DOI 10.1515/cclm-2013-0545 Clin Chem Lab Med 2014; 52(5): 715–723
Hae-il Park, Seungok Lee*, Yonggoo Kim, Dong-Yeok Shin, Changseop Lee, Sunmi Han, Chanil Chung, Jun Keun Chang and In Bum Seo
Analytical performance of a new one-step quantitative prostate-specific antigen assay, the FRENDTM PSA Plus Abstract Background: We evaluated the analytical performance of a new one-step rapid quantitative sandwich immunoassay for total prostate-specific antigen (tPSA), the FRENDTM PSA Plus (FREND PSA) (NanoEnTek Inc., Seoul, Korea). Methods: The imprecision, linearity, hook effect, detection limit (LoD), and interference were evaluated and trueness verification and matrix validation were performed. For method comparison, 79 patient specimens were analyzed with FREND PSA and two comparative tPSA assays (Architect® total PSA and cobas® total PSA assay). Results: Total CVs of the imprecision for low (0.208 ng/ mL), medium (4.051 ng/mL), and high PSA levels (5.469 ng/mL) were 15.9%, 6.4%, and 9.1%, respectively. Linearity was observed from 1.01 to 19.15 ng/mL and the hook phenomenon was absent up to 171.48 ng/mL. The LoD was 0.094 ng/mL. The regression equations between FREND (y) and Architect or cobas were as follows: y = 0.0133+1.054x (r = 0.973), y = –0.2144+1.066x (r = 0.977), respectively. Differences between FREND PSA and the comparative methods at a medical decision level of 4.0 ng/mL were less than the optimum specification bias (9.3%). The percentage biases from the trueness verification and interference test were less than the desirable specifications for bias (18.7%). The plasma tPSA level measured with lithium heparin or K2EDTA was comparable to that in the serum. Conclusions: The FREND PSA provided reliable analytical performance and test results in comparison to two widely used tPSA assays. It is a simple and rapid test for tPSA and can be applied in point-of-care testing. Keywords: FRENDTM PSA Plus; rapid quantitative immunoassay; total prostate-specific antigen. *Corresponding author: Seungok Lee, Department of Laboratory Medicine, Incheon St. Mary′s Hospital, The Catholic University of Korea, 56 Dongsu-ro, Bupyeong-gu, Incheon, 403-720, Republic of Korea, Phone: +82 32 2805512, Fax: +82 32 2805520, E-mail: [email protected]
Hae-il Park: Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea; and Laboratory Development and Evaluation Center, Clinical Research Coordinating Center, The Catholic University of Korea Catholic Medical Center, Seoul, Republic of Korea Seungok Lee: Department of Laboratory Medicine, The Catholic University of Korea, Seoul, Republic of Korea Yonggoo Kim: Department of Laboratory Medicine, The Catholic University of Korea, Seoul, Republic of Korea; and Laboratory Development and Evaluation Center, Clinical Research Coordinating Center, The Catholic University of Korea Catholic Medical Center, Seoul, Republic of Korea Dong-Yeok Shin: Department of Laboratory Medicine, Incheon St. Mary’s Hospital, The Catholic University of Korea, Incheon, Republic of Korea Changseop Lee, Sunmi Han, Chanil Chung and Jun Keun Chang: Immunoassay Team, R&D Division, NanoEnTek, Inc., Seoul, Republic of Korea In Bum Seo: Department of Laboratory Medicine, Kangwon National University Hospital, Choonchun, Republic of Korea
Introduction Total prostate-specific antigen (tPSA) is the most useful tumor marker for early detection, staging, prognosis prediction, and monitoring of prostate cancer [1, 2]. Although tPSA levels increase in patients with benign prostatic diseases such as prostatic hyperplasia and prostatitis as well as in those with prostate cancer and can be influenced by age and race [2], tPSA measurements and digital rectal examination are widely used to detect prostate cancer in men above 50 years of age [3]. tPSA ≥ 4 µg/L is generally accepted as the cut-off value for prostatic biopsy, although this threshold remains the subject of debate [1, 2, 4]. There are two forms of PSA in the circulating blood: free PSA (fPSA) and complexed PSA (cPSA) containing α1-antichymotrypsin (ACT), which is the major form of macroglobulin [2, 5]. tPSA is the sum of fPSA and cPSA, and can be measured by immunoassay [2, 5]. Two WHO reference materials for PSA, 96/670 (90:10) and 96/668, were introduced in 2000
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716 Park et al.: Performance of the FRENDTM PSA Plus to facilitate standardization of PSA testing worldwide [6]. The WHO international standard 96/670 (90:10) consists of 90% purified PSA-ACT and 10% fPSA and can be used for tPSA assays. The international standard 96/668 contains only fPSA; therefore, it can only be used for the fPSA assay. Standard calibration with WHO 96/670 (90:10) is used for metrological traceability in numerous tPSA assays [7, 8]. The tPSA test is typically performed on automated analyzers in clinical laboratories; however, a one-step quantitative tPSA assay, the FRENDTM PSA Plus (FREND PSA) (NanoEnTek Inc., Seoul, Korea), was developed for point-of-care (POC) testing. It consists of a singleuse, disposable FRENDTM test cartridge (Lab-On-a-Chip) and a compact (9.4 × 10.2 × 6.9 in) automated FRENDTM system (Figure 1). The FRENDTM cartridge utilizes blood samples and micro-fluidics lateral flow technology in which the analyte of interest forms immune complexes while moving through the fluidics pathway. The principle for quantifying tPSA is a rapid sandwich immunoassay using fluorescent nano-particles conjugated with PSA antibodies on a test cartridge. Detection of laser-induced fluorescence in the FRENDTM system is used to calculate the analyte concentration (ng/mL) in a blood specimen by comparing the ratio of test to control with a calibration curve. The purpose of this study was to evaluate the analytical performance of the FREND PSA with respect to imprecision, linearity, the hook effect, limits of blank (LoB) and detection (LoD), methods comparison, trueness, interference, and matrix effects according to
Clinical and Laboratory Standards Institute (CLSI) guidelines [9–14].
Materials and methods Materials To evaluate imprecision, three levels of quality control materials were used: Low [lot no 40781], Medium [lot no 40812] (Liquichek Immunoassay Plus Control, BioRad Laboratories, Irvine, CA, USA) and High [lot no. 54542] (Lyphocheck Tumor Marker Plus Control, BioRad). Patient serum specimens with tPSA test orders from the urology department were used to evaluate linearity, LoB, LoD, the hook effect, methods comparison, and interference. Patient specimens associated with various prostatic diseases, including prostate cancer, benign prostatic hyperplasia, and prostatitis, were used for the methods comparison. The WHO international standard PSA (90:10) (NIBSC code: 96/670) was used for trueness verification. For the matrix validation study, 36 matched serum and plasma (anti-coagulated with lithium heparin, K2EDTA, and sodium citrate) specimens were collected. All patient specimens were stored at −70 °C until analysis. This study was performed in accordance with the ethical standards of the Declaration of Helsinki and was approved by the Catholic Medical Center Institutional Review Board (OC11TISI0081). The matrix effect validation study was approved by the Kangwon National University Hospital Institutional Review Board (IRB # K1206-047).
FRENDTM PSA Plus The test cartridges were prepared at room temperature for 15– 30 min before use. First, the FREND PSA code chip card containing
Laser Sample
Fluorescence intensity
Test
Reference
Result of reading
Test zone
Reference zone
Figure 1 Principle of the FRENDTM PSA Plus. (A) The FRENDTM PSA Plus consists of a single-use disposable FRENDTM test cartridge and a portable, automated FRENDTM system (9.4 × 10.2 × 6.9 in). (B) The principle is a rapid sandwich immunoassay for quantifying tPSA on a test cartridge by measuring laser-induced fluorescence in an FRENDTM system. The concentration of tPSA in a sample is calculated by comparing test/control line ratio with a calibration curve (ng/mL).
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Park et al.: Performance of the FRENDTM PSA Plus 717 calibration information based on WHO international PSA (90:10) (NIBSC code: 96/670), specific for the same lot of QC and test cartridges, was inserted and installed in the FRENDTM system according to the assay manual. The QC cartridge was inserted into the cartridge slot and 30 µL of serum was placed the test cartridge, which was inserted into the FRENDTM system. After 6 min, the result was displayed on the screen and the test cartridge was removed.
Precision Imprecision was evaluated according to CLSI guideline EP-5-A2 [9]. Three levels of quality control materials were analyzed in duplicate twice a day for 20 days. Repeatability and the between-run, between-day, and within-laboratory imprecision at each level were obtained. Within-laboratory total imprecision was compared to desirable (9.1%) specifications for imprecision derived from biologic variation [15].
Linearity and hook effect Linearity was evaluated according to CLSI guideline EP-6-A [10]. Two patient specimens with low (L, 1.01 ng/mL) and high (H, 19.15 ng/mL) concentrations were mixed: 0.8*L+0.2*H, 0.6*L+0.4*H, 0.4*L+0.6*H and 0.2*L+0.8*H. Each specimen was measured four times and the mean values were compared to the expected concentrations. The regression equation was obtained in the form of y(measured concentration) = b0 + b1x(expected concentration). The desirable specification for bias derived from biologic variation of 18.7% was considered an allowable nonlinearity in the analysis for linearity [15]. To prepare the samples for evaluation of the prozone effect, a patient specimen with an extremely high tPSA level ( > 154.0 ng/mL without dilution, 8720.0 ng/mL with dilution) was two-fold serially diluted with a pooled serum containing tPSA
Hae-il Park, Seungok Lee*, Yonggoo Kim, Dong-Yeok Shin, Changseop Lee, Sunmi Han, Chanil Chung, Jun Keun Chang and In Bum Seo
Analytical performance of a new one-step quantitative prostate-specific antigen assay, the FRENDTM PSA Plus Abstract Background: We evaluated the analytical performance of a new one-step rapid quantitative sandwich immunoassay for total prostate-specific antigen (tPSA), the FRENDTM PSA Plus (FREND PSA) (NanoEnTek Inc., Seoul, Korea). Methods: The imprecision, linearity, hook effect, detection limit (LoD), and interference were evaluated and trueness verification and matrix validation were performed. For method comparison, 79 patient specimens were analyzed with FREND PSA and two comparative tPSA assays (Architect® total PSA and cobas® total PSA assay). Results: Total CVs of the imprecision for low (0.208 ng/ mL), medium (4.051 ng/mL), and high PSA levels (5.469 ng/mL) were 15.9%, 6.4%, and 9.1%, respectively. Linearity was observed from 1.01 to 19.15 ng/mL and the hook phenomenon was absent up to 171.48 ng/mL. The LoD was 0.094 ng/mL. The regression equations between FREND (y) and Architect or cobas were as follows: y = 0.0133+1.054x (r = 0.973), y = –0.2144+1.066x (r = 0.977), respectively. Differences between FREND PSA and the comparative methods at a medical decision level of 4.0 ng/mL were less than the optimum specification bias (9.3%). The percentage biases from the trueness verification and interference test were less than the desirable specifications for bias (18.7%). The plasma tPSA level measured with lithium heparin or K2EDTA was comparable to that in the serum. Conclusions: The FREND PSA provided reliable analytical performance and test results in comparison to two widely used tPSA assays. It is a simple and rapid test for tPSA and can be applied in point-of-care testing. Keywords: FRENDTM PSA Plus; rapid quantitative immunoassay; total prostate-specific antigen. *Corresponding author: Seungok Lee, Department of Laboratory Medicine, Incheon St. Mary′s Hospital, The Catholic University of Korea, 56 Dongsu-ro, Bupyeong-gu, Incheon, 403-720, Republic of Korea, Phone: +82 32 2805512, Fax: +82 32 2805520, E-mail: [email protected]
Hae-il Park: Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea; and Laboratory Development and Evaluation Center, Clinical Research Coordinating Center, The Catholic University of Korea Catholic Medical Center, Seoul, Republic of Korea Seungok Lee: Department of Laboratory Medicine, The Catholic University of Korea, Seoul, Republic of Korea Yonggoo Kim: Department of Laboratory Medicine, The Catholic University of Korea, Seoul, Republic of Korea; and Laboratory Development and Evaluation Center, Clinical Research Coordinating Center, The Catholic University of Korea Catholic Medical Center, Seoul, Republic of Korea Dong-Yeok Shin: Department of Laboratory Medicine, Incheon St. Mary’s Hospital, The Catholic University of Korea, Incheon, Republic of Korea Changseop Lee, Sunmi Han, Chanil Chung and Jun Keun Chang: Immunoassay Team, R&D Division, NanoEnTek, Inc., Seoul, Republic of Korea In Bum Seo: Department of Laboratory Medicine, Kangwon National University Hospital, Choonchun, Republic of Korea
Introduction Total prostate-specific antigen (tPSA) is the most useful tumor marker for early detection, staging, prognosis prediction, and monitoring of prostate cancer [1, 2]. Although tPSA levels increase in patients with benign prostatic diseases such as prostatic hyperplasia and prostatitis as well as in those with prostate cancer and can be influenced by age and race [2], tPSA measurements and digital rectal examination are widely used to detect prostate cancer in men above 50 years of age [3]. tPSA ≥ 4 µg/L is generally accepted as the cut-off value for prostatic biopsy, although this threshold remains the subject of debate [1, 2, 4]. There are two forms of PSA in the circulating blood: free PSA (fPSA) and complexed PSA (cPSA) containing α1-antichymotrypsin (ACT), which is the major form of macroglobulin [2, 5]. tPSA is the sum of fPSA and cPSA, and can be measured by immunoassay [2, 5]. Two WHO reference materials for PSA, 96/670 (90:10) and 96/668, were introduced in 2000
Brought to you by | University of California - San Francisco Authenticated Download Date | 5/28/15 1:47 AM
716 Park et al.: Performance of the FRENDTM PSA Plus to facilitate standardization of PSA testing worldwide [6]. The WHO international standard 96/670 (90:10) consists of 90% purified PSA-ACT and 10% fPSA and can be used for tPSA assays. The international standard 96/668 contains only fPSA; therefore, it can only be used for the fPSA assay. Standard calibration with WHO 96/670 (90:10) is used for metrological traceability in numerous tPSA assays [7, 8]. The tPSA test is typically performed on automated analyzers in clinical laboratories; however, a one-step quantitative tPSA assay, the FRENDTM PSA Plus (FREND PSA) (NanoEnTek Inc., Seoul, Korea), was developed for point-of-care (POC) testing. It consists of a singleuse, disposable FRENDTM test cartridge (Lab-On-a-Chip) and a compact (9.4 × 10.2 × 6.9 in) automated FRENDTM system (Figure 1). The FRENDTM cartridge utilizes blood samples and micro-fluidics lateral flow technology in which the analyte of interest forms immune complexes while moving through the fluidics pathway. The principle for quantifying tPSA is a rapid sandwich immunoassay using fluorescent nano-particles conjugated with PSA antibodies on a test cartridge. Detection of laser-induced fluorescence in the FRENDTM system is used to calculate the analyte concentration (ng/mL) in a blood specimen by comparing the ratio of test to control with a calibration curve. The purpose of this study was to evaluate the analytical performance of the FREND PSA with respect to imprecision, linearity, the hook effect, limits of blank (LoB) and detection (LoD), methods comparison, trueness, interference, and matrix effects according to
Clinical and Laboratory Standards Institute (CLSI) guidelines [9–14].
Materials and methods Materials To evaluate imprecision, three levels of quality control materials were used: Low [lot no 40781], Medium [lot no 40812] (Liquichek Immunoassay Plus Control, BioRad Laboratories, Irvine, CA, USA) and High [lot no. 54542] (Lyphocheck Tumor Marker Plus Control, BioRad). Patient serum specimens with tPSA test orders from the urology department were used to evaluate linearity, LoB, LoD, the hook effect, methods comparison, and interference. Patient specimens associated with various prostatic diseases, including prostate cancer, benign prostatic hyperplasia, and prostatitis, were used for the methods comparison. The WHO international standard PSA (90:10) (NIBSC code: 96/670) was used for trueness verification. For the matrix validation study, 36 matched serum and plasma (anti-coagulated with lithium heparin, K2EDTA, and sodium citrate) specimens were collected. All patient specimens were stored at −70 °C until analysis. This study was performed in accordance with the ethical standards of the Declaration of Helsinki and was approved by the Catholic Medical Center Institutional Review Board (OC11TISI0081). The matrix effect validation study was approved by the Kangwon National University Hospital Institutional Review Board (IRB # K1206-047).
FRENDTM PSA Plus The test cartridges were prepared at room temperature for 15– 30 min before use. First, the FREND PSA code chip card containing
Laser Sample
Fluorescence intensity
Test
Reference
Result of reading
Test zone
Reference zone
Figure 1 Principle of the FRENDTM PSA Plus. (A) The FRENDTM PSA Plus consists of a single-use disposable FRENDTM test cartridge and a portable, automated FRENDTM system (9.4 × 10.2 × 6.9 in). (B) The principle is a rapid sandwich immunoassay for quantifying tPSA on a test cartridge by measuring laser-induced fluorescence in an FRENDTM system. The concentration of tPSA in a sample is calculated by comparing test/control line ratio with a calibration curve (ng/mL).
Brought to you by | University of California - San Francisco Authenticated Download Date | 5/28/15 1:47 AM
Park et al.: Performance of the FRENDTM PSA Plus 717 calibration information based on WHO international PSA (90:10) (NIBSC code: 96/670), specific for the same lot of QC and test cartridges, was inserted and installed in the FRENDTM system according to the assay manual. The QC cartridge was inserted into the cartridge slot and 30 µL of serum was placed the test cartridge, which was inserted into the FRENDTM system. After 6 min, the result was displayed on the screen and the test cartridge was removed.
Precision Imprecision was evaluated according to CLSI guideline EP-5-A2 [9]. Three levels of quality control materials were analyzed in duplicate twice a day for 20 days. Repeatability and the between-run, between-day, and within-laboratory imprecision at each level were obtained. Within-laboratory total imprecision was compared to desirable (9.1%) specifications for imprecision derived from biologic variation [15].
Linearity and hook effect Linearity was evaluated according to CLSI guideline EP-6-A [10]. Two patient specimens with low (L, 1.01 ng/mL) and high (H, 19.15 ng/mL) concentrations were mixed: 0.8*L+0.2*H, 0.6*L+0.4*H, 0.4*L+0.6*H and 0.2*L+0.8*H. Each specimen was measured four times and the mean values were compared to the expected concentrations. The regression equation was obtained in the form of y(measured concentration) = b0 + b1x(expected concentration). The desirable specification for bias derived from biologic variation of 18.7% was considered an allowable nonlinearity in the analysis for linearity [15]. To prepare the samples for evaluation of the prozone effect, a patient specimen with an extremely high tPSA level ( > 154.0 ng/mL without dilution, 8720.0 ng/mL with dilution) was two-fold serially diluted with a pooled serum containing tPSA
