Investigation of canine hyperadrenocorticism
Introduction
Naturally occurring hyperadrenocorticism (HAC) may be pituitary dependent (PDHAC; 80% of cases) or may be due to adrenal tumours (ADHAC; 20% of cases). Iatrogenic Cushing’s syndrome is discussed under a separate heading. Unfortunately, there is no perfect diagnostic test for hyperadrenocorticism and false positive and false negative test results occur. More than one diagnostic test may be required to establish a confident diagnosis.
Therapy prior to testing
There is a risk that glucocorticoid therapy prior to testing could interfere with interpretation of the tests for adrenal disease. The following should be considered:
- Prednisolone, methylprednisolone and hydrocortisone will cross-react with the cortisol assay and therapy should be withdrawn for 24 hours prior to testing to eliminate the risk of falsely increased result
- Dexamethasone does not cross-react with cortisol assays but use for several days can suppress endogenous cortisol production, giving falsely low results
- Long term steroid therapy may suppress endogenous cortisol production and where possible, therapy should be withdrawn for 4-6 weeks prior to testing
- Treatment with topical agents containing glucocorticoids may be absorbed producing systemic effects and can affect the results of tests. These should be withdrawn prior to testing
Basal cortisol levels
There is considerable overlap between basal cortisol levels in normal dogs and those with HAC. A single basal cortisol is not recommended as a screening test for HAC.
Urine cortisol:creatinine ratio (UCCR)
This is an excellent test to exclude HAC (very few false negatives) but must not be used to diagnose HAC, as non-adrenal illness commonly gives a positive result. A morning urine sample is collected in the animal’s home environment. Normal dogs have a UCCR less than 30 x 106.
Steroid induced alkaline phosphatase
This assay is not a reliable marker for HAC. Although most dogs with HAC have increased enzyme activity, increases are also identified in dogs with primary hepatic pathology, diabetes mellitus and those treated with anticonvulsants.
ACTH stimulation test
This is a good, initial screening test and is the test of choice for diagnosing iatrogenic Cushing’s syndrome and monitoring trilostane therapy. It has a lower false positive rate than the low-dose dexamethasone suppression test (LDDST) but a significant false negative rate. It will reliably diagnose approximately 85% of PDHAC cases but only 50% of ADHAC cases. It is quick and simple to perform and is less affected by stress and non-adrenal illness than the LDDST. The initial values are useful as a reference to monitor effectiveness of treatment.
Protocol
- Take a basal sample (2ml serum)
- Inject 0.25mg synthetic ACTH (Synacthen) i/v or i/m (use 0.125mg for dogs weighing <4.0kg)
- Ensure a minimum dosage of 0.005mg/kg. Dogs >50kg may require additional ACTH
- Take a second sample at 1 hour post injection
- Separate the serum before dispatch to the laboratory and clearly label the tubes ‘pre’ and ‘post’ ACTH
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Interpretation
An increase in cortisol levels post ACTH is expected in all dogs. Normal dogs will show an increase in cortisol levels of up to 450nmol/l post stimulation.
An exaggerated response is expected in animals with pituitary-dependent hyperadrenocorticism (PDH) and cortisol concentrations rise above 600nmol/l and often above 1000nmol/l.
Dogs with an adrenal tumour often have basal cortisol above 250nmol/l with little or no change after stimulation. However the ACTH stimulation test is not as sensitive at detecting adrenal tumours and negative results should be confirmed with a low dose dexamethasone test when strong clinical suspicion remains.
False positive results can be noted with stressful illness. The decision to treat for HAC should not be based upon laboratory results alone. The ACTH stimulation test will identify approximately 85% of PDHAC dogs and approximately 50% of dogs with an adrenal tumour. It will not reliably distinguish between the two conditions.
Concurrent Investigations
If it is necessary for sample collection, the patient can be sedated prior to the ACTH stimulation test.
The test may be combined with a bile acid stimulation test. A baseline sample is followed by feeding and ACTH administration. Two further samples are collected at 1 hour (post ACTH) and 2 hours (post prandial bile acids).
Low dose dexamethasone suppression test
This test is more sensitive than the ACTH stimulation test (sensitivity >95%), therefore false negative results very seldom occur. Unfortunately, false positive results are common, especially when there is concurrent non-adrenal illness or other sources of stress. Positive results should be regarded with suspicion in dogs known to have significant non-adrenal illness. Ideally, the test should be postponed until any identified non-adrenal illnesses have been resolved or stabilised. In some cases, this test will allow differentiation between pituitary and adrenal dependent disease.
Protocol
- Take a basal sample of 2ml clotted or heparinised blood after an overnight fast
- Inject dexamethasone i/v at 0.01mg/kg bodyweight
- Take further 2ml clotted or heparinised blood samples at 3 hours and 8 hours post dexamethasone injection
- Mark the samples clearly and if possible separate the serum or plasma before dispatch to the laboratory
Interpretation
| Normal | PDHAC | PDHAC or Adrenal Tumour | |
|---|---|---|---|
| Basal cortisol | 100% | 100% | 100% |
| 3 hour cortisol | <50% | <50% | >50% |
| 8 hour cortisol | <40mmol/l | >40mmol/l | >40mmol/l |
The low dose dexamethasone screening test is interpreted in two stages. Firstly, the presence or absence of hyperadrenocorticism is established by examining the 8 hour result. A value above 40nmol/l supports the diagnosis. If the 8hr sample is above 40nmol/l then the degree of suppression of cortisol during the test is evaluated. If there is more than 50% suppression from the baseline value at either 3 or 8 hours then the pattern supports the presence of pituitary dependent disease. When there is no, or minimal, evidence of suppression it is necessary to follow-up with a differentiation test. The combination of endogenous (plasma) ACTH and adrenal ultrasonography is ideal but if these tests provide equivocal results then a high dose dexamethasone suppression test may be considered.
17-OH progesterone (17-OHP)
Case reports have suggested that some dogs with adrenal neoplasia do not test positive by conventional ACTH stimulation test, but will show increased concentrations of sex hormones, including 17-OH progesterone, after administration of synthetic ACTH (baseline and 1 hour post ACTH stimulation). The test has also been evaluated in dogs with classical hyperadrenocorticism and most affected dogs showed a significant increase after stimulation. Less than 10% of dogs with classic hyperadrenocorticism had post- ACTH 17-OHP <4.5nmol/l (Chapman et al, 2003 Veterinary Record, 153, 771-5) and fewer than 10% of dogs which do not have classic hyperadrenocorticism had post ACTH 17-OHP >16.7nmol/l.
Differentiating between pituitary and adrenal dependent disease
Low dose dexamethasone suppression test
In approximately 60% of dogs the pattern of results obtained in a low dose dexamethasone suppression test allows differentiation between PDHAC and ADHAC.
Endogenous ACTH
This is currently considered the laboratory test of choice for the differentiation of pituitary dependent hyperadrenocorticism (PDHAC) and adrenal dependent hyperadrenocorticism (ADHAC). Dogs with PDHAC have high (or high-normal) values of ACTH while low concentrations are expected in animals with adrenal neoplasia. Special sampling requirements apply.
Protocol
- Request a transport pack for delivery to the laboratory
- Take a blood sample into a cooled plastic (not glass) EDTA tube kept on ice
- Mix well but gently and centrifuge as quickly as possible (ideally in a refrigerated centrifuge)
- Transfer the plasma into a cooled plastic (not glass) PLAIN tube kept on ice. Immediately freeze (<-10°C) plasma and keep frozen until dispatch in the transport pack
Imaging
Adrenal ultrasonography is useful. Cases of PDHAC generally have bilaterally symmetrical adrenal glands while unilateral enlargement is more likely to reflect adrenal neoplasia. Bilateral adrenal tumours have been reported but are rare.
High dose dexamethasone suppression test
In recent years, the high dose dexamethasone suppression test has become less favoured by some clinicians, but the test is readily available to practitioners. This test must not be used to make an initial diagnosis of HAC.
Protocol
- Take a basal sample of 2ml serum
- Inject soluble dexamethasone i/v at 0.1mg/kg bodyweight
- Take further samples at 3 hours and 8 hours post dexamethasone injection
- Mark the samples clearly and separate the serum before dispatch to the laboratory
Interpretation
Any suppression greater than 50% of the baseline concentration indicates a pituitary source. A failure to suppress by 50% is consistent with either an adrenal tumour or a ‘dexamethasone resistant’ pituitary lesion (about 20% of patients with pituitary dependent disease).
Therapeutic monitoring
The ACTH stimulation test is the test of choice for monitoring the response of dogs with hyperadrenocorticism to therapy. When using trilostane, the timing of the test is critical. The datasheet recommendation is that trilostane should be monitored by performing an ACTH stimulation test (basal and 1hr post ACTH) at 4-6 hours after dosing. Monitoring of biochemistry including electrolyte concentrations is also recommended.
For trilostane therapy, the aim is to achieve pre and post stimulation concentrations of cortisol in the range of 50-200nmol/l. Experience in the field has shown that a post ACTH cortisol of up to 250nmol/l may not require an increase in dosage for continued clinical effect, Basal levels of <50nmol which show little or no response to stimulation suggest over-suppression of cortisol production. The specific management of such cases should be guided by the drug manufacturer’s recommendations.
Recent work suggests that measurement of cortisol immediately pre-trilostane and 3 hours after dosing in combination with a clinical scoring system (available from the drug manufacturer) may correlate better with the clinical response. This may also be helpful given the supply issues regarding synthetic ACTH.
Monitoring of Trilostane should be performed after 10 days, 4 weeks and 12 weeks of therapy (and after any dosage change) and then every 3 months.
Iatrogenic Cushing’s syndrome
Iatrogenic Cushing’s syndrome may arise due to prolonged administration of glucocorticoids. The ACTH stimulation test is the test of choice and affected dogs have a suppressed basal cortisol with little response to administration of ACTH. Prednisolone cross reacts with the cortisol assay and should be withdrawn 24-36 hours prior to testing. Monitoring of this condition is based on clinical response to a reduction in glucocorticoid therapy (where possible).