Lesson 3 Necropsy

 

Introduction

 

This lesson reviews necropsy technique and related topics.

 

Reading Assignment

 

Read Chapter 5 pp. 165-183 in Clincial Textbook for Veterinary Technicians 6th edition by McCurnin and Bassert

 

It is helpful to review anatomy and anatomical terms.

 

Optional: And if you have it you could also read chapter 10 pp 421-451 in Laboratory Procedures for Veterinary Technicians 4th edition (by Hendrix)

 

Writing assignment

 

There is a writing assignment at the end of the lesson. Please turn in your answers by email.

 

Learning Objectives

 

Know indications for necropsy

Understand basic necropsy protocol

Be able to assist in specimen collection, processing, and submission

Know special procedures for rabies suspects

Be aware of necropsy procedures for small animals, horses, ruminants, and exotics

 

Key Words

 

gross pathology

histopathology

lesions

pathogenesis

formalin

mummified

DESC

focal, multifocal, diffuse

morphologic diagnosis

postmortem

antemortem

rigor mortis

necropsy

autopsy

autolysis

prosector

in situ

cosmetic necropsy

sacrificial procedure

DFA


Necropsy

Necropsy is the postmortem evaluation of an animal. A thorough examination of a dead body involves the dissection and study of all the body systems. The term used in human medicine for the same procedure is autopsy. We often use the term autopsy with clients when we really mean necropsy, because autopsy is a familiar term to most people and does not require much explanation.

The purpose of necorpsy is usually to determine a cause of death. However, in research studeis necropsy is also used for data collection. Also, when there are large populations of animals to manage, an animal or animals may be chosen for necropsy procedure as a means to monitor the incidence or cause of disease within the group from which they came. This sacrificial procedure is a technique used in disease surveillance for research such as lab animal colonies, or commercial operations such as that for the poultry industry. 

Necropsy is an important diagnostic technique that is often performed by veterinarians. Usually the role of the veterinary technician is to sterilize and set up instruments, assist in the necropsy procedure, record findings, and to process specimens. However, in some venues, the veterinary technician is responsible for actually performing the necropsy, or at least initiating the dissection. In order to be proficient at necropsy, you must be familiar with normal anatomy and use a standard technique in which the body systems are examined in a sequence so that nothing is overlooked. There is no absolute right or wrong way, just as long as it is standardized, methodical, and systematic. There are many techniques and order of operations used so that the necropsy technique will vary depending upon the prosector's training and preferences as well as the type of subject species. The body is evaluated by gross examination and microscopic examination.

 

Before the necropsy

Before the necropsy, a complete signalment and history should be acquired. The nature of the case discussed to ensure that all supplies needed are available because the history helps guide what tests may be needed. The history should include the date and time of death. An important history fact to have is if the animal bit anyone in the previous 10 days before death. If yes, then the brain must be tested for rabies. Make sure there is written permission to perform the necropsy.

The instruments used in necropsy are somewhat different than you might be used to seeing used for surgery. The instruments are heavier and less delicate. For example, in necropsy a knife is used for dissection, whereas in surgery a scalpel would be used to cut tissue. There are also some instruments specifically used for necropsy such as special shears used to cut bone, such as ribs or vertebrae. A basic necropsy instrument pack includes knives, scalpel with handles, scissors, forceps, saws, and shears. Gauze and paper towels should be available for blotting tissue and fluids.

 

Safety precautions

All work surfaces should be cleaned and disinfected.

The instruments for dissection should be sharp to lessen operator fatigue and to decrease potential damage to any specimens that may be collected. Dull instruments hinder the necropsy procedure and could damage any tissue specimens, perhaps affecting the histopathology results. The instruments should also be disinfected before cleaning, so that you don’t splash infectious material around while cleaning the instruments. Then the instruments are cleaned and sterilized after use, so that contamination of any specimens from another case does not occur when they are used again.  It is ideal to use disposable instruments.  

The prosector and any assistants should wear personal protective equipment such as proper attire (gowns, aprons, shoe covers), eye protection (goggles), mask, and gloves. All personnel working in necropsy should have a current rabies vaccination status.

When collecting specimens, the containers chosen should be of non-breakable material, such as some kind of plastic material.

 

Necropsy technique  
 
The animal first is externally examined for any lesions, and general condition or body scoring is assessed. Small animals may be placed on their back or left side. All other animals are placed on their left side, except the horse, which is often placed on the right side because of the positioning of the digestive tract organs. A ventral midline incision is made from the lips to the pelvis and the internal organs are exposed. Before the thorax is entered, some prosectors puncture the diaphragm with a blade from the abdominal side, and its movement is observed. If air rushes into the thorax to equalize pressures through the incision, the diaphragm will move slightly, and this means that the pleural space was intact and maintained negative pressure during life. Before any organs are handled or removed, the prosector observes the position and condition of the organs as they were found, or “in situ”.  Any specimens for culture are taken aseptically before the organs are disturbed. Then the various organs systems are dissected out and moved onto trays for closer inspection and processing. The organs are examined grossly for any obvious lesions and tissue samples of each organ are submitted for histopathology.  First the tongue, organs of the neck (esophagus, trachea, thyroid), and thoracic organs (lungs and heart) are removed; this dissection grouping is sometimes referred to as the “pluck”. The other organ systems grouped for separate removal and examination are the digestive tracts and urogenital tracts. The nervous system is studied by removal of the eye, brain, spinal cord, and a peripheral nerve section (usually the sciatic), and also cerebrospinal fluid may be collected. A section of bone and muscle may be collected to assess the musculoskeletal system. Joints may be dissected and joint fluid collected for study. A bone marrow sample is harvested to evaluate the hematopoietic system. Lymph nodes are collected to check the lymphatic system. All endocrine glands are collected to study the entire endocrine system. Because the endocrine glands are dispersed throughout the body, and are attached to other organs in various body systems, they are collected as they are encountered. There is so much that needs to be collected, an organ could get overlooked, so it is helpful to use a standard necropsy form to keep track of what needs to be collected.

A standard necropsy form should be used for keeping a complete record of the necropsy. Gross findings should be recorded as they are dictated during the necropsy. There will be descriptions of the size, shape, color, texture, consistency and odor of the tissues regardless if the organ is normal or abnormal. All comments should be recorded. The assessment may be that the organ is normal, has postmortem changes, or is abnormal. Postmortem changes alter the normal appearance of an organ making it more challenging to recognize abnormalities. Even if the assessment is that an organ is normal, most veterinarians are more comfortable stating in the record that the organ “appears normal” or that there are “no visible lesions ("NVL"). It is possible for an organ to appear grossly normal but to exhibit abnormal histopathology. Therefore, samples for histopathology are routinely taken of all organs, whether they appear abnormal or normal.

Sometimes the necropsy is also documented with photography. Properly cataloging the photographs to match up with the case written records is a very important legal and clerical duty.

Ultimately, the gross findings will give a tentative diagnosis, or a list of differential diagnoses, which then will be ruled out or in depending upon the results of any laboratory tests on the specimens collected. A list of the organs that are of clinical importance to the diagnosis is compiled, regardless if the organs were grossly abnormal or normal. The specimens collected and tests requested should also be recorded on the necropsy report. Copies of the necropsy report should be placed in the animal record and should also accompany the specimens to the lab. Once all the results are compiled and a final diagnosis is made, the completed report is sent to the veterinarian requesting the necropsy.

 
Specimen collection
As the organs are examined, representative sections are taken and placed into 10% formalin, even if no lesions are seen. If lesions are found, sections should be taken that include both normal and abnormal tissue so that the pathologist has the two regions to compare. The sections should be no more than 1 cm thick, and the formalin volume to tissue volume should be 10 to 1. Impression smears of tissue sections can be performed for cytology before the tissue is placed in formalin. However, remember that formalin vapors will affect the staining of cytology preparations, making them more basophilic. So if you are preparing cytology slides, do this away from the preparation area containing formalin.

Specimens for virus or bacteria culture should be taken with the same care and aseptic technique as with a live patient. Ideally specimens for culture should be collected before dissection. Microbiological specimens should be protected from contamination with flora, which sometimes is difficult to avoid in a necropsy since they are often handled before the culture is taken. If the tissues to be cultured have already been handled, then a quick way to sterilize the tissue surface is to heat a spatula type instrument and sear the surface of the tissue, effectively sterilizing the surface and eliminating surface contaminants. Then the tissue beneath the seared surface can be excised with sterile instruments and submitted for culture. Even with these precautions, contaminant growth is likely because of postmortem changes. After death, the body starts to break down in an enzymatic process called autolysis, and bacteria from the intestinal tract migrate to other organs. So the bacteria present in a necropsy sample may not be a pathogen, but normal flora instead that has migrated and started the decomposition process.    

If serum is needed, it often can be found in the heart chambers. Fluid from the anterior or posterior chambers of the eye may also be collected to test for urea and electrolytes. Effusions from the pleural or peritoneal cavity, cerebrospinal fluid, synovial fluid are also collected. Depending on the specimen, small amounts of fluids are collected into red top or EDTA tubes. Other fluids, such as urine or ingesta, are collected into jars.  Urine should be collected via needle aspiration. Toxicology specimens typically require serum, ingesta, and urine.

If larval tapeworms are found, extreme caution must be taken, since many tapeworms are zoonotic. If adult parasites are found, they should be preserved in 70% ethanol or formalin. Some internal parasites, such as flukes, will curl up when placed in formalin which will hinder proper identification. For many parasites, it is better to place them in a moist petri dish in the refrigerator for a few hours where the cold will cause them to relax and immobilize. Then they can be placed in preservative and their anatomic shape will be preserved. If feces is to be collected for parasite testing, it can be placed into formalin.

 

Any immunofluoroescent antibody testing usually requires that the specimen be preserved in Michel’s media, and not formalin. Some immunochemistry techniques can use fixed tissue, but conferring with the lab before the necropsy will help you plan what you need in the way of specimen preparation for immunologic testing.

 

Rabies suspects

If an animal had neurologic signs before death, encephalitis due to rabies is always a concern.  Or if the animal bit anyone within 10 days before death, it is required that the brain be tested for rabies. Direct fluorescent antibody (DFA) testing is the standard and most reliable test for postmortem diagnosis of rabies in animals. Fresh brain is needed for DFA testing. Other tests are available that can use frozen or fixed specimens, but you will need to check with the lab in order to give them the specimen they want for the test they will perform.  

Generally, the entire body of small animals such as bats can be submitted for rabies testing. The head and a small portion of the upper neck are submitted from larger animals such as dogs or cats. But you should always check with the lab first to see what kind of specimen they will accept.

Remember rabies is a zoonosis. When working with a rabies suspect, protective equipment is a must. It is rare, but rabies virus can be transmitted through mucosal membranes, and not just through a wound or a bite. Therefore, you should wear protective garments, goggles, mask, shoe covers, and gloves, and follow strict universal precautions when handling tissues from rabies suspects. Never advise a client to prepare and submit a rabies suspect specimen by themselves.

Rabies testing in most jurisdictions is handled through the local health department. However, your regional animal health laboratory can be helpful in matters concerning potential rabies cases. The local animal control departments should be contacted as well for assistance in handling situations involving rabies cases. Persons exposed to rabies suspects should contact their physician for treatment and advice.


Shipping requirements

Many laboratories utilize a courier service for the timely shipment of specimens. If specimens are to be shipped, they must be properly packaged in leak-proof containers so that package handlers or the public are not exposed to any chemicals or infectious agents. Plastic non-breakable containers should be used as a safety precaution. There are strict regulations regarding the shipment of clinical diagnostic specimens.

A diagnostic specimen is defined as "any human or animal material including excreta,
secreta, blood and its components, tissue, and tissue fluids being transported for diagnostic or investigational purposes, but excluding live infected humans or animals." Specimens treated with formalin generally do not contain infectious organisms and are usually exempt from regulations for infectious material. However, formalin specimens must be properly packaged in leak-proof containers and absorbent padding. Diagnostic specimens that are subject to special regulations are listed on the Hazardous Material Tables of Title 49 CFR. For more information you can start by going to http://epa.gov/fedrgstr/EPA-IMPACT/2001/January/Day-22/i92.htm.  

Many laboratories will provide the containers, proper packaging materials, and protocol information for the proper packaging and transport of specimens to their lab.


Disposal

 

There are local ordinances regarding the acceptable disposal of animal cadavers. Even if burial is permitted, the body must be buried deeply enough or in such a way that the remains cannot be dug up by wildlife or roaming pets. There have been several instances in which animals were poisoned by ingesting the remains of animals that had been euthanized. The euthanasia solution stays in a carcass and will be pharmacologically active for some time.

 

Websites of interest

 

Necropsy collection techniques (caution: very graphic)

http://www.radil.missouri.edu/info/teaching/resources.asp

 

Virtual Necropsy of the mouse:

 

http://www3.niaid.nih.gov/labs/aboutlabs/cmb/InfectiousDiseasePathogenesisSection/mouseNecropsy/

 

Avian necropsy:

http://www.vin.com/WebLink.plx?URL=http://edis.ifas.ufl.edu/VM009

 

Sea turtle necropsy

 

http://sacs.vetmed.ufl.edu/wildlife/seaturtletechniques/index.htm

 

Pathology website: Press the continue button and go into the course index. Then within the course, you will find images of pathology specimens with the corresponding descriptive medical term. http://www.msu.edu/~ramosjo/gross.htm

 

Rabies:

 

http://www.cdc.gov/ncidod/dvrd/rabies

 

Rabies Compendium

http://www.nasphv.org/index.html

 

Lesson 3 Writing assignment

 

  1. Name at least 3 reasons for doing a necropsy.
  2. List 5 safety procedures that should be followed when performing a necropsy.
  3. A ruminant should be examined in left or right lateral recumbency?
  4. What is a cosmetic necropsy?
  5. What is a sacrificial procedure?
  6. How is the patency of the bile duct checked in those animals with a gall bladder?
  7. How soon after death should fish be necropsied?
  8. T or F  It is best to freeze cadavers if you can’t get to doing the necropsy right away.
  9. T or F A pet spider lying on its back is dead so you should instruct the client to put the little body into some alcohol to preserve it for necropsy.
  10. What can you do to collect a tissue specimen for bacterial culture if the tissue surface has already been handled and contaminated?
  11. What is the recommended ratio of tissue to formalin?
  12. What is the best specimen for rabies testing using direct immunofluorescence?
  13. What is the “pluck”?
  14. What is the purpose of incising the diaphragm before opening the thoracic cavity?
  15. What procedure should be followed to preserve adult internal parasites?
  16. What percent of formalin is routinely used to fix tissues? Name one exception
  17. List at least 2 precautions when handling formalin or other fixatives.
  18. List the criteria by which lesions should be described.
  19. what is menat by morphologic diagnosis- give an example.
  20. Name at least 3 specimens used for toxicology testing.
  21. What precautions are taken with bird necropsies?