March 2001

TIPS ON SPECIMEN PREPARATION
PART II

[This information first appeared in the November 1997 Antech News and has been updated here.]

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 phenobarbitol 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.

Specimens for blood culture require collection into a specialized vacuum culture bottle containing growth medium. The venipuncture site should be shaved, scrubbed clean with soap and water, and disinfected twice with an iodine preparation. Allow the site to dry. The rubber stopper of the culture bottle should be disinfected with alcohol. Obtain 1-3 mL blood using a needle and syringe, and transfer immediately into the culture bottle. Multiple blood cultures increase the chance of detecting bacteremia. Draw 2-4 bottles taken 1-2 hrs. apart with the animal off all antibiotics for at least 2 days. Leave culture bottles at room temp during transport.

Blood samples for coagulation testing must be drawn into a blue top tube (BTT). The BTT should be filled to at least 75%, but preferably 90% or more capacity, as results will be affected by excess citrate anticoagulant. Centrifugation and separation of plasma from cells is preferred if transportation to the laboratory takes more than 12 hours. Use a plastic pipette or small syringe to transfer the plasma to a clean plastic tube. Cap the plasma tube and keep cold or freeze at -20°C or lower. Freezing the plasma is not necessary unless testing will be delayed for more than 24 hours, but it should always stay cold. Repeated freezing and thawing of plasma denatures coagulation proteins. The ANTECH drivers have coolers to transport local samples. Samples being mailed should be shipped overnight with frozen cold packs.

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

A RTT is the preferred collection tube for urinalysis and urine culture. A Copan swab can also be used for urine culture, but it precludes quantitation of results. Urine for culture is best collected by cystocentesis and transported with a cold pack to prevent bacterial overgrowth.

Aprotonin (protease inhibitor) is added to a LTT to stabilize ACTH so freezing the sample is not needed. [Tubes are available from ANTECH.] The treated plasma should be separated immediately by centrifugation, transferred to a plastic tube, capped and refrigerated. Transport sample to the lab with cold packs.

For avians and reptiles, a blood smear and 2 microhematocrit tubes are preferred. Use microscope slides for blood smears, as cover slips produce less than optimal smears and are easily broken. Smears that are too thick or lack a "feathered-edge" will compromise the accuracy of results obtained. Microhematocrit tubes should be at least half full.

For small mammals, a lavender top (LT) Microtainer™ tube is preferred. Fill the tube between the 2 lines and invert 6-8 times to ensure proper mixing of blood with the EDTA anticoagulant.

The preferred specimen for blood chemistry testing is a spun green top tube (GRT) containing a separator gel. This tube is often referred to as a plasma separator tube. The tube needs to be filled to the top line (0.6 mL) to ensure adequate sample volume, inverted 6-8 times to ensure proper mixing of blood and heparin anticoagulant, and centrifuged immediately.

Other Tests

Serology (antibody testing) can be performed from either a spun GRT with gel or spun Microtainer™ serum separator tube (SST).

PCR (DNA) testing on birds requires whole blood. A Microtainer™ GRT without gel is preferred. A GRT with gel can also be used, but must not be centrifuged.

A Microtainer™ LT is the specimen of choice for measuring lead concentrations. Whole blood in an unspun GRT is also acceptable. For determination of zinc concentrations, submit a spun GRT with gel or a spun Microtainer™ SST.

For other tests, refer to the February 2000 Antech News or contact our Avian and Exotic Client Service representatives.

MICROBIOLOGY SPECIMENS
Aerobic and Anaerobic Cultures

These specimens are best obtained with a Copan swab; one for each culture type. The transport gel in the Copan swab (supplied by Antech) will preserve both aerobic and anaerobic organisms. Older style swabs (culturette) that have ompules to crush are not acceptable for anaerobic cultures. The most productive specimens are obtained by swabbing deep into an infected wound, or obtaining fluid from an actively infected tissue. Bacteria in pus are often nonviable and commonly result in no growth. Anaerobic cultures should be transported at room temp.

Viral Culture

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 with 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.

Mycoplasma

Cultures for Mycoplasma should be submitted on a Copan swab. The swab must be refrigerated or kept with a cold pack until picked up by the Antech courier.

HISTOLOGY AND CYTOLOGY SPECIMENS
Bone Marrow

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 (10-20 cc) rinsed with a little 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.

Biopsy Tissue

Tissue specimens for histology must be preserved and transported in formalin (10 parts formalin to 1 part tissue). The ideal tissue 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 an Antech supplied or FAA approved container, 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. Samples packaged inappropriately may not be picked up by the courier.

Very Large Specimen

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.

Tissue Orientation and Information

Knowing the 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.

Very Small Specimen

Tiny samples, such as from endoscopy, are best preserved if they are first placed in a labeled tissue cassette holder (available from ANTECH) and then dropped into formalin. Small biopsies should not be placed into a container with large tissue, as they are easily lost.

Cytology Fluid

Fluids 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. Do not add fixative or stain to any cytology smears. Urine smears for cytology should be made from fresh unstained urine sediment.

PATIENT DESCRIPTORS
Collection Site/Source, Animal History & Status

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.

Preparation of biopsy samples is a multi-step, labor-intensive process. Generally, Antech’s expected turn around time is 2-4 business days (Monday thru Friday) from the time the sample reaches the laboratory.

Some samples require special preparation and handling such as additional fixation time, decalcification, gross dissection by the pathologist, and/or special staining procedures. These procedures may delay receipt of your final report.