Diagnosis of Spontaneous Canine Hyperadrenocorticism: 2012 ACVIM Consensus Statement (Small Animal)- Part 2 of 3

Test Principles and Diagnostic Accuracy

The ACTH stimulation test assesses adrenocortical reserve and is the gold standard for diagnosis of iatrogenic HAC. Because of its low sensitivity, its diagnostic usefulness as a screening test for spontaneous HAC is inferior to the LDDST.

The sensitivity of the ACTH stimulation test for all forms of spontaneous canine HAC ranges between 57 and 95%. It has been determined that for dogs with HAC because of an AT, sensitivity is 57–63%; for dogs with PDH it is 80‐83%. Specificity ranges between 59 and 93%.6, 21, 36, 43, 48-51

Form, Dosage, and Route of ACTH

Synthetic polypeptides containing the biologically active first 24 amino acids of ACTH are available, eg, Cortrosyn (cosyntropin) or Synacthen (tetracosactrin). The potency of the preparations has not been compared. Recently, Cosyntropin Injection was introduced for IV use only. No differences in cortisol concentrations were found in response to 250 μg Cortrosyn IM or Cosyntropin Injection IV in 18 healthy dogs.52 In some countries, compounded ACTH preparations are available. In 1 study, cortisol concentrations 60 minutes after administration of compounded ACTH (2.2 U/kg IM) were no different than after Cortrosyn (5 μg/kg IV).53

No difference in mean peak cortisol concentration was detected when comparing IV and IM administration of 250 μg Cortrosyn in healthy dogs54; IV and IM administration of 5 μg/kg Cortrosyn in healthy dogs and dogs with HAC55; or IV administration of 250 μg/dog and 5 μg/kg Cortrosyn in dogs with HAC.56, 57 When comparing various cosyntropin dosages (10, 5, 1, 0.5, 0.1, 0.05, 0.01 μg/kg) on cortisol concentrations in healthy dogs56-58, the lowest dosage that stimulated maximal cortisol secretion was 0.5 μg/kg IV.58 Depot tetracosactide (250 μg/kg IM) and cosyntropin (5 μg/kg IV) produced similar cortisol responses at 60 minutes after administration in healthy dogs.59 However, neither cosyntropin dosages below 5 μg/kg nor tetracosactide depot have been assessed in dogs with HAC.

Sample Timing

After administration of Cortrosyn at 5 μg/kg or 250 μg/dog IV or IM, peak cortisol secretion occurs at 60–90 minutes.53-57 After 5 μg/kg IV, no difference was detected between 60‐ and 90‐minute cortisol concentrations.53, 55, 56 Using 4 compounded products (2.2 U/kg IM) in healthy dogs, cortisol concentrations at 60 minutes were similar to each other as well as to concentrations after Cortrosyn (5 μg/kg IV). However, at later times cortisol concentrations varied considerably.53

Effect of Timing and Feeding

Dogs do not exhibit circadian cortisol secretion.23 Similar to the LDDST, the Panel assumes that time of day does not affect test results. Fasting before testing is not necessary unless lipemia affects results of the cortisol assay used.

Cosyntropin Storage

Cosyntropin can be reconstituted and frozen in aliquots at −20°C in plastic syringes for 6 months.60 Whether Synacthen can be frozen has not been investigated; according to the manufacturer, it should be stored at 2–8°C.

Influence of Drugs

In people, cortisol response to ACTH may be decreased by serotonin receptor agonists, progestagens, ketoconazole, and fluconazole and may be enhanced by propranolol.27 In veterinary medicine, the ability of glucocorticoids of any form,30 progestagens29 and ketoconazole61 to suppress cortisol secretion is known. No effect on the ACTH stimulation test was documented overall or individually in healthy dogs treated with phenobarbital for 862 (n = 12) or 29 weeks46 (n = 12) or in epileptic dogs treated for 1 year45 (n = 5) or >2 years62 (n = 5).

Test Principles and Diagnostic Accuracy

The UCCR provides an integrated reflection of corticoid production, adjusting for fluctuations in blood concentrations.

Determination of basal UCCRs can be performed in tandem with a high‐dose dexamethasone suppression test (see below). The combination has the advantage of potentially demonstrating both increased cortisol production and decreased sensitivity to glucocorticoid feedback.

When a single, random urine sample is collected in veterinary hospitals, the reported sensitivity and specificity of the UCCR for diagnosis of HAC ranges from 75–100%21, 63-66 and 20–25%, respectively.21, 63, 64 However, using the protocol below, in dogs with physical and biochemical changes consistent with HAC, the sensitivity of finding 2 basal UCCRs above the cut‐off level was 99% (95% confidence interval [CI], 94–100%) and the specificity was 77% (95% CI, 64–87%).42 In some dogs, considerable day‐to‐day variation exists in the UCCR. In mild cases, a UCCR may be just within the reference range 1 day and increased another day.

Protocol

To avoid the influence of stress,67 urine for UCCR should be collected at home at least 2 days after a visit to a veterinary clinic. Although a UCCR sample can be collected at any time of day,68 morning urine may be preferred because it usually represents several hours of urine production.

Influence of Drugs and Concurrent Disease

Glucocorticoids and other drugs that suppress cortisol secretion, such as progestagens,29 can decrease a UCCR by suppressing endogenous cortisol secretion. Phenobarbital treatment does not affect UCCR.47 Nonadrenal disease may cause endogenous stress and increased cortisol secretion. Therefore, high UCCRs in dogs without a high degree of clinical suspicion of HAC should be interpreted cautiously.

Test Principles

Canine ACTH is secreted from the pituitary gland in an episodic, pulsatile fashion in healthy dogs and those with PDH.23, 71 A circadian rhythm has not been convincingly demonstrated, although 1 study reported higher plasma cACTH concentrations in late afternoon than in the morning.72 Concentrations of cACTH do not differ between healthy dogs and those with PDH, and its measurement is not useful to screen for HAC.73 Measurement of cACTH is the most accurate stand‐alone biochemical test for differentiating PDH from AT.

cACTH Assays

Immunoradiometric assay (IRMA) and chemiluminescent assays have been validated for cACTH measurement.74-77 Measured cACTH concentrations are lower using chemiluminescent technology than RIA.76

The accuracy for differentiation of PDH from AT depends upon analytical sensitivity and the working range of the assay (Table 3). The most common problem with the cACTH assay is poor sensitivity. Some dogs with PDH have cACTH concentrations at or below the sensitivity of the assay, particularly with the Immulite 1000 analyzer. The largest study of cACTH in dogs with HAC used a 2‐site solid‐phase chemiluminescent immunometric assay (Immulite ACTH kit and Immulite 2000 analyzer) and showed excellent discrimination between PDH and AT.75 No dogs with PDH had undetectable cACTH concentrations, likely because of the analytical sensitivity (5 pg/mL), but the range of cACTH concentrations for dogs with PDH was 6–1,250 pg/mL, with many dogs falling close to the lower end of the range. Thus, less sensitive assay systems (eg, Immulite 1000) would likely have poorer discrimination. Intra‐assay and interassay variability (increased at lower cACTH concentrations), pulsatile ACTH secretion, and inappropriate sample handling allowing ACTH degradation increase the likelihood of a falsely low value in dogs with PDH.

• ACTH, adrenocorticotrophic hormone; cACTH, canine ACTH; HAC, hyperadrenocorticism; IRMA, immunoradiometric assay; PDH, pituitary‐dependent hyperadrenocorticism; AT, adrenal tumor.

Timing of Sample Collection

No clear evidence exists that the specific time of sample collection affects results or discriminatory power of the test.

Sample Handling

Plasma proteases degrade cACTH rapidly if samples are not cooled appropriately. Blood should be collected into chilled, silicon‐coated glass or plastic tubes containing EDTA, centrifuged within 15 minutes (ideally in a cooled centrifuge), and the plasma transferred to plastic tubes and frozen immediately.74, 76, 78 Samples must stay frozen until analysis; if a courier is used for quick transport to a reference laboratory, ice may be sufficient. If samples are shipped, they should be sent overnight packed in dry ice.

Addition of the protease inhibitor aprotinin (Trasylol) prevents ACTH degradation by plasma proteases.74 With the Immulite assay, aprotinin introduces an artifactual decrease76 and is not recommended.

Discordant Test Results

Discordance between cACTH concentration and results of other differentiating tests sometimes occurs. Episodic cACTH secretion, poor assay sensitivity, and sample degradation are potential explanations. Stress and the presence of multiple adrenal disorders (ie, cortisol‐secreting AT or PDH with pheochromocytoma; cortisol‐secreting AT and PDH) also may influence ACTH concentrations. Ectopic ACTH secretion and food‐stimulated cortisol secretion could also cause discordance.69, 70