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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].

[TABLE 1 OMITTED]

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].

[FIGURE 1 OMITTED]

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.

Acknowledgments

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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[ILLUSTRATION OMITTED]

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[ILLUSTRATION OMITTED]

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Footnotes

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

References

[1] Hudson MA, Bahnson RR, Catalona WJ. Clinical use of prostate specific antigen in patients with prostate cancer. J Urol 1989;142:1011-1017.

[2] Catalona WJ, Richie JP, Ahmann FR, Hudson MA, Scardino PT, Flanigan RC, et al. Comparison of digital rectal examination and serum prostate specific antigen in the early detection of prostate cancer: results of a multicenter clinical trial of 6,630 men. J Urol 1994;151:1283-1290.

[3] Stenman UH, Leinonen J, Alfthan H, Rannikko S, Tuhkanen K, Alfthan O. A complex between prostate-specific antigen and alpha 1-chymotrypsin 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.

[4] Christensson A, Bjork T, Nilsson O, Dahlen U, Marikainen MT, Cockett AT, et al. Serum prostate specific antigen complexed to alpha 1-antichymotrypsin as an indicator of prostate cancer. J Urol 1993;150:100-105.

[5] Catalona WJ, Smith DS, Wolfert L, Wang TJ, Rittenhouse HG, Ratliff TL, Nadler RB. Evaluation of percentage of free serum prostate-specific antigen to improve specificity of prostate cancer screening. JAMA 1995;264:1214-1220.

[6] Elgamel A-AA, Cornillie FJ, Hendrik PV, Van de Voorde WM, McCabe R, Baert LV. Free-to-total for detection of significant stage T1c prostate cancer. J Urol 1996;156:1042-1049.

[7] Oesterling JE. Prostate specific antigen: a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate. J Urol 1991;145:907-923.

[8] Morote RJ, Ruibal MA, Palou RJ, de Torres MJA, Soler RA. Clinical behavior of prostatic specific antigen and prostatic acid phosphatase: a comparative study. Eur Urol 1988;14:360-366.

[9] 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.

[10] Bankson DD, Lyon ME, Costales LV, Haver VM. The response of assays for total prostate specific antigen to changing proportions of "free" and [alpha]1-antichymotrypsin bound PSA [Abstract]. Clin Chem 1994;40:1009.

[11] Strobel S, Smith K, Wolfert R, Rittenhouse H. Role of free PSA in discordance across commercial PSA assays [Tech Brief]. Clin Chem 1996;42:645-646.

[12] Graves HCB. Standardization of immunoassays for prostate specific antigen. Cancer 1993;72:3141-3144.

[13] Sokoloff R, Wolfert RL, Rittenhouse HG. Standardization of PSA immunoassays: proposals and practical limitations. J Clin Ligand Assay 1995;18:86-92.

[14] Graves HCB, Barren R. Progress towards PSA assay standards. In: Cockett ATK, Aso Y, Chatelain C, Denis L, Griffiths K, Khoury S, Murphy G, eds. Proc., Third International Consultation on Benign Prostate Hyperplasia, Monaco June 26-28, 1995. Paris: SCI Ltd., 1996:404-19..

[15] McCormack RT, Wang TJ, Rittenhouse HG, Wolfert RL, Finlay JA, Sokoloff RL, et al. Molecular forms of prostate specific antigen and the human kallikrein gene family: a new era [Review]. Urology 1995;45:729-744.

[16] Sensabaugh GF, Blake ET. Seminal plasma protein p30: simplified purification and evidence for identity with prostate specific antigen. J Urol 1990;144:1523-1526.

[17] Prestigiacomo AF, Chen Z, Stamey TA. A universal calibrator for prostate specific antigen (PSA). Scand J Clin Lab Invest 1995;55(Suppl 221):57-59.

[18] Bankson DD, Petteway JC, Smith AA, Eisenreich CG, Green RE, Haver VM, Brawer MK. A recalibrated CIBA-Corning ACS-180 PSA ([PSA.sub.2]) assay: comparison with the Hybritech Tandem-R and Abbott IMx PSA assays [Abstract]. Clin Chem 1996;42:S266.

[19] Tewari P, Farrington K, Keelan M, Christensen S, Comerci C, Bluestein B, Maimonis P. ACS [PSA.sub.2], a new immunoassay for the measurement of prostate specific antigen (PSA) in serum [Abstract]. Clin Chem 1996;42:S265.

[20] Zhou AM, Tewari PC, Bluestein BI, Caldwell GW, Larsen FL. Multiple forms of prostate-specific antigen in serum: differences in immunorecognition by monoclonal and polyclonal assays. Clin Chem 1993;39:2483-2491.

[21] Strobel SA, Sokoloff RL, Wolfert RL, Rittenhouse HG. Multiple forms of prostate-specific antigen in serum measured differently in equimolar and skewed response assays [Letter]. Clin Chem 1995;41:125-127.

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

(a) author for correspondence: fax 619-536-8058, e-mail abblase@beckman.com
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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
Words:2380
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