The syntheses of new, fourfold alkynylated tetraazaacenoacenes  are reported. This novel heteroacenoacene motif exhibits surprisingly strong electronic coupling between its constituting diazaacene units. Two are better than one: Soluble, alkynylated azaacenoacenes were synthesized through Buchwald–Hartwig coupling of ortho‐diamines with bistriflated/nonaflated dibromonaphthalenes. The two azaacene units of this long‐forgotten bisacene motif exhibited surprisingly strong electronic coupling. Cata‐condensation of two/four (hetero)aromatic rings onto an acene core leads to a situation, in which one or two rings in the larger aromatic system are chemically similar to the central ring of a triphenylene unit; reduced nucleus‐independent chemical shift (NICS) values and blueshifted absorption results. For tetrabenzoheptacene ABA displays an absorption maximum similar to that of hexacene, blueshifted in comparison to that of heptacene B  Buchwald–Hartwig coupling and gel permeation chromatography (toluene) the product was isolated.In a heat‐gun‐dried Schlenk tube, under an atmosphere of argon, was added The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The antimicrobial potential of the lead candidate FU002 for bacterial infection treatments could be demonstrated in an in vivo study. Molecular imaging and biodistribution studies revealed that conjugation engenders superior pharmacokinetics.Multidrug‐resistant bacteria represent one of the biggest challenges facing modern medicine. The increasing prevalence of glycopeptide resistance compromises the efficacy of vancomycin, for a long time considered as the last resort for the treatment of resistant bacteria. To reestablish its activity, polycationic peptides were conjugated to vancomycin. By site‐specific conjugation, derivatives that bear the peptide moiety at four different sites of the antibiotic were synthesized. The most potent compounds exhibited an approximately 1000‐fold increased antimicrobial activity and were able to overcome the most important types of vancomycin resistance. Additional blocking experiments using  Resistance is futile: By site‐specific conjugation polycationic peptides were conjugated to vancomycin. Derivatives that bear the peptide moiety at four different sites of the antibiotic were synthesized. The most potent compounds exhibited an approximately 1000‐fold increased antimicrobial activity and were able to overcome the most important types of vancomycin resistance. At first glance, the treatment of bacterial infections seems to be a straightforward task. As the cellular structures of prokaryotes differ greatly from those of the human host, they can be readily targeted by drugs, and nature has conjured up a multitude of antimicrobials to do just that.d‐Ala‐d‐Ala motif of the pentapeptide of lipid II, the target structure of vancomycin. The most important resistant strains substitute the C‐terminal d‐Ala by its oxa analogue lactic acid or serine.Vancomycin is the critical and decisive antibiotic for the treatment of multidrug‐resistant infections caused by Gram‐positive bacteria.N, VR, VC, and VV . The method of choice for the evaluation of the antimicrobial potential of compounds remains the broth microdilution method. The clinical breakpoint for vancomycin resistance on enterococci is defined as a concentration >4 mg L−1 by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The MIC (minimum inhibitory concentration) values of the lead compound FU002 determined in this study  revealed that the most important types of vancomycin resistance  could be overcome .The site selectivity of the conjugation to the amine functionalities, that is, Vd‐Ala‐d‐Ala motif by the vancomycin moiety of the conjugates could be excluded as FU002 shows no relevant change in MIC values in blocking experiments  nor significant hemolytic activity a general mechanism mainly based on lysis of cells can be excluded. Moreover, synergistic effects on the bacterial cell wall are not likely owing to the lack of activity of co‐administered vancomycin and the peptide moiety (Table S3). Targeting solely the s Figure  B. BesidFortunately, however, FU002 shows promising non‐toxic characteristics as demonstrated by the absence of hemolysis of human blood cells and good cytocompatibility as demonstrated by in vitro cytotoxicity studies using the XTT assay. This tolerability could be demonstrated for all relevant cell lines  at all tested concentrations of FU002 .For therapeutic antimicrobial substances to be of value, their antimicrobial activity must be combined with preferred pharmacokinetics. Unmodified vancomycin, for example, is known to be predominantly excreted via the kidneys. However, in patients with impaired renal function the risk of associated nephrotoxicity is high, which is the major limitation for the application of vancomycin under these conditions.125I or 124I for the studies in female Wistar rats , vancomycin and the lead candidate FU002 were radiolabeled with either Staphylococcus aureus USA300 LAC infected mice, FU002 was shown to be effective, as demonstrated by a stable body weight despite the infection and the significant reduction of the colony forming units  should be addressed to the authors.SupplementaryClick here for additional data file.


The tumor microenvironment is an integral player in cancer initiation, tumor progression, response and resistance to anti‐cancer therapy. Understanding the complex interactions of tumor immune architecture (referred to as ‘immune contexture’) has therefore become increasingly desirable to guide our approach to patient selection, clinical trial design, combination therapies, and patient management. Quantitative image analysis based on multiplexed fluorescence immunohistochemistry and deep learning technologies are rapidly developing to enable researchers to interrogate complex information from the tumor microenvironment and find predictive insights into treatment response. Herein, we discuss current developments in multiplexed fluorescence immunohistochemistry for immune contexture analysis, and their application in immuno‐oncology, and discuss challenges to effectively use this technology in clinical settings. We also present a multiplexed image analysis workflow to analyse fluorescence multiplexed stained tumor sections using the Vectra Automated Digital Pathology System together with FCS express flow cytometry software. The benefit of this strategy is that the spectral unmixing accurately generates and analyses complex arrays of multiple biomarkers, which can be helpful for diagnosis, risk stratification, and guiding clinical management of oncology patients. This review discusses current developments in multiplexed fluorescent immunohistochemistry for immune contexture analysis, and their application in immuno‐oncology, and discusses challenges to effectively use this technology in clinical settings. We also present a multiplexed image analysis workflow to analyse fluorescence multiplexed stained tumor sections using the Vectra Automated Digital Pathology System together with FCS express flow cytometry software. Progression of all solid cancers is directly influenced by complex interactions between immune and non‐immune cells within the tumor microenvironment (TME).Biomarker analysis on a single‐cell basis can be performed by using multi‐parameter detection methods such as genomics/proteomics and flow cytometry on dissociated tissue. However, the spatial information about the location of cells within the tumor core or margin is lost using these technologies.Multiplex fluorescence IHC (mfIHC) uses fluorescent dyes that offer a greater degree of spectral separation than chromogenic dyes and are commonly used for complex phenotyping of immune cells by flow cytometry. Traditional methodologies for labelling tissue with fluorescent probes involve detecting the protein of interest with an antigen‐specific primary antibody followed by a secondary antibody coupled to an organic fluorescent dye. In some cases, it is also possible to use primary antibodies directly conjugated to fluorescence dyes. These techniques are generally effective for labelling abundant proteins such as CD3 or CD8 but may not be effective for detecting low‐abundance proteins as the signal may not be easily detected above background. Dyes such as nanocrystal quantum dots are reported to be brighter and more stable than organic dyes making it easier to detect low‐abundance proteins.X and Y planes without moving out of focus, or acquiring large stacks of images in the z plane. However, tumors often exhibit a significant degree of cellular and spatial heterogeneity . Consequently, there is a growing need for high‐resolution multiplexed analysis across whole tissue sections. As such, the development of microscopes that incorporate MSI and whole slide scanning, or an image stitching feature, can enable researchers to reliably image the whole tumor tissue and quantify multiple proteins, even in the presence of spatial and spectral overlap.® automated quantitative pathology imaging system is currently one of the most widely used quantitative digital pathology imaging systems that uses whole slide scanning and multispectral unmixing to resolve overlapping fluorescent signals.Traditional fluorescence microscopes, even those equipped with multispectral capabilities, have limited scope for movement in the ®,®™,®,Another promising technology for mIHC, namely MultiOmyx™+ and CD8+ T cells, both in the tumor centre and at the periphery of the tumor using digital pathology.® assay in predicting stage II colon cancer patients with high risk of recurrence.® has already outperformed the standard tumor–node–metastasis (TNM) staging as a prognostic test for colorectal cancer.in vitro diagnostic (IVD) Immunoscore® assay for clinical use has also been developed by the HalioDx immuno‐oncology company.® is the first IVD immune scoring diagnostic test of the HalioDx pipeline, that is used by pathology laboratories leveraging advanced image analysis. Immunoscore® is now validated for prognostic and predictive diagnosis of colon cancer. The presence of CD8+ T‐cell infiltrates has also been shown to have prognostic benefit in other cancer types, including melanoma, NSCC, RCC and bladder cancer.®. For example, PD‐L1 is one such clinically relevant prognostic and predictive marker that has received FDA approval for its use with checkpoint inhibitors pembrolizumab and nivolumab.® with additional prognostic immune parameters on multiple cancer types.+ infiltrating T cells correlates with poor clinical outcome in renal cell carcinoma, indicating a possible exhausted phenotype.®, compared to CD8 and/or CD3 staining alone.The utility of mfIHC relies on the ability to profile several markers simultaneously. However, the analysis of multiparametric information from mfIHC can be laborious to perform manually. Recent application of machine learning, deep learning and artificial intelligence in quantitative image analysis‐based multiplexing has enabled detailed representation of tumor tissue by generating quantifiable data that can precisely profile multiple different cell types in the TME.Integration of MSI and advanced digital image analysis technologies in multiplexed IHC samples has the potential to reveal co‐expression of immune molecules, pathway configurations and the spatial relationships between different immune and malignant cells within a particular tissue compartment.While advances in therapeutic strategies for cancer treatments have significantly improved survival in some patients, questions still remain as to why some patients do not respond. Since tumors are highly heterogeneous among individuals, further development of predictive markers may help maximise the clinical benefit and minimise the incidence of adverse events. In particular, the emerging success in immuno‐oncology requires delineation of complex crosstalk between immune‐ and tumor‐related pathways. Current efforts on novel biomarker candidates rely on identification and quantification of different immune cell populations, their spatial relationship, tumor mutation burden and immune gene signature.+ and CD8+ T cells in NSCLC was associated with better survival outcome, and the prognostic impact of CD8+ T‐cell infiltration was independent from age, tumor size, histology, and stage in multivariate analyses.+CD8+) in metastatic colon cancer was associated with improved overall survival. The abundance of infiltrating PD‐L1 expression on antigen‐presenting cells (APCs) in the TME indicates an immunosuppressive environment in these patients.+ cytotoxic T cells was associated with improved patient outcome, and increased infiltration of regulatory T cells into core regions was identified as an independent marker of poor patient outcome in NSCLC.The immune contexture is defined as the density, localisation and organisation of immune cell within solid tumors. Immune contexture analysis using mfIHC revealed high density of T lymphocytes concentrated in the stromal compartment but not in the epithelial compartment in NSCLC.+CD4+) and epithelial and stromal tumor‐associated macrophages (CD68+) were associated with better outcome in patients with NSCLC.+ T‐cell/regulatory T‐cell ratio in the tumor compartment, and a higher intra‐tumoral/stromal ratio of CD8+ effector cell infiltration correlated with better overall survival in patients with NSCLC.+:Foxp3+), and cytotoxic T cells to PD‐L1 (CD8+:PD‐L1+) were also found to be suppressed in the microenvironment of gastric cancer tissues compared to those of normal adjacent gastric tissues.+ expression on both melanoma cells and macrophages was shown to correlate with high levels of intra‐tumoral CD8+ cells but not with intra‐tumoral CD4+ Tregs.+ T cells and regulatory T cells correlated with improved survival.Studies have now reported that not just one cell type, but the relationship between different immune cells in different tumor compartments have prognostic benefit that can impact on patient survival. For example, after neoadjuvant chemotherapy, higher levels of epithelial lymphocytes (CD3+CD163+) and their proximity to tumor cells.The application of mfIHC in immune contexture analysis has become increasingly useful for the performance of spatial distribution analysis of immune and tumor cells in various cancers, including colorectal cancer, pancreatic cancer and breast cancer.+ T cells in comparison with short‐term survivors, a proportion of short‐term survivors displayed higher PD‐L1 expression. These data suggest that combining T‐cell therapy with PD‐1/PD‐L1 blockade may improve overall survival of GBM patients.Multiplex fluorescence IHC has proved to be useful to delineate the immune‐tumor pathways and their spatial relations in stratification of patients for immunotherapy. For example, we recently used quantitative fluorescence multiplexing imaging techniques to demonstrate that the cellular makeup in pre‐treatment tissue from recurring GBM patients can predict the long‐term response following autologous CMV‐specific T‐cell therapy. While the long‐term survivors had significantly reduced number of CD3As the field of immune oncology continues to grow, the need for standardised multiplexed IHC for relevant tissue biomarkers, together with an accurate and reproducible image analysis pipeline, is likely to provide increased support for future clinical decisions. Advances in mfIHC technology, digitisation and automated image analysis have the potential to provide robust and reproducible multiplexed IHC data for personalised treatment of patients with immunotherapy. Despite these technological advancements, there are several factors that impede the implementation of mfIHC‐based quantitative digital pathology in clinical settings. Overcoming current limitations and providing a unified workflow will be essential for developing full, widespread, clinical benefit of this technology. These limitations can be roughly categorised as follows: tissue handling and processing, tissue staining, image acquisition and digital pathology, quantitative image analysis and centralised workflow.The reproducibility of IHC data can be greatly affected by sample handling, which begins with tissue collection (at autopsy or biopsy) until a section is ready to be stained.Staining of FFPE tissue sections can introduce variability during scoring.Several different approaches to fluorescence‐based multiplex IHC have been developed, which can be categorised as sequential or simultaneous depending on the choice of staining.Simultaneous fluorescence mIHC involves using multiple antibodies (conjugated or unconjugated) to label different antigens on the same slide at the same time. This type of staining is more time efficient, is not influenced by sequential rounds of imaging, and causes less tissue damage, but is limited to the number of fluorophores that can be resolved from each other during image acquisition. This method may incur artefacts because of spectral overlap of the dyes, also known as fluorescence bleed through, which can create difficulties in separating discrete fluorescence signals and complicate the evaluation of co‐localisation experiments.One of the biggest challenges in quantification of fluorescence signal in FFPE tissue is tissue autofluorescence.Histopathological evaluation of tumor tissue has been traditionally performed by pathologists using a standard microscope and a semi‐quantitative scoring system.Whole slide imaging can be categorised into three types, brightfield, fluorescent and MSI, depending on the type of scanner used.®, Vectra® Polaris™ and Hamamatsu NanoZoomer. Different scanner models vary in terms of their features and functionality,WSI scanners that allow for high‐speed digitisation of fluorescence IHC glass slides are available from many different vendors.z‐stacking for 3‐D reconstruction, image resolution, image quality, file compression methods, formats and file size when selecting a slide scanner for digitisation of FFPE slides.z‐stack capabilities,Most WSI scanners for fluorescence IHC comprise a software‐driven, robotically controlled microscope with high‐quality objective lenses, high‐quality monochrome cameras, and multiple filter cubes for single‐plex or multiplex imaging.Multispectral fluorescent WSI requires capturing and storing images at various wavelengths that are later processed to account for bleed through of the individual dyes and autofluorescence. The number of wavelengths captured increases the overall scan time and file size. Technical limitations in scanning can lead to imaging artefacts, such as lower image resolution, heterogeneous staining intensities and patterns, poorly focussed scans, improper stitching of lines or tiles, or overlapping signals from multiplexed spectra, all of which directly affect data accuracy and reproducibility.® requires an optimal biomarker study to be hypothesis driven, reproducible, with prognostic and/or predictive power and cost‐effective.Digital pathology has been successfully implemented around the world for education, clinical pathology conferences and research purposes.Digital pathology systems that support multiplexing have not yet been adopted in clinical pathology laboratories; however, they are widely used in early discovery and clinical studies. Although the widespread adoption of mfIHC in the clinic may still be futuristic, contract research organisations (CROs) are currently offering high‐quality image acquisition and analysis based on mfIHC to clinic and research which may be helpful in patient management Table . Becausein situ in the intact spatial context of tissues.Multiplexed IHC allows simultaneous detection and co‐localisation analysis of multiple markers ® and Vectra® Polaris™,in situ hybridisation (chromogenic or fluorescent), nuclear/cytoplasm/membrane biomarker identification, co‐localisation studies and spatial distribution analysis. There are now an increasing number of laboratories that are incorporating IHC image analysis software into their workflow for single‐cell‐based quantitative pathology. Each software package varies in terms of their flexibility, complexity, application and its ability to work on whole slides versus tiled (static) images, brightfield versus fluorescent images and different file formats.There are already a growing number of image acquisition platforms in the market that combine image analysis with automated slide scanning to support fluorescence multiplexed staining. These include the Akoya Biosciences VectraBased on the level of training required, image analysis software can be classified into two groups: ‘Unsupervised’ and ‘Supervised’.® automated quantitative digital pathology imaging platform. This integration allows for efficient exploration and quantification of large, multi‐parameter single‐cell image datasets while allowing access to the original images for visualisation. This method is useful to analyse cellular phenotypes and their relationships within spatial context of the TME in intact FFPE tissue sections. A typical data analysis pipeline for immune contexture analysis that utilises image cytometry software like FCS Express 6 for multiplexed digital images from the Vectra automated quantitative digital pathology platform is presented in Figure P‐values) and high‐dimensional data reduction tools such as tSNE, SPADE and K‐means, which can be applicable for high‐throughput analysis of multispectral data generated from multiplexed IHC platform.In collaboration with De Novo Software, we have integrated the ability to use FCS Express 6 Image Cytometry software to open and analyse fluorescence multiplexed images that were acquired using the VectraIn the setting of pathology, deep learning methods have been widely used in H&E‐ or IHC‐stained whole slide images for detection of metastatic loci, tumor classification and prediction of gene mutations.Several commercial image analysis software packages that integrate the workflow exist for non‐clinical settings. The workflow of such commercial systems still carries limitations and challenges before its routine implementation in clinical decision‐making. Thereby, in addition to the issues explained above, the challenges in image analysis workflow also need optimisation and standardisation for its effective integration and translation into clinic.Some of the most common image analysis artefacts includes cell segmentation errors (e.g. over segmentation or under segmentation of nuclei) and tissue classification errors (e.g. classifying tumor as stroma and vice versa). Therefore, an appropriate quality control measure must be included to assess potential artefacts throughout the IHC and image analysis workflow. It is important that pathologists are involved in reviewing the quality control step of the image analysis workflow that will otherwise influence the results.In the area of image data analysis, there has been much advancement in tissue and cell segmentation; however, the current software packages still have limitations in phenotyping cells of different sizes and morphologies, distinguishing cells in close proximity, and processing spatial distribution analysis. Different software packages use tools such as deep learning or threshold‐based methods (like pixel intensity) to identify and label objects. These tools are effective for identification of single‐cell type; however, it is challenging to perform co‐expression and multivariate biomarker analysis.One of the biggest challenges in translating tissue‐based diagnostic biomarkers in clinical decision is to determine a clear threshold for patient stratification based on expression of single or multiple markers. Since the staining results of all parameters are provided in a continuous variable, appropriate cut‐off values for each marker should be designated depending on the distribution of staining intensity, absolute number or both. The ‘optimal’ cut‐off point is defined as that threshold value of the continuous covariate distribution, which best separates low‐ and high‐risk patients with response to outcome.Automated image analysis software generates large volumes of high‐quality labelled data with raw information about the individual fluorescence profile in each cellular compartment. These data become more complicated to analyse when multiple samples are batch processed and compared simultaneously. Thus, downstream analytics software such as Excel, Spotfire, R studio, GraphPad Prism, or FCS express is required to inspect, analyse or graphically represent these data and also to perform standard set of statistics which can be associated with the clinical‐pathological parameters. Quantitative imaging informatics also requires tools that can allow simultaneous image visualisation while analysing and reporting on complex image data sets. However, most analytical software can import image data but does not support viewing and exploring of digital images. Different laboratories are incorporating different downstream software into their workflow to analyse and represent their image data. However, a centralised image analysis pipeline that integrates image acquisition, image processing, image analysis algorithms and data visualisation is critical to help improve scientific reproducibility.One of the important hurdles in clinical adoption of digital pathology or image analysis is the integration and interoperability of digital pathology. Clinical adoption of digital pathology or image analysis has become increasingly expensive and is labour intensive and time consuming to install and adjust with regard to standards and practices. With this rapid evolution in technology, it is vital that these new hardware and software platforms can be easily implemented and integrated within existing LISs in pathology workflows, and as such that they do not become outdated very quickly. In addition to integration and implementation, there are issues concerning interoperability between the different vendors. Commercial software that comes bundled with a microscope is often convenient to use. Closed hardware and software systems offer less flexibility for third‐party integration. Opening up a hardware and software technology with universal standard for data movement and management may facilitate more rapid biomarker identification, although more closed systems may be required for integration of these workflows in digital pathology. This in turn will play a larger role in enhancing the pathology workflow and most importantly improving patient outcome.® Digital Spatial Profiling (DSP),®,Several new solutions that perform simultaneous high‐throughput fluorescence multiplexing have been developed, each with its own limitations in terms of time, cost, throughput, flexibility and scalability Table . Some ofThere is a huge clinical need to understand immune contexture, particularly in cases where patients are refractory to current immunotherapy, or relapse. Characterising immune cells requires complex phenotyping of various markers that is currently beyond the capabilities of chromogenic IHC which is widely used in the clinic for diagnosis of disease. Advances in fluorescence IHC have made it possible to view multi‐parameter data on the same slide, and advances in analysis have made it possible to provide clinically relevant data from these images. It is also possible to present these data in a format that is currently palatable to clinicians and immunologists, and to use complex phenotypes to identify new potential biomarkers for treatment and tracking of disease progression. With current advances in technology driven by research laboratories and CROs, there is little hampering in the translation of such technologies to meaningful patient outcomes. A new era will see production of better dyes, faster technology, more biomarkers, better segmentation and a faster more reproducible overall workflow.Reshma Shakya: Conceptualization; Methodology; Visualization; Writing‐original draft; Writing‐review & editing. TamHong Nguyen: Methodology; Visualization; Writing‐review & editing. Nigel Waterhouse: Conceptualization; Methodology; Visualization; Writing‐review & editing. Rajiv Khanna: Conceptualization; Methodology; Supervision; Visualization; Writing‐review & editing.RS is Ambassador for De Novo Software in Australia as a consequence of their collaboration on developing FCS Express Image Plus to open fluorescence files acquired using the Vectra Automated Pathology Platform. RK holds international patents on adoptive immunotherapy and vaccine development, which have been licensed to Atara Biotherapeutics; RK acts as consultants for Atara Biotherapeutics and is on the Scientific Advisory Board of Atara Biotherapeutics.


Varicella-zoster virus (VZV) is a common cause of viral central nervous system (CNS) infection, and patients may suffer from severe neurological sequelae. The biomarker neurofilament light chain (NFL) is used for assessment of neuronal damage and is normally measured in cerebrospinal fluid (CSF). Novel methods have given the possibility to measure NFL in serum instead, which could be a convenient tool to estimate severity of disease and prognosis in VZV CNS infections. Here, we investigate the correlation of serum and CSF NFL in patients with VZV CNS infection and the association of NFL levels in serum and CSF with different VZV CNS entities. NFL in serum and CSF was measured in 61 patients who were retrospectively identified with neurological symptoms and VZV DNA in CSF detected by PCR. Thirty-three herpes zoster patients and 40 healthy blood donors served as control groups. NFL levels in serum and CSF correlated strongly in the patients with VZV CNS infection. Encephalitis was associated with significantly higher levels of NFL in both serum and CSF compared with meningitis and Ramsay Hunt syndrome. Surprisingly, herpes zoster controls had very high serum NFL levels, comparable with those shown in encephalitis patients. We show that analysis of serum NFL can be used instead of CSF NFL for estimation of neuronal injury in patients with VZV CNS infection. However, high levels of serum NFL also in patients with herpes zoster, without signs of CNS involvement, may complicate the interpretation. Varicella-zoster virus (VZV) is a neurotropic virus in the herpes family, and it is one of the most common viral agents causing infection in the central nervous system (CNS) , glial fibrillary acidic protein and S-100B protein  method, which is a digital enzyme-linked immunosorbent assay (ELISA)  in relation to onset of disease. Thirty-one out of the 33 patients were sampled within − 2–15 days in relation to onset of disease. The second control group consisted of 40 healthy blood donors.The study was approved by the medical ethics committee at Gothenburg University, and informed consent was obtained for inclusion in the study.r = 0.9984, p < 0.001, no offset), which was verified in a Passing-Bablok analysis. Serum NFL concentration was measured using an in-house digital ELISA on the Simoa platform as previously described in detail   or geometric mean and 95% confidence interval. Comparison of raw NFL concentrations in CSF and serum as well as age-adjusted CSF NFL , CSF NFL only (n = 5)). Both serum and CSF samples were drawn a median 4 days (range 0–90) after onset of disease. The patient who was sampled 90 days after onset of symptoms was a 38-year-old man who presented with slowly progressive bilateral proximal weakness in arms and legs for the last 3 months. After treatment with acyclovir and cortisone, the symptoms slowly regressed. Details about the study groups and comparison of clinical data are shown in Table p < 0.01 for all).Sixty-one consenting patients with detectable VZV DNA in CSF and neurological symptoms related to VZV infection were included in the study. Nine of the included patients were only analysed for either serum NFL or CSF NFL due to insufficient sample volumes (serum NFL only (n = 4), polyneuropathy (n = 1), radiculitis (n = 1) and cranial nerve ganglionitis (n = 1). Two patients with peripheral facial palsy that also reached criteria for meningitis were categorised as Ramsay Hunt syndrome. One patient with encephalitis presented with subarachnoid haemorrhage due to an aneurysm which was regarded as a VZV manifestation.Seven patients who did not meet the criteria for any predefined group were categorised as “other neurological symptoms” which consisted of the following diagnoses: encephalopathy (n = 3), systemic lupus erythematosus (n = 2), leukaemia (n = 2), essential thrombocytosis (n = 1), lung malignancy (n = 1), rheumatoid arthritis (n = 1) and Crohn’s disease (n = 1)). Of the herpes zoster controls, 17 patients were immunocompromised (leukaemia (n = 6), lymphoma (n = 3), HIV (n = 3), rheumatoid arthritis (n = 1), Wilm’s tumour (n = 1), liver transplant (n = 1), kidney transplant (n = 1) and ulcerative colitis (n = 1)). All of these patients received immunosuppressing treatment except for six patients in the herpes zoster control group (HIV (n = 3), chronic lymphocytic leukaemia (n = 2), and lymphocytic lymphoma (n = 1)).Eleven of the patients with VZV CNS infection were immunocompromised  . The patients with “other neurological symptoms” were not analysed separately as only four patients had available paired samples.There was a strong correlation between concentrations of NFL in serum and CSF in the patients with VZV CNS infection (p = 0.043) , Ramsay Hunt syndrome , and the healthy controls   and healthy controls (p < 0.001), but not compared with the encephalitis patients (p = 0.22).The herpes zoster controls had higher serum NFL concentrations than the VZV CNS infection group as a whole  Fig. . The herp values for group comparisons are shown in Table No age-related cutoffs have been established for serum NFL. To adjust for age differences between the study groups in our material, we performed an analysis of covariance with age as a covariate variable and an interaction term between age and group. Since the interaction was not significant, we dropped the interaction term and assumed parallel regression lines Fig. . For comp < 0.001) and Ramsay Hunt syndrome , but not compared with the patients with “other neurological symptoms”  , but not to those with Ramsay Hunt syndrome (p = 0.062).The concentrations of NFL in CSF were significantly higher in the encephalitis patients  compared with the patients with meningitis  Fig. . The comp = 0.139) . In addition, no significant difference was shown when comparing the herpes zoster controls with the patients that had a rash in the VZV CNS infection group . Thus, based on these results, we cannot exclude an association between rash and higher serum NFL concentrations in patients with VZV infection.To investigate if the presence of a rash affects serum NFL concentration, we divided the VZV CNS infection group into patients with or without rash. Twenty-eight out of the 56 patients with VZV CNS infection (50%) that had been analysed for serum NFL had a rash. These patients had a tendency towards higher concentrations of serum NFL compared with those lacking a rash (28 out of 56 patients) although not significant  or in serum  in the patients with VZV CNS infection. Furthermore, multiple comparison of viral load between the different patient groups with VZV CNS infection showed significantly higher viral load only when comparing the patients with encephalitis and “other neurological symptoms” (p = 0.039) . Yet, a scatter plot indicated a possible influence of two outliers . However, further subgroup analysis showed that there was a significant negative correlation in the meningitis patients . The CSF NFL concentrations and albumin ratios correlated , whereas a less strong but significant correlation was found between the serum NFL concentrations and albumin ratios .To evaluate if the serum NFL concentrations were depending on the degree of blood-brain barrier damage in the patients with VZV CNS infections, the serum/CSF NFL ratios were analysed for correlation with the albumin ratios. No correlation was found when including all values . In the herpes zoster control group, the concentrations of serum NFL were not significantly different between immunocompromised and immunocompetent patients .The influence of immunosuppression on serum NFL concentrations was also evaluated. Within the VZV CNS infection group, the immunocompromised patients presented higher concentrations of serum NFL  versus the immunocompetent patients  (This study is to our knowledge the first to examine serum NFL concentrations in patients with VZV CNS infection. Our results indicate that analysis of serum NFL may be used instead of analysis of CSF for estimation of neuronal injury in patients with VZV CNS infection and possibly also in other viral CNS infections. The finding of a strong correlation between serum and CSF NFL is in agreement with recent studies including patients with CNS injury in HIV infection (Gisslen et al. Both CSF and serum NFL concentrations correlated with increasing blood-brain barrier damage measured by the CSF/serum albumin ratio, as previously shown in studies of dementias and in CNS injury in HIV (Gisslen et al. The encephalitis patients had the highest serum NFL concentrations when compared with the patients with meningitis and Ramsay Hunt syndrome. Encephalitis is considered the most severe manifestation of VZV CNS infection. Indeed, higher concentrations of serum NFL have been correlated to increased disease activity in several neurological diseases such as multiple sclerosis (Disanto et al. In conjunction with high levels of serum NFL, the patients with encephalitis also demonstrated higher concentrations of CSF NFL compared with the patients with meningitis and Ramsay Hunt syndrome. After age adjustment, this difference remained in comparison with that of the meningitis patients. Encephalitis patients have been reported to have high concentrations of CSF NFL also in a previous study (Grahn et al. The influence of immunosuppression on levels of NFL is unclear. In our material, the immunocompromised patients in the VZV CNS infection group showed higher levels of serum NFL than immunocompetent patients. In contrast, this was not shown in the herpes zoster control group. As the inflammatory response differ between the CNS and the peripheral nervous system in patients with CNS infections caused by herpes simplex virus and VZV (Lind et al. Finally, as the serum NFL assay means much less harm for the patient by avoiding a lumbar puncture, this assay can be a convenient tool for assessing the prognosis and for monitoring of disease in patients with VZV CNS infection and probably also in other viral CNS infections. However, longitudinal studies are warranted for evaluation of this purpose.In conclusion, our findings demonstrate a strong correlation between serum and CSF NFL, suggesting that analysis of serum NFL could be used instead of CSF NFL for evaluation of neuronal injury in VZV CNS infection. However, high levels of serum NFL were also seen in patients with herpes zoster without signs of CNS involvement, indicating an influence of peripheral nerve damage on serum NFL, which could complicate the interpretation. In addition, we found significantly higher concentrations of NFL in both serum and CSF in patients with encephalitis compared with that of meningitis which strengthens the argument of using the serum NFL assay in prognostic purpose in VZV CNS infections.


To explore the clinical effect of endometrial injury (EI) on the third day of the menstrual cycle before frozen–thawed embryo transfer (frozen–thawed ET) on patients experienced two or more implantation failures.n = 100 in each group). Patients in the EI group received local EI with a Pipelle catheter on the third day of the menstrual cycle before frozen–thawed ET. Primary outcomes were live birth, clinical pregnancy and implantation rates. Secondary outcomes were biochemical, multiple and ectopic pregnancy rates and abortion rates.A total of 200 patients who suffered at least two failed hormone‐replacement therapies and frozen–thawed ET were randomly divided into two groups: EI group and control group (P = 0.032). Clinical pregnancy and implantation rates in EI group were significantly higher comparing to control group . The rate of multiple pregnancy in EI group (37.50%) was significantly higher than that of control group (18.75%) (P = 0.031). No significant difference in ectopic pregnancy rate and abortion rate was observed between EI group and control group.The rate of live birth in EI group (51.00%) was significantly higher than that of control group (36.00%) (Applying EI to patients experienced two or more implantation failures on the third day of the menstrual cycle before frozen–thawed ET can improve clinical outcomes. Successful implantation depends on both embryo quality and endometrial environment.There are increasing studies focus on performing endometrial injury (EI) in luteal phase to improve endometrial receptivity. However, there is no consensus in the field up to date and the benefit of EI prior to embryo transfer is controversial. Specifically, some studies demonstrated that performing EI in luteal phase prior to in vitro fertilization (IVF)/ICSI improved implantation and clinical pregnancy rates,Frozen–thawed embryo transfer (frozen–thawed ET) is a procedure that involves endometrial preparation and the transfer of thawed embryos obtained through a previous IVF cycle. It mainly applies to patients with supernumerary embryos and canceled fresh embryo transfer due to the risks such as ovarian hyperstimulation syndrome and thin endometrium.et al. suggested that EI had a positive effect on pregnancy rate prior to frozen–thawed ET cycles in patients suffered repeated implantation failures (RIF).The effect of EI before using frozen–thawed ET cycles has not been well explored. With limited proceeding studies, it seems like EI did not have any beneficial effects on an unselected group of women undergoing frozen–thawed ET.Current studies in the field mainly focused on performing EI in luteal phase prior to frozen–thawed ET. This conventional procedure usually takes two menstrual cycles which brings more individual differences and other uncertainties to the situation. Comparing to luteal phase, we hypothesized that the early stage of menstrual cycle might be a better time point to perform EI. This new procedure only requires one menstrual cycle and a more consistent and better‐controlled injury due to a thinner one endometrium.Therefore, we proposed a prospective randomized controlled trial to evaluate the effectiveness of EI in infertility women undergoing frozen–thawed ET cycle. The purpose of our study is to explore the clinical effect of performing EI on the third day of the menstrual cycle before undergoing frozen–thawed ET of patients suffered two or more implantation failures.http://www.clinicaltrials.gov was ChiCTR‐IPR‐17014013. All participants recruited to the study were fully counseled and consented.This prospective randomized controlled trial conducted at Reproductive Center of Anhui Province Maternity and Child Health Hospital between October 2017 and February 2018. All participants were followed‐up to 1 year. This study was approved by the Institutional Ethics Committee Review Board of Anhui Medical University affiliated Maternity and Child Health Hospital. The registration number on 2, hydrosalpinx, endometriosis and receiving oral contraception drugs recently.The inclusion criteria were: patients indicated for frozen–thawed ET, with serum progesterone level < 1.2 ng/mL on the third day of the menstrual cycle, at least two or more previous implantation failures, normal morphology of uterine cavity, The exclusion criteria were: history with pelvic surgery history, history with difficult ET and aged more than 40 years; intrauterine abnormality , body mass index (BMI) > 27 kg/mThe study was explained to all eligible patients who were given a patient information sheet. Randomization was done simply using sealed envelopes before undergoing EI. After randomization, physicians and participants were aware of the trail‐group assignments.In the EI group, patients received endometrial soft scratch on the third day of the menstrual cycle proceeding frozen–thawed ET cycle. EI was performed following standard approach using a Pipelle catheter (Beijing Saipu Jiuzhou Science and Technology Development Company). The Pipelle catheter was introduced through the cervix up to the uterine cavity, and then rotated 360° and moved up and down four times after withdrawing the piston. Simultaneously, endometrial tissue was stained with hematoxylin and eosin, and examined under microscope in order to evaluate the size and level of the injury introduced by the endometrial sampler and to verify the proliferative state of endometrium. The patients in control groups received no intervention. All procedures were performed by two specifically trained senior doctors.All patients from both groups followed our standard endometrial preparation protocol for frozen–thawed ET cycles with hormone‐replacement therapy for approximately 14 days. Specifically, patients started estradiol valerate 6 mg daily from day 3 of the cycle. Transvaginal ultrasound evaluation of the endometrium and serial serum estradiol measurements was monitored. The dose of estradiol valerate would be increased to 8 mg daily if the endometrial thickness was less than 8 mm. When the thickness of endometria was ≥8 mm and the concentration of estradiol exceeded 200 pg/mL, the patients began to receive intramuscular progesterone 60 mg daily and frozen–thawed ET was performed 3 or 5 days later, depending on the embryo's stage of development.The patterns of endometrium were classified according to Zhao's grading.The cleavage embryos were classified according to Steer's grading.All embryo transfers were performed under ultrasound guiding. Patients were asked to fill their bladder to provide an acoustic window for uterus visualization. The catheter tip  was placed 1.0–1.5 cm below the apex of the uterus cavity. In order to prevent uterine contraction, care should be taken to avoid contact of the transfer catheter with the uterine fundus. Hormonal treatment was continued at least till pregnancy test was performed on 14 days after transfer.The primary outcomes were live birth, clinical pregnancy and implantation rates and secondary outcomes were biochemical and multiple pregnancy, miscarriage and ectopic pregnancy rates. Live birth rate was defined by number of deliveries that resulted in a live born after embryo transfer. Clinical pregnancy rate was defined by ultrasound confirmation of gestational sac approximately 5 weeks after embryo transfer. Implantation rate was achieved by dividing the sacs number observed on transvaginal ultrasound scan by the number of transferred embryos. Biochemical pregnancy rate was defined as positive hCG test 14 days after embryo transfer. Multiple pregnancy rate was defined as the number of multiple pregnancies divided by total number of clinical pregnancies. Miscarriage rate was defined as miscarriages number before 20 weeks divided by the number of clinical pregnancies. Ectopic pregnancy rate was defined as the number of patients with ectopic pregnant divided by the number of all confirmed pregnancies.et al. with 30% difference in clinical pregnancy rate demonstrated that our study required 49 patients per group to reach the significance of 5% and a power of 80% in this prospective randomized control design.A power analysis suggested by Barash n = 145) or declining to participate (n = 5). The remaining 220 participants were randomly assigned into two groups: EI group (n = 110) and control group (n = 110). After examination, 10 patients from each group were excluded because of following conditions: endometrial thickness was less than 7 mm, uterine cavity effusion and did not have good quality – according to Veeck classification, embryos for transfer. At the beginning of the study, 100 patients in each group were recruited and analyzed respectively . Student's t‐test was used for comparing quantitative variables and Chi‐square test was used to compare categorical data. P < 0.05 was considered statistically significant.All statistical calculations were performed by n = 100) and control group (n = 100). Baseline characteristics of each group including age, duration of infertility, BMI, basal FSH, endometrial thickness at progesterone initiation day, the pattern A/B of endometrium at progesterone initiation day, number of transferred embryos, ratio of cleavage stage and quality of embryos are shown in Table In our study, a total of 200 participants who received frozen–thawed ET after two or more implantation failures were divided into two groups: EI group n = 00 and coP = 0.032). In addition, clinical pregnancy and implantation rates were significantly increased in EI group compared to control group . Furthermore, the multiple pregnancy rate in EI group (37.50%) was also significantly enhanced than that of control group (18.75%) (P = 0.031). At last but not the least, the biochemical pregnancy rate in EI group (66.00%) was significantly higher when comparing to control group (49.00%) (P = 0.015). In addition, our study suggested that there was no difference in ectopic pregnancy rate and abortion rate between two groups  was significantly higher than control group (36.00%) P = 0.03. In addiThe endometrial tissues that were obtained by the Pipelle catheter on the third day of the menstrual cycle when introducing the EI showed only superficial layer of the endometrium and proliferative endometrium. The endometrial tissue was confined to the functional zone Figure .The results of our study suggested that introducing EI during menstruation cycle proceeding frozen–thawed ET cycle has a positive effect on clinical pregnancy and implantation rates for patients who experienced two or more implantation failures.Successful embryo implantation is a pivotal step in reproduction. This important event can be affected by numerous aspects including several cytokines and growth factors, along with a dialogue between embryo and endometrium..The initial evidence that EI may be helpful for implantation came from in vivo studies.It has been known that EI can remove irregular hyperplasia of endometrial tissue, promote epithelial cells, stromal cells proliferation and differentiation, and enrich the endometrial blood flow.It is important to point out that the key element that differentiates our study from previous studies is the time of performing EI. Comparing to the conventional method which introduce EI at luteal phase, we performed EI on the third day of the menstrual cycle which shortened the whole process significantly and ensured a more consistent injury due to a thinner endometrial. Another difference was all patients in EI group were subjected to endometrium pathological examination in order to check if the endometrial have corresponded to the early follicular phase and the injury confined to the functional zone. The third difference was that all transferred blastocysts were screened for chromosome abnormality before the transfer. The above differences ensured us obtaining a more consistent cohort compared to previous studies.et al. showed that performing EI in oocyte retrieval day could have a hazardous effect on the result of ART.et al. compared performing EI in the luteal phase and in the proliferative and suggested that there was no significant difference in terms of clinical outcomes.There is no consensus regarding the optimal time to perform EI. Most studies performed EI in the luteal phase, proliferative phase or oocyte retrieval. But Karimzade Currently, there are three main hypotheses explain the mechanism of how EI improve the endometrium receptivity: (i) EI might be able to induce endometrial decidualization and pinopode to increase implantation potential.et al. conducted a pragmatic, multicenter, open‐label, randomized, controlled trail including 1364 patients, which showed that performing EI between day 3 of the cycle preceding the IVF cycle and day 3 of the IVF cycle did not result in a higher rate of live birth compared to control group for patients treated with IVF.In is important to point out that in 2019, Lensen As far as we are aware, our study is the first to investigate the impact of performing EI during menstruation cycle for patients undergoing frozen–thawed ET after two or more implantation failures. However, due to the limited sample size, some questions such as whether performing EI can reduce the abortion rate were left unanswered. Another possible criticism of our study is that uterine cavity operation on the third day of the menstrual cycle might increase the risk of surgical complications. In order to avoid complications, the operation was kept aseptic. In our study, no patient in the experimental group experienced significant vaginal bleeding, no fever, abdominal pain and other complications were reported. Another important limitation is that both our physicians and patients were not blinded to randomization and the placebo effect is worth considering. Even though our study suggested that performing EI during menstruation cycle is beneficial to the patients, further studies that including performing EI on different menstrual stages are needed to identify the best timepoint of introducing EI.In summary, our study suggested introducing EI during menstruation cycle proceeding frozen–thawed ET cycle has a positive effect on clinical pregnancy and implantation rates for patients who experienced two or more implantation failures. Our study indicates that introducing EI in menstrual phase undergoing frozen–thawed ET cycle is a feasible, safe and effective method that can improve the clinic outcome. Further studies are necessary to investigate the related mechanisms. Large blinded prospective multicenter studies are also needed to confirm these findings.None declared.Figure S1 Supporting informationClick here for additional data file.


We demonstrate a case, in which endoscopic ultrasonography‐guided fine‐needle biopsy (EUS‐FNB) was useful for determining the diagnosis of lesions of retroperitoneal fibrosis. In our case, accessing the retroperitoneal lesions by conventional percutaneous biopsy procedures was not feasible due to the difficulty of avoiding the inferior vena cava and ureter. We believe that our case demonstrates a unique approach for performing histological analysis in a challenging case. A 67‐year‐old man presented with back pain. Computed tomography (CT) revealed right hydronephrosis caused by a soft‐tissue mass surrounding the abdominal aorta and bilateral common iliac arteries Fig. . PositroRPF is a rare disease characterized by the presence of a fibroinflammatory tissue, which usually surrounds the abdominal aorta and the iliac arteries and extends into the retroperitoneum to envelop neighboring structures, such as the ureters.


Routine quality assurance (QA) of cone‐beam computed tomography (CBCT) scans used for image‐guided radiotherapy is prescribed by the American Association of Physicists in Medicine Task Group (TG)‐142 report. For CBCT image quality, TG‐142 recommends using clinically established baseline values as QA tolerances. This work examined how image quality parameters vary both across machines of the same model and across different CBCT techniques. Additionally, this work investigated how image quality values are affected by imager recalibration and repeated exposures during routine QA.t‐test. The effect of artifacts appearing on CBCTs taken in rapid succession was characterized and an approach to reduce their appearance was evaluated. Additionally, a set of baselines and tolerances for all image quality metrics was presented.Cone‐beam computed tomography scans of the Catphan 604 phantom were taken on four TrueBeam® and one Edge™ linear accelerator using four manufacturer‐provided techniques. TG‐142 image quality parameters were calculated for each CBCT scan using SunCHECK Machine™. The variability of each parameter with machine and technique was evaluated using a two‐way ANOVA test on a dataset consisting of 200 CBCT scans. The impact of imager calibration on image quality parameters was examined for a subset of three machines using an unpaired Student’s P < 0.05) and all imaging parameters except slice thickness varied with machine (P < 0.05). Imager calibration can change the expected value of all imaging parameters, though it does not consistently do so. While changes are statistically significant, they may not be clinically significant. Finally, rapid acquisition of CBCT scans can introduce image artifacts that degrade CBCT uniformity.All imaging parameters except geometric distortion varied with technique (This work characterized the variability of acquired CBCT data across machines and CBCT techniques along with the impact of imager calibration and rapid CBCT acquisition on image quality. QA of CBCT systems on linear accelerators, including safety, mechanicals, and image quality, is outlined in the American Association of Physicists in Medicine (AAPM) Task Group (TG)‐142Establishing a monthly CBCT image quality QA protocol that aligns with TG‐142 requires determining a baseline value of each QA metric and the acceptable tolerances for measurements that are different from baseline. Image quality metrics will fluctuate between scansThe studies listed above are specific to the machine types and imaging techniques that are investigated. There is limited published data on the fluctuations expected with newer TrueBeam® CBCT scans. Additionally, modern linear accelerators often have multiple CBCT techniques applicable for different anatomical sites and clinical situations and many institutions have multiple machines of the same model in clinical use. Previously published studies have not investigated how baselines and tolerances vary across machines and techniques. This information is needed to make an informed decision on whether baselines and tolerances should be technique‐dependent and to determine if departments with multiple machines will require machine‐specific or institution‐wide baselines. There are no published datasets and statistical analyses  of measurements of image quality parameters across multiple imaging techniques on modern TrueBeam® accelerators. This information could be used by clinical physicists for comparison to their local data when establishing their own quantitative QA program with comprehensive baselines and tolerances. Finally, none of these studies has investigated the impact of imager recalibration on CBCT image quality QA results. As clinical physicists may recalibrate the kV imager at a set interval as part of machine maintenance, the information is important for determining if baselines need adjustment post‐calibration.The purpose of this work was to address these limitations in the literature. This work investigated quantitative image quality results for five modern Varian machines (four TrueBeam® and one Edge®) and evaluated intra‐machine variability and inter‐machine variability. Baselines and tolerances were determined for each image quality metric based on technique, institutional data, and manufacturer recommendations. During data collection and analysis, it was found that there are additional, not previously reported, scenarios that may affect the measured image quality metrics. These included: (a) the impact of CBCT calibration  on image quality parameters and (b) the effect of monthly QA procedures on the image detector panel. The effect these scenarios have on monthly CBCT image quality results was quantified to enable other clinical users to incorporate the results into their monthly QA procedures.2All CBCT acquisitions were performed using a Catphan® 604 phantom . This phantom is comparable to the Catphan® 504 phantom with some manufacturing differences that allow for additional test objects.Images were analyzed using SunCHECK Machine™ manufactured by Sun Nuclear Corporation . Figure image, in a given ROI [Fig. reference, which is set by the user. Each HU ROI is generated on a single image plane, is centered within the HU plug, and has a diameter of approximately 60% that of the HU plug. The maximum HU deviation, σHU, determined by Eq. , and SPSS .2.ATwo hundred CBCTs were taken across five linear accelerators and using four CBCT techniques over the course of 12 months. These CBCTs were taken as part of routine monthly QA procedures with additional CBCTs taken specifically for this work. Ten CBCTs were taken with each technique on each accelerator. No image calibrations were done on any of the linear accelerators during the period the CBCTs were taken. All monthly QA CBCTs taken during the year time frame were included in this analysis except for nine scans that did not follow institutional protocols for acquisition . This set of CBCT acquisitions were used to determine the variability of each image quality parameter as a function of machine and technique. The difference in the mean values of each image quality parameter across machines and techniques was evaluated using two‐way ANOVA tests.2.BThe TrueBeam® 2.7 MR3 imaging system undergoes a calibration by the user before clinical use, after major service or part replacement, and at a user‐determined frequency thereafter. For CBCT techniques, the image calibration begins with a dark and flood field calibration of the dynamic gain kV mode (the kV mode used for CBCT scans). This is followed by a rotating air normalization calibration, which uses the same settings as the clinical CBCT technique, but the gantry is rotated through full clockwise and counter‐clockwise rotations. This scan is used to generate a fluence map of the radiation incident on a patient. Finally, the densitometry module of the Catphan® 604 is scanned to create an attenuation to HU curve. Individual calibration of any one component is possible without invalidating existing calibrations within the calibration set.The impact of this imaging calibration on monthly imaging baselines was evaluated for three of the TrueBeam® machines. Image quality data collected before and after three of the machines underwent a new, full image calibration were analyzed to determine if the calibration altered the image quality data. SunCHECK Machine™ was used to analyze all data collected. Data from before the new image calibration were taken from routine monthly QA CBCTs and, for some of the machines, supplemented with additional CBCTs that were taken in between the monthly scans as part of other testing. Data from after the new image calibration were taken from routine monthly QA CBCTS and from additional CBCTs taken specifically to test the post‐calibration image quality. No monthly CBCT scans acquired with either the original or new calibration were excluded from the study. The number of CBCTs in each dataset varied with machine and technique, but ranged from 5 to 12.2.CInitial data analysis showed that some CBCTs taken for routine machine QA exhibited a cylindrical artifact of lower HU values in the center of the phantom Fig. . It was 2.DBaselines and tolerances were set for each image quality parameter based on institutional data. The dataset of CBCT measurements described in Section 33.AImage quality parameters display a range of trends when evaluated graphically as a function of technique and machine Fig. . As expeP < 0.05. The test also reports if there is an interaction between the machine and technique terms. When statistically significant, the interaction term indicates that the expected mean of a parameter is dependent on both the machine and the technique. Test statistics, degrees of freedom, and individual P‐values are reported in Supplemental Table P < 0.05) and all imaging parameters except slice thickness varied with machine (P < 0.05). The interaction term was significant for all image quality parameters except for uniformity, noise, and slice thickness.A two‐way ANOVA test (with machine and CBCT technique as factors) was performed in SPSS to test if image quality parameters were machine‐ and/or technique‐dependent. The two‐way ANOVA evaluated the hypothesis that the mean of each image parameter varied with the machine and technique used to measure it. Statistical significance was taken as having 3.BP < 0.05. The differences in the means between the pre‐ and post‐calibration data are shown in Table p‐values are reported in Tables Cone‐beam computed tomographies were acquired pre‐ and post‐imager calibration with a total of 185 CBCTs in the dataset and a range from 5 to 12 CBCT image sets in each group (machine and technique). For each image quality parameter studied, a two‐tailed, unpaired Student’s t‐test was used to test the hypothesis that the mean value of that image quality parameter was different between data collected before and after the CBCT calibration procedure. Differences were considered significant if The change in the expected value of the image quality parameters after calibration was compared to the machine‐to‐machine variability shown in Fig. 3.CThe artifact displayed in Fig. As an example of the appearance and fading of the artifact, Fig. This artifact was not seen when a maximum of two CBCT scans were acquired in one session with the CBCTs having the same fan type . In‐house QA procedures were adjusted to follow this two scan, same fan‐type limit in order to minimize the presence of this artifact. Any CBCTs with this artifact were excluded from the analysis completed in other sections of this work if it was verified that the CBCTs with the artifact had been acquired rapidly after other CBCT scans.3.DThe CBCT dataset analyzed in Section The spread and standard deviation from Section 4During the course of investigation, it was found that certain QA procedures affected the variability of CBCT image quality metrics. First, acquiring CBCTs in rapid succession during QA has the potential to produce a low‐HU artifact in the center of the reconstructed image. This artifact did not match, in intensity and shape, many previously characterized artifacts including cupping, streak, ring, aliasing, bar, halo, and speckle.It was also found that image quality measurements pre‐ and post‐CBCT calibrations were statistically different. As baselines and tolerances are often set using CBCT data, the user could potentially run into an issue where current baselines and tolerances do not appropriately match the measurements that are performed using a new CBCT calibration. To prevent this, the user would need to characterize any changes in image quality metrics and adjust baselines and tolerances accordingly after completing a CBCT calibration.There has been limited guidance on establishing CBCT QA programs across multiple machines and techniques of the same type. Results from the two‐way ANOVA test Section .A indica90P is the 90th percentile of HU values in the ROIs and 10P is the 10th percentile of all HU values in the ROIs:Image quality parameters measured in this work are specific to the calculation algorithms used for analysis. For example, SunCHECK Machine™ computes uniformity by looking at the difference in the average HU values across different ROIs. On the other hand, DoseLab Pro  first adds 1000 to each HU value to convert the HU scale to only positive numbers. Similar ROIs to those used in SNC are defined for evaluating uniformity, but uniformity is computed by using the following formula, where Because of the variability in calculation and units for reporting uniformity, the user should understand the algorithm used for calculation of each image quality parameter. This will avoid setting tolerances that may be either too stringent and produce failures that result from measurement variability, or too lenient and not be able to discern degradation in image quality.The relationship between image quality QA and the incidence of clinically recognizable image quality issues was investigated by Manger et al.This work began as a departmental initiative to determine data‐driven baselines and tolerances in order to improve our Linac QA imaging program. Initial results showed variation across all metrics and machines prompting a complete reassessment of the CBCT QA program in order to understand the sources of variability and standardize the process. Differences were found in phantom setup and acquisition , phantom used (Catphan 504 vs Catphan 604), the number of phantoms used (each machine used its own Catphan), analysis programs (DoseLab Pro vs SNC Machine), position of analysis ROIs, and test frequency/timing. Staff were retrained, all Catphan 504s were taken out of use, each site used a single Catphan 604, and a single automated CBCT analysis program was installed. The baselines and tolerances determined in this work were only possible after this standardization initiative improved the quality of collected data.5This work focused on the standardization of a CBCT image QA program in an institution with multiple machines and multiple CBCT techniques in clinical use requiring QA. A series of statistically driven studies were completed in order to determine meaningful baselines and tolerances that would be able to identify degradation in image quality while also absorbing routine, expected variability in the measurements. These baselines and tolerances also followed recommendations provided by the AAPM, published in TG‐142 and TG‐179. Two‐way ANOVA tests of CBCT scans from five machines using four imaging techniques showed that, for Varian TrueBeam® machines, CBCT image quality parameters are almost all machine‐ and technique‐dependent indicating that careful consideration is needed when applying institutional‐wide CBCT baselines and tolerances.During the course of the standardization initiative, two QA scenarios were identified that influence measurement stability: taking multiple CBCTs in rapid succession and performing a CBCT calibration. This work showed that for routine QA protocols that require multiple CBCT acquisitions, the order in which the scans are taken needs to be evaluated. If not, multiple, rapid CBCT acquisitions have the potential to degrade the image quality of QA CBCT scans and can artificially skew QA results into indicating that a machine is out of manufacturer tolerance when it is not. Additionally, this work showed that recalibrating the kV imaging system could change the expected value of image quality parameters. Clinical physicists should be aware of this potential and should consider confirming or re‐establishing image quality baselines after image calibration. Finally, this work presented the baselines and tolerances developed from the institutional standardization initiative. Though the specific values of image quality parameters measured in this work may vary based on the institution, a key conclusion of this work is that the expected value of the parameter can vary: across machines of the same model; with the imaging technique used; on a given machine when the imager is recalibrated; and with the order with which scans are taken. This information can be used as a roadmap at other institutions for what to evaluate when attempting to establish a quantitative CBCT QA program across multiple machines. Furthermore, this work provides a basis for comparison to results measured by other institutions as part of establishing a QA program.David L. Barbee reports Sun Nuclear Corporation paid for conference travel to speak. None of the other authors has any conflict of interest to disclose.Table S1. Detailed statistical results for two‐way ANOVA test used to evaluate if image quality parameters are machine or technique dependent. For each main effect  and for the interaction term between the two factors the following are reported: test statistic (F), degrees of freedom (df), and P‐value (p).Table S2. Detailed statistical results for unpaired Student's T‐test used to evaluate difference in mean between the pre‐calibration and the post‐calibration data for the head cone‐beam computed tomography technique. Test statistic (t), degrees of freedom (df) and P‐value (p) are reported.Table S3. Detailed statistical results for unpaired Student's T‐test used to evaluate difference in mean between the pre‐calibration and the post‐calibration data for the spotlight cone‐beam computed tomography technique. t is the test statistic and df is the degrees of freedom.Table S4. Detailed statistical results for unpaired Student's T‐test used to evaluate difference in mean between the pre‐calibration and the post‐calibration data for the thorax cone‐beam computed tomography technique. t is the test statistic and df is the degrees of freedom.Table S5. Detailed statistical results for unpaired Student's T‐test used to evaluate difference in mean between the pre‐calibration and the post‐calibration data for the pelvis cone‐beam computed tomography technique. T is the test statistic and df is the degrees of freedom.Table S6. Institutional baselines and tolerances information for each image quality parameter.Click here for additional data file.


Oral health is important for well‐being and overall health. Older peoples′ oral health is well described in the residential care context, but remains understudied in short‐term care.The aim of this study was to describe oral health, daily oral care and related factors among older people in short‐term care and to compare self‐perceived oral health with professional assessment.ROAG). The older peoples’ perceptions of their own oral health were measured with a global question on self‐perceived oral health. Self‐care ability was assessed with Katz Index of Activities of Daily Living (Katz‐ADL).This cross‐sectional study included 391 older people in 36 short‐term units in 19 Swedish municipalities. Oral health was assessed professionally by clinical oral assessment and the Revised Oral Assessment Guide . This study indicates that in order to improve older peoples′ oral health and oral care we need to provide person‐centred oral care and to develop a close collaboration between nursing and dental staff.Professionals’ assessments of oral health differed considerably from the older peoples′ own assessments. A higher risk of oral problems and more occurrence of coating or food debris or broken teeth were seen among those dependent on help with self‐care ( The number and proportion of older people in Sweden is increasing, as in many other countries Good oral health is important for people's well‐being, nutrition, proper healing, self‐esteem, social satisfaction and quality of life as well as overall health Although it can be assumed that clinical observations alone do not fully indicate how people experience their oral health, assessment of oral health is mostly performed by dental professionals and rarely takes the person's subjective perceptions and satisfaction with oral health into consideration Approximately 20% of the population in Sweden are 65 years or older. In 2016, there were almost 9% receiving home help services in their own home, about 4% lived in special housing, and the number of people in short‐term care was almost 1% Swedish municipalities are responsible for health care in special accommodation and short‐term care for older people. Short‐term care is intended to meet the temporary care needs of older people following hospitalisation, awaiting a decision on permanent special accommodation or providing intermittent care, recurrent relief for family caregivers, rehabilitation and palliative care To obtain a holistic perspective on oral health and provide more person‐centred care, it is important to recognise people's perceptions of their own oral health and not just clinical indicators of oral disease There are several studies describing oral and dental health among older people living in special accommodation In conclusion, knowledge is limited about the oral health of older people and whether their oral care needs are met in short‐term care settings. The aim of this study was to describe oral health, daily oral care and related factors among older people in short‐term care and to compare the older people's self‐perceived oral health with professional assessment of oral health.This descriptive cross‐sectional study was carried out within the framework of an ongoing research project conducted in five counties: SOFIA . The overall aim of the SOFIA project is to describe and analyse oral health and oral health‐related quality of life, swallowing and eating ability, nutritional risk, care quality in relation to oral health and eating and to study the effectiveness of a swallowing training programme among older persons who are admitted to short‐term care Older people admitted to the selected short‐term care units during a 3‐year period were eligible for study participation. The inclusion criteria were being 65 years or older, having spent at least 3 days at the short‐term care unit, being able to understand Swedish and having sufficient cognitive ability (judged by the nurse in charge) to answer questions A convenience sample of five out of 21 Swedish counties was asked to participate. After approval by the head of social welfare of elderly care in each municipality, heads of unit were contacted to provide information on the study and request approval to visit the short‐term unit. The Registered Nurse in charge at the accommodation made an initial assessment about which older persons fulfilled the inclusion criteria and could be invited to participate in the study. The research assistants [eight registered dental hygienists (RDHs) and one speech language pathologist] informed the participants both orally and in writing about the purpose of the study and the procedures involved in participating, clarified the matter of confidentiality and obtained written consent. Questions about the older people's main medical diagnoses were answered by the Registered Nurse, and questions about the older people's self‐care ability were answered by the licensed practical nurse or the Registered Nurse. The RDHs carried out a clinical assessment using a mouth mirror and a flashlight and collected self‐reported questionnaire data by asking the participants questions The data collection was conducted according to ethical principles and included informed consent, confidentiality and the right to withdraw from participation at any time without presenting a reason. If a severe oral health problem was detected, the research assistant informed the participant and the responsible nurse about the need to make contact with dental care for treatment. The study was approved by the Regional Ethical Review Board, Uppsala University, Sweden (Dnr 2013/100).Self‐care ability was assessed with Katz Index of Activities of Daily Living (Katz‐ADL) The oral assessment performed by the RDHs included recording the number of natural teeth, presence of bridges, partial or full dentures and implants, need for dental care and an estimation of oral hygiene in terms of three categories from good to poor. Additionally, one question was asked about the person's ability to brush their own teeth, with three response options: 1 = ‘Yes, completely able’, 2 = ‘Receive some help’ and 3 = ‘No, receive help entirely’ Oral health was measured using an adapted version of ROAG: the Revised Oral Assessment Guide‐Jönköping (ROAG‐J) A global question was used to assess self‐perceived oral health: ‘Are you generally pleased with your mouth and your teeth?’. There were four response alternatives, ranging from ‘very satisfied’ to ‘not at all satisfied’ Descriptive results are shown as frequencies with percentages or means with standard deviations (SD). The ADL index was divided into three categories: A = independent, B–D = partly dependent and E–G = completely dependent Percentage agreement and Cohen's kappa coefficient (k) were calculated to measure the agreement between the clinical oral assessment  and the older people's self‐perceived oral health (satisfied vs. not satisfied). Overall percentage agreement was calculated by taking the number of agreements between the two measurements, dividing this by the total number of readings and multiplying the result by 100. Agreement was considered to occur when either both the older person and the RDH assessed oral health as good  or when both assessed oral health as poor . Cohen's kappa coefficient adjusts for agreements due to chance, and values <0.2 are considered as poor, 0.21–0.40 as fair, 0.41–60 as moderate, 0.61–0.80 as good and >0.80 as very good agreement ibm spss software package .Two separate multivariate logistic regression analyses were conducted, yielding adjusted odds ratios (ORs) with 95% confidence intervals (CIs). The dependent variable in the first analysis was self‐perceived oral health , and the dependent variable in the second analysis was oral problems based on clinical assessment . The independent variables in both analyses were gender, age, education, number of teeth, removable dentures, oral self‐care, need for dental care and ADL index. Statistical significance was set at p < 0.05. Data were analysed using version 22 of the The results are based on 391 older people from 36 short‐term care units in five counties. Their ages ranged from 65 years to 100 years , and they comprised 209 (53%) women and 182 (47%) men  older people were completely edentulous, and 167 (43%) had 20 teeth or more. The presence of removable dentures (full or partly), implants and bridges was indicated if they were observed either in one jaw or both jaws. Two people had missing data regarding dental status  of the older people. Older people with implants assessed having coating or food debris are graded in category teeth. Numbers and percentages of identified oral health problems are shown in Table Score on Katz's ADL index was associated with the ability to brush one's own teeth (p < 0.001). None (0%) of the older people with grade A  received either partial or total help, while the corresponding figures for grades B–D (dependence in one to three ADL) and E–G  were 12 (7.3%) and 61 (33%), respectively. ADL index was also associated with dental status according to ROAG (p = 0.002). Seven (33%) of the older people with grade A had local/general coating or food debris or broken teeth, while the corresponding figures for grades B–D and E–G were 70 (52%) and 105 (67%), respectively.A majority of the older people  reported being very satisfied or generally satisfied with their oral health. However, the assessment based on ROAG found oral problems in 297 (77%) of the total group.k = 0.047), and the overall percentage agreement between professional assessment and the older people's self‐perceived oral health was only 34%. Overall, 21% of the older people were both satisfied with their oral health and clinically assessed as being without oral problems. Oral health was assessed by RDHs as being worse than the participants’ perceptions in 64% of all assessments and better than the participants’ perceptions in 2% of the assessments. The percentage agreements between professional assessment and the older people's self‐perceived oral health are presented in Fig. When comparisons were made between the assessment based on ROAG and the older people's self‐perceived oral health, a low level of agreement was found. The kappa coefficient showed very poor agreement  compared to participants with no teeth . The odds for having oral problems were nearly five times higher for participants with a need for dental care compared to those with no such need . Finally, participants with ADL index E‐G  had 3.4 times higher odds of having oral problems compared to participants who were independent (ADL index A) in all activities . There were no other statistically significant results.This study shows that professional assessments of oral health differed considerably from the self‐perceived oral health of older people in short‐term care settings. Although the majority of the older people had oral health problems, only 19% received help with daily oral care. Older people who were dependent on help with self‐care according to Katz's ADL index had around a sixfold higher risk of oral problems and a doubled risk for presence of local/general coating or food debris or broken teeth, according to ROAG.k = 0.047). These findings are in agreement with a previous study which showed that older people's self‐perceived oral health often differed from health professionals’ oral assessments, with nursing staff assessing oral health as being poorer than the patients did To our knowledge, this is the first study comparing self‐perceived oral health and clinical assessment among frail older people in the context of short‐term care. A large majority (85%) of the older people reported themselves as being very satisfied or generally satisfied with their oral health, while the RDHs only identified 23% to be without oral problems. The older people in this study had a high number of remaining natural teeth, which confirms findings from recent studies in Sweden Almost half of the older people were assessed as having less good to poor oral hygiene, and according to the ROAG assessment, coating or food debris was present locally or generally on both teeth and dentures in almost 60% of the older persons. This indicates a lack of proper oral care and points to the importance of regular oral assessments. Despite the observed care needs, only a fifth of the older persons received any help from care staff with their daily oral care. This low level of help with oral care seems inadequate, considering the large number of older people in the sample who were highly dependent on help with activities of daily living. According to the ADL assessment, half of the sample were dependent on help with four to six activities such as bathing, dressing and undressing, going to the toilet, mobilisation, controlling bowel and bladder, and food intake. Only about one‐third of the older people in this group received partial or total help with oral self‐care. A study from a geriatric ward in Sweden found that patients depending on help with ADL also had poor oral health status, which indicates that older people who depend on support with personal hygiene should also be assumed to need help with oral self‐care The combination of natural teeth, removable dentures and bridges also leads to more complex oral care needs Interventions to improve oral care among older people in special accommodation should include both nursing staff and dental care staff to foster teamwork Factors such as gender, age, number of teeth, removable dentures, ability to perform oral self‐care and dependence on help with ADL did not influence the older people's self‐perceived oral health in this study. However, older people who were not satisfied with their oral health had higher educational level and higher need of dental care. We also examined which factors may affect older people's oral problems based on clinical assessment with ROAG. Older people who were dependent on help with self‐care  had around a sixfold higher risk of oral problems. This result is in line with previous studies that also found increasing dependency to be associated with oral health problems among older people in need of care An experience gained during the data collection, as well as recognised from daily practice, is that some older people are reluctant to accept assistance with their oral care, although this is offered. The reason for the imbalance between oral care needs and care provided should to be further studied. Qualitative studies should be conducted to find out why older people do not always receive, or accept, help with oral care and to explore their experiences of receiving oral care. It is also important to increase our knowledge about the different factors that impact older people's self‐perceived oral health and oral health‐related quality of life.These results can be useful for both dental and nursing staff to improve older peoples′ oral health, by ensuring good daily oral care. It also shows that it is not enough to just ask an older person about oral health, an assessment of the oral health also needs to be performed. Oral health and the ability to independently perform daily oral care should be examined in a similar way as ADL capacity.There is a lack of studies conducted in the short‐term care context, which may be due to methodological and ethical problems related to obtaining informed consent and controlling for confounding factors. It is challenging to include older people with weak health and functional disabilities The research assistants read the questions to the participants in order to make it easier to understand and answer all the questions. Answering questionnaires might be exhausting for older people, and misunderstandings could be corrected by supporting the participants in completing the task. On the other hand, there might be a risk of bias if older people are given support in reading and interpreting the questions. The strength of the assessments was that the research assistants who collected data on oral health were registered dental hygienists with relevant clinical experience in communicating with older people and assessing their oral health. The examinations were either conducted in the morning, after lunch or in the afternoon and sometimes divided over days, dependent on the older person's ability to participate. This might reduce the risk of bias as the timing in relation to food intake and oral care and oral examinations varied. All research assistants were trained in using the different instruments and met regularly with the research group to ensure consistency in assessments. The duration of the study was approximately two and a half years due to the inclusion of one more county in order to achieve sufficient power for the larger SOFIA study, which this study was part of There was poor agreement between professional clinical assessment of oral health and self‐perceived oral health among older people in short‐term care. The majority of the participants was satisfied with their oral health although the clinical assessment often showed poor oral health. Those dependent on help with self‐care (ADL) had much higher risk of having oral problems and more occurrence of coating or food debris or broken teeth. This all together demonstrates the importance of providing person‐centred oral care and that close collaboration between nursing and dental staff must increase in order to improve older peoples′ oral health and oral care.SK designed the study, collected data, analysed data and drafted the paper. AF contributed with statistical analyses and draft of the paper. LO, KS and AE contributed in designing, analysing and drafting of the paper.Ethical approval was obtained from the Regional Ethical Review Board, Uppsala University, Sweden (Dnr 2013/100).This study was supported by grants from Örebro County; the Regional Research Board of Uppsala‐Örebro; the Swedish Research Council for Health, Working Life, and Welfare (Forte); the Kamprad Family Foundation for Entrepreneurship, Research, and Charity. This study was accomplished within the context of the Swedish National Graduate School for Competitive Science on Ageing and Health (SWEAH), funded by the Swedish Research Council.