International Survey on High– and Low–Dose Synacthen Test and Assessment of Accuracy in Preparing Low–Dose Synacthen

Alexandra S. Cross; E. Helen Kemp; Anne White; Leanne Walker; Suzanne Meredith; Pooja Sachdev; Nils P. Krone; Richard J. Ross; Neil P. Wright; Charlotte J. Elder


Clin Endocrinol. 2018;88(5):744-751. 

In This Article

Materials and Methods

International Survey

A thirteen–question online survey (Appendix S1) was distributed to the members of 6 endocrine learned societies with a total of 6744 members: the USA–based Pediatric Endocrine Society (PES, n = 1381), the UK–based Society for Endocrinology (SfE, n = 1188), European Society of Endocrinology (ESE, n = 1540), European Society for Paediatric Endocrinology (ESPE, n = 1239), The Endocrine Society of Australia (ESA, n = 1100) and the Australasian Paediatric Endocrine Group (APEG, n = 296). The survey sought to ascertain the following: the popularity of various diagnostic tests for adrenal insufficiency; the indications for choosing the low–dose (LDT) or high–dose (HDT) SST in preference to the other; LDT dose, administration route of synacthen, cortisol sampling times and cortisol thresholds for test interpretation.

Survey invitations were sent via the e–mailing list or communications bulletin of the societies between March 2016 and January 2017. A follow–up reminder was sent after the initial email. Respondents were given the choice of completing the survey using an online surveying platform ( or an emailed Microsoft Word™ document. Minor changes were made to the survey to meet the various stipulations of the societies.

Low–dose Synacthen Dilution Study

Results from the 2012 survey of British paediatric endocrinologists were used to investigate precision and accuracy of the ten most commonly employed dilution methods for making up 1 μg low–dose synacthen (Table 1).[3] Each dilution protocol was followed and the resultant solution made up 5 times to evaluate intramethod variability. In the 9 methods yielding a sufficient final solution, three 1 mL samples were taken (from the top, middle and bottom of the bag of diluent or the syringe) to assess any variation that may be caused by insufficient mixing. Samples were extracted from the superior quarter of the sample bag/final mL of the syringe (top samples), the vertical halfway point of the sample bag/middle mL of the syringe (middle samples) or taken from the sample bag port/first mL ejected from the sample syringe (bottom samples). All samples were prepared on a single day, by 1 of 3 investigators, with each method made up by the same investigator.

Medical ward equipment (syringes, fluid bags, needles) was used in preference to laboratory equipment to simulate clinical conditions. The 1 mL synacthen ampoules containing 250 μg/mL (Mallinckrodt Pharmaceuticals, Dublin, Ireland) was all from the same manufacturing batch. Synacthen is an inherently unstable drug, rapidly degrading in natural light and at room temperature; therefore, ampoules were refrigerated until use.[12,14] New needles were used for each dilution step to avoid cross–contamination with more concentrated samples. Syringes were reflushed 3 times when injecting into bags of diluent. Mixing was performed by slowly inverting the sample bag or the syringe 5 times, replicating typical ward–based practice. All samples containing the required final concentration of synacthen were frozen immediately at −80°C.

Synacthen ELISA

Synacthen concentrations were estimated using an ELISA format. Unless otherwise stated, all reagents were from Sigma–Aldrich (Poole, UK). NUNC MaxiSorp™ high protein–binding capacity 96–well ELISA plates (ThermoFisher Scientific Inc., Waltham, MA, USA) were coated with anti–ACTH mouse monoclonal antibody A1A12 (which recognises ACTH 1–24) at 2.5 μg/mL in coating buffer (103 mmol/L sodium chloride; 41 mmol/L di–potassium hydrogen phosphate; 8.75 mmol/L potassium dihydrogen phosphate; pH 7.4). Standards were prepared in 0.9% saline at 0–10 000 pg/mL using solid synacthen (Bachem, Bubendorf, Switzerland). Samples containing synacthen were diluted in 0.9% saline to a concentration that was within the standard linear dynamic range (1000–7500 pg/mL) of the ELISA. To assess any variation or reduction in synacthen dose resulting from the laboratory dilutions necessary for the ELISA quantification, 2 vials of synacthen (250 μg/mL) were diluted as required and analysed in the ELISA.

A 100 μL aliquot of sample diluent (phosphate–buffered saline, pH 7.4; 4% bovine serum albumin; 0.05% Tween 20) was added to each well followed by 100 μL of synacthen standard or test sample in duplicate. Plates were incubated at room temperature for 10 minutes, and then washed 3 times with washing buffer (150 mmol/L sodium chloride; 8.5 mmol/L di–potassium hydrogen phosphate; 1.75 mmol/L potassium dihydrogen phosphate; 0.025% Tween 20; 0.0125% ProClin 300; pH 7.0). A 200 μL (1 μg/mL) aliquot of anti–ACTH (7–23) antibody conjugated to HRP (Bioss Antibodies, Woburn, MA, USA) was applied to each well, and plates incubated for 30 minutes at room temperature. Subsequent to washing 3 times, 200 μL of 3,3′,5·5′–tetramethylbenzidine substrate reagent (Europa Bioproducts Ltd., Cambridge, UK) was added to each well. Following incubation at room temperature for 45 minutes, the reaction was stopped by the addition of 100 μL of 0.5 mol/L hydrochloric acid. A Labtech LT4500 spectrophotometer (Labtech International Ltd., Uckfield, UK) was used to read absorption of the wells at 450 nm. Synacthen concentrations (pg/mL) were estimated from standard curves and corrected by the appropriate dilution factor (50–1000 times) to give the expected concentration in the synacthen solution used to deliver a 1 μg dose (Table 1). All samples were assayed 4 to 6 times, and the mean synacthen concentration determined.

The intra–assay coefficient of variation (CV) was 1.70% at 2500 pg/mL, 1.69% at 5000 pg/mL and 2.35% at 7500 pg/mL. The interassay CV was 4.54% at 5000 pg/mL.

Statistical Analyses

Summary statistics of frequency (%) and mean were used to analyse survey data. Free–text responses detailing the clinical scenarios in which the HDT or LDT were used were categorised into themes using content analysis. For each of the ten dilution methods studied in the low–dose synacthen dilution analysis, intramethod and intrabag/syringe variance was calculated and expressed as mean, SD and CV. Method 7 was excluded from intrabag/syringe variance calculations due to an insufficient final volume. Unpaired t tests with Welch's correction were employed to compare components of the different methods, including number of dilution steps, volume of diluent and initial volume of synacthen used. A threshold of ± 10% (0.9–1.1 μg) was chosen as the acceptable range for deliverable synacthen dose values to fall within, reflecting standard laboratory practice.