Clinical Chemistry 43: 1588-1594, 1997;
(Clinical Chemistry. 1997;43:1588-1594.)
© 1997 American Association for Clinical Chemistry, Inc.
Comparison of prostate-specific antigen (PSA) measured by four combinations of free PSA and total PSA assays
Ralf Junker,
Burkhard Brandta,
Christian Zechel and
Gerd Assmann
Institut für Klinische Chemie und Laboratoriumsmedizin, Westfälische Wilhelms-Universität Münster, Albert Schweitzer-Straße 33, 48129 Münster, Germany.
a Author for correspondence. Fax 49-251-837226; e-mail brandt{at}uni- muenster.de.
 |
Abstract
|
|---|
We compared prostate-specific antigen (PSA) assay systems [i.e., free
PSA (f-PSA) and the corresponding total PSA (t-PSA) assay] from four
different manufacturers as well as the f-PSA/t-PSA ratios with regard
to their ability to discriminate between benign prostate hyperplasia
(BPH) and prostate cancer (PCA). ROC analysis showed similar areas
under the curves (AUCs) with different assay systems. For the entire
patient population the AUCs of the f-PSA/t-PSA ratio were not or
slightly increased compared with the sole measurement of t-PSA (t-PSA,
0.792–0.820; f-PSA/t-PSA ratio, 0.685–0.859). In contrast, for only
those patients who showed t-PSA concentrations within the diagnostic
gray area of 4–25 µg/L t-PSA, the AUCs were greater for the
f-PSA/t-PSA ratio than for measurement of t-PSA alone (t-PSA,
0.608–0.647; f-PSA/t-PSA ratio, 0.690–0.806). These results were
confirmed by the predictive values of the negative results (NPVs) of
the t-PSA assays and the f-PSA/t-PSA ratios (assay thresholds
corresponding to a 95% detection limit). Compared with the sole t-PSA
measurement there was no mentionable increase in the NPVs due to the
f-PSA/t-PSA ratio for the entire patient population, but an increase up
to 49% when limited to t-PSA concentrations within 4–25 µg/L. We
therefore conclude that the f-PSA/t-PSA ratio may be helpful for
differential diagnosis of BPH and PCA within the diagnostic gray area
of 4–25 µg/L t-PSA.
|
Introduction
|
|---|
Prostate-specific antigen
(PSA)1
is a serine protease with a chymotrypsin-like activity
(1)(2). The 33-kDa single-chain glycoprotein
is produced by the secretory epithelium of the prostate
(3). PSA is released from the normal prostate and appears
at low serum concentrations in healthy men. Studies with reverse
transcription-PCR have shown that PSA also is expressed at a low
concentration in peripheral blood cells and other tissues
(4). High serum concentrations can be detected in patients
with advanced prostate cancer (PCA) (5). Therefore PSA is
applied as a tumor marker for the clinical management of PCA
(6). However, increased PSA concentrations in serum also
occur in patients with benign prostate hyperplasia (BPH)
(7). Hence the goal is to discriminate clearly between BPH
and PCA in the clinical laboratory to spare the patient invasive
diagnostic procedures, such as a prostate biopsy.
In human serum PSA occurs in two forms: free PSA (f-PSA) and complexed
PSA. The major form is a complex of PSA and
1-antichymotrypsin (ACT). The fraction of f-PSA was
shown to be substantially smaller in patients with untreated PCA than
in patients with BPH. Therefore combined measurements of f-PSA and
total PSA (t-PSA) may lead to a better discrimination between BPH and
PCA (8)(9)(10). Some recent studies have already
shown that the f-PSA/t-PSA ratio is helpful in the differential
diagnosis of BPH and PCA (11)(12).
In this study we investigated assay systems (i.e., f-PSA and the
corresponding t-PSA assay) from four different manufacturers. Our aim
was to compare these assays with regard to their differences in
discriminating between BPH and PCA as well as to show the diagnostic
value of the f-PSA/t-PSA ratio.
|
Materials and Methods
|
|---|
Assays.
Assay systems performed were Enzymun-Test
PSA/Enzymun-Test PSAf (Boehringer Mannheim), Cispack PSA
2-US/FPSA-RIACT (Cis, Bagnols/Cèze, France), Immulite
PSA/Immulite Free PSA [Diagnostic Products Corp. (DPC)], and Tandem-E
PSA/Tandem-R free PSA (Hybritech). Additionally, in the calibrator
PSA-ACT was assessed by the PSA-ACT assay (Cell Diagnostica,
Münster, Germany). With the exception of the Cis and Hybritech
f-PSA assays and the PSA-ACT assay, which were performed manually, the
measurements were performed automatically on Boehringer ES 600, Cispack
4200, DPC Immulite, or Hybritech Photon Era, respectively. All
measurements were performed in duplicate. Controls were measured in
each assay run. Assay characteristics are shown in Table 1
.
Patients.
Sera from 80 patients were investigated: 30 patients
with BPH, ages 47–80 years, prostate volume 26–105 cm3,
determined by transrectal ultrasound with a method described by
Semjonow et al. (13); 50 patients with PCA, ages 40–88
years, stages T1 (n = 3), T2 (n = 7), T3 (n = 35), T4
(n = 5), N0-2, M0-1, G1-3. In all cases diagnosis was confirmed
histologically. All patients were untreated until blood collection.
Blood samples.
Blood was collected by venipuncture into a
10-mL Sarstedt serum monovette before rectal examination. Serum was
obtained by centrifugation at 400g for 15 min and
immediately frozen at -70 °C.
Calibrator.
The application of PSA measurements for clinical
monitoring of PCA leads to misinterpretations because of differences of
the commercially available PSA assays. To highlight this matter, we
used reference material (PSA-Bioref-17; Bioref, Mömbris, Germany)
with four different concentrations of t-PSA (referred to below as
ultralow, low, mid, and high preparation) as an assay-independent
control material.
Statistical analysis.
Linear regression and Pearson
correlation analyses were performed to compare PSA measurement by
different assays. For the entire patient population as well as for only
those patients who showed t-PSA concentrations within the range of
4–25 µg/L, ROC analysis [including calculation of the area under
the curve (AUC) and 95% confidence limits] was performed with the
MedCalc software package (MedCalc Software, Maria-kerke, Belgium).
AUCs were compared for the entire patient population. When P
<0.05, differences of the AUCs were defined as significant. The
predictive values of the negative results (NPV) of the t-PSA assays and
the f-PSA/t-PSA ratios were calculated at the assay thresholds
corresponding to a 95% detection limit. The difference between the NPV
of the f-PSA/t-PSA ratio and the sole measurement of t-PSA represents
the number of possibly eliminated prostate biopsies because of the
f-PSA/t-PSA ratio. Confidence intervals of NPVs were estimated by SAS
Makro cibinom for exact confidence limits for binomial proportion (SAS
Institute).
|
Results
|
|---|
Calibrator.
The t-PSA and f-PSA results varied among
assays, with higher differences at higher PSA concentrations. Because
the concentration of PSA-ACT was not specified by the manufacturer of
the calibrators, we determined it and found it to be 0.0–0.5 µg/L
(Table 2
).
Assay comparisons.
Comparing the results of the Tandem-E PSA
assay (x) with the results of the other t-PSA assays
(y) gave slopes that varied from 0.79 to 1.10 (intercept
0.01–2.37 µg/L). With the results of the Tandem-R free PSA
assay (x) vs the results of the other f-PSA assays
(y), the slopes were from 0.62 to 1.09 (intercept 0.11–0.75
µg/L) (Fig. 1
).
View larger version (27K):
[in this window]
[in a new window]
|
Figure 1. Cross-plots and linear regression analysis of the results
of PSA measurements.
Comparison of the Hybritech assay with the other assays. t-PSA values
up to 50 µg/L and f-PSA values up to 5 µg/L, which represent >95%
of the data, were taken into account.
|
|
The slopes, intercepts, and Pearson correlations of the regression
curves between the corresponding f-PSA and t-PSA assays are shown in
Fig. 2
. The slopes ranged from 0.03 (Immulite Free PSA/Immulite PSA)
to 0.10 (Enzymun-Test PSAf/Enzymun-Test PSA) and the intercepts from
0.36 (Enzymun-Test PSAf/Enzymun-Test PSA) to 0.62 µg/L (Immulite Free
PSA/Immulite PSA). The correlation coefficients varied from 0.43
(Immulite Free PSA/Immulite PSA) to 0.75 (all other combinations).
View larger version (28K):
[in this window]
[in a new window]
|
Figure 2. Cross-plots and linear regression analysis of the results
of PSA measurements.
Comparison of the corresponding f-PSA and t-PSA assays. t-PSA values up
to 50 µg/L and f-PSA values up to 5 µg/L, which represent >95% of
the data, were taken into account.
|
|
Diagnostic performance of t-PSA and f-PSA measurements and the
f-PSA/t-PSA ratio.
For ROCs, AUCs of the t-PSA assays varied
between 0.792 (Cispack PSA 2-US) and 0.833 (Immulite PSA). For f-PSA
measurements the areas were from 0.634 (Immulite Free PSA) to 0.692
(Enzymun-Test PSAf), for the f-PSA/t-PSA ratio from 0.685 (Boehringer)
to 0.859 (DPC) (Fig. 3
, Table 3
). With regard to
thedifferences in PSA measurement by different assays, the frequently
discussed diagnostic gray area between 4 and 10 µg/L for the
differential diagnosis between BPH and PCA was extended to 4–25 µg/L
t-PSA in our study. Limited to patients with t-PSA values of 4–25
µg/L, the AUCs obtained from ROC analysis for the t-PSA measurement
were low and varied from a minimum of 0.608 (Enzymun-Test PSA) to a
maximum of 0.647 (Immulite PSA). The corresponding areas of the
f-PSA/t-PSA ratios were higher for all assay systems and varied from
0.690 (Boehringer) to 0.806 (Hybritech).
For the entire patient population differences between the assays' AUCs
were not significant except for the differences between the Hybritech
and DPC t-PSA measurement and the Hybritech and DPC f-PSA/t-PSA ratios
(in both cases P <0.05) (Table 3
).
The overall diagnostic performance of an assay may be well described by
the AUC, but for the clinical use of tumor markers, the NPVs at a given
detection limit are more important. Within the entire t-PSA range the
NPVs at a 95% detection limit were similar for all t-PSA assays: from
0.78 (Tandem-E PSA) to 0.82 (Cispack PSA 2-US). For the f-PSA/t-PSA
ratio the NPVs varied from 0.75 (DPC and Hybritech) to 0.83 (Cis).
Compared with the sole measurement of t-PSA, there was no mentionable
increase in the percentage of possibly eliminated prostate biopsies by
calculating the f-PSA/t-PSA ratio. For patients with t-PSA
concentrations of 4–25 µg/L the NPVs at a 95% detection limit
ranged from a minimum of 0.18 (Enzymun-Test PSA) to a maximum of 0.76
(Tandem-E PSA). In the calculation of the f-PSA/t-PSA ratio the NPVs
varied from 0.62 (Boehringer, DPC) to 0.85 (Hybritech). Within this
range the number of possibly eliminated prostate biopsies as a result
of the f-PSA/t-PSA ratio compared with the sole t-PSA measurement
varied from 9% (Hybritech) to 49% (Cis) (Table 4
).
|
Discussion
|
|---|
The application of PSA measurements for clinical monitoring of PCA
leads to misinterpretations because of differences among commercially
available PSA assays. Similarly, PSA results of calibrators varied
among assays; in our study, we applied calibrators containing virtually
only f-PSA. The portion of PSA-ACT was <0.5 µg/L in all calibrator
preparations, as determined with the PSA-ACT assay. For the Cis and DPC
systems the measurement with the t-PSA assay led to higher values than
with the corresponding f-PSA assay. In contrast, the results of t-PSA
and f-PSA measurement in the calibrators with the Boehringer and
Hybritech assays led to similar results for both t-PSA and f-PSA (Table 2
). A possible source of these discrepancies might be the
overestimation of f-PSA by some t-PSA assays. At this point the absence
of PSA-ACT is a limitation for the use of this calibrator as a control
for PSA measurements. Because in patients' serum the major form of PSA
is the PSA-ACT complex, a calibrator with a defined portion of PSA-ACT
is required. However, as shown by Chen et al. (14), the
use of material containing defined portions of PSA-ACT and f-PSA as
calibrators resulted in good agreement between different assays for
just this f-PSA/t-PSA ratio. The results of the method comparison
confirm the finding of differences in PSA measurement by different
assays. Although the correlation coefficient exceeded 0.92 in all assay
comparisons (Hybritech t-PSA and f-PSA vs all other assays), there were
remarkable differences in slopes and intercepts (Fig. 1
). Additionally
the detection limits of the t-PSA assays for f-PSA differed
considerably as demonstrated by the slopes of the regression curves of
the corresponding f-PSA and t-PSA assays (Fig. 2
).
A major aim of PSA determination is to investigate the necessity of a
prostate biopsy. However, this is not always easy, because in the case
of BPH, t-PSA can reach high concentrations, whereas in the case of PCA
sometimes low values are measured. In our investigation we compared the
diagnostic value of the t-PSA assays and the f-PSA/t-PSA ratio by ROC
analysis and by calculating differences of the NPVs at a detection
limit of 95%.
Within the entire t-PSA range the AUCs were similar for the four t-PSA
assays. A slight increase of the AUC was found for the f-PSA/t-PSA
ratio only with the Cis and DPC systems. The increase of the NPVs for
the f-PSA/t-PSA ratio was not remarkable when the entire t-PSA range
was used. Compared with the sole measure- ment of t-PSA, the
f-PSA/t-PSA ratio allowed the potential elimination of prostate
biopsies in up to 3% more cases.
With regard to the differences in PSA measurement by different assays,
the frequently discussed diagnostic gray area between 4 and 10 µg/L
was extended up to 4–25 µg/L t-PSA in our study. For patients with
t-PSA concentrations of 4–25 µg/L, AUCs were smaller than within the
entire range, indicating the poor discriminating power of t-PSA between
BPH and PCA. However, in all assay systems the AUCs increased for the
f-PSA/t-PSA ratio. Moreover, compared with the sole measurement of
t-PSA, the prostate biopsy would possibly be eliminated in up to 49%
more cases because of the f-PSA/t-PSA ratio (Table 4
). That within the
4–25 µg/L t-PSA range the Hybritech system showed an increase of
only 9% may be a result of the already high NPV of the Hybritech t-PSA
assay. We therefore suggest that a lack of specificity for
patients showing t-PSA concentrations within the diagnostic gray area
of 4–25 µg/L can be overcome by calculating the f-PSA/t-PSA ratio.
The decreasing or the poor increasing, respectively, of the AUCs of the
f-PSA/t-PSA ratio when applied to the entire spectrum of t-PSA values
can be attributed to low sensitivities of the ratio at low and high
concentrations of t-PSA. In early cancer stages and at low
concentrations of t-PSA, PSA measurement is influenced mainly by
immunological cross-reactions and by PSA of nonprostatic origin (e.g.,
perirectal and periurethral glands, blood cells) (6). On
the other hand, ACT is produced by the tumor itself, and therefore the
production of PSA and ACT depends not only on the tumor size, but also
on the grading of the tumor (15). With higher grading,
tumor cells presumably lose their ability to produce ACT, and therefore
tissue heterogeneity contributes to variation in PSA measurement.
We conclude that the f-PSA/t-PSA ratio may be a helpful
instrument for the differential diagnosis of BPH and PCA within the
diagnostic gray area of 4–25 µg/L t-PSA. Because of strong variation
in t-PSA measurements, the diagnostic gray area of t-PSA will have to
be evaluated for each assay.
|
Acknowledgments
|
|---|
We thank Antje Altekruse, Pia Becker, Francoise Flamong, Marion
Hartmann, Agnes Hülsmann, and Beate Pepping-Schefers for their
excellent technical assistance, Helmut Schulte for his help with
statistical analysis, Axel Semjonow for the patients' data, and H.
Keller, Zürich, Switzerland, for helpful suggestions.
|
Footnotes
|
|---|
1 Nonstandard abbreviations: PSA, prostate-specific antigen; f-PSA, free PSA; t-PSA, total PSA; ACT, 1-antichymotrypsin; AUC, area under the curve; BPH, benign prostate hyperplasia; NPV, predictive value of the negative result; PCA, prostate cancer. 
|
References
|
|---|
-
Christensson A, Laurell CB, Lilja H. Enzymatic activity of prostate-specific antigen and its reactions with extracellular serine proteinase inhibitors. Eur J Biochem 1990;194:755-763.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Watt KW, Lee PJ, M'Timkulu T, Chan WP, Loor R. Human prostate-specific antigen: structural and functional similarity with serine proteases. Proc Natl Acad Sci U S A 1986;83:3166-3170.
[Abstract/Free Full Text]
-
Wang MC, Valenzuela LA, Murphy GP, Chu TM. Purification of a human prostate specific antigen. Invest Urol 1979;17:159-163.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Smith MR, Biggar S, Hussain M. Prostate-specific antigen messenger RNA is expressed in non-prostate cells: implications for detection of micrometastases. Cancer Res 1995;55:2640-2644.
[Abstract/Free Full Text]
-
Wang MC, Papsidero MC, Kuriyama M, Valenzuela LA, Murphy GP, Chu TM. Prostate antigen: a new potential marker for prostate cancer. Prostate 1981;2:89-96.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Ruckle HC, Klee GG, Oesterling JE. Prostate specific antigen: concepts for staging prostate cancer and monitoring response to therapy. Mayo Clin Proc 1994;69:69-79.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Glenski WJ, Malek RS, Myrtle JF, Oesterling JE. Sustained, substantially increased concentration of prostate-specific antigen in the absence of prostatic malignant disease: an unusual clinical scenario. Mayo Clin Proc 1992;67:249-252.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Lilja H, Christensson A, Dahlen U, Matikainen MT, Nilsson O, Pettersson K, Lovgren T. Prostate-specific antigen in serum occurs predominantly in complex with 1-antichymotrypsin. Clin Chem 1991;37:1618-1625.
[Abstract/Free Full Text]
-
Christensson A, Bjork T, Nilsson O, Dahlen U, Matikainen MT, Cockett AT, et al. Serum prostate specific antigen complexed to 1-antichymotrypsin as an indicator of prostate cancer. J Urol 1993;150:100-105.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Stenman UH, Leinonen J, Alfthan H, Rannikko S, Tuhkanen K, Alfthan O. A complex between prostate-specific antigen and 1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.
[Abstract/Free Full Text]
-
Luderer AA, Chen YT, Soriano TF, Kramp WJ, Carlson G, Cuny-C, et al. Measurement of the proportion of free to total prostate-specific antigen improves diagnostic performance of prostate-specific antigen in the diagnostic gray zone of total prostate-specific antigen. Urology 1995;46:187-194.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Catalona WJ, Smith DS, Wolfert RL, et al. Evaluation of percentage of free serum prostate-specific antigen to improve specificity of prostate cancer screening. JAMA 1995;274:1214-1220.
[Abstract/Free Full Text]
-
Semjonow A, Hamm M, Rathert P, Hertle L. Prostate-specific antigen corrected for prostate volume improves differentiation of benign prostatic hyperplasia and organ-confined prostatic cancer. Br J Urol 1994;73:538-543.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Chen Z, Prestigiacomo A, Stamey TA. Purification and characterization of prostate-specific antigen (PSA) complexed to 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays. Clin Chem 1995;41:1273-1282.
[Abstract/Free Full Text]
-
Bjork T, Bjartell A, Abrahamsson PA, Hulkko S, di Sant'Agnese A, Lilja H. 1-Antichymotrypsin production in PSA-producing cells is common in prostate cancer but rare in benign prostatic hyperplasia. Urology 1994;43:427-434.
[Web of Science][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
|
|
|
|
 
K. Jung, U. Elgeti, M. Lein, B. Brux, P. Sinha, B. Rudolph, S. Hauptmann, D. Schnorr, and S. A. Loening
Ratio of Free or Complexed Prostate-specific Antigen (PSA) to Total PSA: Which Ratio Improves Differentiation between Benign Prostatic Hyperplasia and Prostate Cancer?
Clin. Chem.,
January 1, 2000;
46(1):
55 - 62.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. P. Fox, A. A. Reilly, and E. Schneider
Effect of the Ratio of Free to Total Prostate-specific Antigen on Interassay Variability in Proficiency Test Samples
Clin. Chem.,
August 1, 1999;
45(8):
1181 - 1189.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. J. Allard, Z. Zhou, and K. K. Yeung
Novel immunoassay for the measurement of complexed prostate-specific antigen in serum
Clin. Chem.,
June 1, 1998;
44(6):
1216 - 1223.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
