Supplemental Data Table 4: Summary of included studies on PTH stability in plasma and serum measured by second generation assays.
Studies in which anticoagulated plasma and serum were compared directly against each other 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; *** Table of results unclear on the specific time point at which PTH is considered
significantly different compared to baseline in serum. BD: Becton Dickinson; CKD: chronic kidney disease; ECLIA: electrochemiluminescence
immunoassay; EDTA: ethylenediaminetetraacetic acid; GST: gel separator tube; ICMA: immunochemiluminometric assay; IRMA:
immunoradiometric assay; PTH: parathyroid hormone; PST: plasma separator tube; RT: room temperature; ULN: upper limit of normal.
Reference
Anderson
et al, 2001,
32
Number/nature/
concentration
range** of
samples
Sample type
studied/tube
manufacturer
n=9, CKD patients,
predialysis patients
Serum (plain, GST)
(Sarsted and plain
glass tubes)
Storage of samples
Comparator
Blood centrifuged
after
venepuncture
and separated
plain serum
stored at -20oC
before analysis
Test
GST serum
separated after
venepuncture
and stored at 20oC before
analysis
Assay type
(analyser,
manufacturer)
ICMA (Immulite,
Siemens)
Overall conclusion
Statistical
analysis
ANOVA with
Tukey-Kramer
post-test
comparison
PTH concentration higher (13%) in
plain than in GST serum, suggesting
stability over 20 min at RT better in
plain serum.
Brinc et al,
2012, 44
Children of 0-18
years of age
Serum (GST)
Blood allowed to
clot for 20 min,
centrifuged and
stored at 4oC
until baseline
measurement (no
baseline
measurement
performed for “all
ages” and “1-30
days” pools)
Serum stored at 80oC until
analysis. Sample
analysed once
monthly over 1013 month period
ICMA (Immulite,
Siemens)
Stability of
analytes at
monthly intervals
after storage was
assessed by
calculating the
percentage change
from baseline
(tbaseline) as
follows:
((tx−tbaseline)/tbas
eline)*100;
x=storage time in
months. Linear
regression of
analyte
concentration vs.
time was also
performed and
examined for
significant slope
PTH unstable at -80 oC; up to 16%
decrease after 2 months and 27.2%
after 10 months.
Serum (GST),
plasma (EDTA)
(BD)
Blood centrifuged
and
serum/plasma
assayed
immediately
Serum and
plasma stored at
-20oC for 24 and
120 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; PTH stable in serum and
plasma for 24 h at -20oC but after 120 h
greater loss in EDTA than serum (10.8% vs -7.2%) occurs
Sample pooled into
a single pool or into
age-group specific
pool (3-30 days, 1
month – 1 year, 15 years, 6-10
years, 11-14 years
and 15-18 years)
Cavalier et
al, 2007,
16
n=16, hemodialysis
patients, predialysis
samples, 0800h, 298.5 pmol/L
Evans et
al, 2001,
33
minimum of n=6,
non-fasting, 08.0010.00 h, 9-6 pmol/L
Serum (plain),
plasma (EDTA,
lithium heparin,
sodium
fluoride/potassium
oxalate) (Greiner;
BD)
EDTA blood
centrifuged
immediately and
plasma stored at
-20oC
Separation
immediately
(plasma) or 10
min (serum) after
collection.
Storage of
serum/plasma
aliquots at 4oC
for 24 h, 4oC for
120 h, 30oC for
24 h or 30oC for
120 h.
IRMA (Nichols
Institute)
A difference of
10% was selected
because changes
of less than this
were deemed
unlikely to be
clinically significant
At 30oC PTH stable for 3 h (serum), 16
h (fluoride plasma), 33 h (heparin
plasma) and 36 h (EDTA plasma). At
4oC PTH stable for 55 h (serum) and 5
days (heparin, fluoride and EDTA).
Overall conclusion: PTH more stable in
EDTA plasma than serum at both 30oC
and 4oC
Glendennin
g et al,
2002, 34
n=36, patients, 0.927 pmol/L
Plasma (EDTA ),
serum (GST) (BD)
EDTA plasma
assayed within 3
h of collection
Serum assayed
within 3 h of
collection and
serum and EDTA
plasma assayed
after 72 h at RT
ICMA (Immulite,
Siemens)
ANOVA and
Dunnett’s post hoc
test
PTH concentration 35% lower in serum
than plasma at 3 h and 60% lower at
72 h; PTH concentration EDTA plasma
did not change significantly over 72 h.
Overall conclusion: PTH more stable in
EDTA plasma than serum
Hermsen et
al, 2002,
35
n=15, healthy
individuals and
CKD patients
Serum, plasma
(EDTA, heparin)
Aliquots of serum
and plasma
frozen at -20oC at
baseline
Aliquots of serum
and plasma
stored at RT or
4oC for varying
lengths of time up
to 168 h, then
frozen at -20oC
ECLIA (Elecsys,
Roche)
Stability was
defined as <10%
difference between
the initial PTH
concentration and
the measured PTH
concentration
PTH stable for at least 8 h (serum), 24
h (heparin plasma), 48 h (EDTA
plasma) at RT; stable for at least 24 h
(serum, heparin plasma) and 72 h
(EDTA plasma) at 4oC.
Overall conclusion: PTH more stable in
EDTA plasma than serum
Holmes et
al, 2005,
36
n=31, CKD
patients, predialysis
samples, 08.0009.30 h, 29.1-57.8
pmol/L (95% range)
Serum (GST),
Plasma (EDTA)
(BD)
2 tubes (serum,
plasma) collected
on ice and
centrifuged
immediately at
4oC, 2 tubes
(serum, plasma)
collected at RT
and centrifuged
at RT, 1 tube
(serum) collected
at RT and
centrifuged at
4oC
(manufacturer
indications);
Analysed within 3
h
Analysis at 24 h,
48 h and 72 h for
each of the five
conditions/
sample types
ICMA (Immulite,
Siemens)
Joly et al,
2008, 37
n=34, hemodialysis
patients, predialysis
samples, 1.6-136.1
pmol/L
serum (plain),
plasma (EDTA,
citrate) (BD)
All samples
separated and
frozen at -20oC in
aliquots within
2.5-4 h of
venepuncture.
After storage,
aliquots thawed
and assayed
immediately.
Same aliquots reassayed after 18
h at RT. For each
sample type and
each assay,
results were
compared
against their
equivalent that
had been
assayed
immediately after
thawing.
ECLIA (Elecsys,
Roche), ICMA
(ADVIA Centaur,
Siemens), ICMA
(Liaison,
DiaSorin), ICMA
(Immulite,
Siemens), ICMA
(Architect,
Abbott), ICMA
(Access,
Beckman
Coulter)
ANOVA with
Bonferroni
corrections for
multiple
comparisons
Not stated
Higher PTH concentration in plasma at
RT compared to 4oC (9.8% higher at
RT at baseline);
PTH stable over time in EDTA at 4oC
(at least for 72 h), increases slightly in
EDTA at RT (8.3% increase at 24 h
and 8.8% at 48 h): concentration at 72
h not different basline);
PTH stable over time in serum sample
kept at 4oC (at least for 72 h), stable in
serum at RT for 24 h (significant
decrease by -17.3% at 48 h and 24.9% at 72 h from baseline)
Across methods, mean change of PTH
after 18 h +3.3% in thawed EDTA
plasma, -9.5% in serum, +2.8% in
citrate plasma . Concentrations in
citrate plasma lower than EDTA plasma
or serum. Concentrations in serum
higher than EDTA plasma with some
assays (Elecsys, Liaison, Architect,
Beckman) and lower with others
(Immulite, Centaur); PTH more stable
in EDTA plasma after thawing than in
serum.
Levin GE
et al,
1994***, 38
n=9, patients with
primary
hyperparathyroidis
m (8 with CRF),
n=8, healthy
volunteers, n=3
patients with acute
pancreatitis (mean
concentration 13.5
pmol/L)
Serum (plain),
plasma (protease
inhibitors, EDTA,
lithium heparin)
(Nichols Institute)
Samples
separated after
collection
(refrigerated
centrifugation)
and immediately
frozen at -20oC
Separated serum
and plasma kept
at RT for 6, 24,
48 or 72 h and
then frozen at 20oC
IRMA (Nichols
Institute)
Wilcoxon signedrank test
PTH concentration decreased
significantly in serum upon storage at
RT (26% loss after 24 h) but not in any
of the plasmas tested. PTH
concentration higher in serum than
plasma at 0 h.
Morales
Garcia et
al, 2009,
19
n=49, CKD patients
(249 blood
samples), 08.0009.00 h, fasting,
predialysis
samples, 21.5 ±
21.2 pmol/L
Plasma (EDTA)
Blood separated
after collection
(refrigerated
centrifugation)
and plasma
immediately
frozen
IRMA (total intact
assay,
Scantibodies)
Paired t-test
including the
Bonferroni
correction
PTH stable in all storage conditions
tested; PTH stable for 24 h in EDTA
plasma at 4oC
Newman et
al, 1988,
20
n=4 normal
volunteers, n=4
hyperparathyroid
patients
Serum
1 aliquot clotted
for 30 min before
centrifugation,
then serum
frozen (-20oC)
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
Serum kept at
4oC or 23oC for
18 h before
freezing
IRMA (Nichols
Institute)
Not stated
10% loss in serum incubated at 4oC.
Greater increases in serum stored at
23oC (14%).
Overall conclusion: PTH unstable in
serum
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
intra-individual
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 6 h in serum
(plain and GST), and 72 h in EDTA
plasma; At 4oC PTH stable for 72 h in
EDTA plasma and serum (plain and
GST)
Parent et
al, 2008,
31
Parent et
al, 200, 22
(1) n=53,
hemodialysis
patients (1.6-89.4
pmol/L), n=46,
renal calculi
patients (1.5-11.1
pmol/L); (2) n=17
(2.7-93.4 pmol/L) +
21 (1.2-93.4
pmol/L)
hemodialysis
patients; predialysis
samples
n=31, hemodialysis
patients, predialysis
samples 6.6-129.4
pmol/L
(1) Serum (plain),
plasma (EDTA); (2)
Serum (plain, GST)
(BD)
(1) plain serum
separated at 4oC
within 30 min and
stored at -20oC;
(2) plain serum
separated within
30 min and
stored at -20oC
(1) EDTA plasma
separated at 4oC
within 30 min and
stored at -20oC;
(2) Plain serum
and spun GST
tubes left for 4 h
at RT or 4oC
before separation
and freezing at 20oC
ECLIA (Elecsys,
Roche)
Student t test
(1) PTH concentration in plasma and
serum equivalent;
(2) Lower PTH concentration in plain
serum kept at RT; No effect of
temperature on PTH concentration in
GST serum; PTH stable in plain serum
for 4 h at 4oC but not at RT; stable for 4
h in GST serum at both 4oC and RT.
Serum (plain,
GST), plasma
(EDTA, EDTA +
antiprotease =
aprotinin) (BD)
Plain serum,
separated and
frozen at -20oC
immediately
GST serum spun
and frozen
immediately or
kept at RT or 4oC
for 18 h before
freezing; EDTA
and aprotinin
plasma either
centrifuged
immediately and
separated and
frozen, or left as
whole blood at
RT or 4oC for 18
h and then
separated and
frozen
ECLIA (Elecsys,
Roche), ICMA
(Immulite,
Siemens), ICMA
(Liaison,
DiaSorin)
Wilcoxon non
parametric test
No effect of assay used on PTH
concentration (plain serum); 30-35%
bias between Elecsys and Immulite
with EDTA and aprotinin tubes;
PTH stable in samples separated
immediately for all tube types
(Elecsys), lower PTH concentration
with EDTA or aprotinin tubes (Liaison),
higher PTH concentration with EDTA or
aprotinin tubes (Immulite); At 4oC, PTH
stable in serum for 18 h; at RT
significant losses in serum after 18 h.
Ratcliffe et
al, 1989,
23
n=6, primary hyper
parathyroidism
patients, 11.1
pmol/L (mean)
Polypropylene tube
(whole blood,
serum) (Sarstedt)
Serum frozen
immediately
Whole blood or
serum kept at
20oC for up to 30
h before storage
at -20oC and
analysis
IRMA (N-tact,
INCSTAR)
Compared
individual means
by Student’s t-test,
adjusted according
to the Bonferroni
method for multiple
comparisons
PTH stable in serum kept at 20oC for
up to 6 h, after 24 h 64% of PTH
concentration remains.
Russell et
al, 1995,
24
n=18, hyperparathyroidism or
renal failure
patients, 1.9-23.7
pmol/L
Serum (GST)
Serum allowed to
clot for 2 h and
then centrifuged,
separated and
stored frozen at 20oC.
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 analysis.
All samples
divided into 2
groups: samples
analysed within
2-6 h or within
24-30 h
ICMA (Magic
Lite, Ciba
Corning
Diagnostics)
Paired t-test
Serum stored separated 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
serum for at least 6 h after collection
and for longer at 4oC.
Scharnhors
t et al,
2004, 39
n=137, volunteers,
09.00-13.00 h, 2.08.3 pmol/L (95%
reference interval)
Serum (GST),
plasma (EDTA)
(BD)
1 EDTA tube kept
on ice
(centrifuged at
4oC), 1 EDTA
tube and 1 GST
kept at RT;
centrifugation,
separation and
analysis within
2.5 h after
collection
Serum and
plasma kept at
4oC, analysis 48
h after initial
measurement
ICMA (Immulite,
Siemens)
Wilcoxon signedranks test
PTH more stable in plasma than
serum; PTH concentration decreases
over 48 h in serum (2.6 to 2.4 pmol/L),
increases over 48 h in plasma (3.8 to
4.1 pmol/L), stable in EDTA plasma
kept on ice (3.2 pmol/L)
Stokes et
al, 2011*,
25
n=18, metabolic
bone disease
patients (divided
into 3 groups of 6)
Serum, plasma
(lithium heparin,
EDTA) (Sarstedt)
Immediate
separation,
storage of
serum/plasma at
-70oC
Immediate
separation and
storage at RT for
2, 4, 8, 24, 48 h
or 7 days;
Immediate
separation and
storage at 2-8oC
for 24, 48 h, 7
days, 14 days, 28
days; 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
Teal et al,
2003, 40
n=13, hemodialysis
patients, predialysis
samples (2.1-115.8
pmol/L)
Serum (plain,
GST), plasma
(EDTA) (BD)
Aliquots of serum
and plasma
centrifuged,
separated and
frozen rapidly at 20oC after
venepuncture
Aliquots of serum
and plasma kept
at RT for 2, 4, 8,
12, 24 and 48 h
and then frozen
at -20oC
ICMA (Nichols
Institute)
1-way ANOVA and
Wilcoxon’s
matched pairs
signed ranks test
In separated plasma/serum PTH stable
for 48 h (lithium heparin, EDTA) or 24 h
(serum);
In separated plasma/serum at 2-8oC
PTH stable 28 days (lithium heparin,
EDTA), 7 days (serum); [PTH] not
different from baseline at 1, 2, 4 h at
RT and 2-8oC (serum, plasma).
Overall conclusion: PTH more stable in
EDTA plasma than serum. PTH stable
in clotted whole blood at RT for only 4
h. Hence measure PTH on plasma
(EDTA, lithium heparin), on serum if
sample centrifuged immediately and
frozen until analysis.
PTH concentration at 0 h similar in
serum and plasma; median PTH
concentration 4% lower in plasma;
PTH unstable after 2 h (plain serum), at
4 h (GST), stable in plasma for 48 h.
PTH concentration decreases after 48
h by 47% (plain serum), 26% (GST)
Wood,
1992, 27
n=6, normal
subjects and
primary hyper
parathyroidism
patients, 3.9-56.6
pmol/L
serum
Whole blood
samples
centrifuged and
serum frozen at 20oC as soon as
possible
Serum stored at
RT for 2 and 4 h,
1, 2 or 4 days
before freezing at
-20oC
Not stated (but
can assume
second
generation)
Not stated
A delay in freezing of serum for 2 or 4 h
caused a loss of PTH concentration of
4% and 8% respectively.
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