STANDARD OPERATING PROTOCOL (SOP) FOR

"THE NECROPSY"

DR. SHUBHAGATA DAS

Lecturer

Department of Pathology and Parasitology

Faculty of Veterinary Medicine

Chittagong Veterinary and Animal Sciences University, Khulshi, Chittagong 4202

Mob: +88 01717935112

E mail: shubho85@gmail.com

INTRODUCTION:

The word “necropsy” comes from the Greek “nekros dead body + opsis sight”. Necropsy may

be defined as the systematic examination of an animal carcass aimed to search for lesions. It

is an important diagnostic tool and supports other procedures performed in the diagnosis of

disease cases in a herd of flock. There is a literary paradox about the difference between a

necropsy and an autopsy, but the Greek word “Auto” refers to “self” so autopsy is “self

study.” So an autopsy is technically a necropsy, but because a “human is performing it on a

human” it is called an autopsy. The examination of dead or terminally ill animals offers

opportunities in studying the processes involved in disease situations. Although various

medical imaging techniques have evolved in recent years providing adequate information on

the morphologic alterations of organs and tissues following disease; necropsy still provides a

first hand look on what really happened along the course of the disease, particularly in poorly

understood disease situations, tissue alterations resulting from or as a reaction to the disease

process which may or may not be detected during clinical examination. Morphological

changes when correctly recorded and interpreted provide a basis for correlating functional

changes seen in a particular disease process. or functional disturbances. . Morgagni De

Sedibus, 1761 said “Physicians who either performed many autopsies themselves or who

regularly witnessed post mortem examinations, learnt at least to have their doubts. Those,

however, who are not themselves dealing with the very often depressing findings of

autopsy material, are floating in the clouds of uncontrolled optimism”. Ill-performed

necropsy thus confuses the understanding of a disease process. A working routine is desirable

so that adequate information is gathered that will aid in the formulation of a diagnosis. So, a

systematic approach in necropsy is required to so that appropriate and adequate information

be gathered during the examination.

NECROPSY OBJECTIVES:

1. Expose all foci of disease/abnormality in the carcass.

2. Seek lesions to explain clinical and laboratory findings.

3. Identify the sequence of disease events.

i. e. A conscious necropsy investigation includes

1. Systemic observation and dissection.

2. Collection and preservation of appropriate samples (tissue, fluids, etc) for

histologic, cytologic, microbiologic, serologic, chemical, toxicologic, parasitologic,

and/or radiologic evaluation.

3. Record findings logically, accurately, and completely.

4. Interpretation of findings to detect:

a) Immediate cause of death.

b) Contributory causes.

c) Other findings of clinical importance.

d) Incidental findings.

GENERAL CONSIDERATIONS:

TIME:

The necropsy should be performed as soon as possible, immediately after death of an

animal, because post mortem processes of decomposition (autolysis) follow at a fairly

rapid rate that obscures subtle changes in organs and tissues.

If histopathological examination of the diseased organs and tissues is anticipated, it is

best to examine the cadaver immediately and collect the required specimens the

soonest possible time.

If examination of the gastrointestinal tract is anticipated, it is recommended to

euthanasia the moribund animal and should examine right away as because the gut

flora accelerates the autolysis process, and may make the isolation of the causative

agent in question with difficulty or even impossible, especially in suspected bacterial

infections.

If necropsy will be delayed for some reason or another (example the cadaver will be

shipped to a distant laboratory and will take considerable time before it reaches its

destination), freeze the whole carcass solid. This is done to delay the process of post

mortem decomposition. Pack it in dry ice before shipping, observing the pertinent

rules and regulation in the transport of suspected biological hazards.

Freezing/thawing will make gross observations difficult and severely hamper

histopathological analysis as the ice crystals damage the tissues. However, viral or

bacterial isolations and some toxicological analyses can be conducted on frozen

samples.

PLACE:

There are several requirements in the selection of the place for necropsy. The place should

have adequate light, water, ventilation, drainage, provisions for cadaver disposal, and

provisions in lowering the chances of contaminating the surroundings. Animals that died of

suspected transmissible, zoonotic or exotic diseases require that the examination be done in a

laboratory. Usually, a clinical diagnosis will aid in deciding the site for necropsy, for

example, a clinical diagnosis of Anthrax does not warrant necropsy at all for the potential of

contamination is great. Extreme care should be practiced in selecting possible sites for

necropsy, especially in the field. The selected site should be away from sources of feed,

forage and water for the rest of the herd or flock. Avoid those sites that will be frequented by

other animals in the herd in gaining access to other places. Insects, predators and other

biological vectors of diseases should be warded off from the examination site.

NECROPSY EQUIPMENTS:

Usually varies greatly with the species, location of cadaver, etc. But some common

instruments and apparatus must be there for necropsy:

Metallic Instruments:

Sharp knife and sharpening equip (steel/stone)

Scalpel

Tissue forceps and scissors

Saw, cleaver, osteotome, bone cutter, shears, axe,

Metric ruler, scale

Soap, water, brushes for cleaning

Personal Protective Equipment (PPE) for the Pathologist:

Rubber Boots

Coveralls/ aprons

Cut resistant gloves

Latex or Nitrile gloves

Sterile gloves

Duct tape

Safety glasses or goggles

Disposable bouffant cap (hair net)

Surgical or procedural mask

Lined animal tissue waste containers

Autoclave

Fixative and appropriate containers:

Sterile syringes

Needles, swabs

Plastic bags

Paper plates

Microscope slides

Tags

Dissecting Glass

VETERINARY NECROPSY TECHNIQUE:

Procedural detail may vary, but a consistent technique aids in a thorough observation. To the

beginner, necropsy techniques appear unreasonably cumbersome and regimented. The

purpose is to methodically expose all organs and tissues to minimize the chance of missing or

"destroying" a lesion. The following is a brief outline of the technique that is to be used when

performing postmortem examinations.

PRENECROPSY EVALUATION:

1. Identify the animal to ensure that the correct animal is being necropsied.

2. Read the clinical history carefully.

3. Examine the necropsy request form for the following:

a. special organs or systems clinicians may want examined.

b. whether or not this is a cosmetic necropsy.

c. special requests (ie, cultures, photos, etc).

4. Fill containers 75% full with 10% phosphate buffered formalin.

5. Label container with necropsy number, species and initials of pathologist on duty.

6. Weigh the animal - if possible.

NB:

# Do not begin the necropsy until a permission sheet signed by the owner is in your

possession; A signed owner’s release form must accompany any animal to be

euthanized.

## Remember that the entire carcass, including all systems and organs, must be carefully

examined. Lesions may appear anywhere and care should be taken to expose and examine all

lesions. Examine each of the paired organs.

### Every animal should be weighed and/or measured (i.e., crown-rump length for aborted

feti) prior to prosection.

NECROPSY EVALUATION

1. EXTERNAL EXAMINATION:

a) Note any abnormal external findings, eg;

Body Condition: muscle mass / fat stores, decomposition, rigor mortis.

Skin and hair coat: parasites, dehydration, tumours, wounds, scars.

Discharges from body orifices: hemorrhage, nasal exudate, diarrhetic feces.

Eyes: corneal opacities, unequal dilated pupils, exudates, ulcers, hemorrhages.

Ears: parasites, tumours, discharges.

Mucous membranes: colour, ulcers.

b) Clinical pathology : Take appropriate samples for culture, histology, cytology, etc.

2. POSITIONING AND OPENING THE BODY CAVITY OF CARCASS:

[may vary from pathologist to pathologist and from animal to animal]

a) Position the animal on its left side down.

b) Reflect the left front and left rear legs.

To save the cutting edge of knife, insert knife through skin and cut the skin by pulling

out.

The femoral head should be removed from the acetabulum by cutting the ligament of

the head of femur.

c) Connect the two incisions with an incision along the ventral midline extending from

mandibular symphysis to anus. Do not damage the udder

To avoid cutting hair, incise the skin from the subcutaneous side

Raise the front leg and scapula and dissect and reflect dorsally

Remove the remaining skin between the excised front and rear limb to the

level of the spinal column and reflect dorsally

Examine the exposed superficial lymph nodes and jugular veins

Excise through the “up” rear limb (at the level of the pelvis) and continue to

incise through the coxofemoral joint and reflect the rear limb dorsally

d) Reflect skin dorsally.

e) Open abdominal cavity by incising through the dorsal abdominal musculature and

extending your incision downward following the rib cage.

f) Puncture a hole through the diaphragm and listen for air to enter the thoracic cavity (if no

air enters - pneumothorax).

g) Make an incision in diaphragm from the sternum dorsally.

h) Remove the ribs with pruning shears, or a smaller instrument, depending on animal size.

i) Examine viscera. At this time sample organs for microbes (eg bacteria, viruses, parasites)

Examination of the mammary glands or testes:

Mammary glands and mammary lymph nodes are completely cut away from the body

• Examine for symmetry, swellings, tumors, atrophy

• Examine the lymph nodes and incise them

• Incise the gland through the cistern and teat canal, examining each portion

• Palpate for thickenings, fibrosis, and tumors.

5. Examine prepuce and penis

6. Make a paracostal incision through the abdominal wall just behind and parallel to the last

rib

• Extend the incision dorsally to the vertebrae and ventrally to the midline

• Raise the body wall to avoid cutting viscera.

7. Make a paralumbar incision through the abdominal wall caudally to the pelvis

• Reflect the muscle wall ventrally and expose the abdominal cavity

8. Cut the diaphragm on the right side in an arc from the sternum along its costal attachments

to the vertebral column

• Listen for an in rush of air indicative of negative pressure in the pleural cavity

9. Sever the ribs at their sternal and vertebral ends with a pruning shear or other suitable

instrument and lift off the thoracic wall, thus exposing the entire thoracic cavity.

Gross Examination of the Thoracic and Abdominal Cavities:

Examine both cavities and all contents carefully with minimal movement of the

viscera Note transudates, exudates, and hemorrhage

Open the pericardial sac

Note amount, color, and consistency of abnormal fluid accumulations

Examine for adhesions, displacements, absence of organs, and size and symmetry of

organs in situ

Record lesions of organs and perform detailed examination of organs prior to removal

Take initial samples for microbiology, especially exudates in body cavities

Examination of the Thoracic Viscera:

1. Separate the mandibles at the symphysis

• Cut along the lingual surface of both sides of the mandible

• Remove the tongue and pull it down between the rami

• Disarticulate the hyoid bones. The tongue, larynx, trachea and esophagus are

dissected ventrally back to the thoracic inlet

• Lift up viscera and detach heart and lungs from the body wall by cutting dorsal and

ventral mediastinum

• Severe the aorta, post cava and esophagus back to about 2-3 cm anterior to the

diaphragm

• Sever and remove the thoracic viscera (“pluck”)

2. Examine thyroid, parathyroid, and thymus glands

• Note size, shape, and consistency

• Incise glands examining for lesions

3. Arrange the organs in approximately normal position

• Examine tongue by incising transversely

• Open esophagus and examine carefully

• Examine bronchial lymph nodes by palpating and incising

• Observe and palpate lungs for consolidation, emphysema or other abnormal

consistency

Open the larynx, trachea, bronchi and small bronchioles

Note exudates, hemorrhage, foreign bodies or lung worms in bronchial tree

Examine areas of consolidation and other abnormal lung tissue by incising.

4. Examine the heart

Observe any disproportion of parts (dilation, hypertrophy, anomalies) and alterations in

shape; note presence of normal adipose tissue

Open heart;

Cut through the right atrial free wall (including the auricle) horizontally

Examine the endocardium and vena cava

Examine the atrial side of the right A-V valve

Check for sufficiency of valve if indicated

Cut through the right A-V valve and wall of the right ventricle, keeping the incision near the

interventricular septum

Continue the incision around the right ventricle through the pulmonic valve

and pulmonary artery

Examine for patent ductus arteriosis

Open the left atrium and examine in the same manner as the right atrium

Cut through the left A-V valve, incising the ventricle through the midportion

of the free wall, Continue the incision to the apex

Make a horizontal incision in the ventricle approximately mid-way between

the coronary groove and the apex, incising from the first cut to the septum

At the septum, cut upward through the aortic valve and aorta

This process should result in a small flap of left heart with aortic valve on one

side and left A-V valve on the other

Examine vessels, valves and septa for anomalies

Examine endocardium and myocardium

Examination of Abdominal Viscera

1. Remove the spleen; examine grossly and incise several times

2. Examine the pancreas grossly

3. Make a small incision into the duodenum at the level of the pancreatic duct and apply

manual pressure to the gall bladder to see if bile enters the intestine

4. Remove and examine the liver

• Examine the peritoneal surface for fibrosis or adhesions

• Excise the liver from the diaphragm

• Note the size, shape, weight, color and consistency

5. Open the gall bladder and the larger bile ducts and Examine for stones, inflammation,

flukes, thickening of the wall

6. Palpate and incise the liver liberally from the abdominal surface; observe for necrosis,

fibrosis, abscesses, etc.

7. Examine the adrenal glands (prior to removing the kidneys); Cut adrenals in cross- section

and note cortical-medullary ratio

8. Remove urinary organs as a unit, including both kidneys, ureters and urinary bladder

• Cut each kidney longitudinally in half from the convex surface to the hilus and note

alterations in color, consistency, size, etc.

• Strip off capsule and examine the kidney surface ;note the ease with which the

capsule comes off

• Open and inspect the ureters, bladder and urethra; inspect all mucous and serous

surfaces

• Open vagina, cervix and uterine horns along their dorsal borders and examine

carefully all surfaces

• Examine ovaries for cysts, corpora lutea, atrophy, etc.

• Examine male accessory sex organs; observe size, consistency, inflammation,

etc.

9. Remove the stomach and intestines to the rectum

• Place the rectum over the lumbar area when it is cut so that the abdomen will not be

contaminated

• Free the intestine from the mesentery as it is removed and observe its lymph nodes

The examination of the gastrointestinal tract should be in the last so that instruments

and other tissues are not contaminated by gastrointestinal flora.

Examination of the Musculoskeletal System

1. Open the stifle, hock and humero-scapular joints

• To open the stifle, cut the straight patellar ligament 1/3 of the way proximal to the

tibial tuberosity and medial to the trochlea of the femur, and reflect the patella

• Observe synovia, articular surfaces, articular cartilages, and synovial membranes

2. Examination of the muscular system; examine and incise the muscles of various parts of

the body, especially lumbar and thigh muscles; check development, color, etc.

3. Examination of the skeletal system

• Examine body for broken bones or healed fractures

• For marrow inspection, remove femoral head with shears and crack femur

longitudinally.

Examination of the Eyes

1. Remove the eyeball from the orbit if indicated (not routine)

• Incise periorbital tissues and avoid direct contact with the eye

• Look for corneal opacities, cataracts, tumors, etc.

Examination of the Central Nervous System

1. Remove the head from the body at the atlanto-occipital articulation

• Incise the spinal cord before excessive traction is placed on the skull

2. Reflect skin and muscles of the head and examine skull for traumatic lesions

3. Remove the brain as described below

• Make a transverse cut behind the orbits (exact location varies in species) using a

hacksaw

• Make lateral cuts from the ends of the transverse cuts just medial to the occipital

condyles (leave room for brain to be removed intact)

• Lift off bony cap carefully with a chisel

• Incise the dura over the dorsal brain surface and incise the tentorium cerebelli

• Hold the skull with the nose pointing upward and tap it gently on the table; carefully

cut the olfactory tracts and other cranial nerves and allow the brain to slip out. Avoid

traction on the brain

• Remove the pituitary gland by cutting diaphragmatic sella on both sides, clipping

the bony projection posterior to the gland, and cutting soft tissues around the gland

with scissors

4. Observe the dura matter.

5. Incise the brain transversely (1-cm slices) and look for lesions

• When entire brain is to be fixed, make only one transverse cut into lateral ventricles

so fixative may enter tissues.

Examination of the Gastrointestinal Tract

1. The esophagus has been opened

2. Open the stomach along the greater curvature

• Observe the mucosal and serosal surfaces; ingesta must be removed

• Examine for hemorrhage, parasites, foreign bodies, abnormal ingesta, etc.

3. Open the small intestine

• Observe all surfaces and ingesta

• Leave 1-inch segments closed for histopathology

4. Open the cecum and colon back to the anus, and examine carefully .

Species-Specific Procedures

1. Horse:

• When the abdomen is opened, move the left parts of the large colon cranially so

that the pelvic flexure is lying anterior; move the cecum dorsocranially, the small

Intestine over the right flank, and the small colon posterior and down

• The mucosa of the guttural pouches is examined when the head is disarticulated

• The cranial mesenteric artery should be opened from the aorta past the ilealcecal

and colic artery bifurcations

2. Ruminants:

• When the abdomen is opened, place small intestine and colon over the right lumbar

area; examine the forestomachs and abomasums for position and adhesions

• Remove forestomachs and abomasums as a unit; separate serosal attachments to

stretch the organs out. Open and examine each organ. Remove ingesta and rinse the

rumen mucosa with water to examine.

TISSUE PRESERVATION FOR HISTOLOGIC EVALUATION:

1. 10% buffered neutral formalin (10% BNF)

Probably the best routine fixative though penetrates tissue slowly (~5 mm/24 hours).

Commercial Formaldehyde (37-40%) 100 mls

Distilled water 900 mls

sodium phosphate monobasic 4.0 g

sodium phosphate dibasic (anhydrous) 6.5 g (pH should be 7.2 ± 0.5)

Fix tissue slices 24 - 48 hours at room temperature.

2. Bouins fluid - picric acid base

USES: Endocrine tissues (especially pancreas and pituitary)

Viral Diseases - Demonstrate viral inclusion bodies

Eyes, uterine biopsies - rapid fixation

DISADVANTAGES: Tissues become brittle after 24 hours (tissues need to be washed to

remove picric acid and then placed in 50% ethanol).

3. Alcohol

Causes severe dehydration and is not a good fixative.

Notes:

a) Regardless of particular fixative, consider tissue thickness and total fixative volume: 6 to

10 mm maximum thickness (except eye, brain, spinal cord - fix whole)

b) Fixative volume 10 times tissue volume.

c) Handle tissue carefully prior to fixation; do not stretch, squeeze, cut with dull instruments

or rinse excessively with tap water.

d) Freezing - the size of ice crystals formed in tissue is proportional to the length of time

necessary to freeze; ie, snap frozen specimens (liquid nitrogen) have few freezing artifacts

and are useful for histochemical staining, but carcasses that freeze outdoors or in home

freezers result in moderate freezing artifacts ("making interpretation difficult").

GUIDELINES FOR PACKING AND SHIPPING OF SAMPLES

1. Label all sample containers with the following information, using indelible ink:

• Client name

• Animal name

• Case number (if used)

• Date of collection

• Site of collection (e.g. liver, right kidney)

2. Ship in plastic containers whenever possible.

3. Be sure that lids are tight on containers that contain liquid. The ratio of formalin to soft

tissue should be 10:1. If the sample is bone, the ratio should be 20:1.

4. Containers with liquid should be placed into zippered plastic bags separate from

submission forms to prevent forms from becoming damaged and unreadable in case of leaks

5. Complete submission forms, including all requested information

6. Include address, phone number and FAX number for your clinic to facilitate return of

results.

7. Pack container and submission form in box for mailing, allowing room for packing

materials such as foam peanuts, bubble plastic, or newspaper as appropriate.

8. Check with lab to determine if sample must be received at room temperature, cool, or

frozen and include coolant source if needed.

PREPARATION OF THE NECROPSY REPORT

The general rule in making a necropsy report is to be objective in interpreting lesions,

with the finished report being descriptive.

The common fault in recording necropsy findings is the tendency of the examiner to

interpret the lesions observed, than describing the changes seen.

Report must be prepared by the one who examined the specimen.

This is particularly true if another person other than the one who examined the

specimen will synthesize the findings and formulate the diagnosis.

The finished report should be descriptive enough allowing other to clearly visualize

what were observed during the examination to enable them to make their own

interpretation and possible diagnosis.

Relevant sections of the necropsy document dealing with the case identification,

specimen identification, owner's identification, and clinical history should be filled

with the required information before necropsy.

This may be done when the specimen is received for examination. Without these data,

particularly the clinical history of the case, the search for lesions would be particularly

tedious. It may even result to undue trouble to the examiner in terms of the danger

posed by examining a specimen where necropsy should not be done at all (example:

cases of anthrax).

A MODEL NECROPSY REPORT SHEET IS GIVEN BELOW:

SAFETY PROTOCOL FOR W ORKING WITHIN THE NECROPSY ROOM:

A. Work within the necropsy room poses many hazards; so all staff must be competent or be

supervised.

B. Immediately prior to beginning necropsy of all large animals (such as cattle, sheep, deer,

etc.), one gallon of household bleach (5% sodium hypochlorite) will be poured into each

of the floor drains.

C. Sharps boxes will be available where required. Used scalpel and razor blades, syringes

and needles will be discarded into sharps boxes and not left on surfaces, especially near

the carcass.

D. Walk carefully around the necropsy room, as the floor may be slippery. Do not rush.

E. Pick up any dropped fat or tissue from the necropsy room floor and keep boots free of

fat/tissue as this increases the risk of staff slipping.

F. Special care will be taken while working with or carrying sharp items, uncovered, around

the necropsy room.

G. When finished using equipment leave any sharp items and other instruments clearly

visible at a predetermined location in the necropsy room.

H. Use of a cart to transport large items around the post mortem room, eg. Bovine head,

sample crates, is recommended.

I. If a staff sustains a cut/injury while working in the necropsy room, they should

immediately take the following action (with assistance if required): Move to a clean part

of the necropsy room. Remove gloves/clothing to reveal and investigate injury.

CUTS: Encourage blood to flow from the cut to flush out the wound. Immerse the cut in

sodium hypochlorite (bleach) at concentrations ≥20,000 ppm for 1 minute, then wash and

dry the cut area. Apply direct pressure to limit bleeding if necessary.

OTHER INJURIES: All contaminated outer clothing must be removed before staff leave

the post-mortem room – unless to do so would endanger life or risk further injury.

For species specific necropsy procedures, the following

references are recommended:

a. Feldman, D. B. and Seely J.C., Necropsy Guide: Rodents and the Rabbit. CRC

Press Inc., Boca Raton Fl, 1988.

b. Pathology of Domestic Animals 3rd ed., vols. 1-3 Academic Press Inc., 1985.

c. Devor, D.E., Henneman, J.R., Kurata, Y., et al. Pathology Procedures in

Laboratory Animal Carcinogenesis Studies. In Waalkes, M.P. and Ward, J.M.

(eds.), Carcinogenesis, New York: Raven Press, pp. 429-466, 1994. d.

Sundberg, J.P. and Boggess, B. Systematic Approach to Evaluation of

Mouse Mutations. Boca Raton: CRC Press p. 199, 1999.

REFERENCES

Benjamin, L.E. 1985. Veterinary clinical pathology. Kallyani publication, new delhi.

Calnek, B.W.1997. Diseases of poultry. 10th Edition lowa state University press, Ames, Lowa

Coles, E. H. 1986. Veterinary Clinical Pathology. 4th Edn..: W. B. Saunders. Co.Inc.

Philadelphia. pp. 486-488.

GEERING WA, FORMAN AJ, NUNN MJ, Exotic Diseases of Animals, Aust Gov

Publishing Service, Canberra, 1995, p.173-181

Luna, L.G. 1968. Histopathologic Technic and Practical Histochemistry, 3rd ed. Sounders

Co.ltd, London.pp.17-120.

NADC Guidelines for accidental personal injury

NADC Procedures for Incineration and Operation of the Necropsy Facility. (Sept. 2004)

Veterinary necropsy protocol for milliatary working dogs and pathological sample

submission guidelines. 2001, Headquarters, department of US army.

WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. 1999