The aggregation of amyloid β (Aβ) is central in the pathogenesis of Alzheimer’s disease (AD). Recently it has been shown that specifically, larger, Thioflavin T-binding Aβ aggregates are associated with increased neuroinflammation and cytokine release. This study was aimed to quantify fibrillary amyloid aggregates, so-called nanoplaques, and investigate their relationship with cytokines in the cerebrospinal fluid (CSF).CSF was collected from 111 patients assessed for cognitive complaints at the Oslo University Hospital Memory Clinic. The patients were grouped based on their amyloid status. The CSF nanoplaque concentration was quantified with the Thioflavin T-fluorescence correlation spectroscopy (ThT-FCS) assay. The levels of nine cytokines  were quantified with a magnetic bead-based multiplex assay and read on a Luminex IS 200 instrument.There were 49 amyloid-negative and 62 amyloid-positive patients in the cohort; none of the cytokines differed significantly between the amyloid groups. The increased nanoplaque levels were associated with levels of MIP-1β below the lower limit of quantification, and with decreased levels of MIP-1α and IL-8. The associations remained significant when adjusted for age, sex, cognitive function, apolipoprotein ε4 status and CSF core biomarker levels.The cytokine levels were not associated with amyloid status in this cohort. The nanoplaque levels were negatively associated with MIP-1β, MIP-1α and IL-8, which is in line with recent findings suggesting that the upregulation of some cytokine markers has a protective role and is negatively associated with AD progression. Alzheimer’s disease (AD) is the leading cause of dementia globally. This neurodegenerative disorder is characterised by extracellular amyloid β (Aβ) deposits, intracellular tau neurofibrillary tangles and pervasive synaptic loss . These n42 and cytokines have resulted in inconsistent findings [Activated astrocytes and microglia release cytokines, which are signalling proteins with both pro- and anti-inflammatory effects . In AD, findings –11.42 assays primarily recognise monomeric Aβ, the Thioflavin T-fluorescence correlation spectroscopy (ThT-FCS) assay quantifies structured amyloidogenic aggregates, so-called nanoplaques, with high specificity [Importantly, Aβ aggregates are not uniform, and range from small oligomers to larger protofibrils and fibrils , 13. Thecificity –19. As sIn the current study, we set out to investigate the relationship between CSF nanoplaque levels and nine cytokines in a memory clinic cohort, including both patients with AD pathology and patients with non-AD disorders.One hundred and eleven patients from the Norwegian Registry of Persons Assessed for Cognitive Symptoms (NorCog), who were assessed at the Oslo University Hospital Memory Clinic, were included in this study. The patients were included in NorCog between June 2014 and November 2018.APOE) genotyping, magnetic resonance imaging , 18F-FDG positron emission tomography  and 18F-flutemetamol PET (n = 54) brain scans. The core CSF biomarkers for AD were analysed for all patients (n = 111) at the Department of Interdisciplinary Laboratory Medicine and Medical Biochemistry at Akershus University Hospital with enzyme-linked immunosorbent assays using the Innotest kit . Cognitive and functional impairment on the Clinical Dementia Rating (CDR) scale were scored post-hoc by experienced CDR raters (ABK and THE). The CDR sum-of-boxes (CDR-SOB), which has been found to accurately separate MCI and dementia [The patients were assessed according to an established research protocol . The patdementia , was usepost-hoc by experienced memory clinicians (ABK and THE). Subjective cognitive decline (SCD) was diagnosed according to the Subjective Cognitive Decline Initiative-criteria [Clinical diagnoses were made criteria , whilst criteria , 24. Clicriteria , 24. Thecriteria , frontotcriteria , and MCI42 and/or 18F-flutemetamol results for A; CSF phosphorylated tau (P-tau) for T; and CSF total tau (T-tau), 18F-FDG PET and/or MRI results for N. The 18F-flutemetamol PET scans were visually classified as amyloid-positive or negative according to a validated electronic image reader programme [42 > 700 pg/ml, P-tau < 80 pg/ml, and T-tau < 300 pg/ml for those < 50 years, < 450 pg/ml for those aged 50–70 years and < 500 pg/ml for those > 70 years [In line with the 2018 NIA-AA research framework, patients were categorised based on three groups of biomarker data: A – reflecting Aβ pathology, T – reflecting tau pathology and N – reflecting neurodegeneration . The patrogramme . The FDGrogramme , 30. For70 years . In the fseo), directly reflects the concentration of nanoplaques in the CSF.The ThT-FCS assay procedure has been described in detail by Tiiman et al. . BrieflyAll CSF samples underwent analysis of cytokines using a custom-made nine-plex kit  containing eotaxin-1, granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, IL-7, IL-8, interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, and MIP-1β. The nine cytokines were selected based on a screening of a representative set of samples with the human cytokine 27 plex kit . A 10% bovine serum albumin  solution in PBS  was added to all CSF samples to a final concentration of 0.5% and vortexed. Then the samples were centrifuged at 10 000×g for 10 min at 4 °C and 50 μL of the supernatant was loaded onto the assay plate. The CSF cytokine levels were measured on a Luminex IS 200 instrument (Bio-Rad). An in-house control was used to observe both intra percentage coefficient of variation and longitudinal (inter) percentage coefficient of variation. Cytokine concentrations below the lower limit of quantification (LLOQ) or above the upper limit of quantification extrapolated by the analysis software were also included in the statistical analysis.t-test) or non-parametric (Mann-Whitney test) test depending on the data distribution. As the distributions of CSF nanoplaque levels and most of the cytokines were markedly right-skewed, the results are presented as medians and quartiles. The correlation analyses were reported with Spearman’s ρ correlation coefficient. Bonferroni-corrected P-values are presented for the correlations between nanoplaque levels and cytokines. Significant associations between nanoplaque levels and cytokines were further explored in multiple linear regression analyses adjusted for sex, age, CDR-SOB and APOE ε4 carrier status (Model 1), and biomarkers CSF Aβ42 and CSF P-tau (Model 2). The natural logarithm transformed nanoplaque variable log (fseo) was used in the regression analysis in order to achieve normally distributed standardised residuals. Statistical analyses were conducted with STATA 15.1  and R 3.4.4 .Group differences were assessed by parametric (P = 0.34). In the A+ group, 28 patients (45%) were T+ and 58 patients (94%) were N+, whereas in the A- group, 8 patients (16%) were T+ and 31 patients (63%) were N+. The clinical diagnosis was consistent with the amyloid status: 89% of the A+ patients had Alzheimer’s clinical syndrome, and 84% of the A- patients had clinical non-AD.Patient characteristics are presented in Table 42 levels were negatively associated with the levels of CSF nanoplaques , CSF T-tau  and CSF P-tau . Moreover, the levels of CSF P-tau and CSF T-tau were strongly correlated with each other . The CSF nanoplaque levels were not associated with CSF P-tau (ρ = 0.001) or CSF T-tau (ρ = − 0.01), P > 0.05.The CSF Aβn = 4, 3.6%), IL-6  and MIP-1α  were below the LLOQ, and therefore included as zero. In the majority of samples, the levels of MIP-1β  and IL-7  were below the LLOQ, so MIP-1β and IL-7 were dichotomised as detectable/not detectable.The markers eotaxin-1, IL-8, MCP-1 and IP-10 were detectable in all patient CSF samples. In a minority of samples, the levels of G-CSF (P > 0.05).The CSF levels of cytokines are presented in Table P = 0.01). There was no difference in the nanoplaque levels between those with non-detectable levels of IL-7  and those with detectable levels of IL-7 .The distributions of nanoplaque levels in the patients with non-detectable/detectable levels of MIP-1β and IL-7 are presented in Fig. ρ = − 0.34, P = 0.007) and MIP-1α levels , but not associated with any of the other cytokines , and for Aβ42 and P-tau levels (P = 0.003) in two regression models  and for Aβ42 and P-tau levels (P = 0.002) in the two regression models (Table P = 0.006). In the second model . The decreased nanoplaque levels were the strongest predictor for increased MIP-1α levels in both models.Similarly, the negative association between MIP-1α and nanoplaque levels remained significant when adjusting for the covariates sex, age, cognitive function and ls Table . In the ls Table , increasThere has been mounting evidence suggesting that inflammatory mediators released by microglia and astrocytes, such as cytokines, contribute to AD neuropathology . Amyloid42 [42 and P-tau levels. Although amyloid aggregates have been shown to increase the release of cytokines [It has been hypothesised that larger amyloid protofibrils in the CSF of AD patients cause an increase in inflammation . The nan42 , the assytokines , 15, thiytokines . As suchIndeed, the inflammatory activity in AD is complex and may change across disease stages. It has been proposed that there are two peaks of inflammatory  activity in AD: an early, potentially neuroprotective peak, and a later, neurotoxic peak . In line, microglia, astrocytes and neurons have been shown to release IL-8 in response to other pro-inflammatory cytokines such as IL-1β [In the central nervous system, cytokines can be produced by microglia, astrocytes and neurons. In vitroas IL-1β , 38. Simas IL-1β . Howeveras IL-1β . In the as IL-1β . Moreoveas IL-1β ; G-CSF ias IL-1β , whereasas IL-1β , 45. As Inflammatory markers have been extensively studied as biomarkers for AD, both in the CSF and in peripheral samples , 47. A rAPOE-genotyping. By including other disease controls rather than cognitively healthy controls, this study can inform on the overlapping inflammatory mechanisms across different disorders and the use of inflammatory markers, or rather lack thereof, in differential diagnosis of memory clinic patients. A further strength is the application of the highly sensitive ThT-FCS assay. The ThT-FCS assay has the ultimate sensitivity and permits the detection of single aggregated particles in small sample volumes, within a wide range of molecular concentrations. This is achieved without the use of signal-amplification, protein separation or immune probes.The current study was strengthened by the inclusion of a comprehensively characterised memory clinic cohort with extensive clinical and biological data, including multiple biomarker measurements and The current study has several limitations. First, the ThT-FCS method cannot identify the amino acid sequence of the polypeptides in the nanoplaques: ThT selectively binds aggregates with a β-sheet secondary structure. In addition to Aβ, several other proteins can aggregate to this structure and bind ThT , 56. TheFuture research should assess whether the nanoplaques and cytokines are also associated in serum samples. Moreover, studies on microvascular pathology, a common feature of AD , are anoIn conclusion, this study suggests a negative relationship between CSF amyloidogenic nanoplaques and cytokines MIP-1β, MIP-1α and IL-8. This finding contrasts with in vitro studies that have shown increased inflammation in response to aggregated amyloid from the CSF. However, the direction of this association is in line with recent evidence suggesting a protective role of the upregulation of certain cytokines. The relationship between nanoplaques and inflammatory markers should be further explored across the different stages of the AD pathophysiological process, in order to inform on the dynamics between aggregated amyloid and inflammation through the disease.


Background: Automated molecular panels are attractive tools for improving early meningitis diagnosis. This study assessed the Eazyplex® CSF direct M panel (EP), a multiplex real-time Loop-Mediated Isothermal Amplification assay. Methods: From December 2016 to December 2019, cerebrospinal fluid (CSF) samples were routinely tested with the EP V1.0. CSF parameters and microbiological and clinical data were retrospectively collected. Results: Out of 230 CSF samples, the EP yielded positive, negative, and invalid results for 32 (13.9%) , 182 (79.1%), and 16 (7%) samples, respectively. Among the positive samples, 14 (44%) remained negative in culture (antibiotic therapy before lumbar puncture (n = 11), meningococcal meningitis (n = 3)). High CSF protein concentrations and cellularity were associated with LAMP inhibition, counteracted by centrifugation. The automated software yielded 13 false positive and five false negative results. Amplification curve analysis was necessary and enabled the attainment of positive (PPA) and negative percentage agreement and positive and negative predictive values of 91.4%, 100%, 100%, and 98.3%. Three false negative results remained (two E. coli and one N. meningitidis). E. coli presented the poorest PPA (50%). Conclusion: This work confirms the strong performance of the EP, of particular interest in cases of antibiotic therapy before lumbar puncture. To date, very little data is available concerning this panel , 100*[TN/(TN + FP)], 100*[TP/(TP + FP)], and [TN/(TN + FN)] respectively. The terms “PPA” and “NPA” were used instead of “sensitivity” and “specificity” because a “non-gold standard” assay was used for the comparator analysis.t test ). p < 0.05 was conventionally used for statistical significance.CSF protein concentration, WBC and RBC counts were compared between the CSF samples with successful or failed internal inhibition control using an unpaired n = 101) and 56% male (n = 129). The age distribution included 171 (74%) adults 16 years of age or older and 59 (26%) pediatric patients distributed as followed: 24 infants of ≤ 2 months of age, six between 3 and 11 months old, and 29 children between 1 and 15 years old. CSF specimens were sampled in patients from the emergency care units (n = 85), intensive care units (n = 52), medical care units (n = 59), or from neighborhood hospitals (n = 34).During the study period, 7505 CSF specimens were analyzed, of which 236 samples from 225 patients were subjected to EP testing. Redundant samples from six patients were excluded. Thus, a total of 230 CSF samples from 225 patients were included in the analysis . Patient® analysis. Inhibition control came back successful in 15 out of the 17 reanalyzed CSF samples. Nineteen CSF were not reanalyzed: five of them presented a positive target, and 14 an insufficient volume.The internal control failed on the first attempt for 36 of the 230 CSF tested, suggesting a LAMP inhibition (inhibition rate of 15.6%). The median (interquartile range (IQR)) CSF protein concentration was 0.82 g/L (0.53–1.71) in CSF with valid inhibition control versus 3.22 g/L (0.96–6.54) in CSF with invalid inhibition control. The median (IQR) WBC count was 124 cells/µL (19–738) in CSF with valid inhibition control versus 405 cells/µL (59–3870) in CSF with invalid inhibition control. The median (IQR) RBC count was 8 cells/µL (1–186) in CSF with valid inhibition control versus 29 cells/µL (2–520) in CSF with invalid inhibition control. Statistical analysis revealed CSF protein concentration and WBC count were significantly higher in CSF with invalid inhibition control . Over thH. influenzae (n = 8), E. coli (n = 4), and L. monocytogenes (n = 1). Biological expertise of the amplification curves and rates concluded that the observed amplifications were non-specific  and S. pneumoniae (n = 2)), not mentioned by the software because internal control failed. In such situations, the software automatically provided the result “invalid” for all the panel analyses, irrespective of other target results.Amplification curve analysis also allowed the rectification of five false negative results (N. meningitidis (n = 16) and S. pneumoniae (n = 9). Gram stain was negative for 11 (34%) of the 32 EP positive samples. Cultures remained negative for 14 (44%) of the 32 EP positive samples: antimicrobial therapies were administered before lumbar puncture in 11 cases, and the last three cases were meningococcal meningitis.Overall, after biological analysis and exclusion of the false positive and negative results generated by the software, the EP testing yielded positive, negative, and invalid results for 32 (13.9%), 182 (79.1%), and 16 (7%) CSF samples, respectively. The highest detection rates were in the pediatric age groups. The relative prevalence of each pathogen among the positive samples is presented in E. coli meningitis and one N. meningitidis meningitis. Internal inhibition control was valid in the three cases. No FP results were observed (results of the discrepancy investigation are presented in E. coli (PPA = 50%).Nineteen samples were excluded from the performance analysis: three due to missing biological and clinical data, and 16 to invalid results (inhibition control failures). Discrepancy investigations remained inconclusive for six samples. Overall, EP performance was evaluated on 205 CSF analysis . Discrep® meningitis/encephalitis (FA-ME) panel have been assessed several times [® CSF direct M panel assay, a multiplex real-time LAMP assay, provides another alternative for the rapid diagnosis of bacterial meningitis. However, clinical validation data of this panel are still scarce, as only one study has assessed its performance, without conducting any discrepant analysis [Syndromic multiplex molecular tests are promising tools for the rapid etiological diagnosis of bacterial meningitis. Accuracy and impact on the patients’ care of the FilmArrayal times ,10,11,12analysis .We reported here the strong performance of the EP in meningeal pathogen detection , comparable to that reported in the only other study already published  . These pn = 11), or in meningococcal meningitis (n = 3). These conclusions were consistent with the literature since (i) antibiotic treatments before lumbar puncture decreased the yield of CSF culture by 10 to 20%, and (ii) CSF positivity differed with causative microorganisms, with the lowest positivity rates in meningococcal meningitis [Among 32 patients with meningitis, in 14 cases microbiological documentation was only based on the EP results while cultures remained negative. The EP was of particular interest in cases of antibiotic intake before lumbar puncture . We thus suggest centrifugation should be performed before analysis whenever chemistry and cytological parameters are high.E. coli suggests a limitation of the panel regarding this species, and indicates the need for cautious interpretation of negative results in cases of clinically suspected E. coli meningitis. Over 135 CSF samples analyzed, D’Inzeo et al. did not mention any FN results generated by the EP. They reported only four cases due to microorganisms not included in the panel [E. coli detection, the literature on the FA-ME did not underscore lower PPA; only one study reported one FN result [Furthermore, the low PPA obtained with he panel . Howeverhe panel . RegardiN result .S. pneumoniae (17.5%) followed by S. agalactiae (15.4%). S. pneumoniae FP results were mostly attributed to samples or laboratory contaminations. No mention was made of the amplification curve analysis. The availability of the amplification curves and rates appeared as a significant added value of the Eazyplex® technology and we strongly recommend their systematic analysis, including when internal control fails.Our work also highlighted the need for systematic biological expertise of the amplification curves to avoid false positive and negative results generated by the software analysis. According to a systematic review conducted on the accuracy of the FA-ME panel, 4% (32/807) of the positive results generated by the FA-ME panel were considered FP results after discrepant analysis . The hig® CSF direct M panel. The manufacturer has recently marketed a new version of this test (V1.1), notably including an additional reagent to reduce the inhibitory influence of CSF components. Moreover, the new manufacturer’s instructions recommend confirming positive reactions with another technology when the time to positivity is greater than 25 min. This recommendation would have reduced the FP results generated by the automated software.This work was the largest evaluation of the EP performance to date, and relies on a well-conducted discrepancy analysis, including retrospective assessment of medical files by infectious diseases specialists. However, some limitations should be acknowledged. Firstly, this work was limited by the low percentage of positive results. Secondly, invalid results were excluded from the performance assessment, which could lead to an overestimation of these performances. Thirdly, this work was performed with the first version (V1.0) of the EazyplexCryptococcus neoformans/gattii), the EP detects fewer targets but offers the benefits of a focus on bacterial meningitis diagnosis and a lower price [When compared with the FA-ME panel  confirmed the strong performance of the panel, of particular interest in cases of antibiotic intake before lumbar puncture, (ii) underlined the significant impact of CSF protein concentration and WBC counts in LAMP inhibition, (iii) demonstrated the need for systematic biological expertise of the amplification curves, and (iv) observed the limitation of the panel for


Converging lines of evidence from several models, and post‐mortem human brain tissue studies, support the involvement of the kynurenine pathway (KP) in Huntington's disease (HD) pathogenesis. Quantifying KP metabolites in HD biofluids is desirable, both to study pathobiology and as a potential source of biomarkers to quantify pathway dysfunction and evaluate the biochemical impact of therapeutic interventions targeting its components. In a prospective single‐site controlled cohort study with standardised collection of cerebrospinal fluid (CSF), blood, phenotypic and imaging data, we used high‐performance liquid‐chromatography to measure the levels of KP metabolites—tryptophan, kynurenine, kynurenic acid, 3‐hydroxykynurenine, anthranilic acid and quinolinic acid—in CSF and plasma of 80 participants . We investigated short‐term stability, intergroup differences, associations with clinical and imaging measures and derived sample‐size calculation for future studies. Overall, KP metabolites in CSF and plasma were stable over 6 weeks, displayed no significant group differences and were not associated with clinical or imaging measures. We conclude that the studied metabolites are readily and reliably quantifiable in both biofluids in controls and HD gene expansion carriers. However, we found little evidence to support a substantial derangement of the KP in HD, at least to the extent that it is reflected by the levels of the metabolites in patient‐derived biofluids. Converging lines of evidence from non‐human models support the involvement of the kynurenine pathway in Huntington's disease. However, evidence in living humans is limited. In a prospective study with standardised collection of cerebrospinal fluid and blood, we used high‐performance liquid‐chromatography to analyse this pathway. We concluded that the selected metabolites are readily and reliably quantifiable. Yet, we found little evidence to support a substantial derangement of the kynurenine pathway in Huntington's disease, at least to the extent that it is reflected by the levels of the metabolites in patient‐derived biofluids. 3‐HK, 3‐hydroxykynurenine; KYNA, kynurenic acid; QUIN, quinolinic acid. Inherited in an autosomal dominant manner, the polyglutamine expansion results in the ubiquitous expression of a mutant form of huntingtin protein (mHTT)  is an invariably fatal neurodegenerative disease caused by CAG repeat expansions in the Early animal models used excitotoxins such as quinolinic acid (QUIN), injected into the striatum, to recapitulate specific cellular pathology and phenotypic features of HD  receptors, QUIN is a potent free radical generator  of tryptophan (TRP) degradation Figure  Maddiso, ultimatDrosophila , psychiatric disorders , as well as cancer and autoimmune diseases  are accessible biofluids that offer insights into comparative derangements in the periphery and CNS and are sources of useful biomarkers in HD  prospective cohort of gene expansion carriers and matched controls  . Recruitment figures were based on a priori sample size calculations to detect cross‐sectional differences in CSF mHTT—the primary outcome measure of the HD‐CSF study—between healthy controls and gene expansion carriers, and to investigate longitudinal changes over 2 years, based on effect sizes from previous reports . Prior to undertaking study procedures, all participants gave informed consent which was obtained by clinical staff.2.3HTT CAG repeat count ≥ 36. PreHD participants had CAG ≥ 40 and DCL < 4. Healthy controls were age‐ and gender‐matched to gene expansion carriers, mostly not at‐risk spouses  or gene‐negative siblings of HD gene expansion carriers and with no neurological signs or symptoms. Other inclusion criteria were: 18–75 years of age, inclusive; capacity to provide consent and comply with study procedures; Enroll‐HD study (NCT01574053) participation. Exclusion criteria included: concurrent participation in a clinical trial; alcohol or recreational drug intoxication or abuse; inappropriate or unstable use of medications to treat HD symptoms in the previous 30 days; significant medical, neurological or psychiatric co‐morbidity likely interfere with sample quality or safety of study procedures as assessed by the medical history and physical exam; needle phobia, frequent headaches, significant lower spine deformity or major surgery; antiplatelet or anticoagulant use in the previous 14 days; abnormal full blood count, coagulation parameters or C‐reactive protein >2× the upper limit of normal.All potentially eligible participants were consecutively approached in the National Hospital for Neurology & Neurosurgery Huntington's Disease Multidisciplinary Clinic or via the University College London Huntington's Disease Centre research databases, until the study was fully recruited. Manifest HD participants were defined as adults having a Unified Huntington's Disease Rating Scale (UHDRS) diagnostic confidence level (DCL) of 4 and 2.4https://www.enroll‐hd.org) within the 2 months prior to screening. We employed a calibrated iteration of the composite UHDRS (cUHDRS) . Chromatographic separation was performed using a reversed phase analytical column, configured as per Table KP metabolites were quantified by Charles River Laboratories (the Netherlands) using a high‐performance liquid‐chromatography (HPLC) with tandem mass spectroscopy (MS/MS) detection method (LC‐MC) published previously  to confirm performance over time. Each sample was measured once. The acceptance criteria for CSF assays was ±25% accuracy for the low limits of quantification (LoQ) calibrator and QC‐low, and ±20% accuracy for all other calibrators and QC‐mid and ‐high. For plasma assays, acceptance criteria were 5% wider. Individual runs were accepted when >66% of QC's were within criteria as described above . The LoQ, frequency of samples below LoQ, and frequency of rejected samples due to quality criteria are shown in Table 2.7T1‐weighted MRI data were acquired on a 3T Siemens Prisma scanner using a protocol optimized for this study. Images were acquired using a 3D magnetization‐prepared radio‐frequency pulses and rapid gradient‐echo (MPRAGE) sequence with 2 s repetition time, 2.05 ms echo time, 850 ms inversion time, flip angle of 8 degrees, and matrix size 256 × 240 mm; 256 coronal partitions were collected to cover the entire brain with a slice thickness of 1 mm. Parallel imaging acceleration  was used and 3D distortion correction was applied to all images.2.8All T1‐weighted scans passed visual quality control check for the presence of significant motion or other artefacts before processing. Bias correction was performed using the N3 procedure .t tests, assuming a 5% type 1 error  rate, 20% type 2 error  rate, and adapting effect sizes reported in Heyes et al.,  using 2‐sided Student  et al.,  for KYNA et al., , resulteTo minimise type 1 errors  resulting from multiplicity, we predefined CSF levels of KYNA, 3‐HK and QUIN as primary outcomes, hypothesising that quantifiable differences would be apparent if the KP is dysregulated in HD. The ratios of CSF 3‐HK:KYNA, KYNA:KYN and 3‐HK:KYN were secondary outcomes, hypothesised to reflect the overall balance of the key neuroprotective and neurotoxic metabolites, the activity of the KATs, and the activity of KMO respectively ; and categorical variables as absolute (n) and relative frequencies (%).Measurements below the LoD for the metabolites of interest were assumed as missing—no imputation was used. Analyte distributions were visually assessed and if necessary, arithmetical transformations were applied to meet model assumptions. We provide a graphical depiction of the distributions before and after transformations Figures . Of noteAssociations with age , gender, CAG repeat count (gene expansion carriers only) and haemoglobin (in case of CSF metabolites only) and time in freezer storage were visually assessed for all outcomes Figures . Due to HTT CAG repeat count, models including gene expansion carriers only were run with age and CAG repeat count as covariates. To investigate intergroup differences in outcomes of interest, we applied generalised linear regression models estimated via ordinary least squares. Models were fitted with the variable of interest as the dependent variable, and first with age and group membership, and then with age and CAG and group membership as independent variables. We report results for the omnibus group membership main effect test and relevant contrasts . Other comparisons  were not undertaken as we treat HD as a biological continuum. To investigate alterations unique to later stages of the disease we used clinical measures of disease progression (cUHDRS) and/or cumulative mHTT toxicity (DBS). Tests were not adjusted for multiple comparisons, but the statistical approach was designed to minimise multiplicity within each question of independent interest.As a planned second‐level analysis, to assess associations with measures beyond the known combined effect of age and t tests were performed to inform future studies using the detected effect sizes adjusted for age in primary and secondary outcomes, and assuming a 5% type 1 error, 20% type 2 error (80% power) and equal‐sized groups. No adjustments were made for multiplicity .We used Pearson's partial correlations adjusted for age, and for age and CAG to study associations between outcomes of interest and DBS, clinical and imaging measures in gene expansion carriers. Pearson's correlations were used to study associations between metabolites in CSF and plasma. Bias‐corrected and accelerated bootstrapped 95% confidence intervals (95% CI) were calculated for correlation coefficients. We followed Cohen  to quali2.10Funders had no role in study design, data collection, analysis, or interpretation, or writing of the report. The corresponding author had full access to data and final responsibility for the decision to submit for publication.33.1Eighty participants were recruited: all contributed CSF; 2 (3%) manifest HD were missing plasma. Full cohort characteristics are presented in Table 3.2All three primary outcomes—CSF KYNA, 3‐HK and QUIN—were associated with age in healthy controls, but not with gender, CAG repeat count (gene expansion carriers only), CSF haemoglobin or time in freezer storage Figure . Age‐ anAssociations between age‐adjusted CSF levels of KYNA, 3‐HK or QUIN and DBS, clinical and imaging measures were negligible to weak Figure .Within‐subject short‐term stability was good for CSF KYNA  and QUIN , and moderate for 3‐HK . No differences were found for 3‐HK:KYN.None of the three predefined ratios of interest—CSF 3‐HK:KYNA, KYNA:KYN and 3‐HK:KYN—were associated with age, gender, CAG repeat count, CSF haemoglobin or time in the freezer Figure . Age‐ anAssociations between age‐adjusted CSF ratios and DBS, clinical and imaging measures were negligible to weak for 3‐HK:KYNA, weak for KYNA:KYN, and weak to moderate for 3‐HK:KYN Figure .Within‐subject short‐term stability was good for 3‐HK:KYNA  and 3‐HK:KYN , and weak to moderate for KYNA:KYN .The matched CSF and plasma analytes were associated, with the exception of KYNA Figure . The str4In this comprehensive study of KP metabolites in CSF and plasma in HD, we found that TRP, KYN, KYNA, 3‐HK, AA and QUIN were readily and reliably quantifiable in both biofluids in controls and gene expansion carriers. However, we found little evidence to support a substantial derangement of the KP in HD, at least to the extent that it is reflected by the levels of the metabolites in patient‐derived biofluids.Reports on HD  be one reason why previous studies reported some alterations, albeit inconsistently. Careful examination and adjustment for confounding variables is especially important given that HD is characterised by an extended premanifest period followed by a long period of manifest disease. This tends to make premanifest cohorts younger than those with manifest HD, and makes it very challenging to recruit a control group that matches the HD groups well for age. We previously showed that an apparent increase in CSF TREM2 in HD was in fact an artefact of its relationship with age  and higher KYNA:KYN (shown as a higher log(ratio)), differences that remained significant after adjustment for age and CAG. Note that for outcomes where a transformation includes a reciprocal , an increase in the raw measurement corresponds to a decreased in the transformed value and vice‐versa. This suggests that the activity of KATs may be increased in HD, an increase that appears to grow as the disease progresses. This may reflect a primary effect of the HD mutation on KATs, or compensatory overactivity because of alterations in the central KP. In a post hoc exploratory analysis of KYN:TRP ratio, which reflects the activity of IDO1/2 and TDO enzymes, we did not see group differences or associations with other disease markers.As expected, the CSF and plasma levels of each metabolite were significantly associated, and the plasma levels of each were higher, in line with previous reports in Alzheimer's disease and depression  INHIBITORS, AND USES AND COMPOSITIONS THEREOF’. FBR has provided consultancy services to GLG and F. Hoffmann‐La Roche Ltd. LMR has provided consultancy services to GLG, F. Hoffmann‐La Roche Ltd, Genentech and Annexon. RIS has undertaken consultancy services for Ixico Ltd. EJW reports grants from Medical Research Council (MRC), CHDI Foundation, and F. Hoffmann‐La Roche Ltd during the conduct of the study; personal fees from Hoffman La Roche Ltd, Triplet Therapeutics, PTC Therapeutics, Shire Therapeutics, Wave Life Sciences, Mitoconix, Takeda, Loqus23. All honoraria for these consultancies were paid through the offices of UCL Consultants Ltd., a wholly owned subsidiary of University College London. University College London Hospitals NHS Foundation Trust has received funds as compensation for conducting clinical trials for Ionis Pharmaceuticals, Pfizer and Teva Pharmaceuticals.EJW designed the study with the input of FG. FBR and LMB were involved in participant recruitment. Eligibility, clinical examinations and sample collection were performed by FBR, LMB, and RT. Imaging assessments were conceived RIS, EBJ and EDV, data were acquired by EDV, MA, EBJ, and processed by RIS and EBJ. MM and GF processed and analysed the patient samples. FBR developed and performed the statistical analysis; FBR, AJL and EJW interpreted the data and wrote the manuscript; and all authors contributed to reviewing the manuscript.Supplementary MaterialClick here for additional data file.


A large share of the primary energy is consumed to provide space heating. Geothermal energy offers a regenerative alternative. For reasons of efficiency and environmental protection, it is important to ensure the system integrity of a borehole heat exchanger (BHE). Previous investigations have focused on the individual components of the BHE or on the grout and pipe systems’ integrity. This study focused on the analysis of the hydraulic system integrity of the complete subsoil–grout–pipe system as well as possible thermally induced changes. For this purpose, a pilot-scale experiment was built to test a 1-m section of a typical BHE under in situ pressure, hydraulic and temperature conditions. During the tests the hydraulic system permeability of the soil and the BHE was measured continuously and separately from each other. In addition, the temperature monitoring array was installed in a 50-cm cross-sectional area. Significant temperature-related fluctuations in the sealing performance could be observed. Hydraulic conductivity limits required by VDI 4640-2  were exceeded without frost action. The succeeding application of freeze–thaw cycles further enhances the system permeability. The study shows that the thermally induced effects on the system integrity of the BHE are larger and more significant than the subsequent frost-induced effects. The hydrophobic character of the high-density polyethylene (PE-HD) pipes as well as its high coefficient of thermal expansion seem to be the main points of weakness in the system. Optimization research should focus on the interface connection between grout and pipe, whereby hydrophilic pipe materials such as stainless steel or aluminum should also be considered as well as manipulation of the pipe surface properties of PE-HD. Typically, drilling depths for such systems range from 70 to 200 m. These so-called shallow BHEs are in the focus of this study  on a complete BHE system including the surrounding soil have not been investigated experimentally to date. This study presents a new experiment that applies FTC on an in situ BHE specimen at pilot scale  under realistic soil mechanical and hydrogeological conditions. The sampling and monitoring design of this newly developed triaxial vessel is able to research integrity changes for individual construction components as well as the hydraulic performance of the whole system. The inclusion of the surrounding soil as well as the possibility to determine individual flows within the sample are a novelty to previous investigations of a BHE system.Investigations of BHE system integrity and freeze–thaw cycles present a complex issue. The development of a pilot-scale test on this topic must take into account the implementation of theoretical approaches, guideline requirements, and existing small-scale laboratory tests. These fundamentals are reviewed in more detail in the following sections.According to the German guideline VDI 4640-2 , BHE sysThe formation of ice in porous media such as grouting material or soil is a complex process resulting from coupled heat and mass transfer of the porous media’s solid and fluid components  of the main construction components of the BHE and the surrounding soil.In order to achieve the highest possible validation of the experiment, the following requirements for the design of the experiment were formulated in advance:These specifications led to the development of the experimental setup described below.The central device of the pilot-scale experiment Fig.  is a seaA latex sleeve between the tank wall and the specimen body allows to apply radial earth pressure. Furthermore, the sleeve seals off the soil sample to avoid interflow between specimen and vessel wall. At the outside of the steel wall, a spiral copper pipe heat exchanger is installed and connected to a thermostat. The tempering of the steel wall as outer boundary condition is handled with four separated counterflow circuits. The inlet and return flow of the BHE are connected to a high-performance thermostat each, so that the asymmetric heat extraction can be carried out under field-like conditions. The thermostat´s regulation range is from − 20 to 40 °C. Experimental operation took place between − 5 and 12 °C. This range can be regarded as realistic for typical BHE operation on site.Øout 150 mm) was centered as a standpipe in the vessel first. The previously selected soil was filled in the annulus around the standpipe and compacted at proctor water content to achieve a reasonable consolidation state. As especially in aquitard horizons, vertical hydraulic integrity is essential for the permissible operation of BHE, a loamy soil consisting of silt, clay and fine sand  with low hydraulic conductivity was selected to represent these conditions. Four BHE pipes of PE-HD (Øout 32 mm) were placed inside the standpipe. This represents the most common double U-pipe BHE configuration in Germany  and through the grout (QBHE) can be determined independently .Three cylinders using compressed air were used to apply the hydraulic gradient to the sample. They are equipped with a displacement sensor on the stamp so that the piston position can be recorded and logged. In this study, the hydraulic gradient was build up by using the back-pressure method  is calculated with Eq. αT corrects the temperature-related variation of water viscosity according to ASTM D-5084-16a (Ksys is the hydraulic conductivity of the system (m s−1); L is the sample length (m); ∆Vsoil is the measured water flow volume in the soil fraction (m3); ∆VBHE is the measured water flow volume in the BHE fraction (m3); αT is the temperature correction factor for water viscosity (–); ∆hw is the height difference of the water column between outlet and inlet (mwh); ∆t is the time (s); Asoil is the sample area of the soil fraction (m2); ABHE is the sample area of the BHE fraction (m2).An important functionality of the pilot-scale experiment is that the individual shares of her Eqs.  and 3. T5084-16a :4\documeThe accuracy of the hydraulic conductivities determined with the tank experiment was calculated according to the Gaussian error propagation based on the individual uncertainties of the respective input variables. Table KBHE) have the most significant uncertainties in this experiment. However, this has only a minor impact on the overall system hydraulic conductivity Ksys.In particular, the uncertainty of the water volume measurement has a major impact on the accuracy of the hydraulic conductivity results. Thus, maximum deviations apply especially to measurements of low hydraulic conductivities due to the lower volumetric water flow. Figure Type T thermocouples  were installed Fig.  in the spin 200 kPa, pout 140 kPa, cell pressure 250 kPa) was applied and a water flow was initiated to saturate the specimen. Within the next 30 days, stationary flow conditions were established and full saturation conditions achieved. From day 87 on, the actual investigations presented in this study started.Before the actual measurements started, a stationary hydraulic regime of sample hardening and water saturation under constant physical conditions had been established for at least 86 days. The first 56 days of that time span, the grout was allowed to cure and simultaneously the specimen was watered gravitationally using demineralized and de-aired water according to ASTM D-5084-16a  and DIN Prior to the start of the first FTC measurements with a freezing of the specimen, the effect of temperature changes on the hydraulic integrity of the system above 0 °C were investigated. Thus, effects could be detected and quantified under ice-free operation. Differences of the coefficient of thermal expansion of the BHE pipe and grouting material in combination with temperature changes caused mechanical movements at the grout/pipe interface triggering proposed effects onto the hydraulic system conductivity Allan . Thus, bKi value for each FTC.To investigate the influence of FTC on the hydraulic system integrity of BHE, a sequence of FTC then was applied in the pilot-scale experiment. Such a sequence is defined by a row of six single FTC in accordance to VDI 4640-2 . The resKBHE for the different temperature levels. It is obvious that the hydraulic system conductivity of the specimen in the pilot experiment is strongly dependent on the overall temperature level.First of all, investigations were conducted to detect experimentally the influence of temperature changes on the hydraulic integrity of BHE under non-freezing conditions. Figure –10 m s−1, which is in the range of the expected magnitude and therefore in good agreement with the results for the initial state (black) from the norm experiments (E). When cooling the vessel (black marks) to 10 °C, an initial significant increase of KBHE up to 2 × 10−8 m s−1 was observed. Ongoing cooling of the BHE pursued this trend and increased the system conductivity linear on the logarithmic scale up to (B) 2 × 10–7 m s−1 at 3 °C. The subsequent heating of the experiment in steps from 3 °C up to 12 °C confirmed this trend in reversed order. An exponential behavior of the hydraulic system conductivity depending on the overall temperature level was confirmed and resulted in KBHE (C) ~ 2.5 × 10–8 m s−1 at 12 °C at least. However, a difference between the hydraulic system conductivity after the thermal loading and the initial hydraulic system conductivity of the BHE remained and resulted in an irreversible hysteresis of 1.5 magnitudes.The initial hydraulic conductivity (A) at 12 °C was 7.7 × 10–8 m s−1 at 12 °C . The results fit perfectly into the extrapolated course of the retrograde path of the pilot-scale experiment without FTC exposure. Consequently, the thermal conditions are expected to have a significant influence on the measured results for the Anbergen experiment as well.Simultaneously, an experiment according to Anbergen et al.  and VDI To double-check these results an additional experiment with the same pipe product but deviating grouting material was performed. For this purpose, three samples were cured for 56 days at a temperature of 10 °C according to VDI 4640-2 . The genConsidering the presented results, it is quite obvious that the annular space between PE-pipe and grout varies regularly as a result of differential thermal contraction and expansion of both materials. Thus, an increase of the system hydraulic conductivity by just regular operation of the BHE and without of any application of freeze–thaw stress seems to be very likely.3), because in this FTC the main features are particularly prominent.To investigate the influence of freezing and thawing on the hydraulic integrity as well as the temperature distribution of a complete BHE system, consisting of heat exchanger pipes, grout and surrounding subsoil, the pilot experiment was subjected to six FTC. Although the individual test procedures are identical for the different FTC, the observed characteristics are not equally pronounced in each test. Thus, the results are presented and discussed exemplarily using the third FTC  and caused a minor increase of KBHE to approximately 7 × 10–7 m s−1. After 5 h of cooling, the freezing of the specimen begun, which can be recognized by the temperature in sensor Skin zone S and N falling below 0 °C. After 20 h, ice formation initiates a reduction of the KBHE, which decreases by about three orders of magnitude in the following hours.FTCKBHE value began to rise again, approaching almost its initial state at the end of the FTC (after about 48 h). In contrast to KBHE, the soil hydraulic conductivity (KSOIL) only underwent minor changes over almost the entire pilot-scale experiment, with one exception: during the thawing of the sample column at around 42 h a moderate increase in KSOIL was observed, very likely due to an opening of ice-clogged flow paths. The course of the hydraulic system conductivity (KSYS) indicates clearly that the changes to be observed depend solely on increase or decrease of KSOIL. This is no surprise, since in terms of the relevant water flow areas, the proportion of ASOIL is significantly larger than that of ABHE.When the thawing process was initiated after 40 h, the KBHE. A plausible explanation is that a lower heat transfer resistance between pipe and grout might exist, because the thermal conductivity of ice is four times higher than that of water. Furthermore, the convective heat transfer by water transport through the pores might be blocked by ice.The first temperature decrease at the beginning of the freezing operation ends after 8 to 10 h. From this point on, a plateau phase was observed for approx. 12 h. Afterwards (beginning after 20 h), the temperature further decreases simultaneously with the reduction in After a test duration of approx. 30 h, a sudden rise in temperature occurred in the skin zone, which was also detected in sensors 1 to 3 in an attenuated and delayed manner. This phenomenon is interpreted herein as a latent heat transfer effect. Due to the strong nature of the effect in the area of the skin zone, it is assumed that a certain amount of water was frozen in this area, which consequently suggests that there were distinct flow paths in this area. However, such a sudden temperature rise cannot be observed in the direction of the BHE return flow Fig. B. Here tAs mentioned above, while freezing, a radial attenuation of the temperature rise induced by latent heat effects is obvious. In contrast, when the active cooling was stopped after 40 h, a sudden temperature drop was detected by all temperature sensors. This behavior may not be explained with latent heat effects. It is assumed that an unintended influence on the cold junction of the thermocouples occurred due to a decrease in the laboratory temperature of 3 K after switching off the active cooling. After this sudden temperature drop, a gradual temperature rise was established.While on the return side Fig. B the temDuring further warming, the skin zone temperature shows a steeper rise than the temperatures in the soil. This is another indication for an increased permeability in the skin zone, which leads to an increased convective heat transfer with the water fed into the specimen at a temperature of 20 °C that acts as a thermal tracer. These results indicate that the increased permeability in the skin zone region is highly relevant to system considerations.Comparing the values for the hydraulic conductivity of the soil and the BHE measured after various FTCs Table , only miThe increase in soil conductivity during cooling from 12 to 3 °C was caused by the loss of flexibility of the latex cover as the temperature had decreased. As a result, a circumferential flow developed on the outside of the sample, which could be proven afterwards with uranine markings.After finalizing the thermal experiments, a highly concentrated uranine solution was injected into the inlet of the sample flow to serve as a fluorescent marker for a qualitative assessment of the experiment. Under black light, preferred flow paths of the uranine tracer fluoresces can be visualized Fig. .Fig. 11PSpecial attention should be paid to the skin zone, where uranine penetrated approx. 15–20 mm into the soil body, radially. This indicates that the soil in the vicinity of the BHE had a considerably increased hydraulic conductivity. The annular volume, which is affected by uranine impregnation, is congruent with the depth of the freezing front penetration as measured during the FTC tests. Consequently, freezing effects have an unfavorable influence on the hydraulic conductivity, not only on the BHE itself, but also on the surrounding natural subsurface.Furthermore, the uranine method highlighted additional preferential flow paths in the grouted backfill of the BHE Fig. : residueIf the observed increases in permeability in the skin zone gained a penetrative character, brittle material failure (cracks) caused by freeze–thaw effects were detected as a disintegration of the grout body. In particular, these effects occur between the supply and return pipes of the BHE, where the mechanical stresses are considered at a maximum due to the high temperature differences. Moreover, the effects of tensile stress were observed on the largest section of the specimen height, but the intensity was variable.In conclusion, the experimental data, especially the hydraulic investigations, were confirmed by the qualitative visual observations using the uranine tracer.Like with any laboratory experiment, the one presented in this study was subject to simplifications and uncertainties, which impair the transferability of results.The test procedure itself represented a major simplification of reality. It was characterized by defined boundary conditions, such as the starting temperature and the temperatures for heat extraction under cyclic loads. In reality, FTC are subject to natural fluctuations. However, reproducing natural fluctuations in such an experiment would cause severe noise, which would obscure important findings and render a reproduction of the results as impossible.T of 6 K in the presented experiment was rather gentle compared to the Anbergen experiment (ΔT = 30 K). Moreover, it is expected that the freezing process was still slowed down, enough to produce comparable freeze effects.Another point of discussion is the sudden cooling at the beginning of the frost phase. In a real BHE operation, the cooling proceeds more moderately. Nevertheless, the maximum ΔFurthermore, the active control of the vessel temperature with constant 3 °C did not necessarily correspond to the transient temperature behavior around a real BHE. This temperature is also based on previous modeling of a 100-m BHE and corresponds to the temperature distribution in a realistic reference scenario after 10 years of operation. The soil temperature was determined at peak loads within a radius of approx. 25 cm from the center of the BHE. This surely had an effect on the spread of the frost front because of an unnaturally large heat input over the vessel wall. Consequently, it is feasible that the propagation of the frost front was underestimated in this experiment. Nevertheless, an external tempering was necessary to ensure transferable conditions according to reality during the FTC and thawing from the outside to the inside. Moreover, assuming steady-state conditions and taking the soil thermal conductivity into account, the temperature gradient from the vessel wall to BHE induced a radial heat flow of approximately 40 to 50 W/m. This is very well within the range of a real BHE in peak load operation.The hydraulic conditions for the test were determined according to DIN EN ISO 17892-11  as well Another limitation of the experiment relates to the pressure conditions. While a real BHE extends over several tens of meters and thus a wide range of pressure conditions, the experiment only represents a certain depth section. Of course, it would be of utmost interest to conduct multiple experiments with different boundary conditions representing different depth sections, however, the time-consuming preparation and performance of the experiment are defining operational and financial limits to the execution.The test liquid, which perfused the specimen, was kept at laboratory temperature (20 °C) in the hydraulic cylinders. This in turn meant an unnaturally high heat input into the specimen, which resulted in an underestimation of the frost front propagation. However, the comparatively high temperatures of the test liquid rendered it to be a thermal tracer. In conjunction with the sensor distribution, it provided valuable information about internal water paths in the sample.–8 m s−1. In contrast, the initial measurement in the pilot plant at 12 °C was 2 × 10–8 m s−1. Moreover, the conductivity increased to 6 × 10–8 m s−1 at a temperature of 3 °C. This is attributed to the fact that the latex sleeve was too inflexible and had, especially at lower temperatures, a limited sealing function. Nevertheless, the spread of the uranine marker on the outside of the specimen was clearly subordinate in intensity compared to the marker in the skin zone. Thus, it can be expected that the results of the experiment are valid.When the sample was removed, uranine tracer was visually detected on the lateral surface of the sample. This proved the previous assumption that there had been a circumferential flow on the outside of the sample. The hydraulic conductivity of the soil itself was determined in a laboratory experiment to 1 × 10As mentioned before, one major drawback of such an elaborate experiment is its extremely time-consuming nature. One experiment requires at least half a year excluding the time needed for planning, manufacturing and construction of the experiment’s infrastructure. It requires dimensions and a cost like a field survey. Nevertheless, it is planned to repeat the experiment with further configurations of deviating heat exchanger pipe and grout materials.The paper presents a novel pilot-scale testing system to investigate the hydraulic integrity of a borehole heat exchanger under in situ conditions. For the first time, not only the borehole heat exchanger pipes and the backfill material, but also the skin zone and the surrounding unconsolidated rock are included in the consideration.The experiments carried out within this study focused on the investigation of thermal effects on the hydraulic system permeability of a BHE installed in a low-permeability soil. Two different operational states were regarded.In a first experimental phase, the behavior of the system in a regular operation with fluid temperatures above the freezing point of water was investigated. An important finding is that already this regular non-freezing operation of a BHE within temperatures between 0 and 12 °C can lead to a perceptible increase of the system’s vertical hydraulic conductivity. This is attributed to a relatively high thermal expansion factor of the PE-HD pipes compared to the cured grout body. A certain portion of this hydraulic conductivity gain is irreversible and has been experimentally identified as permeability hysteresis.In a second experimental phase, an operation with cyclic loading and regular fluid temperature drops below − 3 °C inducing freezing and thawing processes in the vicinity of the BHE was experimentally reproduced. This operation led to further significant permeability increases, which are attributed to impairments caused by the formation of ice in the contact zone between pipe and grout, and the pores of the grout.More importantly, zone-related permeability measurements showed that the FTC-induced permeability increase is not limited to the BHE body itself, but also extends into the soil body. Subsequent uranine marking of the flow paths revealed that this increase was basically concentrated in the skin zone.When the permeability measurements of the BHE are conducted at temperatures just above the freezing point, the main flow still takes place on the thermal expansion-induced annular gap. Consequently, the FTC-induced impairment is only measurable at increasing temperatures, when the main flow gap has been closed again by the pipe expansion.The standard test according to the VDI 4640-2  is conduAn important disadvantage of the standard test is that the permeability measurements are usually carried out at laboratory temperature. Such a practice does not reflect the permeability increase caused by the thermal expansion of the materials as described above. Consequently, it underestimates the system’s hydraulic conductivity at in situ temperature conditions. Therefore, it is highly recommended to adapt the standard test procedure and prescribe more realistic temperature conditions.Comparing the results gained in the pilot-scale experiment to results of the standard test for hydraulic system integrity after FTC according to the VDI 4640-2  guidelinThe results reveal the disadvantages of combining a rigid backfill and a “flexible” BHE pipe material. The standard grouting materials for geothermal applications usually are a mixture of cement, sand and bentonite. They shall combine the highest possible compressive strength with a minimal hydraulic and enhanced thermal conductivity. In contradiction the backfill has to be resistant against temperature fluctuations and freezing–thawing behavior. Additionally, a long-term plasticity of the backfill would be desirable. Grouting products which combine the aforementioned properties with plasticity maintained over the long term are not presented yet. Such a development can be a possible future research task.An additional scientific challenge may be the improvement of the system integrity by improving the grout–pipe interface. One track in this direction can be the reduction of the hydrophobicity of the BHE pipes. Another track can be the structural change of the pipe’s surface to extend the flow paths. A third research track could investigate the material-specific thermal expansion coefficients. Also, using aluminum or stainless-steel pipes are of a certain interest because of high thermal conductivities and less hydrophobic properties. However, this would put the corrosion issue on the future research agenda and also raise cost issues.


Method and Materials. This study was a retrospective investigation, and patients treated with screw-retained monolithic or partial veneer FPDs on dental implants were selected. Periapical radiographs were taken at baseline and at the 3-year follow-up. Complications were recorded and classified as technical and biological ones.  The introduction of CAD/CAM and the development of zirconia-based restorations have allowed clinicians to use less expensive materials and faster manufacturing procedures. The purpose of the study was to analyze the differences, in terms of mechanical and biological complication, in multiunit zirconia fixed dental prosthesis (FPDs) on posterior implants produced using a digital workflow.  The study population included 25 patients. The occlusal and interproximal corrections were not clinically significant. In the study sample, the survival rate and success rate of the FPDs after 3 years were 100% and 96%, respectively. One implant failed immediately after placement.  Monolithic zirconia FPDs and partial veneer FPDs showed a 100% survival rate, presenting an interesting alternative to metal ceramic restorations. The partial veneer FPDs had a higher technical complication rate than the monolithic FPDs; however, no statistically significant difference was found. Over the years, a variety of new digital technologies and restorative materials for implant-supported prostheses have been developed . Metal–cThe introduction of computer-aided design/manufacturing (CAD/CAM) allowed clinicians to use cheaper materials and faster manufacturing procedures, increasing the efficiency of the prosthetic treatments . The devAccordingly, zirconia-based restorations have become a popular choice in implant dentistry thanks to its mechanical properties such as high flexural strength and the unique crack-inhibitory material properties of yttria-stabilized tetragonal zirconium dioxide polycrystals (3Y-TZP) , 21, as However, conventional 3Y-TZP zirconia is too opaque for monolithic restorations because of its high internal light scattering , 23. TheRecently, changes in composition, structure, and production methods have led to the development of more translucent types of monolithic zirconia, reducing the need for veneering and ensuring higher biocompatibility, higher esthetics, and better biomechanical properties than traditional materials . Thus, iTherefore, the purpose of this retrospective clinical study was to analyze the clinical outcomes of multiunit zirconia FPDs on posterior implants produced using a digital workflow.A total of 25 patients who received screw-retained monolithic or partial veneer FPDs on dental implants between January 1, 2016, and January 1, 2017, were selected for this cross-sectional study.All procedures took place at a private dental practice in Ascoli Piceno, Italy. All procedures were performed according to the Declaration of Helsinki guidelines on experimentation involving human subjects. Each participant enrolled in the study received explanations on the study design and objectives and provided written informed consent.Due to the retrospective nature of this study, it was granted an exemption in writing by the local ethics committee.Patients treated with an implant-supported FPD to rehabilitate a Kennedy Class IIAge ≥ 18 yearsType 3 implant placement Presence of antagonist's teeth Sufficient mesiodistal and interocclusal spaceAt least 3 years of follow-upPatients satisfying the following inclusion criteria were selected for the study:Untreated periodontitisNo residual keratinized tissue at experimental areaDiagnosis of temporomandibular joint disorders assessed using the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) Systemic diseases that could hamper normal healing processesSmokingExcessive alcohol consumptionAn American Society of Anesthesiologists physical status classification of ≥IIIThe exclusion criteria were as follows:From an initial sample of 32 eligible patients, 7 patients did not complete the three-year follow-up; therefore, 25 patients constituted the study sample.The decision as to which of the two protocols to perform was made after a discussion with the patient, explaining the advantages and disadvantages as well as the cost of each treatment.Patients' charts were obtained, and data  were extracted.Prosthesis delivery was considered as baseline. All rehabilitations were carried out by the same trained clinician and dental technician.A complete periodontal examination and a preoperative CBCT were performed to complete the surgical and prosthetic planning. The presence of bruxism was also assessed preoperatively following the bruxism's international consensus definition .Implant fixtures  were placed under local anesthesia, according to the manufacturer's guidelines. Preoperative prosthetic digital planning was performed for all cases, and when indicated, a guided surgery approach was used to facilitate the positioning of the access holes in the preferred areas Figures –3. After2, while interproximal and occlusal contact tightness at 0.10 mm.The dental technician used a software program  to design and fabricate each FPD. During the FPD design and the digital modelling, a minimum of 0.5 mm thickness for the monolithic zirconia was observed in all areas; also, the minimum connector dimension was set at 12 mmFull-contoured prostheses were made of acrylic resin and tried in intraorally.The adopted CAD/CAM monolithic zirconia  is a material that presents higher flexural strength in the cervical region (1200 MPa), where more mechanical strength is needed, and lower flexural strength (600 MPa) in the incisal region, where more translucency is preferred. Zirconia was milled , and then, monolithic restorations were infiltrated with the brush infiltration technique . The infiltrated FPDs were dried thoroughly before sintering.Partial veneer zirconia restorations were designed digitally with a cut-back procedure of 0.4 mm to provide adequate space for the veneering in the buccal surfaces of the zirconia frameworks. Anatomic reduction was performed only in the regions without occlusal contacts. The FPDs were manually veneered using ceramic veneer  and finally stained and glazed.All FPDs were bonded to the metal substructures with an adhesive luting composite resin  following the manufacturer's guidelines to obtain one-piece hybrid cement-screw-retained FPDs. The passive fit of the FPDs was checked intraorally using the Sheffield test. Interproximal contact tightness was evaluated intraorally by assessing the resistance to the passage of dental floss . Occlusion was evaluated with shimstock foil.When required, adjustments were performed using ceramic-specific diamond rotary instruments while being water-cooled and then polished . All FPDs were inserted and screwed in with a manual torque control ratchet at 35 Ncm. The retorque application after 10 minutes was used to increase the screw loosening torque. The screw access holes were closed using polytetrafluoroethylene tape and composite resin Figures  and 5.μ articulating paper using a two-tone representation of static and dynamic occlusion  [The interproximal contact tightness was evaluated at delivery by assessing the resistance to the passage of dental floss , and the occlusion was assessed with shimstock foil. During prosthetic placement, any interproximal or occlusal surface adjustment was recorded with a score ranging from 1 to 3. The occlusal surface adjustment was based on the analysis of the area after the occlusal registration with a 40 equired) .The interproximal contact adjustment was based on the resistance to the passage of dental floss .The esthetic of the restoration was then analyzed with the white esthetic score (WES) .The patients were visited as part of their standard follow-up after 1 week and every 6 months from the prosthesis delivery. At these appointments, if deemed necessary, an ultrasonic supragingival debridement procedure was undertaken.Bleeding On Probing (BOP). At six sites  of each implantProbing Pocket Depths (PPD). At six sites  of each implantDuring the 1-, 2-, and 3-year follow-ups, the following peri-implant parameters were measured with a UNC-15 periodontal probe:Peri-implant parameters were recorded by a single clinician (P.D.A.).Periapical radiographs were taken with the long cone parallel technique at baseline (prosthesis delivery) and at the 3-year follow-up. A silicone bite was placed in the holding system, allowing precise repositioning during each follow-up visit. The magnification factor was measured on each radiograph by dividing the known diameter of the implant with the diameter measured on the radiograph. Linear measurements (mm) on the digital images were performed to record the distances of the most coronal points in the mesial and distal ridge aspects from the implant shoulder and were then adjusted by the magnification factor.Also, at the 3-year follow-up, the full-mouth plaque score (FMPS) and the full-mouth bleeding score (FMBS) were measured.The survival and success rates of the prosthesis were assessed; the survival indicated whether the FPDs were physically in the mouth, while the success was defined as the absence of any complications. Complications were classified as mechanical/technical and biological ones.Fracture of an occlusal screwLoosening of an occlusal screwLoosening of an abutmentFracture of an abutmentFracture of an implantMechanical complications included the following:Loss of retentionFracture and/or chipping of ceramicFracture of the frameworkTechnical complications included the following:The technical complications were also classified as major, medium, and minor complications, as proposed by Lang et al. . The majU Test. All analyses were conducted using Stata . Two-tailed probabilities are reported, and nominal statistical significance was defined using an observed significance level of 0.05.The descriptive statistics used for the continuous factors included the mean, standard deviation (SD), and median; for categorical factors, absolute and relative frequencies (%) were utilized. The technical complication rate according to the presence of bruxism, type of opposing dentition, and the number of implants was analyzed with Fisher's exact test. The WES between monolithic and veneered restoration was compared with the Mann–Whitney The study sample consisted of 25 patients . All participants received implant-supported FPD restoration; 14 FPDs were located in the upper jaw while 11 in the lower jaw. Eleven FPDs were porcelain-veneered (44%) while 14 FPDs were monolithic (56%). Eighteen FPDs were supported by two implants (72%) while seven FPDs were supported by three implants (28%). Thirteen FPDs (52%) had opposing natural dentitions while 12 (48%) had tooth- or implant-supported fixed prostheses. Two patients treated with veneered FPDs (18%) and four patients treated with monolithic restorations (29%) were affected by bruxism. The occlusal and interproximal corrections at prosthesis delivery were not clinically significant, with a mean of 1.2 ± 0.5 for occlusal adjustments and a mean of 1.1 ± 0.3 for interproximal adjustments. Only two FPDs required mild interproximal corrections (8%), while three FPDs (12%) required occlusal corrections (two cases of mild corrections and one case of significant correction). At the end of the follow-up period, all FPDs were in use, and none had a major technical complication causing restoration failure. After 3 years of clinical service, the survival rate of the FPDs in the study sample was 100%. The only technical recorded for the FPDs was a case of ceramic chipping on the buccal cusp of the inferior first premolar (4%), in a patient affected by bruxism which occurred 31 months after prosthesis delivery in a partially veneered restoration, with natural dentition as the antagonist . This wap = 0.240), the presence of a partial veneer (p = 0.440), the type of opposing dentition  (p = 1), and the number of implants (2 or 3) (p = 0.280) had no statistically significant effect on the technical complication rate of the FPDs.Bruxism ; however, the patients treated using monolithic zirconia had a clinically successful value in all cases treated.A mean overall white esthetic score of 8.4 ± 0.9 was recorded, 8.8 ± 0.7 for the partial veneer FPDs, and 8.1 ± 0.9 for the monolithic FPDs. A higher mean esthetic score was recorded in the partial veneer FPDs, and there was a statistically significant difference , while three FPDs (12%) required occlusal corrections (two cases of mild corrections and one case of moderate correction).Despite the promising results of implant-supported zirconia prostheses, the sample size of the present study is small; there was no control group, and follow-up was short-term. Therefore, long-term prospective clinical investigations with a larger population are still necessary for determining the clinical reliability and viability of CAD/CAM technology.Within the limitations of the present study, monolithic zirconia FPDs and partial veneer FPDs showed a 100% survival rate after 3 years of follow-up, indicating that they are a promising alternative to metal–ceramic restorations. The partial veneer FPDs had a higher technical complication rate than monolithic FPDs; however, there was no statistically significant difference. Monolithic restorations, given their esthetic capabilities and lower rate of complication, are the most appropriate choice for a posterior restoration. Further clinical medium- and long-term outcomes are required to validate this choice of treatment.


Tests were completed using a calibrated direct drive indoor trainer for the standing, seated, and vibration GXTs, and a friction belt cycle ergometer for the high-workload step protocol. Power output (PO) and cadence were collected from three different brand, new pedal units against the gold-standard SRM crankset. The three units of the Assioma Favero exhibited very high within-test reliability and an extremely high agreement between 100 and 250 W, compared to the gold standard . Greater PO produced a significant underestimating trend , with pedals showing systematically lower PO than SRM (1–3%) but producing low bias for all GXT tests and conditions (1.5–7.4 W). Furthermore, vibrations ≥ 30 Hz significantly increased the differences up to 4% , whereas peak and mean PO differed importantly between devices during the sprints . These results demonstrate that the Assioma Favero power meter pedals provide trustworthy PO readings from 100 to 650 W, in either seated or standing positions, with vibrations between 20 and 40 Hz at cadences of 70, 85, and 100 rpm, or even at a free chosen cadence.This study aimed to examine the validity and reliability of the recently developed Assioma Favero pedals under laboratory cycling conditions. In total, 12 well-trained male cyclists and triathletes (VO The use of power meters in cycling has been on the rise in recent years, making accessible, valuable information for training, that was only available with impractical and expensive ergometers ,2. PortaThe development of wearable power meters with micro-sensors attached to the bicycle crank, pedals or wheel, constitutes a milestone for cycling, giving rise to the creation of new devices, which can track cyclists’ performance in real settings. The first approach was the SRM  crankset (strain gauges), which remains as the Gold-Standard to measure the bicycle power output (PO) outside the laboratory ,11,12. SIn practice, the main goal of tracking PO is to quantify the real effort incurred during training or competition, and also to determine changes in performance throughout the season . For thiTherefore, considering the practical advantages that the pedals power meter would provide to the PO prescription and monitoring, as well as the need to comprehensively analyze the suitability of this type of technologies to be used on the daily basis, this study aimed to examine the validity and reliability of the recently developed Assioma Favero pedals under laboratory cycling conditions.This study followed a repeated measures design to determine the validity and test–retest reliability of three units of the new power meter pedals Assioma Favero against the gold-standard SRM crankset. After a familiarization session, each participant completed the following cycling tests: three counterbalanced, graded exercises tests (GXT) at different cadences  and sub-maximal workloads  in a seated position, three GXT at four sub-maximal workloads  in a standing position, and a ramp vibration protocol (from 20 to 40 Hz) at constant workload . Finally, all cyclists performed a high-workload step protocol , as well as an 8-s maximal sprint test.2max = 65.7 ± 8.7 mL·kg−1·min−1 [A total of 12 well-trained male cyclists and triathletes volunteered to take part in this study. . All suAll tests were performed in the same facilities under standardized conditions . For the seated and standing GXTs, as well as the vibration tests, the SRM 172.5 mm crank power meter was fixed on a medium-size road bicycle . The rear wheel of the bicycle was removed and attached to a calibrated Cycleops Hammer  device wSubjects visited the laboratory on four separate occasions to test the three Assioma Favero power meter pedals. All tests began with a standardized warm-up of 5 min at 75 W with a free chosen cadence and the Hammer set in the hyperbolic mode. Thereafter, subjects performed three randomized and counterbalanced 1-min GXT in seated position, one for each selected fixed cadence , at six sub-maximal workloads , separated by 4 min of recovery at 75 W with free chosen cadence  Figure . The ordRecords for PO (W) and cadence (rpm) were collected at 1 Hz using a Garmin 520 cycling computer for the Assioma Favero pedals and the Power Control VIII  for the SRM crankset. Data for GXT and vibration tests included the 15th to the 55th s of each 60 s steps, to allow the ergometer enough time to stabilize the assigned breaking load . Similarp ≤ 0.05. Analyses were performed using GraphPad Prism 6.0 , SPSS software version 19.0 , and Microsoft Excel 2016 .Standard statistical methods were used for the calculation of means, standard deviations (SD), coefficient of variation (CV), and standard error of measurement (SEM) . Intraclp < 0.05, ES > 0.22), with Favero showing from 1 to 3% lower PO than SRM consistently. In turn, all devices showed similar PO during [p < 0.05, ES > 0.24). Peak and mean PO differed importantly between devices during the sprints . Bland–Altman plots  comparable to the SRM (CV differences < 2%), and high ICC (from 0.741 to 0.999). SRM crankset and the three Favero Assioma showed similar PO in most conditions , with exO during , the GXTan plots  confirmeThe results of this study indicate that the Assioma Favero Pedals are a highly suitable tool for monitoring cycling performance in a wide range of workloads (100 to 650 W) and cadences , different pedaling positions (seated and standing), and under vibration stress . Importantly, the pedals slightly underestimated the PO compared with SRM readings, but errors are low enough to be handled in practice. To the best of our knowledge, this is the first study examining the validity and reliability of the recently commercialized Assioma Favero pedals. Stemming from this comprehensive research, coaches and researchers may be confident in using these portable power meters for cycling training and testing and benefit from their practical advantages. The SRM crankset constitutes the best alternative available to laboratory cycle ergometers, with extremely low variability  . AccordiAn important contribution of the present study is that we examined a large variety of testing conditions, allowing us to conclude the effects of three big cycling concerns: pedaling positions, vibration, and extremely high loads. Whereas previous studies have included some of these conditions ,17,21,34The fact that the Assioma Favero pedals produce errors of ~2% compared to the SRM suggests that they are sufficiently accurate to track performance changes over time . This reThis study confirms that the new Assioma Favero pedals are valid and reliable mobile power meters to measure PO in cyclists. This portable power meter provides an alternative to more expensive laboratory ergometers while allowing cyclists to use their bicycles for testing, training, or competition purposes. The results demonstrate that the Assioma Favero power meter pedals provide trustworthy PO readings from 100 to 650 W, in either seated or standing positions, with vibrations between 20 and 40 Hz at cadences of 70, 85, and 100 rpm, or even at a free chosen cadence. Of note, pedals consistently underestimated the SRM readings by up to 4%, with differences depending on the cycling condition.