Journal of Endocrinological Investigation

https://doi.org/10.1007/s40618-023-02062-y

ORIGINAL ARTICLE

Morning serum cortisol role in the adrenal insufficiency diagnosis

with modern cortisol assays
A. F. D. Fragoso Perozo1,2,3   · R. Fontes2,3 · F. P. Lopes2 · P. B. Araújo1,2 · Y. Scrank2 · D. M. V. Gomes2 · A. B. Moraes4 ·
L. Vieira Neto1

Received: 28 October 2022 / Accepted: 7 March 2023

© The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE) 2023

Abstract
Purpose  To investigate the accuracy of cutoff values of the morning serum cortisol (MSC) using the cortisol stimulus test
(CST) insulin tolerance test (ITT) and 250 mcg short Synacthen test (SST) as the reference standard tests, to better define
its clinical role as a tool in the diagnostic investigation of adrenal insufficiency (AI) AI.
Methods  An observational study was conducted with a retrospective analysis of MSC in adult patients who had been submit-
ted to a CST to investigate AI between January 2014 and December 2020. The normal cortisol response (NR) to stimulation
was defined based on the cortisol assay.
Results  371 patients underwent CST for suspected AI, 121/371 patients (32.6%) were diagnosed with AI. ROC curve analysis
showed an area under the curve (AUC) for MSC of 0.75 (95% CI 0.69 – 0.80). The best MSC cutoff values to confirm AI
were < 3.65, < 2.35 and < 1.5 mcg/dL with specificity of 98%, 99%, and 100%, respectively. MSC > 12.35, > 14.2 and > 14.5
mcg/dL had sensitivity of 98%, 99%, and 100%, respectively, being the best cutoff values to exclude AI. Almost 25% of
patients undergoing CST for possible AI had MSC values between < 3.65 mcg/dL (6.7% of patients) and > 12.35 mcg/dL
(17.5% of patients), making the formal CST testing unnecessary if we consider these cutoff values.
Conclusion  With the most modern cortisol assays, MSC could be used as a diagnostic tool, with high accuracy to confirm
or exclude AI, avoiding unnecessary CST; thus, reducing expenses and safety risks during AI investigation.

Keywords  Morning serum cortisol · Adrenal insufficiency · Diagnosis · Cortisol assays · Diagnostic accuracy studies

Introduction

The diagnosis of adrenal insufficiency (AI) still presents

* A. F. D. Fragoso Perozo
andreafragosoperozo@gmail.com
1 erly diagnosed and treated. On the other hand, unnecessary
Department of Internal Medicine and Endocrine Unit,
Federal University of Rio de Janeiro, School of Medicine,
Clementino Fraga Filho University Hospital, 255 Professor
Rodolpho Paulo Rocco Street, ground floor, University City,
Rio de Janeiro, RJ ZC 21941‑617, Brazil
2
Diagnósticos da América SA (DASA), Rio de Janeiro, RJ,
Brazil
3
Instituto Estadual de Diabetes e Endocrinologia Luiz
Capriglione, Rio de Janeiro, Brazil
4
Department of Clinical Medicine and Endocrine Unit,
Federal Fluminense University, School of Medicine, Antônio
Pedro University Hospital, Niterói, RJ, Brazil
several challenges, even after more than 150 years since the
first description of this clinical syndrome in 1856 by Thomas
Addison [1].
An unequivocal diagnosis is essential since the syndrome
imposes a high morbidity and risk of death when not prop-
glucocorticoid replacement is far from being harmless [2–4].
As it is a rare disease, which implies a low pre-test prob-
ability, it is essential to have adequate diagnostic tools for
an accurate diagnosis [3, 5–7]. In addition to its rarity, its
insidious course and absence of specific clinical criteria with
predominantly nonspecific signs and symptoms make the
diagnostic suspicion of AI a challenge [6, 7].
The most recent guidelines recommend using a cortisol
stimulation test (CST) to exclude or confirm the condi-
tion in most cases. The dynamic tests considered reference

13
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standards to the AI diagnosis are the two CST called stand-
ard dose (250 mcg) short Synacthen test (SST) and the insu-
lin tolerance test (ITT) [8, 9]. Despite the ITT is no longer
considered the reference standard diagnostic test for primary
AI, replaced by the SST in the last consensus [8], the rea-
son for such change was due to the risks associated with
ITT rather than accuracy. Nevertheless, for the purpose of
our study (diagnostic accuracy study), we considered that,
as the ITT is the most available CST in our country and
also presents excellent accuracy, so both could be included
as reference standard diagnostic tests. The two CST also
present some limitations. SST depends on the administra-
tion of 250 mcg of cosyntropin, a drug not manufactured in
some countries and imported from Europe (Synacthen®) or
North America (Cortrosyn®) at a very high cost. Moreo-
ver, owing to its risks and contraindications, the ITT is no
longer the test of first choice for investigation in some cent-
ers, being restricted to young patients without contraindica-
tions [10–12].
To date, the use of morning serum cortisol (MSC) as an
AI diagnostic tool prior to CST does not have a well-defined
role or uniform cutoff values. In fact, previous studies and
the current consensus statements have emphasized limita-
tions of this tool and argued about how to use the MSC and
which cutoff points should be chosen to confirm or exclude
AI [8, 9, 13–21]. Nevertheless, to validate the MSC as a
diagnostic tool to avoid the need for CST in the AI diag-
nostic flowchart would have several advantages for patients
and providers, resulting in a more widely available and less
expensive approach.
Taking all these considerations together, we aimed to
investigate the accuracy of multiple cutoffs of the index test
MSC using both SST and ITT as the reference standard tests,
to better define its clinical role as a tool in the diagnostic
investigation of AI.
Materials and methods
Patients and eligibility criteria
An observational study was conducted with a retrospective
analysis of baseline MSC measurement in adult patients who
had been submitted to a CST, either the conventional 250
mcg SST or the ITT to investigate AI in one of the labo-
ratories of Diagnostics of America SA (DASA) in Rio de
Janeiro, Brazil, from January 2014 to December 2020. The
study was approved by the Research Ethics Committee of
the Clementino Fraga Filho University Hospital (HUCFF)
of the Federal University of Rio de Janeiro (UFRJ).
Inclusion criteria were adult patients (≥ 18 years), who
have been submitted to one of the two CST (SST or ITT).
CST was performed at the clinical discretion of the attending
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Journal of Endocrinological Investigation
physicians, according to their clinical suspicions of AI,
based on the signs and symptoms. We extract the patient
data from the Diagnostics of America SA (DASA) database
records. Unfortunately, we did not have access to details of
the clinical criteria that motivated the attending physicians
to request the CST because this information was not avail-
able in the database to which we had access. However, it was
an information that the physician responsible for carrying
out the CST had access and could define the dose of insulin
to be applied in the case of the ITT.
We excluded patients with database records of conditions
that could interfere in serum cortisol levels as follows: preg-
nancy, cirrhosis, hypoproteinemia, or use of oral contracep-
tives, menopausal hormone therapy or glucocorticoids. We
also excluded patients who had 17OH progesterone dosage
during the SST. After the exclusion criteria, 371 patients
were included in the study and classified according to the
CST response (Fig. 1).
Testing protocols
General protocol
All SST and ITT were performed by a well-trained team of
physicians and phlebotomists in one of DASA laboratories.
The tests were conducted with the starting time between
08:00 and 10:00 am. All patients had an intravenous scalp
inserted during the procedures to facilitate the samples col-
lection at the specific times depending on the CST.
The normal cortisol response to stimulation in either the
SST or the ITT was defined based on the cortisol assay [22],
in which an adequate response to SST or ITT for Beckman
Access Cortisol was a peak cortisol level > 408 nmol/L (14.8
mcg/dL) at any time after stimulus and for Roche Elecsys
Cortisol generation II was a peak cortisol level > 403 nmol/L
(14.6 mcg/dL) at any time after stimulus. Peak cortisol level
below these cutoffs was considered diagnostic of AI. There-
fore, based on the CST response the patients were classified
Fig. 1  Recruitment flowchart of patients. ITT, insulin tolerance test;
SST, 250 mcg short Synacthen test; AI, adrenal insufficiency patients;
NR, normal response patients
Journal of Endocrinological Investigation
as participants of 2 defined groups: The AI group and nor-
mal response (NR) group.
An MSC was always collected as the first time (time 0,
baseline cortisol) of the CST, since they were performed in
the morning (between 08:00 and 10:00 am), the proper time
for MSC collection. We also considered that the greater the
proximity between the day of the index test (MSC) and the
day of the reference standard (CST), the greater the chance
of patients being under the same conditions and with a lower
risk of bias.
Standard (250 mcg) short synacthen test (SST) protocol
The manufactured drug to be injected—Cosyntropin (Syn-
acthen® or Cortrosyn®) was always obtained from pharma-
cies that import medicines, and delivered to the laboratory
on the SST day, properly packaged, according to the manu-
facturer's instructions. The drug's expiration date and con-
servation status were verified by the physician responsible
for carrying out the test.
The total serum cortisol samples were collected before
(time 0 – baseline cortisol—MSC), and after 30 and 60 min
of the intravenous administration of 250 mcg cosyntropin
(Synacthen® or Cortrosyn®).
Insulin tolerance test (ITT) protocol
All patients had their weight checked on the day of the test.
The drug to be administered was regular insulin (RI) at a
dose of 0.05 to 0.15 IU/kg weight, intravenously. The dose
was defined by the physician responsible for carrying out
the test.
Usually, an initial dose of 0.075 to 0.1 IU/kg body weight
was applied. In case of suspected pan-hypopituitarism, how-
ever, the initial dose was lower, around 0.05 IU/kg weight.
On the other hand, in patients with signs of insulin resist-
ance (for example: obese, acromegalic) the dose of 0.10 or
even 0.15UI/kg was chosen. The stimulus to validate the test
was hypoglycemia (glycemia < 40 mg/dL). Capillary blood
glucose was monitored throughout the entire test. The first
capillary blood glucose measurement was 20 min after RI
administration, or earlier if the patient presented signs and/or
symptoms of hypoglycemia and then verified every 10 min
until blood glucose was < 40 mg/dL. Once it occurred, the
second timing of collection was performed. Hypoglycemia
was reversed with the administration of breakfast (including,
in general, juice from sweetened fruit and sweet biscuits)
and sugar. Hypoglycemia correction by oral administration
of carbohydrates ensures the maintenance of hypoglycemia
for about 10 min, which is important to validate the stimulus.
Samples were collected 4 times: Before insulin adminis-
tration (time 0 – baseline cortisol—MSC), then at the time
of hypoglycemia, 30 and 60 min after hypoglycemia, defined
by capillary blood glucose assessment < 40 mg/dL.
Biochemical evaluation
The total serum cortisol dosages were analyzed by Beckman
Access Cortisol (Beckman Coulter, City, CA) assay from
January 2014 to October 2019, and then by Roche Elecsys
Cortisol generation II (Roche Diagnostics, City, IN) assay,
since November 2019. The correlation between the two sys-
tems was good as shown from the method comparison data
(Pearson's correlation coefficient r = 0.991).
The Beckman Access Cortisol intra-assay imprecision (%
coefficient of variation) was 7.9% in the lower range (6 mcg/
dL) and 6.4% in the higher range (38 mcg/dL).
The Roche Elecsys Cortisol generation II intra-assay
imprecision was 2.8% in the lower range (1.4 mcg/dL) and
2.3% in the higher range (20 mcg/dL).
Statistical analysis
The statistical analyses were performed using SPSS ver-
sion 20.0 for MacOS (SPSS Inc., Chicago, IL, USA). In the
descriptive analysis, categorical variables were expressed as
counts (%); numerical variables were expressed as median
(minimum – maximum). The sample Kolmogorov–Smirnov
test was performed to analyze the distribution pattern of
numerical variables, and all presented non-normal distribu-
tion. The Mann–Whitney U test was performed to compare
the numerical variables between the two groups based on
the result of the CST performed (NR vs. AI). Correlations
between numerical variables were analyzed using Spear-
man’s test. The chi-square test or Fisher’s exact test was
applied to compare categorical variables, as appropriate.
A receiver operating characteristic (ROC) curve analysis
was used to assess the accuracy of MSC to confirm and to
exclude the diagnosis of AI. A p value < 0.05 was considered
statistically significant.
We also analyzed the sensitivity, specificity, posi-
tive, and negative predictive values, positive and negative
likelihood ratio of two MSC cutoff levels proposed in the
literature (8, 9, 13 – 20] to confirm AI (< 3, < 5 mcg/dL
– < 85, < 140 nmol/L, respectively) and two MSC cutoff
values proposed in the literature [9, 13, 14, 16, 17, 19–21]
to exclude AI (> 10, > 15 mcg/dL – > 275, > 415 nmol/L,
respectively) based on the CST result. For this accuracy
analysis, we created 2 × 2 tables, in which the patients with
AI were classified as AI by the CST and the patients without
AI were the patients classified as NR by the CST. Taking all
these in consideration, the higher the specificity, the better
the lower cutoff (confirmatory) values, and the higher the
sensitivity, the better the higher cutoff (exclusion) values
(Fig. 2).
13

Fig. 2  The 2 × 2 tables for the accuracy analyses of cutoff values of
Morning serum cortisol. MSC, morning serum cortisol; AI, adrenal
insufficiency CST, cortisol stimulus test; ITT, insulin tolerance test;
SST, 250 mcg short Synacthen test
We also used the ROC curve to propose better cutoff val-
ues to either confirm or exclude AI with higher accuracy
(higher specificity for the confirmatory cutoff values and
higher sensitivity for the excluding cutoff values).
Results
We included a total of 371 patients undergoing CST for sus-
pected AI. The accuracy analysis was initially performed
considering the total cohort (371 patients), regardless of the
cortisol assay, and later separately according to the cortisol
assay.
Total cohort results
Overall, 121/371 patients (32.6%) had AI and gender was
equally distributed in the AI group and the NR group (73.3%
of women in each group). The age and gender had no statisti-
cally significant difference between the groups (Table 1).
ROC curve analysis presented an area under curve
(AUC) for MSC of 0.75 (CI 95% 0.69 – 0.80) (Fig. 3). The
best cutoff values to confirm AI were MSC < 3.65, < 2.35,
and < 1.5 mcg/dL with specificity of 98%, 99%, and 100%,
respectively. The best cutoff values to exclude AI were
MSC > 12.35, > 14.2, and > 14.5 mcg/dL with sensitivity of
98%, 99%, and 100%, respectively (Tables 2 and 3).
Table 1  Baseline characteristics of adrenal insufficiency and normal
response groups
Characteristics Normal response Adrenal insuf-
group (n = 250) ficiency group
(n = 121)
Age (years) 41.5 (18–84) 41 (18–89)
(median/min–max)
Gender (M/F) (%) 26.7/ 73.3 26.7/ 73.3
M male, F female
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Journal of Endocrinological Investigation
Fig. 3  Receiver operating characteristic (ROC) curve for AI diagnosis
based on CST using MSC as the predictor. ROC, Receiver operating
characteristic; MSC, morning serum cortisol; AUC, area under curve;
Se, Sensitivity; Sp, Specificity
The sensitivity, specificity, positive, and negative pre-
dictive values, positive and negative likelihood ratios of
the two MSC cutoff values proposed in the literature to
confirm AI (< 3, < 5 mcg/dL – < 85, < 140 nmol/L, respec-
tively) and the MSC cutoff values with the best accuracy
assessed by the ROC curve in the present study to con-
firm AI (< 3.65, < 2.35, and < 1.5 mcg/dL – < 100, < 65,
and < 42 nmol/L, respectively) are in Table 2. The same
characteristics were evaluated for the performance of MSC
to exclude AI, evaluating the two cutoff values of MSC sug-
gested in the literature to exclude AI (> 10, > 15 mcg/dL
– > 275, > 415 nmol/L, respectively) and MSC cutoff val-
ues in the current study with the best accuracy by the ROC
curve to exclude AI (> 12.35, > 14.2, and > 14.5 mcg/dL
– > 340, > 392, and > 400 nmol/L, respectively), as shows
in Table 3.
Applying the lower MSC cutoff values suggested to con-
firm AI in our total cohort, we would avoid performing CST
from 11 patients (3% of the cohort) to 25 patients (6.7% of
the cohort) if we had chosen a MSC with 100% specificity
(< 1.5 mcg/dL) or a MSC with 98% specificity (< 3.65 mcg/
dL), respectively. Applying the higher MSC cutoff values
to exclude AI, we would avoid performing CST from 39
patients (10.5% of the cohort) to 65 patients (17.5% of the
cohort) if we had selected a MSC with 100% sensitivity
(> 14.5 mcg/dL) or a MSC with 98% sensitivity (> 12.35
mcg/dL), respectively.
p value Results according to the cortisol assay
The Beckman Access Cortisol was the assay performed in
0.998 most of the total cohort (342/371 patients, 92%) because
it was performed for all cortisol measurements between
0.994
January 2014 and October 2019. Overall, similar to the
total cohort, 110/342 patients (32.2%) had AI. The ROC
Journal of Endocrinological Investigation

Table 2  Performance of a Accuracy analyses MSC to confirm AI

morning serum cortisol cutoff
values proposed to confirm  < 1.5 mcg/dL  < 2.35 mcg/dL  < 3 mcg/dL  < 3.65 mcg/dL  < 5 mcg/dL
adrenal insufficiency
Patients (number, % of total) 11 (3%) 17 (4.6%) 20 (5.4%) 25 (6.7%) 50 (13.5%)
Specificity (%) 100 99 98.8 98 92.4
Sensitivity (%) 9.1 12.4 14 16.5 25.6
Positive predictive value
 In the study population (AI 100 88.2 85 80 62
prevalence = 32.6%)
Negative predictive value
 In the study population 69.4 70 70.4 70.8 72
(AI prevalence = 32.6%)
Positive likelihood ratio ∞ 15.5 11.7 8.3 3.4
Negative likelihood ratio 0.9 0.88 0.87 0.85 0.8

AI adrenal insufficiency, MSC morning serum cortisol

Table 3  Performance of a morning serum cortisol cutoff values proposed to exclude adrenal insufficiency
Accuracy analyses MSC to exclude AI
 ≥ 10 mcg/dL  ≥ 12.35 mcg/dL  ≥ 14.2 mcg/dL  ≥ 14.5 mcg/dL  ≥ 15 mcg/dL

Patients (number, % of total) 126 (34%) 65 (17.5%) 43 (11.6%) 39 (10.5%) 34 (9.2%)

Specificity (%) 4.3 2.5 1.7 1.6 1.36
Sensitivity (%) 85.1 98 99 100 100
Positive predictive value
 In the study population (AI 42 38.9 36.6 36.4 35.9
prevalence = 32.6%)
Negative predictive value
 In the study population 85.7 97 97.7 100 100
(AI prevalence = 32.6%)
Positive likelihood ratio 1.5 1.3 1.2 1.18 1.16
Negative likelihood ratio 0.34 0.07 0.05 0 0

AI adrenal insufficiency, MSC morning serum cortisol

curve analysis for the Beckman Access Cortisol assay also
presented an AUC for MSC of 0.75 (CI 95% 0.69 – 0.80)
(Fig. 4) and the best cutoff values to confirm and exclude AI
were the same as for the total cohort. Except for the lower
cutoff with specificity of 98%, which was achieved with a
MSC < 3.35 mcg/dL in this assay (Table 4; Fig. 4).
The Roche Elecsys Cortisol II has been the assay per-
formed since November 2019 and therefore was performed
in only 29 patients (8% of the total cohort). As it is the most
modern immunoassay currently used in the DASA labora-
tory, we decided to analyze its results separately. Further-
more, an excellent correlation was demonstrated between the
results of this assay and the Beckman assay, which justifies
analyzing them together. Overall, 8/29 patients (28%) had
AI. The ROC curve analysis for the Roche Elecsys Cortisol
II assay presented an AUC for MSC of 0.73 (95% CI 0.54
– 0.92) (Fig. 4) and, given the smaller cohort, we considered
only the best lower and upper cutoff values with specificity
of 100% and sensitivity of 100% that were achieved with
a MSC of < 2.35 mcg/dL and ≥ 9.7 mcg/dL, respectively.
(Table 4 and Fig. 4).
Discussion
The results of the present study showed that the MSC may
have excellent accuracy to confirm and exclude AI depend-
ing on the chosen cutoff values and if performed in a popula-
tion with clinical suspicion of AI, which means indication
of CST, as was the case in our cohort. The choice of a lower
cutoff value of < 1.5 or < 3.65 mcg/dL allowed confirming
AI with 100% or 98% specificity, which would have avoided
performing CST in 3% to 6.7% of patients, respectively. The
choice of a cutoff value > 14.5 or > 12.35 mcg/dL allowed
the exclusion of AI with 100% or 98% sensitivity, which
would have avoided performing CST in 10.5% to 17.5%

13

Fig. 4  Receiver operating char-
acteristic (ROC) curve for AI
diagnosis based on CST using
MSC as the predictor (A) for
Beckman Access Cortisol assay
(B) for Roche Elecsys Cortisol
II assay. ROC, Receiver operat-
ing characteristic
Table 4  Performance of MSC Accuracy analyses MSC to confirm AI
cutoffs to confirm or exclude AI
according to cortisol assays Beckman access corti-
sol assay
Cortisol (mcg/dL)  < 1.5  < 3.35  < 3.65  < 2.35
Specificity (%) 100 99
Sensitivity (%) 10 15.5
AI adrenal insufficiency, MSC morning serum cortisol
of patients, respectively. Therefore, a total of up to almost
25% of CST in our cohort would have been avoided if we
had considered the highly accurate MSC cutoff values as
suggested in the current study. These findings support the
existing evidence in the literature [13–21] that the MSC, an
inexpensive and widely available tool around the world, may
still be used as a diagnostic test in the flowchart investiga-
tion of AI.
This has practical implications, since the CST are tests
with limitations of practical applicability in some countries,
either for cost or safe reasons. Our results also confirm the
assay-dependent variability in cortisol assessment [22] and
therefore support the importance of emphasizing that the
data from the present study apply to the more modern cor-
tisol immunoassays Beckman Access cortisol assay or the
Roche Elecsys cortisol II assay.
The two reference tests in AI diagnostic flowchart do
not have wide applicability in clinical practice in some
countries, including Brazil. The SST is scarcely avail-
able, and it is an expensive test, depending on suitable
stations for performing dynamic tests with a well-trained
team and demanding the administration of 250 mcg of
cosyntropin, a drug imported from Europe (Synacthen®)
or North America (Cortrosyn®) at a very high cost (about
13
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MSC to exclude AI
Roche elecsys Beckman access cortisol Roche elecsys
cortisol II assay assay cortisol II
assay
 ≥ 12.35  ≥ 14.2  ≥ 14.5  ≥ 9.7
98 100 25 17 16 38
17.3 12.5 98 99 100 100
US$100–300). Similar cost has been reported in other
studies [18]. The ITT is a laborious test and with limited
applicability because of safety issues with many contrain-
dications and risks due to hypoglycemia. Therefore, the
ITT is no longer the first choice for investigation in many
centers, being restricted to young patients without con-
traindications [10–12].
In our study, we included a large cohort of adult popula-
tion who underwent to one of the two reference tests for
suspected AI and had cortisol measurements performed by
modern cortisol immunoassays. In this context, we found
that MSC cutoff values < 1.5–3.65 mcg/dL (42–100 nmol/L)
had excellent performance to confirm AI (100–98% speci-
ficity) when applied in the appropriate context of clini-
cal suspicion of AI. In contrast, a MSC cutoff < 5 mcg/dL
(140 nmol/L) recommended by one of the recent consensus
statements [8] did not had a good performance to confirm
AI in our study, with a specificity < 95%, not being reliable
to be used in our population due to the risk of many false
positives, which would lead to unnecessary glucocorticoid
treatments. When we analyzed the data from upper MSC
cutoffs, values > 12.35–14.5 mcg/dL (340–400 nmol/L) had
excellent performance to exclude AI (98–100% sensitivity).
Other studies [13–20] that evaluated the role of MSC in the
Journal of Endocrinological Investigation
diagnosis of AI showed similar results to ours and details are
depicted in the Table 5.
Most importantly, almost 25% of patients in our
study undergoing CST for possible AI had MSC values
between < 3.65 mcg/dL (6.7% of patients) and > 12.35
mcg/dL (17.5% of patients), making the formal CST testing
unnecessary if we consider these cutoff values. Several pre-
vious studies [13, 17–20] also calculated the percentage of
patients who would not have to undergo CST if the proposed
MSC cutoff values were considered, which ranged from 21
to 57% as depicted in the Table 5.
In our study, we also calculated the positive and negative
likelihood ratios of the best MSC cutoff values. According
to Simundic et al., the positive likelihood ratio (PLR) is the
best indicator for the confirmatory capacity of a diagnos-
tic test and the negative likelihood ratio (NLR) is the best
indicator for its ability to exclude a disease. The higher the
PLR the more it can confirm and the lower the NLR the
more it is able to exclude the disease. Excellent diagnos-
tic tests present PLR > 10 and NLR < 0.1 [23]. The lower
MSC cutoff values < 3 mcg/dL (85 nmol/L), < 2.35 mcg/
dL (65 nmol/L), and < 1.5 mcg/dL (42 nmol/L), had PLR
of 11.7, 15.5, and infinite, respectively. This means that a
MSC < 3 mcg/dL is more than 11 times more likely in peo-
ple with AI than without AI and an MSC < 1.5 mcg/dL will
never happen without AI. On the other hand, upper MSC
cutoff values > 12.35 mcg/dL (340 nmol/L), > 14.2 mcg/
dL (392  nmol/L), and > 14.5 mcg/dL (400  nmol/L) had
NLR of 0.07, 0.05, and 0, respectively. Which means that
a MSC > 12.35 mcg/dL is more than 14 times more likely
in people without AI than with AI and MSC > 14.5 mcg/dL
will never happen in a person with AI.
This study aimed to assess the accuracy of MSC and
propose lower MSC cutoffs to confirm AI and higher MSC
cutoffs to exclude it in the context of clinical suspicion. We
were able to compare the MSC index test with the two refer-
ence standard tests (ITT and SST) and, in addition, we were
careful to consider the post-stimulus cortisol cutoff value
specific for the cortisol assay [22], as recommended by the
most recent consensus [8, 9]. Sbardella et al. also proposed
MSC cutoff values that could predict AI for three different
cortisol immunoassays, but they did not include either the
Beckman Access cortisol assay or the Roche Elecsys cortisol
II assay [18].
In the recent years, more specific, modern cortisol immuno-
assays with less cross-reactivity to other endogenous steroids
have replaced older, less specific assays. Cortisol concentra-
tions are approximately 20% lower in these more specific
assays [22, 24]. In the separate analysis according to the cor-
tisol assay used, the MSC showed good clinical utility in both
assays, as the AUC of the ROC curve remained between 0.7
and 0.8 [23]. The Roche Cortisol II assay is the most mod-
ern cortisol assay with less cross-reactivity to other steroids
and possibly because of this, we found lower MSC values that
allowed us to exclude AI with 100% sensitivity (MSC ≥ 9.7
mcg/dL).
We recognize some limitations in our study. First, its obser-
vational retrospective cross-sectional design. Thus, our results
need to be validated in a prospective study. Second, CST was
performed at the clinical discretion of the attending physicians,
according to their clinical suspicions of AI, based on signs and
symptoms. We did not have access to clinical details that raised
the suspicion of AI. However, we know that physicians who
request CST in our country are mostly endocrinologists, which
makes us value the suspicion. We also did not have access
to clinical data other than those registered in the database,
so there was limited information on potentially confounding
comorbidities. For the same reason, a mixed population with
clinical suspicions of primary or secondary AI was included.
On the one hand, this restricts generalizability. On the other
hand, the results are valid for subgroups and therefore repre-
sent daily practice. Another limitation is due the type of this
study: this is a diagnostic accuracy study in which a diagnostic
test (in this case, the MSC) is evaluated against the standard
clinical reference diagnostic test (in this case, the CST). For
this reason, we considered the result of the CST to be clearly
true (whether it excluded or confirmed AI). Nevertheless,
some cases of suspected secondary AI such as partial AI, the
Synacthen test at a dose of 250mcg may be a supraphysiologi-
cal stimulation that results in a false negative result and, in fact,
would not allow us to rule out AI. Therefore, we reinforce that
it is mandatory to consider the pre-test probability to interpret
the result of a diagnostic test. In case of suspected secondary
AI, knowledge of pre-test probability and accurate clinical
judgment are essential to rule out AI in case of MSC > 14.5
mcg/dL or CST with normal cortisol response.
Finally, the small number of patients (51/371, 14%) at
the 2 extremes of classically suggested cutoff points (< 3
mcg/dL and > 15 mcg/dL) makes us suppose that the pre-
test probability was neither so high nor so low. We recog-
nize that some specialists, in a high clinical suspicion and
MSC < 3 mcg/dL already confirm AI (given the difficulties
of CST applicability). On the other hand, in a low clinical
suspicion and MSC > 15 mcg/dL they already exclude it.
This could also configure a limitation of our study, but in
our understanding, it values the data even more since our
results would apply precisely to the AI suspected population
that in daily practice would end up in a CST due to doubtful
pre-test probability.
Conclusions
The MSC is a widely available, safe, simple, and low-cost
test, and therefore has good applicability in clinical practice.
With the most modern cortisol assays (Beckman Access and
13


Table 5  Previous studies that evaluated the use of morning serum cortisol in the diagnosis of adrenal insufficiency
Author (year) Study design Number of par- Reference standard exam Serum cortisol Suggested morning serum cortisol (MSC) cutoff values % of patients who
ticipants assay would not have

13
Type of CST Indication MSC (mcg/dL) MSC (mcg/dL) Accuracy to undergo CST
to confirm AI to exclude AI if MSC cutoff
suggested was
considered

Le Roux et al. Prospective 210 SST Clinical suspi- ELISA (Roche)  < 3.5  > 18 MSC > 18 21%
[13] cions of AIA (Se = 100%)
(Mixed popula- MSC < 3.5
tion) (Sp = 100%)
Lopez Schmidt Prospective 53 ITT Clinical sus- CLIA (ADVIA  < 3.5  > 10 MSC > 10 Not informed
et al. [14] picions of Centaur) (Se = 100%)
secondary AI MSC < 3.5
(Sp = 100%)
Kazlauskaite Meta-Analysis 635 (12 studies) ITT Clinical sus- Several  < 5  > 13 Not informed Not informed
et al. [15] picions of
secondary AI
Varadhan et al. Observational 346 SST Clinical suspi- Siemens Immu- 1) < 5 (low 1) > 14.5 (inde- MSC > 14.5 43%
[17] retrospective cions of AI lite 2000 degree of pendent of (Se = 100%)
(Mixed popula- suspicion) the degree of MSC < 5
tion) 2) < 6 (high suspicion) (Sp = 100%)
degree of
suspicion)
Sbardella et al. Observational (1) 1019 (Advia SST Clinical suspi- (1) CLIA 1) < 2 (Centaur) 1) > 13 (Centaur) 1) MSC > 13 1) 57%
[18] retrospective Centaur—Sie- cions of AI (ADVIA Cen- 2) < 3 (Architect) 2) > 12 (Archi- (Se = 100%) 2) 35%
mens) (Mixed popula- taur) 3) < 3.5 (Roche) tect) and MSC < 2 3) 28%
(2) 449 Architect tion) (2) Architect 3) > 18 (Roche) (Sp = 100%)
-Abbott) (Abbott) (3) 2) MSC > 12
(3) 2050 (Roche ECLIA (Roche (Se = 100%)
Modular) Modular) and MSC < 3
(Sp = 100%)
3) MSC > 18
(Se = 100%)
and MSC < 3.5
(Sp = 99%)
Struja et al. [19] Observational 804 SST and Syn- Clinical suspi- CLIA (ADVIA  < 3.5  > 16 MSC < 3.5 38%
retrospective acthen 1mcg cions of AI Centaur) (Sp = 9.2%)
(Mixed popula- MSC > 16
tion) (Se = 98.4%)
Manosroi et al. Observational 416 SST and Syn- Clinical suspi- ECLIA (Roche)  < 3.3  > 14 MSC < 3.3 30%
[20] retrospective acthen 1mcg cions of AI (Sp = 99.7%)
(Mixed popula- MSC > 14
tion) (Se = 98.9%)
Journal of Endocrinological Investigation
 Journal of Endocrinological Investigation

Roche Elecsys II), in a context of clinical suspicion of AI, a

AI adrenal insufficiency, CST cortisol stimulus test, CLIA chemiluminescence assay, ECLIA electrochemiluminescence assay, ELISA enzyme-linked immunosorbent assay, ITT insulin tolerance
% of patients who MSC < 1.5 mcg/dL, < 2.35 mcg/dL and < 3.65 mcg/dL might
to undergo CST
would not have

suggested was
if MSC cutoff

Not informed
confirm AI with high specificity and a MSC > 12.35 mcg/

considered
dL, > 14.2 mcg/dL and > 14.5 mcg/dL might exclude AI with
high sensitivity, avoiding unnecessary CST; thus reducing
expenses and safety risks during AI investigation. However,
it is important to emphasize that both MSC and CST are
Suggested morning serum cortisol (MSC) cutoff values

(Se = 98.6%)

(Sp = 100%)
complementary exams and, therefore, clinical suspicion
MSC < 2 must always be valued in the interpretation of their results.
MSC > 10
Accuracy

Acknowledgements  This study was made possible thanks to grants

from the Brazilian National Council for Scientific and Technological
Development (CNPq) to LVN.
MSC (mcg/dL)
to exclude AI

Author contributions  All authors contributed to the study conception

and design. Material preparation, data collection and analysis were
performed by AFDFP and LVN. The first draft of the manuscript was
 > 10

written by AFDFP and all authors commented on previous versions of

the manuscript. All authors read and approved the final manuscript.
MSC (mcg/dL)
to confirm AI

Data availability  The datasets generated during and/or analysed dur-

ing the current study are available from the corresponding author on
reasonable request.
 < 2

Declarations 
Serum cortisol

Conflict of interest  The authors report no conflict of interest in this

work.
(Abbott)
Architect

Ethical approval  All procedures involving human participants were

assay

performed in accordance with the ethical standards of the Institutional

test, MSC morning serum cortisol, Se sensibility, Sp specificity, SST 250 mcg short Synacthen test

Ethics Committee at which the studies were conducted. This paper does
(Mixed popula-

not contain any studies with animals performed by any of the authors.
Clinical suspi-
cions of AI

Informed consent  For this type of study, formal consent is not required.
Indication

tion)
Reference standard exam

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