BJU International (1999), 84, 810–814
The stability of free and bound prostate-specific antigen
J.J . CART LEDGE, D . THOMP SON*, H. VER RIL†, P . C LARK SON* and I. EARD LEY
Pyrah Department of Urology and *Department of Chemical Pathology & Immunology, The General Infirmary at Leeds, Leeds, and
†Department of Clinical Biochemistry, Edinburgh Royal Infirmary, Edinburgh, UK
Objective To determine if the assay for free prostate
specific antigen (fPSA) and the calculated ratio of fPSA
to total PSA (f/tPSA) is stable in conditions likely to
be met in routine clinical practice.
Materials and methods Two blood samples were
obtained from 27 patients attending a routine urology
clinic. Sample 1 was centrifuged immediately, assayed
for fPSA and tPSA, and the f/tPSA calculated. This
sample was then stored at 4°C for 24 h, 48 h and
1 week, or at −20°C for 24 h, 1 week and 1 month
before the assays for fPSA and tPSA were repeated.
The second sample was left at room temperature for
24 h before assay and processing, as for sample 1.
Results tPSA is a highly stable analyte; if whole blood
samples are processed immediately, fPSA is stable for
24 h at 4°C and 1 month at −20°C. There was a
significant reduction in the calculated f/tPSA in
samples stored for 24 h at 4°C (P<0.01); if the
sample was stored at −20°C the calculated f/tPSA
was stable. After 24 h storage at room temperature,
Introduction
PSA is a serine protease produced by the epithelium of
the prostate gland [1]. In conditions leading to enlarge-
ment of the prostate [2] and in prostate cancer [3],
serum levels of PSA are increased. In benign enlarge-
ments of the gland the level of PSA is elevated because
the epithelial cell mass is greater, but in carcinoma of
the prostate it is probably elevated because tissue is
destroyed and there is increased leakage into the circu-
lation [4].
In 1986 it was shown that PSA was not wholly free
in the plasma but was mostly bound to other serum
proteins, particularly a -antichymotrypsin (ACT) and a -
1 2
macroglobulin [5]. The former is produced predomi-
nantly in the liver and circulates in a 104−105 molar
excess over PSA. Prostate cancer cells, but not BPH cells,
are also able to produce ACT, which forms complexes at
the site of excretion [6]. Early assays of PSA did not
diCerentially measure free and protein-bound PSA; it
Accepted for publication 6 July 1999
810
fPSA decreased by 6.3% and f/tPSA by 6.4%.
Subsequent storage of serum at 4°C for 1 week resulted
in a 25% decrease from the baseline value. After
1 month at −20°C the fPSA value was 13% lower
than the baseline value.
Conclusion These results indicate that if there is to be
confidence in the accuracy of the f/tPSA value, then
blood samples must be handled and processed cor-
rectly. Total PSA is suBciently stable to permit whole
blood samples to remain at room temperature for 24 h
before serum is separated. If fPSA is to be determined
accurately then the whole blood sample must be
centrifuged promptly. As the fPSA values in blood
samples left at room temperature for 24 h are up to
25% lower than those on immediate assay, and the
subsequent f/tPSA 29% lower, then for the optimum
use of this test, these samples should also be handled
appropriately.
Keywords Prostate specific antigen, free, total, assay,
stability
was only with the development of two-site immuno-
metric assays that measure total PSA (tPSA), PSA bound
to ACT and free PSA (fPSA) that the potential advantages
of this assay became apparent [7].
Total PSA is the predominant tumour marker used in
the diagnosis and monitoring of prostate cancer, but as
it may be elevated in other disease states its poor
sensitivity and specificity makes it a less than ideal
tumour marker [8]. Taking a threshold of 4.0 ng/mL as
the upper limit of normal in a screening programme, a
false-positive rate of 65% and a false-negative rate of
20% were recorded [9]. ECorts have been made to
improve the usefulness of PSA as a marker for prostate
cancer, including serial PSA measurements [10], esti-
mates of PSA density [11], and applying age- and race-
specific reference ranges [12]. More recent work indicates
that the measurement of fPSA and tPSA and the calcu-
lation of the ratio (f/tPSA) allows a better discrimination
of prostate cancer from BPH than the measurement of
tPSA alone [9,13–17].
Most of the studies on PSA have used stored plasma
or serum; there is well-documented evidence that PSA
© 1999 BJU International

is stable [18–20] and evidence about the stability of
fPSA [21–24], but these data derive from studies in
carefully controlled settings. There are very few data on
the stability of fPSA and the calculated f/tPSA value in
conditions likely to be met in routine clinical practice.
Thus we devised a study to provide information on the
stability of fPSA and the accuracy of f/tPSA under such
conditions, where a central hospital laboratory oCers a
routine service for the measurement of fPSA and tPSA
to both local hospitals and GPs.
Materials and methods
Two 6 mL clotted blood samples were obtained (via a
single venepuncture) from 27 patients attending a gen-
eral urology outpatient clinic. All patients had either
been referred by a local GP for further assessment
because of an elevated PSA level, or had a previous
diagnosis of prostate cancer and were being followed up
routinely. Blood samples were collected by the phleb-
otomy service and received in the chemical pathology
laboratory for processing within 2 h.
One of the specimens (sample 1) was centrifuged
immediately upon receipt (at 1500 g for 5 min) and the
serum fraction separated into seven aliquots. One aliquot
was assayed immediately for fPSA and tPSA, and the
remaining aliquots were placed in either a refrigerator
at 4°C or in a freezer at −20°C. An individual aliquot
was removed for assay after storage at 4°C for 24 h,
48 h and 1 week, and after storage at −20°C for 24 h,
1 week and 1 month. A single aliquot was assayed at
each stage; there was no re-freezing of samples.
The second blood sample (sample 2) was left to stand
undisturbed on the laboratory bench at room tempera-
ture (20°C) for 24 h before processing. After storage at
room temperature this sample was centrifuged, stored
and assayed in the same way as sample 1. It was
assumed that the immediate PSA values for individual
samples 1 and 2 were identical.
Total and fPSA were measured with an AutoDelfia
system, using the Prostatus@ dual-label Total/Free PSA
kit (Wallac UK, Milton Keynes, UK). The results of the
immediate assays of fPSA, tPSA and the calculated
f/tPSA on sample 1 were compared with results of assays
measured at all other times. Data were analysed using
a one-way anova for repeated measures, with a diCerence
considered significant if P<0.05.
Results
For sample 1 there was no significant diCerence in tPSA
values between the samples analysed immediately after
receipt and those stored at any temperature for any of
the time intervals. The fPSA results for the samples
© 1999 BJU International 84, 810–814
PR OSTATE-SP EC IFIC ANTIG EN STABILIT Y 811
stored at 4°C for 24 h were also not significantly diCerent
from the immediate sample, but when the samples were
stored at this temperature for 48 h and 1 week the
results were significantly lower (P<0.01; Fig. 1a). The
f/tPSA ratio for these samples was significantly (P<0.01)
decreased after storage for 24 h, 48 h and for 1 week
(Fig. 1a). The aliquots of sample 1 which had been stored
at −20°C before analysis showed no alteration in fPSA
or f/tPSA (Fig. 1b) from the values of the immediate
analysis.
The results for assays on sample 2 show that tPSA is
stable under any of the storage conditions tested.
Although fPSA values were not significantly diCerent
between the blood sample stored for 24 h as whole blood
before analysis and the sample analysed immediately
(P=0.1), the calculated f/tPSA ratio was significantly
lower (P<0.001). Subsequent storage of aliquots of
sample 2 at 4°C led to a significant decrease in measured
fPSA values for all storage times (Fig. 1c). This decrease
was mirrored in the calculated f/tPSA ratio (Fig. 1c).
The f/tPSA ratio from individual samples stored under
these conditions was clearly decreased in most samples
(Fig. 2). Measurement of fPSA after storage at −20°C
for 24 h gave values significantly lower than those found
in samples assayed immediately, whereas values
recorded after the analysis of samples stored for 1 week,
although lower, were not significantly so (P=0.07).
There was also a significant decrease in fPSA levels in
samples stored for 1 month at −20°C (P=0.01)
(Fig. 1d). The calculated f/tPSA value for this sample
was significantly lower than the immediate f/tPSA after
storage at room temperature for 24 h, and after further
storage at −20°C for 24 h, 1 week and 1 month
(P<0.01; Fig. 1d).
Discussion
These results give important information about the rou-
tine use of an assay for fPSA and tPSA. It is clear from
this evidence that the conditions met in routine practice
must be carefully considered when requesting this test,
and should highlight the caution needed when assessing
published evidence about the use of fPSA and f/tPSA.
The present results confirm other published reports that
tPSA is a highly stable analyte [18–20]. If whole blood
samples remain at room temperature for up to 24 h
before processing, the results of subsequent assays on
serum samples stored at either 4°C or −20°C are
unaCected. However, this is not so of the assay for fPSA
and the subsequent f/tPSA value. After a period of
storage of whole blood at room temperature for 24 h,
there was a 6.3% decrease in mean fPSA and a 6.4%
decline in f/tPSA. The magnitude of this reduction in
fPSA is similar to that observed by Jung et al. [21], who
812 J. J. C AR TLED GE et al.

a b
3 * * 20 3 20
18 18
2.5 16 2.5 16
*
f-PSA (ng/mL)

f-PSA (ng/mL)
2 14 2 14

f/t-PSA (%)

f/t-PSA (%)
* 12 12
1.5 * 10 1.5 10
8 8
1 6 1 6
0.5 4 0.5 4
2 2
0 0 0 0
Immediate 24 h 48 h 1 week Immediate 24 h 1 week 1 month

c d
3 * 20 3 * 20
* * *
* 18 * 18
2.5 16 2.5 16
*
f-PSA (ng/mL)

f-PSA (ng/mL)
2 * 14 2 14
f/t-PSA (%)

f/t-PSA (%)
* *
12 12
*
1.5 10 1.5 10
8 8
1 6 1 6
0.5 4 0.5 4
2 2
0 0 0 0
Immediate 24 h 24 h 48 h 1 week Immediate 24 h 24 h 1 week 1 month
room 4°C 4°C 4°C room –20°C –20°C –20°C
temperature temperature

Fig. 1. The mean (sem) fPSA (red) and f/tPSA (green) of: a, sample 1 stored at 4°C for up to 1 week; b, sample 1 stored at −20°C for up
to 1 month; c, sample 2 stored at 4°C for up to 1 week, where the immediate assay result is that from the identical sample 1 at the time
of venepuncture and sample 2 was then stored undisturbed at room temperature for 24 h before processing and separation into aliquots
for further storage at 4°C, before assay as indicated; d, sample 2 stored at −20°C for up to 1 month, where the immediate assay result is
that from the identical sample 1 at the time of venepuncture and sample 2 was then stored undisturbed at room temperature for 24 h
before processing and separation into aliquots for further storage at −20°C, before assay as indicated. In a, * indicates a significant
diCerence (P∏0.01) between the immediate assay and subsequent assays, and in b–d the first assay of that sample and subsequent assays.

reported a 5% reduction in fPSA after storing whole
blood for 8 h at room temperature, and an 8% decrease
after 24 h. In addition to confirming the results of that
study, it was also clear that subsequent storage of serum
at either 4°C or −20°C led to a continued reduction in
fPSA. After a further week at 4°C the value of fPSA
declined by 25% from baseline, and after an additional
month at −20°C, by almost 14%. The total overall
reduction in fPSA after immediate processing and storage
of serum samples at 4°C for 1 week was also 25%, and
after storage for 1 month at −20°C was 13%. The
equivalence of this decrease in fPSA values despite the
diCerent initial storage conditions suggests that the
causes are both time- and temperature-dependent.
The precise reasons for the time- and temperature-
dependent reduction in fPSA are unknown, but it is
probable that several factors are important. Interference
from serum substances such as haemoglobin, bilirubin
and lipids may be excluded, as they have been shown
to have no eCect on repeat fPSA assays at diCering
concentrations [22]. Pettersson et al. [23] showed that
fPSA alone, in vitro, is stable for up to 4 weeks at 35°C,
whereas PSA complexed with ACT dissociated at this
temperature; this dissociation was impaired at lower
temperatures. This implies that a greater concentration
of fPSA should be expected with time. However, this is
not so, and would suggest that there is either an
alteration in fPSA to a form not measurable by the
assay, or that with time fPSA forms complexes with
serum substances other than protease inhibitors that are
not measurable by immunoassay [21]. Woodrum et al.
[25] suggested that the fPSA-specific epitope on the PSA
molecule may be less stable at higher temperatures over
time, making fPSA less measurable.
Despite the uncertainty about the causes in the
reduction of fPSA and f/tPSA with time, it is clear that
it is important. With mounting evidence that the use of
f/tPSA rather than tPSA alone increases the sensitivity
and specificity for diagnosing prostate cancer, it is likely
that the use of this test will increase, and in particular

© 1999 BJU International 84, 810–814


35
30
25
20
f/t-PSA (%)
15
10
5 6 Bjork T, Bjartell A, Lilja H, Abrahamsson PA, di Sant
0 1
Immediate 24 h 1 wk
room temperature 4°C
Fig. 2. f/tPSA results for all samples stored undisturbed at room
temperature for 24 h and subsequently for 1 week at 4°C (green)
and the mean for all samples (red). The result of an assay on a
single sample (immediate 44.9%, 1 week 45.2%) is excluded
for clarity.
its use as a screening test for cancer will increase. The
results presented here indicate that if there is to be
confidence in the accuracy of the results of assays of
samples sent to a central chemical pathology laboratory,
then the samples should be processed appropriately.
Total PSA is suBciently stable to allow whole blood
samples to remain at room temperature for 24 h before
serum separation and storage in a refrigerator at 4°C for
up to 1 week, or a domestic freezer at −20°C for
1 month. If fPSA is to be determined accurately, then
the whole blood sample must be centrifuged promptly
and the serum can then be stored for 24 h at 4°C or
1 month at −20°C. As assay results from samples older
than this give a fPSA value up to 25% lower than on
immediate assay, and a f/tPSA value 29% lower, it is
clear that to use this test confidently, samples should be
handled correctly.
We suggest that clinicians should ensure rapid delivery
of blood samples from their clinic to the laboratory and
if the age of a blood sample arriving in a laboratory for
© 1999 BJU International 84, 810–814
PR OSTATE-SP EC IFIC ANTIG EN STABILIT Y 813
determination of f/tPSA cannot be confidently known,
then only tPSA should be reported.
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Authors
J.J. Cartledge, FRCS(Ed), Specialist Registrar.
D. Thompson, PhD, Principal Biochemist.
H. Verril, MSc, Senior Biochemist.
P. Clarkson, BSc, Senior MLSO.
I. Eardley, Mchir, FRCS(Urol), Consultant.
Correspondence: Mr J. Cartledge, Specialist Registrar in Urology,
Pyrah Department of Urology, The General Infirmary at Leeds,
Great George Street, Leeds LS1 3EX, UK. e-mail: j.cart-
ledge@leeds.ac.uk
© 1999 BJU International 84, 810–814