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Clinical Chemistry 49: 887-894, 2003; 10.1373/49.6.887
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(Clinical Chemistry. 2003;49:887-894.)
© 2003 American Association for Clinical Chemistry, Inc.

Determination of Non-{alpha}1-Antichymotrypsin-complexed Prostate-specific Antigen as an Indirect Measurement of Free Prostate-specific Antigen: Analytical Performance and Diagnostic Accuracy

Sebastian Wesseling1,1, Carsten Stephan1,1, Axel Semjonow3, Michael Lein1, Brigitte Brux2, Pranav Sinha2, Stefan A. Loening1 and Klaus Jung1,a

Departments of
1 Urology and
3 Laboratory Medicine and Pathobiochemistry, University Hospital Charité, Humboldt University, D-10098 Berlin, Germany.

2 Department of Urology, Westfälische Wilhelms-University, D-48129 Münster, Germany.

aAddress correspondence to this author at: Department of Urology, University Hospital Charité, Humboldt University Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany. Fax 49-30-450-515904; e-mail klaus.jung{at}charite.de.


   Abstract
Top
 Abstract
Introduction
 Participants and Methods
 Results
 Discussion
 References
 
Background: A new assay measures prostate-specific antigen (PSA) not complexed to {alpha}1-antichymotrypsin (nACT-PSA) after removing PSA complexed to ACT by use of anti-ACT antibodies. We evaluated nACT-PSA and its ratio to total PSA (tPSA) as alternatives to free PSA (fPSA) and its ratio to tPSA in differentiating prostate cancer (PCa) and benign prostatic hyperplasia (BPH) in patients with tPSA of 2–20 µg/L.

Methods: PSA in serum of 183 untreated patients with PCa and 132 patients with BPH was measured retrospectively on the chemiluminescence immunoassay analyzer LIAISON® (Byk-Sangtec Diagnostica) with the LIAISON tPSA and LIAISON fPSA assays. The nACT-PSA fraction was determined with a prototype assay measuring the residual PSA after precipitation of ACT-PSA with an ACT-precipitating reagent.

Results:nACT-PSA was higher than fPSA in samples with fPSA concentrations <1 µg/L but lower in samples with >1 µg/L fPSA. The median ratios of fPSA/tPSA and of nACT-PSA/tPSA were significantly different between patients with BPH and PCa (19.4% vs 12.2% and 17.4% vs 13.0%, respectively). Within the tPSA ranges tested (2–20, 2–10, and 4–10 µg/L), areas under the ROC curves for the fPSA/tPSA ratios were significantly larger than those for nACT-PSA/tPSA. In the tPSA ranges <10 µg/L, the areas under the ROC curves for fPSA/tPSA were significantly larger than those for tPSA, whereas the areas for nACT-PSA/tPSA were not. At decision limits for 95% sensitivity and specificity, both ratios significantly increased specificity and sensitivity, respectively, compared with tPSA, but the fPSA/tPSA ratio showed higher values.

Conclusions: nACT-PSA and its ratio to tPSA provide lower diagnostic sensitivity and specificity than fPSA/tPSA. The fPSA/tPSA ratio represents the state-of-the-art method for differentiating between PCa and BPH.


   Introduction
Top
 Abstract
 Introduction
Participants and Methods
 Results
 Discussion
 References
 
Prostate-specific antigen (PSA) 2 is the most useful marker for early detection of prostate cancer (PCa). The established PSA cutoff for differentiating cancer and nonmalignant prostatic diseases (4 µg/L) gives a false-positive rate of 65% because serum PSA is also increased in benign prostatic hyperplasia (BPH) and inflammatory prostatic diseases (1). Differentiation between BPH and PCa can be improved, however, by separate measurements of the molecular forms of PSA in serum (2)(3)(4)(5).

PSA occurs in serum in different molecular forms. Approximately 70–90% of total PSA (tPSA) is complexed with {alpha}1-antichymotrypsin (ACT), and smaller amounts (<5%) are complexed with {alpha}1-protease inhibitor and inter-{alpha}-trypsin inhibitor (2)(3)(4)(6). Some PSA is complexed with {alpha}2-macroglobulin and cannot be measured by conventional PSA assays because the PSA is hidden in the complex and the epitopes are not accessible for the PSA antibodies (7). Approximately 10–30% of tPSA is not bound to serum proteins and is called free PSA (fPSA). Numerous studies have demonstrated a lower ratio of fPSA to tPSA (fPSA/tPSA) in PCa patients, calculated as a percentage of fPSA (5)(8). This ratio has been considered a promising tool for differentiating between PCa and BPH.

fPSA measurements are based on an antibody that recognizes fPSA but not the PSA-ACT complex. Although most fPSA assays work well, the low specificities of the antibodies used for these assays (9)(10)(11) and the partial masking of the fPSA-specific binding site by ACT (12) can impair accurate fPSA measurements (13). Two alternative methods have been proposed to circumvent these possible analytical difficulties: measurement of the complexed PSA fraction instead of fPSA, or measurement of fPSA after elimination of the complexed PSA fraction. The pros and cons of measuring complexed PSA have recently been reported (14)(15)(16), and this report is not the place for further statements. On the other hand, the alternative method involving measurement of fPSA after elimination of the complexed PSA fraction would have the advantage of requiring only one immunologic test for determination of the fPSA/tPSA ratio and could eliminate potential differences between two immunologic assays (17).

In 2000, two independent groups reported preliminary results obtained with this new technology for determining fPSA (18)(19). Both groups used the same ACT-precipitating reagent containing antibodies against ACT (Scantibodies PR Technology). The residual PSA after elimination of the ACT-PSA complex consists of the true fPSA and the minor complexed forms. Thus, this PSA fraction has been named "non-ACT-complexed PSA" (nACT-PSA) and has been considered an indirect measurement of fPSA. The preliminary studies showed promising data with regard to the feasibility of this approach to determine fPSA, but included only a limited number of clinical samples and did not give detailed information. A fully automated prototype assay for nACT-PSA has since been developed that is based on previously described chemiluminescent technology for measuring PSA (20). The aims of the present study were (a) to evaluate this new assay in two centers to characterize its analytical performance and its diagnostic accuracy for differentiating PCa and BPH based on the ratio nACT-PSA/tPSA and (b) to compare the data obtained with this indirect method for fPSA with the results obtained with a conventional fPSA assay for calculating the ratios of fPSA and nACT-PSA, respectively, to the same tPSA value.


   Participants and Methods
Top
 Abstract
 Introduction
 Participants and Methods
Results
 Discussion
 References
 
study groups
The study included 315 patients with an established diagnosis of BPH or PCa and tPSA of 2–20 µg/L. Of these, 195 were investigated at the Department of Urology of the University Hospital Charité in Berlin, and 120 were investigated at the Department of Urology of the University Hospital in Münster. We used all archived sera collected between December 1997 and July 2001 (1997, n = 5; 1998, n = 74; 1999, n = 62; 2000, n = 72; 2001, n = 102) and available as surplus serum (neither thawed nor refrozen) with sufficient volume for additional measurements in this retrospective study. Because the patients with PCa and BPH investigated in the two centers did not differ (P >0.05, t-test) with regard to age, tPSA, and nACT-PSA between the two centers, the corresponding patients were combined in one group each. The study was approved by the local ethics boards of the hospitals. Consent was obtained from each patient.

BPH group.
The BPH group included 132 patients (median age, 64 years; range, 23–82 years): 64 (median age, 62 years; range, 44–78 years) who were investigated at the University of Münster and 68 (median age, 65 years; range, 23–82 years) who were investigated at the University Hospital Charité. In the Berlin group, the diagnosis of BPH was established clinically by digital rectal examination and/or transrectal ultrasonography, and in 23 cases the BPH was histologically confirmed based on prostate tissue biopsy. In the Münster group, the diagnosis was histologically confirmed in all cases. Because there were no differences regarding tPSA and the fPSA/tPSA ratio between the two BPH subgroups diagnosed by clinical or histologic examination, both groups were considered as one group. Of the 132 patients with tPSA concentrations between 2 and 20 µg/L, 106 had tPSA values between 2 and 10 µg/L, 26 had values between 10 and 20 µg/L, and 63 had values between 4 and 10 µg/L.

PCa group.
The PCa group included 183 patients (median age, 64 years; range, 48–77 years): 56 (median age, 65 years; range, 48–77 years) who were investigated at the University of Münster and 127 (median age, 64 years; range, 49–77 years) who were investigated at the University Hospital Charité. PCa was diagnosed histologically with blood samples taken before different treatment regimens (radical prostatectomy, radiotherapy, or hormone therapy) (21). Of these 183 patients, 144 had tPSA values between 2 and 10 µg/L, 116 had values between 4 and 10 µg/L, and 39 had values between 10 and 20 µg/L.

sample collection
Blood samples were taken before diagnostic procedures or at least 4 weeks (at the earliest) after digital examination, prostate biopsy, or transrectal ultrasound. Thus, possible errors caused by the differential release of fPSA and complexed PSA from the prostate and the different elimination kinetics of both forms from blood were avoided (22). Blood samples were centrifuged at 1600g for 15 min at 4 °C. The sera were frozen within 2 h after venipuncture and stored at -80 °C until analysis.

psa assays
After the serum samples were thawed at room temperature, all measurements of tPSA, fPSA, and nACT-PSA for each sample were performed within 5 h by experienced laboratory scientists and technicians (three in Berlin and two in Münster) who were blinded to the clinical origin of the samples.

PSA measurements were performed on the fully automated chemiluminescence immunoassay analyzer LIAISON® (Byk-Sangtec Diagnostica) with the LIAISON tPSA and LIAISON fPSA assays exactly according to the manufacturer’s instructions. The analytical details have been described elsewhere (20)(23). In addition, the nACT-PSA fraction was determined with a newly developed prototype assay (Byk-Sangtec Diagnostica) that measures the residual PSA after precipitation of ACT-PSA with an ACT-precipitating reagent (Scantibodies Laboratory PR Technology) containing polyclonal anti-ACT antibodies. This prototype assay uses the components of the established LIAISON PSA assay, a two-step, two-site immunoluminometric assay that incorporates two highly specific monoclonal antibodies against PSA for the equimolar detection of free PSA and the PSA-ACT complex. Addition of the precipitating reagent to the reaction mixture completely removes the ACT-bound PSA from the reaction, allowing specific detection of nACT-PSA. Briefly, 50 µL of serum is incubated with PSA antibody-coated paramagnetic particles and the ACT-precipitating reagent. After a wash step, the isoluminol-labeled tracer PSA antibody is added, and an immunocomplex is formed. The chemiluminescent signal is generated by adding the starter reagents. Sample concentrations are then calculated using a stored master curve that is recalibrated with two calibrators.

Additional comparative PSA measurements were performed with the Immulite fPSA assay (Diagnostic Product Corp.) in Berlin and the Access Hybritech fPSA assay (Beckman-Coulter) in Münster.

statistical analysis
Data were analyzed with the statistical software MedCalc 7.01.0 (MedCalc Software) and GraphPad Prism 3.03 for Windows (GraphPad Software). The methodologies were evaluated according to the procedures of Passing and Bablok (24) and Bland and Altman (25). The software GraphRoc for Windows was used to analyze the ROC curves (26). Differences were considered statistically significant at P <0.05.


   Results
Top
 Abstract
 Introduction
 Participants and Methods
 Results
Discussion
 References
 
analytical performance
Before the beginning of the study, the precision and accuracy of the LIAISON tPSA, LIAISON fPSA, and nACT-PSA assays were ascertained. The PSA concentrations measured in three control sera (Lyphocheck; Bio-Rad Laboratories) were not different in the two centers and were within the target range so that the analytical precondition was fulfilled to perform measurements at the two study sites. During the actual study (March 2001 to July 2001) when the patients’ samples were measured in series (Berlin, n = 11; Münster, n = 5), the mean values for tPSA, fPSA, and nACT-PSA obtained in the three control sera were also not different at both study sites (P >0.05, t-test). The tPSA, fPSA, and nACT-PSA values obtained for control serum L1 were 0.74, 0.27, and 0.24 µg/L, respectively, in Münster and 0.78, 0.28, and 0.28 µg/L, respectively, in Berlin; for control serum L2, the values obtained were 2.21, 1.60, and 1.26 µg/L, respectively, in Münster and 2.18, 1.60, and 1.29 µg/L, respectively, in Berlin; and for control serum L3, the values obtained were 20.6, 11.6, and 12.0 µg/L, respectively. in Münster and 20.2, 11.6, and 11.9 µg/L, respectively. in Berlin. Thus, data from both sites were combined for analysis. The interassay imprecision data for both centers during the study are summarized in Fig. 1 . The new nACT-PSA assay had slightly higher imprecision than fPSA and tPSA assays in the PSA range <1 µg/L.



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  		Figure 1. Interassay imprecision profiles for tPSA, fPSA, and nACT-PSA.

Shown are mean CV values calculated from 16 separate analyses (Berlin, n = 11; Münster, n = 5) of three control sera (Lyphocheck; Bio-Rad Laboratories) during the study. As stated in the text, the mean PSA concentrations measured in the control sera were not different between study sites.

To further characterize the new nACT-PSA assay, we assessed the analytical performance by evaluating assay linearity, the dilution characteristics, the recovery rate, the limit of quantification, and interference by serum components and drugs as described previously (20). Dilution experiments showed that the nACT-PSA assay was linear up to at least 90 µg/L. To test the linearity on dilution, we diluted six serum samples with nACT-PSA concentrations between 3 and 90 µg/L with the assay buffer (volume fraction of the samples: 0.2, 0.4, 0.6, and 0.8). The mean (SD) recovery for each dilution step in relation to the theoretical value was 109.5 (7.1)%. The recovery calculated from the measured data of three mixed serum samples with high and low concentrations was 100 (9.7)%. The limit of detection, defined as the concentration corresponding to the mean + 3 SD of 20 replicate measurements of the zero calibrator, was 0.021 µg/L. There was no high-dose hook effect with serum samples containing up to 38 311 µg/L nACT-PSA. Bilirubin (125 mg/L), triglycerides (12.5 g/L), hemoglobin (500 g/L), and the drugs doxorubicin (98 mg/L), cyclophosphamide (324 mg/L), and methotrexate (12.8 mg/L) did not interfere with the nACT-PSA assay (recovery rate of nACT-PSA, 94–106%). In addition, various amounts of total serum protein (52–95 g/L) had no influence on measurement of nACT-PSA (recovery, 94–109%).

method comparison
We performed method comparisons between the LIAISON fPSA and nACT-PSA assays according to the regression procedure of Passing and Bablok (24) and with use of Bland-Altman plots (25). Although the median fPSA (x) and nACT-PSA (y) concentrations did not differ (0.86 vs 0.89 µg/L; P >0.05), the equation for the regression line [y = 0.81 (0.78–0.84)x + 0.16 (0.14–0.18) µg/L (95% confidence intervals in parentheses); Fig. 2A ] and the percentage difference plot (Fig. 2B ) showed clear differences. Separate regression analyses for PCa and BPH patients yielded similar equations for the regression line, and it can be concluded that these differences depend on the fPSA concentration. In samples with fPSA concentrations <1 µg/L, nACT-PSA was higher than fPSA, whereas in samples with >1 µg/L fPSA, nACT-PSA was lower than fPSA.



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  		Figure 2. Method comparison according to Passing and Bablok (24) (A) and Bland and Altman (25) (B).

Additional details are given in the text.

We also compared the nACT-PSA values (y) in randomized samples with fPSA (x) values determined with the Immulite fPSA assay [y = 0.97 (0.91–1.03)x + 0.07 (0.01–0.13) µg/L; r = 0.979; n = 51] and the Access fPSA assay [y = 0.87 (0.81–0.94)x + 0.12 (0.03–0.18) µg/L; r = 0.933; n = 100].

clinical evaluation of liaison psa, liaison FPSA, NACT-PSA, and their ratios in the study groups
Scatter plots and the medians of the LIAISON assays for tPSA, fPSA, nACT-PSA, and their ratios for the PCa and BPH patients are shown in Fig. 3 . PCa patients had somewhat higher tPSA (Fig. 3A ) but slightly lower fPSA and nACT-PSA concentrations than BPH patients (Fig. 3 , B and C). As expected, the PCa patients had significantly lower fPSA/tPSA and nACT-PSA/tPSA ratios, respectively, compared with BPH patients (Fig. 3 , D and E).



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  		Figure 3. Scatter plots for tPSA (A), fPSA (B), nACT-PSA (C), and the percentage ratios of fPSA/tPSA (D) and nACT-PSA/tPSA (E) in patients with BPH (n = 132) and PCa (n = 183).

PSA measurements were performed on a LIAISON chemiluminescence immunoassay analyzer (Byk-Sangtec Diagnostica) with the LIAISON tPSA and LIAISON fPSA assays and a prototype assay for the nACT-PSA fraction. Horizontal lines indicate mean values. Statistical differences were tested by the Student t-test.

roc analysis and diagnostic validity
ROC analyses were performed for all patients with tPSA values between 2 and 20 µg/L and separately for patients with tPSA concentrations <10 µg/L (Fig. 4 ). When all patients within the tPSA range 2–20 µg/L were considered, areas under the ROC curves for the ratios fPSA/tPSA and nACT-PSA/tPSA showed statistically significant differences compared with the area under the curve of tPSA (Fig. 4A ; P = 0.0002 and 0.007, respectively). The area under the fPSA/tPSA ROC curve was higher than that under the nACT-PSA/tPSA curve (P = 0.006). In the tPSA range 2–10 µg/L (Fig. 4B ), the area under the ROC curve for fPSA/tPSA was significantly higher than that of tPSA (P = 0.029), but the area under the curve for nACT-PSA/tPSA was not (P = 0.226). The area under the ROC curve for fPSA/tPSA was significantly higher than the area under the ROC curve for nACT-PSA/tPSA. In the tPSA range 4–10 µg/L (Fig. 4C ), we observed results similar to those for 2–10 µg/L tPSA (fPSA/tPSA and nACT-PSA/tPSA compared with tPSA: P = 0.010 and 0.103, respectively; fPSA/tPSA compared with nACT-PSA/tPSA: P = 0.010).



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  		Figure 4. ROC curves for all patients with tPSA between 2 and 20 µg/L (A) and patients with tPSA concentrations in the ranges 2–10 µg/L (B) and 4–10 µg/L (C).

The analysis included 132 patients with BPH and 183 with PCa, of whom 106 BPH and 144 PCa patients had 2–10 µg/L tPSA and 63 BPH and 116 PCa patients had 4–10 µg/L tPSA. The curves for tPSA and the ratios fPSA/tPSA and nACT-PSA/tPSA were calculated. Areas under the curves (±SE) are given. Additional details are given in the text.

Listed in Table 1 are the sensitivities and specificities of tPSA and the fPSA/tPSA and nACT-PSA/tPSA ratios at the decision limits for 95% sensitivity and specificity in the tPSA range 2–20 µg/L. The specificity and the sensitivity of the two ratios were significantly higher than the corresponding values for tPSA.


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  		Table 1. Diagnostic sensitivity and specificity of tPSA and the fPSA/tPSA and nACT-PSA/tPSA ratios for differentiating PCa and BPH patients.13


   Discussion
Top
 Abstract
 Introduction
 Participants and Methods
 Results
 Discussion
References
 
Assaying of nACT-PSA as an alternative of fPSA is based on the fact that the PSA complexed to ACT is the main fraction of complexed PSA. Thus, after elimination of this fraction, as performed in this study by precipitation with anti-ACT antibodies, the residual fraction would consist mainly of fPSA. All analytical performance characteristics of the nACT-PSA assay presented in this study, such as interassay imprecision, analytical recovery, linearity, and limit of quantification, demonstrated that this prototype assay gives acceptable analytical results, but at fPSA concentrations <1 µg/L, the nACT-PSA assay had higher imprecision than the conventional fPSA assay. In addition, nACT-PSA was frequently higher than fPSA in samples with fPSA concentrations <1 µg/L, and was lower that fPSA in samples with >1 µg/L fPSA (Fig. 2BUp ). These results are remarkable, but they do not preclude use of the nACT-PSA assay as an indirect measure of fPSA for better differentiation of patients with PCa from those with BPH. We therefore believe testing the clinical utility of this new assay was justified because preliminary data did not provide a clear answer (18)(19).

The proposed assay allows measurement of nACT-PSA and tPSA with the same assay and could therefore eliminate the differences that might exist when two types of assays are being used (11)(27)(28). Our results in Fig. 3Up confirmed that the fPSA/tPSA ratio was lower in PCa than in BPH patients. Similar differences were observed when nACT-PSA was measured. At 95% sensitivity, the fPSA/tPSA and nACT-PSA/tPSA ratios produced comparable cutoffs of 24.8% and 23.2%, respectively (Table 1Up ). These cutpoints correspond to data from other authors (29)(30)(31) and were shown previously with the LIAISON fPSA assay (23). In this respect, our additional comparisons with other fPSA assays (Immulite DPC and Access Hybritech) also showed similar differences with respect to the LIAISON fPSA assay.

The determination of nACT-PSA and use of the nACT-PSA/tPSA ratio provide better differentiation of patients with PCa and those with BPH than do tPSA measurements alone. The nACT-PSA could thus be considered an alternative to fPSA. However, more detailed ROC analysis of the PCa and BPH patients in this study revealed better clinical differentiation with the fPSA/tPSA ratio than with the nACT-PSA/tPSA ratio in the tPSA ranges <10 µg/L (Fig. 2Up , B and C). Although we observed no statistical differences at the 95% sensitivity and specificity cutoffs between the two ratios (Table 1Up ), the values obtained with the fPSA/tPSA ratio were higher. These results demonstrate the diagnostic advantage of the fPSA/tPSA ratio compared with the nACT-PSA/tPSA ratio. Thus, at least from a diagnostic point of view, the nACT-PSA assay needs further optimization if it is to replace fPSA determinations in clinical use.

The approach using nACT-PSA did not meet our expectations for two reasons. The first reason is that the nACT-PSA value includes fPSA and minor complexed PSA forms, but exact data are known only for the fraction of PSA bound to {alpha}1-protease inhibitor. In patients with 4–20 µg/L tPSA, the proportion of {alpha}1-protease inhibitor-bound PSA was significantly higher in BPH patients than in PCa patients (median, 4.1% vs 3.2%) (6). This parallel behavior with fPSA should produce an even higher nACT-PSA fraction in BPH patients that might improve differentiation between BPH and PCa patients. The reason for the opposite result obtained in the present study is not clear but could result from other minor, interfering complexed forms of PSA. The second, and more probable, reason is that the higher imprecision of the nACT-PSA assay in the range <1 µg/L may contribute to the lower diagnostic sensitivity and specificity of the nACT-PSA/tPSA ratio compared with the fPSA/tPSA ratio.

After we had completed our study, we became aware that two other companies (Biotrol Diagnostic and Eurogenetics) used a similar approach to measure the PSA fraction after precipitation of ACT-PSA. To our knowledge, there are no data on the clinical utility of these assays in the literature.

In conclusion, measurement of the nACT-PSA fraction after precipitation of ACT-PSA and use of the nACT-PSA/tPSA ratio provided lower diagnostic sensitivity and specificity for differentiating between PCa and BPH than the fPSA/tPSA ratio. A change of methods should therefore be carefully evaluated by each laboratory, taking into consideration the benefits and disadvantages of either method.


   Acknowledgments
 
This study includes parts of the doctoral thesis of S.W. and was supported in part by the following sources and grants: funds of the German Chemical Industry (no. 400770 to K.J.) and SONNENFELD-Stiftung (to K.J. and P.S.). The LIAISON PSA and LIAISON fPSA reagent sets and the prototype nACT-PSA assay (not commercially available) were provided free of charge by Byk-Sangtec Diagnostica (Dietzenbach, Germany).


   Footnotes
 
1 These authors contributed equally to this work.

2 Nonstandard abbreviations: PSA, prostate-specific antigen; PCa, prostate cancer; BPH, benign prostatic hyperplasia; tPSA and fPSA, total and free PSA, respectively; ACT, {alpha}1-antichymotrypsin; and nACT-PSA, non-ACT-complexed PSA.


   References
Top
 Abstract
 Introduction
 Participants and Methods
 Results
 Discussion
 References
 

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