The collection of quality diagnostic samples is the most important step in receiving accurate laboratory results. Below are some recommendations on how to collect and store samples as well as best practices for packaging and shipping them to us.
Guidelines for sample collection and submission
Performed on fresh tissue or liquids, or swabs. Collect sample aseptically. Overnight shipping with ice packs is highly recommended. Do not freeze.
Place each tissue specimen in separate leak-proof containers with just enough sterile saline to keep the tissue moist. If using whirl-pak bags wrap the tissue in a piece of sterile moistened gauze. If possible, provide a 2-5 cm specimen, which includes the lesion. Fresh tissue is more desirable than a swab. Keep intestines separate from other organs. Tie off the end of intestines if an anaerobic infection is suspected.
Liquid specimens (milk and urine) should be collected aseptically in a leak-proof container. Collect 2-5 ml of urine, mid-stream, for culture. Cystocentesis is recommended if anaerobic infections are suspected or from patients for which a definite culture has not been obtained.
- Fresh Tissue
Collect all tissues aseptically placing each tissue in separate leak-proof containers with just enough sterile saline to keep the tissue moist. If using whirl-pak bags, wrap the tissue in a piece of sterile moistened gauze. If possible, provide a 2-5 cm specimen that includes the lesion. Fresh tissue is more desirable than a swab. Keep intestines separate from other organs. Tie off the end of intestines if an anaerobic infection is suspected.
- Liquid specimens
All liquid specimens should be collected aseptically in a leak-proof container. Collection of 2 to 5 ml mid-stream urine is acceptable for culture. Cystocentesis is recommended if anaerobic infections are suspected or from patients for which a definite culture has not been obtained.
- Label all specimens clearly including animal ID and tissue or site of sample collection.
- Select the appropriate sample. Following appropriate submission guidelines for clinical samples is critical for obtaining quality results.
- Provide a brief history and include the names of any antimicrobial drugs administered and the time of withdrawal.
- Provide a short list of differential diagnoses.
- Overnight shipping with ice packs is highly recommended. Do not freeze.
- Anaerobic Culture Requests
Refrigeration of specimens for anaerobic cultures is not preferred since refrigeration may affect the viability of some anaerobes. Remember that oxygen is toxic to many anaerobes. Aspirates or biopsy specimen are more desirable than swabs. Swabs should be submitted in an anaerobic transport media.
Collect serum specimens using whole blood in plain red top Vacutainer® tubes. Serum is necessary for a majority of biochemical, hormonal, and drug tests. A minimum of 1 ml of serum should be refrigerated (on ice) and shipped overnight. When ordering multiple tests, 2ml of clear serum is required. Serum must be spun down and separated from the clot and submitted separately in a sterile tube. Serum must be separated from the blood clot to minimize hemolysis. This is critical for most chemical testing.
Blood for glucose testing should be submitted either in fluoride tubes (grey top) or as serum that has been separated from the clot within 1 hour of collection prior to shipping.
Submit either an EDTA and a lithium heparin tube along with two unstained slides
EDTA Whole Blood
Whole blood is necessary for hematological analysis. Collect blood in purple top Vacutainer® tubes with EDTA anticoagulant (2-3 ml draw) and ship refrigerated with ice packs overnight (do not place tubes directly on ice or hemolysis may occur). Two fresh-drawn, unstained blood smears are required with complete CBC’s (air dried and protected from the ice pack). For avian and exotic CBCs, submit both an EDTA and a lithium heparin tube, along with two unstained slides.
Lithium Heparinized Blood
Lithium heparinized blood is recommended for most exotic species and for certain biochemical tests (e.g., ionized calcium, ammonia for which plasma is separated and submitted frozen, and for blood gases). Collect specimens in green top Vacutainer® tubes with lithium heparin anticoagulant (liquid/powder) and ship refrigerated (on ice) overnight. A mini Avian panel can be done with 0.25 ml and calcium can be done with 0.5 ml. For non-exotic species, separation of plasma is encouraged. Lithium heparinized plasma for all chemistry testing should be collected by cetrifugation, separated from the blood cells, and submitted in a sterile tube.
Citrated Whole Blood
Collect at least 0.5ml or greater of citrated whole blood in a light blue top Vacutainer® tube for coagulation tests. Samples should be shipped refrigerated (with ice pack) overnight for a coagulation profile. Interpretation of coagulation tests is most accurate when blood from a control animal of similar age and breed is submitted along with the patient sample(s).
Turnaround times provided are for samples that arrive prior to 2:30 p.m. in the laboratory where the test will be performed and do not include time associated with shipping. Requests sent to the Athens laboratory for tests that are performed only in the Tifton laboratory (folate/vitamin B12, fructosamine, iron, LH, toxins, and pharmaceutical drugs) require an additional 2 days for shipping. Whenever possible, specimens for tests performed only by the Tifton laboratory should be sent directly to Tifton. All specimens shipped to the Tifton laboratory will be analyzed the day received and results sent out the same day.
Cytology slides should be clearly labeled. Due to limited space on slides and difficulty reading handwritten labels, a key (ex: 1, 2, 3, 4 or A, B, C, D) is highly recommended. Please provide key on submission form. Label slides using a method where the label is not easily dissolved or smeared (frosted slides marked with a pencil work best and do not interfere with staining). Avoid using tape wrapped around the slide, which prevents slides from sitting flat on the microscope stage.
Gross hemolysis and lipemia should be AVOIDED. When heavy lipemia is present, use a larger gauge needle to collect blood specimens. This will minimize hemolysis. Interference by hemolysis, lipemia, and icterus can invalidate certain results.
Lipemia interferes with evaluation of bile acids, bilirubin, ammonia, ALT, AST, magnesium, and glucose.
Hemolysis interferes with the evaluation of ALT, AST, bilirubin, potassium (horses, cattle, and certain dog breeds), LDH, magnesium, phosphorous, protein, urine creatinine/protein ratios, ammonia, creatine kinase, bile acids, GGT, and bicarbonate.
Icterus interferes with the evaluation of cholesterol, triglycerides, lipase, total protein and glucose.
We recommend the submission of a complete set of tissue, both formalin fixed and fresh tissue. Alternatively, fresh tissue may be saved, refrigerated or frozen if microbial culture or toxicological testing is not needed. Definitive diagnosis of infectious diseases and toxins often requires ancillary testing (i.e. culture, virus isolation, fluorescent antibody testing, electron microscopy, etc.) that cannot be performed on formalin fixed tissues.
There is a flat rate for mail-in necropsies regardless of the number of organs collected – a full set of tissues is recommended.
Suggested tissues to be collected during practitioner necropsies include:
- Brain and/or spinal cord
- Small intestine
- Large Intestine
- Lymph nodes
- Urinary bladder
- Thymus on young animals
- Bone marrow
- Skeletal muscle
- Adrenal glands
- Any affected tissue based on history, clinical signs and/or gross findings not listed above such as thyroid gland, reproductive organs, etc.
Submit fresh feces refrigerated, with ice packs – overnight or same day delivery. A minimum sample of 2g is required for each qualitative and quantitative tests for small ruminants. Quantitative tests for large ruminants and horses require 4-5g samples.
Collect samples under aseptic conditions in a sterile container.
Place in vials or bags with clear identification/label. Remove extra air if possible
Provide a history for each submission as it can assist diagnosis
A minimum of 1ml of serum, plasma, or whole blood in appropriate vacutainer tubes
Specimens preserved in 70% ethanol (ideal) or 10% formalin for parasite identification
Overnight shipping with ice packs is highly recommended. Do not freeze.
Contact the Parasitology Lab if you have any questions regarding tests, submission requirements and to discuss results.
Punch biopsies of at least 6mm in size of multiple affected areas are recommended. Please submit at least 4-6 skin punch biopsies with primary dermatological disease (there is single charge, regardless of number of punch biopsies submitted). Primary lesions are most diagnostic, so biopsy early lesions (e.g., papules, pustules, vesicles, bullae, wheals, etc.) rather than chronic ones. Ulcerated lesions are typically not rewarding.
If you are unsure how to appropriately sample a lesion, please call with questions prior to biopsy.
Submit specimens in neutral buffered 10% formalin solution in wide-mouthed, leak/shatter-proof containers with a volume ratio of tissue to formalin at least 1:10. Tissues fixed for 24 hours in an appropriate amount of formalin prior to submission may be subsequently submitted in a smaller container with a reduced amount of fixative, thus decreasing the size and weight of the container used for shipping.
Label containers with names of the veterinarian, owner, and animal, the date collected and information about the sample submitted. Use a pencil if there is a chance that the label will come in contact with formalin. When multiple biopsies are submitted with one case, each tissue should be submitted in a separate container and labeled appropriately.
For neoplasms, inking of surgical margins with India ink is recommended for the most accurate assessment of completeness of excision.
Improper handling or fixation of tissues can induce artifacts that may result in non-diagnostic specimens. Examples of improper handling include:
Failure to place tissues in formalin immediately after collection – Unfixed specimens subjected to dehydration, autolysis, and proliferation of saprophytic bacteria. Refrigeration slows, but does not prevent these changes.
Inadequate fixation – Using an inadequate volume of fixative, or attempting to fix large specimens (whole kidney,large mass) will result in incomplete fixation and autolysis. This will delay processing because unfixed tissues must be placed in a larger container with fresh formalin for additional fixation prior to routine processing. Formalin penetrates tissue at roughly 18 mm per day; the rate of penetration slows with the thickness of the tissue. It takes approximately 100 hours (4+ days) for formalin to penetrate 36 mm.
Improper fixatives – Alcohol, disinfectants, and saline are unsuitable for transport or fixation of specimens for histopathology and usually result in non-diagnostic samples.
Thermal dehydration – Electrocautery and laser surgery cooks tissues and may destroy small tumors and skin biopsies. Use a scalpel or punch instead. Please include the means of collection on the submission form.
Chemical dehydration – Disinfectants and medications applied to the skin or residues on instruments that are chemically sterilized may damage tissues.
Freezing – Ice formation in/around cells causes cell lysis and disrupts normal tissue architecture; this results in non-diagnostic samples. Additionally, autolysis may occur during thawing.
Excessive pressure – Excessive pressure from forceps or digits can rupture and/or compress cells (e.g., crush artifact), making them unidentifiable. Small pieces of tissue, such as skin punch biopsies, can be manipulated with a needle rather than forceps to prevent crush artifact.
Exposure to Formalin if not a biopsy – Formalin is a powerful fixative and the fumes can damage other samples including cytology samples and cultures. Double-bag all formalin containers separately from all other diagnostic samples.
Strict adherence to these guidelines is necessary to assure that samples imported to the US by our laboratory meet the requirements of the CITES permit under which we operate. Failure to follow these guidelines will result in non-clearance of your samples at the US port of entry or rejection of the samples by our laboratory in the event the samples accidentally slip through the port of entry.
- A validated CITES export permit is required for any foreign entity wishing to submit samples to the Athens Veterinary Diagnostic Laboratory (AVDL). Most countries require an inspection of samples and secondary signature or validation of their CITES export permit prior to shipping the samples. This signature should be obtained when declaring the samples for export to your country’s regulatory body. This will strongly be enforced by the U.S. Fish and Wildlife Service (USFWS) upon import. Samples are subject to confiscation if your export permit does not have a required secondary signature or validation. A copy of the permit may be submitted to our laboratory by email or fax to 706.542.5977 for verification prior to the specimen leaving your country. On your CITES export permit:
- The exporter must be “your organization”
- The recipient (”importer” or “destination”) must be “National Marine Fisheries Service, c/o Dr. Jeremiah T. Saliki, Athens Veterinary Diagnostic Laboratory, Athens, GA 30602, USA”
- Prior to shipping or transporting specimens, blocks 9 (Description), 11 (Quantity) and block 12 (Country of Origin) of our CITES import permit (which will be emailed to you after confirmation that you have a valid CITES export permit) must be completed for the specimens. Click here for a sample completed CITES import permit, which can also be e-mailed to you upon request.
- Before shipping, complete USFWS Form 3-177. Click here for instructions on completing that form. Block 1 4a must indicate your complete name, address (in your country), and telephone number. Do not forget to date, sign, and write or type your full names in section 22 of the form. Send a copy of the form by email
(email@example.com and firstname.lastname@example.org) to arrive at least 24 hours (preferably several days) before the shipment is scheduled to arrive at the US port of entry. We strongly encourage you to complete the form online at:
- Packaging and shipping the samples:
- Package all samples following IATA guidelines for international transportation of dangerous goods.
- Include two (2) copies of the following with the package: one set of copies in a sealed Ziploc bag inside the package and one set of copies taped in the packing slip on top of the box, your completed and validated CITES export permit, our completed CITES permit, completed and signed Form 3-177.
- Notify us by email (email@example.com with copy to firstname.lastname@example.org) on the date and time of arrival of samples at Atlanta airport. In your notification email, attach scanned copies of your completed and validated CITES export permit, our completed CITES permit, completed and signed Form 3-177.