1.0 OBJECTIVE
1.1
To lay down a standard procedure to be followed for Necropsy and collection, weighing & fixation of organs/tissues in the laboratory
animals.
2.0
SCOPE
2.1
This Standard Operating Procedure (SOP) shall be
applicable for Necropsy and collection, weighing &
fixation of organs/tissues in Laboratory animals in Animal House Facility.
3.0
RESPONSIBILITY
3.1
Study Director / In-charge - Animal House Facility
3.2
All personnel involved in Necropsy and collection,
weighing & fixation of organs/tissues in laboratory animals.
4.0
DEFINITIONS
4.1
Nil
5.0
PROCEDURE
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5.1
General
Collection Guidelines
5.1.1
As a Necropsy and gross observations should be
performed by a trained person and should be performed as soon as possible to
ensure tissue integrity by fixing them in 10% Neutral Buffered Formalin (NBF)
after the death of the animal in a manner that avoids the occurrence of artifacts
or post mortem changes in the collected tissues/organs.
5.2
Materials and equipment
5.2.1
Sterile gloves
5.2.2
Animal tissue waste containers
5.2.3
10% Neutral Buffered Formalin
5.2.4
Normal Saline
5.2.5
Tissue containers
5.2.6
Sterile Petri plate
5.2.7
Sterile Swabs
5.2.8
Surgical Instruments (Scissors, Forceps, Surgical
blade, etc.)
5.2.9
Operation Theater Light
5.3
Necropsy
Preparation
5.3.1
Necropsy shall be planned in advance to ensure that the
evaluation is thorough, efficient, and appropriate to the aims of the study plan
(SP).
5.3.2
Procedure shall be performed as soon as possible for
any experimental animal death during a study in animal house.
5.3.3
For experimental related procedure, the necropsy shall
be planned in advance to ensure that the evaluation is thorough, efficient, and
appropriate to the aims of the Study Plan (SP).
5.3.4
Ensure required volumes of fixative, 10% NBF shall be
prepared before starting the procedure.
5.3.5
All the specimen vials, for sample submission to a
laboratory and or for archival purposes must be properly labeled and filled
with the required volume of 10% NBF.
5.3.6
Petri dish shall be filled with normal saline for the
collection of organs/tissue to be weighed.
5.3.7
Animals shall be identified by animal numbering, group
and sex.
5.4
Procedure for
External Examination
5.4.1
Prior to conduct the external examination, animal(s)
shall be euthanized, as per the current running version of SOP.
5.4.2
Once the animal is euthanized, examine the animal
externally.
5.4.3
Observe the condition of hair coat and underlying skin:
e.g. normal (i.e. smooth and silky), ragged and rough, dry and alopecia,
superficial skin lesion and their location, soiling of hair coat with urine
feces etc.
5.4.4
Eyes: Normal (clean and moist), inflamed membranes
(conjunctivitis), discharge cloudy or opaque, cataract, corneal ulceration and
corneal opacity.
5.4.5
Mouth and lips: e.g., normal (smooth and shining) a cracked, dry, alignment of jaws and teeth.
5.4.6
Nose: e.g. Normal (smooth moist and glistening), dry
crusted, inflamed, fresh or dried exudates, type of discharge-watery, purulent
or colored.
5.4.7
Anus: Normal (small and tightly closed), enlarged,
inflamed, ulcerated, perineum soiled with feces.
5.4.8
External genitalia: For male –abnormal number of
testes, equal in size, enlarged, inflamed, ulcerated, perineum soiled with
feces. For female – vulvae size –small, enlarged, inflamed, and no discharge.
5.4.9
Tumor or mass: Any subcutaneous or outwardly visible
growth. A description of color consistency appearance and location shall be
recorded.
5.4.10
Ear/pinna and canal: Injury, encrustation, inflammation
and discharge.
5.4.11
Record the findings in the format to the current
running version of SOP.
5.5
Procedure for Visceral
/ Internal Examination
5.5.1
After the external examination, the carcass shall be
cut open and examined the tissues/organs for gross pathology as per SP and
record the observations in the currently running version of the format.
5.5.2
Organs and tissues shall be examined in situ before
dissecting; abnormalities shall be recorded in the format.
5.6
Dissection
5.6.1
The detailed procedure for internal examination common
to all species of rodents is described as follows:
5.6.2
Lay the animal on its back and with the help of
forceps, grab hold of the skin anterior to the urethral opening.
5.6.3
Using the scissors, cut along the ventral midline from
the groin to the chin, being careful to cut only the skin and not the muscle
wall underneath.
5.6.4
Next, make an incision from the start of the first incision to down near the knee on both sides of the animal, an incision that
looks very similar to an upside-down Y.
5.6.5
Reflect the skin by blunt dissection from the
underlying tissues and examine for any abnormalities e.g. hemorrhages,
growth/mass, enlargement of superficial lymph nodes or mammary glands and
subcutaneous abscess.
5.6.6
Cut the underlying muscles along the midline penetrating
the diaphragm and cut through the ribs, take care not to damage the visceral
organs.
5.6.7
Observe the following organs/tissues for any visible
sign of abnormality:
5.6.7.1
Sternum: Separate the sternum from ribs on either
side and collect it.
5.6.7.2
Eyes: If required,
dissect out eyes as soon as possible using bent forceps and place them in
suitable fixative as per SP
5.6.7.3
Heart: Isolate the heart
by cutting the blood vessels and examine for shape, size, color, dilation,
flabbiness and hemorrhages.
5.6.7.4
Lungs: Dissect out the
lungs and examine them for lesions like emphysema, collapse, hemorrhage,
congestion, consolidation, and pneumonic changes.
5.6.7.5
Liver: Remove the liver
and examine all the lobes (with gall bladder in mice, if required) for change
in color, size, consistency, and hemorrhage. Gall bladder (in mice) should be examined for the patency of bile
duct, thickening, adhesion, smoothness or unevenness, stones, parasites and
discoloration or as defined in SP.
5.6.7.6
Adrenal
Glands: Dissect out the left and right adrenal glands and examine for color,
size and hemorrhage.
5.6.7.7
Kidney: Dissect out the
left and right kidney and examine for the change in color, size, smoothness, or the unevenness of surface (scars), discoloration, congestion, mass or growth,
dilated pelvis, hydronephrosis, and cyst.
5.6.7.8
Spleen: Remove the spleen
and examine it for changes in size, consistency, color, hemorrhages, smoothness
and unevenness.
5.6.7.9
Testes: In male animals
push up the testes gently and draw them up into the abdominal cavity with
epididymis. Collect epididymis along with testes or separately from the testes.
Trim off the excess fat and examine for changes like size, discoloration,
adhesion, consistency, mass/ growth, streaks, hemorrhage, and congestion, if
required.
5.6.7.10
Ovary: In females
dissect out the ovaries with oviducts and examine them for cysts, ovarian and
Para ovarian foci, infection, mass/growth adhesion, hemorrhage, and congestion.
5.6.7.11
Thoracic
cavity: Examine the thoracic cavity for hemorrhage, fluid, and adhesion. Examine
the trachea, esophagus, thyroids, thymus, and larynx for inflammation,
hemorrhage, and enlargement. Free all these organs along with the heart, lungs, and
lymph nodes from a point anterior to the larynx and remove them from the thoracic cavity
after cutting the esophagus near the diaphragm.
5.6.7.12
Examine the mammary tissue for enlargement etc.
Collect the pelvic mammary glands along with teats and skin, from each female
rat and any males showing lesions.
5.6.7.13
Abdominal
Cavity: Collect stomach with a piece of the duodenum. Cut open the stomach along its
long greater curvature, wash out the ingesta and examine the mucus membrane for
ulcer, erosion, or hemorrhage.
5.6.7.14
Collect the entire length of intestines by gently
flushing out the contents.
5.6.7.15
Examine the entire length of small and large
intestine for parasites, ulcer, erosion, hemorrhage, adhesion, inflammation,
tumor mass and discoloration, also enlargement of payer’s patches if required.
5.6.7.16
Examine pancreas for color size/ consistency, mass /
tumor adhesion.
5.6.7.17
From
Males – collect the prostate, seminal vesicles, and coagulating glands and
examine them for size, consistency, and presence of secretions, inflammatory
changes and edema.
5.6.7.18
From
Females – Free the uterine horn from the mesentery and cut the pubic symphysis,
separate the vagina from the rectum by gentle traction and transect below the cervix.
Examine uterus for dilation, distension, cysts, uneven thickness, infection,
mass or growth and polyps.
5.6.7.19
To collect the brain, make a circumferential
incision in the cranium using a bone cutter. Liftoff the roof of the cranium to
expose the meninges carefully.
5.6.7.20
Then holding the head with the nose pointing upward,
the anterior part of the brain is lifted to expose the cranial nerves. Cut
close to the skull whereby the brain should fall into the handheld underneath.
5.6.7.21
Pituitary
Gland: collect the pituitary from the floor of the cranial cavity by
penetrating the thin membrane covering the pituitary fossa with a sharp
scalpel, if required.
5.6.7.22
Femur:
Remove
either side of the femur along with the knee joints, if required.
5.6.7.23
Make a Bone marrow smear by cutting the femur bone
and collecting the marrow with a fine artist’s paintbrush. Smear thin lines
onto a clean glass micro slide parallel to its long axis and allow it to dry
before fixing with methanol for 30 minutes, if required.
5.6.7.24
Sciatic
nerve: Beneath the femur is the sciatic nerve, which is visibly white in color.
Dissect out the sciatic nerves as long as possible, if required.
5.6.7.25
Vertebra
and Spinal Cord: When all the tissues in the list are collected separate the vertebral column
up to the base of the tail from the rest of the carcass.
5.6.7.26
The necropsy prosecutor must sign with date in the
data records.
5.7
Organ Weighing
5.7.1
Switch on the weighing balance and check the calibration
status of weighing balance.
5.7.2
Use only calibrated weighing balance for weighing.
5.7.3
Put an empty Petri dish/weighing bowl on balance.
5.7.4
Make the reading Zero by pressing the “TARE / ZERO”
button and make ensure “0” on the display.
5.7.5
Trim the extra fat or adjoining tissue from the desired
organs before weighing. Gently handle the organs to avoid damage.
5.7.6
Gently place the organ to be weighed on the weighing
bowl. Paired organs shall be weighed together.
5.7.7
Make the organ weight recording in the format of the current
running version of SOP.
5.7.8
When display stabilizes, record the weight of the
organ.
5.7.9
Tare the balance by pressing the “TARE” button to bring
display to Zero again.
5.7.10
Repeat the steps to weigh the next tissue/organ.
5.7.11
Once the weighing of organs/tissues of one animal is
completed, the whole organ tissue should be transferred to the respective tub /
container with proper labeling.
5.7.12
The person weighing the organs/tissues shall sign
with date.
5.7.13
Remove the weighing bowl after completion of weighing
procedure and clean the balance.
5.7.14
Update the weighing balance logbook after completion of
the procedure.
5.8
Trimming and
Fixation
5.8.1
To ensure rapid and even penetration of fixative, large
or encapsulated organs should be trimmed prior to fixation.
5.8.2
Heart:
Remove blood clots by giving them a cut along the coronary artery.
5.8.3
Lungs:
Inflate the lungs by injecting the formalin into them taking care to inflate
all the lobes. They are collected as such without trimming.
5.8.4
Liver: Lay
the lobes flat and separate them from each other. Make transverse cuts of 3mm
thickness across the entire width of the lobes.
5.8.5
Spleen: make
two or three parallel cuts transversely.
5.8.6
Kidneys:
Make two complete parallel cuts from cortex to pelvis (transversely) in one
kidney. In other kidneys make a longitudinal cut to divide it into two halves.
5.8.7
Brain: Fix
the brain without trimming.
5.8.8
Pituitary and
Adrenal Glands: place as such in fixative.
5.8.9
Ovaries:
place as such in formalin.
6.0
ENCLOSURES
6.1
Formats
|
Sr. No. |
Format Title |
Format No. |
No. of pages |
|
1 |
Record
for Gross Pathological Observations |
F………………….. |
01 |
7.0
ABBREVIATIONS
7.1
SOP : Standard Operating Procedure
7.2
QAD : Quality Assurance Department
7.3
AHF : Animal House Facility
7.4
SP : Study plan
8.0
REFERENCES
8.1
The Laboratory Rat – Second Edition, Academic Press, Edited By – Mark A
Suckow, 2006.
8.2
The Laboratory Mouse – Elsevier Academic Press, edited by Hans Hedrich,
2004.
9.0
REVISION HISTORY
|
Sr. No. |
Change Control No. |
Reason for change |
|
|
|
|
END OF
DOCUMENT
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