– Overnight protocol: 1 mg of dexamethasone is administered at 11.00 pm, and the serum cortisol levels are measured at 8.00 am the following morning.
– Two-day protocol: serum cortisol levels are measured at 8.00 am and 0.5 mg of dexamethasone is administered every six hours (9.00 am, 3.00 pm, 9.00 pm, 3.00 am) for two days, totalling 4 mg. Serum cortisol levels are then measured at 9.00 am, six hours after the last dose has been delivered.
– Overnight protocol: baseline serum cortisol or 24-hour urinary free cortisol (UFC) is measured in the morning, and 8 mg of dexamethasone is given at 11.00 pm. Cortisol level in blood is then measured at 8.00 am the following morning.
– Two-day protocol: Baseline serum cortisol or 24-hour UFC is measured at 8.00 am; 2 mg of dexamethasone is administered every six hours (9.00 am, 3.00 pm, 9.00 pm, 3.00 am) for two days, totalling 16 mg, in tandem with collection of urine for UFC measurements. Serum cortisol levels are measured at 9.00 am, six hours after the last dose has been delivered.
Patients whose pituitary glands produce too much ACTH will have an abnormal response to the low-dose test, but a normal reaction to the high-dose test. For LDDST, the cortisol levels should decrease in response to dexamethasone administration, and a value below 18 ng/mL is the recommended cut-off value to separate a healthy from an unhealthy reaction. In the case of HDDST, a reduction in UFC or serum cortisol greater than 50% indicates that the patient has ACTH-dependent Cushing’s syndrome. This rule applies to both the overnight LDDST and two-day HDDST approaches.
Measuring cortisol levels
Chemiluminescence immunoassay (CLIA) is the most common method for measuring cortisol and other steroids, as it is easy to use, has a workflow that can be fully automated, and offers good sensitivity. Unfortunately, this technique has several drawbacks, the main one being cross-reactivity that can lead to an over-estimation of target analyte levels. Other flaws are the lack of standardization between different kits on the market, and the fact that it can only measure one analyte per analysis. This can be problematic as several studies suggest that measuring dexamethasone in tandem with cortisol can help to reduce the number of false-positive results for DSTs [3,4] and improve interpretability  – as it is hard to predict how a patient’s body will react to the synthetic steroid. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) has become a popular alternative to CLIA for DSTs, owing to its ability to measure several analytes at once and its much higher specificity. Target analytes are separated through LC, and their concentrations are measured using MS. Triple quadrupole MS set-ups are commonly used for this purpose, providing not only the ability to measure several analytes at once, but also higher accuracy and sensitivity than CLIA.
Increased ease-of-use and accuracy
A group in the Division of Endocrinology, Diabetes and Metabolism at the University of Turin has created an LC-MS/MS method for simultaneous quantification of cortisol, cortisone, dexamethasone and six additional exogenous corticosteroids in serum/plasma samples . The method can be easily implemented in any clinical laboratory with a mass spectrometer, and has proven useful for DSTs, enabling accurate measurements of the target analytes in a single chromatographic run (Fig. 1). The method is outlined on the next page.
Sample preparation (1 hour)
1. Dilute 200 μL of the serum/plasma sample with 200 μL of water.
2. Perform supported liquid extraction, transferring 400 μL of sample manually to a microplate.
3. Apply positive pressure using Tecan Resolvex® A200 automated positive pressure processor.
4. Elute with 700 μL of methyl tert-butyl ether.
5. Evaporate and reconstitute in H2O/MeOH (1:1, v/v).
LC-MS/MS analysis (10 minutes)
• LC column: C18 (100 × 2.1 mm, 1.7 μm)
• flow rate: 400 μL/min
• temperature: 30 °C
• injection volume: 20 μL
• mobile phase A: H2O + 0.2 mM ammonium fluoride
• mobile phase B: acetonitrile
• elution programme: see Table 1.
The results showed good correlation with measurements performed using a commercially available CE IVD-marked Steroid Panel LC-MS kit* (Tecan). This kit can measure dexamethasone, cortisol and cortisone simultaneously, and includes all essential components for easy implementation, such as calibrators and controls. The samples are prepared using solid-phase extraction (SPE), which can be performed semi-automatically on a Resolvex® A200 positive pressure processor (Tecan). Thanks to the effective SPE process, the kit can also measure 15 additional steroids in the core steroid metabolism pathway.