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  Clinical Abstracts





January 2011

Michael Bissell, MD, PhD, MPH

Study of [-2]proPSA as a tumor marker Study of [-2]proPSA as a tumor marker

A number of approaches have been proposed to improve the clinical utility of prostate-specific antigen for detecting prostate cancer in the early stages. Among them are using prostate-specific antigen (PSA) velocity, PSA density, age-specific reference ranges, artificial neural networks, models and nomograms, and the molecular forms of PSA. Studies have shown less free PSA and more PSA bound to protease inhibitors among men with prostate cancer. These observations led to the development of commercial assays for free PSA and complexed PSA. Although free PSA can improve on total PSA for cancer detection in the 4 to 10 ng/mL total PSA range, it is an imperfect marker, perhaps because it consists of several isoforms that are associated with prostate cancer or benign prostatic hyperplasia. Proenzyme PSA (proPSA) is a cancer-associated form of free PSA found primarily in the peripheral zone of the prostate and the circulation. It contains a seven-amino acid leader peptide sequence and is enzymatically inactive. Enzymatically active PSA results from cleavage of this leader peptide by human kallikrein 2 and trypsin. ProPSA forms with amino acids of varying lengths, including [-2]proPSA, a stable form that resists activation to mature PSA, also exist in serum. An automated assay for [-2]proPSA has received European Union regulatory approval for prostate cancer detection. In the United States, the FDA is reviewing the [-2]proPSA assay for clinical use. The assay previously was examined for the early detection of prostate cancer, including via a retrospective study by the National Cancer Institute Early Detection Research Network. Other assays and proPSA forms have also been studied. The purpose of the study reviewed herein was to further characterize the potential clinical utility of [-2]-proPSA for prostate cancer detection as well as its association with aggressive cancer in a prospective multi-center study. From among 669 subjects in the study, which was conducted at four National Cancer Institute Early Detection Research Network clinical validation centers, 566 were eligible. Serum PSA, free PSA, and [-2]proPSA were measured using the Beckman Coulter Access 2 analyzer. The authors found that 245 (43 percent) of the 566 participants had prostate cancer on biopsy. At 70 percent specificity, the sensitivity of %[-2]proPSA ([-2]proPSA/fPSA) was 54 percent (95 percent confidence interval [CI], 48–61 percent; null hypothesis, 40 percent). Including %[-2]proPSA in a multivariate prediction model incorporating PSA and %fPSA improved performance (P<0.01). In the 2 to 4 ng/mL PSA range, %[-2]proPSA outperformed %fPSA (receiver operator characteristic areas under the curve, 0.73 versus 0.61; P=0.01). At 80 percent sensitivity, %[-2]proPSA had significantly higher specificity (51.6 percent; 95 percent CI, 41.2–61.8 percent) than PSA (29.9 percent; 95 percent CI, 21–40 percent) and %fPSA (28.9 percent; 95 percent CI, 20.1–39.0). In the 2 to 10 ng/mL PSA range, a multivariate model had significant improvement (area under the curve, 0.76) over individual PSA forms (P<0.01 to <0.0001). At 80 percent sensitivity, the specificity of %[-2]proPSA (44.9 percent; 95 percent CI, 38.4–51.5 percent) was significantly higher than PSA (30.8 percent; 95 percent CI, 24.9–37.1 percent) and relatively higher than %fPSA (34.6 percent; 95 percent CI, 28.5–41.4 percent). The %[-2]proPSA increased with increasing Gleason score (P<0.001) and was higher in aggressive cancers (P=0.03). The authors concluded that in this prospective study, %[-2]proPSA showed potential clinical utility for improving prostate cancer detection and was related to the risk of aggressive disease. Adding %[-2]proPSA could affect the early detection of prostate cancer.

Sokoll LJ, Sanda MG, Feng Z, et al. A prospective multicenter, National Cancer Institute Early Detection Research Network study of [-2]proPSA: improving prostate cancer detection and correlating with cancer aggressiveness. Cancer Epidemiol Biomarkers Prev. 2010;19:1193–1200.

Correspondence: Lori J. Sokoll at

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An assay for vascular endothelial growth factor An assay for vascular endothelial growth factor

Vascular endothelial growth factor, an endothelial cell mitogen, is a key factor implicated in angiogenesis and increased vascular permeability. Increased levels are observed in several ocular ischemic conditions, including diabetic retinopathy, retinal vascular occlusions, and age-related macular degeneration. Vascular endothelial growth factor (VEGF) levels in ocular fluids, both aqueous and vitreous humor, have shown direct correlation with severity of the disease process. The most widely used method for estimating VEGF levels in aqueous humor is enzyme-linked immunosorbent assay (ELISA), which is based on a two-site sandwich assay principle in which one antibody is used to capture cytokine antigens and the other is used to detect suitably labeled antibody. However, ELISA measures only one analyte and requires a sample volume of at least 100 µL to detect proteins such as VEGF. The quantification of VEGF from a low-volume biologic sample, such as aqueous humor, using ELISA is technically difficult. Recent studies have measured VEGF levels in aqueous humor using bead-based immunoassay with flow cytometry. However, these measurements have not been validated with that of ELISA, the standard method for estimating proteins in biologic samples. The authors conducted a study in which they evaluated the accuracy of measurement of a known concentration of VEGF using a cytometric bead-based assay in microsamples (50 µL) and compared it with that obtained by ELISA. A known concentration of VEGF samples was prepared with the addition of VEGF (100 pg/mL) to 1 mL of Hank-balanced salt solution. This preparation was further diluted serially to 50, 25, and 12.5 pg/mL. VEGF estimation was done on each of these samples using the Luminex cytometric bead-based assay and enzyme-linked immunosorbent assay. The assay was replicated three times at each dilution. The authors found that the mean concentrations of VEGF measured using Luminex were 97.7±6.2, 47.3±8.1, 24.9±2.2, and 14.2±1.8 pg/mL at 100, 50, 25, and 12.5 pg/mL dilutions, respectively. Similarly, the mean concentrations of VEGF measured using enzyme-linked immunosorbent assay for equivalent dilutions were 100.6±24.4, 53.8±16.8, 44.8±35.5, and 14.19±0.7 pg/mL, respectively. The authors concluded that bead-based assay accurately measures VEGF concentration in a microsample and has better validity and reliability than enzyme-linked immunosorbent assay.

Chalam KV, Balaiya S, Murthy RK. Accurate estimation of vascular endothelial growth factor levels in microsamples with a low-cost bead-based assay. Retina. 2010;30:815–819.

Correspondence: Dr. Kakarla V. Chalam at

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New assay for hepatitis C virus core antigen quantification New assay for hepatitis C virus core antigen quantification

The virological diagnosis of infection with hepatitis C virus is based on detecting specific anti-HCV antibodies. But because anti-hepatitis C virus (HCV) immunoassays cannot distinguish between acute, past, and persistent infections, screening for HCV RNA is regarded as the me-thod of choice for confirming an active infection in immunocompetent patients who are anti-HCV positive and immunocompromised individuals who may not mount an adequate antibody response. Assays for amplifying HCV RNA are expensive and time-consuming and require sophisticated technical equipment and highly trained personnel. However, these constraints do not apply to detecting HCV core antigen, which is easy to perform in an immunoassay format, provides results in a comparably short time frame, and, theoretically, is less prone to sample carryover and contamination than assays based on nucleic acid amplification. During the past decade, several HCV core antigen tests were developed as potential alternatives to HCV RNA testing. Use of these assays in clinical laboratory settings documented that HCV core antigen can be detected in the serum of individuals during the window period of acute infection. Furthermore, it was conclusively shown that core antigen levels correlate well with HCV RNA concentrations and that quantification of the HCV core protein consequently may be a marker of disease progression or could be used to monitor response to antiviral therapy in chronically infected patients. Given the generally favorable performance characteristics of HCV core antigen assays, it seemed reasonable to further refine this analytical format. To this end, Abbott Diagnostics (Wiesbaden, Germany) designed a chemiluminescent magnetic particle-based assay that allows the highly sensitive detection of free HCV core antigen prior to the formation of anti-HCV antibodies. Addition of an immune complex-dissociating reaction resulted in a test for quantifying free and previously antibody-bound (total) HCV core antigen. The authors conducted a study with the Abbott HCV antigen assay to establish comprehensively its intrinsic analytical performance characteristics, including the correlation of HCV core antigen and HCV RNA concentrations; determine its potential clinical utility in managing HCV-infected patients; and demonstrate its general usefulness for analyzing HCV replication in cell culture systems. The authors found that the Abbott Architect HCV antigen assay showed a specificity of 100 percent. The intra- and interassay coefficients of variation ranged from 3.6 to 8.0 percent and from 4.7 to 9.5 percent, respectively. Except for HCV genotype 2 isolates, the analytical sensitivity was always less than 10 fmol core antigen/L, corresponding to approximately 500 to 3,000 IU of HCV RNA/mL. Linearity was guaranteed throughout the dynamic range (10 to 20,000 fmol/L). When seroconversion panels were tested, the assay was not inferior to HCV RNA detection and reduced the preseroconversion period by four to 16 days. The results obtained by core antigen and HCV RNA quantification for 385 clinical specimens were correlated by regression analysis (r=0.857), but the calculated conversion equation differed significantly from the line of identity. Monitoring viral kinetics using core antigen or RNA concentrations in 38 HCV-infected patients undergoing antiviral combination therapy resulted in very similarly shaped curves in all cases. Finally, the Architect HCV Ag assay was also shown to provide high-throughput screening of in vitro HCV RNA replication. The authors concluded that the Architect HCV antigen assay proved to be a specific, reproducible, highly sensitive, and clinically applicable test format that will find a place in virological HCV diagnostics.

Ross RS, Viazov S, Salloum S, et al. Analytical performance characteristics and clinical utility of a novel assay for total hepatitis C virus core antigen quantification. J Clin Micro-biol. 2010;48:1161–1168.

Correspondence: R. S. Ross at

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Accuracy of calculated free testosterone in men Accuracy of calculated free testosterone in men

Growing pressure to justify prescribing testosterone for aging men and women has led to escalating demand to measure so-called free testosterone in endocrine clinical practice and research. Free testosterone (FT) measurement is recommended as part of the clinical evaluation for androgen deficiency in consensus best practice guidelines published by major national and international endocrine societies. Despite demand, laboratory equi-l-ibrium dialysis (ED) measurement of FT is not widely available because the classical manual and ultra-centrifuge-accelerated methods using tracer or nontracer testosterone measurement by mass spectrometry remain labor-ious, slow, costly, and nonautomatable. To meet demand, workarounds have included a modified androgen analog immunoassay to measure FT directly in unprocessed serum without dialysis, or they have included calculations to estimate FT based on equilibrium-binding theory or empirical equations using immunoassay measurement of total testosterone and sex hormone binding globulin. While the direct FT (analog) assay has proven analytically invalid, calculations of FT are increasingly widely used on Web sites and in spreadsheets. Yet calculated FT (cFT) estimates have not been widely validated in extended practical use such as envisaged by their inclusion in clinical best practice guidelines. This requires comparing these convenient, calculated estimates with laboratory FT measurement by the reference ED method. The original equations were evaluated in small numbers of human samples within single experiments in research laboratories and suggested good agreement with ED. Previous comparisons between cFT methods, undertaken without concurrent laboratory FT measurements, were unable to evaluate accuracy of the formulae. While large-scale evaluation in a routine diagnostic labor-a-tory led to questions about the accuracy of some cFT formulae in predicting FT when compared with centrifuge-accelerated ED, the performance of cFT equations has not been evaluated relative to the original standard reference method for ED. Therefore, the authors designed a large-scale evaluation study to critically evaluate the predictive accuracy of five published cFT equations—two based on equilibrium-binding formulae and three as empirical estimates—compared with laboratory FT measurements by traditional (non-centrifugal) ED. Further-more, they aimed to determine whether correcting for serum albumin improved the accuracy of equilibrium-binding formulae and whether the quantitative discrepancies of the formulae were due to obesity, ethnicity, or gonadal status. The authors found that cFT formulae show systematic discrepancies from the two equilibrium-binding formulae. One empirical formula overestimated FT relative to ED measurements, whereas two newer empirical cFT formulae were more concordant. These discrepancies persisted after correcting for serum albumin and were not influenced by obesity, ethnicity, or gonadal status. The authors concluded that commonly used cFT formulae significantly overestimate FT relative to laboratory measurement by ED in male serum samples. The accuracy of the formulae are not influenced by correcting for serum albumin, obesity, ethnicity, or gonadal status. Such inaccuracy relative to the reference method renders some cFT estimates unreliable for evaluating androgen deficiency as rec-o-m-men--ded by clinical best practice guidelines.

Ly LP, Sartorius G, Hull L, et al. Accuracy of calculated free testosterone formulae in men. Clin Endocrinol. 2010;73:382–388.

Correspondence: David J. Handelsman at

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Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.