Is the WHO 90:10 Prostate-Specific Antigen (PSA) First International Reference Standard Really 90% α1-Antichymotrypsin–Bound PSA and 10% Free PSA?

To the Editor: Several factors can contribute to differences among immunoassay methods. Such differences have led to a recognition of the need for the standardization of prostate-specific antigen (PSA)1 assays, and 2 Stanford conferences on the international standardization of PSA immunoassays were held in the early 1990s (1, 2). In 1999, the WHO Expert Committee on Biological Standardization established the First International Standards for PSA with materials that emanated from recommendations of these conferences. These materials consisted of lyophilized 1-μg preparations of 100% free PSA (96/668), and 90% α1-antichymotrypsin–bound PSA (PSA-ACT) and 10% free PSA (96/670) (3) (www.nibsc.ac.uk). A preliminary study found that when total and free PSA were measured in solutions of the 90% PSA-ACT and 10% free PSA (90:10 PSA) material from Stanford/WHO, the slope of the regression line that compared the measured free PSA to measured total PSA was 0.15 instead of the expected 0.10 (unpublished data). The objective of the current study was to further investigate this observation by comparing PSA standard materials from 2 sources (Stanford/WHO and Scripps Laboratories) in 4 total and free PSA assays. The Stanford/WHO reference materials (PSA-ACT standard and 90:10 PSA standard; lyophilized, 1 μg/vial) were purchased from Stanford University. Vials were reconstituted with 2 mL deionized water. Free PSA and PSA-ACT in 10 mmol/L Tris buffer (pH 8) were purchased from Scripps Laboratories. The Scripps 90:10 PSA material was prepared per CLSI document I/LA19-A (4). Dilutions of the Stanford and Scripps materials were prepared in buffer (20 mmol/L PBS, 10 g/L BSA, pH 7.4) (4) at the following concentrations and then frozen at −70 °C: 4, 10, and 50 μg/L PSA-ACT; 4, 10, 20, and 50 μg/L 90:10 PSA. Samples were analyzed in duplicate with the Abbott ARCHITECT®, Beckman Coulter Access® Hybritech, Roche Elecsys® 2010, and Siemens Dimension® RxL assays for total and free PSA. Table 1 presents comparisons of the observed total PSA concentration as a mean percentage (SD) of the expected (theoretical) concentration for 4 concentrations of the 90:10 PSA Stanford/WHO reference standard and the prepared Scripps 90:10 PSA material. Total-PSA methods standardized with the WHO 90:10 PSA standard (ARCHITECT and Elecsys assays) or the WHO free-PSA standard (Dimension RxL) produced results close to 100% in the Stanford/WHO standard. The Access assay with Hybritech standardization produced results that were a mean (SD) of 127.7% (2.4)% of the expected result. This pattern was similar to that for the Scripps 90:10 PSA preparation, although the observed values were lower, possibly due to the preparation of the Scripps material. Data are presented as the mean (SD). Data are presented as the mean (SD). The mean measured percentage of free PSA (free PSA/total PSA × 100) in the 90:10 PSA preparations was 15.6% for the Stanford/WHO material and 11.5% for the Scripps material, whereas the expected percentage was 10%. Table 1 also shows the mean percentage of measured free PSA over 3 concentrations of the 2 PSA-ACT materials, in which, theoretically, no free PSA should be present. The Stanford/WHO PSA-ACT material contained 5.0%–6.9% free PSA, whereas the Scripps material contained only 0.5%–1.2% free PSA. Thus, our results from this study confirmed an observation that the 90:10 PSA Stanford/WHO reference standard appears to be composed of approximately 85% PSA-ACT and 15% free PSA, not 90% PSA-ACT and 10% free PSA. In 2000, Rafferty et al. (3) evaluated the Stanford 100% free PSA and 90:10 PSA preparations as potential WHO international standards. The potency of the 90:10 PSA preparation was estimated at 1.11 μg PSA/vial, of which 0.11 μg/vial was free PSA. They predicted that the degradation of total PSA stored at −20 °C would be limited to 0.027%/year. Recoveries from the present study did not indicate degradation, but rather an increase in free PSA due to dissociation in the PSA-ACT material. A 2006 study reported method-dependent percentages of free PSA of 10%–16% in the 90:10 PSA WHO 96/670 reference preparation (5). PSA-ACT has been reported to be unstable in aqueous solution, and storage at a slightly acidic pH (6.8–7.4); addition of a 100- to 1000-fold molar excess of native ACT, and lower storage temperatures were found to help prevent the dissociation of PSA from the PSA-ACT complex (6). In response to the report by Pettersson et al. (6), Chen et al., who produced the Stanford/WHO standards, claimed that lyophilization is the most important stabilization mechanism for PSA-ACT. In a preliminary study, Chen et al. (7) showed that the lyophilized PSA-ACT standard was stable for up to 3 months at room temperature. The Stanford PSA-ACT standard was estimated to be at least 8 years old when the current study was performed. Chen et al. have also suggested that the PSA-ACT standard be used within 8 h of reconstitution or refrigerated when not in use. In the present study, the Stanford/WHO standards were analyzed on one platform within 1–3 h of reconstitution, and the results were similar to those of samples frozen at −70 °C (data not shown). In summary, the results of our present study suggest that the WHO 90:10 PSA material, with an expected relative abundance of 90% PSA-ACT and 10% free PSA, is actually approximately 85% PSA-ACT and approximately 15% free PSA. This difference is due to the approximately 5% free PSA in the Stanford PSA-ACT material used to prepare the 90:10 PSA standard. Users of this material should be aware of this potential issue and take it into consideration accordingly. Further study is needed to assess any potential impact of this observation on PSA assays and measurements. prostate-specific antigen α1-antichymotrypsin–bound PSA 90% PSA-ACT and 10% free PSA.