NECROPSY PROCEDURES:
GENERAL CONSIDERATIONS



EM Cabana, DVM (CLSU, Phil '84), MVSt (UQ, Aus '91)
Asst Professor - Veterinary Pathology
College of Veterinary Science and Medicine
Central Luzon State University
Nueva Ecija 3120, Philippines
http://www2.mozcom.com/~emcdvm

INTRODUCTION

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. The necropsy procedure employed by veterinary students and practitioner alike varies from examiner to examiner and from specimen to specimen. The conduct of a particular routine depends largely on the individual preferences of the examiner, the availability of the materials and equipment for the examination, the condition or state of the carcass, the extent of the examination required, and the mode of examination requested by the client or owner. It is often observed that necropsy done by the uninitiated or the untrained is characterised not by the voluminous information gathered that have little importance to the diagnosis of a particular case in question. It is the absence of information vital to the formulation of a diagnosis. 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. One factor to consider in the formulation of a diagnosis is the accuracy of the data gathered. A systematic approach in necropsy is required to so that appropriate and adequate information be gathered during the examination.

This manual will describe the standard procedures for the necropsy examination of domestic animals adaptable to most laboratories and in field conditions. It is hoped that it will help practicing veterinarians and students alike to adapt a working routine in performing necropsy.

GENERAL PRINCIPLES

IMPORTANCE OF NECROPSY

The examination of dead or terminally ill animals offers opportunities in studying the processes involved in disease situations. Various medical imaging techniques have evolved in recent years providing adequate information on the morphologic alterations of organs and tissues following disease. However, necropsy still provides a first hand look on what really happened along the course of the disease. In poorly understood disease situations, tissue alterations resulting from or as a reaction to the disease process may or may not be detected during clinical examination. Results of clinical examination alone may not be sufficient to define the process involved. Thus, gross and microscopic examination of diseased organs and tissues may lend valuable information in understanding the pathogenesis of a disease.

Morphological changes when correctly recorded and interpreted provide a basis for correlating functional changes seen in a particular disease process. Not all disease processes will show dramatic morphologic alterations in organs and tissues. Some clues may be derived however from necropsy examination that will provide valuable information in the recognition of such functional disturbances. The challenge is on the examiner to recognise these clues.

While most students and veterinary practitioners would regard necropsy as purely of academic interest, the purpose of necropsy does not end on the recognition of the lesions alone. Skillfully performed necropsy with all the information gathered, accurately recorded and interpreted will provide valuable assistance in the formulation of animal health and production strategies aimed to prevent and control animal diseases in a herd or flock. The primary aims of necropsy are to uncover the cause of death of an animal by defining possible aetiology and pathogenesis to arrive at a diagnosis. Yet, the usefulness of necropsy resides on the application of the information gathered in the formulation of appropriate treatment, control and disease prevention measures.

TIME FOR NECROPSY

The best time for necropsy is immediately after death of an animal. This is because post mortem processes of decomposition (autolysis) follow at a fairly rapid rate that obscures subtle changes in organs and tissues. Also, post mortem invasion of the organs and tissues by normal microbial flora of the gut may make the isolation of the causative agent in question with difficulty or even impossible, especially in suspected bacterial infections.

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. This is particularly true if histopathological examination of the gastrointestinal tract is required. Its content of microorganisms, enzymes and digesta make its decomposition more rapid than the other parts of the animal cadaver. Thus, if examination of the gastrointestinal tract is anticipated, it is recommended to euthanase the moribund animal. Do the examination right away, than wait until the animal dies and miss and opportunity to have it examined in its fresh state.

Cool the cadaver rapidly in a refrigerator or freezer if it is not feasible to examine the animal immediately after death due to some constraints. This is done to delay the process of post mortem decomposition. For specimens submitted during off-hours or during weekends, cut open the abdomen and remove the viscera, particularly the segments of the gastrointestinal tract and examine the parts right away. The other parts of the cadaver may then be saved in a refrigerator and examined the next instance or day, although some degree of post mortem changes must be anticipated. 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 cadaver solid. Pack it in dry ice before shipping, observing the pertinent rules and regulation in the transport of suspected biological hazards. Never pack the cadaver with ordinary ice if it is anticipated that the specimen may not reach the laboratory within one or two hours. Even then, the whole specimen should be packed in a plastic bag and put enough ice packs above and below the cadaver in a Styrofoam container. Send the cadaver the soonest possible time, with the necessary information as will described later.

PLACE FOR NECROPSY

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.

Exercise extreme care 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.

DISPOSAL OF THE CARCASS

In the disposal of the animal cadaver after examination, incineration is still the best method for the disposal of small animal cadaver. Practical reason dictates that this procedure may not be suitable for large animals. The amount of time involved in the incineration and the amount of fuel required turning the large heap of flesh and bones into ash preclude this procedure. For both small and large animals, burying deep into the ground may be practical. However, the pit should be dug deep enough so that astray animals and other predators cannot have easy access to the disposed cadaver. Also, consider the chances of contaminating the surrounding and the odor that may emanate from it should the pit be not deep and large enough to fully hold and cover the whole carcass. Exercise caution in selecting the site for the pit. The site should not be away from sources of feed or where underground water is being pumped. All dead animal cadaver should be considered possible sources of contamination that should be disposed responsibly.

BASIC EQUIPMENT

The choice of equipment for necropsy depends on the size of the animal, the type of examination requested (whether routine or cosmetic necropsy), and the individual preferences of the examiner. For most purposes, two sharp knives, a pair of scissors and forceps, a metal probe, and an ordinary mechanic hacksaw will be sufficient. Preferences for the size of the knives depend on the size of the cadaver, ease of handling and safety. A steel rod or sharpening stone to keep the knives sharp is essential. The forceps could be a lockable scissors type or a lifting forceps with rat-toothed or serrated tips that grasp tissues without slipping. A mechanic hacksaw will prove useful for cutting bones and other hard structures. A metal probe made of stainless steel, copper or bronze, or an ordinary galvanised iron wire gauge 12 and about 10-12 inches long is useful in probing connections and patency of openings. Other useful equipment includes a small axe, mallet and chisel (for cutting bones especially in large animal cadaver), and an ordinary pruning shears (instead of a costotome) for cutting the rib cage, mandibular symphysis, and pelvic bones. Other tools or instruments may be included, as they prove useful and saves manual labor during the examination.

Weighing scales and measuring instruments like a millimeter rule and graduated cylinders or measuring cups are essential for accurately recording dimensions and volumes. Two specimen bottles, one half-filled with 10% neutral buffered formalin are required for containing samples of tissues and body fluids for laboratory examination. Sterile swabs and petri dishes for the collection of samples for microbiological examination (if required) should be made available. Other materials that may be needed include disposable syringes and needles, glass slides, and ordinary fishing twine or thread for tying up hollow organs.

PROTECTIVE CLOTHING

The wearing of protective clothing is not meant to prevent soiling and/or preserve the appearance of what is worn by the examiner underneath. It should protect the examiner from contamination with blood, tissues and body fluids from the cadaver that are potential carrier of infectious particles. The recommended protective clothing should provide comfort to the examiner while not compromising protection from possible contamination. The wearing of cotton coverall, rubber boots, gloves, and butcher's plastic vest is recommended and provide ample protection from contamination. These articles must be washed clean and disinfected after each use.

The common laboratory gown may used. However, the hanging flaps may easily soak with the cadaver's blood and body fluids with the examiner not noticing it, and thus exposing the examiner to contaminants. Although lacking any protective clothing, the laboratory gown will be sufficient. A pair of ordinary garden latex gloves of appropriate size is useful for necropsy. Compared with the surgeon's latex glove, the latter are less expensive, more durable and provide equal protection. Necropsy without wearing any gloves is an open invitation for contamination. The gloves should fit the hands and fingers of the examiner without interfering with manual dexterity and causing numbness. Loose, also very tight fitting gloves may cause undue interference in handling organs and tissues, not to mention the awkward look and the uncomfortable feeling it may render to the examiner.

THE NECROPSY RECORD

FORMS

The style and type of necropsy report forms vary from laboratories to laboratories depending on the mode of document storage and retrieval system in use. The current trend in computerised data storage and retrieval most often uses forms structured to suit the particular software or database program being used. Yet, the structured entries no matter how the data are processed and stored essentially contain the basic items that include the following:

1) Case Identification - includes the assigned case number, the case code (example degenerative condition, bacterial disease, viral disease, etc.), and the date of submission and examination;

2) Owner's Identification - includes the name, address, and phone number of the owner.

3) Specimen Identification - includes the species, breed, age, weight, sex, coat colour, animal number/name, and animal classification (example breeder, draft, pet, etc.), and identifying markings (example: brands, cowlicks, ear-notch number).

4) Clinical History - includes the details of clinical findings, signs and symptoms observed, clinical diagnosis, the referring veterinarian's name and address, and the type of necropsy requested (routine or cosmetic necropsy); It shall also include among other the number of dead and affected animals in the herd or flock. Describe the manner of spread of the disease in the herd or flock, and the type and standard of husbandry before and after such a condition was noted. Include also the date of first cases and subsequent losses, and prior treatment given to the animal;

5) Necropsy Findings - includes the results of the necropsy examination that may be arranged by organs/system or by the sequence of examination;

6) Results of Laboratory Examination - includes the samples collected during necropsy and the results of the examination done;

7) Diagnosis - the outcome of the examination;

8) Examiner's Information - includes the name, qualifications and signature of the one who did the examination and formulated the diagnosis.

RECORDING INFORMATION

During necropsy, it is best to record all findings as the examination progresses. Yet, thorough examination of the animal cadaver will consume a considerable amount of time. An assistant that will record the findings as the examination progresses may not be always present. Thus, it is recommended that the findings be recorded immediately after the examination and before the cadaver is disposed. If an organ or systems have been inadvertently missed during the examination, or the examiner for one reason of another forgot some details of the lesions, the specimen may still be available for reexamination. In the laboratory, the use of a tape recording unit is ideal if many specimens will be examined in a single day. This unit is practical for a busy laboratory with large necropsy specimen accession. Models of tape recording units that are particularly suitable include those with a microphone that can be pinned to the lapel and with either a foot switch or a wireless microphone, or that of a voice-activated recording unit. Although this will involve additional investment, the accuracy in recording information may far outweigh the cost should necropsy accession be large enough.

It is recommended to take at least a photograph of the animal and/or lesions noted in cases where legal proceeding are anticipated. In these cases, carefully record the location and appearance of identifying markings of the animal such brands, cowlicks, and ear notches. These data are particularly sought in establishing the identity of the animal particularly in insurance claims.

PREPARATION OF THE REPORT

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).

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. This may be condoned if the one who examined the specimen will also be the one who shall formulate the diagnosis. Even then, this will limit the usefulness and of the finished report, for necropsy findings are subject to many interpretations and that one examiner may or may not agree with the interpretation of another examiner. 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 visualise what were observed during the examination to enable them to make their own interpretation and possible diagnosis.

DESCRIBING LESIONS

Recognition of lesions requires a sound background in anatomy and pathology. Veterinary students should be aware of the appearance of normal structures and the species difference within and between species. However, as one gain experience in examining various animal species, recognition of the normal structures and what form an abnormality in the structure (or the lesion) becomes routine. Students of necropsy may find it useful to pay a visit to the local abattoir to observe the appearance of organs and tissues of domestic animal species. However, one must be aware that specimens submitted for necropsy most often shows post mortem changes that may mask or alter the appearance of otherwise normal structures. The recognition of these changes requires knowledge of pathology and considerable experience.

It is not uncommon for students and the uninitiated to have a problem in describing lesions. Although veterinary pathology is rich in descriptive terms that describe the forms and appearance of any given lesion, the use of adjectives from ordinary conversational language is desirable than having no descriptions at all. It is recommended to write plainly in the report that no remarkable alterations were noted in the organ/tissue examined should that be the case, than leaving it not mentioned in the report. Also, include in the report the organs not examined for one reason or another. For most purposes, the description of any given lesion should include the following information:

A. Solid organs:

1) Organs/tissue involved
2) Position, relations, and involvement of adjacent structures;
3) Size and shape;
4) Weight;
5) Colour (shade, tint, hues);
6) Appearance;
7) Consistency;
8) Texture or intact and cut surface;
9) Odour;

B. Hollow Organs:

1) Organs involved
2) Appearance;
3) Texture of intact and opened surfaces;
4) Contents, which should be qualified as to:

a) Nature
b) Volume/Amount
c) Consistency
d) Transparency
e) Colour;
f) Odour;


In describing lesions, it is best to keep the descriptions in as few but very descriptive words as possible. Refrain from using verbose descriptions that tend to distract attention and present no clear meaning. Weights and measure should be recorded using the metric system of notation. The approximation of the weights and measure is desirable than comparing what was observed to common or ordinary articles. For example, it is far better to approximate the size of a tumour as "about 4-5 centimeter in diameter" than recording it as "about the size of a 25-cent coin in diameter." Although the latter may be considered more acceptable than having no description at all, its use in the necropsy report should be avoided.

COLLECTION AND SUBMISSION OF SPECIMENS FOR LABORATORY EXAMINATION

SPECIMEN SUBMISSION

Necropsy lends support to other ancillary laboratory diagnostic procedures, and those specimens for laboratory examination may be routinely collected as the examination progresses. Different laboratories have different requirements on the type of specimen required and the modes of specimen preparation and preservation. Contact the laboratory where the specimen will be sent to ensure that appropriate and adequate samples are taken during the necropsy examination.

The specimen collected should be appropriately labeled for proper identification. Information required for the identification of the specimen intended for submission include the following:

1) The species identification
2) The details of clinical history
3) The relevant necropsy findings
4) Nature of the sample collected and the mode employed in the collection and preservation
5) The form or type of examination that is requested

It is the responsibility of the examiner to notify relevant government offices in cases where the specimen is collected from suspected cases of highly contagious, zoonotic or exotic disease. Adequately label specimens taken from such cases to warn others about the potential of spreading the infection or pose danger to the biological system, or to the courier of the specimen should improper handling occur. The full descriptions of the methods employed in specimen collection and preservation are beyond the scope of this work, and the readers are referred to the appropriate text in clinical pathology. However, a short guide will be discussed in the proceeding section.

Submission for Histopathological Examination

Routinely fix specimens intended for histopathological examinations in 10% neutral buffered formalin. The pieces of organs and tissues should be collected the soonest possible time, and should not be more than 0.5 cm thick. Collect the tissue block using a sharp knife or a razor blade, exercising care not to crush or allow the tissue blocks to dry. Crushing the specimen or allowing it to dry will cause undue distortions on the morphology of cells and tissues. Moreover, the tissue block should be selected and should include both the normal and abnormal portion of the organ or tissue. Fix the collected tissue blocks immediately in 10 times the tissue volume of 10% neutral buffered formalin. Wash the specimen in physiological saline solution before fixing it in formalin if it is heavily soiled with debris.

Fix specimens of the brain by cannulating the carotid artery and pumping the formalin until what flows in the jugular vein and the carotid artery at the other side is clear of blood. Alternatively, the whole brain may be immersed in a large volume of the fixative. Allow it to harden for 24 hours, and then slice the brain and take the desired sections. Segments of the gastrointestinal tract should be taken the soonest possible time and immediately after opening the cadaver to minimise post mortem changes. Cut open the segment of the gastrointestinal tract longitudinally before putting the segment in the fixative to ensure adequate and prompt preservation of the mucosal lining, and to increase the surface area for the penetration of the fixative.

Submission for Microbiological Examination

Collect specimens intended for microbiological examination aseptically. It is recommended to sear the surface of the organ or tissue with a hot spatula, then incise and collect the required material from the deeper portion of solid organs, abscess, or coagulated masses. From this incision, sterile swabs, tissue fragments, and aspirates may then be taken. Place sterile swabs and aspirates in a special transport media, especially if the suspect organism is a fastidious one. The choice of transport media depends largely on the microorganism suspected to be present in the specimen. Should sterile swabs be required to be taken from body cavities or openings, the swabs should be taken immediately before fully opening such part of the animal cadaver. Hollow organs such as segments of the gastrointestinal tract are best handled by obtaining a loop tied at both ends and placed in a sterile petri dish.

Submission for Toxicological Examination

Materials for toxicological examination should be taken free from any contaminating chemicals being used during necropsy. Chemicals that may contaminate the specimen include fixatives, detergents and disinfectants routinely used during necropsy. Although different toxicants require specific samples for chemical analyses, the following samples may be taken if toxicological examination is anticipated:

1) Whole blood and sera
2) Tissue blocks (about 100 grams) of both liver and kidneys
3) Urine
4) Stomach and intestinal contents

Contact the toxicology laboratory where the samples will be sent to ensure that the right specimen and amount are collected and the adequate precaution on handling and preservation is observed.

Submission for Parasitological Examination

Occasionally, ectoparasites and endoparasites are collected during necropsy for identification. Collect samples of ectoparasite before the cadaver is cut open for examination. Ticks, fleas and lice should be carefully brushed off from the fur or feather and fixed in formalin. To disable these organisms, wet the fur or feather of the animal with a detergent solution. In collecting ticks, prevent the mouthparts from damage, by wiping the body part of the tick with ether. This will kill the tick and allow it to drop. Fix the collected specimens in 70% ethyl alcohol or in 10% formalin. Information about the degree of ectoparasite infestation should be provided along with the submission. Collect mange mites by scrapping the affected skin deeply, and put the scrapings in a glass slide with a drop of mineral oil.

Roundworms collected from intestinal segments may be fixed in formalin immediately after collection. Allow it to relax by dipping in menthol solution or lukewarm water before fixation to prevent curling of the specimen. Tapeworm segments collected should include both mature and immature segments, with the scolex still intact. Never lift the tapeworm from its attachment for it will break the scolex. The scolex is important in species identification. Excise the part where the scolex is attached and fix them in formalin. Press specimens of cestodes between two glass slides held together by a rubber band, a piece of twine or paper clips before immersion in the fixative. For total worm count in ruminants, tie the abomasum at both ends and save all its contents. Scrapping deeply the mucosa of the affected intestinal segment and examining the scrapings as a wet smear may do the diagnosis of coccidial infection in poultry.

Submission for Cytological Examination

Smears from tumour tissues are prepared occasionally for cytological evaluation. It is often possible to have a diagnosis by preparing and examining smears (from cut surfaces of the tumour), and aspirates even before tissue blocks of the tumour tissues are processed by conventional paraffin embedding technique. Allow the smears to dry immediately after collection to preserve the cell architecture. Fixation may be done by heat treatment, or by dipping in absolute methanol. Some workers suggested the use of hair spray, which presumably contain alcohol in a concentration that is sufficient to fix the cells in the smears.

Smears may be prepared from body fluids by putting a few drops onto the glass slide. The fluid may be collected and centrifuged and the cell pellet at the bottom of the tube fixed in formalin or methanol and the smears prepared. Alternatively, use gelatin or albumin to suspend the cell pellet and then fix the mixture in formalin. This preparation may then be submitted for routine paraffin embedding technique similar to a tissue block.

Submission of Blood and Body Fluids

Blood samples should be collected prior to euthanasia of moribund animals. In some cases, blood samples may still be obtained in animals that have been dead from three to four hours. This is done by aspirating the blood contained in the heart prior to detachment and dissection of the chambers. The addition of anticoagulants (e.g., ethylenediaminetetraacetic acid or EDTA) should be considered if plasma is to be extracted. If sera are required, it is best to collect blood in a glass receptacle to promote clotting. Alternatively, glass beads may be added.

The general rule in collecting body fluid is to obtain samples free from contaminants. Body fluids should be collected as the examination progresses if it is anticipated that such examination is required. Ascitic fluid should be qualified as to amount, colour and turbidity. Aspirating directly from the urinary bladder may collect urine. Cerebrospinal fluid should be collected prior to opening the brain. This should be done by aspirating the fluid through the aid of syringe and needle at the cisterna magna.