Regulatory T cells contribute to the immunosuppressive phenotype of neutrophils in a mouse model of chronic lymphocytic leukemia.

BACKGROUND: Impaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL. METHODS: In this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg. RESULTS: The obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the "healthy" phenotype of neutrophils. CONCLUSIONS: Altogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response.

Secretomes of M1 and M2 macrophages decrease the release of neutrophil extracellular traps.

The release of neutrophil extracellular traps (NETs) can be either beneficial or detrimental for the host, thus it is necessary to maintain a balance between formation and clearance of NETs. Multiple physiological factors eliciting NET release have been identified, yet the studies on natural signals limiting NET formation have been scarce. Accordingly, our aim was to analyze whether cytokines or immune cells can inhibit NET formation. To that end, human granulocytes were incubated with interleukin (IL)-4, IL-10, transforming growth factor beta-2 or adenosine and then stimulated to release NETs. Additionally, neutrophils were cultured in the presence of natural killer (NK) cells, regulatory T cells (Tregs), pro-inflammatory or anti-inflammatory macrophages (M1 or M2 macrophages), or in the presence of NK/Tregs/M1 macrophages or M2 macrophages-conditioned medium and subsequently stimulated to release NETs. Our studies showed that secretome of M1 and M2 macrophages, but not of NK cells and Tregs, diminishes NET formation. Co-culture experiments did not reveal any effect of immune cells on NET release. No effect of cytokines or adenosine on NET release was found. This study highlights the importance of paracrine signaling at the site of infection and is the first to show that macrophage secretome can regulate NET formation.

Trapalyzer: a computer program for quantitative analyses in fluorescent live-imaging studies of neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs), pathogen-ensnaring structures formed by neutrophils by expelling their DNA into the environment, are believed to play an important role in immunity and autoimmune diseases. In recent years, a growing attention has been put into developing software tools to quantify NETs in fluorescent microscopy images. However, current solutions require large, manually-prepared training data sets, are difficult to use for users without background in computer science, or have limited capabilities. To overcome these problems, we developed Trapalyzer, a computer program for automatic quantification of NETs. Trapalyzer analyzes fluorescent microscopy images of samples double-stained with a cell-permeable and a cell-impermeable dye, such as the popular combination of Hoechst 33342 and SYTOX™ Green. The program is designed with emphasis on software ergonomy and accompanied with step-by-step tutorials to make its use easy and intuitive. The installation and configuration of the software takes less than half an hour for an untrained user. In addition to NETs, Trapalyzer detects, classifies and counts neutrophils at different stages of NET formation, allowing for gaining a greater insight into this process. It is the first tool that makes this possible without large training data sets. At the same time, it attains a precision of classification on par with state-of-the-art machine learning algorithms. As an example application, we show how to use Trapalyzer to study NET release in a neutrophil-bacteria co-culture. Here, after configuration, Trapalyzer processed 121 images and detected and classified 16 000 ROIs in approximately three minutes on a personal computer. The software and usage tutorials are available at https://github.com/Czaki/Trapalyzer.

The Activity of Vitamin C Against Ovarian Cancer Cells Is Enhanced by Hyperthermia.

BACKGROUND/AIM: Vitamin C is essential for the proper functioning of the human body and plays a crucial role in many biological processes as a cofactor for enzymes. The anticancer activity of vitamin C has been indicated for years. Hyperthermia used in clinics allows increasing the effectiveness of anticancer therapies and may also be useful in enhancing the action of other substances. The purpose of this study was to enhance the anticancer activity of vitamin C through hyperthermia against ovarian cancer cells. MATERIALS AND METHODS: The ovarian cancer cell lines Caov-3, NIH:OVCAR-3, and human fibroblasts CCD-1064Sk were tested in the present study. Vitamin C was used in the following concentrations: 0.24, 2.50 and 5.25 mM. Each of the selected concentrations was combined with the different temperatures (37°C, 40°C and 43°C). Cell survival, adhesion and changes at the molecular level were assessed. RESULTS: The obtained results revealed that hyperthermia enhances the anticancer activity of vitamin C. Ovarian cancer cells showed greater sensitivity to vitamin C at elevated temperatures. Cells may have different sensitivity to vitamin C due to the activation of different gene signatures associated with redox reactions and apoptosis, therefore we examined the following genes: BCAP31, BCL2L13, BID, CASP7, FADD and HTRA2. The increase in expression of these genes in cancer cells generated a stronger proapoptotic response. CONCLUSION: The present study showed that hyperthermia enhanced the anticancer activity of vitamin C in vitro.

Lack of Functional P110δ Affects Expression of Activation Marker CD80 but Does Not Influence Functions of Neutrophils.

Neutrophils are specialized immune cells that are essential constituents of the innate immune response. They defend the organism against pathogens through various mechanisms. It was reported that phosphatidylinositols are key players in neutrophil functions, especially in the activity of class-I phosphoinositide 3-kinases (PI3Ks). P110δ, one of the PI3K subunits, is mostly expressed in immune cells, and its activity plays an important role in inflammatory responses. The aim of this study was to investigate the role of p110δ in neutrophil antimicrobial functions, activation status and cytokine production. To this end, we used bone marrow and splenic neutrophils isolated from a murine model expressing catalytically inactive p110δD910A/D910A. The level of phagocytosis and degranulation, the expressions of activation markers and cytokine production were determined by flow cytometry. ROS generation and NET release were assessed by fluorometry and fluorescent microscopy. We observed a significantly higher percentage of CD80-positive cells among the splenic granulocytes and found granulocytes subpopulations of differing phenotypes between WT and p110δD910A/D910A mice by multiparametric tSNE analysis. Moreover, we detected some differences in the expressions of activation markers, intracellular production of cytokines and bacterial killing. However, we did not observe any alterations in the selected neutrophil functions in p110δ mutant mice. Altogether, our data suggest that the catalytic p110 subunit(s), other than p110δ, is a key player in most neutrophil functions in mice. A follow-up study to correlate these in vitro results with in vivo observations is highly recommended.

Iron excess affects release of neutrophil extracellular traps and reactive oxygen species but does not influence other functions of neutrophils.

Neutrophils apply several antimicrobial strategies including degranulation, phagocytosis, the generation of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs) to fight pathogens. Iron is considered to be an invaluable constituent of host immune defense and plays a dual role in immunity. It is a well-known component of antimicrobial proteins and is a necessary microelement for pathogen survival. The aim of this study was to broaden the knowledge regarding the impact of iron on the function of neutrophils. Neutrophils from healthy blood donors and patients with mild iron-deficiency anemia and HL-60 cells differentiated toward granulocyte-like cells were incubated with Fe2+ , Fe3+ or holo-transferrin (holo-Tf). Moreover, we isolated murine neutrophils of HFE gene knockout (KO) mice and mice fed iron-deficient, iron-equivalent and high-iron diets. We analyzed the release of NETs, phagocytosis, degranulation of azurophilic granules, ROS release, bactericidal activity of granulocytes against Escherichia coli and neutrophil elastase (NE) activity. We show that holo-Tf inhibits the release of NETs stimulated by phorbol 12-myristate 13-acetate by inhibiting NE activity. Studies performed in mice models reveal that iron overload inhibits the release of NETs and ROS production in neutrophils isolated from HFE KO mice and mice fed a high-iron diet. No impact of a low-iron diet on neutrophil phagocytosis, ROS production or release of NETs was observed. Our study underscores the physiological significance of iron in neutrophil function, specifically in the release of NETs.

Influence of iron- and zinc-chelating agents on neutrophil extracellular trap formation.

Release of neutrophil extracellular traps (NETs) is one of the neutrophils' mechanisms involved in the response to infection. NETs are released from the cell in response to a biological or synthetic stimulus to entrap, immobilize and kill pathogens. Metal ions and metal binding proteins were identified in the structure of NETs, but their role in NET release remains unclear. The aim of this study was to assess how lack of iron and zinc generated by ion sequestration using chelators affects NET release. Neutrophils were isolated from whole blood or buffy coats of healthy blood donors by density gradient centrifugation and incubated with zinc chelators: 20 µM N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), 40 µM diethylenetriaminepentaacetic acid (DTPA) or iron chelators: 400 µM deferoxamine mesylate salt (DFO) and 50 µM iminodiacetic acid (IDA). Next, 100 nM phorbol 12-myristate 13-acetate (PMA) was added to stimulate release of NETs. The amount of released DNA was measured by fluorometry and NETs were visualized by immunofluorescence microscopy. This study demonstrates that iron and zinc chelators are able to modulate NET release. Here we show that preincubation of neutrophils with TPEN and IDA inhibits NET release in cells stimulated with PMA. On the other hand, DFO stimulates NET release. Incubation of cells with DTPA does not affect release of NETs.

Zinc Supplementation Modulates NETs Release and Neutrophils' Degranulation.

Zinc plays an important physiological role in the entire body, especially in the immune system. It is one of the most abundant microelements in our organism and an essential component of enzymes and antibacterial proteins. Zinc levels were reported to be correlated with the intensity of innate immunity responses, especially those triggered by neutrophils. However, as the results are fragmentary, the phenomenon is still not fully understood and requires further research. In this study, we aimed to perform a comprehensive assessment and study the impact of zinc on several basic neutrophils' functions in various experimental setups. Human and murine neutrophils were preincubated in vitro with zinc, and then phagocytosis, oxidative burst, degranulation and release of neutrophil extracellular traps (NETs) were analyzed. Moreover, a murine model of zinc deficiency and zinc supplementation was introduced in the study and the functions of isolated cells were thoroughly studied. We showed that zinc inhibits NETs release as well as degranulation in both human and murine neutrophils. Our study revealed that zinc decreases NETs release by inhibiting citrullination of histone H3. On the other hand, studies performed in zinc-deficient mice demonstrated that low zinc levels result in increased release of NETs and enhanced neutrophils degranulation. Overall, it was shown that zinc affects neutrophils' functions in vivo and in vitro. Proper zinc level is necessary to maintain efficient functioning of the innate immune response.

Overexpression of ATG5 Gene Makes Granulocyte-Like HL-60 Susceptible to Release Reactive Oxygen Species.

Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils' antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.

Design and in vitro evaluation of electrospun shape memory polyurethanes for self-fitting tissue engineering grafts and drug delivery systems.

Integration of multiple features including shape memory, biodegradation, and sustained drug delivery in a single material offers the opportunity to significantly improve the abilities of implantable devices for cardiovascular system regeneration. Two types of shape memory polyurethanes (SMPUs): PU-PLGA and PU-PLLA/PEG differing in soft segments composition that comprising blends of various biodegradable polyols, i.e. D,l-lactide-co-glycolide diol (o-PLGA), poly(e-caprolactone) diols (o-PCL) with various molecular weights, poly-l-lactide diol (o-PLLA), polyethylene glycol (o-PEG) were synthesized and further utilized to electrospun nanofibrous - rapamycin (Rap) delivery system. Structure characterization by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DCS) and hydrophilicity measurements were performed to gain more insights on the influence of the particular units of the softs segments on the transition temperature (Ttrans), shape recovery, degradation profile, and drug release kinetics. In vitro study in PBS solution revealed that incorporation of o-PLGA segments to SMPUs is favorable over o-PEG as increased shape memory performance was observed. Moreover, presence of PLGA in PU-PLGA gave more predictable degradation profile in comparison to PU-PLLA/PEG system. Human Cardiac Fibroblasts (HCF) viability tests in vitro confirmed that the amount of Rap released from evaluated PU-PLLA/PEG/Rap and PU-PLGA/Rap drug delivery systems was sufficient to inhibit cells growth on the surface of the tested materials.

Convolutional Neural Networks-Based Image Analysis for the Detection and Quantification of Neutrophil Extracellular Traps.

Over a decade ago, the formation of neutrophil extracellular traps (NETs) was described as a novel mechanism employed by neutrophils to tackle infections. Currently applied methods for NETs release quantification are often limited by the use of unspecific dyes and technical difficulties. Therefore, we aimed to develop a fully automatic image processing method for the detection and quantification of NETs based on live imaging with the use of DNA-staining dyes. For this purpose, we adopted a recently proposed Convolutional Neural Network (CNN) model called Mask R-CNN. The adopted model detected objects with quality comparable to manual counting-Over 90% of detected cells were classified in the same manner as in manual labelling. Furthermore, the inhibitory effect of GW 311616A (neutrophil elastase inhibitor) on NETs release, observed microscopically, was confirmed with the use of the CNN model but not by extracellular DNA release measurement. We have demonstrated that a modern CNN model outperforms a widely used quantification method based on the measurement of DNA release and can be a valuable tool to quantitate the formation process of NETs.

Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps.

Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.

The Brain Entangled: The Contribution of Neutrophil Extracellular Traps to the Diseases of the Central Nervous System.

Under normal conditions, neutrophils are restricted from trafficking into the brain parenchyma and cerebrospinal fluid by the presence of the brain-blood barrier (BBB). Yet, infiltration of the central nervous system (CNS) by neutrophils is a well-known phenomenon in the course of different pathological conditions, e.g., infection, trauma or neurodegeneration. Different studies have shown that neutrophil products, i.e., free oxygen radicals and proteolytic enzymes, play an important role in the pathogenesis of BBB damage. It was recently observed that accumulating granulocytes may release neutrophil extracellular traps (NETs), which damage the BBB and directly injure surrounding neurons. In this review, we discuss the emerging role of NETs in various pathological conditions affecting the CNS.

Influence of Different Bacteria Strains Isolated from Septic Children on Release and Degradation of Extracellular Traps by Neutrophils from Healthy Adults.

Neutrophils are the first line of immune defense against pathogens. They use three major antimicrobial mechanisms: phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). NETs are structures which consist of nuclear DNA conjugated with antibacterial proteins. They are formed to entrap and kill pathogens. The aim of the study was to evaluate the influence of Escherichia coli (E. coli), Streptococcus pneumoniae (S. pneumoniae), Stenotrophomonas maltophilia (S. maltophilia), and Pseudomonas aeruginosa (P. aeruginosa), isolated from the peripheral blood of children with sepsis, on the release and degradation of NETs by neutrophils isolated from blood healthy adult subjects. Neutrophils were stimulated with the bacterial strains outlined above. The quantitative and qualitative analyses of NETs release were performed by fluorometric measurement and immunofluorescence, respectively. The ability of bacteria to degrade NETs was studied qualitatively. Oxidative burst was assessed by flow cytometry. Histone H3 citrullination was evaluated by Western blot. We found that NETs were formed only when neutrophils were incubated with S. pneumoniae. E. coli, P. aeruginosa, and S. maltophilia did not induce the release of the NETs. P. aeruginosa, S. pneumoniae, and E. coli induced the production of reactive oxygen species (ROS) by neutrophils. Two studied bacterial strains (S. pneumoniae and E. coli) were able to degrade NETs. However, none of the strains induced the citrullination of histone H3. We conclude that the ability of bacteria to induce and degrade NETs depends on the specific bacterial strain.

The influence of agents differentiating HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps.

Studies on neutrophil extracellular traps (NETs) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL-60) cells toward granulocyte-like cells able to release NETs. HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NETs release were measured fluorometrically and analyzed microscopically. LC3-II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL-60 cells toward granulocyte-like cells. ATRA-dHL-60 cells released NETs only upon PMA stimulation, DMSO-dHL-60 cells only post CI stimulation, while DMF-dHL-60 cells formed NETs in response to both stimuli. Oxidative burst was induced in ATRA-, DMSO- and DMF-dHL-60 cells post PMA stimulation and only in DMF-dHL-60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO- and DMF-, but not in ATRA-dHL-60 cells. The calcium influx was diminished in ATRA-dHL-60 cells. Significant increase in autophagosomes formation was observed only in PMA-stimulated DMF-dHL-60 cells. Phagocytic index was higher in ATRA-dHL-60 cells than in control, DMSO- and DMF-dHL-60 cells. We conclude that ATRA, DMSO and DMF differentiate HL-60 in different mechanisms. DMF is the best stimulus for HL-60 cell differentiation for NETs studies.

The Diagnostics of Human Steroid Hormone Disorders.

Disturbances of the steroidogenesis or altered peripheral metabolism of steroids may result in severe clinical manifestations. Therefore, prompt diagnosis and initiation of medical treatment are desirable. The diagnostics of disorders of steroid hormone production, metabolism, and action have been previously based on immunoassay tests. However, in a modern medical laboratory, due to low accuracy of immunoassays, this technique is continuously replaced by chromatographic separation methods coupled to mass spectrometric detection systems. In this review we present current advances in the diagnostics of adrenal gland disorders, focusing on the role of mass spectrometry in prenatal and newborn screening, and in the diagnostics of sexual maturation disorders.

Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release.

Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Antibiotics have an immunomodulating effect, and they can influence the properties of numerous immune cells, including neutrophils. The aim of this study was to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of NETs by neutrophils. Neutrophils were isolated from healthy donors by density-gradient centrifugation method and incubated for 1 h with the studied antibiotics at different concentrations (0.5, 10 and 50 µg/mL-azithromycin and 10 and 50 µg/mL-chloramphenicol). Next, NET release was induced by a 3 h incubation with 100 nM phorbol 12-myristate 13-acetate (PMA). Amount of extracellular DNA was quantified by fluorometry, and NETs were visualized by immunofluorescent microscopy. Degranulation, apoptosis and respiratory burst were assessed by flow cytometry. We found that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of NETs. Moreover, azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils. Chloramphenicol did not affect degranulation, apoptosis nor respiratory burst. It can be concluded that antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity.

UriSed - Preliminary reference intervals and optimal method for urine sediment analysis in newborns and infants.

OBJECTIVES: The aim of this study was to establish reference intervals for urine sediment in newborns and infants in the second month of life for the UriSed automated analyser and for bright field microscopy. We also aimed to provide an optimal protocol for UriSed analysis, which best corresponds to the results of manual microscopy. DESIGN AND METHODS: Urine sediment analyses of 75 healthy newborns and infants in the second month of life were performed by manual microscopy and UriSed automated analyser (two modes: 15 and 20 images per sample). Images were then reviewed and manually corrected by an operator when needed. RESULTS: We observed statistically significant differences between bright-field microscopy and UriSed (when manual correction was not performed) for squamous epithelial cells and red blood cells counts (P<0.0001). There were no differences based on the number of images per sample (P>0.05). Upper reference values for bright-field microscopy and UriSed analyser taking 15 images per sample with manual correction (method we recommend) were as follows: squamous epithelial cells: microscope 8.7×10(6)/l, UriSed 6.4×10(6)/l, non-squamous epithelial cells: microscope 4.3×10(6)/l, UriSed 3.9×10(6)/l; erythrocytes: microscope 5.9×10(6)/l, UriSed: 4.6×10(6)/l; leukocytes: microscope 8.6×10(6)/l, UriSed 9.9×10(6)/l; hyaline casts: microscope 0×10(6)/l, UriSed (no correction) 0.7×10(6)/l. CONCLUSIONS: We established preliminary reference intervals for urine sediment analysis in newborns and infants for UriSed and bright-field microscopy. We concluded that for routine laboratory examination of non-pathological urine it is enough to use the faster mode, with 15 images per sample, followed by a manual correction.