Papers by Marie Korabecna
Optimized cutting laser trajectory for laser capture microdissection
Biologia, Apr 3, 2019
Laser capture microdissection (LCM) is an excellent tool using a laser beam for specific selectio... more Laser capture microdissection (LCM) is an excellent tool using a laser beam for specific selection and harvesting of cells or cell populations from heterogeneous tissue sections prepared on special slides. The aim of our study was to apply mathematical methods for optimizing the planning of laser trajectory in LCM combining positive and negative selection criteria with adjustable size, weight, importance, and security borders. We developed and tested software tool named CutPlanner to be used in a transparent overlay superimposed to the live camera image independently from the manufacturer of the LCM device. Once optimized, quantified and approved by the user, the resulting outline of the region of interest is directly copied to the laser beam cutting trajectory. The software is made publicly available for non-commercial use to the scientific community and provides a versatile tool for effectively minimizing the length of the laser trajectory to obtain the selected cells without destroying surrounding cells and tissue structures. Saving all the settings allows for performing repeating tasks under similar conditions, especially in uniform and routinely performed LCM applications.
Vážená redakce, ráda bych tímto textem reagovala na inspirativní příspěvek autorů Jaroslava Louck... more Vážená redakce, ráda bych tímto textem reagovala na inspirativní příspěvek autorů Jaroslava Louckého a Michala Zemánka publikovaný formou Dopisu redakci o názvu "Neinvazivní prenatální diagnostika nejčastějších chromozomálních aneuploidií -od teorie k praxi" 5. 11. 2012 ve vašem časopise (1). Není pochyb o tom, že metody neinvazivní prenatální diagnostiky založené na vyšetření fetální volné DNA cirkulující v oběhu těhotné představují absolutní vrchol současných technologických možností při kombinaci recentních vědeckých poznatků, nejnovějších sekvenačních technologií a přístupů bioinformatiky. Zkusme se však podívat na uváděná čísla charakterizující tyto nové metody skutečně z pohledu praxe. Pro názornost v našem příkladu budeme uvažovat hodnoty sensitivity testu ve vztahu k detekci Downova syndromu 99,1 % a specificity 99,9 % v souladu s literaturou (2) citovanou autory příspěvku. Za předpokladu, že by tento test měl být nasazen jako skríningový, musíme uvažovat populační prevalenci Downova syndromu v souladu se studiemi zabývajícími se evropskou populací 1:686 (3), eventuálně 1:549 (4). Pro demonstraci v příkladu zvolíme zaokrouhlenou hodnotu ze studie zabývající se i naší populací 1:550 (4). Za těchto předpokladů se dostáváme k hodnotám uvedeným v Tab. 1. Na základě této tabulky můžeme potom vypočítat prediktivní pozitivní hodnotu testu (PPV -positive predictive value), tedy pravděpodobnost, že pacient skutečně trpí chorobou, jestliže výsledek testu vyšel pozitivně. Ta v tomto případě činí 64,28 % (vypočteno jako
InTech eBooks, Dec 7, 2011
nucleic acids in human plasma present very heterogeneous material with heterogeneous function -th... more nucleic acids in human plasma present very heterogeneous material with heterogeneous function -there are for example fragments of genomic DNA, mitochondrial DNA, but also mRNAs and microRNA. The forms in which they circulate are studied also with regard to the development of effective extraction methods for clinical laboratories. From the methodological point of view, it is difficult to exactly distinguish between the apoptotic derived fraction of cell-free DNA and the necrotic-derived one in plasma. Veiko et al. (2008) developed a method for in vivo evaluation of cell death in patients with acute and/or chronic heart disease. The main parameters evaluated in the study by Veiko et al. were: total concentrations of cell-free DNA (cfDNA) in the blood (or serum), concentration of serum ribosomal repeat (rDNA), content of rDNA in total cfDNA, but also factors involved in clearance of cfDNA such as nuclease activity and anti-DNA antibodies. The authors clearly demonstrated significant increase in the concentration of rDNA in the cfDNA pool in patients with acute myocardial infarction. Such an accumulation of rDNA within cfDNA may be caused by the resistance of rDNA to the fragmentation by serum endonucleases, because the nuclease activities in the serum of both acute and chronic coronary disease patients were elevated in comparison to healthy individuals. The titers of anti-DNA antibodies were also higher in the patients group. The anti-DNA antibodies were predominantly bound to cfDNA. It seems that the release of rDNA fragments into the blood may reflect cellular death in the body (Veiko et al., 2008). Another clinical situation connected with massive cellular death is represented by the multiple-organ dysfunction syndrome (MODS). In MODS, the initial insult damages target organs and leads to tissue necrosis. The necrosis induces a systemic inflammatory response and an alteration of hemodynamics, microcirculation and oxygen metabolism. As a consequence, distant organs may be damaged by necrosis or apoptosis. The prognostic role of elevated levels of plasma cfDNA in critically ill patients was demonstrated by Wijeratne et al. (2004). developed an assay allowing to distinguish between DNA released from apoptotic and necrotic cells. The assay is based on electrophoretic separation of isolated plasma cfDNA fragments on agarose gel. The DNA from apoptotic cells (aDNA) is represented by fragments of typical size resembling the ladder on an electrophoretic gel, but DNA derived from necrotic cells (genomic -gDNA) does not provide this typical pattern when subjected to electrophoretic separation. The authors applied their assay on the samples of plasma cfDNA obtained from intensive care unit patients. They found that the contribution of aDNA to the amount of total plasma DNA in the critically ill patients was 16 fold greater than the contribution of gDNA from necrotic cells. The levels of aDNA were highest on the day of admission and declined thereafter, but the levels of gDNA altered in the opposite manner. The concentration of apoptotic DNA in samples collected from patients on the day of admission significantly differentiated survivors and non-survivors . The study by Pachl confirmed the results of previous research performed on rats . The most surprising fact in this context is represented by the finding of the highest concentration of cfDNA of apoptotic origin at the time of patient admission to the intensive care unit. The possible explanation for this fact can be found in the induction of apoptosis by the activation of the intrinsic pathway caused by the affection of mitochondria as a consequence of cellular damage caused by primary insult .
Digital PCR system development accelerator—A methodology to emulate dPCR results
Sensors and Actuators B-chemical, May 1, 2022
Trends in Analytical Chemistry, Sep 1, 2020
Since January 2020 Elsevier has created a COVID-19 resource centre with free information in Engli... more Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre -including this research content -immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
Quantification of circulating fetal DNA as a tool for potential monitoring of pregnant patients with antiphospholipid antibodies
Autoimmunity, May 15, 2014
Apoptosis of tissues of fetal origin is thought to be one of the main sources of cell-free fetal ... more Apoptosis of tissues of fetal origin is thought to be one of the main sources of cell-free fetal DNA (cffDNA) in maternal circulation, impaired apoptosis is also involved in the mechanisms contributing to recurrent spontaneous miscarriages (RSM) associated with antiphospholipid syndrome (APS). The APS increases the risk for preeclampsia nine times. In preeclampsia, the elevated levels of cffDNA were described by different authors. To our knowledge, cffDNA in pregnant patients with APS was never studied. In our pilot study, we focused on the levels of cffDNA in four pregnant patients with treated primary APS and compared them with values obtained in twenty-one healthy subjects of comparable gestation age (the third trimester of pregnancy). We supposed that the increase of cffDNA concentration in our treated patients would signalize the elevated apoptosis of fetal tissues as in other pathological changes of placentation. The aim of our pilot study was to determine cffDNA concentrations in patients with treated APS and to compare them with values detected in healthy pregnant women of comparable gestation age in order to discover potential non-physiological elevations in patients. The elevated values of cffDNA were not observed in our patients (p value = 0.4363, Mann-Whitney test). All patients delivered healthy children. The measurement of concentrations of cffDNA seems to be a promising tool for monitoring of therapy effectiveness in pregnant women with APS but evaluation of randomized controlled trials would be necessary to determine the specificity and the sensitivity of this test.
Comparison of MicroRNA Content in Plasma and Urine Indicates the Existence of a Transrenal Passage of Selected MicroRNAs
Advances in Experimental Medicine and Biology, 2016
MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples f... more MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples from 70 individuals (45 males, 25 females, mean age 65 years, range 20-84 years). Of the urine donors, 15 were healthy volunteers, 5 were patients with non-cancer diseases, 50 were patients with different stages of bladder cancer. To examine the spectrum of miRNAs in the cell-free fraction of urine, TaqMan Human miRNA Array Card A v.2.1 was used. A set of 30 miRNAs were found that are constantly present in urine supernatants independently of sex, age and health status of the subjects. We compared this set with miRNAs found in plasma, expressed in kidney and genito-urinary tract. Our results indicate that some miRNA could be transferred from the circulation into urine.
Quantification of plasminogen activator inhibitor type 1 in the aortic wall
International Angiology, Feb 1, 2009
Aim: Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in regulation of fibrinolyti... more Aim: Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in regulation of fibrinolytic system, cell-associated proteolysis and migration of smooth muscle cells (SMC). This study is focused on the types of PAI-1 expressing cells, quantification of PAI-1 expression in the walls of aneurysmatic abdominal aortas (AAA) and correlation between histological and clinical findings. Methods: A group of nine patients who underwent surgery for AAA: asymptomatic (aAAA), symptomatic (sAAA) and ruptured (rAAA) and one control specimen (CA) were included in the study. Samples underwent histological processing and immunohistochemistry in comparison with in situ hybridisation. In order to assess the PAI-1 area fraction in histological sections through the aortic wall the Line System module of Ellipse software was used. PAI-1 expressing cells were measured in CA and AAA: endothelium, SMC, and foam cells. Photomicrographs with a total area of 0.7 mm(2) for each specimen were analysed by two independent observers. Mean values of PAI-1 positive components per section area were calculated as average values. Results: The results of both observers are as follows: 28.6% in CA; 18.1% in aAAA; 10.9% in sAAA; 11.0% in rAAA. During the progression of AAA, the SMC (PAI-1 expression was found mainly in them) became less abundant in agreement with the values of PAI-1 area fraction. In rAAA immunohistochemistry detected PAI-1 in necrotic centres of atheromathous plaques. Conclusions: AAA may be evaluated as the result of gradual changes in regulation of fibrinolysis that is observed as redistribution of cells expressing PAI-1. The area fraction of PAI-1 positive components correlates with clinical classification of AAA.
The impact of standard chemotherapy on miRNA signature in plasma in AML patients
Leukemia Research, Dec 1, 2015
In our pilot study, we used plasma samples as liquid biopsy to search for miRNA signatures in pat... more In our pilot study, we used plasma samples as liquid biopsy to search for miRNA signatures in patients with acute myeloid leukemia (AML) at diagnosis and in remission achieved after standard chemotherapy before planned transplantation. We examined 10 plasma samples from healthy volunteers and 8 paired samples from patients with AML at diagnosis and in remission using TaqMan MicroRNA Arrays. The results were validated using single-target qPCR reactions run in triplicates. We selected 6 miRNAs with expressions significantly sensitive to therapy: miR-199b-5p, miR-301b, miR-326, miR-361-5p, miR-625 and miR-655. All selected miRNAs were not or very weakly expressed in healthy individuals. They were abundant in plasma in patients at diagnosis but their levels decreased after chemotherapy. We detected a therapy sensitive miRNA signature in plasma of patients with AML.
BMC Nephrology, Mar 15, 2013
Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of inher... more Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of inherited kidney disease that results in renal failure. ADPKD is a systemic disorder with cysts and connective tissue abnormalities involving many organs. ADPKD caused by mutations in PKD1 gene is significantly more severe than the cases caused by PKD2 gene mutations. The large intra-familial variability of ADPKD highlights a role for genetic background. Case presentation: Here we report a case of ADPKD family initially appearing unlinked to the PKD1 or PKD2 loci and the influence of mosaicism and hypomorphic allele on the variability of the clinical course of the disease. A grandmother with the PKD1 gene mutation in mosaicism (p.Val1105ArgfsX4) and with mild clinical course of ADPKD (end stage renal failure at the age of 77) seemed to have ADPKD because of PKD2 gene mutation. On the other hand, her grandson had a severe clinical course (end stage renal disease at the age of 45) in spite of the early treatment of mild hypertension. There was found by mutational analysis of PKD genes that the severe clinical course was caused by PKD1 gene frameshifting mutation inherited from his father and mildly affected grandmother in combination with inherited hypomorphic PKD1 allele with described missense mutation (p.Thr2250Met) from his clinically healthy mother. The sister with two cysts and with PKD1 hypomorphic allele became the kidney donor to her severely affected brother. We present the first case of ADPKD with the influence of mosaicism and hypomorphic allele of the PKD1 gene on clinical course of ADPKD in one family. Moreover, this report illustrates the role of molecular genetic testing in assessing young related kidney donors for patients with ADPKD.
Forenzní vědy, právo, kriminalistika
Dotykové stopy patří ve forenzní genetice k jedněm z nejobtížněji analyzovatelných materiálů, což... more Dotykové stopy patří ve forenzní genetice k jedněm z nejobtížněji analyzovatelných materiálů, což je dáno velmi malým množstvím analyzovatelných molekul v nich obsažených. Ačkoliv obsahují buňky pocházející z povrchu lidského těla, DNA v těchto buňkách je přirozeně fragmentována. Shrnujeme dosud publikované zkušenosti s genetickou analýzou dotykových stop, které často neposkytují plnohodnotný DNA profil vhodný k individuální identifikaci v procesu dokazování. Nově se rozvíjející vědní obor olfaktroniky však skýtá potenciál k získání dalších informací z těchto stop. Dotykové stopy však představují extrémně variabilní materiál, takže nelze vždy zaručit úspěch genetické analýzy. Souběžná olfaktronická analýza pak může poskytnout důležité operativní informace týkající se skupinových vlastností zůstavitele stopy.
Digital polymerase chain reaction duplexing method in a single fluorescence channel
Analytica Chimica Acta
We investigated the possible associations between leukocyte telomere length, therapy outcomes, an... more We investigated the possible associations between leukocyte telomere length, therapy outcomes, and clinicopathological features in patients with colorectal cancer. Aditionally, telomerase reverse transcriptase (TERT) expression was evaluated.Telomere length was measured using singleplex qPCR in 478 consecutive leukocyte DNA samples from 198 patients. Blood was drawn at diagnosis prior to any therapy, and then at 6-month intervals for 18 months.Following diagnosis, the telomeres gradually shortened during the course of the treatment regardless of the patient's age. The most pronounced decrease was observed 12 months after the diagnosis (p < 0.0001). Based on tumor localization, the decrease in telomere length one year after the diagnosis followed different trajectories (p = 0.03). In patients treated with adjuvant therapy, telomere length was correlated with the time elapsed after completion of therapy (p = 0.03). TERT expression did not correlate with the telomere length; how...
Investigative genetic genealogy - new approach to detect relatedness of individuals
Soudni lekarstvi, 2021
The development of molecular genetic techniques, coupled with the development of bioinformatic ap... more The development of molecular genetic techniques, coupled with the development of bioinformatic approaches and the expansion of genealogy as a hobby, also brings new possibilities for forensic genetics. The review article compares todays classical methods of genotyping and determination of kinship based on the analysis of polymorphisms such as short tandem repeats, with the possibilities of investigative genetic genealogy, which uses genotyping based on analysis of hundreds of thousands of single nucleotide polymorphisms, bioinformatics and sharing individual profiles within public databases. We describe the principles of these laboratory techniques and the interpretation of their results. We compare these new approaches with the current practice in forensic laboratories. We stress the importance of the introduction of whole genome sequencing into forensic genetics. Whole genome sequencing allows due its ability to work with degraded DNA samples the preparation of proper material for...
The examples of using microscopy image deconvolution by meansof the Heugens system
Optimization of diagnostic strategy for non‐invasive cell‐free foetal RHD determination from maternal plasma
Vox Sanguinis, 2021
Background and objectivesThe aim of the study was to optimize routine non‐invasive prenatal detec... more Background and objectivesThe aim of the study was to optimize routine non‐invasive prenatal detection of fetal RHD gene from plasma of RhD‐negative pregnant women (the median of gestational age was 25 weeks, range 10–38) to detect RhD materno‐fetal incompatibility and to avoid the redundant immunoprophylaxis.Materials and methodsInitially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real‐time PCR method. Detection of Y‐chromosomal sequence DYS‐14 and five X‐chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence‐specific primer PCR in nine samples.ResultsWhen only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7,...
Telomerase and non-Telomerase Mechanisms of Telomere Maintenance, 2019
Placenta is a transient organ ensuring the intrauterine development of the individual. To meet fe... more Placenta is a transient organ ensuring the intrauterine development of the individual. To meet fetal requirements, rapid and continuous cell proliferation enlarges the areas of tissues maintaining maternofetal transport. The cell division in placenta is accompanied with shortening of telomeres leading to cell senescence. Telomerase activity, on the other hand, ensures replication of telomeres and allows the organ to serve till the end of pregnancy. This balanced process may be negatively influenced by unfavorable circumstances. Here, we summarize available data on telomere length as well as telomerase activity in placentas from normal and complicated pregnancies; attention is also paid to the comparison of methods used in relevant studies.
Placenta, 2017
We applied qPCR in order to compare relative telomere length in terminal villi microdissected fro... more We applied qPCR in order to compare relative telomere length in terminal villi microdissected from term control placentas and placentas of patients suffering from type 1 diabetes. Significant differences were found in the relative T/S ratios neither between placental groups nor between the diabetic placentas affected and non-affected with chorangiosis. We hypothesize that there is no relationship between decreased placental proliferative ability in maternal diabetes type 1 and telomere shortening.
Inflammation research : official journal of the European Histamine Research Society ... [et al.], 2017
Neutrophil extracellular traps (NETs) are formed by activated neutrophils during the process of N... more Neutrophil extracellular traps (NETs) are formed by activated neutrophils during the process of NETosis in which the nuclear material is released into extracellular space, including DNA molecules, citrullinated histones, and neutrophil granule enzymes, such as elastase. This material forms networks that are able not only to physically entrap bacteria but also to provide elevated concentration of bactericidal components. Over the last years, it has become clear that NETs can also be formed under numerous sterile inflammatory conditions, i.e., thrombosis, cancer, SLE, atherosclerosis, and diabetes. We reviewed studies published until July 2016 to find possible associations between elevated cell-free DNA levels in dialyzed patients and the process of NETosis and its consequences. The process of NETosis, its elevated activation, or impaired clearance provides the link between clinical conditions and elevated levels of cell-free DNA found in plasma after the hemodialytic procedure which ...
Comparison of MicroRNA Content in Plasma and Urine Indicates the Existence of a Transrenal Passage of Selected MicroRNAs
Advances in Experimental Medicine and Biology, 2016
MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples f... more MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples from 70 individuals (45 males, 25 females, mean age 65 years, range 20-84 years). Of the urine donors, 15 were healthy volunteers, 5 were patients with non-cancer diseases, 50 were patients with different stages of bladder cancer. To examine the spectrum of miRNAs in the cell-free fraction of urine, TaqMan Human miRNA Array Card A v.2.1 was used. A set of 30 miRNAs were found that are constantly present in urine supernatants independently of sex, age and health status of the subjects. We compared this set with miRNAs found in plasma, expressed in kidney and genito-urinary tract. Our results indicate that some miRNA could be transferred from the circulation into urine.
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Papers by Marie Korabecna