SOP Number: CP006.00 Title: Tissue Sampling, Processing ... PDF/Non-Survival/Tissue Sampling...SOP...

SOP Number: CP006.00

Title: Tissue Sampling, Processing, and Storage

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Author: Dylan Lennon MAL Director: Dr. Peggy Borum FSHN Chair: Dr. Neil Shay

Uncontrolled Copy CONTROLLED DOCUMENT-DO NOT DUPLICATE Controlled Copy No._______

G:\GLP_G\Carnitine SOPs\Piglet Experiment\SOPs in PDF Form\Non-Survival\Tissue Sampling, Processing, and Storage.GLP.docx

I 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. 0.9% saline (approximately 9L per piglet).

V.B. Ice.

V.C. White-capped plastic scintillation vials (appropriately labeled).

V.D. Large weigh boats.

V.E. Small weigh boats.

V.F. 1, 20cc syringe.

V.G. 1, 5cc syringe.

V.H. 1, 22G needle.

V.I. Paper towels.

V.J. Black string.

V.K. Scissors.

V.L. Forceps.

V.M. Hemostats.

V.N. Cutting boards.

V.O. Ice buckets.

V.P. Liver perfusion tube.

VI EQUIPMENT



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VII 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



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sure to note which end is the beginning and which is the end of the

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



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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

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 1/3-

1/2 full so that there is adequate rooms for the organs to expand when they freeze.



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Additional vials for these organs will need to be prepared (Refer to SOP# CP094.00 for

suggested amount of vials needed per organ).

IX.c.3. The vial is then stored on dry ice (See SOP# CP002.00).



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X 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 Sample Codes

Tissue/Fluid: Code: Tissue/Fluid: Code:

Heart H Large Intestine Contents LICON

Lungs LU Red Blood Cells (heart) HRBC

Stomach STO Plasma (heart) HP

Small Intestine (top) TI Red Blood Cells (portal) PRBC

Small Intestine (middle) MI Plasma (portal) PP

Small Intestine (bottom) BI Red Blood Cells (baseline) BRBC

Large Intestine LI Plasma (baseline) BP

Kidney K Urine U

Skeletal Muscle M Bile B

Cerebrum CB Liver L

Cerebellum CBL Adrenal AD

Stomach Contents STOCON Spleen SP

Small Intestine Contents SICON



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X.B. The following layout for MAL personnel is suggested during organ collection in the

surgery room.

SOP Number: CP006.00 Title: Tissue Sampling, Processing ... PDF/Non-Survival/Tissue Sampling...SOP...

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