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Five PSA methods compared by assaying samples with defined PSA ratios.

Prostate-specific antigen (PSA) has been established as a marker to aid in detection and monitoring of prostate cancer (1][2]. PSA in serum exists predominantly in three forms: free, uncomplexed PSA; PSA covalently complexed to 1-antichymotrypsin (PSA-ACT); and PSA covalently complexed to 2-macroglobulin (PSA-MG] [3]. Immunoassays available today recognize free PSA and PSA-ACT but not PSA-MG, and their result for "total PSA" refers to the sum of the free and ACT-bound forms of PSA as measured by the immunoassay.

In general, the proportion of free PSA relative to PSA-ACT is lower in prostate cancer patients than in normal subjects or in patients with noncancerous prostatic disease [4]. The percentage of free PSA ranges from 5% to 50% when total serum PSA is 4-10 [micro]g/L [5][6]. However, increased serum concentrations of PSA do not necessarily indicate prostate cancer because such values can also occur in cases of benign prostate hyperplasia or prostatitis [7][8][9].

Assays of total serum PSA differ from one another in an important respect: They do not recognize the free and ACT-bound species of PSA equivalently [10][11]. "Equimolar-response assays" measure equal molar concentrations of free PSA and PSA-ACT equivalently; "skewed-response assays" measure these PSA forms differently [12][13][14].

The total PSA concentration measured by an equimolar assay depends on only the total concentration of free PSA plus PSA-ACT and is independent of their relative proportions. The interpretation of results reported by skewed-response assays, however, may be misleading because (a) the proportion of free PSA is generally higher in noncancer patients, and (b) the molar response of skewed-response assays for free PSA is typically higher than that for PSA-ACT. Because the average proportion of free PSA is higher in noncancer patients than in cancer patients [4], values reported by skewed-response PSA assays for noncancer patients may well be higher than predicted by equimolar PSA assays. Moreover, if the ratio of free PSA to total PSA changes between serial PSA measurements but the total PSA concentration is unchanged, the skewed-response assay might nonetheless report a change in the PSA value [15].

We compared PSA assays by use of a set of "defined PSA ratio" samples, prepared with specific proportions of free PSA and PSA-ACT. We purified PSA from seminal fluid as described by Sensabaugh and Blake [16] and incubated it with a sixfold molar excess of ACT (purchased from Athens Research and Technology, Athens, GA) for 18 h at 37[degrees]C in Tris-buffered saline, pH 7.4. The PSA-ACT complex and the unreacted free PSA were isolated by hydrophobic interaction chromatography [16]. The fractions corresponding to PSA-ACT complex and to free PSA were pooled separately. After buffer exchange with 100 mmol/L ammonium acetate buffer, the concentration of each pool was determined spectrophotometrically at 280 nm [17]. We prepared four working concentrations of PSA, using as a diluent a bovine protein matrix containing no detectable PSA (PSA-R Zero diluent, provided in the Tandem[R]-R PSA kit; Hybritech, San Diego, CA). Mixtures of PSA-ACT and free PSA in various ratios were prepared by combining the free PSA solution with the corresponding PSA-ACT solution at the same working concentration. In all, 20 solutions were prepared, representing all possible combinations of four combinations (total PSA: 20, 10, 5, and 2.5 [micro]g/L) at five molar ratios of free:complexed PSA (0:100, 25:75, 50:50, 75:25, and 100:0). Adjustments to the concentrations were based on the spectrophotometric values measured. Each solution was divided into aliquots, which were flash-frozen in liquid nitrogen and stored at -70[degrees]C. Assigned concentrations were not based on immunoassay results.

Each of the 20 concentration-ratio combination samples was tested in duplicate at a single, independent laboratory with each of the following assays: Tandem-E PSA for the Photon ERA (Tandem PSA ERA), a semiautomated, dual-monoclonal immunoenzymometric assay (Hybritech); IMx PSA, an automated polyclonal/monoclonal enzymoimmunoassay formatted for use in the IMx system (Abbott Labs., Abbott Park, IL); ACS:180 [PSA.sub.1] and ACS:180 [PSA.sub.2], both automated polyclonal/monoclonal immunochemiluminometric assays formatted for use in the ACS:180 system (Chiron Diagnostics, East Walpole, MA); and AIA-PACK PA, an automated dual-monoclonal immunoenzymometric assay formatted for use with the AIA 600 or 1200 analyzers (Tosoh, Tokyo, Japan). The ACS:180 [PSA.sub.2] assay was recently released as a recalibrated version of the [PSA.sub.1] assay (18][19].

For each assay format, we calculated the ratio of the determined PSA concentration to the assigned concentration ([R.sub.t] = [[PSA].sub.determined]/[[PSA].sub.assigned]) for each sample (Table 1). The data were subjected to general linear models (GLM) and analysis of variance (ANOVA) routines in SAS (Statistical Analysis Software, Cary, NC). Using the GLM procedure, we performed Duncan's Multiple Range test to compare the results from each manufacturer. According to this analysis ( = 0.05), Tandem PSA ERA and Tosoh AIA results were not significantly different from each other, but IMx PSA, ACS [PSA.sub.1], and ACS [PSA.sub.2] differed significantly from each of the other assays. To determine the effect of free PSA proportion and the effect of total PSA concentration on [R.sub.t], we performed a separate ANOVA for each manufacturer's assay. The data ([R.sub.t]) were grouped according to the proportion of free PSA in the sample (0%, 25%, 50%, 75%, and 100%, with each group containing four data points) and the concentration of total PSA (2.5, 5, 10, and 20 [micro]g/L, with each group containing five data points). At [alpha] = 0.05, the effect of PSA concentration was not significant for any of the assay formats: The P-values were 0.47 (Tandem PSA ERA), 0.74 (Tosoh AIA), 0.17 (IMx PSA), 0.37 (ACS [PSA.sub.1]), and 0.26 (ACS [PSA.sub.2]). However, the proportion of free PSA significantly affected [R.sub.t] values for all formats except Tandem PSA ERA: The P-values were 0.40 for Tandem PSA ERA, 0.02 for Tosoh AIA, and <0.01 for IMx PSA, ACS [PSA.sub.1], and ACS [PSA.sub.2].


The extent of this effect is illustrated in Fig. 1 , which shows that the PSA values reported by the Tandem PSA ERA were unaffected by the proportion of free PSA. Although our analysis suggested a statistically significant effect of the proportion of free PSA on [R.sub.t] for the Tosoh AIA-PACK PA assay, Fig. 1 shows that any such effect was barely observable. Thus, for all practical purposes the Tandem PSA ERA and Tosoh AIA-PACK PA both corresponded to the definition of an equimolar assay [12]. In contrast, values reported by the IMx PSA, ACS [PSA.sub.1], and ACS [PSA.sub.2] assays increased with increasing proportions of free PSA, corresponding to the definition of a skewed assay [12].


These results reinforce earlier reports that IMx PSA [11] and ACS [PSA.sub.1] [20][21] are skewed-response assays; i.e., they measure the free PSA and PSA-ACT forms differently. Our study showed that ACS [PSA.sub.2] also displays a skewed response. Although the recalibrated ACS [PSA.sub.2] assay has been reported to give results in concordance with those determined with the Tandem PSA assay [18][19], this study demonstrated that the former displays a degree of skewing similar to that of the IMx PSA assay.

The intent of this study was to investigate the analytical differences between various assays in a controlled manner. For this purpose, very defined samples were created by using purified material. Analyzing these samples offers an insight into one source of the differences in reported values between assays, even when the same samples are tested. Accordingly, under some circumstances, the disparities between assays seen here could translate into clinically significant differences. This possibility, however, requires documentation by further clinical study.


We thank Barb Allen and Kurt Norton for technical assistance, Steve Mikolajczyk for assistance in preparing the samples, and Bob Parson for assistance with the statistical analyses.

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Hybritech Incorporated, PO Box 269006, San Diego, CA 92196-9006


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Amy B. Blase (a), Roger L. Sokoloff and Katie M. Smith

(a) author for correspondence: fax 619-536-8058, e-mail
COPYRIGHT 1997 American Association for Clinical Chemistry, Inc.
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Title Annotation:Technical Briefs
Author:Blase, Amy B.; Sokoloff, Roger L.; Smith, Katie M.
Publication:Clinical Chemistry
Date:May 1, 1997
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