Supplemental Information to Manuscript Submitted to Modern Pathology_revised
Gerald Batist
Detailed procedure for liver NCB procurement and processing for study Q-CROC-01
Liver NCB Procurement
Percutaneous ultrasound guided biopsies were performed as per institutional guidelines by a
staff interventional radiologist or interventional radiology fellow under direct supervision.
The approach (anterior, lateral, anterolateral, intercostal or sub costal) was determined by the
site of the lesion. Ultrasound settings were adjusted to optimally demonstrate the lesion and
anticipated needle tract, mostly using the tissue harmonic imaging mode. Conscious sedation
was used as required and typically consisted of intravenous midazolam administered as per
institutional guidelines. After sterile draping of the entry site, local anaesthetic (1% lidocaine)
was applied to the prospective biopsy tract with particular emphasis on skin, peritoneum and
liver capsule. The biopsy was performed during suspended respiration to avoid liver capsule
lacerations. Only lesions deemed by the interventional radiologist as safely accessible and
sufficiently large in size were biopsied, and the stroke length of the biopsy needle was
adjusted accordingly. The interventional radiologist specifically aimed at including 100%
tumor tissue in the specimen. Three tumour tissue cores were removed using primarily a 16G
or 18G BioPince™ end-cutting biopsy device. Cores were placed on sterile saline-soaked
gauze. Using sterile and RNAse free tweezers, two cores were placed in cryovials containing
RNAlater solutions. Cryovials were gently inverted to ensure completely immersion into the
solution. A third core was placed in formalin. Samples were shipped immediately to a Central
Pathology Laboratory at 4oC for processing. Post-biopsy procedure, patients were transferred
to the recovery area and monitored for 4-6 hours by nursing staff.
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Supplemental Information to Manuscript Submitted to Modern Pathology_revised
Gerald Batist
NCB Processing
1x PBS was prepared fresh by diluting 10x PBS (Fisher) with RNAse-free DEPC water
(Ambion) and placed on dry ice to maintain the solution as cold as possible. RNAlater
solution (Qiagen) was removed from the cryovial containing the biopsy by pipetting the
solution into a petri dish in case any part of the biopsy was inadvertently pipetted, and 1mL
of ice-cold PBS was immediately added. The vial was placed on dry ice and gently rolled to
allow the whole biopsy to be in contact with the PBS. Care was taken to avoid freezing by
removing the vial occasionally from the dry ice, alternating between gentle inversion and
rolling on dry ice. The wash was performed for two minutes and repeated, for a total of two
PBS washes. The PBS was removed between washes by pipetting out the solution into a petri
dish.
To embed the biopsy in OCT, RNAse free tweezers were used to remove the biopsy from the
cryovial containing PBS and place it in the center of 15 mm x 15 mm mm Tissue Tek
disposable cryomold (Somagen). OCT compound (Surgipath) was gently poured in the
cryomold until the specimen was completely covered. Care was taken when pouring the OCT
so that no bubbles were formed and the biopsy remained flat at the bottom of the cryomold in
an elongated position. The cryomold was gently submerged for 30 seconds into a beaker
containing 2-Methyl butane (Fisher Scientific) pre-cooled on dry ice, using tweezers to hold
the cryomold to ensure that the sample remained horizontal. After the OCT solidified, the
OCT block was immediately stored at -80oC. Preparation of tissue cryosections was
performed using standard pathology laboratory procedures. Briefly, OCT blocks were placed
for at least 30 minutes inside the cryostat set at -20oC prior to cutting to ensure the block’s
temperature was optimal for cryosectioning. Using a new clean cryostat blade, tweezers and
brushes, sections 4-5  thick were cut and put on a SuperFrost glass slide (Fisher). Slides
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Supplemental Information to Manuscript Submitted to Modern Pathology_revised
Gerald Batist
were stained with hematoxilin and eosin and submitted to a designated pathologist for
histological assessment.
Isolation of genomic material
All procedures were conducted in an RNAse free environment. A 2 mL mortar and pestle
(Wheaton Tissue Grinder Tenbro CS-2 2ML) were soaked for at least 30 minutes with
RNAse away, rinsed with ethanol, followed by distilled water and placed on ice. The OCT
blocks were placed on RNAse free petri dishes with the biopsy surface facing up. If
macrodissection was recommended by the pathologist as marked on the H&E slide, it
performed on dry ice with a RNAse free sterile blade, using the H&E slide as a reference to
cut the OCT block in the region delineated by the pathologist (area containing neoplastic cells
with little stroma or necrosis). Only the macrodissected tissue containing the area of interest
was then used to isolate genomic material. RNAlater (approximately 1 mL) was dispensed on
the blocks ensuring that the biopsy surface was continuously covered with RNAlater until the
OCT softened. Using clean tweezers and/or the end of a sterile and RNAse/DNAse-free
pipette tip, the biopsy tissue was gently separated from the OCT and immediately placed in
the mortar. 600 L of RLT Plus with β-ME (DNA/RNA extraction kit, Qiagen) was
immediately dispensed into the mortar and the tissue was grinded with the pestle by gently
going up and down in the mortar kept on ice. DNA and RNA were simultaneously isolated
using AllPrep DNA/RNA extraction kit (Qiagen). The quantity of RNA and DNA was
assessed using a NanoDrop® spectrophotometer by measuring absorbance at 230, 260 and
280 nm. The integrity of the RNA was assessed using the Agilent Bioanalyzer 2100, which
consisted of the RIN and electrophoretic profile. For electrophoresis, 1 l of DNA was
loaded onto a 1% agarose gel stained with ethidium bromide. A 1 kilobase DNA ladder was
run as a marker in each gel.
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