SOP Number: CP006.00
Title: Tissue Sampling, Processing, and Storage
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MAL Director: Dr. Peggy Borum
Author: Dylan Lennon
I
FSHN Chair: Dr. Neil Shay
PURPOSE
The purpose of tissue sampling, processing, and storage is to examine tissues in addition to blood
and urine samples and gain more of a whole picture perspective on what is being researched.
II
SCOPE
This procedure provides instructions for tissue sampling, processing, and storage.
III
RESPONSIBILITIES
It is the responsibility of Metabolic Assessment Laboratory personnel to follow this procedure.
It is the responsibility of supervisory personnel to ensure compliance with this procedure and to
train employees and students responsible for performing this procedure. Students will report
accidents to the principal investigators immediately.
IV
REFERENCES
N/A
V
REAGENTS AND MATERIALS
V.A.
V.B.
V.C.
V.D.
V.E.
V.F.
V.G.
V.H.
V.I.
V.J.
V.K.
V.L.
V.M.
V.N.
V.O.
V.P.
VI
0.9% saline (approximately 9L per piglet).
Ice.
White-capped plastic scintillation vials (appropriately labeled).
Large weigh boats.
Small weigh boats.
1, 20cc syringe.
1, 5cc syringe.
1, 22G needle.
Paper towels.
Black string.
Scissors.
Forceps.
Hemostats.
Cutting boards.
Ice buckets.
Liver perfusion tube.
EQUIPMENT
N/A
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SAFETY PRECAUTIONS
VII.A. Members of the MAL have been trained extensively in the procedures described in this
SOP.
VII.B. Members of the MAL have been approved to work with human blood and piglet blood,
tissues, and urine.
VIII
DEFINITIONS
VIII.A. Standard Operating Procedure (SOP) – Standard Operating Procedure is a document that
provides instructions for completing a specific task in the lab.
VIII.B. Metabolic Assessment Laboratory (MAL) – The Metabolic Assessment Laboratory is the
laboratory that will use this SOP.
VIII.C. Piglet Neonatal Intensive Care Unit (PNICU) – The PNICU is a unit where the piglet is
monitored by 24 hour care and routine check-up parameters using PNICU SOPs
conducted by the MAL.
VIII.D. Piglet Facility—The Piglet Facility is comprised of three rooms: the PNICU used for
piglet care; a surgery room used for surgical procedures; and a transition/entry room used
by staff to prepare items for care or surgery and to transfer the piglet from surgery to
care.
IX
PROCEDURE
IX.A. Tissue collection. The primary focus of removing tissues is to do it as quickly as possible
without damaging the organ.
IX.A.1. Heart: The heart is the first to be removed from the body cavity. It might still
be beating. The heart atria (smaller top right and left) are cleaved from the heart
leaving the ventricles (See Figure 1). The ventricles will be cut open and
dunked in chilled 0.9% saline to remove the remaining blood. The heart will
then be blotted dry with paper towels, trimmed of fat, connective tissue, or
vasculature (See Figure 2). See X.B and X.C. for further information on
additional processing information.
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Figure 1.
Figure 2.
IX.A.2. Lungs: The lungs will be removed from the body cavity and then trimmed of
vasculature, bronchi, major bronchioles, and excess tissue (See Figure 3). The
bronchioles extend deep into the base of the lungs. Butterfly each lung in half to
cut out the larger bronchioles. A healthy piglet will have whitish-pink lungs;
while a piglet with respiratory problems may have darker lungs. The lungs are
then dunked in chilled 0.9% saline and blotted dry. See X.B and X.C. for further
information on additional processing information.
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Figure 3.
IX.A.3. Liver and Gallbladder: First, the liver and gallbladder will be removed as one
entity from the body cavity. The diaphragm may still be attached to the liver and
will need to be removed, using forceps and a pair of scissors. Then the
gallbladder is removed from the liver by grabbing its long, skinny part with a
pair of forceps and pulling up gently. Gravity will gently remove the gallbladder
from the liver. If the gallbladder is accidentally cut open, weigh it as is
immediately and place into a vial. Reweigh the empty weigh boat and
subtract it from the original weight to get the total weight collected. Note
the accident on the medical record. The liver itself is trimmed of vasculature,
fat and the diaphragm (See Figure 4). When removing the diaphragm, it is
important not to cut through any blood vessels (which will make the perfusion
more difficult). Expose and identify the hepatic blood vessels before perfusion.
Next, the liver will be perfused with chilled 0.9% saline to remove any hepatic
blood.
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Figure 4.
IX.A.3.a. The liver is perfused using the perfusion tube attached to
a 20cc syringe over the sink in the entry room. It helps to
hold the liver in a vertical position, so that the perfusion
tube may be maneuvered more easily.The tip is inserted
carefully into the liver openings to perfuse the remaining
blood out the liver. Insert the tube gently by feeling for
the tubules inside the liver (See Figure 5). This is done to
each lobe of the liver. Avoid disrupting or destroying any
of the tubules by gentle guidance. A disrupted liver will
have a “mushy” texture and broken vessels. Repeat
process until the liver is clear of blood. Moving quickly is
key; use best judgment to determine end of perfusion.
After perfusion, the liver will become a lighter color (See
Figure 6) and can be further processed according to X.B.
and X.C.
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Figure 5.
Figure 6.
IX.A.4. Bile/gallbladder collection: The entire gallbladder will be dissected from the
liver prior to liver perfusion (See IX.A.3.). The gallbladder will be stored whole
and will not need to be cut into strips.
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IX.A.5. Spleen: The spleen is removed before the stomach and will be trimmed of fat
(See Figure 7). See X.B. and X.C. for further processing information.
Figure 7.
IX.A.6. Stomach: The stomach will be removed from the GI tract (cut near the cardiac
and pyloric sphincters). The stomach will have hemostats at the top and bottom
in order to not lose stomach contents (See Figure 8). Fatty and membranous
tissue will be trimmed from the stomach. The stomach and stomach contents
will be weighed. Remove the stomach contents by gently cutting the outer edges
of the stomach with the scissors (See Figure 9). The stomach contents will be
gently removed using a scoopula or spatula, avoiding disruption of the mucosa.
See X.B. and X.C. for further processing information. The weight of the
stomach contents will be calculated from the difference of the total stomach
weight with contents and the stomach weight with the contents removed.
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Figure 8.
Figure 9.
IX.A.7. Intestines will be removed as a single mass and placed on ice until separated.
Once all the other organs are processed, everyone works on processing the
intestines.
IX.A.7.a. Intestines: Hemostats are attached to the top and the bottom of the
intestines so that the contents do not seep out after it is removed. Be
sure to note which end is the beginning and which is the end of the
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GI tract. Cut the membranous tissue connecting the intestines and
uncoil them, using gravity for assistance (See Figure 10). While in
the process of removing the connective tissue, the distinction
between the large and the small intestine will be apparent from the
presence of an outcropping of tissue that is the appendix. The large
intestine and the small intestine need to be separated. Two hemostats
will be attached tightly around the junction. These locations should
be close enough to cut between and prevent the contents from
seeping out. Cut to separate the large and small intestines.
Figure 10.
IX.A.7.b. Small intestine: The small intestine is cleaned of membranous tissue
and lined in a zigzag formation to allow for easy measurement of the
length using black string (Figure 11). Lay the small intestine parallel
to the longer side of the board to minimize the number of turns in the
formation. Black string is lined beside the small intestine (in the
zigzag formation) and then cut at the end of the small intestine. The
length of the black string is now equal to the length of the small
intestine. The black string is then divided into thirds. The three equal
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portions of the string represent the top small intestine (duodenum),
the middle small intestine (jejunum), and the bottom small intestine
(ileum). The top small intestine is going to be the section that was
originally most closely attached to the stomach. Each section of the
small intestine is then clamped and cut as in the method to separate
the small and large intestine. Each small intestine section, with the
contents intact, will be trimmed of any additional extraneous tissue
and weighed. Now, the contents of all three small intestine sections
will be gently removed. First, the intestine is cut lengthwise. Then
the contents are gently removed using a spatula, without scraping the
mucosal lining (See Figure 12). Avoid disruption of the mucosa. The
total weight of small intestine contents will be calculated from the
difference between the tissue’s weight with and without the contents.
Figure 11.
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Figure 12.
IX.A.7.c. Large Intestine: Fatty and membranous tissue is trimmed from the
large intestine so that it can be straightened out. The large intestine
and its contents will be weighed. Now, the contents of the large
intestine will be removed. First, the intestine is cut lengthwise. Then,
the contents are gently removed using a spatula. Avoid disruption of
the mucosa. The weight of the large intestine contents will be
calculated from the difference between tissue’s weight with and
without the contents. When dunking the intestines, ensure that all
contents are removed. The large intestines may also be divided so
that more people can work on them.
IX.A.8. Adrenals: The adrenals are easy to clean, but difficult to get out (See Figure X.
They are removed from the kidneys, so no damage in placed on the intestines.
See X.B. and X.C. for further processing information.
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Figure X
IX.A.9. Kidneys: The kidneys will be removed from the body cavity and trimmed of fat
and membranes (See Figure X). Kidney tubules may go deep into the organ, but
for time’s sake, only the tubes seen at the tip of the bean will need to be
removed. See X.B. and X.C. for further processing information.
Figure X.
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IX.A.10. Skeletal muscle: A sample of skeletal muscle will be skinned from the
hindquarter in the gastrocnemius region, trimmed of subcutaneou fat. See X.B.
and X.C. for further processing information.
IX.A.11. Brain: The head will be removed and the brain exposed by cutting up through
the skull from the foramen magnum. Various portions of the brain will be
collected.
IX.B. Dunking in Saline
IX.B.1. Refill the saline after processing an organ. Waste saline may be placed in liquid waste.
When refilling, try using a minimal amount so as to conserve saline.
IX.B.2. Do not leave the organs dunked in the saline for too long. The organ will absorb the saline
and skew the weight reading.
IX.B.3. Minimize the amount of time handling the organ. The heat from hands will disintegrate
the molecule more quickly, so it is crucial to keep the organs on ice as long as possible.
IX.B.4. It is preferable that each organ is dunked no more than two times in the same saline.
IX.C. Tissue Storage
IX.C.1. Upon processing the organ, the organ is also weighed on the scale in the operating room.
(See SOP# CP092.00).
IX.C.2. The organ is then cut into long strips and placed in a pre-labeled vial. Only fill vials 1/31/2 full so that there is adequate rooms for the organs to expand when they freeze.
Additional vials for these organs will need to be prepared (Refer to SOP# CP094.00 for
suggested amount of vials needed per organ).
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IX.c.3. The vial is then stored on dry ice (See SOP# CP002.00).
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ATTACHMENTS
X.A. For a list of materials and their locations, refer to SOP# CG098.00.
X.B. The following are tissue and fluid codes that can be used for shorthand notation:
Tissue/Fluid:
Heart
Lungs
Stomach
Small Intestine (top)
Small Intestine (middle)
Small Intestine (bottom)
Large Intestine
Kidney
Skeletal Muscle
Cerebrum
Cerebellum
Stomach Contents
Small Intestine Contents
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Tissue & Fluid Sample Codes
Code:
Tissue/Fluid:
H
Large Intestine Contents
LU
Red Blood Cells (heart)
STO
Plasma (heart)
TI
Red Blood Cells (portal)
MI
Plasma (portal)
BI
Red Blood Cells (baseline)
LI
Plasma (baseline)
K
Urine
M
Bile
CB
Liver
CBL
Adrenal
STOCON
Spleen
SICON
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Code:
LICON
HRBC
HP
PRBC
PP
BRBC
BP
U
B
L
AD
SP
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X.B. The following layout for MAL personnel is suggested during organ collection in the
surgery room.
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