Avoiding False-Positive Adrenal Insufficiency Diagnoses in Children: Insights from Cortisol Kinetics During Pediatric Low-Dose ACTH Stimulation Test

Avoiding False-Positive Adrenal Insufficiency Diagnoses in Children: Insights from Cortisol Kinetics During Pediatric Low-Dose ACTH Stimulation Test | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Avoiding False-Positive Adrenal Insufficiency Diagnoses in Children: Insights from Cortisol Kinetics During Pediatric Low-Dose ACTH Stimulation Test Derya Tepe, Sirmen Kizilcan Cetin, İrem Gokdemir, Pınar Kocaay, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8694799/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background: The low-dose ACTH stimulation test (LDST) is widely used to evaluate hypothalamic–pituitary–adrenal (HPA) axis function in children; however, optimal cortisol sampling times and interpretation strategies remain controversial. Reliance on early or single time-point measurements may lead to false-positive diagnoses of adrenal insufficiency (AI). Objective: To characterize the timing of peak cortisol responses during LDST in children without AI and to assess the incremental diagnostic contribution of extended sampling and time-specific cortisol thresholds. Methods: We retrospectively analyzed 177 pediatric patients who underwent LDST for suspected central adrenal insufficiency at a single tertiary center. Serum cortisol was measured at baseline and at 15, 30, 45, and 60 minutes following intravenous administration of 1 µg ACTH. Adrenal sufficiency was defined as a peak cortisol ≥ 18 µg/dL. Peak timing distribution, basal predictors, incremental diagnostic contribution of additional time points, number needed to test (NNT), and false-positive rates using fixed versus time-specific cut-offs were evaluated. Results: Peak cortisol occurred most frequently at 15 minutes (48.6%), followed by 30 minutes (28.8%), baseline (10.7%), 45 minutes (9.0%), and 60 minutes (2.8%). Termination of testing at 30 minutes would have misclassified 11.9% of patients as insufficient despite normal later responses. Extension to 45 minutes provided meaningful diagnostic improvement, whereas routine extension to 60 minutes yielded only marginal additional benefit (NNT = 30). Higher basal cortisol levels were independently associated with earlier peak responses (p = 0.021), while demographic and auxological factors showed no association. Application of time-specific, percentile-based cortisol thresholds reduced false-positive classifications nearly five-fold at 30 minutes compared with a uniform 18 µg/dL cut-off. Conclusions: LDST cortisol responses in children show substantial interindividual variability in peak timing. Extension of sampling to 45 minutes and use of time-specific interpretation thresholds significantly improve diagnostic accuracy and reduce false-positive AI diagnoses in pediatric practice. low-dose acth stimulation test pediatric adrenal insufficiency cortisol kinetic hypothalamic–pituitary–adrenal axis diagnostic accuracy Figures Figure 1 Introduction The low-dose Synacthen® (adrenocorticotropic hormone, ACTH) stimulation test (LDST) is widely used for the assessment of hypothalamo–pituitary–adrenal (HPA) axis function in children with suspected central adrenal insufficiency (AI) ( 1 – 3 ). By administering a physiological dose of ACTH, the LDST is thought to better approximate endogenous adrenal stimulation and to minimize the supraphysiological cortisol responses associated with the standard-dose ACTH test. ( 1 – 3 ). Accordingly, the LDST has been widely adopted in pediatric endocrinology. However, significant uncertainties remain concerning optimal protocols and the interpretation of test results. Traditionally, cortisol measurement at a single post-stimulation time point, most commonly at 30 minutes, has been used for the interpretation of the LDST ( 4 , 5 ). Morning basal serum cortisol measurement is frequently used as an initial screening tool in the diagnostic evaluation of AI. Very low morning cortisol concentrations (< 3 µg/dL [83 nmol/L]) are strongly suggestive of AI, whereas values exceeding 13 µg/dL (365 nmol/L) indicate preserved HPA axis function. When basal cortisol levels fall within the intermediate range, dynamic stimulation testing is required to assess adrenal reserve accurately ( 6 ). However, accumulating pediatric evidence increasingly challenges the adequacy of this approach. Large pediatric cohorts have demonstrated that the timing of peak cortisol response during LDST is highly variable ( 7 – 11 ). Despite decades of clinical use, the optimal timing for cortisol measurement following tetracosactide (Synacthen®) administration during the LDST remains a subject of ongoing debate ( 8 , 10 , 12 , 13 ). The clinical consequences of false-positive AI diagnoses in childhood are substantial. Misclassification may result in unnecessary lifelong glucocorticoid replacement therapy, with potential adverse effects on growth, pubertal development, metabolic health, bone density, and overall quality of life ( 8 ). In this context, there is a clear need to establish evidence-based, pediatric-specific LDST sampling strategies that reduce diagnostic error. This study aims to (i) characterize peak cortisol timing during LDST in a large, real-world pediatric cohort, (ii) explore clinical and biochemical correlates of peak timing, and (iii) assess the additional diagnostic benefit provided by extended cortisol sampling beyond a single time point. Materials and Methods Study Design and Participants This retrospective observational study evaluated pediatric patients who underwent a LDST for suspected adrenal insufficiency at a tertiary pediatric endocrinology center. Patients with a peak cortisol response < 18 µg/dL, consistent with central AI, were excluded. A total of 177 children and adolescents were included in the analysis. Indications for LDST included abnormal clinical findings, the use of medications (exposure to exogenous steroids) affecting the HPA axis, hypothalamo–hypophyseal disorders, prior cranial radiotherapy, laboratory abnormalities suggestive HPA axis dysfunction, and basal morning cortisol insufficiency (< 13 µg/dL [350 nmol/L]). Demographic data (age, sex), anthropometric measurements (height, weight, body mass index [BMI], and SDS values), pubertal status according to Tanner staging, and relevant baseline laboratory parameters were retrospectively obtained from medical data. The study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the local ethics committee (approval number: TABED 2/918/2025). Low-Dose ACTH Stimulation Test Protocol All LDSTs were performed in the morning hours following an overnight fast. After placement of an intravenous catheter, baseline blood samples were obtained for serum cortisol and plasma ACTH measurements (0th minute). A 1 µg intravenous bolus of synthetic ACTH (tetracosactide; Cortrosyn®) was administered, and serum cortisol levels were measured at 15, 30,45 and 60 minutes following stimulation, in accordance with our institutional protocol. The peak cortisol response was defined as the highest cortisol concentration observed at any sampled time point during the test. Patients were classified according to the time point at which the peak cortisol response occurred (0, 15, 30, 45, or 60 minutes). Laboratory Measurements Serum cortisol concentrations were measured using polyclonal antibody immunoassays using the Siemens Atellica® immunoassay. Statistical Analysis Statistical analyses were performed using SPSS software version 25.0 (IBM Corp., Armonk, NY, USA). Normality of continuous variables was assessed using the Shapiro–Wilk test. Continuous variables were expressed as mean ± standard deviation (SD) or median (interquartile range; Q1–Q3), depending on distribution. Categorical variables were presented as counts and percentages. To compare cortisol levels across multiple time points within individuals, the Friedman test was used. For comparisons among more than two independent groups with non-normal distribution, the Kruskal–Wallis test was applied, followed by Bonferroni-corrected post hoc analyses when appropriate. Categorical variables were compared using the chi-square test. Associations between the timing of peak cortisol response and clinical or biochemical parameters were evaluated using Spearman correlation analysis, with correlation strength classified as weak (r = 0.00–0.29), moderate (r = 0.30–0.49), strong (r = 0.50–0.69), or very strong (r ≥ 0.70). To identify independent predictors of peak cortisol response, multiple linear regression analysis was performed. Age, height SDS, and basal cortisol levels were entered into the model using the Enter method. Multicollinearity was assessed using the variance inflation factor (VIF) and tolerance values. The incremental diagnostic contribution of additional cortisol sampling time points was evaluated to determine the added value of extending the LDST protocol. Specifically, the probability that an additional cortisol measurement at later time points (e.g., 30, 45, or 60 minutes) would reclassify an initially failed LDST result as a passed result was calculated. The number needed to test (NNT) was derived as the reciprocal of this probability to estimate how many additional extended tests would be required to prevent one false-positive LDST classification. NNT values were rounded to the nearest whole number and reported with corresponding 95% confidence intervals. A p value < 0.05 was considered statistically significant. Results A total of 177 patients (%48, n=85 female) who underwent LDST were included in the study. The mean age at testing was 8.81 ± 5.92 years. The most common indication for LDST was insufficient basal morning cortisol identified during laboratory evaluation (69.5%, n = 123). Clinical histories suggestive of adrenal insufficiency—including symptoms such as prolonged jaundice, fatigue, or hypoglycemia—were present in 53.1% of patients (n = 94). Additionally, 25.4% (n = 45) had underlying intracranial pathology, and 20.3% (n = 36) had a history of exogenous glucocorticoid or stimulant exposure. Baseline laboratory characteristics, including ACTH and cortisol levels, are summarized in Table 1. Analysis of peak cortisol responses during LDST (Figure 1) demonstrated that the most frequent peak occurred at 15 minutes (48.6%, n = 86). This was followed by the 30-minute peak (28.8%, n = 51), baseline (0 minute) (10.7%, n = 19), 45 minutes (9.0%, n = 16), and 60 minutes (2.8%, n = 5). Median cortisol concentrations differed significantly across peak time points (p < 0.001). Patients exhibiting a peak cortisol response at 15 minutes had a median cortisol level of 19.2 µg/dL, whereas the highest median cortisol concentration was observed in patients peaking at 60 minutes (27.3 µg/dL). Nearly half of patients reached peak cortisol concentrations at 15 minutes, while fewer than 3% peaked at 60 minutes, highlighting substantial interindividual variability in cortisol response timing. Detailed cortisol measurements at all sampling time points are presented in Table S.1, and the distribution of peak responses is illustrated in Figure 1. Data are presented as number (percentage), mean ± standard deviation (SD) with range, and median with interquartile range (Q1–Q3). Peak cortisol was defined as the highest serum cortisol concentration observed at any sampling point during the low-dose ACTH stimulation test. Basal serum cortisol concentrations differed significantly according to the timing of the peak cortisol response following low-dose ACTH stimulation (p = 0.001). Post-hoc multiple comparison analyses revealed that patients who achieved their peak cortisol response at baseline (0 min) and 15 minutes had significantly higher basal cortisol levels compared with those peaking at 45 minutes (both p = 0.002). Basal cortisol levels differed significantly according to peak response timing, with higher basal cortisol observed in patients peaking at 0 and 15 minutes compared with those peaking at 45 minutes, whereas basal ACTH levels did not differ across groups(Table 2). No significant associations were observed between peak response timing and age, sex, pubertal status, height SDS, weight SDS, or BMI SDS, indicating that the temporal pattern of cortisol response was independent of demographic and auxological characteristics (Table 3). Following ACTH stimulation, serum cortisol concentrations increased progressively over time. Paired Wilcoxon signed-rank tests demonstrated statistically significant increases between all consecutive sampling points: 15 → 30 minutes: median increase +4.1 µg/dL ( p ≈ 1.6 × 10⁻²⁹) 30 → 45 minutes: median increase +1.8 µg/dL ( p ≈ 3.1 × 10⁻¹⁶) 45 → 60 minutes: median increase +1.7 µg/dL ( p ≈ 2.4 × 10⁻¹⁵) These results indicate a rapid early rise in cortisol levels, followed by a plateau beyond 45 minutes, suggesting a diminishing incremental diagnostic contribution of later sampling A multiple linear regression model including age, body height, and basal cortisol levels significantly explained the variability in peak cortisol response ( F (3,37) = 3.65, p = 0.021; adjusted R ² = 0.17). Among the covariates, only basal cortisol level was independently associated with peak cortisol response, demonstrating a significant inverse relationship (B = −0.075, 95% CI −0.139 to −0.012; p = 0.021). Age and body height were not significantly associated with peak cortisol response (Table 4). Higher basal cortisol levels were independently associated with a blunted peak cortisol response, supporting a ceiling effect of adrenal stimulation during LDST. Cortisol concentrations increased consistently at each successive sampling point . When applying the conventional cortisol sufficiency threshold of ≥18 µg/dL, the proportion of patients meeting diagnostic adequacy rose markedly over time, from 10.7% at baseline to 96.6% at 45 minutes and 98.9% at 60 minutes. At 30 minutes, only 88.6% of patients had achieved cortisol levels ≥18 µg/dL, indicating that a clinically relevant subgroup exhibited delayed yet sufficient adrenal responses. Termination of the LDST at 30 minutes would have resulted in the misclassification of 21 patients (11.9%) as having inadequate adrenal function, despite demonstrating normal cortisol responses at later time points (Table 5). The diagnostic contribution of extending sampling time points during LDST to prevent false-positive classifications is summarized in Table 6. Sequential extension of sampling demonstrated variable added diagnostic contribution depending on the time interval evaluated. Extension from 15 to 30 minutes corrected an initial insufficient response in 28.8% of patients, corresponding to a NNT of 3. Similarly, extending sampling from 15 to 45 minutes improved diagnostic classification in 37.3% of cases (NNT = 3). In contrast, extending sampling from 30 to 45 minutes resulted in correction in only 9.0% of patients (NNT = 11). In pediatric patients without adrenal insufficiency, serum cortisol levels typically reach diagnostic thresholds by 30 minutes following low-dose ACTH stimulation. Extending sampling beyond 45 minutes does not provide significant additional diagnostic value. The diagnostic contribution of a 60-minute sample was limited. Extension from 45 to 60 minutes corrected cortisol classification in only 3.4% of cases, yielding an NNT of 30. When comprehensive sampling from 0–45 minutes was already performed, adding a 60-minute sample provided minimal additional diagnostic information (2.8% improvement; NNT = 35). When both baseline (0 min) and 15-minute samples were available, adding a 30-minute sample corrected 28.2% of initially insufficient results (NNT = 4), whereas adding a 45-minute sample corrected 36.7% (NNT = 3). Overall, the greatest diagnostic contribution was observed with extension to 45 minutes, whereas routine extension beyond this time point yielded marginal additional benefit(Table S2) . Among patients without adrenal insufficiency, application of a uniform cortisol cut-off of 18 µg/dL at early sampling times resulted in substantial false-low classifications (Table 6). False-low rates were highest at 15 minutes (40.3%) and remained clinically relevant at 30 minutes (11.4%), indicating a pronounced risk of overestimating adrenal insufficiency when early sampling points are interpreted in isolation (Table S3). To address time-dependent cortisol kinetics, lower reference limits based on the 2.5th percentile (P2.5) were applied (Table 7). Use of time-adjusted cut-offs markedly reduced false-low rates across all sampling points. At 30 minutes, the false-low rate decreased nearly five-fold when a time-specific threshold of 16.1 µg/dL was used instead of the conventional 18 µg/dL (2.3% vs. 11.4%). Basal cortisol concentrations differed significantly according to peak response timing (p = 0.001). Post-hoc analyses demonstrated that patients peaking at 0 and 15 minutes had significantly higher basal cortisol levels than those peaking at 45 minutes (p = 0.002 for both). No significant associations were identified between peak timing and age, sex, pubertal status, or anthropometric parameters. Discusion The low-dose ACTH stimulation test (LDST) remains one of the most widely used tools in pediatric practice. Despite its extensive use, substantial uncertainty persists regarding optimal sampling time points and appropriate cortisol thresholds. In this study, we systematically evaluated the temporal dynamics of cortisol responses during LDST in a pediatric cohort ultimately classified as not having AI and demonstrated that both sampling time and threshold selection critically influence diagnostic interpretation. Existing literature on the LDST (Table S4) demonstrates substantial heterogeneity with respect to ACTH dosing, sampling time points, and diagnostic cortisol thresholds. Although most pediatric studies recommend a standard 1-µg ACTH dose, sampling strategies vary widely, with many protocols limited to baseline and 30-minute measurements and only selected studies incorporating additional 15- or 60-minute samples. Consistent with prior pediatric and adult studies (Table S4), serum cortisol concentrations increased progressively following ACTH stimulation, with the most pronounced rise occurring between 15 and 30 minutes, followed by smaller yet statistically significant increments thereafter( 1 , 7 , 10 , 12 – 17 ). Although the majority of patients achieved diagnostic cortisol thresholds by 30 minutes, a clinically meaningful subgroup reached ≥ 18 µg/dL only at 45 or 60 minutes. These delayed responders represent physiological variation rather than pathology and would have been misclassified as having adrenal insufficiency if testing had been terminated prematurely. Historically, many centers have relied on baseline and 30-minute cortisol measurements during LDST, based on early reports suggesting that peak cortisol responses typically occur within this window( 12 , 17 ). However, accumulating evidence indicates that exclusive reliance on early sampling leads to a non-negligible false-positive rate. In our cohort, termination of testing at 30 minutes would have resulted in misclassification of nearly 12% of patients, a finding that closely mirrors rates reported by Gill et al.( 10 ), Vaiani et al.( 15 ) and Cartarya et al.( 18 ), who demonstrated that omission of later samples misclassified approximately 10–15% of children with normal adrenal function. Studies in neonates consistently demonstrate delayed peak responses, often at 60 minutes( 13 ). Despite these observations, the majority of published protocols continue to apply a time-independent cortisol cut-off of ≥ 18 µg/dL (500 nmol/L) across all sampling points. Our findings quantitatively demonstrate that this approach leads to substantial overestimation of AI, particularly at 15 and 30 minutes, where false-low rates reached 40.3% and 11.4%, respectively. The present study extends prior work by systematically integrating sampling time and cut-off selection. We demonstrate that extending sampling to 45 minutes provides meaningful diagnostic benefit by capturing delayed yet physiologically normal cortisol responses, whereas routine extension to 60 minutes offers only marginal additional value. This incremental pattern is compatible with probability-based analyses in prior cohorts, which demonstrated diminishing diagnostic returns beyond the mid-to-late sampling window ( 14 , 19 ). These data support 45 minutes as a pragmatic upper limit for routine LDST sampling in most pediatric patients. Moreover, by introducing time-specific, percentile-based thresholds, we show that false-positive classifications can be dramatically reduced without compromising diagnostic safety. This approach directly addresses a major gap in the literature, where time-dependent cortisol physiology has been acknowledged but rarely operationalized into practical diagnostic criteria. A key mechanistic finding of our study is the association between basal cortisol levels and peak response timing. Patients exhibiting early peak responses (0–15 minutes) had significantly higher basal cortisol concentrations compared with those peaking at 45 minutes, while basal ACTH levels did not differ across groups. This pattern suggests that baseline adrenal tone rather than central ACTH drive modulates response kinetics, consistent with a physiological ceiling effect. Multivariable regression analysis further supported this interpretation by demonstrating an independent inverse association between basal cortisol levels and peak cortisol increment. Significantly, peak response timing was independent of age, sex, pubertal status, and anthropometric parameters, indicating that delayed cortisol responses cannot be predicted based on routine clinical characteristics. One of the most clinically relevant observations concerns the widespread use of a uniform cortisol cut-off of 18 µg/dL across all sampling times. Our data clearly demonstrate that applying this threshold at early time points—particularly at 15 and 30 minutes—results in substantial overestimation of AI. At 15 minutes, more than 40% of patients without AI would have been misclassified, and at 30 minutes, the false-low rate remained above 10%. Similar concerns have been raised in multiple cohorts, particularly with the adoption of more specific modern immunoassays that provide systematically lower cortisol values compared with older polyclonal assays( 7 , 16 , 20 ). To address this limitation, we explored time-specific, percentile-based cortisol thresholds derived from the distribution of responses in patients without AI. This approach markedly reduced false-low classifications at all sampling points. At the clinically pivotal 30-minute mark, use of a time-adjusted threshold reduced false-low rates nearly five-fold compared with the conventional 18 µg/dL cut-off. This strategy is statistically robust, physiologically plausible, and consistent with calls in the literature for assay- and context-specific interpretation of dynamic endocrine tests (Table S4). An additional noteworthy observation relates to patients who achieved a sufficient cortisol response at 0 minutes (10.7%). Because cortisol results were not available in real time during test administration, these patients could not be excluded a priori. Notably, this subgroup comprised patients with insufficient morning basal cortisol levels who nevertheless demonstrated an adequate response at the initial LDST sampling point, further illustrating the limitations of relying on a single basal cortisol measurement and reinforcing the value of dynamic testing. Taken together, our findings support a stepwise LDST interpretation framework: early termination is appropriate when cortisol sufficiency is achieved at baseline or 30 minutes; mandatory extension to 45 minutes should be performed in patients with subthreshold 30-minute values; and 60-minute sampling should be reserved for selected cases with persistently low intermediate values or high clinical suspicion. Incorporation of time-adjusted thresholds may further enhance diagnostic accuracy and reduce unnecessary lifelong treatment. Several limitations warrant consideration. The analysis focused on patients ultimately classified as not having AI, precluding direct sensitivity estimates for true adrenal failure. Second, cortisol measurements were performed using a polyclonal antibody-based immunoassay, and confirmatory testing with more specific methods such as liquid chromatography–tandem mass spectrometry (LC–MS/MS) was not available. However, this reflects real-world clinical practice, as polyclonal immunoassays remain widely used in many centers due to their greater accessibility and lower cost, despite the superior specificity of monoclonal assays. Consequently, while assay-related variability cannot be excluded, the use of a commonly employed method may enhance the generalizability of our findings to routine pediatric endocrinology settings. In conclusion, LDST responses in children exhibit substantial temporal variability driven primarily by basal cortisol levels rather than demographic or auxological factors. Reliance on early sampling combined with uniform cortisol cut-offs substantially overestimates adrenal insufficiency. Extension of sampling to 45 minutes and adoption of time-specific, percentile-based thresholds provide a more physiologically sound and diagnostically accurate framework for LDST interpretation in pediatric practice. Statements Written informed consent was obtained from the parent/legal guardian of participants prior to the study. Statement of Ethics Written informed consent was obtained from the parent/legal guardian of participants prior to the study for the publication of details such as age, gender, illness. This study was reviewed and approved by Ankara Bilkent City Hospital’s Ethic Committee, approval number TABED2/918/2025, decision date 19.02.2025. Declarations Conflict of Interest Statement The authors have no conflicts of interest to declare. Abbreviations LDST, low-dose ACTH stimulation test; NNT, number needed to test. Funding Sources None. Author Contribution Study design: DT, PK. Data collection: DT,SKC, IG, DD,PK,FG. Data analysis: DT,IG, DD. Data interpretation: DT,IG, DD. Drafting of the manuscript: DT,SKC and ZS; revision of content: DT,FG, SKC; and approval of the final version: DT, SKC, IG, DD, PK, FG. Data Availability The data that support the findings of this study are not publicly available due to privacy concerns, as they include detailed genetic information of individual patients. However, these data are available from References Weintrob N, Sprecher E, Josefsberg Z, Weininger C, Aurbach-Klipper Y, Lazard D et al (1998) Standard and Low-Dose Short Adrenocorticotropin Test Compared with Insulin-Induced Hypoglycemia for Assessment of the Hypothalamic-Pituitary-Adrenal Axis in Children with Idiopathic Multiple Pituitary Hormone Deficiencies. J Clin Endocrinol Metabolism 83(1):88–92 Raux Demay MC, Magny JP, Idrès N, Grimfeld A, Le Bouc Y (2006) Use of the Low-Dose Corticotropin Stimulation Test for the Monitoring of Children with Asthma Treated with Inhaled Corticosteroids. Hormone Res Paediatrics 66(2):51–60 Wildi-Runge S, Deladoëy J, Bélanger C, Deal CL, Van Vliet G, Alos N et al (2013) A Search for Variables Predicting Cortisol Response to Low-Dose Corticotropin Stimulation Following Supraphysiological Doses of Glucocorticoids. The Journal of Pediatrics. ;163(2):484-8.e1 Ozbek OY, Turktas I, Bakırtas A, Bideci A (2006) Evaluation of Hypothalamie-Pituitary-Adrenal Axis Suppression by Low-dose (0.5 µg) and Standard-dose (250 µg) Adrenocorticotropic Hormone (ACTH) Tests in Asthmatic Children Treated with Inhaled Corticosteroid. J Pediatr Endocrinol Metab. ;19(8) Nyunt O, Cotterill AM, Archbold SM, Wu JY, Leong GM, Verge CF et al (2010) Normal Cortisol Response on Low-Dose Synacthen (1 µg) Test in Children with Prader Willi Syndrome. J Clin Endocrinol Metabolism 95(12):E464–E7 Reznik Y, Barat P, Bertherat J, Bouvattier C, Castinetti F, Chabre O et al (eds) (2018) SFE/SFEDP adrenal insufficiency French consensus: introduction and handbook. Annales d'endocrinologie. Elsevier Tosun BG, Turan S, Haklar G, Bereket A, Guran T (2025) Optimal Low-Dose Synacthen Stimulation Test Sampling Time for Diagnosis of Adrenal Insufficiency Using Monoclonal Antibody Immunoassay. Hormone Research in Paediatrics Kazlauskaite R, Evans AT, Villabona CV, Abdu TA, Ambrosi B, Atkinson AB et al (2008) Corticotropin tests for hypothalamic-pituitary-adrenal insufficiency: a metaanalysis. J Clin Endocrinol Metabolism 93(11):4245–4253 DICKSTEIN G, SHECHNER C, NICHOLSON WE, ROSNER I, SHEN-ORR Z, ADAWI F et al (1991) Adrenocorticotropin stimulation test: effects of basal cortisol level, time of day, and suggested new sensitive low dose test. J Clin Endocrinol Metabolism 72(4):773–778 Gill H, Barrowman N, Webster R, Ahmet A (2019) Evaluating the Low-Dose ACTH Stimulation Test in Children: Ideal Times for Cortisol Measurement. J Clin Endocrinol Metabolism 104(10):4587–4593 Yo WS, Toh LM, Brown SJ, Howe WD, Henley DE, Lim EM (2014) How good is a morning cortisol in predicting an adequate response to intramuscular synacthen stimulation? Clin Endocrinol 81(1):19–24 Mushtaq T, Shakur F, Wales JK, Wright NP (2008) Reliability of the Low Dose Synacthen Test in Children Undergoing Pituitary Function Testing. J Pediatr Endocrinol Metab. ;21(12) LeDrew R, Bariciak E, Webster R, Barrowman N, Ahmet A (2020) Evaluating the Low-Dose ACTH Stimulation Test in Neonates: Ideal Times for Cortisol Measurement. J Clin Endocrinol Metabolism 105(12):e4543–e50 Karlsson R, Kallio J, Toppari J, Kero P (1999) Timing of Peak Serum Cortisol Values in Preterm Infants in Low-Dose and the Standard ACTH Tests. Pediatr Res 45(3):367–369 Vaiani E, Lazzati JM, Ramirez P, Costanzo M, Gil S, Dratler G et al (2019) The Low-Dose ACTH Test: Usefulness of Combined Analysis of Serum and Salivary Maximum Cortisol Response in Pediatrics. J Clin Endocrinol Metabolism 104(10):4323–4330 Cortez S, Arbeláez AM, Wallendorf M, McNerney K (2023) Peak Serum Cortisol Cutoffs to Diagnose Adrenal Insufficiency Across Different Cortisol Assays in Children. J Clin Res Pediatr Endocrinol 15(4):375–379 Ciancia S, van den Berg SAA, van den Akker ELT (2023) The Reliability of Salivary Cortisol Compared to Serum Cortisol for Diagnosing Adrenal Insufficiency with the Gold Standard ACTH Stimulation Test in Children. Children 10(9):1569 Cartaya J, Misra M (2015) The low-dose ACTH stimulation test: is 30 minutes long enough? Endocr Pract 21(5):508–513 Rasmuson S, Olsson T, Hägg E (1996) A low dose ACTH test to assess the function of the hypothalamic–pituitary–adrenal axis. Clin Endocrinol 44(2):151–156 Grassi G, Morelli V, Ceriotti F, Polledri E, Fustinoni S, D'Agostino S et al (2020) Minding the gap between cortisol levels measured with second-generation assays and current diagnostic thresholds for the diagnosis of adrenal insufficiency: a single-center experience. Horm (Athens) 19(3):425–431 Tables Table 1. Baseline Demographic, Auxological, and Laboratory Characteristics of the Study Population (n = 177) Mean ± SD (Range) Median (Q1–Q3) Age (years) 8.81 ± 5.92 (0.06–23.00) 9.7 (2.9–13.6) Sex(F/M), n (%) 85 (48.0)/ 92 (52.0) — Height SDS −0.59 ± 1.83 (−5.62–3.10) −0.38 (−1.58–0.64) BMI SDS 0.35 ± 1.62 (−6.04–4.53) 0.46 (−0.69–1.41) BMI category, n (%) Underweight / Normal weight 130 (73.4) — Overweight / Obese 35 (19.8) — Pubertal stage (Tanner), n (%) Stage 1 89 (50.3) — Stage 2 15 (8.5) — Stage 3 25 (14.1) — Stage 4 5 (2.8) — Stage 5 34 (19.2) — Basal ACTH (pg/mL) 18.71 ± 21.13 (0–257) 14.70 (10.38–22.03) Basal cortisol (µg/dL) 5.76 ± 2.16 (1.0–11.8) 5.85 (4.27–7.61) Table 2. Comparison of Basal ACTH and Basal Cortisol Levels According to Timing of Peak Cortisol Response During LDST Peak 0 minᵃ Peak 15 minᵇ Peak 30 minᶜ Peak 45 minᵈ Peak 60 minᵉ p value Basal ACTH (pg/mL), median (Q1–Q3) 18.7 (10.8–28.6) 12.9 (9.8–21.4) 14.4 (11.0–21.2) 14.6 (11.2–20.5) 18.5 (15.0–22.2) 0.394† Basal cortisol (µg/dL), median (Q1–Q3) 6.7 (5.6–11.7) 6.4 (4.7–8.6) 5.2 (3.7–6.6) 4.5 (3.5–5.7) 4.6 (3.0–4.9) 0.001† Data are presented as median (interquartile range, Q1–Q3). † Kruskal–Wallis test was used for overall group comparisons. Post-hoc pairwise comparisons were performed using the Bonferroni-corrected Mann–Whitney U test ; a corrected significance threshold of p < 0.01 (0.05/5) was applied. LDST, low-dose ACTH stimulation test. Post-hoc comparisons (Bonferroni-corrected): Peak 0 min vs Peak 45 min: p = 0.002, Peak 15 min vs Peak 45 min: p = 0.002 Table 3. Demographic, Auxological, and Clinical Characteristics Across Different Peak Cortisol Response Times During LDST Peak 0 min n:19 (10.7%) Peak 15 min n:86 (48.6%) Peak 30 min n:51 (28.8%) Peak 45 min n:16 (9.0%) Peak 60 min n:5 (2.8%) p Age (years), median (Q1–Q3) 6.3 (1.6–11.7) 9.0 (1.5–13.3) 10.9 (5.4–14.1) 11.8 (7.2–13.5) 10.4 (7.5–15.8) 0.441† Age category, n (%) 0.318‡ <5 years 9 (47.4) 34 (39.5) 12 (24.0) 2 (12.5) 1 (20.0) 5–9 years 4 (21.1) 14 (16.3) 12 (24.0) 3 (18.8) 1 (20.0) 10–14 years 2 (10.5) 22 (25.6) 17 (34.0) 8 (50.0) 1 (20.0) ≥15 years 4 (21.1) 16 (18.6) 9 (18.0) 3 (18.8) 2 (40.0) Sex, n (%) 0.726‡ Female 9 (47.4) 44 (51.2) 24 (47.1) 7 (43.8) 1 (20.0) Male 10 (52.6) 42 (48.8) 27 (52.9) 9 (56.2) 4 (80.0) Height SDS, median (Q1–Q3) 0.5 (−1.4–1.3) −0.2 (−1.4–0.6) −0.8 (−2.1–1.1) 0.2 (−1.6–1.0) 0.0 (−0.7–0.9) 0.251† Weight SDS, median (Q1–Q3) 0.2 (−1.0–1.8) 0.1 (−1.3–1.3) −0.4 (−1.6–0.9) 0.4 (−1.5–1.1) 1.8 (0.5–2.0) 0.298† BMI SDS, median (Q1–Q3) −0.03 (−0.41–1.55) 0.62 (−0.52–1.60) −0.04 (−0.80–1.10) 0.76 (−0.45–1.28) 1.80 (0.69–2.39) 0.305† BMI category, n (%) 0.408‡ Underweight / Normal 13 (76.5) 60 (75.9) 43 (86.0) 12 (80.0) 2 (50.0) Overweight / Obese 4 (23.5) 19 (24.1) 7 (14.0) 3 (20.0) 2 (50.0) Pubertal stage (Tanner), n (%) 0.469‡ Stage 1 12 (66.7) 44 (53.0) 24 (49.0) 7 (50.0) 2 (50.0) Stage 2 1 (5.6) 7 (8.4) 6 (12.2) 1 (7.1) 0 (0.0) Stage 3 2 (11.1) 12 (14.3) 7 (28.6) 4 (0.0) 0 (14.9) Stage 4 1 (5.6) 1 (1.2) 1 (2.0) 1 (7.1) 1 (25.0) Stage 5 2 (11.1) 19 (22.4) 11 (7.1) 1 (25.0) 1 (20.2) Table 4. Multiple Linear Regression Analysis of Factors Associated with Peak Cortisol Response Variable B (SE) Standardized β 95% CI for B p value Intercept 1.652 (0.314) — 1.016 to 2.287 <0.001 Basal cortisol −0.075 (0.031) −0.367 −0.139 to −0.012 0.021 Age (years) 0.022 (0.020) 0.165 −0.019 to 0.064 0.278 Table 5. Incremental Diagnostic Contribution of Extended Sampling Duration During the Low-Dose ACTH Stimulation Test Maximum Sampling Time (min) Patients Reaching Cortisol ≥18 µg/dL, n (%) Incremental Gain vs Previous Time Point, n (%) Cumulative False Positives if Test Terminated Earlier, n (%) NNT* 0 19 (10.7) — — — 15 106 (59.9) +87 (49.2) — — 30 157 (88.6) +51 (28.8) — — 45 171 (96.6) +14 (7.9) 20 (11.3) 13 60 175 (98.9) +4 (2.3) 21 (11.9) 8 Abbreviations: LDST, low-dose ACTH stimulation test; NNT, number needed to test. * NNT calculated as the reciprocal of the incremental diagnostic yield between 30 and 60 minutes (1 / 0.119 ≈ 8.4, rounded to 8). Table 6. Reclassification Probability and NNT for Extended Sampling During LDST Proposed Additional Test Probability Fail Changes to Pass With Additional Test, % (95% CI) Number of Patients Who Would Need Additional Test to Prevent One False Positive LDST Result, n (95% CI) 30 min in addition to 15 28.8% (22.3%–36.1%) 3 (3–4) 45 min in addition to 15 37.3% (30.1%–44.9%) 3 (2–3) 45 min in addition to 30 9.0% (5.3%–14.3%) 11 (7–19) 60 min in addition to 45 3.4% (1.3%–7.2%) 30 (14–80) 30 min in addition to 0 and 15 28.2% (21.7%–35.5%) 4 (3–5) 45 min in addition to 0 and 15 36.7% (29.6%–44.3%) 3 (2–3) 60 min in addition to 0–15–30–45 2.8% (0.9%–6.5%) 35,4 (15–108) Table 7. Time-Specific Cortisol Cut-offs Based on the 2.5th Percentile Time Point P2.5 Cut-off (µg/dL) ≥ Cut-off (%) False-Low (%) 15 min 12.0 97.5 2.5 30 min 16.1 97.7 2.3 45 min 17.1 97.8 2.2 60 min 18.5 98.9 1.1 Additional Declarations No competing interests reported. Supplementary Files SuplementaryTableS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Apr, 2026 Reviews received at journal 22 Apr, 2026 Reviews received at journal 08 Apr, 2026 Reviewers agreed at journal 28 Mar, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers agreed at journal 13 Mar, 2026 Reviews received at journal 24 Feb, 2026 Reviewers agreed at journal 17 Feb, 2026 Reviewers invited by journal 11 Feb, 2026 Editor assigned by journal 09 Feb, 2026 Submission checks completed at journal 09 Feb, 2026 First submitted to journal 25 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8694799","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592403649,"identity":"287b6182-f674-463b-88e0-4fd125b4cd7e","order_by":0,"name":"Derya Tepe","email":"","orcid":"","institution":"Ankara City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Derya","middleName":"","lastName":"Tepe","suffix":""},{"id":592403652,"identity":"d78b1158-68c8-4457-a9af-b6841881a6c4","order_by":1,"name":"Sirmen Kizilcan Cetin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIie3PvQrCMBDA8SuB63LUtUV9h0rA0b6KUOgUXVwUBwfBl/A9gqMg2KU6K12sgruCoJtJnVz6sQnmPxw33A8SAJPpF1vnsw8OgHVSGzmVCQIwXxOsQ9DVaylx0vh6P66iAO3ddvIQvRYCy86HAuLtBV8OEsGQhlHalqF6GHIuCoifEGODxRjRpW7qSaYIYbOY2BdNSJORJ2dVCHBFhKuJdZObcuIlpEnkIwnetGRMyEr+4nweFgYNO+ncXnKqlnl2KSJfMcpn1XOd9axzbTKZTH/TG9TdO8WoSoqxAAAAAElFTkSuQmCC","orcid":"","institution":"Ankara City Hospital","correspondingAuthor":true,"prefix":"","firstName":"Sirmen","middleName":"Kizilcan","lastName":"Cetin","suffix":""},{"id":592403653,"identity":"c325431d-cc97-4572-9ac9-5d6dd4daeca8","order_by":2,"name":"İrem Gokdemir","email":"","orcid":"","institution":"Ankara City Hospital","correspondingAuthor":false,"prefix":"","firstName":"İrem","middleName":"","lastName":"Gokdemir","suffix":""},{"id":592403656,"identity":"8b100ffa-3780-4da3-addb-1d7ac0f6476a","order_by":3,"name":"Pınar Kocaay","email":"","orcid":"","institution":"Ankara City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pınar","middleName":"","lastName":"Kocaay","suffix":""},{"id":592403657,"identity":"1b81e703-7aba-431a-a4d7-02c9d6aa23d7","order_by":4,"name":"Duygu Deligozoglu","email":"","orcid":"","institution":"Ankara City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Duygu","middleName":"","lastName":"Deligozoglu","suffix":""},{"id":592403659,"identity":"6c319541-2783-4cdd-8221-5a79fa64d3a0","order_by":5,"name":"Fatih Gurbuz","email":"","orcid":"","institution":"Ankara City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Fatih","middleName":"","lastName":"Gurbuz","suffix":""}],"badges":[],"createdAt":"2026-01-25 20:38:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8694799/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8694799/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102854358,"identity":"89c8e3cd-cf99-4757-ac97-f7e91792add5","added_by":"auto","created_at":"2026-02-17 14:48:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":205432,"visible":true,"origin":"","legend":"\u003cp\u003eSerum cortisol concentrations at baseline and at 15, 30, 45, and 60 minutes following stimulation with Cortrosyn™. In each panel, the horizontal line represents the median, the shaded box indicates the interquartile range (25th–75th percentiles), and the whiskers extend to 1.5× the interquartile range. The dashed horizontal line denotes the conventional diagnostic threshold for cortisol sufficiency (18 µg/dL).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8694799/v1/bca5cc4a62074c1d3488c3e4.png"},{"id":102964218,"identity":"7c2e67cd-9e39-41ff-9f01-64c255266747","added_by":"auto","created_at":"2026-02-19 04:21:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1762600,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8694799/v1/cdbcf811-2af0-421f-98d7-7f6415e3e62b.pdf"},{"id":102854357,"identity":"27cdafa7-ffc2-4819-840e-6d4ada85a7f8","added_by":"auto","created_at":"2026-02-17 14:48:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20626,"visible":true,"origin":"","legend":"","description":"","filename":"SuplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8694799/v1/fc46f41c4b44960db41405b1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Avoiding False-Positive Adrenal Insufficiency Diagnoses in Children: Insights from Cortisol Kinetics During Pediatric Low-Dose ACTH Stimulation Test","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe low-dose Synacthen\u0026reg; (adrenocorticotropic hormone, ACTH) stimulation test (LDST) is widely used for the assessment of hypothalamo\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis function in children with suspected central adrenal insufficiency (AI) (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). By administering a physiological dose of ACTH, the LDST is thought to better approximate endogenous adrenal stimulation and to minimize the supraphysiological cortisol responses associated with the standard-dose ACTH test. (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Accordingly, the LDST has been widely adopted in pediatric endocrinology. However, significant uncertainties remain concerning optimal protocols and the interpretation of test results.\u003c/p\u003e \u003cp\u003eTraditionally, cortisol measurement at a single post-stimulation time point, most commonly at 30 minutes, has been used for the interpretation of the LDST (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Morning basal serum cortisol measurement is frequently used as an initial screening tool in the diagnostic evaluation of AI. Very low morning cortisol concentrations (\u0026lt;\u0026thinsp;3 \u0026micro;g/dL [83 nmol/L]) are strongly suggestive of AI, whereas values exceeding 13 \u0026micro;g/dL (365 nmol/L) indicate preserved HPA axis function. When basal cortisol levels fall within the intermediate range, dynamic stimulation testing is required to assess adrenal reserve accurately (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, accumulating pediatric evidence increasingly challenges the adequacy of this approach. Large pediatric cohorts have demonstrated that the timing of peak cortisol response during LDST is highly variable (\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Despite decades of clinical use, the optimal timing for cortisol measurement following tetracosactide (Synacthen\u0026reg;) administration during the LDST remains a subject of ongoing debate (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe clinical consequences of false-positive AI diagnoses in childhood are substantial. Misclassification may result in unnecessary lifelong glucocorticoid replacement therapy, with potential adverse effects on growth, pubertal development, metabolic health, bone density, and overall quality of life (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In this context, there is a clear need to establish evidence-based, pediatric-specific LDST sampling strategies that reduce diagnostic error.\u003c/p\u003e \u003cp\u003eThis study aims to (i) characterize peak cortisol timing during LDST in a large, real-world pediatric cohort, (ii) explore clinical and biochemical correlates of peak timing, and (iii) assess the additional diagnostic benefit provided by extended cortisol sampling beyond a single time point.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Participants\u003c/h2\u003e \u003cp\u003eThis retrospective observational study evaluated pediatric patients who underwent a LDST for suspected adrenal insufficiency at a tertiary pediatric endocrinology center. Patients with a peak cortisol response\u0026thinsp;\u0026lt;\u0026thinsp;18 \u0026micro;g/dL, consistent with central AI, were excluded. A total of 177 children and adolescents were included in the analysis.\u003c/p\u003e \u003cp\u003eIndications for LDST included abnormal clinical findings, the use of medications (exposure to exogenous steroids) affecting the HPA axis, hypothalamo\u0026ndash;hypophyseal disorders, prior cranial radiotherapy, laboratory abnormalities suggestive HPA axis dysfunction, and basal morning cortisol insufficiency (\u0026lt;\u0026thinsp;13 \u0026micro;g/dL [350 nmol/L]).\u003c/p\u003e \u003cp\u003eDemographic data (age, sex), anthropometric measurements (height, weight, body mass index [BMI], and SDS values), pubertal status according to Tanner staging, and relevant baseline laboratory parameters were retrospectively obtained from medical data.\u003c/p\u003e \u003cp\u003e The study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the local ethics committee (approval number: TABED 2/918/2025).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLow-Dose ACTH Stimulation Test Protocol\u003c/h3\u003e\n\u003cp\u003eAll LDSTs were performed in the morning hours following an overnight fast. After placement of an intravenous catheter, baseline blood samples were obtained for serum cortisol and plasma ACTH measurements (0th minute). A 1 \u0026micro;g intravenous bolus of synthetic ACTH (tetracosactide; Cortrosyn\u0026reg;) was administered, and serum cortisol levels were measured at 15, 30,45 and 60 minutes following stimulation, in accordance with our institutional protocol.\u003c/p\u003e \u003cp\u003eThe peak cortisol response was defined as the highest cortisol concentration observed at any sampled time point during the test. Patients were classified according to the time point at which the peak cortisol response occurred (0, 15, 30, 45, or 60 minutes).\u003c/p\u003e\n\u003ch3\u003eLaboratory Measurements\u003c/h3\u003e\n\u003cp\u003eSerum cortisol concentrations were measured using polyclonal antibody immunoassays using the Siemens Atellica\u0026reg; immunoassay.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS software version 25.0 (IBM Corp., Armonk, NY, USA). Normality of continuous variables was assessed using the Shapiro\u0026ndash;Wilk test. Continuous variables were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) or median (interquartile range; Q1\u0026ndash;Q3), depending on distribution. Categorical variables were presented as counts and percentages. To compare cortisol levels across multiple time points within individuals, the Friedman test was used. For comparisons among more than two independent groups with non-normal distribution, the Kruskal\u0026ndash;Wallis test was applied, followed by Bonferroni-corrected post hoc analyses when appropriate. Categorical variables were compared using the chi-square test. Associations between the timing of peak cortisol response and clinical or biochemical parameters were evaluated using Spearman correlation analysis, with correlation strength classified as weak (r\u0026thinsp;=\u0026thinsp;0.00\u0026ndash;0.29), moderate (r\u0026thinsp;=\u0026thinsp;0.30\u0026ndash;0.49), strong (r\u0026thinsp;=\u0026thinsp;0.50\u0026ndash;0.69), or very strong (r\u0026thinsp;\u0026ge;\u0026thinsp;0.70). To identify independent predictors of peak cortisol response, multiple linear regression analysis was performed. Age, height SDS, and basal cortisol levels were entered into the model using the Enter method. Multicollinearity was assessed using the variance inflation factor (VIF) and tolerance values.\u003c/p\u003e \u003cp\u003eThe incremental diagnostic contribution of additional cortisol sampling time points was evaluated to determine the added value of extending the LDST protocol. Specifically, the probability that an additional cortisol measurement at later time points (e.g., 30, 45, or 60 minutes) would reclassify an initially failed LDST result as a passed result was calculated. The number needed to test (NNT) was derived as the reciprocal of this probability to estimate how many additional extended tests would be required to prevent one false-positive LDST classification. NNT values were rounded to the nearest whole number and reported with corresponding 95% confidence intervals. A p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of\u0026nbsp;177 patients (%48, n=85 female)\u0026nbsp;who underwent LDST were included in the study. The mean age at testing was 8.81 ± 5.92 years. The most common indication for LDST was insufficient basal morning cortisol identified during laboratory evaluation (69.5%, n = 123). Clinical histories suggestive of adrenal insufficiency—including symptoms such as prolonged jaundice, fatigue, or hypoglycemia—were present in 53.1% of patients (n = 94). Additionally, 25.4% (n = 45) had underlying intracranial pathology, and 20.3% (n = 36) had a history of exogenous glucocorticoid or stimulant exposure. Baseline laboratory characteristics, including ACTH and cortisol levels, are summarized in Table 1.\u003c/p\u003e\n\u003cp\u003eAnalysis of peak cortisol responses during LDST (Figure 1) demonstrated that the most frequent peak occurred at 15 minutes (48.6%, n = 86). This was followed by the 30-minute peak (28.8%, n = 51), baseline (0 minute) (10.7%, n = 19), 45 minutes (9.0%, n = 16), and 60 minutes (2.8%, n = 5). Median cortisol concentrations differed significantly across peak time points (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003ePatients exhibiting a peak cortisol response at 15 minutes had a median cortisol level of 19.2 µg/dL, whereas the highest median cortisol concentration was observed in patients peaking at 60 minutes (27.3 µg/dL). Nearly half of patients reached peak cortisol concentrations at 15 minutes, while fewer than 3% peaked at 60 minutes, highlighting substantial interindividual variability in cortisol response timing.\u003c/p\u003e\n\u003cp\u003eDetailed cortisol measurements at all sampling time points are presented in Table S.1, and the distribution of peak responses is illustrated in Figure 1.\u003c/p\u003e\n\u003cp\u003eData are presented as number (percentage), mean ± standard deviation (SD) with range, and median with interquartile range (Q1–Q3).\u003cbr\u003e\u0026nbsp;Peak cortisol was defined as the highest serum cortisol concentration observed at any sampling point during the low-dose ACTH stimulation test.\u003c/p\u003e\n\u003cp\u003eBasal serum cortisol concentrations differed significantly according to the timing of the peak cortisol response following low-dose ACTH stimulation (p = 0.001). Post-hoc multiple comparison analyses revealed that patients who achieved their peak cortisol response at baseline (0 min) and 15 minutes had significantly higher basal cortisol levels compared with those peaking at 45 minutes (both p = 0.002). Basal cortisol levels differed significantly according to peak response timing, with higher basal cortisol observed in patients peaking at 0 and 15 minutes compared with those peaking at 45 minutes, whereas basal ACTH levels did not differ across groups(Table 2).\u003c/p\u003e\n\u003cp\u003eNo significant associations were observed between peak response timing and age, sex, pubertal status, height SDS, weight SDS, or BMI SDS, indicating that the temporal pattern of cortisol response was independent of demographic and auxological characteristics (Table 3).\u003c/p\u003e\n\u003cp\u003eFollowing ACTH stimulation, serum cortisol concentrations increased progressively over time. Paired Wilcoxon signed-rank tests demonstrated statistically significant increases between all consecutive sampling points:\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003e15 → 30 minutes:\u003c/strong\u003e median increase \u003cstrong\u003e+4.1 µg/dL\u003c/strong\u003e (\u003cem\u003ep\u003c/em\u003e ≈ 1.6 × 10⁻²⁹)\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003e30 → 45 minutes:\u003c/strong\u003e median increase \u003cstrong\u003e+1.8 µg/dL\u003c/strong\u003e (\u003cem\u003ep\u003c/em\u003e ≈ 3.1 × 10⁻¹⁶)\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003e45 → 60 minutes:\u003c/strong\u003e median increase \u003cstrong\u003e+1.7 µg/dL\u003c/strong\u003e (\u003cem\u003ep\u003c/em\u003e ≈ 2.4 × 10⁻¹⁵)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThese results indicate a rapid early rise in cortisol levels, followed by a plateau beyond 45 minutes, suggesting a diminishing incremental diagnostic contribution of later sampling\u003c/p\u003e\n\u003cp\u003eA multiple linear regression model including age, body height, and basal cortisol levels significantly explained the variability in peak cortisol response (\u003cem\u003eF\u003c/em\u003e(3,37) = 3.65, \u003cem\u003ep\u003c/em\u003e = 0.021; adjusted \u003cem\u003eR\u003c/em\u003e² = 0.17). Among the covariates, only basal cortisol level was independently associated with peak cortisol response, demonstrating a significant inverse relationship (B = −0.075, 95% CI −0.139 to −0.012; \u003cem\u003ep\u003c/em\u003e = 0.021). Age and body height were not significantly associated with peak cortisol response (Table 4). Higher basal cortisol levels were independently associated with a blunted peak cortisol response, supporting a ceiling effect of adrenal stimulation during LDST.\u003c/p\u003e\n\u003cp\u003eCortisol concentrations increased consistently at each successive sampling point . When applying the conventional cortisol sufficiency threshold of ≥18 µg/dL, the proportion of patients meeting diagnostic adequacy rose markedly over time, from 10.7% at baseline to 96.6% at 45 minutes and 98.9% at 60 minutes.\u003c/p\u003e\n\u003cp\u003eAt 30 minutes, only 88.6% of patients had achieved cortisol levels ≥18 µg/dL, indicating that a clinically relevant subgroup exhibited delayed yet sufficient adrenal responses.\u003c/p\u003e\n\u003cp\u003eTermination of the LDST at 30 minutes would have resulted in the misclassification of 21 patients (11.9%) as having inadequate adrenal function, despite demonstrating normal cortisol responses at later time points (Table 5).\u003c/p\u003e\n\u003cp\u003eThe diagnostic contribution of extending sampling time points during LDST to prevent false-positive classifications is summarized in Table 6. Sequential extension of sampling demonstrated variable added diagnostic contribution depending on the time interval evaluated.\u003c/p\u003e\n\u003cp\u003eExtension from 15 to 30 minutes corrected an initial insufficient response in 28.8% of patients, corresponding to a NNT of 3. Similarly, extending sampling from 15 to 45 minutes improved diagnostic classification in 37.3% of cases (NNT = 3). In contrast, extending sampling from 30 to 45 minutes resulted in correction in only 9.0% of patients (NNT = 11).\u003c/p\u003e\n\u003cp\u003eIn pediatric patients without adrenal insufficiency, serum cortisol levels typically reach diagnostic thresholds by 30 minutes following low-dose ACTH stimulation. Extending sampling beyond 45 minutes does not provide significant additional diagnostic value.\u003c/p\u003e\n\u003cp\u003eThe diagnostic contribution of a 60-minute sample was limited. Extension from 45 to 60 minutes corrected cortisol classification in only 3.4% of cases, yielding an NNT of 30. When comprehensive sampling from 0–45 minutes was already performed, adding a 60-minute sample provided minimal additional diagnostic information (2.8% improvement; NNT = 35).\u003c/p\u003e\n\u003cp\u003eWhen both baseline (0 min) and 15-minute samples were available, adding a 30-minute sample corrected 28.2% of initially insufficient results (NNT = 4), whereas adding a 45-minute sample corrected 36.7% (NNT = 3). Overall, the greatest diagnostic contribution was observed with extension to 45 minutes, whereas routine extension beyond this time point yielded marginal additional benefit(Table S2) .\u003c/p\u003e\n\u003cp\u003eAmong patients without adrenal insufficiency, application of a uniform cortisol cut-off of \u003cstrong\u003e18 µg/dL\u003c/strong\u003e at early sampling times resulted in substantial false-low classifications (Table 6). False-low rates were highest at 15 minutes (40.3%) and remained clinically relevant at 30 minutes (11.4%), indicating a pronounced risk of overestimating adrenal insufficiency when early sampling points are interpreted in isolation (Table S3).\u003c/p\u003e\n\u003cp\u003eTo address time-dependent cortisol kinetics, lower reference limits based on the 2.5th percentile (P2.5) were applied (Table 7). Use of time-adjusted cut-offs markedly reduced false-low rates across all sampling points. At 30 minutes, the false-low rate decreased nearly five-fold when a time-specific threshold of 16.1 µg/dL was used instead of the conventional 18 µg/dL (2.3% vs. 11.4%).\u003c/p\u003e\n\u003cp\u003eBasal cortisol concentrations differed significantly according to peak response timing (p = 0.001). Post-hoc analyses demonstrated that patients peaking at 0 and 15 minutes had significantly higher basal cortisol levels than those peaking at 45 minutes (p = 0.002 for both). No significant associations were identified between peak timing and age, sex, pubertal status, or anthropometric parameters.\u003c/p\u003e"},{"header":"Discusion","content":"\u003cp\u003eThe low-dose ACTH stimulation test (LDST) remains one of the most widely used tools in pediatric practice. Despite its extensive use, substantial uncertainty persists regarding optimal sampling time points and appropriate cortisol thresholds. In this study, we systematically evaluated the temporal dynamics of cortisol responses during LDST in a pediatric cohort ultimately classified as not having AI and demonstrated that both sampling time and threshold selection critically influence diagnostic interpretation.\u003c/p\u003e \u003cp\u003eExisting literature on the LDST (Table S4) demonstrates substantial heterogeneity with respect to ACTH dosing, sampling time points, and diagnostic cortisol thresholds. Although most pediatric studies recommend a standard 1-\u0026micro;g ACTH dose, sampling strategies vary widely, with many protocols limited to baseline and 30-minute measurements and only selected studies incorporating additional 15- or 60-minute samples. Consistent with prior pediatric and adult studies (Table S4), serum cortisol concentrations increased progressively following ACTH stimulation, with the most pronounced rise occurring between 15 and 30 minutes, followed by smaller yet statistically significant increments thereafter(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Although the majority of patients achieved diagnostic cortisol thresholds by 30 minutes, a clinically meaningful subgroup reached\u0026thinsp;\u0026ge;\u0026thinsp;18 \u0026micro;g/dL only at 45 or 60 minutes. These delayed responders represent physiological variation rather than pathology and would have been misclassified as having adrenal insufficiency if testing had been terminated prematurely.\u003c/p\u003e \u003cp\u003eHistorically, many centers have relied on baseline and 30-minute cortisol measurements during LDST, based on early reports suggesting that peak cortisol responses typically occur within this window(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, accumulating evidence indicates that exclusive reliance on early sampling leads to a non-negligible false-positive rate. In our cohort, termination of testing at 30 minutes would have resulted in misclassification of nearly 12% of patients, a finding that closely mirrors rates reported by Gill et al.(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), Vaiani et al.(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and Cartarya et al.(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), who demonstrated that omission of later samples misclassified approximately 10\u0026ndash;15% of children with normal adrenal function. Studies in neonates consistently demonstrate delayed peak responses, often at 60 minutes(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Despite these observations, the majority of published protocols continue to apply a time-independent cortisol cut-off of \u0026ge;\u0026thinsp;18 \u0026micro;g/dL (500 nmol/L) across all sampling points. Our findings quantitatively demonstrate that this approach leads to substantial overestimation of AI, particularly at 15 and 30 minutes, where false-low rates reached 40.3% and 11.4%, respectively.\u003c/p\u003e \u003cp\u003eThe present study extends prior work by systematically integrating sampling time and cut-off selection. We demonstrate that extending sampling to 45 minutes provides meaningful diagnostic benefit by capturing delayed yet physiologically normal cortisol responses, whereas routine extension to 60 minutes offers only marginal additional value. This incremental pattern is compatible with probability-based analyses in prior cohorts, which demonstrated diminishing diagnostic returns beyond the mid-to-late sampling window (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). These data support 45 minutes as a pragmatic upper limit for routine LDST sampling in most pediatric patients. Moreover, by introducing time-specific, percentile-based thresholds, we show that false-positive classifications can be dramatically reduced without compromising diagnostic safety. This approach directly addresses a major gap in the literature, where time-dependent cortisol physiology has been acknowledged but rarely operationalized into practical diagnostic criteria.\u003c/p\u003e \u003cp\u003eA key mechanistic finding of our study is the association between basal cortisol levels and peak response timing. Patients exhibiting early peak responses (0\u0026ndash;15 minutes) had significantly higher basal cortisol concentrations compared with those peaking at 45 minutes, while basal ACTH levels did not differ across groups. This pattern suggests that baseline adrenal tone rather than central ACTH drive modulates response kinetics, consistent with a physiological ceiling effect. Multivariable regression analysis further supported this interpretation by demonstrating an independent inverse association between basal cortisol levels and peak cortisol increment. Significantly, peak response timing was independent of age, sex, pubertal status, and anthropometric parameters, indicating that delayed cortisol responses cannot be predicted based on routine clinical characteristics.\u003c/p\u003e \u003cp\u003eOne of the most clinically relevant observations concerns the widespread use of a uniform cortisol cut-off of 18 \u0026micro;g/dL across all sampling times. Our data clearly demonstrate that applying this threshold at early time points\u0026mdash;particularly at 15 and 30 minutes\u0026mdash;results in substantial overestimation of AI. At 15 minutes, more than 40% of patients without AI would have been misclassified, and at 30 minutes, the false-low rate remained above 10%. Similar concerns have been raised in multiple cohorts, particularly with the adoption of more specific modern immunoassays that provide systematically lower cortisol values compared with older polyclonal assays(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). To address this limitation, we explored time-specific, percentile-based cortisol thresholds derived from the distribution of responses in patients without AI. This approach markedly reduced false-low classifications at all sampling points. At the clinically pivotal 30-minute mark, use of a time-adjusted threshold reduced false-low rates nearly five-fold compared with the conventional 18 \u0026micro;g/dL cut-off. This strategy is statistically robust, physiologically plausible, and consistent with calls in the literature for assay- and context-specific interpretation of dynamic endocrine tests (Table S4).\u003c/p\u003e \u003cp\u003eAn additional noteworthy observation relates to patients who achieved a sufficient cortisol response at 0 minutes (10.7%). Because cortisol results were not available in real time during test administration, these patients could not be excluded a priori. Notably, this subgroup comprised patients with insufficient morning basal cortisol levels who nevertheless demonstrated an adequate response at the initial LDST sampling point, further illustrating the limitations of relying on a single basal cortisol measurement and reinforcing the value of dynamic testing.\u003c/p\u003e \u003cp\u003eTaken together, our findings support a stepwise LDST interpretation framework: early termination is appropriate when cortisol sufficiency is achieved at baseline or 30 minutes; mandatory extension to 45 minutes should be performed in patients with subthreshold 30-minute values; and 60-minute sampling should be reserved for selected cases with persistently low intermediate values or high clinical suspicion. Incorporation of time-adjusted thresholds may further enhance diagnostic accuracy and reduce unnecessary lifelong treatment.\u003c/p\u003e \u003cp\u003eSeveral limitations warrant consideration. The analysis focused on patients ultimately classified as not having AI, precluding direct sensitivity estimates for true adrenal failure. Second, cortisol measurements were performed using a polyclonal antibody-based immunoassay, and confirmatory testing with more specific methods such as liquid chromatography\u0026ndash;tandem mass spectrometry (LC\u0026ndash;MS/MS) was not available. However, this reflects real-world clinical practice, as polyclonal immunoassays remain widely used in many centers due to their greater accessibility and lower cost, despite the superior specificity of monoclonal assays. Consequently, while assay-related variability cannot be excluded, the use of a commonly employed method may enhance the generalizability of our findings to routine pediatric endocrinology settings.\u003c/p\u003e \u003cp\u003eIn conclusion, LDST responses in children exhibit substantial temporal variability driven primarily by basal cortisol levels rather than demographic or auxological factors. Reliance on early sampling combined with uniform cortisol cut-offs substantially overestimates adrenal insufficiency. Extension of sampling to 45 minutes and adoption of time-specific, percentile-based thresholds provide a more physiologically sound and diagnostically accurate framework for LDST interpretation in pediatric practice.\u003c/p\u003e\n\u003ch3\u003eStatements\u003c/h3\u003e\n\u003cp\u003e Written informed consent was obtained from the parent/legal guardian of participants prior to the study.\u003c/p\u003e\n\u003ch3\u003eStatement of Ethics\u003c/h3\u003e\n\u003cp\u003e Written informed consent was obtained from the parent/legal guardian of participants prior to the study for the publication of details such as age, gender, illness. This study was reviewed and approved by Ankara Bilkent City Hospital\u0026rsquo;s Ethic Committee, approval number TABED2/918/2025, decision date 19.02.2025.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAbbreviations\u003c/h2\u003e \u003cp\u003eLDST, low-dose ACTH stimulation test; NNT, number needed to test.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Sources\u003c/h2\u003e \u003cp\u003eNone.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eStudy design: DT, PK. Data collection: DT,SKC, IG, DD,PK,FG. Data analysis: DT,IG, DD. Data interpretation: DT,IG, DD. Drafting of the manuscript: DT,SKC and ZS; revision of content: DT,FG, SKC; and approval of the final version: DT, SKC, IG, DD, PK, FG.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are not publicly available due to privacy concerns, as they include detailed genetic information of individual patients. However, these data are available from\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeintrob N, Sprecher E, Josefsberg Z, Weininger C, Aurbach-Klipper Y, Lazard D et al (1998) Standard and Low-Dose Short Adrenocorticotropin Test Compared with Insulin-Induced Hypoglycemia for Assessment of the Hypothalamic-Pituitary-Adrenal Axis in Children with Idiopathic Multiple Pituitary Hormone Deficiencies. J Clin Endocrinol Metabolism 83(1):88\u0026ndash;92\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaux Demay MC, Magny JP, Idr\u0026egrave;s N, Grimfeld A, Le Bouc Y (2006) Use of the Low-Dose Corticotropin Stimulation Test for the Monitoring of Children with Asthma Treated with Inhaled Corticosteroids. Hormone Res Paediatrics 66(2):51\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWildi-Runge S, Delado\u0026euml;y J, B\u0026eacute;langer C, Deal CL, Van Vliet G, Alos N et al (2013) A Search for Variables Predicting Cortisol Response to Low-Dose Corticotropin Stimulation Following Supraphysiological Doses of Glucocorticoids. The Journal of Pediatrics. ;163(2):484-8.e1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzbek OY, Turktas I, Bakırtas A, Bideci A (2006) Evaluation of Hypothalamie-Pituitary-Adrenal Axis Suppression by Low-dose (0.5 \u0026micro;g) and Standard-dose (250 \u0026micro;g) Adrenocorticotropic Hormone (ACTH) Tests in Asthmatic Children Treated with Inhaled Corticosteroid. J Pediatr Endocrinol Metab. ;19(8)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNyunt O, Cotterill AM, Archbold SM, Wu JY, Leong GM, Verge CF et al (2010) Normal Cortisol Response on Low-Dose Synacthen (1 \u0026micro;g) Test in Children with Prader Willi Syndrome. J Clin Endocrinol Metabolism 95(12):E464\u0026ndash;E7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReznik Y, Barat P, Bertherat J, Bouvattier C, Castinetti F, Chabre O et al (eds) (2018) SFE/SFEDP adrenal insufficiency French consensus: introduction and handbook. Annales d'endocrinologie. Elsevier\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTosun BG, Turan S, Haklar G, Bereket A, Guran T (2025) Optimal Low-Dose Synacthen Stimulation Test Sampling Time for Diagnosis of Adrenal Insufficiency Using Monoclonal Antibody Immunoassay. Hormone Research in Paediatrics\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKazlauskaite R, Evans AT, Villabona CV, Abdu TA, Ambrosi B, Atkinson AB et al (2008) Corticotropin tests for hypothalamic-pituitary-adrenal insufficiency: a metaanalysis. J Clin Endocrinol Metabolism 93(11):4245\u0026ndash;4253\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDICKSTEIN G, SHECHNER C, NICHOLSON WE, ROSNER I, SHEN-ORR Z, ADAWI F et al (1991) Adrenocorticotropin stimulation test: effects of basal cortisol level, time of day, and suggested new sensitive low dose test. J Clin Endocrinol Metabolism 72(4):773\u0026ndash;778\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGill H, Barrowman N, Webster R, Ahmet A (2019) Evaluating the Low-Dose ACTH Stimulation Test in Children: Ideal Times for Cortisol Measurement. J Clin Endocrinol Metabolism 104(10):4587\u0026ndash;4593\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYo WS, Toh LM, Brown SJ, Howe WD, Henley DE, Lim EM (2014) How good is a morning cortisol in predicting an adequate response to intramuscular synacthen stimulation? Clin Endocrinol 81(1):19\u0026ndash;24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMushtaq T, Shakur F, Wales JK, Wright NP (2008) Reliability of the Low Dose Synacthen Test in Children Undergoing Pituitary Function Testing. J Pediatr Endocrinol Metab. ;21(12)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeDrew R, Bariciak E, Webster R, Barrowman N, Ahmet A (2020) Evaluating the Low-Dose ACTH Stimulation Test in Neonates: Ideal Times for Cortisol Measurement. J Clin Endocrinol Metabolism 105(12):e4543\u0026ndash;e50\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarlsson R, Kallio J, Toppari J, Kero P (1999) Timing of Peak Serum Cortisol Values in Preterm Infants in Low-Dose and the Standard ACTH Tests. Pediatr Res 45(3):367\u0026ndash;369\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaiani E, Lazzati JM, Ramirez P, Costanzo M, Gil S, Dratler G et al (2019) The Low-Dose ACTH Test: Usefulness of Combined Analysis of Serum and Salivary Maximum Cortisol Response in Pediatrics. J Clin Endocrinol Metabolism 104(10):4323\u0026ndash;4330\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCortez S, Arbel\u0026aacute;ez AM, Wallendorf M, McNerney K (2023) Peak Serum Cortisol Cutoffs to Diagnose Adrenal Insufficiency Across Different Cortisol Assays in Children. J Clin Res Pediatr Endocrinol 15(4):375\u0026ndash;379\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiancia S, van den Berg SAA, van den Akker ELT (2023) The Reliability of Salivary Cortisol Compared to Serum Cortisol for Diagnosing Adrenal Insufficiency with the Gold Standard ACTH Stimulation Test in Children. Children 10(9):1569\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCartaya J, Misra M (2015) The low-dose ACTH stimulation test: is 30 minutes long enough? Endocr Pract 21(5):508\u0026ndash;513\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRasmuson S, Olsson T, H\u0026auml;gg E (1996) A low dose ACTH test to assess the function of the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal axis. Clin Endocrinol 44(2):151\u0026ndash;156\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrassi G, Morelli V, Ceriotti F, Polledri E, Fustinoni S, D'Agostino S et al (2020) Minding the gap between cortisol levels measured with second-generation assays and current diagnostic thresholds for the diagnosis of adrenal insufficiency: a single-center experience. Horm (Athens) 19(3):425\u0026ndash;431\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e \u003cstrong\u003eBaseline Demographic, Auxological, and Laboratory Characteristics of the Study Population (n = 177)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" width=\"606\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026plusmn; SD (Range)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMedian (Q1\u0026ndash;Q3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.81 \u0026plusmn; 5.92 (0.06\u0026ndash;23.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.7 (2.9\u0026ndash;13.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSex(F/M), n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85 (48.0)/ 92 (52.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eHeight SDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.59 \u0026plusmn; 1.83 (\u0026minus;5.62\u0026ndash;3.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.38 (\u0026minus;1.58\u0026ndash;0.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBMI SDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.35 \u0026plusmn; 1.62 (\u0026minus;6.04\u0026ndash;4.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.46 (\u0026minus;0.69\u0026ndash;1.41)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBMI category, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Underweight / Normal weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e130 (73.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Overweight / Obese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35 (19.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePubertal stage (Tanner), n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e89 (50.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15 (8.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25 (14.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34 (19.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBasal ACTH (pg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.71 \u0026plusmn; 21.13 (0\u0026ndash;257)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.70 (10.38\u0026ndash;22.03)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBasal cortisol (\u0026micro;g/dL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.76 \u0026plusmn; 2.16 (1.0\u0026ndash;11.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.85 (4.27\u0026ndash;7.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Comparison of Basal ACTH and Basal Cortisol Levels According to Timing of Peak Cortisol Response During LDST\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 0 minᵃ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 15 minᵇ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 30 minᶜ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 45 minᵈ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 60 minᵉ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBasal ACTH (pg/mL), median (Q1\u0026ndash;Q3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.7 (10.8\u0026ndash;28.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.9 (9.8\u0026ndash;21.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.4 (11.0\u0026ndash;21.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.6 (11.2\u0026ndash;20.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.5 (15.0\u0026ndash;22.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.394\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBasal cortisol (\u0026micro;g/dL), median (Q1\u0026ndash;Q3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.7 (5.6\u0026ndash;11.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.4 (4.7\u0026ndash;8.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.2 (3.7\u0026ndash;6.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.5 (3.5\u0026ndash;5.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.6 (3.0\u0026ndash;4.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u0026dagger;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData are presented as median (interquartile range, Q1\u0026ndash;Q3).\u003cbr\u003e\u0026dagger;\u0026nbsp;\u003cem\u003eKruskal\u0026ndash;Wallis test\u003c/em\u003e was used for overall group comparisons.\u003cbr\u003ePost-hoc pairwise comparisons were performed using the\u0026nbsp;\u003cem\u003eBonferroni-corrected Mann\u0026ndash;Whitney U test\u003c/em\u003e; a corrected significance threshold of p \u0026lt; 0.01 (0.05/5) was applied.\u003cbr\u003e LDST, low-dose ACTH stimulation test.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePost-hoc comparisons (Bonferroni-corrected): Peak 0 min vs Peak 45 min: \u003cem\u003ep\u003c/em\u003e = 0.002, \u0026nbsp;Peak 15 min vs Peak 45 min: \u003cem\u003ep\u003c/em\u003e = 0.002\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Demographic, Auxological, and Clinical Characteristics Across Different Peak Cortisol Response Times During LDST\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 0 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en:19 (10.7%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 15 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en:86 (48.6%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 30 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en:51 (28.8%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 45 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en:16 (9.0%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak 60 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en:5 (2.8%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years), median (Q1\u0026ndash;Q3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.3 (1.6\u0026ndash;11.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.0 (1.5\u0026ndash;13.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.9 (5.4\u0026ndash;14.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.8 (7.2\u0026ndash;13.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.4 (7.5\u0026ndash;15.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.441\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAge category, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.318\u0026Dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026lt;5 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9 (47.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34 (39.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12 (24.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (20.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; 5\u0026ndash;9 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4 (21.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14 (16.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12 (24.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3 (18.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (20.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; 10\u0026ndash;14 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2 (10.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e22 (25.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17 (34.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (20.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026ge;15 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4 (21.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16 (18.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9 (18.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3 (18.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2 (40.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSex, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.726\u0026Dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9 (47.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44 (51.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24 (47.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7 (43.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (20.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 (52.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42 (48.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27 (52.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9 (56.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4 (80.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eHeight SDS, median (Q1\u0026ndash;Q3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5 (\u0026minus;1.4\u0026ndash;1.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.2 (\u0026minus;1.4\u0026ndash;0.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.8 (\u0026minus;2.1\u0026ndash;1.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.2 (\u0026minus;1.6\u0026ndash;1.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0 (\u0026minus;0.7\u0026ndash;0.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.251\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eWeight SDS, median (Q1\u0026ndash;Q3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.2 (\u0026minus;1.0\u0026ndash;1.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.1 (\u0026minus;1.3\u0026ndash;1.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.4 (\u0026minus;1.6\u0026ndash;0.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.4 (\u0026minus;1.5\u0026ndash;1.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.8 (0.5\u0026ndash;2.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.298\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBMI SDS, median (Q1\u0026ndash;Q3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.03 (\u0026minus;0.41\u0026ndash;1.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.62 (\u0026minus;0.52\u0026ndash;1.60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.04 (\u0026minus;0.80\u0026ndash;1.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.76 (\u0026minus;0.45\u0026ndash;1.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.80 (0.69\u0026ndash;2.39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.305\u0026dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBMI category, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.408\u0026Dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Underweight / Normal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13 (76.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e60 (75.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43 (86.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12 (80.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Overweight / Obese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4 (23.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19 (24.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7 (14.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3 (20.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePubertal stage (Tanner), n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.469\u0026Dagger;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12 (66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44 (53.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24 (49.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7 (8.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6 (12.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (7.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2 (11.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12 (14.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7 (28.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0 (14.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (1.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (2.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (7.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (25.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Stage 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2 (11.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19 (22.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11 (7.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (25.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1 (20.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Multiple Linear Regression Analysis of Factors Associated with Peak Cortisol Response\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" width=\"569\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eB (SE)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eStandardized \u0026beta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI for B\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIntercept\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.652 (0.314)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.016 to 2.287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBasal cortisol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.075 (0.031)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.367\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.139 to \u0026minus;0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.022 (0.020)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026minus;0.019 to 0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.278\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eIncremental Diagnostic Contribution of Extended Sampling Duration During the Low-Dose ACTH Stimulation Test\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMaximum Sampling Time (min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePatients Reaching Cortisol \u0026ge;18 \u0026micro;g/dL, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIncremental Gain vs Previous Time Point, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCumulative False Positives if Test Terminated Earlier, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNNT*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19 (10.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e106 (59.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+87 (49.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e157 (88.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+51 (28.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e171 (96.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+14 (7.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20 (11.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e175 (98.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+4 (2.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e21 (11.9)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e LDST, low-dose ACTH stimulation test; NNT, number needed to test.\u003cbr\u003e* \u003cstrong\u003eNNT calculated as the reciprocal of the incremental diagnostic yield between 30 and 60 minutes\u003c/strong\u003e (1 / 0.119 \u0026asymp; 8.4, rounded to 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6. Reclassification Probability and NNT for Extended Sampling During LDST\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"641\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProposed Additional Test\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProbability Fail Changes to Pass With Additional Test, % (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of Patients Who Would Need Additional Test to Prevent One False Positive LDST Result, n (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e30 min in addition to 15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e28.8% (22.3%\u0026ndash;36.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003e3 (3\u0026ndash;4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e45 min in addition to 15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e37.3% (30.1%\u0026ndash;44.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003e3 (2\u0026ndash;3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e45 min in addition to 30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e9.0% (5.3%\u0026ndash;14.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003e11 (7\u0026ndash;19)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e60 min in addition to 45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e3.4% (1.3%\u0026ndash;7.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003e30 (14\u0026ndash;80)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e30 min in addition to 0 and 15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e28.2% (21.7%\u0026ndash;35.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003e4 (3\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e45 min in addition to 0 and 15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e36.7% (29.6%\u0026ndash;44.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003e3 (2\u0026ndash;3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e60 min in addition to 0\u0026ndash;15\u0026ndash;30\u0026ndash;45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e2.8% (0.9%\u0026ndash;6.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 214px;\"\u003e\n \u003cp\u003e35,4 (15\u0026ndash;108)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7. Time-Specific Cortisol Cut-offs Based on the 2.5th Percentile\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTime Point\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eP2.5 Cut-off (\u0026micro;g/dL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ge; Cut-off (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFalse-Low (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e15 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e30 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e45 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e60 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpe","sideBox":"Learn more about [European Journal of Pediatrics](https://www.springer.com/journal/431)","snPcode":"431","submissionUrl":"https://submission.nature.com/new-submission/431/3","title":"European Journal of Pediatrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"low-dose acth stimulation test, pediatric adrenal insufficiency, cortisol kinetic, hypothalamic–pituitary–adrenal axis, diagnostic accuracy","lastPublishedDoi":"10.21203/rs.3.rs-8694799/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8694799/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eThe low-dose ACTH stimulation test (LDST) is widely used to evaluate hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis function in children; however, optimal cortisol sampling times and interpretation strategies remain controversial. Reliance on early or single time-point measurements may lead to false-positive diagnoses of adrenal insufficiency (AI).\u003c/p\u003e\u003ch2\u003eObjective:\u003c/h2\u003e \u003cp\u003eTo characterize the timing of peak cortisol responses during LDST in children without AI and to assess the incremental diagnostic contribution of extended sampling and time-specific cortisol thresholds.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eWe retrospectively analyzed 177 pediatric patients who underwent LDST for suspected central adrenal insufficiency at a single tertiary center. Serum cortisol was measured at baseline and at 15, 30, 45, and 60 minutes following intravenous administration of 1 \u0026micro;g ACTH. Adrenal sufficiency was defined as a peak cortisol\u0026thinsp;\u0026ge;\u0026thinsp;18 \u0026micro;g/dL. Peak timing distribution, basal predictors, incremental diagnostic contribution of additional time points, number needed to test (NNT), and false-positive rates using fixed versus time-specific cut-offs were evaluated.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003ePeak cortisol occurred most frequently at 15 minutes (48.6%), followed by 30 minutes (28.8%), baseline (10.7%), 45 minutes (9.0%), and 60 minutes (2.8%). Termination of testing at 30 minutes would have misclassified 11.9% of patients as insufficient despite normal later responses. Extension to 45 minutes provided meaningful diagnostic improvement, whereas routine extension to 60 minutes yielded only marginal additional benefit (NNT\u0026thinsp;=\u0026thinsp;30). Higher basal cortisol levels were independently associated with earlier peak responses (p\u0026thinsp;=\u0026thinsp;0.021), while demographic and auxological factors showed no association. Application of time-specific, percentile-based cortisol thresholds reduced false-positive classifications nearly five-fold at 30 minutes compared with a uniform 18 \u0026micro;g/dL cut-off.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eLDST cortisol responses in children show substantial interindividual variability in peak timing. Extension of sampling to 45 minutes and use of time-specific interpretation thresholds significantly improve diagnostic accuracy and reduce false-positive AI diagnoses in pediatric practice.\u003c/p\u003e","manuscriptTitle":"Avoiding False-Positive Adrenal Insufficiency Diagnoses in Children: Insights from Cortisol Kinetics During Pediatric Low-Dose ACTH Stimulation Test","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 14:48:08","doi":"10.21203/rs.3.rs-8694799/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-24T17:19:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T22:24:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-08T14:50:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113992225966549381999662047977133758462","date":"2026-03-29T00:24:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321388506886401222784285873696447945611","date":"2026-03-27T14:08:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280095598590797289459677457424298488179","date":"2026-03-17T15:41:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151021879848000517779839938410265709709","date":"2026-03-14T02:36:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-24T16:34:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136424000007418922979955929003241446933","date":"2026-02-18T03:02:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-11T20:18:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-10T01:44:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-10T00:36:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Pediatrics","date":"2026-01-25T20:30:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpe","sideBox":"Learn more about [European Journal of Pediatrics](https://www.springer.com/journal/431)","snPcode":"431","submissionUrl":"https://submission.nature.com/new-submission/431/3","title":"European Journal of Pediatrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e57009cc-2104-4a66-b535-8e9de295da8d","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T07:10:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 14:48:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8694799","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8694799","identity":"rs-8694799","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}