Supplemental Data Table 3: Summary of included studies on PTH stability in whole blood measured by second generation assays. Studies in
which anticoagulated and clotted blood samples were compared directly against each other at room temperature are highlighted in blue.
* Clarification of published data were sought directly from corresponding author; ** Unless indicated otherwise, values are the range of
concentrations observed on EDTA plasma reference samples. BD: Becton Dickinson, Cl: confidence interval; CKD: chronic kidney disease;
CRF: chronic renal failure; ECLIA: electrochemiluminescence immunoassay; EDTA: ethylenediaminetetraacetic acid; GST: gel separator tube;
ICMA: immunochemiluminometric assay; IRMA: immunoradiometric assay; PTH: parathyroid hormone; RT: room temperature.
Reference
Anderson et
al, 2003, 15
Number/nature/
concentration range**
of samples
Sample type
studied/tube
manufacturer
n=11, CRF patients,
predialysis samples,
20.7-65.6 pmol/L (95%
CI)
Serum (plain
+/- aprotinin),
plasma
(EDTA)
(Sarsted)
Storage of samples
Comparator
Blood centrifuged
20 min after
venepuncture and
serum/plasma
separated and
frozen at -20oC.
Test
Whole blood
samples kept at RT
before
centrifugation,
separation and
storage at -20oC
after 1, 2, 4, 8 or 24
h
Assay type
(analyser,
manufacturer)
ICMA (Immulite,
Siemens)
Overall conclusion
Statistical
analysis
ANOVA with
Tukey-Kramer
post-test
comparison
PTH stable in EDTA whole
blood for at least 24 h at RT,
but only stable in clotted blood
for 8 h
Cavalier et
al, 2007, 16
n=16, hemodialysis
patients, predialysis
samples, 08.00 h, 2.098.5 pmol/L
Serum (GST),
plasma
(EDTA) (BD)
Blood centrifuged
and serum/plasma
separated and
assayed
immediately
Whole EDTA and
GST blood stored at
RT for 8 and 24 h
ECLIA (Elecsys,
Roche)
Applied the
criterion of the
Royal Australasian
College of
Pathologists
Quality Assurance
Program (<25%
difference between
the sample and the
target)
GST and EDTA values
equivalent at baseline. At RT,
PTH more stable in EDTA
whole blood than clotted whole
blood (-4.1% vs -8.4% after 8 h
and -4.1% vs -20.5% after 24
h)
Ellis et al,
2003, 17
n=10, healthy
volunteers, non-fasting,
08.00-10.00 h, 1.4-5.9
pmol/L
Plasma (EDTA
)
Blood centrifuged
and plasma
separated
immediately and
stored at <-15oC
Whole blood stored
at RT or 4oC for 0.5
h, 6 h, 24 h before
separating plasma
and freezing at <15oC
IRMA (Nichols
Institute)
Stability was
defined as a
change of more
than 10% relative
to baseline
concentrations.
PTH stable in EDTA whole
blood at RT and 4oC for 24 h
English et
al, 2007, 18
n=17, hemodialysis
patients, predialysis
samples
Serum (GST),
plasma
(potassium
EDTA)
EDTA and GST
blood samples
centrifuged within
30 min of
venepuncture and
serum/plasma
separated and
stored at -20oC
EDTA and GST
blood samples kept
at RT for 4, 8 or 20 h
after collection and
then centrifuged,
separated and
stored at -20oC
ICMA (ADVIA
Centaur, Siemens)
2-way ANOVA and
Student’s paired
and unpaired ttests.
Baseline PTH concentration
higher in plasma (7% in dialysis
patients; 27% in healthy
controls) than serum. PTH
unstable in clotted blood over
time (10% decrease at 8 h,
22% at 20 h) but stable in
EDTA blood. Overall
conclusion: PTH stable in
EDTA whole blood for at least
20 h at RT but only stable in
clotted blood for 4 h
Morales
Garcia et al,
2009, 19
n=49, CKD patients
(249 blood samples),
08.00-09.00 h, fasting,
predialysis samples,
21.5 ± 21.2 pmol/L
Plasma
(EDTA)
Blood separated
after collection
(refrigerated
centrifugation) and
plasma
immediately frozen
Sample left 1 h at
RT after collection,
then separation and
aliquoting.
1 tube frozen, 1 tube
kept 8 h at 4oC then
frozen, 1 tube kept >
24 h at 4oC then
frozen;
Sample left > 3 h at
RT after collection,
then separation and
aliquoting;
1 tube frozen, 1 tube
kept 8 h at 4oC then
frozen
IRMA (total intact
assay, Scantibodies)
Paired t-test
including the
Bonferroni
correction
PTH stable in all storage
conditions tested; PTH stable
in EDTA whole blood for at
least 3 h at RT
Newman et
al, 1988, 20
n=4, normal
volunteers, n=4,
hyperparathyroidism
patients
Serum
Blood clotted for 30
min before
centrifugation, then
serum frozen at 20oC
Whole blood kept at
RT for 18 h before
separation and
freezing
IRMA (Allegro,
Nichols Institute)
Not stated
Increase of PTH concentration
in blood at RT (32%); PTH
unstable in clotted whole blood
Oddoze et
al, 2012, 29
n=50 healthy
volunteers
Serum (plain
and GST),
plasma
(EDTA)
Sample centrifuged
30 min after blood
clotting (serum)
and immediately
after collection
(plasma)
Samples were
stored at 4oC or RT
for 2 h, 4 h, 6 h, 24 h
or 72 h.
ICMA (Cobas,
Roche)
Combined
analytical and intraindividual
imprecisions by the
estimation of the
square root of the
sum of the squared
analytic and
biologic
imprecisions,
defined as the total
change limit (TCL).
If the results for an
analyte had a mean
percentage
difference
which exceeded
the TCL, then the
difference was
judged to be
significant and not
to meet the stability
criteria.
At RT PTH stable for 72 h in
EDTA whole blood and 6 h in
plain whole blood; At 4oC PTH
stable for 72 h in EDTA and
plain whole blood
Omar et al,
2001, 21
n=36, stone forming
patients, 0.9-10.9
pmol/L
Serum (plain),
plasma
(EDTA) (BD)
Serum and EDTA
plasma samples
separated after
collection and
immediately frozen.
Frozen EDTA
plasma obtained
from centrifuged
whole blood at 0 h
and after 48 h at RT.
ICMA (Immulite,
Siemens)
Wilcoxon paired
signed ranks test.
PTH unstable at RT for 48 h in
EDTA whole blood (14.8%
lower than at 0 h); PTH
concentration 19.5% higher in
plasma than serum (sample
frozen immediately) at 0 h.
Parent et al,
2009, 22
n=31, hemodialysis
patients, predialysis
samples, 6.6-129.4
pmol/L
Serum (plain,
and GST),
plasma (EDTA
and EDTA +
antiprotease
[aprotinin])
(BD)
Plain serum,
separated and
frozen at -20oC
immediately
Ratcliffe et
al, 1989, 23
n=6, primary
hyperparathyroidism
patients, 11.1 pmol/L
(mean)
Polypropylene
tube (whole
blood, serum)
(Sarstedt)
Serum frozen
immediately
Russell et
al, 1995, 24
n=18,
hyperparathyroidism or
renal failure patients,
1.9-23.7 pmol/L
Serum (GST)
Blood allowed to
clot for 2 h and
then centrifuged,
serum separated
and frozen at 20oC.
GST blood
centrifuged and
separated. Serum
either frozen
immediately or kept
at RT or 4oC for 18 h
before freezing;
EDTA and aprotinin
blood either
centrifuged
immediately and
plasma separated
and frozen, or left as
whole blood at RT or
4oC for 18 h and
then centrifuged,
separated and
frozen
Whole blood or
serum kept at 20oC
for up to 30 h before
storage at -20oC and
analysis
ECLIA (Elecsys,
Roche),
ICMA (Immulite,
Siemens), ICMA
(Liaison, DiaSorin )
Student t-test
Overall conclusion: At 4oC,
PTH stable in EDTA whole
blood for 18 h; at RT EDTA
whole blood stable, but
increase seen with Immulite
assay.
IRMA (N-tact,
INCSTAR)
Compared
individual means by
Student’s t-test,
adjusted according
to the Bonferroni
method for multiple
comparisons.
PTH stable in whole blood kept
at 20oC for up to 6 h, after 24 h
80% of PTH concentration
remains.
Two tubes
centrifuged after 2 h
at RT and then
stored at RT or 4oC;
and two tubes left
uncentrifuged at RT
or 4oC.
Serum samples
removed before
ICMA (Magic Lite,
Ciba Corning
Diagnostics)
Paired t-test.
Serum stored within clotted
whole blood stable for 6 h at
both RT and 4oC. Significant
losses seen in samples after
24-30 h at RT but not at 4oC.
Overall conclusion: PTH stable
in clotted whole blood for at
least 6 h after collection and for
longer at 4oC.
analysis. All samples
divided into 2
groups: samples
analysed within 2-6
h or within 24-30 h
Stokes et al,
2011*, 25
n=18, metabolic bone
disease patients
(divided into 3 groups
of 6)
Serum,
plasma
(lithium
heparin,
EDTA)
(Sarstedt)
Immediate
centrifugation,
separation and
storage of
serum/plasma at 70oC
Whole blood sample
stored at 1, 2, 4, 8,
24, 48 h before
separation.
Serum/plasma
samples then stored
at -70oC until
analysis
ECLIA (Elecsys,
Roche)
1-way ANOVA with
Bonferronicorrected post hoc
t-tests to compare
analyte
concentrations at
each time point
with the baseline
concentration. PTH
stability was
defined by the
amount of time a
change of <10%
occurred.
In whole blood PTH stable for 8
h (serum), 48 h (lithium heparin
and EDTA)
Walker et
al, 2000, 26
n=8, dialysis patients,
08.30 h, predialysis
samples, 3.1-83.2
pmol/L
Serum (GST),
plasma
(EDTA)
Blood samples
centrifuged and
serum/plasma
separated and
frozen immediately
at -20oC
Whole blood
samples stored at
RT for 3, 6 or 12 h
before
centrifugation,
separation and
freezing
ICMA (Immulite,
Siemens)
Paired t-test.
PTH stable with time (plasma),
reduced at 3, 6 and 12 h
(serum) with a decrease of
10% at 12 h (serum). Overall
conclusion: PTH more stable in
EDTA whole blood than clotted
whole blood
Wood,
1992, 27
n=6, normal subjects
and primary
hyperparathyroidism
patients, 3.9-56.6
pmol/L
Serum
Blood samples
centrifuged and
serum frozen at 20oC as soon as
possible
Whole blood stored
at RT for 2 or 4 h
before centrifugation
and freezing of
serum at -20oC
Not stated (but can
assume second
generation)
Not stated
Delay in separation of whole
blood for 2 or 4 h caused a 6%
drop in PTH concentration.
Overall conclusion: clotted
blood samples taken for PTH
estimation should be allowed to
clot for 30 min and then the
serum separated and frozen
without delay
Zwart et al,
2009, 28
n=11, healthy
volunteers, fasting, 7.5
+/- 2.9 pmol/L
Serum (GST)
(BD)
Serum separated
and centrifuged
after 45 min at RT
and stored -80oC
Blood centrifuged
and serum
separated after 2.5,
5 and 24 h at RT.
Aliquots frozen at 80oC
Not stated
The Bonferroni t
test was used post
hoc to determine
main-effect
differences
between the first
time point (45–60
min) and each of
the other time
points.
PTH stable for at least 5 h in
whole blood. PTH
concentration lower after 24 h
in whole blood.