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In this article, a high-resolution acoustic emission sensor, accelerometer, and broadband seismometer array data set is made available and described in detail from in situ experiments performed at Äspö Hard Rock Laboratory in May and June 2015. The main goal of the hydraulic stimulation tests in a horizontal borehole at 410 m depth in naturally fractured granitic rock mass is to demonstrate the technical feasibility of generating multi-stage heat exchangers in a controlled way superiorly to former massive stimulations applied in enhanced geothermal projects. A set of six, sub-parallel hydraulic fractures is propagated from an injection borehole drilled parallel to minimum horizontal in situ stress and is monitored by an extensive complementary sensor array implemented in three inclined monitoring boreholes and the nearby tunnel system. Three different fluid injection protocols are tested: constant water injection, progressive cyclic injection, and cyclic injection with a hydraulic hammer operating at 5 Hz frequency to stimulate a crystalline rock volume of size 30 m × 30 m × 30 m at depth. We collected geological data from core and borehole logs, fracture inspection data from an impression packer, and acoustic emission hypocenter tracking and tilt data, as well as quantified the permeability enhancement process. The data and interpretation provided through this publication are important steps in both upscaling laboratory tests and downscaling field tests in granitic rock in the framework of enhanced geothermal system research. Data described in this paper can be accessed at GFZ Data Services under https://doi.org/10.5880/GFZ.2.6.2023.004 (Zang et al., 2023).

Between 1986 and 1995, 429 hydrofracturing tests have been carried out in six now-abandoned coal mines and two coal bed methane boreholes at depths between 600 and 1950 m within the greater Ruhr region in western Germany. From these tests, stress magnitudes and orientations of the stress tensor are derived. The majority of hydrofracturing tests were carried out from mine galleries away from mine workings in a relatively undisturbed rock mass. These data along with detailed information have been disclosed recently. In combination with already published material, we provide the first comprehensive stress database of the greater Ruhr region. Our study summarises the results of the extensive in situ stress test campaign and assigns quality to each data record using the established quality ranking schemes of the World Stress Map project. The stress magnitudes suggest predominantly strike-slip stress regime, where the magnitude of the minimum horizontal stress, Shmin, is half of the magnitude of the maximum horizontal stress, SHmax, implying that the horizontal differential stress is high. We observe no particular change in the stress gradient at depth throughout the Carboniferous layers and no significant difference between tests carried out in coal mines and deep boreholes. The mean SHmax orientation varies between 133 ± 13∘ in the westernmost located Friedrich Heinrich coal mine and 168 ± 23∘ in the easternmost located Westphalia coal mine. The mean SHmax orientation, based on 87 data records from this and already published studies, of 161 ± 43∘ is in good agreement with the regional stress orientation observed in northwestern Europe. The presented public database provides in situ stress magnitude and stress orientation data records that are essential for the calibration of geomechanical numerical models on regional and/or reservoir scales for, among others, assessing stability issues of borehole trajectories, caverns, and georeservoirs in general. For an application example of this database, we estimate slip and dilation tendencies of major geological discontinuities, discovered during the 700-year-long coal mining activities in the region. The result, although burdened by high uncertainties, shows that the discontinuities striking in the N–S and NW–SE directions have a higher slip tendency compared to the ones striking ENE–WSW and NNW–SSE, whereas a high dilation tendency is observed for discontinuities striking NNW–SSE and a low dilation tendency for the ones striking ENE–WSW. The stress orientation database is available under https://doi.org/10.24406/fordatis/200 (Kruszewski et al., 2022a), the stress magnitude database is available under https://doi.org/10.24406/fordatis/201 (Kruszewski et al., 2022b), whereas the hydrofracturing test reports are available under https://doi.org/10.24406/fordatis/222 (Kruszewski et al., 2022c).

Cardiovascular disease (CVD) susceptibility differs between men and women and varies with ethnicity. This variability is not entirely explained by conventional CVD risk factors. We examined differences in circulating levels of 47 novel protein markers of CVD in 2561 men and women of African-American (AA) and non-Hispanic White (NHW) ethnicity, enrolled at geographically distinct sites.Participants (1,324 AAs, mean age 63.5 y, 71% women; 1,237 NHWs, mean age 58.9 y, 57% women) belonged to sibships ascertained on the basis of hypertension. Solid-phase immunoassays and immunoturbidometric, clot-based, chromogenic, and electrophoretic assays were used to measure the 47 protein markers in plasma or serum. Marker levels were log transformed and outliers were adjusted to within 4 SD. To identify markers independently associated with sex or ethnicity, we employed multivariable regression analyses that adjusted for conventional risk factors, prior history of CVD, medication use and lifestyle factors (physical activity, alcohol consumption and education). Generalized estimating equations were used to correct for intrafamilial correlations. After adjustment for the above covariates, female sex was associated with higher levels of 29 markers and lower levels of 6 markers. Female sex was independently associated with higher levels of several inflammatory markers as well as lipoproteins, adipokines, natriuretic peptides, vasoconstrictor peptides and markers of calcification and thrombosis. AA ethnicity was associated with higher levels of 19 markers and lower levels of 6 markers, including higher levels of several inflammatory makers, higher leptin and lower adiponectin levels, lower levels of vasodilator-natriuretic peptides, higher levels of vasoconstrictor-antidiuretic peptides and markers of calcification and thrombosis.Plasma levels of several novel protein markers of CVD differ significantly in the context of sex and ethnicity. These results have implications for individualized CVD risk assessment.

BACKGROUND:Many men develop a rising PSA after initial therapy for prostate cancer. While some of these men will develop a local or metastatic recurrence that warrants further therapy, others will have no evidence of disease progression. We hypothesized that an expression biomarker panel can predict which men with a rising PSA would benefit from further therapy. METHODOLOGY/PRINCIPAL FINDINGS:A case-control design was used to test the association of gene expression with outcome. Systemic (SYS) progression cases were men post-prostatectomy who developed systemic progression within 5 years after PSA recurrence. PSA progression controls were matched men post-prostatectomy with PSA recurrence but no evidence of clinical progression within 5 years. Using expression arrays optimized for paraffin-embedded tissue RNA, 1021 cancer-related genes were evaluated-including 570 genes implicated in prostate cancer progression. Genes from 8 previously reported marker panels were included. A systemic progression model containing 17 genes was developed. This model generated an AUC of 0.88 (95% CI: 0.84-0.92). Similar AUCs were generated using 3 previously reported panels. In secondary analyses, the model predicted the endpoints of prostate cancer death (in SYS cases) and systemic progression beyond 5 years (in PSA controls) with hazard ratios 2.5 and 4.7, respectively (log-rank p-values of 0.0007 and 0.0005). Genes mapped to 8q24 were significantly enriched in the model. CONCLUSIONS/SIGNIFICANCE:Specific gene expression patterns are significantly associated with systemic progression after PSA recurrence. The measurement of gene expression pattern may be useful for determining which men may benefit from additional therapy after PSA recurrence.

Despite the dramatic improvement in both overall survival (OS) and progression-free survival (PFS) in patients with metastatic melanoma treated with immune checkpoint inhibitors, up to 60% will develop treatment resistance and 50% will die from their disease. Therefore, although dacarbazine is no longer a mainstay of modern melanoma management, we examined the extent to, and in which context, it may still play a role. A retrospective analysis of electronic medical records of patients who had received dacarbazine treatment between October 2014 and October 2021, following innate or acquired resistance to immune checkpoint inhibitors, was performed to determine PFS and OS and examine tolerability. Nine patients with locally advanced ( n  = 1) or metastatic melanoma ( n  = 8) were identified (average age: 74 years, 4 males and 5 females). The number of cycles of dacarbazine ranged from 2 to 45 (mean = 12). One-third of patients developed a complete ( n  = 2) or partial ( n  = 1) response, two-thirds did not respond to treatment. The median PFS time was 90 days. Common adverse events included blood dyscrasias; one patient developed a grade 3 hepatitis, although it was unclear if this was due to the chemotherapy or the preceding combined immunotherapy. Dacarbazine may still be a valid option in the setting of treatment for refractory, relapsed, or progressive disease. Future studies should focus on the immunomodulatory effects of dacarbazine on the tumor microenvironment, which could be harnessed to potentially restore sensitivity to immune checkpoint-based therapy., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Despite the dramatic improvements in recurrence-free survival in patients with metastatic melanoma treated with immune checkpoint inhibitors (ICI), a number of patients develop metastases during adjuvant therapy. It is not currently possible to predict which patients are most likely to develop disease recurrence due to a lack of reliable biomarkers. Thus, we retrospectively analyzed the case records of all patients who commenced adjuvant ICI therapy between January 2018 and December 2021 in a single university skin cancer center (n = 46) (i) to determine the rates of disease recurrence, (ii) to examine the utility of established markers, and (iii) to examine whether re-challenge with immunotherapy resulted in clinical response. Twelve out of forty-six (26%) patients developed a relapse on adjuvant immunotherapy in our cohort, and the median time to relapse was 139 days. Adjuvant immunotherapy was continued in three patients. Of the twelve patients who developed recurrence during adjuvant immunotherapy, seven had further disease recurrence within the observation period, with a median time of 112 days after the first progress. There was no significant difference comparing early recurrence (<180 days after initiation) on adjuvant immunotherapy to late recurrence (>180 days after initiation) on adjuvant immunotherapy. Classical tumor markers, including serum lactate dehydrogenase (LDH) and S-100, were unreliable for the detection of disease recurrence. Baseline lymphocyte and eosinophil counts and those during immunotherapy were not associated with disease recurrence. Interestingly, patients with NRAS mutations were disproportionately represented (60%) in the patients who developed disease recurrence, suggesting that these patients should be closely monitored during adjuvant therapy.

Combined immunotherapy is associated with a significant risk of severe and potentially fatal immune-related adverse events (irAEs). Therefore, we retrospectively analyzed the side profile and efficacy of low-dose ipilimumab (1 mg/kg, IPI1) combined with anti-PD-1 immunotherapy in patients who progressed after anti-PD-1 monotherapy. Nine patients with unresectable stage III or IV melanoma treated with combined low-dose ipilimumab (1 mg/kg, IPI1) and anti-PD-1 immunotherapy, following progression after anti-PD-1 treatment, were identified. Treatment response and irAEs were recorded. Grade 3 irAEs occurred in one-third of patients. Interestingly, there were no grade 4 or 5 irAEs. In fact, four out of the nine patients experienced no irAEs at all. One patient discontinued combined immunotherapy due to immune-related colitis. The mean time to the onset of grade 3 irAEs was 14.3 weeks. The objective response rate was 33.3% and a disease control rate of 66.7% was achieved. Median progression-free survival (PFS) was 5.7 months and median overall survival (OS) was 21.6 months. The median PFS when IPI1 and anti-PD-1 treatment was administered in the second-line setting was not reached, but only 2.8 months when used in subsequent treatment settings. Combined IPI1 and anti-PD-1 immunotherapy was well tolerated. Its use in the third-line or above setting was associated with a significantly poorer prognosis than in the second-line setting. Larger, prospective studies are required to evaluate the safety and efficacy of this dosing regimen following anti-PD-1 treatment failure., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: Merkel cell carcinoma (MCC) is a rare, highly aggressive neuroendocrine skin cancer, which typically affects elderly and immunocompromised and/or immunosuppressed patients. The checkpoint inhibitor avelumab, a mAb targeting the anti-programmed cell death ligand 1 (anti-PD-L1), has revolutionized the treatment of metastatic MCC, achieving dramatic improvements in disease control and overall survival. However, checkpoint inhibitors are associated with the development of immune-related adverse events, such as exacerbation of pre-existing RA. Although most immune-related adverse events can be managed successfully with CSs, their frequent and/or long-term use runs the risk of undermining the efficacy of immune checkpoint inhibition., Methods: We report two cases of MCC, in which immunosuppressive therapy for the management of RA was administered., Results: Immunosuppression for (i) pre-existing and (ii) immune checkpoint inhibitor-exacerbated RA was associated with progression of metastatic MCC., Conclusion: Any decision to initiate immunosuppressive treatment for RA in patients receiving immune checkpoint inhibitor therapy should include careful consideration of the risk of potentially fatal cancer progression and be taken after consultation with the patient's oncologist and rheumatologist. When the immunosuppressive treatment is required, it should be administered for as short a time as possible and under strict clinical and radiological surveillance., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

With more than 82 million cases worldwide and almost two million deaths, the Covid-19 global pandemic shows little sign of abating. However, its effect on quality of life (QoL) in skin cancer patients has not been systematically evaluated to date. Given that QoL impairments may be associated with increased psychological morbidity, and may interfere with engagement with cancer therapy and follow-up, we prospectively evaluated quality of life in skin cancer patients using the Covid-19 Emotional Impact Survey (C-19EIS) and the EORTC QLQ-C30 questionnaires. 101 patients (48 females and 53 males) completed both questionnaires. The mean C-19EIS score was 3.8 on a scale from 0 (no impact) to 12 (severe impact). Patients undergoing systemic therapy showed significantly impaired physical (p = 0.006) and social functioning (p = 0.003). However, when compared to the published normative EORTC QLQ-C30 data, there was no evidence that the Covid-19 pandemic had significantly impacted upon overall quality of life. Subscales of the EORTC QLQ-C30 were significantly inversely correlated with the C-19EIS, validating its use in skin cancer patients. Despite the Covid-19 pandemic, skin cancer patients in our tertiary referral center were surprisingly resilient. However, given the geographical variations in the rates of Sars-CoV-2 infection it is possible that the low incidence in Northern Germany may have resulted in a lack of general QoL impairments. Multi-center studies are required to further determine the impact of Covid-19 on psychological wellbeing in skin cancer patients in order to develop supportive interventions and to ensure that engagement with cancer care services is maintained in order to enable early detection of cancer progression and/or recurrence., Competing Interests: The authors have read the journal’s policy and have the following competing interests: GK reports speaker’s honoraria from Novartis and Kyowa Kirin. EAL reports personal fees and non-financial support from BristolMyersSquibb, personal fees and non-financial support from Novartis, meeting and travel support from Curevac and advisory board fees from Sun Pharma. PT reports speaker’s honoraria from BMS, Novartis, MSD, Pierre-Fabre, CureVac and Roche, consultant’s honoraria from BMS, Novartis, Pierre-Fabre, Merck Serono, Sanofi und Roche and travel support from BMS, Pierre-Fabre and Roche. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare. The other authors report no competing interests.

Competing Interests: Competing interests: None declared.

Introduction: Heterogeneity of nephrotic diseases and a lack of validated biomarkers limits interventions and reduces the ability to examine outcomes. Urinary CD80 is a potential biomarker for minimal change disease (MCD) steroid-sensitive nephrotic syndrome (NS). We investigated and validated a CD80 enzyme-linked immunosorbent assay (ELISA) in urine in a large cohort with a variety of nephrotic diseases., Methods: A commercial CD80 ELISA was enhanced and analytically validated for urine. Patients were from Mayo Clinic (307) and Nephrotic Syndrome Study Network Consortium (NEPTUNE; 104) as follows: minimal change disease (MCD, 56), focal segmental glomerulosclerosis (FSGS, 92), lupus nephritis (LN, 25), IgA nephropathy (IgAN, 20), membranous nephropathy (MN, 49), autosomal dominant polycystic kidney disease (ADPKD, 10), diabetic nephropathy (DN; 106), pyuria (19), and controls (34). Analysis was by Kruskal-Wallis test, generalized estimating equation (GEE) models, and receiver operating characteristic (AUC) curve., Results: Urinary CD80/creatinine values were highest in MCD compared to other glomerular diseases and were increased in DN with proteinuria >2 compared to controls (control = 36 ng/g; MCD = 139 ng/g, P  < 0.01; LN = 90 ng/g, P  < 0.12; FSGS = 66 ng/g, P  = 0.18; DN = 63, P  = 0.03; MN = 69 ng/g, P  = 0.33; ng/g, P = 0.07; IgA = 19 ng/g, P  = 0.09; ADPKD = 42, P  = 0.36; and pyuria 31, P  = 0.20; GEE, median, P vs. control). In proteinuric patients, CD80 concentration appears to be independent of proteinuria levels, suggesting that it is unrelated to nonspecific passage across the glomeruli. CD80/creatinine values were higher in paired relapse versus remission cases of MCD and FSGS ( P  < 0.0001, GEE)., Conclusion: Using a validated ELISA, urinary CD80 levels discriminate MCD from other forms of NS (FSGS, DN, IgA, MN) and primary from secondary FSGS., (© 2020 International Society of Nephrology. Published by Elsevier Inc.)

Introduction: The volatile endogenous mediator hydrogen sulfide (H2S) is known to impair thrombus formation by affecting the activity of human platelets. Beside platelets and coagulation factors the endothelium is crucial during thrombogenesis., Objective: This study evaluates the effect of the H2S donor GYY4137 (GYY) on human umbilical vein endothelial cells (HUVECs) in vitro., Methods: Flow cytometry of resting, stimulated or GYY-treated and subsequently stimulated HUVECs was performed to analyse the expression of E-selectin, ICAM-1 and VCAM-1. To study a potential reversibility of the GYY action, E-selectin expression was further assessed on HUVECs that were stimulated 24 h after GYY exposure. A WST-1 assay was performed to study toxic effects of the H2S donor. By using the biotin switch assay, protein S-sulfhydration of GYY-exposed HUVECs was assessed. Further on, the effects of GYY on HUVEC migration and von Willebrand factor (vWF) secretion were assessed., Results: GYY treatment significantly reduced the expression of E-selectin and ICAM-1 but not of VCAM-1. When HUVECs were stimulated 24 h after GYY treatment, E-selectin expression was no longer affected. The WST-1 assay revealed no effects of GYY on endothelial cell viability. Furthermore, GYY impaired endothelial migration, reduced vWF secretion and increased protein S-sulfhydration., Conclusions: Summarizing, GYY dose dependently and reversibly reduces the activity of endothelial cells.

Introduction: Neointima formation is closely linked to vascular stenosis and occurs after endothelial damage. Hydrogen sulfide is an endogenous pleiotropic mediator with numerous positive effects on the cardio vascular system., Objective: This study evaluates the effect of the slow releasing hydrogen sulfide donor GYY4137 (GYY) on neointimal formation in vivo., Methods: The effect of GYY on neointimal formation in the carotid artery was studied in the FeCl3 injury model in GYY- or vehicle-treated mice. The carotid arteries were studied at days 7 and 21 after treatment by means of histology and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and alpha smooth muscle actin (α-SMA)., Results: GYY treatment significantly reduced the maximal diameter and the area of the newly formed neointima on both days 7 and 21 when compared to vehicle treatment. GYY additionally reduced the number of PCNA- and α-SMA-positive cells within the neointima on day 21 after FeCl3 injury of the carotid artery., Conclusions: Summarizing, single treatment with the slow releasing hydrogen sulfide donor GYY reduced the extent of the newly formed neointima by affecting the cellular proliferation at the site of vascular injury.

Free human kallikrein 2 (free-hK2) and hK2 pro-form (pro-hK2) have been found to be increased in tumor tissues and serum from patients with prostate cancer. We established semiautomatic assays for free-hK2 and pro-hK2 using a research version of the Beckman Coulter ACCESS2 system. Serum samples from a cohort of 189 men undergoing radical prostatectomy for known high-risk disease were assayed for Prostate-Specific Antigen (PSA), free-PSA, free-hK2, and pro-hK2. Univariate Cox regression and multivariate models were used to predict both Gleason scores and progression-free survival (PFS). Free-hk2 levels ≥80 ng/L were predictive of both Gleason scores ≥7 ( p = 0.04) and PFS ( p = 0.03). PSA ≥8.0 µg/L also was predictive of PFS ( p = 0.02). However, neither % free-PSA nor pro-hK2, when treated as continuous or cutoff variables were associated with Gleason score or PFS. Multivariable models showed that clinical stage T1c versus T2/T3, Gleason score ≥7, and PSA ≥8.0 µg/L or clinical stage T1c versus T2/T3, Gleason scores ≥7, and free-hK2 ≥80 ng/L were among the best models predicting PFS. Both free-hK2 and PSA in conjunction with clinical stage and Gleason score are good predictors of PFS in prostate cancer., Competing Interests: Declaration of conflicting interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: This study was conducted in compliance with Mayo Clinic conflict of interest policies. Mayo Clinic and Dr Klee have received royalties of less than the federal threshold for significant financial interest from the licensing of technology to Beckman Coulter, Inc.

Objective: To examine the use of the Prostate Health Index (PHI) as a continuous variable in multivariable risk assessment for aggressive prostate cancer in a large multicentre US study., Materials and Methods: The study population included 728 men, with prostate-specific antigen (PSA) levels of 2-10 ng/mL and a negative digital rectal examination, enrolled in a prospective, multi-site early detection trial. The primary endpoint was aggressive prostate cancer, defined as biopsy Gleason score ≥7. First, we evaluated whether the addition of PHI improves the performance of currently available risk calculators (the Prostate Cancer Prevention Trial [PCPT] and European Randomised Study of Screening for Prostate Cancer [ERSPC] risk calculators). We also designed and internally validated a new PHI-based multivariable predictive model, and created a nomogram., Results: Of 728 men undergoing biopsy, 118 (16.2%) had aggressive prostate cancer. The PHI predicted the risk of aggressive prostate cancer across the spectrum of values. Adding PHI significantly improved the predictive accuracy of the PCPT and ERSPC risk calculators for aggressive disease. A new model was created using age, previous biopsy, prostate volume, PSA and PHI, with an area under the curve of 0.746. The bootstrap-corrected model showed good calibration with observed risk for aggressive prostate cancer and had net benefit on decision-curve analysis., Conclusion: Using PHI as part of multivariable risk assessment leads to a significant improvement in the detection of aggressive prostate cancer, potentially reducing harms from unnecessary prostate biopsy and overdiagnosis., (© 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.)

Objectives: The IFCC Committee on Reference Intervals and Decision Limits coordinated a global multicenter study on reference values (RVs) to explore rational and harmonizable procedures for derivation of reference intervals (RIs) and investigate the feasibility of sharing RIs through evaluation of sources of variation of RVs on a global scale., Methods: For the common protocol, rather lenient criteria for reference individuals were adopted to facilitate harmonized recruitment with planned use of the latent abnormal values exclusion (LAVE) method. As of July 2015, 12 countries had completed their study with total recruitment of 13,386 healthy adults. 25 analytes were measured chemically and 25 immunologically. A serum panel with assigned values was measured by all laboratories. RIs were derived by parametric and nonparametric methods., Results: The effect of LAVE methods is prominent in analytes which reflect nutritional status, inflammation and muscular exertion, indicating that inappropriate results are frequent in any country. The validity of the parametric method was confirmed by the presence of analyte-specific distribution patterns and successful Gaussian transformation using the modified Box-Cox formula in all countries. After successful alignment of RVs based on the panel test results, nearly half the analytes showed variable degrees of between-country differences. This finding, however, requires confirmation after adjusting for BMI and other sources of variation. The results are reported in the second part of this paper., Conclusion: The collaborative study enabled us to evaluate rational methods for deriving RIs and comparing the RVs based on real-world datasets obtained in a harmonized manner., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: The intent of this study, based on a global multicenter study of reference values (RVs) for serum analytes was to explore biological sources of variation (SVs) of the RVs among 12 countries around the world., Methods: As described in the first part of this paper, RVs of 50 major serum analytes from 13,396 healthy individuals living in 12 countries were obtained. Analyzed in this study were 23 clinical chemistry analytes and 8 analytes measured by immunoturbidimetry. Multiple regression analysis was performed for each gender, country by country, analyte by analyte, by setting four major SVs (age, BMI, and levels of drinking and smoking) as a fixed set of explanatory variables. For analytes with skewed distributions, log-transformation was applied. The association of each source of variation with RVs was expressed as the partial correlation coefficient (r p )., Results: Obvious gender and age-related changes in the RVs were observed in many analytes, almost consistently between countries. Compilation of age-related variations of RVs after adjusting for between-country differences revealed peculiar patterns specific to each analyte. Judged fromthe r p , BMI related changes were observed for many nutritional and inflammatory markers in almost all countries. However, the slope of linear regression of BMI vs. RV differed greatly among countries for some analytes. Alcohol and smoking-related changes were observed less conspicuously in a limited number of analytes., Conclusion: The features of sex, age, alcohol, and smoking-related changes in RVs of the analytes were largely comparable worldwide. The finding of differences in BMI-related changes among countries in some analytes is quite relevant to understanding ethnic differences in susceptibility to nutritionally related diseases., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Context: Prostate-specific antigen (PSA) is a 34-kDa glycoprotein with chymotrypsin-like enzyme activity that circulates both in free forms and complexed to various enzyme inhibitors including antichymotrypsin and α2-macroglobulin. Prostate-specific antigen bound to α2-macroglobulin is not detected by commercial PSA immunoassays., Objective: To develop a mass spectrometry assay that detects the same forms of PSA as the immunoassays, which could serve as a reference for harmonizing PSA immunoassays., Design: Prostate-specific antigen was immune extracted from serum, trypsin was digested, and the LSEPAELTDAVK peptide was quantitated on an API 5000 spectrometer. Calibrators were made by adding 10% free and 90% antichymotrypsin-bound PSA to female sera. The assay was standardized to the World Health Organization 96/670 reference standard. Validation of clinical utility and comparisons with 2 immunoassays (Roche cobas and Beckman Access) were performed using frozen sera aliquots from 100 men undergoing prostate biopsy (50 negative, 50 with cancer) and 5 serial samples collected over time from 5 men with advanced prostate cancer., Results: The antibody extraction efficiency was greater than 99%. The assay has an analytic range from 1.2 to 76 ng/mL, with precision ranging from 8.6% at 1.5 ng/mL to 5.4% at 27 ng/mL. The mass spectrometry assay correlated well with 2 immunoassays. All 3 assays showed statistically equivalent separation of prostate cancer from benign disease using receiver operating characteristic curve analysis., Conclusions: This mass spectrometry assay can reliably measure PSA concentrations in human serum and could serve as a reference standard for harmonizing PSA immunoassays.

Purpose: Clinicopathologic features and biochemical recurrence are sensitive, but not specific, predictors of metastatic disease and lethal prostate cancer. We hypothesize that a genomic expression signature detected in the primary tumor represents true biological potential of aggressive disease and provides improved prediction of early prostate cancer metastasis., Methods: A nested case-control design was used to select 639 patients from the Mayo Clinic tumor registry who underwent radical prostatectomy between 1987 and 2001. A genomic classifier (GC) was developed by modeling differential RNA expression using 1.4 million feature high-density expression arrays of men enriched for rising PSA after prostatectomy, including 213 who experienced early clinical metastasis after biochemical recurrence. A training set was used to develop a random forest classifier of 22 markers to predict for cases--men with early clinical metastasis after rising PSA. Performance of GC was compared to prognostic factors such as Gleason score and previous gene expression signatures in a withheld validation set., Results: Expression profiles were generated from 545 unique patient samples, with median follow-up of 16.9 years. GC achieved an area under the receiver operating characteristic curve of 0.75 (0.67-0.83) in validation, outperforming clinical variables and gene signatures. GC was the only significant prognostic factor in multivariable analyses. Within Gleason score groups, cases with high GC scores experienced earlier death from prostate cancer and reduced overall survival. The markers in the classifier were found to be associated with a number of key biological processes in prostate cancer metastatic disease progression., Conclusion: A genomic classifier was developed and validated in a large patient cohort enriched with prostate cancer metastasis patients and a rising PSA that went on to experience metastatic disease. This early metastasis prediction model based on genomic expression in the primary tumor may be useful for identification of aggressive prostate cancer.

Background: In a planned International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) worldwide study on reference intervals (RIs), a common panel of serum samples is to be measured by laboratories from different countries, and test results are to be compared through conversion using linear regression analysis. This report presents a validation study that was conducted in collaboration with four laboratories., Methods: A panel composed of 80 sera was prepared from healthy individuals, and 45 commonly tested analytes (general chemistry, tumor markers, and hormones) were measured on two occasions 1 week apart in each laboratory. Reduced major-axis linear regression was used to convert reference limits (LL and UL). Precision was expressed as a ratio of the standard error of converted LL or UL to the standard deviation (SD) comprising RI (approx. 1/4 of the RI width corresponding to between-individual SD). The allowable and optimal levels of error for the SD ratio (SDR) were set as ≤0.250 and ≤0.125, respectively, in analogy to the common method of setting limits for analytical bias based on between-individual SD., Results: The values for the calculated SDRs depended upon the distribution patterns of test results: skewness toward higher values makes SDRLL lower and SDRUL higher. However, the CV of the regression line slope, CV(b), is less affected by skewness. The average of SDRLL and SDRUL (aveSDR) correlates closely with CV(b) (r=0.995). The aveSDRs of ≤0.25 and ≤0.125 corresponds approximately to CV(b) values of ≤11% and ≤5.5%, respectively. For all results (i.e., n=80), conversion was allowable (optimal) in 98% (89%) of the analytes, as judged by CV(b). Resampling studies using random subsets of data with a data size (n) of 70 to 20 revealed that SDRs and CV(b) gradually increase with reduction of n, especially with n ≤30., Conclusions: CV(b) is a robust estimator for judging the convertibility of reference values among laboratories, even with a skewed distribution. Assuming 40 sera to be a practical size for the panel, reference values of 89% (80%) of analytes examined were made comparable by regression analysis with the allowable (optimal) level of precision.

Although manufacturers are compelled by the European IVD Directive, 98/79/EC, to have traceability of the values assigned to their calibrators if suitable higher order reference materials and/or procedures are available, there is still no equivalence of results for many measurands determined in clinical laboratories. The adoption of assays with metrological traceable results will have a significant impact on laboratory medicine in that results will be equivalent across different laboratories and different analytical platforms. The IFCC WG on Allowable Errors for Traceable Results has been formed to define acceptable limits for metrological traceability chains for specific measurands in order to promote the equivalence of patient results. These limits are being developed based on biological variation for the specific measurands. Preliminary investigations have shown that for some measurands, it is possible for manufacturers to assign values to assay calibrators with a measurement uncertainty that allows the laboratory enough combined uncertainty for their routine measurements. However, for other measurands, e.g., plasma sodium, current assays are too imprecise to fulfil limits based on biological variation. Although an alternative approach based on probability theory is being investigated, the most desirable approach would be for industry to improve measurement methods so that they meet clinical requirements.

The reference intervals (RIs) given in laboratory reports have an important role in aiding clinicians in interpreting test results in reference to values of healthy populations. In this report, we present a proposed protocol and standard operating procedures (SOPs) for common use in conducting multicenter RI studies on a national or international scale. The protocols and consensus on their contents were refined through discussions in recent C-RIDL meetings. The protocol describes in detail (1) the scheme and organization of the study, (2) the target population, inclusion/exclusion criteria, ethnicity, and sample size, (3) health status questionnaire, (4) target analytes, (5) blood collection, (6) sample processing and storage, (7) assays, (8) cross-check testing, (9) ethics, (10) data analyses, and (11) reporting of results. In addition, the protocol proposes the common measurement of a panel of sera when no standard materials exist for harmonization of test results. It also describes the requirements of the central laboratory, including the method of cross-check testing between the central laboratory of each country and local laboratories. This protocol and the SOPs remain largely exploratory and may require a reevaluation from the practical point of view after their implementation in the ongoing worldwide study. The paper is mainly intended to be a basis for discussion in the scientific community.

Objective: We sought to determine whether, and if so the mechanism by which, testosterone replacement improves carbohydrate tolerance., Research Design and Methods: Fifty-five elderly men with relative testosterone deficiency ingested a labeled mixed meal and underwent a frequently sampled labeled intravenous glucose tolerance test before and after either placebo or treatment with testosterone patch (5 mg/day) for 2 years., Results: Despite restoring bioavailable testosterone to values observed in young men, the change (24 months minus baseline values) in fasting and postprandial glucose, insulin, and C-peptide concentrations and meal appearance, glucose disposal, and endogenous glucose production were virtually identical to those observed after 2 years of placebo. The change over time in insulin and C-peptide concentrations post-intravenous glucose injection also did not differ. Furthermore, the change over time in insulin action and glucose effectiveness (measured with the unlabeled and labeled "oral" and "intravenous" minimal models), as well as insulin secretion and hepatic insulin clearance (measured with the C-peptide model), did not differ in the testosterone and placebo groups., Conclusions: We conclude that 2 years of treatment with testosterone in elderly men does not improve carbohydrate tolerance or alter insulin secretion, insulin action, glucose effectiveness, hepatic insulin clearance, or the pattern of postprandial glucose metabolism. Thus, testosterone deficiency is unlikely the cause of the age-associated deterioration in glucose tolerance commonly observed in elderly men.

To determine if dehydroepiandrosterone (DHEA) replacement improves insulin secretion, insulin action, and/or postprandial glucose metabolism, 112 elderly subjects with relative DHEA deficiency ingested a labeled mixed meal and underwent a frequently sampled intravenous glucose tolerance test before and after 2 years of either DHEA or placebo. Despite restoring DHEA sulphate concentrations to values observed in young men and women, the changes over time in fasting and postprandial glucose concentrations, meal appearance, glucose disposal, and endogenous glucose production were identical to those observed after 2 years of placebo. The change over time in postmeal and intravenous glucose tolerance test insulin and C-peptide concentrations did not differ in men treated with DHEA or placebo. In contrast, postmeal and intravenous glucose tolerance test change over time in insulin and C-peptide concentrations were greater (P < 0.05) in women after DHEA than after placebo. However, since DHEA tended to decrease insulin action, the change over time in disposition indexes did not differ between DHEA- and placebo-treated women, indicating that the slight increase in insulin secretion was a compensatory response to a slight decrease in insulin action. We conclude that 2 years of replacement of DHEA in elderly men and women does not improve insulin secretion, insulin action, or the pattern of postprandial glucose metabolism.

To determine the effects of age and sex on the regulation of postprandial glucose metabolism, glucose turnover, insulin secretion, insulin action, and hepatic insulin extraction were concurrently measured in 145 healthy elderly (aged 70 +/- 1 years) and in 58 young (aged 28 +/- 1 years) men and women before and after ingestion of a mixed meal containing [1-(13)C]glucose. At the time of meal ingestion, [6-(3)H]glucose and [6,6-(2)H(2)]glucose were infused intravenously to enable concurrent measurement of the rates of postprandial endogenous glucose production (EGP), meal appearance, and glucose disappearance. Fasting and postprandial glucose concentrations were higher (P < 0.001) in both elderly women and elderly men compared with young individuals of the same sex. The higher postprandial glucose concentrations in the elderly than young women were caused by higher rates of meal appearance (P < 0.01) and slightly lower (P < 0.05) rates of glucose disappearance immediately after eating. In contrast, higher glucose concentrations in the elderly than young men were solely due to decreased (P < 0.001) glucose disappearance. Although postprandial glucose concentrations did not differ in elderly women and elderly men, rates of meal appearance and glucose disappearance rates both were higher (P < 0.001) in the women. Fasting EGP was higher (P < 0.05) in elderly than young subjects of both sexes and in women than men regardless of age. On the other hand, postprandial suppression of EGP was rapid all groups. Insulin action and secretion were lower (P < 0.001) in the elderly than young men but did not differ in the elderly and young women. This resulted in lower (P < 0.001) meal disposition indexes in elderly than young men but no difference in elderly and young women. Total meal disposition indexes were lower (P < 0.05) in elderly men than elderly women, indicating impaired insulin secretion, whereas disposition indexes were higher (P < 0.05) in young men than young women. Hepatic insulin clearance was greater (P < 0.001) in the elderly than young subjects of both sexes but did not differ between men and women regardless of age. In contrast, the ability of glucose to facilitate its own uptake (glucose effectiveness) was higher (P < 0.001) in women than men but did not differ in elderly and young subjects. Thus, age and sex impact on insulin secretion, insulin action, hepatic insulin extraction, and glucose effectiveness, resulting in substantial differences in the regulation of postprandial glucose metabolism in men and women and in elderly and young subjects.

A group of investigators met at a Specialized Programs of Research Excellence Workshop to discuss key issues in the translation of biomarker discovery to the development of useful laboratory tests for cancer care. Development and approval of several new markers and technologies have provided informative examples that include more specific markers for prostate cancer, more sensitive tests for ovarian cancer, more objective analysis of tissue architecture and an earlier indication of response to treatment in breast cancer. Although there is no clear paradigm for biomarker development, several principles are clear. Marker development should be driven by clinical needs, including early cancer detection, accurate pretreatment staging, and prediction of response to treatment, as well as monitoring disease progression and response to therapy. Development of a national repository that uses carefully preserved, well-annotated tissue specimens will facilitate new marker development. Reference standards will be an essential component of this process. Both hospital-based and commercial laboratories can play a role in developing biomarkers from discovery to test validation. Partnering of academe and industry should occur throughout the process of biomarker development. The National Cancer Institute is in a unique position to bring together academe, industry, and the Food and Drug Administration to (a) define clinical needs for biomarkers by tumor type, (b) establish analytic and clinical paradigms for biomarker development, (c) discuss ways in which markers from different companies might be evaluated in combination, (d) establish computational methods to combine data from multiple biomarkers, (e) share information regarding promising markers developed in National Cancer Institute-supported programs, and (f) exchange data regarding new platforms and techniques that can accelerate marker development.

Eighty-five to 95% of esophageal cancer patients suffer dysphagia. Yet, few studies have focused on this symptom, and four 'myths' persist: (i) dysphagia cannot be measured; (ii) chemotherapy cannot palliate it; (iii) dysphagia predicts a poor prognosis; (iv) dysphagia is associated with a frustratingly insatiable appetite. Forty-four patients with metastatic esophageal cancer participated in this quality of life/translational component of a previously reported clinical trial. All were monitored for chemotherapy efficacy and toxicity and completed questionnaires on dysphagia and appetite at baseline and every 6 weeks. The appetite hormones, leptin and neuropeptide y, were also assessed. Forty-five per cent of patients could easily swallow solid foods; all others had varying dysphagia, thus enabling exploration of these four 'myths.' First, a single-item visual analog scale (Swallowing Scale), demonstrated excellent agreement with a previously validated questionnaire (81% at baseline), thus reminding us that dysphagia is measurable. Second, chemotherapy was associated with a trend towards improved dysphagia (P = 0.059). Third, dysphagia did not predict tumor response or survival. Fourth, dysphagia was not associated with appetite, leptin or neuropeptide y. This study helps to dispel these four 'myths' and underscores the need for further quality of life research on dysphagia.

Objective: To identify the sources of analytical variation for cyclosporine and tacrolimus in a 3-year longitudinal study., Design: Two pools of whole blood were spiked with cyclosporine and tacrolimus, respectively. One aliquot of cyclosporine and 2 of the tacrolimus pool were distributed in the first and last mailing for years 1999 to 2001. For both drugs, the total variance for each method was partitioned into within- and between-laboratory components., Setting: The A and C mailings of the 1999, 2000, and 2001 AACC/CAP [American Association for Clinical Chemistry/College of American Pathologists] Immunosuppressive Drugs (CS) Monitoring Survey., Main Outcome Measures: For each drug, total variance was partitioned into specimen, mailing, year, and interlaboratory effects for each analytical method., Participants: The 292 laboratories for cyclosporine and 177 laboratories for tacrolimus enrolled in the survey from 1999 to 2001., Results: For both cyclosporine and tacrolimus, the major source of imprecision came from within-laboratory factors, which accounted for nearly 85% (range, 77% to 90%) of the total variance. For cyclosporine, the major component of within-laboratory variance was between-mailing, within-year effect, whereas for tacrolimus it was the between-year, within-laboratory variation., Conclusion: The major source of long-term survey imprecision for cyclosporine and tacrolimus is within-laboratory factors. The finding that 85% of the total variance was due to within-laboratory variation is similar to other therapeutic drugs.

The spun packed cell volume (PCV, hematocrit) is a key measurement on which are based hematology instrument calibration, reference range determination, and assignment of values to calibrators/controls. In 2001, the International Council for Standardization in Haematology (ICSH) recommended a Reference PCV method, which is fully traceable to the ICSH reference hemoglobin method. Because of its complexity, however, this method is impractical for occasional use in routine laboratories and is therefore intended primarily for use by manufacturers of capillary microhematocrit tubes, liquid calibrators, and multichannel analyzers. In response to the need for a simpler method--accessible to all routine laboratories--the ICSH offers this "Surrogate Reference" PCV procedure. It is traceable to the original ICSH Reference PCV method and is based on spun PCVs obtained using borosilicate capillary tubes with an already-known relationship to this reference procedure. This ICSH "Surrogate Reference" PCV method is substantially simpler, thus putting it within the reach of most routine hematology laboratories.

This study was designed to assess whether extrinsic neural regulation of antroduodenal motor function is present in preterm neonates. Antroduodenal motor patterns were recorded in 48 infants during fasting and after a 10 ml/kg/15 min orograstric feeding. Plasma concentrations of pancreatic polypeptide were determined before and after each feeding. Of 48 infants, 24 had adult like duodenal motor responses to feeding, and they released pancreatic polypeptide postprandially. When 18 of these 24 infants were given atropine, their heart rates increased. Feedings, however, now failed to trigger adultlike duodenal motor responses or postprandial release of pancreatic polypeptide. Those 24 infants who lacked adultlike responses failed to release pancreatic polypeptide postprandially. When a subset of 10 of these infants was given atropine, feedings failed to trigger adultlike motor responses or release of pancreatic polypeptide. We conclude that extrinsic neural regulation of duodenal motor patterns is present in only about half of preterm infants.

Biochemical markers of bone turnover provide a means of evaluating skeletal dynamics that complements static measurements of bone mineral density (BMD). This review evaluates the use of commercially available bone turnover markers as aids in diagnosis and monitoring response to treatment in patients with osteoporosis. High within-person variability complicates but does not preclude their use. Elevated bone resorption markers appear to be associated with increased fracture risk in elderly women, but there is less evidence of a relationship between bone formation markers and fracture risk. The critical question of predicting fracture efficacy with treatment has not been answered. Changes in bone markers as currently determined do not predict BMD response to either bisphosphonates or hormone replacement therapy. Single measurements of markers do not predict BMD cross-sectionally (except possibly in the very elderly), or change in BMD in individual patients, either treated or untreated. On the other hand, research applications of bone turnover markers are of value in investigating the pathogenesis and treatment of bone diseases. Markers have potential in the clinical management of osteoporosis, but their use in this regard is not established. Additional studies with fracture endpoints and information on negative and positive predictive value are needed to evaluate fully the utility of bone turnover markers in individual patients.

This document is intended to assist towards the WHO objective that external quality assessment (EQA) schemes be established at national and/or regional levels world-wide. Quality assurance is defined as all steps taken by the director of a laboratory to ensure reliability of laboratory results and to increase accuracy, reproducibility and between-laboratory comparability. This includes the use of internal quality control procedures and participation in external quality assessment. Internal quality control provides the means for evaluation of analytic test results at the time of testing in order to decide whether they are reliable enough to be released to the requesting clinicians. EQA, on the other hand, refers to a system of retrospective and objective comparison of results from different laboratories by means of proficiency testing (PT) organised by an external agency. The main purpose is to establish between-laboratory and between-method (including between-instrument) comparability, and agreement with a reference standard where one exists. Internal quality control and EQA complement each other and must never be considered as alternatives.

In adults, migrating motor complexes (MMCs) appear to be partially under hormonal modulation by motilin and pancreatic polypeptide. Preterm infants do not exhibit MMCs until 32 wk of gestation. Although plasma concentrations of motilin are similar in infants and adults, it is not known if actual hormonal modulation of MMCs is present in infants. In the first study we assessed whether plasma concentrations of motilin and pancreatic polypeptide surge with the occurrence of MMCs in term infants. In the second study we assessed whether erythromycin, a motilin receptor agonist, could induce migrating motor activity in preterm and term infants. In the first study we recorded motor activity in nine term infants who had never been fed. We determined plasma concentrations of motilin and pancreatic polypeptide in the presence and absence of MMCs. In the second study we gave the motilin agonist erythromycin intragastrically to 21 infants at a range of 24-42 wk of gestation to assess whether migrating activity could be induced via the motilin receptor. In the first study, plasma concentrations of motilin were similar during the presence and absence of MMCs, as were plasma concentrations of pancreatic polypeptide. In the second study, the administration of erythromycin induced the appearance of migrating activity in 7 of 14 infants who were older than 32 wk but in none of the infants who was younger than 32 wk. Although the motilin receptor appears to be functionally present beyond 32 wk of gestation, as assessed by in indirect pharmacologic challenge, hormonal modulation of migrating activity in the neonate by plasma motilin and pancreatic polypeptide is absent.

A conceptual model is proposed for defining analytic bias limits utilizing the variations found in cumulative test value distributions. The model is based on the propositions that changes in analytic bias are more important than analytic imprecision in medical diagnoses and that analytic bias alters clinical specificity more than clinical sensitivity. The rationale for these propositions are presented along with a step-by-step procedure for estimating bias tolerance limits. These concepts are illustrated with an example using prostate-specific antigen. A second protocol is provided to define analytic imprecision tolerance limits, based on the quality control performance characteristics required to maintain the bias tolerance limits. This model can be applied to most chemistry, immunoassay, and hematologic quantitative assays. The relationship of this procedure to the published procedures using biologic variation for defining analytic tolerance limits is discussed.

Objectives: Comparison of a new monoclonal digoxin assay with three polyclonal digoxin assays for their cross-reactivity to digoxin-like immunoreactive factors (DLIF) and digoxin metabolites., Design and Methods: Sixty-six nondigitalized patient samples from 5 different groups: neonates, women in 3rd trimester pregnancy, and patients with liver or renal diseases, or undergoing organ transplants, and 139 samples from digoxin-treated patients of 4 categories (hospital sick, liver, renal, and outpatients) were compared in 4 different digoxin assays: (a) ACS Digoxin (ACS) developed for the automated chemiluminescent Ciba Corning ACS 180 system, (b) Baxter Stratus (Stratus, a fluoroimmunoassay), (c) Ciba-Corning Magic (Magic, a radioimmunoassay), and (d) an in-house radioimmunoassay (RIA). The ACS and RIA were also compared for their cross-reactivity to four principal digoxin metabolites., Results and Conclusion: Among the nondigitalized specimens, no significant DLIF interference was found for all 4 assays among the pregnant women or liver and transplant patients. However, the neonates registered high DLIF interference with Magic and RIA, but none for ACS or Stratus. DLIF interference in renal samples was highest in the Magic assay and lowest in RIA. Among the specimens with digoxin, a higher number of discrepant samples were found from the sick patients than from outpatients. In 75% of such discrepant samples, the ACS result was less than other assay results, suggesting DLIF as the probable cause. The two assays differed most in their cross-reactivity to the deglycated metabolites, digoxigenin and its mono-digitoxoside.

Tryptophan (TRP) depletion was used to study serotonin because the ratio of TRP to large neutral amino acids (TRP/LNAA) determines the quantity of TRP that enters the brain. Because TRP is not universally available, a modified technique of TRP depletion was developed where a 1/4 strength preparation of an amino acid mixture (AAM) replaces TRP as the placebo. Seven healthy subjects could not differentiate between the preparations in this double-blind, placebo-controlled study. Urinary 6-hydroxymelatonin sulfate (6-MS) was monitored as a biochemical marker of serotonin. The TRP/LNAA ratio (GG = 0.001) and 6-MS secretion (GG = 0.024) were decreased, but placebo TRP levels (GG = 0.062) were not altered significantly. This modified technique facilitates the use of TRP depletion in clinical research.