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Letters |
Cancer Care Diagnostics Product Devel., Chiron Diagnostics, 333 Coney St., E. Walpole, MA 02032-1597
a Author for correspondence. Fax (508) 660-4591; prakash.tewari@chirondiag. com.
To the Editor:
The Technical Brief by Blase et al. (1) reported
some troubling clinical conclusions while describing analytical
differences among various immunoassays of prostate-specific antigen
(PSA). Their study used samples consisting of free PSA and PSA
complexes prepared in vitro. The free PSA molecule represents a very
heterogeneous population, including pro-PSA, cleaved ("nicked") PSA
(2), PSA that can complex with
1-antichymotrypsin (ACT), and PSA that cannot complex
with ACT but complexes with 2-macroglobulin
(3). In addition, heterogeneity in the carbohydrate part
of the PSA molecule results in several isoforms, ranging from
nonglycosylated to fully glycosylated. These variations of the free PSA
molecule also affect its immunological characteristics, and for that
reason, results of comparison studies done with mixtures of free PSA
from seminal plasma do not represent the results that would be obtained
with serum.
PSA assays included in the study by Blase et al. (1) were: Hybritech Tandem-ERA, Tosoh AIA, and ACS:180 PSA2. The designs of these assays differ, in that the Tandem-ERA and Tosoh assays use two monoclonal antibodies, whereas the ACS:180 PSA2 uses a combination of monoclonal and polyclonal antibodies. The terms "equimolar" and "skewed" are descriptive only of analytical characteristics in PSA assay design and do not take into consideration differences in assay calibration.
In 1996 Stamey and colleagues (4) distributed to several
manufacturers a set of nine control serum samples containing various
proportions of free PSA. Six of these samples contained serum-derived
free PSA; the remaining three contained free PSA purified from seminal
fluid. These samples were tested with Tandem-R, Tosoh, and ACS:180
PSA2 assays. Results from the specimens with serum free PSA
demonstrated that changes in the proportion of free PSA in patients'
sera do not cause a marked difference in the total PSA values measured
by poly/monoclonal- and dual-monoclonal-based assays (Table 1
). Results from the second set of samples, which contained free
PSA isolated from seminal fluid added to serum, demonstrated
substantially different total PSA values obtained by the
poly/monoclonal- and dual-monoclonal-based assays (Table 1
). These
differences cannot be explained by the difference in the assay
calibration or in the tracer antibodies. The results indicate notable
differences in immunoreactivity between free PSA isolated from seminal
fluid and free PSA in serum. Furthermore, because patients' sera with
>50% free PSA is extremely unusual (5), use of any
purified sample containing >50% free PSA adds another degree of error
to a hypothetical situation that has little relevance in clinical
practice.
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Reports in the literature describing the equimolar response of various PSA assays may be used to refute claims made by Blase et al. (1). The dual-monoclonal antibody-based assay, Tandem-R, demonstrated different affinity and reaction kinetics with free PSA isolated from serum and that from seminal fluid (3). Blase et al. (1) did not explain why in the Tandem-ERA assay the ratio of PSA determined by Tandem-ERA and the PSA assigned value ranged from 1.13 to 1.76 for 100% free PSA. The Tandem-ERA value is 71% higher than the Tosoh value (1.76 vs 1.03). Stamey (6) used an experimental protocol similar to that of Blase et al. and compared the Tosoh and Tandem-R PSA assays. The response of the Tosoh assay was consistent with the changing proportion of free PSA, but the Tandem-R values decreased with increasing proportion of free PSA, especially when free PSA made up >50% of the total PSA (6). These observations indicate that the molar response of the Tandem-ERA and Tandem-R assays, which use the identical antibody pair, may be affected by the assay architecture or reaction kinetics.
Studies conducted with the poly/monoclonal assay format have demonstrated excellent clinical correlation, showing linear increases in PSA concentration proportional to increases in tumor volume (7)(8). This was especially evident for serum PSA changes from patients with organ-confined tumors grouped according to tumor volumes (as determined histologically after radical prostatectomy) into ranges of either 1–4 cm or 4–8 cm (7). Stamey et al. (9) compared Tandem-R and ACS:180 PSA assays, using sera from patients with biopsy-confirmed benign prostate hyperplasia or prostate cancer, and demonstrated that an "equimolar" assay did not add any substantial value to clinical practice over a polyclonal-based assay format. Another report suggests that dual-monoclonal-based assays, because of epitope restriction, are highly sensitive to immune complexes of PSA and IgG, whereas commercial assays using a polyclonal detection system were able to detect important amounts of PSA (10).
Our experience in immunodiagnostics has taught us the near-impossibility of making two assays with different architectures and configurations provide identical results. Paired patients' data from Hall et al. (11), who used Tandem-R and Tandem-E assays, serve to highlight this point. In this instance, two assays produced by the same manufacturer with a substantially similar architecture (coated bead) and using the same antibodies gave a 43% difference in the cutoff value for healthy subjects, that for Tandem-E being higher than that for Tandem-R: 3.8 vs 2.7 µg/L. This type of difference is common in immunoassay measurement, as evidenced by the variable absolute values seen in CA 19–9 assays, even when identical antibodies are used and are standardized to the same antigen preparations (12).
Current PSA immunoassays are designed to measure total PSA in serum. To evaluate differences in such assays, one should use serum samples containing different proportions of free and complexed PSA. Free PSA isolated from seminal fluid differs in immunoreactivity from the free PSA present in serum, and it is difficult to draw clinical conclusions from a study examining purified control material. As Blase et al. (1) note, "... The recalibrated ACS PSA2 assay has been reported to give results in concordance with those determined with the Tandem PSA assay ... " Indeed, a recent publication (13) reports substantial equivalence between ACS:180 PSA2 assay and Hybritech Tandem-R PSA assay. Laboratories continue to select PSA assays on the basis of accurate results in clinical specimens and the workflow requirements of the cost-conscious clinical laboratory.
Note: A published correction (14) notes the
contradictory data in Fig. 1
and Table 1
of Blase et al. and confirms
that the correct data are depicted in Fig. 1
(i.e., that the values of
the ACS:180 PSA2 assay most closely correspond to values
from the other manufacturers' assays).
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References
1-antichymotrypsin: influence of cancer volume, location and therapeutic selection of resistant clones. J Urol 1994;152:1510-1514.
[ISI][Medline]
[Order article via Infotrieve]
Hybritech Incorporated, Subsidiary of Beckman Instruments, PO Box 269006, San Diego, CA 92196-9006
a Author for correspondence. Fax (619) 536-8058; e-mail abblase{at}beckman.com.
To the Editor:
We agree that the biochemistry of PSA is complicated and not completely understood; however, the arguments of Tewari and Williams actually support the conclusion that PSA assays are not interchangeable. Our study explored differences between PSA assays in order to develop useful information for the physician and laboratorian in selecting a particular PSA test (1).
The majority of PSA assays use antibodies developed against PSA
isolated from seminal fluid (2). Several investigators,
including Brawer (3), Strobel (4), and
Semjonow (5)(6), have shown that the
"skewed" phenomenon observed in analytically nonequimolar assays is
also strongly reflected in patients' sera (Fig. 1
). Thus, equimolar response remains an important clinical issue.
PSA from seminal fluid does, indeed, differ from PSA in serum. Recently, Mikolajczyk et al. demonstrated that pro-PSA is present in serum, and clipped forms represent only a small portion of total PSA; conversely, in seminal fluid, clipped forms represent a substantial portion, and pro-PSA is undetectable (7). Nevertheless, Hybritech's Tandem® PSA assay detects these various PSA forms equivalently (RL Wolfert and HG Rittenhouse, personal communication). The report cited by Tewari and Williams describing variations in immunorecognition between various PSA assay formats (8) provides further support that assays measure the various forms of PSA differently.
We agree with Tewari and Williams that equimolarity is a consideration independent of calibration. Equimolarity is an intrinsic quality of an immunoassay. Because our study (1) compared response ratios (observed value divided by expected value) across the assays, absolute calibration was removed as a variable. Moreover, "expected" PSA values were assigned to samples on the basis of spectrophotometric determinations and published absorptivity values (9), thereby remaining independent of any immunoassay or its calibration. Thus, the analytical conclusions stand regardless of differences in immunoassay calibration.
We agree with Tewari and Williams that it is nearly impossible to make two assay systems of different architectures provide identical results. However, in choosing to compare the upper end of a population reference range (mean 2 SD) established by Hall et al. (10), Tewari and Williams suggest a difference between assay formats that was not seen by those authors, who in fact concluded there was no significant difference between the compared methods (10).
Equimolarity is not merely an analytical issue but also a potentially important clinical issue. Recent studies have demonstrated that free PSA makes up 5–50% of the total PSA in serum, with occasional samples outside this range (11). This is an important finding, given that skewed assays report a total PSA value that can vary according to the proportion of free PSA in the specimen. Therefore, clinical parameters such as positive and negative predictive values, reference intervals, and cutpoints established for one assay cannot necessarily be transferred to another assay (2)(12)(13)(14)(15). Although several assay systems are approved for use in monitoring prostate cancer, only Hybritech's Tandem PSA assays have undergone extensive clinical trials and are approved by the FDA for detection of prostate cancer, in conjunction with digital rectal examination (16)(17).
From the discussion presented by this letter and reply, it is clear that additional clinical studies are needed to define each assay's particular performance and reference intervals with respect to the intended use. This also underscores the necessity for the laboratorian to understand that clinical utility data and reference intervals for one PSA assay do not necessarily apply to assays from other manufacturers (3)(4)(5)(6).
References
The following articles in journals at HighWire Press have cited this article:
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  S. A.R. Kort, F. Martens, H. Vanpoucke, H. L. van Duijnhoven, and M. A. Blankenstein Comparison of 6 Automated Assays for Total and Free Prostate-Specific Antigen with Special Reference to Their Reactivity toward the WHO 96/670 Reference Preparation Clin. Chem., August 1, 2006; 52(8): 1568 - 1574. [Abstract] [Full Text] [PDF]
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A. Semjonow, F. Oberpenning, C. Weining, M. Schon, B. Brandt, G. De Angelis, A. Heinecke, M. Hamm, P. Stieber, L. Hertle, et al. Do Modifications of Nonequimolar Assays for Total Prostate-specific Antigen Improve Detection of Prostate Cancer? Clin. Chem., August 1, 2001; 47(8): 1472 - 1475. [Full Text] [PDF]
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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]
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C. D. Cheli, M. Marcus, J. Levine, Z. Zhou, P. H. Anderson, D. D. Bankson, J. Bock, S. Bodin, C. Eisen, M. Senior, et al. Variation in the Quantitation of Prostate-specific Antigen in Reference Material: Differences in Commercial Immunoassays, Clin. Chem., July 1, 1998; 44(7): 1551 - 1553. [Full Text] [PDF]
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