Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Diagnóza cystické fibrózy (CF) novorozeneckým screeningem je důležitá, protože umožňuje včasné zahájení léčby, genetického poradenství a specializované terapie. Novorozenecký screening se skládá z analýzy imunoreaktivního trypsinogenu (IRT), genetické analýzy DNA a obsahu chloridů pomocí potního testu. Potní test je považován za tzv. zlatý standard v diagnostice CF. Výsledky potního testu v tzv. šedé zóně (Cl-: 30-60 mmol/L) a genetické analýzy ovšem někdy vedou k nejednoznačnému diagnostickému závěru (CFSPID). V tomto projektu navrhujeme validaci nové metody analýzy potu, založené na neinvazivním, rychlém a jednoduchém stěru předloktí. Ve vzorku budou analyzovány poměry vybraných iontů (Cl-, K+, Na+, HCO3-) které mají významný potenciál pro zlepšení diagnostiky CF. Je plánováno na statisticky významném souboru pacientů a zdravých kontrol vyhodnotit důležité parametry (citlivost, specificita, opakovatelnost, atd.) a vytvořit databázi iontových poměrů , určení korelace s typem genetické mutace, vážností onemocnění a dalšími symptomy, umožňujícímu zlepšení klasifikace CF pacientů.; An early diagnosis of cystic fibrosis (CF) through newborn screening programmes (NBSP) is very important to initiate the treatment, genetic counseling, and specialized medical services. NBSP consists of immunoreactive trypsinogen (IRT) analysis, DNA analysis and sweat test. Sweat test is a golden standard, but many patients fall into the inconclusive diagnosis (CFSPID) due to the intermediate chloride levels (30-60 mmol/L) and unclear DNA analysis. We propose the validation of an improved method, based on non-invasive skin wipe sampling and analysis of all ions in the samples. We propose the use of ion ratios of selected ions (Cl-, K+, Na+, HCO3-) as a relevant diagnostic parameter and aim at evaluation of sensitivity, selectivity and repeatability of the method. We further propose creation of a ion ratio database of a large cohort of subjects during the NBSP as well as adult CF patients and controls to identify possible correlations between the type of mutation, severity and other symptoms with data obtained from the skin wipe samples to improve classification of CF patients.
- MeSH
- chloridy MeSH
- cystická fibróza diagnóza MeSH
- genetické testování MeSH
- ionty MeSH
- klinické chemické testy MeSH
- novorozenec MeSH
- novorozenecký screening MeSH
- pot MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- novorozenec MeSH
- Konspekt
- Pediatrie
- NLK Obory
- perinatologie a neonatologie
- genetika, lékařská genetika
- pneumologie a ftizeologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
A simple and fast method for the analysis of lactate from a single drop of blood was developed. The finger-prick whole blood sample (10 µL) was diluted (1:20) with a 7% (w/v) solution of [tris(hydroxymethyl)methylamino] propanesulfonic acid and applied to a blood plasma separation device. The device accommodates a membrane sandwich composed of an asymmetric polysulfone membrane and a supporting textile membrane that allows the collection of blood plasma into a narrow glass capillary in less than 20 s. Separated and simultaneously diluted blood plasma was directly injected into a capillary electrophoresis instrument with a contactless conductivity detector (CE-C4D) and analyzed in less than one minute. A separation electrolyte consisted of 10 mmol/L l-histidine, 15 mmol/L dl-glutamic acid, and 30 µmol/L cetyltrimethylammonium bromide. The whole procedure starting from the finger-prick sampling until the CE-C4D analysis was finished, took less than 5 min and was suitable for monitoring lactate increase in blood plasma during incremental cycling exercise. The observed lactate increase during the experiments measured by the developed CE-C4D method correlated well with the results from a hand-held lactate analyzer (R = 0.9882). The advantage of the developed CE method is the speed, significant savings per analysis, and the possibility to analyze other compounds from blood plasma.
- MeSH
- cvičení * MeSH
- elektrická vodivost * MeSH
- elektroforéza kapilární metody MeSH
- kyselina mléčná krev MeSH
- lidé MeSH
- limita detekce MeSH
- sportovci psychologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Open source paradigm is becoming widely accepted in scientific communities and open source hardware is finding its steady place in chemistry research. In this review article, we provide the reader with the most up-to-date information on open source hardware and software resources enabling the construction and utilization of an "open source capillary electrophoresis instrument". While CE is still underused as a separation technique, it offers unique flexibility, low-cost, and high efficiency and is particularly suitable for open source instrumental development. We overview the major parts of CE instruments, such as high voltage power supplies, detectors, data acquisition systems, and CE software resources with emphasis on availability of the open source information on the web and in the scientific literature. This review is the first of its kind, revealing accessible blueprints of most parts from which a fully functional open source CE system can be built. By collecting the extensive information on open source capillary electrophoresis in this review article, the authors aim at facilitating the dissemination of knowledge on CE within and outside the scientific community, fosters innovation and inspire other researchers to improve the shared CE blueprints.
Inorganic species still remain of secondary importance when CE is used as an analytical separation tool. Despite considerable efforts of great many groups over last 20 years, plenty of competition, mostly from ion chromatography and elemental MS, restrains the routine use of CE in inorganic analysis. If there is yet hope clinging to make CE a viable alternative in the field, success would ultimately be attained through research activities on negating the disadvantages and amplifying the advantages of the method. Being the latest update of a series of reviews covering the time period from 1990 to 2010, this paper will comment on how and to what extent these challenges of inorganic CE analysis have been addressed in two recent years.
A new method for rapid determination of toxic metabolites after methanol and ethylene glycol intoxication - oxalate, formate and glycolate in various body fluid samples (blood serum, saliva, urine, exhaled breath condensate) by capillary electrophoresis with contactless conductometric detection was developed. A selective separation of the three target analytes from other constituents present in the analyzed biological matrices was achieved in less than 6min in a fused silica capillary of 25μm I.D. using an electrolyte comprising 50mM l-histidine and 50mM 2-(N-morpholino)ethanesulfonic acid at pH 6.1. The only sample preparation was dilution with deionized water. The limits of detection were 0.4, 0.6 and 1.3μM and limits of quantitation 1.3, 1.9 and 4.2μM for oxalate, formate and glycolate, respectively. The method provides a simple and rapid diagnostic test in suspected intoxication and is able to distinguish the ingested liquid, based on its metabolite trace. The method presents a fast screening tool that can be applicable in clinical practice.
- MeSH
- elektroforéza kapilární metody MeSH
- elektrolyty MeSH
- formiáty izolace a purifikace MeSH
- glykoláty izolace a purifikace MeSH
- lidé MeSH
- tělesné tekutiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A novel approach for diagnosis of cystic fibrosis is presented. A simple and fast procedure to obtain sweat sample was developed. It consists of repeatedly wiping the skin of the forearm with deionized water moisturized cotton swab and extraction in 1mL of deionized water. Double opposite end injection capillary electrophoresis with contactless conductometric detection is used for the analysis of the extract. Chloride, sodium and potassium as the three target ions that participate in the ion transfer across the cellular membranes, and are affected by CF, are simultaneously determined in approximately 3min in a background electrolyte containing 20mM 2-(N-morpholino)ethanesulfonic acid, 20mM l-histidine and 2mM 18-crown-6. By using the target ion ratios rather than the concentrations of each individual ion combined with principal component analysis, the diagnosis of CF can be made more accurately and greatly reduce the number of false positive or negative results as is often the case when single ion (chloride) is analyzed.
- MeSH
- analýza hlavních komponent MeSH
- chloridy metabolismus MeSH
- cystická fibróza diagnóza metabolismus MeSH
- draslík metabolismus MeSH
- elektroforéza kapilární metody MeSH
- falešně pozitivní reakce MeSH
- lidé MeSH
- pot metabolismus MeSH
- sodík metabolismus MeSH
- studie případů a kontrol MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A new method for rapid, direct determination of formate in blood serum samples by capillary electrophoresis with contactless conductometric detection is presented. A selective separation of formate was achieved in approximately 1 min using an electrolyte system comprising 10 mM L-histidine, 15 mM glutamic acid and 30 μM cetyltrimethylammonium bromide at pH 4.56. The only sample preparation was dilution (1:100) with deionized water. The limit of detection and limit of quantitation was 2.2 μM and 7.3 μM, respectively, which corresponds to 0.22 mM and 0.73 mM in undiluted blood serum. The method provides a simple and rapid diagnostic test in suspected methanol intoxication cases. The method has been successfully tested on determination of formate in blood of a patient admitted to the hospital under acute methanol intoxication. The peak concentration of formate detected in the patient blood serum was 12.4mM, which is 10- to 100-fold higher than the normal values in healthy population. The developed method presents the fastest test currently available to detect formate in blood samples.
- MeSH
- elektrická vodivost MeSH
- elektroforéza kapilární metody MeSH
- formiáty krev MeSH
- kyselina glutamová chemie MeSH
- lidé MeSH
- methanol krev otrava toxicita MeSH
- otrava alkoholem krev MeSH
- otrava krev MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
CE methods for the speciation of inorganic and organomercury compounds are reviewed. Sample preparation, separation conditions and detection modes are discussed. Efficient separation and sensitive determination of mercury species by CE typically involves complexation with various thiols, chromogenic and other chelating agents; however, some methods do not require complexation. Spectrophotometric detection based on UV-visible absorption is by far the most commonly used. Hyphenated techniques, such as CE/inductively coupled plasma (ICP)-MS, hydride generation coupled to ICP-MS or atomic fluorescence spectrometry and CE/atomic absorption spectrometry are gaining popularity due to their high sensitivity and selectivity. Last, but not least, the potential and applications of electrochemical methods for detection of separated mercury species are outlined.
A new method for the electrophoretic separation of nine phenolic acids (derivatives of benzoic and cinnamic acids) with contactless conductometric detection is presented. Based on theoretical calculations, in which the mobility of the electrolyte co- and counterions and mobility of analytes are taken into consideration, the electrolyte composition and detection mode was selected. This approach was found to be especially valuable for optimization of the electrolyte composition for the separation of analytes having medium mobility. Indirect conductometric detection mode was superior to the direct mode as predicted theoretically. The best performance was achieved with 150 mM 2-amino-2-methylpropanol electrolyte at pH 11.6. The separation was carried out in a counter-electroosmotic mode and completed in less than 6 min. The LODs achieved were about 2.3-3.3 microM and could be further improved to 0.12-0.17 microM by using a sample stacking procedure. The method compares well to the UV-Vis detection.
