Východisko. Cílem bylo zavést kvantifikaci virové nálože viru Epsteina a Baarové (EBV) u dětských pacientů po alogenní transplantaci hematopoetických kmenových buněk (HSCT), charakterizace průběhu virové nálože u čtyř fatálních případů lymfoproliferativní nemoci (EBV-LPD) a testování rizikových faktorů pro reaktivaci EBV. Metody a výsledky. Retrospektivně jsme kvantifikovali EBV v sérii vzorků od čtyř dětí zemřelých na potransplantační EBV-LPD v roce 2000. Následně jsme v letech 2001–2004 prospektivně sledovali 72 dětí po alogenní HSCT. Virus jsme vyšetřovali pomocí kvantitativní real-time PCR z krve odebírané v týdenních intervalech první tři měsíce po HSCT, později při ambulantních kontrolách. Retrospektivní vyšetření vzorků zemřelých pacientů ukázalo, že všichni překročili hladinu 1 milionu kopií EBV (normalizováno na 100 000 lidských genomových ekvivalentů) a že první detekce reaktivovaného EBV předcházela úmrtí o 24–91 dní. Z prospektivně sledovaných pacientů byla překročena hladina 100 normalizovaných kopií EBV u 48 (67 %) dětí, 1000 u 13 (18 %) pacientů. Hladinu 10 000 kopií překročili 4 pacienti, u tří z nich byla prokázána EBV-LPD, a byli úspěšně léčeni monoklonální protilátkou proti CD20. Výskyt EBV po HSCT nelze spolehlivě predikovat ze žádných parametrů primárního onemocnění nebo transplantace. Závěry. Kvantifikace EBV v pravidelných časových intervalech po alogenní HSCT je vhodnou metodou pro časné odhalení růstu nálože viru. Hladina 10 000 virových kopií/100 000 lidských genomových ekvivalentů dobře predikuje EBV-LPD, avšak je stále bezpečná pro časnou specifickou terapii.

Background. Patients undergoing allogeneic hematopoietic stem cell transplantation (AHSCT) are endangered by developing Epstein-Barr virus-related post-transplant lymfoprolipherative disease (EBV-LPD). The aims of the study were to retrospectively characterise the viral loads in four patients who died of this complication, and to test possible risk factors for EBV reactivation in a prospectively observed cohort of children after AHSCT. Methods and Results. Serial DNA samples extracted from whole blood from four patients who died of post-transplant EBV-LPD in year 2000 were retrospectively analysed for EBV load using quantitative real-time PCR. First detection of EBV activation preceded death by 24-91 days. All four patients exceeded a viral load of one million EBV copies per 100,000 human genome equivalents. A cohort of 72 children undergoing AHSCT between 2001-2004 was prospectively followed-on using the same quantification method from regularly obtained samples of whole blood, and clinical and laboratory data were recorded on a weekly basis, totalling at 3,896 person-weeks of observation. Approximately one half of the cohort experienced at least one episode of EBV reactivation during the first 100 days after AHSCT, four of the episodes being accompanied with viral loads higher than our provisional threshold of 10,000 copies per 100,000 human genome equivalents. Three of the four patients developed EBV-LPD and were successfully treated by intravenous administration of anti-CD20 antibody. Testing of possible clinical and laboratory predictors of EBV reactivation did not reveal any clinically useful association. Conclusions. The cornerstone of predicting EBV-LPD in AHSCT is a regular monitoring of EBV viral load using quantitative methods. Using this strategy with a threshold of 10,000 EBV copies per 100,000 human genome equivalents was proved to be effective, as shown by no death of EBV for the study period, compared to four cases in the year before the quantitative monitoring.

• MeSH
• Child MeSH
• DNA, Viral diagnostic use   isolation & purification   blood   MeSH
• Research Support as Topic MeSH
• Epstein-Barr Virus Infections diagnosis   complications   therapy   MeSH
• Humans MeSH
• Lymphoproliferative Disorders etiology   MeSH
• Prospective Studies MeSH
• Graft vs Host Reaction MeSH
• Hematopoietic Stem Cell Transplantation MeSH
• Check Tag
• Child MeSH
• Humans MeSH

U pacientů s vlasatobuněčnou leukémií, léčených 2-chlorodeoxyadenosinem nebo 2-deoxycoformicinem, je dosahováno velmi vysokého počtu terapeutických odpovědí. Při dosažení kompletní remise - minimální reziduálni nemoci nelze prokázat aktivitu onemocnění, splenomegalii a lymfadenopatii; dochází k normalizaci koncentrace hemoglobinu, počtu granulocytů a krevních destiček. Současně nelze standardním barvením prokázat leukemické buňky v periferní krvi a v kostní dřeni. K jejich průkazu lze použít imunohistochemického vyšetření pomocí protilátky DBA.44 na intenzitu imunohistochemické reakce a zároveň morfologii vlasatých buněk. Při kvantifikaci leukemických buněk je používána počítačová analýza obrazu LUCIA-M.

Hairy cell leukemia patients treated with 2-chlorodeoxyadenosine or 2-deoxycoformicin achieve a veiy high number of therapeutic responses. After complete remission, i.e. minimal residual disease, we cannot demonstrate the disease activity, splenomegaly, or lymphadenopathy; moreover, there comes to normalization of hemoglobin concentration, leukocyte count, and platelet count. No leukemic cells in peripheral blood or bone marrow smears can be seen with the use of staining techniques. They can be demonstrated immunohistochemically in decalcified trephine bone marrow biopsies with the use of DBA.44 antibody together with their morphologic features. For quantification of leukemic cells we use LUCIA-M image analysis.

• MeSH
• Immunohistochemistry methods   utilization   MeSH
• Biopsy, Needle methods   utilization   MeSH
• Humans MeSH
• Antibodies, Monoclonal diagnostic use   MeSH
• Leukemia, Hairy Cell diagnosis   immunology   MeSH
• Check Tag
• Humans MeSH

• MeSH
• Carcinoma etiology   MeSH
• Humans MeSH
• Risk Factors MeSH
• Check Tag
• Humans MeSH
• Female MeSH

Hodnocení minimální reziduální nemoci u akutních lymfoblastických leukemií pomocí kvantifikace klonálně specifických přestaveb genů pro imunoglobuliny a T-buněčné receptory je v současné době považováno za standardní laboratorní postup. Výsledky této metody jsou stále častěji využívány v léčebných protokolech pro stratifikaci pacientů do rizikových skupin s různě intenzívní terapií nebo přímo pro volbu konkrétních léčebných postupů. Vzhledem k náročnosti metodiky je třeba dodržovat technická a interpretační kritéria, která umožňují plnou reprodukovatelnost metody a zaručují klinickou validitu výsledků. Autoři shrnují současný pohled na provádění a interpretaci této moderní laboratorní metody a upozorňují na možná rizika plynoucí z nedodržení těchto kritérií.

Currently, qualification of clonal immunoglobulin and T-cell receptor rearrangements is considered to be a standard laboratory investigation to evaluate minimal residual disease in acute lymphoblastic leukemia. Benefit of this method contributes more often to therapeutic protocols that stratify patients into the groups according to the need of differently intensive therapy or particular therapeutic regimen. Regarding complexity of the method, it is necessary to follow technical and interpretative criteria that enable reproducibility and clinical validity of the method. The authors summarize current view on design and interpretation of this modern laboratory method. They also notice possible risks when these criteria were broken.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma diagnosis   MeSH
• Research Support as Topic MeSH
• Immunoglobulins diagnostic use   genetics   MeSH
• Leukemia, T-Cell genetics   MeSH
• Humans MeSH
• Neoplasm, Residual diagnosis   MeSH
• Check Tag
• Humans MeSH

Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners).

• MeSH
• Cell Culture Techniques MeSH
• Protein Array Analysis methods   MeSH
• Cytokines metabolism   MeSH
• Immunoassay methods   MeSH
• Stem Cells physiology   MeSH
• Culture Media, Conditioned metabolism   MeSH
• Cells, Cultured MeSH
• Cell Communication * MeSH
• Intercellular Signaling Peptides and Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Correct determination of the instantaneous level and changes of relevant proteins inside individual cells is essential for correct interpretation and understanding of physiological, diagnostic, and therapeutic events. Thus, single-cell analyses are important for quantification of natural cellular heterogeneity, which cannot be evaluated from averaged data of a cell population measurements. Here, we developed an original highly sensitive and selective instrumentation and methodology based on homogeneous single-step bioluminescence assay to quantify caspases and evaluate their heterogeneity in individual cells. Individual suspended cells are selected under microscope and reliably transferred into the 7 μl detection vials by a micromanipulator. The sensitivity of the method is given by implementation of photomultiplying tube with a cooled photocathode working in the photon counting mode. By optimization of our device and methodology, the limits of detection and quantitation were decreased down to 2.1 and 7.0 fg of recombinant caspase-3, respectively. These masses are lower than average amounts of caspase-3/7 in individual apoptotic and even non-apoptotic cells. As a proof of concept, the content of caspase-3/7 in single treated and untreated HeLa cells was determined to be 154 and 25 fg, respectively. Based on these results, we aim to use the technology for investigations of non-apoptotic functions of caspases.

• MeSH
• Apoptosis * MeSH
• HeLa Cells MeSH
• Caspase 3 MeSH
• Caspases * MeSH
• Humans MeSH
• Technology MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Renal cell carcinoma (RCC) represents 2.2% of all cancer incidences; however, prognostic or predictive RCC biomarkers at protein level are largely missing. To support proteomics research of localized and metastatic RCC, we introduce a new library of targeted mass spectrometry assays for accurate protein quantification in malignant and normal kidney tissue. Aliquots of 86 initially localized RCC, 75 metastatic RCC and 17 adjacent non-cancerous fresh frozen tissue lysates were trypsin digested, pooled, and fractionated using hydrophilic chromatography. The fractions were analyzed using LC-MS/MS on QExactive HF-X mass spectrometer in data-dependent acquisition (DDA) mode. A resulting spectral library contains 77,817 peptides representing 7960 protein groups (FDR = 1%). Further, we confirm applicability of this library on four RCC datasets measured in data-independent acquisition (DIA) mode, demonstrating a specific quantification of a substantially increased part of RCC proteome, depending on LC-MS/MS instrumentation. Impact of sample specificity of the library on the results of targeted DIA data extraction was demonstrated by parallel analyses of two datasets by two pan human libraries. The new RCC specific library has potential to contribute to better understanding the RCC development at molecular level, leading to new diagnostic and therapeutic targets.

• MeSH
• Chromatography, Liquid MeSH
• Carcinoma, Renal Cell * MeSH
• Humans MeSH
• Kidney Neoplasms * MeSH
• Proteome metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cell quantification is widely used both in basic and applied research. A typical example of its use is drug discovery research. Presently, plenty of methods for cell quantification are available. In this review, the basic techniques used for cell quantification, with a special emphasis on techniques based on fluorescent DNA dyes, are described. The main aim of this review is to guide readers through the possibilities of cell quantification with various methods and to show the strengths and weaknesses of these methods, especially with respect to their sensitivity, accuracy, and length. As these methods are frequently accompanied by an analysis of cell proliferation and cell viability, some of these approaches are also described.

• MeSH
• Biological Assay MeSH
• DNA analysis   chemistry   metabolism   MeSH
• Fluorescent Dyes * MeSH
• Cell Count methods   MeSH
• Cell Proliferation MeSH
• Cell Survival MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Comparative Study MeSH

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.

• MeSH
• Acetyl Coenzyme A analysis   MeSH
• Adenosine Triphosphate analysis   MeSH
• Chemistry Techniques, Analytical methods   standards   MeSH
• Cytidine Triphosphate analysis   MeSH
• Embryonic Stem Cells chemistry   MeSH
• Guanosine Triphosphate analysis   MeSH
• Humans MeSH
• Limit of Detection MeSH
• Paracoccus denitrificans chemistry   MeSH
• Reproducibility of Results MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Cytogenetics MeSH
• Molecular Diagnostic Techniques MeSH
• Polymerase Chain Reaction MeSH
• Preimplantation Diagnosis MeSH
• Infertility, Female therapy   MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Biotechnologie. Genetické inženýrství
• NML Fields
• biologie
• genetika, lékařská genetika