Test 2- Necropsy and lab techniques Flashcards
Necropsy / Post-mortem exam
A necropsy is a procedure, and like all procedures, there is a system to use. If you always remember the basic SYSTEM of necropsy, you will always do a thorough job, and won’t miss any key lesions.
There are 6 steps to a successful necropsy:
- Get the history
- Do an external examination
- Open the body
- Remove the organs
- Examine and sample the organs
- Write the report
We do this to determine the cause of death or the changes produced by the disease
General biosafety considerations for necropsy areas,
General biosafety considerations for necropsy areas, minimizing the potential for zoonotic infection
PERSONAL PROTECTION AND HEALTH CONCERNS
- Some animal diseases are zoonotic.
- It is important to protect the operator and any people in the vicinity from
these hazards.
Necropsy procedures pose the greatest risk of disseminating infectious agents to humans because of
Necropsy procedures pose the greatest risk of disseminating infectious agents to humans because of the large amount of tissues and body fluids exposed during dissection
Biosafety/biosecurity precautions during necropsy:
• Personal protective equipment -
• Personal protective equipment -
– Gloves
– Apron
– Boots
Persons not adequately protected will keep
Persons not adequately protected will keep a distance from the carcass
Procedural Considerations
all necropsies should “universal precautions” (all specimens should be handled and treated as though they were known to present risk of zoonotic disease!)
No eating, drinking, grooming are permitted in necropsy areas.
Rabies vaccination
Tools should be used to the extent feasible to manipulate tissues to avoid cut hazards (i.e., forceps).
Hand tools are preferred to power tools.
Carcass disposal
- Burial
- Incineration
- Composting(poultry,sheepandgoatcarcasses)
Dispose of carcasses appropriately, away from scavengers that might drag parts to other locations and inadvertently expose people.
For some diseases, such as rabies,
For some diseases, such as rabies, a vaccine is available for humans and only those people who are vaccinated should be opening the carcass and sampling tissues.
What should you wear in terms of foot wear during a necropsy?
Wear rubber boots during necropsy;sanitizethe boots by washing in disinfectant so as not to spread infectious agents beyond the site of the necropsy.
What kind of instruments are needed for a necropsy?
We use sharp instruments. Take your time, don’t rush. Be sure your knife is sharp. Most accidents with knives happen because the knife is dull.
SUSPECT ANTHRAX CASES
SUSPECT ANTHRAX CASES – DO NOT OPEN CARCASS
Collect blood from percutaneous cardiac puncture, jugular vein, or other peripheral blood vessel into both a red-top and a purple- top (EDTA) blood collection tube.
Submit sample for blood smear examination and blood culture.
Outer package must be labeled appropriately DO NOT PROCEED WITH NECROPSY!
Special Steps to examine the heart
- open right ventricle
- then left ventricle
- go into aortia
Steps to look at the brain
cut from external
Steps to look at the spinal cord
Blood Sampling
Obtain heart blood. Using a 12 ml syringe with an 18 G 1 1⁄2 in. needle, attempt to secure 3 to 10 ml of heart blood.
It may be necessary to carefully incise the left ventricle and aspirate blood from the surface. (Pleural and Peritoneal fluids may be used if blood is not available).
Express blood into a green-topped (heparinized) blood collection tube.
If possible, obtain a second 10 ml of blood and fill a red-topped blood collection tube.
If heart blood is clotted and no body cavity fluid is available, a blood clot can also be collected and placed into one of the zipper-lock tissue bags.
Urine sampling
• Obtain urine. Using another 12 ml syringe with an 18 G 1 1⁄2 in. needle, aspirate up to 10 ml of urine from the urinary bladder and put the urine into a red-top blood collection tube
Brain Sampling
Collect appropriate specimens for the state rabies laboratory before taking any other samples or dividing brain (send to state rabies lab)
Place 1”x1”portions of cerebellum, brainstem and cerebrum into each of 2 different labeled zipper lock bags for microbiology and toxicology.
The remainder of the brain needs to be placed in the largest formalin jar.
For livestock, it may be necessary to split the brain between the two
largest containers.
The best fixation will be achieved if the brain is sliced every 0.5 cm most of the way through (“bread-loafed”), leaving about 0.5-1.0 cm of tissue unsliced along the ventral portion to keep the parts together.
Skin (bovine)
Skin (bovine): 1/4 – 1/2” piece of ear skin. Place into plastic snap-top vial containing phosphate buffered saline
Adipose tissue (fat) sampling:
Adipose tissue (fat): obtain a sample 2” x 3”, if possible. Place in labeled zipper lock bag.
Liver sampling
Liver: submit a large portion (1” x 2”). Place in labeled zipper lock bag.
Kidney Sampling:
Kidney: submit a large portion (1” x 2”). Place in labeled zipper lock bag. Usually it will be trimmed in triangular shape
Eyeball sampling
Eyeball: submit intact eyeball. Place in labeled zipper lock bag.
Sampling Colon Contents
Colon contents: Collect approximately 2 teaspoons of colon contents for parasitology. Place in labeled fecal cup (do
not fill more than 1/3 full).
Sampling Stomach contents
Stomach contents: Collect approximately 2 tablespoons
of simple stomach or rumen contents. Place in labeled zipper lock bag. If possible, freeze stomach contents ASAP prior to packaging for shipment.
Additional fresh tissues
Depending on disease presentation.
For example, collect lung if respiratory disease is suspected.
Collect multiple samples if lesions are noted or if disease may affect different parts of the same organ system, such as small intestine, cecum and large intestine.
Small lymph nodes can be submitted whole. Larger lymph nodes can be sampled for microbiology, such as the retropharyngeal, bronchial, mesenteric or peripheral lymph nodes, by collecting 1⁄2 - 1” pieces.
Place all fresh tissues in labeled zipper lock bags
Whenever anaerobic cultures are indicated,
Whenever anaerobic cultures are indicated, to avoid bacterial contamination and overgrowth when samples cannot be shipped promptly, or when the prosector wants to direct the exact site of sampling for culture (for example, intestine or muscle when Clostridial infection is suspected).
This media will also support aerobic and fungal culture.
When sampling from solid tissues,
When sampling from solid tissues, the surface of the tissue sample should be decontaminated by searing with a heated blade or flaming with alcohol, and then a stab incision is made with a sterile scalpel blade before inserting a sterile swab.
When sampling hollow organs
When sampling hollow organs such as loops of bowel, it may be necessary to open a segment with a clean scalpel or scissors and swab the interior.
Septicemias
Septicemias are best defined by culturing the same organism from more than one site (for example, GI tract and liver or lymph nodes).
If the carcass is noted to be severely autolysed,
If the carcass is noted to be severely autolysed, culture may not be worthwhile.
Example of how to assemble fresh specimens
Label and place anaerobic transport media, if used, in its own Styrofoam mailer to protect from breakage and temperature extremes.
Place this inside a pouch and place in a shipping box (DO NOT chill or pack in contact with the freezer packs).
Place labeled blood tubes, and urine and skin sample vials, into slots in absorbent pouch.
Roll up. Place it, with any inoculated and labeled transport media, into a labeled zipper lock bag.
Place all fresh tissue bags and the fecal cup into the second, labeled zipper lock bag. Place these packets inside the largest 95kPa-rated Specimen Pouch. Seal and place pouch inside the insulated foil pouch with 2 frozen ice packs, and zip shut.
Sample Collection for Suspected Toxicology Cases
The classic necropsy fresh/frozen sample set for toxicological investigation includes:
• Brain
• Liver—without gall bladder– WHERE MOST TOXINS ARE
• Kidney
• Fat
• Urine
• Aqueous humor or intact eyeball
• Skin
• Heart blood (collected into heparinized tube/green top vacuum blood collection tube)
• Collect these last
- Stomach/rumen content
- Intestinal content/feces
Each tissue type should be placed in a separate container.
Sample Collection for Suspected Toxicology Cases
If a particular toxin or class of toxins is suspected of being involved in animal morbidity or mortality, you may also want to collect various other samples, depending on possible routes of exposure:
Environmental samples
Feed samples
Water samples
Heparinized whole blood (20 mls or more, green-topped blood collection tubes) from live animals
Urine from live animals
You may also want to consult with the laboratory or toxicologist about
any special sampling requirements.
In most cases, toxicology samples should be stored frozen until tested.
Rabies Submission Information
rabies virus may only be detected unilaterallyif submissions include only half the brain, on a longitudinal section, the diagnosis of rabies could be missed
FOR ALL CASES OF POSSIBLE RABIES, YOU SHOULD NOTIFY YOUR COUNTY PUBLIC HEALTH DEPARTMENT AND SUBMIT THE RABIES EXPOSURE HISTORY FORM WITH THE SPECIMEN
Small Animals and Wildlife – whole head (cerebellum and brainstem are vital)
Livestock – If the only testing is to rule out rabies and BSE/Scrapie, the brain stem and cerebellum can be removed through the foramen magnum
Sampling for histopathology exam Fixative:
Sampling for histopathology exam Fixative: formalin
Sampling for histopathology exam Brain
Brain: put the remainder of the brain, into the formalin jar(s) if not already done (The best fixation will be achieved if the brain is sliced every 0.5 cm most of the way through, ie “bread-loafed,” leaving about 0.5-1.0 cm of tissue unsliced along the ventral portion to keep the parts together). It may be necessary to divide it for fixation and shipping into two jars (large livestock).
Peripheral nerves: sampling
Peripheral nerves: collect a segment (1”)of a peripheral nerve, such as the sciatic.
Spinal cord sampling
Spinal Cord: collect one or more sections, from cervical, thoracis, and lumbar areas, by disarticulating or sawing through the spinal column at various levels.
Other tissues
Cut thin (1⁄4”/0.5-1.0 cm) sections of each tissue and place in jar of formalin.
Where lesions are recognized, include sections that incorporate both normal and abnormal appearing tissue, when available.
Check to make sure each formalin container
Check to make sure each formalin container is tightly closed and not leaking.
Place each formalin jar, with
Place each formalin jar, with an absorbent sheet, inside a heavy zipper lock bag
Containment Considerations
Containment Considerations
At a minimum, necropsy must be conducted in accordance with BSL-2 containment principles and practices
Review the animal’s history to determine if BSL-2+ practices are recommended based on the likelihood of zoonotic agents (i.e., Q fever, Rabies, Psittacosis, West Nile Virus, SE, etc.).
Necropsy of small animals should be conducted within a biosafety cabinet.
Unessential personnel and others should not be allowed in the necropsy area
You want to make sure the samples for histopathology are cut very…
THIN, so that the formalin will penetrate throughout the sample
What is the order that you want to collect organs?
First: brain, pancreas, other endocrine organs, GI
