To Örebro University

Sudden cardiac death (SCD) is a tragic and traumatic event. SCD is often associated with hereditary genetic disease and in such cases, sequencing of stored formalin fixed paraffin embedded (FFPE) tissue is often crucial in trying to find a causal genetic variant. This study was designed to compare two massive parallel sequencing assays for differences in sensitivity and precision regarding variants related to SCD in FFPE material. From eight cases of SCD where DNA from blood had been sequenced using HaloPlex, corresponding FFPE samples were collected six years later. DNA from FFPE samples were amplified using HaloPlex HS, sequenced on MiSeq, representing the first method, as well as amplified using modified Twist and sequenced on NextSeq, representing the second method. Molecular barcodes were included to distinguish artefacts from true variants. In both approaches, read coverage, uniformity and variant detection were compared using genomic DNA isolated from blood and corresponding FFPE tissue, respectively. In terms of coverage uniformity, Twist performed better than HaloPlex HS for FFPE samples. Despite higher overall coverage, amplicon-based HaloPlex technologies, both for blood and FFPE tissue, suffered from design and/or performance issues resulting in genes lacking complete coverage. Although Twist had considerably lower overall mean coverage, high uniformity resulted in equal or higher fraction of genes covered at ≥ 20X. By comparing variants found in the matched samples in a pre-defined cardiodiagnostic gene panel, HaloPlex HS for FFPE material resulted in high sensitivity, 98.0% (range 96.6-100%), and high precision, 99.9% (range 99.5-100%) for moderately fragmented samples, but suffered from reduced sensitivity (range 74.2-91.1%) in more severely fragmented samples due to lack of coverage. Twist had high sensitivity, 97.8% (range 96.8-98.7%) and high precision, 99.9% (range 99.3-100%) in all analyzed samples, including the severely fragmented samples.

Forensic molecular autopsies have emerged as a tool for medical examiners to establish the cause of death. It is particularly useful in sudden unexplained deaths where the cause of death cannot be determined with a regular medical autopsy. We provide the first study of exome data from formalin-fixed paraffin-embedded samples (FFPE) paired with data from high-quality blood samples in forensic applications. The approach allows exploration of the potential to use FFPE samples for molecular autopsies and identify variants in extensive exome data. We leverage the high uniformity of the hybridization capture approach provided by Twist Bioscience to target the complete exome and sequence the libraries on a NextSeq 550. Our findings suggest that exome sequencing is feasible for 24 out of a total of 35 included FFPE samples. When successful, the coverage across the exome is comparatively high (> 90% covered to 20X) and uniform (fold80 below 1.5). Detailed variant comparisons for matched FFPE and blood samples show high concordance with few false variants (positive predictive value of 0.98 and a sensitivity of 0.97) with no distinct FFPE artefacts. Ultimately, we apply carefully constructed forensic gene panels in a stepwise manner to find genetic variants associated with the clinical phenotype and with relevance to the sudden unexplained death.

Sudden unexpected death (SUD) is an unexpected event that in many cases are caused by diseases with an underlying genetic background. Forensic molecular autopsy is an approach that has gained wide-spread attention, in part explained by the rapid progress of DNA sequencing techniques. The approach leverages genetic data in combination with medical autopsy findings in post-mortem samples to explore a potential underlying genetic cause of death. Traditional forensic approaches to molecular autopsy focus on a small panel of genes, say <200 genes, with strong association to heart conditions whereas clinical genetics tend to capture entire exomes while subsequently selecting targeted panels bioinformatically. The drop in price and the increased throughput has promoted wider exome sequencing as a viable method to discover genetic variants. We explore a targeted gene panel consisting of 2422 genes, selected based on their broad association to sudden unexplained death. A hybridization capture approach from Twist Bioscience based on double stranded DNA probes was used to target exons of the included genes. We selected and sequenced a total of 98 post-mortem samples from historical forensic autopsy cases where the cause of death could not be unambiguously determined based on medical findings and that had a previous negative molecular autopsy. In the current study, we focus on the performance of the hybridization capture technology on a 2422 gene panel and explore metrics related to sequencing success using a mid-end NextSeq 550 as well as a MiSeq FGx platform. With the latter we demonstrate that our sequence data benefits from 2×300 bp sequencing increasing coverage, in particular, for difficult regions where shadow coverage, i.e. regions outside the probes, are utilized. The results further illustrate a highly uniform capture across the panel of genes (mean fold80=1.5), in turn minimizing excessive sequencing costs to reach sufficient coverage, i.e. 20X. We outline a stepwise procedure to select genes associated with SUD through virtual bioinformatical panels extracting tier of genes with increasing strength of association to SUD. We propose some prioritization strategies to filter variants with highest potential and show that the number of high priority genetic variant requiring manual inspections is few (0-3 for all tiers of genes) when all filters are applied.

We investigated differences in circulating cell-free DNA (cfDNA) methylation between patients with cancer and those presenting with severe, nonspecific symptoms. Plasma cfDNA from 229 patients was analyzed, of whom 37 were diagnosed with a wide spectrum of cancer types within 12 months. Samples underwent enzymatic conversion, library preparation, and enrichment using the NEBNext workflow and Twist pan-cancer methylation panel, followed by sequencing. Methylation analysis was performed with nf-core/methylseq. Differentially methylated regions (DMRs) were identified with DMRichR. Machine learning with cross-validation was used to classify cancer and controls. The classifier was applied to an external validation set of 144 controls previously unseen by the model. Cancer samples showed higher overall CpG methylation than controls (1.82% vs. 1.34%, p < 0.001). A total of 162 DMRs were detected, 95.7% being hypermethylated in cancer. Machine learning identified 20 key DMRs for classification between cancer and controls. The final model achieved an AUC of 0.88 (83.8% sensitivity, 83.8% specificity), while mean cross-validation performance reached an AUC of 0.73 (57.1% sensitivity, 77.5% specificity). The specificity of the classifier on unseen control samples was 79.2%. Distinct methylation differences and DMR-based classification support cfDNA methylation as a robust biomarker for cancer detection in patients with confounding conditions.

Purpose: The aim of this systematic literature review was to obtain an overview of when to administer the diuretics in relation to the radiopharmaceutical during a diuresis renography.

Methods: A systematic literature search was performed in three different databases (Embase, PubMed/Medline and Cochrane Library) together with an information specialist. The review question was: when should diuretics be administered in relation to the radiopharmaceutical in a diuresis renography? Studies of adults were included together with guidelines published in collaboration with an organization.

Results: Seventeen articles and four guidelines were retrieved in the literature search. The F - 15 method (diuretics administered 15 min before the radiopharmaceutical) was the one that was studied and described most and was compared with other time points for diuretic administration. The retrieved articles and guidelines report of advantages with different time points for diuretics. Both F - 15 and F + 0 are reported to clarify washout in equivocal cases compared to F + 20.

Conclusion: No consensus could be found for a preferred time point of diuretics administration during a diuresis renography.

OBJECTIVE: The F+10 method for diuretic renography (diuretics given 10 min after the radiopharmaceutical) could be a time-conserving method. This method involves a 30-min dynamic acquisition where diuretics are administered only when necessary by the Nuclear Medicine technologist performing the examination. The purpose of this study was to assess the method's performance and to discover the optimal threshold of residual activity for a diuretic administration 7 min into the F+10 renography by reprocessing raw data from prior performed examinations with 20-min acquisitions without diuretics.

METHODS: Retrospectively, raw data from 320 original examinations of adult patients performed from 2013 to 2015 were reprocessed into 7-min series and categorized as requiring diuretic or not. The diuretic decisions made by an expert panel were used as a reference. A receiver-operating characteristic curve was drawn to assess the optimal cutoff value for the residual renal activity. Sensitivity, specificity, positive and negative predictive values, as well as the Youden J index were calculated.

RESULT: The experts classified 50% (160 examinations) as in need of diuretics. The receiver-operating characteristic curve demonstrated the theoretical optimal cutoff value at 7 min to be 94% of maximum activity (sensitivity 0.93, specificity 0.81, Youden J index 0.73). A clinically acceptable threshold is suggested to be 85% (sensitivity 0.99, specificity 0.59, Youden J index 0.58).

CONCLUSION: Tc-mercaptoacetyltriglycine renography with the F+10 method and the threshold 85% for diuretic decision 7 min into the renography is a feasible and acceptable method in clinical practice.

INTRODUCTION: Diuresis renography is a nuclear medicine examination that can distinguish between obstructive and non-obstructive uropathy. Renography has been investigated before, but not from a patient perspective. The aim of this study was to gain knowledge and understanding of patient experiences with the diuresis renography process.

METHODS: The study had a qualitative descriptive design; data was collected through semi-structured interviews with 17 adult patients that had recently undergone diuresis renography. The transcripts were analysed using inductive qualitative content analysis to identify their manifest and latent content.

RESULTS: One main theme was identified: 'Not smooth all the way through, but still in good hands'. The participants understood the necessity of the performed renography and endured the examination despite its worrying elements. They were bothered by diuretic effects, worried about radioactivity and felt isolated during the imaging. The technologists' caring and informative approach eased their experience. The main theme was identified from three subthemes: 'I experienced inconvenience', 'I was worried' and 'I felt safe'.

CONCLUSION: The participants experienced worry throughout the renography process but were still satisfied with the examination, mostly because of the technologists being involved. Communication and interaction between patient and technologist are important for creating a good experience. Improved knowledge of patient experiences during renographies could be used to develop patient information and increase technologists' awareness of factors patients may experience as troubling.

IMPLICATIONS FOR PRACTICE: This study describes issues that contribute to both negative and positive experiences. Improved professional awareness and knowledge about these issues can contribute to the development of patient information and professional encounters, and may improve patients' compliance and overall experience.

PURPOSE: To assess the accuracy of conventional MRI with T1- and T2-weighted sequences in detecting lymphatic nodal spread (N1) in intermediate- and high-risk prostate cancer (PCa) patients via morphological criteria alone, extended pelvic lymph node dissection (ePLND) was used as the reference standard.

METHODS: This prospective observational study included patients between 2009 and 2016 with intermediate- and high-risk PCa according to the D'Amico criteria and an estimated risk of N1 > 20% on the basis of the Briganti nomogram. All patients underwent MRI prior to ePLND. Interobserver analysis was conducted across three centers.

RESULTS: Ninety-nine men, mean age 67 (5.7 SD), 93% high-risk PCa patients and 39.4% with N1 disease, according to ePLND, were evaluated. The pooled sensitivity of MRI for detecting N1 was 24.6% (95% CI: 16.3-35.1), whereas the pooled specificity was 95% (95% CI: 85.3-98.8). Interobserver agreement was moderate (Fleiss' κ = 0.56). All readers failed to identify patients with high-volume N1, and the identification of those with a high number of N1 events was inconsistent across readers. The strengths of this study include the high number of N1 cases, with a median of 17 (6-40) harvested lymph nodes per participant. Limitations include the time interval between MRI and ePLND (median of 44 days) and the lack of standardized lymph node evaluation criteria, reflecting real-world clinical practice.

CONCLUSION: MRI using only T1W and T2W sequences has demonstrated limited effectiveness in lymph node staging for intermediate- and high-risk prostate cancer, even in high-volume metastatic disease. Additionally, interobserver analysis shows only moderate agreement.

AIM: To prospectively validate 3 T magnetic resonance imaging (MRI) including diffusion-weighted imaging (DWI) for preoperative lymph node (LN) staging in a clinical setting, in intermediate- and high-risk prostate cancer (PCa) patients using laparoscopic extended LN dissection (ePLND) as the reference standard.

MATERIALS AND METHODS: Between August 2011 and May 2013, 40 newly diagnosed intermediate and high-risk PCa patients underwent preoperative LN staging with 3 T MRI DWI using histopathology of ePLND as the reference standard. The sensitivity, specificity, and accuracy of MRI DWI were calculated. A subgroup analysis of proven LN-positive patients was made to investigate differences in PSA, Gleason score, number, and size of LN metastases, estimated risk of LN involvement, and if curative treatment was indicated, between the true-positive and the false-negative groups.

RESULTS: A total of 728 LN were harvested from six anatomical regions per patient (external, obturator, internal) with a mean number of 18 LNs per patient (range 11-40). Twenty patients had histologically proven LN-positive disease. MRI DWI was true positive in 11 patients, false negative in nine patients, false positive in two patients, and true negative in 18 patients, resulting in 90% specificity, 55% sensitivity, and 72.5% accuracy. The true-positive patients had significantly more involved LNs (mean 6.9 versus 2.7, p=0.017), with larger diameter (mean 12.3 versus 5.2 mm, p=0.048) and fewer were treated with curative intent (six versus nine, p=0.03), compared with the false-negative group.

CONCLUSION: MRI DWI LN staging has a low sensitivity but high specificity. The true-positive patients have a considerably higher burden of LN metastases compared to false-negative patients.

OBJECTIVES: To evaluate the accuracy of the radiopharmaceutical [(11) C]-acetate combined with positron emission tomography/computer tomography (acetate-PET/CT) in lymph node (LN) staging in newly diagnosed prostate cancer cases. A second aim was to evaluate the potential discriminative properties of acetate-PET/CT in clinical routine.

PATIENTS AND METHODS: In a prospective comparative study, from July 2010 to June 2013, 53 men with newly histologically diagnosed intermediate- or high-risk prostate cancer underwent acetate-PET/CT investigation at one regional centre before laparoscopic extended pelvic LN dissection (ePLND) at one referral centre. The sensitivity, specificity and accuracy of acetate-PET/CT were calculated. Comparisons were made between true-positive and false-negative PET/CT cases to identify differences in the clinical parameters: PSA level, Gleason status, lymph metastasis burden and size, calculated risk of LN involvement, and curative treatment decisions.

RESULTS: In all, 26 patients had surgically/histologically confirmed LN metastasis (LN+). Acetate-PET/CT was true positive in 10 patients, false positive in one, false negative in 16, and true negative in 26. The individual sensitivity was 38%, specificity 96%, and accuracy 68%. The acetate-PET/CT positive cases had significantly more involved LNs (mean 7.9 vs 2.4, P < 0.001) with larger cancer diameters (14.1 vs 4.9 mm, P = 0.001) and fewer eventually had treatment with curative intent (40% vs 94%, P <0.005), although we lack long-term outcome data.

CONCLUSION: Acetate-PET/CT has too low a sensitivity for routine LN staging but the specificity is high. The acetate-PET/CT positive cases have a very high burden of LN spread.

Background: The prevalence of disabling hearing loss is increasing worldwide. However, previous studies on hearing loss prevalence have enrolled small populations or only provided estimates.

Aim: To establish the prevalence of severe-to-profound hearing loss (STPHL) in the adult Swedish population and compare it with the cochlear implantation rate in Sweden.

Material and methods: We established a database containing over 15 million audiograms obtained from regions covering > 99% of the Swedish population by extracting audiogram data from the computer software application, Auditbase. We used this database to calculate the percentage of adult patients with bilateral hearing levels >= 70 dB. We collected data regarding cochlear implantations in Sweden from the National Board of Welfare and Health.

Results: The prevalence of STPHL in the adult Swedish population was 0.28%. There were regional variations in the prevalence and rate of cochlear implantation; however, there was no association between both parameters.

Conclusions: This study presents an updated and reliable prevalence figure for STPHL in Sweden.

Significance: Patients with STPHL have extensive rehabilitation requirements; accordingly, it is important to determine the accurate prevalence of STPHL to inform the allocation of adequate resources.

Severe-to-profound hearing loss (STPHL) can affect a person negatively in many ways. Audiological rehabilitation is important for these patients. Patients receiving cochlear implants make up less than 10% of this group but have been studied extensively. In 2005, a national registry for adult patients with STPHL was introduced in Sweden. Its purpose was to evaluate and improve rehabilitation for all patients with STPHL. Data from the Swedish registry for adult patients with STPHL were used to evaluate variables affecting the audiological rehabilitation. Previous published data from the registry were reviewed, and new data from the follow-up questionnaire were presented. More than 90% of patients rehabilitated with hearing aids experienced a good or very good benefit of audiological rehabilitation. Tinnitus and vertigo affected quality of life negatively and were reported by many patients with STPHL (41% and 31%) at follow-up. To maintain the high number of patients who find audiological rehabilitation beneficial, individualized treatment plans and timely re-evaluations are crucial. Tinnitus and vertigo need to be addressed repeatedly in the rehabilitation process.

OBJECTIVE: Comparison of asymmetric hearing in patients with severe-to-profound hearing loss (STPHL) and those with less pronounced or no hearing loss. The influence of asymmetric hearing on cochlear implantation (CI) was also analyzed.

STUDY DESIGN: Retrospective cohort.

SETTING: Hearing clinics across all regions in Sweden. PATIENTS: This study included 898,421 adult patients identified using the audiometric software Auditbase and 4286 adult patients from the Swedish National Quality Register for STPHL.

MAIN OUTCOME MEASURES: Proportion of patients with asymmetric hearing according to 11 different asymmetry criteria. Adjusted odds ratios for the presence of cochlear implants, adjusted for age, sex, and the highest level of education.

RESULTS: Across the different criteria, asymmetric hearing ranged from 20% to 59% in STPHL patients and 14% to 46% in those with better hearing. Adjusted odds ratios showed that cochlear implants were more common in patients with >15 dB asymmetry in the pure tone average at the base and mid-range frequencies.

CONCLUSION: This study of national audiogram data from Sweden showed that asymmetric hearing was more common in patients with STPHL. The higher proportion of CI in patients with asymmetric hearing indicates that asymmetric hearing is a complicating factor that must be considered in the rehabilitation of STPHL.

This study examines how a tinkering task in visual programming environments can provide opportunities for developing problem solving skills. We pursue this by analyzing eleven students in year 8 working with a tinkering task in a visual programming environment during a mathematics class. They were asked to create repeating patterns. Their work and discussions were analyzed through a lens of abstraction and decomposition, two elements of computational thinking. The analysis reveals how some students became thoroughly engaged in problem solving while others had a shallower experience of randomly manipulating the pre-made code. Since it was not the difficulty of the task but rather the random outcome of their manipulation of the code that determined whether they became engaged or not suggests that there is a need for support structures to fully tap into the potential of tinkering tasks to elicit problem solving.

Visual programming has become a popular way to provide young students with the opportunity to engage in complex problem-solving, often referred to as computational thinking. Most frameworks for computational thinking are comprehensive yet lack a fine-grained perspective on young students' engagement in programming. Therefore, this paper aims to unpack young students’ enacted problem-solving as they engage in open-ended programming in a visual programming environment (VPE). Through the lens of abstracting and decomposing, we contribute with a close-up understanding of how young students tackle complex problems in a VPE. Data generated in a Swedish first-grade classroom consisted of screen recordings and plans from 13 student pairs, working during four lessons to produce animated stories in ScratchJr. Through the analysis, six enactment patterns were constructed, showing that students as young as seven to eight years old can solve problems involving multiple components, arranging them into cohesive solutions. There were major differences in the problem-solving process involving whether or not the students followed their plans, captured in plan-driven and in-the-moment enactment patterns. Plan-driven enactment patterns elicited shifts within abstracting and decomposing practices, which we consider essential for programming since such enactment patterns more often elicit complex problem-solving. The results suggest that young students should be encouraged to use plans extensively when working in a VPE.

There is a lack of knowledge in research about the intersection between programming and mathematics in an educational context relevant to young students. This paper aims to contribute to the research field with a theoretical framework that captures this intersection. The intersection is explored by interpreting open programming tasks in a visual programming environment through the lens of Bishop’s six mathematical activities, resulting in a framework that shows how mathematical activities intersect with either a context-independent programming process or context-dependent programming processes.