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Test Protocols

Adrenal Gland Disease

Bile Acid Simulation

Reproductive Function Tests

Therapeutic Drug Monitoring

Adrenal Gland Disease

Dogs

Pre-Trilostane Cortisol

Used for monitoring Vetoryl (trilostane) treatment, in combination with the clinical signs, from 4 weeks after commencement of trilostane treatment.

Collect a blood sample immediately before the trilostane is administered.

Suitable for:

  • Dogs on once- or twice-daily Vetoryl dosing
  • Adrenal and pituitary-dependent hyperadrenocorticism
  • Clinically well dogs (with or without signs of hyperadrenocorticism)
  • Calm dogs.

Unsuitable for:

  • Aggressive or stressed dogs, or unwell dogs, in which case an ACTH stimulation test should be performed.

Canine ACTH stimulation test

This test is used for the diagnosis of hypo- and hyperadrenocorticism, for monitoring dogs on treatment of hyperadrenocorticism and for diagnosis of iatrogenic hyperadrenocorticism.

  • Collect basal sample to provide 0.5 ml serum, label ‘pre’
  • Inject synthetic ACTH (Synacthen) IV.  The dose is 0.25 mg for dogs > 5 kg and 0.125 mg for dogs < 5 kg
  • Take a second sample 60 minutes later, label ‘post’
  • Separate the serum.

When used to monitor the effectiveness of trilostane therapy, the test is performed 4‒6 hours post-capsule. Monitoring is usually performed 10‒14 days, 30 days and 90 days after starting therapy and then every 3‒6 months.
NB This test is not useful for monitoring Addison’s disease once this has been diagnosed.

Low-dose dexamethasone suppression test, dogs.

This test is used to identify hyperadrenocorticism.  It is more sensitive than the ACTH stimulation test although it has a higher incidence of false positive results due to non-adrenal illness.

  • Collect basal sample to provide more 0.5 ml serum, label the separated serum ‘pre’
  • Inject 0.01 mg/kg soluble dexamethasone IV
  • Take samples at 4 and 8 hours post-dexamethasone, label the separated serum with times.

SHAP Test (17OHP pre- and post-ACTH) in dogs

This is used to evaluate cases of alopecia suspected to be due to sex hormone excess. It is also used to evaluate animals with suspected Cushing’s, but which have normal ACTH stim and low-dose dexamethasone tests. In such cases there may be a derangement of the normal steroid production pathway with accumulation of cortisol precursors including 17-hydroxyprogesterone (17-OHP).  The method is identical to the ACTH stimulation test: 17-OHP is measured pre- and 1 hour post-ACTH.

 

Cats

The best test for diagnosing feline hyperadrenocorticism has not been established. The ACTH stim test has a reported sensitivity of 50‒80%. The low-dose dexamethasone suppression test may be more sensitive, but too few cases have been assessed. Sometimes both tests are performed.

Low-dose dexamethasone tests, cats

  • Collect basal sample to provide more than 0.5 ml serum, label the separated serum ‘pre’
  • Inject 0.1 mg/kg soluble dexamethasone IV (note higher dose than used in dogs)
  • Take samples at 4 and 8 hours post-dexamethasone, label the separated serum with sample times.

Feline ACTH stimulation test

The time of peak response to ACTH is variable and so multiple samples are taken post-ACTH.

NB this test has been reported to be less sensitive than the low-dose dexamethasone test in cats

  • Collect basal sample to provide more than 0.5 ml serum, label the separated serum ‘pre’
  • Inject synthetic ACTH (Synacthen) IV.  The dose is 125 μg for cats < 5 kg and 250 μg for cats > 5 kg
  • Take samples at 1 and 3 hours post-ACTH, label the separated serum samples with sample times.

Bile Acid Simulation

  • Starve the patient for 12 hours
  • Collect 1‒2 ml blood into a serum gel tube
  • Feed a small amount of food ‒ ideally puppy/kitten food. If the patient is not eating liquidize the food and give by syringe
  • Collect a second sample 2 hours post-feeding, into a serum gel tube
  • Label the tube with patient details and time of sampling.

Reproductive Function Tests

Ovulation detection for timing of mating in female dogs

Since ovulation may occur at varying times after the onset of vulval bleeding, progesterone measurement can be used to determine the optimum time for mating. For most bitches, blood sampling for progesterone measurement should begin around 7 days after the onset of vulval swelling and bleeding. Bitches are then re-tested every 2–3 days until ovulation occurs.

Samples should be collected in a plain serum tube. Gel tubes should be avoided where possible or if used, the serum should be separated and removed from the gel tube as soon as possible.

Tests for detection of ovarian remnant in the bitch

  1. When the bitch is showing signs of oestrus there are several tests, which can detect ovarian tissue:
  • Vaginal smear ‒ cytological changes in vaginal epithelium can be used to identify ovarian activity
  • Measurement of oestrogen ‒ increases in late anoestrus, pro-oestrus and early oestrus, but then declines
  • Measurement of progesterone ‒ increases in late oestrus and remains high in dioestrus (i.e., for 60 days post-ovulation).
  1. When there are no signs of oestrus:

Anti-Mullerian Hormone (AMH) is useful for detection of ovarian remnant in bitches > 6 months of age, after they have reached sexual maturity. AMH levels remain constant throughout the cycle although they may fall transiently around ovulation. Animals that have been neutered will have undetectable levels of AMH. A positive AMH test indicates the presence of ovarian tissue, but a negative result does not fully exclude the presence of ovarian remnant, especially if the sample is taken around the time of ovulation. If a low result is found when there are signs of oestrus, measurement of progesterone is recommended.

Please note this test is not sensitive for detecting ovarian remnant in bitches < 6 months old.

Detection of ovarian tissue in female cats

AMH combined with progesterone is recommended to identify ovarian remnant and distinguish between spayed and ovarian intact queens after they have reached the age of sexual maturity. Cats are generally considered to be induced ovulators but some queens can ovulate spontaneously, and this can result in falsely negative AMH results. Around ovulation the AMH falls and progesterone increases. Since it can be difficult to know at the time of sample collection if an ovarian remnant’s primary secretory product is AMH or progesterone, it is prudent to evaluate both hormones at the same time.

Tests for detection of testicular tissue in cats and dogs

Anti-Mullerian Hormone is a reliable test for detecting the presence of testicular tissue in both cats and dogs, including cryptorchid males, and young males prior to puberty. AMH levels should be very low (undetectable) in correctly castrated animals.

Therapeutic Drug Monitoring

DWR’s Diagnostic Laboratory powered by Antech, based at Dick White Referrals, is a well-established laboratory providing a comprehensive range of diagnostic testing

Phenobarbitone

Sample into a plain serum tube. Separator gel can absorb the drug, so it is important to separate the serum of the gel as soon as possible. Alternatively use a plain tube without a gel separator. However, samples are more likely to be haemolysed when a gel separator is not used, and this can also affect the results. Moderately to markedly haemolysed samples should not be sent.

Potassium Bromide

Sample into a plain serum tube. Separator gel can absorb the drug, so it is important to separate the serum off the gel as soon as possible. Alternatively use a plain tube without a gel separator. However, samples are more likely to be haemolysed when a gel separator is not used, and this can also affect the results. Moderately to markedly haemolysed samples should not be sent.

Digoxin

Digoxin pharmacokinetics and dynamics may vary considerably from dog to dog. Time to ‘peak’ serum level is 2‒4 hours after oral dosage with time to ‘peak clinical effect’ being 6‒8 hours. ‘Trough’ serum levels usually occur after 8‒12 hours. The elimination half-life is variably reported between 14 and 56 hours in dogs.

A standard starting dose is 3‒4 micrograms/kg p.o. q12hrs for dogs ≤20kg and 0.22mg/m2 body surface area p.o. q12hrs for dogs >20 kg. A dose of 250 micrograms per dose should not be exceeded as an initial dose in any dog and this drug should be used with caution in breeds pre-disposed to ventricular ectopy such as Dobermann pinschers. Decreased doses or an increase in dosing intervals may be required in geriatric patients, obese animals or those with significant renal dysfunction.

Digoxin levels should be measured in serum without a gel separator 5‒7 days after initiation or dose-adjustment. The timing of the sample depends on the requirement to either detect a minimum effective level (in which case a ‘trough’ sample at 8‒12 hours post-pill would be sensible) or whether the clinical answer desired relates to potential for toxicity (in which case a ‘peak’ sample at 2‒5 hours post-pill would be sensible).

Trough samples of 0.6–1.2 ng/ml are usually sufficient to document adequate therapeutic digoxinaemia. However, most cardiologists will base assessment of adequacy of ventricular rate control in patients with atrial fibrillation on history, clinical findings, an in-clinic ventricular response rate <150 bpm, a 24 hour mean ventricular rate determined by Holter monitoring or an at-home ventricular response rate <110 bpm.

If toxicity is suspected or risk of toxicity evaluated for then levels exceeding 2.4 ng/ml should prompt dosage adjustment.

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