification • To enter a new sample shipment, select “add shipment” from the toolbar

36. Shipment Notification • Enter your shipping information • Select the date and time of shipment from the drop-down menus. Time may be left blank • Site ID: Enter your 3 digit site number • Site Name: Enter your site name (e.g., Anytown Medical Center) • Operator Name: Enter your name • Operator Email: Enter your email address • Operator number:Leave blank • Courier: Select FedEx or Other • Tracking Number: Enter the tracking number provided by the overnight carrier

37. Shipment Notification • Select “Save” at the bottom of the page to save this shipment information. A new folder with the shipment date is now located in the shipments folder.

38. Shipment Notification • Next, add specific patient information to the shipment folder. You may include more than one patient in the shipment. • Select “add patient” from the toolbar.

39. Shipment Notification • Patient Number: Enter the patient’s 6-digit study ID number assigned by WebEZ at randomization • Use the checkboxes to indicate the sample types included in this shipment. In the Patient Notes section, list the number of vials for each sample type if > 1 (e.g., if there are 3 vials of plasma at baseline)

40. Shipment Notification • After you have entered all information for this patient, click “save” at the bottom of the page. • To add an additional patient to this shipment, return to the shipment folder and repeat these steps.

41. Shipment Notification • Once you have added all patients, change the “state” from ‘pending’ to ‘shipped’ by selecting ‘shipped’ from the drop down menu in the shipment folder. • The EPC Lab receives a notice to expect your shipment. • Once the EPC Lab receives your shipment, the status will change to “received” and you will receive email notification.