November 1997

This tube stabilizes the specimen’s glucose component during transport. It must be at least 50% filled or the high concentration of sodium fluoride will interfere with glucose testing methods. The preferred method to stabilize a specimen for glucose and other chemistry assays is to collect a serum separator tube (SST), and centrifuge it after it clots to separate the serum from the blood cells. Alternately, draw a plain red top tube (RTT), centrifuge it, and transfer the serum to another RTT.

Drugs such as digoxin and phenobarbital are most stable in blood samples drawn into a RTT, as the silicone gel of SST will absorb many drugs. Vitamin B12 and folate levels also may be affected by the SST gel, although elemental therapies such as bromides do not appear to be altered.

Plasma von Willebrand factor can be measured in samples collected in either BTT or lavender top tube (LTT), following the guidelines above.

Specimens should be collected into Microtainer devices. Ideally, in avians, the EDTA Microtainer is used for CBC and heparinized Microtainer for blood chemistries. In reptiles, only heparinized Microtainers should be submitted, as EDTA causes hemolysis and clotting. Fresh blood smears should always be submitted, so that cellular morphology is preserved. Coverslip smears are discouraged. If only a very small quantity of blood is obtained, the most versatile specimen is the heparinized Microtainer. As a general guideline for avians and reptiles, 10% of the body weight is blood, and 10% of that can be safely phlebotomized (roughly 1% of the body weight or 0.1 ml/10 gm).

Collection of urine should be performed in such a way to permit both urinalysis (UA) and culture. Although normal urine is sterile, it is a wonderful medium for growth of both pathogenic bacteria and normal flora. A single contaminating bacterium can overgrow a urine specimen in 2 to 4 hours. Stabilizers are available to preserve a urine specimen for culture, however they render the specimen unsuitable for routine UA. Therefore, urine specimens are best split, with a portion submitted in a RTT for UA and another submitted in a urine culture stabilizing tube. All urine specimens, whether for UA or culture, should be refrigerated and transported with a cold pack to prevent bacterial overgrowth and stabilize cellular components.

Specimens for blood culture require collection into a specialized vacuum tube containing growth medium. The area should be shaved, scrubbed clean with soap and water, and disinfected twice with an iodine preparation, beginning at the venipuncture site and working outward. Allow the site to dry. The rubber stopper of the growth medium tube should be disinfected with alcohol. It will draw up to 4 ml of blood, and even though a small sample can provide diagnostic information, a larger volume will more likely permit recovery of an infectious organism. Draw 2 tubes taken 1 to 2 hours apart with the animal off all antibiotics for at least 2 days. Leave the culture tubes at room temperature during transport.

Aprotonin (protease inhibitor) is added to LTT to stabilize ACTH so that the sample does not need to be frozen. [These tubes are available from ANTECH.] The treated plasma is separated by centrifugation, transferred to a plastic tube, capped and refrigerated. Transport the sample to the lab with cold packs.

These samples can be transported on a swab immersed in either Stuarts or Cary-Blair transport gel. The gel will keep the specimen moist and keep anaerobes away from the ambient air. Culture containers that provide crush-ampules of liquid transport medium, while acceptable for aerobic cultures, are not acceptable for anaerobes. Swabs that contain mostly pus will often provide no-growth on cultures, due to the toxic effect of the white blood cells. The most productive specimens are obtained by swabbing deep into an infected wound or obtaining fluids from an actively infected tissue. Culture swabs in media should be stored and transported at room temperature, as some fastidious organisms will be lost otherwise.

Specimens for viral culture are best transported in liquid viral culture media. Small liquid specimens can be dropped directly into the transport medium. Others are collected on a clean, dry dacron swab having a plastic shaft (not wood or paper). The swab is broken off into the vial of transport medium and submitted to the lab. Store at refrigerator temperature and transport with cold packs. ANTECH can provide a Viral Transport Pack that includes a tube of transport medium and the swab.

Cultures for Mycoplasma should be submitted on a clean, dry dacron swab with a plastic shaft. The culture must be refrigerated and transported with a cold pack to arrive within 24 hours. If delayed beyond 24 hours, the culture swab must be frozen and stored at -20"C.

HISTOLOGY AND CYTOLOGY SPECIMENS

Tissue specimens for histology must be preserved and transported in formalin (10 parts formalin to 1 part tissue). The ideal specimen is less than an inch thick. OSHA and Transportation Safety Regulations limit the size and quantity of formalin containers that can be shipped. Use a container with a tightly sealed lid, place it in a ziplock plastic bag, and then in a second outer bag that contains the requisition. Also, do not enclose cytology samples in bags containing formalin-fixed tissues.

Several (preferably 3 or more) representative sections of large tissues or organs should be selected, preserved and transported for histology. The remainder should be placed in a large plastic container of formalin, refrigerated, and retained at the clinic in case additional samples are needed.

Knowing orientation and other facts about the tissue mass is critical to the pathologist. A diagram on the requisition form is helpful. Borders and areas of interest on the mass can be marked with colored or numbered sutures. Please state if the entire mass has been excised, if all is being submitted, or if it had to be divided into sections before submission.

Fluid for cytology should be submitted in both RTT and LTT. For very small samples, a LTT is preferred. Cellular morphology is preserved by making 2-4 fresh smears on glass slides and allowing them to air dry. Urine smears for cytology should be made from fresh urine sediment. Do not add fixative or stain to any cytology smears.

Specimens of bone marrow will yield the most information if both a core biopsy and aspirate slides are submitted. The biopsy should be cut first, and the core placed in a tissue processing cassette, labeled and dropped into a formalin container. The aspirate needle then can be placed into the same puncture site as the biopsy needle. After placing the needle, attach a large bore syringe (10-20cc) rinsed with a lit- tle liquid EDTA to help control clotting. Pull quickly and forcefully to aspirate 0.5-1.0 ml of sample into the needle. More is not better here as the negative pressure will rapidly rupture capillaries in the marrow, causing influx of peripheral blood. Place a portion of the aspirate into a LTT, which prevents clotting and preserves cellular morphology. Make the remainder into fresh smears. If hemodilution occurred, dispense the aspirate onto a glass surface and pick out spicule material to make smears. Send aspirate specimens in separate bags from the core biopsy to avoid formalin contamination.

Information about the animal is extremely important to the microbiologist in processing a culture and seeking certain pathogens, and to the pathologist in viewing a histology or cytology specimen. If the specimen comes from a necropsy, this should be stated clearly on the requisition, along with the site of collection, type of specimen (wound, abscess, body fluid, tissue, etc.) and relevant clinical history. Please note reference numbers or dates of previous reports from ANTECH that will allow these materials to be reviewed.