Original Article
Clinical validation for the aldosterone-to-renin ratio
and aldosterone suppression testing using simultaneous
fully automated chemiluminescence immunoassays
Jenny Manolopoulou a, Evelyn Fischer b, Anna Dietz b, Sven Diederich c, Daniel Holmes d,
Riia Junnila b, Philipp Grimminger b, Martin Reincke b, Alberto Morganti e, and Martin Bidlingmaier b
Background: As larger numbers of hypertensive patients
are screened for primary aldosteronism with the
aldosterone-to-renin ratio (ARR), automated analyzers
present a practical solution for many laboratories. We
report the method-specific ARR cutoff determined with
direct, automated chemiluminescence immunoassays
allowing the simultaneous measurement of plasma
aldosterone concentrations (PACs) and plasma renin
concentrations (PRCs).
Methods: Method comparisons to commonly employed
assays and tandem mass spectrometry were undertaken.
Patients were previously diagnosed based on the local ARR
cutoff of 1.2 (ng/dl)/(mIU/ml) in samples collected in
upright seated position. Lack of aldosterone suppression in
response to salt load to less than 5 ng/dl confirmed
primary aldosteronism. For the new assays, the optimal
ARR cutoff was established in 152 patients with essential
hypertension, 93 with primary aldosteronism and 147
normotensive patients. Aldosterone suppression was
assessed in 73 essential hypertensive and 46 primary
aldosteronism patients.
Results: PAC and PRC were significantly correlated to
values determined with currently available methods
(P < 0.001). In patients with primary aldosteronism,
patients with essential hypertension and controls, mean
(95% confidence interval) PAC was 28.4 (25.4–31.8), 6.4
(5.9–6.9) and 6.2 (5.6–6.9) ng/dl, respectively. In the same
groups, PRC was 6.6 (5.6–7.7), 12.9 (11.2–14.8) and 26.5
(22.2–31.5) mIU/ml. An ARR cutoff of 1.12 provided
98.9% sensitivity and 78.9% specificity. Employing the
new assay aldosterone suppression confirmed the
diagnosis of primary aldosteronism and essential
hypertension using the cutoff of 5 ng/dl.
Conclusion: Our data demonstrate that the new assays
present a convenient alternative for the measurement of
PAC and PRC on a single automated analyzer. Availability
of these simultaneous assays should facilitate screening
and diagnosis of primary aldosteronism.
Keywords: aldosterone, aldosterone-to-renin ratio,
automated immunoassay, confirmatory testing, cutoff,
primary aldosteronism, renin
2500 www.jhypertension.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Abbreviations: APA, aldosterone-producing adenoma;
ARR, aldosterone-to-renin ratio; BAH, bilateral adrenal
hyperplasia; IDMS, isotope dilution mass spectrometry;
IRMA, immunoradiometric assay; LC-MS/MS, liquid
chromatography tandem mass spectrometry; PAC, plasma
aldosterone concentration; PRA, plasma renin activity; PRC,
plasma renin concentration; RIA, radioimmunoassay; ROC,
receiver-operating characteristic
INTRODUCTION
T
he measurement of plasma aldosterone and renin
has become more common in recent years since the
recognition that primary aldosteronism is a more
frequent cause of hypertension than previously thought,
estimated at more than 10% of the hypertensive population
[1–5]. The plasma aldosterone-to-plasma renin ratio is
recommended to screen for primary aldosteronism in
high-prevalence patient groups, as concurrent measure-
ment of aldosterone and renin is a more sensitive indicator
than either marker alone and a definitive diagnosis can
lead to a cure [6,7]. Following a positive aldosterone-to-
renin ratio (ARR) screening outcome, measurement of
aldosterone during a saline infusion test is one of the most
widely used confirmatory tests to establish the diagnosis
[8,9].
Traditionally, aldosterone was most frequently deter-
mined by use of radioimmunoassay (RIA), with few pub-
lished results of automated chemiluminescence methods
Journal of Hypertension 2015, 33:2500–2511
a
Immunodiagnostic Systems Ltd, Boldon, Tyne and Wear, United Kingdom,
b
Medizinische Klinik und Poliklinik IV, Klinikum der Ludwig Maximilians Universitaet,
Munich, cEndokrinologikum Berlin, Friedrichstr, Berlin, Germany, dDepartment of
Pathology and Laboratory Medicine, The University of British Columbia, Vancouver,
British Columbia, Canada and eCentro di Fisiologia Clinica e Ipertensione, Università di
Milano, Ospedale Policlinico,Via Francesco Sforza, Milano, Italy
Correspondence to Martin Bidlingmaier, Endokrinologisches Labor, Medizinische
Klinik und Poliklinik IV, Klinikum der Universität München, Ziemssenstr. 1, 80336
Munich, Germany. Tel: +49 89 44005 2277; fax: +49 89 44005 4457; e-mail:
martin.bidlingmaier@med.uni-muenchen.de
Received 9 March 2015 Revised 23 July 2015 Accepted 23 July 2015
J Hypertens 33:2500–2511 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights
reserved.
DOI:10.1097/HJH.0000000000000727
Volume 33
Number 12
December 2015
 Automated aldosterone and renin assays, ARR and sodium suppression cutoffs
[10]. Most commercially available immunoassays use
polyclonal antisera with various affinities and specificities
to this steroid hormone. As is often the case with immuno-
assays, these differences along with discrepancies in cali-
bration lead to a generally accepted, poor interlaboratory
reproducibility of aldosterone measurements [11,12]. This
makes it difficult to define cutoff values for screening and
confirmatory test methods. Automated methods for measure-
ment of plasma renin concentrations (PRCs) have also
become available in recent years [10,13], replacing the man-
ual immunoradiometric assays (IRMAs) and the more labour-
intensive method of plasma renin activity (PRA). PRA does
still remain a well established approach, preferred by many
as it offers advantages such as the extension of incubation
times to increase sensitivity down to less than 1 ng/ml/h,
important in the low renin state of primary aldosteronism
[14]. However, a recent study has shown better interlabor-
atory agreement for immunoreactive PRC levels as compared
with enzymatic PRA [15]. In addition, PRC is simpler, faster
and calibrated to an International Reference Standard pre-
paration.
As significant variability exists between assays for
aldosterone and renin, especially at low concentrations,
it is recommended that laboratories establish their own
reference ranges [16,17]. Moreover, method-specific cutoffs
should be established for the ARR and confirmatory testing
[18]. The published recommended cutoff for the ARR with
plasma aldosterone concentrations (PACs) in ng/dl and
PRC in mIU/ml is 3.7 (2.4–4.9) [8]. However, there is no
clear evidence that this cutoff is the optimal to achieve the
desirable balance between sensitivity and specificity for this
screening test. Furthermore, we have no information about
which assays were actually used to assign the suggested
cutoffs.
In this study we present data on the clinical validation of
two recently developed chemiluminescence assays for PAC
and PRC and provide the ARR in normotensive, essential
hypertensive and primary aldosteronism patients. We
report a proposed cutoff using the ARR to discriminate
between primary aldosteronism and essential hyperten-
sives with optimized sensitivity while maintaining satisfac-
tory specificity. In addition, we demonstrate the cutoff at
which aldosterone suppression in response to saline infu-
sion can differentiate primary and non-primary aldosteron-
ism patients, as compared with a commonly employed
method. The availability of these cutoffs as well as PAC
and PRC from a single sample and on a single analyzer
could provide a faster and more practical tool for larger
scale screening studies across multiple centres.
PATIENT AND METHODS
Subjects and samples
All studies were approved by the local Institutional Review
Board and informed consent was obtained from partici-
pants as appropriate. To evaluate the new assays, we made
use of samples from hypertensive patients recruited in our
unit between 2010 and 2013 within the German Conn’s
Registry – Else Kröner-Fresenius Hyperaldosteronismus
Registry (www.conn-register.de). Case detection and sub-
type identification of primary aldosteronism, as described
Journal of Hypertension
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
before [17,19–24], were carried out according to the institu-
tional guidelines and in accordance with the Endocrine
Society Guidelines [8]. For all diagnostic procedures, anti-
hypertensive medications were withheld whenever clini-
cally possible or switched to drugs having little or no effect
on plasma renin and aldosterone levels (e.g. calcium chan-
nel blockers and a-blockers). Patients requiring mineralo-
corticoid receptor antagonists (MRAs) and b-blockers were
excluded. If present, hypokalemia was controlled by oral
supplementation. Biochemical diagnostic workup was
done using our laboratory’s routine methods [Coat-A-Count
Aldosterone RIA (Siemens Healthcare Diagnostics Ltd.,
Camberley, UK) and Liaison Direct Renin (DiaSorin
Deutschland GmbH, Dietzenbach, Germany)]. Primary
aldosteronism was defined by elevated ARR [cutoff 1.2
(aldosterone ng/dl)/(renin mIU/ml)] and a lack of suppres-
sion of aldosterone after the infusion of 2000 ml saline (0.9%
NaCl) over 4 h (cutoff 5 ng/dl). Subsequent subtype differ-
entiation was performed, and the diagnosis of aldosterone-
producing adenoma (APA) was made based on the follow-
ing criteria: biochemical diagnosis of primary aldosteron-
ism, lateralization of aldosterone production during adrenal
vein sampling (AVS), histological confirmation of adreno-
cortical adenomas and normalization of hypokalaemia,
hypertension and ARR after adrenalectomy [20]. Successful
selective AVS was assumed with a selectivity index (adrenal
vein cortisol to peripheral cortisol) of at least 2 on both the
sides. The lateralization ratio was calculated as the highest
adrenal aldosterone-to-cortisol ratio divided by the lowest
one. We used a cutoff of 4 to categorize the patients as
lateralized or not. Essential hypertension was diagnosed if
primary aldosteronism or other causes of secondary arterial
hypertension had been excluded. Blood samples for the
measurement of aldosterone and renin concentrations were
collected between 0800 and 1200 h in an upright seating
position from an antecubital vein into potassium-EDTA
plasma tubes. All samples were spun down at room
temperature and then rapidly frozen and stored at 208C
or less until the time of measure. The only additional
selection criterion for samples to be used in the current
study was the availability of sufficient quantities of plasma
to perform the parallel assessment by different laboratory
methods.
Samples used to evaluate the aldosterone-to-renin
ratio using the new assays
Samples from 93 patients [30 women, 63 men; average age
54.6 years (26–82)], who underwent saline infusion sup-
pression testing and were confirmed to have primary
aldosteronism as described earlier, could be included.
The ARR values from these confirmed cases were used in
the receiver-operating characteristic (ROC) analysis com-
parison with ARR from essential hypertensive patients
(n ¼ 152) to define the optimum cutoff to be applied with
the newly developed iSYS assays. All of the 152 essential
hypertensive cases who had a positive ARR above the 1.2
cutoff (n ¼ 38, 23.5%) had aldosterone suppression testing
demonstrating normal suppression (<5 ng/dl). Seventy-
nine of the 93 confirmed primary aldosteronism cases
(see flow diagram in Fig. 1) underwent successful AVS to
have a differential diagnosis [APA; n ¼ 46, 18 women, 28
www.jhypertension.com 2501
Manolopoulou et al.

Primary aldosteronism patients (93)

Positive ARR screening, confirmed with saline-infusion suppression test

AVS unsuccessful or refused AVS succssful

(14) (79)

Bilateral adrenal
Aldosterone- producing
hyperplasia
adenoma (46)
(33)

Adrenalectomy
(42)

Mineralocorticoid Mineralocorticoid Mineralocorticoid

receptor antagonist receptor antagonist receptor antagonist
(7) (3) (27)

Antihypertensive Anti-hypertensive Anti-hypertensive

medication* medication* medication*
(7) (1) (6)

FIGURE 1 Primary aldosteronism cohort: subtype differentiation and subsequent treatments in patients with samples included in the evaluation of the aldosterone-to-renin
ratio (ARR) using the new assays. AVS, adrenal vein sampling. Antihypertensive medications were not given at the time of screening. For details, refer to section heading
‘Samples used to evaluate the aldosterone-to-renin ratio using the new assays’.

men, aged 36–72 years; bilateral adrenal hyperplasia
(BAH); n ¼ 33, eight women, 25 men, aged 35–81 years].
For the remaining 14 cases, AVS was either unsuccessful or
refused by the patient. Adrenalectomy with histological
confirmation was subsequently performed on 42 of the
APA patients. After diagnosis and subtype differentiation,
three of the APA patients, 27 of the BAH patients and seven
of the patients without AVS went on to take an MRA with
improved control of blood pressure. The remaining
patients did not tolerate or refused to take MRAs and were
handled with standard antihypertensive medication. In
addition to the primary aldosteronism and essential hyper-
tensive patients, we also determined ARR in a group of 147
normotensive patients investigated under the same con-
ditions [two (1.4%) with ARR > 1.2).
Samples used to evaluate the cutoff for
confirmatory suppression testing using the new
assays
In a subset of 46 confirmed primary aldosteronism patients
who underwent aldosterone suppression testing, there was
sufficient plasma remaining after measuring aldosterone
with the Siemens RIA to additionally measure aldosterone
with the iSYS method. The same re-evaluation was per-
formed in available samples from 73 hypertensive patients
who were tested with aldosterone suppression and con-
firmed negative for primary aldosteronism.
Assay methodology, validation and
characterization
Limits of detection (LoD) and quantification (LoQ) [25],
precision [26], linearity [27] and recovery were determined
for the two iSYS assays according to the Clinical and
Laboratory Standards Institute recommendations or
modifications thereof. The potential impact of high
amounts of haemoglobin (Lampire, Pipersville, Pennsylva-
nia, USA), bilirubin (Merck Millipore, Middlesex, UK) and
triglycerides (Sigma-Aldrich, Cambridge, UK) were also
analysed according to the Clinical and Laboratory Standards
Institute guidelines for interference testing [28]. The com-
parability of the results obtained on different instruments
was also investigated. Cross-reactivity to endogenous and
synthetic steroids with close structural homology was tested
in the aldosterone assay using the Abraham’s method [29]
(Table 1). Further methodological details are given in the
Supplemental text, http://links.lww.com/HJH/A520.
iSYS aldosterone
A highly specific monoclonal antibody (A2E11) [30] is used
in the iSYS aldosterone competitive immunoassay. A first
incubation of 200 ml of the sample with the biotinylated
anti-aldosterone antibody is followed by a second short
incubation with an aldosterone-acridinium-conjugate
tracer. The final step includes the addition of streptavi-
din-coupled magnetic particles. The particles are captured
using a magnet and undergo a series of three washing
cycles to separate the bound from any unbound analyte
in the sample. The addition of trigger reagents, NaOH and
H2O2, causes light to be emitted by the acridinium label.
The aldosterone assay has been calibrated against the
accredited (DIN EN ISO/IEC 17043) Reference Institute
for Bioanalytics (RfB; Bonn, Germany) isotope dilution
mass spectrometry (IDMS) method.
iSYS direct renin
A set of two monoclonal antibodies raised against human
renin, a biotinylated capture antibody (REN3E8) and an
acridinium-labelled detection antibody (REN116), were

2502 www.jhypertension.com Volume 33
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 Automated aldosterone and renin assays, ARR and sodium suppression cutoffs

TABLE 1. IDS-iSYS aldosterone and direct renin assay characteristics

IDS-iSYS aldosterone IDS-iSYS direct renin
b
Assay range (analytical sensitivity–upper limit) 2.0–132 ng/dl (55.4–3656 pmol/l) 1.8–550 mIU/ml (1.1–330 pg/ml) conversion factor 1.67
Standardization methodb Isotope dilution mass spectrometry (Reference Traceable to the NIBSC IS 68/356
Institute for Bioanalytics)
Limit of detectiona 3.3 ng/dl Analytical sensitivity 0.50 mU/ml
Limit of quantitationa 3.8 ng/dl 5.0 mU/ml
Intraassaya 6.4 ng/dl (10.1%) to 142.1 ng/dl (4%) 17.3 mU/ml (3.5%) to 432 mU/ml (2.1%)
Interassaya 5.9 ng/dl (12%) to 70.9 ng/dl (3%) 17.3 mU/ml (6.5%) to 432 mU/ml (3.4%)
Linearitya 4.4 ng/dl (102%) to 121.4 ng/dl (105%) 83.9 mU/ml (104.9%) to 747 mU/ml (99.9%)
Recoveryb 80–107% (3.8–30.6 ng/dl) 93–117% (68.3–181.2 mU/ml)
Specificityb Androstenedionea (30 mg/ml), <0.001% Prorenin (0.2 ng/ml), 2.8%
Androsterone (30 mg/ml), <0.001% b-2-Microglobulin (50 mg/ml), 0%
Cortisone (30 mg/ml), <0.001% Cathepsin B (0.1 U/ml), 3%
Corticosterone (30 mg/ml), <0.001% Cathepsin D (0.5 U/ml), 0%
18-OH Corticosterone, 10 mg/ml, 0.2% Captopril (50 mg/ml), 3%
Dexamethasone (30 mg/ml), <0.001% Renitec (enalapril maleate) (50 mg/ml), 2%
11-Deoxycortisol (30 mg/ml), <0.001% Loxen (nicardipine HCl) (50 mg/ml), 0%
Doxazosin mesylate (10 mg/ml), <0.001% Lasix (furosemide) (50 mg/ml), 0%
DHEAa (30 mg/ml), <0.001% Trypsin (1.5 mg/ml), 1%
b-Estradiol (30 mg/ml), <0.001% Plasmin (100 mg/ml), 0%
Estrone (30 mg/ml), <0.001%
Fludrocortisone (30 mg/ml), <0.001%
Hydrocortisone (30 mg/ml), <0.001%
Prazosin HCl (10 mg/ml), <0.001%
Prednisolone (30 mg/ml), <0.001%
Prednisone (30 mg/ml), <0.001%
Pregnenolone (30 mg/ml), <0.001%
Progesterone (30 mg/ml), <0.001%
21-OH Progesterone (30 mg/ml), <0.001%
Spironolactone (30 mg/ml), <0.001%
3a, 5b-Tetrahydroaldosterone (10 mg/ml), 3.1%
Testosteronea (30 mg/ml), <0.001%
Verapamil HCl (30 mg/ml), <0.001%
a
As obtained at the Munich laboratory.
b
As provided by the manufacturer.

selected for use in the automated two-site immunoassay
recognizing two different epitopes on the renin molecule.
REN3E8 recognizes both renin and prorenin whereas
REN116 is directed against the active site of renin, as shown
by enzyme inhibition studies [31,32]. One hundred and
ninety microlitres of sample are incubated with the acridi-
nium-labelled antibody, the biotinylated antibody and two
buffer reagents. Streptavidin-coated magnetic particles are
then added and incubated before being ‘captured’ by a
magnet and washed to remove any unbound material.
Trigger solutions activate the chemiluminescence reaction
whereby signal is directly proportional to the amount of
renin present in the sample. The renin assay has been
calibrated using the National Institute for Biological Stand-
ards and Control (NIBSC) International Standard reference
preparation 68/356.
Comparison to existing methods
The two iSYS assays were compared to existing assays for
measurement of aldosterone and renin by parallel assess-
ment of EDTA plasma samples. Commercial assays were
performed according to the respective manufacturer’s
instructions for use. The PAC comparison studies included
correlation to the Coat-A-Count Aldosterone RIA (Siemens
Healthcare Diagnostics Ltd., Camberley, UK) carried out at
the University of Munich (n ¼ 275) and correlation to ring-
trial samples from the RfB (Bonn, Germany) IDMS method
(n ¼ 15) carried out at Immunodiagnostic Systems Ltd
(IDS), Boldon, Tyne and Wear, UK. In addition, samples
(n ¼ 75) with aldosterone values using a published liquid
chromatography tandem mass spectrometry (LC-MS/MS)
method [33] from St. Paul’s Hospital, Vancouver, British
Columbia, Canada, were measured at IDS Boldon for
comparison to the iSYS. The PRC direct assay was com-
pared with the Renin III generation (Cis-bio Bioassays,
Codolet, France) (n ¼ 164) and the Liaison Direct Renin
(DiaSorin Deutschland GmbH, Dietzenbach, Germany)
(n ¼ 132). A correlation of the PRC iSYS method to PRA
according to the method of Poulsen and Jorgensen [34] was
carried out in 33 samples at St. Paul’s Hospital, Vancouver.
Statistics
Method comparison between the various aldosterone and
renin (or PRA) methods was assessed using Deming
regression and weighted Deming regression in the case
of PRA using the cp-R interface to the R statistical program-
ming language [35]. Results below a detection level were
set to each assay’s respective analytical sensitivity value
for comparative purposes. Percentage differences were
assessed with Bland–Altman analysis [mean (standard
deviation, SD) bias, 95% confidence interval (CI)] [35].
Geometric mean levels (95% CI) of PAC and PRC of the

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Manolopoulou et al.

TABLE 2. Plasma aldosterone and plasma renin concentrations in patients with primary aldosteronism, with essential hypertension and in
control patients measured with the iSYS and two commercially available methods
Plasma aldosterone concentration (PAC; ng/dl) Plasma renin concentration (PRC; mIU/ml)
PA EH Control PA EH Control
iSYS aldosterone 28.4 6.4 6.2 iSYS renin 6.6 12.9 26.5
95% CI 25.4–31.8 5.9–6.9 5.6–6.9 95% CI 5.6–7.7 11.2–14.8 22.2–31.5
P value – P < 0.0001 P < 0.0001y P value – P < 0.0001 P < 0.0001y
P ¼ nsz P < 0.0001z
Siemens aldosterone 26.3 6.9 6.3 Liaison renin 4.9 10.9 27.8
95% CI 23.6–29.2 6.4–7.4 5.7–6.9 95% CI 4.2–5.9 9.4–12.8 23.1–33.4
P value – P < 0.0001 P < 0.0001y P value – P < 0.0001 P < 0.0001y
P ¼ nsz P < 0.0001z

CI, confidence interval; EH, essential hypertension; ns: not significant; PA, primary aldosteronism.

PA vs. EH.
y
PA vs. control.
z
EH vs. control.

primary aldosteronism, essential hypertensive and normo-
tensive groups (Table 2) were compared using Kruskal–
Wallis nonparametric one-way analysis of variance with
Dunn’s multiple comparison post-test using Prism 5 for
Windows (Version 5.01). Comparisons with a P value less
than 0.05 were taken as statistically significant. ROC curve
analysis was applied to the ARR in the primary aldosteron-
ism and essential hypertensive groups to ascertain levels of
sensitivity, specificity and suggested cutoff in each case.
The area under the ROC curve (AUC) was used to sum-
marize the diagnostic discrimination between the two
patient groups. ROC curve analysis was carried out using
Prism 5.
RESULTS
Assay validation
A detailed report of the validation experiments carried out
for both assays can be found in the online Supplemental
text, ‘Sensitivity’, http://links.lww.com/HJH/A520. The LoD
and LoQ at 20% coefficient of variation of the aldosterone
assay at our laboratory were found to be 3.3 and 3.8 ng/dl,
respectively (Supplemental Table 1d and Supplemental
Figure 1d, http://links.lww.com/HJH/A520). For the PRC
assay, we obtained an analytical sensitivity and LoQ at 0.5
and 5.0 mIU/ml, respectively (Supplemental Table 7d and
Supplemental Figure 3d, http://links.lww.com/HJH/A520).
The results for LoB, LoD and LoQ for the two assays as
determined by the manufacturer are provided in Supple-
mental Tables 1 and 7, and Figures 1 and 3, http://link-
s.lww.com/HJH/A520. The most relevant characteristics of
the iSYS aldosterone and renin assays are reported in Table
1. Intra and interassay precision as determined by the
manufacturer and verified in our hands are shown in
Supplemental Tables 2 and 8, http://links.lww.com/HJH/
A520. Linearity as determined by the manufacturer and
verified in our hands is shown in Supplemental Tables 3
and 9, http://links.lww.com/HJH/A520. Results of recov-
ery, interference and cross-reactivity experiments as
assessed by the manufacturer are provided in Supplemental
Tables 4, 5, 6, 10, 11 and 12, and Supplemental Figure 2,
http://links.lww.com/HJH/A520. A summary of the main
assay characteristics according to the various providers’
(IDS, Siemens and Diasorin) Instructions For Use is
provided in Supplemental Table 14, http://links.lww.com/
HJH/A520.
Correlation of the iSYS aldosterone to the
Siemens radioimmunoassay, isotope dilution
mass spectrometry and liquid chromatography
tandem mass spectrometry
Good correlations were observed between the automated
iSYS aldosterone, the manual Siemens RIA assay and the
LC-MS/MS method both overall and in the low range of
samples (Fig. 2a, b, d and e), and the RfB (Fig. 2 g and h).
Bland–Altman analysis showed an overall mean bias (95%
limits) for the PAC difference of the iSYS minus Siemens
(Fig. 2c) of 11.1% (72 to 50), corresponding to an
absolute value of 0.12 ng/dl (10 to 10). In 47 of the 152
essential hypertensive patients, PAC had values equal to or
below 3.7 ng/dl with the iSYS assay, whereas 23 were at or
below the detection limit of 3.5 ng/dl with the Siemens
assay. Exclusion of samples at very low concentrations
(below 10 ng/dl) from the iSYS comparison to the Siemens
assay gave a mean bias of 4% (48 to 57), corresponding to
1.9 ng/dl (13 to 17). The difference of the iSYS minus
LC-MS/MS (Fig. 2f) gave a bias of 14% (15 to 43), corre-
sponding to 3.6 ng/dl (5.9 to 13). The difference of the
iSYS minus the RfB IDMS resulted (Fig. 2h) in a bias of 2.9%
(14 to 20), corresponding to 1.0 ng/dl (4.3 to 6.3). In a
subanalysis of the samples tested with the iSYS against the
LC-MS/MS method shown in Fig. 2, we had enough volume
to additionally test the Siemens assay (Supplemental Figure
4, http://links.lww.com/HJH/A520). The regression plot
shows that the Siemens assay measures approximately
20% higher than the LC-MS/MS [slope 1.23 (CI 1.14–
1.43); slope iSYS vs. LC-MS/M 1.01 (CI 0.901–1.144)] with
a similar variability in bias (from 20 to 55%) for both assays
[Siemens vs. LC-MS/MS mean difference 11.04% (SD differ-
ence 17.24%) and iSYS vs. LC-MS/MS mean difference
14.9% (SD difference 15.8%)].
Correlation of the iSYS direct renin to the Diasorin
Liaison, Cis-bio and plasma renin activity
Results obtained by the iSYS automated PRC assay com-
pared with those obtained by Liaison chemiluminescence
(Fig. 3a) and Cis-bio IRMA (Fig. 3c) assays showed good

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 Automated aldosterone and renin assays, ARR and sodium suppression cutoffs

(a) (b) (c)

Regression all data Low range Difference plot

Difference of methods (%)

20

100
IDS = 1.14 x RIA - 2.1
R 2 = 0.942
IDS-iSYS (ng/dl)

IDS-iSYS (ng/dl)
20 40 60 80 100

n = 275

15

50
Method: Deming

10

0
−100 −50
5
Regression Regression
Identity Identity

0
0

0 20 40 60 80 100 120 0 5 10 15 20 0 20 40 60 80 100 120

Siemens coat-a-count RIA (ng/dl) Siemens coat-a-count RIA (ng/dl) Average of methods (ng/dl)
(d) (e) (f)
Regression all data Low range Difference plot

Difference of methods (%)

100

20

100
IDS = 1.11 x LC-MS/MS + 0.78
IDS-iSYS (ng/dl)

IDS-iSYS (ng/dl)

R 2 = 0.928
80

n = 75
15

50
Method: Deming
60

10

0
40

−100 −50
5
20

Regression Regression
Identity Identity
0

0 20 40 60 80 100 0 5 10 15 20 0 20 40 60 80 100
LC-MS/MS (ng/dl) LC-MS/MS (ng/dl) LC-MS/MS (ng/dl)

(g) (h)
Regression all data Difference plot
Difference of methods (%)
50

100

IDS = 1.05 x IDMS - 0.35

2
R = 0.964
IDS-iSYS (ng/dl)
40

n = 15
50

Method: Deming
30

0
20

−100 −50
10

Regression
Identity
0

0 10 20 30 40 50 0 10 20 30 40 50
RfB IDMS (ng/dl) IDMS (ng/dl)
FIGURE 2 Correlation to existing aldosterone assay methods: the iSYS aldosterone automated immunoassay was compared with a commonly used (a–c) radioimmunoassay
method, to (d–f) LC-MS/MS in routine clinical samples and in a (g and h) set of ring trial samples from the Reference Institute for Bioanalytics (RfB). Bias (%) of the iSYS
assay relative to the RIA and to LC-MS/MS is expressed using Bland–Altman plots. IDMS, isotope dilution mass spectrometry; LC-MS/MS, liquid chromatography tandem
mass spectrometry; RIA, radioimmunoassay.

agreement. In a separate set of samples, the iSYS PRC as compared with the remaining patients (P < 0.0001),
compared with a PRA method (Fig. 3e) also showed good whereas there was some overlap within the controls and
concordance. Bland–Altman analysis showed a mean bias essential hypertensive patients. Comparison of PAC and
(95% limits) of 17.3% (56 to 91) for the iSYS minus the PRC determined with the iSYS method and Siemens and
Liaison results (Fig. 3b) corresponding to a mean bias of Liaison assays are reported in Table 2.
5.4 mIU/ml (42 to 52), and 4.0% (55 to 63) for the iSYS
minus the Cis-bio results (Fig. 3d), corresponding to Determination of the aldosterone-to-renin ratio
2.9 mIU/ml (17 to 23). cutoff on the IDS-iSYS
The currently used ARR screening cutoff for primary aldos-
Clinical studies teronism vs. essential hypertensive using the Siemens and
Liaison assay combination is at 1.2 (ng/dl)/(mIU/ml). ARR
iSYS plasma aldosterone concentration and plasma values observed with the iSYS assays in primary aldoster-
renin concentration in primary aldosteronism, onism, essential hypertensive and controls are shown in
essential hypertensive and normotensive controls Fig. 4a. ROC curve analysis with Siemens–Liaison combi-
Mean PAC was significantly higher and PRC significantly nation of a cohort of the 93 confirmed primary aldosteron-
lower in primary aldosteronism patients (n ¼ 93) as ism cases and 152 essential hypertensives at this cutoff gave

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Manolopoulou et al.

(a) (b)
Regression - log/log scale Difference plot - linear scale

100 150
Difference of methods (%)
IDS = 1.10 x Liaison + 0.99

500
R 2 = 0.920

IDS-iSYS (µU/ml)
n = 132
Method: Deming

50
50

0
−50
5

Regression

−150
Identity
1

1 5 10 50 500 0 100 200 300 400

Diasorin liaison (µlU/ml) Average of methods (µlU/ml)
(c) (d)
Regression - log/log scale Difference plot - linear scale

100 150
Difference of methods (%)
IDS = 1.04 x IRMA + 1.20
500

R 2 = 0.978
IDS-iSYS (µU/ml)

n = 164
Method: Deming

50
50

0
−50
5

Regression
−150

Identity
1

1 5 10 50 500 0 50 100 150 200 250 300 350

Cis-bio IRMA (µIU/ml) Average of methods (µIU/ml)
(e)
Regression - log/log scale

IDS = 103.7 x PRA - 2.1

500

2
R = 0.673
IDS-iSYS (µU/ml)

n = 33
Method: Weighted deming
50
5

Regression
1

0.05 0.20 0.50 2.00 5.00

RIA for PRA (ng/l/s)
FIGURE 3 Correlation to existing renin and plasma renin activity assay methods: results obtained by the iSYS direct renin assay were compared with those using (a and b)
the Liaison renin assay, the (c and d) Cis-bio IRMA and (e) a plasma renin activity method in clinical routine samples. IRMA, immunoradiometric assay; PRA, plasma renin
activity; RIA, radioimmunoassay.

a sensitivity of 98.9% and specificity of 76.3% (AUC; 95% CI
0.947, 0.922–0.971; graph not shown). In the same samples,
the iSYS aldosterone and renin assay combination gave
similar sensitivity and specificity (98.9 and 78.95%) but at a
slightly lower cutoff of 1.12 (AUC 0.952, 0.928–0.976)
(Fig. 4b). Exclusion of samples of essential hypertensive
patients with PAC values at or below the detection limit for
Siemens and iSYS assays yielded a small drop in specificity
of 11.2% for Siemens and 7.6% for iSYS at the respective
ARR cutoffs of 1.2 and 1.12. At these cutoffs, the two assays
combinations were concordant in correctly indicating pres-
ence of primary aldosteronism in all but two patients (one
false negative each). For the essential hypertensive group,
we found 33 out of the 152 patients with an ARR above the
cutoff when screened with the iSYS/iSYS combination,
whereas 46 of those same patients were above the cutoff
with the Siemens/Diasorin assay combination.
As separation of essential hypertensive vs. primary
aldosteronism can be particularly challenging at lower
renin concentrations, we carried out a subanalysis of the
ARR cutoff only in patients with PRC levels 20 mIU/ml or
less. In this subanalysis (93 primary aldosteronism patients,
112 essential hypertensives), we found that at the same
cutoff of 1.12, the same sensitivity of 98.9% gave a lower
specificity of 71.4% on the iSYS (67.9% with Siemens/
Liaison assay combination). If only the 42 histologically

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 Automated aldosterone and renin assays, ARR and sodium suppression cutoffs

(a) IDS-iSYS aldosterone to renin ratio (b) ROC of iSYS/iSYS ARR (c) Aldosterone to renin ratio
[(ng/dl)/(µIU/ml)] (ng/dl and µIU/ml) APA BAH and EH
1000 100
100 100
[(ng/dl)/(µIU/ml)]

10

(ng/dL)/(µU/ml)
10 80

%Sensitivity
ARR

1 60 1

0.1 40
0.1
0.01 20 Identity%
0.001 0 0.01
PA (n = 93) EH (n = 152) Healthy (n = 147) 0 20 40 60 80 100

2)
46

33

15
=
=

=
100%-Specificity%

(n
(n

(n
H
PA

EH
BA
A
FIGURE 4 (a and b) Aldosterone-to-renin ratio (ARR) using the iSYS aldosterone and direct renin assays: in a total cohort of 93 primary aldosteronism (PA) and 152
essential hypertension (EH) patients, the iSYS assays gave a cutoff at 1.12 with 98.9 and 78.9% sensitivity and specificity, respectively (AUC 0.959, 0.941–0.977). The ARR
in APA and BAH compared to essential hypertensive patients at the same cut-off (Figure 4c). APA, aldosterone-producing adenoma; AUC, area under the receiver-
operating characteristics curve; BAH, bilateral adrenal hyperplasia.

confirmed APA patients are taken into account (instead of method (Fig. 5b), whereas the disease was excluded in
all primary aldosteronism patients), the ARR cutoff of 1.12 another group of 73 hypertensive patients admitted to the
gave sensitivity and specificity of 100% (91.6–100) and hospital and initially suspected of primary aldosteronism
78.9% (71.6–85.1), respectively. (3.6 ng/dl; 3.5–3.6; Fig. 5d). In all of the primary aldoster-
onism patients, we also found PAC greater than 5 ng/dl at
Discrimination between aldosterone-producing 4 h post-aldosterone suppression when using the new
adenoma and bilateral adrenal hyperplasia IDS-iSYS assay (18.4 ng/dl; 15.1–22.5; Fig. 5a), whereas
Among the 79 confirmed primary aldosteronism patients, all samples in non-primary aldosteronism patients were
AVS revealed APA in 46 and BAH in 33. At screening, mean below 5 ng/dl with one sample very close to the cutoff
PAC levels were not significantly different between the APA using both the assays (3.6 ng/dl; 3.6–3.7; Fig. 5c).
and BAH group, regardless of method used, whereas mean
levels of PRC were significantly lower in the APA group DISCUSSION
with both the methods (P < 0.0001) (Table 3). As a result,
mean ARR was significantly higher in the APA group as The ARR is currently accepted as the most reliable method
compared with the BAH group (Table 3, Fig. 4c), a finding for detecting cases of primary aldosteronism [8]. Primary
observed with both method combinations. Using the iSYS aldosteronism is associated with an increased risk of
assays, the ARR values in the APA group ranged from 1.14 to cardiovascular morbidity in comparison with essential hy-
52.1 whereas in the BAH group, they were in the range of pertension [36–38]. Studies in community-based settings
1.1 up to 11.2. including nonhypertensive individuals have shown that
increased baseline ARR values are associated with
Plasma aldosterone cutoff during the saline- increased risk of BP rises, arterial stiffness, sustained hy-
infusion test pertension, and ultimately, cardiovascular events [39–47].
Plasma aldosterone concentrations above 5 ng/dl post- In addition, those patients of the general hypertensive
aldosterone suppression (geometric mean; 95% CI population with an elevated ARR may also be responsive
14.7 ng/dl; 12.2–17.8) were used to establish the diagnosis to treatment using MRAs [48], and it has been suggested that
of primary aldosteronism in the 46 patients by the Siemens a wider ARR screening not limited to specialist centres

TABLE 3. Plasma aldosterone and plasma renin concentrations in patients with APA and BAH measured with the iSYS and two
commercially available methods
Plasma aldosterone concentration Plasma renin concentration Aldosterone-to-renin ratio
(PAC; ng/dl) (PRC; mIU/ml) [(ng/dl)/(mIU/ml)]
APA BAH APA BAH APA BAH
iSYS aldosterone 30.3 25.4 iSYS renin 4.5 9.7 6.7 2.6
95% CI 25.6–35.9 20.9–30.9 95% CI 3.8–5.4 7.4–12.8 5.1–8.7 2.1–3.3
P value ns P value P < 0.0001y P < 0.0001z
Siemens aldosterone 25.3 26.1 Liaison renin 3.5 7.9 7.3 3.3
95% CI 21.2–30.1 22.3–30.5 95% CI 2.9–4.1 5.8–10.7 5.7–9.3 2.7–4.1
P value ns P value P < 0.0001y P < 0.0001z

APA, aldosterone-producing adenoma; BAH, bilateral adrenal hyperplasia; CI, confidence interval.

Aldosterone: APA vs. BAH.
y
Renin: APA vs. BAH.
z
ARR: APA vs. BAH.

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Manolopoulou et al.

(a) (b)
PA patients (IDS-iSYS) PA patients (Siemens RIA)
256 256

128 128

Aldosterone (ng/dl)

Aldosterone (ng/dl)
64 64

32 32

16 16

8 8
5 5
4 4

2 2
iSYS pre-AS iSYS post-AS RIA pre-AS RIA post-AS

(c) (d)
non-PA patients (IDS-iSYS) non-PA patients (Siemens RIA)
256 256

128 128
Aldosterone (ng/dl)

Aldosterone (ng/dl)
64 64

32 32

16 16

8 8
5 5
4 4

2 2
iSYS pre-AS iSYS post-AS RIA pre-AS RIA post-AS
FIGURE 5 Aldosterone suppression (AS) testing using the Siemens and iSYS assays in primary aldosteronism and essential hypertensive patients: plasma aldosterone levels
were measured at the end of saline-infusion test in samples from (a and b) 46 primary aldosteronism (PA) patients and in (c and d) 73 other hypertensive patients admitted
to the hospital. RIA, radioimmunoassay.

would be beneficial [49]. Widespread screening across
many primary care centres requires well validated, cost-
effective and easy to perform methods that measure reliably
in the clinically important ranges.
The performance of the new iSYS PAC and PRC assays
was evaluated in our laboratories showing overall good
correlation of both assays to existing methods, good pre-
cision across the assay range and acceptable sensitivity at
the low end (https://www.westgard.com/biodataba-
se1.htm). The iSYS aldosterone assay was compared with
a commonly used RIA method and found to have good
agreement in terms of slope but with a negative bias of
approximately 11% (Fig. 2). As has previously been
described [11], the scatter at concentrations below 8 ng/dl
and near the functional sensitivity of many aldosterone
assays renders comparison between methods at these levels
less meaningful. The inherent limitation of aldosterone
immunoassays in the low range of values is also apparent
from our studies. This must be taken into account when
measuring low-aldosterone low-renin disorders. Discrep-
ancies between immunoassays may arise because of several
reasons. An important factor to consider especially in the
case of steroid assays is that of antibody specificity. The
iSYS aldosterone assay was developed using a well estab-
lished and highly selective monoclonal antibody [30] with
low cross-reactivity to endogenous and synthetic steroids
including spironolactone and fludrocortisone. Our experi-
ence with an extensive number of clinical samples using
this assay was that a slightly larger number of samples
below the detection limit of the assay could be observed
compared with that using some available RIAs. We believe
that the very specific nature of the antibody may explain the
difference in concentrations particularly evident at the low
measurement range and the overall negative bias of the
iSYS assay as compared with the Siemens RIA.
Another major source of variability is that of calibration
which can lead to a dramatic bias between the reported
values. This is a fairly common phenomenon reported in
external quality assessment scheme results, such as those of
the RfB showing that certain assays measure systematically
higher or lower than others [50]. The iSYS aldosterone assay
was standardized using primary calibrators value assigned
by the accredited RfB (Bonn, Germany) IDMS method [51].
In the context of the widely accepted variability between
immunoassays, a more reliable method of assessing the
accuracy of a new assay in a large set of plasma samples is
comparison to mass spectrometry methods. Although more
laborious and perhaps less applicable to large-scale screen-
ing studies, mass spectrometry methods nearly eliminate
interference from other substances present in the sample. A
set of survey samples from the RfB was measured using the
iSYS assay showing excellent agreement to the target IDMS

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 Automated aldosterone and renin assays, ARR and sodium suppression cutoffs
value. Furthermore, a larger set of samples was measured
using another published LC-MS/MS method showing con-
cordant values across the range of the assay and down to
concentrations of approximately 7 ng/dl, with a slight
positive bias of 14% in the iSYS assay owing to a difference
in calibrator value assignment between the two assays.
Good correlation was observed with the iSYS PRC assay
to existing chemiluminescence and IRMA methods. Pre-
cision was good across the assay range of the renin kit,
whereas minimum levels of cross-reactivity to prorenin
levels were observed. Sensitivity is important in renin
assays for two reasons: first, the ARR is mathematically
highly dependent on renin values in the denominator
[52], and second, because of the suppressed renin levels
present in primary aldosteronism as a consequence of the
autonomous hypersecretion of aldosterone. For these
reasons, PRC assays should reliably measure as low as
possible; recommendations by Endocrine Society guide-
lines state 2.0 mIU/ml [8]. In our laboratory, the functional
sensitivity of the iSYS renin assay was determined at
5.0 mIU/ml, close to that recommended by the guidelines
and the observed sensitivity of other available renin chem-
iluminescence assays [13]. Although PRA is a well validated
and trusted technique [53], the complexity of the method
has led to poorer interlaboratory reproducibility [54]. As for
aldosterone assays, chemiluminescent PRC offers the
advantage of avoiding radiotracers and results that are
available within the hour. It is of course important to know
if there is agreement between PRA and PRC overall. More-
over, our regression analysis obtained with the PRC and a
previously published method for PRA show a correlation
that is consistent with the previous studies [54].
The Endocrine Society guidelines offer recommen-
dations for the cutoff to be used with the ARR using various
units for aldosterone and renin. It is, however, important to
realize that these are general suggestions and that the
appropriate cutoff to differentiate primary aldosteronism
cases from cases of essential hypertension should be
defined on a method-specific basis. One important factor
in the cohort included in our study was that the patients
were not on interfering medications (MRAs, renin–
angiotensin system antagonists). This is of course the ideal
scenario but it is not always possible to apply these con-
ditions as the effect can be harmful [24]. Many authors
suggest that apart from MRAs, the other major influencing
class of drug to be avoided are b-blockers [45,55,56]. Indeed
the exclusion of patients with this class of drugs may
explain why in our study the levels of PRC found in
essential hypertensive were relatively high, causing overall
lower ARR values. We carried out a ROC curve analysis
using samples from confirmed primary aldosteronism and
essential hypertensive patients to determine the ARR cutoff
providing highest possible sensitivity with a reasonable
compromise of false-positive tests. At a slightly lower
ARR cutoff than that used with current methods of
1.12 (ng/dl)/(mIU/ml), 98.9% of all suspected primary
aldosteronism patients would be included for further diag-
nosis along with approximately 21% of essential hyper-
tensive patients, this percentage being higher (29%) in
patients with PRC in the low-median range. These findings
show that with the iSYS assays, a similar cutoff to that
Journal of Hypertension
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
currently used could be employed, with the equivalent
sensitivity and specificity compared with currently used
methods. We performed the evaluation in samples taken
from a well defined cohort of primary aldosteronism and
essential hypertensive patients recruited in the context of
the German Conn’s registry. All patients underwent the
same standardized diagnostic procedure based on current
guidelines. However, because we evaluated the new assays
with a patient group definition based on measurements
made by the current routine assays, it remains an inherent
limitation of our study that the reliability of the diagnosis of
primary aldosteronism cannot be superior to the reliability
provided by the group definition in the underlying cohort.
Given that the ARR is a screening test in which a high
sensitivity is desirable, a false-positive rate of approximately
20–30% might be a reasonable ‘compromise’. However,
this certainly has to be considered in the context of the
invasiveness and costs of confirmatory tests in a large
segment of the hypertensive population. To allow this,
we provided a more extensive table illustrating the relation-
ship between sensitivity and specificity at various cutoffs
(see Supplemental Table 13, http://links.lww.com/HJH/
A520). Our retrospective subanalysis of the ARR values at
screening seen in APA vs. BAH patients revealed signifi-
cantly higher mean ARR in the APA due to the significantly
lower PRC levels in that group. Although in our study,
very high ARR values, above 10, are almost exclusively seen
in APA, there was considerable overlap between the ARRs
of both the groups, bringing about the need of AVS to
discriminate with certainty between these two conditions
[57].
A further part of our clinical validation of the iSYS
aldosterone assay included an analysis of a large set of
samples during the aldosterone suppression test. The Endo-
crine Society guidelines recommend an interpretation
whereby post-saline infusion PAC values less than 5 ng/
dl make diagnosis unlikely and levels above 10 ng/dl a very
probable sign of primary aldosteronism, with levels
between 5 and 10 ng/dl considered a grey zone [8,58].
Our study demonstrates that the cutoff of below 5 ng/dl
applied with Siemens method to separate primary aldoster-
onism from non-primary aldosteronism patients holds
equally with the iSYS method.
In summary, our studies confirmed that technical per-
formance and analytical specificity of the new automated
iSYS assays allow meaningful clinical application. The avail-
ability of these automated simultaneous measurements of
PAC and PRC from the same sample would provide a faster,
single-sample method for higher throughput to facilitate the
diagnosis and screening for primary aldosteronism in larger
populations. According to the outcome of our investigation,
an ARR cutoff at above 1.12 (ng/dl)/(mIU/ml) is indicative
of further confirmatory testing for primary aldosteronism
such as the saline infusion test, wherein aldosterone levels
above 5 ng/dl confirm diagnosis.
ACKNOWLEDGEMENTS
Previous presentation: A part of the work presented in this
manuscript, that is a preliminary version of the clinical
validation of the ARR and suppression testing, has been
www.jhypertension.com 2509
Manolopoulou et al.
shown as a poster presentation at the International Society
of Hypertension meeting in October 2012.
Funding: The patient samples of this study were col-
lected within the German Conn’s Registry – Else Kröner-
Fresenius-Hyperaldosteronism Registry supported by a
grant of the Else Kröner-Fresenius-Stiftung, Germany, to
M.R.
Conflicts of interest
E.F., A.D., S.D., D.H., R.J, P.G. and M.R. have no conflicts of
interest to declare. J.M. is an employee of IDS. M.B and A.M.
currently provide consultancy services to IDS.
REFERENCES
1. Rossi GP, Bernini G, Caliumi C, Desideri G, Fabris B, Ferri C, et al. A
prospective study of the prevalence of primary aldosteronism in 1,125
hypertensive patients. J Am Coll Cardiol 2006; 48:2293–2300.
2. Mulatero P, Stowasser M, Loh KC, Fardella CE, Gordon RD, Mosso L, et
al. Increased diagnosis of primary aldosteronism, including surgically
correctable forms, in centers from five continents. J Clin Endocrinol
Metab 2004; 89:1045–1050.
3. Hannemann A, Bidlingmaier M, Friedrich N, Manolopoulou J, Spyr-
oglou A, Volzke H, et al. Screening for primary aldosteronism in
hypertensive subjects: results from two German epidemiological
studies. Eur J Endocrinol 2012; 167:7–15.
4. Stowasser M, Taylor PJ, Pimenta E, Ahmed AH, Gordon RD. Laboratory
investigation of primary aldosteronism. Clin Biochem Rev 2010; 31:39–
56.
5. Calhoun DA. Hyperaldosteronism as a common cause of resistant
hypertension. Annu Rev Med 2013; 64:233–247.
6. Alvarez-Madrazo S, Padmanabhan S, Mayosi BM, Watkins H, Avery P,
Wallace AM, et al. Familial and phenotypic associations of the aldoster-
one renin ratio. J Clin Endocrinol Metab 2009; 94:4324–4333.
7. Tiu SC, Choi CH, Shek CC, Ng YW, Chan FK, Ng CM, et al. The use of
aldosterone-renin ratio as a diagnostic test for primary hyperaldoster-
onism and its test characteristics under different conditions of blood
sampling. J Clin Endocrinol Metab 2005; 90:72–78.
8. Funder JW, Carey RM, Fardella C, Gomez-Sanchez CE, Mantero F,
Stowasser M, et al. Case detection, diagnosis, and treatment of patients
with primary aldosteronism: an endocrine society clinical practice
guideline. J Clin Endocrinol Metab 2008; 93:3266–3281.
9. Schirpenbach C, Seiler L, Maser-Gluth C, Rudiger F, Nickel C, Beus-
chlein F, et al. Confirmatory testing in normokalaemic primary aldos-
teronism: the value of the saline infusion test and urinary aldosterone
metabolites. Eur J Endocrinol 2006; 154:865–873.
10. Perschel FH, Schemer R, Seiler L, Reincke M, Deinum J, Maser-Gluth C,
et al. Rapid screening test for primary hyperaldosteronism: ratio of
plasma aldosterone to renin concentration determined by fully auto-
mated chemiluminescence immunoassays. Clin Chem 2004; 50:1650–
1655.
11. Schirpenbach C, Seiler L, Maser-Gluth C, Beuschlein F, Reincke M,
Bidlingmaier M. Automated chemiluminescence-immunoassay for
aldosterone during dynamic testing: comparison to radioimmuno-
assays with and without extraction steps. Clin Chem 2006; 52:1749–
1755.
12. Stowasser M, Gordon RD. Aldosterone assays: an urgent need for
improvement. Clin Chem 2006; 52:1640–1642.
13. Dorrian CA, Toole BJ, Alvarez-Madrazo S, Kelly A, Connell JM, Wallace
AM. A screening procedure for primary aldosteronism based on the
Diasorin Liaison automated chemiluminescent immunoassay for direct
renin. Ann Clin Biochem 2010; 47:195–199.
14. Sealey JE. Plasma renin activity and plasma prorenin assays. Clin Chem
1991; 37:1811–1819.
15. Morganti A. European study group for the validation of DiaSorin LDRA.
A comparative study on inter and intralaboratory reproducibility of
renin measurement with a conventional enzymatic method and a new
chemiluminescent assay of immunoreactive renin. J Hypertens 2010;
28:1307–1312.
16. Taylor PJ, Cooper DP, Gordon RD, Stowasser M. Measurement of
aldosterone in human plasma by semiautomated HPLC-tandem mass
spectrometry. Clin Chem 2009; 55:1155–1162.
2510 www.jhypertension.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
17. Fischer E, Reuschl S, Quinkler M, Rump LC, Hahner S, Bidlingmaier M,
et al. Assay characteristics influence the aldosterone to renin ratio as a
screening tool for primary aldosteronism: results of the German Conn’s
registry. Horm Metab Res 2013; 45:526–531.
18. Campbell DJ, Nussberger J, Stowasser M, Danser AH, Morganti A,
Frandsen E, et al. Activity assays and immunoassays for plasma renin
and prorenin: information provided and precautions necessary for
accurate measurement. Clin Chem 2009; 55:867–877.
19. Weigel M, Riester A, Hanslik G, Lang K, Willenberg HS, Endres S, et al.
Postsaline infusion test aldosterone levels indicate severity and out-
come in primary aldosteronism. Eur J Endocrinol 2015; 172:443–450.
20. Osswald A, Fischer E, Degenhart C, Quinkler M, Bidlingmaier M,
Pallauf A, et al. Lack of influence of somatic mutations on steroid
gradients during adrenal vein sampling in aldosterone-producing
adenoma patients. Eur J Endocrinol 2013; 169:657–663.
21. Riester A, Fischer E, Degenhart C, Reiser MF, Bidlingmaier M, Beus-
chlein F, et al. Age below 40 or a recently proposed clinical prediction
score cannot bypass adrenal venous sampling in primary aldosteron-
ism. J Clin Endocrinol Metab 2014; 99:E1035–E1039.
22. Fischer E, Hanslik G, Pallauf A, Degenhart C, Linsenmaier U, Beus-
chlein F, et al. Prolonged zona glomerulosa insufficiency causing
hyperkalemia in primary aldosteronism after adrenalectomy. J Clin
Endocrinol Metab 2012; 97:3965–3973.
23. Fischer E, Beuschlein F, Degenhart C, Jung P, Bidlingmaier M, Reincke
M. Spontaneous remission of idiopathic aldosteronism after long-term
treatment with spironolactone: results from the German Conn’s Regis-
try. Clin Endocrinol (Oxf) 2012; 76:473–477.
24. Fischer E, Beuschlein F, Bidlingmaier M, Reincke M. Commentary on
the Endocrine Society Practice Guidelines: Consequences of adjust-
ment of antihypertensive medication in screening of primary aldoster-
onism. Rev Endocr Metab Disord 2011; 12:43–48.
25. EP17-A N. Protocols for determination of limits of detection and limits
of quantitation; approved guideline. NCCLS document EP17-A (ISBN 1-
56238-551-8) 2004; 24.
26. EP5-A2 N. Evaluation of precision performance of quantitative
measurement methods; approved guideline: second edition. NCCLS
document EP5-A2 (ISBN 1-56238-542-9) 2004; 24.
27. EP6-A N. Evaluation of the linearity of quantitative measurement
procedures: a statistical approach; approved guideline. NCCLS docu-
ment EP6-A (ISBN 1-56238-498-8) 2003; 23.
28. EP7-A2 N. Interference testing in clinical chemistry; approved guide-
line: second edition. CLSI document EP7-A2 (ISBN 1-56238-584-4)
2005; 25.
29. Abraham GE. Radioimmunoassay of steroids in biological fluids. Clin
Biochem 1974; 7:193–201.
30. Gomez-Sanchez CE, Foecking MF, Ferris MW, Chavarri MR, Uribe L,
Gomez-Sanchez EP. The production of monoclonal antibodies against
aldosterone. Steroids 1987; 49:581–587.
31. Simon D, Hartmann DJ, Badouaille G, Caillot G, Guyenne TT, Corvol P,
et al. Two-site direct immunoassay specific for active renin. Clin Chem
1992; 38:1959–1962.
32. Galen FX, Devaux C, Atlas S, Guyenne T, Menard J, Corvol P, et al. New
monoclonal antibodies directed against human renin. Powerful tools
for the investigation of the renin system. J Clin Invest 1984; 74:723–
735.
33. Van Der Gugten JG, Crawford M, Grant RP, Holmes DT. Supported
liquid extraction offers improved sample preparation for aldosterone
analysis by liquid chromatography tandem mass spectrometry. J Clin
Pathol 2012; 65:1045–1048.
34. Poulsen K, Jorgensen J. An easy radioimmunological microassay of
renin activity, concentration and substrate in human and animal plasma
and tissues based on angiotensin I trapping by antibody. J Clin
Endocrinol Metab 1974; 39:816–825.
35. Holmes DT. cp-R, an interface the R programming language for clinical
laboratory method comparisons. Clin Biochem 2015; 48:192–195.
36. Reincke M, Fischer E, Gerum S, Merkle K, Schulz S, Pallauf A, et al.
Observational study mortality in treated primary aldosteronism: the
German Conn’s registry. Hypertension 2012; 60:618–624.
37. Milliez P, Girerd X, Plouin PF, Blacher J, Safar ME, Mourad JJ. Evidence
for an increased rate of cardiovascular events in patients with primary
aldosteronism. J Am Coll Cardiol 2005; 45:1243–1248.
38. Savard S, Amar L, Plouin PF, Steichen O. Cardiovascular complications
associated with primary aldosteronism: a controlled cross-sectional
study. Hypertension 2013; 62:331–336.
Volume 33
Number 12
December 2015
 Automated aldosterone and renin assays, ARR and sodium suppression cutoffs
39. Newton-Cheh C, Guo CY, Gona P, Larson MG, Benjamin EJ, Wang TJ,
et al. Clinical and genetic correlates of aldosterone-to-renin ratio and
relations to blood pressure in a community sample. Hypertension 2007;
49:846–856.
40. Vasan RS, Evans JC, Larson MG, Wilson PW, Meigs JB, Rifai N, et al.
Serum aldosterone and the incidence of hypertension in nonhyper-
tensive persons. N Engl J Med 2004; 351:33–41.
41. Olivieri O, Ciacciarelli A, Signorelli D, Pizzolo F, Guarini P, Pavan C,
et al. Aldosterone to Renin ratio in a primary care setting: the Busso-
lengo study. J Clin Endocrinol Metab 2004; 89:4221–4226.
42. Tomaschitz A, Maerz W, Pilz S, Ritz E, Scharnagl H, Renner W, et al.
Aldosterone/renin ratio determines peripheral and central blood
pressure values over a broad range. J Am Coll Cardiol 2010; 55:2171–
2180.
43. Lieb W, Larson MG, Benjamin EJ, Yin X, Tofler GH, Selhub J, et al.
Multimarker approach to evaluate correlates of vascular stiffness: the
Framingham Heart Study. Circulation 2009; 119:37–43.
44. Shapiro Y, Boaz M, Matas Z, Fux A, Shargorodsky M. The association
between the renin-angiotensin-aldosterone system and arterial stiff-
ness in young healthy subjects. Clin Endocrinol (Oxf) 2008; 68:510–
512.
45. Ahmed AH, Gordon RD, Taylor P, Ward G, Pimenta E, Stowasser M.
Effect of atenolol on aldosterone/renin ratio calculated by both plasma
renin activity and direct renin concentration in healthy male volun-
teers. J Clin Endocrinol Metab 2010; 95:3201–3206.
46. Kisaka T, Ozono R, Ishida T, Higashi Y, Oshima T, Kihara Y. Associ-
ation of elevated plasma aldosterone-to-renin ratio with future cardi-
ovascular events in patients with essential hypertension. J Hypertens
2012; 30:2322–2330.
47. Terata S, Kikuya M, Satoh M, Ohkubo T, Hashimoto T, Hara A, et al.
Plasma renin activity and the aldosterone-to-renin ratio are associated
with the development of chronic kidney disease: the Ohasama Study.
J Hypertens 2012; 30:1632–1638.
Reviewers’ Summary Evaluations
Reviewer 1
Manolopoulou et al. have carefully evaluated a new auto-
mated chemiluminescence assay allowing the simultaneous
measurement of renin (expressed in mIU/ml) and aldoster-
one (expressed in ng/dl). This allows the rapid calculation
of the aldosterone-to-renin ratio (ARR) in (ng/dl)/(mIU/ml).
Although the results obtained with the new assays correlate
well those obtained with existing methods, the authors
propose yet another cut-off (1.12) to distinguish between
essential hypertension and primary aldosteronism. This
value differs 3–4-fold from the published recommended
cut-off for the ARR (3.7), and will only apply when using the
new assay. Combined with the large intra-individual vari-
ation in ARR (up to 10-fold), also this assay, despite being
Journal of Hypertension
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
48. Funder JW. Ultimately we are in furious agreement. J Hypertens 2012;
30:1903–1905.
49. Stowasser M. Primary aldosteronism: rare bird or common cause of
secondary hypertension? Curr Hypertens Rep 2001; 3:230–239.
50. Diederich S. Endocrine hypertension – diagnosis and treatment of
hormone-induced blood pressure disorders, 1st ed. Bremen: UNI-MED
Verlag AG; 2013.
51. Siekmann L. Determination of steroid hormones by the use of isotope
dilution – mass spectrometry: a definitive method in clinical chemistry.
J Steroid Biochem 1979; 11:117–123.
52. Holmes DT, Buhr KA. Error propagation in calculated ratios. Clin
Biochem 2007; 40:728–734.
53. Sealey JE, Gordon RD, Mantero F. Plasma renin and aldosterone
measurements in low renin hypertensive states. Trends Endocrinol
Metab 2005; 16:86–91.
54. Morganti A, Pelizzola D, Mantero F, Gazzano G, Opocher G, Piffanelli
A. Immunoradiometric versus enzymatic renin assay: results of the
Italian Multicenter Comparative Study. Italian Multicenter Study for
Standardization of Renin Measurement. J Hypertens 1995; 13:19–26.
55. Lonati C, Bassani N, Gritti A, Biganzoli E, Morganti A. Measurement of
plasma renin concentration instead of plasma renin activity decreases
the positive aldosterone-to-renin ratio tests in treated patients with
essential hypertension. J Hypertens 2014; 32:627–634.
56. Mulatero P, Rabbia F, Milan A, Paglieri C, Morello F, Chiandussi L, et al.
Drug effects on aldosterone/plasma renin activity ratio in primary
aldosteronism. Hypertension 2002; 40:897–902.
57. Rossi GP, Auchus RJ, Brown M, Lenders JW, Naruse M, Plouin PF, et al.
An expert consensus statement on use of adrenal vein sampling for the
subtyping of primary aldosteronism. Hypertension 2014; 63:151–160.
58. Rossi GP, Belfiore A, Bernini G, Desideri G, Fabris B, Ferri C, et al.
Prospective evaluation of the saline infusion test for excluding primary
aldosteronism due to aldosterone-producing adenoma. J Hypertens
2007; 25:1433–1442.
fast and not requiring radioactivity, will have to prove itself
in the clinic.
Reviewer 2
J. Manolopoulou et al. have shown a good performance
and analytical specificity of a new fast automated immuno-
assay allowing the simultaneous determination of plasma
aldosterone and renin with some minor bias towards estab-
lished methods, imposing to define a new cut-off for the
active renin to aldosterone ratio to detect primary aldoster-
onism (PA). This assay may facilitate high throughput
screening for PA. Until LCMSMS methods to measure
plasma aldosterone are made available in all laboratories,
this immunoassay may be an alternative to diagnose with
patients with PA since radioimmunoassay are no more
available.
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