Článek se zabývá popisem a kritickým hodnocením vývoje přístrojové techniky pro semiinvazivní a neinvazivní stanovení glukózy v letech 1996 až 2001. Jsou uvedeny techniky odběru vzorku a vysvětleny principy optických a elektrochemických měřících metod používaných v prototypech přístrojů. Zatím nelze doporučit neinvazivní nebo minimálně invazivní metody analýzy glukózy jako náhradu monitorování krevní glukózy pacientem či biochemickou laboratoří.
The paper deals with description and critical evaluation of the instrumentation trends towards semiinvasive and non-invasive glucose determination from 1996 to 2001. The sample collection modes are mentioned and the principles of optical and electrochemical measuring methods used in prototypes of instruments are explained. The use of non-invasive or minimal invasive glucose analysis methods instead of blood glucose monitoring by the patient or the biochemical lab cannot be recommended yet.
The discovery of presence of fetal non cell DNA in mother's blood and possibility of their measurement in 1997 (Lo ,USA) opened perspectives of safe prenatal screening of aneuploidies. Invasive diagnostics procedures like chorionic villus sampling and amniocentesis which have been widely implemented since 70's and 80's present risk of pregnancy loss in amount 1:100 or 1: 50. This approach of invasive and risky prenatal screening in time of demographic disaster in Europe is no longer unsustainable. NIPT on the contrary represents method which is completely safe and achieves the same accuracy regarding screening of trisomy 21 (Down syndrome). Results Our centre has performed about 100 NIPT's (MaterniT21, Harmony, Panorama Prenascan) since 2012. NIPT is offered only to those pregnant women who have low or intermediate risk of aneuploidies after first trimester screening. High percentage of pregnant after assisted reproduction procedures are among those tested by NIPT. The NIPT is a good choice for pregnant women after IVF who are already in advanced age in comparison to normal population. Invasive procedures like CVS or amniocentesis are recommended in case of high risk of aneuploidies ( calculation risk 1:10 resp. 1:50) or ultrasound abnormal findings. We have experience that running NIPT technology is very easy, and has been accepted our patients. Conclusions Our experience with NIPT is very positive. Main advantage of this new technology is safety and we are sure that NIPT is ideal for using in gynaecological practise. The first trimester ultrasound skills and NIPT technology allows gynaecologist to detect majority of aneupliodies and congenital anomalies of fetus.
- MeSH
- Amniocentesis adverse effects MeSH
- Aneuploidy MeSH
- Pregnancy Trimester, Second blood MeSH
- Genetic Diseases, Inborn blood ultrasonography MeSH
- Genetic Counseling methods standards MeSH
- Genetic Testing * methods MeSH
- Humans MeSH
- Prenatal Diagnosis * methods MeSH
- Pregnancy Trimester, First * blood MeSH
- Pregnancy MeSH
- Ultrasonography, Prenatal methods instrumentation MeSH
- Check Tag
- Humans MeSH
- Pregnancy MeSH
- Female MeSH
Where microbes colonizing skin surface may help maintain organism homeostasis, those that invade living skin layers cause disease. In bats, white-nose syndrome is a fungal skin infection that affects animals during hibernation and may lead to mortality in severe cases. Here, we inferred the amount of fungus that had invaded skin tissue of diseased animals. We used simulations to estimate the unobserved disease severity in a non-lethal wing punch biopsy and to relate the simulated pathology to the measured fungal load in paired biopsies. We found that a single white-nose syndrome skin lesion packed with spores and hyphae of the causative agent, Pseudogymnoascus destructans, contains 48.93 pg of the pathogen DNA, which amounts to about 1560 P destructans genomes in one skin lesion. Relating the information to the known UV fluorescence in Nearctic and Palearctic bats shows that Nearctic bats carry about 1.7 µg of fungal DNA per cm2, whereas Palearctic bats have 0.04 µg cm-2 of P. destructans DNA. With the information on the fungal load that had invaded the host skin, the researchers can now calculate disease severity as a function of invasive fungal growth using non-destructive UV light transillumination of each bat's wing membranes. Our results will enable and promote thorough disease severity assessment in protected bat species without the need for extensive animal and laboratory labor sacrifices.
- MeSH
- Ascomycota * metabolism pathogenicity MeSH
- Chiroptera microbiology MeSH
- Dermatomycoses * microbiology prevention & control therapy veterinary MeSH
- Hibernation * MeSH
- Wings, Animal microbiology MeSH
- Skin microbiology MeSH
- Ultraviolet Rays * MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
V této prospektivní studii shrnujeme naše první zkušenosti s neinvazivní prenatální RH genotypizací plodu u aloimunizovaných těhotenství s využitím PCR v reálném čase, specifických primerů a TaqMan sond navržených pro RHD a RHCE geny. Celkem bylo testováno 22 aloimunizovaných těhotných žen (15 anti-D, 5 anti-D+C, 2 anti-E) v rozmezí 11. až 33. týdne gravidity. Výsledky neinvazivní prenatální RHD a RHCE genotypizace plodu provedené na DNA extrahované z periferní krve matek jsme korelovali s výsledky imunohematologické analýzy pupečníkové krve. Výsledky RHD a RHCE genotypizace plodu byly ve shodě s vyšetřením pupečníku u všech vyšetřených aloimunizovaných těhotných žen. Neinvazivní prenatální RHD a RHCE genotypizace plodu umožňuje identifikaci plodů v riziku hemolytického onemocnění. Detekce negativních plodů v současné graviditě může vyloučit potřebu invazivního prenatálního vyšetření.
In this prospective study, we described our first experience with non-invasive foetal RH genotyping in alloimmunised pregnancies by analysis of DNA extracted from maternal plasma samples by using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analysed 22 alloimmunised pregnant women (15 anti-D, 5 anti-D+C, 2 anti-E) within 11th and 33rd week of pregnancy and correlated the results with serological analysis of cord blood. Non-invasive prenatal foetal RHD and RHCE genotyping analysis of maternal plasma samples was in complete concordance with the analysis of cord blood in all alloimmunised pregnancies. Non-invasive foetal RHD and RHCE genotyping enables the identification of foetuses at risk of haemolytic disease of the newborn. An identification of negative foetuses may exclude the demand of invasive prenatal procedures.
- MeSH
- Anticoagulants pharmacology classification therapeutic use MeSH
- Publication type
- Review MeSH
- Practice Guideline MeSH
In accordance with the 3 Rs principle (to replace, reduce and refine) animal models in biomedical research, we have developed and applied a new approach for sampling and analyzing hair follicles in various experimental settings. This involves use of a convenient device for non-invasive collection of hair follicles and processing methods that provide sufficient amounts of biological material to replace stressful and painful biopsies. Moreover, the main components of hair follicles are live cells of epithelial origin, which are highly relevant for most types of malignant tumors, so they provide opportunities for studying aging-related pathologies including cancer. Here, we report the successful use of the method to obtain mouse hair follicular cells for genotyping, quantitative PCR, and quantitative immunofluorescence. We present proof of concept data demonstrating its utility for routine genotyping and monitoring changes in quality and expression levels of selected proteins in mice after gamma irradiation and during natural or experimentally induced aging. We also performed pilot translation of animal experiments to human hair follicles irradiated ex vivo. Our results highlight the value of hair follicles as biological material for convenient in vivo sampling and processing in both translational research and routine applications, with a broad range of ethical and logistic advantages over currently used biopsy-based approaches.
- MeSH
- Fluorescent Antibody Technique MeSH
- Genotyping Techniques MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Tail pathology MeSH
- DNA Damage * radiation effects MeSH
- Aging pathology physiology MeSH
- Hair Follicle anatomy & histology metabolism physiology radiation effects MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Provedli jsme modifikaci a optimalizaci postupů pro izolaci extracelulární DNA z mateřské cirkulace pro neinvazivní určení pohlaví plodu u těhotenství s rizikem X-vázaných onemocnění u plodu a pro neinvazivní RHD a RHCE genotypizaci plodu u aloimunizovaných těhotenství s rizikem hemolytického onemocnění plodu. V této studii srovnáváme výsledky amplifikace SRY, RHD a RHCE genu na extracelulární DNA izolované z mateřské periferní krve pomocí QIAamp DSP Virus kitu a QIAamp DNA Blood Mini kitu. Výsledky našich analýz prokázaly, že QIAamp DSP Virus kit zvyšuje výtěžnost extracelulární fetální DNA přítomné v mateřské plazmě, což je klíčové zejména u amplifikace paternálně zděděných alel, které se liší od alel maternálních pouze v jednom nukleotidu. Doporučujeme proto provádět detekci paternální Rhc alely a RhE alely RHCE genu na extracelulární DNA izolované z mateřké plazmy pouze pomocí QIAamp DSP Virus kitu. Pro amplifikaci SRY a RHD genu dostačuje extracelulární DNA extrahovaná z mateřské plazmy prostřednictvím QIAamp DNA Blood Mini kitu. V případě sporných výsledků, tj. při nálezu pozdních amplifikačních cyklů (po 40. cyklu), doporučujeme rovněž provedení PCR analýzy na extracelulární DNA izolované z mateřské plazmy pomocí QIAamp DSP Virus kitu. Spolehlivá detekce plodů negativních na aglutinogeny, vůči nimž je matka aloimunizována, vylučuje riziko fetální erytroblastózy. Spolehlivá detekce plodů ženského pohlaví u těhotenství s rizikem X-vázaných onemocnění může vyloučit potřebu invazivního prenatálního vyšetření.
We carried out the modification and optimalisation of extracellular fetal DNA isolation from maternal circulation for non-invasive fetal sex determination in pregnancies at risk of X-linked disorders and for non-invasive fetal RHD and RHCE genotyping in alloimmunized pregnancies at risk of haemolytic disease of newborn. In this study, we compared the results of fetal SRY, RHD and RHCE genotyping by analysis of DNA extracted from maternal plasma samples by using QIAamp DSP Virus kit and QIAamp DNA Blood Mini kit. We showed that QIAamp DSP Virus kit enhanced the recovery of extracellular fetal DNA from maternal plasma that is crucial especially for the detection of those paternally-inherited alleles that differ from maternal alleles only in one nucleotide. Therefore we recommend to perform the detection of fetal Rhc allele and RhE allele of RHCE gene by analysis of extracellular DNA extracted from maternal plasma samples by using QIAamp DSP Virus kit only. We showed that the use of extracellular DNA extracted from maternal plasma samples by using QIAamp DNA Blood Mini kit is sufficient for non-invasive fetal SRY and RHD genotyping. In case of controversial results due to the late amplification (Ct ≥ 40) we recommend to perform real-time PCR analysis on extracellular DNA extracted from maternal plasma samples by using QIAamp DSP Virus kit. Reliable non-invasive detection of negative foetuses in alloimmunized pregnancies may exclude the risk of haemolytic disease of newborn. Reliable non-invasive detection of female foetuses in pregnancies at risk of X-linked disorders may exclude the performance of invasive prenatal diagnostic procedures.
- MeSH
- Sex Determination Analysis methods MeSH
- Research Support as Topic MeSH
- Genetic Diseases, X-Linked prevention & control MeSH
- Erythroblastosis, Fetal prevention & control MeSH
- Humans MeSH
- Blood Specimen Collection utilization MeSH
- Prenatal Diagnosis methods MeSH
- Reagent Kits, Diagnostic utilization MeSH
- Sequence Analysis, DNA MeSH
- Check Tag
- Humans MeSH
- Publication type
- Evaluation Study MeSH
Cíl studie: Koncentrace jaderných erytroblastů cirkulujících v periferní krvi gravidních žen proúčely neinvazivní prenatální diagnostiky.Typ studie: Pilotní studie.Název a sídlo pracoviště: II. dětská klinika 2. LF UK, Fakultní nemocnice Motol, Praha, Českárepublika.Metodika: Z 13 - 28 ml periferní krve 78 gravidních žen v rozmezí 13. - 37. týdnu gravidity bylyizolovány mononukleární buňky. Mateřské leukocyty byly odstraněny z frakce mononukleárníchbuněk pomocí anti-CD14 a anti-CD45 monoklonálních protilátek značených superparamagnetic-kými částicemi metodou magnetické separace buněk (MACS) na zařízení VarioMACS. Jadernéerytroblasty byly následně koncentrovány z CD14 - /CD45 - frakce pozitivní selekcí pomocí anti-CD71 monoklonální protilátky na zařízení MiniMACS. Jednotlivé frakce byly analyzovány tříba-revnou analýzou na průtokovém cytometru (FACS).Výsledky: U 68 těhotných žen z 78 testovaných (87 %) byly nalezeny po dvojité magnetické separa-ci buněk (doubleMACS) zahrnující depleci leukocytů a pozitivní selekci jaderné erytroblastyv rozmezí 2x10 5 - 1,02 x 10 6 . Faktor celkového zkoncentrování se pohyboval v rozmezí 4,2 - 526,0(Ć 138,4). Z našeho souboru vyšetřených je patrné, že počet CD71 + jaderných erytroblastů koncen-trovaných metodou doubleMACS je značně individuální. Jaderné erytroblasty cirkulující v krev-ním oběhu gravidních žen bylo možno zkoncentrovat již v 13. týdnu gravidity.Závěr: Naším cílem je vypracovat a standardizovat metodu koncentrace jaderných erytroblastůcirkulujících v periferii gravidních žen a ověřit možnost použití tohoto alternativního zdroje proúčely neinvazivní prenatální diagnostiky.
Objective: Enrichment of nucleated red blood cells (NRBCs) from maternal blood for non-invasiveprenatal diagnosis.Design: Pilot study.Setting: 2 nd Clinic of Paediatrics, University Hospital Motol, Prague, Czech Republic.Methods: Mononuclear cells were isolated from 13 - 28 ml of peripheral maternal blood between 13and 37 weeks of gestation. Leukocytes from maternal peripheral blood were depleted from mono-nuclear cells by treatment with anti-CD14 and anti-CD45 microbeads and high-gradient magneticcell separation (MACS) on VarioMACS. NRBCs were sorted from CD14 - /CD45 - fraction by positiveselection using anti-CD71 microbeads on MiniMACS. All sorting steps were analysed by three-co-lour cytometric analysis with FACScan flow cytometer.Results: In 68 out of 78 pregnant woman (87 %) NRBCs were found in range 2x10 5 - 1,02 x 10 6.NRBC were enriched with an average enrichment rate of 138-fold ranging from 4 - 526 fold. In ourcohort of pregnant woman the number of isolated NRBCs was individual. We identified NRBCsfrom the 13 th week of gestation.Conclusion: The aim of the study is to establish and standardise the method of enrichment ofNRBCs from maternal blood samples and verify the applicability of this alternative source fornon-invasive prenatal diagnosis.
- MeSH
- Biological Transport MeSH
- Adult MeSH
- Erythroblasts blood MeSH
- Humans MeSH
- Maternal-Fetal Exchange MeSH
- Antibodies, Monoclonal diagnostic use MeSH
- Prenatal Diagnosis MeSH
- Flow Cytometry methods instrumentation MeSH
- Pregnancy MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Pregnancy MeSH
- Female MeSH
- Publication type
- Review MeSH
V této prospektivní studii shrnujeme naše první zkušenosti s neinvazivní prenatální RHc genotypizací plodu s využitím PCR v reálném čase, specifických primerů a TaqMan sondy navržených pro c alelu RHCE genu. Celkem bylo testováno 13 nealoimunizovaných a 1 anti-c aloimunizovaných těhotných žen v rozmezí 12. až 33. týdne gravidity. Výsledky neinvazivní prenatální RHc genotypizace plodu provedené na DNA extrahované z periferní krve matek jsme korelovali s výsledky imunohematologické analýzy pupečníkové krve. Výsledky RHc genotypizace plodu byly ve shodě s vyšetřením pupečníku u všech vyšetřených těhotných žen. Neinvazivní prenatální RHc genotypizace plodu umožňuje identifikaci plodů v riziku hemolytického onemocnění. Detekce Rhc negativních plodů u anti-c aloimunizovaných těhotenství může vyloučit potřebu invazivního prenatálního vyšetření.
In this prospective study, we described our first experience with non-invasive foetal RHc genotyping by analysis of DNA extracted from maternal plasma samples by using real-time PCR, specific primers and TaqMan probe targeted toward c allele of RHCE gene. We analysed 13 non-alloimmunised and 1 anti-c alloimmunised pregnant woman within 12th and 33rd week of pregnancy and correlated the results with serological analysis of cord blood. Non-invasive prenatal foetal RHc genotyping analysis of maternal plasma samples was in complete concordance with the analysis of cord blood in all pregnancies. Non-invasive foetal RHc genotyping enables the identification of foetuses at risk of haemolytic disease of the newborn. An identification of RHc negative foetuses in anti-c alloimmunized pregnancies may exclude the demand of invasive prenatal procedures.
BACKGROUND: In endemic areas of zoonotic leishmaniosis caused by L. infantum, early detection of Leishmania infection in dogs is essential to control the dissemination of the parasite to humans. The aim of this study was to evaluate the serological and/or molecular diagnostic performance of minimally and non-invasive samples (conjunctiva cells (CS) and peripheral blood (PB)) for monitoring Leishmania infection/exposure to Phlebotomus perniciosus salivary antigens in dogs at the beginning and the end of sand fly seasonal activity (May and October, respectively) and to assess associated risks factors. METHODS: A total of 208 sheltered dogs from endemic areas of leishmaniosis were screened. Leishmania DNA detection in PB on filter paper and CS was performed by nested-PCR (nPCR), while the detection of anti-Leishmania antibodies was performed using IFAT and ELISA. The exposure to P. perniciosus salivary antigens (SGH, rSP01 and rSP03B + rSP01) was measured by ELISA. RESULTS: Ninety-seven (46.6%) and 116 (55.8%) of the 208 dogs were positive to Leishmania antibodies or DNA by at least one test at the beginning and end of the sand fly season, respectively. IFAT and ELISA presented a substantial agreement in the serodiagnosis of leishmaniosis. Discrepant PB nPCR results were obtained between sampling points. Leishmania DNA was detected in CS of 72 dogs at the end of the phlebotomine season. The presence of antibodies to the parasite measured by ELISA was significantly higher in dogs presenting clinical signs compatible with leishmaniosis at both sampling points. Phlebotomus perniciosus salivary antibodies were detected in 179 (86.1%) and 198 (95.2%) of the screened dogs at the beginning and end of the phlebotomine season, respectively. CONCLUSIONS: The association between ELISA positivity and clinical signs suggests its usefulness to confirm a clinical suspicion. CS nPCR seems to be an effective and non-invasive method for assessing early exposure to the parasite. PB nPCR should not be used as the sole diagnostic tool to monitor Leishmania infection. The correlation between the levels of antibodies to P. perniciosus saliva and Leishmania antibodies suggests the use of a humoral response to sand fly salivary antigens as biomarkers of L. infantum infection.
- MeSH
- Antigens, Protozoan immunology MeSH
- Endemic Diseases prevention & control MeSH
- Insect Vectors parasitology MeSH
- Insect Proteins immunology MeSH
- Immunoglobulin G blood MeSH
- Conjunctiva cytology parasitology MeSH
- Insect Bites and Stings MeSH
- Leishmania infantum isolation & purification MeSH
- Leishmaniasis blood immunology veterinary MeSH
- Dog Diseases parasitology prevention & control transmission MeSH
- Phlebotomus parasitology MeSH
- Antibodies, Protozoan blood MeSH
- Protozoan Proteins immunology MeSH
- Dogs MeSH
- Risk Factors MeSH
- Serologic Tests MeSH
- Salivary Proteins and Peptides immunology MeSH
- Animals MeSH
- Check Tag
- Dogs MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
INTRODUCTION: The methods for diagnosing compartment syndrome non-invasively remain under debate. Bioimpedance measurements offer a promising avenue in clinical practice, detecting subtle changes in organ impedance due to volume shifts. This study explores bioimpedance measurement as a novel, painless method for diagnosing compartment syndrome, potentially enabling continuous monitoring. OBJECTIVE: This work aims to develop a prototype device for non-invasive diagnosis of compartment syndrome based on bioimpedance changes and assess initial results through in vitro experiments on inanimate biological material. We assume a change in the bioimpedance value after the application of physiological solution. MATERIALS AND METHODS: Between 2018 and 2022, a prototype device for diagnosing limb compartment syndrome was collaboratively developed with the Department of Cybernetics and Biomedical Engineering at the Technical University of Ostrava, Czech Republic. This device operates by comparing bioimpedance between two compartments, one of which is pathologically affected (experiencing compartment syndrome). The Bioimpedance Analyzer for Compartment Syndrome (BACS) has been utilized to conduct measurements on inanimate biological material in laboratory settings. Two samples of duck and chicken tissue, as well as piglets, were employed for these experiments. According to the size of sample was compartment syndrome simulated by injecting 20-120 mL saline into one limb (breast) while leaving the other as a control. Invasive intramuscular pressure measurements were conducted post-saline injection using a conventional device (Stryker). Changes in bioimpedance were evaluated following saline application. RESULTS: The non-invasive bioimpedance measurement instrument has been developed. It meets the safety requirements of European standard EN 60601-1. Measurement of accuracy showed minimal deviation for both channels (1.08% for the left channel and 1.84% for the right channel) when measuring on resistors. Ten measurements were conducted using the BACS prototype - two on chicken legs, two on duck breasts, two on duck legs, and four on piglets. Compartment syndrome simulation was achieved for all 10 measurements (IMP variance 31-45 mmHg). Following saline application, a notable decrease in bioimpedance was observed in the compartment simulating compartment syndrome (decrease by 12-78 Ω). CONCLUSION: Non-invasive methods could revolutionize limb compartment syndrome diagnosis, offering advantages such as non-invasiveness and continuous monitoring of compartment swelling.
- Publication type
- Journal Article MeSH
