Helpful Tips to Improve the Accuracy of the Low Dose Dexamethasone Suppression Test

Of the 3 major screening tests for Cushing's syndrome, the low-dose dexamethasone suppression test (LDDST) is considered by many to be the test of choice for the diagnosis of hyperadrenocorticism in dogs (1-3).

In this post, I want to address some of the common mistakes I see veterinarians make when performing this test. Addressing a few small but important issues in dosing and timing of sampling can greatly improve the diagnostic accuracy of this test.

Advantages of the LDDST as a diagnostic test
Compared with the ACTH stimulation test, the LDDST is much more sensitive in confirming Cushing's syndrome in dogs. The sensitivity of the LDDST is excellent, approximately 90% to 95% in dogs with pituitary-dependent hyperadrenocorticism (PDH) and virtually 100% in dogs with a cortisol-secreting adrenal tumor (1-3).

Thus, this test will fail to confirm hyperadrenocorticism in only about 5% of dogs with the disease. That's not too bad.

Disadvantages of the LDDST as a diagnostic test
In contrast to the ACTH stimulation test, the LDDST is not helpful in the detection of iatrogenic hyperadrenocorticism. The test is also affected by more variables than the ACTH stimulation test, takes 8 hours to complete, and does not provide pre-treatment information that may be used in monitoring the effects of mitotane or trilostane therapy (1-3). For more information about when to use the ACTH stimulation test, see my previous post entitled, Diagnosing Canine Cushing's Disease: Should the ACTH Stimulation Test Ever Be Used?

The specificity of the LDDST can be low (40% to 50%), especially when measured in a population of sick dogs (4).  Because of the low specificity of this test, diagnosis of Cushing's syndrome should never be based on results of an LDDST alone, especially in a dog with nonadrenal disease. It is best to delay testing for hyperadrenocorticism until the dog has recovered from the concurrent illness (4).

Published protocols for the LDDST
Two similar protocols have been described for the LDDST in dogs, both of which yield similar patterns of cortisol suppression (1-3,5). One of these protocols used the veterinary-labeled preparation, dexamethasone in polyethylene glycol (Azium® Solution; 2 mg/ml; Schering-Plough).  Unfortunately, after merger with Merck in 2009, this Azium preparation has been discontinued and is no longer available (6).

The other test protocol for the LDDST —which I currently recommend— uses dexamethasone sodium phosphate (4 mg/ml) as the form of glucocorticoid administered.

Test Protocol & Sampling times
The protocol and steps that I take when performing a LDDST on a dog suspected of suffering from hyperadrenocorticism is as follows:

1. Hospitalize the dog at least 1 hour before the start of test
2. Carefully measure dog's body weight
3. Calculate the dog's dexamethasone sodium phosphate dose to be administered (0.010 mg/kg)
4. Collect a basal sample for serum or plasma cortisol concentration
5. Inject the dexamethasone, IV or IM
6. Collect additional serum or plasma cortisol samples at 4 and 8 hours

Calculating the dose: Remember that only 75% is active dexamethasone
Again, with this LDDST protocol, the dose of dexamethasone to inject is 0.010 mg/kg, IV or IM (1-3,5). A common mistake made by many veterinarians is to miscalculate the dose of dexamethasone to inject for the test.  When using dexamethasone sodium phosphate, one must remember that the label states a concentration of 4 mg/ml of the dexamethasone salt, but that is equivalent to only 3 mg/ml of active dexamethasone (7).

In other words, if you use dexamethasone sodium phosphate, we must use 3 mg/ml for the calculation of how much to inject. Even though it says 4 mg/ml on the label, only 3 mg/ml of it is actually dexamethasone (7).

Best to dilute in dexamethasone in small breed dogs
In smaller breed dogs, it is very important to dilute the dexamethasone (with saline solution) in order to administer an accurate dosage to the dog. Otherwise, too-much or too-little dexamethasone might be administered, which could result in false-negative or false-positive test results.

Stress and other factors to avoid when performing the LDDST
It is important to remember that "stress"activates the hypothalamic-pituitary-adrenal axis, leading to increased secretion of both ACTH and cortisol. In some normal dogs, the stress response may override the suppressive effects of the LDDST to produce a false-positive test result.

Each dog's threshold to stress is different, and it is sometimes difficult to judge the severity of stress in some dogs.  To minimize the effect of stress on this test, remember the following guidelines:

• Allow the dog at least 1 hour to relax and acclimate to the hospital environment before collecting any blood or starting the test.
• Avoid performing other diagnostic procedures on the day of the test, such as radiographs or an abdominal ultrasound. If one must do one of these procedures on the same day that the LDDST is run, the radiography or ultrasonography should be performed a minimum of 2 hours before the start of the LDDST to allow the cortisol concentrations to return to baseline values (8).
• Even more importantly, anesthesia for even minor procedures (e.g., dental, biopsies) must not be done on the day of the LDDST (9-11).
• In dogs that become highly stressed while in the hospital, tranquilizers and sedatives can not be administered before or during the LDDST. Any of the commonly used tranquilizers may skew the cortisol results and make the LDDST invalid.
• In highly stressed or agitated dogs, one option is to have the owner stay with the hospitalized dog in a quiet area of the hospital during the testing day. Alternatively, a house call veterinarian can perform the LDDST on the dog in the home environment.  If neither of those options are feasible, the owner can take the dog out of the hospital after one collects the basal cortisol sample and injects the dexamethasone, then returns at 4 hours and again at 8 hours for the post-dexamethasone cortisol samples.

Interpreting the results of the LDDST
If the LDDST fails to adequately suppress circulating cortisol concentrations in a dog with compatible clinical signs, this is consistent with a diagnosis of Cushing's syndrome (see Figure below).  Although each laboratory should establish their own diagnostic "cut-off" limits, most labs have the suppression set at 1.4-1.5 μg/dl (40 nmol/L).

Although basal and 8-hour post-dexamethasone samples are most important for test interpretation, one or more samples taken at intermediate times (e.g., 4 hours) during the test period may also be very helpful. Approximately 30% of dogs with PDH exhibit serum cortisol suppression at 4 hours (<1.4 μg/dl or <40 nmol/L), with a rise in cortisol concentration 8 hours after dexamethasone administration. This escape from suppression is diagnostic for PDH, and further tests to determine the cause of hyperadrenocorticism are not necessary (1-3,5).

Figure 3: Patterns of serum cortisol suppression seen with the LDDST in normal dogs and dogs with hyperadrenocorticism.  
PDH = pituitary-dependent hyperadrenocorticisim
ADH = adrenal-dependent hyperadrenocorticism (i.e., adrenal tumor).
Figure from reference (3).

Failure to show suppression (serum cortisol remaining above 1.4 μg/dl with less than 50% of basal cortisol concentration at both 4 and 8 hours) is diagnostic for Cushing's syndrome. However, these results do not aid in determining the cause of the disorder. Further tests, such as use of abdominal ultrasound, high-dose dexamethasone suppression testing, or determination of plasma ACTH concentration, must be used to determine the cause of the Cushing's syndrome in those cases (1-3).

References:

1. Melián C, M. Pérez-Alenza, D, Peterson ME. Hyperadrenocorticism in dogs, In: Ettinger SJ (ed): Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat (Seventh Edition). Philadelphia, Saunders Elsevier, 2010;1816-1840.
2. Peterson ME. Diagnosis of hyperadrenocorticism in dogs. Clinical Techiques in Small Animal Practice 2007;22:2-11.
3. Herrtage ME, Ramsey IK. Canine hyperadrenocorticism. In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Quedgeley, Gloucester: British Small Animal Veterinary Association; 2012:167-189.
4. Kaplan AJ, Peterson ME, Kemppainen RJ. Effects of disease on the results of diagnostic tests for use in detecting hyperadrenocorticism in dogs. Journal of the American Veterinary Medical Association 1995; 207:444-451. 
5. Mack RE, Feldman EC. Comparison of two low-dose dexamethasone suppression protocols as screening and discrimination tests in dogs with hyperadrenocorticism. Journal of the American Veterinary Medical Association 1990;15;197:1603-1606.
6. Merck/Schering-Plough Merger. Merckpr.com website. 
7. Plumb DC. Plumb's Veterinary Drug Handbook. Seventh Edition. Wiley-Blackwell; 2011.
8. May ER, Frank LA, Hnilica KA, et al. Effects of a mock ultrasonographic procedure on cortisol concentrations during low-dose dexamethasone suppression testing in clinically normal adult dogs. American Journal of Veterinary Research 2004;65:267-270. 
9. Fox SM, Mellor DJ, Firth EC, et al. Changes in plasma cortisol concentrations before, during and after analgesia, anaesthesia and anaesthesia plus ovariohysterectomy in bitches. Research in Veterinary Science 1994;57:110-118. 
10. Church DB, Nicholson AI, Ilkiw JE, et al. Effect of non-adrenal illness, anaesthesia and surgery on plasma cortisol concentrations in dogs. Research in Veterinary Science 1994;56:129-131. 
11. Fox SM, Mellor DJ, Lawoko CR, et al. Changes in plasma cortisol concentrations in bitches in response to different combinations of halothane and butorphanol, with or without ovariohysterectomy. Research in Veterinary Science 1998;65:125-133.