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, contactDr. 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 supplies10 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
3 Collecting the SampleObtain venous blood in an 8 mL cell preparation tube (CPT). A minimum of 5 mL blood is requiredGently invert tube 8–10 timesMaintain at room temperatureProcess within 2 hours (sooner is better)
4 Processing the SampleIf 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 Full CPT tubes before spin Processing the SampleFull 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 PlasmaBuffy CoatPolyester GelRed Cells
8 Processing the Sample—After Spin PlasmaBuffy CoatPolyester GelRed Cells
9 Processing the Sample3 cc syringe and 16g needle
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 SamplePlasmaBuffy CoatPolyester GelRed Cells
13 Processing the Sample Plasma in cryovial Do not add more than 1.5 mL per vialPlace 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 PBSCap the tube and invert 2 times
18 Processing the Sample50 mL conical tube with cells and wash buffer
19 Processing the Sample EPC Washing Balance centrifuge with another conical tube with equal volume of waterLabel tubesCentrifuge 50 mL conical tube at 500g for 10 minutes
20 Processing the Sample EPC Washing The EPCs will be at the bottom of the tube
22 Processing the Sample EPC Washing Carefully pour off (decant) and discard the supernatantDo not disrupt the cells!
23 Processing the SampleCells 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 SampleThis 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 SampleCells 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 SamplesStore 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 SamplesShip only on Monday–Thursday using the provided FedEx airbills
32 Shipping the SamplesFollow all IATA requirements for shipping of clinical specimens
33 Shipment Notification Log on to the REVEAL Website to notify the EPC Lab of the shipmentFrom the Home page, select “Reveal Shipments”
34 Shipment Notification Select your site folder. You will only have access to shipping information for your siteSelect 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 informationSelect the date and time of shipment from the drop-down menus. Time may be left blankSite ID: Enter your 3 digit site numberSite Name: Enter your site name (e.g., Anytown Medical Center)Operator Name: Enter your nameOperator Enter your addressOperator number: Leave blankCourier: Select FedEx or OtherTracking 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 randomizationUse 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 notification.