STANDARD OPERATING PROCEDURE FOR SAMPLE COLLECTION AND
TRANSPORTATION
Prepared by Aung Myat Oo and Shanthi Wasser
Please read the complete SOP, especially the safety considerations, before
starting on the experiments. Ensure that all reagents and equipment are available
in sufficient quantities before starting experiment.
General safety considerations
Human tissue collected from the Operating Theatre may contain HBV which is a BSL2
agent. However, HBV is not transmitted by the aerosol route and there is an effective
vaccine against it. All personnel involved in this project (including NUH Operating
Theatre and housekeeping personnel) will be prophylactically vaccinated against HepB.
Antibody status will be checked every 5 years and staff sent for re-vaccination/booster
shots if required.
HepB waste: All consumables that come into contact with the human tissue will be
decontaminated in 10% bleach. As per Public Health Agency of Canada's MSDS
(http://www.phac-aspc.gc.ca/msds-ftss/msds76e.html), HBV can be decontaminated with
1% sodium hypochlorite or 70% ethanol. Alternatively, decontamination can be carried
out with 1% VirKon®S solution. Refer to SOP on Biological Spill Cleanup for detailed
procedures.
Biohazard waste at Dept of Medicine is disposed of by NUH contract service provider.
A. Instruments Checklist & Preparation










2 plastic cryotubes for snap frozen HCC tissues (tumor and surrounding nonaffecting area).
2 plastic cryotubes 2/3 filled with William’s E solution for carrying HCC tissues
(T & N) for subsequent processing to get OCT and paraffin blocks.
2 large plastic tubes 2/3 filled with William’s E solution for carrying HCC tissues
(T & N) for subsequent processing of hepatocytes cell culture.
2 blood tubes (plain and heparin).
Styrofoam box (Secondary container) for carrying liquid nitrogen container and
ice box.
Liquid nitrogen container (1.5 liters).
Small Styrofoam cup to be used in snap freezing procedure. Long forceps.
All plastic tubes and plastic cryotubes containing William’s E solution to be
placed in the ice box filled with ice.
2 blood tubes placed in Blood tube carrier.
Liquid nitrogen filled into liquid nitrogen container to a maximum of 2/3 volume
of container. Place container into carrying Styrofoam carrying box.
1
B. Collection







OT coat, mask, gloves and cap are to be worn before entering operating theatre.
Patient consent form and Id sticky label collected from Theatre staff.
Patient blood collected into 2 blood tubes by theatre nurse, placed on rack in
Blood tube carrier.
Liver tissues to be cut by pathologist in theatre.
Large pieces of liver tissues (tumor and surrounding non-affecting area) for
subsequent processing of hepatocytes cell culture are placed into 2 large plastic
tubes 2/3 filled with William’s E solution. Place on ice box for transport to lab.
2 small pieces of liver tissues (tumor and surrounding non-affecting area) for
subsequent processing to get OCT and paraffin blocks are placed into 2 small
plastic cryotubes 2/3 filled with William’s E solution. Place in ice box for
transport to lab.
Liver tumor and non-affecting surrounding liver tissues are cut into 0.5mm3 small
pieces by pathologist. Small pieces of tissues are picked up with long forceps and
snap frozen in liquid nitrogen. Snap frozen tissues are collected in plastic
cryotubes and placed in liquid nitrogen container for transport.
SAFETY MEASURES: Before preparation procedure, wear gloves, lab coat (long
sleeve) and safety glasses. When moving between floors, use the staircase, never use
elevator. This is so that in case of accidental spills, the nitrogen is not trapped in an
enclosed area, thereby displacing air and causing asphyxiation of the occupants of the lift.
In case of accidental spillage of liquid nitrogen, evacuate the area immediately, prohibit
anyone from entering the area and call for safety officer. Safety Officer is to report all
accidents/incidents on OSHE AIRS.
In case of skin contact: Do not rub frozen parts as tissue damage may result. As soon as
practical, place the affected area in a warm water bath which has a temperature of not
more than 40°C. Call a physician or go to NUH A&E as soon as possible.
2. PROCESSING AND STORING
A. Instruments Checklist










Small aluminum foil cups (8 numbers).
2 plastic caskets
10% formalin filled small bottle.
2 plastic cryotubes for storage of OCT tissue block.
OCT compound.
Small plastic cup.
Large Styrofoam cup.
Isopentane bottle.
Surgical blade.
Long forceps.
2


Serum tubes.
Permanent marker
B. Serum and plasma preparation and storing








Wear glove, lab coat (long sleeve) and eye protective gear.
Put the blood at 37° for 30-60 min to clot.
Invert the tube to pulls free the clot from the walls of the tube.
Set the tubes at 4° overnight to allow the clot to contract.
Decant the serum away from the clot into a new centrifuge tube.
Spin the serum 3,000 RPM for 10 min to remove the remaining clots/red blood
cells and other insoluble material.
Aliquots the serum into 1.5 ml serum tubes, label, record the number of tubes and
store in serum boxes in -80° freezer.
Inventorise the number of serum tubes, date of collection, patient id number and
patient consent form number.
SAFETY MEASURE: Before preparation procedure, wear gloves, lab coat (long sleeve)
and eye protective gear (safety glasses). Transfer of blood products and aliquoting of
serum are to be done in the Biosafety Cabinet. All materials in contact with or
contaminated by blood are disposed of into designated biohazard containers. All
equipment and work surfaces were disinfected after use with freshly prepared bleach
diluted 1:10 with water or with 1% VirKon solution.
Spill of blood: Please refer to SOP on Biological Spill Cleanup.
Accidental cut or stab with items contaminated with blood: Wash the wound with soap
and running water immediately and continue for at least 10 minutes. Squeeze or scrub the
wound to induce bleeding if possible. Report and get medical assistance.
For eye exposures, flush eyes under eyewash for at least 10 minutes. Report and get
medical assistance.
C. OCT and tissue preparing and storing






Wear gloves, lab coat (long sleeve) and eye protective gear (safety glasses).
All procedures are to be carried out in the Biological Safety Cabinet.
2 small pieces of liver tissues (tumor and surrounding non-affecting area) carried
in William E solutions are cut into 5 pieces each.
1 piece each of tumor and surrounding non-affecting area are placed in a plastic
gasket and placed into 10% formaldehyde bottle (screw capped) over night and
transfer to ethanol bottle.
Place isopentane into small plastic cup and place into nitrogen filled Styrofoam
cup. Wait till the lower part of isopentane is frozen.
4 pieces each of tumor and surrounding non-affecting area are placed in small
aluminum foil cups covered with OCT compound.
3





Slowly freeze tissues in OCT compound in isopentane.
Place OCT blocks separately in plastic cryotubes labelled tumor and non-tumor
Label the tubes and store in the -80° freezer.
Snap frozen tissues in plastic cryotubes are taken out from liquid nitrogen
container and stored at -80° freezer
Inventorise the number of tissues block, date of collection, patient id number and
patient consent form number.
SAFETY MEASURE: Before preparation procedure, wear gloves, lab coat (long sleeve)
and eye protective gear (safety glasses). All processes of tissue cutting and processing
procedures are to be carried out in the biological safety cabinet. All materials in contact
with or contaminated with tissue were disposed of into designated biohazard containers.
All equipment and work surfaces were disinfected after use with freshly diluted 10%
bleach solution or 1% VirKon solution.
Accidental cut or stab with items contaminated with blood: Wash the wound with soap
and running water immediately and continue for at least 10 minutes. Squeeze or scrub the
wound to induce bleeding if possible. Report and get medical assistance.
For eye exposures, flush eyes under eyewash for at least 10 minutes. Report and get
medical assistance.
4