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Autotransporters constitute a large family of natural proteins that are essential for delivering many types of proteins and peptides across the outer membrane in Gram-negative bacteria. In biotechnology, autotransporters have been explored for display of recombinant proteins and peptides on the surface of Escherichia coli and have potential as tools for directed evolution of affinity proteins. Here, we investigate conditions for AIDA-I autotransporter-mediated display of recombinant proteins. A new expression vector was designed and engineered for this purpose, and a panel of proteins from different affinity-protein classes were subcloned to the vector, followed by evaluation of expression, surface display and functionality. Surface expression was explored in ten different E. coli strains together with assessment of transformation efficiencies. Furthermore, the most promising strain and expression vector combination was used in mock library selections for evaluation of magnetic-assisted cell sortings (MACS). The results demonstrated dramatically different performances depending on the type of affinity protein and choice of E. coli strain. The optimized MACS protocol showed efficient enrichment, and thus potential for the new AIDA-I display system to be used in methods for directed evolution of affinity proteins., QC 20240202

Many methods of single-cell analysis are currently used, and each method provides different characteristics of the cell, such as dielectric and physical properties. In this thesis we investigate the use of optical analysis in tandem with dielectrophoresis analysis in a high throughput flow cytometer system. To examine the viability of combining optical and dielectrophoretic analysis methods, this thesis proposes an optical model using Mie scattering theory, and a data extraction and particle classification algorithm that employs both analysis modalities. The optical model presented here examines the effects of different experimental variables such as particle size and refractive index, the refractive index of the medium, and cytometer components like LED wavelength. The data extraction and particle classification algorithm presented extracts both optical and DEP data and is used to identify and sort particles in mixed population experiments. Combining the information gathered from examining the optical data from particles as well as the dielectric properties from dielectrophoretic measurements provides a more robust and confident classification of particles. In this thesis, the combination of the two methods was applied to both polystyrene polymer sphere (PSS) beads and Chinese hamster ovary (CHO) cell experiments. Particle size data was successfully extracted using optical analysis and used to provide more confidence and robustness to the particle classification conducted using DEP analysis. The findings of this thesis provide a starting point for further use of optical analysis in conjunction with DEP analysis in a high through put flow cytometer system.

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms. Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration. Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

Background: The therapeutic use of gingival mesenchymal stem cells (GMSCs) as autologous cells may pose the challenge of alterations inflicted by the hyperglycemic environment. Objective: This study aims to assess the effects of hyperglycemia on the characteristics of GMSCs in diabetics. Materials and Methods:10 patients who consented and fulfilled the criteria for inclusion and exclusion were recruited and categorized as test (HbA1c > 6.5) and control (HbA1c < 6.0). Gingival explants were obtained from gingival collar of teeth, washed, digested and cultured. The cells were subjected to microscopic observation to assess phenotype characteristics, and flow cytometry and qRT-PCR to assess differentiation potential. Stem cell markers CD90, CD73, CD105, CD34, CD45, HLA DR & HLA ABC, osteogenic differentiation markers RUNX2 & OCN, adipogenic differentiation markers PPARG2 & FABP4 and chondrogenic differentiation markers SOX9 & AGCN were evaluated. Results: Microscopic appearance of spindle shaped cells was found to be comparable in both groups. Flow cytometry results demonstrated comparable expressions with both groups, samples being positive for CD90, CD73, CD105, HLA ABC and negative for CD34, CD45 & HLA DR. There were variations in the expression of markers when assessed for differentiation potentials. Conclusions: The hyperglycemic environment did not manifest any changes in the phenotypic characteristics of GMSCs among diabetics. However, the expression of certain differentiation markers was significantly altered in the diabetic test population included. Further research is being conducted to understand the GMSCs in a hyperglycemic environment with an aim to develop strategies to optimize its clinical implications., Antecedentes: El uso terapéutico de células madre mesenquimales gingivales (GMSC) como células autólogas puede plantear el desafío de las alteraciones infligidas por el entorno hiperglucémico. Objetivo: Este estudio tiene como objetivo evaluar los efectos de la hiperglucemia sobre las características de las GMSC en diabéticos. Materiales y Métodos: Se reclutaron y categorizaron 10 pacientes que dieron su consentimiento y cumplieron los criterios de inclusión y exclusión como prueba (HbA1c > 6,5) y control (HbA1c < 6,0). Los explantes gingivales se obtuvieron del cuello gingival de los dientes, se lavaron, digirieron y cultivaron. Las células se sometieron a observación microscópica para evaluar las características fenotípicas y a citometría de flujo y qRT-PCR para evaluar el potencial de diferenciación. Se evaluaron los marcadores de células madre CD90, CD73, CD105, CD34, CD45, HLA DR y HLA ABC, marcadores de diferenciación osteogénica RUNX2 y OCN, marcadores de diferenciación adipogénica PPARG2 y FABP4 y marcadores de diferenciación condrogénica SOX9 y AGCN. Resultados: Se encontró que la apariencia microscópica de las células fusiformes era comparable en ambos grupos. Los resultados de la citometría de flujo demostraron expresiones comparables en ambos grupos, siendo las muestras positivas para CD90, CD73, CD105, HLA ABC y negativas para CD34, CD45 y HLA DR. Hubo variaciones en la expresión de los marcadores cuando se evaluaron los potenciales de diferenciación. Conclusiones: El entorno hiperglucémico no manifestó ningún cambio en las características fenotípicas de las GMSC entre los diabéticos. Sin embargo, la expresión de ciertos marcadores de diferenciación se alteró significativamente en la población de prueba de diabetes incluida. Se están realizando más investigaciones para comprender las GMSC en un entorno hiperglucémico con el objetivo de desarrollar estrategias para optimizar sus implicaciones clínicas

Veterans returning from deployment in the Middle East have displayed chronic asthmatic-like symptoms after exposure to burn pits and sandstorms. These soldiers also encounter stressful environments during deployment, introducing another factor that could alter their immune response. In this thesis, we have investigated the impact of acute stress on lung inflammation in murine models, focusing on the role of innate lymphoid cells (ILCs) during exposure to the allergen Alternaria alternata (Alt) and burn pit constituents (BPCs) which include dioxins, hydrocarbons, and particulate matter. Our previous studies have shown that combinatory exposure to Alt and BPCs induces a mixed type 2 and non-type 2 inflammatory response, characterized by increased neutrophilia and a shift from ILC2 to ILC1-like populations. Single-cell RNA sequencing (scRNA-seq) of lung samples showed upregulation of neutrophilia-associated genes and stress-related genes in ILCs from Alt+BPC-challenged mice, indicating that stress exacerbates inflammatory responses. Here, we present a novel model to reflect an acute stress event followed by inhalation of BPCs or Alt focusing on analysis of lung granulocytes and innate lymphoid cells. Results indicated a significant decrease in different ILC subpopulations of stressed mice after either allergen or BPC exposure. This decline reflects reduced neutrophilia in non-type 2 inflammation and trending decreases in eosinophilia in type 2 inflammation. This study concludes that acute stress alters the immune response to lung inflammation. These findings suggest that stress, combined with BPC exposure, might contribute to the respiratory issues observed in exposed military personnel.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

Flow cytometry, single-cell RNA sequencing, and other analyses enable us to capture immune profiles of the tumor microenvironment. Here, we present a protocol to characterize the immune profile of tumor-bearing mice. We describe steps for establishing mouse models and preparing single-cell suspensions from tumor tissue and other immune-related organs, which can be further analyzed by flow cytometry and other omics assays. We then detail procedures for staining, flow cytometry analysis, and phenotyping of the immune cell populations. For complete details on the use and execution of this protocol, please refer to Miyauchi et al.1.

Urinvägsinfektion (UVI) är en av de vanligaste bakteriella infektionerna bland människor. Urinodling är standardmetoden för att identifiera bakterier och kvantifiera mängden av dem i urinen. Flödescytometri ger möjligheten att snabbare analysera urinprover för att utesluta urinvägsinfektion och därmed minska den onödiga antibiotikabehandlingen. Den omfattande användningen av antibiotika utgör en stor orsak till antimikrobiell resistens (AMR) och har resulterat i en stor efterfrågan av metoder för att genomföra snabbare tester för antibiotikaresistens. Målet med projektet blev därför att utvärdera om flödescytometern UF-5000 kan användas för att snabbare diagnostisera urinvägsinfektioner. Urinprover kördes tre gånger genom UF-5000 med olika sköljprogram tillsammans med tre olika efterföljande sterila koksaltlösningsprover och utifrån en formel beräknades carryover rate (överföringsgrad). Sterila koksaltlösningaproverna odlades sedan för att se hur stor korskontaminationen blev. Dessutom genomfördes standard resistensbestämning (AST) och direkt resistensbestämning (RAST) på både kontrollstammar och patientprover. Carryover rate av bakterier blev mindre när sköljprogram med fler antal sköljningar användes. Sköljprogrammet [0-0-1-3-4] översteg aldrig gränserna för tillåten carryover rate. Resultat gick att avläsa för de flesta proverna efter RAST på sex timmar men inte på fyra timmar. Det var skillnader mellan resultaten erhållna efter AST och RAST. Vi kom fram till att det mest tidseffektiva sköljprogrammet som inte resulterade i någon betydlig påverkan på efterföljande prover var sköljprogrammet [0-0-1-3-4] i UF-5000. Vi kom även fram till att resistensbestämning på fyra timmar var svår att göra men att efter sex timmar skulle man kunna utesluta vilka antibiotika man inte ska använda för behandling., Urinary tract infection (UTI) is one of the most common bacterial infections among humans. Urine culture is the standard method for identifying and quantifying the amount of bacteria in the urine. Flow cytometry offers the possibility to analyze urine samples more quickly to rule out urinary tract infection, thereby reducing unnecessary antibiotic treatment. The extensive use of antibiotics is a major cause of antimicrobial resistance (AMR) and has resulted in a high demand for methods to perform faster tests for antibiotic resistance. The aim of the project was therefore to evaluate whether the flow cytometer UF-5000 can be used to diagnose urinary tract infections more quickly. Urine samples were run three times through the UF-5000 with different rinse programs, along with three different subsequent sterile saline samples, and the carryover rate was calculated using a formula. The sterile saline samples were then cultured to determine the extent of cross-contamination. Additionally, antimicrobial susceptibility testing (AST) and rapid antimicrobial susceptibility testing (RAST) were performed on both control strains and patient samples. The carryover rate of bacteria was lower when rinse programs with a higher number of rinses were used. The rinse program [0-0-1-3-4] never exceeded the limits for the allowed carryover rate. Results could be read for most samples after RAST at six hours but not at four hours. There were differences between the results obtained after AST and RAST. We concluded that the most time-efficient rinse program that did not result in any significant impact on subsequent samples was the rinse program [0-0-1-3-4] in the UF-5000. We also found that resistance determination at four hours was difficult to perform, but after six hours, it would be possible to rule out which antibiotics should not be used for treatment.

För att bedöma mäns spermiekvalitet och därmed pars chanser till en graviditet har standardparametrar tidigare använts som fertilitetsindikator, men har visat sig vara en mindre tillförlitlig metod. Därför har en ny metod som bygger på flödescytometri och kallas Sperm Chromatin Structure Assay (SCSA) utvecklats som fokuserar på spermiernas arvsmassa, och som då har visat sig vara en mycket mer tillförlitlig metod. Syftet med denna studie var att jämföra DNA Fragmentations Index (DFI) -värde mellan svenska och danska män och se om en signifikant skillnad finns. För att uppfylla detta späddes och färgades spermaproven in och analyserades med SCSA, som därefter gav hur stor andel av spermiernas DNA som är defekt, vilket presenterades som ett procentuellt DFI-värde. Resultatet visade en signifikant skillnad av medianvärdet mellan svenska och danska män med avseende på DFI, där svenska män hade högre medianvärde än de danska männen. Detta i kombination med tidigare forskning som gjordes mellan svenska och danska män och som visade att svenska män hade bättre kvalitet än danska män med avseende på standardparametrarna, stödjer tidigare forskning som visar på att det ej finns ett samband mellan standardparametrar och DFI. Dock hade ytterligare forskning av skillnaderna mellan svenska och danska män behövts, för att validera fynden i denna studie., To assess men's sperm quality and thus couples' chances of pregnancy, standard parameters have previously been used as a fertility indicator, but have proven to be a less reliable method. Therefore, a new method based on flow cytometry called Sperm Chromatin Structure Assay (SCSA) has been developed which focuses on the sperm's genetic mass, and which has been shown to be a much more reliable method. The purpose of this study was to compare the DNA Fragmentation Index (DFI) value between Swedish and Danish men and see if a significant difference exists. To fulfill this, the sperm samples were diluted and stained and analyzed with SCSA, which then gave the ratio of the sperm's DNA that is defective, which was presented as a percentage DFI value. The result showed a significant difference in the median value between Swedish and Danish men with regard to DFI, where Swedish men had a higher median value than the Danish men. This, in combination with previous research that has been done between Swedish and Danish men which showed that Swedish men had better quality than Danish men in regards to the standard parameters, supports previous research that shows that there is no connection between standard parameters and DFI. However, further research into the differences between Swedish and Danish men is needed to validate the findings in this study.

To reduce the risk of transfusion related complications, blood components should, according to European guidelines, contain less than 1 x 106 leukocytes per unit. To verify that these guidelines are upheld, residual leukocytes are measured in randomly selected blood components as means of quality control. At Uppsala University Hospital, the method currently used for this is flow cytometry (FCM). However, the hospital recently purchased a new instrument, ADAM rWBC2, for this purpose. The aim of this study was to verify ADAM rWBC2 as a replacement method for FCM and investigate whether the type of test tube chosen for the instrument (EDTA or micro test tube) would affect the leukocyte concentration. To conduct the study, 30 red blood cell units (RBCs), 30 platelet units (PLTs) and 30 plasma units were analyzed on both the ADAM rWBC2 instrument and with FCM. In addition to this, each RBC and PLT unit was allocated into both an EDTA tube and a micro test tube before analysis on the ADAM rWBC2 instrument. Results from both methods and tubes were compared using statistical analysis. The results from ADAM rWBC2 tended to be higher than the results from FCM, and the difference turned out to be statistically significant (p<0,001). No significant difference could be detected between the results from the different test tubes. The assessment is that ADAM rWBC2 will replace FCM for quality control of residual leukocytes in blood components. According to the results, the type of test tube used does not affect the leukocyte concentration.

In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro. IMPORTANCE Although most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofil

Extensively drug-resistant (XDR) Klebsiella pneumoniae inflict a notable burden on healthcare worldwide. Of specific concern are strains producing carbapenem-hydrolyzing enzymes, as the therapeutic options for these strains are still very limited. Specific sequence types of K. pneumoniae have been noted for their epidemic occurrence globally, but the mechanisms behind the success of specific clones remain unclear. Herein, we have characterized 20 high-risk clones (HiRCs) and 10 non-HiRCs of XDR K. pneumoniae, exploring factors connected to the epidemiological success of some clones. Isolates were subjected to core genome multilocus sequence typing analysis to determine the clonal relationships of the isolates and subsequently characterized with regard to features known to be linked to overall bacterial fitness and virulence. The genomes were analyzed in silico for capsule types, O antigens, virulence factors, antimicrobial resistance genes, prophages, and CRISPR-Cas loci. In vitro growth experiments were conducted to retrieve proxies for absolute and relative fitness for 11 HiRC and 9 non-HiRC isolates selected based on the clonal groups they belonged to, and infections in a Galleria mellonella insect model were used to evaluate the virulence of the isolates in vivo. This study did not find evidence that virulence factors, prophages, CRISPR-Cas loci, or fitness measured in vitro alone would contribute to the global epidemiological success of specific clones of carbapenemase-producing XDR K. pneumoniae. However, this study did find the HiRC group to be more virulent than the non-HiRC group when measured in vivo in a model with G. mellonella. This suggests that the virulence and epidemiological success of certain clones of K. pneumoniae cannot be explained by individual traits investigated in this study and thus warrant further experiments in the future.

Complete blood count (CBC) analysis, often provided by an automated hematology analyzer, is a fundamental diagnostic test for evaluating a patient’s overall health status as a tool in diagnosing various medical conditions. CBC provides insights into the composition of the blood cells with parameters such as Mean Corpuscular Volume (MCV) and Hemoglobin (HGB). MCV represents the average size and volume of red blood cells, while HGB indicates the oxygen-carrying capacity of the blood. Different technologies are used in hematology analyzers, such as the impedance method and spectrophotometry, to achieve precise measurements of MCV and HGB. However, exploring other methodologies is of interest to potentially reduce instrument complexity and cost. Flow cytometry, based on light scatter, provides detailed information on the characteristics of individual cells and is commonly used in CBC analysis to differentiate white blood cells and reticulocytes. However, while the potential of this method for investigating MCV and HGB levels is well established, it is of significant interest to determine if the measurement techniques can be streamlined from three to one by solely using flow cytometry in this prototype analyzer. In this thesis, the possible use of measuring MCV and HGB using a flow cytometry system based on optical scatter data from a prototype hematology analyzer has been examined. The need to sphere the red blood cells before the measurements have also been investigated to evaluate reagent needs. The results have been evaluated based on the correlation factor, accuracy and precision of the proposed optical method. It is shown that the optical method in this thesis, can be used to measure MCV and HGB. However, the necessity of sphering the cells remains. Furthermore, a comparison is made between the optical method and the Sysmex XN-1000 to evaluate the accuracy of the obtained values. Finally, possible improvements and future work are suggested., Blodstatus, utförd av automatiserade hematologiinstrument, är ett grundlägggande diagnostiskt test för att utvärdera en patients allmänna hälsotillstånd som ett verktyg för att diagnostisera olika medicinska tillsånd. Blodstatus ger inblick i blodets sammansättning med parameterar som Medelvolym av röda blodkroppar (MCV) och Hemoglobin (HGB). MCV representerar den genomsnittliga storleken och volymen av röda blodkroppar, medan HGB indikerar blodets syrebärande förmåga. Olika metoder används i hematologiinstrument, såsom impedansmetoden och spektrofotometri för att mäta MCV och HGB värden. Undersökning med hjälp av andra metoder för mätning av dessa parameter är dock utav intresse för att potentiellt minska instrument komplexitet och kostnader. Flödescytometri, baserad på ljusspridning, ger detaljerad information om egenskaperna hos enskilda celler och används ofta för att differentiera vita blodkroppar och reticulocyter. Även om potentialen att undersöka MCV- och HGB-nivåer med denna metod redan är väletablerad, är det av betydande intresse att undersöka om man kan minska mätmetoderna från tre till en, genom att enbart använda flödescytometri i detta prototypinstrument. I detta examensarbete har möjligheten att mäta MCV och HGB med hjälp av flödescytometri baserat på optisk spridningsdata från en prototyp hematologiinstrument undersökts. Behovet av att ”sfära” de röda blodkropparna innan mätningarna har också undersökts för att utvärdera reagensbehov. Resultaten har utvärderats utifrån den förslagna optiska metodens korrelationsfaktor, noggrannhet och precision. Resultatet visar att den optiska metoden som presenteras i denna uppsats, kan användas för att mäta MCV och HGB. Dock kvarstår behovet av att ”sfära” cellerna. Dessutom görs en jämförelse mellan den optiska metoden och Sysmex XN-1000 för att utvärdera noggrannheten hos de erhållna värdena. Till sist ges förslag på möjliga förbättringar och vidareutveckling av den optiska metoden för framtiden.

Die Partner des MoDiCon-Projekts haben einen innovativen Ansatz für ein vollautomatisiertes System zur Überwachung und Vorhersage der Trinkwasserqualität in Versorgungsnetzen entwickelt. Der Schwerpunkt lag auf dem Einsatz moderner Sensortechnologien, wie Fluoreszenzspektroskopie und Durchflusszytometrie, zur Bestimmung der Wasserqualität. Eine Schnittstelle zwischen der physischen Überwachung und den Simulations- sowie Optimierungswerkzeugen ermöglicht es Wasserversorgern, die potenziell Sicherheit ihrer Verteilungssysteme zu erhöhen. Für die reale Umsetzung ist eine praktische Testphase und entsprechende Investitionen in einem realen Wasserversorgungssystem erforderlich. Herausforderungen bei der praktischen Umsetzung des MoDiCon-Ansatzes umfassen notwendige Anpassungen in den Bereichen Sensorik, Aktoren (z.B. Ventile, Schieber und Pumpen) und weiteren Steuerelementen (z. B. Hydranten und Behälter). Zudem müssen verschiedene Druckzonen im Netz sowie Notfallentnahmen, wie etwa bei Feuerhydranten, Berücksichtigung finden., The partners of the MoDiCon project have developed an innovative approach for a fully automated monitoring and prediction system for drinking water quality in supply networks. The focus was on utilizing advanced sensor technologies, such as fluorescence spectroscopy and flow cytometry, to determine water quality. The interface developed between physical monitoring and simulation and optimization tools potentially enables water suppliers to ensure the safety of their distribution systems. For a practical implementation, a test phase in a real water supply system and corresponding investments are necessary. Challenges for the application of the MoDiCon approach include required modifications in sensor technology, actuators (such as valves, gates, pumps), and other control elements (e.g., hydrants, tanks). Additionally, the implementation necessitates consideration of different pressure zones in the network and the management of emergency withdrawals (e.g., fire hydrants), which have not yet been addressed., Bundesministerium für Bildung und Forschung (BMBF)

Flow cytometry with dual staining allows an exact quantification of live and dead cells. It is a potential tool for gaining new information on the mode-of-action of disinfection chemicals and supporting efficient microbial control in recirculating aquaculture systems (RAS). Here, we used a dual-staining flow cytometry to examine the disinfection efficiency of peracetic acid (PAA) against i) the fish pathogenic bacterium Aeromonas salmonicida and ii) on microbes present in RAS. The method allowed us to accurately quantify the A. salmonicida cell response to different PAA concentrations (1, 2, and 10 mg/L) in time and to calculate the PAA concentration needed to achieve 95% cell mortality (LC95 estimates). With the high PAA dose of 10 mg/L, LC95 was reached in <15 min, while with the intermediate dose of 2 mg/L, LC95 was reached only in the end of the 60 min trial and with the low 1 mg/L dose, the disinfection efficiency remained low. The addition of soluble organic matter increased the PAA decay and decreased the disinfection efficiency. In RAS water, disinfection efficiency increased with increasing PAA concentration (from 1 to 10 mg/L) but was significantly reduced with increasing organic matter concentration (TCOD from 7.6 to 30.3 mg O2/L). The increase in the proportion of dead cells was found to be a reliable proxy for disinfection efficiency in RAS water. In the pure cell culture, both the increase in the proportion of dead cells and the fraction of reduced live cells reflected the disinfection efficiency of PAA. Altogether, the study demonstrated the PAA mode-of-action on aquaculture microbes and showed that flow cytometry with dual staining allows fast and precise examination of microbial viability under different conditions.

Allium sect. Codonoprasum represents an evolutionarily young and rapidly radiating group ofbulbous geophytes, with a significant proportion of polyploids and minor morphological differencesbetween species. In the last 20 years, dozens of new species have been described from the Mediterranean.However, very little new information has been obtained on most of them since their description, especiallyin terms of cytogenetic, molecular and distribution data. Allium goumenissanum is a recently describedspecies of this section, known only from three nearby localities in northern Greece. During the last 10 years,we collected population samples of an unknown species of this section from the southern Balkans, whichseemed to be representative of A. goumenissanum. A variety of methods (cytogenetic, molecular,morphological and micromorphological) were used to compare the populations of an unknown species withthe morphologically similar species A. goumenissanum and A. stamatiadae. AFLP, ITS sequencing andgenome size analyses showed that all populations of the unknown species were in fact A. goumenissanumdistinct from A. stamatiadae. The characterization of the morphology and reproduction of A.goumenissanum is completed, and micromorphological and anatomical characteristics of the leaves of bothspecies are newly published. The distribution of A. goumenissanum, based on new data, now includes amore extensive area of north central Greece, and the species is found as a new taxon for Bulgaria.

Due to the high wastepaper recyclability and water-loop system closure, packaging paper mills struggle with increased fines, causing runnability issues. Cellulase enzymes are a preferred treatment choice for the improvement of the pulp refining in stock preparation area but are not widely used or easy to introduce in the production process. Different cellulase enzymes were tested, and those with the highest activity were introduced to the white-water (WW) samples with the aim to reduce fines content as potentially new enzyme applications on the paper machine. The first portion of the study involved the development of an experiment model to find and confirm the optimal enzyme process parameters (40 °C, pH 5.7, reaction time 3 h, and 0.18% v/v enzyme addition) for laboratory made white-water. The second portion of the study included turbidity, colloidal charge, flow cytometry (FCM), and chemical oxygen demand (COD) analysis on industrial and laboratory made white-water samples at optimized process parameters. Obtained results corresponded to reduced fines content in white-water samples, which justified commercial usage of cellulase enzymes on recycled paper machine short loop and potentially increased machine runnability without negative influence on wastewater treatment plant.

Our purpose was to characterize the pattern of B-cell subsets in children with a combined diagnosis of type 1 diabetes (T1D) and celiac disease (C) since children with single or double diagnosis of these autoimmune diseases may differ in peripheral B-cell subset phenotype patterns. B-cells were analyzed with flow cytometry for the expression of differentiation/maturation markers to identify transitional, naive and memory B-cells. Transitional (CD24hiCD38hiCD19+) and memory Bregs (CD24hiCD27+CD19+, CD1d+CD27+CD19+, CD5+CD1d+CD19+) were classified as B-cells with regulatory capacity. Children with a combined diagnosis of T1D and C showed a pattern of diminished peripheral B-cell subsets. The B-cells compartment in children with combined diagnosis had higher percentages of memory B subsets and Bregs, including activated subsets, compared to children with either T1D or C. Children with combined diagnosis had a lower percentage of naive B-cells (CD27-CD19+; IgD+CD19+) and an increased percentage of memory B-cells (CD27+CD19+; IgD-CD19+). A similar alteration was seen among the CD39+ expressing naive and memory B cells. Memory Bregs (CD1d+CD27+CD19+) were more frequent, contrary to the lower percentage of CD5+ transitional Bregs in children with a combined diagnosis. In children with either T1D or C, the peripheral B-cell compartment was dominated by naive cells. Differences in the pattern of heterogenous peripheral B-cell repertoire subsets reflect a shifting in the B-cell compartment between children with T1D and/or C. This is an immunological challenge of impact for the pathophysiology of these autoimmune diseases.

Chimeric Antigen Receptor (CAR) T cell therapy has produced revolutionary success in hematological cancers such as leukemia and lymphoma. Nonetheless, its translation to solid tumors faces challenges due to manufacturing complexities, short-lived in vivo persistence, and transient therapeutic impact. We introduce 'Drydux' - an innovative macroporous biomaterial scaffold designed for rapid, efficient in-situ generation of tumor-specific CAR T cells. Drydux expedites CAR T cell preparation with a mere three-day turnaround from patient blood collection, presenting a cost-effective, streamlined alternative to conventional methodologies. Notably, Drydux-enabled CAR T cells provide prolonged in vivo release, functionality, and enhanced persistence exceeding 150 days, with cells transitioning to memory phenotypes. Unlike conventional CAR T cell therapy, which offered only temporary tumor control, equivalent Drydux cell doses induced lasting tumor remission in various animal tumor models, including systemic lymphoma, peritoneal ovarian cancer, metastatic lung cancer, and orthotopic pancreatic cancer. Drydux's approach holds promise in revolutionizing solid tumor CAR T cell therapy by delivering durable, rapid, and cost-effective treatments and broadening patient accessibility to this groundbreaking therapy.

Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI—median fluorescence intensity—and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status.

Changes in the temperature or salinity of ocean waters can affect marine organisms at multiple trophic levels. Both environmental variables could have an impact on marine microalgae populations. Therefore, the effect of the combination of three levels of temperature (20, 24 and 28 °C), and three levels of salinity (33, 36, and 39 PSU) were evaluated on the growth of a multispecies community of five common species of phytoplankton: (one cyanobacteria, Synechococcus sp., and four microalgae, Chaetoceros gracilis, Amphidinium carterae, Pleurochrysis roscoffensis and Rhodomonas baltica). The co-culture was monitored by flow cytometry under controlled conditions in a 96 h study. The effect of both variables on dissolved oxygen concentrations was measured using the SDR SensorDish Reader system. The results demonstrated that Synechococcus sp., C. gracilis, and A. carterae displayed a high growth at the temperature of 28 °C combined with the lowest salinity assayed. However, salinity increases negatively affected the growth of P. roscoffensis and R. baltica. Decreased salinity combined with decreased temperature exhibited a higher net O2 production. The interaction of two environmental factors related to global change such as temperature and salinity can cause structural (community growth) and functional (net oxygen production) changes in a phytoplanktonic community.

Every living cell is surrounded by a cell membrane that is made up of amphipathicphospholipids, separating the aqueous solutions inside and outside the cell with ahydrophobic barrier. This compartmentalization is a prerequisite for life, but so arethe many molecules that are floating in the membrane, altering its properties andconnecting the inside with the outside. An important group are the transportproteins, that open ways for molecules and ions, that would normally be too large,too charged, or too polar to pass the membrane. The transport proteins are eitheractive transporters – like pumps – or passive transporters – like channels. In thisthesis, I put the spotlight on two types of channel proteins: Transient ReceptorPotential ion channels, that let ions pass when activated by temperature or pungentchemicals, and Aquaporins (AQP), that are mainly responsible for letting watercross the membrane.In my work, I have made an effort to study the structure of one TRP member inparticular, known as TRPA1 from pine weevil (Hylobius abietis). I had to find thebest possible conditions to solubilize the protein in detergents, and I have alsoinvestigated other tools such as nanodiscs to keep the protein stable in solution. Onemajor hurdle has always been the low yields, and it was therefore that a GFP-tag(Green fluorescent Protein) was added to the protein construct, to facilitate thetracking of the protein and evaluation of purification methods. Coupled with flowcytometry, a method for measuring fluorescence and scattering of individual cells,this proved very useful in designing an expression and purification protocol.The purified protein was used for Cryo-EM (Electron Microscopy), but theprotein was difficult to freeze on grids with a good homogeneous spread ofindividual particles. The use of SRCD (Synchrotron Radiation Circular Dichroism)proved more successful, and confirmed the secondary structure of the protein, andgave information on the temperature stability of the pro

This chapter is an extension of the original publication by Schraivogel et al. (Science 375:315–320, 2022) which described, for the first time, image-enabled and high-speed cell sorting based on the BD CellView technology. It summarizes the technical aspects of the instrument in an easy-to-digest form and provides example-based guidance toward implementation of the CellView-based image cell sorting technology. As an example, it explains how to use the image-enabled cell sorter to analyze the chemically induced fragmentation of the Golgi apparatus in HeLa cells—an experiment that was alluded to in the original publication but was not included in the manuscript due to space constraints. The chemically induced Golgi fragmentation sort illustrates an elegant example of the utility of image-enabled cell sorting as a significant expansion of the single-cell toolbox. It is such a striking phenotype when analyzed with image cytometry but undetectable when using conventional flow cytometry. Described in a straightforward and concise manner, this experiment serves as a standard system assurance for image-based cell sorters.

Diagnosing immediate drug hypersensitivity reactions (IDHRs) can pose a significant challenge and there is an urgent need for safe and reliable tests. Evidence has emerged that the basophil activation test (BAT), an in vitro assay that mirrors the in vivo response, can be a complementary test for many drugs. In this position paper, members of Task Force (TF) “Basophil activation test in the evaluation of Drug Hypersensitivity Reactions” from the European Academy of Allergy and Clinical Immunology (EAACI) present the data from a survey about the use and utility of BAT in IDHRs in Europe. The survey results indicate that there is a great interest for using BAT especially for diagnosing IDHRs. However, there are still main needs, mainly in the standardization of the protocols. Subsequently consensus-based recommendations were formulated for: (i) Technical aspects of BAT in IDHRs including type of sample, management of drugs, flow cytometry protocols, interpretation of the results; and (ii) Drug-specific aspects that should be taken into account when performing BAT in relation to betalactams, neuromuscular blocking agents, fluoroquinolones, chlorhexidine, opioids, radio contrast media, chemotherapeutics, biological agents, nonsteroidal anti-inflammatory drugs, COVID vaccine, and excipients. Moreover, aspects in the evaluation of pediatric population have also been considered. All this indicates that BAT offers the clinician and laboratory a complementary tool for a safe diagnostic for IDHRs, although its place in the diagnostic algorithm depends on the drug class and patient population (phenotype, geography, and age). The standardization of BAT is important for generalizing this method beyond the individual laboratory., Diagnosing immediate drug hypersensitivity reactions (IDHRs) can pose a significant challenge and there is an urgent need for safe and reliable tests. Evidence has emerged that the basophil activation test (BAT), an in vitro assay that mirrors the in vivo response, can be a complementary test for many drugs. In this position paper, members of Task Force (TF) “Basophil activation test in the evaluation of Drug Hypersensitivity Reactions” from the European Academy of Allergy and Clinical Immunology (EAACI) present the data from a survey about the use and utility of BAT in IDHRs in Europe. The survey results indicate that there is a great interest for using BAT especially for diagnosing IDHRs. However, there are still main needs, mainly in the standardization of the protocols. Subsequently consensus-based recommendations were formulated for: (i) Technical aspects of BAT in IDHRs including type of sample, management of drugs, flow cytometry protocols, interpretation of the results; and (ii) Drug-specific aspects that should be taken into account when performing BAT in relation to betalactams, neuromuscular blocking agents, fluoroquinolones, chlorhexidine, opioids, radio contrast media, chemotherapeutics, biological agents, nonsteroidal anti-inflammatory drugs, COVID vaccine, and excipients. Moreover, aspects in the evaluation of pediatric population have also been considered. All this indicates that BAT offers the clinician and laboratory a complementary tool for a safe diagnostic for IDHRs, although its place in the diagnostic algorithm depends on the drug class and patient population (phenotype, geography, and age). The standardization of BAT is important for generalizing this method beyond the individual laboratory.

This study investigates the anti-cancer properties of a novel composite coating comprising amorphous calcium phosphate (ACP), chitosan oligosaccharide lactate, and germanium (Ge) on titanium (Ti) substrates. The coating was applied using an in situ anodization/anaphoretic deposition process. The primary focus is on the germanium layer's effectiveness in targeting and eliminating cancerous cells. Experimental analysis included MTT assays and flow cytometry to evaluate cytotoxicity and cellular uptake, respectively. MTT assays were conducted on SCC-25 cancer cell lines and dental pulp stem cells (DPSC) over 1, 3, and 7 days. Results indicated a significant reduction in SCC-25 cell viability, particularly notable with the germanium-enhanced coating, which drastically reduced the number of cancerous cells. In contrast, DPSC showed minimal cytotoxic effects, demonstrating the selective nature of the coating. Flow cytometry further confirmed these findings, revealing that the germanium coating was effective in reducing cancer cell viability without significant penetration into healthy cells. This study suggests that germanium-coated Ti substrates with ACP and chitosan oligosaccharide lactate are highly effective in targeting cancer cells while preserving healthy cells, making this composite coating a promising candidate for anti-cancer therapies. Future research will focus on optimizing the coating process and expanding in vivo studies to further validate these findings.

Acute myeloid leukemia (AML) is a malignancy affecting bone marrow, characterized by abnormal cell maturation and proliferation. Initial treatment involves intensive chemotherapy aiming for complete remission (CR), followed by consolidation therapy. Consolidation options include chemotherapy alone, autologous stem cell transplant (auto-SCT), or allogeneic stem cell transplant (allo-SCT). Selecting the appropriate consolidation therapy balances anti-leukemic efficacy with safety concerns. While allo-SCT reduces relapse risk, it also carries significant morbidity, prompting cautious use. Measurable residual disease (MRD) is increasingly influential in AML management, indicating higher relapse risk when detected pre-consolidation. Measuring MRD is subject to strict protocols and guidelines to ensure consistent and accurate identification and detection of MRD. In addition, it is important that results are harmonized between centers so that results can be compared. Within Europe, this harmonization process is led by the European LeukemiaNet (ELN). Chapter 2 discusses the technical aspects of flow MRD, which is a consensus on the entire process from administration to implementation of MRD. In addition, when MRD is used for clinical decision-making, it must comply with reliability standards and rules set by the European Union, called in vitro diagnostics rules (IVDR). In Chapter 3 we show that our MRD assay meets all requirements set by the IVDR and therefore qualifies for clinical use. While using MRD to guide treatment remains complex, following these guidelines already provides a solid basis for use in practice. In the HOVON/SAKK-132 study in Chapter 4, the choice for SCT is based on the MRD result after initial chemotherapy. With an MRD-positive result in AML patients with an intermediate-risk, there is an increased chance of the disease relapsing and an allo-SCT is advised. Whereas, when a patient is classified as MRD-negative, de-escalation is done by omitting an all

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method., Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field., Ministerio de Ciencia, Innovación e Universidades, Agenda Estatal de Investigación, Fondo Europeo de Desarrollo Regional, Depto. de Inmunología, Oftalmología y ORL, Fac. de Medicina, TRUE, pub

This study examined the dynamic response of Spermatozoa DNA Fragmentation after sex selection in bulls using a MoFlo® SX (Beckman Coulter, Miami FL) spermatozoa sorter. The dynamic response of spermatozoa DNA fragmentation refers to the changing values of SDF, i.e., rate of SDF (rSDF), when analyzed periodically over a set incubation time at 37 °C. A dynamic assessment of SDF using non-sorted and sex-sorted spermatozoa samples during 72 h of incubation at 37 °C was performed. Results showed a reduced DNA longevity in sex-sorted frozen-thawed spermatozoa, with spermatozoa DNA damage appearing between 24 h and 48 h. The baseline SDF level was higher in conventional frozen-thawed than in sex-sorted frozen-thawed spermatozoa samples; while the reverse occurred for the rSDF. The afore-mentioned result produced a crossover point between both dynamic tendencies of SDF for sex-sorted versus conventional samples. We defined this crossover point as the Crossover Positioning Time (CPT) or the time (in hours) where both curves crossover after a period of spermatozoa incubation at 37 °C. The point at which the CPT occurs could be used as an indicator of the rSDF for individual bulls after X- and Y-chromosome bearing spermatozoa selection. CPT values produced a window of SDF ranging between 24 h and 48 h in the present experiment. It is proposed that higher values for CPT are indicative of bulls presenting chromatin that is more resistant to the external stressors affecting spermatozoa DNA after spermatozoa sorting., Ministerio de Ministerio de Educación y Ciencia, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

This study examined the static response of Spermatozoa DNA Fragmentation (SDF) after sex selection in bulls using a MoFlo® SX (Beckman Coulter, Miami FL) spermatozoa sorter to produce three different subpopulations: 1) Spermatozoa bearing X- chromosomes with a purity of 95%, 2) Spermatozoa bearing Y-chromosomes with a purity of 95%, and 3) non-viable spermatozoa. The static response of SDF refers to the baseline values observed for DNA damage when analyzed pre- and post sex-sorting. Results showed that while the baseline level SDF in pre-sorted bull spermatozoa samples ranged from 5.3% to 11% with an average of 7.9% ± 2.1%, the level of SDF obtained in X- and Y-chromosome sorted samples was much lower (3.1% ± 1.9%) and statistical differences were obtained after comparing both groups (P < 0.01). Spermatozoa containing a fragmented DNA molecule tend to be accumulated in the non-viable subpopulation. The baseline SDF level in X- and Y-chromosome sorted subpopulations is reduced, by 63% on average when compared to the values obtained in the neat semen sample. Different bulls exhibit unique SDF reduction efficiencies via the X- and Y-chromosome sex selection process., Ministerio de Ministerio de Educación y Ciencia, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Three novel triphenyltin(IV) compounds with modulated azo-carboxylato ligands: triphenylstannyl (E)-4-((2-hydroxynaphthalen-1-yl)diazenyl)benzoate, 1, triphenylstannyl (E)-4-((4-hydroxyphenyl)diazenyl)benzoate, 2, and triphenylstannyl (E)-4-((4-(dimethylamino)phenyl)diazenyl)benzoate, 3, were synthesized and characterized by elemental analysis, FTIR and NMR (1H, 13C, 119Sn) spectroscopy. The structures and spectra of compounds were predicted by Density Functional Theory (DFT) methods at B3LYP-D3BJ/6–311++G(d,p)(H,C,N,O)/LanL2DZ(Sn) level of theory. Furthermore, the antitumor potential of ligand precursors, HL1–HL3, and appropriate organotin(IV) compounds 1–3 was evaluated across mouse melanoma B16F1, human breast adenocarcinoma MCF-7, human colorectal HT-29 and human prostate PC3 cell lines using MTT and CV assays. The organotin(IV) compounds exhibit enhanced cellular uptake and efficacy in reducing viable cell numbers when compared to free acids. Specifically, compound 3 demonstrates a notable impact at lower nanomolar concentrations on all tested cell lines. Moreover, 3 induces cell death in MCF-7 cells by inhibiting cell division and promoting the overproduction of cellular nitric oxide (NO), ultimately leading to caspase-independent apoptosis. Importantly, this process occurs without concurrent activation of autophagy or the generation of ROS/RNS species. The binding affinity of 1–3 with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy and molecular docking simulations, suggesting their capacity to interact with these biomolecules.

Atypical chronic lymphocytic leukemia (CLL) is still defined according to morphological criteria. However, deviance from the typical surface immunological profile suggests an atypical immunological-based CLL. A large cohort of patients with CLL was retrospectively evaluated aiming at assessing morphological (FAB criteria), immunophenotypical (two or more discordances from the typical profile), and clinical–biological features of atypical CLL. Compared to typical cases, morphologically atypical CLL showed a greater percentage of unmutated IgVH and CD38 positivity, and a higher expression of CD20. Immunophenotypically atypical CLL was characterized by more advanced clinical stages, higher expression of CD20, higher rate of FMC7, CD79b and CD49d positivity, and by an intermediate–high expression of membrane surface immunoglobulin, compared to typical cases. When patients were categorized based on immunophenotypic and morphologic concordance or discordance, no difference emerged. Finally, morphological features better discriminated patients’ prognosis in terms of time-to-first treatment, while concordant atypical cases showed overall a worse prognosis. Discordant cases by immunophenotype and/or morphology did not identify specific prognostic groups. Whether—in the era of molecular markers used as prognostic indicators—it does make sense to focus on morphology and immunophenotype features in CLL is still matter of debate needing further research.