Významnou součástí současné medicíny jsou léčivé přípravky moderní terapie připravované úpravami genotypu a/nebo fenotypu vlastních buněk pacienta nebo buněk dárce. Historie terapeutického využití krevních složek sahá až k počátkům transfuzní medicíny a k prvním experimentům s transfuzemi. Navazující výzkum pak pomohl nalézt metody pro přesnější oddělení jednotlivých krevních elementů a jejich aplikaci v imunoterapii. Nové objevy a na ně navazující potřeba státních regulací léčebného využití buněk izolovaných z krve představují pak důležitou součást moderní hematoonkologie.
Modern medicinal products prepared by manipulating genotype and/or phenotype of cells from a patient or a donor represent an important part of contemporary medicine. The history of therapeutic utilization of blood components reaches as far as the origins of transfusion medicine and the first experiments with transfusions. Subsequent research helped with developing methods for more precise separation of indivdual blood components and their application in immunotherapy. New findings and the related need for state regulation of medicinal usage of cells isolated from blood represent an important part of modern hematooncology.
The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (μMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the μMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.
- MeSH
- Escherichia coli izolace a purifikace MeSH
- imunoglobulin G chemie MeSH
- imunomagnetická separace přístrojové vybavení metody MeSH
- laboratoř na čipu MeSH
- mikrofluidní analytické techniky přístrojové vybavení metody MeSH
- mikrosféry MeSH
- mléko chemie MeSH
- Salmonella cytologie imunologie izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Molecular characterization of tumors could be a key to therapeutic decision-making with regards to targeted therapies in castration-resistant prostate cancer (CRPC). A convenient solution may be non-invasive liquid biopsy testing of circulating tumor cells (CTCs). For this reason, CTC-enriched samples obtained by immunomagnetic separation (AdnaTest®) were studied as a source material for high-throughput gene expression analysis using BioMark™. PATIENTS AND METHODS: CTC-enriched samples from 41 CRPC patients previously determined to be CTC positive using the AdnaTest® were retrospectively re-analysed for androgen receptor (AR) messenger RNA (mRNA), using the updated AdnaTest®. Blood samples were drawn two times from each patient: at the time of CRPC diagnosis and after the third docetaxel cycle. A gene expression panel of 27 genes related to CRPC therapeutic decision-making, including AR full length (ARFL) and splice variant 7 (ARV7), was retrospectively analyzed on a BioMark™ platform in 29 of 41 patients. RESULTS: The AdnaTest® detected AR mRNA in three-quarters of CTC-positive samples taken at the time of CRPC diagnosis and after the third docetaxel cycle. AR detection was associated with a shorter disease-specific survival (45.0 vs. 20.4 months) at the time of CRPC diagnosis. ARFL expression at the time of CRPC diagnosis, measured on the BioMark™ platform, was associated with a lower decrease of serum level of prostate-specific antigen (sPSA) (p = 0.029), i.e., worse therapy response. ARV7 was found in 38% of the ARFL--positive samples at both analyzed timepoints. CONCLUSION: Detection of AR expression by AdnaTest® in CTC-enriched samples may help predict patients' survival. These AdnaTest® CTC-enriched samples can be used in a high-throughput quantitative polymerase chain reaction (qPCR) analysis of gene expression, provided that the specificity of the assay for each individual gene is properly validated. The BioMark™ platform can be used for the simultaneous detection of ARFL and ARV7 and other genes in CTC-enriched samples from CRPC patients.
- MeSH
- genetické testování metody MeSH
- imunomagnetická separace MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery * MeSH
- nádorové cirkulující buňky metabolismus patologie MeSH
- nádory prostaty rezistentní na kastraci diagnóza genetika MeSH
- následné studie MeSH
- rychlé screeningové testy MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stanovení celkové genové exprese * metody MeSH
- transkriptom MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
1 svazek : ilustrace, tabulky ; 30 cm
The aim of the project is to detect circulating tumor cells (CTC) in peripheral blood of patients with metastatic prostate cancer before and during the cytotoxic therapy. Detection will be performed by the method of immunomagnetic separation and RNA isolation from this fraction. Then we will perform the cultivation of the detected cells including the cultivation in milieu of cytotoxic agents. In the next stage the genetic profile of the detected cells will be assessed (a panel of 24 genes) and a similar profile will be evaluated also in histological specimen of a primary tumor. Analysis results will be correlated to the clinical course of disease and the prognostic factors of treatment effect on CTC count decline and patients‘ survival will be determined.
Cílem projektu je detekovat cirkulující nádorové buňky (CTC) v periferní krvi u pacientů s metastatickým karcinomem prostaty před cytotoxickou léčbou a v jejím průběhu. Detekce bude provedena metodou imunomagnetické separace buněk a izolací RNA z této frakce. Dále proběhne kultivace části detekovaných buněk včetně kultivace v prostředí cytotoxických látek. V další fázi bude hodnocen genetický profil zjištěných buněk (stanovení panelu 24 genů) a obdobný profil také u histologického preparátu primárního tumoru. Výsledky analýz budou vztaženy na klinický průběh onemocnění a budou stanoveny prognostické faktory účinku léčby na pokles CTC a přežití pacientů.
- MeSH
- analýza přežití MeSH
- diagnostické techniky molekulární MeSH
- exprese genu MeSH
- imunomagnetická separace MeSH
- individualizovaná medicína MeSH
- nádorové buněčné linie cytologie MeSH
- nádorové cirkulující buňky MeSH
- nádory prostaty rezistentní na kastraci diagnóza MeSH
- prognóza MeSH
- prostatický specifický antigen analýza MeSH
- RNA analýza MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biologie
- andrologie
- onkologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
BACKGROUND: The presence of circulating tumor cells (CTC) has been reported in patients with advanced colorectal cancer. Monitoring CTC (also known as a liquid-biopsy) has recently become the center of interest for low-invasive monitoring of cancer progression and predictive biomarkers testing. Along with high-cost technology and a complex methodology, a straightforward method based on magnetic beads enrichment followed by RT-PCR is set to allow for routine CTC analysis in colorectal cancer patients. OBJECTIVES: The main purpose of this study was to evaluate the possibility of CTC detection in routine monitoring of patients starting before and continuing after surgery. MATERIAL AND METHODS: The investigated group consisted of 30 patients mainly in advanced stages of colorectal cancer. In all patients, CTC detection was performed prior to surgery, in a subset of 14 patients additional sampling was done during and after surgery. In all cases, peripheral blood was processed using AdnaTest ColonCancer kit, which relies on enriching CTCs using EpCAM-functionalized magnetic beads and subsequently identifying tumorspecific CEA, EGFR and GA733-2 mRNA transcripts. RESULTS: Out of all the tested samples, CTC were found in one patient suffering from advanced disease with lung and liver metastases. There, however, the positive finding was confirmed in 3 consecutive samples acquired before, during and shortly after palliative R2 resection. CONCLUSIONS: The presence of CTC may be used to observe post-operative disease development. Due to the overall low CTC detection, further technology development may be necessary before its universal applicability to manage colorectal cancer patients.
- MeSH
- imunomagnetická separace metody MeSH
- kolorektální chirurgie * MeSH
- kolorektální nádory patologie chirurgie MeSH
- lidé MeSH
- nádorové cirkulující buňky patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- senioři MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
A nanobiosensor based on the use of porous magnetic microspheres (PMM) as efficient capturing/pre-concentrating platform is presented for detection of Alzheimer's disease (AD) biomarkers. These PMMs prepared by a multistep swelling polymerization combined with iron oxide precipitation afford carboxyl functional groups suitable for immobilization of antibodies on the particle surface allowing an enhanced efficiency in the capturing of AD biomarkers from human serum samples. The AD biomarkers signaling is produced by gold nanoparticle (AuNP) tags monitored through their electrocatalytic effect towards hydrogen evolution reaction (HER). Novel properties of PMMs in terms of high functionality and high active area available for enhanced catalytic activity of the captured AuNPs electrocatalytic tags are exploited for the first time. A thorough characterization by scanning transmission electron microscope in high angle annular dark field mode (STEM-HAADF) demonstrates the enhanced ability of PMMs to capture a higher quantity of analyte and consequently of electrocatalytic label, when compared with commercially available microspheres. The optimized and characterized PMMs are also applied for the first time for the detection of beta amyloid and ApoE at clinical relevant levels in cerebrospinal fluid (CSF), serum and plasma samples of patients suffering from AD.
- MeSH
- Alzheimerova nemoc diagnóza metabolismus MeSH
- amyloidní beta-protein analýza MeSH
- apolipoproteiny E analýza MeSH
- barvení a značení MeSH
- biologické markery analýza MeSH
- imunomagnetická separace metody MeSH
- katalýza MeSH
- konduktometrie metody MeSH
- kovové nanočástice chemie ultrastruktura MeSH
- lidé MeSH
- mikrosféry MeSH
- poréznost MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.
- MeSH
- design vybavení MeSH
- elektrochemické techniky přístrojové vybavení metody MeSH
- elektrody MeSH
- imobilizační protilátky chemie metabolismus MeSH
- imunoglobulin G chemie metabolismus MeSH
- imunoglobuliny chemie metabolismus MeSH
- imunomagnetická separace přístrojové vybavení metody MeSH
- králíci MeSH
- kur domácí MeSH
- lidé středního věku MeSH
- lidé MeSH
- metalothionein krev MeSH
- nádory hlavy a krku krev MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The emergence of drug-resistant bacteria and new or changing infectious pathogens is an important public health problem as well as a serious socioeconomic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. Nanomaterial-based techniques including fluorescent labeling by quantum dots as well as immunoextraction by magnetic particles are excellent tools for such purposes. Moreover, the combination with capillary electrophoresis in miniaturized microchip arrangement brings numerous benefits such as fast and rapid analysis, low sample consumption, very sensitive electrochemical and fluorescent detection, portable miniaturized instrumentation, and rapid and inexpensive device fabrication. Here the use of superparamagnetic particle-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II)-containing proteins (Zn-proteins) is reported using a robotic pipetting system speeding up the sample preparation and enabling to analyze 48 real samples within 6 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with the sufficient yield for SDS-PAGE (several tens ng of proteins).
- MeSH
- bakteriální proteiny izolace a purifikace MeSH
- elektroforéza kapilární metody MeSH
- elektroforéza mikročipová metody MeSH
- imunomagnetická separace metody MeSH
- kvantové tečky * MeSH
- Staphylococcus aureus chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Karcinom prostaty (KP) je nejčastější nádorové onemocnění u mužů. Prognóza pacientů s metastatickým onemocněním je nepříznivá, ačkoliv v tomto stadiu existují velké rozdíly v kvalitě života a době přežívání. Definice prognózy je nezbytná pro volbu léčebného postupu, který respektuje individuální riziko progrese onemocnění. Mezi základní prognostické parametry u metastatického onemocnění patří Gleasonovo skóre nádoru, hladina prostatického specifického antigenu (PSA), výkonnostní stav a další laboratorní markery. V posledních letech se v predikci odpovědi na protinádorovou léčbu uplatňuje detekce cirkulujících nádorových buněk (CTC), které jsou nezbytnou součástí metastatického procesu. Stanovení CTC využívá znalostí nádorově specifických antigenů na povrchu buněk. Jednotlivé metody stanovují CTC s různou citlivostí a zatím nejsou využitelné u lokalizovaného stadia KP. Pouze metoda imunomagnetické separace a semiautomatické vizualizace (CellSearchTM) byla validována a schválena FDA pro použití v diagnostice a léčbě metastatického KP. Stanovení počtu CTC přímo koreluje s prognózou pacientů s kastračně rezistentním KP a je dynamičtějším markerem celkového přežití než hladina PSA. Změna v hodnotách CTC v průběhu léčby také výrazně zpřesňuje odhad rizika úmrtí. Nové postupy kultivace a genového profilování CTC mohou přispět k individualizaci léčby podobně jako u karcinomu prsu. Autoři předkládají přehledné informace o teorii, metodách detekce a klinickém využití CTC u kastračně rezistentního KP.
Prostate cancer (PC) is the most common malignant disease in men. Prognosis of patients with metastatic PC is generally unfavourable; however there are significant differences in survival at this stage of the disease. The definition of prognosis is essential for the selection of therapy, respecting an individual risk. In recent years, the association between circulating tumor cells (CTC) detection and response to PC treatment has been widely investigated. Detection of CTC is based on a metastatic process theory and uses well-known tumor-specific antigens on the cell surface. Individual methods assess CTC with different sensitivity and are not yet efficient at the localised PC stage. Only the method of immunomagnetic separation and semi-automatic visualisation (CellSearchTM) has been validated and approved for the use in the PC management. Assessment of the CTC count directly correlates with the prognosis of patients with castration-resistant PC. Change in the CTC count during the therapy also considerably improves risk estimation and represents a marker of overall survival. New methods of CTC cultivation and gene profiling may contribute to individualisation of the treatment similarly to breast cancer. The authors present a review article about theory, methods of detection and clinical use of CTC in castration-resistant PC.
- MeSH
- DNA nádorová MeSH
- imunomagnetická separace * metody MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé MeSH
- nádorové biomarkery * krev MeSH
- nádorové cirkulující buňky * MeSH
- nádory prostaty * krev MeSH
- prognóza MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
