Validation of a New Automated Renin Assay
Rene ´ A. de Bruin,1Angelique Bouhuizen,1Sven Diederich,2Frank H. Perschel,3
Frans Boomsma,1and Jaap Deinum1,4*
Background: Measurement of plasma renin is impor-
tant for the treatment of patients with congenital adre-
nal hyperplasia (CAH) and in the evaluation of pa-
tients with suspected hyperaldosteronism. Immuno-
logic assays for plasma renin offer easier implementa-
tion and standardization than enzyme-kinetic assays for
plasma renin activity, but their sensitivity and specific-
ity have been questioned. We studied a renin immuno-
chemiluminescence assay on an automated platform.
Methods: Renin was measured by an enzymatic assay,
by IRMA, and by the new Nichols Advantage Specialty
System immunochemiluminometric assay (ICMA), in
plasmas from unselected individuals from our outpa-
tient departments and in samples from patients with
Results: The detection limit in the ICMA was 0.1 mU/L.
The recovery was >90%, and the imprecision (CV) was
generally <9%. Mean (SD) concentrations measured by
ICMA were 32 (21)% lower than those measured by
IRMA. Renin concentrations as measured by ICMA
were identical in serum and EDTA-, heparin-, and
citrate-anticoagulated plasmas. Prolonged incubation of
whole blood at room temperature before centrifugation
did not affect renin concentrations. The central 95%
interval for 80 healthy adults was 6–85.5 mU/L. Plasma
renin as assessed by ICMA in patients with primary
hyperaldosteronism was <0.2 mU/L.
Conclusions: The performance characteristics of the
new renin ICMA allow its use for patients with CAH
and for the diagnosis of mineralocorticoid hypertension.
In view of the variability of renin concentrations, use
for other forms of hypertension or physiologic re-
search calls for the development of uniform sampling
© 2004 American Association for Clinical Chemistry
Measurement of plasma renin content is generally
thought to be the most practical method to determine the
activity of the renin-angiotensin system. Previously, renin
was measured by its enzymatic, angiotensin I-generating
activity on its endogenous substrate, the so-called plasma
renin activity (PRA).5This assay is laborious and has poor
intra- and interlaboratory reproducibility. This may be
one of the reasons that renin measurements have been
established only for the clinical diagnosis of primary
hyperaldosteronism and the treatment of congenital ad-
renal hyperplasia (CAH).
For the past 15 years, direct measurement of renin by
IRMAs has also been possible (1, 2). These IRMAs have
several advantages over the enzymatic kinetic assays:
they are less laborious and have the potential of better
standardization because IRMAs are much less prone to
interlaboratory variation. On the other hand, the specific-
ity of the IRMA method has been questioned because the
inactive precursor of renin, prorenin, may be converted
on prolonged incubation at room temperature into a
renin-like conformation that leads to comeasurement of
prorenin as renin (2, 3). This problem can be overcome by
shorter incubation at a higher temperature (4).
The problem of overestimation of renin arises espe-
cially at low renin concentrations, which occur in states of
salt loading and in primary hyperaldosteronism. Here the
PRA method may have an advantage because low renin
activity might still be measured by extending the incuba-
tion time. However, angiotensin I production in this assay
is not linear over time, probably because of loss of
angiotensin I on prolonged incubation (5). Here we report
the performance of a new immunometric method on an
1Department of Internal Medicine, Erasmus Medical Center, Rotterdam,
2Medizinische Klinik IV (WE28) and3Institut fu ¨r Klinische Chemie und
Pathobiochimie, Freie Universita ¨t Berlin, Klinikum Benjamin Franklin, Berlin,
4Department of Medicine, University Medical Center Nijmegen St. Rad-
boud, Nijmegen, The Netherlands.
*Address correspondence to this author at: Department of Medicine-541,
University Medical Center St. Radboud, Geert Grooteplein 8, 6525 GA Nijme-
gen, The Netherlands. Fax 31-24-3541734; e-mail email@example.com.
Received January 27, 2004; accepted July 8, 2004.
Previously published online at DOI: 10.1373/clinchem.2004.032052
5Nonstandard abbreviations: PRA, plasma renin activity; CAH, congenital
adrenal hyperplasia; NASS-ICMA, Nichols Advantage Specialty System im-
munochemiluminometric renin assay; and ND-IRMA, Nichols Diagnostics
Clinical Chemistry 50:11
automated multianalyzer system. This method uses a
short incubation, which makes comeasurement of prore-
nin unlikely. We compared the results of this method with
those of a PRA assay and a conventional IRMA that is
resistant to prorenin comeasurement.
Materials and Methods
nichols advantage specialty system for renin
The Nichols Advantage Specialty System (NASS) is an
automated immunoanalyzer. The antigen of interest is
sandwiched between a magnetic particle solid-phase
capture antibody and an acridinium-labeled tag antibody.
Quantification is by acridinium ester chemiluminescence
detectionfor this immunochemiluminometric
(ICMA). For the renin assay (NASS-ICMA), the solid-
phase antibody is biotinylated R3-36-16, which is bound
to streptavidin-coated magnetic particles during the incu-
bation, and the second, acridinium-labeled antibody is
R1-20-5. R3-36-16 recognizes both prorenin and renin,
but R1-20-5 is specific for renin. Detailed information on
the antibodies is given in Ref. (6). All materials for the
NASS-ICMA were provided by the manufacturer. The
instrument uses 200 ?L of the plasma sample per test and
requires a dead volume of 200 ?L. Briefly, 200 ?L of the
plasma sample and the two anti-renin antibodies (30 ?L
each) are added to a disposable cuvette and incubated for
20 min at 37 °C. Afterward, 20 ?L of the magnetic
particles (suspended in phosphate-buffered saline, ?0.95
g/L sodium azide, and ProClin-300) and 50 ?L of the
assay buffer (normal saline, sheep serum, ?0.95 g/L
sodium azide, and ProClin-300) are added and incubated
at 37 °C for another 10 min. After a washing step, the
acidic (hydrogen peroxide and nitric acid, both in a final
concentration of 10 mL/L) and alkaline (sodium hydrox-
ide in a final concentration of 10–30 g/L) trigger solutions
are injected into the cuvette. The emitted light, expressed
in relative light units, is measured by the integrated
system luminometer and is directly proportional to the
concentration of renin (mU/L). The system is calibrated
by a two-point recalibration against a stored master curve.
First results are available after 40 min (20 ? 10 min of
incubation and 10 min of pipetting, transport, and mea-
suring steps), and the throughput is 85 results per hour.
The maximum loading capacity of the Advantage system
is 120 samples.
The IRMA for renin (ND-IRMA) was purchased from
Nichols Diagnostics and was performed as described by
Deinum et al. (4). This assay uses the same antibodies as
the Advantage specialty system. Measurements were per-
formed in duplicate. To 200-?L aliquots of untreated
plasma or renin calibrators we added 100 ?L of a 1:1
mixture of biotinylated monoclonal antibody R3-36-16
(0.5 mg/L) and radiolabeled (?250 000 cpm) monoclonal
antibody R1-20-5. This was followed by incubation of this
mixture with an avidin-coated polystyrene bead for 6 h at
37 °C (IRMA37 °C, 6h). This incubation minimizes comea-
surement of prorenin as renin (4). After incubation, the
beads were washed three times and transferred to a clean
tube. Radioactivity of bound antibody was counted for 5
min in a gamma counter. In our hands, this assay has a
detection limit of 1.3 mU/L and a recovery and impreci-
sion (CV) generally ?90% and ?10%, respectively (4).
For the PRA assay we used a modification of the method
proposed by Sealey (7). The method includes blank
subtraction, and the 18-h incubation step for low renin
samples is eliminated. In this assay, 50 ?L of maleic acid
(pH 5.7) and 12.5 ?L of a protease inhibitor solution
consisting of 1 volume of 0.287 mol/L phenylmethylsul-
fonyl fluoride in ethanol, 2 volumes of 0.5 mol/L diso-
dium EDTA, and 2 volumes of 100 g/L neomycin sulfate
were added to 0.5 mL of plasma. The mixture was then
incubated at 37 °C for at least two time periods between
0.5 and 3 h to check for linear angiotensin I generation.
The generated angiotensin I was quantified by RIA (8).
Results are expressed as nmol ? L?1? h?1. The lower limit
of detection was 0.08 nmol angiotensin I ? L?1? h?1.
To carry out our performance studies for the NASS-
ICMA, we used plasma samples selected on the basis of
their renin concentration and stock volume.
Samples were from unselected individuals recruited from
our outpatient hypertension clinic. In addition, samples
were studied from patients with specific diagnoses, such
as patients with diabetes (n ? 17; age range, 19–50 years),
patients with renal artery stenosis (not on beta-blockers or
angiotensin-converting enzyme inhibitors; n ? 9; age
range, 48–66 years), from pregnant women with (n ? 11;
age range, 24–39 years) or without (n ? 11; age range,
20–32 years) preeclampsia, and from women on oral
contraceptives (n ? 7; age range, 24–37 years). Renin was
measured for diagnostic purposes in three individuals
(32, 62, and 60 years of age) with primary hyperaldoste-
ronism. Renin was also measured in 14 children and
young adults with CAH (age range 4–20 years) to titrate
mineralocorticoid dose. To determine the central 95%
interval, we studied 80 individuals (age range, 16–75
years; 25 males) with normal blood pressure and without
medication or specified salt intake. All participants gave
informed consent, and procedures observed the rules
imposed by the Helsinki Declaration. Blood was taken
from an indwelling venous catheter after the individual
had been in the supine position for at least 45 min. The
normotensive controls were sampled when they were in a
seated position. Anticoagulation was with citrate (0.2 mL
of 0.646 mol/L per 10 mL of blood) or, for sample type
de Bruin et al.: New Rapid Renin Assay
comparison with plasma, in heparin- or EDTA-containing
tubes (Vacutainer, BD Pre-analytical Solutions).
performance of the nass-icma
Sensitivity. The limits of detection, defined as the mean
result of the assay plus 3 SD in two series of 20 runs of the
sample diluent, were 0.013 and 0.094 mU/L, respectively.
The functional sensitivity, defined as the lowest renin
concentration at which the CV in four series of five assay
runs was ?20%, was 2.65 mU/L (not shown). Although
no official conversion factor between U and moles for
renin has been established, this amounts to a functional
sensitivity of ?65 amol/L (1 mU ? 1 pg of renin of Mr
Precision. The intraassay variation of three samples with
various renin concentrations ranged from 1.7% to 5.3%.
Interassay variation in seven samples with renin concen-
trations from 10 to 466.5 mU/L was 2.7–8.2% (Table 1).
Linearity. Dilution of four plasma samples with the sam-
ple diluent gave parallel lines whose slopes did not differ
(Fig. 1). Measurement of renin in two series of mixtures
prepared from two plasma samples with low and high
renin content, respectively, yielded renin values that did
not differ from expected values (Table 2).
Plasma samples from 102 individuals were assessed for
renin concentration by ND-IRMA and by NASS-ICMA
and for PRA by enzyme kinetic assay (Figs. 2 and 3). The
rank correlation coefficient for the PRA and NASS-ICMA
was 0.88 (95% confidence interval, 0.83–0.92) and for the
ND-IRMA and NASS-ICMA was 0.92 (0.89–0.95). Bland–
Altman analysis (not shown) showed that the results
obtained with the NASS-ICMA were 32% lower than
those obtained with the ND-IRMA over the entire range
of values. The NASS-ICMA results for controls and spe-
cific patient groups are shown in Fig. 4. As expected, renin
values were low in three patients with primary hyperal-
dosteronism (0.01, 0.01, and 0.02 mU/L, respectively) and
highly increased in patients with heart failure. The central
95% interval for healthy adults, sampled in the sitting
position, was 6–85.5 mU/L.
In whole blood left at room temperature, the plasma renin
content remained stable for a period of 72 h (Fig. 5).
Stability was less if blood was centrifuged immediately
and serum was left at room temperature.
sample type comparison
Measurement of simultaneously taken plasma and serum
samples yielded comparable results, although variance
Fig. 1. Linearity of the NASS-ICMA.
Dilution test using four plasmas. Y axis shows the renin concentration as
determined with the NASS-ICMA.
Table 1. Intra- and interassay CVs for the NASS-ICMA.
Renin concentration, mU/L
5.3 (n ? 20)
1.7 (n ? 15)
1.8 (n ? 10)
8.2 (n ? 12)
7.5 (n ? 12)
3.5 (n ? 20)
6.2 (n ? 12)
2.7 (n ? 20)
2.8 (n ? 20)
3.3 (n ? 12)
aND, not determined.
Table 2. Recovery of renin in two plasma samples.a
aIn experiment I, a plasma containing 30.3 mU/L renin was mixed with a
plasma pool containing 387.2 mU/L, and in II, a plasma with 59.4 mU/L renin
was mixed with a plasma pool containing 350 mU/L. The volume-to-volume
ratios in both experiments were 1:6, 2:4, 1:1, 4:2, and 6:1, respectively.
Clinical Chemistry 50, No. 11, 2004
increased at very low concentrations (Fig. 6). This indi-
cates that the NASS-ICMA provides similar results with
serum and plasma, the latter irrespective of type of
Previously, we demonstrated that renin could be reliably
measured with an IRMA (ND-IRMA) that used the same
antibodies as the NASS-ICMA (4). One prerequisite was
that the incubation was performed only for 6 h and at
37 °C to prevent a slow room-temperature-induced con-
formational change in prorenin, the enzymatically inac-
tive precursor of renin, which would cause prorenin to be
measured as renin. Theoretically, the risk of comeasure-
ment of prorenin as renin in the NASS-ICMA is very low
because of the very short processing time (30-min incu-
bation at 37 °C and 10 min for transport and measure-
ment). This is confirmed by the good agreement between
the assay results obtained with the NASS-ICMA and the
ND-IRMA. The NASS-IRMA is therefore useful for clini-
cal management of patients with CAH and for the diag-
nosis of primary hyperaldosteronism. The sample-pro-
cessing procedure is not critical, as evidenced by the
stability of the result after prolonged storage of whole
blood at room temperature. The NASS-ICMA yields iden-
Fig. 2. Relationship between renin values measured by the PRA
method and by the NASS-ICMA (n ? 102).
Fig. 3. Relationship between renin values measured by the ND-IRMA
and by the NASS-ICMA (n ? 102).
The dashed line represents the line of identity.
Fig. 4. Renin concentrations in plasma samples from patients in
different clinical categories.
cah, congenital adrenal hyperplasia (n ? 12); hf, heart failure (n ? 5); dm,
diabetes mellitus, insulin-dependent (n ? 17); oc, women on oral contraceptives
(n ? 7); preecl, preeclamptic women (n ? 11); pregn, pregnant women (n ? 11);
pha, primary hyperaldosteronism (n ? 3); ras, renal artery stenosis (n ? 9); co,
normotensive individuals (n ? 80). The central 95% interval for controls is
Fig. 5. Effect of prolonged incubation at room temperature on renin
concentration (NASS-ICMA) in whole blood (f) and serum (Œ).
Results are the means of samples from three individuals.
de Bruin et al.: New Rapid Renin Assay
tical results for serum and plasma. The sensitivity and
linearity of the NASS-ICMA are excellent.
Of some concern may be the observation that the
NASS-ICMA yielded lower values than the ND-IRMA.
This may be caused by use of a different reference sample,
but we did not evaluate the cause of this discrepancy
because it was consistent over the entire range of renin
values. Moreover, it is not the major obstacle to interlabo-
ratory comparison of ICMA results at this moment; uni-
form sampling conditions are more important (see be-
The NASS-ICMA also correlated well with PRA, al-
though this does not mean that it can replace the PRA
assay in all circumstances. For example, oral contracep-
tives lower plasma renin concentrations (see Fig. 4) but
generally do not influence PRA (9). This divergence
between plasma renin concentration and PRA is ex-
plained by the large increase in angiotensinogen during
contraceptive use. Because the PRA assay is based on the
action of renin on endogenous angiotensinogen, which
usually circulates at a concentration close to the Km, the
increase in angiotensinogen and the decrease in renin
during contraceptive use appear to balance out. This
suggests that the PRA is regulated and that with changes
in angiotensinogen concentration, such as during contra-
ceptive use or pregnancy (9), the PRA may be more
Immunologic renin assays have better potential for
international standardization than do PRA methods (10).
This standardization is much in need because its lack has
seriously hampered use of renin measurements in clinical
situations and also makes the contribution of plasma
renin to disease difficult to study (11). For example, it has
been claimed that the PRA assay can be used as an aid for
making therapeutic choices in hypertension (12). Al-
though this concept is attractive, the development of
guidelines based on PRA results has not been possible,
undoubtedly in part because of the considerable inter-
and intralaboratory variation for the PRA assay (10).
Renin immunometric assays have several properties
that may lead them to become the standard method in
cases where angiotensinogen concentrations are normal
and the renin concentration and PRA correlate well. They
can be calibrated against an international reference stan-
dard. The NASS-ICMA for renin may have additional
advantages over the ND-IRMA in that the assay car-
tridges are prepared centrally and the assay is executed
on a single type of automated instrument. The latter
ensures uniform procedures and permits high-through-
There are two issues that need to be addressed. The
first issue involves the establishment of a calibrator for
renin immunoassays. We found a consistent proportional
difference between the results obtained with the IRMA
and the ICMA, which suggests that both assays use a
different calibrator. The second, more important issue is
the blood sampling protocol. Renin concentrations vary
with posture, activity, and medication use (13); therefore,
interlaboratory comparisons (or establishment of univer-
sal reference values) may not be possible when sampling
conditions differ. In our own laboratory, we have individ-
uals rest for 45 min, but in an outpatient clinic this may
not be practical. It is therefore highly recommended that
laboratories adopt standardized blood-sampling proto-
cols. With these protocols, reference values can be devel-
oped for healthy and hypertensive individuals that may
serve in the diagnosis and treatment of patients with CAH
In summary, the new rapid renin NASS-ICMA has a
performance that at least equals that of an optimized
ND-IRMA. The ICMA is easy to use and seems suitable
for use in the clinical setting as well as for physiology
Nichols Institute Diagnostika GmbH (Bad Vilbel, Ger-
many) made available the Nichols Advantage Specialty
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