COLORECTAL CANCER FAMILY REGISTRY
UNIVERSITY
OF SOUTHERN CALIFORNIA NORRIS CANCER CENTER
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COLORECTAL CANCER FAMILY REGISTRY
UNIVERSITY OF SOUTHERN CALIFORNIA NORRIS CANCER CENTER
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PROTOCOL MANUAL for
HANDLING OF BIOLOGICAL SPECIMENS
BIOSPECIMEN PROCESSING
Banking Flow Chart
Biospecimen Availability
A. Peripheral blood
Flow Chart
About blood collection
Guideline for receiving blood
After the blood arrives
Summary
1. Blood Spot Preparation
2. Plasma and Buffy Coat Separation
3. Ficoll Separation
B. Frozen tissue
C. Paraffin-embedded tissue
D. Others: Buccal Smears, etc.
· Supplies
FINAL CELLULAR AND GENETIC MATERIAL PREPARATION
A. DNA
1. from fresh whole blood
2. from buffy coat
3. from dry blood stains
4. from frozen tissue
5. from paraffin-embedded tissue
6. from others (plasma, urine, buccal smear...)
B. RNA
1. from fresh whole blood
2. from buffy coat
3. from cells
C. Lymphoblastoid cell line
· Supplies
Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine Revised Nov 23, 1998 AD
24
STORAGE, PACKING AND SHIPPING
A. Storage
Location of all storage
Preventive maintenance
B. Packing and shipping
· Supplies
SPECIMEN DATABASE
A. Scheme for specimen ID assignment
B. Scheme for location assignment of biospecimens
C. Biospecimen labeling guideline
D. Biospecimen data records and report
E. Biospecimen database tables
· Supplies
QUALITY CONTROL
Summary of Routine QC for all specimens
A. Routine QC for DNA
B. Routine QC for RNA
C. Routine QC for Cell Cultures
D. Special QC for DNA
E. QC Exchange Plan
General Protocol for Lab QC and QA
· Supplies
STANDARDIZED LAB WORKSHEETS
USC COORDINATING LABORATORY SETUP
A. Resource and Environment
B. Safety Requirement
1. Biosafety
2. Radiation safety
C. Contamination Control
1. For PCR-based experiment
2. For RNA-based experiment
3. For tissue culture work
D. USC Safety Resource
REFERENCES
Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine Revised Nov 23, 1998 AD
24
BIOSPECIMEN PROCESSING
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CFR
Biospecimen Availability
(Draft modified from CFRBCS and CFRCCS)
Biospecimen products available to researchers from CFR participants
Product / Original Amount / Aliquots / Storage Condition / Available to researchers? / CommentsPlasma / 10-12mL / 18-24
@ 0.5mL each / -70°C / Yes / Nonrenewable product
Blood Spots / 44 spots of 20ul each / 2 cards of 22 spots each / Room temperature / No / Nonrenewable;
For QC and back up
DNA / 0.4-0.5mg from EDTA
more from EBV / Upon request / -20°C / Yes / Preferred product for distribution; Inexpensive
Ficoll-separated WBCs from 10mL blood / ~4 aliquots
@ 2x106 cells each / Liquid N2 / No / Source of EBV lines and non-EBV RNA; Nonrenewable product
EBV-transformed WBC / Theoretically unlimited / NA / Liquid N2 / Yes / Provides DNA and RNA;
Only renewable product in CFR.
H&E stained paraffin-embedded tissue / 2 slides per block (possible multi-blocks per case) / 2 slides @ 5 um thickness / Room temperature / No / Nonrenewable;
Upon request
Paraffin-embedded tissue on ProbeOn Plus slides / 10 sections per block (possible multi-blocks per case) / 10 slides @ 5 um thickness / Room temperature / Yes / Nonrenewable;
For immunohistochemical analyses
Paraffin-embedded tissue on regular slides / 16-26 sections per block (possible multi-blocks per case) / 16-26 slides @
5 um thickness / Room temperature / Yes / Nonrenewable;
Provides DNA.
Paraffin-embedded tissue in 1.5 ml microcentrifuge tubes / 4 sections per block (possible multi-blocks per case) / 4 sections @ 5 um thickness in 4 1.5- ml tubes unmounted on any slides / Room temperature / Yes / Nonrenewable;
Provides DNA.
Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine Revised December 30, 2002
24
A. Processing the peripheral blood
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Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine Revised Nov 23, 1998 AD
24
About blood collection
Blood will be drawn from Sunday to Thursday and shipped from Monday to Thursday. Please allow 72 hours notice for shipments that occur under special circumstances, such as shipments that will arrive on a weekend or a holiday. Any blood not received by the lab within 24 hours of it being drawn will be rendered unusable for ficoll separation. In this case, the blood will be used for plasma and buffy coat. All blood should be shipped at room temperature using the “Two-Layer Insulation Method”. If the temperature of the blood exceeds room temperature during shipping, ice packs should be used. The blood should never be in direct contact with these ice packs. If drawing an amount below the minimal request, a second draw should be set up, if feasible. Once received, the blood should be kept at room temperature until processed. All samples sent to the lab will use study ID numbers only.
Priority of collecting blood samples
The goal of the phlebotomy visit is to collect 4 tubes of blood in the following order:
-1 ACD tube (~8.5cc blood each): Shipped to Coriell by USC after ficoll separation for future establishment of cell lines.
-3 EDTA tubes (~7.5cc to 10cc blood each) Shipped to USC lab to be used for DNA extraction.
· When conducting the blood draw, always begin with filling the EDTA tubes.
· Only after all 3 EDTA tubes are filled should the ACD tube be filled.
· In all cases, filling of the EDTA tubes take precedence.
Filling the blood collection tubes
Tubes should be filled to the indicated, colored line on the manufacturer’s label, which is about ¼” below the stopper.
Time between blood draw and arrival at lab
The goal is to have blood samples arrive at the lab within 24 hours of the blood draw. Any blood not received by the lab within 24 hours of being drawn will be used for DNA isolation only, not for plasma. If the time between the blood draw and shipping is delayed, so that more than 24 hours elapse between draw and processing at the lab, a second draw should be requested if feasible.
No samples are ever to be discarded even in a blood drawing error!
Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine Revised Dec 30, 2002 AD
24
Guideline for receiving blood
Between Coordinating Centers and Processing Lab in USC
· As soon as a blood draw is scheduled, please inform immediately all lab staffs who work for this study (give at least 72 hours advance notice). They will primarily oversee and process the biological specimen for the registry.
· Once the blood is sent out, inform the lab the date and the number of subjects of the shipment.
· If the lab is to expect the shipment on a weekend or a holiday, please also include the tracking number of the shipment and sent directly to the lab.
· If no confirmation is returned from the lab about the receiving schedule, please find to inform them in person.
· After the shipment arrives, bring it to the lab at room 5427 where a section of Thanh’s bench is designated to receive samples for the registry. Unpack and leave the blood tubes standing upright in the assigned rack. Information such as the subject’s study ID, date and time (Pacific time) drawn must be provided.
· Finally, the lab should be informed promptly if there is any cancellation.
e-mail address work number emergency contact
Anh Diep (323) 865-0713 (818) 361-7503
Ehab El-Khouly (323) 865-0582 (818) 265-9431
Thanh Diep (323) 865-0583 (818) 365-8095
Evgenia Ter-Karapetova (323) 865-0714 (626) 296-2766
Lab address
(when a shipment is to be sent on a weekend or a holiday)
Anh T. Diep c/o Haile’s Lab
USC SCH of MED-Norris Cancer Center
Topping Tower 5427, 5429
1441 Eastlake Ave.
Los Angeles, CA 90033
Lab Telephone Line #1: (323) 865-0585
Lab Telephone Line #2: (323) 865-0586
Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine Revised July 8, 1999 AD
24
Blood Delivery Notification Procedure
1. As soon as a blood draw is scheduled, please inform the following people:
Email address / Work number / Emergency contactAnh Diep / / (323) 865-0713 / (818) 361-7503
Evgenia Ter-Karapetova / / (323) 865-0714 / (626) 296-2766
Thanh Diep / / (323) 865-0583 / (661) 799-9726
2. Ship the blood directly to our processing lab in USC. The attention line and the address are:
Anh Diep c/o Haile’s Lab
USC Norris Cancer Center
1441 Eastlake Ave, NOR 5427
Los Angeles, CA 90033
3. The labels for blood tubes should be preprinted with these information:
Study ID / Date DrawnTime Drawn / Time Zone / (EST, CST, PST, ...)
Initial of phlebotomist
Stick the labels on top of the writing area of the tube.
4. Once the blood draw is confirmed, email all the above three personnel using the following format:
Name of Center......
Study ID......
Drawn In...... (name of state the blood was drawn in)
Date Drawn......
FedEx Tracking #......
Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted July 11, 1999 TD
University of Southern California, Department of Preventive Medicine Revised July 8, 1999 AD
24
Special instructions for shipments that will arrive on a weekend
· Please provide 72 hours notice for shipments that will arrive on a weekend.
· Please follow the special instructions for these types of deliveries:
When filling out the FedEx forms for weekend delivery of bloods, you must:
1. Sign the air bill so shipments can be dropped off even without obtaining a signature from the lab.
2. Write a note for FedEx (next to our address on the forms) which says:
"Please do not deliver before 10:30 am (PST)."
· It is imperative that this is done because FedEx only makes one delivery attempt for a Saturday delivery, and we can't afford to miss it.
· USC observes 14 holidays in 2001 - please do not schedule bloods to arrive at USC on these days:
New Year’s Day / Monday, January 1, 2001Martin Luther King, Jr. Day / Monday, January 15, 2001
President’s Day / Monday, February 19, 2001
Memorial Day Observed / Monday, May 28, 2001
Independence Day / Wednesday, July 4, 2001
Labor Day / Monday, September 3, 2001
Thanksgiving Day / Thursday, November 22, 2001
Day after Thanksgiving Day / Friday, November 23, 2001
Christmas Eve / Monday, December 24, 2001
Christmas / Tuesday, December 25, 2001
Between Christmas and New Year’s Day / December 26, 27 and 28, 2001
New Year’s Eve (observed) / Monday, December 31, 2001
Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted July 11, 1999 TD
University of Southern California, Department of Preventive Medicine Revised July 8, 1999 AD
24
Labeling blood collection tubes
Each blood collection tube must be labeled with the following information:
Study ID
Date of draw,
Time of draw and time zone (see Time Zone Table)
Phlebotomist’s initials
Study identifier: CFR
The label must be placed vertically along the length of the tube covering the writing area of the tube so that lab will be able to view the level of the blood in the tube. Avery Labels Style 5667 (0.5” x 1.75”, height x length) is suggested for use.
All samples sent to the lab will be labeled with study ID numbers only.
Do not include any personal identifiers such as name or address.
*111111* / 01/02/01Time Drawn______EDT
Phlebotomist: 1234 / CFR
If possible, blood tube labels should be pre-printed at the data collection centers using the Excel blood collection tube label generator. These labels include the Study ID and corresponding bar code, date of draw, time of draw and time zone (see Time Zone Table), phlebotomist’s ID, and the study identifier, CFR.
In cases where the phlebotomist or the date of draw is unknown or may change, leave the respective fields blank in the label generator and the label shown at left will be generated: The date of draw and phlebotomist ID can be handwritten in just prior to the blood draw.
US Time Zone Table
The following table contains time zones in Standard and Daylight Savings Time for each center’s registry catchment’s areas. If the blood draw is done by a physician or outside phlebotomy service in another time zone, then that time zone should be recorded on the tube label.
US Center / Standard TimeThrough 3/31/01
10/28/01-4/6/02
10/27/02-4/5/03 / Daylight Savings Time
4/1/01-10/27/01
4/7/02-10/26/02
4/6/03-10/25/04
Arizona / PST / PDT
Cleveland / EST / EDT
Minnesota / CST / CDT
North Carolina / EST / EDT
Colorado / MST / MDT
Dartmouth / PST / PDT
USC / PST / PDT
Colorectal Cancer Family Registry - Coordinating Center Laboratory Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine Revised Nov 22, 2001 AD
24
Packaging the blood collection tubes for shipment
“Two-layer insulation packing method”
Supplies required:
5-tube foam carrier
Ziploc bags
Insulated foam carton
Cardboard box
Absorbent wadding material
Tape for sealing boxes
Biohazard label
Express shipping air bill
1. Blood collection tubes are placed in the 5-tube foam carrier, which is then taped shut.
2. The sealed 5-tube foam carrier is placed in a Ziploc bag with absorbent material.
3. The 5-tube carrier is then placed in the large insulated box with additional absorbent material.
4. The foam box is securely taped shut and placed in the cardboard outer box.
5. Affix a bright orange biohazard label to the cardboard outer box.
6. The cardboard box is then securely taped shut and the completed air bill is affixed.
Blood shipment temperature
All blood should be shipped at room temperature using the two-layer insulation packing method. This system plus sufficient taping is the best blood shipment method. Do not include ice packs when using this system.