Introduction

Necropsy can be an invaluable tool for the exotic animal practitioner. Relative to traditional pet species, non-domestic patients may be less amenable to antemortem diagnostics due to size and temperament constraints and in some cases, necropsy may be the only method of obtaining information regarding these patients' medical conditions. Practicing clinicians can use the necropsy to identify the cause of death, to aid diagnostic investigations in "herd-health" situations or to monitor "herds" for subclinical disease processes (e.g., nutritional or parasitic), to self-educate following difficult or unusual clinical disease courses, or to help document novel diseases. Additionally, the gross postmortem examination can serve to reacquaint the clinician with the anatomy of species not extensively covered in traditional veterinary training programs. Regardless of the inciting cause for performing the necropsy, it serves as an educational tool for the practitioner and his/her staff and can help improve the care of future patients.

Necropsy Preparation

Patient Information

Prior to starting a necropsy, basic information should be obtained. Necessary historical information is similar to what you would ask an owner during an appointment. This includes housing and diet parameters, inter- or intraspecific contact, travel history, source (captive born or wild caught), and symptoms of disease and their duration. Additionally, the prosector should be informed of diagnostic test results and clinical suspicions. When sending carcasses or tissues out for pathology, such suspicions are occasionally omitted, potentially impairing the pathologist's ability to interpret the entire case and at the expense of the final report or even pathologist safety in the case of zoonotic diseases. Site visits can add historical information in mortalities suspected to be related to environmental hazards, including intoxications or husbandry aberrations. For aquatic species (including certain amphibians and reptiles), complete water quality parameters are an essential component of the history.

Instrumentation and Personal Protective Equipment

Most instrumentation required for performing a necropsy, especially for small exotic species, can be found in many veterinary hospitals and/or local hardware stores (Table 1).

Protection of the prosector during postmortem examinations is of upmost importance. This includes paying careful attention to avoid accidental injury. For all cases, a surgical mask, latex gloves and an apron or dedicated outer clothing is recommended and eye protection should be worn while cutting bone. For cases in which a zoonotic disease is suspected, in-house necropsy is not recommended and veterinarians should contact the state animal diagnostic lab for assistance. The necropsy of venomous animals carries its own inherent hazards. For these cases, consider wrapping the head in elastic surgical wrap or tape and decapitating the animal prior to necropsy. Immersion in formalin deactivates most components of venom from many species; the possibility of incomplete inactivation encourages further handling be performed by experienced individuals with appropriate protective gear.

Documentation

It is important to document all findings during the necropsy. This includes detailed descriptions of any lesion or presumed abnormality. A good description includes size, shape, texture/firmness, color and potentially odor. It may include the percentage of the organ affected. All measurements should include three dimensions, except flat areas of discoloration. Modifiers such as "severely" or "mildly" should be avoided; more objective descriptors including dimensions or volume are preferred. A basic gross necropsy form and a tissue checklist to help maintain organized record-keeping are available as supplements to this manuscript.

Photographic documentation is of utmost importance for the novice prosector to assist pathologists in determining lesion significance. Included in all photographs should be a scale and animal identification. Dirt and excess blood should be removed. The lesion is ideally placed in the center of the image and efforts should be made to reduce glare. It is often helpful to take an overview or low magnification image for orientation before zooming in to the desired subject. Photographing the lesion in situ and again isolated on a solid background is helpful and makes for more visually appealing images, particularly for publication. Keep in mind that it is easier to digitally enhance underexposed images than overexposed ones.

Performing the Necropsy Exam

Introduction

The necropsy procedure is similar across species and taxa, in that the goal is to identify and document lesions and collect tissues for histopathology. Collecting a complete set of tissues is ideal, especially in cases where the cause of death cannot be determined grossly (see the tissue checklist) - this will be the only opportunity you will have to collect samples. Please consult the references provided^1-6 for description of necropsy techniques in different taxa and consider the following general tips:

  Hollow organs should be opened and solid organs incised multiple times ("bread-loafed")

  Incise lesions of adequate size to determine parenchymal invasion and cut section appearance

  For formalin samples, at least one dimension should not exceed 1.0 cm and is ideally 0.5 cm

  Tissue to formalin ratio should not exceed 1:10

Lesion Interpretation

Knowing the normal appearance of the internal viscera greatly assists the prosector in performing a good necropsy. Given that we treat countless different species across multiple taxa, the best way to build this knowledge is by performing postmortem exams. One of the most difficult aspects of performing a necropsy is determining the significance of the changes found. Although additional diagnostic modalities may be still required to obtain a final diagnosis (e.g., histopathology, cytology, culture), a basic understanding of why tissues take on different appearances can narrow your list of differential diagnoses and help guide intelligent decision-making regarding ancillary diagnostics.

Color changes are some of the more obvious aberrations detected at necropsy. However, changes in color can be misleading, so care must be taken not to assign too much emphasis on color alone. The prosector should consider other physical attributes, such as texture/firmness, before deciding if the change is a true lesion. Possible causes of diffuse pallor of an organ include increased lipid, increased fibrous connective tissue (scarring), edema, diffuse neoplastic infiltration (typically lymphoma or other blood cancer), or reduced perfusion including anemia. Multifocal areas of pale tan discoloration can be due to inflammatory infiltrates, neoplastic processes or necrosis. Differentiation of these processes requires integration of other factors such as distribution, size and texture/firmness. Alternatively, organs can appear diffusely darker than normal, although this occurrence is less common. This is typically the result of atrophy, but can also be due to increased blood flow. Red discoloration generally indicates increased blood flow or hemorrhage, the former often being the result of inflammation. In the lungs, redness can indicate inflammation or hemorrhage, but can also be due to reduced aeration (atelectasis, postmortem diffusion) due to superimposition of alveolar vasculature. Multifocal hemorrhages (petechiae, ecchymoses) can be due to primary coagulation abnormalities and can also appear in septic patients. Causes of multifocal black or dark brown discoloration include melanomacrophage hyperplasia (reptiles, amphibians and fish), pigmented neoplasia (melanoma), necrosis, or in the lungs, anthracosis. Green discoloration is often the result of postmortem leaching of bile from the gall bladder to adjacent tissues. Less common causes of green discoloration include chronic hemorrhage in avian species (biliverdin) and eosinophilic inflammation. Specific color changes include orange discoloration in the liver due to diffuse amyloidosis, pale yellow coloration of steroid-producing tumors, brownish discoloration of the lungs in some heart failure patients, and the diffuse yellow pallor of icterus in mammals.

In addition to color, alterations in normal organ size can be an important indication of disease. Enlargement of organs can be diffuse or nodular. Regardless of distribution, increased size indicates infiltration, typically by fluid or additional cells. This includes inflammatory cells, neoplastic cells, edema or hemorrhage. It may also be the result of an increase in the number (hyperplasia) or size (hypertrophy) of normal cells. While some neoplastic processes can cause diffuse enlargement of an organ (lymphoma and other blood cancers), this change is often due to inflammatory or hyperplastic conditions. Acute processes are more likely to result in enlargement of a tissue due to influx of inflammatory cells, edema and/or hemorrhage. Physiologic enlargement of some organs can be a normal process - mammary development during pregnancy and seasonal changes in testicular size (which can be marked in birds, reptiles and amphibians) are examples. There are multiple ways to determine if organ size is excessive, even if preexisting knowledge is absent. Organs with typically sharp margins (liver, mammalian spleen) will become rounded at the edges and encapsulated organs may have a turgid appearance or feel. Additionally, enlarged tissues may bulge from the cut surface due to release of accumulated pressure resulting from infiltration. Certain tissues can have specific appearances when enlarged beyond normal. In birds, for example, identification of hepatomegaly can be made if the liver extends far beyond the caudal aspect of the keel. In non-lissencephalic animals, enlargement of the brain (typically the result of edema if diffuse) results in shallow sulci. Finally, assessment of paired organs for asymmetry can indicate unilateral changes in size. Reduction in normal size is less common than enlargement and, if diffuse, can be the result of atrophy or fibrosis. Fibrosis can also result in focal contraction, as in chronic renal infarcts.

Alterations in texture and firmness can also help with the interpretation of lesions. Increased firmness typically indicates fibrosis or atrophy (due to a relative increase in connective tissue). While overly soft tissues can imply necrosis, I find that softness most often indicates autolysis. Tissues that are easily broken, especially the liver, may contain excessive lipid or amyloid. A cobblestone surface on solid organs often indicates fibrosis which may be accompanied by nodular regeneration. When examining mucosal surfaces, change in texture from shiny and moist to dry or granular indicates interruption of the mucosal epithelium. This can be the result of erosion or ulceration, extensive inflammation or metaplastic transformation. Alterations from the normal spongy texture of the lungs are important to assess. Firm or rubbery lungs indicate filling by something other than air, i.e., fluid or inflammation, and cut section may release accumulated fluid in those cases. Severely affected lungs may sink in formalin.

The final aspect of lesion interpretation is distribution. It is important to note if a change affects a portion of an organ (focally extensive), the entire organ (diffuse) or is randomly located throughout the organ (multifocal or segmental). Depending on the tissue under consideration, the distribution can offer helpful insight into the pathogenesis. For example, lesions affecting the cranioventral portions of the lungs of quadrupedal species are often the result of an inhaled process (e.g., viral or bacterial pneumonia and aspiration), while those affecting the caudal areas are more likely the result of hematogenous spread. Multifocal areas of inflammation or necrosis (tan foci) in the liver, kidney or spleen are also typically the result of hematogenous dissemination.

Euthanasia Artifact

Pentobarbital-based euthanasia solutions can impart gross changes in tissues. Tissues at the injection site often appear brown to black and occasionally green. Blood accumulating in the heart and vasculature often becomes red-brown and has a clay-like texture. The surface of the heart can rarely have white crystalline deposits on the surface (most often in birds). If sufficient euthanasia solution is given, multifocal areas of tan discoloration in solid organs can appear and close examination may reveal these foci to be vasocentric.

Ancillary Diagnostics

In addition to gross and histopathological examination, other diagnostic modalities can be employed to assist in diagnosis. This includes radiology, which can help document trauma (fractures and gunshots). As became apparent above, multiple processes can result in similar gross appearances. In these situations, cytology can help make the diagnosis, whether slides be read in-house or sent to a pathologist. Tissues should be blotted repeatedly to prevent overly thick smears and blood contamination and multiple slides should be made for potential special staining. Touch preps of avian liver, spleen and lung can identify hemoparasites. Although serum chemistry evaluation is not typically possible postmortem, limited biochemical information can be obtained from analysis of the aqueous humor. Liver, kidney and adipose are commonly requested by toxicology labs and as much of these tissues as possible should be saved when toxins are suspected. Other considerations, depending on the case, include fecal analysis; fluid analysis for effusions, synovial fluid, or CSF; and urinalysis. Parasites can be collected and sent for identification and areas of presumed infection should be collected for culture. For suspected cases of sepsis, sterile collection of heart blood or a piece of spleen is ideal for microbiology. Consider freezing small pieces of major organs from every case; these tissues can be used at a later date for microbiology, molecular or toxicology assays if indicated by histopathology. In general, many diagnostics performed for an antemortem case investigation can also be pursued at necropsy.

Table 1. Necropsy instrumentation

Essential equipment:

  Forceps

  Scalpel handle, blades (or knife)

  Scissors

  Bone cutter (rongeurs, shears, loppers, oscillating saw, handsaw)

  Tape measure / ruler

  Formalin and container

Non-essential equipment:

  Needles, syringes

  Culturettes, transport media

  Cryovials / Whirl-Paks

  Slides (cytology)

  Tissue identification tags

  Matches

  Camera

Gross Necropsy Checklist

Gross Necropsy Report

References

1.  Berzins, I. K., and R. Smolowitz. 2006. Diagnostic techniques and sample handling. In: Lewbart, G. A. (ed.). Invertebrate Medicine. Blackwell Publishing, Ames, Iowa. Pp. 263–274.

2.  Feldman, D. B., and J. C. Seely. 1988. Necropsy Guide: Rodents and the Rabbit. CRC Press, Boca Raton, Florida.

3.  Latimer, K. S., and P. M. Rakich. 1994. Necropsy examination. In: Ritchie, B. W., G. J. Harrison, and L. R. Harrison (eds.). Avian Medicine: Principles and Application. Wingers Publishing, Inc., Lake Worth, Florida. Pp. 355–379.

4.  Nichols, D. K. 2001. Necropsy. In: Wright, K. M., and B. R. Whitaker (eds.). Amphibian Medicine and Captive Husbandry. Krieger Publishing Co., Malabar, Florida. Pp. 331–334.

5.  Noga, E. J. 2010. Postmortem techniques. In: Fish Disease: Diagnosis and Treatment. 2nd ed. Wiley-Blackwell, Ames, Iowa. Pp. 49–64.

6.  Terrell, S. P., and B. A. Stacy. 2007. Reptile necropsy techniques. In: Jacobson, E. R. (ed.). Infectious Diseases and Pathology of Reptiles: Color Atlas and Text. CRC Press, Boca Raton, Florida. Pp. 219–256.