for the diagnosis
of
hemodialyzed
patients.
J
crinol
Metab 1991;73:516-24.
6. Blind E, Schmidt-Gayk
H,
al. Two-site
assay
of intact
hormone
in
bone disease
Clin Endo-
Scharla S, et
parathyroid
investigation
of primary
and other disorders
in the
hyperparathyroidism
of calcium
metabolism
hormone
compared
with
a
midregion
assay. J Clin Endocrinol
Metab
1988;67:353-60.
7. Bouillon R, Coopmans,
W, Degroote DE,
Radoux D, Eliard PH. Iminunoradiometnic
assay
of parathyrin
with polyclonal
and
monoclonal region-specific
antibodies.
Clin
Chem 1990;36:271-6.
8. Schmidt-Gayk
H, Schmitt-Fiebig
M,
Hitzler W, Armbruster
FP, Mayer E. Two
homologous
radioimmunoassays
for parathynin compared
and applied to disorders
ofcalcium
metabolism.
Clin Chem 1986;32:
57-62.
9. Newman
III, Ashby JP. Clinical
and
laboratory
noradiometric
evaluation
of a two-site immufor intact parathyroid
Ann Chin Biochem
1988;25:654assay
hormone.
60.
10. Dilena
BA, White
GH. Interference
with measurement
of intact parathyrin
in
serum from renal dialysis
patients.
Chin
Chem 1989;35:1543-4.
11. Bonnin MR, Gonzalez MT, Huguet J,
Navarro
MA. Intact parathynin
measured
in serum from patients
with chronic
renal
failure [Letter]. Clin Chem 1990;36:175-.6.
Eberhard
Friedheim
Helmut
Blind’
Raue’
Reichel2
1 Endocrinol.
nd2
Div. of Nephrol.
Dept. oflntern.
Med. I.
Univ. of Heidelberg
Bergheimer
Str. 58
D 6900 Heidelberg,
Germany
Heinnich
3
Im Breitspiel
Heidelberg,
Schmidt-Gayk3
15
Germany
Interference
immunoassay
in the Allegro
Is Collected
in Silicone-Coated
System
When
per liter,
in blood drawn
25 and
45 days after the birth ofa premature
infant
before
treatment
for primary
hypothyroidism.
Both capillary
blood
samples
were collected
in Microtainer
Tube
serum-separator
collection
tubes (no. 5960; Becton Dickinson
VamIU
Blood
Tubes
To the Editor:
Many
bioanalytical
systems
have
incorporated
the high-affinity
avidinbiotin binding
interaction
as a critical
step in measuring
a target
molecular
complex
(1, 2). We report here extensive interference
in a commercially
available
immunoradiometric
assay
system
(Allegro;
Nichols
Institute
Diagnostics,
San Juan Capistrano,
CA)
in which
avidin-biotin
binding
is
used to separate
bound and free radioactively
labeled
antibody
(3).
Interference
in the Allegro
system
was noted after
we observed
normal
thyrotropin
(TSH) values,
3.9 and 1.8
cutainer
Systems,
Rutherford,
NJ).
However,
above-normal
TSH values
(42 and 23 mIU/L)
had been reported
from
dried
capillary
blood specimens
from blood
drawn
18 and
45 days
after
birth
and submitted
on filter
paper
to the
Wisconsin
Newborn
Screening
Program.
Furthermore,
in
the Allegro
system,
recovery
of added
TSH was decreased
from
100% to only
30% whenever
serum
specimens
obtamed
from capillary
blood collected
in Microtainer
no. 5960 were mixed
in equal
parts with a human
serum
pool
that
contained
TSH
at 21.6
mIUIL.
We also tested
“no additive”
Microtainer
Tubes
(no. 5962), placing
0.3 mL of the above
human
serum
pool directly
in tubes from two different lot numbers;
recovery
of TSH
ranged
from
14% to 24%. The Microtamer
no. 5962 Collection
Tube does
not contain
silicone
serum
separator
but does contain
the same
interior
water-soluble
silicone
polymer
coating found in the Microtainer
no. 5960
Serum
Tube
(personal
communication,
Becton
Dickinson
Technical
Services).
The
inhibiting
material,
presumably
the
water-soluble
silicone polymer
found in the above collection
devices,
was
completely
removed
by washing
the interior
of the
tube once with distilled
water.
We also tested
the following
bloodcollection
tubes
for the presence
of
inhibitor:
Becton
Dickinson
glass Vacutainer
Tube
(no. 6490);
Samplette
(no. HRI 8881-008034)
and Monoject
glass
tubes
(no. HRI
8881-301314;
Sherwood
Medical,
St. Louis,
MO);
and
Terumo
Capiject
product
T-M
(Terumo,
Elkton,
MD). These
bloodcollection
tubes
do not contain
additives but also have
a silicone-coated
interior.
When
0.3 mL of human
serum
pool
(TSH
21.6
mIU/L)
was
placed
in these tubes, recovery
of TSH
measured
by the
Allegro
system
ranged
from only 12% (Sherwood
no.
HRI 8881-008034)
to 70% (Sherwood
no.
Hill
8881-301314).
We also tested the blood-droplet
collection
devices
found
on the Microtamer
no. 5962
and the Sherwood
Samplette
no.
HRI
8881-008034.
When the Allegro
TSH assay is used,
sampling
0.3 mL ofhuman
serum pool
(TSH 21.6 mIU/L)
with the collectors
of these two products
decreases
recovery from 100% to 15% (Sherwood
col-
CLINICAL
hector) and 52% (Becton
Dickinson
colhector).
The Allegro
human
choriomc
gonadotropin
(hCG) and prolactin
assays
also yielded
<30% recovery
from human serum
placed
in Microtainer
no.
5960.
These
Allegro
assays
also use
avidin-biotin
binding
as a final step to
separate
bound
radiolabeled
complex
from free radiolabeled
antibody.
However, the Allegro
intact
parathyroid
hormone
assay,
which
does not use
avidin-biotin
separation,
was not a!fected.
We investigated
the nature
of the
interference
by exposing
the 0.8-cm
avidin-coated
polystyrene
bead
used
in the Allegro
separation
step to dilutions of the assumed
inhibitor
(inhibitem solution
was prepared
by rinsing
the Microtainer
no. 5962
collection
tube with 0.3 mL of distilled
water).
Allegro
avidin-coated
beads
were
shaken
for 30 mm at room temperature
with
0.1 mL of distilled
water
containing
0, 1:1, 1:4, 1:10, 1:20, and
1:50 dilutions
of inhibitor
solution;
each solution
was tested
in duplicate.
The inhibitor
solution
was removed,
and the beads were put into new test
tubes to eliminate
any residual
fluid.
We then used the beads according
to
the manufacturer’s
instructions
to assay a quality-control
pool of human
serum
previously
determined
to have
a TSH
value
of 21.6 mIUIL.
Undiluted, 1:1, 1:4, 1:10, 1:20, and 1:50
dilutions
ofinhibitor
gave TSH recoveries of 3%, 8%, 22%, 49%, 70%, and
85%, respectively.
Beads
treated
as
described
above
with distilled
water
containing
no inhibitor
yielded
85%
recovery
of TSH.
We also added inhibitor
to the AiMgre TSH assay
at the end of the incabation
step, when
the avidin-biotin
binding
reaction
is complete.
Adding
0.1 mL of undiluted
inhibitor
solution
at this point in the assay rather
than
at the start ofthe incubation
increased
recovery
ofTSH
from 24% to 77%.
We conclude
that a water-soluble
silicone
polymer
currently
in use in
several
commercially
available
bloodcollection
devices
can,
if
present
in
high
with
and
least
enough
concentration,
interfere
the avidin-biotin
binding
step
greatly
reduce
recovery
in at
three
different
Allegro
assays.
Although
we have
not tested
all of the
Allegro
products
that
use
avidincoated
beads,
we suspect
that others
may be affected
by this interference.
The interference
appears
to reduce
the ability
of the avidin
bead to bind
radiolabeled
complex
and competes
with biotin
for access
to the binding
sites on the coated
beads.
Manufac-
CHEMISTRY,
Vol. 38, No. 11, 1992
2347
do use different
concentrations
ofsilicone
polymer
to coat the interior
ofblood-collection
tubes. Becton Dickinson
Technical
Service
indicates
that
their
Microtainer
Capillary
Blood-Collection
Tubes
(nos.
5960
and 5962)
are coated
with
10 g/L
solutions
of silicone
polymer
rather
than the 4 g/L solution
used in some
of the glass
Vacutainer
Tube
products. Until
this problem
is resolved,
we prefer
to use blood-collection
dovices that are not silicone
coated.
Because
manufacturers
of blood-collection
devices
are not required
to
state
that
interior
silicone
coating
is
used, laboratories
that use the Allegro
assay system
should
contact
the manufacturer
ofthe collection
devices
they
use and confirm
that the interior
of
these
products
is not coated
with
a
silicone
polymer
that
could
interfere
with the assay.
Alternatively,
bloodturers
collection
interference
collect
devices
should
before
they
patients’
samples.
be tested
are used
for
to
References
1_ Diamandis
EP, Christopoulo
TK. The
biotin.-(strept)avidin
system:
principles
and applications
in biotechnology
(Review]. Chin Chem 1991;37:625-36.
2. Wilchek
M, Bayer
EA. The avidin-biotin complex
in bioanalytical
applications
[Review]. Anal Biochem
1988;171:1-32.
3. Odehi WD, Griffin J, Zahradnik
R. Two
monoclonal-antibody
sandwich-type
assay
for thyrotropin,
with use of an avidinbiotin separation
technique.
Clin Chem
1986;32:1873-8.
Gary
Robert
Elizabeth
Gundersen
LaCrosse
La Crosse,
Clinic/Lutheran
after
Hosp.
WI 54601
Chromogranin
Plasma
G. Wickus
J. Mordan
A. Mathews
A Concentrations
of Physically
Acute
Active
in
Men
Exercise
To the Editor:
Chromogranin
A is an acidic monomeric
protein
that
is present
along
with catecholamines
in storage
vesides in sympatho-chromaffin
cells in
the adrenal
medulla
and elsewhere
(1-3).
Chromogranin
A is coreleased
from these
vesicles
along
with catscholamines
(1, 4). Recently,
measurement
of chromograrnn
A has been
used to investigate
patients
with pheochromocytoma
and other forms of hypertension
(5). Before the commercial
availability
ofchromogranin
A assays,
screening
of hypertensive
patients
2348
CLINICAL
CHEMISTRY,
thought
to have
pheochromocytoma
was based on measurement
of urinary
concentrations
of catecholamines
and
their metabohites
(6) or measurement
of serum
catecholamines
(7, 8). Because catecholamine
concentrations
in
plasma
are greatly
influenced
by postune, physical
activity,
stress,
and numerous
medications
(9),
screening
tests for pheochromocytomas
that rely
on measurement
of urine
or plasma
catechohamine
and their metabolites
must
take
into account
and exclude
these interfering
factors.
Plasma
chromogranin
A concentrations are not significantly
influenced
by posture,
venipuncture,
or many
medications
that
interfere
with mensurement
of catecholamines
or their
metabohites
(10). The effect of physical
activity
on plasma
chromogranin
A
concentrations
has not been fully investigated,
but physical
activity
is
known
to increase
concentrations
of
plasma
catecholamines.
We studied
the effect of strenuous
physical
activity on plasma chromogranin
A concentrations
in physically
active
male volunteers,
specifically
evaluating
the
rise in chromogranin
A after
such oxercise for comparison
with previously
reported
values
for patients
with essential
hypertension
and pheochromoc-s.
Eight men, ages 20-42 years (mean
± SD, 26.6 ± 7.1 years),
participated
in the study. They weighed
58-94.5
kg
(mean ± SD, 70.6 ± 13.0 kg) and were
154-185.4
cm tall (mean
± SD, 192.9
± 10.4 cm). Although
serum
creatinine
concentrations
were
not meastired,
no volunteers
had a history
of
renal
impairment.
All studies
were
performed
between
0930 and 1000 a!ten an overnight
fast.
All exercises
were performed
on a
motorized
treadmill
(Marathon
7000;
Cal-Mod,
Brea, CA). Ventilatory
mensurements
during
exercise
were made
on a breath-by-breath
basis,
and values were averaged
and reported
for
each 15-s interval.
Subjects
breathed
through
a Model 2700B
valve (HansRudolph,
Kansas
City,
MO;
dead
space
90 mL). Expired
air was sampled just distal
to the valve and was
routed
via capillary
tubing
into a mass
spectrometer
(Model
1100;
PerkinElmer,
Pomona,
CA) at the rate of
45-60 mlimin.
The Hans-Rudolph
valve
was connected
to a pneumotachograph
(Model
50MC2-2;
Meriam,
Cleveland,
OH)
that
was connected
to a differentialpressure transducer and a carrier
demodulator
(Model
CD-15;
Validyne,
Northridge,
CA). Analog
signals,
based
on samphingevery
15 ms, were digitized
Vol. 38, No. 1 1 1992
,
(Keithly
System
Model 570; Data Acquisition
and Control,
Cleveland,
OH).
The digitized
signals
were
sent
to a
computer
(IBM-AT
clone;
Micro
Express 286, Santa Ana, CA), where
metabolic measurements
were calculated
in
real time and analyzed.
The flow signal
from the pneumotachograph
was integrated
to determine expired
minute
ventilation
(V5),
oxygen
uptake
W92), and carbon dioxide production
(VCO2).
Respiratory
exchange
ratios were calculated
from
VE and exhaled
gas measurements.
Cardiac
rhythm
was
continuously
monitored
with a simultaneous
threelead electrocardiogram
(Model
ECG
3A;
Brentwood
Instruments,
Tonrance, CA).
Exercise
testing
was performed
by
using
the Bruce
multistage
protocol
with
a 3-mm
preexercise
baseline
(standing)
stage
followed
by a 3-mm
warm-up
at 0.765
rn/s 5% grade
(11).
An electrocardiogram
strip was taken
20 s before
the end of each
minute
of
the test. The Borg scale of rating
of
perceived
exertion
(RPE)
(12),
indicated with finger signals
from 6 to 20,
was obtained
near the beginning
and
end of each stage.
The mean
total
exercise
time,
excluding
warm-up,
was 12.5-17
mm (mean ± SD, 14.4 ±
1.4 miii).
All eight
volunteers
cornpleted at least four stages ofthe Bruce
protocol.
Perceived
exertion
at the
conclusion
of exercise
was
19-20.
Blood
was removed
for hormone
mensurements
within
1 ruin after exercise.
Peak
V02
(VO2max)
was
obtained
from measurements
during
the maximum power level achieved.
After
30 mm of recumbency,
blood
was removed
from
the antecubital
vein
via
a hepaninized
indwelling
catheter
for measurement
of baseline
plasma
concentrations
of chromogranin A. Blood was then removed
immediately
after exercise
and at 15, 30, 60,
90, and 180 mm after
exercise.
The
plasma
was separated
and stored
at
-70 #{176}C
until
assayed.
Plasma
chromogranin
A was
measured
by radioimmunoassay
by using
lasIlabeled
chromogranin
A and rabbit
antisera
to purified
chromogranin
A (13). Separation
ofbound
from free chromogranin A was accomplished
by using
a
second
antibody
(goat
anti-rabbit
y-globulin).
The
lower
limit
of sensitivity of the assay
is 1.5 gfL
at 90%
BIB0. Intra-assay
variation
was <8%
and interassay
variation
was <12%.
Serum
lactic acid concentration
was
measured
by
standard
laboratory
techniques
(14) in all subjects
within
30 s after the completion
of exercise.
Statistical
analysis
was performed