Clinical Biochemistry 48 (2015) 377–387

Contents lists available at ScienceDirect

Clinical Biochemistry
journal homepage: www.elsevier.com/locate/clinbiochem

Review

Laboratory challenges in primary aldosteronism screening and diagnosis

Muhammad Rehan a,1, Joshua E. Raizman b,1, Etienne Cavalier c,
Andrew C. Don-Wauchope a, Daniel T. Holmes d,⁎
a
Department of Pathology and Molecular Medicine, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4L8, Canada
b
Department of Pathology and Laboratory Medicine, University of Toronto, Medical Science Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
c
Department of Clinical Chemistry, University of Liège, CHU Sart-Tilman, Bât B35, 4000 Liège, Belgium
d
Department of Pathology and Laboratory Medicine, University of British Columbia, Rm. G227-2211 Wesbrook Mall, Vancouver, BC V6T 2B5, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Background and objective: The laboratory has a critical role to play in the screening and diagnosis of primary
Received 12 December 2014 aldosteronism. This review highlights some of the important analytical considerations and the new develop-
Received in revised form 8 January 2015 ments in the determination of aldosterone and renin.
Accepted 12 January 2015 Methods: The review considered the published literature and clinical practice guidelines in the area of prima-
Available online 22 January 2015
ry aldosteronism.
Results: A brief introduction to primary aldosteronism is provided. A detailed description of the pre-
Keywords:
Aldosterone
analytical, analytical and post-analytical considerations for the laboratory determination of aldosterone, renin
Plasma renin activity and the aldosterone to renin ratio follows.
Primary aldosteronism Conclusions: The lack of internationally accepted standardized methodologies and standard reference mate-
Mineralocorticoid rial has impeded screening and diagnosis of primary aldosteronism. The development of more accurate and sen-
Hypertension sitive methods by LC–MS/MS has improved the reliability of aldosterone and renin testing and the availability of
Secondary commercial chemiluminescent assays may improve the standardization of reporting. Laboratorians need to un-
derstand the strengths and weaknesses of their analytical approach and ensure that their interpretative reports
are appropriate to their assays.
© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Definition of primary aldosteronism and its causes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Sporadic forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Familial forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Clinical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Biochemical screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Laboratory analytical issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Aldosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Renin determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Prorenin activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Prorenin interference with renin determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Plasma renin activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Incubation period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Angiotensinase inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

⁎ Corresponding author.
E-mail addresses: muhammad.rehan@medportal.ca (M. Rehan), josh.raizman@mail.utoronto.ca (J.E. Raizman), etienne.cavalier@chu.ulg.ac.be (E. Cavalier), donwauc@mcmaster.ca
(A.C. Don-Wauchope), dtholmes@mail.ubc.ca (D.T. Holmes).
1
Authors who made equal contribution.

http://dx.doi.org/10.1016/j.clinbiochem.2015.01.003
0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
378 M. Rehan et al. / Clinical Biochemistry 48 (2015) 377–387

PRA by LC–MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

Renin mass assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Aldosterone to renin ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
PRA vs DRC in the aldosterone to renin ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Diagnostic use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Minimum aldosterone concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Method combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Patient preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Diagnostic tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Subtype classification with adrenal venous sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Preanalytical pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Analytical issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Costs and benefits of ACTH stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

Introduction
Primary aldosteronism (PA), first described by Jerome Conn [1], is by
far the most common form of secondary hypertension accounting for
approximately 10% of hypertension in all comers [2] with higher rates
in younger hypertensive populations. Hypertension is a common chron-
ic condition affecting 22–23% of the population in Canada and close to
1 billion people worldwide [3]. Given the well-known risks of athero-
sclerosis, myocardial infarction, peripheral vascular disease, stroke and
chronic renal impairment, there are compelling reasons to identify
and treat curable forms of hypertension such as PA.
In the past, prevalence estimates of PA among hypertensives have
been as low as 1%. The discrepancy between historical (1%) and contem-
porary (10%) estimates has been attributed to differences in study pop-
ulations, diagnostic approaches, cutoff thresholds of screening tests, and
the choice of confirmatory tests [4]. For example, one important differ-
ence is the recognition that hypokalemia is not essential for the diagno-
sis of PA [5].
Specific identification of PA is essential because affected patients
have higher cardiovascular morbidity and mortality than age, sex, and
blood pressure (BP) matched patients with essential hypertension [6,
7]. The increased rate of cardiovascular events is thought to be related
to effects of aldosterone on inflammation, fibrosis at the level of various
target organs [8]. When detected early, unilateral forms of PA can be
treated successfully by surgical removal of the tumour often resulting
in normalization of BP [2,9–12]. Pharmacological intervention with al-
dosterone receptor antagonists is an effective alternative in patients
who refuse or are not candidates for surgery [13–16]. Early detection
is associated with improved patient outcome and reduced cardiovascu-
lar risk [6,17].
The aldosterone to renin ratio (ARR) is currently the recommended
biochemical screening tool for PA in those for whom there is clinical sus-
picion. Development and improvement in performance of aldosterone
and renin determinations have permitted more reliable screening and
diagnosis of PA. It should be mentioned that the ARR results of a popu-
lation of 1172 normotensive and hypertensive subjects from 248 fami-
lies identified by probands were continuously distributed and did not
suggest a sub-group that would have PA [18]. This observation under-
lines the point that the ARR may not always cleanly separate PA from es-
sential hypertension and that the individuals selected for ARR should
have clinical features suggestive of PA.
This review will discuss the laboratory challenges of screening, diag-
nosis and tumour localization in PA and outline the critical role that
laboratorians play in developing and maintaining an effective PA diag-
nostic programme. Analytical challenges and their effect on the ARR
will also be discussed along with some of the recent advancements in
the measurement of aldosterone and plasma renin activity (PRA)
using liquid chromatography and tandem mass spectrometry (LC–MS/
MS).
Definition of primary aldosteronism and its causes
PA is characterized by production of aldosterone independent of or
out of proportion to angiotensin II (AngII) stimulation. By definition,
PA is resistant to sodium loading, which would normally suppress aldo-
sterone secretion, PRA, angiotensin I (AngI) and AngII.
Sporadic forms
Idiopathic adrenal hyperplasia (IAH) accounts for approximately
60% of PA cases, aldosterone producing adenomas (APA) or Conn's syn-
drome accounts for approximately 30%, and unilateral adrenal hyper-
plasia accounts for another 2–3% [2]. Approximately 1% of PA is caused
by aldosterone producing carcinoma, which can result in very high
concentrations of a number of adrenal steroids leading to PA, Cushing
syndrome, virilization or feminization. Adrenal carcinoma is often me-
tastasized at the time of diagnosis [19].
Ectopic expression of luteinizing hormone receptor [20] and gonad-
otropin releasing hormone receptor [21] in APAs suggests a possible
role for these hormones in the excess production of aldosterone. It has
also been shown that human adipocytes produce aldosterone secre-
tagogues unrelated to angiotensinogens [22]. This supports the clin-
ical observation that obesity is associated with higher aldosterone
concentrations [23,24].
Familial forms
Familial forms of PA are rare (b 1% of patients) and include familial
hyperaldosteronism type I (glucocorticoid-remediable aldosteronism,
GRA), type II (the familial occurrence of aldosterone-producing adeno-
ma or bilateral idiopathic hyperplasia or both), and type III (familial
non-glucocorticoid-remediable hyperaldosteronism).
Familial aldosteronism type I (GRA) is an autosomal dominant
condition caused by a fusion gene between CYP11B2 (aldosterone
synthase) and CYP11B1 (11-β-hydroxylase) which causes excessive al-
dosterone production in response to adrenocorticotropin (ACTH) [25].
This condition was identified clinically [26] and described genetically
some time ago [27]. More recently, kindreds showing low-renin
hyperaldosteronism with an autosomal dominant pattern of inheri-
tance (familial hyperaldosteronism type III) have been described [28].
This condition has been shown to be caused by germ-line mutations
in the G protein-activated inward rectifier potassium channel 4
 M. Rehan et al. / Clinical Biochemistry 48 (2015) 377–387
(GIRK4) which is encoded by the KCNJ5 gene [29,30]. Those affected by
the first-described mutation, T158A, have a severe phenotype as do
those with G151R and I157S. G151E is associated with a milder pheno-
type [31]. Importantly, somatic mutations in KCNJ5 cause some sporadic
non-syndromic APAs [29]. The cause of familial hyperaldosteronism
type II is a matter of ongoing investigation [32].
Clinical features
Patients with PA are typically diagnosed in their 3rd to 6th decade
and are often found incidentally because of their resistance to antihy-
pertensives [2]. The clinical features of PA come as a direct result of in-
appropriate production of aldosterone and subsequent activation of
the mineralocorticoid receptor (MR). MR is expressed in a variety of tis-
sues such as the renal tubular epithelium, smooth muscle cells, cardiac
myocytes, endothelial progenitor cells, and neutrophils [4]. The re-
sponse of unregulated aldosterone on the distal renal tubule is excessive
sodium and water retention, as well as potassium excretion, leading to
volume expansion, hypertension, and, variably, hypokalemia [33,34].
PRA is characteristically low or suppressed due to feedback inhibition
from sodium excess and increased blood pressure.
Hypertension in PA is often poorly responsive to angiotensin
converting enzyme inhibitors, angiotensin receptor blockers, calcium
channel blockers and beta blockers, which in itself is a diagnostic clue
[5]. Additionally patients with PA can sometimes present with hyper-
tensive crisis, though this is not typical [35].
Hypokalemia is found in 37% of patients diagnosed with PA but is
likely a more common presentation when the disease has progressed
[36,37]. Half of patients with APA and 17% of those with IAH were
found to have serum potassium concentrations less than 3.5 mmol/L
[37]. Potassium is often within the normal range in GRA. For these rea-
sons, hypokalemia is an insensitive screening tool for PA. Clinically, hy-
pokalemia in severe cases may result in muscle weakness, particularly
in the lower extremities, muscle cramps, palpitations and, rarely, hypo-
kalemic paralysis. Treatment of hypokalemia in PA may require large
doses of oral potassium until aldosterone antagonists are prescribed or
surgery is undertaken.
Chronically PA causes all of the expected sequelae of hypertension
and a number of cardiac [6,7,38] and renal [39,40] effects that appear
to be specific to aldosterone excess. PA is also associated with bone de-
mineralization [41] and may even be seen concomitantly with primary
hyperparathyroidism [42]. Associations of PA and diabetes mellitus
have been observed in some cohorts [43] but not in others [44] and
the pathophysiological role of aldosterone in glucose metabolism is an
area of ongoing investigation.
Screening
Guidelines
In 2008, the Endocrine Society published clinical practice guidelines
for primary hyperaldosteronism [5]. For screening, it identified four sub-
groups of individuals with the presence of hypertension who have high
prevalence of PA: 1) Patients with severe hypertension N 160 mm Hg
systolic or N100 mm Hg diastolic or resistant hypertension defined by
suboptimal control on a three-drug regimen; 2) the presence of hyper-
tension and spontaneous or diuretic-induced hypokalemia; 3) the pres-
ence of hypertension with adrenal incidentaloma; and 4) the presence
of hypertension and a family history of early onset hypertension or ce-
rebrovascular accident at a young age (b40 years). It is recommended
that all hypertensive first-degree relatives of patients with PA should
be screened. Additionally, some investigators suggest screening in the
following high risk categories of patients with hypertension, namely:
1) those with evidence of target organ damage (e.g., left ventricular hy-
pertrophy, microalbuminuria) that appears out of proportion to the
379
degree of blood pressure elevation; 2) obese individuals (BMI N 35)
and 3) those with obstructive sleep apnea syndrome [23,24].
Some have argued in favour of screening all hypertensive patients
for PA [45–47]. This opinion is rooted in the observation that PA is far
more common than previously believed, patients are often normokale-
mic and that significant and preventable end-organ damage can be
avoided with early detection. Although indirect evidence demonstrates
that screening for PA improves patient outcomes, there are no clinical
trials or prospective studies that clearly measure the impact of screen-
ing on morbidity, mortality, or quality of life [5]. Additionally, screening
of hypertensive all comers may not be economically or practically feasi-
ble [48]. Further the lack of a clear biochemical delineation of PA from
other hypertensive populations suggests that targeted screening is
preferable [18].
Biochemical screening
The accepted approach to screening for PA in at-risk populations is
to measure serum/plasma aldosterone and plasma renin and to calcu-
late the ARR [5,49]. The ratio can be calculated using serum aldosterone
concentration (SAC) and either the PRA or direct renin concentration
(DRC) and has superior test characteristics to either SAC or PRA alone
[50]. The ARR has further advantages over SAC in relation to
preanalytical factors including lower intra- and inter-day variation,
and less dependence on salt intake, diuretic exposure, and posture
prior to collection [51]. However, analytical challenges in the determi-
nation of both the numerator and denominator make identification of
an appropriate screening threshold a challenge [49] and, as with all cal-
culated quantities, error propagation is an issue [52].
Laboratory analytical issues
Aldosterone
Aldosterone concentration in healthy individuals is present in low
quantities in serum (100–600 pmol/L), and there are numerous
compounds that can potentially interfere with analysis making aldoste-
rone a technically challenging analyte to accurately quantify. Urine is a
cleaner matrix with much higher analyte concentrations but requires
24 h collections to produce clinically useful results. About 0.5% of uri-
nary aldosterone is excreted as parent compound, while aldosterone-
18-glucuronide accounts for 5–15% and 3α-5β-tetrahydroaldosterone
accounts for 15–40% [53]. Urinary aldosterone methods require acid or
enzymatic hydrolysis to convert the glucuronide to aldosterone before
analysis by a variation of the serum method. In our laboratories, SAC re-
quests outnumber urine aldosterone analyses by 40:1 so we will not
discuss urine analysis further.
Historically, aldosterone was analysed in serum by radioimmunoas-
say after a chromatographic clean-up [54]. This allowed for the separa-
tion of aldosterone from potentially cross-reacting compounds such as
aldosterone-18-glucuronide and tetrahydroaldosterone [55]. However,
the need for increased throughput led to homogenous RIAs and more
recently, chemiluminescent immunoassays (CLIA) [56] with automa-
tion [57–59]. The increase in convenience obtained by the automated
CLIA methods has not mitigated the problematic aspects of aldosterone
estimation by immunoassays (IAs). In particular it has been shown that
aldosterone assays provide numeric results that vary by a factor of ≈2
between methods [59,60]. Use of automated CLIA approaches may
have improved analytical variability but has not changed antibody
cross-reactivity.
It has been known for many years that in the setting of renal impair-
ment, the use of homogenous IAs results in marked overestimation
of aldosterone [55]. This phenomenon is eliminated by methods
employing solvent extraction [55] and/or chromatographic separation,
which in the modern context represents those performed by liquid
chromatography and tandem mass spectrometry (LC–MS/MS). Fig. 1
380 M. Rehan et al. / Clinical Biochemistry 48 (2015) 377–387
Fig. 1. A: Comparison of two LC–MS/MS methods for aldosterone on samples collected from patients with dialysis dependent renal failure (n = 27). SPH = St. Paul's Hospital, Vancouver,
Canada. CHU = Centre Hospitalier Universitaire de Liège, Belgium. B: Comparison on the same samples between the Diasorin Liaison and LC–MS/MS. Analysis at SPH was performed using
the method described in reference [61]. Regression analysis was performed with open-source software, cp-R [62]. The black line is the line of regression. The dashed line is the line of
identity.
shows a three-way comparison of 27 serum samples by two separate
LC–MS/MS aldosterone methods and an automated commercial IA
performed on patients with chronic kidney disease. The two LC–
MS/MS methods provide comparable results in the measuring
range but IA demonstrates a median positive bias of 127%, presum-
ably because of antibody cross-reactivity with uncleared aldosterone
metabolites.
Developments in LC–MS/MS have allowed aldosterone to be quanti-
fied in a consistently accurate manner in routine clinical laboratories
[63]. Existing approaches include those using protein precipitation and
solid phase extraction (SPE), liquid–liquid extraction (LLE), and
supported liquid extraction (SLE). SPE methods have employed
electrospray ionization (ESI) negative mode [63,64] or atmospheric
pressure photoionization (APPI) negative ion mode [65]. LLE methods
have also used ESI negative mode [61,66,67] as did SLE methods [68,
69]. The intermethod comparisons for LC–MS/MS methods [68,66] rep-
resent a stark improvement over comparisons between pairs of IAs [59,
60] and between IA and LC–MS/MS [61,66]. By using LC–MS/MS it is en-
tirely achievable to have intermethod coefficients of variation (R) great-
er than 0.99 and median biases less than 5% in the analytical range of
interest for the screening and diagnosis of PA (100–1000 pmol/L) [68].
An additional benefit of LC–MS/MS is the ability to measure aldo-
sterone by two different multiple reaction monitoring (MRM) transi-
tions which are 359.1 → 188.9 amu for the quantifier ion and
359.1 → 331.1 amu for the qualifier ion in negative electrospray
mode. The use of two MRMs permits independent confirmation of the
aldosterone concentration and the calculation of “ion ratios” to confirm
the absence of analytical interference. In one of our laboratories, this has
enabled the identification of interference from inappropriate sample
collection in gel containing tubes, a phenomenon well-known for LC–
MS/MS testosterone determination [70].
However, the financial outlay for an LC–MS/MS system is substantial
and the assay development and troubleshooting can be technically de-
manding. For this reason, RIA and automated IAs will continue to exist
and laboratorians will have to balance the tensions between finances,
convenience, and the desire for analytical accuracy.
It is probable that standardization efforts will improve the accuracy
of both IAs and LC–MS/MS assays for aldosterone. However, at present,
there is no LC–MS/MS reference method published nor is there a stan-
dard reference material. There is gas chromatography and tandem
mass spectrometry (GC–MS) assigned serum-based material available
from Referenzinstitut für Bioanalytik [71].
Renin determination
Prorenin activation
Prorenin is the precursor of renin and can be activated by an irre-
versible proteolytic mechanism or non-proteolytically by unfolding of
the pro-segment [72]. At physiological temperatures and pH, up to 2%
of prorenin is in its open conformation and therefore catalytically active
[73].
It has been repeatedly observed that the storage of plasma at refrig-
eration and ambient temperatures can lead to the activation of prorenin
and therefore spuriously increase PRA and direct renin concentration
(DRC). It is therefore advised that specimens for renin determination
be frozen immediately and stored frozen, rapidly thawed and rapidly
analysed. While these precautions are sound from the standpoint of
good laboratory practice, the phenomenon of prorenin cryoactivation
may sometimes be overstated since it has been shown that it takes
12 h of incubation at 4 °C for 5% activation of recombinant prorenin
[74]. Though prorenin activation may not be as serious a problem as
once thought, rapid freezing is still advisable because renin may con-
sume angiotensinogen substrate at ambient temperatures potentially
leading to substrate depletion, larger AngI concentrations in the blank,
and lower PRA results than would have been obtained with appropriate
specimen handling [73].
Prorenin interference with renin determination
Accidental detection of prorenin is particularly a concern because of
its concentration relative to renin. Prorenin is secreted constitutively
from the kidneys and in healthy individuals plasma concentrations are
10-fold higher than active renin concentrations [75,76] but this in-
creases up to 100-fold in conditions where renin is suppressed such as
in anephric patients but more importantly, in those with PA [77,76].
Therefore, DRC assay cross reactivity with non-target molecules has
the potential to be most significant in the very context of interest, name-
ly, the low-renin state. Naturally, PRA could be likewise affected by this
same phenomenon but analytical overestimation is expected to be alle-
viated by incubation at 37 °C in the AngI generation step which pro-
motes refolding of prorenin to its catalytically inactive form.
Plasma renin activity
Traditionally, renin was assessed using PRA assays that quantified
AngI by RIA. In appropriately buffered conditions, endogenous renin
 M. Rehan et al. / Clinical Biochemistry 48 (2015) 377–387
(EC 3.4.23.15) acts upon endogenous angiotensinogen to produce AngI
over a fixed incubation period at 37 °C. This is sometimes chosen to be
1 h, most often 3 h and in low PRA samples, 18 h [78,79]. Simultaneous-
ly, a second aliquot of the specimen is incubated in identical buffering
conditions and for the same duration but on ice, so that renin has no ac-
tivity. At the end of incubation, AngI is measured in both the 37 °C and
the 4 °C (“blank”) aliquots and PRA is calculated by Eq. (1)
½AngI37 °C −½AngI4 °C
PRA ¼ ; ð1Þ
Δt
where Δt is the incubation time. Unlike many enzyme activity assays,
there is no IFCC guideline for a standardized approach and, preanalytical
issues aside, several key factors affect the numerical results that are pro-
duced. These include the duration of incubation, the choice of
angiotensinase inhibition strategy [79,80], the pH of incubation [79],
the analytical performance of the AngI assay and the purity of the
calibrators.
Incubation period
Prolonged incubation periods (18 h) have long been recommended
for low-renin specimens [79]. The motivation is to raise the concentra-
tion of AngI in the sample so that analytical accuracy could be achieved
and blank concentration, inaccurate by virtue of its relatively low level,
would become mathematically insignificant. While it has been noted
that prolonged incubation periods have little effect on the numerical
value of the reported result for low renin samples (except to improve
their accuracy) [79,81], at higher PRA levels, shorter incubation periods
yield higher numerical results [81]—likely because of higher mean sub-
strate (i.e., angiotensinogen) concentrations.
pH
The pH at which renin is highest in activity is about 5.5–6.0 which
leads to PRA results that are approximately 2-fold higher than those
afforded by assays buffered to pH = 7.4 [79], as is required of antibody
capture assays [82]. PRA shows substantial drop-off below pH = 4 and
above pH = 8.
Angiotensinase inhibition
AngI cleaved from angiotensinogen is vulnerable to in vitro proteo-
lytic degradation by non-specific proteases and serum angiotensin
converting enzyme (ACE). ACE inhibitors employed have included
EDTA, 8-hydroxyquinolone hemi-sulfate, 2,3-dimercaptopropanol (“di-
mercaprol”, “British anti-Lewisite”, “BAL”), diisopropyl fluorophos-
phate, and phenylmethanesulfonylfluoride (PMSF) [80]. Sealey also
recommended the addition of neomycin sulfate to prevent bacterial
peptidases from consuming generated AngI [79].
However, the choice and concentration of ACE inhibitor can alter the
recovery of generated AngI [79] and can inhibit renin itself [80]. To fur-
ther complicate matters, it has been shown that there is a small sub-
population of subjects in whom AngI is unexpectedly rapidly degraded
during the incubation process yielding spuriously low results [83].
Oddly enough, all previous PRA methods employing chemical inhibition
strategies may have been similarly affected. An AngI protection strategy
that would have been theoretically less vulnerable was the antibody-
capture approach where AngI was antibody-bound [82] or bound to cat-
ion exchange resins as it was generated [80]. It is unclear whether the
rapid degradation of AngI occurs in vivo and is therefore a physiological
phenomenon or if it is purely an analytical issue, though bacterial con-
tamination appears to be ruled out [84]. It is also unclear what, if any,
are the clinical ramifications. Nevertheless, samples affected by rapid
AngI degradation can be detected by the addition of a second stable iso-
topically labelled standard (SIS) AngI during the incubation phase and
subsequent independent monitoring to confirm the presence of the ex-
pected signal relative to the SIS employed for AngI quantitation [83].
381
PRA by LC–MS/MS
While RIAs and ELISAs for PRA are still available, LC–MS/MS assays
are becoming more prevalent [81,83,85,86] and have a number of ana-
lytical advantages. First, they have linear, rather than sigmoidal AngI
calibration curves and accordingly wider dynamic ranges [87,81]. Addi-
tionally LC–MS/MS methods have improved analytical specificity by
virtue of the mass filtering technology meaning that non-specific im-
munoreactivity in the blank [79] is not an issue. This likely accounts
for the fact that these assays do not appear to require blank subtraction
[81,88]. Additionally, the wide dynamic range facilitates the reporting of
results in patients with high PRA, including infants, those with renal ar-
tery stenosis and pregnant females, without the use of sample dilution
which causes unpredictable recoveries on a patient-specific basis [79,
89]. LC–MS/MS methods also have the benefit of being free of the use
of radiotracers and do not require duplicate incubations/analyses for
each of the 37 °C and 4 °C aliquots. Because of the technical challenges
of HPLC, there have been significant efforts made to develop
chromatography-free matrix-assisted laser desorption/ionization
(MALDI) mass spectrometric assays for PRA [87,88]. Although there is
no internationally recognized standardized reference material for AngI
or PRA, there is a non-WHO reference material for AngI available from
the National Institute for Biological Standards and Control (NIBSC)
[90] which allows a common high-quality calibrant.
While LC–MS/MS has improved AngI determination, the challenge
to standardize PRA assays is still unmet. However, given that accurate
and sensitive determination of AngI over wide dynamic ranges is now
achievable using mass spectrometry, an internationally agreed-upon
protocol for PRA buffering conditions and ACE inhibition makes stan-
dardization achievable, particularly since AngI is a small, well-defined
analyte. It is also a reasonable suggestion that LC–MS/MS may be sensi-
tive enough to explore screening using aldosterone and directly-
measured angiotensins [91].
Renin mass assays
Sandwich assays for the active form of the renin enzyme have been
available for approximately 20 years. While these were originally
immunoradiometric, there are now automated CLIA approaches. The
detection antibody in DRC assays is generally targeted at an epitope re-
gion on the active site of renin when the pro-segment is absent, or when
prorenin is in an open conformation [73]. Hence, the “active” renin im-
munoassay is the sum of both renin and prorenin in open conformation.
Unfolding of the pro-segment occurs preferentially at ambient and
refrigeration temperatures. For example it has been shown in one DRC
assay using room-temperature incubation that DRC is over-estimated
by about 25% in pooled human plasma because of quantitation of
spontaneously activated prorenin [92]. In these conditions, 5% of
prorenin was quantified as renin. This problem was mitigated by a
shorter, 37 °C assay incubation. Nevertheless, there is concern that the
very binding of antibody to the active site may lock prorenin into its
open-conformation leading to spurious elevation in samples that have
been stored at ambient or refrigeration temperatures prior to analysis
[73].
Aldosterone to renin ratio
PRA vs DRC in the aldosterone to renin ratio
There is an abundance of literature in support for ARR calculated
with PRA. However, because the correlation of PRA with DRC is poor
[93,94] to absent [95] in the low-renin state, it should not necessarily
be inferred that the same literature supports DRC. Simply stated: PRA
and DRC are different metrics of the renin angiotensin aldosterone sys-
tem (RAAS). Therefore, it is not surprising that positivity rates in screen-
ing are different [96]. False positives have been noted when DRC is used
in lieu of PRA in females in the luteal phase of their menstrual cycle [97]
and those on oral contraceptive agents [98]. We also note that those
with undetectable PRA may have DRC well within the normal range
382 M. Rehan et al. / Clinical Biochemistry 48 (2015) 377–387

[94]. For these reasons, numerous experts remain committed to the use
of PRA over DRC notwithstanding the inconveniences [76,99]. However,
screening with the SAC/DRC ratio can be effective [95,100,101].
From a laboratorian's perspective, advantages of DRC include
walk-away automation, better turnaround time, better stability at
freezer temperatures [102] and instrument communication with
the laboratory information system without the use of third party or cus-
tom middleware required for LC–MS/MS systems [103]. Advantages of
PRA are better low-end precision, a wider body of supporting literature,
a more complete assessment of RAAS tone, and a well-defined analyti-
cal target.
Diagnostic use
There are a number of considerations in the interpretation of the
SAC/PRA and SAC/DRC ratio, the most important being the establish-
ment of clinically useful thresholds.
venous sampling eventually revealed a left-dominant lateralization
index of 12.3 enabling left adrenalectomy and cure for this patient.
Method combinations
There are a pantheon of method combinations for aldosterone and
renin, each with intermethod biases for the numerator and denomina-
tor. This underlines the responsibility of each laboratory to adopt an
ARR threshold from literature only if they are using the identical aldo-
sterone and renin methods. This ARR threshold should be validated in
their population. Alternatively the laboratory should perform full meth-
od comparisons for each of their assays against the same method used
in the original literature report. Once the laboratory has determined a
putative threshold for the ARR, it should then evaluate it in their popu-
lation. This approach is becoming more challenging as some of the ana-
lytical methods are no longer available. However, the availability of CLIA
assays on semi-automated instruments may facilitate the transference
of established ARR thresholds.

Minimum aldosterone concentration Units

An elevated ARR reflects the pathophysiology of PA since high levels To compound the confusion, different laboratories report SAC in dif-
of aldosterone are secreted independent of the RAAS causing volume ferent units, including pg/mL, ng/dL, ng/L, and pmol/L while PRA can be
expansion and suppressed renin. It has been suggested that an elevated reported in ng/mL/h, pmol/L/min, nmol/L/h, and nmol/L/s and DRC in
ARR should be considered only when the SAC exceeds a minimum con- ng/L or mU/L. This leads to published ARR thresholds appearing widely
centration. The motivation for this recommendation is that even in low discrepant to medical students, residents, and physicians who fail to
aldosterone states, if renin is undetectable, the ARR may still be high, consider the system of units of the numerator and denominator.
depending on the lower reportable limit of the renin assay, leading to Laboratories should be vigilant in educating their users to employ
false positives [104,105]. While Young recommended a minimum SAC the ratio thresholds established by the reporting lab and displayed on
of 415 pmol/L (15 ng/dL) before a high ARR be considered a positive the report rather than consulting a textbook or online reference. Setting
screen for PA [106,107], it has been observed that in bona fide cases of a universal ARR threshold that will work across methods is not possible
PA, the ambulatory aldosterone level may be as low as 250 pmol/L given the analytical challenges described. This is underlined by a sys-
[108] and that about one-third of PA patients have ambulatory aldoste- tematic review of prospective trials using ARR as a screening tool
rone levels b415 pmol/L [109]. We have observed that if screening oc- where differences in patient populations, hypertensive medication
curs in the setting of ongoing intermittent hypokalemia (which it use, diet, posture during blood collection, analytical methods, and vary-
often does), a patient's ambulatory aldosterone could even go below ing confirmatory diagnostic strategies confounded interpretations and
200 pmol/L. Table 1 shows results observed in one case where adrenal prevented any generalizations about the most appropriate ARR; thresh-
olds employed or determined in the studies reviewed varied by more
than 10-fold [49]. It is therefore entirely appropriate that suggested
ranges for the ARR threshold are provided in the Endocrine Society
Guideline rather than any definitive threshold: 20–40 ng/dL:ng/mL/h
Table 1
ARR screening data and adrenal venous sampling (AVS) data on a patient who presented
for SAC/PRA and 3.8–7.7 ng/dL:ng/L for SAC/DRC [5]. In Canada, the
with hypertension and hypokalemia. It is noted that ambulatory aldosterone levels were most commonly reported units for SAC and PRA are pmol/L and
substantially lower than the minimum threshold of 415 pmol/L which has been recom- ng/mL/h respectively. In these units, the ARR ranges quoted above con-
mended [2]. “Pre” samples refer to those collected prior to administration of 250 μg ACTH vert to 550–1100 pmol/L:ng/mL/h for SAC/PRA or 66–135 pmol/L:mU/L
IV. Reference interval for aldosterone: 70–660 pmol/L. Reference interval for PRA: 0.36–
and 105–210 pmol/L:ng/L respectively for SAC/DRC [5].
4.00 ng/mL/h. Reference interval for ARR: b400 pmol/L:ng/mL/h. SI = selectivity index
(Eq. (2)). LI = lateralization index (Eq. (3)). Importantly, for specific methods, these thresholds may differ sub-
stantially from the Endocrine Society recommendations. For example,
Screening data
for the Diasorin Liaison instrument, an ARR cutoff of 35 pmol/L:mU/L
Day SAC PRA ARR K Post saline SAC (58 pmol/L:ng/L) has been suggested [95] (along with a minimum
(pmol/L) (ng/mL/h) (mmol/L) (pmol/L) SAC of 300 pmol/L) while for the Immunodiagnostic Systems iSYS,
0 312 b0.18 N1733 – – 30.5 pmol/L:mU/L (51 pmol/L:ng/L) has been found [110].
9 130 b2a N65a 4.0 – Given all the analytical complexities described, the ARR should al-
63 375 b0.18 N2083 3.8 –
ways be interpreted in the context of the full clinical picture: medical
41 – – – 3.3 111
71 209 b0.18 N1161 3.8 – and family history, severity of hypertension, resistance to antihyperten-
82 414 b0.18 N2300 – – sives, presence of adrenal adenoma, absolute aldosterone concentra-
182 – – – 3.4 98 tion, the presence of electrolyte abnormalities and any demonstrated
responsiveness to aldosterone antagonists.
Adrenal venous sampling data

Location SAC Cortisol SI A/C LI Patient preparation

(pmol/L) (nmol/L)
The preanalytical effects of diet, posture, time of collection and anti-
Pre-R 302 400 3.4 0.8 – hypertensives on the ARR are significant and a topic of frequent inquiry
Pre-L 2258 244 2.1 9.3 12.3
to laboratorians. However, the matter is thoroughly reviewed else-
Pre-IVC 102 119 – 0.9 –
Post-R 11,004 19,506 54.8 0.6 – where [5,99] and will not be discussed except to mention a few
Post-L 65,977 15,754 44.3 4.2 7.4 recommendations:
Post-IVC 277 356 – 0.8 –
a
Measured using renin mass assay (ng/L). Reference interval: 5–75 ng/L. Reference • SAC and renin should be collected in the morning before 10:00 am and
interval for ARR: b100 pmol/L:ng/L. after 1 h or more of ambulation.
 M. Rehan et al. / Clinical Biochemistry 48 (2015) 377–387
• Diet should be unrestricted.
• Antihypertensive medications cause both false negatives and false
positives. Medication free periods prior to screening should be consid-
ered if it is safe to do so [5]. In the specific case of potassium sparing di-
uretics (spironolactone, eplerenone, amiloride and triamterene) a
medication free period of 1 month is mandatory as they make the
ARR almost uniformly uninterpretable. Alpha blockers and verapamil
have the smallest effect on the RAAS and make suitable substitutes
during biochemical workup.
• Oral contraceptive agents [97], antidepressants [111], and certain
physiological states (phase of menstrual cycle [97], pregnancy [112],
untreated HIV [113]) may all variably affect the ARR depending on
whether PRA or DRC is used and results must ultimately be
interpreted with the clinical setting in mind.
Diagnostic tests
The ARR is only a screening tool and PA needs to be confirmed by a
diagnostic test before proceeding to subtype classification. Thorough
discussions of diagnostic testing protocols are available elsewhere [2,5,
114–116]. In brief, protocols are designed to suppress aldosterone
below a specific threshold, usually by expansion of the plasma volume.
These are: 1) the saline suppression test; 2) the fludrocortisone sup-
pression test; 3) the captopril suppression test and; 4) the oral salt load-
ing test. In the first three cases a post suppression SAC is used for
diagnostic purposes and in the oral salt load a 24 h urinary aldosterone
excretion is generally used.
For the purposes of this discussion, the challenge for the laboratorian
is assigning the diagnostic threshold for result reporting and clinician
consultation. The problem, which is frequently overlooked in clinical
studies and review articles, is that aldosterone assays have intermethod
biases of approximately 100% [59,60]. Renal impairment will further
confound the matter with analytical interferences. Many physicians
rely on literature sources for interpretation and may not understand
the analytical context adequately.
Taking the saline suppression test (SST) as a prototype of the diag-
nostic tests, it is not surprising that reported post-saline diagnostic
thresholds for SAC range from 138 pmol/L (5 ng/dL) [117] to 194
pmol/L (7 ng/dL) [118] and as high as 277 pmol/L (10 ng/dL) [119,
116]. It therefore behoves the clinical laboratorian to find or perform
studies specific to their analytical SAC methodology. Interestingly, in a
study exploring the recumbent vs SST test employing LC–MS/MS for
post-saline SAC analysis, it was observed that all (n = 11 of 11 in a
cohort of 24 PA) cases of IAH had post-saline levels of SAC below
140 pmol/L for a traditional recumbent SST. This seated version of the
SST appeared to have better test characteristics than the traditional re-
cumbent form [120].
While a thorough discussion of medication effects on confirmatory
tests is again unnecessary here, it must be understood that the diagnos-
tic thresholds described were established in the context of patients who
were free of medications that physiologically interfere with the renin
angiotensin aldosterone system [5,109]. They were also supplemented
with potassium and maintained eukalemic during investigations be-
cause hypokalemia suppresses aldosterone secretion even in the con-
text of PA.
Finally, it is important to emphasize that no matter which diagnostic
test is employed, the gold-standard for the diagnosis of PA is retrospec-
tive and clinical in nature, relying on response to treatment, consistent
histopathological findings, and post-operative biochemical resolution.
Subtype classification with adrenal venous sampling
Adrenal venous sampling (AVS) is the gold standard for the identifi-
cation of unilateral, surgically curable forms of PA. Blood samples from
383
the right adrenal vein (RAV), left adrenal vein (LAV) and inferior vena
cava (IVC) are collected and analysed for aldosterone and cortisol to
identify lateralized forms of PA based on calculations shown below.
Some sites administer ACTH infusion during collection while others col-
lect pre- and post-250 μg intravenous administration of ACTH.
From a technical standpoint, adrenal venous sampling is challenging
owing to the anatomy of the right adrenal vein (RAV). Unlike the left ad-
renal vein (LAV) which can be easily identified by its drainage into the
superior aspect of the left renal vein, the RAV drains directly into the
IVC in the neighbourhood of a number of small vessels: accessory hepat-
ic, phrenic or retroperitoneal [121]. The hepatic branches particularly
can mimic the RAV when injected with radiographic contrast [121].
Additionally, the RAV is small in calibre and short making it difficult
for radiological catheters to remain in situ during blood collection and
the low flow of the drainage means that aspiration may collapse the
vein. For this reason, catheters with side-holes may be preferable for
the collection [121,122].
Preanalytical pitfalls
Numerous samples are collected in radiology as part of the proce-
dure. Samples from both adrenal veins and the vena cava potentially
pre- and post-ACTH stimulation must be labelled in radiology, barcode
labels may be added in the laboratory and subsequently re-barcoded if
they are sent-out for analysis. The lab performing aldosterone and cor-
tisol analyses must be very careful to ensure that accurate labelling
has occurred at every step of the pre-analytical process. The samples
are typically all named similarly, all arrive simultaneously, and may
have identical or similar collection times. Review of the original requisi-
tions is frequently necessary. It may be helpful for the laboratory to es-
tablish a standard protocol with the radiology department to facilitate
correct sample handling.
Analytical issues
Adrenal venous (AV) aldosterone results are typically in the 104–
6
10 pmol/L range depending on whether the sampled adrenal gland is
normal, hyperplastic or tumour-bearing and whether ACTH was used
while AV cortisol results are in the range of 102–104 nmol/L. The results
will frequently but not predictably be outside the analytical range of any
IA or LC–MS/MS assay. For this reason, samples will need to be run neat
and at two other dilutions (such as 1:10 and 1:50 or similar) [122] be-
fore analysis. Obtaining absolute aldosterone and cortisol results is gen-
erally necessary. Non-serum diluents may introduce significant matrix
effects in IA methods which should be investigated before offering ana-
lytical services. All samples, neat and diluted, which may number 12
from AV (RAV and LAV, pre and post ACTH, at three dilutions each)
and two from the IVC, mean that there is significant room for sample
mixup in the analytical or post analytical phase.
LC–MS/MS methods offer the benefit of simultaneous analysis of al-
dosterone and cortisol on a routine basis and numerous related adrenal
steroids on a research basis [123,124]. However, care must be taken
during the drydown phase of liquid–liquid extraction methods because
of the phenomenon of “hotspot carryover” where wells with high con-
centration can contaminate adjacent wells with low concentration by
analyte dissolution in the solvent vapour [125]. Naturally, elimination
of carryover at all stages of the automated immunoassay or LC–MS/MS
process also needs to be excluded as part of method validation. AV sam-
ples with aldosterone levels of 104–106 pmol/L are commonplace after
ACTH stimulation—well outside the expected working conditions of au-
tomated IA.
It is worth noting that occasionally one notes putative RAV sam-
ples that are markedly depleted in aldosterone relative to the IVC,
with cortisol relatively spared. This finding is typical of accidental
cannulation of a hepatic accessory vein [126] and renders the proce-
dure uninterpretable.
384 M. Rehan et al. / Clinical Biochemistry 48 (2015) 377–387
Calculations
Technical success of the AVS procedure is gauged by the calculation
of the ratio of the right and left AV cortisol results to those of the IVC, as
shown in Eq. (2).
½Cort AV
SI ¼
½Cort IVC
[Cort]AV and [Cort]IVC are the concentrations of cortisol in the AV and
IVC respectively. This ratio, or “gradient” is called the selectivity index
(SI). A typical threshold for the definition of successful cannulation or
“selectivity” is 2 for unstimulated collections and 3–5 for ACTH-
stimulated collections [127,128]. Once bilateral selectivity is confirmed,
the lateralization index (LI) can be calculated using Eq. (3). The LI is the
ratio of the larger AV (“dominant”) aldosterone:cortisol ratio to the
smaller (“non-dominant”) aldosterone:cortisol ratio.


½AldoDom =½Cort Dom
LI ¼
ð3Þ
½AldoNon−Dom =½Cort Non−Dom
[Aldo]Dom and [Aldo]Non−Dom represent the aldosterone concentra-
tions in the dominant and non-dominant AVs respectively. Cortisol con-
centrations are abbreviated analogously. While the interpretation of the
LI is a matter of some debate [127,128], LI N 4 is generally accepted to
represent clinically significant lateralization indicating the presence of
a Conn's adenoma or unilateral hyperplasia. Results between 2 and 4
may be considered lateralized or indeterminate depending on the
study and whether or not ACTH was used [128]. LI b 2 is accepted to rep-
resent bilateral adrenal overproduction of aldosterone.
Laboratorians play a critical role in providing accurate results for the
calculations in Eqs. (2) and (3). Since both aldosterone and cortisol are
run neat and at 2 dilutions, the results whose diluted values are closest
to the middle of the analytical ranges of the assays used should be re-
ported. For IA assays, which suffer from matrix-related inaccuracy
more than LC–MS/MS assays, it is preferable to report results from the
LAV and RAV at the same dilution if possible.
Costs and benefits of ACTH stimulation
Although some specialty centres have reported technical success
rates for AVS N 95% [129], this has not been the case in smaller centres
where success rates may be as low as 0–10% [130,131] at sites
performing few procedures. The use of ACTH, by infusion [132,122] or
250 μg bolus [133], has been shown to increase the rate of biochemically
proven selectivity of the RAV and LAV because it exaggerates the pro-
duction of both aldosterone and cortisol bilaterally and has the added
benefit of preventing misleading spikes in aldosterone and cortisol re-
lated to patient anxiety. In this sense ACTH rescues a significant propor-
tion of AVS procedures from uninterpretability [131]. The down-side of
ACTH is that in cases of unilateral adrenal overproduction, it stimulates
the unaffected gland leading to spurious decreases in lateralization
[131,134,135]. For this reason, it is our practice [131] and that of others
[133,136,134,137] to collect from the AVs both pre and 10–15 min post
250 μg ACTH stimulation to have the best opportunity for interpretable
results.
An alternative approach to improve the accuracy of the cannulation
is the use of intra-procedural cortisol analysis to confirm bilateral selec-
tivity before completion of the AVS procedure [138–140], but this is not
easily accessible in most hospital environments.
Recently, there has been some interest in analysis of other adrenal
steroids which show promise in being more sensitive markers of selec-
tivity which may obviate the need for ACTH simulation in the future
[123,124]. Use of catecholamines as markers of selectivity has also
been advocated for PA [141] having been previously used in cases of
ACTH-independent Cushing Syndrome for patients with bilateral adre-
nal adenomas [142].
Conclusion
The use of SAC and renin for the screening and diagnosis of aldoste-
ronism is an important area for close interaction between the laborato-
ry, the endocrinologist/hypertension specialist and the interventional
radiologist. The laboratory plays a critical role in providing suitable test-
ð2Þ
ing with appropriate interpretative information for the specific methods
used in the laboratory. There are many nuances and caveats to the anal-
ysis of SAC and renin and careful attention to detail is required in the
handling and processing of samples. The development of clinical LC–
MS/MS methods has improved the analytical reliability and the avail-
ability of commercial CLIA assays may improve the standardization of
reference intervals and thresholds. This review has highlighted these is-
sues and will hopefully enable laboratories and clinicians to better un-
derstand these important tests.
Funding
None.
Acknowledgements
DH would like to thank J Grace Van Der Gugten, Suzette Walsh,
Lynne Coleman, Tracey Ayotte, Eden Tiu, Margaret McLean, and Bill
Prest for their outstanding contribution to the St. Paul's Hospital Mass
Spectrometry Laboratory and to BC's PA screening programme. Teresa
Feduszczak is acknowledged for her tireless work in keeping RIA assays
running and for carefully supervising the transition to automated
chemiluminescent assays for the Hamilton Regional Laboratory Medi-
cine Program.
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