881. Assessment of the real-time PCR and different digital PCR platforms for DNA quantificationJernej Pavšič, Jana Žel, Mojca Milavec, 2016, original scientific article Abstract: Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.
Keywords: real-time PCR, molecular diagnostics, human cytomegalovirus, DNA quantification, digital PCR
Published in DiRROS: 25.07.2024; Views: 286; Downloads: 177
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882. Digital PCR for direct quantification of viruses without DNA extractionJernej Pavšič, Jana Žel, Mojca Milavec, 2016, original scientific article Abstract: DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.
Keywords: molecular diagnostics, direct quantification, viruses, virus reference materials, human cytomegalovirus, digital PCR
Published in DiRROS: 25.07.2024; Views: 252; Downloads: 212
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883. Assessment of short-term effect of platelet-rich plasma treatment of tendinosis using texture analysis of ultrasound imagesKarlo Pintarić, Vladka Salapura, Žiga Snoj, Andrej Vovk, Mojca Božič, Jernej Vidmar, 2023, original scientific article Keywords: texture analysis, ultrasonography, tendinosis
Published in DiRROS: 25.07.2024; Views: 284; Downloads: 116
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885. The effects of normobaric and hyperbaric oxygenation on MRI signal intensities in T1-weighted, T2-weighted and FLAIR images in human brainVida Velej, Ksenija Cankar, Jernej Vidmar, 2023, original scientific article Abstract: Dissolved oxygen has known paramagnetic effects in magnetic resonance imaging (MRI). The aim ofthis study was to compare the effects of normobaric oxygenation (NBO) and hyperbaric oxygenation (HBO) on hu-man brain MRI signal intensities.Patients and methods. Baseline brain MRI was performed in 17 healthy subjects (mean age 27.8 ± 3.2). MRI wasrepeated after exposure to the NBO and HBO at different time points (0 min, 25 min, 50 min). Signal intensities in T1-weighted, T2-weighted images and fluid attenuated inversion recovery (FLAIR) signal intensities of several intracranialstructures were compared between NBO and HBO.Results. Increased T1-weighted signal intensities were observed in white and deep grey brain matter, cerebrospinalfluid (CSF), venous blood and vitreous body after exposure to NBO as well as to HBO compared to baseline (Dunnett’stest, p < 0.05) without significant differences between both protocols. There was also no significant difference in T2-weighted signal intensities between NBO and HBO. FLAIR signal intensities were increased only in the vitreous bodyafter NBO and HBO and FLAIR signal of caudate nucleus was decreased after NBO (Dunnett’s test, p < 0.05). Thestatistically significant differences in FLAIR signal intensities were found between NBO and HBO (paired t-test, p < 0.05)in most observed brain structures (paired t-test, p < 0.05).Conclusions. Our results show that NBO and HBO alters signal intensities T1-weighted and FLAIR images of humanbrain. The differences between NBO and HBO are most pronounced in FLAIR imaging.
Keywords: hyperbaric oxygen, normobaric oxygen, magnetic resonance, human brain
Published in DiRROS: 25.07.2024; Views: 262; Downloads: 108
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886. Local control and survival after stereotactic body radiation therapy of early-stage lung cancer patients in SloveniaKarmen Stanič, Jasna But-Hadžić, Jan Žagar, Martina Vrankar, 2023, original scientific article Abstract: Background. Stereotactic body radiation therapy (SBRT) precisely and non-invasively delivers ablative radiationdose to tumors in early-stage lung cancer patients who are not candidates for surgery or refuse it. The aim of researchwas to evaluate local control, overall survival (OS), local progression free survival (LPFS), distant metastases free survival(DMFS), disease free survival (DFS) and toxicity in early-stage lung cancer patients treated with SBRT in a single tertiarycancer centre.Patients and methods. We retrospectively evaluated medical records and radiation treatment plan parametersof 228 tumors irradiated in 206 early-stage lung cancer patients between 2016 and 2021 at the Institute of OncologyLjubljana.Results. After 25 months of median follow up, 68 of 206 (33%) patients died. Median OS was 46 months (CI 36 −56),1-year, 2-year and 3-year OS were 87%, 74% and 62% and 5-year OS was 31%. A total of 45 disease progressions havebeen identified in 41 patients. Local progress only was noticed in 5 (2%) patients, systemic progress in 32 (16%) andcombined systemic and local in 4 (2%) patients. Local control rate (LCR) at 1 year was 98%, at 2 and 3 years 96%and 95% at 5 years. The 1-, 2- and 3-year LPFS were 98%, 96% and 94%, respectively and 5-year LPFS was 82%. One,2-, 3- and 5-year DFS w ere 89%, 81%, 72% and 49%, respectively. Among 28 toxicities recorded only one was Grade4 (pneumonitis), all others were Grade 1 or 2. No differences in LCR, LPFS, DFS were found in univariate analysis com-paring patient, tumor, and treatment characteristics. For OS the only statistically significant difference was found inpatients with more than 3 comorbidities compared to those with less comorbidities.Conclusions. Early lung cancer treated with SBRT at single tertiary cancer centre showed that LCR, LPFS, DFS, DMFSand OS were comparable to published studies. Patients with many comorbidities had significantly worse overallsurvival compared to those with less comorbidities. No other significant differences by patient, tumor, or treatmentcharacteristics were found for DMFS, LPFS, and DFS. Toxicity data confirmed that treatment was well tolerated.
Keywords: stereotactic body radiotherapy, early-stage lung cancer, lung cancer
Published in DiRROS: 25.07.2024; Views: 263; Downloads: 133
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887. Breast cancer risk assessment and risk distribution in 3,491 Slovenian women invited for screening at the age of 50 : a population-based cross-sectional studyKatja Jarm, Vesna Zadnik, Mojca Birk, Miloš Vrhovec, Kristijana Hertl, Žan Klaneček, Andrej Studen, Cveto Šval, Mateja Krajc, 2023, original scientific article Abstract: Background. The evidence shows that risk-based strategy could be implemented to avoid unnecessary harm in mammography screening for breast cancer (BC) using age-only criterium. Our study aimed at identifying the uptake of Slovenian women to the BC risk assessment invitation and assessing the number of screening mammographies in case of risk-based screening.Patients and methods. A cross-sectional population-based study enrolled 11,898 women at the age of 50, invited to BC screening. The data on BC risk factors, including breast density from the first 3,491 study responders was col-lected and BC risk was assessed using the Tyrer-Cuzick algorithm (version 8) to classify women into risk groups (low, population, moderately increased, and high risk group). The number of screening mammographies according to risk stratification was simulated. Results. 57% (6,785) of women returned BC risk questionnaires. When stratifying 3,491 women into risk groups, 34.0% were assessed with low, 62.2% with population, 3.4% with moderately increased, and 0.4% with high 10-year BC risk. In the case of potential personalised screening, the number of screening mammographies would drop by 38.6% com-pared to the current screening policy. Conclusions. The study uptake showed the feasibility of risk assessment when inviting women to regular BC screen-ing. 3.8% of Slovenian women were recognised with higher than population 10-year BC risk. According to Slovenian BC guidelines they may be screened more often. Overall, personalised screening would decrease the number of screening mammographies in Slovenia. This information is to be considered when planning the pilot and assessing the feasibility of implementing population risk-based screening.
Keywords: breast cancer screening, personalised screening, risk assessment, mammography
Published in DiRROS: 25.07.2024; Views: 259; Downloads: 230
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888. Correlation of t(14;18) translocation breakpoint site with clinical characteristics in follicular lymphomaMatej Panjan, Lučka Boltežar, Srdjan Novaković, Ira Koković, Barbara Jezeršek Novaković, 2023, original scientific article Abstract: Background: t(14;18)(q32;q21) translocation is an important genetic feature of follicular lymphoma resulting in antiapoptotic B-cell lymphoma 2 (BCL2) protein overexpression. On chromosome 18 breakpoint-site variation is high but does not affect BCL2. Breakpoint most commonly occurs at major breakpoint region (MBR) but may happen at minor cluster region (mcr) and between MBR and mcr at 3'MBR and 5'mcr. The aim of this study was to analyze the correlation of t(14;18)(q32;q21) breakpoint site with clinical characteristics in follicular lymphoma. Patients and methods: We included patients diagnosed with follicular lymphoma who received at least 1 cycle of systemic treatment and had the t(14;18)(q32;q21) translocation detected by polymerase chain reaction (PCR) at MBR, mcr or 3'MBR prior to first treatment. Among patients with different breakpoints, sex, age, disease grade, stage, B-symptoms, follicular lymphoma international prognostic index (FLIPI), presence of bulky disease, progression free survival and overall survival were compared. Results: Of 84 patients, 63 had breakpoint at MBR, 17 at mcr and 4 at 3'MBR. At diagnosis, the MBR group had a significantly lower disease stage than the mcr group. Although not significant, in the MBR group we found a higher progression-free survival (PFS) and overall survival (OS), lower grade, age, FLIPI, and less B-symptoms. Conclusions: Compared to patients with mcr breakpoint, those with MBR breakpoint seem to be characterised by more favourable clinical characteristics. However, a larger study would be required to support our observation.
Keywords: clinical characteristics, follicular lymphoma, t(14, 18) translocation
Published in DiRROS: 25.07.2024; Views: 256; Downloads: 156
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889. Awake craniotomy for operative treatment of brain gliomas - experience from University Medical Centre LjubljanaTilen Žele, Tomaž Velnar, Blaž Koritnik, Roman Bošnjak, Jasmina Markovič Božič, 2023, original scientific article Abstract: Background. Awake craniotomy is a neurosurgical technique that allows neurophysiological testing with patient cooperation during the resection of brain tumour in regional anaesthesia. This allows identification of vital functional (i.e. eloquent) brain areas during surgery and avoidance of their injury. The aim of the study was to present clinical experience with awake craniotomy for the treatment of gliomas at the University Medical Centre Ljubljana from 2015 to 2019.Patients and methods. Awake craniotomy was considered in patients with a gliomas near or within the language brain areas, in all cases of insular lesions and selected patients with lesions near or within primary motor brain cortex. Each patient was assessed before and after surgery.Results. During the 5-year period, 24 awake craniotomies were performed (18 male and 6 female patients; average age 41). The patient’s cooperation, discomfort and perceived pain assessed during the awake craniotomy were in majority of the cases excellent, slight, and moderate, respectively. After surgery, mild neurological worsening was observed in 13% (3/24) of patients. Gross total resection, in cases of malignant gliomas, was feasible in 60% (6/10) and in cases of low-grade gliomas in 29% (4/14). The surgery did not have important negative impact on functional status or quality of life as assessed by Karnofsky score and Short-Form 36 health survey, respectively (p > 0.05). Conclusions. The results suggest that awake craniotomy for treatment of gliomas is feasible and safe neurosurgical technique. The proper selection of patients, preoperative preparation with planning, and cooperation of medical team members are necessary for best treatment outcome.
Keywords: awake craniotomy, surgery of gliomas, intraoperative neurophysiological testing, primary brain tumours, clinical experiences
Published in DiRROS: 25.07.2024; Views: 226; Downloads: 374
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890. ADC values as a biomarker of fetal brain maturationLucija Kobal, Katarina Šurlan Popović, Jernej Avsenik, Tina Vipotnik-Vesnaver, 2023, original scientific article Abstract: Background. During the period of fetal development, myelination plays a key role and follows specific time and spa-tial sequences. The water content in the brain is inversely proportional to myelination – the more myelinated the brain, the lower the water content in it. The diffusion of water molecules can be quantitatively assessed using the apparent diffusion coefficient (ADC). We were interested in whether, by determining the ADC values, we could quantitatively evaluate the development of the fetal brain. Patients and methods. The study included 42 fetuses with gestational age 25 to 35 weeks. We manually selected 13 regions on diffusion-weighted images. Statistically significant differences between ADC values were checked using one-way analysis of variance and Tukey’s post hoc test. The relationship between the ADC values and the gestational age of the fetuses was then assessed using linear regression. Results. The average gestational age of the fetuses was 29.8 ± 2.4 weeks. ADC values in the thalami, pons and cerebellum differed significantly among each other and from the ADC values in other brain regions. In the thalami, pons and cerebellum, linear regression showed a significant decrease in ADC values with increasing gestational age. Conclusions. ADC values change with the increasing gestational age of the fetus and differ among different brain regions. In the pons, cerebellum and thalami, the ADC coefficient could be used as a biomarker of fetal brain matura-tion since ADC values decrease linearly with increasing gestational age.
Keywords: myelination, fetal brain maturation, ADC, biomarker, diffusion-weighted imaging, diffusion
Published in DiRROS: 25.07.2024; Views: 269; Downloads: 186
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