Standardization of PSA measures: a reappraisal and an experience with WHO calibration of Beckman Coulter Access Hybritech total and free PSA

Prostate-specific antigen (PSA) is the serum biomarker most widely used in prostate diseases. Since there is significant variation in PSA results among non-equimolar assays, the 90:10 ratio of complexed PSA to free PSA (the Stanford standard) was proposed as standardization; this became the basis for the PSA mass standards WHO 96/670 for tPSA and 96/668 for fPSA. Nevertheless, recent publications underlined the lack of interchangeability between different commercial assays, all claimed to be equimolar and calibrated to the WHO standard. Importantly, the WHO calibration yields about 16-20% lower PSA results. Manufacturers that have chosen to calibrate existing assays to the mass value of WHO 96/760 have introduced a significant negative bias compared to the Hybritech assay calibration; this bias is transferred to clinical evaluation if the cutoff of 4 ng/mL, clinically validated for the Hybritech assay, is maintained with the WHO calibration. Beckman Coulter recently provided the option of calibrating the Access Hybritech PSA and Free PSA assays to the WHO standard introducing a different clinical cutoff. Using two different reagent lots, we tested about 200 routine patients for tPSA and fPSA with both calibrations; we also calculated the f/tPSA ratio with both calibrated methods. Moreover, we verified the analytical sensitivity and inter- and intra-assay variability. In accordance with the claim of the manufacturer, the results obtained with the WHO calibration showed a negative bias of about 25% and, as expected, no significant difference was found for % f/tPSA. The same bias was found when retesting samples of the External Quality Assessment Scheme of the Institute of Clinical Physiology of the National Research Council in Pisa. Based on this experience we decided for the moment to keep the Hybritech calibration, in order to avoid cutoff changes during patient follow-up. Moreover, we have started to provide information to clinicians aimed at the alignment of our results with the WHO standardization.     

Prostate specific antigen levels after acute myocardial infarction

Prostate Specific Antigen (PSA), a member of kallikrein family, is a specific serine protease of prostatic tissue. In some case reports, changes in PSA levels after acute myocardial infarction (AMI) have been reported. In this study we evaluated variations in PSA levels post-AMI. Twenty-six male patients who had PSA levels within reference limits were included in the study. The diagnosis of AMI was confirmed by clinical findings, ECG (electrocardiogram) and cardiac marker studies. Serum total PSA (tPSA) and free PSA (fPSA) levels were measured at days 0 (day of admission), 1, 2 and 3 after AMI. PSA/albumin ratio was also calculated in order to evaluate the effect of dilution. A statistical analysis of the results of all patients revealed significant decrease in tPSA levels and tPSA/Albumin ratio at day 2 when compared to days 0 and 3, which showed a similar pattern. Changes of fPSA and fPSA/ Albumin ratio according to days were not found significant. In only four patients we found increased levels of tPSA and increased fPSA levels in three of them. These patients displayed severe problems such as renal failure, cardiac failure, ventricular aneurysm and cerebral ischemia due to cardiac arrest. The lower tPSA levels on day 2 suggest that tPSA can be eliminated rapidly from the circulation on days 1 and 2, probably through the formation of complexes of tPSA with acute phase proteins.     

Five-year stability study of free and total prostate-specific antigen concentrations in serum specimens collected and stored at -70 degrees C or less

The stability of total (t) and free (f) prostate-specific antigen (PSA) in male serum specimens stored at -70 degrees C or lower temperature for 4.7 to 4.9 years was studied. Until now, the stability of these analytes in serum has not been evaluated systematically beyond 2 years of storage at -70 degrees C. Aliquots of frozen serum were thawed in 2001 and 2006 and assayed for tPSA and fPSA using a Dade Behring Dimension(R) RxL analyzer and reagents. tPSA values ranged from 0.07 to 69.94 and 0.00 to 69.83 ng/mL in 2001 and 2006, respectively, whereas fPSA values for the tested specimens ranged from 0.02 to 5.72 and 0.00 to 5.92, respectively. Deming regression analyses showed agreement in assay values over time as tPSA values yielded a slope of 1.0112 and a y-intercept of 0.0195; fPSA values produced a slope 1.0538 and a y-intercept of -0.0442; f/tPSA values yielded a slope of 0.9631 and a y-intercept of 0.1195. A Bland-Altman analysis of the data demonstrated analyte and ratio stability over this time period. We conclude that serum, when collected properly and stored at -70 degrees C or lower temperature, may be used for tPSA and fPSA clinical studies for at least 5 years after collection.     

Age-Specific Prostate Specific Antigen Cutoffs for Guiding Biopsy Decision in Chinese Population

Background

Age-specific prostate specific antigen (PSA) cutoffs for prostate biopsy have been widely used in the USA and European countries. However, the application of age-specific PSA remains poorly understood in China.

Methods

Between 2003 and 2012, 1,848 men over the age of 40, underwent prostate biopsy for prostate cancer (PCa) at Huashan Hospital, Shanghai, China. Clinical information and blood samples were collected prior to biopsy for each patient. Men were divided into three age groups (≤60, 61 to 80, and >80) for analyses. Digital rectal examination (DRE), transrectal ultrasound (prostate volume and nodule), total PSA (tPSA), and free PSA (fPSA) were also included in the analyses. Logistic regression was used to build the multi-variate model.

Results

Serum tPSA levels were age-dependent (P = 0.008), while %fPSA (P = 0.051) and PSAD (P = 0.284) were age-independent. At a specificity of 80%, the sensitivities for predicting PCa were 83%, 71% and 68% with tPSA cutoff values of 19.0 ng/mL (age≤60),21.0 ng/mL (age 61–80), and 23.0 ng/mL (age≥81). Also, sensitivities at the same tPSA levels were able to reach relatively high levels (70%–88%) for predicting high-grade PCa. Area (AUC) under the receive operating curves (ROCs) of tPSA, %fPSA, PSAD and multi-variate model were different in age groups. When predicting PCa, the AUC of tPSA, %fPSA, PSAD and multi-variate model were 0.90, 0.57, 0.93 and 0.87 respectively in men ≤60 yr; 0.82, 0.70, 0.88 and 0.86 respectively in men 61–80 yr; 0.79, 0.78, 0.87 and 0.88 respectively in men>80 yr. When predicting Gleason Score ≥7 or 8 PCa, there were no significant differences between AUCs of each variable.

Conclusion

Age-specific PSA cutoff values for prostate biopsy should be considered in the Chinese population. Indications for prostate biopsies (tPSA, %fPSA and PSAD) should be considered based on age in the Chinese population.     

Outcomes and Trends of Prostate Biopsy for Prostate Cancer in Chinese Men from 2003 to 2011

Background

Prostate-specific antigen (PSA) screening is growing in popularity in China, but its impact on biopsy characteristics and outcomes are poorly understood.

Objective

Our objective was to characterize prostate biopsy outcomes and trends in Chinese men over a 10-year period, since the increasing use of PSA tests.

Methods

All men (n = 1,650) who underwent prostate biopsy for PCa at Huashan Hospital, Shanghai, China from 2003–2011 were evaluated. Demographic and clinical information was collected for each patient, including age, digital rectal examination (DRE), transrectal ultrasound (prostate volume and nodule), total prostate-specific antigen (tPSA) levels and free PSA ratio (fPSA/tPSA) prior to biopsy. Prostate biopsy was performed using six cores before October 2007 or ten cores thereafter. Logistic regression and multivariate analysis were used to evaluate our data.

Results

The overall positive rate of prostate biopsy for PCa was 47% and the rate decreased significantly over the years from 74% in 2003 to 33% in 2011 (P-trend = 0.004) . Age at diagnosis was slightly increased (P-trend = 0.04) while fPSA/tPSA was significantly decreased (P-trend = 1.11×10-5). A statistically significant trend was not observed for tPSA levels, prostate volume, or proportion of positive nodule. The model including multiple demographic and clinical variables (i.e., age, DRE, tPSA, fPSA/tPSA and transrectal ultrasound results) (AUC = 0.93) statistically outperformed models that included only PSA (AUC = 0.85) or fPSA/tPSA (AUC = 0.66) to predict PCa risks (P<0.05). Similar results were observed in a subgroup of men whose tPSA levels were lower than 20 ng/mL (AUC = 0.87, vs. AUC of tPSA  = 0.62, P<0.05).

Conclusions

Detection rates of PCa and high-grade PCa among men that underwent prostate biopsy at the institution has decreased significantly in the past 10 years, likely due to increasing use of PSA tests. Predictive performance of demographic and clinical variables of PCa was excellent. These variables should be used in clinics to determine the need for prostate biopsy.     

Long Distance Bicycle Riding Causes Prostate-Specific Antigen to Increase in Men Aged 50 Years and Over

Objectives

To investigate whether bicycle riding alters total prostate-specific antigen (tPSA) serum concentrations in healthy older men.

Methods

129 male participants, ranging in age from 50 to 71 years (mean 55 years), rode in a recreational group bicycle ride of between 55 and 160 kilometers. Blood samples for tPSA analysis were drawn within 60 minutes before starting, and within 5 minutes after completing the ride. The pre-cycling and post-cycling tPSA values were log transformed for normality and compared using paired t-tests. Linear regression was used to assess the relationship between changes in tPSA with age and distance cycled.

Results

Bicycle riding caused tPSA to increase by an average of 9.5% (95% CI = 6.1–12.9; p<0.001) or 0.23 ng/ml. The number of participants with an elevated tPSA (using the standard PSA normal range cut-off of 4.0 ng/ml) increased from two pre-cycle to six post-cycle (or from five to eight when using age-based normal ranges). Univariate linear regression analysis revealed that the change in tPSA was positively correlated with age and the distance cycled.

Conclusions

Cycling causes an average 9.5% increase in tPSA, in healthy male cyclists ≥50 years old, when measured within 5 minutes post cycling. We considered the increase clinically significant as the number of participants with an elevated PSA, according to established cut-offs, increased post-ride. Based on the research published to date, the authors suggest a 24–48 hour period of abstinence from cycling and ejaculation before a PSA test, to avoid spurious results.     

Reaction Pattern in Vertebrate Cells (RiV): Studies of the Course of Tumor Therapy using an anti‐RiV ELISA (Initial Results)

Detection of anti‐RiV antibodies by ELISA can be used to follow a patient's response to treatments such as cancer surgery or RiV therapy. The initial results of the authors demonstrated that it is necessary to optimize the sensitivity of the ELISA. Known tumor markers such as prostate specific antigen (PSA), especially the ratio of free PSA (fPSA)/total PSA (tPSA) or neurones‐specific enolase (NSE) detect the therapeutic effect of treatment with RiV particle preparation (RiV‐PP) more rapidly and with greater sensitivity than does the anti‐RiV antibody assay. However, a continuous decrease of anti‐RiV‐antibody titers seems to indicate a good prognosis. RiV therapy improved the quality of life and achieved an apparent prolongation of life of cancer patients. After treatment with 12 mL of RiV‐PP, a patient with a prostate hyperplasia of uncertain genesis became free of symptoms. This monotherapy with an adequate dose of RiV‐PP resulted in a decrease of tPSA and an increase of fPSA in the first 200 days. The general value of RiV ELISA is emphasized by the fact that it could detect RiV antigen in urinary samples offering a simple means of early diagnosis and monitoring. Since chemo‐ or/and radiotherapy, both of which are immunosuppressive, are frequently used to treat cancer patients, an ELISA for the detection of RiV (particles as) antigen may be more informative than one designed to detect anti RiV antibodies.     

Association of Alcohol Consumption with Markers of Prostate Health and Reproductive Hormone Profiles: A Multi-Center Study of 4535 Men in China

Background

The effect of alcohol consumption on prostate health and reproductive hormone profiles has long been investigated and currently, no consensus has been reached. Additionally, large studies focusing on this topic are relatively rare in China.

Purpose

To investigate the association of alcohol consumption with prostate measurements and reproductive hormone profiles in Chinese population; and to examine the relationship between hormone levels and prostate measurements.

Methods

This cross-sectional study included 4535 men from four representative provinces of China. Demographic details, family history of prostate disease, tobacco and alcohol consumption, as well as International Prostate Symptom Score (I-PSS) were collected through a questionnaire. Total prostate specific antingen (total PSA), free PSA, free PSA/total PSA ratio (f/tPSA), and reproductive hormones were measured in serum. Multi-variable regression models were used to test for association of alcohol consumption with markers of prostate health, used to test for association of alcohol consumption with reproductive hormones, and reproductive hormones with markers of prostate health.

Results

Alcohol consumption had no obvious impact on total PSA concentration and I-PSS. Current drinkers had lower level of free PSA (β = -0.11, p = 0.02) and f/tPSA (β = -0.03, p = 0.005), former drinkers also had lower level of free PSA (β = -0.19, p = 0.02) when compared with never drinkers. Lower Luteinizing hormone (LH) (β = -1.05, p = 0.01), sex hormone-binding globulin (SHBG) (β = -4.71, p = 0.01) and higher estradiol (β = 7.81, p = 0.01) was found in current drinkers than never drinkers, whereas higher LH (β = 1.04, p = 0.04) and free testosterone (FT) (β = 0.03, p = 0.02) was detected in former drinkers than never drinkers. Furthermore, LH was positively associated with f/tPSA (β = 0.002, p = 0.006), SHBG was also positively related with free PSA (β = 0.003, p = 0.003) and f/tPSA (β = 0.0004, p = 0.01). Both total testosterone (TT) and FT were inversely related with I-PSS (OR = 0.97, 95% CI, 0.95–0.98; OR = 0.23, 95% CI, 0.11–0.45, respectively).

Conclusions

Alcohol consumption could affect serum free PSA concentration and also f/tPSA ratio, and also acts as an endocrine disruptor on the male reproductive hormone profiles. LH and SHBG were positively related with fPSA and f/tPSA, and higher level of TT and FT may be helpful for improving participants'' subjective symptoms.     

Multicenter evaluation of the performance and clinical utility in longitudinal monitoring of the Bayer Immuno 1 complexed PSA assay.

We conducted a multicenter evaluation of the analytical and clinical performance of the automated Bayer Immuno 1 complexed PSA (cPSA) assay, and compared assay performance to the Bayer Immuno 1 PSA assay. We sought to determine whether measurements of cPSA could be of clinical utility in the management of patients with prostate cancer. Results of the 10-day imprecision across three evaluation sites produced total CV < 2.50% and an analytical sensitivity of 0.02 microgram/L. There was an increased trend in clinical sensitivity for prostate cancer with increasing stage of disease (71-86%). Clinical specificity for patients with benign urogenital disease was 74.8%, and for other nonprostate diseases ranged from 91.1-100%. Retrospective serial monitoring of 155 patients with prostate cancer demonstrated concordance of cPSA measurements to clinical status for 97% of the patients analyzed. Results from the clinical studies using the Bayer Immuno 1 cPSA assay were comparable to results obtained with the Bayer Immuno 1 PSA assay. The Bayer Immuno 1 cPSA assay demonstrates analytical performance and clinical effectiveness in the management of prostate cancer patients during the course of disease and therapy.     

Explication of the roles of prostate health index (PHI) and urokinase plasminogen activator (uPA) as diagnostic and predictor tools for prostate cancer in equivocal PSA range of 4–10 ng/mL

BackgroundProstate cancer (PCa) is one of the most commonly encountered cancers and the leading cause of death worldwide. Currently used biomarkers accounts difficulties in discriminating benign from malignant cases or predicting outcome, so investigating new biomarkers performance is needed.ObjectivesAssessment of diagnostic and predictor roles of prostate health index (PHI) and urokinase plasminogen activator (uPA) in PCa.Methods194 males with initial tPSA of 4–10 ng/mL were categorized into three groups: PCa, benign prostatic hyperplasia (BPH) and healthy control. Serum levels of tPSA, fPSA, p2PSA, and uPA were performed by ELISA with calculation of PHI as (p2PSA/fPSA) × √PSA.ResultsPHI and uPA were significantly higher in PCa patients relevant to BPH and healthy control (p ≤ 0.001). Both markers outperformed all assessed biomarkers and showed the highest area under the curve (AUC) in ROC curve analysis. Both were significantly higher in PCa patients with {Gleason score ≥ 7, late stages (cT2b,c; T3), LN extension and distant metastasis}relative to their counterparts. Additionally, PHI and uPA and were independent predictors of distant metastasis and Gleason score ≥ 7, while PHI was predictor of LN invasion (β = 0.25, p = 0.004).ConclusionPHI and uPA would be of potential value in discriminating between PCa, BPH and healthy men in addition, both are promising as independent predictors of adverse pathological features.     

Analysis of PSA velocity in 1666 healthy subjects undergoing total PSA determination at two consecutive screening rounds

The study purpose was to assess PSA velocity (PSAV) in healthy subjects in order to establish a reliable cutoff for the differential diagnosis of prostate cancer in a screening setting. We studied a series of 1666 healthy men aged 55 to 74 years undergoing two total PSA determinations at a four-year interval within a population-based randomized screening trial at the Centro per lo Studio e la Prevenzione Oncologica of Florence. First and second screening round PSA assays (PSA1 and PSA2) were carried out with the same method and by the same laboratory. PSAV (PSA1-PSA2/year) was determined in non-cancer subjects in the overall series or in specific age and PSA subgroups, and in subjects with cancer detected at the second screening round. Average PSAV in 1648 non-cancer subjects was 0.07 ng/mL/year (range -2.18+5.99, 95% CI 0.05-0.09); at least one third of subjects showed a decrease in PSA (negative PSAV), mostly of limited magnitude and in the low PSA range. Average PSAV in the 18 cancer patients was 1.16 ng/mL/year (range 0.10-5.6, 95% CI 0.56-1.77), which is significantly higher (p<0.01) than in non-cancer subjects. None of the cancer patients showed a PSA decrease over time. Whatever cutoff was taken for PSAV, its power to discriminate cancer was limited: in particular the previously used PSAV cutoff of 0.75 ng/mL/year would have included only 42 of the 1648 non-cancer subjects (specificity 97.5%) but excluded eight of the 18 cancer patients (sensitivity 55.5%). At best, with the adopted screening protocol PSAV (cutoff 0.10 ng/mL/year) could have spared 27.9% of non-cancer subjects with PSA > or =2.5 ng/mL further diagnostic assessment and 22.7% of non-cancer subjects with PSA > or =4 ng/mL random sextant biopsy, while missing no cancers. This study provides a reliable estimate of PSAV based on a large unbiased population sample. PSAV is widely variable over time, particularly at low PSA values. PSAV might be of value as an indicator for diagnostic assessment and random sextant biopsy in a screening setting.     

Measurement of aberrant glycosylation of prostate specific antigen can improve specificity in early detection of prostate cancer

Introduction

We previously identified prostate cancer (PCa)-associated aberrant glycosylation of PSA, where α2,3-linked sialylation is an additional terminal N-glycan on free PSA (S2,3PSA). We then developed a new assay system measuring S2,3PSA using a magnetic microbead-based immunoassay. We compared the diagnostic accuracy of conventional PSA and percent-free PSA (%fPSA) tests.

Methods

We used MagPlex beads to measure serum S2,3PSA levels using anti-human fPSA monoclonal antibody (8A6) for capture and anti-α2,3-linked sialic acid monoclonal antibody (HYB4) for detection. We determined the cutoff values in a training test and measured serum S2,3PSA levels in 314 patients who underwent biopsy, including 138 PCa and 176 non-PCa patients with PSA of <10.0 ng/ml. Serum S2,3PSA levels were presented as mean fluorescence intensity (MFI). Receiver operating characteristic curves were used to evaluate the diagnostic accuracy of total PSA, %fPSA, and S2,3PSA.

Results

We determined an MFI cutoff value of 1130 with a sensitivity of 95.0% and specificity of 72.0% for the diagnosis of PCa in the training test. In the validation study, the area under the curve for the detection of PCa with S2,3PSA was 0.84, which was significantly higher than that with PSA or %fPSA.

Conclusions

Although the present study is small and preliminary, these results suggest that the measurement of serum S2,3PSA using a magnetic microbead-based immunoassay may improve the accuracy of early detection of PCa and reduce unnecessary prostate biopsy.     

Prevalence of Prostate Cancer in High Boron-Exposed Population: A Community-Based Study

We investigated the possible relationship between boron exposure and prostate cancer (PCa) for men living and being employed at boron mines in villages with rich boron minerals. Out of 456 men studied, 159 were from villages with rich boron sources and boron levels in drinking water of >1?mg?L(-1) and these men formed the study group, while 63 from villages with rich boron sources and boron levels in drinking water of <1?mg?L(-1) were enrolled into control group?1. A further 234 subjects from other villages with no boron mines were considered as control group?2. Prostate specific antigen (PSA) levels could be obtained from a total of 423 men. Urinary boron concentration as an indicator of boron exposure in 63 subjects, prostatic volumes by transrectal ultrasonography in 39 subjects, and prostatic biopsies in 36 subjects were obtained for study and control groups. The daily boron exposure was calculated according to urinary boron levels. Although there was no significant difference among the groups in terms of total PSA levels, the number of subjects with tPSA ≥2.5 and tPSA ≥10.0?ng?dL(-1) prostatic volumes in men whose prostates were biopsied (p?相似文献   

A mechanistic insight into the anti-metastatic role of the prostate specific antigen

AimSince its discovery Prostate Specific Antigen (PSA), also referred to as kallikrein-3 (KLK3), has been used as standard circulating biomarker for prostate cancer (PCa). However, its specificity remains not adequate and its mechanism of action still elusive. Therefore, deciphering PSA role throughout PCa-pathobiology would be relevant in improving both cancer diagnosis and outcome prediction. We investigated the possible role played by PSA on/in the tumor microenvironment and over the first steps of cancer invasion.MethodsFresh PCa-specimens and cell lines were used for ex-vivo/in-vitro invasion assays and assessment of prostate tissue-PSA (tPSA), type 1 collagen (COL1A1) and ß1-integrin expression. Tissue Cancer Genome Atlas (TCGA) and Decipher® datasets were considered to estimate tPSA clinical relevance.ResultsA more precise, inverse, correspondence between tPSA and clinical/pathological parameters was found than for circulating PSA. KLK3 combined with Gleason grade and pathologic stage, better predicted cancer-related mortality. Consistently, we demonstrated that PSA inhibits prostate extracellular-matrix (ECM) invasion by PCa cells. As for the mechanism of action, we provided novel information that PSA is able to cleave COL1A1, a main component of the ECM. Finally, ß1-integrin, a crucial COL1A1 transducing-receptor involved in tumor adhesion/invasion, resulted to be downregulated in PCa specimens with higher levels of tPSA.ConclusionsBy interfering with type 1 collagen and its downstream targets, PSA may hamper adhesion and path of the cancer cells through ECM and their migration ability, thus explaining the inverse correlation highlighted between prostate tPSA levels and clinically significant disease.     

Prostate Health Index (Phi) and Prostate Cancer Antigen 3 (PCA3) Significantly Improve Prostate Cancer Detection at Initial Biopsy in a Total PSA Range of 2–10 ng/ml

Many efforts to reduce prostate specific antigen (PSA) overdiagnosis and overtreatment have been made. To this aim, Prostate Health Index (Phi) and Prostate Cancer Antigen 3 (PCA3) have been proposed as new more specific biomarkers. We evaluated the ability of phi and PCA3 to identify prostate cancer (PCa) at initial prostate biopsy in men with total PSA range of 2–10 ng/ml. The performance of phi and PCA3 were evaluated in 300 patients undergoing first prostate biopsy. ROC curve analyses tested the accuracy (AUC) of phi and PCA3 in predicting PCa. Decision curve analyses (DCA) were used to compare the clinical benefit of the two biomarkers. We found that the AUC value of phi (0.77) was comparable to those of %p2PSA (0.76) and PCA3 (0.73) with no significant differences in pairwise comparison (%p2PSA vs phi p = 0.673, %p2PSA vs. PCA3 p = 0.417 and phi vs. PCA3 p = 0.247). These three biomarkers significantly outperformed fPSA (AUC = 0.60), % fPSA (AUC = 0.62) and p2PSA (AUC = 0.63). At DCA, phi and PCA3 exhibited a very close net benefit profile until the threshold probability of 25%, then phi index showed higher net benefit than PCA3. Multivariable analysis showed that the addition of phi and PCA3 to the base multivariable model (age, PSA, %fPSA, DRE, prostate volume) increased predictive accuracy, whereas no model improved single biomarker performance. Finally we showed that subjects with active surveillance (AS) compatible cancer had significantly lower phi and PCA3 values (p<0.001 and p = 0.01, respectively). In conclusion, both phi and PCA3 comparably increase the accuracy in predicting the presence of PCa in total PSA range 2–10 ng/ml at initial biopsy, outperforming currently used %fPSA.     

ADVIA Centaur HER-2/neu shows value in monitoring patients with metastatic breast cancer

INTRODUCTION:The proteolytic breakdown product corresponding to the extracellular domain (ECD) of the HER-2/neu oncoprotein p185 is found in the circulation of healthy individuals and patients having cancers of epithelial origin. For the current evaluation we sought to determine the analytical performance as well as the clinical utility of the newly developed ADVIA Centaur HER-2/neu assay (Bayer HealthCare LLC, Diagnostics Division, Tarrytown, NY, USA) in monitoring patients with metastatic breast cancer during the course of disease and treatment and to compare the obtained results with those of CA 15-3. METHODS: The analytical performance (including precision, normal range, interfering substances, minimum detectable concentration, dilution recovery, spiking recovery and high-dose hook effect) were determined. HER-2/neu and CA 15-3 values were measured in retrospective samples obtained from 59 patients with metastatic breast cancer undergoing treatment over a 6-12 month period. Serial changes in serum HER-2/neu and CA 15-3 were correlated with changes in clinical status on a visit-to-visit basis. For each pair of serial measurements, changes of equal to or greater than, or less than 15% for HER-2/neu and 21% for CA 15-3 were considered to indicate progression or lack of progression, respectively. RESULTS: The ADVIA Centaur HER-2/neu assay demonstrated within-run imprecision and total imprecision ranging from 3.0-5.6% and from 3.2-5.7%, respectively. The upper limit of normal was 15.2 ng/mL (90% CI: 14.2-17.0 ng/mL). No significant interference (<5%) was seen with bilirubins, hemoglobin, triglycerides and cholesterol or therapeutic drugs commonly present in the sera of breast cancer patients. The minimum detectable concentration (analytical sensitivity) was found to be 0.5 ng/mL. The patient population in the clinical study included breast cancer patients who responded to therapy (stable, partial or complete response) or had disease progression. HER-2/neu levels showed a concordance of 78.1% (82/105 restaging time points) with the clinical course of disease, whereas CA 15-3 levels showed a concordance of 76.2% (80/105 restaging time points). The concordance with clinical status increased to 85.7% (90/105 restaging time points) when both results were used in combination as a series test. CONCLUSIONS: The ADVIA Centaur HER-2/neu assay provides excellent analytical performance for serial testing of serum HER-2/neu levels. The clinical data demonstrate the usefulness of serum HER-2/neu in monitoring metastatic breast cancer patients during treatment. Furthermore, the results indicate that serum HER-2/neu and CA 15-3 may be useful in identifying disease progression or therapeutic response in different subgroups of women with metastatic breast cancer.     

Analysis of serum total and free PSA using immunoaffinity depletion coupled to SRM: correlation with clinical immunoassay tests

Recently, selected reaction monitoring mass spectrometry (SRM-MS) has been more frequently applied to measure low abundance biomarker candidates in tissues and biofluids, owing to its high sensitivity and specificity, simplicity of assay configuration, and exceptional multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. Low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed good correlation in several independent clinical serum sample sets. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring candidate biomarkers in human blood, without the need to develop affinity reagents. Furthermore, the simultaneous measurement of multiple biomarkers, including the free and bound forms of PSA, can be performed in a single multiplexed analysis using high-resolution liquid chromatographic separation coupled with SRM-MS. This article is part of a Special Issue entitled: Translational Proteomics.     

Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals

This study directly demonstrates that cardiac troponin I (cTnI) is a sensitive, specific, and persistent biomarker in laboratory animals. Histopathological and pathophysiological cardiac changes in dogs, rats and mice correlated with increased serum cTnI with various cardiac inotropic agents, and cardiotoxic drugs and with cardiac arrhythmias, tachycardia, cardiac effusion with dyspnoea, and ageing. A comparison of six immunoassays for cTnI and cardiac troponin T (cTnT) to detect and monitor cardiac injury in a rodent model indicated that enzyme-linked immunosorbent (Life Diagnostics Inc and TriChem Resources Inc, West Chester, Philadelphia, USA) and Immulite (Diagnostic Products Corporation, Llanberis, UK) assays had low sensitivity and less than 1% of the dynamic range of Centaur (Bayer Healthcare Diagnostics, Newbury, UK) cTnI and Elecsys (Roche Diagnostics, Basel, Switzerland) and M8 (Bioveris Europe, Whitney, UK) cTnT assays. In dogs, however, the Immulite assay was effective and correlated with the Centaur. Serum concentrations were highly correlated but 10-fold lower for cTnT compared with cTnI with cardiac injury. Centaur assay also detected cTnI in myocardium from marmosets, swine, cattle, and guinea pigs, indicating it to be candidate cardiac biomarker for these species as well. Purified rat cTnI was 50% more reactive than purified human cTnI in the Centaur assay. In the rat, an age- and gender-dependent variation in serum cTnI was found. Male rats aged six and eight months had a 10-fold greater serum cTnI than age-matched females and three-month-old rats. These increases correlated with minimal histopathological change. Isoproterenol-induced serum cTnI increased up to 760-fold the minimal detectable concentration of 0.07 microg/L, within 4-6 h and decreased with a half-life of 6 h, with an expected return to baseline of 60 h. Severity of histopathological change correlated with serum cTnI during the ongoing injury.     

Evaluation of three different assays for the assessment of Epstein Barr Virus immunological status

Various attempts have been made to improve Epstein Barr Virus serodiagnosis by developing convenient methods. The present study evaluated the performance of multiplexed bead assays and immunoblot based assays on automated platforms by comparing them with immunofluorescence based assays for the determination of EBV immune status. A total of 45 serum samples were included in the study. Serum samples were tested by multiplexed bead EBV assays (AtheNA Multi-Lyte, Zeus Scientific,USA) and immunoblot based assays (Euroline, Euroimmun AG, Germany) on automated platforms. Assay systems were evaluated by comparing them with immunofluorescence based assays (Zeus Scientific, USA). For EBV anti-VCA IgM, anti-VCA IgG, anti-EA and anti-EBNA, the kappa values reflecting agreements of AtheNA and IFA were 0.20, 0.54, 0.92 and 0.95 for anti-EA, anti-VCA IgG, anti-VCA IgM and anti-EBNA respectively and the agreements of Euroline and IFA were 0.53, 0.67, 0.81 and 1.000 for anti-VCA IgG, anti-EA, anti-VCA IgM and anti-EBNA respectively. The results of the study performed on a limited number of serum samples demonstrated that the multiplexed bead assays and immunoblot assays agree with the standard IFA assay for anti-EBNA IgG and anti-VCA IgM detection while the agreement is less for anti-EA and anti-VCA IgG.