Collection and Submission of Laboratory Samples from Animals

Each veterinary diagnostic laboratory offers a unique set of diagnostic tests that is subject to frequent changes as new and improved tests become available. The protocols for sample collection and submission are therefore also subject to change.

The practitioner and diagnostic laboratory staff must maintain good communication to complete their diagnostic efforts efficiently and provide optimal service to the client. Practitioners must be specific and clear in their test requests. The laboratory staff will provide guidance regarding sample collection and handling protocols, as well as assistance in the interpretation of test results.

Most diagnostic laboratories publish user guidelines with preferred protocols for sample collection and submission; however, the following broad recommendations are fairly standard.

A detailed case history should be included with submitted samples. This information is particularly critical for the interpretation of samples submitted for cytologic and histologic evaluation. The submission form should be protected in a waterproof bag to avoid damage from any fluids in the packaged materials.

A detailed case history should include the following elements :

• owner's first and last name
• species
• breed or size/weight (if breed unknown)
• sex
• age
• animal identification
• relevant clinical signs
• gross appearance (including size and anatomic location) of lesions
• previous treatment, including response to treatment (if any)
• results of relevant diagnostic testing, such as CBC, serum biochemical analysis, cytologic and histologic evaluation, and imaging studies
• for population health studies, morbidity and mortality in the group

If a zoonotic disease is suspected, this should also be clearly indicated on the submission form to alert laboratory personnel.

Packaging of Samples from Animals for Shipment

The shipment of biological specimens should comply with protocols established by the courier or shipping service used. In some instances, air transport requires compliance with International Aviation and Transportation Association (IATA) regulations for hazardous materials.

These regulations include restrictions on the volume of formalin that can be shipped in a container: no more than 30 mL of free liquid solution in each inner container, and no more than 1 L in the entire outer package. Rather than shipping large volumes of formalin, tissue samples can be fixed in-house for at least 24 hours in an appropriate volume and then transferred into a smaller volume of fresh formalin for shipment.

Shipments with fresh tissue samples are required to be clearly labeled:

• UN2814, Infectious substances, affecting humans
• UN2900, Infectious substances, affecting animals
• UN3373, Biological substances, Category B

Most veterinary diagnostic samples are classified as Category B (an infectious substance not in a form generally capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals when exposure to it occurs). If high-risk or reportable diseases are suspected, it is essential to contact state and federal veterinary agencies regarding necessary shipment precautions.

Further details can be found at the International Air Transport Association website and the US Postal Service Postal Explorer website as well as commercial courier websites.

A fundamental approach to packaging samples involves a 3-layer barrier to protect them:

• The sample is placed in an appropriate primary container (sealed jar, bag, or tube).
• The primary container is then enclosed within a secondary container , which also includes absorbent material.
• The secondary container is then placed in the shipping box ( tertiary container ), which often houses refrigerant or cold packs as well as various cushioning materials (eg, polystyrene foam) to protect the sample. Ideally, the tertiary container is a sturdy polystyrene refrigerator box or a cardboard box lined with a fitted polystyrene lining.

Items such as syringes, obstetrical gloves, and containers without sealable orifices are not suitable for shipment. Under no circumstances should needles, scalpel blades, or any other potentially injurious item (eg, broken microscope slides with sharp edges) be submitted to the lab.

Liquid samples should not ship in plastic bags; instead, a sealable jar should be used. Waterproof markers should be used when labeling specimen bags and containers; at a minimum, patient identification and specimen type (eg, urine, serum, plasma) are critical labeling information.

Coolant materials should be sealed in separate plastic bags to prevent condensation damage. Refrigerant or cold packs should not be placed in direct contact with samples, such as tubes of whole blood, that could suffer adverse effects if frozen in transit.

The suitably protected, completed submission form must be included.

If dry ice is used, this should be noted on the cardboard box label, and the lid should not be sealed with tape. Otherwise, CO ₂ released from the dry ice could increase pressure and damage the package or contents.

Preparation of Samples from Animals for Histologic Evaluation

Tissues for microscopic examination collected either via biopsy or during necropsy can be critical to obtaining a diagnosis. Immunohistochemical (IHC) and other advanced tests that can be applied to formalin-fixed tissue further reinforce the utility of histologic evaluation as diagnostic technique.

Autolyzed tissues are generally useless for histologic evaluation; prompt necropsy and organ sampling are critical.

Tissue should not be frozen prior to fixation.

Other than CNS tissues, samples collected for histologic evaluation should never be > 1 cm thick (preferably 5–7 mm) and must be immediately placed in ≥ 10 times their volume of phosphate-buffered 10% formalin to ensure adequate fixation.

Tissues should be representative of any lesions present and, in the case of cutaneous punch biopsies and biopsies obtained via endoscopic collection, be centered directly on the grossly visible lesions.

Wedge biopsies or tissue samples collected at necropsy should include some of the apparently normal surrounding tissue; the interface between normal and abnormal tissue may provide key information.

Excisional biopsies of small tumors (

Rather than shipping large volumes of formalin, tissue samples can be fixed in-house for at least 24 hours in an appropriate volume and then transferred into a smaller volume of fresh formalin for shipment.

• Rather than shipping large volumes of formalin, tissue samples can be fixed in-house for at least 24 hours in an appropriate volume and then transferred into a smaller volume of fresh formalin for shipment.

Submission of tissue that has been stored in formalin is acceptable within any time frame, although quality of sample will degrade over time. Prolonged formalin fixation can adversely affect advanced testing; for example, immunohistochemical testing is not recommended for tissue that has been stored in formalin for > 7 weeks because results may be invalid.

Samples should be shipped in laboratory-approved unbreakable containers with screw-top lids and packed in a manner that prevents spillage or freezing during shipment (see Packaging of Samples for Shipment ).

Specific tissues collected at necropsy require additional attention:

• Because the GI mucosa decomposes rapidly, short sections of gut collected at necropsy should be opened lengthwise to allow adequate fixation.
• If the spinal cord is submitted, the dura mater should be carefully incised lengthwise to permit more rapid penetration to the cord.
• Fixing the brain poses a special dilemma, especially if a neuroanatomical location of the lesions within the organ could not be determined antemortem.
  • Ideally, a whole, intact, fixed brain is required for complete histologic evaluation.
  • Immersion of the brain for many days in a large volume of formalin is required for adequate fixation; consequently, brains are commonly transported in an only partially fixed state. If the specimen can be shipped by overnight delivery, it may be acceptable to send a chilled, carefully packaged, unfixed brain, which can then be processed at the diagnostic laboratory.
  • Often, the brain is halved longitudinally and one half is sent unfixed (fresh), properly refrigerated, for microbiological testing, while the other half partially fixes in transit. This method can prove unsatisfactory if a solitary unilateral lesion is involved. Slicing the brain into widths suitable for rapid fixation introduces considerable fixation artifact and should be avoided if possible; fixing the intact (or halved) brain in a large volume of formalin for > 24 hours is preferred.

  Preparation of Samples from Animals for Microbiological Testing

  Any specific infectious agents that are suspected clinically should be noted on the submission form; some organisms have specific growth requirements (eg, anaerobic culture, special media) that may not be routinely used in laboratories unless the pathogen was cited as a differential diagnosis.

  Laboratory techniques and capabilities for microbiological testing vary; however, most tests rely on either the growth or visualization of intact viable organisms or the detection of the nucleic acids and proteins of these pathogens. Therefore, unfixed specimens (tissue, fluid, etc) should be collected aseptically and shipped promptly to avoid degradation.

  If microbial culture of a fluid specimen such as urine or a cavitary effusion is a possibility, a separate aliquot of the fluid in a sterile, additive-free container should be submitted. Tubes containing EDTA or other preservatives are generally ideal for cytologic evaluation but unacceptable for culturing.

  If PCR assays are to be performed, it is particularly important to avoid cross contamination between multiple animals in a submission; this principle applies to tissues, fluids, and even dissection instruments.

  Furthermore, swabs for PCR analysis should not be placed in agar or charcoal-based transport media, and calcium alginate swabs should be avoided. Instead, sterile cotton- or polyester-tipped applicators should be shipped in a tube, either dry (without transport media) or with a few drops of sterile saline solution or viral transport media, depending on the individual laboratory's requirements.

  Requirements for PCR assay can vary widely by specific PCR test, and consulting the recipient laboratory before obtaining the sample is strongly encouraged.

  Some test protocols may permit pooling of organ specimens from an individual; however, it is generally preferable that each tissue be collected into separate, clearly labeled sterile bags or tubes for shipping. Gut samples must never be pooled in a container with other tissue samples.

  Tissues and fluids for most microbiological assays may be frozen before shipment, but freezing is generally undesirable if samples can be chilled and delivered directly to the laboratory within 24 hours after collection.

  Exceptions to this rule include analysis for certain toxins, such as those of Clostridium perfringens and C botulinum , in which degradation of the toxin must be prevented by prompt freezing after collection. Adequate coolant should be provided so that samples remain chilled (or frozen) until they reach the laboratory.

  Fecal samples for parasitological testing should be submitted chilled in appropriately sealed containers. Freezing may have little impact on routine flotation or sedimentation tests but will negate the possibility of Baermann analysis for nematode larvae. Ectoparasites or nematodes being submitted for identification should be submitted in vials containing 70% alcohol.

  Preparation of Samples from Animals for Toxicological Testing

  If a known toxin is suspected, a specific analysis should always be requested—laboratories cannot just “check for poisoning.” A complete description of clinical and epidemiological findings may help differentiate poisoning from infectious diseases that mimic poisoning.