European Journal of (2010) 162 109–113 ISSN 0804-4643

CLINICAL STUDY Technical details influence the diagnostic accuracy of the 1 mg ACTH stimulation test Matthew Wade1, Smita Baid1, Karim Calis2, Hershel Raff3, Ninet Sinaii4 and Lynnette Nieman1 1Program in Reproductive and Adult Endocrinology, National Institute of Child Health and Human Development and 2Pharmacy Department, National Institutes of Health, Clinical Center, Building 10, CRC, 1 East, Room 1-3140, 10 Center Drive, MSC 1109, 53215 Bethesda, Maryland, USA, 3Endocrine Research Laboratory, Medical College of Wisconsin, Aurora St Luke’s Medical Center, Milwaukee, Wisconsin, USA and 4Biostatistics and Clinical Epidemiology Service, National Institutes of Health, Clinical Center, Bethesda, Maryland, USA (Correspondence should be addressed to L Nieman; Email: [email protected])

Abstract Objective: To examine the factors causing inadequate responses to the 1 mg ACTH stimulation test. Design: Random test assignment (by age and gender) at 0800 or 1600 h. Methods: We recruited 20 healthy adults to each of the three age groups (!40 years, 40–55 years, and O55 years; half females in each group). ACTH stimulation tests were performed in an outpatient clinic at the NIH Clinical Research Center. Plasma cortisol was measured just before, and 30 and 60 min after the administration of 1 mg ACTH (1–24). The ACTH concentration in diluted and administered solutions was measured. Results: Twenty-five volunteers (19 at 1600 h) had a subnormal cortisol response (peak cortisol 10.4–17.5 mg/dl), using a criterion !18 mg/dl (497 nmol/l), for a specificity of 58% (confidence interval (CI) 45–71%). Afternoon testing had a significant impact on failure rates (odds ratio 6.98, CI 2.17–22.43), while gender and age did not. The stock solution contained 1 mg ACTH, but after administration through tubing it contained only 0.5–0.8 mg. Conclusions: The high rate of abnormal results, especially in the afternoon, and loss of ACTH through tubing suggest that morning testing and minimal tubing should be adopted to avoid an inappropriate diagnosis of adrenal insufficiency. Earlier time points and standardized protocols would facilitate comparison of studies.

European Journal of Endocrinology 162 109–113

Introduction Therefore, to clarify the optimal protocol for the 1 mg ACTH test, we evaluated factors associated with a falsely Cosyntropin (ACTH 1–24, ACTH) stimulation is abnormal response in 60 healthy volunteers aged commonly used to diagnose adrenal insufficiency. 22–71 years. Initially, the test was performed with a 250 mg dose (1). However, in response to concerns that patients with mild or recent secondary adrenal insufficiency may respond normally to this supraphysiologic dose, inves- Methods tigators developed a 1 mg ‘low dose’ test (2–4). Based on a report that 3% of adults are prescribed Volunteers and tests high-dose corticosteroids, the population’s potential risk We recruited healthy adults from three age groups of secondary adrenal insufficiency is significant (5). (!40 years, 40–55 years, and O55 years, evenly split However, there is no consensus about which diagnostic by gender) using community flyers. Exclusion criteria test is optimal (6–9). Two meta-analyses have agreed included uncontrolled illnesses, abnormal cell blood that the 1 mg test has better sensitivity, but only one count or electrolytes, pregnancy, lactation, recent use meta-analysis found a worse specificity (79 vs 94%) for of imidazole or medications, or the central adrenal insufficiency (7, 10). presence of signs or symptoms of adrenal insufficiency Since a falsely positive test may lead to unnecessary (unintentional weight loss, nausea, fatigue, or joint life-long glucocorticoid replacement therapy, with its pain). Well-controlled chronic illnesses (e.g. hyperten- attendant risks, it is important to enhance the sion) were allowed. Subjects received $100 (USD) upon specificity. Few studies have examined the effects of study completion. The NICHD IRB approved the study time or method of administration of the agent or the (ClinicalTrials.gov identifier NCT00156767). Subjects influence of age or gender on the test responses. provided written informed consent.

q 2010 European Society of Endocrinology DOI: 10.1530/EJE-09-0746 Online version via www.eje-online.org

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An outpatient visit at the NIH Clinical Center ensured An alternative dilution technique was evaluated an eligibility based on medical history and measure- whereby 250 mg ACTH was added to 250 ml of 0.9% ment of blood count and chemistries and, in women, saline. Samples a–d were collected for each dilution b-human chorionic gonadotropin. method, flash frozen, and stored at K80 8C. A single Within each age group, volunteers were evenly sample for the two dilutions at steps a–d was assayed in allocated to outpatient testing at 0800 or 1600 h. At duplicate for ACTH (below), and the results were averaged. least 30 min after the insertion of an i.v. line, 1 mg ACTH 1–24 (Cortrosyn, Amphastar Pharmaceuticals, ACTH RIA Rancho Cucamonga, CA, USA) was given, followed by 10 ml saline. Serum cortisol was measured just before ACTH levels were measured by RIA with reagents the ACTH administration, and 30 and 60 min later. purchased from MP Biomedicals (Orangeburg, NY, USA). The rabbit anti-porcine ACTH antibody has 100% cross-reactivity with ACTH (1–24) verified Preparation of ACTH using synthetic, HPLC-purified ACTH 1–24 (Phoenix Pharmaceuticals, Belmont, CA, USA). The tracer was In US, ACTH is available only in vials containing 250 mg 125 sterile lyophilized powder. A 1 mg dose was prepared just synthetic human ACTH– I. Samples were assayed in before administration as follows: 2.5 ml of sterile 0.9% duplicate at dilutions of 1:5000 and 1:10 000 using saline was injected into the vial, yielding a 100 mg/ml zero ACTH standard as a diluent to give an assayed solution, and 0.1 ml was injected into a vial containing ACTH result of 22–300 pg/ml (standard curve range: 9.9 ml of 0.9% saline, yielding a 1 mg/ml solution for 11–1050 pg/ml). Intra-assay and inter-assay CV are administration. 4.1–6.8 and 3.9–10.7% respectively. Data analysis I.v. tubing Clinical characteristics were summarized using descrip- The routine i.v. tubing used for endocrine testing in our tive statistics. clinic included a 2.5 cm catheter with attached A published criterion for a normal cortisol response to polyurethane tubing and hub (ProtectIV Plus, Medex, ACTH (497 nmol/l (18 mg/dl) or greater at 30 min) was Inc., Carlsbad, CA, USA) connected to a 20.3 cm tubing chosen to minimize falsely abnormal results, and it was with an injection port (Baxter Healthcare Corp., used to classify the results (11–13). Deerfield, IL, USA). Resultsarepresentedasmean(S.D). Logistic regression analysis determined the influence of time of test, gender, and age on the ‘pass’ or ‘fail’ rate. Odds Assays ratios (ORs) and their 95% confidence interval (CI) (14) The NIH Department of Laboratory Medicine were determined from logistic regression analyses, and measured cortisol using a chemiluminescent competi- specificity and Fisher’s exact 95% CIs were computed. All statistical analyses were two-tailed, with signi- tive immunoassay (Siemens, Los Angeles, CA, USA). % The inter-assay and intra-assay coefficients of varia- ficance defined as a P value 0.05. Data were analyzed tion (CV) are !11.1 and 7.4% respectively. The using SAS system software, release 9.1 (SAS Institute, Inc., Cary, NC, USA). functional detectable value is 28 nmol/l. Corticoste- roid-binding globulin (CBG) was measured by RIA (Quest Diagnostics Nichols Institute). The inter-assay Results and intra-assay CV are !22.4 and 5.8% respectively. The least detectable value is 0.1 mg/l (2 nmol/l). Volunteers Characteristics of the subjects are presented in Table 1. In vitro study Four women (13%) were taking oral contraceptives (nZ3) or -containing hormone replacement To investigate a higher failure rate than anticipated therapy (nZ1); 12 were postmenopausal. No other failure rate (see Results), we evaluated if ACTH was subjects were receiving medication known to affect diluted correctly, absorbed by the i v. tubing, or degraded cortisol or CBG levels. Some had well-controlled chronic by delayed administration. This was done by measuring illnesses, including hypertension (nZ4), hyperlipidemia ACTH levels in the following samples: (a) 1 mg/ml stock (nZ6), diabetes (nZ2), depression/anxiety (nZ7), solution prepared as described, (b) 1 ml aliquot of the migraine headache (nZ2), sleep apnea (nZ1), gastro- stock solution and the saline flush after pushing esophageal reflux disease (GERD) (nZ3), seasonal through the i.v. tubing; samples (c) and (d) repeated allergies (nZ6), osteopenia (nZ3), and osteoarthritis these steps after leaving the stock solution syringe at (nZ1). room temperature for 60 min. All subjects completed the test without adverse events.

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Table 1 Characteristics of healthy volunteers.

Age group (years) !40 40–55 O55

Number 20 20 20 Female/male 10/10 10/10 10/10 Mean age (S.D.) 27.5 (4.1) 46.7 (4.7) 62.1 (4.9) Age range 22–38 40–53 56–71 Race/ethnicity (n) White (43) 14 13 16 Black (11) 3 6 2 Figure 2 Absolute change (d TF) in total cortisol at 30 min from Asian (6) 3 1 2 baseline total cortisol (TF) at 0 min of the 1 mg ACTH stimulation test Hispanic (3) 1 2 0 performed in the morning (AM) or afternoon (PM). The line divides subjects who passed or failed based on the criterion of a response of at least 497 nmol/l (18 mg/dl). Cortisol responses Surprisingly, in this group of healthy volunteers without cortisol concentration at 30 minutes was significantly signs or symptoms of adrenal insufficiency, 25/60 (42%, higher with morning testing (AM: 20.5C4.0 mg/dl vs CI 29–55%) had cortisol responses !497 nmol/l PM: 16.9C3.5mg/dl, PZ0.0006). (range 287–483 nmol/l; 10.4–17.5 mg/dl) (Fig. 1). The time of testing influenced the failure rate: 19/30 CBG results (63%, CI 44–80%) volunteers tested at 1600 h and 6/30 (20%, CI 8–39%) tested at 0800 h had abnormal Basal CBG levels were higher in women taking estrogen responses (Fig. 1). Afternoon testing had a significant (50.0G25.5 mg/dl, nZ2) than in women not taking impact on failure rates (OR 6.98, CI 2.17–22.43), while estrogen (34.7G13.0 mg/dl, nZ17), or men (26.3G5.7, gender and age did not. nZ22) (PZ0.003). As a result, total cortisol values were The absolute cortisol increment (30-min value minus significantly greater in the three women taking oral con- 0-min value) was not affected by the time of the test traceptives than in the other women who were tested in (AM: 10.6G3.5 mg/dl (292G97 nmol/l) versus PM: the morning (20.9G5.9 vs 8.5G3.2 mg/dl, PZ0.002). 10.2G3.7 mg/dl (281G102 nmol/l, PZ0.74)), but Basal CBG values were slightly higher in subjects who was marginally significant by pass/fail status (pass: passed the ACTH stimulation test than in those who 11.1G4.0 mg/dl (306G110 nmol/l) versus fail: 9.4 failed, but this did not reach statistical significance G G Z G2.5 mg/dl (259G69 nmol/l, PZ0.049; Fig. 2)). (32.0 9.8 vs 29.4 14.1 mg/dl, P 0.0524). However, the absolute change in cortisol did not predict a pass or fail outcome for individual patients (Fig. 2). In vitro ACTH levels Those who failed the test had lower baseline values but similar incremental results (Fig. 2). The absolute serum The stock solutions contained at least 1 mg/ml ACTH (range 1.1–1.6 mg/ml). Values were similar in samples kept at room temperature for 60 min (standard dilution method: 1.6 mg/ml, alternative: 1.15 mg/ml) or pro- cessed immediately (standard: 1.25 mg/ml, alternative: 1.1 mg/ml). Values in samples pushed through the i.v. tubing were lower (immediately collected: standard 0.047 mg/ml, alternative 0.074 mg/ml; kept at room temperature: standard 0.070 mg/ml, alternative 0.082 mg/ml). Thus, among samples pushed through the i.v. tubing, 21.6–58.6% of ACTH was not recovered, corresponding to the administration of 0.5–0.8 mg of ACTH.

Discussion This study of healthy volunteers revealed a surprisingly high rate of abnormal responses to the 1 mg ACTH test Figure 1 Cortisol responses at 30 min after 1 mg cortrosyn when a cortisol value of !18 mg/dl (497 nmol/l) was administration between 0800 and 0900 h (AM) and 1600 and 1700 h (PM). Open circles denote subjects who passed the test, considered abnormal. This 58% specificity (CI 45–71%) and closed circles show those who failed the test based on a is less than that reported by most others (mean 79%, criterion of response of at least 497 (18 mg/dl). CI 74–84%) (10). Further analysis attributed these

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Downloaded from Bioscientifica.com at 10/01/2021 04:18:00PM via free access 112 M Wade and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 162 abnormal results to afternoon testing and loss of However, the 30-min time point may not be optimal cosyntropin in the i.v. tubing, suggesting a need for as up to 20% of the healthy volunteers fail the test at the standardization of the testing protocol. Age and this time point (6, 22–24). Other studies demonstrated gender did not influence outcome. that the peak cortisol response in healthy subjects In this study, afternoon testing was associated with a occurs 20–30 min after the administration of low-dose sevenfold increased likelihood of failing the 1 mg test. ACTH (1 mg or 500 ng/1.73 m2) (6, 12), and another However, time of day did not explain all the abnormal study recommends the measurement of cortisol at both responses since 6/30 (20%) failed morning testing. times (15). In another study, no responses were The incremental (delta) cortisol response was similar abnormal when both 20- and 30-min values were at both times of the day, but both the 0- and 30-min used. Notably, in patients with adrenal insufficiency, the values were lower in the afternoon. times of peak total cortisol response tended to occur Our findings of abnormal afternoon responses to 1 mg later than in the healthy volunteers, further supporting ACTH differ from those of Park et al., who reported lower the use of earlier measurement intervals (12). peak cortisol values in the afternoon than in the morning, Differences among cortisol assays and different cut-off but a normal response in all eight volunteers (12).The points might lead to either a ‘fail’ or a ‘pass’, and might larger size of the current study may underlie these underlie the variability in published diagnostic differences. Another study found no influence of after- accuracy. A recent meta-analysis found the 1 mg test noon testing in ten subjects (3). Perhaps, as a result of superior to the 250 mg test only after converting plasma this variability, there is no published consensus on the cortisol levels to serum values (7). Additionally, cortisol optimal time for testing. For example, one report suggests threshold criteria have been proposed for various time that the test may be performed at any time of the day (15). points of the 1 mg (500–600 nmol/l; 18–21.9 mg/dl) The overall cortisol increment above baseline was and 250 mg (500–725 nmol/l; 18–26.3 mg/dl) tests similar at each time of the day, but values for individual (15, 20). It is not clear whether this variability results subjects did not predict the pass or fail outcome. As a result, although the incremental cortisol response is not from differing assays, study populations, or ‘gold influenced by the time of testing, these data do not standard’ criteria for disease. Taken together, this support the use of this value for diagnostic purposes. variability suggests that it is important to validate What might explain the 20% failure rate with diagnostic threshold criteria at each center. morning testing? The cortisol dose–response curve is As has been reported, higher CBG levels are extremely steep when ACTH is delivered as a s.c. dose of associated with higher cortisol measurements, and 2.5–10 mg (16). Thus, possible loss of ACTH during oral can significantly increase CBG levels dilution or administration might sharply reduce the (25). Thus, estrogen administration is another con- cortisol response. As reported earlier, we found that founding factor that might cause a falsely normal test short-term exposure to room temperature did not response. degrade ACTH (3). Although the lack of a commercial In conclusion, we identified two potential reasons for 1 mg preparation might lead to dilution errors, in this abnormal cortisol stimulation after the administration study loss of ACTH due to adherence to plastic tubing of 1 mg ACTH in healthy volunteers: afternoon testing appeared to be more significant. These findings and long plastic tubing. Previous publications suggest corroborate the results of Murphy who showed losses that the use of a single 30-min endpoint may also lead up to 70% when ACTH was passed through a plastic to false results. Presumably, these factors would 30 cm scalp vein set (17), but they contrast with the influence falsely abnormal results in patients suspected report of Wood, who found that storage in a plastic tube of having adrenal insufficiency, especially those with did not decrease the delivered dose (3). The importance long indwelling lines. We suggest that morning testing of minimizing or eliminating catheter length was with 20- and 30-min time points, minimal i.v. tubing, emphasized in one recent review (18), but was not and standardized dilution methods be adopted routinely, mentioned in others (7, 19). Relatively few papers and that the potential influence of CBG be considered. If explicitly cite catheter length; of those that do, both these requirements cannot be met, it may be prudent to short (12) and long (14) catheters were used. We did consider alternative ‘gold standard’ tests for the not investigate the possibilities that the type of plastic diagnosis, such as stimulation and insulin and the length of tubing independently influence this stimulation, in patients suspected of having a recent or effect. Taken together, these data suggest that the mild secondary adrenal insufficiency (26). Alternatively, shortest possible length of tubing, or direct venous the standard 250 mg test works well in the majority of injection, should be used for ACTH administration. the patients, even in those with ACTH deficiency (27). Most previous studies used a 30-min endpoint based on data showing similar cortisol values at 20 and 30 min (20) or similar 30-min values after the Declaration of interest administration of 1 and 250 mg ACTH (21), and a The authors declare that there is no conflict of interest that could be recent meta-analysis preferred this time point (7). perceived as prejudicing the impartiality of the research reported.

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