Establishing reference intervals for soluble urokinase plasminogen activator receptor in Northern European adults.

Objectives: Soluble urokinase plasminogen activator receptor (suPAR) may have untapped potential in clinical diagnostics. Previous studies determined reference intervals using an enzyme-linked immunoassay, but there is a need for reference intervals using a faster assay if the analysis is to be used in emergency medicine. The current study aims to determine reference intervals for suPAR using a fully automated particle-enhanced turbidimetric immunoassay (PETIA) according to the Clinical and Laboratory Standards Institute guideline A28-A3c. Design and methods: Blood samples were prospectively collected from Danish blood donors. Plasma suPAR was analyzed on the cobas 8000 module c502 in an open channel using a PETIA. Sex-partitioned reference intervals were determined using a parametric quantile approach. Results: The study included 241 participants-123 females and 118 males. The common reference interval for suPAR was 1.56-4.11 ng/mL (95% confidence intervals (CI) for the lower and upper limits were 1.56-1.63 and 3.81-4.47, respectively). The reference interval for females was 1.59-4.65 ng/mL (95% CIs 1.48-1.70 and 4.09-5.48, respectively) and for males, 1.56-3.59 ng/mL (95% CIs 1.47-1.65 and 3.31-3.93, respectively). Conclusions: Our results support using sex-partitioned reference intervals for suPAR and provide a basis for future studies using the PETIA method.

Establishing Sex-Dependent Reference Intervals for KL-6 in Danish Adults.

Krebs von den Lungen-6 (KL-6) is a promising biomarker for the diagnosis and prognosis of interstitial lung disease. However, reference intervals in Northern Europeans remain to be established using a latex-particle-enhanced turbidimetric immunoassay. The participants were Danish blood donors subjected to strict health requirements. Analyses were performed using the Nanopia KL-6 reagent on the cobas 8000 module c502. Sex-partitioned reference intervals were determined using a parametric quantile approach according to the Clinical and Laboratory Standards Institute guideline EP28-A3c. The study included 240 participants-121 females and 119 males. The common reference interval was 59.4-398.5 U/mL (95% confidence intervals (CI) for the lower and upper limits were 47.3-71.9 and 369.5-430.1, respectively). For females, the reference interval was 56.8-324.0 U/mL (95% CIs for the lower and upper limits were 36.1-77.6 and 303.3-344.7, respectively). For males, the reference interval was 51.5-448.7 U/mL (95% CIs for the lower and upper limits were 32.8-71.2 and 397.3-508.1, respectively). These results emphasize the importance of sex partitioning when evaluating KL-6 reference intervals. The reference intervals increase the clinical applicability of the KL-6 biomarker and provide a basis for future scientific studies of its utility in patient management.

Using fecal immunochemical test values below conventional cut-off to individualize colorectal cancer screening.

Background and study aims Of the participants in the Danish screening program, 89.9 % to 92.5 % have fecal immunochemical test (FIT) values < 10 µg/g feces (equivalent to 50 ng hemoglobin/mL buffer). This study aimed to investigate the risk of interval colorectal cancer (CRC) in this group before the next biennial screening round. Patients and methods This cohort study included all citizens from the region of Southern Denmark who participated in the Danish bowel screening program from 2014 trough 2016 and had a FIT value < 10 µg/g feces. Individuals receiving a CRC diagnosis were identified through the national CRC registry, with a follow up of 2 years corresponding to the current screening interval. We also examined the 3-year CRC incidence. Hazard ratios (HRs) were estimated using univariate and multivariate Cox proportional hazard regression models. Results Data from 185,654 citizens presenting with a FIT value < 10 µg/g feces were eligible for analysis. Overall, interval CRC incidence was 0.07 % within 2 years with HRs of 4.16 (95 % confidence interval [CI] 2.67;6.48) and 5.8 (95 % CI 3.34;10.05) for FIT values of 4 to 6.9 µg/g feces and 7 to 9.9 µg/g feces, respectively, compared to those having a FIT value below the limit of quantification of 4 µg/g feces. After 3 years, the overall CRC incidence increased to 0.14 %; however, this was not significant. Conclusions This study demonstrates a positive correlation between FIT value and risk of interval cancer even for very low values. It further suggests that an increase in the screening interval could be reasonable in the low FIT categories.

Higher free thyroxine associated with PFAS exposure in first trimester. The Odense Child Cohort.

BACKGROUND: Perfluoroalkyl substances (PFAS) are endocrine disrupting chemicals with elimination half-lives ranging from four to eight years. Experimental studies found PFAS able to interfere with thyroid hormone-binding proteins. During the first 20 weeks of gestation (GW), the fetus is reliant on placental transfer of maternal thyroid hormones, mainly free thyroxine (FT4). However, previous studies investigating associations between exposure to PFAS and thyroid hormone status mainly focused on blood samples from late pregnancy or umbilical cord with mixed findings. OBJECTIVES: To investigate associations between serum-PFAS concentrations and thyroid hormone status in early pregnancy as reflected by FT4 and thyroid-stimulating hormone (TSH). METHODS: In the Odense Child Cohort, a single-center study, we measured maternal pregnancy serum concentrations of five PFAS: perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA); and FT4 and TSH in 1048 pregnant women at median GW 12 (25th, 75th percentile: 10, 15). Multivariate linear regression models were performed to estimate associations between PFAS exposure and thyroid hormone status. RESULTS: A doubling in PFOS, PFOA, and PFNA concentrations was associated with an increment in FT4 concentration by 1.85% (95% CI: 0.66%, 3.05%), 1.29% (95% CI: 0.21%, 2.39%), and 1.70% (95% CI: 0.48%, 2.94%), respectively, in adjusted analyses. A statistically significant dose-response relationship was observed across exposure quartiles for PFOS, PFOA, and PFNA in the association with FT4. No association was found between concentrations of PFAS and TSH in adjusted analyses. CONCLUSION: Exposure to PFOS, PFOA, and PFNA was associated with higher FT4 concentrations in women during early pregnancy. The potential clinical implications of these findings remain to be clarified.

Evaluation of the point-of-care devices KetoSureTM and StatStrip Express® blood ketone tests using ß-hydroxybutyrate spiked samples.

Measurement of ß-hydroxybutyrate (BHB) in blood is used clinically as a measure of ketosis when diabetic ketoacidosis is expected. With the introduction of point-of-care testing (POCT) for blood-BHB, nonproductive time is reduced to a minimum in a potential critical situation; however, studies have observed inferior quality of POCT-BHB. Recently, the POCT device KetoSure (Roche) has been introduced to the clinic. In this study, we evaluated the imprecision and linearity of KetoSure and compared this to the established StatStrip Express (Nova Biomedical) based on spiked full blood samples. We found comparable imprecision for KetoSure and StatStrip Express. However, linearity was only observed in the lower part of the measuring range for both devices. In a method comparison, higher values of BHB were measured by KetoSure than by an enzymatic endpoint spectrophotometric reference method (mean bias: 21% (95% confidence interval (CI): 4%-37%)). Conversely, StatStrip Express returned lower values of BHB (mean bias: -16% (95% CI: -38%-7%), while the widely applied POCT device FreeStyle Precision Neo (Abbott) returned values equivalent with the reference method (mean bias: 5% (95% CI: -14%-24%). In samples with concentrations of BHB above the measuring range, the POCT devices could be provoked to return falsely low results. In conclusion, the quality of KetoSure is in line with other established POCT devices; however, the KetoSure measures higher concentrations than other POCT devices. As, linearity only was observed in the lower part of the measuring range and as falsely low measures could be provoked, we advise users to interpret results with precaution.

Colon capsule endoscopy in colorectal cancer screening: Interim analyses of randomized controlled trial CareForColon2015.

Background and study aims The Danish CareForColon2015 trial, launched in 2020 as part of the Danish Colorectal Cancer Screening program, is the largest randomized controlled trial to date on colon capsule endoscopy (CCE). This paper presents the interim analysis with the objective of ensuring the safety of patients in the intervention group and evaluating the clinical performance of the trial's predefined clinical parameters. Patients and methods We evaluated the initial 234 CCEs according to quality, safety, and completion. The participation rates and preference distribution of all individuals invited were analyzed and sample size calculations were adjusted. Results Fecal immunochemical test and diagnostic participation rates were 62.1 % and 91.1 %, respectively. The completion rate for CCEs was 67.9 % and the rate of conclusive investigations was 80.3 %. The polyp detection rate (PDR) was high (73.5 %), only two (0.85 %) technical failures in 234 videos were observed, and six suspected cancers were identified (2.6 %). No major adverse events were recorded. The required number of invitations had been underestimated due to inaccurate assumptions in sample size calculations. Conclusions The trial was efficient and safe in terms of CCE quality and time to diagnostic investigation. Participation rates and PDRs were high. The proportion of suspected cancers was lower than expected and will be followed. The completion rate for CCEs was acceptable but lower than expected and the CCE procedure was reviewed for potential improvements and Resolor was added to the regime. The number of invitations for the intervention group of the trial has been adjusted from 62,107 to 185,153.

Free thyroxine in early pregnancy is an independent negative predictor of 3rd trimester HbA1c. Odense child cohort.

BACKGROUND: Lower thyroid function outside pregnancy is associated with an increased risk of type 2 diabetes mellitus. The relationship between thyroid function in early pregnancy and glucose status in 3rd trimester has not been investigated. AIMS: To study the association between 1st trimester thyroid function and 3rd trimester glucose status. DESIGN: In the prospective study Odense Child Cohort (OCC), 1,041 women had 1st trimester blood samples analysed for thyroid-stimulating hormone (TSH), free T4 (FT4), thyroid peroxidase antibody and HbA1c. Third trimester (week 28) fasting blood samples included plasma glucose, insulin and HbA1c. Oral glucose tolerance test (OGTT, 75 g glucose) was performed in 509 women. First trimester FT4 was dichotomized >vs. ≤ the 25th percentile (25p = 12.9 pmol/L). Homeostatic model assessment-insulin resistance (HOMA)-IR and HOMA-ß were calculated. RESULTS: Women with FT4 ≤25p had significantly higher HbA1c in 1st and 3rd trimesters and higher 3rd trimester fasting glucose, insulin, HOMA-IR and HOMA-ß compared to women with FT4 >25p. In multiple regression analyses, FT4 was an independent negative predictor of 3rd trimester HbA1c. FT4 levels in 3rd and 4th quartiles (high-normal FT4 levels) showed closest inverse associations with HbA1c (p-trend <.001). TSH was not associated with 3rd trimester HbA1c. CONCLUSION: Women with lower levels of FT4 in early pregnancy had higher HbA1c in 3rd trimester and FT4 was an independent negative predictor of 3rd trimester HbA1c.

An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key Intermediate for Polymerization Revealed by Hydrogen/Deuterium-Exchange Mass Spectrometry.

Antithrombin deficiency is associated with increased risk of venous thrombosis. In certain families, this condition is caused by pathogenic polymerization of mutated antithrombin in the blood. To facilitate future development of pharmaceuticals against antithrombin polymerization, an improved understanding of the polymerogenic intermediates is crucial. However, X-ray crystallography of these intermediates is severely hampered by the difficulty in obtaining well-diffracting crystals of transient and heterogeneous noncovalent protein assemblies. Furthermore, their large size prohibits structural analysis by NMR spectroscopy. Here, we show how hydrogen/deuterium-exchange mass spectrometry (HDX-MS) provides detailed insight into the structural dynamics of each subunit in a polymerization-competent antithrombin dimer. Upon deuteration, this dimer surprisingly yields bimodal isotope distributions for the majority of peptides, demonstrating an asymmetric configuration of the two subunits. The data reveal that one subunit is very dynamic, potentially intrinsically disordered, whereas the other is considerably less dynamic. The local subunit-specific deuterium uptake of this polymerization-competent dimer strongly supports a ß4A-ß5A ß-hairpin runaway domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts toward future pharmacological intervention with protein polymerization and associated diseases.

Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins.

Protein glycosylation is the most frequent post-translational modification and is present on more than 50% of eukaryotic proteins. Glycosylation covers a wide subset of modifications involving many types of complex oligosaccharide structures, making structural analysis of glycoproteins and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution. N-linked glycoproteins however pose a challenge for HDX-MS. HDX information can typically not be obtained from regions of the glycoprotein that contain the actual N-linked glycan as glycan heterogeneity combined with pepsin digestion yields a large diversity of peptic N-glycosylated peptides that can be difficult to detect. Here, we present a novel HDX-MS workflow for analysis of the conformational dynamics of N-linked glycoproteins that utilizes the enzyme PNGase A for deglycosylation of labeled peptic N-linked glycopeptides at HDX quench conditions, i.e., acidic pH and low temperature. PNGase A-based deglycosylation is thus performed after labeling (post-HDX) and the utility of this approach is demonstrated during analysis of the monoclonal antibody Trastuzumab for which it has been shown that the native conformational dynamics is dependent on the N-linked glycan. In summary, the HDX-MS workflow with integrated PNGase A deglycosylation enables analysis of the native HDX of protein regions containing N-linked glycan sites and should thus significantly improve our ability to study the conformational properties of glycoproteins.

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding.

The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2'-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA-PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 2'-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.

Biochemical properties of plasminogen activator inhibitor-1.

PAI-1 is a Mr ~50,000 glycoprotein, which is the primary physiological inhibitor of the two plasminogen activators uPA and tPA. PAI-1 belongs to the serpin protein family. Studies of PAI-1 have contributed significantly to the elucidation of the protease inhibitory mechanism of serpins, which is based on a metastable native state becoming stabilised by insertion of the RCL into the central beta-sheet A and formation of covalent complexes with target proteases. In PAI-1, this insertion can occur in the absence of the protease, resulting in generation of a so-called latent, inactive form of the protein. PAI-1, in its active state, also binds to the extracellular protein vitronectin. When in complex with its target proteases, it binds with high affinity to endocytosis receptors of the low density receptor family.