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

Endothelial Progenitor Cell (EPC)Endothelial Progenitor Cell (EPC)Core Laboratory InstructionsCore Laboratory Instructions

Version 5.0 March 29, 2008Version 5.0 March 29, 2008

For questions regarding collection, processing, For questions regarding collection, processing, storage, or shipping of EPC core lab samples, storage, or shipping of EPC core lab samples, contact contact

Dr. Tom PovsicDr. Tom Povsic ((919) 681-XXXX (M–F, 8–5 ET)919) 681-XXXX (M–F, 8–5 ET)

(919) 970-xxxx (pager)(919) 970-xxxx (pager)

Equipment and SuppliesEquipment and SuppliesEquipment and SuppliesEquipment 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)

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)

Obtain venous blood in an 8 mL cell Obtain venous blood in an 8 mL cell preparation tube (CPT). preparation tube (CPT). A minimum of 5 mL A minimum of 5 mL blood is requiredblood is required

Gently invert tube 8Gently invert tube 8––10 times10 times

Maintain at room temperature Maintain at room temperature

Process within 2 hours Process within 2 hours (sooner is better)(sooner is better)

Collecting the SampleCollecting the SampleCollecting the SampleCollecting the Sample

If this is the baseline sample, If this is the baseline sample, remove RPMI-FCS DMSO remove RPMI-FCS DMSO solution (freezing buffer) from solution (freezing buffer) from the freezer to thaw. the freezer to thaw.

At room temperature it will thaw At room temperature it will thaw in 20–30 min. in 20–30 min.

Store remainder of DSMO Store remainder of DSMO solution in refrigerator until all solution in refrigerator until all three samples are processed.three samples are processed.

If this is the baseline sample, If this is the baseline sample, remove RPMI-FCS DMSO remove RPMI-FCS DMSO solution (freezing buffer) from solution (freezing buffer) from the freezer to thaw. the freezer to thaw.

At room temperature it will thaw At room temperature it will thaw in 20–30 min. in 20–30 min.

Store remainder of DSMO Store remainder of DSMO solution in refrigerator until all solution in refrigerator until all three samples are processed.three samples are processed.

Processing the SampleProcessing the SampleProcessing the SampleProcessing the Sample

Full CPT tubes before

spin

Full CPT tubes before

spin


Separate the plasmaSeparate the plasma Invert CPT 8–10 times Invert CPT 8–10 times

before centrifuging at before centrifuging at 1500g for 30 minutes1500g for 30 minutes


Plasma

Buffy Coat

Red Cells

Processing the Sample—After SpinProcessing the Sample—After SpinProcessing the Sample—After SpinProcessing the Sample—After Spin

Polyester Gel

Plasma

Buffy Coat

Red Cells

Polyester Gel

Processing the Sample—After SpinProcessing the Sample—After SpinProcessing the Sample—After SpinProcessing the Sample—After Spin

3 cc syringe and 16g needle


~ ¼ inch

Processing the SampleProcessing the SampleProcessing the SampleProcessing the SampleRemove the plasmaRemove the plasma Carefully remove ~ ½ of the plasma (top Carefully remove ~ ½ of the plasma (top

yellowish layer) using a 3 cc syringe with 16g yellowish layer) using a 3 cc syringe with 16g needle. needle.

Do not disturb the Do not disturb the buffy coat layer.buffy coat layer.

Polyester gelPolyester gel


Remove the plasmaRemove the plasma If consent signed for plasma If consent signed for plasma

storage, divide plasma evenly storage, divide plasma evenly into cryo vials (no more than 1.5 into cryo vials (no more than 1.5 mL per vial). Label each vial.mL per vial). Label each vial.

If no consent for plasma storage, If no consent for plasma storage, discard the plasma according to discard the plasma according to your institutional guidelines for your institutional guidelines for disposal of blood and body fluid.disposal of blood and body fluid.

EPC BL 24h 48 h Cells Plasma Patient Study Number: __ __ __ - __ __ __ Patient Initials: __ __ __ Date of Collection: __ __ / __ __ / __ __ Time of Collection: __ __ : __ __ (24 hr clock)

Plasma

Buffy Coat

Red Cells

Polyester Gel


Plasma in cryovial

Do not add more than 1.5 mL per vial

Place label on each vial

Plasma in cryovial

Do not add more than 1.5 mL per vial

Place label on each vial




EPC WashingEPC Washing Measure ~ 40 mL of PBS into a 50 mL Measure ~ 40 mL of PBS into a 50 mL

conical tubeconical tube

Processing the SampleProcessing the SampleProcessing the SampleProcessing the SampleRemove the buffy coatRemove the buffy coat Use the same needle/syringe to Use the same needle/syringe to

remove the buffy coat layer and remove the buffy coat layer and remaining plasma. Place the remaining plasma. Place the needle in the middle of the buffy needle in the middle of the buffy coat layer, being careful not to coat layer, being careful not to touch the polyester gel layer. touch the polyester gel layer.

Carefully aspirate the buffy coat Carefully aspirate the buffy coat and remaining plasma. and remaining plasma.

Discard the CPT and red blood Discard the CPT and red blood cells cells

CPT tube after removal CPT tube after removal of buffy coatof buffy coat

Discard according to Discard according to your institutional your institutional guidelines for disposal guidelines for disposal of blood and body fluid of blood and body fluid

CPT tube after removal CPT tube after removal of buffy coatof buffy coat

Discard according to Discard according to your institutional your institutional guidelines for disposal guidelines for disposal of blood and body fluid of blood and body fluid



EPC WashingEPC Washing Inject the buffy coat into Inject the buffy coat into

the 50 mL conical the 50 mL conical tube containing the PBStube containing the PBS

Cap the tube and invert Cap the tube and invert 2 times2 times

50 mL conical tube with 50 mL conical tube with cells and wash buffercells and wash buffer

50 mL conical tube with 50 mL conical tube with cells and wash buffercells and wash buffer



EPC WashingEPC Washing Balance centrifuge with another conical tube Balance centrifuge with another conical tube

with equal volume of water with equal volume of water

Label tubesLabel tubes

Centrifuge 50 mLCentrifuge 50 mLconical tube at 500g conical tube at 500g for 10 minutesfor 10 minutes


EPC WashingEPC Washing The EPCs will be at the bottom of the tubeThe EPCs will be at the bottom of the tube

Processing the SampleProcessing the SampleProcessing the SampleProcessing the SampleEPC WashingEPC Washing Carefully pour off (decant) and discard the Carefully pour off (decant) and discard the

supernatantsupernatant Do not disrupt the cells!Do not disrupt the cells!

Cells at the bottom of the conical tube

Cells at the bottom of the conical tube


Processing the SampleProcessing the SampleProcessing the SampleProcessing the SampleEPC FreezingEPC Freezing Using a clean 3 cc syringe with a 16g needle, add Using a clean 3 cc syringe with a 16g needle, add

~ 2 mL of thawed RPMI-FCS DMSO freezing solution ~ 2 mL of thawed RPMI-FCS DMSO freezing solution to the 50 mL conical tube with the EPCs. to the 50 mL conical tube with the EPCs.

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

This is what the thawed thawed RPMI-FCS DMSORPMI-FCS DMSO (freezing buffer) tubes should look like

This is what the thawed thawed RPMI-FCS DMSORPMI-FCS DMSO (freezing buffer) tubes should look like



EPC FreezingEPC Freezing Resuspend EPCs by aspirating and injecting Resuspend EPCs by aspirating and injecting

the solution 4–5 timesthe solution 4–5 times

Cells are now suspended in buffer

Cells are now suspended in buffer



EPC FreezingEPC Freezing Aspirate the solution into the syringe. Some Aspirate the solution into the syringe. Some

froth will remain in the bottom of the tubefroth will remain in the bottom of the tube

Label and freezeLabel and freeze Immediately transfer EPCs into cryovial(s). Do Immediately transfer EPCs into cryovial(s). Do

not add more than 1.5 mL of EPCs per vial.not add more than 1.5 mL of EPCs per vial. Label vial(s) and place in a -80 (or -70) freezer or Label vial(s) and place in a -80 (or -70) freezer or

on dry ice.on dry ice.



Storing the SamplesStoring the SamplesStoring the SamplesStoring 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.

Shipping the SamplesShipping the Samples

Ship only on Monday–Thursday using the provided FedEx airbills

Follow all IATA requirements for shipping of clinical specimens

Shipping the SamplesShipping the Samples

Shipment NotificationShipment Notification

Log on to the REVEAL Website to notify the EPC Lab of the shipment

From the Home page, select “Reveal Shipments”

Select your site folder. You will only have access to shipping information for your site

Select the Shipments folder within your site folder

Select your site folder. You will only have access to shipping information for your site

Select the Shipments folder within your site folder


To enter a new sample shipment, select To enter a new sample shipment, select “add shipment” from the toolbar“add shipment” from the toolbar


Enter your shipping informationEnter your shipping information Select the date and time of shipment from the Select the date and time of shipment from the

drop-down menus. Time may be left blankdrop-down menus. Time may be left blank Site ID: Enter your 3 digit site numberSite ID: Enter your 3 digit site number Site Name: Enter your site name (e.g., Anytown Site Name: Enter your site name (e.g., Anytown

Medical Center)Medical Center) Operator Name: Enter your nameOperator Name: Enter your name Operator Email: Enter your email address Operator Email: Enter your email address Operator number:Operator number: Leave blankLeave blank Courier: Select FedEx or Other Courier: Select FedEx or Other Tracking Number: Enter the tracking number Tracking Number: Enter the tracking number

provided by the overnight carrierprovided by the overnight carrier


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


Next, add specific patient information to the Next, add specific patient information to the shipment folder. You may include more than shipment folder. You may include more than one patient in the shipment.one patient in the shipment.

Select “add patient” from the toolbar.Select “add patient” from the toolbar.


Patient Number: Enter the patient’s 6-digit study Patient Number: Enter the patient’s 6-digit study ID number assigned by WebEZ at randomizationID number assigned by WebEZ at randomization

Use the checkboxes to indicate the sample Use the checkboxes to indicate the sample types included in this shipment. In the Patient types included in this shipment. In the Patient Notes section, list the number of vials for each Notes section, list the number of vials for each sample type if > 1 (e.g., if there are 3 vials of sample type if > 1 (e.g., if there are 3 vials of plasma at baseline)plasma at baseline)


After you have entered all information for this After you have entered all information for this patient, click “save” at the bottom of the page.patient, click “save” at the bottom of the page.

To add an additional patient to this shipment, To add an additional patient to this shipment, return to the shipment folder and repeat these return to the shipment folder and repeat these steps.steps.


Once you have added all patients, change the Once you have added all patients, change the “state” from ‘pending’ to ‘shipped’ by selecting “state” from ‘pending’ to ‘shipped’ by selecting ‘shipped’ from the drop down menu in the ‘shipped’ from the drop down menu in the shipment folder.shipment folder.

The EPC Lab receives a notice to expect your The EPC Lab receives a notice to expect your shipment.shipment.

Once the EPC Lab receives your shipment, the Once the EPC Lab receives your shipment, the status will change to “received” and you will status will change to “received” and you will receive email notification.receive email notification.


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

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