1. Study Contact Information

CIBMTR Study Coordinators:

Mike Tierney

763-406-8245

Hati Kobusingye, MS, CCRP

763-406-4385

13-TLEC Laboratory Sample Manual

Version 5.0

January 2018

Page 1

  1. Sample Collection Schedule

SAMPLE TYPE / SAMPLE COLLECTION TIME POINTS
Pre-HCT / Day 30
+/- 10 days / Day 100
+/- 28 days / Day 180
+/- 45 days / 1-year
+/- 60 days / 2-year
+/- 60 days
NMDP Research Sample Repository whole blood sample (Future Genomics Research) / X
13-TLEC Study Recipient plasma samples
(Future Proteomics Research) / X / X / X / X / X
Urine Elafin samples / X / X / X / X / X / X

Note: pre-HCT assessment must occur within 30 days of the start of the conditioning regimen. If greater than 30 days, assessments must be repeated unless granted an exemption by the Principal Investigator.

13-TLEC Laboratory Sample Manual

Version 5.0

January 2018

Page 1

  1. Overview of Required Samples

This manual provides detailed instruction for the collection, processing, and shipping for the various sample types required for participation on the RCI BMT 13-TLEC/PBMTC LTE1401 protocol.

  1. SAMPLE TYPES
  1. NMDP Research SampleRepository whole blood sample (Future GenomicsResearch)

a)Collected under the National Marrow Donor Program® (NMDP) Protocol for a Research Sample Repository for Allogeneic Hematopoietic Stem Cell Transplantation and Marrow Toxic Injuries.

b)Co-enrollment on this protocol is required for participation on the RCI BMT 13-TLEC protocol.

c)Sites must also have current IRB approval for sample collection from unrelated and related donors and recipients.

d)Drawn prior to transplant and at no other time points.

  1. 13-TLEC Study Recipient plasma samples(Future Proteomics Research)

a)Blood samples for plasma aliquot storage will be collected pre-transplant (prior to the start of conditioning) and post-transplant at days +30, +100, +365 and +730.

b)Blood samples are to be collected and processed locally at the study site and may occur any day of the week.

c)Blood must beprocessed within 60 minutes of collection, and plasma sample aliquots placed into labeled cryovials and frozen as quickly as possible at ≤ -70°C.

d)Processed samples will be stored frozen at the study site and periodically batch shipped to the NMDPResearch Sample Repository in accordance with a schedule mutually determined and agreed upon by the site and CIBMTR study coordinators.

e)Monitoring of sample submission will be performed by CIBMTR study coordinators.

  1. Urine Elafin samples

a)Urine samples for elafin evaluation will be collected pre-transplant (prior to the start of conditioning), and post-transplant at days +30, +100, +180, +365 and +730.

b)Urine samples aliquots will be placed into labeled cryovials, within 60 minutes of collection, promptly frozen at -80°C, and stored until batch-shipped to project laboratory (Fred Hutchinson Cancer Research Center).

c)Urine samples will be batch shipped directly from the site to the project laboratory (Fred Hutchinson Cancer Research Center) in accordance with a schedule mutually determined and agreed upon by the site and CIBMTR study coordinators.

d)Monitoring of sample submission will be performed by CIBMTR study coordinators.

  1. NMDP Research Sample Repository whole blood sample (Future Genomics Research)

1. Recipient samples

For complete instructions on recipient research blood sample collection volumes and tubes, sample shipment, and contact information, please reference the Research Sample RepositoryCritical Facts Sheet found on the NMDP Network Website.

  1. Related Donor samples

Sites must also have IRB approval for the collection and submission of related donor samples if the transplant utilizes a related donor for a recipient enrolled on the 13-TLEC study.

  1. 13-TLEC StudyRecipient plasma samples (Future Proteomics Research)

1. KIT COMPONENTS AND SAMPLE COLLECTION

  1. Required Materials

a)Collection/Processing Supplies (shipped to sites from NMDP Research Sample Repository)

  • Cryovials
  • Cryovial Boxes
  • EDTA (ethylene-diamine-tetra-acetic) lavender top tubes (5.0 mL)

b)Collection/Processing Supplies (shipped to sites from CIBMTR)

  • Cryovial Labels
  • Pre-filled FedEx air bills

c)Dry Ice Sample Shipping Kit (shipped to sites from Inmark)

  • Biohazard bag containing absorbent
  • Insulated box(EPS foam panels)
  • Outer shipping box
  • Ice tray
  • Plastic liner bag

d)A set number of Collection/Processing Supplies, determined by estimated patient accrual at a given site, will be shipped to site upon site activation and as needed throughout studyupon site request.

e)To request additional collection/processing supplies, sites may email the CIBMTR study coordinators .

f)Shipment of Dry Ice Sample Shipping Kits will be arranged by CIBMTR study coordinators upon scheduling of batch shipment of plasma samples.

  1. Sample Collection

a)Collect patient peripheral blood samples at the following time points:

  • Prior to transplant (within 30 days of start of conditioning regimen)
  • Day 30 (+/- 7 days)
  • Day 100 (+/- 14 days)
  • 1 Year (+/- 30 days)
  • 2 Year (+/- 60 days)

b)Whenever possible, collect enough blood to result in a minimum of 7 mL plasma.

NOTE: In the event it is not possible to collect this minimum volume (e.g. for smaller subjects), any lesser volume is acceptable.

c)Follow these steps to process each tube collected:

  • Collect samples using lavender top (EDTA) tubes at each time point.
  • Keep all blood tubes at ambient temperature prior to processing.
  • Gently mix blood with EDTA by inverting the tube 8-10 times.
  • Promptly process the blood tubes within 60 minutes of collection.
  • Centrifuge the blood tube at 1000 – 1300 x g (approximately 2000 rpm) for at least 10 minutes to separate the plasma (supernatant) form the red and white blood cells.
  • Remove the plasma using caution not to disrupt the cell layer.
  • Pipette a 0.7 mL aliquot of plasma into up to 10 cryovials (maximum of 7 mL total).
  • Pipette any volume of plasma in excess of 7 mL into up to 2 additional cryovials (up to 1.8 mL per cryovial).
  • Any additional plasma remaining after aliquoting up to 10 cryovials total (as described in previous two bullet points) may be discarded.
  • Label each cryovial with the one of the provided study cryovial labels following the procedure outlined in the attachedAppendixB.(NOTE: each cryovial label corresponds to a unique sample aliquot ID. Be sure that all cryovials associated with any given patient visit are all labeled with a different sample aliquot specimen label.)
  • Store cryovials promptly at ≤ -70°C in a scientific-grade freezer until batch-shipped to the NMDP Research Sample Repository.
  1. Completion ofshipping manifest

a)The sample manifest is located inAppendix Aand also in the Laboratory Documents section on the 13-TLEC study web page.

b)The manifest must be completed in its entirety with all requested information.

c)Whenever possible,cryovial barcode labels should be scanned to populate the “Barcode Label ID” column.

d)Even though samples will be batch shipped to the NMDP Research Sample Repository, it is highly recommended that all information be added to the manifest at the time each sample is collected. This will both expedite the data entry process and ensure accuracy of the information being recorded.

2. SAMPLE PACKAGING AND SHIPPING INSTRUCTIONS

CIBMTR study coordinators will work with the site to arrange mutually agreeable dates for shipment of plasma samples to the NMDP Research Sample Repository. Frequency of shipments may vary between sites contingent upon factors such as rate of subject accrual and freezer storage capacity at each site.

  1. Batch ship samples Monday through Wednesday only, as instructed by CIBMTRstudy coordinators.
  2. Ship all frozen samples on dry ice using the approved shipping kits provided by Inmark (see previous SAMPLE COLLECTION AND KIT COMPONENTS section).
  3. IMPORTANT! Prior to shipment of samples, send a scanned, electronic copy of all sample manifests (Appendix A) corresponding to the shipment to: .
  4. Follow instructions below (aswell as outlined in Appendix C) for batch shipping of cryovial boxes of processed plasma samples.
  5. Prepare the cryovial aliquots for shipment

a)Insert the cryovials into the divider grid of the cryovial box in chronological order starting with the top left slot and moving left to right and row to row as depicted below and in the diagram on the upper right-hand corner of the shipping manifest.

b)Leave the last slot of in the lower right hand corner of each box empty. This will provide “orientation” for the labs so they will know where the first sample in the box is.

c)Number the cryovial boxes and record on the shipping manifest which cryovial box the manifest corresponds to.

d)Put the top on the box.

e)Place the cryovial box in the biohazard bag containing absorbent.

f)IMPORTANT: Bleed as much air as possible out of the bag prior to sealing.

g)Peel the tape from the top of the biohazard bag.

h)To seal the bag, fold the top of the bag over AT THE SLIT and if present orient lines onto corresponding lines. GENTLY tack together, working outward from the center. PRESS HARD from the CENTER working outward, making sure the bag is completely sealed.

i)Repeat all of above steps with any remaining cryovial box(es).

  1. Stack bagged and sealed cryovial box(es) in a corner of the insulated box. Fill the cavity to the top of the bagged cryovial box(es) with dry ice (gently shaking the box so pelletized dry ice fills the corners of the cavity).
  2. Fold the sides of the ice tray UP. Slip the ice tray into the box until it is resting on top of the cryovial box(es). Fill try with dry ice and continue filling until cavity is filled within 1-1/2” of the top.
  3. Place a piece of white EPS foam on the top of the dry ice. It must be flush with, or below, the side of the EPS foam.
  4. IMPORTANT: be sure to include paper copies of all sample manifests corresponding to the shipment inside the shipping box on top of the white EPS foam used as the lid.
  5. LOOSELY fold the liner bag on top of the EPS foam lid. Do NOT seal it. Gas from the dry ice must be able to escape.
  6. Close the inner and outer box flaps. Use a 2” tape strip to seal the top seam of the box. Make sure the seam if fully sealed across the top with excess tape running down the sides.
  7. Use the 2” tape strips to seal both side seams making sure that half the tape in on the top of the box and the other half is on the side of the box.
  8. Fill in Dry Ice Label with the amount of dry ice used. Without covering any markings on the box, apply the label to the panel bearing the UN marking.
  9. Again without covering any other markings, attach the supplied “Biological Substance, Category B” label to the box.
  10. Attach the shipping address sticker to the carton.
  1. Confirm the following shipment information is pre-filled on the Fed Ex air bill to ensure the correct one is being used:

Jon Van Hulzen

NMDP Research Sample Repository

National Marrow Donor Program

711 5th Street SW

Suite 6

New Brighton, MN 55112

Phone: 651-746-5008

  1. CONTACT INFORMATION(FOR QUESTIONS REGARDING SAMPLE COLLECTION, PROCESSING OR SHIPMENT OF PLASMA SAMPES)

Mike Tierney

763-406-8245

Hati Kobusingye, MS, CCRP

763-406-4385

13-TLEC Laboratory Sample Manual

Version 5.0

January 2018

Page 1

  1. Urine Elafin samples

1. KIT COMPONENTS AND SAMPLE COLLECTION

  1. Required Materials

a)Collection/Processing Supplies (shipped to sites from NMDP Research Sample Repository)

  • Cryovials
  • Cryovial Boxes
  • Urine collection cups

b)Collection/Processing Supplies (shipped to sites from CIBMTR)

  • Cryovial Labels
  • Pre-filled FedEx air bills

c)Dry Ice Sample Shipping Kit (shipped to sites from Inmark)

  • Biohazard bag containing absorbent
  • Insulated box (EPS foam panels)
  • Outer shipping box
  • Ice tray
  • Plastic liner bag

d)A set number of Collection/Processing Supplies, determined by estimated patient accrual at a given site, will be shipped to site upon site activation and as needed throughout study upon site request.

e)To request additional collection/processing supplies, sites may email the CIBMTR study coordinators or .

f)Shipment of Dry Ice Sample Shipping Kits will be arranged by CIBMTR study coordinators upon scheduling of batch shipment of urine samples.

B. Sample Collection

a)Collect patient urine samples at the following time points:

  • Prior to transplant (within 30 days of start of conditioning regimen)
  • Day 30 (+/- 7 days)
  • Day 100 (+/- 14 days)
  • Day 180 (+/- 21 days)
  • 1 Year (+/- 30 days)
  • 2 Year (+/- 60 days)

b)Collect 8 -10 mL at each time point.

NOTE: Collection of this volume is crucial so as not to result in exclusion of patients from some analyses. In the event it is not possible to collect this volume, any lesser volume is acceptable. However, this must be reported as a protocol deviation.

c)Follow these steps to process each urine sample collected:

  • Preferably, collection should occur in the morning between the hours of 8 and 10 AM.
  • Collect urine in the collection cup provided and immediatelyplace on ice.
  • Process urine within one hour of collection.
  • Affix a unique pre-printed sample ID label to each cryovial prior to aliquoting. (NOTE: each label corresponds to a unique patient sample ID. Be sure that all cryovials associated with any given patient visit are all labeled with a unique sample aliquot ID.)
  • Using a transfer pipet, aliquot approximately 1.8 mL urine into a maximum of six2 mL cryovials.
  • Store samples for batch shipment in -80°C freezer until ready to batch ship to the project laboratory at Fred Hutchinson Cancer Research Center.

C. Completion of shipping manifest

a) The sample manifest is located in Appendix A and also in the Laboratory Documents section on the 13-TLEC study web page.

b) The manifest must be completed in its entirety with all requested information.

c) Whenever possible, cryovial barcode labels should be scanned to populate the “Barcode Label ID” column.

d) Even though samples will be batch shipped to the NMDP Research Sample Repository, it is highly recommended that all information be added to the manifest at the time each sample is collected. This will both expedite the data entry process and ensure accuracy of the information being recorded.

2. SAMPLE PACKAGING AND SHIPPING INSTRUCTIONS

CIBMTR study coordinators will work with the site to arrange mutually agreeable dates for shipment of plasma samples to the NMDP Research Sample Repository. Frequency of shipments may vary between sites contingent upon factors such as rate of subject accrual and freezer storage capacity at each site.

  1. Batch ship samples Monday through Wednesday only, as instructed by CIBMTR study coordinators.
  2. Ship all frozen samples on dry ice using the approved shipping kits provided by Inmark (see previous SAMPLE COLLECTION AND KIT COMPONENTS section).
  3. IMPORTANT! Prior to shipment of samples, send a scanned, electronic copy of all sample manifests (Appendix A) corresponding to the shipment to: .
  4. Follow instructions below (as well as outlined in Appendix C) for batch shipping of cryovial boxes of processed urine samples.
  5. Prepare the cryovial aliquots for shipment

a) Insert the cryovials into the divider grid of the cryovial box in chronological order starting with the top left slot and moving left to right and row to row as depicted below and in the diagram on the upper right-hand corner of the shipping manifest.

b) Leave the last slot of in the lower right hand corner of each box empty. This will provide “orientation” for the labs so they will know where the first sample in the box is.

c) Number the cryovial boxes and record on the shipping manifest which cryovial box the manifest corresponds to.

d) Put the top on the box.

e) Place the cryovial box in the biohazard bag containing absorbent.

f) IMPORTANT: Bleed as much air as possible out of the bag prior to sealing.

g) Peel the tape from the top of the biohazard bag.

h) To seal the bag, fold the top of the bag over AT THE SLIT and if present orient lines onto corresponding lines. GENTLY tack together, working outward from the center. PRESS HARD from the CENTER working outward, making sure the bag is completely sealed.

i) Repeat all of above steps with any remaining cryovial box(es).

  1. Stack bagged and sealed cryovial box(es) in a corner of the insulated box. Fill the cavity to the top of the bagged cryovial box(es) with dry ice (gently shaking the box so pelletized dry ice fills the corners of the cavity).
  2. Fold the sides of the ice tray UP. Slip the ice tray into the box until it is resting on top of the cryovial box(es). Fill try with dry ice and continue filling until cavity is filled within 1-1/2” of the top.
  3. Place a piece of white EPS foam on the top of the dry ice. It must be flush with, or below, the side of the EPS foam.
  4. IMPORTANT: be sure to include paper copies of all sample manifests corresponding to the shipment inside the shipping box on top of the white EPS foam used as the lid.
  5. LOOSELY fold the liner bag on top of the EPS foam lid. Do NOT seal it. Gas from the dry ice must be able to escape.
  6. Close the inner and outer box flaps. Use a 2” tape strip to seal the top seam of the box. Make sure the seam if fully sealed across the top with excess tape running down the sides.
  7. Use the 2” tape strips to seal both side seams making sure that half the tape in on the top of the box and the other half is on the side of the box.
  8. Fill in Dry Ice Label with the amount of dry ice used. Without covering any markings on the box, apply the label to the panel bearing the UN marking.
  9. Again without covering any other markings, attach the supplied “Biological Substance, Category B” label to the box.
  10. Attach the shipping address sticker to the carton.
  11. Confirm the following shipment information is pre-filled on the Fed Ex air bill to ensure the correct one is being used:

Fred Hutchinson Cancer Research Center

1100 Fairview Ave N. D2-281

Seattle Washington, 98109

ATTENTION: Emily Pao