True North Veterinary Diagnostics Inc

320-6325 204 Street
Langley, BC V2Y3B3

(604)539-5550

www.tnvd.ca

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ACTH RESPONSE TEST (HYPERADRENOCORTICISM SCREENING TEST)

ACTIVATED CLOTTING TIME (ACT)

BAL-BRONCHIOALVEOLAR LAVAGE

BILE ACID PROTOCOL

BIOPSY-BONE

BIOPSY-LIVER

BONE MARROW ASPIRATE

BONE MARROW CORE BIOPSY

CEREBROSPINAL FLUID-CSF

COAGULATION TEST SAMPLE COLLECTION

DEXAMETHASONE SUPPRESSION TEST (EQUINE HYPERADRENOCORTICISM)

DEXAMETHASONE SUPPRESSION TEST-HIGH DOSE (CANINE HYPERADRENOCORTICISM DISCRIMINATORY TEST)

DEXAMETHASONE SUPRESSION TEST-LOW DOSE (CANINE HYPERADRENOCORTICISM SCREENING TEST)

FINE NEEDLE ASPIRATES

FREEZING PROTOCOL FOR TRANSPORT OF LABILE SAMPLES

GLUCOSE TOLERANCE TEST

HCG RESPONSE TEST (TESTOSTERONE)

IMPRESSION SMEARS

JOINT FLUID ASPIRATION

NASAL FLUSH

NASAL ASPIRATION BIOPSY

ORAL SUGAR TEST

PROSTATIC WASH

T3 SUPPRESSION TEST (FELINE HYPERTHYROIDISM)

TRH RESPONSE TEST (CANINE HYPOTHYROIDISM)

TRH RESPONSE TEST (FELINE HYPERTHYROIDISM)

TSH RESPONSE TEST (CANINE HYPOTHYROIDISM)

TRANSTRACHEAL WASH

ACTH RESPONSE TEST (HYPERADRENOCORTICISM SCREENING TEST)

PROCEDURE

Note: Sedation of the animal can be done but is not recommended as it can interfere with the outcome of the test.

CANINE: Collect a baseline sample in a green or red top or SST tube and separate the serum or plasma as soon as possible.

  1. Inject Cortrosyn at .25mg/dog or 5 ug/kg IV or IM. IV is advised if patient is dehydrated. 
  2. If using Synacthen Depot 1mg/ml - Inject Intramuscularly 0.5 mg or 1/2 vial for dogs under 10-15 kg and 1mg or a full vial for dogs weighing more  than 10-15 kg.  Note in normal dogs (weighing 13.5 to 33 kg) Synacthen Depot administered at a dose of .25mg (or 250 ug) IM resulted in an equivalent  adrenal cortical stimulation (AJVR 2012). A specific protocol has not been researched in clinically Cushingoid patients. Previous  internal data at CVL noted a 90 minute post is appropriate for Synacthen Depot while a two hour post is often cited in other sources. Gel products  are generally dosed at 2.2 IU/kg. For e.g. Bexco ACTH gel (40U/ml). Compounded gel products are generally not advised.
  3. Collect a post sample at 1 hour with the Cortrosyn,90 minutes or up to two hours with Synacthen Depot and at 1 and hours with a gel product.
  4. Separate the sample as soon as possible.
  5. Submit the original tubes and the spun off serum/plasma (0.5 ml) to the lab. Make sure the tubes are labeled correctly as pre or post.

COMBINED BILE ACIDS/ACTH STIMULATION TEST: ACTH and bile acids panels can be run simultaneously however varying protocols apply. There is also the rare report of food-dependent Cushing’s to consider. If combining a bile acids tests with Cortrosyn, take the pre-sample, administer Cortrosyn according to previously mentioned protocol. Feed the dog appropriately (higher fat meal if not contraindicated), wait one hour then collect the post ACTH sample and then wait another hour to collect the post bile acids sample. If using ACTH gel products ideally a one hour and a two hour post should be collected along with the two hour post prandial bile acids. If using Synacthen Depot a two hour post cortisol along with the post-prandial bile acids can be collected. Alternatively a 90 minute post ACTH and an additional 2 hr post prandial bile acids can be collected. 

MONITORING RESPONSE TO TRILOSTANE THERAPY:

  1. Only an ACTH stimulation can be used to monitor a response to Trilostane. A LDDST cannot be used for monitoring response to Trilostane.
  2. Note the drug can have additive effects and maximal effect may take up to 30 days.
  3. Trilostane should be given with food. Trilostane can be given with food at home and the ACTH stimulation test started 2-4 hours afterwards in the clinic. (Griebsch JVIM 2014)
  4. Importantly the ACTH stimulation test should be initiated at the same time every time when monitoring/comparing ACTH stimulation tests. 
  5. If short duration of action is suspected one can consider checking a 24 hour post Trilostane ACTH stimulation. Short duration of action is a consideration when the ACTH stimulation appears to be within an optimal range but clinical signs persist.   A 24 hour post Trilostane UCCR collected at home may be helpful in determining short duration of action. 
  6. If a dose adjustment is indicated a 10-20% change up to a maximum increase of 25% is advised.  If moving from once a day to BID dosing - the original once a day dose should be split as a starting point. 

 

FELINE: Procedure is the same as for dogs however IV is the preferred method for administration of Cortrosyn. The dose is .125 mg or 125 ug/cat IV. Post samples should be collected a 30 and 60 minutes. 

Synacthen Depot has not been well evaluated in cats. An ACTH stimulation is less sensitive than a LDDST for the diagnosis of hyperadrenocorticism in the cat. A UCCR is also a good screening test in cats for Cushing’s. Note Urine for a UCCR should be collected at home and it should be 48-72 hours since the last veterinary visit. 

 

FERRETS: Procedure is the same as for cats; samples should be collected at 0, 30, and 60 minutes. Please note: It may be helpful in both ferrets and cats to measure pre and post progesterone as well as cortisol.

PLEASE NOTE: The diagnosis of adrenal tumors in ferrets is difficult and recent information suggests that measurement of adrenal metabolites may be the best way to determine adrenal neoplasia. Concentrations of androstenedione, estradiol and 17-hydroxyprogesterone are elevated.

Comments: Protocols for performing the test differ between species and within a species. Refer to the protocol section of the manual. Corticosteroids will interfere with this test and should be withheld a minimum of 6-8 weeks prior to testing. This may not be the best or first choice for diagnosis of adrenal tumor(s) in ferrets. See discussion of adrenal imbalance and adrenal metabolite panel with regards to non Cushing’s adrenocortical disease.

 

ACTIVATED CLOTTING TIME (ACT)

Used as a crude screen of coagulation.

  1. A tube containing diatomaceous earth is filled with 2 ml of blood
  2. The tube is inverted 5 times
  3. Keep tube at body temperature (e.g. under arm) checking every thirty seconds for complete clot formation.

 

BAL-BRONCHIOALVEOLAR LAVAGE

PROCEDURE

  1. Anesthetize animal.
  2. Place an endotracheal tube.
  3. Place animal in lateral recumbency.
  4. Pass a 14-gauge catheter with stylet; remove metal stylet before catheter reaches the end of the endotracheal tube.  Extend catheter beyond the tube.
  5. Use the end of the catheter to collect scrapings from the bronchi.  Lavage fluid can also be used in small amounts-5 ml aliquots.  Collect samples into EDTA (lavender top) tubes for cytology and red top tubes for culture.
  6. Alternatively use a fiber optic bronchoscope with brush attachments.

 

BILE ACID PROTOCOL

PROCEDURE

The patient should be fasted for 12 hours in order to allow for filling of the gall bladder. Shorter fasting may result in a gall bladder that is not completely full and therefore an inadequate challenge. ACTH and bile acid panels can be run simultaneously however varying protocols apply. For example, if using Cortrosyn take the pre sample, administer Cortrosyn according to ACTH protocol, feed the dog a fat rich meal, wait one hour then collect the post ACTH sample and then wait another hour to collect the post bile acids sample. If using ACTH gel products the same pre and post samples could be used as the collection times are the same for both ACTH and the bile acids panel-two hours post.   When running a bile acids profile on an animal with pancreatitis or prone to pancreatitis, feed the animal its regular meal (forgo the high fat meal) and indicate in the history the fact that the animal is prone to pancreatitis or has had pancreatitis in the past and the pathologist will interpret the results based on that information. 1-2 tbsp. of vegetable oil can be added to the food to increase fat content if necessary.

  1. Collect a baseline blood sample (0.5 ml serum).
  2. Feed a fat rich meal (any commercial diet with a high fat content or add 1-3 tsp of vegetable oil).
  3. Collect a second blood sample (0.5 ml serum) 2 hours after the meal was fed.

In order for the test to be successful the animal must have a full gall bladder at the beginning of the test and the gall bladder should empty after the meal to provide a challenge to the liver via the enterohepatic circulation. Problems with the test include incomplete fasting, premature gall bladder contraction (excitement, smell of food coupled with habitual feeding times), intestinal malabsorption and difficulty with feeding (anorexia, failure to eat adequately, insufficient fat in meal).

 

BIOPSY-BONE

PROCEDURE

Choose the site most representative of the condition. Remember that bone neoplasia may be accompanied by a marked periosteal bone reaction that is non-neoplastic. Biopsies that are too superficial often only sample this reactive tissue rather than the underlying lesion. The biopsy should focus on lytic areas and penetrate through to the opposite cortex (ie. a core of tissue that spans the entire lesion). Radiographs taken after the biopsy will demonstrate the location of the biopsy site. Reference: JAAHA, 1981; 17:889.

 

BIOPSY-LIVER

NOTE: It is recommended that an ACT test be done in the clinic and, if abnormal, a coagulation screen should be done prior to surgery since liver disease may be accompanied by coagulopathies.  Trucut biopsies may be too small to adequately assess liver pathology.

PROCEDURE

Trucut needles can be purchased through Travenol Laboratories or AVP (BC), listed as "biopsy need, trucut". A 6" needle is recommended for most small animal procedures.

SMALL ANIMAL LIVER BIOPSY

  1. Fast patient for 8-12 hours.
  2. Feed small quantity of a fatty meal orally 1/2 to 1 hour prior to biopsy to stimulate gall bladder contraction, (e.g., ice cream).
  3. Restrain by sedation and local anesthesia.

Method:

    1. Clip and surgically prepared ventral abdomen.
    2. Position in dorsal recumbency tilting right side slightly towards table-30 degrees.
    3. Locate biopsy site at level at level of xiphoid, 1/2 distance from midline to costal arch.
    4. Take biopsy from left side to prevent penetration of the gall bladder.
    5. Infuse local anesthetic down to peritoneum.
    6. Make stab skin incision.
    7. Advance trucut needle in closed position into liver, directing needle's longitudinal axis 2-30 degrees craniodorsal and slightly left of midline.
    8. Advance the needle 1-3 cm into the liver.
    9. Inner obturator should be quickly and firmly thrust into liver parenchyma.
    10. Withdraw entire needle.
    11. Gently tease biopsy specimen off the trucut needle.  A swab for culture can be obtained from one end of the biopsy.  It is helpful to place the needle biopsy in a section of lens paper, which is then folded around the biopsy and placed into 10% buffered formalin.
    12. Place the patient in sternal recumbency for 5-10 minutes to allow body weight to compress biopsy site.

EQUINE LIVER BIOPSY

  1. Clip and surgically scrub area.  Site is located in the 14th right intercostal space at the intersection of the line drawn between the tuber coxae and the point of the shoulder.
  2. Use local anesthetic and a skin stab incision.  Direct needle cranioventral.
  3. Proceed as with instructions for canine biopsy.

These approaches can also be used for liver aspirates.

 

BONE MARROW ASPIRATE

It is impossible to accurately assess a bone marrow without a current CBC-please submit with a CBC if one has not been performed within the previous 24 hours.

PROCEDURE

SMALL ANIMAL

  1. Proximal femur-enter trochanteric fossa between greater trochanter and femoral head, needle entry parallel to and in the center of the femoral shaft. Advance the needle far enough to go through the cortex and into the cancellous bone of the proximal femur. It is possible to tell when the needle is seated as the needle will move with the femur. Do not advance too far into the medullary cavity as active marrow tissue is confined to the cancellous bone.
  2. Iliac crest-palpate and locate prominence of the iliac crest. With the stylet in place the needle is advanced to the bone with needle entry perpendicular to the long axis of the ilium. Advance needle by rotation until seated firmly in bone.

The humerus can also be used in smaller animals, cats.

HORSES

STERNUM: Palpate midline of sternum. Site of entry should be the intersection of the midline and point of the elbow. Needle should enter perpendicular to the sternum and can be tapped into the bone using a hammer.

ASPIRATION AND SLIDE PREPARATION

  1. Once needle is correctly positioned, remove stylet and attach 6-20 cc syringe.
  2. Aspirate with full negative pressure.
  3. As soon as marrow appears in syringe barrel, release negative pressure.
  4. Detach syringe, express marrow into a cold plastic dish containing 1-2 drop of EDTA. Swirl marrow to prevent clotting. Can also place droplets directly on slides and smear. Compounding pharmacists can make EDTA solutions or EDTA can be tapped out of lavender top tubes.
  5. Using forceps or a Pasteur pipette, pick up spicules and a small amount of blood and place onto slide.
  6. Gently place another slide over top at right angles and pull top slide over bottom slide making a smear. 8-10 slides should be made. Do not press. Allow the surface tension of the blood/fluid to provide the pressure.
  7. All remaining blood and spicules should be placed into LT tube and submitted with the slides. Avoid placing bone marrow slides in the same packaging as formalin samples.

 

BONE MARROW CORE BIOPSY

Core biopsies of bone can be taken with a trephine to provide information on marrow architecture.

PROCEDURE

Use Jamshidi 11 ga x 4” needle (or similar needle); the wing of the ilium is the preferred site. The needle is inserted at right angles to the ilium and using a firm twisting motion the cannula portion is drilled through the entire width of the ilium.

 

CEREBROSPINAL FLUID-CSF

Collect using a spinal needle, 1 ½” preferred in small dogs and cats, 2 ½” in larger dogs.

PROCEDURE

  1. General anesthesia and intubation are mandatory.
  2. Right lateral recumbency-the caudal skull and proximal neck are shaved and surgically prepared.
  3. Flex neck so the nose is perpendicular to long axis of the spine, head and neck are parallel to the tabletop.
  4. External occipital protuberance is palpated on the skull, marking the midline.
  5. The most anterior margins of the wings of the atlas are located. An imaginary line is drawn between these two and the needle inserted where this line intersects with the midline.
  6. The needle should be inserted through the skin, muscle and fascia. Then advance the needle 1-2 mm at a time, removing the stylet often to check for CSF. If the needle strikes bone, withdraw completely and redirect.
  7. Collect CSF by allowing it to drip freely into the EDTA tube and RT. RT is required for glucose/protein and culture or serology, if needed. EDTA is used for cytology.

 

COAGULATION TEST SAMPLE COLLECTION

PROCEDURE

It is imperative that proper collection procedures are followed for coagulation test results to be accurate. Contamination with tissue thromboplastin, prolonged collection, improper mixing and improper anticoagulant: blood ratios will all adversely affect coagulation tests. If necessary, sedation or anesthesia may be required in difficult patients. Avoid the jugular if serious coagulopathy is suspected. Clean venipuncture of large caliber blood vessels is optimal. If initial venipuncture is less than optimal, the first 1-2 ml of blood that may be contaminated with tissue thromboplastin can be discarded or placed in EDTA for a CBC before a clean syringe is used to collect the coagulation sample. The sodium citrate tube (BT) must be properly filled with blood; our tubes require 2.7 ml whole blood. The vacuum in the tube is designed to draw the correct amount of blood into the tube to achieve the optimal ratio with the anticoagulant. The tube is spun immediately. The plasma is removed and placed into another blue top tube which has had the sodium citrate removed by shaking it out of the tube. If not available, a plastic tube can be used. Do not use glass (RT) tube-only siliconized glass or plastic can be used to transport citrated plasma for coagulation tests. The RT tubes currently supplied by the lab are siliconized but please verify this before using the tube for transport of citrate plasma. Citrated plasma is required for running tests of coagulation such as PT, PTT, D-Dimer , Von Willebrand factor and other factor analyses. The separated plasma should be transported to the lab on ice. Freeze the samples if they will not reach the lab within 6 hours of collection.

Note: An instructional video is available at https://ahdc.vet.cornell.edu/Sects/Coag/samplinginstructionsinstructions.cfm This test can be ordered in conjunction with a prothrombin time at a cost of $42.45

Reporting time: Same day.

 

DEXAMETHASONE SUPPRESSION TEST (EQUINE PPID)

PROCEDURE

  1. At 5 pm draw a pre-injection blood sample into a labeled green top or RT/SST tube. Inject 40 ug dexamethasone/kg of body weight IM.
  2. At 8 am (15 hours) draw a second blood sample into a labeled tube (optional).
  3. At 12 noon (19 hours) draw a third blood sample.
  4. All blood samples should be spun as soon as possible after collection and then both the spun tubes and the separated samples submitted to the laboratory. There should be a minimum of 0.5 ml serum or plasma per sample. Times should be clearly labeled on all tubes.

 

DEXAMETHASONE SUPPRESSION TEST-HIGH DOSE (CANINE HYPERADRENOCORTICISM DISCRIMINATORY TEST)-HDDST

This test is designed to differentiate pituitary dependent Cushing’s from functional adrenocortical neoplasia (adrenal dependent Cushing’s) in the dog. A suppressive response to a high dose of dexamethasone is seen in about 70% of pituitary dependent Cushing’s and very few dogs with functional adrenal tumors. This leaves about 30% of dogs with pituitary dependent Cushing’s which do not suppress and which cannot be differentiated from dogs with functional adrenal tumors by this method. In cats the percentage is reported to be even lower, between 50-65%. The high dose test is used after the diagnosis of Cushing’s disease is established. The test is an aid in differentiating pituitary based Cushing’s from adrenal tumors. It is based on the premise that adrenal tumors are autonomous producers of cortisol and will not discontinue production when an exogenous source of corticosteroid is given (loss of negative feedback).   Endogenous ACTH can also be used as a screening test (refer to page 26 for more information).

 

PROCEDURE

CANINE  

  1. Collect a baseline sample in a RT/SST or green top tube and separate as soon as possible.
  2. Inject 0.1 mg/kg dexamethasone IM or IV (IV preferred) in dogs regardless of size..
  3. Collect a sample 8 hours post injection. Separate as soon as possible and submit both original tubes and spun samples to the laboratory. Make sure the samples are correctly labeled as to pre and 8 hours post. A minimum of 0.5 ml of serum or plasma per sample is required.

FELINE

 

  1. Collect a baseline sample in a RT/SST or green top tube and separate as soon as possible.
  2. Administer 1 mg/kg dexamethasone IM or IV (IV preferred).
  3. Collect samples at 4+8 hours post dexamethasone.

Comment: Please label the tubes as to time of collection, 0 and 8 hours.

 

DEXAMETHASONE SUPRESSION TEST-LOW DOSE (CANINE HYPERADRENOCORTICISM SCREENING TEST)-LDDST

PROCEDURE

CANINE

Make sure to calculate dose based on active ingredient unique to certain preparations such as Dexamethasone sodium phosphate. Consider dilution in saline to ensure accurate dosing in small dogs.

 

  1. Collect a baseline sample in RT/SST or green top tube.
  2. Inject 0.01 mg/kg dexamethasone IM or IV (IV preferred).
  3. Collect 4 and 8 hour samples if possible or alternatively only an 8 hour sample.
  4. Separate as soon as possible and send all tubes clearly labeled as to collection times to the laboratory. A minimum of 0.5 ml serum or plasma is needed for each sample.

FELINE

  1. Collect a baseline sample in RT/SST or green top tube.
  2. Administer 0.1 mg/kg dexamethasone IM or IV (IV preferred).
  3. Collect samples at 4 and 8 hours post dexamethasone.

FERRET

  1. Collect a baseline sample.
  2. Inject 0.2 mg dexamethasone IM or IV (IV preferred).

Collect 3 and 5 hours post injection. Note: Protocols for doing dexamethasone suppression testing have been described for ferrets but this is not considered the test of choice in this species.  

 

Note: Animals must be off all topical and systemic steroids for a minimum of 4-6 weeks prior to running this test.

 

See www.endocrinevet.info/2012/05/helpful-tips-to-improve-accuracy-of-low.html

 

FINE NEEDLE ASPIRATES

The objective is to obtain a monolayer of intact cells from the tissue/lesion of interest spread onto a slide and allowed to air dry. Due to the highly variable nature of the lesions sampled, technique must be modified to suit each case. Avoid overly thick or highly blood contaminated samples as cells will be shrunken and distorted which inhibits interpretation.

PROCEDURE

  1. A small gauge needle (typically 22 or 25 g) is inserted into the lesion and moved in a forward and back motion while at the same time redirecting the needle into multiple areas of the lesion, loosening cells and filling the needle.
  2. After the needle is withdrawn, a syringe with a small quantity of air (1-2 cc) is attached and the contents of the needle are gently blown onto a glass slide.
  3. The sample is then spread onto the slide using the most appropriate technique to form a monolayer. Fluid/bloody samples are typically spread using a similar technique to a blood smear i.e., touching a second slide to the sample, allowing it to spread along the edge and moving it slowly forward. “Chunky “samples can be squashed by pressing two slides together and either pulling them directly apart (vertical technique) or by moving the two slides in opposite directions (horizontal technique). Care must be taken to avoid disrupting the cells, especially with the horizontal technique.
  4. Soft or bloody lesions typically require smaller gauge needles for optimal sampling and firm or fibrous lesions may require larger gauge needles. In some cases 18 to 21 g needles may be used if deemed appropriate. A syringe can be attached to the needle during the aspiration procedure and suction applied to obtain cells from a lesion that is not exfoliating cells. Suction must be stopped prior to withdrawing the needle to avoid sucking the sample into the barrel of the syringe.

 

FREEZING PROTOCOL FOR TRANSPORT OF LABILE SAMPLES

Some analytes are labile and will deteriorate quickly at room temperature so they must be frozen and maintained in a frozen state until testing. When the distance the sample must travel to reach the lab is long, keeping a sample frozen during transport can be difficult. Most labile analytes are also sensitive to glass and frequently need to be placed in plastic tubes.

 

    1. Separate samples as soon as possible. Plasma samples can be spun immediately while serum samples will need to clot approximately 20 minutes before they can be spun and separated.
    2. Place the spun sample in a plastic tube or small, sealable plastic container (siliconized glass may also be acceptable). Do not overfill the tube-allow for expansion during freezing.
    3. Place the sample in a styrofoam cup partly filled with water. There should be sufficient water to completely cover the serum or plasma in the tube but the entire tube should never be completely submerged.
    4. Freeze the sample so the sample tube is partly embedded within a block of ice. The ice should completely cover the serum or plasma. Place a lid on the cup and seal with para film. Place in a ziploc bag.
    5. Package the sample with ice packs and insulating material such as styrofoam chips or bubble pack.
  • Inform the lab that you are sending the sample, its ETA and provide any tracking information for the shipment.

 

GLUCOSE TOLERANCE TEST

PROCEDURE

CANINE

Following a 12 hour fast, draw a blood sample. Then give 1 g glucose per 2.2 kg body weight per os. Draw samples at 30, 60, 120 and 180 minutes.

EQUINE

Following a 12 hour fast, draw a sample for blood glucose. Then give 1 g dextrose/kg body weight by stomach tube. Collect samples at 10, 30, 60, 120 and 160 minutes.

INTERPRETATION: Normal dogs are supposed to reach 8.8 mmol/l @ 60 minutes and return to baseline values by 120-180 minutes. In horses with malabsorption there is a flat curve, indicative of poor absorption.

 

HCG RESPONSE TEST (TESTOSTERONE)

PROCEDURE

  1. Collect a baseline sample (1 ml of serum).
  2. Inject 10,000 to 20,000 iu of HCG, intravenously.
  3. Collect a sample 30 and 60 minutes post injection (1 ml serum each).

INTERPRETATION: Males with functional testicular tissue will have a marked increase in testosterone levels.

 

IMPRESSION SMEARS

PROCEDURE

Pat the surface of the sample dry with gauze or paper towel. Take a scalpel and score the surface in a crisscross pattern for firm lesions. Gently dry the surface again if the sample was scored and then lightly touch the slide to the surface of the tissue. A single slide can be used for 3 or 4 impressions depending on the size of the original mass.

 

JOINT FLUID ASPIRATION

PROCEDURE

Small animal joint aspiration can be from any joint but commonly the stifle or carpus is used. In large animals, procedures are described for almost all joints. In general, flexion or extension of the joint to open the joint space and choosing an entry site where the joint space is most accessible is preferred.

CARPUS

Use a 22 to 25 gauge needle of adequate length to enter the joint. Attach 3 ml syringe.

Enter the carpus on the anterior aspect and slightly medial, between the radiocarpal bone and radius, or in an intercarpal joint space. Flexing the carpus may help determine the location of the joint space.

STIFLE

Flex joint, insert needle lateral to the straight patellar ligament, direct needle medial and proximal towards the cruciate attachment ensuring a sufficient length of needle to pass through the patellar fat pad.

The restraint required will depend on the patient-general anesthesia or tranquilization is typically required. If only a drop of fluid is obtained then make an air dried smear similar to a blood smear, taking into account the viscosity of the sample. More viscous samples require slower forward movement of the spreader slide. Please record color, amount and viscosity at the time of collection.

Reference: Compendium, 1979, 1:11;855.

 

NASAL FLUSH

PROCEDURE

  1. A stiff plastic catheter (#10 French) is used.
  2. General anesthesia is required and the animal must be intubated with a cuffed endotracheal tube.
  3. The animal is placed in lateral recumbency and the table is tilted so the animal’s head is down.
  4. The catheter is measured from the external nares to the medial canthus of the eye and cut to this length with a 45 degree bevel at the tip.
  5. Place gauze sponges behind the soft palate.
  6. The syringe is attached to the catheter after it is placed into the nose.
  7. Flush the catheter with sterile saline or ringer’s solution while at the same time forcefully moving the catheter within the nasal cavity.
  8. Flush approximately 200-300 ml of fluid through the turbinates.
  9. Collect some of the fluid in lavender top tubes. Larger pieces of tissue and the tissue collected on the gauze sponges should be transferred to 10% formalin for histopathologic evaluation.
  10. Samples for bacteriology or fungal cultures should be taken from the first fluid flushed through the nose and placed into a RT tube.
  11. At the end of the flush, 10-20 ml of Betadine solution may be flushed into the nostril.

Reference: JAAHA, 1977:13;704.

 

NASAL ASPIRATION BIOPSY

This can be used if a mass is identified radiographically within the nasal cavity.

PROCEDURE

A large bore, 7 mm, plastic tube, which can be the outside protective covering of an IV catheter, or plastic protective covering from a spinal needle, is used.

  1. This tube is advanced into the nasal cavity and then negative pressure applied by syringe (12 cc).
  2. The tube should be redirected and advanced to obtain more than one fragment.
  3. The tube is withdrawn, while negative pressure is maintained on the syringe. Air is flushed through the tube to force the biopsy out onto a gauze sponge.
  4. The tissue and gauze are placed in 10% formalin.

Reference: JAAHA, 1985: 21;851.

 

ORAL SUGAR TEST

 

This test is more sensitive than a single insulin baseline for detecting EMS. The horse must be fasted overnight before performing this test.

 

  1. Collect a baseline (or Pre) blood specimen into either a plain red top or lavender top collection tube and follow specimen processing as described below (Sample Collection and Processing Guidelines)
  2. Give 0.15 mL/kg (approximately 75 ml) Karo Light corn syrup orally.
  3. Collect additional blood specimens at 60 and 90 minutes after administration using the same type of collection tubes used in Step 1. Process the specimens and submit all samples together as described below.

Sample Collection and Processing Guidelines:

  1. Collect blood into a plain red top collection tube for serum or a lavender top tube (EDTA) for plasma
  2. For plasma, gently invert the lavender top tube several times immediately after collection to mix anti-coagulant and refrigerate specimen until centrifugation.
  3. For serum, allow the red top blood specimen adequate time to clot prior to centrifugation to avoid fibrin formation and ensure sufficient serum yield. This can be at room temperature or refrigerate if longer than one hour will be needed before centrifugation.
  4. After centrifugation, immediately transfer serum or plasma into a vial suitable for shipping and frozen storage.
  5. Frozen storage is recommended unless samples are being shipped the day taken. A frozen sample is not necessary but the samples should arrive chilled. Ship samples with cold packs using an overnight courier service.

*Avoid use of serum collection tubes with additives (ie. separator gels, clot activators, inhibitors, etc. ) due to potential assay interference.

 

PROSTATIC WASH

PROCEDURE

  1. Catheterize bladder and empty urine.
  2. Withdraw catheter and place a clean catheter to the level of the prostate.
  3. Wash urethra with 2 to 5 ml of saline while at the same time massaging the prostate per rectum.
  4. Aspirate fluid and place small amount in RT tube for culture and remainder in LT tube for cytology.

 

T3 SUPPRESSION TEST (FELINE HYPERTHYROIDISM)

PROCEDURE

  1. Baseline T3 and T4 levels are obtained on 0.75 ml of serum.  Refrigerate.
  2. The cat is given Cytobin (oral T3) at a dose of 25 ug three times daily for 2 days.  On the morning of the third day a seventh dose of Cytobin is given and a serum sample collected 2 to 4 hours after the Cytobin administration.  0.75 ml of serum is required for T3 and T4 levels.

Both the pretest and the post T3 sera should be submitted at the same time.

INTERPRETATION: Cytobin will cause marked suppression of T4 levels in normal cats.

TRH RESPONSE TEST (CANINE HYPOTHYROIDISM)

PROCEDURE

  1. Collect a resting sample (0.25 ml serum).
  2. Inject 0.2 mg of TSH IV.  This is 1 vial of Relefact.
  3. Collect samples at 4 to 6 hours post TRH.  (0.25 ml serum).
  4. If TSH is being measured in addition to T4, collect 0.75 ml serum at each sampling as well as an additional sample 30 minutes post TRH.

INTERPRETATION: Some dogs will have the maximum response at 4 hours, others at 6 hours.  Dogs with normal thyroid function will have a response, which is equal to or greater than 1.6 times the baseline values, and the actual value should exceed 39 nmol/l.  The concurrent measurement of TSH results in a slight increase in the sensitivity and specificity of the test and is necessary to identify type II hypothyroidism (primary TSH deficiency).

 

TRH RESPONSE TEST (FELINE HYPERTHYROIDISM)

PROCEDURE

  1. Collect baseline serum sample (0.5 ml serum) for T4 measurement.
  2. Inject Relefact 0.1 mg/kg intravenously (1/2 vial).  Never exceed one vial per cat.
  3. Collect a 4 hour post sample (0.5 ml serum) for T4 measurement.

Interpretation: Cats with hyperthyroidism will have little change in their T4 levels, whereas normal cats have a consistent rise in T4 levels.

Cats often vomit after TRH injection.  This can be prevented by pretreating with dolasetron, ondansetron or Maropitant.

 

TSH RESPONSE TEST (CANINE HYPOTHYROIDISM)

PROCEDURE

  1. Collect a resting sample.
  2. Inject 0.1 iu/kg IV, up to a maximum of 5 units.
  3. Collect a sample 4 hours post injection.

INTERPRETATION: Dogs with normal thyroid function will have a post value at least double the baseline value and results should exceed 64 nmol/l.

 

TRANSTRACHEAL WASH

PROCEDURE

SMALL ANIMAL

Use a through the needle IV catheter (ie. Intracath), 18-20 gauge according to the size of the dog or cat.

  1. Restrain in sternal recumbency with head extended. Use mild sedation (narcotics are contraindicated) to allow preservation of the cough reflex. Run finger along the trachea and palpate for the first prominent ring ventral to the larynx, which is the cricoid cartilage. The cricothyroid ligament is immediately dorsal to the cricoid cartilage. This ligament will feel like an open space between firm cartilage rings.
  2. Clip and surgically prepare the area, use a local anesthetic. Insert the IV catheter with the point directed towards the rear of the animal and into the lumen of the trachea. Advance the catheter down to the tracheal bifurcation.
  3. Remove the needle from skin; apply the needle guard to prevent the catheter from being cut off. Remove stylet and infuse 2-4 ml of saline; as the animal coughs, vigorously aspirate the fluid. Additional fluid can be injected until an adequate aspirate is obtained. Be sure to flush any material from the catheter after it is removed. Bandage the site as this should help prevent excessive bleeding and subcutaneous emphysema.
  4. The sample can be obtained using a catheter through a sterile endotracheal tube similar to the BAL procedure, however it is difficult to maintain a cough reflex and contamination of the sample with oropharyngeal bacteria is common.
  5. Place samples for cytology in an EDTA (lavender top) tube and culture samples onto a culture swab or into a red top tube.

Reference: JAAHA, 1979:10;219.