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Endothelial Progenitor Cell (EPC) Core Laboratory Instructions Version 5.0 March 29, 2008 For questions regarding collection, processing, storage, or shipping.

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Presentation on theme: "Endothelial Progenitor Cell (EPC) Core Laboratory Instructions Version 5.0 March 29, 2008 For questions regarding collection, processing, storage, or shipping."— Presentation transcript:

1 Endothelial Progenitor Cell (EPC) Core Laboratory Instructions Version 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 n 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 ) n 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) n 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 ) n 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 n Obtain venous blood in an 8 mL cell preparation tube (CPT). A minimum of 5 mL blood is required n Gently invert tube 8–10 times n Maintain at room temperature n Process within 2 hours (sooner is better) Collecting the Sample

4 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. 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. Processing the Sample

5 Full CPT tubes before spin Processing the Sample

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

7 Plasma Buffy Coat Red Cells Processing the SampleAfter Spin Polyester Gel

8 Plasma Buffy Coat Red Cells Polyester Gel Processing the SampleAfter Spin

9 3 cc syringe and 16g needle Processing the Sample

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

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

12 Plasma Buffy Coat Red Cells Polyester Gel Processing the Sample

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

14 EPC Washing n Measure ~ 40 mL of PBS into a 50 mL conical tube

15 Processing the Sample Remove the buffy coat n 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. n Carefully aspirate the buffy coat and remaining plasma. n Discard the CPT and red blood cells

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

17 EPC Washing n Inject the buffy coat into the 50 mL conical tube containing the PBS n Cap the tube and invert 2 times

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

19 EPC Washing n Balance centrifuge with another conical tube with equal volume of water n Label tubes n Centrifuge 50 mL conical tube at 500g for 10 minutes

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

21 Processing the Sample

22 EPC Washing n Carefully pour off (decant) and discard the supernatant n Do not disrupt the cells!

23 n Cells at the bottom of the conical tube Processing the Sample

24 EPC Freezing n 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. n Store remaining RPMI-FCS DMSO solution in a refrigerator and use with next sample for this patient. Discard remainder after all samples collected.

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

26 EPC Freezing n Resuspend EPCs by aspirating and injecting the solution 4–5 times

27 n Cells are now suspended in buffer Processing the Sample

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

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

30 Storing the Samples n 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 n n Ship only on Monday–Thursday using the provided FedEx airbills

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

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

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

35 n To enter a new sample shipment, select add shipment from the toolbar Shipment Notification

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

37 n 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. Shipment Notification

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

39 n Patient Number: Enter the patients 6-digit study ID number assigned by WebEZ at randomization n 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) Shipment Notification

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

41 n 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. n The EPC Lab receives a notice to expect your shipment. n Once the EPC Lab receives your shipment, the status will change to received and you will receive email notification. Shipment Notification


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