BACKGROUND: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco-Phosphatidyl-Inositol (GPI)-linked cell surface structures. Fluorescent proaerolysin (FLAER-Alexa488) is increasingly used to detect GPI-deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER-based and non-FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER. METHODS: We have used a single tube approach with a 7-color assay comprising FLAER-CD157-CD15-CD64-CD24-CD14-CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10-color Navios or 8-color FACSCanto II platforms. The 6-color (minus CD14) and 5-color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5-color assay comprising FLAER-CD157PE-CD64ECD-CD15PC5-CD45PC7. RESULTS/CONCLUSIONS: CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R2  = 1) and monocytes (R2  = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6-, and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. © 2018 International Clinical Cytometry Society.

• MeSH
• CD antigeny analýza   MeSH
• imunofenotypizace přístrojové vybavení   metody   MeSH
• lidé MeSH
• paroxysmální hemoglobinurie diagnóza   MeSH
• průtoková cytometrie přístrojové vybavení   metody   MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.

• MeSH
• imunofenotypizace přístrojové vybavení   normy   MeSH
• lidé MeSH
• průtoková cytometrie přístrojové vybavení   normy   MeSH
• referenční standardy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Funkční hyposplenizmus je stav provázející řadu chorob, vč. autoimunitních onemocnění a lymfomů. Hyposplenizmus se běžně vyskytuje také u celiakie (až ve 20 % nekomplikované a 80 % komplikované nemoci). Hyposplenizmus je spojen se zvýšeným rizikem závažných infekcí (Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae). Cílem této prospektivní pilotní studie bylo vyšetřit paměťové B lymfocyty coby nepřímý biomarker funkčního hyposplenizmu. Bylo vyšetřeno 42 pacientů s celiakií (11 mužů, 31 žen, průměrný věk 49 ± 14 let) a 10 kontrolních osob, dárců krve (2 muži, 8 žen, průměrný věk 39 ± 7 let). Pomocí panelu DuraClone IM byly průtokovou cytometrií (Navios, Beckman Coulter) vyšetřeny jednotlivé subpopulace B lymfocytů v periferní krvi. Imunoglobulin (Ig) M a IgD paměťové B lymfocyty byly u nemocných s celiakií signifikantně nižší ve srovnání s kontrolními osobami. Dysfunkce paměťových B lymfocytů může být zodpovědná za zvýšené riziko bakteriálních infekcí u celiakie. Pacienti s celiakií s prokázanou dysfunkcí paměťových B lymfocytů jsou indikováni k pneumokokové vakcinaci.

Functional hyposplenism is a condition accompanying many diseases including autoimmune disorders and lymphomas. Hyposplenism is also commonly found in adult coeliac disease (up to 20% of non-complicated and up to 80% of complicated disease). Hyposplenism is associated with an increased risk of severe infections (Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae). The aim of this prospective study was to investigate memory B lymphocytes as an indirect biomarker of functional hyposplenism. A total of 42 patients with coeliac disease (11 men, 31 women; mean age 49 ± 14 years) and 10 healthy controls, blood donors (2 men, 8 women; mean age 39 ± 7 years) were enrolled into the study. None of the patients had a history of splenectomy and none of them suffered. from immunodeficiency. The DuraClone IM panel was used to identify B lymphocytes subpopulations in peripheral blood samples by flow cytometry Navios (Beckman Coulter) with software analysis using Kaluza version 1.2. Patients with coeliac disease and controls did not differ in basic parameters of leukocyte and total lymphocyte blood count. Switched memory B lymphocytes (CD19+CD27+IgD-), non-switched memory/marginal-zone-like B lymphocytes (CD19+CD27+IgD+) and immunoglobulin (Ig) M memory B lymphocytes (CD19+CD27+IgM++) were significantly lower in patients with coeliac compared to controls. Follicular (naïve) B lymphocytes were not significantly different between coeliac disease and controls. In conclusion, dysfunction of memory B lymphocytes can be responsible for an increased risk of severe bacterial infections in coeliac disease. Patients with coeliac disease with dysfunction of memory B lymphocytes are clearly indicated for pneumococcal vaccination.

• Klíčová slova
• funkční hyposplenizmus, paměťové B lymfocyty,
• MeSH
• B-lymfocyty * MeSH
• celiakie komplikace   krev   MeSH
• lidé MeSH
• pilotní projekty MeSH
• pneumokokové vakcíny MeSH
• průtoková cytometrie MeSH
• slezina * imunologie   patofyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Cílem výzkumu bylo vyhodnocení vlivu deklastrovaného kultivačního media RPMl na životnost a proliferační potenciál nádorových buněk akutní leukémie a mnohočetného myelomu (myeloma multiplex). Analýza procesu apoptózy byla provedena s použitím průtokového cytometru Navios firmy Beckman Coulter. V případě sedmi pozorovaných buněčných rad leukemie: HL-60, HL-60/MXl, HL-60/MX2, CCRF/ CEM, CEM/Cl, J 45 a U266 byl zjištěn narůst apoptózy výsledkem kultivace v médiu RPMI pod vlivem působení technologie ZiP

• MeSH
• apoptóza * MeSH
• buněčná smrt MeSH
• nádorové buňky kultivované účinky léků   MeSH
• nanostruktury využití   MeSH
• nanotechnologie MeSH
• voda MeSH

A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.

• MeSH
• hematologické nádory diagnóza   MeSH
• imunofenotypizace přístrojové vybavení   normy   MeSH
• lidé MeSH
• průtoková cytometrie přístrojové vybavení   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVE: The aim of the study is to analyze the absolute count of leukocytes, neutrophils, monocytes, eosinophils, T cells, natural killer cells, B cells and to evaluate the expression of functionally important CD23 and CD200 molecules on B cells in patients suffering from atopic dermatitis (AD), (with and without dupilumab therapy). MATERIALS AND METHODS: We examined 45 patients suffering from AD - 32 patients without dupilumab treatment (10 men, 22 women, average age 35.0 years), 13 patients with dupilumab treatment (7 men, 6 women, average age 43.4 years) and 30 healthy control (10 men, 20 women, average age 44.7 years). Immunophenotype was examined by flow cytometry (Navios Flow Cytometer - Beckman Coulter). The blood count was examined with a Sysmex XN 3000, Sysmex SP10, microscope DI60 for digital morphology evaluating cell division and microscope Olympus BX40. We compared the absolute count of leukocytes and their subsets, T cells (CD4, CD8), natural killers cells, absolute and relative count of B lymphocytes and expression of surface molecules CD23 and CD200 on B cells in AD patients and in control group. Non-parametric Kruskal-Wallis one-factor analysis of variance with post-hoc (follow-up multiple comparison) and Dunn's test with Bonferroni modification of significance level were used for statistical analysis. RESULTS: We confirmed the significantly higher number of neutrophils, monocytes and eosinophils and higher expression of CD23 and CD200 on B cells in peripheral blood of AD patients (either with or without dupilumab) therapy. We demonstrated the lower number of CD8+ T cells. CONCLUSION: We demonstrated the difference in the count of white blood cells populations in patients suffering from AD compared with healthy control. There were a differences in the expression of immunoregulatory molecules CD23 and CD200 on B cells in AD patients (either with or without dupilumab therapy) in comparison to healthy controls.

• MeSH
• atopická dermatitida * farmakoterapie   MeSH
• B-lymfocyty MeSH
• dospělí MeSH
• humanizované monoklonální protilátky farmakologie   terapeutické užití   MeSH
• lidé MeSH
• monocyty MeSH
• stupeň závažnosti nemoci MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

"A imaginação de Morgan Rice não tem limites. Em mais uma série que promete ser tão cativante como as anteriores, UM TRONO PARA IRMÃS apresenta-nos o conto de duas irmãs (Sophia e Kate), órfãs, que lutam para sobreviver no mundo cruel e exigente de um orfanato. Um sucesso imediato. Mal posso esperar para por as mãos no segundo e no terceiro livro!"-- Books and Movie Reviews, Roberto MattosDe Morgan Rice, Bestseller #1, chega uma nova série inesquecível de fantasia. Em UMA ENDECHA PARA PRÍNCIPES (Um Trono para Irmãs-Livro Quatro), Sophia, de 17 anos, luta pela sua vida, tentando recuperar do ferimento feito por Lady D'Angélica. Serão os novos poderes da sua irmã Kate suficientes para a trazer de volta?O navio navega com as irmãs para as terras distantes e exóticas do tio delas, que é a sua última esperança e única ligação que conhecem com os seus pais. No entanto, a viagem é traiçoeira, e mesma se elas o encontrarem, as irmãs não sabem se a receção será calorosa ou hostil.Kate, contratada ao orfanato pela bruxa, dá por si numa situação desesperada que não para de aumentar - até encontrar uma feiticeira que pode deter o segredo para a sua liberdade.Sebastian regressa à corte, de coração desfeito, desesperado para saber se Sophia está viva. Quando a sua mãe o obriga a casar com Lady D'Angélica, ele sabe que chegou o momento de arricar tudo.UMA ENDECHA PARA PRÍNCIPES (Um Trono para Irmãs - Livro Quatro) é o quarto livro de uma deslumbrante nova série de fantasia repleta de amor, desgosto, tragédia, ação, aventura, magia, espadas, feitiçaria, dragões, destino e suspense de tirar o fôlego. Um livro que não se quer parar de ler. Está cheio de personagens que vão fazer com que você se apaixone, e com um mundo que você nunca vai esquecer. Livro #5 na série está agora igualmente disponível!"[UM TRONO PARA IRMÃS] é um começo poderoso de uma série que irá produzir uma combinação de protagonistas corajosos e circunstâncias desafiantes para envolver completamente não apenas jovens adultos, mas também fãs de fantasia adulta que procuram histórias épicas alimentadas por amizades e adversários poderosos"--Midwest Book Review (Diane Donovan)