Histopathology

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General:

1. All specimens should be submitted in 10% neutral buffered formalin. 10% phosphate buffered formalin fixation RECIPE (makes 1L):
   1. Commercial Formaldehyde (37- 40%) 100 ml Distilled water 900 ml
   2. Sodium phosphate monobasic 4.0 g Sodium phosphate dibasic (anhydrous) 6.5 g (pH should be 7.2 ± 0.5)
2. Do not allow the formalin solution and/or fixed tissues to freeze.
3. Unless masses are very large (ie > 6 cm in greatest thickness), please do not section or incise samples. When submitting larger masses, then sectioning in half, or possibly in quarters may be necessary before placing samples in formalin.
4. Aquatic Specimens:
   1. The width of fin-fish and crustacean tissues should not exceed 1 cm, this will ensure proper fixation.
   2. Bivalve Shellfish – remove one valve from the animal and immerse whole animal in 10% formalin made up in filtered seawater (35 ppt).
5. Fix samples in adequate amounts of formalin. Ideally, the volume ratio of formalin to tissue would be 10:1. If this is not feasible, a minimum of 5-7:1 would suffice. Place tissue samples immediately in the large volume of formalin for 24 hours, then transfer to a smaller sample containing less formalin for shipping.
   1. Alternatively, following fixation in large volumes of formalin solution for 24 – 48 hours, tissues may be removed, wrapped in formalin-soaked gauze sponge, placed in a plastic bag, and sealed for shipment. This technique decreases the probability of spillage and leakage of formalin during mailing. Do not submit these samples completely dry, as this can create artifactual changes.
6. Ideally each sample should be submitted in a separate container labelled as to the anatomical location of excision.
7. Do not send samples in glass bottles (they often break, leaking formalin en route). Plastic screw cap bottles should be placed in a sealable, plastic bag. Include enough material to absorb the entire volume of formalin in case of breaks or spills.
8. Send submission sheets with clinical history, signalment and working diagnoses in a separate plastic bag.
9. Digital images of lesions are always welcome and heartily encouraged. These images may be printed off in clinic and attached to the submission form or may be emailed directly to Diagnostic Services (avcdiagnostics@upei.ca). When emailing images, please include the owner and patients name and what test you have requested so that these images can be forwarded to the persons working on your case.

Carcass, Whole Fish and Shellfish Submissions:

Tissue Submissions:
Avian Submissions
Suggested Standard Submissions from Aborted Fetuses
Suggested Standard Submission for Neonatal Scours
Histology – Research Submissions
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General Information

Whenever possible, submission of the entire carcass is preferred. Under ideal circumstances, the carcass should be submitted as soon as possible after death. If a period of time must elapse prior to submission (<3-4 days), the carcass should be kept cool but not frozen. If at all possible, please avoid freezing the carcass as this produces severe artifactual changes which can make interpretation of subtle lesions difficult to impossible. However, if you cannot get the carcass to the lab in this time period or cannot keep it cool, freezing of the carcass may be necessary. Please include a complete clinical history with all submissions. Please feel free to call and talk to a pathologist regarding case material prior to submission if you have questions regarding shipping, sampling or specific tests.

For Fish and Shellfish submission live animals are preferable if at all possible, please contact the Diagnostic Lab in advance to ensure proper holding facilities are available for live submissions.

Herd Health Problems

In cases involving a herd problem, especially neonatal diarrhea, the best specimen for submission is a live, acutely affected, untreated animal. If this option is considered, please call the lab prior to sending in the animal as arrangements of euthanasia will need to be made, which may entail an extra charge. When submission of a live animal is not possible, the next best submission would be samples obtained by the submitting veterinarian at the time of field necropsy. Prior to euthanasia, collect blood in red top and lavender top tubes.

Centrifuge the red-top tube, harvest the serum and save in another tube. Perform a complete post- mortem and submit specimens for microbiology and histopathology as indicated. Submit these samples and a complete history to the laboratory.

Euthanasia Policies

The post-mortem laboratory does not offer euthanasia services. The only exceptions are animals being submitted through AVC Farm Services and Aquatic Diagnostic services. Live animals must be brought in during normal working hours (8-4 pm, Mon to Fri). Submitters must call the post-mortem office (566-0864) prior to receiving these animals to make appropriate arrangements for euthanasia.

Cosmetic Necropsies, Animal Remains, and Disposal Service Policies

Cosmetic necropsies are not performed. The release of any remains from carcasses to owners is prohibited. Reasons for this policy include the possibility of disease transmission from the laboratory to either the owner or the owners’ other animals, and misunderstandings by the owner about the condition of the returned carcass. If the submitting veterinarian wishes to perform a cosmetic post- mortem we will gladly do histopathology, microbiology, etc., for you.

Disposal services are limited to those animals necropsied at Diagnostic Services.

Insurance/Legal Necropsy Policies

It is the responsibility of the consignor to alert the laboratory to cases involving insurance or potential legal aspects to insure full documentation. There is an additional charge associated with these cases.

Legal cases are defined as those requiring lab personnel to maintain chain of evidence in order for any information obtained to be admissible as evidence in legal court proceedings.

Tissue Submissions:

Carcass, Whole Fish and Shellfish Submissions
Avian Submissions
Suggested Standard Submissions from Aborted Fetuses
Suggested Standard Submission for Neonatal Scours
Histology – Research Submissions
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1. Place tissues in fixative as soon as possible. Fixation is typically complete within 24-48 hrs for most tissues.
2. Please avoid crushing and squeezing samples to be sent for histopathology. Such trauma induces significant artifactual changes that can significantly alter the microscopic appearance of tissues and limit the pathologist’s ability to interpret lesions.
3. Please do not use electocautery or lasers to remove samples for histology. These instruments “cook” the tissues and can obscure microscopic lesions.
4. Use wide-mouthed, leak-proof containers. Please DO NOT USE GLASS JARS. Also, please avoid using narrow necked containers. Fresh tissues are soft and pliable and can easily be put into a narrow-mouthed jar, but following fixation tissue becomes firm, rigid and cannot be easily removed.
5. Provide thorough a clinical history
   1. Record lesions, include size, extent, colour, consistency
   2. Report negative findings if relevant. An example could be the absence of gross brain lesions in an animal which had neurological signs.
   3. Report important treatment information (e.g. the dog is currently on antibiotics or steroids….) and important clinical findings (e.g. hypoalbuminemia, hypercalceimia, etc.).
6. Tips and considerations on tissue submissions: The quality of your diagnostic submission is critical in allowing the pathologist to recognize and accurately interpret lesions, and thus provide you and your client with a diagnosis. The following are some suggestions on how to handle many common, types of submissions:
   1. Eyes – please do not puncture or incise the eye.Remove all extraocular soft tissues (eyelids, muscles, etc.), leaving the orb bare, ideally with a small stump of optic nerve intact. If there is something of concern within these soft tissues that you would like examined, please submit them in a separate container.

      For a canine eyeball, inject approximately 1 ml of formalin into the eye, through the sclera, and then place the eye in formalin for fixation. For smaller feline eyeballs, approximately 0.5 ml of formalin will be sufficient, while larger equine eyeballs may require injection of 2 ml of formalin for adequate fixation

   2. Muscle biopsiesWhen considering submitting muscle biopsies for evaluation, there are several factors to take into account. Diagnostic Services can perform routine histopathologic evaluation of formalin fixed muscle samples. For more specific histochemical and immunohistochemical testing, which generally requires evaluation of fresh (not frozen) muscle and possibly nerve samples, we would forward such samples to specialized neuromuscular laboratories for evaluation. Please call the lab before sampling, to make arrangements for the handling of these samples, to get appropriate submissions forms, to arrange for import permits if testing is to be forwarded to an American lab and to get a quote for costs, which vary depending on the tests required and the referring lab.

      When considering performing muscle biopsy, the selection of which muscle to sample and the testing required depends on the clinical presentation and the presumptive/ or differential diagnoses that you are considering.

      For clinical signs restricted to the muscle mastication in dogs (i.e. masticatory muscle myositis – MMM), the temporalis muscle is the optimal sampling site. Be sure to retract the more superficial caudoauricular/or frontalis muscle which lies just under the skin (and is not affected by MMM), and incise the thick fascia that covers the temporalis muscle to sample the underlying muscle. Often, this disease can be diagnosed by the detection of antibodies in the serum alone.

      When clinical signs are generalized (usually in small animals) and a myopathic disorder is considered, proximal limb muscles, such as the vastus lateralis or biceps femoris from the pelvic limb and/or triceps muscle from the thoracic limb, are frequently suggested sampling sites.

      In the horse, for the most commonly encountered muscular diseases, the gluteal or semimembranosus muscle are recommended sampling sites but this might vary depending on the disease suspected. For more information please check out this website : www.cvm.msu.edu/research/faculty-research/valberg-laboratory/for- veterinarians/obtaining-and-submitting-a-biopsy#information-on-submitting-a-sample.

Suggestions when sampling skeletal muscle:

1. Ideally, the sample should be 1 cm long, by 0.5 cm wide, and 0.5 cm deep and removed with as little trauma as possible (no cautery please)
2. The sample should be dissected along the long axis of the muscle so that the muscle fibers in the sample are oriented lengthwise
3. Fresh samples are the best samples in most instances, but when considering other differential diagnoses (tumors, etc.) or when there may be significant delays in getting fresh samples to the lab, taking an additional sample to be placed in formalin would be prudent.
4. Fresh samples on which all histochemical and immunohistochemical staining can be applied. Please notify the lab before sending these samples. Wrap the sample in saline moistened (not dripping wet) gauze and placed in a dry, water tight, specimen jar or red top tube. Keep the sample refrigerated. For overnight shipping to the lab, sandwich the sample between 2cold packs and label the package “refrigerate upon arrival”. Do not ship the samples Thursdays or Fridays or before long weekends/holidays.
5. Formalin fixed samples – if the sample is to be shipped immediately, place it directly in a water-tight container containing 10% buffered formalin. If the sample is to be shipped the following day, to minimize artifact associated with muscle contraction, immediately following sampling you can pin the muscle sample lengthwise to a piece of tongue depressor using 2 small bore needles, one positioned at each end of the sample. The following day, prior to packaging for shipping REMOVE THE NEEDLES, then send the sample in formalin (still attached to the tongue depressor), as you would any other biopsy sample.
6. Frequently used, referral veterinary neuromuscular labs include, but are not limited to: UC Davis, Neuromuscular Disease Laboratory (www.vetneuromuscular.ucsd.edu) Equine Neuromuscular Diagnostic Laboratory, Michigan State University, (www.cvm.msu.edu/research/faculty-research/valberg-laboratory)
7. Animal Health Laboratory, Guelph, Ontario (www.guelphlabservices.com)

Bone lesions – please provide a description of radiographic findings. Bone samples are generally hard and require time to soak in decalcification solution following fixation to soften sufficiently for subsequently trimming. Depending on the size of these lesions, this may take several days to weeks to complete, so be prepared for delays in receiving results in these cases.

Very small tissue samples (e.g. endoscopic biopsies, core needle biopsies etc.) – should be placed directly in special histology cassettes immediately following sampling, then the cassette is placed in formalin. These cassettes have small sieve like openings which allow formalin to enter but tissues cannot escape. This keeps these small samples safe and minimally traumatized. Please contact us should you like more information regarding these cassettes.

Hollow Organs: e.g. intestine, urinary bladder, uterus

Cut open longitudinally (with care) prior to placing in formalin to ensure fixation of mucosal surfaces

Tumour Submissions

Please indicate the size, location of the mass. Whenever possible, include information such as: How long has it been present? Is it increasing in size? Does it invade surrounding tissues? or does it have a capsule?

Margin assessment – we highly recommend the use of surgical dyes to mark specific surgical margins. These dyes are relatively inexpensive, and different colors are available which can be used to identify different areas of interest. Black, yellow, purple and green are generally preferred. Unlike using sutures, these dyes do not cause tissue damage in areas of interest, and the dye remains in place following fixation and sectioning.

1. Ideally inking should be performed as soon as possible following resection (within 30 min at most) and prior to placement of tissue in formalin.
2. A cotton swab or wooden applicator should be used to place ink on specific areas of interest or true surgical margins (you should avoid submerging the whole sample in ink)
3. To prevent the ink from washing off, allow it dry 5-10 min before placing the tissue in formalin
4. For larger samples that will require sectioning, first ink tissues, allow it to dry and then section before placing them in formalin. This will help to minimize the dye from staining unimportant areas.

Skin Biopsies:

Do not shave biopsy sites, as the hairs are useful guidelines for proper plane of section.

When investigating multifocal or generalized skin conditions, taking multiple skin punch biopsies or samples is recommended. Take samples from the most recently developing lesions and/or from the margins of more chronic lesions, including the adjacent more normal appearing skin where possible. Avoid sampling the center of very chronic, ulcerated lesions. If a bullous skin disease (pemphigus) is suspected the active lesion (such as an actual blister) is required for definitive histological diagnosis.

Providing a thorough clinical history, including signalment (including breed, sex, distribution of lesions, etc.) is vital in dermatology cases.

Flattening the biopsy on a piece of cardboard and floating it upside down in formalin (do not use needles to pin the samples), will result in a fixed tissue that can be properly orientated by the pathologist for sectioning.

Immunohistochemistry staining can be done on tissue blocks either in-house or sent to a referral laboratory. There is an additional fee for this testing.

Avian Submissions:

Carcass, Whole Fish and Shellfish Submissions
Tissue Submissions:
Suggested Standard Submissions from Aborted Fetuses
Suggested Standard Submission for Neonatal Scours
Histology – Research Submissions
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FOR THE DIAGNOSIS OF A DISEASE PROBLEM WITHIN A FLOCK

Submit 4 to 5 freshly dead birds that have died of the condition, preferably refrigerated and kept cool until they arrive at the laboratory. Time is important as small carcasses decompose quickly.

Submit 4 to 5 live, sick birds that are clinically affected with the condition. Take care not to select cull birds from the flock which do not represent the disease condition. These specimens should be shipped by express in containers suitable for live birds.

Suggested Standard Submissions from Aborted Fetuses:

Carcass, Whole Fish and Shellfish Submissions
Tissue Submissions:
Avian Submissions
Suggested Standard Submission for Neonatal Scours
Histology – Research Submissions
Top

Comments

Histopathology:Fix 5 mm thick portions of tissue in l0 volumes formalin per volume tissue; do not freeze if at all possible.

Bacteriology:Submit tissues in whirl-pack bags labelled “Bact”, put each tissue in a separate bag. Fluids should be submitted in labelled red-top vacutainers. These specimens should be at refrigerator temperature but NOT FROZEN.

Virology:Submit tissues in whirl-pack bags labelled “viro”, freezing is acceptable.

Serology:Collect serum at the time of abortion and 2-3 weeks later; remove serum from clot and store frozen at your clinic; paired sera can then be submitted as indicated. Never freeze clotted whole blood with the expectation of using it for serology later as this will interfere with serological tests.

Suggested Standard Submission for Neonatal Scours:

Carcass, Whole Fish and Shellfish Submissions
Tissue Submissions:
Avian Submissions
Suggested Standard Submissions from Aborted Fetuses
Histology – Research Submissions
Top

Comments

Histopathology: Fix 5 mm thick portions of tissue in l0 volumes of formalin per volume of tissue; do not freeze; either open intestine longitudinally or fill lumen with formalin and ligate ends.

Bacteriology: Submit each tissue in a separate, labelled whirl-pack bag. Smears for Gram stain may be made from a composite ileal/ jejunal swab (not the sections for culture) and air-dried.

Virology: Tissues may be submitted frozen, clearly labelled “Virology”.

Parasitology: To make ileal mucosal smears transfer ileal mucosal scrapings to a glass slide and air dry. Fecal material should arrive at the laboratory in refrigerated condition (not frozen) as soon as possible after the sample is taken.

Alternatively, feces maybe preserved in 2.5% potassium dichromate solution (l volume feces to l volume K₂Cr₂O₇ solution).

Histology – Research Submissions:

Carcass, Whole Fish and Shellfish Submissions
Tissue Submissions:
Avian Submissions
Suggested Standard Submissions from Aborted Fetuses
Suggested Standard Submission for Neonatal Scours
Top

-Call (902) 566-0883 for submission recommendations and scheduling.