Pre-analytical in-vitro stability of [-2]proPSA in blood and serum

https://doi.org/10.1016/j.clinbiochem.2010.04.062Get rights and content

Abstract

Objectives

[-2]proPSA may discriminate prostate cancer from benign biopsy results. We characterized the pre-analytical stability of [-2]proPSA.

Design and methods

22 volunteers, total PSA of 4.5–19.3 µg/L, had blood drawn simultaneously. Baseline measurements were performed and samples were stored under various conditions prior to measurements. Freeze–thaw cycles were performed. [-2]proPSA was measured with the p2PSA automated research use only immunoassay on the Access analyzer.

Results

Mean [-2]proPSA increases with clotting time, exceeding 10% change in recovery after 3 h. In serum, [-2]proPSA values decline over time under investigated storage conditions. Serum samples kept frozen show less than 10% variation in recoveries over the course of 2 freeze–thaw cycles.

Conclusions

For proper measurement of [-2]proPSA, blood samples should be centrifuged within 3 h of blood draw. Serum may be stored at RT or refrigerated (+ 4 °C) for a maximum of 48 h and should be frozen if stored for a longer period. Two freeze–thaw cycles have no effect on [-2]proPSA stability.

Introduction

The measurement of serum prostate-specific antigen (PSA, Enzyme Nomenclature 3.4.21.77) is widely used for detection of prostate cancer (PCa). Serum total PSA (tPSA) values are significantly associated with the risk of PCa. However, the measurement of tPSA suffers from a lack of specificity because various benign prostatic conditions can also lead to increased serum tPSA, and the ratio of free PSA (fPSA) to tPSA (%fPSA) has been established as a means to improve the clinical specificity of tPSA. Efforts are being made to discover new markers for PCa. Recently examination of the fPSA fraction resulted in the identification of several distinctive precursor isoforms of fPSA.

The precursor proPSA has been found in serum as native [-7]proPSA and with truncated pro-leader peptides as [-5], [-4] and [-2]proPSA [1], [2]. The [-2]proPSA has been shown to be the primary form present in tumor extracts [3] and the p2PSA immunoassay has been developed by Beckman Coulter to measure [-2]proPSA in serum. The p2PSA assay results are combined with tPSA and fPSA to calculate the Prostate Health Index (phi), a risk assessment tool available in Europe. In studies of men with biopsy confirmed prostate cancer, [-2]proPSA in the > 2.0 to < 10 µg/L tPSA range has been shown to improve the specificity for cancer detection relative to %fPSA alone [4], [5], [6], [7], [8], whereas [-5,-7]proPSA could not be shown to improve discrimination between PCa and benign prostate biopsies [9].

Molecular forms of PSA may differ in their in-vitro stability properties and it has been shown that the recovery [10] of free PSA decreases approximately by 1% per hour of clotting time [10]. Stability information is therefore essential for proper clinical interpretation [11]. This study aimed at characterizing the stability of [-2]proPSA in whole blood and serum under a variety of sample storage conditions.

Section snippets

Material and methods

Blood samples containing 4.46–19.31 µg/L (mean 10.5, median 10.3) tPSA (Beckman Coulter Access Hybritech, Beckman Coulter, Inc. Brea, CA 92822-8000 www.beckmancoulter.com) were drawn from 18 Caucasian men scheduled for prostate biopsy or at least 3 months after the last biopsy and from 4 men enrolled in an active surveillance protocol for prostate cancer [12] at the Prostate Center of the University Clinic Münster in March 2008. 18 men had benign biopsy results of which 6 had high grade prostatic

Results

Results are summarized in Table 1. [-2]proPSA concentrations ranged from 9 to 41 pg/mL. When whole blood was allowed to clot at RT, the mean [-2]proPSA percent recovery increased with time during 8 h, exceeding 10% over-recovery after 3 h (Fig. 1). At 3 h, the mean [-2]proPSA percent recovery was 112.7% (95% CI: 109.7% to 115.6%) encompassing the acceptance criterion of 110%. In serum, [-2]proPSA declined over time under the investigated storage conditions. However, the mean [-2]proPSA percent

Discussion

Our study suggests that centrifugation of samples within 3 h of blood draw and storage of serum samples at 21 or 4 °C for up to 48 h is acceptable for accurate measurement of [-2]proPSA. Serum samples retained for longer periods should be stored frozen at −20 °C or lower temperature prior to [-2]proPSA testing. An interesting result of this study was the finding that [-2]proPSA concentration as measured with the Access p2PSA assay increased with clotting time at RT. The linear relationship of this

Acknowledgments

The authors acknowledge the assistance of Martin Bögemann, Reinhard Conrad, Andreas Ebbing, Jürgen Grünebaum, Reemt Hinkelammert, and Mustafa Nakiboglu in the punctually simultaneous blood drawing in March 2008.

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