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V 20TH ANNIVERSARY Vol. 21, No.

10 October 1999

CE Refereed Peer Review

A Practitioner’s Guide
FOCAL POINT to Necropsy
★A systematic necropsy helps Auburn University
ensure that each body system Eva A. Sartin, DVM, PhD
suspected to be involved in Joseph S. Spano, DVM, PhD
illness or death will be examined Terri L. Hathcock, BS, MS
effectively and that appropriate
samples are collected. ABSTRACT: A well-performed necropsy can provide useful information to veterinarians and
pet owners. This article provides an overview of the necropsy examination procedure using an
organ system approach, which allows for variation depending on the clinical signs seen in the
KEY FACTS live animal and lesions observed during the necropsy. Proper specimen collection for microbi-
ology, cytology, and histopathology is emphasized.
■ Necropsy should be performed as

N
soon after death as possible or ecropsies frequently answer many questions that veterinarians and pet own-
within 24 hours if the body has ers have concerning an animal’s illness or death, such as the cause, reason
been refrigerated. for the lack of response to therapy, and legal issues. In addition, knowledge
gained from a necropsy performed on an animal with a poorly defined clinical illness
■ Microbiologic specimens should may offer helpful insight into the treatment of other animals with similar conditions.
be generous in size or volume This article provides an overview of a systematic necropsy technique for small
and placed in commercial animals, with emphasis on sample collection for further diagnostic evaluation.
transport containers. Specific comments on some gross changes are made to help direct practitioners
in the examination process. Excellent detailed information on normal and ab-
■ Samples taken for histopathology normal tissue characteristics, necropsy technique, and indications for sample col-
should be no thicker than 3/8 inch lection is available in the literature.1–4
and preserved at a 10:1 volume
ratio of 10% buffered formalin: GETTING STARTED
tissue. It is best to perform a necropsy immediately after death. When this is not pos-
sible, the body must be refrigerated immediately after death and the necropsy
■ Bone marrow evaluation performed within 24 hours. If refrigeration is not available, euthanasia can be
should be conducted using planned for a time when necropsy can be performed without delay. Otherwise,
touch impressions taken as soon tissues will autolyze and become difficult or impossible to evaluate.
after death as possible. Before beginning the necropsy, practitioners must first anticipate their needs
for the procedure and collect the appropriate materials. The safety of veterinari-
■ Historical, clinical, laboratory, ans and live patients is important, so common sense must be used; gloves and
and necropsy findings are protective eye wear and clothing are needed. Instruments for the gross examina-
essential for interpretation of tion include a knife or scalpel, scissors, forceps, and bone cutters. Other require-
samples and should accompany ments include specimen transport tubes and sterile instruments (for microbiolo-
specimens submitted for gy samples), clean glass slides and EDTA tubes (for cytologic evaluation), and a
diagnosis. large-mouth container of 10% buffered formalin (for histopathology samples).

SAMPLE COLLECTION AND PREPARATION


The results of laboratory examination of specimens depend on the quality of
Compendium October 1999 20TH ANNIVERSARY Small Animal/Exotics

submitted specimens; proper technique is therefore vi- normal skin should be collected for histopathology.
tal. Using collection techniques that minimize contami- When multiple lesions are present, samples of early-
nation of microbiology samples improves the quality of stage lesions as well as progressing lesions should be ob-
the results. Urine, fluids, and aspirates can be collected tained. More information is obtained by examining
using a syringe and needle. Microbiologic specimens early and progressing lesions than those that are chronic
should be generous in size or volume and placed in and healing. All mammary glands and the lymph nodes
commercial transport containers. The buffering agents that drain them should be examined and then sec-
in the transport media preserve the integrity of speci- tioned to reveal masses or other abnormalities.
mens and survival of organisms without contaminating
overgrowth. Samples should be transported immediate- Reflecting Limbs and Skin
ly after collection; however, when a delay is unavoid- Necropsies can be performed with animals in dorsal
able, the samples can be refrigerated at 4˚C. If shipping or lateral recumbency. Lateral recumbency is easier for
is necessary, overnight delivery should be used and cool large, deep-chested dogs. Limbs can be reflected by in-
packs added to the shipping container. cising the skin and soft tissues at the axillae and cox-
Cytologic evaluation of some necropsy specimens can ofemoral joints of the uppermost limbs (for animals in
be an important adjunct to postmortem diagnosis. Liv- lateral recumbency) or of all four limbs (for animals in
er, kidney, lymph node, spleen, and bone marrow are a dorsal recumbency). Skin should be reflected from the
few tissues that can be easily sampled for cytologic eval- abdomen and thorax.
uation and provide useful, sometimes diagnostically de-
cisive, information. The freshly cut surface of the tissue Peritoneal Cavity
being sampled should be lightly blotted with a paper The abdomen should be opened prior to the thorax
towel or gauze sponge to remove excess fluids. A clean to check the position of the diaphragm before releasing
slide should then be gently touched to the tissue’s sur- the negative pressure of the thorax. The abdomen is
face, lifted straight up (avoiding side-to-side move- opened from the xiphoid to the pubis and the abdomi-
ment), and quickly air-dried. Body cavity effusions, nal wall reflected to obtain maximum exposure of its
synovial fluid, and cerebrospinal fluid collected at contents. The color, consistency, transparency, and
necropsy can also provide important diagnostic clues. A odor of the peritoneal fluid should be noted. Peritoneal
fluid sample should be collected in an anticoagulant fluid abnormalities may result from peritonitis, rup-
(preferably EDTA) for routine cell count, protein de- tured visceral or vascular structure, or neoplasia. This
termination, and differentiation of cell types. Slides determination can be aided by microbiologic and cyto-
must not be exposed to formalin fumes because they logic examination of fluid (see Sample Collection and
greatly impair staining quality. Any commercially avail- Preparation section). Samples for creatinine analysis of
able quick stain can be used. Details of cytologic exam- peritoneal fluid and blood may also be helpful. After
ination and diagnosis have been reviewed elsewhere.2 fluid samples are obtained, organs should be checked
Histopathology samples should be no thicker than 3/8 for displacement.
inch and preserved in 10% buffered formalin at a for-
malin:tissue volume ratio of 10:1. After 2 to 3 days, Abdominal and Pelvic Viscera
samples fixed at this ratio can be placed in a smaller The position of the diaphragm must be checked be-
volume of formalin for shipment. Frozen specimens are fore removing the abdominal viscera. A diaphragm that
not suitable for histologic examination. is not convex indicates there is loss of negative thoracic
pressure, a space-occupying lesion, or increased thoracic
NECROPSY PROCEDURE fluid. As the diaphragm is punctured, an inrush of air
Like the clinical workup, the necropsy evaluation in- occurs as negative pressure is lost and the diaphragm re-
cludes a general examination with emphasis on organ laxes.
systems that have been noted to be abnormal. Unex- Before removing the gastrointestinal tract, the paten-
pected findings discovered during necropsy may change cy of the bile duct should be checked by squeezing the
the original focus of the examination. The examination gallbladder and observing the duodenum for filling.
begins by noting the animal’s body condition, skin ab- The bile duct can then be transected and the duode-
normalities, discharges, diarrhea, discoloration, nod- num freed from the liver. Removing the gastrointestinal
ules, masses, or anomalies. viscera involves transecting the abdominal esophagus,
freeing the stomach and spleen from their attachments,
Integumentary System and removing the small and large intestine from the
Samples of cutaneous lesions that include adjacent mesentery. The distal colon can then be transected and

COLLECTION TECHNIQUES ■ CYTOLOGIC EVALUATION ■ REMOVING GASTROINTESTINAL VISCERA


Small Animal/Exotics 20TH ANNIVERSARY Compendium October 1999

the entire intestinal tract removed. If a pelvic cavity le- from the mandible and the hyoid bones and soft palate
sion is suspected, the pelvic floor can be removed by cut to free the larynx. The oral, cervical, and thoracic
cutting through the bone on each side of the pelvic viscera can then be easily removed together.
symphysis. The colon and urogenital tract can be freed
from the pelvic cavity by cutting the soft tissue around Respiratory System
the anus and external genitalia. This approach is help- The nasal cavities, larynx, external surfaces of the
ful when evaluating intestinal, reproductive, and lower lungs, and mediastinum should be examined for nod-
urinary tracts. The adrenals should be removed and ules, masses, and pulmonary consolidation. The lungs
carefully sectioned to determine the cortical:medullary ra- must be palpated gently because just one well-inten-
tio (which should be approximately 2:1). The adrenals tioned squeeze can induce many artifacts. The larynx,
must then be immediately placed into formalin. The trachea, bronchi, and smaller airways should be opened
liver, kidneys, and diaphragm can then be removed. from proximal to distal. The content of the airway lu-
mens should be examined for fluid and exudate before
Oral, Cervical, and Thoracic Viscera rinsing with water. Color change, which is a common
The first step in examination of the thoracic cavity is postmortem change in lung tissue, must correlate with
to remove the ribs and take samples of pleural fluid for palpable abnormalities to be considered significant.
culture and/or cytology if indicated. Next, the skin Opened airways and vessels should be examined for ex-
should be reflected laterally from the mandibular sym- udate, thrombi, and/or parasites. Samples containing
physis to the thoracic inlet and the ventral cervical mus- lesions should also include normal tissue.
culature removed to expose the thyroid and parathyroid
glands. After these glands have been removed, they Cardiovascular System
should be placed into fixative without sectioning, unless After death, the lower portions of the body (i.e., the
they are greatly enlarged. The tongue should be freed “down side”) will become darker (hypostatic congestion)

Opening the Heart


■ Open the right atrium (RA) completely
with scissors.
■ (A) Open the right ventricle (RV)
by placing scissors through the
atrioventricular (AV) valve next to the
interventricular septum and cutting
the free wall of the RV adjacent to the
septum, following a line from the AV
valve to the apex.
■ (B) Continue opening the RV by
cutting the free wall of the RV along
the septum through the pulmonic A B

valve and well into the pulmonary Figures A and B— The heart should be opened along the dotted lines in the
right side of the heart (as shown in A), starting with the right atrium (1) and
artery.
continuing into the right ventricle (2) and (as shown in B) beginning the pul-
■ Open the left atrium. (B) Place scissors monary artery (3). The left heart can then be opened along the dotted lines
into this opening, through the left AV (as shown in B) in the left atrium and continuing into the left ventricle (4).
valve, and into the left ventricle (LV).
Balance the convexity of the LV across the blade, and cut to open the entire LV. To examine the aortic valve, cut
through the septal leaflet of the left AV valve.
■ Gently rinse away blood to inspect the chambers, valves, and vessels. Check endocardial surfaces, valves, and
vessels for abnormalities. Make several sections into the myocardium, and look for areas of discoloration or
masses. The LV:RV wall-thickness ratio should be approximately 3:1 in adults and 1:1 in neonates.

REMOVING ADRENALS ■ PALPATING LUNGS ■ COLOR CHANGE IN LUNG TISSUE


Compendium October 1999 20TH ANNIVERSARY Small Animal/Exotics

than the upper portions. Clin- creatic and mesenteric fat are
ical findings and lesions (e.g., characteristic of fat necrosis, as
petechiae, ecchymoses, severe is seen with pancreatitis.
hemorrhage, edema, icterus)
can be used as clues for fo- Urinary System
cusing dissections. Clinicians The length of the kidney
should look for evidence of should approximate the length
thrombi or hemorrhage as- of three vertebrae. It should
sociated with vessels they be sectioned midsagitally and
transect. the cut surface examined (the
The pericardial sac should normal cortex:medullary ra-
be examined, the amount tio in the sagittal plane is
and characteristics of its con- approximately 1:2 to 1:3).
tent noted, and samples col- The capsule is removed and
lected for culture or cytology Figure 1—Obtaining a sample of a tubular organ without the subcapsular surface in-
if indicated. The inner sur- damaging the mucosal surface. Note that the forceps do not spected for depressions/scars,
face of the pericardium should come into contact with the mucosa of the sample to be sub- nodules, petechiae, abscess-
mitted (i.e., between the dotted lines).
be examined and the peri- es, infarcts, or other lesions.
cardial sac removed, noting Samples should be collected
the shape of the heart and for culture or cytology if in-
examining the epicardial sur- dicated. Samples for histo-
face. The heart can be opened pathology should extend
following the path of normal from the outer cortex to the
blood flow (see Opening the pelvis (Figure 2).
Heart). The ureters, urinary blad-
der, and urethra should be
Digestive System examined externally and then
The oral cavity and exter- the urinary bladder partially
nal surfaces of the alimentary opened to note the charac-
tract should be examined be- teristics of the urine. Sam-
fore opening the esophagus, ples for culture and/or cy-
stomach (along the greater tology should be taken if
curvature), and entire intes- Figure 2—Obtaining a kidney sample that contains all parts indicated. The opening in
of the nephron: The sample should extend from the outer
tine. Microbiologic fecal speci- cortex to the pelvis. the urinary bladder can then
mens can be collected at this be enlarged, extending from
time and placed in a trans- the fundus through the
port container specific for stool specimens. The mucosa neck and into the urethra (including the prostatic ure-
should be rinsed gently with a stream of water because thra in males; Figure 3). Bladder tissue samples are ob-
spraying or rubbing the mucosa will cause loss of mucosa tained for histopathology as shown in Figure 1. If there
and possibly a significant lesion. Samples for histopathol- is evidence of obstruction, the entire urethra and
ogy are obtained with forceps and scissors (Figure 1), ureters should be opened.
again taking care not to rub the mucosa. Normal tissue
should be included with the abnormal samples. Genital System
The liver should be examined for scars or nodules. The external examination should reveal any abnor-
Nodules could be abscesses, granulomas, hyperplasia, or malities of the genitalia. The size and external and cut
metastatic neoplasia, and thus samples for culture, cytology, surfaces of the gonads of sexually intact animals should
and histopathology should be collected if indicated. Be- be examined for cysts, nodules, exudate, and scars. In
cause diffuse liver lesions may be subtle, samples for females, the reproductive tract should be opened and
histopathology should be obtained even if lesions are not any samples needed for culture or cytology collected
visible. The next step is to open and examine the gallblad- before the mucosa is examined. Samples for histo-
der and bile duct. The pancreas should be examined for ev- pathology should be obtained as shown in Figure 1. In
idence of hemorrhage or inflammation. Small (i.e., 2 to 4 males, the size, shape, color, and consistency of the
mm), sharply defined, white to yellow foci in the peripan- prostate must be noted. The prostate can be sectioned

PERICARDIAL SAC ■ LIVER LESIONS ■ OBTAINING KIDNEY SAMPLES


Small Animal/Exotics 20TH ANNIVERSARY Compendium October 1999

as shown in Figure 3 and Musculoskeletal System


checked for masses, discol- Joints should be opened
oration, or exudate. by carefully incising the cap-
sule to avoid contamination.
Hematopoietic and If there are abnormalities in
Lymphatic Systems the synovial fluid or mem-
Cytologic examination of brane, samples for culture,
bone marrow specimens can cytology, and histopathology
provide important clues in de- should be obtained as indi-
termining the cause of hema- cated. The articular cartilage
tologic abnormalities. Sam- should be examined for dis-
ples collected as soon after coloration and irregularities.
death as possible are of the Determining which por-
greatest diagnostic value. A Figure 3—Opening the urinary bladder and prostatic urethra. tions of bone to select when
bone marrow sample can be (Top) Note that the incision in the bladder extends from the sampling bone lesions can be
obtained by cracking open a bladder to the prostatic urethra. (Bottom) Prostatic samples difficult. If possible, the en-
long bone, scooping the mar- taken along the double row of dotted lines will allow histo- tire bone—or at least a por-
row from the extremities of logic examination of urethral mucosa in the prostate. tion containing the lesion with
the shaft, and making touch adjacent normal bone—should
impressions. Interpretation of be collected for histopatholo-
stained bone marrow speci- gy. Areas of necrosis and pe-
mens is reviewed elsewhere.2 riosteal proliferation may
A second sample of bone mar- complicate sample selection,
row can be placed in forma- so the sample must consist of
lin for histopathology. viable tissue and extend deep
Lymph nodes should be ex- into subcortical bone and
amined as they are exposed marrow.
during the necropsy proce- Breaking strength and de-
dure. Enlarged nodes and gree of calcification can be
those draining sites of inflam- tested by isolating and bend-
mation or neoplasia should be ing a rib. Ribs that bend easily
sectioned to check the cortical indicate poor calcification and
and medullary architecture. suggest dietary mineral imbal-
The spleen and thymus are ance, severe renal disease, or
examined by making multiple parathyroid disease. When
sections into the splenic and generalized bone disease is
thymic parenchyma and look- suspected, samples of flat and
ing for abnormalities. Histo- long bones should be taken.
pathology and cytology sam- If there is clinical evidence
ples of lymph node, thymus, of generalized muscle dis-
or spleen should be taken ease, random incisions in var-
from areas that are free of ex- ious muscle groups, includ-
cessive blood or necrosis. ing the diaphragm, tongue,
Figure 4—Opening the calvarium to remove the brain. Bilat- and heart, should be made
Endocrine System eral cuts begin at the foramen magnum and dorsomedial to to look for abnormalities.
Thyroids, parathyroids, and each occipital condyle and extend to just behind the orbits. A Multiple samples should be
adrenals are examined when third cut that joins the two previous cuts is then made. taken from various sites, even
the viscera are removed. If if they appear normal. Sam-
the adrenal cortex:medullary ratio is not approximately ples of focal lesions should include normal tissue.
2:1, then the pituitary should be examined following
removal of the brain. The pancreas and adjacent Eyes and Nervous System
mesenteric adipose tissue should be examined for evi- The eyes should be removed and peripheral nerves ex-
dence of hypoplasia, neoplasia, or inflammation. amined if indicated by clinical findings. Eyes are removed

BONE MARROW SPECIMENS ■ SELECTING BONE SAMPLES ■ TESTING CALCIFICATION


Small Animal/Exotics 20TH ANNIVERSARY Compendium October 1999

by routine enucleation. The extraocular muscles should be be placed in formalin.


trimmed carefully, without puncturing the globe, and the If the animal had clinical signs of spinal cord disease,
globe placed in fixative intact. Peripheral nerves can be ex- the epaxial muscles and dorsal vertebral arches should
amined as they are exposed during necropsy. Clinical find- be removed as is done with a dorsal laminectomy. The
ings will determine the extent of the peripheral nerve ex- spinal cord should be carefully removed by gently
amination. If nerves contain or are associated with masses grasping the dura with forceps, lifting slightly, and
or nodules, samples should be obtained for histopathology. transecting each spinal nerve root as progress is made
If indicated by the history, the brain and spinal cord from cranial to caudal. Care must be taken to not bend
are removed. The brain is easier to remove if the head is the spinal cord sharply during removal because this will
disarticulated at the atlanto-occipital joint. The temporal induce artifacts. The entire spinal cord should be
muscles can be removed and the cranium opened as placed in formalin.
shown in Figure 4. Bilateral cuts are made through the
cranium, beginning at the foramen magnum on the dor- COMMENTS
somedial aspect of each occipital condyle and extending Samples that are easily folded and placed into a con-
to just posterior to the orbits in the cranium. One cut is tainer with a small opening become hardened with fixa-
then made into the cranium just behind the orbits, join- tion and can be very difficult to remove. In some cases,
ing the two previous cuts. The detached portion of the the sample may be damaged or destroyed when its ex-
calvarium should be pried upward and carefully re- traction from the container is difficult. With this in
moved. The overlying dura mind, specimens should be placed in containers with
and tentorium cerebelli are wide openings. Questions about sample submission
ENDIU
MP then gently removed. The should be directed to the laboratory that will be receiv-
M’

20th
 CO

head should be held upside ing the specimens. A good history with accurate descrip-
S

9 9 9
1 9 7
9 - 1

ANNIVERSARY
down while the cranial nerves tions of gross lesions is an essential part of sample sub-
are transected from caudal mission to a laboratory. Even if data are limited, this
to rostral. The brain should information significantly aids diagnostic examination
A LookBack be allowed to fall into the and interpretation.
clinician’s (or assistant’s) hand
Although the necropsy procedure as its weight and gentle pres-
has several variations, the basic sure pull it downward. ACKNOWLEDGMENT
techniques have not changed The pituitary should be The authors thank Lisa Makarchuck, Biomedical Commu-
significantly over the past 20 removed if there is an adreno- nications, College of Veterinary Medicine, Auburn Uni-
years. What have changed are cortical abnormality or clini- versity, for the excellent illustrative artwork.
the diagnostic tools that can be cal evidence of pituitary
applied to collected specimens. dysfunction. This can be
Advances in
done by cutting through its REFERENCES
dural attachment to the 1. Strafuss AC: Necropsy, in Pratt PW (ed): Laboratory Proce-
immunohistochemistry and dures for Veterinary Technicians, ed 3. St. Louis, Mosby, 1997,
floor of the cranial cavity.
molecular technology (e.g., pp 505–534.
The pituitary is very friable 2. Petrites-Murphy M: User’s guide to pathology services.
polymerase chain reaction, in and thus must be immedi- JAVMA 212:362–364, 1998.
situ hybridization) have ately placed in fixative. 3. Cowell RL, Tyler RD, Meinkotch JH: Diagnostic Cytology of
empowered veterinarians to If rabies is a diagnostic the Dog and Cat, ed 2. St. Louis, Mosby, 1999.
make great strides in diagnostic differential along with sev- 4. Hudson LC, Hamilton P: Atlas of Feline Anatomy for Veteri-
narians. Philadelphia, WB Saunders Co, 1993.
interpretation of specimens and eral other neurologic condi-
in understanding the tions, appropriate precau-
pathogenesis for many tions must be taken. The
infectious, neoplastic, metabolic, brain should be sectioned
sagittally down the midline About the Authors
and genetic disorders. Drs. Sartin and Spano and Ms. Hathcock are affiliated with
and half of it submitted for
rabies evaluation; the other the Department of Pathobiology, College of Veterinary
half is fixed in formalin for Medicine, Auburn University, Alabama. Drs. Sartin and
histopathology. If rabies is Spano are Diplomates of the American College of Veteri-
not suspected, the entire nary Pathologists.
unsectioned brain should

REMOVING THE BRAIN ■ RABIES ■ SPINAL CORD DISEASE